U.S. patent application number 15/385357 was filed with the patent office on 2017-09-14 for methods for regulating cell mitosis by inhibiting serine/threonine phosphatase.
This patent application is currently assigned to LIXTE BIOTECHNOLOGY, INC.. The applicant listed for this patent is John S. Kovach. Invention is credited to John S. Kovach.
Application Number | 20170259081 15/385357 |
Document ID | / |
Family ID | 43356659 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170259081 |
Kind Code |
A1 |
Kovach; John S. |
September 14, 2017 |
METHODS FOR REGULATING CELL MITOSIS BY INHIBITING SERINE/THREONINE
PHOSPHATASE
Abstract
Disclosed herein are methods of inhibiting proliferation of a
cancer cell or inducing apoptosis of a cancer cell, which does not
overexpress N--CoR. Also disclosed herein are methods of inhibiting
proliferation or inducing apoptosis of a cancer cell that
overexpresses TCTP and methods for determining whether a compound
is effective in inducing cell death.
Inventors: |
Kovach; John S.; (East
Setauket, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kovach; John S. |
East Setauket |
NY |
US |
|
|
Assignee: |
LIXTE BIOTECHNOLOGY, INC.
EAST SETAUKET
NY
|
Family ID: |
43356659 |
Appl. No.: |
15/385357 |
Filed: |
December 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13870763 |
Apr 25, 2013 |
9526915 |
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15385357 |
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13378623 |
Feb 17, 2012 |
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PCT/US2010/000279 |
Feb 1, 2010 |
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13870763 |
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PCT/US2009/004108 |
Jul 16, 2009 |
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13378623 |
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61269101 |
Jun 18, 2009 |
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61269101 |
Jun 18, 2009 |
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61137715 |
Aug 1, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/20 20130101;
C07D 493/08 20130101; A61K 31/44 20130101; A61K 31/496 20130101;
A61K 38/212 20130101; A61K 39/39558 20130101; A61K 45/06 20130101;
A61K 31/337 20130101; A61N 5/10 20130101; A61K 38/50 20130101 |
International
Class: |
A61N 5/10 20060101
A61N005/10; A61K 31/496 20060101 A61K031/496; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395; A61K 38/50 20060101
A61K038/50; A61K 38/21 20060101 A61K038/21; A61K 38/20 20060101
A61K038/20; A61K 31/337 20060101 A61K031/337; A61K 31/44 20060101
A61K031/44; C07D 493/08 20060101 C07D493/08 |
Claims
1-23. (canceled)
24. A method for reducing the amount of TCTP in a cell comprising
contacting the cell with an effective amount of a protein
phosphatase 2A inhibitor, thereby reducing the amount of TCTP in
the cell.
25. The method of claim 24, wherein the cell is a cancer cell.
26. The method of claim 25, wherein the cancer is adrenocortical
cancer, bladder cancer, osteosarcoma, cervical cancer, esophageal,
gallbladder, head and neck cancer, Hodgkin lymphoma, non-Hodgkin
lymphoma, renal cancer, melanoma, pancreatic cancer, rectal cancer,
thyroid cancer, throat cancer, breast cancer, lung cancer and
prostate cancer.
27. The method of claim 24, wherein the protein phosphatase 2A
inhibitor has the structure: ##STR00028## or a salt, enantiomer or
zwitterion of the compound.
28. A method for determining whether a compound is effective in
inducing cell death in a cancer cell comprising: (a) contacting a
first cancer cell with the compound; (b) determining the level of
expression of TCTP in the first cancer cell; (c) contacting a
second cancer cell with a protein phosphatase 2A inhibitor; (d)
determining the level of expression of TCTP in the second cancer
cell; (e) comparing the level of expression of TCTP determined in
step (b) with the level determined in step (d), wherein, when the
level of expression determined in step (b) is equal to, or lower
than, the level of expression determined in step (d), the compound
is effective to induce cell death.
29. The method of claim 28, wherein the cancer is adrenocortical
cancer, bladder cancer, osteosarcoma, cervical cancer, esophageal,
gallbladder, head and neck cancer, Hodgkin lymphoma, non-Hodgkin
lymphoma, renal cancer, melanoma, pancreatic cancer, rectal cancer,
thyroid cancer, throat cancer, breast cancer, lung cancer and
prostate cancer.
30. The method of claim 28, wherein the protein phosphatase 2A
inhibitor has the structure: ##STR00029## or a salt, enantiomer or
zwitterion of the compound.
31. A method for predicting whether treatment of a subject with an
agent will be successful in treating a subject suffering from
cancer comprising: (a) obtaining a sample comprising cancer cells
from the subject; (b) culturing the cancer cells; (c) determining
the level of expression of TCTP in the cancer cells; (d) contacting
the cancer cells with the agent; (e) determining the level of
expression of TCTP in the cancer cells; (f) comparing the level of
expression of TCTP determined in step (c) with the level of
expression determined in step (e), wherein, when the level of
expression determined in step (c) is lower than the level of
expression determined in step (e) predicts that treatment of the
subject with the agent will be successful in treatment of the
cancer.
Description
[0001] This application is a continuation of U.S. Ser. No.
13/870,763, filed Apr. 25, 2013, now allowed, which is a
continuation of U.S. Ser. No. 13/378,623, filed Feb. 17, 2012, now
abandoned, which is a .sctn.371 national stage of PCT International
Application No. PCT/US2010/000279, filed Feb. 1, 2010, which claims
benefit of PCT International Application No. PCT/US2009/004108,
filed Jul. 16, 2009, which claims benefit of each of U.S.
