U.S. patent application number 12/949034 was filed with the patent office on 2011-05-19 for implantable bone graft materials.
This patent application is currently assigned to AFFINERGY, INC.. Invention is credited to Mora Carolynne Melican.
Application Number | 20110117166 12/949034 |
Document ID | / |
Family ID | 44011447 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110117166 |
Kind Code |
A1 |
Melican; Mora Carolynne |
May 19, 2011 |
IMPLANTABLE BONE GRAFT MATERIALS
Abstract
Compositions and methods are provided for promoting bone growth.
An implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide. In addition,
an implantable bone graft material is provided consisting
essentially of a resorbable .beta.-TCP and a resorbable polymer,
wherein the .beta.-TCP has a total porosity of about 50% or greater
and wherein the .beta.-TCP has a particle size ranging from about
100 micron to about 300 micron. The implantable bone graft
materials are useful for promoting bone growth in a subject.
Inventors: |
Melican; Mora Carolynne;
(Cary, NC) |
Assignee: |
AFFINERGY, INC.
Research Triangle Park
NC
|
Family ID: |
44011447 |
Appl. No.: |
12/949034 |
Filed: |
November 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61262353 |
Nov 18, 2009 |
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61368849 |
Jul 29, 2010 |
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61370723 |
Aug 4, 2010 |
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Current U.S.
Class: |
424/423 ;
514/16.7 |
Current CPC
Class: |
A61L 2300/414 20130101;
A61L 27/425 20130101; A61L 27/54 20130101; A61P 19/00 20180101;
A61K 47/42 20130101; A61P 19/08 20180101; A61P 21/00 20180101; A61K
38/10 20130101; A61K 9/0024 20130101; A61K 47/02 20130101; A61L
2430/02 20130101; A61K 47/6957 20170801; A61P 17/02 20180101; A61L
27/58 20130101; A61P 9/00 20180101; A61P 17/00 20180101; A61P 19/04
20180101; A61P 43/00 20180101; A61K 47/64 20170801; A61L 27/427
20130101 |
Class at
Publication: |
424/423 ;
514/16.7 |
International
Class: |
A61F 2/00 20060101
A61F002/00; A61K 38/00 20060101 A61K038/00; A61P 19/08 20060101
A61P019/08 |
Goverment Interests
GRANT STATEMENT
[0002] The invention was made with government support under Grant
No. 5R44GM077753-03 awarded by the National Institute of General
Medical Sciences and under Grant No. 5R44DE020760-03 and Grant No.
2R44DE018071-02 awarded by the National Institute of Dental and
Craniofacial Research. The government has certain rights in the
invention.
Claims
1. An implantable bone graft material, wherein the implantable
material consists essentially of a resorbable .beta.-TCP and a
resorbable polymer, wherein the .beta.-TCP has a total porosity of
about 50% or greater and wherein the .beta.-TCP has a particle size
ranging from about 100 micron to about 300 micron.
2. The implantable bone graft material of claim 1, wherein the
resorbable .beta.-TCP and the resorbable polymer are in the form of
a composite.
3. The implantable bone graft material of claim 2, wherein the
composite is in the form of a sponge, a granulized sponge, a putty,
or a strip.
4. The implantable bone graft material of claim 1, wherein the
total porosity of the .beta.-TCP is about 70%.
5. The implantable bone graft material of claim 1, wherein the
diameter of the pores in the .beta.-TCP is less than 100
micron.
6. The implantable bone graft material of claim 1, wherein the
.beta.-TCP and the polymer are present at a weight ratio ranging
from about 10:1 .beta.-TCP to polymer to about 2:1 .beta.-TCP to
polymer.
7. The implantable bone graft material of claim 1, wherein the
polymer is selected from the group consisting of collagen,
fibrillar collagen, Type I collagen, bovine collagen, keratin,
silk, polysaccharides, dextran, cellulose derivatives, oxidized
cellulose, oxidized regenerated cellulose, carboxymethylcellulose,
hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid,
aliphatic polyesters, polyanhydrides, poly(orthoester)s,
poly(glycolide), poly(lactide), poly(.epsilon.-caprolactone),
poly(trimethylene carbonate), poly(p-dioxanone),
poly(lactide-co-glycolide),
poly(.epsilon.-caprolactone-co-glycolide),
poly(glycolide-co-trimethylene carbonate),
poly(lactide-co-.epsilon.-caprolactone), tyrosine-based
polycarbonates, tyrosine-based polyarylates, and copolymers and
derivatives thereof.
8. The implantable bone graft material of claim 7, wherein the
polymer is bovine Type 1 fibrillar collagen.
9. The implantable bone graft material of claim 7, wherein the
polymer is collagen and the weight ratio of .beta.-TCP to collagen
is about 4:1 (80% .beta.-TCP to about 20% collagen).
10. The implantable bone graft material of claim 7, wherein the
polymer is bovine Type 1 fibrillar collagen, wherein the weight
ratio of the .beta.-TCP to the collagen is about 4:1 (80%
.beta.-TCP to about 20% collagen), and wherein the total porosity
of the .beta.-TCP is about 70%.
11. The implantable bone graft material of claim 7, wherein the
polymer is bovine Type 1 fibrillar collagen, wherein the weight
ratio of the .beta.-TCP to the collagen is about 4:1 (80%
.beta.-TCP to about 20% collagen), wherein the total porosity of
the .beta.-TCP is about 70%, and wherein the diameter of the pores
in the .beta.-TCP is less than 100 micron.
12. A method for promoting bone growth in a subject, the method
comprising delivering the implantable bone graft material of claim
1 to a subject, wherein the presence of the bone graft material
promotes bone growth.
13. The method of claim 12, wherein the resorbable .beta.-TCP and
the resorbable polymer are in the form of a composite.
14. The method of claim 13, wherein the composite is in the form of
a sponge, a granulized sponge, a putty, or a strip.
15. The method of claim 12, wherein the .beta.-TCP and the polymer
are present at a weight ratio ranging from about 10:1 .beta.-TCP to
polymer to about 2:1 .beta.-TCP to polymer.
16. The method of claim 12, wherein the polymer is selected from
the group consisting of collagen, fibrillar collagen, Type I
collagen, bovine collagen, keratin, silk, polysaccharides, dextran,
cellulose derivatives, oxidized cellulose, oxidized regenerated
cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose,
chitosan, chitin, hyaluronic acid, aliphatic polyesters,
polyanhydrides, poly(orthoester)s, poly(glycolide), poly(lactide),
poly(.epsilon.-caprolactone), poly(trimethylene carbonate),
poly(p-dioxanone), poly(lactide-co-glycolide),
poly(.epsilon.-caprolactone-co-glycolide),
poly(glycolide-co-trimethylene carbonate),
poly(lactide-co-.epsilon.-caprolactone), tyrosine-based
polycarbonates, tyrosine-based polyarylates, and copolymers and
derivatives thereof.
17. The method of claim 16, wherein the polymer is bovine Type 1
fibrillar collagen.
18. The method of claim 16, wherein the polymer is collagen and the
weight ratio of .beta.-TCP to collagen is about 4:1 (80% .beta.-TCP
to about 20% collagen).
19. The method of claim 16, wherein the polymer is bovine Type 1
fibrillar collagen, wherein the weight ratio of the .beta.-TCP to
the collagen is about 4:1 (80% .beta.-TCP to about 20% collagen),
wherein the diameter of the pores in the .beta.-TCP is less than
100 micron, and wherein the total porosity of the .beta.-TCP is
about 70%.
20. A method for promoting spinal fusion in a subject, the method
comprising delivering the implantable bone graft material of claim
1 to a subject, wherein the presence of the graft material promotes
spinal fusion.
21. The method of claim 20, wherein the resorbable .beta.-TCP and
the resorbable polymer are in the form of a composite.
22. The method of claim 21, wherein the composite is in the form of
a sponge, a granulized sponge, a putty, or a strip.
23. The method of claim 20, wherein the .beta.-TCP and the polymer
are present at a weight ratio ranging from about 10:1 .beta.-TCP to
polymer to about 2:1 .beta.-TCP to polymer.
24. The method of claim 20, wherein the polymer is selected from
the group consisting of collagen, fibrillar collagen, Type I
collagen, bovine collagen, keratin, silk, polysaccharides, dextran,
cellulose derivatives, oxidized cellulose, oxidized regenerated
cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose,
chitosan, chitin, hyaluronic acid, aliphatic polyesters,
polyanhydrides, poly(orthoester)s, poly(glycolide), poly(lactide),
poly(.epsilon.-caprolactone), poly(trimethylene carbonate),
poly(p-dioxanone), poly(lactide-co-glycolide),
poly(.epsilon.-caprolactone-co-glycolide),
poly(glycolide-co-trimethylene carbonate),
poly(lactide-co-.epsilon.-caprolactone), tyrosine-based
polycarbonates, and tyrosine-based polyarylates, and copolymers and
derivatives thereof.
25. The method of claim 24, wherein the polymer is bovine Type I
fibrillar collagen.
26. The method of claim 24, wherein the polymer is collagen and the
weight ratio of .beta.-TCP to collagen is about 4:1 (80% .beta.-TCP
to about 20% collagen).
27. The method of claim 24, wherein the polymer is bovine Type I
fibrillar collagen, wherein the weight ratio of the .beta.-TCP to
the collagen is about 4:1 (80% .beta.-TCP to about 20% collagen),
wherein the diameter of the pores in the .beta.-TCP is less than
100 micron, and wherein the total porosity of the .beta.-TCP is
about 70%.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/262,353, filed Nov. 18, 2009; U.S. Provisional
Application No. 61/368,849, filed Jul. 29, 2010; and U.S.
Provisional Application No. 61/370,723, filed Aug. 4, 2010, each of
which is hereby incorporated in its entirety by reference
herein.
FIELD
[0003] The presently disclosed subject matter relates to
implantable bone graft materials for promoting bone growth.
BACKGROUND
[0004] Certain growth factors have shown clinical benefit in
treatment of bone defects, injuries, disorders, or diseases. In
particular, the bone morphogenic proteins (BMP), including BMP-2
and BMP-7, have shown clinical benefit in the treatment of bone
fractures and spine fusions. Back pain is one of the leading
reasons for physician visits in the United States and in many cases
requires surgical intervention. In 2009 in the United States, there
were 425,000 spinal fusion surgeries, and the frequency of these
surgeries is projected to grow 6% per year. The gold standard for
bone graft in spinal fusion is autograft from the iliac crest;
however, the use of autograft presents multiple challenges
including donor site morbidity, blood loss, limited availability,
prolonged operating times, and pseudarthrosis due to a slow rate of
fusion. Bone marrow aspirate (BMA) contains osteoinductive factors
and can be harvested at point-of-care without the complications of
harvesting autogenous bone. However, the current bone graft
substitutes are not adequate for the retention and release of
osteoinductive factors from BMA over the length of the healing
cycle. As a result, there is a large effort to develop bone graft
substitutes or extenders that can not only reduce or replace the
need for harvest of autogenous bone but also accelerate the rate of
fusion (arthrodesis). Tricalcium phosphate (TCP)-based bone graft
substitutes often containing collagen are used commonly in lumbar
spinal fusion because TCP is resorbed over several months as bone
heals. Ceramic bone graft substitutes, such as the MASTERGRAFT and
VITOSS line of products, have been used successfully in spinal
fusion surgeries (Miyazaki et al., Eur Spine J, 2009, 18:783-99;
Khan et al., Am Acad Orthop Surg, 2005, 13:129-37; Neen et al.,
Spine, 2006, 31:E636-40; Epstein, Spine J, 2009, 9:630-8; Carter,
Spine J, 2009, 9:434-8; Epstein, J Spinal Disord Tech, 2006,
19:424-9; Birch, N. and W. L. D'Souza, J Spinal Disord Tech, 2009,
22:434-8; Lerner, T., V., Eur Spine J, 2009, 18:170-9; Knop. et
al., Arch Orthop Trauma Surg, 2006, 126:204-10; Epstein, Spine J,
2008, 8:882-7), in particular when used in combination with the
recombinant BMP-2--containing product INFUSE (Glassman et al.,
Spine J, 2007, 7:44-9; Boden et al., Spine, 2002, 27:2662-73;
Glassman, Spine, 2005, 30:1694-8). Recombinant BMP-2 is effective
but carries a high cost and serious safety risks (Cahill et al.,
JAMA, 2009, 302:58-66), in part because of leakage away from its
carrier and the high dose required to achieve therapeutic levels
(Poynton, A. R. and J. M. Lane, Spine, 2002, 27:S40-8).
