The present invention relates to W-Peptide analogues comprising a W-Peptide, such as the hexapeptide W-Peptide (SEQ ID NO:1 ) and functional variants thereof, and one or more branched amino acid probes.

Background

Proteins and peptides are widely employed for therapeutic purposes whether in their native forms, variant forms or analogues thereof. Protein therapeutics tend to be specific for their targets, leading to potentially fewer side effects, but often with lower bioavailability, poorer membrane permeability, and metabolic instability, as compared to small molecules. Protein-based drugs are generally referred to as 'biologies' and include molecules such as insulin, growth factors, and engineered antibodies.

Proteinaceous molecules typically require injection; nevertheless, biologies have been an extremely successful class of therapeutics including antibodies for treatment of arthritis and various cancers, soluble proteins for diabetes, myelosuppression and renal anemia; as well as short injectable peptides for multiple sclerosis, cancers,

endometriosis and fibroids and acromegaly.

Peptides represent a class of molecules that have the specificity and potency of larger protein biologies, but are smaller in size and more accessible and cheaper to manufacture using chemical methods, thus potentially combining some of the advantages of proteins with those of small molecules.

Protein and peptide compounds can be modified in various ways in order to improve one or more features of the compound, or address one or more potential draw-backs of the compound. For example, a stabilizing peptide sequence may be added to the N- and/or C-terminus of pharmacologically active peptides potentially making them less susceptible to degradation (WO 99/46283). Further, a linear amino acid probe of 6 amino acids selected from Lys or Glu added to the N-terminus of a-MSH potentially increases efficacy compared to the native peptide (WO 07/22774). Known peptide-drug conjugates further include addition of polycationic peptides CPP (cell-penetrating peptides) to improve transport across the cell lipid bi-layer. Analogues of omelanocyte- stimulating hormone (a-MSH) and γ-melanocyte stimulating hormone (γ-MSH) comprising an N-terminally branched amino acid probe is disclosed in WO

2014/060606 and EP 2722340. Summary

The present invention provides W-Peptide analogues comprising one or more branched amino acid probes (abbreviated BAP herein). The present invention also provides W-Peptide analogues comprising butyric acid residues. In some embodiments, the W-Peptide analogues provided herein have one or more improved properties compared to the corresponding W-Peptide without one or more BAPs ('native' W-peptide). For example, in some embodiments, addition of one or more branched amino acid probes to a W-Peptide potentially improves one or more features of the peptide, such as

- improve or increase an inherent effect of the W-Peptide (including for example increasing the activity, affinity and/or efficacy of a pharmacologically active peptide; improved binding to and/or activation of one or more relevant receptors);

alter an inherent effect of the W-Peptide (including for example an altered receptor binding profile), or

improve or alter an external effect of the W-Peptide (including for example increased stability, reduced degradation, altered configuration and/or altered solubility). It is an aspect of the invention to provide a W-Peptide analogue comprising a W- Peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino alkyl amino acid residue,

said first amino alkyl amino acid residue optionally being covalently linked to a second amino alkyl amino acid residue, or to a second and a third amino alkyl amino acid residue, to form a linear chain of 2 or 3 amino alkyl amino acid residues,

wherein the side chain of one or more of said first, second and/or third amino alkyl amino acid residues are each modified by attaching to the side chain amino group a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) _p -AAA _q ; AAA _q -(aa ₃ ) _P ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P ;

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala,

wherein said first amino alkyl amino acid residue is covalently linked to the N-terminus of said W-Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide,

with the proviso that said branched amino acid probe consists of 2 to 9 amino acid residues.

A W-Peptide according to the present invention is any functional W-Peptide, including W-Peptide (SEQ ID NO:1 ) and any functional variant thereof.

The present invention also encompasses pharmaceutical compositions comprising the W-Peptide analogues of the present invention, as well as the analogues of the present invention for use as a medicament. The invention is in one embodiment directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic condition, an inflammatory condition, an infection and/or a metabolic condition.

Description of Drawings

Figure 1 : Schematic representation of the branched amino acid probe Ac-(Ac-Lys-

Lys)Lys-, showing the first amino alkyl amino acid residue being a lysine residue (Lysi), covalently linked to the N-terminus of a 'peptide sequence' via a regular peptide bond, said first lysine being acetylated (COCH ₃ ), and said first lysine modified by attaching to the ε-amino group of said first lysine residue two further lysine residues wherein one is also acetylated (the outermost).

Figure 2: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #1 : Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet-NH2 and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3) . Figure 3: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #2: Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-NH2 and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3).

Figure 4: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #3: Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Abu-DMet-NH2; where Abu is 2-Aminobutyric acid and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3).

Figure 5: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #4: Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet-NH2 and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3.

Figure 6: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #5: Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-NH2 and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3). Figure 7: Binding affinity (a) and receptor efficacy (b) against the human FPR2 receptor of W-peptide analogue #6: Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Abu-DMet-NH2; and the control hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (for details see Example 2 and 3).

Detailed description

W-Peptide analogues

Human neutrophils and monocytes are highly specialised for their primary function, i.e. phagocytosis and destruction of microorganisms. Phagocytic leukocyte recruitment to sites of inflammation and infection is dependent upon the presence of a gradient of locally produced chemotactic factors. The bacterial peptide N-formyl-methionyl-leucyl- phenylalanine (fMLP) was one of the first of these to be identified and is a highly potent leukocyte chemoattractant. It interacts with receptors on the neutrophil membrane, activating these cells through a G-protein-coupled pathway.

The formyl peptide receptors (FPR) belong to a class of G protein-coupled receptors involved in chemotaxis. These receptors were originally identified by their ability to bind N-formyl peptides such as N-formylmethionine produced by the degradation of either bacterial or host cells. Hence formyl peptide receptors are involved in mediating immune cell response to infection. These receptors may also act to suppress the immune system under certain conditions.

In humans, there are three formyl peptide receptor isoforms, each encoded by a separate gene FPR1 , FPR2 and FPR3.

The synthetic leukocyte-activating hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH ₂ denoted W-Peptide (WKYMVM; SEQ ID NO: 1 ) as well as its D-conformer Trp-Lys-Tyr-Met-Val- DMet-NH ₂ (WKYMVm) are known to activate FPRs. W-Peptide variants have been disclosed, e.g. the tritiated peptide WK[3,5 ³ H ₂ )]YMVM (Christophe et al., J Biol Chem. 2001 ) and hexapeptides with the consensus-sequence XKYX(PA )M optionally comprising Proline at position 5 (wherein X is any amino acid) (Baek et al., J Biol Chem 1996).

It is an aspect of the present invention to provide a W-Peptide modified by addition of one or more branched amino acid probes. Thus in one embodiment the W-Peptide analogues are conjugates comprising a W-Peptide and one or more branched amino acid probes. The W-Peptide of the invention comprise W-Peptide (SEQ ID NO:1 ) and functional variants thereof.

In some embodiments, the W-Peptide analogues as provided herein have certain improved properties compared to the corresponding native or unconjugated W-Peptide. In one embodiment the W-Peptide analogues provided herein have increased binding affinity and/or activation of one or more relevant receptors, such as FPRs. In another embodiment, the W-Peptide analogues provided herein are more stable, such as less susceptible to proteases. Still further, in one embodiment the W-Peptide analogues have higher solubility.

It is an aspect of the invention to provide a W-Peptide analogue comprising a W- Peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino alkyl amino acid residue, said first amino alkyl amino acid residue optionally being covalently linked to a second amino alkyl amino acid residue, or to a second and a third amino alkyl amino acid residue, to form a linear chain of 2 or 3 amino alkyl amino acid residues,

wherein the side chain of one or more of said first, second and/or third amino alkyl amino acid residues are each modified by attaching to the side chain amino group a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) _p -AAA _q ; AAA _q -(aa ₃ ) _P ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P ;

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala,

wherein said first amino alkyl amino acid residue is covalently linked to the N-terminus of said W-Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide,

with the proviso that said branched amino acid probe consists of 2 to 9 amino acid residues.

In one embodiment the N-terminal amino acid residue of the molecule is acetylated at the alpha amino group.

In one embodiment said first amino alkyl amino acid residue is linked by a peptide bond (amide) formed by a reaction of the carboxylic acid, or a derivative thereof, of said first amino alkyl amino acid with the alpha amino group of the N-terminal amino acid residue of said W-Peptide; linked by a peptide bond to the C-terminal amino acid residue of said W-Peptide formed by reacting the alpha amino group of said amino alkyl amino acid residue with the carboxylic acid, or derivative thereof, of said C- terminal amino acid residue; and/or linked to an amino alkyl amino acid residue within said W-Peptide by an amide formed by a reaction of the carboxylic acid, or a derivative thereof, of said first amino alkyl amino acid residue with the alkyl amino group of the amino alkyl amino acid residue.

In one embodiment said first amino alkyl amino acid residue is covalently linked to the N-terminal Trp of said W-Peptide, covalently linked to the C-terminal Met or DMet of said W-Peptide, and/or covalently linked to the side chain amino group of a Lys or Orn residue within said W-Peptide, such as covalently linked to Lys at position 2 of said W- Peptide.

According to the invention an amino acid residue being covalently linked to further amino acid residues and/or a peptide in one embodiment means that a peptide bond is present. In another embodiment an amino acid residue being covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide means that an amide bond is present. A peptide bond (amide bond) is a covalent chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, causing the release of a molecule of H ₂ 0. The process usually occurs between amino acids. If the branched amino acid probe is to be covalently linked to the N-terminus of said W- Peptide, the N-terminal amino alkyl amino acid residue of the backbone of the branched amino acid probe is preferably acetylated.

If the branched amino acid probe is to be covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide, the N-terminal amino alkyl amino acid residue of the backbone of the branched amino acid probe is preferably acetylated.

If the branched amino acid probe is to be covalently linked to the C-terminus of said W- Peptide, the C-terminal amino alkyl amino acid residue of the backbone of the branched amino acid probe is preferably a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide; most preferably amidated.

The amino alkyl amino acid residues (or AAA) and the amino acid residues (aa ₃ ) according to the invention may each be the same (identical) or different (non-identical).

Branched amino acid probe

Amino alkyl amino acid residue

According to the present invention an 'amino alkyl amino acid residue' (or AAA) is an amino acid having the conventional amine (-NH ₂ ) and carboxylic acid (-COOH) functional groups, and a side chain covalently linked to the first (alpha-) carbon atom, wherein the side-chain comprises an amino alkyl group (-CnH2nNH ₂ ).

Thus an amino alkyl amino acid residue (or AAA) is an amino acid with a side chain comprising or consisting of an amino alkyl group (-CnH2nNH ₂ ), in one embodiment denoted a side chain amino alkyl group.

In one embodiment the side chain alkyl group is derived from the group consisting of methyl (CH ₃ -), ethyl (C ₂ H ₅ -), propyl (C ₃ H ₇ -), butyl (C ₄ H ₉ -), pentyl (C ₅ H _i ), hexyl (C ₆ H ₁₃ - ), heptyl (C ₇ H ₁₅ -), octyl (C ₈ H ₁₇ -), nonyl (C ₉ H ₁₉ -), decyl (Ci ₀ H ₂ i-), undecyl (CnH ₂₃ -) and dodecyl (Ci ₂ H ₂₅ -). When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl and t-butyl.

In one embodiment the side chain amino group (NH ₂ ) of said amino alkyl amino acid residue is the amine of methylamine, the amine of ethylamine, the amine of propylamine, the amine of n-butylamine, the amine of pentylamine, the amine of n- hexylamine, the amine of heptylamine, the amine of octylamine, the amine of nonylamine, the amine of decylamine, the amine of undecylamine or the amine of dodecylamine.

In one embodiment the side chain amino alkyl group according to the invention is selected from the group consisting of methylamine (-CH ₂ NH ₂ ), ethylamine (-C ₂ H ₄ NH ₂ ), propylamine (-C ₃ H ₆ NH ₂ ), n-butylamine (-C ₄ H ₈ NH ₂ ), pentylamine (-C ₅ H ₁ oNH ₂ ), n- hexylamine (-C ₆ H ₁₂ NH ₂ ), heptylamine (-C ₇ H ₁₄ NH ₂ ), octylamine (-C ₈ H ₁₆ NH ₂ ), nonylamine (.C ₉ H ₁₈ NH ₂ ), decylamine (-CioH ₂₀ NH ₂ ), undecylamine (-CnH ₂₂ NH ₂ ) and dodecylamine (. C"| ₂ H ₂₄ NH ₂ ). In one embodiment the side chain amino group (NH ₂ ) of said first, second and/or third amino alkyl amino acid residues are each modified by attaching a molecule thereto.

In one embodiment the side chain amino group of said amino alkyl amino acid residue is selected from the group consisting of

the β (beta) amino group (1 methylene in the side chain; methylamine); the Y (gamma) amino group (2 methylenes in the side chain, ethylamine);

the δ (delta) amino group (3 methylenes in the side chain, propylamine); = ornithine the ε (epsilon) amino group (4 methylenes in the side chain; n-butylamine); = lysine the ζ (zeta) amino group (5 methylenes in the side chain; pentylamine);

the η (eta) amino group (6 methylenes in the side chain; n-hexylamine);

the Θ (theta) amino group (7 methylenes in the side chain; heptylamine);

the I (iota) amino group (8 methylenes in the side chain; octylamine);

the K (kappa) amino group (9 methylenes in the side chain; nonylamine);

the λ (lambda) amino group (10 methylenes in the side chain; decylamine);

the μ (mu) amino group (1 1 methylenes in the side chain; undecylamine); and the v (nu) amino group (12 methylenes in the side chain; dodecylamine).

For example, the ε-amino group is covalently linked to the fifth carbon beginning from (including) the ocarbon, which ocarbon is covalently linked to the carboxyl (C=OOH) group.

An amino alkyl amino acid residue wherein the side chain is n-butylamine and the side chain amino group is the ε (epsilon) amino group is lysine (Lys, K). Likewise, the δ-amino group is covalently linked to the fourth carbon beginning from the a-carbon.

An amino alkyl amino acid residue wherein the side chain is propylamine and the side chain amino group is the δ (delta) amino group is ornithine (Orn).

Ornithine is formed in cells by deguanidation of arginine. While it is not used in proteinogenesis in vivo it is a participant in several enzyme pathways and appears to play a role in nitrogen balance in vivo as it can be gaunidated enzymatically to form arginine.

Any amino acid according to the present invention may be in the L- or D-configuration. If nothing is specified, reference to the L-isomeric form is preferably meant. It follows that the amino alkyl amino acid residues of the invention in one embodiment are individually in the L- or D- configuration. In one embodiment the amino alkyl amino acid residues are in the L- configuration. In one embodiment the amino alkyl amino acid residues comprised in the branched amino acid probe are individually selected from the group consisting of lysine and ornithine.

In one embodiment the amino alkyl amino acid residues of the invention are selected from the group consisting of lysine and D-lysine. In a particular embodiment the amino alkyl amino acid residues of the invention are lysine residues.

In one embodiment the amino alkyl amino acid residues of the invention are selected from the group consisting ornithine and D-ornithine.

In one embodiment there is provided a W-Peptide analogue comprising a W-Peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino acid residue selected from lysine and ornithine,

said first amino acid residue optionally being covalently linked to a second, or to a second and a third amino acid residue selected from lysine or ornithine, to form a linear chain of 2 or 3 lysine or ornithine residues,

wherein the side chain of one or more of said first, second and/or third lysine or ornithine residues are modified by attaching to the δ-amino group (ornithine) or the ε- amino group (lysine) a molecule independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _P -Lys _q ; Lys _q -(aa ₃ ) _P ; [(aa ₃ )-Lys] _p ; [Lys-(aa ₃ )] _P ;

Orn _q -Orn; (aa ₃ ) _p -Orn _q ; Orn _q -(aa ₃ ) _p ; [(aa ₃ )-Orn] _p and [Orn-(aa ₃ )] _p ;

Orn _p -Lys _p ; Lys _p -Orn _p ; [Orn-Lys] _p and [Lys-Orn] _p ;

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala,

wherein said first lysine or ornithine residue is covalently linked to the N-terminus of said W-Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the ε-amino group of a lysine residue or the δ-amino group of an ornithine residue within said W-Peptide,

with the proviso that said branched amino acid probe consists of 2 to 9 amino acid residues.

In one embodiment there is provided a W-Peptide analogue comprising a W-Peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first lysine residue,

said first lysine residue optionally being covalently linked to a second, or to a second and a third lysine residue, to form a linear chain of 2 or 3 lysine residues,

wherein the side chain of one or more of said first, second and/or third lysine residues are modified by attaching to the ε-amino group of said lysine a molecule independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) -Lys _q ; Lys _q -(aa ₃ ) ; [(aa ₃ )-Lys] _p ; [Lys-(aa ₃ )] _P ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala,

wherein said first lysine residue is covalently linked to the N-terminus of said W- Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the ε-amino group of a lysine or δ-amino group of an ornithine residue within said W- Peptide,

with the proviso that said branched amino acid probe consists of 2 to 9 amino acid residues.

Branching the probe

A branched amino acid probe according to the present invention in one embodiment consists of 2 to 9 amino acid residues.

In one embodiment each of said one or more branched amino acid probe consist of from 2 to 3 amino acid residues, such as from 3 to 4 amino acid residues, for example from 4 to 5 amino acid residues, such as from 5 to 6 amino acid residues, for example from 6 to 7 amino acid residues, such as from 7 to 8 amino acid residues, for example from 8 to 9 amino acid residues. In one embodiment each of said one or more branched amino acid probe consist of 2 amino acid residues, such as 3 amino acid residues, for example 4 amino acid residues, such as 5 amino acid residues, for example 6 amino acid residues, such as 7 amino acid residues, for example 8 amino acid residues, such as 9 amino acid residues. In a particular embodiment each of said one or more branched amino acid probes consist of 3 amino acid residues.

In one embodiment the branched amino acid probe comprises a first amino alkyl amino acid residue (also denoted AAA-i), which first amino alkyl amino acid residue is connected to a W-Peptide to provide a W-Peptide analogue according to the invention.

In one embodiment the first amino alkyl amino acid of (each of) the one or more branched amino acid probe(s) is covalently linked to the N-terminus of said W-Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In one embodiment the first amino alkyl amino acid residue of (each of) the one or more branched amino acid probe(s) is covalently linked to the N-terminal Trp of said W-Peptide, covalently linked to the C-terminal Met or DMet of said W-Peptide, and/or covalently linked to the side chain of a Lys or Orn residue within said W-Peptide, such as covalently linked to Lys at position 2 of W-Peptide.

In one embodiment the branched amino acid probe comprises a first amino alkyl amino acid residue. In one embodiment the side chain of said first amino alkyl amino acid residue is modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the first amino alkyl amino acid of the branched amino acid probe is acetylated at the alpha amino group. In one embodiment the N-terminus of the first amino alkyl amino acid residue of the branched amino acid probe is acetylated.

In one embodiment the N-terminus of the first amino alkyl amino acid residue of the branched amino acid probe is acetylated when the branched amino acid probe comprising said first amino alkyl amino acid residue is covalently linked to the N- terminus of the W-Peptide; or when the branched amino acid probe comprising said first amino alkyl amino acid residue is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In one embodiment the C-terminus of the first amino alkyl amino acid residue of the branched amino acid probe is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ). In a preferred embodiment the C-terminus of the first amino alkyl amino acid residue is amidated.

In one embodiment the C-terminus of the first amino alkyl amino acid residue of the branched amino acid probe is an amide when the branched amino acid probe comprising said first amino alkyl amino acid residue is covalently linked to the C- terminus of the W-Peptide.

In one embodiment said first amino alkyl amino acid residue is covalently linked to a second amino alkyl amino acid residue to form a linear chain of 2 amino alkyl amino acid residues. In one embodiment the alpha-amino group of the second amino alkyl amino acid residue of the branched amino acid probe is acetylated. In one embodiment the N-terminus of the second amino alkyl amino acid residue of the branched amino acid probe is acetylated.

