U.S. patent application number 12/023801 was filed with the patent office on 2015-12-10 for positive modulator of bone morphogenic protein-2.
The applicant listed for this patent is Xinhua Lin, Louis A. Pena, Kazuyuki Takahashi, Paul O. Zamora. Invention is credited to Xinhua Lin, Louis A. Pena, Kazuyuki Takahashi, Paul O. Zamora.
Application Number | 20150353615 12/023801 |
Document ID | / |
Family ID | 34910841 |
Filed Date | 2015-12-10 |
United States Patent
Application |
20150353615 |
Kind Code |
A9 |
Zamora; Paul O. ; et
al. |
December 10, 2015 |
POSITIVE MODULATOR OF BONE MORPHOGENIC PROTEIN-2
Abstract
Compounds of the present invention of formula I and formula II
are disclosed in the specification and wherein the compounds are
modulators of Bone Morphogenic Protein activity. Compounds are
synthetic peptides having a non-growth factor heparin binding
region, a linker, and sequences that bind specifically to a
receptor for Bone Morphogenic Protein. Uses of compounds of the
present invention in the treatment of bone lesions, degenerative
joint disease and to enhance bone formation are disclosed.
Inventors: |
Zamora; Paul O.;
(Gaithersburg, MD) ; Pena; Louis A.; (Poquott,
NY) ; Lin; Xinhua; (Plainview, NY) ;
Takahashi; Kazuyuki; (Germantown, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zamora; Paul O.
Pena; Louis A.
Lin; Xinhua
Takahashi; Kazuyuki |
Gaithersburg
Poquott
Plainview
Germantown |
MD
NY
NY
MD |
US
US
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20080166392 A1 |
July 10, 2008 |
|
|
Family ID: |
34910841 |
Appl. No.: |
12/023801 |
Filed: |
January 31, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11064039 |
Feb 22, 2005 |
7482427 |
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12023801 |
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60547012 |
Feb 20, 2004 |
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Current U.S.
Class: |
424/422 ;
424/426; 514/8.8; 530/324 |
Current CPC
Class: |
C07H 21/04 20130101;
C07K 14/51 20130101; A61K 38/00 20130101; C07K 14/503 20130101;
A61K 47/64 20170801; A61K 38/1709 20130101; C07K 14/475 20130101;
A61P 19/02 20180101; A61P 19/08 20180101; A61K 38/1875 20130101;
A61P 43/00 20180101; A61L 27/34 20130101; C07K 14/4705 20130101;
C07K 2319/00 20130101; A61K 31/727 20130101; A61L 27/34 20130101;
C08L 89/00 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07K 14/475 20060101 C07K014/475; A61K 38/18 20060101
A61K038/18; A61K 38/17 20060101 A61K038/17; A61P 19/08 20060101
A61P019/08 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] This invention was made with United States Government
support under contract number DE-AC02-98CH10886, awarded by the
U.S. Department of Energy. The United States Government has certain
rights in the invention.
Claims
1. A compound of formula: ##STR00015## wherein: X is a peptide
chain that (i) has a minimum of three amino acid residues, (ii) has
a maximum of about fifty amino acid residues, and (iii) binds
specifically to a Bone Morphogenic Protein-2 receptor; R.sub.1 is
independently hydrogen, such that the terminal group is NH.sub.2,
an acyl group with a linear or branched C.sub.1 to C.sub.17 alkyl,
aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or is amino acid, a dipeptide or a tripeptide
with an N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group; R.sub.2
is independently a trifunctional alpha amino acid residue, wherein
X is covalently bonded through a side chain of R.sub.2; R.sub.3 is
independently a linker comprising a chain from 0 to about 15
backbone atoms covalently bonded to R.sub.2; R.sub.4 is OH such
that the terminal group is a carboxyl, NH.sub.2, an acyl group with
a linear or branched C.sub.1 to C.sub.17 alkyl, aryl, heteroaryl,
alkene, alkenyl or aralkyl chain including an N-terminus NH.sub.2,
NH.sub.3.sup.+, or NH group or a corresponding acylated derivative,
or NH--R.sub.1; Y is a linker comprising a chain from 0 to about 50
backbone atoms covalently bonded to R.sub.2 and Z; and Z is a
non-signaling peptide chain that includes a heparin binding domain
comprising an amino acid sequence that comprises (i) a minimum of
one heparin binding motif, (ii) a maximum of about ten heparin
binding motifs, and (iii) a maximum of about thirty amino
acids.
2. The compound of claim 1 wherein Y further comprises a linker
that (i) is hydrophobic, (ii) comprises a chain of a minimum of
about 9 and a maximum of about 50 backbone atoms, and (iii) is not
found in Bone Morphogenic Protein-2.
3. The compound of claim 1 wherein R.sub.2 is an L- or D-diamine
amino acid residue.
4. The compound of claim 3 wherein the L- or D-diamine amino acid
residue is 2,3 diamino propionyl amino acid, 2,4 diamino butylic
amino acid, lysine or ornithine.
5. The compound of claim 1 wherein the X is covalently bonded to
R.sub.2 and wherein the covalent bonds comprise an amide,
disulfide, thioether, Schiff base, reduced Schiff base, imide,
secondary amine, carbonyl, urea, hydrazone or oxime bond.
6. The compound of claim 1 wherein X is covalently bonded to
R.sub.3 when R.sub.3>0 atoms and wherein the covalent bond
comprises an amide, disulfide, thioether, Schiff base, reduced
Schiff base, imide, secondary amine, carbonyl, urea, hydrazone or
oxime bond.
7. The compound of claim 1 wherein Y comprises a straight chain
amino carboxylic acid.
8. The compound of claim 1 wherein X is selected from the group
consisting of SEQ ID NO:7 to SEQ ID NO:44.
9. The compound of claim 1 wherein Z is selected from the group
consisting of SEQ ID NO:1 to SEQ ID NO:6.
10. The compound of claim 1 having the following structure:
##STR00016##
11. A bioactive implant having at least one coating comprising the
compound of claim 1.
12. The use of the compound of claim 1 in the manufacture of a
medicament for the therapeutic and/or prophylactic treatment of
treat bone lesions or degenerative joint conditions.
13. A pharmaceutical composition comprising the compound of claim 1
or a pharmaceutically acceptable salt thereof and a pharmaceutical
carrier.
14. The pharmaceutical composition of claim 13 further comprising a
Bone Morphogenic Protein-2.
15. The use of the compound of claim 1 in the manufacture of a
medicament for the therapeutic or prophylactic treatment of treat
bone lesions or degenerative joint conditions.
16. A method to enhance bone formation or to treat bone lesions or
to treat degenerative joint conditions in a vertebrate animal,
which method comprises administering to a vertebrate subject in
need of such treatment an effective amount of a compound that
augments Bone Morphogenic Protein-2 activity wherein the compound
is a synthetic peptide having a non-growth factor heparin binding
region, a linker and a sequence that binds specifically to a Bone
Morphogenic Protein-2 receptor.
17. The method of claim 16 wherein the compound is of formula:
##STR00017## wherein: X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to a Bone
Morphogenic Protein-2 receptor; R.sub.1 is independently hydrogen,
such that the terminal group is NH.sub.2, an acyl group with a
linear or branched C.sub.1 to C.sub.17 alkyl, aryl, heteroaryl,
alkene, alkenyl or aralkyl chain including an N-terminus NH.sub.2,
NH.sub.3.sup.+, or NH group or a corresponding acylated derivative,
or is amino acid, a dipeptide or a tripeptide with an N-terminus
NH.sub.2, NH.sub.3.sup.+, or NH group; R.sub.2 is independently a
trifunctional alpha amino acid residue, wherein X is covalently
bonded through a side chain of R.sub.2; R.sub.3 is independently a
linker comprising a chain from 0 to about 15 backbone atoms
covalently bonded to R.sub.2; R.sub.4, is OH such that the terminal
group is a carboxyl, NH.sub.2, an acyl group with a linear or
branched C.sub.1 to C.sub.17 alkyl, aryl, heteroaryl, alkene
alkenyl or aralkyl chain including an N-terminus NH.sub.2,
NH.sub.3.sup.+, or NH group or a corresponding acylated derivative,
or NH--R.sub.1; Y is a linker comprising a chain from 0 to about 50
backbone atoms covalently bonded to R.sub.2 and Z; and Z is a
non-signaling peptide chain that includes a heparin binding domain
comprising an amino acid sequence that comprises (i) a minimum of
one heparin binding motif, (ii) a maximum of about ten heparin
binding motifs, and (iii) a maximum of about thirty amino
acids.
18. The method of claim 16 wherein the compound is of formula
##STR00018## wherein: X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to a Bone
Morphogenic Protein-2 receptor; R.sub.1 is independently hydrogen,
such that the terminal group is NH.sub.2 an acyl group with a
linear or branched C.sub.1 to C.sub.17 alkyl, aryl, heteroaryl,
alkene, alkenyl or aralkyl chain including an N-terminus NH.sub.2,
NH.sub.3.sup.+, or NH group or a corresponding acylated derivative,
or is amino acid, a dipeptide or a tripeptide with an N-terminus
NH.sub.2, NH.sub.3.sup.+, or NH group; R.sub.6 is a linker
comprising a chain from 0 to about 15 backbone atoms covalently
bonded to R.sub.5 when the linker is greater than 0 backbone atoms;
R.sub.5 is a trifunctional alpha amino acid residue, wherein X is
covalently bonded through a side chain of R.sub.3; R.sub.4 is OH
such that the terminal group is a carboxyl, NH.sub.2, an acyl group
with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or NH--R.sub.1; Y is a linker comprising a
chain from 0 to about 50 backbone atoms covalently bonded to
R.sub.5 and Z; and Z is a non-signaling peptide chain that includes
a heparin binding domain comprising an amino acid sequence that
comprises (i) a minimum of one heparin binding motif, (ii) a
maximum of about ten heparin binding motifs, and (iii) a maximum of
about thirty amino acids.
19. The method of claim 16, which further comprises administering
to the subject one or more agents that promote bone growth.
20. The method of claim 18, wherein the agents that promote bone
growth are selected from the group consisting of bone morphogenetic
factors, anti-resorptive agents, osteogenic factors,
cartilage-derived morphogenetic proteins, growth hormones,
estrogens, bisphosphonates, statins and differentiating factors.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/164,039, entitled "Positive Modulator of Bone
Morphogenic Protein-2," filed on Feb. 22, 2005, which in turn is a
continuation-in-part application of U.S. patent application Ser.
No. 10/644,703, entitled "Synthetic Heparin-Binding Growth Factor
Analogs," filed on Aug. 19, 2003, which in turn is a
continuation-in-part of U.S. Pat. No. 7,166,574, entitled
"Synthetic Heparin-Binding Growth Factor Analogs," issued on Jan.
23, 2007, and the specification thereof of each is incorporated
herein by reference.
[0002] This application claims the benefit of the filing of U.S.
Provisional Patent Application Ser. No. 60/547,012, entitled
"Positive Modulator of Bone Morphogenic Protein-2," filed on Feb.
20, 2004 and the specification thereof is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention (Technical Field)
[0005] The present invention relates to synthetic growth factor
modulator compositions, particularly modulators of the Bone
Morphogenic Protein (BMP) family. Compositions of the present
invention are of the formulas disclosed herein with a single or
dual chain peptide sequence having specific binding affinity to a
BMP-2 receptor, a linker, optionally a hydrophobic linker, and a
non-growth factor heparin-binding sequence, and methods of use of
synthetic growth factor modulators.
[0006] 2. Background Art
[0007] Note that the following discussion refers to a number of
publications by author(s) and year of publication, and that due to
recent publication dates certain publications are not to be
considered as prior art vis-a-vis the present invention. Discussion
of such publications herein is given for more complete background
and is not to be construed as an admission that such publications
are prior art for patentability determination purposes.
[0008] Bone Morphogenic Proteins (BMPs) are a group of proteins
involved in the development of a wide range of organs and tissues
from embryonic through adult stages, (Wozney J M 2002, Spine 27(16
Suppl 1):S2-8). BMPs also play important roles in tissue repair and
remodeling processes following injuries. Certain BMPs induce
ectopic bone formation and enhance healing of critical-sized
segmental bone defects in animal models. Clinical studies show that
recombinant human BMPs (rhBMPs) are safe and effective alternatives
to autologous bone grafting. rhBMP-2 and rhBMP-7 are approved for
human use in spinal fusion and recalcitrant long-bone nonunions,
respectively. (Kleeman et al. 2001, Spine 26(24):2751-6. Burkus et
al. 2002, Spine 27(21):2396-408. McKay et al. 2002, Spine 27(16
suppl 1):S66-85. Poynton et al. 2002, Spine 27(16 suppl
1):S40-8.)
[0009] The effectiveness of rhBMP-2 seems to heavily depend on the
dose. Significantly higher-than-physiological doses are required
for therapeutic effect in vivo. For example, levels in the
neighborhood of 1 mg/mL of rhBMP-2 are used in spinal fusion cages
(up to 8 mg/cage), an amount three orders of magnitude higher than
what is typically found endogenously. (McKay et al. 2002, Spine
27(16 suppl 1):S66-85.) Administration of such a high dose of
recombinant protein is not only costly, but may also be associated
with adverse effects such as bony overgrowth and immunological
reactions. Therefore, the development of positive modulators of
BMP-2 to enhance BMP activities is of clinical significance.
[0010] BMP-2 signaling involves two types of transmembrane
serine/threonine kinase receptors, namely type I (BRI) and type II
(BRII). (Hoodless et al. 1996, Cell 85(4):489-500. Kawabata et al.
