U.S. patent application number 17/058884 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 | 20210214408 17/058884 |
Document ID | / |
Family ID | 1000005536218 |
Filed Date | 2021-07-15 |
United States Patent
Application |
20210214408 |
Kind Code |
A1 |
Low; Philip Stewart ; et
al. |
July 15, 2021 |
TARGETING ANABOLIC DRUGS FOR ACCELERATED FRACTURE REPAIR
Abstract
The targeted delivery of growth factors, vasoactive peptides and
other representative anabolic peptide drugs from different
signaling cascades to bone fracture for accelerated healing is
disclosed herein.
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 |
US |
|
|
Family ID: |
1000005536218 |
Appl. No.: |
17/058884 |
Filed: |
May 30, 2019 |
PCT Filed: |
May 30, 2019 |
PCT NO: |
PCT/US2019/034759 |
371 Date: |
November 25, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62678016 |
May 30, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/575 20130101;
C12N 9/12 20130101; C07K 14/50 20130101; C12N 9/54 20130101; C07K
14/51 20130101; C07K 14/4721 20130101; C07K 14/79 20130101; C07K
14/65 20130101; C07K 14/49 20130101; C07K 14/47 20130101; C12N
9/6429 20130101 |
International
Class: |
C07K 14/51 20060101
C07K014/51; C07K 14/65 20060101 C07K014/65; C07K 14/50 20060101
C07K014/50; C07K 14/79 20060101 C07K014/79; C07K 14/575 20060101
C07K014/575; C07K 14/47 20060101 C07K014/47; C12N 9/74 20060101
C12N009/74; C07K 14/49 20060101 C07K014/49; C12N 9/12 20060101
C12N009/12; C12N 9/54 20060101 C12N009/54 |
Claims
1-21. (canceled)
22. A compound having a structure of: X-Y-Z wherein: X is a growth
factor, a growth factor fragment, an agent that activates a growth
factor receptor, or an agent that activates a mitogen-activated
protein kinase (MAPK) pathway; Y is absent or a linker; and Z is a
bone-targeting molecule, or a pharmaceutically acceptable salt
thereof.
23. The compound of claim 22, wherein Z comprises a
polypeptide.
24. The compound of claim 22, wherein Z comprises not less than 4
and not more than 40 amino acid residues.
25. The compound of claim 24, wherein at least one amino acid is
aspartic acid or glutamic acid.
26. The compound of claim 22, wherein Z comprises not less than 6
and not more than 20 glutamic acid residues or not less than 6 and
not more than 20 aspartic acid residues.
27. The compound of claim 22, wherein Z is 10 D-glutamic acid
residues or 10 D-aspartic acid residues.
28. The compound of claim 22, 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, an ether, and an
amide.
31. The compound of claim 28, wherein Y comprises one or more
ethylene glycol unit.
32. The compound of claim 31, wherein Y comprises 2-8 oxyethylene
units.
33. The compound of claim 22, wherein the growth factor is a bone
morphogenic protein (BMP), an insulin like growth factor (IGF), or
a fibroblast growth factor.
34. The compound of claim 33, wherein the BMP is selected from the
group consisting of P4 (BMP2), BFP (BMP7), pBMP9 (BMP9), and B2A
(BMP2/TGF beta).
35. The compound of claim 33, wherein the IGF is selected from the
group consisting of preptin (IGF-II) and mechano growth factor
(MGF) (IGF-1).
36. The compound of claim 33, wherein the fibroblast growth factor
is selected from the group consisting of fibroblast growth factor 2
(F119) and F2A.
37. The compound of claim 22, wherein the growth factor fragment is
lactoferrin or ghrelin.
38. The compound of claim 37, wherein the agent that activates the
MAPK pathway is a c-Jun N-terminal kinase 3 (JNK3) agonist.
39. The compound of claim 38, wherein the JNK3 agonist is selected
from the group consisting of JNK3 and Annexin 1.
40. A compound having a structure of: X-Y-Z wherein: X is a
vasoactive compound or an angiogenic compound; Y is absent or a
linker; and Z is a bone-targeting molecule, or a pharmaceutically
acceptable salt thereof.
41. The compound of claim 40, wherein the vasoactive compound or
the angiogenic compound is selected from the group consisting of
C-type natriuretic peptide (CNP), thrombin peptide (TP508),
vascular endothelial growth factor (VEGF) mimetic QK, and platelet
derived growth factor 2A (P2A).
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: Bone Morphogenic Protein(s)
(BMP), Insulin Like Growth Factors (IGF), fibroblast growth
factors, peptide, hormones, hormone releasing agents, lactoferrin,
Ghrelin, c-Jun N-terminal kinase 3 agonists (JNK3 agonists),
vasoactive peptide(s), any growth factor, and any active portion of
any growth factor; [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. 23) and a polypeptide comprising 10 glutamic
acid residues (SEQ ID NO. 24). [0028] 6. The compound according to
claims 1-4, wherein Z is selected from the group consisting of: 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 one 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 BMP is at least one compound selected from
the group consisting of: P4 (BMP2), BFP (BMP7), pBMP9 (BMP9), and
B2A (BMP2/TGF beta). [0036] 14. The compound according to claims,
1-9, wherein the Insulin Like Growth Factor is at least one
compound selected from the group consisting of: Preptin (IGF-II),
and Mechano Growth Factor (MGF) (IGF-1). [0037] 15. The compound
according to claims, 1-9, wherein the Fibroblast Growth Factor, is
at least one compound selected from the group consisting of:
Fibroblast Growth Factor 2 (F119) and F2A. [0038] 16. The compound
according to claims, 1-9, wherein the Osteogenic peptide is histone
h4. [0039] 17. The compound according to claims, 1-9, wherein X is
selected from the group consisting of: parathyroid hormone-related
protein (PTHrP) [107-139], and Osteogenic peptide. [0040] 18. The
compound according to claims, 1-9, wherein the JNK3 agonist is,
JNK3, or Annexin 1. [0041] 19. The compound according to claims,
1-9, wherein the Vasoactive peptide is at least one compound
selected from the group consisting of: C-Type Natriuretic peptide
(CNP), Thrombin peptide (TP508), VEGF mimetic (QK) and platelet
derived growth factor 2A (P2A). [0042] 20. Use of a compound
according to any of claims 1-19, for the manufacture of a
medicament for therapeutic application. [0043] 21. A method of
treating a patient, comprising the step of administering at least
one dose of a compound according to claims 1-9.
[0044] 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
[0045] 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 .mu.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.