In one embodiment the C-terminus of the second amino alkyl amino acid residue of the branched amino acid probe is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ). In a preferred embodiment the C-terminus of the second amino alkyl amino acid residue is amidated.

In one embodiment said first amino alkyl amino acid residue is covalently linked to a second and (covalently linked to) a third amino alkyl amino acid residue to form a linear chain of 3 amino alkyl amino acid residues. In one embodiment the alpha-amino group of the third amino alkyl amino acid residue of the branched amino acid probe is acetylated. In one embodiment the N-terminus of the third amino alkyl amino acid residue of the branched amino acid probe is acetylated. In one embodiment the C-terminal of the third amino alkyl amino acid residue of the branched amino acid probe is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ). In a preferred embodiment the C-terminus of the third amino alkyl amino acid residue is amidated.

In one embodiment the first amino alkyl amino acid residue have both the second and third amino alkyl amino acid residues attached at its amine group. In one embodiment the first amino alkyl amino acid residue have both the second and third amino alkyl amino acid residues covalently linked to its carboxylic acid group. In one embodiment the first amino alkyl amino acid residue have the second amino alkyl amino acid residue attached at its amine group and the third amino alkyl amino acid residue attached at its carboxylic acid group.

The second and third amino alkyl amino acid residues may be denoted AAA ₂ and AAA ₃ , respectively.

In one embodiment each of said first, second and/or third amino alkyl amino acid residues is an amino acid having a side chain amino alkyl group selected from the group consisting of methylamine (-CH ₂ NH ₂ ), ethylamine (-C ₂ H ₄ NH ₂ ), propylamine (. C ₃ H ₆ NH ₂ ), n-butylamine ( _-- C ₄ H ₈ NH ₂ ), pentylamine (-C ₅ H ₁ oNH ₂ ), n-hexylamine (.

C ₆ H ₁₂ NH ₂ ), heptylamine (_C ₇ H ₄ N H ₂ ), octylamine (.C ₈ H ₁₆ NH ₂ ), nonylamine (.C ₉ H ₁₈ NH ₂ ), decylamine (-CioH ₂₀ NH ₂ ), undecylamine (-Cn H ₂₂ NH ₂ ) and dodecylamine (.Ci ₂ H ₂₄ NH ₂ ).

In one embodiment each of the first, second and/or third amino alkyl amino acid residues of the branched amino acid probe are individually selected from the group consisting of lysine, D-lysine, ornithine and D-ornithine.

In one embodiment each of the first, second and third amino alkyl amino acid residues of the branched amino acid probe are lysine residues (including L-lysine and D-lysine).

In one embodiment the first, the second or the third amino alkyl amino acid residues of the branched amino acid probe are acetylated at the alpha amino group (Ac-AAA) (COCHs). In one embodiment, the first, the first and second, and the first, second and third amino alkyl amino acid residues of the branched amino acid probe are referred to as the amino alkyl amino acid backbone of the branched amino acid probe (AAA-i, AAA _1-2 , AAA _1-3 ).

In one embodiment the first, the first and second, and the first and second and third amino alkyl amino acid residues are each lysine residues. In one embodiment the first, the first and second, and the first, second and third lysine residues of the branched amino acid probe are referred to as the lysine backbone of the branched amino acid probe (Lysi, Lysi _-2 , Lysi _-3 ).

In one embodiment the first lysine residue, or the second lysine residue, or the third lysine residue of the lysine backbone of the branched amino acid probe is acetylated at the alpha-amino group (Ac-Lys).

In one embodiment the side chain of one of said first, second and/or third amino alkyl amino acid residues are modified by attaching to the side chain amino group a molecule as defined herein. In one embodiment the branched amino acid probe comprises a first amino alkyl amino acid residue, wherein the side chain of said first amino alkyl amino acid residue is modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the branched amino acid probe comprises a first and a second amino alkyl amino acid residue, wherein the side chain of said first amino alkyl amino acid residue is modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the branched amino acid probe comprises a first and a second amino alkyl amino acid residue, wherein the side chain of said second amino alkyl amino acid residue is modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the branched amino acid probe comprises a first and a second amino alkyl amino acid residue, wherein the side chains of said first and second amino alkyl amino acid residue are modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the side chain of two of said first, second and/or third amino alkyl amino acid residues are modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the side chain of all three of the first, second and third amino alkyl amino acid residues are modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the side chain of i) the first amino alkyl amino acid residue, ii) the second amino alkyl amino acid residue, iii) the third amino alkyl amino acid residue, iv) the first and the second amino alkyl amino acid residues, v) the first and the third amino alkyl amino acid residues, vi) the second and the third amino alkyl amino acid residues, or vii) the first, the second and the third amino alkyl amino acid residues, are modified by attaching to the side chain amino group a molecule as defined herein.

In one embodiment the first lysine residue, or the second lysine residue, or the third lysine residue, or the first and the second lysine residues, or the first and the third lysine residues, or the second and the third lysine residues, or the first, the second and the third lysine residues of the lysine backbone of the branched amino acid are modified by attaching a molecule to the ε-amino group. In one embodiment the side chain of one or more of each of said first, second and/or third amino alkyl amino acid residues is modified by attaching to the side chain amino group a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) -AAA _q ; AAA _q -(aa ₃ ) ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal AAA or (aa) ₃ of the molecule is acetylated at the alpha amino group. In one embodiment the side chain of one or more of each of said first, second and/or third amino alkyl amino acid residues is modified by attaching to the side chain amino group a molecule independently selected from the group consisting of

Lys _q -Lys; (aa ₃ ) _P -Lys _q ; Lys _q -(aa ₃ ) _P ; [(aa ₃ )-Lys] _p ; [Lys-(aa ₃ )] _P ;

Orn _q -Orn; (aa ₃ ) _p -Orn _q ; Orn _q -(aa ₃ ) _p ; [(aa ₃ )-Orn] _p and [Orn-(aa ₃ )] _p ;

Orn _p -Lys _p ; Lys _p -Orn _p ; [Orn-Lys] _p and [Lys-Orn] _p ;

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal Lys, Orn or (aa) ₃ of the molecule is acetylated at the alpha amino group.

In one embodiment the side chain of one or more of each of said first, second and/or third amino alkyl amino acid residues is modified by attaching to the side chain amino group a molecule independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _p - Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p and [Lys-(aa ₃ )] _p ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; Lys is a lysine residue selected from L-Lys and D-Lys; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal Lys or (aa) ₃ of the molecule is acetylated at the alpha amino group.

In one embodiment the side chain of one or more of each of said first, second and/or third lysine residues of the lysine backbone is modified by attaching to the ε-amino group of the side chain a molecule independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _p -Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p and [Lys-(aa ₃ )] _p ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; Lys is a lysine residue selected from L-Lys and D-Lys; and (aa ₃ ) is an amino acid residue

independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal Lys or (aa) ₃ of the molecule is acetylated at the alpha amino group. In one embodiment the side chain of one or more of each of said first, second and/or third lysine residues of the lysine backbone are modified by attaching to the ε-amino group of the side chain a molecule being Lys _q -Lys; wherein q is a number selected from 0, 1 , 2 and 3. In one embodiment the N-terminal Lys of the molecule is acetylated at the alpha amino group. In one embodiment, the molecule to be covalently linked to the ε-amino group of the one or more lysine residues of the lysine backbone of the branched amino acid probe are independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) -Lys _q ; Lys _q - (aa ₃ ) ; [(aa ₃ )-Lys] _p and [Lys-(aa ₃ )] _P , wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3, and (aa ₃ ) is an amino acid residue

independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal Lys or (aa) ₃ of the molecule is acetylated at the alpha amino group.

It follows that in one embodiment the first lysine residue, or the second lysine residue, or the third lysine residue, or the first and the second lysine residues, or the first and the third lysine residues, or the second and the third lysine residues, or the first, the second and the third lysine residues of the branched amino acid probe are each modified by attaching to the ε-amino group(s) a molecule independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _p -Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p and [Lys-(aa ₃ )] _p , wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3, and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala. In one embodiment the N-terminal Lys or (aa) ₃ of the molecule is acetylated at the alpha amino group. In a particular embodiment (aa ₃ ) is an amino acid residue independently selected from Gly and Ala. In a further embodiment, (aa ₃ ) is Gly.

In one embodiment, the molecules to be covalently linked to the side chain amino group(s) of said first, second and/or third alkyl amine amino acid residue are acetylated at the alpha amino group of the N-terminal amino acid residue.

In one embodiment the molecules are independently selected from the group consisting of Ac-AAA _q -AAA; Ac-(aa ₃ ) _p -AAA _q ; Ac-AAA _q -(aa ₃ ) _p ; Ac-[(aa ₃ )-AAA] _p and Ac- [AAA-(aa ₃ )] _p ; and/or AAA _q -AAA; (aa ₃ ) _p -AAA _q ; AAAq-(aa ₃ ) _p ; [(aa ₃ )-AAA] _p and [AAA- (aa ₃ )] _P .

In one embodiment the molecules are independently selected from the group consisting of Ac-Orn _q -Orn; Ac-(aa ₃ ) _p -Orn _q ; Ac-Orn _q -(aa ₃ ) _p ; Ac-[(aa ₃ )-Orn] _p ; Ac-[Orn- (aa ₃ )] _p ; Ac-Orn _p -Lys _p ; Ac-Lys _p -Orn _p ; Ac-[Orn-Lys] _p and Ac-[Lys-Orn] _p ; and/or Orn _q -Orn; (aa ₃ ) _P -Orn _q ; Orn _q -(aa ₃ ) _P ; [(aa ₃ )-Orn] _p and [Orn-(aa ₃ )] _P ; Orn _p -Lys _p ; Lys _p -Orn _p ; [Orn-Lys] _p and [Lys-Orn] _p .

It follows that the molecules are in one embodiment independently selected from the group consisting of Ac-Lys _q -Lys; Ac-(aa ₃ ) _p -Lys _q ; Ac-Lys _q -(aa ₃ ) _p ; Ac-[(aa ₃ )-Lys] _p and Ac- [Lys-(aa ₃ )] _p ; and/or Lys _q -Lys; (aa ₃ ) _p -Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p and [Lys-(aa ₃ )] _p.

In a particular embodiment, the molecule to be covalently linked to the side chain amino group(s) is Ac-AAA _q -AAA or AAA _q -AAA, wherein q is a number selected from 0, 1 , 2 and 3.

It follows that in one embodiment the branched amino acid probe consists of 2 to 9 amino alkyi amino acid residues. In one embodiment said 2 to 9 amino alkyi amino acid residues are individually selected from the group consisting of lysine, D-lysine, ornithine and D-ornithine.

In a particular embodiment, the molecule to be covalently linked to the side chain amino group(s) is Ac-Lys _q -Lys or Lys _q -Lys, wherein q is a number selected from 0, 1 , 2 and 3.

It follows that in one embodiment the branched amino acid probe consists of 2 to 9 lysine residues.

In one embodiment, the branched amino acid probe comprises a maximum of 1 , 2, 3 or 4 amino acids selected from Arg, His, Gly and Ala (aa ₃ ), wherein the remaining amino acids are amino alkyi amino acid residues. In another embodiment, the branched amino acid probe comprises a maximum of 1 Arg residue, and/or comprises a maximum of 1 His residue, and/or comprises a maximum of 1 Gly residue, and/or comprises a maximum of 1 Ala residue.

In one embodiment, the molecule to be covalently linked to the side chain amino group(s) of one or more of the first, second and/or third amino alkyi amino acid residues is selected from the group consisting of AAA, Ac-AAA, AAA-AAA, Ac-AAA- AAA, AAA-AAA-AAA, Ac-AAA-AAA-AAA, AAA-AAA-AAA-AAA, Ac-AAA-AAA-AAA- AAA, AAA-Gly-AAA, Ac-AAA-Gly-AAA, AAA-AAA-Gly, Ac-AAA-AAA-Gly, AAA-Gly, Ac- AAA-Gly, AAA-Ala-AAA, Ac-AAA-Ala-AAA, AAA-AAA-Ala, Ac-AAA-AAA-AI a , AAA-Ala, Ac-AAA-Ala, AAA-His-AAA, Ac-AAA-His-AAA, AAA-AAA-His, Ac-AAA-AAA-His, AAA- His, Ac-AAA-His, AAA-Arg-AAA, Ac-AAA-Arg-AAA, AAA-AAA-Arg, Ac-AAA-AAA-Arg , AAA-Arg and Ac-AAA-Arg; wherein AAA is an amino alkyl amino acid residue as specified herein. The above-mentioned AAA, Gly, Ala, His and Arg amino acid residues may each be in the L- or D-conformation.

In one embodiment, the molecule to be covalently linked to the side chain amino group(s) of one or more of the first, second and/or third amino alkyl amino acid residues is selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys,

Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac- Lys-Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys- Ala-Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His- Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In a particular embodiment, the molecule to be covalently linked to the ε-amino group(s) of one or more of the first, second and/or third lysine residues is selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly, Ac- Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys, Lys-Lys-Ala, Ac-Lys- Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac- Lys- His- Lys, Lys-Lys-His, Ac-Lys-Lys- His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In a particular embodiment, the branched amino acid probe comprise or consist of a first lysine residue selected from Lys and D-Lys, said first lysine residue being optionally N-terminally acetylated or C-terminally amidated, wherein said first lysine residue is modified by attaching to the ε-amino group of said first lysine residue a molecule selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys- Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys- Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala- Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys- Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg. In a particular embodiment, the branched amino acid probe comprise or consist of a first and a second lysine residue each selected from Lys and D-Lys, said second lysine residue being optionally N-terminally acetylated or C-terminally amidated, wherein i) said first lysine residue, ii) said second lysine residue, or iii) said first and second residue are each modified by attaching to the ε-amino group of said lysine residue a molecule selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys- Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys- Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala- Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys- Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In a particular embodiment, the branched amino acid probe comprise or consist of a first, a second and a third lysine residue each selected from Lys and D-Lys, said third lysine residue being optionally N-terminally acetylated or C-terminally amidated, wherein i) said first lysine residue, ii) said second lysine residue, iii) said third lysine residue, iv) said first and second lysine residue, v) said first and third lysine residue, vi) said second and third lysine residue, or vii) said first, second and third lysine residues are each modified by attaching to the ε-amino group of said lysine residue a molecule selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys, Lys- Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys- His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In one embodiment the branched amino acid probe comprises or consists of the formula: Ac-(Ac-Lys-Lys)Lysr (identical to Ac-(Ac-Lys-Lys)Lys-), wherein Lysi is the first lysine residue, which is acetylated, covalently linked to the N-terminus of a peptide such as W-Peptide, and (Ac-Lys-Lys) is the molecule covalently linked to the ε-amino group of said first lysine residue Lysi. Figure 1 illustrates this formula/structure.

In one embodiment Ac-(Ac-Lys-Lys)Lys- is covalently linked to the N-terminal of the W- peptide and/or to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide. In one embodiment the branched amino acid probe comprises or consists of the formula: Ac-(Ac-Lys)Lysr. In one embodiment the branched amino acid probe comprises or consists of the formula: (Ac-Lys-Lys)LysrNH ₂ (identical to (Ac-Lys-Lys)Lys-NH ₂ ), wherein Lysi is the first lysine residue, which is amidated (-NH ₂ ) at the C-terminal, and (Ac-Lys-Lys) is the molecule attached to the ε-amino group of said first lysine residue Lysi. In one embodiment (Ac-Lys-Lys)LysrNH ₂ is attached to the C-terminal of the W-peptide.

In one embodiment the branched amino acid probe comprises or consists of a formula selected from the group consisting of (AAA)AAA , (AAA-AAA)AAA , (AAA-AAA- AAA)AAA , (AAA-AAA-AAA-AAA)AAAr, (AAA-Gly-AAA)AAA , (AAA-AAA-Gly)AAA , (AAA-Gly)AAA-i-, (AAA-Ala-AAA)AAA , (AAA-AAA-Ala)AAA , (AAA-Ala)AAA , (AAA- His-AAA)AAA , (AAA-AAA-His)AAA , (AAA-His)AAA , (AAA-Arg-AAA)AAA , (AAA- AAA- Arg)AAA , and (AAA-Arg)AAA . In one embodiment said first amino alkyl amino acid reside (AAA ) is N-terminally acetylated or C-terminally amidated.

In one embodiment the branched amino acid probe comprises or consists of a formula selected from the group consisting of (Lys)Lysr, (Lys-Lys)Lysr, (Lys-Lys-Lys)Lysr, (Lys-Lys-Lys-Lys)Lysr, (Lys-Gly-Lys)Lysr, (Lys-Lys-Gly)Lysr, (Lys-Gly)Lysr, (Lys- Ala-Lys)Lysr, (Lys-Lys-Ala)Lysr, (Lys-Ala)Lysr, (Lys-His-Lys)Lysr, (Lys-Lys- His)Lysr, (Lys-His)Lysr, (Lys-Arg-Lys)Lysr, (Lys-Lys-Arg)Lysr, and (Lys-Arg)Lysr. In one embodiment said first lysine reside (Lysr) is N-terminally acetylated or C- terminally amidated.

In one embodiment the branched amino acid probe comprises or consists of a formula selected from the group consisting of Ac-(Ac-Lys)Lysr, Ac-(Ac-Lys-Lys)Lysr, Ac-(Ac- Lys-Lys-Lys)Lysr, Ac-(Ac-Lys-Lys-Lys-Lys)Lysr, Ac-(Ac-Lys-Gly-Lys)Lysr, Ac-(Ac- Lys-Lys-Gly)Lysr, Ac-(Ac-Lys-Gly)Lys , Ac-(Ac-Lys-Ala-Lys)Lys , Ac-(Ac-Lys-Lys- Ala)Lysr, Ac-(Ac-Lys-Ala)Lysr, Ac-(Ac-Lys-His-Lys)Lysr, Ac-(Ac-Lys-Lys-His)Lysr, Ac-(Ac-Lys-His)Lysr, Ac-(Ac-Lys-Arg-Lys)Lysr, Ac-(Ac-Lys-Lys-Arg)Lysr, and Ac- (Ac-Lys-Arg)Lysr. In one embodiment the branched amino acid probe comprises or consists of a formula selected from the group consisting of (Ac-Lys)LysrNH ₂ , (Ac-Lys-Lys)LysrNH ₂ , (Ac- Lys-Lys-Lys)Lysi-NH ₂ , (Ac-Lys-Lys-Lys-Lys)LysrNH ₂ , (Ac-Lys-Gly-Lys)Lys NH ₂ , (Ac- Lys-Lys-Gly)Lys NH ₂ , (Ac-Lys-Gly)Lys NH ₂ , (Ac-Lys-Ala-Lys)Lys NH ₂ , (Ac-Lys-Lys- Ala)Lys NH ₂ , (Ac-Lys-Ala)Lys NH ₂ , (Ac-Lys-His-Lys)LysrNH ₂ , (Ac-Lys-Lys-His)Lys NH ₂ , (Ac-Lys-His)LysrNH ₂ , (Ac-Lys-Arg-Lys)Lys NH ₂ , (Ac-Lys-Lys-Arg)Lys NH ₂ , and (Ac- Ly s-Arg ) Ly s 1 - N H ₂ .

More specifically, in one embodiment the branched amino acid probe comprises or consists of a formula selected from the group consisting of Ac-(Ac-Lys)Lysi-, Ac-(Ac- Lys-Lys)Lysi-, Ac-(Ac-Lys-Lys-Lys)Lysi-, Ac-(Ac-Lys-Lys-Lys-Lys)Lysi-, Ac-(Ac-Lys- Gly-Lys)Lysr, Ac-(Ac-Lys-Lys-Gly)Lysr and Ac-(Ac-Lys-Gly)Lysr.

