Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6429—Thrombin (3.4.21.5)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21005—Thrombin (3.4.21.5)

Definitions

• the present invention lies within the field of peptides for treatment of inflammation and/or infection.
• the invention provides peptides with good stability, high anti-inflammatory activity and/or anti-microbial activity.
• LPS Lipopolysaccharide sensing by Toll-like receptor 4 (TLR4) is crucial in early responses to infection, and the subsequent NFkB activation causes a variety of biological effects associated with sepsis and ARDS, including release of cytokines, chemokines, and subsequent detrimental hemostatic disturbances, leading to consumption of coagulation factors and other mediators.
• spike glycoprotein the major extracellular protein of SARS-CoV-2, boosts LPS responses in vitro and in animal models, providing a molecular explanation to the ARDS seen during COVID-19 (Petruk et al., JMCB, 2020), where an uncontrolled LPS response gives rise to excessive localised inflammation, but also in severe systemic responses to infection. Therefore, although sensing of LPS is important for initial host defence responses, clearance and control of this molecule is critical in order to avoid excessive inflammation and organ damage.
• 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.
• HDPs host-defence peptides
• TCP-25 SEQ ID NO: 12
• 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., PLOS One, 2011).
• TCP-25 binds to LPS and interacts directly with monocytes and macrophages and interferes with CD14 signalling 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., Nat Comm, 2018).
• TCPs apart from their interactions with bacterial membranes and LPS, also bind to the LPS-binding groove of CD14 (Saravanan et al., Nat Comm, 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.
• TCP-25 is however degraded by endogenous proteases (Puthia et al., 2020).
• endogenous proteases for diseases such as sepsis and ARDS, which require systemic or inhalation applications, a rapidly degraded peptide would require prohibitively large doses and frequent administration.
• an improved affinity to its target receptor CD14 is desirable, and would reduce the effective concentration needed.
• an improved solubility would be an advantage from a drug delivery perspective.
• HNE human neutrophil elastase
• PE Pseudomonas elastase
• high anti-inflammatory activity as for example determined by reduced release of inflammatory cytokines, such as e.g. TNF-a and/or IL-i p or reduced NF-KB activity
• anti-microbial activity for example bactericidal activity against Gram negative and/or Gram positive bacteria
• the peptides of the invention have a low hemolytic activity in blood at a concentration where the peptides have high anti-inflammatory activity.
• Preferred peptides of the invention have all of the aforementioned properties.
• the high in vivo stability, increase stability to proteases as well as the low hemolytic activity render the peptides of the invention particularly useful for systemic administration.
• the present invention provides peptides, which have a low hemolytic activity against red blood cells (RBC) at concentrations where they have high anti-inflammatory activity.
• RBC red blood cells
• 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.
• 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.
• 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.
• 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.
• NMR nuclear magnetic resonance spectroscopy
• the invention shows that an increase of charge, and in particular an increase in charge by +2 may yield an optimum efficacy and a high therapeutic index, contrasting to peptides with longer cationic stretches which may be highly toxic and have reduced anti-inflammatory activity.
• Stapling of peptides may improve their proteolytic stability, however, surprisingly, stapling of certain thrombin derived peptide also led to undesired effects.
• 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.
• 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.
• 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.
• the invention further shows that peptides comprising additional positively charged amino acids have even better anti-inflammatory effect.
• 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.
• the invention provides peptides 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 acid substitutions, wherein said peptides: i) have a total length between 10 and 40 amino acids; 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 ; 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.
• Right panels cytokines released from human blood stimulated with 100 ng ml -1 E. coli LPS in the presence or the absence of increasing concentrations of HVF18 and sHVF18, 24 h post stimulation. Results are presented as mean ⁇ SEM.
• Fig. 6 shows the anti-inflammatory activity of linear and stapled HVF18.
• a NF-KB activation and cell viability in THP1-XBIue-CD14 reporter cells stimulated with 100 ng ml -1 of E. coli LPS (LPSEC), 1 gg ml’ 1 S. aureus LTA (LTA Sa ), 1 gg ml’ 1 E. coli PGN (PGNEB), 1 j g ml -1 S. aureus PGN (PGNsa), 10 ig ml -1 S. cerevisiae zymosan (Zymsc) in the presence or the absence of 10 iM of linear and stapled HVF18 20 h post stimulation.
• Fig. 8 shows the effects of stapled peptide on endotoxin responses in experimental mouse models, a, representative in vivo inflammation imaging by IVIS in NF-KB reporter mice.
• HVF18 or sHVF18 were mixed with LPS immediately before subcutaneous injection on the back of transgenic BALB/c Tg(NF-KB-RE-luc)-Xen reporter mice.
• P values were determined using a Mann-Whitney II test, b, cytokine release from plasma collected after 8 and 20 hours from C57BL/6 mice stimulated with sublethal dose of LPS administrated intraperitoneally (i.p.) and then treated with sHVF18 i.p. Data are presented as the means ⁇ SEM (each circle represent a single mouse). P values were determined using ordinary one-way ANOVA following Dunnett's multiple comparisons tests.
• Fig. 9 shows the effects of linear and stapled peptide on endotoxin responses in experimental mouse models.
• P values were determined using a Mann-Whitney II test, b, cytokines release from plasma collected after 20 hours from C57BL/6 mice stimulated with sublethal dose of LPS given i.p. and then treated with increasing doses of sHVF18 i.p. Data are presented as the means ⁇ SEM (each circle represent a single mouse). P values were determined using ordinary one-way ANOVA following Dunnett's multiple comparisons tests.
• Fig. 10 shows the effects of stapling on antimicrobial activity of HVF18.
• c The killing effect of sHVF18 on S.
• Figure 11 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.
• Figure 12 shows evaluation of hemolytic property of different stapled peptides in vitro.
• 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).
• the hemolytic activity of GKY25, sGKY25 and 2sGKY25 on whole blood are reported.
• Data are the means ⁇ SD of two independent experiments.
• dashed line represents the hemolytic activity of 100 .M sHVF18, whereas the dotted line corresponds to 10 % of lysis.
• Figure 13 shows evaluation of anti-inflammatory activity of sHVF18 and its K and R variants THP-1-XBIue-CD14 reporter cells.
• Figure 14 shows evaluation of anti-inflammatory activity of stapled peptides in human blood.
• Figure 15 shows evaluation of anti-inflammatory activity of sHVF18 and its K and R variants in mouse endotoxin model.
• C57BL/6 mice were stimulated with sublethal dose of LPS and after 30 min treated with 10 jig of SHVF18, SKVF18, SKKVF18, SRVF18 or SRRVF18.
• FIG 16 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.
• Figure 17 shows evaluation of antibacterial activity of sHVF18 K and R variants in solution.
• the bactericidal effect of sH VF 18 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).
• Figure 18 shows the effect of different peptides on coagulation.
• Figure 19 shows the hydrodynamic radii (Rh) of different peptides as a measure of oligomerisation.
• Peptides were resuspended in 10 mM Tris at pH 7.4 or in 10 mM NaOAc at pH 5.0 at 1 mM as the final concentration.
• 30 pL of each sample were used to measure the hydrodynamic radii (in nm) of particles in solution.
• P-values were determined using a one-way ANOVA with T ukey’s multiple comparisons test. **P ⁇ 0.01 , ****p ⁇ 0.0001.
• Figure 20 shows a selection of stapled peptide with improved anti-inflammatory activity
• 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.