1995, J Biol Chem 270(10):5625-30. Nohno et al. 1995, J Biol Chem
270(38):22522-6. Rosenzweig et al. 1995, Proc Natl Acad Sci USA
92(17):7632-6.) An active ligand/receptor complex consists of
BMP-2, BRI, and BRII in a 2:2:2 ratio. (Reddi A H 2001, J Bone
Joint Surg Am 83-A Suppl 1 (Pt 1):S1-6.) Both type I and type II
receptors are required for BMP-2 to exert its biological functions.
Upon BMP-2 binding, BRI kinase is activated as a result of
phosphorylation by BRII. BRII would not bind to BMP-2 without the
presence of BRI and the complex of BMP-2 and BRII is not capable of
initiating signaling in the absence of BRI. The serine/threonine
kinase in the BRI receptor is believed to be responsible for the
phosphorylation of Smad1, Smad5, and Smad8, which in turn assemble
into heteromeric complexes with Smad4 and translocate into the
nucleus to regulate transcription of target genes. (Massague et al.
2000, Genes Dev 14(6):627-44. Attisano et al. 2000, Curr Opin Cell
Biol 12(2):235-43.) In addition, the activated receptor complexes
can activate the p38 MAP kinase pathway independent of the Smad
pathway. (Iwasaki et al. 1999, J Biol Chem 274(37):26503-10.
Miyazono K 2000, J Cell Sci 113(Pt 7):1101-9.) Currently there are
thought to be two modes for BMP-2 to initiate signaling. Gilboa and
colleagues showed that multiple BMP receptor oligomers are present
at the cell surface prior to ligand binding. (Gilboa et al. 2000,
Mol Biol Cell 11(3):1023-35.) Nohe and colleagues then showed that
the pre-formed receptor complexes are responsible for the BMP-2
induced Smad pathway activation, and BMP-2-induced receptor
complexes initiate the p38 kinase pathway. (Nohe et al. 2002, J
Biol Chem 277(7):5330-8.)
[0011] Some efforts have been made to generate heparin-binding
growth factor analogs. For example, natural platelet-derived growth
factors (PDGF) occur as an A chain and a B chain arranged in
head-to-head (AA or BB) homodimers, or (AB or BA) heterodimers.
Thus, U.S. Pat. No. 6,350,731 to Jehanli et al. discloses PDGF
analogs in which two synthetic PDGF receptor-binding domains are
covalently linked through a polyglycine or an
N-(4-carboxy-cyclohexylmethyl)-maleimide (SMCC) chain to mimic the
natural active polypeptide dimer.
[0012] U.S. Pat. No. 6,235,716 to Ben-Sasson discloses analogs of
angiogenic factors. The analogs are branched multivalent ligands
that include two or more angiogenic homology regions connected by a
multilinker backbone.
[0013] U.S. Pat. No. 5,770,704 (the '704 patent) to Godowski
discloses conjugates for activating receptor tyrosine kinases,
cytokine receptors and members of the nerve growth factor receptor
superfamily. The conjugates include at least two ligands capable of
binding to the cognate receptor, so that the binding of the
respective ligands induces oligomerization of these receptors. The
ligands disclosed in the '704 patent are linked by covalent
attachment to various non-proteinaceous polymers, particularly
hydrophilic polymers, such as polyvinylalcohol and
polyvinylpyrrolidone, and the polyvinylalkene ethers, including
polyethylene glycol and polypropylene glycol. The ligands include
hepatocyte growth factor (HGF) peptide variants that each bind HGF
receptor, thereby causing receptor dimerization and activation of
the biological activity of the HGF receptor dimer.
[0014] U.S. Pat. No. 6,284,503 (the '503 patent) to Caldwell et al.
discloses a composition and method for regulating the adhesion of
cells and biomolecules to hydrophobic surfaces and hydrophobic
coated surfaces for cell adhesion, cell growth, cell sorting and
biological assays. The composition is a biomolecule conjugated to a
reactive end group activated polymer. The end group activated
polymer includes a block copolymer surfactant backbone and an
activation or reactive group. The block copolymer may be any
surfactant having a hydrophobic region capable of adsorbing onto a
hydrophobic surface, and a hydrophilic region which extends away
from the surface when the hydrophobic region is adsorbed onto the
hydrophobic surface. The '503 patent discloses that the
biomolecules that may be conjugated to the end group activated
polymer include natural or recombinant growth factors, such as
PDGF, EGF, TGF.alpha., TGF.beta., NGF, IGF-I, IGF-II, GH and GHRF,
as well as multi-CSF(II-3), GM-CSF, G-CSF, and M-CSF.
[0015] Other workers have described compositions that include
homologs and analogs of fibroblast growth factors (FGFs). See for
example U.S. Pat. No. 5,679,673 to Lappi and Baird; U.S. Pat. No.
5,989,866 to Deisher et al. and U.S. Pat. No. 6,294,359 to Fiddes
et al. These disclosures relate to FGF homologs or analogs that are
either conjugated to a toxic moiety and are targeted to the FGF
receptor-bearing cells; or are homologs or analogs that modulate
the biological pathways through the signal transduced by the FGF
receptor upon binding by the FGF homolog or analog.
[0016] A series of patent applications to Kochendoerfer et al.
disclose polymer-modified proteins, including synthetic chemokines
and erythropoiesis stimulating proteins. See, for example,
International Publications WO 02/04105, WO 02/19963 and WO
02/20033. These include chemically ligated peptide segments of a
polypeptide chain of a synthetic erythropoiesis protein, such that
a polypeptide chain results, with a water soluble polymer attached
at one or more glycosylation sites on the protein. These
applications also disclose synthetic chemokines, which are also
polymer modified, and are asserted to be antagonists. However,
heparin-binding domains are not disclosed. Other erythropoietin
mimetics are known, such as those disclosed in U.S. Pat. Nos.
5,773,569 and 5,830,851 to Wrighton et al.
[0017] International Publication WO 00/18921 to Ballinger and
Kavanaugh discloses a composition consisting of fusion proteins
having FGF receptor affinity linked to an "oligomerization domain",
either directly or through a linking group. The oligomerization
domain ranges in length from about 20 to 300 residues, and includes
constructs such as transcription factors, Fc portions of IgG,
leucine zippers and the like. The oligomerization domains disclosed
are homodimeric domains, wherein a single FGF receptor affinity
fusion protein is linked to a single domain, such as a leucine
zipper, which in turn is linked to a similar molecule by means of
cysteine residues at both the amino and carboxy termini of the
leucine zippers, such that two parallel leucine zippers, each with
a single FGF receptor affinity fusion protein, are cross-linked by
means of disulfide bonds. It is also disclosed that fusion proteins
may include a heparin binding domain, such as the use of jun as a
multimerization domain, which is asserted to be a heparin binding
domain. Thus the compositions disclosed by Ballinger and Kavanaugh
are all composed of a single receptor-binding sequence covalently
attached to an oligomerization domain, whereby two or more similar
oligomerization domains, each with a single receptor-binding
sequence, are conjoined by means of either an association provided
by the oligomerization domain, or alternatively, are chemically
cross-linked to provide for the covalent bonding of the individual
components.
[0018] The above described homologs, analogs, conjugates or ligands
each include a receptor-binding domain. However, none of the
disclosed compounds or compositions further include both a linker,
providing for the linking of two receptor-binding domains to a
dipeptide sequence, and further providing a single non-signaling
peptide containing a heparin-binding domain. Moreover, none of
these or other known heparin-binding growth factor analogs provide
the advantages described herein below. Further, the prior art does
not disclose modulators which, through a synergistic effect,
increase or enhance the efficacy of a naturally occurring growth
factor, such as BMP-2.
BRIEF SUMMARY OF THE INVENTION
[0019] Compounds of the present invention are partial agonists of
bone morphogenic protein 2 (BMP-2), and particularly human BMP-2.
As used herein, "BMP-2" includes specifically human BMP-2, but is
not limited to human BMP-2. Compounds of the present invention
substantially augment the bioactivity of BMP-2. Among other
applications, compounds of the present invention can be employed as
an additive to demineralized bone matrix (DBM) and bone graft
materials to maximize the bioactivity of BMP-2. Compounds of the
present invention augment the bioactivity of BMP-2 found in DBM
(exogenous) and in bone undergoing repair (endogenous). Compounds
of the present invention are preferably made by solid phase peptide
chemistry. The clinical use of compounds of the present invention
provide a new and novel treatment strategy applicable to
accelerating bone repair, among other uses.
[0020] Compounds of the present invention substantially increase
the bio-effectiveness of BMP-2 and significantly decrease the BMP-2
dose threshold. Compounds of the present invention plus BMP-2
result in significant increases of alkaline phosphatase (ALP)
activity at sub-threshold concentrations of BMP-2. Compounds of the
present invention interact directly with BMP receptor isoforms, and
the combination of compounds of the present invention and BMP-2
causes a synergistic repression of mitogen-activated protein kinase
(MAP kinase) and a synergistic increase of Smad activation. The
synergistic increase of Smad activation is hypothesized to be
largely responsible for the observed effect or action of these
compounds on a system. Compounds of the present invention may be
supplied with DBM, for example, with enhanced bone repair
accordingly resulting from a) the augmentation BMP-2 found in DBM,
and b) augmentation of host BMP-2 known to be unregulated in
bone-repair. Similarly, if compounds of the present invention are
supplied in concert with classic osteoconductive materials such as
tricalcium phosphate or calcium sulfate, it can augment host BMP-2
and lead to osteoinduction and increased cellular migration into
the bone fill material. Both approaches take advantage of the fact
that BMP-2 and its receptors are up-regulated during bone repair
processes.
[0021] One embodiment of the present invention is a compound of
formula I:
##STR00001##
[0022] wherein: [0023] X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to a Bone
Morphogenic Protein-2 receptor; [0024] R.sub.1 is independently
hydrogen, such that the terminal group is NH.sub.2, an acyl group
with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or is amino acid, a dipeptide or a tripeptide
with an N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group; [0025]
R.sub.2 is independently a trifunctional alpha amino acid residue,
wherein X is covalently bonded through a side chain of R.sub.2;
[0026] R.sub.3 is independently a linker comprising a chain from 0
to about 15 backbone atoms covalently bonded to R.sub.2; [0027]
R.sub.4 is OH such that the terminal group is a carboxyl, NH.sub.2,
an acyl group with a linear or branched C.sub.1 to C.sub.17 alkyl,
aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or NH--R.sub.1; [0028] Y is a linker
comprising a chain from 0 to about 50 backbone atoms covalently
bonded to R.sub.2 and Z; and [0029] Z is a non-signaling peptide
chain that includes a heparin binding domain comprising an amino
acid sequence that comprises (i) a minimum of one heparin binding
motif, (ii) a maximum of about ten heparin binding motifs, and
(iii) a maximum of about thirty amino acids.
[0030] Yet another embodiment of the present invention is a
bioactive implant containing a coating of formula I. Yet another
embodiment of the present invention is a medicament for the
therapeutic or prophylactic treatment of treat bone lesions or
degenerative joint conditions made from formula I. Still another
embodiment of the present invention is a compound of formula I used
in a pharmaceutical composition and or a pharmaceutically
acceptable salt thereof and a pharmaceutical carrier.
[0031] Another embodiment of the present invention is a compound of
formula II
##STR00002##
[0032] wherein: [0033] X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to a Bone
Morphogenic Protein-2 receptor; [0034] R.sub.1 is independently
hydrogen, such that the terminal group is NH.sub.2, an acyl group
with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or is amino acid, a dipeptide or a tripeptide
with an N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group; [0035]
R.sub.6 is independently a linker comprising a chain from 0 to
about 15 backbone atoms covalently bonded to R.sub.3 when the
linker is greater than 0 atoms; [0036] R.sub.5 is a trifunctional
alpha amino acid residue, wherein X is covalently bonded through a
side chain of R.sub.3; [0037] R.sub.4 is OH such that the terminal
group is a carboxyl, NH.sub.2, an acyl group with a linear or
branched C.sub.1 to C.sub.17 alkyl, aryl, heteroaryl, alkene,
alkenyl or aralkyl chain including an N-terminus NH.sub.2,
NH.sub.3.sup.+, or NH group or a corresponding acylated derivative,
or NH--R.sub.1; [0038] Y is a linker comprising a chain from 0 to
about 50 backbone atoms covalently bonded to R.sub.5 and Z; and
[0039] Z is a non-signaling peptide chain that includes a heparin
binding domain comprising an amino acid sequence that comprises (i)
a minimum of one heparin binding motif, (ii) a maximum of about ten
heparin binding motifs, and (iii) a maximum of about thirty amino
acids.
[0040] Another embodiment of the present invention is a bioactive
implant having at least one coating containing the compound of
formula II.
[0041] Yet another embodiment of the present invention is a
pharmaceutical composition containing the compound of formula II or
a pharmaceutically acceptable salt thereof and a pharmaceutical
carrier.
[0042] Yet another embodiment of the present invention is a method
to enhance bone formation or to treat bone lesions or to treat
degenerative joint conditions in a vertebrate animal, which method
comprises administering to a vertebrate subject in need of such
treatment an effective amount of a compound of formula I or formula
II that augments Bone Morphogenic Protein-2 activity wherein the
compound is a synthetic peptide having a non-growth factor heparin
binding region, a linker and a sequence that binds specifically a
to Bone Morphogenic Protein-2 Receptor.
[0043] One aspect of the present invention provides a synthetic
growth factor modulator.
[0044] Another aspect of the present invention provides a compound
that is a synthetic growth factor analog which is a positive
modulator of BMP-2 activity in vivo.
[0045] Yet another aspect of the present invention provides a
compound that is a positive modulator of BMP-2 activity in
vitro.
[0046] Still another aspect of the present invention provides a
compound that reduces the effective dose of exogenously applied
BMP-2 for therapeutic purposes.
[0047] Another aspect of the present invention is to reduce the
therapeutically effective dose of recombinant BMP delivered to a
subject in need thereof.
