U.S. patent application number 14/310619 was filed with the patent office on 2014-12-11 for chemical inducers of neurogenesis.
This patent application is currently assigned to The Board of Regents of the University of Texas System. The applicant listed for this patent is The Board of Regents of the University of Texas System. Invention is credited to Douglas Frantz, Jenny Hsieh, Steven L. McKnight, Joseph M. Ready, Jay Schneider.
Application Number | 20140364467 14/310619 |
Document ID | / |
Family ID | 40455226 |
Filed Date | 2014-12-11 |
United States Patent
Application |
20140364467 |
Kind Code |
A1 |
Schneider; Jay ; et
al. |
December 11, 2014 |
CHEMICAL INDUCERS OF NEUROGENESIS
Abstract
The present invention relates to compounds and methods for
inducing neuronal differentiation in normal neural stem cells and
brain cancer stem cells. The methods may take place in vitro, such
as in isolates from the adult mammalian brain, or in vivo.
Compounds and methods described herein may find use in the
treatment of neurodegenerative and psychiatric diseases, the repair
and regeneration of the nervous system, and in treatment of
neurologic malignancy.
Inventors: |
Schneider; Jay; (Coppell,
TX) ; Hsieh; Jenny; (Irving, TX) ; Frantz;
Douglas; (Flower Mound, TX) ; McKnight; Steven
L.; (Dallas, TX) ; Ready; Joseph M.;
(Carrollton, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Regents of the University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
The Board of Regents of the
University of Texas System
Austin
TX
|
Family ID: |
40455226 |
Appl. No.: |
14/310619 |
Filed: |
June 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13487963 |
Jun 4, 2012 |
8778940 |
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14310619 |
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11974479 |
Oct 12, 2007 |
8193225 |
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13487963 |
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60953182 |
Jul 31, 2007 |
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60829338 |
Oct 13, 2006 |
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Current U.S.
Class: |
514/378 ;
435/377 |
Current CPC
Class: |
A61K 31/427 20130101;
C12N 5/0618 20130101; C07D 413/14 20130101; C07D 417/04 20130101;
A61K 31/42 20130101; C12N 5/0657 20130101; A61K 31/4155 20130101;
C07D 403/04 20130101; A61K 31/496 20130101; C07D 261/18 20130101;
A61K 31/422 20130101; C07D 405/04 20130101; A61P 25/00 20180101;
C07C 311/49 20130101; A61P 29/00 20180101; C07D 261/08 20130101;
C12N 2501/999 20130101; A61K 31/4439 20130101; C07D 409/04
20130101; C07D 231/12 20130101; A61P 35/00 20180101; C07D 413/04
20130101; A61K 31/5377 20130101; A61K 31/415 20130101 |
Class at
Publication: |
514/378 ;
435/377 |
International
Class: |
C07D 413/04 20060101
C07D413/04; C12N 5/079 20060101 C12N005/079; C07D 261/18 20060101
C07D261/18 |
Claims
1-13. (canceled)
14. A method of inducing neuronal differentiation in a stem cell
comprising contacting said stem cell with a compound of formula
(II): ##STR00043## wherein: R.sub.1 is substituted or unsubstituted
thiophenyl or a substituent of formula (A): ##STR00044## wherein:
R.sub.A, R.sub.B and R.sub.C are each independently selected from
the group consisting of hydrogen, halogen, alkyl (e.g.,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl), aralkyl, aryl,
cyano, nitro, and a carbonyl group; and G is O, --NH, or S; R.sub.2
is hydrogen, hydroxy, halogen, nitro, aryl, alkyl, alkoxy, alkenyl,
alkenyloxy, alkynyl, alkynyloxy, aralkyl, --CHO, --C(O)R.sub.9,
--OC(O)R.sub.9, --OC(O)OR.sub.9, --O(CN)OR.sub.9,
--C(O)NR.sub.9R.sub.10, --OC(O)NR.sub.9R.sub.10,
--NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9; wherein R.sub.9 and
R.sub.10 are each independently hydrogen, alkyl, aryl, or aralkyl;
R.sub.3 is --NH--O-alkyl, --NH--OH, --OR.sub.11 or
--NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 are each
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, or aralkyl;
or R.sub.11 and R.sub.12 together form a cyclic group; or R.sub.11
and R.sub.12 together with the nitrogen to which they are bound
form a cyclic group; X is O or --NR.sub.13, wherein R.sub.13 is
hydrogen, alkyl, aryl, or aralkyl; or a stereoisomer, solvate,
hydrate, or pharmaceutically acceptable salt thereof.
15. The method of claim 14, wherein any alkyl group comprised in
any of R.sub.2, R.sub.9, R.sub.10, R.sub.11, R.sub.12, or R.sub.13
is lower alkyl.
16. The method of claim 15, wherein the cyclic group formed by
R.sub.11 and R.sub.12 is cyclopropyl, cyclobutyl, or
cyclopentyl.
17. The method of claim 14, wherein the compound of formula (II) is
further defined as a compound of formula (III): ##STR00045##
wherein: R.sub.1 and R.sub.2 are both hydrogen; or R.sub.1 is
hydrogen and R.sub.2 is selected from the group consisting of
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl and
benzyl; or R.sub.1 and R.sub.2 may be joined together to form a
ring selected from azetidinyl, pyrrolidinyl, piperidinyl,
piperazinyl and morpholinyl; R.sub.2, R.sub.3 and R.sub.4 are
independently selected from the group consisting of hydrogen,
halogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl,
substituted or unsubstituted aromatic or heteroaromatic ring,
cyano, nitro, and a carbonyl group; X is O, NH, or S; and Y is O,
NH, or S, or a stereoisomer, solvate, hydrate, or pharmaceutically
acceptable salt thereof.
18. The method of claim 17, wherein Y is S and R.sub.2 is a
substituted or unsubstituted C.sub.1-C.sub.6 alkyl.
19. The method of claim 17, wherein Y is S and R.sub.2 is a
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl.
20. The method of claim 17, wherein Y is O and R.sub.2 is a
substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl.
21. The method of claim 17, wherein Y is O and R.sub.2 is a
substituted or unsubstituted C.sub.1-C.sub.6 alkyl.
22. The method of claim 17, wherein Y is S and R.sub.2 is a
substituted or unsubstituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl or benzyl.
23. The method of claim 17, wherein Y is O and R.sub.2 is a
substituted or unsubstituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl or benzyl.
24. The method of claim 17, wherein R.sub.1 is H.
25. The method of claim 17, wherein said stem cell is located in an
animal subject.
26. The method of claim 17, wherein said stem cell is contacted ex
vivo.
27. The method of claim 14, wherein the compound of formula (II) is
further defined as a compound of formula (IV): ##STR00046##
wherein: R.sup.1 is selected from C.sub.1-6alkyl and a 5- or
6-membered ring containing atoms independently selected from the
group consisting of C, N, O and S, wherein R.sup.1 is optionally
substituted with one or more substituents selected from the group
consisting of hydroxy, halo, nitro, aryl, heteroaryl,
C.sub.1-6alkylhalo, OC.sub.1-6alkylhalo, OC.sub.1-6alkyl,
C.sub.2-6alkenyl, OC.sub.2-6alkenyl, C.sub.2-6alkynyl,
OC.sub.2-6alkynyl, C.sub.0-6alkylC.sub.3-6cycloalkyl,
OC.sub.0-6alkylC.sub.3-6cycloalkyl, C.sub.1-6alkylaryl,
OC.sub.0-6alkylaryl, CHO, (CO)R.sup.4, O(CO)R.sup.4, O(CO)OR.sup.4,
O(CN)OR.sup.4, C.sub.1-6alkylOR.sup.4, OC.sub.2-6alkylOR.sup.4,
C.sub.1-6alkyl(CO)R.sup.4, OC.sub.1-6alkyl(CO)R.sup.4,
C.sub.0-6alkylCO.sub.2R.sup.4, OC.sub.1-6alkylCO.sub.2R.sup.4,
C.sub.0-6alkylcyano, OC.sub.2-6alkylcyano,
C.sub.0-6alkylNR.sup.4R.sup.5, OC.sub.2-6alkylNR.sup.4R.sup.5,
C.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
OC.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)R.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)R.sup.5, C.sub.0-6
alkylNR.sup.4(CO)NR.sup.4R.sup.5, C.sub.0-6alkylSR.sup.4,
OC.sub.2-6alkylSR.sup.4, C.sub.0-6alkyl(SO)R.sup.4,
OC.sub.2-6alkyl(SO)R.sup.4, C.sub.0-6alkylSO.sub.2R.sup.4,
OC.sub.2-6alkylSO.sub.2R.sup.4, C.sub.0-6
alkyl(SO.sub.2)NR.sup.4R.sup.5, OC.sub.1-6alkyl(S
O.sub.2)NR.sup.4R.sup.5, C.sub.0-6 alkylNR.sup.4(SO.sub.2)R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
(CO)NR.sup.4R.sup.5, O(CO)NR.sup.4R.sup.5, NR.sup.4OR.sup.5,
C.sub.0-6alkylNR.sup.4(CO)OR.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)OR.sup.5 and SO.sub.3R.sup.4; R.sup.2 is
selected from the group consisting of hydrogen, hydroxy, halo,
nitro, aryl, heteroaryl, C.sub.1-6alkylhalo, OC.sub.1-6alkylhalo,
C.sub.1-6alkyl, OC.sub.1-6alkyl, C.sub.2-6alkenyl,
OC.sub.2-6alkenyl, C.sub.2-6alkynyl, OC.sub.2-6alkynyl,
C.sub.0-6alkylC.sub.3-6cycloalkyl,
OC.sub.0-6alkylC.sub.3-6cycloalkyl, C.sub.1-6alkylaryl,
OC.sub.0-6alkylaryl, CHO, (CO)R.sup.4, O(CO)R.sup.4, O(CO)OR.sup.4,
O(CN)OR.sup.4, C.sub.1-6alkylOR.sup.4, OC.sub.2-6alkylOR.sup.4,
C.sub.1-6alkyl(CO)R.sup.4, OC.sub.1-6 alkyl(CO)R.sup.4,
C.sub.0-6alkylCO.sub.2R.sup.4, OC.sub.1-6alkylCO.sub.2R.sup.4,
C.sub.0-6alkylcyano, OC.sub.2-6alkylcyano,
C.sub.0-6alkylNR.sup.4R.sup.5, OC.sub.2-6alkylNR.sup.4R.sup.5,
C.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
OC.sub.1-6alkyl(CO)NR.sup.4R.sup.5, C.sub.0-6
alkylNR.sup.4(CO)R.sup.5, OC.sub.2-6alkylNR.sup.4(CO)R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)NR.sup.4R.sup.5, C.sub.0-6alkylSR.sup.4,
OC.sub.2-6alkylSR.sup.4, C.sub.0-6alkyl(SO)R.sup.4,
OC.sub.2-6alkyl(SO)R.sup.4, C.sub.0-6alkylSO.sub.2R.sup.4,
OC.sub.2-6alkylSO.sub.2R.sup.4,
C.sub.0-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)R.sup.5, C.sub.0-6
alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
(CO)NR.sup.4R.sup.5, O(CO)NR.sup.4R.sup.5, NR.sup.4OR.sup.5,
C.sub.0-6alkylNR.sup.4(CO)OR.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)OR.sup.5 and SO.sub.3R.sub.4; R.sup.3 is
selected from the group consisting of OR.sup.4, NR.sup.4R.sup.5,
and NR.sup.7R.sup.8, wherein R.sup.7 and R.sup.8, together with the
nitrogen atom to which they are bound, combine to form a 5- to
6-member ring optionally containing one or more of S, O, and NH;
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylaryl,
C.sub.1-6alkylheteroaryl, C.sub.3-7cycloalkyl and aryl; and X is O
or NR.sup.6, wherein R.sup.6 is selected from the group consisting
of hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylaryl, C.sub.1-6
alkylheteroaryl, C.sub.3-7 cycloalkyl and aryl; or a stereoisomer,
solvate, hydrate, or pharmaceutically acceptable salt thereof.
28-44. (canceled)
Description
[0001] This application is a divisional of U.S. Continuation
application Ser. No. 13/487,963 filed Jun. 4, 2012 which claims the
benefit of U.S. application Ser. No. 11/974,479 filed Oct. 12, 2007
and issued as U.S. Pat. No. 8,193,225 on Jun. 5, 2012, which claims
the benefit of the filing dates of U.S. Provisional Application
60/829,338, filed Oct. 13, 2006 and U.S. Provisional Application
60/953,182, filed Jul. 31, 2007, the entire contents of each of
these applications being hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
cell biology, developmental biology and neurobiology. More
particularly, it concerns methods and compositions relating to the
induction of neural differentiation in stem cells.
[0004] 2. Description of Related Art
[0005] Extensive evidence suggests that new neurons originate from
stem cells in the adult mammalian hippocampus, a region of the
brain that is important for learning and memory. The
differentiation of stem cells into neurons begins within a month of
the birth of a cell and continues throughout the adult life of the
mammal (Gage et al. 1995). Hippocampal neurogenesis is a mechanism
for maintaining cellular homeostasis in the adult brain and plays
an important functional role in higher cerebral activities like
learning and memory. While exercise and exposure to an enriched
environment promote adult hippocampal neurogenesis, chronic stress,
depression, sleep deprivation and aging can decrease neural
stem/progenitor cell proliferation in the adult hippocampus.
[0006] In the mammalian central nervous system, adult multipotent
neural progenitor cells cultured in the presence of retinoic acid
(RA) differentiate into neurons (Palmer et al., 2001; Ray and Gage,
2006). By contrast, when such cells are cultured in the presence of
insulin-like growth factor I (IGF-I) or leukemia inhibitory factor
(LIF) plus bone-morphogenetic protein (BMP), they differentiate
into oligodendrocytes and astrocytes, respectively (Hsieh et al.
2004). The cellular and molecular mechanisms that control the
differentiation of adult hippocampal neural progenitor cells into
neurons, oligodendrocytes and astrocytes are not completely
understood.
[0007] Compounds that selectively direct stem cell fate could be
useful for the treatment of neurodegenerative and psychiatric
diseases and in the repair and regeneration of the nervous system.
Chemicals can be identified that not only strongly favor neuronal
differentiation, but also actively suppress astrocyte and
oligodendrocyte differentiation. In addition, neurons whose
differentiation has been induced in vitro could be used for stem
cell grafting and transplantation. Ultimately, the study of
multipotent neural progenitor cells in culture can be applied to
studies of neurogenesis and gliogenesis in vivo, both in normal and
in diseased and malignant states. Accordingly, potent inducers of
neuronal differentiation of neural stem cells merit
investigation.
[0008] As a corollary to the ability of a chemical compound to
induce neurogenesis in a neural stem/progenitor cell, such a
compound might also be an effective differentiation-inducing
anti-neoplastic agent. Increasing evidence indicates that stem
cells lie at the root of brain tumors like glioblastoma multiforme
(GBM). Small-molecules that are active in neural stem/progenitor
cells might therefore also have bioactivity against the brain tumor
stem cell. Thus, small-molecules that induce neural stem cell
differentiation might also be useful for arresting growth, killing,
or differentiating GBM cancer stem cells, currently thought to be
the cause of one of the most devastating and incurable of human
malignancies.
[0009] Moreover, evidence is accumulating that primitive cancerous
stem cells for hematopoietic cancers and several types of solid
tumors exist. See, e.g., Cooper; 1992; Bonnet and Dick, 1997; Park
et al., 1971; Hamburger and Salmon, 1977; and U.S. Pat. No.
4,411,990. Current methods for diagnosing or treating cancer,
removing cancer cells from transplant grafts prior to injection
into a patient, or methods to screen the efficacy of anti-cancer
agents in completely eliminating cancer cells, do not account for
the presence of cancer stem cells, which can propagate,
differentiate into mature cancer cells and self-renew, thereby
reforming cancers and leading to remissions. Accordingly, there
exists a need for new methods for treating cancer which account for
and/or are specifically directed to cancer stem cells.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery that certain
compounds induce neuronal differentiation of stem cells.
Accordingly, the present invention provides compounds,
pharmaceutical compositions and methods relating to the induction
of neuronal differentiation in a variety of stem cells. The
compounds, compositions and methods of the present invention may be
employed in vitro or in vivo. Neurologic malignancies,
neurodegenerative and psychiatric diseases may be treated using the
compounds and methods described herein. Certain cancers and cancer
stem cells may also be targeted using compounds and methods of the
present invention. Aspects of the present invention may also find
use in the repair and regeneration of the nervous system.
[0011] Accordingly, certain aspects of the present invention
contemplate a compound of formula (I):
##STR00001##
wherein X is O or NH, Y is S or O and R is H, a substituted or
unsubstituted alkyl, such as C.sub.1-C.sub.6 alkyl or
C.sub.3-C.sub.6 cycloalkyl, or a substituted or unsubstituted
alkenyl, such as C.sub.2-C.sub.6 alkenyl, a substituted or
unsubstituted alkynyl, such as C.sub.2-C.sub.6 alkynyl, or a
stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt
thereof. In certain embodiments regarding compounds of formula (I),
the proviso exists such that with the provisos that if X is O, then
R must be a substituted or unsubstituted C.sub.3-C.sub.6
cycloalkyl; and/or if X is NH, then R must not be pyrazinyl
substituted C.sub.1-C.sub.6 alkyl. Compounds of formula (I) may be
employed in methods of the present invention, such as a method of
inducing neuronal differentiation or treating cancer.
[0012] In certain embodiments regarding compounds of formula (I), Y
is S and R is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl.
In certain embodiments, Y is S and R is a substituted or
unsubstituted C.sub.3-C.sub.6 cycloalkyl. In certain embodiments, Y
is O and R is a substituted or unsubstituted C.sub.3-C.sub.6
cycloalkyl. In certain embodiments, Y is O and R is a substituted
or unsubstituted C.sub.1-C.sub.6 alkyl. In certain embodiments, R
is a substituted or unsubstituted C.sub.2-C.sub.6 alkenyl. In
certain embodiments, R is a substituted or unsubstituted
C.sub.2-C.sub.6 alkynyl. In certain embodiments, R is H. In certain
embodiments, X is O, Y is O and R is a substituted or unsubstituted
cycloalkyl. In certain embodiments, X is O, Y is S and R is a
substituted or unsubstituted cycloalkyl.
[0013] Other aspects of the present invention contemplate a
compound of formula (Ia), (Ib), or (Ic):
##STR00002##
wherein R.sub.1 is substituted or unsubstituted phenyl,
unsubstituted pyrrolyl, unsubstituted pyridyl, unsubstituted
furanyl, unsubstituted thienyl, unsubstituted benzofuranyl,
unsubstituted benzo[b]thiophenyl, or unsubstituted thiazolyl. Any
of these R.sub.1 substituents may be substituted as well. Any one
or more of compounds (Ia), (Ib) and (Ic) are contemplated in
methods of the present invention, such as methods of inducing
neuronal differentiation or treating cancer.
[0014] Also contemplated by the present invention are methods of
inducing neuronal differentiation in a stem cell comprising
contacting said stem cell with a compound of formula (II):
##STR00003##
wherein: R.sub.1 is substituted or unsubstituted thiophenyl or a
substituent of formula (A):
##STR00004##
wherein: R.sub.A, R.sub.B and R.sub.C are each independently
selected from the group consisting of hydrogen, halogen,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl, aryl, cyano,
nitro, and a carbonyl group; and G is O, --NH, or S; R.sub.2 is
hydrogen, hydroxy, halogen, nitro, aryl, alkyl, alkoxy, alkenyl,
alkenyloxy, alkynyl, alkynyloxy, aralkyl, --CHO, --C(O)R.sub.9,
--OC(O)R.sub.9, --OC(O)OR.sub.9, --O(CN)OR.sub.9,
--C(O)NR.sub.9R.sub.10, --OC(O)NR.sub.9R.sub.10,
--NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9; wherein R.sub.9 and
R.sub.10 are each independently hydrogen, alkyl, aryl, or aralkyl;
R.sub.3 is --NH--O-alkyl, --NH--OH, --OR.sub.11 or
--NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 are each
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, or aralkyl;
or R.sub.11 and R.sub.12 together form a cyclic group; or R.sub.11
and R.sub.12 together with the nitrogen to which they are bound
form a cyclic group; X is O or --NR.sub.13, wherein R.sub.13 is
hydrogen, alkyl, aryl, or aralkyl; or a stereoisomer, solvate,
hydrate, or pharmaceutically acceptable salt thereof.
[0015] As used herein, the phrase "method of inducing neuronal
differentiation in a stem cell" refers to, in certain embodiments,
morphological change in a neural stem/progenitor cell or other cell
contemplated by the present invention (described herein),
flattening of such cells, and/or the extension of neuronal-like
processes as associated with the contacting of such cells with a
compound of the present invention. Induction of neuronal
differentiation may also be associated with increased neuronal
expression as detected by, for example, mRNA levels, protein
levels, and/or neuronal markers. The terms "neuronal
differentiation" and "neurogenic differentiation" are used
interchangeably herein.
[0016] In certain embodiments regarding compounds of formula (II),
any alkyl group comprised in any of R.sub.2, R.sub.9, R.sub.10,
R.sub.11, R.sub.12, or R.sub.13 (such as alkyl, alkoxy, aralkyl,
etc.) is lower alkyl. In certain embodiments, higher alkyls are
contemplated in this context. In certain embodiments, any aryl
group within a compound of formula (II) is mono-substituted. In
certain embodiments, any aryl group within a compound of formula
(II) is di-substituted (e.g., R.sub.1, R.sub.A, R.sub.B, R.sub.C,
R.sub.2, R.sub.9, R.sub.10, R.sub.11, R.sub.12, and/or R.sub.13).
In certain embodiments, any aryl group within a compound of formula
(II) is tri-substituted. In certain embodiments, any aryl group
within a compound of formula (II) is tetra-substituted. In certain
embodiments, the cyclic group formed by R.sub.11 and R.sub.12 is
cyclopropyl, cyclobutyl, or cyclopentyl. In certain embodiments,
the cyclic group formed by R.sub.11 and R.sub.12 and the nitrogen
to which it is bound to the rest of the molecule is piperazinyl or
a salt thereof, or morpholino. In certain embodiments, R.sub.3 is
--OH. In certain embodiments, R.sub.3 is alkoxy, such as ethoxy. In
certain embodiments, R.sub.3 is --NH.sub.2. In certain embodiments,
R.sub.3 is --NH-alkyl-OH. In embodiments, R.sub.3 is --NH-lower
unsubstituted alkyl, including lower unsubstituted cycloalkyl
(e.g., cyclopropyl). In certain embodiments, R.sub.3 is
--NH-substituted lower alkyl; in certain embodiments, such
substitution may be by a hydroxy group, an --NH.sub.2 group, or a
lower alkoxy group. In certain embodiments, any aralkyl group of
the compound of formula (II) is --CH.sub.2-aryl. In certain
embodiments, R.sub.3 is --NH--O-alkyl.
[0017] In particular embodiments, a compound of formula (II) is
further defined as a compound of formula (III):
##STR00005##
wherein: R.sub.1 and R.sub.2 are both hydrogen; or R.sub.1 is
hydrogen and R.sub.2 is selected from the group consisting of
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl and
benzyl; or R.sub.1 and R.sub.2 may be joined together to form
cyclic group, such as a ring selected from azetidinyl,
pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl; R.sub.2,
R.sub.3 and R.sub.4 are independently selected from the group
consisting of hydrogen, halogen, alkyl, such as C.sub.1-C.sub.6
alkyl or C.sub.3-C.sub.6 cycloalkyl, aryl (such as substituted or
unsubstituted aromatic or heteroaromatic ring), cyano, nitro, and a
carbonyl group; X is O, NH, or S; and Y is O, NH, or S, or a
stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt
thereof.
[0018] In certain embodiments regarding compounds of formula (III),
Y is S and R.sub.2 is a substituted or unsubstituted
C.sub.1-C.sub.6 alkyl. In certain embodiments, Y is S and R.sub.2
is a substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl. In
certain embodiments, Y is O and R.sub.2 is a substituted or
unsubstituted C.sub.3-C.sub.6 cycloalkyl. In certain embodiments, Y
is O and R.sub.2 is a substituted or unsubstituted C.sub.1-C.sub.6
alkyl. In certain embodiments, Y is S and R.sub.2 is a substituted
or unsubstituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl
or benzyl. In certain embodiments, Y is O and R.sub.2 is a
substituted or unsubstituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl or benzyl. In certain embodiments, R.sub.1
is H.
[0019] In certain embodiments regarding methods of the present
invention involving stem cells, said stem cell is located in an
animal subject, such as mammal (e.g., mouse, human). In certain
embodiments regarding methods of the present invention involving
stem cells, said stem cell is contacted ex vivo. In any method
involving stem cells, the stem cell may be, for example, an
embryonic stem cell, an autologous embryonic stem cells generated
through therapeutic cloning, an adult stem cell, a neural stem
cell, a brain cancer stem cell, a cardiogenic stem cell, or a bone
marrow stromal cells.
[0020] In certain embodiments, the compound of formula (II) is
further defined as a compound of formula (IV):
##STR00006##
wherein: R.sup.1 is selected from alkyl, alkoxy, alkenoxy,
alkynoxy, acyl, acyloxy, alkylamino, alkenylamino, alkynylamino,
aryl and aralkyl. An alkyl group comprised in compounds of formula
(IV) may be lower alkyl in certain embodiments, or higher alkyl in
certain embodiments. Methods of inducing neuronal differentiation
and treating cancer, for example, are methods of the present
invention that may employ compounds of formula (IV).
