Note: Descriptions are shown in the official language in which they were submitted.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
METHODS FOR SPECIFICALLY INHIBITING HISTONE
DEACETYLASE-4
(Case No. MET-002PC)
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the fields of molecular biology and
medicine. More specifically, the invention relates to the fields of gene
expression and oncology.
Summary of the Related Art
Chromatin is the complex of proteins and DNA in the nucleus of
eukaryotes. Chromatin proteins provide structural and functional
organization to nuclear DNA. The nucleosome is the fundamental unit of
structural organization of chromatin. The nucleosome principally consists of
(1) the core histones, termed H2A, H2B, H3, and H4, which associate to form a
protein core particle, and (2) the approximately 146 base pairs of DNA
wrapped around the histone core particle. The physical interaction between
the core histone particle and DNA principally occurs through the negatively
charged phosphate groups of the DNA and the basic amino acid moieties of
the histone proteins. (Csordas, Biochem. J., 286:23-38 (1990)) teaches that
histones are subject to posttranslaHonal acetylation of their epsilon-amino
groups of N-terminal lysine residues, a reaction that is catalyzed by histone
acetyl transferase (HAT). The posttranslational acetylation of histones has
both structural and functional, i.e., gene regulatory, consequences.
Acetylation neutralizes the positive charge of the epsilon-amino groups
of N-terminal lysine residues, thereby influencing the interaction of DNA
with the histone core particle of the nucleosome. Thus, histone acetylaHon
and histone deacetylation (HDAC) are thought to impact chromatin structure
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
and gene regulation. For example, Taunton et al., Science, 272:408-411 (1996),
teaches that access of transcription factors to chromatin templates is
enhanced
by histone hyperacetylation. Taunton et al. further teaches that an enrichment
in underacetylated histone H4 has been found in transcriptionally silent
regions of the genome.
Studies utilizing known HDAC inhibitors have established a link
between acetylation and gene expression. Yoshida et al, Cancer Res. 47:3688-
3691 (1987) discloses that (R)-Trichostatin A (TSA) is a potent inducer of
differentiation in murine erythroleukemia cells. Yoshida et al., J. Biol.
Chem.
265:17174-17179 (1990) teaches that TSA is a potent inhibitor of mammalian
HDAC.
Numerous studies have examined the relationship between HDAC and
gene expression. Taunton et al., Science 272:408-411 (1996), discloses a human
HDAC that is related to a yeast transcriptional regulator. Cress et al., J.
Cell.
Phys. 184:1-16 (2000), discloses that, in the context of human cancer, the
role of
HDAC is as a coreprescor of transcription. Ng et al., TIBS 25:March (2000),
discloses HDAC as a pervasive feature of transcriptional repressor systems.
Magnaghi-Jaulin et al., Prog. Cell Cycle Res. 4:41-47 (2000), discloses HDAC
as a
transcriptional co-regulator important for cell cycle progression.
The molecular cloning of gene sequences encoding proteins with
HDAC activity has established the existence of a set of discrete HDAC
enzyme isoforms. Grozinger et al., Proc. Natl. Acad. Sci. USA, 96:4868-4873
(1999), teaches that HDACs may be divided into two classes, the first
represented by yeast Rpd3-like proteins, and the second represented by yeast
Hda1-like proteins. Grozinger et al. also teaches that the human HDAC-1,
HDAC-2, and HDAC-3 proteins are members of the first class of HDACs, and
discloses new proteins, named HDAC-4, HDAC-5, and HDAC-6, which are
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
members of the second class of HDACs. Kao et al., Gene ~ Development 14:55-
66 (2000), discloses an additional member of this second class, called HDAC-7.
More recently, Hu, E. et al. J. Bio. Chem. 275:15254-13264 (2000) discloses
the
newest member of the first class of histone deacetylases, HDAC-8. It has been
unclear what roles these individual HDAC enzymes play.
Known inhibitors of mammalian HDAC have been used to probe the
role of HDAC in gene regulation for some time. Yoshida et al., J. Biol. Chem.
265:17174-17179 (1990) discloses that (R)-Trichostatin A (TSA) is a potent
inhibitor of mammalian HDAC. Yoshida et al, Cancer Res. 47:3688-3691 (1987)
discloses that TSA is a potent inducer of differentiation in murine
erythroleukemia cells.
Known inhibitors of histone deacetylase are all small molecules that
inhibit histone deacetylase activity at the protein level. Moreover, all of
the
known histone deacetylase inhibitors are non-specific for a particular histone
deacetylase isoform, and more or less inhibit all members of both the histone
deacetylase families equally. (Grozinger, C.M., et al., Proc. Natl. Acad. Sci.
U.S.A. 96:4868-4873 (1999)). For example, see Marks et al., J. National Cancer
Inst. 92:1210-1216 (2000), which reviews histone deacetylase inhibitors and
their role in studying differentiation and apoptosis.
Therefore, there remains a need to develop reagents for inhibiting
specific histone deacetylase isoforms. There is also a need for the
development of methods for using these reagents to modulate the activity of
specific histone deacetylase isoforms and to identify those isoforms involved
in tumorigenesis and other proliferative diseases and disorders.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
BRIEF SUMMARY OF THE INVENTION
The invention provides methods and reagents for modulating the
activity of histone deacetylase (HDAC) isoforms . For example, the invention
provides methods and reagents for inhibiting HCAC isoforms, particularly
HDAC-1 and HDAC-4, by inhibiting expression at the nucleic acid level or
enzymatic activity at the protein level. The invention provides for the
specific
inhibition of specific histone deacetylase isoforms involved in tumorigenesis
and thus provides a treatment for cancer. The invention further provides for
the specific inhibition of particular HDAC isoforms involved in cell
proliferation, and thus provides a treatment for cell proliferative diseases
and
disorders.
The inventors have made the surprising discovery that the specific
inhibition of HDAC-4 dramatically induces apoptosis and growth arrest in
cancerous cells. Accordingly, in a first aspect, the invention provides agents
that inhibit the activity of the HDAC-4 isoform.
In certain preferred embodiments of the first aspect of the invention,
the agent that inhibits the HDAC-4 isoform is an oligonucleotide that inhibits
expression of a nucleic acid molecule encoding the HDAC-4 isoform. The
nucleic acid molecule encoding the HDAC-4 isoform may be genomic DNA
(e.g., a gene), cDNA, or RNA. In some embodiments, the oligonucleotide
inhibits transcription of mRNA encoding the HDAC-4 isoform. In other
embodiments, the oligonucleotide inhibits translation of the HDAC-4 isoform.
In certain embodiments the oligonucleotide causes the degradation of the
nucleic acid molecule.
In a preferred embodiment thereof, the agent of the first aspect of the
invention is an antisense oligonucleotide complementary to a region of RNA
that encodes a portion of HDAC-4 or to a region of double-stranded DNA
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
that encodes a portion of HDAC-4. In one embodiment thereof, the antisense
oligonucleotide is a chimeric oligonucleotide. In another embodiment
thereof, the antisense oligonucleotide is a hybrid oligonucleotide. In another
embodiment thereof, the antisense oligonucleotide has a nucleotide sequence
of from about 13 to about 35 nucleotides selected from the nucleotide
sequence of SEQ ID N0:4. In still yet another embodiment thereof, the
antisense oligonucleotide has a nucleotide sequence of from about 15 to about
26 nucleotides selected from the nucleotide sequence of SEQ ID N0:4. In
another embodiment.thereof, the antisense oligonucleotide has a nucleotide
sequence of from about 20 to about 26 nucleotides selected from the
nucleotide sequence of SEQ ID N0:4. In another embodiment thereof, the
antisense oligonucleotide has a nucleotide sequence of from about 13 to about
35 nucleotides and which comprises the nucleotide sequence of SEQ ID
N0:11. In still yet another embodiment thereof, the antisense oligonucleotide
has a nucleotide sequence of from about 15 to about 26 nucleotides and which
comprises the nucleotide sequence of SEQ ID N0:11. In another embodiment
thereof, the antisense oligonucleotide has a nucleotide sequence of from about
to about 26 nucleotides and which comprises the nucleotide sequence of
SEQ ID N0:11. In another embodiment thereof, the antisense oligonucleotide
20 is SEQ ID N0:11. In another embodiment thereof, the antisense
oligonucleodde has one or more phosphorothioate internucleoside linkages.
In another embodiment thereof, the antisense oligonucleotide further
comprises a length of 20-26 nucleotides. In still another embodiment thereof,
the antisense oligonucleotide is modified such that the terminal four
nucleotides at the 5' end of the oligonucleotide and the terminal four
nucleotides at the 3' end of the oligonucleotide each have 2' -O- methyl
groups
attached to their sugar residues.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In certain preferred embodiments of the first aspect, the agent that
inhibits the HDAC-4 isoform in a cell is a small molecule inhibitor that
inhibits expression of a nucleic acid molecule encoding HDAC-4 isoform or
activity of the HDAC-4 protein.
In a second aspect, the invention provides a method for inhibiting
HDAC-4 activity in a cell, comprising contacting the cell with a specific
inhibitor of HDAC-4, whereby HDAC-4 activity is inhibited. In an
embodiment thereof, the invention provides method for inhibiting the
HDAC-4 isoform in a cell, comprising contacting the cell with an anHsense
oligonucleotide complementary to a region of RNA that encodes a portion of
HDAC-4 or to a region of double-stranded DNA that encodes a portion of
HDAC-4, whereby HDAC-4 activity is inhibited. In one embodiment thereof,
the cell is contacted with an HDAC-4 anHsense oligonucleotide that is a
chimeric oligonucleotide. In another embodiment thereof, the cell is
contacted with an HDAC-4 antisense oligonucleotide that is a hybrid
oligonucleotide. In another embodiment thereof, the antisense
oligonucleotide has a nucleotide sequence of from about 13 to about 35
nucleotides selected from the nucleotide sequence of SEQ ID N0:4. In still yet
another embodiment thereof, the antisense oligonucleotide has a nucleotide
sequence of from about 15 to about 26 nucleotides selected from the
nucleotide sequence of SEQ ID N0:4. In another embodiment thereof, the
antisense oligonucleotide has a nucleotide sequence of from about 20 to about
26 nucleotides selected from the nucleotide sequence of SEQ ID N0:4. In yet
another embodiment thereof, the cell is contacted with an HDAC-4 antisense
oligonucleotide that has a nucleotide sequence length of from about 13 to
about 35 nucleotides and which comprises the nucleotide sequence of SEQ ID
N0:11. In another embodiment thereof, the cell is contacted with an HDAC-4
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
antisense oligonucleotide that has a nucleotide sequence length of from about
15 to about 26 nucleotides and which comprises the nucleotide sequence of
SEQ ID N0:11. In another embodiment thereof, the cell is contacted with an
HDAC-4 antisense oligonucleotide that is SEQ ID N0:11. In another
embodiment thereof, the inhibition of HDAC-4 activity leads to the inhibition
of cell proliferation in the contacted cell. In another embodiment thereof,
the
inhibition of HDAC-4 activity in the contacted cell further leads to growth
retardation of the contacted cell. In another embodiment thereof, the
inhibition of HDAC-4 activity in the contacted cell further leads to growth
arrest of the contacted cell. In another embodiment thereof, the inhibition of
HDAC-4 activity in the contacted cell further leads to programmed cell death
of the contacted cell. In another embodiment thereof, the inhibition of
HDAC-4 activity in the contacted cell further leads to necrotic cell death of
the
contacted cell. In certain embodiments thereof, the cell is a neoplastic cell
which may be in an animal, including a human, and which may be in a
neoplastic growth. In certain preferred embodiments, the method further
comprises contacting the cell with an HDAC-4 small molecule inhibitor that
interacts with and reduces the enzymatic activity of the HDAC-4 histone
deacetylase isoform. In some embodiments thereof, the histone deacetylase
small molecule inhibitor is operably associated with the andsense
oligonucleotide.
In a third aspect, the invention provides a method for inhibiting
neoplastic cell proliferation in an animal, comprising administering to an
animal having at least one neoplastic cell present in its body a
therapeutically
effective amount of a specific inhibitor of HDAC-4, whereby neoplastic cell
proliferation is inhibited in the animal. In an embodiment thereof, the
invention provides a method for inhibiting neoplastic cell growth in an
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
animal, comprising administering to an animal having at least one neoplastic
cell present in its body a therapeutically effective amount of the antisense
oligonucleotide of the first aspect of the invention with a pharmaceutically
acceptable carrier for a therapeutically effective period of time. In an
embodiment thereof, the animal is administered a chimeric HDAC-4 antisense
oligonucleotide. In another embodiment thereof, the animal is administered a
hybrid HDAC-4 antisense oligonucleotide. In another embodiment thereof,
the antisense oligonucleotide has a nucleotide sequence of from about 13 to
about 35 nucleotides selected from the nucleotide sequence of SEQ ID N0:4.
In still yet another embodiment thereof, the antisense oligonucleotide has a
nucleotide sequence of from about 15 to about 26 nucleotides selected from
the nucleotide sequence of SEQ ID N0:4. In another embodiment thereof, the
antisense oligonucleotide has a nucleotide sequence of from about 20 to about
26 nucleotides selected from the nucleotide sequence of SEQ ID N0:4. In
another embodiment thereof, the animal is administered an HDAC-4
antisense oligonucleotide having a nucleotide sequence of from about 13 to
about 35 nucleotides and which comprises the nucleotide sequence of SEQ ID
N0:11. In another embodiment thereof, the animal is administered an
HDAC-4 antisense oligonucleotide having a nucleotide sequence of from
about 15 to about 26 nucleotides and which comprises the nucleotide
sequence of SEQ ID N0:11. In another embodiment thereof, the animal is
administered an HDAC-4 antisense oligonucleotide that is SEQ ID N0:11. In
another embodiment thereof, the animal is a human. In another embodiment
thereof, the method further comprises administering to an animal a
therapeutically effective amount of an antisense oligonucleotide
complementary to a region of RNA that encodes a portion of HDAC-1 or
double-stranded DNA that encodes a portion of HDAC-1. In an embodiment
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
thereof, the animal is administered a chimeric HDAC-1 antisense
oligonucleotide. In another embodiment thereof, the animal is administered a
hybrid HDAC-1 antisense oligonucleotide. In another embodiment thereof,
the antisense oligonucleotide has a nucleotide sequence of from about 13 to
about 35 nucleotides selected from the nucleotide sequence of SEQ ID N0:2.
In still yet another embodiment thereof, the antisense oligonucleotide has a
nucleotide sequence of from about 15 to about 26 nucleotides selected from
the nucleotide sequence of SEQ ID N0:2. In another embodiment thereof, the
antisense oligonucleotide has a nucleotide sequence of from about 20 to about
26 nucleotides selected from the nucleotide sequence of SEQ ID N0:2. In
another embodiment thereof, the animal is administered an HDAC-1
antisense oligonucleotide having a nucleotide sequence of from about 13 to
about 35 nucleotides and which comprises the nucleotide sequence of SEQ ID
N0:5. In another embodiment thereof, the animal is administered an HDAC-
1 antisense oligonucleotide having a nucleotide sequence of from about 15 to
about 26 nucleotides and which comprises the nucleotide sequence of SEQ ID
N0:5. In yet another embodiment thereof, the animal is administered an
HDAC-1 antisense oligonucleoHde that is SEQ ID N0:5.
In fourth aspect, the invention provides a method for inhibiting
HDAC-4 activity in a cell, comprising contacting the cell with a small
molecule inhibitor of HDAC-4, wherein HDAC-4 activity is inhibited.