[0048] Another aspect of the present invention provides a method
for treating a subject having a bone injury, by providing a
compound of the present invention in combination with a recombinant
member of the BMP family to a fracture site.
[0049] Another aspect of the present invention provides a method
for treating a subject having a bone injury, by providing a
compound of the present invention to a fracture site.
[0050] Another aspect of the present invention provides a method
for treating a subject in need of bone growth, by providing a
compound of the present invention in combination with a recombinant
member of the BMP family to a site in a subject in need of
treatment.
[0051] Another aspect of the present invention provides a method
for treating a subject in need of bone growth, by providing
compound of the present invention to a site in a subject in need of
treatment.
[0052] Another aspect of the present invention provides for kits
containing a compound of the present invention.
[0053] Another aspect of the present invention provides for kits
containing a composition of the present invention.
[0054] Another aspect of the present invention is a bioactive
implantable device containing a compound of the present
invention.
[0055] Other aspects, advantages and novel features, and further
scope of applicability of the present invention will be set forth
in part in the detailed description to follow, taken in conjunction
with the accompanying drawings, and in part will become apparent to
those skilled in the art upon examination of the following, or may
be learned by practice of the invention.
[0056] The objects and advantages of the invention may be realized
and attained by means of the instrumentalities and combinations
particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0057] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate one or more
embodiments of the present invention and, together with the
description, serve to explain the principles of the invention. The
drawings are only for the purpose of illustrating one or more
preferred embodiments of the invention and are not to be construed
as limiting the invention. In the drawings:
[0058] FIGS. 1A and 1B are graphs illustrating that B2A2 enhances
BMP-2 induction of alkaline phosphatase (ALP) activity in C3H10T1/2
cells.
[0059] FIG. 2 is a graph illustrating that B2A2 enhances the
activity of recombinant human BMP-2 obtained from CHO cell and E.
coli commercial production methods.
[0060] FIG. 3 is a graph illustrating that the synergistic effect
of B2A2 was specific to BMP-2.
[0061] FIG. 4 is a graph illustrating the induction of ALP activity
despite the temporal separation of the addition of B2A2 and BMP-2
to the C2C12 cell line.
[0062] FIG. 5 is a graph illustrating that B2A2-coated surfaces
enhanced BMP-2 activity. Surfaces of a variety of compositions were
first coated with silyl heparin under sterile conditions in tissue
culture dishes (a 1% solution in acid ethanol incubated 30 min at
37.degree. C., rinsed with H.sub.2O, dried at 56.degree. C.).
[0063] FIG. 6 is a graph illustrating the relative density from
radiographic image analysis from athymic rats implanted at 3
weeks.
[0064] FIG. 7 is a graph illustrating the relative number of L6
cells in culture after treatment with cytotoxic agents or
B2A2-K--NS.
[0065] FIG. 8 is a graph illustrating the induction of osteogenic
differentiation in C2C12 cells with varying concentrations of
B2A2-K--NS in the presence and absence of BMP-2.
[0066] FIG. 9 is a graph comparing the area of explants excised
from an area implanted with matrigel containing B2A2-K--NK analog
with and without BMP-2.
[0067] FIG. 10 is a graph illustrating Alcian staining of
chondrogenic pathway proteins in C3H10T1/2 cells whose expression
was stimulated by B2A2-K--NS treatment.
[0068] FIG. 11 is a graph illustrating the induction of osteogenic
differentiation in C2C12 cells by B2A7-K--NS in the presence and
absence of suboptimal concentration of BMP-2.
[0069] FIG. 12 is a graph illustrating the specific binding between
a compound of the present invention and BMP-2 Receptor.
[0070] FIG. 13 is a graph illustrating the synergistic action of
BMP-2 and B2A2 binding to BMP-2 receptor.
DETAILED DESCRIPTION OF THE INVENTION
[0071] In a clinical setting, compounds of the present invention
may be supplied with DBM, for example, with enhanced bone repair
accordingly resulting from a) the augmentation BMP-2 found in DBM,
and b) augmentation of host BMP-2 known to be upregulated in
bone-repair. Similarly, if compounds of the present invention are
supplied in concert with classic osteoconductive materials such as
tricalcium phosphate, it can augment host BMP-2 and lead to
osteoinduction and increased cellular migration into the bone fill
material. Both approaches take advantage of the fact that BMP-2 and
its receptors are up-regulated during bone repair processes.
[0072] In keeping with the known activation pathway of BMP-2, it is
hypothesized that compounds of the present invention interact
directly with BMP receptor isoforms (BRI and BRII), and that the
combination of a compound of the present invention and BMP-2 causes
a synergistic repression of mitogen-activated protein kinase (MAP
kinase) and a synergistic increase of Smad activation compared to
using BMP-2 alone. While BMP-2 inhibitors are known, these are the
first known BMP-2 enhancers that functions in the physiological
range.
[0073] Compounds of the present invention interact directly with
BMP receptors to positively modulate BMP-2 induced events leading
to osteogenic differentiation. Synergistic effects between
compounds of the present invention and BMP-2 were observed in two
multipotent cell lines, C3H10T1/2 and C2C12, as determined by at
least two osteogenic differentiation markers, ALP activity and
phosphorylation of Smad. The augmentation of ALP activity at any
given concentration of BMP-2 was generally a 5-20 fold increase.
While researchers have identified other BMP-2 modulators that have
either been negative regulators or agents that fail to work under
normal physiological conditions, compounds of the present invention
are the first peptide specific regulators that positively modulate
BMP-2.
[0074] Recently several BMP-specific antagonists have been
identified. Noggin, chordin, and gremlin have been shown to bind to
BMPs with the same affinity as BMP receptors, and thus
competitively inhibit BMPs. (Zimmerman et al. 1996, Cell
86(4):599-606. Hsu et al. 1998, Mol Cell 1(5):673-83.) In a rat
marrow cell culture, bFGF has been shown to act synergistically
with BMP (Hanada et al. 1997, J Bone Miner Res 12(10):1606-14. Wang
et al. 1993, Acta Orthop Scand 64(5):557-61.), however, higher
doses of bFGF caused profound inhibitory effect in vivo.
Spinella-Jaegle and colleagues reported that Sonic hedgehog (Shh)
enhanced BMP-2 effects in C3H10T1/2 and ST2 cells, but it failed to
enhance BMP-2 activity in analogous osteoprogenitor cells C2C12 and
a preosteoblast cells MC3T3-E1. They further showed that the
enhancing effect appeared to be a priming effect in which Shh
increased the percentage of cells responding to BMP-2
(Spinella-Jaegle S, et al. 2001, J Cell Sci 114(Pt 11):2085-94),
whereas Shh itself is able to induce ALP activity in C3H10T1/2.
(Nakamura et al. 1997, Biochem Biophys Res Commun 237(2):465-9.
Kinto et al. 1997 FEBS Lett 404(2-3):319-23. Katsuura et al. 1999,
FEBS Lett 447(2-3):325-8. Yuasa et al. 2002, J Cell Physiol
193(2):225-32.)
[0075] In another line of investigation, attempts to generate
peptides that possess BMP activity have been less than
satisfactory. Osteoinductive effects were reported by Dee and
colleagues for a stretch of BMP-7 sequence (White et al. 2001, vol.
BED-Vol. 50. American Society of Mechanical Engineers, Snowbird,
Utah, pp 201-202.), and also by Suzuki & Tanihara for two
overlapping stretches of BMP-2 sequence (Saito et al. 2003, Biochim
Biophys Acta 1651(1-2):60-7. Suzuki et al. 2000, J Biomed Mater Res
50(3):405-9.). These results, however, were obtained in supranormal
experimental systems with peptides at extremely high concentrations
and/or covalently attached to a substrate that kept them in contact
with cells for a period of weeks. For example, the linear peptide
reported to have the highest BMP-2-like activity (Saito et al.
2003, Biochim Biophys Acta 1651(1-2):60-7.) works only at
concentrations .about.2,000 times higher than BMP-2--at this level
it completely displaces BMP-2 from cell surface receptors and is
thus a competitor of BMP-2.
[0076] In contrast to prior-art peptides, compounds of the present
invention enhance the activity of BMP-2 and do so in a
concentration range of BMP-2 that can be anticipated in
physiological settings.
[0077] Different sources of BMPs present different attributes to
consider for human applications. BMPs have been purified from bone,
but with very low yields, and potential health risks associated
with isolation from allogenic donor bone also limit clinical
application of BMP from this source. Most of the BMP in clinical
use is recombinant protein obtained from eukaryotic cell culture.
Complications of post-translation modification and low yield result
in a very high cost of these recombinant proteins. Moreover, the
amounts required for efficacy in human applications turned out to
be unexpectedly high (McKay et al. 2002, Spine 27(16 suppl
1):S66-85. Poynton et al. 2002, Spine 27(16 suppl 1):S40-8.).
[0078] A BMP-specific enhancer, such as that disclosed herein, has
unique clinical significance. A BMP-2 enhancer may be used to
reduce the amounts of BMP-2 required. This is of medical and
practical significance because as a synthetic peptide, compounds of
the present invention are (a) less expensive to produce, (b) vastly
more chemically stable, and (c) easy to chemically modify for
enhanced drug delivery. Biologically, there are also other
advantages. For example, the process of spinal fusion involves a
sequence of events associated with a temporal and spatial pattern
of osteogenic-related gene expression. Morone and colleagues
(Morone et al. 1998, Clin Orthop (351):252-65.) studied the
expression of the mRNA of several BMPs in spinal fusion and found
that BMP-2 and others were increased at different levels at
different times. It is daunting to match exogenous application
recombinant BMP-2 to the biologically optimal schedule. Similarly,
BMPs can occur as homo- and heterodimers. A BMP enhancer may thus
be effective by augmenting the natural endogenous expression of
BMPs as they occur in situ.
[0079] Compounds of the present invention can thus be used to
reduce the effective dose of recombinant BMP-2 on or associated
with medical devices, to maximize the biological activity of
biological preparations like demineralized bone matrix (DMB), and
to augment the endogenous levels of BMP-2 generated by host tissue
during bone healing process.
[0080] DBM is one alternative material that is bone-derived and
widely used in clinical practice. DBM is processed from human bone
via solvent and acid treatments, and in its final form contains
collagens and low levels of growth factors. DBM is available from a
number of companies and organizations, including Wright Medical
Technologies, Osteotech, the American Red Cross, and Innova. DBM,
via the collagen component, provides a scaffold on which new bone
forms and it also has some osteoinductive potential via its low
levels of growth factors. It may also elicit some activation of
mesenchymal stem cells from the surrounding area that differentiate
into osteoblasts.
[0081] The osteoinductive potential of DBM is low, however, and
varies widely from lot-to-lot and manufacturer-to-manufacturer.
Since the growth factors in DBM are expected to have their most
pronounced effect on osteoprogenitor cells, the availability of
osteoprogenitor cells is critical when demineralized bone matrix is
used. The limited ability of DBM to elicit a robust osteoinduction
is widely seen as a limiting factor in the use of this
material.
[0082] Among the calcium-rich bone graft materials, there are a
large number of commercially available products bone filler agents
that are not derived from human sources, including Pro Osteon.TM.
(coralline hydroxyappatite, Interpore Cross International),
Bioglass.TM. (bioactive glass implant, US Biomaterials Corp.),
Collagraft.TM. (hydroxyapatite/tricalcium phosphate and pure bovine
fibrillar collagen, Zimmer), Cellplex.TM. (tricalcium phosphate,
synthetic cancellous bone, Wright Medical Technologies, Inc.), and
a number of calcium phosphate and calcium phosphate fillers. All of
these materials are osteoconductive and support the in-growth of
capillaries, perivascular tissues, and osteoprogenitor cells from a
host into an implant or graft. They are not, however,
osteoinductive.
[0083] Among the biologics, a number of companies have developed
bone fill products that are intended to be used with autologous
bone marrow cells or platelet concentrates. These products are
intended to increase the number of stem cells in a graft or to
increase the amount of growth factors, respectively.
[0084] In a related vein, the InFuse.TM. spinal cage product
(Sofamor-Danek, a division of Medtronic) is an example of a device
that combines a osteoconductive material (collagen) with an
osteoinductive agent. InFuse.TM. is indicated for use in
conjunction with spinal fusion procedures, and a similar product is
being developed for fresh fracture repair.
[0085] The success of InFuse.TM., and to a lesser extent, Stryker
Corporation's OP-1.TM. for use in tibial non-unions, has led to a
high level of interest in recombinant growth factor approaches.
Numerous additional growth factors are being evaluated in the
orthopedic and ortho-biologic fields. Yet among the various BMPs,
BMP-2 appears to be the factor with the highest degree of
osteoinduction.
[0086] There is thus an increasing clinical demand for bone graft
materials and a high level of interest in alternatives to growth
factors or improvements in existing bone graft materials.
DEFINITIONS
[0087] As used here and elsewhere, the following terms have the
meanings given.
[0088] The term "alkene" includes unsaturated hydrocarbons that
contain one or more double carbon-carbon bonds. Examples of such
alkene groups include ethylene, propene, and the like.
[0089] The term "alkenyl" includes a linear monovalent hydrocarbon
radical of two to six carbon atoms or a branched monovalent
hydrocarbon radical of three to six carbon atoms containing at
least one double bond; examples thereof include ethenyl,
2-propenyl, and the like.
[0090] The "alkyl" groups specified herein include those alkyl
radicals of the designated length in either a straight or branched
configuration. Examples of such alkyl radicals include methyl,
ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl,
isopentyl, hexyl, isohexyl, and the like.
