Hydrophobic modified peptides for liver specific diagnosis

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.

Claims

1. A hydrophobic modified peptide of the formula
[XPYR.sub.o]A.sub.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, ?-amino glycine, ?,?-diaminobutyric acid, ornithine, and ?,?-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 .sup.18F, .sup.51Cr, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.99mTe, .sup.140La, .sup.175Yb, .sup.153Sm, .sup.166Ho, .sup.88Y, .sup.90Y, .sup.149Pm, .sup.177Lu, .sup.47Sc, .sup.142Pr, .sup.159Gd, .sup.212Bi, .sup.72As, .sup.72Se, .sup.109Pd, .sup.105Rh, .sup.101m15Rh, .sup.119Sb, .sup.128Ba, .sup.123I, .sup.124I, .sup.131I, .sup.197Hg, .sup.211At, .sup.169Eu, .sup.203Pb, .sup.212Pb, .sup.64Cu, .sup.67Cu, .sup.188Re, .sup.186Re, .sup.198Au and .sup.199Ag.

22. The hydrophobic modified peptide according claim 1, wherein the hydrophobic modified peptide is selected from the group consisting of: TABLE-US-00004 (SEQIDNO:15) stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP, (SEQIDNO:15) stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP, (SEQIDNO:15) stearoy1-[K(DOTA[.sup.111In])].sub.3-NLSTSNPLGFFPDHQLDP, (SEQIDNO:15) stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP)-amide, (SEQIDNO:15) stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide, and (SEQIDNO:15) stearoyl-[K(DOTA[.sup.111In])].sub.3-NLSTSNPLGFFPDHQLDP-amide.

23. The hydrophobic modified peptide according to claim 1, wherein the hydrophobic modification is by acylation with carboxylic acid.

24. The hydrophobic modified peptide according to claim 1, wherein the fatty acids are C.sub.8 to C.sub.22 fatty acids.

25. The hydrophobic modified peptide according to claim 1, wherein the synthetic polymer is PEG.

26. A pharmaceutical composition comprising: at least one hydrophobic modified peptide according to claim 1; and, optionally a pharmaceutically acceptable carrier and/or excipient.

27. A method of specifically delivering a label to the liver by administering to a patient the hydrophobic modified peptide according to claim 1 or a pharmaceutical composition comprising said peptide.

28. The method according to claim 27, wherein the hydrophobic modified peptide is administered to the patient in a dosage ranging from 10 pmol per kg body weight to 20 ?mol per kg body weight.

29. The method according to claim 28, wherein the hydrophobic modified peptide is administered to the patient in the dosage ranging from 100 nmol per kg body weight to 2 ?mol per kg body weight.

30. The method according to claim 27, wherein the hydrophobic modified peptide is administered to the patient subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or by suppository.

31. A method of medical imaging comprising administering to a patient the hydrophobic modified peptide according to claim 1 or a pharmaceutical composition comprising said peptide and conducting a medical imaging method selected from the group consisting of X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), X-ray computed tomography (CT) and combinations thereof.

32. The method according to claim 31, wherein the hydrophobic modified peptide is administered to the patient in a dosage ranging from 10 pmol per kg body weight to 20 ?mol per kg body weight.

33. The method according to claim 31, wherein the hydrophobic modified peptide is administered to the patient subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or by suppository.

34. A method of intraoperative visualization comprising administering to a patient the hydrophobic modified peptide according to claim 1 or a pharmaceutical composition comprising said peptide.

35. The method according to claim 34, wherein the hydrophobic modified peptide is administered to the patient in a dosage ranging from 10 pmol per kg body weight to 20 pmol per kg body weight.

36. The method according to claim 34, wherein the hydrophobic modified peptide is administered to the patient subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or by suppository.

37. A method of diagnosing or monitoring the treatment of a liver disease or disorder comprising administering to a patient the hydrophobic modified peptide according to claim 1 or a pharmaceutical composition comprising said peptide.

38. The method according to claim 37, wherein the liver disease or disorder is selected from hepatitis, cirrhosis, haemochromatosis, a disease which involves a liver stadium of a virus or a non-viral pathogen, a tropical disease, malaria, schistosomiasis, leishmaniasis, a liver tumor, liver metastases, and a metabolic disease.

39. The method according to claim 37, wherein the hydrophobic modified peptide is administered to the patient in a dosage ranging from 10 pmol per kg body weight to 20 pmol per kg body weight.

