Peptides with multivalent effects for topical use

Abstract

The present invention lies within the field of peptides for topical treatment. In particular, the invention provides peptides with good stability, high anti-microbial activity, high anti-fungal activity and high anti-inflammatory activity for treatment of treatment or prevention of a disorder or condition of the skin or the mucosa.

Description

[0001] Peptides with multivalent effects for topical use

[0002] Technical field

[0003] The present invention lies within the field of peptides for topical treatment. In particular, the invention provides peptides with good stability, high anti-microbial activity, high antifungal activity and high anti-inflammatory activity.

[0004] Background

[0005] In normal conditions, wounds heal in a sequenced and timely manner, characterized by four major phases (haemostasis, inflammation, proliferation, and remodelling). This is a complex process involving chemokines, growth factors, cytokines, proteases and antiproteases, and multiple cell types that all work in a controlled manner during the healing process. If this timely process is interrupted at various stages and by different factors, a dysfunctional inflammatory phase occurs, which may be associated with disruption in wound healing. Toll-like receptors (TLR), crucial for normal wound repair, can become over-activated and induce local dysfunctional inflammation which may delay wound healing. Many wounds are often complicated by bacterial infection or hyper-colonization, where bacteria and their proinflammatory products (also denoted PAMPs), such as endotoxins (lipopolysaccharide / LPS) from Gram-negative bacteria, lipoteichoic acid (LTA) and peptidoglycan (PGN) from Gram-positive bacteria, activate the TLR signalling and induce an excessive non-controlled inflammation with high tumour necrosis factor-alfa (TNF-a), interleukin (IL)-6, and I L-113> levels yielding a high level of proteolysis, which may lead to delayed healing. PAMPs may lead to TLR- mediated nuclear factor (NF)KB activation, which in turn may induce local cytokine release and secondary tissue damage.

[0006] Current treatments based on antibiotics and antivirals target only the microbes and not the accompanying inflammation. Further, as antimicrobial resistance increases, common antibiotics and some antiseptics are becoming even less effective also in treatment of wound infections. Many of the existing antimicrobial treatments, especially silver-containing creams, are harmful for the environment as well as toxic to the patient if used long-term. Therefore, there is a great need for new and cost-effective treatments that will improve healing. Treatment concepts based on Nature’s own innate defence strategies, aiming at not only targeting bacteria, but also the inflammation, could therefore have a significant therapeutic potential. Thrombin-derived C-terminal peptides (TCP) of roughly 2 kDa have been demonstrated to exert anti-endotoxic functions in vitro and in vivo. Such smaller peptides belong to the diverse family of host-defence peptides (HDPs), which includes neutrophil-derived a-defensins and the cathelicidin LL-37, all known to exhibit immunomodulatory activities TCP-25 (SEQ ID NO: 12) encompassing sequences of natural TCPs has been shown to neutralise LPS in vitro and protect against P. aeruginosa sepsis and LPS-mediated shock in experimental animal models, mainly via reduction of systemic cytokine responses (Kalle et al., 2011).

[0007] TCP-25 binds to LPS and interacts directly with monocytes and macrophages and interferes with CD14 signaling and TLR4 / MD2 dimerization thus inhibiting TLR4- and TLR2-induced NF-kB activation in response to microbe-derived agonists and intact bacteria (Saravanan et al., 2018). TCPs, apart from their interactions with bacterial membranes and LPS, also bind to the LPS-binding groove of CD14 (Saravanan et al., 2018). The fact that TCPs exert multiple and relatively weak affinities, all in the iM range to LPS and CD14, enables a modulation of host responses to infection. Sharing many characteristics with transient drugs, defined by their multivalency, multiple targets, high-off-rates and Kd values at iM levels, TCPs are therefore of interest in the development of novel anti-inflammatory therapies inspired by Nature.

[0008] Like many peptide-based therapeutics, TCP-25 is however degraded by endogenous proteases (Puthia et al., 2020). For treatment and skin diseases such as for example chronic wounds, which require extended administration of stable and active medicament, a rapidly degraded peptide would require prohibitively large doses and frequent administration. Moreover, from a pharmacological perspective, an improved affinity to its target receptor CD14 is desirable, and would reduce the effective concentration needed. Finally, as TCP-25 forms oligomers and aggregates at higher concentrations, an improved solubility would be an advantage from a drug delivery perspective.

[0009] Summary

[0010] Surprisingly, the inventors have demonstrated that compositions comprising a peptide as described herein provides both anti-inflammatory and antibacterial effects when applied topically. This makes them advantageous for the treatment of blisters, wounds and other skin disorders and conditions having a component of inflammation, infection or both inflammation and infection.

[0011] Accordingly, the present invention provides peptides with several advantageous properties including one or more of the following:

[0012] • high in vivo stability

[0013] • increased stability in the presence of proteases, such as human neutrophil elastase (HNE), Pseudomonas elastase (PE), and / or trypsin

[0014] • high anti-inflammatory activity, as for example determined by reduced release of inflammatory cytokines, such as e.g. TNF-a and / or I L-1 p or reduced NF-KB activity

[0015] • anti-microbial activity, for example bactericidal activity against Gram negative and / or Gram positive bacteria

[0016] • low hemolytic activity in blood

[0017] • low hemolytic activity against RBC

[0018] • low toxicity

[0019] In particular, the peptides of the invention have a low hemolytic activity in blood at a concentration where the peptides have high anti-inflammatory activity and high antibacterial activity.

[0020] Preferred peptides of the invention have all of the aforementioned properties. The high in vivo stability, high anti-bacterial activity, high anti-inflammatory activity and increased stability to proteases as well as the low hemolytic activity render the peptides of the invention particularly useful for topical administration.

[0021] More specifically the peptides of the invention are based on thrombin derived peptides, the structure of which have been locked by a covalent linkage between two nonneighbouring amino acids. Interestingly, the peptides of the invention have several - and preferably all - of the aforementioned advantageous properties. Many linear, thrombin derived peptides have both anti-inflammatory and antimicrobial activity, however, in general they have low in vivo stability. Having a stabilized structure, the peptides of the invention in general comprise helical structure(s). They may have a stabilized protease resistant structure, exert antimicrobial activity, and in general have an improved anti-inflammatory efficacy. The peptides of the invention are therefore interesting lead anti-inflammatory peptide mimetics. The peptides in general have lower tendency to oligomerise compared to native TCPs. Oligomerisation of drugs is a well-known phenomenon, which can cause aggregation, reduce efficacy, and increase the risk for delayed immune reactions. Thus, the peptides of the invention in general show significantly less oligomerization, which is advantageous from a drug perspective.

[0022] The endogenous TCP HVF18 exerts a higher affinity to LPS at low pH. Preferred peptides of the invention have increased polarity and charge at the N-terminus, e.g. by additions of cationic K and R residues.

[0023] Stapling of peptides may improve their proteolytic stability, however, surprisingly, stapling of certain thrombin derived peptide also led to undesired effects. For example, stapling of GKY25 at a single position results in a peptide with high hemolytic activity, which is undesirable. Stapling GKY25 at a single position further results in a peptide with reduced anti-inflammatory effect compared to unstapled GKY25.

[0024] Surprisingly, the present invention discloses that in contrast to longer peptides, such as GKY25, shorter thrombin derived peptides having a total length of 10 to 23 amino acids, such as 13 to 23 amino acids have most, and often all, of the aforementioned advantageous properties.

[0025] Furthermore, the present invention discloses that stapling of longer thrombin derived peptides having a length of 24 to 40 amino acids at at least two positions renders a peptide with some of the aforementioned advantageous properties.

[0026] The invention further shows that peptides comprising additional positively charged amino acids have even better anti-inflammatory effect. Furthermore, the peptides of the invention may also have anti-coagulant activity. The invention shows that peptides comprising additional positively charged amino acids may have even better anti-coagulant activity.

[0027] Thus, in one aspect, the present invention provides a composition for use in a method of treatment or prevention of a disorder or condition in an individual in need thereof, wherein said method comprises topical administration of the composition, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0028] In another aspect, the present invention provides a composition for use in a method of treatment or prevention of a disorder or condition of the skin or the mucosa in an individual in need thereof, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0029] In another aspect, the present invention provides a composition for use in a method of treatment or prevention of fungal infection in an individual in need thereof, the composition comprising a compound comprising a peptide comprising: a. a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; or b. the amino acid sequence

[0030] X1-X2-X3-X4-X5-X6-W-X8-X9-X10, wherein

[0031] X4, 6, 9 is any standard amino acid,

[0032] Xi is I, L or V,

[0033] X2 is any standard amino acid except C,

[0034] X3is A, E, Q, R or Y,

[0035] X5 is any standard amino acid except R,

[0036] Xs is I or L,

[0037] X10 is any standard amino acid except H, wherein said peptide has a length of from 10 to 100 amino acid residues.

[0038] In one aspect, the present disclosure provides for a composition selected from: a gel, an ointment, a cream, a paste, a lotion, or a liquid; the composition comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0039] In one aspect, the present disclosure provides for a device comprising a composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0040] Description of Drawings

[0041] Figure 1 shows the effects of stapling on antimicrobial activity of HVF18. A, the heatmaps show the antimicrobial activity of increasing concentrations of HVF18 and sHVF18 determined by RDA. The activity was evaluated on E. coli, P. aeruginosa 01 , and S. aureus, both in the absence and the presence of NaCI. Data are presented as the zones of clearance. Greyscale and values in each box represent mean values (n = 4). B, the killing effect of HVF18 and sHVF18 in Tris buffer, alone or complemented with NaCI or 25% of human plasma, evaluated by VGA. Data are presented as the means ± SEM (n=4). C The killing effect of sHVF18 on S. aureus in Tris buffer evaluated by VGA. Data are presented as the means ± SEM (n=4). D Transmission electron microscopy (TEM) showing that both P. aeruginosa 01 and S. aureus lost their membrane integrity after incubation with both linear and stapled HVF18. E-F, MIC values of HVF18 and sHVF18 for E. coli, P. aeruginosa 01 , and S. aureus (E) and clinical isolates for . aeruginosa 01 , and S. aureus (F). Experiment was repeated 4 times with similar results (n=4). G SDS-PAGE of intact and digested peptides with different proteases for different length of time. One representative image from 3 independent experiments is shown (n=3).

[0042] Figure 2 shows evaluation of secondary structure of sHVF18 and its K and R variants. All peptides were diluted in 10 mM Tris at pH 7.4 at final concentration of 10 .M from 1 mM stock solution. The spectra were acquired at 25 °C. Results are presented as the mean of three different experiments.

[0043] Figure 3 shows evaluation of hemolytic property of different stapled peptides in vitro.

[0044] The histograms show the hemolytic activity of different concentrations of sHVF18 K and R variants on erythrocytes (A) or whole blood (B). Data are the means ± SD of four independent experiments (shown as dots). In (C) the hemolytic activity of GKY25, sGKY25 and 2sGKY25 on whole blood are reported. Data are the means ± SD of two independent experiments. In all graphs dashed line represents the hemolytic activity of 100 .M sHVF18, whereas the dotted line corresponds to 10 % of lysis.

[0045] Figure 4 shows evaluation of anti-inflammatory activity of sHVF18 and its K and R variants THP-1-XBIue-CD14 reporter cells. NF-KB activation and cell viability in THP-1 monocytes stimulated with 100 ng ml-1E. coli LPS in the presence or the absence of increasing concentrations of K (A) and R (B) sHVF18 variants, 20 h post stimulation. Results are presented as means ± SD of 4 experiments (n=4). 10 iM sHVF18 in the presence of LPS was used for comparison.

[0046] Figure 5 shows evaluation of anti-inflammatory activity of stapled peptides in human blood. Cytokines released from human blood stimulated with 100 ng ml-1E. coli LPS in the presence or the absence of increasing concentrations of K (A) and R (B) sHVF18 variants, or GKY25 and its stapled variants (C), 24 h post stimulation. Results are presented as mean ± SEM. Blood from a different donor was used each time (n=4).

[0047] Figure 6 shows evaluation of antibacterial activity of sHVF18 K and R variants. The heatmaps show the antimicrobial activity of increasing concentrations of the peptides determined by RDA. The activity was evaluated on E. coli, P. aeruginosa 01 (PAO1), and S. aureus, both in the absence (A) and the presence (B) of NaCI. Data are presented as the zones of clearance expressed in mm. Greyscale and values in each box represent mean values (n = 4).

[0048] Figure 7 shows evaluation of antibacterial activity of sHVF18 K and R variants in solution. The bactericidal effect of sHVF18 and its variants in 10 mM Tris at pH 7.4, alone (A) or complemented with NaCI (B) evaluated by VGA on E. coli, P. aeruginosa 01 (PAO1), and S. aureus. Data are presented as the means ± SEM (n=4).

[0049] Figure 8 shows a selection of stapled peptide with improved anti-inflammatory activity.

[0050] A, the heatmaps show the hemolytic activity of the peptides on whole blood or erythrocytes (RBCs). Data from experiments performed on erythrocytes or blood from 4 different donors are presented as mean (n=4). B, the heatmaps show the cytokines released from human blood stimulated with 100 ng ml-1E. coli LPS in the presence or the absence of increasing concentrations of different peptides, 24 h post stimulation. Results are presented as mean. Blood from a different donor was used each time (n=4). C, graph obtained combining data from (A) and (B), and represents hemolytic activity of the peptides in function of their IC50 for different cytokines as indicated. For peptides with IC50 >10 hemolytic activity at 20 pM is shown. D, hemolytic activity of the peptides in function of their IC50 for TNF-a. The graph summarises results obtained as described in Examples 1 and 2. For peptides with IC50 >10 pM, where the exact IC50 is unknown, it was chosen to show the hemolytic activity at 50 pM.

[0051] Figure 9 shows the hemolytic and anti-inflammatory activity of K and R variants of sKKW13. A, the heatmaps show the hemolytic activity of the peptides on whole blood. Data from experiments performed on blood from 4 different donors are presented as mean (n=4). B, the heatmaps show the cytokines released from human blood stimulated with 100 ng ml-1E. coli LPS in the presence or the absence of increasing concentrations of different peptides, 24 h post stimulation. Results are presented as mean. Blood from a different donor was used each time (n=4).

[0052] Figure 10 shows the efficacy of treatment with SK-KVF18 formulated in a hydrogel (TP- 03 gel) in a mouse model of S. aureus infected acute wounds. Acute excisional wounds were made using a biopsy punch and infected with S. aureus. Wounds were treated with either control or TP-03 gel and dressings were applied. (A) Representative photographs showing wounds after 5 days of control or TP-03 gel treatment. (B) Bar chart showing percent wound closure after 5 days of treatment. (C) Luminescence I VIS in vivo imaging showing in situ wound infection. The dotted line shows the location of the wound. (D) Bacterial CFU analysis from wound swabs and dressings.

[0053] Figure 11 shows in silico analysis of staple positions. (A) HVF18 was docked on CD14 and the N-terminal GKYGFYT residues were modelled to form GKY25 peptide. The binding energy of GKY25 to CD14 was calculated using MMPBSA. Indicated amino acids were substituted with pentenyl alanine, and a staple was added to connect residues i and i+3 along the sequence of the peptide. MMPBSA was used to calculate the binding energy of the stapled peptide to CD14. The graph shows the binding energy difference between non-stapled and stapled GKY25 for all staple positions. Positive values indicate worse binding, while negative values indicate more favorable binding for the stapled peptide. (B) A similar analysis was performed by adding the staple to connect residues i and i+4. (C and D) A similar analysis was also performed for the shorter HVF18 peptide.

[0054] Figure 12 shows the antimicrobial effect of sHVF18, and its K and R variants on different Candida strains. The heatmaps show the antifungal activity of increasing concentrations of the peptides determined by RDA. The activity was evaluated on C. albicans, C. parapsilosis, C. lipolytica and C. krusei. Data are presented as the zones of clearance expressed in mm. Greyscale and values in each box represent mean values (n > 3).

[0055] Figure 13 shows the antimicrobial activity of sHVF18 against E. faecalis and B. subtilis.

[0056] Figure 14. (A) Photographs of wound on day 6 show improved wound healing with topical sHVF18 gel treatment. (B) Histology of wound tissues shows improved wound histology score on topical sHVF treatment.

[0057] Figure 15. Microbiological analysis of wounds. Wound swab and dressing CFU count shows a decrease in bacterial numbers on topical sHVF18 treatment.

[0058] Figure 16. Proinflammatory cytokine analysis of wound fluid. Topical sHVF18 treatment reduces proinflammatory cytokines in wound fluid.

[0059] Definitions

[0060] In this specification, unless otherwise specified, “a” or “an” means “one or more”.

[0061] As used herein the term “approximately” or “about” when used in relation to a numerical value refers to + / -10%, preferably + / - 5%, more preferably to + / - 1%.

[0062] The term “amino acid”, as used herein, includes the twenty standard amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural ‘L’ form), omega-amino acids other naturally-occurring amino acids, unconventional amino acids (e.g., a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

[0063] The term “blistering disease or blistering condition” as used herein refers to any disease or condition characterized by one or more blisters, in particular one or more blisters on the skin. A blister is a pocket of body fluid, commonly within the upper layers of skin. Blisters may be filled with serum, plasma, blood or, if infected, with pus. When, or if, a blister bursts or ruptures, wounds may be formed.

[0064] The term “cream” as used herein refers to a soft solid or a thick liquid intended for topical use.

[0065] The term “covalent linkage” between two side chains of amino acids as used herein refers to either a covalent bond between said side chains or to that said side chains are bound covalently to each end of a linker, so that all bonds connecting the side chains are covalent.

[0066] The term “EDTA” as used herein refers to ethylenediaminetetraacetic acid.

[0067] The term “excessive wound exudate” as used herein refers to a high amount of wound exudate emitted from a wound. In particular, an excessive (i.e. high amount of) wound exudate may hinder or slow down wound healing. It may further result in that the individual suffering from excessive wound must change wound dressing very frequently, for example because wound fluid quickly saturates or even leaks from the wound dressing. Dressing leakage may increase the risk of infection.

[0068] The term “flow point” as used herein refers to the value of the shear stress at the crossover point G' = G", wherein G’ is the storage modulus and G” is the loss modulus at 1 Hz frequency and 25°C. For example, the flow point can be determined using a Kinexus Pro rheometer (Malvern Panalytical Ltd., Malvern, UK), equipped with a plateplate geometry and a gap of 1mm. A shear strain from 0.001 to 10 strain is applied to determine the linear viscoelastic region (LVR), and flow point (shear stress at G' and G" crossover) at 1 Hz frequency and 25C. The flow point is determined directly by the rheometer. In some instances the flow point is provided as the strain at the G' and G" crossover, however if nothing else is indicated the flow point is the shear stress at G' and G" crossover, typically in Pa.

[0069] The term “gel” as used herein refers to a semi-solid that can have properties ranging from soft and weak to hard and tough. Generally, gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still diffuse through this system.

[0070] The term “hydrocarbon staple” as used herein refer to an alkyl or alkenyl moiety linking to amino acid side chains. Typically, the “hydrocarbon staple” is a Ce-16 alkenyl moiety comprising one or more double bonds.

[0071] The term "hydrogel" as used herein refers to a continuous phase of an aqueous solution and a hydrophilic polymer that is capable of swelling on contact with water. The "hydrogel" comprises nanostructures formed of said polymer and water, and typically contain more than 90% water. Hydrogels are typically transparent or translucent, regardless of their degree of hydration. Hydrogels are generally distinguishable from hydrocolloids, which typically comprise a hydrophobic matrix that contains dispersed hydrophilic particles. Hydrogels typically have a flow point of at least 10 Pa, such as at least 15 Pa, for example in the range of 10 to 80 Pa, such as in the range of 40 to 60 Pa.

[0072] As used herein the term “hydrophilic polymer” refers to a polymer that is characterized by being soluble in and compatible with water. Typically, a hydrophilic polymer possesses a polymer backbone composed of carbon and hydrogen, and generally possesses a high percentage of oxygen in either the main polymer backbone or in pendent groups substituted along the polymer backbone.

[0073] The term “internal” as used herein in relation to amino acids within a peptide, refers to that the amino acids is neither not positioned as the most N-terminal nor as the most C- terminal amino acids in the primary sequence of the peptide.

[0074] The term "local administration" as used herein refers to any form of administration of the compositions of the invention directly at the intended region of the body to be treated. Frequently, said local administration will be topical administration directly to the site of the disorder. By way of example, if the disorder is a wound, local administration implies that the composition is applied directly on the wound.

[0075] The term “mucosa” as used herein refers to the moist, inner lining of some organs and body cavities (such as the nose, mouth, lungs, and stomach). It may also be referred to as mucous membrane. In particular, the term "mucosa" refers to a moist tissue that covers some organs and body cavities. Typically, the mucosa tissue secretes the mucous material. Examples of mucosa tissue include, but are not limited to, oral mucosa e.g. buccal and sublingual; nasal mucosa; eye mucosa; genital mucosa; rectal mucosa; aural mucosa; lung mucosa; bronchial mucosa; gastric mucosa; intestinal mucosa; olfactory mucosa; uterine mucosa; and esophageal mucosa.

[0076] As used herein the term “non-ionic polymer” refers to a polymer which in a protic solvent under at room temperature and 1 atm pressure substantially bears no structural units having cationic or anionic groups needing to be offset by counterions to maintain electrical neutrality. In particular, a “non-ionic polymer” according to the invention may be a hydrophilic polymer which does not comprise monomeric units having ionizable functional groups, such as acidic or basic groups. Such a polymer will be uncharged in aqueous solution.

[0077] The term “non-neighbouring” as used herein in relation to amino acids within a peptide, refers to that two amino acids are not positioned next to each other in the primary sequence of the peptide.

[0078] The term “ointment” as used herein refers to an oil-based topical formulation with a semi-solid texture which can be applied topically. Ointments commonly comprise medicament.

[0079] The term “paste” as used herein refers to a soft, wet, sticky mixture of a substance and a liquid, which can be spread easily.

[0080] As used herein the term “polymer capable of forming a hydrogel” refers to a hydrophilic polymer that is capable of swelling on contact with water. Useful polymers will absorb at least 10 times, preferably at least 50 times, such as in the range of 50 to 200 times the amount of water compared to the polymer’s weight in an anhydrous state. The term “powder” as used herein refers to a dry mass of very small fine pieces or grains.

[0081] The term “sequence identity” as used herein refers to the % of identical amino acids or nucleotides between a candidate sequence and a reference sequence following alignment. Thus, a candidate sequence sharing 80% amino acid identity with a reference sequence requires that, following alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is determined by aid of computer analysis, such as, without limitations, the Clustal Omega computer alignment program for alignment of polypeptide sequences (Sievers et al. (2011 October 11) Molecular Systems Biology 7 :539, PMID: 21988835; Li et al. (2015 April 06) Nucleic Acids Research 43 (W1) :W580-4 PMID: 25845596; McWilliam et al., (2013 May 13) Nucleic Acids Research 41 (Web Server issue) :W597-600 PMID: 23671338, and the default parameters suggested therein. The Clustal Omega software is available from EMBL- EBI at https: / / www.ebi.ac.uk / Tools / msa / clustalo / . Using this program with its default settings, the mature (bioactive) part of a query and a reference polypeptide are aligned. The number of fully conserved residues are counted and divided by the length of the reference polypeptide. The MUSCLE or MAFFT algorithms may be used for alignment of nucleotide sequences. Sequence identities may be calculated in a similar way as indicated for amino acid sequences. Sequence identity as provided herein is thus calculated over the entire length of the reference sequence.

[0082] The term "standard amino acid" refers to any of the twenty genetically-encoded amino acids commonly found in naturally occurring peptides. The standard amino acids are referred to herein both by their IUPAC 1 -letter code and 3-letter code. The term “standard amino acid” is used to refer both to free standard amino acids, as well as standard amino acids incorporated into a peptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation.

[0083] The term “stapled peptide” as used herein refer to a peptide comprising at least one covalent linkage between the side chains of two non-neighbouring, internal amino acids. In particular, a stapled peptide may comprise a hydrocarbon staple. The term "topical administration" or "topically administering" as used herein refers to the application of a composition to the external surface of a patient, notably to the skin or mucosa. Desirably, the external surface is the skin and topical administration involves application of the composition to intact skin, to broken skin, to raw skin, to a blister or to an open skin wound.

[0084] The term “treatment” as used herein refers to any type of treatment or prevention of a disorder, including improvement in the disorder of the subject (e.g., in one or more symptoms), delay in the progression of the disorder, delay the onset of symptoms or slowing the progression of symptoms. Treatment may also be ameliorating or curative treatment. As such, the term "treatment" also includes prophylactic treatment of the individual to prevent the onset of symptoms, however treatment is preferably curative treatment, ameliorating treatment or treatment to alleviate the effect of one or more symptoms.

[0085] The term “wound exudate” as used herein refers to any fluid that filters from the circulatory system into wounds or blisters. Wound exudate may be serous, serosanguineous, sanguineous, haemorrhaging and / or purulent. It may comprise plasma, serum and / or various cell types such as neutrophils. The terms “exudate”, “wound exudate”, wound leakage” and “wound fluid” are used interchangeably herein. Wound exudate may be quantified by visual inspection of the wound dressing; by weighing the wound dressing; and / or by measuring the total protein content in the wound dressing, for example using the Bradford protein assay.

[0086] The term “position n” as used herein in relation to amino acids within a peptide refers to position in the primary sequence, wherein the most N-terminal amino acids has position n.