Provisional Application Nos. 61/269,101, filed Jun. 18, 2009 and
61/137,715, filed Aug. 1, 2008, the contents of each of which in
its entirety is hereby incorporated by reference.
[0002] Parts of this invention were created in collaboration with
the National Institutes of Health. The Government of the United
States has certain rights in the invention.
[0003] Throughout this application, certain publications are
referenced. Full citations for these publications may be found
immediately preceding the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to describe more fully the
state-of-the art to which this invention relates.
BACKGROUND OF THE INVENTION
[0004] Most current strategies for pharmacologic treatment of
cancers are based on developing drugs or biologicals, primarily
antibodies and anti-sense RNAs that specifically inhibit the
activity of an enzyme in a signaling pathway or a gene encoding an
enzyme upon which the cancer cell is dependent for growth and
survival (Shoshan and Linder 2008). Dependence of a particular type
of cancer on excessive activity of a specific signaling pathway has
been termed "oncogene addiction" (Lim et al 2008). Interference
with the function or abundance of an addicting oncogene may inhibit
growth and, in some cases, result in the death of cancer cells that
are dependent upon the pathway. Inhibition of a single oncogene,
however, is usually insufficient for complete inhibition of a
cancer and inhibition is overcome by mutation leading to drug
resistance. Older approaches to cancer treatment have involved
primarily the use of non-specific agents alone and in combinations
of drugs with non-overlapping toxicities to normal tissues that
damage DNA or interfere with cell metabolic pathways including
modulation of microtubule stability.
[0005] A variety of mechanisms maintain the integrity of the genome
of normal cells in the face of stress. DNA-damage response
mechanisms, however, may also protect cancer cells from killing by
chemotherapy and radiation, allowing cancers to recur despite
aggressive treatment. Cell responses to DNA-damage are mediated in
part by polo-like kinase 1 (Plk-1) (Strebhardt and Ullrich, 2006),
Akt-1 (protein kinase B) (Brazil et al, 2004) and p53 (Vogelstein
et al 2000; Vazquez et al 2008), pathways, which lead to cell cycle
arrest, senescence, or apoptosis. Because many cancers over-express
Plk-1 (Lei and Erikson, 2008; Olmos et al, 2008; Liu et al, 2006)
and Akt-1 (Garcia-Echeverria and Sellers, 2008; Hirose et al 2005)
or have acquired p53 (Vazquez et al, 2008) genetic defects,
inhibition of Plk-1 (Strebhardt and Ullrich, 2006; Olmos et al,
2008; Liu et al, 2006) and Akt-1 (Garcia-Echeverria and Sellers,
2008; Hirose et al, 2005) and the restoration of p53 function
(Vazquez et al, 2008) are being widely investigated as cancer
treatments.
[0006] Translationally controlled tumor protein (TCTP) is one of
the most highly conserved and most abundant proteins in eukaryotic
cells (Bommer and Thiele, 2004). TCTP is associated with many
cellular functions and is essential for fetal development (Bommer
and Thiele, 2004; Chen et al, 2007B). TCTP is also essential to
cancer cell growth but is not critical to the survival of normal
adult (untransformed) cells (Chen et al, 2007). Disclosed herein is
that targeting of TCTP with a pharmacologic intervention may be an
effective means for disrupting cancer cell division and therefore
for treating cancers in general.
SUMMARY OF THE INVENTION
[0007] The invention provides a method of inhibiting proliferation
of a cancer cell or inducing apoptosis of a cancer cell, which
cancer cell does not overexpress N--CoR, comprising administering
to the subject a compound, wherein the compound has the
structure
##STR00001##
wherein bond .alpha. is present or absent; R.sub.1 and R.sub.2 is
each independently H, O.sup.- or OR.sub.9, where R9 is H, alkyl,
alkenyl, alkynyl or aryl, or R.sub.1 and R.sub.2 together are
.dbd.O; R.sub.3 and R.sub.4 are each different, and each is OH,
O.sup.-, OR.sub.9, SH, S.sup.-, SR.sub.9,
##STR00002##
where X is O, S, NR.sub.10, or N.sup.+R.sub.10R.sub.10, where each
R.sub.10 is independently H, alkyl, substituted C.sub.2-C.sub.12
alkyl, alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl where the substituent
is other than chloro when R.sub.1 and R.sub.2 are .dbd.O,
##STR00003##
--CH.sub.2CN, --CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11,
--NHR.sub.11 or --NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is
independently alkyl, alkenyl or alkynyl, each of which is
substituted or unsubstituted, or H; R.sub.5 and R.sub.6 is each
independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and R.sub.7 and R.sub.8 is each independently H, F, Cl, Br,
SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, where R.sub.12 is H,
aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl,or
a salt, enantiomer or zwitterion of the compound, in an amount
effective to inhibit the proliferation or to induce apoptosis of
the cancer cell.
