Protease Stabilized, Pegylated Insulin Analogues

Abstract

Novel PEGylated insulin analogues exhibiting resistance towards proteases can, effectively, be administered pulmonary or orally. The insulin analogues contain B25H and A14E or A14H. The PEGylation is at B29K.

1 . A PEGylated insulin analogue wherein, in the daughter insulin, at least two hydrophobic amino acids have been substituted with hydrophilic amino acids relative to the parent insulin, wherein the substitutions are within or in close proximity to two or more protease cleavage sites of the parent insulin and wherein such daughter insulin optionally further comprises one or more additional mutations, and wherein the PEG moiety, via a linker, is attached to the ε amino group of the lysine residue in position B29 in said daughter insulin. 
     
     
         2 . A PEGylated insulin analogue according to  claim 1  wherein, in the daughter insulin, the amino acid in position A14 is Glu or His, the amino acid in position B25 is His and which optionally further comprises one or more additional mutations, and wherein the PEG moiety, via a linker, is attached to the c amino acid in the lysine residue in position B29. 
     
     
         3 . A PEGylated insulin analogue according to  claim 1  wherein the daughter insulin comprises the A14E mutation. 
     
     
         4 . A PEGylated insulin analogue according to  claim 1  wherein the daughter insulin analogue comprises the desB30 mutation. 
     
     
         5 . A PEGylated insulin analogue, according to  claim 1 , comprising the moiety
 —(OCH 2 CH 2 ) n —, wherein n is in integer in the range from 2 to about 1000, from 2 to about 500, from 2 to about 250, from 2 to about 125, from 2 to about 50, from 2 to about 25, or from 2 to about 12.   
     
     
         6 . A PEGylated insulin analogue, according to  claim 1 , possible claims, wherein the polyethylene glycol moiety has a nominal molecular weight in the range from about 200 to about 40,000, preferably from about 200 to about 30,000, from about 200 to about 20,000, from about 200 to about 10,000, from about 200 to about 5,000, from about 200 to about 2,000, from about 200 to about 1,000, or from about 200 to about 750. 
     
     
         7 . A PEGylated insulin analogue, according to  claim 1 , wherein the polyethylene glycol moiety is monodisperse. 
     
     
         8 . A PEGylated insulin analogue, according to  claim 1 , which is
 A14E, B25H, B29K(N ε -3-mdPEG 24 -propionyl), desB30 human insulin;   A14E, B25H, B29K(N ε -3-mPEG2.000-propionyl), desB30 human insulin;   A14E, B25H, B29K(N ε 3-{mPEG750}-propionylcarbamoyl), desB30 human insulin;   A14E, B25H, B29K(N ε -3-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}propionyl), desB30 human insulin;   B25H, B29K(N ε -3-(mdPEG 12 )-propionyl) human insulin;   A14E, B25H, B29K(N ε mdPEG 12 -dPEG 24 -propionyl), desB30 human insulin;   A14E, B25H, B29K(N ε (mdPEG 24 -yl-dPEG 24 -yl), desB30 human insulin;   A14E, B25H, B29K(N ε (mdPEG 12 )-3-dPEG 4 -yl), desB30 human insulin;   A14E, B25H, B29K(N ε (mdPEG 12 )-3-dPEG 4 -yl-dPEG 12 -yl), desB30 human insulin;   A14E, B25H, B29K(N ε (mdPEG 12 )-3-dPEG 4 -yl-dPEG 24 -yl), desB30 human insulin;   A14E, B25H, B29K(N ε -3-mdPEG 4×4 -propionyl), desB30 human insulin;   A14E, B25H, B29K(N ε -3-mdPEG 4×4 -dPEG 12 -propionyl), desB30 human insulin or A14E, B25H, B29K(N ε -3-mdPEG 4×4 -dPEG 24 -propionyl), desB30 human insulin.   
     
     
         9 . A pharmaceutical composition comprising a PEGylated insulin according to  claim 1  and one or more pharmaceutically acceptable carriers or diluents. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A method for the treatment, prevention or alleviation of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, syndrome X or dyslipidemia in a subject comprising administering to a subject in need thereof an insulin analogue according to  claim 1  or a pharmaceutical composition thereof. 
     
     
         14 . (canceled) 
     
     
         15 . A pharmaceutical composition according to  claim 9 , wherein the pharmaceutical composition is formulated in an oral dosage form. 
     
     
         16 . A pharmaceutical composition according to  claim 9 , wherein the pharmaceutical composition is formulated in a pulmonary dosage form. 
     
     
         17 . A method for the treatment of diabetes comprising administering to a subject in need thereof, a pharmaceutically effective amount of an insulin analogue according to  claim 1 , or a pharmaceutical composition thereof.