[0087] The terms “sK-KVF18” and “sKKVF18” are used interchangeably herein. The terms “sKK-KVF18” and “sKKKVF18” are used interchangeably herein. The terms “sKKK- KVF18” and “sKKKKVF18” are used interchangeably herein.

[0088] The terms “sR-RVF18” and “sRRVF18” are used interchangeably herein. The terms “sRR-RVF18” and “sRRRVF18” are used interchangeably herein. The terms “sR- RRRVF18” and “sRRRRVF18” are used interchangeably herein. Detailed description

[0089] Medical use

[0090] In one aspect, the present invention provides a composition for use in a method of treatment or prevention of a disorder or condition in an individual in need thereof, wherein said method comprises topical administration of the composition, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0091] In one embodiment, the disorder or condition is a disease or condition of the skin or the mucosa.

[0092] In another aspect, the present invention provides a composition for use in a method of treatment or prevention of a disorder or condition of the skin or the mucosa in an individual in need thereof, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1. In another aspect, the present invention provides a composition for use in a method of treatment or prevention of fungal infection in an individual in need thereof, the composition comprising a compound comprising a peptide comprising: a. a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; or b. the amino acid sequence

[0093] X1-X2-X3-X4-X5-X6-W-X8-X9-X10, wherein X4, 6, 9 is any standard amino acid, Xi is I, L or V, X2 is any standard amino acid except C, X3is A, E, Q, R or Y, X5 is any standard amino acid except R, Xs is I or L, X10 is any standard amino acid except H, wherein said peptide has a length of from 10 to 100 amino acid residues.

[0094] In one embodiment, the peptide of b. has a length of 18 to 35 amino acids, preferably 18-25 amino acids, and comprises or consists of any of the amino acid sequences: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 12), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 15), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 34), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 35) GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 36) GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 37), or a sequence having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity thereto. In one embodiment, the fungal infection is an infection by one or more fungi selected from the group consisting of Candida, Aspergillus, Basidiobolus, Blastomyces, Cladophialophora, Coccidioides, Fonsecaea, Phialophora, Pneumocystis, Microsporum, Trichophyton, Microsporum, Epidermophyton, Hitoplasma, Lacazia, Paracoccidioides, Scedosporium, Sporothrix and Emmonsia.

[0095] In preferred embodiments, the fungal infection is a Candida infection, such as Candidiasis. The Candida infection may for example be an infection by Candida albicans, Candida parapsilosis, Candida lipolytica and / or Candida krusei.

[0096] In one embodiment, the method of treatment comprises topical administration of the composition.

[0097] In one embodiment, the method of treatment comprises local administration of the composition.

[0098] In one embodiment, said treatment is selected from the group consisting of an ameliorating treatment, a curative treatment and a preventive treatment.

[0099] In one embodiment, the composition for use as described herein is selected from the group consisting of: a gel, an ointment, a cream, a paste, a lotion and a liquid.

[0100] Treatment of disorder or conditions of the skin

[0101] In one embodiment, disorder or condition is skin conditions or skin disorder.

[0102] In some embodiments, the disorder or condition is selected from the group consisting of: epidermolysis bullosa (EB), burn-induced blisters, chilblains, cryotherapy-induced blisters, virus-induced blisters, such as chickenpox, shingles, herpes simplex, atypical enterovirus infection, hand, foot and mouth disease, eczema herpeticum, impetigo, bullous impetigo and staphylococcal scalded skin syndrome, pemphigus, such as pemphigus foliaceous, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus erythematosus or pemphigus vegetans, pemphigoid, such as bullous pemphigoid, cicatricial pemphigoid, 200k pemphigoid or mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, porphyria cutanea tarda, bullous lupus erythematosus,

[0103] Hailey-Hailey disease and

[0104] Steven Johnson syndrome (toxic epidermal necrolysis).

[0105] In one embodiment, the disease or condition is selected from the group consisting of impetigo, chronic skin ulcers, infected acute wound and burn wounds, acne, external otitis, fungal infections, pneumonia, eye infections and nasal infections.

[0106] In one embodiment, the disease or condition is an inflammatory skin condition.

[0107] In one embodiment, the disease or condition is selected from the group consisting of rosacea, pityriasis rosea, psoriasis, eczema, microbial eczema, atopic dermatitis, seborrheic dermatitis and contact dermatitis.

[0108] In one embodiment, the disease or condition is selected from the group consisting of candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, pityriasis, acne vulgaris, dermatophytosis.

[0109] In one embodiment, the disease or condition is selected from the group consisting of folliculitis, hidradenitis suppurativa, balanitis, pearly penile papules, pyoderma gangrenosum.

[0110] In one embodiment, the disease or condition is cutaneous T cell lymphoma, such as mycosis fungoides or Sezary syndrome.

[0111] In some embodiments, the skin conditions or skin disorder is selected from the group consisting of: a disease or condition characterized by blistering, a disease or condition characterized by excessive wound exudate, and scars. In one embodiment, the skin condition or skin disorder is characterized by blistering. In one embodiment, the skin condition or skin disorder is one or more blister(s).

[0112] In some embodiments, the skin disorder or skin condition is selected from the group consisting of: burn wounds, chronic skin ulcers, infected acute wounds, pressure ulcers and chronic wounds such as venous ulcers or diabetic leg ulcers.

[0113] In some embodiments, the disorder or condition characterized by blistering is selected from the group consisting of: epidermolysis bullosa (EB), burn-induced blisters, chilblains, cryotherapy-induced blisters, virus-induced blisters, such as chickenpox, shingles, herpes simplex, atypical enterovirus infection, hand, foot and mouth disease, eczema herpeticum, bullous impetigo and staphylococcal scalded skin syndrome, pemphigus, such as pemphigus foliaceous, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus erythematosus or pemphigus vegetans, pemphigoid, such as bullous pemphigoid, cicatricial pemphigoid, 200k pemphigoid or mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, porphyria cutanea tarda, bullous lupus erythematosus, Hailey-Hailey disease and

[0114] Steven Johnson syndrome (toxic epidermal necrolysis).

[0115] In some embodiments, the disease or condition characterized by blistering is burn- induced blisters, such as blisters from second-degree superficial partial thickness burns or second-degree deep partial thickness burns.

[0116] In one embodiment, the blistering disease or condition is characterized by excessive wound exudate, such that leakage of wound fluid is a symptom of the blistering disease or condition. In one embodiment, the blistering disease or condition is characterized by a dysregulated skin microbiome, such that dysregulated skin microbiome is a symptom of the blistering disease or condition.

[0117] In one embodiment, the blistering disease or condition is characterized by scars, such that scar formation is a symptom of the blistering disease or condition.

[0118] Epidermolysis bullosa

[0119] In preferred embodiments, the disease or condition characterized by blistering is epidermolysis bullosa (EB). Thus, in a preferred embodiment the present invention relates to the compound comprising a peptide described herein for use in a method for treatment of EB.

[0120] EB is a rare genetic disease characterized by blistering and wound formation in the skin and mucous membranes following minimal trauma. There are an estimated 500 000 cases worldwide, which are caused by different mutations. At least 20 different genes coding for components contributing to the adhesion and anchorage of the skin and mucous membrane have been described to be involved in different kinds of EB. There is no cure for the condition, and treatment may instead focus on wound care and treating symptoms associated with the wounds such as pain, itch, or infection.

[0121] There are several different types of EB. The peptides of the invention may be used for treatment of any type of EB. Thus, in some embodiments of the invention, the EB is inherited epidermolysis bullosa. In other embodiments, the EB is acquired epidermolysis bullosa. In some embodiments, the EB is epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa, such as dominant dystrophic epidermolysis bullosa or recessive dystrophic epidermolysis bullosa, Kindler syndrome or epidermolysis bullosa acquisita.

[0122] Blisters may occur in all types of EB following friction and / or relatively minor trauma. They may be present anywhere on the skin and the mucous membranes. Commonly, blister formation in EB is the first sequence in wound generation, the blisters containing exudate. The wounds formed after the blisters open may produce an excessive amount of exudate. Dysregulated skin microbiome and scarring, as well as infection and inflammation of the wounds, may also be prevalent features of EB. The methods of the invention may be used to treat any such blisters, and / or to reduce excessive amount of exudate and / or to restore a healthy microbiome in EB patients.

[0123] In some embodiments, the method of treatment disclosed herein, in particular in the section “Medical use”, reduces the size of one or more blisters or wounds in an individual suffering from EB.

[0124] EB may be associated with a pain, such that pain is a symptom of EB. In some embodiments, treatment reduces the pain experienced by the individual, such as wherein said treatment reduces the pain of one or more wound or blister.

[0125] Pain may be measured using a pain scale. In some embodiments, pain is measured using a self-reporting pain scale. In other embodiments, pain is measured using an observer pain scale. In other words, in some embodiments, treatment reduces the pain experienced by the individual, wherein said reduction in pain is measured using a pain scales.

[0126] In some embodiments, pain is measured using a pain scale selected from the group consisting of Neonatal / lnfants Pain Scale (NIPS), Children’s Hospital of Eastern Ontario Pain Scale (CHEOPS), Face, Legs, Activity, Crying, Consolability (FLACC) scale, Faces Pain Scale (Wong-Baker Faces Pain Rating Scale), Coloured Analogue Scale, Visual Analog Scale, Verbal Numerical Rating Scale and Wharton Impairment and Pain Scale.

[0127] In some embodiments, pain is reduced at least one step or score on a pain scale, such as at least two steps or scores, such as at least three steps or scores, such as at least four steps or scores, such as at least five steps or scores, such as at least six steps or scores, such as at least seven steps or scores, such as at least eight steps or scores, such as at least nine steps or scores, such as wherein the pain is reduced ten steps or scores on a pain scale, such as on any one of the pain scales mentioned herein above.

[0128] In some embodiments, pain is reduced between one and ten steps or scores on a pain scale, such as between one and nine steps or scores, such as between one and eight steps or scores, such as between one and seven steps or scores, such as between one and six steps or scores, such as between one and five steps or scores, such as between one and four steps or scores, such as between one and three steps or scores, such as between one and two steps or scores, such as between two and ten steps or scores, such as between two and nine steps or scores, such as between two and eight steps or scores, such as between two and seven steps or scores, such as between two and six steps or scores, such as between two and five steps or scores, such as between two and four steps or scores, such as between two and three steps or scores, such as between three and ten steps or scores, such as between three and nine steps or scores, such as between three and eight steps or scores, such as between three and seven steps or scores, such as between three and six steps or scores, such as between three and five steps or scores, such as between three and four steps or scores, such as between four and ten steps or scores, such as between four and nine steps or scores, such as between four and eight steps or scores, such as between four and seven steps or scores, such as between four and six steps or scores, such as between four and five steps or scores, such as between five and ten steps or scores, such as between five and nine steps or scores, such as between four and eight steps or scores, such as between four and seven steps or scores, such as between four and six steps or scores, such as between five and six steps or scores, such as between six and ten steps or scores, such as between six and nine steps or scores, such as between six and eight steps or scores, such as between six and seven steps or scores, such as between seven and ten steps or scores, such as between seven and nine steps or scores, such as between seven and eight steps or scores, such as between eight and ten steps or scores, such as between eight and nine steps or scores, such as wherein pain is reduced between nine and ten steps or scores on a pain scale.

[0129] EB may also be associated with itch, such that itch is a symptom of EB. In some embodiments, treatment reduces the itch experienced by the individual, such as wherein said treatment reduces the itch of one or more wound or blister.

[0130] Itch may be determined using subjective patient reporting or by objective measurement of scratching activity or scratching induced skin changes. In some embodiments, itch is measured using the Leuven Itch Scale. In some embodiments, itch is measured using the 5-D itch scale.

[0131] EB may also be associated with excessive wound leakage, such that leakage from the EB wounds is a symptom of EB. In particular embodiments, the individual suffering from EB has one or more wounds that leak fluid, i.e. one or more wounds with excessive wound exudate, such as described herein, in particular in the section “Disease or condition characterized by wound exudate”.

[0132] Thus, in one embodiment the treatment reduces wound leakage in a patient suffering from EB, for example as described herein, in particular in the section “Disease or condition characterized by wound exudate”.

[0133] EB may also be associated with scars, such that scar formation is a symptom of EB. In particular embodiments, the individual suffering from EB has one or more scars that have been formed from the one or more wounds, such as described herein, in particular in the section “Scars”.

[0134] Thus, in one embodiment treatment reduces scar formation in a patient suffering from EB, for example as described herein, in particular in the section “Scars”.

[0135] Disease characterized by wound exudate

[0136] Wound exudate or wound fluid may be any fluid that filters from the circulatory system into wounds or blisters. Wound exudate may be serous, serosanguineous, sanguineous, haemorrhaging and / or purulent. It may comprise plasma, serum and / or various cell types such as neutrophils. In certain wounds, the amount of wound exudate may be at such a high level that it impairs healing of the wound. In other words, excessive wound exudate may slow down or worsen wound healing. Thus, decreasing the amount of wound exudate may improve healing of the wound.

[0137] The amount of wound exudate emitted from a wound may be independent of the amount of bacteria colonizing the wound. This is for example evident by the fact that the peptides of the present invention decreases bacterial levels in a wound in a doseindependent manner, while decrease of wound leakage appears to be dosedependent.

[0138] The amount of fluid leaking from a wound may be reduced in any individual in need thereof, such as in any individual suffering from a disease or condition characterized by excessive wound exudate. The individual may for example suffer a disease or condition characterized by blistering, as described herein in the section “Disease or condition characterized by blistering”. In one embodiment, the individual suffers from epidermolysis bullosa as described herein in the section “Epidermolysis bullosa”.

[0139] In some embodiments, however, the individual suffers from a skin disease or skin condition selected from the group consisting of burn wounds, chronic skin ulcers, infected acute wounds, pressure ulcers and chronic wounds such as venous ulcers, venous leg ulcers or diabetic leg ulcers.

[0140] In some embodiments, treatment reduces the amount of fluid that exudes from one or more blister or wound, such as wherein treatment reduces the amount of wound exudate.

[0141] In some embodiments, treatment decreases the amount of fluid that exudes from a wound or blister by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the amount of wound exudate is decreased by at least 95%.

[0142] In some embodiments, treatment decreases the amount of fluid that exudes from a wound or blister by between 10% and 95%, such as between 10% and 90%, such as between 15% and 80%, such as between 10% and 40%, such as between 30% and 80%, such as between 50% and 90%, such as between 20% and 50%, such as between 40% and 60%, such as between 10% and 25%, such as between 30% and 70%, such as between 80% and 90%, such as between 70% and 80% such as between 50% and 70% such as between 20% and 70%.

[0143] In some embodiments, the decrease is as compared to before starting treatment of the wound or blister. In some embodiments, the decrease is as compared to a wound or blister of similar size and character which was not treated with the compound comprising the peptide and which before treatment had a similar amount of wound exudate as the treated wound or blister.

[0144] In order to quantify wound exudate, the wound exudate may be collected in wound dressing(s), and the amount collected in said wound dressing(s) may be quantified. In some embodiments, the amount of wound exudate is estimated by visual inspection of the wound dressing.

[0145] In some embodiments, the amount of wound exudate is measured by imaging of the wound dressing.

[0146] In some embodiments, the amount of wound exudate is measured by weighing the wound dressing.

[0147] In some embodiments, the amount of wound exudate is measured by measuring the protein content in the wound dressing, such as the total protein content in the wound dressing. Thus, the total protein content in a wound dressing is correlated to wound exudate and can therefore be used as a quantification of wound exudate. In some embodiments, the protein content is measured using the Bradford protein assay and / or using any other method well known in the art.

[0148] Thus, in some embodiments, treatment decreases the total protein content that exudes from a wound or blister.

[0149] Excessive leakage of wound fluid from one or more wounds may result in that the individual suffering from said one or more wounds must change wound dressing at regular intervals. Changing wound dressing may, in particular for individuals suffering from EB or other painful wounds, be associated with a high degree of pain and suffering. Reducing the amount of fluid leaking from one or more wounds may result in that the dressing does not need to be changed as often.

[0150] Thus, in one embodiment, treatment reduces the amount of times that wound dressings need to be changed within a set time period, such as wherein treatment increases the amount of days passed before the wound dressings must be changed, such as wherein treatment results in wound dressings not needing to be changed as often as compared to before starting treatment.

[0151] The timepoint for changing the wound dressing may be determined by any person familiar with determining said timepoint, such as a caregiver, a physician, a nurse and / or the individual suffering from the disease or condition. Thus, in some embodiments, the timepoint for changing the wound dressing is determined by visual inspection of the wound dressing, such as by visual inspection by a person familiar with determining when the wound dressing needs to be changed.

[0152] In some embodiments, the timepoint for changing the wound dressing is determined based on the amount of exudate present in the dressing.

[0153] In some embodiments the timepoint for changing the wound dressing is determined to reduce the risk of inflammation and / or infection, for example wherein the timepoint is predetermined in order to reduce the risk of inflammation and / or infection.

[0154] Scars

[0155] A scar or scar tissue is an area of fibrous tissue that replaces normal skin after injury, such as after a wound. Many wounds, in particular wounds where the dermis is damaged, result in scarring. Generally, improving wound healing may decrease scar formation or prevent scars from being formed.

[0156] Scars may be treated in any individual in need thereof using the peptides disclosed herein, such as in any individual suffering from a disease or condition characterized by scar formation. The individual may for example suffer a disease or condition characterized by blistering, as described herein in the section “Disease or condition characterized by blistering”.

[0157] In some embodiments, however, the individual suffers from a skin disease or skin condition selected from the group consisting of burn wounds, chronic skin ulcers, infected acute wounds, pressure ulcers and chronic wounds such as venous ulcers, venous leg ulcers or diabetic leg ulcers, wherein said wounds are characterized by scar formation. In some embodiments, the individual suffers from an infected wound, such as a wound infected with pathogenic bacteria, for example S. aureus.

[0158] In some embodiments, treatment reduces scar formation.

[0159] In some embodiments, treatment reduces the size of an existing scar.

[0160] In some embodiments, the scar is a fibrotic scar. In some embodiments, treatment reduces the size of the scar by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein treatment reduces scar formation by at least 95%.

[0161] In some embodiments, said reduction is as compared to the size of a scar formed from a wound of similar size and character which was not treated.

[0162] In some embodiments, treatment reduces the size of the scar by between 10% and 95%, such as between 10% and 90%, such as between 15% and 80%, such as between 10% and 40%, such as between 30% and 80%, such as between 50% and 90%, such as between 20% and 50%, such as between 40% and 60%, such as between 10% and 25%, such as between 30% and 70%, such as between 80% and 90%, such as between 70% and 80% such as between 50% and 70% such as between 20% and 70%.

[0163] Wounds

[0164] The disorder or condition to be prevented or treated with the compositions of the invention may be a wound.

[0165] For example, the disorder may be disorder of the skin, ears, eyes or nose. Thus, in one embodiment the disease or condition is a disease or condition of the skin, ears, eyes or nose. In one embodiment, the treatment involves topical administration to the skin or the mucosa.

[0166] In one embodiment the method of treatment involves topical administration, for example topical administration to the skin.

[0167] The compositions described herein may be used for the treatment of wounds. Thus in one embodiment, the disorder or condition is a wound.

[0168] As shown in the examples the compositions of the invention are antibacterial and reduces inflammation and provides a faster and better wound healing compared to prior art techniques and products.

[0169] In one embodiment, the wound is selected from: burn wounds and non-healing ulcers. In one embodiment, the wound is a surgical wounds.

[0170] In one embodiment the disorder or condition is a chronic skin ulcer.

[0171] In one embodiment, the disorder or condition is a chronic wound. In one embodiment the chronic wound is caused by a disorder selected from diabetes, cancer and vasculitis. For example, the disorder or condition may be a venous ulcer, venous leg ulcer or diabetic leg ulcer.

[0172] In one embodiment, the wound is selected from the group consisting of a radiation- induced wound, a laser-induced wound and a cryotherapy-induced wound.

[0173] The composition or product may for example be applied directly to the wound, or be applied in the form of any of the products described herein, e.g. as a bandage, or suture, etc for treating the surgical wound.

[0174] These types of wound may require special measures, and the compositions of the invention are particularly useful for treatment of such wounds, because they provide both an antibacterial effect and an anti-inflammatory effect.

[0175] The disorder to be treated may in particular be a disorder comprising an inflammation or a disorder associated with an inflammation or a disorder at risk of contracting an inflammation. In particular, the disorder may be a disorder comprising or associated with a local inflammation.

[0176] The disorder to be treated may in particular be a disorder comprising an infection or a disorder associated with an infection or a disorder at risk of contracting an infection. In particular, the disorder may be a disorder comprising or associated with a local infection. Said infection may in particular be an infection by bacteria, i.e. a bacterial infection.

[0177] Said bacteria may be any infectious bacteria. For example, the bacteria may be Gram, negative or Gram positive bacteria. Thus, the bacteria may be of a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter, Haemophilus, Pseudomonas, Bacillus and Citrobacter. In one embodiment, the bacteria are selected from the group consisting of S. aureus, P. aeruginosa, E. faecalis and B. subtilis, for example the bacteria are selected from the group consisting of S. aureus and P. aeruginosa. In another embodiment, the bacteria are gram negative bacteria.

[0178] Said bacteria may even be multiresistant bacteria. Surprisingly, the compositions of the invention (and accordingly the products) are capable of providing an antibacterial effect against several multiresistant bacteria, i.e. bacteria which are resistant to several known antibiotics. The composition thus provides an additional way of treating these bacteria, including treating wounds infected by these bacteria.

[0179] In one embodiment, the treatment reduces the total amount of microorganisms present in one or more wound(s) or blister(s) by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the total amount of microorganisms present in one or more wound or blister is reduced by at least 95% as compared to the amount of bacteria present before treatment, optionally wherein the amount of microorganisms is measured by measuring the amount of colony-forming units (CFU) present in a wound dressing and / or in a wound or blister on the skin, wherein said measurement takes place before, during and / or after treatment.

[0180] The individual in need of treatment may be any individual. Typically, said individual is a mammal, and preferably said individual is a human being. In one embodiment the individual is an individual suffering from diabetes, arterial insufficiency or venous insufficiency. Individuals suffering from diabetes, arterial insufficiency or venous insufficiency frequently also suffers from non-healing ulcers, and the disorder may thus be a non-healing ulcer of an individual suffering from diabetes, arterial insufficiency or venous insufficiency. In one embodiment, the composition or product is for use in a method of treatment of a disorder of the skin, ears, eyes or nose, e.g. for treatment of a wound. Said disorder may for example be selected from the group consisting of atopic dermatitis, impetigo, chronic skin ulcers, infected acute wound and burn wounds, acne, external otitis, fungal infections, pneumonia, seborrhoic dermatitis, candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, eye infections and nasal infections. Furthermore, the disorder may be burn wounds, surgical wounds or skin trauma.

[0181] Since aforementioned disorder are often accompanied with complications such as bacterial infection and / or inflammation, the anti-infectious and anti-inflammatory treatment provided by the compositions of the invention is beneficial.

[0182] The treatment as described herein may be preventive. Thus, in one embodiment, the compositions for use as described herein may be administered to a wound in order to reduce the risk of infection and / or inflammation in said wound. The compositions of the invention may however also be administered to individuals already suffering from a local infection and / inflammation.

[0183] Preferably, treatment with the peptides of the invention significantly improves wound healing. In one embodiment, treatment reduces the size of one or more blister or wound by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the treatment reduces the size of one or more blister or wound by at least 95%.

[0184] In one embodiment, treatment reduces the size of one or more blister or wound with between 20% and 90%, such as between 30% and 80%, such as between 40% and 60%.

[0185] In one embodiment, treatment reduces the size of one or more blister or wound by 100%, such that the treatment results in that one or more blister or wound heals completely.

[0186] The reduction in size of the blister or wound may for example be as compared to the size of the one or more blister or wound before treatment. Alternatively, the reduction in size of the blister or wound may be as compared to the size of a blister or wound which was not treated with the compound comprising the peptide, and which before treatment was of similar size and character as the one or more treated blister or wound.

[0187] The peptide may be topically administered in any dose and formulation suitable.

[0188] In one embodiment, at least 1 .5 mg / cm2peptide is administered to the wound or blister, such as at least 2 mg / cm2peptide, such as at least 2.5 mg / cm2peptide, such as at least 3 mg / cm2peptide, such as at least 4 mg / cm2peptide, such as at least 5 mg / cm2peptide.

[0189] In one embodiment, between 1 and 5 mg / cm2peptide is administered to the wound or blister, such as between 1.5 and 4 mg / cm2peptide, such as between 2 and 3 mg / cm2peptide.

[0190] Stapled peptide

[0191] In one embodiment, the peptide according to the present disclosure comprises a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptides: i) have a total length between 10 and 40 amino acids, preferably a total length between 10 and 23, more preferably a total length between 13 and 23; ii) comprise at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprise at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0192] In one embodiment, the peptide according to the present disclosure comprises a consecutive sequence of in the range of 10 to 40 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptides: i) have a total length between 10 and 40 amino acids, preferably a total length between 10 and 23, more preferably a total length between 13 and 23; ii) comprise at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprise at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0193] In one embodiment, the peptide according to the present disclosure comprises or even consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 10 and 23 amino acids; ii) comprises at least one covalent linkage between the side chains of two nonneighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2 herein; and iii) comprises at least amino acids K247, K248, K252 of thrombin of SEQ ID NO: 1.