[0008] The invention provides a method of inhibiting proliferation
or inducing apoptosis of a cancer cell which overexpresses TCTP
comprising administering to the subject a compound, wherein the
compound had the structure
##STR00004##
wherein bond .alpha. is present or absent; R.sub.1 and R.sub.2 is
each independently H, O.sup.- or OR.sub.9, where R.sub.9 is H,
alkyl, alkenyl, alkynyl or aryl, or R.sub.1 and R.sub.2 together
are .dbd.O; R.sub.3 and R.sub.4 are each different, and each is OH,
O.sup.-, OR.sub.9, SH, S.sup.-, SR.sub.9,
##STR00005##
where X is O, S, NR.sub.10, or N.sup.+R.sub.10R.sub.10, where each
R.sub.10 is independently H, C.sub.2-C.sub.12alkyl, substituted
C.sub.2-C.sub.12 alkyl, alkenyl, substituted C.sub.4-C.sub.12
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where
the substituent is other than chloro when R.sub.1 and R.sub.2 are
.dbd.O,
##STR00006##
--CH.sub.2CN, --CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11,
--NHR.sub.11 or --NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is
independently alkyl, alkenyl or alkynyl, each of which is
substituted or unsubstituted, or H; R.sub.5 and R.sub.6 is each
independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and R.sub.7 and R.sub.8 is each independently H, F, Cl, Br,
SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, where R.sub.12 is H,
aryl or a substituted or unsubstituted alkyl, alkenyl or alkynyl,or
a salt, enantiomer or zwitterion of the compound, in an amount
effective to inhibit the proliferation or to induce apoptosis of
the cancer cell.
[0009] The invention provides a method of inhibiting proliferation
or inducing apoptosis of a cancer cell that overexpresses TCTP by
administering to the subject a compound, wherein the compound has
the structure
##STR00007##
wherein bond .alpha. is present or absent; R.sub.1 and R.sub.2 is
each independently H, O.sup.- or OR.sub.9, where R.sub.9 is H,
alkyl, alkenyl, alkynyl or aryl, or R.sub.1 and R.sub.2 together
are .dbd.O; R.sub.3 and R.sub.4 are each different, and each is OH,
O.sup.-, OR.sub.9, SH, S.sup.-, SR.sub.9,
##STR00008##
where X is O, S, NR.sub.10, or N.sup.+R.sub.10R.sub.10, where each
R.sub.10 is independently C.sub.2-C.sub.12 alkyl, substituted
C.sub.2-C.sub.12 alkyl, alkenyl, substituted C.sub.4-C.sub.12
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where
the substituent is other than chloro when R.sub.1 and R.sub.2 are
.dbd.O, --CH.sub.2CN, --CH.sub.2CO.sub.2R.sub.11,
--CH.sub.2COR.sub.11, --NHR.sub.11 or --NH.sup.+(R.sub.11).sub.2,
where each R.sub.11 is independently alkyl, alkenyl or alkynyl,
each of which is substituted or unsubstituted, or H; R.sub.5 and
R.sub.6 is each independently H, OH, or R.sub.5 and R.sub.6 taken
together are .dbd.O; and R.sub.7 and R.sub.8 is each independently
H, F, Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, or a salt, enantiomer or zwitterions of the
compound, in an amount effective to inhibit the proliferation or to
induce apoptosis of the cancer cell.
[0010] This invention provides a method for determining whether a
compound is effective in inducing cell death comprising (a)
contacting a first cancer cell with the compound; (b) determining
the level of expression of TCTP in the first cancer cell; (c)
contacting a second cancer cell with a protein phosphatase 2A
inhibitor (d) determining the level of expression of TCTP in the
second cancer cell; (e) comparing the level of expression of TCTP
determined in step (b) with the level determined in step (d),
wherein, when the level of expression determined in step (b) is
equal to, or lower than, the level of expression determined in step
(d) indicates that the compound is effective to induce cell
death.
[0011] This invention provides a method for determining whether a
compound is effective in inducing cell death in a cancer cell
comprising (a) contacting a cancer cell with the compound; (b)
determining the level of expression of TCTP in the cancer cell; (c)
determining the level of expression of TCTP in a non-cancerous
cell; (e) comparing the level of expression of TCTP determined in
step (b) with the level determined in step (d), wherein, when the
level of expression determined in step (b) is lower than, the level
of expression determined in step (d) indicates that the compound is
effective to induce cell death in the cancer cell.
[0012] This invention provides a method for determining whether
treatment of a subject with an agent will be successful in treating
a subject suffering from cancer comprising (a) obtaining a first
sample from the subject prior to treatment;
(b) determining the level of expression of TCTP in the sample; (c)
administering to the subject the agent; (d) obtaining a second
sample from the subject after treatment with the agent; (e)
determining the level of expression of TCTP in the second sample
obtained; wherein, when the level of expression determined in step
(b) is lower than the level of expression determined in step (e)
indicates that the treatment of the subject with the agent be
successful.
[0013] This invention provides a method for predicting whether
treatment of a subject with an agent will be successful in treating
a subject suffering from cancer comprising (a) obtaining a sample
comprising cancer cells from the subject; (b) culturing the cancer
cells; (c) determining the level of expression of TCTP in the
cancer cells (d) contacting the cancer cells with the agent; (e)
determining the level of expression of TCTP in the cancer cells;
(f) comparing the level of expression of TCTP determined in step
(c) with the level of expression determined in step (e); wherein,
when the level of expression determined in step (c) is lower than
the level of expression determined in step (e) predicts that
treatment of the subject with the agent will be successful in
treatment of the cancer.
[0014] This invention provides a method for reducing the amount of
TCTP in a cell comprising contacting the cell with an effective
amount of protein phosphatase inhibitor, thereby reducing the
amount of TCTP in the cell.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Inhibition of Protein Phosphatase 2 A (PP2A) in DAOY
cell line by Compound 102.
[0016] Cultured DAOY cells were plated in 175 cm.sup.3 flasks. When
the cells were 80% confluent, the media was replaced with media
containing either 0.15 .mu.M Compound 102, 0.25 .mu.M Compound 102,
0.3 .mu.M Compound 102, or an equivalent volume of PBS vehicle.