In one embodiment thereof, the cell is contacted with a small molecule
inhibitor having the structure
Cy-CH(OMe)-Yl-C(O)-NH-Z (1)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; Yl is a C4 - C6 alkylene, wherein said alkylene may
be optionally substituted and wherein one of the carbon atoms of the alkylene
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
optionally may be replaced by a heteroatom moiety selected from the group
consisting of O; NRI, Rl being alkyl, acyl or hydrogen; S; S(O); or S(O)z; and
Z
is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl and -
O-
M, M being H or a pharmaceutically acceptable canon, wherein the anilinyl or
pyridyl or thiadiazolyl may be optionally substituted.
In another embodiment thereof, the invention provides a method
wherein the cell is contacted with a small molecule inhibitor having the
structure
Cy-Yz-C(O)NH-Z
(2)
wherein Cy is cycloallcyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; Yz is Cs - C~ alkylene, wherein said alkylene may
be
optionally substituted and wherein one of the carbon atoms of the alkylene
optionally may be replaced by a heteroatom moiety selected from the group
consisting of O; NRl, Rl being alkyl, acyl or hydrogen; S; S(O); or S(O)z; and
Z
is anilinyl or pyridyl, or thiadiazolyl, any of which may be optionally
substituted.
In another embodiment thereof, the invention provides a method
wherein the cell is contacted with a small molecule inhibitor having the
structure
Cy-B-Y3-C(O)-NH-Z
(3)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; B is selected from the group consisting of -
CH(OMe), ketone and methylene; Y3 is a C4 - C6 alkylene, wherein said
alkylene may be optionally substituted and wherein one of the carbon atoms
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
of the alkylene optionally may be replaced by a heteroatom moiety selected
from the group consisting of O; NRl, Rl being alkyl, acyl or hydrogen; S;
S(O); or S(O)z; and Z is selected from the group consisting of anilinyl,
pyridyl, thiadiazolyl and -O-M, M being H or a pharmaceutically acceptable
canon, wherein the anilinyl or pyridyl or thiadiazolyl may be optionally
substituted.
In another embodiment thereof, the invention provides a method
wherein the cell is contacted with a small molecule inhibitor having the
structure
Cy-L1-Ar-Y1-C(O)-NH-Z (4)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; L~ is -(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W
is selected from the group consisting of -C(O)NH-, -S(O)zNH-, -NHC(O)-,
-NHS(O)z-, and -NH-C(O)-NH-; Ar is arylene, wherein said arylene
optionally may be additionally substituted and optionally may be fused to
an aryl or heteroaryl ring, or to a saturated or partially unsaturated
cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Yl is a chemical bond or a straight- or branched-chain saturated alkylene,
wherein said alkylene may be optionally substituted; and Z is selected from
the group consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, M being H
or a pharmaceutically acceptable canon; provided that when L~ is -
C(O)NH-, Yl is -(CH2)~ , n being 1, 2, or 3, and Z is -O-M, then Cy is not
aminophenyl, dimethylaminophenyl, or hydroxyphenyl; and further
provided that when Ll is -C(O)NH- and Z is pyridyl, then Cy is not
substituted indolinyl.
11
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In another embodiment thereof, the invention provides a method
wherein the cell is contacted with a small molecule inhibitor having the
structure
Cy-Lz-Ar-Y2-C(O)NH-Z (5)
wherein Cy is cycloallcyl, aryl, heteroaryl, or heterocyclyl, any of which may
be
optionally substituted, provided that Cy is not a .
(spirocycloalkyl)heterocyclyl; LZ is C,-C6 saturated alkylene or CZ-C6
alkenylene, wherein the alkylene or alkenylene optionally may be
substituted, provided that LZ is not -C(O)-, and wherein one of the carbon
atoms of the alkylene optionally may be replaced by a heteroatom moiety
selected from the group consisting of O; NR', R' being alkyl, aryl, or
hydrogen; S; S(O); or S(O)z; Ar is arylene, wherein said arylene optionally
may be additionally substituted and optionally may be fused to an aryl or
heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or
heterocyclic ring, any of which may be optionally substituted; and Yz is a
chemical bond or a straight- or branched-chain saturated alkylene, which
may be optionally substituted, provided that the alkylene is not substituted
with a subsntuent of the formula -C(O)R wherein R comprises an a-amino
aryl moiety; and Z is selected from the group consisting of anilinyl, pyridyl,
thiadiazolyl, and -O-M, M being H or a pharmaceutically acceptable canon;
provided that when the carbon atom to which Cy is attached is oxo
substituted, then Cy and Z are not both pyridyl.
In another embodiment thereof, the invention provides a method
wherein the cell is contacted with a small molecule inhibitor has the
structure
Cy-L3-Ar-Y3-C(O) NH-Z (6)
12
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted, provided that Cy is not a
(spirocycloalkyl)heterocyclyl; L3 is selected from the group consisting of (a)
-(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W is selected from the group
consisting of -C(O)NH-, -S(O)zNH-, -NHC(O)-, -NHS(O)z-, and
-NH-C(O)-NH-; and (b) C,-C6 alkylene or CZ-C6 alkenylene, wherein the
alkylene or alkenylene optionally may be substituted, provided that L3 is not
-C(O)-, and wherein one of the carbon atoms of the alkylene optionally may
be replaced by O; NR', R' being alkyl, aryl, or hydrogen; S; S(O); or S(O)z;
Ar
is arylene, wherein said arylene optionally may be additionally substituted
and optionally may be fused to an aryl or heteroaryl ring, or to a saturated
or partially unsaturated cycloalkyl or heterocyclic ring, any of which may be
optionally substituted; and Y3 is Cz alkenylene or Cz alkynylene, wherein
one or both carbon atoms of the alkenylene optionally may be substituted
with alkyl, aryl, alkaryl, or aralkyl; and Z is selected from the group
consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, M being H or a
pharmaceutically acceptable cation; provided that when Cy is unsubstituted
phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each
other.
In another embodiment thereof, the invention provides a method wherein
the cell is contacted with a small molecule inhibitor having the structure
selected from the group consisting of
0
\ N.OH
H
S~N
0 H
13
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
o /
~ N ~ I
H
NHz
n
/ S~N /
o H (8) and
g I~
p H / ~ / N
O I /
(9).
In another embodiment therein, the invention provides a method
wherein the inhibition of HDAC-4 activity in the contacted cell further leads
to an inhibition of cell proliferation in the contacted cell. In another
embodiment therein, the invention provides a method wherein inhibition of
HDAC-4 activity in the contacted cell further leads to growth retardation of
the contacted cell. In another embodiment therein, the invention provides a
method wherein inhibition of HDAC-4 activity in the contacted cell further
leads to growth arrest of the contacted cell. In another embodiment therein,
the invention provides a method wherein inhibition of HDAC-4 activity in the
contacted cell further leads to programmed cell death of the contacted cell.
In
another embodiment therein, the invention provides a method wherein
inhibition of HDAC-4 activity in the contacted cell further leads to necrotic
cell death of the contacted cell. In another embodiment, thereof, the
contacted
cell is a human cell.
In fifth aspect, the invention provides a method for inhibiting
neoplastic cell proliferation in an animal, comprising administering to an
animal having at least one neoplastic cell present in its body a
therapeutically effective amount of a small molecule inhibitor of HDAC-4,
whereby neoplastic cell proliferation is inhibited. In one embodiment
thereof, the animal is administered a small molecule inhibitor having the
14
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
structure
Cy-CH(OMe)-Y1-C(O)-NH-Z (1)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; Yl is a Cø - C6 alkylene, wherein said alkylene may
be optionally substituted and wherein one of the carbon atoms of the alkylene
optionally may be replaced by a heteroatom moiety selected from the group
consisting of O; NRI, Rl being alkyl, aryl or hydrogen; S; S(O); or S(O)z; and
Z
is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl and -
O-
M, M being H or a pharmaceutically acceptable canon, wherein the anilinyl or
pyridyl or thiadiazolyl may be optionally substituted. In
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
another embodiment thereof, the invention provides a method wherein the
animal is administered a small molecule inhibitor having the structure
Cy-YZ-C(O)NH-Z
(2)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; Y2 is Cs - C~ alkylene, wherein said alkylene may
be
optionally substituted and wherein one of the carbon atoms of the alkylene
optionally may be replaced by a heteroatom moiety selected from the group
consisting of O; NRl, Ri being alkyl, aryl or hydrogen; S; S(O); or S(O)2; and
Z
is anilinyl or pyridyl or thiadiazolyl, any of which may be optionally
substituted. In another embodiment thereof, the invention provides a method
wherein the animal is administered a small molecule inhibitor having the
structure
Cy-B-Y3-C(O)-NH-Z (3)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; B is selected from the group consisting of -
CH(OMe), ketone and methylene; Y3 is a C4 - C6 alkylene, wherein said
alkylene may be optionally substituted and wherein one of the carbon atoms
of the alkylene optionally may be replaced by a heteroatom moiety selected
from the group consisting of O; NRl, Rl being alkyl, acyl or hydrogen; S;
S(O); or S(O)2; and Z is selected from the group consisting of anilinyl,
pyridyl, thiadiazolyl and -O-M, M being H or a pharmaceutically acceptable
canon, wherein the anilinyl or pyridyl or thiadiazolyl may be optionally
substituted. In another embodiment thereof, the invention provides a
method wherein the animal is administered a small molecule inhibitor
having the structure
16
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Cy-Ll-Ar-Yl-C(O)-NH-Z (4)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted; L' is -(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W
is selected from the group consisting of -C(O)NH-, -S(O)zNH-, -NHC(O)-,
-NHS(O)z-, and -NH-C(O)-NH-; Ar is arylene, wherein said arylene
optionally may be additionally substituted and optionally may be fused to
an aryl or heteroaryl ring, or to a saturated or partially unsaturated
cycloalkyl or heterocyclic ring, any of which may be optionally substituted;
Yl is a chemical bond or a straight- or branched-chain saturated alkylene,
wherein said alkylene may be optionally substituted; and Z is selected from
the group consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, M being H
or a pharmaceutically acceptable canon; provided that when L~ is -
C(O)NH-, Yl is -(CHz)~ , n being 1, 2, or 3, and Z is -O-M, then Cy is not
aminophenyl, dimethylaminophenyl, or hydroxyphenyl; and further
provided that when L~ is -C(O)NH- and Z is pyridyl, then Cy is not
substituted indolinyl. In another embodiment thereof, the invention
provides a method wherein the animal is administered a small molecule
inhibitor having the structure
Cy-Lz-Ar-YZ-C(O)NH-Z (5)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted, provided that Cy is not a
(spirocycloalkyl)heterocyclyl; Lz is C,-C6 saturated alkylene or Cz-C6
alkenylene, wherein the alkylene or alkenylene optionally may be
substituted, provided that Lz is not -C(O)-, and wherein one of the carbon
atoms of the alkylene optionally may be replaced by a heteroatom moiety
selected from the group consisting of O; NR', R' being alkyl, aryl, or
17
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
hydrogen; S; S(O); or S(O)z; Ar is arylene, wherein said arylene optionally
may be additionally substituted and optionally may be fused to an aryl or
heteroaryl ring, or to a saturated or partially unsaturated cycloalkyl or
heterocyclic ring, any of which may be optionally substituted; and Yz is a
chemical bond or a straight- or branched-chain saturated alkylene, which
may be optionally substituted, provided that the alkylene is not substituted
with a substituent of the formula -C(O)R wherein R comprises an a-amino
acyl moiety; and Z is selected from the group consisting of anilinyl, pyridyl,
thiadiazolyl, and -O-M, M being H or a pharmaceutically acceptable canon;
provided that when the carbon atom to which Cy is attached is oxo
substituted, then Cy and Z are not both pyridyl. In another embodiment
thereof, the invention provides a method wherein the animal is
administered a small molecule inhibitor having the structure
Cy-L3-Ar-Y3-C(O)NH-Z (6)
wherein Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
be optionally substituted, provided that Cy is not a
(spirocycloalkyl)heterocyclyl; L3 is selected from the group consisting of (a)
-(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W is selected from the group
consisting of -C(O)NH-, -S(O)zNH-, -NHC(O)-, -NHS(O)z-, and
-NH-C(O)-NH-; and (b) C,-C6 alkylene or Cz-C6 alkenylene, wherein the
alkylene or alkenylene optionally may be substituted, provided that L3 is not
-C(O)-, and wherein one of the carbon atoms of the alkylene optionally may
be replaced by O; NR', R' being alkyl, aryl, or hydrogen; S; S(O); or S(O)z;
Ar
is arylene, wherein said arylene optionally may be additionally substituted
and optionally may be fused to an aryl or heteroaryl ring, or to a saturated
or partially unsaturated cycloallcyl or heterocyclic ring, any of which may be
optionally substituted; and Y3 is Cz alkenylene or Cz alkynylene, wherein
18
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
one or both carbon atoms of the alkenylene optionally may be substituted
with alkyl, aryl, alkaryl, or aralkyl; and Z is selected from the group
consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, M being H or,a
pharmaceutically acceptable canon; provided that when Cy is unsubsntuted
phenyl, Ar is not phenyl wherein L3 and Y3 are oriented ortho or meta to each
other. In another embodiment thereof, the invention provides a method
wherein the animal is
19
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
administered a small molecule inhibitor having the structure selected from
the group consisting of
0
\ N.OH
O I \ ~ H
0 H
O
\ N ~ I
R I \ ~ N~
i
p\H
(8) and
o I
\ / N
O H / \ H \
o I ~ (9).
In another embodiment thereof, the invention provides a method
wherein the animal administered a small molecule inhibitor is a human.
In a sixth aspect, the invention provides a method for inhibiting the
induction of cell proliferation, comprising contacting a cell with an
antisense
oligonucleotide that inhibits the expression of HDAC-4 and/or contacting a
cell with a small molecule inhibitor of HDAC-4. In certain preferred
embodiments, the cell is a neoplastic cell, and the induction of cell
proliferation is tumorigenesis.
In a seventh aspect, the invention provides a method for identifying a
small molecule histone deacetylase inhibitor that inhibits the HDAC-4
isoform, the isoform being required for the induction of cell proliferation.
The
method comprises contacting the HDAC-4 isoform with a candidate small
molecule inhibitor and measuring the enzymatic activity of the contacted
histone deacetylase isoform, wherein a reduction in the enzymatic activity of
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
the contacted HDAC-4 isoform identifies the candidate small molecule
inhibitor as a small molecule histone deacetylase inhibitor of the HDAC-4
isoform.
In an eighth aspect, the invention provides a method for identifying a
small molecule histone deacetylase inhibitor that inhibits HDAC-4 isoform,
which is involved in the induction of cell proliferation. The method
comprises contacting a cell with a candidate small molecule inhibitor and
measuring the enzymatic activity of the contacted histone deacetylase
isoform, wherein a reduction in the enzymatic activity of the HDAC-4 isoform
identifies the candidate small molecule inhibitor as a small molecule histone
deacetylase inhibitor of HDAC-4.
In a ninth aspect, the invention provides a small molecule histone
deacetylase inhibitor identified by the method of the seventh or the eighth
aspect of the invention. Preferably, the histone deacetylase small molecule
inhibitor is substantially pure.
In a tenth aspect, the invention provides a method for inhibiting cell
proliferation in a cell comprising, contacting a cell with at least two
reagents
selected from the group consisting of an antisense oligonucleotide that
inhibits expression of HDAC-4 isoform, a small molecule histone deacetylase
inhibitor that inhibits expression or activity of HDAC-4 isoform, an antisense
oligonucleodde that inhibits expression of the HDAC-1 isoform, a small
molecule histone deacetylase inhibitor that inhibits the expression or the
activity of the HDAC-1 isoform, an andsense oligonucleotide that inhibits
expression of a DNA methyltransferase, and a small molecule DNA
methyltransferase inhibitor. In certain embodiments, the inhibition of cell
growth of the contacted cell is greater than the inhibition of cell growth of
a
cell contacted with only one of the reagents. In certain embodiments, each of
21
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
the reagents selected from the group is substantially pure. In preferred
embodiments, the cell is a neoplastic cell. In yet additional embodiments, the
reagents selected from the group are operably associated.
