U.S. patent application number 17/058891 was filed with the patent office on 2021-07-15 for targeting anabolic drugs for accelerated fracture repair.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Philip Stewart Low, Stewart A. Low, Jeffery Jay Howard Nielsen.
Application Number | 20210213135 17/058891 |
Document ID | / |
Family ID | 1000005538431 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210213135 |
Kind Code |
A1 |
Low; Philip Stewart ; et
al. |
July 15, 2021 |
TARGETING ANABOLIC DRUGS FOR ACCELERATED FRACTURE REPAIR
Abstract
Aspects of the disclosure include materials and methods for the
targeted delivery of growth factors, and other compounds that
stimulate bone growth and in some aspect bone healing. Some aspects
of the disclosure include methods for synthesizing and testing
these compounds. Some aspects of the invention include methods of
using the compounds disclosed herein to treat bone fractures and
bone defects.
Inventors: |
Low; Philip Stewart; (West
Lafayette, IN) ; Low; Stewart A.; (West Lafayette,
IN) ; Nielsen; Jeffery Jay Howard; (West Lafayette,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
|
IN |
|
|
Family ID: |
1000005538431 |
Appl. No.: |
17/058891 |
Filed: |
May 30, 2019 |
PCT Filed: |
May 30, 2019 |
PCT NO: |
PCT/US2019/034767 |
371 Date: |
November 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62678016 |
May 30, 2018 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6435 20170801;
A61K 47/60 20170801; A61K 47/645 20170801; A61K 38/1875
20130101 |
International
Class: |
A61K 47/64 20060101
A61K047/64; A61K 47/60 20060101 A61K047/60; A61K 38/18 20060101
A61K038/18 |
Claims
1-19. (canceled)
20. A compound having a structure of: X-Y-Z wherein: X is an
extracellular matrix component, a fragment of an extracellular
matrix component, or an agent that activates an extracellular
matrix component; Y is absent or a linker; and Z is a
bone-targeting molecule; or a pharmaceutically acceptable salt
thereof.
21. The compound of claim 20, wherein Z comprises a
polypeptide.
22. The compound of claim 20, wherein Z comprises not less than 4
and not more than 40 amino acid residues.
23. The compound of claim 22, wherein at least one amino acid is
aspartic acid or glutamic acid.
24. The compound of claim 20, wherein Z comprises not less than 6
and not more than 20 D-glutamic acid residues.
25. The compound of claim 24, wherein Z is 10 D-glutamic acid
residues.
26. The compound of claim 20, wherein Z comprises not less than 6
and not more than 20 D-aspartic acid residues.
27. The compound of claim 26, wherein Z is 10 D-aspartic acid
residues.
28. The compound of claim 20, wherein Y is a releasable linker or a
non-releasable linker.
29. The compound of claim 28, wherein the releasable linker
comprises at least one releasable linker group, each releasable
linker group being independently selected from the group consisting
of a disulfide (S-S), an ester, and a protease-specific amide
bond.
30. The compound of claim 28, wherein the non-releasable linker
comprises at least one non-releasable linker group, each
non-releasable linker group being independently selected from the
group consisting of a carbon-carbon bond and an amide.
31. The compound of claim 28, wherein Y comprises one or more
ethylene glycol units.
32. The compound of claim 31, wherein Y comprises 2-8 oxyethylene
units.
33. The compound of claim 20, wherein the extracellular matrix
component is selected from the group consisting of a laminin, a
fibronectin, and an osteopontin fragment.
34. The compound of claim 33, wherein the laminin is selected from
the group consisting of a laminin fragment (IKVAV) and Ln2-P3.
35. The compound of claim 33, wherein the fibronectin is selected
from the group consisting of PHSRN and ITGA.
36. The compound of claim 33, wherein the osteopontin fragment is
selected from the group consisting of a collagen binding motif, a
osteopontin derived peptide, and a collagen binding domain.
37. The compound of claim 20, wherein the agent that activates the
extracellular matrix component is an integrin ligand.
38. The compound of claim 37, wherein the integrin ligand is
selected from the group consisting of ITGaS cys, ITGA stb-KD, ITGA
stb-KE, ITGA stb-DAPE, and ITGA stb-DAPD.
39. The compound of claim 20, wherein the extracellular matrix
component is selected from the group consisting of chemotatic
collagen (CTC), P15, and DGEA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/678,016, filed on May 30, 2018. This application
is incorporated herein by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] Aspects of the present disclosure relate to the materials
and methods for treating bone fractures and bone defects.
BACKGROUND
[0003] Src tyrosine kinase plays a crucial role in bone metabolism:
despite its ubiquitous expression profile, the only apparent
phenotypical abnormality in a sarcoma-knockout (Src-KO) mouse
strain was osteopetrosis. Although Src inhibitors inhibit both the
formation and activity of osteoblasts (OBs) in vitro, the number of
osteoclasts (OCs) derived from Src-KO mice were actually elevated
in Src-KO mice, measuring more than twice that in wild-type (WT)
mice. Also, a marked increase in both osteoblast number and
activity was observed in vivo in Src-KO mice. These results confirm
that the osteopetrosis phenotype of Src-KO mice was not a result of
reduced osteoclast formation, but rather of boosted osteoblast
activity as well as reduced osteoclast function. Moreover,
osteoblasts derived from Src-KO mice demonstrated unremarkable
morphological features compared to those harvested from WT mice,
and were able to fully regulate normal osteoclast differentiation
via the receptor activator of nuclear factor kappa-B
ligand/receptor activator of nuclear factor kappa-B/osteoprotegerin
(RANKL/RANK/OPG) pathway. Thus, this bone-resorption defect should
be easily alleviated by restoring normal Src functionality in
osteoclasts, reducing potential risks on the musculoskeletal system
involved in long-term use of Src inhibitors for fracture
healing.
[0004] Broadly, peptide anabolic drugs include different categories
of protein or the fragments thereof. They are represented by bone
morphogenetic protein pathway signaling peptides including P4, bone
forming peptide (BFP) and peptide from Bone morphogenetic protein 9
(pBMP9); insulin-like growth factor (IGF) derived peptides
including mechano-growth factor (MGF) and Preptin; bone stimulatory
neuropeptides including Substance P and vasoactive intestinal
peptide (VIP); and peptides enhancing vascular functions, including
C-type Natriuretic peptide (CNP), thrombin fragment or targeted
prothrombin peptide (TP508) and VIP. Each of these peptides may
have its own unique mechanism working to regulate bone growth, as
will be outlined in the detailed description.
[0005] Current clinical treatment of fractures generally does not
include the use of site-specific anabolic drugs. In fact, the only
drugs approved for clinical use on such fractures are bone
morphogenic protein (BMP)-2 (approved for use only in tibial
trauma) and BMP-7 (discontinued), which are applied locally and
generally used in the treatment of open long bone fractures and
spinal fusions. The need for broader application of anabolic drugs
to treat bone maladies such as osteoporotic fractures with efficacy
is evident.
[0006] Therefore, it is desirable to have a fracture treatment drug
that is administered systemically yet targets the fracture site
with evident efficacy.
SUMMARY
[0007] A first aspect of the present disclosure includes at least
one compound of the formula X-Y-Z, or a pharmaceutically acceptable
salt thereof, or a metabolite thereof, wherein X is at least one
agent that improves bone density, mechanical strength, bone
deposition, or quality; Z is at least one bone-targeting molecule;
and Y is a linker that joins and/or links X and Z. In some aspects,
X is at least one agent that enhances the activity or one agent
that improves bone density, mechanical strength, bone deposition or
otherwise promotes bone healing and/or growth. Consistent with some
of these aspects, Z is at least one negatively charged oligopeptide
or an equivalent thereof that binds to hydroxyapatite and/or raw
bone.
[0008] The second aspect includes the compound according to the
first aspect, wherein when X is a polypeptide, any polypeptide
having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%
identity to X can be used to practice the invention.
[0009] In some aspects, Y is at least one polypeptide comprising at
least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% sequence
identity to amino acid residues 35-40, 35-41, 35-42, 35-43, 35-44,
35-45, 35-46, 35-47, 35-48, 35-49, 35-50, 35-51, 35-52, 35-55,
35-84, 41-44, 41-45, 41-46, 41-47, 41-48, 41-49, 41-50, and/or
41-84 of a full length parathyroid hormone related peptide or
parathyroid hormone, and/or at least one Cathepsin K sensitive
polypeptide.
[0010] In some aspects, Z is at least one polypeptide comprising
about 4 or more, from about 4 to about 100, from about 4 to about
50, from 4 to about 20, from about 4 to about 15, from about 4 to
about 10 acidic amino acid residues, polyphosphate,
2-aminohexanedioic (aminoadipic) acid or derivatives thereof,
and/or alendronate or derivatives thereof. In some aspects, Z is at
least one polypeptide comprising about 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, and/or 30 acidic amino acid residues, polyphosphate,
2-aminohexanedioic acid or derivatives thereof, and/or alendronate
or derivatives thereof. In some aspects, Z is at least one
negatively charged oligopeptide or an equivalent thereof that binds
to hydroxyapatite and/or raw bone.
[0011] The targeted delivery strategy recited in some aspects of
the invention enable the delivery of Src inhibitors specifically to
bone fracture surfaces thereby facilitating fracture healing. This
in vivo efficacy is shown by the acceleration of fracture healing
observed using the Src inhibitors Dasatinib and E738.
[0012] In addition to Src inhibitors, a group of peptides targeted
specifically to the fracture surfaces also demonstrates an enhanced
ability to facilitate fracture healing. These peptides include
osteopontin derived fragments such as osteopontin-derived peptide
(ODP), collagen binding motif (CBM); BMP fragments such as P4, BFP,
pBMP7; IGF fragments such as MGF and Preptin; neuropeptides such as
Substance P and VIP; Vasoconstrictive fragments such as CNP, TP508
and VIP; and other anabolic drugs such as osteogenic growth peptide
(OGP).
[0013] The in vivo efficacy of these peptides for accelerated
fracture healing are demonstrated herein. All peptide conjugates
are produced by solid phase synthesis.
[0014] Some aspects of this disclosure include compounds
comprising: a compound of the formula X-Y-Z, wherein X is at least
one agent that modulates bone growth, such as activity of Src
tyrosine kinase; Z is at least one bone-targeting molecule; and Y
is a linker that joins and/or links X and Z; or a pharmaceutically
acceptable salt thereof, or a metabolite thereof. In some aspects,
Z is at least one polypeptide comprising 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 and/or 20 acidic amino acid residues. In
some aspects, X is selected from the group consisting of Dasatinib
and E738. In some aspects, Y is a releasable linker selected from
disulfide; ester; or protease specific amide bond. In some aspects,
Y is a nonreleasable bond selected from carbon-carbon bond; or
amide bond.
[0015] In some aspects, this disclosure includes a compound of the
formula X-Y-Z, wherein X is at least one peptide or a fragment
thereof that modulates activity of bone and cartilage formation; Z
is at least one bone-targeting molecule; and Y is a linker that
joins and/or links X and Z; or a pharmaceutically acceptable salt
thereof, or a metabolite thereof. In some aspects, Y is a
releasable linker selected from disulfide; ester; or protease
specific amide bond. In some aspects, Y is a nonreleasable bond
selected from carbon-carbon bond; or amide bond. In some aspects, Y
is a peptide belonging to the natural sequence of Z. In some
aspects, Y is a polyethylene glycol (PEG) linker. In some aspects Y
is a PEG linker comprised of 2-8 oxyethylene units. In some
aspects, Z comprises at least 10 aspartic or glutamic acids
conjugated to X. In some aspects, Z comprises at least 20 aspartic
or glutamic acids conjugated to X. In some aspects, the compound
may be produced by solid phase synthesis.
[0016] In some aspects, X is a bone anabolic peptide derived from
BMP. In some aspects, X is a bone anabolic peptide derived from
IGF. In some aspects, X is a bone anabolic peptide derived from a
neuropeptide. In some aspects, X is a bone anabolic peptide that
improves vascular function and/or vascularization. In some aspects,
X is osteogenic growth peptide (OGP). In some aspects, the peptide
is BFP, P4, or pBMP9. In some aspects, the peptide is MGF or
Preptin. In some aspects, the peptide is Substance P or VIP. In
some aspects, the peptide is TP508, VIP, or CNP. Unless indicated
otherwise, the invention may be practiced by combining any X with
any Z and optionally any suitable linking group Y. [0017] 1. A
compound comprising: [0018] a compound of the formula X-Y-Z,
wherein [0019] X is at least one agent that modulates activity
selected from the group consisting of: Components of the
Extracellular Matrix, Integrin alpha 5 ligands, Laminins,
fibronectins, P3, and fragments of Osteopotin: [0020] Z is at least
one bone-targeting molecule; and [0021] Y is an optional linker
that joins and/or links X and Z; [0022] or a pharmaceutically
acceptable salt thereof, or a metabolite thereof. [0023] 2. The
compound according to claim 1, wherein [0024] Z is at least one
polypeptide comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 and/or 20 acidic amino acid residues. [0025] 3. The compound
according to claims, 1-2 wherein Z includes multiple aspartates
and/or multiple glutamates. [0026] 4. The compound according to
claim 3, wherein Z is comprised of at least one polypeptide
selected from the group consisting of: at least 5 aspartic acids,
at least 5 glutamic acids, at least 10 aspartic acids, at least 10
glutamic acids, at least 20 aspartic acids, at least 20 glutamic
acids. [0027] 5. The compounds according to claims 1-4, wherein Z
is selected from the group consisting of: a polypeptide comprising
10 aspartic acid residues (SEQ ID NO. 33) and a polypeptide
comprising 10 glutamic acid residues (SEQ ID NO. 34). [0028] 6. The
compound according to claims 1-4, wherein Z is at least one
polypeptide comprising 4 or more acidic amino acid residues,
polyphosphate, aminohexanedioic acid or derivatives thereof, and/or
alendronate or derivatives thereof. [0029] 7. The compound
according to claims 1-6, wherein Y is selected from the group
consisting of: releasable linkers and non-releasable linkers.
