U.S. patent application number 14/482524 was filed with the patent office on 2014-12-25 for methods for using synthetic triterpenoids in the treatment of bone or cartilage diseases or conditions.
The applicant listed for this patent is Rutgers, The State University of New Jersey, The United States of America, as represented by the Secretary, Dept. of Health and Human Services, Trustees of Dartmouth College, The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Gordon W. Gribble, Karen T. Liby, Damian Medici, Pamela Gehron Robey, Michael B. Sporn, Nanjoo Suh.
Application Number | 20140377235 14/482524 |
Document ID | / |
Family ID | 48430091 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377235 |
Kind Code |
A1 |
Sporn; Michael B. ; et
al. |
December 25, 2014 |
METHODS FOR USING SYNTHETIC TRITERPENOIDS IN THE TREATMENT OF BONE
OR CARTILAGE DISEASES OR CONDITIONS
Abstract
The present invention features the use of a synthetic
triterpenoid to induce gene expression and differentiation of stem
or progenitor cells in the treatment of bone/cartilage diseases or
conditions.
Inventors: |
Sporn; Michael B.;
(Tunbridge, VT) ; Liby; Karen T.; (West Lebanon,
NH) ; Gribble; Gordon W.; (Lebanon, NH) ; Suh;
Nanjoo; (Bridgewater, NJ) ; Medici; Damian;
(Boston, MA) ; Robey; Pamela Gehron; (Bethesda,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Dartmouth College
Rutgers, The State University of New Jersey
The United States of America, as represented by the Secretary,
Dept. of Health and Human Services |
Hanover
Somerset
Rockville |
NH
NJ
MD |
US
US
US |
|
|
Family ID: |
48430091 |
Appl. No.: |
14/482524 |
Filed: |
September 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13466473 |
May 8, 2012 |
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14482524 |
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11941723 |
Nov 16, 2007 |
8299046 |
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13466473 |
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61629298 |
Nov 15, 2011 |
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60866344 |
Nov 17, 2006 |
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Current U.S.
Class: |
424/93.7 ;
435/375; 514/399; 514/519 |
Current CPC
Class: |
C12N 5/0663 20130101;
A61K 38/20 20130101; A61K 38/22 20130101; C12N 5/0655 20130101;
A61K 35/32 20130101; A61P 19/00 20180101; C12N 5/0662 20130101;
A61K 31/4164 20130101; A61K 38/18 20130101; A61K 38/20 20130101;
A61K 38/18 20130101; C12N 2501/00 20130101; A61K 31/26 20130101;
A61K 35/32 20130101; A61K 31/277 20130101; A61K 31/122 20130101;
A61K 38/22 20130101; C12N 2501/999 20130101; A61K 35/28 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.7 ;
435/375; 514/519; 514/399 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 31/277 20060101 A61K031/277; A61K 31/4164 20060101
A61K031/4164; C12N 5/0775 20060101 C12N005/0775 |
Claims
1. A method for producing a stem or progenitor cell with induced
gene expression comprising contacting a stem or progenitor cell
with an effective amount of a synthetic triterpenoid to induce the
expression of one or more of SOX9 (Sex determining region Y-box 9),
COL2A1 (Type II Collagen (alpha1)), TGF (Transforming Growth
Factor)-.beta.1, TGF-.beta.2, TGF-.beta.3, BMP (Bone Morphogenic
Protein) 2, BMP4, BMPRII (Bone Morphogenic Protein Receptor II),
SMAD (Small Mothers Against Decapentaplegic) 3, SMAD4, SMAD6,
SMAD7, TIMP (Tissue Inhibitor of Metalloproteinase)-1 or TIMP-2 in
the stem or progenitor cell, wherein the stem or progenitor cell is
a mesenchymal stem cell, adipose tissue-derived mesenchymal stem
cell, embryonic stem cell, stem cell from exfoliated deciduous
teeth, periosteum cell, osteoprogenitor cell, or growth plate
progenitor cell.
2. The method of claim 1, wherein the synthetic triterpenoid has
the structure of Formula I: ##STR00005## wherein, X' and X.sup.2
are independently hydrogen, OR.sup.a, NR.sup.aR.sup.b, or SR.sup.a,
wherein R.sup.a is a hydrogen, cyano, --CF.sub.3, nitro, amino, or
substituted or unsubstituted heteroaryl group; R.sup.b is hydrogen,
hydroxyl, alkyl, aryl, aralkyl, acyl, alkoxy, aryloxy, acyloxy,
alkylamino, arylamino, amido, or a substituted version of any of
these groups; or a substituent convertible in vivo to hydrogen;
provided that R.sup.a is absent when the atom to which it is bound
is part of a double bond, further provided that when R.sup.a is
absent the atom to which it is bound is part of a double bond; Y is
CH.sub.2 or CH.sub.2--CH.sub.2; Z is a covalent bond,
--C(.dbd.O)--, alkanediyl, alkenediyl, alkynediyl, or a substituted
version of any of these groups; the dashed bonds can be
independently present or absent; R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are each independently a hydrogen, hydroxyl, alkyl,
substituted alkyl, alkoxy or substituted alkoxy group; R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are independently
hydrogen, hydroxyl, halo, cyano, --C.dbd.CR.sup.a,
--CO.sub.2R.sup.a, --COR.sup.a, alkyl, alkenyl, alkynyl, aryl,
aralkyl, heteroaryl, heteroaralkyl, acyl, alkoxy, aryloxy, acyloxy,
alkylamino, arylamino, nitro, amino, amido, --C(O)R.sup.c or a
substituted version of any of these groups, wherein R.sup.c is
hydrogen, hydroxy, halo, amino, hydroxyamino, azido or mercapto; or
C.sub.1-C.sub.15-alkyl, C.sub.2-C.sub.15-alkenyl,
C.sub.2-C.sub.15-alkynyl, C.sub.6-C.sub.15-aryl,
C.sub.7-C.sub.15-aralkyl, C.sub.1-C.sub.15-heteroaryl,
C.sub.2-C.sub.15-heteroaralkyl, C.sub.1-C.sub.15-acyl,
C.sub.1-C.sub.15-alkoxy, C.sub.2-C.sub.15-alkenyloxy,
C.sub.2-C.sub.15-alkynyloxy, C.sub.6-C.sub.15-aryloxy,
C.sub.7-C.sub.15-aralkyloxy, C.sub.1-C.sub.15-heteroaryloxy,
C.sub.2-C.sub.15-heteroaralkyloxy, C.sub.1-C.sub.15-acyloxy,
C.sub.1-C.sub.15-alkylamino, C.sub.2-C.sub.15-dialkylamino,
C.sub.1-C.sub.15-alkoxyamino, C.sub.2-C.sub.15-alkenylamino,
C.sub.2-C.sub.15-alkynylamino, C.sub.6-C.sub.15-arylamino,
C.sub.7-C.sub.15-aralkylamino, C.sub.1-C.sub.15-heteroarylamino,
C.sub.2-C.sub.15-heteroaralkylamino,
C.sub.1-C.sub.15-alkylsulfonylamino, C.sub.1-C.sub.15-amido,
C.sub.1-C.sub.15-alkylsilyloxy, or substituted versions of any of
these groups; R.sup.5 and R.sup.6, R.sup.7 and R.sup.8, or R.sup.9
and R.sup.10 are independently taken together as .dbd.O; R.sup.11
and R.sup.12 are each independently hydrogen, hydroxyl, halo, alkyl
, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, acyl,
alkoxy, aryloxy, aralkoxy, heteroaryloxy, hetero-aralkoxy, acyloxy,
alkylamino, dialkylamino, arylamino, aralkylamino, heteroarylamino,
heteroaralkylamino, amido, or a substituted version of any of these
groups, or R.sup.11 and R.sup.12 are taken together and are
alkanediyl, alkenediyl, arenediyl, alkoxydiyl, alkenyloxydiyl,
alkylaminodiyl, alkenylaminodiyl, or alkenylaminooxydiyl; R.sup.13
is hydrogen, hydroxy or oxo; R.sup.14 is hydrogen or hydroxyl; and
R.sup.15 is a hydrogen, hydroxyl, --NR.sup.dR.sup.e, cyano, halo,
azido, phosphate, 1,3-dioxoisoindolin-2-yl, mercapto, silyl or
--COOH group, substituted or unsubstituted versions of
C.sub.1-C.sub.15-alkyl, C.sub.2-C.sub.15-alkenyl,
C.sub.2-C.sub.15-alkynyl, C.sub.6-C.sub.15-aryl,
C.sub.7-C.sub.15-aralkyl, C.sub.1-C.sub.15-heteroaryl,
C.sub.2-C.sub.15-heteroaralkyl, C.sub.1-C.sub.15-acyl,
C.sub.1-C.sub.15-alkoxy, C.sub.2-C.sub.15-alkenyloxy,
C.sub.2-C.sub.15-alkynyloxy, C.sub.6-C.sub.15-aryloxy,
C.sub.7-C.sub.15-aralkyloxy, C.sub.1-C.sub.15-heteroaryloxy,
C.sub.2-C.sub.15-heteroaralkyloxy, C.sub.1-C.sub.15-acyloxy,
C.sub.1-C.sub.15-alkylamino, C.sub.2-C.sub.15-alkenylamino,
C.sub.2-C.sub.15-alkynylamino, C.sub.6-C.sub.15-arylamino,
C.sub.7-C.sub.15-aralkylamino, C.sub.1-C.sub.15-heteroarylamino,
C.sub.2-C.sub.15-heteroaralkylamino, C.sub.1-C.sub.15-amido,
C.sub.1-C.sub.15-alkylthio, C.sub.2-C.sub.15-alkenylthio,
C.sub.2-C.sub.15-alkynylthio, C.sub.6-C.sub.15-arylthio,
C.sub.7-C.sub.15-aralkylthio, C.sub.1-C.sub.15-heteroarylthio,
C.sub.2-C.sub.15-heteroaralkylthio, C.sub.1-C.sub.15-acylthio,
C.sub.1-C.sub.12-thioacyl, C.sub.1-C.sub.12-alkylsulfonyl,
C.sub.2-C.sub.12-alkenylsulfonyl, C.sub.2-C.sub.12-alkynylsulfonyl,
C.sub.6-C.sub.12-arylsulfonyl, C.sub.7-C.sub.12-aralkylsulfonyl,
C.sub.1-C.sub.12-heteroarylsulfonyl,
C.sub.1-C.sub.12-heteroaralkylsulfonyl,
C.sub.1-C.sub.12-alkylsulfinyl, C.sub.2-C.sub.12-alkenylsulfinyl,
C.sub.2-C.sub.12-alkynylsulfinyl, C.sub.6-C.sub.12-aryl sulfinyl,
C.sub.7-C.sub.12-aralkylsulfinyl,
C.sub.1-C.sub.12-heteroarylsulfinyl,
C.sub.1-C.sub.12-heteroaralkylsulfinyl,
C.sub.1-C.sub.12-alkylphosphonyl, C.sub.1-C.sub.12-alkylphosphate,
C.sub.2-C.sub.12-dialkylphosphate, C.sub.1-C.sub.12-alkylammonium,
C.sub.1-C.sub.12-alkylsulfonium, C.sub.1-C.sub.15-alkylsilyl, or a
substituted version of any of these groups, a --CO.sub.2Me,
carbonyl imidazole, --CO--D-Glu(OAc).sub.4, --CONH.sub.2,
--CONHNH.sub.2, --CONHCH.sub.2CF.sub.3, or --C(.dbd.O)-heteroaryl
group, or Z and R.sup.15 form a three to seven-membered ring, such
that Z and R.sup.15 are further connected to one another through
one or more of --O-- and alkanediyl, further wherein Z is --CH--
and R.sup.15 is --CH.sub.2-- or Z, R.sup.15, and carbon numbers 13,
17 and 18 form a ring such that R.sup.15 is bound to carbon 13,
wherein Y is methanediyl or substituted methanediyl and R.sup.15 is
--O--, wherein R.sup.d and R.sup.e are independently hydrogen,
hydroxyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, acyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy,
aralkoxy, heteroaryloxy, heteroaralkoxy, thioacyl, alkylsulfonyl,
alkenylsulfonyl, alkynylsulfonyl, arylsulfonyl, aralkylsulfonyl,
heteroarylsulfonyl, or heteroaralkylsulfonyl, or a substituted
version of any of these groups.
3. The method of claim 1, wherein the stem or progenitor cell is
multipotent.
4. A stem or progenitor cell produced by the method of claim 1.
5. A method for treating a degenerative disease or injury
comprising transplanting into a patient suffering from a
degenerative disease or injury a stem or progenitor cell of claim 4
thereby treating the patient's degenerative disease or injury,
wherein the degenerative disease or injury is bone injury,
cartilage injury, joint injury, dental disease, osteoarthritis,
rheumatoid arthritis, degenerative disc disease, kyphosis, or
osteopenia.
