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US 200901 11743A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0111743 A1 Takahashi (43) Pub. Date: Apr. 30, 2009

(54) CYSTEINE-BRANCHED HEPARIN-BINDING Related U.S. Application Data ANALOGS (60) Provisional application No. 60/656,713, filed on Feb. 25, 2005. (75) Inventor: Kazuyuki Takahashi, O O Germantown, MD (US) Publication Classification (51) Int. Cl. Correspondence Address: A638/08C07K 700 30.82006.O1 201 THIRD STREET, N.W., SUITE 1340 A638/16 (2006.01) ALBUQUEROUE, NM 87102 (US) A638/10 (2006.01) A638/00 (2006.01) (73) Assignee: BioSurface Engineering (52) U.S. Cl...... 514/12:530/330; 530/329; 530/328; Technologies, Inc., Rockville, MD 530/327: 530/326; 530/325; 530/324; 514/17; (US) 514/16; 514/15: 514/14: 514/13 (57) ABSTRACT (21) Appl. No.: 11/362,137 The present invention provides a heparin binding growth factor analog of any of formula I-VIII and methods and uses (22) Filed: Feb. 23, 2006 thereof. US 2009/011 1743 A1 Apr. 30, 2009

CYSTENE-BRANCHED HEPARN-BINDING 0007 HBGFs useful in prevention or therapy of a wide GROWTH FACTOR ANALOGS range of diseases and disorders may be purified from natural sources or produced by recombinant DNA methods, however, CROSS-REFERENCE TO RELATED Such preparations are expensive and generally difficult to APPLICATIONS prepare. 0008. Some efforts have been made to generate heparin 0001. This application claims priority to and the benefit of binding growth factor analogs. For example, natural PDGF the filing of U.S. Provisional Patent Application Ser. No. occurs as an A chain and a B chain arranged in head-to-head 60/656,713,570 entitled “Cysteine-Branched Heparin-Bind (AA or BB) homodimers, or (AB or BA) heterodimers. Thus, ing Growth Factor Analogs' filed on Feb. 22, 2005 and the U.S. Pat. No. 6,350,731 to Jehanli et al. discloses PDGF specification and claims thereof are incorporated herein by analogs in which two synthetic PDGF receptor-binding reference. domains are covalently linked through a polyglycine or an N-(4-carboxy-cyclohexylmethyl)-maleimide (SMCC) chain INTRODUCTION to mimic the natural active polypeptide dimer. 0009 U.S. Pat. No. 6,235,716 to Ben-Sasson discloses 0002 The invention relates to the field of synthetic pep analogs of angiogenic factors. The analogs are branched mul tides and analogs of heparin-binding growth factors, includ tivalent ligands that include two or more angiogenic homol ing homodimeric and heterodimeric chain synthetic heparin ogy regions connected by a multilinker backbone. binding growth factor analogs wherein a linear homodimeric 0010 U.S. Pat. No. 5,770,704 (the 704 patent) to or heterodimeric sequence is covalently bonded to a heparin Godowski discloses conjugates for activating receptor binding sequence by means of a side chain in the tyrosine kinases, cytokine receptors and members of the homodimeric or heterodimeric sequence. The invention fur receptor Superfamily. The conjugates ther relates to the clinical uses of Such analogs as soluble include at least two ligands capable of binding to the cognate drugs and as coatings for medical devices. receptor, so that the binding of the respective ligands induces oligomerization of these receptors. The ligands disclosed in BACKGROUND OF THE INVENTION the 704 patent are linked by covalent attachment to various 0003) Note that the following discussion refers to a num nonproteinaceous polymers, particularly hydrophilic poly ber of publications by author(s) and year of publication. Dis mers, such as polyvinylalcohol and polyvinylpyrrolidone, cussion of Such publications herein is given for more com and the polyvinylalkene ethers, including polyethylene gly plete background and is not to be construed as an admission col and polypropylene glycol. The ligands include hepatocyte that such publications are prior art for patentability determi growth factor (HGF) peptide variants that each bind HGF nation purposes. receptor, thereby causing receptor dimerization and activa 0004. The heparin-binding growth factors (HBGFs) con tion of the biological activity of the HGF receptor dimer. stitute a large class of growth factors that includes the 23 (0011 U.S. Pat. No. 6.284,503 (the 503 patent) to Cald fibroblast growth factors identified to date (FGFs 1-23), well et al. discloses a composition and method for regulating HBBM (heparin-binding brain mitogen), HB-GAF (heparin the adhesion of cells and biomolecules to hydrophobic sur binding growth associated factor), HB-EGF (heparin-binding faces and hydrophobic coated surfaces for cell adhesion, cell EGF-like factor) HB-GAM (heparin-binding growth associ growth, cell sorting and biological assays. The composition is ated molecule), TGF-C. (transforming growth factor-C), a biomolecule conjugated to a reactive end group activated TGF-Bs (transforming growth factor-?s), PDGF (platelet polymer. The end group activated polymer includes a block derived growth factor), EGF (), copolymer Surfactant backbone and an activation or reactive VEGF (vascular endothelial growth factor), IGF-1 ( group. The block copolymer may be any Surfactant having a like growth factor-1), IGF-2 (insulin-like growth factor-2), hydrophobic region capable of adsorbing onto a hydrophobic HGF (), IL-1 (-1), IL-2 Surface, and a hydrophilic region which extends away from (interleukin-2), IFN-C. (interferon-C), IFN-Y (interferon-Y), the surface when the hydrophobic region is adsorbed onto the TNF-C. (tumor necrosis factor-O.), SDGF (Schwannoma-de hydrophobic surface. The 503 patent discloses that the bio rived growth factor) and the many other growth factors, molecules that may be conjugated to the end group activated cytokines, lymphokines and chemokines that have an affinity polymer include natural or recombinant growth factors. Such for heparin. as PDGF, EGF, TGFC, TGFB, NGF, IGF-I, IGF-II, GH and 0005 Peptides from natural HBGFs that bind heparin GHRF, as well as multi-CSF (II-3), GM-CSF, G-CSF, and binding growth factor receptors have been identified. See for M-CSF. example Ray et al., Proc. Natl. Acad. Sci. USA94:7047-7052 0012. Other workers have described compositions that (1997). These authors demonstrated that two amino acid include homologs and analogs of fibroblast growth factors sequences from FGF-2 are sufficient to block the mitogenic (FGFs). See for example U.S. Pat. No. 5,679,673 to Lappiand activity of FGF-2 on neural progenitor cells. The first peptide Baird; U.S. Pat. No. 5,989,866 to Deisher et al. and U.S. Pat. is a ten amino acid sequence, from amino acids 65-74, the No. 6,294,359 to Fiddes et al. These disclosures relate to FGF second peptide extends from amino acids 115-129. homologs or analogs that are either conjugated to a toxic 0006. In an alternative approach, an artificial peptide that moiety and are targeted to the FGF receptor-bearing cells; or binds aheparin-binding (HBGFR) was are homologs or analogs that modulate the biological path identified by a phage display method. Ballinger et al., Nature ways through the signal transduced by the FGF receptor upon BioTechnology 17:1199-1204 (1999) used this technique to binding by the FGF homolog or analog. isolate a 28 amino acid peptide called C19, binds FGF-2 0013. A series of patent applications to Kochendoerfer et receptors, but by itself fails to stimulate biological activity. al. disclose polymer-modified , including synthetic The peptide has no amino acid sequence identity with any chemokines and erythropoiesis stimulating proteins. See, for known FGF. example, International Publications WO 02/04105, WO US 2009/011 1743 A1 Apr. 30, 2009

02/19963 and WO 02/20033. These include chemically ing growth factoragonists useful for coating medical devices ligated peptide segments of a polypeptide chain of a synthetic and as soluble biologics, and as pharmaceutical agents for erythropoiesis , such that a polypeptide chain results, treating a variety of conditions. with a water soluble polymer attached at one or more glyco Sylation sites on the protein. These applications also disclose synthetic chemokines, which are also polymer modified, and SUMMARY OF THE INVENTION are asserted to be antagonists. However, heparin-binding domains are not disclosed. Other mimetics are 0018. One aspect of the present invention provides a hep known, such as those disclosed in U.S. Pat. Nos. 5,773,569 arin-binding growth factor analog of formula I: and 5,830,851 to Wrighton et al. 0014. A series of applications with some inventors incom mon, including U.S. patent application Ser. No. 10/644.703, entitled Synthetic Heparin-Binding Growth Factor Analogs, filed on Aug. 19, 2003, and U.S. patent application Ser. No. R3 10/224.268, entitled Synthetic Heparin-Binding Growth Fac R-R-AA AA2 Y-Z tor Analogs, filed on Aug. 20, 2002, disclose constructs in which two receptor-binding domains are branched from an R3 R3 R3 amine of a backbone amino acid through a peptide bond. X X X 0015 The above described homologs, analogs, conjugates pi iii. or ligands each include a receptor-binding domain. However, none of the disclosed compositions further include both a wherein: linker, providing for the linking of at least two receptor 0019 each X a peptide chain that (i) has a minimum of binding domains through Sulfur complexation. three amino acid residues, (ii) has a maximum of about fifty 0016 International Publication WOO0/18921 to Ballinger and Kavanaugh discloses a composition consisting of fusion amino acid residues, and (iii) binds a heparin-binding growth proteins having FGF receptor affinity linked to an "oligomer factor receptor (HBGFR): ization domain', either directly or through a linking group. 0020 R is a trifunctional amino acid residue covalently The oligomerization domain ranges in length from about 20 bonded to one X through the N terminus amine of R and the to 300 residues, and includes constructs such as transcription remaining X through the C terminus carboxyl of R: factors, Fc portions of IgG, leucine zippers and the like. The 0021 R is a linker comprising a chain from 3 to about 20 oligomerization domains disclosed are homodimeric atoms covalently bonded to the side chain of R and Y when domains, wherein a single FGF receptor affinity fusion pro n=0, or to the side chain of R and to the N terminus amine of tein is linked to a single domain, such as a leucine Zipper, AA when n=1; which in turn is linked to a similar molecule by means of 0022. Each R is independently from 0 to about 3 amino cysteine residues at both the amino and carboxy termini of the acid residues which are the same or different; leucine Zippers, such that two parallel leucine Zippers, each 0023 Y is a linker comprising a chain from 0 to about 50 with a single FGF receptor affinity fusion protein, are cross atoms covalently bonded to R and Zwhen n=0, or to AA and linked by means of disulfide bonds. It is also disclosed that Z when n=1 and m=0, or to AA and Z when n=1 and m=1: fusion proteins may include a heparin binding domain, Such 0024 Z is a non-signaling peptide chain that includes a as the use of jun as a multimerization domain, which is heparin binding domain, comprising an amino acid sequence asserted to be a heparin binding domain. Thus the composi that comprises (i) a minimum of one heparin binding motif. tions disclosed by Ballinger and Kavanaugh are all composed of a single receptor-binding sequence covalently attached to (ii) a maximum of about ten heparin binding motifs, and (iii) an oligomerization domain, whereby two or more similar a maximum of about thirty amino acids; oligomerization domains, each with a single receptor-binding 0025 AA and AA are each independently a trifunctional sequence, are conjoined by means of either an association amino acid residue, wherein X is covalently bonded through provided by the oligomerization domain, or alternatively, are the side chain of AA and AA, and chemically cross-linked to provide for the covalent bonding 0026 n is 0 or 1, and when n is 1 m is 0 or 1, and when n of the individual components. is 0, m is 0. 0017. The above described homologs, analogs, conjugates 0027. Another aspect of the present invention provides a or ligands each include a receptor-binding domain. However, heparin-binding growth factor analog of formula II: none of the disclosed compositions further include both a linker, providing for the linking of at least two receptor II binding domains to the linker through a side chain of the X receptor-binding domains, and further providing a single non-signaling peptide containing a heparin-binding domain. ck-y Z Moreover, none of these or other known heparin-binding X growth factor analogs provide the advantages described herein below. There is still a need for new peptide analogs of HBGFs, particularly for those that function as agonists, and wherein: preferably those that contain two receptor-binding domains 0028 Cys is cysteine, and R is a linker consisting of a specific for a HBGFR. In particular, there is still a need for sulfhydryl reactive homo-bifunctional cross-linker and cost-effective synthetic peptide agonists of heparin-binding a second Cys or comprising a hetero-bifunctional cross growth factor receptors, particularly synthetic heparin-bind linker. All other features are as indicated for formula I. US 2009/011 1743 A1 Apr. 30, 2009

0029. One aspect of the present invention provides a hep m=1, comprise an amide, disulfide, thioether, Schiff base, arin-binding growth factor analog of formula III: reduced Schiffbase, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. 0037. One aspect of the present invention provides a hep III arin-binding growth factor analog of formula I-IV wherein the side chains of AA and AA comprise reactive carboxyl Rs groups. 0038 Yet another aspect of the present invention provides X a heparin-binding growth factor analog of formula I-IV al O O wherein the side chain of R comprises a reactive sulfhydryl group. CH-C-S NH2 0039. One aspect of the present invention provides a hep | H. N-R-N arin-binding growth factor analog of formula I-IV wherein R OR S-C-CH is an L- or D-3-mercapto amino acid. He 0040 Still another aspect of the present invention provides a heparin-binding growth factor analog of formula I-IV i O O =o wherein the L- or D-3-mercapto amino acid is L- or D-cys R6 teine, L- or D-penicillamine, 3-mercaptophenylalanine, or a Z derivative of any of the foregoing. 0041. One aspect of the present invention provides a hep arin-binding growth factor analog of formula I-IV wherein X is any of SEQID NO:5 to SEQID NO:55 and Z is RKRLD RIAR (SEQ ID NO:58), RKRKLERIAR (SEQ ID NO:2) wherein: RKRKLGRIAR (SEQID NO:3) or RKRKLWRARA (SEQ ID NO:4). 0030 R is a linker comprising a chain of between 1 and 0042. Yet another aspect of the present invention provides about 10 backbone atoms selected from carbon, oxygen, a heparin-binding growth factor analog of formula I-IV Sulfur and nitrogen or mixtures thereof; wherein one or more of R is between one and about three 0031 Rs is NH, an acyl group with a linear or branched amino acid residues selected from the group consisting of C to C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl glycine, 6-aminohexanoic acid, 7-aminoheptanoic acid, 9-aminononanoic acid and mixtures thereof. chain including an N-terminus NH2, NH, or NH group or a 0043. One aspect of the present invention provides a hep corresponding acylated derivative; arin-binding growth factor analog of formula I-IV wherein X 0032 R is OH, NH, or NH Rs; and comprises an amino acid sequence found in any of FGF-1, 0033 R, is NH, an acyl group with a linear or branched FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF C to C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl 9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, chain including an N-terminus NH2, NH, or NH group or a FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF corresponding acylated derivative. All other features are as 22, FGF-23, HBBM (heparin-binding brain mitogen), HB indicated for formula I. GAF (heparin-binding growth associated factor), HB-EGF (heparin-binding EGF-like factor) HB-GAM (heparin-bind 0034 Still another aspect of the present invention provides ing growth associated molecule, also known as , a heparin-binding growth factor analog of formula IV: PTN, HARP). TGF-C. (transforming growth factor-C.), TGF Bs (transforming growth factor-3s), VEGF (vascular endot helial growth factor), EGF (epidermal growth factor), IGF-I IV (insulin-like growth factor-1), IGF-2 (insulin-like growth fac tor-2), PDGF (platelet derived growth factor), RANTES, NH SDF-1, secreted frizzled-related protein-1 (SFRP-1), small O HN O inducible cytokine A3 (SCYA3), inducible cytokine subfam NH ily A member 20 (SCYA20), inducible cytokine subfamily B CH-C-S 1n 1-1N member 14 (SCYB14), inducible cytokine subfamily D | H2 N S-C-CH member 1 (SCYD1), stromal cell-derived factor-1 (SDF-1), O= O H =o thrombospondins 1, 2, 3 and 4 (THEBS1-4), platelet factor 4 X (PF4), lens epithelium-derived growth factor (LEDGF), mid O Y ikine (MK), macrophage inflammatory protein (MIP-1), NH2 moesin (MSN), hepatocyte growth factor (HGF, also called SF), placental growth factor, IL-1 (interleukin-1), IL-2 (inter NH2 leukin-2), IL-3 (interleukin-3), IL-6 (interleukin-6), IL-7 (in terleukin-7), IL-10 (interleukin-10), IL-12 (interleukin-12), wherein: IFN-O. (interferon-C.), IFN-Y (interferon-Y), TNF-C. (tumor necrosis factor-O.), SDGF (Schwannoma-derived growth fac 0035 X is any of SEQID NOS:6-55;Y is between two tor), nerve growth factor, neurite growth-promoting factor 2 and about five amino acid residues selected from the (NEGF2), , BMP-2 (bone morphogenic protein group consisting of 6-aminohexanoic acid, 7-aminohep 2), OP-1 (osteogenic protein 1, also called BMP-7), kerati tanoic acid, 9-aminononanoic acid and mixtures thereof, nocyte growth factor (KGF), interferon-Y inducible protein and Z is any of SEQID NOS:2-5 or SEQID NO:58. 20, RANTES, and HIV-tat-transactivating factor, amphiregu 0036 Yet another aspect of the present invention provides lin (AREG), angio-associated migratory cell protein a heparin-binding growth factor analog of formula I-IV (AAMP), , (BTC), connective tissue wherein the covalent bonds between the side chain of R and growth factor (CTGF), cysteine-rich angiogenic inducer 61 R and between R and Z when n=0, or between AA and Z (CYCR61), , fractalkine/neuroactin, glial derived when n=1 and m=0, or between AA and Z when n=1 and neurotrophic factor (GDNF), GRO2, hepatoma-derived US 2009/011 1743 A1 Apr. 30, 2009