[0194] In one embodiment, the peptide according to the present disclosure comprises or even consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 13 and 23 amino acids; ii) comprises at least one covalent linkage between the side chains of two nonneighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2 herein; and iii) comprises at least amino acids K247, K248, K252 of thrombin of SEQ ID NO: 1.

[0195] In one embodiment, the peptide is as disclosed herein above with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two non-neighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0196] In some embodiments, the peptides of the invention comprise at least amino acids

[0197] R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1 The internal covalent linkage may be as described herein below in the section “Internal covalent linkage”.

[0198] Preferably, the peptide has one or more of the advantageous properties described in the “Summary” herein above and in the section “Peptide Function” below.

[0199] The peptides are useful for treatment of inflammation and / or infection as disclosed herein, in particular as described in the section “Method of treatment”.

[0200] The sequence of prothrombin is given herein as SEQ ID NO: 16. Prothrombin may be cleaved at Arg271. This cleavage produces two fragments known as Fragment 1 *2, comprising the first 271 residues of prothrombin and the intermediate prethrombin 2, which is made up of residues 272-579. Fragment 1*2 is released as an activation peptide, and prethrombin 2 is cleaved at Arg320, yielding active thrombin. The sequence of active thrombin is given herein as SEQ ID NO: 1.

[0201] Internal covalent linkage

[0202] One hallmark of the peptides of the invention is that they contain a covalent linkage between the side chains of two non-neighbouring, internal amino acids. Thus, said covalent linkage is either a direct covalent bond between the side chains of said amino acids or the side chains are linked covalently to each other through a linker. In other words, covalent linkages in the peptide back-bone are not considered “a covalent linkage between the side chains of two non-neighbouring, internal amino acids” according to the invention.

[0203] Shorter peptides

[0204] Whereas it is possible that the peptide contains more than one such covalent linkage, it is preferred that the peptide contains only one covalent linkage between two nonneighbouring, internal amino acids if said peptide is between 10 and 23 amino acids. The amino acids having an internal linkage between the side chains are also denoted Xi and X2 herein.

[0205] Whereas Xi and X2 are bound to each other by a covalent linkage in the peptides of the invention, Xi and X2 may be described in their free, unbound form herein. The skilled person will understand that even if Xi and X2 are described in their unbound form, in the final peptides of the invention, they will have formed the relevant covalent linkage. By way of example, Xi and X2 may each be described as “(S)-2-(4’-pentenyl)-alanines”, however in the peptide of the invention, the pentenyl groups will have reacted - typically by ring closing metathesis - to form a linker consisting of an 8 carbon long alkenyl with one double bond only.

[0206] Peptides comprising a covalent linkage between two non-neighbouring, internal amino acids are also known as “stapled” peptides.

[0207] There are many different ways of forming stapled peptides known to the skilled person and the peptides of the invention may comprise any kind of covalent linkage between two non-neighbouring, internal amino acids useful for peptide stapling. For example, the peptide may comprise any of the staples described in Li et al, 2020 or in international patent application WO2019018499 both of which are incorporated herein by reference in their entirety.

[0208] It is preferred that when amino acid Xi is positioned at position n, then amino acid X2 is positioned at position n+3, or at position, n+4, or at position n+5, or at position n+6, or at position n+7, or at position n+8, or at position n+9, or at position n+10, or at position n+11, wherein n is an integer. More preferably, when amino acid Xi is positioned at position n, then amino acid X2 is positioned at position n+3, or at position n+4, or at position n+7, or at position n+11, wherein n is an integer. Even more preferably, when amino acid Xi is positioned at position n, then amino acid X2 is positioned at position at position n+4, or at position n+7, wherein n is an integer. The latter positioning pattern is especially useful for supporting an a-helical structure of the peptide. Typically, n is an integer in the range of 2 to 18, however n must be chosen such that Xi is not positioned at the very N-terminus, more preferably, neither Xi nor X2 is positioned at the very N-terminus or the very C-terminus.

[0209] In principle, Xi can be positioned at any position within the peptide apart from at the very N-terminus. However, certain positions within the peptide may be more favourable than others. The peptides of the invention comprises a consecutive sequence of amino acids from Thrombin of SEQ ID NO: 1 or from GKY25 of SEQ ID NO: 12. In the following, the position of the amino acids is given in relation to the amino acid numbering of GKY25 of SEQ ID NO: 12. Thus, any amino acid having the same position as a given amino acid in GKY25 of SEQ ID NO: 12 following an alignment, is referred to as “aligning to” said amino acid of GKY25.

[0210] In one embodiment it is preferred that following alignment of the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then: a. X2 does not align to a Lys in GKY25 of SEQ ID NO: 12; and b. X2 does not align to a Gin, when Xi aligns to a Lys.

[0211] In one embodiment it is preferred that following alignment of the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then: i) Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 ii) Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12 iii) X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12; and iv) X2 does not align to Lys 18 in GKY25 of SEQ ID NO: 12; and v) X2 does not align to Gln22, when Xi aligns to Lys18 of SEQ ID NO: 12.

[0212] In one embodiment, amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+3, wherein n is an integer in the range of 2 to 18, and following alignment of the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then: i) Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 ii) Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12; and iii) X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12.

[0213] In one embodiment, amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, and following alignment of of the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then: i) Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 ii) Xi does not align to Leu12 in GKY25 of SEQ ID NO: 12 iii) Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12 iv) X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12; v) Xi does not align to Lys 18 in GKY25 of SEQ ID NO: 12; and vi) X2 does not align to Lys 18 in GKY25 of SEQ ID NO: 12. In a preferred embodiment, amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+3, wherein n is an integer in the range of 2 to 18, and wherein when aligning the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds to: Val9 and Leu12; or Phe10 and Lys13; or Leu12 and Trp15; or Lys13 and Ile16; or Ile16 and Val19; or Gln17 and lle20; or Lys18 and Asp21 ; or Val19 and Gln22; or lle20 and Phe23; or Asp21 and Gly24; or Gln22 and Glu25 of SEQ ID NO: 12.

[0214] In another preferred embodiment, amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, wherein when aligning the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds to: Val9 and Lys13; or Lys13 and Gln17; or Trp15 and Val19; or Ile16 and lle20; or Gln17 and Asp21 ; or Val19 and Phe23; or lle20 and Gly24; or Asp21 and Glu18 of SEQ ID NO: 12.

[0215] In a very preferred embodiment, amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, wherein when aligning the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds Gln17 and Asp21 , respectively.

[0216] In one embodiment, Xi and X2 are canonical amino acids before reaction to form the covalent linkage. For example, Xi and X2 may before reaction to form the covalent linkage be selected from the group consisting of: i) Xi is Lys and X2 is selected from the group consisting of Asp, Glu, Lys, Cys and Tyr; ii) Xi is Cys and X2 is selected from the group consisting of Cys, Lys and Met; iii) Xi is Asp and X2 is Lys; iv) Xi is Glu is X2 is selected from the group consisting of Lys and Glu; v) Xi is Tyr and X2 is selected from the group consisting of Lys, Phe and Trp; vi) Xi is Met and X2 is selected from the group consisting of Met and Cys; vii) Xi is His and X2 is His; viii) Xi is Phe and X2 is selected from the group consisting of Phe, Tyr, Ala and Trp; ix) Xi is Ala and X2 is Phe or Tyr; x) Xi is Trp and X2 is selected from the group consisting of Trp, Phe and

[0217] Tyr.

[0218] In one embodiment, Xi and X2 may before reaction to form the covalent linkage be as follows:

[0219] Xi is Lys and X2 is Asp, Glu, Cys or Lys or vice versa.

[0220] Xi and X2 are Cys.

[0221] The covalent linkage may be formed by a direct reaction between the side chains of the canonical amino acids, and it may be formed via a cross linker. When Xi and X2 are Cys the covalent linkage may be a disulphide bridge or it may be formed via a crosslinker, wherein the crosslinker for example is a bis-alkylator, such as linker comprising at least two (bromomethyl) substituents. In a preferred embodiment, Xi and X2 are derivatised canonical amino acids. Before reaction to form the covalent linkage, Xi and / or X2 may for example be selected from the group consisting of Ser derivatives and Ala derivatives.

[0222] In one embodiment, the covalent linkage is formed by linking two non-canonical amino acids. For example, the covalent linkage may be formed by linking two non-canonical amino acids, which have substituted two native amino acids of the consecutive sequence from thrombin.

[0223] In one embodiment the covalent linkage is a hydrocarbon staple.

[0224] In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage are alkenylated amino acids, such as two C-alkenylated amino acids, such as two a- substituted alkenyl amino acids and / or a, a-disubstituted alkenyl amino acids, and the covalent linkage is an olefin tether formed between said alkenyl residues.

[0225] Said alkenylated amino acids may be amino acids native to thrombin, which have been alkenylated. Alternatively, said alkenylated amino acids may be amino acids substituting amino acids native to thrombin.

[0226] In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage may individually be selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe. In such cases the covalent linkage is an olefin tether formed between said alkenyl residues.

[0227] In one preferred embodiment, one of Xi and X2 before reaction to form the covalent linkage may be alkenylated Ala and the other may be selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe. In such cases the covalent linkage is an olefin tether formed between said alkenyl residues.

[0228] In one preferred embodiment, wherein Xi and X2 are a-alkenyl olefin-terminated amino acids and / or a, a-disubstituted alkenyl olefin-terminated amino acids. In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage may be alkenylated alanine, preferably a-substituted alkenyl or a,a-disubstituted alkenylated alanine In such cases the covalent linkage is an olefin tether formed between said alkenyl residues.

[0229] In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage may be alkenylated Ser, such as O-alkenylated Ser.

[0230] Said alkenylated amino acids comprise 2 to 10 carbons in the alkenyl chain, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, preferably 4, 5 or 6 carbons. Said alkenylated amino acids may contain one or more double bonds, however preferably only one double bond. It is further preferred that the double bond is positioned at the free end of the alkenyl. Two double bonds positioned at the free end of two alkenyl residues can react by RCM to form an olefin tether.

[0231] Thus, it is preferred that Xi and X2 are amino acids, which are linked by an olefin tether formed between said alkenyl residues. Said olefin tether may be a Ce-16 alkenyl, such as a Cs-14 alkenyl, for example a Cs or a C14 alkenyl tether. Said tether may contain one or more double bonds, preferably one double bond.

[0232] In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage may be a,a-disubstituted S- or R-pentenylalanine (S5 or R5) or S- or R-octenylalanine (S8 or R8) alanine. Thus, the internal hydrocarbon staple may be formed by linking two a,a-disubstituted S- or R-pentenylalanine (S5 or R5) or S- or R-octenylalanine (S8 or R8) alanine.

[0233] In one preferred embodiment, Xi and X2 before reaction to form the covalent linkage may be (S)-2-(4’-pentenyl)-alanines. Thus, the internal hydrocarbon staple may be formed by linking two (S)-2-(4’-pentenyl)-alanines.

[0234] In one embodiment the covalent bond is established through ring-closing, such as through ring closing metathesis (RCM). In one preferred embodiment, one of Xi and X2 before reaction to form the covalent linkage may non-canonical azido terminated amino acid and the other a non-canonical yne-terminated amino acid.

[0235] Longer peptides

[0236] It is preferred that peptides of the invention which are 24 amino acids or longer comprise at least two staples. In other words, it is preferred that peptides of the invention which are 24 amino acids or longer, such as between 24 and 40 amino acids, comprise at least two internal covalent linkages between the side chains of two nonneighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and are as described above, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0237] Thus, in one embodiment, the composition comprises a peptide as described herein, with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two nonneighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0238] It is also preferred that peptides comprising the 4 most N-terminal amino acids of GKY25 of SEQ ID NO: 12 also comprises at least two staples, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and are as described above, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0239] X3 and X4 may be as described above in the section “Shorter peptides”. Preferably, X3 and X4 are closer to the N-terminal as compared to Xi and X2. In particular, X3 and X4 may be positioned so that X3 is positioned at one of the first 4 amino acids. If the peptide comprises the 4 most N-terminal amino acids of GKY25 of SEQ ID NO: 12, it is preferred that X3 is positioned at a position corresponding to one of the 4 most N- terminal amino acids of GKY25 of SEQ ID NO: 12.

[0240] In some embodiments, amino acid X3 is positioned at position n, and amino acid X4 is positioned at position n+3, or at position, n+4, or at position n+5, wherein n is an integer. In preferred embodiments, amino acid X3 is positioned at position n, and amino acid X4 is positioned at position, n+4, wherein n is an integer in the range of 1 to 18, preferably in the range of 1 to 10, more preferably in the range of 1 to 5. Thus, whereas it is preferred that Xi is not positioned at the very N-terminus, X3 may very well be positioned at the very N-terminus.

[0241] If X3 is positioned at the very N-terminus, the staple may be formed between the N- terminal -NH2 group and the side chain of an amino acid, preferably with the side chain of a Glu or an Asp, more preferably a Glu.

[0242] In certain embodiments, amino acid X3 is positioned at the very N-terminus of the peptide. In preferred embodiments, amino acid X3 is positioned at the very N-terminus and amino acid X4 is positioned at position n+4. In very preferred embodiments, when aligning the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then X3 and X4 corresponds to Gly 1 and Phe5 of GKY25 of SEQ ID NO: 12, wherein Phe5 is substituted with either Glu or Asp, preferably Glu. The latter positioning pattern of X3 and X4 is especially useful for protecting or masking protease site(s) of the peptide.

[0243] The covalent linkage of X3 and X4 may for example be an amide bond formed between a carboxylic acid moiety and an amine. In some embodiments, the carboxylic acid moiety is a glutamic acid side chain. In some embodiments, the amine is an amino acid side chain. In other embodiments, the amine is an N-terminal amine group of a peptide backbone. In some embodiments, the covalent linkage of X3 and X4 is a lactam bridge formed between an N-terminal amine group and a side chain carboxylic acid of said respective amino acids.

[0244] In preferred embodiments, Phe5 of GKY25 of SEQ ID NO: 12 is substituted by Glu5. In such embodiments, X3 and X4 corresponds to Gly 1 and Glu5 of SEQ ID NO: 14.

[0245] Peptide properties

[0246] The peptides of the invention may have one or more of the following properties.

[0247] The invention shows that stapled, longer thrombin derived peptides, such as GKY25, containing a single staple have several less desirable properties. Thus, GKY25 containing one staple has high hemolytic activity and low anti-inflammatory activity in blood, which therefore precludes in vivo use.

[0248] In contrast, stapled shorter thrombin derived peptides, such as stapled HVF18 have relatively low hemolytic activity in blood and high anti-inflammatory activity in blood.

[0249] Furthermore, double-stapled longer thrombin derived peptides, such as GKY25 with two staples, have lower hemolytic activity in blood compared to single stapled GKY25, and higher stability compared to non-stapled GKY25.

[0250] Shorter peptides

[0251] Accordingly, it is preferred that the peptide of the invention has an appropriate length. Thus, preferably the peptide of the invention has a length between 10 and 23 amino acids, such as between 13 and 18 amino acids, such as between 12 and 22 amino acids, such as between 14 and 22 amino acids, such as between 15 and 21 amino acids, such as between 16 and 20 amino acids, such as between 17 and 20 amino acids.

[0252] In particular, the peptide may have a length of 18 to 20 amino acids, such as 18 or 19 amino acids.

[0253] The length of the peptide indicates the total length of the peptide. Thus, even if the peptide may be conjugated to one or more additional moieties, the peptide preferably does not comprise more than the indicated number of amino acids.

[0254] Furthermore, it is also preferred that the peptide of the invention comprises a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , which has an appropriate length. Thus, preferably the peptide of the invention comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 10 to 23 amino acids, such as between 13 and 23 amino acids, such as between 13 and 18 amino acids, such as between 14 and 22 amino acids, such as between 15 and 21 amino acids, such as between 16 and 20 amino acids, such as between 17 and 20 amino acids, preferably between 17 and 18 amino acids, more preferably between 18 and 19 amino acids. The peptide according to the invention comprises or even consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , wherein up to 6 amino acids may be exchanged. In other words, the peptide may comprise or consist of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , however from 1 to 6 of the amino acids within said consecutive sequence may be substituted for another amino acid. It is however important that the peptide comprises at least amino acids K247, K248, K252 of thrombin of SEQ ID NO: 1. In other words, said amino acids should not be substituted.

[0255] In some embodiments, the peptide comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0256] The peptide according to the invention may in particular have a total length between 10 and 23 amino acids and comprise or even consist of a consecutive sequence of amino acids from GKY25 of SEQ ID NO: 12, wherein up to 6 amino acids may be exchanged. In other words, the peptide may comprise or consist of a consecutive sequence of amino acids from GKY25 of SEQ ID NO: 12, however from 1 to 6 of the amino acids within said consecutive sequence may be substituted for another amino acid. It is however important that the peptide comprises at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12. In other words, said amino acids should not be substituted.

[0257] In some embodiments, the peptide comprises at least amino acids R11, K13, K14 and K18 of GKY25 of SEQ ID NO: 12.

[0258] The peptide according to the invention may preferably comprise or even consist of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 16 to 21 amino acids, more preferably of in the range of 17 to 18 amino acids.

[0259] The consecutive sequence may contain up to 6 amino acid substitutions. For example, said peptide may comprise at least 2 amino acid substitutions, such as 3 amino acid substitutions, such as 4 amino acid substitutions, such as 5 amino acids substitutions compared to the consecutive sequence of thrombin. Preferably, the consecutive sequence may comprise up to 4 amino acid substitutions, even more preferably the consecutive sequence may comprise up to 2 amino acid substitutions. One or more of said substitutions may be conservative substitutions, for example 1 , 2, 3 or 4 amino acid substitutions may be conservative substitutions.

[0260] In a very preferred embodiment, the peptides comprise or even consist of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , wherein one amino acid has been substituted with amino acid Xi (e.g. any of Xi described in the section “Internal covalent linkage”) and another amino acid has been substituted with X2 (e.g. any of X2 described in the section “Internal covalent linkage”). Said amino acids, which are substituted are preferably positioned in relation to each other as described for amino acids Xi and X2 in the section “Internal covalent linkage”.

[0261] In addition, to the consecutive sequence of SEQ ID NO: 1, the peptides of the invention may comprise one or more additional amino acids. Preferably, the peptide may contain up to 4 additional amino acids, for example up to 3 additional amino acids, such as 2 additional amino acids. Said additional amino acids may for example be any of the amino acids described in the section “Positively charged amino acids” below.

[0262] In one embodiment, the peptide according to the invention comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 17 to 18 amino acids containing up to 2 amino acid substitutions, where the peptide may comprise up to 4 additional amino acids.

[0263] In one embodiment, the peptide according to the invention comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 17 to 18 amino acids containing a substitution of one amino acid for amino acid Xi and a substitution of one amino acid for amino acid X2, wherein the peptide may comprise up to 4 additional amino acids.

[0264] Thus, in one embodiment, the peptide of the invention consists of in the range of 15 to 20 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids has been substituted with Xi and X2, wherein Xi and X2 are alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 5, preferably 2 additional N-terminal amino acids. In one embodiment, the peptide of the invention consists of in the range of 16 to 18 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids has been substituted with Xi and X2, wherein Xi and X2 are alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 5 additional N-terminal amino acids, preferably 2 additional N-terminal amino acids.

[0265] In one embodiment, the peptide of the invention consists of 17 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids have been substituted with Xi and X2, wherein Xi and X2 are alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 3 additional N-terminal amino acids.

[0266] Said additional N-terminal amino acids are preferably positively charged amino acids, for example Lys or Arg.

[0267] The peptide of the invention may further comprise one or more moieties conjugated to said peptide. Said moieties may optionally be linked to the peptide via a linker. Said one or more conjugated moieties may for example be selected from the group consisting of alkyls, aryls, heteroaryls, olefins, fatty acids, polyethylene glycol (PEG), saccharides, and polysaccharides.

[0268] Longer peptides

[0269] However, the peptide of the invention may also be longer. In some embodiments, the peptide of the invention has a length between 24 and 40 amino acids, such as between 25 and 35 amino acids, such as between 25 and 30 amino acids, such as between 28 and 34 amino acids.

[0270] In particular, the peptide may have a length of 24 to 28 amino acids, such as 25 or 26 amino acids.

[0271] Peptides of the invention which are 24 amino acids or longer comprise at least two staples.

[0272] Peptides of the invention with a length of 24 to 40 amino acids comprise a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1. Thus, preferably, longer peptides of the invention comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 24 to 40 amino acids, such as between 25 and 35 amino acids, such as between 25 and 30 amino acids, such as between 28 and 34 amino acids, preferably between 24 and 28 amino acids, even more preferably between 25 and 26 amino acids.

[0273] Longer peptides of the invention may be as defined above in the section “Peptide properties - Shorter peptides”.

[0274] Thus, longer peptides according to the invention comprises or even consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , wherein up to 6 amino acids may be exchanged. It is however important that the peptide comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1 . In other words, said amino acids should not be substituted. In some embodiments, the longer peptide according to the invention comprises R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0275] In a very preferred embodiment, the longer peptides of the invention comprise or even consist of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , wherein one amino acid has been substituted with amino acid Xi (e.g. any of Xi described in the section “Internal covalent linkage”) and another amino acid has been substituted with X2 (e.g. any of X2 described in the section “Internal covalent linkage”), and another amino acid has been substituted with X3 (e.g. any of X3 described in the section “Internal covalent linkage”), and another amino acid has been substituted with X4 (e.g. any of X4 described in the section “Internal covalent linkage”). Said amino acids, which are substituted are preferably positioned in relation to each other as described for amino acids Xi and X2, and X3 and X4, in the section “Internal covalent linkage”.

[0276] Positively charged amino acids

[0277] Interestingly, the present invention shows that insertion of one or more positively charged amino acids may significantly enhance the antibacterial and anti-inflammatory effect of the peptides.

[0278] Thus, peptide according to the invention may in preferred embodiments comprise between 1 and 5, such as between 1 and 4, for example between 1 and 3, for example between 1 and 2, such as 2 positively charged amino acids inserted at or close to the end of the peptide.

[0279] In particular, said positively charged amino acids may be inserted at or close to the N- terminal, such as at a position selected from positions 1 , 2, 3, 4 and / or 5 relative to the N-terminal of the peptide.

[0280] In a preferred embodiment, the positively charged amino acids are inserted at the N- terminal.

[0281] In one embodiment, the peptide of the invention comprises 2 positively charged amino acids inserted at or close to the N-terminal, such as at a position selected from positions 1 , 2, and / or 3 relative to the N-terminal of the peptide.

[0282] Said positively charged amino acids may preferably be selected form the group consisting of arginine, lysine and histidine. Preferably, the positively charged amino acids are arginine and / or lysine. In a preferred embodiment, the positively charged amino acids are arginine. In an equally preferred embodiment, the positively charged amino acids are lysine.

[0283] Peptide sequence

[0284] In some embodiments of the invention, the peptide may have one of the peptide sequences described herein in this section. In addition to the peptide having the sequences described in this section, it is preferred that the peptide also:

[0285] • comprises a consecutive sequence of in the range of 10 to 23 amino acids, preferably in the range of 13 to 23 amino acids from thrombin of SEQ ID NO: 1

[0286] • has a total length of between 10 to 40 amino acids

[0287] • comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1 , more preferably comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1 ;

[0288] In some embodiments, a peptide having a total length between 24 to 40 amino acids comprises at least two internal covalent linkages between the side chains of two nonneighbouring, internal amino acids. Shorter peptides

[0289] In some embodiments of the invention, the peptide may have one of the peptide sequences described herein in this section. In addition to the peptide having the sequences described in this section, it is preferred that the peptide also:

[0290] • comprises a consecutive sequence of in the range of 10 to 23 amino acids, preferably in the range of 13 to 23 amino acids from thrombin of SEQ ID NO: 1

[0291] • has a total length of between 10 to 23 amino acids, preferably between 13 and 23 amino acids

[0292] • comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1, more preferably comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0293] In some embodiments of the invention, the peptide may have one of the peptide sequences described herein in this section. In addition to the peptide having the sequences described in this section, it is preferred that the peptide also:

[0294] • comprises a consecutive sequence of in the range of 10 to 23 amino acids, preferably in the range of 13 to 23 amino acids from GKY25 of SEQ ID NO: 12

[0295] • has a total length of between 10 to 23 amino acids, preferably between 13 and 23 amino acids

[0296] • comprises at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12, more preferably comprises at least amino acids R11, K13, K14 and K18 of GKY25 of SEQ ID NO: 12.

[0297] Xi and X2 in the sequences of this section may for example be as described herein above in the section “Internal covalent linkage”.

[0298] II in the sequences of this section may for example be His, Arg or Lys, preferably Arg or Lys.

[0299] Z in the sequences of this section may individually be any canonical amino acid. In preferred embodiments most or even all Z of a sequence are selected to correspond to the amino acids of GKY25 of SEQ ID NO: 12.

[0300] In one embodiment, the peptide according to the invention comprises the sequence: -K-K-Z-Z-X1-K-Z-Z-X2-Z wherein

[0301] Z is any canonical amino acid; and

[0302] Xi and X2are amino acids, the side chains of which are linked by a covalent linkage.