6. A method for treating a congenital disorder comprising
transplanting into a patient suffering from a congenital disorder a
stem or progenitor cell of claim 4 thereby treating the patient's
congenital disorder, wherein the congenital disorder is scoliosis
or a skeletal disorder.
7. A method for treating a degenerative disease or injury
comprising administering to a patient suffering from a degenerative
disease or injury an effective amount of a synthetic triterpenoid
thereby treating the patient's degenerative disease or injury,
wherein the degenerative disease or injury is bone injury,
cartilage injury, joint injury, degenerative disc disease, dental
disease, osteoarthritis, rheumatoid arthritis, kyphosis, or
osteopenia.
8. A method for treating a congenital disorder comprising
administering to a patient suffering from a congenital disorder an
effective amount of a synthetic triterpenoid thereby treating the
patient's congenital disorder, wherein the congenital disorder is
scoliosis or a skeletal disorder.
Description
INTRODUCTION
[0001] This patent application is a continuation of U.S.
application Ser. No. 13/466,473 filed May 8, 2012, which claims the
benefit of priority from U.S. Provisional Application Ser. No.
61/629,298, filed Nov. 15, 2011, and is a continuation-in-part
application of U.S. application Ser. No. 11/941,723, filed Nov. 16,
2007, now issued as U.S. Pat. No. 8,299,046, which claims the
benefit of priority from U.S. Provisional Application Ser. No.
60/866,344, filed Nov. 17, 2006, the content of each of which is
herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Triterpenoids, biosynthesized in plants by the cyclization
of squalene, are used for medicinal purposes in many Asian
countries; and some, like ursolic and oleanolic acids, are known to
exhibit anti-inflammatory and anti-carcinogenic activity (Huang, et
al. (1994) Cancer Res. 54:701-708; Nishino, et al. (1988) Cancer
Res. 48:5210-5215). However, the biological activity of these
naturally-occurring molecules is relatively weak, and therefore the
synthesis of new analogs to enhance their potency has been
undertaken (Honda, et al. (1997) Bioorg. Med. Chem. Lett.
7:1623-1628; Honda, et al. (1998) Bioorg. Med. Chem. Lett.
8(19):2711-2714). An ongoing effort for the improvement of
anti-inflammatory and antiproliferative activity of oleanolic and
ursolic acid analogs led to the discovery of
2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid (CDDO) and
related compounds (Honda, et al. (1997) supra; Honda, et al. (1998)
supra; Honda, et al. (1999) Bioorg. Med. Chem. Lett.
9(24):3429-3434; Honda, et al. (2000) J. Med. Chem. 43:4233-4246;
Honda, et al. (2000) J. Med. Chem., 43:1866-1877; Honda, et al.
(2002) Bioorg. Med. Chem. Lett. 12:1027-1030; Suh, et al. (1998)
Cancer Res. 58:717-723; Suh, et al. (1999) Cancer Res.,
59(2):336-341; Suh, et al. (2003) Cancer Res. 63:1371-1376; Place,
et al. (2003) Clin. Cancer Res. 9:2798-2806; Liby, et al. (2005)
Cancer Res. 65:4789-4798). Several potent derivatives of oleanolic
acid were identified, including
methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO-Me).
[0003] CDDO-Me suppresses the induction of several important
inflammatory mediators, such as iNOS, COX-2, TNF.alpha., and
IFN.gamma., in activated macrophages. CDDO-Me has also been
reported to activate the Keap1/Nrf2/ARE signaling pathway resulting
in the production of several anti-inflammatory and antioxidant
proteins, such as heme oxygenase-1 (HO-1). These properties have
made CDDO-Me a candidate for the treatment of neoplastic and
proliferative diseases, such as cancer. Moreover, synthetic
triterpenoids have been found to induce apoptosis and
differentiation and inhibit proliferation in human leukemia cells
(Ikeda, et al. (2003) Cancer Res. 63:5551-5558; Konopleva, et al.
(2002) Blood 99(1):326-335; Suh, et al. (1999) supra; Ito, et al.
(2000) Mech. Dev. 97:35-45), induce osteoblastic differentiation in
osteosarcoma cells (Ito, et al. (2001) Antimicrob. Agents
Chemother. 45:1323-1336), enhance neuronal growth factor-induced
neuronal differentiation of rat PC12 pheochromocytoma cells, and
induce adipogenic differentiation of fibroblasts into adipocytes
(Suh, et al. (1999) supra). CDDO-Me has also been found an
effective drug for improving kidney function in patients suffering
for renal/kidney disease using CDDO-Me (U.S. Pat. No.
8,129,429).
SUMMARY OF THE INVENTION
[0004] The present invention encompasses the finding that certain
triterpenoid compounds induce stem or progenitor cell
differentiation. The invention encompasses the specific finding
that certain such triterpenoid compounds alter gene expression in
stem or progenitor cells. The invention therefore provides methods
and reagents for inducing stem or progenitor cell differentiation,
in a variety of important contexts and applications.
[0005] Among other things, the present invention provides methods
for producing a stem or progenitor cell with induced gene
expression by contacting a stem or progenitor cell with an
effective amount of a synthetic triterpenoid to induce the
expression of one or more of SOX9 (Sex determining region Y-box 9),
COL2A1 (Type II Collagen (alpha1)), TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3, BMP2, BMP4, BMPRII (Bone Morphogenic Protein Receptor
II), SMAD (Small Mothers Against Decapentaplegic) 3, SMAD4, SMAD6,
SMAD7, TIMP (Tissue Inhibitor of Metalloproteinase)-1 or TIMP-2 in
the stem or progenitor cell, wherein the stem or progenitor cell is
a mesenchymal stem cell, adipose tissue-derived mesenchymal stem
cell, embryonic stem cell, stem cell from exfoliated deciduous
teeth, periosteum cell, osteoprogenitor cell, or growth plate
progenitor cell. In one embodiment, the stem or progenitor cell is
multipotent.
[0006] A stem or progenitor cell produced by the method is also
provided, as are methods of using such cells in the treatment of a
degenerative disease, disorder, condition, or injury such as bone
injury, cartilage injury, joint injury, dental disease,
osteoarthritis, rheumatoid arthritis, degenerative disc disease,
kyphosis, or osteopenia. In addition, treatment of a congenital
disorder such as scoliosis or a skeletal disorder is also
provided.
[0007] Methods for treating a degenerative disease or injury or a
congenital disorder by administering to a patient an effective
amount of a synthetic triterpenoid are also provided.
DETAILED DESCRIPTION OF THE INVENTION
[0008] It has now been shown that certain synthetic triterpenoid
compounds induce the expression of SOX9, COL2A1, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, BMP2, BMP4, BMPRII, SMAD3, SMAD4, SMAD6,
SMAD7, TIMP-1 and/or TIMP-2 in stem cells. For example, the
synthetic triterpenoids CDDO-Im and CDDO-EA induce the expression
of each of the above-referenced genes in mesenchymal stem cells and
induce chondrogenesis in newborn mouse calvaria. In this respect,
the present invention provides methods of using such synthetic
triterpenoid compounds to produce stem/progenitor cells with
altered expression of one or more of SOX9, COL2A1, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, BMP2, BMP4, BMPRII, SMAD3, SMAD4, SMAD6,
SMAD7, TIMP-1 or TIMP-2, as compared to a control (e.g., a cell not
contacted with the synthetic triterpenoid), and application of such
cells in the prevention or treatment of disease, where the cells
can contacted with the triterpenoid in situ, in vivo, ex vivo or in
vitro. In some embodiments, expression of one or more of said genes
is indicative of differentiation of the stem/progenitor cell.
[0009] As is known in the art, stem cells are primal cells found in
all multi-cellular organisms. They retain the ability to renew
themselves through mitotic cell division and can differentiate into
a diverse range of specialized cell types. Mammalian stem cells
include embryonic stem cells, derived from blastocysts; adult stem
cells, which are found in adult tissues; and cord blood stem cells,
which are found in the umbilical cord. In a developing embryo, stem
cells can differentiate into all of the specialized embryonic
tissues. In adult organisms, stem cells and progenitor cells act as
a repair system for the body, replenishing specialized cells. As
stem cells can be grown and transformed into specialized cells with
characteristics consistent with cells of various tissues such as
muscles or nerves through cell culture, stem cells are of use in
medical therapies.
[0010] "Potency" specifies the differentiation potential (the
potential to differentiate into different cell types) of the stem
cell. Totipotent stem cells are produced from the fusion of an egg
and sperm cell. Cells produced by the first few divisions of the
fertilized egg are also totipotent. These cells can differentiate
into embryonic and extraembryonic cell types. Pluripotent stem
cells are the descendants of totipotent cells and can differentiate
into cells derived from any of the three germ layers. Multipotent
stem/progenitor cells can produce only cells of a closely related
family of cells (e.g., hematopoietic stem cells differentiate into
red blood cells, white blood cells, platelets, etc.).
[0011] The term "differentiation" or "differentiated," as used
herein, refers to the developmental process wherein an
unspecialized or less specialized cell becomes more specialized for
a specific function, such as, for example, the process by which a
mesenchymal stem cell becomes a more specialized cell such as a
cartilage cell, a bone cell, a muscle cell, or a fat cell.
Differentiation can be assessed by identifying lineage-specific
markers, such as, for example, aggrecan, a proteoglycan specific
for cartilage.
[0012] Embryonic stem (ES) cell lines are cultures of cells derived
from the epiblast tissue of the inner cell mass (ICM) of a
blastocyst. A blastocyst is an early stage embryo, approximately 4
to 5 days old in humans and composed of 50-150 cells. ES cells are
pluripotent, and give rise during development to all derivatives of
the three primary germ layers: ectoderm, endoderm and mesoderm. In
other words, they can develop into each of the more than 200 cell
types of the adult body when given sufficient and necessary
stimulation for a specific cell type. They do not contribute to the
extra-embryonic membranes or the placenta.
[0013] A human embryonic stem cell is defined by the presence of
several transcription factors and cell surface proteins. The
transcription factors Oct-4, Nanog, and Sox2 form the core
regulatory network, which ensures the suppression of genes that
lead to differentiation and the maintenance of pluripotency. The
cell surface proteins most commonly used to identify hES cells are
the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens
Tra-1-60 and Tra-1-81. Because of the combined ability of unlimited
expansion and pluripotency, embryonic stem cells remain a potential
source for regenerative medicine and tissue replacement after
injury or disease. Therefore, the present invention contemplates
the use of embryonic stem cells. Cells derived from embryonic
sources may include embryonic stem cells or stem cell lines
obtained from a stem cell bank or other recognized depository
institution.
[0014] An adult or somatic stem cell, a cell which is found in a
developed organism, has two properties: the ability to divide and
create another cell like itself, and also divide and create a cell
more differentiated than itself. Adult stem cell treatments have
been used for many years to successfully treat leukemia and related
bone/blood cancers through bone marrow transplants. Therefore, in
particular embodiments of the present invention, adult stem cells
are used in the methods of the disclosed herein. Pluripotent adult
stem cells are rare and generally small in number but can be found
in a number of tissues including umbilical cord blood. Most adult
stem cells are lineage restricted (multipotent). Therefore, in
certain embodiments, the adult stem cells of the invention are
multipotent.
[0015] Adult stem cells have been identified in many organs and
tissues, including brain, bone marrow, peripheral blood, blood
vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian
epithelium, and testis, and are thought to reside in a specific
area of each tissue (called a "stem cell niche"). In this respect,
stem cells are generally referred by the tissue from which they are
derived, e.g., cardiac stem cells are stems that naturally reside
within the heart, adipose-derived stem cells are stem cells found
in fat tissue, myoblasts are muscle stem cells, dental pulp stem
cells are found in the teeth, etc.
[0016] Stem cells give rise to a number of different cell types.
For example, mesenchymal stem cells (CD105.sup.+, CD90.sup.+,
CD11b.sup.+, CD34.sup.+ and CD45.sup.+) give rise to bone cells
(osteocytes), cartilage cells (chondrocytes), fat cells
(adipocytes), and other kinds of connective tissue cells such as
those in tendons. Hematopoietic stem cells (Lin.sup.-, CD34.sup.+,
CD90.sup.+, CD34.sup.- and CD45.sup.-) give rise to all the types
of blood cells including red blood cells, B lymphocytes, T
lymphocytes, natural killer cells, neutrophils, basophils,
eosinophils, monocytes, and macrophages. Neural stem cells in the
brain (CD133.sup.+, FA-1.sup.+, CD34.sup.- and CD45.sup.-) give
rise to nerve cells (neurons), astrocytes and oligodendrocytes.
