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 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 endorphins, adrenocorticotropic hormone (ACTH), melanotan II, melanocyte stimulating hormone (MSH), alpha-MSH, and any modified or truncated analogues thereof, and   wherein Z is a stabilizing 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 . The 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 . The peptide conjugate according to  claim 1 , wherein X is alpha-MSH or a modified or truncated analogue thereof. 
     
     
         4 . The peptide conjugate according to  claim 1 , wherein X comprises at least 10 amino acid residues. 
     
     
         5 . The peptide conjugate according to  claim 3 , wherein X is alpha-MSH. 
     
     
         6 . The peptide conjugate according to  claim 3 , wherein X is a modified or truncated analogue of alpha-MSH. 
     
     
         7 . The peptide conjugate according to  claim 1 , wherein X is a modified or truncated analogue in which the amino acid sequence of a pharmacologically active peptide sequence selected from the group consisting of endorphins, adrenocorticotropic hormone (ACTH), melanotan II, melanocyte stimulating hormone (MSH), alpha-MSH, has been modified by changing and/or deleting one or more amino acid residues. 
     
     
         8 . The peptide conjugate according to  claim 7 , wherein X has been modified by altering the side chain stereochemistry and/or backbone in said one amino acid residue. 
     
     
         9 . The peptide conjugate according to  claim 7  wherein a peptide bond has been changed into a reduced form. 
     
     
         10 . The peptide conjugate according to  claim 1 , wherein Z consists of 4-10 amino acid units. 
     
     
         11 . The peptide conjugate according to  claim 1 , wherein Z consists of 4-7 amino acid units. 
     
     
         12 . The peptide conjugate according to  claim 1 , wherein Z consists of 6 amino acid units. 
     
     
         13 . The peptide conjugate according to  claim 12 , wherein Z is Lys 6 . 
     
     
         14 . The peptide conjugate according to  claim 1 , wherein the amino acid units in Z are Lys. 
     
     
         15 . The peptide conjugate according to  claim 14 , wherein Z is selected from the group consisting of Lys 4 , Lys 5 , and Lys 6 . 
     
     
         16 . 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 immobilized N-α-protected amino acid,   b) removing the N-α-protecting group, thereby forming an immobilized 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 immobilized 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,   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 immobilized peptide fragment, and repeating the removal/coupling step procedure in step b) and d) until the desired peptide sequence Z is obtained, and then   e) cleaving off the peptide conjugate from the solid support material.   
     
     
         17 . 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.   
     
     
         18 . 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.   
     
     
         19 . A composition comprising a pharmacologically active peptide conjugate as defined in  claim 1  and a pharmaceutical acceptable carrier. 
     
     
         20 . The composition of  claim 19 , wherein said composition is formulated for oral, subcutaneous, parenteral, intramuscular, rectal, epidural, intratracheal, intranasal, vaginal, buccal, ocular, direct brain, pulmonary or topical administration. 
     
     
         21 . A method for treating a disease or disorder, wherein the peptide sequence X, when not bound to Z, is able to interact with a receptor or a receptor system involved in the condition or disorder in question, and where the interaction between X, when not bound to Z, and the receptor or receptor system has a therapeutic or prophylactic effect on the condition or disorder, said method comprising administering to a patient in need thereof a peptide conjugate as defined in  claim 1 . 
     
     
         22 . A method for treating a disease or disorder selected from the group consisting of HIV infection, cancer, diabetes, incontinence, hypertension, amnesias, Alzheimer's disease, fever, depression, sex hormone regulation, eating, schizophrenia, osteoporosis, and insomnia, said method comprising administering to a patient in need thereof a peptide conjugate as defined in  claim 1 .