[0046] 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.
[0047] FIG. 3 depicts structures for Dasatinib and E738.
[0048] FIG. 4 depicts structures for targeted conjugates of
Dasatinib and E738.
[0049] FIG. 5 depicts peak load of Fractured Femurs after 2
weeks.
[0050] FIG. 6 depicts BV/TV two weeks after fractured femur
received various concentration of Preptin D10 treatment.
[0051] FIG. 7 depicts TbTh (the trabecular thickness of the 100
thickest micro CT slices of the fracture callus) two weeks after
fractured femur received various concentration of Preptin D10
treatment.
[0052] 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.
[0053] FIG. 9 depicts BV/TV two weeks after fractured femur
received various concentration of OGP D10.
[0054] FIG. 10 depicts TbTh two weeks after fractured femur
received various concentration of OGP D10.
[0055] 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.
[0056] FIG. 12 depicts BV/TV two weeks after fractured femur
received various concentration of BFP D10.
[0057] FIG. 13 depicts TbSp two weeks after fractured femur
received various concentration of BFP D10.
[0058] 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.
[0059] FIG. 15 depicts BV/TV four weeks after fractured femur
received various concentration of Ghrelin D10.
[0060] FIG. 16 depicts BV four weeks after fractured femur received
various concentration of pBMP9 D10.
[0061] FIG. 17 depicts BV/TV four weeks after fractured femur
received various concentration of pBMP9 D10.
[0062] FIG. 18 depicts BV/TV four weeks after fractured femur
received various concentration of CNP D10.
[0063] FIG. 19 depicts BV/TV four weeks after fractured femur
received 1 nmol/day of ODP D10.
[0064] 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).
[0065] FIG. 21 depicts BV/TV four weeks after fractured femur
received various concentrations of P4 D10.
[0066] FIG. 22 depicts BV four weeks after fractured femur received
1 nmol/day of P4 D10.
[0067] FIG. 23 depicts BV/TV four weeks after fractured femur
received various concentrations of MGF D10.
[0068] FIG. 24 depicts BV/TV four weeks after fractured femur
received various concentrations of TP508_D10.
[0069] FIG. 25 depicts BV/TV four weeks after fractured femur
received 1 nmol/day of VIP_D10.
[0070] FIG. 26 depicts TbTh four weeks after fractured femur
received 1 nmol/day of VIP_D10.
[0071] FIG. 27 depicts the structure for BMP9 (SEQ ID NO: 14).
[0072] FIG. 28 depicts the structure for Ghrelin D10 (SEQ ID NO:
15).
[0073] FIG. 29 depicts the structure for Preptin D10 (SEQ ID NO:
3).
[0074] FIG. 30 depicts the structure for CNP-D10 (SEQ ID NO:
16).
[0075] FIG. 31 depicts the structure for VIP D10 (SEQ ID NO:
17).
[0076] FIG. 32 depicts the structure for Substance P with 4 mini
PEG conjugated to D10 (SEQ ID NO: 18).
[0077] FIG. 33 depicts the structure for CBM D10 (SEQ ID NO:
9).
[0078] FIG. 34 depicts the structure for ODP D10 (SEQ ID NO:
19).
[0079] FIG. 35 structure of B2A_AHX3_e10 (SEQ ID NO: 27).
[0080] FIG. 36. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of B2A_AHX_e10.
[0081] FIG. 37. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
B2A_AHX_e10.
[0082] FIG. 38. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of B2A_AHX_e10.
[0083] FIG. 39. Work to Fracture (mJ) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
B2A_AHX_e10.
[0084] FIG. 40. Structure of F2A_mp4_e10 (SEQ ID NO: 28).
[0085] FIG. 41. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of F2A_mp4_e10
[0086] FIG. 42. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
F2A_mp4_e10.
[0087] FIG. 43. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of F2A_mp4_e10
[0088] FIG. 44. Work to Fracture (mJ) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
F2A_mp4_e10.
[0089] FIG. 45. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
F119_mp4_e10_stb.
[0090] FIG. 46. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of F119_mp4_e10_stb.
[0091] FIG. 47. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of F119_mp4_e10_stb.
[0092] FIG. 48. Work to Fracture (mJ) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
F119_mp4_e10_stb.
[0093] FIG. 49. Structure of JNK3_mp4_e10 (SEQ ID NO: 21).
[0094] FIG. 50. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of JNK3_mp4_e10.
[0095] FIG. 51. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
JNK3_mp4_e10.
[0096] FIG. 52. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of JNK3_mp4_e10.
[0097] FIG. 53. Work to Fracture (mJ) after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of JNK3_mp4_e10.
[0098] FIG. 54. Structure of Lactoferrin_mp4_e10 (SEQ ID NO:
22).
[0099] FIG. 55. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Lactoferrin_mp4_e10_stb.
[0100] FIG. 56. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Lactoferrin_mp4_e10_stb.
[0101] FIG. 57. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Lactoferrin_mp4_e10_stb.
[0102] FIG. 58. Work to Fracture (mJ) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Lactoferrin_mp4_e10_stb.
[0103] FIG. 59. Structure of Osteostatin_mp4_(D)E.sub.10 (SEQ ID
NO: 23).
[0104] FIG. 60. TbTh measured after treatment with saline, or 0.1
nmol/day, 1 nmol/day or 10 nmol/day of
Osteostatin_mp4_(D)E.sub.10.
[0105] FIG. 61. Max Load(N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Osteostatin_mp4_(D)E.sub.10.
[0106] FIG. 62. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Osteostatin_mp4_(D)E.sub.10.
[0107] FIG. 63. Stiffness (N/mm) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Osteostatin_mp4_(D)E.sub.10.
[0108] FIG. 64. Work to Fracture (mJ) measured after treatment with
saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
Osteostatin_mp4_(D)E.sub.10.
[0109] FIG. 65. Structure of P2A-mp4_e10 (SEQ ID NO: 29).
[0110] FIG. 66. Bone Volume measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of P2A-mp4-e10
[0111] FIG. 67. Bone Volume/Total Volume measured after treatment
with saline, or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of
P2A-mp4-e10.
[0112] FIG. 68. Max Load (N) measured after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of P2A-mp4-e10
[0113] FIG. 69. Work to Fracture (mJ) after treatment with saline,
or 0.1 nmol/day, 1 nmol/day or 10 nmol/day of P2A-mp4-e10.
[0114] FIG. 70. Structure of Preptin (1-34)_mp4_e10 (SEQ ID NO:
24).