Epithelial stem cells in the lining of the digestive tract occur in
deep crypts and give rise to several cell types including
absorptive cells, goblet cells, paneth cells, and enteroendocrine
cells. Skin stem cells occur in the basal layer of the epidermis
and at the base of hair follicles, and give rise to keratinocytes,
which migrate to the surface of the skin and form a protective
layer. The follicular stem cells can give rise to both the hair
follicle and to the epidermis. Neural crest stem cells (p75
receptor.sup.+, .alpha..sub.4 integrin receptor.sup.+, CD29.sup.+
and CD9.sup.+) differentiate into the cells of the peripheral
nervous system. In some embodiments, the invention includes the use
of mesenchymal stem cells, adipose tissue-derived mesenchymal stem
cells, embryonic stem cells or stem cells from exfoliated deciduous
teeth.
[0017] Progenitor cells are cells that are direct descendants of
stem cells, are typically less potent than stem cells, and have
diminished capacity for self-renewal relative to stem cells, but
retain the ability to become at least one, if not multiple, cell
types. In this respect, multipotent progenitor cells are also
encompassed within the scope of the present invention. Multipotent
progenitor cells are similar to mesenchymal stem cells as they have
the ability to differentiate, in vitro, into cells with phenotypic
characteristics of cells from all three germ cell layers (mesoderm,
ectoderm and endoderm). Multipotent progenitor cells can be
isolated in a fashion similar to mesenchymal stem cells, with
adherence to plastic as an initial staple property; however
additional separation techniques including magnetic activated cell
sorting for CD45.sup.-/TER119.sup.- and 96-well single cell
isolation/expansion on fibronectin in enriched media are required
(Breyer, et al. (2006) Exp. Hematol. 34:1596-601). Multipotent
progenitor cells possess certain cell surface markers (distinct
from mesenchymal stem cells) including CD13, CD31 and SSEA-1 and
lack markers including CD3, CD11b, CD19, CD34, CD44, CD45, MHC I
and MHC II (Breyer et al. (2006) supra; Jiang, et al. (2002) Exp.
Hematol. 34:809). Progenitor cells include satellite cells found in
muscles; intermediate progenitor cells formed in the subventricular
zone; bone marrow stromal cells; periosteum cells, which includes
progenitor cells that develop into osteoblasts or chondroblasts;
pancreatic progenitor cells; angioblasts or endothelial progenitor
cells; and blast cells involved in the generation of B- and
T-lymphocytes. In some embodiments, the invention includes the use
of periosteum cells, osteoprogenitor cells or growth plate
progenitor cells.
[0018] The invention applies to cells of an embryonic or adult
origin in mammals, both human and non-human mammals, including but
not limited to human and non-human primates, ungulates, ruminants
and rodents. Ungulate species include, but are not limited to,
cattle, sheep, goats, pigs, horses. Rodent species include, but are
not limited to, rats and mice. The invention may also find
application in other mammalian species such as rabbits, cats and
dogs. Examples of preferred stem/progenitor cell populations which
can be used in accordance with the methods of the present invention
include primate stem/progenitor cells, such as human
stem/progenitor cells. Such cells include adult human mesenchymal
stem cells. As will be appreciated by one of skill in the art, a
stem or progenitor cell referred to herein generally refers to a
population of stem or progenitor cells.
[0019] Stem cells of the present invention can be isolated by
conventional methods including Fluorescence Activated Cell Sorting
(FACS), microbead separation, or affinity chromatograph using
antibodies specific to cell surface antigens. Alternatively, stem
cells can be isolated by enzymatic digestion of source tissue and
gradient separation with, e.g., PERCOLL or HISTOPAQUE. Once
isolated, the stem cells can be maintained and propagated in vitro
under controlled conditions. Cells may be cultured in a variety of
types of vessels constructed of, for example, glass or plastic. The
surfaces of culture vessels may be pre-treated or coated with, for
example, collagen, polylysine, or components of the extracellular
matrix, to facilitate the cellular adherence. In addition, layers
of adherent cells or feeder cells, which are used to support the
growth of cells with more demanding growth requirements, may be
used.
[0020] Cells are normally cultured under conditions designed to
closely mimic those observed in vivo. In order to mimic the normal
physiological environment, cells are generally incubated in a
CO.sub.2 atmosphere with semi-synthetic growth media. Culture media
is buffered and contains, among other things, amino acids,
nucleotides, salts, vitamins, and also a supplement of serum such
as fetal calf serum (FCS), horse serum or even human serum. Culture
media may be further supplemented with growth factors and
inhibitors such as hormones, transferrin, insulin, selenium, and
attachment factors.
[0021] In certain aspects of the instant invention, cells are
cultured prior to contact with a synthetic triterpenoid. They may
also be cultured after contact, i.e., after they have been induced
to express one or more of the genes disclosed herein or
differentiate toward a given or specific phenotype. Cells will be
cultured under specified conditions to achieve particular types of
differentiation, and provided with various factors necessary to
facilitate the desired differentiation.
[0022] In some embodiments, cells contacted with a synthetic
triterpenoid are also contacted with one or more cell growth and
differentiation factors. Cell growth and differentiation factors
are molecules that stimulate cells to proliferate and/or promote
differentiation of cell types into functionally mature forms. In
some embodiments of the invention, cell growth and differentiation
factors may be administered in combination with synthetic
triterpenoids of the invention in order to direct the administered
cells to proliferate and differentiate in a specific manner. One of
ordinary skill would recognize that the various factors may be
administered prior to, concurrently with, or subsequent to the
administration of one or more synthetic triterpenoids of the
present invention. In addition, administration of the growth and/or
differentiation factors may be repeated as needed.
[0023] It is envisioned that a growth and/or differentiation factor
may constitute a hormone, cytokine, hematapoietin, colony
stimulating factor, interleukin, interferon, growth factor, other
endocrine factor or combination thereof that act as intercellular
mediators. Examples of such intercellular mediators are
lymphokines, monokines, growth factors and traditional polypeptide
hormones. Included among the growth factors are growth hormones
such as human growth hormone, N-methionyl human growth hormone, and
bovine growth hormone; parathyroid hormone; thyroxine; insulin;
proinsulin; relaxin; prorelaxin; glycoprotein hormones such as
follicle stimulating hormone (FSH), thyroid stimulating hormone
(TSH), and luteinizing hormone (LH); hepatic growth factor;
prostaglandin, fibroblast growth factor; prolactin; placental
lactogen, OB protein; tumor necrosis factors -.alpha.and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon.alpha., .beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte/macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18. As used herein, the term growth and/or
differentiation factors include proteins from natural sources or
from recombinant cell culture and biologically active equivalents
of the native sequence, including synthetic molecules and
mimetics.
[0024] The present disclosure provides methods of inducing the
expression of one or more of SOX9, COL2A1, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, BMP2, BMP4, BMPRII, SMAD3, SMAD4, SMAD6,
SMAD7, TIMP-1 or TIMP-2, such as in a stem/progenitor cell, and the
prevention or treatment of disease. In some embodiments, the
expression of one or more of SOX9, COL2A1, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, BMP2, BMP4, BMPRII, SMAD3, SMAD4, SMAD6,
SMAD7, TIMP-1 or TIMP-2 in the stem/progenitor cell induces
differentiation. For example, the results presented below
demonstrate that CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide
(CDDO-EA) induce expression of each of the above-reference genes
and induce chondrogenesis in mesenchymal stem cells and organ
cultures of newborn mouse calvaria. Accordingly, cells exposed to
synthetic triterpenoids under in vitro, in vivo, or ex vivo
conditions are of use in the treatment of diseases, conditions or
disorders wherein altered gene expression has been shown to provide
an advantage and/or transplantation of differentiated cells would
provide a benefit. Examples of synthetic triterpenoid-mediated gene
induction in particular stem cells and use of the same are as
follows.
Synthetic Triterpenoid-Mediated Induction of SOX9
[0025] Sox9 is a member of the Sox gene family, which is
characterized by the presence of an HMG box with more than 50%
homology to the sex determining gene Sry (Schepers, et al. (2002)
Dev. Cell 3:167-170). The HMG box can bind and bend DNA, and it has
been proposed that Sox genes encode architectural DNA binding
proteins. In addition to the HMG box, Sox9 possesses a
transactivation domain at its C terminus (Sudbeck, et al. (1996)
Nat. Genet. 13:230-232), and it has been shown that it can activate
the genes Mis in testis (De Santa Barbara, et al. (1998) Mol. Cell
Biol. 18:6653-6665; Arango, et al. (1999) Cell 99:409-419) and
Col2aI (Bell, et al. (1997) Nat. Genet. 16:174-178) during
chondrogenesis in vitro and in vivo. Moreover, in vitro studies
suggest that Sox genes may also have a role in RNA splicing (Ohe,
et al. (2002) Proc. Natl. Acad. Sci. USA 99:1146-51).
[0026] Degeneration of the intervertebral disc, often called
"degenerative disc disease" (DDD) of the spine, is a condition that
can be painful and can greatly affect the quality of one's life.
While disc degeneration is a normal part of aging and for most
people is not a problem, for certain individuals a degenerated disc
can cause severe constant chronic pain. Etopic expression of Sox9
has been shown to induce adipose tissue-derived stem cells (ASCs)
to function as real nucleus pulposus (NP) cells in vitro (Yang, et
al. (2011) J. Orthop. Res. 29:1291-7). Sox-9-engineered ASCs
(ASCs/Sox-9) were induced for the chondrocyte-like cell
differentiation by 3D culture in alginate beads and TGF-.beta.3 for
2 weeks. After induction, type II collagen and proteoglycan levels
significantly increased in the ASCs/Sox-9 compared to the ASCs. In
addition, co-culture of induced ASCs/Sox-9 with matured NP cells
resulted in enhanced increase in proteoglycan and type II collagen
production (Yang, et al. (2011) supra). Therefore, synthetic
triterpenoid-mediated induction of Sox9 in ACSs could be of use in
differentiating ASCs into chondrocyte-like cells, which may be
potentially used as a stem cell-based therapeutic tool for the
treatment of degenerative disc diseases.
[0027] The chondrogenic effect of Sox9 on bone marrow mesenchymal
stem cells (BMSCs) in vitro and its application in articular
cartilage repair in vivo have been evaluated. Rabbit BMSCs were
transduced with adenoviral vector containing Sox9. The results
showed that Sox9 could induce chondrogenesis of BMSCs both in
monolayer and on PGA scaffold effectively. A rabbit model with
full-thickness cartilage defects was established and then repaired
by PGA scaffold and rabbit BMSCs with or without Sox9 transduction.
This analysis indicated that better repair, including more
newly-formed cartilage tissue and hyaline cartilage-specific
extracellular matrix and greater expression of several
chondrogenesis marker genes were observed in PGA scaffold and BMSCs
with Sox9 transduction, compared to that without transduction (Cao,
et al. (2011) Biomaterials 32:3910-20). Therefore, synthetic
triterpenoid-mediated induction of Sox9 in BMSCs could be of use in
the repair of cartilage defects in vivo and in tissue
engineering.
Synthetic Triterpenoid-Mediated Induction of BMP-2
[0028] BMP-2 can drive embryonic stem cells to the cartilage,
osteoblast or adipogenic fate depending on supplementary co-factors
(zur Nieden, et al. (2005) BMC Dev. Biol. 5:1). TGF-.beta.1,
insulin and ascorbic acid have been identified as signals that
together with BMP-2 induce a chondrocytic phenotype that is
characterized by increased expression of cartilage marker genes in
a timely co-ordinated fashion. BMP-2 induced chondrocytes undergo
hypertrophy and begin to alter their expression profile towards
osteoblasts. Supplying mineralization factors such as
beta-glycerophosphate and vitamin D3 with the culture medium can
facilitate this process. Moreover, gene expression studies show
that adipocytes can also differentiate from BMP-2 treated ES cells.
Ultimately, embryonic stem cells can be successfully triggered to
differentiate into chondrocyte-like cells, which can further alter
their fate to become hypertrophic, and adipocytes (zur Nieden, et
al. (2005) supra). Therefore, this analysis and the results
presented herein indicate that synthetic triterpenoid-mediated
induction of BMP-2 in embryonic stem cells is of use as therapy in
joint injury and disease.
[0029] Most spine fusion procedures involve the use of prosthetic
fixation devices combined with autologous bone grafts rather than
biological treatment. However, it has been shown that spine fusion
can be achieved by injection of bone morphogenetic protein-2
(BMP-2)-expressing mesenchymal stem cells (MSCs) into the
paraspinal muscle. BMP-2-expressing MSCs were injected bilaterally
into paravertebral muscles of the mouse lumbar spine. Bone bridging
of the targeted vertebrae was observed in the BMP-2-expressing MSC
group, whereas no bone formation was noted in any control group.
