U.S. patent application number 12/710540 was filed with the patent office on 2010-09-02 for methods compositions and article of manufacture for modulating bone growth.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Itai Bab, Raphael Mechoulam, Esther Shohami, Andreas Zimmer.
Application Number | 20100222438 12/710540 |
Document ID | / |
Family ID | 33479219 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100222438 |
Kind Code |
A1 |
Bab; Itai ; et al. |
September 2, 2010 |
METHODS COMPOSITIONS AND ARTICLE OF MANUFACTURE FOR MODULATING BONE
GROWTH
Abstract
Novel methods and pharmaceutical compositions suitable for
modulating bone growth and remodeling, preventing bone diseases,
inducing bone growth or repair by cannabinoid receptor-mediated
effects on bone cells is disclosed. Methods of identifying bone
growth modulating agents are also disclosed.
Inventors: |
Bab; Itai; (Karmei Yossef,
IL) ; Mechoulam; Raphael; (Jerusalem, IL) ;
Zimmer; Andreas; (Bonn, DE) ; Shohami; Esther;
(Mevasseret Zion, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
|
Family ID: |
33479219 |
Appl. No.: |
12/710540 |
Filed: |
February 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11003939 |
Dec 6, 2004 |
7749953 |
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12710540 |
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PCT/IL03/00480 |
Jun 8, 2003 |
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11003939 |
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60385881 |
Jun 6, 2002 |
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60410276 |
Sep 13, 2002 |
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Current U.S.
Class: |
514/719 ; 435/29;
435/6.1; 435/6.18 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/663 20130101; A61K 38/1875 20130101; A61P 1/02 20180101;
A61P 19/10 20180101; A61K 31/00 20130101; A61P 19/00 20180101; A61P
19/08 20180101; A61K 38/553 20130101; A61K 31/232 20130101; A61K
31/232 20130101; A61K 31/454 20130101; A61K 38/1825 20130101; A61K
31/353 20130101; A61K 38/1825 20130101; A61K 38/27 20130101; A61K
38/29 20130101; A61K 38/1875 20130101; G01N 33/566 20130101; A61K
38/29 20130101; A61K 38/27 20130101; A61K 31/353 20130101; A61P
43/00 20180101; A61K 31/663 20130101; A61K 31/454 20130101; A61K
2300/00 20130101; A61K 38/553 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/719 ; 435/29;
435/6 |
International
Class: |
A61K 31/085 20060101
A61K031/085; A61P 19/00 20060101 A61P019/00; C12Q 1/02 20060101
C12Q001/02; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of identifying a bone growth modulating agent,
comprising screening a plurality of molecules to thereby uncover a
molecule capable of regulating an expression or activity of at
least one cannabinoid receptor, said molecule being the bone growth
modulating agent.
2. The method of claim 1, further comprising determining an ability
of said molecule to modify bone formation rate and/or altering bone
mineralization perimeter.
3. The method of claim 1, wherein said screening is effected by
exposing bone cells to said plurality of molecules and determining
said expression of said at least one cannabinoid receptor in said
bone cells.
4. The method of claim 1, wherein said cannabinoid receptor is
selected from the group consisting of a CB1 receptor, a CB1-like
receptor, a CB2 receptor, a CB2-like receptor, and a non-CB1
non-CB2 receptor.
5. The method of claim 1, wherein said at least one cannabinoid
receptor is a bone cell or bone cell progenitor cannabinoid
receptor.
6. The method of claim 5, wherein said bone cell progenitor is an
osteogenic cell.
7. The method of claim 5, wherein said bone cell progenitor is a
stromal cell.
8. The method of claim 5, wherein said bone cell progenitor is a
bone resorbing cell progenitor.
9. The method of claim 3, wherein said bone cell is a cultured bone
cell.
10. The method of claim 5, further comprising exposing said bone
cells to an osteoclast differentiating factor.
11. The method of claim 1, wherein said expression of said
cannabinoid receptor is determined by RT-PCR.
12. A method of identifying and producing a pharmaceutical
composition for modulating bone growth, comprising screening a
plurality of molecules to thereby uncover a molecule capable of
regulating an expression or activity of at least one cannabinoid
receptor, said molecule being the bone growth modulating agent, and
producing a pharmaceutical composition comprising said bone growth
modulating agent and a pharmaceutically acceptable carrier.
13. The method of claim 12, wherein said screening is effected by
exposing bone cells to said plurality of molecules and determining
said expression of said at least one cannabinoid receptor in said
bone cells.
14. The method of claim 12, wherein said cannabinoid receptor is
selected from the group consisting of a CB1 receptor, a CB1-like
receptor, a CB2 receptor, a CB2-like receptor, and a non-CB1
non-CB2 receptor.
15. The method of claim 12, wherein said at least one cannabinoid
receptor is a bone cell or bone cell progenitor cannabinoid
receptor.
16. The method of claim 15, wherein said bone cell progenitor is an
osteogenic cell.
17. The method of claim 15, wherein said bone cell progenitor is a
stromal cell.
18. The method of claim 15, wherein said bone cell progenitor is a
bone resorbing cell progenitor.
19. The method of claim 13, wherein said bone cell is a cultured
bone cell.
20. The method of claim 12, wherein said expression of said
cannabinoid receptor is determined by RT-PCR.
Description
[0001] This is a continuation-in-part of PCT/IL03/00480, filed Jun.
8, 2003, which claims the benefit of priority of U.S. provisional
patent application No. 60/410,276, filed Sep. 13, 2002 and of U.S.
provisional patent application No. 60/385,881, filed Jun. 6, 2002.
Priority is claimed of all of the above-identified
applications.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods of modulating bone
growth and, remodeling, methods of treating or preventing bone
diseases, methods of inducing and stimulating bone growth and peak
bone mass or repair, pharmaceutical compositions for modulating
bone growth, articles of manufacturing and methods of identifying
bone growth modulating agents. Specifically, the present invention
employs regulating the expression or activity of cannabinoid
receptors in bone cells which in turn modulates bone growth and
remodeling.
[0003] Naturally occurring cannabinoids may be divided into two
categories, plant-derived and endogenous. Plant-derived
cannabinoids are known to elicit dramatic psychobehavioral effects,
exemplified by the well-known .DELTA..sup.9-tetrahydrocannabinol
(THC), the psychotropic principle in marijuana. They are also known
to have complex cardiovascular effects, a prominent component of
which is hypotension [Vollmer et al., J. Pharm. Pharmacol.
26:186-198 (1974)]. Endogenous cannabinoids (endocannabinoids) are
a class of lipid-like molecules that share receptor binding sites
with plant-derived cannabinoids and mimic many of their
neurobehavioral effects [Mechoulam et al., Adv. Exp. Bio. Med.
402:95-101 (1996)]. Two endocannabinoids have been characterized in
some detail: arachidonyl ethanolamide (anandamide) [Devane et al.,
Science 258:1946-1949 (1992); Felder et al., Proc. Natl. Acad. Sci.
USA. 90:7656-7660 (1993)] and 2-arachidonoyl glycerol (2-AG)
[Mechoulam et al., Biochem. Pharmacol 50:83-90 (1995)].
[0004] Additional natural or synthetic cannabinoids are described
in U.S. Pat. Nos. 4,371,720, 5,013,387, 5,081,122, 5,292,736,
5,461,034, 5,618,955, 6,166,066 and 6,531,636; International Patent
applications WO 01/9773, WO 97/29079, WO 99/02499, WO 98/41519, and
WO 94/12466; European Patent Nos. EP 0570920 and EP 0444451; French
Patent No. FR 2735774; and Israeli Pat. Nos. IL 01/00551 and IL
99/00187; Gaoni and Mechoulam, J. Amer. Chem. Soc. 93, 217 (1971);
Mechoulam et al., Science 169, 611 (1970); Edery et al., Ann. N.Y.
Acad. Sci., 191, 40 (1971); Mechoulam et al., J. Amer. Chem. Soc.,
94, 7930 (1972); R. Mechoulam (ed.), "Marijuana: Chemistry,
Metabolism, Pharmacology, and Clinical Effects" Academic Press,
1973, New-York; Houry et al., J. Med. Chem., 17, 287 (1974); Houry
et al., J. Med. Chem., 18, 951 (1975); Mechoulam et al., Chem.
Reviews, 76, 75 (1976); Mechoulam et al., J. Med. Chem., 23, 1068
(1980); Srebnik et al., J. Chem. Soc., Perkin Trans. I, 2881
(1984); Mechoulam et al., Tetrahedron: Asymmetry, 1, 315 (1990);
Devane et al., Science, 258, 1946 (1992); Burstein et al., J. Med.
Chem., 35, 3135 (1992); Hanus et al., J. Med. Chem., 36, 3032
(1993); Mechoulam et al., Biochem. Pharmacol., 50, 83 (1995);
Sheskin et al., J. Med. Chem., 40, 659 (1997); Rhee et al., J. Med.
Chem. 40, 3228 (1997); and Hanus et al., PNAS, 98, 3662 (2001).
[0005] Endocannabinoids exert their effects by binding to specific
receptors thereby activating neurotransmitters and hormone
regulators [Piomelli et al., Trends Pharmacol. Sci. 21: 218-224
(2000); Petwee, R. G., Curr. Med. Chem. 6:635-664 (1999); and
Devane et al., J. Med. Chem. 35: 2065-2069)]
[0006] To date, two types of high-affinity cannabinoid receptors
have been identified by molecular cloning: (i) CB1 receptors,
present mostly in brain [Devane et al., Mol. Pharmacol. 34:605-613
(1988); Matsuda et al., Nature 346:561-564 (1990)] but also in some
peripheral tissues [Shire et al., J. Biol. Chem. 270:3726-3731
(1995); Ishac et al., Br. J. Pharmacol. 118:2023-2028 (1996)], and
(ii) CB2 receptors, present on macrophages in the spleen [Munro et
al., Nature 365:61-65 (1993)]. Other types or subtypes of
cannabinoid receptors have been recently described, designated
CB1-like receptors, CB2-like receptors, and non-CB1 non-CB2
receptors [Hanus et al., J. Pharmacol. Exper. Therapeutics 54:
161-202 (2002)].
[0007] The physiological roles of endogenous cannabinoids and the
pathways of endocannabinoid signaling are the subject of intense
investigation and have been reported to affect processes in the
nervous, cardiovascular, immune, and reproductive systems
[Mechoulam et al., Eur. J. Pharmacol. 359: 1-18 (1998); Axelrod and
Felder, Neurochem. Res. 23: 575-581 (1998); Wagner et al., J. Mol.
Med. 76: 824-836 (1999); and Klein et al., Immunol. Today 19:
373-381 (1998)].
[0008] Accordingly, cannabinoids or cannabinoids receptor ligands
have been used or described as useful therapeutic agents for
treating a variety of medical disorders.
[0009] Thus, THC has been extensively used to prevent excessive
weight loss by cancer or AIDS patients [Mechoulam et al., E. Prog.
Med. Chem. 35: 199-243 (1998)].
[0010] U.S. Pat. No. 5,939,429 discloses use of agonists of CB1
receptors as well as other cannabinoid receptors to treat
cardiovascular conditions, including hemorrhagic shock and in other
conditions associated with excessive vasoconstriction, such as
hypertension, peripheral vascular disease, cirrhosis of the liver,
and certain forms of angina pectoris. In addition it teaches use of
antagonists of CB1 and other cannabinoid receptors for treating
hypotension which is caused by endotoxin activation of
macrophages.
[0011] U.S. Pat. No. 6,166,066 discloses use of cannabinoids which
are selective for the CB2 receptor as immunosuppressive agents for
preventing tissue rejection in organ transplant patients and for
treating autoimmune associated diseases
[0012] U.S. application Ser. No. 09/779,109 discloses use of
cannabinoids receptor modulators for treating respiratory or
non-respiratory leukocyte-activation associated diseases. Exemplary
non-respiratory cannabinoid receptor-mediated diseases include
transplant rejection, rheumatoid arthritis, multiple sclerosis,
inflammatory bowel disease, lupus, graft v. host disease, T-cell
mediated hypersensitivity disease, psoriasis, Hashimoto's
thyroiditis, Guillain-Barre syndrome, cancer, contact dermatitis,
allergic rhinitis, and ischemic or reperfusion injury.
