U.S. patent application number 14/438894 was filed with the patent office on 2015-09-17 for cellular compositions used to restore stem cell or progenitor cell function and methods related thereto.
The applicant listed for this patent is EMORY UNIVERSITY. Invention is credited to Ji Woong Han, Young-Sup Yoon.
Application Number | 20150259649 14/438894 |
Document ID | / |
Family ID | 50685111 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259649 |
Kind Code |
A1 |
Yoon; Young-Sup ; et
al. |
September 17, 2015 |
CELLULAR COMPOSITIONS USED TO RESTORE STEM CELL OR PROGENITOR CELL
FUNCTION AND METHODS RELATED THERETO
Abstract
This disclosure relates to compounds, compositions and methods
of epigenetically transforming cells. In certain embodiments, the
disclosure relates to methods of generating epigenetically altered
cells comprising mixing isolated cells with compositions disclosed
herein under conditions such that epigenetically altered cells are
formed. In certain embodiments, methods of treating or preventing
vascular or diabetic diseases or conditions are contemplated. In
certain embodiments, epigenetically reprogramming adult bone
marrow-derived stem or progenitor cells including mesenchymal stem
cells (MSCs) or endothelial progenitor cells (EPCs) for autologous
treatments are contemplated.
Inventors: |
Yoon; Young-Sup; (Atlanta,
GA) ; Han; Ji Woong; (Decatur, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMORY UNIVERSITY |
Atlanta |
GA |
US |
|
|
Family ID: |
50685111 |
Appl. No.: |
14/438894 |
Filed: |
November 5, 2013 |
PCT Filed: |
November 5, 2013 |
PCT NO: |
PCT/US2013/068505 |
371 Date: |
April 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61723846 |
Nov 8, 2012 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/377 |
Current CPC
Class: |
C07D 401/14 20130101;
C07D 239/95 20130101; A61K 35/44 20130101; A61K 35/28 20130101;
C12N 5/0663 20130101; C07D 401/12 20130101; C12N 2501/065 20130101;
C12N 2501/40 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A61K 35/28 20060101 A61K035/28; A61K 35/44 20060101
A61K035/44 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under Grant
1DP3DK04346-01 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of epigenetically modifying progenitor cells comprising
mixing stem or progenitor cells and compositions comprising a
quinazoline compound under conditions such that cells with enhanced
angiogenic gene expression are produced.
2. The method of claim 1, wherein the conditions are such that
reduced DNA methylation in the promoters of angiogenic genes
occurs.
3. The method of claim 1, wherein the angiogenic genes are one or
more or all of the genes selected from Akt1, Hgf, Mapk14, Sphk1,
Vegfc, Nudt6, Kdr, Vegfa, and Pten.
4. The method of claim 1, wherein the stem or progenitor cells are
mesenchymal stem cells (MSCs) or endothelial progenitor cells
(EPCs), cardiac stem cells, myoblasts, adult bone marrow-derived
cells, umbilical cord blood cells, hematopoietic stem cells,
fibroblasts, or peripheral blood CD34.sup.+ cells.
5. The method of claim 4, wherein the cells endothelial progenitor
cells are bone marrow derived.
6. The method of claim 5, wherein the cells are obtained from a
subject diagnosed with diabetes and/or cardiovascular disease.
7. The method of claim 1, wherein the composition further
comprising a DNA methyltransferase inhibitor, a histone deacetylase
(HDAC) inhibitor, DNA methylation inhibitor, a Rho-associated
kinase (ROCK) inhibitor, Wnt inhibitor, GSK-3beta inhibitor, or a
dihydropyridine.
8. The method of claim 7, wherein the DNA methyltransferase
inhibitor is N-phthalyl-L-tryptophan (RG 108).
9. The method of claim 7, wherein the DNA methylation inhibitor is
5-azacitidine or decitabine.
10. The method of claim 7, wherein the HDAC inhibitor is
vorinostat-suberoylanilide hydroxamic acid (SAHA), trichostatin A
(TSA), or valproic acid (VPA).
11. The method of claim 7, wherein the ROCK inhibitor is
4-(1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (Y-27632)
or salt thereof.
12. The method of claim 7, wherein the dihydropyridine is
1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine-
carboxylic acid, methyl ester (BayK8644), ester, derivative, or
salt thereof.
13. The method of claim 7, wherein the GSK-3beta inhibitor is
6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2
pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99201)
or salt thereof.
14. The method of claim 1, wherein the quinazoline compound has
formula I, ##STR00011## or salts thereof wherein, R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each the same or
different hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
optionally substituted with one or more, the same or different,
R.sup.7; R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8; and R.sup.8 is halogen, nitro, cyano, hydroxy,
trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
15. The method of claim 14, wherein the quinazoline compound has
Formula IA: ##STR00012## or salts thereof wherein, R.sup.1,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each the same or
different hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
optionally substituted with one or more, the same or different,
R.sup.7; R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8; R.sup.8 is halogen, nitro, cyano, hydroxy,
trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;
R.sup.9 and R.sup.10 are each the same or different hydrogen,
alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,
carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,
(alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,
carbocyclyl, aryl, or heterocyclyl, wherein each R.sup.9 and
R.sup.10 are optionally substituted with one or more, the same or
different, R.sup.11; or R.sup.9 and R.sup.10 come together to form
a heterocyclyl optionally substituted with one or more, the same or
different, R.sup.11; R.sup.11 is alkyl, halogen, nitro, cyano,
hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl,
alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl,
alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,
wherein R.sup.11 is optionally substituted with one or more, the
same or different, R.sup.12; and R.sup.12 is halogen, nitro, cyano,
hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
16. The method of claim 1, wherein the quinazoline compound is
selected from:
N-(1-benzylpiperidin-4-yl)-2-(4-methylpiperazin-1-yl)quinazolin-4-a-
mine,
N-(1-benzylpiperidin-4-yl)-2-(4-methyl-1,4-diazepan-1-yl)quinazolin--
4-amine,
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-
-1-yl)quinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methylpiperazin-1-yl)quinaz-
olin-4-amine,
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-morpholinoquinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-2-thiomorpholinoquinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-2-(piperidin-1-yl)quinazolin-4-amine,
2-(azepan-1-yl)-N-(1-benzylpiperidin-4-yl)quinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-2-(4-methylpiperidin-1-yl)quinazolin-4-amine,
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(piperidin-1-yl)quinazoline-2,4--
diamine,
N-(1-benzylpiperidin-4-yl)-2-(pyrrolidin-1-yl)quinazolin-4-amine,
N-(1-benzylpiperidin-4-yl)-2-(4-(pyridin-2-yl)piperazin-1-yl)quinazolin-4-
-amine,
(4-(4-((1-benzylpiperidin-4-yl)amino)quinazolin-2-yl)piperazin-1-y-
l)(phenyl)methanone,
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(3-(dimethylamino)propyl)quinazo-
line-2,4-diamine,
2-((4-((1-benzylpiperidin-4-yl)amino)quinazolin-2-yl)amino)ethanol,
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(4-methylpiperazin-1-yl)quinazol-
ine-2,4-diamine,
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-morpholinoquinazoline-2,4-diamin-
e,
N-(1-benzylpiperidin-4-yl)-2-(2-methylpiperidin-1-yl)quinazolin-4-amine-
,
7-((5-aminopentyl)oxy)-N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(3-(dime-
thylamino)propyl)-6-methoxyquinazoline-2,4-diamine,
N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine,
and
7-((5-aminopentyl)oxy)-N.sup.4-(1-(5-aminopentyl)piperidin-4-yl)-N.su-
p.2-(3-(dimethylamino)propyl)-6-methoxyquinazoline-2,4-diamine or
salts thereof.
