WO2016205902A2 - Compositions et méthodes pour le traitement de biofilms - Google Patents

Compositions et méthodes pour le traitement de biofilms Download PDF

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WO2016205902A2
WO2016205902A2 PCT/BE2016/000028 BE2016000028W WO2016205902A2 WO 2016205902 A2 WO2016205902 A2 WO 2016205902A2 BE 2016000028 W BE2016000028 W BE 2016000028W WO 2016205902 A2 WO2016205902 A2 WO 2016205902A2
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seq
biofilm
peptide
fungal
antifungal agent
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PCT/BE2016/000028
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English (en)
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WO2016205902A8 (fr
WO2016205902A3 (fr
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Bruno Cammue
Tanne COOLS
Jan Wouter Drijfhout
Karin Thevissen
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Katholieke Universiteit Leuven Ku Leuven Research & Development
Academisch Ziekenhuis Leiden
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Priority claimed from GBGB1511051.3A external-priority patent/GB201511051D0/en
Priority claimed from GBGB1511447.3A external-priority patent/GB201511447D0/en
Application filed by Katholieke Universiteit Leuven Ku Leuven Research & Development, Academisch Ziekenhuis Leiden filed Critical Katholieke Universiteit Leuven Ku Leuven Research & Development
Publication of WO2016205902A2 publication Critical patent/WO2016205902A2/fr
Publication of WO2016205902A3 publication Critical patent/WO2016205902A3/fr
Publication of WO2016205902A8 publication Critical patent/WO2016205902A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • compositions and methods for the treatment or prevention of microbial biofilms preferably a fungal biofilm, more preferably a Candida biofilm. More in particular, said compositions and methods comprise combining an antifungal agent, with a potentiating compound, preferably a plant defensin or peptide derivatives thereof, for increasing the antibiofilm activity of the antifungal agent, and for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation in a subject and/or on a surface or other medium susceptible to biofilm formation.
  • a potentiating compound preferably a plant defensin or peptide derivatives thereof
  • Biofilms of microbial pathogens consist of dense layers of microorganisms surrounded by an extracellular polymeric matrix, adherent to a surface, thereby protecting the microbes from the action of antimicrobial agents, and are believed to be involved in at least 80% of human microbial infections.
  • biofilms are critical to the development of clinical infections in general. Due to the increasing number of immunocompromised patients, combined with the advances in medical technology, fungi have emerged as a major cause of infectious disease, with Candida sp., particularly C. albicans being the major pathogen.
  • Candida sp. are known to form biofilms upon contact with various surfaces.
  • C. albicans cells are able to colonize and subsequently form biofilms on both host cutaneous or mucosal surfaces and on indwelling medical implants and devices, such as (dental) implants, intravascular and urinary catheters, (voice) prostheses and heart valves.
  • C. albicans is an opportunistic human fungal pathogen, causing not only superficial infections, but also life-threatening systemic diseases.
  • C. albicans is now recognized as the fourth most common cause of bloodstream infections in the United States, with a high attributable mortality rate, namely 20-40%.
  • Candida sp. also play a predominant role in mixed-species fungal biofilms. Such biofilm cells are tolerant towards most conventional antimycotics and there are only few novel agents that can be used to treat biofilm-related infections. To date, only miconazole, caspofungin, anidulafungin and liposomal formulations of amphotericin B are used to effectively treat these infections, and hence, there is a need to identify novel antibiofilm compounds. All the currently marketed antifungal drugs have major drawbacks, including no broad- spectrum activity, no per oral absorption, side-effects, low antifungal activity, no fungicidal activity, drug-drug interactions and/or high costs.
  • Persisters are antibiotic-tolerant cells that survive treatments with high antibiotic concentrations. Because they start growing again when the antibiotic pressure drops, persisters are considered as one of the most important reasons for the recurrence of biofilm-associated infections.
  • Plant defensins are present in all plant families, including the Brassicaceae, Fabaceae and Solanaceae. Plant defensins are small, basic, Cysteine-rich peptides with a length of approximately 45-54 amino acids. Their structure typically comprises a conserved structure known as a Cysteine-stabilized ⁇ -motif with a prominent a-helix and a triple-stranded antiparallel ⁇ -sheet that is stabilized by four disulphide bridges. Plant defensins exhibit antimicrobial activity against a broad range of microorganisms, whereas they are in general non-toxic to human cells.
  • ROS reactive oxygen species
  • HsAFPI is a plant defensin found in coral bells ("Heuchera sanguinea"), which was previously characterized by Osborn and colleagues (2). HsAFPI inhibits the growth of various plant pathogenic fungi, including Botrytis cinerea, Verticillium albo-atrum and Fusarium culmorum, and causes swelling of germ tubes and hyphae in the latter (2). In addition, it was reported that HsAFPI shows antifungal activity against Saccharomyces cerevisiae and the human pathogen C. albicans, and induces apoptosis in the latter (3). Furthermore, it was shown that HsAFPI has a low in vitro frequency of resistance occurrence in planktonic C. albicans cultures (i.e.
  • the present invention addresses the increasing problems of biofilms, in particular of fungal biofilms on different surfaces including host tissue and medical devices, outside or within the human body and which escape conventional antifungal treatment.
  • the present invention provides novel compositions and methods with improved antibiofilm properties by enhancing the efficacy of antifungal drugs, such as by increasing the susceptibility and sensitivity of biofilms, particularly of fungal biofilms to said drugs and/or by a continued, highly localised treatment with said drugs.
  • plant defensin HsAFPI (SEQ ID No. 1 ), and/or peptide derivatives (SEQ ID Nos. 2 to 17) thereof, are biofilm inhibiting agents and potentiating compounds, which increase the antibiofilm inhibiting and eradicating activity of antifungal agents, preferably of the echinocandin or polyene class, more preferably caspofungin, anidulafungin, micafungin, or amphotericin B, against Candida biofilms, preferably against C. albicans biofilms.
  • biofungal agents preferably of the echinocandin or polyene class, more preferably caspofungin, anidulafungin, micafungin, or amphotericin B, against Candida biofilms, preferably against C. albicans biofilms.
  • the present invention relates to compounds of a peptidic nature which inhibit microbial biofilm formation and development, for use in suppressing, reducing, inhibiting, controlling, treating or preventing microbial biofilm formation, and therefore can be formulated into antibiofilm compositions for administration to humans and animals and for application to inert surfaces susceptible to infection by microbial biofilms.
  • the present invention also provides a method for suppressing, reducing, inhibiting, controlling, treating or preventing the development of a microbial biofilm on a biotic or abiotic surface or in a subject, which comprises the step of exposure or administration of such a peptide or composition on said surface or to the subject.
  • An isolated peptide wherein said peptide comprises an amino acid sequence with at least 70% sequence identity to amino acid sequences GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No. 10) or a derivative thereof wherein X stands for Cystein or a-aminobutyric acid, wherein said peptide consists of 12 to 44 amino acids, and wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • the isolated peptide according to statement 1 wherein said peptide comprises a sequence with at least 70% sequence identity to the amino acid sequences GAXHYQFPSVKX (SEQ ID No. 8), GAXHYQFPSVKXFXKR (SEQ ID No. 11 ) or AYGGAXHYQFPSVKX (SEQ ID No. 12), or a derivative thereof and wherein said peptide consists of 12 to 24 amino acids.
  • FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or a derivative thereof.
  • EH FAYGGAXH YQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof.
  • compositions for use in treatment of prevention of a fungal biofilm associated condition or infection in a human or animal subject comprising at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.
  • composition of statement 9 wherein said at least one antifungal agent is selected from the group of echinocandins or polyenes.
  • composition of statement 9, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B.
  • composition of any one of the statements 9 to 11 further comprising one or more pharmaceutically acceptable compounds, carries and/or adjuvants.
  • composition of statement 13 wherein said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.
