Single branch heparin-binding growth factor analogs

Abstract

A heparin-binding growth factor (HBGF) analog having two substantially similar sequences (homodimeric sequences) branched from a single amino acid residue, where the sequences are analogs of a particular HBGF that binds to a heparin-binding growth factor receptor (HBGFR), or alternatively that bind to a HBGFR without being an analog of any particular HBGF. The homodimeric sequences may be derived from any portion of a HBGF. The synthetic HBGF analog may be an analog of a hormone, a cytokine, a lymphokine, a chemokine or an interleukin, and may bind to any HBGFR. Further provided are preparations for medical devices, pharmaceutical compositions and methods of using the same.

1 . A heparin-binding growth factor analog of formula I: 
       
         
           
           
               
               
           
         
       
       wherein:
 each X is a peptide chain that (i) has a minimum of three amino acid residues, (ii) has a maximum of about fifty amino acid residues, and (iii) binds a heparin-binding growth factor receptor (HBGFR); 
 R 1  is a single trifunctional amino acid residue covalently bonded to each X; 
 Each R 2  is independently a linker comprising a chain from 0 to about 20 backbone atoms including carbon, oxygen, sulfur, nitrogen and mixtures thereof covalently bonded to R 1  and X; 
 Each R 3  is hydrogen (H) such that the terminal group is NH 2 , or is an acyl group with a linear or branched C 1  to C 17  alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an N-terminus NH 2 , NH 3   + , or NH group or a corresponding acylated derivative; 
 R 4  is OH such that the terminal group is a carboxyl, NH 2 , an acyl group with a linear or branched C 1  to C 17  alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an N-terminus NH 2 , NH 3   + , or NH group or a corresponding acylated derivative, or NH-R 3 ; 
 Y is a linker comprising a chain from 0 to about 50 backbone atoms covalently bonded to R 1  and Z; and 
 Z is a non-signaling peptide chain that includes a heparin binding domain, comprising an amino acid sequence that comprises (i) a minimum of one heparin binding motif, (ii) a maximum of about ten heparin binding motifs, and (iii) a maximum of about thirty amino acids. 
 
     
     
         2 . The heparin-binding growth factor analog of  claim 1  wherein X and Z are synthetic peptide chains. 
     
     
         3 . The heparin-binding growth factor analog of  claim 1  wherein Y further comprises a linker that (i) is hydrophobic, (ii) comprises a chain of a minimum of about 9 and a maximum of about 50 atoms, and (iii) is not found in the natural ligand of the heparin-binding growth factor receptor (HBGFR) which X binds. 
     
     
         4 . The heparin-binding growth factor analog of  claim 1  wherein the heparin-binding growth factor analog has an avidity for heparin such that the synthetic heparin-binding growth factor analog binds heparin in 0.15 M NaCl, but is eluted by 1 M NaCl. 
     
     
         5 . The heparin-binding growth factor analog of  claim 1  of formula II: 
       
         
           
           
               
               
           
         
       
       wherein R 1  is a diamine amino acid. 
     
     
         6 . The heparin-binding growth factor analog of  claim 1  wherein R 1  is an L- or D-diamine amino acid residue selected from the group consisting of 2,3 diamino propionyl amino acid, 2,4 diamino butylic amino acid, lysine and ornithine. 
     
     
         7 . The heparin-binding growth factor analog of  claim 5  of formula III: 
       
         
           
           
               
               
           
         
       
       wherein:
 C is carbon, H is hydrogen, N is nitrogen and O is oxygen. 
 
     
     
         8 . The heparin-binding growth factor analog of  claim 1  wherein the construct is of formula IV: 
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  is a trifunctional amino acid wherein the side chain of R 1  comprises a reactive sulfhydryl; and 
 R 2  comprises a trifunctional amino acid wherein the side chain comprises a reactive sulfhydryl, wherein R 2  is covalently bonded to R 1  by a disulfide bond. 
 
     
     
         9 . The heparin-binding growth factor analog of  claim 8  wherein R 1  and R 2  are each independently an L- or D-3-mercapto amino acid selected from the group consisting of L- or D-cysteine, L- or D-penicillamine, 3-mercapto phenylalanine, and a derivative of any of the foregoing. 
     
     
         10 . The heparin-binding growth factor analog of  claim 8  of formula V: 
       
         
           
           
               
               
           
         
       
       wherein:
 Prg Grp is OH or a carboxy terminus protecting group; and 
 C is carbon, H is hydrogen, N is nitrogen, O is oxygen and S is sulfur. 
 
     
     
         11 . The heparin-binding growth factor analog of  claim 1  wherein the construct is of formula VI: 
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  is a trifunctional amino acid wherein the side chain comprises a first reactive group; and 
 R 2  comprises a trifunctional amino acid wherein the side chain comprises a second reactive group, wherein R 2  is covalently bonded to R 1  by a covalent bond between the first reactive group and the second reactive group. 
 
     
     
         12 . A heparin-binding growth factor analog comprising a synthetic peptide having two sequences branched from a single residue, the two sequences being the same and binding specifically to a heparin-binding growth factor receptor, and a sequence comprising a non-growth factor heparin-binding sequence covalently bonded to the single residue. 
     
     
         13 . The heparin-binding growth factor analog of  claim 12  wherein the non-growth factor heparin-binding sequence is covalently bonded to the single residue by means of a linker. 
     