• IC50 IC50 >10 hemolytic activity at 20 pM
• 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.
• Figure 21 shows the hemolytic and anti-inflammatory activity of K and R variants of sKKW13.
• Figure 22 shows intravenous administration of sHVF18 ameliorated septic like conditions and hindered development of severe states of ARDS in pigs.
• the figure shows results from pigs with acute lung injury and ARDS treated with and without sHVF18.
• Figure 23 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 CD 14.
• the graph shows the binding energy difference between non-stapled and stapled GKY25 for all staple positions.
• amino acid refers to any amino acid, such as any canonical and non-canonical amino acid.
• canonical amino acid refers to a proteinogenic amino acid.
• the proteinogenic amino acid is one of the 20 amino acids encoded by the standard genetic code.
• IIIPAC one and three letter codes are used to name amino acids.
• 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.
• hydrocarbon staple refers to an alkyl or alkenyl moiety linking to amino acid side chains.
• the “hydrocarbon staple” is a Ce- alkenyl moiety comprising one or more double bonds.
• 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.
• non-neighbouring refers to that two amino acids are not positioned next to each other in the primary sequence of the peptide.
• position n refers to position in the primary sequence, wherein the most N-terminal amino acids has position n.
• stapled peptide refers to a peptide comprising at least one covalent linkage between the side chains of two non-neighbouring, internal amino acids.
• a stapled peptide may comprise a hydrocarbon staple.
• the present invention provides peptides 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 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 ; 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
• the present invention further provides peptides comprising 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 ; 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
• the invention further provides peptides comprising or even consisting 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.
• the invention further provides peptides comprising or even consisting 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.
• the peptides of the invention comprise at least amino acids
• the internal covalent linkage may be as described herein below in the section “Internal covalent linkage”.
• the peptide has one or more of the advantageous properties described in the “Summary” herein above or the section “Peptide Function” below.
• the peptides are useful for treatment of inflammation and/or infection, e.g. as described in the section “Method of treatment”.
• prothrombin The sequence of prothrombin is given herein as SEQ ID NO: 16.
• Prothrombin may be cleaved at Arg 271 . 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 Arg 320 , yielding active thrombin.
• the sequence of active thrombin is given herein as SEQ ID NO: 1.
• peptides of the invention contain a covalent linkage between the side chains of two non-neighbouring, internal amino acids.
• 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.
• 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.
• 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.
• 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.
• 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.
• Peptides comprising a covalent linkage between two non-neighbouring, internal amino acids are also known as “stapled” peptides.
• the peptides of the invention may comprise any kind of covalent linkage between two non-neighbouring, internal amino acids useful for peptide stapling.
• 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.
• amino acid Xi 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.
• amino acid Xi 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.
• 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.
• Xi can be positioned at any position within the peptide apart from at the very N-terminus.
• 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.
• the position of the amino acids is given in relation to the amino acid numbering of GKY25 of SEQ ID NO: 12.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• Xi and X2 are canonical amino acids before reaction to form the covalent linkage.
• 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 Met
• Xi and X2 may before reaction to form the covalent linkage be as follows:
• Xi is Lys and X2 is Asp, Glu, Cys or Lys or vice versa.
• Xi and X2 are Cys.
• 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.
• 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.
• 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.
• the covalent linkage is formed by linking two non-canonical amino acids.
• 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.
• the covalent linkage is a hydrocarbon staple.
• 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.
• Said alkenylated amino acids may be amino acids native to thrombin, which have been alkenylated.
• said alkenylated amino acids may be amino acids substituting amino acids native to thrombin.
• 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.
• the covalent linkage is an olefin tether formed between said alkenyl residues.
• 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.
• the covalent linkage is an olefin tether formed between said alkenyl residues.
• Xi and X2 are a-alkenyl olefin-terminated amino acids and/or a,a-disubstituted alkenyl olefin-terminated amino acids.
• 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.
• Xi and X2 before reaction to form the covalent linkage may be alkenylated Ser, such as O-alkenylated Ser.
• 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.
• 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- 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.
• 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.
• 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.
• Xi and X2 before reaction to form the covalent linkage may be (S)-2-(4’-pentenyl)-alanines.
• the internal hydrocarbon staple may be formed by linking two (S)-2-(4’-pentenyl)-alanines.
• the covalent bond is established through ring-closing, such as through ring closing metathesis (RCM).
• 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.
• peptides of the invention which are 24 amino acids or longer comprise at least two staples.
• 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.
• 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 X 4 .
• X3 and X 4 may be as described above in the section “Shorter peptides”. Preferably, X3 and X 4 are closer to the N-terminal as compared to Xi and X2. In particular, X3 and X 4 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.
• amino acid X3 is positioned at position n
• amino acid X 4 is positioned at position n+3, or at position, n+4, or at position n+5, wherein n is an integer.
• amino acid X3 is positioned at position n
• amino acid X 4 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.
• Xi is not positioned at the very N-terminus
• X3 may very well be 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.
• 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.
• X3 and X4 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.
• the covalent linkage of X3 and X4 may for example be an amide bond formed between a carboxylic acid moiety and an amine.
• the carboxylic acid moiety is a glutamic acid side chain.
• the amine is an amino acid side chain.
• the amine is an N-terminal amine group of a peptide backbone.
• 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.
• Phe5 of GKY25 of SEQ ID NO: 12 is substituted by Glu5.
• X3 and X4 corresponds to Gly 1 and Glu5 of SEQ ID NO: 14.
• the peptides of the invention may have one or more of the following properties.
• the invention shows that stapled, longer thrombin derived peptides, such as GKY25, containing a single staple have several less desirable properties.
• GKY25 containing one staple has high hemolytic activity and low anti-inflammatory activity in blood, which therefore precludes in vivo use.
• stapled shorter thrombin derived peptides, such as stapled HVF18 have relatively low hemolytic activity in blood and high anti-inflammatory activity in blood.
• 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.
• the peptide of the invention has an appropriate length.
• 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.
• the peptide may have a length of 18 to 20 amino acids, such as 18 or 19 amino acids.
• the length of the peptide indicates the total length of the peptide.
• the peptide preferably does not comprise more than the indicated number of amino acids.
• the peptide of the invention comprises a consecutive sequence of amino acids from thrombin of SEQ ID NO: 1 , which has an appropriate length.
• 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.
• 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.
• the peptide comprises at least amino acids R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.
• 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.
• 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.
• the peptide comprises at least amino acids R11, K13, K14 and K18 of GKY25 of SEQ ID NO: 12.
• 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.
• the consecutive sequence may contain up to 6 amino acid substitutions.
• 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.
• 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.
• 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.
• 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”.
• the peptides of the invention may comprise one or more additional amino acids.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Said additional N-terminal amino acids are preferably positively charged amino acids, for example Lys or Arg.
• 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.
• the peptide of the invention may also be longer.
• 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.
• the peptide may have a length of 24 to 28 amino acids, such as 25 or 26 amino acids.
• Peptides of the invention which are 24 amino acids or longer comprise at least two staples.
• 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.
• 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.
• Longer peptides of the invention may be as defined above in the section “Peptide properties - Shorter peptides”.
• 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.
• the longer peptide according to the invention comprises R245, K247, K248 and K252 of thrombin of SEQ ID NO: 1.
• 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”.
• the present invention shows that insertion of one or more positively charged amino acids may significantly enhance the anti-inflammatory effect of the peptides.
• 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.
• 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.
• the positively charged amino acids are inserted at the N- terminal.