[0091] The term "aryl" includes a monovalent or bicyclic aromatic
hydrocarbon radical of 6 to 12 ring atoms, and optionally
substituted independently with one or more substituents selected
from alkyl, haloalkyl, cycloalkyl, alkoxy, alkylhio, halo, nitro,
acyl, cyano, amino, monosubstituted amino, disubstituted amino,
hydroxy, carboxy, or alkoxy-carbonyl. Examples of an aryl group
include phenyl, biphenyl, naphthyl, 1-naphthyl, and 2-naphthyl,
derivatives thereof, and the like.
[0092] The term "aralkyl" includes a radical--R.sup.aR.sup.b where
R.sup.a is an alkylene (a bivalent alkyl) group and R.sup.b is an
aryl group as defined above. Examples of aralkyl groups include
benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the
like. The term "aliphatic" includes compounds with hydrocarbon
chains, such as for example alkanes, alkenes, alkynes, and
derivatives thereof.
[0093] The term "acyl" includes a group RCO--, where R is an
organic group. An example is the acetyl group CH.sub.3CO--.
[0094] A peptide or aliphatic moiety is "acylated" when an alkyl or
substituted alkyl group as defined above is bonded through one or
more carbonyl {--(C.dbd.O)--} groups. A peptide is most usually
acylated at the N-terminus.
[0095] An "amide" includes compounds that have a trivalent nitrogen
attached to a carbonyl group (--CO.NH.sub.2).
[0096] An "amine" includes compounds that contain an amino group
(--NH.sub.2).
[0097] A "diamine amino acid" is an amino acid or residue
containing two reactive amine groups and a reactive carboxyl group.
Representative examples include 2,3 diamino propionyl amino acid,
2,4 diamino butylic amino acid, lysine or ornithine.
[0098] A "trifunctional amino acid" is an amino acid or residue
with three reactive groups, one the N-terminus amine, a second the
C-terminus carboxyl, and the third comprising all or a part of the
side chain. Trifunctional amino acids thus include, by way of
example only, diamine amino acids; amino acids with a reactive
sulfhydryl group in the side chain, such as mercapto amino acids
including cysteine, penicillamine, or 3-mercapto phenylalanine;
amino acids with a reactive carboxyl group in the side chain, such
as aspartic acid and glutamic acid; and amino acids with a reactive
guanadium group in the side chain, such as arginine.
Compounds of the Present Invention
[0099] According to one embodiment of the present invention,
compounds are of formula I:
##STR00003##
[0100] wherein: [0101] X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to Bone
Morphogenic Protein-2 receptor; [0102] R.sub.1 is independently a
hydrogen, such that the terminal group is NH.sub.2, an acyl group
with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or is amino acid, a dipeptide or a tripeptide
with an N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group; [0103]
R.sub.2 is independently a trifunctional amino acid residue,
wherein X is covalently bonded through a side chain of R.sub.2;
[0104] R.sub.3 is independently a linker comprising a chain from 0
to about 15 backbone atoms covalently bonded to R.sub.2; [0105]
R.sub.4 is OH such that the terminal group is a carboxyl, NH.sub.2,
an acyl group with a linear or branched C.sub.1 to C.sub.17 alkyl,
aryl, heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or NH--R.sub.1; [0106] Y is a linker
comprising a chain from 0 to about 50 backbone atoms covalently
bonded to R.sub.2 and Z; and [0107] Z is a non-signaling peptide
chain that includes a heparin binding domain comprising an amino
acid sequence that comprises (i) a minimum of one heparin binding
motif, (ii) a maximum of about ten heparin binding motifs, and
(iii) a maximum of about thirty amino acids. According to another
embodiment of the present invention compounds are of formula
II:
##STR00004##
[0108] wherein: [0109] X is a peptide chain that (i) has a minimum
of three amino acid residues, (ii) has a maximum of about fifty
amino acid residues, and (iii) binds specifically to Bone
Morphogenic Protein-2 receptor; [0110] R.sub.1 is independently a
hydrogen, such that the terminal group is NH.sub.2, an acyl group
with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or is amino acid, a dipeptide or a tripeptide
with an N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group; [0111]
R.sub.6 is independently a linker comprising a chain from 0 to
about 15 backbone atoms covalently bonded to R.sub.5; [0112]
R.sub.5 is a trifunctional amino acid residue, wherein a first X is
covalently bonded through a side chain of R.sub.5 and a second X is
covalently bonded through the N-terminus amine; [0113] R.sub.4 is
OH such that the terminal group is a carboxyl, NH.sub.2, an acyl
group with a linear or branched C.sub.1 to C.sub.17 alkyl, aryl,
heteroaryl, alkene, alkenyl or aralkyl chain including an
N-terminus NH.sub.2, NH.sub.3.sup.+, or NH group or a corresponding
acylated derivative, or NH--R.sub.1; [0114] Y is a linker
comprising a chain from 0 to about 50 atoms covalently bonded to
R.sub.2 and Z; and [0115] Z is a non-signaling peptide chain that
includes a heparin binding domain comprising an amino acid sequence
that comprises (i) a minimum of one heparin binding motif, (ii) a
maximum of about ten heparin binding motifs, and (iii) a maximum of
about thirty amino acids.
[0116] In each of formula I and formula II, the covalent bonds can
be, for example, a peptide bond or other amide bond, a thioether
bond or ester bond. A group is covalently bonded to another group
when it is, directly or through one or more other groups or atoms
comprising covalent bonds, covalently bonded.
[0117] The chain of atoms of the Y region of formula I is
covalently attached to R.sub.2 and to sequence Z, and in formula II
the Y region is covalently attached to R.sub.5 and to sequence Z.
The covalent bonds can be, for example, peptide, amide, thioether
or ester bonds. Particularly preferred is a peptide bond.
Preferably, the Y region includes a chain of a minimum of about
nine backbone atoms. More preferably, the Y region includes a chain
of a minimum of about twelve backbone atoms. Most preferably, the Y
region includes a chain of a minimum of about fifteen backbone
atoms. For example, the Y region may be formed from a chain of at
least four, at least five or at least six amino acids.
Alternatively, the Y region may be formed from a chain of at least
one, at least two, or at least three amino carboxylic acids, such
as aminohexanoic acid residues. Particularly preferred are
embodiments in which Y is one or more straight chain amino
carboxylic acids, such as where Y comprises
[NH.sub.2--(CH.sub.2).sub.pCO].sub.q wherein p is from 1 to about
10 and q is from 1 to about 20. Examples of straight chain amino
carboxylic acids that may be employed include 6-aminohexanoic acid,
7-aminoheptanoic acid, 9-aminononanoic acid and the like.
[0118] Preferably, the Y region includes a chain of a maximum of
about fifty atoms. More preferably, the Y region includes a chain
of a maximum of about forty-five atoms. Most preferably, the Y
region includes a chain of a maximum of about thirty-five atoms.
For example, the Y region may be formed from a chain of up to about
twelve, up to about fifteen, or up to about seventeen amino
acids.
[0119] The amino acid sequence of the Y region 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 BMP receptor.
[0120] In a particular embodiment, the Y region includes a
hydrophobic amino acid residue, or a chain of hydrophobic amino
acid residues. The Y region can, for example, include one or more
amino carboxylic acid residues, such as one, two, three or more
aminohexanoic acid residues. In another alternative embodiment, the
Y region can include a combination of amino acid hydrophobic
residues.
[0121] In another particular embodiment, the Y region of the
molecule can include a branched or unbranched, saturated or
unsaturated alkyl chain of between one and about twenty carbon
atoms. In a further embodiment, the Y region can include a chain of
hydrophilic residues, such as for instance, ethylene glycol
residues. For instance, the Y region can include at least about
three, or at least about four, or at least about five ethylene
glycol residues.
[0122] The Z region of the molecule of formula I and formula II is
a heparin-binding region and can include one or more
heparin-binding motifs, BBxB or BBBxxB as described by Verrecchio
et al. J. Biol. Chem. 275:7701 (2000). Alternatively, the Z region
can include both BBxB and BBBxxB motifs (where B represents lysine,
arginine, or histidine, and x represents a naturally occurring, or
a non-naturally occurring amino acid). For example, the
heparin-binding motifs may be represented by the sequence
[KR][KR][KR]X(2)[KR] (SEQ ID NO:1), designating the first three
amino acids as each independently selected from lysine or arginine,
followed by any two amino acids and a sixth amino acid which is
lysine or arginine.
[0123] The number of heparin-binding motifs is variable. For
instance, the Z region may include at least one, at least two, at
least three or up to at least five heparin-binding motifs. Where
there are more than one heparin-binding motifs, the motifs may be
the same or different. Alternatively, the Z region includes up to a
maximum of about ten heparin-binding motifs. In another alternative
embodiment, the Z region includes at least four, at least six or at
least eight amino acid residues. Further, in certain embodiments
the Z region includes up to about twenty, up to about twenty-five,
or up to about thirty amino acid residues. It is to be realized
that, in part, the avidity of the Z region for heparin is
determined by the particular heparin-binding motifs selected and
the number of such motifs in Z. Thus for particular applications
both the selection and number of such motifs may be varied to
provide optimal heparin binding of the Z region.
[0124] In a preferred embodiment, the amino acid sequence of the Z
region is RKRKLERIAR (SEQ ID NO:2). In another embodiment, the
amino acid sequence of the Z region is RKRKLGRIAR (SEQ ID NO:3). In
yet another embodiment, the amino acid sequence of the Z region is
RKRKLWRARA (SEQ ID NO:4). In yet another embodiment, the amino acid
sequence of the Z region is RKRLDRIAR (SEQ ID NO:5), providing a
heparin-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 Oncoprotein-mediated Transcription. J. Cell Biol.
116:31-42, 1992). In yet another embodiment, the amino acid
sequence of the Z region is RKRKLERIARC (SEQ ID NO:6). The presence
of a terminal cysteine residue optionally affords the opportunity
to link other molecules, including detection reagents such as
fluorochromes, radioisotopes and other detectable markers, to the Z
region, as well as the opportunity to link toxins, immunogens and
the like.
[0125] The synthetic bone morphogenic protein analogs of the
present invention, including those of formulas I and II, include
embodiments wherein the X region is all or a portion, or a homolog
of all or a portion, of any of the following amino acid
sequences:
[0126] AISMLYLDENEKVVL (SEQ ID NO:7)
[0127] ISMLYLDENEKVVLKNY (SEQ ID NO:8),
[0128] LYFDESSNVILKK (SEQ ID NO:9),
[0129] LYVDFSDVGWNDW (SEQ ID NO:10),
[0130] EKWLKNYQDMVVEG (SEQ ID NO:11),
[0131] CAISMLYLDENEKVVL (SEQ ID NO:12),
[0132] AFYCHGECPFPLADHL (SEQ ID NO:13),
[0133] PFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:14), or
In a preferred embodiment the X region is the amino acid sequence
ISMLYLDENEKVVLKNY (SEQ ID NO:8). More preferably the X region is
the amino acid sequence LYFDESSNVILKK (SEQ ID NO:9). More
preferably still, the X region is the amino acid sequence
AISMLYLDENEKVVL (SEQ ID NO:7).
[0134] The inventors have surprisingly and advantageously found
that in the compounds of the present invention, including those of
formulas I and II, the X region may be synthesized in a reverse
direction, such that considering the sequence AISMLYLDENEKVVL (SEQ
ID NO:7) illustrated in the conventional N.fwdarw.C orientation,
and using formula I, the first amino acid bound to the R.sub.2 side
chains is the N-terminus amino acid residue, the second amino acid
bound to the N-terminus amino acid residue is the 2 position
residue, and so on, and the compounds nonetheless retain biological
activity and specifically bind to a BMP receptor. It may be seen
that such a construct has, based on a conventional N.fwdarw.C
orientation, a reverse sequence, in that it is the carboxyl group
of the conventional N-terminus amino acid residue that forms a
peptide bond with the epsilon amine where R.sub.2 is a diamine
amino acid. Thus again employing a conventional N.fwdarw.C
orientation, the foregoing sequences may be employed in a reverse
orientation, and the resulting compound of present invention is
biologically active and may be employed as described herein.
According to a preferred embodiment, the X region is the sequence
LVVKENEDLYLMSIA (SEQ ID NO:15) (again considering the sequence in
the conventional N.fwdarw.C orientation), as disclosed in Example 2
herein. As described in Example 2, the C-terminus alanine (A) is
bound to the epsilon amine of a lysine (K) in the R.sub.2 position
of formula I, the isoleucine (I) is bound by a peptide bond to the
alanine, and so on. Thus the following sequence is provided, and is
biologically active, as described herein:
##STR00005##
[0135] Other reverse sequences that may be employed, in whole or in
part, including homologs thereto, in addition to LVVKENEDLYLMSIA
(SEQ ID NO:15), include but are not limited to YNKLVVKENEDLYLMSI
(SEQ ID NO:16), KKLIVNSSEDFYL (SEQ ID NO:17), WDNWGVDSFDVYL (SEQ ID
NO:18), GEVVMDQYNKLWKE (SEQ ID NO:19), LHDALPFPCEGHCYFA (SEQ ID
NO:20), VSNVLTQVIAHNTSNLHDALPFP (SEQ ID NO:21), and
LVVKENEDLYLMSIAC (SEQ ID NO:22).