In one embodiment the branched amino acid probe comprises or consists of the formula: Ac-(Ac-Lys)Lys ₂ -Lysi-, wherein Lysi is the first lysine residue, Lys ₂ is the second lysine residue which is acetylated and covalently linked to Lysi via a peptide bond, and (Ac-Lys) is the molecule covalently linked to the ε-amino group of said second lysine residue Lys ₂ . In one embodiment the branched amino acid probe comprises or consists of the formula: Ac-Lys ₂ -(Ac-Lys)Lysi-, wherein the molecule (Ac-Lys) is covalently linked to the ε-amino group of said first lysine residue Lysi.

In one embodiment the branched amino acid probe(s) is selected from the group consisting of

Ac-(Ac-Lys)Lys-Lys-, (Ac-Lys)Lys-Lys-, Ac-(Lys)Lys-Lys-, (Lys)Lys-Lys-, (Ac-Lys)Lys- Lys-NH ₂ , (Lys)Lys-Lys-NH ₂ ;

Ac-Lys-(Ac-Lys)Lys-, Lys-(Ac-Lys)Lys-, Ac-Lys-(Lys)Lys-, Lys-(Lys)Lys- Lys-(Ac-Lys)Lys-NH ₂ , Lys-(Lys)Lys-NH ₂ ;

Ac-(Ac-Lys-Lys)-Lys-, (Ac-Lys-Lys)-Lys-, Ac-(Lys-Lys)-Lys- and (Lys-Lys)-Lys- (Ac-Lys-Lys)-Lys-NH ₂ , and (Lys-Lys)-Lys-NH ₂ .

In one embodiment the branched amino acid probe(s) is selected from the group consisting of Ac-(Ac-Lys)Lys-, Ac-(Lys)Lys-, (Ac-Lys) Lys-NH ₂ , (Lys)Lys-NH ₂ and (Lys)Lys-. In one embodiment the branched amino acid probe is selected from the group consisting of Ac-(Ac-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys-Gly)Lys ₂ - Lysr, Ac-(Ac-Lys-Lys-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys-Lys-Lys-Lys)Lys ₂ -Lysr, Ac-Lys ₂ -(Ac- Lys)-Lysi-, Ac-Lys ₂ -(Ac-Lys-Lys)-Lysr, Ac-Lys ₂ -(Ac-Lys-Gly)-Lysr, Ac-Lys ₂ -(Ac-Lys- Lys-Lys)-Lysi-, Ac-Lys ₂ -(Ac-Lys-Lys-Lys-Lys)-Lysr, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-)-Lysr, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysr, and Ac-(Ac-Lys-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysr.

More specifically, in one embodiment the branched amino acid probe is selected from the group consisting of Ac-(Ac-Lys)Lys ₂ -Lysi-, Ac-(Ac-Lys-Lys)Lys ₂ -Lysi-, Ac-(Ac-Lys- Gly)Lys ₂ -Lysi-, Ac-Lys ₂ -(Ac-Lys)-Lysr, Ac-Lys ₂ -(Ac-Lys-Lys)-Lysr, Ac-Lys ₂ -(Ac-Lys- Gly)-Lysr, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-)-Lysr, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysr, and Ac-(Ac-Lys-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysi-. In one embodiment the branched amino acid probe is selected from the group consisting of Ac-Lys ₃ - Lys _2- (Ac-Lys)Lysr, Ac-Lys ₃ -(Ac-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys)Lys ₃ - Lys ₂ -Lysi-, Ac-Lys ₃ -(Ac-Lys)Lys ₂ -(Ac-Lys)Lysr, Ac-(Ac-Lys)Lys ₃ -(Ac-Lys)Lys ₂ -Lysr, and Ac-(Ac-Ly s ) Ly s ₃ - Ly s ₂ -(Ac- Ly s ) Ly s i - . In a particular embodiment the branched amino acid probe is selected from the group consisting of Ac-(Ac-Lys)Lysr, Ac-(Ac-Lys-Lys)Lysi-, Ac-(Ac-Lys-Lys-Lys)Lysi-, Ac- (Ac-Lys-Lys-Lys-Lys)Lysr, Ac-(Ac-Lys-Gly-Lys)Lysi-, Ac-(Ac-Lys-Lys-Gly)Lysi-, Ac- (Ac-Lys-Gly)Lysr, Ac-(Ac-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys-Lys)Lys ₂ -Lysr, Ac-(Ac-Lys- Gly)Lys ₂ -Lysr, Ac-Lys ₂ -(Ac-Lys)-Lysr, Ac-Lys ₂ -(Ac-Lys-Lys)-Lysr, Ac-Lys ₂ -(Ac-Lys- Gly)-Lysi-, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-)-Lysr, Ac-(Ac-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysr, Ac- (Ac-Lys-Lys)Lys ₂ -(Ac-Lys-Lys-)-Lysi-, Ac-Lys ₃ - Lys _2- (Ac-Lys)Lysr, Ac-Lys ₃ -(Ac- Lys)Lys ₂ -Lysr, Ac-(Ac-Lys)Lys ₃ -Lys ₂ -Lysr, Ac-Lys ₃ -(Ac-Lys)Lys ₂ -(Ac-Lys)Lysr, Ac- (Ac-Lys)Lys ₃ -(Ac-Lys)Lys ₂ -Lysr, and Ac-(Ac-Lys)Lys ₃ -Lys ₂ -(Ac-Lys)Lysr. In one embodiment said branched amino acid probe is covalently linked to the N-terminal of the W-peptide and/or to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In a particular embodiment, the branched amino acid probe consists of 2 or 3 lysine residues (selected from Lys and D-Lys). In a particular embodiment, the branched amino acid probe consists of 3 lysine residues. In another embodiment, the branched amino acid probe consists of 2 lysine residues. In a particular embodiment, the branched amino acid probe consists of a first and a second lysine residue selected from Lys and D-Lys, wherein one or both of the first and second lysine residues are modified by attaching to the ε-amino group of said first and/or second lysine residue one lysine residue selected from Lys and D-Lys; wherein each of said lysine residues are optionally acetylated at the alpha amino group.

In a particular embodiment, the branched amino acid probe consists of a first lysine residue selected from Lys and D-Lys, wherein said first lysine residue is modified by attaching to the ε-amino group of said first lysine residue two lysine residues selected from Lys and D-Lys; wherein each of said lysine residues are optionally acetylated at the alpha amino group.

Linking the branched amino acid probes and the W-Peptide

According to the invention, the first amino alkyl amino acid residue of each of the one or more branched amino acid probes is covalently linked to the N-terminus of a W- Peptide, covalently linked to the C-terminus of a W-Peptide, and/or covalently linked to the side chain amino group of an amino alkyl amino acid residue within a W-Peptide to be modified according to the invention.

Attaching one or more branched amino acid probes to a W-Peptide yields a W- Peptide/BAP-conjugate.

The term covalently linked to the N-terminus of said W-Peptide means that the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the alpha amino group of the most N-terminal amino acid residue of W-Peptide.

The term covalently linked to the C-terminus of said W-Peptide means that the alpha amino group of the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the most C-terminal amino acid residue of W-Peptide. Furthermore, it is understood that a branched amino acid probe in one embodiment is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide. In one particular embodiment said amino alkyl amino acid residue within said W-

Peptide sequence is selected from the group consisting of an ornithine residue and a lysine residue. In one particular embodiment said amino alkyl amino acid residue within said peptide sequence is a lysine residue. In one embodiment the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the δ-amino group of an ornithine residue within said W- Peptide or the ε-amino group of a lysine residue within said W-Peptide.

In one embodiment the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the ε-amino group of a lysine residue within said W- Peptide.

In one embodiment the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the ε-amino group of the lysine residue at position 2 of said W-Peptide.

In one embodiment the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the N-terminal Trp of said W-Peptide. In one embodiment the first amino alkyl amino acid residue of the branched amino acid probe is covalently linked to the C-terminal Met or DMet of said W-Peptide.

It is understood that an amino alkyl amino acid residue within said peptide sequence means that the amino alkyl amino acid residue does not form part of the branched amino acid probe per se, but is a residue occurring within the existing amino acid sequence of the W-Peptide. Said amino alkyl amino acid residue can be positioned at any position of the W-Peptide.

According to the invention, a peptide analogue comprising one or more branched amino acid probes means that the peptide analogue in one embodiment comprises 1 branched amino acid probe, such as 2 branched amino acid probes, for example 3 branched amino acid probes, such as 4 branched amino acid probes, for example 5 branched amino acid probes, such as 6 branched amino acid probes. In principle the peptide analogue can comprise any number of branched amino acid probes provided they can be covalently linked to the peptide (N-terminally, C-terminally and/or one or more amino alkyl amino acid residues within said W-Peptide).

In one embodiment the W-Peptide analogue comprises 1 branched amino acid probe.

In one embodiment the W-Peptide analogue comprises 1 branched amino acid probe, which branched amino acid probe is covalently bound to the N-terminus of the W- Peptide. In one embodiment the W-Peptide analogue comprises 1 branched amino acid probe, which branched amino acid probe is covalently bound to the C-terminus of the W- Peptide.

In one embodiment the W-Peptide analogue comprises 1 branched amino acid probe, which branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In one embodiment the W-Peptide analogue comprises more than one (two or more) branched amino acid probe(s). In the embodiments wherein the W-Peptide analogue comprises more than one branched amino acid probe it is understood that the more than one branched amino acid probes may individually be the same (identical) or different (non-identical).

In one embodiment the W-Peptide analogue comprises 2 branched amino acid probes.

In one embodiment the W-Peptide analogue comprises 2 branched amino acid probes, wherein one branched amino acid probe is covalently bound to the N-terminus of the W-Peptide and another branched amino acid probe is covalently bound to the C- terminus of the W-Peptide. In one embodiment the W-Peptide analogue comprises 2 branched amino acid probes, wherein one branched amino acid probe is covalently bound to the N-terminus of the W-Peptide and another branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In one embodiment the W-Peptide analogue comprises 2 branched amino acid probes, wherein one branched amino acid probe is covalently bound to the C-terminus of the W-Peptide and another branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide.

In one embodiment the peptide analogue comprises 2 branched amino acid probes, wherein each of the two branched amino acid probes are covalently linked to the side chain amino group of different (or separate) amino alkyl amino acid residues within said W-Peptide.

In one embodiment the W-Peptide analogue comprises 3 branched amino acid probes.

In one embodiment the W-Peptide analogue comprises 3 branched amino acid probes, wherein the first branched amino acid probe is covalently bound to the N-terminus of the W-Peptide, the second branched amino acid probe is covalently bound to the C- terminus of the W-Peptide and the third branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W- Peptide. In one embodiment the W-Peptide analogue comprises 3 branched amino acid probes, wherein the first branched amino acid probe is covalently bound to the N-terminus of the W-Peptide, and the second and third branched amino acid probes are each covalently linked to the side chain amino group of different amino alkyl amino acid residues within said W-Peptide.

In one embodiment the W-Peptide analogue comprises 3 branched amino acid probes, wherein the first branched amino acid probe is covalently bound to the C-terminus of the W-Peptide, and the second and third branched amino acid probes are each covalently linked to the side chain amino group of different amino alkyl amino acid residues within said W-Peptide. W-Peptide

The W-Peptide analogue according to the present invention comprises a W-peptide as defined herein and one or more branched amino acid probes.

A W-Peptide according to the invention comprises any functional W-Peptide, including W-Peptide (SEQ ID NO:1 ) and any functional variant thereof.

As used herein, each amino acid of the hexapeptide W-peptide (SEQ ID NO:1 ) is referred to as Trp (position 1 ) - Lys (position 2) - Tyr (position 3) - Met (position 4) - Val (position 5) - Met (position 6).

In one embodiment a W-Peptide according to the present invention is Trp-Lys-Tyr-Met- Val-Met (SEQ ID NO:1 ) or a functional variant thereof. In one embodiment a W-Peptide according to the present invention is Trp-Lys-Tyr-Met-Val-DMet or a functional variant thereof.

A W-Peptide, or a functional variant thereof, includes any W-Peptide which binds to one or more of the formyl peptide receptors, including Formyl Peptide Receptor 1 (FPR1 )(Uniprot P21462), Formyl Peptide Receptor 2 (FPR2)(Uniprot P25090) and Formyl Peptide Receptor 3 (FPR3)(Uniprot 25089).

A functional W-Peptide in one embodiment is a W-Peptide which activates and/or stimulates one or more of Formyl Peptide Receptor 1 (FPR1 ), Formyl Peptide Receptor 2 (FPR2) and Formyl Peptide Receptor 3 (FPR3).

A functional W-Peptide in one embodiment is a W-Peptide which is a ligand and/or agonist of one or more of Formyl Peptide Receptor 1 (FPR1 ), Formyl Peptide Receptor 2 (FPR2) and Formyl Peptide Receptor 3 (FPR3).

The term "agonist" in the present context refers to a W-Peptide as defined herein, capable of binding to, or in some embodiments, capable of binding to at least some extent and/or activating a receptor, or in some embodiments, activating a receptor to at least some extent. For example, a FPR2 agonist is thus capable of binding to and/or activating the FPR2. An agonist may be an agonist of several different types of receptors, and thus capable of binding and/or activating several different types of receptors. Said agonist can also be a selective agonist which only binds and activates one type of receptor. The term "antagonist" in the present context refers to a substance capable of inhibiting the effect of a receptor agonist.

Full agonists bind (have affinity for) and activate a receptor, displaying full efficacy at that receptor. "Partial agonists" in the present context are peptides able to bind and activate a given receptor, but having only partial efficacy at the receptor relative to a full agonist. Partial agonists can act as antagonists when competing with a full agonist for receptor occupancy and producing a net decrease in the receptor activation compared to the effects or activation observed with the full agonist alone. "Selective agonists" in the present context are compounds which are selective and therefore predominantly bind and activates one type of receptor. Thus a selective FPR2 agonist is selective for the FPR2.

W-Peptides according to the present invention are in one embodiment capable of binding and activating to some extent one or several formyl peptide receptors and can have different binding affinities and/or different receptor activation efficacy for different receptors. Affinity refers to the number and size of intermolecular forces between a peptide ligand and its receptor, and residence time of the ligand at its receptor binding site; and receptor activation efficacy refers to the ability of the peptide ligand to produce a biological response upon binding to the target receptor and the quantitative magnitude of this response. In some embodiments, such differences in affinity and receptor activation efficacy are determined by receptor binding/activation studies which are conventional in the art, for instance by generating EC ₅₀ and Emax values for stimulation of ligand binding in cells expressing one or several types of receptors as mentioned herein, or on tissues expressing the different types of receptors. High affinity means that a lower concentration of a ligand is needed to obtain a binding of 50% of the receptors compared to ligand peptides which have lower affinity; high receptor activation efficacy means that a lower concentration of the peptide is needed to obtain a 50% receptor activation response (low EC ₅ o value), compared to peptides which have lower affinity and/or receptor activity efficacy (higher EC ₅₀ value). In one embodiment, the peptides have differing affinities and/or receptor activation efficacies for two or more of the receptors selected from FPR1 , FPR2 and FPR3.

The receptor activation potency of peptide agonists can also be measured in p(A ₅₀ ) values which is a conventional method for determining the receptor activation efficacy of an agonist.

In a particular embodiment, a functional W-Peptide is a W-Peptide which has binding affinity and/or receptor efficacy for the Formyl Peptide Receptor 2 (FPR2). This may be tested using conventional methods, or as outlined in examples 2 and 3.

In one particular embodiment, the W-Peptide is capable of binding to and activating FPR2. In a further embodiment said peptide is a full agonist of FPR2.

In one embodiment a W-Peptide is capable of one or more of

a) binding to one or more of the formyl peptide receptors, including FPR1 , FPR2 and FPR3,

b) activating and/or stimulating one or more of the formyl peptide receptors, including FPR1 , FPR2 and FPR3,

c) binding and/or activating FPR2,

d) activating immune cells

e) activating leukocytes, such as phagocytic leukocytes,

f) activating neutrophils and/or monocytes

g) activate phagocytic leukocytes' effector functions, such as inducing

neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase, and/or h) inducing chemotaxis in phagocytic leukocytes.

Specific W-Peptides

In one embodiment a W-peptide of the present invention is selected from Trp-Lys-Tyr- Met-Val-Met (SEQ ID NO:1 ) and a functional variant thereof.

In one embodiment a W-peptide of the present invention is selected from Trp-Lys-Tyr- Met-Val-Met (SEQ ID NO:1 ) and Trp-Lys-Tyr-Met-Val-DMet and a functional variant thereof. In one embodiment the C-terminal of a W-Peptide is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ) or a secondary amide. In one embodiment a W-peptide is C-terminally amidated (-NH ₂ ). In one embodiment a W-peptide of the present invention is not C-terminally amidated, in particular when a C- terminal branched amino acid probe is attached. In one embodiment a W-peptide of the present invention is C-terminally amidated, in particular when a C-terminal branched amino acid probe is not attached.

In one embodiment a W-peptide is N-terminally acetylated (COCH ₃ or Ac-). In one embodiment a W-peptide of the present invention is not N-terminally acetylated, in particular when an N-terminal branched amino acid probe is attached. In one embodiment a W-peptide of the present invention is N-terminally acetylated, in particular when an N-terminal branched amino acid probe is not attached.

In one embodiment a W-peptide is selected from Trp-Lys-Tyr-Met-Val-Met-NH ₂ and Trp-Lys-Tyr-Met-Val-DMet-NH ₂ and a functional variant thereof. In one embodiment a W-peptide is selected from Ac-Trp-Lys-Tyr-Met-Val-Met and Ac- Trp-Lys-Tyr-Met-Val-DMet and a functional variant thereof.

In one embodiment a W-peptide is selected from Trp-Lys-Tyr-Met-Val-Met-NH ₂ and Ac- Trp-Lys-Tyr-Met-Val-DMet-NH ₂ and a functional variant thereof.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having one amino acid substitution. One amino acid substitution means that the amino acid differs between the original sequence and the variant sequence at one position.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having two amino acid substitutions.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having three amino acid substitutions. In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 1 . In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 2.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 3.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 4.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 5.

In one embodiment a W-peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 6. In one embodiment a W-peptide comprises an ornithine residue. In one embodiment a W-peptide comprises one ornithine residue. In one embodiment a W-peptide comprises two ornithine residues. In one embodiment a W-peptide of the present invention comprises an ornithine residue at position 2 of W-Peptide (whereby lysine is substituted with ornithine). In one embodiment the W-peptide is Trp-Orn-Tyr-Met-Val- Met (SEQ ID NO:2) or Trp-Orn-Tyr-Met-Val-DMet.

In one embodiment a W-peptide comprises a proline residue. In one embodiment a W- peptide comprises one proline residue. In one embodiment a W-peptide comprises two proline residues. In one embodiment a W-peptide of the present invention comprises a proline residue at position 5 of W-Peptide. In one embodiment the W-peptide is Trp- Lys-Tyr-Met-Pro-Met (SEQ ID NO:3) or Trp-Lys-Tyr-Met-Pro-DMet.

In one embodiment a W-peptide comprises an isoleucine residue. In one embodiment a W-peptide comprises one isoleucine residue. In one embodiment a W-peptide comprises two isoleucine residues. In one embodiment a W-peptide of the present invention comprises an isoleucine residue at position 4 of W-Peptide (whereby Met is substituted with isoleucine). In one embodiment the W-peptide is Trp-Lys-Tyr-lle-Val- Met (SEQ ID NO:4) or Trp-Lys-Tyr-lle-Val-DMet. In one embodiment a W-peptide comprises a phenylalanine residue. In one embodiment a W-peptide comprises one phenylalanine residue. In one embodiment a W-peptide comprises two phenylalanine residues. In one embodiment a W-peptide of the present invention comprises a phenylalanine residue at position 3 of W-Peptide (whereby Tyr is substituted with phenylalanine). In one embodiment the W-peptide is Trp-Lys-Phe-Met-Val-Met (SEQ ID NO:5) or Trp-Lys-Phe-Met-Val-DMet.