[0115] FIG. 71. Bone Volume/Total Volume measured after treatment
with saline, or Preptin (1-34)_mp4_e10_50.times..
[0116] FIG. 72. Bone Volume measured after treatment with saline,
or Preptin (1-34)_mp4_e10_50.times..
[0117] FIG. 73. Max Load (N) measured after treatment with saline,
or Preptin (1-34)_mp4_e10_50.times..
[0118] FIG. 74. Post Yield Displacement (mm) measured after
treatment with saline, or Preptin (1-34)_mp4_e10_50.times..
[0119] FIG. 75. Work to Fracture (mJ) measured after treatment with
saline, or Preptin (1-34)_mp4_e10_50.times..
[0120] FIG. 76. Structure of QK_mp4_e10 (SEQ ID NO: 25).
[0121] FIG. 77. Bone Volume measured after treatment with saline,
or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of QK_mp4_e10.
[0122] FIG. 78. Bone Volume/Total Volume measured after treatment
with saline, or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of
QK_mp4_e10.
[0123] FIG. 79. Max Load (N) measured after treatment with saline,
or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of QK_mp4_e10.
[0124] FIG. 80. Work to Fracture (mJ) measured after treatment with
saline, or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of
QK_mp4_e10.
[0125] FIG. 81 depicts the structure for Annexin 1 (SEQ ID NO:
26).
[0126] FIG. 82 Max Load (N) measured after treatment with saline,
or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of Annexin 1.
[0127] FIG. 83 Work to Fracture (mJ) measured after treatment with
saline, or 1.0 nmol/day, 10 nmol/day or 100 nmol/day of Annexin
1.
[0128] FIG. 84 depicts the structure for F119 (SEQ ID NO: 20).
BRIEF DESCRIPTION OF THE SEQUENCES
[0129] SEQ ID NO: 1: bone forming peptide conjugated with 10
aspartate acids (BFP D10).
[0130] SEQ ID NO: 2: osteogenic growth peptide conjugated with 10
aspartate acids (OGP D10).
[0131] SEQ ID NO: 3: Preptin conjugated with 10 aspartate acids
(Preptin D10).
[0132] SEQ ID NO: 4: substance P with 4 mini PEG linker and
conjugated with 10 aspartate acids (substance P 4 mini PEG
D10).
[0133] SEQ ID NO: 5: Ghrelin D10 with Ser-3 replaced with
diaminopropinoic acid.
[0134] SEQ ID NO: 6: BMP9 D10.
[0135] SEQ ID NO: 7: C-type Natriuretic peptide (CNP) conjugated
with 10 aspartate acids (CNP 10).
[0136] SEQ ID NO: 8: Vasoactive intestinal peptide (VIP) conjugated
with D10.
[0137] SEQ ID NO: 9: collagen binding motif conjugated with 10
aspartate acids (CBM D10).
[0138] SEQ ID NO: 10: P4 conjugated with 10 aspartate acids (P4
D10).
[0139] SEQ ID NO: 11: Mechano-growth factor conjugated with 10
aspartate acids (MGF D10).
[0140] SEQ ID NO: 12: Thrombin fragment TP508 conjugated with 10
aspartate acids (TP 508 D10).
[0141] SEQ ID NO: 13: Osteopontin-derived peptide conjugated with
10 aspartate acids (ODP D10).
[0142] SEQ ID NO: 14: BMP9 with 10 aspartate acids (BMP9).
[0143] SEQ ID NO: 15: Ghrelin with 10 aspartate acids (Ghrelin
D10).
[0144] SEQ ID NO: 16: CNP with 10 aspartate acids (CNP-D10).
[0145] SEQ ID NO: 17: VIP with 10 aspartate acids (VIP D10).
[0146] SEQ ID NO: 18: 4 mini PEG D10.
[0147] SEQ ID NO: 19: ODP with 10 aspartate acids (ODPD10).
[0148] SEQ ID NO: 20: F119 with 10 glutamic acids (F119).
[0149] SEQ ID NO: 21: JNK with 10 glutamic acids
(JNK3_mp4_e10).
[0150] SEQ ID NO: 22: Lactoferrin with 10 glutamic acids
(Lactoferrin_mp4_e10).
[0151] SEQ ID NO: 23: Osteostatin with 10 glutamic acids
(Osteostatin_mp4_(D)E.sub.10).
[0152] SEQ ID NO: 24: Preptin with 10 glutamic acids (Preptin
(1-34)_mp4_e10).
[0153] SEQ ID NO: 25: QK with 10 glutamic acids (QK_mp4_e10).
[0154] SEQ ID NO: 26: Annexin 1 with 10 glutamic acids (Annexin
1).
[0155] SEQ ID NO: 27: B2A with 10 glutamic acids
(B2A_AHX3_e10).
[0156] SEQ ID NO: 28: F2A with 10 glutamic acids (F2A_mp4_e10).
[0157] SEQ ID NO: 29: P2A with 10 glutamic acids (P2A-mp4_e10).
[0158] SEQ ID NO: 30: Targeting group consisting of a polypeptide,
DDDDDDDDDD.
[0159] SEQ ID NO: 31: Targeting group consisting of a polypeptide,
EEEEEEEEEE.
DETAILED DESCRIPTION
[0160] 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 aspects are shown and described and that
all changes and modifications that come within the spirit of the
disclosure are desired to be protected.
[0161] 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.
[0162] 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
[0163] 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, .about.65% of all wounds associated
with military conflicts since World War I (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.
[0164] 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.
[0165] 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.
[0166] 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, 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.
[0167] 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).
[0168] 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
[0169] 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.
[0170] The term "BV/TV" means the bone volume divided by total
volume of the 100 thickest micro CT slices of the fracture
callus.
[0171] The term "TbTh" means the trabecular thickness of the 100
thickest micro CT slices of the fracture callus.
[0172] The term "BV" means the overall bone volume of the 100
thickest micro CT slices of the fracture callus.
[0173] The term "TbSp" means the spacing between the trabecula in
the 100 thickest micro CT slices of the fracture callus.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] The term "P4" means a fragment that represents the knuckle
epitope in hBMP-2.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] The term "STB" at the end of any name represents that the
compound represents a chemically more stable version of its natural
version.
