WO2016003364A1 - Peptides et leurs utilisations - Google Patents

Peptides et leurs utilisations Download PDF

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Publication number
WO2016003364A1
WO2016003364A1 PCT/SG2014/000316 SG2014000316W WO2016003364A1 WO 2016003364 A1 WO2016003364 A1 WO 2016003364A1 SG 2014000316 W SG2014000316 W SG 2014000316W WO 2016003364 A1 WO2016003364 A1 WO 2016003364A1
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WIPO (PCT)
Prior art keywords
peptide
keratitis
seq
peptides
amino acids
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PCT/SG2014/000316
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English (en)
Inventor
Jackie Y. Ying
Hong Wu
Yi Yan Yang
Zhan Yuin ONG
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Agency For Science, Technology And Research
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Publication date
Application filed by Agency For Science, Technology And Research filed Critical Agency For Science, Technology And Research
Priority to PCT/SG2014/000316 priority Critical patent/WO2016003364A1/fr
Priority to CN201480080301.8A priority patent/CN106470674B/zh
Priority to SG11201610918RA priority patent/SG11201610918RA/en
Priority to JP2016575724A priority patent/JP6506317B2/ja
Publication of WO2016003364A1 publication Critical patent/WO2016003364A1/fr

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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • 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
    • A61P31/10—Antimycotics
    • 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
    • A61P31/12—Antivirals

Definitions

  • the present invention generally relates to antimicrobial peptides and methods for their use.
  • AMPs membrane active antimicrobial peptides
  • Naturally occurring antimicrobial peptides also known as 'host defence peptides', were first discovered as components of the innate immunity, forming the first line of defence against invading pathogens in all living organisms.
  • 'host defence peptides' were first discovered as components of the innate immunity, forming the first line of defence against invading pathogens in all living organisms.
  • the majority of the cationic antimicrobial peptides exert their activities via physical disruption of the more negatively charged microbial membrane lipid bilayers to induce leakage of cytoplasmic components leading to cell death.
  • the physical nature of membrane disruption is believed to result in a lower likelihood for drug resistance development as it becomes metabolically 'costlier' for the microorganism to mutate or to repair its membrane components at the same rate as the damage is being inflicted.
  • antimicrobial peptides As the majority of the antimicrobial peptides exert their antimicrobial activities through a rapid and direct membrane lytic mechanism, they possess an inherent advantage in overcoming conventional mechanisms of antibiotics resistance such as the increased expression of drug efflux pumps on microbial membranes, production of drug degradation enzymes or alteration to drug interaction sites acquired by microbes against small molecular antibiotics targeting specific biosynthetic pathways.
  • Significant barriers limiting the successful clinical translation of antimicrobial peptides include high systemic toxicities as a result of poor microbial membrane selectivities, relatively high manufacturing cost (for long peptide sequences) and susceptibility to degradation by proteases present in biological fluids such as blood serum, wound exudates or lacrimal fluids.
  • Keratitis continues to be an important cause of ocular morbidity, and it is also a major eye disease that leads to blindness. Symptoms of keratitis include pain and redness in the eye, blurred vision, sensitivity to light, and excessive tearing or discharge. It may lead to severe vision loss and scars on the cornea. Moreover, the incidence of keratitis has increased over the past 30 years mainly due to the frequent use of topical corticosteroids and antibacterial agents in the treatment of patients with keratitis, as well as the rise in the number of patients who are immunocompromised.
  • causes of keratitis include herpes simplex virus type 1, varicella zoster, and adenoviruses, bacteria, parasites, fungi and vitamin A deficiency.
  • An outbreak of keratitis was reported in 2005 and 2006 in the United States and also in France, Hong Kong and Singapore. Most of the cases in the United States involved soft contact lens wear, which was mainly caused by fungal infections.
  • Keratitis remains a diagnostic and therapeutic challenge to the ophthalmologist due to several reasons. The main challenge is fast and accurate diagnosis. Diagnosis of keratitis is typically conducted by culture or corneal biopsy. However, isolating and identifying the species of organism in the laboratory take time, leading to late diagnosis frequently. Furthermore, as keratitis can be caused by various microorganisms, the different types of keratitis are often misdiagnosed as one another. For example, fungal keratitis is often misdiagnosed as bacterial keratitis as clinicians often consider fungal keratitis only after a presumed bacterial keratitis worsens during antibiotic therapy.
  • anti-fungal sensitivity testing is unreliable, and correlates poorly with clinical efficacy. Even if the diagnosis is made accurately, management of the condition remains a challenge because of the poor corneal penetration and the limited commercial availability of agents useful in treating keratitis.
  • azole compounds such as funconazole
  • polyenes such as amphopterin B, nystatin and natamycin
  • the azoles are fungistatic, which function by enzyme inhibition and are prone to drug resistance development.
  • azoles available in the art are extremely unstable; their topical solution must be kept refrigerated for no more than 48 hours and protected from light.
  • Another antifungal drugs known in the art are the polyenes, which function via disrupting the permeability of ions through the cell membrane, are rather expensive, poorly water soluble and quite unstable in aqueous, acid or alkaline media, or when exposed to light and excessive heat. All these limit their clinical applications.
  • an amphiphilic peptide comprising (XiY]X2Y2) n (Formula I), wherein the C-terminal end of the peptide is amidated; X] and X 2 is independently of each other a hydrophobic amino acid; Y] and Y 2 is independently of each other a cationic amino acid and wherein n is at least 1.5, wherein the peptide is capable of self-assembly into b-sheet structure in the manufacture of a medicament for treating keratitis in a subject.
  • a method of treating keratitis in a subject comprises the administration of a pharmaceutically effective amount of a peptide as described herein.
  • a method of removing a biofilm from a cornea of a subject comprises the administration of a peptide as described herein.
  • Figure 1 shows design features of synthetic ⁇ -sheet forming peptides.
  • A) shows exemplary peptide of the present disclosure.
  • B) shows an example of the peptide of the present disclosure presented as a linear molecule.
  • C) shows a schematic diagram of possible membrane disruption caused by the peptide of the present disclosure.
  • the peptides exist as monomelic random coils due to electrostatic repulsion between the protonated Arg and/or Lys residues.
  • the peptides readily assemble into ⁇ -sheet secondary structures stabilized by electrostatic interactions between the positively charged residues and the negatively charged phospholipids, followed by the insertion of their hydrophobic residues into the lipid bilayer to mediate membrane disruption.
  • Figure 2 shows circular dichroism spectra of (a) (VRVK) 2 -NH 2 (SEQ ID NO: 11), (b) (IRIR) 2 -NH 2 (SEQ ID NO: 12), (c) (IKIK) 2 -NH 2 (SEQ ID NO: 13), (d) (IRIK) 2 -NH 2 (SEQ ID NO: 17), (e) (IRVK) 2 -NH 2 (SEQ ID NO: 14), (f) (FRFK) 2 -NH 2 (SEQ ID NO: 15), (g) (VRVK) 3 -NH 2 (SEQ ID NO: 20), (h) (IRIK) 3 -NH 2 (SEQ ID NO: 21), (i) (IRVK) 3 -NH 2 (SEQ ID NO: 22) in deionized water and 25 mM SDS micelles solution.
  • Figure 2 demonstrates how the peptides of the present disclosure readily self-assembled into ⁇ -sheet secondary structures in a microbial membrane mimicking
  • Figure 3 shows haemo lytic activities of the synthetic antimicrobial peptides.
  • Figure 3 shows that the peptides of the present disclosure induced minimal or no haemolysis against rat red blood cells at various minimum inhibitory concentration (MIC) values.
  • MIC minimum inhibitory concentration
  • Figure 4 shows plot of viable microbe colony-forming units (CFU) after treatment with representative peptide (IRIK) 2 -NH 2 (SEQ ID NO: 17) at various concentrations (i.e. 0, 0.5 x minimum inhibitory concentration (MIC), MIC and 2 x MIC).
  • Figure 4 shows that the peptides of the present disclosure are bactericidal at MIC values and above.
  • Figure 5 shows FE-SEM images of (a) Escherichia coli and (b) Staphylococcus aureus treated with broth containing 10% (by volume) of water as control and representative peptides (IRIK) 2 -NH 2 (SEQ ID NO: 17) and (IRVK) 3 -NH 2 (SEQ ID NO: 22) for 2 h.
  • Figure 5 shows that the peptides of the present disclosure can cause membrane lysis in Escherichia coli and Staphylococcus aureus.
  • Figure 6 shows cell viability of Staphylococcus aureus in biofilms treated with various concentrations of (IRIK) 2 -NH 2 (SEQ ID NO: 17) and (IRVK) 3 -NH 2 (SEQ ID NO: 22) for 24 h as determined using the XTT assay. * P ⁇ 0.01 relative to (IRVK) 3 -NH 2 .
  • Figure 6 shows that the peptides of the present disclosure demonstrated a dose-dependent killing of Staphylococcus aureus residing in biofilms.
  • Figure 1 shows changes in biomass of pre-formed Staphylococcus aureus biofilms treated with various concentrations of (IRIK) 2 -NH 2 (SEQ ID NO: 17) and (IRVK) 3 -NH 2 (SEQ ID NO: 22) for 24 h as determined by crystal violet staining.
  • Figure 7 demonstrates that the peptides of the present disclosure can be used to remove biofilms.
  • Figure 8 demonstrates that the peptides of the present invention are effective in disrupting lipopolysaccharide (LPS) aggregates.
  • LPS lipopolysaccharide
  • Figure 9 shows the inhibitory effects of the peptides of the present disclosure on LPS- stimulated NO production in NR8383 rat macrophage cell line.
  • Figure 9 demonstrates that the peptides of the present disclosure are effective in neutralizing the effect of LPS.
  • Figure 10 shows cell viability of NR8383 rat macrophage cell line after 18 h incubation with the peptides of the present disclosure at various concentrations.