After 1 hour, the cells were washed three times in a 0.9% normal
saline solution. T-PER solution was added to the cells, and cells
were prepared for protein extraction. Lysates from each treatment
group containing 300 .mu.g of protein were applied to a spin column
(Catch and Release v2.0 Reversible Immunoprecipitation System,
Millipore, Billerica, Mass.) for immunoprecipitation of PP2A/Akt-1
kinase protein complexes using polyclonal anti-rabbit Akt-1
antibody (Cell Signaling Technology, Danvers, Mass.). PP2A activity
from the immunoprecipitated complexes was assayed using a Malachite
Green Phosphatase Assay specific for serine/threonine phosphatase
activity (Ser/Thr Phosphatase Assay Kit 1, Millipore, Billerica,
Mass.).
[0017] FIG. 2: Inhibition of serine/threonine phosphatase activity
by compound 102.
[0018] Inhibition of PP2A and PP1 activity by compound 102 on
purified PP1 and PP2A (mean and s.d.; n=3) (Ser/Thr Phosphatase
Assay Kit 1, Millipore, Billerica, Mass.).
[0019] FIG. 3: Inhibition of proliferation of U87 cells by compound
102.
[0020] PP2A activity in U87 s.c. xenografts (blue) and in normal
brain tissue (yellow) of SCID mice at different times after i.p.
injection of 1.5 mg/kg compound 102 (one mouse per point; each
lysate was measured in triplicate: mean and s.d.)
[0021] FIG. 4: Inhibition of U87 glioblastoma multiforme cells
grown as subcutaneous xenografts in SCID mice by Compound 100.
[0022] SCID mice were implanted with 5.times.10.sup.6 U87 cells
subcutaneously. On day 7 treatment was begun on half of the
animals. The size of the subcutaneous mass of tumor cells was
measured weekly until the animals were sacrificed on day 26.
[0023] FIG. 5: Inhibition of DAOY medulloblastoma cells grown as
subcutaneous xenografts in SCID mice by Compound 100 and Compound
102.
[0024] DAOY medulloblastoma cells were implanted subcutaneously
into the flanks of SCID mice. On day 6, mice were divided into 3
groups, one group receiving Compound 100, one group receiving
Compound 102, and one group receiving vehicle alone. The
subcutaneous tumor masses were measured on day 6, day 13, day 18,
and on day 23 when all animals were sacrificed. Both compounds led
to marked regression of the tumor by day 23. In this model DAOY
cells, when untreated, reached their maximum growth about 2 weeks
after implantation with slow regression thereafterwards.
[0025] FIG. 6: Effect of compound 102 on U87 cells in vitro at 1
uM; 2 uM; 5 uM; and 10 uM.
[0026] Viable cells were counted (mean and s.d.; n=3; Coulter
particle counter).
[0027] FIG. 7: Activation of Plk-1 and disruption of alpha tubulin
in DAOY medulloblastoma cells in culture by Compound 100.
[0028] DAOY cells growing in tissue culture were exposed to 5 .mu.M
Compound 100 for 4 hours. The cells were rinsed, fixed, and stained
for immunofluoroescent recognition of alpha-tubulin and Plk-1.
Control cells at the left show in the upper left panel diffuse
staining for alpha-tubulin distributed throughout the cytoplasm.
The upper right panel shows nuclear staining by the DNA binding
agent DAPI. The lower left panel shows that control cells have no
detectable Plk-1. The lower right panel, stained for Plk-1,
alpha-tubulin, and DNA show the almost pure extra nuclear location
of homogeneously distributed alpha-tubulin. The right panel
consists of 4 elements showing the effects of exposure to Compound
100. In the upper left, staining for alpha-tubulin reveals marked
distortion of the homogeneous distribution seen in the control
cells, with multiple clumps of alpha-tubulin irregularly
distributed in the cytoplasm. The upper right panel shows
disordered chromatin undergoing cell division. At the lower left,
staining for Plk-1 shows chromatin as two dense masses, which can
be seen to be located in the remnant of the remaining bridge
between dividing cells.
[0029] FIG. 8: Reduction in concentration of TCTP after treatment
with Compound 100 in U87 glioblastoma multiformed cells grown as
subcutaneous xenografts in SCID mice, detected by 2-dimensional gel
electrophoretic analysis.
[0030] SCID mice were implanted with 5.times.10.sup.6 U87 cells
subcutaneously. On day 26, the mice were given 1.5 mg/kg Compound
100 by IP injection. The animals were sacrificed after 4 hours
treatment and the subcutaneous mass of tumor cells were removed for
2-dimensional gel electrophoretic analysis. There was a comparable
group mice exposed to vehicle. In the left panel, TCTP subsequently
identified by LC-MS-MS is circled and shown in an enlargement of
the gel. The lysate from Compound 100 treated cells reveals a
diminution in TCTP.
[0031] FIG. 9: Reduction in concentration of TCTP and activation of
Plk-1 after treatment with Compound 100 in DAOY medullublastoma
cells in culture detected by western blot analysis of cell
lysates.
[0032] DAOY cells in culture were exposed to Compound 100 for 4
hours and for 24 hours, and stained for TCTP, p-Plk and total Plk
on western blots. As early as 4 hours, there is a decrease in the
TCTP and an increase of Plk-1 phosphorylation and at 24 hours, no
TCTP is detectable at loading of comparable concentrations of total
cell protein.
[0033] FIG. 10A-C: Compound 100 enhances the cytotoxic activity of
standard cytotoxic chemotherapeutic drugs as assessed after 7 days
of growth in culture.