In one embodiment a W-peptide has the sequence Trp-Lys-Tyr-Met-aa-Met (SEQ ID NO:10) or Trp-Lys-Tyr-Met-aa-DMet, wherein aa is an amino acid having the structure R ¹ R ² C(NH ₂ )-COOH, wherein R ¹ is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyl and cycloalkenyl, and wherein R ² is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkenyl.

In one embodiment a W-peptide comprises an alpha-amino n-butyric acid residue. In one embodiment a W-peptide comprises one alpha-amino n-butyric acid residue. In one embodiment a W-peptide comprises two alpha-amino n-butyric acid residues. In one embodiment a W-Peptide comprises an alpha-amino n-butyric acid residue at position 5 of W-Peptide. In one embodiment a W-peptide comprises an alfa-amino butyric acid residue (2- Aminobutanoic acid, Abu), such as comprises an alfa-amino butyric acid residue at position 5 of W-Peptide.

In one embodiment a W-peptide comprises an alfa-amino isobutyric acid residue (alfa- methylalanine or 2-methylalanine, Aib), such as comprises an alfa-amino isobutyric acid residue at position 5 of W-Peptide.

In one embodiment the W-peptide is Trp-Lys-Tyr-Met-Abu-Met (SEQ ID NO:6) or Trp- Lys-Tyr-Met-Abu-DMet. In one embodiment the W-peptide is Trp-Lys-Tyr-Met-Aib-Met (SEQ ID NO:7) or Trp- Lys-Tyr-Met-Aib-DMet.

In one embodiment a W-peptide is X-Lys-Tyr-X-Val-Met (SEQ ID NO:8) or X-Lys-Tyr- X-Pro-Met (SEQ ID NO:9), wherein X is individually any amino acid. In one

embodiment X is selected from Trp and Met.

In one embodiment a W-peptide is Trp - X2 - X3 - X4 - X5 - X6 (SEQ ID NO:1 1 ), wherein

X2 is selected from Lys and Orn,

X3 is selected from Tyr and Phe,

X4 is selected from Met and lie

X5 is selected from Val, Pro, Abu, Aib and aa

X6 is selected from Met and DMet,

wherein aa is an amino acid having the structure R1 R2C(NH2)-COOH, wherein R1 is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyi and cycloalkenyl, and wherein R2 is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyi and cycloalkenyl, In one embodiment a W-peptide is selected from the group consisting of

Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ),

Trp-Lys-Tyr-Met-Val-DMet,

Trp-Orn-Tyr-Met-Val-Met (SEQ ID NO:2),

Trp-Orn-Tyr-Met-Val-DMet,

Trp-Lys-Tyr-Met-Pro-Met (SEQ ID NO:3),

Trp-Lys-Tyr-Met-Pro-DMet,

Trp-Lys-Tyr-lle-Val-Met (SEQ ID NO:4),

Trp-Lys-Tyr-lle-Val-DMet,

Trp-Lys-Phe-Met-Val-Met (SEQ ID NO:5),

Trp-Lys-Phe-Met-Val-DMet,

Trp-Lys-Tyr-Met-Abu-Met (SEQ ID NO:6)

Trp-Lys-Tyr-Met-Abu-DMet,

Trp-Lys-Tyr-Met-Aib-Met (SEQ ID NO:7)

Trp-Lys-Tyr-Met-Aib-DMet,

X-Lys-Tyr-X-Val-Met, wherein X is any amino acid (SEQ ID NO:8), X-Lys-Tyr-X-Pro-Met, wherein X is any amino acid (SEQ ID NO:9), Trp-Lys-Tyr-Met-aa-Met (SEQ ID NO: 10) and Trp-Lys-Tyr-Met-aa-DMet, wherein aa is an amino acid having the structure R ¹ R ² C(NH ₂ )-COOH, wherein R ¹ is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyl and cycloalkenyl, and wherein R ² is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkenyl,

Trp - X2 - X3 - X4 - X5 - X6 (SEQ ID NO:1 1 ), wherein X2 is selected from Lys and Orn, X3 is selected from Tyr and Phe, X4 is selected from Met and lie, X5 is selected from Val, Pro, Abu, Aib and aa; and X6 is selected from Met and DMet,

or a functional variant thereof.

A variant of a W-peptide can in principle have one or more substitutions at one or more positions. Individual amino acid residues in the W-Peptide can be substituted with any given proteinogenic or non-proteinogenic amino acid.

The genetic code specifies 20 standard amino acids naturally incorporated into polypeptides (proteinogenic): Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Tyr, Thr, Trp, Val, and 2 which are incorporated into proteins by unique synthetic mechanisms: Sec (selenocysteine, or U) and Pyl (pyrrolysine, O). These are all L-stereoisomers.

Aside from the 22 standard or natural amino acids, there are many other non-naturally occurring amino acids (non-proteinogenic or non-standard They are either not found in proteins, or are not produced directly and in isolation by standard cellular machinery. Non-standard amino acids are usually formed through modifications to standard amino acids, such as post-translational modifications. Examples of unnatural amino acid residues are Abu, Aib, NIe (Norleucine), DOrn (D-ornithine, deguanylated arginine), Nal (beta-2-naphthyl-alanine), D-Nal (beta-2-naphthyl-D-alanine), DArg, DTrp, DPhe and DVal.

Any amino acids according to the present invention may be in the L- or D-configuration. If nothing is specified, reference to the L-isomeric form is preferably meant. The term peptide also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art. Such post- translational modifications can be introduced prior to partitioning, if desired. Also, functional equivalents may comprise chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids, which do not normally occur in human proteins.

W-Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention. Functional equivalents also comprise glycosylated and covalent or aggregative conjugates formed with the same molecules, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in a fragment including at any one or both of the N- and C-termini, by means known in the art.

In some embodiments, the W-Peptides according are modified by acetylation, such as N-terminal acetylation. In some embodiments the W-Peptides are modified by C- terminal amidation. W-Peptide comprising alpha-amino n-butyric acid

It is also an aspect to provide a W-Peptide of sequence Trp-Lys-Tyr-Met-aa-Met (SEQ ID NO:10) or Trp-Lys-Tyr-Met-aa-DMet, wherein aa is an amino acid having the structure R ¹ R ² C(NH ₂ )-COOH,

wherein R ¹ is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyl and cycloalkenyl, and

wherein R ² is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkenyl.

R ¹ and R ² may each be the same (identical) or different (non-identical).

As used herein, "alkyl" refers to linear, branched or cyclic hydrocarbon structures preferably having from 1 to 20 carbon atoms (a "C1 -C20 alkyl") e.g., 1 to 10 carbon atoms or 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, t- butyl, n-heptyl, octyl, cyclobutylmethyl, cyclopropylmethyl and the like. "Unsubstituted alkyl" refers to an alkyl group that is not substituted with any additional substituents. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl and t-butyl. As used herein, "alkenyl" refers to linear, branched or cyclic hydrocarbon structures preferably having from 2 to 20 carbon atoms (a "C1 -C20 alkenyl") and more preferably 2 to 10 carbon atoms or 2 to 6 carbon atoms and having at least 1 site of alkenyl unsaturation. As used herein, "unsubstituted alkenyl" refers to an alkenyl group that is not substituted with any additional substituents. When an alkenyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed. This term is exemplified by groups such as propen-3-yl (-CH2- CH=CH ₂ ), 3-methyl-but-2-enyl and (=CH ₂ ). The group represented by =CH2 indicates connectivity from, e.g., an sp2 hybridized carbon atom of a parent structure to CH ₂ via a double bond.

As used herein, "Cycloalkyi" refers to a 3-, 4-, 5-, 6-, 7- or 8-membered saturated non- aromatic monocyclic cycloalkyi ring. Representative groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. In one embodiment, the cycloalkyi group is substituted with one or more alkyl or alkenyl.

As used herein, " cycloalkenyl" refers to a 3-, 4-, 5-, 6-, 7- or 8-membered non- aromatic monocyclic carbocyclic ring having at least one endocyclic double bond, but which is not aromatic. Representative cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, 1 ,3-cyclobutadienyl, cyclopentenyl, 1 ,3- cyclopentadienyl, cyclohexenyl, 1 ,3-cyclohexadienyl, cycloheptenyl, 1 ,3- cycloheptadienyl, 1 ,4-cycloheptadienyl, 1 ,3,5- cycloheptatrienyl, cyclooctenyl, 1 ,3- cyclooctadienyl, 1 ,4-cyclooctadienyl, or 1 ,3,5-cyclooctatrienyl. In one embodiment, the cycloalkenyl group is substituted with one or more alkyl or alkenyl.

In one embodiment there is provided a W-Peptide of sequence Trp-Lys-Tyr-Met-aa- Met-NH ₂ (SEQ ID NO:10), wherein aa is an amino acid having the structure

R ¹ R ² C(NH ₂ )-COOH, wherein R ¹ is H and R ² is selected from the group consisting of methyl (C1 alkyi), ethyl (C2 alkyi), propyl (C3 alkyi), butyl (C4 alkyi), pentyl (C5 alkyi) and hexyl (C6 alkyi).

In one embodiment R ¹ is H, R ² is methyl and the amino acid aa of SEQ ID NO:10 is alanine.

In a particular embodiment R ¹ and R ² are methyl and the amino acid aa of SEQ ID NO:10 is 2-amino isobutyric acid; (CH ₃ ) ₂ CH(NH ₂ )-COOH (Aib). In a particular embodiment R ¹ is H, R ² is ethyl (C2 alkyi) and the amino acid aa of SEQ ID NO:10 is 2-amino n-butyric acid; CH ₃ CH ₂ CH(NH ₂ )-COOH (Abu).

In one embodiment R ¹ is H and R ² is selected from the group consisting of propyl (C3 alkyi) including n-propyl and isopropyl. Examples of amino acids aa include valine and norvaline.

In one embodiment R ¹ is H and R ² is selected from the group consisting of butyl (C4 alkyi) including n-butyl, sec-butyl, isobutyl and t-butyl. Examples of amino acids aa include leucine, isoleucine and norleucine.

In one embodiment there is provided a novel W-Peptide variant comprising an alpha- amino n-butyric acid residue. Alpha-amino n-butyric acid may exist in its L- or D- conformation (S- or R-conformation). In one embodiment there is provided a W-Peptide comprising one or more alfa-amino butyric acid residues. Alfa-amino butyric acid (AABA) or 2-Aminobutanoic acid (Abu), also known as homoalanine, is a non-proteinogenic alpha amino acid with chemical formula CH ₃ CH ₂ CH(NH ₂ )-COOH. In one embodiment there is provided a W-Peptide comprising one or more alfa-amino isobutyric acid residues, also known as alfa-methylalanine or 2-methylalanine (Aib), with structural formula (CH ₃ ) ₂ CH(NH ₂ )-COOH. In one embodiment the W-Peptide comprises an alpha-amino n-butyric acid at one or more positions, such as at one or more positions selected from position 1 , position 2, position 3, position 4, position 5 and position 6 of W-Peptide. In one embodiment the W-Peptide comprises an alpha-amino n-butyric acid residue at more than one position, such as at two positions or three positions.

In one embodiment the W-Peptide comprises alpha-amino n-butyric acid at position 5 of W-Peptide. In this embodiment Met of SEQ ID NO:1 is substituted with an alpha- amino n-butyric acid.

In one embodiment the W-Peptide is selected from the group consisting of

Trp-Lys-Tyr-Met-Abu-DMet-NH ₂ , Trp-Lys-Tyr-Met-Abu-Met-NH ₂ (SEQ ID NO:6), Trp- Lys-Tyr-Met-Aib-DMet-NH ₂ and Trp-Lys-Tyr-Met-Aib-Met-NH ₂ (SEQ ID NO:7).

In one embodiment the W-Peptide comprising an alpha-amino n-butyric acid further comprises one or more branched amino acid probes.

Methods of preparation

The W-Peptide analogues may be prepared by any suitable methods known in the art. Thus, in some embodiments the W-Peptide and the branched amino acid probe are each prepared by standard peptide-preparation techniques, such as solution synthesis or solid phase peptide synthesis (SPPS) such as Merrifield-type solid phase synthesis. The W-Peptide analogues are in one embodiment prepared by solid phase synthesis by first constructing the pharmacologically active W-Peptide sequence, using well- known standard protection, coupling and de-protection procedures, thereafter sequentially coupling the branched amino acid probe onto the active W-Peptide in a manner similar to the construction of the active W-Peptide, and finally cleaving off the entire W-Peptide analogue from the carrier. This strategy yields a W-Peptide, wherein the branched amino acid probe is covalently bound to the pharmacologically active W- Peptide at the N-terminal nitrogen atom of the W-Peptide. In one embodiment, the alpha nitrogen on the final amino acid in the branched amino acid sequence is capped with acetyl, using standard acylation techniques, prior to or after coupling of the branched amino acid sequence on the active W-Peptide. Reactive moieties at the N- and C-termini, which facilitates amino acid coupling during synthesis, as well as reactive side chain functional groups, can interact with free termini or other side chain groups during synthesis and peptide elongation and negatively influence yield and purity. Chemical groups are thus developed that bind to specific amino acid functional groups and block, or protect, the functional group from

nonspecific reactions. Purified, individual amino acids are reacted with these protecting groups prior to synthesis and then selectively removed during specific steps of peptide synthesis. Examples of N-terminal protecting groups are t-Boc and Fmoc, commonly used in solid-phase peptide synthesis. C-terminal protecting groups are mostly used in liquid-phase synthesis. Because N-terminal deprotection occurs continuously during peptide synthesis, protecting schemes have been established in which the different types of side chain protecting groups (benzyl;Bzl or tert-butyl;tBu) are matched to either Boc or Fmoc, respectively, for optimized deprotection.

In a particular embodiment, when preparing the branched amino acid probe, exemplified by Ac(Ac-Lys-Lys)Lys-, the protection group for Lys is Mtt, which as Fmoc protected amino acid is commercially available (Fmoc-Lys(Mtt)-OH; N - a - Fmoc - N - ε - 4 - methyltrityl - L - lysine, CAS# 167393-62-6). Lys(Mtt) allows for capping Lys with acetyl or extending the sequence at the alpha amino group of lysine as it is not cleaved under the conditions that cleave Fmoc, and may be removed without cleavage of other side chain protection groups.

In a particular embodiment, when preparing the branched amino acid probe, exemplified by (Ac-Lys-Lys)Lys-NH ₂ , the protection group for Lys is ivDde, which as Fmoc protected amino acid is commercially available (Fmoc-Lys(ivDde)-OH; N - a - Fmoc-N-e-1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl-L-lysine, CAS# 204777-78-6). Lys(ivDde) allows for extending the sequence at the alpha amino group of lysine or capping Lys with acetyl as it is not cleaved under the conditions that cleave Fmoc, and may be removed without cleavage of other side chain protection groups. The method of preparation is in some embodiments optimized by routine methods in the art that may increase the yield and/or quality of the thus prepared synthetic W- Peptide. For instance, use of pseudoproline (oxazolidine) dipeptides in the Fmoc SPPS of serine- and threonine-containing peptides may lead to improvements in quality and yield of crude products and may help avoid unnecessary repeat synthesis of failed sequences. These dipeptides are easy to use: simply substitute a serine or threonine residue together with the preceding amino acid residue in the peptide sequence with the appropriate pseudoproline dipeptide. The native sequence is regenerated on cleavage and deprotection.

In one embodiment the sequence of the pharmacologically active W-Peptide and the branched amino acid probe (or parts thereof) are each prepared separately by for example solution synthesis, solid phase synthesis, recombinant techniques, or enzymatic synthesis, followed by coupling of the (at least) two sequences by well- known segment condensation procedures, either in solution or using solid phase techniques, or a combination thereof.

In one embodiment, the W-Peptide is prepared by recombinant DNA methods and the branched amino acid probe is prepared by solid or solution phase synthesis. The conjugation of the W-Peptide and the branched amino acid probe is in one embodiment carried out by using chemical ligation. This technique allows for the assembling of totally unprotected peptide segments in a highly specific manner. In another

embodiment, the conjugation is performed by protease-catalysed peptide bond formation, which offers a highly specific technique to combine totally unprotected peptide segments via a peptide bond.

In one embodiment, the C-terminal amino acid of the branched amino acid probe or the C-terminal amino acid of the W-Peptide is covalently linked to the solid support material by means of a common linker such as 2,4-dimethoxy-4'-hydroxy-benzophenone, 4-(4- hydroxy-methyl-3-methoxyphenoxy)-butyric acid, 4-hydroxy-methylbenzoic acid, 4- hydroxymethyl- phenoxyacetic acid, 3-(4-hydroxymethylphenoxy)propionic acid, or p - {(R,S) - a - [1 - (9H - Fluoren - 9 - yl) - methoxyformamido] - 2,4 - dimethoxybenzyl} - phenoxyacetic acid (Rink amide linker). Examples of suitable solid support materials (SSM) are e.g., functionalised resins such as polystyrene, polyacrylamide, polydimethylacrylamide, polyethyleneglycol, cellulose, polyethylene, polyethyleneglycol grafted on polystyrene, latex, dynabeads, etc. The produced W-Peptide analogues of the invention are in some embodiment cleaved from the solid support material by means of an acid such as trifluoracetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogen chloride, hydrogen fluoride, etc. optionally in combination with one phenol, thioanisole, etc., or the peptide conjugate are in other embodiments cleaved from the solid support by means of a base such as ammonia, hydrazine, an alkoxide, such as sodium ethoxide, an hydroxide, such as sodium hydroxide, etc.

In one embodiment the produced W-peptide analogues are isolated as salts, such as an acetate salt or maleate salt or any other salt known to the skilled person.

In other embodiments, the W-Peptide analogues may be prepared or produced by recombinant techniques. Thus, in one aspect of the present invention the peptide is produced by host cells comprising a first nucleic acid sequence encoding the W- Peptide or W-Peptide analogue operably associated with a second nucleic acid capable of directing expression in said host cells. In some embodiments the second nucleic acid sequence comprises or even consists of a promoter that will direct the expression of protein of interest in said cells. A skilled person will be readily capable of identifying useful second nucleic acid sequences (e.g. vectors and plasmids) for use in a given host cell.

The process of producing a recombinant peptide in general comprises the steps of: providing a host cell, preparing a gene expression construct comprising a first nucleic acid encoding the peptide operably linked to a second nucleic acid capable of directing expression of said protein of interest in the host cell, transforming the host cell with the construct and cultivating the host cell, thereby obtaining expression of the peptide. In one embodiment , the recombinantly produced peptide is excreted by the host cells. The host cell include any suitable host cell known in the art, including prokaryotic cells, yeast cells, insect cells and mammalian cells. In one embodiment, the recombinant peptide thus produced is isolated by any conventional method and may be linked via conventional peptide bond forming chemistry to any suitably protected branched amino peptide moiety. The skilled person will be able to identify suitable protein isolation steps for purifying the peptide.

Methods of treatment

It is an aspect to provide W-Peptide analogues as defined according to the present invention for use as a medicament. In another aspect, the present invention provides methods for treatment, prevention or alleviation of a medical condition. Such methods according to the present invention in one embodiment comprise one or more steps of administration or release of an effective amount of a W-Peptide analogue according to the present invention, or a pharmaceutical composition comprising one or more such W-Peptides, to an individual in need thereof. In one embodiment, such steps of administration or release according to the present invention are simultaneous, sequential or separate.

An individual in need as referred to herein, is in one embodiment an individual that benefits from the administration of a W-Peptide analogue or pharmaceutical composition according to the present invention. Such an individual in one embodiment suffers from a disease or condition or is at risk of suffering therefrom. The individual is in one embodiment any human being, male or female, infant, middle-aged or old. The disorder to be treated or prevented in the individual in one embodiment relates to the age of the individual, the general health of the individual, the medications used for treating the individual and whether or not the individual has a prior history of suffering from diseases or disorders that may have or have induced the condition in the individual.