  • a method for reducing, eradicating, inhibiting or preventing fungal biofilms or fungal biofilm formation characterized in that a surface or medium outside the body of a human or animal subject carrying said fungal biofilm or susceptible to said fungal biofilm formation, is exposed to at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1.
  • said at least one antifungal agent is selected from the group of echinocandins or polyenes. 19. The method of statement 17, wherein said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B.
  • a method for the treatment or prevention of a condition or infection associated with fungal biofilm development in a human or animal subject comprising administering to said human or animal subject a composition comprising at least one peptide according to any one of the statements 1 to 6, or according to SEQ ID No. 1 and at least one antifungal agent.
  • Figure 1 shows the sequence alignment of HsAFPI with other plant defensins.
  • A Amino acid sequence alignment of NaD1 , Psd1 , MtDef4, RsAFPI , RsAFP2 and HsAFPI , matching their Cysteine residues (numbered l-VIII). Multiple alignment was performed using the COBALT alignment tool. Cysteine-pairing is shown at the top of the figure. Highly conserved residues are shown in vertical boxes; (-) denote gaps in the alignment. Horizontal boxes represent peptide fragments that exhibit antifungal activity similar to the parental peptide, and hence, are important for antifungal activity. The large box indicates the position of the v- core.
  • Fig. 2 shows the secondary shift analysis of rHsAFPI , pH 4.0 at 298 K. Regions of a-helix and ⁇ -strand are indicated at the top of the figure.
  • Fig. 3 shows the three-dimensional structure of rHsAFPI .
  • A A family of 20 lowest energy structures superimposed over all backbone heavy atoms;
  • B A ribbon representation with disulfide bonds shown by arrows. The termini are labeled as N and C. Diagrams were generated using MOLMOL.
  • Fig. 4 shows scanning electron microscopy images of 4 hours-old biofilms, grown in the presence or absence (untreated) of 11.8 ⁇ rHsAFPI . Images at multiple magnifications (500x, 1000x and 2000x) are presented.
  • Fig. 6 shows cell viability and cell proliferation of HepG2 cells treated with rHsAFPI .
  • HepG2 cells were treated with water (control treatment) or rHsAFPI (0.01 ⁇ - 42 ⁇ ) for 24 hours.
  • Cell viability and cell proliferation were determined by XTT staining and BrdU staining, respectively, and results were expressed relative to cells receiving control treatment. Mean and SEM of three experiments in quadruplicate is shown(Unpaired Student t-test; P ⁇ 0.05 was defined as statistically significant).
  • Fig. 7 shows representation of the HsLin peptides imposed on the rHsAFPI structure, according to the amino acid sequence.
  • HsLin peptides are shown as a thick line in the same orientation as rHsAFPI ; other residues of rHsAFPI , not present in the HsLin peptide, are shown as a thin line.
  • the Cysteine residues are replaced by a-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSa3 scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFPI .
  • Fig. 10 shows HsLin06_18 potentiation of (A) caspofungin, (B) micafungin and (C) anidulafungin for C. albicans biofilm formation inhibition, using microtiter plates.
  • Dose- response curves of the echinocandin (caspofungin, micafungin and anidulafungin) with a concentration series of HsLin06_18 were presented respectively in A, B and C, with the different connecting lines corresponding to the HsLin06_18 concentration.
  • Black arrows represent HsLin06_18's potentiation effect.
  • Means ⁇ SEM of triplicates are represented.
  • Fig. 12 shows HsLin06_18 does not affect HepG2 cell viability.
  • HepG2 cells were treated with water (control treatment) or HsLin06_18 (0.09 ⁇ - 93 ⁇ ) for 24 hours.
  • Cell viability was determined by MTT staining, and results were expressed relative to cells receiving the control treatment. Mean and SEM of two experiments in quadruplicate is shown. No statistically significant differences were found in cell viability between untreated (control treatment) and HsLin06_18-treated cells up to the highest tested HsLin06_18 concentration (i.e. 93 ⁇ ) (One way ANOVA-Tukey test; P ⁇ 0.05 was defined as statistically significant).
  • Fig. 13 shows potentiation of caspofungin with (A) HsLin06, (B) HsLin06_18 and (C) HsLin06_18_13 for C. albicans biofilm formation inhibition.
  • Dose response curves of caspofungin in the presence of subinhibitory concentrations of HsLin06, HsLin06_18 or HsLin06_18_13 are represented.
  • the lines and corresponding numbers in the graphs indicate the different HsLin doses for each curve ( ⁇ ).
  • the inventors have shown that the plant defensin HsAFPI (SEQ ID No. 1 ) or peptide derivatives (SEQ ID Nos. 2 to 17) thereof show inhibitory activity against microbial biofilms and that said plant defensin and peptide derivatives thereof are potentiating compounds which act synergistically with antifungal agents, such as caspofungin, anidulafungin, micafungin and amphotericin B, against microbial biofilms and/or planktonic cells, increasing the susceptibility and sensitivity of microbial biofilms and/or of planktonic cells, in particular fungal biofilms or cells, such as Candida biofilms, to said antifungal drugs.
  • antifungal agents such as caspofungin, anidulafungin, micafungin and amphotericin B
  • the present invention provides an isolated peptide or derivatives thereof, or a composition comprising said isolated peptide, and at least one antifungal agent, preferably an echinocandin or a polyene, more preferably caspofungin, anidulafungin, micafungin, or amphotericin B, for use in the treatment or prevention of fungal biofilms or fungal biofilms associated infections or diseases, particularly Candida biofilms, more particularly C. albicans biofilms.
  • the compositions provide means for carrying out the methods of the invention.
  • the present invention therefore overcomes the increasing problems of biofilms, in particular of fungal biofilms on different biotic and abiotic surfaces such as on medical devices outside or within the human body and which escape conventional antifungal treatment.
  • reducing means complete or partial inhibition (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of microorganisms or biofilm formation (in the term of number of remaining cells) and/or development and also includes within its scope the reversal of microorganisms or biofilm development or processes associated with microorganisms or biofilm formation and/or development. Further, inhibition may be permanent or temporary.
  • microorganisms or biofilm formation and/or development may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days).
  • the inhibition of microorganisms or a biofilm is complete and/or permanent (no persisters) ("eradicating").
  • preventing or the like in reference to microorganisms or a biofilm or biofilm formation means complete or partial prevention (more than 50%, preferably more than 90%, still more preferably more than 95% or even more than 99%) of microorganisms or biofilm formation (in the term of number of remaining cells) and also includes within its scope processes associated with microorganisms or biofilm formation. Further, prevention may be permanent or temporary. In terms of temporary prevention, microorganisms or biofilm formation may be inhibited for a time sufficient to produce the desired effect (for instance at least 5 days, preferably at least 10 days). Preferably, the prevention of microorganisms or biofilm is complete and/or permanent.
  • exposing means administering to, or otherwise bringing into contact with.
  • a microorganism or biofilm may be exposed to an active agent directly or indirectly.
  • direct exposure refers to administration of the agent to the microorganism or biofilm to be treated or otherwise bringing the microorganism or biofilm into contact with the agent itself.
  • indirect exposure refers to the administration of a precursor of the active agent or a compound or molecule capable of generating, either solely or in reaction with other compounds or molecules, the active agent to the microorganism or biofilm or otherwise bringing the microorganism or biofilm into contact therewith.
  • the terms “treat” and “treating” and variations thereof as used herein mean administering to, or otherwise bringing into contact with.
  • microorganism sometimes referred to as a microbe, is any organism too small to be visible to the naked eye. Bacteria, viruses, protozoans, fungi and some algae are microorganisms.
  • antifungal means that a compound or composition of the present invention reduces, eradicates, inhibits or prevents the growth or proliferation of a fungus.
  • biofilm refers to a mode of microbial growth comprising sessile cells, usually within a complex and highly heterogeneous matrix of extracellular polymers, and characterized by a reduced sensitivity to antifungal agents.