[0021] In certain embodiments regarding compounds of formula (IV),
R.sup.1 is selected from C.sub.1-6alkyl and a 5- or 6-membered ring
containing atoms independently selected from the group consisting
of C, N, O and S, wherein R.sup.1 is optionally substituted with
one or more substituents selected from the group consisting of
hydroxy, halo, nitro, aryl, heteroaryl, C.sub.1-6alkylhalo, alkyl,
alkoxy, aralkyl, OC.sub.1-6alkylhalo, OC.sub.1-6alkyl,
C.sub.2-6alkenyl, OC.sub.2-6alkenyl, C.sub.2-6alkynyl,
OC.sub.2-6alkynyl, C.sub.0-6alkylC.sub.3-6cycloalkyl,
OC.sub.0-6alkylC.sub.3-6cycloalkyl, C.sub.1-6alkylaryl,
OC.sub.0-6alkylaryl, CHO, (CO)R.sup.4, O(CO)R.sup.4, O(CO)OR.sup.4,
O(CN)OR.sup.4, C.sub.1-6alkylOR.sup.4, OC.sub.2-6alkylOR.sup.4,
C.sub.1-6alkyl(CO)R.sup.4, OC.sub.1-6alkyl(CO)R.sup.4,
C.sub.0-6alkylCO.sub.2R.sup.4, OC.sub.1-6alkylCO.sub.2R.sup.4,
C.sub.0-6alkylcyano, OC.sub.2-6alkylcyano,
C.sub.0-6alkylNR.sup.4R.sup.5, OC.sub.2-6alkylNR.sup.4R.sup.5,
C.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
OC.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)R.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)NR.sup.4R.sup.5, C.sub.0-6alkylSR.sup.4,
OC.sub.2-6alkylSR.sup.4, C.sub.0-6alkyl(SO)R.sup.4,
OC.sub.2-6alkyl(SO)R.sup.4, C.sub.0-6alkylSO.sub.2R.sup.4,
OC.sub.2-6alkylSO.sub.2R.sup.4,
C.sub.0-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.1-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
(CO)NR.sup.4R.sup.5, O(CO)NR.sup.4R.sup.5, NR.sup.4OR.sup.5,
C.sub.0-6alkylNR.sup.4(CO)OR.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)OR.sup.5 and SO.sub.3R.sup.4; R.sup.2 is
selected from the group consisting of hydrogen, hydroxy, halo,
nitro, aryl, heteroaryl, C.sub.1-6alkylhalo, OC.sub.1-6alkylhalo,
C.sub.1-6alkyl, OC.sub.1-6alkyl, C.sub.2-6alkenyl,
OC.sub.2-6alkenyl, C.sub.2-6alkynyl, OC.sub.2-6alkynyl,
C.sub.0-6alkylC.sub.3-6cycloalkyl,
OC.sub.0-6alkylC.sub.3-6cycloalkyl, C.sub.1-6alkylaryl,
OC.sub.0-6alkylaryl, CHO, (CO)R.sup.4, O(CO)R.sup.4, O(CO)OR.sup.4,
O(CN)OR.sup.4, C.sub.1-6alkylOR.sup.4, OC.sub.2-6alkylOR.sup.4,
C.sub.1-6alkyl(CO)R.sup.4, OC.sub.1-6alkyl(CO)R.sup.4,
C.sub.0-6alkylCO.sub.2R.sup.4, OC.sub.1-6alkylCO.sub.2R.sup.4,
C.sub.0-6alkylcyano, OC.sub.2-6alkylcyano,
C.sub.0-6alkylNR.sup.4R.sup.5, OC.sub.2-6alkylNR.sup.4R.sup.5,
C.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
OC.sub.1-6alkyl(CO)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)R.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)R.sup.5,
C.sub.0-6alkylNR.sup.4(CO)NR.sup.4R.sup.5, C.sub.0-6alkylSR.sup.4,
OC.sub.2-6alkylSR.sup.4, C.sub.0-6alkyl(SO)R.sup.4,
OC.sub.2-6alkyl(SO)R.sup.4, C.sub.0-6alkylSO.sub.2R.sup.4,
OC.sub.2-6alkylSO.sub.2R.sup.4,
C.sub.0-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkyl(SO.sub.2)NR.sup.4R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)R.sup.5,
C.sub.0-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
OC.sub.2-6alkylNR.sup.4(SO.sub.2)NR.sup.4R.sup.5,
(CO)NR.sup.4R.sup.5, O(CO)NR.sup.4R.sup.5, NR.sup.4OR.sup.5,
C.sub.0-6alkylNR.sup.4(CO)OR.sup.5,
OC.sub.2-6alkylNR.sup.4(CO)OR.sup.5 and SO.sub.3R.sub.4; R.sup.3 is
selected from the group consisting of OR.sup.4, NR.sup.4R.sup.5,
and NR.sup.7R.sup.8, wherein R.sup.7 and R.sup.8, together with the
nitrogen atom to which they are bound, combine to form a 5- to
6-member ring optionally containing one or more of S, O, and NH;
R.sup.4 and R.sup.5 are independently selected from the group
consisting of hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylaryl,
C.sub.1-6alkylheteroaryl, C.sub.3-7cycloalkyl and aryl; and X is O
or NR.sup.6, wherein R.sup.6 is selected from the group consisting
of hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylaryl,
C.sub.1-6alkylheteroaryl, C.sub.3-7cycloalkyl and aryl; or a
stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt
thereof.
[0022] Also contemplated by the present invention are methods of
inducing neuronal differentiation in a stem cell comprising
contacting said stem cell with a compound having formula (V):
##STR00007##
wherein: the ABD ring comprises two non-adjacent double bonds; A, B
and D are each independently S, N, O, C, --NR.sub.14, --CR.sub.15,
or --CR.sub.15R.sub.16, wherein R.sub.14 is hydrogen, halogen,
alkyl, aryl, or aralkyl; and R.sub.15 and R.sub.16 are each
independently hydrogen, hydroxy, halogen, nitro, aryl, alkyl,
alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, aralkyl, --CHO,
--C(O)R.sub.9, --OC(O)R.sub.9, --OC(O)OR.sub.9, --O(CN)OR.sub.9,
--C(O)NR.sub.9R.sub.10, --OC(O)NR.sub.9R.sub.10,
--NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9; wherein R.sub.9 and
R.sub.10 are each independently hydrogen, alkyl, aryl, or aralkyl,
provided that at least two of A, B and D comprise S, N, or O;
R.sub.1 is alkyl, --CH.dbd.CH-aryl, or aryl; and R.sub.3 is alkyl,
--C(O)alkyl, aryl, --C(O)aryl, aralkyl, --C(O)aryl, --OR.sub.4,
--C(O)R.sub.4, --NR.sub.4R.sub.5, or --C(O)NR.sub.4R.sub.5,
wherein: R.sub.4 and R.sub.5 are each independently hydrogen,
alkyl, aryl, or aralkyl; or R.sub.4 and R.sub.5 together form a
cyclic group; or R.sub.4 and R.sub.5 together with the nitrogen to
which they are bound form a cyclic group. In certain embodiments,
the D atom and R.sub.3 come together to form a cyclic group.
[0023] In certain embodiments regarding compounds of the present
invention, such as compounds of formula (V), the proviso exists
such that compounds of formula (V.sub.a) are excluded:
##STR00008##
wherein R.sub.18 is alkyl, such as lower alkyl or cyclopentyl, or
alkenyl, such as lower alkenyl or allyl, and G is O or S. In
certain embodiments regarding compounds of formula (V), any alkyl
group comprised in R.sub.1, R.sub.4, R.sub.5, R.sub.9, R.sub.10,
R.sub.14, R.sub.15, and/or R.sub.16 is lower alkyl. Higher alkyls
are also contemplated in this respect, in certain embodiments.
Regarding R.sub.1, the aryl group of either aryl-containing R.sub.1
substituent may, in certain embodiments, be substituted or
unsubstituted pyranyl, thiophenyl, furanyl, thiazolyl, or
pyridinyl. In certain embodiments, R.sub.4 and R.sub.5 together
form a cyclopropyl group. In certain embodiments, R.sub.3 is
--NR.sub.4R.sub.5.
[0024] It is specifically contemplated that, in certain
embodiments, ABD rings of the present invention are substituted at
R.sub.1 and R.sub.3, but not at any other available ring atom of
the ABD ring.
[0025] In certain embodiments, the compound of formula (V) is a
compound of formula (V.sub.b):
##STR00009##
wherein: R.sub.1 and R.sub.2 are each independently hydrogen,
alkyl, --CH.dbd.CH-aryl, aralkyl, or aryl; and R.sub.3 is alkyl,
aryl, aralkyl, --OR.sub.4, or --NR.sub.4R.sub.5, wherein: R.sub.4
and R.sub.5 are each independently hydrogen, alkyl, aryl, or
aralkyl; or R.sub.4 and R.sub.5 together form a cyclic group; or
R.sub.4 and R.sub.5 together with the nitrogen to which they are
bound form a cyclic group. In certain embodiments regarding
compounds of formula (V.sub.b), the proviso exists such that
compounds of formula (V.sub.a) are excluded:
##STR00010##
wherein R.sub.18 is alkyl, such as lower alkyl or cyclopentyl, or
alkenyl, such as lower alkenyl or allyl, and G is O or S.
[0026] In certain embodiments, a compound of formula (V) is further
defined as a compound of formula (VI):
##STR00011##
wherein: R.sub.1 is aryl or --CH.dbd.CH-aryl; R.sub.3 is --OR.sub.4
or --NR.sub.4R.sub.5, wherein: R.sub.4 and R.sub.5 are each
independently hydrogen, alkyl, aryl, or aralkyl; and R.sub.4 and
R.sub.5 together form a cyclic group; or R.sub.4 and R.sub.5
together with the nitrogen to which they are bound form a cyclic
group; X is O, S, or --NR.sub.6, wherein R.sub.6 is hydrogen,
alkyl, aryl, or aralkyl; and Y is N or --CR.sub.7, wherein R.sub.7
is hydrogen, alkyl, aryl, or halogen, or a stereoisomer, solvate,
hydrate or pharmaceutically acceptable salt thereof.
[0027] In certain embodiments, a compound of formula (V) is further
defined as a compound of formula (VII):
##STR00012##
wherein: R.sub.1 is aryl or --CH.dbd.CH-aryl; R.sub.3 is --OR.sub.4
or --NR.sub.4R.sub.5, wherein: R.sub.4 is hydrogen, alkyl, aryl, or
aralkyl; and R.sub.5 is alkyl, aryl, or aralkyl; or R.sub.4 and
R.sub.5 together form a cyclic group; or R.sub.4 and R.sub.5
together with the nitrogen to which they are bound form a cyclic
group; W is N or --CR.sub.8, wherein R.sub.8 is hydrogen, hydroxy,
halogen, nitro, aryl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl,
alkynyloxy, aralkyl, --CHO, --C(O)R.sub.9, --OC(O)R.sub.9,
--OC(O)OR.sub.9, --O(CN)OR.sub.9, --C(O)NR.sub.9R.sub.10,
--OC(O)NR.sub.9R.sub.10, --NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9;
wherein R.sub.9 and R.sub.10 are each independently hydrogen,
alkyl, aryl, or aralkyl; X is O, S, or --NR.sub.6, wherein R.sub.6
is hydrogen, alkyl, aryl, or aralkyl; and Y is N or --CR.sub.7,
wherein R.sub.7 is hydrogen, alkyl, aryl, or halogen, or a
stereoisomer, solvate, hydrate or pharmaceutically acceptable salt
thereof.
[0028] In particular embodiments regarding compounds of formula
(V), the ABD ring is selected from the group consisting of:
##STR00013##
wherein the substituent pictured to the left is R.sub.1 and the
substituent pictured to the right is R.sub.3. In certain
embodiments, R.sub.1 and R.sub.3 are reversed in this regard. In
particular embodiments, the ABD ring is
##STR00014##
In certain embodiments, any one or more of these ABD rings is
excluded from the present invention. In particular embodiments, the
following ABD ring is excluded:
##STR00015##
wherein R.sub.1 and R.sub.3 may be attached at any of this ABD
ring's 3 available ring carbon atoms.
[0029] Specific compounds, such as those shown below, are also
contemplated by the present invention. Such compounds may be
compounds of formula (II) and/or (V), for example.
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028##
[0030] In particular embodiments, a compound of formula (V) is not
any one or more of the following compounds:
##STR00029##
[0031] Other general aspects of the present invention contemplate a
method of treating a brain tumor comprising administrating to a
patient a compound of the present invention, such as a compound of
formula (I), (Ia), (Ib), (Ic), (II) or (V). The brain tumor may be
of any type known to those of skill in the art. For example, the
brain tumor may be selected from the group consisting of a glioma,
a glioblastoma, an astrocytoma, an oligodendroglioma, an
ependymoma, a meningioma, or a medulloblastoma. In certain
embodiments, the brain tumor is glioblastoma multiforme, anaplastic
astrocytoma, infiltrative astrocytoma, pilocytic astrocytoma, mixed
oligoastrocytoma, or mixed glioma. In treating a patient with a
brain tumor, a compound of the present invention (e.g., a compound
of formula (I), (Ia), (Ib), (Ic), (II) or (V)) is administered
intratumorally. Methods of treatment as described herein may
further comprise resection of the brain tumor. A compound of the
present invention (e.g., a compound of formula (I), (Ia), (Ib),
(Ic), (II) or (V)) may be administered before or after such
resection. For example, after a tumor has been resected, a compound
of the present invention may be administered, wherein such
administration comprises administration to the tumor bed.
[0032] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a compound of the
invention is delivered to a target cell or is placed in direct
juxtaposition with the target cell.
[0033] The term "effective," as that term is used in the
specification and/or claims (e.g., "an effective amount," means
adequate to accomplish a desired, expected, or intended result.
[0034] "Treatment" and "treating" as used herein refer to
administration or application of a therapeutic agent to a subject
or performance of a procedure or modality on a subject for the
purpose of obtaining a therapeutic benefit of a disease or
health-related condition.
[0035] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well-being of the subject with
respect to the medical treatment of a condition. This includes, but
is not limited to, a reduction in the frequency or severity of the
signs or symptoms of a disease.
[0036] It is specifically contemplated that any limitation
discussed with respect to one embodiment of the invention may apply
to any other embodiment of the invention. Furthermore, any
composition of the invention may be used in any method of the
invention, and any method of the invention may be used to produce
or to utilize any composition of the invention.
[0037] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternative are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0038] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device and/or method being employed to determine the value.
[0039] As used herein the specification, "a" or "an" may mean one
or more, unless clearly indicated otherwise. As used herein in the
claim(s), when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more.
[0040] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
[0042] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0043] FIGS. 1A-F--Isx mediate an instructive fate signal in
uncommitted NSCs and increase proliferation of committed
neuroblasts. (FIG. 1A) Isx-9 treatment induce at least a 8-fold
increase in both neuroDand gluR2-luc reporters, and exceed the gold
standard, retinoic acid and forskolin (RA/FSK) in HCN cells (FIG.
1B) Isx-9 promotes robust neuronal differentiation of HCN cells and
dominantly blocks gliogenesis in 4 d cultures. Scale bar: 25 .mu.m.
Quantification of Tuj1+ cells over 4 d is shown. (FIG. 1C)
Synthetic scheme of 3,5-disubstituted isoxazoles. (FIG. 1D) RT-PCR
gene expression profiles and protein blotting analysis of
neuronal-specific genes and p27.sup.KIP1 levels in HCN cells. GAPDH
is used as a normalization control. (FIG. 1E) Proliferation
dynamics in Isx-9-treated cultures. Shown is a representative field
of vehicle- or 20 .mu.M Isx-9-treated HCNs. Scale bar: 5 .mu.m.
Shown in FIG. 1F is a summary of HCN response to Isx-9 versus
vehicle at early (0-48 h) and later timepoints (48-96 h) in terms
of proliferation, differentiation, and cell death. Values in FIGS.
1B and 1E represent average of 2 replicates +SD of one
representative experiment from three independent experiments.
[0044] FIGS. 2A-D--Activity of isoxazoles (Isx) against brain
cancer stem cells. Isx treatment blocks the ability of human glioma
cells (U87MG cell line from ATCC) to proliferate and form colonies
in soft agar (FIG. 2A) and causes death of lung adenocarcinoma
cells from human patients, but not control immortalized bronchial
epithelial cells from the same patient (FIG. 2B). Within three days
of Isx treatment (30 mM), there are profound morphological changes
indicative of neuronal differentiation, confirmed by IHC staining
(Tuj1, an early neuronal marker) (green) and protein expression
(Tuj1 and DCX, both markers of immature neurons). Isx also blocks
the growth of astrocytoma cells, demonstrated by the rarity of
BrdU-positive cells (red) in these cultures (FIG. 2C). Finally, Isx
rapidly induces chromatin modification (hyperacetylation of histone
113 and 114) within 24 hours in human glioblastoma cancer stem
cells, and these changes are associated with marked neuronal
differentiation (Map2ab and Tuj1 are two neuronal markers) (FIG.
2D).
[0045] FIGS. 3A-C--Isx activate Ca.sup.2+ influx in NSCs. (FIG. 3A)
Representative images showing Fura-2 340/380 ratios in HCN cells.
Scale bar: 5 .mu.m. (FIG. 3B) Average 340/380 ratio traces of four
representative fields (n=40-50 cells/field) corresponding to Veh-
(dark blue), Isx-9-(red), Isx-9/cocktail inhibitor-(light blue), or
Isx-9/MK801- (light green) treated HCNs. (FIG. 3C) 50 .mu.M
cocktail inhibitor or 100 .mu.M MK801 in Isx-9-treated HCNs
attenuated neuroD-luc activity after 24 h. Values represent average
of 12 replicates .+-.SD; *: P=0.0002 and **: P=0.0003, Student's
t-test.
[0046] FIGS. 4A-F--Isx signaling leads to phosphorylation and
export of HDAC5 and MEF2-dependent gene expression in NSCs. (FIG.
4A) Dose-dependent activation of a MEF2 reporter gene (3XMRE-luc)
in Isx-9-treated HCN cells. (FIG. 4B) MEF2C is up-regulated in
Isx-9-treated cultures compared to vehicle controls. (FIG. 4C)
Increased HDAC5 phosphorylation over time with Isx-9 treatment,
normalized to GAPDH. (FIG. 4D) Accumulation of phospho-HDAC5 in the
cytoplasm of 2-d Isx-9 treated HCNs. (FIG. 4E) Shown are
representative fields of live-cell GFP fluorescence in vehicle or
20 .mu.M Isx-9-treated HCN cells expressing wild-type GFP-HDAC5
[AdGFP-HD5 (WT)] or 5259/498A mutant GFP-HDAC5 [AdGFP-HDAC5 (S-A)].
Scale bar: 5 .mu.m. Quantification of the percentage of HCN cells
showing GFP-HDAC5 localization in nuclear (Nuc), cytoplasmic
(Cyto), or both compartments (Nuc/Cyto) is shown. (FIG. 4F)
Expression of signal-resistant mutant HDAC5 (SA) and dominant
negative MEF2-engrailed repressor fusion protein abrogated
Isx-9-mediated activation of the NR1-luc reporter gene, normalized
to a control GFP plasmid. Values in FIGS. 4A, 4E and 4F represent
the average of 12 replicates +SD.
[0047] FIGS. 5A-F--CaMK is the major HDAC kinase activated by Isx
small-molecules in NSCs. (FIG. 5A) 5 .mu.M KN93 (CaMK inhibitor)
blocked Isx-9-mediated MEF2 reporter activation, while 200 nM
G66976 (PKC inhibitor) was completely ineffective. (FIG. 5B) 5
.mu.M KN93 decreased phospho-HDAC5 and active phospho-CaMKII levels
in Isx-9-treated NSCs, and (FIG. 5C) MEF2 reporter activity, while
5 .mu.M KN92, an analog of KN93 that blocks potassium channels, had
no effect. The doses of KN93 and KN92 in reporter assays were 10,
5, and 1 .mu.M. (FIG. 5D) A schematic diagram of a mammalian
2-hybrid luciferase assay system (pHDAC5:14-3-3-luciferase) which
is dependent on the interaction of HDAC5 and 14-3-3. KN93, and not
KN92, completely blocked HDAC5 kinase activity in Isx-9-treated
NSCs. (FIG. 5E) Shown are representative images and quantification
of the percentage of Isx-9-treated HCNs showing GFP-HDAC5
localization in nuclear (Nuc), cytoplasmic (Cyto), or both
compartments (Nuc/Cyto) with and without KN93 or KN92 treatment
after 24 h. (FIG. 5F) Blocking CaMK with KN93 resulted in an
inhibition of Isx-9 neurogenic activity in HCN cells in 2 d
cultures. Scale bar: 5 .mu.m. Values in FIGS. 6A, 6C and 6D
represent the average of 12 replicates .+-.SD. Values in FIG. 5E
represent the average of 2 replicates +SD of one representative
experiment from two independent experiments.
[0048] FIGS. 6A-D--Isx are a novel class of neurogenic
small-molecules. (FIG. 6A) Select 3,5-disubstituted isoxazoles
obtained from the primary high throughput screen (Sadek et al.,
manuscript submitted) and assayed for neuroD-luc activity in P19CL6
or HCN cells. Values represent the average of 12 replicates +SD.
(FIG. 6B) Initial 5 hits from the primary screen and select analogs
from secondary structure activity relationship (SAR) studies. (FIG.
6C) Isx-9 promotes maximum Tuj1+ differentiation (>50% by 4 d)
in HCN cells. This was confirmed by increased Tuj1 protein
((.beta.IIITub) and decreased expression of NSC-enriched
transcription factor, SOX2. (FIG. 6D) Activation of neuroD-luc in
HCNs treated with various Isx for 24 h. All Isx were 20 .mu.M,
unless otherwise noted.
[0049] FIGS. 7A-C--Isx is a neurogenic small-molecule in a variety
of stem cells. (FIG. 7A) Blocking cell death with the caspase
inhibitor Q-VD-Oph does not further increase Tuj1+ cells in 20
Isx-9-treated HCNs, suggesting that selective effects on survival
of pre-committed progenitors is not a major contribution to the net
increased neurogenesis. (FIG. 7B) Confirmation that 2 .mu.M
Q-VD-Oph blocks cell death in Isx-9-treated HCNs. (FIG. 7C) Isx-9
promotes neuronal differentiation in adult mouse whole brain (MWB)
neural progenitor cells and P19CL6 cells in 4 d cultures. Scale
bar: 25 and 50 .mu.m, respectively. Values in FIGS. 7A and 7B
represent average of 2 replicates +SD of one representative
experiment from three independent experiments.
[0050] FIGS. 8A-C--Epigenetic regulation of MEF2 activity triggered
by Isx-9 in NSCs. (FIG. 8A) Protein blotting time-course analysis
of MEF2 isoforms revealed that MEF2A and MEF2C levels are unchanged
with Isx-9-induced neurogenesis. GAPDH served as a normalization
control. (FIG. 8B) Total MEF2 binding to MEF2 response element
within the NR1 gene is similar between Isx-9-treated HCN cells
versus vehicle-treated cells. (FIG. 8C) Confirmation of cytoplasmic
enrichment of phospho-HDAC5 by immunoprecipitating/protein blotting
of nuclear and cytoplasmic extracts in control (Veh) and
Isx-9-treated HCN cells infected with wild-type Flag-HDAC5 for 2 d.
NSC lysates were immunoprecipated with the Flag antibody and
blotted with phospho-specific HDAC5 and Flag antibodies. CREB and
GFP served as normalization controls.
[0051] FIGS. 9A-H--Isoxazoles specifically target the CD133(+)
glioblastoma cancer stem cell to adopt a neuronal fate program.
Isx-9 triggers a pro neuronal-differentiation effect on human
CD133(+) brain tumor stem cells (BTSCs) within 4 days. (FIG. 9A)
Relative to growth factors alone (20 ng/ml FGF/EGF), (FIG. 9C)
Isx-9 treatment (20 .mu.M) leads to more differentiated (adherent
and phase-dark with neuronal-like processes) cells. (FIG. 9B) BTSCs
cultured with DMSO (vehicle) (1% vol./vol.). (FIG. 9D)
Isx-9-treated BTSCs stained with neuronal markers Tuj1 (red) and
Map2ab (green), Dapi-stained nuclei is blue. Scale bar=25 .mu.m.
(FIG. 9E) Dose-dependent activation of two neuron-specific reporter
genes, NeuroD- and (FIG. 9F) NMDA receptor 1-(NR1) luciferase (luc)
in Isx-9-treated BTSCs compared to vehicle control after 24 hours.
An unrelated class of small-molecules (sulfonyl hydrazone, Shz) did
not significantly induce reporter gene activity. (FIGS. 9G and 9H)
RT-PCR and protein blotting analysis of gene expression in
Isx-9-treated BTSCs. (FIG. 9G) Up-regulation of NeuroD in BTSCs
treated with Isx-9 for 2 days which remains elevated when FGF/EGF
is added back (without Isx-9) for another 2 days. The
Kruppel-family zinc finger transcriptional regulator and
proto-oncogene neuron-restrictive silencer factor (NRSF) and CD133
are both downregulated with Isx-treatment. (FIG. 9H)NRSF protein is
downregulated with longer Isx-9 timepoints, compared to the
chromatin-remodeling enzymes histone deacetylases (HDAC)-4 and -5,
which remain relatively unchanged. GAPDH is used as a normalization
control. Despite a dramatic change in cell morphology and
attachment mediated by Isx-9 treatment, overall levels of neuronal
proteins, such as .beta.TujIII and glutamate receptor 2 (GluR2) do
not significantly differ between control and Isx-9-treated
BTSCs.
[0052] FIGS. 10A-N--Isoxazoles induce growth arrest and inhibit the
tumorigenic potential of human BTSCs in vitro and in vivo. (FIG.
