Urocortin peptides

Abstract

Urocortin (Ucn) is a native mammalian peptide generally related to Urotensin I and Corticotropin Releasing Factor (CRF). Human Ucn has the formula: Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 (SEQ ID NO: 15). Rat-derived Ucn is identical but for 2 substitutions, Asp2 for Asn2 and Pro4 for Ser4. Ucn or analogs thereof or pharmaceutically acceptable salts can be administered to humans and other mammals to achieve substantial elevation of ACTH, beta-endorphin, beta-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone. They can also be used to lower blood pressure over an extended period of time, as stimulants to elevate mood and to improve memory and learning performance, as well as diagnostically. Shortened fragments may be administered to release endogenous CRF and/or Ucn in the brain and peripherally. Ucn antagonists can be used to block the action of Ucn and/or CRF, as can antibodies to Ucn. Labelled Ucn agonists and antagonists can be used in drug screening assays along with CRF receptors; they may also be used diagnostically along with Ucn antibodies.

What is claimed is:  
     
         1 . A peptide having SEQ ID NO: 15 of human urocortin (Ucn) or an analogous sequence having only conservative substitutions to the amino acid residues therein or an N-terminally shortened fragment of either which is biologically active to increase ACTH production.  
     
     
         2 . Antibodies which bind specifically to a urocortin peptide or to a fragment thereof according to  claim 1 .  
     
     
         3 . Antibodies according to  claim 2  which specifically bind to and biologically inactivate said urocortin peptide so it no longer increases production of ACTH.  
     
     
         4 . A method for screening for ligands for CRF receptors, which method comprises carrying out a competitive binding assay with a CRF receptor, a peptide according to  claim 1  which contains a suitable label, and a candidate ligand and 
 determining the ability of said candidate ligand to displace said labelled peptide.  
 
     
     
         5 . The screening method according to  claim 4  wherein said CRF receptor is CRF Receptor 2 and said labelled peptide is  125 I-Tyr ∘ -Ucn.  
     
     
         6 . A method for stimulating secretion of ACTH and β-endorphin-like-activities (β-END-LI) in mammals comprising 
 peripherally administering to said mammal an effective amount of the peptide according to  claim 1  or a nontoxic salt thereof and a pharmaceutically acceptable carrier therefor.  
 
     
     
         7 . A method of modifying blood flow and/or blood pressure which comprises administering an effective amount of the peptide according to  claim 1  or of an N-terminally shortened antagonist peptide thereof.  
     
     
         8 . A method according to  claim 7  for modulating blood flow in a desired vascular bed which comprises peripherally administering said effective peptide amount.  
     
     
         9 . A method of increasing coronary blood flow which comprises peripherally administering an effective amount of the peptide according to  claim 1 .  
     
     
         10 . A method of decreasing swelling and/or inflammation and/or vascular permeability which comprises parenterally administering an effective amount of the peptide according to  claim 1 .  
     
     
         11 . A pharmaceutical composition which comprises an effective amount of the peptide according to  claim 1  in combination with a pharmaceutically acceptable carrier, which amount is effective to modulate the transactivation of CRF receptors, or to increase intestinal transit rate.  
     
     
         12 . A method of treatment comprising peripherally administering to a mammal an effective amount of the peptide according to  claim 1  or a nontoxic salt thereof and a pharmaceutically acceptable carrier therefor, which amount is effective to stimulate secretion of ACTH and β-endorphin-like-activities (β-END-LI) in such mammal.  
     
     
         13 . A Ucn antagonist peptide which comprises the following amino acid sequence: 
 Asp-Leu-R 10 -R 11 -His-Leu-Leu-Arg-Thr-Leu-Leu-R 19 -Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-R 29 -Ala-Glu-R 32 -Asn-Arg-Ile-R 36 -Phe-R 38 -Ser-Val-NH 2  (residues 90-122 SEQ ID NO: 15), wherein R 10  is Pro, D-Pro, Thr or D-Tyr; R 11  is D-Phe or D-Tyr or another D-amino acid; R 19  is Glu or Ala; R 29  is Arg or Glu; R 32  is Gln, Lys or Orn; R 36  is Ile, C α MeIle or C α MeLeu; R 38 . is Asp or Ala; provided that when R 29  is Glu, R 32  is either Lys or Orn and the side chains thereof are linked by an amide bond and provided further that Glu in the 31-position can be D-Glu or another D-amino acid and that the N-terminus can be shortened by 1, 2 or 3 residues.    
     
     
         14 . The Ucn antagonist peptide according to  claim 13  which is (cyclo 29-32)D-Phe 11 ,Glu 29 ,Lys 32 -Ucn(11-40).  
     
     
         15 . A method for screening for antagonists for CRF receptors which bind with high affinity to such receptors which method comprises 
 carrying out a competitive binding assay with a CRF receptor, with the peptide according to  claim 14  which contains a suitable label, and with a candidate antagonist and    determining the ability of said candidate antagonist to displace said labelled peptide.    
     
     
         16 . A compound useful for blocking CRF-binding protein (CRF-BP) to thereby increase availability of endogenous CRF and/or Ucn, which compound is a peptide having the amino acid sequence: 
 Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln (residues 85-114 of SEQ ID NO: 15), or a biologically active fragment thereof which is formed by deleting 1 to 8 residues consecutively from the N-terminus, or 1 to 5 residues consecutively from the C-terminus, or both or which has at least 80% homology with said peptide or with said fragment and binds to CRF-BP.    
     
     
         17 . The compound according to  claim 16  selected from the group consisting of Ucn(5-32), Ucn(8-32) and hUcn(3-27).  
     
     
         18 . A method for increasing the in vivo level of CRF and/or Ucn, which method comprises administering an effective amount of the compound according to  claim 16 .  
     
     
         19 . The method according to  claim 18  wherein said effective amount is sufficient to promote parturition in a pregnant female.  
     
     
         20 . The method according to  claim 18  wherein said amount of said compound administered is effective so as to result in an increase in free endogenous CRF and/or Ucn in the brain which causes (a) improvement in short to medium term memory in a subject afflicted with Alzheimer's disease; (b) relief from chronic fatigue syndrome; (c) suppression of appetite; (d) stimulation of the respiratory system, (e) improvement in learning performance; (f) improvement in memory; (g) improvement in alertness; (h) reduction of depression and/or (i) lessening of anxiety.  
     
     
         21 . The method according to  claim 20  wherein said compound is administered so that it reaches the brain.