U.S. patent application number 11/653855 was filed with the patent office on 2007-10-04 for valproic acid and derivatives thereof as histone deacetylase inhibitors.
This patent application is currently assigned to Georg-Speyer-Haus. Invention is credited to Martin Gottlicher, Bernd Groner, Thorsten Heinzel, Peter Herrlich.
Application Number | 20070232696 11/653855 |
Document ID | / |
Family ID | 8169134 |
Filed Date | 2007-10-04 |
United States Patent
Application |
20070232696 |
Kind Code |
A1 |
Gottlicher; Martin ; et
al. |
October 4, 2007 |
Valproic acid and derivatives thereof as histone deacetylase
inhibitors
Abstract
The present invention relates to the use of the drug valproic
acid and derivatives thereof as inhibitors of enzymes having
histone deacetylase activity. The invention also relates to the use
of those compounds for the manufacture of a medicament for the
treatment of diseases which are associated with hypoacetylation of
histones or in which induction of hyperacetylation has a beneficial
effect for example by induction of differentiation and/or apoptosis
in transformed cells.
Inventors: |
Gottlicher; Martin;
(Stutensee, DE) ; Heinzel; Thorsten; (Frankfurt am
Main, DE) ; Groner; Bernd; (Frankfurt am Main,
DE) ; Herrlich; Peter; (Karlsruhe, DE) |
Correspondence
Address: |
REED SMITH LLP
3110 FAIRVIEW PARK DRIVE
FALLS CHURCH
VA
22042
US
|
Assignee: |
Georg-Speyer-Haus
|
Family ID: |
8169134 |
Appl. No.: |
11/653855 |
Filed: |
January 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10332353 |
Aug 11, 2003 |
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PCT/EP01/07704 |
Jul 5, 2001 |
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11653855 |
Jan 17, 2007 |
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Current U.S.
Class: |
514/546 ;
435/6.1; 435/7.23; 514/558; 514/625 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61P 35/02 20180101; A61K 31/19 20130101; A61P
5/02 20180101 |
Class at
Publication: |
514/546 ;
514/558; 514/625; 435/007.23; 435/006 |
International
Class: |
A61K 31/22 20060101
A61K031/22; G01N 33/574 20060101 G01N033/574; A61K 31/16 20060101
A61K031/16; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 7, 2000 |
EP |
00.114088.8 |
Claims
1-23. (canceled)
24. The use of a compound of Formula I as an inhibitor of an enzyme
having histone deacetylase activity, ##STR2## wherein R.sup.1 and
R.sup.2 independently are a linear or branched, saturated or
unsaturated, aliphatic C.sub.2-25 hydrocarbon chain which may be
substituted with a hydroxyl, amino, alkoxy, aryl or heterocyclic
group, R.sup.3 is hydroxyl, halogen, alkoxy or an optionally
alkylated amino group; or a pharmaceutically acceptable salt of a
compound of the Formula I; or a prodrug or pharmaceutically active
metabolite of a compound of the Formula I, or a pharmaceutically
acceptable salt of a prodrug or metabolite thereof.
25. The use according to claim 24, wherein R.sup.1 and R.sup.2
independently are a linear or branched, saturated or unsaturated,
C.sub.2-10 hydrocarbon chain.
26. The use according to claim 24, wherein the compound is selected
from the group consisting of VPA, S-4-yn VPA, 2-ethyl hexanoic acid
and pharmaceutically acceptable salts, prodrugs or pharmaceutically
active metabolites thereof.
27. The use according to anyone of claims 24 to 26, wherein the
enzyme having histone deacetylase activity is mammalian, preferably
human histone deacetylase.
28. The use according to claim 27, wherein the human histone
deacetylase is selected from the group consisting of histone
deacetylases HDAC 1-3 (class I) and HDAC 4-8 (class II).
29. The use according to claim 24, wherein the compound is used for
the induction of differentiation of cells.
30. The use according to claim 29, wherein the compound is used for
the induction of differentiation of transformed cells.
31. The use according to claim 24, wherein the compound is used for
the induction of apoptosis of transformed cells.
32-49. (canceled)
50. A method of treating a disease or disorder associated with the
hyperacetylation of histones, comprising administering to a subject
in need of such treatment, an agent capable of inhibiting histone
deacetylase, selected from the group consisting of valproic acid,
S-4-yn valproic acid, and 2-ethyl-hexanoic acid or a
pharmaceutically acceptable salt; or a prodrug or pharmaceutically
active metabolite, or a pharmaceutically acceptable salt of a
prodrug or metabolite thereof, wherein the disease is an endocrine
disorder associated with induction of histone deacetylase.
51. The method of claim 50, wherein the disease is thyroid
resistance syndrome.
Description
[0001] The present invention relates to the use of the drug
valproic acid and derivatives thereof as inhibitors of enzymes
having histone deacetylase activity. The invention also relates to
the use of those compounds for the manufacture of a medicament for
the treatment of diseases which are associated with hypoacetylation
of histones or in which induction of hyperacetylation has a
beneficial effect for example by induction of differentiation
and/or apoptosis in transformed cells.
[0002] Local remodelling of chromatin is a key step in the
transcriptional activation of genes. Dynamic changes in the
nucleosomal packaging of DNA must occur to allow transcriptional
proteins contact with the DNA template. One of the most important
mechanisms contributing to chromatin remodelling is the
posttranslational modification of histones by acetylation. Change
in electrostatic attraction for DNA and steric hindrance introduced
by the hydrophobic acetyl group leads to destabilisation of the
interaction of histones with DNA. As a result, acetylation of
histones disrupts nucleosomes and allows the DNA to become
accessible to the transcriptional machinery. Removal of the acetyl
groups allows the histones to bind more tightly to DNA and to
adjacent nucleosomes and thus maintain a transcriptionally
repressed chromatin structure. Acetylation is mediated by a series
of enzymes with histone acetyltransferase (HAT) activity.
Conversely, acetyl groups are removed by specific histone
deacetylase (HDAC) enzymes. Disruption of these mechanisms gives
rise to transcriptional misregulation and may lead to leukemic
transformation.
[0003] Nuclear hormone receptors are ligand-dependent transcription
factors that control development and homeostasis through both
positive and negative control of gene expression. Defects in these
regulatory processes underlie the causes of many diseases and play
an important role in the development of cancer.
[0004] Several members of the nuclear receptor superfamily have
been reported to interact with basal transcription factors,
including TFIIB. However, numerous lines of evidence indicate that
nuclear receptors must interact with additional factors to mediate
both activation and repression of target genes. A number of
cofactors that associate with the ligand binding domains of
estrogen (ER), retinoic acid (RAR), thyroid hormone (T3R), retinoid
X (RXR), and other nuclear receptors have recently been identified.