• 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.
• Said positively charged amino acids may preferably be selected form the group consisting of arginine, lysine and histidine.
• the positively charged amino acids are arginine and/or lysine, even more preferably wherein the positively charged amino acids are lysine.
• 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:
• • 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
• • 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 ; 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.
• the peptide may have one of the peptide sequences described herein in this section.
• the peptide also: • 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
• • 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.
• 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:
• • 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
• Xi and X2 in the sequences of this section may for example be as described herein above in the section “Internal covalent linkage”.
• 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.
• the peptide according to the invention comprises the sequence:
• Z is any canonical amino acid; and Xi and X 2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide according to the invention comprises or consists of the amino acid sequence:
• peptide has a total length between 10 to 23 amino acids; and each Z is individually any canonical amino acid;
• n is an integer in the range of 0 to 10
• 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 peptide of the invention comprises or consists of the sequence:
• Z is any canonical amino acid
• n is an integer in the range of 0 to 10 and
• Xi and X 2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide of the invention comprises or consists of the sequence:
• Z is any canonical amino acid
• Xi and X 2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide of the invention comprises or consists of the sequence:
• Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide comprises or consists of the sequence:
• Z is any canonical amino acid
• Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide comprises or consists of the sequence:
• Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• 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 wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• 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 wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• 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 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.
• 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 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 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 wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide comprises or consists of the sequence as set forth in: v) SEQ ID NO: 3 vi) SEQ ID NO: 5 vii) SEQ ID NO: 6; or viii) SEQ ID NO: 7 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.
• the peptide comprises or consists of the sequence as set forth in: ix) SEQ ID NO: 3 x) SEQ ID NO: 5 xi) SEQ ID NO: 6; or xii) SEQ ID NO: 7 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 peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5; or wherein Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• the peptide comprises or consists of the sequence as set forth in: i) SEQ ID NO: 3 ii) SEQ ID NO: 5; or 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.
• the peptide comprises or consists of the sequence as set forth in: iii) SEQ ID NO: 3 iv) SEQ ID NO: 5; or 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 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.
• the peptide also:
• • 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
• the peptide of the invention is a longer peptide that comprises at least two staples.
• the peptide also:
• 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 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”.
• 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.
• the peptides may have one or more of the following functions.
• the peptide has high in vivo stability.
• 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.
• In vivo stability may for example may be determined by determining anti-inflammatory activity in a mouse after administration of the peptide over time. If significant antiinflammatory activity is maintained after e.g. 24 h, peptides have high in vivo stability.
• the anti-inflammatory effect of the peptide may preferably be maintained 24 h in vivo after systemic administration of the peptide.
• the in vivo stability can for example be determined as described in Example 1 herein below.
• the peptide of the invention has increased protease resistance.
• 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.
• 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.
• HNE human neutrophil elastase
• PE Pseudomonas elastase
• trypsin trypsin
• Said resistance to protease may for example be determined as described in Example 1 below.
• the peptide of the invention has a high anti-inflammatory activity. Even more preferably, the peptide of the invention has both anti-microbial activity and antiinflammatory activity.
• 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.
• the peptide according to the invention reduces the secretion of pro-inflammatory cytokines in the presence of one or more endotoxins, such as LPS.
• the peptide decreases secretion of pro-inflammatory cytokines in vivo in blood comprising one or more endotoxins, such as LPS
• 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).
• the inflammatory cytokines are TNF-a and/or IL-1 p.
• 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.
• TLR toll-like receptor
• a concentration of 10 pM of the peptide of the invention 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 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.
• 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.
• the anti-inflammatory activity may be tested by one of the methods described herein below in Examples 1 and 2.
• the peptides of the invention preferably has anti-microbial activity. Whereas the peptides may not have improved anti-microbial activity compared to other thrombin derived peptides, it is preferred that they have at least some anti-microbial activity combined with increased anti-inflammatory activity.
• the peptides of the invention may have bactericidal activity against Gram negative and/or Gram positive bacteria.
• 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.
• the peptides may have bactericidal activity mediated through the peptide being capable of killing bacteria by damaging the bacterial membrane.
• the peptide of the invention has bactericidal effect against one or more bacteria selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.
• the peptide has bactericidal effect against all of the aforementioned bacteria.
• 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.
• 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 systemic administration.
• a hemolytic activity up to 10% is acceptable.
• 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%.
• the hemolytic activity of the peptides of the invention is less than 10% at a peptide concentration with effective anti-inflammatory activity.
• 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.
• 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%.
• 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%.
• Release of TNF-a in LPS stimulated blood may preferably be determined as described in Example 1 or 2 herein below.
• 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%.
• the hemolytic activity is at the most 5%, such as at the most 4%.
• 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.
• a detergent such as Tween-20.
• the hemolytic activity is provided as the % hemolysis compared to the control.
• the hemolytic activity may in particular be determined as described herein below in Examples 1 and 2.
• the peptides of the invention preferably also have low toxicity allowing systemic administration of the peptides.
• 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).
• CD14 cluster differentiation 14
• Alpha helical secondary structure in aqueous solution is preferably determined by circular dichroism spectroscopy.
• 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.
• 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.
• Said hydrodynamic radii is preferably determined by dynamic light scattering as described herein below in Example 4.
• the peptides of the invention may also have anti-coagulant activity.
• the peptides of the invention have an ability to increase the time for blood to form clots.
• 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).
• aPTT Activity Partial Thromboplastin Clotting Time
• the peptide of the invention increases the clotting time as determined by aPPT by at least 100% at a concentration of 60 pM.
• 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.
• Said clotting time may in particular be determined by aPTT as described herein below in Example 3.
• the peptides of the invention preferably have a low haemolytic activity on purified RBC at a peptide concentration with effective anti-inflammatory activity.
• 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-
• Said haemolytic activity on purified RBCs may preferably be determined as described in Example 1 in the section Hemolysis Assay.
• the peptides of the invention are useful for treatment of various clinical conditions. Accordingly, the invention provides the peptides disclosed herein for use as a medicament.
• the peptides of the invention may be for use in a method of treatment and/or prevention of inflammation in an individual in need thereof.
• Said method usually comprise administering a therapeutically effective amount of the peptide to said individual.
• the peptides of the invention may also be for use in a method of treatment and/or prevention of infection in an individual in need thereof.
• the peptides of the invention are useful for combined treatment or prevention of inflammation and infection, for example for treatment of inflammation associated with an infection in an individual in need thereof.
• the peptides may be administered by any useful manner, however, the peptides are particularly useful for systemic administration, such as parenteral administration.
• the peptides may be for subcutaneous or intravenous administration.
• the peptides may also be administrated by pulmonary administration, e.g. by inhalation.
• Other administration routes include intratecheal and intraperitoneal administration.
• the individual to be treated may be any individual in need thereof, for example a human being.
• the infection to be treated with the peptide of the invention may be infection with a microorganism, such as infection with a microorganism selected from the group consisting of bacteria, fungi, virus and protozoa.
• a microorganism selected from the group consisting of bacteria, fungi, virus and protozoa.
• the peptides may be for use in treatment of bacterial infection, fungal infection, or a viral infection or treatment of conditions associated with such infections.
• the individual may suffer from a bacterial infection.
• the bacterial infection may be an acute or chronic bacterial infection.
• Non-limiting examples of conditions to be treated with the peptides of the invention includes acute respiratory distress syndrome (ARDS), pneumonia or sepsis.
