Pharmacologically active peptide conjugates having a reduced tendency towards enzymatic hydrolysis

Abstract

The invention is directed to a pharmacologically active peptide conjugate having a reduced tendency towards enzymatic cleavage including a pharmacologically active peptide sequence (X) and a stabilising peptide sequence (Z) covalently bound to X.

1 . A method for the preparation of a pharmacologically active peptide conjugate (X-Z), comprising the steps of:
 a) coupling an additional N-α-protected amino acid in the carboxyl activated form or an N-α-protected dipeptide in the C-terminal activated form to an immobilized peptide sequence H-Z-SSM, thereby forming an immobilized N-α-protected peptide fragment,   b) removing the N-α-protecting group, thereby forming an immobilized 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 immobilized peptide fragment, and repeating the removal/coupling step procedure in steps b) and c) until the desired peptide sequence X is obtained, and   d) cleaving off the peptide conjugate from the solid support material, wherein X is a pharmacologically active peptide sequence from 2 to 10 amino acid residues in length, Z is a stabilizing peptide consisting of 4 to 15 amino acid residues covalently bound by its N terminus to the C terminus end of X, wherein Z is a sequence selected from:   (i) Lys 4-10 ,   (ii) (Lys-Xaa) m ,   (iii) (Xaa-Lys) m ,   (iv) Lys p -Xaa q ,   (v) Xaa p -Lys q ,   (vi) Xaa 1 -Lys-Xaa 2 -Lys,   (vii) Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 ,   (viii) Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 -Lys,   (ix) Xaa 1 -Xaa 2 -Lys-Xaa 2 ,
 (x) Xaa 1 -Xaa 2 -Lys-Xaa 2 -Lys, 
 (xi) Xaa 1 -Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 , 
 (xii) Lys-Xaa 2 -Lys-Xaa 1 , 
 (xiii) Lys-Xaa 2 -Lys-Xaa 2 -Xaa 1 , 
 (xiv) Lys-Xaa 2 -Lys-Xaa 2 -Lys-Xaa 1 , 
 (xv) Xaa 2 -Lys-Xaa 2 -Xaa 1 , 
 (xvi) Xaa 2 -Lys-Xaa 2 -Lys-Xaa 1 , and 
 (xvii) Xaa 2 -Lys-Xaa 1 -Lys-Xaa 2 -Xaa 1 , 
   
       wherein 
       each of Xaa, Xaa 1 , and Xaa 2  is, independently, selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, and Met; 
       each of p and q is, independently, an integer from 1 to 14, with the proviso that p+q is from 4 to 15; and 
       m is an integer in the range from 2 to 7. 
     
     
         2 . The method of  claim 1 , wherein Z is Lys 4  (SEQ ID NO: 55), Lys 5  (SEQ ID NO: 56) or Lys 6  (SEQ ID NO: 62). 
     
     
         3 . The method of  claim 1 , wherein X is selected from growth hormone (GH)-releasing peptide-6 (GHRP-6) (His-DTrp-Ala-Trp-DPhe-Lys-NH2) (SEQ ID NO: 21), beta-Interleukin I (163-171) (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys) (SEQ ID NO: 22), Melanotan-II (MT-II, alpha-MSH4-10-NH 2 , or Ac-N1le-4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10) (SEQ ID NO: 32), octreotide (201-995) (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol; disulfide bridge: Cys2-Cys7) (SEQ ID NO: 35), endomorphin-1 Tyr-Pro-Trp-Phe-NH2 (SEQ ID NO: 37); endomorphin-2 Tyr-Pro-Phe-Phe-NH 2  (SEQ ID NO: 38), antiarrhytmic peptide (AAP) (Gly-Pro-Hyp-Gly-Ala-Gly) (SEQ ID NO: 42), Antagonist G (Arg-DTrp-(nMe)Phe-DTrp-Leu-Met-NH 2 ), PD-145065 (Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp) (SEQ ID NO: 47), PD-142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp) (SEQ ID NO: 48), GR 83074 (Boc-Arg-Ala-DTrp-Phe-DPro-Pro-Nle-NH2) (SEQ ID NO:51), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) (SEQ ID NO: 52), parathyroid hormone related peptide (107-111) (Thr-Arg-Ser-Ala-Trp) (SEQ ID NO: 53), Leupeptin (Ac-Leu-Leu-Arg-CHO), kyotorphin, LHRH, eptifibatide, gonadotropin-releasing hormone, growth hormone, oxytocin, vasopressin, vasotocin, and truncated analogues thereof. 
     
     
         4 . A method for the preparation of a pharmacologically active peptide conjugate (X-Z), comprising the steps of:
 a) coupling an additional N-α-protected amino acid in the carboxyl activated form or an N-α-protected dipeptide in the C-terminal activated form to an immobilized peptide sequence H-Z-SSM, thereby forming an immobilized N-α-protected peptide fragment,   b) removing the N-α-protecting group, thereby forming an immobilized 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 immobilized peptide fragment, and repeating the removal/coupling step procedure in steps b) and c) until the desired peptide sequence X is obtained, and   d) cleaving off the peptide conjugate from the solid support material, wherein X is a pharmacologically active peptide, Z is a stabilizing peptide consisting of 4 to 15 amino acid residues covalently bound by its N terminus to the C terminus end of X, wherein Z is a sequence selected from:   (i) LyS 4-10 ,   (ii) (Lys-Xaa) m ,   (iii) (Xaa-Lys) m ,   (iv) Lys p -Xaa q ,   (v) Xaa p -Lys q ,   (vi) Xaa 1 -Lys-Xaa 2 -Lys,   (vii) Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 ,   (viii) Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 -Lys,   (ix) Xaa 1 -Xaa 2 -Lys-Xaa 2 ,   (xi) Xaa 1 -Xaa 1 -Lys-Xaa 2 -Lys-Xaa 2 ,   (xii) Lys-Xaa 2 -Lys-Xaa 1 ,   (xiii) Lys-Xaa 2 -Lys-Xaa 2 -Xaa 1 ,   (xiv) Lys-Xaa 2 -Lys-Xaa 2 -Lys-Xaa 1 ,   (xv) Xaa 2 -Lys-Xaa 2 -Xaa 1 ,   (xvi) Xaa 2 -Lys-Xaa 2 -Lys-Xaa 1 , and   (xvii) Xaa 2 -Lys-Xaa 1 -Lys-Xaa 2 -Xaa 1 ,   
       wherein 
       each of Xaa, Xaa 1 , and Xaa 2  is, independently, selected from the group consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, and Met; 
       each of p and q is, independently, an integer from 1 to 14, with the proviso that p+q is from 4 to 15; and 
       m is an integer in the range from 2 to 7, 
       and wherein X is selected from human parathyroid hormone (1-34), glucagon, peptide YY, exendin-4, bivalirudin, hirulog-1, AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) (SEQ ID NO: 14), Interleukin II (60-70) (Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala) (SEQ ID NO: 24), vasoactive intestinal peptideexendin-3, C-type natriuretic peptide (1-53), cortistatin 14, EMP-1, Atrial natriuretic peptide, human brain natriuretic peptide, gastric inhibitory polypeptide, glucagon-like peptide-1, glucagon-like peptide-2, pituitary adenylate cyclase activating polypeptide, pancreatic polypeptide, somatostatin, calcitonin, and truncated analogues thereof. 
     
     
         5 . The method of  claim 4 , wherein Z is Lys 4  (SEQ ID NO: 55), Lys 5  (SEQ ID NO: 56) or Lys 6  (SEQ ID NO: 62).