[0034] Exposure to Compound 100 enhances the inhibition of the
human glioblastoma cell line, U373, by cisplatin (A), doxorubicin
(B), and Taxol (C). Cells were exposed to vehicle alone (control);
Compound 100 at 2.5uM; cisplatin at 0.1 uM; doxorubicin at 0.01 uM;
or taxol at 0.3 nM alone or to the combination of Compound 100 plus
each of the standard agents at the same concentrations. In each
case the addition of Compound 100 enhanced the effect of the
cytotoxic agent at 7 days to an extent greater than that expected
form the activity of each agent used alone. The expected percent
inhibition at 7 days is the product of the inhibition by each agent
alone. For cisplatin and Compound 100 expected inhibition at 7 days
was 66% (93 5 for cisplatin alone.times.71% for LB-1 alone) versus
the actual extent of inhibition by the combination of 50% (A). For
doxorubicin and Compound 100 expected inhibition at 7 days was 53%
(75.7 5 for doxorubicin alone.times.71% for Compound 100 alone)
versus the actual extent of inhibition by the combination of 42.3%
For Taxol and Compound 100 expected inhibition at 7 days 80% (114%
for Taxol alone.times.71% for Compound 100 alone) versus the actual
extent of inhibition by the combination of 61% (C).
[0035] FIG. 11A-G: Cellular and molecular changes in U87 cells
induced by compound 102 at 2.5 uM after 24 hour exposure (A-D, F,
G) and after 3 hours (G).
[0036] A, Nuclear changes in U87 cells in unsynchronized
logarithmic growth (upper panel, green immunofluorescence [IFS] GFP
labeled-actin a and lower panel, blue DAPI staining). Numerous
irregular nuclei with clumped chromatin in compound 102 treated
cells are indicated by arrows. B, Disordered microtubules (green
IFS tubulin-a and red IFS pPlk-1-Tre 210) and irregular clumped
chromatin (blue DAPI) C, Western blots of U87 lysates: pAkt-1,
total Akt-1, and .beta.-actin. D, Western blots of U87 lysates:
TCTP, pPlk (Tre-210), total Plk, and .beta.-actin. E, IFS of TCTP
in U87 cells, F, Western blots p53 (ser-15), pMDM2 (ser-166), and
.beta.-actin and G, IFS of p53 (ser-15).
[0037] FIG. 12A-H: Synergistic anti-cancer activity of compound 102
combined with TMZ.
[0038] 5.times.10.sup.6 U87MG cells were inoculated s.c. into each
flank of 20 SCID mice. When the xenografts were 0.5+/-0.1 cm (day
0), 5 animals each received i.p. vehicle alone days 1-12 (50%
DMSO/H20); compound 102 alone at 1.5 mg/kg days 1-3, 5-7, &
9-11; TMZ alone at 80 mg/kg days 4, 8, 12; or both drugs at the
same doses and schedules. If xenografts reached 1800 mm.sup.3,
animals were sacrificed. A, U87 xenografts in controls grew rapidly
requiring sacrifice at 3 weeks; compound 102 treated slowed growth
with sacrificed between week 4-5. TMZ treated had complete
regression of all xenografts by week 5 but with recurrence
requiring sacrifice of all 5 animals week 7-9. compound 102 plus
TMZ treated had complete regression of all xenografts by week 5
with recurrence of 1/2 xenografts in 3 mice at weeks 7, 11, 13
requiring sacrifice at weeks between week 11-15 and the other 2
mice had no recurrence of either xenograft for more than 7 months.
Average tumor volume is shown through week 9 the last time point
when xenograft volume could be determined in all 10 xenografts in
the 2-drug combination group (mean and s.d., n=10 per treatment
group). B, Survival curve combining the data from the study shown
in FIG. 12A, with a second identical study involving a total of 10
animals with two xenografts each. Disease-free survival was defined
as no recurrence of either xenografts. Kaplan-Meier analysis
revealed that survival following compound 102 plus TMZ was
significantly greater than with compound 102 alone and TMZ alone
(logrank, p <0.001, % no xenograft recurrence, n=20). C, Tumor
regression of SH-SY5Y xenografts. As in 12A, except that xenografts
of SH-SYSY cells were implanted in one flank only and animals were
treated with one additional cycle of drugs, i.e., compound 102 on
days 13-15, TMZ, on day 16, and compound 102 plus TMZ on the same
schedules. Growth of all xenografts in control animals required
sacrifice by week 3; TMZ alone delayed growth but approached the
maximum allowable volume by week 7; compound 102 alone was more
inhibitory with no growth until week 3 with progression thereafter,
reaching about half the size of the TMZ alone treated xenografts by
week 7. Compound 102 plus TMZ completely inhibited xenograft growth
but with a slight residual tumor mass present at week 7. All
xenografts in treatment arms ulcerated by week 7, necessitating
sacrifice (mean and s.d., n=5 per treatment group). D, Histologic
features (H & E staining) of U87 xenografts_24 hours after i.p.
vehicle (upper left), compound 102 at 1.5 mg/kg (upper right), TMZ
at 80 mg/kg (lower left), and both drugs (lower right). E, Western
blots of U87 cells in culture 24 hours after exposure to compound
102 at 2.5 uM, TMZ at 25 uM or DOX at 2 uM, and compound 102 plus
TMZ or DOX. F, Western blots of U373 cells 24 hours after exposure
to compound 102 at 2.5 uM, doxorubicin at 2 uM, and both drugs. G,
Western blots of U373 cells 24 hours after exposure to TMZ at 25
uM, okadaic acid at 2 nM, and both drugs. H, IFS of p-p53 (red) and
nuclear morphology (DAPI, blue) in U373 cells after 24 hour
exposure to vehicle (upper left), compound 102 at 2.5 uM (upper
right), TMZ at 25 uM (lower left), and both drugs (lower
panel).
