Hydrophobic modified peptides for liver specific diagnosis

Patent metadata

Field	Value
Publication number	US-10363323-B2
Application number	US-201213984715-A
Country	US
Kind code	B2
Filing date	Feb 10, 2012
Priority date	Feb 10, 2011
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

The present invention relates to hydrophobic modified peptides for the specific delivery of labels to the liver, preferably to hepatocytes, in vitro as well as in vivo. The present invention relates to pharmaceutical compositions comprising said hydrophobic modified peptide(s) and the label(s) to be specifically delivered to the liver. The present invention furthermore relates to the diagnostic use of the inventive hydrophobic modified peptides as well as to a method for the diagnosis of liver diseases or disorders.

First claim

The invention claimed is:

1. A hydrophobic modified peptide of the formula
[X—P—Y—R o ]A p ,

wherein
X is an amino acid sequence having a length of m amino acids,
wherein m is at least 4, and
wherein the N-terminus of X comprises one or more amino acids comprising an amino group in the side chain,
wherein the one or more of the amino acids comprising an amino group in the side chain comprises a hydrophobic modification selected from acylation with carboxylic acids, fatty acids, and amino acids with lipophilic side chains or addition of a hydrophobic moiety selected from the group consisting of cholesterol, derivatives of cholesterol, a phospholipid, a glycolipid, a glycerol ester, steroids, a ceramide, an isoprene derivative, adamantane, farnesol, an aliphatic group, and a polyaromatic compound; and
wherein the one or more amino acids comprising the amino group in the side chain is coupled to one or more labels selected from the group consisting of a fluorescent dye, a fluorescence emitting isotope, a radioisotope, and a contrast agent
P is a peptide comprising the amino acid sequence NPLGFXaaP (SEQ ID NO: 1), wherein Xaa is an arbitrary amino acid;
Y is an amino acid sequence having a length of n amino acids, wherein n is 0 or at least 1;
m+n≥ 11

R is a C-terminal modification of said hydrophobic modified peptide, which protects from degradation selected from the group consisting of an amide, a D-amino acid, a modified amino acid, a cyclic amino acid, an albumin, a natural polymer, a synthetic polymer, and a glycane; o is 0 or at least 1; and
A is an anchor group selected from the group consisting of an ester, an ether, a disulfide, an amide, a thiol, and a thioester; p is 0 or at least 1.

2. The hydrophobic modified peptide according to claim 1 , wherein m is 4 to 10 and/or n is 0 to 78.

3. The hydrophobic modified peptide according to claim 1 , wherein Xaa is F or L.

4. The hydrophobic modified peptide according to claim 3 , wherein Xaa is F.

5. The hydrophobic modified peptide according to claim 1 , wherein the one or more amino acids is coupled to a label via a linker or spacer.

6. The hydrophobic modified peptide according to claim 5 , wherein the linker or spacer is cleaved by a liver protein.

7. The hydrophobic modified peptide according to claim 6 , wherein the linker or spacer is cleaved by an enzyme selected from a cytochrome, a protease of the endocytic pathway, a lyase of the endocytic pathway, matrix-metallo-protease 1 (MMP1), matrix-metallo-protease 2 (MMP2), matrix-metallo-protease 7 (MMP7), matrix-metallo-protease 9 (MMP9), and matrix-metallo-protease 12 (MMP12).

8. The hydrophobic modified peptide according to claim 7 , wherein the linker or spacer comprises the amino acid sequence selected from GCHAK (SEQ ID NO: 19) and RPLALWRS (SEQ ID NO: 20).

9. The hydrophobic modified peptide according to claim 1 , wherein the one or more amino acids of X comprising the amino group in the side chain is selected from the group consisting of lysine, α-amino glycine, α,γ-diaminobutyric acid, ornithine, and α,β-diaminopropionic acid.

10. The hydrophobic modified peptide according to claim 1 , wherein the N-terminus comprises 2 to 11 amino acids comprising an amino group in the side chain.

11. The hydrophobic modified peptide according to claim 10 , wherein the one or more amino acids of X comprising the amino group in the side chain is coupled to the label via an activated ester.

12. The hydrophobic modified peptide according to claim 10 , wherein the N-terminus of X comprises 1 to 3 amino acids having an amino group in a side chain.

13. The hydrophobic modified peptide according to claim 1 , wherein the one or more labels are coupled to the amino acids of X comprising the amino group in the side chain by using one or more methods selected from
formation of amides by the reaction of an amine and activated carboxylic acids, NHS-esters, or carbodiimides;
disulfide linkage using two thiols or one thiol that specifically reacts with pyridyl disulfides;
thioether formation using maleimides or haloacetyls and a thiol component;
amidine formation using an imidoester and an amine;
hydrazide linkage using carbonyls and hydrazides;
amine linkage using carbonyls and amines under reductive conditions;
triazol formation using nitriles and azides;
thiourea formation using isothiocyanates and amines;
formation of esters by the reaction of an alcohol and activated carboxylic acids, acid chlorides, or carbodiimides; and
formation of ethers by the reaction of an alcohol and alkyl halides.

14. The hydrophobic modified peptide according to claim 13 , wherein the carbonyls used in the hydrazide linkage are aldehydes.

15. The hydrophobic modified peptide according to claim 1 , wherein the hydrophobic modification is by acylation selected from acylation with myristoyl (C14), palmitoyl (C16) or stearoyl (C18).

16. The hydrophobic modified peptide according to claim 15 , wherein the hydrophobic modification is by acylation with myristoyl (C14).

17. The hydrophobic modified peptide according to claim 15 , wherein the hydrophobic modification is by acylation with stearoyl (C18).

18. The hydrophobic modified peptide according to claim 1 , wherein the label comprises a chelating agent selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, Diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid (DTPA) and 6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

19. The hydrophobic modified peptide according to claim 18 , wherein the chelating agent is 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA).

20. The hydrophobic modified peptide according to claim 1 , wherein
(a) the contrast agent comprises a paramagnetic agent;
(b) the radioisotope/fluorescence emitting isotope is selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, and fluorescence emitting isotopes; and
(c) the fluorescent dye is selected from the group consisting of the following classes of fluorescent dyes: xanthens, acridines, oxazines, cynines, styryl dyes, coumarins, porphines, metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenes, phtalocyanines, conjugates thereof and combinations thereof.

21. The hydrophobic modified peptide according to claim 20 , wherein the radioisotope/fluorescence emitting isotope is selected from the group consisting of 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Te, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 72 As, 72 Se, 109 Pd, 105 Rh, 101m15 Rh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 169 Eu, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au and 199 Ag.

22. The hydrophobic modified peptide according claim 1 , wherein the hydrophobic modified peptide is selected from the group consisting of:

(SEQ ID NO: 15)

stearoyl-[K(DOTA[Gd])] 3 -NLSTSNPLGFFPDHQLDP,

(SEQ ID NO: 15)

stearoyl-[K(DOTA[ 68 Ga])] 3 -NLSTSNPLGFFPDHQLDP,

Assignees

• Mier Walter
• Urban Stephan
• Mehrle Stefan
• Haberkorn Uwe
• Mueller Thomas
• Askoxylakis Vasileios
• Univ Heidelberg Ruprecht Karls

Inventors

• Mier Walter
• Urban Stephan
• Mehrle Stefan
• Haberkorn Uwe
• Müller Thomas
• Askoxylakis Vasileios

Classifications

• A61K49/085

  conjugated systems · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• A61K49/14

  Peptides, e.g. proteins · CPC title

• C07K2319/50

  containing protease site · CPC title

• A61K49/0056

  Peptides, proteins, polyamino acids · CPC title