U.S. patent application number 15/062834 was filed with the patent office on 2016-06-30 for compositions and methods for treating bone conditions.
The applicant listed for this patent is CASE WESTERN RESERVE UNIVERSITY. Invention is credited to James E. Dennis, Feng Lin, Zhidan Tu.
Application Number | 20160185872 15/062834 |
Document ID | / |
Family ID | 48903084 |
Filed Date | 2016-06-30 |
United States Patent
Application |
20160185872 |
Kind Code |
A1 |
Lin; Feng ; et al. |
June 30, 2016 |
COMPOSITIONS AND METHODS FOR TREATING BONE CONDITIONS
Abstract
A method of treating a degenerative bone condition of a subject
includes administering to hematopoietic progenitor cells or
osteoclast progenitor cells of the subject at least one agent that
substantially reduces the interaction of at least one of C3a or C5a
with the C3a receptor (C3aR) and/or C5a receptor (C5aR), a
STAT3/IL-6 signaling pathway antagonist, and a combination thereof,
the agent being administered to the hematopoietic progenitor cells
or osteoclast progenitor cells at an amount effective to inhibit
osteoclast differentiation of hematopoietic progenitor cells or
osteoclast progenitor cells.
Inventors: |
Lin; Feng; (Cleveland,
OH) ; Dennis; James E.; (Cleveland, OH) ; Tu;
Zhidan; (Cleveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CASE WESTERN RESERVE UNIVERSITY |
Cleveland |
OH |
US |
|
|
Family ID: |
48903084 |
Appl. No.: |
15/062834 |
Filed: |
March 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13572141 |
Aug 10, 2012 |
9289467 |
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15062834 |
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61521847 |
Aug 10, 2011 |
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Current U.S.
Class: |
424/158.1 ;
424/172.1; 424/173.1; 424/178.1; 435/375; 514/44A |
Current CPC
Class: |
C07K 2317/76 20130101;
A61K 31/7088 20130101; A61K 38/1725 20130101; A61K 47/6849
20170801; A61K 47/6843 20170801; C07K 16/40 20130101; A61K 38/177
20130101; C12N 15/1138 20130101; C07K 16/2896 20130101; C12N
2310/351 20130101; C07K 16/18 20130101; C12N 2310/14 20130101; A61K
39/3955 20130101; A61K 47/6871 20170801; A61K 31/713 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/40 20060101 C07K016/40; A61K 47/48 20060101
A61K047/48; C07K 16/18 20060101 C07K016/18; C12N 15/113 20060101
C12N015/113 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
No. 5R01NS052471 awarded by The National Institute of Neurological
Disorders and Stroke (NINDS). The United States government may have
certain rights to the invention.
Claims
1. A method of treating a degenerative bone condition, the method
comprising: administering to hematopoietic progenitor cells or
osteoclast progenitor cells of the subject at least one agent that
substantially reduces the interaction of at least one of C3a or C5a
with the C3a receptor (C3aR) and/or C5a receptor (C5aR), a
STAT3/IL-6 signaling pathway antagonist, and a combination thereof,
the agent being administered to the hematopoietic progenitor cells
or osteoclast progenitor cells at an amount effective to inhibit
osteoclast differentiation of hematopoietic progenitor cells or
osteoclast progenitor cells.
2. The method of claim 1, the agent comprising at least one
complement antagonist selected from the group consisting of a small
molecule, a polypeptide, and a polynucleotide.
3. The method of claim 2, the at least one complement antagonist
selected from the group consisting of DAF or an antibody directed
against at least one of C3, C5, C3 convertase, C5 convertase, C3a,
C5a, C3aR, or C5aR.
4. The method of claim 2, the polynucleotide comprising a small
interfering RNA directed against a polynucleotide encoding at least
one of C3, C5, C3aR, or C5aR.
5. The method of claim 3, the at least one complement antagonist
including an antibody directed against C5aR and an antibody
directed against C3aR.
6. The method of claim 3, the at least one complement antagonist
including an antibody directed against C5a and an antibody directed
against C3a.
7. The method of claim 3, the at least one complement antagonist
including an antibody directed against C5 and an antibody directed
against C3.
8. The method of claim 1, the agent being administered to the
hematopoietic progenitor cells or osteoclast progenitor cells in
vitro.
9. The method of claim 1, the agent being administered locally to
the hematopoietic progenitor cells or osteoclast progenitor cells
at the site of the bone condition.
10. The method of claim 1, the agent being conjugated to a
targeting moiety that targets hematopoietic progenitor cells or
osteoclast progenitor cells.
11. The method of claim 1 wherein the bone condition comprises
osteopenia or osteoporosis.
12. A method of treating post-menopausal osteoporosis, the method
comprising: administering to hematopoietic progenitor cells or
osteoclast progenitor cells of the subject at least one complement
antagonist that substantially reduces the interaction of at least
one of C3a or C5a with the C3a receptor (C3aR) and/or C5a receptor
(C5aR), the agent being administered to the hematopoietic
progenitor cells or osteoclast progenitor cells at an amount
effective to inhibit osteoclast differentiation of hematopoietic
progenitor cells.
13. The method of claim 12, the at least one complement antagonist
selected from the group consisting of DAF or an antibody directed
against at least one of C3, C5, C3 convertase, C5 convertase, C3a,
C5a, C3aR, or C5aR.
14. The method of claim 12, the at least one complement antagonist
including an antibody directed against C5aR and an antibody
directed against C3aR or an antibody directed against C5a and an
antibody directed against C3a.
15. The method of claim 12, the agent being administered to the
hematopoietic progenitor cells or osteoclast progenitor cells in
vitro.
16. The method of claim 12, the agent being administered locally to
the hematopoietic progenitor cells or osteoclast progenitor cells
of the bone marrow.
17. The method of claim 12, the agent being conjugated to a
targeting moiety that targets hematopoietic progenitor cells or
osteoclast progenitor cells of the bone marrow.
18. A method of treating post-menopausal osteoporosis of a subject,
the method comprising: administering locally to bone marrow of the
subject at least one complement antagonist that substantially
reduces the interaction of at least one of C3a or C5a with the C3a
receptor (C3aR) and C5a receptor (C5aR), the agent being
administered locally to the bone marrow cells of the subject at an
amount effective to inhibit osteoclast differentiation of
hematopoietic progenitor cells or osteoclast progenitor cells.
19. The method of claim 18, the at least one complement antagonist
selected from the group consisting of DAF or an antibody directed
against at least one of C3, C5, C3 convertase, C5 convertase, C3a,
C5a, C3aR, or C5aR.
20. The method of claim 19, the at least one complement antagonist
including an antibody directed against C5aR and an antibody
directed against C3aR or an antibody directed against C5a and an
antibody directed against C3a.
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application No. 61/521,847, filed Aug. 10, 2011, the subject matter
of which is incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] This application relates to compositions and methods of
treating bone conditions, and particularly relates to compositions
and methods of treating osteoporosis and/or osteopenia.
BACKGROUND
[0004] Osteoporosis and particularly osteoporosis-related fractures
are a major health problem in the United States. Approximately 10
million Americans are at risk for osteoporosis-related fractures
and there are an estimated 1.5 million osteoporosis-related
fractures per year. While there are several currently-approved
therapies for the treatment of osteoporosis, including
bisphosphonates, calcitonin, estrogen, selective estrogen receptor
modulators (SERMS) and intermittent parathyroid hormone (PTH)
treatments, each of these has drawbacks. Estrogen treatment or
hormone replacement therapy has fallen out of favor due to the
increased risk of breast cancer. There are a range of
bisphosphonates available that show relatively good tolerance, but
there remain issues with respect to osteonecrosis of the jaw,
atypical bone fragility, gastrointestinal discomfort and some cases
of influenza-like illnesses. Importantly, while most
bisphosphonates show good to excellent efficacy for decreasing
vertebral fracture risk, none of the bisphosphonate treatments
shows particularly good efficacy in preventing peripheral fractures
(<30% decrease in risk). Calcitonin treatments are limited
because they have yet to show a reduction in non-vertebral fracture
risk. PTH is the only approved anabolic treatment for osteoporosis
and is the only treatment that is moderately effective for reducing
peripheral fracture risk, but there is an increased risk of
osteosarcoma which limits PTH treatments to no more than 2 years.
Because of lingering issues of side effects and clinical efficacy,
significant efforts continue to be made in an attempt to develop
more effective drugs for treating or preventing osteoporosis.
SUMMARY
[0005] This application relates to a method of treating a
degenerative bone condition of a subject. The method includes
administering to hematopoietic progenitor cells or osteoclast
progenitor cells of the subject at least one agent that
substantially reduces the interaction of at least one of C3a or C5a
with the C3a receptor (C3aR) and/or C5a receptor (C5aR), a
STAT3/IL-6 signaling pathway antagonist, and a combination thereof.
The agent can be administered to the hematopoietic progenitor cells
or osteoclast progenitor cells at an amount effective to inhibit
osteoclast differentiation of hematopoietic progenitor cells or
osteoclast progenitor cells.
[0006] In some embodiments, the agent can include at least one
complement antagonist selected from the group consisting of a small
molecule, a polypeptide, and a polynucleotide. The at least one
complement antagonist can be selected from the group consisting of
DAF or an antibody directed against at least one of C3, C5, C3
convertase, C5 convertase, C3a, C5a, C3aR, or C5aR. The at least
one complement antagonist can also be a small interfering RNA
directed against a polynucleotide encoding at least one of C3, C5,
C3aR, or C5aR.
[0007] In other embodiments, the at least one complement antagonist
can include an antibody directed against C5aR and an antibody
directed against C3aR, an antibody directed against C5a and an
antibody directed against C3a, and/or an antibody directed against
C5 and an antibody directed against C3.
[0008] In some embodiments, the agent can be administered to the
hematopoietic progenitor cells or osteoclast progenitor cells in
vitro. In other embodiments, the agent be administered locally to
the hematopoietic progenitor cells or osteoclast progenitor cells
at the site of the bone condition. The agent can also be conjugated
to a targeting moiety that targets hematopoietic progenitor cells
or osteoclast progenitor cells.
[0009] In some embodiments, the degenerative bone condition can
include osteopenia or osteoporosis, such as post-menopausal
osteopenia or post-menopausal osteoporosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic illustration showing that complement
regulates bone balance in osteoporosis by modulating OC and OB
differentiation.
[0011] FIG. 2 illustrates a graph showing the number of TRAP.sup.+
cells of WT and factor D.sup.-/- BM cells subjected to OC
differentiation conditions.
[0012] FIG. 3(A-C) illustrates images showing illustrates
TRAP-positive cells from (A) WT and (B)C3.sup.-/- BM cells produced
from aliquots of 2.times.10.sup.6 WT and C3.sup.-/- BM cells
cultured in .alpha.-MEM/10% heat-inactivated FBS media in each well
of a 24-well plate together with 1.times.10.sup.-8M 1.25(OH).sub.2
vitamin D.sub.3; and (C) a graph showing total mononuclear
(mono)/multinucleated (multi) TRAP-positive cells in each well.
[0013] FIG. 4(A-B) illustrates graph showing C3-deficient BM cells
produce decreased amounts of M-CSF and failed to up-regulate RANKL
during differentiation. (A) M-CSF levels were measured by ELISA in
WT and C3.sup.-/- BM cell-conditioned media during differentiation.
(B) RANKL/OPG expression levels were quantified by qRT-PCR in WT
and C3.sup.-/- BM cells after 1.25(OH)2 vitamin D3 (VD)
stimulation.
[0014] FIG. 5 illustrates a graph showing the hemolysis percent
using C5-depleted sera plus 1:5 diluted control media or BM
cell-conditioned media of an E.sup.sha hemolytic assay.
[0015] FIG. 6(A-C) illustrates images showing TRAP-positive cells
from (A) WT and (B) factor D.sup.-/- BM cells of aliquots of
2.times.10.sup.6 WT and factor D.sup.-/- BM cells cultured in
.alpha.-MEM/10% heat-inactivated FBS media plate together with
1.times.10.sup.-8M 1.25(OH).sub.2 vitamin D.sub.3; and (C) a graph
showing representative TRAP-positive cells from, and total
mononuclear (mono)/multinucleated (multi) TRAP-positive cells in
each well.