In an eleventh aspect, the invention provides a method of inhibiting
neoplastic cell growth, comprising contacting a cell with at least two
reagents
selected from the group consisting of an antisense oligonucleotide that
inhibits expression of HDAC-4 isoform, a small molecule histone deacetylase
inhibitor that inhibits the expression or the activity of HDAC-4 isoform, an
antisense oligonucleotide that inhibits expression of the HDAC-1 isoform, a
small molecule histone deacetylase inhibitor that inhibits expression or
activity of the HDAC-1 isoform, an antisense oligonucleotide that inhibits
expression of a DNA methyltransferase, and a small molecule DNA
methyltransferase inhibitor. In some embodiments, the inhibition of cell
growth of the contacted cell is greater than the inhibition of cell growth of
a
cell contacted with only one of the reagents. In certain embodiments, each of
the reagents selected from the group is substantially pure. In preferred
embodiments, the cell is a neoplastic cell. In yet additional preferred
embodiments, the reagents selected from the group are operably associated.
22
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 AS1 and AS2 can inbibit HDAC4 expression at RNA level in a dose-
dependent manner. Human cancer A549 cells were treated with
escalating doses of AS1, AS2 or MM2 oligos for 24 hours. Total RNAs
were harvested for Northern analysis.
Fig. 2 AS1 and AS2 can inbibit HDAC4 expression at protein level. Human
cancer A549 cells were treated with AS1, AS2 or MM2 oligos for 48
hours. Whole cell lysates were analyzed by Western blotting using
antibodies specific against human HDAC4.
Fig. 3 Growth curve of human cancer cells A549 treated with HDAC4 AS1 or
AS2. Cells were plated at 2.5X105/10 cm dish at 0 hour time point.
Cells were treated with 50 nM oligos at 24 and 48 hours. Cells were
counted at 24, 48 and 72 hours by trypan blue exclusion.
Fig. 4 Growth curve of human cancer cells Du145 treated with HDAC4 AS1
or AS2. Cells were plated at 2.5X105/10 cm dish at day 0: Cells were
treated with 50 nM oligos at day 1, day 2 and day 3. Cells were
counted at day 1, day 2, day 3 and day 4 by trypan blue exclusion.
Fig. 5 Graphic representation demonstrating the apoptotic effect of
HDAC isotype-specific antisense oligos on human A549 cancer cells.
Figure 6 is a a graphic representation demonstrating the cell cycle
blocking effect of HDAC-4 antisense oligos on human A549 cancer cells.
Figure 7 is a representation of an RNAse protection assay
demonstrating the effect of HDAC isotype-specific antisense oligos on HDAC
isotype mRNA expression in human A549 cells.
23
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Figure 8 is a representation of a Western blot demonstrating that
treatment of human A549 cells with HDAC-4 antisense oligos induces the
expression of the p21 protein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The patent and scientific literature referred to herein establishes
knowledge that is available to those with skill in the art. The issued
patents,
applications, and references, including GenBank database sequences, that are
cited herein are hereby incorporated by reference to the same extent as if
each
was specifically and individually indicated to be incorporated by reference.
The invention provides methods and reagents for modulating histone
deacetylase (HDAC) isoforms , particularly HDAC-1 and HDAC-4, by
inhibiting expression at the nucleic acid level or by inhibiting enzymatic
activity at the protein level. The invention provides for the specific
inhibition
of specific histone deacetylase isoforms involved in tumorigenesis, and thus
provides a treatment for cancer. The invention further provides for the
specific inhibition of specific HDAC isoforms involved in cell proliferation
and thus provides a treatment for cell proliferative disorders.
The inventors have made the surprising discovery that the specific
inhibition of HDAC-4 dramatically induces apoptosis and growth arrest in
cancerous cells. This discovery has been exploited to develop the present
invention which, in a first aspect, provides agents that inhibit the HDAC-4
isoform.
In certain preferred embodiments of the first aspect of the invention,
the agent that inhibits the HDAC-4 isoform is an oligonucleotide that inhibits
expression of a nucleic acid molecule encoding HDAC-4 isoform. The HDAC-
4 nucleic acid molecule may be genomic DNA (e.g., a gene), cDNA, or RNA.
24
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In some embodiments, the oligonucleoHde inhibits transcription of mRNA
encoding the HDAC-4 isoform. In other embodiments, the oligonucleotide
inhibits translation of the HDAC-4 isoform. In certain embodiments the
oligonucleotide causes the degradation of the nucleic acid molecule.
Preferred andsense oligonucleotides have potent and specific antisense
activity at nanomolar concentrations.
In certain preferred embodiments, the agent that inhibits the HDAC-4
isoform is a small molecule inhibitor that inhibits expression of a nucleic
acid
molecule encoding HDAC-4 isoform or activity of the HDAC-4 protein.
The term "small molecule" as used in reference to the inhibition of
histone deacetylase is used to identify a compound having a molecular
weight preferably less than 1000 Da, more preferably less than 800 Da, and
most preferably less than 600 Da, which is capable of interacting with a
histone deacetylase and inhibiting the expression of a nucleic acid molecule
encoding an HDAC isoform or activity of an HDAC protein. Inhibiting
histone deacetylase enzymatic activity means reducing the ability of a histone
deacetylase to remove an acetyl group from a histone. In some preferred
embodiments, such reduction of histone deacetylase activity is at least about
50%, more preferably at least about 75%, and still more preferably at least
about 90%. In other preferred embodiments, histone deacetylase activity is
reduced by at least 95% and more preferably by at least 99%. In a particularly
preferred embodiment, the small molecule inhibitor of HDAC is an inhibitor
of HDAC-1 and/or HDAC-4. Most prefered are small molecule inhibitors of
HDAC-4.
Preferably, such inhibition is specific, i.e., the histone deacetylase
inhibitor reduces the ability of a histone deacetylase to remove an acetyl
group from a histone at a concentration that is lower than the concentration
of
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
the inhibitor that is required to produce another, unrelated biological
effect.
Preferably, the concentration of the inhibitor required for histone
deacetylase
inhibitory activity is at least 2-fold lower, more preferably at least 5-fold
lower, even more preferably at least 10-fold lower, and most preferably at
least 20-fold lower than the concentration required to produce an unrelated
biological effect.
Preferred agents that inhibit HDAC-4 inhibit growth of human cancer
cells, independent of their p53 status. These agents induce apoptosis in
cancer
cells and cause growth arrest. They also can induce transcription of
p21~~'~~'1
(a tumor suppressor gene), Bax, an extremely important gene involved in
apoptosis regulation and GADD45, a stress-induced gene and important
regulator of cell growth. These agents may exhibit both in vitro and in vivo
anti-tumor activity. Inhibitory agents that achieve one or more of these
results are considered within the scope of this aspect of the invention.
The antisense oligonucleotides according to the invention are
complementary to a region of RNA or to a region of double-stranded DNA
that encodes a portion of one or more histone deacetylase isoforms (taking
into account that homology between different isoforms may allow a single
antisense oligonucleotide to be complementary to a portion of more than one
isoform). For purposes of the invention, the term "oligonucleotide" includes
polymers of two or more deoxyribonucleosides, ribonucleosides, or any
combination thereof. Preferably, such oligonucleotides have from about 6 to
about 50 nucleoside residues, and most preferably from about 12 to about 30
nucleoside residues. The nucleoside residues may be coupled to each other
by any of the numerous known internucleoside linkages. Such
internucleoside linkages include without limitation phosphorothioate,
phosphorodithioate, alkylphosphonate, alkylphosphonothioate,
26
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester,
acetamidate, carbamate, thioether, bridged phosphoramidate, bridged
methylene phosphonate, bridged phosphorothioate, and sulfone
internucleotide linkages. These internucleoside linkages preferably are
phosphotriester, phosphorothioate, or phosphoramidate linkages, or
combinations thereof.
Preferably, the oligonucleotides may also contain 2'-O-substituted
ribonucleotides. For purposes of the invention the term "2'-O-substituted"
means substitution of the 2' position of the pentose moiety with an -O-lower
alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an
-O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl, or
allyl group may be unsubstituted or may be substituted, e.g., with halo,
hydroxy, trifluoromethyl, cyano, rutro, aryl, acyloxy, alkoxy, carboxyl,
carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy
group (to produce a ribonucleoside), an amino or a halo group, but not with a
2'-H group. The term "alkyl" as employed herein refers to straight and
branched chain aliphatic groups having from 1 to 12 carbon atoms, preferably
1-8 carbon atoms, and more preferably 1-6 carbon atoms, which may be
optionally substituted with one, two or three substituents. Unless otherwise
apparent from context, the term "alkyl" is meant to include saturated,
unsaturated, and partially unsaturated aliphatic groups. When unsaturated
groups are particularly intended, the terms "alkenyl" or "alkynyl" will be
used. When only saturated groups are intended, the term "saturated alkyl"
will be used. Preferred saturated alkyl groups include, without limitation,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
pentyl,
and hexyl.
27
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
The term oligonucleotide also encompasses such polymers having
chemically modified bases or sugars and/or having additional substituents
including, without limitation, lipophilic groups, intercalating agents,
diamines, and adamantane. The term oligonucleotide also encompasses such
polymers as PNA and LNA.
For purposes of the invention, the term "complementary" means
having the ability to hybridize to a genomic region, a gene, or an RNA
transcript thereof, under physiological conditions. Such hybridization is
ordinarily the result of base-specific hydrogen bonding between
complementary strands, preferably to form Watson-Crick or Hoogsteen base
pairs, although other modes of hydrogen bonding, as well as base stacking
can lead to hybridization. As a practical matter, such hybridization can be
inferred from the observation of specific gene expression inhibition, which
may be at the level of transcription or translation (or both).
Particularly preferred antisense oligonucleotides utilized in this aspect
of the invention include chimeric oligonucleotides and hybrid
oligonucleotides.
For purposes of the invention, a "chimeric oligonucleotide" refers to an
oligonucleotide having more than one type of internucleoside linkage. One
preferred embodiment of such a chimeric oligonucleotide is a chimeric
oligonucleotide comprising internucleoside linkages, phosphorothioate,
phosphorodithioate, internucleoside linkages and phosphodiester, preferably
comprising from about 2 to about 12 nucleotides. Some useful
oligonucleotides of the invention have an alkylphosphonate-linked region
and an alkylphosphonothioate region (see e.g., Pederson et al. U.S. Patent
Nos.
5,635,377 and 5,366,878). Preferably, such chimeric oligonucleotides contain
28
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
at least three consecutive internucleoside linkages that are phosphodiester
and phosphorothioate linkages, or combinations thereof.
For purposes of the invention, a "hybrid oligonucleotide" refers to an
oligonucleotide having more than one type of nucleoside. One preferred
embodiment of such a hybrid oligonucleotide comprises a ribonucleotide or
2'-O-substituted ribonucleotide region, preferably comprising from about 2 to
about 12 2'-O-substituted nucleotides, and a deoxyribonucleotide region.
Preferably, such a hybrid oligonucleotide contains at least three consecutive
deoxyribonucleosides and contains ribonucleosides, 2'-O-substituted
ribonucleosides, or combinations thereof (see e.g., Metelev and Agrawal, U.S.
Patents Nos. 5,652,355 and 5,652,356).
The exact nucleotide sequence and chemical structure of an antisense
oligonucleotide utilized in the invention can be varied, so long as the
oligonucleotide retains its ability to modulate expression of the target
sequence, e.g., the HDAC-4 or the HDAC-1 isoform. This is readily
determined by testing whether the particular antisense oligonucleotide is
active by quantitating the amount of mRNA encoding the HDAC-4 or the
HDAC-1 isoform, quantitating the amount of the HDAC-4 or the HDAC-1
isoform protein, quantitating the the HDAC-4 or the HDAC-1 isoform
enzymatic activity, or quantitating the ability of the the HDAC-4 or the
HDAC-1 isoform, for example, to inhibit cell growth in a an in vitro or in
vivo
cell growth assay, all of which are described in detail in this specification.
The
term "inhibit expression" and similar terms used herein are intended to
encompass any one or more of these parameters.
Antisense oligonucleotides according to the invention may
conveniently be synthesized on a suitable solid support using well-known
chemical approaches, including H-phosphonate chemistry, phosphoramidite
29
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
chemistry, or a combination of H-phosphonate chemistry and
phosphoramidite chemistry (i.e., H-phosphonate chemistry for some cycles
and phosphoramidite chemistry for other cycles). Suitable solid supports
include any of the standard solid supports used for solid phase
oligonucleotide synthesis, such as controlled-pore glass (CPG) (see, e.g.,
Pon,
R. T., Meth. Molec. Biol. 20:465-496,1993).
Antisense oligonucleotides according to the invention are useful for a
variety of purposes. For example, they can be used as "probes" of the
physiological function of specific histone deacetylase isoforms by being used
to inhibit the activity of specific histone deacetylase isoforms in an
experimental cell culture or animal system and to evaluate the effect of
inhibiting such specific histone deacetylase isoform activity. This is
accomplished by administering to a cell or an animal an antisense
oligonucleotide that inhibits one or more histone deacetylase isoform
expression according to the invention and observing any phenotypic effects.
In this use, the anHsense oligonucleotides used according to the invention are
preferable to traditional "gene knockout" approaches because they are easier
to use, and because they can be used to inhibit specific histone deacetylase
isoform activity at selected stages of development or differentiation.
Preferred antisense oligonucleotides of the invention inhibit either the
transcription of a nucleic acid molecule encoding the the HDAC-4 or the
HDAC-1 isoform, and/or the translation of a nucleic acid molecule encoding
the the HDAC-4 or the HDAC-1, and/or lead to the degradation of such
nucleic acid molecules. HDAC-4- or HDAC-1-encoding nucleic acid
molecules may be RNA or double stranded DNA regions and include,
without limitation, intronic sequences, untranslated 5' and 3' regions, intron-
exon boundaries, as well as coding sequences from the HDAC-4 or the
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
HDAC-1 isoform genes. For human sequences, see e.g., Yang et al., Proc. Natl.
Acad. Sci. USA 93(23):2845-12850,1996; Furukawa et al., Cytogenet. Cell Genet.
73(1-2):130-133,1996; Yang et al., J. Biol. Chem. 272(44):28001-28007,1997;
Betz
et al., Genomics 52(2):245-246,1998; Taunton et al., Science 272(5260):408-
411,
1996; and Dangond et al., Biocliem. Biophys. Res. Commun. 242(3):648-
652,1998).
Antisense oligonucleotides for human HDAC isotype polynucleotides
may be designed from known HDAC isotype sequence data. For example,
the following amino acid sequences are available from GenBank for HDAC-1,
HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8:
AAC50475, AAC50814, AAC98927, BAA22957, AB011172, AAD29048,
AAF63491, and AAF73076, respectively, and the following nucleotide
sequences are available from GenBank for HDAC-1, HDAC-2, HDAC-3,
HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8: U50079, U31814,
AF039703, AB006626, AF039691, AJ011972, AF239243, and AF230097,
respectively.
Particularly preferred non-limiting examples of antisense
oligonucleotides of the invention are complementary to a region of RNA or to
a region of double-stranded DNA encoding the HDAC-4 or the HDAC-1
isoform, (see e.g., GenBank Accession No. U50079 for human HDAC-1 (Fig.