  • Figure 10 shows that the anti-inflammatory property of the peptides of the present disclosure was independent of their effects on cell viability and that the peptides were not cytotoxic at concentrations required for antimicrobial and anti-inflammatory activities.
  • Figure 11 shows circular dichroism spectra of (a) IK8 enantiomers, (b) IK12 enantiomers, (c) IK8 stereoisomers and (d) control peptides in microbial membrane- mimicking conditions (25 mM SDS micelles solution).
  • Figure 11 shows the peptides of the present disclosure readily self-assembled to form ⁇ -sheet secondary structure in microbial membrane mimicking conditions.
  • Figure 12 shows haemolytic activities of (a) IK8 stereoisomers, (b) IK12 enantiomers and (c) IK8 ⁇ - ⁇ -sheet forming peptide controls in rabbit red blood cells.
  • Figure 12 shows the D-stereoisomers of the peptides of the present disclosure exhibit minimal or no haemolysis at MIC values, with high selectivity for microbial membranes.
  • Figure 13 shows antimicrobial activities of IK8-all L and IK8-all D against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa after 6 h treatment with proteases (a) trypsin and (b) proteinase K.
  • Figure 13 shows all the D enantiomers of the peptides of the present disclosure are protease resistant.
  • Figure 14 shows plot of viable colony-forming units (CFU) of (a) Staphylococcus epidermidis, (b) Staphylococcus aureus, (c) Escherichia coli, (d) Pseudomonas aeruginosa, and (e) Candida albicans after 18 h treatment with IK8-all D at various concentrations (i.e. 0, 0.5 x minimum inhibitory concentration (MIC), MIC and 2 ⁇ MIC).
  • the designed peptide achieved more than 3 log reductions in colony counts (> 99.9% kill) for each of the microorganisms at MIC and 2 ⁇ MIC values, indicating a bactericidal mechanism of action.
  • Figure 14 demonstrates that similar to the L-enantiomers, the D-enantiomers of the peptide of the present disclosure have bactericidal mechanisms.
  • Figure 15 shows FE-SEM images of (a) Staphylococcus aureus and (b) Pseudomonas aeruginosa treated for 2 h with 125 mg L "1 of IK8-all D and MHBII containing 10% (by volume) HPLC water.
  • Figure 15 demonstrates the D-enantiomers of the peptides of the present disclosure can cause membrane lysis in Staphylococcus aureus and Pseudomonas aeruginosa.
  • Figure 16 shows drug resistance development profiles of (a) Escherichia coli and (b) Staphylococcus aureus exposed to sub-minimum inhibitory concentrations (MIC) of IK8-all D and various clinically used antibiotics.
  • Figure 16 suggests that the peptide of the present disclosure does not induce any development of drug resistance within the timeframe tested.
  • Figure 17 shows intracellular killing of Staphylococcus aureus mediated by IK8-all D in infected mouse macrophage cell line RAW264.7 (MOI 10).
  • Figure 17 demonstrates that the peptides of the present disclosure have potent antimicrobial properties against bacteria present within infected cells.
  • Figure 18 shows the circular dichroism spectra of (a) IK8 enantiomers, (b) IK8 stereoisomers, (c) IK12 enantiomers and (d) control peptides in deionized water.
  • Figure 18 demonstrates that both the enantiomers and stereoisomers of the peptides of the present disclosure remained as random coils and did not adopt any secondary structures in deionized water.
  • Figure 19 shows MALDI-TOF mass spectra demonstrating proteolytic activity of trypsin and proteinase K on (a) (IRIK) 2 -NH 2 (SEQ ID NO: 17) and (b) (irik) 2 -NH 2 (SEQ ID NO: 18). Arrow indicates predominant enzyme cleavage site.
  • Figure 19 shows that in contrast to L-enantiomers, the treatment of proteases on D-enantiomer peptides of the present disclosure does not lead to degradation of the D-enantiomer peptides.
  • Figure 20 shows minimum inhibitory concentration (MIC) determination of (a) ciprofloxacin- and (b) gentamicin-resistant Escherichia coli after 18 h treatment with the synthetic antimicrobial peptide IK8- all D.
  • Figure 20 demonstrates that D-enantiomers of the peptides of the present disclosure is able to inhibit gentamicin- and ciprofloxacin-resistant Escherichia coli at the same concentration as that of wild-type (non-drug resistant) Escherichia coli.
  • Figure 21 shows cytotoxicity study of IK8-all D against mouse alveolar macrophage RAW264.7 and human foetal lung fibroblast WI-38 cell lines.
  • Figure 21 demonstrates that the D-enantiomers of the peptides of the present disclosure are only cytotoxic at concentrations that are well above the antimicrobial concentrations.
  • Figure 22 shows that the peptides of the present disclosure selectively kills cancer cells (HeLa) and are less cytotoxic towards non-cancerous rat alveolar macrophages (NR8383) and human dermal fibroblast cell lines.
  • HeLa cancer cells
  • NR8383 non-cancerous rat alveolar macrophages
  • Figure 23 shows a graph illustrating the killing kinetics of peptide (IKIK) 2 -NH 2 towards C. albicans.
  • Figure 23 shows significant decreased in C. albicans numbers after 2 hours treatment at MIC level. Most fungi were eradicated at 4 hours at MIC and at 1 hour at 2 x MIC or 4 x MIC.
  • Figure 23 illustrates the peptide of the present disclosure to be a useful antifungal agent against C. albicans.
  • Figure 24 shows a graph showing the percentage cell viability of C. albicans after treatment with peptide (IKIK) 2 -NH 2 for 24 hours.
  • Figure 24A shows 90% of C. albicans cells were killed in 24 hours after a single treatment of 500 mg/L of peptide (IKIK) 2 -NH 2 .
  • Figure 24B shows significantly decreased C. albicans biomass after the treatment of the same peptide at the same concentration.
  • Figure 24 shows the peptide of the present disclosure to be useful in inducing death of C. albicans and in decreasing C. albicans biomass.
  • Figure 25 shows an field emission scanning electron micrograph images of the morphological state of C. albicans biofilm before and after treatment with peptide (IKIK) 2 - N3 ⁇ 4. C. albicans cell morphology was deformed and biomass was significantly reduced after 24 hours treatment. Thus, Figure 25 shows the peptide of the present disclosure to be useful in deforming the morphology of C. albicans and in decreasing C. albicans biomass.
  • Figure 26 shows histological images of the cornea after topical administration of peptide (IKIK) 2 -NH 2 (i.e. peptide 1) and peptide (IRIK) 2 -NH 2 (i.e. peptide 2).
  • FIG. 26 shows the peptides of the present disclosure to be nontoxic to the eyes when used as eye drops.
  • Figure 27 shows photographical images of mouse eyes with C. albicans keratitis after treatment with H 2 0 (control), amphotericin B (AMB) (1000 mg/L), peptide (IKIK) 2 -NH 2 (i.e. peptide 1) (3000 mg/L) and peptide (IRIK) 2 -NH 2 (i.e. peptide 2) (3000 mg/L).
  • AMB amphotericin B
  • IKIK peptide
  • IRIK peptide
  • Figure 27 shows that the treatment efficacy of the peptides of the present disclosure was comparable with that of amphotericin B.
  • Figure 28 shows a bar graph depicting clinical slit lamp scores ( ⁇ standard deviation) for keratitis before and after treatment with four topical eye drop solutions: H 2 0 (control), amphotericin B (AMB) (1000 mg/L), peptide (IKIK) 2 -NH 2 (i.e. peptide 1) (3000 mg/L) and peptide (IRIK) 2 -NH 2 (i.e. peptide 2) (3000 mg/L).
  • the clinical scores of the amphotericin B- treated and peptide-treated eyes were significantly lower than that of the eyes in the control group (P ⁇ 0.01).
  • Figure 28 shows that there was no significant difference in the scores of the amphotericin B-treated and peptides-treated eyes.
  • Figure 29 shows histological images of corneas treated with the specified solutions.
  • the cornea samples were embedded in paraffin and serially sectioned to be stained with PAS; fungi were stained in pink.
  • Scale bar 200 ⁇ .
  • Fungal hyphae extended into the stroma of cornea in the control group, while the treatments with amphotericin B (AMB), peptide 1 and peptide 2 reduced the maximal depth of hyphal invasion into the corneas.
  • AMB amphotericin B
  • Figure 30 shows a bar graph depicting microbiology counts of C. albicans in mice cornea after treatment with amphotericin B (AMB), peptide (IKIK) 2 -NH 2 (i.e. peptide 1) and peptide (IRIK) 2 -NH 2 (i.e. peptide 2).
  • AMB amphotericin B
  • peptide (IKIK) 2 -NH 2 i.e. peptide 1
  • IRIK peptide
  • Six cornea samples from each group were collected and analyzed.
  • the peptides were as effective as amphotericin B in reducing the number of C. albicans colonies in the eyeballs, and ⁇ 90% fungal cells were eradicated from the biofilms after the treatments, as compared to the untreated control group.
  • Figure 30 shows that the peptides of the present disclosure were effective in eradicating the sessile keratitis-related fungi and their biofilms in mouse eyes.
  • Table 1 shows the characterization of the peptides of the present disclosure.
  • Table 2 shows the minimum inhibitory concentrations (MICs) and selectivity indices of the peptides of the present disclosure.
  • Table 3 shows the design and characterization of the peptides of the present disclosure.
  • Table 4 shows the minimum inhibitory concentrations (MICs) and selectivity indices of the peptides of the present disclosure.
  • Table 5 shows the minimum microbicidal concentrations (MBCs) of the peptides of the present disclosure against clinically isolated drug resistant microorganisms.
  • Table 6 shows the minimum inhibitory concentrations (MICs) of the peptides of the present disclosure against clinically isolated Mycobacterium tuberculosis.