  • biofilms can contain single species (e.g. a fungi/yeast such as C. albicans) or multiple species microorganisms (such as C. albicans, C. glabrata and other microorganisms, preferably yeasts and/or fungi or even prokaryotes).
  • said biofilm is a fungal biofilm, more preferably a Candida species biofilm, comprising C. albicans, C. glabrata, and/or C. /cruse/, an Aspergillus species (e.g. A. flavus, A. fumigatus, A. clavatus) biofilm or a Fusarium species (e.g. F.
  • fungal biofilm refers to a biofilm comprising fungal species
  • Candida albicans biofilm refers to a biofilm comprising Candida species.
  • C. albicans refers to a percentage (number of C. albicans celhtotal cell) in the biofilm. Preferably the percentage is above 50%, more preferably above 75%, still more preferably above 90% and/or this term refers to the fact that C. albicans is present in a concentration sufficient to provoke the biofilm.
  • the biofilms are associated with microbial infection (e.g., burns, wounds or skin ulcers) or a disease condition including, without limitation, dental caries, periodontal disease, prostatitis, osteomyelitis, septic arthritis, and cystic fibrosis.
  • microbial infection e.g., burns, wounds or skin ulcers
  • a disease condition including, without limitation, dental caries, periodontal disease, prostatitis, osteomyelitis, septic arthritis, and cystic fibrosis.
  • biofilms are associated with a microbial (fungal) infection on medical devices like indwelling intravascular catheters and in the oral cavity (e.g. on dental implants).
  • Biofilms may be associated with a surface, e.g., a solid support surface.
  • Such surface can be the surface of any structure in animals or humans.
  • such surface can be any epithelial surface, mucosal surface, or any host surface associated with microbial infection, e.g. persistent and chronic microbial infections.
  • the surface can also include any surface of a bio-device in an animal or human, including without limitation, bio- implants such as dental implants, bone prostheses, heart valves, pacemakers and indwelling catheters.
  • the microbial or fungal biofilm can also be associated with the oral cavity, including the surface of dental implants or speech prostheses.
  • the surfaces can also be any surface associated with industrial biofilm formation.
  • the surfaces being treated can be any surface associated with biofouling of pipelines, heat exchangers, air filtering devices, or contamination of computer chips or water-lines in surgical units like those associated with dental hand-pieces.
  • controlled release refers to a relatively slow or delayed or prolonged release of a bio-active compound from a device in its environment. Particularly, an 80% release of the bio-active compound into an aqueous fluid at a pH between 1.0 and 8.0 is only obtained when a period of time of at least 30 minutes, preferably at least 60 min, at least 24 hours, or at least 48 hours has passed, even more preferably when a period of time lasting several hours, days, weeks or even months has passed (i.e. 20% (or more) of the bio- active compound remains in the device after at least 30, 60 min, 24 h, 48 h or even several days).
  • the term "effective amount” or “effective concentration” includes within its meaning a non-toxic but sufficient amount or concentration of an agent to provide the desired effect.
  • the exact amount/concentration required will vary depending on factors such as the species of microorganism(s) being treated, the extent, severity and/or age of a biofilm being treated, whether the biofilm is surface-associated or suspended, the particular agent(s) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • Plant defensin refers to small, cationic peptides with a length of approximately 45-54 amino acids.
  • CRISPR Cysteine-stabilized ⁇ -motif
  • Plant defensins are present in all plant families, including the Brassicaceae, Fabaceae and Solanaceae. These peptides were primarily found in the seeds, but leaves and flowers are also common sources. They are either constitutively expressed in storage and reproductive organs or produced upon pathogenic attack or injury as part of a systemic defence response.
  • Plant defensins exhibit antimicrobial activity against a broad range of microorganisms, whereas they are in general non-toxic to human cells. Their activity is primarily directed against fungi, but bactericidal and insecticidal actions have also been reported. To date, several fungal targets have been identified, including membrane sphingolipids and phospholipids. Upon interaction with the fungal membrane, plant defensins are either internalized into the cell and interact with cytosolic or nuclear proteins, or they remain localized at the cell wall or membrane of the fungus. The mechanisms by which plant defensins induce fungal cell death are diverse, but common aspects are observed. These include the production of reactive oxygen species and the induction of apoptosis.
  • HsAFPI refers to a plant defensin from coral bells ⁇ "Heuchera sanguinea") with SEQ ID No. 1 which was previously characterized by Osborn and colleagues (2).
  • the present invention also includes analogs and derivatives of the plant defensins of the present invention, provided that the analog or derivative has a detectable antimicrobial, more preferably a detectable antifungal activity. It is not necessary that the analog or derivative has activity identical to the activity of the plant defensin from which the analog or derivative variant is derived.
  • potentiating compounds refers to compounds that increase the susceptibility and sensitivity of biofilms (and/or sessile cells) of microorganisms, in particular of fungal species, to antifungal drugs, even in situations where the potentiating compounds alone, or the antifungal drugs alone, do not reduce, eradicate, inhibit or prevent biofilm formation and/or development.
  • the combination of both the potentiating compound and an antimicrobial agent synergizes against microorganisms in the form of biofilms (and/or of sessile cells) in reducing the number of microbial cells, in particular in reducing the number of fungal species, preferably Candida species, more preferably Candida albicans, or of the frequency of infections.
  • combination therapy comprising one or more potentiating compounds and one or more antimicrobial agents, preferably antifungal agents, preferably antifungal agents selected from the group consisting of echinocandins or polyenes, more preferably caspofungin, anidulafungin, micafungin or amphotericin B, allows reducing the antifungal dose of the antifungal agent, or even applying an antifungal concentration of the antifungal agent which would be sub-lethal for a microorganism in the form of a biofilm and/or of sessile cells in the absence of the potentiating compound.
  • antifungal agents preferably antifungal agents selected from the group consisting of echinocandins or polyenes, more preferably caspofungin, anidulafungin, micafungin or amphotericin B
  • combination therapy comprising one or more potentiating compounds and one or more antimicrobial agents, preferably antifungal agents, allows reducing the antifungal dose of the antifungal agent, thereby also lowering the potential side effects of the antifungal agents, preferably antifungal agents selected from the group consisting of echinocandins or polyenes, more preferably caspofungin, anidulafungin, micafungin or amphotericin B.
  • the activity of said antifungal agents can be improved by combining said antifungal agent(s) with a potentiating compound, and thus concomitantly resulting in a reduction of its minimal inhibitory concentration (MIC) against a microorganism infection, especially a fungal infection and/or Candida (C. albicans) in the form of a biofilm.
  • MIC minimal inhibitory concentration
  • the potentiating compound may also be used in combination with one or more antifungal agent(s) against a fungal species, and/or yeast, such as Candida infection in the form of a biofilm at a reduced amount, which is the amount effective against this (fungal) infection in planktonic form (non sessile, non forming a biofilm).
  • peptide or “polypeptide” as used herein refer to a polymer of amino acid residues, including D-amino acids, typically L-amino acids, and to variants and synthetic analogues of the same, encompassing native peptides (including synthetically synthesized or recombinant peptides), modified peptides and peptidomimetics (typically, synthetically synthesized peptides and peptide analogues).
  • native peptides including synthetically synthesized or recombinant peptides
  • modified peptides and peptidomimetics typically, synthetically synthesized peptides and peptide analogues.
  • amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • a peptide of the present invention may also be produced by recombinant expression in prokaryotic and eukaryotic engineered cells other than plant cells, such as bacteria, fungi, or animal cells. Suitable expression systems are known to those skilled in the art.
  • recombinant (poly)peptide is meant a (poly)peptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
  • Suitable expression protocols and strategies are known to the skilled person and can be retrieved e.g. from Sambrook, 2001.
  • the polypeptide When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the peptide preparation.
  • peptide as used herein also refers to modified peptides wherein the modifications render the peptides even more stable e.g. while in a body.