[0039] FIG. 13: Compound 102 in combination with doxorubicin causes
regression of subcutaneous xenografts.
[0040] SCID mice implanted with 5 million U87 cells divided into
four groups of 10 were treated starting at time 0 when average
tumor volume was approximately 60 cubic millimeters by i.p.
injection of vehicle alone (100 uL of 50% DMSO in PBS), compound
102 alone, doxorubicin alone, or compound 102 and doxorubicin at
the concentrations shown in the inset. Vehicle was given on days 1,
2, 4, 5, 7, 8; compound 102 on days 1, 4, 7; doxorubicin on days 2,
5, and 8; and, each drug of the combination on the same schedule as
when used alone.
[0041] FIG. 14A-C: Cell cycle distribution of U87 and U373 cells
exposed for 48 hours to compound 102, TMZ or DOX, and compound 102
plus TMZ or DOX and schema of mechanisms of action.
[0042] Cells in unsynchronized logarithmic growth were exposed to
vehicle or drug and adherent cells and cells in the media were
collected. Harvested cells were fixed with 70% cold ethanol for 20
min at -20 degrees, washed with PBS, and stained with 10 ug/ml of
PI and 1 ug/ml RNAse in TBS for 30 minutes and analyzed by FACS. A,
Flow cytometry profiles of U87 cells after exposures to DMSO only,
compound 102 only, 5 uM; TMZ only, 25 uM; compound 102 with TMZ
combination, doxorubicin only, 2.0 uM and compound 102 with
doxorubicin combination. B, Flow cytometry profiles of U373 cells.
Exposures as in A, and C, Schema of proposed mechanisms of compound
102 enhancement of cancer chemotherapy.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The invention provides a method of inhibiting proliferation
of a cancer cell or inducing apoptosis of a cancer cell, which
cancer cell does not overexpress N--CoR, comprising administering
to the subject a compound, wherein the compound has the
structure
##STR00009## [0044] wherein [0045] bond .alpha. is present or
absent; [0046] R.sub.1 and R.sub.2 is each independently H, O.sup.-
or OR.sub.9, [0047] where R.sub.9 is H, alkyl, alkenyl, alkynyl or
aryl, or R.sub.1 and R.sub.2 together are .dbd.O; [0048] R.sub.3
and R.sub.4 are each different, and each is OH, O.sup.-, OR.sub.9,
SH, S.sup.-, SR.sub.9,
[0048] ##STR00010## [0049] where X is O, S, NR.sub.10, or
N.sup.+R.sub.10R.sub.10, [0050] where each R.sub.10 is
independently H, alkyl, substituted C.sub.2-C.sub.12 alkyl,
alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl, substituted
alkynyl, aryl, substituted aryl where the substituent is other than
chloro when R.sub.1 and R.sub.2 are .dbd.O,
[0050] ##STR00011## [0051] --CH.sub.2CN,
--CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11, --NHR.sub.11 or
--NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is independently
alkyl, alkenyl or alkynyl, each of which is substituted or
unsubstituted, or H; [0052] R.sub.5 and R.sub.6 is each
independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and [0053] R.sub.7 and R.sub.8 is each independently H, F,
Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, [0054] where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, [0055] or a salt, enantiomer or zwitterion of
the compound, in an amount effective to inhibit the proliferation
or to induce apoptosis of the cancer cell.
[0056] In an embodiment of the above method, when X is
N.sup.+R.sub.10R.sub.10 and one R.sub.10 is CH.sub.3, then the
other R.sub.10 is [0057] alkyl, substituted C.sub.2-C.sub.12 alkyl,
alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl, substituted
alkynyl, aryl, substituted aryl where the substituent is other than
chloro when R.sub.1 and R.sub.2 are .dbd.O,
[0057] ##STR00012## [0058] --CH.sub.2CN,
--CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11, --NHR.sub.11 or
--NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is independently
alkyl, alkenyl or alkynyl, each of which is substituted or
unsubstituted, or H.
[0059] This invention provides a method of inhibiting proliferation
or inducing apoptosis of a cancer cell which overexpresses TCTP
comprising administering to the subject a compound, wherein the
compound had the structure
##STR00013## [0060] wherein [0061] bond .alpha. is present or
absent; [0062] R.sub.1 and R.sub.2 is each independently H, O.sup.-
or OR.sub.9, [0063] where R.sub.9 is H, alkyl, alkenyl, alkynyl or
aryl, or R.sub.1 and R.sub.2 together are .dbd.O; [0064] R.sub.3
and R.sub.4 are each different, and each is OH, O.sup.-, OR.sub.9,
SH, S.sup.-, SR.sub.9,
[0064] ##STR00014## [0065] where X is O, S, NR.sub.10, or
N.sup.+R.sub.10R.sub.10, [0066] where each R.sub.10 is
independently H, C.sub.2-C.sub.12alkyl, substituted
C.sub.2-C.sub.12 alkyl, alkenyl, substituted C.sub.4-C.sub.12
alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl where
the substituent is other than chloro when R.sub.1 and R.sub.2 are
.dbd.O,
[0066] ##STR00015## [0067] --CH.sub.2CN,
--CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11, --NHR.sub.11 or
--NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is independently
alkyl, alkenyl or alkynyl, each of which is substituted or
unsubstituted, or H; [0068] R.sub.5 and R.sub.6 is each
independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and [0069] R.sub.7 and R.sub.8 is each independently H, F,
Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, [0070] where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, [0071] or a salt, enantiomer or zwitterion of
the compound, in an amount effective to inhibit the proliferation
or to induce apoptosis of the cancer cell.