  • Table 7 shows the clinical grading and scoring of in vivo keratitis treatment.
  • Table 8 shows the minimum inhibitory concentrations (MICs) of the peptides of the present disclosure.
  • amphipathic ⁇ -sheet peptides have been found to be less haemolytic while possessing comparable antimicrobial activities to their a-helical counterparts of equal charge and hydrophobicity.
  • Keratitis is a leading cause of ocular morbidity.
  • the different causes of keratitis are frequently misdiagnosed as one another, thus causing a delay in proper treatment.
  • treatment of keratitis remains a challenge.
  • antimicrobial peptides AMPs
  • the inventors found that synthetic ⁇ -sheet forming peptide are useful for in vivo keratitis treatment in comparison with a ⁇ - ⁇ -sheet forming peptide and the commercially available amphotericin B.
  • an amphophilic peptide comprising (XiYiX2Y 2 ) n (Formula I), wherein the C-terminal end of the peptide is amidated; Xi and X 2 may be individually selected from the group consisting of hydrophobic amino acids; Yi and Y 2 may be individually selected from the group consisting of cationic amino acids; and n may be at least 1.5, wherein the peptide is capable of self-assembly into ⁇ -sheet structure, in the manufacture of a medicament for treating keratitis in a subject.
  • the inventors of the present disclosure designed short synthetic ⁇ -sheet folding peptides consisting of short recurring (XiY)X 2 Y 2 ) n -NH 2 sequences based upon several basic design principles from naturally occurring ⁇ -sheet folding AMPs including, but not limited to: 1) the common occurrence of amphipathic dyad repeats in membrane-spanning ⁇ -sheets, 2) the requirement for hydrophobic residues (such as, but not limited to Val, He, Phe and Trp) and cationic (such as, but not limited to Arg and Lys) and to interact and perturb microbial cell walls and membranes, and 3) the strong ⁇ -sheet folding propensities of Val, He, Phe and Trp.
  • amphiphilic peptide refers to a peptide that possesses both hydrophilic and lipophilic properties, which is conferred by the peptide having cationic amino acids attached to hydrophobic amino acids.
  • hydrophobic amino acid refers to amino acid residues that are not soluble in water.
  • hydrophobic amino acid may include, but is not limited to alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), methionine (M), tryptophan (W), cysteine (C), tyrosine (Y), histidine (H), threonine (T), serine (S), proline (P) and glycine (G).
  • the hydrophobic amino acid may be alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C).
  • the hydrophobic amino acid may be isoleucine (I) or valine (V).
  • the term "cationic amino acid” refers to amino acid residues which are soluble in water.
  • the cationic amino acid may include, but is not limited to arginine (R), lysine (K) and histidine (H).
  • the amphiphilic peptide is an isolated peptide.
  • isolated' refers to a peptide free of or substantially free of proteins, lipids, nucleic acids, for example, with which it is naturally associated.
  • the amphiphilic peptide may consists of (XiYiX 2 Y2)n (Formula I), wherein Xi and X 2 may be independently of each other a hydrophobic amino acid; Yi and Y 2 may be independently of each other a cationic amino acid.
  • X] and X 2 may be the same or different amino acids and Yi and Y 2 may be the same or different amino acids.
  • n may not be 1 because peptides with four amino acids as described herein (for example XiY]X 2 Y 2 (SEQ ID NO: 1)) do not have antimicrobial activities and do not form ⁇ -sheets in microbial membrane environments. Accordingly, n may be between about 1.5 to 5. In one example, n may be 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5.
  • the amphiphilic peptide may include, but is not limited to X1Y1X2Y2X3Y3 (SEQ ID NO: 2), XiY,X 2 Y2X3Y3X4Y4 (SEQ ID NO: 3), X,YiX 2 Y2X3Y3X4Y4X 5 Y5 (SEQ ID NO: 4), XiY,X 2 Y 2 X3Y3X4Y4X5Y5X6Y6 (SEQ ID NO: 5), X1Y1X2Y2X3Y3X4Y4X5Y5X6Y6X7Y7 (SEQ ID NO: 6),
  • X, , X 2 , X3, X4, X5, X 6 , X7 and X 8 may be the same or independently different amino acids and Yi, Y2, Y3, Y4, Y 5 , Y 6 , Y7 and Y 8 , may be the same or independently different amino acids.
  • the peptides of the present disclosure may have C-terminal end that is amidated.
  • C-terminal end is used herein in accordance to its definition as commonly known in the art, that is, can be used interchangeably with any of the following terminologies such as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminus or COOH-terminus, which refer to the end of an amino acid chain, terminated by a free carboxyl group (-COOH).
  • the peptides as described herein are presented as C-terminal end on the right and N-terminal end on the left.
  • C-terminal end is amidated refers to the substitution of the hydroxyl group of C-terminal end of the peptide of the present disclosure with a primary or secondary amine group .
  • the peptides as described herein may be (XiYiX2Y2)n, which may be (X]Y
  • the peptides of the present disclosure may be amidated by process commonly known in the art. Without wishing to be bound by theory, it is believed that C- terminal amidation enhances antimicrobial activities presumably due to the reinforcement of cationic character; the peptides designed for this study were amidated at the C-terminal to confer a high net positive charge.
  • the peptides of the present disclosure does not require secondary structure stabilizer.
  • secondary structure stabilizer refers to molecules that are capable of holding local spatial arrangement of a polypeptide's backbone atoms without regard to the conformations of its side chains.
  • the secondary structure stabilizer as used herein may include, but is not limited to hydrophobic effect, electrostatic interactions and chemical crosslinks such as disulphide bonds within and between polypeptide chains or metal ions internal cross-linking.
  • the peptides of the present disclosure are distinct from existing ⁇ -sheet peptides known in the art in that they do not require disulphide bridges or other covalent bond constraints to stabilize the secondary structure.
  • the peptides of the present disclosure may be expected to exist as monomers due to electrostatic repulsion between the protonated Arg and/or Lys residues.
  • the peptides of the present disclosure readily fold into secondary ⁇ -sheet structures stabilized by electrostatic interactions between the positively charged residues and the negatively charged phospholipids, followed by the insertion of its hydrophobic residues into the lipid bilayer.
  • the peptide may be capable of self-assembly into ⁇ -sheet structure.
  • the term "self-assembly” refers to a type of process wherein a disordered system of pre-existing components forms an organized structure or pattern as a consequence of specific, local interactions among the components themselves, without external direction.
  • the peptides may spontaneously form ⁇ -sheet structures in microbial membrane environments.
  • microbial membrane environments refers to microenvironment in the membrane of target microbes. In one example, this environment may be simulated by the presence of the anionic surfactant SDS. In one example, the peptides may not spontaneously form ⁇ -sheet structures in deionised water or aqueous solution.
  • ⁇ -sheet or “ ⁇ -pleated sheet structure” refers to a regular secondary structure of peptides that may comprise at least one ⁇ -strand, which is a stretch of polypeptide chain typically 3 to 12amino acid long with backbone in an almost fully extended formation.
  • the ⁇ -sheet structure of the present invention consists of at least one ⁇ -strand.
  • the arrangement of the cationic and hydrophobic amino acids within the beta-sheet forming sequence may not be changed.
  • the peptides of the present disclosure may include: 1) choice of cationic amino acid residue (i.e. Arg vs. Lys vs. combination of both), 2) degree of polarity and bulkiness of the hydrophobic side chain, and 3) a specific length of peptide sequence.
  • the various possible configurations of peptides of the present disclosure were systematically investigated for effects on antimicrobial and haemolytic activities.
  • peptides containing Arg have been found to have stronger antimicrobial properties compared to Lys-containing.
  • each repeating unit n of Formula I comprises independently of each other 1, or 2, or 3 or 4 D-amino acids with the remaining amino acids being L-amino acids.
  • the repeating unit n of Formula I may comprise independently of each other 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 D-amino acids with the remaining amino acids being L-amino acids.
  • the repeating unit n of Formula I may comprise independently of each other 2 or 4 or 8 D-amino acids with the remaining amino acids being L-amino acids.
  • L-amino acids and “D-amino acids” refer to the two isomers that can occur in every amino acid.
  • small underlined residues represent D-amino acids while capital non-underlined represents L-amino acid.
  • L-amino acids refer to the amino acid isomer which are manufactured in cells and incorporated into proteins.
  • D-amino acids refers to isomeric modification to amino acids of the peptides of the present disclosure.
  • each repeating unit n of Formula I may comprise independently of each other 1 or 2 D-amino acids with the remaining amino acids being L-amino acids.
  • the distribution of D-amino acids in each repeating unit n of Formula I may be identical or different from each other.
  • the peptide as described herein, wherein n may be 2 and amino acids in position 4 and 6 may be D-amino acids while the remaining amino acids are L-amino acids.
  • D-amino acid substitution at positions 4, 6, enabled the retention of secondary structure forming propensity (see Figure 11c).
  • the peptides as described herein may comprise or consists of the sequence (IYiIY2) n -NH 2 . In one example, the peptides as described herein may comprise or consists of the sequence (IRX 2 ) n -NH 2 .
  • the peptide as described herein when represented by general formula X1Y1X2Y2X3Y3 may include, but is not limited to irikir-NH? (SEQ ID NO: 10), wherein small underlined residues represent D-amino acids.
  • the peptide as described herein when represented by general formula X]YiX 2 Y 2 X3Y 3 X4Y4 may include, but is not limited to VRVKVRVK-NH 2 (SEQ ID NO: 11), IRIRIRIR-NH 2 (SEQ ID NO: 12) , IKIKIKIK-NH 2 (SEQ ID NO: 13), IRVKIRVK-NH 2 (SEQ ID NO: 14), FRFKFRFK-NH 2 (SEQ ID NO: 15), WRWKWRWK-NH 2 (SEQ ID NO: 16), IRIKIRIK-NH 2 (SEQ ID NO: 17) , irikirik-NH?