17. The method of claim 1, wherein the quinazoline compound has
Formula II: ##STR00013## or salts thereof wherein, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each the same or
different hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are optionally
substituted with one or more, the same or different, R.sup.7;
R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto,
formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8; R.sup.8 is halogen, nitro, cyano, hydroxy,
trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;
R.sup.13 and R.sup.14 are each the same or different hydrogen,
alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,
carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino,
(alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,
carbocyclyl, aryl, or heterocyclyl, wherein each R.sup.13 and
R.sup.14 are optionally substituted with one or more, the same or
different, R.sup.15; R.sup.15 is alkyl, halogen, nitro, cyano,
hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl,
alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl,
alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,
wherein R.sup.15 is optionally substituted with one or more, the
same or different, R.sup.16; and R.sup.16 is halogen, nitro, cyano,
hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
18. The method of claim 17, wherein the quinazoline compound is
selected from:
7-((5-aminopentyl)oxy)-N.sup.2-(3-(dimethylamino)propyl)-6-methoxy--
N.sup.4,N.sup.4-dimethylquinazoline-2,4-diamine and
7-((5-aminopentyl)oxy)-6-methoxy-N.sup.2,N.sup.2,N.sup.4,N.sup.4-tetramet-
hylquinazoline-2,4-diamine or salts thereof.
19. A method of treating or preventing vascular disease or
condition comprising: mixing progenitor cells from a subject and a
composition as provided in claim 1, under conditions such that
epigenetically modified cells with enhanced angiogenic gene
expression are produced; and administering an effective amount of a
composition comprising the epigenetically modified cells or cells
cultured therefrom to a subject in need thereof.
20. The method of claim 19, wherein the progenitor cells are bone
marrow derived cells, endothelial progenitor cells, or mesenchymal
stem cells.
21. The method of claim 20, wherein the progenitor cells were
obtained from the subject receiving the administered
composition.
22. The method of claim 20, wherein the vascular disease or
condition is peripheral vascular disease, myocardial ischemia,
heart failure, or stroke.
23. A method of treating or preventing a diabetic disease or
conditions comprising: mixing progenitor cells from a subject and a
composition as provided in claim 1 under conditions such that
epigenetically modified cells with enhanced angiogenic gene
expression are produced; and administering an effective amount of a
composition comprising the epigenetically modified cells or cells
cultured therefrom to a subject in need thereof.
24. The method of claim 23, wherein the progenitor cells are bone
marrow derived cells, endothelial progenitor cells, or mesenchymal
stem cells.
25. The method of claim 24, wherein the progenitor cells were
obtained from the subject receiving the administered
composition.
26. The method of claim 23, wherein the diabetic disease or
condition is diabetic wounds or diabetic neuropathy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 371 U.S.C. of International
Application No. PCT/US2013/068505 filed Nov. 5, 2013, which claims
the benefit of priority to U.S. provisional application No.
61/723,846 filed Nov. 8, 2012, which applications are hereby
incorporated by reference in their entireties.
BACKGROUND
[0003] Obesity and old age are risk factors for cardiovascular
disease, diabetes, and chronic kidney disease. See Osher &
Stern, Diabetes Care, 2009, 32(2):S398-S402. Often they occur
together indicating a mechanistic relationships. The migration of
circulating bone-marrow-derived stem cells to the site of damaged
tissue is typically not sufficient to restore the myocardium after
a heart attack. Typical cardiovascular compounds such as
beta-blockers, diuretics, and angiotensin-converting enzyme (ACE)
inhibitors do not restore function to damaged tissue. Thus, there
is a need to identify improved methods of treating cardiovascular
disease.
[0004] One strategy to treat cardiovascular disease is by restoring
damaged tissue through regeneration. Embryonic and adult-derived
stem cells for have been investigated. Orlic et al., Nature, 2001,
410:701-705, report that bone marrow cells regenerate infarcted
myocardium. See also Condorelli et al. Proc Natl Acad Sci USA.,
2001, 98:10733-10738. Wu et al., Stem Cells, 2007, 25:2648-2659,
report that mesenchymal stem cells enhance wound healing through
differentiation and angiogenesis.
[0005] Shi et al., Cell Stem Cell, 2008, 2(6):525-8, report that
BIX01294 and BayK8644, in combination with two factors (Oct4 and
Klf4), enhanced the reprogramming efficiency of mouse neural
progenitors and mouse embryonic fibroblasts. Upadyay et al., J Mol
Biol, 2012, 416(3):319-27, report that an analog of BIX-01294
selectively inhibits a family of histone H3 lysine 9 Jumonji
demethylases.
[0006] References cited herein are not an admission of prior
art.
SUMMARY
[0007] This disclosure relates to compounds, compositions and
methods of epigenetically transforming cells. In certain
embodiments, the disclosure relates to methods of generating
epigenetically altered cells comprising mixing isolated cells with
compositions disclosed herein under conditions such that
epigenetically altered cells are formed. In certain embodiments,
methods of treating or preventing vascular or diabetic diseases or
conditions are contemplated.
[0008] In certain embodiments, the disclosure contemplates methods
of epigenetically modifying stem or progenitor cells comprising
mixing the stem or progenitor cells and compositions comprising
compounds disclosed wherein such as a quinazoline compound,
5-aza-2'-deoxycytidine, or/and N-Phthalyl-L-tryptophan (RG108),
under conditions such that cells with enhanced angiogenic gene
expression are produced. In certain embodiments, the conditions are
such that reduced DNA methylation in the promoters of angiogenic
genes occurs. In certain embodiments, the angiogenic genes are one
or more or all of the genes selected from Akt1, Hgf, Mapk14, Sphk1,
Vegfc, Nudt6, Kdr, Vegfa, and Pten.
[0009] In certain embodiments, the stem or progenitor cells are
mesenchymal stem cells (MSCs) or endothelial progenitor cells
(EPCs), cardiac stem cells, myoblasts, adult bone marrow-derived
cells, umbilical cord blood cells, fibroblasts, or peripheral blood
CD34.sup.+ cells. In certain embodiments, the endothelial
progenitor cells are bone marrow derived. In certain embodiments,
the cells are obtained from a subject diagnosed with diabetes
and/or cardiovascular disease.
[0010] In certain embodiments, the composition comprise mixtures of
compounds disclosed herein. I certain embodiments, the composition
comprises a quinazoline compound optionally in combination with DNA
methyltransferase inhibitor, a histone deacetylase (HDAC)
inhibitor, DNA methylation inhibitor, a Rho-associated kinase
(ROCK) inhibitor, Wnt inhibitor, GSK-3beta inhibitor, and/or a
dihydropyridine. In certain embodiments, the DNA methyltransferase
inhibitor is N-phthalyl-L-tryptophan (RG 108). In certain
embodiments, the DNA methylation inhibitor is 5-azacitidine or
decitabine. In certain embodiments, the HDAC inhibitor is
vorinostat--suberoylanilide hydroxamic acid (SAHA), trichostatin A
(TSA), or valproic acid (VPA). In certain embodiments, the ROCK
inhibitor is
4-(1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide (Y-27632)
or salt thereof. In certain embodiments, the dihydropyridine is
1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine-
carboxylic acid, methyl ester (BayK8644), ester, derivative, or
salt thereof. In certain embodiments, the GSK-3beta inhibitor is
6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2
pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99201)
or salt thereof. In certain embodiments, the quinazoline compound
has formula I, formula IA, or formula II.
[0011] In certain embodiments, the disclosure contemplates methods
of treating or preventing vascular disease or condition comprising:
mixing progenitor cells from a subject and a composition as
provided herein under conditions such that epigenetically modified
cells with enhanced angiogenic gene expression are produced; and
administering an effective amount of a composition comprising the
epigenetically modified cells or cells cultured therefrom to a
subject in need thereof. In certain embodiments, the progenitor
cells are bone marrow derived cells, endothelial progenitor cells,
or mesenchymal stem cells. In certain embodiments, the progenitor
cells were obtained from the subject receiving the administered
composition. In certain embodiments, the vascular disease or
condition is peripheral vascular disease, myocardial ischemia,
cardiovascular disease, heart failure, or stroke.
[0012] In certain embodiments, the disclosure contemplates methods
of treating or preventing a diabetic disease or conditions
comprising: mixing progenitor cells from a subject and a
composition as provided herein under conditions such that
epigenetically modified cells with enhanced angiogenic gene
expression are produced; and administering an effective amount of a
composition comprising the epigenetically modified cells or cells
cultured therefrom to a subject in need thereof. In certain
embodiments, the progenitor cells are bone marrow derived cells,
endothelial progenitor cells, or mesenchymal stem cells. In certain
embodiments, the progenitor cells were obtained from the subject
receiving the administered composition. In certain embodiments, the
diabetic disease or condition is diabetic wounds or diabetic
neuropathy.