growth factor (HDGF), and granulocyte-macrophage colony wherein: stimulating factor (GMCSF), a homolog of any amino acid 0054 each X and each Wis a peptide chain differing by at sequence found in the foregoing, a reverse sequence of any least one amino acid residue that (i) has a minimum of three amino acid sequence found in the foregoing, or a homolog of amino acid residues, (ii) has a maximum of about fifty amino a reverse sequence of any amino acid residue found in the acid residues, and (iii) binds a heparin-binding growth factor foregoing. receptor (HBGFR): 0044 Another aspect of the present invention provides a heparin-binding growth factor analog of formula I-IV 0055 R is a trifunctional amino acid residue covalently wherein Y comprises between one and about thirty-three eth bonded to one X through the N terminus amine of R and the ylene glycol units. remaining X through the C terminus carboxyl of R: 0045 Still another aspect of the present invention provides 0056 R is a linker comprising a chain from 3 to about 20 a heparin-binding growth factor analog of formulas I-IV atoms covalently bonded to the side chain of R and Y when wherein Y comprises a branched or unbranched, saturated or n=0, or to the side chain of R and to the N terminus amine of unsaturated alkyl chain of between one and about twenty AA when n=1; carbon atoms. 0057 Each R is independently from 0 to about 3 amino 0046. One aspect of the present invention provides a hep acid residues which are the same or different; arin-binding growth factor analog of formulas I-IV whereinY comprises (NH2 (CH2)CO), wherein p is from 1 to about 0.058 Y is a linker comprising a chain from 0 to about 50 10 and q is from 1 to about 20. atoms covalently bonded to R and Zwhen n=0, or to AA and 0047 Another aspect of the present invention provides a Z when n=1 and m=0, or to AA and Z when n=1 and m=1: heparin-binding growth factor analog of formulas I-IV 0059 Z is a non-signaling peptide chain that includes a wherein Y comprises a peptide sequence comprising from heparin binding domain, comprising an amino acid sequence one to about 16 Gly residues. that comprises (i) a minimum of one heparin binding motif. 0048 One aspect of the present invention provides a hep (ii) a maximum of about ten heparin binding motifs, and (iii) arin-binding growth factor analog of formula I-IV wherein a maximum of about thirty amino acids; each heparin binding motif of Z is BxBB or BBBXxB, 0060 AA and AA are each independently a trifunctional wherein each B is independently lysine, arginine, ornithine, amino acid residue, wherein X and W are covalently bonded or histidine, and each X is a independently a naturally occur ring amino acid. In another aspect, Z of the heparin-binding through the side chain of AA and AA, respectively; and, growth factor analog may comprise at least two heparin 0061 n is 0 or 1, and when n is 1 m is 0 or 1, and when n binding motifs. is 0, m is 0. 0049 Still another aspect of the present invention provides 0062 Yet another aspect of the present invention provides a pharmaceutical composition comprising the heparin-bind a heparin-binding growth factor analog of formula V wherein ing growth factor analog of formulas I-IV or a pharmaceuti X, W and Z are synthetic peptide chains. cally acceptable salt thereof and a pharmaceutical carrier. 0063. One aspect of the present invention provides a hep 0050. One aspect of the present invention provides a hep arin-binding growth factor analog of formula V wherein Y arin-binding growth factor analog of any of formulas I-IV further comprises a linker that (i) is hydrophobic, (ii) com wherein X and Z are synthetic peptide chains. prises a chain of a minimum of about 9 and a maximum of 0051 Yet another aspect of the present invention provides about 50 atoms, and (iii) is not found in the natural ligand of a heparin-binding growth factor analog of any of formulas the heparin-binding growth factor receptor (HBGFR) which I-IV having a linker that (i) is hydrophobic, (ii) comprises a X binds. chain of a minimum of about 9 and a maximum of about 50 0064. Still another aspect of the present invention provides atoms, and (iii) is not found in the natural ligand of the a heparin-binding growth factor analog of formula V wherein heparin-binding growth factor receptor (HBGFR) which X the heparin-binding growth factor analog has an avidity for binds. heparin Such that the synthetic heparin-binding growth factor 0052 Still another aspect of the present invention provides a heparin-binding growth factor analog of any of formulas analog binds heparin in 0.15 M NaCl, but is eluted by 1 M I-IV having an avidity for heparin such that the synthetic NaCl. heparin-binding growth factor analog binds heparin in 0.15 M 0065. Yet still another aspect of the present invention pro NaCl, but is eluted by 1 M NaCl. vides a heparin-binding growth factor analog of formula VI: 0053) One aspect of the present invention provides a hep arin-binding growth factor analog of formula V: VI

W

R-R-AA AA-HY-Z wherein: R3 R3 R3 0.066 Cys is cysteine; and 0067 R is a linker consisting of a sulfhydryl reactive X X pi W iii. homo-bifunctional cross-linker and a second Cys or comprising a hetero-bifunctional cross-linker. All other features are as represented for formula V. US 2009/011 1743 A1 Apr. 30, 2009

0068. One aspect of the present invention provides a hep when n=1 and m=0, or between AA and Z when n=1 and arin-binding growth factor analog of formula VII: m=1, comprise an amide, disulfide, thioether, Schiff base, reduced Schiffbase, imide, secondary amine, carbonyl, urea, hydrazone or oxime bond. VII 0077. Another aspect of the present invention provides a heparin-binding growth factor analog of formula I-IV Rs wherein XandW each independently comprise anamino acid sequence found in any of FGF-1, FGF-2, FGF-3, FGF-4, y FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, I FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF CH-C-S NH2 18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, HBBM(he H N-R-N parin-binding brain mitogen), HB-GAF (heparin-binding OEC S-C-CH growth associated factor), HB-EGF (heparin-binding EGF H2 like factor) HB-GAM (heparin-binding growth associated O O =o molecule, also known as pleiotrophin, PTN, HARP). TGF-C. R6 (transforming growth factor-O.), TGF-Bs (transforming growth factor-Bs), VEGF (vascular endothelial growth fac Z tor), EGF (epidermal growth factor), IGF-1 (insulin-like growth factor-1), IGF-2 (insulin-like growth factor-2), PDGF R7 (platelet derived growth factor), RANTES, SDF-1, secreted frizzled-related protein-1 (SFRP-1), small inducible cytokine wherein: A3 (SCYA3), inducible cytokine subfamily A member 20 0069 R is a linker comprising a chain of between 1 and (SCYA20), inducible cytokine subfamily B member 14 about 10 backbone atoms selected from carbon, oxygen, Sul (SCYB14), inducible cytokine subfamily D member 1 fur and nitrogen or mixtures thereof; (SCYD1), stromal cell-derived factor-1 (SDF-1), thrombo 0070 Rs is NH, an acyl group with a linear or branched spondins 1,2,3 and 4 (THEBS1-4), platelet factor 4 (PF4), lens C to C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl epithelium-derived growth factor (LEDGF), midikine (MK), chain including an N-terminus NH, NH, or NH group or a macrophage inflammatory protein (MIP-1), moesin (MSN), corresponding acylated derivative; hepatocyte growth factor (HGF, also called SF), placental (0071 R is OH, NH, or NH Rs; and growth factor, IL-1 (interleukin-1), IL-2 (interleukin-2). IL-3 0072 R, is NH, an acyl group with a linear or branched (interleukin-3), IL-6 (interleukin-6), IL-7 (interleukin-7), C to C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl IL-10 (interleukin-10), IL-12 (interleukin-12), IFN-C. (inter feron-C.), IFN-Y (interferon-Y), TNF-C. (tumor necrosis factor chain including an N-terminus NH2, NH, or NH group or a O), SDGF (Schwannoma-derived growth factor), nerve corresponding acylated derivative. All other features are as growth factor, neurite growth-promoting factor 2 (NEGF2). presented for formula V. neurotrophin, BMP-2 (bone morphogenic protein 2), OP-1 0073 Yet another aspect of the present invention provides (osteogenic protein 1, also called BMP-7), keratinocyte a heparin-binding growth factor analog of formula VIII: growth factor (KGF), interferon-Y inducible protein-20, RANTES, and HIV-tat-transactivating factor, (AREG), angio-associated migratory cell protein (AAMP), VIII angiostatin, betacellulin (BTC), connective tissue growth fac tor (CTGF), cysteine-richangiogenic inducer 61 (CYCR61), H. endostatin, fractalkine/neuroactin, glial derived neurotrophic y O factor (GDNF), GRO2, hepatoma-derived growth factor HN O (HDGF), and granulocyte-macrophage colony stimulating NH2 CH-C-S 11a-N factor (GMCSF), a homolog of any amino acid sequence H2 N S-C-CH found in the foregoing, a reverse sequence of any amino acid O= H2 CEO sequence found in the foregoing, or a homolog of a reverse X O O sequence of any amino acid residue found in the foregoing. Y 0078. Other aspects, objects, advantages and novel fea NH2 tures, and further scope of applicability of the present inven 1. tion will be set forth in part in the detailed description to NH2 follow, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by wherein: practice of the invention. The objects and advantages of the 0074 X and W are each independently selected from invention may be realized and attained by means of the instru any of SEQID NOS:6-55; mentalities and combinations particularly pointed out in the 0075 Y is between two and about five amino acid resi appended claims. dues selected from the group consisting of 6-aminohex 0079 Additional objects and advantages of the present anoic acid, 7-aminoheptanoic acid, 9-aminononanoic invention will be apparent in the following detailed descrip acid and mixtures thereof; and Z is any of SEQ ID tion read in conjunction with the accompanying drawing fig NOS:2-5 or SEQ ID NO:58. All other features are as U.S. represented for formula V. DETAILED DESCRIPTION OF THE INVENTION 0076 Still another aspect of the present invention provides a heparin-binding growth factor analog of formula V-VIII 0080. In one embodiment, each synthetic HBGF analog of wherein the covalent bonds between the side chain of R and the invention contains two identical sequences that are ana R and between R and Z when n=0, or between AA and Z logs of a particular HBGF that binds to a HBGFR, or alter US 2009/011 1743 A1 Apr. 30, 2009 natively each synthetic HBGF analog of the invention con I0083. The amino acid sequences of many of these and tains two sequences, both of which bind to a HBGFR. The other HBGFs are available from the National Library of homodimeric sequences may be derived from any portion of Medicine Protein Database at the internet site accessible a HBGF. The synthetic HBGF analog may be an analog of a through the worldwide web address ncbi.nlm.nih.gov/entrez. hormone, a cytokine, alymphokine, a chemokine or an inter These HBGF amino acid sequences on the foregoing internet leukin, and may bind to any HBGFR for any of the foregoing. site are hereby incorporated by reference. The use of syn 0081. In one aspect the synthetic HBGF analog of the thetic HBGF analogs incorporating the amino acid sequences present invention is a molecule of any one of formulas I to IV. of the receptor binding domains from these and other HBGFs HBGFs include any growth factor that binds selectively to is specifically contemplated in the present invention. heparin. For example, the HBGF can be any of the known I0084. In particular embodiments of the present invention, FGFs (FGF-1 to FGF-23), HBBM (heparin-binding brain the synthetic HBGF analog of the present invention consists mitogen), HB-GAF (heparin-binding growth associated fac essentially of the molecule of any one of formulas I to VIII, tor), HB-EGF (heparin-binding EGF-like factor) HB-GAM i.e. the molecule of any one of formula I to VIII is the major (heparin-binding growth associated molecule, also known as active component in the synthetic HBGF analog composition. pleiotrophin, PTN, HARP), TGF-C. (transforming growth The Heparin-Binding Growth Factors of Formulas I to IV factor-O.), TGF-Bs (transforming growth factor-?s), VEGF (vascular endothelial growth factor), EGF (epidermal growth I0085. The regions X and Z of the synthetic HBGF analogs factor), IGF-1 (insulin-like growth factor-1), IGF-2 (insulin of formulas I to IV include amino acid residues, and option like growth factor-2), PDGF (platelet derived growth factor), ally the region Yincludes amino acid residues. An amino acid residue is defined as NHRCO , where R can be hydrogen RANTES, SDF-1, secreted frizzled-related protein-1 (SFRP or any organic group. The amino acids can be D-amino acids 1), small inducible cytokine A3 (SCYA3), inducible cytokine or L-amino acids. Additionally, the amino acids can be subfamily A member 20 (SCYA20), inducible cytokine sub C.-amino acids, B-amino acids, y-amino acids, or Ö-amino family B member 14 (SCYB14), inducible cytokine subfam acids and so on, depending on the length of the carbon chain ily D member 1 (SCYD1), stromal cell-derived factor-1 of the amino acid. (SDF-1), thrombospondins 1, 2, 3 and 4 (THEBS1-4), platelet I0086. The amino acids of the X, Y and Z component factor 4 (PF4), lens epithelium-derived growth factor regions of the synthetic HBGF analogs of the invention can (LEDGF), midikine (MK), macrophage inflammatory pro include any of the twenty amino acids found naturally in tein (MIP-1), moesin (MSN), hepatocyte growth factor (HGF, proteins, i.e. alanine (Ala., A), arginine (Arg, R), asparagine also called SF), placental growth factor, IL-1 (interleukin-1), (ASn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic IL-2 (interleukin-2), IL-3 (interleukin-3), IL-6 (interleukin acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine 6), IL-7 (interleukin-7). IL-10 (interleukin-10), IL-12 (inter (His, H), isoleucine, (Ile, I), leucine (Leu, L), lysine (LyS, K). leukin-12), IFN-C. (interferon-C), IFN-Y (interferon-Y), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, TNF-C. (tumor necrosis factor-O.), SDGF (Schwannoma-de P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W). rived growth factor), nerve growth factor, neurite growth tyrosine (Tyr, Y), and valine (Val, V). promoting factor 2 (NEGF2), neurotrophin, BMP-2 (bone 0087 Furthermore, the amino acids of the X, Y and Z morphogenic protein 2), OP-1 (osteogenic protein 1, also component regions of the synthetic HBGF analogs of the called BMP-7), keratinocyte growth factor (KGF), inter invention can include any of the naturally occurring amino feron-Y inducible protein-20, RANTES, and HIV-tat-transac acids not found naturally in proteins, e.g. B-alanine, betaine tivating factor, amphiregulin (AREG), angio-associated (N.N.N-trimethylglycine), homoserine, homocysteine, migratory cell protein (AAMP), angiostatin, betacellulin y-aminobutyric acid, ornithine, and citrulline. (BTC), connective tissue growth factor (CTGF), cysteine I0088 Additionally, the amino acids of the X, Y and Z rich angiogenic inducer 61 (CYCR61), endostatin, fractalk component regions of the synthetic HBGF analogs of the ine/neuroactin, or glial derived neurotrophic factor (GDNF), invention can include any of the non-biological amino acids, GRO2, hepatoma-derived growth factor (HDGF), granulo i.e. those not normally found in living systems, such as for cyte-macrophage colony Stimulating factor (GMCSF), and instance, a straight chain amino-carboxylic acid not found in the many growth factors, cytokines, and chemok nature. Examples of straight chain amino-carboxylic acids ines that have an affinity for heparin. It is also contemplated not found in nature include 6-aminohexanoic acid, 7-amino that agents of the invention can be modified through the heptanoic acid, 9-aminononanoic acid and the like. introduction of appropriate binding sequences to direct ana I0089. In formula I, two X regions are covalently linked to logs of growth factors, cytokines, interleukins, and chemok R, either directly or through an R group, where R is a ines, which do not normally bind to heparin, to have heparin trifunctional amino acid residue, preferably a trifunctional binding affinity. alpha amino acid residue. It is to be appreciated that Such 0082 In another aspect the synthetic HBGF analog of the covalent bonds may be to any chemically permitted func present invention is a molecule of any one of formulas V to tional group. Where the trifunctional amino acid residue is an VIII, which is a heterodimeric construct including at least one amino acid with a reactive Sulfhydryl side chain, such as X region and one Wregion, wherein the XandWregions vary cysteine, it is possible and contemplated that one X is by at least one residue. In a preferred embodiment, each of the covalently bonded through the N-terminus amine group, the X and W regions are different sequences derived from differ remaining X is covalently bonded through the C-terminus ent regions of the same growth factor. X and W regions may carboxyl group, and the cysteine is bound to R through the be derived in that they are identical to or homologous with a reactive sulfhydryl side chain. Similarly, one X may be sequence within a growth factor. It is to be understood that covalently bonded to an R group, such as between one and any definition of X contained herein is also equally applicable three glycines, which R group is covalently bonded through to W, and W is nothereafter defined. the C-terminus carboxyl group of R and the other X is simi US 2009/011 1743 A1 Apr. 30, 2009