[0030] 8. The compound according to claim 7, wherein the releasable
linker includes at least of the following groups: a disulphide, an
ester, or a Protease specific amide bond. [0031] 9. The compound
according to claim 7, the non-releasable linker includes at least
one of the following groups; a carbon-carbon bond, or an amide.
[0032] 10. The compound according to claims 1-6, wherein Y is
polyethylene glycol (PEG). [0033] 11. The compound according to
claim 10, wherein the PEG linker is comprised of 2-8 oxyethylene
units. [0034] 12. The compound according to claims 1-6, wherein Y
is a peptide belonging to the natural sequence of Z. [0035] 13. The
compound according to claims, 1-9, wherein the Components of the
Extracellular Matrix are selected from the group consisting of:
Chemotatic Collagen (CTC), P15, and DGEA. [0036] 14. The compound
according to claims, 1-9, wherein the Integrin alpha 5 ligands are
selected from the group consisting of: ITGaS_cys, ITGA_stb-KD,
ITGA_stb-KE, ITGA_stb-DAPE, and ITGA_stb-DAPD. [0037] 15. The
compound according to claims, 1-9, wherein the Laminins, are
selected from the group consisting of: Laminin Fragment (IKVAV) and
Ln2-P3. [0038] 16. The compound according to claims, 1-9, wherein
the fibronectin is the cell binding peptide PHSRN. [0039] 17. The
compound according to claims, 1-9, wherein the Osteopontin
fragments are selected from the group consisting of: Collagen
Binding Motif (1-28), Collagen Binding Motif (1-19), Osteopontin
Derived Peptide, and Collagen Binding Domain. [0040] 18. Use of a
compound according to any of claims 1-17, for the manufacture of a
medicament for therapeutic application. [0041] 19. A method of
treating a patient, comprising the step of administering at least
one dose of a compound according to claims 1-17.
[0042] These and other features, aspects and advantages of the
present disclosure will become better understood with reference to
the following figures, associated descriptions and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 depicts the bone volume divided by total volume of
the 100 thickest micro computed tomography (CT) slices of the
fracture callus bone density ("BV/TV") using a Dasatinib and
targeted Dasatinib conjugate (both 10 mol/kg). Both were
subcutaneously dosed daily to fracture-bearing Notre Dame breed
(ND4) of Swiss Webster mice. Bone density of the fracture callus
from the targeted Dasatinib group is twice as dense as the saline
group, and 50% denser than the free Dasatinib group.
[0044] FIG. 2 depicts BV/TV and Trabecular Thickness using targeted
E738 conjugate (1 .mu.mol/kg), subcutaneously dosed every-other-day
to fracture-bearing Charles River's breed (CFW) of Swiss Webster
mice. Targeted E738 conjugate significantly improved the bone
density and trabecular thickness at the fracture callus.
[0045] FIG. 3 depicts structures for Dasatinib and E738.
[0046] FIG. 4 depicts structures for targeted conjugates of
Dasatinib and E738.
[0047] FIG. 5 depicts peak load of Fractured Femurs after 2
weeks.
[0048] FIG. 6 depicts BV/TV two weeks after fractured femur
received various concentration of Preptin D10 treatment.
[0049] FIG. 7 depicts TbTh (the trabecular thickness of the 100
thickest micro computed tomography (CT) slices of the fracture
callus) two weeks after fractured femur received various
concentration of Preptin D10 treatment.
[0050] FIG. 8 depicts BV (the overall bone volume of the 100
thickest micro CT slices of the fracture callus) two weeks after
fractured femur received various concentration of Preptin D10
treatment.
[0051] FIG. 9 depicts BV/TV two weeks after fractured femur
received various concentration of OGPD10.
[0052] FIG. 10 depicts TbTh two weeks after fractured femur
received various concentration of OGP D10.
[0053] FIG. 11 depicts TbSp (the spacing between the trabecula in
the 100 thickest micro CT slices of the fracture callus) two weeks
after fractured femur received various concentration of OGP
D10.
[0054] FIG. 12 depicts BV/TV two weeks after fractured femur
received various concentration of BFPD10.
[0055] FIG. 13 depicts TbSp two weeks after fractured femur
received various concentration of BFPD10.
[0056] FIG. 14A depicts BV/TV four weeks after a fractured femur
received various concentration of substance P4 mini peg D10 (P4
D10); FIG. 14B depicts the max load of substance P4 D10 four weeks
after a fractured femur received the max load of substance P4
D10.
[0057] FIG. 15 depicts BV/TV four weeks after fractured femur
received various concentration of Ghrelin D10.
[0058] FIG. 16 depicts BV four weeks after fractured femur received
various concentration of pBMP9 D10.
[0059] FIG. 17 depicts BV/TV four weeks after fractured femur
received various concentration of pBMP9 D10.
[0060] FIG. 18 depicts BV/TV four weeks after fractured femur
received various concentration of CNP D10.
[0061] FIG. 19 depicts BV/TV four weeks after fractured femur
received 1 nmol/day of ODP D10.
[0062] FIG. 20 depicts BV/TV three weeks after fractured femur
received various concentrations of CBM D10 as compared to a
fractured femur that received parathyroid hormone 1-34 (PTH).
[0063] FIG. 21 depicts BV/TV four weeks after fractured femur
received various concentrations of P4 D10.
[0064] FIG. 22 depicts BV four weeks after fractured femur received
1 nmol/day of P4 D10.
[0065] FIG. 23 depicts BV/TV four weeks after fractured femur
received various concentrations of MGF D10.
[0066] FIG. 24 depicts BV/TV four weeks after fractured femur
received various concentrations of TP 508_D10.
[0067] FIG. 25 depicts BV/TV four weeks after fractured femur
received 1 nmol/day of VIP_D10.
[0068] FIG. 26 depicts TbTh four weeks after fractured femur
received 1 nmol/day of VIP_D10.
[0069] FIG. 27 depicts the structure for BMP9.
[0070] FIG. 28 depicts the structure for Ghrelin D10.
[0071] FIG. 29 depicts the structure for Preptin D10.
[0072] FIG. 30 depicts the structure for CNP-D10.
[0073] FIG. 31 depicts the structure for VIP D10.
[0074] FIG. 32 depicts the structure for Substance P with 4 mini
PEG conjugated to D10.
[0075] FIG. 33 depicts the structure for CBM D10.
[0076] FIG. 34 depicts the structure for ODP D10.
[0077] FIG. 35 depicts the structure of CTC_peg 10_e10 (SEQ ID NO:
20).
[0078] FIGS. 36-37 depict in vivo fracture healing efficacy of
CTC_peg10_(D)E.sub.10 conjugate.
[0079] FIG. 38 depicts the structure of CTC_MP4_e10 (SEQ ID NO:
21).
[0080] FIGS. 39-41 depict in vivo fracture healing efficacy of
CTC_mp4_(D)E.sub.10 conjugate.
[0081] FIG. 42 depicts the structure of P15 (SEQ ID NO: 22).
[0082] FIGS. 43-44 depict In vivo fracture healing efficacy of
P15_(D)E.sub.10 conjugate.
[0083] FIG. 45 depicts the structure of P15_mp4_e10 (SEQ ID NO:
23).
[0084] FIGS. 46-49 depict In vivo fracture healing efficacy of
P15_mp4_(D)E.sub.10 conjugate.
[0085] FIG. 50 depicts the structure of DGEA_mp4_e10 (SEQ ID NO:
24).
[0086] FIGS. 51-54 depict In vivo fracture healing efficacy of
DGEA_mp4_(D)E.sub.10 conjugate.
[0087] FIG. 55 depicts the structure of ITGA5 (SEQ ID NO: 25).
[0088] FIGS. 56-64 depict In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate.
[0089] FIG. 65 depicts a structure with a stabilized ring of ITGA
(SEQ ID NO: 25).
[0090] FIGS. 66a and 66b depict In vivo fracture healing efficacy
of ITGA_mp4_(D)E.sub.10_Stb(stable) conjugate.
[0091] FIG. 67 depicts the structure of ITGA_mp4_e10_DAPE (SEQ ID
NO: 25).
[0092] FIGS. 68-71 depict In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate.
[0093] FIG. 72 depicts the structure of ITGA_mp4_e10_DAPD (SEQ ID
NO: 25).
[0094] FIGS. 73-76 depict In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPD conjugate.
[0095] FIG. 77 depicts the structure of ITGA_mp4_e10_KD (SEQ ID NO:
25).
[0096] FIGS. 78-81 depict In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KD conjugate.
[0097] FIG. 82 depicts the structure of ITGA_mp4_e10_KE (SEQ ID NO:
25).
[0098] FIGS. 83-86 depict In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KE conjugate.
[0099] FIG. 87 depicts the structure of IKVAV_mp4_e10 (SEQ ID NO:
26).
[0100] FIGS. 88-91 depict In vivo fracture healing efficacy of
IKVAV_mp4_(D)E.sub.10 conjugate.
[0101] FIG. 92 depicts the structure of LN2_P3_mp4_e10 (SEQ ID NO:
27).
[0102] FIGS. 93-96 depict In vivo fracture healing efficacy of
LN2_P3_mp4_(D)E.sub.10 conjugate.
[0103] FIG. 97 depicts the structure of PHSRN_mp4_e10 (SEQ ID NO:
28).
[0104] FIGS. 98-101 depict In vivo fracture healing efficacy of
PHSRN_mp4_(D)E.sub.10 conjugate.
[0105] FIG. 102 depicts the structure of P3_mp4_e10 (SEQ ID NO:
29).
[0106] FIGS. 103-106 depict In vivo fracture healing efficacy of
P3_mp4_(D)E.sub.10 conjugate.
[0107] FIG. 107 depicts the structure of SPARC 113_mp4_e10 (SEQ ID
NO: 30).
[0108] FIGS. 108-111 depict In vivo fracture healing efficacy of
SPARC113_mp4_(D)E.sub.10 conjugate.
[0109] FIG. 112 depicts the structure of CBM(1-19)-D10 collagen
binding motif (SEQ ID NO: 31).
[0110] FIGS. 113-115 depict In vivo fracture healing efficacy of
CBM(1-19)_D.sub.10 conjugate.
[0111] FIG. 116 depicts the structure of CBD_MP4_e10 (SEQ ID NO:
32).
[0112] FIGS. 117-119 depict In vivo fracture healing efficacy of
CBD_MP4_(D)E.sub.10 conjugate.
BRIEF DESCRIPTION OF THE SEQUENCES
[0113] SEQ ID NO: 1: bone forming peptide conjugated with 10
aspartate acids (BFP D10).
[0114] SEQ ID NO: 2: osteogenic growth peptide conjugated with 10
aspartate acids (OGP D10).
[0115] SEQ ID NO: 3: Preptin conjugated with 10 aspartate acids
(Preptin D10).
[0116] SEQ ID NO: 4: substance P with 4 mini PEG linker and
conjugated with 10 aspartate acids (substance P 4 mini PEG
D10).
[0117] SEQ ID NO: 5: Ghrelin D10 with Ser-3 replaced with
diaminopropinoic acid.
[0118] SEQ ID NO: 6: BMP9 D10.
[0119] SEQ ID NO: 7: C-type Natriuretic peptide (CNP) conjugated
with 10 aspartate acids (CNP 10).
[0120] SEQ ID NO: 8: Vasoactive intestinal peptide conjugated with
D10.
[0121] SEQ ID NO: 9: collagen binding motif conjugated with 10
aspartate acids (CBM D10).
[0122] SEQ ID NO: 10: P4 conjugated with 10 aspartate acids (P4
D10).
[0123] SEQ ID NO: 11: Mechano-growth factor conjugated with 10
aspartate acids (MGF D10).
[0124] SEQ ID NO: 12: Thrombin fragment TP508 conjugated with 10
aspartate acids (TP 508 D10).
[0125] SEQ ID NO: 13: Osteopontin-derived peptide conjugated with
10 aspartate acids (ODP D10).
[0126] SEQ ID NO: 14: BMP9 (BMP9).
[0127] SEQ ID NO: 15: Ghrelin D10 (Ghrelin D10).
[0128] SEQ ID NO: 16: CNP-D10.
[0129] SEQ ID NO: 17: VIP D10.
[0130] SEQ ID NO: 18: 4 mini PEG D10.
[0131] SEQ ID NO: 19: ODP D10.
[0132] SEQ ID NO: 20: CTC conjugated with 10 glutamic acids
(CTC_peg 10_e10).
[0133] SEQ ID NO: 21: CTC conjugated with 10 glutamic acids
(CTC_MP4_e10).