[0303] In one embodiment, the peptide according to the invention comprises or consists of the amino acid sequence:

[0304] -U-U-(Z)n-l-Q-K-V-l-D-Q-(Z)m- wherein the peptide has a total length between 10 to 23 amino acids; and each Z is individually any canonical amino acid; and

[0305] II is His, Lys or Arg; and n is an integer in the range of 0 to 10; and m is an integer in the range of 0 to 5, and wherein two of the amino acids have been substituted for alkenylated amino acids, the side chains of which are linked by a covalent linkage.

[0306] In one embodiment, the peptide of the invention comprises or consists of the sequence:

[0307] U-U-(Z)n-K-K-Z-Z-Xi-K-Z-Z-X2-Z, or the sequence: U-U-Z-Z-R-Z-K-K-Z-Z-X1-K-Z-Z-X2-Z wherein

[0308] Z is any canonical amino acid;

[0309] II is His, Lys or Arg; n is an integer in the range of 0 to 10 and

[0310] Xi and X2are amino acids, the side chains of which are linked by a covalent linkage.

[0311] In one embodiment, the peptide of the invention comprises or consists of the sequence:

[0312] U-V-F-R-L-K-K-W-I-X1-K-V-I-X2-Z-F-G-Z wherein

[0313] Z is any canonical amino acid;

[0314] II is His, Lys or Arg;

[0315] Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0316] In one embodiment, the peptide of the invention comprises or consists of the sequence:

[0317] V-F-R-L-K-K-W-I-X1-K-V-I-X2-Q-F-G-E wherein

[0318] Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0319] In one embodiment, the peptide comprises or consists of the sequence:

[0320] U-U-V-F-R-L-K-K-W-I-X1-K-V-I-X2-Z-F-G-Z wherein

[0321] Z is any canonical amino acid;

[0322] II is His, Lys or Arg; and

[0323] Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0324] In one embodiment, the peptide comprises or consists of the sequence:

[0325] U-U-V-F-R-L-K-K-W-I-X1-K-V-I-X2-Q-F-G-E wherein

[0326] II is His, Lys or Arg; and

[0327] Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0328] In one embodiment of the invention, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 6 v) SEQ ID NO: 7 vi) SEQ ID NO: 8; vii) SEQ ID NO: 9; viii) SEQ ID NO: 10; ix) SEQ ID NO: 11 x) SEQ ID NO: 17; xi) SEQ ID NO: 18; xii) SEQ ID NO: 19; xiii) SEQ ID NO: 20; xiv) SEQ ID NO: 21; xv) SEQ ID NO: 22; xvi) SEQ ID NO: 23; xvii) SEQ ID NO: 24; xviii) SEQ ID NO: 25; or xix) SEQ ID NO: 26; or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to xix); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0329] In one embodiment of the invention, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 8; or v) SEQ ID NO: 9 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to v); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0330] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 8; or v) SEQ ID NO: 9 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to v); wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether.

[0331] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 8; or v) SEQ ID NO: 9 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to v); wherein Xi and X2 are Ala, the side chains of which are covalently bound to each other by a Cs alkenyl tether comprising one double bond.

[0332] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5 iii) SEQ ID NO: 6; or iv) SEQ ID NO: 7 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to iv); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0333] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5 iii) SEQ ID NO: 6; or iv) SEQ ID NO: 7 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to iv); wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether.

[0334] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5 iii) SEQ ID NO: 6; or iv) SEQ ID NO: 7 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to iv); wherein Xi and X2 are Ala, the side chains of which are covalently bound to each other by a Cs alkenyl tether comprising one double bond.

[0335] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5; or or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0336] In one embodiment, the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5; or or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether.

[0337] In one embodiment, the peptide comprises or consists of the sequence as set forth in: iii) SEQ ID NO: 3 iv) SEQ ID NO: 5; or or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are Ala, the side chains of which are covalently bound to each other by a Cs alkenyl tether comprising one double bond.

[0338] Longer peptides

[0339] In some embodiments of the invention, the peptide of the invention is a longer peptide that comprises at least two staples and between 24 and 40 amino acids from thrombin of SEQ ID NO: 1 and may have one of the peptide sequences described herein in this section. In addition to the peptide having the sequences described in this section, it is preferred that the peptide also:

[0340] • comprises a consecutive sequence of in the range of 24 to 40 amino acids, preferably in the range of 25 to 30 amino acids from thrombin of SEQ ID NO: 1

[0341] • has a total length of between 24 to 40 amino acids, preferably between 25 and 30 amino acids

[0342] • comprises at least amino acids K247, K248 and K252 of SEQ ID NO: 1, preferably comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.

[0343] In some embodiments of the invention, the peptide of the invention is a longer peptide that comprises at least two staples. In addition to the peptide having the sequences described in this section, it is preferred that the peptide also:

[0344] • comprises a consecutive sequence of in the range of 24 to 25 to amino acids from GKY25 of SEQ ID NO: 12

[0345] • has a total length of between 24 to 40 amino acids, preferably between 25 and 30 amino acids comprises at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12, preferably comprises at least amino acids R11, K13, K14 and K18 of GKY25 of SEQ ID NO: 12.

[0346] In a preferred embodiment, the longer peptide of the invention comprises or consists of the sequence as set forth in: i) SEQ ID NO: 14; or ii) SEQ ID NO: 26 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage; and further wherein X3 and X4 are amino acids, the side chains of which are linked by a covalent linkage. Xi, X2, Xsand X4 in the sequences of this section may for example be as described herein above in the section “Internal covalent linkage”. Preferably, Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether, and X3 and X4 are Gly and Glu, respectively, which have been reacted with each other to form a lactam bridge.

[0347] Peptide function

[0348] As mentioned herein above, the peptides may have one or more of the following functions.

[0349] High stability

[0350] Preferably, the peptides of the invention have high stability. Thus, the peptide according to the invention preferably has increased stability in vivo and / or in vitro compared to a peptide with the same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions.

[0351] In vivo stability may for example may be determined by determining antibacterial and / or anti-inflammatory activity in a mouse wound model after topical administration of the peptide over time. If significant antibacterial and / or anti-inflammatory activity is maintained after e.g. 24 h, peptides have high in vivo stability. The in vivo stability can for example be determined as described in Example 4 herein below.

[0352] Increased protease resistance

[0353] Preferably, the peptides of the invention have increased protease resistance. Thus, the peptide may have increased resistance to one or more proteases compared to a peptide with the same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage.

[0354] Preferably, the peptide of the invention has increased stability in the presence of a protease, such as human neutrophil elastase (HNE), Pseudomonas elastase (PE), and / or trypsin, compared to a peptide of same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions.

[0355] High anti-inflammatory activity

[0356] Preferably the peptides of the invention have high anti-inflammatory activity. Even more preferably, the peptide of the invention has both anti-microbial activity and antiinflammatory activity.

[0357] The anti-inflammatory activity may be determined in different ways, however, it may in particular be determined by inducing inflammation in a controlled way, and determining whether inflammation is reduced. The inflammation may for example be induced by contacting a reporter cell or a blood sample in vitro with LPS or by administering LPS to an animal. Inflammation may for example determined by measuring the levels of one or more proinflammatory cytokines or by determining NF-KB activity.

[0358] Thus, it is preferred that the peptide according to the invention reduces the secretion of pro-inflammatory cytokines in the presence of one or more endotoxins, such as LPS. In particular, it is preferred that the peptide decreases secretion of pro-inflammatory cytokines in vivo in blood comprising one or more endotoxins, such as LPS.

[0359] Said pro-inflammatory cytokines may for example be selected from the group consisting of tumour necrosis factor a (TNF-a), interleukin p (IL-1 P), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon (IFN-Y) and / or monocyte chemoattractant protein-1 (MCP-1). Preferably, the inflammatory cytokines are TNF-a and / or I L-1 p.

[0360] It is preferred that the peptide according to the invention decreases NF-kB activation in the presence of a toll-like receptor (TLR)-agonist, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), Staphylococcus aureus peptidoglycan (SA-PGN) and / or zymosan.

[0361] It is preferred that a concentration of 10 pM of the peptide of the invention reduces secretion of TNF-a and or I L-1 after incubation in fresh blood in the presence of LPS by at least 50%, such as by at least 60%, for example by at least 70%, compared to the level in the presence of a peptide of same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions.

[0362] In one embodiment the peptides of the invention are capable of reducing release of TNF-a in LPS stimulated blood by 50% at a concentration of less than 10 pM, such as less than 7 pM, for example in the range of 1 to 10 pM, such as at a concentration in the range of 1 to 7 pM.

[0363] The anti-inflammatory activity may be tested by one of the methods described herein below in Examples 2 and 3.

[0364] High anti-microbial activity

[0365] Preferably, the peptides of the invention have high anti-microbial activity.

[0366] Thus, the peptides of the invention may have bactericidal activity against Gram negative and / or Gram positive bacteria. Interestingly, the peptides of the invention have been shown effective against Gram negative bacteria. Without being bound by theory it is believed that this effect is mediated by binding of the peptides to LPS and / or other membranes structures. The peptides of the invention have also been shown effective against Gram positive bacteria. Without being bound by theory it is believed that this effect is mediated by binding of LTA and / or other membranes structures. Thus, the peptides may have bactericidal activity mediated through the peptide being capable of killing bacteria by damaging the bacterial membrane.

[0367] In particular, it is preferred that the peptides of the invention have bactericidal effect against one or more bacteria selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Preferably, the peptide has bactericidal effect against all of the aforementioned bacteria.

[0368] Further, it is preferred that the peptides of the invention have the peptides of the invention have bactericidal effect against one or more, and preferably all, of the bacteria selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli both in the absence and the presence of NaCI and / or in plasma.

[0369] It is preferred that a concentration of 5 pM of the peptide of the invention inhibits the growth of one or more bacteria selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by at least 0.5 mm, such as at least 1 mm, such as at least 2 mm, in the presence of NaCI.

[0370] Anti-microbial activity or bactericidal activity may for example be determined by radial diffusion assay or by a viable count assay. Said assays may for example be performed as described herein below in Examples 1 and 2.

[0371] Anti-fungal activity

[0372] Preferably, the peptides of the invention have high anti-fungal activity. Thus, the peptides of the invention may fungicidal activity against any type of fungi, in particular against clinically relevant fungal species such as Aspergillus, Basidiobolus, Blastomyces, Cladophialophora, Coccidioides, Fonsecaea, Phialophora, Pneumocystis, Microsporum, Trichophyton, Microsporum, Epidermophyton, Hitoplasma, Lacazia, Paracoccidioides, Scedosporium, Sporothrix and Emmonsia.

[0373] In particular, it is preferred that the peptides of the invention have effect against Candida, such as for example Candida albicans, Candida parapsilosis, Candida lipolytica and / or Candida krusei Preferably, the peptides have fungicidal effect against all of the aforementioned fungi.

[0374] Anti-fungal activity or fungicidal activity may for example be determined by radial diffusion assay or by a viable count assay. Said assays may for example be performed as described herein below in Example 6.

[0375] Low hemolytic activity in blood

[0376] It is very preferred that the peptides of the invention have low hemolytic activity in blood. Peptides having high hemolytic activity may be toxic and are thus less suitable for in vivo use in humans.

[0377] In general, a hemolytic activity up to 10% is acceptable. Thus, it is preferred that the hemolytic activity of the peptides of the invention is less than 10%, in particular, the hemolytic activity of the peptides of the invention in fresh, whole blood is preferably less than 10%.

[0378] More preferably, the hemolytic activity of the peptides of the invention is less than 10% at a peptide concentration with effective anti-inflammatory activity. In a preferred embodiment, the peptide of the invention has a hemolytic activity of less than 10%, at a peptide concentration which reduces anti-inflammatory activity by 50%. Said antiinflammatory activity is preferably determined as release of TNF-a in LPS stimulated blood.

[0379] Thus, it is preferred that the hemolytic activity of the peptides of the invention is less than 10% at a peptide concentration, where said peptide is capable of reducing release of TNF-a in LPS stimulated blood by 50%.

[0380] In one embodiment, the hemolytic activity of the peptides of the invention is less than 5% at a peptide concentration, where said peptide is capable of reducing release of TNF-a in LPS stimulated blood by 50%.

[0381] Release of TNF-a in LPS stimulated blood may preferably be determined as described in Example 2 or 3 herein below. In some embodiments, when 50 pM of the peptide is incubated in fresh, whole blood, the hemolytic activity is at the most 5%, such as at the most 4%, for example at the most 3%, such as at the most 2%.

[0382] In some embodiments, when 20 pM of the peptide is incubated in 25% fresh, whole blood, the hemolytic activity is at the most 5%, such as at the most 4%.

[0383] The hemolytic activity is preferably determined by incubating fresh blood with the peptide of the invention and determining the hemolysis and comparing to hemolysis in fresh blood in a control prepared by incubating fresh blood with a detergent, such as Tween-20. Preferably, the hemolytic activity is provided as the % hemolysis compared to the control.

[0384] The hemolytic activity may in particular be determined as described herein below in Examples 2 or 3 herein below

[0385] Low toxicity

[0386] The peptides of the invention preferably also have low toxicity allowing topical administration of the peptides.

[0387] Structure

[0388] The peptides of the invention preferably has a substantially alpha helical secondary structure in aqueous solution. Said substantially alpha helical secondary structure is preferably maintained upon binding a target molecule, such as cluster differentiation 14 (CD14).

[0389] Alpha helical secondary structure in aqueous solution is preferably determined by circular dichroism spectroscopy.

[0390] Furthermore, the peptides preferably have a low degree of oligomerisation. The hydrodynamic radii is an indication of the oligomerisation, and thus the peptides of the invention preferably have a low hydrodynamic radii. Preferably, the peptides of the invention have hydrodynamic radii of less than 150 nm at pH 7.4 and / or less than 100 nm at pH 5. In some embodiments, it is preferred the peptides of the invention have hydrodynamic radii of less than 100 nm at pH 7.4 and / or less than 80 nm at pH 5. Anti-coagulant activity

[0391] The peptides of the invention may also have anti-coagulant activity. Thus, preferably the peptides of the invention have an ability to increase the time for blood to form clots. In particular, it is preferred that the peptides are able to increase the clotting time of the intrinsic pathway of coagulation. Said clotting time may preferably be determined by determining the “Activated Partial Thromboplastin Clotting Time” (aPTT). Preferably, the peptide of the invention increases the clotting time as determined by aPPT by at least 100% at a concentration of 60 pM. In some embodiments, the peptide of the invention increases the clotting time as determined by aPPT by at least 90% at a concentration of 40 pM. peptide. Said increase in the clotting time is compared to the clotting time in the absence of peptide.

[0392] Reduced haemolytic activity on purified RBCs

[0393] The peptides of the invention preferably have a low haemolytic activity on purified RBC at a peptide concentration with effective anti-inflammatory activity. Thus, it is preferred that the peptides of the invention have a haemolytic activity on purified RBCs of less than 75%, more preferably less than 65%, for example less than 60% at a concentration corresponding to IC50 in terms of TNF-a release and / or IL-p release in LPS stimulated blood. Said haemolytic activity on purified RBCs may preferably be determined as described in Example 2 in the section Hemolysis Assay.

[0394] Preparation of peptide

[0395] Methods for the production of peptides are well known in the art.

[0396] Peptides may be produced by recombinant methods well known in the art (see e.g. Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).

[0397] Alternatively, peptides may be chemically synthesized, e.g. by linking multiple amino acids via amide bonds. Typically, peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. Protecting group strategies may be used to prevent undesirable side reactions with the various amino acid side chains. Well known liquid-phase or solid phase peptide synthesis techniques are known to the skilled person (such as standard f-Boc or Fmoc solid-phase peptide synthesis).

[0398] The covalently linkage of the side chains of two, non-neighbouring internal amino acids may be introduced by any method known to the skilled person, such as for example by any of the methods described by Li et al., 2020.

[0399] When Xi and X2 are alkenylated amino acids, such as olefin terminated amino acids, then the hydrocarbon staple may be introduced using ring-closing metathesis (RCM), for example by ruthenium catalysed ring-closing metathesis or by RCM using Grubbs’ first-generation catalyst in 1 ,2-dichloroethane. RCM may be performed in solution or on solid supports, and multiple ways are described in Li et al., 2020.

[0400] Similarly, when X3 and X4 are alkenylated amino acids, such as olefin terminated amino acids, then the hydrocarbon staple may be introduced using ring-closing metathesis (RCM), for example by ruthenium catalysed ring-closing metathesis or by RCM using Grubbs’ first-generation catalyst in 1 ,2-dichloroethane. RCM may be performed in solution or on solid supports, and multiple ways are described in Li et al., 2020.

[0401] When X3 is an N-terminal amino acid and / or an amino acid comprising an amine group, and X4 is an amino acid comprising a carboxylic acid side chain, then the covalent linkage may be an amide bond between said amine and said carboxylic acid. In other words, the covalent linkage may be an amide bond, i.e. a lactam bridge, between said amine and carboxylic acid. Such cyclization may be performed on solid phase.

[0402] Peptides according to the invention can also be ordered from companies specialised in producing custom made peptides, for example from AmbioPharm Inc. (US).

[0403] Formulation

[0404] The compositions according to the present invention may be formulated for suitable application in methods of treatment as described herein.

[0405] The compound may be formulated in any way suitable for the route of administration. In some embodiments, the compound is formulated in an ointment, cream, emulsion, lotion, powder, spray, solution, viscous solution or gel, such as a hydrogel. Preferably, the compound is formulated in a hydrogel.

[0406] In one aspect, the present disclosure provides for a composition selected from: a gel, an ointment, a cream, a paste, a lotion, or a liquid; the composition comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1 ; optionally with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two non-neighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted X1 and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0407] In one embodiment, the peptide is as described elsewhere herein, such as in the section “Stapled peptide”.

[0408] In one embodiment, the composition is for use in a method of treatment of a disease or condition as describe elsewhere herein, such as in the section “Treatment of disorder or condition”.

[0409] In one embodiment, the composition as described herein is comprised in one selected from the group consisting of: a patch, a dry patch, a wound irrigation liquid, a contact lens liquid, a plaster, a bandage, a suture, a prothesis, an implant, a film, a dressing, a wound dressing, a catheter a skin graft or a skin substitute.

[0410] In one embodiment, the composition comprises: i. a polymer, for example a non-ionic polymer, capable of forming a hydrogel when mixed with an aqueous solution, and ii. an aqueous solution.

[0411] In one embodiment, the composition comprises: i. the compound and / or the peptide at a concentration in the composition of at least 0.08 wt% and / or ii. a polymer, for example a non-ionic polymer present in said composition at a concentration of at least 0.05 wt%.

[0412] The composition may preferably be in the form of a hydrogel or a viscous solution. The form of the composition depends on the intended use or area of application. Preferably, the composition is a hydrogel. Hydrogels are useful for local administration, and may be directly useful for topical administration. Furthermore, hydrogels are particularly suitable for use in the methods of treatment of the invention due to the high water content. Where the composition is a viscous solution, the composition may be suitable for eye, ear or nose drops or sprays. If the composition is a viscous solution it may for example also be applied to a product or absorbed by a product.

[0413] In embodiments of the invention, wherein the composition is a hydrogel, said hydrogel preferably has a flow point of at least 15 Pa, more preferably of at least 25 Pa, such as in the range of 40 to 60 Pa. It is advantageous that a hydrogel has a suitable flow point in order to be particularly useful for local administration. Thus, it is frequently preferred that the hydrogel is sufficiently thick to largely remain at the site of administration.

[0414] The compositions may be used on its own and may for example be administered locally directly to the site of the disorder to be treated. In particular, the composition may be administered topically. The composition may alternatively be used together with a product.

[0415] The composition should preferably be pharmaceutically acceptable, i.e. not toxic, and may thus be provided as a pharmaceutical composition. However, it is contemplated that, where the composition is used in a way in which it does not come into contact with human or animal tissue, such as for disinfecting an object, the composition can also be non-pharmaceutically acceptable. The composition may be subjected to conventional pharmaceutical operations such as sterilisation and / or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

[0416] It will be appreciated by persons skilled in the art that the composition of the invention may be administered locally. Routes of administration include topical, ocular, nasal, buccal, oral, vaginal and rectal administration. In preferred embodiments the compositions of the invention are for use in methods of treatment by topical administration.

[0417] The composition is preferably administered to a patient in a pharmaceutically effective amount. By "pharmaceutically effective amount" is meant an amount that is sufficient to produce the desired effects in relation to the condition for which it is administered, i.e. to provide a desired wound healing, antibacterial effect and / or anti-inflammatory effect.

[0418] In one embodiment, the compound or peptide is present in said composition in a concentration of at least 0.01 wt%, more preferably in a concentration of 0.01 to 5 wt%, such as 0.08 to 3 wt%, for example in the range of 0.1 to 2%.

[0419] In some embodiments, the concentration of the compound in the composition is between 0.05 wt% and 5 wt%, such as between 0.08 wt% and 1 wt%, such as between 0.08 wt% and 0.3 wt%, such as between 0.25 wt% and 0.9 wt%.

[0420] In some embodiments, the concentration of the peptide or compound in the composition is 0.086 wt%, 0.29 wt% or 0.86 wt%.

[0421] In some embodiments, between 0.075 mg / cm2and 600 mg / cm2of the compound is administered to the individual affected by the skin condition or skin disorder, such as between 0.1 mg / cm2and 1.5 mg / cm2.

[0422] In some embodiments, 0.145 mg / cm2, 0.29 mg / cm2, 0.43 mg / cm2or 0.86 mg / cm2of the peptide or compound is administered to the individual affected by the skin condition or skin disorder. Polymer capable of forming a hydrogel

[0423] The invention provides compositions comprising a polymer capable of forming a hydrogel. The polymer may for example be a non-ionic polymer.

[0424] In the context of the present invention a non-ionic polymer is understood to encompass a polymer which in a protic solvent under at room temperature and 1 atm pressure substantially bears no structural units having cationic or anionic groups needing to be offset by counterions to maintain electrical neutrality. Cationic groups include for example quaternized ammonium groups and protonated amines. Anionic groups include for example carboxyl and sulfonic acid groups.

[0425] The non-ionic polymer, also termed non-ionic hydrogel polymer, is a polymer capable of forming a hydrogel when mixed with an aqueous solution or aqueous buffer. Depending on the concentration of the polymer the composition may be in the form of a viscous liquid or a hydrogel, i.e. gel.

[0426] The non-ionic polymer should be hydrophilic, and accordingly, the non-ionic polymer is preferably hydroxylated.

[0427] Examples of suitable nonionic polymers for use in the present method are polyallylalcohol, polyvinylalcohol, polyacrylamide, polyethylene glycol (PEG), polyvinyl pyrrolidone, starches, such as corn starch and hydroxypropylstarch, alkylcelluloses, such as Ci-Ce-alkylcelluloses, including methylcellulose, ethylcellulose and n- propylcellulose; substituted alkylcelluloses, including hydroxy-alkylcelluloses, preferably hydroxy-Ci-Ce-alkylcelluloses and hydroxy-Ci-Ce-alkyl-Ci-Ce-alkylcelluloses, such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxypropylmethylcellulose, and ethylhydroxyethylcellulose. Mixtures of the aforementioned may also be employed.

[0428] In one embodiment the non-ionic polymer is selected from the group consisting of hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), Hydroxypropyl Methylcellulose (HPMC), poly(vinyl)alcohol (PVA), polyacrylamide (PA), polyethylene glycol (PEG) and polyvinyl pyrrolidone, and mixtures thereof, In one embodiment the non-ionic polymer is selected from the group consisting of hydroxyalkyl celluloses, preferably hydroxy-Ci-Ce-alkylcelluloses or hydroxy-Ci-Ce- alkyl-Ci-Ce-alkylcelluloses.

[0429] Among the non-ionic polymers, hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC) are preferred.

[0430] In preferred embodiments, the concentration of the non-ionic polymer in the compositions of the invention is sufficient to obtain a composition with a flow point of at least 10 Pa, such as at least 15 Pa, for example in the range of 10 to 80 Pa, such as in the range of 40 to 60 Pa.

[0431] The compositions of the invention may in particular comprise the non-ionic polymer in a concentration of at least 0.05 wt%, for example in a concentration of at least 0.09 wt%, such as in a concentration of at least 0.1 wt%, for example in a concentration of at least 0.5 wt%, such as in a concentration of at least 0.8 wt%, for example in a concentration of at least 1.0 wt%, for example in the range of 0.2 to 1.5 wt% for example in the range of 0.09 to 4 wt%, such as in the range of 0.1 to 3 wt%, more preferably in the range of 1.0 to 2.5 wt%. This may for example be the case, when the non-ionic polymer is HEC.

[0432] In one embodiment the concentration of non-ionic polymer is sufficient to obtain a viscosity of the composition of at least 10 mPas, and preferably no more than 100000 mPas, such as 14000 mPas, for example in the range of 100 mPas to 25000 mPas, for example in the range of 10000 mPas to 20000mPas. At 10mPas the composition is typically in the form of a viscous solution, whereas at 100000 mPAs the composition may be in the form of a dense gel.

[0433] EDTA

[0434] In one embodiment, the composition comprises: i. EDTA and ii. an aqueous buffer, wherein the composition has a pH of at the most 8.

[0435] In some embodiments, it is preferred that compositions comprising TCP peptide and

[0436] EDTA have a pH of at the most 7, though in other embodiments, compositions comprising TCP peptide and EDTA may have any useful pH, for example a pH of at the most 8 or a pH in the range of 3 to 10, such as in the range of 3 to 8, such as in the range of 3.5 to 8, for example in the range of 5 to 8.