• ARDS acute respiratory distress syndrome
• pneumonia pneumonia or sepsis.
• the peptides of the invention are for use in the treatment of an inflammatory disease.
• Said inflammatory disease may for example be selected from the group consisting of acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), severe acute respiratory syndrome (SARS), gastroenteritis, and pulmonary inflammation, e.g. pneumonitis or inflammation of the lung tissue.
• the infection to be treated with the peptides of the invention may be infection may any infectious bacteria.
• the bacteria may be Gram, negative or Gram positive bacteria.
• the bacteria may for example be of a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter, Haemophilus, Pseudomonas and Citrobacter.
• the individual to be treated has an increased level of endotoxin, such as an increased level of LPS, LTA, zymosan and/or SA-PGN.
• Said individual may have an increased level of endotoxin in one or more body fluids.
• Said body fluid may for example be selected from the group consisting of blood, serum, saliva, nasopharyngeal swab samples and bronchoalveolar lavage (BAL) samples.
• Said endotoxin may in particular be LPS.
• Said increased level of LPS may be a level of at least 50 pg/ml, such as a serum level of LPS of at least 50 pg/ml.
• the individual may also suffer from a viral infection, e.g. infection with a Spike glycoprotein containing virus (also referred to as “S protein virus” herein), for example a virus of the Coronaviridae family, such as a virus selected from the group consisting of the virus is selected from the group consisting of:
• a viral infection e.g. infection with a Spike glycoprotein containing virus (also referred to as “S protein virus” herein)
• S protein virus also referred to as “S protein virus” herein
• a virus of the Coronaviridae family such as a virus selected from the group consisting of the virus is selected from the group consisting of:
• the peptides of the invention may be administered alone or in combination with other therapeutic agents, such as antibiotic, anti-inflammatory or antiseptic agents such as anti-bacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents.
• therapeutic agents such as antibiotic, anti-inflammatory or antiseptic agents such as anti-bacterial agents, anti-fungicides, anti-viral agents, and anti-parasitic agents.
• the present invention concerns both humans and other mammal such as horses, dogs, cats, cows, pigs, camels, among others.
• the peptides of the invention are for use in both human therapy and veterinary applications.
• the objects, suitable for such a treatment may be identified by well-established hallmarks of an infection, such as fever, puls, culture of organisms, and the like.
• Infections that may be treated with the molecules include those caused by or due to microorganisms. Examples of microorganisms include bacteria (e.g.
• Gram-positive or Gram-negative fungi
• fungi e.g., yeast and molds
• parasites e.g., protozoans, nematodes, cestodes and trematodes
• viruses and prions and mixtures thereof.
• specific organisms in these classes are well known (see for example, Davis et al., Microbiology, 3. sup. rd edition, Harper & Row, 1980).
• the peptides disclosed herein are for use in treatment or prevention of a disease, condition or indication, which for example may be any of the diseases, conditions or indications described below.
• Acute systemic inflammatory disease with or without an infective component, such as systemic inflammatory response syndrome (SIRS), ARDS, sepsis, severe sepsis, urosepsis, and septic shock.
• SIRS systemic inflammatory response syndrome
• ARDS a systemic inflammatory response syndrome
• sepsis severe sepsis
• urosepsis a systemic inflammatory response syndrome
• septic shock Other invasive infective and inflammatory disease, including meningitis, arthritis, toxic shock syndrome, diverticulitis, appendicitis, pancreatitis, cholecystitis, colitis, pneumonia, urinary tract infections and peritonitis.
• Chronic inflammatory and or infective diseases including cystic fibrosis, COPD and other pulmonary diseases, gastrointestinal disease including chronic stomach ulcerations.
• Inflammatory and coagulative disorders including thrombosis or disseminated intravascular coagulation (DIC).
• DIC disseminated intravascular coagulation
• vasculitis related inflammatory disease as well as allergy, including allergic rhinitis and asthma.
• Inflammation in relation to, but not limited to, stroke, extracorporeal circulation procedures such as ECMO, cardiopulmonary bypass, or ex vivo lung perfusion process.
• Excessive contact activation and/or coagulation in relation to, but not limited to, stroke, extracorporeal circulation procedures such as ECMO, cardiopulmonary bypass, or ex vivo lung perfusion process.
• the peptides of the invention may be for use in the treatment or prevention of an acute inflammation, sepsis, acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, allergic and other types of rhinitis, vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).
• ARDS acute respiratory distress syndrome
• SIRS systemic inflammatory response syndrome
• COPD chronic obstructive pulmonary disease
• cystic fibrosis asthma, allergic and other types of rhinitis, vasculitis, thrombosis and/or disseminated intravascular coagulation (DIC).
• ARDS acute respiratory distress syndrome
• SIRS systemic inflammatory response syndrome
• COPD chronic obstructive pulmonary disease
• cystic fibrosis asthma, allergic and other types of rhinitis, vasculitis, thrombosis and/or diss
• the peptides of the invention exhibits both anti-inflammatory and anti-coagulant activity and may be used in the concomitant treatment or prevention of inflammation and coagulation.
• Such peptides may be particularly suited to the treatment and prevention of conditions where the combined inhibition of both inflammatory and coagulant processes is desirable, such as ARDS, sepsis, chronic obstructive pulmonary disorder (COPD), thrombosis, DIC and acute respiratory distress syndrome (ARDS).
• COPD chronic obstructive pulmonary disorder
• ARDS acute respiratory distress syndrome
• other diseases associated with excessive inflammation and coagulation changes may benefit from treatment by the peptides, such as cystic fibrosis, asthma, allergic and other types of rhinitis, and vasculitis.
• 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).
• peptides may be chemically synthesized, e.g. by linking multiple amino acids via amide bonds.
• 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.
• 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.
• 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 as well as in Example 1.
• 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 as well as in Example 1.
• the covalent linkage may be an amide bond between said amine and said carboxylic acid.
• 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.
• Peptides according to the invention can also be ordered from companies specialised in producing custom made peptides, for example from AmbioPharm Inc. (US). Examples
• the peptides GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) (SEQ ID NO: 12), HVF18 (HVFRLKKWIQKVIDQFGE) (SEQ ID NO: 2), and their respective stapled versions denoted as sGKY25 (GKYGFYTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 13), 2sGKY25 (cyclo[GKYGE]YTHVFRLKKWIXKVIXQFGE) (SEQ ID NO: 14) and SHVF18 (HVFRLKKWIXKVIXQFGE) (SEQ ID NO: 3) were synthesised by AmbioPharm, Inc. (USA).
• Venous blood was collected from healthy donors, after written informed consent was obtained. After collection, whole blood or its fraction, such as plasma and serum, were used immediately or were stored at -80 °C. The use of blood was approved by the Ethics Committee at Lund University, Lund, Sweden (Permit Number: DNR2015/801).
• the spectra were recorded between 190- 260 nm (scan speed: 20 nm min -1 ) as an average of 5 measurement in a 0.2 cm quartz cuvette (Hellma, GmbH & Co, Germany).
• the baseline (10 mM Tris pH 7.4 ⁇ 100 pg mL' 1 LPS) was subtracted from each spectra and the final signal was converted to mean residue ellipticity, 0 (mdeg cm 2 dmol -1 ) as reported by Morrissette et al..