[0013] U.S. application Ser. No. 10/032,163 discloses a method of
increasing the activity of a cannabinoid agonist that binds
specifically to an endogenous cannabinoid receptor, so as to
protect the cells against glutamate-induced neurotoxicity.
[0014] Yet, while cannabinoids or cannabinoid receptor ligands have
been suggested for use as therapeutic agents, application of
cannabinoids for treating or preventing bone-related diseases has
never been described nor suggested in prior art.
[0015] While reducing the present invention to practice, the
inventors of the present invention surprisingly uncovered the major
role of endocannabinoids in modulating bone growth and remodeling,
thus indicating the potential benefits of using cannabinoids or
cannabinoids receptor ligands as therapeutic agents for treating
bone diseases and injuries as well as promoting bone formation or
inhibiting bone resorption.
[0016] Bone is subject to constant breakdown and resynthesis in a
complex process mediated by osteoblasts, which produce new bone,
and osteoclasts, which destroy bone. This process is referred to as
bone remodeling. The activities of these cells are regulated by a
large number of cytokines, hormones and growth factors, many of
which have now been identified and cloned.
[0017] There is a plethora of conditions which are characterized by
the need to promote bone formation and/or to inhibit bone
resorption. Perhaps the most obvious is the case of bone fractures,
where it would be desirable to stimulate bone growth and to hasten
and complete bone repair. Agents that enhance bone formation would
also be useful in endosseous implants and facial reconstruction
procedures, and of great importance in the growing field of
prosthetic and therapeutic bone implants. Other bone deficit
conditions include bone segmental defects, periodontal disease,
metastatic bone disease, osteolytic bone disease and conditions
where connective tissue repair would be beneficial, such as healing
or regeneration of cartilage defects or injury. Also of great
significance is the chronic condition of osteoporosis, including
age-related osteoporosis and osteoporosis associated with
post-menopausal hormone status. Other conditions characterized by
the need for bone growth include primary and secondary
hyperparathyroidism, disuse osteoporosis, diabetes-related
osteoporosis, osteoporosis associated with depression and
hypogonadism and glucocorticoid-related osteoporosis.
[0018] On the other hand, there are conditions which are
characterized by the need to inhibit bone formation or to promote
bone resorption. These include certain stages of Paget's disease,
blastic metastatic bone cancer, Hodgkin's lymphoma, degenerative
sclerosis and osteomyelitis. Agents known to be effective in
inhibition of bone growth, and in bone resorption, are the
cyclooxygenase inhibitors, 1, 25 (OH).sub.2 vitamin D3, the
glucocorticoids, omeprazole, the serum protein fetuin, noggin,
blockers of beta adrenergic receptors, chordin and DAN proteins and
high concentrations of TGF-beta. However, all of the abovementioned
agents (particularly the glucocorticoids and other hormones) are
known to exert their influence on a wide variety of tissues, and as
such are unsuited for pharmacological applications in bone
diseases.
[0019] Various therapeutic agents and approaches to treatment of
bone related diseases have been disclosed in patent
publications.
[0020] U.S. Pat. No. 5,461,034 discloses osteogenic growth
polypeptides identified from regenerating bone marrow, for the
enhancement of bone formation and bone marrow in preparation for
bone marrow transplant. U.S. Pat. No. 5,280,040 discloses
antiestrogenic, oral contraceptive compounds, 3,4-diarylchromans,
described as useful in the treatment of osteoporosis. U.S. Pat. No.
6,352,973 discloses a recombinant protein containing a bone
morphogenic polypeptide of the TGF-beta superfamily of cytokines
originally isolated from blood serum, for enhancing bone growth.
U.S. Pat. No. 6,462,019 discloses inhibitors of proteasomal
activity and production for inhibiting osteoclastic activity and
stimulating bone growth, based on the observation that mice lacking
proteasomal activity develop the condition of excess bone formation
known as osteopetrosis.
[0021] International patent application No. 92/15615 discloses a
protein derived from a porcine pancreas which acts to depress serum
calcium levels for treatment of bone disorders that cause elevation
of serum calcium levels.
[0022] International patent application No. 92/14481 discloses a
composition for inducing bone growth which contains activin and
bone morphogenic protein.
[0023] European Patent Application No. 504 938 discloses the use of
di- or tripeptides which inhibit cysteine protease in the treatment
of bone diseases.
[0024] European Patent Application No. 499 242 discloses the use of
cell growth factor compositions thought to be useful in bone
diseases involving bone mass reduction because they cause
osteoblast proliferation.
[0025] European Patent Application No. 451 867 discloses
parathyroid hormone peptide antagonists for treating dysbolism
associated with calcium or phosphoric acid, such as
osteoporosis.
[0026] Yet, currently no satisfactory pharmaceutical approaches to
managing bone defects are available. Bone fractures are still
treated exclusively using casts, braces, anchoring devices and
other strictly mechanical means. Further bone deterioration
associated with osteoporosis has been treated with estrogens or
bisphosphonates, which may have drawbacks for some individuals.
[0027] Although the Bone Morphogenic Proteins (BMPs) are potent
stimulators of bone formation in vitro and in vivo, there are
disadvantages to their use as therapeutic agents to enhance bone
healing. Receptors for the bone morphogenetic proteins have been
identified in many tissues, and the BMPs themselves are expressed
in a large variety of tissues in specific temporal and spatial
patterns. This suggests that BMPs may have effects on many tissues
in addition to bone, potentially limiting their usefulness as
therapeutic agents when administered systemically. Moreover, since
they are peptides, they would have to be administered by injection.
These disadvantages impose severe limitations to the development of
BMPs as therapeutic agents.
[0028] The fluorides, suggested also for the purpose of enhancing
bone formation, have a mode of action which may be related to
tyrosine phosphorylation of growth factor receptors on osteoblasts,
as described, for example, Burgener et al. J Bone Min Res (1995)
10:164-171, but administration of fluorides is associated with
increased bone fragility, presumably due to adverse effects on bone
mineralization.
[0029] Parathyroid hormone, currently considered the leading agent
for metabolic enhancement of bone formation, is inherently
problematic, since it is only administered by injection.
[0030] Thus, although various approaches have been tried, such as
described above, there remains a need for additions to the
repertoire of agents which can be used to treat these
conditions.
[0031] There is thus a widely recognized need for, and it would be
highly advantageous to have novel effective bone growth modulating
agents acting through normal signaling pathways, which can be used
to treat these conditions. Accordingly, the present invention
provides novel methods, pharmaceutical compositions and articles of
manufacture for modulating bone growth and for treating or
preventing bone defects based on regulating cannabinoid
receptors.
SUMMARY OF THE INVENTION
[0032] According to one aspect of the present invention there is
provided a method of modulating bone growth and/or bone remodeling,
comprising regulating an expression or activity of at least one
cannabinoid receptor, thereby modulating bone growth and/or bone
remodeling.
[0033] According to another aspect of the present invention there
is provided a method of treating or preventing a bone disease in a
subject in need thereof, comprising regulating an expression or
activity of at least one cannabinoid receptor of the subject,
thereby treating or preventing the bone disease in the subject.
[0034] According to yet another aspect of the present invention
there is provided a method of inducing bone growth and/or repair in
a subject in need thereof, comprising: (a) isolating bone cells;
(b) regulating an expression or activity of at least one
cannabinoid receptor of the bone cells; and (c) administering the
bone cells resulting from step (b) to the subject, thereby inducing
bone growth or repair in the subject.
[0035] According to further features in preferred embodiments of
the invention described below, the subject is a vertebrate.
[0036] According to yet further features in preferred embodiments
of the invention described below, the vertebrate is a human.
[0037] According to further features in preferred embodiments of
the invention described below, the molecule which prevents
activation or ligand binding of the bone cell or bone cell
progenitor cannabinoid receptor is SR-141761A.
[0038] According to still further features in preferred embodiments
of the invention described below, the subject suffers from a
condition or disease selected from the group consisting of
osteoporosis, bone fracture or deficiency, primary or secondary
hyperparathyroidism, osteoarthritis, periodontal disease or defect,
an osteolytic bone disease, post-plastic surgery, post-orthopedic
implantation, and post-dental implantation.
[0039] According to further features in preferred embodiments of
the invention described below, the method further comprising
administering to the subject at least one compound capable of
promoting bone formation and/or inhibiting bone resorption.
[0040] According to yet further features in preferred embodiments
of the invention described below, the at least one compound is
selected from the group consisting of a bone morphogenetic protein,
an anti-resorptive agent, an osteogenic factor, a cartilage-derived
morphogenetic protein, a parathyroid hormone, IGF1, FGF, a noggin,
an osteogenic growth peptide, a growth hormone, an estrogen, a
bisphosphonate, a statin and a differentiating factor.
[0041] According to still further features in preferred embodiments
of the invention described below, the subject suffers from a
condition or disease selected from the group consisting of Paget's
disease, osteoblastic bone disease, blastic metastatic bone cancer,
metastatic bone disease, Hodgkin's lymphoma, degenerative sclerosis
and osteomyelitis.
[0042] According to still further features in preferred embodiments
of the invention described below, the method further comprising
administering to the subject at least one compound capable of
inhibiting bone formation and/or promoting bone resorption.
[0043] According to still another aspect of the present invention
there is provided a pharmaceutical composition for modulating bone
growth and/or bone remodeling, comprising an agent capable of
regulating an expression or activity of at least one cannabinoid
receptor of a bone cell, a compound capable of modulating bone
growth and/or bone remodeling, and a pharmaceutically acceptable
carrier.
[0044] According to yet another aspect of the present invention
there is provided an article-of-manufacturing, comprising a
packaging material and a therapeutically effective amount of a
pharmaceutical composition being identified for the treatment of a
bone disease or a bone defect, the pharmaceutical composition
including an agent capable of regulating activity or expression of
at least one cannabinoid receptor and a pharmaceutically acceptable
carrier.
[0045] According to further features in preferred embodiments of
the invention described below, the at least one compound is
selected from the group consisting of a bone morphogenetic protein,
an anti-resorptive agent, an osteogenic factor, a cartilage-derived
morphogenetic protein, a parathyroid hormone, IGF1, FGF, a noggin,
an osteogenic growth peptide, a growth hormone, an estrogen, a
bisphosphonate, a statin and a differentiating factor.
[0046] According to yet further features in preferred embodiments
of the invention described below, the pharmaceutical composition
comprising at least one compound capable of inhibiting bone
formation or promoting bone resorption.
[0047] According to an additional aspect of the present invention
there is provided a method of identifying a bone growth modulating
agent, comprising screening a plurality of molecules to thereby
uncover a molecule capable of regulating an expression or activity
of at least one cannabinoid receptor, the molecule being the bone
growth modulating agent.
[0048] According to further features in preferred embodiments of
the invention described below, the method further comprising
determining an ability of the molecule to modify bone formation
rate and/or altering bone mineralization perimeter.
[0049] According to yet further features in preferred embodiments
of the invention described below the screening is effected by
exposing bone cells to the plurality of molecules and determining
the expression of at least one cannabinoid receptor in the bone
cells.
[0050] According to further features in preferred embodiments of
the invention described below, the at least one cannabinoid
receptor is a bone cell or bone cell progenitor cannabinoid
receptor.
[0051] According to yet further features in preferred embodiments
of the invention described below, expression of the cannabinoid
receptor is determined by RT-PCR or real time RT-PCR.
[0052] According to yet further features in preferred embodiments
of the invention described below, the bone cell progenitor is an
osteogenic cell, a stromal cell or a bone resorbing cell
progenitor.
[0053] According to still further features in preferred embodiments
of the invention described below, the regulating is upregulating,
wherein the upregulating of the expression or activity is effected
by an agent, or administering to the subject at least one agent
selected from the group consisting of (a) an exogenous
polynucleotide sequence designed and constructed to express at
least a functional portion of the at least one cannabinoid
receptor; (b) a compound which increases an expression of an
endogenous DNA or mRNA encoding the at least one cannabinoid
receptor; and (c) a molecule which activates the at least one
cannabinoid receptor.