  • the peptide as described herein when represented by general formula X 1 Y 1 X 2 Y 2 X 3 Y 3 X4Y4X5Y5X6Y6 may include, but is not limited to VRVKVRVKVRVK-NH 2 (SEQ ID NO: 20), IRIKIRIKIRIK-NH 2 (SEQ ID NO: 21), IRVKIRVKIRVK-NH 2 (SEQ ID NO: 22), and irvkirvkirvk-NI (SEQ ID NO: 23), wherein small underlined residues represent D-amino acids while capital non-underlined represent L-amino acid.
  • the peptides as described herein may be selected from the group consisting of VRVKVRVK-NH 2 (SEQ ID NO: 11), VRVKVRVKVRVK-NH 2 (SEQ ID NO: 20), IRIRIRIR-NH 2 (SEQ ID NO: 12), IKIKIKIK-NH 2 (SEQ ID NO: 13), IRVKIRVK-NH 2 (SEQ ID NO: 14), FRFKFRFK-NH 2 (SEQ ID NO: 15), WRWKWRWK- NH 2 (SEQ ID NO: 16), IRIKIRIK-NH 2 (SEQ ID NO: 17), IRIKIRIKIRIK-NH 2 (SEQ ID NO: 21), irikir-NH 2 (SEQ ID NO: 10), irikirik-NH?
  • an amphiphilic peptide for treating keratitis in a subject wherein the peptide comprises: (XiYiX 2 Y 2 ) n (Formula I), wherein the C-terminal end of the peptide is amidated; Xj and X 2 is independently of each other a hydrophobic amino acid Yi and Y 2 is independently of each other a cationic amino acid; and n is at least 1.5, wherein the peptide is capable of self-assembly into ⁇ -sheet structure.
  • the amphiphilic peptide as disclosed in the present disclosure may be used as a broad range antimicrobial agent that may be used to treat keratitis caused by microbes.
  • the term "microbes” or “microorganism” is used in its broadest sense and is therefore not limited in scope to prokaryotic organisms. Rather, the term “microorganism” includes within its scope bacteria, archaea, virus, yeast, fungi, protozoa and algae.
  • the keratitis as disclosed in the present disclosure is a fungal keratitis, a viral keratitis or a bacterial keratitis.
  • the bacteria may be gram positive or gram negative bacteria.
  • bacterial infections that may be treated include, but not limited to, those caused by bacteria from the genus of Acetobacter, Acinetobacter, Actinomyces, Agrobacterium spp., Azorhizobium, Azotobacter, Anaplasma spp., Bacillus spp., Bacteroides spp., Bartonella spp., Bordetella spp., Borrelia, Brucella spp., Burkholderia spp., Calymmatobacterium, Campylobacter, Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Coxiella, Ehrlichia, Enterobacter, Enterococcus spp., Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus spp.,
  • the bacteria may include, but are not limited toAcetobacter aurantius, Acinetobacter baumannii, Actinomyces Israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma phagocytophilum, Anaplasma marginale, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaminogenicus (Prevotella melaminogenica), Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi
  • the bacteria may include, but is not limited to Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus spp., Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa and Mycobacterium tuberculosis.
  • the bacterial infection may cause conditions such as, but are not limited to pneumonia, tuberculosis, meningitis, diarrhoeal diseases, formation of biofilm, sepsis, listeriosis, gastroenteritis, toxic shock syndrome, hemorrhagic colitis; hemolytic uremic syndrome, Lyme Disease, gastric and duodenal ulcers, human ehrlichiosis, pseudomembranous colitis, cholera, salmonellosis, cat scratch fever, necrotizing fasciitis (GAS), streptococcal toxic shock syndrome, nosocomial and community associated infections, atherosclerosis, sudden infant death syndrome (SIDS), ear infections, respiratory tract infections, urinary tract infections, skin and soft tissue infections, nail bed infections, wound infection, septicemia, gastrointestinal disease, hospital-acquired endocarditis and blood stream infections.
  • the bacteria may be drug resistant bacteria.
  • amphiphilic peptide as disclosed in the present disclosure may be for treating bacterial keratitis that may be caused by Pseudomonas aeruginosa, Streptococcus pneumonia, Staphylococcus aureus or Staphylococcus epidermidis.
  • the viral infectious diseases may be caused by virus including, but not limited to infections or infectious diseases caused by adenoviruses, herpes viruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, paramyxoviruses, papillomaviruses, retroviruses (such as Human Immunodeficiency Virus) and hepadnaviruses.
  • viruses including, but not limited to infections or infectious diseases caused by adenoviruses, herpes viruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, paramyxoviruses, papillomaviruses, retroviruses (such as Human Immunodeficiency Virus) and hepadnaviruses.
  • the viral infectious disease may include, but is not limited to common cold, influenza, chickenpox, cold sores, Ebola, AIDS, avian influenza, SARS, dengue, herpes, shingles, measles, mumps, rubella, rabies, human papillomavirus, viral hepatitis, coxsackie virus, Epstein Barr virus and the like.
  • amphiphilic peptide as disclosed in the present disclosure may also be used for treating viral keratitis that may be caused by varicella zoster, adenoviruses, Herpes Simplex Virus-1 (HSV-1).
  • HSV-1 Herpes Simplex Virus-1
  • fungi' includes, but is not limited to, references to organisms (or infections due to organisms) of the following genus: Absidia, Ajellomyces, Arthroderma, Aspergillus, Blastomyces, Candida, Cladophialophora, Coccidioides, Cryptococcus, Cunninghamella, Epidermophyton, Exophiala, Filobasidiella, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea, Issatschenkia, Madurella, Malassezia, Microsporum, Microsporidia, Mucor, Nectria, Paecilomyces, Paracoccidioides, Penicillium, Pichia, Pneumocystis, Pseudallescheria, Rhizopus, Rhodotorula, Scedosporium, Schizophyllum, Sporothr
  • fungal infections caused by species such as, but is not limited to Absidia corymbifera, Ajellomyces capsulatus, Ajellomyces dermatitidis, Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae and Arthroderma vanbreuseghemii, Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida pelliculosa, Cladophialophora carrionii, Coccidioides immitis and Coccidioides posadasii, Cryptococcus neoformans, Cunninghamella Sp, Epidermophyton floccosum, Exophiala derma derma
  • the fungal infection as described herein may cause by C. albicans. In one example, the fungal infection may be caused by a drug resistant fungus. Exemplary use of the peptides as described herein for inhibiting, treating or removing fungus is provided in Tables 2 and 4.
  • amphiphilic peptide as disclosed herein may be used for treating fungal keratitis that may be caused by filamentous and/or yeast-like fungi.
  • the yeast-like fungi may be Candida albicans.
  • the filamentous fungi may include Aspergillus flavus, Aspergillus fumigatus, Fusarium spp., Alternaria spp., and Paecilomyces lilacinus.
  • Sepsis refers to the major cause of mortality in intensive care units worldwide, which is triggered by the release of lipopolysaccharide molecules from the outer wall of gram- negative bacteria.
  • the peptide of the present disclosure may be used for preventing sepsis.
  • LPS lipopolysaccharide
  • LPS-binding plasma proteins LBPs
  • LPS-LBP complexes which stimulate the host monocytes and macrophages to secrete various cytokines (e.g. TNF-a, IL-6, IL-8) and proinflammatory mediators (e.g. NO and reactive oxygen species).
  • the anionic amphiphilic lipid A domain which is structurally conserved across most Gram-negative genera, is well-regarded as the active moiety of LPS.
  • the inventors of the present disclosure demonstrates that cationic antimicrobial peptide amphiphiles as described herein present a particularly useful candidate for binding and neutralizing of LPS via electrostatic interactions with the anionic head group by cationic lysine or arginine residues, and dissociation of LPS aggregates via hydrophobic interactions between the alkyl chains of LPS and non-polar amino acid side chains.
  • a method of neutralizing endotoxins comprising administration of a pharmaceutically effective amount of a peptide of the present disclosure.
  • the endotoxins may be bacterial endotoxins or fungal endotoxins.
  • the endotoxins may be polysaccharides, lipoteichoic acid, lipopolysaccharide or lipooligosaccharide.
  • Figure 9 demonstrates that the peptides of the present disclosure effectively reduce the effects of LPS on macrophages.
  • biofilms Another problem that bacteria may pose towards human is the formation of biofilms.
  • the formation of biofilms is a significant problem that is implicated in a variety of both the medical field and the non-medical field.
  • Biofilm formation occurs when microbial cells adhere to each other and are embedded in a matrix of extracellular polymeric substance (EPS) on a surface.
  • EPS extracellular polymeric substance
  • the growth of microbes in such a protected environment that is enriched with biomacromolecules (e.g. polysaccharides, nucleic acids and proteins) and nutrients allow for enhanced microbial cross-talk and increased virulence.
  • biomacromolecules e.g. polysaccharides, nucleic acids and proteins
  • a method of removing a biofilm comprising administration of a pharmaceutically effective amount of the peptide of the present disclosure.
  • the biofilm may occur on surfaces.
  • the interface between fluid and solid can be intermittent, and can be caused by flowing or stagnant fluid, aerosols, or other means for air-borne fluid exposure.
  • a surface refers, in some examples, to a plane whose mechanical structure is compatible with the adherence of bacteria or fungi.
  • the terminology "surface” encompasses the inner and outer aspects of various instruments and devices, both disposable and non-disposable, medical and non-medical.
  • non-medical uses include the hull of a ship, dockyard, food processors, mixers, machines, containers, water tanks, water filtrations, purification systems, preservatives in food industries, personal care products such as shampoo, cream, moisturizer, hand sanitizer, soaps and the like.
  • medical uses include the entire spectrum of medical devices.
