Growth Hormones with Prolonged In-Vivo Efficacy

Abstract

The invention relates to growth hormone compounds with a protracted profile. The effect is obtained by linking an albumin binding residue via a hydrophilic spacer to growth hormone variants. Further described are methods of preparing and using such compounds. These growth hormone compounds are based on there althered profile considered particular useful in therapy.

1 . A growth hormone conjugate which comprises a growth hormone compound (GH) having
 a) a single Cys mutation,   b) an additional disulfide bridge, or   c) a single Cys mutation and an additional disulfide bridge,   wherein an albumin binding residue via a hydrophilic spacer is linked to said GH, or   a pharmaceutically acceptable salt thereof.   
     
     
         2 . The conjugate of  claim 1  wherein the growth hormone conjugate has the formula (I):
   A-W-B-GH  (I)
 
 wherein 
 GH represents the growth hormone compound; 
 B represents a hydrophilic spacer; 
 W is a chemical group linking A and B; and 
 A represent an albumin binding residue; 
 and pharmaceutically acceptable salts thereof. 
 
     
     
         3 . The conjugate of  claim 1  wherein the hydrophilic spacer is linked to
 a) the N-terminal or Gln40 or Gln141 of the growth hormone compound or 
 b) the sulphur residue of a single Cys mutation present in the growth hormone compound selected from the group consisting of T3C, P5C, S7C, D11C, H18C, Q29C, E30C, E33C, A34C, Y35C, K38C, E39C, Y42C, S43C, D47C, P48C, S55C, S57C, P59C, S62, E65C, Q69C, E88C, Q91C, S95C, A98C, N99C, S100C, L101C, V102C, Y103C, D107C, S108C, D112C, Q122C, G126C, E129C, D130C, G131C, P133C, T135C, G136C, T142C, D147C, N149C, D154C, A155C, L156C, R178C, E186C, G187C and G190C of SEQ ID NO: 1. 
 
     
     
         4 . The conjugate of  claim 1 , wherein the GH has an additional disulfide bridge between at least one of the amino acid pairs in the positions selected from the group consisting of R16C/L117C, A17C/E174C, H21C/M170C, D26C/V102C, D26C/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, P61C/E66C, P61C/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and V185C/S188C in SEQ ID NO: 1. 
     
     
         5 . The conjugate of  claim 2 , wherein A is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein * denotes the attachment to B through W. 
       
     
     
         6 . The conjugate of  claim 2 , wherein W has the formula
   —W 7 —Y—,
   
       wherein
 Y is —(CH 2 ) l7 —C 3-10 -cycloalkyl-W 8 — or a valence bond; 
 l7 is 0-6; 
 W 7  is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH 2 —, —CH 2 NHC(O)—, —C(O)NHS(O) 2 —, —S(O) 2 NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH 2 —, —CH 2 C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH 2 ) s3 —, —C(O)—, —C(O)O—, —OC(O)—, or a valence bond; wherein s3 is 0 or 1; 
 W 8  is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH 2 —, —CH 2 NHC(O)—, —C(O)NHS(O) 2 —, —S(O) 2 NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH 2 —, —CH 2 C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH 2 ) s4 —, —C(O)—, —C(O)O—, —OC(O)—, or a valence bond; wherein s4 is 0 or 1. 
 
     
     
