Beta-lipotropin and uses thereof

Abstract

The invention provides isolated nucleic acids, vectors, transformed host cells, analogs, functional fragments, and fusion proteins, related to beta-lipotropin. Also provided are pharmaceutical compositions comprising beta-lipotropin or fragments and/or analogs thereof, and methods for treating diabetes and complications associated therewith by administration of an effective amount of beta-lipotropin.

We claim:  
     
         1 . A substantially pure protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:7.  
     
     
         2 . An isolated nucleic acid compound encoding a protein of  claim 1 .  
     
     
         3 . A vector comprising an isolated nucleic acid compound of  claim 2 .  
     
     
         4 . A vector as in  claim 3 , wherein said isolated nucleic acid compound is operably-linked to a promoter sequence.  
     
     
         5 . A host cell containing a vector of  claim 4 .  
     
     
         6 . A method for constructing a recombinant host cell having the potential to express beta-lipotropin, said method comprising introducing into said host cell by any suitable means a vector of  claim 5 .  
     
     
         7 . A method for expressing beta-lipotropin in a recombinant host cell of  claim 6 , said method comprising culturing said recombinant host cell under conditions suitable for gene expression.  
     
     
         8 . A pharmaceutical formulation comprising as an active ingredient beta-lipotropin, analog, or functional fragment thereof, associated with one or more pharmaceutically acceptable carriers, excipients, or diluents therefor.  
     
     
         9 . A pharmaceutical formulation as in  claim 8  wherein said beta-lipotropin is human beta-lipotropin.  
     
     
         10 . Beta-lipotropin, analog, or functional fragment thereof, for use in treating diabetes or complication thereof.  
     
     
         11 . A method for treating diabetes in a mammal comprising administration of a therapeutically effective amount of beta-lipotropin or analog thereof.  
     
     
         12 . A method for treating diabetes in a mammal comprising administration of a therapeutically effective amount of a functional fragment of beta-lipotropin.  
     
     
         13 . A method, as in  claim 11  wherein said beta-lipotropin is human beta-lipotropin, having a sequence identified herein as SEQ ID NO:8.  
     
     
         14 . A method as in  claim 11  wherein said beta-lipotropin is recombinant human beta-lipotropin.  
     
     
         15 . A method as in  claim 11  wherein said diabetes is Type 1 or Type 2 diabetes.  
     
     
         16 . A method as in  claim 11  wherein said mammal is a human.  
     
     
         17 . A method for treating complications of diabetes in a patient in need thereof comprising administering a therapeutically effective amount of beta-lipotropin, analog, or functional fragment thereof.  
     
     
         18 . A method for lowering blood glucose levels in a mammal by administering an effective amount of beta-lipotropin, analog, or functional fragment thereof.  
     
     
         19 . A method for treating hyperglycemia in a mammal in need thereof by administering an effective amount of beta-lipotropin, analog, or functional fragment thereof.  
     
     
         20 . A method for treating hyperinsulinemia in a mammal by administering an effective amount of beta-lipotropin, analog, or functional fragment thereof.  
     
     
         21 . A method for enhancing insulin sensitivity in a mammal by administering an effective amount of beta-lipotropin, analog, or functional fragment thereof.  
     
     
         22 . A solid-phase synthetic method for synthesizing beta-lipotropin, analog, or fragment thereof, in a single run, comprising the steps of: 
 a) activating an amino acid having its α-amino group, and alternatively, side chain functional group, protected to form a reactive ester;    b) attaching said activated amino acid to an inert solid support;    c) activating a second protected amino acid to an ester wherein the α-amino group of the first amino acid is de-protected yielding a reactive amine, and the second activated amino acid is reacted with said first amino acid to form a di-peptide on the solid support;    d) repeating of steps (a) to (c);    e) removing the peptide from the solid support and deprotecting side chain functional groups;    f) separating the peptide from the solid support; and    g) purifiying the peptide by any suitable means; wherein,    h) where the BLT sequence contains a ser-pro-pro tripeptide segment, multiple-coupling of the pro residues is carried out;    i) after completion of the coupling step, any unreacted, deprotected peptide is blocked to prevent chain lengthening of a deletion peptide; and    j) where the BLT sequence contains an asp-gly dipeptide sequence, utilization of an N-α protecting group at each glycine residue preceding an asp residue.    
     
     
         23 . A process for preparing beta-lipotropin as claimed in  claim 1  comprising: 
 a. transforming a suitable host with an expression vector wherein said vector encodes a beta-lipotropin;  
 b. culturing said transformed host under conditions that enable expression of said beta-lipotropin;  
 c. purifying said beta-lipotropin by any suitable means.  
 