[0016] FIG. 7(A-B) illustrates: (A) TRAP-positive cells of
2.times.10.sup.6 WT, C3aR.sup.-/-, C5aR.sup.-/-, and
C3aR.sup.-/-C5aR.sup.-/- BM cells cultured in .alpha.-MEM/10%
heat-inactivated media of a 24-well plate together with
1.times.10.sup.-8M 1.25(OH).sub.2 vitamin D.sub.3; and (B)
TRAP-positive cells of WT BM cells (2.times.10.sup.6) cultured in
.alpha.-MEM/10% heat-inactivated FBS media of a 24-well plate
together with 1.times.10.sup.-8M 1.25(OH).sub.2 vitamin D.sub.3 in
the presence of placebo (control), C3aRA, C5aRA, or
C3aRA.cndot.C5aRA.
[0017] FIG. 8(A-D) illustrates complement graphs showing: (A)
quantification of IL-6 levels in supernatants cultured with WT,
C3.sup.-/-, and C3aR.sup.-/-C5aR.sup.-/- BM cells during
differentiation. BM cells (2.times.10.sup.6) were cultured in
differentiation media in each well of a 24-well plate together with
1.times.10.sup.-8M 1.25(OH).sub.2 vitamin, and IL-6 levels were
measured in supernatants on day 1; (B) supplementing IL-6 into
C3.sup.-/- BM cell cultures restored their OC differentiation
capabilities; (C) exogenous C3a/C5a augmented OC differentiation
from WT BM cells, while neutralization of IL-6 abolished the
stimulating effect; and (D) exogenous C3a/C5a augmented OC
differentiation from C3.sup.-/- BM cells, while neutralization of
IL-6 abolished the stimulating effect.
[0018] FIG. 9(A-C) illustrates graphs showing (A) EshA-hemolytic
assays using C5-depleted sera plus 1:5 diluted control media or BM
cell-conditioned media, showing that BM cell-conditioned media
compensated the absence of C5, therefore inducing C5b-9-mediated
hemolysis; (B) human BM cells were incubated with
1.times.10.sup.-8M 1.25(OH).sub.2 vitamin D.sub.3 in the presence
of placebo (control), C3aRA, C5aRA, or C3aRA.cndot.C5aRA, showing
that efficient OC differentiation in humans also requires C3aR/C5aR
as in mice. Representative results of 2 independent experiments;
and (C) Both mesenchymal cells and OC progenitors are involved in
the complement-regulated OC differentiation. Samples of
2.times.10.sup.4 primary WT or C3.sup.-/- calvarial OBs were
cultured with 2.times.10.sup.6 WT and C3.sup.-/- plenocytes (as
source of OC progenitors) together with 1.times.10.sup.-8M
1.25(OH).sub.2 vitamin D.sub.3.
DETAILED DESCRIPTION
[0019] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises, such as
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates). Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the present invention pertains. Commonly
understood definitions of molecular biology terms can be found in,
for example, Rieger et al., Glossary of Genetics: Classical and
Molecular, 5th Edition, Springer-Verlag: New York, 1991, and Lewin,
Genes V, Oxford University Press: New York, 1994. The definitions
provided herein are to facilitate understanding of certain terms
used frequently herein and are not meant to limit the scope of the
application described herein.
[0020] As used herein, the term "polypeptide" refers to an
oligopeptide, peptide, or protein sequence, or to a fragment,
portion, or subunit of any of these, and to naturally occurring or
synthetic molecules. The term "polypeptide" also includes amino
acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres, and may contain any type of
modified amino acids. The term "polypeptide" also includes peptides
and polypeptide fragments, motifs and the like, glycosylated
polypeptides, and all "mimetic" and "peptidomimetic" polypeptide
forms.
[0021] As used herein, the term "polynucleotide" refers to
oligonucleotides, nucleotides, or to a fragment of any of these, to
DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin
which may be single-stranded or double-stranded and may represent a
sense or antisense strand, to peptide nucleic acids, or to any
DNA-like or RNA-like material, natural or synthetic in origin,
including, e.g., iRNA, siRNAs, microRNAs, and ribonucleoproteins.
The term also encompasses nucleic acids, i.e., oligonucleotides,
containing known analogues of natural nucleotides, as well as
nucleic acid-like structures with synthetic backbones.
[0022] As used herein, the term "antibody" refers to whole
antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), and
includes fragments thereof which are also specifically reactive
with a target polypeptide. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility
and/or interaction with a specific epitope of interest. Thus, the
term includes segments of proteolytically-cleaved or
recombinantly-prepared portions of an antibody molecule that are
capable of selectively reacting with a certain polypeptide.
Non-limiting examples of such proteolytic and/or recombinant
fragments include Fab, F(ab')2, Fab', Fv, and single chain
antibodies (scFv) containing a V[L] and/or V[H] domain joined by a
peptide linker. The scFv's may be covalently or non-covalently
linked to form antibodies having two or more binding sites. The
term "antibody" also includes polyclonal, monoclonal, or other
purified preparations of antibodies, recombinant antibodies,
monovalent antibodies, and multivalent antibodies. Antibodies may
be humanized, and may further include engineered complexes that
comprise antibody-derived binding sites, such as diabodies and
triabodies.
[0023] As used herein, the term "complementary" refers to the
capacity for precise pairing between two nucleobases of a
polynucleotide and its corresponding target molecule. For example,
if a nucleobase at a particular position of a polynucleotide is
capable of hydrogen bonding with a nucleobase at a particular
position of a target polynucleotide (the target nucleic acid being
a DNA or RNA molecule, for example), then the position of hydrogen
bonding between the polynucleotide and the target polynucleotide is
considered to be complementary. A polynucleotide and a target
polynucleotide are complementary to each other when a sufficient
number of complementary positions in each molecule are occupied by
nucleobases, which can hydrogen bond with each other. Thus,
"specifically hybridizable" and "complementary" are terms which can
be used to indicate a sufficient degree of precise pairing or
complementarity over a sufficient number of nucleobases such that
stable and specific binding occurs between a polynucleotide and a
target polynucleotide.
[0024] As used herein, the term "subject" refers to any
warm-blooded organism including, but not limited to, human beings,
rats, mice, dogs, goats, sheep, horses, monkeys, apes, rabbits,
cattle, etc.
[0025] As used herein, the terms "complement polypeptide" or
"complement component" refer to a polypeptide (or a polynucleotide
encoding the polypeptide) of the complement system that functions
in the host defense against infections and in the inflammatory
process. Complement polypeptides constitute target substrates for
the complement antagonists provided herein.
[0026] As used herein, the term "complement antagonist" refers to a
polypeptide, polynucleotide, or small molecule capable of
substantially reducing or inhibiting the activity of a complement
component.
[0027] A complement component can include any one or combination of
interacting blood polypeptides or glycoproteins. There are at least
30 soluble plasma polypeptides, in addition to cell surface
receptors, which can bind complement reaction products and which
can occur on inflammatory cells and cells of the immune system. In
addition, there are regulatory membrane proteins that can protect
host cells from accidental complement attack. Complement components
can include polypeptides that function in the classical pathway,
such as C2, polypeptides that function in the alternative pathway,
such as Factor B, and polypeptides that function in the lectin
pathway, such as MASP-1.
[0028] Complement components can also include: any of the "cleavage
products" (also referred to as "fragments") that are formed upon
activation of the complement cascade; complement polypeptides that
are inactive or altered forms of complement polypeptides, such as
iC3 and C3a-desArg; and components indirectly associated with the
complement cascade. Examples of such complement components can
include, but are not limited to, C1q, C1r, C1s, C2, C3, C3a, C3b,
C3c, C3dg, C3g, C3d, C3f, iC3, C3a-desArg, C4, C4a, C4b, iC4,
C4a-desArg, C5, C5a, C5a-des-Arg, C6, C7, C8, C9, MASP-1, MASP-2,
MBL, Factor B, Factor D, Factor H, Factor I, CR1, CR2, CR3, CR4,
properdin, C1Inh, C4bp, MCP, DAF, CD59 (MIRL), clusterin, HRF, and
allelic and species variants of any complement polypeptide.
[0029] As used herein, the terms "treatment," "treating," or
"treat" refers to any specific method or procedure used for the
cure of, inhibition of, prophylaxis of, reduction of, elimination
of, or the amelioration of a bone condition or degenerative bone
condition, such as osteopenia, osteoporosis, post-menopausal
osteopenia, and post-menopausal osteoporosis.
[0030] As used herein, the term "effective amount" refers to a
dosage of an agent described herein administered alone or in
conjunction with any additional therapeutic agents that are
effective and/or sufficient to provide treatment of a bone
condition or degenerative bone condition, such as osteopenia,
osteoporosis, post-menopausal osteopenia, and post-menopausal
osteoporosis. The effective amount can vary depending on the
subject, the disease being treated, and the treatment being
affected.
[0031] As used herein, the term "therapeutically effective amount"
refers to that amount of an agent described herein administered
alone and/or in combination with additional therapeutic agents that
results in amelioration of symptoms associated with a bone
condition or degenerative bone condition, such as osteopenia,
osteoporosis, post-menopausal osteopenia, and post-menopausal
osteoporosis.
[0032] As used herein, the terms "parenteral administration" and
"administered parenterally" refers to modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intraventricular, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal and intrasternal injection
and infusion.
[0033] As used herein, the terms "pharmaceutically or
pharmacologically acceptable" refer to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate. Veterinary uses are equally included within the
invention and "pharmaceutically acceptable" formulations include
formulations for both clinical and/or veterinary use.
[0034] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. For human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards. Supplementary active
ingredients can also be incorporated into the compositions.
[0035] As used herein, "Unit dosage" formulations are those
containing a dose or sub-dose of the administered ingredient
adapted for a particular timed delivery. For example, exemplary
"unit dosage" formulations are those containing a daily dose or
unit or daily sub-dose or a weekly dose or unit or weekly sub-dose
and the like.
[0036] Embodiments of this application relate to methods and
compositions for modulating osteoclast (OC) and/or osteoblast (OB)
differentiation and to methods and compositions for treating treat
diseases, disorders, and conditions where inhibition and/or
promotion of osteoclast differentiation and/or osteoblast
differentiation is desired. The methods can include administering
to osteoclast progenitors (e.g., hematopoietic progenitors or
hematopietic stem cells) or osteoblast progenitors (e.g.,
mesenchymal stem cells) at least one agent that modulates (e.g.,
inhibits or promotes) C3aR and/or C5aR signaling of the cells.
[0037] It was found that complement deprivation has a protective
effect on estrogen deficiency-driven osteoporosis in an animal
model of post-menopausal osteoporosis, and that complement
regulates mouse and human osteoclast (OC) and osteoblast (OB)
differentiation through C3aR/C5aR-driven IL-6 production. The
bone-resorbing OCs and the bone-forming OBs maintain the dynamic
balance of bone. While OCs are differentiated from hematopoietic
stem cells (HSCs), OBs are derived from mesenchymal stem (or
stromal) cells (MSCs), and MSCs differentiate into OBs at the
expense of other potential differentiation lineages, e.g.
adipocytes. An array of factors including RANKL, OPG, IL-6,
TNF-.alpha. and IL-1 regulate OC differentiation from HSCs, while
Run.times.2 and PPAR-.gamma. are the transcription factors that
regulate MSC differentiation along the OB and adipocyte lineages.
In post-menopause osteoporosis, both OC and OB numbers increase
after estrogen deprivation. However, the increased OC numbers
overwhelm the increased OB numbers, leading to net bone loss.
Complement, locally produced by bone marrow (BM) cells, regulates
both human and mouse OC differentiation through C3aR/C5aR-driven
IL-6 production. C3aR and C5aR are the two receptors for the
complement activation products C3a and C5a, which are expressed in
a broad spectrum of cells. It was also found that C3.sup.-/- mice
are protected from bone loss after ovariectomy (OVX) despite the in
vitro results that C3.sup.-/- MSC have decreased capacity of
differentiating to OBs in vitro, suggesting that complement has a
major role in the regulation of skeletal homeostasis (FIG. 1) and
that that C3aR and C5aR antagonists can be an effective treatment
modality for osteoporosis. The complement system can therefore be
use as a target for the treatment and prevention of a bone
condition, such a degenerative bone condition including osteopenia,
osteoporosis, post-menopausal osteopenia, post-menopausal
osteoporosis and other degenerative bone conditions, such as in
autoimmune arthritis.
[0038] Accordingly, based at least in part on these findings, in
some embodiments of the application hematopoietic progenitor cells,
such as hematopoietic stem cells, or osteoclast progenitor cells,
for example, found in bone marrow, can be contacted (e.g., directly
or locally) with a therapeutically effective amount of an agent
that modulates (e.g., inhibits or promotes) C3aR and/or C5aR
signaling of the cells and modulates (e.g., inhibits or promotes)
osteoclast differentiation.