[0072] In an embodiment of the above method, the cancer cell does
not overexpress N--CoR.
[0073] In another embodiment of any of the above methods, the
compound has the structure
##STR00016## [0074] wherein [0075] bond .alpha. is present or
absent; [0076] R.sub.1 and R.sub.2 is each independently H, O.sup.-
or OR.sub.9, [0077] where R.sub.9 is H, alkyl, alkenyl, alkynyl or
aryl, or R.sub.1 and R.sub.2 together are .dbd.O; [0078] R.sub.3
and R.sub.4 are each different, and each is OH, O.sup.-, OR.sub.9,
SH, S.sup.-, SR.sub.9,
[0078] ##STR00017## [0079] where X is O, S, NR.sub.10, or
N.sup.+R.sub.10R.sub.10, [0080] where each R.sub.10 is
independently C.sub.2-C.sub.12 alkyl, substituted C.sub.2-C.sub.12
alkyl, alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl where the substituent
is other than chloro when R.sub.1 and R.sub.2 are .dbd.O, [0081]
--CH.sub.2CN, --CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11,
--NHR.sub.11 or --NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is
independently alkyl, alkenyl or alkynyl, each of which is
substituted or unsubstituted, or H; [0082] R.sub.5 and R.sub.6 is
each independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and [0083] R.sub.7 and R.sub.8 is each independently H, F,
Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, [0084] where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, [0085] or a salt, enantiomer or zwitterion of
the compound.
[0086] In an embodiment of any of the above methods the cancer is
adrenocortical cancer, bladder cancer, osteosarcoma, cervical
cancer, esophageal, gallbladder, head and neck cancer, Hodgkin
lymphoma, non-Hodgkin lymphoma, renal cancer, melanoma, pancreatic
cancer, rectal cancer, thyroid cancer and throat cancer.
[0087] This invention provides a method of inhibiting proliferation
or inducing apoptosis of a cancer cell that overexpresses TCTP by
administering to the subject a compound, wherein the compound has
the structure
##STR00018## [0088] wherein [0089] bond .alpha. is present or
absent; [0090] R.sub.1 and R.sub.2 is each independently H, O.sup.-
or OR.sub.9, [0091] where R.sub.9 is H, alkyl, alkenyl, alkynyl or
aryl, or R.sub.1 and R.sub.2 together are .dbd.O; [0092] R.sub.3
and R.sub.4 are each different, and each is OH, O.sup.-, OR.sub.9,
SH, S.sup.-, SR.sub.9,
[0092] ##STR00019## [0093] where X is O, S, NR.sub.10, or
N.sup.+R.sub.10R.sub.10, [0094] where each R.sub.10 is
independently C.sub.2-C.sub.12 alkyl, substituted C.sub.2-C.sub.12
alkyl, alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl where the substituent
is other than chloro when R.sub.1 and R.sub.2 are .dbd.O, [0095]
--CH.sub.2CN, --CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11,
--NHR.sub.11 or --NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is
independently alkyl, alkenyl or alkynyl, each of which is
substituted or unsubstituted, or H; [0096] R.sub.5 and R.sub.6 is
each independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and [0097] R.sub.7 and R.sub.8 is each independently H, F,
Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, [0098] where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, [0099] or a salt, enantiomer or zwitterions of
the compound, in an amount effective to inhibit the proliferation
or to induce apoptosis of the cancer cell.
[0100] In an embodiment of any of the above methods, the cancer
cell is in a subject. In a further embodiment, the subject is
mammal.
[0101] In an embodiment of any of the above methods, the cancer
cell is a neural cell. In another embodiment, the cancer cell is a
lymphoid cell.
[0102] Another embodiment of the above methods further comprises
administering an anti-cancer agent in an amount effective to
inhibit the proliferation or to induce apoptosis of the cancer
cell. In a further embodiment, the anticancer agent is
chemotherapeutic agent, a DNA intercalating agent, a spindle poison
or a DNA damaging agent.
[0103] Another embodiment of the above methods further comprises
administering a retinoid receptor ligand in an amount such that any
of the compounds described above and the retinoid receptor ligand
is effective to inhibit the proliferation or to induce apoptosis of
the cancer cell.
[0104] In the method of the invention, the retinoid receptor ligand
may be a retinoid, such as a retinoic acid, e.g. cis retinoic acid
or trans retinoic acid. The cis retinoic acid may be 13-cis
retinoic acid and the trans retinoic acid may be all-trans retinoic
acid. In the preferred embodiment, the retinoic acid is all-trans
retinoic acid (ATRA).
[0105] Retinoid receptor ligands used in the method of the
invention include vitamin A (retinol) and all its natural and
synthetic derivatives (retinoids).
[0106] Another embodiment of the above method further comprises
administering a histone deacetylase ligand in an amount such that
the any of the compounds described above and the histone
deacetylase ligand is effective to inhibit the proliferation or to
induce apoptosis of the cancer cell.