Putative coactivator proteins include SRC-1/NCoA-1,
GRIP1/TIF2/NCoA-2, p/CIP/ACTR/AIB1, CBP and a variety of other
factors (reviewed in Xu et al., 1999, Curr Opin Genet Dev 9,
140-147). Interestingly, SRC proteins as well as CBP have been
shown to harbor intrinsic histone acetyltransferase activity and to
exist in a complex with the histone acetylase P/CAF.
[0005] Many nuclear receptors, including T3R, RAR and PPAR, can
interact with the corepressors N-CoR and SMRT in the absence of
ligand and thereby inhibit transcription. Furthermore, N-CoR has
also been reported to interact with antagonist-occupied
progesterone and estrogen receptors. N-CoR and SMRT have been shown
to exist in large protein complexes, which also contain mSin3
proteins and histone deacetylases. Thus, the ligand-induced switch
of nuclear receptors from repression to activation reflects the
exchange of corepressor and coactivator complexes with antagonistic
enzymatic activities.
[0006] The N-CoR corepressor complex not only mediates repression
by nuclear receptors, but also interacts with additional
transcription factors including Mad-1, BCL-6 and ETO. Many of these
proteins play key roles in disorders of cell proliferation and
differentiation. T3R for example was originally identified on the
basis of its homology with the viral oncogene v-erbA, which in
contrast to the wildtype receptor does not bind ligand and
functions as a constitutive repressor of transcription.
Furthermore, mutations in RARs have been associated with a number
of human cancers, particularly acute promyelocytic leukemia (APL)
and hepatocellular carcinoma. In APL patients RAR fusion proteins
resulting from chromosomal translocations involve either the
promyelocytic leukemia protein (PML) or the promyelocytic zinc
finger protein (PLZF). Although both fusion proteins can interact
with components of the corepressor complex, the addition of
retinoic acid dismisses the corepressor complex from PML-RAR,
whereas PLZF-RAR interacts constitutively. These findings provide
an explanation why PML-RAR APL patients achieve complete remission
following retinoic acid treatment whereas PLZF-RAR APL patients
respond very poorly. The hypothesis that corepressor-mediated
aberrant repression may be causal for pathogenesis in APL is
supported by the finding that trichostatin A, which inhibits
histone deacetylase (HDAC) function is capable of overcoming the
differentiation block in cells containing the PLZF-RAR fusion
protein. Furthermore, a PML-RAR patient who had experienced
multiple relapses after treatment with retinoic acid has recently
been treated with the HDAC inhibitor phenylbutyrate, resulting in
complete remission of the leukemia (Warrell et al., 1998, J. Natl.
Cancer Inst. 90, 1621-1625).
[0007] Additional evidence that histone acetylation plays a role in
cancer comes from studies on the AML1-ETO oncoprotein and on
chromosomal rearrangements involving the MLL locus (Redner et al.,
1999, Blood 94, 417-428).
[0008] WO 99/37150 discloses a transcription therapy for cancer
comprising administering a retinoid substance and an inhibitor of
histone deacetylase.
[0009] Several compounds are known to be HDAC inhibitors. Butyric
acid, or butyrate, was the first HDAC inhibitor to be identified.
In millimolar concentrations, butyrate is not specific for HDAC, it
also inhibits phosphorylation and methylation of nucleoproteins as
well as DNA methylation. Its analogue phenylbutyrate acts in a
similar manner. More specific are trichostatin A (TSA) and trapoxin
(TPX). TPX and TSA have emerged as potent inhibitors of histone
deacetylases. TSA reversibly inhibits, whereas TPX irreversibly
binds to and inactivates HDAC enzymes. Unlike butyrate, nonspecific
inhibition of other enzyme systems has not yet been reported for
TSA or TPX. TSA and TPX, however, exhibit considerable toxicity and
are poorly bioavailable. Therefore they are of limited therapeutic
use.
[0010] It is one object of the present invention to provide
substances which can induce differentiation and/or apoptosis in a
wide variety of transformed cells and therefore can be useful in
the treatment of cancer.
[0011] The invention relies on the novel finding that valproic acid
(VPA; 2-n-propylpentanoic acid) is capable of inhibiting histone
deacetylases.
[0012] Valproic acid is a known drug with multiple biological
activities which depend on different molecular mechanisms of
action. [0013] VPA is an antiepileptic drug. [0014] VPA is
teratogenic. When used as antiepileptic drug during pregnancy VPA
can induce birth defects (neural tube closure defects and other
malformations) in a few percent of born children. In mice, VPA is
teratogenic in the majority of mouse embryos when properly dosed.
[0015] VPA activates a nuclear hormone receptor (PPAR8). Several
additional transcription factors are also derepressed but some
factors are not significantly derepressed (glucocorticoid receptor,
PPAR.alpha.). [0016] VPA is hepatotoxic, which may depend on poorly
metabolized esters with coenzyme A.
[0017] The use of VPA derivatives allowed to determine that the
different activities are mediated by different molecular mechanisms
of action. Teratogenicity and antiepileptic activity follow
different modes of action because compounds could be isolated which
are either preferentially teratogenic or preferentially
antiepileptic (Nau et al., 1991, Pharmacol. Toxicol. 69, 310-321).
Activation of PPAR8 was found to be strictly correlated with
teratogenicity (Lampen et al., 1999, Toxicol. Appl. Pharmacol. 160,
238-249) suggesting that, both, PPAR.delta. activation and
teratogenicity require the same molecular activity of VPA. Also,
differentiation of F9 cells strictly correlated with PPAR.delta.
activation and teratogenicity as suggested by Lampen et al., 1999,
and documented by the analysis of differentiation markers (Werling
et al., 2001, Mol. Pharmacol. 59, 1269-1276).
[0018] It is shown in the present application, that PPAR.delta.
activation is caused by the HDAC inhibitory activity of VPA and its
derivatives. Furthermore it is shown that the established HDAC
inhibitor TSA activates PPAR.delta. and induces the same type of F9
cell differentiation as VPA. From these results we conclude that
not only activation of PPAR.delta. but also induction of F9 cell
differentiation and teratogenicity of VPA or VPA derivatives are
most likely caused by HDAC inhibition.
