Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis
The invention is directed to a pharmacologically active peptide conjugate having a reduced tendency towards enzymatic cleavage comprising a pharmacologically active peptide sequence (X) and a stabilising peptide sequence (Z) of 4-20 amino acid residues covalently bound to X.
1. A pharmacologically active peptide conjugate having a reduced tendency towards enzymatic cleavage comprising X and Z,
wherein X is a pharmacologically active peptide sequence selected from the group consisting of adrenocorticotropic hormone (ACTH), melanotan II, melanocyte stimulating hormone (MSH), and alpha-MSH, and
wherein Z is a peptide sequence of 4-15 amino acid units covalently bound to X via a peptide bond to the C-terminal carbonyl function of X and/or to the N-terminal nitrogen atom of X, and wherein Z consists of residues selected from Glu, Lys, and Met, and
wherein the ratio between the half-life of said peptide conjugate and the half-life of the corresponding pharmacologically active peptide sequence X, when treated with carboxypeptidase A or leucine aminopeptidase in about 50 mM phosphate buffer solution at about pH 7.4 at about 37° C. or in serum or plasma is at least about 2, or wherein said peptide conjugate has a half-life in human or mice serum or plasma at 37° C. of at least about 10 minutes.
2. A peptide conjugate according to claim 1 , wherein Z is covalently bound to X via a peptide bond to the N-terminal nitrogen atom of X.
3. A peptide conjugate according to claim 1 , wherein X is alpha-MSH.
4. A peptide conjugate according to claim 1 , wherein Z consists of 4-10 amino acid units.
5. A peptide conjugate according to claim 1 , wherein Z consists of 4-7 amino acid units.
6. A peptide conjugate according to claim 1 , wherein Z consists of 6 amino acid units.
7. A peptide conjugate according to claim 6 , wherein Z is Lys 6 .
8. A peptide conjugate according to claim 1 , wherein the amino acid units in Z are Lys.
9. A peptide conjugate according to claim 8 , wherein Z is selected from the group consisting of Lys 4 , Lys 5 , and Lys 6 .
10. A method for producing a peptide conjugate according to claim 1 , comprising
a) introducing a nucleic acid sequence encoding said conjugate into a host cell;
b) culturing said host cell and
c) isolating said conjugate from the culture.
11. A method for producing a peptide conjugate according to claim 1 , comprising
a) culturing a recombinant host cell comprising a nucleic acid sequence encoding said conjugate under conditions permitting the production of said conjugate; and
b) isolating said conjugate from the culture.
12. A composition comprising a pharmacologically active peptide conjugate as defined in claim 1 and a pharmaceutical acceptable carrier.
13. The peptide conjugate of claim 1 , wherein said pharmacologically active peptide sequence is melanotan II.
14. The peptide conjugate of claim 1 , wherein X comprises at least 10 amino acid residues.
15. The composition of claim 12 , wherein said composition is formulated for oral, subcutaneous, parenteral, intramuscular, rectal, epidural, intratracheal, intranasal, vaginal, buccal, ocular, direct brain, pulmonary or topical administration.
16. The composition of claim 12 , wherein X comprises at least 10 amino acid residues.
17. The composition of claim 12 , said composition being in a form selected from the group consisting of a capsule, a tablet, an aerosol, a solution, a suspension or a topical application.
18. The composition of claim 12 , wherein Z is covalently bound to the N-terminal nitrogen atom of X.
19. The composition of claim 12 , wherein X is alpha-MSH.
20. The composition of claim 12 , wherein Z consists of 4-10 amino acid units.
21. The composition of claim 12 , wherein Z consists of 4-7 amino acid units.
22. The composition of claim 12 , wherein Z consists of 6 amino acid units.
23. The composition of claim 12 , wherein the amino acid units in Z are lysine residues.
24. The composition of claim 12 , wherein Z is selected from the group consisting of Lys 4 , Lys 5 , and Lys 6 .
25. The composition of claim 24 , wherein Z is Lys 6 .
26. A method for the preparation of a pharmacologically active peptide conjugate (Z—X) as defined in claim 2 , comprising the steps of:
a) coupling an N-α-protected amino acid, or an N-α-protected dipeptide to a solid support material (SSM), thereby forming an immobilised N-a-protected amino acid;
b) removing the N-α-protecting group, thereby forming an immobilised amino acid or peptide fragment having an unprotected N-terminal end;
c) coupling an additional N-α-protected amino acid in the carboxyl activated form, or an additional N-α-protected dipeptide in the C-terminal activated form to the N-terminal end of the immobilised amino acid or peptide fragment, and repeating the removal/coupling step procedure in step b) and c) until the desired peptide sequence X is obtained, wherein X is a pharmacologically active peptide sequence selected from the group consisting of adrenocorticotropic hormone (ACTH), melanotan II, melanocyte stimulating hormone (MSH), and alpha-MSH;
d) coupling an additional N-α-protected amino acid in the carboxyl activated form, or an additional N-α-protected dipeptide in the C-terminal activated form to the N-terminal end of the immobilised peptide fragment, and repeating the removal/coupling step procedure in step b) and d) until the desired peptide sequence Z is obtained, wherein Z is a peptide sequence of 4-15 amino acid units and consists of residues selected from Glu, Lys, and Met; and then
e) cleaving off the peptide conjugate from the solid support material.