     
         14 . The heparin-binding growth factor analog of  claim 13  wherein the linker comprises a backbone chain from 2 to about 50 atoms. 
     
     
         15 . The heparin-binding growth factor analog of  claim 12  wherein the single residue comprises a trifunctional amino acid residue. 
     
     
         16 . The heparin-binding growth factor analog of  claim 1  wherein Y comprises between one and about thirty-three ethylene glycol units. 
     
     
         17 . The heparin-binding growth factor analog of  claim 1  wherein Y comprises a branched or unbranched, saturated or unsaturated alkyl chain of between one and about twenty carbon atoms. 
     
     
         18 . The heparin-binding growth factor analog of  claim 1  wherein Y comprises [NH 2 -(CH 2 ) p CO] q  wherein p is from 1 to about 10 and q is from 1 to about 20. 
     
     
         19 . The heparin-binding growth factor analog of  claim 1  wherein Y comprises a peptide sequence comprising from one to about 16 Gly residues. 
     
     
         20 . The heparin-binding growth factor analog of  claim 1  wherein each heparin binding motif of Z is BxBB or BBBxxB, wherein each B is independently lysine, arginine, ornithine, or histidine, and each x is a independently a naturally occurring amino acid. 
     
     
         21 . The heparin-binding growth factor analog of  claim 1  wherein Z comprises at least two heparin-binding motifs. 
     
     
         22 . The heparin-binding growth factor analog of  claim 1  wherein the covalent bonds between R 1  and Y comprise an amide, disulfide, thioether, Schiff base, reduced Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. 
     
     
         23 . The heparin-binding growth factor analog of  claim 1  wherein the covalent bonds between R 1  and each X comprise an amide, disulfide, thioether, Schiff base, reduced Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. 
     
     
         24 . The heparin-binding growth factor analog of  claim 1  wherein the covalent bonds between Y and Z comprise an amide, disulfide, thioether, Schiff base, reduced Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. 
     
     
         25 . The heparin-binding growth factor analog of  claim 1  wherein X is any of SEQ ID NO:7 to SEQ ID NO:107, a portion thereof, a homolog thereof, or a homolog of a portion thereof, and Z comprises any of SEQ ID NO: 1 to SEQ ID NO:6. 
     
     
         26 . The heparin-binding growth factor analog of  claim 1  wherein R 2  comprises between one and about three amino acid residues selected from the group consisting of glycine, a straight chain amino carboxylic acid, a bifunctional amino-PEG-acid spacer and combinations thereof. 
     
     
         27 . The heparin-binding growth factor analog of  claim 1  wherein Y comprises between one and about ten amino acid residues selected from the group consisting of glycine, a linear chain amino carboxylic acid, a bifunctional amino-PEG-acid spacer and combinations thereof. 
     
     
         28 . The heparin-binding growth factor analog of  claim 1  wherein X comprises an amino acid sequence found in any of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, HBBM (heparin-binding brain mitogen), HB-GAF (heparin-binding growth associated factor), HB-EGF (heparin-binding EGF-like factor) HB-GAM (heparin-binding growth associated molecule, also known as pleiotrophin, PTN, HARP), TGF-α (transforming growth factor-α), TGF-βs (transforming growth factor-βs), VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), IGF-1 (insulin-like growth factor-1), IGF-2 (insulin-like growth factor-2), PDGF (platelet derived growth factor), RANTES, SDF-1, secreted frizzled-related protein-1 (SFRP-1), small inducible cytokine A3 (SCYA3), inducible cytokine subfamily A member 20 (SCYA20), inducible cytokine subfamily B member 14 (SCYB14), inducible cytokine subfamily D member 1 (SCYD1), stromal cell-derived factor-1 (SDF-1), thrombospondins 1, 2, 3 and 4 (THBS1-4), platelet factor 4 (PF4), lens epithelium-derived growth factor (LEDGF), midikine (MK),macrophage inflammatory protein (MIP-1), moesin (MSN), hepatocyte growth factor (HGF, also called SF), placental growth factor, IL-1 (interleukin-1), IL-2 (interleukin-2), IL-3 (interleukin-3), IL-6 (interleukin-6), IL-7 (interleukin-7), IL-10 (interleukin-10), IL-12 (interleukin-12), IFN-α (interferon-α), IFN-γ (interferon-γ), TNF-α (tumor necrosis factor-α), SDGF (Schwannoma-derived growth factor), nerve growth factor, neurite growth-promoting factor 2 (NEGF2), neurotrophin, BMP-2 (bone morphogenic protein 2), OP-1 (osteogenic protein 1, also called BMP-7), keratinocyte growth factor (KGF), interferon-γ inducible protein-20, RANTES, and HIV-tat-transactivating factor, amphiregulin (AREG), angio-associated migratory cell protein (AAMP), angiostatin, betacellulin (BTC), connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYCR61), endostatin, fractalkine/neuroactin, glial derived neurotrophic factor (GDNF), GRO2, hepatoma-derived growth factor (HDGF), and granulocyte-macrophage colony stimulating factor (GMCSF), or a homolog of an amino acid sequence found in any of the foregoing. 
     
     
         29 . A pharmaceutical composition comprising the heparin-binding growth factor analog of any of  claims 1 ,  5 ,  7 ,  8 ,  10  or  11  or a pharmaceutically acceptable salt thereof and a pharmaceutical carrier.