[0005] Therefore, there is an unmet clinical need in bone repair
and spinal fusion surgery for a safe, cost-effective bone graft
substitute that can maintain a sustained dose of osteoinductive
factors throughout the healing process. The presently disclosed
subject matter provides such a bone graft substitute.
SUMMARY
[0006] The presently disclosed subject matter provides compositions
and methods for promoting bone growth. In one embodiment, the
presently disclosed subject matter provides an implantable bone
graft material, wherein the implantable material consists
essentially of a resorbable .beta.-TCP and a resorbable polymer,
wherein the .beta.-TCP has a total porosity of about 50% or greater
and a particle size ranging from about 100 micron to about 300
micron. In one embodiment, the presently disclosed subject matter
provides an implantable bone graft material comprising a resorbable
ceramic and a resorbable polymer, wherein the polymer comprises a
covalently attached BMP binding peptide.
[0007] In one embodiment, the presently disclosed subject matter
provides a method for promoting bone growth in a subject, the
method comprising delivering an implantable bone graft material to
a subject, wherein the graft material consists essentially of a
.beta.-TCP and a resorbable polymer, and wherein the presence of
the graft material promotes bone growth. In one embodiment, the
presently disclosed subject matter provides a method for promoting
bone growth in a subject, the method comprising delivering an
implantable bone graft material to a subject, wherein the graft
material comprises a resorbable ceramic and a resorbable polymer,
wherein the polymer comprises a covalently attached BMP binding
peptide, and wherein the presence of the graft material having
attached BMP binding peptide promotes bone growth. In one
embodiment, the presently disclosed subject matter provides a
method for capturing BMP onto an implantable bone graft material,
the method comprising contacting a sample comprising BMP with the
graft material, wherein the graft material comprises a resorbable
ceramic and a resorbable polymer, wherein the polymer comprises a
covalently attached BMP binding peptide, and wherein the BMP
comprised in the sample is captured onto the graft material through
binding to the attached BMP binding peptide. In one embodiment, the
presently disclosed subject matter provides a method for promoting
bone growth in a subject, the method comprising contacting a sample
comprising BMP with an implantable bone graft material, wherein the
graft material comprises a resorbable ceramic and a resorbable
polymer, wherein the polymer comprises a covalently attached BMP
binding peptide, wherein the BMP comprised in the sample is
captured onto the graft material through binding to the attached
BMP binding peptide; and delivering to the subject the graft
material comprising the captured BMP, wherein the presence of the
captured BMP promotes bone growth in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a schematic diagram depicting one method for
covalently attaching a BMP binding peptide to a substrate
comprising amino functional groups.
[0009] FIG. 2 is a schematic diagram depicting one method for
covalently attaching a BMP binding peptide to a substrate
comprising amino functional groups.
[0010] FIG. 3 is a schematic diagram depicting methods for
covalently attaching a BMP binding peptide to a substrate having an
amino functional group.
[0011] FIG. 4 is a schematic diagram depicting one method for
covalently attaching a BMP binding peptide to a substrate
comprising amino functional groups.
[0012] FIG. 5 is a schematic diagram depicting one method for
covalently attaching a BMP binding peptide to a substrate
comprising amino functional groups.
[0013] FIG. 6 is a schematic diagram depicting the chemistry for
covalently attaching a BMP binding peptide to a polyanhydride
polymer, polymaleic anhydride (PMA), through the reactive amines on
the peptide.
[0014] FIG. 7 is a schematic diagram depicting exemplary chemistry
for covalently attaching a BMP binding peptide to chitosan.
[0015] FIG. 8 is a schematic diagram depicting exemplary chemistry
for covalently attaching a BMP binding peptide to chitosan.
[0016] FIG. 9 is a schematic diagram depicting exemplary chemistry
for covalently attaching a BMP binding peptide to hyaluronic
acid.
[0017] FIG. 10 is a schematic diagram depicting exemplary chemistry
for covalently attaching a BMP binding peptide to hyaluronic
acid.
[0018] FIG. 11 is a schematic diagram depicting exemplary chemistry
for introducing an amino functional group on cellulose for
subsequent covalent attachment of a BMP binding peptide.
[0019] FIG. 12 is a schematic diagram depicting exemplary chemistry
for covalently attaching a BMP binding peptide to oxidized
cellulose.
[0020] FIG. 13 is a schematic diagram depicting one method for
covalently attaching more than one BMP binding peptide to a
substrate comprising amino functional groups.
[0021] FIGS. 14A-14C are bar graphs showing binding of BMP Binding
Peptides for BMP-2, BMP-5, BMP-6, BMP-7, and GDF-7 (BMP-12). BMP
binding peptide SEQ ID NO: 1 is denoted as P9 and BMP binding
peptide SEQ ID NO: 2 is denoted as P10 in FIGS. 14A-14C. BMP
binding peptides SEQ ID NOs: 5-7 are denoted as P11-P13,
respectively, in FIG. 14C.
[0022] FIG. 15 is a graph showing BMP-2 release from collagen
sponges modified with BMP binding peptides. Each of the BMP Binding
peptides SEQ ID NO: 2 (Peptide 1) and SEQ ID NO: 1 (Peptide 2) were
covalently attached to a collagen sponge. The peptide-modified
sponges were loaded with BMP-2, and then challenged with repeated
plasma changes. Release of BMP-2 into the plasma was measured at 1,
3, and 7 h by ELISA. Peptide-modified sponges retained
significantly more BMP-2 than the unmodified sponge.
[0023] FIG. 16 is a bar graph showing that BMP binding peptide (SEQ
ID NO: 2) does not affect BMP-2 activity. BMP-2 biological activity
was measured by alkaline phosphatase secretion by C2C12 cells.
C2C12 cells were incubated with BMP-2 and a range of BMP binding
peptide concentrations. The data show that the BMP binding peptide
does not interfere with the biological activity of BMP-2 and the
BMP binding peptide does not have BMP-2 activity on its own.
[0024] FIG. 17 is a graph showing BMP-2 release from a BMP binding
peptide-modified collagen/TCP composite after 6 weeks of incubation
in plasma. Composites modified with either a low or a high density
of BMP binding peptide (SEQ ID NO: 2) retained significantly more
BMP-2 than unmodified composite, and the composite with a higher
peptide density retained more BMP-2 than the low density
composite.
[0025] FIG. 18 is a graph showing BMP-2 capture from BMP-2 spiked
plasma by unmodified and BMP binding peptide-modified collagen/TCP
composites. Composites modified with a low (Low-peptide
collagen/TCP) and a high (High-peptide collagen/TCP) density of BMP
binding peptide SEQ ID NO: 2 were compared to control groups
without a composite (No collagen/TCP) or with a composite without
peptide (No-peptide collagen/TCP). The amount of BMP-2 that
remained in the plasma was measured at each time-point. BMP binding
peptide-modified composites captured significantly more BMP-2 from
the plasma than either the unmodified composite or the no-composite
control.
[0026] FIGS. 19A-19D show 12 week histology results for the bone
graft substitute BMP binding peptide-modified collagen/TCP
composite bone graft substitute in the rat calvarial defect model.
The data shown in Panels A-D are for the BMA-hydrated groups. Panel
A) Representative images. Panel B) New bone area. Panel C) New bone
maturity. Panel D) Osteogenic cellular activity. Sections from the
group treated with peptide-modified composite had higher scores for
new bone area, new bone maturity, and osteogenic cellular activity.
Data are presented as mean.+-.SEM. *, **, ***, p<0.05, 0.01,
0.001, respectively, vs. BMP binding peptide-modified
composite.
[0027] FIG. 20 is a composite of images taken from one animal
treated with the peptide-modified product candidate in the rat
calvarial defect model for 12 weeks. Four of the eight animals
treated with the peptide-modified product candidate had mature bone
bridging the entire gap at 12 weeks. There were no animals in the
other experimental groups with contiguous bone across the gap.
[0028] FIGS. 21A-21B are graphs of the results at 4 weeks of a bone
healing model in the rat calvaria. Treatment groups included a
TCP/collagen composite without attached BMP binding peptide
(unmodified), a TCP/collagen composite with attached BMP binding
peptide (modified), and two commercially available bone void
fillers MEDTRONIC MASTERGRAFT putty (MG) and SYNTHES CHRONOS
granules. Control defects (void) were included. In the study the
bone void fillers were hydrated with sterile saline or autologous
bone marrow aspirate (BMA) harvested from the tibia prior to
implantation into the defect. The graphs in FIGS. 21A & 21B
show the results of micro-computed tomography (.mu.CT) for the
saline hydrated samples for bone volume (mm.sup.3) (FIG. 21A) and
bone mass (bone volume.times.bone density) (FIG. 21B) in units of
mg HA-hydroxyapatite.
[0029] FIGS. 22A-22C are graphs of the results at 4 weeks of a bone
healing model in the rat calvaria. Treatment groups included a
TCP/collagen composite without attached BMP binding peptide
(unmodified), a TCP/collagen composite with attached BMP binding
peptide (modified), and two commercially available bone void
fillers MEDTRONIC MASTERGRAFT putty (MG) and SYNTHES CHRONOS
granules. Control defects (void) were included. In the study the
bone void fillers were hydrated with sterile saline or autologous
bone marrow aspirate (BMA) harvested from the tibia prior to
implantation into the defect. The graphs in FIGS. 22A-22C show the
results of histology for the saline hydrated samples for osteogenic
cellular activity (FIG. 22A), bone area (FIG. 22B), and bone
maturity (FIG. 22C; scored on a scale from 1-4).
[0030] FIGS. 23A-23B are graphs of the results at 4 weeks of a bone
healing model in the rat calvaria. Treatment groups included a
TCP/collagen composite without attached BMP binding peptide
(unmodified), a TCP/collagen composite with attached BMP binding
peptide (modified), and two commercially available bone void
fillers MEDTRONIC MASTERGRAFT putty (MG) and SYNTHES CHRONOS
granules. Control defects (void) were included. In the study the
bone void fillers were hydrated with sterile saline or autologous
bone marrow aspirate (BMA) harvested from the tibia prior to
implantation into the defect. The graphs in FIGS. 23A-23B show the
results of micro-computed tomography (.mu.CT) for the BMA hydrated
samples for bone volume (mm.sup.3) (FIG. 23A) and bone mass (bone
volume.times.bone density) (FIG. 23B) in units of mg
HA-hydroxyapatite.
[0031] FIGS. 24A-24C are graphs of the results of a bone healing
model in the rat calvaria. Treatment groups included a TCP/collagen
composite without attached BMP binding peptide (unmodified), a
TCP/collagen composite with attached BMP binding peptide
(modified), and two commercially available bone void fillers
MEDTRONIC MASTERGRAFT putty (MG) and SYNTHES CHRONOS granules.
Control defects (void) were included. In the study the bone void
fillers were hydrated with sterile saline or autologous bone marrow
aspirate (BMA) harvested from the tibia prior to implantation into
the defect. The graphs in FIGS. 24A-24C show the results of
histology for the BMA hydrated samples for osteogenic cellular
activity (FIG. 24A), bone area (FIG. 24B), and bone maturity (FIG.
24C).
DETAILED DESCRIPTION
[0032] The presently disclosed subject matter provides compositions
and methods for promoting bone growth. The compositions and methods
of the presently disclosed subject matter are described in greater
detail herein below.
DEFINITIONS
[0033] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the presently disclosed
subject matter.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently disclosed subject
matter belongs.
[0035] Following long-standing patent law convention, the terms
"a", "an", and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a BMP binding peptide" or reference to "a 1 unit polyethylene
glycol ("mini-PEG" or "MP")" includes a plurality of such BMP
binding peptides or such polyethylene glycol units, and so
forth.