• HVF18 and its stapled version (2.5 pg) was injected in reverse-phase C18-column (Phenomenex Kinetex 50x2.1 mm 2.6 pM, 100 A pore size, California, USA) by using Agilent 1260 Infinity System following the protocol reported by Petruk et al. Biomolecules (2020). Briefly, the column was equilibrated using 95% of buffer A containing 0.25% of TFA in MilliQ and 5% of Buffer B containing 0.25% of TFA in Acetonitrile. The peptide was pre-mixed with Buffer A (1 :3) 5 min before being loaded on the column.
• Peptides were digested with different proteases for different lengths of time as described below, and then injected in reverse-phase C18-column. The analysis was performed as above. Samples from two different digestions were analysed.
• Peptides were resuspended in endotoxin free water at concentration of 1 mM. Then, 20 pg of GKY25 and sGKY25 or 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.
• HNE human neutrophil elastase
• PE P. aeruginosa elastase
• PE Calbiochem®, Merk KGaA, Darmstadt, Germany
• Glutamyl- C endopeptidase EC 3.4.2.11.9
• the NMR experiment was performed on a 700 MHz Bruker Avance III HD spectrometer (Swedish NMR Centre, Gothenburg, Sweden), equipped with QCI cryo-probe and pulse field gradients. Sample was prepared by dissolving 1.6 mM sHVF18 in 50% 2,2,2- Trifluoroethanol (TFE), supplemented with 10% D2O, 200 pM DSS, 0.02 vol% NaNs at pH 4.5. 1 H spectra were acquired from the sample at 298 K.
• TFE 2,2,2- Trifluoroethanol
• THP1-XBIue-CD14 reporter cells THP1-XBIue-CD14 reporter cells
• 180,000 cells well -1 were seeded in 96 well plates in phenol red RPMI media, supplemented with 10% (v v’ 1 ) heat-inactivated FBS and 1% (v v 1 ) Antibiotic-Antimycotic solution (AA).
• 100 ng mL -1 LPS (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.
• Abs 450 nm (Sample- control) Abs 450 nm (Posivite control-control)
• 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.
• a NanoT emper Monolith NT.115 apparatus (Nano T emper T echnologies, Germany) was used to performe Microscale thermophoresis (MST).
• a Monolith NT Protein labelling kit RED - NHS (Nano Temper Technologies, Germany) was used to label 687 pL (20 pM) of recombinant hCD14 according to the manufacturer’s protocol.
• hCD14 (5 pL of 21 nM) was incubated with increasing concentrations of GKY25, HVF18 and their stapled versions (0.03-1000 pM) in 10 mM Tris at pH 7.4 with or w/o 150 mM NaCI in a ration 1 :1.
• the sample was loaded into standard glass capillaries (Monolith NT Capillaries, Nano Temper Technologies), and the MST analysis was performed (settings for the light-emitting diode and infrared laser were 80%). Results shown are mean values ⁇ SD of six measurements.
• 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 -1 of 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.
• the blood was stimulated with 100 ng mL -1 LPS and after 30 min incubation at 37 °C was treated with increasing doses of four peptides.
• 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 -1 of LPS.
• Plasma obtained from blood experiment 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 at least four independent experiments all performed in duplicate.
• TNF- a 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.
• Mouse inflammation model 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.
• Mouse inflammation model 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.
• mice The immunomodulatory effects of HVF18 and sHVF18 were studied in BALB/c tg(NF- KB-RE-Luc)-Xen reporter mice (Taconic, 10-12 weeks old).
• the peptide 50 pg mouse- 1
• E. coli LPS 25 pg mouse -1
• mice were carefully shaved and cleaned. After injection, animals were immediately transferred to individually ventilated cages and imaged 3 h later.
• E. coli 0111 :B4 LPS was resuspended in 10 mM Tris pH 7.4. Then sublethal dose (6 mg per kg of body weight) were injected intraperitoneally (i.p.) into male C57BL/6 mice (11- 12 weeks, 22+/-5 g). Thirty minutes after, 10, 20, 50, 100 or 500 pg SHVF18 (in 10 mM NaOAc pH 5) per mouse were injected i.p. into the mice. After 8 and 20 h post LPS challenge, mice were deeply anesthetized by isoflurane and the blood was collected by cardiac puncture and stored at -80 °C until further analysis.
• E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa 01 as well as of 8 clinical isolates of S. aureus and 7 of P. aeruginosa was inoculated in 5 mL of Todd-Hewitt (TH) medium overnight at 37 °C with shaking. The day after, the bacteria cell culture was diluted 1 :50 in fresh TH media and left to grow to mid-logarithmic phase. Then, the bacteria were centrifuged at 3500 g for 10 min, washed and subsequently resuspended in 10 mM Tris pH 7.4 at final concentration of 2 x 10 9 colony forming units (CFU) mL -1 .
• TH Todd-Hewitt
• RDA Radial diffusion assay
• Bacteria were grown and prepared as described above.
• the microorganisms (4 10 6 CFU) were then added to 15 mL of the 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 required concentration were 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 the experiments were performed at least 4 times.
• VGA Viable count assay
• Bacteria were grown and prepared as described above. Next, the bacterial suspension was diluted 1 :1000 in 10 mM Tris pH 7.4 at concentration 2 x 10 6 CFU mL -1 . Bacteria (50 pL) were incubated with different concentrations of GKY25, sGKY25, HVF18 and sH F 18 (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 respective buffer were used as a control. All the experiments were performed at least 4 times. Data shown are mean values ⁇ SEM.
• the permeability of the bacteria membrane was evaluated by LIVE/DEAD BacLightTM Bacterial Viability kit (Invitrogen, Molecular Probes, Carlsbad, CA, USA) as previously described 24 . Briefly, the bacterial suspension was prepared as for VGA. S. aureus and P. aeruginosa 01 suspension (50 pL) was then treated by 1 pM or 5 pM HVF18 and its stapled version in 10 mM Tris at pH 7.4, respectively. The buffer was used as negative control. After 2 h, the samples were mixed with 1 pL of the dye mixture for each mL of the bacterial suspension, as reported on the manufacturer’s protocol, and incubated in the dark at room temperature for 15 min.
• TEM Transmission electron microscopy
• 5 .L bacterial suspension from VGA were adsorbed onto carbon coated grids (Copper mesh, 400) for 60 s and stained with 7 L of 2% uranyl acetate for 30 s.
• the grids were rendered hydrophilic via glow discharge at low air pressure. Analysis was done on 10 view fields (magnification '4200) of the mounted samples on the grid (pitch 62 pm) from three independent experiments.
• 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 BBLTM 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 concentration ranging from 2.5-320 pM. The plates were then incubated at 37 °C for 24 h.
• MBC minimal bactericidal concentration
• Thrombin C-terminal peptide GKY25 has a proven dual-action that targets both bacterial infection and the associated TLR-driven inflammation.
• One approach to increase peptide stability is peptide hydrocarbon stapling which is a modification that stabilizes peptide secondary structure with a side-chain covalent hydrocarbon bridge. Stapling has been applied to other peptides, although the effect of stapling is difficult to predict.
• a hydrocarbon staple moiety was introduced into the sequence of GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) by substituting certain amino acids with (S)-2- (4’pentenyl)-alanine. The position of stapling was decided based on the information for GKY25 summarized in Table 1.
• centrally located lysine residues (K13, K14 and K18 - numbers in this section refers to the GKY25 sequence (SEQ ID NO: 12)) may be important for GKY25’s antimicrobial activity.
• protonation of H8 at pH 5.5 increases the antibacterial activity of GKY25 against Gram-negative Escherichia coli by membrane disruption.