[0437] In some embodiments it is preferred that compositions comprising EDTA also contain TCP peptides at a high concentration, i.e. a concentration of at least 0.08 wt%, such as a concentration of at least 0.1 wt%, such as a concentration in the range of 0.08 to 3 wt%.

[0438] The compositions may comprise any useful amount of EDTA. Preferably, the compositions may comprises EDTA at a concentration of at least 1 mM, such as in the range of 1 to 100 mM, preferably at least 1.5 mM, such as at least 2 mM, for example in the range of 2 to 100 mM, such as in the range of 2 to 50 mM, such as in the range of 2 to 25 mM.

[0439] Aqueous solution

[0440] The compositions of the present invention may comprise an aqueous solution. In particular, said aqueous solution may be an aqueous buffer.

[0441] An aqueous solution or an aqueous buffer contains water. When the aqueous solution is an aqueous buffer it also comprise components of a buffer system, i.e. weak bases or acids and their conjugate acids and bases, for obtaining an aqueous buffer capable of providing a generally stable pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, Acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0442] In one embodiment it is preferred that the pH of the composition is lower than 7, preferably lower than 6, more preferably 5.5 or lower, and higher than 3, such at least 3.5. Thus, the pH may be in the range of 3 to 6, such as approx. 5. This is advantageous because it has surprisingly been shown that a lower pH significantly increases the antibacterial effect of the hydrogel composition, in particular in the presence of EDTA. Furthermore, a pH lower than 7 also increases the solubility of TCP peptides. Solubility may for example be determined by visual inspection as described in Example 4. A desired pH may be obtained by using a suitable aqueous buffer, having the desired pH, as discussed above.

[0443] In such embodiments the aqueous solution or aqueous buffer may be an Acetate buffer comprising Acetate, preferably at a concentration of 5 to 50 mM, more preferably at a concentration of in the range of 10 to 30 mM. Said Acetate buffer may have a pH in the range of 3 to 6, such in the range of 3.6 to 5.8, for example approx. 5.

[0444] An Acetate buffer, such as a sodium Acetate buffer, is particularly suitable as buffer for obtaining a pH of 3.6 to 5.8. Typically, for a 10 mM sodium Acetate buffer, a pH of 5 is obtained, see example 2. However, as explained above, other aqueous buffers can be used to obtain the desired pH.

[0445] In an alternative embodiment the pH of the composition is between 7 and 8, preferably 7.4.

[0446] A neutral pH may in some cases preferred as this provides the composition with a pH close to that of the human or animal body, and hence decreases the risk of irritation from the composition when administered to a human or animal.

[0447] In such embodiments the aqueous solution or aqueous buffer may be a Trisaminomethane (Tris) buffer comprising Trisaminomethane, preferably at a concentration of 5 to 50 mM, such as in the range of 10 to 30 mM.

[0448] A Tris buffer may for example be used to obtain a pH of approx. 7.4, However, as explained above, other aqueous buffers can be used to obtain the desired pH.

[0449] The aqueous solution or aqueous buffer may additionally or alternatively comprise further components such as diluents, adjuvants, tonicity regulators and / or excipients. Generally such further components should be pharmaceutically acceptable.

[0450] The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the peptide in the composition. The diluent may be one or more of saline, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil). The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the peptide. The adjuvant may be one or more of colloidal silver, or zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and Acetates of different acyl composition. The adjuvant may also be a compound with antibacterial and / or antiinflammatory properties.

[0451] The “excipient” may be any useful excipient, such as one or more of polymers, lipids and minerals.

[0452] The term “tonicity regulator” refers to a compound capable of regulating the tonicity of the composition. In general, it is preferred that the compositions of the invention are either isotonic or somewhat hypotonic. A hypotonic composition may for example have a tonicity, which is 50 to 99% of isotonic. The tonicity regulator for example be a salt or glycerol.

[0453] In one embodiment the composition further comprises glycerol, preferably at a concentration of 1 to 2.5%, preferably at a concentration of 1.2 to 2.2 vol%. The concentration is provided as the concentration in the composition, e.g. the concentration in the hydrogel. In general, a composition comprising 2% glycerol will be isotonic, and thus in some embodiments, the composition may comprise approx. 2% glycerol. However, in other embodiments, the composition is hypotonic, in which case it may comprise in the range of 1.2 to 1.9% glycerol.

[0454] Compositions of the invention may have any desirable pH, e.g. a pH in the range of pH 4 to pH 8, such as in the range of pH 5 to pH 8, for example in the range of 7 to 8.

[0455] Compositions having a pH of more than 6, such as a pH of more than 7, such as a pH in the range of 6 to 8, for example a pH in the range of 7 to 8 may be particularly stable - even in the absence of EDTA. The composition may be administered by single administration or by multiple administrations. The composition may be administered alone or in combination with other therapeutic agents. A device

[0456] The invention also provides devices comprising the compositions according to the present disclosure.

[0457] In one aspect, the present disclosure provides for a device comprising a composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1 ; optionally with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two non-neighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted X1 and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0458] In one embodiment, the device comprises the composition as described herein. In one embodiment, the device comprises the peptide as described in the section “Stapled peptide”.

[0459] In one embodiment, the device is for use in a method of treatment of a disorder or condition in an individual in need thereof. In one embodiment, the method of treatment, the disorder or the individual are as described in the section “Medical use” or “Treatment of disorder or condition”. Preferably, the device is a medical device.

[0460] The device may for example be a device that can aid the local administration of the compositions of the invention. Thus in one embodiment, the device as described herein is administered topically.

[0461] In some embodiments, the device may be selected from a patch, a powder, a dry patch, a wound irrigation liquid, a plaster, a bandage, a suture, a prosthesis, an implant, a film, a dressing, a wound dressing, a catheter, a skin graft or a skin substitute.

[0462] Drops and sprays may for example be configured, (i.e. formulated) for applying the composition to ears, eyes, or the nose. The composition may for example be formulated as a viscous liquid easily applicable to eyes or ears, and may alternatively be formulated as hydrogel for easy application to ears.

[0463] Devices like, e.g. hydrogels, drops, sprays, wound dressings, plasters, skin substitutes and bandages may be configured or formulated for administration of the composition to the skin or to other epithelial surfaces or to a wound.

[0464] In general, the composition and devices described herein may be formulated for local administration, and in particular for topical administration.

[0465] In such embodiments, the composition may be coated, painted, or sprayed onto the product, or the composition may be adsorbed or absorbed by the device.

[0466] In so doing, the composition may impart antibacterial and anti-inflammatory properties to the device.

[0467] The term 'coated' as used herein refers to the composition being applied to the surface of the product. Thus, the product may be painted or sprayed with a solution comprising the composition. Alternatively, the product may be dipped in a reservoir of the composition.

[0468] Advantageously, the product is impregnated with the composition. By 'impregnated' is meant that the composition is absorbed or adsorbed with the product.

[0469] Items

[0470] 1. A composition for use in a method of treatment or prevention of a disorder or condition in an individual in need thereof, wherein said method comprises topical administration of the composition, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1. The composition for use according to item 1 , wherein the disorder or condition is a disease or condition of the skin or the mucosa. A composition for use in a method of treatment or prevention of a disorder or condition of the skin or the mucosa in an individual in need thereof, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1. A composition for use in a method of treatment or prevention of fungal infection in an individual in need thereof, the composition comprising a compound comprising a peptide comprising: a. a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1; or b. the amino acid sequence:

[0471] X1-X2-X3-X4-X5-X6-W-X8-X9-X10, wherein X4, 6, 9 is any standard amino acid, Xi is I, L or V,

[0472] X2 is any standard amino acid except C, X3is A, E, Q, R or Y,

[0473] X5 is any standard amino acid except R, Xs is I or L,

[0474] X10 is any standard amino acid except H, wherein said peptide has a length of from 10 to 100 amino acid residues. The composition according to item 4, wherein the peptide of b. has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and comprises or consists of any of the amino acid sequences:

[0475] GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 12), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 15), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 34), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 35) GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 36) GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 37),

[0476] Or a sequence having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to any of the aforementioned peptides. The composition according to item 4, wherein the fungal infection is an infection by one or more fungi selected from the group consisting of Candida, Aspergillus, Basidiobolus, Blastomyces, Cladophialophora, Coccidioides, Fonsecaea, Phialophora, Pneumocystis, Microsporum, Trichophyton, Microsporum, Epidermophyton, Hitoplasma, Lacazia, Paracoccidioides, Scedosporium, Sporothrix and Emmonsia.

[0477] 7. The composition according to item 4, wherein the fungal infection is an infection by Candida, such as for example an infection by Candida albicans, Candida parapsilosis, Candida lipolytica and / or Candida krusei.

[0478] 8. The composition for use according to any one of items 3 to 7, wherein the method of treatment comprises topical administration of the composition.

[0479] 9. The composition for use according to any one of the preceding items, wherein the method of treatment comprises local administration of the composition.

[0480] 10. The composition for use according to any one of the preceding items, wherein said treatment is selected from the group consisting of an ameliorating treatment, a curative treatment and a preventive treatment.

[0481] 11. The composition for use according to any one of the preceding items, wherein the composition is selected from the group consisting of an ointment, cream, paste, liquid, emulsion, lotion, powder, spray, solution, viscous solution or gel, such as a hydrogel..

[0482] 12. The composition for use according to any one of the preceding items, with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two non-neighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4.

[0483] 13. The composition for use according to any one of the preceding items, wherein said skin disorder or skin condition is selected from the group consisting of a disease or condition characterized by blistering, a disease or condition characterized by excessive wound exudate, and scars. The composition for use according to any one of the preceding items, wherein the disorder is a blister. The composition for use according to any one of the preceding items wherein the individual suffers from a skin disease or skin condition selected from the group consisting of burn wounds, chronic skin ulcers, infected acute wounds, pressure ulcers and chronic wounds such as venous ulcers, venous leg ulcers or diabetic leg ulcers. The composition for use according to any one of the preceding items wherein the disorder or condition is a chronic skin ulcer or a chronic wound, for example a venous ulcer, venous leg ulcer or diabetic leg ulcer. The composition for use according to any one of the preceding items, wherein the disease or condition is selected from the group consisting of: epidermolysis bullosa (EB), burn-induced blisters, chilblains, cryotherapy-induced blisters, virus-induced blisters, such as chickenpox, shingles, herpes simplex, atypical enterovirus infection, hand, foot and mouth disease, eczema herpeticum, bullous impetigo and staphylococcal scalded skin syndrome, pemphigus, such as pemphigus foliaceous, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus erythematosus or pemphigus vegetans, pemphigoid, such as bullous pemphigoid, cicatricial pemphigoid, 200k pemphigoid or mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, porphyria cutanea tarda, bullous lupus erythematosus, Hailey-Hailey disease and

[0484] Steven Johnson syndrome (toxic epidermal necrolysis). The composition for use according to any one of the preceding items, wherein the disease or condition is epidermolysis bullosa (EB). The composition for use according to any one of the preceding items, wherein the disease or condition is EB, wherein EB is epidermolysis bullosa simplex, junctional epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, Kindler syndrome or epidermolysis bullosa acquisita. The composition for use according to any one of the preceding items, wherein the disease or condition is selected from the group consisting of impetigo, external otitis, fungal infections, pneumonia, eye infections and nasal infections. The composition for use according to any one of the preceding items, wherein the disease or condition is selected from the group consisting of rosacea, pityriasis rosea, psoriasis, eczema, microbial eczema, atopic dermatitis, seborrheic dermatitis and contact dermatitis. The composition for use according to any one of the preceding items, wherein the disease or condition is selected from the group consisting of candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, pityriasis, acne vulgaris, dermatophytosis. The composition for use according to any one of the preceding items, wherein the disease or condition is selected from the group consisting of folliculitis, hidradenitis suppurativa, balanitis, pearly penile papules, pyoderma gangrenosum. The composition for use according to any one of the preceding claims, wherein the disease or condition is cutaneous T cell lymphoma, such as mycosis fungoides or Sezary syndrome. The composition for use according to any one of the preceding items wherein the individual suffers from eczema, such as of atopic dermatitis. The composition for use according to item 13 or 14, wherein the disease or condition characterized by blistering is selected from the group consisting of: epidermolysis bullosa (EB), burn-induced blisters, chilblains, cryotherapy-induced blisters, virus-induced blisters, such as chickenpox, shingles, herpes simplex, atypical enterovirus infection, hand, foot and mouth disease, eczema herpeticum, bullous impetigo and staphylococcal scalded skin syndrome, pemphigus, such as pemphigus foliaceous, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus erythematosus or pemphigus vegetans, pemphigoid, such as bullous pemphigoid, cicatricial pemphigoid, 200k pemphigoid or mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, porphyria cutanea tarda, bullous lupus erythematosus, Hailey-Hailey disease and

[0485] Steven Johnson syndrome (toxic epidermal necrolysis). The composition for use according to any one of the preceding items, wherein the disease or condition characterized by blistering is epidermolysis bullosa (EB). The composition for use according to any one of the preceding items, wherein the disease or condition characterized by blistering is EB, and wherein EB is inherited epidermolysis bullosa or acquired epidermolysis bullosa. The composition for use according to any one of the preceding items, wherein the disease or condition characterized by blistering is EB, wherein EB is epidermolysis bullosa simplex, junctional epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, Kindler syndrome or epidermolysis bullosa acquisita. The composition for use according to any one of the preceding items, wherein said treatment reduces the size of one or more blisters or wounds. The composition for use according to any one of the preceding items, wherein treatment reduces the size of one or more blister or wound by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the treatment reduces the size of one or more blister or wound by at least 95% as compared to the size of the one or more blister or wound before treatment, or as compared to the size of a blister or wound which was not treated with the compound comprising the peptide, and which before treatment was of similar size and character as the one or more treated blister or wound. The composition for use according to any one of the preceding items, wherein treatment reduces the size of one or more blister or wound by 100%, such that the treatment results in that one or more blister or wound heals completely. The composition for use according to any one of the preceding items, wherein said treatment reduces the pain experienced by the individual, such as wherein said treatment reduces the pain of one or more wound or blister, optionally wherein the pain is measured using a pain scale selected from the group consisting of Neonatal / lnfants Pain Scale (NIPS), Children’s Hospital of Eastern Ontario Pain Scale (CHEOPS), Face, Legs, Activity, Crying, Consolability (FLACC) scale, Faces Pain Scale (Wong-Baker Faces Pain Rating Scale), Coloured Analogue Scale, Visual Analog Scale, Verbal Numerical Rating Scale and Wharton Impairment and Pain Scale. The composition for use according to any one of the preceding items, wherein the pain is reduced at least one step or score on a pain scale, such as at least two steps or scores, such as at least three steps or scores, such as at least four steps or scores, such as at least five steps or scores, such as at least six steps or scores, such as at least seven steps or scores, such as at least eight steps or scores, such as at least nine steps or scores, such as wherein the pain is reduced ten steps or scores on a pain scale, optionally wherein the pain is measured using a pain scale selected from the group consisting of Neonatal / lnfants Pain Scale (NIPS), Children’s Hospital of Eastern Ontario Pain Scale (CHEOPS), Face, Legs, Activity, Crying, Consolability (FLACC) scale, Faces Pain Scale (Wong-Baker Faces Pain Rating Scale), Coloured Analogue Scale, Visual Analog Scale, Verbal Numerical Rating Scale and Wharton Impairment and Pain Scale.

[0486] 35. The composition for use according to any one of the preceding items, wherein said treatment reduces the itch experienced by the individual, such as the itch of one or more wound or blister, optionally wherein the itch is evaluated via subjective patient reporting or by objective measurement of scratching activity or scratching induced skin changes, optionally wherein the itch is measured using the Leuven Itch Scale, optionally wherein the itch is measured using the 5-D itch scale.

[0487] 36. The composition for use according to any one of the preceding items, wherein treatment reduces the amount of fluid that exudes from one or more blister or wound, such as wherein treatment reduces the amount of wound exudate.

[0488] 37. The composition for use according to any one of the preceding items, wherein treatment decreases the amount of fluid that exudes from a wound or blister by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the amount of wound exudate is decreased by at least 95%, optionally wherein the amount of wound exudate is estimated by visual inspection of the wound dressing, optionally wherein the amount of wound exudate is measured by imaging of the wound dressing, optionally wherein the amount of wound exudate is measured by weighing the wound dressing, optionally wherein the amount of wound exudate is measured by measuring the protein content in the wound dressing, such as the total protein content in the wound dressing, optionally wherein the protein content is measure using the Bradford protein assay, optionally wherein the decrease is as compared to before starting treatment of the wound or blister, optionally wherein the decrease is as compared to a wound or blister of similar size and character which was not treated with the compound comprising the peptide and which before treatment had a similar amount of wound exudate as the treated wound or blister. The composition for use according to any one of the preceding items, wherein treatment decreases the total protein content that exudes from a wound or blister. The composition for use according to any one of the preceding items, wherein treatment reduces the amount of times that wound dressings need to be changed within a set time period, such as wherein treatment increases the amount of days passed before the wound dressings must be changed, such as wherein treatment results in wound dressings not needing to be changed as often as compared to before starting treatment, optionally wherein the timepoint for changing the wound dressing is determined based on the amount of exudate present in the dressing, optionally wherein the timepoint for changing the wound dressing is determined to reduce the risk of inflammation and / or infection. The composition for use according to any one of the preceding items, wherein treatment increases the amount of days passed between changing wound dressings with half a day, such as with one day, such as with 1.5 days, such as with 2 days, such as with 2.5 days, such as with 3 days, such as with 3.5 days, such as with 4 days, such as with 4.5 days, such as with 5 days, such as wherein treatment increases the amount of days passed between changing wound dressings with 6 days, optionally wherein treatment results in that the wound dressing only needs to be changed every third day, such as every fourth day, such as every fifth day, such as every sixth day, such as wherein treatment results in that wound dressing only needs to be changed once per week. The composition for use according to any one of the preceding items, wherein treatment reduces the size of the scar. 42. The composition for use according to any one of the preceding items, wherein treatment reduces scar formation.

[0489] 43. The composition for use according to any one of the preceding items, wherein treatment reduces the size of the scar by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein treatment reduces scar formation by at least 95% as compared to the size of a scar formed from a wound of similar size and character which was not treated.

[0490] 44. The composition for use according to any one of the preceding items, wherein the disorder is a disorder of the skin, ears, eyes or nose.

[0491] 45. The composition for use according to any one of the preceding items, wherein the disorder is a wound.

[0492] 46. The composition for use according to any one of the preceding items, wherein the wound is selected from the group consisting of burns and non-healing ulcers.

[0493] 47. The composition for use according to any one of the preceding items, wherein the wound is a surgical wound.

[0494] 48. The composition for use according to any one of the preceding items, wherein the wound is a chronic wound.

[0495] 49. The composition for use according to any one of the preceding items, wherein the chronic wound is caused by a disorder selected from diabetes, cancer and vasculitis.

[0496] 50. The composition for use according to any one of the preceding items, wherein the wound is selected from the group consisting of a radiation-induced wound, a laser-induced wound and a cryotherapy-induced wound. 51. The composition for use according to any one of the preceding items wherein the disorder comprises an inflammation or is associated with an inflammation.

[0497] 52. The composition for use according to any one of the preceding items wherein the disorder comprises an infection by bacteria or is associated with infection by bacteria.

[0498] 53. The composition for use according to any one of the preceding items wherein the bacteria are multiresistant bacteria.

[0499] 54. The composition for use according to any one of the preceding items, wherein the bacteria is Gram negative or Gram positive.

[0500] 55. The composition for use according to any one of the preceding items, wherein the bacteria is Gram negative.

[0501] 56. The composition for use according to any one of the preceding items, wherein the bacteria are of a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter , Haemophilus, Pseudomonas and Citrobacter.

[0502] 57. The composition for use according to any one of the preceding items, wherein the bacteria are selected from the group consisting of S. aureus and P. aeruginosa.

[0503] 58. The composition for use according to any one of the preceding items, wherein said individual is suffering from diabetes, arterial insufficiency or venous insufficiency.

[0504] 59. The composition for use according to any one of the preceding items, wherein treatment reduces the total amount of microorganisms present in one or more wound or blister, such as the total amount of bacteria present in one or more wound or blister. 60. The composition for use according to any one of the preceding items, wherein treatment reduces the total amount of microorganisms present in one or more wound or blister by at least 10%, such as at least 20%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as wherein the total amount of microorganisms present in one or more wound or blister is reduced by at least 95% as compared to the amount of bacteria present before treatment, optionally wherein the amount of microorganisms is measured by measuring the amount of colony-forming units (CFU) present in a wound dressing and / or in a wound or blister on the skin, wherein said measurement takes place before, during and / or after treatment.

[0505] 61. The composition for use according to any one of the preceding items, wherein the individual is a human.

[0506] 62. The composition for use according to any one of the preceding items, wherein said individual suffers from a bacterial infection.

[0507] 63. The composition for use according to any one of the preceding items, wherein the peptide comprises a consecutive sequence in the range of 10 to 40 amino acids from thrombin of SEQ ID NO: 1.

[0508] 64. The composition for use according to any one of items 1 to 62, wherein the peptide comprises a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 10 and 23 amino acids; ii) comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprises at least amino acid K247, K248 and K252 of thrombin of SEQ ID NO: 1. The composition for use according to any one of items 1 to 62, wherein the peptide comprises a consecutive sequence of in the range of 10 to 23 amino acids from GKY25 of SEQ ID NO: 12 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 10 and 23 amino acids; ii) comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprises at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12. The composition for use according to any one the preceding items, wherein the peptide comprises a consecutive sequence of in the range of 13 to 23 amino acids from thrombin of SEQ ID NO: 1 or GKY25 of SEQ ID NO: 12. The composition for use according to any one of items 1 to 62, wherein the peptide comprises or consists of the amino acid sequence:

[0509] -U-U-(Z)n-l-Q-K-V-l-D-Q-(Z)m- wherein the peptide has a total length between 10 to 23 amino acids; and each Z is individually any canonical amino acid; and

[0510] II is His, Lys or Arg; and n is an integer in the range of 0 to 10; and m is an integer in the range of 0 to 5, and wherein two of the amino acids have been substituted for alkenylated amino acids, the side chains of which are linked by a covalent linkage. The composition for use according to any one of the preceding items, wherein the peptide has a total length of 13 to 23 amino acids. The composition for use according to any one of items 1 to 62, wherein the peptide comprises a consecutive sequence of in the range of 13 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 13 and 23 amino acids; ii) comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprises at least amino acids R245, K247, K248, K252 of thrombin of SEQ ID NO: 1.

[0511] 70. The composition for use according to any one of items 1 to 62 and 56, wherein the peptide comprises at least amino acids R245, K247, K248, K252 of thrombin of SEQ ID NO: 1.

[0512] 71. The composition for use according to any one of items 1 to 62, wherein the peptide comprises a consecutive sequence of in the range of 13 to 23 amino acids from GKY25 of SEQ ID NO: 12 containing up to 6 amino acid substitutions, wherein said peptide: i) has a total length between 13 and 23 amino acids; ii) comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii) comprises at least amino acids K13, K14 and K18 of GKY25 of SEQ ID NO: 12.

[0513] 72. The composition for use according to item 71 , wherein the peptide comprises at least amino acids R11, K13, K14 and K18 of thrombin of SEQ ID NO: 12.

[0514] 73. The composition for use according to any one of the preceding items, wherein the peptide contains only one internal covalent linkage between two nonneighbouring, internal amino acids.

[0515] 74. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position n+3, or at position, n+4, or at position n+5, or at position n+6, or at position n+7, or at position n+8, or at position n+9, or at position n+10, or at position n+11, wherein n is an integer in the range of 2 to 18. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position n+3, or at position n+4, or at position n+7, or at position n+11 , wherein n is an integer in the range of 2 to 18. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+3 or at position n+4, or at position n+7, wherein n is an integer in the range of 2 to 18. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, or at position n+7, wherein n is an integer in the range of 2 to 18. The composition for use according to any one of the preceding items, with the proviso that when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then a. X2 does not align to a Lys in GKY25 of SEQ ID NO: 12; and b. X2 does not align to a Gin, when Xi aligns to a Lys. The composition for use according to any one of the preceding items, with the proviso that when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then a. Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 b. Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12 c. X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12; and d. X2 does not align to Lys 18 in GKY25 of SEQ ID NO: 12; and e. X2 does not align to Gln22, when Xi aligns to Lys18 of SEQ ID NO: 12. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+3, wherein n is an integer in the range of 2 to 18, with the proviso that when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then a. Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 b. Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12; and c. X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12.

[0516] 81. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, with the proviso that when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then a. Xi does not align to Arg11 in GKY25 of SEQ ID NO: 12 b. Xi does not align to Leu12 in GKY25 of SEQ ID NO: 12 c. Xi does not align to Lys14 in GKY25 of SEQ ID NO: 12 d. X2 does not align to Lys14 in GKY25 of SEQ ID NO: 12; e. Xi does not align to Lys 18 in GKY25 of SEQ ID NO: 12; and f. X2 does not align to Lys 18 in GKY25 of SEQ ID NO: 12.

[0517] 82. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+3, wherein n is an integer in the range of 2 to 18, wherein when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds to Val9 and Leu12; or Phe10 and Lys13; or Leu12 and Trp15; or Lys13 and Ile16, or Ile16 and Val19; or Gln17 and lle20; or Lys18 and Asp21 ; or Val19 and Gln22; or lle20 and Phe23; or Asp21 and Gly24; or Gln22 and Glu25 of SEQ ID NO: 12.