  • modified peptides include peptides comprising D-amino acids, retro modified peptides, inverso modified peptides, retro-inverso modified peptides or cyclic peptides.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
  • derivative(s) of a peptide refers to peptides or polypeptides which, compared to the amino acid of a peptide according to this invention, may comprise: (i) substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues (including D-amino acids); (ii) amino acid residues that are substituted by corresponding naturally or non-naturally altered amino acids; (iii) naturally occurring altered, (such as glycosylated, acylated, myristoylated or phosphorylated amino acids) or non-naturally occurring amino acid residues (such as biotinylated amino acids, or amino acids modified after CNBr treatment); (iv) peptides carrying post-translational modifications.
  • a derivative may also be a retro, inverso or retro-inverso modified form of any of the peptides according to this invention.
  • a derivative may also comprise one or more non-amino acid substituents compared to the amino acid from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid such as, for example, a reporter molecule which is bound to facilitate its detection.
  • amino acid substitutions comprise conservative amino acid substitutions.
  • One or more amino acid residues may be introduced into a predetermined site in said peptide of the present invention. Insertions can comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • amino- or carboxy- terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag* 00 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S-transferase-tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase dihydrofolate reductase
  • a “retro modified” peptide is a peptide that is made up of amino acids in which the amino acid residues are assembled in opposite direction to the native peptide with respect to which it is retro modified. Where the native peptide comprises L-amino acids, the “retro modified” peptide will also comprise L-amino acids. However, where the native peptide comprises D- amino acids, the “retro modified” peptide will comprise D-amino acids.
  • An “inverso modified” peptide is a peptide in which the amino acid residues are assembled in the same direction as the native peptide with respect to which it is inverso modified, but the chirality of the amino acids is inverted.
  • the "inverso modified” peptide will comprise D-amino acids.
  • the “inverso modified” peptide will comprise L-amino acids.
  • a “retro-inverso modified” peptide refers to a peptide that is made up of amino acid residues which are assembled in the opposite direction and which have inverted chirality with respect to the native peptide to which it is retro-inverso modified.
  • a retro-inverso analogue has reversed termini and reversed direction of peptide bonds while approximately maintaining the topology of the side chains as in the native peptide sequence.
  • retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, incorporated herein by reference.
  • Partial retro-inverso peptide analogues are polypeptides in which only part of the sequence is reversed and replaced with enantiomeric amino acid residues. Processes for making such analogues are described in Pessi, A., Pinori, M., Verdini, A. S. & Viscomi, G. C. (1987) "Totally solid phase synthesis of peptide(s)- containing retro-inverted peptide bond, using crosslinked sarcosinyl copolymer as support", European Patent 97994-B.
  • non-natural amino acid refers to a non coded, non proteinogenic amino acid, or a post-translationally modified variant thereof that is not naturally encoded or found in the genetic code of any organisms.
  • the term refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine, selenocysteine or N-formylmethionine, or post- translationally modified variants thereof.
  • sequence identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Sequence identity is generally determined by aligning the residues of the two sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical residues, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met or a non-naturally altered amino acid) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met or a non-naturally altered amino acid
  • sequence identity between two amino acid sequences is determined by comparing said sequences using the Blastp program, available at http://blast.ncbi.nlm.nih.gov/Blast.cgi.
  • Blastp program available at http://blast.ncbi.nlm.nih.gov/Blast.cgi.
  • Similarity refers to the percentage number of amino acids that are identical or constitute conservative substitutions.
  • pharmaceutically acceptable carrier refers to any material, substance, or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals.
  • the pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi- steps procedure, with the selected carrier material and, where appropriate, the other additives.
  • the present invention presents an isolated peptide (also herein referred to as "peptide of the present invention") comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide consists of 12 to 54 amino acids.
  • a peptide of the present invention consists of 12 to 44 amino acids. More preferably, a peptide of the present invention comprises at least 12 amino acids, for instance at least 14, 16, 18, 20, 22, 24 or at least 28 amino acids. It is further preferred that a peptide of the present invention does not comprise more than 54 amino acids, for instance not more than 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 amino acids.
  • said isolated peptide does not comprise more than 44 amino acids. More preferably, said isolated peptide consists of 24 amino acids. More preferably, said isolated peptide consists of 19, 20, 21 , 22 or 23 amino acids.
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of GAXHYQFPSVKX (SEQ ID No. 8), GAXHYQFPSVKXFXKR (SEQ ID No. 1 1 ) or AYGGAXHYQFPSVKX (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), or FAYGGAXHYQFPSVKXFXK (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).
  • said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No.
  • said isolated peptide is FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17) or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18).
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3), WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),
  • SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or EHFAYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide is selected from the group consisting of DGVKLCDVPSGTWSGHCGSSSKCSQQCKDREHFAYGGACHYQFPSVKCFCKRQC (SEQ ID No. 1 ), DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3), WSGHXGSSSKXSQQXKDREHFAYG (SEQ ID No. 4),
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • the peptides of the present invention as described herein are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject.
  • said microbial biofilm associated condition or infection is a fungal or yeast biofilm associated condition or infection, more preferably said biofilm comprises Candida species, even more preferably said biofilm comprises Candida albicans, even more preferably said biofilm consists essentially of Candida albicans.
  • composition of the present invention comprising an isolated peptide of the present invention comprising an amino acid sequence which is at least 70% or 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to amino acid sequence GAXHYQFPSVKX (SEQ ID No. 8), QQXKDREHFAYG (SEQ ID No. 9) or WSGHXGSSSKXS (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide consists of 12 to 54 amino acids.
  • a peptide comprised in the composition of the second object of the present invention consists of 12 to 44 amino acids. More preferably, said peptide comprises at least 12 amino acids, for instance at least 14, 16, 18, 20, 22, 24 or at least 28 amino acids. It is further preferred that a peptide of the present invention does not comprise more than 54 amino acids, for instance not more than 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52 amino acids.
  • said isolated peptide does not comprise more than 44 amino acids. More preferably, said isolated peptide consists of 24 amino acids. More preferably, said isolated peptide consists of 19, 20, 21, 22 or 23 amino acids.
  • said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of GAXHYQFPSVKX (SEQ ID No. 8), G AXH YQ FPS VKXFXKR (SEQ ID No. 11) or AYGGAXHYQFPSVKX (SEQ ID No.
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No.
  • the isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15) or FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).
  • said isolated peptide comprises an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequence FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17).
  • said composition comprises an isolated peptide selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), AYGGAXHYQFPSVKXFXKRQX (SEQ ID No. 14), YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 15), HFAYGGAXHYQFPSVKXFXK (SEQ ID No. 16), FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17), or FAYGGAXHYQFPAVKXFXK (SEQ ID No.
  • X stands for Cystein or ot-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said isolated peptide is selected from the group consisting of EHFAYGGAXHYQFPSVKXFXKRQ (SEQ ID No. 13), YGGAXHYQFPSVKXFXKRQX (SEQ ID No.
  • said isolated peptide is FAYGGAXHYQFPSVKXFXK (SEQ ID No. 17) or FAYGGAXHYQFPAVKXFXK (SEQ ID No. 18).
  • said composition comprises an isolated peptide comprising an amino acid sequence which is at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 97%, 99% or 100% identical to the amino acid sequences selected from the group consisting of DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),
  • SSSKXSQQXKDREHFAYGGAXHYQ (SEQ ID No. 5), QQXKDREHFAYGGAXHYQFPSVKX (SEQ ID No. 6), or EHFA YGGAXHYQFPSVKXFXKRQX (SEQ ID No. 7), or a derivative thereof, wherein X stands for Cystein or a-aminobutyric acid, and wherein said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • said composition comprises an isolated peptide selected from the group consisting of DGVKLCDVPSGT SGHCGSSSKCSQQCKDREHFAYGGACHYQFPSVKCFCKRQC (SEQ ID No. 1 ), DGVKLXDVPSGTWSGHXGSSSKXS (SEQ ID No. 2), DVPSGTWSGHXGSSSKXSQQXKDR (SEQ ID No. 3),
  • X stands for Cystein or a-aminobutyric acid
  • said isolated peptide has microbial antibiofilm activity, particularly wherein said isolated peptide inhibits microbial biofilm formation and development and/or potentiates the effect of an antifungal agent.