     
     
         24 . An assay for beta-lipotropin activity comprising the steps of: 
 a) administering to a mammal that exhibits insulin insensitivity and elevated blood glucose levels a test protein; and    b) testing by any suitable means for blood glucose and insulin levels after step (a).    
     
     
         25 . A method, as in  claim 11  wherein said beta lipotropin is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8.  
     
     
         26 . A peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.  
     
     
         27 . A peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.  
     
     
         28 . A peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.  
     
     
         29 . A method for treating diabetes comprising administration of a therapeutically effective amount of at least one peptide selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.  
     
     
         30 . A method for treating diabetes comprising administration of a therapeutically effective amount of at least one peptide selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.  
     
     
         31 . A pharmaceutical formulation comprising as an active ingredient a peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.  
     
     
         32 . A functional analog of beta-lipotropin.  
     
     
         33 . A fragment of beta-lipotropin having insulinotropic activity in vitro or in vivo.  
     
     
         34 . A functional analog of beta-lipotropin that is about 90% to 95% identical with SEQ ID NO:8, wherein amino acid substitutions are selected from the group consisting of: 
 the residue at position 1 is alternatively Glu, Ala, Asp or Gln;    the residue at position 2 is alternatively Leu, Ile, Val, or Met;    the residue at position 3 is alternatively Thr, Ala, Glu, Ser, Pro, or Gly;    the residue at position 4 is alternatively Gly, Arg, Ala, Leu, Pro, or Ser;    the residue at position 5 is alternatively Glu, Gln, Asp, Asn, or Ala;    the residue at position 6 is alternatively Arg, Glu, Leu, Lys, Gln, or Ala;    the residue at position 7 is alternatively Leu, Pro, Asp, Val, Ile, or Met;    the residue at position 8 is alternatively Arg, Glu, Ala, Tyr, Leu, Lys, Pro, Gln, or Trp;    the residue at position 9 is alternatively Glu, Ala, Pro, Asp, Asn, or Gln;    the residue at position 10 is alternatively Gly, Ala, Ser, or Asp;    the residue at position 11 is alternatively Asp, Arg, Pro, Asn, Gln, Ala, or Glu;    the residue at position 12 is alternatively Gly, Ala, Ser, or Met;    the residue at position 13 is alternatively Pro, Glu, Gly, or Val;    the residue at position 14 is alternatively Asp, Glu, Asn, Gln, or Gly;    the residue at position 15 is alternatively Gly, Ala, Ser, or Glu;    the residue at position 16 is alternatively Pro, Gln, Leu, Gly, or Glu;    the residue at position 17 is alternatively Ala, Asp, Ser, or Gly;    the residue at position 18 is alternatively Asp, Glu, Gln, or Asn;    the residue at position 19 is alternatively Asp, Glu, Asn, or Gln;    the residue at position 20 is alternatively Gly, Ser, or Ala;    the residue at position 21 is alternatively Ala, Gly, Ser, or Phe;    the residue at position 22 is alternatively Gly, Ala, Ser, or Lys;    the residue at position 23 is alternatively Ala, Phe, Thr, Gly, Ser, or Leu;    the residue at position 24 is alternatively Gln, Arg, Asp, Asn, Leu, or Val;    the residue at position 25 is alternatively Ala, Leu, Asp, Ile, Gly, Ser, or Thr;    the residue at position 26 is alternatively Asp, Glu, Gly, Asn, Gln, or Lys;    the residue at position 27 is alternatively Leu, Ala, Ile, Met, or Val;    the residue at position 28 is alternatively Glu, Gln, Asn, or Asp;    the residue at position 29 is alternatively His, Asn, Tyr, Ala, Gln or Glu;    the residue at position 30 is alternatively Ser, Gly, Glu, Ala, Leu, or Asp;    the residue at position 31 is alternatively Leu, Ala, Val, Met, or Ile;    the residue at position 32 is alternatively Leu, Ala, Val, Ile, Met, or Pro;    the residue at position 33 is alternatively Val, Ala, Glu, Leu, Ile, Met, or Arg;    the residue at position 34 is alternatively Ala, Ser, Pro, Glu, or Gly;    the residue at position 35 is alternatively Ala, Asp, Gly, Ser, or Leu;    the residue at position 36 is alternatively Glu, Ala, Thr, Leu, Asp, Asn, or Gln;    the residue at position 37 is alternatively Lys, Glu, Thr, Arg, Gln, or Asp;    