40. The method according to claim 37, wherein the hydrophobic modified peptide is administered to the patient subcutaneously, intravenously, orally, nasally, intramuscularly, transdermally, by inhalation or by suppository.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic representation of the HBV particle and the HBV L-, M- and S-proteins. The partially double stranded DNA is covalently associated with the viral polymerase complex, consisting of the terminal protein, (TP), the reverse transcriptase (RT) and the RNaseH. The genome is encapsulated by an icosahedral shell, built of 120 core-protein dimers. The 3 HBV surface proteins L-, M- and S- are embedded into an ER-derived lipid bilayer. The L- and M-proteins contain the complete S-domain serving as a membrane anchor. Schematic representation of the partially double stranded DNA genome of HBV; C=Core protein forming the viral capsid; X=X Protein, a pleiotropic transactivator with undefined function; P=Viral Polymerase; preS1/preS2/S combinations of these form the large (preS1/preS2/S) HBV surface protein (L Protein) the medium (preS2/S) HBV surface protein and the small (S) HBV surface protein.

(2) FIG. 2: Schematic diagram of the general formula of SEQ ID NO: 1, the hydrophobic modified peptide of the present invention (i.e., NPLGFXaaP) and an example of SEQ ID NO: 21, a hydrophobic modified peptide of the invention (i.e., KKKNLSSNPLGFFPDHQLDP).

(3) FIG. 3: Schematic diagram of the binding of a hydrophobic modified peptide of the invention to the surface of a hepatocyte.

(4) FIG. 4. PET image of a tumor bearing rat. FIG. 4A Specific enrichment of 400 nmol/kg stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 in the liver of a WAG/Rji rat five minutes post i.v. injection. FIG. 4B Counterstain using .sup.18F-FDG 24 hours after the initial measurement .sup.18F-FDG is enriched in organs consuming high amounts of glucose, in the picture the heart (grey mass on the top) and the tumour enrich glucose (.sup.18F-FDG enriched in the brain is not shown for technical reasons). FIG. 4C Merge of both images demonstrating the specificity of the peptides staining only liver tissue but not the tumor tissue.

(5) FIG. 5 shows a representative MRI image series of a Rat before injection of 600 nmol/kg body weight stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP of SEQ ID NO: 15 (FIG. 5A) as well as 20 (FIG. 5B) and 30 minutes (FIG. 5C) post injection.

(6) FIG. 6 shows the contrast enhacement determinded by MRI measurement in liver, muscle tissue and heart.

(7) FIG. 7: R1 relaxivity of the peptide stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP of SEQ ID NO: 15 (Peptide X), Primovist?, Magnevist? and Vasovist? dissolved in PBS in equimolar amounts (1 mM).

(8) FIG. 8: R2 realxivity of the peptide stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP of SEQ ID NO: 15 (Peptide X), Primovist?, Magnevist? and Vasovist? dissolved in PBS in equimolar amounts (1 mM).

(9) FIG. 9: Organ distribution of .sup.111In labelled hydrophobic modified peptide in mice.

(10) FIG. 10: Stability of stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 in non-inactivated human serum.

EXAMPLES

(11) In the following examples hydrophobic peptides carrying Gd, .sup.68Ga or .sup.111In as labels complexed with DOTA (stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP-amide, stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide or stearoyl-[K(DOTA[.sup.111In])].sub.3-NLSTSNPLGFFPDHQLDP-amide) of SEQ ID NO: 15, were used as exemplary products of the present invention. If not indicated otherwise, the in vivo experiments using different medical imaging methods have been conducted by using WAG/Rij rats.

Example 1

(12) Synthesis of the Hydrophobic Modified Peptide

(13) The synthesis of the peptides was carried out by using the Fmoc method as described in (10) Gripon, P. et al. J Virol 79, 1613-1622 (2005).

(14) Synthesis DOTA-DFP

(15) Diisopropylcarbodiimide (5 mmol, 631 mg, 774 ?l) was dissolved in pyridine (15 ml) and dropped over 10 min to a solution of DOTA (5 mmol, 2.02 g) and difluoro phenole (5 mmol, 650 mg) in water (60 ml) while stirring. 30 min after addition, the reaction mixture was extracted three times with dichlormethane and the aqueous phase was evaporated to dryness by using a rotating evaporator. The crude product was dissolved in a mixture of water (11 ml) and acetonitrile (3 ml) and was purified via preparative RP-HPLC. The HPLC fractions containing the product were concentrated by lyophilisation. Yield: 1.0633 g (41%).

(16) Coupling to the Peptide

(17) The peptide stearoyl-KKKNLSTSNPLGFFPDHQLDP-amide or stearoyl-SEQ ID NO: 15-amide (140 mg, 0.055 mmol) was dissolved in 5 ml DMF. DOTA-DFP (129 mg, 0.25 mmol) was added and, furthermore, DIPEA (410 ?l, 2.5 mmol) was added. The mixture was stirred over night at 50? C. Diethylether was added until precipitation; the precipitate was separated by using a centrifuge and was washed twice with diethylether. The crude product was purified by using RP-HPLC. The purification was effected by using a gradient of water and acetonitrile, both comprising 0.1% trifluoroacetic acid. The HPLC fractions containing the product were concentrated by lyophilisation. Yield: 112 mg (55%).

