Glucagon-like peptide-2 analogs

Abstract

Analogs of glucagon-like peptide 2, a product of glucagon gene expression, have been identified as intestinal tissue growth factors. Their formulation as pharmaceutical, and therapeutic use in treating disorders of the small bowel, are described.

What is claimed is:  
     
         1 . A GLP-2 analog which is characterized by intestinotrophic activity and which conforms to Formula 1:  
       R1-(Y1)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(P1)-Leu14-Asp15-Asn16-Leu17- Ala18-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile27-Gln-28-Thr29-Lys30-(P3)-(Y2)n-R2,  
       wherein 
 X1 is His or Tyr  
 X2 is Ala or an Ala-replacement amino acid conferring on said analog resistance to DPP-IV enzyme;  
 X3 is Pro, HPro, Asp or Glu;  
 X4 is Gly or Ala;  
 P1 is Glu-X10-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr;  
 X10 is Met or an oxidatively stable Met-replacement amino acid;  
 X19 is Ala or Thr;  
 X20 is Arg, Lys, His or Ala;  
 P2 is Ile-Asn, Ile-Ala or Val-Gln;  
 P3 is a covalent bond, or is Ile, Ile-Thr or Ile-Thr-Asn;  
 R1 is H or an N-terminal blocking group;  
 R2 is OH or a C-terminal blocking group;  
 Y1 is one or two basic amino acids selected from the group Arg, Lys, and His;  
 Y2 is one or two basic amino acids selected from the group Arg, Lys, and His; and  
 m and n, independently, are 0 or 1; and  
 wherein at least one of X1, X2, X3, X4, P1, X10, X19, X20, P2 and P3 is other than a wild type, mammalian GLP-2 residue.  
 
     
     
         2 . The GLP-2 analog according to    claim 1   , wherein said analog incorporates at least one amino acid substitution selected from: 
 a) incorporation at X2 of an amino acid which renders said intestinotrophic GLP-2 analog resistant to cleavage by human DPP-IV enzyme;    b) incorporation at X10 of an oxidatively stable, Met-replacement amino acid; and    c) incorporation at X20 of a basic amino acid selected from His or Lys.    
     
     
         3 . The GLP-2 analog according to    claim 2   , wherein X2 is substituted with an amino acid which confers on said analog resistance to digestion by DPP-IV enzyme.  
     
     
         4 . The GLP-2 analog according to    claim 3   , wherein X2 is selected from the group consisting of D-hPr, D-Pro, D-Ala, Gly, Val, Glu, Lys, Arg, Leu, and Ile.  
     
     
         5 . The GLP-2 analog according to    claim 3    which incorporates at least one amino acid substitution at the following positions: X1, X3, X4, X10, X19, X20 and P2.  
     
     
         6 . The GLP-2 analog according to    claim 2   , wherein X10 is substituted with an amino acid chosen from the group consisting of Val, Ile, Asn, Glx, Tyr, Phe, Leu, Nle, Ala, Ser, and Gly.  
     
     
         7 . The GLP-2 analog according to    claim 1    selected from the group consisting of: [Tyr 1 ]rGLP-2; [Ala 4 ]rGLP-2; [Val 23 Gln 24 ]hGLP-2 and [Asn 33 ]hGLP-2(1-33).  
     
     
         8 . A pharmaceutical composition comprising a therapeutically effective amount of a GLP-2 analog according to    claim 1    and a pharmaceutically acceptable carrier.  
     
     
         9 . A method for promoting growth of small bowel tissue in a patient in need thereof, comprising the step of delivering to the patient the pharmaceutical composition of    claim 8   .  
     
     
         10 . A method for treating a gastrointestinal disease, wherein the method comprises administering to a patient having the gastrointestinal disease a therapeutically effective amount of a GLP-2 analog according to    claim 1   , together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the gastrointestinal disease.  
     
     
         11 . The method of    claim 10   , wherein the gastrointestinal disease is selected from the group consisting of ulcers, digestion disorders, malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), small intestinal damage due to toxic or other chemotherapeutic agents, and short bowel syndrome.  
     