[0054] According to further features in preferred embodiments of
the invention described below, the molecule which activates the at
least one cannabinoid receptor is a cannabinoid.
[0055] According to still further features in preferred embodiments
of the invention described below, the cannabinoid is selected from
the group consisting of .DELTA..sup.9-THC, .DELTA..sup.8-THC,
.DELTA..sup.9-THC-dimethylheptyl, HU-210, 5'-F-.DELTA..sup.8-THC,
11-OH-cannabinol, .DELTA..sup.8-THC-1'-oic-dimethylheptyl acid,
JWH-051, 11-Hydroxy THCs, desacetyl-L-nantradol,
11-OH-cannabinol-dimethylheptyl, cannabinol-dimethylheptyl-11-oic
acid, HU-308, HU 243, L-759633, L-759656, L-768242, JWH-133,
JWH-139, JWH-051, JWH-015, CP55940, CP47497, CP55244,
R-(+)-WIN55212, ACEA, ACPA, O-1812, anandamide, 2AG,
2-arachidonoylglyceryl ether and methanandamide.
[0056] According to still further features in preferred embodiments
of the invention described below, the cannabinoid is HU-308.
[0057] According to still further features in preferred embodiments
of the invention described below, the cannabinoid is 2AG.
[0058] According to yet further features in preferred embodiments
of the invention described below, the regulating is downregulating,
wherein the downregulating of the expression or activity is
effected by an agent, or administering to the subject at least one
agent selected from the group consisting of: (a) a molecule which
binds the at least one cannabinoid receptor; (b) an enzyme which
cleaves the at least one cannabinoid receptor; (c) an siRNA
molecule capable of inducing degradation of mRNA transcripts of the
at least one cannabinoid receptor; (d) a DNAzyme which specifically
cleaves mRNA transcripts or DNA of the at least one cannabinoid
receptor; (e) an antisense polynucleotide capable of specifically
hybridizing with an mRNA transcript encoding the at least one
cannabinoid receptor; (f) a ribozyme which specifically cleaves
mRNA transcripts encoding the at least one cannabinoid receptor;
(g) a non-functional analogue of at least a binding portion of the
at least one cannabinoid receptor; and (h) a molecule which
prevents activation or ligand binding of the at least one
cannabinoid receptor.
[0059] According to still further features in preferred embodiments
of the invention described below, the regulating of the expression
or activity is effected by upregulating a first cannabinoid
receptor of the at least one cannabinoid receptor and
downregulating a second cannabinoid receptor of the at least one
cannabinoid receptor.
[0060] According to further features in preferred embodiments of
the invention described below, the at least one cannabinoid
receptor is selected from the group consisting of a CB1 receptor, a
CB1-like receptor, a CB2 receptor, a CB2-like receptor and a
non-CB1 non-CB2 receptor.
[0061] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
methods of modulating bone growth and, remodeling, methods of
treating or preventing bone diseases, methods of inducing bone
growth and peak bone mass or repair by regulation of the expression
or activity of cannabinoid receptors in bone cells. Specifically,
the cannabinoid receptor-mediated effects, acting on both
osteoblast (bone forming) and osteoclast (bone resorbing)
activities, provide new methods for prevention as well as
therapeutic intervention in diverse bone diseases. Also provided
are pharmaceutical compositions for modulating bone growth and/or
bone remodeling, articles of manufacturing and methods of
identifying bone growth modulating agents based on cannabinoid
receptor-mediated effects.
[0062] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0064] In the drawings:
[0065] FIGS. 1A-B illustrate qualitative micro-computed tomography
(ICT) of femora of male CB1.sup.-/- [CB1 cannabinoid receptor
knockout (deficient) mice] and of the wild type control (WT). FIGS.
1A and 1B show three dimensional .mu.CT images indicating a marked
decrease of trabecular bone-volume density in the distal femoral
metaphysis of CB1.sup.-/- (FIG. 1A), as well as diminishing of the
trabecular connectivity in the CB1.sup.-/- mice (FIG. 1A). The
images were taken from representative femora with median trabecular
bone volume density values.
[0066] FIGS. 2A-E illustrate comparative morphometric analyses of
micro-computed tomography (.mu.CT) of femora of male CB1.sup.-/-
[CB1 cannabinoid receptor knockout (deficient) mice] and of the
wild type control (WT). FIG. 2A (top left) shows a significant
increase of cortical thickness in CB1.sup.-/- mice (p=0.012); FIG.
2B (top right) shows a significant decrease in medullary space
volume in CB1.sup.-/- mice (p=0.003); FIG. 2C (bottom left) shows a
significant decrease of trabecular bone volume in CB1.sup.-/- mice
(p=0.018); FIG. 2D (bottom middle) shows a significant decrease of
trabecular number decrease in CB1.sup.-/- mice (p=0.0003); and FIG.
2E (bottom right) shows a significant decrease of trabecular
connectivity in CB1.sup.-/- mice (p=0.0002). The error bars
indicate .+-.standard error.
[0067] FIGS. 3A-D illustrate micro computed-tomographic (.mu.CT)
morphometric analyses of femora of female CB1 receptor-knockout
(deficient) mice (CB1.sup.-/-) in comparison with female wild type
control (WT). The error bars indicate .+-.standard error and the
asterisks indicate statistical significance. FIG. 3A (top left)
shows a significant decrease of trabecular bone volume in
CB1.sup.-/- mice; FIG. 3B (top right) shows a non-significant
decrease of trabecular connectivity in CB1.sup.-/- mice; FIG. 3C
(bottom left) shows a significant decrease of trabecular number in
CB1.sup.-/- mice; while FIG. 3D (bottom right) shows a significant
increase of trabecular spacing in CB1.sup.-/- mice.
[0068] FIGS. 4A-D illustrate .mu.CT morphometric analyses of femora
of male FAAH.sup.-/- [fatty-acid amide hydrolase (FAAH) knockout
(deficient) mice] and of the wild type control (WT). The error bars
indicate .+-.standard error. FIG. 4A (top left) shows a significant
decrease the cortical thickness in FAAH.sup.-/- mice (p=0.049);
FIG. 4B (top right) shows a non-significant decrease of trabecular
bone volume FAAH.sup.-/- mice; while FIG. 4C (bottom left) show a
significant increase of medullary space volume in FAAH.sup.-/-
mice. FIG. 4D shows a linear regression analysis between the bone
cortical thickness (Ct. Th.) and the medullary space volume
(MV/TV), indicating a significant inverse correlation (r=-0.985,
p=0.005).
[0069] FIG. 5 illustrates RT-PCR expression analyses of CB2
cannabinoid receptor, fatty acid amide hydrolase (FAAH),
parathyroid hormone receptor (PTHRc1) and tissue-nonspecific
alkaline phosphatase (ALP), in differentiating osteoblast
progenitor cells. Note that the RT-PCR analyses of ST2 bone-marrow
derived stromal progenitor cells (left panel) reveals CB2 gene
expression from as early as 5 days in osteogenic medium, while the
RT-PCR analyses of MC3T3 E1 calvaria-derived osteoblast cells
(right panel) indicate a much later appearance of CB2 receptor
expression (10 and 20 days).
[0070] FIGS. 6A-C illustrate expression of the cannabinoid receptor
CB2 and of fatty-acid amide hydrolase (FAAH) in differentiating
osteoclasts. FIG. 6A (left panel) is a micrograph of femoral
monocytes cultured in osteoclast differentiation medium containing
M-CSF and RANKL (osteoclast differentiating factors). FIG. 6B
(right panel) is a micrograph of differentiating osteoclasts
stained with tartarate-resistant acid phosphatase. Differentiated
osteoclasts are stained red to pink red color. FIG. 6C (bottom) is
a RT-PCR analysis which illustrates a positive expression of CB2
and of FAAH in both cultured monocytes and differentiated
osteoclasts.
[0071] FIGS. 7A-D illustrate qualitative and histomorphometric
analyses of mice treated or untreated with the endocannabinoid
2-arachidonoyl glycerol (2AG). FIG. 7A (top left) shows a
significant positive dose response effect of 2AG on bone formation
rate (p=0.04). FIG. 7B (top right) shows representative fluorescent
histological images of 2AG treated (2AG) and untreated (vehicle)
mice, revealing the incorporation of calcein staining into sites of
bone formation. Note the increased density of fluorescent calcein
staining, indicating the higher density of mineralization fronts,
in the 2AG-treated mice (right panel). The bone tissue of a 2AG
treated mouse (right image) appears substantially denser than a
similar bone tissue of an untreated mouse (left image). FIG. 7C
(bottom left) shows a similar significant positive dose response
effect of 2AG on mineralizing perimeter (p=0.019). However, no
significant effect of 2AG on mineral appositional rate was evident
(FIG. 7D, bottom right).
[0072] FIGS. 8A-F illustrate low trabecular bone mass/high bone
turnover phenotype in CB2.sup.-/- mice. FIG. 8A is a .mu.CT
analysis of a distal femoral metaphysis showing trabecular bone
volume density as percent trabecular network of total metaphyseal
volume (BV/TV). Horizontal lines indicate proximal and distal
borders of metaphyseal reference compartment. FIG. 8B shows a
tri-dimensional trabecular bone structure in 51 week old male mice.
FIG. 8C shows trabecular number per mm.sup.3 of metaphyseal
reference volume (Tb.N). Empty circles, CB2.sup.-/- mice; filled
circles, control mice. FIGS. 8D-F illustrate histomorphometric
analyses performed in distal femoral metaphysis of 8-week old
female mice. FIG. 8D shows osteoclast number per trabecular
mm.sup.2 of trabecular surface area. Osteoclasts were identified
using TRAP staining. FIG. 8E shows a trabecular mineral
appositional rate (MAR). FIG. 8F shows bone formation rate (BFR).
Parameters in FIGS. 8E-F were determined using vital double
labelling with calcein fluorochrome. Quantitative data are mean
value: .+-.SE; * indicates significant difference at p<0.05.
[0073] FIG. 9 is a .mu.CT analysis illustrating cortical expansion
in femoral mid-diaphysis of 8-week old CB2.sup.-/- mice.
Quantitative data are mean values.+-.SE; * indicates significant
difference at p<0.05.
[0074] FIGS. 10A-C illustrate a CB2 expression in normal bone. FIG.
10A is a real-time RT-PCR for CB2 of osteoblast differentiation
markers in stromal cells derived from murine femoral diaphyseal
bone marrow undergoing osteoblastic differentiation in osteogenic
medium (9). NOM, cells grown for 20 days in non-osteogenic medium;
RUNX2, runt-related transcription factor 2; TNSALP, tissue
non-specific alkaline phosphatase; PTHRc1 parathyroid
hormone/parathyroid hormone-related protein receptor 1. FIGS. 10B-C
illustrate immunohistochemical localization of CB2-positive
osteoblasts (arrowheads), osteocytes (double arrowhead) and
osteoclasts (arrows) in distal femoral metaphysis of WT mice (FIG.
10B), but not of CB2.sup.-/- mice (FIG. 10C).
[0075] FIGS. 11A-B illustrate the mitogenic effect of the CB2
specific agonist HU-308 on partially differentiated preosteoblasts.
FIG. 11A shows diaphyseal derived bone marrow stromal cells.
Osteogenic growth peptide is a osteoblastic mitogen (22); noladin
ether is a specific CB1 agonist (17). FIG. 11B shows pertussis
toxin (PTX)-induced inhibition DNA synthesis of a MC3T3 E1 cell DNA
24 her following HU-308 treatment. Cells were grown for 10 days in
osteogenic medium prior to HU-308 treatment. Data are mean
values.+-.SE obtained in triplicate culture wells per
condition.
[0076] FIGS. 12A-C illustrate the stimulating effect of CB2
specific agonist HU-308 on osteoblastic activity of mature MC3T3 E1
cells. FIGS. 12A-C show cells DNA content (indicative of cell
density), TNSALP activity and accumulation of external minerals
(indicative of osteoblastic activity), respectively. Cells were
grown for 20 days in osteogenic medium supplemented with HU-308
during the last 14 days of incubation. Data are mean values.+-.SE
obtained in triplicate culture wells per condition.