(18) Complexing of Gd.sup.3+

(19) The peptide stearoyl-[K(DOTA)].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 (112 mg, 0.030 mmol) was dissolved in 0.4 M sodium acetate buffer (pH 5) and GdCl.sub.3*6 H.sub.2O (335 mg, 0.90 mmol) was added. The mixture was heated for 1 h in a water bath while stirring. The resulting mixture of products was purified by using RP-HPLC. The purification was effected by using a gradient of water and acetonitrile, both comprising 0.1% trifluoroacetic acid. The HPLC fractions containing the product (stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15) were concentrated by lyophilisation. Yield: 94 mg (77%).

Example 2

(20) Imaging in PET

(21) The peptide stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 was dissolved in citrate buffer (pH 8.0)+4% BSA and injected i.v. in the tail vene of tumour bearing rats. The rats were orthopically injected with 1?10.sup.6 syngenic colon carcinoma cells (CC531 cells) 10 days prior to the measurements. At the day of measurement the rats received the peptide in a concentration of 400 nmol/kg body weight. During the experiments the rats were anaesthetised by isoflurane and kept at 37? C. PET imaging was performed using a Inveon small animal PET from Siemens, imaging was started immediately after i.v. injection of the peptide 24 h after the initial measurement using stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15, the rats were injected with .sup.18F-FDG (Fluodeoxyglucose(.sup.18F) in a concentration of 5 millicuries as a control. FIG. 4A-C shows a representative PET image of a tumor bearing rat. FIG. 4A Specific enrichment of 400 nmol/kg stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 in the liver of a WAG/Rji rat five minutes post i.v. injection. FIG. 4B Counterstain using .sup.18F-FDG 24 hours after the initial measurement .sup.18F-FDG is enriched in organs consuming high amounts of glucose, in the picture the heart (grey mass on the top) and the tumour enrich glucose (.sup.18F-FDG enriched in the brain is not shown for technical reasons). FIG. 4C Merge of both images demonstrating the specificity of the peptides staining only liver tissue but not the tumor tissue.

Example 3

(22) Imaging in MRI

(23) The peptide stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 was dissolved in citrate buffer (pH 8.0)+4% BSA and injected i.v. in the tail vene of the rats in a concentration of 600 nmol/kg body weight. During the experiments the rats were anaesthetised by isoflurane. Upon application of the peptide, the rats were examined in MRI with T1 contrast sensitive sequences (Siemens ViBE?). The measurement was conducted on a Siemens Avanto 1.5 T MRI Scanner. FIG. 5 shows a representative image of a healthy rate before, as well as 20 and 30 minutes after i.v. injection.

Example 4

(24) Dose Escalation

(25) Isoflurane anaesthetised rats received increasing amounts of stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 in subsequent i.v. injections. The interval between subsequent injections was 20 min. In the time between two injections, continued MRI measurements of the liver with T1 contrast sensitive sequences (Siemens ViBE?) were made and the enhancement of the contrast in liver, muscle tissue and heart was determined. In parallel, contrast values of the clinical relevant dosage of Primovist? were determined. The result is shown in FIG. 6.

Example 5

(26) Determining the R1/R2 Relaxivity by NMR

(27) The peptide stearoyl-[K(DOTA[Gd])].sub.3-NLSTSNPLGFFPDHQLDP of SEQ ID NO: 15 (Peptide X; 1 mM), Primovist? (1 mM), Magnevist? (1 mM) as well as Vasovist? (1 mM) were dissolved in PBS in equimolar amounts at room temperature and the R1/r2 relaxitivies were measured in a Varian 300 Mhz NMR. By using Fourier transformation, the single contrast values were determined from the data resulting from the experiment. The results are shown in FIG. 7 and FIG. 8.

Example 6

(28) Organ Distribution Studies

(29) For examining the organ distribution .sup.111In labelled peptide stearoyl-[K(.sup.111In DOTA)].sub.3-NLSTSNPLGFFPDHQLDP of SEQ ID NO: 15 was dissolved in citrate buffer (pH 8.0)+4% BSA and injected i.v. in the tail vene of mice in a concentration of 400 nmol/kg body weight. The mice were euthanized at the given time points and blood and organs were taken. The radioactive signal of each organ was determined by using a Gamma-Counter. The results are shown in FIG. 9.

Example 7

(30) Stability in the Serum

(31) Stearoyl-[K(DOTA[.sup.68Ga])].sub.3-NLSTSNPLGFFPDHQLDP-amide of SEQ ID NO: 15 was incubated at 37? C. in non-inactivated human serum from healthy voluntary donors over the indicated period. The detection of degradation products was conducted by purification of the peptides by using HPLC affinity chromatography and subsequent radioactivity determination was performed on a gamma counter at the indicated time points. The correct mass of the radioactive peak fractions eluted from the column was confirmed by mass spectrometry (data not shown). The results are shown in FIG. 10. Only radioactive decay but no cleavage was observed.

(32) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

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