larly bonded through the N-terminus amine group of R 0.095 The R regions of formulas I or II can include a chain through an R group, again Such as one to three glycines. of atoms or a combination of atoms that form a chain. Typi Similar approaches may be employed with other trifunctional cally, the chains are chains of carbon atoms, that may also amino acid residues, using cross-linkers as hereafter optionally include oxygen, nitrogen or Sulfur atoms, such as described. for example chains of atoms formed from amino acids (e.g. 0090 The amino acids AA and AA can be any trifunc amino acids found in proteins, as listed above; naturally tional amino acid residue, preferably a trifunctional alpha occurringamino acids not found in proteins, such as ornithine amino acid residue. In one preferred embodiment, the trifunc and citrulline; or non natural amino acids, such as amino tional amino acid residue is a diamine amino acid, Such as for hexanoic acid; or a combination of any of the foregoing amino instance lysine or ornithine, or any other amino acid having acids). It is also contemplated that agents such as polyethyl two amino groups. ene glycol (PEG), polyethylene oxide (PEO), amino polyeth 0091. The term “homologous', as used herein refers to ylene glycol, bis-amine-PEG, and other variants of polyeth peptides that differ in amino acid sequence at one or more amino acid positions when the sequences are aligned. For ylene glycol known to those skilled in the art can similarly be example, the amino acid sequences of two homologous pep used. tides can differ only by one amino acid residue within the 0096. The chain of atoms of the R region of formula I and aligned amino acid sequences of five to ten amino acids. IV is covalently attached to R and AA if n=1, or to R and Y Alternatively, two homologous peptides of ten to fifteen if n=0. The covalent bonds can be, for example, peptide, amino acids can differ by no more than two amino acid amide, thioether or ester bonds. Preferably, the R region residues when aligned. In another alternative, two homolo includes a chain of a minimum of about three atoms. For gous peptides of fifteen to twenty or more amino acids can example, where the covalent bonds are peptide bonds, the R differ by up to three amino acid residues when aligned. For region may beformed from a chain of at least one, at least two longer peptides, homologous peptides can differ by up to or at least three amino acids. However, where other than approximately 5%, 10%, 20% or 25% of the amino acid peptide bonds are employed, the R region may further residues when the amino acid sequences of the two peptide include a cross-linking moiety. For example, informula II the homologs are aligned. R region is a linker consisting of a sulfhydryl reactive homo 0092 Particularly useful amino acid sequences as X bifunctional cross linker and a second Cys, or alternatively regions of formulas I to IV include homologs of fragments of includes a hetero-bifunctional cross-linker. naturally occurring HBGFs that differ from the amino acid sequences of natural growth factor in only one or two or a very 0097. In one preferred embodiment, two X regions form a few positions. Such sequences preferably include conserva single linear peptide construct, joined by an R group that is a tive changes, where the original amino acid is replaced with trifunctional amino acid residue, and optional one or two R. an amino acid of a similar character according to well known groups. The trifunctional amino acid residue may, for principles; for example, the replacement of a non-polaramino example, have a reactive Sulfhydryl group in the side chain, acid such as alanine with valine, leucine, isoleucine or pro Such as an L- or D-3-mercapto amino acid, including but not line; or the substitution of one acidic or basic amino acid with limited to L- or D-cysteine, L- or D-penicillamine, 3-mer another amino acid of the same acidic or basic character. captophenylalanine, or a derivative of any of the foregoing. 0093. In another alternative, the X regions of the synthetic The R trifunctional amino acid residue is covalently bonded HBGF analog can include an amino acid sequence that shows to the X regions by peptide bonds, such that the single linear no detectable homology to the amino acid sequence of any peptide construct is, by way of example only, X-C X or HBGF. Peptides or growth factor analogs useful as compo X-R-C-R X, where C is L- or D-cysteine, and each X nents of the X region of the synthetic analogs of the present is covalently linked to C by peptide bonds, directly in the case invention, that have little or no amino acid sequence homol of X-C X, and indirectly in the case of X-R C R - ogy with the cognate growth factor and yet bind HBGFRs X. Similarly, the R group can include a trifunctional amino may be obtained by any of a wide range of methods, including acid residue with a reactive sulfhydryl group in the side chain, for instance, selection by phage display. See as an example: again covalently bonded to the Y region by an ordinary pep Sidhu et al. Phage display for selection of novel binding tide bond. In one generalized description, this includes the peptides. Methods Enzymol. 328:333-63 (2000). following general formula: 0094. The X region of the synthetic HBGF analogs of the invention can have any length that includes an amino acid sequence that effectively binds an HBGFR. Preferably, the X NH2 regions of the synthetic HBGF analogs have a minimum k length of at least approximately three amino acid residues. More preferably, the X regions of the synthetic HBGF ana t NH2 logs have a minimum length of at least approximately six amino acid residues. Most preferably the X regions of the i- S-s-Homo-bifunctional cross-linkers-- h synthetic HBGF analogs have a minimum length of at least ORC CEO approximately ten amino acid residues. The X regions of the k synthetic HBGF analogs of the invention preferably also have a maximum length of up to approximately fifty amino acid l, Y residues, more preferably a maximum length of up to approximately forty amino acid residues, and most preferably , a maximum length of up to approximately thirty amino acid residues. US 2009/011 1743 A1 Apr. 30, 2009 or alternatively: dosalicylamidobutylamine, or an amino group and a guana dium group that is present in the side chain of arginine, for example p-azidophenylglyoxal monohydrate. 0101 Preferably, the R2 region includes a chain of a maxi mum of about twenty backbone atoms. The amino acid sequence of the R region, if amino acid residues are employed therein, is preferably an artificial sequence, i.e. it does not include any amino acid sequence of four or more amino acid residues found in a natural ligand of a HBGF. CH-C-S-Hono-bifunctional cross-linker-S-C - CH 0102. In the synthetic HBGF analogs of the present inven H H tion, in one preferred embodiment the Y region of any of EO formulas I to IV is a linker that is sufficiently hydrophobic to O = non-covalently bind the HBGF analog to a polystyrene or R3 polycaprolactone Surface, or the like. In addition, the Y region may bind to other hydrophobic surfaces, particularly the hydrophobic surfaces formed from materials used in medical H devices. Such surfaces are typically hydrophobic Surfaces. Examples of suitable surfaces include but are not limited to those formed from hydrophobic polymers such as polycar 0098. In these formulas, the “homo-bifunctional cross bonate, polyester, polypropylene, polyethylene, polystyrene, linker forms a part of R, together with the C residue to polytetrafluoroethylene, expanded polytetrafluoroethylene, which Y is covalently bonded. Any sulfhydryl reactive homo polyvinyl chloride, polyamide, polyacrylate, polyurethane, bifunctional crosslinking agent may be employed, such as for polyvinyl alcohol, polyurethane, poly ethyl vinyl acetate, example a maleimide cross-linker, a haloacetyl cross-linker poly(butyl methacrylate), poly(ethylene-co-vinyl acetate), or a pyridyl disulfide cross-linker. A large number of Such polycaprolactone, polylactide, polyglycolide and copoly Sulfhydryl cross-linkers, such as maleimide cross-linkers, are mers of any two or more of the foregoing; siloxanes Such as known. 2,4,6,8-tetramethylcyclotetrasiloxane; natural and artificial For example, in maleimide cross-linkers of the general for rubbers; glass; and metals including stainless steel, titanium, mula: platinum, and nitinol. Preferably, the binding of the HBGF analogs to the hydrophobic Surface is of Sufficient quantity to be detected by an analytical method such as an enzyme-linked immunoassay or a biological assay. 0103) According to one embodiment of the invention, the Y region of formulas I to IV includes a chain of atoms or a combination of atoms that form a chain. Typically, the chains are chains of carbon atoms, that may also optionally include oxygen, nitrogen or Sulfur atoms, such as for example chains of atoms formed from amino acids (e.g. amino acids found in proteins, as listed above; naturally occurring amino acids not R may be a C to Cs alkyl chain, such as for example 1.2- found in proteins, such as ornithine and citrulline; or non bis-maleimidoethane, 1,4-bis-maliimidobutane or 1,6-bis natural amino acids, such as amino hexanoic acid; or a com maleimidohexane, or may be an aryl group Such as phenyl, bination of any of the foregoing amino acids). Such as for example 1.4-phenylene dimaleimide or 1.2-phe 0104. The chain of atoms of the Y region of formula Ito IV nylene dimaleimide, or may be an aliphatic chain containing is covalently attached to either R or, if provided, AA or AA, one or more oxygen (O), Sulfur (S) or nitrogen (N) chain and to peptide Z. The covalent bonds can be, for example, members, and optionally a ketone, such as for example dithio peptide, amide, thioether or ester bonds. Preferably, the Y bis-maleimidoethane, maleimidopropionic acid maleimi region includes a chain of a minimum of about nine atoms. domethyl ester, bis-maleimidomethylether, 1.11-bis-male More preferably, the Y region includes a chain of a minimum imido-(PEO), 1.8-bis-maleimido-(PEO), and so on. of about twelve atoms. Most preferably, the Y region includes 0099. In yet another embodiment, any of a number of a chain of a minimum of about fifteen atoms. For example, the homo- or hetero-functional electrophilically-activated PEGs Y region may be formed from a chain of at least four, at least may be employed, including those that contain functional five or at least sixamino acids. Alternatively, the Y region may groups such as Succinimidyl propionate, Succinimidyl be formed from a chain of at least one, at least two, or at least butanoate, N-hydroxysuccinimide, benzotriazol carbonate, three aminohexanoic acid residues. aldehydes, acetaldehyde diethyl acetal, or vinylsulfone, and 0105 Preferably, the Y region includes a chain of a maxi others known to those skilled in the art. mum of about fifty atoms. More preferably, the Y region 0100. In yet another embodiment, a hetero-bifunctional includes a chain of a maximum of about forty-five atoms. cross-linker is employed. Hetero-bifunctional reagents which Most preferably, the Y region includes a chain of a maximum cross-link by two different coupling moieties can be particu of about thirty-five atoms. For example, the Y region may be larly useful. Thus, the coupling moiety on R is a cysteine formed from a chain of up to about twelve, up to about fifteen, residue and, on either Y or a part of R, a residue or other or up to about seventeen amino acids. moiety with an amino group. Such that a cross-linker for an 0106 The amino acid sequence of the Y region is prefer amino group and Sulfhydryl group is employed, for example ably an artificial sequence, i.e. it does not include any amino m-maleimidobenzoyl-N-hydroxysuccinimide ester. Alterna acid sequence of four or more amino acid residues found in a tively the cross-linker reagent links two amino groups, for natural ligand of a HBGF. example N-5-azido-2-nitrobenzoyloxysuccinimide, an 0107. In a particular embodiment, the Y region includes a amino group and a carboxyl group, for example 4-p-azi hydrophobic amino acid residue, or a chain of hydrophobic US 2009/011 1743 A1 Apr. 30, 2009

amino acid residues. The Y region can, for example, include low salt concentrations, up to about 0.15 MNaCl, optionally one or more aminohexanoic acid residues. Such as one, two, up to about 0.48 MNaCl, forming a complex between heparin three or more aminohexanoic acid residues. Alternatively, the and the Z region of the factor analog. The complex can be Y region can include up to about twelve, up to about fifteen, dissociated in 1 MNaCl to release the synthetic HBGF analog or up to about seventeen ethylene glycol residues. In another from the heparin complex. alternative embodiment, the Y region can include a combina 0114. The Z region is a non-signaling peptide. Accord tion of amino acid hydrophobic residues. ingly, when used alone the Z region binds to heparin which 0108. In another particular embodiment, the Y region of can be bound to a receptor of a HBGF, but the binding of the the molecule can include a branched or unbranched, saturated Z region peptide alone does not initiate or block signaling by or unsaturated alkyl chain of between one and about twenty the receptor. carbon atoms. In a further embodiment, the Y region can 0115 The C-terminus of the Z region may be blocked or include a chain of hydrophilic residues, such as for instance, free. For example, the C terminus of the Z region may be the ethylene glycol residues. For instance, the Y region can free carboxyl group of the terminal amino acid, or alterna include at least about three, or at least about four, or at least tively, the C terminus of the Z region may be a blocked about five ethylene glycol residues. carboxyl group. Such as for instance, an amide group. 0109 The Z region of the molecule of any of formulas I to IV is a heparin-binding region and can include one or more The Heparin-Binding Growth Factors of Formulas V to VIII heparin-binding motifs, BBxB or BBBXxB as described by Verrecchio et al. J. Biol. Chem. 275:7701 (2000). Alterna 0116. The regions X and Wregions of the synthetic HBGF tively, the Z region can include both BBxB and BBBXXB analogs of formulas V to VIII are as defined above for each X motifs (where B represents lysine, arginine, or histidine, and region. All other assigned values and descriptions, including X represents a naturally occurring, or a non-naturally occur without limitation cross-linkers, Y, and Z, are as defined for ring amino acid). For example, the heparin-binding motifs the heparin-binding growth factors of formulas I to IV. may be represented by the sequence KRKRKRX(2) KR (SEQID NO:1), designating the first three amino acids DEFINITIONS as each independently selected from lysine or arginine, fol lowed by any two amino acids and a sixth amino acid which 0117. As used here and elsewhere, the following terms is lysine or arginine. have the meanings given. 0110. The number of heparin binding motifs is variable. 0118. The term “alkene' includes unsaturated hydrocar For instance, the Z region may include at least one, at least bons that contain one or more double carbon-carbon bonds. two, at least three or at least five heparin-binding motifs. Examples of such alkene groups include ethylene, propene, Where there are more than one heparin-binding motifs, the and the like. motifs may be the same or different. Alternatively, the Z 0119 The term “alkenyl' includes a linear monovalent region includes up to a maximum of about ten heparin-bind hydrocarbon radical of two to six carbonatoms or a branched ing motifs. In another alternative embodiment, the Z region monovalent hydrocarbon radical of three to six carbon atoms includes at least four, at least six or at least eight amino acid containing at least one double bond; examples thereof include residues. Further, in certain embodiments the Z region ethenyl, 2-propenyl, and the like. includes up to about twenty, up to about, twenty-five, or up to 0.120. The “alkyl groups specified herein include those about thirty amino acid residues. It is to be realized that, in alkyl radicals of the designated length in either a straight or part, the avidity of the Z region for heparin is determined by branched configuration. Examples of Such alkyl radicals the particular heparin-binding motifs selected and the number include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, ter of such motifs in Z. Thus for particular applications both the tiary butyl, pentyl, isopentyl, hexyl, isohexyl, and the like. selection and number of such motifs may be varied to provide I0121 The term “aryl' includes a monovalent or bicyclic optimal heparin binding of the Z region. aromatic hydrocarbon radical of 6 to 12 ring atoms, and 0111. In a preferred embodiment, the amino acid sequence optionally substituted independently with one or more sub of the Zregion is RKRKLERIAR (SEQID NO:2). In another stituents selected from alkyl, haloalkyl, cycloalkyl, alkoxy, embodiment, the amino acid sequence of the Z region is alkylthio, halo, nitro, acyl, cyano, amino, monosubstituted RKRKLGRIAR (SEQID NO:3). In yet another embodiment, amino, disubstituted amino, hydroxy, carboxy, or alkoxy the amino acid sequence of the Z region is RKRKLWRARA carbonyl. Examples of an aryl group include phenyl, biphe (SEQ ID NO:4). In yet another embodiment, the amino acid nyl, naphthyl, 1-naphthyl, and 2-maphthyl, derivatives sequence of the Z region is RKRKLERIARC (SEQ ID thereof, and the like. NO:5). The presence of a terminal cysteine residue optionally (0122) The term “aralkyl” includes a radical-R'R' where affords the opportunity to link other molecules, including R" is an alkylene (a bivalent alkyl) group and R is an aryl detection reagents such as fluorochromes, radioisotopes and group as defined above. Examples of aralkyl groups include other detectable markers, to the Z region, as well as the benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, opportunity to link toxins, immunogens and the like. and the like. The term “aliphatic' includes compounds with 0112 Heparin-binding domains that bear little or no hydrocarbon chains, such as for example alkanes, alkenes, sequence homology to known heparin-binding domains are alkynes, and derivatives thereof. also contemplated in the present invention. As used herein the I0123. The term “acyl includes a group RCO , where R term “heparin-binding means binding to the NHSO and is an organic group. An example is the acetyl group Sulfate modified polysaccharide, heparin, and also binding to CHCO-. the related modified polysaccharide, heparan. Such domains 0.124. A peptide oraliphatic moiety is “acylated when an are contemplated to exhibit binding in physiological solu alkyl or substituted alkyl group as defined above is bonded tions including 0.15 MNaCl, and are expected to uncomplex through one or more carbonyl —(C=O)—groups. A pep at salt concentrations greater than 0.5 M NaCl. tide is most usually acylated at the N-terminus. 0113. The Z region of the synthetic HBGF analogs of the 0.125. An "amide' includes compounds that have a triva present invention confers the property of binding to heparin in lent nitrogen attached to a carbonyl group (-CO.NH2). US 2009/011 1743 A1 Apr. 30, 2009