[0077] FIGS. 13A-B illustrate the inhibiting effect of CB2 specific
agonist HU-308 on osteoclastogenesis. FIG. 13A shows TRAP-positive
multinucleated osteoclastogenic-primary bone marrow derived
monocytes which have been cultured for 5 days in medium
supplemented with M-CSF and RANKL. FIG. 13B shows RAW 264.7 cells
which have been cultured for 7 days in growth medium supplemented
with RANKL. Data are mean value: .+-.SE obtained in triplicate
culture wells per condition.
[0078] FIGS. 14A-D illustrate the attenuating effect of CB2
specific agonist HU-308 on OVX-induced femoral bone loss in
sexually mature C3H mice. HU-308 was administered to mice at 10
mg/Kg/day over a 4 week period commenced at the time of
ovariectomy. FIG. 14A is a .mu.CT analysis of trabecular bone
volume density. FIG. 14B is a histomorphometric analysis of
osteoclast number. FIG. 14C is a histomorphometric analysis of bone
formation rate. FIGS. 14A-C were analysed in the distal femoral
metaphysis. FIG. 14D shows a mid-diaphyseal .mu.CT analysis (top)
and a histomorphometric analysis. Quantitative microtomograpic and
histomorphometric parameters are as defined in FIGS. 8A-F. Data are
mean values .+-.SE; * indicates significant difference at
p<0.05.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0079] The present invention relates to methods and pharmaceutical
compositions suitable for modulating bone growth and remodeling,
preventing bone diseases and inducing bone growth or repair. The
present invention also relates to methods of identifying bone
growth modulating agents. The principles and operation of the
present invention may be better understood with reference to the
drawings and accompanying descriptions.
[0080] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0081] Cannabinoid receptors belong to the superfamily of G-protein
coupled receptors. They are classified into the predominantly
neuronal CB1 receptors and the predominantly peripheral CB2
receptors. While the effects of CB1 receptors are principally
associated with the central nervous system, CB2 receptors are
believed to have peripheral effects related to immunomodulation and
inflammation. In addition to the CB1 and CB2 receptors, recent
pharmacological evidence indicates possible existence of additional
types of cannabinoid receptors [e.g., Breivogel et al., Mol
Pharmacol 60: 155-163 (2001); Calignano A., Eur J Pharmacol 419:
191-198 (2001); Jarai et al., Proc Natl Acad Sci USA 96:
14136-14141 (1999); and Di Marzo et al., J. Neurochem 75: 2434-2444
(2000)].
[0082] Upregulation of CB1 receptors inhibits transmitter release,
while upregulation of CB2 receptors inhibits monocyte/macrophage
activity and the release of inflammatory cytokines [Howlett et al.,
Pharmacol. Rev. 54:161-202 (2002)]. Accordingly, ligands of
cannabinoids receptors have been described as therapeutic agents
for treating a range of diseases or disorders which relate to this
described function of these receptors.
[0083] While reducing the present invention to practice the present
inventors surprisingly and unexpectedly discovered that cannabinoid
receptors are expressed in bone cells and participate in regulation
of bone formation, remodeling and bone growth (see Examples 1-4 of
the Examples section which follows).
[0084] As used herein, the term "bone growth" is defined as
including all processes resulting in a maintenance of or positive
increase in amount and integrity of bone tissue. Particularly, bone
growth includes bone remodeling, bone formation, mineralization,
etc.
[0085] The expression of cannabinoid receptors in bone tissue, the
regulatory effect of cannabinoid receptors on bone growth and
remodeling and the potential benefit of manipulating expression or
activity of cannabinoid receptors for treating bone diseases have
not been described, nor suggested, in prior art.
[0086] Thus, according to one aspect of the present invention there
is provided a method of modulating bone growth and remodeling. The
method according to this aspect is effected by regulating an
expression or activity of one or more cannabinoid receptors.
[0087] The cannabinoid receptor of the present invention is
preferably a bone cell or bone cell progenitor receptor. As used
herein, the phrase "bone cell" refers to a skeletal tissue cell,
such as, bone, cartilage, tendon, ligament, marrow stroma and
connective tissue cells, including bone resorbing cells such as
marrow monocyte-derived osteoclasts, macrophages and scavenger
cells. As used herein, the term "bone cell progenitor" refers to a
cell that can become committed, or partially committed, to a bone
cell differentiation pathway, including stem cells and bone
resorbing cell progenitors, but does not generally express markers
or function as a mature, fully differentiated cell.
[0088] Preferably, the bone cell progenitor is a stromal or
osteogenic cell, or a bone resorbing cell progenitor. As used
herein, the term "stromal cell" refers to a pluripotent progenitor
cell which is capable of dividing many times, and whose progeny
will give rise to skeletal tissues, including cartilage, bone,
tendon, ligament, marrow stroma and connective tissue [see A.
Caplan J. Orthop. Res. 9:641-50 (1991)]. It will be noted that the
term "stromal cell" also includes mesenchymal cells. As used
herein, the term "osteogenic cell" refers to an osteoblast or a
progenitor osteoblast cell, which give rise to a bone tissue.
[0089] The cannabinoid receptor of the present invention, can be,
for example, a CB1 or CB2 receptor, a CB1-like, or CB2-like
receptor or any other type of cannabinoid receptor [Howlett et al.
Pharmacol. Rev. 54:161-202 (2002)].
[0090] As used herein, the phrase "regulating an expression or
activity" refers to either upregulation or downregulation of
receptor expression or activity, or in select instances
upregulation of one cannabinoid receptor expression or activity and
downregulation of another cannabinoid receptor expression or
activity.
[0091] As is further described hereinunder, up or down regulation
of cannabinoid receptor expression or activity can be utilized to
treat a variety of bone diseases or disorders. Such regulation can
be achieved using a variety of agents and approaches well known to
the ordinary skilled artisan. The section below provides several
examples of such agents starting with a description of agents which
can be used to upregulate activity of cannabinoid receptors.
[0092] One example of an agent capable of upregulating activity of
cannabinoid receptors is a cannabinoid molecule. The term
"cannabinoid" refers to any natural or synthetic agonist of a
cannabinoid receptor, or an analogs or derivative thereof.
[0093] Presently known cannabinoids include, for example,
.DELTA..sup.9-tetrahydrocannabinol (.DELTA..sup.9-THC),
.DELTA..sup.8-THC, .DELTA..sup.9-THC-dimethylheptyl,
11-hydroxy-.DELTA..sup.8-THC-dimethylheptyl (HU-210),
5'-F-.DELTA..sup.8-THC, 11-OH-cannabinol,
.DELTA..sup.8-THC-11-oic-dimethylheptyl acid,
1-deoxy-11-OH-.DELTA..sup.8-THC-dimethylheptyl (JWH-051),
11-Hydroxy THCs, desacetyl-L-nantradol,
11-OH-cannabinol-dimethylheptyl, cannabinol-dimethylheptyl-11-oic
acid, HU-308, HU 243, L-759633, L-759656, L-768242, JWH-133,
JWH-139, JWH-051, JWH-015, CP55940, CP47497, CP55244,
R--H-WIN55212, ACEA, ACPA, 0-1812, arachidonyl ethanolamide
(anandamide), 2-arachidonoylglycerol (2AG), 2-arachidonoylglyceryl
ether, and methanandamide, and analogs or derivatives thereof.
[0094] HU-308
[(+)-(1-a-H-(3-H,5-a-H)-4-(2,6-dimethoxy-4-(1,1-dimethylhept-yl)phenyl-6,-
6 dimethylbicyclo[3.1.1]hept-2-ene-2-carbinol] is having the
formula I described below and can be synthesized as described by
Hanus et al. (Proc. Natl. Acad. Sci. U.S.A. 96: 14228-14233, 1999)
and in U.S. patent application Ser. No. 10/133,153.
##STR00001##
[0095] Additional cannabinoids are described in the references
cited in the background section above.
[0096] Another method of upregulating receptor activity is by
inhibiting metabolism of the endogenous (or exogenous) ligand, as
in the SSRI (selective serotonin reuptake inhibitors) class of
antidepressants or methylxanthine phosphodiesterase inhibitors
upregulation of cAMP-dependent receptor activity. Inhibition of
FAAH, for example (as described in the Examples section below), can
effectively increase cannabinoid receptor activity.
[0097] An agent capable of upregulating expression of a cannabinoid
receptor may be an exogenous polynucleotide sequence designed and
constructed to express at least a functional portion of the
receptor. Accordingly, the exogenous polynucleotide sequence may be
a DNA or RNA sequence encoding a cannabinoid receptor molecule,
capable of modulating bone growth and/or bone remodeling.
[0098] Cannabinoid receptors CB I and CB2 have been cloned from
human, rat and mouse sources [Chakrabarti et al., DNA Sequence 5:
385-388 (1995); Gerard et al., Nucleic Acids Res 18: 7142 (1990);
Griffin et al., J Pharmacol Exp Ther 292: 886-894 (2000); Shire et
al., Bioch Biophys Acta 1307: 132-136 (1996); and Munro et al.,
Nature 365: 61-65 (1993)]. Thus, coding sequences information for
both CB1 and CB2 is available from several databases including the
GenBank database available through
http://www4.ncbi.nlm.nih.gov/.
[0099] To express exogenous cannabinoid receptors in mammalian
cells, a polynucleotide sequence encoding a cannabinoid receptor
(for example, CB1 receptor cDNA: GenBank Accession No. NM007726;
CB2 receptor cDNA: GenBank Accession No. NM001841) is preferably
ligated into a nucleic acid construct suitable for mammalian cell
expression. Such a nucleic acid construct includes a promoter
sequence for directing transcription of the polynucleotide sequence
in the cell in a constitutive or inducible manner. A suitable
promoter can be, for example, a human osteocalcin gene promoter
which is capable of directing bone specific gene expression (see
U.S. Pat. No. 5,948,951), or the human collagenase 1 (MMP-1)
promoter (GenBank Accession No. AF023338). The nucleic acid
construct of the present invention can further include additional
polynucleotide sequences such as for example, sequences encoding
selection markers or reporter polypeptides, sequences encoding
origin of replication in bacteria, sequences that allow for
translation of several proteins from a single mRNA (IRES),
sequences for genomic integration of the promoter-chimeric
polypeptide encoding region and/or sequences generally included in
mammalian expression vector such as pcDNA3, pcDNA3.1(+/-),
pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto,
pCR3.1, which are available from Invitrogen, pCI which is available
from Promega, pBK-RSV and pBK-CMV which are available from
Stratagene, pTRES which is available from Clontech, and their
derivatives.
[0100] An agent capable of upregulating a cannabinoid receptor may
also be any compound which is capable of increasing the
transcription and/or translation of an endogenous DNA or mRNA
encoding the cannabinoid receptor.
[0101] As is mentioned hereinabove, the method according to this
aspect of the present invention also provides downgulation of
expression or activity of at least one cannabinoid receptor.
[0102] One example of an agent capable of downregulating a
cannabinoid receptor is an antibody or antibody fragment capable of
specifically binding a cannabinoid receptor. Preferably, the
antibody specifically binds at least one epitope of a cannabinoid
receptor. Preferably, this epitope resides in an extracellular
portion or most preferably, a ligand binding portion of the
cannabinoid receptor. Examples of anti-cannabinoid receptor
antibodies suitable for use in downregulation of cannabinoid
receptor activity are the specific antibodies for CB1 described by
Katona et al (J. Neurosci 1999; 19:4544-58).
[0103] As used herein, the term "epitope" implies any antigenic
determinant on an antigen to which the paratope of an antibody
binds.
[0104] Epitopic determinants usually consist of chemically active
surface groupings of molecules such as amino acids or carbohydrate
side chains and usually have specific three-dimensional structural
characteristics, as well as specific charge characteristics.