1B), and GenBank Accession No. AB006626 for human HDAC-4 (Fig. 2B)).
The sequences encoding histone deacetylases from many non-human
animal species are also known (see, for example, GenBank Accession Nos.
X98207 (murine HDAC-1) and AF006602 (murine HDAC-4)). Accordingly,
the antisense oligonucleotides of the invention may also be complementary to
a region of RNA or to a region of double-stranded DNA that encode the
HDAC-4 or the HDAC-1 isoform from non-human animals. Antisense
31
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
oligonucleotides according to these embodiments are useful as tools in animal
models for studying the role of specific histone deacetylase isoforms.
Particularly, preferred oligonucleotides have nucleotide sequences of
from about 13 to about 35 nucleotides which include the nucleotide sequences
shown in Table I below.
These oligonucleotides have nucleotide sequences of from about 15 to
about 26 nucleotides of the nucleotide sequences shown below in Table I.
Most preferably, the oligonucleoHdes shown below have phosphorothioate
backbones, are 20-26 nucleotides in length, and are modified such that the
terminal four nucleotides at the 5' end of the oligonucleotide and the
terminal
four nucleotides at the 3' end of the oligonucleotide each have 2' -O- methyl
groups attached to their sugar residues.
Table 1: HDAC isotype-specific antisense and
mismatch oligos
AccessionNucleotide
Oligo Target NumberPositionSequence position
within
Gene
HDAC1 Human U500791585-16045'- 3'-UTR
AS1 HDAC1 GAAACGTGAGGGACTCAGCA-3'
HDAC1 Human U500791565-15845'- 3'-UTR
AS2 HDAC1 GGAAGCCAGAGCTGGAGAGG-
3'
HDAC1 Human U500791585-16045'- 3'-UTR
MM HDAC1 GTTAGGTGAGGCACTGAGGA-3'
HDAC2 Human U318141643-16225'-GCTGAGCTGTTCTGATTTGG-3'-UTR
AS HDAC2 3'
HDAC2 Human U318141643-16225'-CGTGAGCACTTCTCATTTCC-3'-UTR
MM HDAC2 3'
HDAC3 Human AF0397031276-12955'-CGCTTTCCTTGTCATTGACA-3'-UTR
AS HDAC3 3'
HDAC3 Human AF0397031276-12955'-GCCTTTCCTACTCATTGTGT-3'-UTR
MM HDAC3 3'
HDAC4 Human AB006626514-33 5- 5'-UTR
AS1 HDAC4 GCTGCCTGCCGTGCCCACCC-3'
HDAC4 Human AB006626514-33 5'- 5'-UTR
MM1 HDAC4 CGTGCCTGCGCTGCCCACGG-
3'
HDAC4 Human A80066267710-295'-TACAGTCCATGCAACCTCCA-3'-UTR
32
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
AS2 HDAC4 3'
HDAC4 Human AB0066267710-295'-ATCAGTCCAACCAACCTCGT-3'-UTR
MM2 HDAC4 3'
HDACS Human BE7949121-20 5'- 5'-UTR
AS1 HDACS GCAGCGGCGGCAGCACCTCC-
3'
HDACS Human AF0396912663-26825'-CTTCGGTCTCACCTGCTTGG-3'-UTR
AS2 HDAC5 3'
HDAC6 Human AJ0119723791-38105'- 3'-UTR
AS HDAC6 CAGGCTGGAATGAGCTACAG-3'
HDAC6 Human AJ0119723791-38105'- 3'-UTR
MM HDAC6 GACGCTGCAATCAGGTAGAC-3'
HDAC7 Human AF23924365-84 5'-CAGGCTCACTTGACAATGGC-5'-UTR
AS1 HDAC7 3'
HDAC7 Human AF2392432896-29155'- 3'-UTR
AS2 HDAC7 CTTCAGCCAGGATGCCCACA-3'
HDAC8 Human AF23009751-70 5'-CTCCGGCTCCTCCATCTTCC-5'-UTR
AS1 HDAC8 3'
HDAC8 Human AF2300971328-13475'- 3'-UTR
AS2 HDAC8 AGCCAGCTGCCACTTGATGC-3'
The antisense oligonucleotides according to the invention may
optionally be formulated with any of the well known pharmaceutically
acceptable carriers or diluents (see preparation of pharmaceutically
acceptable
formulations in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, ed.
A. Gennaro, Mack Publishing Co., Easton, PA,1990), with the proviso that
such carriers or diluents not affect their ability to modulate HDAC activity.
In certain preferred embodiments, the agent that inhibits the HDAC-4
and/or HDAC-1 isoform is a small molecule. In certain preferred
embodiments, the small molecule inhibits the enzymatic activity of the
HDAC-4 or HDAC-1 isoform.
33
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Certain preferred small molecule inhibitors of the HDAC-4 and/or
HDAC-1 isoform include compounds having the formula (1):
Cy-CH(OMe)-Yl-C(O)-NH-Z (1)
wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
optionally be substituted;
Yl is a C4 - C6 alkylene which optionally may be substituted and
wherein one of the carbon atoms of the alkylene optionally may be replaced
by a heteroatom moiety such as O, NR1 (R1 being alkyl, aryl or hydrogen) S,
S(O), or S(O)z; and
Z is selected from the group consisting of arulinyl, pyridyl, thiadiazolyl
and -O-M, M being H or a pharmaceutically acceptable canon, wherein the
anilinyl or pyridyl or thiadiazolyl may be optionally substituted.
An "alkylene" group is an alkyl group, as defined hereinabove, that is
positioned between and serves to connect two other chemical groups.
Preferred alkylene groups include, without limitation, methylene, ethylene,
propylene, and butylene.
The term "cycloalkyl" as employed herein includes saturated and
partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,
preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the
cycloalkyl group additionally may be optionally substituted. Preferred
cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and
cyclooctyl.
An "aryl" group is a Cb-C,4 aromatic moiety comprising one to three
aromatic rings, which may be optionally substituted. Preferably, the aryl
34
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
group is a C6-C,o aryl group. Preferred aryl groups include, without
limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An "aralkyl" or
"arylalkyl" group comprises an aryl group covalently linked to an alkyl
group, either of which may independently be optionally substituted or
unsubsHtuted. Preferably, the aralkyl group is (C,-C6)alk(C.~-C,o)aryl,
including, without limitation, benzyl, phenethyl, and naphthylmethyl. An
"alkaryl" or "alkylaryl" group is an aryl group having one or more alkyl
substituents. Examples of alkaryl groups include, without limitation, tolyl,
xylyl, mesityl, ethylphenyl, tert-butylphenyl, and methylnaphthyl.
An "arylene" group is an aryl group, as defined hereinabove, that is
positioned between and serves to connect two other chemical groups.
Preferred arylene groups include, without limitation, phenylene and
naphthylene. The term "arylene" is also meant to include heteroaryl bridging
groups, including, but not limited to, benzothienyl, benzofuryl, quinolyl,
isoquinolyl, and indolyl.
A "heterocyclyl" or "heterocyclic" group is a ring structure having from
about 3 to about 8 atoms, wherein one or more atoms are selected from the
group consisting of N, O, and S. The heterocyclic group may be optionally
substituted on carbon at one or more positions. The heterocyclic group may
also independently be substituted on nitrogen with alkyl, aryl, aralkyl,
alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl,
aralkoxycarbonyl, or on sulfur with oxo or lower alkyl. Preferred heterocyclic
groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranyl,
pyrrolidinyl, piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl,
oxazolidinonyl, and morpholino. In certain preferred embodiments, the
heterocyclic group is fused to an aryl or heteroaryl group. Examples of such
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
fused heterocyles include, without limitation, tetrahydroquinoline and
dihydrobenzofuran.
As used herein, the term "heteroaryl" refers to groups having 5 to 14
ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6,10, or 14 ~
electrons
shared in a cyclic array; and having, in addition to carbon atoms, between one
and about three heteroatoms selected from the group consisting of N, O, and
S. Preferred heteroaryl groups include, without limitation, thienyl,
benzothienyl, furyl, benzofuryl, dibenzofuryl, pyrrolyl, imidazolyl,
pyrazolyl,
pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl,
tetrazolyl, oxazolyl, thiazolyl, and isoxazolyl.
As employed herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl,
or heterocyclic group is one having between one and about four, preferably
between one and about three, more preferably one or two, non-hydrogen
substituents. Suitable substituents include, without limitation, halo,
hydroxy,
rutro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino,
acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl,
carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,
arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and
ureido groups.
The term "halogen" or "halo" as employed herein refers to chlorine,
bromine, fluorine, or iodine.
As herein employed, the term "acyl" refers to an alkylcarbonyl or
arylcarbonyl subsdtuent.
The term "acylamino" refers to an amide group attached at the nitrogen
atom. The term "carbamoyl" refers to an amide group attached at the
carbonyl carbon atom. The nitrogen atom of an acylamino or carbamoyl
substituent may be additionally substituted. The term "sulfonamido" refers to
36
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
a sulfonamide substituent attached by either the sulfur or the nitrogen atom.
The term "amino" is meant to include NH2 alkylamino, arylamino, and cyclic
ammo groups.
The term "ureido" as employed herein refers to a substituted or
unsubstituted urea moiety.
In another embodiment, the small molecule inhibitors of the HDAC-4
and/or HDAC-1 isoform are represented by formula (2):
Cy-Y2-C(O)NH-Z
(2)
wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
optionally be substituted;
YZ is Cs - C~ alkylene which may be optionally substituted and wherein
one of the carbon atoms of the alkylene optionally may be replaced by a
heteroatom moiety such as O, NRl (R1 being alkyl, acyl or hydrogen), S, S(O),
or S(O)2; and
Z is anilinyl or pyridyl or thiadiazolyl, any of which may optionally be
optionally substituted. In another embodiment, preferred small molecule
inhibitors of the HDAC-4 and/or HDAC-1 isoform include compounds
having the formula (3):
Cy-B-Y3-C(O)-NH-Z (3)
wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may
optionally be substituted;
B is -CH(OMe), ketone, or methylene;
37
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Y3 is a C4 - C6 alkylene which may be optionally substituted, and
wherein one of the carbon atoms of the alkylene optionally may be replaced
by a heteroatom moiety such as O, NRl (R1 being alkyl, acyl or hydrogen), S,
S(O), or S(O)z; and
Z is anilinyl, pyridyl, thiadiazolyl or -O-M (M being H or a
pharmaceutically acceptable canon), wherein the anilinyl or pyridyl or
thiadiazolyl optionally may be substituted.
In another embodiment, the inhibitors of the HDAC-4 and/or HDAC-1
isoform are represented by formula (4):
Cy-L~-Ar-Yi-C(O)-NH-Z (4)
wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which
optionally may be substituted;
L~ is -(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W is -C(O)NH-, -
S(O)zNH-, -NHC(O)-, -NHS(O)z-, or -NH-C(O)-NH-;
Ar is arylene which may be additionally substituted and optionally
may be fused to an aryl or heteroaryl ring, or to a saturated or partially
unsaturated cycloalkyl or heterocyclic ring, any of which optionally may be
substituted;
Yl is a chemical bond or a straight- or branched-chain saturated
alkylene, which optionally may be substituted; and
Z is anilinyl, pyridyl, thiadiazolyl, or -O-M (M being H or a
pharmaceutically acceptable canon);
provided that when L~ is -C(O)NH-, Y1 is -(CHZ)~ (n being 1, 2, or 3),
and Z is -O-M, then Cy is not aminophenyl, dimethylaminophenyl, or
38
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
hydroxyphenyl; and further provided that when L~ is -C(O)NH- and Z is
pyridyl, then Cy is not substituted indolinyl.
In another embodiment, the inhibitors of the HDAC-4 and/or HDAC-1
isoform are represented by formula (5):
Cy-Lz-Ar-YZ-C(O)NH-Z (5)
wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which
optionally may be substituted, provided that Cy is not a
(spirocycloalkyl)heterocyclyl;
Lz is C,-C6 saturated alkylene or CZ-C6 alkenylene, wherein the alkylene
or alkenylene optionally may be substituted, provided that LZ is not -C(O)-,
and wherein one of the carbon atoms of the alkylene optionally may be
replaced by a heteroatom moiety such as O, NR' (R' being alkyl, aryl, or
hydrogen), S, S(O), or S(O)z;
Ar is arylene which optionally may be additionally substituted and
optionally may be fused to an aryl or heteroaryl ring, or to a saturated or
partially unsaturated cycloalkyl or heterocyclic ring, any of which optionally
may be substituted; and
Yz is a chemical bond or a straight- or branched-chain saturated
alkylene which may be optionally substituted, provided that the alkylene is
not substituted with a substituent of the formula -C(O)R ,wherein R
comprises an a-amino aryl moiety; and
Z is anilinyl, pyridyl, thiadiazolyl, or -O-M (M being H or a
pharmaceutically acceptable cation);
provided that when the carbon atom to which Cy is attached is oxo-
substituted, then Cy and Z are not both pyridyl.
39
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In another embodiment, the inhibitors of the HDAC-4 and/or HDAC-1
isoform are represented by formula (6):
Cy-L3-Ar-Y3-C(O) NH-Z (6)
n wherein:
Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which
optionally may be substituted, provided that Cy is not a
(spirocycloalkyl)heterocyclyl;
L3 is:
(a) -(CHz)m-W-, where m is 0,1, 2, 3, or 4, and W is -
C(O)NH-, S(O)zNH-, -NHC(O)-, -NHS(O)z-, or -NH-C(O)-NH-; or
(b) C,-C6 alkylene or CrCb alkenylene, wherein the alkylene
or alkenylene optionally may be substituted, provided that L3 is not -
C(O)-, and wherein one of the carbon atoms of the alkylene optionally
may be replaced by O; NR', R' being alkyl, acyl, or hydrogen; S; S(O);
or S(O)z;
Ar is arylene which optionally may be additionally substituted and
optionally may be fused to an aryl or heteroaryl ring, or to a saturated or
partially unsaturated cycloalkyl or heterocyclic ring, any of which optionally
may be substituted; and
Y3 is Cz alkenylene or Cz alkynylene, wherein one or both carbon atoms
of the alkenylene optionally may be substituted with alkyl, aryl, alkaryl, or
aralkyl; and
Z is anilinyl, pyridyl, thiadiazolyl, or -O-M (M being H or a
pharmaceutically acceptable canon);
provided that when Cy is unsubsntuted phenyl, Ar is not phenyl
wherein L3 and Y3 are oriented ortho or meta to each other.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In another embodiment, the small molecule inhibitors of the HDAC-4
and/or HDAC-1 isoform have the structure selected from the group
consisting of
0
\ N.OH
O I ~ ~ H
I O~H
(7)
o /
\ N ~ I
o I ~ ~ H
NHp
~~N /
~ I o H (8) and
o ~\
/ I SAN / \ / N NHz
O H \
o I /
(9).
Non-limiting examples of small molecule inhibitors for use in the
methods of the invention are presented in Table 2.
41
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Table 2: Properties of Selected MG
Anilides in vitro and in vivo shown in uM
Enzyme IC50 cell p21 % inh. of tumor
(uM) cycl formation in vivo
a
MG Structure HDA HDA HDA HDA HDA H MT arre inducti colon lun prost
# C1 C2 C3 C4 C6 4- T st on g ate
Ac EC
24 ~ I 25 21 23 >50 1 1 2 3
29 ' ' ' N '
N°
~N
I
G1,
36 ° " "'~ 4 >20 23 >50 10 5 9 10 53(40,
50 ~ ~ ° I ~ iP)
I,
37 ° ~~ ~ 3 22 45 28 >50 5 4 2 2 55(40,
' N' T
63 I ' g\N ' ~ "~ iP)
38 ~ ~ 8 18 13 >50 5 5 3 5
69 ' " '
W I J y I,
note: for in vivo antitumor studies, numbers outside brackets indicate % of
inhibition of tumor formation in vivo;
numbers in brackets indicate daily dose of inhibitor used
(mg/kg body weight/day);
oral (PO) or intraperitoneal (1P) administration is
indicated in brackets.