  • Such "surfaces" may include the inner and outer aspects of various instruments and devices, whether disposable or intended for repeated uses.
  • Examples include the entire spectrum of articles adapted for medical use, including scalpels, needles, scissors and other devices used in invasive surgical, therapeutic or diagnostic procedures; implantable medical devices, including artificial blood vessels, catheters and other devices for the removal or delivery of fluids to patients, artificial hearts, artificial kidneys, orthopaedic pins, plates and implants; catheters and other tubes (including urological and biliary tubes, endotracheal tubes, peripherally insertable central venous catheters, dialysis catheters, long term tunnelled central venous catheters, peripheral venous catheters, short term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinary devices (including long term urinary devices, tissue bonding urinary devices, artificial urinary sphincters, urinary dilators), shunts (including ventricular or arterio-venous shunts); prostheses (including breast implants, penile prostheses, vascular grafting prostheses, heart valve
  • Surfaces found in the medical environment also include the inner and outer aspects of pieces of medical equipment, medical gear worn or carried by personnel in the health care setting. Such surfaces can include counter tops and fixtures in areas used for medical procedures or for preparing medical apparatus, tubes and canisters used in respiratory treatments, including the administration of oxygen, of solubilised drugs in nebulisers and of aesthetic agents. Also included are those surfaces intended as biological barriers to infectious organisms in medical settings, such as gloves, aprons and face-shields. Commonly used materials for biological barriers may be latex-based or non- latex based. An example for a non-latex based biological barrier material may include vinyl.
  • Such surfaces can include handles and cables for medical or dental equipment not intended to be sterile. Additionally, such surfaces can include those non-sterile external surfaces of tubes and other apparatus found in areas where blood or body fluids or other hazardous biomaterials are commonly encountered.
  • the biofilm may be comprised on catheters and medical implants.
  • a method of treating keratitis in a subject comprising administration of a pharmaceutically effective amount of a peptide of the present disclosure.
  • the terms "treat,” “treatment,” and grammatical variants thereof, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease or obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e.
  • Treatment includes eliciting a cellular response that is clinically significant, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • a method of removing a biofilm from a cornea in a subject comprising administration of a pharmaceutically effective amount of the peptide of the present disclosure.
  • the terms “decrease” , “reduced”, “reduction” , “decrease”, “removal” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “"reduced”, “reduction” or “decrease”, “removal”, or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease ( e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level (e.g., in the absence of a peptide as described herein).
  • a method of treating proliferative diseases may comprise administration of a pharmaceutically effective amount of a peptide as described herein.
  • the proliferative diseases may include, but are not limited to tumour, cancer, malignancy or combinations thereof.
  • the proliferative diseases may include, but are not limited to of colorectal cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangio-endotheliosarcoma, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
  • a peptide of the present disclosure in the manufacture of a medicament for treating bacterial infection, or removing bacteria, or neturalising endotoxins, or treating viral based infectious diseases or treating a fungal infection or infestations, or removing fungus or treating proliferative diseases.
  • the use may further comprise providing the peptide of the present disclosure for administration into a subject in need thereof.
  • the medicament is to be administered into a subject in need thereof.
  • the subject or patient may be an animal, mammal, human, including, without limitation, animals classed as bovine, porcine, equine, canine, lupine, feline, murine, ovine, avian, piscine, caprine, corvine, acrine, or delphine.
  • the patient may be a human.
  • the peptide as described herein may be provided as a composition or a pharmaceutical composition.
  • the compositions as described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration may be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) or systemic such as oral, and/or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. In one example, the route of administration may be selected from the group consisting of systemic administration, oral administration, intravenous administration and parenteral administration.
  • the composition may be administered for local treatment.
  • the composition may be provided as composition for topical administration such as eye drops, creams, foams, gels, lotions and ointments.
  • compositions and formulations for topical administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions as described herein include, but are not limited to, solutions, pastes, ointment, creams, hydrogels, emulsions, liposome-containing formulations, foams, eye drops and coatings. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • formulations as described herein may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions as described herein may be formulated into any of many possible dosage forms including, but not limited to tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions as described herein may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • compositions as described herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritic, astringents, local anaesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as buffer, dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers or combination thereof appropriate for use with the pharmacologically active agent that may be added to solution in any concentration suitable for use in eye drops.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as buffer, dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers or combination thereof appropriate for use with the pharmacologically active agent that may be added to solution in any concentration suitable for use in eye drops.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colourings, flavourings and/or aromatic substances and the like which do not deleteriously interact with the peptide(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colourings, flavourings and/or aromatic substances and the like which do not deleteriously interact with the peptide(s) of the formulation.
  • the term "pharmaceutically effective amount" as used herein includes within its meaning a sufficient but non-toxic amount of the compound as described herein to provide the desired effect, that is, causing a Log reduction in the number of microorganisms of at least 1.0, which means that less than 1 microorganism in 10 remains.
  • the modified peptides of the present disclosure may provide Log reductions in the number of microorganisms of at least about 2.0, or at least about 3.0, or at least about 4.0, or at least about 5.0, or at least about 6.0, or at least about 7.0.
  • the exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, 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.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.
  • Optimum dosages may vary depending on the relative potency of the composition, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 16 mg/ml to 5000 mg/ml and may be given once or more daily, weekly, monthly or yearly.
  • the protocol for use of the resulting eye drops to treat or to alleviate the symptoms of ocular keratitis may include placing drops in the affected eye 3 times daily until the severity of the symptoms is reduced to an acceptable level. In particularly severe cases, more frequent applications may be necessary, and in less severe cases, a single daily dose may be sufficient.
  • the composition may be administered in an amount of between any one of about 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg.
  • the concentration of the administered composition is about 1 to about 100 mg/Kg of body weight of the patient, about 5 to about 100 mg/Kg of body weight of the patient, about 10 to about 100 mg/Kg of body weight of the patient, about 20 to about 100 mg/Kg of body weight of the patient, about 30 to about 100 mg/Kg of body weight of the patient, about 1 to about 50 mg/Kg of body weight of the patient, about 5 to about 50 mg/Kg of body weight of the patient and about 10 to about 50 mg/Kg of body weight of the patient.
  • the term "about”, in the context of amounts or concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • the invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation.
  • the term "consisting essentially of” refers to those elements required for a given example. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that example of the invention.
  • Peptides used in this study were synthesized by GL Biochem (Shanghai, China) and purified to more than 95% using analytical reverse phase (RP)-HPLC.
  • the molecular weights of the peptides were further confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS, Model Autoflex II, Bruker Daltonics Inc., U.S.A.) using a-cyano-4-hydroxycinnamic acid (4-HCCA) as matrix.
  • 4-HCCA was purchased from Sigma-Aldrich (Singapore) and used in saturated acetonitrile/water (1 : 1 volume ratio) after re-crystallization.
  • Phosphate-buffered saline (10 x PBS) was purchased from 1 st Base (Singapore) and diluted to the intended concentration before use.
  • Mueller-Hinton Broth II (MHB II) and yeast mould broth (YMB) powders were purchased from BD Diagnostics (Singapore) and re-constituted according to the manufacturer's instructions.
  • Staphylococcus epidermidis ATCC No. 12228
  • Staphylococcus aureus ATCC No. 29737
  • Escherichia coli ATCC No. 25922
  • Pseudomonas aeruginosa ATCC No. 9027
  • yeast Candida albicans ATCC No.
  • LPS Lipopolysaccharide
  • FITC-conjugated LPS from E. coli 0111 :B4 were purchased from Sigma-Aldrich.
  • Griess reagent system was obtained from Promega (U.S.A.) and used according to the manufacturer's protocol.
  • Each peptide was first dissolved at 0.5 mg mL " ' in deionized (DI) water alone or DI water containing 25 niM of SDS surfactant.
  • the CD spectra were recorded at room temperature with a CD spectropolarimeter (JASCO Corp. J-810), using a quartz cell with 1.0 mm path length.
  • CD spectra were acquired with solvent subtraction from 190 to 240 nm at 10 nm/min scanning speed and were averaged from 5 runs per peptide sample.
  • the acquired CD spectra were converted to mean residue ellipticity using the following equation:
  • ⁇ ⁇ refers to the mean residue ellipticity (deg-cm 2 -dmol "1 ), is the observed ellipticity corrected for DI water at a given wavelength (mdeg), MRW is the residue molecular weight (M w /number of amino acid residues), c is the peptide concentration (mg mL 1 ), and / is the path length (cm).
  • Haemolysis (%) [(O.D.576 nm of treated sample - O.D. 576 n m of negative control)/(O.D.576 n m of positive control - O.D.576 nm of negative control)] ⁇ 100. Data are expressed as mean ⁇ standard deviations of 4 replicates.
  • E. coli suspension at ⁇ 3 10 8 CFU ml "1 (100 ⁇ ) was incubated with an equal volume of broth containing 20%> (by volume) of water and a lethal dose (250 mg L "1 ) of a representative peptide in a 96 well-plate for 2 h.
  • Eight replicates of each condition was pooled into a microfuge tube, pelleted down at 5000 ⁇ g for 5 min, and rinsed twice with PBS.
  • the samples were then fixed with 4% formaldehyde at room temperature for 15 min, followed by rinsing with DI water. Dehydration of the cells was performed using a series of graded ethanol solutions (35, 50, 75, 90, 95 and 100%).
  • the samples were allowed to air-dry, mounted on carbon table, and sputter coated with platinum for imaging with a FE-SEM setup (JEOL JSM-7400F, Japan).
