Antagonists of intestinotrophic GLP-2 peptides

Abstract

Antagonists of glucagon-like peptide 2, have been identified. Their effects on the growth of gastrointestinal tissue are described. Its formulation as a pharmaceutical, and its therapeutic and related uses in treating bowel tissue, are described. Also described are methods of identifying antagonists of glucagon-like peptide 2.

What is claimed is:  
     
         1 . A polypeptide which comprises an amino acid sequence corresponding to that of a first reference mammalian GLP-2, which polypeptide is mutated so that: (i) from one to four of any of the first four N-terminal residues are deleted; or (ii) at least one amino acid selected from the amino acid positions corresponding to the amino acid positions of human GLP-2 at Asp 15 , Phe 22 , Thr 29 , Thr 32  and Asp 33  is substituted with an amino acid which does not naturally occur at that position in the reference GLP-2; or (iii) position Ala 2  is substituted with an amino acid selected from the group consisting of Leu, Cys, Glu, Arg, Trp, and PO 3 -Tyr 2 ; or (iv) a combination of (i), (ii), and/or (iii) is mutated; said polypeptide exhibiting GLP-2 antagonist activity.  
     
     
         2 . A peptide as defined in  claim 1 , wherein the reference mammalian GLP-2 is selected from the group consisting of human GLP-2, degu GLP-2, ox GLP-2, porcine GLP-2, guinea pig GLP-2 and hamster GLP-2.  
     
     
         3 . A peptide as defined in  claim 1 , wherein the reference mammalian GLP-2 is human GLP-2.  
     
     
         4 . A peptide as defined in  claim 1 , said peptide having a substitution at a position selected from the group consisting of Asp 15 , Phe 22 , Thr 29 , Thr 32 , and Asp 33 .  
     
     
         5 . A peptide as defined in  claim 1 , said peptide selected from the group consisting of [Leu 2 ]GLP-2, [Glu 2 ]GLP-2, [Arg 2 ]GLP-2, [Trp 2 ]GLP-2, [PO 3 -Tyr 2 ]GLP-2, and [Cys 2 ]GLP-2.  
     
     
         6 . A peptide as defined in  claim 1 , which is GLP-2(2-33).  
     
     
         7 . A peptide as defined in  claim 1 , which is GLP-2(3-33).  
     
     
         8 . A peptide as defined in  claim 1 , which is GLP-2(4-33).  
     
     
         9 . A peptide as defined in  claim 1 , which is GLP-2(5-33).  
     
     
         10 . A peptide as defined in  claim 1 , which peptide further comprises one or more substitutions selected from the group consisting of: (i) substitution of Ala 2  with Val, Gly, or D-Ala; (ii) substitution of Met 10  with Leu, Ile, Nle, or Ala; (iii) an amino terminal blocking group; and (iv) a carboxy terminal blocking group.  
     
     
         11 . A peptide as defined in  claim 10 , selected from the group consisting of: [Gly 2 , Ala 15 ]GLP-2; [Gly 2 , Ala 22 ]GLP-2; [Gly 2 , Ala 29 ]GLP-2; [Gly 2 , Ala 32 ]GLP-2; and [Gly 2 , Ala 33 ]GLP-2.  
     
     
         12 . A pharmaceutical composition comprising a therapeutically effective amount of a peptide according to  claim 1 , and a pharmaceutically acceptable carrier.  
     
     
         13 . A method for suppressing growth of small bowel tissue in a subject, comprising the step of administering to the subject an effective amount of a funtional antagonist of GLP-2.  
     
     
         14 . A method of reducing hyperplasia or inducing hypoplasia of small bowel tissue in a subject, comprising the step of delivering to the subject a therapeutically effective amount of a peptide according to  claim 1 , which results in the reduction of hyperplasia or induction of hypoplasia of small bowel tissue.  
     
     
         15 . The method of  claim 14 , wherein the subject suffers from obesity or the subject requires bowel rest prior to treatment with chemotherapy or radiotherapy.  
     
     
         16 . A method of treating a gastrointestinal disease, wherein the method comprises administering to a subject having the gastrointestinal disease a therapeutically effective amount of a peptide according to  claim 1 , which results in the amelioration of a pathological effect or symptom of the gastrointestinal disease.  
     
     
         17 . The method of  claim 16 , wherein the gastrointestinal disease is selected from the group consisting of small bowel cancer, cholera, irritable bowel, bowel motility disorders, and chronic diarrhea.  
     
     
         18 . A method of identifying a GLP-2 antagonist comprising the steps of: 
 (1) obtaining a GLP-2 analog having a structural alteration selected from (i) a deletion of at least one amino acid, and (ii) a substitution of at least one amino acid position with an amino acid which does not naturally occur at that position, and (iii) a combination of (i) and (ii);    (2) treating a mammal with said analog using a regimen capable of eliciting a decrease in small bowel weight; and    (3) determining the effect of said analog on small bowel weight relative to a control mammal receiving vehicle only, whereby said functional antagonist of GLP-2 is identified as an analog which elicits a decrease in said small bowel weight.    
     
     
         19 . The method as defined in  claim 18 , wherein the GLP-2 analog structural alteration is a deletion of from one to four of any of the first four N-terminal residues.  
     
     
         20 . The method as defined in  claim 18 , wherein the GLP-2 analog structural alteration is a substitution at a position selected from Asp 15 , Phe 22 , Thr 29 , Thr 32 , and Asp 33 .  
     
     
         21 . A GLP-2 antagonist identified by the method of  claim 18 .  
     
     
         22 . A method of identifying GLP-2 antagonists comprising the steps of: 
 (1) obtaining a GLP-2 analog having a structural alteration selected from (i) a deletion of at least one amino acid, and (ii) a substitution of at least one amino acid position with an amino acid which does not naturally occur at that position, and (iii) a combination of (i) and (ii);    (2) treating a mammal with said analog co-administered with GLP-2 or an intestinotrophic analog of GLP-2, using a regimen capable of eliciting a reduction in the small bowel weight increase seen when GLP-2 is administered alone; and    (3) determining the effect of said analog on small bowel weight relative to a control animal, receiving GLP-2 alone or a intestinotrophic analog of GLP-2 alone, whereby said functional antagonist is identified as an analog which elicits a reduction in the increase in said bowel weight.    
     
     
         23 . The method as defined in  claim 22 , wherein the GLP-2 analog structural alteration is a deletion of from one to four of any of the first four N-terminal residues.  
     
     
         24 . The method as defined in  claim 22 , wherein the GLP-2 analog structural alteration is a substitution at a position selected from Asp 15 , Phe 22 , Thr 29 , Thr 32 , and Asp 33 .  
     
     
         25 . A GLP-2 antagonist identified by the method of  claim 22.