The biomechanical tests showed that MSC-mediated spinal fusion was
as effective as stainless steel pin-based fusion and significantly
more rigid than the control groups. (Sheyn, et al. (2010) Tissue
Eng. Part A 16:3679-86). See also, Fu, et al. (2009) J. Orthop.
Res. 27:380-4 and Hasharoni, et al. (2005) J. Neurosurg. Spine
3:47-52. These findings in combination with the results presented
herein indicate that synthetic triterpenoid-mediated induction of
BMP-2 in mesenchymal stem cells is of use in spinal fusion.
[0030] The regeneration of the periodontal attachment apparatus
remains clinically challenging because of the involvement of three
tissue types and the complexity of their relationship. It has been
demonstrated that a combination of ex vivo autologous bone marrow
MSCs engineered by replication-defective adenovirus to express the
BMP-2 gene and Pluronic F127 (PF127) can be used to regenerate the
periodontal attachment apparatus (Chen, et al. (2008) Gene Ther.
15:1469-77). Periodontal defects were surgically created in New
Zealand white rabbits and treated with PF127 and MSCs expressing
BMP-2. This approach regenerated not only cementum with Sharpey's
fiber insertion, but also statistically significant quantities of
bone, re-establishing a more normal relationship among the
components of the regenerated periodontal attachment apparatus,
which is beneficial for the maintenance of periodontal health
(Chen, et al. (2008) supra). These findings in combination with the
results presented herein indicate that synthetic
triterpenoid-mediated induction of BMP-2 in mesenchymal stem cells,
in combination with PF127, may represent an alternative means for
periodontal alveolar bone graft in clinical settings.
[0031] Adult stem cells have therapeutic potential in bone
regeneration. In this respect, it has been demonstrated that ex
vivo modified MSC enhance bone density in an immunocompetent mouse
model of osteopenia (Kumar, et al. (2010) Gene Ther. 17:105-16).
MSC were transduced ex vivo with a recombinant adeno-associated
virus 2 expressing BMP-2 under the transcriptional control of
collagen type-1alpha promoter. The modified MSC were systemically
administered to ovariectomized, female C57BL/6 mice. Results
indicated that mice transplanted with MSC expressing BMP-2 showed
significant increase in bone mineral density and bone mineral
content with relatively better proliferative capabilities of bone
marrow stromal cells and higher osteocompetent pool of cells
compared to control animals. Micro-CT analysis of femora and other
bone histomorphometric analyses indicated more trabecular bone
following MSC-BMP-2 therapy. Moreover, production of BMP-2 from
transplanted MSC also influenced the mobilization of endogenous
progenitors for new bone formation (Kumar, et al. (2010) supra).
Therefore, this analysis and the results presented herein indicate
that synthetic triterpenoid-mediated induction of BMP-2 in
mesenchymal stem cells is of use in increasing bone mineral density
and content, and stimulating new bone formation in subjects with
osteopenia.
Synthetic Triterpenoid-Mediated Induction of BMP-4
[0032] The potential of different growth factors (basic fibroblast
growth factor (bFGF), TGF-.beta.1, activin-A, BMP-4, hepatocyte
growth factor (HGF), epidermal growth factor (EGF), beta nerve
growth factor (.beta.NGF), and retinoic acid) to direct the
differentiation of human ES-derived cells in vitro has been
analyzed (Schuldiner, et al. (2000) PNAS 97:11307-12).
Differentiation of the cells was assayed by expression of 24
cell-specific molecular markers that cover all embryonic germ
layers and 11 different tissues. Each growth factor had a unique
effect that may have resulted from directed differentiation and/or
cell selection, and could be divided into three categories: growth
factors that mainly induce mesodermal cells (Activin-A and
TGF-.beta.1); factors that activate ectodermal and mesodermal
markers (retinoic acid, EGF, BMP-4, and bFGF); and factors that
allow differentiation into the three embryonic germ layers,
including endoderm (NGF and HGF) (Schuldiner, et al. (2000) supra).
Therefore, synthetic triterpenoid-mediated induction of BMP-4
and/or TGF-.beta.1 in human ES cells may be of use in generating
mesodermal cells and/or ectodermal cells.
Synthetic Triterpenoid-Mediated Induction of BMPRII
[0033] It is known that stem cells from exfoliated deciduous teeth
(SHED) can be induced to differentiate into odontoblasts. SHED
express the BMP receptors BMPRIA, BMPRIB, and BMPRII, and blockade
of BMP-2 signaling inhibits the expression of markers of
odontoblastic differentiation by SHED cultured in tooth
slice/scaffolds. (Casagrande, et al. (2010) J. Dent. Res.
89:603-8). See also Yang, et al. (2007) Tissue Eng. 13:2803-12.
Therefore, synthetic triterpenoid-mediated induction of BMPRII in
SHED may be of use in combination with BMP-2 to facilitate
differentiation into odontoblasts.
Synthetic Triterpenoid-Mediated Induction of TGF-.beta.2
[0034] Combinations of growth factors that induce effective
chondrogenesis from adipose tissue-derived mesenchymal stem cells
(ATMSCs) have been evaluated (Kim & Im (2009) Tissue Eng. Part
A 15:1543-51). This analysis indicated that the combination of
TGF-.beta.2 and BMP-7 most effectively induced chondrogenesis from
ATMSCs (Kim & Im (2009) supra). Therefore, synthetic
triterpenoid-mediated induction of TGF-.beta.2 in ATMSCs, in
combination with BMP-7, can be used to enhance chondrogenesis of
ATMSCs in cartilage tissue engineering.
Synthetic Triterpenoid-Mediated Induction of Smad4
[0035] The growth in height of the bone plate is a result of
endochondral proliferation in epiphyseal growth plates and the
conversion of chondrocytes into new bone. The control of
chondrogenic differentiation and hypertrophy is critical for these
processes. It has been demonstrated that treatment of ATDC5 cells
with Genkwadaphnin induces cartilaginous nodules that are greater
in number and larger in size than control cultures (Choi, et al.
(2011) Eur. J. Pharmacol. 655:9-15). Genkwadaphnin increased the
synthesis of matrix proteoglycans, induced the activation of
alkaline phosphatase, as well as the expression of chondrogenic
marker genes such as type II collagen, aggrecan, type I collagen,
type X collagen, osteocalcin, and bone sialoprotein in ATDC5 cells.
The expression of signaling molecules involved in chondrogenesis
including Smad4, Sox9, and .beta.-catenin were also induced by
treatment of ATDC5 cells with Genkwadaphnin. Furthermore,
Genkwadaphnin induced the activation of extracellular
signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). To
analyze the role of Genkwadaphnin in growth plate chondrocyte in
vivo, mice were treated with Genkwadaphnin and chondrogenesis was
analyzed. This analysis showed a significant expansion in growth
plate and hypertrophic zone and numerous numbers of chondrocyte
positive cells in hypertrophic and proliferative bone areas (Choi,
et al. (2011) supra). Given these results and the results
exemplified herein, synthetic triterpenoid-mediated induction of
Smad4, Sox9, and type II collagen in cells of the growth plate may
be of use in new therapeutic avenues to treat a variety of skeletal
diseases, such as dwarfism.
[0036] As indicated herein, synthetic triterpenoids induce gene
expression and stem/progenitor cell differentiation. In this
respect, the present invention also provides a method for the
treatment of a patient suffering from a degenerative disease or
injury by transplantation into said patient of a population of
stem/progenitor cells treated with a synthetic triterpenoid.
Transplantation can be achieved using any conventional stem cell
transplant procedure with or without a scaffold or matrix. In
accordance with some embodiments, degenerative diseases or injuries
that may be treated in accordance with this method of the
invention, include but are not limited to, bone and cartilage
injury, joint injury, dental disease, osteoarthritis, rheumatoid
arthritis, degenerative disc disease, kyphosis, or osteopenia. In
addition, the synthetic triterpenoids of the instant invention find
application in the treatment of congenital disorders. In some
embodiments, the congenital disorder is scoliosis or a skeletal
disorder such as dwarfism. Embodiments of this method of the
invention, therefore extend to the use of cells prepared according
the present invention in the preparation of a medicament for the
treatment of a degenerative disease, injury or congenital
disorder.
[0037] Exemplary stem/progenitor cells, which can be treated with a
synthetic triterpenoid, the result of treatment, and the
therapeutic use of said cells are listed in Table 1.
TABLE-US-00001 TABLE 1 Result of Treatment with a Synthetic
Stem/Progenitor Cell Triterpenoid Therapeutic Uses Adipose tissue-
Induce chondrogenesis Cartilage tissue derived engineering
mesenchymal Treatment of stem cells degenerative disc diseases
Growth plate Bone formation Treatment of progenitors skeletal
diseases Embryonic stem Induce chondrogenesis Treatment of joint
cells injury and disease Mesenchymal Induce chondrogenesis Repair
of stem cell cartilage defects Induce bone formation Spinal fusion
Periodontal bone Periodontal formation alveolar bone graft in
periodontal defects or disease Increase bone mineral Treatment of
density and content Osteopenia Stem cells from Induce
differentiation Treatment of exfoliated into odontoblasts dental
caries deciduous teeth
[0038] For the purposes of the present invention, "prevention" or
"preventing" includes inhibiting the onset of a disease in a
subject or patient which may be at risk and/or predisposed to the
disease but does not yet experience or display any or all of the
pathology or symptomatology of the disease, and/or slowing the
onset of the pathology or symptomatology of a disease in a subject
or patient which may be at risk and/or predisposed to the disease
but does not yet experience or display any or all of the pathology
or symptomatology of the disease.
[0039] "Treatment" or "treating" includes inhibiting a disease in a
subject or patient experiencing or displaying the pathology or
symptomatology of the disease (e.g., arresting further development
of the pathology and/or symptomatology), ameliorating a disease in
a subject or patient that is experiencing or displaying the
pathology or symptomatology of the disease (e.g., reversing the
pathology and/or symptomatology), and/or effecting any measurable
decrease in a disease in a subject or patient that is experiencing
or displaying the pathology or symptomatology of the disease.
[0040] An "effective amount," "therapeutically effective amount" or
"pharmaceutically effective amount" means that amount which, when
administered to a subject or patient for treating a disease, is
sufficient to effect such treatment for the disease.