the residue at position 38 is alternatively Lys, Arg, Gin, or Glu;    the residue at position 39 is alternatively Asp, Ala, Asn, Glu, Gln, or Lys;    the residue at position 40 is alternatively Glu, Ser, Asp, Asn, Gln, or Gly;    the residue at position 41 is alternatively Gly, Ala, or Ser;    the residue at position 42 is alternatively Pro, Gly, Ser, or Asn;    the residue at position 43 is alternatively Tyr, Phe, or Trp;    the residue at position 44 is alternatively Arg, Lys, Gin, or Glu;    the residue at position 45 is alternatively Met, Ile, Ser, or Val;    the residue at position 46 is alternatively Glu, Gln, Asp, Asn, His, Arg or Gly;    the residue at position 48 is alternatively Tyr or Trp;    the residue at position 49 is alternatively Arg or Lys;    the residue at position 50 is alternatively Trp, Tyr, or Phe;    the residue at position 51 is alternatively Gly, Ala, Ser, or Gln;    the residue at position 52 is alternatively Ser, Thr, Asn, or Ala;    the residue at position 53 is alternatively Pro or Gly;    the residue at position 54 is alternatively Pro, Ala, Gly, Arg, Leu, or Thr;    the residue at position 55 is alternatively Lys, Arg, Gln, or Ala;    the residue at position 56 is alternatively Asp, Asn, Glu, Gln, Ala, Gly, or Ile;    the residue at position 57 is alternatively Lys, Gln, or Arg;    the residue at position 58 is alternatively Arg, Gln, or Lys;    the residue at position 59 is alternatively Tyr, Phe or Trp;    the residue at position 60 is alternatively Gly, Ala, or Ser;    the residue at position 61 is alternatively Gly, Ala, or Ser;    the residue at position 62 is alternatively Phe, Tyr, or Trp;    the residue at position 63 is alternatively Met, Leu, Ile, or Val;    the residue at position 64 is alternatively Thr, Ala, Ser, or Lys;    the residue at position 65 is alternatively Ser, Ala, Thr, or Pro;    the residue at position 66 is alternatively Glu, Asp, Asn, Lys, or Gln;    the residue at position 67 is alternatively Lys, Arg, or Gln;    the residue at position 68 is alternatively Ser, Ala, Thr, or Gly;    the residue at position 69 is alternatively Gln, Glu, Asp, Asn, Arg, or His;    the residue at position 70 is alternatively Thr, Ser, Ala, or Lys;    the residue at position 71 is alternatively Pro or Gly;    the residue at position 72 is alternatively Leu, Ile, Met, or Val;    the residue at position 73 is alternatively Val, Leu, Ile, or Met;    the residue at position 74 is alternatively Thr, Ala, or Ser;    the residue at position 75 is alternatively Leu, Ile, Met, or Val;    the residue at position 76 is alternatively Phe, Tyr, Trp, or Leu;    the residue at position 77 is alternatively Lys, Gln, or Arg;    the residue at position 78 is alternatively Asn, Asp, Glu, Gln, or His;    the residue at position 79 is alternatively Ala, Gly, Ser, Ile or Val;    the residue at position 80 is alternatively Ile, Leu, Met, Val, or Thr;    the residue at position 81 is alternatively Ile, Met, Val, Thr, or Leu;    the residue at position 82 is alternatively Lys, Gln, or Arg;    the residue at position 83 is alternatively Asn, Asp, Glu, Gln, or Ser;    the residue at position 84 is alternatively Ala, Val, Ser, Gly, or Glu;    the residue at position 85 is alternatively Tyr, Phe, Trp, or His;    the residue at position 86 is alternatively Lys, Gln, or Arg;    the residue at position 87 is alternatively Lys, Gln, or Arg;    the residue at position 88 is alternatively Gly, Ala, or Ser;    the residue at position 89 is alternatively Glu, Gln, Asp, Asn, or His.    
     
     
         35 . A functional analog of beta-lipotropin as in  claim 34  wherein said analog is about between 95% to 99% identical with SEQ ID NO:8.  
     
     
         36 . A peptide having insulinotropic activity selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22.  
     
     
         37 . A Method as in  claim 22 , wherein at step: 
 h)where the BLT sequence contains a ser-pro-pro tripeptide segment, double-coupling of the pro and ser residues is carried out;    i) after completion of the coupling step, any unreacted, deprotected peptide is blocked with an anhydride to prevent chain lengthening of a deletion peptide.    
     
     
         38 . A method as in  claim 37 , wherein at step: 
 j) said N-α protection comprising N-α-Hmb or methyl benzoate.    
     
     
         39 . A functional analog of beta-lipotropin that is at least 90% identical with SEQ ID NO:8.