[0105] The term "antibody" as used in this invention includes
intact molecules as well as functional fragments thereof, such as
Fab, F(ab').sub.2, and Fv that are capable of binding to
macrophages. These functional antibody fragments are defined as
follows: (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain; (2) Fab', the
fragment of an antibody molecule that can be obtained by treating
whole antibody with pepsin, followed by reduction, to yield an
intact light chain and a portion of the heavy chain; two Fab'
fragments are obtained per antibody molecule; (3) (Fab').sub.2, the
fragment of the antibody that can be obtained by treating whole
antibody with the enzyme pepsin without subsequent reduction;
F(ab').sub.2 is a dimer of two Fab' fragments held together by two
disulfide bonds; (4) Fv, defined as a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains; and (5)
Single chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain and the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule.
[0106] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference). Specifically, several cannabinoid CB.sub.1 and CB.sub.2
receptor-specific antibodies have been successfully developed and
described by Egertova et al., J Comp Neurol 422: 159-171 (2000);
Tsou et al., Neuroscience 83: 393-411 (1998); Daaka et al., J
Pharmacol Exp Ther 276: 776-783 (1996); Sinha et al., J
Neuroimmunol 82: 13-21 (1998); Waksman et al., J Pharmacol Exp Ther
288: 1357-1366; Galiegue et al., Eur J Biochem 232: 54-61 (1995);
and Carayon et al., Blood 92: 3605-3615 (1998).
[0107] Antibody fragments according to the present invention can be
prepared by proteolytic hydrolysis of the antibody or by expression
in E. coli or mammalian cells (e.g. Chinese hamster ovary cell
culture or other protein expression systems) of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly. These methods are described, for example, by
Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references
contained therein, which patents are hereby incorporated by
reference in their entirety. See also Porter, R. R. [Biochem. J.
73: 119-126 (1959)]. Other methods of cleaving antibodies, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0108] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al.
[Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by [Whitlow and Filpula, Methods
2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et
al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778,
which is hereby incorporated by reference in its entirety.
[0109] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry [Methods, 2: 106-10
(1991)].
[0110] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0111] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0112] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0113] Downregulating of a cannabinoid receptor may also be
effected by an enzyme which cleaves the cannabinoid receptor.
[0114] Another agent capable of downregulating a cannabinoid
receptor is a small interfering RNA (siRNA) molecule. RNA
interference is a two step process. the first step, which is termed
as the initiation step, input dsRNA is digested into 21-23
nucleotide (nt) small interfering RNAs (siRNA), probably by the
action of Dicer, a member of the RNase III family of dsRNA-specific
ribonucleases, which processes (cleaves) dsRNA (introduced directly
or via a transgene or a virus) in an ATP-dependent manner.
Successive cleavage events degrade the RNA to 19-21 by duplexes
(siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore
Curr. Opin. Genetics and Development 12:225-232 (2002); and
Bernstein Nature 409:363-366 (2001)].
[0115] In the effector step, the siRNA duplexes bind to a nuclease
complex to from the RNA-induced silencing complex (RISC). An
ATP-dependent unwinding of the siRNA duplex is required for
activation of the RISC. The active RISC then targets the homologous
transcript by base pairing interactions and cleaves the mRNA into
12 nucleotide fragments from the 3' terminus of the siRNA
[Hutvagner and Zamore Curr. Opin. Genetics and Development
12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119
(2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the
mechanism of cleavage is still to be elucidated, research indicates
that each RISC contains a single siRNA and an RNase [Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
[0116] Because of the remarkable potency of RNAi, an amplification
step within the RNAi pathway has been suggested. Amplification
could occur by copying of the input dsRNAs which would generate
more siRNAs, or by replication of the siRNAs formed. Alternatively
or additionally, amplification could be effected by multiple
turnover events of the RISC [Hammond et al. Nat. Rev. Gen.
2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and
Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For
more information on RNAi see the following reviews Tuschl
ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599
(2002); and Brantl Biochem. Biophys. Act. 1575:15-25 (2002).
[0117] Synthesis of RNAi molecules suitable for use with the
present invention can be effected as follows. First, the
cannabinoid receptor mRNA sequence is scanned downstream of the AUG
start codon for AA dinucleotide sequences. Occurrence of each AA
and the 3' adjacent 19 nucleotides is recorded as potential siRNA
target sites. Preferably, siRNA target sites are selected from the
open reading frame, as untranslated regions (UTRs) are richer in
regulatory protein binding sites. UTR-binding proteins and/or
translation initiation complexes may interfere with binding of the
siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will
be appreciated though, that siRNAs directed at untranslated regions
may also be effective, as demonstrated for GAPDH wherein siRNA
directed at the 5' UTR mediated about 90% decrease in cellular
GAPDH mRNA and completely abolished protein level
(www.ambion.com/techlib/tn/91/912.html).
[0118] Second, potential target sites are compared to an
appropriate genomic database (e.g., human, mouse, rat etc.) using
any sequence alignment software, such as the BLAST software
available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/).
Putative target sites which exhibit significant homology to other
coding sequences are filtered out.
[0119] Qualifying target sequences are selected as template for
siRNA synthesis. Preferred sequences are those including low G/C
content as these have proven to be more effective in mediating gene
silencing as compared to those with G/C content higher than 55%.
Several target sites are preferably selected along the length of
the target gene for evaluation. For better evaluation of the
selected siRNAs, a negative control is preferably used in
conjunction. Negative control siRNA preferably include the same
nucleotide composition as the siRNAs but lack significant homology
to the genome. Thus, a scrambled nucleotide sequence of the siRNA
is preferably used, provided it does not display any significant
homology to any other gene.
[0120] Another agent capable of downregulating a cannabinoid
receptor is a DNAzyme molecule capable of specifically cleaving an
mRNA transcript or DNA sequence of the cannabinoid receptor.
DNAzymes are single-stranded polynucleotides which are capable of
cleaving both single and double stranded target sequences (Breaker,
R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S.
W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A
general model (the "10-23" model) for the DNAzyme has been
proposed. "10-23" DNAzymes have a catalytic domain of 15
deoxyribonucleotides, flanked by two substrate-recognition domains
of seven to nine deoxyribonucleotides each. This type of DNAzyme
can effectively cleave its substrate RNA at purine:pyrimidine
junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci.
USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol
Ther 4:119-21 (2002)].
[0121] Examples of construction and amplification of synthetic,
engineered DNAzymes recognizing single and double-stranded target
cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to
Joyce et al. DNAzymes of similar design directed against the human
Urokinase receptor were recently observed to inhibit Urokinase
receptor expression, and successfully inhibit colon cancer cell
metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am
Soc Gen Ther www.asgt.org). In another application, DNAzymes
complementary to bcr-abl oncogenes were successful in inhibiting
the oncogenes expression in leukemia cells, and lessening relapse
rates in autologous bone marrow transplant in cases of CML and
ALL.
[0122] Downregulation of a cannabinoid receptor can also be
effected by using an antisense polynucleotide capable of
specifically hybridizing with an mRNA transcript encoding the
cannabinoid receptor.
[0123] Design of antisense molecules which can be used to
efficiently downregulate a cannabinoid receptor must be effected
while considering two aspects important to the antisense approach.
The first aspect is delivery of the oligonucleotide into the
cytoplasm of the appropriate cells, while the second aspect is
design of an oligonucleotide which specifically binds the
designated mRNA within cells in a way which inhibits translation
thereof.
[0124] The prior art teaches of a number of delivery strategies
which can be used to efficiently deliver oligonucleotides into a
wide variety of cell types [see, for example, Luft J Mol Med 76:
75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et
al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys
Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem
Biophys Res Commun 231: 540-5 (1997)]. Of particular interest is
the method described by Erikkson (U.S. Pat. No. 6,525,030) for
periosteal transformation using microinjection of DNA at the bone
surface.
[0125] In addition, algorithms for identifying those sequences with
the highest predicted binding affinity for their target mRNA based
on a thermodynamic cycle that accounts for the energetics of
structural alterations in both the target mRNA and the
oligonucleotide are also available [see, for example, Walton et al.
Biotechnol Bioeng 65: 1-9 (1999)].
[0126] Such algorithms have been successfully used to implement an
antisense approach in cells. For example, the algorithm developed
by Walton et al. enabled scientists to successfully design
antisense oligonucleotides for rabbit beta-globin (RBG) and mouse
tumor necrosis factor-alpha (TNF alpha) transcripts. The same
research group has more recently reported that the antisense
activity of rationally selected oligonucleotides against three
model target mRNAs (human lactate dehydrogenase A and B and rat
gp130) in cell culture as evaluated by a kinetic PCR technique
proved effective in almost all cases, including tests against three
different targets in two cell types with phosphodiester and
phosphorothioate oligonucleotide chemistries.
[0127] In addition, several approaches for designing and predicting
efficiency of specific oligonucleotides using an in vitro system
were also published (Matveeva et al., Nature Biotechnology 16:
1374-1375 (1998)].
[0128] Several clinical trials have demonstrated safety,
feasibility and activity of antisense oligonucleotides. For
example, antisense oligonucleotides suitable for the treatment of
cancer have been successfully used [Holmund et al., Curr Opin Mol
Ther 1:372-85 (1999)], while treatment of hematological
malignancies via antisense oligonucleotides targeting c-myb gene,
p53 and Bcl-2 had entered clinical trials and had been shown to be
tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306
(1999)].
[0129] More recently, antisense-mediated suppression of human
heparanase gene expression has been reported to inhibit pleural
dissemination of human cancer cells in a mouse model [Uno et al.,
Cancer Res 61:7855-60 (2001)].
[0130] Thus, the current consensus is that recent developments in
the field of antisense technology which, as described above, have
led to the generation of highly accurate antisense design
algorithms and a wide variety of oligonucleotide delivery systems,
enable an ordinarily skilled artisan to design and implement
antisense approaches suitable for downregulating expression of
known sequences without having to resort to undue trial and error
experimentation.
[0131] Another agent capable of downregulating a cannabinoid
receptor is a ribozyme molecule capable of specifically cleaving an
mRNA transcript encoding a cannabinoid receptor. Ribozymes are
being increasingly used for the sequence-specific inhibition of
gene expression by the cleavage of mRNAs encoding proteins of
interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The
possibility of designing ribozymes to cleave any specific target
RNA has rendered them valuable tools in both basic research and
therapeutic applications. In the therapeutics area, ribozymes have
been exploited to target viral RNAs in infectious diseases,
dominant oncogenes in cancers and specific somatic mutations in
genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71
(1998)]. Most notably, several ribozyme gene therapy protocols for
HIV patients are already in Phase 1 trials. More recently,
ribozymes have been used for transgenic animal research, gene
target validation and pathway elucidation. Several ribozymes are in
various stages of clinical trials. ANGIOZYME was the first
chemically synthesized ribozyme to be studied in human clinical
trials. ANGIOZYME specifically inhibits formation of the VEGF-r
(Vascular Endothelial Growth Factor receptor), a key component in
the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well
as other firms have demonstrated the importance of
anti-angiogenesis therapeutics in animal models. HEPTAZYME, a
ribozyme designed to selectively destroy Hepatitis C Virus (HCV)
RNA, was found effective in decreasing Hepatitis C viral RNA in
cell culture assays (Ribozyme Pharmaceuticals, Incorporated--WEB
home page).
[0132] Another agent capable of downregulating a cannabinoid
receptor can be a non-functional analogue of a binding portion of
the cannabinoid receptor. Examples include truncated CB1 or CB2
sequences (lacking for example the N-terminal portion).
[0133] Yet another agent capable of downregulating a cannabinoid
receptor is a molecule which can prevent activation of, or ligand
binding on, the cannabinoid receptor. The molecule may be a
cannabinoid antagonist or an inverse agonist, such as, for example,
SR141716A, SR144528, AM251, AM281, SR144528, LY320135, AM630,
WIN56098, WIN54461, O-1184 and O-1238 [see in Howlett et al.,
Pharmacol. Rev. 54:161-202 (2002)].
[0134] Cannabinoid receptor activity can also be downregulated by
specifically targeting the natural ligand of the cannabinoid
receptor, such as anandamine, 2AG or any other endocannabinoid
capable of binding a cannabinoid receptor in a bone cell.
