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 peptide conjugate comprising X and Z, wherein X is a pharmacologically active peptide sequence consisting of 2 to 75 amino acid residues, Z is a stabilizing peptide consisting of 4 to 15 amino acid residues covalently bound by its C terminus to the N 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, 
 
       and salts of said peptide conjugate. 
     
     
         2 . The peptide conjugate 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 peptide conjugate of  claim 1 , wherein X is AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) (SEQ ID NO: 14), insulin-like growth factor I (57-70) (Ala-Leu-Leu-Glu-Thr-Tyr-Cys-Ala-Thr-Pro-Ala-Lys-Ser-Glu) (SEQ ID NO: 15), insulin-like growth factor I (30-41) (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr) (SEQ ID NO: 16), insulin-like growth factor I (24-41) (Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr) (SEQ ID NO: 17), insulin-like growth factor II (33-40) (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) (SEQ ID NO: 18), insulin-like growth factor II (33-40) (Tyr-Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) (SEQ ID NO: 19), insulin-like growth factor II (69-84) (Asp-Val-Ser-Thr-Pro-Pro-Thr-Val-Leu-Pro-Asp-Asn-Phe-Pro-Arg-Tyr) (SEQ ID NO: 20), 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), beta-Interleukin II (44-56) (Ile-Leu-Asn-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu) (SEQ ID NO: 23), Interleukin II (60-70) (Leu-Thr-Phe-Lys-Phe-Tyr-Met-Pro-Lys-Lys-Ala) (SEQ ID NO: 24), exendin-4 (GLP-1 analog) (His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2) (SEQ ID NO: 25), exendin-3 (GLP-I analog) (His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser) (SEQ ID NO: 26), epidermal growth factor (20-31) Cys(Acm)-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys(Acm) (SEQ ID NO: 27), bivalirudin (Hirulog) (D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu) (SEQ ID NO: 28), hirulog-1  D -Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu (SEQ ID NO: 29), C-type natriuretic peptide (1-53) (CNP) (Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys; Disulfide bridge: Cys37-Cys53) (SEQ ID NO: 30), “Mini ANP” (Met-Cys-His-cyclohexylAla-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys-Tyr-Arg, disulfide bridge cys2-cys13) (SEQ ID NO: 31), Melanotan-II (MT-II, alpha-MSH4-110—NH2, or Ac-N11e4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys 10) (SEQ ID NO: 32), thymosin alpha 1 (TA1) (Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn) (SEQ ID NO: 33), Cys-Phe-Ile-Gln-Asn-Cys-Pro-Orn-Gly-NH2, Disulfide bridge: Cys1-Cys6) (SEQ ID NO: 34), octreotide (201-995) (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol; disulfide bridge: Cys2-Cys7) (SEQ ID NO: 35), calcitonin gene-related peptide (CGRP) Ala-Cys-Asp-Thr-Ala-Thr-Cys-yla-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH2; Disulfide bridge: Cys2-Cys7) (SEQ ID NO: 36), endomorphin-1 Tyr-Pro-Trp-Phe-NH2 (SEQ ID NO: 37); endomorphin-2 Tyr-Pro-Phe-Phe-NH2 (SEQ ID NO: 38), nociceptin (also known as Orphanin FQ, Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln) (SEQ ID NO: 39), angiotensinogen (1-13) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His) (SEQ ID NO: 40), adrenomodullin (1-12) (Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg) (SEQ ID NO: 41), antiarrhytmic peptide (AAP) (Gly-Pro-Hyp-Gly-Ala-Gly) (SEQ ID NO: 42), Antagonist G (Arg-DTrp-(nMe)Phe-DTrp-Leu-Met-NH 2 ), indolicidin (Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2) (SEQ ID NO: 43), osteocalcin (37-49) (Gly-Phe-Gln-Glu-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val) (SEQ ID NO: 44), cortistatin 29 (1-13) (Glp)-Glu-Arg-Pro-Pro-Leu-Gln-Gln-Pro-Pro-His-Arg-Asp) (SEQ ID NO: 45), cortistatin 14 Pro-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys; disulfide bridge: Cys2-Cys 13 (SEQ ID NO: 46), 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), fibrinogen binding inhibitor peptide (His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) (SEQ ID NO: 49), leptin (93-105) (Asn-Val-Ile-Gln-Ile-Ser-Asn-Asp-Leu-Glu-Asn-Leu-Arg) (SEQ ID NO: 50), 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), angiotensinogen (1-14) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn (SEQ ID NO: 54), Leupeptin (Ac-Leu-Leu-Arg-CHO), enkephalin, Leu-enkephalin, Met-enkephalin, angiotensin I, angiotensin II, vasopressin, endothelin, vasoactive intestinal peptide, neurotensin, endorphins, insulin, gramicidin, paracelsin, delta-sleep inducing peptide, gonadotropin-releasing hormone, human parathyroid hormone (1-34), EMP-1, Atrial natriuretic peptide, human brain natriuretic peptide, cecropin, kinetensin, neurophysins, elafin, guamerin, atriopeptin I, atriopeptin II, atriopeptin III, deltorphin I, deltorphin II, vasotocin, bradykinin, dynorphin, dynorphin A, dynorphin B, growth hormone release factor, growth hormone, growth hormone releasing peptide, oxytocin, calcitonin, calcitonin gene-related peptide, calcitonin gene-related peptide II, growth hormone releasing peptide, tachykinin, adrenocorticotropic hormone, cholecystokinin, corticotropin releasing factor, diazepam binding inhibitor fragment, FMRF-amide, galanin, gastric releasing polypeptide, gastric inhibitory polypeptide, gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, LHRH, melanin concentrating hormone, melanocyte stimulating hormone, alpha-MSH,-morphine modulating peptides, motilin, neurokinin A, neurokinin B, neuromedin B, neuromedin C, neuromedin K, neuromedin N, neuromedin U, neuropeptide K, neuropeptide Y, pituitary adenylate cyclase activating polypeptide, pancreatic polypeptide, peptide YY, peptide histidine-methionine amide, secretin, somatostatin, substance K, thyrotropin-releasing hormone, kyotorphin, eptifibatide, and melanostatin. 
     
     
         4 . The peptide conjugate of  claim 1 , wherein X is selected from gonadotropin releasing hormone, human parathyroid hormone (1-34), growth hormone, oxytocin, 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), vasopressin, vasoactive intestinal peptide, vasotocin, growth hormone (GH)-releasing peptide-6 (GHRP-6) (His-DTrp-Ala-Trp-DPhe-Lys-NH2) (SEQ ID NO: 21), exendin-3, LHRH, kyotorphin, 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, octreotide (201-995) (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol; disulfide bridge: Cys2-Cys7) (SEQ ID NO: 35), calcitonin, 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), eptifibatide, Melanotan-II (MT-II, alpha-MSH4-10—NH2, or Ac-N1le4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10) (SEQ ID NO: 32), adrenocorticotropic hormone, melanocyte stimulating hormone, alpha-MSH, and truncated analogues thereof.