[0019] The present invention is based on the finding that VPA and
the derivatives described in this application are inhibitors of
histone deacteylases. The finding of this novel mechanism of action
of VPA and compounds derived thereof, i.e. the inhibition of
enzymes with histone deacetylase activity led us to the proposition
that VPA due to its HDAC-inhibitory activity should be useful to
induce differentiation and/or apoptosis in a wide variety of cancer
cells for two reasons: (1) these enzymes are present in all cells
and (2) pilot studies with model compounds such as butyrate or TSA
which are different from those described in this invention had
shown that HDAC inhibitors induce differentiation in a wide variety
of cells.
[0020] The activity to induce differentiation and/or apoptosis in a
wide variety of transformed cells is a much more complex biological
activity than only the inhibition of proliferation. In the latter
case it would not be evident, why only the proliferation of
transformed (tumor) but not of normal cells should be inhibited.
The induction of apoptosis, differentiation or more specifically
re-differentiation in dedifferentiated tumor cells provides a
rationale why the compounds of this invention have beneficial
effects in a wide variety of tumors by induction of differentiation
and/or apoptosis. This proposition was confirmed in a wide variety
of tumor cells (see examples).
[0021] Antiepileptic and sedating activities follow different
structure activity relationships and thus obviously depend on a
primary VPA activity distinct from HDAC inhibition.
[0022] The mechanism of hepatotoxicity is poorly understood and it
is unknown whether it is associated with formation of the VPA-CoA
ester. The use according to the invention, e.g. HDAC inhibition,
however, appears not to require CoA ester formation.
[0023] U.S. Pat. No. 5,672,746 and WO 96/06821 disclose the use of
VPA and derivatives thereof for the treatment of neuredegenerative
and neuroproliferative disorders.
[0024] One aspect of the present invention is the use of VPA and
derivatives thereof as an inhibitor of enzymes having histone
deacetylase activity. Derivatives of VPA are .alpha.-carbon
branched carboxylic acids as described by formula I ##STR1##
wherein R.sup.1 and R.sup.2 independently are a linear or branched,
saturated or unsaturated aliphatic C.sub.2-25, preferably
C.sub.3-25 hydrocarbon chain which optionally comprises one or
several heteroatoms and which may be substituted, R.sup.3 is
hydroxyl, halogen, alkoxy or an optionally alkylated amino
group.
[0025] Different R.sup.1 and R.sup.2 residues give rise to chiral
compounds. Usually one of the stereoisomers has a stronger
teratogenic effect than the other one (Nau et al., 1991, Pharmacol.
Toxicol. 69, 310-321) and the more teratogenic isomer more
efficiently activates PPAR.delta. (Lampen et al, 1999). Therefore,
this isomer can be expected to inhibit HDACs more strongly (this
invention). The present invention encompasses the racemic mixtures
of the respective compounds, the less active isomers, and in
particular the more active isomers.
[0026] The hydrocarbon chains R.sup.1 and R.sup.2 may comprise one
or several heteroatoms (e.g. O, N, S) replacing carbon atoms in the
hydrocarbon chain. This is due to the fact that structures very
similar to that of carbon groups may be adopted by heteroatom
groups when the heteroatoms have the same type of hybridization as
a corresponding carbon group.
[0027] R.sup.1 and R.sup.2 may be substituted. Possible
substituents include hydroxyl, amino, carboxylic and alkoxy groups
as well as aryl and heterocyclic groups.
[0028] Preferably, R.sup.1 and R.sup.2 independently comprise 2 to
10, more preferably 3 to 10 or 5 to 10 carbon atoms. It is also
preferred that R.sup.1 and R.sup.2 independently are saturated or
comprise one double bond or one triple bond. In particular, one of
the side chains (R.sup.1) may preferably contain sp.sup.1
hybridized carbon atoms in position 2 and 3 or heteroatoms which
generate a similar structure. This side chain should comprise 3
carbon or heteroatoms but longer chains may also generate
HDAC-inhibiting molecules. Also inclusion of aromatic rings or
heteroatoms in R.sup.2 is considered to generate compounds with
HDAC inhibitory activity because the catalytic site of the HDAC
protein apparently accommodates a wide variety of binding
molecules. With the novel observation that teratogenic VPA
derivatives are HDAC inhibitors, also compounds which have
previously been disregarded as suitable antiepileptic agents are
considered as HDAC inhibitors under this invention. In particular,
but not exclusively, compounds having a propinyl residue as R.sup.1
and residues of 7 or more carbons as R.sup.2, are considered
(Lampen et al, 1999).
[0029] Preferably, the group "COR.sup.3" is a carboxylic group.
Also derivatization of the carboxylic group has to be considered
for generating compounds with potential HDAC inhibitory activity.
Such derivatives may be halides (e.g. chlorides), esters or amides.
When R.sup.3 is alkoxy, the alkoxy group comprises 1 to 25,
preferably 1-10 carbon atoms. When R.sup.3 is a mono- or
di-alkylated amino group, the alkyl substituents comprise 1 to 25,
preferably 1-10 carbon atoms. An unsubstituted amino group,
however, is preferred.
[0030] According to the present invention also pharmaceutically
acceptable salts of a compound of formula I can be used. According
to the present invention also substances can be used which are
metabolized to a compound as defined in formula I in the human
organism or which lead to the release of a compound as defined in
formula I for example by ester hydrolysis.
[0031] In a particular embodiment, the invention concerns the use
of an .alpha.-carbon branched carboxylic acid as described by
formula I or of a pharmaceutically acceptable salt thereof as an
inhibitor of an enzyme having histone deacetylase activity wherein
R.sup.1 is a linear or branched, saturated or unsaturated,
aliphatic C.sub.5-25 hydrocarbon chain, R.sup.2 independently is a
linear or branched, saturated or unsaturated, aliphatic C.sub.2-25
hydrocarbon chain, but not --CH.sub.2--CH.dbd.CH2,
--CH.sub.2--C.ident.CH or --CH.sub.2--CH.sub.2--CH.sub.3, R.sup.1
and R.sup.2 are optionally substituted with hydroxyl, amino,
carboxylic, alkoxy, aryl and/or heterocyclic groups, and R.sup.3 is
hydroxyl.
[0032] In yet another embodiment the invention concerns the use of
an .alpha.-carbon branched carboxylic acid as described by formula
I or of a pharmaceutically acceptable salt thereof as an inhibitor
of an enzyme having histone deacetylase activity wherein R.sup.1 is
a linear or branched, saturated or unsaturated, aliphatic
C.sub.3-25 hydrocarbon chain, and R.sup.2 independently is a linear
or branched, saturated or unsaturated, aliphatic C.sub.2-25
hydrocarbon chain, R.sup.1 or R.sup.2 comprise one or several
heteroatoms (e.g. O, N, S). replacing carbon atoms in the
hydrocarbon chain, R.sup.1 and R.sup.2 are optionally substituted
with hydroxyl, amino, carboxylic, alkoxy, aryl and/or heterocyclic
groups, and R.sup.3 is hydroxyl.