[0036] The term "substrate" is used, for the purposes of the
specification and claims, to refer to any material that is
biologically compatible with a BMP and to which a BMP binding
peptide can be attached for the purpose of capturing BMP onto the
substrate. In one embodiment the substrate is in the form of an
implantable device. Therefore, the terms "substrate", "implantable
device", "implantable bone graft material", "bone void filler", and
"bone graft substitute" are herein used interchangeably for the
purposes of the specification and claims to refer to an implantable
medical device for promoting bone formation. In one embodiment, the
implantable bone graft material comprises a resorbable ceramic and
a resorbable polymer, wherein the polymer comprises a covalently
attached BMP binding peptide. Accordingly, the term "substrate" is
used interchangeably herein with the term "polymer" for the
purposes of the specification and claims when referring to the
attachment of a BMP binding peptide to a "substrate" it is meant
that attachment of the BMP binding peptide is to a polymer
comprised in the substrate. Therefore, the attachment of a BMP
binding peptide to a "substrate" is referring to attachment of the
BMP binding peptide to the polymer comprised in the substrate.
[0037] In one embodiment, the implantable bone graft material of
the presently disclosed subject matter is a composite of a
resorbable ceramic (e.g., TCP) and a resorbable polymer and,
therefore, the terms "implantable device", "implantable bone graft
material", "bone void filler", "bone graft substitute",
"composite", and "collagen/TCP composite" are also in some cases
used interchangeably for the purposes of the specification and
claims. In one embodiment, the implantable bone graft material
comprises a composite of a resorbable ceramic and a resorbable
polymer. In one embodiment, the ceramic and the polymer are present
at a weight ratio ranging from about 10:1 ceramic to polymer to
about 2:1 ceramic to polymer. In one embodiment, the weight ratio
of the ceramic to the polymer is from about 2:1 (about 66% ceramic
to about 33% polymer), from about 3:1 (about 75% ceramic to about
25% polymer), from about 4:1 (about 80% ceramic to about 20%
polymer), from about 9:1 (about 90% ceramic to about 10% polymer),
from about 10:1 (about 99% ceramic to about 1% polymer).
[0038] The term "resorbable ceramic" is herein used interchangeably
for the purposes of the specification and claims with the term
"ceramic". The term "resorbable ceramic" is used herein, for the
purposes of the specification and claims, to refer to particulate
ceramic mineral or inorganic filler useful for promoting bone
formation. The term "resorbable ceramic" is herein used
interchangeably, for the purposes of the specification and claims,
with the terms "ceramic" and "inorganic fillers". The ceramics of
the presently disclosed subject matter include, by non-limiting
example, synthetic and naturally occurring inorganic fillers such
as alphatricalcium phosphate, beta-tricalcium phosphate,
tetra-tricalcium phosphate, dicalcium phosphate, calcium carbonate,
barium carbonate, calcium sulfate, barium sulfate, hydroxyapatite,
biphasic calcium phosphate (e.g., composite between HA and
.beta.-TOP), bioglass, bone particles, and combinations and
mixtures thereof. In certain embodiments the ceramic comprises a
polymorph of calcium phosphate. Preferably, the ceramic is
beta-tricalcium phosphate.
[0039] The term "resorbable polymer" is herein used interchangeably
for the purposes of the specification and claims with the term
"polymer". The term "resorbable polymer" is used herein, for the
purposes of the specification and claims, to refer to a natural
resorbable polymer or a synthetic resorbable polymer suitable for
use in the implantable medical device of the presently disclosed
subject matter. Natural resorbable polymers of the of the presently
disclosed subject matter include, by non-limiting example,
collagen, fibrillar collagen, Type I collagen, bovine collagen,
porcine collagen, human recombinant collagen, keratin, silk,
polysaccharides, dextran, cellulose derivatives, oxidized
cellulose, oxidized regenerated cellulose, carboxymethylcellulose
(CMC), hydroxypropylmethylcellulose (HPMC), chitosan, chitin, and
hyaluronic acid. In some embodiments, a BMP binding peptide is
covalently attached to the natural resorbable polymer. In some
cases, the term "resorbable polymer" is used herein, for the
purposes of the specification and claims, to refer to a synthetic
resorbable polymer. Synthetic resorbable polymers of the presently
disclosed subject matter include, by non-limiting example,
aliphatic polyesters, polyanhydrides and poly(orthoester)s, and
homopolymers, such as, for example, poly(glycolide) (PGA),
poly(lactide) (PLLA), poly(.epsilon.-caprolactone),
poly(trimethylene carbonate) and poly(p-dioxanone), and copolymers,
such as for example poly(lactide-co-glycolide)(PLGA),
poly(.epsilon.-caprolactone-co-glycolide),
poly(glycolide-co-trimethylene carbonate), tyrosine-based
polycarbonates, and tyrosine-based polyarylates. The synthetic
resorbable polymers of the presently disclosed subject matter can
be derivatives of the foregoing polymers, and/or statistically
random copolymers, segmented copolymers, block copolymers, or graft
copolymers of the foregoing polymers. In some embodiments, a BMP
binding peptide is attached to the synthetic resorbable polymer of
the presently disclosed subject matter. Synthetic resorbable
polymers of the presently disclosed subject matter to which a BMP
binding peptide can be attached include, by non-limiting example,
aliphatic polyesters, polyanhydrides, and poly(orthoester)s. The
synthetic resorbable polymers of the presently disclosed subject
matter to which a BMP binding peptide can be attached can be
derivatives of the foregoing polymers, and/or statistically random
copolymers, segmented copolymers, block copolymers, or graft
copolymers of the foregoing polymers. In one example, a synthetic
"resorbable polymer" to which a BMP binding peptide can be
attached, for the purposes of the specification and claims, means a
polyanhydride polymer where the anhydride groups are not present in
the backbone of the polymer and the portion of the polyanhydride
polymer chain that will not be hydrolyzed in vivo is small enough
to allow efficient clearance through the renal system. Polymaleic
anhydride (PMA) having molecular weight of about 5,000 Dalton or
less is one example of a resorbable polyanhydride polymer for the
purposes of the specification and claims. In another example, the
synthetic resorbable polymer to which the BMP binding peptide is
attached is a block co-polymer of polymaleic anhydride having
molecular weight of about 5,000 Dalton or less and a co-polymer
comprising a biodegradable functionality, wherein the co-polymer is
selected from the group consisting of polylactic acid (PLA),
polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA),
polycaprolactone, poly-3-hydroxybutyrate, poly(p-dioxanone) and
copolymers thereof, polyhydroxyalkanoate, poly(propylene fumarate),
poly(ortho esters), and polyanhydrides, and combinations
thereof.
[0040] The term "BMP binding peptide" is used herein, for the
purposes of the specification and claims, to refer to an amino acid
chain comprising a peptide that can bind to a bone morphogenic
protein (BMP) (i.e., the BMP is the binding "target" of the BMP
binding peptide). The BMPs are members of the transforming growth
factor beta (TGF-.beta.) superfamily that share a set of conserved
cysteine residues and a high level of sequence identity overall.
Over 15 different BMPs have been identified, and most BMPs
stimulate the cascade of events that lead to new bone formation and
are considered to be osteoinductive factors (see, e.g., U.S. Pat.
Nos. 5,013,649; 5,635,373; 5,652,118; and 5,714,589; also reviewed
by Reddi and Cunningham (1993) J. Bone Miner. Res. 8 Supp. 2:
S499-S502; Issack and DiCesare (2003) Am. J. Orthop. 32: 429-436;
and Sykaras & Opperman (2003) J. Oral Sci. 45: 57-73). The
BMP's, including BMP-2 and BMP-7, have shown clinical benefit in
the treatment of bone fractures and spine fusions. Preferably, the
BMP binding peptides of the presently disclosed subject matter bind
to one or more of BMP-2, BMP-4, BMP-6, or BMP-7. In one embodiment,
the BMP binding peptide is set forth in US Patent Application
Publication No. US20060051395A1. In one embodiment, the BMP binding
peptide is set forth in US Patent Application Publication No.
US20060051395A1 and is identified therein as one of SEQ ID No's:
11-28, 44-74, or 77-94 (i.e., these are the SEQ ID NO identifiers
for the previously published patent application rather than this
current one). In one embodiment, the BMP binding peptide is set
forth in US Patent Application Publication No. US20090098175A1. In
one embodiment, the BMP binding peptide is set forth in US Patent
Application Publication No: US20090098175A1 and is identified
therein as one of SEQ ID No's: 1-12 (i.e., these are the SEQ ID NO
identifiers for the previously published patent application rather
than this current one). In one embodiment, the BMP binding peptide
is set forth in US Patent Application Publication No. US
2006/0051395A1 or US 2009/0098175A1, and is any one of SEQ ID NOs:
1-10 (see Table 1 herein below). The BMP binding peptides of the
presently disclosed subject matter can include naturally occurring
amino acids, synthetic amino acids, genetically encoded amino
acids, non-genetically encoded amino acids, and combinations
thereof; however, an antibody is specifically excluded from the
scope and definition of a BMP binding peptide of the presently
disclosed subject matter. A BMP binding peptide used in accordance
with the presently disclosed subject matter can be produced by
chemical synthesis, recombinant expression, biochemical, or
enzymatic fragmentation of a larger molecule, chemical cleavage of
larger molecule, a combination of the foregoing or, in general,
made by any other method in the art, and preferably isolated.
[0041] BMP binding peptides useful in the presently disclosed
subject matter also include peptides having one or more
substitutions, additions, and/or deletions of residues relative to
the sequence of an exemplary binding peptide shown in US Patent
Application Publication No. US 2006/0051395A1 or US 2009/0098175A1,
as long as the binding properties of the exemplary BMP binding
peptides to their BMP targets are substantially retained. Thus, the
BMP binding peptides include those that differ from the exemplary
sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids,
and include BMP binding peptides that share sequence identity with
the exemplary peptide of at least 65%, 66%, 67%, 68%, 69%, 70%,
71%; 72%, 73%, 74%, 75%, 80%, 81%; 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%; 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
greater sequence identity. Sequence identity can be calculated
manually or it can be calculated using a computer implementation of
a mathematical algorithm, for example, GAP, BESTFIT, BLAST, FASTA,
and TFASTA, or other programs or methods known in the art.
Alignments using these programs can be performed using the default
parameters. A BMP binding peptide can have an amino acid sequence
consisting essentially of a sequence of an exemplary BMP binding
peptide or a BMP binding peptide can have one or more different
amino acid residues as a result of substituting an amino acid
residue in the sequence of the exemplary binding peptide with a
functionally similar amino acid residue (a "conservative
substitution"); provided that the peptide containing the
conservative substitution will substantially retain the BMP binding
activity of the exemplary BMP binding peptide not containing the
conservative substitution. Examples of conservative substitutions
include the substitution of one non-polar (hydrophobic) residue
such as alanine, isoleucine, valine, leucine, or methionine for
another; the substitution between asparagine and glutamine, the
substitution of one large aromatic residue such as tryptophan,
tyrosine, or phenylalanine for another; the substitution of one
small polar (hydrophilic) residue for another such as between
glycine, threonine, serine, and proline; the substitution of one
basic residue such as lysine, arginine, or histidine for another;
or the substitution of one acidic residue such as aspartic acid or
glutamic acid for another. Accordingly, BMP binding peptides useful
in the presently disclosed subject matter include those peptides
that are conservatively substituted variants of the BMP binding
peptides in US Patent Application Publication Nos. US
2006/0051395A1 and US 2009/0098175A1, and those peptides that are
variants having at least 65% sequence identity or greater to the
BMP binding peptides in US Patent Application Publication Nos. US
2006/0051395A1 and US 2009/0098175A1, wherein all of the variant
BMP binding peptides useful in the presently disclosed subject
matter substantially retain the ability to bind to BMP. In one
embodiment of the presently disclosed subject matter, a useful BMP
binding peptide comprises a sequence selected from the group
consisting of SEQ ID NOs: 1-7 (see Table 1 herein below),
conservatively substituted variants of SEQ ID NOs: 1-7, and
variants having at least 65% sequence identity to SEQ ID NOs: 1-7,
wherein the variant BMP binding peptide substantially retains the
ability to bind BMP.