10A-E) Phase-bright images of brain tumor stem cells (BTSCs)
treated with EGF/FGF (FIG. 10A) or Isx-9 (FIG. 10B) for 7 days
before returning to EGF/FGF (FIG. 10C) or EGF/FGF plus Isx-9
conditions (FIG. 10D) for an additional 2 days. (FIG. 10E) BTSCs
labeled with CAG-red fluorescent protein (RFP) in vitro. (FIG. 10F)
7-day pretreatment of BTSCs with DMSO (vehicle), EGF/FGF, or Isx-9,
dissociated and re-plated to form secondary neurospheres (1000
cells/well) under various conditions [N2/B27 media alone (white
bars), N2/B27 plus EGF/FGF (black bars), and N2/B27 plus EGF/FGF
plus Isx-9 (gray bars)] for 7 additional days. Shown in FIG. 10F is
a graphical representation of the average number of neurospheres
(per 3 wells) and the average area of each neurosphere
(.mu.m.sup.2). (FIGS. 10G-J) Isx-9 pre-treatment for 7 days leads
to decreased DNA synthesis/proliferation (BrdU uptake with a 1 hour
pulse prior to fixation) compared to EGF/FGF or vehicle
pre-treatment, even when EGF/FGF is added back for 24 hours. (FIGS.
10G-I) BrdU staining in fixed cultures; BrdU (red) and Dapi-stained
nuclei (blue). (FIG. 10J) Graphical representation of the percent
of BrdU(+) cells (out of the total Dapi(+) cells). (FIGS. 10K-N)
7-day exposure of RFP-labeled human CD133(+) BTSCs to 20 ng/ml
EGF/FGF (FIGS. 10K and 10L) or Isx-9 (20 .mu.M) (FIGS. 10M and 10N)
in vitro, before transplantation into the striatum of NOD/scid
mice, dramatically reduces tumor-initiating ability. Scale bar=200
.mu.m.
[0053] FIG. 11--Schematic of reporter transgene with luciferase
inserted by homologous recombination into the Nkx2.5 locus on an
.about.178 kb mouse BAC.
[0054] FIG. 12A-I--Results of neuronal differentiation induction by
various compounds of the present invention (see Example 4).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0055] The present invention overcomes the deficiencies of the
prior art by providing compounds that induce neuronal
differentiation and methods relating thereto.
[0056] Increasing clinical success of stem cell therapy for
neuronal regeneration purposes and addressing neurological problems
is coupled to the need for better understanding of neural cell fate
mechanisms. Here, the inventors report the identification of
small-molecules involved in neuronal fate by screening a chemical
library for activators of the signature gene, Nkx2.5, using a
luciferase knock-in bacterial artificial chromosome (BAC) in mouse
P19CL6 pluripotent stem cells. This family of isoxazole-derived
small molecules can be used to trigger neuronal mRNA and protein
expression in a variety of embryonic and adult stem/progenitor
cells (NSCs). Small-molecule enhanced NSCs engrafted into the brain
or spinal cord may assist with the recovery of neuronal function
following injury or disease. Isoxazoles and their derivatives are
thus promising drugs to promote neuronal repair/regeneration by
activating neuronal differentiation in stem cells.
[0057] Over the past several years the concept of cancer stem cells
has been extended from hematological malignancies to epithelial
cancers including gliomas. Although the mechanistic understanding
of cancer stem cells remains limited, the concept raises
fundamental issues about the cellular origins of cancer as well as
tumor progression and maintenance, and therefore has important
implications for the development of therapeutics. At its core, the
cancer stem cell hypothesis proposes that a small (<1-5%)
fraction of the tumor cells are exclusively responsible for
maintaining tumor burden and represent the most recalcitrant cell
types to conventional radiotherapy and chemotherapy. The clear
implications are that effective cancer therapies must be able to
target and destroy the cancer stem cells. Discovery of this
isoxazole-based small molecule family provides both an important
mechanistic clue to the cancer stem cell hypothesis as it applies
to gliomas, and provides for development of novel chemotherapeutic
agents that can specifically target cancer stem cells.
[0058] These, and other aspects of the invention, are set out in
detail below.
B. Compounds of the Present Invention
[0059] Compounds of the present invention may be considered as
derived from isoxazoles. The following compounds are representative
of certain compounds of the present invention:
[0060] a compound of formula (I):
##STR00030##
wherein X is O or NH, Y is S or O and R is H, a substituted or
unsubstituted alkyl, such as C.sub.1-C.sub.6 alkyl or
C.sub.3-C.sub.6 cycloalkyl, or a substituted or unsubstituted
alkenyl, such as C.sub.2-C.sub.6 alkenyl, a substituted or
unsubstituted alkenyl, such as C.sub.2-C.sub.6 alkynyl, or a
stereoisomer, solvate, hydrate, or pharmaceutically acceptable salt
thereof. In certain embodiments regarding compounds of formula (I),
the proviso exists such that with the provisos that if X is O, then
R must be a substituted or unsubstituted C.sub.3-C.sub.6
cycloalkyl; and/or if X is NH, then R must not be pyrazinyl
substituted C.sub.1-C.sub.6 alkyl;
[0061] a compound of formula (Ia), (Ib), or (Ic):
##STR00031##
wherein R.sub.1 is substituted or unsubstituted phenyl,
unsubstituted pyrrolyl, unsubstituted pyridyl, unsubstituted
furanyl, unsubstituted thienyl, unsubstituted benzofuranyl,
unsubstituted benzo[b]thiophenyl, or unsubstituted thiazolyl. Any
of these R1 substituents may be substituted as well;
[0062] a compound of formula (II):
##STR00032##
wherein: R.sub.1 is substituted or unsubstituted thiophenyl or a
substituent of formula (A):
##STR00033##
wherein: R.sub.A, R.sub.B and R.sub.C are each independently
selected from the group consisting of hydrogen, halogen,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.6 cycloalkyl, aryl, cyano,
nitro, and a carbonyl group; and G is O, --NH, or S; R.sub.2 is
hydrogen, hydroxy, halogen, nitro, aryl, alkyl, alkoxy, alkenyl,
alkenyloxy, alkynyl, alkynyloxy, aralkyl, --CHO, --C(O)R.sub.9,
--OC(O)R.sub.9, --OC(O)OR.sub.9, --O(CN)OR.sub.9,
--C(O)NR.sub.9R.sub.10, --OC(O)NR.sub.9R.sub.10,
--NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9; wherein R.sub.9 and
R.sub.10 are each independently hydrogen, alkyl, aryl, or aralkyl;
R.sub.3 is --NH--O-alkyl, --NH--OH, --OR.sub.11 or
--NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 are each
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, or aralkyl;
or R.sub.11 and R.sub.12 together form a cyclic group; or R.sub.11
and R.sub.12 together with the nitrogen to which they are bound
form a cyclic group; X is O or --NR.sub.13, wherein R.sub.13 is
hydrogen, alkyl, aryl, or aralkyl; or a stereoisomer, solvate,
hydrate, or pharmaceutically acceptable salt thereof;
[0063] and a compound having formula (V):
##STR00034##
wherein: the ABD ring comprises two non-adjacent double bonds; A, B
and D are each independently S, N, O, C, --NR.sub.14, --CR.sub.15,
or --CR.sub.15R.sub.16, wherein R.sub.14 is hydrogen, halogen,
alkyl, aryl, or aralkyl; and R.sub.15 and R.sub.16 are each
independently hydrogen, hydroxy, halogen, nitro, aryl, alkyl,
alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, aralkyl, --CHO,
--C(O)R.sub.9, --OC(O)R.sub.9, --OC(O)OR.sub.9, --O(CN)OR.sub.9,
--C(O)NR.sub.9R.sub.10, --OC(O)NR.sub.9R.sub.10,
--NR.sub.9OR.sub.5, or --SO.sub.3R.sub.9; wherein R.sub.9 and
R.sub.10 are each independently hydrogen, alkyl, aryl, or aralkyl,
provided that at least two of A, B and D comprise S, N, or O;
R.sub.1 is alkyl, --CH.dbd.CH-aryl, or aryl; and R.sub.3 is alkyl,
aryl, aralkyl, --OR.sub.4, or --NR.sub.4R.sub.5, wherein: R.sub.4
and R.sub.5 are each independently hydrogen, alkyl, aryl, or
aralkyl; or R.sub.4 and R.sub.5 together form a cyclic group; or
R.sub.4 and R.sub.5 together with the nitrogen to which they are
bound form a cyclic group. In certain embodiments regarding
compounds of formula (V), the proviso exists such that compounds of
formula (V.sub.a) are excluded:
##STR00035##
wherein R.sub.18 is alkyl, such as lower alkyl or cyclopentyl, or
alkenyl, such as lower alkenyl or allyl, and G is O or S.
C. Chemical Definitions
[0064] As used herein, the term "amino" means --NH.sub.2; the term
"nitro" means --NO.sub.2; the term "halo" designates --F, --Cl,
--Br or --I; the term "mercapto" or "thiol" means --SH; the term
"cyano" means --CN; the term "azido" means --N.sub.3; the term
"silyl" means --SiH.sub.3, and the term "hydroxy" means --OH.
[0065] The term "alkyl" includes straight-chain alkyl,
branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl,
heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl,
heteroatom-unsubstituted C.sub.n-alkyl, and heteroatom-substituted
C.sub.n-alkyl. In certain embodiments, lower alkyls are
contemplated. The term "lower alkyl" refers to alkyls of 1-6 carbon
atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). Higher alkyls are
contemplated in certain embodiments. The term "higher alkyl" refers
to alkyls of 6 carbon atoms or higher, such as 6-15 carbon atoms.
The term "heteroatom-unsubstituted C.sub.n-alkyl" refers to a
radical, having a linear or branched, cyclic or acyclic structure,
further having no carbon-carbon double or triple bonds, further
having a total of n carbon atoms, all of which are nonaromatic, 3
or more hydrogen atoms, and no heteroatoms. For example, a
heteroatom-unsubstituted C.sub.1-C.sub.10-alkyl has 1 to 10 carbon
atoms. The groups, --CH.sub.3 (Me), --CH.sub.2CH.sub.3 (Et),
--CH.sub.2CH.sub.2CH.sub.3 (n-Pr), --CH(CH.sub.3).sub.2 (iso-Pr),
--CH(CH.sub.2).sub.2 (cyclopropyl),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu),
--CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl),
--CH.sub.2CH(CH.sub.3).sub.2 (iso-butyl), --C(CH.sub.3).sub.3
(tert-butyl), --CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl), cyclobutyl,
cyclopentyl, and cyclohexyl, are all non-limiting examples of
heteroatom-unsubstituted alkyl groups. The term
"heteroatom-substituted C.sub.n-alkyl" refers to a radical, having
a single saturated carbon atom as the point of attachment, no
carbon-carbon double or triple bonds, further having a linear or
branched, cyclic or acyclic structure, further having a total of n
carbon atoms, all of which are nonaromatic, 0, 1, or more than one
hydrogen atom, at least one heteroatom, wherein each heteroatom is
independently selected from the group consisting of N, O, F, Cl,
Br, I, Si, P, and S. For example, a heteroatom-substituted
C.sub.1-C.sub.10-alkyl has 1 to 10 carbon atoms. The following
groups are all non-limiting examples of heteroatom-substituted
alkyl groups: trifluoromethyl, --CH.sub.2F, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2OH, --CH.sub.2OCH.sub.3,
--CH.sub.2OCH.sub.2CF.sub.3, --CH.sub.2OC(O)CH.sub.3,
--CH.sub.2NH.sub.2, --CH.sub.2NHCH.sub.3,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2CH.sub.2Cl,
--CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2OC(O)CH.sub.3,
--CH.sub.2CH.sub.2NHCO.sub.2C(CH.sub.3).sub.3, and
--CH.sub.2Si(CH.sub.3).sub.3. The term "cycloalkyl" refers to,
e.g., cyclopropyl, cyclopentyl, cyclohexyl, etc., as well as
heteroatom-substituted cycloalkyl (e.g., pyrrolidinyl,
cyclohexanol). The term "alkyl" refers also to a straight- or
branched-chain alkyl group that terminates in a cycloalkyl
group.
[0066] The term "alkenyl" includes straight-chain alkenyl,
branched-chain alkenyl, cycloalkenyl, cyclic alkenyl,
heteroatom-unsubstituted alkenyl, heteroatom-substituted alkenyl,
heteroatom-unsubstituted C.sub.n-alkenyl, and
heteroatom-substituted C.sub.n-alkenyl. In certain embodiments,
lower alkenyls are contemplated. The term "lower alkenyl" refers to
alkenyls of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon
atoms). The term "heteroatom-unsubstituted C.sub.n-alkenyl" refers
to a radical, having a linear or branched, cyclic or acyclic
structure, further having at least one nonaromatic carbon-carbon
double bond, but no carbon-carbon triple bonds, a total of n carbon
atoms, three or more hydrogen atoms, and no heteroatoms. For
example, a heteroatom-unsubstituted C.sub.2-C.sub.10-alkenyl has 2
to 10 carbon atoms. Heteroatom-unsubstituted alkenyl groups
include: --CH.dbd.CH.sub.2 (vinyl), --CH.dbd.CHCH.sub.3,
--CH.dbd.CHCH.sub.2CH.sub.3, --CH.sub.2CH.dbd.CH.sub.2 (allyl),
--CH.sub.2CH.dbd.CHCH.sub.3, and --CH.dbd.CH--C.sub.6H.sub.5. The
term "heteroatom-substituted C.sub.n-alkenyl" refers to a radical,
having a single nonaromatic carbon atom as the point of attachment
and at least one nonaromatic carbon-carbon double bond, but no
carbon-carbon triple bonds, further having a linear or branched,
cyclic or acyclic structure, further having a total of n carbon
atoms, 0, 1, or more than one hydrogen atom, and at least one
heteroatom, wherein each heteroatom is independently selected from
the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For
example, a heteroatom-substituted C.sub.2-C.sub.10-alkenyl has 2 to
10 carbon atoms. The groups, --CH.dbd.CHF, --CH.dbd.CHCl and
--CH.dbd.CHBr, are non-limiting examples of heteroatom-substituted
alkenyl groups.
[0067] The term "alkynyl" includes straight-chain alkynyl,
branched-chain alkynyl, cycloalkynyl, cyclic alkynyl,
heteroatom-unsubstituted alkynyl, heteroatom-substituted alkynyl,
heteroatom-unsubstituted C.sub.n-alkynyl, and
heteroatom-substituted C.sub.n-alkynyl. In certain embodiments,
lower alkynyls are contemplated. The term "lower alkynyl" refers to
alkynyls of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon
atoms). The term "heteroatom-unsubstituted C.sub.n-alkynyl" refers
to a radical, having a linear or branched, cyclic or acyclic
structure, further having at least one carbon-carbon triple bond, a
total of n carbon atoms, at least one hydrogen atom, and no
heteroatoms. For example, a heteroatom-unsubstituted
C.sub.2-C.sub.10-alkynyl has 2 to 10 carbon atoms. The groups,
--C.ident.CH, --C.ident.CCH.sub.3, and --C.ident.CC.sub.6H.sub.5
are non-limiting examples of heteroatom-unsubstituted alkynyl
groups. The term "heteroatom-substituted C.sub.n-alkynyl" refers to
a radical, having a single nonaromatic carbon atom as the point of
attachment and at least one carbon-carbon triple bond, further
having a linear or branched, cyclic or acyclic structure, and
having a total of n carbon atoms, 0, 1, or more than one hydrogen
atom, and at least one heteroatom, wherein each heteroatom is
independently selected from the group consisting of N, O, F, Cl,
Br, I, Si, P, and S. For example, a heteroatom-substituted
C.sub.2-C.sub.10-alkynyl has 2 to 10 carbon atoms. The group,
--C.ident.CSi(CH.sub.3).sub.3, is a non-limiting example of a
heteroatom-substituted alkynyl group.
[0068] The term "aryl" includes heteroatom-unsubstituted aryl,
heteroatom-substituted aryl, heteroatom-unsubstituted C.sub.n-aryl,
heteroatom-substituted C.sub.n-aryl, heteroaryl, heterocyclic aryl
groups, carbocyclic aryl groups, biaryl groups, and single-valent
radicals derived from polycyclic fused hydrocarbons (PAHs). The
term "heteroatom-unsubstituted C.sub.n-aryl" refers to a radical,
having a single carbon atom as a point of attachment, wherein the
carbon atom is part of an aromatic ring structure containing only
carbon atoms, further having a total of n carbon atoms, 5 or more
hydrogen atoms, and no heteroatoms. For example, a
heteroatom-unsubstituted C.sub.6-C.sub.10-aryl has 6 to 10 carbon
atoms. Non-limiting examples of heteroatom-unsubstituted aryl
groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl,
--C.sub.6H.sub.4CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH.sub.2CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH(CH.sub.3).sub.2,
--C.sub.6H.sub.4CH(CH.sub.2).sub.2,
--C.sub.6H.sub.3(CH.sub.3)CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH.dbd.CH.sub.2, --C.sub.6H.sub.4CH.dbd.CHCH.sub.3,
--C.sub.6H.sub.4C.ident.CH, --C.sub.6H.sub.4C.ident.CCH.sub.3,
naphthyl, and the radical derived from biphenyl. The term
"heteroatom-substituted C.sub.n-aryl" refers to a radical, having
either a single aromatic carbon atom or a single aromatic
heteroatom as the point of attachment, further having a total of n
carbon atoms, at least one hydrogen atom, and at least one
heteroatom, further wherein each heteroatom is independently
selected from the group consisting of N, O, F, Cl, Br, I, Si, P,
and S. For example, a heteroatom-unsubstituted
C.sub.1-C.sub.10-heteroaryl has 1 to 10 carbon atoms. Non-limiting
examples of heteroatom-substituted aryl groups include the groups:
--C.sub.6H.sub.4F, --C.sub.6H.sub.4Cl, --C.sub.6H.sub.4Br,
--C.sub.6H.sub.4I, --C.sub.6H.sub.4OH, --C.sub.6H.sub.4OCH.sub.3,
--C.sub.6H.sub.4OCH.sub.2CH.sub.3, --C.sub.6H.sub.4OC(O)CH.sub.3,
--C.sub.6H.sub.4NH.sub.2, --C.sub.6H.sub.4NHCH.sub.3,
--C.sub.6H.sub.4N(CH.sub.3).sub.2, --C.sub.6H.sub.4CH.sub.2OH,
--C.sub.6H.sub.4CH.sub.2OC(O)CH.sub.3,
--C.sub.6H.sub.4CH.sub.2NH.sub.2, --C.sub.6H.sub.4CF.sub.3,
--C.sub.6H.sub.4CN, --C.sub.6H.sub.4CHO, --C.sub.6H.sub.4CHO,
--C.sub.6H.sub.4C(O)CH.sub.3, --C.sub.6H.sub.4C(O)C.sub.6H.sub.5,
--C.sub.6H.sub.4CO.sub.2H, --C.sub.6H.sub.4CO.sub.2CH.sub.3,
--C.sub.6H.sub.4CONH.sub.2, --C.sub.6H.sub.4CONHCH.sub.3,
--C.sub.6H.sub.4CON(CH.sub.3).sub.2, furanyl, thienyl, pyridyl,
pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, indolyl, and
imidazoyl.
[0069] The term "aralkyl" includes heteroatom-unsubstituted
aralkyl, heteroatom-substituted aralkyl, heteroatom-unsubstituted
C.sub.n-aralkyl, heteroatom-substituted C.sub.n-aralkyl,
heteroaralkyl, and heterocyclic aralkyl groups. In certain
embodiments, lower aralkyls are contemplated. The term "lower
aralkyl" refers to aralkyls of 7-12 carbon atoms (that is, 7, 8, 9,
10, 11 or 12 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-aralkyl" refers to a radical, having a single saturated
carbon atom as the point of attachment, further having a total of n
carbon atoms, wherein at least 6 of the carbon atoms form an
aromatic ring structure containing only carbon atoms, 7 or more
hydrogen atoms, and no heteroatoms. For example, a
heteroatom-unsubstituted C.sub.7-C.sub.10-aralkyl has 7 to 10
carbon atoms. Non-limiting examples of heteroatom-unsubstituted
aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl. The term
"heteroatom-substituted C.sub.n-aralkyl" refers to a radical,
having a single saturated carbon atom as the point of attachment,
further having a total of n carbon atoms, 0, 1, or more than one
hydrogen atom, and at least one heteroatom, wherein at least one of
the carbon atoms is incorporated an aromatic ring structures,
further wherein each heteroatom is independently selected from the
group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example,
a heteroatom-substituted C.sub.2-C.sub.10-heteroaralkyl has 2 to 10
carbon atoms.
[0070] The term "acyl" includes straight-chain acyl, branched-chain
acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl,
heteroatom-substituted acyl, heteroatom-unsubstituted C.sub.n-acyl,
heteroatom-substituted C.sub.n-acyl, alkylcarbonyl, alkoxycarbonyl
and aminocarbonyl groups. In certain embodiments, lower acyls are
contemplated. The term "lower acyl" refers to acyls of 1-6 carbon
atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-acyl" refers to a radical, having
a single carbon atom of a carbonyl group as the point of
attachment, further having a linear or branched, cyclic or acyclic
structure, further having a total of n carbon atoms, 1 or more
hydrogen atoms, a total of one oxygen atom, and no additional
heteroatoms. For example, a heteroatom-unsubstituted
C.sub.1-C.sub.10-acyl has 1 to 10 carbon atoms. The groups, --CHO,
--C(O)CH.sub.3, --C(O)CH.sub.2CH.sub.3,
--C(O)CH.sub.2CH.sub.2CH.sub.3, --C(O)CH(CH.sub.3).sub.2,
--C(O)CH(CH.sub.2).sub.2, --C(O)C.sub.6H.sub.5,
--C(O)C.sub.6H.sub.4CH.sub.3, --C(O)C.sub.6H.sub.4CH.sub.2CH.sub.3,
and --COC.sub.6H.sub.3(CH.sub.3).sub.2, are non-limiting examples
of heteroatom-unsubstituted acyl groups. The term
"heteroatom-substituted C.sub.n-acyl" refers to a radical, having a
single carbon atom as the point of attachment, the carbon atom
being part of a carbonyl group, further having a linear or
branched, cyclic or acyclic structure, further having a total of n
carbon atoms, 0, 1, or more than one hydrogen atom, at least one
additional heteroatom, in addition to the oxygen of the carbonyl
group, wherein each additional heteroatom is independently selected
from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For
example, a heteroatom-substituted C.sub.1-C.sub.10-acyl has 1 to 10
carbon atoms. The groups, --C(O)CH.sub.2CF.sub.3, --CO.sub.2H,
--CO.sub.2CH.sub.3, --CO.sub.2CH.sub.2CH.sub.3,
--CO.sub.2CH.sub.2CH.sub.2CH.sub.3, --CO.sub.2CH(CH.sub.3).sub.2,
--CO.sub.2CH(CH.sub.2).sub.2, --C(O)NH.sub.2 (carbamoyl),
--C(O)NHCH.sub.3, --C(O)NHCH.sub.2CH.sub.3,
--CONHCH(CH.sub.3).sub.2, --CONHCH(CH.sub.2).sub.2,
--CON(CH.sub.3).sub.2, and --CONHCH.sub.2CF.sub.3, are non-limiting
examples of heteroatom-substituted acyl groups.
[0071] The term "alkoxy" includes straight-chain alkoxy,
branched-chain alkoxy, cycloalkoxy, cyclic alkoxy,
heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy,
heteroatom-unsubstituted C.sub.n-alkoxy, and heteroatom-substituted
C.sub.n-alkoxy. In certain embodiments, lower alkoxys are
contemplated. The term "lower alkoxy" refers to alkoxys of 1-6
carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-alkoxy" refers to a group, having
the structure --OR, in which R is a heteroatom-unsubstituted
C.sub.n-alkyl, as that term is defined above.
Heteroatom-unsubstituted alkoxy groups include: --OCH.sub.3,
--OCH.sub.2CH.sub.3, --OCH.sub.2CH.sub.2CH.sub.3,
--OCH(CH.sub.3).sub.2, and --OCH(CH.sub.2).sub.2. The term
"heteroatom-substituted C.sub.n-alkoxy" refers to a group, having
the structure --OR, in which R is a heteroatom-substituted
C.sub.n-alkyl, as that term is defined above. For example,
--OCH.sub.2CF.sub.3 is a heteroatom-substituted alkoxy group.
[0072] The term "alkenyloxy" includes straight-chain alkenyloxy,
branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy,
heteroatom-unsubstituted alkenyloxy, heteroatom-substituted
alkenyloxy, heteroatom-unsubstituted C.sub.n-alkenyloxy, and
heteroatom-substituted C.sub.n-alkenyloxy. In certain embodiments,
lower alkenyloxys are contemplated. The term "lower alkenyloxy"
refers to alkenyloxys of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkenyloxy" refers to a group, having the structure --OR,
in which R is a heteroatom-unsubstituted C.sub.n-alkenyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkenyloxy" refers to a group, having the structure --OR,
in which R is a heteroatom-substituted C.sub.n-alkenyl, as that
term is defined above.
[0073] The term "alkynyloxy" includes straight-chain alkynyloxy,
branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy,
heteroatom-unsubstituted alkynyloxy, heteroatom-substituted
alkynyloxy, heteroatom-unsubstituted C.sub.n-alkynyloxy, and
heteroatom-substituted C.sub.n-alkynyloxy. In certain embodiments,
lower alkynyloxys are contemplated. The term "lower alkynyloxy"
refers to alkynyloxys of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkynyloxy" refers to a group, having the structure --OR,
in which R is a heteroatom-unsubstituted C.sub.n-alkynyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkynyloxy" refers to a group, having the structure --OR,
in which R is a heteroatom-substituted C.sub.n-alkynyl, as that
term is defined above.
[0074] The term "aryloxy" includes heteroatom-unsubstituted
aryloxy, heteroatom-substituted aryloxy, heteroatom-unsubstituted
C.sub.n-aryloxy, heteroatom-substituted C.sub.n-aryloxy,
heteroaryloxy, and heterocyclic aryloxy groups. The term
"heteroatom-unsubstituted C.sub.n-aryloxy" refers to a group,
having the structure --OAr, in which Ar is a
heteroatom-unsubstituted C.sub.n-aryl, as that term is defined
above. A non-limiting example of a heteroatom-unsubstituted aryloxy
group is --OC.sub.6H.sub.5. The term "heteroatom-substituted
C.sub.n-aryloxy" refers to a group, having the structure --OAr, in
which Ar is a heteroatom-substituted C.sub.n-aryl, as that term is
defined above.