     
         12 . An analog of a human GLP-2 peptide, wherein the GLP-2 analog comprises at least one amino acid substitution selected from the group consisting of: 
 a) incorporation at position X2 and/or X3 of a replacement amino acid which renders the analog resistant to cleavage by DPP-IV enzyme;    b) incorporation at position X10 of a replacement amino acid which is oxidatively stable; and    c) incorporation at position X20 of a replacement amino acid other than Arg.    
     
     
         13 . The GLP-2 analog according to    claim 12    which incorporates an amino acid substitution at position X2, and wherein the amino acid substituted at position X2 is chosen from the group consisting of D-hPr, D-Pro, D-Ala, Gly, Val, Glu, Lys, Arg, Leu, and Ile.  
     
     
         14 . The GLP-2 analog of    claim 13   , wherein the analog is selected from the group consisting of: 
 [Gly 2 ]hGLP-2;    [D-Ala 2 Thr 19 ]hGLP-2;    [Gly 2 Thr 19 ]hGLP-2;    [Ala 1 Gly 2 ]hGLP-2;    [Gly 2 Ala 3 ]hGLP-2;    [Gly 2 Ala 4 ]hGLP-2;    [Gly 2 Ala 5 ]hGLP-2;    [Gly 2 Ala 6 ]hGLP-2;    [Gly 2 Ala 7 ]hGLP-2;    [Gly 2 Ala 8 ]hGLP-2;    [Gly 2 Ala 9 ]hGLP-2;    [Gly 2 Ala 10 ]hGLP-2;    [Gly 2 Ala 11 ]hGLP-2;    [Gly 2 Ala 12 ]hGLP-2;    [Gly 2 Ala 13 ]hGLP-2;    [Gly 2 Ala 16 ]hGLP-2;    [Gly 2 Ala 17 ]hGLP-2;    [Val 2 Thr 19 ]hGLP-2;    [Gly 2 Ala 20 ]hGLP-2    [Gly 2 Ala 12 ]hGLP-2;    [Gly 2 Ala 24 ]hGLP-2;    [Gly 2 Ala 27 ]hGLP-2;    [Gly 2 Ala 28 ]hGLP-2; and    [Gly 2 Ala 31 ]hGLP-2.    
     
     
         15 . The GLP-2 analog according to    claim 12    which incorporates an amino acid substitution at position X10, wherein the amino acid substituted at position X10 is chosen from the group consisting of Val, Ile, Asn, Glx, Tyr, Phe, Leu, Nle, Ala, Ser, and Gly.  
     
     
         16 . The GLP-2 analog according to    claim 15   , wherein the analog is selected from the group consisting of [Ser 10 ]hGLP-2(1-33); [Nle 10 ]hGLP-2(1-33); [Ala 10 ]hGLP-2(1-33); [Leu 10 ]rGLP-2(1-33); [Nle 10 ]ratGLP-2(1-33); [Gly 2 Ala 10 ]hGLP-2(1-33); [Met()) 10 ]ratGLP-2(1-33) and [Tyr 9 Ser 10 Lys 11 Tyr 12  (desIle 13 ) ]hGLP-2(1-33).  
     
     
         17 . The GLP-2 analog according to    claim 12    wherein the nalog is selected from the group consisting of [Pro 3 ]hGLP-2; [HPr 3 ]hGLP-2; [Glu 3 Thr 19 ]hGLP-2; and [Thr 19 Lys 20  ]hGLP-2.  
     
     
         18 . The GLP-2 analog of    claim 12   , wherein the analog is elected from the group consisting of: 
 [Ser 2 ,Gln 3 ]hGLP-2(1-33);    [Gly 2 , Ala 24 ]hGLP-2(1-33);    [Gly 2 , Ala 26 ]hGLP-2(1-33);    [Gly 2 , Ala 14 ]hGLP-2(1-33);    [Gly 2 , Ala 23 ]hGLP-2(1-33);    [Gly 2 , Ala 30 ]hGLP-2(1-33);    [Tyr 1 , Gly 2 ]hGLP-2(1-33);    [Gly 2 , Arg 34 ]hGLP-2(1-34);    [Gly 2 , Tyr 34 ]hGLP-2(1-34);    [tBuGly 2 ]hGLP-2;    [Asp 2 ]hGLP-2 (1-33);    [Glu 2 ]hGLP-2 (1-33);    [Phe 2 ]hGLP-2(1-33);    [His 2 ]hGLP-2(1-33) ;    [Ile 2 ]hGLP-2(1-33);    [Lys 2 ]hGLP-2 (1-33);    [Met 2 ]hGLP-2(1-33);    [Asn 2 ]hGLP-2(1-33);    [Pro 2 ]hGLP-2(1-33)    [Gln 2 ]hGLP-2 (1-33);    [Ser 2 ]hGLP-2 (1-33);    [Thr 2 ]hGLP-2(1-33);    [Val 2 ]hGLP-2(1-33);    [Tyr 2 ]hGLP-2(1-33);    [D-Ala 2 ]hGLP-2 (1-33);    [Pen 2 ]hGLP-2(1-33);    [bAla 2 ]hGLP-2(1-33);    [aAbu 2 ]hGLP-2 (1-33);    [Nval 2 ]hGLP-2 (1-33);    [PhGly 2 ]hGLP-2(1-33); and    [Aib 2 ]hGLP-2(1-33).    
     