[0136] Alternatively, in another particular aspect the invention
provides synthetic BMP, TGF or GDF (growth differentiation factor)
peptide analogs with sequences as shown in Table 1 wherein the
transforming growth factor family member peptides are particularly
useful in augmenting the activity of endogenous or artificial BMP
peptides or TGF peptides, wherein is shown (under the heading
"preferred X receptor binding domain") the sequence forming all or
part of the X region of constructs of any of I or II. It is to be
understood that some or only a portion of any sequence listed under
the heading "preferred X receptor binding domain" may be employed,
and thus the X region employed may be a subset of any sequence
listed below. It is further to be understood that the X sequence
need not be identical to all or a portion of a sequence listed
below, and may be homologous with all or a portion, such as a
sequence that is 80% to 95% homologous.-*
TABLE-US-00001 TABLE 1 CYTOKINE PREFERRED X RECEPTOR BINDING DOMAIN
TGF-.beta.1 IVYYVGRKPKVEQLSNMIVRS (SEQ ID NO:23) TGF-.beta.2
TILYYIGKTPKIEQLSNMIVKS (SEQ ID NO:24) TGF-.beta.3
LTILYYVGRTPKVEQLSNMVV (SEQ ID NO:25) BMP-2 AISMLYLDENEKVVLKNYQDMVV
(SEQ ID NO:26) BMP-3 SSLSILFFDENKNVVLKVYPNMTV (SEQ ID NO:27)
BMP-.beta.3 NSLGVLFLDENRNVVLKVYPNMSV (SEQ ID NO:28) BMP-4
AISMLYLDEYDKVVLKNYQEMVV (SEQ ID NO:29) BMP-5
AISVLYFDDSSNVILKKYRNMVV (SEQ ID NO:30) BMP-6
AISVLYFDDNSNVILKKYRNMVV (SEQ ID NO:31) BMP-7
AISVLYFDDSSNVILKKYRNMVV (SEQ ID NO:32) BMP-8
ATSVLYYDSSNNVILRKARNMVV (SEQ ID NO:33) BMP-9
ISVLYKDDMGVPTLKYHYEGMSV (SEQ ID NO:34) BMP-10
ISILYLDKGVVTYKFKYEGMAV (SEQ ID NO:35) BMP-11 INMLYFNDKQQIIYGKIPGMVV
(SEQ ID NO:36) BMP-12 ISILYIDAANNVVYKQYEDMVV (SEQ ID NO:37) BMP-13
ISILYIDAGNNVVYKQYEDMVV (SEQ ID NO:38) BMP-14 ISILFIDSANNVVYKQYEDMVV
(SEQ ID NO:39) BMP-15 ISVLMIEANGSILYKEYEGMIA (SEQ ID NO:40) GDF-1
ISVLFFDNSDNVVLRQYEDMVV (SEQ ID NO:41) GDF-3 ISMLYQDNNDNVILRHYEDMVV
(SEQ ID NO:42) GDF-8 INMYLFNGKEQIIYGKIPAMVV (SEQ ID NO:43) GDF-9
LSVLTIEPDGSIAYKEYEDMIA (SEQ ID NO:44)
[0137] The term "homologous", as used herein refers to peptides
that differ in amino acid sequence at one or more amino acid
positions when the sequences are aligned. For example, the amino
acid sequences of two homologous peptides can differ only by one
amino acid residue within the aligned amino acid sequences of five
to ten amino acids. Alternatively, two homologous peptides of ten
to fifteen amino acids can differ by no more than two amino acid
residues when aligned. In another alternative, two homologous
peptides of fifteen to twenty or more amino acids can differ by up
to three amino acid residues when aligned. For longer peptides,
homologous peptides can differ by up to approximately 5%, 10%, or
20% of the amino acid residues when the amino acid sequences of the
two peptide homologs are aligned.
[0138] Particularly useful amino acid sequences as X regions of
formulas I or II include homologs of fragments of naturally
occurring sequences that differ from the amino acid sequences of
natural growth factor in only one or two or a very few positions.
Such sequences preferably include conservative changes, where the
original amino acid is replaced with an amino acid of a similar
character according to well known principles; for example, the
replacement of a non-polar amino acid such as alanine with valine,
leucine, isoleucine or proline; or the substitution of one acidic
or basic amino acid with another amino acid of the same acidic or
basic character.
[0139] The R.sub.3 regions of formula I or the R.sub.6 regions of
formula II can include a chain of atoms or a combination of atoms
that form a chain. Typically, the chains are chains primarily of
carbon atoms, that may also optionally include oxygen or nitrogen
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 found in proteins, such as ornithine and
citrulline; or non-natural amino acids, such as aminohexanoic acid;
or a combination of any of the foregoing amino acids). It is also
contemplated that agents such as polyethylene glycol (PEG),
polyethylene oxide (PEO), amino polyethylene glycol, bis-amine-PEG,
and other variants of polyethylene glycol known to those skilled in
the art can similarly be used. Particularly preferred for the
R.sub.3 or R.sub.6 regions are chains which include an amino
terminal and a carboxyl terminal, such that the chains may be
utilized in standard peptide synthesis methodologies. Examples
include any amino acids, amino carboxylic acids, preferably
straight chain amino carboxylic acids, and bifunctional
amino-PEG-acid spacers. Among amino acids, glycine is
preferred.
[0140] In certain embodiments of the invention, each of the R.sub.3
regions of formula I or each of the R.sub.6 regions of formula II
can be different, although in most embodiments it is preferred that
the regions be identical. However, it is contemplated that such
regions may differ; for example, in formula II the R.sub.5 may be a
diamine amino acid, such as lysine. It is possible to utilize an
orthogonal protecting group during synthesis to protect either the
alpha amine or epsilon amine, to thereafter add one or amino acid
residues or other groups to form an R.sub.6 group, and then to
remove the orthogonal protecting group, and proceed with parallel
synthesis of the X groups from the deprotected amine on R.sub.5 and
the terminal amine on R.sub.6. Similar methods may be employed with
formula I.
Methods of Synthesizing the Compounds of the Present Invention
[0141] The synthesis of the compounds of the present invention can
be achieved by any of a variety of chemical methods well known in
the art. Such methods include bench scale solid phase synthesis and
automated peptide synthesis in any one of the many commercially
available peptide synthesizers. Preferably, the synthesizer has a
per cycle coupling efficiency of greater than 99 percent.
[0142] The compounds of the present invention can be produced by
stepwise synthesis or by synthesis of a series of fragments that
can be coupled by similar well known techniques. See, for instance,
Nyfeler, Peptide synthesis via fragment condensation. Methods Mol.
Biol. 35:303-16 (1994); and Merrifield, Concept and early
development of solid-phase peptide synthesis. Methods in Enzymol.
289:3-13 (1997). These methods are routinely used for the
preparation of individual peptides. It is possible to assemble the
analogs of the present invention in component parts, such as
peptides constituting the X, Y and Z components thereof, and to
thereafter couple such component parts to assemble the analog. See,
for instance, Dawson and Kent, Synthesis of native proteins by
chemical ligation. Annu. Rev. Biochem. 69:923-960 (2000); and Eom
et al., Tandem ligation of multipartite peptides with
cell-permeable activity. J. Am. Chem. Soc. 125:73-82 (2003).
However, in a preferred embodiment the compounds of the present
invention are synthesized by solid phase synthesis, with the
C-terminus residue of the Z region of formulas I or II bound to
resin, and the synthesis proceeding stepwise. Conventional
protecting groups are employed as required, with deprotection
either prior to, during or following cleavage of the peptide from
the resin. By way of example only, for compounds of the present
invention containing one or more lysine residues in addition to any
at the R.sub.2 position of formula I or the R.sub.5 position of
formula II, such additional lysine residues are conventionally
protected with a protecting group, and deprotected following
synthesis.
Methods of Use of the Compounds of the Present Invention
[0143] The compounds of the present invention provide a cost
effective source of biologically active molecules that are useful
in a number of ways, including as soluble prophylactic or
therapeutic pharmaceutical agents.
[0144] The compounds of the present invention are also useful as
biologically active agents as components of medical devices and for
coating of medical devices, such as for instance, sutures, implants
and medical instruments to promote biological responses, for
instance, to stimulate growth and proliferation of cells, or
healing of wounds.
[0145] In one aspect, the invention provides a method and
compositions for treating a mammal with bone injury, by providing a
compounds of the present invention, such as an analog of BMP-2. For
example, such compounds of the present invention may be
administered as a pharmaceutical agent, or may be employed as an
additive to bone matrix or bone graft materials.
[0146] The term "medical device" as used herein means a device that
has one or more surfaces in contact with an organ, tissue, blood or
other bodily fluid in an organism, preferably a mammal,
particularly, a human. Medical devices include, for example,
extracorporeal devices for use in surgery such as blood
oxygenators, blood pumps, blood sensors, tubing used to carry
blood, and the like which contact blood that is returned to the
patient. The term can also include endoprostheses implanted in
blood contact in a human or animal body, such as vascular grafts,
stents, pacemaker leads, heart valves, aneurism coils, and the like
that are implanted in blood vessels or in the heart. The term can
further include devices for temporary intravascular use such as
catheters, guide wires, and the like that are placed in blood
vessels or the heart for purposes of monitoring or repair. The term
can further include nerve electrodes, muscle electrodes,
implantable pulse generators, implantable drug pumps, and
defibrillators. Moreover, the term medical device can include
sutures, graft materials, wound coverings, nerve guides, bone wax,
embolization particles, microbeads, dental implants, bone
prostheses, bone graft materials, spinal fusion cages, bone
fillers, orthopedic devices, tissue scaffolds, artificial joints or
controlled release drug delivery devices.
[0147] The surface of the medical device can be formed from any of
the commonly used materials suitable for use in medical devices,
such as for instance, stainless steel, titanium, platinum,
tungsten, ceramics, polyurethane, polytetrafluoroethylene, extended
polytetrafluoroethylene, polycarbonate, polyester, polypropylene,
polyethylene, polystyrene, polyvinyl chloride, polyamide,
polyacrylate, polyurethane, polyvinyl alcohol, polycaprolactone,
polylactide, polyglycolide, polysiloxanes (such as
2,4,6,8-tetramethylcyclotetrasiloxane), natural rubbers, or
artificial rubbers, or block polymers or copolymers thereof.
[0148] Methods for coating biological molecules onto the surfaces
of medical devices are known. See for instance U.S. Pat. No.
5,866,113 to Hendriks et al., the specification of which is hereby
incorporated by reference. Tsang et al. in U.S. Pat. No. 5,955,588
teach a non-thrombogenic coating composition and methods for using
the same on medical devices, and is incorporated herein by
reference. Zamora et al. in U.S. Pat. No. 6,342,591 teach an
amphipathic coating for medical devices for modulating cellular
adhesion composition, and is incorporated herein by reference.
[0149] The compounds of the present invention can be used for as an
active ingredient in pharmaceutical compositions for both medical
applications and animal husbandry or veterinary applications.
Typically, the compound of the present invention or pharmaceutical
composition is used in humans, but may also be used in other
mammals. The term "patient" is intended to denote a mammalian
individual, and is so used throughout the specification and in the
claims. The primary applications of this invention involve human
patients, but this invention may be applied to laboratory, farm,
zoo, wildlife, pet, sport or other animals.
[0150] The compounds of the present invention may be in the form of
any pharmaceutically acceptable salt. The term "pharmaceutically
acceptable salts" refers to salts prepared from pharmaceutically
acceptable non-toxic bases or acids including inorganic or organic
bases and inorganic or organic acids. Salts derived from inorganic
bases include aluminum, ammonium, calcium, copper, ferric, ferrous,
lithium, magnesium, manganic salts, manganous, potassium, sodium,
zinc, and the like. Particularly preferred are the ammonium,
calcium, lithium, magnesium, potassium, and sodium salts. Salts
derived from pharmaceutically acceptable organic non-toxic bases
include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines, and basic ion exchange resins, such as
arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine,
diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
ethanolamine, ethylenediamine, N-ethyl-morpholine,
N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine,
piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, tromethamine, and
the like.
[0151] When the compounds of the present invention are basic, acid
addition salts may be prepared from pharmaceutically acceptable
non-toxic acids, including inorganic and organic acids. Such acids
include acetic, benzenesulfonic, benzoic, camphorsulfonic,
carboxylic, citric, ethanesulfonic, formic, fumaric, gluconic,
glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic,
malic, mandelic, methanesulfonic, malonic, mucic, nitric, pamoic,
pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric,
p-toluenesulfonic acid, trifluoroacetic acid, and the like. Acid
addition salts of the compounds of the present invention are
prepared in a suitable solvent for the compound and an excess of an
acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric,
acetic, trifluoroacetic, citric, tartaric, maleic, succinic or
methanesulfonic acid. The acetate salt form is especially useful.
Where the compounds of the present invention include an acidic
moiety, suitable pharmaceutically acceptable salts may include
alkali metal salts, such as sodium or potassium salts, or alkaline
earth metal salts, such as calcium or magnesium salts.
[0152] The invention provides a pharmaceutical composition that
includes a compounds of the present invention and a
pharmaceutically acceptable carrier. The carrier may be a liquid
formulation, and in one embodiment a buffered, isotonic, aqueous
solution. Pharmaceutically acceptable carriers also include
excipients, such as diluents, carriers and the like, and additives,
such as stabilizing agents, preservatives, solubilizing agents,
buffers and the like, as hereafter described.
[0153] Thus the compounds of the present invention may be
formulated or compounded into pharmaceutical compositions that
include at least one compounds of the present invention together
with one or more pharmaceutically acceptable carriers, including
excipients, such as diluents, carriers and the like, and additives,
such as stabilizing agents, preservatives, solubilizing agents,
buffers and the like, as may be desired. Formulation excipients may
include polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
PEG, PEO, mannitol, sodium chloride or sodium citrate, as well as
any number of simple sugars, including sucrose, dextrose, lactose
and the like, and combinations of the foregoing. For injection or
other liquid administration formulations, water containing at least
one or more buffering constituents is preferred, and stabilizing
agents, preservatives and solubilizing agents may also be employed.
For solid administration formulations, any of a variety of
thickening, filler, bulking and carrier additives may be employed,
such as starches, sugars, fatty acids and the like. For topical
administration formulations, any of a variety of creams, ointments,
gels, lotions and the like may be employed. For most pharmaceutical
formulations, non-active ingredients will constitute the greater
part, by weight or volume, of the preparation. For pharmaceutical
formulations, it is also contemplated that any of a variety of
measured-release, slow-release or time-release formulations and
additives may be employed, so that the dosage may be formulated so
as to effect delivery of a compounds of the present invention over
a period of time.