Small molecule inhibitors of the invention of the formulae Cy-
CH(OMe)-Yl-C(O)-NH-Z, Cy-Yz-C(O)NH-Z and Cy-B-Y3-C(O)-NH-Z, which
may be conveniently prepared according to the following schemes 1-3 or
using other art-recognized methods.
Scheme 1
A dialkyl acetal I is treated with 1-trimethylsilyloxy-1,3-butadiene or
with 1-trimethylsilyloxy-2,4-dimethyl-1,3-butadiene in the presence of zinc
42
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
bromide to yield the aldehyde II. Wittig reaction of II with a carboalkoxy
phosphorous yield such as ethyl (triphenylphosphoranylidene)acetate yields
the dime ester III. Hydrolysis of the ester function in III can be effected by
treatment with a hydroxide base, such as lithium hydroxide, to yield the
corresponding acid IV.
The acid IV is converted to the corresponding acid chloride V
according to standard methods, e.g., by treatment with sodium hydride and
oxalyl chloride. Treatment of V with 1,2-phenylenediamine and a tertiary
base such as N-methylinorpholine, preferably in dichloromethane at reduced
temperature, then yields the anilinylamide VI. Alternatively, the acid
chloride
OMe
1I + R
/\ -0SiMc,
Cy~OMa
1 R
R=F4Mc
ZnBrZ CHzCI=, rt
OMe O OMe O
O ~ H Ph,P~H=CO~EI OY ~ \ OEt
R R R R
11
LiOH ~ McOH-Hz0
OMe O
OMe O
Null,
cy ~ c~ (C~)= c ~ \ off
r
R R V
R R IV
NHS ~ ~ NH=
of
I. CD1, 7NF
Ihcn 4N HCI NH-tB0 / NN~
2I ~ NHS
.CF~CO011
OMe O
NH=
CY ~ ~ H
R R NHZ
VI
V may be treated with a mono-protected 1,2-phenylenediamine, such as 2-(t-
BOC-amino)aniline, followed by deprotection, to yield VI.
43
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In an alternative procedure, the acid IV may be activated by treatment
with carbonyldiimidazole (CDI), followed by treatment with 1,2-
phenylenediamine and trifluoroacetic acid, to yield the arulinyl amide VI.
Compounds of formula Cy-y2-C(O)-NH2 (VII), wherein Yz is:
O
Me Me
may be prepared from the corresponding methoxy-substituted
compounds (VI) by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ), as illustrated in Scheme 2
OMe O
C \ \ N \ ~
Y
R R ,,, H N H2
DDQ
O
C \ \ N \ ~
y
R R ,," H NH2
44
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Compounds of formula Cy-yz-C(O)-NHz, wherein yz has the structure
w
Me Me
may be prepared as shown in Scheme 3. The methoxy substituted
dime ester III, prepared as described in Scheme 1, is treated with
triethylsilane and boron trifluoride etherate to yield the deoxygenated
compound VIII. Conversion of VIII to the anilinylamide X is accomplished
by procedures analogous to those described in Scheme 1.
Scheme 3
OMe O O
fa,Sifl
Cy ~ ~ OEt ~Cy ~ \ OEt
R R ... II~,.ala, R R
L.OII
O
Cy ~ \ OH
R R Ix
I. (;I)1.'I'IIF z.
~NH2
NHz
O
Cy ~ \ N
R R X NHz
Compounds of formula Cy-L'-Ar-Y1-C(O)-NH-O-M, wherein L~ is
-S(O)ZNH-, may be prepared according to the synthetic routes depicted in
Schemes 4-6. In certain preferred embodiments, compounds XI are preferably
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
prepared according to the general synthetic route depicted in Scheme 4. A
sulfonyl chloride (XII) is treated with an amine (XIII) in a solvent such as
methylene chloride in the presence of an organic base such as triethylamine.
Treatment of the crude product with a base such as sodium methoxide in an
alcoholic solvent such as methanol effects cleavage of any dialkylated
material and affords the sulfonamide (XIV). Hydrolysis of the ester function
in XIV can be effected by treatment with a hydroxide base, such as lithium
hydroxide, in a solvent mixture such as tetrahydrofuran and methanol to
yield the corresponding acid (XV).
Scheme 4
0
Cy-S02CI + H NiAr~Y~~C02Me 1. Et3N IS Ar ~C02Me
CY II\N~ ~Y1
2. NaOMe, MeOH O H
XII XIII XIV
LiOH
O Method A: O
II NH20THP, DCC II
/S~ ,Arm ~~C(O)NHOTHP /S~ jAr~ ~~C02H
CY II H Y Method D: CY II N Y
O XVI NH20THP, EDC, O H XV
HOBt
Method C:
1. COCIp
2. NH20TMS
O 3. 1 N HCI .
II
CSA, MeOH Cy~II\N~Ar~Y~~C(O)NHOH
O H
Method B:
XI NH20H,
NaOMe, MeOH
46
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In some embodiments, conversion of the acid XVto the hydroxamic
acid XI is accomplished by coupling XV with a protected hydroxylamine,
such as tetrahydropyranylhydroxylamine (NHzOTHP), to yield the protected
hydroxamate XVI, followed by acidic hydrolysis of XVI to provide the
hydroxamic acid XI. The coupling reaction is preferably accomplished with
the coupling reagent dicyclohexylcarbodiimide (DCC) in a solvent such as
methylene chloride (Method A), or with the coupling reagent 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide in presence of N-hydroxy
benzotriazole in an aprotic solvent such as dimethylformamide (Method D).
Other coupling reagents are known in the art and may also be used in this
reaction. Hydrolysis of XVI is preferably effected by treatment with an
organic acid such as camphorsulforuc acid in a proHc solvent such as
methanol.
Alternatively, in some other embodiments, acid XV is converted to the
corresponding acid chloride, preferably by treatment with oxalic chloride,
followed by the addition of a protected hydroxylamine such as O-
trimethylsilylhydroxylamine in a solvent such as methylene chloride, which
then provides the hydroxamic acid XI upon workup (Method C).
In still other embodiments, the ester XIV is treated with
hydroxylamine in a solvent such as methanol in the presence of a base such as
sodium methoxide to furnish the hydroxamic acid XI directly (Method B).
47
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
SCHEME 5 x=COOH ~ NHOH
1. Coupling
0
reagent
NHzOR ArSOyNH XX
2. Deprotection
1) ArSO2Cl X=CHpOH ~ CO Et
X TPAP
Et3N ~ ~ pd~p) M.S. 4A NMO
CHyCl2
~X ~ . CH3CN lhr. I / ,.
HpN 2) N80Me q~p=N ~ ~ ~ ph3P- ~O NH/ / XXI
MBOH ArSOzNH
XVII XVIII XIX COzEt
CH3CN, 50°C
X=COOH
O
O
L_iOH / \ off O 1. Coupling
/ reagent ~ HHOH
T~ ~ MeOHd/C ( ~ off NHZOR
za
\HHSOzAr \NHSOzAr 2. Deprotection NHSO r
XXII XXIII XXIV
Compounds of formula XX and XXIV preferably are prepared
according to the general procedure outlined in Scheme 5 above.
An aminoaryl halide (XVII) is treated with a sulfonyl chloride in
presence of a base such as triethylamine, followed by treatment with an
alkoxide base, to furnish the sulfonamide XVIII. One of skill in the art will
recognize that reverse sulfonamide analogs can be readily prepared by an
analogous procedure, treating a haloarenesulfonyl halide with an arylamine.
Compound XVIII is coupled with a terminal acetylene or olefiruc
compound in the presence of a palladium catalyst such as
tetrakis(triphenylphosphine)palladium(0) in a solvent such as pyrrolidine to
yield XIX.
Oxidation of the compound of formula XIX (X=CHZOH), followed by
homologation of the resulting aldehyde (using a Wittig type reagent such as
carbethoxymethylenetriphenylphosphorane in a solvent such as acetonitrile),
yields the compound of formula XXI. Basic hydrolysis of XXI, such as by
treatment with lithium hydroxide in a mixture of THF and water, provides
48
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
the acid XXII. Hydrogenation of XXII may preferably be performed over a
palladium catalyst such as Pd/C in a protic solvent such as methanol to yield
the saturated acid XXIII. Coupling of the acid XXIII with an O-protected
hydroxylamine such as O-tetrahydropyranylhydroxylamine is effected by
treatment with a coupling reagent such as 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide in the presence of N-hydroxybenzotriazole (HOBT), or
N,N-dicyclohexylcarbodiimide (DCC), in a solvent such as DMF, followed by
deprotection to furnish the compound of general formula XXIV.
The acid XIX, wherein X=COOH, may be coupled directly with an
O-protected hydroxylamine such as O-tetrahydropyranylhydroxylamine,
followed by deprotection of the hydroxy protecting group to furnish the
hydroxamic acid XX.
Compounds of formula Cy-L~-Ar-Yl-C(O)-NH--O-M, wherein L~ is
-C(O)NH-, preferably may be prepared according to the synthetic routes
analogous to those depicted in Schemes 4-5, substituting acid chloride
starting
materials for the sulfonyl chloride starting materials in those schemes.
49
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Scheme 6
0 0 0
+ Base
Cy"CH3 H~A~ C02R - Cy~~Ar C02R
R-H, Me
XXV XXVI XXVI I
H2
R = H NH20R~,
R = Me coupling reagent
O . O
C ~~A~ C02Me C ~ Af C(O)NHORi
Y Y
XXX XXVI II
1. LiOH
2. NH20R~,
coupling reagent H
O 3. H+ O
Cy~~~Ar C(O)NHOH Cy~~~A' C(O)NHOH
XXXI XXIX
Compounds of the formula Cy-Lz-Ar-YZ-C(O)-NH-O-M are
preferably prepared according to the synthetic routes outlined in Schemes 6-8.
Accordingly, in certain preferred embodiments, compounds of formulae
XXIX and XXXI (Lz = -C(O)-CH=CH- or -C(O)-CHzCHz-) preferably are
prepared according to the route described in Scheme 6. Thus, a substituted
aryl acetophenone (XXV) is treated with an aryl aldehyde (XXVI) in a proHc
solvent such as methanol in the presence of a base such as sodium methoxide
to afford the enone XXVII.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
The acid substituent of XXVII (R = H) is coupled with an O-protected
hydroxylamine such as O-tetrahydropyranylhydroxylamine (R, _
tetrahydropyranyl) to yield the O-protected-N-hydroxybenzamide XXVIII.
The coupling reaction is preferably performed by treating the acid and
hydroxylamine with dicyclohexylcarbodiimide in a solvent such as methylene
chloride or with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the
presence of N-hydoxybenzotriazole in a solvent such as dimethylformamide.
Other coupling reagents are known in the art and may also be used in this
reaction. O-Deprotection is accomplished by treatment of XXVIII with an
acid such as camphorsulfonic acid in a solvent such as methanol to afford the
hydroxamic acid XXIX (L2 = -C(O)-CH=CH-)
Saturated compounds of formula XXXI (L2 = -C(O)-CHzCHz-) are
preferably prepared by hydrogenation of XXVII (R = Me) over a palladium
catalyst, such as 10% Pd/C, in a solvent such as methanol-tetrahydrofuran.
Basic hydrolysis of the resulting saturated ester XXX with lithium hydroxide,
followed by N-hydroxy amide formation and acid hydrolysis as described
above, then yields the hydroxamic acid XXXI.
51
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Scheme 7
R2 R2
Pd(II) or Pd(0)
X
Cy~~~ + ~A C02H Cy'~Ar C02H
o ~P o
XXXII XXXIII XXXIV P
H2
R2 1. NH20R~, R
2
coupling reagent
Cy~~~\~Ar C(O)NHOH
o + Cy~~~ ~~Ar C02H
XXXVI 'P 2. H o /
XXXV P
Compounds of formula XXXVI (L2 = -(CHZ)o+2-) are preferably
prepared by the general procedures described in Scheme 7. Thus, in some
embodiments, a terminal olefin (XXXII) is coupled with an aryl halide
(XXXIII) in the presence of a catalytic amount of a palladium source, such as
palladium acetate or tris(dibenzylideneacetone)dipalladium(0), a phosphine,
such as triphenylphosphine, and a base, such as triethylamine, in a solvent
such as acetonitrile to afford the coupled product XXXIV. Hydrogenation,
followed by N-hydroxyamide formation and acid hydrolysis, as described
above, yields the hydroxamic acid XXXVI.
52
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Scheme 8
- o
Br
~C02H B-a ~ iC02H
Cy~PPhg H Ar Cy~~~\~Ar
0 0
XXXVII XXXVIII XXXIV
H2
1. NH20R~,
~C(O)NHOH coupling reagent ~~~ %~ C02H
Cy Ar E Cy Ar
0 0
2. H+ XXXV
Alternatively, in some other embodiments, a phosphonium salt of
formula XXXVII is treated with an aryl aldehyde of formula XXXVIII in the
presence of base, such as lithium hexamethyldisilazide, in a solvent, such as
tetrahydrofuran, to produce the compound XXXIV. Hydrogenation, followed
by N-hydroxyamide formation and acidic hydrolysis, then yields the
compounds XXXVI (Scheme 8).
Compounds of formula Cy-L-Ar-Y-C(O)-NH-Z, wherein L is L~ or
Lz,as previously described herein, Y is Yl or Y2, as previously described
herein, and Z is anilinyl or pyridyl or thiadiazolyl, are preferably prepared
according to synthetic routes outlined in Scheme 9.
53
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Scheme 9
NaH, (COCI)2 O
Cy-L-Ar-Y-C02H Cy-L-Ar-Y
CI
XXXIX XL
HZN ~ HpN
I
N / /
HzN
O OR
Cy-L-Ar-Y--
HN HpN
\ 1. ~
N t BOC-NH- v
XLI
2. H CI
O
Cy-L-Ar-Y-
HN
H2N
XLII
1. CDI, THF O
Cy-L-Ar-Y-C02H Cy-L-Ar-Y-
HpN ~ H N
XXXIX 2. H2N ~ / ,
CF3C02H H2N
XLII
An acid of formula Cy-L-Ar-Y-C(O)-OH (XXXIX), prepared by one of
the methods shown in Schemes 4-8, is converted to the corresponding acid
chloride XL according to standard methods, e.g., by treatment with sodium
hydride and oxalyl chloride. Treatment of XL with 2-aminopyridine and a
tertiary base such as N-methylmorpholine, preferably in dichloromethane at
54
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
reduced temperature, then yields the pyridyl amide XLI. In a similar fashion,
the acid chloride XL may be treated with 1,2-phenylenediamine to afford the
anilinyl amide XLII. Alternatively, the acid chloride XL may be treated with a
mono-protected 1,2-phenylenediamine, such as 2-(t-BOC-amino)aniline,
followed by deprotection, to yield XLII.
In an alternative procedure, the acid XXXIX may be activated by
treatment with carbonyldiimidazole (CDI), followed by treatment with 1,2-
phenylenediamine and trifluoroacetic acid to afford the anilinyl amide XLII.