[0039] In some embodiments, osteoclast differentiation of
hematopoietic progenitor cells or osteoclast progenitor cells, can
be inhibited by administering to the hematopoietic progenitor cells
or osteoclast progenitor cells an agent that inhibits C3aR and/or
C5aR signaling of the cells. The agent can be selected from the
group consisting of a complement antagonist that inhibits or
substantially reduces the interaction of at least one of C3a or C5a
with the C3a receptor (C3aR) and C5a receptor (C5aR), an IL-6/STAT3
signaling pathway antagonist, and combinations thereof.
[0040] By inhibiting or substantially reducing the activity of a
complement component, it is meant that the activity of the
complement component may be entirely or partly diminished. For
example, an inhibition or reduction in the functioning of a C3/C5
convertase may prevent cleavage of C5 and C3 into C5a and C3a,
respectively. An inhibition or reduction in the functioning of C5,
C3, C5a and/or C3a polypeptides may reduce or eliminate the ability
of C5a and C3a to bind C5aR and C3aR, respectively. An inhibition
or reduction in Factor B, Factor D, properidin, Bb, Ba and/or any
other protein of the complement pathway that is used in the
formation of C3 convertase, C5 convertase, C5, C3, C5a and/or C3a
may reduce or eliminate the ability of C5a and C3a to be formed and
bind to C5aR and C3aR, respectively. Additionally, an inhibition or
reduction in the functioning of a C5aR or C3aR may similarly reduce
or eliminate the ability of C5a and C3a to bind C5aR and C3aR,
respectively.
[0041] In an aspect of the application, the at least one complement
antagonist can include an antibody or antibody fragment directed
against a complement component that can affect or inhibit the
formation of C3a and/or C5a (e.g., anti-Factor B, anti-Factor D,
anti-C5, anti-C3, anti-C5 convertase, and anti-C3 convertase)
and/or reduce C5a/C3a-C5aR/C3aR interactions (e.g., anti-C5a,
anti-C3a, anti-C5aR, and C3aR antibodies). In one example, the
antibody or antibody fragment can be directed against or
specifically bind to an epitope, an antigenic epitope, or an
immunogenic epitope of a C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase. The term "epitope" as used herein
can refer to portions of C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase having antigenic or immunogenic
activity. An "immunogenic epitope" as used herein can include a
portion of a C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3
convertase that elicits an immune response in a subject, as
determined by any method known in the art. The term "antigenic
epitope" as used herein can include a portion of a polypeptide to
which an antibody can immunospecifically bind as determined by any
method well known in the art.
[0042] Examples of antibodies directed against C5, C3, C3a, C5a,
C5aR, C3aR, C5 convertase, and/or C3 convertase are known in the
art. For example, mouse monoclonal antibodies directed against C3aR
can include those available from Santa Cruz Biotechnology, Inc.
(Santa Cruz, Calif.). Monoclonal anti-human C5aR antibodies can
include those available from Research Diagnostics, Inc. (Flanders,
N.J.). Monoclonal anti-human/anti-mouse C3a antibodies can include
those available from Fitzgerald Industries International, Inc.
(Concord, Me.). Monoclonal anti-human/anti-mouse C5a antibodies can
include those available from R&D Systems, Inc. (Minneapolis,
Minn.).
[0043] In some embodiments, the complement antagonist can include a
purified polypeptide that is a dominant negative or competitive
inhibitor of C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3
convertase. As used herein, "dominant negative" or "competitive
inhibitor" refers to variant forms of a protein that inhibit the
activity of the endogenous, wild type form of the protein (i.e.,
C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase).
As a result, the dominant negative or competitive inhibitor of a
protein promotes the "off" state of protein activity. In the
context of the present invention, a dominant negative or
competitive inhibitor of C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase is a C5, C3, C3a, C5a, C5aR, C3aR,
C5 convertase, and/or C3 convertase polypeptide, which has been
modified (e.g., by mutation of one or more amino acid residues, by
posttranscriptional modification, by posttranslational
modification) such that the C5, C3, C3a, C5a, C5aR, C3aR, C5
convertase, and/or C3 convertase inhibits the activity of the
endogenous C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3
convertase.
[0044] In some embodiments, the competitive inhibitor of C5, C3,
C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase can be a
purified polypeptide that has an amino acid sequence, which is
substantially similar (i.e., at least about 75%, about 80%, about
85%, about 90%, about 95% similar) to the wild type C5, C3, C3a,
C5a, C5aR, C3aR, C5 convertase, and/or C3 convertase but with a
loss of function. The purified polypeptide, which is a competitive
inhibitor of C5, C3, C3a, C5a, C5aR, C3aR, C5 convertase, and/or C3
convertase, can be administered to a cell expressing C5aR and/or
C3aR.
[0045] It will be appreciated that antibodies directed to other
complement components used in the formation of C5, C3, C5a, C3a, C5
convertase, and/or C3 convertase can be used in accordance with the
method described herein to reduce and/or inhibit interactions C5a
and/or C3a with C5aR and C3aR. The antibodies can include, for
example, known Factor B, properdin, and Factor D antibodies that
reduce, block, or inhibit the formation of C5a and/or C3a.
[0046] In some embodiments, the complement antagonist can include
RNA interference (RNAi) polynucleotides to induce knockdown of an
mRNA encoding a complement component. For example, an RNAi
polynucleotide can comprise a siRNA capable of inducing knockdown
of an mRNA encoding a C3, C5, C5aR, or C3aR polypeptide.
[0047] RNAi constructs comprise double stranded RNA that can
specifically block expression of a target gene. "RNA interference"
or "RNAi" is a term initially applied to a phenomenon observed in
plants and worms where double-stranded RNA (dsRNA) blocks gene
expression in a specific and post-transcriptional manner. Without
being bound by theory, RNAi appears to involve mRNA degradation,
however the biochemical mechanisms are currently an active area of
research. Despite some mystery regarding the mechanism of action,
RNAi provides a useful method of inhibiting gene expression in
vitro or in vivo.
[0048] As used herein, the term "dsRNA" refers to siRNA molecules
or other RNA molecules including a double stranded feature and able
to be processed to siRNA in cells, such as hairpin RNA
moieties.
[0049] The term "loss-of-function," as it refers to genes inhibited
by the subject RNAi method, refers to a diminishment in the level
of expression of a gene when compared to the level in the absence
of RNAi constructs.
[0050] As used herein, the phrase "mediates RNAi" refers to
(indicates) the ability to distinguish which RNAs are to be
degraded by the RNAi process, e.g., degradation occurs in a
sequence-specific manner rather than by a sequence-independent
dsRNA response.
[0051] As used herein, the term "RNAi construct" is a generic term
used throughout the specification to include small interfering RNAs
(siRNAs), hairpin RNAs, and other RNA species, which can be cleaved
in vivo to form siRNAs. RNAi constructs herein also include
expression vectors (also referred to as RNAi expression vectors)
capable of giving rise to transcripts which form dsRNAs or hairpin
RNAs in cells, and/or transcripts which can produce siRNAs in
vivo.
[0052] "RNAi expression vector" (also referred to herein as a
"dsRNA-encoding plasmid") refers to replicable nucleic acid
constructs used to express (transcribe) RNA which produces siRNA
moieties in the cell in which the construct is expressed. Such
vectors include a transcriptional unit comprising an assembly of
(I) genetic element(s) having a regulatory role in gene expression,
for example, promoters, operators, or enhancers, operatively linked
to (2) a "coding" sequence which is transcribed to produce a
double-stranded RNA (two RNA moieties that anneal in the cell to
form an siRNA, or a single hairpin RNA which can be processed to an
siRNA), and (3) appropriate transcription initiation and
termination sequences.
[0053] The choice of promoter and other regulatory elements
generally varies according to the intended host cell. In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of "plasmids" which refer to circular double
stranded DNA loops, which, in their vector form are not bound to
the chromosome. In the present specification, "plasmid" and
"vector" are used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors which serve
equivalent functions and which become known in the art subsequently
hereto.
[0054] The RNAi constructs contain a nucleotide sequence that
hybridizes under physiologic conditions of the cell to the
nucleotide sequence of at least a portion of the mRNA transcript
for the gene to be inhibited (i.e., the "target" gene). The
double-stranded RNA need only be sufficiently similar to natural
RNA that it has the ability to mediate RNAi. The number of
tolerated nucleotide mismatches between the target sequence and the
RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in
10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs.
Mismatches in the center of the siRNA duplex are most critical and
may essentially abolish cleavage of the target RNA. In contrast,
nucleotides at the 3' end of the siRNA strand that is complementary
to the target RNA do not significantly contribute to specificity of
the target recognition.
[0055] Sequence identity may be optimized by sequence comparison
and alignment algorithms known in the art (see Gribskov and
Devereux, Sequence Analysis Primer, Stockton Press, 1991, and
references cited therein) and calculating the percent difference
between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript.
[0056] Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques.
Endogenous RNA polymerase of the treated cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vitro. The RNAi constructs may include
modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of a nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general response to dsRNA Likewise, bases may be
modified to block the activity of adenosine deaminase. The RNAi
construct may be produced enzymatically or by partial/total organic
synthesis, any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis.
[0057] Methods of chemically modifying RNA molecules can be adapted
for modifying RNAi constructs (see, for example, Heidenreich et al.
(1997) Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol
Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668;
Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61).
Merely to illustrate, the backbone of an RNAi construct can be
modified with phosphorothioates, phosphoramidate,
phosphodithioates, chimeric methylphosphonate-phosphodie-sters,
peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers
or sugar modifications (e.g., 2'-substituted ribonucleosides,
a-configuration).
[0058] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective inhibition, while lower doses may also be
useful for specific applications. Inhibition is sequence-specific
in that nucleotide sequences corresponding to the duplex region of
the RNA are targeted for genetic inhibition.
[0059] In certain embodiments, the subject RNAi constructs are
"small interfering RNAs" or "siRNAs." These nucleic acids are
around 19-30 nucleotides in length, and even more preferably 21-23
nucleotides in length, e.g., corresponding in length to the
fragments generated by nuclease "dicing" of longer double-stranded
RNAs. The siRNAs are understood to recruit nuclease complexes and
guide the complexes to the target mRNA by pairing to the specific
sequences. As a result, the target mRNA is degraded by the
nucleases in the protein complex. In a particular embodiment, the
21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.
[0060] The siRNA molecules can be obtained using a number of
techniques known to those of skill in the art. For example, the
siRNA can be chemically synthesized or recombinantly produced using
methods known in the art. For example, short sense and antisense
RNA oligomers can be synthesized and annealed to form
double-stranded RNA structures with 2-nucleotide overhangs at each
end (Caplen, et al. (2001) Proc Natl Acad Sci USA, 98:9742-9747;
Elbashir, et al. (2001) EMBO J, 20:6877-88). These double-stranded
siRNA structures can then be directly introduced to cells, either
by passive uptake or a delivery system of choice, such as described
below.
[0061] In certain embodiments, the siRNA constructs can be
generated by processing of longer double-stranded RNAs, for
example, in the presence of the enzyme dicer. In one embodiment,
the Drosophila in vitro system is used. In this embodiment, dsRNA
is combined with a soluble extract derived from Drosophila embryo,
thereby producing a combination. The combination is maintained
under conditions in which the dsRNA is processed to RNA molecules
of about 21 to about 23 nucleotides.
[0062] The siRNA molecules can be purified using a number of
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column
chromatography, can be used to purify the siRNA. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, affinity purification with antibody can be
used to purify siRNAs.
[0063] Examples of a siRNA molecule directed to an mRNA encoding a
C3a, C5a, C5aR, or C3aR polypeptide are known in the art. For
instance, human C3a, C3aR, and C5a siRNA is available from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Additionally, C5aR
siRNA is available from Qiagen, Inc. (Valencia, Calif.). siRNAs
directed to other complement components, including C3 and C5, are
known in the art.
[0064] In other embodiments, the RNAi construct can be in the form
of a long double-stranded RNA. In certain embodiments, the RNAi
construct is at least 25, 50, 100, 200, 300 or 400 bases. In
certain embodiments, the RNAi construct is 400-800 bases in length.
The double-stranded RNAs are digested intracellularly, e.g., to
produce siRNA sequences in the cell. However, use of long
double-stranded RNAs in vivo is not always practical, presumably
because of deleterious effects, which may be caused by the
sequence-independent dsRNA response.