[0013] In certain embodiments, the epigenetically modified cells
may be cultured, expanded, or replicated in order to provide
enhanced concentrations upon administration/transplantation and the
modified cells may be autologous (i.e., derived from the person on
whom they are used) or allogeneic (i.e., originating from another
person) in origin. In certain embodiments, methods include those
subjects that are co-morbid with a vascular disease or condition
and a diabetic disease or condition.
[0014] In certain embodiments, the disclosure contemplates
intravenous injection and direct infusion into the coronary
arteries. In certain embodiments, the methods can be used in
subject whose blood flow has been restored to their hearts after a
heart attack. In certain embodiments, the compositions are injected
directly into the ventricular wall of the subject, i.e.,
endo-myocardial injection or into the peritoneal cavity, and may be
carried out either via a catheter or during open-heart surgery.
[0015] In certain embodiments, the disclosure contemplates methods
of treating diseases or conditions disclosed herein comprising
administering effective amounts of pharmaceutical compositions
disclosed herein comprising a compound or mixture of compounds to a
subject in need thereof.
[0016] In certain embodiments, treating or preventing kidney
disease or wound healing by methods disclosed herein are
contemplated.
[0017] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present disclosure to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
[0018] It should be emphasized that the embodiments of the present
disclosure, particularly, any "preferred" embodiments, are merely
possible examples of the implementations, merely set forth for a
clear understanding of the principles of the disclosure. Many
variations and modifications may be made to the above-described
embodiment(s) of the disclosure without departing substantially
from the spirit and principles of the disclosure. All such
modifications and variations are intended to be included herein
within the scope of this disclosure, and the present disclosure and
protected by the following claims.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows data suggesting the restoration of gene
expression of angiogenic genes in diabetic MSCs with compounds.
[0020] FIG. 2 shows data on impaired proliferation of D-MSCs.
[0021] FIG. 3 shows data on the restoration of impaired
proliferation of D-MSCs with compounds.
[0022] FIG. 4 shows data on the restoration of impaired cell
survival of diabetic MSCs with compounds.
[0023] FIG. 5 shows data on the restoration of impaired adhesion of
diabetic MSCs with compounds.
[0024] FIG. 6 shows data on the restoration of impaired cell
movement of diabetic MSCs with compounds.
[0025] FIG. 7 shows data on the restoration of impaired tube
formation of diabetic MSCs with compounds.
[0026] FIG. 8 shows data on the improvement of hindlimb ischemia by
implantation of reprogrammed D-MSCs.
[0027] FIG. 9 shows data on the increased capillary density by
implantation of reprogrammed D-MSCs.
[0028] FIG. 10 shows data on the increased angiogenic factors by
implantation of reprogrammed D-MSCs.
[0029] FIG. 11A shows data on the restoration of impaired global
gene expressions in diabetic MSCs by epigenetic compounds. Heat map
of differentially down-regulated genes in D-MSCs which were
significantly increased in RD-MSC. The color key represents FPKM
normalized log 2 transformed counts. Yellow indicates high
expression; orange indicates intermediate expression and red
indicates no expression.
[0030] FIG. 11B shows a Bar plot subset of representative 350 genes
which are straightforward to identify highly expressed genes in
RD-MSCs which were treated with epigenetic drug. Blue bar
represented genes expressed in N-MSC, and red bar indicated for
genes in D-MSC. And restored gene expressions in RD-MSC were showed
with green bar plot. Abbreviations: FPKM, fragment per kilobase of
exon per million fragments mapped.
[0031] FIG. 12 shows microarray results of mRNA expression of
angiogenic genes.
[0032] FIG. 13 shows gene expression patterns (top) and DNA
methylation (bottom) at the promoter regions of angiogenic genes in
EPCs.
DETAILED DESCRIPTION
[0033] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, and as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
disclosure will be limited only by the appended claims.
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0035] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0036] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0037] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of medicine, organic chemistry,
biochemistry, molecular biology, pharmacology, and the like, which
are within the skill of the art. Such techniques are explained
fully in the literature.
[0038] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
[0039] Prior to describing the various embodiments, the following
definitions are provided and should be used unless otherwise
indicated.
[0040] As used herein, the term "induced pluripotent stem cells"
refers to cells induced to a state that can differentiate into at
least one cell of the endoderm, mesoderm and ectoderm. Such
characteristics include the expression of certain genes and
proteins, chromatic methylation patterns, doubling time, embryoid
body formation, teratoma formation, and differentiability. Induced
pluripotent stem cells typically express alkaline phosphatase, Oct
4, Sox2, Nanog, and other pluripoteny-promoting factors. It is not
intended that the cells be entirely identical to embryonic cells.
Induced pluripotent stem cells may not necessarily be capable of
differentiating into any type of cell. SSEA-1 is a mouse ESC/iPSC
specific marker; SSEA-3 and -4 are not expressed in mouse ESC/iPSC.
However, human ESC/iPSC express SSEA-3 and SSEA-4, not SSEA-1.
SSEA-1 is mouse iPSC specific. SSEA-3, SSEA-4 is human iPSC
specific. TRA-1-60 and TRA-1-8 are usually used to identify human
PSC.
[0041] As used herein, "alkyl" means a noncyclic straight chain or
branched, unsaturated or saturated hydrocarbon such as those
containing from 1 to 10 carbon atoms, while the term "lower alkyl"
or "C.sub.1-4alkyl" has the same meaning as alkyl but contains from
1 to 4 carbon atoms. The term "higher alkyl" has the same meaning
as alkyl but contains from 7 to 20 carbon atoms. Representative
saturated straight chain alkyls include methyl, ethyl, n-propyl,
n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the
like; while saturated branched alkyls include isopropyl, sec-butyl,
isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls
contain at least one double or triple bond between adjacent carbon
atoms (referred to as an "alkenyl" or "alkynyl", respectively).
Representative straight chain and branched alkenyls include
ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, and the like; while representative straight
chain and branched alkynyls include acetylenyl, propynyl,
1-butyryl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,
and the like.
[0042] Non-aromatic mono or polycyclic alkyls are referred to
herein as "carbocycles" or "carbocyclyl" groups. Representative
saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and the like; while unsaturated carbocycles include
cyclopentenyl and cyclohexenyl, and the like.
[0043] "Heterocarbocycles" or heterocarbocyclyl" groups are
carbocycles which contain from 1 to 4 heteroatoms independently
selected from nitrogen, oxygen and sulfur which may be saturated or
unsaturated (but not aromatic), monocyclic or polycyclic, and
wherein the nitrogen and sulfur heteroatoms may be optionally
oxidized, and the nitrogen heteroatom may be optionally
quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl,
pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl,
oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, and the like.
[0044] "Aryl" means an aromatic carbocyclic monocyclic or
polycyclic ring such as phenyl or naphthyl. Polycyclic ring systems
may, but are not required to, contain one or more non-aromatic
rings, as long as one of the rings is aromatic.
[0045] As used herein, "heteroaryl" refers an aromatic
heterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and polycyclic ring systems. Polycyclic ring systems
may, but are not required to, contain one or more non-aromatic
rings, as long as one of the rings is aromatic. Representative
heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl,
pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,
isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,
imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that
the use of the term "heteroaryl" includes N-alkylated derivatives
such as a 1-methylimidazol-5-yl substituent.
[0046] As used herein, "heterocycle" or "heterocyclyl" refers to
mono- and polycyclic ring systems having 1 to 4 heteroatoms
selected from nitrogen, oxygen and sulfur, and containing at least
1 carbon atom. The mono- and polycyclic ring systems may be
aromatic, non-aromatic or mixtures of aromatic and non-aromatic
rings. Heterocycle includes heterocarbocycles, heteroaryls, and the
like.
[0047] "Alkylthio" refers to an alkyl group as defined above
attached through a sulfur bridge. An example of an alkylthio is
methylthio, (i.e., --S--CH.sub.3).
[0048] "Alkoxy" refers to an alkyl group as defined above attached
through an oxygen bridge. Examples of alkoxy include, but are not
limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,
s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy. Preferred alkoxy
groups are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy,
s-butoxy, t-butoxy.