[0041] Synthetic triterpenoids of use in the instant methods
include synthetic oleanane triterpenoids. In particular
embodiments, the synthetic triterpenoids of use in the instant
methods have the structure of Formula I, which includes hydrates,
isomers, prodrugs or pharmaceutically acceptable salts of Formula
I:
##STR00001##
wherein, [0042] X.sup.1 and X.sup.2 are independently hydrogen,
OR.sup.a, NR.sup.aR.sup.b, or SR.sup.a, wherein [0043] R.sup.a is a
hydrogen, cyano, --CF.sub.3, nitro, amino, or substituted or
unsubstituted heteroaryl group; [0044] R.sup.b is hydrogen,
hydroxyl, alkyl, aryl, aralkyl, acyl, alkoxy, aryloxy, acyloxy,
alkylamino, arylamino, amido, or a substituted version of any of
these groups; [0045] or a substituent convertible in vivo to
hydrogen; [0046] provided that R.sup.a is absent when the atom to
which it is bound is part of a double bond, further provided that
when R.sup.a is absent the atom to which it is bound is part of a
double bond; [0047] Y is CH.sub.2 or CH.sub.2--CH.sub.2; [0048] Z
is a covalent bond, --C(.dbd.O) alkanediyl, alkenediyl, alkynediyl,
or a substituted version of any of these groups; [0049] the dashed
bonds can be independently present or absent; [0050] R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each independently a hydrogen,
hydroxyl, alkyl, substituted alkyl, alkoxy or substituted alkoxy
group; [0051] R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are independently hydrogen, hydroxyl, halo, cyano,
--C.ident.CR.sup.a, --CO.sub.2R.sup.a, --COR.sup.a, alkyl, alkenyl,
alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, acyl, alkoxy,
aryloxy, acyloxy, alkylamino, arylamino, nitro, amino, amido,
--C(O)R.sup.c or a substituted version of any of these groups,
wherein [0052] R.sup.c is hydrogen, hydroxy, halo, amino,
hydroxyamino, azido or mercapto; or C.sub.1-C.sub.15-alkyl,
C.sub.2-C.sub.15-alkenyl, C.sub.2-C.sub.15-alkynyl,
C.sub.6-C.sub.15-aryl, C.sub.7-C.sub.15-aralkyl,
C.sub.1-C.sub.15-heteroaryl, C.sub.2-C.sub.15-heteroaralkyl,
C.sub.1-C.sub.15-acyl, C.sub.1-C.sub.15-alkoxy,
C.sub.2-C.sub.15-alkenyloxy, C.sub.2-C.sub.15-alkynyloxy,
C.sub.6-C.sub.15-aryloxy, C.sub.7-C.sub.15-aralkyloxy,
C.sub.1-C.sub.15-heteroaryloxy, C.sub.2-C.sub.15-heteroaralkyloxy,
C.sub.1-C.sub.15-acyloxy, C.sub.1-C.sub.15-alkylamino,
C.sub.2-C.sub.15-dialkylamino, C.sub.1-C.sub.15-alkoxyamino,
C.sub.2-C.sub.15-alkenylamino, C.sub.2-C.sub.15-alkynylamino,
C.sub.6-C.sub.15-arylamino, C.sub.7-C.sub.15-aralkylamino,
C.sub.1-C.sub.15-heteroarylamino,
C.sub.2-C.sub.15-heteroaralkylamino,
C.sub.1-C.sub.15-alkylsulfonylamino, C.sub.1-C.sub.15-amido,
C.sub.1-C.sub.15-alkylsilyloxy, or substituted versions of any of
these groups; or [0053] R.sup.5 and R.sup.6, R.sup.7 and R.sup.8,
or R.sup.9 and R.sup.10 are independently taken together as .dbd.O;
[0054] R.sup.11 and R.sup.12 are each independently hydrogen,
hydroxyl, halo, alkyl , alkenyl, alkynyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, acyl, alkoxy, aryloxy, aralkoxy,
heteroaryloxy, hetero-aralkoxy, acyloxy, alkylamino, dialkylamino,
arylamino, aralkylamino, heteroarylamino, heteroaralkylamino,
amido, or a substituted version of any of these groups, or [0055]
R.sup.11 and R.sup.12 are taken together and are alkanediyl,
alkenediyl, arenediyl, alkoxydiyl, alkenyloxydiyl, alkylaminodiyl,
alkenylaminodiyl, or alkenylaminooxydiyl; [0056] R.sup.13 is
hydrogen, hydroxy or oxo; [0057] R.sup.14 is hydrogen or hydroxyl;
and [0058] R.sup.15 is [0059] a hydrogen, hydroxyl,
--NR.sup.dR.sup.e, cyano, halo, azido, phosphate,
1,3-dioxoisoindolin-2-yl, mercapto, silyl or --COOH group, [0060]
substituted or unsubstituted versions of C.sub.1-C.sub.15-alkyl,
C.sub.2-C.sub.15-alkenyl, C.sub.2-C.sub.15-alkynyl,
C.sub.6-C.sub.15-aryl, C.sub.7-C.sub.15-aralkyl,
C.sub.1-C.sub.15-heteroaryl,
C.sub.2-C.sub.15-heteroaralkyl,C.sub.1-C.sub.15acyl,
C.sub.1-C.sub.15-alkoxy, C.sub.2-C.sub.15-alkenyloxy,
C.sub.2-C.sub.15-alkynyloxy, C.sub.6-C.sub.15-aryloxy,
C.sub.7-C.sub.15-aralkyloxy, C.sub.1-C.sub.15-heteroaryloxy,
C.sub.2-C.sub.15-heteroaralkyloxy, C.sub.1-C.sub.15-acyloxy,
C.sub.1-C.sub.15-alkylamino, C.sub.2-C.sub.15-alkenylamino,
C.sub.2-C.sub.15-alkynylamino, C.sub.6-C.sub.15-arylamino,
C.sub.7-C.sub.15-aralkylamino, C.sub.1-C.sub.15-heteroarylamino,
C.sub.2-C.sub.15-heteroaralkylamino, C.sub.1-C.sub.15-amido,
C.sub.2-C.sub.15-alkenylthio, C.sub.2-C.sub.15-alkynylthio,
C.sub.2-C.sub.15- alkynylthio, C.sub.6-C.sub.15-arylthio,
C.sub.7-C.sub.15-aralkylthio, C.sub.1-C.sub.15-heteroarylthio,
C.sub.2-C.sub.15-heteroaralkylthio, C.sub.1-C.sub.15-acylthio,
C.sub.1-C.sub.12-thioacyl, C.sub.1-C.sub.12-alkylsulfonyl,
C.sub.2-C.sub.12-alkenylsulfonyl, C.sub.2-C.sub.12-alkynylsulfonyl,
C.sub.6-C.sub.12-arylsulfonyl, C.sub.7-C.sub.12-aralkylsulfonyl,
C.sub.1-C.sub.12-heteroarylsulfonyl,
C.sub.1-C.sub.12-heteroaralkylsulfonyl,
C.sub.1-C.sub.12-alkylsulfinyl, C.sub.2-C.sub.12-alkenylsulfinyl,
C.sub.2-C.sub.12-alkynylsulfinyl, C.sub.6-C.sub.12-aryl sulfinyl,
C.sub.7-C.sub.12-aralkylsulfinyl,
C.sub.1-C.sub.12-heteroarylsulfinyl,
C.sub.1-C.sub.12-heteroaralkylsulfinyl,
C.sub.1-C.sub.12-alkylphosphonyl, C.sub.1-C.sub.12-alkylphosphate,
C.sub.2-C.sub.12-dialkylphosphate, C.sub.1-C.sub.12-alkylammonium,
C.sub.1-C.sub.12-alkylsulfonium, C.sub.1-C.sub.15-alkylsilyl, or a
substituted version of any of these groups, [0061] a --CO.sub.2Me,
carbonyl imidazole, --CO--D-Glu(OAc).sub.4, --CONH.sub.2,
--CONHNH.sub.2, --CONHCH.sub.2CF.sub.3, or --C(=O)-heteroaryl
group, or [0062] Z and R.sup.15 form a three to seven-membered
ring, such that Z and R.sup.15 are further connected to one another
through one or more of --O--and alkanediyl, further wherein Z is
--CH-- and R.sup.15 is --CH.sub.2-- or Z, R.sup.15, and carbon
numbers 13, 17 and 18 form a ring such that R.sup.15 is bound to
carbon 13, wherein Y is methanediyl or substituted methanediyl and
R.sup.15 is --O--, wherein [0063] R.sup.d and R.sup.e are
independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl,
aralkyl, heteroaryl, heteroaralkyl, acyl, alkoxy, alkenyloxy,
alkynyloxy, aryloxy, aralkoxy, heteroaryloxy, heteroaralkoxy,
thioacyl, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl,
arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, or
heteroaralkylsulfonyl, or a substituted version of any of these
groups.
[0064] In certain embodiments, the bond between C.sub.2 and C.sub.3
in the A-ring is a double bond. In other embodiments, the bond
between C.sub.2 and C.sub.3 in the A-ring is a single bond.
[0065] Exemplary compounds of use in the methods of the present
invention include CDDO, CDDO-Me, CDDO-MA, CDDO-TFEA, CDDO-EA,
CDDO-Im and compound 1.
##STR00002## ##STR00003##
[0066] As used herein, "hydrogen" means --H; "hydroxyl" means --OH;
"oxo" means .dbd.O; "halo" or "halogen" means independently --F,
--Cl, --Br or --I; "hydroxyamino" means --NHOH; "nitro" means
--NO.sub.2; "cyano" means --CN; "azido" means --N.sub.3; "mercapto"
means --SH; "thio" means .dbd.S; "sulfonyl" means --S(O).sub.2--
(see additional definitions of groups containing the term sulfonyl,
e.g., alkylsulfonyl); and "silyl" means --SiH.sub.3 (see additional
definitions of group(s) containing the term silyl, e.g.,
alkylsilyl).
[0067] For the groups below, the following parenthetical subscripts
further define the groups as follows: "(Cn)" defines the exact
number (n) of carbon atoms in the group. For example,
"C.sub.1-C.sub.15-alkoxy" designates those alkoxy groups having
from 1 to 15 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
etc. or any range derivable therein (e.g., 3-10 carbon atoms)).
[0068] The term "alkyl" refers to a non-aromatic monovalent group
with a saturated carbon atom as the point of attachment, a linear
or branched, cyclo, cyclic or acyclic structure, no carbon-carbon
double or triple bonds, and no atoms other than carbon and
hydrogen. The groups, --CH.sub.3, --CH.sub.2CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.3, --CH(CH.sub.3).sub.2,
--CH(CH.sub.2).sub.2, --CH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--CH(CH.sub.3)CH.sub.2CH.sub.3, ---CH.sub.2CH(CH.sub.3).sub.2,
--C(CH.sub.3).sub.3, --CH.sub.2C(CH.sub.3).sub.3, cyclobutyl,
cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting
examples of alkyl groups.
[0069] The term "alkanediyl" refers to a non-aromatic divalent
group, wherein the alkanediyl group is attached with two
.sigma.-bonds, with one or two saturated carbon atom(s) as the
point(s) of attachment, a linear or branched, cyclo, cyclic or
acyclic structure, no carbon-carbon double or triple bonds, and no
atoms other than carbon and hydrogen. The groups, --CH.sub.2--
(methylene), --CH.sub.2CH.sub.2--,
--CH.sub.2C(CH.sub.3).sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2-- are non-limiting examples of
alkanediyl groups.
[0070] The term "alkenyl" refers to a monovalent group with a
nonaromatic carbon atom as the point of attachment, a linear or
branched, cyclo, cyclic or acyclic structure, at least one
nonaromatic carbon-carbon double bond, no carbon-carbon triple
bonds, and no atoms other than carbon and hydrogen. Non-limiting
examples of alkenyl groups include: --CH.dbd.CH.sub.2,
--CH.dbd.CHCH.sub.3, --CH.dbd.CHCH.sub.2CH.sub.3,
--CH.sub.2CH.dbd.CH.sub.2, --CH.sub.2CH.dbd.CHCH.sub.3, and
--CH.dbd.CH--C.sub.6H.sub.5.
[0071] The term "alkenediyl" refers to a nonaromatic divalent
group, wherein the alkenediyl group is attached with two
.sigma.-bonds, with two carbon atoms as points of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, at least
one nonaromatic carbon-carbon double bond, no carbon-carbon triple
bonds, and no atoms other than carbon and hydrogen. The groups,
--CH.dbd.CH--, --CH.dbd.C(CH.sub.3)CH.sub.2--, and
--CH.dbd.CHCH.sub.2-- are non-limiting examples of alkenediyl
groups.
[0072] The term "alkynyl" refers to a monovalent group with a
nonaromatic carbon atom as the point of attachment, a linear or
branched, cyclo, cyclic or acyclic structure, at least one
carbon-carbon triple bond, and no atoms other than carbon and
hydrogen. The groups, --C.ident.CH, --C.ident.CCH.sub.3,
--C.ident.CC.sub.6H.sub.5 and --CH.sub.2C.ident.CCH.sub.3, are
non-limiting examples of alkynyl groups.
[0073] The term "alkynediyl" refers to a nonaromatic divalent
group, wherein the alkynediyl group is attached with two
.sigma.-bonds, with two carbon atoms as points of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, at least
one carbon-carbon triple bond, and no atoms other than carbon and
hydrogen. The groups, --C.ident.C--, --C.ident.CCH.sub.2--, and
--C.ident.CCH(CH.sub.3)-- are non-limiting examples of alkynediyl
groups.
[0074] The term "aryl" refers to a monovalent group with an
aromatic carbon atom as the point of attachment, said carbon atom
forming part of a six-membered aromatic ring structure wherein the
ring atoms are all carbon, and wherein the monovalent group is
composed of carbon and hydrogen. Non-limiting examples of aryl
groups include phenyl, methylphenyl, (dimethyl)phenyl,
-ethylphenyl, propylphenyl, --C.sub.6H.sub.4CH(CH.sub.3).sub.2,
--C.sub.6H.sub.4CH(CH.sub.2).sub.2, methylethylphenyl, vinylphenyl,
naphthyl, and the monovalent group derived from biphenyl.
[0075] The term "arenediyl" refers to a divalent group, wherein the
arenediyl group is attached with two .sigma.-bonds, with two
aromatic carbon atoms as points of attachment, said carbon atoms
forming part of one or more six-membered aromatic ring structure(s)
wherein the ring atoms are all carbon, and wherein the monovalent
group is composed of carbon and hydrogen. Non-limiting examples of
arenediyl groups include:
##STR00004##
[0076] The term "aralkyl" refers to the monovalent
group--alkanediyl-aryl, in which the terms alkanediyl and aryl are
each used in a manner consistent with the definitions provided
above. Non-limiting examples of aralkyls include 1-phenyl-ethyl,
2-phenyl-ethyl, indenyl and 2,3-dihydro-indenyl, provided that
indenyl and 2,3-dihydro-indenyl are only examples of aralkyl in so
far as the point of attachment in each case is one of the saturated
carbon atoms.