Scheme 10
O O
~R + H~Ar~CO2R3 Bas C ~~jAr~C02R3
CY ~ ~~Jo 1~IP R = H, Me Y ~o
R2 R~ R2
XLIII ~ XLIV
XLV R3 = H
H2
R3 = Me NH20H,
coupling reagent
O O
CY~~Ar.(yCO2R3 ~Ar~(~- CONHOH
o p CY~~~o 'I' p
R~ RZ R~ R2
XLVII 1. LiOH
XLVI
2. NH20H,
coupling reagent
O
CY~~~ Ar( yC(O)NHOH
'_/o ~T~ p
R~ R2
XLVIII
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Compounds of formula XLVIII (L2 = -C(O)-alkylene-) preferably are
prepared according to the general procedure depicted in Scheme 10. Thus,
Aldol condensation of ketone XLIII (Rl = H or alkyl) with aldehyde XLIV
affords the adduct XLV. The adduct XLV may be directly converted to the
corresponding hydroxamic acid XLVI. Hydrogenation of XLV may yield the
saturated compound XLVII and which is then converted to the hydroxamic
acid XLVIII.
Scheme 11
R R~
OH
y~ ~ P OH ~ ~ P
-C 7~ + ~ I o
C oSH ~ I ~ O Cy oS
XLI
XXXIX XL
1. mCPBA / DCM,
2. CH2Nz /Et20, DCM
R~ 3. LiOH.H20, MeOH, THF
NHOH
P
CY,(-~.a5 w O Ri
OH
XLIV O ~ I P
Te02 ~ i ~ ~ O
35 % HpOz / H20 CY os
MeOH O XLII
R~
NHOH
P
I o
CY o0 R~
XLV / NHOH
~O W I PO
Cy oS
O
XLIII
56
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Compounds of formula (5), wherein one of the carbon atoms in Lz is
replaced with S, S(O), or S(O)2 preferably are prepared according to the
general procedure outlined in Scheme 11. Thus, thiol XLIX is added to olefin
L to produce LI. The reaction is preferably conducted in the presence of a
radical initiator such as 2,2'-azobisisobutyronitrile (AIBN) or
1,1'-azobis(cyclohexanecarbonitrile) (VAZOTM). Sulfide oxidation, preferably
by treatment with m-chloroperbenzoic acid (mCPBA), affords the
corresponding sulfone, which is conveniently isolated after conversion to the
methyl ester by treatment with diazomethane. Ester hydrolysis then affords
the acid LII, which is converted to the hydroxamic acid LIII according to any
of the procedures described above. The sulfide LI also may be converted
directly to the corresponding hydroxamic acid LIV, which then may be
selectively oxidized to the sulfoxide LV, for example, by treatment with
hydrogen peroxide and tellurium dioxide.
T'he reagents according to the invention are useful as analytical tools
and as therapeutic tools, including gene therapy tools. The invention also
provides methods and compositions which may be manipulated and fine-
tuned to fit the conditions) to be treated while producing fewer side effects.
The invention also provides method for inhibiting HDAC-4 activity in
a cell, comprising contacting the cell with a specific inhibitor of HDAC-4,
whereby HDAC-4 activity is inhibited. As used herein, the term "specific
inhibitor" means any molecule or compound that decreases the amount of
HDAC RNA, HDAC protein, and/or HDAC protein activity in a cell.
Particularly preferred specific inhibitors decrease the amount of RNA,
protein, and/or protein activity in a cell for HDAC-1 and/or HDAC-4.
In an embodiment thereof, the invention provides a method for
inhibiting the HDAC-4 isoform in a cell comprising contacting the cell with an
57
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
antisense oligonucleotide of the first aspect of the invention. Preferably,
cell
proliferation is inhibited in the contacted cell. In preferred embodiments,
the
cell is a neoplastic cell which may be in an animal, including a human, and
which may be in a neoplastic growth. In certain preferred embodiments, the
method of the second aspect of the invention further comprises contacting the
cell with HDAC-4 small molecule inhibitor that interacts with and reduces the
enzymatic activity of the HDAC-4 isoform. In some embodiments, the
histone deacetylase small molecule inhibitor is operably associated with the
antisense oligonucleotide.
Thus, the antisense oligonucleotides according to the invention are
useful in therapeutic approaches to human diseases, including benign and
malignant neoplasms, by inhibiting cell proliferation in cells contacted with
the antisense oligonucleoHdes. The phrase "inhibiting cell proliferation" is
used to denote an ability of HDAC-4 anHsense oligonucleotide or a small
molecule HDAC-4 inhibitor (or combination thereof) to retard the growth of
cells contacted with the oligonucleotide or small molecule inhibitor, as
compared to cells not contacted.
An assessment of cell proliferation can be made by counting cells that
have been contacted with the oligonucleotide or small molecule of the
invention and compare that number with the number of non-contacted cells
using a Coulter Cell Counter (Coulter, Miami, FL) or a hemacytometer.
Where the cells are in a solid growth (e.g., a solid tumor or organ), such an
assessment of cell proliferation can be made by measuring the growth of the
tissue or organ with calipers, and comparing the size of the growth of
contacted cells with non-contacted cells. Preferably, the term includes a
retardation of cell proliferation that is at least 50% of non-contacted cells.
More preferably, the term includes a retardation of cell proliferation that is
58
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
100% of non-contacted cells (i.e., the contacted cells do not increase in
number
or size). Most preferably, the term includes a reduction in the number or size
of contacted cells, as compared to non-contacted cells. Thus, HDAC-4
antisense oligonucleotide or HDAC-4 small molecule inhibitor that inhibits
cell proliferation in a contacted cell may induce the contacted cell to
undergo
growth retardation, growth arrest, programmed cell death (i.e., to apoptose),
or necrotic cell death.
The cell proliferation inhibiting ability of the antisense oligonucleotides
according to the invention allows the synchronization of a population of a-
synchronously growing cells. For example, the antisense oligonucleotides of
the invention may be used to arrest a population of non-neoplasHc cells
grown in vitro in the G1 or G2 phase of the cell cycle. Such synchronization
allows, for example, the identification of gene and/or gene products
expressed during the G1 or G2 phase of the cell cycle. Such a synchronization
of cultured cells may also be useful for testing the efficacy of a new
transfection protocol, where transfection efficiency varies and is dependent
upon the particular cell cycle phase of the cell to be transfected. Use of the
antisense oligonucleotides of the invention allows the synchronization of a
population of cells, thereby aiding detection of enhanced transfection
efficiency.
The anti-neoplastic utility of the antisense oligonucleotides according
to the invention is described in detail elsewhere in this specification.
In yet other preferred embodiments, the cell contacted with HDAC-4
antisense oligonucleodde is also contacted with HDAC-4 small molecule
inhibitor.
59
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
As used herein, the term "histone deacetylase small molecule inhibitor"
denotes an active moiety capable of interacting with one or more specific
histone deacetylase isoforms at the protein level and reducing the activity of
that histone deacetylase isoform. Particularly preferred are histone
deacteylase small molecule inhibitors that inhibit the HDAC-1 and/or the
HDAC-4 isoform. An HDAC-1 small molecule inhibitor is a molecule that
reduces the activity of the HDAC-1 isoform. An HDAC-4 small molecule
inhibitor is a molecule that reduces the activity of the HDAC-4 isoform. In a
preferred embodiment, the reduction of activity is at least 5-fold, more
preferably at least 10-fold, most preferably at least 50-fold. In another
embodiment, the activity of the histone deacetylase isoform is reduced 100-
fold. As discussed below, a preferred histone deacetylase small molecule
inhibitor is one that interacts with and reduces the enzymatic activity of
HDAC-4 and/or the HDAC-1 isoform that is involved in tumorigenesis.
In a few preferred embodiments, the histone deacetylase small
molecule inhibitor is operably associated with the antisense oligonucleoHde.
As mentioned above, the antisense oligonucleotides according to the
invention may optionally be formulated well known pharmaceutically
acceptable carriers or diluents. This formulation may further contain one or
more one or more additional histone deacetylase antisense oligonucleotide(s),
and/or one or more histone deacetylase small molecule inhibitor(s), or it may
contain any other pharmacologically active agent.
The term "operably associated with" or "operable association'
includes any association between the antisense oligonucleotide and the
histone deacetylase small molecule inhibitor which allows an antisense
oligonucleotide to inhibit one or more specific histone deacetylase isoform-
encoding nucleic acid expression and allows the histone deacetylase small
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
molecule inhibitor to inhibit specific histone deacetylase isoform enzymatic
activity. One or more antisense oligonucleotide of the invention may be
operably associated with one or more histone deacetylase small molecule
inhibitor. In some preferred embodiments, an antisense oligonucleotide of
the invention that targets one particular histone deacetylase isoform (e.g.,
HDAC-4) is operably associated with an small molecule inhibitor which
targets the same histone deacetylase isoform (e.g., HDAC-4). A preferred
operable association is a hydrolyzable. Preferably, the hydrolyzable
association is a covalent linkage between the antisense oligonucleotide and
the histone deacetylase small molecule inhibitor. Such a covalent linkage is
hydrolyzable, for example, by esterases and/or amidases. Examples of such
hydrolyzable associations are well known in the art. Phosphate esters are
particularly preferred.
In certain preferred embodiments, the covalent linkage may be directly
between the antisense oligonucleotide and the histone,deacetylase small
molecule inhibitor so as to integrate the histone deacetylase small molecule
inhibitor into the backbone of the oligonucleotide. Alternatively, the
covalent
linkage may be through an extended structure and may be formed by
covalently linking the anHsense oligonucleotide to the histone deacetylase
small molecule inhibitor through coupling of both the antisense
oligonucleotide and the histone deacetylase small molecule inhibitor to a
carrier molecule such as a carbohydrate, a peptide, a lipid or a glycolipid.
Another useful operable associations include lipophilic association, such as
the formation of a liposome containing an antisense oligonucleotide and the
histone deacetylase small molecule inhibitor covalently linked to a lipophilic
molecule. Such lipophilic molecules include, without limitation,
phosphoHdylcholine, cholesterol, phosphatidylethanolamine, and synthetic
61
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
neoglycolipids, such as syalyllacNAc-HDPE. In certain preferred
embodiments, the operable association may not be a physical association, but
simply a simultaneous co-existence in the body, for example, when the
antisense oligonucleotide is associated with one liposome and the small
molecule inhibitor is associated with another liposome.
In a third aspect, the invention provides a method for inhibiting
neoplastic cell proliferation in an animal, comprising administering to an
animal having at least one neoplastic cell present in its body a
therapeutically
effective amount of a specific inhibitor of HDAC-4, whereby neoplastic cell
proliferation is inhibited in the animal. In an embodiment thereof, the
invention provides a method for inhibiting neoplastic cell growth in an
animal. In this method, a therapeutically effective amount of the antisense
oligonucleotide of the invention is administered to an animal having at least
one neoplastic cell present in its body, the oligonucleotide being
administered
with a pharmaceutically acceptable carrier for a therapeutically effective
period of time. Preferably, the animal is~a mammal, particularly a
domesticated mammal. Most preferably, the animal is a human.
The term "neoplastic cell" is used to denote a cell that shows aberrant
cell growth. A neoplastic cell may be a hyperplastic cell, a cell that shows a
lack of contact inhibition of growth in vitro, a benign tumor cell that is
incapable of metastasis in vivo, or a cancer cell that is capable of
metastases in
vivo and that may recur after attempted removal. The term "tumorigenesis"
is used to denote the induction of uncharacteristic or untimely cell
proliferation that leads to the development of a neoplastic growth.
As used herein, the term "therapeutically effective amount" means the
total amount of each active component of the pharmaceutical composition or
method that is sufficient to show a meaningful patient benefit, i.e.,
inhibiting
62
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
HDAC activity, particularly HDAC-1 and/or HDAC-4 activity or to inhibit
neoplastic growth or for the treatment of proliferative diseases and
disorders.
When applied to an individual active ingredient, administered alone, the
term refers to that ingredient alone. When applied to a combination, the term
refers to combined amounts of the active ingredients that result in the
therapeutic effect, whether administered in combination, serially or
simultaneously.
Administration of the synthetic oligonucleotide of the invention used
in the pharmaceutical composition or to practice the method of the present
invention can be carried out in a variety of conventional ways, such as
intraocular, oral ingestion, inhalation, or cutaneous, subcutaneous,
intramuscular, or intravenous injection.
When a therapeutically effective amount of synthetic oligonucleotide
of the invention is administered orally, the synthetic oligonucleotide will be
in
the form of a tablet, capsule, powder, solution or elixir. When administered
in tablet form, the pharmaceutical composition of the invention may
additionally contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% synthetic
oligonucleoHde and preferably from about 25 to 90% synthetic
oligonucleotide. When administered in liquid form, a liquid carrier such as
water, petroleum, oils of animal or plant origin such as peanut oil, mineral
oil,
soybean oil, sesame oil, or synthetic oils may be added. The liquid form of
the pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol. When administered in liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by
63
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
weight of the synthetic oligonucleotide and preferably from about 1 to 50%
synthetic oligonucleotide.
When a therapeutically effective amount of synthetic oligonucleotide
of the invention is administered by intravenous, subcutaneous, intramuscular,
intraocular, or intraperitoneal injection, the synthetic oligonucleotide will
be
in the form of a pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable solutions, having due regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, subcutaneous, intramuscular,
intraperitoneal, or intraocular injection should contain, in addition to the
synthetic oligonucleotide, an isotonic vehicle such as Sodium Chloride
Injection, Ringei s Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, Lactated Ringer s Injection, or other vehicle as known in
the art. The pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other additives
known to those of skill in the art.
The amount of synthetic oligonucleotide in the pharmaceutical
composition of the present invention will depend upon the nature and
severity of the condition being treated, and on the nature of prior treatments
which the patent has undergone. Ultimately, the attending physician will
decide the amount of synthetic oligonucleotide with which to treat each
individual patient. Initially, the attending physician will administer low
doses of the synthetic oligonucleotide and observe the patient's response.
Larger doses of synthetic oligonucleotide may be administered until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the present
64
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
invention should contain about 10 wg to about 20 mg of synthetic
oligonucleotide per kg body or organ weight.
The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the severity of
the disease being treated and the condition and potential idiosyncratic
response of each individual patient. Ultimately the attending physician will
decide on the appropriate duration of intravenous therapy using the
pharmaceutical composition of the present invention.
In a preferred embodiment, the therapeutic composition of the
invention is administered systemically at a sufficient dosage to attain a
blood
level of antisense oligonucleotfde from about 0.01 ~,M to about 20 ~M. In a
particularly preferred embodiment, the therapeutic composition is
administered at a sufficient dosage to attain a blood level of antisense
oligonucleotide from about 0.05 ~M to about 15 ~,M. In a more preferred
embodiment, the blood level of antisense oligonucleotide is from about 0.1
~.M to about 10 ~M.
For localized administration, much lower concentrations than this may
be therapeutically effective. Preferably, a total dosage of anHsense
oligonucleotide will range from about 0.1 mg to about 200 mg oligonucleotide
per kg body weight per day. In a more preferred embodiment, a total dosage
of antisense oligonucleotide will range from about 1 mg to about 20 mg
oligonucleotide per kg body weight per day. In a most preferred
embodiment, a total dosage of antisense oligonucleotide will range from
about 1 mg to about 10 mg oligonucleotide per kg body weight per day. In a
particularly preferred embodiment, the therapeutically effective amount of
HDAC-4 antisense oligonucleotide is about 5 mg oligonucleotide per kg body
weight per day.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
The method may further comprise administering to the animal a
therapeutically effective amount of an HDAC-4 small molecule inhibitor with
a pharmaceutically acceptable carrier for a therapeutically effective period
of
time. In some preferred embodiments, the histone deacetylase small molecule
inhibitor is operably associated with the antisense oligonucleotide, as
described supra.