[0075] The term "aralkyloxy" includes heteroatom-unsubstituted
aralkyloxy, heteroatom-substituted aralkyloxy,
heteroatom-unsubstituted C.sub.n-aralkyloxy, heteroatom-substituted
C.sub.n-aralkyloxy, heteroaralkyloxy, and heterocyclic aralkyloxy
groups. In certain embodiments, lower aralkyloxys are contemplated.
The term "lower aralkyloxy" refers to alkenyloxys of 7-12 carbon
atoms (that is, 7, 8, 9, 10, 11, or 12 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-aralkyloxy" refers to a group,
having the structure --OAr, in which Ar is a
heteroatom-unsubstituted C.sub.n-aralkyl, as that term is defined
above. The term "heteroatom-substituted C.sub.n-aralkyloxy" refers
to a group, having the structure --OAr, in which Ar is a
heteroatom-substituted C.sub.n-aralkyl, as that term is defined
above.
[0076] The term "acyloxy" includes straight-chain acyloxy,
branched-chain acyloxy, cycloacyloxy, cyclic acyloxy,
heteroatom-unsubstituted acyloxy, heteroatom-substituted acyloxy,
heteroatom-unsubstituted C.sub.n-acyloxy, heteroatom-substituted
C.sub.n-acyloxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. In
certain embodiments, lower acyloxys are contemplated. The term
"lower acyloxy" refers to acyloxys of 1-6 carbon atoms (that is, 1,
2, 3, 4, 5 or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-acyloxy" refers to a group, having the structure --OAc, in
which Ac is a heteroatom-unsubstituted C.sub.n-acyl, as that term
is defined above. For example, --OC(O)CH.sub.3 is a non-limiting
example of a heteroatom-unsubstituted acyloxy group. The term
"heteroatom-substituted C.sub.n-acyloxy" refers to a group, having
the structure --OAc, in which Ac is a heteroatom-substituted
C.sub.n-acyl, as that term is defined above. For example,
--OC(O)OCH.sub.3 and --OC(O)NHCH.sub.3 are non-limiting examples of
heteroatom-unsubstituted acyloxy groups.
[0077] The term "alkylamino" includes straight-chain alkylamino,
branched-chain alkylamino, cycloalkylamino, cyclic alkylamino,
heteroatom-unsubstituted alkylamino, heteroatom-substituted
alkylamino, heteroatom-unsubstituted C.sub.n-alkylamino, and
heteroatom-substituted C.sub.n-alkylamino. In certain embodiments,
lower alkylaminos are contemplated. The term "lower alkylamino"
refers to alkylaminos of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkylamino" refers to a radical, having a single nitrogen
atom as the point of attachment, further having one or two
saturated carbon atoms attached to the nitrogen atom, further
having a linear or branched, cyclic or acyclic structure,
containing a total of n carbon atoms, all of which are nonaromatic,
4 or more hydrogen atoms, a total of 1 nitrogen atom, and no
additional heteroatoms. For example, a heteroatom-unsubstituted
C.sub.1-C.sub.10-alkylamino has 1 to 10 carbon atoms. The term
"heteroatom-unsubstituted C.sub.n-alkylamino" includes groups,
having the structure --NHR, in which R is a
heteroatom-unsubstituted C.sub.n-alkyl, as that term is defined
above. A heteroatom-unsubstituted alkylamino group would 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, --N(CH.sub.3).sub.2,
--N(CH.sub.3)CH.sub.2CH.sub.3, --N(CH.sub.2CH.sub.3).sub.2,
N-pyrrolidinyl, and N-piperidinyl. The term "heteroatom-substituted
C.sub.n-alkylamino" refers to a radical, having a single nitrogen
atom as the point of attachment, further having one or two
saturated carbon atoms attached to the nitrogen atom, no
carbon-carbon double or triple bonds, further having a linear or
branched, cyclic or acyclic structure, further having a total of n
carbon atoms, all of which are nonaromatic, 0, 1, or more than one
hydrogen atom, and at least one additional heteroatom, that is, in
addition to the nitrogen atom at the point of attachment, wherein
each additional heteroatom is independently selected from the group
consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.1-C.sub.10-alkylamino has 1 to 10
carbon atoms. The term "heteroatom-substituted C.sub.n-alkylamino"
includes groups, having the structure --NHR, in which R is a
heteroatom-substituted C.sub.n-alkyl, as that term is defined
above.
[0078] The term "alkenylamino" includes straight-chain
alkenylamino, branched-chain alkenylamino, cycloalkenylamino,
cyclic alkenylamino, heteroatom-unsubstituted alkenylamino,
heteroatom-substituted alkenylamino, heteroatom-unsubstituted
C.sub.n-alkenylamino, heteroatom-substituted C.sub.n-alkenylamino,
dialkenylamino, and alkyl(alkenyl)amino groups. In certain
embodiments, lower alkenylaminos are contemplated. The term "lower
alkenylamino" refers to alkenylaminos of 1-6 carbon atoms (that is,
1, 2, 3, 4, 5 or 6 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-alkenylamino" refers to a
radical, having a single nitrogen atom as the point of attachment,
further having one or two carbon atoms attached to the nitrogen
atom, further having a linear or branched, cyclic or acyclic
structure, containing at least one nonaromatic carbon-carbon double
bond, a total of n carbon atoms, 4 or more hydrogen atoms, a total
of one nitrogen atom, and no additional heteroatoms. For example, a
heteroatom-unsubstituted C.sub.2-C.sub.10-alkenylamino has 2 to 10
carbon atoms. The term "heteroatom-unsubstituted
C.sub.n-alkenylamino" includes groups, having the structure --NHR,
in which R is a heteroatom-unsubstituted C.sub.n-alkenyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkenylamino" refers to a radical, having a single nitrogen
atom as the point of attachment and at least one nonaromatic
carbon-carbon double bond, but no carbon-carbon triple bonds,
further having one or two carbon atoms attached to the nitrogen
atom, further having a linear or branched, cyclic or acyclic
structure, further having a total of n carbon atoms, 0, 1, or more
than one hydrogen atom, and at least one additional heteroatom,
that is, in addition to the nitrogen atom at the point of
attachment, wherein each additional heteroatom is independently
selected from the group consisting of N, O, F, Cl, Br, I, Si, P,
and S. For example, a heteroatom-substituted
C.sub.2-C.sub.10-alkenylamino has 2 to 10 carbon atoms. The term
"heteroatom-substituted C.sub.n-alkenylamino" includes groups,
having the structure --NHR, in which R is a heteroatom-substituted
C.sub.n-alkenyl, as that term is defined above.
[0079] The term "alkynylamino" includes straight-chain
alkynylamino, branched-chain alkynylamino, cycloalkynylamino,
cyclic alkynylamino, heteroatom-unsubstituted alkynylamino,
heteroatom-substituted alkynylamino, heteroatom-unsubstituted
C.sub.n-alkynylamino, heteroatom-substituted C.sub.n-alkynylamino,
dialkynylamino, alkyl(alkynyl)amino, and alkenyl(alkynyl)amino
groups. In certain embodiments, lower alkynylaminos are
contemplated. The term "lower alkynylamino" refers to alkynylaminos
of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The
term "heteroatom-unsubstituted C.sub.n-alkynylamino" refers to a
radical, having a single nitrogen atom as the point of attachment,
further having one or two carbon atoms attached to the nitrogen
atom, further having a linear or branched, cyclic or acyclic
structure, containing at least one carbon-carbon triple bond, a
total of n carbon atoms, at least one hydrogen atoms, a total of
one nitrogen atom, and no additional heteroatoms. For example, a
heteroatom-unsubstituted C.sub.2-C.sub.10-alkynylamino has 2 to 10
carbon atoms. The term "heteroatom-unsubstituted
C.sub.n-alkynylamino" includes groups, having the structure --NHR,
in which R is a heteroatom-unsubstituted C.sub.n-alkynyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkynylamino" refers to a radical, having a single nitrogen
atom as the point of attachment, further having one or two carbon
atoms attached to the nitrogen atom, further having at least one
nonaromatic carbon-carbon triple bond, further having a linear or
branched, cyclic or acyclic structure, and further having a total
of n carbon atoms, 0, 1, or more than one hydrogen atom, and at
least one additional heteroatom, that is, in addition to the
nitrogen atom at the point of attachment, wherein each additional
heteroatom is independently selected from the group consisting of
N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.2-C.sub.10-alkynylamino has 2 to 10
carbon atoms. The term "heteroatom-substituted
C.sub.n-alkynylamino" includes groups, having the structure --NHR,
in which R is a heteroatom-substituted C.sub.n-alkynyl, as that
term is defined above.
[0080] The term "arylamino" includes heteroatom-unsubstituted
arylamino, heteroatom-substituted arylamino,
heteroatom-unsubstituted C.sub.n-arylamino, heteroatom-substituted
C.sub.n-arylamino, heteroarylamino, heterocyclic arylamino, and
alkyl(aryl)amino groups. The term "heteroatom-unsubstituted
C.sub.n-arylamino" refers to a radical, having a single nitrogen
atom as the point of attachment, further having at least one
aromatic ring structure attached to the nitrogen atom, wherein the
aromatic ring structure contains only carbon atoms, further having
a total of n carbon atoms, 6 or more hydrogen atoms, a total of one
nitrogen atom, and no additional heteroatoms. For example, a
heteroatom-unsubstituted C.sub.6-C.sub.10-arylamino has 6 to 10
carbon atoms. The term "heteroatom-unsubstituted C.sub.n-arylamino"
includes groups, having the structure --NHR, in which R is a
heteroatom-unsubstituted C.sub.n-aryl, as that term is defined
above. The term "heteroatom-substituted C.sub.n-arylamino" refers
to a radical, having a single nitrogen atom as the point of
attachment, further having a total of n carbon atoms, at least one
hydrogen atom, at least one additional heteroatoms, that is, in
addition to the nitrogen atom at the point of attachment, wherein
at least one of the carbon atoms is incorporated into one or more
aromatic ring structures, further wherein each additional
heteroatom is independently selected from the group consisting of
N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.6-C.sub.10-arylamino has 6 to 10
carbon atoms. The term "heteroatom-substituted C.sub.n-arylamino"
includes groups, having the structure --NHR, in which R is a
heteroatom-substituted C.sub.n-aryl, as that term is defined
above.
[0081] The term "aralkylamino" includes heteroatom-unsubstituted
aralkylamino, heteroatom-substituted aralkylamino,
heteroatom-unsubstituted C.sub.n-aralkylamino,
heteroatom-substituted C.sub.n-aralkylamino, heteroaralkylamino,
heterocyclic aralkylamino groups, and diaralkylamino groups. In
certain embodiments, lower aralkylaminos are contemplated. The term
"lower aralkylamino" refers to aralkylaminos of 7-12 carbon atoms
(that is, 7, 8, 9, 10, 11, or 12 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-aralkylamino" refers to a
radical, having a single nitrogen atom as the point of attachment,
further having one or two saturated carbon atoms attached to the
nitrogen atom, further having a total of n carbon atoms, wherein at
least 6 of the carbon atoms form an aromatic ring structure
containing only carbon atoms, 8 or more hydrogen atoms, a total of
one nitrogen atom, and no additional heteroatoms. For example, a
heteroatom-unsubstituted C.sub.7-C.sub.10-aralkylamino has 7 to 10
carbon atoms. The term "heteroatom-unsubstituted
C.sub.n-aralkylamino" includes groups, having the structure --NHR,
in which R is a heteroatom-unsubstituted C.sub.n-aralkyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-aralkylamino" refers to a radical, having a single nitrogen
atom as the point of attachment, further having at least one or two
saturated carbon atoms attached to the nitrogen atom, further
having a total of n carbon atoms, 0, 1, or more than one hydrogen
atom, at least one additional heteroatom, that is, in addition to
the nitrogen atom at the point of attachment, wherein at least one
of the carbon atom incorporated into an aromatic ring, further
wherein each heteroatom is independently selected from the group
consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.7-C.sub.10-aralkylamino has 7 to 10
carbon atoms. The term "heteroatom-substituted
C.sub.n-aralkylamino" includes groups, having the structure --NHR,
in which R is a heteroatom-substituted C.sub.n-aralkyl, as that
term is defined above.
[0082] The term "amido" includes straight-chain amido,
branched-chain amido, cycloamido, cyclic amido,
heteroatom-unsubstituted amido, heteroatom-substituted amido,
heteroatom-unsubstituted C.sub.n-amido, heteroatom-substituted
C.sub.n-amido, alkylcarbonylamino, arylcarbonylamino,
alkoxycarbonylamino, aryloxycarbonylamino, acylamino,
alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups.
The term "heteroatom-unsubstituted C.sub.n-amido" refers to a
radical, having a single nitrogen atom as the point of attachment,
further having a carbonyl group attached via its carbon atom to the
nitrogen atom, further having a linear or branched, cyclic or
acyclic structure, further having a total of n carbon atoms, 1 or
more hydrogen atoms, a total of one oxygen atom, a total of one
nitrogen atom, and no additional heteroatoms. For example, a
heteroatom-unsubstituted C.sub.1-C.sub.10-amido has 1 to 10 carbon
atoms. The term "heteroatom-unsubstituted C.sub.n-amido" includes
groups, having the structure --NHR, in which R is a
heteroatom-unsubstituted C.sub.n-acyl, as that term is defined
above. The group, --NHC(O)CH.sub.3, is a non-limiting example of a
heteroatom-unsubstituted amido group. The term
"heteroatom-substituted C.sub.n-amido" refers to a radical, having
a single nitrogen atom as the point of attachment, further having a
carbonyl group attached via its carbon atom to the nitrogen atom,
further having a linear or branched, cyclic or acyclic structure,
further having a total of n aromatic or nonaromatic carbon atoms,
0, 1, or more than one hydrogen atom, at least one additional
heteroatom in addition to the oxygen of the carbonyl group, wherein
each additional heteroatom is independently selected from the group
consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.1-C.sub.10-amido has 1 to 10 carbon
atoms. The term "heteroatom-substituted C.sub.n-amido" includes
groups, having the structure --NHR, in which R is a
heteroatom-unsubstituted C.sub.n-acyl, as that term is defined
above. The group, --NHCO.sub.2CH.sub.3, is a non-limiting example
of a heteroatom-substituted amido group.
[0083] The term "alkylthio" includes straight-chain alkylthio,
branched-chain alkylthio, cycloalkylthio, cyclic alkylthio,
heteroatom-unsubstituted alkylthio, heteroatom-substituted
alkylthio, heteroatom-unsubstituted C.sub.n-alkylthio, and
heteroatom-substituted C.sub.n-alkylthio. In certain embodiments,
lower alkylthios are contemplated. The term "lower alkylthio"
refers to alkylthios of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or
6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkylthio" refers to a group, having the structure --SR, in
which R is a heteroatom-unsubstituted C.sub.n-alkyl, as that term
is defined above. The group, --SCH.sub.3, is an example of a
heteroatom-unsubstituted alkylthio group. The term
"heteroatom-substituted C.sub.n-alkylthio" refers to a group,
having the structure --SR, in which R is a heteroatom-substituted
C.sub.n-alkyl, as that term is defined above.
[0084] The term "alkenylthio" includes straight-chain alkenylthio,
branched-chain alkenylthio, cycloalkenylthio, cyclic alkenylthio,
heteroatom-unsubstituted alkenylthio, heteroatom-substituted
alkenylthio, heteroatom-unsubstituted C.sub.n-alkenylthio, and
heteroatom-substituted C.sub.n-alkenylthio. In certain embodiments,
lower alkenylthios are contemplated. The term "lower alkenylthio"
refers to alkenylthios of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkenylthio" refers to a group, having the structure --SR,
in which R is a heteroatom-unsubstituted C.sub.n-alkenyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkenylthio" refers to a group, having the structure --SR,
in which R is a heteroatom-substituted C.sub.n-alkenyl, as that
term is defined above.
[0085] The term "alkynylthio" includes straight-chain alkynylthio,
branched-chain alkynylthio, cycloalkynylthio, cyclic alkynylthio,
heteroatom-unsubstituted alkynylthio, heteroatom-substituted
alkynylthio, heteroatom-unsubstituted C.sub.n-alkynylthio, and
heteroatom-substituted C.sub.n-alkynylthio. In certain embodiments,
lower alkynylthios are contemplated. The term "lower alkynylthio"
refers to alkynylthios of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkynylthio" refers to a group, having the structure --SR,
in which R is a heteroatom-unsubstituted C.sub.n-alkynyl, as that
term is defined above. The term "heteroatom-substituted
C.sub.n-alkynylthio" refers to a group, having the structure --SR,
in which R is a heteroatom-substituted C.sub.n-alkynyl, as that
term is defined above.
[0086] The term "arylthio" includes heteroatom-unsubstituted
arylthio, heteroatom-substituted arylthio, heteroatom-unsubstituted
C.sub.n-arylthio, heteroatom-substituted C.sub.n-arylthio,
heteroarylthio, and heterocyclic arylthio groups. The term
"heteroatom-unsubstituted C.sub.n-arylthio" refers to a group,
having the structure --SAr, in which Ar is a
heteroatom-unsubstituted C.sub.n-aryl, as that term is defined
above. The group, --SC.sub.6H.sub.5, is an example of a
heteroatom-unsubstituted arylthio group. The term
"heteroatom-substituted C.sub.n-arylthio" refers to a group, having
the structure --SAr, in which Ar is a heteroatom-substituted
C.sub.n-aryl, as that term is defined above.
[0087] The term "aralkylthio" includes heteroatom-unsubstituted
aralkylthio, heteroatom-substituted aralkylthio,
heteroatom-unsubstituted C.sub.n-aralkylthio,
heteroatom-substituted C.sub.n-aralkylthio, heteroaralkylthio, and
heterocyclic aralkylthio groups. In certain embodiments, lower
aralkylthios are contemplated. The term "lower aralkylthio" refers
to aralkylthios of 7-12 carbon atoms (that is, 7, 8, 9, 10, 11, or
12 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-aralkylthio" refers to a group, having the structure --SAr,
in which Ar is a heteroatom-unsubstituted C.sub.n-aralkyl, as that
term is defined above. The group, --SCH.sub.2C.sub.6H.sub.5, is an
example of a heteroatom-unsubstituted aralkyl group. The term
"heteroatom-substituted C.sub.n-aralkylthio" refers to a group,
having the structure --SAr, in which Ar is a heteroatom-substituted
C.sub.n-aralkyl, as that term is defined above.
[0088] The term "acylthio" includes straight-chain acylthio,
branched-chain acylthio, cycloacylthio, cyclic acylthio,
heteroatom-unsubstituted acylthio, heteroatom-substituted acylthio,
heteroatom-unsubstituted C.sub.n-acylthio, heteroatom-substituted
C.sub.n-acylthio, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. In
certain embodiments, lower acylthios are contemplated. The term
"lower acylthio" refers to acylthios of 1-6 carbon atoms (that is,
1, 2, 3, 4, 5 or 6 carbon atoms). The term
"heteroatom-unsubstituted C.sub.n-acylthio" refers to a group,
having the structure --SAc, in which Ac is a
heteroatom-unsubstituted C.sub.n-acyl, as that term is defined
above. The group, --SCOCH.sub.3, is an example of a
heteroatom-unsubstituted acylthio group. The term
"heteroatom-substituted C.sub.n-acylthio" refers to a group, having
the structure --SAc, in which Ac is a heteroatom-substituted
C.sub.n-acyl, as that term is defined above.
[0089] The term "alkylsilyl" includes straight-chain alkylsilyl,
branched-chain alkylsilyl, cycloalkylsilyl, cyclic alkylsilyl,
heteroatom-unsubstituted alkylsilyl, heteroatom-substituted
alkylsilyl, heteroatom-unsubstituted C.sub.n-alkylsilyl, and
heteroatom-substituted C.sub.n-alkylsilyl. In certain embodiments,
lower alkylsilyls are contemplated. The term "lower alkylsilyl"
refers to alkylsilyls of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5
or 6 carbon atoms). The term "heteroatom-unsubstituted
C.sub.n-alkylsilyl" refers to a radical, having a single silicon
atom as the point of attachment, further having one, two, or three
saturated carbon atoms attached to the silicon atom, further having
a linear or branched, cyclic or acyclic structure, containing a
total of n carbon atoms, all of which are nonaromatic, 5 or more
hydrogen atoms, a total of 1 silicon atom, and no additional
heteroatoms. For example, a heteroatom-unsubstituted
C.sub.1-C.sub.10-alkylsilyl has 1 to 10 carbon atoms. An alkylsilyl
group includes dialkylamino groups. The groups,
--Si(CH.sub.3).sub.3 and --Si(CH.sub.3).sub.2C(CH.sub.3).sub.3, are
non-limiting examples of heteroatom-unsubstituted alkylsilyl
groups. The term "heteroatom-substituted C.sub.n-alkylsilyl" refers
to a radical, having a single silicon atom as the point of
attachment, further having at least one, two, or three saturated
carbon atoms attached to the silicon atom, no carbon-carbon double
or triple bonds, further having a linear or branched, cyclic or
acyclic structure, further having a total of n carbon atoms, all of
which are nonaromatic, 0, 1, or more than one hydrogen atom, and at
least one additional heteroatom, that is, in addition to the
silicon atom at the point of attachment, wherein each additional
heteroatom is independently selected from the group consisting of
N, O, F, Cl, Br, I, Si, P, and S. For example, a
heteroatom-substituted C.sub.1-C.sub.10-alkylsilyl has 1 to 10
carbon atoms.
[0090] The term "phosphonate" includes straight-chain phosphonate,
branched-chain phosphonate, cyclophosphonate, cyclic phosphonate,
heteroatom-unsubstituted phosphonate, heteroatom-substituted
phosphonate, heteroatom-unsubstituted C.sub.nphosphonate, and
heteroatom-substituted C.sub.n-phosphonate. The term
"heteroatom-unsubstituted C.sub.n-phosphonate" refers to a radical,
having a single phosphorous atom as the point of attachment,
further having a linear or branched, cyclic or acyclic structure,
further having a total of n carbon atoms, 2 or more hydrogen atoms,
a total of three oxygen atom, and no additional heteroatoms. The
three oxygen atoms are directly attached to the phosphorous atom,
with one of these oxygen atoms doubly bonded to the phosphorous
atom. For example, a heteroatom-unsubstituted
C.sub.0-C.sub.10-phosphonate has 0 to 10 carbon atoms. The groups,
--P(O)(OH).sub.2, --P(O)(OH)OCH.sub.3, --P(O)(OH)OCH.sub.2CH.sub.3,
--P(O)(OCH.sub.3).sub.2, and --P(O)(OH)(OC.sub.6H.sub.5) are
non-limiting examples of heteroatom-unsubstituted phosphonate
groups. The term "heteroatom-substituted C.sub.n-phosphonate"
refers to a radical, having a single phosphorous atom as the point
of attachment, further having a linear or branched, cyclic or
acyclic structure, further having a total of n carbon atoms, 2 or
more hydrogen atoms, three or more oxygen atoms, three of which are
directly attached to the phosphorous atom, with one of these three
oxygen atoms doubly bonded to the phosphorous atom, and further
having at least one additional heteroatom in addition to the three
oxygen atoms, wherein each additional heteroatom is independently
selected from the group consisting of N, O, F, Cl, Br, I, Si, P,
and S. For example, a heteroatom-unsubstituted
C.sub.0-C.sub.10-phosphonate has 0 to 10 carbon atoms.
[0091] The term "phosphinate" includes straight-chain phosphinate,
branched-chain phosphinate, cyclophosphinate, cyclic phosphinate,
heteroatom-unsubstituted phosphinate, heteroatom-substituted
phosphinate, heteroatom-unsubstituted C.sub.n-phosphinate, and
heteroatom-substituted C.sub.n-phosphinate. The term
"heteroatom-unsubstituted C.sub.n-phosphinate" refers to a radical,
having a single phosphorous atom as the point of attachment,
further having a linear or branched, cyclic or acyclic structure,
further having a total of n carbon atoms, 2 or more hydrogen atoms,
a total of two oxygen atom, and no additional heteroatoms. The two
oxygen atoms are directly attached to the phosphorous atom, with
one of these oxygen atoms doubly bonded to the phosphorous atom.
For example, a heteroatom-unsubstituted
C.sub.0-C.sub.10-phosphinate has 0 to 10 carbon atoms. The groups,
--P(O)(OH)H, --P(O)(OH)CH.sub.3, --P(O)(OH)CH.sub.2CH.sub.3,
--P(O)(OCH.sub.3)CH.sub.3, and --P(O)(OC.sub.6H.sub.5)H are
non-limiting examples of heteroatom-unsubstituted phosphinate
groups. The term "heteroatom-substituted C.sub.n-phosphinate"
refers to a radical, having a single phosphorous atom as the point
of attachment, further having a linear or branched, cyclic or
acyclic structure, further having a total of n carbon atoms, 2 or
more hydrogen atoms, two or more oxygen atoms, two of which are
directly attached to the phosphorous atom, with one of these two
oxygen atoms doubly bonded to the phosphorous atom, and further
having at least one additional heteroatom in addition to the two
oxygen atoms, wherein each additional heteroatom is independently
selected from the group consisting of N, O, F, Cl, Br, I, Si, P,
and S. For example, a heteroatom-unsubstituted
C.sub.0-C.sub.10-phosphinate has 0 to 10 carbon atoms.
[0092] Modifications or derivatives of the compounds, agents, and
active ingredients disclosed throughout this specification are
contemplated as being useful with the methods and compositions of
the present invention. Derivatives may be prepared and the
properties of such derivatives may be assayed for their desired
properties by any method known to those of skill in the art.