[0049] "Alkylamino" refers an alkyl group as defined above attached
through an amino bridge. An example of an alkylamino is
methylamino, (i.e., --NH--CH.sub.3).
[0050] "Alkanoyl" refers to an alkyl as defined above attached
through a carbonyl bride (i.e., --(C.dbd.O)alkyl).
[0051] "Alkylsulfonyl" refers to an alkyl as defined above attached
through a sulfonyl bridge (i.e., --S(.dbd.O).sub.2alkyl) such as
mesyl and the like, and "Arylsulfonyl" refers to an aryl attached
through a sulfonyl bridge (i.e.,--S(.dbd.O).sub.2aryl).
[0052] "Alkylsulfinyl" refers to an alkyl as defined above attached
through a sulfinyl bridge (i.e. --S(.dbd.O)alkyl).
[0053] The term "substituted" refers to a molecule wherein at least
one hydrogen atom is replaced with a substituent. When substituted,
one or more of the groups are "substituents." The molecule may be
multiply substituted. In the case of an oxo substituent (".dbd.O"),
two hydrogen atoms are replaced. Example substituents within this
context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano,
oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl,
heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, --NR.sub.aR.sub.b, --NR.sub.aC(.dbd.O)R.sub.b,
--NR.sub.aC(.dbd.O)NR.sub.aNR.sub.b, --NR.sub.aC(.dbd.O)OR.sub.b,
--NR.sub.aSO.sub.2R.sub.b, --C(.dbd.O)R.sub.a, --C(.dbd.O)OR.sub.a,
--C(.dbd.O)NR.sub.aR.sub.b, --OC(.dbd.O)NR.sub.aR.sub.b,
--OR.sub.a, --SR.sub.a, --SOR.sub.a, --S(.dbd.O).sub.2R.sub.a,
--OS(.dbd.O).sub.2R.sub.a and --S(.dbd.O).sub.2OR.sub.a. R.sub.a
and R.sub.b in this context may be the same or different and
independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl,
amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,
heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl,
heteroaryl, heteroarylalkyl.
[0054] The term "optionally substituted," as used herein, means
that substitution is optional and therefore it is possible for the
designated atom to be unsubstituted.
[0055] As used herein, "salts" refer to derivatives of the
disclosed compounds where the parent compound is modified making
acid or base salts thereof. Examples of salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines, alkylamines, or dialkylamines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like. In
preferred embodiment the salts are conventional nontoxic
pharmaceutically acceptable salts including the quaternary ammonium
salts of the parent compound formed, and non-toxic inorganic or
organic acids. Preferred salts include those derived from inorganic
acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,
phosphoric, nitric and the like; and the salts prepared from
organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the
like.
[0056] "Subject" refers any animal, preferably a human patient,
livestock, rodent, monkey or domestic pet.
[0057] As used herein, the term "derivative" refers to a
structurally similar compound that retains sufficient functional
attributes of the identified analogue. The derivative may be
structurally similar because it is lacking one or more atoms,
substituted, a salt, in different hydration/oxidation states, or
because one or more atoms within the molecule are switched, such
as, but not limited to, replacing an oxygen atom with a sulfur or
nitrogen atom or replacing an amino group with a hydroxyl group or
vice versa. The derivative may be a prodrug. Derivatives may be
prepare by any variety of synthetic methods or appropriate
adaptations presented in synthetic or organic chemistry text books,
such as those provide in March's Advanced Organic Chemistry:
Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007)
Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley
(2006) Lutz F. Tietze hereby incorporated by reference.
[0058] As used herein, the term "combination with" when used to
describe administration with an additional treatment means that the
agent may be administered prior to, together with, or after the
additional treatment, or a combination thereof.
Epigenetic Regulation and Methods of Restoration of Diabetic Cell
Function
[0059] Gene expression is affected by chromatin. Chromatin is the
complex of DNA and the histone proteins. Chromatin remodeling
occurs through post translational modification of the histone
proteins. Chromatic remodeling may also occur with the addition of
methyl groups to the DNA. Cytosines are often converted to
5-methylcytosine in CG sequences of DNA, often referred to as
methylated "CpGs." 5-Methylcytosine, like cytosine, pairs with
guanine Highly methylated DNA tends to be less transcriptionally
active. Histone acetylation, methylation, ubiquitylation, and
phosphorylation modifications typically occur at the N-termini of
histones. For example, acetylation of the K14 and K9 lysines of the
tail of histone H3 by histone acetyltransferase enzymes (HATs) has
typically been correlated with transcriptional competence.
Methylation of lysine 9 of histone H3 has typically been associated
with constitutively transcriptionally silent chromatin.
[0060] Adult bone marrow-derived stem or progenitor cells including
mesenchymal stem cells (MSCs) or endothelial progenitor cells
(EPCs) are very useful for repairing cardiovascular disease;
however, autologous cells derived from diabetic patients are not as
effective as those from non-diabetic or healthy volunteers. As many
cardiovascular disease patients who are candidates for cell therapy
have diabetes as co-morbid disease which reduce the therapeutic
effects of stem or progenitor cells derived from patient own
bodies. Studied disclosed herein indicate that EPCs and MSCs have
significant epigenetic changes in the promoter regions of
angiogenic factors and these defects reduce the functionality of
the cells. Certain disclosed herein are epigenetic modulators can
rescue gene expression of these progenitor and stem cells and thus
restore their tissue repairing and angiogenic capacities. The
candidate diseases that are applied by this new cell therapy
include peripheral vascular disease, myocardial ischemia, heart
failure, diabetic wounds, diabetic neuropathy, and stroke.
[0061] Reversing epigenetic alterations with small molecules can
restore gene expression and cell biologic function of diabetic bone
marrow-derived cells (D-BMCs). Studies herein indicate that
diabetic EPCs (D-EPCs) have high methylation in the promoter
regions of angiogenic genes and compounds disclosed herein can
reverse these changes, increase gene expression and improve
cellular function. In certain embodiments, the disclosure
contemplates composition comprising compounds or combinations of:
DNA methyltransferase inhibitors (e.g., 5-Aza-2'-Deoxycytidine,
5-Aza or RG1 08), histone deacetylase (HDAC) inhibitors (e.g.,
valproic acid, VPA), and histone methyltransferase (HMT) inhibitors
(e.g., compounds of formula I for G9a and GLP HMTs).
Quinazoline Compounds
[0062] In certain embodiments, the disclosure contemplates the use
of quinazoline compounds disclosed herein to generate
epigenetically altered cells. Although it is not intended that
certain embodiments of the disclosure be limited by any particular
mechanism, it is believed that the compounds typically have the
ability to inhibit the function of methyltransferases such as G9a
methyltransferase.
[0063] In certain embodiments, contemplated quinazoline compounds
have Formula I:
##STR00001##
[0064] or salts thereof wherein,
[0065] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
each the same or different hydrogen, alkyl, halogen, nitro, cyano,
hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl,
alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl,
alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,
wherein each R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are optionally substituted with one or more, the same or
different, R.sup.7;
[0066] R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8; and
[0067] R.sup.8 is halogen, nitro, cyano, hydroxy, trifluoromethoxy,
trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,
sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,
methylamino, ethylamino, dimethylamino, diethylamino,
N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,
N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,
N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,
ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,
ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,
N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
[0068] In certain embodiments, R.sup.4 is alkoxy optionally
substituted with one or more, the same or different, R.sup.7.
[0069] In certain embodiments, R.sup.2 is alkylamino optionally
substituted with one or more, the same or different, R.sup.7.
[0070] In certain embodiments, R.sup.2 is a heterocyclyl optionally
substituted with one or more, the same or different, R.sup.7.
[0071] In certain embodiments, R.sup.1 is alkyl or benzyl
optionally substituted with one or more, the same or different,
R.sup.7.