[0135] As is mentioned hereinabove, regulation of cannabinoid
receptor expression or activity can be upregulation of one or more
cannabinoid receptors, downregulation of one or more cannabinoid
receptors or upregulation of one receptor and downregulation of
another. While the latter scenario has not yet been described in
prior art in any application related to cannabinoid receptor
activity, the results of Example 4 of the Examples section that
follows, suggest that such a case exists with the CB1
cannabinoid-receptor antagonist SR-141761A which may also act as an
agonist of another yet unknown cannabinoid receptor.
[0136] Regulation of cannabinoid receptor expression or activity
may be effected ex vivo by exposing cultured bone cells to an
upregulating or downregulating agent, or in vivo by administering
such an agent to a subject.
[0137] Thus, according to another aspect of the present invention,
there is provided a method of inducing bone growth or repair in a
subject of need thereof.
[0138] The term "subject" used herein refers to human as well as
other animal species, such as, for example, canine, feline, bovine,
porcine, rodent, and the like.
[0139] The phrase "treating or preventing" used herein refers to a
postponement of development of bone deficit symptoms and/or a
reduction in the severity of such symptoms that will or are
expected to develop. These further include ameliorating existing
bone or cartilage deficit symptoms, preventing additional symptoms,
ameliorating or preventing the underlying metabolic causes of
symptoms, preventing or reversing bone resorption and/or
encouraging bone growth. Thus, the phrase denotes that a beneficial
result has been conferred on a vertebrate subject with a cartilage,
bone or skeletal deficit, or with the potential to develop such
deficit. The phrase further refers to a postponement of development
of bone overgrowth symptoms and/or a reduction in the severity of
such symptoms that will or are expected to develop.
[0140] The method can be effected using two alternative approaches.
In a first approach, bone cells are isolated from the subject or an
allogeneic or syngeneic donor and expression or activity of one or
more cannabinoid receptors of these bone cells is either
downregulated or preferably upregulated (or both) as described
above. Once expression or activity is either upregulated or
downregulated cells displaying modified cannabinoid receptor
activity are administered to the subject (preferably via local
injection).
[0141] In a second approach, the agent is directly administered to
the subject via one of several alternative administration modes
(further described hereinbelow).
[0142] The above described approaches can be utilized to treat a
variety of bone related diseases or disorders. For example, agents
capable of upregulating cannabinoid receptor expression or activity
can be used for treating or preventing any bone deficit-related
disease or condition such as, for example, preventing bone defects
and deficiencies in closed, open and non-union fractures;
augmenting bone mass in young individuals at risk; prophylactic
treatment in young individuals by enhancing peak bone mass in
closed and open fracture reduction; promotion of bone healing in
plastic surgery; stimulation of bone ingrowth into non-cemented
post orthopedic and dental implants; elevation of peak bone mass in
pre-menopausal women; treatment of growth deficiencies; treatment
of primary or secondary hyperparathyroidism; treatment of
osteolytic bone disease such as cancer; treatment of periodontal
disease and defects, and other tooth repair processes; increase in
bone formation during distraction osteogenesis; and treatment of
other skeletal disorders, such as age-related osteoporosis,
post-menopausal osteoporosis, glucocorticoid-induced osteoporosis
or disuse osteoporosis and arthritis, osteoarthritis or any
condition that benefits from stimulation of bone formation, on the
one hand, and inhibition of bone resorption, on the other. The
agents of the present invention can also be useful in repair of
congenital, trauma-induced or surgical resection of bone (for
instance, for cancer treatment), and in cosmetic surgery. Further,
the compounds of the present invention can be used for limiting or
treating cartilage defects or disorders, and may be useful in wound
healing or tissue repair.
[0143] Agents capable of downregulating cannabinoid receptors, such
as described hereinabove, can be used for treating or preventing
any bone overgrowth-related disease or condition such as, for
example, certain stages of Paget's disease, an osteoblastic bone
disease, a metastatic bone disease such as breast cancer and
prostate cancer, a blastic metastatic bone cancer, Hodgkin's
lymphoma, degenerative sclerosis and osteomyelitis.
[0144] The agents of the present invention can be in therapy per se
or as part (active ingredient) of a pharmaceutical composition.
[0145] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0146] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0147] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0148] A pharmaceutical composition which includes one or more
cannabinoid receptor upregulating agents may also include one or
more compounds which promote bone formation and/or inhibiting bone
resorption, such as, for example, a bone morphogenic factors, bone
morphogenic protein, parathyroid hormone, noggin, osteogenic growth
peptide, anti-resorptive agents, osteogenic factors,
cartilage-derived morphogenic proteins, growth hormones, cytokines
such as fibroblast growth factor (FGF), insulin-like growth
factor-I (IGF-I), transforming growth factors, estrogens,
bisphosphonates, statin, calcitonin, dihydroxy vitamin D.sub.3, and
calcium preparations are preferred for this purpose.
[0149] Alternatively, a pharmaceutical composition which includes
one or more cannabinoid receptor downregulating agents may also
include one or more compounds which inhibit bone formation and/or
promote bone resorption.
[0150] Further, up- or downregulating agents can be targeted to
bone or other specific sites of activity using targeting
molecules.
[0151] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0152] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0153] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
bone tissue region of a patient.
[0154] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0155] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredients into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0156] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. One route of
administration which is suited for the pharmaceutical compositions
of the present invention is sub-periosteal injection, as described
in U.S. Pat. No. 6,525,030 to Erikkson. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0157] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. As used herein, the term "oral
administration" includes administration of the pharmaceutical
compound to any oral surface, including the tongue, gums, palate,
or other buccal surfaces. Addition methods of oral administration
include provision of the pharmaceutical composition in a mist,
spray or suspension compatible with tissues of the oral
surface.
[0158] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0159] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0160] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0161] For administration by nasal inhalation, the active
ingredients for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation
from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in a dispenser may be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
[0162] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0163] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0164] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0165] The pharmaceutical composition of the present invention may
also be formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides.
[0166] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount of active ingredients (e.g. antisense
oligonucleotide) effective to prevent, alleviate or ameliorate
symptoms of a disorder (e.g., mammary tumor progression) or prolong
the survival of the subject being treated.
[0167] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0168] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in an animal model, such as the murine Neu
model [Muller et al., Cell 54, 105-115 (1988)], to achieve a
desired concentration or titer. Other such exemplary model system
suitable for use with the methods of the present invention are
differentiating osteoclasts (see Example 3 hereinbelow), and
differentiating cultured osteogenic cells (see Example 2
hereinbelow). Such information can be used to more accurately
determine useful doses in humans.
[0169] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).
[0170] Dosage amount and interval may be adjusted individually to
levels of the active ingredient which are sufficient to, for
example, retard tumor progression in the case of blastic metastases
(minimal effective concentration, MEC). The MEC will vary for each
preparation, but can be estimated from in vitro data. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. Detection assays can
be used to determine plasma concentrations.
[0171] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or diminution of the disease state is
achieved.
[0172] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0173] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition, as if further detailed
above.
[0174] In order to facilitate practice of the methods described
hereinabove, and/or production of pharmaceutical compositions and
articles of manufacture as described hereinabove, the present
invention further provides a method of identifying novel bone
growth and remodeling modulating agents.
[0175] The method of identifying a drug candidate includes
screening a plurality of molecules for a molecule capable of
regulating an expression or activity of one or more cannabinoid
receptors of bone cells. Screening may be accomplished in vitro by
exposing cultured bone cell progenitors, such as murine bone
marrow-derived osteoprogenitor cells (ST2 cell line) or
calvaria-derived osteoblastic cells (MC3T3 E1 cell line) to test
molecules followed by Reverse Transcription Polymerase Chain
Reaction (RT-PCR) analysis for cannabinoid receptors expression,
using a standard RT-PCR procedure, such as described in Example 2
of the Examples section which follows. Selected molecules may be
further evaluated for a bone-growth modulating activity in vivo by
administering selected test molecules to laboratory animals
followed by determining their effect on bone growth in the treated
animals. For example, a test molecule may be dissolved in 1:1:18
ethanol:emulfor:saline (v/v/v) vehicle and injected
intraperitoneally to a C3H (Harlan) mouse, using a protocol such as
described in Example 4 of the Examples section below. The efficacy
of the test molecules may be determined by comparing the bone
growth rate and/or bone mineralization perimeter in the treated
mice with the bone growth parameters in similar untreated mice.
Molecules which induce significant stimulation, or inhibition, of a
bone growth parameter become candidate for additional evaluations
as bone-growth modulating agents.
[0176] Thus, the present invention provides novel methods,
compositions and articles of manufacture for use in treatment or
prevention of bone diseases. Since the present invention is based
on natural specific mechanisms of modulating new bone growth, it
can be applied to treat or to prevent a wide range of bone
deficit-related as well as bone overgrowth-related diseases safely
and effectively.
[0177] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0178] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Example 1
Effect of CB1 Cannabinoid Receptor Expression in Bone Cells: CB1
Expression Regulates Bone Growth and Remodeling
[0179] Materials and Methods
[0180] Animals: C57BL/6J mice [Zimmer, A. et al. Proc. Natl. Acad.
Sci. USA. 96: 5780-5785 (1999)] were used as wild type control (WT)
and were compared with CB1 receptor knockout mice (CB1.sup.-/-)
(Ledent et al., Science 283: 401-404 (1999)] or with fatty-acid
amide hydrolase knockout mice (FAAH.sup.-/-) [Cravat et al., PNAS
USA 2001; 198:9371-76 (2001)].
[0181] Micro-computed tomographic (.mu.CT) analysis: Whole femora
were examined by a .mu.CT system (.mu.CT 40, Scanco Medical AG,
Basserdorf, Switzerland) equipped with a 5 .mu.M focal spot
microfocus X-ray tube as a source. A two-dimensional CCD, coupled
to a thin scintillator as a detector permitted parallel acquisition
of stacks including 20 tomographic images. The long axis of the
femur was set parallel to the plane of the X-ray beam axis. The
X-ray tube was operated at 50 KVp and 160 .mu.A. The integration
time was set to 100 ms. The scans were performed at a resolution of
20 .mu.m in all three spatial dimensions (medium resolution mode).
Two-dimensional CT images were reconstructed in 1024.times.1024
pixel matrices from 1000 projections using a standard
convolutionbackprojection procedure with a Shepp and Logan filter.
Images were stored in 3-D arrays with an isotropic voxel size of 20
.mu.m. A constrained 3-D Gaussian filter (width: .sigma.=0.8,
support: one voxel width) was used to partly suppress the noise in
the volumes. The samples were binarized using a global thresholding
procedure (12). The threshold was set to 22.4% and 16.0% of the
maximal gray scale value for cortical bone and trabecular bone,
respectively. Morphometric parameters were determined using a
direct 3-D approach (13). Trabecular bone parameters were measured
in a metaphyseal segment, extending proximally from the proximal
tip of the primary spongiosa to the proximal border of the distal
femoral quartile. Cortical bone parameters were determined in a
diaphyseal segment extending 1.12 mm distally from the midpoint
between the femoral ends.
[0182] Femora obtained from ten week old CB1-/- and WT mice were
sampled from ten weeks old mice since the WT mice reach their peak
trabecular bone mass at this age. The representative .mu.CT images
displayed were obtained from mice with median bone volume density
or cortical thickness values, and were thus representative. Data
shown are mean.+-.standard error (SE) obtained from 8 mice
replications.
[0183] Results
[0184] Comparative three-dimensional .mu.CT images of the secondary
spongiosa in male CB1 knockout (deficient) (CB1.sup.-/-) and
wild-type (WT) mice are illustrated in FIGS. 1A and B. These images
show a substantial decrease of trabecular network density and a
substantial increase of bone-marrow spaces in the CB1.sup.-/-
mice.