[0183] Compounds which effect bone growth and may be used to
practice aspects of the present disclosure include but are not
limited to the following: growth factors, fragments of growth
factors, or synthetic peptides or small molecules that mimic the
action of growth factors. Examples of which may include but are not
limited to: transforming growth factors(alpha or beta), insulin,
insulin-like growth factors, fibroblast growth factor (1-23),
erythropoietin, epidermal growth factor, colony-stimulating factors
(including macrophage colony-stimulating factor, granulocyte
colony-stimulating factor, granulocyte macrophage
colony-stimulating factor), bone morphogenetic proteins (BMP)
(1-8a, 8b, 10, 11, 15), angiopoietin, vascular endothelial growth
factor, megakaryocyte growth and development factor,
adrenomedullin, autocrine motility factor, ciliary neurotrophic
factor, leukemia inhibitory factor, interleukin(1-7), ephrins(A1-5,
B1-3), Foetal Bovine Somatotrophin, glial cell line-derived
neurotrophic factor, neurturin, persephin, artemin, growth
differentiation factor-9, hepatocyte growth factor,
hepatoma-derived growth factor, keratinocyte growth factor,
migration-stimulating factor, macrophage-stimulating protein,
myostatin, neuregulins(1-4), neurotrophins, brain-derived
neurotrophic factor, nerve growth factor, neurotrophin-3,
neurotrophin-4, placental growth factor, platelet-derived growth
factor, renalase, T-cell growth factor, tumor necrosis
factor-alpha, Wnt signaling activators, parathyroid hormone-related
peptide, parathyroid hormone, growth differentiation factor, Growth
Hormone (GH), thyroid hormone, calcitonin, vitamin D, Apelin,
Annexin, Bmp fragments including: OPD (BMP2), P-1(Bmp2), P24(bmp2),
P4(BMP) HBD(bmp4), Bone Forming Peptide (BFP), BFP-1(BMP-7),
BFP-2(BMP-7), BFP-4(BMP-7), BFP-7 derived peptide, (bmp7), peptide
b(BMP-7), BMP-9-derived peptide(bmp-9), pBMP-9(BMP-9),
SpBMP-9(BMP-9), BMP2-L51P(BMP2), BMP2 108(BMP2), mBMP (BMP2),
osteopromotive domain (OPD) (BMP2), PEP7 (BMP2), AB204(BMP2/ACTIVIN
A), AB204-1103Y(BMP2/ACTIVIN A), AB211(BMP2/ACTIVIN A),
AB215((BMP2/ACTIVIN A), BMP2/BMP9 chimera BB29, BMP6/BMP7 chimera
80, BMP7-E60K BMP6, THR-123 (BMP7), MB109 (BMP9), GDF5-S94N,
GDF5-N445K, GDF5-N445T, GDF5-V453/V456, BMP2/6, BMP2/7, BMP4/7,
Casein kinase 2 (CK2), CK2.1, CK2.2, CK2.3; Fibroblast growth
factor 2 fragments: F105, F119, F36, F77; Glucagon like peptide,
Exendin 4, exenatide, liraglutide, lixisenatide, albiglutide,
dulaglutide, semaglutide, taspoglutide, Preptin, E peptide, mechano
growth factor, Cathelicidin, Endothelin-1, OGP (osteogenic growth
peptide), cyclic OGP10-14, OGP10-14, Ostabolin, Ostabolin-C,
ZP2307, TP-508 Chrysalin, PBA2-1c, QK, Amylin, ghrelin, GHRP-6,
hexarelin, ipamorelin, amylin, human c peptide, vessel dilator,
CNP, BMN 11, Osteoblast activating peptide, fMLF, hepcidin, PSI,
Epoxomicin, bortezomib, Carfizmib, oprzomib, Epiregulin,
Betacellulin, amphiregulin, Neuregulin, Stem cell factor, Agrin,
Ephrin, Glial cell line-derived neurotrophic factor, neurturin,
artemin, Angiopoietins SPARC-113, SPARC-118, Osteoactivin, or a
molecule stimulates JNK3 upregulation: such as JNK3, and arrestin 3
fragments, or annexin1.
[0184] Still other compounds which effect bone growth and may be
used to practice aspects of the present disclosure include but are
not limited to an angiogenic factor or a vasoactive compound that
either stimulates angiogenesis or hematopoiesis, effects
hematopoiesis, is released by or affects cells of a hematopoietic
lineage, such as red blood cells, leukocytes, platelets, monocytes,
or endothelial cells, or is a protein, peptide or other small
molecule release in response to injury to blood vessel, or the full
protein, peptide or fragments of such molecules or synthetic or
small molecules that mimic their actions. Examples of which may
include but are not limited to: VEGF, QK, SPARC 113, VIP, PACAP,
Dobutamine, dopamine, isoprenaline, endothelin 1, phenylphrine,
adrenaline, terlipressin, Angeotensin II, Ace inhibitors, FGF2,
PDGF, Angiopoietin, Hypoxia inducable factors, MMP2,
metaloproteases, sokotrasterol sulfate, 0-sitosterol, oversulfated
exopolysaccharide, IGF-1fumagillin, Vatanib, Axtinib, Placental
growth factor, neuropilin, VE-cadherin, Alpha-v beta 3, afibercept,
HIF1A, cediranib, Shingosinel-phosphate, 2 methoxyesterdiol,
vandetanib, semanib, ephrin, ramucirumab, TIE1, Platelet activating
vactor, Thrombin, TP508, fibrin, fibrinogen, von Willebrand factor,
protease-activated receptor, serotonin, platlet activating factor,
ATP, ADP, Thromboxane A2, factor X, factor VII, Factor IX, Hangeman
factor, factor I, Factor VIII, Vitamin K, platelet factor 4,
endothelium derived hyperpolarizing factor, prostaglandins,
leukotrienes, notchagonists, LIF, Jagged, and memaquinone.
Bone Growth Modifiers and Delivery Peptides
TABLE-US-00002 [0185] (SEQ ID NO: 1) BFP1D10
DDDDDDDDDDGQGFSYPYKAVFSTQ
[0186] 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
[0187] 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
[0188] 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
[0189] 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
[0190] 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
[0191] 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 (OSX), 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
[0192] 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
[0193] 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
[0194] 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 are not completely elucidated for this molecule
its believed that it works in a similar mechanism as CBM. The
structure of ODP D10 is depicted in FIG. 34.
TABLE-US-00003 (SEQ ID NO: 2) OGP-D10 DDDDDDDDDDALKRQGRTLYGFGG
[0195] 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
[0196] 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
[0197] 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.
B2A Peptide
[0198] B2A is a synthetic, multidomain peptide with two 19 amino
acid branches derived from the region which is similar to the
TGF-P/BMP-binding region of fetuin, a member of the cystatin family
of protease inhibitors. The 19 amino acid branch acts as a BMP
receptor ligand. BMP receptor activation is well known to
osteogenic as BMP2 is currently approved to treat bone fractures.