0126 An "amine' includes compounds that contain an acid sequence of the X region is YRSRKYTSWYVALKR amino group (-NH2). (SEQ ID NO:6) from FGF-2. In yet another particular 0127 A'diamine amino acid” is an amino acid or residue embodiment, the present invention provides a synthetic FGF containing two reactive amine groups and a reactive carboxyl analog wherein the amino acid sequence of the X region is group. Representative examples include 2.3 diamino propio NRFHSWDCIKTWASDTFVLVCYDDGSEA (SEQ ID nyl amino acid residue, 2.4 diaminobutylic amino acid resi NO:7). In yet another particular embodiment, the present due, lysine or ornithine. invention provides a synthetic FGF-2 analog wherein the 0128. A “trifunctional amino acid' is an amino acid or amino acid sequence of the X region is residue with three reactive groups, one the N-terminus amine, HIKLQLQAEERGVVS (SEQID NO:8). a second the C-terminus carboxyl, and the third comprising 0.137 Inayetfurther particular embodiment, the invention all or a part of the side chain. Trifunctional amino acids thus provides a synthetic FGF analog of FGF-1, wherein the X include, by way of example only, diamine amino acids; amino region is YISKKHAEKNWFVGLKK (SEQID NO:9). This acids with a reactive Sulfhydryl group in the side chain, Such sequence is derived from amino acids bridging the beta 9 and as mercaptoamino acids including cysteine, penicillamine, or beta 10 loop of FGF-1. In yet another particular embodiment, 3-mercapto phenylalanine; amino acids with a reactive car an FGF-1 analog is provided wherein the X region is boxyl group in the side chain, Such as aspartic acid and HIQLQLSAESVGEVY (SEQ ID NO:10), corresponding to glutamic acid; and amino acids with a reactive guanadium amino acids derived from the B-4 and B-5 region of FGF-1. group in the side chain, Such as arginine. 0.138 Inayetfurther particular embodiment, the invention provides a synthetic FGF analog of FGF-7, wherein the X FGFSynthetic Analogs region is YASAKWTHNGGEMFVALNOK (SEQ ID 0129. In another particular aspect, the invention provides a NO:11). In yet another embodiment of a synthetic FGF ana synthetic FGF peptide analog. The synthetic FGF analogs log of FGF-7, the X region is the amino acid sequence represented by any of formulas I to IV above, wherein X is an YNIMEIRTVAVGIVA (SEQID NO:12). FGF analog which can be any FGF, such as any of the known (0.139. Other FGF receptor binding domains, derived FGFs, including all 23 FGFs from FGF-1 to FGF-23. largely from targeting sequences in the C-terminus of human 0130. The X region of the molecule of formulas I to IV can FGF, include the following sequences shown in Table 1: include an amino acid sequences found in an FGF, such as for instance FGF-2 or FGF-7. Alternatively, the X regions can TABL E 1. include sequences not found in the natural ligand of the FGFR bound by the molecule. PREFERRED X RECEPTOR 0131 The Y region of the synthetic FGF peptide analogs CYTOKINE BINDING DOMAIN of any of formulas I to IV are not necessarily hydrophobic, FGF-10 YASFNWOHNGROMYVALNOK and thus, if present, can be polar, basic, acidic, hydrophilic or (SEQ ID NO:13) hydrophobic. Thus, the amino acid residues of the Y region of synthetic FGF peptide analogs can include any amino acid, or FGF-22 YASORWRRRGOPNLALDRR polar, ionic, hydrophobic or hydrophilic group. (SEQ ID NO:14) 0132) The X region of synthetic FGF peptide analogs can FGF-9 YSSNLYKHWDTGRRYYWALNK include an amino acid sequence that is 100% identical to an (SEQ ID NO:15) amino acid sequence found in a oran FGF-16 YASTLYKHSDSEROYVALNK amino acid sequence homologous to the amino acid sequence (SEQ ID NO:16) of a fibroblast growth factor. For instance, the X region can include an amino acid sequence that is at least about 50%, at FGF-2O YSSNIYKHGDTGRRFWALNK least about 75%, or at least about 90% homologous to an (SEO ID NO:17) amino acid sequence from a fibroblast growth factor. The fibroblast growth factor can be any fibroblast growth factor, FGF-4 YESYKYPGMFIALSKN including any of the known or yet to be identified fibroblast (SEQ ID NO:18) growth factors. FGF- 6 YESDLYOGTYILSKYGR 0133. In a particular embodiment, the synthetic FGF ana (SEQ ID NO:19) log of the invention is an agonist of the HBGFR. When bound to the HBGFR, the synthetic HBGF analog initiates a signal FGF-12 YSSTLYROOESGRAWFLGNK by the HBGFR. (SEQ ID NO: 2O) 0134. In a further particular embodiment, the synthetic FGF-14 YSSMLYROOESGRAWFLGLINK FGF analog of the invention is an antagonist of the HBGFR. (SEQ ID NO:21) When bound to the HBGFR, the synthetic HBGF analog FGF-13 YSSMIYROOOSGRGWYLGLINK blocks signaling by the HBGFR. (SEQ ID NO: 22) 0135) In another particular embodiment of the present invention, the synthetic FGF analog is an analog of FGF-2 FGF-11 YASALYRORRSGRAWYLDK (also known as basic FGF, or bFGF). In another particular (SEQ ID NO:23) embodiment of the present invention, the binding of the syn thetic FGF analog to an FGF receptor initiates a signal by the FGF receptor. In a further particularembodiment, the binding VEGF Synthetic Analogs of the synthetic FGF analog to the FGF receptor blocks sig naling by the FGF receptor. 0140. In another particular aspect, the invention provides a 0136. In a yet further particular embodiment, the present synthetic VEGF peptide analog. The synthetic VEGF analogs invention provides a synthetic FGF analog of FGF-2. In represented include, in one embodiment, a VEGF analog another particular embodiment, the present invention pro wherein the amino acid sequence of the X region is APMAE vides a synthetic FGF analog of FGF-2, wherein the amino GGGQNHHEVVKFMDV (SEQ ID NO:24). In another US 2009/011 1743 A1 Apr. 30, 2009 embodiment, there is provided a synthetic VEGF peptide analog wherein the amino acid sequence of the X region is TABLE 2 - continued GATWLPPNPTK (SEQ ID NO:25). In yet another embodi ment, there is provided a synthetic VEGF peptide analog PREFERRED X RECEPTOR wherein the amino acid sequence of the X region is CYTOKINE BINDING DOMAIN NFLLSWVHWSLALLLYLHHA (SEQID NO:26). BMP-11 NMLYFNDKOOIIYGKIPGMVV BMP Synthetic Analogs (SEO ID NO: 47) BMP-12 SILYIDAANNVVYKOYEDMVV 0141. In another particular aspect, the invention provides a (SEQ ID NO: 48) synthetic BMP peptide analog. The synthetic bone morpho genic protein analogs include embodiments wherein the X BMP-13 SILYIDAGNNVVYKOYEDMVV region includes the amino acid sequence LYVDFSDVG (SEQ ID NO: 49) WNDW (SEQID NO:27), AISMLYLDENEKVVL(SEQID BMP-14 SILFIDSANNVVYKOYEDMVV NO:28), ISMLYLDENEKVVLKNY (SEQ ID NO:29), (SEO ID NO : 5O) EKVVLKNYQDMVVEG (SEQID NO:30), LVVKENED BMP-15 SWLMIEANGSILYKEYEGMIA LYLMSIAC (SEQ ID NO:31), AFYCHGECPFPLADHL (SEQ ID NO:51) (SEQ ID NO:32), or PFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:33). GDF- SWLFFDNSDNVVLROYEDMVV 0142. Alternatively, in another particular aspect the inven (SEQ ID NO: 52) tion provides synthetic BMP. TGF or GDF (growth differen GDF-3 SMILYODNNDNVILRHYEDMVV tiation factor) peptide analogs as shown in Table 2 wherein (SEO ID NO : 53) the transforming growth factor family member peptides are particularly useful in augmenting the activity of endogenous GDF-8 NMYLFNGKEOIIYGKIPAMVV or artificial BMP peptides or TGF peptides, wherein is shown (SEQ ID NO:54) (under the heading “preferred receptor binding domain”) the GDF-9 LSWLTIEPDGSIAYKEYEDMIA sequence forming all or part of the X region of constructs of (SEO ID NO : 55) any of formulas I to IV.

TABLE 2 Methods of Synthesizing the Heparin-Binding Growth Factor Analogs PREFERRED X RECEPTOR CYTOKINE BINDING DOMAIN 0143. The synthesis of the analogs of the invention can be achieved by any of a variety of chemical methods well known TGF-B1 IWYYVGRKPKVEOLSNMIVRS in the art. Such methods include bench scale solid phase (SEQ ID NO:34) synthesis and automated peptide synthesis in any one of the TGF-B2 TILYYIGKTPKIEOLSNMIVKS many commercially available peptide synthesizers. Prefer (SEO ID NO:35) ably, the synthesizer has a per cycle coupling efficiency of greater than 99 percent. TGF-B3 LTILYYVGRTPKVEOLSNMVV 0144. The analogs of the present invention can be pro (SEQ ID NO:36) duced by stepwise synthesis or by synthesis of a series of BMP-2 AISMLYLDENEKVVLKNYODMVV fragments that can be coupled by similar well known tech (SEO ID NO:37) niques. See, for instance, Nyfeler, Peptide synthesis via frag ment condensation. Methods Mol. Biol. 35:303-16 (1994): BMP-3 SSLSILFFDENKNWWLKWYPNMTW and Merrifield, Concept and early development of solid (SEQ ID NO:38) phase peptide synthesis. Methods in Enzymol. 289:3-13 (1997). These methods are routinely used for the preparation BMP-3B NSLGWLFLDENRNWWLKWYPNMSW of individual peptides. It is possible to assemble the analogs (SEO ID NO:39) of the present invention in component parts, such as peptides BMP-4. AISMLYLDEYDKWVLKNYOEMVV constituting the X,Y and Z components thereof, and to there (SEQ ID NO: 4 O) after couple Such component parts to assemble the analog. See, for instance, Dawson and Kent, Synthesis of native pro BMP-5 AISWLYFDDSSNWILKKYRNMWW teins by chemical ligation. Annu. Rev. Biochem. 69:923-960 (SEQ ID NO: 41) (2000); and Eom et al., Tandem ligation of multipartite pep BMP-6 AISWLYFDDNSNWILKKYRNMWW tides with cell-permeable activity. J. Am. Chem. Soc. 125:73 (SEQ ID NO: 42) 82 2003). 0145 Peptide libraries that can be used to screen for a BMP-7 AISWLYFDDSSNWILKKYRNMWW desired property, such as binding to an HBGFR, can be pre ( : 43) pared by adaptations of these methods. See for instance, Fox, BMP-8 ATSWLYYDSSNNWILRKARNMWW Multiple peptide synthesis, Mol. Biotechnol. 3:249-58 (SEQ ID NO:44) (1995); and Wade and Tregear, Solid phase peptide synthesis: recent advances and applications. Austral. Biotechnol. BMP-9 ISWLYKDDMGWPTLKYHYEGMSW 3:332-6 (1993). (SEQ ID NO: 45) 0146 In a particular embodiment, the synthetic HBGF BMP-10 ISILYLDKGWWTYKFKYEGMAW analog of the invention is an agonist of the HBGFR. When (SEQ ID NO:46) bound to the HBGFR, the synthetic HBGF analog initiates a signal by the HBGFR. US 2009/011 1743 A1 Apr. 30, 2009