[0154] In practical use, the compounds of the present invention can
be combined as the active ingredient in an admixture with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, for example, oral, parenteral (including
intravenous), urethral, vaginal, nasal, buccal, sublingual, or the
like. In preparing the compositions for oral dosage form, any of
the usual pharmaceutical media may be employed, such as, for
example, water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like in the case of oral
liquid preparations, such as, for example, suspensions, elixirs and
solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid
preparations such as, for example, powders, hard and soft capsules
and tablets. The pharmaceutical forms suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that it may be administered by
syringe. The form must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, a polyol, for example glycerol, propylene
glycol or liquid polyethylene glycol, suitable mixtures thereof,
and vegetable oils.
[0155] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
[0156] Materials. C2C12 cells and C3H10T1/2 cells were purchased
from American Type Culture Collection (Manassas, Va.). E. coli or
Chinese hamster ovary (CHO) cell-derived recombinant human BMP-2
were purchased from R&D Systems (Minneapolis, Minn.). Soluble
BMP-2 receptors as the recombinant BRI-Fc chimeric proteins were
also obtained from R&D Systems. Endostatin-Fc, FGF-2, and VRGF
were supplied by through the Biological Resources Branch of
Developmental Therapeutics Program, National Cancer Institute.
TGF-beta1 was purchased from Sigma Aldrich Chemical Company. Bovine
serum albumin (BSA), anti-phosphorylated MAP kinase antibody, and
anti-human Fc antibody conjugated to horseradish peroxidase were
from Sigma (St. Louis, Mo.). Fetal bovine serum (FBS), calf bovine
serum (CBS), DMEM/F12 medium, and penicillin/streptomycin were
purchased from Invitrogen (Carlsbad, Calif.). Silyl-heparin is
benzyl-tetra(dimethylsilylmethyl)oxycarbamoyl-heparin and was
synthesized as detailed elsewhere (Zamora et al. 2002, Bioconjug
Chem 13(5):920-6.). In brief, silyl-heparin is made by reacting the
hydrophobic group
benzyl-tetra(dimethylsilylmethyl)-oxycarbamoyl-succinimide with
heparin thereby resulting in an amphipathic heparin derivative that
can be adsorbed onto hydrophobic surfaces. For coating purposes,
silyl-heparin was used as a 1% solution in 70% acidified, aqueous
ethanol.
[0157] Alkaline phosphatase (ALP) Activity Assay. C2C12 cells and
C3H10T1/2 cells were cultured at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 and 95% air, with DMEM/F12 medium
supplemented with 10% serum, penicillin/streptomycin. For the BMP-2
induced ALP assay, cells were plated in 96-well
(1.times.10.sup.4/well) dishes in regular growth medium.
Twenty-four hours later, when the cells formed a confluent
monolayer, medium was replaced with DMEM/F12, supplemented with 2%
serum and containing indicated concentration of BMP-2 and/or B2A2.
At 4-5 days post induction, ALP activity was determined as
described by Akiyama and colleagues (Akiyama et al. 1997, Exp Cell
Res 235(2):362-9.) with modifications. Briefly, cells were washed
once with phosphate-buffered saline (PBS) and lysed with 0.1%
Triton X 100 in 10 mM Tris HCl, pH 9.0. Protein concentration was
determined using the BCA Protein Assay Kit (Pierce Biotechnology,
Rockford, Ill.). Then ALP activity was measured by adding ALP
buffer (1 M diethanolamine, 0.5 mM MgCl.sub.2, 1 mg/mL
p-nitrophenylphosphate, pH 9.0), incubating in 37.degree. C., and
absorbance (405 nm) read at 15, 30 and 60 minutes using a
microplate spectrophotometer (Molecular Devices, Sunnyvale,
Calif.). The activity was expressed as O.D. per mg protein per
hour.
[0158] Peptide synthesis and preparation. The peptides B2A2 and
B2A2-K--NS were synthesized by conventional solid phase synthesis
and purified by reverse phase HPLC on C-18, as described in Example
2 and 9.
[0159] Fractions of HPLC purified peptide were pooled, lyophilized,
and stored frozen. Aliquots of the lyophilized bulk material were
used to determine the peptide content, which was determined using a
commercially available kit (BCA, Pierce Endogen, Inc.). For most
further purposes, the peptide was dissolved in 5.5% glucose
containing 0.05% Pluronic 127 to a final concentration of 0.5 mg/mL
or 1 mg/mL, sterilize filtered through a 0.22 micron filter, and
lyophilized in aliquots containing 50 or 100 .mu.g.
[0160] Receptor binding assays. Binding to BMP receptors in a solid
phase binding assay. B2A2 was absorded onto ELISA plates to
saturation, soluble BMP receptor-immunoglobin Fc fusion proteins
were added, and bound receptor was detected by HRP-conjugated
anti-Fc antibody and colorimetric assay, values shown are
background substracted. Specific binding of B2A2 to different
receptor isoforms of the BMPR and Activin Receptor family were
tested, employing the receptor-Fc chimeras shown. Negative controls
establishing specificity included unrelated polypeptide (e.g.
insulin) adsorbed to the plates, and incubation of unrelated
chimeric protein (Endostatin-Fc), neither of which resulted in
binding of B2A2. Apparent two stage binding to BMPR-Ib was revealed
by receptor displacement experiments. Bound receptor was displaced
by the addition of rhBMP-2 at the levels indicated.
[0161] Cell growth. L6 rat skeletal myoblasts and cells from a
human fetal osteoblast cell line (hFOB) (5) were used as target.
Aliquots of cells (1-5.times.10.sup.3 cells) were seeded into wells
of 96 well plates and allowed to attach for 6-24 hours. The medium
was replaced with serum low (2%) medium containing peptide.
Paclitaxel (100 ng/mL) and sodium azide (0.01%), if used, were
included as reference materials known to induce cytotoxicity.
Cultures were incubated typically for 3 days after which time the
relative cell number was assessed using the tetrazolium salt
MTS.
[0162] Cell migration. For studies involving migration across a
wound margin, the cells were grown in vitro and used when
approximately 90% confluent. A simulated wound was made by scraping
cells away from the cultureware surface. The cultures were rinsed
to remove unbound cells, and then incubated in DMEM:F12 medium
containing 2% newborn calf serum with or without peptide. FGF-2 (50
ng/mL) was used as a positive control reference material. The cells
were allowed to migrate for 6 hours after which the cells were
fixed in buffered formalin. Migration was monitored via phase
contrast microscopy. Migrating cells were those that had migrated
across the site of the simulated wound margin.
[0163] In vivo Matrigel plug assay. The in vivo model involved
subcutaneous implant in young adult Fisher 344 of growth
factor-reduced Matrigel with and without BMP-2 and B2A2. Animals
were anesthetized before all procedures by intra peritoneal
injections of ketamine (50 mg/kg) and xylazine (5 mg/kg). Growth
factor reduced Matrigel at 4.degree. C. (liquid state) was mixed
with saline (control), BMP-2 (R&D Systems, Minneapolis, Minn.),
B2A2-K--NS, or B2A2-K--NS plus BMP-2. Aliquots of 0.5 mL of
Matrigel with additives as above were injected subcutaneously on
the upper flanks of the rats. The injection sites were clipped with
stainless steel clips to prevent leakage. The Matrigel was kept on
ice until the time of injection, as were the needle and syringe (to
prevent gelling in the needle). The animals were subsequently
euthanized after 14 days, the gel surgically removed, measured with
calipers, and fixed in buffered formalin. Most of the explants had
a generally elliptical shape and the surface area of the ellipse
was determined using the equation:
Area=.pi.ab
where a and b are 1/2 the width and height of the ellipse.
[0164] The fixed specimens were processed for histological
examination and stained with either hemotoxylin and eosin or
toluidine blue O (Histoserv, Inc., Germantown, Md.).
EXAMPLE 2
[0165] A compound of the present invention was synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP-2 receptor binding amino acid sequence having
the sequence AISMLYLDEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was
stepwise synthesized in parallel from R.sub.2 trifunctional amino
acids of formula I wherein each R.sub.2 is lysine. R.sub.3 is 0
backbone atoms. The resulting synthetic growth modulator analog is
of the following specific structure:
##STR00006##
and is sometimes referred to as B2A2. In the foregoing structure,
"Ahx" is 6-amino hexanoic acid, sometimes also called "6-Ahx" or
"Hex". The single letters are standard amino acid single letter
abbreviations for the naturally coded amino acids. The two chains
of SEQ ID NO:7 link to lysine of the R.sub.2 position via a peptide
bond with the epsilon amines of the lysine side chains.
EXAMPLE 3
[0166] The compound of Example 2 (B2A2) was tested in cell
osteogenic differentiation studies to determine the analog's
ability to stimulate osteogenic activity. B2A2 binds to BMP
receptors, and that receptor activation is associated with the
expression of the osteogenic transcription factor Smad and
repression of MAPK followed by a phenotypic transformation in which
ALP is induced. Referring now to FIG. 1A, the induction of
osteogenic differentiation in C3H10T1/2 cells by BMP-2 in the
presence and absence of B2A2 is illustrated. Treatment of C3H10T1/2
cells with B2A2 alone (up to 10 .mu.g/mL) only slightly increases
alkaline phosphatase (ALP) activity. However, B2A2 plus BMP-2 at
suboptimal concentrations (100 ng/mL) results in significant
increases of ALP activity. The EC.sub.50 for BMP-2 is typically 300
ng/mL.
[0167] C3H10T1/2 cells were seeded onto 96-well plates, treated
with BMP-2 alone or in combination with B2A2 at different
concentrations (solid circles represent BMP-2 at 100 ng/mL, solid
squares represent BMP-2 at 50 ng/mL and unshaded squares represent
samples with no BMP-2). The cells were incubated for 5 days, and
then assayed for ALP activity. ALP activity was assayed by
conversion of para-nitrophenol phosphate (PNPP).
[0168] B2A2 alone had little if any effect on ALP activity in the
dose range between about 0.075-10.0 .mu.g/mL as illustrated in FIG.
1A. The induction of ALP activity was enhanced when cells are
treated with 100 ng/mL of BMP-2 together with B2A2. Co-treatment
was not additive, but was synergistic. Thus B2A2 is a partial
agonist of BMP-2.
[0169] Referring now to FIG. 1B, the synergistic effect of B2A2 and
BMP-2 is illustrated under conditions where the B2A2 concentration
is constant at about 1000 ng/mL while the concentration of BMP-2 is
varied. Using a fixed concentration of B2A2 (1 .mu.g/mL),
augmentation of ALP activity was seen from as low as 25 ng BMP-2/mL
to as high as 1000 ng BMP-2/mL. The threshold for BMP-2 induction
of ALP starts at .about.30 ng/mL but in the presence of 1000 ng/mL
B2A2 the threshold was lowered to about 3 ng/mL BMP-2.
EXAMPLE 4
[0170] B2A2 was tested to determine whether B2A2 enhanced the
biological effects of CHO-produced rhBMP-2. Referring now to FIG.
2, induction of ALP activity in C2C12 cells by recombinant BMP-2
protein (rh-BMP-2) and B2A2 is illustrated. Rh-BMP-2 is
commercially available from either E. coli or mammalian CHO cell
production methods with slightly different potencies, yet B2A2
augments both types of rhBMP-2. Mouse C2C12 cells were seeded onto
96 well plates, treated with B2A2 in combination with human BMP-2
derived from different sources ( /.smallcircle. CHO versus
.box-solid./.quadrature. E. coli), incubated for 4 days, and then
assayed for ALP activity as described. B2A2 was applied at 1000
ng/mL, and BMP-2 at the concentrations indicated in the graph. B2A2
increased the efficacy of E. coli-derived BMP-2 to levels similar
to that of CHO cell-derived BMP-2, and the efficacy of CHO-derived
BMP-2 is further increased by B2A2. Points represent means of
quintuplicate determinations .+-.SD.
EXAMPLE 5
[0171] B2A2 was tested in combination with other growth factors
including FGF-2, VEGF, and TGF-.beta.1 for induction of ALP in
C2C12 cells. Referring now to FIG. 3, induction of ALP activity by
BMP-2 but not various other growth factors in the presence of B2A2
is illustrated. Treatments of FGF-2, TGF-B, VEGF alone failed to
induce ALP in C2C12 cells in the presence of B2A2 demonstrating
that BMP-2 is the effector in the combination of B2A2 and BMP-2.
Mouse C2C12 cells were cultured as described for FIG. 1A, treated
with a combination of various growth factors plus or minus B2A2,
incubated for 3 days, and then assayed for ALP activity as
described for FIG. 1A. FGF-2 was used at 50 ng/mL, VEGF at 25
ng/mL, TGF-.beta.1 at 50 pg/mL, BMP-2 at 50 ng/mL, and B2A2 at 1000
ng/mL. Bars represent means of quintuplicate determinations
.+-.SD.
EXAMPLE 6
[0172] B2A2 was tested to determine whether temporal dissociation
of B2A2+BMP-2 administered to cells affected the BMP-2 induction of
osteogenic activity by the cell. Referring now to FIG. 4, the
induction of ALP activity is illustrated despite the temporal
separation of the addition of B2A2 and BMP-2 to the C2C12 cell
line. Co-administration of the agents is not required since serial
addition of B2A2 followed by washout and addition of BMP-2 in
intervals up to one hour was effective in inducing ALP activity.
Mouse C2C12 cells were cultured as before and B2A2 (1000 ng/mL) was
added to some wells. After a 45 minute incubation all wells were
rinsed with fresh medium and the medium was replaced. To one set of
wells, BMP-2 (200 ng/mL) was added, another set was incubated an
additional 30 min and then BMP-2 added, and finally yet another set
was incubated an additional 60 minutes and then BMP-2 added. After
5 days ALP activity was measured. The synergistic effect was still
observed despite the temporal separation of B2A2 and BMP-2
administration and the washout in between. Data is the means of
triplicates .+-.SD.