However existing strategies rely on the use of a full recombinant
human protein. B2A is a dimer peptide that achieves similar levels
of BMP receptor activation with a much mass. Bone morphogenetic
protein receptor (BMPR) activation is canonically associated with
activation of the small mothers against decapentaplegic (Smads)
pathway BMPR ligands can, activate non smad signaling via p38,
extracellular signal-regulated kinases 1 and 2 (ERK1/2),
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK),
and protein kinase B (Akt/PKB). non smad signaling is key in cell
survival, nutution and metabolism and migration. B2A has the
ability to bind both type I and II receptors for BMP. In the
presence of BMP-2, B2A augments osteo-differentiation. In the
absence of BMP-2, B2A-induced proliferation, aggrecan synthesis,
and collagen accumulation in mesenchymal stem cells. Here by
targeting B2A to the fracture, the osteogenic signal that locally
applied BMP2 can be specifically localize to allow a much smaller
much less invasive to apply form. The structure of B2A is depicted
in FIG. 35.
F2A (Growth Factor)
[0199] F2A_mp4_e10 is made up of two copies of the fibroblast
growth factor (FGF) receptor targeting sequence from FGF2 branching
from a single lysine then 4 minipegs to act a linker and spacer and
10 D glutamic acids to target to the bone. F2A's receptor targeting
sequence is YRSRKYSSWYVALKR derived from derived from native FGF-2
(residues from 106 to 120). It has been shown to be nearly as
potent as the full native protein at stimulating angiogenesis and
osteogenesis in its dimer form. FGF2 is an anabolic growth factor
heavily involved in wound healing and tissue repair. It Is both
angiogenic and osteogenic. Fgf2 has shown promise as a protein
treatment for wound healing. The advantage of F2A e10 is its small
size allows for better perfusion and the e10 allows it to
chemically home to the damaged tissue site from a noninvasive
injection at a distal site. The structure of F2A is depicted in
FIG. 40.
F119 (Growth Factor)
[0200] F119 is a heparin binding fragment from growth factor
fibroblast growth factor 2. Fibroblast growth factor (FGF)-2
regulates a variety of cellular functions, such as proliferation
and differentiation, by binding to cell surface FGF receptors
(FGFRs) in the presence of heparin proteoglycans F119 a heprerin
binding site for fgf2 which corresponds to 119-135 of fgf2 (F119,
KRTGQYKLGSKTGPGQK). F119 interacts with cell-surface heparan
sulfate proteoglycans. In addition, osteoblast differentiation,
confirmed by ALPase activity and mineralization, is increased by
F119. The structure of F119 is depicted in FIG. 84.
c-Jun NH2-terminal Protein Kinases (JNK3)
[0201] The c-Jun NH2-terminal protein kinases (JNKs) belong to the
MAPK family. JNKs regulate normal physiological processes of cell
proliferation, apoptosis, differentiation, and migration7. JNKs
were also implicated in many diseases, from cancer to neurological
and immunological disorders recently they were implicated as one of
reasons adults bones don't have the same response to mechanical
stimuli. It was found that increasing JNK3 activity stimulated more
responsiveness to osteoinductive activity Arrestin-3 facilitates
INK activation in cells, and a short 25-residue arrestin-3 peptide
was identified as the critical JNK3-binding element.sup.2. It was
demonstrated that this 25 residue peptide, JNK3, also binds
mitogen-activated protein kinase 4 (MKK4), MKK7, and apoptosis
signal-regulating kinase 1 (ASK1), which are upstream
JNK3-activating kinases. This peptide is sufficient to enhance JNK3
activity in cells. The increases the responsiveness osteogenic
signals that occur during bone fracture repair. Thus restoring the
regenerative abilities of youth to adults. The structure of JNK3 is
depicted in FIG. 49.
Lactoferrin
[0202] Lactoferrin is an iron-binding glycoprotein that belongs to
the transferrin family. It is present in breast milk, in epithelial
secretions, and in the secondary granules of neutrophils. In
healthy subjects lactoferrin circulates at concentrations of
2-7.times.10-6 g/ml. Lactoferrin is a pleiotropic factor with
potent antimicrobial and immunomodulatory activities. It has shown
that lactoferrin can also promote bone growth. Lactoferrin is
stimulatory towards osteoblasts and prevents osteoclastogensis. It
has been shown that that lactoferrins interactions with osteoblast
like cells is primarily through LRP1, a member of the family of
low-density lipoprotein receptor-related proteins that are
primarily known as endocytic receptors. Lactoferrin also induces
activation of p42/44 MAPK signaling in primary osteoblasts, but the
two pathways seem to operate independently as activation of MAPK
signaling, but not endocytosis, is necessary for the mitogenic
effect of lactoferrin. Lactoferrin is too large to act as a good
therapeutic. But several active fragments of lactoferrin have been
identified. Lactoferricin which is a loop region from the n
terminus of Lactoferrin has been shown to have both antimicrobial
and osteogenic effects..sup.4,5, Our targeted compound comes from
residues 17-31 from the n terminus followed by a 4 minipeg spacer
and linker and 10 D glutamic acids to home it to bone. The
structure of lactoferrin is depicted in FIG. 54.
Osteostatin (PTHrP[107-139])
[0203] Osteostatin (PTHrP[107-139]) is a fragment of Parathyroid
hormone related protein(PTHrP) that has been reported to be more
anabolic than 1-34 of PTHrP. PTHrP is anabolic on bone both because
of an inhibitory effect on osteoclast but mostly a stimulatory
effect on osteoblasts. Some of its effect is believed to function
like PTHrP though WNT signaling and also through interactions with
the vascular endothelial growth factor (VEGF) receptor to increase
survival of osteoblasts. The structure of osteostatin
(PTHrP[107-139]) is depicted in FIG. 59.