0147 In another particular embodiment, the synthetic lular retention on blood-contacting Surfaces of medical HBGF analog of the invention is an antagonist of the HBGFR. devices. In one embodiment, a homodimeric HBGF analog of When bound to the HBGFR, the synthetic HBGF analog VEGF of the present invention is applied on vascular graft blocks signaling by the HBGFR. materials such that the bound analog recruits and binds cir 0148. In a particular aspect, the invention provides a culating endothelial stem cells from the blood, thereby result method for stimulating growth factor receptor signaling in a ing in endothelialization of the graft Surface with resultant cell by contacting the cell with an effective amount of a long-term thromboresistance being imparted to the graft. synthetic HBGF analog according to formulas I to IV. The 0.155. In another aspect, the invention provides a method effective amount can be readily determined by one of skill in and compositions to increase and provide for membrane the art. The signaling can result in cytokine release from the guided tissue growth. cell, stimulation or inhibition of proliferation or differentia 0156. In another aspect, the invention provides a method tion of the cell, chemotaxis of the cell, stimulation or inhibi and composition for treatment of difficult-to-treat dermal tion of the immune system of the mammal. wounds, including ulcers. In one embodiment, a homodimeric HBGF analog of TGF-B1 is applied topically in Methods of Use of the HBGFs of the Invention a pharmaceutically acceptable cream or gel for treatment of 014.9 The HBGF analogs of the invention provide a cost ulcerated bed sores and similar difficult-to-treat dermal effective and potentially unlimited source of biologically wounds. active molecules that are useful in a number of ways, includ 0157. In yet another aspect, the invention provides a ing as soluble prophylactic or therapeutic pharmaceutical method and compositions to selectively increase cellular agents, such as for instance for administration as a soluble populations in vitro. For example, a homodimeric HBGF drug for prevention or treatment of various diseases, includ analog of TGF-B1 is formulated in a tissue culture medium to ing for example, uses in cancer therapy and radioprotection. specifically stimulate the growth of chondrocytes, stem cells 0150. The synthetic HBGF analogs of present invention which give rise to chondrocytes, or pluripotent cells which are also useful as biologically active agents for coating of give rise of chondrocytes. Similarly, a homodimeric HBGF medical devices, such as for instance, Sutures, implants and analog of VEGF may be employed to stimulate the growth of medical instruments to promote biological responses, for endothelial cells. instance, to stimulate growth and proliferation of cells, or 0158. The term “medical device' as used herein means a healing of wounds. device that has one or more Surfaces in contact with an organ, 0151. In one aspect, the present invention provides a tissue, blood or other bodily fluid in an organism, preferably method and compositions for treating a mammal that has a mammal, particularly, a human. Medical devices include, been exposed to a harmful dose of radiation. The method for example, extracorporeal devices for use in Surgery such as includes administering an effective dose of a synthetic HBGF blood oxygenators, blood pumps, blood sensors, tubing used analog of the invention which is an FGF analog to the mam to carry blood, and the like which contact blood that is mal. The treatment is particularly useful in the prevention or returned to the patient. The term can also include endopros treatment of mucositis, gastrointestinal syndrome (G.I. Syn theses implanted in blood contact in a human or animal body, drome), or radionecrosis such as can result from exposure to Such as vascular grafts, stents, pacemaker leads, heart valves, radiation. The HBGF analog can be administered parenter and the like that are implanted in blood vessels or in the heart. ally, orally, or topically. Alternatively, the HBGF analog can The term can further include devices for temporary intravas be delivered loco-regionally, e.g. on an analog coated medical cular use Such as catheters, guide wires, and the like that are device. In a related embodiment, the present invention pro placed in blood vessels or the heart for purposes of monitor vides a method for treating a mammal that has been admin ing or repair. The term can further include nerve electrodes, istered a dose of a chemotherapeutic agent, to ameliorate the muscle electrodes, implantable pulse generators, implantable toxicity of the chemotherapeutic agent to the mammal. In a drug pumps, and defibrillators. Moreover, the term medical particular embodiment of the above-described methods, the device can include Sutures, graft materials, wound coverings, mammal is a human. In another particular embodiment of the nerve guides, bone wax, aneurysm coils, embolization par method, the HBGF analog is an FGF-2 analog or an FGF-7 ticles, microbeads, dental implants, bone prostheses, tissue analog. scaffolds, artificial joints or a controlled release drug delivery 0152. In another aspect, the invention provides a method devices. and compositions for treating a mammal with bone injury, by 0159. The surface of the medical device can be formed providing a HBGF analog of the present invention having an from any of the commonly used materials suitable for use in X region reactive with a BMPHBGFR, such as an analog of medical devices, such as for instance, stainless Steel, titanium, BMP-2. For example, such HBGF analogs of the present platinum, tungsten, ceramics, polyurethane, polytetrafluoro invention may be administered as a pharmaceutical agent, or ethylene, extended polytetrafluoroethylene, polycarbonate, may be employed as an additive to bone matrix or bone graft polyester, polypropylene, polyethylene, polystyrene, polyvi materials. nyl chloride, polyamide, polyacrylate, polyurethane, polyvi 0153. In another aspect, the invention provides a method nyl alcohol, polycaprolactone, polylactide, polyglycolide, and compositions for preparation of cell or organ implant polysiloxanes (such as 2,4,6,8-tetramethylcyclotetrasilox sites. In one embodiment, a homodimeric HBGF analog of ane), natural rubbers, or artificial rubbers, or block polymers FGF-2 of the present invention is administered by a percuta or copolymers thereof. neous route to stimulate localized prior to 0160 Methods for coating biological molecules onto the implant of insulin-secreting pancreatic cells, and thereby surfaces of medical devices are known. See for instance U.S. improve the survival of the implanted cells. Similarly, a Pat. No. 5,866,113 to Hendriks et al., the specification of homodimeric HBGF analog of FGF-2 of the present inven which is hereby incorporated by reference. Tsang et al. in tion is administered into ischemic heart tissue prior to the U.S. Pat. No. 5,955,588 teach a non-thrombogenic coating implant of myocte stem cells. composition and methods for using the same on medical 0154) In another aspect, the invention provides a method devices, and is incorporated herein by reference. Zamora et and compositions to increase cellular attachment to and cel al. in U.S. Pat. No. 6,342,591 teach an amphipathic coating US 2009/011 1743 A1 Apr. 30, 2009 for medical devices for modulating cellular adhesion compo p-toluenesulfonic acid, trifluoroacetic acid, and the like. Acid sition, and is incorporated herein by reference. addition salts of the HBGF analogs of this invention are 0161 In one embodiment, the invention provides a prepared in a suitable solvent for the HBGF analog and an method for delivering an active peptide to a mammal, the excess of an acid, Such as hydrochloric, hydrobromic, Sulfu method includes (i) providing a medical device coated on its ric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic, surface with a synthetic HBGF analog of formulas I to IV, the Succinic or methanesulfonic acid. The acetate salt form is synthetic HBGF analog being bound to the surface of the especially useful. Where the HBGF analogs of this invention medical device by non-covalent bonds; and (ii) placing the include an acidic moiety, Suitable pharmaceutically accept medical device onto a surface of, or implanting the medical able salts may include alkali metal salts, such as Sodium or device into, the mammal. potassium salts, or alkaline earth metal salts, such as calcium 0162. In a particular embodiment of the above method, the or magnesium salts. non-covalent bonds are associations between the heparin 0167. The invention provides a pharmaceutical composi binding domain of the synthetic HBGF analog and a heparin tion that includes a HBGF analog of this invention and a containing compound bound to the Surface of the medical pharmaceutically acceptable carrier. The carrier may be a device. The heparin-containing compound bound to the Sur liquid formulation, and in one embodiment a buffered, iso face of the medical device can be any heparin-containing tonic, aqueous solution. Pharmaceutically acceptable carriers compound, such as for instance, benzyl-bis(dimethylsilylm also include excipients, such as diluents, carriers and the like, ethyl)oxy carbamoyl-heparin. and additives, such as Stabilizing agents, preservatives, solu 0163. In another particular embodiment of the above bilizing agents, buffers and the like, as hereafter described. method, the medical device is not pre-coated with a heparin 0168 Thus the HBGF analog compositions of this inven containing compound before being coated with the synthetic tion may be formulated or compounded into pharmaceutical HBGF analog of formulas I to IV. compositions that include at least one HBGF analog of this invention together with one or more pharmaceutically accept Heparin-Binding Growth Factor Analog Pharmaceutical able carriers, including excipients, such as diluents, carriers Applications and the like, and additives, such as stabilizing agents, preser 0164. The HBGF analogs of this invention can be used for Vatives, solubilizing agents, buffers and the like, as may be as an active ingredient in pharmaceutical compositions for desired. Formulation excipients may include polyvinylpyr both medical applications and animal husbandry or veteri rolidone, gelatin, hydroxy cellulose, acacia, PEG. PEO, man nary applications. Typically, the HBGF analog orpharmaceu nitol, Sodium chloride or sodium citrate, as well as any num tical composition is used in humans, but may also be used in ber of simple Sugars, including sucrose, dextrose, lactose and other mammals. The term “patient' is intended to denote a the like, and combinations of the foregoing. For injection or mammalian individual, and is so used throughout the speci other liquid administration formulations, water containing at fication and in the claims. The primary applications of this least one or more buffering constituents is preferred, and invention involve human patients, but this invention may be stabilizing agents, preservatives and solubilizing agents may applied to laboratory, farm, Zoo, wildlife, pet, sport or other also be employed. For Solid administration formulations, any animals. of a variety of thickening, filler, bulking and carrier additives 0.165. The HBGF analogs of this invention may be in the may be employed, such as starches, Sugars, fatty acids and the form of any pharmaceutically acceptable salt. The term like. For topical administration formulations, any of a variety “pharmaceutically acceptable salts' refers to salts prepared of creams, ointments, gels, lotions and the like may be from pharmaceutically acceptable non-toxic bases or acids employed. For most pharmaceutical formulations, non-active including inorganic or organic bases and inorganic or organic ingredients will constitute the greater part, by weight or Vol acids. Salts derived from inorganic bases include aluminum, ume, of the preparation. For pharmaceutical formulations, it ammonium, calcium, copper, ferric, ferrous, lithium, magne is also contemplated that any of a variety of measured-release, sium, manganic salts, manganous, potassium, Sodium, Zinc, slow-release or time-release formulations and additives may and the like. Particularly preferred are the ammonium, cal be employed, so that the dosage may be formulated so as to cium, lithium, magnesium, potassium, and Sodium salts. Salts effect delivery of a HBGF analog of this invention over a derived from pharmaceutically acceptable organic non-toxic period of time. bases include salts of primary, secondary, and tertiary amines, 0169. In practical use, the HBGF analogs of the invention Substituted amines including naturally occurring Substituted can be combined as the active ingredient in an admixture with amines, cyclic amines, and basic ion exchange resins, such as a pharmaceutical carrier according to conventional pharma arginine, betaine, caffeine, choline, N,N'-dibenzylethylene ceutical compounding techniques. The carrier may take a diamine, diethylamine, 2-diethylaminoethanol, 2-dimethy wide variety of forms depending on the form of preparation laminoethanol, ethanolamine, ethylenediamine, N-ethyl desired for administration, for example, oral, parenteral (in morpholine, N-ethylpiperidine, glucamine, glucosamine, cluding intravenous), urethral, vaginal, nasal, buccal, Sublin histidine, hydrabamine, isopropylamine, lysine, methylglu gual, or the like. In preparing the compositions for oral dosage camine, morpholine, piperazine, piperidine, polyamine res form, any of the usual pharmaceutical media may be ins, procaine, purines, theobromine, triethylamine, trimethy employed, such as, for example, water, glycols, oils, alcohols, lamine, tripropylamine, tromethamine, and the like. flavoring agents, preservatives, coloring agents and the like in 0166 When the HBGF analog of the present invention is the case of oral liquid preparations, such as, for example, basic, acid addition salts may be prepared from pharmaceu Suspensions, elixirs and solutions; or carriers such as tically acceptable non-toxic acids, including inorganic and starches, Sugars, microcrystalline cellulose, diluents, granu organic acids. Such acids include acetic, benzenesulfonic, lating agents, lubricants, binders, disintegrating agents and benzoic, camphorsulfonic, carboxylic, citric, ethanesulfonic, the like in the case of oral Solid preparations such as, for formic, fumaric, gluconic, glutamic, hydrobromic, hydro example, powders, hard and Soft capsules and tablets. chloric, isethionic, lactic, maleic, malic, mandelic, methane 0170 The pharmaceutical forms suitable for injectable Sulfonic, malonic, mucic, nitric, pamoic, pantothenic, phos use include sterile aqueous Solutions or dispersions and ster phoric, propionic, Succinic, Sulfuric, tartaric, ile powders for the extemporaneous preparation of sterile US 2009/011 1743 A1 Apr. 30, 2009 injectable solutions or dispersions. In all cases, the form must The human FGF-5 oncogene encodes a novel protein related be sterile and must be fluid to the extent that it may be to fibroblast growth factors. Mol. Cell. Biol. 8:3487-3495 administered by syringe. The form must be stable under the (1988)). conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as 0177 FGF-6 is also known as HBGF-6, and sometimes bacteria and fungi. The carrier can be a solvent or dispersion called hist-2 or oncogene hst-1 related growth factor, see Iida medium containing, for example, water, ethanol, a polyol, for et al. Human hst-2 (FGF-6) oncogene. cINA cloning and example glycerol, propylene glycol or liquid polyethylene characterization. Oncogene 7:303-9 (1992); and Marics et al. glycol, Suitable mixtures thereof, and vegetable oils. Characterization of the HST-related FGF-6 , a new mem 0171 If the HBGF analog pharmaceutical composition is ber of the fibroblast growth factor gene family. Oncogene administered by injection, the injection may be intravenous, 4:335-40 (1989). Subcutaneous, intramuscular, intraperitoneal or other means 0.178 FGF-7 or K-FGF is also known as KGF or kerati known in the art. The HBGF analogs of this invention may nocyte growth factor (See Aaronson et al. Keratinocyte alternatively be formulated by any means known in the art, growth factor. A fibroblast growth factor family member with including but not limited to formulation as tablets, capsules, unusual target cell specificity. Annals NY Acad. Sci. 638:62 caplets, Suspensions, powders, lyophilized preparations, Sup 77 (1991); Finch et al. Human KGF is FGF-related with positories, ocular drops, skin patches, oral soluble formula properties of a paracrine effector of epithelial cell growth. tions, sprays, aerosols and the like, and may be mixed and Science 245:752-5 (1989); Marchese et al. Human kerati formulated with buffers, binders, excipients, stabilizers, anti nocyte growth factor activity on proliferation and differentia oxidants and other agents known in the art. In general, any tion of human keratinocytes: differentiation response distin route of administration by which the HBGF analogs of inven guishes KGF from EGF family. J. Cellular Physiol. 144: tion are introduced across an epidermal layer of cells may be 326-32 (1990)). employed. Administration means may thus include adminis 0179 FGF-8 was found to be identical to androgen-in tration through mucous membranes, buccal administration, duced growth factor, AIGF and has been well studied (See oral administration, dermal administration, inhalation admin Blunt et al. Overlapping expression and redundant activation istration, nasal administration, urethral administration, vagi of mesenchymal fibroblast growth factor (FGF) receptors by nal administration, and the like. alternatively spliced FGF-8 ligands. J. Biol. Chem. 0172. In general, the actual quantity of HBGF analog of 272:3733-8 (1997); Dubrulle et al. FGF signaling controls this invention administered to a patient will vary between Somite boundary position and regulates segmentation clock fairly wide ranges depending upon the mode of administra control of spatiotemporal Hox gene activation. Cell 106:219 tion, the formulation used, and the response desired. The 232 (2001); Gemel et al. Structure and sequence of human dosage for treatment is administration, by any of the forego FGF8. Genomics 35:253-257 (1996); Tanaka et al. A novel ing means or any other means known in the art, of an amount isoform of human fibroblast growth factor 8 is induced by sufficient to bring about the desired therapeutic effect. androgens and associated with progression of esophageal carcinoma. Dig. Dis. Sci. 46:1016-21 (2001)). Heparin-Binding Growth Factors 0180 FGF-9 was originally called glia activating factor, or HBGF-9. See Miyamoto et al. Molecular cloning of a novel 0173 The fibroblast growth factors, FGFs, constitute a cytokine cDNA encoding the ninth member of the fibroblast family of related proteins controlling normal growth and dif growth factor family, which has a unique secretion pattern. ferentiation of mesenchymal, epithelial, and neuroectoder mal cell types. Homologs have been foundina wide variety of Mol. Cell. Biol. 13:4251-9 (1993); and Naruo et al. Novel species. FGFs show a very high affinity to heparin and are secretory heparin-binding factors from human glioma cells therefore also referred to as heparin-binding growth factors (glia-activating factors) involved in glial cell growth. J. Biol. (HBGFs). As used herein, the term HBGFs includes all FGFs. Chem. 268: 2857-64 (1993). 0.174. Two main types of FGF are known. The first type of 0181 FGF-10 is also called KGF-2. keratinocyte growth FGF was isolated initially from brain tissue. It was identified factor-2 (see Kok et al. Cloning and characterization of a by its proliferation-enhancing activities for murine fibro cDNA encoding a novel fibroblast growth factor preferen blasts, such as 3T3 cells. Due to its basic p the factor was tially expressed in human heart. Biochem. Biophys. Res. named basic FGF (bFGF, or HBGF-2, heparin-binding Comm. 255:717-721, (1999)). growth factor-2) and is now generally referred to as FGF-2. 0182. Several FGF-related factors have been described as This is the prototype of the FGF family. fibroblast growth factor homologous factors (FHFs) and are (0175. Another type of FGF, also initially isolated from also referred to as FGF-11 (FHF-3), FGF-12 (FHF-1), FGF brain tissues, is acidic FGF (aFGF, also known as HBGF-1, 13 (FHF-2, see Greene et al. Identification and characteriza heparin-binding growth factor-1 or HBGF-C. heparin-bind tion of a novel member of the fibroblast growth factor family. ing growth factor-O.), now generally referred to as FGF-1. It Eur. J. Neurosci. 10:1911-1925 (1998)), and FGF-14 (FHF was identified by its proliferation-enhancing activity for 4). myoblasts. 0183 FGF-15 is expressed in the developing nervous sys 0176) Other fibroblast growth factors belonging to the tem and was identified as a gene regulated by transcription same family include FGF-3 (or HBGF-3, heparin-binding factor E2A-PbX1. McWhirter et al. A novel fibroblast growth growth factor-3, originally called int-2; see Fekete, Trends in factor gene expressed in the developing nervous system is a Neurosci. 23:332 (2000)), FGF-4 (HBGF-4, heparin-binding downstream target of the chimeric homeodomain oncopro growth factor-4, initially recognized as the product of the tein E2A-PbX1. Development 124:3221-3232 (1997). oncogene hst; see Sakamoto et al., Proc. Natl. Acad. Sci. USA 0.184 FGF-16 was isolated as a cDNA clone from ratheart 91:12368-72), and FGF-5 (originally called HBGF-5, see by homology-based polymerase chain reaction expressing an Bates et al. Biosynthesis of human fibroblast growth factor 5. FGF of 207 amino acids. FGF-16 is 73% identical to FGF-9. Mol. Cell. Biol. 11:1840-1845 (1991); Burgess and Maciag, Miyake et al. Structure and expression of a novel member, The heparin-binding (fibroblast) growth factor family of pro FGF-16, of the fibroblast growth factor family. Biochem. teins. Ann. Rev. Biochem.58:575-606 (1989); and Zhanet al. Biophys. Res. Commun. 243:148-152 (1998). US 2009/011 1743 A1 Apr. 30, 2009