  • said isolated peptide potentiates the effect of an antifungal agent on a fungal biofilm.
  • compositions according to the present invention comprise an isolated peptide of the present invention, and at least one antimicrobial (such as an antibacterial, antiprotozoal or antifungal) agent, preferably in an effective amount thereof.
  • at least one antimicrobial agent is selected from the following compounds: antifungal agents, such as polyenes (e.g. amphotericin B, nystatin, natamycin); azoles (e.g. miconazole, fluconazole, itraconazole, voriconazole); allylamines (e.g. terbinafine); echinocandins (e.g.
  • said antimicrobial agent is an antifungal agent, more preferable said antimicrobial agent is an antifungal agent selected from the group of echinocandins or polyenes, even more preferably said antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B. Even more preferably, said antifungal agent is caspofungin.
  • the compositions according to the present invention are used in the treatment or prevention of a microbial biofilm associated condition or infection in a human or animal subject.
  • said microbial biofilm associated condition or infection is a fungal or yeast biofilm associated condition or infection, more preferably said biofilm comprises Candida species, even more preferably said biofilm comprises Candida albicans, even more preferably said biofilm consists essentially of Candida albicans.
  • the compositions according to the present invention are used for treatment or prevention of a fungal biofilm associated condition or disease in a human or animal subject, wherein said subject has been implanted with a medical device, which is infected or at risk of being infected with a fungal biofilm.
  • said medical device is selected from the group consisting of catheters, stents, surgical plates, prostheses, valves or pins, artificial joints, pacemakers, contact lenses and bio-implants.
  • composition of the present invention may, depending upon the desired mode of administration or application, be formulated in very different forms such as, but not limited to, liquids, gels, foams, semi-solids and solids.
  • Said antibiofilm composition may further comprise one or more non-active pharmaceutically acceptable carriers, adjuvants, excipients and/or diluents (i.e. ingredients that do not interfere with the antibiofilm function of the active peptide).
  • a pharmaceutically acceptable carrier is a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Pharmaceutically acceptable excipients, ingredients, adjuvants and carriers are well known, and one skilled in the pharmaceutical art can easily select them for any particular route of administration (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).
  • composition of the present invention may be formulated for topical administration (e.g., as a lotion, cream, spray, gel, or ointment).
  • topical formulations are useful in treating or inhibiting microbial and/or fungal and/or biofilm presence or infections on the eye, skin, and mucous membranes such as mouth, vagina and the like.
  • formulations in the market place include topical lotions, creams, soaps, wipes, and the like. It may be formulated into liposomes to reduce toxicity or increase bioavailability. Other methods of administration will be known to those skilled in the art.
  • Preparations for parenteral administration of a composition of the present invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate.
  • aqueous carriers include water, saline, and buffered media, alcoholic/aqueous solutions, and emulsions or suspensions.
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as, other antimicrobials, anti-oxidants, cheating agents, inert gases and the like also can be included.
  • composition of the present invention may also be in the form of a kit wherein each agent is kept separate until effective use.
  • the present invention presents methods for reducing, eradicating, inhibiting or preventing microbial biofilms or biofilm formation, preferably fungal biofilms or biofilm formation, on a solid support surface or other medium susceptible to biofilm formation, preferably on a surface or medium outside the body of a human or animal subject, wherein said surface or medium carrying said biofilm or susceptible to said biofilm formation is exposed to at least one isolated peptide of the present invention or a derivative thereof, or to a composition of the present invention.
  • said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent.
  • said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin, or amphotericin B, even more preferably said at least one antifungal agent is caspofungin.
  • said biofilm is a Candida biofilm.
  • said method further comprises the step of exposing the surface or medium carrying said biofilm or susceptible to said biofilm formation to at least one antimicrobial agent before, after or concurrent with the isolated peptide of the present invention or a derivative thereof.
  • the present invention also relates to a method for inhibiting biofilm formation and/or development, wherein said surface or medium susceptible to biofilm formation is treated with at least one peptide of the present invention or a derivative thereof, or a with a composition of the present invention, or wherein said peptide of the present invention is incorporated in said surface or medium susceptible to biofilm formation.
  • said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent.
  • said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said at least one antifungal agent is caspofungin.
  • said biofilm is a Candida biofilm.
  • the present invention also relates to a method for the treatment or prevention of a condition or infection associated with microbial biofilm development, preferably fungal biofilm development, in a human or animal subject comprising administering to said subject an effective amount of at least one peptide of the present invention or of the composition of the present invention.
  • a condition or infection associated with microbial biofilm development preferably fungal biofilm development
  • said composition comprises at least one isolated peptide of the present invention or a derivative thereof, and at least one antimicrobial agent.
  • said at least one antimicrobial agent is an antifungal agent, more preferably said antifungal agent is selected from the groups consisting of echinocandins or polyenes, even more preferably said at least one antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said at least one antifungal agent is caspofungin.
  • said fungal biofilm is a Candida biofilm.
  • each component of the composition of the present invention may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect.
  • the components may be formulated together in a single dosage unit as a combination product.
  • Another related aspect of the present invention is the combination of a medical device, particularly an implantable medical device, and a composition of the present invention comprising at least one peptide of the present invention or potentiating compound, medicament or bio-active agents for preventing or suppressing microbial biofilms in a patient, preferably a human or animal subject.
  • a medical device particularly an implantable medical device
  • a composition of the present invention comprising at least one peptide of the present invention or potentiating compound, medicament or bio-active agents for preventing or suppressing microbial biofilms in a patient, preferably a human or animal subject.
  • said microbial biofilm is a fungal biofilm, more preferably said fungal biofilm is a Candida biofilm.
  • Another related aspect relates to the preparation (e.g. by coating, incorporation) of a solid support surface, such as a medical device, like implants, plastics or (subcutaneous) catheters, with the addition of a sufficient concentration (or dose) of an isolated peptide of the present invention upon this solid support surface (or inside the solid support), for reducing, eradicating, inhibiting or preventing microbial biofilms, preferably fungal biofilms, more preferably Candida biofilms, in combination with at least one antifungal agent, preferably said antifungal agent is an echinocandin or a polyene, more preferably said antifungal agent is caspofungin, anidulafungin, micafungin or amphotericin B, even more preferably said antifungal agent is caspofungin.
  • a solid support surface such as a medical device, like implants, plastics or (subcutaneous) catheters
  • the composition may be an anti-fouling composition, incorporated in a medical device or component thereof, a coating for a medical device or a pharmaceutical composition.
  • the composition of the present invention or one or more components thereof is used in coating medical devices, including implantable medical devices, including but not limited to venous catheters, urinary catheters, stents, prostheses such as artificial joints, hearts, heart valves or other organs, pacemakers, dental implants, surgical plates and pins, dialysis equipment and contact lenses.
  • said implantable medical devices may have the capacity to release the composition on the surface of such medical device, thus imparting a highly localised treatment with said composition.
  • Methods and compositions of the invention also find application in the management of infectious diseases.
  • infections associated with (fungal) biofilm formation may be treated with methods and compositions of the invention, such as urinary tract infections, pulmonary infections, dental plaque, dental caries and infections associated with surgical procedures or burns.
  • dosages of antifungal agents may be reduced but remain effective in reducing, eradicating, inhibiting, or preventing biofilms.
  • treatment may be continued during a defined period of treatment, at a preferably constant and/or localised dose, without antifungal pressure drops so that development of resistance, persisters and recurrence of biofilm associated conditions or infections can be prevented.
  • the antifungal agents used in the composition of the present invention are preferably used at a concentration effective against the planktonic form of the cells, which is a concentration too low to be effective against microorganisms in biofilms (in sessile form).