[0093] In certain aspects, "derivative" refers to a chemically
modified compound that still retains the desired effects of the
compound prior to the chemical modification. "Isoxazole
derivatives," therefore, refers to a chemically modified compound
that still retains the desired effects of the parent isoxazole
prior to its chemical modification. Such effects may be enhanced
(e.g., slightly more effective, twice as effective, etc.) or
diminished (e.g., slightly less effective, 2-fold less effective,
etc.) relative to the parent isoxazole, but may still be considered
an isoxazole derivative. Such derivatives may have the addition,
removal, or substitution of one or more chemical moieties on the
parent molecule. Non-limiting examples of the types modifications
that can be made to the compounds and structures disclosed herein
include the addition or removal of lower unsubstituted alkyls such
as methyl, ethyl, propyl, or substituted lower alkyls such as
hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl
groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate,
sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate,
phosphono, phosphoryl groups, and halide substituents. Additional
modifications can include an addition or a deletion of one or more
atoms of the atomic framework, for example, substitution of an
ethyl by a propyl; substitution of a phenyl by a larger or smaller
aromatic group. Alternatively, in a cyclic or bicyclic structure,
heteroatoms such as N, S, or O can be substituted into the
structure instead of a carbon atom.
[0094] Prodrugs and solvates of the compounds of the present
invention are also contemplated herein. The term "prodrug" as used
herein, is understood as being a compound which, upon
administration to a subject, such as a mammal, undergoes chemical
conversion by metabolic or chemical processes to yield a compound
any of the formulas herein, or a salt and/or solvate thereof
(Bundgaard, 1991; Bundgaard, 1985). Solvates of the compounds of
the present invention are preferably hydrates.
[0095] The term "pharmaceutically acceptable salts," as used
herein, refers to salts of compounds of this invention that are
substantially non-toxic to living organisms. Typical
pharmaceutically acceptable salts include those salts prepared by
reaction of a compound of this invention with an inorganic or
organic acid, or an organic base, depending on the substituents
present on the compounds of the invention.
[0096] Non-limiting examples of inorganic acids which may be used
to prepare pharmaceutically acceptable salts include: hydrochloric
acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic
acid, phosphorous acid and the like. Examples of organic acids
which may be used to prepare pharmaceutically acceptable salts
include: aliphatic mono- and dicarboxylic acids, such as oxalic
acid, carbonic acid, citric acid, succinic acid,
phenyl-heteroatom-substituted alkanoic acids, aliphatic and
aromatic sulfuric acids and the like. Pharmaceutically acceptable
salts prepared from inorganic or organic acids thus include
hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate,
bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide,
hydrofluoride, acetate, propionate, formate, oxalate, citrate,
lactate, p-toluenesulfonate, methanesulfonate, maleate, and the
like.
[0097] Suitable pharmaceutically acceptable salts may also be
formed by reacting the agents of the invention with an organic base
such as methylamine, ethylamine, ethanolamine, lysine, ornithine
and the like.
[0098] Pharmaceutically acceptable salts include the salts formed
between carboxylate or sulfonate groups found on some of the
compounds of this invention and inorganic cations, such as sodium,
potassium, ammonium, or calcium, or such organic cations as
isopropylammonium, trimethylammonium, tetramethylammonium, and
imidazolium.
[0099] 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, Selection and Use (P. H. Stahl
& C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002),
which is incorporated herein by reference.
[0100] As used herein, the term "cyclic group" refers to a
carbocycle group (e.g., cyclopropyl, cyclohexyl), a heterocycle
group (e.g., pyrrolidinyl), an aryl group, or any combination
thereof (e.g., fused bicyclic group).
[0101] As used herein, "protecting group" refers to a moiety
attached to a functional group to prevent an otherwise unwanted
reaction of that functional group. Protecting groups are well-known
to those of skill in the art. Non-limiting exemplary protecting
groups fall into categories such as hydroxy protecting groups,
amino protecting groups, sulfhydryl protecting groups and carbonyl
protecting groups. Such protecting groups may be found in Greene
and Wuts, 1999. Compounds of the present invention are specifically
contemplated wherein one or more functional groups are protected by
a protecting group.
[0102] Compounds of the present invention may contain one or more
asymmetric centers and thus can occur as racemates and racemic
mixtures, single enantiomers, diastereomeric mixtures and
individual diastereomers. In certain embodiments, a single
diastereomer is present. All possible stereoisomers of the
compounds of the present invention are contemplated as being within
the scope of the present invention. However, in certain aspects,
particular diastereomers are contemplated. The chiral centers of
the compounds of the present invention can have the S- or the
R-configuration, as defined by the IUPAC 1974 Recommendations. In
certain aspects, certain compounds of the present invention may
comprise S- or R-configurations at particular carbon centers.
[0103] Solvent choices for the synthetic preparation of compounds
of the present invention will be known to one of ordinary skill in
the art. Solvent choices may depend, for example, on which one(s)
will facilitate the solubilizing of all the reagents or, for
example, which one(s) will best facilitate the desired reaction
(particularly when the mechanism of the reaction is known).
Solvents may include, for example, polar solvents and non-polar
solvents. Solvents choices include, but are not limited to,
tetrahydrofuran, dimethylformamide, dimethylsulfoxide, dioxane,
methanol, ethanol, hexane, methylene chloride and acetonitrile.
More than one solvent may be chosen for any particular reaction or
purification procedure. Water may also be admixed into any solvent
choice. Further, water, such as distilled water, may constitute the
reaction medium instead of a solvent.
[0104] Persons of ordinary skill in the art will be familiar with
methods of purifying compounds of the present invention. One of
ordinary skill in the art will understand that compounds of the
present invention can generally be purified at any step, including
the purification of intermediates as well as purification of the
final products. In preferred embodiments, purification is performed
via silica gel column chromatography or HPLC.
[0105] In view of the above definitions, other chemical terms used
throughout this application can be easily understood by those of
skill in the art. Terms may be used alone or in any combination
thereof. The preferred and more preferred chain lengths of the
radicals apply to all such combination.
D. Stem Cells
[0106] 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. Research in the human stem cell field grew
out of findings by Canadian scientists Ernest A. McCulloch and
James E. Till in the 1960s.
[0107] The three broad categories of mammalian stem cells are:
embryonic stem cells, derived from blastocysts, and adult stem
cells, which are found in adult tissues. 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. In
particular embodiments, neural stem cells isolated from the adult
mammalian brain and embryonic stem cells are contemplated by the
present invention. Human bone marrow stromal cells are also
contemplated, in certain embodiments.
[0108] 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, their use in
medical therapies has been proposed. In particular, embryonic cell
lines, autologous embryonic stem cells generated through
therapeutic cloning, and highly plastic adult stem cells from
mature organs of the body.
[0109] "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 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.). Unipotent cells can
produce only one cell type, but have the property of self-renewal
which distinguishes them from non-stem cells.
[0110] 1. Embryonic Stem Cells
[0111] Embryonic stem cell lines (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 consisting 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.
[0112] Nearly all research to date has taken place using mouse
embryonic stem cells (mES) or human embryonic stem cells (hES).
Both have the essential stem cell characteristics, yet they require
very different environments in order to maintain an
undifferentiated state. Mouse ES cells are grown on a layer of
gelatin and require the presence of Leukemia Inhibitory Factor
(LIF). Human ES cells are grown on a feeder layer of mouse
embryonic fibroblasts (MEF's) and require the presence of basic
Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture
conditions or genetic manipulation embryonic stem cells will
rapidly differentiate.
[0113] A human embryonic stem cell is also 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. The molecular definition of a stem cell
includes many more proteins and continues to be a topic of
research.
[0114] After 20 years of research, there are no approved treatments
or human trials using embryonic stem cells. Their tendency to
produce tumors and malignant carcinomas, cause transplant
rejection, and form the wrong kinds of cells are just a few of the
hurdles that embryonic stem cell researchers still face. Many
nations currently have moratoria on either ES cell research or the
production of new ES cell lines. Because of their combined
abilities of unlimited expansion and pluripotency, embryonic stem
cells remain a theoretically potential source for regenerative
medicine and tissue replacement after injury or disease.
[0115] The present invention contemplates the use of embryonic stem
cells. Table 1 lists cellular markers that can be used to identify
and separate embryonic stem cells for use in accordance with the
present invention.
TABLE-US-00001 TABLE 1 Pluripotent Stem Cell Markers Cell Marker
Cell Type Significance Alkaline Phosphatase Embryonic Elevated
expression of stem (ES), this enzyme is embryonal associated with
carcinoma (EC) undifferentiated pluripotent stem cell (PSC)
Alpha-fetoprotein (AFP) Endoderm Protein expressed during
development of primitive endoderm; reflects endodermal
differentiation Pluripotent Stem Cells Bone morphogenetic Mesoderm
Growth and protein-4 differentiation factor expressed during early
mesoderm formation and differentiation Brachyury Mesoderm
Transcription factor important in earliest phases of mesoderm
formation and differentiation; used as the earliest indicator of
mesoderm formation Cluster designation 30 ES, EC Surface receptor
(CD30) molecule found specifically on PSC Cripto (TDGF-1) ES,
cardiomyocyte Gene for growth factor expressed by ES cells,
primitive ectoderm, and developing cardiomycyte GATA-4 gene
Endoderm Expression increases as ES differentiates into endoderm
GCTM-2 ES, EC Antibody to a specific extracellular-matrix molecule
that is synthesized by undifferentiated PSCs Genesis ES, EC
Transcription factor uniquely expressed by ES cells either in or
during the undifferentiated state of PSCs Germ cell nuclear factor
ES, EC Transcription factor expressed by PSCs Hepatocyte nuclear
factor- Endoderm Transcription factor 4 (HNF-4) expressed early in
endoderm formation Nestin Ectoderm, Intermediate filaments neural
and within cells; characteristic of pancreatic primitive
neuroectoderm progenitor formation Neuronal cell-adhesion Ectoderm
Cell-surface molecule molecule (N-CAM) that promotes cell-cell
interaction; indicates primitive neuroectoderm formation Oct-4 ES,
EC Transcription factor unique to PSCs; essential for establishment
and maintenance of undifferentiated PSCs Pax6 Ectoderm
Transcription factor expressed as ES cell differentiates into
neuropithelium Stage-specific embryonic ES, EC Glycoprotein
antigen-3 (SSEA-3) specifically expressed in early embryonic
development and by undifferentiated PSCs Stage-specific embryonic
ES, EC Glycoprotein antigen-4 (SSEA-4) spcecifically expressed in
early embryonic development and by undifferentiated PSCs Stem cell
factor (SCF or c- ES, EC, Membrane protein that Kit ligand) HSC,
MSC enhances proliferation of ES and EC cells, hematopoietic stem
cell (HSCs), and mesenchymal stem cells (MSCS); binds the receptor
c-Kit Telomerase ES, EC An enzyme uniquely associated with immortal
cell lines; useful for identifying undifferentiated PSCs TRA-1-60
ES, EC Antibody to a specific extracellular matrix molecule is
synthesized by undifferentiated PSCs TRA-1-81 ES, EC Antibody to a
specific extracellular matrix molecule normally synthesized by
undifferentiated PSCs Vimentin Ectoderm, Intermediate filaments
neural and within cells; pancreatic characteristic of progenitor
primitive neuroectoderm formation
[0116] 2. Adult Stem Cells
[0117] Adult stem cells, a cell which is found in a developed
organism, have two properties: the ability to divide and create
another cell like itself, and also divide and create a cell more
differentiated than itself. 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) and are generally referred to by
their tissue origin (mesenchymal stem cell, adipose-derived stem
cell, endothelial stem cell, etc.). A great deal of adult stem cell
research has focused on clarifying their capacity to divide or
self-renew indefinitely and their differentiation potential.
[0118] While embryonic stem cell potential remains untested, adult
stem cell treatments have been used for many years to successfully
treat leukemia and related bone/blood cancers through bone marrow
transplants. The use of adult stem cells in research and therapy is
not as controversial as embryonic stem cells, because the
production of adult stem cells does not require the destruction of
an embryo. Consequently, more US government funding is being
provided for adult stem cell research.
[0119] The present invention contemplates, in particular
embodiments, neural stem/progenitor cells that act as precusor
cells for the mature cells of the central nervous system (e.g.,
neurons, oligodendrocytes, and astrocytes).
[0120] i. Cardiogenic Stem Cells
[0121] Evidence for potential stem cell-based therapies for heart
disease has been provided by studies showing that human adult stem
cells, taken from the bone marrow, are capable of giving rise to
vascular endothelial cells when transplanted into rats. Such stem
cells demonstrated plasticity, meaning that they become cell types
that they would not normally be. The cells were used to form new
blood vessels in the damaged area of the rats' hearts and to
encourage proliferation of preexisting vasculature following the
experimental heart attack.
[0122] Like the mouse stem cells, human hematopoietic stem cells
can be induced under the appropriate culture conditions to
differentiate into numerous tissue types, including cardiac muscle.
When injected into the bloodstream leading to the damaged rat
heart, these cells prevented the death of hypertrophied or
thickened but otherwise viable myocardial cells and reduced
progressive formation of collagen fibers and scars. Furthermore,
hematopoietic cells can be identified on the basis of highly
specific cell markers that differentiate them from cardiomyocyte
precursor cells, enabling such cells to be used alone or in
conjunction with myocyte-regeneration strategies or pharmacological
therapies.
[0123] Table 2, below, lists cell surface markers that can be used
to identify cardiogenic stem cells. In particular, Flk1.sup.+ cells
are contemplated.
TABLE-US-00002 TABLE 2 Cardiac Progenitor Markers Cell Marker Cell
Type Significance MyoD and Pax7 Myoblast, myocyte Transcription
factors that direct differentiation of myoblasts into mature
myocytes Myogenin and MR4 Skeletal myocyte Secondary transcription
factors required for differentiation of myoblasts from muscle stem
cells Myosin heavy chain Cardiomyocyte A component of structural
and contractile protein found in cardiomyocyte Myosin light chain
Skeletal myocyte A component of structural and contractile protein
found in skeletal myocyte
[0124] ii. Neural Stem Cells
[0125] The adult mammalian central nervous system (CNS) is composed
primarily of three differentiated cell types--neurons, astrocytes
and oligodendrocytes. Astrocytes and oligodendrocytes provide a
critical supporting role for neuronal function. Neurons responsible
for forming connections and are the communicating cells of the
nervous system. The limits on adult mammals' ability to replace
non-functional CNS tissue makes CNS death due to injury or disease
devastating. Thus, research on brain repair has traditionally
focused on keeping neurons alive following injury and promoting
their ability to extend processes and establish functional cell
connections. This focus was based on the belief that the adult
mammalian CNS was incapable of generating new brain cells. However,
the early 1990's brought the discovery of stem cells existed in the
embryonic and adult CNS, opening up the way for research on the use
of these cells in neuronal therapies and CNS tissue repair.
[0126] Neural stem cells have been isolated from nearly all regions
of the embryonic mouse CNS, including the septum, cortex, thalamus,
ventral mesencephalon and spinal cord. Cells from all these CNS
regions exhibit the same general features--extensive proliferative
ability, self-renewal and differentiation of the progeny into
neurons, astrocytes and oligodendrocytes. In the adult mouse,
neural stem cells appear to be located primarily in the
sub-ventricular zone (SVZ) of the forebrain and in the sub-granular
layer zone (SGZ) of the dentate gyrus of the hippocampal formation.
A recent study indicates the cells from the SGZ may have a more
limited proliferative potential, and that the hippocampal stem
cells lie dorsal to the hippocampus in a collapsed ventricle.
[0127] While the role of neural stem cells in vivo is poorly
understood, they do appear to exhibit properties similar to other
stem cells. In the sub-ventricular region, for example, stem cells
can be induced to proliferate and to repopulate the sub-ventricular
zone following irradiation. Stem cell-derived subependymal
progenitor cells are the source of new neurons in the olfactory
bulb of rodents and in the association cortex of nonhuman primates
under normal conditions. And recently, it has been shown that stem
cell progeny in the hippocampal region are able to compensate for
behavioral deficits following ischemic injury in rodents.
[0128] Neural stem/progenitor cells are normally grown in
serum-free medium (N2/B27 or N2 supplement in DMEM:F12) and
supplemented with growth factors, such as fibroblast growth factor
and/or epidermal growth factor. Under these conditions, stem cells
remain undifferentiated and do not give rise to neurons. To induce
neuronal differentiation, cells may be trypsinized (to dissociate
into single cells) and re-plated in serum-free medium containing
"differentiating agents," such as isoxazole-derived small-molecules
described herein. Within 24 hours, cells will undergo a rapid and
vast morphological change, flattening and extension of
neuronal-like processes. By 4 days, there is increased neuronal
gene expression by RT-PCR (for mRNA), protein blotting (for
protein), and staining for neuronal markers (by
immunohistochemistry). Under certain conditions when stem cells are
co-cultured with astrocytes, the stem cell can differentiate into
mature neuronal cells that fire action potentials.
[0129] Table 3, below, lists various cell markers for neural stem
cells.
TABLE-US-00003 TABLE 3 Neurogenic Progenitor Markers Cell Marker
Cell Type Significance CD133 Neural stem cell, Cell-surface protein
that identifies HSC neural stem cells, which give rise to neurons
and glial cells Glial fibrillary acidic Astrocyte Protein
specifically produced by protein (GFAP) astrocyte Microtubule-
Neuron Dendrite-specific MAP; protein associated protein-2 found
specifically in dendritic (MAP-2) branching of neuron Myelin basic
protein Oligodendrocyte Protein produced by mature (MPB)
oligodendrocytes; located in the myelin sheath surrounding neuronal
structures Nestin Neural progenitor Intermediate filament
structural protein expressed in primitive neural tissue Neural
tubulin Neuron Important structural protein for neuron; identifies
differentiated neuron Neurofilament (NF) Neuron Important
structural protein for neuron; identifies differentiated neuron
Neuroshpere Embryoid body Cluster of primitive neural cells in
(EB), ES culture of differentiating ES cells; indicates presence of
early neurons and glia Noggin Neuron A neuron-specific gene
expressed during the development of neurons O4 Oligodendrocyte
Cell-surface marker on immature, developing oligodendrocyte O1
Oligodendrocyte Cell-surface marker that characterizes mature
oligodendrocyte Synaptophysin Neuron Neuronal protein located in
synapses; indicates connections between neurons Tau Neuron Type of
MAP; helps maintain structure of the axon
E. Detection of Cell Surface Markers
[0130] In accordance with the present invention, one will seek to
obtain various stem cell populations by screening of cell
populations for appropriate cell surface markers, as discussed
above. Generally, this is performed by labeling or physically
selecting cells that are bound by antibodies to cell determinants
that identify the cells as stem, pluripotent or totipotent stem
cells. It is particularly contemplated that antibodies will be of
particular use in the various cell separation techniques described
below.
[0131] 1. Antibody Constructs
[0132] Antibodies directed against the various cell surface
antigens are readily available from commercial sources. While
available from commercial sources, it is also contemplated that
monoclonal or polyclonal antibodies for use in the context of the
invention may be constructed by a person of ordinary skill
[0133] As used herein, the term "antibody" is intended to refer
broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD
and IgE. Generally, IgG and/or IgM are preferred because they are
the most common antibodies in the physiological situation and
because they are most easily made in a laboratory setting.
[0134] The term "antibody" is used to refer to any antibody-like
molecule that has an antigen binding region, and includes antibody
fragments such as Fab', Fab, F(ab')2, single domain antibodies
(DABs), Fv, scFv (single chain Fv), and the like. The techniques
for preparing and using various antibody-based constructs and
fragments are well known in the art. Means for preparing and
characterizing antibodies are also well known in the art (see,
e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988; incorporated herein by reference).
[0135] Monoclonal antibodies (MAbs) are recognized to have certain
advantages, e.g., reproducibility and large-scale production, and
their use is generally preferred. The invention thus provides
monoclonal antibodies of the human, murine, monkey, rat, hamster,
rabbit and even chicken origin. Due to the ease of preparation and
ready availability of reagents, murine monoclonal antibodies will
often be preferred.
[0136] 2. Antibody Conjugates
[0137] The instant invention provides for the use of antibodies
against various cell surface antigens which are generally of the
monoclonal type, and that may be linked to at least one agent to
form an antibody conjugate. It is conventional to link or
covalently bind or complex at least one desired molecule or moiety.
Such a molecule or moiety may be, but is not limited to a reporter
molecule. A reporter molecule is defined as any moiety which may be
detected using an assay. Non-limiting examples of reporter
molecules which have been conjugated to antibodies include enzymes,
radiolabels, haptens, fluorescent labels, phosphorescent molecules,
chemiluminescent molecules, chromophores, luminescent molecules,
photoaffinity molecules, colored particles or ligands, such as
biotin.
[0138] Any antibody of sufficient selectivity, specificity or
affinity may be employed as the basis for an antibody conjugate.
Such properties may be evaluated using conventional immunological
screening methodology known to those of skill in the art. Sites for
binding to biological active molecules in the antibody molecule, in
addition to the canonical antigen binding sites, include sites that
reside in the variable domain that can bind pathogens, B-cell
superantigens, the T cell co-receptor CD4 and the HIV-1 envelope
(Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995;
Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993;
Kreier et al., 1991). In addition, the variable domain is involved
in antibody self-binding (Kang et al., 1988), and contains epitopes
(idiotopes) recognized by anti-antibodies (Kohler et al.,
1989).
[0139] Certain examples of antibody conjugates are those conjugates
in which the antibody is linked to a detectable label. "Detectable
labels" are compounds and/or elements that can be detected due to
their specific functional properties, and/or chemical
characteristics, the use of which allows the antibody to which they
are attached to be detected, and/or further quantified if
desired.
[0140] Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies (see, e.g., U.S. Pat.
Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein
by reference). The imaging moieties used can be paramagnetic ions;
radioactive isotopes; fluorochromes; NMR-detectable substances;
X-ray imaging.
[0141] In the case of paramagnetic ions, one might employ, by way
of example, ions such as chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium
(III), samarium (III), ytterbium (III), gadolinium (III), vanadium
(II), terbium (III), dysprosium (III), holmium (III) and/or erbium
(III), with gadolinium being particularly preferred. Ions useful in
other contexts, such as X-ray imaging, include but are not limited
to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0142] In the case of radioactive isotopes for therapeutic and/or
diagnostic application, one might employ, for example,
.sup.211astatine, .sup.14carbon, .sup.51chromium, .sup.36chlorine,
.sup.57cobalt, .sup.58cobalt, copper.sup.67, .sup.152Eu,
gallium.sup.67, .sup.3hydrogen, iodine.sup.123, iodine.sup.125,
iodine.sup.131, indium.sup.111, .sup.59iron, .sup.32phosphorus,
rhenium.sup.186, rhenium.sup.188, .sup.75selenium, .sup.35sulphur,
.sup.99mtechnicium and/or .sup.90yttrium. .sup.125I is often being
preferred for use in certain embodiments, and .sup.99mtechnicium
and/or indium.sup.111 are also often preferred due to their low
energy and suitability for long range detection. Radioactively
labeled monoclonal antibodies of the present invention may be
produced according to well-known methods in the art. For instance,
monoclonal antibodies can be iodinated by contact with sodium
and/or potassium iodide and a chemical oxidizing agent such as
sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Monoclonal antibodies according to the invention
may be labeled with technetium.sup.99m by ligand exchange process,
for example, by reducing pertechnate with stannous solution,
chelating the reduced technetium onto a Sephadex column and
applying the antibody to this column. Alternatively, direct
labeling techniques may be used, e.g., by incubating pertechnate, a
reducing agent such as SNCl.sub.2, a buffer solution such as
sodium-potassium phthalate solution, and the antibody. Intermediary
functional groups which are often used to bind radioisotopes which
exist as metallic ions to antibody are
diethylenetriaminepentaacetic acid (DTPA) or ethylene
diaminetetracetic acid (EDTA).
[0143] Among the fluorescent labels contemplated for use as
conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,
Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX,
6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin,
ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
[0144] Another type of antibody conjugate contemplated in the
present invention are those intended primarily for use in vitro,
where the antibody is linked to a secondary binding ligand and/or
to an enzyme (an enzyme tag) that will generate a colored product
upon contact with a chromogenic substrate. Examples of suitable
enzymes include urease, alkaline phosphatase, (horseradish)
hydrogen peroxidase or glucose oxidase. Preferred secondary binding
ligands are biotin and/or avidin and streptavidin compounds. The
use of such labels is well known to those of skill in the art and
are described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each
incorporated herein by reference.
[0145] 3. Methods of Conjugation
[0146] If desired, the compound of interest may be joined to an
antibody via a biologically-releasable bond, such as a
selectively-cleavable linker or amino acid sequence. Certain
linkers will generally be preferred over other linkers, based on
differing pharmacologic characteristics and capabilities. For
example, linkers that contain a disulfide bond that is sterically
"hindered" are to be preferred, due to their greater stability in
vivo, thus preventing release of the moiety prior to binding at the
site of action.
[0147] Additionally, any other linking/coupling agents and/or
mechanisms known to those of skill in the art can be used to
combine to components or agents with antibodies of the present
invention, such as, for example, avidin biotin linkages, amide
linkages, ester linkages, thioester linkages, ether linkages,
thioether linkages, phosphoester linkages, phosphoramide linkages,
anhydride linkages, disulfide linkages, ionic and hydrophobic
interactions, or combinations thereof.
[0148] Cross-linking reagents are used to form molecular bridges
that tie together functional groups of two different molecules,
e.g., a stablizing and coagulating agent. However, it is
contemplated that dimers or multimers of the same analog can be
made or that heteromeric complexes comprised of different analogs
can be created. To link two different compounds in a step-wise
manner, hetero-bifunctional cross-linkers can be used that
eliminate unwanted homopolymer formation.
[0149] U.S. Pat. No. 4,680,338, describes bifunctional linkers
useful for producing conjugates of ligands with amine-containing
polymers and/or proteins, especially for forming antibody
conjugates with chelators, drugs, enzymes, detectable labels and
the like. U.S. Pat. Nos. 5,141,648 and 5,563,250 disclose cleavable
conjugates containing a labile bond that is cleavable under a
variety of mild conditions. This linker is particularly useful in
that the agent of interest may be bonded directly to the linker,
with cleavage resulting in release of the active agent. Preferred
uses include adding a free amino or free sulfhydryl group to a
protein, such as an antibody, or a drug.