• GKY25 binds to LPS and the LPS-binding hydrophobic pocket of CD14 and the residues responsible for LPS and CD14 interaction have been indicated in Table 1.
• sGKY25 The helicity of stapled GKY25 (herein denoted as sGKY25) in comparison to its native versions were analysed using circular dichroism (CD).
• CD circular dichroism
• sGKY25 showed an a-helical structure and the content of the helicity was comparable to GKY25 when it is bound to LPS. In case of sGKY25 with LPS, the a-helical content remained unchanged.
• SDS-PAGE SDS-PAGE
• stapling was enhancing the stability of GKY25 in the presence of HNE up to 6 h.
• the stability was also increased against trypsin. Indeed, it was still possible to detect intact sGKY25 after 6 h of digestion.
• none of the peptides was susceptible to V8 digestion, even though its cleavage site is present in the sequence.
• LC-MS/MS To understand which regions were released from the digested sGKY25 in comparison to its native form, we employed LC-MS/MS. As expected, linear peptide showed wide variety of fragments released already after 30 min of digestion. On the other hand, the stapling was conferring a partial protection to sGKY25.
• sGKY25 showed significant improvement with respect to linear peptide, but also a high toxicity at higher concentrations (Fig. 2a).
• a more physiological condition i.e. in blood, the stapled sGKY25 peptide completely failed in blocking inflammation induced by LPS (Fig. 2b).
• HVF18 The helicity of stapled HVF18 (sHVF18) was confirmed by CD and in a hydrophobic environment such as that characteristic of RP-HPLC. sHVF18 showed longer retention time (9.42 min) if compared with linear HVF18 (8.03min), since the functional binding surface was increased with the locking. Subsequently we tested the resistance to proteases cleavage by SDS-PAGE (Fig. 1 b), HPLC and LC-MS/MS. Stapling was enhancing the stability of HVF18 up to 18 h with HNE and PE. The stability was also increased against trypsin. Indeed, it was still possible to detect intact sHVF18 even after 18 h.
• 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. Notably, sHVF18 showed lower hemolytic activity if compared with sGKY25 (compare Figure 4b with Figure 4a). In particular, sHVF18 showed significantly lower hemolytic activity in whole blood compared to sGKY25.
• sHVF18 was dissolved in 50% TFE, that is known to increase the secondary structure and then TOCSY, NOESY, ROESY, 13 C-HSQC and 15 N-SOFAST- HMQC spectra were collected. Secondary structure estimations were done using the DANGLE dihedral angle and chemical shift index (CSI) module in the CCPNMR suite, which estimates that sHVF18 contains an a-helix consisting of residues 7 to 14.
• CSI chemical shift index
• the 13 C HSQC spectrum shows well-dispersed peaks.16 cross peaks corresponding to amide backbone atoms could be detected in the 15 N SOFAST-HMQC spectra as well as side chain cross peaks for 8Trp and 15Gln.
• the 15 N HMQC and 13 C HSQC spectra indicate that sHVF18 samples have a well-defined conformational state under these conditions.
• the presence of multiple HN-HN (I ,i+2) and HN-Ha (I, i+2/3) indicates the presence of a well-defined secondary structure. Assignments were done and 97% of the available 1 H resonances could be identified.
• the N- terminal and C-terminal a-helices are structurally well-defined, however there is a relatively high amount of variability in the orientation of the two a-helices with respect to one another.
• sHVF18 in TFE is compared to HVF18 in the presence of LPS, it is possible to observe a similar L-shaped tertiary structure with a backbone RMSD of 2.2 A.
• the main differences are seen for the N-terminal part of HVF18, where the a-helix seen in sHVF18 is not observed. This can, however, be due to the TFE inducing sHVF18 into a more helical structure than LPS inducing HVF18, as showed by CD analysis.
• Microscale thermophoresis was used to determine Kd.
• the Kd Of sHVF18 to CD14 was markedly decreased, even in the presence of salt (Fig. 5).
• THP1-XBIue-CD14 reporter cells were stimulated with LPS in the presence of increasing concentration of linear and stapled HVF18 and the NF-KB activation was evaluated.
• Fig. 3 clearly shows that the anti-inflammatory activity of sHVF18 was significantly improved.
• other TLR-agonists such as LTA and PGN form S. aureus, PGN from E. coli and Zymosan from S. cerevisiae (Fig. 6a).
• sHVF18 In TH P-1 cells a stronger inhibition of LPS-induced NF-KB/AP-1 activation by sHVF18 was observed compared to its linear form (Fig. 3). 25% fresh venous human blood was incubated with stapled HVF18 or linear HVF18 and simultaneously stimulated with LPS for 24 h. We found that sHVF18 efficiently and in dose-dependent manner reduced TNF-a and IL-ip secretion, particularly when the peptide was added before stimulation with LPS or together (Fig. 3 and Fig. 7a). The inhibition of sHVF18 was lower, but still significant when the blood was first stimulated with LPS for 30 min and then treated with increasing doses of the peptide (Fig. 7b).
• sHVF18 was evaluated in a mouse model of endotoxin-induced shock.
• C57BL/6 mice were injected i.p. with a sublethal dose of LPS and after 30 min treated with increasing doses of sHVF18.
• mice were sacrificed, and cytokine levels were analyzed in blood samples (Fig. 9b).
• pro-inflammatory cytokines such as TNF-a, IL-6, IFN-y and MCP-1
• SHVF18 at 500 pug completely failed to reduce the cytokine levels.
• the effect of 50 and 100 pg of sHVF18 was investigated (Fig. 8b). A lower, but still significant reduction of cytokines levels was observed for both concentrations of sH VF 18.
• sHVF18 was active in the same manner independently of the conditions, i.e., w/o or with NaCI, whereas the linear peptide showed better bacterial killing in experiments w/o salt. Indeed, a decrease in activity was observed in the presence of NaCI (Fig. 10a). Testing the antimicrobial activity in solution, it was found that sHVF18 was more bactericidal than HVF18 on all strains evaluated (Fig. 10b, left panels). Moreover, it showed marked affinity for S. aureus, killing it at concentration lower than 1 pM (Fig. 10c).
• sHVF18 The antimicrobial activity of sHVF18 was further confirmed in a standard minimum inhibitory concentration (MIC) assay (Fig. 10 d, e). As in the VGA, sHVF18 showed a stronger activity towards Gram-positive bacteria.
• MIC minimum inhibitory concentration
• 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.
• 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.
• E. coli 0111 :B4 LPS was resuspended in 10 mM Tris at pH 7.4. Then a sublethal dose (6 mg per kg of body weight) was injected intraperitoneally (i.p.) into male C57BL/6 mice (11-12 weeks, 22+/-5 g). Thirty minutes after, 10 pg of SHVF18, SKVF18, SKKVF18, sRVF18 or SRRVF18 (in 10 mM NaOAc at pH 5) per mouse were injected i.p. into the mice. For untreated mice, 100 pL of 10 mM Tris at pH 7.4 were injected before and 100 pL of 10 mM NaOAc at pH 5 after 30 min. After 20 h post LPS challenge, mice were deeply anesthetized by isoflurane and the blood was collected by cardiac puncture and stored at -80 °C until further analysis.
• 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.
• another variant of stapled GKY25 (2sGKY25) was made, 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. Since sGKY25 failed to block LPS-induced inflammation in complex environment as human blood as described in Example 1 (see Fig. 2b), the purpose with the doublestaple was to evaluate if 2sGKY25 was able to show any anti-inflammatory activity.