[0033] In yet another embodiment of the invention R.sup.1 and
R.sup.2 do not comprise an ester group (--CO--O--). The atom of
R.sup.1 which is next to the .alpha.-carbon of the carboxylic acid
(derivative) of formula I and covalently linked to said
.alpha.-carbon may be a carbon atom. The atom of R.sup.2 which is
next to the .alpha.-carbon of the carboxylic acid (derivative) of
formula I and covalently linked to said .alpha.-carbon may be a
carbon atom. R.sup.1 and R.sup.2 may be hydrocarbon chains
comprising no heteroatoms O, N or S.
[0034] The compounds which are most preferably used according to
the present invention are VPA, S-4-yn VPA, 2-EHXA (2-Ethyl-hexanoic
acid).
[0035] The compounds are useful for inhibiting mammalian (for use
of cell lines in in vitro assays and animal models systems) and in
particular human (in vivo and in vitro) histone deacetylases HDAC
1-3 (class I) and HDAC 4-8 (class II).
[0036] The compounds may be used to induce the differentiation
and/or apoptosis of cells such as undifferentiated tumour cells.
Presumably, this reflects a general mechanism, as differentiation
can be induced in F9 teratocarcinoma cells, MT 450 breast cancer
cells, HT-29 colon carcinoma cells and several leukemia cell lines
as assessed by morphological alterations and specific marker gene
or protein expression. Furthermore, for example MT450 cells can be
induced to undergo apoptosis (see example 6).
[0037] The invention also concerns the use of a compound of formula
I for the induction of differentiation and/or apoptosis of
transformed cells.
[0038] Another aspect of the present invention is the use of a
compound of formula I for the manufacture of a medicament for the
treatment of a disease which is associated with gene-specific
hypoacetylation of histones. There are a number of diseases which
are associated with aberrant repression of specific genes which
correlates with a local level of histone acetylation below the
regular level.
[0039] Yet another aspect of the invention is the use of a compound
of formula I for the manufacture of a medicament for the treatment
of a disease in which the induction of hyperacetylation of histones
has a beneficial effect resulting in differentiation and/or
apoptosis of a patient's tumor cells and thus causing a clinical
improvement of the patient's condition. Examples of such diseases
are skin cancer, estrogen receptor-dependent and independent breast
cancer, ovarian cancer, prostate cancer, renal cancer, colon and
colorectal cancer, pancreatic cancer, head and neck cancer, small
cell and non-small cell lung carcinoma. The induction of
hyperacetylation may also be beneficial by reverting inappropriate
gene expression in diseases based on aberrant recruitment of
histone deacetylase activity such as thyroid resistance
syndrome.
[0040] The compounds and salts thereof can be formulated as
pharmaceutical compositions (e.g. powders, granules, tablets,
pills, capsules, injections, solutions, foams, enemas and the like)
comprising at least one such compound alone or in admixture with
pharmaceutically acceptable carriers, excipients and/or diluents.
The pharmaceutical compositions can be formulated in accordance
with a conventional method. Specific dose levels for any particular
patient will be employed depending upon a variety of factors
including the activity of specific compounds employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, rate of excretion, drug combination, and
the severity of the particular disease undergoing therapy. The
active ingredient will preferably be administered in an appropriate
amount, for example, selected from the range of about 10 mg/kg to
100 mg/kg body weight a day orally or intravenously. The dose
levels are not specifically restricted as long as serum levels of
0.05 mM to 3 mM, preferably of about 0.4 mM to 1.2 mM are
achieved.
[0041] Another aspect of the invention is a method for the
identification of substances having histone deacetylase inhibitory
activity which comprises providing a derivative of valproic acid,
determining its histone deacetylase inhibitory activity, and
selecting the substance if the substance has histone deacetylase
inhibitory activity. Valproic acid can serve as a lead substance
for the identification of other compounds exhibiting histone
deacetylase inhibitory activity. Thereby compounds may be selected
which show increased HDAC inhibitory activity at lower doses and
serum levels and have decreased effects on the central nervous
system such as sedating activity. Another parameter that may be
optimised is the appearance of the hepatotoxic effect. Compounds
may be selected which show a reduced liver toxicity. The
derivatives may be provided by synthesising compounds which
comprise additional and/or modified substituents. The HDAC
inhibitory activity may be determined by a state-of-the-art
technology such as transcription repression assay, a Western Blot
which detects acetylation of histone H3 and/or histone H4, or by an
enzymatic assay.
[0042] The transcriptional assay for repressor activity exploits
activation and derepression of a Gal4-dependent reporter gene. This
assay can be performed either by transient transfection of
mammalian cell lines (e.g. HeLa, 293T, CV-1) or with specifically
constructed permanent cell lines. Transcription factors such as
thyroid hormone receptor, PPAR.delta., retinoic acid receptor,
N-CoR and AML/ETO repress transcription when they bind to a
promoter containing UAS elements as fusion proteins with the
heterologous DNA-binding domain of the yeast Gal4 protein. In the
absence of the Gal4-fusion protein the reporter gene has a high
basal transcriptional activity due to the presence of binding sites
for other transcription factors in the thymidine kinase promoter.
The Gal4 fusion proteins repress this activity by up to 140-fold.
HDAC inhibitors induce relief of this repression which can be
detected as an increase in reporter gene activity (e.g. by
luciferase assay).
[0043] Histone deacetylase inhibitors induce the accumulation of
N-terminally hyperacetylated histones H3 and H4. These acetylated
histones can be detected by Western blot analysis of whole cell
extracts or of histone preparations from histone deacetylase
inhibitor-treated cells using antibodies specific for the
acetylated N-terminal lysine residues of histones H3 and H4.
[0044] The enzymatic assay for HDAC activity records the release of
.sup.3H-labeled acetic acid from hyperacetylated substrates.
Sources of HDAC activity can be co-immunoprecipitates with
antibodies directed against N-CoR (or other repressors known to
recruit HDACs) or crude cell extracts containing histone
deacetylases (e.g. HeLa, 293T, F 9). Substrates may be either
chemically .sup.3H-acetylated peptides corresponding to the.