P2A (Platelet-Derived Growth Factor (PDGF))
[0204] Platelet-derived growth factors (PDGFs) are mitogens for
many cells primary of mesenchymal origin and their involvement in
wound healing has lead to the therapeutic use of recombinant human
PDGF-BB, one of PDGF family. PDGF are released by plates at the
site of fractures are chemotactic and mitogenic for osteoblast
lineage cells and osteoblasts and therefore may enhance fracture
healing by attracting osteoprogenitor cells to the fracture by
chemotaxis and amplifying their quantity by mitogenesis. Further,
PDGF-3B upregulates the expression of angiogenic vascular
endothelial growth factor (VEGF), a key molecule for bone
regeneration. PDGF-BB is approved to accelerate ankle fusion. It
was found that a Arg160, Lys161 and Lys162 in PDGF-BB are required
for mitogenic activity and high-affinity receptor. It was shown
that a chain containing a branched dimer of VRKIEIVRKK derived from
residues 153-162 of PDGF-BB was almost as active as the full
PDGF-BB. The targeted construct of the present disclosure consists
of that cyclized branched dimer of VRKIEIVRKK derived from residues
153-162 of PDGF-BB, with a 4 minipeg spacer and 10 (D) glutamic
acid to localize it to the site of the fracture. The structure of
P2A is depicted in FIG. 65.
Preptin(1-34)
[0205] 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(1-34) is depicted in FIG. 70.
QK (Vascular Endothelial Growth Factor (VEGF))
[0206] The vascular endothelial growth factor (VEGF) is the main
regulator of angiogenesis. It elicits its proangiogenic activity by
binding to two membrane receptors (VEGFR1 and VEGFR2) on the
surface of endothelial cells (ECs). The binding of the natural
ligand to VEGFR2 induces receptor dimerization and
autophosphorylation of the intracellular kinase domain, which
activates intracellular pathways ending in proliferation,
migration, survival, and definitively triggering of the
proangiogenic cellular response. Recently a de novo synthesized
VEGF mimetic, named QK that shares the same properties as VEGF.
This mimetic is a 15 amino acid peptide which adopts a very stable
helical conformation in aqueous solution that resembles the 17-25
.alpha.-helical region of VEGF165, and binds both VEGFR-1 and 2. QK
recapitulates all of VEGFs properties of angiogenesis, vasodilation
and most importantly wound healing. It is well established that
VEGF improves the spread at which a wound heals. The quicker the
blood supply is returned to normal the faster the body can repair
itself. Full VEGF is difficult to control and use as drug to treat
internal damage. But the targeted construct of the present
disclosure takes the power of QK and homes it just to the damage
site to control the site of angiogenesis to jus the desired region.
The construct of the present disclosure is made up of the bone
targeting ligand of the present disclosure attached to a mini-peg
spacer and then to QK on its N terminus. The structure of QK is
depicted in FIG. 76.
Annexin-1
[0207] Annexin-1 is a family of phospholipid-binding proteins found
in many tissues including bone marrow.363 The N-terminal fragment
of annexin-1, Anx (2-26) is an endogenous ligand agonist of FPR1.
Anx (2-26) promotes chemotactic migration and OB differentiation of
MSCs via FPR1. The N-formyl peptide receptor (FPR) is a
chemoattractant receptor belonging to the G-protein coupled
receptor family. It is expressed in human bone marrow derived MSCs
and is functionally involved in promoting MSC adhesion to
extracellular matrix protein-coated surfaces as well as migration
to sites of fracture for tissue regeneration. The structure of
Annexin-1 is depicted in FIG. 81.
Material and Methods
Solid Phase Peptide Synthesis
[0208] 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
[0209] 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 (I2) 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 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 phosphate buffered saline (PBS)
at the appropriate dose concentrations for drug delivery.
General Methods for Obtaining Test Data
[0210] 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 ribonucleic acid (RNA)
was purified from the cells. Expression levels of ALP, RUNx2, 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 two weeks, three weeks, 4 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 (TbTh),
trabecular spacing (TbSp), 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
[0211] 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
[0212] 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(1-16) (Preptin D10) Efficacy on Fracture
Healing
[0213] 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
[0214] 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
[0215] 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
[0216] 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 10.times. induced healed
femur can withstand between 30-35 Newtons force.
Example 7. Ghrelin-D10 Effect on Fracture Healing
[0217] 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
[0218] Example 8 indicates pBMP9 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 bone volume in FIG. 16 and BV/TV in FIG. 17 in a dose dependent
manner.
Example 9. CNP D10 Effect on Fracture Healing
[0219] Example 9 indicates C-Type Natriuretic Peptide conjugate
(CNP D10) 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. 18 in a dose dependent manner.
Example 10. ODP D10 Effect on Fracture Healing
[0220] 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
[0221] 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
[0222] 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
[0223] 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
[0224] 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
[0225] 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. B2A_AHX3_e10 Effect on Fracture Healing
[0226] Example 16 indicates B2A (D)E.sub.10 effect on fracture
healing. Referring now to FIG. 36, In vivo fracture healing
efficacy of B2A_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. B2A_mp4_(D)E.sub.10
conjugate raises bone mineralization at the fracture calluses three
weeks post fracture.
[0227] Referring now to FIG. 37, in vivo fracture healing efficacy
of B2A_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. B2A_mp4_(D)E.sub.10
conjugate raises bone density at the fracture calluses three weeks
post fracture.
[0228] Referring now to FIG. 38, in vivo fracture healing efficacy
of B2A_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.
B2A_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
[0229] Referring now to FIG. 39, in vivo fracture healing efficacy
of B2A_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. B2A_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 17. F2A_mp4-e10 Effect on Fracture Healing
[0230] Example 17 indicates F2A (D)E.sub.10 effect on fracture
healing. Referring now to FIG. 41, In vivo fracture healing
efficacy of F2A_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. F2A_mp4_(D)E.sub.10
conjugate raises bone mineralization at the fracture calluses three
weeks post fracture.
[0231] Referring now to FIG. 42, in vivo fracture healing efficacy
of F2A_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. F2A_mp4_(D)E.sub.10
conjugate raises bone density at the fracture calluses three weeks
post fracture.
[0232] Referring now to FIG. 43, in vivo fracture healing efficacy
of F2A_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.
F2A_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
[0233] Referring now to FIG. 44, in vivo fracture healing efficacy
of F2A_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. F2A_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 18. F119: Effect on Fracture Healing
[0234] Example 18 indicates F119 effect on fracture healing.
Referring now to FIG. 45, in vivo fracture healing efficacy of
F119_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as BV/TV. 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., lx and 100.times. are respectively 1
nmol, 10 nmol, and 100 nmol of the conjugate delivered daily by
subcutaneous injection. F119_mp4_(D)E.sub.10 conjugate raises bone
density at the fracture calluses three weeks post fracture.
[0235] Referring now to FIG. 46, in vivo fracture healing efficacy
of F119_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as BV. 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., 10.times. and 100.times. are
respectively 1 nmol, 10 nmol, and 100 nmol of the conjugate
delivered daily by subcutaneous injection. F119_mp4_(D)E.sub.10
conjugate raises bone mineralization at the fracture calluses three
weeks post fracture.