[0185] The .mu.CT morphometric analyses of male CB1.sup.-/- mice as
compared with the WT are summarized in FIGS. 2A-E. These Figures
corroborate the results shown in FIGS. 1A and B: the CB1.sup.-/-
mice developed a significantly higher cortical thickness [FIG. 2A;
p=0.012 (t test)]; a significantly lower medullary space volume
[FIG. 2B; p=0.003 (t test)]; a significantly lower trabecular bone
volume density [FIG. 2C; BV/TV=11.5.+-.1.7% vs. 18.8.+-.2.1%
(mean.+-.SE), p=0.018 (t-test)]; a significantly lower trabecular
number [FIG. 2D; Tb.N.=1.9.+-.0.2 mm-1 vs. 3.0.+-.0.1 mm-1
(mean.+-.SE), p=0.0003 (t-test)]; and a significantly lower
trabecular connectivity [FIG. 2E; bottom right graph;
Conn.D=21.6.+-.3.6 mm-3 vs. 48.5.+-.3.9 mm-3 (mean.+-.SE), p=0.0002
(t-test)] as compared to their age-matched wild type (WT)
controls.
[0186] The .mu.CT morphometric analyses of female CB1.sup.-/- as
compared with the WT are illustrated in FIGS. 3A-D. These analyses
show that the metaphyseal changes observed in the female
CB1.sup.-/- were similar to those seen with male CB1.sup.-/-, i.e.,
having a decreased trabecular bone volume (FIG. 3A), a decreased
trabecular connectivity (FIG. 3B), decreased trabecular number
(FIG. 3C), and an increased trabecular spacing (FIG. 3D).
[0187] The .mu.CT morphometric analyses of male fatty acid amide
hydrolase knockout (deficient) mice (FAAH.sup.-/-), having impaired
endocannabinoid degradation, as compared with the WT are
illustrated in FIGS. 4A-D. The Figures show that the FAAH.sup.-/-
mice developed a significantly lower cortical thickness [FIG. 4A,
top left graph; p=0.049 (t-test)]; and a significantly higher
medullary space volume [FIG. 4C, left bottom graph; p=0.049
(t-test)]. These effects were expectedly the opposite from what was
observed with CB1.sup.-/- mice (see FIGS. 4B and 4C above) since
the FAAH enzyme is known to degrade endocannabinoides (Cravatt et
al., PNAS USA 198:9371-76 (2001)]).
[0188] These results clearly show that CB1 knockout (deficient)
mice have a substantial disruption of the trabecular structural
integrity with possible severe consequences to the bone load
bearing capacity, as compared with wild type controls. Because the
body weight and neurological sign score of the CB1.sup.-/- mice do
not differ from those of their WT littermates, it appears that this
osteopenic phenotype of the CB1 knockout (deficient) mice is not
secondary to impaired food intake or physical activity.
[0189] Hence, these results clearly demonstrate that CB1 receptor
expression regulates bone growth and remodeling in mice, and that
cannabinoids capable of regulating CB1 expression or activity can
thereby modulate the bone growth and remodeling.
Example 2
Expression of Cannabinoid Receptors in Differentiating Cultured
Osteogenic Cells
[0190] Materials and Methods
[0191] Cell cultures: the expression of cannabinoid receptors
(Cnrs) CB1 and CB2, endocannabinoid-degrading enzyme fatty-acid
amide hydrolase (FAAH), osteoblast differentiation-marker alkaline
phosphatase (ALP), and osteoblast differentiation-marker
parathyroid hormone-receptor I (PTH-Rc1), were analyzed in murine
bone marrow-derived osteoprogenitor cells (ST2 cell line) and in
calvaria-derived osteoblastic cells (MC3T3 E1 cell line).
Osteoblastic differentiation of these cells was induced by growing
them in "osteogenic medium" which contains ascorbic acid,
dexamethasone and .beta.-glycerophosphate [Frank et al. J. Cell.
Biochem. 85: 737-746 (2002)].
[0192] Reverse Transcription Polymerase Chain Reaction (RT-PCR):
RNA was extracted from cultured cells after 5, 10, 20 and 30 days
of incubation and was analyzed by RT-PCR. The primers and RT-PCR
methodology and conditions used for analyzing the expression
receptor CB1 were essentially as described by: Noe et al. [Adv. Exp
Med. Biol. 437:223-9 (1998)], and by Noe et al. [Adv. Exp. Med.
Biol. 493:215-21 (2001]. The primers and RT-PCR methodology and
conditions used for analyzing the expression receptor CB2 were
essentially as described by Lee et al. [Eur. J. Pharmacol.
423:235-41 (2001). The primers and RT-PCR methodology and
conditions used for analyzing the expression of osteoblast markers
tissue-nonspecific alkaline phosphatase receptor (ALP) were
essentially as described by Ohkubo et al. [Br J. Pharmacol.
131:1667-1672 (2000). The primers and RT-PCR methodology and
conditions used for analyzing the expression of parathyroid hormone
receptor I (PTH-Rc1; 12) were essentially as described by Kato et
al. [J. Bone Min. Res. 16:1622-1633 (2001), as follows: CB1, sense:
5'-TGGTGTATGATGTCTTTGGG-3' (SEQ ID NO:1), antisense:
5'-ATGCTGGCTGTGTTATTGGC-3' (SEQ ID NO:2); CB2, sense:
5'-AACGGTGGCTTGGAGTTCAAC-3' (SEQ ID NO: 3); antisense:
5-`TAGGTAGCGGTCAACAGCG-GTTAG (SEQ ID NO: 4); FAAH, sense:
5`-GCCTGAAAGCTCTACTGTGTGAGC-3' (SEQ ID NO: 5); antisense:
5'-GAAGGTCCAGACTTGGTTGTGGCT-3' (SEQ ID NO: 6), ALP (Accession No.
J02980), sense: 5'-GACA-CAAGCATTCCCACTAT-3' (967.+-.986) (SEQ ID
NO: 7); antisense: 5'-ATCAG-CAGTAACCACAGTCA-3' (1316.+-.1297) (SEQ
ID NO: 8); parathyroid hormone (PTH-Rc1), sense:
5'-CAAGAAGTGGATCATCCAGGT-3'(SEQ ID NO: 9); antisense:
5'-GCTGCTACTCCCACTTCGTGCTTT-3' (SEQ ID NO: 10).
[0193] Results
[0194] After 5 days in culture with the osteogenic medium, both the
ST2 stromal progenitor calls and the MC3T3 calvaria derived
preosteoblast cells exhibited minimal or no expression of CB1 or
CB2 cannabinoid receptors. This was followed by a temporal increase
to a considerably high steady level of CB2 mRNA expression by day
(FIG. 5). On the other hand, no expression of CB1 mRNA was evident
even in the most differentiated cells (data not shown). The CB2
expression pattern was similar to the temporal expression patterns
of FAAH, ALP and PTH-Rc1 (FIG. 5) which are consistent with
osteoblastic differentiation.
[0195] Hence, the positive expression of cannabinoid receptor CB2,
and FAAH in differentiating progenitor osteoblasts as shown,
indicates that developing osteoblasts become sensitive to
endocannabinoid signaling early in osteogenesis, and that this
upregulation of CB2 receptor expression, and endocannabinoid
receptor activity may be crucial to induction of osteoblast
differentiation and bone formation.
Example 3
Expression of Cannabinoid Receptor CB2 and FAAH in Differentiating
Osteoclasts
[0196] Materials and Methods
[0197] Mouse femoral monocytes were separated on a Ficoll gradient
and grown in culture for 5 days in medium containing the osteoclast
differentiation factors M-CFS and RANKL as described by Zou et al
FASEB J 2002; 16:274-82). At the end of incubation cultures were
analyzed for CB2 and FAAH expression by RT-PCR as described above,
and were stained with an osteoclast marker tartarate-resistant acid
phosphatase for direct observation of differentiated
osteoclasts.
[0198] Results
[0199] As illustrated in FIG. 6A-C both CB2 and FAAH are expressed
in differentiating osteoclasts and their monocyte precursors. Since
it is known that CB2 expression in monocyte/macrophage cells, which
are osteoclast progenitors, inhibits their cellular activity
(Parolaro et al Life Sci 1999; 65:637-44), these results suggest
that CB2 expression in osteoclasts may similarly inhibit
differentiation and cellular activity of the monocyte-derived
osteoclast cells. Thus endocannabinoids, and other cannabinoid
receptor ligands, can activate receptors on both osteoblast and
osteoclast cells, and may on one hand suppress the bone resorptive
activity of osteoclasts, while on the other hand promoting bone
growth and remodeling activity of osteoblasts. Such a ligand,
capable of effectively stimulating bone formation as well as
inhibiting bone resorption, would constitute an ideal therapeutic
agent for treating bone disorders.
Example 4
Effect of Cannabinoids on Bone Formation Activity In Vivo
[0200] Materials and Methods:
[0201] The endocannabinoid 2-arachidonoyl glycerol (2AG) and the
CB1 cannabinoid receptor antagonist
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H--
pyrazole-3-carboxamide hydrochloride (SR-141761A; Panikashvili et
al., Nature 413: 527-531 (2001)] were dissolved in 1:1:18
ethanol:emulfor:saline (v/v/v) vehicle. Different dosages of each
agent were daily injected intraperitoneally to 11 week-old C3H
(Harlan) mice. 2AG was administered at 0 (vehicle control), 0.5, 5,
and 20 mg/Kg body weight per day for 9 days; SR-141761A was
administered at 1 and 10 mg/Kg body weight per day for 9 days.
[0202] To assess the in vivo bone formation activity the mice were
given 15 mg/Kg body weight calcein intrapertoneally, four days and
one day (same day as the last 2AG injection) prior to sacrifice.
Femoral bones were separated immediately after sacrifice and
subjected, undecalcified, to histological processing. Dynamic bone
histomorphometric parameters were analyzed in the distal metaphysis
(secondary spongiosa) in unstained longitudinal mid-saggital
sections, using the procedure described by Parfitt et al. [J. Bone
Miner. Res. 2: 595-610 (1987)]. The data obtained were
statistically analyzed by Mann-Whitney U-test to determine
significance of differences between the treatments.
[0203] Results
[0204] The administration of the endocannabinoid 2AG to mice at up
to 5 mg/Kg/day for 9 days resulted in a dose-dependent stimulation
of bone-formation rate (BFR; an expression of overall osteoblastic
activity), followed by a plateau (FIG. 7A). The mice which were
treated with 2AG at 5 and 20 mg/Kg/day showed substantially (up to
44%) and significantly (p=0.04) higher BFRs than the vehicle
control. The osteogenic effect of 2AG administration is also
evident from comparison of fluorescent histological images of the
bones from mice treated with 2AG (5 mg/Kg/day), compared with their
vehicle-treated controls (FIG. 7C), indicated by the visibly
increased uptake of fluorescently labeled calcein in the treated
animal's bone tissue. Similarly, the administration of 2AG at up to
5 mg/Kg/day resulted in a dose-dependent increase of mineralizing
perimeter (MP; an expression of osteoblast number), followed by a
plateau (FIG. 7B). The treatment of 2AG at 5 and 20 mg/Kg/day also
resulted in substantially and significantly higher MPs than the
control (p=0.019). Thus, the administration of effective dosages of
2AG to mice substantially stimulated bone accrual and bone
formation activity in vivo.
[0205] Administration of the CB1 cannabinoid-receptor antagonist
SR-141761A to mice at up to 10 mg/Kg/day for 9 days also resulted
in higher (but not statistically different) BFR (osteoblastic
activity) and in higher and statistically different MP (osteoblast
number), than the vehicle control (data is not shown).
[0206] These results clearly demonstrate that the administration of
the cannabinoid ligands 2AG and SR-141761A to mice substantially
increased the number of osteoblasts and the bone formation rate,
thereby effectively increasing bone accrual and structural
integrity in the treated mice.
Example 5
Modulation of Bone Mass by Administering a Specific Agonist of
CB2
[0207] Materials and Methods:
[0208] Animals: Mice with a deletion of the CNR2 gene (CB2.sup.-/-
mice) (9) were crossed for 10 generations to wild type C57BL/6J
mice to generate a congenic C57BL/6J CB2.sup.-/- strain. The effect
of CB2 signalling on OVX-induced bone loss was analysed in normal
C3H mice (Harlan, Israel) due to their high femoral bone density
(10), which allows for a substantial amount of bone loss to occur.