• Both these variants showed a higher therapeutic index, i.e. a larger difference between the therapeutic concentration and the dose causing toxicity.
• 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.
• Table 2 The comparative hemolytic, anti-inflammatory and antimicrobial activities of linear and stapled peptides. * For peptides with IC50 >10 pM, where the exact IC50 is unknown, it was chosen to show the hemolytic activity at 50 pM.
• Example 3 Anti-coagulative properties of the peptides
• Coagulometer (Amelung, Lemgo, Germany) was used to measure all clotting times. Freshly collected human citrated plasma was used for all experiment.
• aPTT activated partial thromboplastin time
• 100 pL of a kaolin-containing solution (Dapttin, Technoclone) and plasma-peptide mix were incubated for 200 s at 37 °C , then clot formation was initiated by adding 100 pL of 30 mM fresh CaCh solution.
• Prothrombin clotting time (PT, thromboplastin reagent (Trinity Biotech) was recorded by adding 100 pL clotting reagent to 100 pL pre-warmed (60 sec at 37 °C) plasma-peptide mix.
• Activation of coagulation, inhibition of fibrinolysis, and consumption of coagulation inhibitors lead to a procoagulant state resulting in fibrin deposition in the microvasculature as observed in ARDS and sepsis, diseases which may be complicated by disseminated intravascular coagulation (DIG).
• DIG disseminated intravascular coagulation
• microvascular thrombosis contributes to promotion of organ dysfunction.
• excessive contact activation leads to the release of the pro-inflammatory peptide bradykinin and a subsequent induction of inflammatory reactions, which contribute to serious complications such as hypotension and vascular leakage.
• peptides which in a biologically relevant context modulate several pathways, including inflammation and coagulation as demonstrated for sHVF18 and particularly the KK and RR variants, are of interest in developing future peptide-based treatments for patients presenting with an excessive activation of these pathways, such as seen in ARDS, sepsis and other systemic inflammatory disorders. Moreover, as activation of the contact system occurs in several non-infectious diseases, interference by the peptides may be beneficial in other conditions involving dysfunctional coagulation.
• the hydrodynamic radii of particles in solution were measured using a DynaPro Plate reader (WYATT Technology) equipped with a temperature-controlled chamber (25 °C).
• Peptides HVF18, SHVF18, SKVF18, SKKVF18, SRVF18 and SRRVF18 - sequences provided in the sequence overview below
• 10 mM Tris pH 7.4
• 10 mM NaOAc pH 5 at 1 mM as the final concentration
• HVF18 The particles of HVF18 were larger at pH 7.4 than at pH 5. While sHVF18 showed particles with significantly reduced size compared to HVF18 at both pHs. When analyzing the hydrodynamic radii of K and R variants of sHVF18, the size of particles was even smaller and completely independent of pH, suggesting that both stapling and positive charge make the peptide less prone to oligomerize. Interestingly, no significant difference was observed between K and R variants as well as the number of these positively charged amino acids in the sequence.
• 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.
• Fmoc 9-fluorenylmethyloxycarbonyl
• 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%).
• IV line peripheral intravenous line was placed in the earlobe and general anesthesia was maintained with ketamine (Ketaminol® vet, MSD Animal Health Sweden, Swiss, Sweden), midazolam (Midazolam Panpharma®, Panpharma Nordics AS, Oslo, Norway) and fentanyl (Leptanal®, Piramal Critical Care B.V., Voorschoten, Netherlands) infusions.
• ketamine Ketaminol® vet, MSD Animal Health Sweden, Sweden
• midazolam Middleazolam Panpharma®, Panpharma Nordics AS, Oslo, Norway
• fentanyl Lidamal Critical Care B.V., Voorschoten, Netherlands
• inspiration time was set to 25% with a pause time of 10%, and ventilation was adjusted to maintain carbon dioxide levels (PaCO2) between 33 - 41 mmHg.
• the tidal volume (Vt) was kept at 6 - 8 mL kg -1 .
• the equation (a) was used to determine the dynamic compliance.
• an arterial line (Secalon-TTM, Merit Medical Ireland Ltd, Galway, Ireland) was inserted in the right common carotid artery.
• a pulmonary artery catheter (Swan-Ganz CCOmbo V and Introflex, Edwards Lifesciences Services GmbH, Unterschleissheim, Germany) was placed in the right internal jugular vein.
• E. coli LPS (O111 :B4, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was used intravenously. Prior to administration LPS was diluted in saline solution (2 g kg -1 min -1 ).
• the different ARDS stages were defined based on the measured PaO2 FiO ratio, according to the Berlin definition (Force et al.): Mild ARDS for a ratio between 201 - 300 mmHg, moderate ARDS for a ratio between 101 -200 mmHg, and severe ARDS for a ratio ⁇ 100 mmHg.
• the ARDS state was considered as confirmed, when two separate arterial blood gas measurements, taken within a 15- minute interval, fell within the Berlin definition’s PaO2 FiO range.
• sHVF18 Treatment In the treated cohort, each animal received two intravenous doses of peptide solution (12 mg in 50 mL) over the course of 30 minutes, administrated using the central venous catheter in the superior vena cava.
• Arterial blood gas analysis Arterial blood gas analysis’. Arterial blood was collected every 30 minutes and analyzed with an ABL 90 FLEX blood gas analyzer (Radiometer Medical ApS, Bronshoj, Denmark). According to clinical standards, the measurements were normalized to a blood temperature of 37 °C.
• Hemodynamic measurements The animals were closely observed, hemodynamic parameters were measured and recorded before the start of ARDS-induction and every 30 minutes thereafter, using thermodilution with a Swan-Ganz catheter and an arterial line. Parameters recorded were heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO), systolic pulmonary pressure (SPP), diastolic pulmonary pressure (DPP), mean pulmonary pressure (MPP), pulmonary artery wedge pressure (PAWP), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR).
• HR heart rate
• SBP systolic blood pressure
• DBP diastolic blood pressure
• MAP mean arterial pressure
• CVP central venous pressure
• CO cardiac output
• SPP systolic pulmonary pressure
• DPP diastolic pulmonary pressure
• MPP mean pulmonary pressure
• Biopsies of the right lower lobe were taken after confirmed ARDS. Biopsies were immediately transferred in 10% neutral buffered formalin solution (Sigma Aldrich, St. Louis, Missouri, USA) and fixed at 4 °C overnight. Formalin-fixed tissues were processed with a graded ethanol series (solutions obtained from Histolab Products AB, Gothenburg, Sweden) and clearing solution (Sigma Aldrich) prior to paraffin embedding (Histolab). Sections of 4 pm were cut and transferred to SuperFrost Plus microscopy slides (Thermo Fisher Scientific, Waltham, Massachusetts, US). Sections were dried at room temperature overnight.
• Sections were stained with hematoxylin and eosin (both Merck Millipore, Darmstadt, Germany) after de- paraffinization, followed by dehydration in consecutively graded ethanol and xylene solutions (Histolab). Stained sections were finally mounted with Pertex solution (Histolab). Bright-field images were acquired with an Olympus CKX53 microscope (Olympus, Shinjuku, Tokyo, Japan).
• Example 1 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.
• sGKY25 Double stapled GKY25
• the library of different stapled peptides was generated and then screened for their hemolytic effect (Fig. 20a) versus anti-inflammatory activity (Fig. 20b). 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-ip in LPS-stimulated blood, emerged that in particular sHVF18 was promising (Fig. 20c).