[0236] Referring now to FIG. 47, in vivo fracture healing efficacy
of F119_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as max load. 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. F119_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0237] Referring now to FIG. 48, in vivo fracture healing efficacy
of F119_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as work to fracture. 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.
F119_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
Example 19. JNK3_Mp4_e10 Effect on Fracture Healing
[0238] Example 19 indicates JNK3 effect on fracture healing.
Referring now to FIG. 50, in vivo fracture healing efficacy of
JNK3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as BV. 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.
JNK3_mp4_(D)E.sub.10 conjugate raises bone mineralization at the
fracture calluses three weeks post fracture.
[0239] Referring now to FIG. 51, in vivo fracture healing efficacy
of JNK3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as BV/TV. 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. JNK3_mp4_(D)E.sub.10 conjugate raises bone density at
the fracture calluses three weeks post fracture.
[0240] Referring now to FIG. 52, in vivo fracture healing efficacy
of JNK3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as max load. 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. JNK3_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0241] Referring now to FIG. 53, in vivo fracture healing efficacy
of JNK3_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 3 weeks is reflected as work to fracture. 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.
JNK3_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
Example 20. Lactoferrin_mp4_e10 Effect on Fracture Healing
[0242] Example 20 indicates Lactoferrin effect on fracture healing.
Referring now to FIG. 55, in vivo fracture healing efficacy of
Lactoferrin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks is reflected as BV. 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. Lactoferrin_mp4_(D)E.sub.10 conjugate raises bone
mineralization at the fracture calluses three weeks post
fracture.
[0243] Referring now to FIG. 56, in vivo fracture healing efficacy
of Lactoferrin_mp4_(D)E10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks is reflected as BV/TV.
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. Lactoferrin_mp4_(D)E10 conjugate raises
bone density at the fracture calluses three weeks post
fracture.
[0244] Referring now to FIG. 57, in vivo fracture healing efficacy
of Lactoferrin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks is reflected as work to
fracture. 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. Lactoferrin_mp4_(D)E.sub.10 conjugate raises bone
strength at the fracture calluses three weeks post fracture.
[0245] Referring now to FIG. 58, in vivo fracture healing efficacy
of Lactoferrin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks is reflected as work to
fracture. 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. Lactoferrin_mp4_(D)E.sub.10 conjugate raises bone
strength at the fracture calluses three weeks post fracture.
Example 21. Osteostatin: Osteostatin (PTHrP[107-139])_e10 Effect on
Fracture Healing
[0246] Example 21 indicates Osteostatin effect of fracture healing.
Referring now to FIG. 60, in vivo fracture healing efficacy of
Osteostatin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 3 weeks is reflected as Tbth.
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
Osteostatin_mp4_(D)E.sub.10 conjugate raises bone quality at the
fracture calluses three weeks post fracture.
[0247] Referring now to FIG. 61, in vivo fracture healing efficacy
of Osteostatin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice(n=5) after 3 weeks is reflected as max load.
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. Osteostatin_mp4_(D)E.sub.10 conjugate
raises bone strength at the fracture calluses three weeks post
fracture.
[0248] Referring now to FIG. 62, in vivo fracture healing efficacy
of Osteostatin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice(n=5) after 3 weeks is reflected as BV/TV.
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. Osteostatin_mp4_(D)E.sub.10 conjugate raises bone
density at the fracture calluses three weeks post fracture.
[0249] Referring now to FIG. 63, in vivo fracture healing efficacy
of Osteostatin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice(n=5) after 3 weeks is reflected as stiffness.
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
Osteostatin_mp4_(D)E.sub.10 conjugate raises bone stiffness at the
fracture calluses three weeks post fracture.
[0250] Referring now to FIG. 64, in vivo fracture healing efficacy
of Osteostatin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice(n=5) after 3 weeks is reflected as work to
fracture. 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.
Osteostatin_mp4_(D)E.sub.10 conjugate raises bone strength at the
fracture calluses three weeks post fracture.
Example 22. P2A-Mp4-e10 Effect on Fracture Healing
[0251] Example 22 indicates P2A effect on fracture healing.
Referring now to FIG. 66, In vivo fracture healing efficacy of
P2A_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. P2A_mp4_(D)E.sub.10 conjugate raises bone
mineralization at the fracture calluses three weeks post
fracture.
[0252] Referring now to FIG. 67, in vivo fracture healing efficacy
of P2A_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. P2A_mp4_(D)E.sub.10
conjugate raises bone density at the fracture calluses three weeks
post fracture.
[0253] Referring now to FIG. 68, in vivo fracture healing efficacy
of P2A_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.
P2A_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses three weeks post fracture.
[0254] Referring now to FIG. 69, in vivo fracture healing efficacy
of P2A_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. P2A_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
Example 23. Preptin (1-34) Mp4_e10 Effect on Fracture Healing
[0255] Example 23 indicates Preptin (1-34) effect on fracture
healing. Referring now to FIG. 71, in vivo fracture healing
efficacy of Preptin(1-34)_mp4_(D)E.sub.10 conjugate on cluster of
differentiation-1 (CD-1) male mice fracture-bearing mice (n=10)
after 4 weeks is reflected as BV/TV. 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. 50.times. represents 50 nmol of the conjugate
delivered daily by subcutaneous injection.
Preptin(1-34)_mp4_(D)E.sub.10 conjugate significantly raises bone
density at the fracture calluses four weeks post fracture.
[0256] Referring now to FIG. 72, in vivo fracture healing efficacy
of Preptin(1-34)_mp4_(D)E.sub.10 conjugate on CD-1 male mice
fracture-bearing mice (n=10) after 4 weeks is reflected as BV. 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 50.times. represents 50
nmol of the conjugate delivered daily by subcutaneous injection.
Preptin(1-34) mp4_(D)E.sub.10 conjugate significantly raises bone
mineralization at the fracture calluses four weeks post
fracture.
[0257] Referring now to FIG. 73, in vivo fracture healing efficacy
of Preptin(1-34)_mp4_(D)E.sub.10 conjugate on CD-1 Male mice
fracture-bearing mice(n=10) after 4 weeks is reflected as max load.
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. 50.times. represents 50 nmol of the conjugate delivered
daily by subcutaneous injection. Preptin(1-34)_mp4_(D)E.sub.10
conjugate significantly raises bone strength at the fracture
calluses four weeks post fracture.