The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the W-Peptide analogue for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing" or "prevention" is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications. The patient to be treated is preferably a mammal, in particular a human being. Treatment of animals, such as mice, rats, dogs, cats, cows, horses, sheep and pigs, is, however, also within the scope of the present invention. The patients to be treated according to the present invention can be of various ages, for example, adults, children, children under 16, children age 6-16, children age 2-16, children age 2 months to 6 years or children age 2 months to 5 years.

Medical indications

The invention is in one embodiment directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic condition, an inflammatory condition, an infection and/or a metabolic condition.

The invention is in one embodiment directed to use of a W-Peptide analogue according to the present invention for the manufacture of a medicament for the treatment of an ischemic condition, an inflammatory condition, an infection and/or a metabolic condition.

The invention is in one embodiment directed to a method for treatment of an ischemic condition, an inflammatory condition, an infection and/or a metabolic condition, said method comprising administering an effective amount of a W-Peptide analogue according to the present invention to an individual in need thereof.

The invention is in one embodiment, directed to a W-Peptide analogue for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal.

In one embodiment said treatment is prophylactic, ameliorative and/or curative. In one embodiment, said mammal is a human (homo sapiens).

The invention in certain embodiments is also directed to a method for treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs, said method comprising the step of administering a therapeutically effective amount of a W- Peptide analogue according to the present invention to an individual in need thereof.

In a specific embodiment, the invention is also directed to use of a W-Peptide analogue for manufacturing of a medicament for the treatment of an ischemic and/or

inflammatory condition in the tissue of one or more organs of a mammal.

When referring to the tissue of one or more organs, said organ is in one embodiment selected from the group consisting of kidney, liver, brain, heart, muscles, bone marrow, skin, skeleton, lungs, the respiratory tract, spleen, exocrine glands, bladder, endocrine glands, reproduction organs including the phallopian tubes, eye, ear, vascular system, the gastroinstestinal tract including small intestines, colon, rectum, canalis analis and the prostate gland. In one embodiment, the ischemic and/or inflammatory condition in the tissue of one or more organs is an acute, subacute or chronic condition.

In one embodiment, the ischemic and/or inflammatory condition in the tissue of one or more organs is an ischemic condition. In another embodiment, the ischemic and/or inflammatory condition in the tissue of one or more organs is an inflammatory condition.

In a further embodiment, the ischemic condition in the tissue of one or more organs is secondary ischemia.

Secondary ischemia is ischemia which is caused by an underlying condition such that the ischemia typically is secondary to e.g. stroke, injury, septic shock, systemic hypotension, cardiac arrest due to heart attack, cardiac arrhythmia, atheromatous disease with thrombosis, embolism from the heart or from blood vessel from any organ, vasospasm, aortic aneurysm or aneurisms in other organs, coronary stenosis, myocardial infarction, angina pectoris, pericarditis, myocarditis, myxodemia, or endocarditis.

An aortic aneurysm is in one embodiment thoracal or abdominal or dissecting aortic aneurysm. Systemic hypotension is in one embodiment hypotension due to heart disease, hypotension due to systemic disease including infection or allergic reactions, or hypotension due to one or more toxic compound or poison(s) or drug(s).

In one embodiment said ischemic and/or inflammatory condition in the tissue of one or more organs is due to (or caused by) a condition selected from stroke, injury, septic shock, systemic hypotension, cardiac arrest due to heart attack, cardiac arrhythmia, atheromatous disease with thrombosis, embolism from the heart or from blood vessel from any organ, vasospasm, aortic aneurysm or aneurisms in other organs, coronary stenosis, myocardial infarction, angina pectoris, pericarditis, myocarditis, myxodemia, or endocarditis.

In one embodiment, said ischemic condition is myocardial ischemia.

In one embodiment said ischemic and/or inflammatory condition in the tissue of one or more organs is due to crdiac arrhythmia. In one embodiment, said cardiac arrhythmia is the primary disease or secondary to another condition of the individual, including acute infections particularly those affecting the lungs, pulmonary embolism, hypotension, shock, anoxaemia and anaemia. Cardiac arrhythmias include ventricular or supra ventricular tachyarrhythmias, atrioventricular block, sinus node disease, Wolff-Parkinson- White syndrome, Lenegres disease, Lev's disease any syndrome involving an abnormal myocardial connection between atrium and ventricle. In one embodiment, secondary ischemia can also be observed in connection with a range of other diseases and conditions, including but not limited to diabetes mellitus, hyperlipidaemia, thromboangiitis obliterans, Takayasu 's syndrome, arteritis temporalis, mucocutaneous lymph node syndrome (Kawasaki disease), cardiovascular syphilis, connective tissue disorders such as Raynaud 's disease, phlegmasia coerulae dolens, blood vessel trauma including iatrogene trauma such as cannulation, conditions with increased fasting levels of LDL-Cholesterol, triglycerid, and/or HDL- Cholesterol, retroperitoneal fibrosis, rheumatic diseases, systemic lupus erythematosus, polyarteritis nodosa, scleroderma, polymyositis, dermatomyositis, rheumatoid arthritis, neuromyopathic disorders such as progressive muscular dystrophy of Duchenne, Friedreich 's ataxia, and myotonic dystrophy, anaphylaxis, serum sickness, hemolytic anaemia, allergy, and allergic agranulocytosis. In one embodiment the peptides of the present invention are also be useful in the treatment or prevention of said conditions.

The invention is in one embodiment, directed to a W-Peptide analogue for use in the treatment of infection.

Many infections may have an influence on the tissue and disturb the normal function resulting in decreased performance, which in one embodiment is treated by administration of an effective dose of a W-Peptide analogue . In one embodiment, infections include infections by protozoa, virus, bacteria and fungus and include conditions such as AIDS, bacterial septicemia, systemic fungal infections, Rickettsial diseases, toxic shock syndrome, infectious mononucleosis, chlamydia thrachomatis, chlamydia psittaci, cytomegalovirus infection, Campylobacter, salmonella, influenza, poliomyelitis, toxoplasmosis, Lassa Fever, Yellow Fever, billharziose, colibacteria, enterococcus, preteus, klebsiella, pseudomonas, staphylococcus aureus,

staphylococcus epidermidis, Candida albicans, tuberculosis, mumps, infectious mononucleosis, hepatitis and Coxackie virus.

In one embodiment the condition to be treated is caused by a cancer or a by premalignant disorder having an impact on the organ, e.g. on the respiratory system including lung, bronchiole, upper airways, and/or on the heart and/or on the kidney and/or on the gastrointestinal system, including acute leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, lymphosarcoma, myeloma, metastasizing carcinoma of any origin. In one embodiment the W-Peptide analogue are used in the treatment or prevention of said conditions.

In one embodiment, the ischemic and/or inflammatory condition in the tissue of one or more organs is caused by a physical trauma including electromagnetic radiation. Surgery and transplantation

Major surgical interventions including cardiothoracic surgery, abdominal surgery, surgery on the aorta and other major blood vessels, as well as organ transplantation such as lung or heart or combined lung and heart transplantation, liver transplantation or renal transplantation induce a systemic inflammatory response (SIR; or systemic inflammatory response syndrome SIRS) and is associated with post-surgical organ dysfunction including development of renal failure.

Renal failure is a consequence of the SIR and the reduced blood flow generated during the surgical intervention. The result is post-surgical acute kidney injury (AKI) which for a large fraction deteriorates into chronic renal failure. Currently no efficient treatment modality exists to prevent the development of renal failure. Post-surgical renal failure may be defined as a more than 25% reduction in Glomerular filtration rate (GFR) present 3 month after the surgical intervention.

Major cardiac surgery such as repair of one or more cardiac valves, cardiac artery bypass grafting (CABG), surgery on the aortic root, or aortic branch including the common carotic arteries, or combined cardiac surgery such as valve(s) replacement and CABG and/or aortic root surgery is associated with development of renal impairment that, when present, is associated with increased morbidity and mortality.

In one embodiment, treatment with a W-Peptide analogue according to the present invention reduce the degree of renal impairment. In one embodiment this is achieved by reducing the fall in GFR post-surgery; by reducing the degree of post-surgical increases in serum creatinine or cystatin C or the more immediate increases in urinary excretion of AKI markers NGAL, IL18 or KIM-1 ; and/or or by reducing the degree of post-surgical SIR (for example by reduced circulating levels of IL-6 and other proinflammatory markers). Lung transplantation (LTX) is the ultimate treatment modality for end-stage lung disease. The major challenges associated with LTX are scarcity of donors, acute and chronic rejection of the transplanted lungs and side-effects of immune suppressive treatment including development of chronic renal failure (CRF). While there has been a good development in the treatment of acute rejection by newer immunosuppressive drugs leading to fewer episodes of acute rejection within the first year, fewer organ losses, fewer side effects, fewer infections, and less invasive monitoring methods, the control of chronic organ rejection has not greatly improved and the half-life time in terms of how many years 50% of the patients survive has only marginally improved during the last 2 decades to around 7 years. Side effects of the immunosuppressive treatment are dominated by 2 major challenges: Nephrotoxicity and post-transplant lymphoproliferative diseases (PTLD), where the latter can be considered as a consequence of the degree of immune-suppression needed to avoid chronic organ rejection - "too much" keeps the rejection on distance, but gives infections and PTLD, while giving "too little" puts the patients at an increased risk of rejecting the graft. Neprotoxicity and development of CRF is despite of extensive research during the last 30 years, still a significant problem. Five years after LTX none of the patients retain normal kidney function and 20% of the long term survivors will end with a kidney transplant as well.

Calcineurin inhibitor treatment (Tacrolimus, Cyclosporin A) is the corner-stone in the immune-suppressive treatment strategy for successful solid organ transplantation. The limiting factor in using calcineurin inhibitors is the acute and chronic irreversible nephrotoxicity. Recent data indicate that kidney function (measured as reduction in GFR) is reduced with 40% within the first 14 days after LTX and that this reduction is irreversible.

Heart transplantation (HTX) is the ultimate treatment modality for end-stage heart failure. As for LTX the major challenges associated with HTX are scarcity of donors, acute and chronic rejection of the transplanted hearts and side-effects of immune suppressive treatment including development of CRF. Like for LTX the number of patients with retained kidney function over time is limited or absent and like LTX a major reduction i kidney function is present already two to four weeks post

transplantation.

This dramatic effect on kidney function seen after LTX and HTX is probably not caused by calcineurin inhibitor treatment alone, but is the final result of the surgical and anesthesiological trauma in combination with the organ ischemia and side effects of antibiotic, antiviral, antifungal and immunsuppressive drugs. Consequently, in one embodiment pharmacological intervention by employment of the W-Peptide analogues according to the present invention will reduce the degree of renal impairment associated with organ transplantation, such as LTX and HTX. Surgery, as is outlined herein above in detail, including organ transplantation, may thus be the cause of secondary ischemia.

The invention is thus in one embodiment directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal, wherein said ischemic and/or inflammatory condition is associated with surgery. In one embodiment said surgery is major surgery or major surgical intervention. In one embodiment, said surgery is selected from the group consisting of

cardiothoracic surgery, abdominal surgery, surgery on the aorta and/or other major blood vessels, repair of one or more cardiac valves, cardiac artery bypass grafting (CABG), surgery on the aortic root or the aortic branch including the common carotic arteries, and combined cardiac surgery such as valve(s) replacement and CABG and/or aortic root surgery.

In one embodiment, said surgery encompasses surgical insertion transplants, devices, grafts, prostheses or other biomedical compounds or devices inserted by surgical operations.

In one embodiment, said major surgery comprises organ transplantation. It follows that the invention in one embodiment is directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal, wherein said ischemic and/or inflammatory condition is associated with organ transplantation. In one embodiment, said organ transplantation is solid organ transplantation.

In one embodiment said solid organ transplantation is heart transplantation, lung transplantation, combined heart and lung transplantation, liver transplantation or kidney (renal) transplantation.

The invention in another embodiment is directed to a W-Peptide analogue according to the present invention for use in the treatment of post-surgical systemic inflammatory response syndrome (SIRS), post-surgical organ dysfunction and/or post-surgical renal failure such as acute kidney injury (AKI), neprotoxicity and/or chronic renal failure (CRF).

The invention is in one embodiment directed to a W-Peptide analogue according to the present invention for reducing the degree of renal impairment associated with major surgery, in one embodiment organ transplantation.

Reperfusion injury is tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. The absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.

Reperfusion injuries may occur in connection with surgery, such as major surgical interventions including organ transplantations. It is a primary concern when performing liver transplantations, and also during cardiac surgery.

In a particular embodiment, said ischemic and/or inflammatory condition in the tissue of one or more organs is associated with reperfusion injury. Thus, in one embodiment the present invention is directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal, wherein said ischemic and/or inflammatory condition is associated with reperfusion injury. In some embodiments, the peptides or compositions of the present invention are to be administered before and/or during surgery and/or organ transplantation.

Toxins and drugs

In one embodiment the ischemic and/or inflammatory condition in the tissue of one or more organs as described herein is caused by toxin- or drug-induced cell, tissue or organ failure.

The invention is thus in one embodiment directed to a W-Peptide analogue according to the present invention for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal, wherein said ischemic and/or inflammatory condition is caused (or induced) by toxin- or drug-induced cell, tissue or organ failure.

Said drug includes but are not restricted to cancer chemotherapeutics including cisplatin, carboplatin, dacarbezine, procarbazine, altretamine, semustine, lomustine, carmustine, busulfan, thiotepa, melphalan, cyclophosphamide, chlorambucil, mechlorethamine, azadtidine, cladrrbine, cytorabine, fludarabine, fluorouracil, mercaptopurine, metrotrexate, thioguanine, allopurinol, bleomycin, dactinomycin, daunorubicin, docetaxel, doxorubicin (adriamycin), etoposide, idarubicin, irinotecan, mitomycin, paclitaxel, plicamycin, topotecan, vinblastine, vincristine, vinorelbine, amasacrine, asparaginase, hydroxyurea, mititane, mitoxantrone; Antibiotics as aminoglycosides including streptomycin, neomycin, kanamycin, amikacin, gentamicin, tobramycin, sisomicin and nitilmicin; immunodepressive compounds as cyclosporine; tricyclic antidepressants, lithium salts, prenylamine and phenothizine derivatives.

Inflammatory conditions

Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. Loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to chronic inflammation. Chronic inflammation is extremely relevant in today's modern medicine, as it contributes to the pathogenesis of the most important diseases of the industrialized societies including atherosclerosis, acute and chronic heart failure, cancer, diabetes, and obesity-associated diseases. Recent insight into endogenous anti-inflammatory pathways have identified a number of natural anti-inflammatory and pro-resolving molecules and pathways suitable for pharmacological intervention that would make it possible to develop drugs that mimic the natural course of resolving inflammation. Among these natural anti-inflammatory and pro-resolving pathways is Annexin A1 acting through FPR2 stimulation.

The immune modulating effects of FPR-2 agonists are exerted through inhibition of inflammatory mediators and by inhibition of inflammatory cell migration. FPR-2 agonists exert these effects in a variety of cells including monocytes, macrophages, subtypes of T-cells, endothelial cells and epithelial cells. Joint diseases such as rheumatoid arthritis (RA) and gout are characterized by episodes with acute exacerbations, in RA the exacerbations (often described as flairs) typically develop on top of chronic symptoms and develop despite intense

pharmacological treatment. A similar pattern can be seen in gout, with the major difference that most gout patients are without symptom between the exacerbations. In both conditions significant neutrophil infiltration into the synovial membrane and joint fluid are the primary pathological hallmark of the exacerbations. The most important pro-inflammatory effectors involved include ΙΙ_-1 β, TNF-α, IL-6, IL-8, and COX-2. Resolution of the acute exacerbations to avoid development or deterioration of chronic inflammation involves activation of macrophages to phagocyte the apoptotic neutrophils.

Consequently in one embodiment it would be attractive to apply treatment with a W- Peptide analogue according to the present invention to joint diseases, not at least in order to reduce the severity of exacerbations in existing disease as flairs in rheumatoid arthritis would have major clinical impact. However, not only joint diseases are associated with exacerbations of symptoms. Neurodegenerative diseases such as multiple sclerosis have flair-like exacerbations where treatment with a W-Peptide analogue e according to the present invention in one embodiment could reduce the symptoms and eventually as for Joint diseases reduce the overall deterioration of the patients' functional level.

The invention is thus in one embodiment directed to a W-Peptide analogue according to the present invention for use in the treatment of an inflammatory condition in the tissue of one or more organs of a mammal, wherein said ischemic and/or inflammatory condition is an inflammatory disease.

In one embodiment, said inflammatory disease is Arthritis. In one embodiment, said inflammatory disease is selected from the group consisting of an arthropathy (a disease of a joint, Arthritis (including diseases associated with arthritis), osteoartritis, rheumatoid arthritis; spondylarthropathies (e.g. ankylosing spondilitis), reactive arthritis (including arthritis following rheumatic fever), Henoch-Schonlein purpura, Reiter's disease, Juvenile Chronic arthritis including Still 's disease, juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, psoriasis, osteoarthritis, osteoarthritis secondary to hypermobilty, congenital dysplasias, slipped femoral epiphysis, Perthes' disease, intra-articular fractures, meniscectomy, obesity, recurrent dislocation, repetitive actions, crystal depositions and diseases and metabolic abnormalities of cartilage including pyrophosphate arthropathy, ochronosis, haemochromatosis, avascular necrosis including Sickle Cell disease, therapy with corticoids or other drugs, Caisson disease, septic or infectious arthitis (including tuberculous arthritis, meningococcal arthritis, gonococcal arthritis, salmonella arthritis), infective

endocarditis, viral arthritis, recurrent haemarthrosis, and all kinds of deposition diseases such as Gout, pyrophosphate arthopathy and acute calcific periarthritis. In one embodiment, said inflammatory disease is a connective tissue disorder; in one embodiment selected from the group consisting of systemic lupus erythematosus, polymyositis/dermatomyositis, systemic sclerosis, mixed connective tissue disease, sarcoidosis and primary Sjogrens syndrome including keratoconjunctivitis sicca, polymyalgia rheumatica, and other types of vasculitis, crystal deposition diseases (including gout), pyrophosphate arthropathy, and acute calcific periarthritis.

In one embodiment, said inflammatory disease is a soft-tissue rheumatism including bursitis, tenosynovitis or peritendonitis, enthesitis, nerve compression, periarthritis or capsulitis, muscle tension and muscle dysfunction.

In one embodiment, said inflammatory disease is selected from the group consisting of vasculitis including vasculitis secondary to rheumatoid arthritis, infective vasculitis due to infections with bacterial species including spirochaetal diseses as Lyme disease, syphilis, rickettsial and mycobacterial infections, fungal, viral or protozoal infections, non-infective vasculitis secondary to hypersensibility and leucocytoplastic vasculitis including Serum Sickness and Henoch-Schonlein purpura, Drug induced vasculitis, essential mixed cryoglobulinaemia, hypocomplentaemia, Vasculitis associated with other kinds of malignancy, non-infective vascultitis including Takayasu's

arteritis/disease, Giant Cell Arteritis (Temporal arteritis and polymyalgia rheumatica), Buerger's disease, polyarteritis nodosa, microscopic polyarteritis, Wegener's granulomatose, Churg-Strauss syndrome, and vasculitis secondary to connective tissue diseases including Systemic Lupus Erythematosus, Polymyositis/

Dermatomyositis, Systemic Sclerosis, Mixed Connetive Tissue Disease, sarcoidosis and Primary Sjogrens syndrome. In one embodiment, said inflammatory disease is inflammatory diseases of the gastrointestinal system. Said inflammatory diseases of the gastrointestinal system may be selected from the group consisting of inflammatory bowel disease, coeliac disease, gluten sensitive enteropathy, eosinophilic gastroenteritis, intestinal lympangiectasia, inflammatory bowel disease (including Chrohn's disease and ulcerative colitis), diverticular disease of the colon, radiation enteritis, irritable bowel syndrome, Whipple 's diease, stomatitis of all kinds, salivary gland diseases (such as sarcoidosis, salivary duct obstruction and Sjogrens syndrome), inflammaton of the oesophagus (e.g. due to gastro- oesophagel reflux or infections with Candida species, herpes simplex and cytomegalus virus), inflammatory diseases of the stomach (including acute and chronic gastritis, helicobacter pylori infection and Mentriers disease), and inflammation of the small intestine.