[0150] U.S. Pat. No. 5,856,456 provides peptide linkers for use in
connecting polypeptide constituents to make fusion proteins, e.g.,
single-chain antibodies. The linker is up to about 50 amino acids
in length, contains at least one occurrence of a charged amino acid
(preferably arginine or lysine) followed by a proline, and is
characterized by greater stability and reduced aggregation. U.S.
Pat. No. 5,880,270 discloses aminooxy-containing linkers useful in
a variety of immunodiagnostic and separative techniques.
[0151] Molecules containing azido groups may also be used to form
covalent bonds to proteins through reactive nitrene intermediates
that are generated by low intensity ultraviolet light (Potter &
Haley, 1983). In particular, 2- and 8-azido analogues of purine
nucleotides have been used as site-directed photoprobes to identify
nucleotide binding proteins in crude cell extracts (Owens &
Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides
have also been used to map nucleotide binding domains of purified
proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et
al., 1989) and may be used as antibody binding agents.
[0152] Several methods are known in the art for the attachment or
conjugation of an antibody to its conjugate moiety. Some attachment
methods involve the use of a metal chelate complex employing, for
example, an organic chelating agent such a
diethylenetriaminepentaacetic acid anhydride (DTPA);
ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;
and/or tetrachloro-3.alpha.-6.alpha.-diphenylglycouril-3 attached
to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each
incorporated herein by reference). Monoclonal antibodies may also
be reacted with an enzyme in the presence of a coupling agent such
as glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared in the presence of these coupling agents or by
reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948,
imaging of breast tumors is achieved using monoclonal antibodies
and the detectable imaging moieties are bound to the antibody using
linkers such as methyl-p-hydroxybenzimidate or
N-succinimidyl-3-(4-hydroxyphenyl)propionate.
[0153] In other embodiments, derivatization of immunoglobulins by
selectively introducing sulfhydryl groups in the Fc region of an
immunoglobulin, using reaction conditions that do not alter the
antibody combining site are contemplated. Antibody conjugates
produced according to this methodology are disclosed to exhibit
improved longevity, specificity and sensitivity (U.S. Pat. No.
5,196,066, incorporated herein by reference). Site-specific
attachment of effector or reporter molecules, wherein the reporter
or effector molecule is conjugated to a carbohydrate residue in the
Fc region have also been disclosed in the literature (O'Shannessy
et al., 1987). This approach has been reported to produce
diagnostically and therapeutically promising antibodies which are
currently in clinical evaluation.
F. Cell Separation Techniques
[0154] Methods of separating cell populations and cellular subsets
are well known in the art and may be applied to the cell
populations of the present invention. Cells purified in this
fashion may then be used for stimulation and cell replacement
therapy, such as in tissue regeneration purposes. Embryonic stem
cells and neural stem cells, as well as stem cells for endothelial,
cardiac and other cell types are believed by the inventors to be
useful in accordance with the present invention. Stimulating those
stem cells from a quiescent condition with compounds of the present
invention should promote differentiation. They may also be treated
with particular combinations with previously known growth and
differentiation factors and then cultured to expand and/or
differentiate. The following description sets forth exemplary
methods of separation for stem cells based upon the surface
expression of various markers.
[0155] 1. Fluorescence Activated Cell Sorting (FACS)
[0156] FACS facilitates the quantitation and/or separation of
subpopulations of cells based upon surface markers. Cells to be
sorted are first tagged with a fluorescently labeled antibody or
other marker specific ligand. Generally, labeled antibodies and
ligands are specific for the expression of a phenotype specific
cell surface molecule. The labeled cells are then passed through a
laser beam and the fluorescence intensity of each cell determined.
The sorter distributes the positive and negative cells into
label-plus and label-minus wells at a flow rate of approximately
3000 cells per second.
[0157] The use of multiple fluorescent tags exciting at different
wavelengths allows for sorting based upon multiple or alternate
criteria. Among the fluorescent labels contemplated for use as
conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,
Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX,
6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin,
ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red. Thus, for
example, a single PBMC sample may be analyzed with alternatively
labeled anti-Ig antibody, anti-CD3 antibody, anti-CD8 antibody and
anti-CD4 antibody to screen for the presence of B cells and T cells
within the sample, as well as distinguishing specific T cell
subsets.
[0158] FACS analysis and cell sorting is carried out on a flow
cytometer. A flow cytometer generally consists of a light source,
normally a laser, collection optics, electronics and a computer to
translate signals to data. Scattered and emitted fluorescent light
is collected by two lenses (one positioned in front of the light
source and one set at right angles) and by a series of optics, beam
splitters and filters, which allow for specific bands of
fluorescence to be measured.
[0159] Flow cytometer apparatus permit quantitative multiparameter
analysis of cellular properties at rates of several thousand cells
per second. These instruments provide the ability to differentiate
among cell types. Data are often displayed in one-dimensional
(histogram) or two-dimensional (contour plot, scatter plot)
frequency distributions of measured variables. The partitioning of
multiparameter data files involves consecutive use of the
interactive one- or two-dimensional graphics programs.
[0160] Quantitative analysis of multiparameter flow cytometric data
for rapid cell detection consists of two stages: cell class
characterization and sample processing. In general, the process of
cell class characterization partitions the cell feature into cells
of interest and not of interest. Then, in sample processing, each
cell is classified in one of the two categories according to the
region in which it falls. Analysis of the class of cells is very
important, as high detection performance may be expected only if an
appropriate characteristic of the cells is obtained.
[0161] Not only is cell analysis performed by flow cytometry, but
so too is sorting of cells. In U.S. Pat. No. 3,826,364, an
apparatus is disclosed which physically separates particles, such
as functionally different cell types. In this machine, a laser
provides illumination which is focused on the stream of particles
by a suitable lens or lens system so that there is highly localized
scatter from the particles therein. In addition, high intensity
source illumination is directed onto the stream of particles for
the excitation of fluorescent particles in the stream. Certain
particles in the stream may be selectively charged and then
separated by deflecting them into designated receptacles. A classic
form of this separation is via fluorescent tagged antibodies, which
are used to mark one or more cell types for separation.
[0162] Additional and alternate methods for performing flow
cytometry and fluorescent antibody cell sorting are described in
U.S. Pat. Nos. 4,284,412; 4,989,977; 4,498,766; 5,478,722;
4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; and
4,661,913, herein expressly incorporated by reference.
[0163] 2. Micro-Bead Separation
[0164] Cells in suspension may be separated to very high purity
according to their surface antigens using micro-bead technologies.
The basic concept in micro-bead separations is to selectively bind
the biomaterial of interest (e.g., a specific cell, protein, or DNA
sequence) to a particle and then separate it from its surrounding
matrix. Micro-bead separation involves contacting a cell suspension
with a slurry of microbeads labeled with a cell surface specific
antibody or ligand. Cells labeled with the micro-beads are then
separated using an affinity capture method specific for some
property of the beads. This format facilitates both positive and
negative selection.
[0165] Magnetic beads are uniform, superparamagnetic beads
generally coated with an affinity tag such as recombinant
streptavidin that will bind biotinylated immunoglobulins, or other
biotinylated molecules such as, for example, peptides/proteins or
lectins. Magnetic beads are generally uniform micro- or
nanoparticles of Fe.sub.3O.sub.4. These particles are
superparamagnetic, meaning that they are attracted to a magnetic
field but retain no residual magnetism after the field is removed.
Suspended superparamagnetic particles tagged to a cell of interest
can be removed from a matrix using a magnetic field, but they do
not agglomerate (i.e., they stay suspended) after removal of the
field.
[0166] A common format for separations involving superparamagnetic
nanoparticles is to disperse the beads within the pores of larger
microparticles. These microparticles are coated with a monoclonal
antibody for a cell-surface antigen. The antibody-tagged,
superparamagnetic microparticles are then introduced into a
cellular suspension. The particles bind to cells expressing the
surface antigen of interest and maybe separated out with the
application of a magnetic field. This may be facilitated by running
the suspension over a high gradient magnetic separation column
placed in a strong magnetic field. The magnetically labeled cells
are retained in the column while non-labeled cells pass through.
When the column is removed from the magnetic field, the
magnetically retained cells are eluted. Both, labeled and
non-labeled fractions can be completely recovered.
[0167] 3. Affinity Chromatography
[0168] Affinity Chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule that it can specifically bind to. This is a
receptor-ligand type interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(alter pH, ionic strength, temperature, etc.).
[0169] The matrix should be a substance that itself does not adsorb
molecules to any significant extent and that has a broad range of
chemical, physical and thermal stability. The ligand should be
coupled in such a way as to not affect its binding properties. The
ligand should also provide relatively tight binding. And it should
be possible to elute the substance without destroying the sample or
the ligand. One of the most common forms of affinity chromatography
is immunoaffinity chromatography. The generation of antibodies that
would be suitable for use in accord with the present invention is
discussed elsewhere in this document.
G. Stem/Progenitor/Differentiated Cell Culture
[0170] Cell culture facilitates the maintenance and propagation of
cells 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.
Some sophisticated techniques utilize entire layers of adherent
cells, feeder cells, which are used to support the growth of cells
with more demanding growth requirements.
[0171] 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.
[0172] As a rule, cells grown in vitro do not organize themselves
into tissues. Instead, cultured cells grow as monolayers (or in
some instances as multilayers) on the surface of tissue culture
dishes. The cells usually multiply until they come into contact
with each other to form a monolayer and stop growing when they come
into contact with each other due to contact inhibition.
[0173] Anchorage-dependent cells show the phenomenon of adherence,
i.e., they grow and multiply only if attached to the inert surface
of a culture dish or another suitable support. Such cells cannot
normally be grown without a solid support. Many cells do not
require this solid surface and show a phenomenon known as
Anchorage-independent growth. Accordingly, one variant of growing
these cells in culture is the use of Spinner cultures or suspension
cultures in which single cells float freely in the medium and are
maintained in suspension by constant stifling or agitation. This
technique is particularly useful for growing large amounts of cells
in batch cultures.
[0174] Anchorage-independent cells are usually capable of forming
colonies in semisolid media. Some techniques have been developed
that can be used also to grow anchorage-dependent cells in spinner
cultures. They make use of microscopically small positively-charged
dextran beads to which these cells can attach.
[0175] The starting material for the establishment of a cell
culture typically is tissue from a suitable donor obtained under
sterile conditions. The tissues may be minced and treated with
proteolytic enzymes such as trypsin, collagenase of dispase to
obtain a single cell suspension that can be used to inoculate a
culture dish. In some cases dispersion of tissue is also
effectively achieved by treatment with buffers containing EDTA. A
particular form of initiating a cell culture is the use of tiny
pieces of tissues from which cells may grow out in vitro.
[0176] Primary cell cultures maintained for several passages may
undergo a crisis. Ascrisis is usually associated with alterations
of the properties of the cells and may proceed quickly or extend
over many passages. Loss of contact inhibition is frequently an
indication of cells having lost their normal characteristics. These
cells then grow as multilayers in tissue culture dishes. The most
pronounced feature of abnormal cells is the alteration in
chromosome numbers, with many cells surviving this process being
aneuploid. The switch to abnormal chromosome numbers is usually
referred to as cell transformation and this process may give rise
to cells that can then be cultivated for indefinite periods of time
by serial passaging. Transformed cells give rise to continuous cell
lines.
[0177] In certain aspects of the instant invention, cells are
cultured prior to contact with differentiating agents. They may
also be cultured after contact, i.e., after they have been induced
to differentiate toward a given or specific phenotype. Cells will
be cultured under specified conditions to achieve particular types
of differentiation, and provided various factors necessary to
facilitate the desired differentiation.
H. Stimulatory Factors
[0178] 1. Cell Growth and Differentiation Factors
[0179] 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 compounds of the present 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 compounds
of the present invention. In addition, administration of the growth
and/or differentiation factors may be repeated as needed.
[0180] 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; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; 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.
[0181] 2. Post-Stimulation Purification of Induced Cells
[0182] Following stimulation, it may be desirable to isolate stem
cells that have been induced to undergo differentiation from those
that have not. As discuss above, a variety of purification
procedures may be applied to cells to effect their separation, and
a number of these rely on cell surface markers.
[0183] i. Cardiomyocytes
[0184] U.S. Patent Publication No. 2005/0164382, incorporated
herein by reference, describes methods of obtaining cardiomyoctyes
as well as various cardiomyocyte markers including cTnI, cTNT,
ventricular myosin, connexin43, sarcomeric myosin heavy chain
(MHC), GATA-4, Nkx2.5, N-cadherin, P1-adrenoceptor (.beta.1-AR),
ANF, MEF-2A, MEF-2B MEF-2C, MEF-2D creatine kinase MB (CK-MB),
myoglobin, or atrial natriuretic factor (ANF).
[0185] ii. Neuronal Cells
[0186] A number of neuronal markers have been used to identify
various classes of cells that are of neuronal origin. For example,
glial fibrillary acidic protein and S100 protein are used to
identify astrocytes, GAP-43, microtubule associated protein 2
neuronal specific enolase, synaptophysin, tryptophan hydroxylase,
.beta.-tubulin and vimentin/LN6 are used to identify neuronal cells
generally, and myelin basis protein can be used to identify
oligodendrocytes.
I. Cancer Stem Cells
[0187] Stem cells are functionally characterized by the ability to
self renew and differentiate into distinct cell lineages. It has
been established that embryonic stem (ES) cells, derived from the
inner cell mass of the developing blastocyst, are pluripotent,
undifferentiated cells with the potential to proliferate,
self-renew, and generate new tissues. Such ES cells have now been
isolated from both mouse and human embryos. In addition, stem cells
have been identified within adult, differentiated tissues. These
adult stem cells, sometimes also termed multi-potent adult
progenitor cells (MAPCs), are believed to play essential roles in
growth and tissue regeneration and have been identified in certain
tissues, including the brain, epidermis, lung, breast,
hematopoietic and neural systems. Gage, 2000; Abeyta et al., 2004;
Tumbar et al., 2004; Zepeda et al., 1995; Dontu et al., 2003; Welm
et al., 2002; Gudjonsson et al., 2002; Lagasse et al., 2001;
Ramalho-Santos et al., 2002.
[0188] There is evidence that many common cancers, including skin
and breast cancers, in addition to leukemias, can result from
transforming events that occur in adult stem cells (Perez-Losada
and Balmain, 2003; Al-Hajj et al., 2003; Reya et al., 2001).
Indeed, functional parallels exist between tumorigenic and normal
stem cells. Both cell types demonstrate significant proliferative
potential, the ability to self-renew, and the ability to generate
new tissues. However, tumorigenic stem cells lack the normal growth
regulatory mechanisms that limit the uncontrolled proliferation of
stem cells (Reya et al., 2001).
[0189] Tumorigenic stem cells arise in normal adult stem cell
populations through the accumulation of multiple transforming
mutations. As adult stem cells can persist and self-renew for the
lifespan of the individual, these cells are more likely to accrue
the genetic lesions necessary for malignant transformation. Such
transformed tumorigenic stem cells, arising in normal adult stem
cell populations, can initiate cancer development (Reya et al.,
2001). Furthermore, tumorigenic stem cells may also play important
roles in tumor evolution, metastatic invasion and local recurrence
following treatment.
[0190] Cancer stem cells constitute only a small proportion of a
tumor or a cancerous tissue. But the cancer stem cells have a
unique ability to establish new colonies of cancer cells. For
example, when mouse myeloma cells are obtained from mouse ascites,
separated from normal hematopoietic cells, and put into in vitro
colony-forming assays, only 1 in 10,000 to 1 in 100 cancer cells
were able to form colonies (Park et al., 1971). Even when leukemic
cells were transplanted in vivo, only 1-4% of cells could form
spleen colonies (Bruce et al., 1963; Wodinsky et al., 1967;
Bergsagel et al., 1968). Moreover it has been shown that a subset
of cells from a population of seemingly homogeneous cancer cells is
capable of proliferation and is clonogenic, while the remainder of
cancer cells cannot undergo significant proliferation. Thus,
workers have purified such a proliferative subset of leukemia cells
as CD34.sup.+CD38.sup.- cells from patient samples (Bonnet and
Dick, 1997). Despite the fact that these cells represented a small
and variable proportion of acute myelogenic leukemia cells (0.2% in
one patient), they were the only cells capable of transferring
acute myelogenic leukemia (AML) from human patients to NOD/SCID
mice in the vast majority of cases. Thus, not all AML cells had a
similar clonogenic capacity. Only a small, identifiable subset was
consistently enriched for the ability to proliferate and transfer
disease.
[0191] As used herein, a cancer stem cell is a stem cell that has a
cancerous phenotype. Cancer stem cells lack the normal growth
regulatory mechanisms that limit the controlled proliferation of
stem cells. Cancer stem cells constitute only a subset of cells
from a population of seemingly homogeneous cancer cells. While
cancer stem cells are capable of proliferation and are clonogenic,
most cancer cells in a population of seemingly homogeneous cancer
cells cannot undergo significant proliferation.
[0192] As discussed herein, compounds of the present invention may
be used to treat cancer. For example, compounds of the present
invention may be used to target cancer stem cells. In certain
embodiments, combination therapy may be employed, wherein a
compound of the present invention is administered to a cancer
patient in addition to other therapy, such as surgery, chemotherapy
and/or radiation therapy, wherein the compound of the present
invention targets primarily the cancer stem cells and the chemo- or
radiation therapy targets primarily the non-stem cell cancer cells.
Combination therapy is discussed further below. Compounds of the
present invention may also find use in on-going treatment after
other cancer therapy (e.g., surgery, chemo- and/or radiation
therapy) has been terminated. This approach would be designed to
suppress cancer stem cells from forming cancerous tumors, and/or
encourage stem cells with cancerous predilections to instead form
non-cancerous cells. Determining whether a cancer patient possesses
cancer stem cells, and thus could be a candidate for receiving
compounds of the present invention in therapeutically effective
amounts, could be determined, for example, by methods described in
U.S. Publ. Appl. No. 2005/0277162, wherein Rex-1 is described as a
cancer stem cell marker.
[0193] Other uses of compounds of the present invention in a cancer
context contemplate administration of a compound of the present
invention to a patient having a cancerous tumor. Curative surgery
includes resection in which all or part of cancerous tissue is
physically removed, excised, and/or destroyed. Tumor resection
refers to physical removal of at least part of a tumor.
[0194] In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0195] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0196] Accordingly, a compound of the present invention may be
administered, for example, to treat a primary tumor, and following
resection of the tumor, a compound of the present invention could
continue to be administered to treat any residual, microscopic
disease, be it comprised of cancer stem cells or cancer cells that
are not stem cells. In this regard, treatment with a therapeutic
amount of a compound of the present invention may increase the
resectability of the tumor due to shrinkage at the margins or by
elimination of certain particularly invasive portions. Additional
treatments subsequent to resection may then serve to eliminate
microscopic residual disease at the tumor site. The tumor may be a
brain cancer tumor, for example.
[0197] Moreover, a compound of the present invention may be
administered via placement of the compound directly at the site of
the tumor bed such that the compound is released over time. For
example, a compound of the present invention may be comprised in a
wafer that is left in the tumor bed following resection of the
tumor, wherein the wafer is attached to the edges of the resection
cavity at the conclusion of surgical tumor removal. Such wafers
have been employed in other contexts, such as biodegradable
carmustine (BCNU) wafers for treatment of gliomas. Multiple wafers
may be employed in such therapeutic intervention.
[0198] 1. Combination Therapy
[0199] In certain aspects, a compound of the present invention may
be used in combination with another agent or therapy method, e.g.,
another cancer treatment. Administration of a compound of the
present invention may precede or follow the other agent treatment
by intervals ranging from minutes to weeks. In embodiments where
the other agent and the compound of the present invention are
applied separately, one would generally ensure that a significant
period of time did not expire between the time of each delivery,
such that the agent and the compound of the present invention would
still be able to exert an advantageously combined effect. For
example, in such instances, it is contemplated that one may contact
the cell, tissue or organism with two, three, four or more agents
substantially simultaneously (i.e., within less than about a
minute) with a compound of the present invention. In other aspects,
one or more agents may be administered within about, at least
about, or at most about 1 minute, 5 minutes, 10 minutes, 20 minutes
30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5
hours, 6 hours, 7 hours 8 hours, 9 hours, 10 hours, 11 hours, 12
hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours,
19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25
hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours,
32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38
hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours,
45 hours, 46 hours, 47 hours, to, at least, or 48 hours or more
prior to and/or after administering the compound of the present
invention. In certain other embodiments, an agent may be
administered within about 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,
14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20, to 21
days prior to and/or after administering the compound of the
present invention. In some situations, it may be desirable to
extend the time period for treatment significantly, however, where
several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks or more) lapse
between the respective administrations.
[0200] Various combination regimens of the agents may be employed.
Non-limiting examples of such combinations are shown below, wherein
the compound of the present invention is "A" and the secondary
agent, such as radiation or chemotherapy, is "B":
[0201] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
[0202] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
[0203] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0204] Administration of the compound of the present invention of
the present invention to a patient will follow general protocols
for the administration of chemotherapeutics, taking into account
the toxicity, if any, of the conjugate. It is expected that the
treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the compound of
the present invention. These therapies include but are not limited
to chemotherapy, radiotherapy, immunotherapy, gene therapy and
surgery.
J. Methods of Treatment
[0205] The present invention contemplates a variety of uses for the
compounds of the present invention. In particular, they can be used
to treat individuals that have undergone trauma, injury, disease or
other destruction or damage to tissue, such as neuronal or cardiac
tissue.
[0206] In one embodiment, the invention contemplates the
administration of the compounds directly into an affected subject.
Traditional routes and modes of administration may be utilized
depending the clinical situation and the tissue target of the
therapy. Alternatively, the invention may rely on an ex vivo
approach, where stem cells are stimulated with compounds of the
present invention outside an organism and then administered,
optionally after culturing to expand the cells, to a recipient. The
cells may be heterologous to the recipient, or they may have
previously been obtained from that recipient, i.e. autologous.
[0207] In another embodiment, the present invention contemplates
the use of compounds of the present invention to induce
differentiation in cells that have become pathologically
de-differentiated, i.e., hyper- or neoplastic cells, such as cancer
cells. Particular embodiments of this aspect of the invention
involved the treatment of individuals have neuronal cancers, such
as gliomas and glioblastomas, including glioblastoma
multiforme.
K. Pharmaceutical Compositions
[0208] It is envisioned that, for administration to a host,
compounds of the present invention and stimulated/differentiated
cells will be suspended in a formulation suitable for
administration to a host. Aqueous compositions of the present
invention comprise an effective amount of a compound and/or cells
dispersed in a pharmaceutically acceptable formulation and/or
aqueous medium. The phrases "pharmaceutically and/or
pharmacologically acceptable" refer to compositions that do not
produce an adverse, allergic and/or other untoward reaction when
administered to an animal, and specifically to humans, as
appropriate.
[0209] As used herein, "pharmaceutically acceptable carrier"
includes any solvents, dispersion media, coatings, antibacterial
and/or antifungal agents, isotonic and/or absorption delaying
agents and the like. The use of such media or agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions. For administration to humans,
preparations should meet sterility, pyrogenicity, general safety
and/or purity standards as required by FDA Office of Biologics
standards.
[0210] Compounds and/or cells for administration will generally be
formulated for parenteral administration, e.g., formulated for
injection via the intravenous, intramuscular, subcutaneous,
intralesional, or even intraperitoneal routes. The preparation of
an aqueous composition that contains cells as a viable component or
ingredient will be known to those of skill in the art in light of
the present disclosure. In all cases the form should be sterile and
must be fluid to the extent that easy syringability exists and that
viability of the cells is maintained. It is generally contemplated
that the majority of culture media will be removed from cells prior
to administration.
[0211] Generally, dispersions are prepared by incorporating the
compounds and cells into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients for
maintaining cell viability as well as potentially additional
components to effect proliferation, differentiation or
replacement/grafting in vivo. Upon formulation, solutions will be
administered in a manner compatible with the dosage formulation or
in such amount as is therapeutically effective. Some variation in
dosage will necessarily occur depending on the condition of the
subject being treated. The person responsible for administration
will, in any event, determine the appropriate dose for the
individual subject.
L. Examples
[0212] The following examples are included to demonstrate certain
preferred embodiments of the invention. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples which follow represent techniques discovered by the
inventor to function well in the practice of the invention, and
thus can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
Example 1
Identification of Certain Neurogenic-Inducing Compounds of the
Present Invention
[0213] Chemical Libraries and Medicinal Chemistry.
[0214] The UTSW compound library used in this screen was purchased
in two components: 47,000 unique compounds selected from the
DiverSet.TM. small-molecules library (ChemBridge, Inc; San Diego,
Calif.) and 100,000 unique compounds from Chemical Diversity Labs,
Inc. (San Diego, Calif.). This collection of compounds was
additionally filtered to exclude compounds containing reactive
groups and compounds likely to be insoluble or cytotoxic.
[0215] Assay Development.
[0216] An Nkx2.5-luc transgene was constructed by replacing the two
coding sequence exons of Nkx2.5 locus from the ATG with the coding
sequence of luc (from pGL3-basic vector, Promega) in an
.about.180,000 base pair-long BAC (FIG. 11). The recombinant BAC
DNA was introduced into pluripotent P19CL6 cells using
Lipofectamine-2000 (Invitrogen) and neo.sup.R clones were selected,
and tested for chemically inducible luc activity with sodium
butyrate (NaB). #5-1, a clonal stem cell line with low basal and at
least a 4-fold higher NaB-inducible luciferase activity compared to
vehicle control (DMSO), yielding a Z' value of .about.0.7 in
384-well plate format (Zhang et al. 1999), was chosen for the
HTS.