[0065] In certain embodiments, the RNAi construct is in the form of
a hairpin structure (named as hairpin RNA). The hairpin RNAs can be
synthesized exogenously or can be formed by transcribing from RNA
polymerase III promoters in vivo. Examples of making and using such
hairpin RNAs for gene silencing in mammalian cells are described
in, for example, Paddison et al., Genes Dev, 2002, 16:948-58;
McCaffrey et al., Nature, 2002, 418:38-9; McManus et al., RNA,
2002, 8:842-50; Yu et al., Proc Natl Acad Sci USA, 2002,
99:6047-52). Such hairpin RNAs are engineered in cells or in an
animal to ensure continuous and stable suppression of a desired
gene. It is known in the art that siRNAs can be produced by
processing a hairpin RNA in the cell.
[0066] In yet other embodiments, a plasmid can be used to deliver
the double-stranded RNA, e.g., as a transcriptional product. In
such embodiments, the plasmid is designed to include a "coding
sequence" for each of the sense and antisense strands of the RNAi
construct. The coding sequences can be the same sequence, e.g.,
flanked by inverted promoters, or can be two separate sequences
each under transcriptional control of separate promoters. After the
coding sequence is transcribed, the complementary RNA transcripts
base-pair to form the double-stranded RNA.
[0067] PCT application WO01/77350 describes an exemplary vector for
bi-directional transcription of a transgene to yield both sense and
antisense RNA transcripts of the same transgene in a eukaryotic
cell. Accordingly, in certain embodiments, the a recombinant vector
can have the following unique characteristics: it comprises a viral
replicon having two overlapping transcription units arranged in an
opposing orientation and flanking a transgene for an RNAi construct
of interest, wherein the two overlapping transcription units yield
both sense and antisense RNA transcripts from the same transgene
fragment in a host cell.
[0068] RNAi constructs can comprise either long stretches of double
stranded RNA identical or substantially identical to the target
nucleic acid sequence or short stretches of double stranded RNA
identical to substantially identical to only a region of the target
nucleic acid sequence. Exemplary methods of making and delivering
either long or short RNAi constructs can be found, for example, in
WO01/68836 and WO01/75164.
[0069] Examples RNAi constructs that specifically recognize a
particular gene or a particular family of genes, can be selected
using methodology outlined in detail above with respect to the
selection of antisense oligonucleotide. Similarly, methods of
delivery RNAi constructs include the methods for delivery antisense
oligonucleotides outlined in detail above.
[0070] In some embodiments, a lentiviral vector can be used for the
long-term expression of a siRNA, such as a short-hairpin RNA
(shRNA), to knockdown expression of C5, C3, C5aR, and/or C3aR in
hematopoietic stem cells or bone marrow cells. Although there have
been some safety concerns about the use of lentiviral vectors for
gene therapy, self-inactivating lentiviral vectors are considered
good candidates for gene therapy as they readily transfect
mammalian cells.
[0071] It will be appreciated that RNAi constructs directed to
other complement components used in the formation of C5, C3, C5a,
C3a, C5 convertase, and/or C3 convertase components can be used in
accordance with the method described herein to reduce and/or
inhibit interactions C5a and/or C3a with C5aR and C3aR in
hematopoietic stem cells or bone marrow cells.
[0072] The RNAi constructs can include, for example, known Factor
B, properdin, and Factor D siRNA that reduce expression of Factor
B, properdin, and Factor D.
[0073] Moreover, it will be appreciated that other antibodies,
small molecules, and/or peptides that reduce or inhibit the
formation of C5, C3, C5a, C3a, C5 convertase, and/or C3 convertase
and/or that reduce or inhibit interactions C5a and/or C3a with C5aR
and C3aR in hematopoietic progenitor cells or osteoclast progenitor
cells can be used as a complement antagonist in accordance with the
method described herein. These other complement antagonists can be
administered to the hematopoietic progenitor cells or osteoclast
progenitor cells at amount to inhibit osteoclast
differentiation.
[0074] Many complement antagonists are already in clinical trials
for various human diseases, and one of them, an anti-C5 monoclonal
antibody, has been approved by the FDA for the treatment of
paroxysmal nocturnal hemoglobinuria (PNH), in which patients'
erythrocytes are lysed by activated complement, leading to
hemogloginuria and anemia. The C5aR antagonist JPE-1375 is a
hexameric linear peptidomimetic molecule (M.W. 955) which has been
shown to be effective in ameliorating disease symptoms in many
mouse models where C5aR is integrally involved in the pathogenesis.
JPE-1375 is reportedly more potent than another C5aR antagonist,
PMX205, which has shown promising results in treating murine
disease models such as amyotrophic lateral sclerosis and
Alzheimer's disease. The Examples below show that the JPE-1375
antagonist inhibits mouse and human OC differentiation in vitro.
The C3aR antagonist SB290157 is a synthesized small molecule which
is commercially available from several companies including EMD
Chemicals (Gibbstown, N.J.). It has been successfully used to treat
murine models of neutrophilia, intestinal ischaemia/reperfusion
injury and lupus nephritis. The Examples below demonstrate that
these C3aR and C5aR antagonists significantly reduced TRAP.sup.+
cells in human-derived bone marrow cells in vitro (FIG. 2). The
C3aRA showed a significant decrease in both mononuclear and
multi-nuclear TRAP.sup.+ cells while C5aRA showed a decrease in
both cell types, but only the mono-nuclear cells were at the level
of statistical significance.
[0075] Examples of other complement antagonists include C5aR
antagonists, such as AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg,
prednisolone, and infliximab (Woodruff et al,. The Journal of
Immunology, 2003, 171: 5514-5520), hexapeptide MeFKPdChaWr (March
et al., Mol Pharmacol 65:868-879, 2004), PMX53, and
N-[4-dimethylaminophenyl)methyl]-N-(4-isopropylphenyl)-7-methoxy-1,2,3,4--
tetrahydronaphthalen-1-carboxamide hydrochloride (W-54011)
(Sumichika et al., J. Biol. Chem., Vol. 277, Issue 51, 49403-49407,
Dec. 20, 2002), and a C3aR antagonist, such as SB 290157 (Ratajczak
et al., Blood, 15 Mar. 2004, Vol. 103, No. 6, pp. 2071-2078).
[0076] In other embodiments, the agent that inhibits C3aR and/or
C5aR signaling in the hematopoietic progenitor cells or osteoclast
progenitor cells, can include an IL-6/STAT3 signaling pathway
antagonist that substantially decreases or inhibits the expression
and/or functional activity of a component of the IL-6/STAT3
signaling pathway in the cell. The functional activity of the
IL-6/STAT3 signaling pathway can be suppressed, inhibited, and/or
blocked in several ways including: direct inhibition of the
activity of IL-6 and/or STAT3 (e.g., by using neutralizing
antibodies, small molecules or peptidomimetics, dominant negative
polypeptides); inhibition of genes that express IL-6 and/or STAT-3
(e.g., by blocking the expression or activity of the genes and/or
proteins); activation of genes and/or proteins that inhibit one or
more of the functional activity of IL-6 and/or STAT3 (e.g., by
increasing the expression or activity of the genes and/or
proteins); inhibition of genes and/or proteins that are downstream
mediators of the iNOS expression (e.g., by blocking the expression
and/or activity of the mediator genes and/or proteins);
introduction of genes and/or proteins that negatively regulate one
or more of functional activity of IL-6 and/or STAT3 (e.g., by using
recombinant gene expression vectors, recombinant viral vectors or
recombinant polypeptides); or gene replacement with, for instance,
a hypomorphic mutant of STAT-3 (e.g., by homologous recombination,
overexpression using recombinant gene expression or viral vectors,
or mutagenesis).
[0077] In an embodiment of the application, the IL-6/STAT3
signaling pathway antagonist is an IL-6 antagonist. In some
aspects, the IL-6 antagonist can include a humanized IL-6
receptor-inhibiting monoclonal antibody. In certain aspects, the
IL-6 antagonist is the product tocilizumab (a descriptive name sold
under the trademark ACTEMRA by Roche, Switzerland). In other
aspects, the IL-6 antagonist can include a vaccine that when
administered to a subject generates IL-6 antibodies in the subject.
An example of such a vaccine is disclosed in Fosergau et al.
Journal of Endocrinology (2010) 204, 265-273.
[0078] In another embodiment, the IL-6/STAT3 signaling pathway
antagonist is a tyrosine kinase inhibitor. Exemplary tyrosine
kinase inhibitors for use in the present invention include but are
not limited to tyrphostins, in particular AG-490, and inhibitors of
Jak, Src, and BCR-Abl tyrosine kinases. Other tyrphostins suitable
for use herein include, but are not limited to AG17, AG213
(RGS0864), AG18, AG82, AG494, AG825, AG879, AG1112, AG1296, AG1478,
AG126, RG13022, RG14620, AG555, and related compounds. In certain
aspects, a BCR-Abl tyrosine kinase inhibitor for use herein can
include the product imatinib mesilate (a descriptive name sold
under the trademark GLEEVEC.RTM. by Novartis, Switzerland).
[0079] In a further embodiment, the IL-6/STAT3 signaling pathway
antagonist is an HMG CoA reductase inhibitor
(3-hydroxymethylglutaryl coenzyme A reductase inhibitors) (e.g.,
statin). HMG-CoA (3-hydroxy methylglutaryl coenzyme A) reductase is
the microsomal enzyme that catalyzes the rate limiting reaction in
cholesterol biosynthesis (HMG-CoA Mevalonate.
[0080] Statins that can be used for administration, or
co-administration with other agents described herein include, but
are not limited to, simvastatin (U.S. Pat. No. 4,444,784),
mevistatin, lovastatin (U.S. Pat. No. 4,231,938), pravastatin
sodium (U.S. Pat. No. 4,346,227), fluvastatin (U.S. Pat. No.
4,739,073), atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin,
and numerous others described in U.S. Pat. No. 5,622,985, U.S. Pat.
No. 5,135,935, U.S. Pat. No. 5,356,896, U.S. Pat. No. 4,920,109,
U.S. Pat. No. 5,286,895, U.S. Pat. No. 5,262,435, U.S. Pat. No.
5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat. No. 5,283,256, U.S.
Pat. No. 5,256,689, U.S. Pat. No. 5,182,298, U.S. Pat. No.
5,369,125, U.S. Pat. No. 5,302,604, U.S. Pat. No. 5,166,171, U.S.
Pat. No. 5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No.
5,196,440, U.S. Pat. No. 5,091,386, U.S. Pat. No. 5,091,378, U.S.
Pat. No. 4,904,646, U.S. Pat. No. 5,385,932, U.S. Pat. No.
5,250,435, U.S. Pat. No. 5,132,312, U.S. Pat. No. 5,130,306, U.S.
Pat. No. 5,116,870, U.S. Pat. No. 5,112,857, U.S. Pat. No.
5,102,911, U.S. Pat. No. 5,098,931, U.S. Pat. No. 5,081,136, U.S.
Pat. No. 5,025,000, U.S. Pat. No. 5,021,453, U.S. Pat. No.
5,017,716, U.S. Pat. No. 5,001,144, U.S. Pat. No. 5,001,128, U.S.
Pat. No. 4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat. No.
4,994,494, U.S. Pat. No. 4,992,429, U.S. Pat. No. 4,970,231, U.S.
Pat. No. 4,968,693, U.S. Pat. No. 4,963,538, U.S. Pat. No.
4,957,940, U.S. Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S.
Pat. No. 4,946,860 U.S. Pat. No. 4,940,800, U.S. Pat. No.
4,940,727, U.S. Pat. No. 4,939,143, U.S. Pat. No. 4,929,620, U.S.
Pat. No. 4,923,861, U.S. Pat. No. 4,906,657, U.S. Pat. No.
4,906,624 and U.S. Pat. No. 4,897,402, the disclosures of which
patents are incorporated herein by reference.
[0081] In yet another embodiment, the IL-6/STAT3 signaling pathway
antagonist can be a STAT3 inhibitor. Examples of STAT3 inhibitors
are described in U.S. Patent Application No. 2010/0041685 and can
include
4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoic
acid;
4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}be-
nzoic acid;
4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoic
acid;
3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-
-ethoxyphenoxy}methyl)benzoic acid; methyl
4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}met-
-hyl)benzoate;
4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidiny-
-lidene)methyl]-2-furyl}benzoic acid; a functionally active
derivative thereof and a mixture thereof. Other examples of STAT3
inhibitors are described in WO 2010/118309 and in G. Zinzalla et
al. Bioorg. Med. Chem. Lett. 20 (2010)7029-7032.