• ARDS was induced injecting intravenously E. coli LPS (study outlines are presented in Fig. 22a). All pigs developed hemodynamic instability and requiring inotropic support with norepinephrine following LPS administration (inotropic support refers to the use of agents of dobutamine, and norepinephrine with the clinical purpose of maintaining hemodynamic stability). This hemodynamic instability, but also the differences between treated and not treated pigs over the time course of the experiment, is shown by PaO2/FiO2 ratio (Fig. 22b), cardiac output (Fig. 22c), urine output (Fig. 22d), norepinephrine (NA, Fig. 22e), and lactate levels (Fig. 22f).
• lung tissue samples were taken from right lower lobe and were compared to lung tissue samples from five healthy control pigs.
• Lung biopsy taken from healthy controls for histological analysis appeared normal, with no anomalies (Fig. 22g, left panel).
• All biopsies taken from both treated and not treated animals at the endpoint of the experiment showed infiltration of immune cells and signs of diffuse alveolar damage, including thickening of the alveolar capillary barrier with intra-alveolar haemorrhage, however less in the treated pigs (Fig. 22g, middle and right panels).
• blinded scoring was performed on all pigs by three independent observers.
• 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).
• 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).
• MMPBSA Molecular mechanics Poisson-Boltzmann surface area
• 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 non-stapled GKY25 was then calculated. A similar analysis of peptide staple positions was also performed on the shorter HVF18 peptide.
• the reduced affinities could be caused by the staple perturbing interactions between the peptide and CD14.
• Some staples resulted in improved binding to CD14.
• these staples include I16-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.
• Puthia, M. et al. A dual-action peptide-containing hydrogel targets wound infection and inflammation. Sci Transl Med 2 (2020).
• AMPs antimicrobial peptides
• AMR antimicrobial resistance
• CD circular dichroism
• PAMPs pathogen-associated molecular patterns
• TCP-25 Thrombin C-terminal Peptide of 25 amminoacids
• TEM Transmission electron microscopy
• TLRs Toll-like receptors.
• Xi and X2 are each initially (S)-2-(4’pentenyl)-alanine, which are reacted with each other by RCM to form an olefin tether.
• sequences of the unreacted peptides are given. The skilled person will understand that even though the unreacted sequences are provided, in general, the peptides are used in the stapled format, i.e. after the olefin tether has been formed by RCM.
• X3 and X4 are glycine and glutamic acid, respectively, which are reacted with each other to form a covalent bond in the form of a lactam bridge.
• the invention may further be defined by any one of the following items:
• 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 acid 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; 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.
• peptide according to item 1 wherein the peptide comprises a consecutive sequence in the range of 10 to 40 amino acids from thrombin of SEQ ID NO: 1.
• a peptide comprising 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.
• peptide according to any one of the preceding items, wherein 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.
• a peptide comprising or consisting 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; and
• peptide 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 peptide according to any one of the preceding items, wherein the peptide has a total length of 13 to 23 amino acids.
• a peptide comprising 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.
• the peptide according to item 10 wherein the peptide comprises at least amino acids R11, K13, K14 and K18 of thrombin of SEQ ID NO: 12.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 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.
• 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 I le 16 and I le20; or Gln17 and Asp21 ; or Val19 and Phe23; or I le20 and Gly24; orAsp21 and Glu18 of SEQ ID NO: 12.
• 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.
• 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
• Xi is Lys and X2 is Asp, Glu, Cys or Lys or vice versa.
• Xi and X2 are Cys
• 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.
• Xi and X2 are alkenylated amino acids, such as two C-alkenylated amino acids, such as two ⁇ -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.
• alkenylated amino acids are amino acids native to thrombin, which have been alkenylated, and/or alkenylated amino acids substituting amino acids native to thrombin.
• 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.
• 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.
• alkenylated amino acids are a-substituted alkenyl olefin-terminated amino acids and/or a,a- disubstituted alkenyl olefin-terminated amino acids.
• Xi and/or X2 are a,a-disubstituted S- or R-pentenylalanine (S5 or R5) and S- or R- octenylalanine (S8 or R8) alanine.
• amino acid X3 is positioned at position n
• amino acid X4 is positioned at position n+3, or at position, n+4, or at position n+5, wherein n is an integer.
• amino acid X3 is positioned at position n
• amino acid X4 is positioned at position, n+4.
• amino acid X3 is positioned at the very N-terminus of the peptide and amino acid X4 is positioned at position n+4.
• the internal covalent bond is an amide bond formed by reacting an amine and a carboxylic acid.
• peptide 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.
• peptide 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.
• peptide 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.
• peptide 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.
• peptide 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.
• peptide 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.
• peptide 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.
• peptide 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.
• substitutions are conservative substitutions, such as wherein 1, 2, 3 or 4 amino acid substitutions are conservative substitutions.
• 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.
• peptide 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.
• peptide according 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.
• peptide 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
• peptide 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 peptide 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.
• Xi and X2 are amino acids, the side chains of which are linked by a covalent linkage.
• 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.
• Xi and X2 are amino acids linked by a covalent linkage.
• Xi and X2 are amino acids linked by a covalent linkage.
• Xi and X2 are amino acids linked by a covalent linkage.
• Xi and X2 are amino acids linked by a covalent linkage.
• peptide according to any one of the previous 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 wherein Xi and X2 are amino acids linked by a covalent
• the stabilized peptide according to any one of the previous items, wherein the peptide comprises or consists of the sequence as set forth in: iv) SEQ ID NO: 3 v) SEQ ID NO: 4 vi) SEQ ID NO: 5 vii) SEQ ID NO: 8; or viii) SEQ ID NO: 9 wherein Xi and X2 are amino acids linked by a covalent linkage. .
• peptide 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; 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.
• peptide 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; or 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.
• peptide 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 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.
• peptide 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.
• peptide 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.
• peptide 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.
• 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 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 peptide has anti-coagulant activity.
• 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.
• peptide 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.
• 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).
• TNF-a tumour necrosis factor a
• IL-1 P interleukin p
• IL-6 interleukin 6
• IL-10 interleukin 10
• IFN-Y interferon
• MCP-1 monocyte chemoattractant protein-1
• TLR toll-like receptor
• LPS lipopolysaccharide
• LTA lipoteichoic acid
• SA-PGN Staphylococcus aureus peptidoglycan
• peptide 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 peptide is bactericidal, for example wherein the peptide is capable of killing bacteria by damaging the bacterial membrane.
• the peptide according to item 105 wherein said bacteria is selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. .
• ARDS acute respiratory distress syndrome
• COPD chronic obstructive pulmonary disease
• cystic fibrosis asthma, allergic and other types of rhinitis, vasculitis, thrombosis, disseminated intravascular coagulation (DIC) gastroenteritis, and pulmonary inflammation.
• ARDS acute respiratory distress syndrome
• SARS severe acute respiratory syndrome
• gastroenteritis gastroenteritis
• pulmonary inflammation .
• said increased level of LPS is a level of at least 50 pg/ml, such as a serum level of LPS of at least 50 pg/ml.
• a method of treatment and/or prevention of inflammation and/or infection in an individual in need thereof comprising administering a therapeutically effective amount of the peptide according to any one of items 1 to 107 to said individual. .
• the use according to item 127, wherein the treatment, the inflammation, the infection and/or the individual is a defined in any one of items 110 to 124.
• a pharmaceutical composition comprising the peptide according to any of items 1 to 107.

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