The histone deacetylase small molecule inhibitor-containing
therapeutic composition of the invention is administered systemically at a
sufficient dosage to attain a blood level histone deacetylase small molecule
inhibitor from about 0.01 ~M to about 10 ~.M. In a particularly preferred
embodiment, the therapeutic composition is administered at a sufficient
dosage to attain a blood level of histone deacetylase small molecule inhibitor
from about 0.05 ~M to about 10 ~M. In a more preferred embodiment, the
blood level of histone deacetylase small molecule inhibitor is from about 0.1
~M to about 5 ~M. For localized administration, much lower concentrations
than this may be effective. Preferably, a total dosage of histone deacetylase
small molecule inhibitor will range from about 0.01 mg to about 100 mg
protein effector per kg body weight per day. In a more preferred
embodiment, a total dosage of histone deacetylase small molecule inhibitor
will range from about 0.1 mg to about 50 mg protein effector per kg body
weight per day. In a most preferred embodiment, a total dosage of histone
deacetylase small molecule inhibitor will range from about 0.1 mg to about 25
mg protein effector per kg body weight per day. In a particularly preferred
embodiment, the therapeutically effective synergistic amount of histone
deacetylase small molecule inhibitor (when administered with an antisense
oligonucleotide) is about 5 mg per kg body weight per day.
66
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
When the method of the invention results in an improved inhibitory
effect, the therapeutically effective concentrations of either or both of the
nucleic acid level inhibitor (i.e., antisense oligonucleotide) and the protein
level inhibitor (i.e.,histone deacetylase small molecule inhibitor) required
to
obtain a given inhibitory effect are reduced as compared to those necessary
when either is used individually.
Furthermore, one of skill will appreciate that the therapeutically
effective synergistic amount of either the antisense oligonucleotide or the
histone deacetylase inhibitor may be lowered or increased by fine tuning and
altering the amount of the other component. The invention therefore
provides a method to tailor the administration/treatment to the particular
exigencies specific to a given animal species or particular patient.
Therapeutically effective ranges may be easily determined for example
empirically by starting at relatively low amounts and by step-wise increments
with concurrent evaluation of inhibition.
In a fourth aspect, the invention provides a method for inhibiting
HDAC-4 isoform in a cell comprising contacting the cell with a small
molecule inhibitor of the first aspect of the invention. In certain preferred
embodiments of the fourth aspect of the invention, cell proliferation is
inhibited in the contacted cell. In preferred embodiments, the cell is a
neoplastic cell which may be in an animal, including a human, and which
may be in a neoplastic growth.
In a fifth aspect, the invention provides a method for inhibiting
neoplastic cell growth in an animal comprising administering to an animal
having at least one neoplastic cell present in its body a therapeutically
effective amount of a small molecule inhibitor of the first aspect of the
67
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
invention with a pharmaceutically acceptable carrier for a therapeutically
effective period of time.
The histone deacetylase small molecule inhibitor-containing
therapeutic composition of the invention is administered systemically at a
sufficient dosage to attain a blood level histone deacetylase small molecule
inhibitor from about 0.01 ~ M to about 10 ~M. In a particularly preferred
embodiment, the therapeutic composition is administered at a sufficient
dosage to attain a blood level of histone deacetylase small molecule inhibitor
from about 0.05 ~,M to about 10~M. In a more preferred embodiment, the
blood level of histone deacetylase small molecule inhibitor is from about
0.1~M to about 5~.M. For localized administration, much lower
concentrations than this may be effective. Preferably, a total dosage of
histone
deacetylase small molecule inhibitor ranges from about 0.01 mg to about 100
mg protein effector per kg body weight per day. In a more preferred
embodiment, a total dosage of histone deacetylase small molecule inhibitor
ranges from about 0.1 mg to about 50 mg protein effector per kg body weight
per day. In a most preferred embodiment, a total dosage of histone
deacetylase small molecule inhibitor will range from about 0.1 mg to about 25
mg protein effector per kg body weight per day.
In a sixth aspect, the invention provides a method of inhibiting the
induction of cell proliferation, comprising contacting a cell with an
antisense
oligonucleotide that inhibits the expression of HDAC-4 or contacting a cell
with a small molecule inhibitor of HDAC-4. In certain preferred
embodiments, the cell is a neoplastic cell, and the induction of cell
proliferation is tumorigenesis.
The invention further provides for histone deacetylase small molecule
inhibitors that may be generated which specifically inhibit the histone
68
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
deacetylase isoform(s) required for inducing cell proliferation, e.g., HDAC-1
and HDAC-4, while not inhibiting other histone deacetylase isoforms not
required for inducing cell proliferation. Accordingly, in a seventh aspect,
the
invention provides a method for identifying a small molecule histone
deacetylase inhibitor that inhibits the HDAC-4 isoform and or the HDAC-1
isoform, which is required for the induction of cell proliferation. The method
comprises contacting the HDAC-4 and/or the HDAC-1 isoform with a
candidate small molecule inhibitor and measuring the enzymatic activity of
the contacted histone deacetylase isoform, wherein a reduction in the
enzymatic activity of the contacted histone deacetylase isoform identifies the
candidate small molecule inhibitor as a small molecule histone deacetylase
inhibitor that inhibits the histone deacetylase isoform, i.e., HDAC-4 and/or
HDAC-1.
Measurement of the enzymatic activity of HDAC-4 or HDAC-1 may be
achieved using known methodologies. For example, Yoshida et al. (]. Biol.
Chem. 265:17174-17179,1990) describe the assessment of histone deacetylase
enzymatic activity by the detection of acetylated histones in trichostatin A
treated cells. Taunton et al. (Science 272:408-411,1996) similarly describes
methods to measure histone deacetylase enzymatic activity using endogenous
and recombinant HDAC. Both Yoshida et al. (J. Biol. Chem. 265:17174-17179,
1990) and Taunton et al. (Science 272:408-411,1996) are hereby incorporated by
reference.
Preferably, the histone deacetylase small molecule inhibitor that
inhibits the HDAC-4 and or the HDAC-1 isoform required for induction of
cell proliferation is an HDAC-4 small molecule inhibitor that interacts with
and reduces the enzymatic activity of the HDAC-4 and/or the HDAC-1
isoform.
69
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In an eighth aspect, the invention provides a method for identifying a
small molecule histone deacetylase inhibitor that inhibits the HDAC-4 isoform
involved in the induction of cell proliferation. The method comprises
contacting a cell with a candidate small molecule inhibitor and measuring the
enzymatic activity of the contacted histone deacetylase isoform, wherein a
reduction in the enzymatic activity of the HDAC-4 isoform identifies the
candidate small molecule inhibitor as a small molecule histone deacetylase
inhibitor that inhibits HDAC-4.
In a ninth aspect, the invention provides a small molecule histone
deacetylase inhibitor identified by the method of the seventh or the eighth
aspects of the invention. Preferably, the histone deacetylase small molecule
inhibitor is substantially pure.
In a tenth aspect, the invention provides a method for inhibiting cell
proliferation in a cell comprising contacting a cell with at least two
reagents
selected from the group consisting of an antisense oligonucleotide that
inhibits expression of HDAC-4 isoform, a small molecule histone deacetylase
inhibitor that inhibits expression or activity of HDAC-4 isoform, an antisense
oligonucleotide that inhibits expression of the HDAC-1 isoform, a small
molecule histone deacetylase inhibitor that inhibits the expression or the
activity of the HDAC-1 isoform, an antisense oligonucleotide that inhibits
expression of a DNA methyltransferase, and a small molecule DNA
methyltransferase inhibitor. In one embodiment, the inhibition of cell growth
of the contacted cell is greater than the inhibition of cell growth of a cell
contacted with only one of the reagents. In certain embodiments, each of the
reagents selected from the group is substantially pure. In preferred
embodiments, the cell is a neoplastic cell. In yet additional preferred
embodiments, the reagents selected from the group are operably associated.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In an eleventh aspect, the invention provides a method of inhibiting
neoplasHc cell growth comprising contacting a cell with at least two reagents
selected from the group consisting of an antisense oligonucleotide that
inhibits expression of HDAC-4 isoform, a small molecule histone deacetylase
inhibitor that inhibits the expression or the activity of HDAC-4 isoform, an
antisense oligonucleotide that inhibits expression of the HDAC-1 isoform, a
small molecule histone deacetylase inhibitor that inhibits expression or
activity of the HDAC-1 isoform, an antlsense oligonucleotide that inhibits
expression of a DNA methyltransferase, and a small molecule DNA
methyltransferase inhibitor. In one embodiment, the inhibition of cell growth
of the contacted cell is greater than the inhibition of cell growth of a cell
contacted with only one of the reagents. In certain embodiments, each of the
reagents selected from the group is substantially pure. In preferred
embodiments, the cell is a neoplastic cell. In yet additional preferred
embodiments, the reagents selected from the group are operably associated.
Antisense oligonucleotides that inhibit DNA methyltransferase are
described in Szyf and von Hofe, U.S. Patent No. 5,578,716. DNA
methyltransferase small molecule inhibitors include, without limitation, 5-
aza-2'-deoxycytidine (5-aza-dC), 5-fluoro-2'-deoxycytidine, 5-aza-cyHdine (5-
aza-C), or 5,6-dihydro-5-aza-cytidine.
71
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
EXAMPLES
The following examples are intended to further illustrate certain
preferred embodiments of the invention and are not limiting in nature. Those
skilled in the art will recognize, or be able to ascertain, using no more than
routine experimentation, numerous equivalents to the specific substances and
procedures described herein. Such equivalents are considered to be within
the scope of this invention, and are covered by the appended claims.
Example 1
Synthesis and Identification of Antisense Oligonucleotides
Antisense (AS) and mismatch (MM) oligodeoxynucleoHdes (oligos)
were designed to be directed against the 5'- or 3'-untranslated region (UTR)
of
the targeted gene. Oligos were synthesized with the phosphorothioate
backbone and the 4X4 nucleotides 2'-O-methyl modification on an automated
synthesizer and purified by preparative reverse-phase HPLC. All oligos used
were 20 base pairs in length.
To identify antisense oligodeoxynucleotide (ODN) capable of
inhibiting HDAC-1 expression in human cancer cells, eleven
phosphorothioate ODNs containing sequences complementary to the 5' or 3'
UTR of the human HDAC-1 gene (GenBank Accession No. U50079) were
initially screened in T24 cells at 100 nM. Cells were harvested after 24 hours
of treatment, and HDAC-1 RNA expression was analyzed by Northern blot
analysis. This screen identified HDAC-1 AS as an ODN with antisense
activity to human HDAC-1. HDAC-1 MM oligo was created as a control;
compared to the antisense oligo, it has a 6-base mismatch.
Twenty-four phosphorothioate ODNs containing sequences
complementary to the 5' or 3' UTR of the human HDAC-2 gene (GenBank
72
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Accession No. U31814) were screened as above. HDAC-2 AS was identified
as an ODN with antisense activity to human HDAC-2. HDAC-2 MM was
created as a control; compared to the antisense oligo, it contains a 7-base
mismatch.
Twenty-one phosphorothioate ODNs containing sequences
complementary to the 5' or 3' UTR of the human HDAC-3 gene (GenBank
Accession No. AF039703) were screened as above. HDAC-3 AS was identified
as an ODN with antisense activity to human HDAC-3. HDAC-3 MM oligo
was created as a control; compared to the antisense oligo, it contains a a 6-
base mismatch.
Seventeen phosphorothioate ODNs containing sequences
complementary to the 5' or 3' UTR of the human HDAC-4 gene (GenBank
Accession No. AB006626) were screened as above. HDAC-4 AS was identified
as an ODN with antisense activity to human HDAC-4. HDAC-4 MM oligo
was created as a control; compared to the antisense oligo, it contains a 6-
base
mismatch.
Thirteen phosphorothloate ODNs containing sequences
complementary to the 5' or 3' untranslated regions of the human HDAC-6
gene (GenBank Accession No. AJ011972) were screened as above. HDAC-6
AS was identified as an ODN with antisense activity to human HDAC-6.
HDAC-6 MM oligo was created as a control; compared to the antisense oligo,
it contains a 7-base mismatch.
Example 2
HDAC AS ODNs Specifically Inhibit Expression at the mRNA Level
73
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
In order to determine whether AS ODN treatment reduced HDAC
expression at the mRNA level, Human A549 cells were treated with 50 nM of
antisense (AS) oligo directed against human HDAC-3 or its corresponding
mismatch (MM) oligo for 48 hours, and A549 cells were treated with 50 nM or
100 nM of AS oligo directed against human HDAC-4 or its MM oligo (100 nM)
for 24 hours.
Briefly, human A549 and/or T24 human bladder carcinoma cells were
seeded in 10 cm tissue culture dishes one day prior to oligonucleotide
treatment.
The cell lines were obtained from the American Type Culture Collection (ATCC)
(Manassas, VA) and were grown under the recommended culture conditions.
Before the addition of the oligonucleotides, cells were washed with PBS
(phosphate buffered saline). Next, lipofectin transfection reagent (GIBCO BRL
Mississauga, Ontario, CA), at a concentration of 6.25 ~g/ml, was added to
serum
free OPTIMEM medium (GIBCO BRL, Rockville, MD), which was then added to
the cells. The oligonucleotfdes to be screened were then added directly to the
cells (i.e., one oligonucleotlde per plate of cells). Mismatched
oligonucleoHdes
were used as controls. The same concentration of oligonucleotide (e.g., 50 nM)
was used per plate of cells for each oligonucleotide tested.
Cells were harvested, and total RNAs were analyzed by Northern blot
analysis. Briefly, total RNA was extracted using RNeasy miniprep columns
(QIAGEN). Ten to twenty ~g of total RNA was run on a formaldehyde
containing 1% agarose gel with 0.5 M sodium phosphate (pH 7.0) as the buffer
system. RNAs were then transferred to nitrocellulose membranes and
hybridized with the indicated radiolabeled DNA probes. Autoradiography was
performed using conventional procedures.
As presented in Figures 3A and 3B, respectively, the expression of HDAC-
3 mRNA and HDAC-4 mRNA in human A549 cells was inhibited by treatment
74
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
with the respective antisense oligonucleotides. These results indicate that
HDAC
AS ODNs can specifically inhibit targeted HDAC expression at the mRNA level.
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Example 3
HDAC OSDNs Inhibit HDAC Protein Expression
In order to determine whether treatment with HDAC OSDNs would
inhibit HDAC protein expression, human A549 cancer cells were treated with
50 nM of paired antisense or its mismatch oligos directed against human
HDAC-1, HDAC-2, HDAC-3, HDAC-4 or HDAC-6 for 48 hours. OSDN
treatment conditions were as previously described.
Cells were lysed in buffer containing 1 % Triton X-100, 0.5 % sodium
deoxycholate, 5 mM EDTA, 25 mM Tris-HC1, pH 7.5, plus protease inhibitors.
Total protein was quantified by the protein assay reagent from Bio-Rad
(Hercules, CA).100 ug of total protein was analyzed by SDS-PAGE. Next,
total protein was transferred onto a PVDF membrane and probed with
various HDAC-specific primary antibodies. Rabbit anti-HDAC-1 (H-51), anti-
HDAC-2 (H-54) antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA)
were used at 1:500 dilution. Rabbit anti-HDAC-3 antibody (Sigma, St. Louis,
MO) was used at a dilution of 1:1000. Anti-HDAC-4 antibody was prepared
as previously described (Wang, S.H. et al., (1999) Mol. Cell. Biol.19:7816-
27~,
and was used at a dilution of 1:1000. Anti-HDAC-6 antibody was raised by
immunizing rabbits with a GST fusion protein containing a fragment of
HDAC-6 protein (amino acid #990 to #1216, GenBank Accession
No. AAD29048). Rabbit antiserum was tested and found only to react
specifically to the human HDAC-6 isoform. HDAC-6 antiserum was used at
1:500 dilution in Western blots to detect HDAC-6 in total cell lysates. Horse
Radish Peroxidase conjugated secondary antibody was used at a dilution of
1:5000 to detect primary antibody binding. The secondary antibody binding
was visualized by use of the Enhanced chemiluminescence (ECL) detection kit
(Amersham-Pharmacia Biotech., Inc., Piscataway, NJ).