[0134] SEQ ID NO: 22: P15 conjugated with 10 glutamic acids
(P15).
[0135] SEQ ID NO: 23: P15 conjugated with 10 glutamic acids
(P15_mp4_e10).
[0136] SEQ ID NO: 24: DGEA conjugated with 10 glutamic acids
(DGEA_mp4_e10).
[0137] SEQ ID NO: 25: ITGA conjugated with 10 glutamic acids
(ITGA5).
[0138] SEQ ID NO: 26: IKVAV conjugated with 10 glutamic acids
(IKVAV_mp4_e10).
[0139] SEQ ID NO: 27: LN2 conjugated with 10 glutamic acids
(LN2_P3_mp4_e10).
[0140] SEQ ID NO: 28: PHSRN conjugated with 10 glutamic acids
(PHSRN_mp4_e10).
[0141] SEQ ID NO: 29: P3 conjugated with 10 glutamic acids
(P3_mp4_e10).
[0142] SEQ ID NO: 30: SPARC conjugated with 10 glutamic acids
(SPARC 113_mp4_e10).
[0143] SEQ ID NO: 31: CBM(1-19)-D10 collagen binding motif.
[0144] SEQ ID NO: 32: CBD conjugated with 10 glutamic acids
(CBD_MP4_e10).
[0145] SEQ ID NO: 33: Targeting group consisting of a polypeptide,
DDDDDDDDDD.
[0146] SEQ ID NO: 34: Targeting group consisting of a polypeptide,
EEEEEEEEEE.
DETAILED DESCRIPTION
[0147] While the concepts of the present disclosure are illustrated
and described in detail in the figures and the description herein,
results in the figures and their description are to be considered
as examples and not restrictive in character; it being understood
that only the illustrative embodiments are shown and described and
that all changes and modifications that come within the spirit of
the disclosure are desired to be protected.
[0148] Unless defined otherwise, the scientific and technology
nomenclatures have the same meaning as commonly understood by a
person in the ordinary skill in the art pertaining to this
disclosure.
[0149] Aspects of the fracture targeted technology disclosed herein
can help both civilians and military personnel. Bone fractures
occur at an annual rate of 2.4 per 100 people and cost the US
healthcare system approximately $28 billion per year. Of the 6.3
million bone fractures that occur annually in the US, 300,000
result in delayed union or non-union healing. Approximately 887,679
hospitalizations result each year from fractures. Over half (57%)
of fractures resulting in hospitalizations occur in persons aged 65
and over. Estimated health care costs are indicated in Table 1,
below.
TABLE-US-00001 TABLE 1 Cost without Cost with Fracture Healing time
surgery surgery Leg 10-12 weeks) $2,500 $16,000 Hip 12+ weeks
$11,500 $66,500 Vertebral (8+ weeks) $5,000-15,000 $50,000-150,000
Arm 6-10 weeks $2,500 $16,000
[0150] Currently, a substantial fraction of national defense
outlays is devoted to combat-related medical expenditures, with a
significant proportion of these costs devoted to treatment of
orthopedic injuries. Indeed, 65% of all wounds associated with
military conflicts since WWI have included orthopedic injuries, and
26% of all injuries to an extremity have involved one or more
broken bones. Treatment of bone fractures not only removes a
soldier from service for an extended period of time, but also
requires the attention of multiple additional personnel to treat,
monitor and rehabilitate the injured soldier. Unfortunately, some
orthopedic injuries are so severe that resolution of the damage
never occurs, and the armed services are then obligated to care for
the damaged combatant in perpetuity.
[0151] Fractured bones are not only an adverse consequence of
combat, they also constitute a prominent repercussion of military
training exercises. During the course of a soldier's schooling, a
female recruit will have a 3.4-21% chance of suffering a stress
fracture, while a male recruit will have a 1-7.9% probability of
experiencing the same injury. While such maladies may at first seem
trivial, statistics reveal that they cost the military
.about.$34,000 per soldier which totals up to .about.$100 million
in aggregate per year. Not surprisingly, many affected recruits
eventually leave the military as a consequence of their stress
fracture, which results in further expenses arising from wasted
recruiting and training efforts. Therapies for fractured bones both
within and outside of the military rely almost exclusively on
mechanical stabilization of the damaged bone (i.e. use of a cast,
pin, rod, or plate, etc.). In fact, the only FDA-approved drug for
enhancing fracture repair is a bone anabolic agent that must be
applied topically to the fracture surface during surgery. Needless
to say, such a therapy is inappropriate when the surgery is not
otherwise indicated, can only be administered once (i.e. during the
brief period when the fracture surface is exposed), cannot be
easily adapted for treatment of multiple fractures, and is never
used for therapy of stress fractures. What is critically needed is
obviously a systemically administered bone anabolic agent (i.e. as
drug that can stimulate rapid bone fracture healing) that will
concentrate selectively on the bone fracture surface and induce
accelerated bone formation only at the damaged site. Surprisingly,
nothing of this sort has ever been described in the literature.
[0152] Recognizing the enormous need for a systemically
administered bone fracture-targeted healing agent, peptides and
other molecules with structures that home specifically to bone
fracture surfaces following intravenous or subcutaneous
administration were identified. A second group of bone anabolic
agents (for example, both bone growth stimulating hormones and
cytokines as well as various low molecular weight bone
growth-inducing drugs, etc.), that when linked to one of our bone
fracture-homing peptides, would promote accelerated fracture
repair, were also identified. Fortunately, several
fracture-targeted bone anabolic drugs met all initial requirements
for advancement into large animal studies. That is, the targeted
conjugates were found to: i) reduce the time for fractured femur
repair in mice by roughly half, ii) induce no detectable systemic
toxicity at its effective dose, iii) cause no ectopic bone
formation at either the injection site or elsewhere), iv) lead to
regeneration of bone at the fracture site that was biomechanically
stronger than the contralateral (unbroken) femur, and v) result in
eventual remodeling of the fractured region into normal cortical
bone.
[0153] All in vivo data included herein are from Swiss Webster
mice. All mice received an osteotomy on their right femur and
received subcutaneous drug administration daily for either 2,3 or 4
weeks, or 17 days, as indicated, for each compound. 1.times.
concentration represents 1 nmol/day, 10.times. represents 10
nmol/day, 100.times. represents 100 nmol/day most studies have an n
of 5.
[0154] Aspects of the disclosure include conjugates sometimes
written in the form of X-Y-Z, wherein each conjugate includes at
least one moiety (X) that has the ability to effect bone growth,
development, and/or healing, for example, anabolic agents, and a
targeting moiety (Z) which has an affinity for bone and helps to
direct the conjugate to bone. In some of these conjugates, the X
and Z portions are joined together by a linker region (Y).
[0155] Targeting moieties (Z), many of which are explicit or
implicit disclosed herein, have the potential to target bone
anabolic agents to bone fractures, ostectomies, and osteotomy
sites. The compounds described here are composed of molecules with
high affinity towards hydroxyapatite and a bone anabolic agent.
Although targeting has been exemplified primarily with acidic
oligopeptides, all molecules with affinity towards hydroxyapatite
could be attached to a bone anabolic agent to improve fracture
repair. These molecules include but are not limited to ranelate,
bisphosphonates, tetracyclines, polyphosphates, molecules with
multiple carboxylic acids, calcium chelating molecules, metal
chelators, acidic amino acid chains of either d or L chirality.
Each of the previously listed targeting molecules can be single
units, polymers, dendrimers or multiple units. Other molecules can
also be substituted for the targeting agent. These include
peptides, proteins and manmade molecules that intercalate, bind to,
adsorb to, or hybridize with: collagen, the extracellular matrix,
heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic
acid, elastin, fibronectin, laminin, proteoglycans, basement
membrane, extracellular polymeric substances, integrins, blood
clotting factors, fibrinogen, thrombin, fibrin, and other
extracellular macromolecules. It is also possible to target using a
combination of the listed targeting molecules.
Definitions
[0156] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this disclosure pertains.
[0157] The term "BV/TV" means the bone volume divided by total
volume of the 100 thickest micro CT slices of the fracture
callus.
[0158] The term "TbTh" means the trabecular thickness of the 100
thickest micro CT slices of the fracture callus.
[0159] The term "BV" means the overall bone volume of the 100
thickest micro CT slices of the fracture callus.
[0160] The term "TbSp" means the spacing between the trabecula in
the 100 thickest micro CT slices of the fracture callus.
[0161] The term "Peak Load" means a postmortem 4 point bend of the
healed femur. Peak load represents the maximum force the healed
femur withstood before it refractured.
[0162] The term "D10" at the end of any name represents that the
peptide is targeted to bone by a chain of 10 aspartic acids. D10
can be at the N-terminus or C-terminus of the specified
peptide.
[0163] The term "E10" or "(D)E10" at the end of any name represents
that the peptide is targeted to bone by a chain of 10 (D) glutamic
acids.
[0164] The term "P4" means a fragment that represents the knuckle
epitope in hBMP-2.
[0165] The term "P-4" corresponds to residues 73-92 of BMP-2 in
which Cys-78, Cys-79, and Met-89 are changed to Serine (Ser), Ser,
and Threonine (Thr). BMPs are well known regulators of bone and
cartilage formation. BMPs bind as dimers to type I and type II
Ser/Thr receptor kinases, forming an oligomeric complex that
activates intracellular Smad proteins leading to their
translocation into the nucleus where they serve as transcription
factors to activate different OB differentiation markers (such as
Runx2 (transcription factor for osteoblast differentiation)),
leading to osteoblastogenesis. BMPs have also been shown to
stimulate mesenchymal stem cells (MSC) differentiation to OBs by
promoting recruitment of osteoprogenitor cells.
[0166] The term "AHX" in the middle of any name represents that the
therapeutic is linked to the targeting peptide via a polymer of
6-(amino)hexanoic acid.
[0167] The term "AHX3" in the middle of any name represents that
the therapeutic is linked to the targeting peptide via a polymer of
(6-(amino)hexanoic acid).sub.3.
[0168] The term "mp4" in the middle of any name represents that the
therapeutic is linked to the targeting peptide via a polymer of 4
minipegs as known as 8-Amino-3,6-Dioxaoctanoic Acid.
[0169] The term "STB" at the end of any name represents that the
compound represents a chemically more stable version of its natural
version.
[0170] The term "peg10" in the middle of any name represents that
the therapeutic is linked to the targeting peptide via a polymer of
polyethylene glycol 10.
[0171] The term "DAPD" at the end of a compound denotes that the
compound has been cyclized via a lactam bridge between
diaminopropionic acid (DAP) and aspartic acid (D).
[0172] The term "DAPE" at the end of a compound denotes that the
compound has been cyclized via a lactam bridge between
diaminopropionic acid (DAP) and glutamic acid (E).
[0173] The term "KD" at the end of a compound denotes that the
compound has been cyclized via a lactam bridge between--Lysine
(K)-Aspartic acid (D).
[0174] The term "KE" at the end of a compound denotes that the
compound has been cyclized via a lactam bridge between--Lysine
(K)-Glutamic acid (E).
[0175] The term "Cys" at the end of a compound denotes that the
compound has a disulfide bridge made by two cysteines.
[0176] Compounds which effect bone growth and may be used to
practice aspects of the present disclosure include but are not
limited to the following: extracellular matrix proteins, fragments
of extracellular matrix components, or synthetic peptides or small
molecules that mimic the action of extracellular matrix component.