[0217] HTS for Chemical Inducers of Nkx2.5-luc in P19CL6.
[0218] Approximately 147,000 unique compounds were screened using
clone #5-1 P19CL6 Nkx2.5-luc cells in 384-well white plates. To
ensure pluripotency at the time of compound screening, each P19CL6
#5-1 cell batch was pre-screened for uniform high-level Oct3/4
expression by immunofluorescence cell staining. Cells were plated
using an automated dispenser at 1,200 cells/well in 70 .mu.L
media/well in 10% fetal calf-serum MEMa media. Parallel plating
onto clear-bottom plates was done to ensure the viability and
appropriate cell density for large-scale screens. On day 3, 0.7
.mu.L of library compounds at 5 .mu.M in pure DMSO (1% final DMSO
concentration) was dispersed robotically (384-pin array Biomek FX
high-precision robot) and the plates were incubated for an
additional 48 hours before measuring luc activity.
[0219] Primary and Secondary Screening Hit Selection.
[0220] Screening the entire 147,000 UTSW compound library at 5
.mu.M with clone #5-1 reporter cells resulted in .about.3,000
primary "hits," using a luc activity of >2 times the plate
median as the cut-off for positive score. Secondary screening using
#5-1 cells at 1.7, 5, and 15 .mu.M identified 66% of these hits as
repeat positives, and from these positive hits, the inventors
placed 150 compounds with the best dose response into a candidate
hit list. Among these candidates, 10 major sub-groups with common
core structural motifs emerged, as shown below.
##STR00036## ##STR00037##
Top-10 Hit List
[0221] Each of these compound families was independently identified
multiple times in the primary screen using Nkx2.5-luc P19CL6 cells,
and then re-identified in a secondary, confirmatory screen using
the same reporter cells. X, Y, R, R.sub.1, R.sub.2 are additional
chemical side groups.
Example 2
Synthesis of Certain Compounds of the Present Invention
[0222] Synthesis of 3,5-Disubstituted Isoxazoles.
[0223] Two approaches based on known literature procedures (Jager
and Colinas, 2003; Grunanger and Vita-Finzi, 1991; Cicchi et al.,
2003) were used to synthesize certain compounds of the present
invention.
[0224] General procedure A: 3+2 Cycloaddition Route to Isoxazole
Core.
[0225] To a stirred suspension of ethyl chlorooximido acetate (3.0
mmol) and the corresponding alkyne (1.0 mmol) in THF (5 mL) was
added triethylamine (4 mmol) dropwise at room temperature. The
reaction was monitored by LC/MS analysis for conversion to the
isoxazole. After 24 hrs, the reaction was diluted with water (2 mL)
and ethyl acetate (2 mL). The organic layer was washed with brine
(2 mL) and dried over Na.sub.2SO.sub.4. Concentration gave an oil
that was purified by silica gel chromatography to yield the
corresponding ethyl 5-(aryl)isoxazole-3-carboxylate (30-50%
yield).
[0226] General Procedure B: Cyclodehydration Route to Isoxazole
Core.
[0227] To a stirred solution of dimethyl oxalate (3.3 mmol) and the
corresponding aryl ketone (3.0 mmol) in toluene (10 mL) was added a
1.0M solution of potassium tert-butoxide in THF (3.5 mmol)
dropwise. The corresponding reaction was monitored by LC/MS for
conversion to the corresponding methyl 2,4-dioxo-4-arylbutanoate.
Once the reaction was complete, it was quenched with 1N HCl (5 mL).
The organic layer was washed with brine (10 mL) and dried to a
crude solid. The crude product was then directly treated with
hydroxylamine hydrochloride (4 mmol) in MeOH at 50.degree. C. for
4-6 hours. The resulting methyl 5-(aryl)isoxazole-3-carboxylate was
isolated by direct crystallization using water as the anti-solvent
(60-70% yield).
[0228] Hydrolysis of Isoxazole Esters:
[0229] From either Procedure A or B, the isoxazole esters obtained
were hydrolyzed using 1N LiOH (2 equiv) in THF at 50.degree. C. for
the appropriate amount of time as indicated by LC/MS analysis. Once
the reaction was complete, the reaction was extracted with toluene.
The resulting aqueous layer was then acidified to pH=1 with 1H HCl
and extracted with EtOAc. Concentration gave a crude solid that was
taken directly into the next step.
[0230] Amide Coupling Step:
[0231] The crude isoxazole acids (1 mmol) were dissolved in
dichloromethane (3 mL) along with
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (1.1 mmol) and
1-hydroxybenzotriazole hydrate (HOBt) (1.1 mmol). After stirring
for 10 minutes at room temperature, the corresponding amine (1.2
mmol) was added in dropwise. The reaction was monitored by LC/MS
for amide formation. Once the reaction was complete, the solvent
was removed the crude reaction was partitioned between water (2 mL)
and EtOAc (3 mL). The EtOAc layer was washed with brine (2 mL) and
concentrated to give the crude product. The products can either be
purified by silica gel chromatography or by crystallization from
ethanol/water. Typical yields were 60-80%.
Example 3
Synthesis of Particular Compounds of the Present Invention
N-cyclopropyl-5-(thiophen-2-yl)isoxazole-3-carboxamide
##STR00038##
[0233] To a solution of ethyl
5-(thiophen-2-yl)isoxazole-3-carboxylate (220 mg, 1 mmol) in
absolute ethanol (5 mL) was added cyclopropylamine (285 mg, 5
mmol). The reaction was sealed and heated to 80.degree. C. for 24
hours. The reaction was allowed to cool to room temperature and
water (1 mL) was added. The product was collected by vacuum
filtration as a white crystalline solid (150 mg, 64% yield).
.delta..sub.H (400 MHz, d.sup.6 DMSO): 8.85 (1H, d), 7.83 (1H, d),
7.76 (1H, d), 7.24 (1H, d), 7.18 (1H, s), 2.83 (1H, m), 0.58-0.71
(4H, m).
Synthesis of Certain Isoxazoles and Pyrazoles of the Present
Invention
[0234] Generally:
[0235] The synthesis of certain isoxazoles and pyrazoles starts
with the reaction of an aromatic ketone (i.e., acetophenone) with
an appropriate base (i.e., potassium tertbutoxide) to generate the
corresponding enolate in a suitable solvent such as
tetrahydrofuran. This enolate is trapped in situ with an
electrophile such as dimethyloxalate to yield the corresponding
methyl-2,4-dioxo-phenylbutanoate derivative. This derivative can
then be reacted with hydroxylamine hydrochloride to generate the
corresponding isoxazole or hydrazine to generate the corresponding
pyrazole usually in a polar protic solvent (i.e., methanol). The
methyl ester of either the isoxazole or pyrazole can be hydrolyzed
to the carboxylic acid using an appropriate base such as aqueous
lithium hydroxide and suitable organic solvent (i.e.,
tetrahydrofuran). Under standard peptide type coupling conditions
(EDC, HOBt, amine), the acid can be converted to the desired amide
using a suitable aprotic solvent such as dichloromethane. The
products can then be purified using standard protocols that would
be familiar to those of ordinary skill in the art.
Synthesis of
N-cyclopropyl-5-(2-hydroxyphenylisoxazole-3-carboxamide
Step 1. Synthesis of methyl 4-(2-hydroxyphenyl)-2,4-dioxobutanoate
(2)
##STR00039##
[0237] To a stirred solution of 2-hydroxyacetophenone (1, 2.0 mmol)
and dimethyloxalate (2, 2.2 mmol) in toluene (10 mL) was added
dropwise a solution of potassium tert-butoxide in THF (4.5 mL of a
1.0 M solution in THF) at room temperature. The reaction was
monitored by HPLC analysis for complete consumption of the starting
acetophenone. Once the reaction was complete (.about.60 min), it
was quenched with a 1.0 N solution of HCl (5.0 mL) and the
resulting biphasic mixture was stirred for .about.10 minutes. After
phase separation, the aqueous layer was removed. The organic layer
was then washed with water (2 mL) and brine (2 ml) then dried over
Na.sub.2SO.sub.4. The dried organic layer was then concentrated in
vacuo to give a light yellow solid (350 mg, 79% yield) that was
used "as is" in the next step.
Step 2. Synthesis of methyl
5-(2-hydroxyphenyl)isoxazole-3-carboxylate (4)
##STR00040##
[0239] To a 20 mL flask was added the diketone 3 (110 mg, 0.5 mmol)
and hydroxylamine hydrochloride (35 mg, 0.5 mmol). Methanol was
then added (7 mL) and the mixture was heated to reflux. The
reaction was monitored by HPLC for product formation. Once the
reaction was complete (.about.18 hours), the reaction was
concentrated in vacuo to a crude yellow oil. This oil was
partitioned between EtOAc (5 mL) and water (2 mL). The aqueous
layer was discarded and the organic layer was washed with water (2
mL), brine (2 mL) and then dried over Na.sub.2SO.sub.4. The
solution was then concentrated to an oil which solidified upon
standing to give the desired product 4 (85 mg, 77.6% yield) and
used "as is" in the next step.
Step 3. Synthesis of 5-(2-hydroxyphenyl)isoxazole-3-carboxylic acid
(5)
##STR00041##
[0241] To a stirred solution of methyl ester 4 (32 mg, 0.15 mmol)
in THE (2 mL) was added a solution of 1.0 M LiOH (2 mL) in one
portion. The corresponding reaction was monitored by HPLC for
consumption of the starting material. After 3 hours at room
temperature, the reaction was complete. To the reaction was added
EtOAc (2 mL) and water (2 mL) and the layers were allowed to
separate. The aqueous layer was transferred to clean flask and then
acidified to pH=1 using 2.0 N HCl. This was then extracted with
EtOAc (5 mL) and the aqueous layer was discarded. The organic layer
was washed with water (2 mL) and brine (2 mL) followed by drying
over Na.sub.2SO.sub.4. Concentration gave 20 mg (65% yield) of the
desired product 5 that was used "as is" in the next step.
Step 4. Synthesis of
N-cyclopropyl-5-(2-hydroxyphenyl)isoxazole-3-carboxamide (6)
##STR00042##
[0243] To a 20 mL flask was added the crude acid from step 3 (20
mg, 0.1 mmol), HOBt (16 mg, 0.12 mmol), and EDC (23 mg, 0.12 mmol).
To this was added dichloromethane (3 mL) and the resulting
heterogeneous mixture was stirred for 30 minutes at room
temperature. To the resulting solution was added cyclopropylamine
(9.0 mg, 0.15 mmol) and the reaction was monitored by HPLC for
product formation. After 2 hours, the reaction was complete and
concentrated to a thick oil. This oil was partitioned between EtOAc
(3 mL) and water (2 mL) with stirring for 10 minutes. The layers
were then allowed to separate and the aqueous layer was discarded.
The organic layer was then washed with water (2 mL), brine (2 ml)
and dried over Na.sub.2SO.sub.4. Concentration gave an oil that was
purified by column chromatography (30% EtOAc in hexanes) to give
6.1 mg (25% yield) of the desired isoxazole 6; .sup.1H NMR (400
MHz, d6-DMSO) S 8.85 (d, 1H), 7.78 (d, 1H), 7.32 (t, 1H), 7.08 (s,
1H), 7.01 (d, 1H), 6.94 (t, 1H), 2.82 (m, 1H), 0.67 (m, 2H), 0.59
(m, 2H). LC/MS (electrospray ionization, positive mode)
M+H=245.0.
Example 4
Materials and Methods
[0244] Reporter Gene Assay.
[0245] All reporter gene assays were done in 96-well format, and
each data point represents the average of 12 replicates. Reporter
genes included neuroD-, gIuR2-, MREx3-, NR1-, and
pHDAC5:14-3-3-luciferase and each well contained .about.25,000
cells in a volume of 100 .mu.l. 5.times.10.sup.6 HCN cells were
transfected with 5 .mu.g DNA by electroporation (Amaxa) and plated
in growth media overnight. In the case of pHDAC5:14-3-3-luciferase,
cells were transfected with 2 .mu.g of each plasmid. Compounds were
added the day following transfection and luciferase assays were
performed 24 or 48 h later. The typical dose of Isx-9 in reporter
assays was 20 .mu.M unless noted otherwise.
[0246] Neural Stem Cell Culture, In Vitro Differentiation, and CaMK
Inhibitor Studies.
[0247] The hippocampal neural stem/progenitor (NSC) line used in
this study (HCN cells) were originally isolated from adult (8-10
wks old) female Fisher 344 rats and have been characterized
previously (Gage et al., 1995; Palmer et al., 1997). HCN cells were
cultured in DMEM:F12 (Omega Scientific) with N2 supplement
(Invitrogen) and basic fibroblast growth factor (20 ng/ml FGF-2)
(PeproTech). The mouse whole brain neural stem/progenitors (MWB)
were derived from adult (8-10 wks old) C57BL/6 mice that have been
characterized previously (Ray and Gage, 2006). The P19CL6 cells is
a sub-line of P19EC cells, originally generated by the late Dr.
Habara-Ohkubo (Habara-Ohkubo, 1996), were grown in MEM-.alpha.
(Invitrogen) containing 10% fetal bovine serum (Omega). HCN cells
or MWB neural progenitors were trypsinized and plated into N2
medium (Invitrogen) containing FGF-2 (HCN cells) or
FGF-2/EGF/heparin (MWB) overnight, and switched to fresh N2 medium
without FGF-2 and Isx-9 (20 .mu.M for HCN cells and 5 .mu.M for
MWB) was added for 4 d. To test if Isx-9 could block competing
astrocyte or oligodendrocyte differentiation, standard gliogenic
conditions were used as described previously (Hsieh et al., 2004).
To induce neuronal differentiation of P19CL6 cells, 1 .mu.M
all-trans retinoic acid (RA) was added to the culture media to form
P19CL6 aggregates for 2-4 d, then media was replaced with fresh N2
medium and 10 .mu.M Isx-9 was added for 4 d. In some cultures, BrdU
(10 .mu.M, Sigma) was added to label dividing cells 1 h prior to
fixation, propidium iodide (1 .mu.g/ml, Molecular Probes) or
Hoescht 33342 (1 .mu.g/ml, Sigma) were added to label dead or all
cells, and Q-VD-OPh (2 .mu.M, Enzyme Systems Products) was used to
block caspase-mediated apoptosis. Staining of live (rather than
fixed) cultures was used to avoid underestimating cell death
because of possible detachment of dying and/or dead cells from
culture substrates. For CaMK/PKC inhibitor experiments, HCN cells
were pre-treated with inhibitors (KN93 [10, 5, and 1 .mu.M], KN92
[10, 5, and 1 .mu.M] or G66976 [200 nM]) for 2 h, and 20 .mu.M
Isx-9 was added for 24 or 48 h.
[0248] RT-PCR and Protein Blotting.
[0249] Total RNA was isolated by Trizol reagent (Invitrogen) and
RT-PCR was carried out as previously described (Hsieh et al.,
2004b). Primer sequences are available upon request. For protein
blotting, whole cell lysates were prepared from HCN cells cultured
in undifferentiated conditions (FGF-2 or DMSO vehicle control) or
from differentiating conditions (Isx-B).
[0250] Protein Blotting, Immunoprecipitation, and Gel Shift
Assay.
[0251] For protein blotting analysis, whole cell lysates were
prepared from HCN cells using RIPA buffer (Tris-HCl, 50 mM, pH 7.4;
NP-40, 1%; Na-deoxycholate, 0.25%; NaCl, 150 mM; EDTA, 1 mM; PMSF,
1 mM; aprotinin, leupeptin, pepstatin, 1 mg/ml; Na.sub.3VO.sub.4, 1
mM; NaF, 1 mM). For nuclear and cytoplasmic extracts, HCNs were
lysed in hypotonic buffer (Hepes, 10 mM, pH 8; MgCl.sub.2, 1.5 mM;
KCl, 10 mM; DTT, 1 mM). Nuclei was then isolated and resuspended in
the same buffer with NaCl, 420 mM; EDTA, 0.2 mM; glycerol, 25%;
NP-40, 1%; PMSF, 1 mM; aprotinin, leupeptin, pepstatin, 1 mg/ml.
Protein concentration in centrifugation-clarified cell lysates were
measured by the BCA Protein Assay Kit (Pierce) and equal amounts of
protein were separated on a 4-12% SDS-PAGE and transferred to
Hybond PVDF (Amersham Biosciences). Protein blots were done using
the NuPage gel and transfer system with 4-20% Tris-Bis gels
(Invitrogen). Primary Abs for protein blotting included: rabbit
anti-.beta.TubIII (1:1000; Covance), mouse anti-Map2AB (1:100;
Sigma); goat anti-doublecortin (DCX) (1:100; Chemicon), mouse
anti-GAPDH (1:5000; Chemicon), rabbit anti-MEF2A (1:500; Upstate),
rabbit anti-MEF2C (1:500; Santa Cruz); rabbit anti GluR2/3 (1:250;
Chemicon); rabbit anti-phosphorylated HDAC5 (1:500; gift from T.
McKinsey); rabbit anti-HDAC5 (1:500; Upstate), rabbit anti-HDAC4
(1:500; Cell Signaling), rabbit anti-CREB (1:100; Cell Signaling);
mouse anti-FLAG (1:5000; Sigma), mouse anti-GFP (1:500;
Invitrogen); rabbit anti-phosphorylated CamKII (1:200; Cell
Signaling). Immunoreactive bands were visualized using HRP- or
AP-conjugated secondary antibodies, followed by ECL (Amersham
Biosciences) or BCIP/NBT detection (KPL, Gaithersburg, Md.).
Immunoprecipitation of FLAG-tagged HDAC5 was done with the FLAG
antibody and blotting for phospho-HDAC5 and Flag antibodies, or
CREB and GFP as a normalization control. For electrophoretic
mobility shift assay (EMSA) experiments, labeling of 10 pmol
double-stranded oligonucleotides was performed by Klenow fill in of
overhanging 5' ends with .sup.32P-dCTP and purification with a
Sephadex G50 column. Binding reactions of 1.times. binding buffer
(40 mM KCl, 15 mM HEPES pH 7.0, 1 mM EDTA, 5% glycerol), 0.5 mM
DTT, 10 .mu.g lysate, 1 .mu.g poly dI-dC and competitor DNA
(100-fold excess) were incubated at room temperature for 10 min
before addition of probe, incubated an additional 20 min and
electrophoresed on a 6% polyacrylamide-TBE gel. For visualization,
the gel was dried and exposed with an intensifying screen for 6
hours at -80.degree. C.
[0252] Immunostaining, GFP-HDAC5 Visualization, and Fluorescence
Microscopy.
[0253] Labeled cells were visualized using a Nikon TE2000-U
inverted microscope (Nikon, Inc.) and a CoolSnap digital camera
(Photometrics, Inc.). Quantification of cell phenotype was done by
sampling 6-8 random fields in each well and counting a total of
250-500 cells at 20.times. magnification.
4',6-diamidino-2-phenylindole (DAPI) was used to identify
individual cells. For quantification of live/dead cells, images
were taken of cultures live-stained with propidium iodide and
Hoescht 33342 at 20.times. magnification, and 500-1000 cells were
counted by sampling 6-8 random fields in each well. For
quantification of HDAC5 subcellular localization, 20.times. images
were taken of live cultures, and 40-50 GFP+ cells were counted by
sampling 3 random fields in each well. The following primary
antibodies were used: rabbit anti-Tuj1 (1:7500; Covance), mouse
anti-Map2ab (1:250; Sigma); guinea pig anti-GFAP (1:2500; Advanced
Immunochemical, Inc.) and rat anti-BrdU (1:400; Accurate).
Secondary antibodies were all from Jackson ImmunoResearch and used
at 1:250 dilution. The detection of BrdU in cultured cells required
treatment in 2N HCl at 37.degree. C. for 30 min (Palmer et al.,
1999).
[0254] Adenoviral Infection.
[0255] NPCs or Cos cells were infected with adenovirus at a
multiplicity of infection of 10 particles/cell for 24 or 48 hours.
For reporter gene experiments, NPCs were electroporated with
luciferase constructs and mixed with virus before plating in N2
medium with FGF-2. After 48 hours, cells were replaced with N2
medium containing Isx compounds and cultured for an additional 24
hours. To visualize GFP on glass slides, NPCs were plated in N2
medium with FGF-2 and infected with adenovirus for 48 hours, then
media was replaced with N2 medium plus vehicle or Isx, and cultured
for an additional 24 hours. In some experiments, Cos cells were
plated in IMDM medium (Invitrogen) containing 10% FBS and infected
with adenovirus, and cultured for an additional 24 hours.
[0256] Ca.sup.2+ Imaging.
[0257] HCN cells were loaded with 5 .mu.M Fura2-AM (Molecular
Probes) in artificial cerebrospinal fluid (ACSF) (140 mM NaCl, 5 mM
KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2, 10 mM HEPES (pH 7.3) for 45
min at 37.degree. C. For imaging experiments the coverslips were
mounted onto a recording/perfusion chamber (RC-26G, Warner
Instrument) maintained at 37.degree. C. (PH1, Warner
[0258] Instrument) positioned on the movable stage of an Olympus
IX-70 inverted microscope, and perfused with ACSF media by gravity
flow. Compounds (Isx 20 .mu.M) or DMSO (vol/vol) were added and
recording was started (time 0). For Isx-9 plus inhibitor
experiments, HCNs were pre-treated with a "cocktail" inhibitor (5
.mu.M each of AP5/CNQX/Nifedipine) or 2 .mu.M MK801 for 5 minutes
before Isx-9 treatment. Cells were intermittently excited by 340 nm
and 380 nm UV light (DeltaRAM illuminator, PTI) using a Fura-2
dichroic filter cube (Chroma Technologies) and a 60.times.UV-grade
oil-immersed objective (Olympus) and collected at 10 sec intervals
and shown as 340/380 ratios at timepoints as indicated in FIG.
3.
[0259] GFP-HDAC5 and MEF2C-Engrailed Experiments.
[0260] For GFP-HDAC5 experiments, HCN cells were infected with
adenovirus at a multiplicity of infection of 10 particles/cell for
24 or 48 h. For reporter gene experiments, HCN cells were
electroporated with luciferase constructs and mixed with virus
(GFP-HDAC5 (SA) or CMV-GFP control virus) before plating in N2
medium with FGF-2. After 48 h, cells were replaced with N2 medium
containing 20 .mu.M Isx-9 and cultured for an additional 24 h. To
visualize GFP on glass slides, HCNs were plated in N2 medium with
FGF-2 and infected with adenovirus for 48 h, then media was
replaced with N2 medium plus vehicle or 20 .mu.M Isx-9, and
cultured for an additional 24 h. For MEF2C-engrailed experiments,
HCN cells were co-electroporated with two DNA constructs: 3 .mu.g
of the MEF2C-engrailed plasmid (CAG-MEF2C-engrailed-IRES-GFP) or
GFP control plasmid (CAG-GFP) plus 2 .mu.g of the NR1-luc reporter
plasmid. GFP expression was visualized 48 h after electroporation
to confirm >50% transduction efficiency before adding compounds
and luciferase assays were performed an additional 24 h later.
[0261] Statistical Analysis.
[0262] Results were analyzed for statistical significance using
two-tailed Student's t test and all error bars are expressed as
standard deviations (SD). Values of p<0.05 were considered
significant.
Example 5
Results Relating to Example 4
[0263] Isoxazole-Induced Neuronal Differentiation in Adult
Hippocampal Neural Stem/Progenitor Cells.
[0264] The inventors screened a pre-selected collection of
synthetic small organic molecules (Sadek et al., manuscript
submitted) for compounds that could chemically activate the
neuronal gene program in P19 embryonal carcinoma cells using semi
high-throughput luciferase assays as described in Examples 1 and 3.
Among the candidate small molecules capable of inducing our
neuronal reporter gene, neuroD, a key bHLH transcription factor
involved in neuronal cell fate determination and differentiation,
the inventors identified several compounds belonging to the
structural class of 3,5-disubstituted isoxazole (Isx), molecules
not previously associated with biological activity. Isx treatment
induced at least a 8-fold increase in both NeuroD and GluR2
luciferase reporters, compared to a modest 2-fold increase in
reporter activity with the pleiotropic chemical inducers retinoic
acid and forskolin, until now gold standards for neuronal
induction.
[0265] Isoxazoles at 20 .mu.M concentration were effective inducers
of neuronal differentiation in NPCs within 4 days, inducing
morphological changes such as cell clustering, cell flattening and
extension of processes, and induction of neuronal lineage-specific
markers like TuJ1 and Map2ab, when compared to control cells
treated with vehicle alone. Morphological changes became evident
within hours of drug exposure, while an increase in the percentage
of Tuj1+ neurons dramatically increased between 1 and 4 days
compared to vehicle alone. NPCs that differentiated into definitive
neuronal cells were scored on the basis of morphological criteria
(elaboration of neuronal processes), as well as immunoreactivity
with various neuronal markers (e.g., Tuj1). Isx treatment could
also efficiently convert undifferentiated pluripotent embryonic
stem cells (P19 embryonal carcinoma) as well as adult mouse whole
brain (MWB) neural progenitors, suggesting that neuronal
differentiation by isoxazoles is not just specific to adult rat
hippocampal NPCs.
[0266] In addition to promoting neuronal differentiation, Isx
dominantly suppressed glial differentiation in NPCs, even in the
presence of strong gliogenic signals such as LIF and BMP2. 50 ng/ml
LIF and 50 ng/ml BMP2 normally induced the differentiation of NPCs
in 4-day cultures into Tuj1+ neurons and GFAP+ astrocytes, however
treatment of cells with Isx completely suppressed astrocyte
differentiation, and instead promoted neuronal differentiation.
Moreover, Isx treatment also suppressed IGF-I induced
oligodendrocyte differentiation. Taken together, these data suggest
that Isx is a potent activator of the neurogenic lineage in stem
cells.