  • the biomass of the biofilms was estimated by a crystal violet staining assay. Briefly, the formed biofilms were first treated with the peptides for 24 h as described above. After aspirating the culture medium, the biofilms were washed once with PBS, fixed with methanol for 15 min at room temperature and stained with 100 of 0.1% (weight by volume) crystal violet for 10 min. The excess crystal violet dye was removed by rinsing the wells with DI water for five times. The dye that is associated with the biofilm was extracted using 100 ⁇ ,, of 33% glacial acetic acid per well and quantified by measuring the absorbance at a wavelength of 570 nm using a microplate reader (TECAN, Switzerland). The relative amount of biomass remaining after peptide treatment was expressed as a percentage of the control treated with broth containing 10% (by volume) of water. Data represent mean ⁇ standard deviations of four replicates per concentration.
  • the rat macrophage cell line NR8383 was maintained in FK15 growth medium supplemented with 10% FBS, 100 U mL '1 penicillin and 100 mg mL "1 streptomycin and cultured at 37 °C under an atmosphere of 5% C0 2 and 95% humidified air. Endotoxin Neutralization Assay
  • NR8383 cells were plated at a density of 4 ⁇ 10 4 and stimulated with LPS (100 ng mL "1 ) from E. coli 0111:B4 in the presence (3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 mg L " ') or absence of peptides in each well of a 96-well plate for 18 h at 37 °C. Untreated cells and cells that were stimulated with LPS alone served as the positive and negative controls, respectively.
  • the amount of NO produced was estimated by quantifying the concentration of the stable NO metabolite nitrite in the isolated supernatant fractions using the Griess reagent (1% sulphanilamide, 0.1% N-l-napthylethylenediamine dihydrochloride, 5% phosphoric acid) according to the manufacturer's protocol. The absorbance was measured at 540 nm and nitrite concentrations were determined using a standard reference curve prepared from known concentrations of NaN0 2 solutions.
  • each peptide adopted a random coil structure that is characterized by a minimum at ⁇ 195 nm in aqueous solutions due to intermolecular electrostatic repulsion between the protonated lysine and/or argi ine residues.
  • the synthetic peptides readily self-assembled into ⁇ -sheet secondary structures with characteristic CD spectra showing a maximum at ⁇ 200 nm and minimum at ⁇ 218 nm ( Figure 2).
  • the antimicrobial activities of the synthetic peptides were tested against a representative set of clinically relevant microorganisms including Gram-positive S. epidermidis and S. aureus, Gram-negative E. coli and P. aeruginosa, and yeast C. albicans.
  • the designed peptides displayed broad spectrum antimicrobial activities against the panel of microorganisms tested with geometric mean (GM) minimum inhibitory concentrations (MICs) ranging from 13.3 to 162.7 mg L "1 .
  • GM geometric mean
  • MICs minimum inhibitory concentrations
  • Hemolysis concentration 10% (HC J O ) is defined as the lowest peptide concentration that induces > 10% hemolysis.
  • Sis of the various peptides were calculated as the ratio of the HCio value (defined as the lowest peptide concentration that induces 10% or more haemolysis) to the GM (geometric mean MICs of the 5 microbial strains tested). With the exception of (IRIK) 3 -NH 2 (SEQ ID NO: 21), all of the peptides tested were found to have high Sis of greater than 10, indicating that they are highly attractive candidates suitable for both external and systemic applications in the body.
  • the present disclosure showed the combination of both Arg and Lys residues in (IRIK) 2 -NH 2 (SEQ ID NO: 17) led to marked improvements in the SI from 11.3 and 18.3 of the single cationic amino acid peptide sequences (IRIR) 2 -NH 2 (SEQ ID NO: 12) and (IKIK) 2 - NH 2 (SEQ ID NO: 13), respectively to a value of 44.8.
  • (IRIK) 2 -NH 2 (SEQ ID NO: 17) and (IRVK) 3 -NH 2 (SEQ ID NO: 22) were respectively found to have lethal dose, 50% (LD50; dose required to kill 50% of mice after a specific test period) values of 35.2 mg/kg.
  • the LD50 values of the designed peptides contrast favorably with that reported for polymyxin B (5.4 mg/kg) and gramicidin (1.5 mg/kg).
  • aureus treated with the peptide appeared rough and uneven as compared to -the relatively smooth surface of the control sample treated with broth containing 10% (by volume) of water. This observation is consistent with the membrane lytic mechanism of the various naturally occurring and synthetic AMPs reported in the literature. Compared to conventional antibiotics that inhibit various targets within the biosynthetic pathways of microorganisms, the physical disruption of microbial cell membranes by the peptides is expected to provide an inherent advantage in the clinical setting due to the reduced likelihood for the development of mutations that confer multidrug resistance.
  • FITC-conjugated LPS was conjugated with the various peptides and monitored the changes in fluorescent intensities over 2 h.
  • FITC sequestered within LPS aggregates exhibit self-quenching, resulting in low fluorescent intensities.
  • FITC-LPS aggregates dissociate, the fluorescence increases due to a dequenching effect.
  • the endotoxin neutralizing capabilities of the synthetic peptides was established by estimating the amount of pro-inflammatory nitrogen oxide released via the quantification of the stable NO metabolite nitrite concentration present in the cell culture media after co-incubation of the rat macrophage cell line NR8383 with 100 ng mL "1 LPS and the various peptides.
  • the peptides were found to effectively inhibit LPS-stimulated NO production, with significantly decreased nitrite concentrations compared to the non-peptide treated control even at low peptide concentrations of 15.6 mg L "1 ( Figure 9).
  • the degree of LPS neutralization mediated by the various peptides was in the order of (IRIK) 3 -NH 2 (SEQ ID NO: 21) > (IRVK) 3 -NH 2 (SEQ ID NO: 22) > (VRVK) 3 -NH 2 (SEQ ID NO: 20), which was in close agreement with the trend observed earlier in the FITC-LPS interaction assay.
  • the nitrite concentration was reduced to the control level.
  • the cytotoxicities of the peptides was further evaluated against the NR8383 cell line and it was found that the cell viabilities was in excess of 80% up to concentrations of 62.5, 125 and 125 mg L "1 for (VRVK) 3 -NH 2 (SEQ ID NO: 20), (IRVK) 3 -NH 2 (SEQ ID NO: 22) and (IRIK) 3 -NH 2 (SEQ ID NO: 21), respectively ( Figure 10). These results confirmed that the good anti-inflammatory properties of the synthetic peptides were independent of their effects on cell viability and that the peptides were not cytotoxic at antimicrobial and anti-inflammatory doses, indicating their suitability for systemic administration.
  • the inventors designed a series of short synthetic ⁇ -sheet folding peptides based upon the common occurrence of amphipathic dyad repeats in natural ⁇ -sheet spanning membrane proteins.
  • the designed ⁇ -sheet folding peptides demonstrated broad spectrum antimicrobial activities against Gram-positive S. epidermidis and S. aureus, Gram- negative E. coli and P. aeruginosa as well as yeast C, albicans.
  • Acute in vivo toxicity testing in mice revealed higher intravenous LD50 values for the optimal synthetic peptides as compared to the clinically used polymyxin B and gramicidin. Further to the highly efficient killing of planktonic microbes, the synthetic peptides were also demonstrated to be potent inhibitors of bacterial growth in biofilms.
  • Peptides used in this study were synthesized by GL Biochem (Shanghai, China) and purified to more than 95% using analytical reverse phase (RP)-HPLC.
  • the molecular weights of the peptides were further confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS, Model Autoflex II, Bruker Daltonics Inc., U.S.A.) using a-cyano-4-hydroxycmnamic acid (4-HCCA) as matrix.
  • 4-HCCA was purchased from Sigma-Aldrich (Singapore) and used in saturated acetonitrile/water (1 : 1 volume ratio) after re-crystallization.
  • Phosphate-buffered saline (10 ⁇ PBS) was purchased from 1 st Base (Singapore) and diluted to the intended concentration before use.
  • Cation-adjusted Mueller-Hinton broth II (MHB II) and yeast mould broth (YMB) powders were purchased from BD Diagnostics (Singapore) and reconstituted according to the manufacturer's instructions.
  • Staphylococcus epidermidis ATCC No. 12228
  • Staphylococcus aureus ATCC No'. 6538
  • Escherichia coli ATCC No. 25922
  • Pseudomonas aeruginosa ATCC No. 9027
  • yeast Candida albicans ATCC No. 10231
  • Ciprofloxacin, gentamicin sulfate and penicillin G were obtained from Sigma-Aldrich.
  • Each peptide was first dissolved at 0.5 mg mL "1 in deionized (DI) water alone or DI water containing 25 mM of SDS surfactant.
  • the CD spectra were recorded at room temperature with a CD spectropolarimeter (JASCO Corp. J-810), using a quartz cell with 1.0 mm path length.
  • CD spectra were acquired with solvent subtraction from 190 to 240 nm at 10 nm min "1 scanning speed and were averaged from 5 runs per peptide sample.
  • the acquired CD spectra were converted to mean residue ellipticity using the following equation:
  • ⁇ refers to the mean residue ellipticity (deg cm 2 dmol "1 )
  • 0 O b S is the observed ellipticity corrected for DI water at a given wavelength (mdeg)
  • MRW is the residue molecular weight (Mw ⁇ number of amino acid residues "1 )
  • c is the peptide concentration (mg mL “1 )
  • 1 is the path length (cm).
  • MIC Minimal inhibitory concentration
  • the microbial suspensions were diluted with the appropriate broths and adjusted to give an initial optical density (O.D.) reading of approximately 0.07 at a wavelength of 600 nm on a microplate reader (TECAN, Switzerland).
  • the O.D. reading corresponds to McFarland Standard No. 1 (approximately 3 x 10 8 CFU mL "1 ).
  • the peptides were dissolved in HPLC grade water and subjected to a series of two-fold dilutions using the appropriate broths.
  • microorganism suspension with an initial loading level of 3 10 5 CFU mL "1 was added to an equal volume (100 ⁇ of polymer solutions to achieve final polymer concentrations ranging from 3.9-500 mg L "1 and with water content fixed at 10% (by volume) in each well of a 96-well plate.