Because of the low trabecular bone volume density in C57BL/6J
females (FIG. 8A), the absolute amount of OVX-induced bone loss in
these animals is small and a large sample is required to achieve
statistical significance. In addition, the number of calcein
labelled packets (see below) in OVX C57BL/6J mice is often too
small for the calculation of bone formation parameters in the
trabecular compartment. HU-308, synthetic CB2 specific agonist with
a MW of 414 g/mol, was prepared as described by Hildebrand et al.
(11) and injected intraperitoneally to OVX and control mice once
daily as ethanol/emulphor/saline (1:1:18) solution. To study bone
formation, newly formed bone was vitally labelled in all reported
animals by the fluorochrome calcein (Sigma), injected
intraperitoneally (15 mg/Kg) four days and one day prior to
sacrifice. Groups of 8-10 mice, 8-11 or 51 weeks old, were used in
each experiment. The experimental protocols were approved by the
Institutional Animal Care and Use Committee, Faculty of Medicine,
the Hebrew University of Jerusalem, Israel and by the
Regierungsprasidium Koln for the University of Bonn, Germany.
[0209] Micro-computed tomographic (.mu.CT) analysis: The .mu.CT
Analyses were performed as described in Example 1 above.
[0210] Histomorphometry and immunohistochemistry: After .mu.CT
image acquisition, the specimens were embedded undecalcified in
Technovit 9100 (Heraeus Kulzer, Wehrheim, Germany). Longitudinal
sections through the mid-frontal plane were left unstained for
dynamic histomorphometry based on the vital calcein double
labeling. To identify osteoclasts, consecutive sections were
stained for tartrate-resistant acid phosphatase (TRAP) (14).
Parameters were determined according to a standardized nomenclature
(15). Immunohistochemistry was performed using paraffin-embedded
decalcified sections (16) with a polyclonal first antibody raised
against the human CB2(20-33) peptide (Cayman Ann Arbor, Mich.; Cat.
No. 101550). The same peptide (Cayman Ann Arbor, Mich.; Cat. No.
301550) was used to block protein-antibody complex formation in
control staining. The antibody is highly specific for the human and
mouse CB2 and does not cross react with CB1.
[0211] mRNA analysis: Primary osteoblastogenic cultures of stromal
cells were prepared from femoral and tibial diaphyseal bone marrow
of WT C57BL/6J mice and grown in "osteogenic medium" containing
ascorbic acid, .beta.-glycerophophate and dexamethasone (Sigma) as
described by Frenkel et al. (17). Real-time RT-PCR analysis was
carried out using Applied Biosystems Assay-on-Demand. Data was
normalized to GAPDH. Assay ID: GAPDH, Mm99999915_gl; CB1,
Mm00432621_s1; CB2, Mm00438286_ml; PTHRc1, Mm00441046_ml; RUNX2,
Mm00501578_m 1; TNSALP, Mm00475831_ml. RT-PCR analysis was carried
out using the primers described by Lee et al. (19).
[0212] In vitro effect of HU-308: Differentiating primary bone
marrow stromal cells and MC3T3 E1 osteoblasts were initially
incubated in osteogenic medium for 10 days followed by 2 h serum
starvation. Ligands were dissolved in dimethylsulfoxide (DMSO) and
further diluted to their final concentration using tissue culture
medium. In the primary cultures, cells were counted after 48 h
incubation in .alpha.MEM supplemented with 4% BSA and ligand. BrdU
incorporation was determined in MC3T3 E1 cells after 24 h
incubation with HU-308 preceded by 2 h with pertussis toxin (PTX).
DNA content, tissue non-specific alkaline phosphatase (TNSALP)
activity and calcium were determined in MC3T3 E1 cells grown for 20
days in osteogenic medium and supplemented with HU-308 in the last
14 days. The effect of HU-308 on osteoclast differentiation was
measured in the osteoclastogenic system described above,
supplemented with HU-308 dissolved initially in DMSO and diluted
with medium. HU-308 was also tested in a RAW 264.7 cell grown for 7
days in RANKL-supplemented medium. For osteoclast-like cell counts
the cultures were fixed in ethanol and TRAP-stained.
[0213] Statistical Analyses: Differences between CB2.sup.-/- and WT
mice were analysed by t-test. HU-308 and vehicle treated OVX and
sham-OVX mice were analysed by ANOVA. When significant differences
were indicated by ANOVA, group means were compared using the Tukey
test for pairwise comparisons.
[0214] Results
[0215] Low bone mass phenotype and high bone turnover in
CB2.sup.-/- mice: CB2.sup.-/- mice are healthy, fertile and of size
and weight as their age matched wild type controls (9). The present
skeletal analysis shows a LBM phenotype in both male and female
CB2.sup.-/- mice. The trabecular bone volume density (BV/TV) and
trabecular number density (Tb.N) were significantly lower in these
mice already at the age of 8 weeks (FIGS. 8A and 8C top). The
findings in one year old mice indicate progressive, marked
trabecular bone loss: the BV/TV and Tb.N in these animals were
approximately half compared to age-matched WT controls (FIGS. 8A
bottom, 8B and 8C). The osteoclast number (Oc.N/BS) was almost 40%
higher in the CB2.sup.-/- mice (FIG. 8D). An approximately 20%
increases in mineral appositional rate (MAR) and bone formation
rate (BFR) was observed (FIGS. 8E-F), indicating that the LBM in
these mice is associated with high bone turnover (as observed in
many osteopenic states in humans and experimental animals). Another
feature reminiscent of human osteoporosis is the cortical expansion
(23), consisting of increased total diaphyseal and medullary cavity
diameters (FIG. 9). These observations indicate that the CB2
receptor signaling is essential for in the maintenance of normal
trabecular and cortical bone structure.
[0216] Expression of cannabinoid receptors in osteoblasts: In order
to explore the mechanism involved in the effect of CB2 signaling in
bone, its expression was analysed in mouse bone marrow-derived
stromal cells whose osteoblastic differentiation was promoted using
"osteogenic medium" (17). This system demonstrated progressive
expression of CB2 mRNA which paralleled the expression of the
osteoblastic marker genes TNSALP (encoding tissue non-specific
alkaline phosphatase) and PTHRc1 (encoding PTH receptor 1) and
particularly RUNX2 (24). When the cells were grown in
non-osteogenic medium the CB2 mRNA level was very low (FIG. 3A).
Immunohistochemical analysis in the distal femoral metaphysis
clearly demonstrated CB2 receptors in osteoblasts, osteocytes and
osteoclasts in normal mice (FIG. 10B), but not in CB2.sup.-/- mice
(FIG. 10C).
[0217] Effect of CB2 specific agonist on bone cells: In order to
further investigate how CB2 activation affects bone cells, the
effect of HU-308, a synthetic, highly specific small molecule CB2
agonist (11), was studied in differentiating and mature
osteoblastic cells and in osteoclastogenic culture systems. HU-308
potently increased the number of diaphyseal bone marrow derived
stromal cells that had been grown for 10 days in osteogenic medium
(FIG. 11A) to allow for partial differentiation, thus reaching a
preosteoblastic stage that included an initial increase in CB2
expression (FIG. 10A). Noladin ether, a specific CB1 agonist (27)
had no significant effect (FIG. 11A). At this early differentiation
stage TNSALP activity and matrix mineralization were unaffected by
the CB2 ligand. Thus, the HU-308-induced increase in cell number in
this system is consistent with a CB2-mediated stimulation of
diaphyseal preosteoblasts, implicating preosteoblastic cell pool
expansion as a mechanistic aspect of the endocannabinoid action in
bone.
[0218] The homogenous osteoblastic cell line, MC3T3 E1 was used to
identify the mitogenic signalling pathway triggered by activated
CB2. Short-term exposure of partially differentiated MC3T3 E1
osteoblastic cells to HU-308 resulted in stimulation of DNA
synthesis (FIG. 11B). Since CB2 signals via G.sub.i/o-protein (28),
it was attempted to block the CB2 mitogenic effect by pertussis
toxin (PTX), a specific G.sub.i-protein inhibitor. Indeed, PTX
inhibited the HU-308-induced increase in BrdU uptake by the MC3T3
E1 cells (FIG. 11B), indicating CB2 mitogenic signalling via the
activation of a G.sub.i-protein. The effect of HU-308 on cell
number (measured as DNA content) was milder in mature MC3T3 E1
osteoblasts, grown for a prolonged period of time in HU-308
osteogenic medium supplemented with this agonist (FIG. 12A).
However, in this mature osteoblastic system, TNSALP activity and
especially the accumulation of extracellular mineral, key
osteoblastic functions, were markedly enhanced (FIGS. 12B-C).
[0219] In the diaphyseal bone marrow derived osteoclastic cell
system, which was grown for 5 days in the presence of M-CSF and
RANKL, HU-308 dose dependently inhibited the number of
osteoclast-like cells (FIG. 13). In addition, HU-308 reduced the
number of osteoclast-like cells formed in a 7-day RAW 264.7 cell
culture supplemented with M-CSF, indicating that CB2 is involved in
restraining osteoclast differentiation.
[0220] Attenuation of OVX-induced bone loss by HU-308: In view of
the HU-308 in vitro activity, and because CB2 is only peripherally
expressed, CB2 specific ligands, such as HU-308, could provide an
opportunity to augment bone mass while avoiding the cannabinoid
psychotropic activity. Therefore, C3H mice, known for their high
bone mass (10) and thus expected high amount of OVX-induced bone
loss, were used. Four weeks postoperatively, these mice showed
markedly decreased bone volume density accompanied by high turnover
bone loss (FIGS. 14A and 14C). In the vehicle treated mice the
osteoclast number at this time point was already back to normal and
was further inhibited by HU-308 (FIG. 14B), resulting in
attenuation of the OVX-induced trabecular bone loss (FIG. 14A). In
the trabecular compartment, HU-308 did not stimulate bone
formation, which was already vastly enhanced as part of the high
bone turnover triggered by OVX (FIG. 14C) (29). By contrast, HU-308
induced a marked increase in the cortical thickness, which exceeded
the thickness in either OVX or sham-OVX mice (FIG. 14D top). This
increase was associated with a significant reduction in the size of
the medullary cavity (FIG. 14D top) and a vast stimulation of
endocortical bone formation (FIG. 14D bottom), attributable to the
CB2-induced increase in the diaphyseal preosteoblastic cell pool
(FIG. 11A).
[0221] These results clearly demonstrate that bone growth is
regulated by the cannabinoid CB2 receptor and that administering a
CB2 agonist, such as HU-308, can substantially increase bone mass
in treated animals.
[0222] Hence, when viewed together, the results described
hereinabove show unequivocally that cannabinoid receptors
expression significantly affects bone growth and remodeling. It is
further demonstrated that administering agents capable of
regulating the expression of cannabinoid receptor may effectively
modulate bone formation both in vitro and in vivo.
[0223] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0224] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
LIST OF REFERENCES CITED IN NUMERALS
Additional References are Cited in the Text
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Sequence CWU 1
1
10120DNAArtificial sequenceSingle strand DNA oligonucleotide
1tggtgtatga tgtctttggg 20220DNAArtificial sequenceSingle strand DNA
oligonucleotide 2atgctggctg tgttattggc 20321DNAArtificial
sequenceSingle strand DNA oligonucleotide 3aacggtggct tggagttcaa c
21424DNAArtificial sequenceSingle strand DNA oligonucleotide
4taggtagcgg tcaacagcgg ttag 24524DNAArtificial sequenceSingle
strand DNA oligonucleotide 5gcctgaaagc tctactgtgt gagc
24624DNAArtificial sequenceSingle strand DNA oligonucleotide
6gaaggtccag acttggttgt ggct 24720DNAArtificial sequenceSingle
strand DNA oligonucleotide 7gacacaagca ttcccactat
20820DNAArtificial sequenceSingle strand DNA oligonucleotide
8atcagcagta accacagtca 20921DNAArtificial sequenceSingle strand DNA
oligonucleotide 9caagaagtgg atcatccagg t 211024DNAArtificial
sequenceSingle strand DNA oligonucleotide 10gctgctactc ccacttcgtg
cttt 24
* * * * *
References