[0267] Using semi-quantitative reverse transcriptase polymerase
chain reaction (RT-PCR), the inventors observed increased neuroD,
GluR2, .beta. III-tubulin (Tuj1) and NMDA receptor subunit 1 (NR1)
mRNA levels, all genes associated with neuronal commitment,
differentiation, and/or maturation. Indeed, by mRNA, significant
changes in gene expression were evident even after 3 hours of Isx
treatment. Notably, many of these neuronal transcripts tested were
highest at 1 day, with lower levels by 4 days, suggesting a
temporal regulation of neuronal gene expression by Isx. Gapdh
levels did not change with Isx treatment and was used as an
internal control. Furthermore, the inventors never observed the
expression of astrocytic and oligodendrocytic markers in the
presence of Isx
[0268] Isx treatment also induced a gradual increase in the level
of neuronal protein expression over time, including .beta.TUBIII
and Doublecortin (DCX), which are expressed in immature neurons, as
well as microtubule associated protein-2AB (Map2AB), which is
expressed in more mature neurons. Neuronal protein expression was
normalized for total protein concentration as well as to GAPDH.
These data strongly support that Isx small molecules acts
specifically and dominantly to induce neuronal differentiation and
suppress glial differentiation.
[0269] Isoxazole-Induced Neuronal Differentiation is Due to
Instructive Effects and a Subsequent Proliferation of Committed
Neuroblasts.
[0270] Although these results established Isx as an inducer of
neuronal differentiation of multipotent NPCs, it is important to
assess the instructive versus selective effects of Isx. Stem cells
can self-renew to give rise to more stem cells, or commit to a
particular lineage and differentiate into cells of a mature
phenotype, or die, and Isx may mediate a net increase in neuronal
cells by acting at multiple levels. The results suggest a biphasic
response of NPCs to Isx treatment: (1) the initial cell death
associated with Isx-treatment and FGF-2 withdrawal does not
distinguish between neuronal and non-neuronal cells within the
first two days, and (2) continued Isx treatment might additionally
promote the survival of differentiated neurons, in addition to
inducing neuronal cell fate choice.
[0271] Next, the inventors determined whether proliferation of
progenitors might contribute to the Isx-mediated neuronal
differentiation. There was a gradual decrease in dividing cells in
vehicle-treated cultures over time, most likely due to FGF-2
withdrawal. In contrast, Isx-treatment exhibited an initial drop in
BrdU incorporation compared to vehicle controls (.about.10%
compared to .about.40% on the first day), suggesting that Isx does
not have a major proliferation effect on NPCs. Interestingly,
Isx-treated progenitor cells did show a slight increase in BrdU
cells between 1 and 2 days, suggesting that there might be a
secondary effect of Isx on NPC proliferation.
[0272] To determine if Isx has an effect on cells already committed
to the neuronal lineage but still retained the ability to divide
(neuroblasts), the inventors assessed the proliferation of Tuj1+
cells with Isx treatment. Indeed, an increase in the number of
Tuj1+ cells that were also BrdU+ with Isx during the 4-day period
was observed. Vehicle treated cultures did not produce BrdU+ cells
that were also Tuj1+, while a significant number of Tuj1+ cells in
Isx-treated cultures were BrdU+, suggesting that Isx-mediated
neuronal differentiation is due to instructive effects on neuronal
cell fate, as well as a subsequent proliferation of committed
neuroblasts.
[0273] Isoxazole Treatment Triggers the Release of Intracellular
Ca.sup.2+ in NPCs.
[0274] At the core of the 3,5-disubstituted Isx small molecule is a
chemical structure shared by molecules known to affect
neurotransmission, such as .alpha.-amino-3-hydroxy-5-methyl
isoxazole-4-proprionic acid (AMPA) and
5-aminomethyl-3-hydroxy-isoxazole (Muscimol). Treatment of NPCs
with 25 .mu.M AMPA or 50 .mu.M Muscimol failed to induce
morphologic neuronal differentiation, although there was some
neuronal gene activation as evidenced by an increase in
neuroD-luciferase activity and GluR2/3 protein expression with 15
or 50 .mu.M Muscimol, respectively, suggesting that there may be a
degree of specificity of Isx neurotransmitter-like effects in
stem/progenitor cells. Based on studies by others, the inventors
hypothesized that Isx might also regulate [Ca.sup.2+].sub.i levels
in NPCs. The relatively slow kinetics of [Ca.sup.2+].sub.i release
after Isx treatment, compared to other strong inducers of
[Ca.sup.2+].sub.i such as ionomycin, was reminiscent of
transcription factor activation consistent with "excitation" of
NPCs leading to neuronal gene expression and neurogenesis. Indeed,
the inventors did observe a small but significant decrease in
NeuroD-luciferase in NPCs treated for 24 hours with Isx plus
cocktail inhibitor (P=0.0002) as well as Isx plus MK801 (P=0.0003),
suggesting that the Isx-mediated Ca.sup.2+ release involved the
concerted actions of high-voltage Ca.sup.2+ channels as well as
NMDA receptors.
[0275] Isx-Mediated Neuronal Differentiation is Coupled to MEF2
Activation in NPCs.
[0276] A number of transcription factors, including CREB, NFAT, and
MEF2 are activated by a slow and sustained release of intracellular
Ca.sup.2+ in neuronal cells. Thus, the inventors next assessed Isx
effects on a set of Ca.sup.2+-activated reporter transgenes. NFAT
and MCIP are two cellular transcription factors regulated by the
Ca.sup.2+-activated phosphatase, calcineurin. To monitor NFAT and
MCIP activities, they used luciferase reporters bearing the NFAT
and MCIP regulatory regions (NFAT- and MCIP-luc). They observed a
strong induction of both NFAT- and MCIP-luc with 24 hours of Isx
treatment. The induction of NFAT and MCIP by Isx was dependent on
calcineurin activity, since treatment of NPCs with two calcineurin
inhibitors, FK506 (1.25-20 .mu.M) and CsA (1.25-20 .mu.M), blocked
Isx activation in a dose-dependent manner.
[0277] Isoxazoles were originally identified in a cardiogenic small
molecule screen of pluripotent embryonal carcinoma cells (P19CL6),
based on the activation of the NK-2 class homeodomain protein
(Nkx2.5) (Sadek et al., manuscript submitted). In light of Nkx2.5
role in neuronal cell differentiation, the inventors confirmed that
Isx treatment triggered the activation of a rat Nkx2.5-luciferase
transgene in NPCs. Recently, it was suggested that Nkx2.5 and
myocyte enhancer factor-2C (MEF2C) could reciprocally regulate each
other's expression and induce cardiogenesis in P19EC cells, as well
as neurogenesis in P19 cells. Moreover, Ca.sup.2+ signaling
strongly influences the activity of MEF2 proteins through
voltage-sensitive Ca.sup.2+ channels triggering phosphorylation and
calcineurin-mediated dephosphorylation leading to MEF2-dependent
transcription, and calcineurin has recently been shown to control
neuronal synapse formation via a MEF2-regulated transcriptional
mechanism. The inventors thus examined whether there was a
connection between Isx-mediated neuronal differentiation and MEF2.
Indeed, Isx treatment induced a MEF2 promoter as compared to
vehicle alone, and was blocked by calcineurin inhibitors FK506 and
CsA in a dose-dependent manner similar to what was seen with NFAT
and MCIP.
[0278] To further examine the role of MEF2 proteins in NPCs, the
inventors determined the levels of MEF2 isoforms in NPCs treated
with Isx for 2- and 4-days, compared to vehicle treatment. There
are four MEF2 isoforms in all, with MEF2A, 2C, and 2D having the
highest expression in the adult brain. MEF2C mRNA levels, and to a
lesser extent MEF2A, appeared to be up-regulated with Isx
treatment, suggesting a possible role of MEF2A and 2C in neuronal
lineage progression of NPCs. Despite the increase in mRNA levels,
the inventors did not observe an induction of MEF2A or 2C protein
after Isx treatment up to 64 hours, which is the timeframe that
neuronal differentiation is occurring as evident the expression of
two mature neuronal markers Map2AB and GluR2/3.
[0279] MEF2 Activation is Due to Phosphorylation and Export of
HDAC5.
[0280] Post-transcriptional/translational regulation of MEF2 is
commonly mediated by CaMK-mediated phosphorylation,
calcineurin-dependent dephosphorylation, calreticulin-dependent
translocation into the nucleus, and/or through its interaction with
class II histone deacetylases (HDACs). Unlike MEF2A and 2C mRNA
levels that were induced with Isx treatment, the activation of MEF2
by isoxazoles did not appear to be regulated at the level of
phosphorylation or dephosphorylation or translocation since MEF2A
and MEF2C protein levels are relatively unchanged between nuclear
or cytoplasmic fractions with drug treatment. The inventors thus
considered the possibility that MEF2 activity was controlled by
epigenetic mechanisms, held in a repressed state in NPCs through
its interaction with class II HDACs.
[0281] There are three classes of histone deacetylases (HDACs)
expressed in vertebrates. Class I HDACs are ubiquitiously
expressed, whereas class II HDACs have tissue-specific patterns of
expression with highest levels in brain, heart and skeletal muscle.
Class III HDACs are related to the Sir2 family proteins in yeast.
Importantly, recent findings show that class Ha HDACs (HDAC-4, -5,
-7, and -9) act as signal-responsive repressors of cardiac
hypertrophy and that hypertrophic stimulus induced phosphorylation
of class II HDACs in a Ca.sup.2+/calmodulin kinase (CaMK) dependent
fashion that causes export from the nucleus resulting in
derepression of target gene expression. To examine the subcellular
distribution of HDAC5 in NPCs, the inventors took advantage of a
phospho-specific HDAC5 antibody. NPCs treated with vehicle or Isx
for 1 day have similar levels of phos-HDAC5 in the nucleus, whereas
higher levels of phos-HDAC5 in the cytoplasm are observed with Isx
treatment while total HDAC5 levels are unchanged. Next, the
inventors observed that Isx treatment slightly increased
cytoplasmic accumulation of HDAC4, whereas phospho-HDAC4 (using an
antibody that cross-reacts with phospho XX of HDAC4) did not appear
to change or increase with drug treatment and remained cytoplasmic,
suggesting that phosphorylation of HDAC5 compared to HDAC4 was more
sensitive to Isx effects.
[0282] In addition to MEF2 proteins, another key transcription
factor that is activated by Ca.sup.2+-dependent signaling in
neuronal cells is the nuclear CREB transcription factor, which
remained unchanged between vehicle and Isx-treated cells, and
served as normalization controls for nuclear extracts. GAPDH was
used as a loading control for cytoplasmic extracts.
[0283] To visualize the subcellular distribution of HDAC5, NPCs
were infected with an adenovirus expressing GFP-HDAC5 (AdGFP-HDAC5)
and stimulated with vehicle or Isx for 1 day. In unstimulated
cells, 80-90% of the cells contained GFP-HDAC5 in both nuclear and
cytoplasmic compartments, reflecting a basal level of nuclear
export consistent with what is observed in other cell types. In
contrast, Isx triggered nuclear export of HDAC5 (60% cytoplasmic
localization versus 35% both nuclear and cytoplasmic).
Phosphorylation of serines 259 and 498 in HDAC5 creates docking
sites for 14-3-3 chaperone proteins, which shuttle HDAC5 to the
cytoplasm. Conversely, HDAC5 S259/498A (S-A mutant) was found
mostly in the nuclear compartment regardless of Isx treatment. To
further confirm the subcellular distribution of phospho-HDAC5, NPCs
were co-infected with adenovirus expressing HDAC5-Flag and CMV-GFP
(to monitor transgene expression in live cells). NPC lysates were
immunoprecipitated with a Flag antibody and protein blotted with
phospho-HDAC5 and Flag antibody. Indeed the majority of
overexpressed Flag-HDAC5 was found in the cytoplasmic fraction
after a 2-day Isx treatment, when probed with both the
phospho-HDAC5 and Flag antibodies. Total lysates was also
immunoblotted with CREB and GFP antibodies to verify the separation
of nuclear and cytoplasmic fractions. Finally, to determine whether
Isx treatment could induce hyperphosphorylation of HDAC5 in a
different cell type, the inventors also found a significant
increase in the percentage of Cos cells that exhibited cytoplasmic
localization of HDAC5 after drug treatment (from 80.9% Nuc and
13.6% Cyto in vehicle-treated cells to 39.5% Nuc and 46.2% Cyto in
Isx-treated cells).
[0284] The inventors next directly examined HDAC5 and MEF2 effects
on an endogenous MEF2 target gene, NR1, since the inventors did
observe an induction of NR1 mRNA with Isx treatment. Indeed an NR1
promoter luciferase construct that contains a functional
MEF2-binding site (SEQ ID NO: 1 (TTATTTATTTAG, -805 to -796)) was
induced with Isx treatment in a dose-dependent manner. Adenovirus
over-expression of HDAC5 suppressed NR1-luciferase activity by at
least two-fold compared to infection of a control GFP adenovirus
(AdCMV-GFP), in both vehicle- and Isx-treated NPCs. The HDAC5 S-A
mutant also suppressed NR1 activity in vehicle- and Isx-treated
NPCs. Similar results were seen with an additional MEF2 reporter
3XMRE-luc. NPCs were infected with a control GFP adenovirus at
equivalent titers to the HDAC5-GFP WT and S-A adenoviruses to
confirm a transduction efficiency of nearly 80-90%. These data
suggest Isx de-repression/activation of MEF2 in NPCs is associated
with class II HDAC, such as HDAC5, transcriptional regulation of
neuronal target genes.
[0285] To further confirm this, the inventors co-transfected NPCs
with a construct encoding a repressor form of MEF2C in NPCs, where
the repressor domain of engrailed is fused to MEF2C(CAG-MEF2C-ENG),
there was significantly less activation of NR1-luciferase in
vehicle-treated cultures compared to over-expression of a CAG-GFP
control plasmid. More interestingly, this was sufficient to block
Isx-activation of NR1-luciferase. In addition, the inventors
measured the neuronal reporter gene NeuroD-luciferase. As with
NR1-luciferase, there was significantly less NeuroD activity in
both vehicle- and Isx-treated NPCs when HDAC5 (WT), HDAC5 (S-A), or
the dominant repressor MEF2C-eng was over-expressed, compared to
NPCs that expressed a control GFP plasmid. Taken together, these
results suggest that the initial up-regulation of NR1 and NeuroD,
and potentially other Ca.sup.2+-activated neuronal genes after Isx
treatment is coupled to MEF2 activation in NPCs.
[0286] Isx Triggered HDAC5 Export and Neuronal Differentiation is
CamK-Dependent.
[0287] As mentioned previously, Class II HDACs are considered
signal-responsive repressors during cardiac hypertrophy and
nucleocytoplasmic shuttling of HDACs enables MEF2 to associate with
HATs. Shuttling of class II HDACs is dependent on phosphorylation
of two serine-containing motifs found at their N-termini, when
phosphorylated, these motifs are associated with 14-3-3 that masks
a nuclear localization sequence. The kinase(s) that phosphorylate
class II HDACs and transmit various extracellular signals down to
the genome have been mainly attributed to CaM kinases (CaMK) and
protein kinase D (PKD), which is phosphorylated and activated by
protein kinase C (PKC). To further define the signaling pathways
leading to phosphorylation and nuclear export of HDAC5, the
inventors tested inhibitors of CaMK and PKC for their abilities to
block Isx-induced neuronal differentiation. A specific inhibitor of
CaMK (KN93) was extremely effective in blocking Isx-mediated
reporter gene expression (3XMRE- and NeuroD-luciferase). In
contrast, G66976, a specific inhibitor of the Ca.sup.2+-dependent
PKC isozymes did not significantly affect Isx-induced reporter gene
expression. Most importantly, KN93 blocked HDAC5 phosphorylation in
Isx-treated NPCs, while G66976 did not. Autophosphorylation of CaMK
is usually required for maximal activity and results in the
formation of Ca.sup.2+-independent enzymes that is usually
associated with its ability to respond to different frequencies of
Ca.sup.2+ spikes, which is critical in neuronal cells for learning
and memory processes.
Example 6
Isx-9 Treatment Induces Differentiation and Affects Neuron-Specific
Gene and Protein Expression in Brain Tumor Stem Cells
[0288] CD133+ glioblastoma brain tumor stem cells (BTSCs) from an
explanted human tumor specimen were treated with 20 .mu.M Isx-9
(FIG. 9C) or vol/vol DMSO (vehicle) control (FIG. 9B). FIG. 9D
shows staining of the Isx-9-treated cells. Dose-dependent
activation of two neuron-specific reporter genes, NeuroD- and NMDA
receptor 1-luciferase was observed with Isx-9-treated BTSCs as
opposed to vehicle control-treated cells (FIGS. 9E and 9F). The
effect of 20 ng/mL FGF/EGF growth factors on BTSC differentiation
is shown in FIG. 9A. A compound from an unrelated class of small
molecules (sulfonyl hydrazone, Shz) did not significantly induce
reporter gene activity (FIG. 9F).
[0289] Up-regulation of NeuroD was observed in BTSCs treated with
Isx-9 for 2 days (FIG. 9G). This activity remained following
addition of FGF/EGF without additional Isx-9 addition. The
Kruppel-family zinc finger transcriptional regulator and
proto-oncogene neuron-restrictive silencer factor (NRSF) (FIG. 9H)
and CD133 are both downregulated with Isx-9-treatment.
[0290] Despite a dramatic change in cell morphology and attachment
mediated by Isx-9 treatment, overall levels of neuronal proteins,
such as .beta.TujIII and glutamate receptor 2 (GluR2) do not
significantly differ between control and Isx-9-treated BTSCs (FIG.
9H).
Example 7
Isoxazoles Induce Growth Arrest and Inhibit the Tumorigenic
Potential of Human BTSCs In Vitro and In Vivo
[0291] CD133(+) brain tumor stem cells (BTSCs) from an explanted
human tumor specimen were treated with EGF/FGF (FIG. 10A) or Isx-9
(20 .mu.M) (FIG. 10B) for 7 days before returning to EGF/FGF (FIG.
10C) or EGF/FGF plus Isx-9 conditions (FIG. 10D) for an additional
2 days. FIGS. 10A-D demonstrate the growth inhibition effects of
Isx-9 on these cells. A 7-day exposure to Isx-9 promotes an
irreversible state of cell attachment and differentiation, even
when challenged with growth factors.
[0292] The number and size of neurospheres observed in 7-day
pretreated BTSCs with DMSO (vehicle), EGF/FGF, or Isx-9 that were
dissociated and re-plated to form secondary neurospheres (1000
cells/well) under various conditions is shown in FIG. 10F. Isx-9
pre-treatment mediates a decrease in the average number and size of
each secondary neurosphere (an indicator of stem cell self-renewal
activity) compared to EGF/FGF pre-treatment, even when growth
factors are added back for an additional 7 days.
[0293] Isx-9 pre-treatment for 7 days leads to decreased DNA
synthesis/proliferation (BrdU uptake with a 1 hour pulse prior to
fixation) compared to EGF/FGF or vehicle pre-treatment, even when
EGF/FGF is added back for 24 hours (FIGS. 10G-J).
[0294] Seven-day exposure of RFP-labeled human CD133(+) BTSCs to 20
ng/ml EGF/FGF (FIGS. 10K and 10L) or Isx-9 (20 .mu.M) (FIGS. 11M
and 11N) in vitro, before transplantation into the striatum of
NOD/scid mice, dramatically reduces tumor-initiating ability. Isx-9
pre-treatment significant reduced the number of RFP-labeled BTSCs
in the striatum after 1 month (n=3 mice/group, 30,000 cells
injected/striatum). BTSC transplantation was performed by
stereotactic surgery into the left striatum (coordinates from
Bregma AP -0 mm, ML +2.5 mm, DV -3.5 mm) with a 33-gauge Hamilton
syringe at a flow rate of 30,000 cells/10 minutes.
[0295] All of the methods and apparatuses disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the methods and apparatuses and in the
steps or in the sequence of steps of the methods described herein
without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
REFERENCES
[0296] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0297] U.S. Pat. No. 3,817,837 [0298] U.S. Pat. No. 3,826,364
[0299] U.S. Pat. No. 3,850,752 [0300] U.S. Pat. No. 3,939,350
[0301] U.S. Pat. No. 3,996,345 [0302] U.S. Pat. No. 4,275,149
[0303] U.S. Pat. No. 4,277,437 [0304] U.S. Pat. No. 4,284,412
[0305] U.S. Pat. No. 4,366,241 [0306] U.S. Pat. No. 4,411,990
[0307] U.S. Pat. No. 4,472,509 [0308] U.S. Pat. No. 4,498,766
[0309] U.S. Pat. No. 4,661,913 [0310] U.S. Pat. No. 4,680,338
[0311] U.S. Pat. No. 4,714,682 [0312] U.S. Pat. No. 4,767,206
[0313] U.S. Pat. No. 4,774,189 [0314] U.S. Pat. No. 4,857,451
[0315] U.S. Pat. No. 4,938,948 [0316] U.S. Pat. No. 4,989,977
[0317] U.S. Pat. No. 5,021,236 [0318] U.S. Pat. No. 5,141,648
[0319] U.S. Pat. No. 5,160,974 [0320] U.S. Pat. No. 5,196,066
[0321] U.S. Pat. No. 5,478,722 [0322] U.S. Pat. No. 5,563,250
[0323] U.S. Pat. No. 5,856,456 [0324] U.S. Pat. No. 5,880,270
[0325] U.S. Publ. Appl. No. 2005/0164382 [0326] U.S. Publ. Appl.
No. 2005/0277162 [0327] Abeyta et al., Hum. Mol. Genet.,
13(6):601-608, 2004. [0328] Al-Hajj et al., Proc. Natl. Acad. Sci.
USA, 100:3983-3988, 2003. [0329] Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, 1988. [0330] Atherton et al., Biol.
of Reproduction, 32:155-171, 1985. [0331] Berberian et al.,
Science, 261:1588-1591, 1993. [0332] Bergsagel et al. Cancer Res.,
28:2187-2196, 1968. [0333] Bonnet and Dick, Nature Med., 3,
730-737, 1997. [0334] Bruce et al., Nature, 199:79-80, 1963. [0335]
Bundgaard, Drugs of the Future, 16:443-458, 1991. [0336] Bundgaard,
In: Design of Prodrugs, 7-9; 21-24, Elsevier, Amsterdam, 1985.
[0337] Cicchi et al., In: Progress in Heterocyclic Chemistry,
Elsevier, 2003. [0338] Cleary et al., Trends Microbiol., 4:131-136,
1994. [0339] Cooper, In: Elements Of Human Cancer, Jones and
Bartlett Publishers, ISBN: 0867201916, 1992. [0340] Dholakia et
al., J. Biol. Chem., 264, 20638-20642, 1989. [0341] Dontu et al.,
Genes Dev., 17:1253-1270, 2003. [0342] Gage et al., Proc. Natl.
Acad. Sci. USA, 92:11879-11883, 1995. [0343] Gage, Science,
287:1433-1438, 2000. [0344] Greene and Wuts, In: Protecting Groups
in Organic Synthesis, 3.sup.rd ed., John Wiley & Sons, Inc.,
1999. [0345] Grunanger and Vita-Finzi, In: The Chemistry of
Heterocyclic Compounds: Isoxazoles, Wiley-Interscience, NY, 1991.
[0346] Gudjonsson et al., Genes Dev., 16:693-706, 2002. [0347]
Habara-Ohkubo, A., Cell Struct Funct., 21, 101-110, 1996. [0348]
Hamburger and Salmon, Science, 197: 461-463, 1977. [0349] Handbook
of Pharmaceutical Salts: Properties, Selection and Use, Stahl and
Wermuth (Eds.), Verlag Helvetica Chimica Acta, 2002. [0350] Hsieh
et al., J. Cell Biol., 164:111-122, 2004. [0351] Hsieh et al.,
Proc. Natl. Acad. Sci. USA 101, 16659-16664, 2004. [0352] Kang et
al., Science, 240:1034-1036, 1988. [0353] Khatoon et al., Ann. of
Neurology, 26, 210-219, 1989. [0354] King et al., J. Biol. Chem.,
269, 10210-10218, 1989. [0355] Kohler et al., Methods Enzymol.,
178:3, 1989. [0356] Kreier et al., In: Infection, Resistance and
Immunity, Harper and Row, New York, 1991. [0357] Lagasse et al.
Immunity, 14:425-436, 2001. [0358] Lenert et al., Science,
248:1639-1643, 1990. [0359] O'Shannessy et al., J. Immun. Meth.,
99, 153-161, 1987. [0360] Owens and Haley, J. Biol. Chem.,
259:14843-14848, 1987. [0361] Palmer et al., Mol. Cell Neurosci.,
8:389-404, 1997. [0362] Palmer et al., Nature, 411(6833):42-43,
2001. [0363] Park et al., J. Nat. Cancer Inst., 46:411-422, 1971.
[0364] Perez-Losada and Balmain, Nat. Rev. Cancer, 3:434-443, 2003.
[0365] Potter and Haley, Meth. in Enzymol., 91, 613-633, 1983.
[0366] Ramalho-Santos et al., Science, 298:597-600, 2002. [0367]
Ray and Gage, Mol. Cell Neurosci., 31(3):560-573, 2006. [0368] Reya
et al., Nature, 414:105-111, 2001. [0369] Sasso et al., J.
Immunol., 142:2778-2783, 1989. [0370] Shorki et al., J. Immunol.,
146:936-940, 1991. [0371] Silvermann et al., J. Clin. Invest.,
96:417-426, 1995. [0372] Tumbar et al., Science, 303(5656):359-363,
2004. [0373] Welm et al., Develop. Biology, 245:42-56, 2002. [0374]
Wodinsky et al., Cancer Chemother. Rep., 51:415-421, 1967. [0375]
Zepeda et al., Somat. Cell Mol. Genet., 21:61-73, 1999. [0376]
Zhang et al., J. Biomol. Screen, 4:67-73, 1999.
Sequence CWU 1
1
1112DNAArtificialSynthetic primer 1ttatttattt ag 12
* * * * *