Examples of which may include but are not limited to: integrin
alpha (ITGA), integrin beta(ITGB), very late antigen(VLA),
Fibrinogen receptor, fibronectin, collagen, the extracellular
matrix, heparan sulfate, chondroitin sulfate, keratan sulfate,
hyaluronic acid, elastin, laminin, proteoglycans, basement
membrane, extracellular polymeric substances, integrins, blood
clotting factors, fibrinogen, thrombin, fibrin, Cell adhesion
molecules (CAMs), integrins, immunoglobulin superfamily (IgSF)
CAMs, Selectins, E-selectin, L-selectin, P-selectin, N-CAM (Myelin
protein zero), ICAM (1, 5), VCAM-1, PE-CAM, L1-CAM, Nectin(PVRL1,
PVRL2, PVRL3), Integrins, LFA-1 (CD11a+CD18), Integrin
alphaXbeta2(CD11c+CD18), Macrophage-1 antigen(CD11b+CD18), VLA-4
(CD49d+CD29), Glycoprotein IIB/IIIa(ITGA2B+ITGB3), ITGB1, ITGA7,
ITGAV, CD51, Vitronectin(VNRA), MSK8, ITGA2B, CD41, ITGAX, CD11c,
ITGA7, FLJ25220, ITGA8, ITGA9, RLC, ITGA10, ITGA11, HsT18964,
ITGAD, CD11D, FLJ39841, ITGAE, CD103, HUMINAE, ITGAL, CD11a, LFA1A,
ITGAM, CD11b, Macrophage Antigen(MAC), MAC-1, ITGA1, CD49a, VLA1,
ITGA2, CD49b, VLA2, ITGA3, CD49c, VLA3, ITGA4, CD49d, VLA4, ITGA5,
CD49e, VLA5, ITGA6, CD49f, VLA6, CD29, FNRB, MSK12, MDF2, ITGB2,
CD18, LFA-1, MAC-1, MFI7, ITGB3, CD61, GP3A, GPIIIa, ITGB4, CD104,
ITGB5, ITGB5, FLJ26658, ITGB6, ITGB6, ITGB7, ITGB8, ITGB8,
.alpha.1.beta.1, .alpha.2.beta.1, .alpha.3.beta.1, .alpha.4.beta.1,
.alpha.5.beta.1, .alpha.6.beta.1, .alpha.7.beta.1, .beta.L.beta.2,
.alpha.M.beta.2, Cadherin (CDH), CDH1, CDH2, CDH12, CDH3,
Desmoglein, Desmocollin, Protocadherins, CDH4-R-cadherin (retinal),
CDH5, CDH6, CDH7, CDH8, CDH9, CDH10, CDH11, CDH13, CDH15, CDH16,
CDH17, CDH18, CDH19, CDH20, CDH23, CDH22, CDH24, CDH26, CDH28,
cadherin EGF LAG seven-pass G-type receptor 1(CELSR1), CELSR2,
CELSR3, CLSTN1, CLSTN2, CLSTN3, dachsous homolog 1(DCHS1), DCHS2,
LOC389118, PCLKC, RESDA, RET, .alpha.IIb.beta.3, .alpha.V.beta.1,
.alpha.V.beta.3, .alpha.V.beta.5, .alpha.V.beta.6, .alpha.V.beta.8,
.alpha.6.beta.4, Desmoglein(DSG), DSG1, DSG2, DSG3, DSG4,
Desmocollin(DSC), DSC1, DSC2, DSC3, CD22, CD24, CD44, CD146, CD164,
perlecan, agrin, collagen XVIII, collagen type I, collagen type II,
collagen type III, collagen type IV, collagen type V, collagen type
VI, collagen type VII, collagen type VIII, collagen type IX,
collagen type X, collagen type XI, collagen type XII, collagen type
XIII, collagen type XIV, collagen type XV, collagen type XVI,
collagen type XVII, collagen type XVIII, collagen type XIX,
collagen type XX, collagen type XXI, collagen type XXII, collagen
type XXIII, collagen type XXIV, collagen type XXV, collagen type
XXVI, collagen type XXVII, collagen type XXVIII, collagen type
XXIX, Notch II, Notch I, osteopontin, osteonectin, bone
sialoprotein, Osteoprotegerin, osteocrin, osteocalcin, matrix
extracellular phosphoglycoprotein(MEPE), AC-100, SPARC 113, Sparc
118, CBM, ODP, IKVAV, ITGA5, RRETAWA, YIGSR, PHSRN, RGD, CTC, BCSP,
DGEA, GFOGER, KRSR, pRGD, hBSP278-293, heparin binding domain of
bone sialoprotein, or CB.
Bone Growth Modifiers and Delivery Peptides
TABLE-US-00002 [0177] (SEQ ID NO: 1) BFP1D10
DDDDDDDDDDGQGFSYPYKAVFSTQ
[0178] BFP (bone forming Peptide) a fragment of immature BMP7 is a
15-amino acid peptide corresponding to residues 100-115 of the
immature form of BMP-7 which like BMP2 is involved in osteogenic
differentiation, proliferation, and formation of new bone. This
short peptide also induces osteogenesis calcium content in
MSCs.
BMP-9
[0179] BMP-9 is also a potent regulator of osteogenesis and
chondrogenesis and is a potent inducer of differentiation of
osteoblasts. pBMP9 is a 23-residue peptide derived from residues
68-87 of the knuckle epitope of human BMP-9. The mechanism of
action of this peptide is likely to involve the small mothers
against decapentaplegic (Smad) pathway. The structure of BMP9 is
depicted in FIG. 27.
Ghrelin D10
[0180] Ghrelin is a 28-residue peptide hormone synthesized
primarily by the gastric fundus in response to fasting, and acts as
a ligand of the growth hormone secretagogue (GHS) receptor (GHSR)
to promote growth hormone release from the pituitary. Ghrelin
stimulation at the GHSR leads to the proliferation of osteoblasts
and prevents the apoptosis of osteoblasts through mitogen-activated
protein kinase/extracellular signal-regulated kinases (MAPK/ERK)
and phosphoinositide 3-kinase/protein kinase B (PKB) (PI3K/AKT)
pathways. Ghrelin also stimulates osteoprotegerin (OPG) gene
expression, which inhibits the coupling between the osteoclasts and
osteoblasts, leading to reduced osteoblast-related osteoclast
differentiation. Increased OPG also and decreases osteoclast
activity. Ghrelin is only active when the Ser-3 is acylated with
octanoic acid. Our construct contains a stabilized version of this
where Ser-3 was replaced with diaminopropionic acid. The structure
of Ghrelin D10 is depicted in FIG. 28.
Preptin D10
[0181] Preptin is a 34-residue peptide hormone that is secreted by
the .beta.-cells of the pancreatic islets. This peptide corresponds
to Asp-69 to Leu-102 of the E-peptide of proinsulin-like growth
factor-II (pro-IGF-II). Preptin's anabolic effects on bone are
exerted through its ability to stimulating osteoblasts s
proliferation, differentiation, and promoting their survival.
Preptin's proliferative effect is predicted to be facilitated
through a G-protein-coupled receptor triggering phosphorylation of
p42/44 MAP kinases. Some of preptin's anabolic effects are believed
to be due to it stimulating an increase in a known bone anabolic
connective tissue growth factor. While the native peptide effects
glucose metabolism the first 16 amino acids are important for its
anabolic effects and have no effects on glucose metabolism. The
structure of Preptin D10 is depicted in FIG. 29.
CNP-D10 is a C-Type Natriuretic Peptide Targeted with D10
[0182] C-Type Natriuretic Peptide (CNP) contains 22 residues
stabilized by an intramolecular disulfide linkage between Cys-6 and
Cys-22 it functions as a local regulator of vascular tone, possibly
due to its strong vasorelaxant properties. CNP also acts on the
differentiation and proliferation of OBs, OCs, and chondrocytes via
an autocrine/paracrine process through binding to the natriuretic
peptide receptor B (NPR-B). CNP activates bone turnover and
remodeling. Endochondral ossification is another mechanism of bone
formation affecting chondrocytes. It involves the conversion of an
initial cartilage template into bone such as long bones and
vertebrae. CNP has been shown to be an important anabolic regulator
of endochondral ossification. The structure of CNP-D10 is depicted
in FIG. 30.
VIP D10 is Vasoactive Intestinal Peptide Targeted with D10
[0183] Vasoactive intestinal peptide (VIP), a neuropeptide that
consists of 28 amino acids and originally isolated from porcine
intestine. VIP has several effects however its receptors are
present on the nerves that rapidly innervate the fracture callus.
It has been shown to be an important regulator of bone formation.
VIP exerts its biological effects through the G-protein-coupled
receptors (VPAC1, VPAC2, and PAC1). Signaling through these
receptors also enhanced cell osteoblast differentiation and
proliferation. It also increases expressions of collagen type I,
osterix, and alkaline phosphatase (ALP) through signaling at the
VPAC2 receptor by triggering an increase in intracellular calcium.
VIP also increases the expressions of BMPs and the nuclear presence
of Smad1 transcription factor, which can activate various
bone-specific genes. VIP also enhances osteoblast proliferation and
mineralization through increased gap junction intercellular
communication (GJIC) between osteoblasts. VIP also affects the
differentiation of osteoclasts thus leading to an increase in bone
resorption. The structure of VIP D10 is depicted in FIG. 31.
Substance P with 4 Mini PEG Conjugated to D10
[0184] Substance P- is an 11-amino acid long pro-inflammatory
neuropeptide belonging to the tachykinin family. Substance P
improves mineralization of osteoblasts and the expression of
osteogenic markers at late-stage bone formation, by activating
neurokinin-1 receptor, a G-protein coupled receptor found in the
central and peripheral nervous systems. Also, substance P reduces
osteoclastogenesis and bone resorption. Substance P upregulates the
expressions of collagen type 1, ALP, Runx2 and osteocalcin in
osteoblasts this effect involves the activation of
Wnt/.beta.-catenin signaling pathway. Substance P promotes
differentiation and migration capability of rat bone marrow MSCs
and activates BMP-2 expression in osteoblasts. Some of substance
p's anabolic effects are attributed to in human to increases in
osteoblast proliferation and mineralization through increased gap
junction intercellular communication between osteoblasts. Gap
junction intercellular communication has important roles in
conveying the anabolic effects of hormones and growth factors and
regulating transcription of osteogenic markers. The structure of
Substance P with 4 mini PEG conjugated to D10 is depicted in FIG.
32.
CBMD10- is the Collagen Binding Motif of Osteopontin Targeted by
D10
[0185] CBM-collagen binding motif is the highly conserved
28-residue collagen binding motif (CBM) (residues 150-177) of human
osteopontin. Osteopontin, a glycosylated phosphoprotein prominently
localized in the extracellular matrix (ECM) of mineralized bone
tissue to form a complex with collagen in bone tissue, thereby
inducing mineralization of collagen fibrils. CBM enhances
osteoblast differentiation of human MSC. CBM causes osteogenic
differentiation of human bone marrow MSCs and increases mineralized
of bone. CBM works in human MSCs by increasing extracellular
Ca.sup.2+ influx, which leads to the activation of CaMKII and the
subsequent phosphorylation of ERK1/2, ultimately influencing OB
differentiation. The structure of VIP D10 is depicted in FIG.
33.
ODP D10
[0186] Osteopontin-derived peptide (ODP), a 15-residue peptide
derived from rat osteopontin. ODP like CBM is a fragment of
extracellular protein involved in the mineralization of collagen.
ODP enhanced the differentiation and mineralization of MSCs. ODP
improves the attachment via receptor mediated attachment and
migration of osteoblasts and fibroblasts to the fracture site. ODP
improves the proliferation and migration of osteoblasts. Though the
signaling pathways aren't completely elucidated for this molecule
its believed that it works in a similar mechanism as CBM. The
structure of ODPD10 is depicted in FIG. 34.
TABLE-US-00003 (SEQ ID NO: 2) OGP-D10 DDDDDDDDDDALKRQGRTLYGFGG
[0187] OGP-targeted Osteogenic growth peptide (OGP) is composed of
a 14-AA residue identical to the C-terminus of histone 4 conjugated
to an acidic oligopeptide at the N-terminus. Systemic
administration of free OGP has been shown to improve fracture
repair by improving the mineralization of cartilaginous fracture
callus.
TABLE-US-00004 (SEQ ID NO: 11)
MGF-DDDDDDDDDDYQPPSTNKNTKSQRRKGSTFEEHK
[0188] Targeted Mechano growth factor (MGF E peptide is a splice
variant of insulin-like growth factor I (IGF-I) with a targeting
acidic oligopeptide on the N terminus. MGF causes osteoblast
proliferation through the MAPK-ERK signaling pathway. Local
injections (57 ug/kg) in rabbit bone defects (5 mm) demonstrated
accelerated healing through osteoblast proliferation.
TABLE-US-00005 (SEQ ID NO: 12) TP508-
DDDDDDDDDDAGYKPDEGKRGDACEGDSGGPFV
[0189] Targeted TP-508 is a prothrombin peptide that has been
modified on the N-terminus with an acidic oligopeptide. The
anabolic portion of TP-508 has been used in clinical trials for
repairing foot ulcers. Free tp-508 has a proliferative effect on
osteoblasts. Local injections have demonstrated accelerated
fracture repair in older rats.
Chemotactic Collagen Fragment (CTC) (D)E.sub.10
[0190] The 12-residue chemotactic cryptic peptide (CTC), derived
from the CTX region of collagen type III, has chemotactic activity
for a number of human stem cells. CTX is the C-terminal telopeptide
that can be used as a biomarker in the serum to measure the rate of
bone turnover. In vitro, 0.1 mM CTC increased the expression of
osteogenic genes; ALP activity and mineralization of human
perivascular stem cells (have properties of MSCs and are able to
undergo osteogenesis). In a mouse model of limb amputation, 150 g
CTP caused local bone nodule formation after 2 weeks. In addition,
it has the ability to alter stem cell recruitment and
differentiation at the site of injury. The structure of CTC
(D)E.sub.10 is depicted in FIG. 35.
P-15 Peptide (Collagen Fragment)
[0191] P-15 peptide is a 15-reside peptide corresponding to the
sequence 766-780 of the .alpha.-1 chain of type I collagen, which
is uniquely involved in binding of cells, such as OBs. This peptide
mimics the role of collagen in forming collagenous matrices and
playing a role in cell adhesion and mineralization. Nguyen et al.
fabricated a composite matrix consisting of an organic bone matrix
(ABM), which has the same mineral composition as normal human bone,
coated with P-15 suspended in hyaluronate hydrogel. Implantation of
this composite matrix in vivo led to migration and attachment of
host osteoprogenitor cells to this matrix, followed by development
of mineralized bone. Other reports also showed the peptide's
bone-forming capability when implanted in some form of matrix or
another. A systemically adminsterable conjugate has been developed
that targets the collagen memetic to sites of damaged bone without
the need for invasive surgery. FIG. 42 depicts the structure of
P-15.