[0258] Referring now to FIG. 74, in vivo fracture healing efficacy
of Preptin(1-34)_mp4_(D)E.sub.10 conjugate on CD-1 Male mice
fracture-bearing mice(n=10) after 4 weeks is reflected as post
yield displacement. Post yield displacement represents the total
displacement of the healed femur after the yield point has been
reached in a postmortem 4 point bend analysis Post yield
displacement is a measure of how elastic a material is. 50.times.
represents 50 nmol of the conjugate delivered daily by subcutaneous
injection. Preptin(1-34)_mp4_(D)E.sub.10 conjugate significantly
reduces the bone brittleness at the fracture calluses four weeks
post fracture.
[0259] Referring now to FIG. 75, in vivo fracture healing efficacy
of Preptin(1-34)_mp4_(D)E.sub.10 conjugate on CD-1 Male mice
fracture-bearing mice(n=10) after 4 weeks is reflected as work to
fracture. 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. 50.times. represents 50
nmol of the conjugate delivered daily by subcutaneous injection.
Preptin(1-34)_mp4_(D)E.sub.10 conjugate significantly raises bone
strength at the fracture calluses four weeks post fracture.
Example 24. QK_mp4_e10 Effect on Fracture Healing
[0260] Example 24 indicates QK effect on fracture healing.
Referring now to FIG. 77, in vivo fracture healing efficacy of
QK_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice(n=5) after 17 days is reflected as BV. 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. QK_mp4_(D)E.sub.10
conjugate raises bone mineralization at the fracture calluses 17
days post fracture.
[0261] Referring now to FIG. 78, in vivo fracture healing efficacy
of QK_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice(n=5) after 17 days is reflected as BV/TV. 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. QK_mp4_(D)E.sub.10 conjugate raises bone density at the
fracture calluses 17 days post fracture.
[0262] Referring now to FIG. 79, in vivo fracture healing efficacy
of QK_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice(n=5) after 17 days is reflected as max load (N). 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. QK_mp4_(D)E.sub.10 conjugate raises bone
strength at the fracture calluses 17 days post fracture.
[0263] Referring now to FIG. 80, in vivo fracture healing efficacy
of QK_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 17 days is reflected as work to fracture. 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.
QK_mp4_(D)E.sub.10 conjugate raises bone strength at the fracture
calluses 17 days post fracture.
Example 22. Effect of Annexin-1 on Fracture Healing
[0264] Example 22 indicates Annexin-1 effect on fracture healing.
Referring now to FIG. 82, in vivo fracture healing efficacy of
Annexin_mp4_(D)E.sub.10 conjugate on Swiss Webster fracture-bearing
mice (n=5) after 16 days is reflected as max load (N). Max load
represents the maximum force the healed femur withstood before it
refractured in a postmortem 4 point bend analysis. Max 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. Annexin_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post
fracture.
[0265] Referring now to FIG. 83, in vivo fracture healing efficacy
of Annexin_mp4_(D)E.sub.10 conjugate on Swiss Webster
fracture-bearing mice (n=5) after 16 days is reflected as work to
fracture (mJ). 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. Annexin_mp4_(D)E.sub.10 conjugate raises
bone strength at the fracture calluses three weeks post fracture.
Sequence CWU 1
1
29125PRTArtificial 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 252028PRTArtificial
SequenceF119MISC_FEATURE(18)..(19) 20His Lys Arg Thr Gly Gln Tyr
Lys Leu Gly Ser Lys Thr Gly Pro Gly1 5 10 15Gln Lys Glu Glu Glu Glu
Glu Glu Glu Glu Glu Glu 20 252136PRTArtificial
SequenceJNK3_mp4_e10MISC_FEATURE(26)..(27) 21His Met Gly Glu Lys
Pro Gly Thr Arg Val Phe Lys Lys Ser Ser Pro1 5 10 15Asn Cys Lys Leu
Thr Val Tyr Leu Gly Lys Glu Glu Glu Glu Glu Glu 20 25 30Glu Glu Glu
Glu 352226PRTArtificial
SequenceLactoferrin_mp4_e10MISC_FEATURE(16)..(17) 22His Phe Lys Cys
Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala1 5 10 15Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu 20 252344PRTArtificial
SequenceOsteostatin_mp4_(D)E10)MISC_FEATURE(34)..(35) 23His Thr Arg
Ser Ala Trp Leu Asp Ser Gly Val Thr Gly Ser Gly Leu1 5 10 15Glu Gly
Asp His Leu Ser Asp Thr Ser Thr Thr Ser Leu Glu Leu Asp 20 25 30Ser
Arg Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 35 402442PRTArtificial
SequencePreptin (1-34)_mp4_e10MISC_FEATURE(10)..(11) 24Glu Glu Glu
Glu Glu Glu Glu Glu Glu Glu Val Arg Gly Ala Ser Gln1 5 10 15Arg Trp
Thr Asp Tyr Gln Phe Phe Gly Val Pro Tyr Arg Pro Phe Asp 20 25 30Pro
Leu Val Ala Gln Ser Thr Ser Val Asp 35 402525PRTArtificial
SequenceQK_mp4_e10MISC_FEATURE(10)..(11) 25Glu Glu Glu Glu Glu Glu
Glu Glu Glu Glu Lys Leu Thr Trp Gln Glu1 5 10 15Leu Tyr Gln Leu Lys
Tyr Lys Gly Ile 20 252635PRTArtificial SequenceAnnexin
1MISC_FEATURE(25)..(26) 26Ala Met Val Ser Glu Phe Leu Lys Gln Ala
Trp Phe Ile Glu Asn Glu1 5 10 15Glu Gln Glu Tyr Val Gln Thr Val Tyr
Glu Glu Glu Glu Glu Glu Glu 20 25 30Glu Glu Glu 352726PRTArtificial
SequenceB2A_AHX3_e10MISC_FEATURE(4)..(5)MISC_FEATURE(16)..(17)
27His 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
252826PRTArtificial SequenceF2A_mp4_e10MISC_FEATURE(16)..(17) 28His
Tyr Arg Ser Arg Lys Tyr Ser Ser Trp Tyr Val Ala Leu Lys Arg1 5 10
15Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 20 252920PRTArtificial
SequenceP2A-mp4_e10MISC_FEATURE(10)..(11) 29Val Arg Lys Ile Glu Ile
Val Arg Lys Lys Glu Glu Glu Glu Glu Glu1 5 10 15Glu Glu Glu Glu
20
* * * * *