N-termini of histones H3 or H4 or histone proteins isolated from
metabolically labelled cells which were treated with HDAC
inhibitors. After extraction with ethyl acetate the release of
.sup.3H-labeled acetic acid is detected by liquid scintillation
counting.
[0045] Yet another aspect of the invention is a method for
profiling of the HDAC isoenzyme specificity of a compound as
defined in formula I wherein the binding of the compound to HDAC
and/or the competition for binding to HDAC is measured.
[0046] The method may comprise the following steps: HDACs are
either immune precipitated with HDAC isoform specific antibodies,
with antibodies directed against corepressor complexes, or with
specific antibodies against recombinant HDACs overexpressed in
transgenic cells. The method may further involve determination of
individual HDACs present in these immune precipitates by Western
blot analysis. Radiolabeled VPA or compounds according to formula I
are bound to the immune precipitates. The amount of bound compound
is determined through measurement of bound radioactivity after
appropriate washing steps. A variation of this aspect involves
binding of one labeled HDAC inhibitor such as VPA, TSA or trapoxin
and competition of binding by a compound according to formula I.
Another variation of the method involves the use of alternate
labeling and/or detection procedures.
[0047] It is preferred that compounds are selected which
specifically inhibit only a subset of HDACs.
[0048] Another aspect of the invention is the use of VPA or
derivatives thereof to define genes which are induced by said
compounds in cells such as primary human or rodent cells, leukemic
cells, other cancer cells or tumor cell lines. Methods to define
such genes that are induced by VPA include established technologies
for screening large arrays of cDNAs, expressed sequence tags or
so-called unigene collections. Also the use of subtractive
hybridization techniques is suitable to define genes which are
induced by VPA or derivatives thereof. The use of these methods to
identify potential targets for drug development downstream of
HDAC-inhibition, and furthermore the use of these methods to define
diagnostic means in order to facilitate the therapeutic treatment
of patients with suitable compounds is part of this invention.
Considering the low general toxicity of VPA in the organism
compared to other HDAC-inhibitors it is a specific aspect of this
invention to use VPA or derivatives thereof for defining target
genes which are selectively regulated or not regulated by VPA,
particularly also in comparison to other HDAC inhibitors like
trichostatin A.
[0049] The present invention also concerns a diagnostic method to
identify tumors comprising the step of testing whether a tumor is
responsive to treatment with compounds as defined by formula I. The
method preferably comprises the method for the identification of
genes induced by VPA or a derivative thereof described supra. In a
particular embodiment, the diagnostic method comprises the use of
nucleic acid technology, preferably of hybridization or polymerase
chain reaction for detection. Other types of nucleic acid
technology, however, may be employed. In another embodiment the
method comprises the use of specific antibodies against
differentially regulated proteins for detection. For this purpose
proteins encoded by the genes showing deregulation of their
expression upon VPA treatment would be expressed e.g. in
recombinant expression systems and antibodies directed against
these proteins would be generated. Subsequently such antibodies
could be used (or patterns of antibodies) to characterize the
status of a tumor or tumor cells for diagnostic and/or prognostic
reasons.
[0050] The present invention provides novel possibilities to treat
various cancer diseases. Applicant found that VPA and derivatives
thereof are potent HDAC inhibitors. The HDAC inhibitors known so
far are either nonspecific like butyrate, or toxic or poorly
bioavailable in the whole organism like TSA and TPX. VPA has the
advantage that it is already an approved drug and has been used
over decades for the treatment of epilepsy in human. Thus, a vast
amount of data concerning pharmaceutical acceptability and the lack
of serious side effects are available. Thus VPA should be a
suitable drug for the use in humans to induce differentiation
and/or apoptosis in transformed cells and by that to exert
beneficial effects in a wide variety of patients suffering from
cancer.
[0051] FIG. 1 describes the histone deacetylase inhibitor-like
activation of PPAR.delta. by VPA (example 1).
[0052] FIG. 2 shows that VPA activates several transcription
factors in addition to PPAR.delta. (example 2).
[0053] FIG. 3 shows VPA-induced accumulation of hyperacetylated
histones H3 and H4 (example 3).
[0054] FIG. 4 shows the biochemical analysis of histone deacetylase
activity in the absence or presence of VPA (example 4).
[0055] FIG. 5 shows indicators of VPA induced differentiation in
HT-29 colonic carcinoma cells, F9-teratocarcinoma cells, and RenCa
renal carcinoma cells. The phenotypes of F9-teratocarcinoma cells
differentiated by VPA or the histone deacetylase inhibitor
trichostatin A appear identical (example 5).
[0056] FIG. 6 shows induction of apoptosis in MT450 breast cancer
cells (example 6).
[0057] FIG. 7 shows the the loss of viable cells upon treatment
with valproic acid. Renca-lacZ, Renca-lacZ/EGFR,
Renca-lacZ/EGFRvIII and Renca-lacZ/ErbB2 renal carcinoma cells (A)
or SKOV3 ovarian carcinoma cells, SKBR3, MCF7, MDA-MB453 and
MDA-MB468 breast carcinoma cells, and A431 squamous cell carcinoma
cells (B) were incubated with the indicated concentrations of
valproic acid (VPA). The relative number of viable cells was
determined using the enzymatic MTT assay, measuring cellular
metabolic activity, as described in Example 7. Each point
represents the mean of a set of data determined in triplicate
(example 7).
[0058] FIG. 8 shows the reduction in cellular biomass after
treatment of cell cultures with VPA (example 8)
[0059] The following examples further illustrate the invention.
EXAMPLE 1
[0060] Activation of a PPAR.delta.-Glucocorticoid Receptor Hybrid
Protein by VPA
[0061] A reporter gene cell line for activation of the PPAR.delta.
ligand binding domain was constructed in CHO cells. A subclone of
CHO cells was used which contained a transgenic reporter gene
expressing a secreted form of the human placental alkaline
phosphatase under control of the glucocorticoid receptor-dependent
LTR-promoter of the mouse mammary tumor virus (Gottlicher et al.
(1992) Proc. Natl. Acad. Sci. USA 89, pp. 4653-4657). A hybrid
receptor comprising the amino-terminus of the glucocorticoid
receptor fused to the ligand binding domain of PPAR.delta. was
expressed in these cells essentially as described for the
expression of the corresponding hybrid of PPAR.alpha. (Gottlicher
et al., 1992, ibd.). The ligand binding domain of PPAR.delta. was
used starting at amino acid 138 as deduced from the sequence
published by Amri et al. (J. Biol. Chem. 270 (1995) pp. 2367-2371).