[0077] The term "heteroaryl" refers to a monovalent group with an
aromatic carbon atom or nitrogen atom as the point of attachment,
said carbon atom or nitrogen atom forming part of an aromatic ring
structure wherein at least one of the ring atoms is nitrogen,
oxygen or sulfur, and wherein the monovalent group is composed of
carbon, hydrogen, aromatic nitrogen, aromatic oxygen or aromatic
sulfur. Non-limiting examples of aryl groups include acridinyl,
furanyl, imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl,
imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl,
phenylimidazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl,
quinolyl, quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl,
triazinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl,
pyrrolotriazinyl, pyrroloimidazolyl, chromenyl (where the point of
attachment is one of the aromatic atoms), and chromanyl (where the
point of attachment is one of the aromatic atoms).
[0078] The term "heteroaralkyl" refers to the monovalent group
-alkanediyl-heteroaryl, in which the terms alkanediyl and
heteroaryl are each used in a manner consistent with the
definitions provided above. Non-limiting examples of aralkyls
include pyridylmethyl, and thienylmethyl.
[0079] The term "acyl" refers to a monovalent group with a carbon
atom of a carbonyl group as the point of attachment, further having
a linear or branched, cyclo, cyclic or acyclic structure. The
groups, --CHO, --C(.dbd.O)CH.sub.3, --C(.dbd.O)CH.sub.2CH.sub.3,
--C(.dbd.O)CH.sub.2CH.sub.2CH.sub.3, --C(.dbd.O)CH(CH.sub.3).sub.2,
--C(.dbd.O)CH(CH.sub.2).sub.2, --C(.dbd.O)C.sub.6H.sub.5,
--C(.dbd.O)C.sub.6H.sub.4CH.sub.3, and
--C(.dbd.O)C.sub.6H.sub.4CH.sub.2CH.sub.3 are non-limiting examples
of acyl groups. The term "acyl" therefore encompasses, but is not
limited to groups sometimes referred to as "alkyl carbonyl" and
"aryl carbonyl" groups.
[0080] The term "alkoxy" refers to the group --OR, in which R is an
alkyl, as that term is defined herein. Non-limiting examples of
alkoxy groups include --OCH.sub.3, --OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2CH.sub.3, --OCH(CH.sub.3).sub.2,
--OCH(CH.sub.2).sub.2, --O-cyclopentyl, and --O-cyclohexyl.
[0081] Similarly, the terms "alkenyloxy," "alkynyloxy," "aryloxy,"
"aralkoxy," "heteroaryloxy," "heteroaralkoxy" and "acyloxy," refer
to groups, defined as --OR, in which R is alkenyl, alkynyl, aryl,
aralkyl, heteroaryl, heteroaralkyl and acyl, respectively, as those
terms are defined above.
[0082] The term "alkoxydiyl" refers to a non-aromatic divalent
group, wherein the alkoxydiyl group is attached with two
.sigma.-bonds, with (a) two saturated carbon atoms as points of
attachment, (b) one saturated carbon atom and one oxygen atom as
points of attachment, or (c) two oxygen atoms as points of
attachment, further having a linear or branched, cyclo, cyclic or
acyclic structure, no carbon-carbon double or triple bonds in the
group's backbone, further having no backbone atoms other than
carbon or oxygen and having at least one of each of these atoms in
the group's backbone. The groups, --O--CH.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2CH.sub.2--, --O--CH.sub.2CH.sub.2--O-- and
--O--CH.sub.2--O-- are non-limiting examples of alkoxydiyl
groups.
[0083] The term "alkenyloxydiyl" refers to a divalent group that is
nonaromatic prior to attachment, wherein the alkenyloxydiyl group
is attached with two .sigma.-bonds, which may become aromatic upon
attachment, with (a) two carbon atoms as points of attachment, (b)
one carbon atom and one oxygen atom as points of attachment, or (c)
two oxygen atoms as points of attachment, further having a linear
or branched, cyclo, cyclic or acyclic structure, at least one
carbon-carbon double bond that is non-aromatic at least prior to
attachment, further having no backbone atoms other than carbon or
oxygen and having at least one of each of these atoms in the
group's backbone. The groups, --O--CH.dbd.CH--, --O--CH.dbd.CHO--
and --O--CH.dbd.CHCH.sub.2-- are non-limiting examples of
alkenyloxydiyl groups.
[0084] The term "amino" refers to a moiety of the formula --NRR',
wherein R and R' are independently hydrogen, alkyl, cycloalkyl,
heterocycloalkyl, aryl or heteroaryl.
[0085] The term "alkylamino" refers to the group --NHR, in which R
is an alkyl, as that term is defined above. Non-limiting examples
of alkylamino groups include --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--NHCH.sub.2CH.sub.2CH.sub.3, --NHCH(CH.sub.3).sub.2,
--NHCH(CH.sub.2).sub.2, --NHCH.sub.2CH.sub.2CH.sub.2CH.sub.3,
--NHCH(CH.sub.3)CH.sub.2CH.sub.3, --NHCH.sub.2CH(CH.sub.3).sub.2,
--NHC (CH.sub.3).sub.3, --NH-cyclopentyl, and --NH-cyclohexyl.
[0086] Similarly, the terms "alkoxyamino," "alkenylamino,"
"alkynylamino," "arylamino," "aralkylamino," "heteroarylamino,"
"heteroaralkylamino," and "alkylsulfonylamino" refer to groups,
defined as --NHR, in which R is alkoxy, alkenyl, alkynyl, aryl,
aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl, respectively,
as those terms are defined above. A non-limiting example of an
arylamino group is --NHC.sub.6H.sub.5.
[0087] The term "dialkylamino" refers to the group --NRR', in which
R and R' can be the same or different alkyl groups, or R and R' can
be taken together to represent an alkanediyl having two or more
saturated carbon atoms, at least two of which are attached to the
nitrogen atom. Non-limiting examples of dialkylamino groups include
--NHC(CH.sub.3).sub.3, --N(CH.sub.3)CH.sub.2CH.sub.3,
--N(CH.sub.2CH.sub.3).sub.2, N-pyrrolidinyl, and N-piperidinyl.
[0088] The term "alkylaminodiyl" refers to a non-aromatic divalent
group, wherein the alkylaminodiyl group is attached with two
.sigma.-bonds, with (a) two saturated carbon atoms as points of
attachment, (b) one saturated carbon atom and one nitrogen atom as
points of attachment, or (c) two nitrogen atoms as points of
attachment, further having a linear or branched, cyclo, cyclic or
acyclic structure, no double or triple bonds in the group's
backbone, further having no backbone atoms other than carbon or
nitrogen and having at least one of each of these atoms in the
group's backbone. The groups, --NH--CH.sub.2CH.sub.2--,
--CH.sub.2--NH--CH.sub.2CH.sub.2--, --NH--CH.sub.2CH.sub.2--NH--
and --NH--CH.sub.2--NH-- are non-limiting examples of
alkylaminodiyl groups.
[0089] The term "alkenylaminodiyl" refers to a divalent group that
is nonaromatic prior to attachment, wherein the alkenylaminodiyl
group is attached with two .sigma.-bonds, which may become aromatic
upon attachment, with (a) two carbon atoms as points of attachment,
(b) one carbon atom and one nitrogen atom as points of attachment,
or (c) two nitrogen atoms as points of attachment, further having a
linear or branched, cyclo, cyclic or acyclic structure, at least
one carbon-carbon double bond or carbon-nitrogen double that is
non-aromatic at least prior to attachment, further having no
backbone atoms other than carbon or nitrogen. The groups
--NH--CH.dbd.CH--, --NH--CH.dbd.N-- and --NH--CH.dbd.CH--NH-- are
non-limiting examples of alkenylaminodiyl groups.
[0090] The term "alkenylaminooxydiyl" refers to a divalent group,
wherein the alkenylaminooxydiyl group is attached with two
.sigma.-bonds, which may become aromatic upon attachment, with two
atoms selected from the group consisting of carbon, oxygen and
nitrogen as points of attachment, further having a linear or
branched, cyclo, cyclic or acyclic structure, at least one
carbon-carbon double bond, carbon-nitrogen double, or
nitrogen-nitrogen double bond that is non-aromatic at least prior
to attachment, further having no backbone atoms other than carbon
nitrogen or oxygen and having at least one of each of these three
atoms in the backbone. The group --O--CH.dbd.N--, is a non-limiting
example of an alkenylaminooxydiyl group.
[0091] The term "amido" (acylamino) refers to the group --NHR, in
which R is acyl, as that term is defined herein. A non-limiting
example of an acylamino group is --NHC(.dbd.O)CH.sub.3.
[0092] The term "alkylthio" refers to the group --SR, in which R is
an alkyl, as that term is defined above. Non-limiting examples of
alkylthio groups include ---SCH.sub.3, --SCH.sub.2CH.sub.3,
--SCH.sub.2CH.sub.2CH.sub.3, --SCH(CH.sub.3).sub.2,
--SCH(CH.sub.2).sub.2, --S-cyclopentyl, and --S-cyclohexyl.
[0093] Similarly, the terms "alkenylthio," "alkynylthio,"
"arylthio," "aralkylthio," "heteroarylthio," "heteroaralkylthio"
and "acylthio" refer to groups, defined as --SR, in which R is
alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and
acyl, respectively, as those terms are defined above.
[0094] The term "thioacyl" refers to a monovalent group with a
carbon atom of a thiocarbonyl group as the point of attachment,
further having a linear or branched, cyclo, cyclic or acyclic
structure. The groups --CHS, --C(.dbd.S)CH.sub.3,
--C(.dbd.S)CH.sub.2CH.sub.3, --C(.dbd.S)CH.sub.2CH.sub.2CH.sub.3,
--C(.dbd.S)CH(CH.sub.3).sub.2, --C(.dbd.S)CH(CH.sub.2).sub.2,
--C(.dbd.S)C.sub.6H.sub.5, --(.dbd.S)C.sub.6H.sub.4CH.sub.3,
--C(.dbd.S)C.sub.6H.sub.4CH.sub.2CH.sub.3,
C(.dbd.S)C.sub.6H.sub.3(CH.sub.3).sub.2, and
--C(.dbd.S)CH.sub.2C.sub.6Hs, are non-limiting examples of thioacyl
groups. The term "thioacyl" therefore encompasses, but is not
limited to, groups sometimes referred to as "alkyl thiocarbonyl"
and "aryl thiocarbonyl" groups.
[0095] The term "alkylsulfonyl" refers to the group
--S(.dbd.O).sub.2R, in which R is an alkyl, as that term is defined
above. Non-limiting examples of alkylsulfonyl groups include:
--S(.dbd.O).sub.2CH.sub.3, --S(.dbd.O).sub.2CH.sub.2CH.sub.3,
--S(.dbd.O).sub.2CH.sub.2CH.sub.2CH.sub.3,
--S(.dbd.O).sub.2CH(CH.sub.3).sub.2,
--S(.dbd.O).sub.2CH(CH.sub.2).sub.2, --S(.dbd.O).sub.2-cyclopentyl,
and --S(.dbd.O).sub.2-cyclohexyl.
[0096] Similarly, the terms "alkenylsulfonyl," "alkynylsulfonyl,"
"arylsulfonyl," "aralkylsulfonyl," "heteroarylsulfonyl," and
"heteroaralkylsulfonyl" refer to groups, defined as --S(O).sub.2R,
in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and
heteroaralkyl, respectively, as those terms are defined above.
[0097] The term "alkylsulfinyl" refers to the group --S(.dbd.O)R,
in which R is an alkyl, as that term is defined above. Non-limiting
examples of alkylsulfinyl groups include --S(.dbd.O)CH.sub.3,
--S(.dbd.O)CH.sub.2CH.sub.3, --S(.dbd.O)CH.sub.2CH.sub.2CH.sub.3,
--S(.dbd.O)CH(CH.sub.3).sub.2, --S(.dbd.O)CH(CH.sub.2).sub.2,
--S(.dbd.O)-cyclopentyl, and --S(.dbd.O)-cyclohexyl.
[0098] Similarly, the terms "alkenylsulfinyl," "alkynylsulfinyl,"
"arylsulfinyl," "aralkylsulfinyl," "heteroarylsulfinyl" and
"heteroaralkylsulfinyl" refer to groups, defined as --S(.dbd.O)R,
in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and
heteroaralkyl, respectively, as those terms are defined above.
[0099] The term "alkylammonium" refers to a group, defined as
--NH.sub.2R.sup.+, --NHRR'.sup.+, or --NRR'R''.sup.+, in which R,
R' and R'' are the same or different alkyl groups, or any
combination of two of R, R' and R'' can be taken together to
represent an alkanediyl. Non-limiting examples of alkylammonium
cation groups include --NH.sub.2(CH.sub.3).sup.+,
--NH.sub.2(CH.sub.2CH.sub.3)+,
--NH.sub.2(CH.sub.2CH.sub.2CH.sub.3)+, --NH(CH.sub.3).sub.2.sup.+,
--NH(CH.sub.2CH.sub.3).sub.2.sup.+,
--NH(CH.sub.2CH.sub.2CH.sub.3).sub.2.sup.+, --N
(CH.sub.3).sub.3.sup.+,
--N(CH.sub.3)(CH.sub.2CH.sub.3).sub.2.sup.+,
--N(CH.sub.3).sub.2(CH.sub.2CH.sub.3).sup.+,
--N.sub.2C(CH.sub.3).sub.3.sup.+, --NH(cyclopentyl).sub.2.sup.+,
and --NH.sub.2(cyclohexyl).sup.30.