EXAMPLE 7
[0173] B2A2 was tested to determine whether spatial dissociation of
B2A2 plus BMP-2 administered to cells affected the BMP-2 induction
of osteogenic activity in the cells. Referring now to FIG. 5, the
induction of ALP activity is illustrated despite the spatial
separation of the addition of B2A2 and BMP-2. In FIG. 5A, a
polystyrene surface of 96-well plates were first coated by
silyl-heparin (open bars), followed by a 1 .mu.g/mL solution of
B2A2 (solid bars) in PBS for (1 hr at 37.degree. C.) and rinsed in
PBS and dried at room temperature. C2C12 cells were subsequently
seeded at densities that resulted in confluent monolayers, and
after allowance for attachment (1-2 hrs), BMP-2 at 50 ng/mL was
added to the cultures. ALP activity was measured five days later.
Data is the means of triplicates .+-.SD. While silyl-heparin alone
potentiates BMP-2 activity, the ALP activity induced by B2A2 and
BMP-2 together is more profound.
[0174] In FIG. 5B, stainless steel wafers were first coated with
silyl-heparin (open bars) followed by 100 .mu.g/mL B2A2 in PBS as a
second coating (solid bars) and rinsed in PBS and dried at room
temperature. Wafers were coated separately in wells of a 24-well
plate and transferred to a fresh untreated plate prior to cell
seeding.
[0175] C2C12 cells were subsequently seeded at densities that
resulted in confluent monolayers, and after allowance for
attachment (1-2 hrs), BMP-2 at the concentrations indicated in the
graph were added to the cultures. ALP activity was measured five
days later. Data is the means of triplicates .+-.SD. The results
indicate that the enhancement of BMP-2 by B2A2 on stainless steel
was profound. Similar results were observed for silyl-heparin+B2A2
coating on titanium wafers in the presence of BMP-2.
EXAMPLE 8
[0176] B2A2 was tested to determine if B2A2 could augment
demineralized bone matrix material (DBM) in an ectopic model of
bone formation. Referring now to FIG. 6, the synergistic activity
of B2A2 with DBM for bone formation is illustrated. B2A2 was coated
onto DBM. B2A2 (100 ng/mg or 300 mg/mL) in a small volume of water
(pH 4) was added to DBM (100 .mu.L/g), mixed, and air-dried at
37.degree. C. The resultant DBM was then further dried overnight in
a vacuum oven.
[0177] The B2A2-coated DBM was implanted into the muscle of athymic
rats and the radiographic density of the implant area is examined
after 3 weeks. There was a 250% increase in relative bone density
after 3 weeks in comparison to DBM without B2A2 and a 650% increase
in bone density after 6 weeks in comparison to DBM without B2A2
(data not shown), As indicated in FIG. 6, there was a statistically
significant increase in radiographic density in B2A2 coated-DBM
muscle at both time points.
[0178] B2A2 can be employed as an additive to demineralized bone
matrix (DBM) and bone graft materials to maximize the bioactivity
of BMP-2. B2A2 augments the bioactivity of BMP-2 found in DBM
(exogenous) and in bone undergoing repair (endogenous). The
clinical use of B2A2 provides a new and novel treatment strategy
applicable to accelerating bone repair.
[0179] Table 2 below summarizes the biochemical interactions of
B2A2, and the modulation of alkaline phosphatase, wherein
modulation was monitored using C2C12 cells.
TABLE-US-00002 TABLE 2 Biochemical interactions of B2A2 Interaction
with heparin Yes MAP kinase phosphorylation Yes Positive modulation
of alkaline phosphatase BMP-2 (E. coli) Yes BMP-2 (Chinese hamster
ovary cells) Yes BMP-7 (mammalian cell) No Modulation via a coating
of alkaline phosphatase B2A2 coating, BMP-2 in solution Yes BMP-2
coating, B2A2 in solution Yes Silyl-heparin/BMP-2 coating, B2A2 in
solution Yes
EXAMPLE 9
[0180] A compound of the present invention was synthesized by solid
phase peptide chemistry with the general structure of formula II
wherein X is a BMP-2 receptor binding amino acid sequence having
the sequence AISMLYLDEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was
stepwise synthesized in parallel from the R.sub.5 trifunctional
amino acid of formula II when R.sub.6 is 0 backbone atoms and
R.sub.5 is lysine. The resulting synthetic growth modulator analog
is of the following specific structure:
##STR00007##
and is sometimes called B2A2-K--NS. In the foregoing structure,
"Ahx" is 6-amino hexanoic acid, sometimes also called "6-Ahx" or
"Hex". The single letters are standard amino acid single letter
abbreviations for the naturally coded amino acids. The chain of SEQ
ID NO:7 is grown from the alpha and epsilon amine groups of the
lysine in the R.sub.5 position. The theoretical molecular weight of
B2A2-K--NS is 5486.9.
EXAMPLE 10
[0181] The synthetic growth factor analog of Example 9 (B2A2-K--NS)
was tested for deleterious effect on L6 cells. Referring now to
FIG. 7, the relative number of L6 cells in culture after treatment
with cytotoxic agents or B2A2-K--NS is illustrated. L6 cells were
treated with 100 ng/mL of Paclitaxel or 0.01% sodium azide and the
effects of these cytotoxic agents were compared to L6 cells treated
with varying concentrations of B2A2-K--NS after three days of
treatment. B2A2-K--NS induced cell proliferation above control
values at concentrations between 2-10 .mu.g/mL. Similar results
were observed in human fetal osteoblasts, C3H10T1/2 cells and
MC-3T3-E1 cells.
EXAMPLE 11
[0182] B2A2-K--NS was tested in cell osteogenic differentiation
studies to determine the ability of the synthetic growth analog to
stimulate osteogenic activity. Referring now to FIG. 8, the
induction of osteogenic differentiation in C2C12 cells with varying
concentrations of B2A2-K--NS in the presence and absence of BMP-2
is illustrated. Treatment of C2C12 cells with B2A2-K--NS alone (up
to 10 .mu.g/mL) only slightly increases alkaline phosphatase (ALP)
activity, however, B2A2-K--NS plus BMP-2 results in significant
increases of ALP activity even at normally sub-threshold
concentrations of BMP-2. C2C12 cells were seeded onto 96-well
plates, treated with varying concentrations of B2A2-K--NS in the
presence (solid bars) and absence (open bars) of BMP-2 at 100
ng/mL. The cells were incubated for 4 days, and then assayed for
ALP activity. ALP activity was assayed by conversion of
para-nitrophenol phosphate (PNPP). B2A2-K--NS had no effect on the
induction of ALP activity at concentrations up to about 10
.mu.g/mL. B2A2-K--NS substantially augments ALP activity induced by
suboptimal amounts of BMP-2 (100 ng/mL). Similar results were
obtaining with C3H10T1/2 cells.
EXAMPLE 12
[0183] B2A2-K--NS was tested for its ability to induce cells of
preosteoblast origin to migrate to a stimulated wound margin.
Murine C3H10T1/2, MC3T3 cells or hFOB were grown to near confluency
in vitro. A stimulated wound was made by scraping the cells away
from the substrate. The cells were allowed to migrate for 6 hours
after which migration was monitored via microscopy. Statistical
significance was determined using ANOVA followed by post hoc
testing using multiple comparison versus control group (Dunnett's
Method). FGF-2 was used as a positive control reference material
and induced a significant increase in migrating cells compared to
controls (data not shown). Table 3 summarizes the increase in
migrating cells at the simulated wound margin induced by about 0.2
to 2.0 .mu.g/mL B2A2-K--NS.
TABLE-US-00003 TABLE 3 .mu.g B2A2-K-NS/mL Mean Std Dev % of control
Migrating C3H10T1/2 cells/field 0.0 104.7 18.1 100 0.2 148.4 21.5
142 0.5 177.3 24.3 169 1.0 214.6 34.9 205 2.0 197.4 12.5 188
Migrating MC-3T3 cells/field 0.0 162.8 43.3 100 0.2 251.2 37.2 154
0.5 286.7 24.0 176 1.0 297.7 34.3 183 2.0 254.3 41.4 156 Migrating
hFOB cells/field 0.0 92.4 33.5 100 0.2 149.7 25.3 162 0.5 164.7
28.1 178 1.0 192.4 33.2 208 2.0 165.9 27.6 179
EXAMPLE 13
[0184] B2A2-K--NS analog was tested for its effect in vivo.
Referring now to FIG. 9, a comparison of the area of explants
excised from an area implanted with Matrigel containing B2A2-K--NK
analog with and without BMP-2 is illustrated. Adult rats were
implanted with Matrigel with and without BMP-2 and B2A2-K--NS and
after 14 days the residual gel was surgically removed and measured.
Nearly all of the implant sites that received B2A2-K--NS, BMP-2,
and BMP-2 and B2A2-K--NS had palpable sites upon inspection whereas
the control implant with carrier only had been largely adsorbed.
Further, the explants from sites that had received B2A2-K--NS,
BMP-2 or a combination of B2A2-K--NS plus BMP-2 had significantly
larger explants. The morphology of the explants differed with
differing explant compositions. Animals receiving only carrier had
residual plugs that were small and tended to have morphology with
poor cellular organization. Animals receiving B2A2-K--NS had plugs
with morphologies consistent with fibrocartilage. Animals receiving
BMP-2 treatments developed plugs containing increased numbers of
cells accompanied by a moderate amount of organization that was
consistent with developing membranous ossification. In animals
receiving B2A2-K--NS plus BMP-2, an increase in cell density was
observed along with an organization consistent with developing
membranous ossification. The cell density was greater than observed
for controls or B2A2-K--NS but less than the cell density observed
for explants from animals receiving BMP-2 alone (data not
shown).
EXAMPLE 14
[0185] The B2A2-K--NS analog was tested in cell osteogenic
differentiation studies to determine the synthetic growth analogs
ability to stimulate osteogenic activity. Referring now to FIG. 10,
the induction of osteogenic differentiation in C2C12 cells by
B2A2-K--NS in the presence and absence of suboptimal concentrations
(100 ng/mL) of BMP-2 is illustrated. Treatment of C2C12 cells with
B2A2-K--NS alone (up to 10 .mu.g/mL) only slightly increases
alkaline phosphatase (ALP) activity. However, B2A2-K--NS plus BMP-2
results in significant increases of ALP activity even at normally
sub-threshold concentrations of BMP-2. C2C12 cells were seeded onto
96-well plates, treated with B2A2-K--NS at different concentrations
in the presence (solid bars) or absence (unshaded bars) of 100
ng/mL BMP-2. The cells were incubated for 4 days, and then assayed
for ALP activity. ALP activity was assayed by conversion of
para-nitrophenol phosphate (PNPP).
EXAMPLE 15
[0186] The B2A2-K--NS analog was tested for its ability to induce
phenotypic expression changes in cells independent of BMP-2 (data
not shown). MC3T3 cells were stimulated with B2A2-K--NS and changes
in the expression of osteocalcin, osteoponin, and type II collagen
were observed as measured with specific antibodies to each which
were subsequently detected with secondary antibodies conjugated to
HPRO. The developed membranes were digitized with a scanner and
converted to gray scale with color inversion with software.
[0187] Referring now to FIG. 10, Alcian staining of C3H10T1/2 cells
for chondrogenic pathway derived proteins is illustrated.
B2A2-K--NS increases the amount of Alcian blue stainable material
produced in C3H10T1/2 cells at 10 days after stimulation.
Suboptimal amounts of BMP-2 (50 ng/mL) did not augment the increase
in Alcian blue stainable material.
EXAMPLE 16
[0188] A compound of the present invention was synthesized by solid
phase peptide chemistry with the general structure of formula II
wherein X is a BMP receptor binding amino acid sequence having the
sequence LYFDESSNVILKK (SEQ ID NO:9) wherein SEQ ID NO:9 was
stepwise synthesized in parallel from the R.sub.5 trifunctional
amino acid of formula II when R.sub.6 is 0 atoms and R.sub.5 is a
lysine. In synthesis, side chains of lysine residues other than the
R.sub.5 lysine were protected, as were other reactive side chains,
with selective deprotection following synthesis. The resulting
synthetic growth modulator analog is of the following specific
structure:
##STR00008##
and is sometimes called B7A1-K--NS. In the foregoing structure,
"Ahx" is 6-amino hexanoic acid, sometimes also called "6-Ahx" or
"Hex". The single letters are standard amino acid single letter
abbreviations for the naturally coded amino acids. The chain of SEQ
ID NO:9 is grown from the alpha and epsilon amine groups of the
lysine in the R.sub.5 position.
EXAMPLE 17
[0189] B7A1-K--NS was tested in cell osteogenic differentiation
studies to determine the ability of the synthetic growth analog to
stimulate osteogenic activity. Referring now to FIG. 11, the
induction of osteogenic differentiation in C2C12 cells with varying
concentrations of B7A1-K--NS in the presence and absence of BMP-2
is illustrated. Treatment of C2C12 cells with B2A2 alone (up to 10
.mu.g/mL) did not affect the production of ALP activity. However,
B7A1-K--NS plus BMP-2 results in significant increases of ALP
activity even at normally sub-threshold concentrations (100 ng/mL)
of BMP-2. C2C12 cells were seeded onto 96-well plates, treated with
varying concentrations of B2A2 in the presence (solid bars) and
absence (open bars) of BMP-2 at 100 ng/mL. The cells were incubated
for 4 days, and then assayed for ALP activity. ALP activity was
assayed by conversion of para-nitrophenol phosphate (PNPP).