Activation of the PPAR.delta. ligand binding domain in these cells
induces expression of the alkaline phosphatase reporter gene which
is detectable by an enzymatic assay from the cell culture
supernatant. Similar cells expressing the full length
glucocorticoid receptor served as negative controls for specificity
of receptor activation. For the experiment shown in FIG. 1 the
PPAR.delta. hyrid receptor expressing cells were seeded at 20%
confluency into 24-well culture dishes and treated for 40 h with
the PPAR.delta. ligand carbocyclic prostaglandin I.sub.2 (PGI, 5
.mu.M), VPA (1 or 2 mM), or the histone deacetylase inhibitors
sodium butyrate (0, 2-5 mM) and trichostatin A (TSA, 300 nM).
Reporter gene activity was monitored by an enzymatic assay
(alkaline phosphatase). Values except for butyrate are
means.+-.S.D. from triplicate determinations in 2 independent
experiments which were normalized according to cPGI-induced
activity (FIG. 1). The highly synergistic activation of the
reporter gene by VPA together with the PPAR.delta. ligand cPGI
(P+V) which is similar to the synergistic activation by
Trichostatin A together with cPGI (P+T), and the lack of synergism
with trichostatin (T+V) or butyrate (not shown) indicate that VPA
does not act like a bona fide ligand to PPAR.delta.. VPA rather
affects PPAR.delta. activity by a mechanism which lies in the same
sequence of events by which also the inhibitors of
corepressor-associated histone deacetylases induce transcriptional
activity of PPAR.delta..
EXAMPLE 2
[0062] Activation of Transcriptional Repressors by VPA
[0063] The transcription factors thyroid hormone receptor (TR),
peroxisome proliferator activated receptor .delta. (PPAR.delta.),
retinoic acid receptor (RAR), the corepressor N-CoR and the AML/ETO
fusion protein repress transcription when they bind to a promoter
containing UAS sites (Gal4 response element) as fusion proteins
with the heterologous DNA binding domain of the yeast Gal4 protein.
In the absence of the Gal4 fusion protein a luciferase reporter
gene is transcribed at a high basal level due to the presence of
binding sites for other transcription factors in the thymidine
kinase (TK) promoter. Hela cells were transfected with a UAS TK
luciferase reporter plasmid (Heinzel et al., 1997, Nature 387, pp
43-48) and expression plasmids for the indicated Gal4 fusion
proteins using the calcium phosphate precipitate method. After 24 h
the medium was changed and cells were incubated with histone
deacetylase inhibitors for a further 24 h. Transcriptional
repression is measured as luciferase activity relative to the
baseline of cells transfected with an expression plasmid for the
Gal4 DNA binding domain alone (FIG. 2). The Gal4 fusion proteins
repress this baseline activity by up to 140 fold. VPA at a
concentration of 1 mM (close to the serum levels which are reached
during therapeutic use) induces relief of this- repression which is
indicated as an increase in reporter gene activity. A relief of
repression is also found after treatment with established histone
deacetylase inhibitors (10 nM Trapoxin, 100 nM TSA) as well as
after partial activation of TR and PPAR.delta. by their respective
ligands. A combination of ligand and HDAC inhibitors (including
VPA) results in a synergistic effect, indicating that different
molecular mechanisms are involved. FIG. 2 shows that VPA affects
the activity of several distinct transcription factors and
cofactors. This finding suggests that VPA acts on a common factor
in the regulation of gene expression such as corepressor-associated
histone deacetylases rather than on individual transcription
factors or receptors (e.g. as a ligand).
EXAMPLE 3
[0064] Accumulation of hyperacetylated histones in VPA-treated
cells VPA and established histone deacetylase inhibitors like
sodium butyrate (NaBu) or trichostatin A (TSA) induce the
accumulation of hyperacetylated histones H3 and H4. These
acetylated histones can be detected by Western blot analysis in
cell extracts of appropriately treated cells. FIG. 3 shows the
results of such an analysis from a representative experiment. In
this experiment both the time course of VPA-induced
hyperacetylation (A) and the required VPA concentration (B) were
determined.
[0065] (A) For the time course analysis F9 cells were seeded into
6-well culture dishes 30 h before the intended time point of
analysis. Individual cultures were treated at the indicated time
points before analysis by addition of 10-fold concentrated stock
solutions in culture medium of VPA or trichostatin A. Whole cell
extracts were prepared by rinsing the cell cultures twice in
ice-cold phosphate buffered saline and lysis of cells in 250 .mu.l
of sample buffer for denaturing SDS gel electrophoresis. DNA of
collected samples was sheared by sonication and samples were
separated on a 15% denaturing polyacrylamide gel. Acetylated
histones H3 and histone H4 were detected by Western blot analysis
using commercially available antibodies (Upstate Biotechnology)
specific for the acetylated forms of histones (Ac-H3, Cat-Nr.:
06-599; Ac-H4, Cat-Nr.: 06-598). Equal loading of the lanes was
confirmed by staining a part of the polyacrylamide gel by Coomassie
blue.
[0066] (B) For determination of the required VPA dose F9 cells were
cultured in 6-well culture dishes for 8 h prior to addition of VPA
at the indicated concentrations. Whole cell extracts were prepared
16 h after treatment as described above. Analysis for acetylated
histones H3 and H4 was performed as described in (A). VPA
concentrations in the range of blood serum levels reached during
therapeutic use of VPA as antiepileptic agent in humans induce
hyperactylation of histones H3 and H4. At serum levels only
slightly exceeding those intended for antiepileptic therapy VPA
induces histone hyperacetylation as efficiently as sodium butyrate
or trichostatin A used at concentrations which are expected to have
a maximum effect. This experiment indicates that VPA or a
metabolite formed in F9 cells inhibits histone deacetylase
activity.