[0518] 83. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, wherein when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds Val9 and Lys13; or Lys13 and Gln17; or Trp15 and Val19; or Ile16 and lle20; or Gln17 and Asp21 ; or Val19 and Phe23; or

[0519] I le20 and Gly24; or Asp21 and Glu18 of SEQ ID NO: 12.

[0520] 84. The composition for use according to any one of the preceding items, wherein amino acid Xi is positioned at position n and amino acid X2 is positioned at position at position n+4, wherein n is an integer in the range of 2 to 18, wherein when aligning the peptide sequence to the sequence of GKY25 of SEQ ID NO: 12, then Xi and X2 corresponds Gln17 and Asp21 of SEQ ID NO: 12.

[0521] 85. The composition for use according to any one of the preceding items, wherein Xi and X2 are selected from the group consisting of: i) Xi is Lys and X2 is selected from the group consisting of Asp, Glu, Lys, Cys and Tyr; ii) Xi is Cys and X2 is selected from the group consisting of Cys, Lys and Met; iii) Xi is Asp and X2 is Lys; iv) Xi is Glu is X2 is selected from the group consisting of Lys and Glu; v) Xi is Tyr and X2 is selected from the group consisting of Lys, Phe and Trp; vi) Xi is Met and X2 is selected from the group consisting of Met and Cys; vii) Xi is His and X2 is His; viii) Xi is Phe and X2 is selected from the group consisting of Phe, Tyr, Ala and Trp; ix) Xi is Ala and X2 is Phe or Tyr; x) Xi is Trp and X2 is selected from the group consisting of Trp, Phe and Tyr; xi) Xi is Gly and X2 is Glu.

[0522] 86. The composition for use according to any one of the preceding items, wherein Xi is Lys and X2 is Asp, Glu, Cys or Lys or vice versa.

[0523] 87. The composition for use according to any one of the preceding items, wherein Xi and X2 are Cys, and wherein the covalent linkage is either a direct covalent bond (i.e. a disulphide bridge) or via a crosslinker, wherein the crosslinker for example is a bis-alkylator, such as linker comprising at least two (bromomethyl) substituents.

[0524] 88. The composition for use according to any one of the preceding items, wherein either Xi or X2 is a derivatised canonical amino acid, for example selected from the group consisting of Ser derivatives and Ala derivatives. 89. The composition for use according to any one of the preceding items, wherein the covalent linkage is formed by linking two non-canonical amino acids, optionally wherein said non-canonical amino acids have substituted two native amino acids of thrombin.

[0525] 90. The composition for use according to any one of the preceding items, wherein the covalent linkage is a hydrocarbon staple.

[0526] 91. The composition for use according to any one of the preceding items, wherein Xi and X2 are alkenylated amino acids, such as two C-alkenylated amino acids, such as two a-substituted alkenyl amino acids and / or a,a-disubstituted alkenyl amino acids, and the covalent linkage is an olefin tether formed between said alkenyl residues.

[0527] 92. The composition for use according to item 91 , wherein said alkenylated amino acids are amino acids native to thrombin, which have been alkenylated, and / or alkenylated amino acids substituting amino acids native to thrombin.

[0528] 93. The composition for use according to any one of the preceding items, wherein Xi and X2 individually are selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe.

[0529] 94. The composition for use according to any one of the preceding items, wherein either Xi or X2 is alkenylated Ala and the other is selected from the group consisting of alkenylated Ala, alkenylated Leu, alkenylated Met, alkenylated Ser, alkenylated Tyr, alkenylated Lys, alkenylated Arg and alkenylated Phe.

[0530] 95. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are alkenylated alanine.

[0531] 96. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are a,a-disubstituted alkenylated alanine, and the covalent linkage is an olefin tether formed between said alkenyl residues. 97. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are linked by a tether, wherein the tether is an alkene chain of 10 carbon atoms counting from the C-alpha carbon.

[0532] 98. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are alkenylated alanines, which have been linked to each other by ring closing metathesis forming an alkene tether, wherein the tether is an alkene chain of 10 carbon atoms counting from the C-alpha carbon.

[0533] 99. The composition for use according to any one of items 97 to 98, wherein said alkene chain is an unbranched alkene chain.

[0534] 100. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are alkenylated Ser, such as O-alkenylated Ser.

[0535] 101. The composition for use according to any one of items 91 to 100, wherein said alkenylated amino acids comprise 2 to 10 carbons in the alkenyl chain, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, preferably 4, 5 or 6 carbons.

[0536] 102. The composition for use according to any one of items 91 to 101 , wherein the alkenylated amino acids are a-substituted alkenyl olefin-terminated amino acids and / or a,a-disubstituted alkenyl olefin-terminated amino acids.

[0537] 103. The composition for use according to any one of the preceding items, wherein Xi and / or X2 are a,a-disubstituted S- or R-pentenylalanine (S5 or R5) and S- or R-octenylalanine (S8 or R8) alanine.

[0538] 104. The composition for use according to any one of the preceding items, wherein the internal hydrocarbon staple is formed by linking two (S)-2-(4’- pentenyl)-alanines.

[0539] 105. The composition for use according to any one of the preceding items, wherein the covalent linkage is established through ring-closing. 106. The composition for use according to any one of the preceding items, wherein the covalent linkage is established through ring-closing metathesis (RCM).

[0540] 107. The composition for use according to any one of the preceding items, wherein Xi is a non-canonical azido terminated amino acid and X2 is a non- canonical yne-terminated amino acid or vice versa.

[0541] 108. The composition for use according to any one of the preceding items, wherein X3 and X4 are closer to the N-terminal relative to Xi and X2.

[0542] 109. The composition for use according to any one of the preceding items, wherein amino acid X3 is positioned at position n, and amino acid X4 is positioned at position n+3, or at position, n+4, or at position n+5, wherein n is an integer. In preferred embodiments, amino acid X3 is positioned at position n, and amino acid X4 is positioned at position, n+4.

[0543] 110. The composition for use according to any one of the preceding items, wherein amino acid X3 is positioned at the very N-terminus of the peptide.

[0544] 111. The composition for use according to any one of the preceding items, wherein amino acid X3 is positioned at the very N-terminus of the peptide and amino acid X4 is positioned at position n+4.

[0545] 112. The composition for use according to any one of the preceding items, wherein the internal covalent bond is an amide bond formed by reacting an amine and a carboxylic acid.

[0546] 113. The composition for use according to any one of the preceding items, wherein the internal covalent bond is a lactam bridge formed by reacting an amine and a carboxylic acid.

[0547] 114. The composition for use according to any one of the preceding items, wherein X3 is an N-terminal amino acid harbouring an N-terminal amine group, and X4 is an amino acid harbouring a carboxylic acid side chain, and wherein X3 and X4 are linked by an internal covalent bond formed by reacting said amine with said carboxylic acid. . The composition for use according to any one of the preceding items, wherein when aligning the sequence of the peptide of the invention to the sequence of GKY25 of SEQ ID NO: 12, then X3 and X4 corresponds to Gly 1 and Phe5 of GKY25 of SEQ ID NO: 12. . The composition for use according to any one of the preceding items, wherein X3 and X4 corresponds to Gly 1 and Glu5 of SEQ ID NO: 14. . The composition for use according to any one of the preceding items, wherein the peptide has a length between 14 and 22 amino acids, such as between 15 and 21 amino acids, such as between 16 and 20 amino acids, such as between 17 and 20 amino acids. . The composition for use according to any one of the preceding items, wherein the peptide has a length of 18 to 20 amino acids, such as 18 or 19 amino acids. . The composition for use according to any one of the preceding items, wherein the peptide comprises a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of between 14 and 22 amino acids, such as between 13 and 18 amino acids, such as between 15 and 21 amino acids, such as between 16 and 20 amino acids, such as between 17 and 20 amino acids, preferably between 17 and 18 amino acids. . The composition for use according to any one of the preceding items, wherein the peptide has a length between 24 and 40 amino acids, such as between 25 and 35 amino acids, such as between 25 and 30 amino acids, such as between 28 and 34 amino acids. . The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 16 to 21 amino acids containing up to 6 amino acid substitutions. . The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 17 to 18 amino acids containing up to 2 amino acid substitutions, where in peptide may comprise up to 4 additional amino acids. . The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 of in the range of 17 to 18 amino acids containing a substitution of one amino acid for amino acid Xi and a substitution of one amino acid for amino acid X2, where in peptide may comprise up to 4 additional amino acids. . The composition for use according to any one of the preceding items, wherein said peptide comprises at least 2 amino acid substitutions, such as 3 amino acid substitutions, such as 4 amino acid substitutions, such as 5 amino acids substitutions compared to the consecutive sequence of thrombin. . The composition for use according to any one of the preceding items, wherein one or more of said substitutions are conservative substitutions, such as wherein 1, 2, 3 or 4 amino acid substitutions are conservative substitutions. . The composition for use according to any one of the preceding items, wherein said peptide further comprises one or more moieties conjugated to said peptide, optionally wherein the peptide and the one or more moieties are conjugated to each other by a linker, wherein the one or more moieties are selected from the group consisting of alkyls, aryls, heteroaryls, olefins, fatty acids, polyethylene glycol (PEG), saccharides, and polysaccharides. . The composition for use according to any one of the preceding items, wherein said peptide further comprises between 1 and 5, such as between 1 and 4, for example between 1 and 3, for example between 1 and 2, such as 2 positively charged amino acids inserted at or close to the end of the peptide. . The composition for use according to item 127, wherein said positively charged amino acids are inserted at or close to the N-terminal, such as at a position selected from positions 1 , 2, 3, 4 and / or 5 relative to the N-terminal of the peptide. . The composition for use according to any one of items 127 to 128, wherein said positively charged amino acids are inserted at the N-terminal. . The composition for use according to any one of the preceding items, wherein said peptide comprises 2 positively charged amino acids inserted at or close to the N-terminal, such as at a position selected from positions 1 , 2, and / or 3 relative to the N-terminal of the peptide. . The composition for use according to any one of the preceding items, wherein the peptide consists of in the range of 15 to 20 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids has been substituted with alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 5 additional N-terminal amino acids . The composition for use according to any one of the preceding items, wherein the peptide consists of in the range of 16 to 18 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids has been substituted with alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 5 additional N-terminal amino acids. . The composition for use according to any one of the preceding items, wherein the peptide consists of 17 consecutive amino acids of thrombin of SEQ ID NO: 1 , wherein 2 amino acids has been substituted with alkenylated amino acids forming an internal hydrocarbon staple, and in the range of 2 to 3 additional N-terminal amino acids. 134. The composition for use according to any one of items 127 to 133, wherein the additional N-terminal amino acids are positively charged.

[0548] 135. The composition for use according to any one of items 127 to 134, wherein the positively charged amino acids are selected form the group consisting of arginine, lysine and histidine, preferably wherein the positively charged amino acids are arginine and / or lysine, even more preferably wherein the positively charged amino acids are lysine.

[0549] 136. The composition for use according to any one of items 127 to 134, wherein the positively charged amino acids are selected form the group consisting of arginine, lysine and histidine, preferably wherein the positively charged amino acids are arginine and / or lysine, even more preferably wherein the positively charged amino acids are arginine.

[0550] 137. The composition for use according to any one of the preceding items, wherein the alkenylated amino acids are as defined in any one of items 91 to 104.

[0551] 138. The composition for use according to any one of the preceding items, wherein the peptide comprises the sequence:

[0552] -K-K-Z-Z-X1-K-Z-Z-X2-Z wherein each Z individually is any canonical amino acid; and

[0553] Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0554] 139. The composition for use according to any one of the preceding items, wherein the peptide comprises the sequence: U-U-(Z)n-K-K-Z-Z-Xi-K-Z-Z-X2-Z, wherein each Z individually is any canonical amino acid;

[0555] II is His, Lys or Arg; n is an integer in the range of 0 to 10 and Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.

[0556] 140. The composition for use according to any one of the preceding items, wherein the peptide comprises the sequence:

[0557] Z-R-Z-K-K-Z-Z-X1-K-Z-Z-X2-Z wherein each Z individually is any canonical amino acid; and Xi and X2 are amino acids linked by a covalent linkage.

[0558] 141. The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of the sequence: U-U-Z-Z-R-Z-K-K-Z-Z-X1-K-Z-Z-X2-Z wherein each Z individually is any canonical amino acid;

[0559] II is His, Arg or Lys; and

[0560] Xi and X2 are amino acids linked by a covalent linkage.

[0561] 142. The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of the sequence:

[0562] V-F-R-L-K-K-W-I-X1-K-V-I-X2-Q-F-G-E wherein

[0563] Xi and X2 are amino acids linked by a covalent linkage.

[0564] 143. The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of the sequence: U-U-V-F-R-L-K-K-W-I-X1-K-V-I-X2-Q-F-G-E wherein

[0565] II is His, Arg or Lys; and

[0566] Xi and X2 are amino acids linked by a covalent linkage.

[0567] 144. The peptide according to any one of items 139 to 144, wherein II is Lys or Arg. . The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 6 v) SEQ ID NO: 7 vi) SEQ ID NO: 8; vii) SEQ ID NO: 9; viii) SEQ ID NO: 10; ix) SEQ ID NO: 11 x) SEQ ID NO: 17; xi) SEQ ID NO: 18; xii) SEQ ID NO: 19; xiii) SEQ ID NO: 20; xiv) SEQ ID NO: 21; xv) SEQ ID NO: 22; xvi) SEQ ID NO: 23; xvii) SEQ ID NO: 24; xviii) SEQ ID NO: 25; or xix) SEQ ID NO: 26 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to xix); wherein Xi and X2 are amino acids linked by a covalent linkage, and wherein X3 and X4 are amino acids linked by a covalent linkage. . The composition for use according to any one of the preceding items, wherein the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 8; or v) SEQ ID NO: 9 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to v); wherein Xi and X2 are amino acids linked by a covalent linkage. . The composition for use according to any one of the preceding items, wherein said peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3; ii) SEQ ID NO: 5; iii) SEQ ID NO: 6; or iv) SEQ ID NO: 7; or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to iv); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage, optionally wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether, further optionally wherein Xi and X2 are Ala, the side chains of which are covalently bound to each other by a Cs alkenyl tether comprising one double bond. . The composition for use according to any one of the preceding items, wherein said peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3; or ii) SEQ ID NO: 5; or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage, optionally wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether, further optionally wherein Xi and X2 are Ala, the side chains of which are covalently bound to each other by a Cs alkenyl tether comprising one double bond.

[0568] 149. The composition for use according to any one of the preceding items, wherein said peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 14; or ii) SEQ ID NO: 26 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to ii); wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage; and further wherein X3 and X4 are amino acids, the side chains of which are linked by a covalent linkage, preferably wherein Xi and X2 are (S)-2-(4’-pentenyl)-alanines, the side chains of which have been reacted with each other to form an alkenyl tether, and X3 and X4 are Gly and Glu, respectively, which have been reacted with each other to form a lactam bridge.

[0569] 150. The composition for use according to any one of items 110 to 116, wherein Xi and X2 are as defined in any one of items 74 to 108.

[0570] 151 . The peptide according to any one of the preceding items, wherein X3 and X4 are as defined in any one of items 74 to 116.

[0571] 152. The composition for use according to any one of the preceding items, wherein the peptide contains at least one alpha helical secondary structure unit in aqueous solution.

[0572] 153. The composition for use according to item 152, wherein the alpha helical secondary structure unit is maintained upon binding a target molecule, such as cluster differentiation 14 (CD14).

[0573] 154. The composition for use according to any one of the preceding items, wherein the peptide has a hydrodynamic radii of less than 150 nm, such as less than 100 nm at pH 7.4 and / or less than 100 nm, such as less than 80 nm at pH 5. . The composition for use according to any one of the preceding items, wherein the peptide has increased stability in vivo and / or in vitro compared to a peptide with the same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions. . The composition for use according to any one of the preceding items, wherein the peptide has increased stability in the presence of a serine protease, such as, compared to a peptide of same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions. . The composition for use according to any one of the preceding items, wherein 50 pM of the peptide has a hemolytic activity in fresh, whole blood of at the most 5%, such as at the most 4%, for example at the most 3%, such as at the most 2%. . The composition for use according to any one of the preceding items, the peptide has a hemolytic activity in fresh, whole blood of at the most 10%, preferably of at the most 5% at a peptide concentration reducing release of TNF-a in LPS stimulated blood in vitro by 50%. . The composition for use according to any one of the preceding items, wherein the peptide has anti-coagulant activity. . The composition for use according to any one of the preceding items, wherein the peptide increases the clotting time as determined by aPPT by at least 100% at a concentration of 60 pM and / or increases the clotting time as determined by aPPT by at least 90% at a concentration of 40 pM peptide. . The composition for use according to any one of the preceding items, wherein the peptide reduces inflammation and / or infection. . The composition for use according to any one of the preceding items, wherein the peptide reduces the secretion of pro-inflammatory cytokines in the presence of one or more endotoxins, such as LPS. . The composition for use according to any one of the preceding items, wherein the peptide decreases secretion of pro-inflammatory cytokines in vivo in blood comprising one or more endotoxins, such as LPS . The composition for use according to any one of items 162 or 163, wherein said pro-inflammatory cytokines are selected from the group consisting of tumour necrosis factor a (TNF-a), interleukin p (IL-1 P), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon (IFN-Y) and / or monocyte chemoattractant protein-1 (MCP-1). . The composition for use according to any one of the preceding items, wherein the peptide decreases NF-kB activation in the presence of a toll-like receptor (TLR)-agonists, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), Staphylococcus aureus peptidoglycan (SA-PGN) and / or zymosan. . The composition for use according to any one of the preceding items, wherein 10 pM of peptide reduces secretion of TNF-a and or I L-1 p after incubation in fresh blood in the presence of LPS by at least 50%, such as by at least 60%, for example by at least 70%, compared to a peptide of same sequence except that the amino acids Xi and X2 are exchanged for other amino acids lacking the covalent linkage, when said peptides are tested under the same conditions. . The composition for use according to any one of the preceding items, wherein the peptide is bactericidal, for example wherein the peptide is capable of killing bacteria by damaging the bacterial membrane. . The composition for use according to any one of the preceding items, wherein said bacteria is selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. . The composition for use according to any one of the preceding items, wherein the anti-inflammatory effect of the peptide is maintained 24 h in vivo after topical administration of the peptide. . The composition for use according to any one of the preceding items, wherein the anti-bacterial effect of the peptide is maintained 24 h in vivo after topical administration of the peptide. . A composition selected from: a gel, an ointment, a cream, a paste, a lotion, or a liquid; the composition comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1. . The composition according to item 171 , with the proviso that if the peptide has a total length between 24 to 40 amino acids it comprises at least two internal covalent linkages between the side chains of two non-neighbouring, internal amino acids, wherein the amino acids of the first internal covalent linkage are denoted Xi and X2, and the amino acids of the second internal covalent linkage are denoted X3 and X4. . The composition according to any one of the items 171 to 172, wherein the peptide is as described in any one of items 1 to 169. . The composition according to any one of items 171 to 173, wherein the composition is for use in a method of treatment or prevention of a disorder or condition as described in any one of items 1 to 169. . The composition according to any one of items 171 to 174, or the composition for use according to any one of items 1 to 169, wherein the composition is comprised in a patch, a dry patch, a wound irrigation liquid, a contact lens liquid, a plaster, a bandage, a suture, a prothesis, an implant, a film, a dressing, a wound dressing, a catheter a skin graft or a skin substitute. . The composition according to any one of items 171 to 175, or the composition for use according to any one of items 1 to 169, wherein the composition comprises: i. a polymer, for example a non-ionic polymer, capable of forming a hydrogel when mixed with an aqueous solution, and ii. an aqueous solution. . The composition according to any one of items 171 to 176, or the composition for use according to any one of items 1 to 169, wherein the composition comprises: i. EDTA and ii. an aqueous buffer, wherein the composition has a pH of at the most 8. . The composition according to any one of items 171 to 177, or the composition for use according to any one of items 1 to 169, wherein: iii. the concentration of the compound and / or the peptide in the composition is at least 0.08 wt% and / or iv. the polymer is present in said composition at a concentration of at least 0.05 wt%. . The composition according to any one of items 171 to 178, or the composition for use according to any one of items 1 to 169, wherein the compound is formulated as a gel, such as a hydrogel. . The composition according to any one of items 171 to 179, or the composition for use according to any one of items 1 to 169, wherein the composition is a viscous liquid. 181. The composition according to any one of items 171 to 180, or the composition for use according to any one of items 1 to 169, wherein the nonionic polymer is hydroxylated.

[0574] 182. The composition according to any one of items 171 to 181, or the composition for use according to any one of items 1 to 169, wherein the nonionic polymer is selected from the group consisting of polyallylalcohol, polyvinylalcohol, polyacrylamide, polyethylene glycol (PEG), polyvinyl pyrrolidone, starches, such as corn starch and hydroxypropylstarch, alkylcelluloses, such as Ci-Ce-alkylcelluloses, including methylcellulose, ethylcellulose and n-propylcellulose; substituted alkylcelluloses, including hydroxy-alkylcelluloses, preferably hydroxy-Ci-Ce-alkylcelluloses and hydroxy- Ci-Ce-alkyl-Ci-Ce-alkylcelluloses, such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulosen and mixtures of the aforementioned.

[0575] 183. The composition according to any one of items 171 to 182, or the composition for use according to any one of items 1 to 169, wherein the nonionic polymer is selected from the group consisting of hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), Hydroxypropyl Methylcellulose (HPMC), poly(vinyl)alcohol (PVA), polyacrylamide (PA), polyethylene glycol (PEG) and polyvinyl pyrrolidone, and mixtures thereof,

[0576] 184. The composition according to any one of items 171 to 183, or the composition for use according to any one of items 1 to 169 wherein the nonionic polymer is selected from the group consisting of hydroxyalkyl celluloses, preferably from the group consisting of hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC).

[0577] 185. The composition according to any one of items 171 to 184, or the composition for use according to any one of items 1 to 169, wherein the concentration of the non-ionic polymer in the compositions of the invention is sufficient to obtain a composition with a flow point of at least 10 Pa, such as at least 15 Pa, for example in the range of 10 to 80 Pa, such as in the range of 40 to 60 Pa. 186. The composition according to any one of items 171 to 185, wherein concentration of the non-ionic polymer is sufficient to obtain a viscosity of the composition of at least 10 m Pas, such as between 10 m-Pas and 100,000 m-Pas

[0578] 187. The composition according to any one of items 171 to 186, or the composition for use according to any one of items 1 to 169, wherein the nonionic polymer is present in said composition at a concentration of at least 0.05 wt%, for example in a concentration of at least 0.09 wt%, such as in a concentration of at least 0.1 wt%, for example in a concentration of at least 0.5 wt%, such as in a concentration of at least 0.8 wt%, for example in a concentration of at least 1.0 wt%, preferably a concentration in the range of 0.09 to 4 wt%, more preferably in the range of 1 to 3 wt%.

[0579] 188. The composition according to any one of items 171 to 187, or the composition for use according to any one of items 1 to 169, wherein the nonionic polymer is present in said composition at a concentration of at least 1 wt%.

[0580] 189. The composition according to any one of items 171 to 188, or the composition for use according to any one of items 1 to 169, further comprising glycerol, preferably at a concentration of 1 to 3 vol%, more preferably at concentration of 1 to 2 vol%.

[0581] 190. The composition according to any one of items 171 to 189, or the composition for use according to any one of items 1 to 169, wherein the aqueous solution is an aqueous buffer.

[0582] 191. The composition according to any one of items 171 to 190, or the composition for use according to any one of items 1 to 169, wherein the peptide is capable of simultaneously binding both to lipopolysaccharides and to the LPS-binding hydrophobic pocket of CD14.

[0583] 192. The composition according to any one of items 171 to 191, or the composition for use according to any one of items 1 to 169, wherein the peptide is present in said composition in a concentration of at least 0.08 wt%, for example at least 0.1 wt%.

[0584] 193. The composition according to any one of items 171 to 192, or the composition for use according to any one of items 1 to 169, wherein the peptide is present in said composition in a concentration of at least 0.01 wt%, more preferably in a concentration of 0.01 to 5 wt%, such as 0.08 to 3 wt%.

[0585] 194. The composition according to any one of items 171 to 193, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is at the most 7.

[0586] 195. The composition according to any one of items 171 to 194, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is lower than 7, preferably lower than 6, more preferably 5.5 or lower.

[0587] 196. The composition according to any one of items 171 to 195, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is lower than 7, preferably lower than 6, more preferably 5.5 or lower, and higher than 3, such at least 3.5.

[0588] 197. The composition according to any one of items 171 to 196, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is in the range of 3 to 6, such as approx. 5.

[0589] 198. The composition according to any one of items 171 to 197, or the composition for use according to any one of items 1 to 169, wherein the aqueous buffer is an Acetate buffer comprising Acetate, preferably at a concentration of 10-50 mM, more preferably at a concentration of 25 mM, and having a pH of 3.6 to 5, such as 5.