TABLE-US-00001 TABLE 1 BMP Binding Peptides SEQ ID Amino acid
sequence NO: (single letter code) 1 GGGAWEAFSSLSGSRV 2
GGALGFPLKGEVVEGWA 3 WEAFSSLSG 4 LGFPLKGEV 5 ssGPREIWDSLVGVVNPGWsr 6
ssGGVGGWALFETLRGKEVsr 7 ssVAEWALRSWEGMRVGEAsr 8 WXXFE(S/T)LXGXEX 9
(W/F/Y)XXFX(S/T/A/G)L 10 (L/V)XFPL(K/R)G
[0042] BMP binding peptides can include L-form amino acids, D-form
amino acids, or a combination thereof. Representative
non-genetically encoded amino acids include but are not limited to
2-aminoadipic acid; 3-aminoadipic acid; .beta.-aminopropionic acid;
2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid);
6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid;
3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric
acid; desmosine; 2,2'-diaminopimelic acid; 2,3-diaminopropionic
acid; N-ethylglycine; N-ethylasparagine; hydroxylysine;
allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline;
isodesmosine; allo-isoleucine; N-methylglycine (sarcosine);
N-methylisoleucine; N-methylvaline; norvaline; norleucine;
ornithine; and 3-(3,4-dihydroxyphenyl)-L-alanine ("DOPA").
Representative derivatized amino acids include, for example, those
molecules in which free amino groups have been derivatized to form
amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups can be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine can be derivatized
to form N-im-benzylhistidine.
[0043] Further, a BMP binding peptide according to the presently
disclosed subject matter can include one or more modifications,
such as by addition of chemical moieties, or substitutions,
insertions, and deletions of amino acids, where such modifications
provide for certain advantages in its use, such as to facilitate
attachment to the polymer with or without a spacer or to improve
peptide stability. The term "spacer" is used herein, for the
purposes of the specification and claims, to refer to a compound or
a chemical moiety that is optionally inserted between a BMP binding
peptide and the polymer. In some embodiments, the spacer also
serves the function of a linker (i.e. to attach the BMP binding
peptide to the polymer). Therefore, the terms "linker" and "spacer"
can be used interchangeably herein, for the purposes of the
specification and claims, when performing the dual functions of
linking (attaching) the peptide to the polymer and spacing the BMP
binding peptide from the polymer. In some cases the spacer can
serve to position the BMP binding peptide at a distance and in a
spatial position suitable for BMP binding and capture and/or in
some cases the spacer can serve to increase the solubility of the
BMP binding peptide. Spacers can increase flexibility and
accessibility of the BMP binding peptide to BMP, as well as
increase the BMP binding peptide density on the polymer surface.
Virtually all chemical compounds, moieties, or groups suitable for
such a function can be used as a spacer unless adversely affecting
the binding behavior to such an extent that binding of the BMP to
the BMP binding peptides is prevented or substantially impaired.
Thus, the term "BMP binding peptide" encompasses any of a variety
of forms of BMP binding peptide derivatives including, for example,
amides, conjugates with proteins, conjugates with polyethylene
glycol or other polymers, cyclic peptides, polymerized peptides,
peptides having one or more amino acid side chain group protected
with a protecting group, and peptides having a lysine side chain
group protected with a protecting group. Any BMP binding peptide
derivative that has substantially retained BMP binding
characteristics can be used in the practice of the presently
disclosed subject matter.
[0044] Further, a chemical group can be added to the N-terminal
amino acid of a binding peptide to block chemical reactivity of the
amino terminus of the peptide. Such N-terminal groups for
protecting the amino terminus of a peptide are well known in the
art, and include, but are not limited to, lower alkanoyl groups,
acyl groups, sulfonyl groups, and carbamate forming groups.
Preferred N-terminal groups can include acetyl,
9-fluorenylmethoxycarbonyl (Fmoc), and t-butoxy carbonyl (Boc). A
chemical group can be added to the C-terminal amino acid of a
synthetic binding peptide to block chemical reactivity of the
carboxy terminus of the peptide. Such C-terminal groups for
protecting the carboxy terminus of a peptide are well known in the
art, and include, but are not limited to, an ester or amide group.
Terminal modifications of a peptide are often useful to reduce
susceptibility by protease digestion, and to therefore prolong a
half-life of a BMP binding peptide in the presence of biological
fluids where proteases can be present. In addition, as used herein,
the term "BMP binding peptide" also encompasses a peptide wherein
one or more of the peptide bonds are replaced by pseudopeptide
bonds including but not limited to a carba bond
(CH.sub.2--CH.sub.2), a depsi bond (CO--O), a hydroxyethylene bond
(CHOH--CH.sub.2), a ketomethylene bond (CO--CH.sub.2), a
methylene-oxy bond (CH.sub.2--O), a reduced bond (CH.sub.2--NH), a
thiomethylene bond (CH.sub.2--S), an N-modified bond (--NRCO), and
a thiopeptide bond (CS--NH).
[0045] The BMP binding peptides are covalently attached to the
polymer. The term "attached" in reference to a BMP binding peptide
of the presently disclosed subject matter being attached to a
polymer means, for the purposes of the specification and claims, a
BMP binding peptide being immobilized on the polymer by covalent
attachment by any means that will enable binding of BMP onto the
peptide-modified polymer such that the bound BMP retains biological
growth factor activity. In one embodiment, the linkers/spacers for
use in attaching BMP binding peptides to polymers have at least two
chemically active groups (functional groups), of which one group
binds to the polymer, and a second functional group binds to the
BMP binding peptide or in some cases it binds to the "spacer"
already attached to the BMP binding peptide. Preferably, the
attachment of the BMP binding peptides to the polymer is effected
through a spacer. Virtually all chemical compounds, moieties, or
groups suitable for such a function can be used as a spacer unless
adversely affecting the BMP peptide binding behavior to such an
extent that binding of the BMP to the BMP binding peptides is
prevented or substantially impaired.
[0046] Again, the terms "linker" and "spacer" can be used
interchangeably herein, for the purposes of the specification and
claims, when performing the dual functions of linking (attaching)
the BMP binding peptide to the polymer and spacing the peptide from
the polymer. In many embodiments herein, the linkers used to attach
the BMP binding peptide to the polymer function as both a linker
and a spacer. For example, a linker molecule can have a linking
functional group on either end while the central portion of the
molecule functions as a spacer. The BMP binding peptides of the
presently disclosed subject matter can comprise a functional group
that is intrinsic to the BMP binding peptide (e.g., amino groups on
lysine), or the functional group can be introduced into the BMP
binding peptide by chemical modification to facilitate covalent
attachment of the BMP binding peptide to the polymer. Similarly,
the polymer can comprise a functional group that is intrinsic to
the polymer (e.g., amino groups on collagen), or the polymer can be
modified with a functional group to facilitate covalent attachment
to the BMP binding peptide. The BMP binding peptide can be
covalently attached to the polymer with or without one or more
spacer molecules.
[0047] For example, linkers/spacers are known to those skilled in
the art to include, but are not limited to, chemical compounds
(e.g., chemical chains, compounds, reagents, and the like). The
linkers/spacers may include, but are not limited to,
homobifunctional linkers/spacers and heterobifunctional
linkers/spacers. Heterobifunctional linkers/spacers, well known to
those skilled in the art, contain one end having a first reactive
functionality (or chemical moiety) to specifically link a first
molecule (e.g., polymer), and an opposite end having a second
reactive functionality to specifically link to a second molecule
(e.g., BMP binding peptide). It is evident to those skilled in the
art that a variety of bifunctional or polyfunctional reagents, both
homo- and hetero-functional can be employed as a linker/spacer with
respect to the presently disclosed subject matter such as, for
example, those described in the catalog of the PIERCE CHEMICAL CO.,
Rockford, Ill.; amino acid linkers/spacers that are typically a
short peptide of between 3 and 15 amino acids and often containing
amino acids such as glycine, and/or serine; and wide variety of
polymers including, for example, polyethylene glycol. In one
embodiment, representative linkers/spacers comprise multiple
reactive sites (e.g., polylysines, polyornithines, polycysteines,
polyglutamic acid and polyaspartic acid) or comprise substantially
inert peptide spacers (e.g., polyglycine, polyserine, polyproline,
polyalanine, and other oligopeptides comprising alanyl, serinyl,
prolinyl, or glycinyl amino acid residues). In one embodiment,
representative spacers between the reactive end groups in the
linkers include, by non-limiting example, the following functional
groups: aliphatic, alkene, alkyne, ether, thioether, amine, amide,
ester, disulfide, sulfone, and carbamate, and combinations thereof.
The length of the spacer can range from about 1 atom to 200 atoms
or more. In one embodiment, linkers/spacers comprise a combination
of one or more amino acids and another type of spacer or linker
such as, for example, a polymeric spacer.
[0048] Suitable polymeric spacers/linkers are known in the art, and
can comprise a synthetic polymer or a natural polymer.
Representative synthetic polymer linkers/spacers include but are
not limited to polyethers (e.g., poly(ethylene glycol) ("PEG"), 11
unit polyethylene glycol ("PEG10"), or 1 unit polyethylene glycol
("mini-PEG" or "MP"), poly(propylene glycol), poly(butylene
glycol), polyesters (e.g., polylactic acid (PLA) and polyglycolic
acid (PGA)), polyamines, polyamides (e.g., nylon), polyurethanes,
polymethacrylates (e.g., polymethylmethacrylate; PMMA), polyacrylic
acids, polystyrenes, and polyhexanoic acid, and combinations
thereof. Polymeric spacers/linkers can comprise a diblock polymer,
a multi-block copolymer, a comb polymer, a star polymer, a
dendritic or branched polymer, a hybrid linear-dendritic polymer, a
branched chain comprised of lysine, or a random copolymer. A
spacer/linker can also comprise a mercapto(amido)carboxylic acid,
an acrylamidocarboxylic acid, an acrlyamido-amidotriethylene
glycolic acid, 7-aminobenzoic acid, and derivatives thereof.
[0049] In one embodiment, the binding peptide comprises one or more
modifications to the peptide N-terminus, peptide C-terminus, or
within the peptide amino acid sequence, to facilitate covalent
attachment of the binding peptide to a substrate polymer with or
without a spacer. The binding peptides can comprise one or more
modifications including, but not limited to, addition of one or
more groups such as hydroxyl, thiol, carbonyl, carboxyl, ester,
carbamate, hydrazide, hydrazine, isocyanate, isothiocyanate, amino,
alkene, dienes, maleimide, ,.beta.-unsaturated carbonyl, alkyl
halide, azide, epoxide, N-hydroxysuccinimide (NHS) ester, lysine,
or cysteine. In addition, a binding peptide can comprise one or
more amino acids that have been modified to contain one or more
chemical groups (e.g., reactive functionalities such as fluorine,
bromine, or iodine) to facilitate linking the binding peptide to a
spacer molecule or to the substrate polymer to which the binding
peptide will be attached.
[0050] The BMP binding peptides can be covalently attached to the
substrate polymer through one or more anchoring (or linking) groups
on the substrate polymer and the BMP binding peptide. The BMP
binding peptides of the presently disclosed subject matter can
comprise a functional group that is intrinsic to the BMP binding
peptide, or the BMP binding peptide can be modified with a
functional group to facilitate covalent attachment to the substrate
polymer with or without a spacer. Representative anchoring (or
linking) groups include by non-limiting example hydroxyl, thiol,
carbonyl, carboxyl, ester, carbamate, hydrazide, hydrazine,
isocyanate, isothiocyanate, amino, alkene, dienes, maleimide,
,.beta..sup..about.-unsaturated carbonyl, alkyl halide, azide,
epoxide, NHS ester, lysine, and cysteine groups on the surface of
the substrate polymer. The anchoring (or linking) groups can be
intrinsic to the material of the substrate polymer (e.g., amino
groups on a collagen or on a polyamine-containing polymer) or the
anchoring groups can be introduced into the substrate polymer by
chemical modification.
[0051] By way of non-limiting example, in one embodiment, a BMP
binding peptide is attached to a substrate polymer in a two step
process (see FIG. 1; Mikulec & Puleo, 1996, J. Biomed. Mat.