  • the present invention is based on the surprising finding by the inventors that plant defensins such as HsAFPI and synthetic peptides derived from HsAFPI are potentiating compounds which increase the antibiofilm activity of antifungal drugs such as caspofungin, anidulafungin, micafungin or amphotericin B against fungal biofilms, preferably against Candida biofilms.
  • antifungal drugs such as caspofungin, anidulafungin, micafungin or amphotericin B against fungal biofilms, preferably against Candida biofilms.
  • the present invention shows that the antibiofilm activity of caspofungin, anidulafungin, micafungin or amphotericin B, can be improved by combining the antifungal compound with said plant defensin HsAFPI and/or peptide derivatives thereof.
  • HsAFPI the plant defensin from coral bells, HsAFPI .
  • HsAFPI the plant defensin from coral bells, HsAFPI .
  • HsAFPI has a potent antifungal activity towards C. albicans
  • its potential activity towards C. albicans biofilms was further analyzed.
  • HsAFPI was heterologously expressed using the yeast Pichia pastoris and the solution structure of recombinant (r) rHsAFPI was determined by NMR analysis. Subsequently, the activity of the plant defensin was tested alone and in combination with conventional antimycotics against C. albicans biofilms.
  • Pichia pastoris strain X33 was used for heterologous production of HsAFPI .
  • Fusarium culmorum strain K0311 was used to evaluate the antifungal activity of the recombinant peptide and to compare it with that of native HsAFPI purified from seeds, in a fungal growth inhibitory assay (2).
  • C. albicans strain SC5314 was used in all biofilm experiments. rHsAFPI toxicity testing was performed on HepG2, human hepatoma cells, purchased from ATCC (catalogue number HB-8065; Rockville, MD, USA).
  • HsLin06_18 cytotoxicity tests were performed on HepG2, human hepatoma cells (29), purchased from ATCC (HB-8065; USA).
  • P. pastoris was cultured in YPD (1% yeast extract, 2% peptone and 2% glucose), BMGY (buffered complex glycerol medium; 1 % yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids (Becton Dickinson, UK), 1% glycerol, 100 mM K3P04 pH 6, 4 x 10-5% biotin) or BMMY (buffered complex methanol medium; 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base w/o amino acids (Becton Dickinson, UK), 0.5% methanol, 100 mM K3P04 pH 6, 4 x 10-5% biotin).
  • F. culmorum was grown in half strength PDB (1.2% potato dextrose broth).
  • C. albicans was grown overnight in YPD (1% yeast extract, 2% peptone and 2% glucose), with all compounds purchased from LabM (UK), unless stated otherwise.
  • YPD 1% yeast
  • Biofilm experiments were performed in RPMI-1640 medium (Roswell Park Memorial Institute- 1640 medium; pH 7) with L-glutamine and without sodium bicarbonate (purchased from Sigma Aldrich, St.-Louis, MO, USA), buffered with MOPS (Sigma Aldrich, St.-Louis, MO, USA).
  • Amphotericin B and caspofungin (Cancidas) were purchased from Sigma Aldrich (St. Louis, MO, USA) and Merck (Beeston Nottingham, UK), respectively.
  • HepG2 cells were grown in MEM (Minimal Essential Medium, Gibco, Invitrogen; CA, USA), supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin, and cultured using standard cell culture conditions (37°C, 5% C02, 95% humidity).
  • MEM Minimum Essential Medium
  • L-glutamine 100 U/mL
  • penicillin and 100 pg/mL streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/mL
  • streptomycin 100 U/m
  • Caspofungin cancidas
  • micafungin mycamine
  • anidulafungin ecalta
  • Merck USA
  • Astellas Pharma Europe The Netherlands
  • Pfizer UK
  • Polyurethane triple-lumen intravenous catheters 2.4 mm diameter
  • Fetal Bovine Serum FBS
  • the filtered supernatant was then subjected to automated tangential flow filtration using an automated peristaltic pump (Spectrum Laboratories, CA, USA) and a hollow fiber module with 1 kDa cut-off mPES membranes (Spectrum Laboratories, CA, USA). During the ultrafiltration, the sample was concentrated a 15-fold and subsequently dialyzed against 50 mM sodium acetate pH 5.
  • rHsAFPI was purified by cation exchange chromatography, using 75 mL SP sepharose High Performance resin (GE Healthcare, UK) packed in a XK26/20 column (GE Healthcare) and 50 mM sodium acetate buffers at pH 5. The flow rate was maintained at 5 mUmin. Elution of the peptides was carried out by a washing step with 10% (v/v%) elution buffer (50 mM sodium acetate, 1 M sodium chloride, pH 5) for 10 column volumes (CV), followed by a linear gradient to 50% (v/v%) elution buffer in 15 CV, resulting in a peak at approximately 29% (v/v%) elution buffer.
  • the eluted fraction was further purified by reversed phase chromatography employing a Gemini C18 250x10 column (Phenomenex, CA, USA) and acetonitrile (ACN) for elution of the bound peptides.
  • the flow rate was maintained at 4.6 mL/min.
  • Elution of the peptides was carried out by a washing step at 15% (v/v%) ACN for 1.9 CV, followed by a linear gradient to 35% (v/v%) ACN in 2.3 CV.
  • Elution of rHsAFPI occurred at 28%.
  • the eluted fraction was vacuum dried by centrifugal evaporation (SpeedVac Savant, Thermo Fisher Scientific, MA, USA), re-dissolved in MilliQ water and subjected to a micro bicinchoninic acid assay (Pierce, Thermo Scientific, USA) according to the manufacturer's instructions, to determine the protein concentration.
  • Bovine serum albumin served as a reference protein. At least 40 mg/L of culture of purified rHsAFPI was obtained.
  • Solvent suppression was achieved using excitation sculpting with gradients (11 ). Spectra were acquired with 4096 complex data points in F2 and 512 increments in the F1 dimension. Slowly exchanging amide protons were identified by spectra also recorded in 100% D20.
  • Spectra were processed using TopSpin (Bruker) software. The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Chemical shifts were referenced to internal 2,2-dimethyl-2- silapentane-5-sulfonate (DSS). Processed spectra were analysed and assigned using CcpNmr Alalysis (12). Spectra were assigned using the sequential assignment protocol (13).
  • Structure calculations were based on distance restraints derived from NOESY spectra recorded in both 10% and 100% D20. Initial structures were generated using the program CYANA (14), followed by addition of restraints for the disulfide bonds, hydrogen bonds as indicated by slow D20 exchange and sensitivity of amide proton chemical shift to temperature, chil restraints from ECOSY and NOESY data, and backbone phi and psi dihedral angles restraints generated using the program TALOS+ (15). The structural family was generated using torsion angle dynamics, refinement and energy minimization in explicit solvent and protocols as developed for the RECOORD database (16) within the program CNS (17).
  • the IC50 value which is the concentration required for 50% growth inhibition as compared to control treatment, was determined by measuring the optical density at 490 nm (OD490nm) after 48 hours of incubation and was confirmed microscopically.
  • the antifungal activity of rHsAFPI against C. albicans was subsequently analysed according to the standard CLSI protocol M27-A3 (21 ) with minor modifications: an inoculum of approximately 106 cells/mL was suspended in RPMI-1640 medium and added to a two-fold dilution series of rHsAFPI in water.
  • the DMSO concentration was similar to that in the biofilm assays, i.e. 0.5% DMSO.
  • the MIC50 value i.e. the minimum concentration required to reduce planktonic growth by 50% as compared to control treatment, was determined by measuring the OD490nm after 24 hours of incubation.
  • Biofilm inhibition assay The Biofilm Inhibitory Concentration 50 value (BIC50; the minimum concentration required to reduce biofilm formation by 50% as compared to control treatment) of rHsAFPI or HsLin peptides was determined using the following antibiofilm assay: a C. albicans SC5314 overnight culture, grown in YPD, was diluted to an optical density (600 nm) of 0.1 in RPMI 1640 medium and 100 ⁇ _ of this suspension was added to the wells of a round-bottomed microtitre plate (TPP, Tradingen, Switzerland).