0185. The cDNA encoding FGF-17 was isolated from rat (0191 FGF-23 is most similar to FGF-21 and FGF-19. The embryos and encodes a protein of 216 amino acids. When human FGF-23 gene maps to 12p13 linked to expressed in 3T3 fibroblasts, mouse FGF-17 is transforming. human FGF-6 gene. FGF-23 mRNA is expressed mainly in During embryogenesis, FGF-17 is expressed at specific sites the brain (preferentially in the ventrolateral thalamic nucleus) in forebrain, the midbrain-hindbrain junction, the developing and thymus at low levels. Missense mutations in the FGF-23 skeleton and in developing arteries. See Hoshikawa et al. gene have been found in patients with autosomal dominant Structure and expression of a novel fibroblast growth factor, hypophosphataemic rickets. Overproduction of FGF23 FGF-17, preferentially expressed in the embryonic brain. causes tumor-induced osteomalacia, a paraneoplastic disease Biochem. Biophys. Res. Commun. 244:187-191 (1998); and Xuetal. Genomic structure, mapping, activity and expression characterized by hypophosphatemia caused by renal phos of fibroblast growth factor 17. Mechanisms of Development phate wasting. See Yamashita et al. Identification of a novel 83:165-178 (1999). fibroblast growth factor, FGF-23, preferentially expressed in 0186. The cDNA encoding FGF-18 was isolated from rat the ventrolateral thalamic nucleus of the brain. Biochem. embryos encoding a protein of 207 amino acids. FGF-18 is a Biophys. Res. Commun. 277:494-8 (2000); and Shimada et glycosylated protein and is most similar to FGF-8 and FGF al. Cloning and characterization of FGF23 as a causative 17. Injection of recombinant murine FGF-18 has been shown factor of tumor-induced osteomalacia. Proc. Natl. Acad. Sci. to induce proliferation in tissues of both epithelial and mes (USA) 98:6500-5 (2001). 0.192 HBBM (Heparin-binding brain mitogen) was iso enchymal origin, particularly in liver and Small intestine. lated initially as a heparin binding protein from brain tissues Recombinant rat FGF-18 induces neurite outgrowth in PC12 of several species and is identical to heparin-binding neurite cells. Recombinant murine FGF-18 protein stimulates prolif promoting factor. See Huber et al. Amino-terminal sequences eration in NIH 3T3 fibroblasts in vitro in a heparan sulfate of a novel heparin-binding protein with mitogenic activity for dependent manner. For general information see Hu et al. endothelial cells from human bovine, rat, and chick brain: FGF-18, a novel member of the fibroblast growth factor fam high interspecies homology. Neurochem. Res. 15:435-439 ily, stimulates hepatic and intestinal proliferation. Mol. Cell. (1990). Biol. 18:6063-6074 (1998); and Ohbayashi et al. Structure 0193 HB-GAF (heparin-binding growth associated fac and expression of the mRNA encoding a novel fibroblast tor) is a neurotrophic and mitogenic factor identical to HBNF growth factor, FGF-18. J. Biol. Chem. 273:18161-18164 (heparin-binding neurite-promoting factor). See Kuo et al. (1998). Characterization of heparin-binding growth-associated factor 0187 FGF-19 is related distantly to other members of the receptor in NIH 3T3 cells. Biochem. Biophys. Res. Commun. FGF family. FGF-19 mRNA is expressed in several tissues 182:188-194 (1992). including fetal cartilage, skin, and retina, as well as adult gall (0194 HB-EGF (heparin-binding EGF-like factor) is bladder. It is overexpressed in a colon adenocarcinoma cell found in conditioned media of cell line U937 and is also line. FGF-19 is a high affinity, heparin-dependent ligand for synthesized by macrophages and human vascular Smooth the FGF-4 receptor. See Xie et al. FGF-19, a novel fibroblast muscle cells. HB-EGF is a monomeric heparin-binding growth factor with unique specificity for FGFR4 Cytokine O-glycosylated protein of 86 amino acids and is processed 11:729-735 (1999). from a precursor of 208 amino acids. Several truncated forms 0188 FGF-20 is expressed in normal brain, particularly of HB-EGF have been described. HB-EGF is a potent mito the cerebellum, and in some cancer cell lines. FGF-20 mRNA gen for NIH 3T3 cells, keratinocytes and smooth muscle is expressed preferentially in the Substantia nigra pars com cells, but not for endothelial cells. The mitogenic activity on smooth muscle cells is much stronger than for EGF and pacta. Recombinant FGF-20 protein induces DNA synthesis appears to involve interactions with cell Surface heparan Sul in a variety of cell types and is recognized by multiple FGF fate proteoglycans. HB-EGF is a major growth factor com receptors. FGF-20 functions like an oncogene, causing a ponent of wound fluid and may play an important role in transformed phenotype when expressed in the 3T3 fibroblast wound healing. See Abraham et al. Heparin-binding EGF cell line. These transformed cells are tumorigenic in nude like growth factor: characterization of rat and mouse cDNA mice. See Jeffers et al. Identification of a novel human fibro clones, protein domain conservation across species, and tran blast growth factor and characterization of its role in onco Script expression in tissues. Biochem. BiophyS. Res. Com genesis. Cancer Res. 61:3131-8 (2001); and Ohmachi et al. mun. 190:125-133 (1993); Higashiyama et al. A heparin FGF-20, a novel neurotrophic factor, preferentially expressed binding growth factor secreted by macrophage like cells that in the Substantia nigra pars compacta of rat brain. Biochem. is related to EGF. Science 251:936-9 (1991); and Marikovsky Biophys. Res. Commun. 277:355-60 (2000). et al. Appearance of heparin-binding EGF-like growth factor (0189 FGF-21 was isolated from mouse embryos. FGF in wound fluid as a response to injury. Proc. Natl. Acad. Sci. 21 mRNA is most abundant in the liver with lower levels in the (USA) 90:3889-93. thymus. FGF-21 is most similar to human FGF-19. See Nish 0.195 HB-GAM (heparin-binding growth associated mol imura et al. Identification of a novel FGF, FGF-21, preferen ecule) also referred to as HBNF (heparin-binding neurite tially expressed in the liver. Biochim. Biophys. Acta 1492: promoting factor) is a protein of 15.3 kDa isolated as a hep 203-6 (2000). arin binding protein from brain tissues of several species. (0190. The cDNA encoding FGF-22 (170 amino acids) was HB-GAM promotes growth of SW-13 cells in soft agar. isolated from human . FGF-22 is most similar to Courty et al. Mitogenic properties of a new endothelial cell FGF-10 and FGF-7. Murine FGF-22 mRNA is expressed growth factor related to pleiotrophin. Biochem. Biophys. preferentially in the skin. FGF-22 mRNA in the skin is found Res. Commun. 180: 145-151 (1991); and Hampton et al. preferentially in the inner root sheath of the hair follicle. See Structural and functional characterization of full-length hep Nakatake et al. Identification of a novel fibroblast growth arin-binding growth associated molecule. Mol. Biol. Cell. factor, FGF-22, preferentially expressed in the inner root 3:85-93 (1992). sheath of the hair follicle. Biochim. Biophys. Acta 1517: (0196) TGF-beta (TGF-?3) exists in at least five isoforms, 460-3 (2001). known TGF-B1, TGF-B2, TGF-B3, TGF-B4 and TGF-?35, that US 2009/011 1743 A1 Apr. 30, 2009 are not related to TGF-C. Their amino acid sequences display domain (Busch et al. Trans-Repressor Activity of Nuclear homologies on the order of 70-80 percent. TGF-31 is the Glycosaminoglycans on Fos and Jun/AP-1 Oncoprotein-me prevalent form and is found almost ubiquitously while the diated Transcription. J. Cell Biol. 116:31-42, 1992). other isoforms are expressed in a more limited spectrum of (0199. In the second peptide precursor LYVDFSDVG cells and tissues. WNDWCLYVDFSDVGWNDW-amide (SEQ ID NO:57), 0197) TGF-beta is the prototype of a family of proteins the sequence LYVDFSDVGWNDW (SEQ ID NO:27) is known as the TGF-beta superfamily. This family includes derived from amino acids 19-31 in the human BMP-2 inhibins, ActivinA, MIS (Mullerian activating substance) and sequence and the cysteine residue served to link the precursor BMPs (Bone morphogenic proteins). Burt, Evolutionary to 1,4-bis-maleimidobutane. grouping of the transforming growth factor-beta Superfamily. 0200 Synthetic Scheme 1 shows the general approach for Biochem. Biophys. Res. Commun. 184:590-5 (1992). synthesis. The first precursor CXXXRKRLDRIAR-amide (SEQID NO:56) is reacted in a slightly acidic conditions with EXAMPLES a large molar excess of A, 1.4-bis-maleimidobutane, a homo Example 1 bifunctional, sulfhydryl reactive agent with a four carbon spacer, in a 50% acetonitrile/water adjusted to 5.8 pH by use 0198 A synthetic branched peptide construct of the gen of 0.1M sodium phosphate dibasic buffer. In the depiction of eral formula: NH-LYVDFSDVGWNDWC(bMB the resulting reaction product B, “S” depicts the sulfur atom CXXXXRKRLDRIAR-amide)-LYVDFSDVGWNDW “S” of the side chain of the cysteine residue, shown as “C”. amide, where bMB is 1,4-bis-maleimidobutane and each X is The reaction product B is isolated by preparative HPLC and 6-aminohexanoic acid, is synthesized using two peptide pre the isolated reaction product B is then reacted with LYVDFS CUSOS. The first peptide precursor is DVGWNDWCLYVDFSDVGWNDW-amide (SEQ ID CXXXRKRLDRIAR-amide (SEQ ID NO:56), again NO:57), again in a 50% acetonitrile/water adjusted to 8.0 pH where each X is 6-aminohexanoic acid, while the second by use of 0.1 M sodium phosphate dibasic. Here too in the peptideprecursor is LYVDFSDVGWNDWCLYVDFSDVG depiction of the resulting final product C, “S” depicts the WNDW-amide (SEQID NO:57). Both peptideprecursors are sulfur atom “S” of the side chain of the cysteine residue, synthesized by conventional Solid phase methods and purified shown as “C”, forming a part of SEQ ID NO:57. The final by reverse-phase HPLC. In the first peptide precursor, product C is purified by HPLC, using a 250x10.0 mm Cs CXXXRKRLDRIAR-amide (SEQ ID NO:56), the cys column at a flow rate of 3 mL/min in a solvent gradient of 0 to teine residue serves to link the precursor to 1,4-bis-maleimi 60% of 0.1% trifluoroacetate in acetonitrile where the initial dobutane, the 6-aminohexanoic acid XXX tripeptide solvent is 0.1% trifluoroacetate and water. The resulting final serves as both a spacer and provided a hydrophobic region, product C is a branched peptide construct wherein the two and the sequence RKRLDRIAR (SEQID NO:58) provides a peptide subunits are conjugated via thioether linkages. Mass heparin-binding motif derived from a modification of the spectroscopy is used to confirm the sequence of the final sequence at residues 270-279 of the Jun/AP-1 DNA binding COnStruct.

SYNTHETIC SCHEME 1. NH-CXXXRKRLDRIAR-amide O O N 1n 1a-N O O A

S-CXXXRKRLDRIAR-amide

Nivorsovownwelvorsovows was US 2009/011 1743 A1 Apr. 30, 2009 17

-continued Z. it a.

A.

s5 O Z. O l 2 o-S2 1N1N1 N t S-CXXXRKRLDRIAR-amide a. 2 O O a.

l s E. C

0201 It may thus be seen that the structure of the molecule medium is then replaced with a low serum medium contain may alternatively be depicted as shown below, where amino ing between 0 and 10 ug/mL of the synthetic branched peptide acid residues are shown by bolded single letter designations, construct of Example 1. After 3 days in culture, the relative letter designations that are not bolded refer to atoms, and X cell number is monitored using MTS following the directions is in each instance 6-aminohexanoic acid: of the manufacturer. Statistical significance is determined using ANOVA followed by post-hoc multiple comparisons Z. versus control group (Dunnett's Method). 0203 MC3T3E1 osteoblastic cells, C3H10T1/2 mouse a. pluripotent stem cells, C2C12 murine myoblasts, and cells l from a human fetal osteoblast cell line (hFOB) were obtained l from the American Type Culture Collection (Manassas, Va.) 5 and are maintained in DMEM:F12 medium containing new s Z. born calf serum and antibiotics. MC3T3E1, C3H10T1/2, l C2C12 and hFOB cells are tested for growth induction by the O NH O synthetic branched peptide construct of Example 1. NH2 HC-C-SH2 N 11nu-N S-C-CH Example 3 OEC H2 O q=o s O > 0204. In an alternative method of synthesis, a first peptide l > precursor chain NH C(Npys)XXXRKRLDRIAR amide (SEQID NO:59) is synthesized by conventional pep 5 t tide synthesis methods, where each X is 6-aminohexanoic s t acid and C(Npys) is S-(3-nitro-2-pyridinesulfenyl)-cysteine. Z. 2 The second peptide precursor chain AISMLYLDENEKVV l t 2 t LCAISMLYLDENEKVVL-amide (SEQID NO:60), is syn thesized as in Example 1. The sequence AISM 2. 2. LYLDENEKVVL (SEQ ID NO:28) is derived from amino acids 87-100 in the human BMP-2 sequence. As shown in Synthetic Scheme 2, the two chains are reacted, resulting in a disulfide bond between the two sulfur atoms (S" and S) of the Example 2 cysteine residues of SEQ ID NO. 59 and SEQ ID NO:60, respectively. 0202 The synthetic branched peptide construct of Example 1 is tested in cell growth studies, to determine the ability of the synthetic branched peptide construct to stimu late cell growth. Cell growth is monitored using a commer SYNTHETIC SCHEME 2 cially available (Promega Corporation, Madison, Wis.) NH2-C(Npys)XXXRKRLDRIAR-amide based on a tetrazolium compound (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet It is MLYLDENEKWWL-C-ALSMLYLDENEKWWL-amide razolium, inner salt (MTS). Aliquots of 10 cells are seeded into wells of 96-well plates and allowed to attach. The US 2009/011 1743 A1 Apr. 30, 2009 18

peptide precursor, CXXXRKRLDRIAR-amide (SEQ ID -continued NO:58), the cysteine residue served to link the precursor to 1,4-bis-maleimidobutane, the 6-aminohexanoic acid XXX tripeptide served as both a spacer and provided a hydrophobic region, and the sequence RKRLDRIAR (SEQ ID NO:60) provided aheparin-binding motif derived from a modification of the sequence at residues 270-279 of the Jun/AP-1 DNA binding domain (Busch et al. Trans-Repressor Activity of Nuclear Glycosaminoglycans on Fos and Jun/AP-1 Oncopro tein-mediated Transcription. J. Cell Biol. 116:31-42, 1992). (0206. In the second peptide precursor LYVDFSDVG WNDWCAISMLYLDENEKVVL-amide (SEQ ID NO:59), the sequence LYVDFSDVGWNDW (SEQ ID NO:27) was -S2-S-CXXXRKRLDRIAR-amide derived from amino acids 19-31 in the human BMP-2 sequence, the cysteine residue served to link the precursor to 1,4-bis-maleimidobutane, and the sequence AISM LYLDENEKVVL (SEQID NO:28) was derived from amino acids 87-100 in the human BMP-2 sequence. 0207 Synthetic Scheme 3 shows the general approach for synthesis. The first precursor CXXXRKRLDRIAR-amide (SEQ ID NO:58) was reacted in a slightly acidic conditions with a large molar excess of A, 1.4-bis-maleimidobutane, a homobifunctional, sulfhydryl reactive agent with a four car pys = 3-Nitro-2-pyridinesulfenyl bon spacer, in a 50% acetonitrile/water adjusted to 5.8 pH by use of 0.1M sodium phosphate dibasic buffer. In the depiction of the resulting reaction product B, “S” depicts the sulfur atom “S” of the side chain of the cysteine residue, shown as “C”. The reaction product B was isolated by preparative HPLC and the isolated reaction product B was then reacted Example 4 with LYVDFSDVGWNDWCALSMLYLDENEKVVL amide (SEQ ID NO:59), again in a 50% acetonitrile/water 0205. A synthetic branched peptide construct of the gen adjusted to 8.0 pH by use of 0.1M sodium phosphate dibasic. eral formula: NH-LYVDFSDVGWNDWC(bMB Here too in the depiction of the resulting final product C, “S” CXXXRKRLDRIAR-amide)-AISMLYLDENEKVVL depicts the sulfur atom “S” of the side chain of the cysteine amide, where bMB is 1,4-bis-maleimidobutane and each X is residue, shown as “C”, forming a part of SEQID NO:59. The 6-aminohexanoic acid, was synthesized using two peptide final product C was purified by HPLC, using a 250x10.0 mm precursors. The first peptide precursor was Cs column at a flow rate of 3 mL/min in a solvent gradient of CXXXRKRLDRIAR-amide (SEQ ID NO:58), again O to 60% of 0.1% trifluoroacetate in acetonitrile where the where each X is 6-aminohexanoic acid, while the second initial solvent was 0.1% trifluoroacetate and water. The peptide precursor was LYVDFSDVGWNDWCAISM resulting final product C was a branched peptide construct LYLDENEKVVL-amide (SEQ ID NO:59). Both peptide wherein the two peptide subunits were conjugated via thioet precursors were synthesized by conventional Solid phase her linkages. Mass spectroscopy was used to confirm the methods and purified by reverse-phase HPLC. In the first sequence of the final construct.

SYNTHETIC SCHEME 3. NH-CXXXRKRLDRIAR-amide O O N 1N1\-N O O A US 2009/011 1743 A1 Apr. 30, 2009

-continued O O

N 1N1 Y1 N S-CXXXRKRLDRIAR-amide

O O B Nivorsovowsowcas MLYLDENEKWWL-amide Z. i t K a. l H A. l s5 O lZ. O 2 o-S2 N 1n 1\-N S-CXXXRKRLDRIAR-amide A. sK O tl O 2 C t 7. a. a. t 2.

0208. It may thus be seen that the structure of the molecule 10 ug/mL. C2C12 was negative for growth using the synthetic may alternatively be depicted as shown in FIG. 2, where branched peptide construct of Example 4. amino acid residues are shown by bolded single letter desig nations, letter designations that are not bolded refer to atoms, TABLE 3 and X is in each instance 6-aminohexanoic acid. MEAN % OF Example 5 Compound ABSOR- CONTROL Cell line Ig/mL BANCE S.D. P value VALUE 0209. The synthetic branched peptide construct of FOB O O48 0.08 OO Example 4 was tested in cell growth studies, to determine the O.S O.S8 O.O7 S 21 ability of the synthetic branched peptide construct to stimu- 062 0.04 -0.05 31 late cell growth. Cell growth was monitored using a commer- 2 O.64 O.O3 &O.OS 35 cially available kit (Promega Corporation, Madison, Wis.) 5 O.6O O.O3 &O.OS 27 based on a tetrazolium compound (3-(4,5-dimethylthiazol-2- 10 O.62 0.04 &O.OS 30 yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet- MC3T3E1 O O.72 0.088 OO razolium, inner salt (MTS). Aliquots of 10 cells were seeded O.S O.74 0.100 ns O3 into wells of 96-well plates and allowed to attach. The 1 O.71 O.097 ns 99 medium was then replaced with a low serum medium con- 2 O.99 O.124 &O.OS 38 taining between 0 and 10 ug/mL of the synthetic branched 5 O.96 O. 129 &O.OS 33 peptide construct of Example 4. After 3 days in culture, the 10 O.96 O.234 &O.OS 34 relative cell number was monitored using MTS following the C3H10T1/2 O O.28 O.04 OO directions of the manufacturer. Statistical significance was O.S O41 0.06 &O.OS 47 determined using ANOVA followed by post-hoc multiple l C. g 3. s comparisons versus control group (Dunnett's Method). 5 0.43 003 . 05 56 0210 MC3T3E1 osteoblastic cells, C3H10T1/2 mouse 10 0.40 003 ins 44 pluripotent stem cells, C2C12 murine myoblasts, and cells C2C12 O O.30 O.O2 OO from a human fetal osteoblast cell line (hFOB) were obtained O.S O.30 O.O1 S 99 from the American Type Culture Collection (Manassas, Va.) 1 O.30 O.O1 S O1 and were maintained in DMEM:F12 medium containing 2 O.30 O.O1 S 99 newborn calf serum and antibiotics. MC3T3E1, C3H10T1/2, 5 O.30 O.O1 S 99 and hFOB cells were positive for growth induction by syn- 10 O.27 O.O1 S 90 thetic branched peptide construct of Example 2 as shown in Table 3 below. The effective concentration was between 1 and US 2009/011 1743 A1 Apr. 30, 2009 20