Res., Vol 32, 203-08). In the first step, the anchoring (or
linking) groups (i.e., amino groups on a collagen for example) on
the surface of a substrate polymer are activated by an acylating
reagent (4-nitrophenyl chloroformate). In the second step, a lysine
residue which has been introduced along with a PEG10 spacer at the
C-terminus of a BMP binding peptide is reacted with the activated
chloroformate intermediate on the substrate polymer surface,
resulting in attachment of the BMP binding peptide to the
substrate.
[0052] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to a substrate polymer
comprising an amino functional group (see FIG. 2). FIG. 2
exemplifies attachment of a BMP binding peptide comprising an
aldehyde group at one terminus to a substrate polymer that
comprises an amino functional group. The BMP binding peptide
comprising an aldehyde functional group is treated with the
substrate polymer amino groups under reductive amination conditions
to give attached BMP binding peptide. In another embodiment not
depicted in FIG. 2, a BMP binding peptide comprising an amine
functional group is reacted with the substrate polymer amino groups
via a homobifunctional linker such as, for example, glutaraldehyde,
to yield a covalently attached BMP binding peptide (Simionescu et.
al., 1991, J. Biomed Mater. Res., 25:1495-505).
[0053] By way of non-limiting example, in one embodiment, a
homobifunctional linker possessing N-hydroxysuccinimide esters at
both ends is reacted at one end with the BMP binding peptide having
an amino group (FIG. 3). The BMP binding peptide with attached
linking group is then reacted through the remaining
N-hydroxysuccinimide ester with an amino group on the substrate
polymer to form a peptide-substrate conjugate (FIG. 3). The
homobifunctional N-hydroxysuccinimide ester depicted in FIG. 3 is
BS.sup.3 crosslinking reagent (THERMO SCIENTIFIC, Rockford, Ill.).
As stated herein previously, the length and type of spacer groups
between the two reactive end groups on the NHS ester can vary.
[0054] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to a substrate polymer
having amino functional groups in a two-step process using a
disulfide linkage (see FIG. 4; Hermanson, G. T. Bioconjugate
Techniques; Academic Press: San Diego, 1996; pp. 150-151). First,
the substrate polymer containing amino groups is reacted with
2-iminothiolane resulting in the introduction of thiol groups on
the substrate polymer. Simultaneous addition of
4,4'-dithiodipyridine or 6,6'-dithiodinicotinic acid results in
rapid capping of the newly-introduced thiol as a pyridyl disulfide.
Second, the BMP binding peptide containing a free thiol is attached
covalently to the substrate polymer through a thiol-disulfide
exchange resulting in a disulfide bond between the substrate and
BMP binding peptide.
[0055] By way of non-limiting example, in one embodiment, a BMP
binding peptide is attached covalently to a substrate polymer
comprising amino functional groups in a similar process using a
disulfide linkage (see FIG. 5; Carlsson et al., 1978, Biochem. J.,
173:723-37). The substrate polymer is first functionalized with
amine groups using known methods (if the amino groups are not
intrinsic to the material of the substrate). Next, a
thiol-cleavable, heterobifunctional (amine- and
sulfhydryl-reactive) compound (LC-SPDP; THERMO SCIENTIFIC,
Rockford, Ill.) is reacted with the amino-functionalized substrate
polymer. The BMP binding peptide is reacted with the LC-SPDP
modified substrate polymer.
[0056] By way of non-limiting example, in one embodiment, a BMP
binding peptide is attached covalently to a substrate polymer via a
thioether bond formed by reaction of a thiol and maleimide
(O'Sullivan et al., 1979, Anal. Biochem., 100:100-8). In one
embodiment, the maleimide is added to a substrate polymer
comprising amino functional groups and then the modified substrate
polymer is reacted with a BMP binding peptide having a free thiol
group. Alternatively, in one embodiment, the same chemical scheme
is utilized but with the substrate polymer modified with a thiol
group and the BMP binding peptide modified with the maleimido
group.
[0057] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached through a non-backbone
anhydride group of a polyanhydride polymer, polymaleic acid (PMA),
through a reactive lysine group on the BMP binding peptide shown in
the schematic diagram in FIG. 6 (Pompe, et al., 2003,
Biomacromolecules, 4(4):1072-9).
[0058] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to a chitosan. The chemical
scheme is shown in FIG. 7. First, the amino group on chitosan is
protected with phthaloyl group. The hydroxyl group on chitosan is
then reacted with chloroacetic acid to give an acid handle on
chitosan. The BMP binding peptide amine is coupled to the acid
group on the chitosan to give the BMP binding peptide-chitosan
conjugate. The phthaloyl group is then removed using hydrazine.
[0059] By way of non-limiting example, in one embodiment a BMP
binding peptide is covalently attached to a chitosan. The chemical
scheme is shown in FIG. 8. First, the amino group on chitosan is
protected with a phthaloyl group. The hydroxyl group on chitosan is
then converted to a bromo group under standard halogenation
conditions. The BMP binding peptide amine is reacted with
halogenated chitosan to give the BMP binding peptide-chitosan
conjugate. The phthaloyl group is finally removed by reacting with
hydrazine.
[0060] By way of non-limiting example, in one embodiment a BMP
binding peptide is covalently attached to chitosan through the
amino group on chitosan. For example, a chemical scheme using a
homobifunctional N-hydroxysuccinimide ester, such as that described
for FIG. 3, is useful for attaching the BMP binding peptide through
the amino group on chitosan.
[0061] By way of non-limiting example, in one embodiment a BMP
binding peptide is covalently attached to a hyaluronan (HA). The
chemical scheme is shown in FIG. 9. The hyaluronan is chemically
modified at the carboxylic acid group on the glucuronate units. The
carboxylic group is activated using carbonyl diimidazole (CDI). The
activated HA is then reacted with the amino group of BMP binding
peptide to yield the peptide-HA conjugate.
[0062] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to a hyaluronan (HA). The
chemical scheme is shown in FIG. 10. Hyaluronan is chemically
modified at the carboxylic acid group on the glucuronate units. The
carboxylic group is activated using water soluble carbodiimide such
as 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC) along with
HOBt. The activated HA is coupled with the amino group of a BMP
binding peptide to yield the peptide-HA conjugate.
[0063] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to cellulose. The chemical
scheme is shown in FIG. 11. Hydroxyl groups on the polysaccharide
are first reacted with epichlorohydrin to introduce an epoxide.
Ring opening of the epoxide by reaction with aqueous ammonia
provides free amino groups that can function as anchors for peptide
conjugation using chemistry described in previous embodiments
(Matsumoto, et al. (1980) J. Biochem., 87: 535-540).
[0064] By way of non-limiting example, in one embodiment, a BMP
binding peptide is covalently attached to oxidized cellulose. The
chemical scheme is shown in FIG. 12. Sulfhydryl groups are
introduced by reaction of carboxylates on the oxidized cellulose
with cystamine and EDC followed by reduction with dithiothreitol
(DTT). Activation of sulfhydryls with 6,6'-dithiodinicotinic acid
(DTNA) followed by a sulfhydryl-containing BMP binding peptide
results in covalent attachment of the peptide to the oxidized
cellulose through a disulfide bond. In another embodiment not
depicted in FIG. 12, the sulfhydryl modified oxidized cellulose is
reacted with a maleimide or other Michael acceptor on the BMP
binding peptide resulting in covalent attachment through a
thioether bond. In another embodiment not depicted in FIG. 12,
carboxyl groups on oxidized cellulose are activated with EDC and
1-hydroxybenzotriazole (HOBt) followed by reaction with BMP-2
binding peptide containing a free amine group. This results in
conjugation of peptide to the oxidized cellulose through an amide
bond (this chemistry is exemplified in FIG. 10). In another
embodiment not depicted in FIG. 12, a BMP-2 binding peptide can be
covalently attached to oxidized cellulose through the aldehyde
groups on the oxidized cellulose. In this example, a BMP-2 binding
peptide having a free amine undergoes reductive amination with the
aldehyde group on the polymer substrate to yield an amine bond as
shown in FIG. 2 (the chemistry is the same as that in FIG. 2 except
that the functional groups on the polymer substrate and BMP-2
binding peptide are reversed).
[0065] By way of non-limiting example, in one embodiment, a BMP-2
binding peptide can be covalently attached to an oxidized dextran
polymer substrate by reductive amination as described above for
oxidized cellulose. More specifically, a BMP-2 binding peptide
having a free amine undergoes reductive amination with the aldehyde
group on the polymer substrate to yield an amine bond as shown in
FIG. 2 (the chemistry is the same as that in FIG. 2 except that the
functional groups on the polymer substrate and BMP-2 binding
peptide are reversed).
[0066] By way of non-limiting example, in one embodiment, more than
one BMP binding peptide is attached to a substrate polymer.
Attaching multiple BMP binding peptides to a single substrate
polymer is only limited by practical considerations related to the
method of attachment. For example, in one embodiment, two different
BMP binding peptides are covalently attached to a substrate polymer
using any of the chemical schemes shown in FIGS. 1-12. In each of
the chemical schemes depicted in FIGS. 1-12, the substrate polymer
having a functional group is reacted with two or more different BMP
binding peptides that each comprise a functional group to
covalently attach the two or more BMP binding peptides to the
substrate polymer based on simple competition between the BMP
binding peptides. In particular, for example, in the case of the
chemical schemes depicted in FIGS. 1 and 2, the modified substrate
is reacted with two or more different BMP binding peptides that
each comprise an amino group or an aldehyde group (i.e., the two
different BMP binding peptides replace the single peptide depicted
in FIGS. 1 and 2), to covalently attach the two or more BMP binding
peptides to the substrate polymer through the amino or aldehyde
group, respectively. In the case of the chemical schemes depicted
in FIGS. 4 and 5, the modified substrate polymer is reacted with
two or more different BMP binding peptides that each comprise a
thiol group, to covalently attach the two or more BMP binding
peptides to the substrate polymer through the thiol group (i.e.,
the "HS-Peptide" in FIGS. 4 and 5 in this embodiment represents two
or more different BMP binding peptides).
[0067] By way of non-limiting example, in one embodiment, two
different BMP binding peptides are covalently attached to a
substrate polymer comprising amino groups using the chemical scheme
shown in FIG. 13. In this embodiment, the amino groups on the
substrate polymer are modified with maleimido groups. The modified
substrate polymer is then reacted with a BMP binding peptide
comprising both a thiol group and an aldehyde group to covalently
attach the BMP binding peptide to the substrate polymer through the
thiol group. Next, the substrate-BMP binding peptide conjugate is
reacted with another BMP binding peptide having a hydrazine group,
to give a second covalent bond through the aldehyde-hydrazine (see
FIG. 13). Alternatively, in one embodiment, the same chemical
scheme is utilized but with the substrate polymer modified with a
thiol group and the BMP binding peptide modified with the maleimido
group. In addition to using this scheme to covalently attach
different BMP binding peptides, the scheme is also useful for
attaching the same BMP binding peptide.
[0068] The presently disclosed subject matter provides compositions
and methods for promoting bone growth. In one embodiment, an
implantable bone graft material is provided consisting essentially
of a resorbable .beta.-TCP and a resorbable polymer, wherein the
.beta.-TCP has a total porosity of about 50% or greater and a
particle size ranging from about 100 micron to about 300 micron. In
one embodiment, the resorbable .beta.-TCP and the resorbable
polymer are in the form of a composite. In one embodiment, the
composite is in the form of a sponge, a granulized sponge, a putty,
or a strip. In one embodiment, the total porosity of the .beta.-TCP
is about 70%. In one embodiment, the diameter of the pores in the
.beta.-TCP is less than 100 micron. In one embodiment, the polymer
is selected from the group consisting of collagen, fibrillar
collagen, Type I collagen, bovine collagen, keratin, silk,
polysaccharides, dextran, cellulose derivatives, oxidized
cellulose, oxidized regenerated cellulose, carboxymethylcellulose,
hydroxypropylmethylcellulose, chitosan, chitin, hyaluronic acid,
aliphatic polyesters, polyanhydrides, poly(orthoester)s,
poly(glycolide), poly(lactide), poly(.epsilon.-caprolactone),
poly(trimethylene carbonate), poly(p-dioxanone),
poly(lactide-co-glycolide),
poly(.epsilon.-caprolactone-co-glycolide),
poly(glycolide-co-trimethylene carbonate),
poly(lactide-co-.epsilon.-caprolactone), tyrosine-based
polycarbonates, tyrosine-based polyarylates, and copolymers and
derivatives thereof. In one embodiment, the .beta.-TCP and the
polymer are present at a weight ratio ranging from about 10:1
.beta.-TCP to polymer to about 2:1 .beta.-TOP to polymer. In one
embodiment, the polymer is collagen and the weight ratio of
.beta.-TOP to collagen is about 4:1 (80% .beta.-TOP to about 20%
collagen).