         7 . The conjugate of  claim 2 , wherein B has the formula
   -X 1 -X 2 -X 3 -X 4 -   
       wherein
 X 1  is —W 1 —[(CHR 1 ) l1 —W 2 ] m1 —{[(CH 2 ) n1 E1] m2 —[(CHR 2 ) l2 W 3 ] m3 } n2 —, 
 X 2  is —[(CHR 3 ) l3 —W 4 ] m3 —{[(CH 2 ) n3 E2] m5 —[(CHR 4 ) l4 —W 5 ] m6 } n4 —; 
 X 3  is —[(CHR 5 ) l5 —W 6 ] m7 —; 
 X 4  is F-D1-(CH 2 ) l6 -D2-; 
 l1, l2, l3, l4, l5 and l6 independently are selected from 0-16, such as from 0-6 
 m1, m3, m4, m6 and m7 independently are selected from 0-10, such as from 0-6 
 m2 and m5 independently are selected from 0-25, such as from 0-10 
 n1, n2, n3 and n4 independently are selected from 0-16, such as from 0-10 
 F is aryl, hetaryl, pyrrolidine-2,5-dione or a valence bond, wherein the aryl and hetaryl groups are optionally substituted with halogen, —CN, —OH, —C(O)OH, —C(O)NH 2 , —S(O) 2 OH or C 1-6 -alkyl; 
 R 1 , R 2 , R 3 , R 4  and R 5  independently are selected from hydrogen, —C(O)OH, —C(O)NH 2 , —S(O)OH, —S(O) 2 OH, —NH—C(═NH)—NH 2 , C 1-6 -alkyl, aryl or hetaryl, wherein the alkyl, aryl and hetaryl groups optionally are substituted with halogen, —C(O)OH, —C(O)NH 2 , —S(O)OH, —S(O) 2 OH, —CN or —OH; 
 D1, D2, E1 and E2 independently are selected from —O—, —N(R 6 )—, —N(C(O)R 7 )— or a valence bond, wherein R 6  and R 7  independently represent hydrogen or C 1-6 -alkyl; 
 W 1  to W 5  independently are selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH 2 —, —CH 2 NHC(O)—, —C(O)NHS(O) 2 —, —S(O) 2 NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH 2 —, —CH 2 C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH 2 ) s2 —, —C(O)—, —C(O)O—, —OC(O)—, or a valence bond, wherein s2 is 0 or 1; 
 W 6  is selected from —C(O)NH—, —NHC(O)—, —C(O)NHCH 2 —, —CH 2 NHC(O)—, —C(O)NHS(O) 2 —, —S(O) 2 NHC(O)—, —OC(O)NH—, —NHC(O)O—, —C(O)CH 2 —, —CH 2 C(O)—, —C(O)CH═CH—, —CH═CHC(O)—, —(CH 2 ) s1 —, —C(O)—, —C(O)O—, —OC(O)—, —NHC(O)C 1-6 -alkyl, —C(O)NHC 1-6 -alkyl or a valence bond, wherein s1 is 0 or 1 and the C 1-6 -alkyl group is optionally substituted with oxo, pyrrolidine-2,5-dione, —NHC(O)CH*CH 2 COOH or —NHC(O)CH 2 CH*COOH, wherein (*) indicates the attachment point from the carbon atom of CH to X 4 . 
 
     
     
         8 . The conjugate of  claim 7 , wherein D1 and D2 are independently selected from —O— or —N(R 6 )— or a valence bond. 
     
     
         9 . The conjugate of  claim 7 , wherein E1 and E2 are independently selected from —O— or —N(R 6 )— or a valence bond. 
     
     
         10 . The conjugate of  claim 2 , wherein W 1  through W 8  independently are selected from the group consisting of —C(O)NH—, —NHC(O)—, —CH 2 NHC(O)—, —C(O)NHS(O) 2 —, —S(O) 2 NHC(O)—, —NHC(O)C 1-6 -alkyl or —C(O)NHC 1-6 -alkyl or a valence bond, wherein the alkyl group is optionally substituted with oxo, pyrrolidine-2,5-dione, —NHC(O)CH*CH 2 COOH or —NHC(O)CH 2 CH*COOH, wherein (*) indicates the attachment point from the carbon atom of CH to X 4 . 
     
     
         11 . The conjugate of  claim 7 , wherein R 1 , R 2 , R 3 , R 4  and R 5  independently are selected from hydrogen, —C(O)OH, —C(O)NH 2 , —S(O) 2 OH or C 1-6 -alkyl; wherein the alkyl group optionally is substituted with —C(O)OH, —C(O)NH 2  or —S(O) 2 OH. 
     
     
         12 . The conjugate of  claim 7 , wherein X 4  is a valence bond and W 6  is selected from either pyrrolidine-2,5-dione, —NHC(O)CH*CH 2 COOH or —NHC(O)CH 2 CH*COOH, wherein (*) indicates the attachment point from the carbon atom of CH to GH. 
     
     
         13 . The conjugate of  claim 2 , wherein B is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         14 . The conjugate of  claim 1 , wherein the growth hormone compound (GH) comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 1. 
     
     
         15 . The conjugate of  claim 1 , wherein the growth hormone compound (GH) comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1. 
     
     
         16 . The conjugate of  claim 1 , wherein the growth hormone compound (GH) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1. 
     
     
         17 . A compound of formula (III)
   A-W-B1-U  (III)
   wherein A represent an albumin binding residue;   B1 represents a hydrophilic spacer;   W is a chemical group linking A and B1, and U represent a conjugating moiety wherein the conjugating moiety, U,   a) comprises or consists of an aryl, an heteroaryl, a substituted malimide or a pyrrolidine-2,5-dione,   b) comprises D1-(CH 2 ) l6 -D2, wherein D1 and D2 are independently selected from —O—, —N(R6)-, —NC(O)R7- or a valence bond, wherein R6 and R7 independently represent hydrogen or C 1-6 -alkyl,   c) comprises or consists of a leaving group, such as Cl, Br, I, —OH, —OS(O) 2 Me, —OS(O) 2 CF 3 , —OTs,   d) comprises or consists of an allyl amine (H 2 C═CH—CH2-NH 2 ), or   e) comprises an amine.