DGEA (Collagen Fragment)
[0192] DGEA is a tetrapeptide corresponding to residues 435-438 of
type I collagen. Type 1 collagen is an important component of the
extracellular matrix that is involved in cell attachment and
regulation. This peptide was shown to induce early osteogenic
differentiation of human bone marrow MSCs via binding to the
integrin receptor .alpha.2.beta.1. In vivo, the peptide increased
bone formation the DGEA sequence resulted in enhanced osteogenic
differentiation and increased mineral deposition. This positive
regulator of bone cell development once localized to the fracture
site can assist in creating the correct chemical cue to the
mesenchymal stem cells to differentiate into osteoblast to repair
the defect. The disclosed construct of DGEA is the full natural 4
amino acids with a Serine and Proline on the C terminus. On the N
terminus is 4 minipeg spacers proceeded by ten d glutamic acids.
FIG. 50 depicts the structure of DGEA_mp4_e10.
ITGA5
[0193] The present disclosure demonstrates that numerous components
of the extracellular matrix are anabolic and when delivered to the
fracture site can improve fracture repair with virtually no side
effects. One of the most promising compounds is ITGA5 a synthetic
integrin alpha 5 ligand. Integrin alpha 5 is high expressed on
mesenchymal stem cells as they transition from stem cell to
osteoblast. ITGA5 natural ligand is fibronectin. But ITGA5 or
CRRETAWACITGA 5 was discovered via phage display and has a high
affinity to just ITGA5. ITGA5 is a cyclic 9 amino acids that can be
cyclized with a stable amide bond. Targeted delivery of ITGA5 has
shown impressive anabolic effects so far in repeated experiments.
Peptide-mediated activation of ITGA5 in murine C3H10T1/2
mesenchymal cells in the literature resulted in the generation of
the integrin-mediated cell signals FAK and ERK1/2-MAPKs. It has
been shown that, peptide-based activation of ITGA5 protected from
cell apoptosis but did not affect cell adhesion or replication,
while it enhanced the expression of the osteoblast marker genes
Runx2 and type I collagen and increased extracellular matrix (ECM)
mineralization anabolic effect resulted from decreased cell
apoptosis and increased bone forming surfaces and bone formation
rate (BFR). It has been shown that pharmacological activation of
ITGA5 in mesenchymal cells is effective in promoting de novo bone
formation as a result of increased osteoprogenitor cell
differentiation into osteoblasts and increased cell protection from
apoptosis. Some of ITGAs effect is potentially through
Wnt-.beta.-catenin signaling to promote osteoblast as mediated via
signals FAK and ERK1/2-MAPKs. FIG. 55 depicts the structure of
ITGA5. FIG. 67 depicts the structure of ITGA_mp4_e10_DAPE. FIG. 65
depicts a structure with a stabilized ring of ITGA, which can be
created by substituting the 2 cystines for lysine and glutamic acid
and forming a stabilized amide bone between their side chain. FIG.
72 depicts the structure of ITGA_mp4_e10_DAPD. FIG. 77 depicts the
structure of ITGA_mp4_e10_KD. FIG. 82 depicts the structure of
ITGA_mp4_e10_KE.
IKVAV
[0194] IKVAV is a laminin .alpha.1 which is a 400 kd chain of
lamanin the large glycoprotein that makes up the majority of the
basement membrane of the extracellular matrix. Lamanins contribute
to cell differentiation, cell shape and movement, maintenance of
tissue phenotypes, and promotion of tissue survivaLT IKAV derived
from the largest of the chains of lamanin has been found to drives
osteogenic differentiation of human MSCs. IKVAV induces Runx2 and
ALP expression in human MSCs promotes osteogenesis by integrin
signaling. This is due to the fact that IKVAV the activation of a
range of integrins: a3b1, a4b1, and a6b1 that are expressed on
human mesenchymal stem cells as they undergo the transition to
osteoblasts. By targeting Ikvav to the fracture callus we can
create a stimulatory environment which differentiates the
periosteal stem cells into osteoblasts to promote a more anabolic
repair response. Ikvav e10 also improves cell adhesion at the
damaged site. The osteogenic genes induction of Ikvav and other
related integrin binding peptides is facilitated via the formation
of a focal adhesion. Focal adhesions mediate intracellular changes
beyond just serving as attachment point for the cytoskeleton to the
extracellular matrix via the recruitment of proteins such as focal
adhesion kinase (FAK), Rho (family of GTPases that regulates
cellular function), Rac (a subfamily of the Pho family of GTPases),
and integrin-linked kinase (ILK), are also recruited. The function
of the proteins includes both regulation of cytoskeletal remodeling
and the assembly or disassembly of the focal adhesion complex, but
they also tie into intracellular signaling cascades, such as
mitogen-activated protein kinase and C-Jun N-terminal kinase (INK).
These signaling cascades induce the change in expression of
osteogenic genes such as Runx2 and ALP. FIG. 87 depicts the
structure of IKVAV_mp4_e10.
LN2_P3 (Laminin)
[0195] Laminin in a major protein comment of the extracellular
matrix. They primarily mediate cell attachment to surfaces either
via syndecans or integrins. Human laminin a2 LG1 domain mediates
cell attachment through syndecan-1 by inducing phosphorylation and
membrane localization of protein kinase Cd. LN2_P3 or DLTIDDSYWYRI
is one of the bioactive cores of human laminin a2 chain. It has
been shown to accelerate osteointegration of dental implants and
overall to improve cell attachment and activate osteoblasts. The
conjugate of the present disclosure localized the LN2_p3 motif via
an N terminally attached minipeg 4 spacer and targeting ligand.
FIG. 92 depicts the structure of LN2_P3_mp4_e10.
PHSRN (Fibronectin)
[0196] Fibronectin (FN) is a predominant ECM protein that mediates
the adhesion and spreading of many cell types. Its interactions
with cells mediate and controls, migration, survival and
activation. RGD is the most well studied binding motif form
fibronectin. But fibronectin contains another cell attachment motif
PHSRN. The peptide PHSRN is found in the 9th type III domain of FN,
adjacent to the 10th domain that contains the RGD peptide. Like RGD
PHSRN interacts with the Alpha5Beta1 and AlphaIIbBeta31 Integrin
receptors. Through these cellular interactions it mediates not only
cell attachment but initiates an increase in the activity of the
cells. It has been shown that PHSRN improves the attachment and
activity of osteoblasts. The conjugate of the present disclosure
chemically homes PHSRN to the sight of damaged bone via a (D)e10
targeting ligand attached to PHSRNs N terminus via a minipeg
spacer. FIG. 97 depicts the structure of PHSRN_mp4_e10.
P3 (Bone Sialoprotein)
[0197] Bone sialoprotein is a very common nonmineral component of
bone It is a highly acidic globular protein which plays roles in
cell attachment, angiogenesis. One of it more important roles is in
the nucleation of hydroxyapatite crystals in newly forming bone.
Bone sialoprotein like fibronectin contains the RGD motif (a
sequence made up of arginine, glycine, and aspartic acid) which is
in evolved in cell attachment and proliferation. P3 which
represents residues 278-293 of human bone sialoprotein has been
utilized to improve osteointegration by improving cell
proliferation and attachment via its interaction the alpha5beta1
and alpha3beta3 ligand receptors RIIbd3 receptors integrin
interaction. Rather than having to locally apply it, a targeted
construct of the present disclosure has been developed which
consists of the P3 fragment followed by a 4 minipeg spacer and 10
(D) glutamic acids to home to bone fractures. FIG. 102 depicts the
structure of P3_mp4_e10.
SPARC.sub.113
[0198] SPARC.sub.113 are fragments of the Secreted Protein Acidic
and Rich in Cysteine (SPARC aka osteonectin) which is expressed
during development and in wound repair. SPARC is a copper binding
protein in the extracellular matrix. SPARC is cleaved by a number
of proteases in vivo, which releases domains with a variety of
biological effects. SPARC.sub.113 is residue 113-127 of SPARC and
is from released from the follistatin-like domain and contain the
tripeptide GHK (a sequence made up of glycine, histidine, and
lysine), which promotes angiogenesis in the rabbit cornea assay and
accelerates dermal wound healing in mouse and rat models. GHK of
SPARC.sub.113 binds to copper and initiates a vast host of tissue
repair including, macrophage chemotaxis, angiogenesis, initiates
protein expression of collagen and other growth factors. FIG. 107
depicts the structure of SPARC 113_mp4_e10.
CBM(1-19)-D10 Collagen Binding Motif
[0199] CBM(1-19)-D10 collagen binding motif is the truncated
version (1-19) of the highly conserved 28-residue collagen binding
motif (CBM) (residues 150-177) of human osteopontin. Osteopontin, a
glycosylated phosphoprotein prominently localized in the ECM of
mineralized bone tissue to form a complex with collagen in bone
tissue, thereby inducing mineralization of collagen fibrils. CBM
enhances osteoblast differentiation of human MSC. CBM causes
osteogenic differentiation of human bone marrow MSCs and increases
mineralized of bone. CBM works in human MSCs by increasing
extracellular Ca.sub.2+ influx, which leads to the activation of
CaMKII and the subsequent phosphorylation of ERK1/2, ultimately
influencing OB differentiation. FIG. 112 depicts the structure of
CBM(1-19)-D10 collagen binding motif.
Collagen Binding Domain (CBD) of Osteopontin
[0200] Collagen Binding domain (CBD) of osteopontin, which
corresponds to residues 35-62 of rat osteopontin, was also shown to
stimulate human osteosarcoma cell differentiation into Osteoblasts
in vitro (at concentration of 0.01 mM) as determined by increased
expression of ALP, and type I collagen after 14 days (FIG.
16)..sub.1 CBD's effect on cell differentiation was shown to
involve the activation of MAPK and protein kinase B (Akt) pathways.
The conjugate of the present disclosure is 1-28 of osteopontin
followed by 4 peg 2 spacers followed by ten d glutamic acids to
home it to bone fractures. FIG. 116 depicts the structure of
CBD_MP4_e10.
Material and Methods
Solid Phase Peptide Synthesis
[0201] Unless noted otherwise, the conjugates of the present
disclosure are synthesized using the following synthesis. In a
solid phase peptide synthesis vial capable of bubbling nitrogen,
Wang resin (0.39 mmol/g) was loaded at 0.39 mmol/g with the first
amino acid overnight in dichloromethane (DCM) and diisopropyl ethyl
amine (DIPEA). The resin was then capped with acetic anhydride and
pyridine for 30 minutes, followed by three washes of DCM and
dimethylformamide (DMF), respectively. Following each amino acid
coupling reaction, fluoroenylmethyloxycarbonyl (Fmoc)-groups were
removed by three 10-minute incubations with 20% (v/v) piperidine in
DMF. The resin was then washed 3.times. with DMF prior to the next
amino acid being added. Each amino acid was added in a 5-fold
excess with N,N,N'N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU) in DIPEA. Upon completion of the
synthesis, peptides were cleaved using 95:2.5:2.5 trifluoroacetic
acid:water:triisopropylsilane. Cysteine containing peptides were
cleaved using 95:2.5:2.5 trifluoroacetic
acid:triisopropylsilane:water: and 10 fold
tris(2-carboxyethyl)phosphine (TCEP).
Cyclization Method for Disulfide Bridged Cyclic Peptides
[0202] For Amylin(1-8) and CGRP, the standard synthesis of the
linear form of the cyclic peptides Fmoc Cystine with
Acetamidomethyl protecting group on the sulfur was used. Then, to
cyclize the peptide, the Cys(Acm) On-Resin was suspended the linear
peptide resin in N,N-dimethylformamide (DMF) (approximately 1
mL/gram of resin). Then, the resin was treated with 10 equiv. of
iodine (12) in DMF/H2O 4:1 (v/v), approximately 1 mL/gram of
resin). Then, argon gas was bubbled through the reaction mixture at
room temperature for 40 minutes. Then, the resin was filtered and
washed 3 times with DMF, 2 times with 2% ascorbic acid in DMF, 5
times with DMF, and 3 times with dichloromethane (DCM). Then,
proceeded with normal n terminal fmoc deprotection and cleavage
from the resin with normal cleavage solution with no TCEP added to
preserve the disulfide bond. The peptides were then as all peptides
purified using reverse phase chromatography on an IPLC using a
0-50% 20 mM ammonium acetate:acetonitrile gradient. The product was
then identified from the appropriate fraction using liquid
chromatography/mass spectrometry (LC/MS) and lyophilized to recover
it from the water:acetonitrile mixture. All compounds were
dissolved in sterile phosphate buffered saline (PBS) at the
appropriate dose concentrations for drug delivery.
Cyclization Method for Stabilized Lactam Peptides
[0203] For ITGA_mp4_e10_stb, ITGA_mp4_e10_KD, ITGA_mp4_e10_KE, and
ITGA_mp4_e10_DAPD, ITGA_mp4_e10_DAPE, the standard synthesis of the
linear form of the cyclic peptides Fmoc Glutamic, Asapartic acid,
Diaminopropionic, Lysine acid containing with Allyl ethers or
allyloxycarbonyl (alloc) protecting group on the carboxylic acid or
amine were used at the appropriate points desired for cyclization.