     
         19 . A pharmaceutical composition comprising a therapeutically effective amount of a GLP-2 analog according to    claim 12   , and a pharmaceutically acceptable carrier.  
     
     
         20 . A method for promoting growth of small bowel tissue in a patient in need thereof, comprising the step of delivering to the patient the pharmaceutical composition of    claim 19   .  
     
     
         21 . A method for treating a gastrointestinal disease, wherein the method comprises administering to a patient having the gastrointestinal disease a therapeutically effective amount of a GLP-2 analog according to    claim 12   , together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the gastrointestinal disease.  
     
     
         22 . The method of    claim 21   , wherein the gastrointestinal disease is selected from the group consisting of ulcers, digestion disorders, malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), small intestinal damage due to toxic or other chemotherapeutic agents, and short bowel syndrome.  
     
     
         23 . A method of identifying intestinotrophic analogs of GLP-2, comprising the steps of: 
 a) obtaining a GLP-2 analog according to    claim 1   ;    b) treating a mammal with said sing a regimen capable of eliciting an intestinotrophic effect when utilized for rat GLP-2; and    c) determining the effect of said analog on small bowel weight relative to a mock treated control mammal, whereby said intestinotrophic analog of GLP-2 is identified as an analog which elicits an increase in said weight.    
     
     
         24 . An intestinotrophic GLP-2 analog selected from the group consisting of: 
 ratGLP-2(4-33)    Ac-ratGLP-2(1-33);    ratGLP-2(1-30);    ratGLP-2(1-25);    ratGLP-2(1-33)amide;    [Arg- 2 ,Arg- 1 ]ratGLP-2 (2-33);    [Pro 1 ]hGLP-2 (1-33);    [Gln 20 ]hGLP-2(1-33);    [Asp 1 ]hGLP-2 (1-33);    [Tyr 34 ]hGLP-2(1-34);    [desNH2Tyr 1 ]hGLP-2 (1-33);    [Thr 5 ]hGLP-2(1-33);    [ser 16 , Arg 17 ,Arg 18 ]hGLP-2(1-33);    [Agm 34 ]hGLP-2(1-34);    [Arg 30 ]hGLP-2(1-33);    [Ala 5 , Ala 7 ]hGLP-2 (1-33);    [Glu 33 )hGLP-2(1-33);    [Phe 25 ]hGLP-2(1-33); and    [Tyr 25 ]hGLP-2(1-33).    
     
     
         25 . A pharmaceutical composition comprising a therapeutically effective amount of a GLP-2 analog according to    claim 24   , and a pharmaceutically acceptable carrier.  
     
     
         26 . A method for promoting growth of small bowel tissue in a patient in need thereof, comprising the step of delivering to the patient the pharmaceutical composition of    claim 25   .  
     
     
         27 . A method for treating a gastrointestinal disease, wherein the method comprises administering to a patient having the gastrointestinal disease a therapeutically effective amount of a GLP-2 analog according to    claim 24   , together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the gastrointestinal disease.  
     
     
         28 . The method of    claim 27   , wherein the gastrointestinal disease is selected from the group consisting of ulcers, digestion disorders, malabsorption syndromes, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), small intestinal damage due to toxic or other chemotherapeutic agents, and short bowel syndrome.  
     
     
         29 . The peptide [Gly 2 ]hGLP-2.