EXAMPLE 4
[0067] VPA and Derivatives Inhibit Histone Deacetylase Activity in
vitro
[0068] Immune precipitates from whole cell extracts using
antibodies against the corepressor N-CoR or mSin3 contain histone
deacetylase activity. This enzymatic activity is measured by
incubating the immune precipitates with radioactively acetylated
histone substrates from cells in which histones have been
hyperacetylated in the presence of .sup.3H-acetate. The release of
.sup.3H-acetate is detected as a measure of enzymatic activity by
extraction with ethyl acetate and subsequent liquid scintillation
counting (FIG. 4). Addition of the histone deacetylase inhibitor
trichostatin A (TSA, 10.sup.-7 M) to the reaction in vitro severely
inhibits the enzymatic activity. VPA (from left to right 0.2 mM, 1
mM, 5 mM) and the related compounds ethyl hexanoic acid (EHXA, from
left to right 0.008 mM, 0.04 mM, 0.2 mM, 1 mM, 5 mM), R-4-yn VPA
(from left to right 0.2 mM, 1 mM, 5 mM) and S-4-yn VPA (from left
to right 0.2 mM, 1 mM, 5 mM) were tested for HDAC inhibitory
activity. The assays were performed with N-CoR immunoprecipitates
from 293T cells in duplicate. Immunoprecipitates were pretreated
with HDAC inhibitors for 15 min prior to the addition of substrate
and subsequent incubation for 2.5 h at 37.degree. C. (untreated
enzyme activity 2,205 cpm=100%). Precipitates of a preimmune serum
served as a negative control. EC.sub.50 values are 0.6 mM for VPA,
0.2 mM for EHXA and 0.3 mM for S-4-yn VPA, whereas the stereoisomer
R-4-yn VPA is inactive. These data show that VPA by itself rather
than a cellular metabolite inhibits histone deacetylase
activity.
EXAMPLE 5
[0069] Induction of Cell Differentiation in F9 Teratocarcinoma,
HT-29 Colonic Cancer, and RenCa Renal Carcinoma Cells.
[0070] Histone deacetylase inhibitors and VPA in particular induce
differentiation of dedifferentiated tumorigenic cells. Cell
differentiation is associated with cell cycle arrest, morphological
alterations and the appearance of expression of markers of the
differentiated phenotype. Morphological alterations where
determined by microscopic evaluation of F9 and HT-29 cells. One
parameter of differentiation, the cell cycle arrest, was shown in
F9 teratocacrcinoma, estrogen independent MT-450 breast cancer and
HT-29 colonic carcinoma cells by means of the reduced incorporation
of .sup.3H-thymidine into cultured cells. F9 and HT-29 cells were
cultured for 36 h in the absence or the presence of 1 mM VPA in
96-well culture dishes. 37 kBq of .sup.3H-thymidine were added for
additional 12 h of culture. MT-450 cells were cultured for 72 h
prior to a 1 h .sup.3H-thymidine labelling period. Incorporation of
.sup.3H-thymidine into DNA was determined by automatic cell
harvesting and liquid scintillation counting. VPA pretreatment
reduced the rate of thymidine incorporation by 48.+-.5%, 63.+-.8%,
and 52.+-.8% in F9, MT-450, and HT-29 cells, respectively. The
dose-response for the reduction of thymidine incorporation into
HT-29 cells (FIG. 5A) was determined by the same experimental
procedure. In addition, the induction of a cell differentiation
marker was shown in F9 teratocarcinoma cells (FIG. 5B).
[0071] F9 teratocarcinoma cells were treated for 48 h with VPA (1
mM), sodium butyrate (B, 1 mM) and trichostation A (TSA, 30 nM).
Differentiation was followed by morphological criteria, a reduced
rate in the increase of cell number (e.g. cell cycle arrest, data
not shown), the drop of .sup.3H-thymidine incorporation by 48%
during a 12 h pulse labeling period (see above) and the appearance
of nuclear AP-2 protein (FIG. 5B) as a specific marker of histone
deacetylase inhibitor-induced differentiation of F9 cells. Nuclear
AP-2 protein was detected in nuclear extract which had been
prepared by mild detergent lysis (25 mM Tris, pH 7.5; 1 mM EDTA,
0.05*NP40) of treated or non-treated F9 cells, recovery of nuclei
by centrifugation (3000.times.g, 5 min) and lysis of nuclei in
sample buffer for denaturing SDS gel electrophoresis. Nuclear
extracts were separated on a 9% SDS polyacrylamide gel. AP-2
protein was detected by Western blot analysis using a rabbit
polyclonal antibody (Santa Cruz, Cat.-No.: SC-184) at a dilution of
1/1000 in Tris buffered saline containing 3W non-fat dry milk and
0.05% Tween 20. Both VPA and trichostatin A induce nuclear AP-2
protein whereas the activity of butyrate at the chosen
concentration is weak. Since appearance of AP-2 is a delayed effect
which is only detectable after 36 to 40 h of VPA treatment the weak
activity of butyrate may be caused by efficient metabolism of the
compound. Nevertheless, VPA induces differentiation of the
epithelial F9 cell line in a way indistinguishable from
differentiation by other histone deacetylase inhibitors.
[0072] Induction of differentiation in RenCa-LacZ cells by VPA was
determined by alterations in cell morphology. RenCa-LacZ cells were
cultured for 36 h either in the absence or the presence of 1 mM
VPA. Morphological alterations were observed by phase contrast
microscopy and micrographs of representative fields were taken
(FIG. 5C)
EXAMPLE 6
[0073] Induction of Apoptosis in MT-450 Breast Cancer Cells
[0074] MT-450 cells were cultured for 72 h in the absence or
presence of 1 mM VPA. Apoptotic cells were detected by flow
cytometric analysis after staining of cell surface exposed
phosphatidylserine with FITC-conjugated annexin V (Becton
Dickinson) according to suppliers instructions. Dead cells were
excluded by propidium iodide staining. Cells positive for annexin V
and negative for propidium iodide uptake (lower right quadrant in
FIG. 6) were judged and counted as apoptotic cells.
EXAMPLE 7
[0075] Loss of Viable Tumor Cells Upon Treatment with Valproic Acid
(MTT Tests)
[0076] Cell Lines and Cell Culture
[0077] Human MDA-MB468, MDA-MB453 and SKBR3 breast carcinoma cells,
A431 squamous cell carcinoma cells, and SKOV3 ovarian carcinoma
cells were maintained in Dulbecco's modified Eagle's medium (DMEM,
BioWhittaker, Verviers, Belgium) supplemented with 10% heat
inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100
units/ml penicillin, and 100 .mu.g/ml streptomycin. Human MCF7
breast carcinoma cells were grown in RPMI medium supplemented as
described above.
[0078] Renal cell carcinoma (Renca) cells stably transfected with
plasmid pZeoSV2/lacZ encoding E. coli .beta.-galactosidase
(Renca-lacZ cells) (Maurer-Gebhard et al., Cancer Res. 58:
2661-2666, 1998) were grown in RPMI-1640 medium supplemented with
8% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 0.25 mg/ml Zeocin. Renca-lacZ cells cotransfected
with plasmids pSV2ErbB2N and pSV2neo encoding c-erbB2 and neomycin
resistance (Renca-lacZ/ErbB2) (Maurer-Gebhard et al., Cancer Res.