[0082] The at least one agent that inhibits C3aR and/or C5aR
signaling can be administered to the the hematopoietic progenitor
cells or osteoclast progenitor cells in vivo or in vitro to inhibit
osteoclast differentiation of the cells. The cells can be derived
from a human subject, from a known cell line, or from some other
source.
[0083] In some embodiments, the agent that inhibits at least one of
C3aR and/or C5aR signaling in the hematopoietic progenitor cells or
osteoclast progenitor cells may be used in a method for enhancing
bone formation (i.e., increasing the amount of new bone that is
laid down) and inhibiting bone resorption (i.e., reducing the
amount of bone that is dissolved) simultaneously in a subject in
need thereof by administering to the subject an agent described
herein in an amount effective to enhance bone formation and inhibit
bone resorption simultaneously in said subject. Nonlimiting
examples of subjects for whom such treatment would be indicated
and/or beneficial include women (e.g., postmenopausal;
premenopausal) with osteoporosis or low bone mass, men with
osteoporosis or low bone mass, subjects with a healing fracture,
subjects undergoing prolonged immobilization, subjects who have
been or are immobilized for a prolonged period, subjects likely to
undergo or experience prolonged immobilization, subjects with
estrogen deficiency, etc., as would be known in the art.
[0084] Also provided herein is a method for inducing deposition and
maturation of bone in a subject in need thereof (e.g., a subject
having a compromised bone condition) by administering to the
subject an agent described herein in an amount effective to induce
deposition and maturation of bone in the subject. In some
embodiments, a compromised bone condition is at a targeted site of
the subject. The site may be an intervertebral space, a facet
joint, a site of a bone fracture, bones of the mouth, chin and jaw,
or an implant site.
[0085] Also provided herein is a method for improving bone marrow
reconstitution in a subject in need thereof by administering to the
subject an agent that inhibits at least one of C3aR and/or C5aR
signaling in an amount effective to improve bone marrow
reconstitution (i.e., restoring (e.g., partially or fully) of bone
marrow cells in a subject, which can be, for example, a subject
having chemotherapy, radiation or other treatments that deplete
bone marrow cells. For example, a subject undergoing chemotherapy
with or without radiation would benefit from more rapid restoration
of cells in the bone marrow in order to prevent opportunistic
infections. A subject of these methods can also be a subject having
or suspected of having a hematologic disorder (e.g., aplastic
anemia; myelodysplasia) that depletes bone marrow cells. Such an
improvement or enhancement or increase in bone marrow
reconstitution is in comparison to a subject to whom the agent that
inhibits at least one of C3aR and/or C5aR signaling has not been
administered.
[0086] In some embodiments, the methods described herein can be
employed in methods of ex vivo expansion of stem cells, such as
hematopoietic stem cells, carried out according to protocols known
in the art. Thus, a method of expanding stem cells ex vivo,
comprising contacting the agent that inhibits at least one of C3aR
and/or C5aR signaling with stem cells from a subject, wherein said
stem cells are maintained under conditions whereby they are
reintroduced into the subject.
[0087] For example in some ex vivo embodiments, the stem cells are
obtained from a subject, e.g., a human, e.g., from peripheral
blood, umbilical cord blood, or bone marrow, and the stem cells are
contacted with the agent that inhibits at least one of C3aR and/or
C5aR signaling outside the body of the subject. Ex vivo embodiments
include obtaining stem cells, such as hematopoietic stem cells,
from a subject and culturing the cells for a period of time prior
to use (e.g., for transplantation). In some embodiments, after
contact with the agent that inhibits at least one of C3aR and/or
C5aR signaling, the cells are delivered to a subject, e.g., the
same subject from which the cells were isolated (autologous
donation) or a different subject (non-autologous (e.g., syngeneic
or allogeneic) donation).
[0088] Nonlimiting examples of a subject for whom these methods
would be indicated or beneficial include a subject having or who
has had chemotherapy, a subject having or who has had radiation, a
subject having aplastic anemia, a subject having myelodysplasia,
and any combination thereof.
[0089] Administration of the agent that inhibits at least one of
C3aR and/or C5aR signaling in the hematopoietic progenitor cells or
osteoclast progenitor cells can be by any suitable route, including
intrathecal injection, subcutaneous, cutaneous, oral, intravenous,
intraperitoneal, intramuscular injection, in an implant, in a
matrix, in a gel, or any combination thereof.
[0090] A bone condition that can be treated according to the
methods described herein may be one or more of broken bones, bone
defects, bone transplant, bone grafts, bone cancer, joint
replacements, joint repair, fusion, facet repair, bone
degeneration, dental implants and repair, bone marrow deficits and
other conditions associated with bone and boney tissue. Bone
defects may be a gap, deformation and/or a nonunion fracture in a
bone.
[0091] Bone degeneration may be due to osteopenia or osteoporosis
(e.g., the patient is afflicted with geriatric or senile
osteoporosis, with post-menopausal osteoporosis, etc.), or due to
dwarfism.
[0092] Joint replacements that may be treated include vertebral,
knee, hip, tarsal, phalangeal, elbow, ankle and/or other
articulating joints or replacements thereof. Joint repairs include,
but are not limited to, vertebral, knee, hip, tarsal, phalangeal,
elbow, ankle, and sacroiliac joint repairs.
[0093] In designing appropriate doses of the agents that inhibit at
least one of C3aR and/or C5aR signaling for the treatment of bone
conditions, one may readily extrapolate from the knowledge in the
literature in order to arrive at appropriate doses for clinical
administration. To achieve a conversion from animal to human doses,
one would account for the mass of the agents administered per unit
mass of the experimental animal and, preferably, account for the
differences in the body surface area (m2) between the experimental
animal and the human patient. All such calculations are well known
and routine to those of ordinary skill in the art.
[0094] It will be understood that lower doses may be more
appropriate in combination with other agents, and that high doses
can still be tolerated.
[0095] In some embodiments, the agent that inhibits at least one of
C3aR and/or C5aR signaling can be administered directly to or about
the periphery of the bone condition being treated to inhibit
osteoclast differentiation. In one aspect of the invention, the
agent the agent that inhibits at least one of C3aR and/or C5aR
signaling can be delivered to or about the periphery of the site of
the bone condition being treated by administering the agent neat or
in a pharmaceutical composition to or about the bone. The
pharmaceutical composition can provide localized release of the
agent to the bone marrow or bone marrow cells being treated.
Pharmaceutical compositions will generally include an amount of the
agent the agent that inhibits at least one of C3aR and/or C5aR
signaling admixed with an acceptable pharmaceutical diluent or
excipient, such as a sterile aqueous solution, to give a range of
final concentrations, depending on the intended use. The techniques
of preparation are generally well known in the art as exemplified
by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing
Company, 1980, incorporated herein by reference. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards.
[0096] The pharmaceutical composition can be in a unit dosage
injectable form (e.g., solution, suspension, and/or emulsion).
Examples of pharmaceutical formulations suitable for injection
include sterile aqueous solutions or dispersions and sterile
powders for reconstitution into sterile injectable solutions or
dispersions. The carrier can be a solvent or dispersing medium
containing, for example, water, ethanol, polyol (e.g., glycerol,
propylene glycol, liquid polyethylene glycol, and the like),
suitable mixtures thereof and vegetable oils.
[0097] Proper fluidity can be maintained, for example, by the use
of a coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions
[0098] Additionally, various additives which enhance the stability,
sterility, and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating agents, and
buffers, can be added. Prevention of the action of microorganisms
can be ensured by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. In many cases, it will be desirable to include isotonic
agents, for example, sugars, sodium chloride, and the like.
Prolonged absorption of the injectable pharmaceutical form can be
brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
would have to be compatible with the compounds.
[0099] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0100] Pharmaceutical "slow release" capsules or "sustained
release" compositions or preparations may be used and are generally
applicable. Slow release formulations are generally designed to
give a constant drug level over an extended period and may be used
to deliver the agent. The slow release formulations are typically
implanted in the vicinity of the bone condition, for example, at
the site of the bone condition (e.g., bone marrow).
[0101] Examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
agent that inhibits at least one of C3aR and/or C5aR signaling,
which matrices are in the form of shaped articles, e.g., films or
microcapsule. Examples of sustained-release matrices include
polyesters; hydrogels, for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol);
polylactides, e.g., U.S. Pat. No. 3,773,919; copolymers of
L-glutamic acid and .gamma. ethyl-L-glutamate; non-degradable
ethylene-vinyl acetate; degradable lactic acid-glycolic acid
copolymers, such as the LUPRON DEPOT (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate); and poly-D-(-)-3-hydroxybutyric acid.
[0102] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated agent remain in the body for a long time, and may
denature or aggregate as a result of exposure to moisture at
37.degree. C., thus reducing biological activity and/or changing
immunogenicity. Rational strategies are available for stabilization
depending on the mechanism involved. For example, if the
aggregation mechanism involves intermolecular S--S bond formation
through thio-disulfide interchange, stabilization is achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives,
developing specific polymer matrix compositions, and the like.
[0103] In certain embodiments, liposomes and/or nanoparticles may
also be employed with the agent that inhibits at least one of C3aR
and/or C5aR signaling. The formation and use of liposomes is
generally known to those of skill in the art, as summarized
below.
[0104] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0105] Phospholipids can form a variety of structures other than
liposomes when dispersed in water, depending on the molar ratio of
lipid to water. At low ratios, the liposome is the preferred
structure. The physical characteristics of liposomes depend on pH,
ionic strength and the presence of divalent cations. Liposomes can
show low permeability to ionic and polar substances, but at
elevated temperatures undergo a phase transition which markedly
alters their permeability. The phase transition involves a change
from a closely packed, ordered structure, known as the gel state,
to a loosely packed, less-ordered structure, known as the fluid
state. This occurs at a characteristic phase-transition temperature
and results in an increase in permeability to ions, sugars and
drugs.
[0106] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one may operate at the same time.
[0107] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and such particles may be are easily made.
[0108] In another aspect, the agent that inhibits at least one of
C3aR and/or C5aR signaling can be administered directly to or about
the periphery of the bone condition being treated by introducing an
agent into target cells, such as bone marrow cells or the
hematopoietic progenitor cells or osteoclast progenitor cells, that
causes, increases, and/or upregulates expression of at least one of
C3, C5, C3a, C5a, a C3aR agonist, or C5aR agonist in or about the
periphery of the bone marrow cells or the hematopoietic progenitor
cells or osteoclast progenitor cells. The at least one of at least
one of C3, C5, C3a, C5a, a C3aR agonist, or C5aR agonist expressed
in or about the periphery of the bone condition can be an
expression product of a genetically modified cell. The target cells
can include cells within or about the periphery of the bone
condition or ex vivo cells that are biocompatible with the bone
condition being treated. The biocompatible cells can also include
autologous cells that are harvested from the subject being treated
and/or biocompatible allogeneic or syngeneic cells, such as
autologous, allogeneic, or syngeneic stem cells (e.g., mesenchymal
stem cells), progenitor cells (e.g., multipotent adult progenitor
cells) and/or other cells that are further differentiated and are
biocompatible with the bone condition being treated.
[0109] In other embodiments of the application, osteoblast
differentiaion can be promoted or stimulated by administering to
stromal cells, mesenchymal stem cell (MSC), MAPC, induced
pluripotent stem cell (IPC), or osteoblast progenitor cells an
agent that promotes or stimulates C3aR and/or C5aR signaling of the
cells. The agent can be selected from the group consisting of C3,
C5, C3a, C5a, a C3aR agonist, a C5aR agonist, a DAF antagonist, or
combination thereof. Promotion or stimulation of C3aR and/or C5aR
activation in to a stromal cells, MSCs, MAPCs, IPCs, and osteoblast
progenitor cells can induce osteoblast differentiation and promote
bone regeneration.
[0110] The MSCs can include the formative pluripotent blast or
embryonic cells that differentiate into the specific types of
connective tissues, (i.e., the tissue of the body that support
specialized elements, particularly including adipose, osseous,
cartilaginous, elastic, muscular, and fibrous connective tissues
depending on various in vivo or in vitro environmental influences).
These cells are present in bone marrow, blood, dermis, and
periosteum and can be isolated and purified using various well
known methods, such as those methods disclosed in U.S. Pat. No.
5,197,985 to Caplan and Haynesworth, herein incorporated by
reference, as well as other numerous literature references.