In one embodiment, said inflammatory disease is a neurodegenerative disease, such as a neurodegenerative disease having an inflammatory component, such as multiple sclerosis (MS).

In one embodiment, said inflammatory disease is selected from the group consisting of dermatitis, pemfigus, bulloid pemphigoid, benign mucous membrane pemphigoid, dermatitis herpitiformis, tropical sprue, systemic amyloidosis, primary biliary cirrhosis, Goodpasture syndrome, all kinds of deposition diseases as Gout, pyrophosphate arthopathy and acute calcific periarthritis, pancreatitis, septic discitis, tuberculosis, malignancies (such as matastases, myeloma and others), spinal tumours, ancylosing spondylitis, acute disc prolapse, chronic disc disease/osteoarthritis, osteoporosis, and osteomalacia, Pagets disease, hyperparathyroidism, renal osteodystrophy, spondylolisthesis, spinal senosis congenital abnormalities and fibromyalgia.

In one embodiment, said inflammatory disease is selected from the group consisting of upper and lower airway diseases such as chronic obstructive pulmonary diseases (COPD), allergic and non-allergic asthma, allergic rhinitis, allergic and non-allergic conjunctivitis, allergic and non-allergic dermatitis and lung inflammation. Further active ingredients

In some embodiments, the W-Peptide analogues of the present invention are combined with or comprise one or more further active ingredients which are understood as other therapeutic compounds or pharmaceutically acceptable derivatives thereof.

Methods for treatment according to the present invention in one embodiment thus further comprise one or more steps of administration of one or more further active ingredients, either concomitantly or sequentially, and in any suitable ratios. Methods of treatment according to the present invention in one embodiment include a step wherein the pharmaceutical composition or W-Peptide analogue as defined herein is administered simultaneously, sequentially or separately in combination with one or more further active ingredients. Administration and dosage

According to the present invention, a composition comprising a W-Peptide analogue as defined herein is in one embodiment administered to individuals in need thereof in pharmaceutically effective doses or a therapeutically effective amount. A therapeutically effective amount of a W-Peptide analogue according to the present invention is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount".

In one embodiment of the present invention, the composition is administered in doses of from 1 μg day to 100 mg/day; such as from 1 μg/day to 10 Mg/day, such as 10 Mg/day to 100 Mg/day, such as 100 Mg/day to 250 Mg/day, such as 250 Mg/day to 500 Mg/day, such as 500 Mg/day to 750 Mg/day, such as 750 Mg/day to 1 mg/day, such as 1 mg/day to 2 mg/day, such as 2 mg/day to 5 mg/day, or such as 5 mg/day to 10 mg/day, such as 10 mg/day to 20 mg/day, such as 20 mg/day to 30 mg/day, such as 30 mg/day to 40 mg/day, such as 40 mg/day to 50 mg/day, such as 50 mg/day to 75 mg/day, or such as 75 mg/day to 100 mg/day. In one embodiment of the present invention, one single dose of the composition is administered and may comprise of from 1 μg kg body weight to 100 mg/kg body weight; such as from 1 to 10 μg kg body weight, such as 10 to 100 g/day, such as 100 to 250 μg kg body weight, such as 250 to 500 μg kg body weight, such as 500 to 750 μg kg body weight, such as 750 μg kg body weight to 1 mg/kg body weight, such as 1 mg/kg body weight to 2 mg/kg body weight, such as 2 to 5 mg/kg body weight, such as 5 to 10 mg/kg body weight, such as 10 to 20 mg/kg body weight, such as 20 to 30 mg/kg body weight, such as 30 to 40 mg/kg body weight, such as 40 to 50 mg/kg body weight, such as 50 to 75 mg/kg body weight, or such as 75 to 100 mg/kg body weight.

In one embodiment, a dose according to the present invention is administered one or several times per day, such as from 1 to 6 times per day, such as from 1 to 5 times per day, such as from 1 to 4 times per day, such as from 1 to 3 times per day, such as from 1 to 2 times per day, such as from 2 to 4 times per day, such as from 2 to 3 times per day.

Routes of administration

It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the active ingredient chosen.

Systemic treatment

In one embodiment, the route of administration allows for introducing the peptide analogue into the blood stream to ultimately target the sites of desired action.

In one embodiment the routes of administration is any suitable routes, such as an enteral route (including the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and/or a parenteral route (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal administration). Appropriate dosage forms may be prepared by conventional techniques. Parenteral administration

Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration or subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.

Accordingly, the peptide analogue or composition is in one embodiment administered topically to cross any mucosal membrane of an animal to which the substance or peptide is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, for example the mucosa of the nose, or mouth, and accordingly, parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonal and bronchial administration by inhalation or installation. In some embodiments, the peptide analogue is administered topically to cross the skin.

In one embodiment, the intravenous, subcutaneous and intramuscular forms of parenteral administration are employed.

Local treatment

In one embodiment, the peptide analogue or composition according to the invention is used as a local treatment, i.e. is introduced directly to the site(s) of action. Accordingly, the peptide may be applied to the skin or mucosa directly, or the peptide may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue.

Pharmaceutical formulations

In one embodiment the W-Peptide analogues or pharmaceutically acceptable derivatives thereof are administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical compositions or peptides according to the invention may be formulated with

pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 20 Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2000.

The term "pharmaceutically acceptable derivative" in the present context includes pharmaceutically acceptable salts, which indicate a salt which is not harmful to the patient. Such salts include pharmaceutically acceptable basic or acid addition salts as well as pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts. A pharmaceutically acceptable derivative further includes

pharmaceutically acceptable esters, prodrugs, or other precursors of a compound which may be biologically metabolized into the active compound, or crystal forms of a compound.

The pharmaceutical composition or pharmaceutically acceptable composition may be specifically formulated for administration by any suitable route, such as an enteral route, the oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal, intraperitoneal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route.

Pharmaceutical compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient, such as sustained or prolonged release, according to methods well known in the art. In the same solid dosage form two active ingredients may be combined so as to provide controlled release of one active ingredient and immediate release of another active ingredient.

Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions, as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable formulations are also regarded as being within the scope of the present invention. Other suitable administration forms include suppositories, sprays, ointments, cremes/lotions, gels, inhalants, dermal patches, implants, etc.

In one embodiment, a W-Peptide analogue according to the present invention is generally utilized as the free substance or as a pharmaceutically derivative such as a pharmaceutically acceptable ester or such as a salt thereof. Examples of the latter are: an acid addition salt of a compound having a free base functionality, and a base addition salt of a compound having a free acid functionality. The term "pharmaceutically acceptable salt" refers to a non-toxic salt of a W-Peptide analogue for use according to the present invention, which salts are generally prepared by reacting a free base with a suitable organic or inorganic acid, or by reacting an acid with a suitable organic or inorganic base. When a W-Peptide analogue according to the present invention contains a free base functionality, such salts are prepared in a conventional manner by treating a solution or suspension of the compound with a chemical equivalent of a pharmaceutically acceptable acid. When a W-Peptide analogue according to the present invention contains a free acid functionality, such salts are prepared in a conventional manner by treating a solution or suspension of the compound with a chemical equivalent of a pharmaceutically acceptable base. Physiologically acceptable salts of a W-Peptide analogue with a hydroxy group include the anionic form of the compound in combination with a suitable cation, such as sodium or ammonium ion.

Other salts which are not pharmaceutically acceptable may be useful in the preparation of W-Peptide analogues, and these form a further aspect of the invention.

Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, trifluoroacetate, trichloroacetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1 ,1 '-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

In one embodiment of the present invention, the W-Peptide analogues of the present invention are in crystalline forms, for example co-crystallized forms or hydrates of crystalline forms. The term "prodrug" refers to peptides that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood or by metabolism in cells, such as for example the cells of the basal ganglia. A thorough discussion is provided in T. Higuchi and V Stella, "Pro-drugs as Novel Delivery

Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference. Examples of prodrugs include pharmaceutically acceptable, non-toxic esters of the compounds of the present invention. Esters of the compounds of the present invention may be prepared according to conventional methods "March's Advanced Organic Chemistry, 5 ^th Edition". M. B. Smith & J. March, John Wiley & Sons, 2001.

In one embodiment, for parenteral administration, solutions of W-Peptide analogues according to the present invention in sterile aqueous solution, in aqueous propylene glycol or in sesame or peanut oil are employed. Aqueous solutions should be suitably buffered where appropriate, and the liquid diluent rendered isotonic with, e.g., sufficient saline or glucose. Aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media to be employed are all readily available by standard techniques known to those skilled in the art.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Moreover, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed by combining the compounds according to the present invention and the pharmaceutically acceptable carriers are then readily administered in a variety of dosage forms suitable for the disclosed routes of administration. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy. Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules or tablets, which each contain a predetermined amount of the active ingredient, and which may include a suitable excipient.

Furthermore, the orally available formulations may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in- water or water-in-oil liquid emulsion.

Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatine or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or a soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions may contain the compound for use according to the present invention in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium

carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present.

The pharmaceutical compositions comprising W-Peptide analogues according to the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agent. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compositions may also be in the form of suppositories for rectal administration of the compounds of the invention. These compositions can be prepared by mixing the W- Peptide analogues with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will thus melt in the rectum to release the drug. Such materials include, for example, cocoa butter and polyethylene glycols. W-Peptide analogues of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes may be formed from a variety of phospholipids, such as but not limited to cholesterol, stearylamine or

phosphatidylcholines.

In addition, some W-Peptide analogues of the present invention may form solvates with water or common organic solvents. Such solvates are also encompassed within the scope of the invention. Thus, a further embodiment provides a pharmaceutical composition comprising a W- Peptide analogue according to the present invention, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents. Examples of Sequences

2-amino acid BAPs

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Abu-DMet

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet

Ac-(Ac-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

The C-terminal Met/DMet may be amidated (-NH ₂ ). Trp-Lys-Tyr-Met-Val-DMet-(Ac-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Pro-DMet-(Ac-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Abu-DMet-(Ac-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Aib-DMet-(Ac-Lys)Lys-NH ₂

Trp-Orn-Tyr-Met-Val-DMet-(Ac-Lys)Lys-NH ₂

Trp-Lys-Tyr-lle-Val-DMet-(Ac-Lys)Lys-NH ₂

Trp-Lys-Phe-Met-Val-DMet-(Ac-Lys)Lys-NH ₂

The N-terminal Trp may be acetylated (Ac).

Trp-[(Ac-Lys)Lys]Lys-Tyr-Met-Val-DMet

Trp-[(Ac-Lys)Lys]Lys-Tyr-Met-Pro-DMet

Trp-[(Ac-Lys)Lys]Lys-Tyr-Met-Abu-DMet

Trp-[(Ac-Lys)Lys]Lys-Tyr-Met-Aib-DMet

Trp-[(Ac-Lys)Lys]Orn-Tyr-Met-Val-DMet

Trp-[(Ac-Lys)Lys]Lys-Tyr-lle-Val-DMet

Trp-[(Ac-Lys)Lys]Lys-Phe-Met-Val-DMet

The N-terminal Trp may be acetylated (Ac) and the C-terminal Met/DMet may be amidated (-NH ₂ ).

3-amino acid BAPs

Ac-( Ac- Lys- Lys ) Ly s-Trp- ^■ Lys-Tyr-Met-Val-DMet

Ac-(Ac- Ly s- Lys ) Ly s-Trp- ^■ Lys-Tyr-Met-Pro-DMet

Ac-(Ac- Ly s- Lys ) Ly s-Trp- ^■ Lys-Tyr-Met-Abu-DMet

Ac-( Ac- Ly s- Lys ) Ly s-Trp- ^■ Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys-Lys)Lys-Trp- ^■ Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Lys)Lys-Trp- ^■ Lys-Tyr-lle-Val-DMet

Ac-( Ac- Ly s- Lys ) Ly s-Trp- ^■ Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Tyr-Met-Abu-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Tyr-lle-Val-DMet

Ac-(Ac-Lys)Lys-Lys-Trp- ^■ Lys-Phe-Met-Val-DMet Ac-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac- Ly s-(Ac- Ly s ) Ly s-Trp- Ly s-Ty r- M et- P ro- D M et

Ac-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Abu-DMet

Ac-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet

Ac-Lys-(Ac-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet

Ac-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet

Ac-Lys-(Ac-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

The C-terminal Met/DMet may be amidated (-NH ₂ ).

Trp-Lys-Tyr-Met-Val-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Pro-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Abu-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Aib-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Orn-Tyr-Met-Val-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-lle-Val-DMet-(Ac-Lys-Lys)Lys-NH ₂

Trp-Lys-Phe-Met-Val-DMet-(Ac-Lys-Lys)Lys-NH ₂

The N-terminal Trp may be acetylated (Ac).

Trp-[(Ac-Lys-Lys)Lys]Lys-Tyr-Met-Val-DMet

Trp-[(Ac-Lys-Lys)Lys]Lys-Tyr-Met-Pro-DMet

Trp-[(Ac-Lys-Lys)Lys]Lys-Tyr-Met-Abu-DMet

Trp-[(Ac-Lys-Lys)Lys]Lys-Tyr-Met-Aib-DMet

Trp-[(Ac-Lys-Lys)Lys]Orn-Tyr-Met-Val-DMet

Trp-[(Ac-Lys-Lys)Lys]Lys-Tyr-lle-Val-DMet

Trp-[(Ac-Lys-Lys)Lys]Lys-Phe-Met-Val-DMet

The N-terminal Trp may be acetylated (Ac) and the C-terminal Met/DMet may be amidated (-NH ₂ ).

4-amino acid BAPs

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Abu-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet

Ac-(Ac-Lys-Lys-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys-Gly-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Gly-Lys)Lys- Trp-Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys-Gly-Lys)Lys- Trp-Lys-Tyr-Met-Abu-DMet

Ac-(Ac-Lys-Gly-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys-Gly-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Gly-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet

Ac-(Ac-Lys-Gly-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys-Lys-Gly)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Lys-Gly)Lys- Trp-Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys-Lys-Gly)Lys- Trp-Lys-Tyr-Met-Abu-DMet bo

Ac-(Ac-Lys-Lys-Gly)Lys-Trp-Lys-Tyr-Met-Aib-DMet Ac-(Ac-Lys-Lys-Gly)Lys-Trp-Orn-Tyr-Met-Val-DMet Ac-(Ac-Lys-Lys-Gly)Lys-Trp-Lys-Tyr-lle-Val-DMet Ac-(Ac-Lys-Lys-Gly)Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys-Lys)Lys-Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-( Ac- Lys- Lys ) Lys- Lys- Trp-Lys-Ty r- Met- P ro- D Met Ac-(Ac-Lys-Lys)Lys-Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-(Ac-Lys-Lys)Lys-Lys-Trp-Lys-Tyr-Met-Aib-DMet Ac-(Ac-Lys-Lys)Lys-Lys-Trp-Orn-Tyr-Met-Val-DMet Ac-(Ac-Lys-Lys)Lys-Lys-Trp-Lys-Tyr-lle-Val-DMet Ac-(Ac-Lys-Lys)Lys-Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-Lys-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-Lys-(Ac-Lys-Lys)Lys- Trp-Lys-Tyr-Met-Pro-DMet Ac-Lys-(Ac-Lys-Lys)Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-Lys-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet Ac-Lys-(Ac-Lys-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet Ac-Lys-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet Ac-Lys-(Ac-Lys-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Lys-Tyr-Met-Pro-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Lys-Tyr-Met-Aib-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Orn-Tyr-Met-Val-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Lys-Tyr-lle-Val-DMet Ac-(Ac-Lys)Lys-(Ac-Lys-)Lys- Trp-Lys-Phe-Met-Val-DMet Ac-Lys-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-Lys-Lys-(Ac-Lys)Lys- Trp-Lys-Tyr-Met-Pro-DMet Ac-Lys-Lys-(Ac-Lys)Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-Lys-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Aib-DMet Ac-Lys-Lys-(Ac-Lys)Lys-Trp-Orn-Tyr-Met-Val-DMet Ac-Lys-Lys-(Ac-Lys)Lys-Trp-Lys-Tyr-lle-Val-DMet Ac-Lys-Lys-(Ac-Lys)Lys-Trp-Lys-Phe-Met-Val-DMet

Ac-Lys-(Ac-Lys)Lys-Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Lys-Tyr-Met-Pro-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Lys-Tyr-Met-Aib-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Orn-Tyr-Met-Val-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Lys-Tyr-lle-Val-DMet Ac-Lys-(Ac-Lys)Lys-Lys- Trp-Lys-Phe-Met-Val-DMet

Ac-(Ac-Lys)Lys-Lys-Lys-Trp-Lys-Tyr-Met-Val-DMet Ac-(Ac-Lys)Lys-Lys-Lys- Trp-Lys-Tyr-Met-Pro-DMet Ac-(Ac-Lys)Lys-Lys-Lys- Trp-Lys-Tyr-Met-Abu-DMet Ac-(Ac-Lys)Lys-Lys-Lys-Trp-Lys-Tyr-Met-Aib-DMet Ac-(Ac-Lys)Lys-Lys-Lys-Trp-Orn-Tyr-Met-Val-DMet Ac-(Ac-Lys)Lys-Lys-Lys-Trp-Lys-Tyr-lle-Val-DMet Ac-(Ac-Lys)Lys-Lys-Lys-Trp-Lys-Phe-Met-Val-DMet Ac-(Ac-Lys-Gly)Lys-Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Lys-Tyr-Met-Pro-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Lys-Tyr-Met-Abu-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Lys-Tyr-Met-Aib-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Orn-Tyr-Met-Val-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Lys-Tyr-lle-Val-DMet

Ac-(Ac-Lys-Gly)Lys-Lys- Trp-Lys-Phe-Met-Val-DMet

Ac-Lys-(Ac-Lys-Gly)Lys-Trp-Lys-Tyr-Met-Val-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Lys-Tyr-Met-Pro-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Lys-Tyr-Met-Abu-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Lys-Tyr-Met-Aib-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Orn-Tyr-Met-Val-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Lys-Tyr-lle-Val-DMet

Ac-Lys-(Ac-Lys-Gly)Lys- Trp-Lys-Phe-Met-Val-DMet

The C-terminal Met/DMet may be amidated (-NH ₂ ).

Trp-Lys-Tyr-Met-Val-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Pro-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Abu-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-Met-Aib-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Orn-Tyr-Met-Val-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Lys-Tyr-lle-Val-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

Trp-Lys-Phe-Met-Val-DMet-(Ac-Lys-Lys-Lys)Lys-NH ₂

The N-terminal Trp may be acetylated (Ac).

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Tyr-Met-Val-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Tyr-Met-Pro-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Tyr-Met-Abu-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Tyr-Met-Aib-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Orn-Tyr-Met-Val-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Tyr-lle-Val-DMet

Trp-[(Ac-Lys-Lys-Lys)Lys]Lys-Phe-Met-Val-DMet

The N-terminal Trp may be acetylated (Ac) and the C-terminal Met/DMet may be amidated (-NH ₂ ).

ITEMS

1 . A W-Peptide analogue comprising a W-Peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino alkyl amino acid residue,

said first amino alkyl amino acid residue optionally being covalently linked to a second amino alkyl amino acid residue, or to a second and a third amino alkyl amino acid residue, to form a linear chain of 2 or 3 amino alkyl amino acid residues,

wherein the side chain of one or more of said first, second and/or third amino alkyl amino acid residues are each modified by attaching to the side chain amino group a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) _p - AAA _q ; AAA _q -(aa ₃ ) _P ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P ;

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala,

wherein said first amino alkyl amino acid residue is covalently linked to the N- terminus of said W-Peptide, covalently linked to the C-terminus of said W-Peptide, and/or covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide,

with the proviso that said branched amino acid probe consists of 2 to 9 amino acid residues.