Then, the first step was the removal of Allyl Esters and Aloc
Groups. This was done by swelling the linear peptide-resin in
chloroform (CHCl.sub.3) followed by suspending the swollen resin in
CHCl.sub.3 (approximately 35 mL per gram of resin). Then, acetic
acid (0.5 mL per gram of resin), N-methylmorpholine (2 mL per gram
of resin), and Pd(PPh3)4 (0.3 equivalents based on resin
substitution) were added to the resin. Next, the mixture was
bubbled at room temperature for 24 hours to remove the allyl ethers
and aloc protecting group. Then, the deprotected resin was filter
and wash with dichloromethane (DCM). The deprotected amino acids
were then coupled onto the resin by suspending the resin in
Dimethyl formamide(DMF). Then, they were coupled with 3 equivalents
of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate, 3 equivalents of
6-Chloro-1-hydroxybenzotriazole dihydrate, 3 equivalents of
N,N-Diisopropylethylamine for 24 hours. Next, it was washed with 3
times with DMF, 3 times with DCM, 2 times with methanol, and then
dried with argon gas. Then, the resin was subjected to normal
cleavage conditions as previously described. The peptides were then
as all peptides purified using reverse phase chromatography on an
HPLC using a 0-50% 20 mM ammonium acetate:acetonitrile gradient.
The product was then identified from the appropriate fraction using
LCMS and lyophilized to recover it from the water:acetonitrile
mixture. All compounds were dissolved in sterile PBS at the
appropriate dose concentrations for drug delivery.
General Methods for Obtaining Test Data
[0204] The targeted conjugates were synthesized using standard Fmoc
solid-phase peptide synthesis, as described above. To ensure the
conjugates' activity, mouse pre-osteoblast (MCTC3-E1) cells were
treated with the targeted and untargeted compounds for three days
at concentrations from 1 pM to 100 nM. After three days of
treatment, the cells were harvested, and the RNA was purified from
the cells. Expression levels of ALP, RUNx2 (transcription factor
for osteoblast differentiation), osterix (OSX), osteopontin (OPN),
collagen 1A (Col-1A), OPG, RANKL, sclerostin gene (SOST), and OC
were quantified via quantitative reverse transcription polymerase
chain reaction (RT-qPCR). Once the biological activity of the
conjugates was confirmed, they were tested in vivo in a fracture
model. Aseptic surgical techniques were used to place a 23-gage
needle as in intramedullary nail in the femur of anesthetized,
12-week-old Swiss Webster mice for internal fixation before
fracture. Femur fractures were induced using a drop weight fracture
device from RISystem. The mice received buprenorphine for three
days post fracture. The mice were dosed subcutaneously each day for
three weeks or 17 days. Fracture healing was assessed using microCT
(Scanco Medical Ag). Morphometric parameters were quantified in the
100 widest slices of the fracture callus. Trabecular thickness
(Tb.Th.), trabecular spacing (Tb.Sp.), total volume (TV), and
volume of calcified callus (BV) were calculated. Fractured femurs
were tested for strength in a four-point bend to failure using an
Electro Force TestBench (TA Instruments). Lower supports were 10 mm
apart on the anterior face of the femur in contact with the
proximal and distal diaphysis. Upper supports were 4 mm apart and
spanned the entire fracture callus on the diaphysis. Force was
applied from the posterior face of the femur with a displacement
rate of 0.3 mm/sec. Peak load, yield load, stiffness, displacement
post yield, work to fracture, and deformation data were generated.
Statistical analysis was performed using a two-way analysis of
variance (ANOVA) and a Tukey post-hoc analysis with significance
reported at the 95% confidence level. All animal experiments were
performed in accordance with protocols approved by Purdue
University's Institutional Animal Care and Use Committee
(IACUC).
EXAMPLES
Example 1. Targeted Delivery of Src Kinase Inhibitors to Fracture
Site for Accelerated Healing
[0205] Example 1 shows representative Src kinase inhibitors
Dasatinib and E738 (structures shown in FIGS. 3-4 respectively)
effectively increased the bone density of the fracture callus when
they are conjugated with acidic aspartic acids. See FIGS. 1-2,
where bone density of the fracture callus from the targeted
Dasatinib group is twice as dense as the saline group, and 50%
denser than the free Dasatinib group; targeted E738 conjugate has
significantly improved the bone density and trabecular thickness at
the fracture callus. The structure of CBMD10 is depicted in FIG.
33.
Example 2. Representative Anabolic Peptides on Peak Load of
Fractured Femurs after Two Weeks
[0206] Example 2 provides the maximum force a representative
anabolic peptide induced healed femur can withstand before it
refractured. As shown in FIG. 5, bone morphogenetic protein pathway
signaling peptide BFP-D10 with 100 nmol/day (100.times.) treatment
obtained the maximum peak load, followed by IGF derived peptide of
Preptin-D10 100.times., and Osteogenic growth peptide (OGP-D10
100.times.), as compared to PBS.
Example 3. Preptin D10 Efficacy on Fracture Healing
[0207] Example 3 indicates Preptin D10 effect on healing fractured
bone after 2 weeks of various concentrations application (1
nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times.,
10.times. and 100.times. respectively). The healing was reflected
as BV/TV in FIG. 6, TbTh in FIG. 7 and bone volume in FIG. 8, all
in a dose dependent manner.
Example 4. OGP D10 Efficacy on Fracture Healing
[0208] Example 4 indicates osteogenic growth peptide conjugate
(OGP-D10) effect on healing fractured bone after 2 weeks of various
concentrations application (1 nmol/day, and 100 nmol/day, referred
as 1.times., and 100.times. respectively). The healing was
reflected as BV/TV in FIG. 9, TbTh in FIG. 10 and TbSp in FIG. 11,
all in a dose dependent manner.
Example 5. BFP D10 Efficacy on Fracture Healing
[0209] Example 5 indicates bone forming peptide conjugate (BMP-D10)
effect on healing fractured bone after 2 weeks of various
concentrations application (1 nmol/day, 10 nmol/day and 100
nmol/day, referred as 1.times., 10.times. and 100.times.
respectively). The healing was reflected as BV/TV in FIG. 12, and
TbSp in FIG. 13 in a dose dependent manner.
Example 6. Substance P D10 Effect on Fracture Healing
[0210] Example 6 indicates substance P D10 conjugate effect on
healing fractured bone after 4 weeks of various concentrations
application (1 nmol/day, 10 nmol/day and 100 nmol/day, referred as
1.times., 10.times. and 100.times. respectively). The healing was
reflected as BV/TV in FIG. 14A in dose dependent manner. FIG. 14B
indicates the peak load of substance P D10 0.times. induced healed
femur can withstand between 30-35 Newtons force.
Example 7. Ghrelin-D10 Effect on Fracture Healing
[0211] Example 7 indicates Ghrelin-D10 conjugate effect on healing
fractured bone after 4 weeks of various concentrations application
(nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times.,
10.times. and 100.times. respectively). The healing was reflected
as BV/TV in FIG. 15 in dose dependent manner.
Example 8. pBMP9 D10 Effect on Fracture Healing
[0212] Example 8 indicates pBMP9 D10 conjugate effect on healing
fractured bone after 4 weeks of various concentrations application
(nmol/day, 10 nmol/day and 100 nmol/day, referred as 1.times.,
10.times. and 100.times. respectively). The healing was reflected
as bone volume in FIG. 16 and BV/TV in FIG. 17 in a dose dependent
manner.
Example 9. CNP D10 Effect on Fracture Healing
[0213] Example 9 indicates C-Type Natriuretic Peptide conjugate CNP
D10 effect on healing fractured bone after 4 weeks of various
concentrations application (1 nmol/day and 10 nmol/day referred as
1.times. and 10.times. respectively). The healing was reflected as
BV/TV in FIG. 18 in a dose dependent manner.
Example 10. ODP D10 Effect on Fracture Healing
[0214] Example 10 indicates osteopontin derived peptide conjugate
ODP D10 effect on healing fractured bone after 4 weeks of 1
nmol/day (referred as 1.times.). The healing was reflected as BV/TV
in FIG. 19.
Example 11. CBM D10 Effect on Fracture Healing
[0215] Example 11 indicates collagen binding motif of osteopontin
conjugate CBM D10 effect on healing fractured bone after 3 weeks of
various concentrations application (0.1 nmol/day, 1 nmol/day and 10
nmol/day, referred as 0.1.times., 1.times. and 10.times.
respectively). The healing was reflected as BV/TV in FIG. 20 in a
dose dependent manner. It is worth noting that the lowest does of
CBM D10 has the similar effect of free PTH, an anabolic drug
without specific bone targeting.
Example 12. P4 D10 Effect on Fracture Healing
[0216] Example 12 indicates P4 D10 conjugate effect on healing
fractured bone after 4 weeks of various concentrations application
(nmol/day and 10 nmol/day, referred as 1.times. and 10.times.
respectively). The healing was reflected as BV/TV in FIG. 21 in a
dose dependent manner and bone volume in FIG. 22.
Example 13. MGF D10 Effect on Fracture Healing
[0217] Example 13 indicates mechano growth factor conjugate MGF D10
effect on healing fractured bone after 4 weeks of various
concentrations application (1 nmol/day and 10 nmol/day, referred as
1.times. and 10.times. respectively). The healing was reflected as
BV/TV in FIG. 23 in a dose dependent manner.
Example 14. TP508 D10 Effect on Fracture Healing
[0218] Example 14 indicates thrombin fragment TP508 conjugate TP508
D10 effect on healing fracture after 4 weeks of various
concentrations application (1 nmol/day and 10 nmol/day, referred as
1.times. and 10.times. respectively). The healing was reflected as
BV/TV in FIG. 24 in a dose dependent manner.
Example 15. VIP D10 Effect on Fracture Healing
[0219] Example 15 indicates vasoactive intestinal peptide conjugate
VIP D10 effect on healing fracture after 4 weeks of 1 nmol/day
application (1.times.). The healing was reflected as BV/TV in FIG.
25 and TbTh in FIG. 26.
Example 16. Chemotactic Collagen Fragment(CTC) (D)E10
[0220] Example 16 indicates CTC (D)E.sub.10 conjugate effect on
healing fractures. FIG. 36 depicts in vivo fracture healing
efficacy of CTC_peg10_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV--represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 0.1.times., 1.times., and
10.times. are respectively 1 nmol, 1 nmol, and 10 nmol of the
conjugate delivered daily by subcutaneous injection.
CTC_peg10_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0221] FIG. 37 depicts in vivo fracture healing efficacy of
CTC_peg10_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair 1.times., 10.times., and
100.times. are respectively 1 nmol, 10 nmol, and 100 nmol of the
conjugate delivered daily by subcutaneous injection. The
CTC_peg10_(D)E.sub.10 conjugate improves bone strength at the
fracture calluses three weeks post fracture.
[0222] FIG. 38 depicts the structure of CTC_MP4_e10. FIG. 39
depicts in vivo fracture healing efficacy of CTC_mp4_(D)E.sub.10
conjugate on Swiss Webster fracture-bearing mice (n=10) after 3
weeks. BV/TV--represents the bone volume divided by total volume of
the 100 thickest micro CT slices of the fracture callus and is a
measure of how dense the bone is at the site of fracture repair.
1.times. and 10.times. are respectively 1 nmol, and 10 nmol of the
conjugate delivered daily by subcutaneous injection.
CTC_mp4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0223] FIG. 40 depicts in vivo fracture healing efficacy of
CTC_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=10) after 3 weeks. Max load represents the maximum force
the healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 1.times. and 10.times. are
respectively 1 nmol, and 10 nmol of the conjugate delivered daily
by subcutaneous injection. CTC_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0224] FIG. 41 depicts in vivo fracture healing efficacy of
CTC_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=10) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
1.times. and 10.times. are respectively 1 nmol, and 10 nmol of the
conjugate delivered daily by subcutaneous injection.
CTC_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
Example 17. P-15 (Collagen Fragment)
[0225] Example 17 indicates P-15 conjugate effect on healing
fractures. FIG. 43 depicts in vivo fracture healing efficacy of
P-15_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 3 weeks. BV/TV--represents the bone volume divided by
total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1.times., 1.times., and 10.times. are
respectively 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
delivered daily by subcutaneous injection. The P-15_(D)E10
conjugate raises bone density at the fracture calluses three weeks
post fracture.
[0226] FIG. 44 depicts in vivo fracture healing efficacy of
P-15_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 0.1.times., 1.times. and
10.times. are respectively 0.1 nmol, 1 nmol, and 10 nmol of the
conjugate delivered daily by subcutaneous injection. The
P-15_(D)E10 conjugate raises bone strength at the fracture calluses
three weeks post fracture.
[0227] FIG. 45 depicts the structure of P-15_mp4_e10, a P15
fragment connect via 4 peg2 spacers to (D)E10 bone targeting
ligand. The linker gives it the space it needs to interact with
neighboring cells.
[0228] FIG. 46 depicts in vivo fracture healing efficacy of
P-15_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 1
nmol, 10 nmol, and 100 nmol of the conjugate were delivered daily
by subcutaneous injection. P-15_mp4_(D)E.sub.10 conjugate raises
bone mineralization at the fracture calluses three weeks post
fracture.
[0229] FIG. 47 depicts in vivo fracture healing efficacy of
P-15_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 1 nmol, 10 nmol, and 100 nmol of the conjugate
were delivered daily by subcutaneous injection.
P-15_mp4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0230] FIG. 48 depicts in vivo fracture healing efficacy of
P-15_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 1 nmol, 10 nmol, and 100 nmol of
the conjugate were delivered daily by subcutaneous injection.