[0072] In certain embodiments, contemplated quinazoline compounds
have Formula IA:
##STR00002##
[0073] or salts thereof wherein,
[0074] R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each the
same or different hydrogen, alkyl, halogen, nitro, cyano, hydroxy,
amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,
alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl,
alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,
wherein each R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
optionally substituted with one or more, the same or different,
R.sup.7;
[0075] R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8;
[0076] R.sup.8 is halogen, nitro, cyano, hydroxy, trifluoromethoxy,
trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,
sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,
methylamino, ethylamino, dimethylamino, diethylamino,
N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,
N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,
N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,
ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,
ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,
N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;
[0077] R.sup.9 and R.sup.10 are each the same or different
hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto,
formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each
R.sup.9 and R.sup.10 are optionally substituted with one or more,
the same or different, R.sup.11;
[0078] or R.sup.9 and R.sup.10 come together to form a heterocyclyl
optionally substituted with one or more, the same or different,
R.sup.11;
[0079] R.sup.11 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.11
is optionally substituted with one or more, the same or different,
R.sup.12; and
[0080] R.sup.12 is halogen, nitro, cyano, hydroxy,
trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
[0081] In certain embodiments, contemplated quinazoline compounds
are selected from: [0082]
N-(1-benzylpiperidin-4-yl)-2-(4-methylpiperazin-1-yl)quinazolin-4-amine,
[0083]
N-(1-benzylpiperidin-4-yl)-2-(4-methyl-1,4-diazepan-1-yl)quinazoli-
n-4-amine, [0084]
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)qu-
inazolin-4-amine, [0085]
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-(4-methylpiperazin-1-yl)quinaz-
olin-4-amine, [0086]
N-(1-benzylpiperidin-4-yl)-6,7-dimethoxy-2-morpholinoquinazolin-4-amine,
[0087] N-(1-benzylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine,
[0088]
N-(1-benzylpiperidin-4-yl)-2-thiomorpholinoquinazolin-4-amine,
[0089]
N-(1-benzylpiperidin-4-yl)-2-(piperidin-1-yl)quinazolin-4-amine,
[0090]
2-(azepan-1-yl)-N-(1-benzylpiperidin-4-yl)quinazolin-4-amine,
[0091]
N-(1-benzylpiperidin-4-yl)-2-(4-methylpiperidin-1-yl)quinazolin-4-amine,
[0092]
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(piperidin-1-yl)quinazoli-
ne-2,4-diamine, [0093]
N-(1-benzylpiperidin-4-yl)-2-(pyrrolidin-1-yl)quinazolin-4-amine,
[0094]
N-(1-benzylpiperidin-4-yl)-2-(4-(pyridin-2-yl)piperazin-1-yl)quinazolin-4-
-amine, [0095]
(4-(4-((1-benzylpiperidin-4-yl)amino)quinazolin-2-yl)piperazin-1-yl)(phen-
yl)methanone, [0096]
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(3-(dimethylamino)propyl)quinazo-
line-2,4-diamine, [0097]
2-((4-((1-benzylpiperidin-4-yl)amino)quinazolin-2-yl)amino)ethanol,
[0098]
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(4-methylpiperazin-1-yl)q-
uinazoline-2,4-diamine, [0099]
N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-morpholinoquinazoline-2,4-diamin-
e, [0100]
N-(1-benzylpiperidin-4-yl)-2-(2-methylpiperidin-1-yl)quinazolin--
4-amine, [0101]
7-((5-aminopentyl)oxy)-N.sup.4-(1-benzylpiperidin-4-yl)-N.sup.2-(3-(dimet-
hylamino)propyl)-6-methoxyquinazoline-2,4-diamine, [0102]
N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine,
and [0103]
7-((5-aminopentyl)oxy)-N.sup.4-(1-(5-aminopentyl)piperidin-4-yl)-N.sup.2--
(3-(dimethylamino)propyl)-6-methoxyquinazoline-2,4-diamine or salts
thereof.
[0104] In certain embodiments, contemplated quinazoline compounds
have Formula II:
##STR00003##
[0105] or salts thereof wherein,
[0106] R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each the
same or different hydrogen, alkyl, halogen, nitro, cyano, hydroxy,
amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy,
alkylthio, alkylamino, (alkyl).sub.2amino, alkylsulfinyl,
alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,
wherein each R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
optionally substituted with one or more, the same or different,
R.sup.7;
[0107] R.sup.7 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.7
is optionally substituted with one or more, the same or different,
R.sup.8;
[0108] R.sup.8 is halogen, nitro, cyano, hydroxy, trifluoromethoxy,
trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,
sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,
methylamino, ethylamino, dimethylamino, diethylamino,
N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,
N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,
N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,
ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,
ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,
N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;
[0109] R.sup.13 and R.sup.14 are each the same or different
hydrogen, alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto,
formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each
R.sup.13 and R.sup.14 are optionally substituted with one or more,
the same or different, R.sup.15;
[0110] R.sup.15 is alkyl, halogen, nitro, cyano, hydroxy, amino,
mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkylsulfonyl,
arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R.sup.15
is optionally substituted with one or more, the same or different,
R.sup.16; and
[0111] R.sup.16 is halogen, nitro, cyano, hydroxy,
trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,
carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,
acetyl, acetoxy, methylamino, ethylamino, dimethylamino,
diethylamino, N-methyl-N-ethylamino, acetylamino,
N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,
N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio,
ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl,
methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,
N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,
N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
[0112] In certain embodiments, R.sup.13 and R.sup.14 are hydrogen
or alkyl optionally substituted with one or more, the same or
different, R.sup.15.
[0113] In certain embodiments, R.sup.4 is alkoxy optionally
substituted with one or more, the same or different, R.sup.7.
[0114] In certain embodiments, R.sup.2 is alkylamino optionally
substituted with one or more, the same or different, R.sup.7.
[0115] In certain embodiments, R.sup.2 is a alkyl or heterocyclyl
optionally substituted with one or more, the same or different,
R.sup.7.
[0116] In certain embodiments, contemplated quinazoline compounds
are selected from: [0117]
7-((5-aminopentyl)oxy)-N.sup.2-(3-(dimethylamino)propyl)-6-methoxy-N.sup.-
4,N.sup.4-dimethylquinazoline-2,4-diamine and [0118]
7-((5-aminopentyl)oxy)-6-methoxy-N.sup.2,N.sup.2,N.sup.4,N.sup.4-tetramet-
hylquinazoline-2,4-diamine or salts thereof.
Examples
Restore of Epigenetic Silencing of Angiogenic Genes in Diabetic
MSCs by Compound Epigenetic Regulators
[0119] To reprogram diabetic MSCs with epigenetic compounds,
cultured D-MSCs were treated with HDAC inhibitor: Vorinostat or
suberoylanilide hydroxamic acid (SAHA); DNA methyltransferase
inhibitor: Decitabine (trade name Dacogen), or
5-aza-2'-deoxycytidine (5-aza); multiple histone 3 lysine 9 (H3K9)
methyltransferases G9a and GLP inhibitor: BIX-01294; and GSK3.beta.
inhibitor: CHIR99021 for 7 more days. N-MSCs and D-MSCs were
cultured by a standard MSC culture method. The cultured MSCs are
typically reseeded at day 4 and cultured for another 4 days. To
examine the expression levels of angiogenic genes, we isolated RNAs
from N-, D-, or epigenetic compound treated reprogrammed D-MSCs
(RD-MSCs), and performed real-time RT-PCR with specific primers of
angiogenic genes. The expression of major angiogenic genes of
RD-MSCs were significantly higher than D-MSCs up to N-MSCs or more
(FIG. 1) although different type of inhibitors showed different
pattern of gene expression. This result indicated that defective
expression of angiogenic gene in D-MSCs could be restored by
epigenetic modification by treatment of known epigenetic compounds.
Among inhibitors, HDAC inhibitor showed significant increased
pattern in most of genes, therefore, HDAC inhibitor, SAHA, was
selected for following experiments.
Restore of Impaired Cell Proliferation and Survival of Diabetic
MSCs with Compound
[0120] To determine whether diabetes can affect the cell
proliferation capacity of MSCs, immunocytochemistry was performed
for Ki67, an effective marker for mitosis. The percentage of
Ki67-positive cells was 53% lower in D-MSCs compared to N-MSCs
(FIG. 2). To determine whether this was due to the slow
proliferation of D-MSCs compared to N-MSCs, a colony forming unit
assay was performed, which determines actively proliferating cell
portions. The number of colony forming units (colonies/100 cells)
was 45% smaller in D-MSCs compared to N-MSCs, while RD-MSCs
restored the number of colony forming up to 80% of N-MSCs (FIG. 3),
suggesting that the proportion of actively proliferating cells is
smaller in D-MSCs than N-MSCs which could be restored by the
treatment of compounds.