Example 6 0211. In an alternative method of synthesis, a first peptide precursor chain NH C(Npys)XXXRKRLDRIAR SYNTHETIC SCHEMES amide (SEQID NO:61) is synthesized by conventional pep AISMLYLDENEKVVLK(Dde)XXXRKRLDRIAR-Resin tide synthesis methods, where each X is 6-aminohexanoic acid and C(Npys) is S-(3-nitro-2-pyridinesulfenyl)-cysteine. The second peptide precursor chain LYVDFSDVGWND stain WCAISMLYLDENEKVVL-amide (SEQID NO:59), is syn AISMLYLDENEKVVLK(NH2)XXXRKRLDRIAR-Resin thesized as in Example 4. As shown in Synthetic Scheme 4, the two chains are reacted, resulting in a disulfide bond ins peptide synthesis protocol between the two sulfur atoms (S. and S) of the cysteine residues of SEQID NO. 61 and SEQID NO:59, respectively. AISMLYLDENEKVVLKXXXRKRLDRIAR-Resin LYWDFSDVGWNDW SYNTHETIC SCHEME 4 levue from resin and deprotection NH2-C(Npys)XXXRKRLDRIAR-amide AISMLYLDENEKVVLKXXXRKRLDRIAR-amide NH-LYVDFSDVGWNDW-C-ALSMLYLDENEKVVL-amide LYWDFSDVGWNDW

0212. The preceding examples can be repeated with simi lar Success by Substituting the generically or specifically described peptide sequences, reactants and/or operating con ditions of this invention for those used in the preceding examples. 0213 Although the invention has been described in detail with particular reference to these preferred embodiments, -S-S-CXXXRKRLDRIAR-amide other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above are hereby incorporated by refer CCC. 0214. The present invention has been described interms of preferred embodiments, however, it will be appreciated that pys = 3-Nitro-2-pyridinesulfenyl various modifications and improvements may be made to the described embodiments without departing from the scope of the invention.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 61

<210 SEQ ID NO 1 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin-binding motif &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (1) . . (1) <223> OTHER INFORMATION: Lys or Arg &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: Lys or Arg &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (3) . . (3) <223> OTHER INFORMATION: Lys or Arg &220s FEATURE: US 2009/011 1743 A1 Apr. 30, 2009 21

- Continued <221 NAMEAKEY: misc feature <222> LOCATION: (4) . . (5) <223> OTHER INFORMATION: any naturally or non-naturally occuring amino acid &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (6) . . (6) <223> OTHER INFORMATION: Lys or Arg <4 OO SEQUENCE: 1

Xaa Xala Xala Xala Xala Xala 1. 5

<210 SEQ ID NO 2 <211 LENGTH: 10 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin-binding motif of Z region <4 OO SEQUENCE: 2 Arg Lys Arg Llys Lieu. Glu Arg Ile Ala Arg 1. 5 1O

<210 SEQ ID NO 3 <211 LENGTH: 10 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin-binding motif of Z reqion

<4 OO SEQUENCE: 3 Arg Lys Arg Llys Lieu. Gly Arg Ile Ala Arg 1. 5 1O

<210 SEQ ID NO 4 <211 LENGTH: 10 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin-binding motif of Z region <4 OO SEQUENCE: 4 Arg Lys Arg Llys Lieu. Trp Arg Ala Arg Ala 1. 5 1O

<210 SEQ ID NO 5 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin-binding motif of Z region <4 OO SEQUENCE: 5 Arg Lys Arg Llys Lieu. Glu Arg Ile Ala Arg Cys 1. 5 1O

<210 SEQ ID NO 6 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-2 analog

<4 OO SEQUENCE: 6 US 2009/011 1743 A1 Apr. 30, 2009 22

- Continued Tyr Arg Ser Arg Llys Tyr Thr Ser Trp Tyr Val Ala Lieu Lys Arg 1. 5 1O 15

<210 SEQ ID NO 7 <211 LENGTH: 28 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF analog

<4 OO SEQUENCE: 7 Asn Arg Phe His Ser Trp Asp Cys Ile Llys Thir Trp Ala Ser Asp Thr 1. 5 1O 15 Phe Val Lieu Val Cys Tyr Asp Asp Gly Ser Glu Ala 2O 25

<210 SEQ ID NO 8 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-2 analog

<4 OO SEQUENCE: 8 His Ile Llys Lieu. Glin Lieu. Glin Ala Glu Glu Arg Gly Val Val Ser 1. 5 1O 15

<210 SEQ ID NO 9 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM; artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-1 analog <4 OO SEQUENCE: 9 Tyr Ile Ser Lys Llys His Ala Glu Lys Asn Trp Phe Val Gly Lieu Lys 1. 5 1O 15 Lys

<210 SEQ ID NO 10 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-1 analog

<4 OO SEQUENCE: 10 His Ile Glin Lieu. Glin Lieu. Ser Ala Glu Ser Val Gly Glu Val Tyr 1. 5 1O 15

<210 SEQ ID NO 11 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-7 analog <4 OO SEQUENCE: 11 Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val Ala 1. 5 1O 15 Lieu. Asn Gln Lys 2O US 2009/011 1743 A1 Apr. 30, 2009 23

- Continued

<210 SEQ ID NO 12 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-7 analog

<4 OO SEQUENCE: 12 Tyr Asn. Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala 1. 5 1O 15

<210 SEQ ID NO 13 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-10 analog <4 OO SEQUENCE: 13 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala 1. 5 1O 15 Lieu. Asn Gln Lys 2O

<210 SEQ ID NO 14 <211 LENGTH: 19 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-22 analog <4 OO SEQUENCE: 14 Tyr Ala Ser Glin Arg Trp Arg Arg Arg Gly Glin Pro Asn Lieu Ala Lieu. 1. 5 1O 15 Asp Arg Arg

<210 SEQ ID NO 15 <211 LENGTH: 21 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-9 analog <4 OO SEQUENCE: 15 Tyr Ser Ser Asn Lieu. Tyr Lys His Val Asp Thr Gly Arg Arg Tyr Tyr 1. 5 1O 15 Val Ala Lieu. Asn Llys 2O

<210 SEQ ID NO 16 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-16 analog <4 OO SEQUENCE: 16 Tyr Ala Ser Thr Lieu. Tyr Lys His Ser Asp Ser Glu Arg Glin Tyr Val 1. 5 1O 15 Ala Lieu. Asn Lys 2O US 2009/011 1743 A1 Apr. 30, 2009 24

- Continued

<210 SEQ ID NO 17 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-2O analog

<4 OO SEQUENCE: 17 Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp Thr Gly Arg Arg Phe Val 1. 5 1O 15 Ala Lieu. Asn Lys 2O

<210 SEQ ID NO 18 <211 LENGTH: 16 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-4 analog

<4 OO SEQUENCE: 18 Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala Leu Ser Lys Asn 1. 5 1O 15

<210 SEQ ID NO 19 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF- 6 analog <4 OO SEQUENCE: 19 Tyr Glu Ser Asp Lieu. Tyr Glin Gly Thr Tyr Ile Leu Ser Lys Tyr Gly 1. 5 1O 15 Arg

<210 SEQ ID NO 2 O <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-12 analog <4 OO SEQUENCE: 2O Tyr Ser Ser Thr Lieu. Tyr Arg Glin Glin Glu Ser Gly Arg Ala Trp Phe 1. 5 1O 15 Lieu. Gly Asn Lys 2O

<210 SEQ ID NO 21 <211 LENGTH: 21 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-14 analog <4 OO SEQUENCE: 21 Tyr Ser Ser Met Lieu. Tyr Arg Glin Glin Glu Ser Gly Arg Ala Trp Phe 1. 5 1O 15 Lieu. Gly Lieu. Asn Llys 2O US 2009/011 1743 A1 Apr. 30, 2009 25

- Continued

<210 SEQ ID NO 22 <211 LENGTH: 21 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-13 analog

<4 OO SEQUENCE: 22 Tyr Ser Ser Met Ile Tyr Arg Glin Glin Glin Ser Gly Arg Gly Trp Tyr 1. 5 1O 15 Lieu. Gly Lieu. Asn Llys 2O

<210 SEQ ID NO 23 <211 LENGTH: 19 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic FGF-11 analog

<4 OO SEQUENCE: 23 Tyr Ala Ser Ala Lieu. Tyr Arg Glin Arg Arg Ser Gly Arg Ala Trp Tyr 1. 5 1O 15 Lieu. Asp Llys

<210 SEQ ID NO 24 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic VEGF analog

<4 OO SEQUENCE: 24 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val 2O

<210 SEQ ID NO 25 <211 LENGTH: 11 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic VEGF analog

<4 OO SEQUENCE: 25 Gly Ala Thir Trp Leu Pro Pro Asn Pro Thr Lys 1. 5 1O

<210 SEQ ID NO 26 <211 LENGTH: 2O &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic VEGF analog <4 OO SEQUENCE: 26 Asn Phe Lieu. Lieu. Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. Tyr 1. 5 1O 15

Lieu. His His Ala 2O US 2009/011 1743 A1 Apr. 30, 2009 26

- Continued

<210 SEQ ID NO 27 <211 LENGTH: 13 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog

<4 OO SEQUENCE: 27 Lieu. Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 1. 5 1O

<210 SEQ ID NO 28 <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog <4 OO SEQUENCE: 28 Ala Ile Ser Met Lieu. Tyr Lieu. Asp Glu Asn. Glu Lys Val Val Lieu. 1. 5 1O 15

<210 SEQ ID NO 29 <211 LENGTH: 17 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog <4 OO SEQUENCE: 29 Ile Ser Met Lieu. Tyr Lieu. Asp Glu Asn. Glu Lys Val Val Lieu Lys Asn 1. 5 1O 15 Tyr

<210 SEQ ID NO 3 O <211 LENGTH: 15 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog <4 OO SEQUENCE: 30 Glu Lys Val Val Lieu Lys Asn Tyr Glin Asp Met Val Val Glu Gly 1. 5 1O 15

<210 SEQ ID NO 31 <211 LENGTH: 16 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog

<4 OO SEQUENCE: 31 Lieu Val Val Lys Glu Asn. Glu Asp Lieu. Tyr Lieu Met Ser Ile Ala Cys 1. 5 1O 15

<210 SEQ ID NO 32 <211 LENGTH: 16 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog <4 OO SEQUENCE: 32 US 2009/011 1743 A1 Apr. 30, 2009 27

- Continued

Ala Phe Tyr Cys His Gly Glu. Cys Pro Phe Pro Leu Ala Asp His Leu 1. 5 1O 15

<210 SEQ ID NO 33 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP analog

<4 OO SEQUENCE: 33 Pro Phe Pro Lieu Ala Asp His Lieu. Asn. Ser Thr Asn His Ala Ile Val 1. 5 1O 15

Glin. Thir Lieu. Wall Asn. Ser Wall 2O

<210 SEQ ID NO 34 <211 LENGTH: 21 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic TGF-beta1 analog

<4 OO SEQUENCE: 34 Ile Val Tyr Tyr Val Gly Arg Llys Pro Llys Val Glu Glin Lieu. Ser Asn 1. 5 1O 15 Met Ile Val Arg Ser 2O

<210 SEQ ID NO 35 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic TGF-beta2 analog <4 OO SEQUENCE: 35 Thir Ile Leu Tyr Tyr Ile Gly Lys Thr Pro Lys Ile Glu Glin Leu Ser 1. 5 1O 15 Asn Met Ile Val Lys Ser 2O

<210 SEQ ID NO 36 <211 LENGTH: 21 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic TGF-beta3 analog <4 OO SEQUENCE: 36 Lieu. Thir Ile Leu Tyr Tyr Val Gly Arg Thr Pro Llys Val Glu Gln Leu 1. 5 1O 15

Ser Asn. Met Wal Wall 2O

<210 SEQ ID NO 37 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-2 analog US 2009/011 1743 A1 Apr. 30, 2009 28

- Continued <4 OO SEQUENCE: 37 Ala Ile Ser Met Lieu. Tyr Lieu. Asp Glu Asn. Glu Lys Val Val Lieu Lys 1. 5 1O 15 Asn Tyr Glin Asp Met Val Val 2O

<210 SEQ ID NO 38 <211 LENGTH: 24 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-3 analog

<4 OO SEQUENCE: 38 Ser Ser Lieu. Ser Ile Lieu. Phe Phe Asp Glu Asn Lys Asn Val Val Lieu. 1. 5 1O 15 Llys Val Tyr Pro Asn Met Thr Val 2O

<210 SEQ ID NO 39 <211 LENGTH: 24 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-3beta analog <4 OO SEQUENCE: 39 Asn Ser Lieu. Gly Val Lieu. Phe Lieu. Asp Glu Asn Arg Asn Val Val Lieu 1. 5 1O 15 Llys Val Tyr Pro Asn Met Ser Val 2O

<210 SEQ ID NO 4 O <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-4 peptide analog

<4 OO SEQUENCE: 40 Ala Ile Ser Met Lieu. Tyr Lieu. Asp Glu Tyr Asp Llys Val Val Lieu Lys 1. 5 1O 15 Asn Tyr Glin Glu Met Val Val 2O

<210 SEQ ID NO 41 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-5 analog <4 OO SEQUENCE: 41 Ala Ile Ser Val Lieu. Tyr Phe Asp Asp Ser Ser Asn Val Ile Lieu Lys 1. 5 1O 15 Llys Tyr Arg Asn Met Val Val 2O

<210 SEQ ID NO 42 <211 LENGTH: 23 &212> TYPE: PRT US 2009/011 1743 A1 Apr. 30, 2009 29

- Continued

<213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-6 analog

<4 OO SEQUENCE: 42 Ala Ile Ser Val Lieu. Tyr Phe Asp Asp Asn. Ser Asn Val Ile Lieu Lys 1. 5 1O 15 Llys Tyr Arg Asn Met Val Val 2O

<210 SEQ ID NO 43 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-7 analog

<4 OO SEQUENCE: 43 Ala Ile Ser Val Lieu. Tyr Phe Asp Asp Ser Ser Asn Val Ile Lieu Lys 1. 5 1O 15 Llys Tyr Arg Asn Met Val Val 2O

<210 SEQ ID NO 44 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-8 analog

<4 OO SEQUENCE: 44 Ala Thir Ser Val Lieu. Tyr Tyr Asp Ser Ser Asn. Asn Val Ile Lieu. Arg 1. 5 1O 15 Lys Ala Arg Asn Met Val Val 2O

<210 SEQ ID NO 45 <211 LENGTH: 23 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-9 analog <4 OO SEQUENCE: 45 Ile Ser Val Lieu. Tyr Lys Asp Asp Met Gly Val Pro Thr Lieu Lys Tyr 1. 5 1O 15 His Tyr Glu Gly Met Ser Val 2O

<210 SEQ ID NO 46 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-10 analog <4 OO SEQUENCE: 46 Ile Ser Ile Lieu. Tyr Lieu. Asp Llys Gly Val Val Thr Tyr Llys Phe Lys 1. 5 1O 15 Tyr Glu Gly Met Ala Val 2O US 2009/011 1743 A1 Apr. 30, 2009 30

- Continued

<210 SEQ ID NO 47 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: synthetic BMP-11 analog

<4 OO SEQUENCE: 47 Ile Asn Met Lieu. Tyr Phe Asn Asp Llys Glin Glin Ile Ile Tyr Gly Lys 1. 5 1O 15 Ile Pro Gly Met Val Val 2O

<210 SEQ ID NO 48 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-12 analog

<4 OO SEQUENCE: 48 Ile Ser Ile Lieu. Tyr Ile Asp Ala Ala Asn. Asn Val Val Tyr Lys Glin 1. 5 1O 15 Tyr Glu Asp Met Val Val 2O

<210 SEQ ID NO 49 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-13 analog <4 OO SEQUENCE: 49 Ile Ser Ile Lieu. Tyr Ile Asp Ala Gly Asn. Asn Val Val Tyr Lys Glin 1. 5 1O 15 Tyr Glu Asp Met Val Val 2O

<210 SEQ ID NO 50 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-14 analog <4 OO SEQUENCE: 5 O Ile Ser Ile Lieu. Phe Ile Asp Ser Ala Asn. Asn Val Val Tyr Lys Glin 1. 5 1O 15 Tyr Glu Asp Met Val Val 2O

<210 SEQ ID NO 51 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic BMP-15 analog <4 OO SEQUENCE: 51 Ile Ser Val Lieu Met Ile Glu Ala Asn Gly Ser Ile Lieu. Tyr Lys Glu 1. 5 1O 15 US 2009/011 1743 A1 Apr. 30, 2009 31

- Continued

Tyr Glu Gly Met Ile Ala 2O

<210 SEQ ID NO 52 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic GDF-1 analog

<4 OO SEQUENCE: 52 Ile Ser Val Lieu. Phe Phe Asp Asn. Ser Asp Asn Val Val Lieu. Arg Glin 1. 5 1O 15 Tyr Glu Asp Met Val Val 2O

<210 SEQ ID NO 53 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic GDF-3 analog

<4 OO SEQUENCE: 53 Ile Ser Met Lieu. Tyr Glin Asp Asn. Asn Asp Asn Val Ile Lieu. Arg His 1. 5 1O 15 Tyr Glu Asp Met Val Val 2O

<210 SEQ ID NO 54 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic GDF-8 analog <4 OO SEQUENCE: 54 Ile Asn Met Tyr Lieu. Phe ASn Gly Lys Glu Glin Ile Ile Tyr Gly Lys 1. 5 1O 15

Ile Pro Ala Met Wal Wall 2O

<210 SEQ ID NO 55 <211 LENGTH: 22 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Synthetic GDF-9 analog <4 OO SEQUENCE: 55 Lieu. Ser Val Lieu. Thir Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu 1. 5 1O 15 Tyr Glu Asp Met Ile Ala 2O

<210 SEQ ID NO 56 <211 LENGTH: 13 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin binding motif &220s FEATURE: US 2009/011 1743 A1 Apr. 30, 2009 32