76
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
As shown in Figure 4, the treatment of cells with HDAC-1, HDAC-2,
HDAC-3, HDAC-4 or HDAC-6 ODNs for 48 hours specifically inhibits the
expression of the respective HDAC isotype protein.
In order to demonstrate that the level of HDAC protein expression is
an important factor in the cancer cell phenotype, experiments were done to
determine the level of HDAC isotype expression in normal and cancer cells.
Western blot analysis was performed as described above.
The results are presented in Table 3 clearly demonstrate that HDAC-1,
HDAC-2, HDAC-3, HDAC-4, and HDAC-6, isotype proteins are
overexpressed in cancer cell lines.
77
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Table 3: Expression Level of HDAC Isotypes in
Human
Normal and Cancer Cells
Norm
al TissueCell
or
Cance Desi4naHDA HDA HDA HDA HDA HDA HDA
r Type tion C-6 C-2 C-1 C~ C-4 C-5 C-7
Norma
I BreastHMEC + + - ++ + _ _
Epithet
ial
NormaForesk
I in MRHF + + - + ++ _ ++
Fibrobl
asts
CanceBladde
r r T24 +++ ++ +++ +++ ++ + ++
Cance
r Lung A549 ++ +++ ++ +++ +++ +++ +
Cance
r Colon SW48 +++ +++ +++ +++ +++
Cance
r Colon HCT116 +++ +++ ++++ +++ ++++ + -
Cance
r Colon HT29 +++ +++ +++ +++ +++
Cance NCI-
r Colon H446 ++ ++++ ++ +++ ++++ ++++++
Cance
r CervixHela +++ ++++ +++ +++ +++
CanceProstat
r a DU145 +++. +++ +++ +++ ++++
78
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Cance MDA-
r BreastMB-231 ++++ +++ ++ +++ +++
Cance
r BreastMCF-7 ++ +++ +++ +++ ++
Cance
r BreastT47D +++ +++ +++ +++ ++
Cance
r Kidney293T ++ ++++ +++ ++++ ++ ++++ +
CanceLeuke
r mia K562 ++++ ++++ +++ ++++ ++++
CanceLeuke
r mia Jurkat ++ ++ +++ ++++ ++ ++ +
T
(-): not detectable; (+): detectable; (++): 2X over (+); (+++);
5X over (+); (++++); ~ OX over (+)
Example 4
Effect of HDAC Isotvve Specific OSDNs on Cell Growth and Anovtosis
In order to determine the effect of HDAC OSDNs on cell growth and
cell death through apoptosis, A549 or T24 cells, MDAmb231 cells, and HMEC
cells (ATCC, Manassas, VA) were treated with HDAC OSDNs as previously
described.
For the apoptosis study, cells were analyzed using the Cell Death
Detection ELISA Plus kit (Roche Diagnostic GmBH, Mannheim, Germany)
according to the manufacturer's directions. Typically,10,000 cells were plated
79
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
in 96-well tissue culture dishes for 2 hours before harvest and lysis. Each
sample was analyzed in duplicate. ELISA reading was done using a MR700
plate reader (DYNEX Technology, Ashford, Middlesex, England) at 410 nm.
The reference was set at 490 nm.
For the cell growth analysis, human cancer or normal cells were treated
with 50 nM of paired AS or MM oligos directed against human HDAC-1,
HDAC-2, HDAC-3, HDAC-4 or HDAC-6 for 72 hours. Cells were harvested
and cell numbers counted by trypan blue exclusion using a hemocytometer.
Percentage of inhibition was calculated as (100 - AS cell numbers/control cell
numbers) % .
Results of the study are shown in Figure 5 and Figure 6, and in Table 4
and Table 5. Treatment of human cancer cells by HDAC-4 AS, and to a lesser
extent, HDAC 1 AS, induces growth arrest and apoptosis of various human
cancer cells (Figure 5 and Figure 6, Table 4 and Table 5). The corresponding
mismatches have no effect. The effects of HDAC-4 AS or HDAC-1 AS on
growth inhibition and apoptosis are significantly reduced in human normal
cells. In contrast to the effects of HDAC-4 or HDAC-1 AS oligos, treatment
with human HDAC-3 and HDAC-6 OSDNs has no effect on cancer cell
growth or apoptosis, and treatment with human HDAC-2 OSDN has a
minimal effect on cancer cell growth inhibition. Since T24 cells are p53 null
and A549 cells are p53 wild type, this induction of apoptosis is independent
of
p53 activity.
Table 4: Gene Transcription Altered by HDAC-4 AS1
CDK4 -3
cyclin A2 -3
cyclin B1 -3
p21 4
PLK -4
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
topo II a -5
GADD153 6
GADD45 3
Notch-4 -3
basic FGF 2
Egr-1 3
IL-15 4
IRF 2
Human A549 cells were treated with 50 nM oligos for two days before total RNAs
were harvested for cDNA array analysis;
Fold change on transcriptions was compared to that of HDAC-4
mismatch oligo (MM2) treated cells;.
Expression of 39 genes altered by HDAC-4 AS1 out of 588 genes
Table 5
Effect of HDAC Isotype-Specific OSDNs on Human Normal
and Cancer Cells Apoptosis After 48 Hour Treatment
A549 T24 MDAmb231 HMEC
HDAC-1 + - -
HDAC-2 - - - -
HDAC-3 - - - -
HDAC-4 +++ + ++ -
HDAC-6 - - - -
TSA (100ng/ml)++ ++ ++ +
"-": < = 2x fold over non-specific background; "+": 2-3X fold; "++": 3-
5X fold;
"+++": 5-8X fold; "++++"; 8X fold
Example 5
81
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Inhibition of HDAC Isotypes Induces the Expression of Growth Regulatory
Genes
In order to understand the mechanism of growth arrest and apoptosis
of cancer cells induced by HDAC-1 or HDAC-4 AS treatment, RNase
protection assays were used to analyze the mRNA expression of cell growth
regulators (p21 and GADD45) and proapoptotic gene Bax.
Briefly, human cancer A549 or T24 cells were treated with HDAC
isotype-specific antisense oligonucleotides (each 50 nM) for 48 hours. Total
RNAs were extracted and RNase protection assays were performed to
analyzed the mRNA expression level of p21 and GADD45. As a control, A549
cells were treated by lipofectin with or without TSA (250 ng/ml) treatment for
16 hours. These RNase protection assays were done according to the
following procedure. Total RNA from cells was prepared using "RNeasy
miniprep kit" from QIAGEN following the manufacturer's manual. Labeled
probes used in the protection assays were synthesized using "hStress-1
multiple-probe template sets" from Pharmingen (San Diego, California,
U.S.A.) according to the manufacturer's instructions. Protection procedures
were performed using "RPA IITM Ribonuclease Protection Assay Kit" from
Ambion, (Austin, Tx) following the manufacturer's instructions. Quantitation
of the bands from autoradiograms was done by using CycloneTM Phosphor
System (Packard Instruments Co. Inc., Meriden, CT). The results are shown in
Figure 7 and Table 6.
82
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Table 6
Up-Regulation of p21, GADD45 and Bax After Cell
Treatment with Human HDAC~Isotype-Specific Antisenses
A549 T24
p21 GADD45 Bax p21 GADD45 Bax
HDAC-1 1.7 5.0 0.8 2.4 3.4 0.9
HDAC-2 1.1 1.2 1.0 1.0 1.0 0.9
HDAC-3 0.7 0.9 1.0 0.9 1.0 1.0
HDAC-4 3.1 5.7 2.6 2.8 2.7 1.9
HDAC-6 1.0 1.0 1.0 1.0 0.8 1.1
TSA vs lipofectin2.8 0.6 0.8
Values indicate the fold induction of transcription as measured by RNase
protection analysis for the respective AS vs. MM HDAC isotype-specific
oligos.
As can be seen in Figure 7; the inhibition of HDAC-4 in both A549 and
T24 cancer cells dramatically up-regulates both p21 and GADD45 expression.
Inhibition of HDAC-1 by antisense oligonucleotides induces p21 expression
but more greatly induces GADD45 expression. Inhibition of HDAC-4,
upregulates Bax expression in both A549 and T24 cells. The effect of HDAC-4
AS treatment (50 nM, 48 hrs) on p21 induction in A549 cells is comparable to
that of TSA (0.3 to 0.8 uM,16 hrs).
Experiments were also conducted to examine the affect of HDAC
antisene oligonucleotides on HDAC protein expression. In A549 cells,
83
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
treatment with HDAC-4 antisene oligonucleotides results in a dramatic
increase in the level of p21 protein (Figure 8).
Example 7
Inhibition of HDAC Isotypes by Small Molecules
In order to demonstrate the identification of HDAC small molecule
inhibitors, HDAC small molecule inhibitors were screened in histone
deacetylase enzyme assays using various human histone deacetylase isotypic
enzymes (i.e., HDAC-1, HDAC-3, HDAC-4 and HDAC-6). Cloned
recombinant human HDAC-1, HDAC-3 and HDAC-6 enzymes, which were
tagged with the Flag epitope (Grozinger, C.M., et al., Proc. Natl. Acad. Sci.
U.S.A.
96:4868-4873 ( 1999)) in their C-termini, were produced by a baculovirus
expression system in insect cells.
Flag-tagged human HDAC-4 enzyme was produced in human
embronic kidney 293 cells after transformation by the calcium phosphate
precipitation method. Briefly, 293 cells were cultured in Dulbecco's Modified
Eagle Medium (DMEM) containing 10% fetal bovine serum and antibiotics.
Plasmid DNA encoding Flag-tagged human HDAC-4 was precipitated by
ethanol and resuspend in sterile water. DNA-calcium precipitates, formed by
mixing DNA, calcium choloride and 2XHEPES-buffered saline solution, were
left on 293 cells for 12-16 hours. Cells were return to serum-contained DMEM
84
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
medium and harvested at 48 hour post transfection for purification of Flag-
tagged HDAC-4 enzyme.
HDAC-1 and HDAC-6 were purified on a Q-Sepharose column,
followed by an anti-Flag epitope affinity column. The other I-iDAC isotypes,
HDAC-3 and HDAC-4, were purified directly on an anti-Flag affinity column.
For the deacetylase assay, 20,000 cpm of an [3H]-metabolically-labeled
acetylated histone was used as a substrate. Histones were incubated with
cloned recombinant human HDAC enzymes at 37oC. For the HDAC-1 asasy,
the incubation time was 10 minutes, and for the HDAC-3, HDAC-4 and
HDAC-6 assays, the incubation time was 2 hours. All assay conditions were
pre-determined to be certain that each reaction was linear. Reactions were
stopped by adding acetic acid (0.04 M final concentration) and HCl (250 mM,
final concentration). The mixture was extracted with ethyl acetate, and the
released [3H]-acetic acid was quantified by liquid scintillation counting. For
the inhibition studies, HDAC enzyme was preincubated with test compounds
for 30 minutes at 4oC prior to the start of the enzymatic assay. ICSO values
for
HDAC enzyme inhibitors were identified with dose response curves for each
individual compound and, thereby, obtaining a value for the concentration of
inhibitor that produced fifty percent of the maximal inhibition.
ss
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
Example 8
Inhibition of HDAC Activity in Whole Cells ~ Small Molecules
T24 human bladder cancer cells (ATCC, Manassas, VA) growing in
culture were incubated with test compounds for 16 hours. Histones were
extracted from the cells by standard procedures (see e.g. Yoshida et al.,
supra)
after the culture period. Twenty ~g total core histone protein was loaded
onto SDS/PAGE and transferred to nitrocellulose membranes, which were
then reacted with polyclonal antibody specific for acetylated histone H-4
(Upstate Biotech Inc., Lake Placid, WY). Horse Radish Peroxidase conjugated
secondary antibody was used at a dilution of 1:5000 to detect primary
antibody binding. The secondary antibody binding was visualized by use of
the Enhanced chemiluminescence (ECL) detection kit (Amersham-Pharmacia
Biotech., Inc;, Piscataway, NJ). After exposure to film, acetylated H-4 signal
was quantitated by densitometry.
The results, shown in Table 2 above, demonstrate that small molecule
inhibitors selective for HDAC-1 and/ or HDAC-4 can inhibit histone
deacetylation in whole cells.
Example 9
Inhibition of Cancer Growth by HDAC Small Molecule Inhibitors
Four thousand five hundred (4,500) human colon cancer HCT116 cells
(ATCC, Manassas, VA were used in an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5
diphenyl tetrazolium bromide) assay to quantitatively determine cell
proliferation and cytotoxicity. Typically, HCT116 cells were plated into each
well of the 96-well tissue culture plate and left overnight to attach to the
plate.
Compounds at various concentrations (1 uM, 5 uM and 25 uM, in DMSO)
86
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
were added in triplicate into the culture media (final DMSO concentration
1%) and incubated for 48 hours. MTT solution (obtained from Sigma as
powder) was added and incubated with the cells for 4 hours at 37~C in
incubator with 5% COz. During the incubation, viable cells convert MTT to a
water-insoluble formazan dye. Solubilizing buffer (50% N,N-
dimethylformamide, 20% SDS, pH 4.~ was added to cells and incubate for
overnight at 37C in incubator with 5% C02. Solubilized dye was quantitated
by colorimetric reading at 570 nM using a reference of 630 nM.
The results, shown in Table 2 above, demonstrate that small molecule
inhibitors selective for HDAC-1 and/or HDAC-4 can affect cell proliferation.
Example 10
Inhibition by Small Molecules of Tumor Growth in a Mouse Model
Female BALB/c nude mice were obtained from Charles River
Laboratories (Charles River, NY) and used at age 8-10 weeks. Human
prostate tumor cells (DU145, 2 x 106) or human colon cancer cells (HCT116;
2x106) or small lung core A549 2X106 were injected subcutaneously in the
animal's flank and allowed to form solid tumors. Tumor fragments were
serially passaged a minimum of three times, then approximately 30 mg tumor
fragments were implanted subcutaneously through a small surgical incision
under general anaesthesia. Small molecule inhibitor administration by
intraperotineal or oral administration was initiated when the tumors reached
a volume of 100 mm3. For intraperotineal administration, small molecule
inhibitors of HDAC (40--50 mg/kg body weight/day) were dissolved in 100%
DMSO and administered daily intraperitoneally by injection. For oral
administration, small molecule inhibitors of HDAC (40-50mg/kg body
weight/days) were dissolved in a solution containing 65% polyethylene
87
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
glycol 400 (PEG 400 (Sigma-Aldridge, Mississauga, Ontario, CA, Catalogue
No. P-3265), 5% ethanol, and 30% water. Tumor volumes were monitored
twice weekly up to 20 days. Each experimental group contained at least 6-8
animals. Percentage inhibition was calculated using volume of tumor from
vehicle-treated mice as controls.
The results, shown in Table 2 above, demonstrate that small molecule
inhibitors selective for HDAC-1 and/or HDAC-4 can inhibit the growth of
tumor cells in vivo.
88
CA 02434601 2003-07-10
WO 02/069947 PCT/IB02/02002
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain, using no
more than routine experimentation, many equivalents to the specific
embodi,emts of the invention described herein. Such equivalents are intended
to
be encompasssed by the following claims.
89