[0121] Furthermore, TUNEL assay was conducted to determine the
effects of diabetes on the cell survival of MSCs. Compared to the
N-MSCs, D-MSCs showed significantly higher number of TUNEL positive
cells. However, the percent of TUNEL positive cells in RD-MSCs was
reduced to normal level (FIG. 4). These results suggest that
apoptosis of MSCs observed in diabetes can also rescued by
epigenetic compound treatment.
Restore of Impaired Angiogenic Cellular Functions of Diabetic MSCs
with Compound
[0122] To verify the angiogenic function in vitro, the adhesion
capability of MSCs was compared because cell-adhesion mechanisms
play a fundamental role during angiogenesis. Most of the adherent
cells from normal bone marrow were spindle- or flatten-shaped,
whereas the adherent cells from diabetes were rounded up showing
that they are losing the adhesion capability. But the shape of
adherent reprogrammed cells were similar to normal cells, and the
number of adherent cells in these reprogrammed cells was also
increased (FIG. 5). Also, wound healing assay to was performed
verify the migration activity of different MSCs as the cellular
function for angiogenesis. The distance of migrating D-MSCs was
significantly decreased compared to N-MSCs, while RD-MSCs move
faster than D-MSCs and even more than N-MSCs indicating that
impaired cell migration of diabetic cells was restored (FIG. 6).
Furthermore, the tube formation capability of each MSC were
compared as an in vitro angiogenic index. HUVECs were plated onto
Matrigel together with N-, D-, or RD-MSCs in the absence of
additional angiogenic factors. The tubular structures were not
clearly formed in the D-MSC group, but N- and RD-MSC group formed
the similar tube-like structures suggesting that tube forming
ability was also restored by compound treatment (FIG. 7). Taken
together, these data indicated that epigenetic compounds increase
the cellular function of D-MSCs for angiogenesis.
Reprogrammed D-MSC by Epigenetic Compounds Improved Hindlimb
Ischemia In Vivo
[0123] To test the recovered therapeutic and regenerative effects
of RD-MSCs, three hundred thousand N-, D-, or RD-MSCs were injected
into ischemic hindlimbs and monitored blood flow using LDPI. PBS
injection served as a control. Compared to the PBS-injected group,
only the N- and RD-MSC implanted group showed significantly
improved blood flow (FIG. 8), suggesting that the impaired
regenerative capacity for limb ischemia of D-MSCs was restored by
epigenetic compounds in experimental animal model. To further
determine the neovascularization capability of reprogrammed D-MSCs,
capillary density in the ischemic muscle was quantified after the
cell implantation. Capillary density, determined by BSL-1 staining,
was significantly different between the N-MSC and D-MSC implanted
groups. RD-MSC injected group showed the significant increased
number of capillary compared to D-MSC group (FIG. 9).
Restored Levels of Paracrine Factors in Ischemic Limbs Injected
with RD-MSCs
[0124] To investigate the changes of paracrine factors involved in
neovascularization and apoptosis in vivo by the implantation of
RD-MSCs, various gene expression levels were measured by real-time
RT-PCR using muscles harvested at 2 weeks after cell implantation.
The levels of Vegfa, Vegfc, Fgf2, Pdgfb, and Kdr were significantly
increased in RD-MSC group compared D-MSC group which showed lower
levels than in the N-MSC group (FIG. 10). However, Angp1 levels
were not detected in RD-MSC group as well as D-MSC group. Together,
these results suggest that the defective expression of angiogenic
paracrine factors of D-MSCs is restored to higher levels of certain
angiogenic factors up to N-MSCs by compound treatment.
Global Gene Expression Changes in Diabetic MSCs by Epigenetic
Compounds
[0125] To determine global gene expression changes in diabetic and
epigenetic compound treating conditions in MSCs compared to normal
conditions, the next-generation RNA-sequencing (RNA-seq) was
performed with total RNAs from N-MSCs, D-MSCs, and RD-MSCs with two
biological replicates. A total of 1,303 genes were identified were
differentially and significantly down-regulated in D-MSCs compared
to N-MSCs, and 350 genes out of these down-regulated subset of
genes in D-MSC were significantly up-regulated by epigenetic
compound in RD-MSC (FIG. 11). Down-regulated genes in D-MSCs
compared to N-MSCs were enriched with functional annotation
clusters associated with MAPK signaling pathway, mTOR signaling
pathway, chemokine/cytokine signaling pathway, pathways in cancer,
Jak-STAT signaling pathway, axon guidance as well as gene clusters
involved in cellular carbohydrates metabolism including insulin
signaling pathway. Notably, angiogenesis related gene groups such
as gene clusters in VEGF signaling pathway, chemokine signaling
pathway, and cell adhesion molecules; and immune response related
gene clusters including leukocyte transendothelial migration,
hematopoietic cell lineage, TB cell receptor signaling pathway, Fc
epsilon RI signaling pathway, Toll-like receptor signaling pathway,
NK cell mediated cytotoxicity, and complement/coagulation cascades
were also repressed in diabetic cells.
[0126] Next, the effects of epigenetic compounds on
diabetic-associated gene repressions was evaluated by comparing
RNA-seq data from D-MSC and RD-MSC. Interestingly, only specific
gene sets in D-MSC were restored by epigenetic compound although
the target of this compound was known as general effector in
epigenetic modulators. The most enriched down-regulating gene
clusters in D-MSC such as MAPK signaling pathway, axon guidance,
and VEGF signaling pathway, mTOR signaling pathway, and
adipocytokine signaling pathway, as well as carbohydrate
metabolisms were significantly increased in RD-MSC. However, immune
response related gene sets were not restored by epigenetic compound
except the genes in complementary/coagulation cascades
Methods
Isolation of Mesenchymal Stem Cells (MSCs) and Cell Culture.
[0127] Diabetes was induced in 8 week-old male Fischer 344 rats by
intravenous injection of streptozotocin (5 mg/kg). Blood glucose
levels were measured using an Accu-Check glucometer (Roche,
Indianapolis, Ind.) after one week and two months. If blood glucose
was higher than 200 mg/dl, rats were considered diabetic. Rats with
glucose levels lower than 200 mg/dl were excluded from the study.
Age- and sex-matched normal rats were used as controls. At 2 months
after diabetes, bone marrow mononuclear cells were isolated using
HISTOPAQUE.RTM.-1083 (Sigma-Aldrich, St. Louis, Mo.) according to
the manufacturer's instructions. Whole bone marrow mononuclear
cells from normal rats were plated in low glucose (100 mg/dl)
Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented
with 15% FBS and antibiotics (Antibiotic-Antimycotic, Invitrogen).
For diabetic MSCs, high glucose (450 mg/dl) DMEM supplemented with
15% FBS media was used. After 3 days of incubation in a humidified
incubator at 37.degree. C. with 5% CO.sub.2, non-adherent
hematopoietic cells were removed by changing media. The adherent
MSCs were further cultured. Passage two to four cells were used for
study. At the 4 day of MSC culture, MSCs from diabetic rats were
treated with various combinations of chemicals: 6 .mu.M of SAHA, 1
.mu.M of BIX01294, 2 .mu.M of 5-aza (Dacogen), and 6 .mu.M of
CHIR99021 for 7 days.
Real-Time PCR
[0128] Gene expression was determined by TaqMan.RTM. real-time
quantitative PCR on the 7300 Sequence Detection System (Applied
Biosystems) using TaqMan.RTM. PCR Master Mix (Applied Biosystems).
Total RNA was extracted from tissue or cells with RNeasy Plus Mini
Kit (Qiagen, Carlsbad, Calif.) according to the manufacturer's
instructions. First-strand cDNA was generated using the TaqMan.RTM.
Multiscribe Reverse Transcription Kit (Applied Biosystems, Foster
City, Calif.) primed with a mix of oligo dT and random
hexamers.