P-15_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
[0231] FIG. 49 depicts in vivo fracture healing efficacy of
P-15_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair. 1
nmol, 10 nmol, and 100 nmol of the conjugate were delivered daily
by subcutaneous injection. P-15_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 18. DGEA (Collagen Fragment)
[0232] Example 18 indicates DGEA conjugate effect on healing
fractures. FIG. 51 depicts in vivo fracture healing efficacy of
DGEA_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 1
nmol, 10 nmol, and 100 nmol of the conjugate were delivered daily
by subcutaneous injection. DGEA_mp4_(D)E.sub.10 conjugate raises
bone mineralization at the fracture calluses three weeks post
fracture.
[0233] FIG. 52 depicts in vivo fracture healing efficacy of
DGEA_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 1 nmol, 10 nmol, and 100 nmol of the conjugate
were delivered daily by subcutaneous injection.
DGEA_mp4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0234] FIG. 53 depicts in vivo fracture healing efficacy of
DGEA_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 1 nmol, 10 nmol, and 100 nmol of
the conjugate were delivered daily by subcutaneous injection.
DGEA_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
[0235] FIG. 54 depicts in vivo fracture healing efficacy of
DGEA_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair. 1
nmol, 10 nmol, and 100 nmol of the conjugate were delivered daily
by subcutaneous injection. DGEA_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 19. ITGA5
[0236] Example 19 indicates ITGA5 conjugates effect on healing
fractures. FIG. 56 depicts In vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=10) after 3 weeks. BV/TV--represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 1.times. and 10.times. are
respectively 1 nmol, and 10 nmol of the conjugate delivered daily
by subcutaneous injection. ITGA_mp4_(D)E.sub.10_Cys conjugate
raises bone density at the fracture calluses three weeks post
fracture.
[0237] FIG. 57 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=10) after 3 weeks. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 1.times. and
10.times. are respectively 1 nmol, and 10 nmol of the conjugate
delivered daily by subcutaneous injection. ITGA_mp4_(D)E.sub.10_Cys
conjugate raises bone strength at the fracture calluses three weeks
post fracture.
[0238] FIG. 58 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=10) after 3 weeks. Work to fracture
represents the total amount of energy absorbed by the healed femur
before it refractured in a postmortem 4 point bend analysis. Work
to fracture is a measure of how strong the bone is at the site of
fracture repair. 1.times. and 10.times. are respectively 1 nmol,
and 10 nmol of the conjugate delivered daily by subcutaneous
injection. ITGA_mp4_(D)E.sub.10_Cys conjugate raises bone strength
at the fracture calluses three weeks post fracture.
[0239] FIG. 59 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV--represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 0.1.times., 1.times. and
10.times. are respectively 0.1 nmol, 1 nmol, and 10 nmol of the
conjugate delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_Cys conjugate raises bone density at the
fracture calluses three weeks post fracture.
[0240] FIG. 60 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV represents the bone
volume of the 100 thickest micro CT slices of the fracture callus
and is a measure of how much bone has mineralized at the site of
fracture repair. 0.1.times., 1.times. and 10.times. are
respectively 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
delivered daily by subcutaneous injection. ITGA_mp4_(D)E.sub.10_Cys
conjugate raises bone mineralization at the fracture calluses three
weeks post fracture.
[0241] FIG. 61 depicts in vivo fracture healing efficacy of of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Tbth--represents The
trabecular thickness of the 100 thickest micro CT slices of the
fracture callus and is a measure the quality of the bone at the
site of fracture repair. 0.1.times., 1.times. and 10.times. are
respectively 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
delivered daily by subcutaneous injection. The of
ITGA_mp4_(D)E.sub.10_Cys conjugate raises bone quality at the
fracture calluses three weeks post fracture.
[0242] FIG. 62 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Stiffness represents the
Youngs modulus of the healed femur as it was measured before it
refractured in a postmortem 4 point bend analysis. Peak load is a
measure of how stiff the bone is at the site of fracture repair.
0.1.times., 1.times. and 10.times. are respectively 0.1 nmol, 1
nmol, and 10 nmol of the conjugate delivered daily by subcutaneous
injection. The of ITGA_mp4_(D)E.sub.10_Cys conjugate raises bone
stiffness at the fracture calluses three weeks post fracture.
[0243] FIG. 63 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 0.1.times.,
1.times. and 10.times. are respectively 0.1 nmol, 1 nmol, and 10
nmol of the conjugate delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_Cys conjugate raises bone strength at the
fracture calluses three weeks post fracture.
[0244] FIG. 64 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Cys conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Work to fracture
represents the total amount of energy absorbed by the healed femur
before it refractured in a postmortem 4 point bend analysis. Work
to fracture is a measure of how strong the bone is at the site of
fracture repair. 0.1.times., 1.times. and 10.times. are
respectively 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
delivered daily by subcutaneous injection. ITGA_mp4_(D)E.sub.10_Cys
conjugate raises bone strength at the fracture calluses three weeks
post fracture.
[0245] FIG. 66a depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Stb(stable) conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV represents the bone
volume of the 100 thickest micro CT slices of the fracture callus
and is a measure of how much bone has mineralized at the site of
fracture repair. 1.times., 1.times. and 100.times. are respectively
1 nmol, 10 nmol, and 100 nmol of the conjugate delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_Stb conjugate raises
bone mineralization at the fracture calluses three weeks post
fracture.
[0246] FIG. 66b depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_Stb(stable) conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/tv represents the
bone volume of the total volume of 100 thickest micro CT slices of
the fracture callus and is a measure of bone density at the site of
fracture repair. 1.times., 10.times. and 100.times. are
respectively 1 nmol, 10 nmol, and 100 nmol of the conjugate
delivered daily by subcutaneous injection. ITGA_mp4_(D)E.sub.10_Stb
conjugate raises bone density at the fracture calluses three weeks
post fracture.
[0247] FIG. 68 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV represents the bone
volume of the 100 thickest micro CT slices of the fracture callus
and is a measure of how much bone has mineralized at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_DAPE conjugate raises bone mineralization at
the fracture calluses three weeks post fracture.
[0248] FIG. 69 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10
nmol of the conjugate were delivered daily by subcutaneous
injection. ITGA_mp4_(D)E.sub.10_DAPE conjugate raises bone density
at the fracture calluses three weeks post fracture.
[0249] FIG. 70 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 0.1 nmol, 1
nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_DAPE conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0250] FIG. 71 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Work to fracture
represents the total amount of energy absorbed by the healed femur
before it refractured in a postmortem 4 point bend analysis. Work
to fracture is a measure of how strong the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_DAPE raises bone strength at the fracture
calluses three weeks post fracture.
[0251] FIG. 73 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPD conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV represents the bone
volume of the 100 thickest micro CT slices of the fracture callus
and is a measure of how much bone has mineralized at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_DAPD conjugate raises bone mineralization at
the fracture calluses three weeks post fracture.
[0252] FIG. 74 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPD conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10
nmol of the conjugate were delivered daily by subcutaneous
injection. ITGA_mp4_(D)E.sub.10_DAPD conjugate raises bone density
at the fracture calluses three weeks post fracture.
[0253] FIG. 75 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPD conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 0.1 nmol, 1
nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_DAPD conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0254] FIG. 76 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPE conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Work to fracture
represents the total amount of energy absorbed by the healed femur
before it refractured in a postmortem 4 point bend analysis. Work
to fracture is a measure of how strong the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_DAPE raises bone strength at the fracture
calluses three weeks post fracture.
[0255] FIG. 78 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KD conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 0.1
nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_KD conjugate raises
bone mineralization at the fracture calluses three weeks post
fracture.
[0256] FIG. 79 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KD conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_KD conjugate raises bone density at the
fracture calluses three weeks post fracture.
[0257] FIG. 80 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_DAPD conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 0.1 nmol, 1
nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_DAPD conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0258] FIG. 81 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KD conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
0.1 nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily
by subcutaneous injection. ITGA_mp4_(D)E.sub.10_KD raises bone
strength at the fracture calluses three weeks post fracture.
[0259] FIG. 83 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KE conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 0.1
nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. ITGA_mp4_(D)E.sub.10_KE conjugate raises
bone mineralization at the fracture calluses three weeks post
fracture.
[0260] FIG. 84 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KE conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_KE conjugate raises bone density at the
fracture calluses three weeks post fracture.
[0261] FIG. 85 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KE conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of
the conjugate were delivered daily by subcutaneous injection.
ITGA_mp4_(D)E.sub.10_KE conjugate raises bone strength at the
fracture calluses three weeks post fracture
[0262] FIG. 86 depicts in vivo fracture healing efficacy of
ITGA_mp4_(D)E.sub.10_KE conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
0.1 nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily
by subcutaneous injection. ITGA_mp4_(D)E.sub.10_KE raises bone
strength at the fracture calluses three weeks post fracture.
Example 20. IKVAV (Laminin)
[0263] Example 20 indicates IKVAV conjugate effect on healing
fractures. FIG. 88 depicts In vivo fracture healing efficacy of
Ikvav_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 0.1
nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. IKVAV_mp4_(D)E.sub.10 conjugate raises bone
mineralization at the fracture calluses three weeks post
fracture.
[0264] FIG. 89 depicts in vivo fracture healing efficacy of
IKVAV_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
IKVAV_mp4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0265] FIG. 90 depicts in vivo fracture healing efficacy of
IKVAV_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of
the conjugate were delivered daily by subcutaneous injection.
IKVAV_mp4_(D)E.sub.10 conjugate raises bone strength at the
fracture calluses three weeks post fracture.
[0266] FIG. 91 depicts in vivo fracture healing efficacy of
IKVAV_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
0.1 nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily
by subcutaneous injection. IKVAV_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 21. LN2 (Laminin)
[0267] Example 21 indicates LN2 conjugate effect on healing
fractures. FIG. 93 depicts In vivo fracture healing efficacy of
LN2_P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 0.1
nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. LN2_P3_mp4_(D)E.sub.10 conjugate raises
bone mineralization at the fracture calluses 17 days post
fracture.
[0268] FIG. 94 depicts in vivo fracture healing efficacy of
LN2_P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
Ln2_P3_mp4_(D)E.sub.10 conjugate raises bone density at the
fracture calluses 17 days post fracture.
[0269] FIG. 95 depicts in vivo fracture healing efficacy of
Ln2_P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of
the conjugate were delivered daily by subcutaneous injection.
Ln2_P3_mp4_(D)E.sub.10 conjugate raises bone strength at the
fracture calluses 17 days post fracture.
[0270] FIG. 96 depicts in vivo fracture healing efficacy of
Ln2_P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
0.1 nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily
by subcutaneous injection. LN2_P3_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses 17 days post fracture.
Example 22. PHSRN (Fibronectin)
[0271] Example 22 indicates PHSRN conjugate effect on healing
fractures. FIG. 98 depicts In vivo fracture healing efficacy of
PHSRN_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 0.1
nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. PHSRN_mp4_(D)E.sub.10 conjugate raises bone
mineralization at the fracture calluses 17 days post fracture.
[0272] FIG. 99 depicts in vivo fracture healing efficacy of
PHSRN_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. BV/TV--represents the bone volume divided
by total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
PHSRN_mp4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses 17 days post fracture.
[0273] FIG. 100 depicts in vivo fracture healing efficacy of
PHSRN_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of
the conjugate were delivered daily by subcutaneous injection.
PHSRN_mp4_(D)E.sub.10 conjugate raises bone strength at the
fracture calluses 17 days post fracture.
[0274] FIG. 101 depicts in vivo fracture healing efficacy of
PHSRN_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair.
0.1 nmol, 1 nmol, and 10 nmol of the conjugate were delivered daily
by subcutaneous injection. PHSRN_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses 17 days post fracture.
Example 23. P3 (Bone Sialoprotein)
[0275] Example 23 indicates P3 conjugate effect on healing
fractures. FIG. 103 depicts In vivo fracture healing efficacy of
P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 17 days. BV represents the bone volume of the 100
thickest micro CT slices of the fracture callus and is a measure of
how much bone has mineralized at the site of fracture repair. 1
nmol, 10 nmol, and 100 nmol of the conjugate were delivered daily
by subcutaneous injection. P3_mp4_(D)E.sub.10 conjugate raises bone
mineralization at the fracture calluses 17 days post fracture.
[0276] FIG. 104 depicts in vivo fracture healing efficacy of
P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 17 days. BV/TV--represents the bone volume divided by
total volume of the 100 thickest micro CT slices of the fracture
callus and is a measure of how dense the bone is at the site of
fracture repair. 1 nmol, 10 nmol, and 100 nmol of the conjugate
were delivered daily by subcutaneous injection. P3_mp4_(D)E.sub.10
conjugate raises bone density at the fracture calluses 17 days post
fracture.
[0277] FIG. 105 depicts in vivo fracture healing efficacy of
P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 17 days. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair. 1 nmol, 10 nmol, and 100 nmol of
the conjugate were delivered daily by subcutaneous injection.
P3_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses 17 days post fracture.
[0278] FIG. 106 depicts in vivo fracture healing efficacy of
P3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 17 days. Work to fracture represents the total amount
of energy absorbed by the healed femur before it refractured in a
postmortem 4 point bend analysis. Work to fracture is a measure of
how strong the bone is at the site of fracture repair. 1 nmol, 10
nmol, and 100 nmol of the conjugate were delivered daily by
subcutaneous injection. P3_mp4_(D)E.sub.10 conjugate raises bone
strength at the fracture calluses 17 days post fracture.