[0590] 199. The composition according to any one of items 171 to 198, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is at the most 8, such as in the range of 3 to 8, for example in the range of 3.5 to 8, such as in the range of 5 to 8. . The composition according to any one of items 171 to 199, or the composition for use according to any one of items 1 to 169, wherein the pH of the composition is between 7 and 8, preferably approx. 7.4. . The composition according to any one of items 171 to 200, or the composition for use according to any one of items 1 to 169, wherein the aqueous solution or aqueous buffer is a Trisaminomethane (Tris) buffer comprising Trisaminomethane, preferably at a concentration of 5 to 20 mM, such as approx. 10 mM. . The composition according to any one of items 171 to 201 , or the composition for use according to any one of items 1 to 169, wherein said peptide within the composition has a Tmof at least 30°C, preferably of at least 35°C, even more preferably of at least 40°C. . The composition according to any one of items 171 to 202, or the composition for use according to any one of items 1 to 169, wherein the compound comprising said peptide within the composition has a Tmof at least 30°C, preferably of at least 35°C, even more preferably of at least 40°C. . The composition according to any one of items 171 to 203, or the composition for use according to any one of items 1 to 169, wherein said peptide within the composition has a Cm urea of of at least 0.8 M, preferably of at least 1.0 M, even more preferably of at least 1.1 M. . The composition according to any one of items 171 to 204, or the composition for use according to any one of items 1 to 169, wherein the compound comprising said peptide within the composition has a Cm urea of of at least 0.8 M, preferably of at least 1.0 M, even more preferably of at least 1.1 M. . The composition according to any one of items 171 to 205, or the composition for use according to any one of items 1 to 169, wherein said peptide within the composition has a CmGnd-Hci of at least 0.8 M, preferably of at least 0.9 M.

[0591] 207. The composition according to any one of items 171 to 206, or the composition for use according to any one of items 1 to 169, wherein the compound comprising said peptide within the composition has a CmGnd-Hci of at least 0.8 M, preferably of at least 0.9 M.

[0592] 208. The composition according to any one of items 171 to 207, or the composition for use according to any one of items 1 to 169, wherein the composition comprises at least 90%, such as at least 95% of the initial content of said compound comprising said peptide after storage for 2 months at 37°C.

[0593] 209. The composition according to any one of items 171 to 208, or the composition for use according to any one of items 1 to 169, wherein the composition comprises at least 75%, such as at least 80%, for example at least 90% of the initial content of said compound comprising said peptide after storage for 4 months at 37°C.

[0594] 210. The composition according to any one of items 171 to 209, or the composition for use according to any one of items 1 to 169, wherein the composition comprises at least 70%, such as at least 80%, for example at least 85% of the initial content of said compound comprising said peptide after storage for 6 months at 37°C.

[0595] 211. The composition according to any one of items 171 to 210, or the composition for use according to any one of items 1 to 169, wherein the composition comprises at least 90%, such as at least 95% of initial content of said compound comprising said peptide after storage for 8 months at room temperature.

[0596] 212. A device comprising the composition according to any one of items 171 to 211 , or the composition for use according to any one of items 1 to 169.

[0597] 213. The device according to item 212, wherein the device is selected from a patch, a powder, a dry patch, a wound irrigation liquid, a plaster, a bandage, a suture, a prosthesis, an implant, a film, a dressing, a wound dressing, a catheter, a skin graft or a skin substitute. 214. The device according to any one of items 212 to 213, wherein the composition is coated on the device.

[0598] 215. Use of a composition according to any one of items 171 to 211 or the device according to any one of items 212 to 214, for the preparation of a medicament for treatment of a disorder or condition of the skin or the mucosa in an individual in need thereof.

[0599] 216. A method of treatment of a disorder or condition of the skin or the mucosa, wherein the method comprises administration of a therapeutically effective amount of the composition according to any one of items 171 to 211 , or the device according to any one of items 212 to 214.

[0600] Examples

[0601] Example 1 - Antimicrobial activity of stapled peptide

[0602] Summary

[0603] Herein we demonstrate the effects of stapling on the antimicrobial activity of the peptide HVF18. The study investigated the retention of the antimicrobial activity of HVF18 after stapling and found that the stapled form, sHVF18, showed comparable or stronger bactericidal activity than the linear form in various conditions. The study also demonstrated that sHVF18 displayed greater selectivity for Gram-positive bacteria and that the killing mechanism involved bacterial membrane permeabilization and disruption.

[0604] Results

[0605] The present invention relates to the field of antimicrobial agents and more particularly to a stapled peptide HVF18 and its antimicrobial activity. The study aimed to investigate the effects of stapling on the antimicrobial activity of HVF18 and to determine the retention of its bactericidal activity after stapling.

[0606] The study used radial diffusion assay (RDA) to compare the antimicrobial activity of sHVF18 and HVF18 in different conditions. The results showed that sHVF18 was active in a comparable manner to HVF18, regardless of the presence of NaCI (Fig. 1A- B). In contrast, the linear peptide showed better bacterial killing in the absence of salt, and a dramatic decrease in activity was observed in the presence of NaCI (Fig. 1A-B). Furthermore, the study found that sHVF18 was more bactericidal than HVF18 in solution, particularly against S. aureus, where it showed marked affinity and killing at concentrations lower than 1 M (Fig. 1C).

[0607] To evaluate the activity of sHVF18 in more complex situations, such as the presence of salt or human plasma, the study performed additional tests using radial diffusion assay (RDA). The antimicrobial activity of both peptides decreased with increased complexity of the media, but sHVF18 still showed a stronger killing effect than its linear form (Fig. 1B). To understand the mechanism of antimicrobial activity of sHVF18, the study used transmission electron microscopy (TEM). The results showed that both P. aeruginosa 01 and S. aureus lost their membrane integrity after incubation with both linear and stapled HVF18 (Fig. 1 D), and leakage of electron-dense material and the intracellular content was clearly visible. The study further confirmed the antimicrobial activity of sHVF18 in standard minimum inhibitory concentration (MIC) assay (Fig. 1 E and F). These results demonstrate that sHVF18 retains its antibacterial activity against Gramnegative bacteria, while showing greater selectivity for Gram-positive bacteria (Fig. 1 E and F).

[0608] Stapling enhanced stability of HVF18 up to 18 h with HNE and PE (Fig. 1G). The stability was also increased against trypsin. Indeed, it was still possible to detect intact sHVF18 even after 18 h. Linear and stapled HVF18 variants were not susceptible to V8 digestion. LC-MS / MS results confirmed that stapling of HVF18 was conferring the peptide complete protection to proteolysis. The helix stability of sHVF18 was further tested exposing the peptide to increasing temperatures and analyzing the secondary structure by CD. It was found that even after exposure to 80 °C, sHVF18 still presented an a-helical spectrum, i.e. with two characteristic minimum at 208 and 222 nm.

[0609] In conclusion, the stapled peptide HVF18 demonstrates strong antimicrobial activity against various bacterial strains, particularly Gram-positive bacteria, and the mechanism of action involves bacterial membrane permeabilization and disruption. The present invention provides a new approach to the development of antimicrobial agents with potential therapeutic applications.

[0610] Material and methods

[0611] Peptides

[0612] The peptides HVF18 (HVFRLKKWIQKVIDQFGE) (SEQ ID NO: 2), and the respective stapled version denoted as SHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3) were synthesised by AmbioPharm, Inc. (USA). Briefly, standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPSS) was used. To obtain hydrocarbon stapled peptides, olefin-bearing (S)-2-(4’pentenyl)-alanine was inserted at specified locations in the peptide sequence (denoted by X). Olefin metathesis reaction was performed on solid support using Grubbs’ first-generation catalyst in 1 ,2-dichloroethane. The product peptide was cleaved from the resin and further purified by RP-HPLC. Peptides were provided as acetate salts, and the purity was confirmed with MALDI-TOF MS (>95%).

[0613] Radial Diffusion Assay (RDA)

[0614] To prepare the RDA, bacteria were grown and prepared as described previously. Microorganisms (4 x 106CFU) were then added to 15 mL of underlay agarose gel, consisting of 0.03% (w / v) TSB, 1% (w / v) low electroendosmosis type agarose (Sigma- Aldrich), and 0.02% (v / v) Tween 20 (Sigma-Aldrich). The underlay gel was poured into a 144-mm diameter petri dish. After agarose solidification, 4-mm diameter wells were punched, and 6 pL peptide solution of the required concentration was added to each well. Plates were incubated at 37 °C for 3 h to allow peptide diffusion. The underlay gel was then covered with 15 mL of molten overlay gel (6% TSB and 1% low electroendosmosis type agarose in distilled H2O). The activities of the peptides are presented as clear zone-to-well diameter (excluding the 4 mm well). All experiments were performed at least four times.

[0615] Viable Count Assay (VGA)

[0616] To prepare the VGA, bacteria were grown and prepared as described previously. Next, the bacterial suspension was diluted 1 :1000 in 10 mM Tris pH 7.4 at a concentration of 2 x 106CFU mL-1. Bacteria (50 pL) were incubated with different concentrations of HVF18, and SHVF18 (1-20 pM) in 10 mM Tris pH 7.4 with or without 150 mM NaCI or 25% of human citrate-plasma for 2 h at 37 °C. At the end of incubation, serial dilutions of the samples were plated on TH agar plates, incubated overnight at 37 °C, and the CFU were calculated. Bacteria treated with the respective buffer were used as a control. All experiments were performed at least four times, and the data shown are mean values ± SEM.

[0617] Transmission electron microscopy (TEM)

[0618] The effect of peptides on the membranes of S. aureus and P. aeruginosa 01 was evaluated using transmission electron microscopy (TEM) in combination with negative staining. Specifically, 5 pL of bacterial suspension from VGA were adsorbed onto carbon-coated grids (copper mesh, 400) for 60 seconds and stained with 7 pL of 2% uranyl acetate for 30 seconds. The grids were rendered hydrophilic via glow discharge at low air pressure. Analysis was conducted on 10 view fields (magnification x4200) of the mounted samples on the grid (pitch 62 pm) from three independent experiments.

[0619] The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration

[0620] The minimal inhibitory concentration (MIC) was determined according to the protocol reported by Wiegand et al. Bacteria were grown and diluted as described above. Next, the bacteria were further diluted 1 :1000 in 2x BBL™ Mueller Hinton II (MH), cation- adjusted broth (Becton, Dickinson and Company, Sparks, USA). Bacteria (50 pL) were added to 96-well round bottom polystyrene plates (Corning INC, Kennebunk, USA) containing 50 pL 2x MH broth (control) or 2x MH broth with peptide (HVF18 or sHVF18) at concentrations ranging from 2.5-320 pM. The plates were then incubated at 37°C for 24 hours. MIC was validated as the lowest concentration at which no visible bacterial growth was observed. Following analysis of the MIC, the same plate was used to determine the minimal bactericidal concentration (MBC). For this purpose, the sample for each condition was resuspended using a pipette tip, and a droplet of 10 pL was plated on THA plates and incubated at 37°C overnight. MBC was fixed at the concentration at which no bacterial colony formation was observed.

[0621] Proteolysis of the peptides in vitro

[0622] Peptides were resuspended in endotoxin free water at concentration of 1 mM. Then, 14.7 pg of HVF18 and sHVF18 were incubated with 0.2 pg of human neutrophil elastase (HNE, Calbiochem®, Merk KGaA, Darmstadt, Germany), P. aeruginosa elastase (PE, Calbiochem®, Merk KGaA, Darmstadt, Germany), Glutamyl-C endopeptidase (EC 3.4.2.11.9) from S. aureus V8 (BioCol GmbH, Michendorf, Germany) or trypsin (Try, Promega, Madison, Wl, USA) for different lengths of time (0-18 h) in a final volume of 20 pL. At the end of the incubation, the digestion was evaluated by Tricine SDS-PAGE. All digestions were performed in three independent experiments.

[0623] Example 2 - Activity of additional stapled peptides

[0624] Materials and Methods

[0625] The peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE)(SEQ ID NO: 12), sGKY25 (GKYGFYTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 13), 2sGKY25 (cyclo[GKYGE] YTHVFRLKKWIXKVIXQFGE)( SEQ ID NO: 14), SHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3), SKVF18 (KVFRLKKWIXKVIXQFGE) (SEQ ID NO: 4), SK-KVF18 (SEQ ID NO: 5), SKK-KVF18 (SEQ ID NO: 6), SKKK-KVF18 (SEQ ID NO: 7), SRVF18 (RVFRLKKWIXKVIXQFGE) (SEQ ID NO: 8), SR-RVF18 (SEQ ID NO: 9), SRR-RVF18 (SEQ ID NO: 10) and SRRR-RVF18 (SEQ ID NO: 11) were synthesized and purified by AmbioPharm, Inc. (USA) as described for sHVF18 in Example 1. Cyclization of 2sGKY25 was performed using solid phase conditions, by forming an amide bond, i.e. a lactam bridge between the N-terminal amine of Gly 1 and the carboxylic acid side chain of Glu5 of said peptide. Peptides were provided as acetate salts, and the purity was confirmed with MALDI-TOF MS (>95%).

[0626] Circular dichroism spectroscopy

[0627] The secondary structure of all stapled peptides was assessed by Circular dichroism (CD). The peptides were diluted to 10 pM in 10 mM Tris at pH 7.4 and spectra were measured by a Jasco J-810 spectropolarimeter (Jasco, USA) equipped with a Jasco CDF-426S Peltier set to 25 °C, as reported for sHVF18. Data shown are mean values obtained from three independent experiments.

[0628] NF-KB activation assay

[0629] Anti-inflammatory activity of all stapled peptides was tested on THP1-XBIue-CD14 reporter cells (InvivoGen, San Diego, USA). Briefly, 180,000 cells well-1were seeded in 96 well plates in phenol red RPMI media, supplemented with 10% (v v’1) heat- inactivated FBS and 1% (v v1) Antibiotic-Antimycotic solution (AA). Then 100 ng mL-1LPS (Sigma, USA) with and w / o peptides at different concentrations (1-20 pM) were added. The NF-KB activation was determined after 20 h of incubation according to the manufacturer’s instructions (InvivoGen, San Diego, USA), i.e. by mixing 20 pL of supernatant with 180 pL of SEAP detection reagent (Quanti-BlueTM, Invivo- Gen), followed by absorbance measurement at 600 nm. The final concentration of each peptide was between 1-10 pM. Data shown are mean values ± SEM obtained from four independent experiments all made in triplicate.

[0630] Hemolysis assay

[0631] Fresh venous blood from healthy donors was collected in in lepirudine tubes (50 pg mL'1). Then 50 pL of blood were transfered to round-bottom 96-wells plate containing 150 pL of peptides previously diluted in RPMI-1640- GlutaMAX-l without phenol red (Gibco). 1 :4 diluted blood was used as control. 50 pL of blood mixed with 150 pl 5% Tween-20 were used as positive control. After 1 h incubation at 37 °C and 5% CO2, the plate was centrifuged at 800 g, 150 pL of each sample were transferred to a flat-bottom 96-wells plate and the absorbance at 450 nm was measured. The percentage of hemolysis was calculated following the formula reported below:

[0632] Abs 450 nm (Sample- control) Abs 450 nm (Posivite control-control)

[0633] To evaluate the hemolytic effect of the peptides on erythrocytes the blood was collected as reported above and centrifuged at 250 g for 10 min. Then plasma was discarded, and red blood cells were washed with 150 mM NaCI in 10 mM Tris pH 7.4 for three times. Next, the pellet was dilute 100 times with saline Tris buffer. 100 pL of this solution were added to round-bottom 96-wells plate containing 100 pL of peptides previously diluted in saline Tris buffer. After 1 h incubation at 37 °C and 5% CO2, the plate was centrifuged and the absorbance at 450 nm was measured, then the percentage of erythrocyte lysis was determined using formula reported above. Data shown are mean values ± SEM obtained from at least four independent experiments all made in triplicate. For each experiment blood from a different donor was used.

[0634] Whole blood assay

[0635] Fresh venous blood was collected in the presence of lepirudin (50 pg mL-1) from healthy donors. The blood was diluted 1 :4 in RPMI-1640-GlutaMAX-l (Gibco) and 1 mL of this solution was transferred to 24-well plates and stimulated with 100 ng mL-1of LPS immediately after adding increasing concentrations of GKY25, HVF18 and their stapled versions. After 24 h incubation at 37 °C in 5% CO2, the plate was centrifuged for 5 min at 1000 g and then the supernatants were collected and stored at -80 °C before analysis. The experiment was performed at least 4 times by using blood from different donors each time. To evaluate the healing properties of the peptides, the blood was stimulated with 100 ng mL-1LPS and after 30 min incubation at 37 °C was treated with increasing doses of four peptides. In the last set of experiments, the preventive anti-inflammatory activity of the peptides was evaluated by exposing the blood to increasing concentrations of GKY25, HVF18 and their stapled versions for 30 min. Then the blood was stimulated with 100 ng mL-1of LPS. For each experiment blood from a different donor was used. Cytokine assay

[0636] Plasma obtained from blood experiments was used to evaluate cytokine release. Human inflammation DuoSet® ELISA Kit (R&D Systems) specific for TNF-a and I L-1 p was used according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 450 nm. Data shown are mean values ± SEM obtained from four independent experiments. For 2sGKY25, the data shown are mean values ± SEM obtained from two independent experiments.

[0637] The level of TNF- a, IFN-y, MCP-1 , IL-10 and IL-6 in murine plasma were assessed using the Mouse Inflammation Kit, (Becton Dickinson AB) according to the manufacturer’s instructions.

[0638] Antibacterial activity

[0639] Antibacterial activity of different stapled peptides on E. coli ATCC 25922, S. aureus ATCC 29213 and P. aeruginosa 01, by radial diffusion assay (RDA) or viable count assay (VGA) was performed as for sHVF18. The highest tested concentration of each peptide was 20 and 10 pM for RDA and VGA, respectively.

[0640] Results

[0641] Analysis of the structure and hemolytic activity of stapled peptides

[0642] As shown in Example 1 stapling of HVF18 increased its stability to proteolysis and greatly improved its anti-inflammatory activity in vitro and in vivo), retaining its antimicrobial activity, but turning it mostly against Gram-positive bacteria, while at the same time having a low hemolytic activity. Different variants of sHVF18 were made, where the N-terminal His residue was exchanged with 1 to 4 Lys or Arg residues. In addition, two variants of stapled GKY25 (sGKY25 and 2sGKY25) were made, the latter which had 2 stapled regions, one variant as in sGKY25 in the C-terminal region, but with an extra staple in the N-terminal region.

[0643] The secondary structure of sHVF18 and its variants was evaluated by circular dichroism (CD). In Figure 2 it is possible to observe that K and R variants of sHVF18 have even a more defined a-helical structure, with two minimum one at 208 nm and one at 222 nm. The hemolytic activity of these peptides were compared on red blood cells (RBCs) and whole blood (Figure 3A and B, respectively). It was found that all peptides were hemolytic on RBCs. The difference in amount of K and R residues was not making any difference in this regard (Figure 3A). When the same analysis was performed in whole blood, it was observed that the hemolytic activity of K and R variants was significantly increased when the number of positive residues was 3 or 4 as compared to 1 or 2 (Figure 3B). When analyzing the hemolytic activity of 2sGKY25 in whole blood, it was noted that the hemolytic activity was slightly lower than for sGKY25, but definitely higher than for sHVF18 (Figure 3C - see dashed line). The dotted line in figure 12 indicates 10% hemolytic activity. In general it is preferred that peptides have a hemolytic activity below 10%.

[0644] Evaluation of anti-inflammatory activity of stapled peptides in vitro and in vivo The ability of stapled peptides to counteract the LPS-induced inflammation was first evaluated in THP-1-XBIue-CD14 reporter cells. The data are reported in Figure 4A for variants of HVF18 with K and in Figure 4B for variants with R. All peptides showed improved activity than the original sHVF18 when tested at 10 pM as the final concentration. The variants with 3 and 4 K or R showed even better activity already at 2 and 5 pM, respectively, with respect to 10 pM sHVF18. Then we evaluated the antiinflammatory activity in a more physiological environment, i.e. 25% of whole blood. The blood was stimulated with LPS in the presence or the absence of different peptides, and then the release of TNF-a and I L-1 p in the plasma was quantified by ELISA (Figure 5A and B). Surprisingly, here the activity of the peptides with 3 and 4 K or R was lower than for the peptides with 1 or 2 positively charged residues. Particularly the peptides with 2 K or R showed improved effects. In particular, it was found that these peptides significantly reduced TNF-a release, even at very low concentrations of 1-2 pM, and at 5-10 pM peptide, TNF-a release was almost completely abolished. It was also found that these peptides significantly reduced I L-1 p release at the 2-5 pM. We also tested the activity of double stapled GKY25 with respect to its linear and single stapled version in blood stimulated with LPS (Figure 5C). It was found that closing GKY25 also on N-terminal region was conferring better suppression of LPS-induced TNF-a and I L-1 p release than GKY25, but higher doses were needed when compared with variants of sHVF18. Evaluation of antibacterial activity of stapled peptides in vitro

[0645] The activity of antimicrobial peptides is strongly linked to their specific sequence characteristics. The effect of K and R additions in the stapled sHVF18 variants on their activity against E. coli, P. aeruginosa 01 and S. aureus was therefore determined. The zones of clearance was measured in the absence of NaCI (Figure 6A) and in the presence of 150 mM NaCI (Figure 6B). It was found that in general the peptides with lower positive net charge were more active in the media without salt, and vice versa. Moreover, the ability to clear bacteria on the plate without NaCI of sHVF18 K and R variants in general was better at lower concentrations if compared to the original sHVF18. On the plates with NaCI, which is more comparable to an in vivo situation, particularly the variants with increased number of K showed a significantly improved antimicrobial activity. The bactericidal activity of the peptides in solution, again in the absence (Figure 7A) and in the presence (Figure 7B) of NaCI was investigated. Here it was seen that the sHVF18 with K and R variants were more active on all bacterial strains when compared to original sHVF18, in particular in the presence of salt. In addition, the activity was higher with increasing net charge of the peptides.

[0646] The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of SK-KVF18 on clinically derived isolates of S. aureus and P. aeruginosa as well as reference strains is shown in Table 1. Interestingly, the concentration of SK-KVF18 needed to inhibit the bacterial growth often is enough to kill the bacteria (MIC=MBC), confirming the bactericidal effect of the peptide.

[0647] Table 1. MIC values for SK-KVF18 against clinical isolates and reference strains.

[0648] Bacteria MIC (pM) MBC (pM)

[0649] ATCC 27853 80 80

[0650] PAO1 80 80

[0651] Clinical isolate 10.5 40 80

[0652] P. aeruginosa Clinical isolate 13.2 80 80

[0653] Clinical isolate 23.1 40 80

[0654] Clinical isolate 27.1 40 80

[0655] Clinical isolate 51.1 80 80 Clinical isolate 62.1 40 40

[0656] Clinical isolate 15159 80 80

[0657] Clinical isolate 18488 40 40

[0658] ATCC 29213 40 40

[0659] Clinical isolate 18800 20 40

[0660] Clinical isolate 16065 40 40

[0661] Clinical isolate 18319 40 40

[0662] FDA 486 40 40

[0663] Clinical isolate 1088 20 40

[0664] Clinical isolate 1090 20 20

[0665] S. aureus

[0666] Clinical isolate 13430 40 40

[0667] Clinical isolate 14312 20 40

[0668] Clinical isolate 1779 20 20

[0669] Clinical isolate 2278 10 20

[0670] Clinical isolate 2279 20 20

[0671] Clinical isolate 1781 20 40

[0672] Clinical isolate 1086 20 40

[0673] E. coli ATCC 25922 40 40

[0674] Conclusions

[0675] Overall results for different linear and stapled peptides investigated in Example 1 and Example 2 are summarized in the Table 2 and in Figure 8D. It is demonstrated that the anti-inflammatory activity of the stapled peptides is significantly increased in a complex environment such as human blood. In particular, the variant of sHVF18 with 2 R or K showed superior results. On the other hand, when replacing His with 3 and 4 R or K, the anti-inflammatory effect was lower than for the variants with 1 and 2 R or K, possibly due to the higher toxicity of these peptides, as demonstrated by the hemolysis results. These results indicate that there is an optimal amount of positively charged residues that can be inserted in the peptide structure, which in the present case corresponds to two residues of K or R. Both these variants showed a higher therapeutic index, i.e. a larger difference between the therapeutic concentration and the dose causing toxicity. Regarding the retention of antimicrobial activity, it was observed that replacement of His with one or more Lys or Arg residues enhances the bactericidal effect of these variants if compared with the original sHVF18, particularly in the presence of salt.

[0676] Table 2: The comparative hemolytic, anti-inflammatory and antimicrobial activities of linear and stapled peptides.

[0677]

[0678] Example 3 - Activity of truncated stapled peptides

[0679] Materials and Methods

[0680] Peptides

[0681] The peptide SHVF18 (HVFRLKKWIXKVIXQFGE)(SEQ ID NO: 3), its shorter versions (denoted as SVFR17, SFRL16, SRLK15, SLKK14, SKKW13), variants of SKKW13 (denoted as SKKK14, SKKK15, SRKK14, SRRK15), double stapled GKY25 (cyclo(GKYGFY)THVFRLKKWIXKVIXQFGE) denoted as 2sGKY25, were synthesised by AmbioPharm, Inc. (USA). Briefly, standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPSS) was used. To obtain hydrocarbon stapled peptides, olefin-bearing (S)-2-(4’pentenyl)-alanine was inserted at specified locations in the respective peptide sequences (denoted by X) in SEQ ID NOs: 3 and 13. Olefin metathesis reaction was performed on solid support using Grubbs’ first-generation catalyst in 1,2-dichloroethane. The product peptides were cleaved from the resin and further purified by RP-HPLC. Peptides were provided as acetate salts, and the purity was confirmed with MALDI-TOF MS (>95%).