[0129] Relative mRNA expression of target gene normalized to GAPDH
was calculated by using the formula Relative Expression
Level=2-.DELTA.Ct, where .DELTA.Ct=Ct gene of interest-Ct GAPDH.
The primers and probes were designed using Primer Express 3.0
(Applied Biosystems).
MSC Proliferation and Colony Forming Unit Assay
[0130] For the cell proliferation assay, MSCs were plated at 200
cells/cm.sup.2 and the number of resultant cells was determined. To
evaluate proliferative cells, immunocytochemistry with anti-Ki-67
antibody (Millipore) was performed. For the colony forming unit
assay, one hundred MSCs were plated onto 100 mm dishes and cultured
for 14 days. The cells were stained with crystal violet and the
number of colonies were counted.
Apoptosis Assay
[0131] In situ labeling of fragmented DNA was performed with
terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin
nick end labeling (TUNEL) method with the use of an in situ cell
detection kit (Roche) as previously described.
Cell Adhesion Assay
[0132] Ninety-six well plates were pre-coated with 1 .mu.g/well
vitronectin (Sigma), 10 .mu.g/well collagen (Sigma, MO), 10
.mu.g/well laminin (BD Biosciences), and 20 .mu.g/well fibronectin
(Sigma) respectively at 4.degree. C. overnight. Plates were washed
with PBS and incubated for 1 hour with 1% bovine serum albumin to
block remaining protein binding sites. 2.5.times.104 of bone
marrow-derived rat mesenchymal stem cells; N-MSC, D-MSC, and
RD-MSC, were seeded on 96 well plates and cultured in DMEM medium
for 24 hour at 37.degree. C. and 5% CO2. Cells were gently washed
three-times with PBS and adherent cells were counted under phase
contrast microscopy (Nikon Eclipse Ti).
Wound Healing Assay
[0133] MSCs were plated in confluence in 24-well plates
(1.5.times.10.sup.5 cells/300 .mu.l/well) and starved for 24 h in
serum-free DMEM media. After 24 h the medium was discarded and the
monolayer was scratched with a sterile plastic pipette tip. The
cells were washed three times with DMEM, and 300 .mu.l of DMEM
medium with serum were added. At 0 and 24 h, images were taken
(Nikon Eclipse Ti). The area of each dish was measured using Image
J.
Tube Formation Assay
[0134] Glass chamber slides were coated with Matrigel (Becton
Dickinson) and overlaid with 7.times.10.sup.4 human umbilical vein
endothelial cells together with 2.5.times.10.sup.5 MSCs in
endothelial basal medium (EBM, Cambrex Bio Science) in the absence
of additional growth factors or cytokines and incubated at
37.degree. C., 5% CO.sub.2 for 16 hours. The tube formation was
evaluated using a phase-contrast microscope.
In Vivo Hindlimb Ischemia Experiments
[0135] Unilateral hindlimb ischemia was created in normal F344 rats
by ligation of the femoral artery and removing all arterial
branches. Three hundred thousand DiI-labeled MSCs in 500 .mu.l of
PBS were intramuscularly injected into the ischemic hindlimbs.
Blood flow in the hindlimb was measured using a Laser Doppler
perfusion imager (LDPI, Moor instrument, UK). Mean values of
perfusion was calculated from the stored digital color-coded
images. The level of blood flow of the ischemic (left) limb was
normalized to that of non-ischemic (right) limb to avoid data
variations caused by ambient light and temperature. Four weeks
after the injection of N-, D-, or RD-MSCs into ischemic hindlimbs,
the hindlimb muscles were harvested, fixed with 4% paraformaldehyde
at 4.degree. C. overnight, and frozen-sectioned. To visualize
capillaries, the sections were stained using BS lectin-I and the
capillary density was determined under conventional epifluorescence
microscopy.
Next-Generation RNA Sequencing
[0136] The converted cDNAs from isolated RNAs,
immunoprecipitated/captured DNA, input, and negative control (in
the absence of biotin) were used to generate DNA libraries
following the Illumina protocol. 38-cycle single end sequencing was
performed. Image processing and sequence extraction were done using
the standard Illumina Pipeline. Two independent libraries and runs
(one lane per library) for each biological replicate were
generated. FASTQ sequence files were aligned to reference genome
using Bowtie. The best alignment and reporting option were used for
all conditions, corresponding to no more than 2 bp mismatches
across each 38 bp read. Mapped reads were assembled into RNA
transcripts using the Cufflinks software (version 1.3.0). Cufflinks
was run using the annotation file of known genes and the mapped
reads produced by Tophat. Fragments Per Kilobase of exon model per
Million mapped fragments (FPKM) value (a normalized gene expression
value that are comparable between different samples and genes)
together with confidence intervals were estimated for each
replicate. R/bioconductor packages were used to generate the
heatmap and box plot of the different expressed genes comparing
N-MSC, D-MSC, and RD-MSC. Database for Annotation, Visualization
and Integrated Discovery (DAVID) tool was used to generate the
functional annotation charts for impaired (N-MSC vs. D-MSC) and
restored (D-MSC vs. RD-MSC) gene expressions.
ChIP-PCR
[0137] The cells were grown on the 10-cm plates to 85% confluence.
Formaldehyde was added to a final concentration of 1%, and the
plates were incubated for 10 min at 37.degree. C. The cross-linking
reaction was stopped by the addition of 100 mM glycine containing
protease inhibitors (Complete; RocheApplied Science). Cells were
washed in dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA,
16.7 mMTris-HCl, 150 mMNaCl, pH 8.0 plus protease inhibitors),
resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mMTris-HCl, pH
8.0 plus protease inhibitors) and sonicated to shear the DNA into
0.3.about.3-kb fragments. Following sonication and centrifugation,
sheared chromatin was incubated with anti-H3K9/14 antibodies, that
can detect the changes of histone acetylation as activation mark,
or anti-rabbit IgG antibody (negative control) overnight at
4.degree. C. Then, protein G beads were added and the chromatin was
incubated for 2 hours in rotation. An aliquot of chromatin that was
not incubated with an antibody was used as the input control
sample. Antibody-bound protein/DNA complexes were eluted and
subjected to PCR analysis.
Synthesis of Quinazoline Derivatives
##STR00004##
[0139] Reagents and conditions: (a) Benzyl bromide,
K.sub.2CO.sub.3, dry DMF, rt; (b) HNO.sub.3 69.5%, (Ac).sub.2O,
0.degree. C. then rt; (c) iron dust, NH.sub.4Cl, i-PrOH-H.sub.2O
(5:3), reflux; (d) i) methyl chloroformate, DIPEA, DMF-DCM (2:1),
0.degree. C. then rt, ii) H.sub.2O.sub.2 30%, NaOH, EtOH; (e)
POCl.sub.3, N,N-diethylaniline, reflux; (f) dimethylamine 2M in
THF, dry THF, rt; (g) dimethylamine 2M in THF, dry THF, 110.degree.
C., sealed tube; (h) H.sub.2, Pd-C, dry THF-MeOH (2:1), 1 atm, rt;
(i) tert-butyl(5-hydroxypentyl)carbamate, PPh.sub.3, DIAD, dry THF,
N.sub.2, rt; (j) HCl 4N in dioxane, dry THF, rt; (k) TFA, reflux;
(l) N.sup.1,N.sup.1-dimethylpropane-1,3-diamine, 110.degree. C.,
sealed tube; (m) HCl 4N in dioxane, dry THF-MeOH (1:1), rt.
[0140]
7-((5-aminopentyl)oxy)-N.sup.2-(3-(dimethylamino)propyl)-6-methoxy--
N.sup.4,N.sup.4-dimethylquinazoline-2,4-diamine (E67-2):
.sup.1H-NMR (400 MHz, DMSO) .delta. 1.50 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2O), 1.65 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.81 (m, 2H,
CH.sub.2CH.sub.2O), 2.80 (m, 2H, H.sub.3N.sup.+CH.sub.2CH.sub.2),
3.26 (s, 6H, N(CH.sub.3).sub.2), 3.45 (s, 6H, N(CH.sub.3).sub.2),
3.87 (s, 3H, OCH.sub.3), 4.08 (m, 2H, CH.sub.2CH.sub.2CH.sub.2O),
7.45 (s, 1H, H quinazoline ring), 7.76 (s, 1H, H quinazoline ring),
7.99 (s br, 3H, H.sub.3N.sup.+), 12.1 (s br, 1H, HN.sup.+
quinazoline ring).