[0069] In one embodiment, a method is provided for promoting bone
growth in a subject by delivering the implantable bone graft
material consisting essentially of a .beta.-TOP and a resorbable
polymer to a subject, wherein the presence of the graft material
promotes bone growth. In one embodiment, a method is provided for
promoting spinal fusion in a subject by delivering the implantable
bone graft material consisting essentially of a .beta.-TOP and a
resorbable polymer to a subject, wherein the presence of the graft
material promotes spinal fusion. In one embodiment, the implantable
bone graft material consisting essentially of a .beta.-TOP and a
resorbable polymer is in the form of a composite. In one
embodiment, the composite is in the form of a sponge, a granulized
sponge, a putty, or a strip. In one embodiment, the implantable
bone graft material is mixed or contacted with saline, bone marrow
aspirate (BMA), blood, platelet rich plasma (PRP), or recombinant
BMP, or combinations or derivatives thereof prior to or during
delivery to the subject.
[0070] In one embodiment of the presently disclosed subject matter,
an implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a composite. In one embodiment, the composite is in
the form of a sponge, a granulized sponge, a putty, or a strip. In
one embodiment, the resorbable ceramic and the resorbable polymer
are in the form of an injectable bone graft material. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a moldable cement. In one embodiment, the BMP
binding peptide binds one or more of BMP-2, BMP-4, BMP-6, or
BMP-7.
[0071] In one embodiment of the presently disclosed subject matter,
an implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide, wherein the
ceramic is selected from the group consisting of calcium phosphate,
tetra-tricalcium phosphate, dicalcium phosphate, calcium carbonate,
calcium sulfate, barium carbonate, barium sulfate, alphatricalcium
phosphate (.alpha.-TOP), tricalcium phosphate (TCP), betatricalcium
phosphate (.beta.-TOP), hydroxyapatite (HA), biphasic calcium
phosphate (e.g., composite between HA and .beta.-TOP), bioglass,
bone particles, and combinations and mixtures thereof. In one
embodiment, the polymer is selected from the group consisting of
collagen, fibrillar collagen, Type I collagen, porcine collagen,
human recombinant collagen, bovine collagen, keratin, silk,
polysaccharides, dextran, cellulose derivatives, oxidized
cellulose, oxidized regenerated cellulose, carboxymethylcellulose,
hydroxypropylmethylcellulose, chitosan, chitin, and hyaluronic
acid. In one embodiment, the polymer is a block co-polymer of
polymaleic anhydride having molecular weight of about 5,000 Dalton
or less and a co-polymer comprising a biodegradable functionality,
wherein the co-polymer is selected from the group consisting of
polylactic acid, polyglycolic acid, polylactic-co-glycolic acid,
polycaprolactone, poly-3-hydroxybutyrate, poly(p-dioxanone) and
copolymers thereof, polyhydroxyalkanoate, poly(propylene fumarate),
poly(ortho esters), and polyanhydrides, and combinations
thereof.
[0072] In one embodiment of the presently disclosed subject matter,
an implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide, and wherein
the BMP binding peptide comprises one or more modifications to the
peptide N-terminus, peptide C-terminus, or within the peptide amino
acid sequence to facilitate linkage of the BMP binding peptide to
the polymer with or without a spacer, wherein the modification is
selected from the group consisting of aldehyde group, hydroxyl
group, thiol group, amino group, amino acids, lysine, cysteine,
acetyl group, polymers, synthetic polymers, polyethers,
poly(ethylene glycol) ("PEG"), a 11 unit polyethylene glycol
("PEG10"), and a 1 unit polyethylene glycol ("mini-PEG" or "MP"),
and combinations thereof. In one embodiment, the BMP binding
peptide is attached to the polymer with or without a spacer. In one
embodiment, the BMP binding peptide comprises the mini-PEG
modification and the thiol group modification.
[0073] In one embodiment of the presently disclosed subject matter,
an implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide, and wherein
the ceramic and the polymer are present at a weight ratio ranging
from about 10:1 ceramic to polymer to about 2:1 ceramic to polymer.
In one embodiment, the ceramic is .beta.-TCP and the polymer is
bovine Type I fibrillar collagen. In one embodiment, the weight
ratio of .beta.-TCP to bovine Type I fibrillar collagen is about
4:1 (about 80% .beta.-TCP to about 20% collagen). In one
embodiment, the ceramic is .beta.-TCP having a total porosity of
about 50% or greater and a particle size ranging from about 100
micron to about 300 micron. In one embodiment, the polymer is
collagen, and the ceramic is .beta.-TCP having total porosity of
about 70%, a particle size ranging from about 100 micron to about
300 micron, and a pore diameter less than 100 micron.
[0074] In one embodiment of the presently disclosed subject matter,
an implantable bone graft material is provided comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide, wherein the
covalently attached BMP binding peptide is present at a range of
about 1-200 .mu.mol peptide/gram polymer, at range of about 5-90
.mu.mol peptide/gram polymer, or at a range of about 5-15 .mu.mol
peptide/gram polymer. In one embodiment, the polymer is collagen
and the covalently attached BMP binding peptide is present at a
range of about 1-200 .mu.mol peptide/gram collagen, at range of
about 5-90 .mu.mol peptide/gram collagen, or at a range of about
5-15 .mu.mol peptide/gram collagen.
[0075] In one embodiment of the presently disclosed subject matter,
a method is provided for promoting bone growth in a subject by
delivering the implantable bone graft material comprising a
resorbable ceramic and a resorbable polymer, wherein the polymer
comprises a covalently attached BMP binding peptide to a subject,
wherein the presence of the graft material having attached BMP
binding peptide promotes bone growth. In one embodiment, the
resorbable ceramic and the resorbable polymer are in the form of a
composite. In one embodiment, the composite is in the form of a
sponge, a granulized sponge, a putty, or a strip. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of an injectable bone graft material. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a moldable cement.
[0076] In one embodiment, a method is provided for capturing BMP
onto an implantable bone graft material by contacting a sample
comprising BMP with the graft material comprising a resorbable
ceramic and a resorbable polymer, wherein the polymer comprises a
covalently attached BMP binding peptide, wherein the BMP comprised
in the sample is captured onto the graft material through binding
to the attached BMP binding peptide. In one embodiment, the
resorbable ceramic and the resorbable polymer are in the form of a
composite. In one embodiment, the composite is in the form of a
sponge, a granulized sponge, a putty, or a strip. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of an injectable bone graft material. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a moldable cement. In one embodiment, the sample
comprises autologous bone, allograft bone, xenograft bone, bone
marrow aspirate (BMA), blood, platelet rich plasma (PRP), or
recombinant BMP, or combinations or derivatives thereof.
[0077] In one embodiment, a method is provided for promoting bone
growth in a subject by contacting a sample comprising BMP with the
implantable bone graft material comprising a resorbable ceramic and
a resorbable polymer, wherein the polymer comprises a covalently
attached BMP binding peptide, wherein the BMP comprised in the
sample is captured onto the graft material through binding to the
attached BMP binding peptide, and delivering to the subject the
graft material comprising the captured BMP, wherein the presence of
the captured BMP promotes bone growth in the subject. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a composite. In one embodiment, the composite is in
the form of a sponge, a granulized sponge, a putty, or a strip. In
one embodiment, the resorbable ceramic and the resorbable polymer
are in the form of an injectable bone graft material. In one
embodiment, the resorbable ceramic and the resorbable polymer are
in the form of a moldable cement. In one embodiment, the sample
comprises autologous bone, allograft bone, xenograft bone, bone
marrow aspirate (BMA), blood, platelet rich plasma (PRP), or
recombinant BMP, or combinations or derivatives thereof.
[0078] A wide range of BMP binding peptides are useful in the
compositions and methods of the presently disclosed subject matter.
By way of non-limiting example, the BMP binding peptides described
in US Patent Application Publication No's. US 2006/0051395A1 (the
BMP binding peptides identified therein as one of SEQ ID No's:
11-28, 44-74, or 77-94) and US 200910098175A1 (the BMP binding
peptides identified therein as one of SEQ ID No's: 1-12) are useful
in the presently disclosed subject matter. In particular, the BMP
binding peptides described in US Patent Application Publication
No's. US 2006/0051395 A1 and US 2009/0098175A1 shown herein below
at Table 1 (SEQ ID NOs: 1-10) are useful in the presently disclosed
subject matter. In addition, BMP binding peptides useful in the
presently disclosed subject matter include those peptides that are
conservatively substituted variants of the BMP binding peptides in
US Patent Application Publication Nos. US 2006/0051395A1 and US
2009/0098175A1, and those peptides that are variants having at
least 65% sequence identity or greater to the BMP binding peptides
in US Patent Application Publication Nos. US 2006/0051395A1 and US
2009/0098175A1, wherein all of the variant BMP binding peptides
useful in the presently disclosed subject matter substantially
retain the ability to bind to BMP. In one embodiment of the
presently disclosed subject matter, a useful BMP binding peptide
comprises a sequence selected from the group consisting of SEQ ID
NOs: 1-7 (see Table 1 herein below), conservatively substituted
variants of SEQ ID NOs: 1-7, and variants having at least 65%
sequence identity to SEQ ID NOs: 1-7, wherein the variant BMP
binding peptide substantially retains the ability to bind BMP.
[0079] The following examples are provided to further describe
certain aspects of the presently disclosed subject matter and are
not intended to limit the scope of the presently disclosed subject
matter.
EXAMPLES
Example 1
BMP Binding Peptides
[0080] BMP binding peptides SEQ ID NOs:1-4 & 8-10 were
identified as described in US Patent Application Publication No.
US20060051395A1. Briefly, the peptides were identified by phage
display using immobilized BMP-2 as a substrate for the phage
library selections. Synthetic peptides SEQ ID NOs: 1-2 were
determined to bind to BMP-2 with a relative EC50 value of 0.8 nM
and 0.9 nM, respectively (data not shown). Separately, the relative
binding affinity of peptides SEQ ID NOs: 1-2 for BMP-2 is shown in
FIG. 14A (SEQ ID NO: 1 is denoted as P9 and SEQ ID NO: 2 is denoted
as P10). Briefly, the results shown in FIG. 14A were generated by
immobilizing biotinylated BMP binding peptides SEQ ID NOs:1 & 2
on streptavidin coated plates. Serial dilutions of 200 nM, 20 nM
and 2 nM of BMP-2 were incubated with the immobilized BMP binding
peptides for 45 minutes, washed, and bound BMP quantified using
ELISA. In addition to binding BMP-2 on which the phage selection
was based, panel B in FIG. 14 performed shows that the BMP binding
peptides SEQ ID NOs: 1-2 also bind to BMP-7 (FIG. 14B; SEQ ID NO: 1
is denoted as P9 and SEQ ID NO: 2 is denoted as P10). The results
in FIGS. 14A-14B show that BMP binding peptides SEQ ID NOs: 1-2
have high relative affinity for each of BMP-2 (panel A) and BMP-7
(panel B).
[0081] BMP binding peptides SEQ ID NOs: 5-7 were identified as
described in US Patent Application Publication No. US20090098175A1.