  • BIC50 the minimum concentration required to reduce biofilm formation by 50% as compared to control treatment
  • Biofilm eradication assay The Biofilm Eradicating Concentration 50 value (BEC50; the minimum concentration required to reduce the viability of the cells in a pre-grown biofilm by 50% as compared to control treatment) of rHsAFPI was determined using the BEC50 determination assay as described by De Cremer and co-workers (23). Briefly, a C. albicans SC5314 overnight culture, grown in YPD, was diluted to an optical density (600 nm) of 0.1 in RPMI 1640 medium and 100 ⁇ _ of this suspension was added to the wells of a round- bottomed microtitre plate (TPP, Tradingen, Switzerland).
  • BEC50 Biofilm Eradicating Concentration 50 value
  • the biofilms were washed with 100 ⁇ _ PBS to remove non-adherent cells, followed by addition of 100 ⁇ _ RPMI 1640 medium.
  • the biofilms were allowed to grow for 24 h at 37°C.
  • an rHsAFPI concentration series in RPMI was added to the biofilms.
  • the DMSO concentration was similar to that in the checkerboard assays, i.e. 0.5%.
  • the biofilms were incubated for another 24 h at 37°C, after which they were washed and quantified with CTB as described above. Checkerboard assay. C. albicans biofilms or C. albicans planktonic cultures were grown as described above.
  • SEM Scanning electron microscopy
  • HepG2 cells were seeded at 10.000 cells/well in 96 well- plates and incubated for 24 hours. Subsequently, cells were treated with water (untreated) or rHsAFPI (0.01 ⁇ - 40 ⁇ ) for 24 hours after which cell viability or cell proliferation was determined using the "Cell Proliferation Kit II (XTT)", as described previously (27), or the “Cell Proliferation ELISA BrdU (colorimetric) kit", according to the manufacturer's instructions, respectively.
  • XTT Cell Proliferation Kit II
  • BrdU colorimetric
  • HsAFPI Structure-function analysis of HsAFPI. Synthesis and purification of the 24-mer peptides (HsLin01-HsLin06) spanning the HsAFPI amino acid sequence was performed as described previously (28). Cysteine residues were replaced by a-aminobutyric acid to avoid formation of disulfide bonds.
  • C. albicans biofilms were grown on catheters as described previously (30). After an adhesion phase of 90 minutes, C. albicans biofilms (on catheters) were treated with caspofungin, caspofungin + HsLin06_18 or control treatment (0.5% DMSO +0.5% MQ water) in RPMI for 24 hours, after which biofilm-associated cells were determined via Colony Forming Units (CFU's) counts (30). Averages of 4 technical replicates were used to calculate the CFU/catheter for each treatment in one experiment. Next, data were normalized to the CFU/catheter corresponding to the control treatment.
  • CFU's Colony Forming Units
  • HsLin06 18 toxicity assays Human hepatoma HepG2 cells were incubated for 24 hours in 96 well-plates at 10 000 cells/well. Subsequently, cells were treated with water (control treatment) or HsLin06_18 (0.09 ⁇ - 93 ⁇ ). After 24 hours of treatment, cell viability was determined via the cell proliferation kit II (MTT), as described by van Malenstein and coworkers (27).
  • MTT cell proliferation kit II
  • Unpaired Student t-tests were performed to analyse significant differences between the IC50 value of native HsAFPI and that of recombinant HsAFPI , and between the MIC50, BIC50 and BEC50 of caspofungin or amphotericin B alone and the combination of these compounds with rHsAFPI or its derivatives in the checkerboard assays.
  • rHsAFPI was produced in Pichia pastoris and subsequently purified using cation exchange and reversed phase chromatography. A yield of at least 40 mg/L of culture of purified rHsAFPI was obtained.
  • the antifungal activity of HsAFPI against a broad range of fungi, including the fungus Fusarium culmorum, has been reported previously (2). In this respect, Osborn and colleagues showed that native HsAFPI can inhibit growth of F. culmorum with an IC50 value of 1 pg/mL (2).
  • rHsAFPI we tested the antifungal activity of rHsAFPI and native HsAFPI against F. culmorum according to the method of Osborn (2).
  • rHsAFPI The solution structure of rHsAFPI was solved via NMR analysis, a technique that has been previously used to characterize the structures of other plant defensins, including RsAFPI , MtDef4, Psd1 and NaD1.
  • a sequence alignment of HsAFPI with these peptides and RsAFP2 is presented in Fig. 1 A, showing the disulfide bond pattern common for plant defensins.
  • the NMR spectra of rHsAFPI showed the sample to be of high purity and good dispersion in the amide region was indicative of a highly structured peptide.
  • Two-dimensional spectra were recorded at several temperatures in the range 283 to 303 K to obtain full proton assignments (data not shown).
  • Secondary chemical shift analysis was then used to locate elements of secondary structure. Ha secondary shifts are calculated by subtracting the chemical shift of the alpha proton from "random coil” values. Deviations greater than 0.1 ppm from random coil are indicative of structured peptides, with positive values present for beta type structures and negative values for helical structures.
  • the secondary Ha shifts of rHsAFPI are shown in Fig. 2 and indicate that the solution structure of rHsAFPI consists of both a-helix and ⁇ - strand elements.
  • rHsAFPI The three-dimensional structure of rHsAFPI was calculated from 614 distance restraints, 15 hydrogen bond pairs, and a total of 90 dihedral angle restraints (data not shown).
  • the disulfide connectivities (l-VIII, ll-V, lll-VI, IV-VII) were fully consistent with the NOE data and were included as restraints in the structure calculations.
  • one proline Pro9 is present in the trans configuration and the second (Pro44) has a cis peptide bond.
  • Fig. 3A shows the ensemble of structures superimposed over the backbone heavy-atoms of all residues (rmsd 1.16 ⁇ 0.40 A).
  • a ribbon representation of the lowest energy structure is shown in Fig. 3B.
  • the four disulfide bonds are arranged in a typical Cysteine-stabilized ⁇ motif in that the a-helix is tethered to the ⁇ -sheet by two disulfide bonds to the central strand (Cys23-Cys39 and Cys27-Cys50).
  • EXAMPLE 3 rHsAFPI prevents C. albicans biofilm formation.
  • EXAMPLE 4 rHsAFPI acts synergistically with caspofungin or amphotericin B against C. albicans.
  • BEC50 values were determined by CTB assay; mean ⁇ SEM for n ⁇ 3 independent experiments is presented; BEC50, minimum concentration that is required to reduce viability of 24 hours-old biofilm cells by 50% as compared to control treatment; FICl, Fractional Inhibitory Concentration Index, FICl ⁇ 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds.
  • EXAMPLE 5 rHsAFPI does not affect HepG2 cell viability and proliferation.
  • EXAMPLE 6 The ⁇ -core and adjacent regions are important for rHsAFPI antibiofilm activity.
  • Figure 7 shows a diagram in which the HsLin peptides are imposed on the rHsAFPI structure, according to their amino acid sequence. Note that (i) the Cysteine residues are replaced by a-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSa scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFPI .
  • HsLin06 and rHsAFPI showed antibiofilm activity as well, however, with a 10- to 15-fold higher BIC50 value than that of rHsAFPI or HsLin06.
  • Other fragments did not inhibit biofilm formation up to 175 ⁇ , the highest tested concentration.
  • HsLin06 but also HsLinOI and HsLin05, acted synergistically with caspofungin to inhibit C. albicans biofilm formation in a range of 0.75 ⁇ to 1.5 ⁇ (Fig. 9 and Table 5 for HsLin06 and Fig. 8 for the other HsLin). We did not observe synergistic effects between the other linear fragments and caspofungin for preventing biofilm formation (Fig. 8).