Example 24. SPARC113
[0279] Example 24 indicates SPARC.sub.113 conjugate effect on
healing fractures. FIG. 108 depicts In vivo fracture healing
efficacy of SPARC113_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 17 days. BV represents the bone
volume of the 100 thickest micro CT slices of the fracture callus
and is a measure of how much bone has mineralized at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
SPARC113_mp4_(D)E.sub.10 conjugate raises bone mineralization at
the fracture calluses 17 days post fracture.
[0280] FIG. 109 depicts in vivo fracture healing efficacy of
SPARC_113_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 17 days. BV/TV--represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 0.1 nmol, 1 nmol, and 10
nmol of the conjugate were delivered daily by subcutaneous
injection. SPARC_113_mp4_(D)E.sub.10 conjugate raises bone density
at the fracture calluses 17 days post fracture.
[0281] FIG. 110 depicts in vivo fracture healing efficacy of
SPARC_113_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 17 days. Max load represents the
maximum force the healed femur withstood before it refractured in a
postmortem 4 point bend analysis. Peak load is a measure of how
strong the bone is at the site of fracture repair. 0.1 nmol, 1
nmol, and 10 nmol of the conjugate were delivered daily by
subcutaneous injection. SPARC_113_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses 17 days post fracture.
[0282] FIG. 111 depicts in vivo fracture healing efficacy of
SPARC_113_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 17 days. Work to fracture
represents the total amount of energy absorbed by the healed femur
before it refractured in a postmortem 4 point bend analysis. Work
to fracture is a measure of how strong the bone is at the site of
fracture repair. 0.1 nmol, 1 nmol, and 10 nmol of the conjugate
were delivered daily by subcutaneous injection.
SPARC_113_mp4_(D)E.sub.10 conjugate raises bone strength at the
fracture calluses 17 days post fracture.
Example 25. CBM(1-19)-D10 Collagen Binding Motif
[0283] Example 25 indicates CBM(1-19) collagen binding motif effect
on healing fractures. FIG. 113 depicts in vivo fracture healing
efficacy of CBM(1-19)_D.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV--represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 1.times., 10.times., and
100.times. are respectively 1 nmol, 10 nmol, and 100 nmol of the
conjugate delivered daily by subcutaneous injection. The
CBM(1-19)_D.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0284] FIG. 114 depicts in vivo fracture healing efficacy of
CBM(1-19)_D.sub.10 conjugate on Swiss Webster fracture-bearing mice
(n=5) after 3 weeks. Tbth--represents The trabecular thickness of
the 100 thickest micro CT slices of the fracture callus and is a
measure the quality of the bone at the site of fracture repair.
1.times., 10.times., and 100.times. are respectively 1 nmol, 10
nmol, and 100 nmol of the conjugate delivered daily by subcutaneous
injection. The CBM(1-19)_D.sub.10 conjugate raises bone quality at
the fracture calluses three weeks post fracture.
[0285] FIG. 115 depicts in vivo fracture healing efficacy of
CBM(1-19)_D.sub.10 conjugate on Swiss webster fracture-bearing mice
(n=5) after 3 weeks. Max load represents the maximum force the
healed femur withstood before it refractured in a postmortem 4
point bend analysis. Peak load is a measure of how strong the bone
is at the site of fracture repair 1.times., 10.times., and
100.times. are respectively 1 nmol, 10 nmol, and 100 nmol of the
conjugate delivered daily by subcutaneous injection. The
CBM(1-19)_D.sub.10 conjugate improves bone strength at the fracture
calluses three weeks post fracture.
Example 26. Collagen Binding Domain (CBD) of Osteopontin
[0286] Example 26 indicates CBD (D)E.sub.10 conjugate effect on
healing fractures. FIG. 117 depicts In vivo fracture healing
efficacy of CBD_MP4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks. BV/TV represents the
bone volume divided by total volume of the 100 thickest micro CT
slices of the fracture callus and is a measure of how dense the
bone is at the site of fracture repair. 1.times., 10.times., and
100.times. are respectively 1 nmol, 10 nmol, and 100 nmol of the
conjugate delivered daily by subcutaneous injection. The
CBD_MP4_(D)E.sub.10 conjugate raises bone density at the fracture
calluses three weeks post fracture.
[0287] FIG. 118 depicts in vivo fracture healing efficacy of
CBD_MP4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks. Tbth--represents The trabecular thickness
of the 100 thickest micro CT slices of the fracture callus and is a
measure the quality of the bone at the site of fracture repair.
1.times., 10.times., and 100.times. are respectively 1 nmol, 10
nmol, and 100 nmol of the conjugate delivered daily by subcutaneous
injection. The of CBD_MP4_(D)E.sub.10 conjugate raises bone quality
at the fracture calluses three weeks post fracture.
[0288] FIG. 119 depicts in vivo fracture healing efficacy of
CBD_MP4_(D)E.sub.10 conjugate on Swiss webster fracture-bearing
mice (n=5) after 3 weeks. Work to fracture represents the total
amount of energy absorbed by the healed femur before it refractured
in a postmortem 4 point bend analysis. Work to fracture is a
measure of how strong the bone is at the site of fracture repair
1.times., 10.times., and 100.times. are respectively 1 nmol, 10
nmol, and 100 nmol of the conjugate delivered daily by subcutaneous
injection. The CBD_MP4_(D)E.sub.10 conjugate improves bone strength
at the fracture calluses three weeks post fracture.
Sequence CWU 1
1
32125PRTArtificial SequenceBFP D10 1Asp Asp Asp Asp Asp Asp Asp Asp
Asp Asp Gly Gln Gly Phe Ser Tyr1 5 10 15Pro Tyr Lys Ala Val Phe Ser
Thr Gln 20 25224PRTArtificial SequenceOGP D10 2Asp Asp Asp Asp Asp
Asp Asp Asp Asp Asp Ala Leu Lys Arg Gln Gly1 5 10 15Arg Thr Leu Tyr
Gly Phe Gly Gly 20327PRTArtificial SequencePreptin D10 3His Asp Val
Ser Thr Ser Gln Ala Val Leu Pro Asp Asp Phe Pro Arg1 5 10 15Tyr Asp
Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25421PRTArtificial
Sequencesubstance P 4 mini PEG D10 withMISC_FEATURE(10)..(11)4 mini
PEG linker 4Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Arg Pro Lys Pro
Gln Gln1 5 10 15Phe Phe Gly Leu Met 20538PRTArtificial
SequenceGhrelin D10 with Ser-3 replacementMISC_FEATURE(3)..(3)Ser-3
modified with diaminopropionic acid 5His Gly Ser Phe Leu Ser Pro
Glu His Gln Lys Ala Gln Gln Arg Lys1 5 10 15Glu Ser Lys Lys Pro Pro
Ala Lys Leu Gln Pro Arg Asp Asp Asp Asp 20 25 30Asp Asp Asp Asp Asp
Asp 35633PRTArtificial SequenceBMP9-D10MOD_RES(23)..(24)4 MINI PEG
LINKER 6Cys Gly Gly Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu
Ser1 5 10 15Pro Ile Ser Val Leu Tyr Lys Asp Asp Asp Asp Asp Asp Asp
Asp Asp 20 25 30Asp733PRTArtificial SequenceCNP
D10DISULFID(17)..(33) 7His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
Gly Leu Ser Lys Gly1 5 10 15Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly
Ser Met Ser Gly Leu Gly 20 25 30Cys839PRTArtificial
SequenceVasoactive intestinal peptide with D10 (VIP D10) 8His His
Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys1 5 10 15Gln
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Asp Asp Asp 20 25
30Asp Asp Asp Asp Asp Asp Asp 35939PRTArtificial SequenceCBM D10
9His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu Arg Ser Lys1 5
10 15Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr
Asp 20 25 30Glu Asp Ile Thr Ser His Met 351031PRTArtificial
SequenceP4 D10-a BMP2 fragment 10His Lys Ile Pro Lys Ala Ser Ser
Val Pro Thr Glu Leu Ser Ala Ile1 5 10 15Ser Thr Leu Tyr Leu Asp Asp
Asp Asp Asp Asp Asp Asp Asp Asp 20 25 301134PRTArtificial
SequenceMechano-growth factor (MGF) D10, an IGF-I fragment 11Asp
Asp Asp Asp Asp Asp Asp Asp Asp Asp Tyr Gln Pro Pro Ser Thr1 5 10
15Asn Lys Asn Thr Lys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu
20 25 30His Lys1233PRTArtificial SequenceTP 508_D10 12Asp Asp Asp
Asp Asp Asp Asp Asp Asp Asp Ala Gly Tyr Lys Pro Asp1 5 10 15Glu Gly
Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe 20 25
30Val1325PRTArtificial SequenceODP-D10MISC_FEATURE(10)..(11)4 mini
PEG linker 13Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Val Asp
Val Pro Asp1 5 10 15Gly Arg Gly Asp Ser Leu Ala Tyr Gly 20
251431PRTArtificial SequenceBMP9MISC_FEATURE(21)..(22) 14Gly Gly
Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser Pro1 5 10 15Ile
Ser Val Leu Tyr Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp 20 25
301535PRTArtificial SequenceGhrelin D10 15Phe Leu Ser Pro Glu His
Gln Lys Ala Gln Gln Arg Lys Glu Ser Lys1 5 10 15Lys Pro Pro Ala Lys
Leu Gln Pro Arg Asp Asp Asp Asp Asp Asp Asp 20 25 30Asp Asp Asp
351630PRTArtificial SequenceCNP-D10.MISC_FEATURE(15)..(16) 16Asp
Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu Ser Lys Gly Phe1 5 10
15Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly 20 25
301739PRTArtificial SequenceVIP D10 17His His Ser Asp Ala Val Phe
Thr Asp Asn Tyr Thr Arg Leu Thr Lys1 5 10 15Gln Met Ala Val Lys Lys
Tyr Leu Asn Ser Ile Leu Asn Asp Asp Asp 20 25 30Asp Asp Asp Asp Asp
Asp Asp 351811PRTArtificial Sequence4 mini PEG D10 18His Asp Asp
Asp Asp Asp Asp Asp Asp Asp Asp1 5 101926PRTArtificial
SequenceODPD10MISC_FEATURE(4)..(5)MISC_FEATURE(16)..(17) 19His Ala
Ile Ser Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys1 5 10 15Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu 20 252023PRTArtificial
SequenceCTC_peg 10_e10MISC_FEATURE(11)..(12) 20His Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Ile Ala Gly Val Gly1 5 10 15Gly Glu Lys Ser
Gly Gly Phe 202123PRTArtificial
SequenceCTC_MP4_e10MISC_FEATURE(11)..(12) 21His Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu Ile Ala Gly Val Gly1 5 10 15Gly Glu Lys Ser Gly
Gly Phe 202226PRTArtificial SequenceP15MISC_FEATURE(16)..(17) 22His
Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val1 5 10
15Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 20 252326PRTArtificial
SequenceP15_mp4_e10MISC_FEATURE(16)..(17) 23His Gly Thr Pro Gly Pro
Gln Gly Ile Ala Gly Gln Arg Gly Val Val1 5 10 15Glu Glu Glu Glu Glu
Glu Glu Glu Glu Glu 20 252417PRTArtificial
SequenceDGEA_mp4_e10MISC_FEATURE(11)..(12) 24His Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Asp Gly Glu Ala Ser1 5 10
15Pro2518PRTArtificial SequenceITGAMISC_FEATURE(11)..(12) 25His Glu
Glu Glu Glu Glu Glu Glu Glu Glu Glu Arg Arg Glu Thr Ala1 5 10 15Trp
Ala2625PRTArtificial SequenceIKVAV_mp4_e10MISC_FEATURE(11)..(12)
26His Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Gln Ala Ala Ser1
5 10 15Ile Lys Val Ala Val Ser Ala Asp Arg 20 252723PRTArtificial
SequenceLN2_P3_mp4_e10MISC_FEATURE(11)..(12) 27His Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Asp Leu Thr Ile Asp1 5 10 15Asp Ser Tyr Trp
Tyr Arg Ile 202816PRTArtificial
SequencePHSRN_mp4_e10MISC_FEATURE(11)..(12) 28His Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Pro His Ser Arg Asn1 5 10
152927PRTArtificial SequenceP3_mp4_e10 29His Tyr Glu Ser Glu Asn
Gly Glu Pro Arg Gly Asp Asn Tyr Arg Ala1 5 10 15Tyr Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu 20 253026PRTArtificial SequenceSPARC
113_mp4_e10MISC_FEATURE(11)..(12) 30His Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu Thr Leu Glu Gly Thr1 5 10 15Lys Lys Gly His Lys Leu His
Leu Asp Tyr 20 253130PRTArtificial SequenceCBM(1-19)-D10 collagen
binding motif 31His Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Gly Leu
Arg Ser Lys1 5 10 15Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro
Asp Ala 20 25 303239PRTArtificial SequenceCBD
_MP4_e10MISC_FEATURE(29)..(30) 32His Asn Gly Val Phe Lys Tyr Arg
Pro Arg Tyr Tyr Leu Tyr Lys His1 5 10 15Ala Tyr Phe Tyr Pro His Leu
Lys Arg Phe Pro Val Gln Glu Glu Glu 20 25 30Glu Glu Glu Glu Glu Glu
Glu 35
* * * * *