  • the MIC was taken as the lowest polymer concentration at which no microbial growth was observed visually and with no change in O.D. readings from 0 h.
  • Microbial cells in broth containing 10% (by volume) water as well as pure broth alone were used as the negative controls. To ensure aseptic handling, wells containing pure broth without microbes were included in each experiment. Each test was performed in 6 replicates on at least 2 independent occasions.
  • Dehydration of the cells was performed using a series of graded ethanol solutions (35, 50, 75, 90, 95 and 100%). The samples were mounted on a copper tape, allowed to air-dry and sputter coated with platinum for imaging using a FE-SEM setup (JEOL JSM-7400F, Japan).
  • Fresh rabbit red blood cells were subjected to 25 x dilution with RPMI1640 to obtain an approximate 4 % (by volume) suspension for use in this experiment.
  • 300 ⁇ ⁇ of red blood cells suspension was added to each tube containing equal volume (300 ⁇ ) of peptide solutions in RPMI1640.
  • the tubes were incubated at 37 °C for 1 h before subjected to centrifugation at 1000 x g for 5 min. Aliquots (100 ⁇ ) of supernatant were transferred to each well of a 96-well plate and analysed for haemoglobin release at 576 nm using a microplate reader (TECAN, Switzerland). Red blood cells suspension incubated with RPMI1640 was used as negative control.
  • Haemolysis (%) [(O.D.576 nm of treated sample - O.D.576 nm of negative control)/(O.D.576 nm of positive control - O.D.576 nm of negative control)] x 100. Data are expressed as mean ⁇ standard deviations of 4 replicates.
  • E. coli and S. aureus (initial loading level of 3 x 10 5 CFU mL '1 ) were treated repeatedly for up to 20 passages with various concentrations of the peptide IK8-all D as well as the clinically used ciprofloxacin, gentamicin and penicillin G antibiotics according to the broth microdilution method described earlier. After the end of 18 h incubation at each passage, bacterial cells (at 1 ⁇ 4 MIC of the particular passage) were sub-cultured and allowed to grow to reach mid-logarithmic growth phase before being used in the subsequent MIC testing. By recording the changes in MIC with each passage, i.e.
  • Mouse alveolar macrophage cell line RAW264.7 and human foetal lung fibroblast WI-38 cells were respectively maintained in DMEM and RPMI media, supplemented with 2 mM L-glutamine, 1.5 g L "1 sodium bicarbonate and 10% FBS, and cultured at 37 °C under an atmosphere of 5% C0 2 and 95% humidified air. Intracellular bacteria killing
  • RAW264.7 cells were seeded at a density of 4 10 5 per well of a 12-well plate. Following an overnight incubation, the cells were infected for 1 h with 13.3 ⁇ ⁇ of processed bacteria (3.0 x 10 8 CFU mL "1 ) to achieve a 10:1 multiplicity of infection. The cells were then rinsed twice with 1 ⁇ PBS and incubated with 1 mL of fresh media containing 50 ⁇ g mL "1 gentamicin for 45 min to eliminate extracellular bacteria.
  • the media in each well were removed and the infected cells were incubated with fresh media containing 10% (by volume) water or various concentrations of IK8-all D (2, 3.9, 7.8, 15.6 and 31.3 mg L "1 ) for up to 4 and 8 h.
  • the infected cells were trypsinized, rinsed twice with PBS and lyzed with 800 of sterile water with incubation at room temperature for 10 min, followed by 5 min sonication.
  • the intracellular bacteria count was determined by plating serially diluted cultures onto LB agar and enumerated after 24 h.
  • RAW264.7 and WI-38 cells were respectively seeded at a density of 1.5 x 10 4 and 1 x 10 4 per well of a 96-well plate. Following overnight incubation, the cells were treated with 1.0 to 125 mg L " ' of IK8-all D for 48 h. Subsequently, the incubation media in each well were replaced with 100 ⁇ , of growth media and 10 ⁇ , of MTT solution (5 mg-ml "1 in PBS) and the cells were incubated for 4 h at 37°C according to the manufacturer's directions. Resultant formazan crystals formed in each well were solubilized using 150 of DMSO upon removal of growth media.
  • Example 1 above described the design of short synthetic ⁇ -sheet forming peptides composed of naturally occurring L-amino acids and demonstrated their broad spectrum and highly selective antimicrobial activities against clinically relevant microorganisms.
  • the antimicrobial activities of the designed peptides were evaluated against a panel of clinically relevant microorganisms, including S. epidermidis and S. aureus (Gram- positive), E. coli and P. aeruginosa (Gram-negative) as well as C. albicans (yeast).
  • IK8-4D > 500 > 500 >500 > 500 > 500 - > 2000 -
  • Control-all L 500 > 500 >500 > 500 500 - > 2000 -
  • Control-all D 500 > 500 >500 > 500 250 - > 2000 -
  • Control-4D 125 500 31.3 125 62.5 85.9 d) > 2000
  • Haemolysis concentration 10% (HCio) is defined as the lowest peptide concentration that induces > 10% haemolysis.
  • IK8-all 0 D demonstrated the most potent antimicrobial activities, with a very low geometric mean (GM) MIC value of 4.3 mg L "1 against the various types of microorganisms, which is superior to that obtained for the clinically used lipopeptide antibiotic polymyxin B (41.4 mg L "1 ) under identical testing conditions.
  • GM geometric mean
  • the designed AMPs demonstrated broad-spectrum antimicrobial activities against MRSA, VRE, multidrug resistant A. baumanni, P. aeruginosa and yeast C. neoformans. Consistent with the results observed earlier, IK8-all D induced stronger antibacterial activities, with 4-8 folds reduction in MBCs as compared to its L-enantiomer. Additionally, IK8-all D, IK8-2D and IK12-all L also demonstrated excellent activities against clinically isolated M. tuberculosis, with MICs ranging from 15.6 to 125 mg L "1 (Table 6).
  • MCCs Minimum microbicidal concentrations
  • MICs Minimum inhibitory concentrations
  • the designed peptide effectively mediated 1.2-1.5 log reductions in colony counts at 8 h when compared to the control ( Figure 17).
  • the intracellular killing of S. aureus mediated by IK8-all D was found to be independent of the cytotoxicity of the peptide as more than 80% cell viability was observed at bactericidal doses ( Figure 21).
  • Example 2 investigated the importance of secondary structure formation and effects of stereochemistry on antimicrobial activities and selectivities. It was demonstrated that the self-assembly of the synthetic peptide into ⁇ -sheets under microbial membrane- mimicking conditions was essential for its strong antimicrobial activities.
  • IK8-all D exhibited the most potent antimicrobial activities, with a very high selectivity index of 407.0.
  • the D-enantiomer also displayed enhanced stability in the presence of broad spectrum proteases, trypsin and proteinase K.
  • membrane-lytic activities of IK8-all D provided an effective means to prevent drug resistance development and effectively mediated killing of various clinically isolated multidrug-resistant microorganisms.
  • the synthetic ⁇ - sheet forming peptide IK8-all D also demonstrated efficient killing of intracellular S. aureus.
  • the human cervical cancer HeLa cell line, human dermal fibroblast HDF cell line and rat macrophage NR8383 cell line were respectively maintained in RPMI, DMEM and FK15 growth media supplemented with 10% FBS, 100 U mL "1 penicillin and 100 mg mL-1 streptomycin and cultured at 37 °C under an atmosphere of 5% C02 and 95% humidified air.
  • HeLa, HDF and NR8383 cells were respectively seeded at a densities of 1 x 10 4 , 1 x 10 4 and 4 x 10 4 cells per well of a 96-well plate. Following overnight incubation, the cells were treated with 3.9 to 500 mg L "1 of (IRIK) 3 -NH 2 (SEQ ID NO: 21) and (VRVK) 3 - NH 2 (SEQ ID NO: 20) for 24 h. For the adherent HeLa and HDF cell lines, the incubation media in each well were replaced with 100 of growth media and 10 ⁇ , of MTT solution (5 mg-rnl "1 in PBS). The cells were further incubated for 4 h at 37°C according to the manufacturer's directions.
  • Peptides used in this study were synthesized by GL Biochem (Shanghai, China) and purified to more than 95% using analytical reverse phase (RP)-HPLC.
  • the molecular weights of the peptides were further confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS, Model Autoflex II, Bruker Daltonics Inc., U.S.A.) using a-cyano-4-hydroxycinnamic acid (4-HCCA) as matrix.
  • 4-HCCA was purchased from Sigma-Aldrich (Singapore) and used in saturated acetonitrile/water (1 : 1 volume ratio) after re-crystallization.
  • Phosphate-buffered saline (10 x PBS) was purchased from 1 st Base (Singapore) and diluted to the intended concentration before use.
  • Mueller-Hinton Broth II (MHB II) and yeast mould broth (YMB) powders were purchased from BD Diagnostics (Singapore) and re-constituted according to the manufacturer's instructions.
  • Staphylococcus epidermidis ATCC No. 12228
  • Staphylococcus aureus ATCC No. 29737
  • Escherichia coli ATCC No. 25922
  • Pseudomonas aeruginosa ATCC No. 9027
  • yeast Candida albicans ATCC No.
  • LPS Lipopolysaccharide
  • FITC-conjugated LPS from E. coli 0111:B4 were purchased from Sigma-Aldrich.
  • Griess reagent system was obtained from Promega (U.S.A.) and used according to the manufacturer's protocol.
  • the MICs of peptides were determined against the fungi C. albicans (American Type Culture Collection, ATCC) and E solani (ATCC) by the broth microdilution method.
  • C. albicans were grown in yeast mold broth (YMB), at room temperature under constant shaking at 300 rpm to reach mid-logarithmic growth phase.