- Continued <221 NAMEAKEY: misc feature <222> LOCATION: (2) ... (4) <223> OTHER INFORMATION: 6-aminohexanoic acid

<4 OO SEQUENCE: 56 Cys Xaa Xala Xala Arg Lys Arg Lieu. Asp Arg Ile Ala Arg 1. 5 1O

<210 SEQ ID NO 57 <211 LENGTH: 27 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Peptide precursor <4 OO SEQUENCE: 57 Lieu. Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Cys Lieu. Tyr 1. 5 1O 15 Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 2O 25

<210 SEQ ID NO 58 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Heparin binding motif

<4 OO SEQUENCE: 58 Arg Lys Arg Lieu. Asp Arg Ile Ala Arg 1. 5

<210 SEQ ID NO 59 <211 LENGTH: 14 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Peptide precursor &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: S- (3-nitro-2-pyridinesulfenyl) - cysteine &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (3) . . (5) <223> OTHER INFORMATION: 6-aminohexanoic acid

<4 OO SEQUENCE: 59 Cys Xaa Xala Xala Xaa Arg Lys Arg Lieu. Asp Arg Ile Ala Arg 1. 5 1O

<210 SEQ ID NO 60 <211 LENGTH: 31 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Peptide precursor <4 OO SEQUENCE: 60 Ala Ile Ser Met Lieu. Tyr Lieu. Asp Glu Asn. Glu Lys Val Val Lieu. Cys 1. 5 1O 15 Ala Ile Ser Met Lieu. Tyr Lieu. Asp Glu Asn. Glu Lys Val Val Lieu. 2O 25 3O US 2009/011 1743 A1 Apr. 30, 2009

- Continued <210 SEQ ID NO 61 <211 LENGTH: 14 &212> TYPE: PRT <213> ORGANISM: Artificial &220s FEATURE: <223> OTHER INFORMATION: Peptide precursor &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: S- (3-nitro-2-pyridinesulfenyl) - cysteine &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (3) . . (5) <223> OTHER INFORMATION: 6-aminohexanoic acid

<4 OO SEQUENCE: 61 Cys Xaa Xala Xala Xaa Arg Lys Arg Lieu. Asp Arg Ile Ala Arg 1. 5 1O

What is claimed is: (ii) comprises a chain of a minimum of about 9 and a maxi 1. A heparin-binding growth factor analog of formula I: mum of about 50 atoms, and (iii) is not found in the natural ligand of the heparin-binding growth factor receptor (HB GFR) which X binds. i 4. The heparin-binding growth factor analog of claim 1 R3 wherein the heparin-binding growth factor analog has an avidity for heparin Such that the synthetic heparin-binding R-R-AA AA-HY-Z growth factor analog binds heparin in 0.15 M NaCl, but is R3 R3 R3 eluted by 1 MNaCl. 5. The heparin-binding growth factor analog of claim 1 X X pi X iii. wherein the construct is of formula II: wherein: II each X 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 is a trifunctional amino acid residue covalently bonded wherein: to one X through the N terminus amine of R and the CyS is cysteine; and remaining X through the C terminus carboxyl of R: R is a linker consisting of a sulfhydryl reactive homo R is a linker comprising a chain from 3 to about 20 atoms bifunctional cross-linker and a second Cys or compris covalently bonded to the side chain of R and Y when ing a hetero-bifunctional cross-linker. n=0, or to the side chain of R and to the N terminus 6. The heparin-binding growth factor analog of claim 5 amine of AA when n=1; wherein the construct is of formula III: Each R is independently from 0 to about 3 amino acid residues which are the same or different; III Y is a linker comprising a chain from 0 to about 50 atoms Rs covalently bonded to R and Zwhen n=0, or to AA and Z when n=1 and m=0, or to AA and Z when n=1 and X m=1; r O O Z is a non-signaling peptide chain that includes a heparin CH-C-S NH2 binding domain, comprising an amino acid sequence | H2 N-R-N that comprises (i) a minimum of one heparin binding OEC S-C-CH motif, (ii) a maximum of about ten heparin binding H2 motifs, and (iii) a maximum of about thirty amino acids; O O =o AA and AA are each independently a trifunctional amino acid residue, wherein X is covalently bonded through R6 the side chain of AA and AA, and Z n is 0 or 1, and when n is 1, m is 0 or 1, and when n is 0, m R is 0. 2. The heparin-binding growth factor analog of claim 1 wherein: wherein X and Z are synthetic peptide chains. R is a linker comprising a chain of between 1 and about 10 3. The heparin-binding growth factor analog of claim 1 backbone atoms selected from carbon, oxygen, Sulfur wherein Y further comprises a linker that (i) is hydrophobic, and nitrogen or mixtures thereof; US 2009/011 1743 A1 Apr. 30, 2009 34

Rs is NH, an acyl group with a linear or branched C to 15. The heparin-binding growth factor analog of claim 1 C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl wherein X comprises an amino acid sequence found in any of chain including an N-terminus NH2, NH, or NH FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF group or a corresponding acylated derivative; 8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF R is OH, NH, or NH Rs; and 15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, R, is NH, an acyl group with a linear or branched C to FGF-22, FGF-23, HBBM (heparin-binding brain mitogen), C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl HB-GAF (heparin-binding growth associated factor), HB chain including an N-terminus NH2, NH, or NH EGF (heparin-binding EGF-like factor) HB-GAM (heparin group or a corresponding acylated derivative. binding growth associated molecule, also known as pleiotro 7. The heparin-binding growth factor analog of claim 6 phin, PTN, HARP), TGF-C. (transforming growth factor-C), wherein the construct is of formula IV: TGF-Bs (transforming growth factor-?s), VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), IGF-1 (insulin-like growth factor-1), IGF-2 (insulin-like IV growth factor-2), PDGF (platelet derived growth factor), RANTES, SDF-1, secreted frizzled-related protein-1 (SFRP NH 1), small inducible cytokine A3 (SCYA3), inducible cytokine O subfamily A member 20 (SCYA20), inducible cytokine sub HN O family B member 14 (SCYB14), inducible cytokine subfam NH2 ily D member 1 (SCYD1), stromal cell-derived factor-1 CH-C-SH N 1n 1-N S-C-CH (SDF-1), thrombospondins 1, 2, 3 and 4 (THEBS1-4), platelet O = H2 CEO factor 4 (PF4), lens epithelium-derived growth factor X O O (LEDGF), midikine (MK), macrophage inflammatory pro Y tein (MIP-1), moesin (MSN), hepatocyte growth factor (HGF, NH2 also called SF), placental growth factor, IL-1 (interleukin-1), IL-2 (interleukin-2), IL-3 (interleukin-3), IL-6 (interleukin NH2 6), IL-7 (interleukin-7). IL-10 (interleukin-10), IL-12 (inter leukin-12), IFN-C. (interferon-C), IFN-Y (interferon-Y), wherein: TNF-C. (tumor necrosis factor-C), SDGF (Schwannoma-de X is any of SEQID NOS:6-55; rived growth factor), nerve growth factor, neurite growth Y is between two and about five amino acid residues promoting factor 2 (NEGF2), neurotrophin, BMP-2 (bone Selected from the group consisting of 6-aminohexanoic morphogenic protein 2), OP-1 (osteogenic protein 1, also acid, 7-aminoheptanoic acid, 9-aminononanoic acid and called BMP-7), keratinocyte growth factor (KGF), inter feron-Y inducible protein-20, RANTES, and HIV-tat-transac mixtures thereof, and tivating factor, amphiregulin (AREG), angio-associated Z is any of SEQID NOS:2-5 or SEQID NO:58. migratory cell protein (AAMP), angiostatin, betacellulin 8. The heparin-binding growth factor analog of claim 1 (BTC), connective tissue growth factor (CTGF), cysteine wherein the covalent bonds between the side chain of R and rich angiogenic inducer 61 (CYCR61), endostatin, fractalk R and between R and Z when n=0, or between AA and Z ine/neuroactin, glial derived neurotrophic factor (GDNF), when n=1 and m=0, or between AA and Z when n=1 and GRO2, hepatoma-derived growth factor (HDGF), and granu m=1, comprise an amide, disulfide, thioether, Schiff base, locyte-macrophage colony stimulating factor (GMCSF), a reduced Schiffbase, imide, secondary amine, carbonyl, urea, homolog of any amino acid sequence found in the foregoing, hydrazone or oxime bond. a reverse sequence of any amino acid sequence found in the 9. The heparin-binding growth factor analog of claim 1 foregoing, or a homolog of a reverse sequence of any amino wherein the side chains of AA and AA comprise reactive acid residue found in the foregoing. carboxyl groups. 16. The heparin-binding growth factor analog of claim 1 10. The heparin-binding growth factor analog of claim 1 wherein Y comprises between one and about thirty-three eth wherein the side chain of R comprises a reactive sulfhydryl ylene glycol units. group. 17. The heparin-binding growth factor analog of claim 1 11. The heparin-binding growth factor analog of claim 10 wherein Y comprises a branched or unbranched, saturated or wherein R is an L- or D-3-mercapto amino acid. unsaturated alkyl chain of between one and about twenty 12. The heparin-binding growth factor analog of claim 11 carbon atoms. wherein the L- or D-3-mercapto amino acid is L- or D-cys 18. The heparin-binding growth factor analog of claim 1 teine, L- or D-penicillamine, 3-mercaptophenylalanine, or a wherein Y comprises (NH2 (CH2)CO), wherein p is from derivative of any of the foregoing. 1 to about 10 and q is from 1 to about 20. 13. The heparin-binding growth factor analog of claim 1 19. The heparin-binding growth factor analog of claim 1 wherein X is any of SEQID NO:5 to SEQID NO:55 and Z is wherein Y comprises a peptide sequence comprising from RKRLDRIAR (SEQ ID NO:58), RKRKLERIAR (SEQ ID one to about 16 Gly residues. NO:2) RKRKLGRIAR (SEQID NO:3) or RKRKLWRARA 20. The heparin-binding growth factor analog of claim 1 (SEQ ID NO:4). wherein each heparin binding motif of Z is BxBB or 14. The heparin-binding growth factor analog of claim 1 BBBXxB, wherein each B is independently lysine, arginine, wherein one or more of R is between one and about three ornithine, or histidine, and each X is a independently a natu amino acid residues selected from the group consisting of rally occurring amino acid. glycine, 6-aminohexanoic acid, 7-aminoheptanoic acid, 21. The heparin-binding growth factor analog of claim 20 9-aminononanoic acid and mixtures thereof. wherein Z comprises at least two heparin-binding motifs. US 2009/011 1743 A1 Apr. 30, 2009

22. A pharmaceutical composition comprising the heparin 27. The heparin-binding growth factor analog of claim 23 binding growth factor analog of claim 1 or a pharmaceutically wherein the construct is of formula VI: acceptable salt thereof and a pharmaceutical carrier. VI 23. A heparin-binding growth factor analog of formula V: y V R3 wherein: R-R-AA AA-HY-Z CyS is cysteine; and R is a linker consisting of a sulfhydryl reactive homo R3 R3 R3 bifunctional cross-linker and a second Cys or compris X X W ing a hetero-bifunctional cross-linker. 28. The heparin-binding growth factor analog of claim 27 wherein the construct is of formula VII:

VII wherein: Rs each X and each W is a peptide chain differing by at least one amino acid residue that (i) has a minimum of three y amino acid residues, (ii) has a maximum of about fifty amino acid residues, and (iii) binds a heparin-binding ICH-C-S NH2 growth factor receptor (HBGFR); | H2 N-R-N OEC S-C-CH R is a trifunctional amino acid residue covalently bonded H2 to one X through the N terminus amine of R and the O O =o remaining X through the C terminus carboxyl of R: R6 Y R is a linker comprising a chain from 3 to about 20 atoms covalently bonded to the side chain of R and Y when Z n=0, or to the side chain of R and to the N terminus R amine of AA when n=1; Each R is independently from 0 to about 3 amino acid residues which are the same or different; wherein: Y is a linker comprising a chain from 0 to about 50 atoms R is a linker comprising a chain of between 1 and about 10 covalently bonded to R and Zwhen n=0, or to AA and backbone atoms selected from carbon, oxygen, Sulfur Z when n=1 and m=0, or to AA and Z when n=1 and and nitrogen or mixtures thereof; m=1; Rs is NH2, an acyl group with a linear or branched C to Z is a non-signaling peptide chain that includes a heparin C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl binding domain, comprising an amino acid sequence chain including an N-terminus NH2, NH, or NH that comprises (i) a minimum of one heparin binding group or a corresponding acylated derivative; motif, (ii) a maximum of about ten heparin binding R is OH, NH, or NH Rs; and motifs, and (iii) a maximum of about thirty amino acids; R, is NH2, an acyl group with a linear or branched C to AA and AA are each independently a trifunctional amino C, alkyl, aryl, heteroaryl, alkene, alkenyl or aralkyl acid residue, wherein X and W are covalently bonded chain including an N-terminus NH, NH", or NH through the side chain of AA and AA, respectively; group or a corresponding acylated derivative. and, 29. The heparin-binding growth factor analog of claim 28 n is 0 or 1, and when n is 1, m is 0 or 1, and when n is 0, m wherein the construct is of formula VIII: is 0. VIII 24. The heparin-binding growth factor analog of claim 23 wherein X, W and Z are synthetic peptide chains. H. 25. The heparin-binding growth factor analog of claim 23 y O wherein Y further comprises a linker that (i) is hydrophobic, HN O NH2 (ii) comprises a chain of a minimum of about 9 and a maxi CH-C-S 11a-N mum of about 50 atoms, and (iii) is not found in the natural H2 N S-C-CH ligand of the heparin-binding growth factor receptor (HB O= H2 CEO X O O GFR) which X binds. Y 26. The heparin-binding growth factor analog of claim 23 NH2 wherein the heparin-binding growth factor analog has an 1. avidity for heparin Such that the synthetic heparin-binding NH2 growth factor analog binds heparin in 0.15 M NaCl, but is eluted by 1 M NaCl. US 2009/011 1743 A1 Apr. 30, 2009 36 wherein: frizzled-related protein-1 (SFRP-1), small inducible cytokine X and Ware each independently selected from any of SEQ A3 (SCYA3), inducible cytokine subfamily A member 20 ID NOS:6-55; (SCYA20), inducible cytokine subfamily B member 14 Y is between two and about five amino acid residues (SCYB14), inducible cytokine subfamily D member 1 (SCYD1), stromal cell-derived factor-1 (SDF-1), thrombo Selected from the group consisting of 6-aminohexanoic spondins 1,2,3 and 4 (THEBS1-4), platelet factor 4 (PF4), lens acid, 7-aminoheptanoic acid, 9-aminononanoic acid and epithelium-derived growth factor (LEDGF), midikine (MK), mixtures thereof, and macrophage inflammatory protein (MIP-1), moesin (MSN), Z is any of SEQID NOS:2-5 or SEQID NO:58. hepatocyte growth factor (HGF, also called SF), placental 30. The heparin-binding growth factor analog of claim 23 growth factor, IL-1 (interleukin-1), IL-2 (interleukin-2). IL-3 wherein the covalent bonds between the side chain of R and (interleukin-3), IL-6 (interleukin-6), IL-7 (interleukin-7), R and between R and Z when n=0, or between AA and Z IL-10 (interleukin-10), IL-12 (interleukin-12), IFN-C. (inter when n=1 and m=0, or between AA and Z when n=1 and feron-C.), IFN-Y (interferon-Y), TNF-C. (tumor necrosis factor m=1, comprise an amide, disulfide, thioether, Schiff base, O), SDGF (Schwannoma-derived growth factor), nerve reduced Schiffbase, imide, secondary amine, carbonyl, urea, growth factor, neurite growth-promoting factor 2 (NEGF2). hydrazone or oxime bond. neurotrophin, BMP-2 (bone morphogenic protein 2), OP-1 31. The heparin-binding growth factor analog of claim 1 (osteogenic protein 1, also called BMP-7), keratinocyte wherein XandW each independently comprise anamino acid growth factor (KGF), interferon-Y inducible protein-20, sequence found in any of FGF-1, FGF-2, FGF-3, FGF-4, RANTES, and HIV-tat-transactivating factor, amphiregulin FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, (AREG), angio-associated migratory cell protein (AAMP), FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF angiostatin, betacellulin (BTC), connective tissue growth fac 18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, HBBM(he tor (CTGF), cysteine-richangiogenic inducer 61 (CYCR61), parin-binding brain mitogen), HB-GAF (heparin-binding endostatin, fractalkine/neuroactin, glial derived neurotrophic growth associated factor), HB-EGF (heparin-binding EGF factor (GDNF), GRO2, hepatoma-derived growth factor like factor) HB-GAM (heparin-binding growth associated (HDGF), and granulocyte-macrophage colony stimulating molecule, also known as pleiotrophin, PTN, HARP). TGF-C. factor (GMCSF), a homolog of any amino acid sequence (transforming growth factor-O.), TGF-Bs (transforming found in the foregoing, a reverse sequence of any amino acid growth factor-?s), VEGF (vascular endothelial growth fac sequence found in the foregoing, or a homolog of a reverse tor), EGF (epidermal growth factor), IGF-1 (insulin-like sequence of any amino acid residue found in the foregoing. growth factor-1), IGF-2 (insulin-like growth factor-2), PDGF (platelet derived growth factor), RANTES, SDF-1, secreted c c c c c