  • BIC50 values were determined by CTB assay; mean ⁇ SEM for n ⁇ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment. Unpaired Student t-tests were performed to analyse significant differences between the effect of the linear fragments and rHsAFPI ; the significance level is presented (*, ** and *** represent P ⁇ 0.05, P ⁇ 0.01 and P ⁇ 0.001 , respectively; NS, no significant difference).
  • BIC50 values were determined by CTB assay; mean ⁇ SEM for n ⁇ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment; FICI, Fractional Inhibitory Concentration Index, FICI ⁇ 0.5 indicates synergy between two compounds; NA, not applicable. Values in bold represent synergistic effects between two compounds. Unpaired Student t-tests were performed in case FICI did not indicate synergy to analyse significant differences between the effect of the compound alone and the combination of compound and rHsAFPI ; the significance level is presented (*, ** and *** represent P ⁇ 0.05, P ⁇ 0.01 and P ⁇ 0.001 , respectively; NS, no significant difference).
  • HsAFPI As the C-terminal part of HsAFPI (corresponding to HsLin06, 24 amino acids) was identified as an important region for antibiofilm and caspofungin's synergistic activity, we tested a series of 44 HsLin06-variants (HsLin06_01-44) with N- or C-terminal truncations of HsLin06's sequence, ranging from 16-23 amino acids (Table 6). Their antibiofilm activity in microtiter plates was evaluated by determining BIC50 values. Caspofungin potentiating activity of the HsLin06-variants against C.
  • albicans biofilms was evaluated by comparing the reduction of BIC50 of caspofungin in the presence and absence of the HsL ' in06-variant, represented as fold change values (shown in Table 6). The higher the fold change, the better the potentiating effect of HsLin to caspofungin's antibiofilm activity.
  • HsLin06_01-05;07;08;10-12;15;16;22;29;30;36;38 17 HsLin06-variants (HsLin06_01-05;07;08;10-12;15;16;22;29;30;36;38) as calculated by their BIC50 values (0.5 * BIC50(Hsl_in06) ⁇ BIC50(HsLin) ⁇ 2 * BIC50(HsLin06)) (Table 6).
  • peptides HsLin06_02 and Hsl_in06_10 had both a pronounced caspofungin potentiating and antibiofilm effect.
  • HsLin06-variants were able to potentiate caspofungin's antibiofilm activity at least 5.2-fold (i.e. > 2x F.C. HsLin06), irrespective of their BIC50 values.
  • Five of them namely HsLin06_02;06;10;13 and HsLin06_18 (Table 6), reduced the BIC50 of caspofungin by at least 10-fold.
  • HsLin06_18 being the shortest peptide (containing 19 amino acids) was selected for further in vitro and in vivo experiments.
  • HsLin06_18 is the smallest Hsl_in06-variant with fold change value > 10 and is therefore marked in bolt.
  • HsLin06_03 22 FAYGGAXHYQFPSV XFXKRQX 0,56 2,52
  • HsLin06_04 22 HFAYGGAXHYQFPSVKXFXKRQ 0,38 1,01
  • HsLin06_06 21 AYGGAXHYQFPSVKXFXKRQX 1,16 11,14
  • HsLln06_08 21 HFAYGGAXHYQFPSVKXFXKR 0,48 1,22
  • HsLin06_10 20 YGGAXHYQFPSVKXFXKRQX 0,84 12,49
  • HsLin06_12 20 FAYGGAXHYQFPSVKXFXKR 0,71 1,17
  • HsLin06_15 19 GGAXHYQFPSVKXFXKRQX 0,90 1,04
  • HsLin06_18 Besides caspofungin, other echinocandins such as micafungin and anidulafungin are used in the clinic to treat patients suffering from invasive fungal infections (31 ). Therefore, we examined the effect of HsLin06_18 on caspofungin, anidulafungin and micafungin's antibiofilm activity. We found that HsLin06_18 could potentiate caspofungin's antibiofilm activity, as well as that of anidulafungin (Table 7). Further, some HsLin06_18's potentiation effect on micafungin's antifbiofilm activity could be observed in Figure 10B.
  • Table 7 Potentiation activity of Hsl_in06_18 with different echinocandins: caspofungin (CASPO), micafungin (MICA) and anidulafungin (ANIDULA) in an in vitro microtiter plate biofilm inhibition test.
  • BIC50 values i.e. minimum inhibitory concentration resulting in 50% biofilm inhibition compared to control treatment.
  • Unpaired student t- test were performed to determine significant differences between the single treatment with an echinocandin and the combinational treatment with both the echinocandin and Hsl_in06_18.
  • the significance levels *, *** represents respectively P ⁇ 0.05 and P ⁇ 0.001; NS, not significant.
  • EXAMPLE 9 Caspofungin's potentiating activity ofHsLin06_18 in an in vitro catheter assay
  • the potentiating effect of HsLin06_18 on caspofungin's antibiofilm activity was investigated in an in vitro catheter assay. Therefore, biofilms, adhering on serum pre-incubated catheters, were treated with caspofungin, caspofungin with Hsl_in06_18, HsLin06_18 alone or DMSO (control treatment) after which the survival of the biofilm cells was determined via plating assays.
  • DMSO control treatment
  • Hsl_in06_18 was found not cytotoxic up to 200pg/ml, the highest tested concentration, as presented in Figure 12.
  • HsLin06_18 For its caspofungin potentiating activity, we performed an alanine scan in which every amino acid of HsLin06_18 was replaced by alanine, resulting in 19 HsLin06_18-derived peptides (HsLin06_18_01-19), which were subsequently tested for their ability to potentiate caspofungin's antibiofilm activity. By comparing fold change values (Table ), the amino acids important for HsLin06_18 caspofungin's potentiating activity can be identified. Surprisingly, none of the amino acid replacements in HsLin06_18 resulted in the abolishment of its caspofungin potentiating activity.
  • HsLin06_18 and HsLin06_18_01-19 were in the same range, indicating that the amino acid sequence of HsLin06_18 can be substituted at different positions without losing its caspofungin potentiating effect.
  • HsLin06_18_13 potentiated caspofungin more than HsLin06_18 (Table and Fout! Verwijzingsbron niet gevonden.).
  • Table 8 Antibiofilm activity and caspofungin's potentiating activity of HsLin06_:18- derived peptide fragments, resulting in £ albicans biofilm formation inhibition. Amino acid replacements in HsLin06_18_01-19 are marked in bolt. HsLin + caspofungin

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Abstract

La présente invention concerne une nouvelle composition destinée à être utilisée dans l'inhibition ou le traitement de la formation d'un biofilm microbien, ladite composition comprenant une défensine de plante ou un dérivé peptidique de celle-ci et un composé antifongique, de préférence la caspofungine, l'anidulafungine, la micafungine, ou l'amphotéricine B. Par conséquent, la présente invention concerne l'utilisation de ladite composition pour l'inhibition ou le traitement de biofilms, en particulier des biofilms fongiques, tels que des biofilms de Candida, chez un sujet ou sur une surface ou un autre milieu sensible à la formation de biofilms.
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IT202100018530A1 (it) 2021-07-14 2023-01-14 Clever Bioscience S R L Composizioni antimicrobiche sinergiche contenenti peptidi selezionati e acidi grassi
IT202100018542A1 (it) 2021-07-14 2023-01-14 Clever Bioscience S R L Liposomi contenenti combinazioni antimicrobiche sinergiche a base di peptidi selezionati e acidi grassi

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IT202100018530A1 (it) 2021-07-14 2023-01-14 Clever Bioscience S R L Composizioni antimicrobiche sinergiche contenenti peptidi selezionati e acidi grassi
IT202100018542A1 (it) 2021-07-14 2023-01-14 Clever Bioscience S R L Liposomi contenenti combinazioni antimicrobiche sinergiche a base di peptidi selezionati e acidi grassi
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