  • the microbial suspensions were diluted with the appropriate broths, and adjusted to give an initial optical density (O.D.) reading of ⁇ 0.07 at a wavelength of 600 nm on a microplate reader (TECAN, Switzerland), which corresponds to McFarland Standard No. 1 ( ⁇ 3 ⁇ 10 8 CFU mL-1).
  • the peptides were dissolved in 0.2 ⁇ m-I ⁇ ltered HPLC -grade water, and subjected to a series of two-fold dilutions using the appropriate broths.
  • 100 of microorganism suspension with an initial loading level of 3 ⁇ 10 5 CFU mL "1 was added to an equal volume (100 ⁇ xL) of peptide solution to achieve final concentrations ranging from 3.9 to 500 mg L "1 and with water concentration fixed at 10% (by volume) in each well of a 96-well plate. After 42 h of incubation with shaking at room temperature, the MIC was taken as the lowest peptide concentration at which no microbial growth was observed visually.
  • Yeast cells in broth containing 10% (by volume) of HPLC-grade water, as well as pure broth alone were used as the negative controls.
  • F. solani were cultured on sabouraud dextrose agar plates in incubator at 37°C. To collect conidia following incubation, conidia of F. solani species were harvested and standardized to 1 ⁇ 10 spores/ml before performing the experiments.
  • Sabouraud dextrose broth (90 ⁇ ,) was added to the wells of culture plates.
  • concentrations of peptide were prepared in the wells by 2-fold dilution method, and these wells were inoculated with 10 of spore suspension containing 1 ⁇ 10 6 spores.
  • the killing kinetics test was performed to evaluate the antimicrobial activities of peptides against C. albicans.
  • Yeast solution was re-suspended in YMB (Difco) to achieve from 10 5 to 10 6 CFU ml/ 1 .
  • the initial inoculum was exposed to peptide 1 at concentrations of l x, 2x and 4 MIC for 0, 1, 2, 4 and 24 h, and at these time points, the samples were diluted with various dilution factors. Each dilution (20 uL) was plated on agar plate, and incubated 2 days. Surviving colonies were counted after incubation. An untreated inoculum group was used as a negative control. Tests were conducted in triplicates, and the results were presented as killing efficiency (%). Stability testing
  • Peptides 1 and 2 were dissolved in HPLC-grade water at 2 mg/mL, subjected to a standard steam sterilization autoclave cycle at 121°C for 15 min, and allowed to stand at room temperature for a period of 6 weeks. The integrity of the peptides was determined by evaluating their molecular weights via MALDI-TOF MS before and after treatment.
  • C. albicans were used as representative fungi for testing the antifungal functions of peptide 1.
  • C. albicans suspensions were prepared from fresh overnight cultures in YMB using -80°C-frozen stock cultures. Subsamples of these cultures were grown for another 3 h, and adjusted to an optical density of ⁇ 0.1 at 600 nm, giving a density of 10 7 -10 8 CFU/mL.
  • the biomass of the biofilms was estimated by a crystal violet staining assay. Briefly, the formed biofilms were first treated with the peptide for 24 h as described above. After aspirating the culture medium, the biofilms were rinsed once with PBS, fixed with methanol for 15 min at room temperature and stained with 100 ⁇ ]_, of 0.1% (weight by volume) crystal violet for 10 min. The excess crystal violet dye was removed by rinsing the wells with DI water five times.
  • the dye that is associated with the biofilm was extracted using 100 ⁇ , of 33% glacial acetic acid per well, and ⁇ - ⁇ aliquot from each well was transferred to a new 96-well plate for quantification of absorbance at a wavelength of 570 nm using a microplate reader (TECAN, Switzerland).
  • the relative amount of biomass remaining after peptide treatment was expressed as a percentage of the control treated with broth containing 10 vol% of water. Data represented mean ⁇ standard deviations of 4 replicates for each concentration. Scanning electron microscopy studies on biofilm
  • C. albicans biofilm treated with peptide 1 were observed using field emission scanning electron microscopy (FESEM) (JEOL JSM-7400F) operated at an accelerating voltage of 4.0-6.0 keV.
  • FESEM field emission scanning electron microscopy
  • the C. albicans biofilm was incubated with 1000 mg/L of peptide for 24 h. It was washed with PBS thrice, and then fixed overnight in PBS containing 2.5% of glutaraldehyde. The cells were further washed with DI water, and dehydrated using a series of ethanol washes. Several drops of the suspension were placed on copper tape, and left to dry at room temperature. The samples were coated with platinum prior to FESEM analyses.
  • mice 8-week-old, 18-22 g were used for animal studies. All mice eyes were examined for absence of ocular pathology before experiments were initiated. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Biological Resource Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
  • mice eyes The peptides were tested in mice eyes.
  • the peptides were administered to the mice at a dose of 3000 mg/L every 5 min during the first hour, and every 30 min during the next 7 h. After a pause of 16 h, eye drops were administered at hourly intervals for another 8 h. All mice were sacrificed 16 h after the administration of the last eye drop.
  • the treated eyeballs were collected immediately.
  • the fixed eyeballs were embedded in paraffin, sectioned and stained with hematoxylin and eosin by the standard protocol.
  • Lotrafilcon A contact lenses used in the present study were purchased from CIBA Vision with a power of +1.50 diopters. The contact lenses were washed with PBS, and immersed in YMB overnight before they were punched into smaller pieces of 2 mm in diameter. To grow C. albicans biofilms, the lenses were placed in 6-well tissue culture plates with 4 mL of yeast suspension (107 CFU/mL), and incubated for 3 h at 22°C. The contact lenses were gently washed with PBS to remove non-adherent yeast cells, and immersed in YMB for 48 h at 22°C with shaking at 100 rpm.
  • mice A total of 29 mice were used in this study.
  • a 1-mm filter paper disc moistened with 99% 1 -heptanol (Sigma- Aldrich, Lausanne, Switzerland) was placed on the center of the cornea for 40 s.
  • the corneal epithelium was traumatically wiped out, and the eyes were rinsed with PBS to remove any remaining traces of 1 -heptanol.
  • a 2 mm-diameter punch from the contact lens with C. albicans biofilm was then placed on the denuded cornea surface.
  • the contact lenses were kept inside the eyes by closing the eye lids with silk sutures.
  • mice were immune suppressed by subcutaneous injection of cyclophosphamide (Sigma- Aldrich, 180 g/kg).
  • Table 7 Clinical grading and scoring for in vivo keratitis treatment.
  • Clinical deClear cornea Slightly Cloudy corCloudy corNearly uniscription cloudy cornea; iris and nea; opacity form opacity
  • nea pupil are visnot yet uniof cornea
  • the animals were randomly treated with 4 topical eye drop solutions: water solution (control), 3000 mg/L of peptide 1 and peptide 2, as well as 1000 mg/L of amphotericin B.
  • Eye drops (20 ⁇ each) were administered to the mice every 5 min during the first hour and every 30 min during the next 7 h. After a pause of 16 h, eye drops were administered at hourly intervals for another 8 h. All mice were sacrificed 16 h after the administration of the last eye drop.
  • the treated eyeballs were collected immediately. 3 eyeballs from each group were collected for histology, and the remaining 6 eyeballs were homogenized for quantitative fungal recovery study.
  • the fixed eyeballs were embedded in paraffin, sectioned and stained with Grocott's methenamine silver by the standard protocol.
  • peptides 1 and 2 have high potency against both planktonic keratitis-related fungi and their biofilms, as well as excellent stability. In addition, they were non-toxic to the eyes when used as eye drops.
  • C. albicans keratitis model was established in mice.
  • C. albicans were first cultured on contact lens for biofilm formation.
  • the contact lens containing a layer of C. albicans biofilm was then transferred onto the surface of de-epithelial cornea of mice.
  • the C. albicans biofilm infected the cornea, resulting in the formation of eye ulcers with a leathery, tough, raised surface.
  • Peptide 1 solution (3000 mg/L), peptide 2 solution (3000 mg/L), amphotericin B (1000 mg/L) and water (control) were applied topically as eye drops on the surface of the cornea in 20- ⁇ quantities.
  • significant resolution of the keratitis infection was observed, with the corneas becoming more transparent and the iris being visible, as compared with the control group.
  • the treatment efficacy of the peptides was comparable with that of amphotericin B (Figure 27).
  • Clinical scores of the fungal keratitis before and after treatment were evaluated according to the clinical standard. As shown in Figure 28, the clinical scores of mice corneas in all treatment groups were significantly lower than that in the control group.
  • the treated eyeballs were homogenized and the number of C. albicans cells in each eyeball was determined via agar plating.
  • the peptides were as effective as amphotericin B in reducing the number of C. albicans colonies in the eyeballs, and ⁇ 90% fungal cells were eradicated from the biofilms after the treatments, as compared to the untreated control group ( Figure 30).
  • these synthetic ⁇ -sheet forming peptides were effective in eradicating the sessile keratitis- related fungi and their biofilms in mouse eyes.
  • the inventors of the present disclosure have demonstrated that the ⁇ -sheet forming peptides have strong anti-fungi activities, and are capable of removing fungi biofilm formed both in vitro and in a fungal biofilm-induced keratitis mouse model without causing significant toxicity to the eyes.
  • the peptides are water-soluble, and stable in a autoclaving procedure. They are useful antifungal agents for treating fungal keratitis.

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Abstract

La présente invention concerne l'utilisation de peptides amphiphiles dans la fabrication d'un médicament pour le traitement de la kératite. L'invention concerne également des méthodes de traitement de la kératite et de suppression de biofilm de la cornée.
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Title
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ONG Z. ET AL.: "Effect of stereochemistry, chain length and sequence pattern on antimicrobial properties of short synthetic beta-sheet forming peptide amphiphiles", BIOMATERIALS, vol. 35, no. 4, November 2013 (2013-11-01), pages 1315 - 1325, XP028787920, ISSN: 0142-9612 *

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