[0111] The MAPCs in accordance can include adult progenitor or stem
cells that are capable of differentiating into cells types beyond
those of the tissues in which they normally reside (i.e., exhibit
plasticity). MAPCs express the ES cell-specific transcription
factor Oct3/4 (POU5F1) but not Nanog. FACS analysis demonstrates
that MAPCs do not express class I and II MHC, CD34, CD44, CD45 and
are CD105 (also endoglin, or SH2) negative. Hence, MAPCs differ
from classical MSCs that are Oct4 low/negative but CD44 and MHC
class I positive and differentiate essentially into mesodermal
cells but not cells of endoderm and ectoderm. Compared with
mesoangioblasts, MAPCs do not express CD34 and Flk1 (KDR), and have
a broader differentiation ability. MAPCs differ from hematopoietic
stem cells (HSC) in that MAPCs do not express CD45, CD34, and cKit,
but like HSC, MAPC express Thy1, AC133 (human MAPC) and Sca1
(mouse) albeit at low levels. In the mouse, MAPC express low levels
of stage specific embryonic antigen (SSEA)-1, and express low
levels of the transcription factors Oct4 and Rex1, known to be
important for maintaining embryonic stem (ES) cells
undifferentiated and to be down-regulated when ES cells undergo
somatic cell commitment and differentiation.
[0112] MAPCs can be cultured from mouse brain and mouse muscle. Of
note, the differentiation potential and expressed gene profile of
MAPCs derived from the different tissues appears to be highly
similar. Unlike most adult somatic stem cells, MAPC proliferate
without obvious signs of senescence, and have active telomerase.
Human, mouse and rat MAPCs have been shown to be successfully
differentiated into typical mesenchymal lineage cells, including
osteoblasts, chondroblasts, adipocytes and skeletal myoblasts. In
addition, human, mouse and rat MAPCs can be induced to
differentiate into cells with morphological, phenotypic and
functional characteristics of endothelial cells, and morphological,
phenotypic and functional characteristics of hepatocytes.
[0113] An enriched population of iPCs can formed as described by
known methods described in, for example, Mali P, Ye Z, Hommond H H,
Yu X, Lin J, Chen G, Zou J, Cheng L. Stem Cells. 2008 August;
26(8):1; Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K.
Science. 2008 Nov. 7; 322(5903):945-9; and Park I H, Lerou P H,
Zhao R, Huo H, Daley G Q. Nat Protoc. 2008; 3(7):1180-6.
[0114] In some embodiments, an enriched population of MSCs can be
prepared by isolating bone marrow cells from the femurs of a
subject. Cells can then be separated by Percoll density gradient.
The cells can be centrifuged and washed with PBS supplemented with
penicillin, and streptomycin (Invitrogen, Carlsbad, Calif.). The
cells can then be re-suspended and plated in DMEM-LG (GIBCO,
Invitrogen, Carlsbad, Calif.) with 10% FBS and 1% antibiotic and
antimycotic (GIBCO, Invitrogen, Carlsbad, Calif.) and maintained at
37.degree. C. Non-adherent cells can then be removed by replacing
the medium after 3 days. At this point, adherent cells can then be
detached by incubation with 0.05% trypsin and 2 mM EDTA
(Invitrogen, Carlsbad, Calif.) for 5 minutes and subsequently
re-plated.
[0115] To prevent non-specific selection of monocytes and
macrophages, MSCs Cultures can be immunodepleted of CD45+, CD34+
cells by negative selection using primary PE-conjugated mouse
anti-rat CD45 (BD Biosciences, San Diego, Calif.) and CD34
antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.)
using the EasySep PE selection kit according to the manufacturer's
instruction (Stem Cell technologies). The MSCs can then tested by
FACS and were positive for CD90, CD29 and negative for CD34 and
CD45. The multipotentiality of resulting cells can be subsequently
verified with the use of in vitro assays to differentiate MSCs into
osteogenic (alkaline phosphatase activity), adipogenic (oil red O
staining) and chondrogenic (Alcian Blue) lineages according to
published protocols.
[0116] In some embodiments, the stromal cells, MSCs, MAPCs, IPCs,
or osteoblast progenitor cells treated with an agent that promotes
or stimulates C3aR and/or C5aR signaling of the cells can be
provided in and/or on a substrate, solid support, and/or wound
dressing for cells to a muscloskeletal injury site. As used herein,
the term "substrate," or "solid support" and "wound dressing" refer
broadly to any substrate when prepared for, and applied to, a wound
for protection, absorbance, drainage, etc. The substrate one of the
numerous types of substrates and/or backings that are commercially
available, including films (e.g., polyurethane films),
hydrocolloids (hydrophilic colloidal particles bound to
polyurethane foam), hydrogels (cross-linked polymers containing
about at least 60% water), foams (hydrophilic or hydrophobic),
calcium alginates (non-woven composites of fibers from calcium
alginate), and cellophane (cellulose with a plasticizer).
[0117] In one example, the substrate can be a bioresorbable implant
that includes a polymeric matrix and the stromal cells, MSCs,
MAPCs, IPCs, or osteoblast progenitor cells treated with an agent
that promotes or stimulates C3aR and/or C5aR signaling of the cells
dispersed in the matrix. The polymeric matrix may be in the form of
a membrane, sponge, gel, or any other desirable configuration. The
polymeric matrix can be formed from biodegradable polymer. It will
be appreciated, however, that the polymeric matrix may additionally
comprise an inorganic or organic composite. The polymeric matrix
can comprise anyone or combination of known materials including,
for example, chitosan, poly(ethylene oxide), poly (lactic acid),
poly(acrylic acid), poly(vinyl alcohol), poly(urethane),
poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone) (PVP),
poly(methacrylic acid), poly(p-styrene carboxylic acid),
poly(p-styrenesulfonic acid), poly(vinylsulfonicacid),
poly(ethyleneimine), poly(vinylamine), poly(anhydride),
poly(Llysine), poly(L-glutamic acid), poly(gamma-glutamic acid),
poly(carprolactone), polylactide, poly(ethylene), poly(propylene),
poly(glycolide), poly(lactide-co-glycolide), poly(amide),
poly(hydroxylacid), poly(sulfone), poly(amine), poly(saccharide),
poly(HEMA), poly(anhydride), collagen, gelatin, glycosaminoglycans
(GAG), poly(hyaluronic acid), poly(sodium alginate), alginate,
hyaluronan, agarose, polyhydroxybutyrate (PHB), and the like.
[0118] It will be appreciated that one having ordinary skill in the
art may create a polymeric matrix of any desirable configuration,
structure, or density. By varying polymer concentration, solvent
concentration, heating temperature, reaction time, and other
parameters, for example, one having ordinary skill in the art can
create a polymeric matrix with any desired physical
characteristic(s). For example, the polymeric matrix may be formed
into a sponge-like structure of various densities. The polymeric
matrix may also be formed into a membrane or sheet, which could
then be wrapped around or otherwise shaped to a wound. The
polymeric matrix may also be configured as a gel, mesh, plate,
screw, plug, or rod. Any conceivable shape or form of the polymeric
matrix is within the scope of the present invention. In an example
of the present invention, the polymeric matrix can comprise an
osteoconductive matrix.
[0119] In other aspects, the polymer matrix seeded the stromal
cells, MSCs, MAPCs, IPCs, or osteoblast progenitor cells treated
with an agent that promotes or stimulates C3aR and/or C5aR
signaling of the cells can comprise bone graft or bone graft
substitute. In some embodiments, an osteoconductive matrix can be
used to support the mammalian cells and include collagen fibers
coated with hydroyapatite. In other aspects, the osteoconductive
matrix is saturated with the population of the cells. In one
particular example, the stromal cells, MSCs, MAPCs, IPCs, or
osteoblast progenitor cells treated with an agent that promotes or
stimulates C3aR and/or C5aR signaling of the cells is delivered to
the musculoskeletal injury or to an area proximate the skeletal
injury. The seeded osteoconductive matrix may then be implanted
adjacent to a bone fracture site for the treatment of a skeletal
injury in a subject.
[0120] In another aspect of the application, a therapeutic
composition can include a bone graft, such as an autograft, that
seeded with the stromal cells, MSCs, MAPCs, IPCs, or osteoblast
progenitor cells treated with an agent that promotes or stimulates
C3aR and/or C5aR signaling of the cells. Bone grafting is commonly
used to repair fractured bones. While grafting can include
artificial bone replacement, autografting is often the most
successful type of grafting available. Bones tend to more readily
adhere to one another when a subject's own bone is used. The most
common donor area is the iliac crest, which is located in the
subject's pelvis.
[0121] A bone graft may also include an allograft bone graft.
Typically, an allograft bone graft is bone obtained from cadavers.
An allograft may be sterilized and/or fresh frozen or freeze-dried
prior to grafting. An allograft may also be used as a bone graft
supplement (to the subject's own bone) in subjects.
[0122] In a further aspect, the stromal cells, MSCs, MAPCs, IPCs,
or osteoblast progenitor cells treated with an agent that promotes
or stimulates C3aR and/or C5aR signaling of the cells can be
provided in or on a surface of a medical device used to treat a
musculoskeletal injury. The medical device can comprise any
instrument, implement, machine, contrivance, implant, or other
similar or related article, including a component or part, or
accessory which is recognized in the official U.S. National
Formulary, the U.S. Pharmacopoeia, or any supplement thereof; is
intended for use in the diagnosis of disease or other conditions,
or in the cure, mitigation, treatment, or prevention of disease, in
humans or in other animals; or, is intended to affect the structure
or any function of the body of humans or other animals, and which
does not achieve any of its primary intended purposes through
chemical action within or on the body of man or other animals, and
which is not dependent upon being metabolized for the achievement
of any of its primary intended purposes.
[0123] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLE 1
[0124] In this Example, using knockout mice deficient of C3, factor
D, C3aR, and/or C5aR, we show the role of complement in
1.25(OH).sub.2 vitamin D.sub.3-induced OC differentiation. We found
that BM cells from C3.sup.-/- mice generated significantly
decreased numbers of OC after stimulation. In accordance with these
results, C3.sup.-/- BM cells exhibited reduced receptor activator
of nuclear factor kB ligand (RANKL)/osteoprotegerin (OPG)
expression ratios and produced decreased amounts of macrophage
colonystimulating factor (M-CSF) and IL-6 during OC
differentiation. More importantly, we also found that in addition
to C3, BM cells locally produce factor B, factor D, and C5 after
1.25(OH).sub.2 vitamin D.sub.3 stimulation, and that the
alternative pathway of complement activation is required to
activate C3 for efficient OC differentiation. In addition to the C3
receptors reported before, our data show that C3aR/C5aR are also
integrally involved in OC differentiation, and their regulatory
roles are mediated, at least in part, through modulating local IL-6
production.
Methods
Genetically Engineered Mice
[0125] Wild-type (WT) C57BL/6 and C3.sup.-/- mice were ordered from
The Jackson Laboratory. Factor D.sup.-/- mice were gifts from Dr
Yuanyuan Ma (University of Alabama at Birmingham), and factor
B.sup.-/- mice were kindly provided by Dr Michael Holers
(University of Colorado at Denver). C3aR.sup.-/- and C5aR.sup.-/-
mice were generously provided by Dr Craig Gerard (Harvard
University), and C3aR.sup.-/-C5aR.sup.-/- mice were identified by
polymerase chain reaction (PCR) genotyping after crossing the
C3aR.sup.-/- with C5aR.sup.-/- mice. All mice are on the C57BL/6
background, and all animal studies were performed under an approved
protocol in accordance with the guidelines of the Institutional
Animal Care and Use Committee of Case Western Reserve
University.
BM-Cell Cultures
[0126] Human BM cells from healthy donors were obtained from the
Hematopoietic Stem Cell Core Facility of Case Western Reserve
University. Murine BM cells were isolated from 8- to 12-week-old
female mouse femurs and tibias, washed, and collected in 15-mL
tubes in .alpha.-modified Eagle medium (MEM) containing 10% fetal
bovine serum (FBS) that was heat-inactivated to eliminate
complement activity. For OC differentiation, 2.times.10.sup.6 BM
cells were cultured in complete .alpha.-MEM medium in wells of a
24-well plate together with 1.times.10.sup.-8M 1.25(OH).sub.2
vitamin D.sub.3 (Cayman Chemical) as described before. Cultures
were fed every 3 days with fresh media. For IL-6 or C5aR antagonist
(C5aRA)/C3aR antagonist (C3aRA) supplementation experiments, 20
ng/mL IL-6 (R&D Systems) or 50 .mu.M of each antagonist or both
(C5aRA:JPE-1375; custom synthesized by Anaspec; C3aRA:SB290157;
purchased from BMD Chemicals) were included in the medium. For C3a
and/or C5a supplementation experiments, 50 ng/mL purified C3a (BD
Biosciences) or C5a (Cell Sciences) or both were added daily into
WT and C3.sup.-/- BM-cell cultures. To neutralize IL-6 in samples
treated with C3a/C5a, 10 ng/mL rat anti-IL-6 mAb (Clone 6B4 IGH 54;
eBioscience) were added every 2 days. At day 12, differentiated OCs
were identified by conventional tartrate-resistant acid phosphatase
(TRAP) staining using a kit (Sigma-Aldrich) and following the
protocol provided by the manufacturer. Total numbers of
TRAP-positive cells in each well were counted under a microscope,
and cells containing 3 or more nuclei were categorized as
multinucleated.