58: 2661-2666, 1998), or plasmids pLTR-EGFR or pLTR-EGFRvIII and
pSV2neo encoding epidermal growth factor (EGF) receptor, the
oncogenically activated EGF receptor variant EGFRvIII, and neomycin
resistance (Renca-lacZ/EGFR and Renca-lacZ/EGFRvIII) (Schmidt et
al., Oncogene 18: 1711-1721, 1999) were grown in the same medium
further containing 0.48 mg/ml G418.
[0079] Cell Viability Assays
[0080] Tumor cells were seeded in 96 well plates at a density of
1.times.10.sup.4 cells/well in normal growth medium. Valproic acid
was added at final concentrations of 1 or 3 mM to triplicate
samples and the cells were incubated for 40 h (Renca-lacZ,
Renca-lacZ/ErbB2, Renca-lacZ/EGFR, Renca-lacZ/EGFRvIII, SKBR3 and
SKOV3 cells) or 70 h (A431, MCF7, MDA-MB453 and MDA-MB468 cells).
Control cells were grown in the absence of valproic acid. Ten .mu.l
of 10 mg/ml 3-(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) (Sigma, Deisenhofen, Germany) in PBS were added to
each well and the cells were incubated for another 3 h. Cells were
lysed by the addition of 90 .mu.l of lysis buffer (20% SDS in 50%
dimethyl formamide, pH 4.7). After solubilization of the formazan
product, the absorption at 590 nm was determined in a microplate
reader (Dynatech, Denkendorf, Germany) and the relative amount of
viable cells in comparison to cells grown without the addition of
valproic acid was calculated.
[0081] Results
[0082] The results presented in FIG. 7 show that valproic acid
reduces the viability of breast carcinoma cells, ovarian carcinoma
cells, squamous cell carcinoma cells, renal carcinoma cells, and
renal carcinoma cells expressing at high levels the ErbB2 or EGF
receptor proto-oncogenes, or the oncogenically activated EGF
receptor variant EGFRVIII, in a concentration dependent manner.
These results demonstrate that valproic acid potently reduces the
number and/or viability of a wide variety of tumor cells derived
from solid tumors of epithelial origin. The loss of viability could
indicate a reduction in cell number upon induction of cellular
differentiation and/or induction of cell death. The observation of
changes of cellular morphology suggest that cellular
differentiation is at least responsible for a part of the effect.
This induction of differentiation and/or induction of cell death
suggest that valproic acid and derivatives thereof could be used
for the therapy of such tumors.
EXAMPLE 8
[0083] Reduction in Cellular Biomass After Treatment of Human
Cancer Cell Cultures with Valproic Acid (see FIG. 8).
[0084] VPA induces differentiation and/or cell death in a series of
human cancer cells and reduces the total cellular biomass of human
cancer cell cultures. The reduction in biomass could indicate cell
loss due to cell death and/or differentiation associated cell cycle
arrest. Quantitative parameters, e.g. the loss of biomass, was
determined in 30 human cancer cell lines (FIG. 8e) and twelve
examples of dose-response curves are shown, e.g. BT-549 breast
cancer cells (1), estrogen dependent ZR-75 breast cancer cells (2),
DMS-114 small cell lung cancer cells (3), NCI-H226 non-small cell
lung cancer cells (4), SK-MEL-28 skin cancer cells (5), OVCAR-3
ovarian cancer cells (6), HUP-T3 pancreatic cancer cells (7),
DU-145 prostate cancer cells (8), DETROIT-562 head and neck cancer
cells, LS-174 colon cancer cells (10), A-172 brain cancer cells
(11) and HL-60 leukemia cells (12) (FIG. 8a-d). All cells were
evaluated for morphological signs of cell death and/or
differentiation. All cultures contained an increased number of
dying cells at the highest tested VPA concentration and in some
cultures such as SW-1116 colon cancer cells (FIG. 8e) most cells
were dying already at 1 mM VPA during the experiment. PC-3 (FIG.
8e) and DU-145 (FIG. 8c) cells change their normal round morphology
to a long fibroblast-like shape. Also U87MG (FIG. 8e) cells
increase in length and develop spider-like filamentous
extensions.
[0085] Cells in panels 1 to 9 (FIG. 8a-c) were seeded in 96 well
culture dishes at densities between 3000 and 8000 per well. After
recovery of 24 hours cells were cultured for 48 hours in the
absence or presence of the indicated concentrations of VPA.
Cultures were fixed with TCA by layering 50 .mu.l of cold 50*TCA on
top of the growth medium in each well to produce a final TCA
concentration of 10%. After 1 hour of incubation at 4.degree. C.
the cells were washed five times with tap water and air dried.
Fixed cells were stained for 30 minutes with 0.4% (wt/vol)
Sulforhodamine B dissolved in 1% acetic acid and washed four times
with 1% acetic acid to remove unbound dye.After air drying bound
dye was solubilized with 10 mM unbuffered Tris base (pH 10.5) for 5
minutes on a gyratory shaker. Optical densities were read on a
Titertek Multiskan Plus plate reader at a single wavelength of 550
nm. Six test wells for each dose-response were set in parallel with
12 control wells per cell line. A measure of the cell population
density at time 0 (T.sub.0; the time at which the drug was added)
was also made from 12 extra reference wells of cells fixed with TCA
just prior to drug addition to the test plates. Background OD of
complete medium with 5% FBS fixed and stained as described above
was also determined in 12 separate wells.
[0086] From the unprocessed OD data the background OD measurements
(i.e. OD of complete medium plus stain and OD of cells at T.sub.0)
were subtracted thus giving the reduction of total cellular biomass
of the cells.
[0087] Cells in panels 10 to 12 (FIG. 8d) were cultured 36 to 50
hours as indicated in the absence or presence of the indicated
concentrations of VPA in 96 well dishes. 37 kBq of
.sup.3H-thymidine were added for additional 12 hours of culture.
Incorporation of .sup.3H-thymidine into DNA was determined by
automatic cell harvesting and liquid scintillation counting.
[0088] The graphs in FIG. 8a-d show means.+-.S.D. from sixfold
determinations.
[0089] In addition cancer cells of further organ origins have been
treated with valproic acid in the same way as described for
experiments presented in FIG. 8a-c. FIG. 8e summarizes the
reduction of total cellular biomass of various human cancer cells
by treatment with 1 mM VPA. This reduction could indicate
differentiation associated cell cycle arrest and/or induction of
cell death. Cells were VPA treated for 48 hours. The inhibition was
calculated from six response tests performed in parallel and
reductions of cellular biomass are given in percent of untreated
cells with standard deviations.
* * * * *