[0100] The term "alkylsulfonium" refers to the group --SRR', in
which R and R' can be the same or different alkyl groups, or R and
R' can be taken together to represent an alkanediyl. Non-limiting
examples of alkylsulfonium groups include --SH(CH.sub.3),
--SH(CH.sub.2CH.sub.3), --SH(CH.sub.2CH.sub.2CH.sub.3),
--S(CH.sub.3).sub.2, --S(CH.sub.2CH.sub.3).sub.2,
(CH.sub.2CH.sub.2CH.sub.3).sub.2, --SH(cyclopentyl), and
--SH(cyclohexyl).
[0101] The term "alkylsilyl" refers to a monovalent group, defined
as --SiH.sub.2R, --SiHRR', or --SiRR'R'', in which R, R' and R''
can be the same or different alkyl groups, or any combination of
two of R, R' and R'' can be taken together to represent an
alkanediyl. The groups --SiH.sub.2CH.sub.3, --SiH(CH.sub.3).sub.2,
--Si(CH.sub.3).sub.3 and --Si(CH.sub.3).sub.2C(CH.sub.3).sub.3, are
non-limiting examples of unsubstituted alkylsilyl groups.
[0102] The term "alkylphosphonyl" refers to the group
--OPO(OR).sub.2, where R is alkyl, as defined herein.
[0103] The term "alkylphosphate" refers to the group
--OP(.dbd.O)(OH)(OR), in which R is an alkyl, as that term is
defined above. Non-limiting examples of alkylphosphate groups
include --OP(.dbd.O)(OH)(OMe) and --OP(.dbd.O)(OH)(OEt).
[0104] The term "dialkylphosphate" refers to the group
--OP(.dbd.O)(OR)(OR'), in which R and R' can be the same or
different alkyl groups, or R and R' can be taken together to
represent an alkanediyl having two or more saturated carbon atoms,
at least two of which are attached via the oxygen atoms to the
phosphorus atom. Non-limiting examples of dialkylphosphate groups
include --OP(.dbd.O)(OMe).sub.2, --OP(.dbd.O)(OEt)(OMe) and
--OP(.dbd.O)(OEt).sub.2.
[0105] "Cycloalkyl" means a non-aromatic mono- or multicyclic ring
system including about 3 to about 10 carbon atoms, preferably about
5 to about 10 carbon atoms. Non-limiting examples of suitable
monocyclic cycloalkyls include cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl and the like. Non-limiting examples of
suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl,
adamantyl and the like.
[0106] "Heterocyclyl" or "heterocycloalkyl" means a non-aromatic
saturated monocyclic or multicyclic ring system including about 3
to about 10 ring atoms, preferably about 5 to about 10 ring atoms,
in which one or more of the atoms in the ring system is an element
other than carbon, for example nitrogen, oxygen or sulfur, alone or
in combination. Preferred heterocyclyls contain about 5 to about 6
ring atoms. The prefix aza, oxa or thia before the heterocyclyl
root name means that at least a nitrogen, oxygen or sulfur atom
respectively is present as a ring atom. The nitrogen or sulfur atom
of the heterocyclyl can be optionally oxidized to the corresponding
N-oxide, S-oxide or S,S-dioxide. Non-limiting examples of suitable
monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl,
piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl,
1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, lactam,
lactone, and the like. Non-limiting examples of suitable bicyclic
heterocyclyl rings include decahydro-isoquinoline,
decahydro-[2,6]naphthyridine, and the like.
[0107] Any of the groups described herein may be unsubstituted or
optionally substituted. When modifying a particular group,
"substituted" means that the group the term modifies may, but does
not have to, be substituted. Substitutions typically replace an
available hydrogen with an alkyl, alkenyl, alkynyl, aryl,
heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl,
heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy,
aryloxy, aralkoxy, alkoxyalkoxy, acyl, halo, nitro, cyano, carboxy,
alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl,
arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio,
heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, or
heterocyclyl.
[0108] Any undefined valency on an atom of a structure shown in
this application implicitly represents a hydrogen atom bonded to
the atom.
[0109] The term "hydrate" when used as a modifier to a compound
means that the compound has less than one (e.g., hemihydrate), one
(e.g., monohydrate), or more than one (e.g., dihydrate) water
molecules associated with each compound molecule, such as in solid
forms of the compound.
[0110] An "isomer" of a first compound is a separate compound in
which each molecule contains the same constituent atoms as the
first compound, but where the configuration of those atoms in three
dimensions differs.
[0111] "Pharmaceutically acceptable salts" means salts of compounds
of the present invention which are pharmaceutically acceptable, and
which possess the desired pharmacological activity. Such salts
include acid addition salts formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or with organic acids such as
1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,
2-naphthalenesulfonic acid, 3-phenylpropionic acid,
4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),
4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,
aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids,
aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,
camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,
cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,
glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,
heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,
laurylsulfuric acid, maleic acid, malic acid, malonic acid,
mandelic acid, methanesulfonic acid, muconic acid,
o-(4-hydroxybenzoyl)benzoic acid, oxalic acid,
p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids,
propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic
acid, stearic acid, succinic acid, tartaric acid,
tertiarybutylacetic acid, trimethylacetic acid, and the like.
Pharmaceutically acceptable salts also include base addition salts
which may be formed when acidic protons present are capable of
reacting with inorganic or organic bases. Acceptable inorganic
bases include sodium hydroxide, sodium carbonate, potassium
hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable
organic bases include ethanolamine, diethanolamine,
triethanolamine, tromethamine, N-methylglucamine and the like. It
should be recognized that the particular anion or cation forming a
part of any salt of this invention is not critical, so long as the
salt, as a whole, is pharmacologically acceptable. Additional
examples of pharmaceutically acceptable salts and their methods of
preparation and use are presented in Handbook of Pharmaceutical
Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds.,
Verlag Helvetica Chimica Acta, 2002).
[0112] Compounds of the invention may also exist in prodrug form.
Since prodrugs are known to enhance numerous desirable qualities of
pharmaceuticals, e.g., solubility, bioavailability, manufacturing,
etc., the compounds employed in some methods of the invention may,
if desired, be delivered in prodrug form. Thus, the invention
contemplates prodrugs of compounds of the present invention as well
as methods of delivering prodrugs. Prodrugs of the compounds
employed in the invention may be prepared by modifying functional
groups present in the compound in such a way that the modifications
are cleaved, either in routine manipulation or in vivo, to the
parent compound. Accordingly, prodrugs include, for example,
compounds described herein in which a hydroxy, amino, or carboxy
group is bonded to any group that, when the prodrug is administered
to a patient, cleaves to form a hydroxy, amino, or carboxylic acid,
respectively. For example, a compound comprising a hydroxy group
may be administered as an ester that is converted by hydrolysis in
vivo to the hydroxy compound. Suitable esters that may be converted
in vivo into hydroxy compounds include acetates, citrates,
lactates, phosphates, tartrates, malonates, oxalates, salicylates,
propionates, succinates, fumarates, maleates,
methylene-bis-.beta.-hydroxynaphthoate, gentisates, isethionates,
di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,
benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates,
quinates, esters of amino acids, and the like. Similarly, a
compound comprising an amine group may be administered as an amide
that is converted by hydrolysis in vivo to the amine compound.
[0113] The synthetic triterpenoids of the present disclosure may be
provided to a stem/progenitor cell ex vivo, in vitro or in vivo.
When used under ex vivo or in vivo conditions, the stem/progenitor
cell may be cultured under conventional cell culture conditions in
the presence of a synthetic triterpenoid to induce the expression
of one or more the genes described herein. As described herein,
additional cell growth and differentiation factors may be employed
to facilitate differentiation of the stem/progenitor cells.
Stem/progenitor cells treated in such a manner can then be
transplanted into a subject for therapeutic purposes.
[0114] The present invention also extends to articles coated with
one or more synthetic triterpenoids, such as tissue culture dishes,
multi-well plates (e.g., 1, 2, 4, 8, 24, 48, 96-wells, etc),
PETRI-dishes, tissue culture flasks, fermentors, bioreactors, etc.
for differentiating stem/progenitor cells. Such articles may be
composed of any generally suitable material, such as a plastics
material, for example polypropylene, or other materials such as
glass, metal, etc. Suitable metals include mirror-polished metals,
e.g., mirror-polished stainless steel.
[0115] When used in vivo, i.e., administered directly to a subject,
the instant synthetic triterpenoids can be administered by a
variety of methods, e.g., orally or by injection (e.g.
subcutaneous, intravenous, intraperitoneal, etc.). Depending on the
route of administration, the active compounds may be coated in a
material to protect the compound from the action of acids and other
natural conditions which may inactivate the compound. They may also
be administered by continuous perfusion/infusion into a subject or
wound site.
[0116] To administer the therapeutic compound by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation. For example, the therapeutic compound may be
administered to a patient in an appropriate carrier, for example,
liposomes, or a diluent. Pharmaceutically acceptable diluents
include saline and aqueous buffer solutions. Liposomes include
water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan, et al. (1984) J. Neuroimmunol. 7:27).
[0117] The therapeutic compound may also be administered
parenterally, intraperitoneally, intraspinally, or intracerebrally.
Dispersions can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations may contain a
preservative to prevent the growth of microorganisms.
[0118] Sterile injectable solutions can be prepared by
incorporating the synthetic triterpenoid in the required amount in
an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the synthetic
triterpenoid into a sterile carrier which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (i.e., the synthetic triterpenoid)
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0119] The synthetic triterpenoid can be orally administered, for
example, with an inert diluent or an assimilable edible carrier.
The synthetic triterpenoid and other ingredients may also be
enclosed in a hard or soft shell gelatin capsule, compressed into
tablets, or incorporated directly into the patient's diet. For oral
therapeutic administration, the synthetic triterpenoid may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. The percentage of the synthetic
triterpenoid in the compositions and preparations may, of course,
be varied. The amount of the synthetic triterpenoid in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0120] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
patients to be treated; each unit containing a predetermined
quantity of synthetic triterpenoid calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the synthetic triterpenoid and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such a synthetic
triterpenoid for the treatment of a selected condition in a
patient.
[0121] The synthetic triterpenoid may also be administered
topically to the skin, eye, or mucosa. Alternatively, if local
delivery to the lungs is desired the synthetic triterpenoid may be
administered by inhalation in a dry-powder or aerosol
formulation.
[0122] In yet another embodiment, the synthetic triterpenoid can be
administered as a coating on an article for implantation. Such
articles include polymer scaffolds containing stem/progenitor
cells, stents, shunts, and the like.
[0123] Active compounds are administered at a therapeutically
effective dosage sufficient to treat a condition associated with a
given patient. For example, the efficacy of a synthetic
triterpenoid can be evaluated in an animal model system that may be
predictive of efficacy in treating the disease in humans, such as
the model systems described herein.
[0124] The actual dosage amount of a synthetic triterpenoid of the
present disclosure or composition comprising a synthetic
triterpenoid of the present disclosure administered to a patient
may be determined by physical and physiological factors such as
age, sex, body weight, severity of condition, the type of disease
being treated, previous or concurrent therapeutic interventions,
idiopathy of the patient and on the route of administration. These
factors may be determined by a skilled artisan. The practitioner
responsible for administration will typically determine the
concentration of active ingredient(s) in a composition and
appropriate dose(s) for the individual patient. The dosage may be
adjusted by the individual physician in the event of any
complication.
[0125] An effective amount typically will vary from about 0.001
mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750
mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 1.0
mg/kg to about 250 mg/kg, from about 10.0 mg/kg to about 150 mg/kg
in one or more dose administrations daily, for one or several days
(depending of course of the mode of administration and the factors
discussed above). Other suitable dose ranges include 1 mg to 10000
mg per day, 100 mg to 10000 mg per day, 500 mg to 10000 mg per day,
and 500 mg to 1000 mg per day. In some particular embodiments, the
amount is less than 10,000 mg per day with a range of 750 mg to
9000 mg per day.