Primary Calvarial Osteoblast and Splenocyte Cocultures
[0127] Primary calvarial osteoblasts (OBs) were isolated from
newborn WT and C3.sup.-/- mice, following protocols. In brief,
dissected calvariae from 2- to 3-day-old WT and C3.sup.-/- mice
were digested with 1 mg/mL collagenase D (Roche) and 0.05% trypsin
(Invitrogen) in Hanks buffered salt solution. Cells from second and
third digestions were pooled and grown in .alpha.-MEM with
antibiotics and 10% FBS. At confluence, cells were trypsinized and
counted for coculture experiments. To set up coculture experiments,
2.times.10.sup.4 primary calvarial OBs from WT or C3.sup.-/- mice
were cultured with 2.times.10.sup.6 splenocytes from WT and
C3.sup.-/- mice together with 1.times.10.sup.-8 1.25(OH).sub.2
vitamin D.sub.3. The resultant TRAP-positive cells were counted
after 12 days of coculture.
Complement Assays
[0128] To determine that functional factor B, factor D, and C5 are
produced during OC differentiation, culture supernatants were
collected at day 6 from 2.times.10.sup.6 WT BM cells stimulated
with 1.times.10.sup.-8M 1.25(OH).sub.2 vitamin D.sub.3 and assayed
for complement activities. For functional factor B and D detection,
collected culture supernatants were directly added into zymosan C3
uptake assays with factor B or D deficient mouse sera. In brief,
sera were prepared by bleeding male WT, factor B.sup.-/-, or factor
D.sup.-/- mice through the tail vein. After this, 20% BM
cell-conditioned culture supernatants or control media were added
into 10% of WT, factor B.sup.-/-, or factor D.sup.-/- serum
together with 30 .mu.g/mL zymosan (Sigma-Aldrich) in gelatin
veronal buffer (GVB)/Mg.sup.2+-EGTA (ethylene glycol tetraacetic
acid) buffer and incubated at 37.degree. C. for 30 minutes. After
washing, C3b deposition was assessed by fluorescein isothiocyanate
(FITC)-anti-mouse C3 mAb (Cedarlane) staining, followed by flow
cytometry analysis. For functional C5 detection, BM cell-culture
supernatant or control media was concentrated 10-fold using a
Microcon (Millipore) and added into E.sup.shA-based hemolytic
assays using 10% of human C5-depleted serum (Complement Tech). The
ability of the BM cell-conditioned media to compensate the C5
deficiency in the serum was assessed by measuring OD.sub.541 to
quantify C5b-9 (membrane attack complex)-mediated sheep erythrocyte
hemolysis.
IL-6 and M-CSF ELISA
[0129] For IL-6-level measurements, culture supernatants were
collected at day 12 after 1.25(OH).sub.2 vitamin D3 stimulation,
and standard IL-6 enzymelinked immunosorbent assay (ELISA; R&D
Systems) was used according to the protocol provided by the
manufacturer. For M-CSF assays, culture supernatants were collected
at day 3 after stimulation, concentrated by ultrafiltration using a
centrifugal concentrator (molecular weight [MW] cutoff: 3000;
Millipore), then analyzed by a murine M-CSF ELISA kit (PeproTech),
following the manufacturer-provided protocol.
Assessment of RANKL/OPG Expression Ratios
[0130] To compare RANKL/OPG expression levels in WT and C3.sup.-/-
BM cells during OC differentiation, 2.times.10.sup.6 BM cells were
isolated from WT or C3.sup.-/- mice and cultured in complete
.alpha.-MEM with and without the presence of 1.times.10.sup.-8M
1.25(OH).sub.2 vitamin D.sub.3. At 24 hours, total RNA was purified
from the cells using TRIzol (Invitrogen), and reverse transcribed
using a first-strand cDNA synthesis kit (Invitrogen), following
protocols provided by the manufacturer. The relative expression
levels of RANKL and OPG were assessed by SYBR Green-based
quantitative reverse transcription (RT)-PCR (qRT-PCR; GoTag qPCR
master mix; Promega). In brief, the qRT-PCR was carried out in
triplicate for RANKL, OPG, and .beta.-actin (internal control) of
each sample on an ABI PRISM 7500 machine (Applied Biosystems). The
data were analyzed and normalized against levels of RANKL and OPG
in BM cells without 1.25(OH).sub.2 vitamin D.sub.3 stimulation by
the 7500 SDS Version 1.3 software package (Applied Biosystems).
Dissociation experiments were used to ensure that the fluorescent
signal for each amplicon was derived from the PCR products
only.
Results
[0131] C3.sup.-/- BM Cells Generate Fewer OCs than WT BM Cells
[0132] To examine whether the absence of locally produced C3 from
BM cells will inhibit OC generation we incubated WT and C3.sup.-/-
BM cells with 1.25(OH).sub.2 vitamin D.sub.3 and compared the
number of TRAP-positive cells on day 12. As shown in FIG. 3, after
stimulation, C3.sup.-/- BM cells produced 119.+-.31 TRAP-positive
mononuclear cells and 21.+-.6 multinucleated TRAP-positive cells
per well, while WT BM cells generated 230.+-.26 TRAP-positive
mononuclear cells and 49.+-.10 multinucleated TRAP-positive cells
(P<0.05). These results indicate that the absence of C3 from BM
cells reduces 1.25(OH).sub.2 vitamin D.sub.3-stimulated OC
differentiation by nearly 50%.
C3.sup.-/- BM Cells Produced Reduced Levels of M-CSF and Failed to
Efficiently Up-Regulate RANKL Expression after 1.25(OH)2 Vitamin D3
Stimulation
[0133] We next assessed the levels of M-CSF in WT and C3.sup.-/- BM
cell-culture supernatants using a standard ELISA kit. These assays
showed that, compared with 23.7.+-.2.1 pg/mL M-CSF in WT BM
cell-conditioned media, C3.sup.-/- BM cell-conditioned media
contained only 10.9.+-.1.2 pg/mL of M-CSF (FIG. 4A). Because of the
well-established roles of RANKL and OPG in osteoclast generation,
we also analyzed the expression levels of RANKL and OPG in WT and
C3.sup.-/- BM cells after 1.25(OH).sub.2 vitamin D.sub.3
stimulation by qRT-PCR. Consistent with previous reports by others,
in WT BM cells, 1.25(OH).sub.2 vitamin D.sub.3 stimulation markedly
up-regulated RANKL expression (.about.6-fold), but had little
effect on OPG expression (FIG. 4B). However, the same assays
demonstrated that C3.sup.-/- BM cells failed to significantly
up-regulate RANKL expression (1.6-fold), and that the expression
levels of OPG remained essentially unchanged. These results
indicate that during OC differentiation, C3.sup.-/- BM cells
produced decreased levels of M-CSF and decreased RANKAL/OPG ratios,
which is in accordance with the lower numbers of OCs generated.
BM Cells Locally Produce Functional Factor B, Factor D, and C5 in
Addition to C3 during Differentiation
[0134] For C3 to impact any cell, it first needs to be activated.
To examine how BM cell-generated C3 could be activated to regulate
OC differentiation, we assessed the presence of factor B and factor
D, components essential for the activation of C3 through the
alternative pathway of complement activation, as well as C5, the
component required for C5a generation. The presence of mRNA for
factor B, factor D, and C5 were first confirmed by RT-PCR (data not
shown), and then the presence of complement proteins were tested
with functional assays of the BM cell-conditioned media using sera
deficient of factor B, factor D, or C5. These assays (FIG. 3)
showed that after 1.25(OH).sub.2 vitamin D.sub.3 stimulation, BM
cell-conditioned media compensated the absence of factor B, factor
D, or C5 (FIG. 4) in zymosanbased C3b uptake and E.sup.shA-based
hemolytic assays, indicating that BM cells produce functional
factor B, factor D, and C5 during differentiation. These results
indicate that C3 could be activated through the alternative pathway
during BM cell differentiation, leading to the production of
complement activation products, including ligands for C3 receptors
and the released anaphylatoxins, C3a/C5a.
Alternative Pathway of Complement Activation is Required for
Efficient OC Differentiation
[0135] To determine whether the alternative pathway of complement
activation is required to activate the C3 locally produced by the
BM cells, and thus implicating complement activation in the
regulation of OC differentiation, we compared the numbers of OCs
generated from WT and factor D.sup.-/- BM cells after
1.25(OH).sub.2 vitamin D.sub.3 stimulation. Factor D is essential
for the alternative pathway of complement activation. These
osteoclast assays (FIG. 6) showed that factor D.sup.-/- BM cells
generated 163.+-.16 mononuclear and 25.+-.8 multinucleated
TRAP-positive cells per well, compared with 236.+-.16 mononuclear
and 50.+-.2 multinucleated TRAP-positive cells in wells containing
WT BM cells (P<0.05). These results indicate that the
alternative pathway of complement activation is required to
activate C3 for efficient OC differentiation from BM cells after
1.25(OH).sub.2 vitamin D.sub.3 stimulation.
C3aR and C5aR are Required for Efficient OC Differentiation
[0136] Since BM cells locally produce both C3 and C5 during OC
differentiation, and their activation through the alternative
pathway can generate C3a and C5a, we next examined whether the
receptors for these ligands, C3aR and C5aR, could regulate OC
differentiation. We isolated BM cells from WT, C3aR.sup.-/-,
C5aR.sup.-/-, and C3aR.sup.-/-C5aR.sup.-/- mice, then incubated
them with 1.25(OH).sub.2 vitamin D.sub.3, following the same OC
differentiation protocol. The osteoclast induction assays (FIG. 7A)
demonstrated that, compared with WT BM cells (226.+-.22 mononuclear
and 55.+-.5 multinucleated cells), C3aR.sup.-/- and C5aR.sup.-/- BM
cells generated a reduced number of TRAP-positive cells;
C3aR.sup.-/- BM cells had 115.+-.12 mononuclear and 20.+-.3
multinucleated cells, and C5aR.sup.-/- BM cells had 164.+-.6
mononuclear and 40.+-.5 multinucleated cells, while the
double-knockout C3aR.sup.-/-C5aR.sup.-/- BM cells had the least
number of TRAP-positive cells (91.+-.10 mononuclear and 20.+-.7
multinuclear cells). In complementary experiments, during
1.25(OH).sub.2 vitamin D.sub.3 stimulation, we treated WT BM cells
with C3aRA(SB290157), C5aRA(JPE-1375), or both. In these assays
(FIG. 7B), compared with the placebo (217.+-.25 mononuclear and
42.+-.9 multinuclear cells), C3aRA significantly inhibited OC
generation (156.+-.23 mononuclear and 29.+-.7 multinuclear cells).
While C5aRAappeared to reduce numbers of both mono- and
multinucleated TRAP-positive cells (176.+-.29 mononuclear and
34.+-.8 multinuclear cells), it did not reach a statistical
significance (P=0.068 for mononuclear cells and P=0.12 for
multinucleated cells). However, a combination of C3aRAand C5aRA
inhibited OC generation synergistically (66.+-.4 mononuclear and
16.+-.2 multinuclear cells; P<0.05). These results indicate that
in addition to C3 receptors, C3aR and, possibly, C5aR are also
required for efficient OC generation, in which C3aR may play a more
prominent role than C5aR.
C3aR/C5aR Regulated IL-6 Expression is Involved in OC
Differentiation
[0137] We next explored a potential mechanism underlying the
C3aR/C5aR-mediated effects on OC differentiation, which is based on
previous reports that IL-6 augments OC generation, and that
C3aR/C5aR stimulates IL-6 production in many types of cells. We
measured IL-6 levels by ELISA in WT, C3.sup.-/-, and
C3aR.sup.-/-C5aR.sup.-/- BM cell-conditioned media after
1.25(OH).sub.2 vitamin D.sub.3 stimulation. These measurements
showed that, compared with 357.5.+-.66.4 pg/mL IL-6 in WT BM
cell-conditioned media, there was only 72.8.+-.12.7 or 86.2.+-.16.3
pg/mL IL-6 in C3.sup.-/- or C3aR.sup.-/-C5aR.sup.-/- BM
cell-conditioned media (FIG. 8A).