Briefly, the peptides were identified by phage display using
immobilized GDF-7 (GDF-7 is BMP-12) as a substrate for the phage
library selections. In addition to binding GDF-7 on which the phage
selection was based, a relative EC50 value of 2.0 nM, 1.8 nM, and
1.1 nM, respectively, for BMP-2 was measured for peptides SEQ ID
NOs: 5-7 (data not shown). In addition to binding BMP-2, the data
in FIG. 14C shows that SEQ ID NOs: 1-2 & 5-7 also bind BMP-5
and BMP-6 (SEQ ID NOs: 1-2 are denoted as P9-P10, respectively and
SEQ ID NOs: 5-7 are denoted as P11-P13, respectively). The data
shown in FIG. 14C were collected similarly to that described above
for FIGS. 14A-14B except that the serial dilutions of BMP were 100
nM and 10 nM.
Example 2
Covalent Attachment of BMP Binding Peptide to Collagen
Substrate
[0082] In this experiment, a bone morphogenic protein (BMP) binding
peptide SEQ ID NO: 2 was covalently attached to a collagen
substrate using a disulfide linkage (see FIG. 3). The BMP binding
peptide SEQ ID NO: 2 was modified at the amino terminus with a
spacer and thiol group: HS-Propionyl-MP-MP-(SEQ ID NO:
2)-amide.
[0083] Peptide synthesis. BMP binding peptides (including SEQ ID
NO: 2) were synthesized by solid-phase peptide synthesis techniques
either manually in glass reaction vessels or on a RAININ SYMPHONY
PEPTIDE SYNTHESIZER multiplex automated peptide synthesizer
(PROTEIN TECHNOLOGIES INC., Tucson Ariz.). Homogeneity of each
synthetic peptide was evaluated by analytical RP-HPLC (WATERS
ANALYTICAL/SEMI-PREPARATIVE HPLC), and the identity of the peptide
confirmed with electrospray ionization mass spectrometry.
[0084] Collagen substrate modification. A slurry of fibrillar type
I bovine collagen (1-3% collagen; KENSEY NASH, Exton, Pa.) was
reacted with 2-iminothiolane (0.09-2.0 mg/ml) and either
4,4'-dithiodipyridine or 6,6'-dithiodinicotinic acid (DTNA) ranging
from 0.4-9 mg/ml with gentle agitation at room temperature for
about 24 h. The reaction mixture was isolated from the collagen
using vacuum filtration. The collagen material was washed 4 times
with phosphate buffer pH 8 and 2 times with PBS pH 7.4. For each
wash, the mixture was agitated and then the wash buffer removed by
vacuum.
[0085] Peptide coupling. BMP binding peptide SEQ ID NO: 2 (with
N-terminal spacer and thiol group) at a concentration ranging from
about 0.25-1.0 mg/mL in PBS was added to the activated collagen
substrate described above. The reaction mixture was incubated with
agitation at room temperature for about 24 hours. The resulting
degree of peptide covalent attachment to the collagen ranged from
about 5-90 .mu.mol peptide/g collagen.
[0086] In another example, BMP binding peptides SEQ ID NO: 1 and
SEQ ID NO: 2 were each covalently attached to a collagen matrix
(HELISTAT sponge, INTEGRA LIFE SCIENCES, Plainsboro, N.J.). Amine
groups (principally lysine E-amino) on HELISTAT collagen were
modified with sulfosuccinimidyl
6-(3'-[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP;
THERMO FISHER SCIENTIFIC, Rockford, Ill.) to introduce a
thiol-reactive pyridyl disulfide. After washing, sponges were
reacted directly with the peptides containing a free thiol group
(2.4 mg/mL for both SEQ ID NO: 1 & 2 peptides) resulting in
covalent attachment of the peptide via a disulfide. For
quantification, the peptides were released by reduction and
analyzed by HPLC. The resulting degree of peptide covalent
attachment to the HELISTAT collagen was 102 .mu.mol peptide/g
collagen (SEQ ID NO: 1) and 65 .mu.mol peptide/g collagen (SEQ ID
NO: 2).
Example 3
Retention of BMP-2 by BMP Binding Peptides Attached to Collagen
[0087] The BMP binding peptide-modified HELISTAT collagen sponges
generated in Example 2 were evaluated for ability to bind and
retain BMP-2. BMP-2 was added to sponges (n=3) with or without the
covalently attached BMP binding peptide (SEQ ID NO: 1 and SEQ ID
NO: 2) and then the sponges were challenged with repeated changes
in plasma (1, 3, 7, and 24 h). The amount of BMP-2 released into
the plasma at each time-point was measured by ELISA (QUANTIKINE,
R&D SYSTEMS), and the amount retained on the sponge after 24 h
was estimated by Western blot analysis. After 4 h, a large amount
of the initial load of BMP-2 was released from the unmodified
collagen sponge (FIG. 15). On the other hand, less than 5% of the
BMP-2 was released after 7 h from the SEQ ID NO: 2-modified
(Peptide 1) and SEQ ID NO: 1-modified (Peptide2) sponges. In
addition, the Peptide 1-modified sponge released less than half as
much BMP-2 than the Peptide2-modified sponge after 1 h (0.44 .mu.g
and 1.22 .mu.g, respectively). After 24 h, the sponges were
digested by collagenase, and the amount of BMP-2 retained on the
sponges was quantified by ELISA. Peptide-modified sponges retained
more than 40-fold the amount of BMP-2 as unmodified sponges (data
not shown). Western blot analysis confirmed that only a small
amount of BMP-2 was retained on the unmodified sponge or on a
sponge modified with a scrambled peptide (data not shown). However,
on the Peptide 1- and Peptide 2-modified sponges, 50% of the
initial BMP-2 load was retained (data not shown). These data
indicate that the BMP binding peptides can bind and retain BMP-2 on
collagen.
Example 4
Activity of BMP-2 Retained on BMP Binding Peptide-Modified
Collagen
[0088] In an experiment to test the biological activity of BMP-2
delivered by BMP binding peptide, alkaline phosphatase secreted by
C2C12 cells in response to BMP-2 was measured in the presence and
absence of BMP binding peptide in solution. C2C12 cells were
incubated with BMP-2 (35 nM) and BMP binding peptide at a
concentration ranging from 0-3500 nM. The C2C12 cells were
incubated with the BMP-2 and BMP binding peptide for 3 days and the
alkaline phosphatase assay was performed as described herein above.
The data show that the BMP binding peptide does not interfere with
the biological activity of BMP-2 and the BMP binding peptide does
not have BMP-2 activity on its own (FIG. 16).
Example 5
Collagen/TCP Composite Bone Void Filler
[0089] Tri-calcium phosphate (TCP)-based bone void fillers or
otherwise referred to as bone graft substitutes are used commonly
in long bone applications and lumbar spinal fusion, because the TCP
is absorbed over several months as bone heals. These products often
contain collagen and are often combined with bone marrow aspirate
(BMA) to provide osteoinductive factors. BMA can be harvested at
point-of-care without the same complications of harvesting
autogenous bone. In this Example, collagen/TCP composites were
generated for use as a bone graft substitute. Both an unmodified
collagen/TCP composite and a collagen/TCP composite having BMP
Binding Peptide (SEQ ID NO: 2) covalently attached to the collagen
portion of the composite were generated according to the following
procedure.
[0090] BMP binding peptide SEQ ID NO: 2 was covalently attached to
fibrillar Type I bovine collagen at both a low peptide load density
(ranging from 5-15 .mu.mol peptide/g collagen) and a high peptide
load density (70-100 .mu.mol peptide/g collagen) according to the
procedure described herein above at Example 2 (using
6,6'-dithiodinicotinic acid). A collagen/TCP composite was
generated using both unmodified collagen (i.e. without peptide
attachment) and collagen modified with BMP binding peptide. The TCP
used to make the collagen/TCP composite was .beta.-TCP having about
70% porosity and having a particle size distribution of about 100
.mu.m to about 300 .mu.m (CAP BIOMATERIALS, East Troy, Wis.). The
pore size of the .beta.-TCP ranged from about 0.5 .mu.m to less
than about 70 .mu.m (data not shown).
[0091] For each of the BMP binding peptide-modified and unmodified
collagen/TCP composites, the collagen slurry was homogenized by
hand or in a mixer with the .beta.-TCP at a ratio of about 80% TCP
to about 20% collagen while keeping the homogenate chilled and
keeping the pH in the range of about pH 3-4. In separate
experiments, the collagen was modified with BMP binding peptide
both before and after mixing with the .beta.-TCP.
[0092] After lyophilization, the collagen/TCP composites were in
the form of sponges that are formable into a putty upon hydration.
There was no discernable loss of .beta.-TCP filler from the
composites after hydration in saline (approximately 1.5 .mu.l
saline/mg composite) and puttying, and the composites retained
their form after being shaped. In addition, an experiment was
performed to evaluate pushing the hydrated and puttied composite
through a 4.5 mm tube such as used in a non-invasive spinal fusion
type surgery. Specifically, 117 mg of the BMP peptide-modified
collagen/TCP was hydrated with 175 .mu.l saline to produce a
homogeneous putty. To simulate a cannula, the tip of a 1 ml plastic
syringe was cut off to expose the opening of the barrel, which had
a diameter of 4.5 mm. The putty was loaded into the top of the
syringe and slowly pushed down the barrel with the syringe piston.
When it reached the open bottom of the syringe, the material exited
the syringe as a cylindrical plug of approximately 0.1 cc and 4.5
mm in diameter.
Example 6
BMP-2 Binding/Retention by Collagen/TCP Composite in Putty Form
[0093] The ability of the BMP binding peptide-modified and
unmodified collagen/TCP composites described in Example 5 to retain
BMP-2 after long-term incubation in plasma was tested. Coupons of
the unmodified composite and composite modified with low or high
densities of BMP binding peptide were sterilized by e-beam
sterilization. A 40 .mu.M solution of BMP-2 was added to each of
the sterilized composite coupons until the entire BMP-2 solution
was absorbed into the coupon. The coupons were then transferred to
human plasma and incubated at 37.degree. C. with 100 rpm shaking.
The plasma was changed at regular time-points for 6 weeks, and the
amount of BMP-2 released into the plasma supernatant was assessed
at each time-point by ELISA (FIG. 17). Half of the BMP-2 was
released from the unmodified composite after 6 h, and 2/3 was
released after 1 week. On the other hand, the composite with the
low BMP binding peptide load released only 20% of the BMP-2 after 6
weeks. The composite with the high BMP binding peptide load
released less than 1% of its BMP-2 after 6 weeks. Taken together,
these data show that the BMP binding peptide can retain BMP-2 on
the composite for long periods of time even in challenging
biological fluids such as plasma and the higher peptide density
increases BMP-2 retention.
[0094] The ability of the BMP binding peptide-modified and
unmodified composite sponges from Example 5 to capture BMP-2 from
plasma was also tested. The BMP binding peptide-modified composite
sponges with the low and the high peptide-loading density were
incubated in plasma spiked with 2,600 pg/mL BMP-2 at 37.degree. C.
with shaking for 1 week. This concentration of BMP-2 was chosen
because it is in the range of physiological levels of BMP-2 in bone
marrow aspirate (BMA). At various time-points, 60 .mu.L of plasma
supernatant was removed for analysis of BMP-2 levels by ELISA. The
levels of BMP-2 in the plasma were the same for the unmodified
composite and the no-composite control throughout the course of the
experiment (FIG. 18). Both the low and the high load
peptide-modified composites captured significantly more BMP-2 from
the plasma than the control groups. The low peptide-density
composite captured 90% of the BMP-2 after 7 days, and the high
density composite captured 95% of the BMP-2 after 7 days and 85%
within the first day. These data demonstrate that the BMP binding
peptide-modified composite can capture BMP-2 from complex
biological fluids.