[0141]
7-((5-aminopentyl)oxy)-6-methoxy-N.sup.2,N.sup.2,N.sup.4,N.sup.4-te-
tramethylquinazoline-2,4-diamine (E67-5): .sup.1H-NMR (400 MHz,
DMSO) .delta. 1.50 (m, 2H, CH.sub.2CH.sub.2CH.sub.2O), 1.64 (m, 2H,
CH.sub.2CH.sub.2CH.sub.2CH.sub.2O), 1.81 (m, 2H,
CH.sub.2CH.sub.2O), 1.99 (m, 2H, NHCH.sub.2CH.sub.2), 2.74 (s, 6H,
.sup.+HN(CH.sub.3).sub.2), 2.80 (m, 2H,
H.sub.3N.sup.+CH.sub.2CH.sub.2), 3.11 (m, 2H,
CH.sub.2CH.sub.2N(CH.sub.3).sub.2H.sup.+), 3.47 (s, 6H,
N(CH.sub.3).sub.2), 3.49 (m, 2H, NHCH.sub.2CH.sub.2CH.sub.2), 3.86
(s, 3H, OCH.sub.3), 4.09 (m, 2H, CH.sub.2CH.sub.2CH.sub.2O), 7.15
(s, br, 1H NHCH.sub.2CH.sub.2CH.sub.2), 7.45 (s, 1H, H quinazoline
ring), 7.93 (m, 4H, H quinazoline ring and H.sub.3N.sup.+), 10.6
(s, br, .sup.+HN(CH.sub.3).sub.2), 12.8 (s br, 1H, HN.sup.+
quinazoline ring).
[0142] Additional compound below were prepared by using the same or
similar conditions by substituting appropriate starting
materials.
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
Gene Expression Pattern and DNA Methylation at the Promoter Regions
of Angiogenic Genes in Endothelial Progenitor Cells (EPCs)
[0143] To prepare diabetic endothelial progenitor cells (EPCs)
D-EPCs, streptozotocin (STZ)- was used to induce diabetes in rats
(12 wks after the onset of diabetes). For normal EPCs (N-EPCs),
age- and sex-matched non-diabetic (normal) rats were used. To
culture EPCs, bone marrow (BM)-derived mononuclear cells (MNCs)
isolated from the tibia and femur were plated on cell culture
dishes coated with rat vitronectin and cultured in EBM-2 medium
containing 5% FBS and cytokine cocktail, singleQuots.RTM. for 7
days. differential expression of angiogenic genes between N-EPCs
and DEPCs were investigated by using an Angiogenesis Microarray kit
(Oligo GEArray.RTM. kit, Superarray bioscience) (FIG. 12). The left
panel shows a set of representative mRNA expression profiles
comparing N-EPCs and D-EPCs. The microarray profiles illustrate
some of the increased (>5 fold) angiogenic factors (the black
spots) in N-EPCs v.s., D-EPCs. Gene expression is significantly
suppressed in D-EPCs.
Gene Expression Pattern and DNA Methylation at the Promoter Regions
of Angiogenic Genes in Endothelial Progenitor Cells (EPCs)
[0144] To prepare diabetic endothelial progenitor cells (EPCs)
D-EPCs, streptozotocin (STZ)- was used to induce diabetes in rats
(12 wks after the onset of diabetes). For normal EPCs (N-EPCs),
age- and sex-matched non-diabetic (normal) rats were used. To
culture EPCs, bone marrow (BM)-derived mononuclear cells (MNCs)
isolated from the tibia and femur were plated on cell culture
dishes coated with rat vitronectin and cultured in EBM-2 medium
containing 5% FBS and cytokine cocktail, singleQuots.RTM. for 7
days. differential expression of angiogenic genes between N-EPCs
and DEPCs were investigated by using an Angiogenesis Microarray kit
(Oligo GEArray.RTM. kit, Superarray bioscience) (FIG. 12). The left
panel shows a set of representative mRNA expression profiles
comparing N-EPCs and D-EPCs. The microarray profiles illustrate
some of the increased (>5 fold) angiogenic factors (the black
spots) in N-EPCs v.s., D-EPCs. Gene expression is significantly
suppressed in D-EPCs.
[0145] To test the reversible changes of gene expression and DNA
methylation of such gene promoters in D-EPCs by compound epigenetic
regulators, epigenetic profiling studies on D-EPCs were performed.
STZ-induced diabetic rats 12 wks after the onset of diabetes were
used. Age- and sex-matched rats were used as non-diabetic controls.
At the 4 day of EPC culture, EPCs from diabetic rats were treated
with various combinations of chemicals:
0.5 mM of BIX-01294, 1 mM of VPA, and 5.about.M RG108. Cells were
collected at the 10 day of culture for extraction of genomic DNAs
and RNAs for epigenetic profiling by modified methylation-sensitive
restriction enzyme (MSRE)-PCR assay (EpiMark.TM., NEB) and qRT-PCR,
respectively.
[0146] For MSRE-PCR assay, purified genomic DNAs were treated with
methylation sensitive restriction enzyme, followed by the treatment
of T4-BGT and UDP-Glucose (UDP-Glc) for adding a glucose moiety to
5-hydroxymethylcytosine (5-hmC). Real time PCR of the experimental
(glucosylated) and control (mock glucosylated) target DNA with
specific primers flanking a CCGG site of interest was performed to
measure the amount of DNA methylation at specific promoter region
of target genes.
[0147] The results of MSRE-PCR showed that D-EPCs have
significantly high DNA methylation in the promoters of angiogenic
genes such as Akt1, Hgf, Mapk14, Sphk1, Vegfc, Nudt6, Kdr, Vegfa,
and Pten which were identified by the above microarray studies, and
treatments with compounds reduced DNA methylation at these
promoters and enhance gene expression (FIG. 13). In addition,
reduced cell biologic function such as cell adhesion, migration and
tube formation were all increased.
[0148] Human BM-derived EPCs were cultured similarly. Two
approaches were used for reprogramming diabetic EPCs. Initially we
used an unselected approach employing combinations of compounds.
For the combinatorial approach, combinations of the three classes
of compounds were used and the gene and epigenetic changes were
determined. In the next targeted approach, selective reagents were
chose accordingly to the methylation and histone modification
status of key factors identified.
[0149] Studies indicate that EPC function may be attributed to
VEGF, SDF-1, CXCR4, HIF1.alpha., IGF-1, or Akt12.
[0150] EPCs from diabetic subjects were cultured. The cultured EPCs
are typically reseeded at day 4 and cultured for another 3-7 days.
HDAC inhibitors--SAHA, sodium butyrate, and VPA, two DNA
methylation inhibitor 5-Aza and RG108, and multiple histone 3
lysine 9 (H3K9) methyltransferases G9a and GLP inhibitors of
formula I were used. Compounds were tested to find candidates which
induce the highest cell functionality with minimum toxicity. The
following assays was conducted comparing N-EPCs, D-EPCs, and
rescued diabetic EPCs (RD-EPCs) to determine rescued diabetic
(RD)-EPCs with respect to their biological potency. Cell biological
assays (adhesion, migration, proliferation and tube formation) were
performed as well as angiogenic and neurotrophic gene assays (low
density qRT-PCR array). In some samples, western blot (WB) or ELISA
was performed to confirm results. In vivo vessel forming capability
were tested by Matrigel plug, corneal micropocket, and ear or skin
wound assays.
[0151] RD-EPCs were identified that possess the highest angiogenic
and neurotrophic capacity. In this EPC reprogramming, a short
duration of compound treatment should be sufficient to reverse the
epigenetic modifications incurred by diabetes, and culture
conditions will then guide their fate back to more naive EPCs. This
new reprogramming technology can be applied and expanded to
diabetic bone marrow-derived stem or progenitor cells including
MSCs, EPCs, and even to unfractionated bone marrow-derived
mononuclear cells (BM-MNCs).
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