B7A1-K--NS had no effect on the induction of ALP activity at
concentrations up to about 10 .mu.g/mL. B7A1-K--NS substantially
augments ALP activity induced by suboptimal amounts of BMP-2 (100
ng/mL). Similar results were obtaining with C3H10T1/2 cells.
EXAMPLE 18
[0190] A compound of the present invention is synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP-2 receptor binding amino acid sequence having
the sequence ISMLYLDENEKVVLKNY (SEQ ID NO:8) wherein SEQ ID NO:8 is
stepwise synthesized in parallel from R.sub.2 trifunctional amino
acids of formula I and wherein each R.sub.2 is lysine. The
resulting synthetic growth modulator analog is of the following
specific structure:
##STR00009##
In the foregoing structure, "Ahx" is 6-amino hexanoic acid,
sometimes also called "6-Ahx" or "Hex". The single letters are
standard amino acid single letter abbreviations for the naturally
coded amino acids. The two chains of SEQ ID NO:8 link to lysines in
the R.sub.2 position via a peptide bond with the secondary amine of
the lysine side chains.
EXAMPLE 19
[0191] A compound of the present invention is synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP receptor binding amino acid sequence having the
sequence LYVDFSDVGVVNDW (SEQ ID NO:10) wherein SEQ ID NO:10 is
stepwise synthesized in parallel from the R.sub.2 trifunctional
amino acids of formula I and wherein each R.sub.2 is lysine. The
resulting synthetic growth modulator analog is of the following
specific structure:
##STR00010##
In the foregoing structure, "Ahx" is 6-amino hexanoic acid,
sometimes also called "6-Ahx" or "Hex". The single letters are
standard amino acid single letter abbreviations for the naturally
coded amino acids. The two chains of SEQ ID NO:10 link to lysines
in the R.sub.2 position via a peptide bond with the secondary
amines of the lysine side chains.
EXAMPLE 20
[0192] A synthetic growth modulator analog of the BMP family is
synthesized by solid phase peptide chemistry with the general
structure of formula I wherein X is a BMP receptor binding amino
acid sequence having the sequence CAISMLYLDENEKVVL (SEQ ID NO:12)
wherein SEQ ID NO:12 is stepwise synthesized in parallel from
R.sub.2 trifunctional amino acids of formula I and where R.sub.2
are each lysine. The resulting synthetic growth modulator analog is
of the following specific structure:
##STR00011##
In the foregoing structure, "Ahx" is 6-amino hexanoic acid,
sometimes also called "6-Ahx" or "Hex". The single letters are
standard amino acid single letter abbreviations for the naturally
coded amino acids. The two chains of SEQ ID NO:12 link to lysines
in the R.sub.2 position via a peptide bond with the secondary
amines of the lysine side chains.
EXAMPLE 21
[0193] A compound of the present invention is synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP receptor binding amino acid sequence having the
sequence AFYCHGECPFPLADHL (SEQ ID NO:13) wherein SEQ ID NO:13 is
stepwise synthesized in parallel from R.sub.2 trifunctional amino
acids of formula I and wherein each R.sub.2 is lysine. The
resulting synthetic growth modulator analog is of the following
specific structure:
##STR00012##
In the foregoing structure, "Ahx" is 6-amino hexanoic acid,
sometimes also called "6-Ahx" or "Hex". The single letters are
standard amino acid single letter abbreviations for the naturally
coded amino acids. The two chains of SEQ ID NO:13 link to lysines
in the R.sub.2 position via a peptide bond with the epsilon amines
of the lysine side chains.
EXAMPLE 22
[0194] A compound of the present invention is synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP receptor binding amino acid sequence having the
sequence PFPLADHLNSTNHAIVQTLVNSV (SEQ ID NO:14) wherein SEQ ID
NO:14 is stepwise synthesized in parallel from R.sub.2
trifunctional amino acids of formula I and wherein each R.sub.2 is
a lysine. The resulting synthetic growth modulator analog is of the
following specific structure:
##STR00013##
In the foregoing structure, "Ahx" is 6-amino hexanoic acid,
sometimes also called "6-Ahx" or "Hex". The single letters are
standard amino acid single letter abbreviations for the naturally
coded amino acids. The two chains of SEQ ID NO:14 link to lysines
in the R.sub.2 position via a peptide bond with the secondary
amines of the lysine side chains.
EXAMPLE 23
[0195] A compound of the present invention was synthesized by solid
phase peptide chemistry with the general structure of formula I
wherein X is a BMP-2 receptor binding amino acid sequence having
the sequence AISMLYLDEKVVL (SEQ ID NO:7) wherein SEQ ID NO:7 was
stepwise synthesized in parallel from R.sub.2 trifunctional amino
acids of formula I when R.sub.3 is 0 backbone atoms and each
R.sub.2 is lysine. The resulting synthetic growth modulator analog
is of the following specific structure:
##STR00014##
and is sometimes called B2A2-K2-NS. In the foregoing structure,
"Ahx" is 6-amino hexanoic acid, sometimes also called "6-Ahx" or
"Hex". The single letters are standard amino acid single letter
abbreviations for the naturally coded amino acids.
EXAMPLE 24
[0196] The compound of Example 2 was tested for specific binding to
Bone Morphogenic Protein-2 receptors. Referring now to FIG. 12,
results of solid phase receptor binding assays utilizing purified
receptor/Fc chimeric molecules are illustrated. The chimeras are
recombinant constructs of the soluble ectodomain of various
receptor molecules (BMPR and ActivinR isoforms) fused to the
carboxyl-terminal of the human IgG1 Fc region via a polypeptide
liner. ELISA plates were coated with B2A2 or control compounds,
soluble chimeric receptor/Fc antibody and quantified with a
colorimeteric ELISA. B2A2 was shown to bind preferentially to
BMPR-Ib and ActivinR-II, as well as other isoforms in the following
order: BMPR-Ib=ActR-II>>BMPR-Ia=ActRIIb>BMPR-II. Insulin,
used as a control, did not bind either B2A2 or BMP-2 (data not
shown). Referring now to FIG. 13, B2A2 binding to purified BMP-2
receptor/Fc chimeric molecules in varying concentrations of BMP-2
is illustrated. BMP-2 added in large molar excess with the
receptors blocked binding to B2A2. When BMP-2 was added in varying
concentrations, the resulting displacement curve suggests two-stage
binding kinetics of B2A2 to BMPR-Ib.
[0197] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
[0198] Although the invention has been described in detail with
particular reference to these preferred embodiments, 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 reference.
Sequence CWU 1
1
4416PRTArtificialheparin-binding motif 1Xaa Xaa Xaa Xaa Xaa Xaa1
5210PRTArtificialsynthetic heparin-binding sequence 2Arg Lys Arg
Lys Leu Glu Arg Ile Ala Arg1 5 10310PRTArtificialsynthetic
heparin-binding sequence 3Arg Lys Arg Lys Leu Gly Arg Ile Ala Arg1
5 10410PRTArtificialsynthetic heparin-binding sequence 4Arg Lys Arg
Lys Leu Trp Arg Ala Arg Ala1 5 1059PRTArtificialsynthetic
heparin-binding sequence 5Arg Lys Arg Leu Asp Arg Ile Ala Arg1
5611PRTArtificialsynthetic heparin-binding sequence 6Arg Lys Arg
Lys Leu Glu Arg Ile Ala Arg Cys1 5 10715PRTArtificialsynthetic
BMP-2 analog 7Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val
Val Leu1 5 10 15817PRTArtificialsynthetic BMP-2 analog 8Ile Ser Met
Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn1 5 10
15Tyr913PRTArtificialsynthetic BMP-7 analog 9Leu Tyr Phe Asp Glu
Ser Ser Asn Val Ile Leu Lys Lys1 5 101013PRTArtificialsynthetic
BMP-2 analog 10Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp1
5 101115PRTArtificialsynthetic BMP-2 analog 11Glu Lys Val Val Leu
Lys Asn Tyr Gln Asp Met Val Val Glu Gly1 5 10
151216PRTArtificialsynthetic BMP-2 analog 12Cys Ala Ile Ser Met Leu
Tyr Leu Asp Glu Asn Glu Lys Val Val Leu1 5 10
151316PRTArtificialsynthetic BMP-2 analog 13Ala Phe Tyr Cys His Gly
Glu Cys Pro Phe Pro Leu Ala Asp His Leu1 5 10
151423PRTArtificialsynthetic BMP-2 analog 14Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val1 5 10 15Gln Thr Leu Val Asn
Ser Val201515PRTArtificialsynthetic reverse BMP-2 analog 15Leu Val
Val Lys Glu Asn Glu Asp Leu Tyr Leu Met Ser Ile Ala1 5 10
151617PRTArtificialsynthetic reverse BMP-2 analog 16Tyr Asn Lys Leu
Val Val Lys Glu Asn Glu Asp Leu Tyr Leu Met Ser1 5 10
15Ile1713PRTArtificialsynthetic reverse BMP-2 analog 17Lys Lys Leu
Ile Val Asn Ser Ser Glu Asp Phe Tyr Leu1 5
101813PRTArtificialsynthetic reverse BMP-7 analog 18Trp Asp Asn Trp
Gly Val Asp Ser Phe Asp Val Tyr Leu1 5 101915PRTArtificialsynthetic
reverse BMP-2 analog 19Gly Glu Val Val Met Asp Gln Tyr Asn Lys Leu
Val Val Lys Glu1 5 10 152016PRTArtificialsynthetic reverse BMP-2
analog 20Leu His Asp Ala Leu Pro Phe Pro Cys Glu Gly His Cys Tyr
Phe Ala1 5 10 152123PRTArtificialsynthetic reverse BMP-2 analog
21Val Ser Asn Val Leu Thr Gln Val Ile Ala His Asn Thr Ser Asn Leu1
5 10 15His Asp Ala Leu Pro Phe Pro202216PRTArtificialsynthetic
reverse BMP-2 analog 22Leu Val Val Lys Glu Asn Glu Asp Leu Tyr Leu
Met Ser Ile Ala Cys1 5 10 152321PRTArtificialsynthetic TGF-beta1
analog 23Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu
Ser Asn1 5 10 15Met Ile Val Arg Ser202422PRTArtificialsynthetic
TGF-beta2 analog 24Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro Lys Ile
Glu Gln Leu Ser1 5 10 15Asn Met Ile Val Lys
Ser202521PRTArtificialsynthetic TGF-beta3 analog 25Leu Thr Ile Leu
Tyr Tyr Val Gly Arg Thr Pro Lys Val Glu Gln Leu1 5 10 15Ser Asn Met
Val Val202623PRTArtificialsynthetic BMP-2 analog 26Ala Ile Ser Met
Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys1 5 10 15Asn Tyr Gln
Asp Met Val Val202724PRTArtificialsynthetic BMP-3 analog 27Ser Ser
Leu Ser Ile Leu Phe Phe Asp Glu Asn Lys Asn Val Val Leu1 5 10 15Lys
Val Tyr Pro Asn Met Thr Val202824PRTArtificialsynthetic BMP-3beta
analog 28Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn Arg Asn Val
Val Leu1 5 10 15Lys Val Tyr Pro Asn Met Ser
Val202923PRTArtificialsynthetic BMP-4 analog 29Ala Ile Ser Met Leu
Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys1 5 10 15Asn Tyr Gln Glu
Met Val Val203023PRTArtificialsynthetic BMP-5 analog 30Ala Ile Ser
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys1 5 10 15Lys Tyr
Arg Asn Met Val Val203123PRTArtificialsynthetic BMP-6 analog 31Ala
Ile Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys1 5 10
15Lys Tyr Arg Asn Met Val Val203223PRTArtificialsynthetic BMP-7
analog 32Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile
Leu Lys1 5 10 15Lys Tyr Arg Asn Met Val
Val203323PRTArtificialsynthetic BMP-8 analog 33Ala Thr Ser Val Leu
Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg1 5 10 15Lys Ala Arg Asn
Met Val Val203423PRTArtificialsynthetic BMP-9 analog 34Ile Ser Val
Leu Tyr Lys Asp Asp Met Gly Val Pro Thr Leu Lys Tyr1 5 10 15His Tyr
Glu Gly Met Ser Val203522PRTArtificialsynthetic BMP-10 analog 35Ile
Ser Ile Leu Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys1 5 10
15Tyr Glu Gly Met Ala Val203622PRTArtificialsynthetic BMP-11 analog
36Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys1
5 10 15Ile Pro Gly Met Val Val203722PRTArtificialsynthetic BMP-12
analog 37Ile Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn Val Val Tyr
Lys Gln1 5 10 15Tyr Glu Asp Met Val Val203822PRTArtificialsynthetic
BMP-13 analog 38Ile Ser Ile Leu Tyr Ile Asp Ala Gly Asn Asn Val Val
Tyr Lys Gln1 5 10 15Tyr Glu Asp Met Val
Val203922PRTArtificialsynthetic BMP-14 analog 39Ile Ser Ile Leu Phe
Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln1 5 10 15Tyr Glu Asp Met
Val Val204022PRTArtificialsynthetic BMP-15 analog 40Ile Ser Val Leu
Met Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu1 5 10 15Tyr Glu Gly
Met Ile Ala204122PRTArtificialsynthetic GDF-1 analog 41Ile Ser Val
Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gln1 5 10 15Tyr Glu
Asp Met Val Val204222PRTArtificialsynthetic GDF-3 analog 42Ile Ser
Met Leu Tyr Gln Asp Asn Asn Asp Asn Val Ile Leu Arg His1 5 10 15Tyr
Glu Asp Met Val Val204322PRTArtificialsynthetic GDF-8 analog 43Ile
Asn Met Tyr Leu Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys1 5 10
15Ile Pro Ala Met Val Val204422PRTArtificialsynthetic GDF-9 analog
44Leu Ser Val Leu Thr Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu1
5 10 15Tyr Glu Asp Met Ile Ala20
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