Example 7
Collagen/TCP Composite in Rat Calvarial Defect Model
[0095] The ability of each of the bone void filler collagen/TCP
composites (unmodified and BMP binding peptide modified at 5-15
.mu.mol peptide/g collagen described in Example 5) to speed bone
healing was assessed in a rat calvarial defect model. A defect was
introduced into male Sprague Dawley rats (n=8) as described
previously (Poehling, et. al, J Periodontol, 2006, 77:1582-90). A
full thickness circular defect, 6.8 mm in diameter, was made in the
parietal bone, centered across the midline. The disc of bone was
removed, and the void was filled with bone graft substitute
material hydrated with or without BMA harvested from the tibia
(saline was used for hydration without BMA). Tissue was harvested
at 4, 8, and 12 weeks. The calvaria were removed and analyzed by
micro-computed tomography (.mu.CT) to provide measurements of bone
density (SCANCO MEDICAL; Wayne, Pa.). Following .mu.CT, the
explants were processed for histology, and the slides were stained
with haematoxylin and eosin (H&E). For each sample, 6 sections
evenly distributed across the defect were scored by two independent
observers, blind to the treatment groups. The slides were scored
for osteogenic cellular activity, new bone area, and new bone
maturity according to the following procedure. For osteogenic
cellular activity, fibroblasts/loose connective tissue, immature
cartilage progenitors, immature bone progenitors, giant cells,
osteoclasts, osteoblasts, and osteocytes were counted and scored
according to the following scale: 1=Rare; 2=Few; 3=Moderate; and
4=Dense. New bone cross-sectional area was scored utilizing the
micrometer eye piece to determine the percent of the defect with
new bone formed according to the following scale: 1.1=1-10%;
1.2=11-25%; 2.1=26-35%; 2.2=36-50%; 3.0=51-75%; and 4.0=76-100%.
New bone maturity was scored according to the following scale:
1=Immature/Unorganized; 2=Immature; 3=Mature; and 4=Mature/Well
organized. Data were analyzed by one-way Analysis of Variance
(ANOVA). When the Main effect was significant (p<0.05),
individual groups were compared by post-hoc analyses with Fisher's
PLSD.
[0096] The bone graft substitute with covalently attached BMP
binding peptide was compared to unmodified collagen/TCP composite
(Example 5; no peptide) and two commercially available bone graft
substitutes containing TCP: MASTERGRAFT PUTTY (MEDTRONIC SOFAMOR
DANEK) and CHRONOS granules (SYNTHES). Animals with empty defects
were included as a negative control. Histological analysis revealed
increased bone formation at 4, 8, and 12 weeks. At 4 weeks,
histology showed greater osteogenic cellular activity and new bone
area in the BMP binding peptide-modified group than the unmodified
and comparison product groups (data not shown). At 8 weeks, the BMP
binding peptide-modified group scored higher for bone maturity and
new bone area than any other group. By 12 weeks, none of the empty
defects had healed. Among the treatment groups at 12 weeks, there
were no significant differences in bone volume assessed by .mu.CT;
however, histological analysis revealed marked differences in bone
maturity. The peptide-modified group hydrated with BMA had
significantly more new bone area, mature new bone, and osteogenic
cellular activity than all the other groups (FIGS. 19A-19D; all
Main effects, p<0.001). In addition, only the BMP binding
peptide-modified group had animals (4 of the 8 animals) with bone
bridging the entire defect (FIGS. 19A and 20). Overall, the BMA
groups produced more new bone than the saline groups. Among the
groups hydrated with saline, osteogenic cellular activity and bone
maturity were significantly higher at 12 weeks in the collagen/TCP
composite described in Example 5 than the commercially available
products regardless of whether the collagen/TCP composite was
modified with BMP binding peptide (data not shown; Main effect,
p<0.001; post-hoc analyses, all p's<0.001). Therefore, the
BMP binding peptide does not possess bioactivity itself, but when
the BMP binding peptide is combined with BMA it promotes bone
formation better than the commercially available products.
[0097] These data demonstrate that the BMP binding peptide-modified
collagen/TCP composite not only performs as well as the comparative
commercial products, but it accelerates bone formation and produces
significantly more mature new bone. Furthermore, when combined with
BMA, the presence of the BMP binding peptide resulted in better
bone maturity than the collagen/TCP composite without peptide. This
result indicates that the BMP binding peptide-modified composite's
ability to deliver bioactive growth factors has significant
advantages for bone healing.
[0098] As stated above for the results of the animal study for bone
healing, the unmodified collagen/TCP composite of the presently
described subject matter (no attached BMP binding peptide; Example
5), demonstrated significantly higher osteogenic cellular activity
and bone maturity at 12 weeks when hydrated with saline than the
commercially available products. This was an unexpected result.
Furthermore, in addition to the higher osteogenic cellular activity
and bone maturity observed at 12 weeks, the collagen/TCP composite
without peptide also demonstrated an unexpected acceleration in
bone healing at 4 weeks relative to commercially available
MASTERGRAFT (MG) which is also a collagen/TCP composite. At 4
weeks, the unmodified composite when hydrated with either saline or
BMA showed greater bone healing than MG as measured by each of the
.mu.CT and histological measurements of bone healing examined (see
FIGS. 21-24). The unmodified composite at 4 weeks hydrated with
either saline or BMA showed a statistical increase in osteogenic
cellular activity relative to MG (see FIGS. 22A & 24A).
Similarly, the observed increase in bone maturity for the
unmodified composite relative to MG when hydrated with saline at 4
weeks was also statistically significant (see FIG. 22C). These data
illustrate the unexpected result that the unmodified collagen/TCP
composite of the presently disclosed subject matter accelerates
bone healing relative to a commercially available TCP/collagen
composite.
Example 8
Collagen/TCP Composite in Strip Formulation
[0099] Bone void fillers are used in some cases for promoting bone
growth in spinal fusion applications and numerous animal models of
spine fusion are available (e.g., Kraiwattanapong, C., et al.,
Spine, 2005, 30:1001-7; Magit, D. P., et al., Spine, 2006,
31:2180-8; Choi, Y., et al., Spine, 2007, 32: 36-41; Martin, G. J.,
et al., Spine, 1999, 24:637-45). Bone void fillers are often
formulated into a strip having shape memory when used for spinal
fusion applications. In this example, the BMP binding
peptide-modified and unmodified collagen/TCP composites described
in Example 5 were formulated into a strip having shape memory.
[0100] Various methods are available for cross-linking collagen in
a collagen/ceramic composite to make a strip that meets the
criteria for physical properties in spine fusion applications, such
as with chemical agents (e.g., glutaraldehyde), dehydrothermal
treatment, ultra-violet irradiation, or a combination of these
techniques (see, e.g., Ruijgrok, J. M., et al., Journal of
Materials Science: Materials in Medicine, 1994, 5:80-87; Gorham, S.
D., et al., Int J Biol Macromol, 1992, 14:129-38; Weadock, K. S.,
et al., J Biomed Mater Res, 1995. 29: 1373-9. Lew, D. H., et al., J
Biomed Mater Res B Appl Biomater, 2007, 82:51-6). In this example,
the BMP binding peptide-modified and unmodified collagen/TCP
composites described in Example 5 were formulated into a strip by
dehydrothermal treatment.
[0101] In the first example, a composite from Example 5 (both
peptide-modified and unmodified) was compressed between two
titanium plates (TIMET, Ofallon, Mo.) held in place with two
C-clamps (BESSEY, Leroy, N.Y.). The C-clamps were turned until the
composite was compressed to at least half the original height. The
clamped composite was placed in an ISOTEMP OVEN OV600G (FISHER
SCIENTIFIC, Waltham, Mass.) at 88.degree. C. for 74-112.5 hours.
The sample was removed, cooled to room temperature, and hydrated
with saline. The hydrated composite exhibited strip-like properties
such as shape memory, flexibility, as well as liquid retention and
compression resistance under a 50 g weight. In another example, a
composite generated as described in Example 5 (peptide-modified and
unmodified) was placed in an vacuum oven (SHELDON, Cornelius,
Oreg.) at a temperature ranging from 100-110.degree. C., at vacuum
29.5 inches Hg, and for 48-162 hours. The sample was removed,
cooled to room temperature under vacuum, and hydrated with saline.
The hydrated composite exhibited strip-like properties such as
shape memory, flexibility, as well as liquid retention and
compression resistance under a 50 g weight.
Example 9
BMP-2 Binding/Retention by Collagen/TCP Composite in Strip Form
[0102] The ability of the BMP binding peptide-modified and
unmodified collagen/TCP composites formulated into strips as
described in Example 8 to bind and retain BMP-2 were tested
according to the following procedure. Coupons of each of the BMP
binding peptide-modified and unmodified composites were loaded with
27 .mu.l of a 40 .mu.M solution of BMP-2 (28 .mu.g). The coupons
were then transferred to human plasma and incubated at 37.degree.
C. with 100 rpm shaking. The plasma was changed 1 h, 6 h, and 24 h,
and the amount of BMP-2 released into the plasma supernatant was
assessed at each time-point by ELISA. Both the BMP binding
peptide-modified composites (110.degree. C. for 162 h under vacuum)
and (88.degree. C. for 112 h+compression) bound and retained BMP-2
as compared to unmodified composite controls (data not shown). Each
peptide-modified composite released less than 5% of the BMP-2
originally loaded onto the coupons after 24 h of incubation in
plasma (data not shown).
Example 10
Covalent Attachment of BMP Binding Peptide to Polyanhydride
Polymer
[0103] BMP binding peptide is covalently attached to polymaleic
anhydride (PMA) using established methods (Pompe, et al., 2003,
Biomacromolecules, 4(4):1072-9). First, a spacer, such as for
example, GSSGK, is added to a terminus of the peptide and the
peptide is attached to the PMA anhydride groups through the
reactive terminal lysine amine group on the peptide-spacer. A
schematic diagram of one example of this chemistry is shown in FIG.
6. PMA .about.5,000 MW is dissolved in anhydrous dimethylformamide
(DMF) and peptide is dissolved in DMF with excess
diisopropylethylamine (DIEA). The peptide solution is heated with
the PMA solution at 40.degree. C. overnight, for example, and the
reaction mixture quenched with water. The crude PMA-peptide
conjugate is filtered and analyzed. For example, the extent of
substitution on the polyanhydride polymer can be estimated by
integration of .sup.1H-NMR peaks from the peptide together with the
integrals of key reference peaks on the polymer to provide an
estimate of the level of peptide substitution. In another example,
size exclusion chromatography is used by monitoring the UV
absorption of the peptide along with a known amount of unconjugated
PMA. The degree of peptide substitution is estimated from the mass
of the lyophilized product and the UV absorbance of the peptide
component.
[0104] The foregoing description of the specific embodiments of the
presently disclosed subject matter has been described in detail for
purposes of illustration. In view of the descriptions and
illustrations, others skilled in the art can, by applying current
knowledge, readily modify and/or adapt the presently disclosed
subject matter for various applications without departing from the
basic concept of the presently disclosed subject matter; and thus,
such modifications and/or adaptations are intended to be within the
meaning and scope of the appended claims.
Sequence CWU 1
1
10116PRTArtificial sequenceSynthetic 1Gly Gly Gly Ala Trp Glu Ala
Phe Ser Ser Leu Ser Gly Ser Arg Val1 5 10 15217PRTArtificial
sequenceSynthetic 2Gly Gly Ala Leu Gly Phe Pro Leu Lys Gly Glu Val
Val Glu Gly Trp1 5 10 15Ala39PRTArtificial sequenceSynthetic 3Trp
Glu Ala Phe Ser Ser Leu Ser Gly1 549PRTArtificial sequenceSynthetic
4Leu Gly Phe Pro Leu Lys Gly Glu Val1 5521PRTArtificial
sequenceSynthetic 5Ser Ser Gly Pro Arg Glu Ile Trp Asp Ser Leu Val
Gly Val Val Asn1 5 10 15Pro Gly Trp Ser Arg 20621PRTArtificial
sequenceSynthetic 6Ser Ser Gly Gly Val Gly Gly Trp Ala Leu Phe Glu
Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg 20721PRTArtificial
sequenceSynthetic 7Ser Ser Val Ala Glu Trp Ala Leu Arg Ser Trp Glu
Gly Met Arg Val1 5 10 15Gly Glu Ala Ser Arg 20812PRTArtificial
sequenceSynthetic 8Trp Xaa Xaa Phe Glu Xaa Leu Xaa Gly Xaa Glu Xaa1
5 1097PRTArtificial sequenceSynthetic 9Xaa Xaa Xaa Phe Xaa Xaa Leu1
5107PRTArtificial sequenceSynthetic 10Xaa Xaa Phe Pro Leu Xaa Gly1
5
* * * * *