[0138] To causally link reduced levels of IL-6 to decreased OC
differentiation in C3.sup.-/- BM cells, we repeated the
differentiation experiments with WT and C3.sup.-/- BM cells and,
this time, supplemented 20 ng/mL of IL-6 into the C3.sup.-/- BM
cell-culture daily and counted TRAP-positive cells at 12 days. As
shown in FIG. 6B, supplementation of exogenous IL-6 into C3.sup.-/-
BM cell cultures increased the numbers of TRAP-positive cells from
108.+-.17 (mononuclear) and 31.+-.4 (multinucleated) to 249.+-.39
(mononuclear) and 52.+-.9 (multinucleated) (P<0.05), suggesting
that complement regulates OC differentiation, at least in part,
through modulating local IL-6 production.
[0139] To further verify that C3aR/C5aR are integrally involved in
OC differentiation, and IL-6 is the underlying mechanism, we next
incubated WT or C3.sup.-/- BM cells with purified C3a, C5a, or both
during the differentiation process, and neutralized IL-6 using an
anti-IL-6 mAb in the cultures stimulated with C3a/C5a. These assays
showed that while exogenous C3a increased the numbers of resultant
OC from both WT and C3.sup.-/- BM cells, C5a did not appear to have
a significant effect on TRAP.sup.+ cell formation (FIG. 8C-D).
Neutralizing IL-6 totally ablated the effects of C3a/C5a on
augmenting OC generation from both the WT and C3.sup.-/- BM cells.
Interestingly, exogenous C3a and the combination of C3a/C5a
appeared to have a greater effect on C3.sup.-/- BM cells than WT BM
cells (66.7% increase vs. 36.2% increase of mononuclear cells and
87.5% increase vs. 33.3% increase of multinucleated cells),
possibly due to the lack of endogenous C3a/C5a production in
C3.sup.-/- BM cells, while WT BM cells can still make the baseline
of C3a/C5a during differentiation. Similarly, neutralization of
IL-6 after C3a/C5a treatment in WT BM cells reduced OC numbers
below placebo-treated WT BM cells, while neutralizing IL-6 in
C3.sup.-/- BM cells just ablated C3a/C5a effects without further
reducing OC numbers below the baseline, which is consistent with
previous reports by others that IL-6 is critical in OC
differentiation, and our findings that C3.sup.-/- BM cells only
produce trace amount of IL-6 during OC differentiation.
Locally Produced Complement also Regulates OC Differentiation in
Humans
[0140] To determine whether the above-observed results would apply
to humans, we subjected normal human BM cells to OC differentiation
conditions with and without the C3aR/C5aR antagonists, then
analyzed the conditioned media for the presence of factor B, factor
D, and C5, and quantified the resultant TRAP.sup.+ cells. These
assays showed that, like the results with the mouse system, human
BM cells locally produce functional factor B (FIG. 9A), factor D
(FIG. 9B), and C5 (FIG. 9C) during OC differentiation, and that
blocking C3aR and/or C5aR significantly inhibited OC generation
(FIG. 9D).
EXAMPLE 2
Direct Complement Effects on OC Differentiation
[0141] To maintain bone homeostasis, the differentiation of OC and
OB needs to be tightly regulated to maintain the balance between
bone formation and destruction. The results of Example 1 show a
significant effect of complement on both OC and OB differentiation,
which can then have a profound effect on bone balance, depending
upon which cell type is more severely affected. The results of
Example 1 also strongly indicate that the inhibition of OCs by
complement deprivation has a more profound impact on bone balance
than the diminished osteogenic potential of MSCs, thus leading to a
decreased bone loss in C3.sup.-/- mice. In addition, our in vitro
data suggest that the alternative pathway of complement activation
is important for the complement mediated effects on OC
differentiation.
Bone Balance in C3-/- Mice after OVX
[0142] C3.sup.-/- mice were used to conduct studies into the
possible role of complement in OC and OB differentiation and in in
vivo bone homeostasis. OVX-induced osteoporosis in mice is a
well-established animal model of post-menopausal osteoporosis, in
which estrogen deprivation results in increased OC and OB numbers
with OCs playing a more dominant role, thus leading to net bone
loss. To determine if complement affects bone homeostasis in this
model, WT and C3.sup.-/- mice were OVXed and, after 6 weeks, were
anesthetized, their femurs imaged by microCT, and the acquired
images analyzed for multiple bone parameters (Table 1). The results
indicate a clear bone sparing effect in the C3.sup.-/- mice. For
example, skeletal connectivity (Skel. Conn.) in the WT mice was
down to only one third that of C3.sup.-/- mice, and trabecular
thickness (Tb.Th.) and bone volume (Bone Vol.) were all
significantly lower in WT than C3.sup.-/- mice. Only trabecular
number (Tb.-Num.) did not rise to the level of significance, but
still showed a trend (.rho.=0.09), again, with C3.sup.-/- showing
greater Tb.-Num than WT. The bone mineral density and bone mineral
content parameters showed similar results where, in 3 out of 4
cases where the data reached significance, the C3.sup.-/- mice
showed greater mineral content or mineral density than did WT
mice.
TABLE-US-00001 TABLE 1 Micro CT Bone Parameter Analysis Parameter
C3.sup.-/- WT P value Skel. Conn. 114.3 .+-. 15.4 54.6 .+-. 2.4
0.002 Tb. Th. 81.5 .+-. 3.3 61.7 .+-. 11.7 0.005 Bone Vol. 0.73
.+-. 0.92 0.48 .+-. 0.11 0.002 Tb. Num. 3.00 .+-. 0.21 2.25 .+-.
0.82 0.09 NS Med. BMD 188.4 .+-. 9.0 148.4 .+-. 9.5 <0.001 Med
BMC 0.80 .+-. 0.07 0.62 .+-. 0.05 0.005 Tr. BMC 0.25 .+-. 0.03 0.17
.+-. 0.03 0.006 BMD = Bone Mineral Density; BMC = Bone Mineral
Content; Conn = Connectivity; Tr = Trabecular; Th = thickness; Num
= number; Med = Medullary (entire cavity volume). (n = 3 in each
group)
In Vitro Effect of Complement Deficiency on OC Differentiation
[0143] We showed in Example 1 the impact of complement on OC
formation in vitro using both mouse and human cells. In this study,
the effect of complement on OC differentiation was investigated
using WT and mice deficient in C3, factor D and C3aR/C5aR, and
respective C3aR/C5aR antagonists. The results (Table 2) showed that
BM cells from C3.sup.-/- and C3aR.sup.-/-/C5aR.sup.-/- mice
produced significantly fewer OCs than BM cells from WT, indicating
that complement plays a significant role in OC formation. The role
of C3aR/C5aR was confirmed in an experiment where WT BM cells were
incubated in medium supplemented with C3aR and C5aR antagonists,
which resulted in dramatically reduced OC numbers. The results also
showed that C3.sup.-/- and C3aR.sup.-/-/C5aR.sup.-/- BM cells
produce reduced levels of IL-6, and that supplementing IL-6 into
C3.sup.-/- BM cultures rescued the C3.sup.-/- phenotype, suggesting
that complement regulates OC differentiation through
C3aR/C5aR-driven IL-6 production. The studies using WT and factor
D.sup.-/- BM cells showed that the deficiency of the alternative
pathway of complement activation significantly reduces the numbers
of differentiated TRAP+ OCs, indicating that at least in vitro,
complement is activated through the alternative pathway to regulate
OC differentiation (FIG. 2).
TABLE-US-00002 TABLE 2 Table 2: Complement regulates OC formation
Trap.sup.+ Multi- Bone Marrow Source Nucl. Cells/Well Trap.sup.+
Mono-Nucl. Cells/Well WT 49 .+-. 10 230 .+-. 26 C3.sup.-/- 21 .+-.
6* 119 .+-. 31* WT 50 .+-. 2 236 .+-. 16 C3aR.sup.-/-C5aR.sup.-/-
20 .+-. 7* 91 .+-. 10* WT 55 .+-. 29 226 .+-. 27 WT + C3aR/C5aR 23
.+-. 12* 159 .+-. 23* Antagonists C3.sup.-/- 31 .+-. 4.dagger. 108
.+-. 17.dagger. C3.sup.-/- + IL-6 52 .+-. 9 249 .+-. 39 *.rho. <
0.005 compared to WT .dagger..rho. < 0.05 compared to IL-6
supplemented
[0144] In summary, these studies demonstrated that C3 deficiency
protects mice from bone loss in a model of post-menopausal
osteoporosis, and that the local BM cell-produced complement
components have a significant impact on in vitro
osteoclastogenesis, in which the alternative pathway of complement
activation is important, and C3aR/C5aR-regulated IL-6 production
plays a critical role. These results strongly suggest that
complement directly regulates OC numbers and bone loss in
osteoporosis through C3aR and/or C5aR.
EXAMPLE 3
Mechanisms by Which Complement Regulates OB Differentiation in
Osteoporosis
[0145] In this Example we show C3aR and/or C5aR upregulate OB
differentiation of MSCs by promoting Run.times.2 expression and
inhibiting PPAR.gamma. expression.
[0146] We examined each of the complement receptors on WT MSCs by
flow cytometry following staining with rat anti-mouse CR1, CR2, CR3
or CR4 mAbs and with goat anti-mouse C5aR or C3aR Ab. These
analyses showed that MSCs express both C5aR and C3aR but do not
express detectable levels of CR1/CR2/CR3/CR4. As a result, we now
focus on studying the effect of C5aR and C3aR on OB
differentiation.
[0147] The complement components fB, fD, C3 and C5 are essential to
generate C3a from C3, and C5a from C5 through the alternative
pathway of complement activation. To test whether MSCs locally
produce all these components, we isolated total RNA form WT MCCs
and performed RT-PCR to test for the presence of transcripts of C3,
fB, fD and C5. These analyses showed that MSCs do, indeed, express
C3, fB, fD and C5. Complement functional assaysl using fB, fD or C5
deficient sera and MSC-conditioned media demonstrated that MSC
locally produce functional fB, fD and C5 protein. Consequently, in
principle, MSCs are fully capable of generating C5a and C3a through
local complement activation.
[0148] C3-/- MSCs are Less Efficient in Differentiating into
OBs
[0149] To test whether complement modulates OB generation from
MSCs, WT and C3.sup.-/- MSCs were exposed to osteoblastic
differentiation conditions using established methods. After 3 weeks
of OB differentiation, WT MSCs were highly positive for Alizarin
Red S, indicative of OB mineral deposition; whereas C3.sup.-/- MSCs
showed little if any Alizarin Red S positivity. To verify this
result, we assayed the cells for alkaline phosphatase activity.
Consistent with the Alizarin Red S staining, differentiated WT MSCs
showed significant alkaline phosphatase activity (dark blue
staining), while differentiated C3.sup.-/- MSCs were negative,
indicating that complement locally produced by MSCs is required for
efficient OB differentiation.
[0150] In contrast to decreased OB generation, C3.sup.-/- MSCs
exhibited markedly greater adipocyte generation. At day 21 in
adipogenic conditions, differentiated WT MSCs showed only scattered
small adipocytes stained with Oil Red O while, under the identical
conditions, C3.sup.-/- MSCs showed abundant Oil Red
O-positivity.
C3.sup.-/- MSCs Failed to Increase Levels of Run.times.2 during OB
Differentiation
[0151] Previous studies by others have shown that the runt-related
transcription factor 2 (Run.times.2) is essential for MSC
osteogenesis. To investigate the potential relationship between
Run.times.2 expression and osteoblast differentiation in C3.sup.-/-
mice, we next tested the expression levels of Run.times.2 in
C3.sup.-/- and WT MSCs exposed to osteoblastic and adipogenic
differentiation conditions in vitro. Total RNA was isolated from WT
and MSCs at day 0 and day 20 in osteoblastic or adipocytic
differentiation conditions and assayed by qRT-PCR. These analyses
showed that under OB differentiation conditions WT MSCs had
augmented Run.times.2 expression as reported previously (8.6 fold),
while C3.sup.-/- MSCs failed to significantly upregulate
Run.times.2 expression (1.1 fold).
[0152] From the above description of the invention, those skilled
in the art will perceive improvements, changes and modifications
Such improvements, changes and modifications are within the skill
of the art and are intended to be covered by the appended claims.
All publications, patents, and patent applications cited in the
present application are herein incorporated by reference in their
entirety.
* * * * *