[0682] Hemolysis assay

[0683] The hemolytic effect of stapled peptides on erythrocytes and / or whole blood was tested as reported above. Data shown are mean values ± SEM obtained from at least four independent experiments all made in triplicate. For each experiment blood from a different donor was used.

[0684] Whole blood assay

[0685] The anti-inflammatory effect of different stapled peptides in blood was performed as reported above. For each experiment blood from a different donor was used.

[0686] Cytokine assay

[0687] Plasma obtained from blood experiments was used to evaluate cytokine release. Human inflammation DuoSet® ELISA Kit (R&D Systems) specific for TNF-a and I L-1 p was used according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 450 nm. Data shown are mean values ± SEM obtained from four independent experiments. Results

[0688] Analysis of the hemolytic and anti-inflammatory activity of shorter variants of sHVF18 As shown in Example 1 stapling of HVF18 increased its stability to proteolysis and greatly improved its anti-inflammatory activity in vitro and in vivo), retaining its antimicrobial activity, but turning it mostly against Gram-positive bacteria, while at the same time having a low hemolytic activity. Shorter variants of sHVF18 were made, to evaluate how small can be the active peptide. Double stapled GKY25 (2sGKY25), i.e. one in the C-terminal region as for sGKY25, and one in the N-terminal region, was used for comparison.

[0689] The library of different stapled peptides was generated and then screened for their hemolytic effect (Figure 8A) versus anti-inflammatory activity (Figure 8B). Reporting the percentage of hemolytic activity of all peptides on RBCs in function of the concentration of the same peptide needed to have IC50 of TNF-a and IL-1 p in LPS- stimulated blood, emerged that in particular sHVF18 was promising (Figure 8C).

[0690] Analysis of the hemolytic and anti-inflammatory activity of sKKW13 variants Both sLKK14 and SKKW13 showed low hemolitic activity (Figure 8A), but SLKK14 was was prone to oligomerize once in solution and concomitantly losing its immunomodulatory effect, while sKKW13 was not (Figure 8B). Therefore, we hypothesized that adding one or two K and R residues to sKKW13 could have improved its anti-inflammatory activity. We first compared the hemolytic activity of these new peptides to the original one and to the sHVF18, on whole blood (Figure 9A). The K and R variants showed much higher hemolytic activity. Then we tested their ability to reduce cytokines production in LPS-stimulated blood (Figure 9B). All the K and R variants were less active than sHVF18 and no big improvement was obtained if compared to sKKW13.

[0691] Example 4 - Wound model

[0692] Summary

[0693] This study describes an acute wound model in mice, where punch biopsies are used to generate acute wounds infected with S. aureus. Treatment with SK-KVF18 formulated in a hydrogel twice daily was evaluated for wound closure and bacterial burden. The results provide insight into the efficacy of SK-KVF18 formulated in a hydrogel (TP-03) as a potential treatment for infected wounds.

[0694] Materials and methods

[0695] Animal model

[0696] Female BALB / c mice (7-9 weeks old) were obtained from the Janvier labs. Mice were housed in a temperature-controlled room (22±2°C) with a 12-h light / dark cycle and had free access to food and water. The experimental protocol was approved by the Animal Ethics Committee of Malmb / Lund, Sweden.

[0697] Acute wound model

[0698] The acute wound model was generated by punch biopsy. Briefly, mice were anesthetized with isoflurane (4 % in oxygen), and the dorsal skin was shaved and disinfected with 70% ethanol. A 4 mm biopsy punch was used to create full-thickness wounds on the mouse dorsum. The wounds were infected with 5x105colony-forming units (CFUs) of luminescent S. aureus SAP229 in 5 pL solution.

[0699] Treatment

[0700] After 30 minutes of bacterial inoculation, treatment was applied with the peptide sK- KVF18 formulated in a hydrogel twice daily. The hydrogel was prepared by mixing sK- KVF18 (6.7 mg / ml / 2.8 mM) with HEC (1.5%), glycerol (2%) in 10 mM Tris pH 7. The hydrogel was applied topically to the wound bed in a volume of 50 pL.

[0701] Dressing

[0702] The wounds were covered with a polyurethane foam dressing (Mepilex transfer, Mdlnlycke Health Care, Gothenburg, Sweden). A second film dressing (Tegaderm, 3M) was applied and secured with surgical tape. The dressings were changed every 24 h.

[0703] Evaluation

[0704] The wounds were evaluated daily for 5 days. Wound closure was assessed by measuring the wound area. Bacterial burden was determined by taking swabs from the wound surface and the dressing, and plating TH agar plates. Bacterial luminescent was imaged by luminescence MS in vivo imaging system (Perkin Elmer). The plates were incubated at 37°C overnight and bacterial colonies were counted. Photos of the wounds were taken daily using a digital camera. Data analysis

[0705] Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (version 8.0, GraphPad Software, San Diego, CA, USA) with a student t-test or one-way analysis of variance (ANOVA). Differences were considered significant at p<0.05.

[0706] Results

[0707] As shown in Figure 10 gel containing SK-KVF18 improves overall wound healing in mice infected with luminescent strain of S. aureus. The mice treated with SK-KVF18 gel showed a significant reduction in wound area (Fig. 10B). Additionally, the amount of bacteria was reduced, as demonstrated by measuring bacterial luminescence using an I VIS in vivo imaging system (Fig. 10C) and by performing a bacterial count assay after collecting bacteria from the wound surface with swabs and dressings (Fig. 10D).

[0708] Example 5 - Analysis of staple positions

[0709] Materials and Methods

[0710] In silico analysis of peptide staple positions

[0711] The NMR structure of HVF18 (PDB: 5Z5X) (Saravanan et al., 2018) was docked to the modelled structure of human CD14 using the ClusPro webserver (Kozakov et al., 2017). Similar results were obtained as described in Saravanan et al., 2018, whereby the peptide binds to the N-terminus of CD14. The structure of the top scoring docking pose was selected as a template to model GKY25. The N-terminal GKYGFYT residues were modelled using Modeller version 9.21 and the model with the lowest discrete optimised protein energy score (Shen et al., 2006) was chosen. The GKY25-CD14 complex was solvated with TIP3P water and NaCI salts using the CHARMM-GUI Solution Builder as described in (Jo et al., 2008). A steepest descent energy minimisation and a 125 ps equilibration simulation, whereby positional restraints were applied to the backbone atoms of the protein and peptide, was performed following the standard CHARMM-GUI protocols (Lee et al., 2016). The final snapshot after equilibration was extracted and the binding energy between GKY25 and CD14 was calculated using the Molecular mechanics Poisson-Boltzmann surface area (MMPBSA) method (Kumari et al., 2014). A hydrophobic staple was added to the GKY25 peptide by mutating residues at positions i and i+3 to alanine and linking them with two pentene segments using the CHARMM-GUI Solution Builder (Jo et al., 2008). Stapled GKY25 bound to CD14 were then subjected to the same solvation, minimization and equilibration procedures described above, after which their binding energies were determined using MMPBSA. A similar protocol was performed for the addition of a staple at positions i and i+4. The difference in binding energies to the nonstapled GKY25 was then calculated. A similar analysis of peptide staple positions was also performed on the shorter HVF18 peptide.

[0712] Results

[0713] The design of dual-action peptides with improved proteolytic stability.

[0714] We then performed an in silico analysis of peptide staple positions, whereby a short hydrophobic pentenyl-alanine staple linking either residues i and i+3 or residues i and i+4 was added along the sequence of GKY25. To determine the effect of adding this staple on binding to CD14, we then calculated the difference in binding energies between non-stapled and stapled version of the peptide and the results are shown in Figure 11. We found that the addition of staple at most positions in the longer GKY25 peptide, particularly on the N-terminal region of the peptide, resulted in poorer binding as demonstrated by the positive binding energy differences (see results in Figure 11A and B). The reduced affinities could be caused by the staple perturbing interactions between the peptide and CD14. Some staples resulted in improved binding to CD14. For the i-i+3 configuration, these include 116-V19, V19-Q22, I20-D23, and D21-G24, whereas for the i-i+4 configuration, these include V9-K13 and Q17-D21. All the staples that resulted in a more favourable binding to CD14 comprises hydrophobic residues, which could be important for interaction with LPS, except the Q17-D21 staple.

[0715] In contrast, for the shorter HVF18 peptide, improved affinity was observed for most staples (see Figure 11C and D). For the i-i+3 configuration at least the following staples resulted in improved affinity: V2-L5, F3-K6, L5-W8, K6-I9, I9-V12, Q10-I13, K11-D14, V12-Q15, I13-F16, D14-G17 and Q15-E18. For the i-i+4 configuration, at least the following staples resulted in improved affinity: V2-K6, K6-Q10, W8-V12, I9- 113, Q10-D14, V12-F16, I13-G17 and D14-E18. For the staples that resulted in improved binding, the Q10-D14 (equivalent to Q17-D21 in GKY25) has one of the most negative binding energy differences. Example 6 - Antifungal activity

[0716] Materials and methods

[0717] Culture of yeast

[0718] Candida albicans (900228), C. parapsilosis (90018) and C. krusei (6258), were purchased from the American Type Culture Collection (ATCC), C. lipolytica was kindly provided by Andreas Sonesson (Lund University), All yeast species were maintained by subculturing every 3 weeks on Sabouraud dextrose agar (SDA; Sigma-Aldrich, St. Louis, MO) plates. Before each experiment, one colony of yeast was inoculated in 5 ml of yeast extract-peptone-dextrose (YPD) medium (Sigma-Aldrich) and allowed to grow overnight at 29°C. The day after, the optical density at 620 nm (OD620) was measured using a spectrophotometer (Genesys 20; Thermo Scientific, Rochester, NY). When the culture had reached the midlogarithmic phase, i.e. , ;0.6 OD, the medium was removed by centrifuging the culture at 5,600 xg for 10 min. The pellet was resuspended in 5 ml of 10 mM Tris at pH 7.4 and centrifuged once more. After washing, the pellet was resuspended in 10 mM Tris at pH 7.4 (0.6 OD / ml) to obtain a 1% solution of yeast, which corresponds to approximately 1 % 109CFU / ml.

[0719] Viable count assay

[0720] A 1% yeast solution of C. albicans and C. parapsilosis was prepared as reported above. Thereafter, the yeast suspension was diluted 1 :5 in 10 mM Tris at pH 7.4. Different concentrations of HVF18, sHVF18, and its K and R variants (1 to 20 pM) were added to the yeast suspension (55 pl total volume) and incubated for 2 h at 37°C. Yeasts with only Tris buffer were used as a control. At the end of the incubation time, serial dilutions (1 :10) of all samples were performed in a 96-well plate. Then, 10 pl of each dilution was plated onto SDA plates. The plates were incubated at 30°C for 48 h. The colonies from each dilution were counted, and the CFU / ml was estimated.

[0721] Radial diffusion assay

[0722] As previously described, a 1% solution of different yeasts was prepared and further diluted (1 :10 000) in 10 mM Tris at pH 7.4 to obtain a 1 x 105CFU / ml working suspension. A 10-pl aliquot of this solution was mixed with 5 ml underlay gel (10 g / liter agarose (Sigma-Aldrich), 300 mg / liter tryptic soy broth (Sigma-Aldrich), and 0.02% Tween (Sigma-Aldrich) in 10 mM Tris at pH 7.4) at 42°C. The gel was quickly poured onto a petri dish (100 mm x 15 mm) and, after solidification, wells of 4 mm in diameter were punched in patterns of 5 x 3. Peptides was dissolved in 10 mM Tris pH 7.4 with concentrations of 2, 5, 10, and 20 pM. Aliquots (6 pl) of each sample were then pipetted into the wells. Tris buffer alone was used as a negative control. The petri dish was then incubated at 37°C for 3 h. After incubation, 5 ml of overlay gel (10 g / liter agarose and 60 g / liter tryptic soy broth) at 47°C was poured onto the plate. The plate was then incubated at 30°C for 24 h. After incubation, the diameter of the developed zones was measured, excluding the punch diameter (4 mm).

[0723] Results

[0724] Antifungal activity of stapled peptides

[0725] To determine the activity of various peptides on C. albicans and C. parapsilosis in solution, a viable count assay (VGA) was conducted. All stapled peptides exhibited improved fungicidal activity compared to linear HVF18 (Table 3). Similarly to the findings for bacteria (Fig. 7A-B), peptides with a higher positive net charge showed stronger anti-fungal activity.

[0726] We confirmed the fungicidal activity of all stapled peptides on solid media using a radial diffusion assay (RDA). Furthermore, we tested their activity on two additional Candida species and observed a dose-dependent increase in the zone of clearance in all cases (Fig. 12). The results from both methods demonstrate the antifungal potential of the stapled peptides on the selected Candida species.

[0727] Table 3. Data from viable count assay. Example 7 - Additional antimicrobial activity of stapled peptide

[0728] Material and methods

[0729] The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC)

[0730] The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) was determined as described in Example 1.

[0731] Viable Count Assay (VCA)

[0732] VCA was determined as described in Example 1.

[0733] Results

[0734] Evaluation of antibacterial activity of stapled peptides in vitro sHVF18 shows antimicrobial activity also against two additional strains of gram-positive bacteria (Figure 13), i.e. E. faecalis and B. subtilis.

[0735] The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of sHVF18 on clinically derived isolates of S. aureus is shown in the table below. Stapled peptide is showing better bactericidal activity compared to the linear HVF18 across all tested strains.

[0736] Example 8 - Efficacy of sHVF18 in a mouse model of full thickness wound

[0737] Hair from mouse (8-9 weeks old) dorsum were trimmed and skin was cleaned with sterile gauze. Under anaesthesia, two punch biopsy full-thickness wounds were made on the left and right side. The wounds were infected with a bioluminescent S. aureus (SAP229). One of the wounds was treated topically with sHVF18 gel (8.6 mg / mL), and the other was treated with control gel. A primary dressing was applied on the wound which was followed by a secondary dressing. Swab samples and dressings were collected at dressing change and were analyzed for CFU and cytokines. After 5 days of treatment (DO, 1 , 2, 3, 6), mice were sacrificed on day 8 and skin biopsies were also collected.

[0738] Material and methods

[0739] Animal model

[0740] Female BALB / c mice (8-9 weeks old) were obtained from the Janvier labs. Mice were housed in a temperature-controlled room (22±2°C) with a 12-h light / dark cycle and had free access to food and water. The experimental protocol was approved by the Animal Ethics Committee of Malmb / Lund, Sweden.

[0741] Punch biopsy

[0742] Procedures were performed under isoflurane anaesthesia (4% induction, 2% maintenance). Using a trimmer, hair from the mouse dorsum were trimmed and skin was cleaned with sterile gauze. Using 4 mm biopsy punch, two full-thickness wounds were made on the left and right side (4 mm diameter).

[0743] Treatment

[0744] After 30 minutes of bacterial inoculation, treatment was applied with the peptide sHVF18 formulated in a hydrogel twice daily. The hydrogel was prepared by mixing sHVF18 (6.4 mg / ml) with HEC (1.2%), glycerol (2%) in 10 mM Tris pH 6. The hydrogel was applied topically to the wound bed in a volume of 50 pL. The control gel contained 1.6% HEC, 2% glycerol in 10 mM Tris pH 6.

[0745] Primary and secondary dressing

[0746] A 1.5 x 1.8 cm polyurethane primary dressing (Mepilex Transfer) was applied on each wound which was followed by a secondary dressing (Tegaderm). Finally, an adhesive dressing (Vet tape) was applied to secure dressings underneath.

[0747] CFU and cytokines analysis of swab and dressing

[0748] For CFU analysis, 500 .L of PBS was added to either swab or dressing samples and serial dilutions were plated on THA (Todd-Hewitt Agar) plates and incubated overnight at 37°C. Wound fluid extractions from dressings were analysed for cytokines using V- PLEX Cytokine Panel 1 Mouse Kit (MSD) following manufacturers protocol.

[0749] Skin biopsy

[0750] Wound area including surrounding skin was harvested using a scissors. Harvested skin was placed on absorbent paper to prevent curling, which were fixed overnight in 10% neutral buffered formalin.

[0751] Hematoxylin and eosin (H&E) staining and microscopy

[0752] After serial dehydration, the tissue was embedded in paraffin blocks, sectioned at 5 pm, and stained with hematoxylin and eosin (H&E). Samples were imaged with bright field microscopy (Axioplan2, Zeiss, Germany) under 100x and 200x magnifications.

[0753] Histology score

[0754] For mouse tissue, four random microscopic views (100x) from each H&E-stained skin tissue section were scored. The histological scoring was based on epithelization, inflammatory cells, abscesses and tissue architecture. Scoring was based on a scale of 0-5. For each wound tissue section, 4-5 areas were examined under 100x magnification.

[0755] Data analysis

[0756] Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (version 8.0, GraphPad Software, San Diego, CA, USA) with a student t-test or one-way analysis of variance (ANOVA).

[0757] Differences were considered significant at p<0.05.

[0758] Results

[0759] Topical sHVF18 gel treatment resulted in improved wound healing (Fig. 14A) and in improved histology score (Fig. 14B) as compared to wounds treated with control gel.

[0760] Wound swab and dressing CFU count showed a decrease in bacterial numbers on topical sHVF18 treatment as compared to control (Fig. 15).

[0761] Finally, topical sHVF18 treatment reduced proinflammatory cytokines in wound fluid as compared to control gel (Fig. 16). References

[0762] Jo, S., Kim, T., Iyer, V. & Im, W. CHARMM-GUI: A Web-Based Graphical User Interface for CHARMM. J. Comput. Chem. 29, 1859-1865 (2008).

[0763] Kalle, M. et al. Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis. PLoS One 7, e51313 (2012).

[0764] Kozakov, D. et al. The ClusPro web server for protein-protein docking. Nat. Protoc. 12, 255-278 (2017).

[0765] Kumari, R., Kumar, R. & Lynn, A. G-mmpbsa -A GROMACS tool for high-throughput MM-PBSA calculations. J. Chem. Inf. Model. 54, 1951-1962 (2014).

[0766] Lee, J. etal. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM / OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 12, 405-413 (2016).

[0767] Li, X., et al. Stapled Helical Peptides Bearing Different Anchoring Residues, Chem. Rev. 2020, 120, 10079-10144

[0768] Puthia, M. et al. A dual-action peptide-containing hydrogel targets wound infection and inflammation. Sci Transl Med 2 (2020).

[0769] Puthia, M., et al. A dual-action peptide-containing hydrogel targets wound infection and inflammation. Science translational medicine, 72(524) (2020).

[0770] Saravanan, R., et al. Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides. Nature communications, 9(1), 1-14 (2018).

[0771] Shen, M., Devos, D., Melo, F. & Sali, A. A composite score for predicting errors in protein structure models. Protein Sci. 15, 1653-1666 (2006).

[0772] Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008;3(2): 163-75. doi: 10.1038 / nprot.2007.521. PMID: 18274517. Sequence overview

Claims

Claims1. A composition for use in a method of treatment or prevention of a disorder or condition in an individual in need thereof, wherein said method comprises topical administration of the composition, the composition comprising a compound comprising a peptide comprising a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted Xi and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1.

2. The composition for use according to claim 1 , wherein the disorder or condition is a disease or condition of the skin or the mucosa.

3. The composition for use according to any one of the preceding claims, wherein the individual suffers from a disease or condition selected from the group consisting of a disease or condition characterized by blistering, a disease or condition characterized by excessive wound exudate, and scars.

4. The composition for use according to any of the preceding claims, wherein the individual suffers from a disease or condition selected from the group consisting of burn wounds, chronic skin ulcers, infected acute wounds, pressure ulcers and chronic wounds, such as venous ulcers, venous leg ulcers or diabetic leg ulcers.

5. The composition for use according to claim 3, wherein the disease or condition is selected from the group consisting of: epidermolysis bullosa (EB), burn-induced blisters, chilblains, cryotherapy-induced blisters,virus-induced blisters, such as chickenpox, shingles, herpes simplex, atypical enterovirus infection, hand, foot and mouth disease, eczema herpeticum, bullous impetigo and staphylococcal scalded skin syndrome, pemphigus, such as pemphigus foliaceous, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus erythematosus or pemphigus vegetans, pemphigoid, such as bullous pemphigoid, cicatricial pemphigoid, 200k pemphigoid or mucous membrane pemphigoid, linear IgA bullous dermatosis, dermatitis herpetiformis, porphyria cutanea tarda, bullous lupus erythematosus, Hailey-Hailey disease andSteven Johnson syndrome (toxic epidermal necrolysis).

6. The composition for use according to any one of the preceding claims, wherein the disease or condition is selected from the group consisting of impetigo, external otitis, fungal infection, pneumonia, eye infections and nasal infections.

7. The composition for use according to any one of the preceding claims, wherein the disease or condition is selected from the group consisting of rosacea, pityriasis rosea, psoriasis, eczema, microbial eczema, atopic dermatitis, seborrheic dermatitis and contact dermatitis.

8. The composition for use according to any one of the preceding claims, wherein the disease or condition is selected from the group consisting of candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, pityriasis, acne vulgaris, dermatophytosis.

9. The composition for use according to any one of the preceding claims, wherein the disease or condition is selected from the group consisting of folliculitis, hidradenitis suppurativa, balanitis, pearly penile papules, pyoderma gangrenosum.

10. The composition for use according to any one of the preceding claims, wherein the disease or condition is cutaneous T cell lymphoma, such as mycosis fungoides or Sezary syndrome.

11. The composition for use according to any one of the preceding claims, wherein the peptide comprises or consists of the amino acid sequence:-U-U-(Z)n-l-Q-K-V-l-D-Q-(Z)m- wherein the peptide has a total length between 10 to 23 amino acids; and each Z is individually any canonical amino acid; andII is His, Lys or Arg; and n is an integer in the range of 0 to 10; and m is an integer in the range of 0 to 5, and wherein two of the amino acids have been substituted for alkenylated amino acids, the side chains of which are linked by a covalent linkage.

12. The composition for use according to any one of the preceding claims, wherein Xi and X2 are alkenylated amino acids, such as two C-alkenylated amino acids, such as two a-substituted alkenyl amino acids and / or a,a-disubstituted alkenyl amino acids, and the covalent linkage is an olefin tether formed between said alkenyl residues.

13. The composition for use according to any one of the preceding claims, wherein the peptide comprises or consists of the sequence:V-F-R-L-K-K-W-I-X1-K-V-I-X2-Q-F-G-E whereinXi and X2 are amino acids linked by a covalent linkage.

14. The composition for use according to any one of the preceding claims, wherein the peptide comprises or consists of the sequence:U-U-V-F-R-L-K-K-W-I-XI-K-V-I-X2-Q-F-G-E whereinII is His, Arg or Lys; andXi and X2are amino acids linked by a covalent linkage.

15. The composition for use according to any one of the preceding claims, wherein the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 4 iii) SEQ ID NO: 5 iv) SEQ ID NO: 6 v) SEQ ID NO: 7 vi) SEQ ID NO: 8; vii) SEQ ID NO: 9; viii) SEQ ID NO: 10; ix) SEQ ID NO: 11 x) SEQ ID NO: 17; xi) SEQ ID NO: 18; xii) SEQ ID NO: 19; xiii) SEQ ID NO: 20; xiv) SEQ ID NO: 21 ; xv) SEQ ID NO: 22; xvi) SEQ ID NO: 23; xvii) SEQ ID NO: 24; xviii) SEQ ID NO: 25; or xix) SEQ ID NO: 26 or a sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with any of the sequences of i) to xix); wherein Xi and X2are amino acids linked by a covalent linkage, and wherein X3 and X4 are amino acids linked by a covalent linkage.

16. The composition according to any one of the preceding claims, wherein the compound is formulated as a gel, such as a hydrogel.

17. A device comprising the composition according to any one of the preceding claims.

18. The device according to claim 17, wherein the device is selected from a patch, a powder, a dry patch, a wound irrigation liquid, a plaster, a bandage, a suture, a prosthesis, an implant, a film, a dressing, a wound dressing, a catheter, a skin graft or a skin substitute.

19. A composition for use in a method of treatment or prevention of fungal infection in an individual in need thereof, the composition comprising a compound comprising a peptide comprising: a. a consecutive sequence of in the range of 10 to 23 amino acids from thrombin of SEQ ID NO: 1 containing up to 6 amino acids substitutions, wherein said peptide: i. has a total length between 10 and 40 amino acids; ii. comprises at least one internal covalent linkage between the side chains of two non-neighbouring, internal amino acids, wherein said amino acids are denoted X1 and X2; and iii. comprises at least amino acids K247, K248 and K252 of thrombin of SEQ ID NO: 1 ; or b. the amino acid sequence:X1-X2-X3-X4-X5-X6-W-X8-X9-X10, whereinX4, 6, 9 is any standard amino acid,Xi is I, L or V,X2 is any standard amino acid except C,X3is A, E, Q, R or Y,X5 is any standard amino acid except R,Xs is I or L,X10 is any standard amino acid except H, wherein said peptide has a length of from 10 to 100 amino acid residues.

20. The composition for use according to claim 19, wherein the fungal infection is an infection by Candida, such as for example an infection by Candida albicans, Candida parapsilosis, Candida lipolytica and / or Candida krusei.

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