[0126] The effective amount may be less than 1 mg/kg/day, less than
500 mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day,
less than 50 mg/kg/day, less than 25 mg/kg/day or less than 10
mg/kg/day. It may alternatively be in the range of 1 mg/kg/day to
200 mg/kg/day. In other non-limiting examples, a dose may also
comprise from about microgram/kg/body weight, about 5
microgram/kg/body weight, about 10 microgram/kg/body weight, about
50 microgram/kg/body weight, about 100 microgram/kg/body weight,
about 200 microgram/kg/body weight, about 350 microgram/kg/body
weight, about 500 microgram/kg/body weight, about 1
milligram/kg/body weight, about 5 milligram/kg/body weight, about
10 milligram/kg/body weight, about 50 milligram/kg/body weight,
about 100 milligram/kg/body weight, about 200 milligram/kg/body
weight, about 350 milligram/kg/body weight, about 500
milligram/kg/body weight, to about 1000 mg/kg/body weight or more
per administration, and any range derivable therein. In
non-limiting examples of a derivable range from the numbers listed
herein, a range of about 5 mg/kg/body weight to about 100
mg/kg/body weight, about 5 microgram/kg/body weight to about 500
milligram/kg/body weight, etc., can be administered, based on the
numbers described above.
[0127] Single or multiple doses of the synthetic triterpenoids are
contemplated. Desired time intervals for delivery of multiple doses
can be determined by one of ordinary skill in the art employing no
more than routine experimentation. As an example, patients may be
administered two doses daily at approximately 12 hour intervals. In
some embodiments, the synthetic triterpenoid is administered once a
day.
[0128] The synthetic triterpenoid may be administered on a routine
schedule. As used herein a routine schedule refers to a
predetermined designated period of time. The routine schedule may
encompass periods of time which are identical or which differ in
length, as long as the schedule is predetermined. For instance, the
routine schedule may involve administration twice a day, every day,
every two days, every three days, every four days, every five days,
every six days, a weekly basis, a monthly basis or any set number
of days or weeks there-between. Alternatively, the predetermined
routine schedule may involve administration on a twice daily basis
for the first week, followed by a daily basis for several months,
etc. In other embodiments, the invention provides that the
synthetic triterpenoid may taken orally and that the timing of
which is or is not dependent upon food intake. Thus, for example,
the synthetic triterpenoid can be taken every morning and/or every
evening, regardless of when the patient has eaten or will eat.
[0129] The invention is described in greater detail by the
following non-limiting examples.
EXAMPLE 1
Materials and Methods
[0130] Reagents. The synthesis of the triterpenoids has been
described (Liby, et al. (2007) Nat. Rev. Cancer 7:357-69; Sporn, et
al. (2011) J. Nat. Prod. 74:537-45). All other chemicals were from
Sigma-Aldrich.
[0131] Calvarial Organ Cultures. Details have been published
previously (Garrett, et al. (2003) J. Clin. Invest. 111:1771-82).
Calvaria were cultured in BGJ medium supplemented with 1 mg/mL of
bovine serum albumin (Cohn fraction V), 100 U/ml each of
penicillin/streptomycin, and 0.292 mg/mL of glutamine. On Day 1,
the calvaria were treated with synthetic oleanane triterpenoids. On
Day 4, the medium was replaced with fresh medium, again containing
synthetic oleanane triterpenoids. On Day 7, calvaria were
collected, either stored at -80.degree. C. for further RNA analysis
or fixed in 10% buffered formalin for 24 hours and transferred to
80% ethanol for histologic analysis.
[0132] Histologic Analysis of Calvaria. After fixation for 24
hours, calvaria were decalcified in EDTA, embedded in paraffin and
sectioned at 4 .mu.m. Sections were stained either with modified
hematoxylin and eosin (H&E) or with toluidine blue (1% in 70%
ethanol for 20 minutes, followed by destaining in 70%, 90% and 100%
ethanol for 15 seconds), placed in xylene twice, and then mounted.
Procedures for immunofluorescence staining have been described
(Medici, et al. (2010) Nat. Med. 74:537-45). Primary antibodies
against collagen type II (AB746P, Millipore), were used at 1:100
dilution; ALEXFLUOR secondary antibodies (Invitrogen) at 1:200
dilution. For nuclear staining, To-PRO-3 Iodide (T3605, Invitrogen)
was used.
[0133] Bone Marrow-Derived Stem Cell Culture and Immunoblotting.
Human bone marrow-derived stromal cells, which contain a population
of responsive mesenchymal stem cells (ScienCell Research
Laboratories), were grown in mesenchymal stem cell medium
(ScienCell Research Laboratories). Cells were serum-starved 24
hours prior to all experimental conditions. Immunoblotting was
performed using the following antibodies at concentrations (and
using protocols) recommended by the respective manufacturers: SOX9
(sc-20095, Santa Cruz Biotechnology), collagen II.alpha.1 (sc-7764
and sc-28887, Santa Cruz), aggrecan (ab3778 and ab36861, Abcam),
BMP-2 (ab14933, Abcam), phospho-Smad5 (9516, Cell Signaling
Technology), Smad5 (9517, Cell Signaling Technology), .beta.-actin
(A1978, SigmaeAldrich). HRP-conjugated IgG TRUEBLOT reagents
(18-8814, eBioscience) were used at a dilution of 1:1000.
[0134] Quantitative RT-PCR (qRT-PCR). Detailed procedures for
qRT-PCR are known in the art (Lee, et al. (2006) Biochem.
Pharmacol. 72:332-43). In brief, 30 ng of RNA was reverse
transcribed to cDNA using the random primers and Applied
Biosystems' High Capacity cDNA Archive Kit in a 96-well format
MASTERCYCLER Gradient from EPPENDORF. Subsequently, cDNA was
amplified with ASSAYS-ON-DEMAND Products containing two gene
specific primers and one TAQMAN MGB probe (6-FAM dye-labeled) using
the TAQMAN Universal PCR Master Mix in an ABI PRISM 7000 Sequencing
Detector (Applied Biosystems). All labeled primers were obtained
from Applied Biosystems.
EXAMPLE 2
Gene Expression and Cellular Differentiation
[0135] For the results described here, more than 300 individual
calvarial organ cultures were performed. The analysis presented
herein indicated that both CDDO-Im and CDDO-EA have marked ability
to induce chondrogenesis in newborn mouse calvaria. Because it is
membranous bone, the newborn calvarium does not manifest the
chondrogenic phenotype, except for a very thin margin at suture
lines; care was taken to avoid using suture areas of calvaria in
any of the analyses described below. Treatment with either
triterpenoid (200 nM) for 7 days clearly had a profound
chondrogenic effect on the calvaria. No new cartilage was seen on
control sections stained with either H&E or toluidine blue. In
contrast, the metachromatic toluidine blue purple staining was
indicative of the ability of CDDO-Im and CDDO-EA to induce the
formation of proteoglycans, such as aggrecan, which are
characteristic of cartilage (Roughley (2001) Arthritis Res.
3:342-7). With toluidine blue, bone stained orthochromatically
(blue).
[0136] With respect to dose-response to either synthetic oleanane,
200 nM appeared to be optimal. Treatment with 50 nM triterpenoid
yielded only marginal induction of chondrogenesis, while treatment
with 500 nM synthetic oleanane gave somewhat variable results.
Treatment with 1 mM triterpenoid was invariably toxic to the organ
cultures. Results with 200 nM CDDO-Im and CDDO-EA were obtained in
at least three sets of replicate experiments and
immunohistochemistry showed that CDDO-Im and CDDO-EA (200 nM) both
induced the formation of type II collagen (collagen II.alpha.1),
which was not seen in the control sections.
[0137] In addition to the histologic analysis of the calvarial
cultures, mechanistic aspects of the action of both CDDO-EA and
CDDO-Im were investigated in the calvaria. After 7 days of culture,
RNA was isolated from the calvaria and quantitative RT-PCR analysis
was performed for more than 15 different markers, including: SOX9,
collagen II.alpha.1, all three isoforms of TGF-.beta., BMPs 2 and
4, BMP receptor II, Smads 3, 4, 6, and 7, tissue inhibitors of
metalloproteinases (TIMP-1 and TIMP-2), and matrix
metalloproteinase-9 (MMP-9). Tables 2 and 3 show that essentially
all of these markers (except MMP-9) were significantly up-regulated
by both triterpenoids, when the calvaria were treated at either the
200 or the 500 nM dose. The 50 nM dose was generally ineffective.
In contrast, both triterpenoids were strong inhibitors of the
expression of MMP-9; CDDO-EA (200 nM) caused almost 80% inhibition
of the expression of this metalloproteinase, which is known to be
involved in the degradation of cartilage (Shinoda, et al. (2008) J.
Biol. Chem. 283:24632-9).
TABLE-US-00002 TABLE 2 CDDO-Im Gene 50 nM 200 nM 500 nM SOX9 1.26
.+-. 0.08 1.33 .+-. 0.05 1.83 .+-. 0.13** COL2A1 1.45 .+-. 0.09
1.32 .+-. 0.23 1.72 .+-. 0.39 TGF-.beta.1 0.96 .+-. 0.07 1.08 .+-.
0.12 1.46 .+-. 0.12* TGF-.beta.2 1.17 .+-. 0.05 1.15 .+-. 0.09 1.17
.+-. 0.10 TGF-.beta.3 1.30 .+-. 0.07 1.52 .+-. 0.16** 1.66 .+-.
0.13** BMP-2 1.17 .+-. 0.07 1.60 .+-. 0.19 3.14 .+-. 0.53** BMP-4
0.97 .+-. 0.05 0.98 .+-. 0.06 1.35 .+-. 0.22 BMPRII 1.09 .+-. 0.06
1.22 .+-. 0.11 1.46 .+-. 0.14* Smad3 0.97 .+-. 0.07 0.90 .+-. 0.05
1.35 .+-. 0.19 Smad4 0.96 .+-. 0.06 1.03 .+-. 0.09 1.26 .+-. 0.09
Smad6 0.95 .+-. 0.05 1.23 .+-. 0.14 1.77 .+-. 0.19** Smad7 1.05
.+-. 0.07 1.320.14 1.78 .+-. 0.18** TIMP-1 1.43 .+-. 0.19 1.83 .+-.
0.18 3.00 .+-. 0.65** TIMP-2 1.24 .+-. 0.10 1.67 .+-. 0.19 2.62
.+-. 0.29** MMP-9 0.73 .+-. 0.16 0.44 .+-. 0.12** 0.26 .+-.
0.03**
TABLE-US-00003 TABLE 3 CDDO-ea Gene 50 nM 200 nM 500 nM SOX9 1.64
.+-. 0.17 2.62 .+-. 0.33** 3.12 .+-. 0.38** COL2A1 4.12 .+-. 1.78*
5.45 .+-. 1.13** 3.18 .+-. 0.71 TGF-.beta.1 1.17 .+-. 0.09 1.47
.+-. 0.24* 1.74 .+-. 0.17** TGF-.beta.2 1.36 .+-. 0.13* 1.61 .+-.
0.11** 1.88 .+-. 0.09** TGF-.beta.3 1.39 .+-. 0.07* 1.56 .+-.
0.13** 1.44 .+-. 0.11** BMP-2 1.53 .+-. 0.20 2.52 .+-. 0.29** 5.66
.+-. 0.67** BMP-4 1.28 .+-. 0.12 1.47 .+-. 0.13 2.49 .+-. 0.35**
BMPRII 1.50 .+-. 0.14* 1.71 .+-. 0.19** 2.24 .+-. 0.22** Smad3 1.14
.+-. 0.12 1.35 .+-. 0.07 1.93 .+-. 0.16** Smad4 1.06 .+-. 0.03 1.37
.+-. 0.12** 1.89 .+-. 0.12** Smad6 1.09 .+-. 0.04 1.77 .+-. 0.24**
2.85 .+-. 0.31** Smad7 1.28 .+-. 0.10 1.86 .+-. 0.19** 3.06 .+-.
0.28** TIMP-1 1.88 .+-. 0.34 2.85 .+-. 0.38** 4.65 .+-. 0.38**
TIMP-2 1.78 .+-. 0.18* 2.44 .+-. 0.31** 4.09 .+-. 0.36** MMP-9 0.49
.+-. 0.09** 0.22 .+-. 0.03** 0.19 .+-. 0.06**
[0138] Further studies on mechanism were pursued in human bone
marrow stem cell (BMSC) cultures. Western blot analysis showed that
CDDO-Im and CDDO-EA (each 100 nM for 7 days) induced expression of
the chondrocyte markers, SOX9, collagen type 2, and aggrecan, none
of which were detectable in the control cultured stem cells.
Furthermore, both triterpenoids (also at 100 nM) rapidly induced
expression of both BMP-2 and its relevant signal transduction
protein, phospho-Smad5 (P-Smad5), in these stem cells. BMP-2 is
known to induce chondrogenic lineage development of human
mesenchymal stem cells in culture (Schmitt, et al. (2003)
Differentiation 71:567-77).
* * * * *