The W-Peptide analogue according to item 1 , wherein said amino alkyl amino acid residue is an amino acid with a side chain comprising an amino alkyl group (- C _n H2nNH ₂ ); such as a side chain amino alkyl group selected from the group consisting of methylamine (-CH ₂ NH ₂ ), ethylamine (-C2H4NH2), propylamine (. C ₃ H ₆ NH ₂ ), n-butylamine (-C4H ₈ NH ₂ ), pentylamine (-C ₅ H ₁ oNH2), n-hexylamine (. C ₆ H ₁₂ NH ₂ ), heptylamine (-C ₇ H ₁₄ NH ₂ ), octylamine (-C ₈ H ₁₆ NH ₂ ), nonylamine (.

C ₉ H ₁₈ NH ₂ ), decylamine (-CioH ₂₀ NH ₂ ), undecylamine (-CnH ₂₂ NH ₂ ) and

dodecylamine (-Ci ₂ H ₂₄ NH ₂ ).

The W-Peptide analogue according to the preceding items, wherein the side chain amino group of said amino alkyl amino acid residue is selected from the group consisting of the β-amino group (methylamine); the γ-amino group (ethylamine); the δ-amino group (propylamine), the ε-amino group (n-butylamine); the ζ-amino group (pentylamine); the η-amino group (n-hexylamine); the θ-amino group (heptylamine); the i-amino group (octylamine); the κ-amino group (nonylamine); the λ-amino group (decylamine); the μ-amino group (undecylamine); and the v- amino group (dodecylamine). The W-Peptide analogue according to the preceding items, wherein said branched amino acid probe comprises

a. a first amino alkyl amino acid residue, or

b. a first and a second amino alkyl amino acid residue, or

c. a first, a second and a third amino alkyl amino acid residue.

The W-Peptide analogue according to the preceding items, wherein said branched amino acid probe comprises

a. a first amino alkyl amino acid residue, wherein the N-terminus of said first amino alkyl amino acid residue is acetylated,

b. a first and a second amino alkyl amino acid residue, wherein the N-terminus of said second amino alkyl amino acid residue is acetylated, or

c. a first, a second and a third amino alkyl amino acid residue, wherein the N- terminus of said third amino alkyl amino acid residue is acetylated.

The W-Peptide analogue according to the preceding items, wherein said branched amino acid probe comprises

a. a first amino alkyl amino acid residue, wherein the C-terminus of said first amino alkyl amino acid residue is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ),

b. a first and a second amino alkyl amino acid residue, wherein the C-terminus of said second amino alkyl amino acid residue is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ), or

c. a first, a second and a third amino alkyl amino acid residue, wherein the C- terminus of said third amino alkyl amino acid residue is a carboxylic acid, an aldehyde, an ester, or an amide, such as a primary amide (CONH ₂ ). The W-Peptide analogue according to the preceding items, wherein the N-terminal amino acid residue of the molecule is acetylated at the alpha amino group. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first amino alkyl amino acid residue, said first amino alkyl amino acid residue being optionally N-terminal acetylated or C-terminal amidated, wherein the side chain amino group of said first amino alkyl amino acid residue is modified by attaching a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) _P -AAA _q ; AAA _q -(aa ₃ ) _P ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala, and the N-terminal AAA or (aa) ₃ of the molecule is optionally acetylated. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first amino alkyl amino acid residue covalently linked to a second amino alkyl amino acid residue, to form a linear chain of 2 amino alkyl amino acid residues,

said second amino alkyl amino acid residue being optionally N-terminal acetylated or C-terminal amidated,

wherein the side chain amino group of said first and/or said second amino alkyl amino acid residue is modified by attaching a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) _p -AAA _q ; AAA _q -(aa ₃ ) _p ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _p ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala, and the N- terminal AAA or (aa) ₃ of the molecule is optionally acetylated. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first and a second amino alkyl amino acid residue, wherein the side chain of said first amino alkyl amino acid residue is modified by attaching a molecule to the side chain amino group. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first and a second amino alkyl amino acid residue, wherein the side chain of said second amino alkyl amino acid residue is modified by attaching a molecule to the side chain amino group. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first amino alkyl amino acid residue covalently linked to a second and a third amino alkyl amino acid residue to form a linear chain of 3 amino alkyl amino acid residues,

said third amino alkyl amino acid residue being optionally N-terminal acetylated or C-terminal amidated,

wherein the side chain amino group of said first, second and/or third amino alkyl amino acid residues is modified by attaching a molecule independently selected from the group consisting of AAA _q -AAA; (aa ₃ ) -AAA _q ; AAA _q -(aa ₃ ) ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P ; wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; AAA is an amino alkyl amino acid residue; (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala, and the N- terminal AAA or (aa) ₃ of the molecule is optionally acetylated. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe comprises a first, a second and a third amino alkyl amino acid residue, wherein

a. the side chain of said first amino alkyl amino acid residue is modified by

attaching a molecule to the side chain amino group,

b. the side chain of said second amino alkyl amino acid residue is modified by attaching a molecule to the side chain amino group,

c. the side chain of said third amino alkyl amino acid residue is modified by

attaching a molecule to the side chain amino group,

d. the side chain of said first and second amino alkyl amino acid residue is

modified by attaching a molecule to the side chain amino group,

e. the side chain of said first and third amino alkyl amino acid residue is modified by attaching a molecule to the side chain amino group,

f. the side chain of said second and third amino alkyl amino acid residue is

modified by attaching a molecule to the side chain amino group, or g. the side chain of said first, second and third amino alkyl amino acid residue is modified by attaching a molecule to the side chain amino group. The W-Peptide analogue according to the preceding items, wherein the amino alkyl amino acid residues of the branched amino acid probe are individually selected from the group consisting of lysine and ornithine. 15. The W-Peptide analogue according to the preceding items, wherein each of the first, second and/or third amino alkyl amino acids of the branched amino acid probe are individually selected from the group consisting of lysine and ornithine.

16. The W-Peptide analogue according to the preceding items, wherein each AAA of the molecules AAA _q -AAA; (aa ₃ ) _P -AAA _q ; AAA _q -(aa ₃ ) _P ; [(aa ₃ )-AAA] _p and [AAA-(aa ₃ )] _P are individually selected from the group consisting of lysine and ornithine.

17. The W-Peptide analogue according to the preceding items, wherein said amino acid residues of said branched amino acid probe each are the same or different.

18. The W-Peptide analogue according to the preceding items, wherein said side chain amino group is individually selected from the δ-amino group (ornithine) and the ε-amino group (lysine).

19. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to said side chain amino group is independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _p -Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p ; [Lys- (aa ₃ )] _p ; Orn _q -Orn; (aa ₃ ) _p -Orn _q ; Orn _q -(aa ₃ ) _p ; [(aa ₃ )-Orn] _p and [Orn-(aa ₃ )] _p ; Orn _p -Lys _p ; Lys _p -Orn _p ; [Orn-Lys] _p and [Lys-Orn] _p , wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala; and the N-terminal Lys, Orn or (aa) ₃ amino acid residue is optionally acetylated at the alpha amino group. 20. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to said side chain amino group is independently selected from the group consisting of Lys _q -Lys; Orn _q -Orn; Orn _p -Lys _p ; Lys _p -Orn _p ; [Orn-Lys] _p and [Lys-Orn] _p , wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; and the N-terminal Lys or Orn amino acid residue is optionally acetylated at the alpha amino group.

21 . The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to said side chain amino group is independently selected from the group consisting of Lys _q -Lys; (aa ₃ ) _p -Lys _q ; Lys _q -(aa ₃ ) _p ; [(aa ₃ )-Lys] _p ; and [Lys-(aa ₃ )] _p .; and the N-terminal Lys or (aa) ₃ residue is optionally acetylated at the alpha amino group. 22. The W-Peptide analogue according to the preceding items, wherein the amino alkyl amino acid residues of the branched amino acid probe are lysine residues. 23. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to said side chain amino group is Lys _q -Lys; wherein q is a number selected from 0, 1 , 2 and 3 and the N-terminal Lys residue is optionally acetylated at the alpha amino group. 24. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to said side chain amino group is independently selected from the group consisting of

Ac-AAA _q -AAA; Ac-(aa ₃ ) _P -AAA _q ; Ac-AAA _q -(aa ₃ ) _P ; Ac-[(aa ₃ )-AAA] _p ; Ac-[AAA-(aa ₃ )] _P , Ac-Lys _q -Lys; Ac-(aa ₃ ) _p -Lys _q ; Ac-Lys _q -(aa ₃ ) _p ; Ac-[(aa ₃ )-Lys] _p ; Ac-[Lys-(aa ₃ )] _p ;

Ac-Orn _q -Orn; Ac-(aa ₃ ) _p -Orn _q ; Ac-Orn _q -(aa ₃ ) _p ; Ac-[(aa ₃ )-Orn] _p ; Ac-[Orn-(aa ₃ )] _p ; Ac-

Orn _p -Lys _p ; Ac-Lys _p -Orn _p ; Ac-[Orn-Lys] _p and Ac-[Lys-Orn] _p ,

wherein q is a number selected from 0, 1 , 2 and 3; p is a number selected from 1 , 2 and 3; and (aa ₃ ) is an amino acid residue independently selected from Arg, His, Gly and Ala.

25. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe consist of 2 to 3 amino acid residues, such as 3 to 4 amino acid residues, for example 4 to 5 amino acid residues, such as 5 to 6 amino acid residues, for example 6 to 7 amino acid residues, such as 7 to 8 amino acid residues, for example 8 to 9 amino acid residues.

26. The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe consist of 2 amino acid residues, such as 3 amino acid residues, for example 4 amino acid residues, such as 5 amino acid residues, for example 6 amino acid residues, such as 7 amino acid residues, for example 8 amino acid residues, such as 9 amino acid residues.

27. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to the side chain amino group(s) of one or more of the first, second and/or third amino alkyl amino acid residues is selected from the group consisting of AAA, Ac-AAA, AAA-AAA, Ac-AAA-AAA, AAA-AAA-AAA, Ac-AAA- AAA-AAA, AAA-AAA-AAA-AAA, Ac-AAA-AAA-AAA-AAA, AAA-Gly-AAA, Ac-AAA- Gly-AAA, AAA-AAA-Gly, Ac-AAA-AAA-Gly, AAA-Gly, Ac-AAA-Gly, AAA-Ala-AAA, Ac-AAA-Ala-AAA, AAA-AAA-Ala, Ac-AAA-AAA-AI a , AAA-Ala, Ac-AAA-Ala, AAA- His-AAA, Ac-AAA-His-AAA, AAA-AAA-His, Ac-AAA-AAA-His, AAA-His, Ac-AAA-

His, AAA-Arg-AAA, Ac-AAA-Arg-AAA, AAA-AAA-Arg, Ac-AAA-AAA-Arg, AAA-Arg and Ac-AAA-Arg; wherein AAA is an amino alkyl amino acid residue. The W-Peptide analogue according to the preceding items, wherein the molecule to be covalently linked to the side chain amino group(s) of one or more of the first, second and/or third amino alkyl amino acid residues is selected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys- Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly, Ac- Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys, Lys-Lys-Ala, Ac- Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His, Ac-

Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac- Lys- Lys-Arg, Lys-Arg and Ac-Lys-Arg. The W-Peptide analogue according to the preceding items, wherein said branched amino acid probe is selected from the group consisting of

a. (AAA)AAA-i-, (AAA-AAA)AAA , (AAA-AAA-AAA)AAA , (AAA-AAA-AAA- AAA)AAAi, (AAA-Gly-AAA)AAAr, (AAA-AAA-Gly)AAA , (AAA-Gly)AAA , (AAA-Ala-AAA)AAAr, (AAA-AAA-Ala)AAA , (AAA-Ala)AAA , (AAA- Hi s- AAA)AAA , (AAA-AAA-His)AAAr, (AAA-His)AAA , (AAA-Arg-AAA)AAA , (AAA-AAA-Arg)AAAr, and (AAA-Arg )AAA , wherein said first amino alkyl amino acid reside (AAA ) is optionally N-terminally acetylated or C-terminally amidated; or

b. (Lys)Lysr, (Lys-Lys)Lysr, (Lys-Lys-Lys)Lysr, (Lys-Lys-Lys-Lys)Lysr, (Lys- Gly-Lys)Lysr, (Lys-Lys-Gly)Lysr, (Lys-Gly)Lysr, (Lys-Ala-Lys)Lysr, (Lys- Lys-Ala)Lysr, (Lys-Ala)Lysr, (Lys-His-Lys)Lysr, (Lys-Lys-His)Lysr, (Lys- His)Lysr, (Lys-Arg-Lys)Lysr, (Lys-Lys-Arg)Lysr, and (Lys-Arg) Lysr, wherein said first lysine reside (Lysr) is optionally N-terminally acetylated or C- terminally amidated; or

c. Ac-(Ac-Lys)Lysr, Ac-(Ac-Lys-Lys)Lysr, Ac-(Ac-Lys-Lys-Lys)Lysr, Ac-(Ac- Lys-Lys-Lys-Lys)Lysr, Ac-(Ac-Lys-Gly-Lys)Lysr, Ac-(Ac-Lys-Lys-Gly)Lysr, Ac-(Ac-Lys-Gly)Lysr, Ac-(Ac-Lys-Ala-Lys)Lys , Ac-(Ac-Lys-Lys-Ala)Lys , Ac- (Ac-Lys-Ala)Lysr, Ac-(Ac-Lys-His-Lys)Lysr, Ac-(Ac-Lys-Lys-His)Lysi-, Ac- (Ac-Lys-His)Lysr, Ac-(Ac-Lys-Arg-Lys)Lysr, Ac-(Ac-Lys-Lys-Arg)Lysr, and Ac-(Ac-Lys-Arg)Lysi-, or

d. (Ac-Lys)LysrNH ₂ , (Ac-Lys-Lys)LysrNH ₂ , (Ac-Lys-Lys-Lys)LysrNH ₂ , (Ac-Lys- Lys-Lys-Lys)LysrNH ₂ , (Ac-Lys-Gly-Lys)Lys NH ₂ , (Ac-Lys-Lys-Gly)Lys NH ₂ , (Ac-Lys-Gly)Lys NH ₂ , (Ac-Lys-Ala-Lys)Lys NH ₂ , (Ac-Lys-Lys-Ala)Lys NH ₂ , (Ac-Lys-Ala)Lys NH ₂ , (Ac-Lys-His-Lys)LysrNH ₂ , (Ac-Lys-Lys-His)LysrNH ₂ , (Ac-Lys-His)LysrNH ₂ , (Ac-Lys-Arg-Lys)Lys NH ₂ , (Ac-Lys-Lys-Arg)Lys NH ₂ , and (Ac-Lys-Arg)LysrNH ₂ . The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe is selected from the group consisting of

Ac-(Ac-Lys)Lys-Lys-, (Ac-Lys)Lys-Lys-, Ac-(Lys)Lys-Lys-, (Lys)Lys-Lys-, (Ac- Lys)Lys-Lys-N H ₂ , (Lys)Lys-Lys-N H ₂ ;

Ac-Lys-(Ac-Lys)Lys-, Lys-(Ac-Lys)Lys-, Ac-Lys-(Lys)Lys-, Lys-(Lys)Lys- Lys-(Ac-Lys)Lys-NH ₂ , Lys-(Lys)Lys-NH ₂ ; or

Ac-(Ac-Lys-Lys)-Lys-, (Ac-Lys-Lys)-Lys-, Ac-(Lys-Lys)-Lys- and (Lys-Lys)-Lys- (Ac-Lys-Lys)-Lys-NH ₂ , and (Lys-Lys)-Lys-NH ₂ . The W-Peptide analogue according to the preceding items, wherein the branched amino acid probe is selected from the group consisting of Ac-(Ac-Lys)Lys-, Ac- (Lys)Lys-, (Ac-Lys)Lys-NH ₂ , (Lys)Lys-NH ₂ and (Lys)Lys-. The W-Peptide analogue according to the preceding items, wherein (aa ₃ ) is an amino acid residue selected from Gly and Ala. The W-Peptide analogue according to the preceding items, wherein said first amino alkyl amino acid residue is covalently linked to the N-terminus of said W- Peptide. The W-Peptide analogue according to the preceding items, wherein said first amino alkyl amino acid residue is covalently linked to the N-terminal Trp of said W- Peptide. 35. The W-Peptide analogue according to the preceding items, wherein said first amino alkyl amino acid residue is covalently linked to the side chain amino group of a lysine or ornithine residue within said W-Peptide. 36. The W-Peptide analogue according to the preceding items, wherein said first

amino alkyl amino acid residue is covalently linked to the ε-amino group of a lysine residue within said W-Peptide, such as covalently linked to the ε-amino group of the lysine residue at position 2 said W-Peptide. 37. The W-Peptide analogue according to the preceding items, wherein said first

amino alkyl amino acid residue is covalently linked to the C-terminus of said W- Peptide.

38. The W-Peptide analogue according to the preceding items, wherein said first

amino alkyl amino acid residue is covalently linked to the C-terminal Met or DMet of said W-Peptide.

39. The W-Peptide analogue according to the preceding items comprising 1 branched amino acid probe.

40. The W-Peptide analogue according to the preceding items comprising 1 branched amino acid probe covalently linked to the N-terminus of said W-Peptide.

41 . The W-Peptide analogue according to the preceding items comprising 1 branched amino acid probe covalently linked to the C-terminus of said W-Peptide.

42. The W-Peptide analogue according to the preceding items comprising 1 branched amino acid probe covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide; such as covalently linked to the δ-amino group of an ornithine residue within said W-Peptide or the ε-amino group of a lysine residue within said W-Peptide.

43. The W-Peptide analogue according to the preceding items comprising 2 branched amino acid probes. The W-Peptide analogue according to the preceding items comprising 2 branched amino acid probes, wherein

i) one branched amino acid probe is covalently bound to the N-terminus of the W- Peptide and another branched amino acid probe is covalently bound to the C- terminus of the W-Peptide; or

ii) one branched amino acid probe is covalently bound to the N-terminus of the W- Peptide and another branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide; or

iii) one branched amino acid probe is covalently bound to the C-terminus of the W- Peptide and another branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide; or

iv) each of the two branched amino acid probes are covalently linked to the side chain amino group of separate amino alkyl amino acid residues within said W- Peptide. The W-Peptide analogue according to the preceding items comprising 3 branched amino acid probes. The W-Peptide analogue according to the preceding items comprising 3 branched amino acid probes, wherein

i) the first branched amino acid probe is covalently bound to the N-terminus of the W-Peptide, the second branched amino acid probe is covalently bound to the C- terminus of the W-Peptide and the third branched amino acid probe is covalently linked to the side chain amino group of an amino alkyl amino acid residue within said W-Peptide; or

ii) the first branched amino acid probe is covalently bound to the N-terminus of the W-Peptide, and the second and third branched amino acid probes are each covalently linked to the side chain amino group of different amino alkyl amino acid residues within said W-Peptide; or

iii) the first branched amino acid probe is covalently bound to the C-terminus of the W-Peptide, and the second and third branched amino acid probes are each covalently linked to the side chain amino group of different amino alkyl amino acid residues within said W-Peptide; or iv) each of the first, the second and the third branched amino acid probes are covalently linked to the side chain amino group of different amino alkyl amino acid residues within said W-Peptide.

47. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a functional W-Peptide.

48. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof.

49. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-DMet.

50. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having one amino acid substitution.

51 . The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having two amino acid substitutions.

52. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having three amino acid substitutions.

53. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 1 .

54. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 2.

55. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 3. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having an amino acid substitution at position 4. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having one amino acid substitution at position 5. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ) or a functional variant thereof having one amino acid substitution at position 6. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a variant comprising an ornithine residue. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Orn-Tyr-Met-Val-Met (SEQ ID NO:2) or Trp-Orn-Tyr-Met-Val-DMet. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a variant comprising a proline residue. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Pro-Met (SEQ ID NO:3) or Trp-Lys-Tyr-Met-Pro-DMet. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a variant comprising an isoleucine residue. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-lle-Val-Met (SEQ ID NO:4) or Trp-Lys-Tyr-lle-Val-DMet.

The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a variant comprising a phenylalanine residue. 66. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Phe-Met-Val-Met (SEQ ID NO:5) or Trp-Lys-Phe-Met-Val-DMet.

67. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is a variant comprising an alpha-amino n-butyric acid residue, such as an alfa-amino butyric acid residue (Abu) and/or an alfa-amino isobutyric acid residue (Aib).

68. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Abu-Met (SEQ ID NO:6) or Trp-Lys-Tyr-Met-Abu-DMet.

69. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-Aib-Met (SEQ ID NO:7) or Trp-Lys-Tyr-Met-Aib-DMet.

70. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is X-Lys-Tyr-X-Val-Met (SEQ ID NO:8) or X-Lys-Tyr-X-Pro-Met (SEQ ID NO:9), wherein X is individually any amino acid, such as Trp or Met.

71 . The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp-Lys-Tyr-Met-aa-Met (SEQ ID NO:10) or Trp-Lys-Tyr-Met-aa-DMet, wherein aa is an amino acid having the structure R ¹ R ² C(NH ₂ )-COOH, wherein R ¹ is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyi and cycloalkenyl, and wherein R ² is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyi and cycloalkenyl.

72. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is Trp - X2 - X3 - X4 - X5 - X6 (SEQ ID NO:1 1 ), wherein X2 is selected from Lys and Orn, X3 is selected from Tyr and Phe, X4 is selected from Met and lie, X5 is selected from Val, Pro, Abu, Aib and aa; and X6 is selected from Met and DMet.

73. The W-Peptide analogue according to the preceding items, wherein

Peptide is selected from the group consisting of

Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:1 ),

Trp-Lys-Tyr-Met-Val-DMet,

Trp-Orn-Tyr-Met-Val-Met (SEQ ID NO:2), Trp-Orn-Tyr-Met-Val-DMet,

Trp-Lys-Tyr-Met-Pro-Met (SEQ ID NO:3),

Trp-Lys-Tyr-Met-Pro-DMet,

Trp-Lys-Tyr-lle-Val-Met (SEQ ID NO:4),

Trp-Lys-Tyr-lle-Val-DMet,

Trp-Lys-Phe-Met-Val-Met (SEQ ID NO:5),

Trp-Lys-Phe-Met-Val-DMet,

Trp-Lys-Tyr-Met-Abu-Met (SEQ ID NO:6)

Trp-Lys-Tyr-Met-Abu-DMet,

Trp-Lys-Tyr-Met-Aib-Met (SEQ ID NO:7)

Trp-Lys-Tyr-Met-Aib-DMet,

X-Lys-Tyr-X-Val-Met, wherein X is any amino acid (SEQ ID NO:8), X-Lys-Tyr-X-Pro-Met, wherein X is any amino acid (SEQ ID NO:9), Trp-Lys-Tyr-Met-aa-Met (SEQ ID NO: 10) and Trp-Lys-Tyr-Met-aa-DMet, wherein aa is an amino acid having the structure R ¹ R ² C(NH ₂ )-COOH, wherein R ¹ is a functional group selected from the group consisting of -H, alkyl, alkenyl, cycloalkyl and cycloalkenyl, and wherein R ² is a functional group selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkenyl, and

Trp - X2 - X3 - X4 - X5 - X6 (SEQ ID NO: 1 1 ), wherein X2 is selected from

Lys and Orn, X3 is selected from Tyr and Phe, X4 is selected from Met and lie, X5 is selected from Val, Pro, Abu, Aib and aa; and X6 is selected from Met and DMet,

or a functional variant thereof. The W-Peptide analogue according to the preceding items, wherein said W- Peptide is C-terminally amidated (-NH ₂ ). The W-Peptide analogue according to the preceding items, wherein said W- Peptide is N-terminally acetylated (COCH ₃ or Ac-). The W-Peptide analogue according to the preceding items, wherein said W- Peptide binds to one or more of the formyl peptide receptors, including Formyl Peptide Receptor 1 (FPR1 ), Formyl Peptide Receptor 2 (FPR2) and Formyl Peptide Receptor 3 (FPR3), and/or

activates and/or stimulates one or more of the formyl peptide receptors, including Formyl Peptide Receptor 1 (FPR1 ), Formyl Peptide Receptor 2 (FPR2) and Formyl Peptide Receptor 3 (FPR3), and/or

is a ligand and/or agonist of one or more of the formyl peptide receptors, including Formyl Peptide Receptor 1 (FPR1 ), Formyl Peptide Receptor 2 (FPR2) and Formyl Peptide Receptor 3 (FPR3), and/or

binds, activates and/or is an agonist for FPR2, and/or

activates immune cells, and/or

activates leukocytes, such as phagocytic leukocytes, and/or

activates neutrophils and/or monocytes, and/or

activates phagocytic leukocytes' effector functions, such as inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase, and/or

induces chemotaxis in phagocytic leukocytes.

77. The W-Peptide analogue according to the preceding items selected from the group consisting of:

Ac-(Ac- Ly s- Ly s ) Ly s-Trp- Ly s-Ty r- M et-Va I- D M et- N H ₂

Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-NH ₂

Ac-(Ac- Ly s- Lys ) Ly s-Trp- Ly s-Ty r- M et-Ab u - D M et- N H ₂

Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet-NH ₂

Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et- P ro- D M et- N H ₂ , and

Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et-Ab u - D M et- N H ₂ .

78. A pharmaceutical composition comprising a W-Peptide analogue according to any of the preceding items.

79. A W-Peptide analogue according to any of the preceding items for use as a

medicament.

80. A W-Peptide analogue according to any of the preceding items for use in the

treatment of an ischemic condition, an inflammatory condition, an infection and/or a metabolic condition. 81 . The W-Peptide analogue for use according to any of the preceding items wherein said treatment is prophylactic, ameliorative or curative. 82. The W-Peptide analogue for use according to any of the preceding items for use in the treatment of an ischemic and/or inflammatory condition in the tissue of one or more organs of a mammal.

83. The W-Peptide analogue for use according to any of the preceding items, wherein said mammal is a human (homo sapiens).

84. The W-Peptide analogue for use according to any of the preceding items, wherein said condition is acute, subacute or chronic. 85. The W-Peptide analogue for use according to any of the preceding items, wherein said organ is selected from the group consisting of kidney, liver, brain, heart, muscles, bone marrow, skin, skeleton, lungs, the respiratory tract, spleen, exocrine glands, bladder, endocrine glands, reproduction organs including the phallopian tubes, eye, ear, vascular system, the gastroinstestinal tract including small intestines, colon, rectum, canalis analis and prostate gland.

86. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic condition is secondary ischemia. 87. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemia is due to stroke, injury, septic shock, systemic hypotension, cardiac arrest due to heart attack, cardiac arrhythmia, atheromatous disease with thrombosis, embolism from the heart or from blood vessel from any organ, vasospasm, aortic aneurysm or aneurisms in other organs, coronary stenosis, myocardial infarction, angina pectoris, pericarditis, myocarditis, myxodemia, or endocarditis.

88. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is associated with surgery, such as major surgery.

89. The W-Peptide analogue for use according to any of the preceding items, wherein said surgery is selected from the group consisting of cardiothoracic surgery, abdominal surgery, surgery on the aorta and/or other major blood vessels, repair of one or more cardiac valves, cardiac artery bypass grafting (CABG), surgery on the aortic root or the aortic branch including the common carotic arteries, and combined cardiac surgery such as valve(s) replacement and CABG and/or aortic root surgery.

90. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is associated with organ

transplantation, such as solid organ transplantation.

91 . The W-Peptide analogue for use according to any of the preceding items, wherein said organ transplantation is selected from the group consisting of heart transplantation, lung transplantation, combined heart and lung transplantation, liver transplantation and kidney transplantation.

92. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is post-surgical systemic inflammatory response syndrome (SIRS).

93. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is post-surgical organ dysfunction.

94. The W-Peptide analogue for use according to any of the preceding items, wherein said post-surgical organ dysfunction is post-surgical renal failure such as acute kidney injury (AKI), neprotoxicity and/or chronic renal failure (CRF).

95. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is reperfusion injury.

96. The W-Peptide analogue for use according to any of the preceding items, wherein said ischemic and/or inflammatory condition is an inflammatory disease.

97. The W-Peptide analogue for use according to any of the preceding items, wherein said inflammatory disease is selected from the group consisting of arthropathy (joint disease), rheumatoid arthritis (RA), gout, inflammatory diseases of the gastrointestinal system, and multiple sclerosis. 98. The W-Peptide analogue for use according to any of the preceding items, wherein said peptide is to be administered systemically to an individual in need thereof.

99. The W-Peptide analogue for use according to any of the preceding items, wherein said peptide is to be administered before and/or during surgery and/or organ transplantation.

100. The W-Peptide analogue for use according to any of the preceding items, wherein said systemic administration is intravenous, subcutaneous and

intramuscular administration.

101 . A method for treatment of an ischemic condition, an inflammatory

condition, an infection and/or a metabolic condition, said method comprising administering an effective amount of a W-Peptide analogue according to any of the preceding items to an individual in need thereof.

Examples Example 1 - Synthesis of BAP-modified W-Peptide analogues

BAP modified peptides were synthesized using standard Fmoc chemistry using 1 - [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro- phosphate (HATU) or 2-(6-Chloro-1 H-benzotriazole-1 -yl)-1 ,1 ,3,3-tetramethylaminium hexafluorophosphate (HCTU) as the coupling reagents with Hunig's Base (N,N- diisopropylethylamine, DIPEA). For the lysine branching as described in more detail below, combination of orthogonally protected lysines were used including Fmoc- Lys(MTT)-OH, Fmoc-Lys(ivDde)-OH, and Fmoc-Lys(Boc)-OH. Peptides were cleaved with standard cleavage cocktails including trifluoroacetic acid, triisoproproylsilane, and water and precipitated with ice-cold ether. All crude peptides were purified by reversed- phase chromatography on columns with C-18 functionality and using gradients of acetonitrile, deionized water, and trifluoroacetic acid as running buffers. Purity was determined by high-pressure liquid chromatography and mass (MS) and sequence (tandem MS) information was obtained using a nanospray mass spectrometer. BAP attached in the C-terminus of the sequence

Branching on the C-terminal lysine (METHOD 1 ): N - α - Fmoc - N - ε - 4 - methyltrityl - L - lysine or N - a - Fmoc-N-e-1 -(4,4-dimethyl- 2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl-L-lysine was added to Rink amide resin after piperidine deprotection. The remaining sequence of the target peptide was added and the full length sequence was acetylated with acetic anhydride. The lysine side chain protecting group was then removed using 1 % trifluoroacetic acid in

dichloromethane (MTT) or hydroxylamine hydrochloride/imidazole in NMP (ivDde). Additional Na-Fmoc-Ne-Boc-L-lysine was then added to the side chain and acetylated when desired. Branching on other than the C-terminal lysine: analogously to attaching BAP to lysines in the sequence between the N- and C-termini (METHOD 2).

BAP covalently linked to lysines in the sequence between the N- and C-termini

METHOD 2: N - a - Fmoc - N - ε - 4 - methyltrityl - L - lysine or N - a - Fmoc-N-e-1 - (4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)-3-methylbutyl-L-lysine was added to the peptide sequence, side chain protecting group was removed after finalizing the sequence and optionally N-terminal acetylation. Appropriate lysine analogues such as Fmoc-Lys(MTT)-OH, Fmoc-Lys(ivDde)-OH and Fmoc-Lys(Boc)-OH were sequentially added and selectively deprotected, before acetylation to ensure appropriate side chain and acetyl addition.

BAP was added to other amino alkyl residues than lysine by analogously using Fmoc/4-methyltrityl protected amino alkyl amino acids. BAP attached in the N-terminus of the sequence

Branching on the N-terminal lysine (METHOD 3): N - a - Fmoc - N - ε - 4 - methyltrityl - L - lysine was added to N-terminal of the sequence, Fmoc was removed, the sequence acetylated at the N-terminus, and the metyltrityl group was removed. Additional No Fmoc-Ne-Boc-L-lysine was then added to the side chain and acetylated when desired.

Branching on other than the N-terminal lysine: analogously to attaching BAP to lysines in the sequence between the N- and C-termini (METHOD 2).

Peptides

BAP identified as bold/italic: Control peptide (W-peptide; SEQ I D NO: 1 ): Trp-Lys-Tyr-Met-Val-Met-NH ₂

Analogue 1 (by METHOD 3): ^c-CiAc-Lys-Lys^Lys-Trp-Lys-Tyr-Met-Val-DMet-NHz Purity: 97.2%, MS: 442.2, 662.9

Analogue 2 (by METHOD 3): Ac^Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-N ₂ Purity: 96.9%, MS: 441 .6, 661 .9

Analogue 3 (by METHOD 3): Ac-(Ac-Lys-Lys)Lys-Jrp-Lys-Jyr-Met-Abu-DMet-N ₂ Purity: 97.5%, MS: 437.6, 655.9

Analogue 4 (by METHOD 3): 4c-(flc-LysjLys-Trp-Lys-Tyr-Met-Val-DMet-NH ₂ Purity: 95.3%, MS: 598.8 Analogue 5 (by METHOD 3): c-(4c-LysjLys-Trp-Lys-Tyr-Met-Pro-DMet-NH ₂ Purity: 95%, MS: 597.8

Analogue 6 (by METHOD 3): c-(4c-LysjLys-Trp-Lys-Tyr-Met-Abu-DMet-NH ₂ Purity: 96.2%, MS: 591 .8

Additional W-Peptide analogues:

Ac-Trp-^c-Lys-LysJLys-Tyr-Met-Val-DMet

Ac-Trp-Lys-Tyr-Met-Val-DMet-( ^' iAc-Lys-Lys)Lys-yVH2

A c-(A c-Lys-Lys)Lys-Trp- Ly s-Ty r- M et-Ai b- D M et

c-(flc-Lys-LysjLys-Trp-0rn-Tyr-Met-Val-DMet

A c-(A c-Lys-Lys)Lys-Trp- Ly s-Ty r- 11 e-Va I- D M et

^c-f^c-Lys-LysJLys-Trp-Lys-Phe-Met-Val-DMet

Aib=a-aminoisobutyric acid=alfa-methylalanine

Example 2: Pharmacological characterization of BAP-modified W-Peptide analogues - Binding affinity

Method:

Radioligand competition binding was performed in duplicate in the wells of a 96 well plate (Master Block, Greiner, 786201 ) containing binding buffer (50 mM Tris HCI pH 7.4, saponin 10 μg/ml and 0.5% protease free BSA), FPR2 membrane extracts (Ο.δμς protein/well), 2 nM [125l]-WHYMV(D-met)-NH2 (PerkinElmer, NEX386) and test compound at increasing concentrations. Nonspecific binding was determined by co- incubation with 200-fold excess of non labelled agonist. The samples were incubated in a final volume of 0.1 ml for 60 min at 25°C and then filtered over GF/C filters (Perkin

Elmer, 6005174) , presoaked in binding buffer for 2h at room temperature. Filters were washed six times with 0.5 ml of ice-cold washing buffer (50 mM Tris HCI pH 7.4) and 50 μΙ of Microscint 20 (Packard) were added in each well. The plates were incubated 15 min on an orbital shaker and then counted with a TopCountTM for 1 min/well.

Test compounds, i.e. W-Peptide analogues 1 -6 and the W-Peptide control compound (SEQ ID NO:1 ) were tested in duplicate in a concentration range of 0.01 to 10,000 nM.

W-peptide analogues tested: Analogue 1 ; Analogue 2; Analogue 3; Analogue 4;

Analogue 5; Analogue 6.

Data presented as mean values. The IC50 (i.e. the concentration that induced 50% displacement of radioactive agonist) was determined by best fit analyses after logarithmic transformation using the graph pad software (version 6.0).

Results:

All test compounds as well as the W-peptide control compound induced 100% displacement of radioactive labelled agoinst at the highest concentration tested. The IC50 value for the W-peptide control compound was 96 nM. For all 6 analogues the IC50 determined value was more than one decade lower that for the control compound:

Analogue IC50

1 Ac-(Ac- Ly s- Ly s ) Ly s-Trp- Ly s-Ty r- M et-Va 1- D M et- N H ₂ 2.48 nM

2 Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-NH ₂ 2.14 nM

3 Ac-(Ac- Ly s- Lys ) Ly s-Trp- Ly s-Ty r- M et-Ab u - D M et- N H ₂ 2.59 nM

4 Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet-NH ₂ 2.63 nM

5 Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et- P ro- D M et- N H ₂ 9.12 nM

6 Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et-Ab u - D M et- N H ₂ 8.73 nM

Conl rol (W-Peptide, SEQ ID NO:1 ) 96 nM Example 3: Pharmacological characterization of BAP-modified W-Peptide analogues- Receptor efficacy

Method:

Recombinant CHO-K1 cells co-expressing mitochondrial apo-aequorin and

recombinant human FPR2 receptor were grown 18 hours prior to the test in media without antibiotics. The cells were detached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspended in assay buffer containing DMEM/HAM's F12 with HEPES + 0.1 % BSA (protease free). Cells were incubated at room temperature for at least 4h with coelenterazine h (Molecular Probes).

For agonist testing, 50 μΙ of cell suspension were injected on 50 μΙ of test compound or reference agonist plated in 96-well plates. The resulting emission of light was recorded using the Hamamatsu Functional Drug Screening System 6000 (FDSS 6000). To standardize the emission of recorded light (determination of the "100% signal") across plates and across different experiments, the emission from 100 μΜ digitonin or 20 μΜ ATP leaded wells were used as "signal" standards.

Agonist activity of test compound was expressed as a percentage of the activity of the reference agonist at its EC100 concentration.

Test compounds, i.e. W-Peptide analogues 1 -6 were tested in duplicate in a concentration range of 0.0001 to 1000 nM. The control compound (W-Peptide, SEQ ID NO:1 ) was tested in a range of 0.0001 to 100 nM. W-Peptide analogues tested: Analogue 1 ; Analogue 2; Analogue 3; Analogue 4;

Analogue 5; Analogue 6.

Data is presented as mean values. The EC50 (i.e. the concentration that induced 50% of maximal agonist response) was determined by best fit analyses after logarithmic transformation using the graph pad software (version 6.0).

Results:

Determination of EC50 values confirmed that W-Peptide control compound is a full agonist against the human FPR2 receptor with an EC50 value of 0.2 nM. The BAP-modified W-Peptide analogues all induced full agonist activity (see figures 2b- 7b). The potency as evaluated by EC50 is high for all the tested analogues. The EC50 was obtained at concentration more than 3 decades lower that for the control compound:

Analogue EC50

1 Ac-(Ac- Ly s- Ly s ) Ly s-Trp- Ly s-Ty r- M et-Va 1- D M et- N H ₂ 0.22 pM

2 Ac-(Ac-Lys-Lys)Lys-Trp-Lys-Tyr-Met-Pro-DMet-NH ₂ 0.46 pM

3 Ac-(Ac- Ly s- Lys ) Ly s-Trp- Ly s-Ty r- M et-Ab u - D M et- N H ₂ 0.52 pM

4 Ac-(Ac-Lys)Lys-Trp-Lys-Tyr-Met-Val-DMet-NH ₂ 0.26 pM

5 Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et- P ro- D M et- N H ₂ 0.78 pM

6 Ac-(Ac- Ly s ) Ly s-Trp-Ly s-Ty r- M et-Ab u - D M et- N H ₂ 0.48 pM

Conl rol (W-Peptide, SEQ ID NO:1 ) 0.2 nM