[0107] In the method of the invention, the histone deacetylase
ligand may be an inhibitor, e.g. the histone deacetylase inhibitor
HDAC-3 (histone deacetylase-3). The histone deacetylase ligand may
also be selected from the group consisting of
2-amino-8-oxo-9,10-epoxy-decanoyl,
3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide, APHA Compound
8, apicidin, arginine butyrate, butyric acid, depsipeptide,
depudecin, HDAC-3, m-carboxycinnamic acid bis-hydroxamide,
N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl) aminomethyl]
benzamide, MS 275, oxamfiatin, phenylbutyrate, pyroxamide,
scriptaid, sirtinol, sodium butyrate, suberic bishydroxamic acid,
suberoylanilide hydroxamic acid, trichostatin A, trapoxin A,
trapoxin B and valproic acid. In another embodiment of the
invention, the inhibitor is valproic acid.
[0108] In one embodiment, the methods described above further
comprise administering both a retinoid receptor ligand and a
histone deacetylase ligand each in an amount such that the amount
of each of the compounds described above, the histone deacetylase
ligand and the retinoid receptor ligand is effective to inhibit the
proliferation or to induce apoptosis of the cancer cell.
[0109] In one embodiment of the methods disclosed herein R3 or R4
is
##STR00020##
where X is O, S, NR.sub.10, or N.sup.+R.sub.10R.sub.10.
[0110] This invention provides a method for determining whether a
compound is effective in inducing cell death comprising (a)
contacting a first cancer cell with the compound; (b) determining
the level of expression of TCTP in the first cancer cell; (c)
contacting a second cancer cell with a protein phosphatase 2A
inhibitor; (d) determining the level of expression of TCTP in the
second cancer cell; (e) comparing the level of expression of TCTP
determined in step (b) with the level determined in step (d),
wherein, when the level of expression determined in step (b) is
equal to, or lower than, the level of expression determined in step
(d) indicates that the compound is effective to induce cell
death.
[0111] In one embodiment of the above method, the protein
phosphatase 2A inhibitor is a compound having the structure:
##STR00021## ##STR00022##
[0112] This invention provides a method for determining whether a
compound is effective in inducing cell death in a cancer cell
comprising (a) contacting a cancer cell with the compound; (b)
determining the level of expression of TCTP in the cancer cell; (c)
determining the level of expression of TCTP in a non-cancerous
cell; (e) comparing the level of expression of TCTP determined in
step (b) with the level determined in step (d), wherein, when the
level of expression determined in step (b) is lower than, the level
of expression determined in step (d) indicates that the compound is
effective to induce cell death in the cancer cell.
[0113] This invention provides a method for determining whether
treatment of a subject with an agent will be successful in treating
a subject suffering from cancer comprising (a) obtaining a first
sample from the subject prior to treatment; (b) determining the
level of expression of TCTP in the sample; (c) administering to the
subject the agent; (d) obtaining a second sample from the subject
after treatment with the agent; (e) determining the level of
expression of TCTP in the second sample obtained; wherein, when the
level of expression determined in step (b) is lower than the level
of expression determined in step (e) indicates that the treatment
of the subject with the agent be successful.
[0114] This invention provides a method for predicting whether
treatment of a subject with an agent will be successful in treating
a subject suffering from cancer comprising (a) obtaining a sample
comprising cancer cells from the subject; (b) culturing the cancer
cells; (c) determining the level of expression of TCTP in the
cancer cells; (d) contacting the cancer cells with the agent; (e)
determining the level of expression of TCTP in the cancer cells;
(f) comparing the level of expression of TCTP determined in step
(c) with the level of expression determined in step (e); wherein,
when the level of expression determined in step (c) is lower than
the level of expression determined in step (e) predicts that
treatment of the subject with the agent will be successful in
treatment of the cancer.
[0115] This invention provides a method for reducing the amount of
TCTP in a cell comprising contacting the cell with an effective
amount of protein phosphatase inhibitor, thereby reducing the
amount of TCTP in the cell.
[0116] In one embodiment of the above method, the protein
phosphatase inhibitor is a protein phosphatase 2A inhibitor. In
another embodiment, the protein phosphatase 2A inhibitor is a
compound having the structure
##STR00023## [0117] wherein [0118] bond .alpha. is present or
absent; [0119] R.sub.1 and R.sub.2 is each independently H, O.sup.-
or OR.sub.9, [0120] where R.sub.9 is H, alkyl, alkenyl, alkynyl or
aryl, or R.sub.1 and R.sub.2 together are .dbd.O;
[0121] R.sub.3 and R.sub.4' are each different, and each is OH,
O.sup.-, OR.sub.9, SH, S.sup.-, SR.sub.9,
##STR00024## [0122] where X is O, S, NR.sub.10, or
N.sup.+R.sub.10R.sub.10, [0123] where each R.sub.10 is
independently H, alkyl, substituted C.sub.2-C.sub.12 alkyl,
alkenyl, substituted C.sub.4-C.sub.12 alkenyl, alkynyl, substituted
alkynyl, aryl, substituted aryl where the substituent is other than
chloro when R.sub.1 and R.sub.2 are .dbd.O,
[0123] ##STR00025## [0124] --CH.sub.2CN,
--CH.sub.2CO.sub.2R.sub.11, --CH.sub.2COR.sub.11, --NHR.sub.11 or
--NH.sup.+(R.sub.11).sub.2, where each R.sub.11 is independently
alkyl, alkenyl or alkynyl, each of which is substituted or
unsubstituted, or H; [0125] R.sub.5 and R.sub.6 is each
independently H, OH, or R.sub.5 and R.sub.6 taken together are
.dbd.O; and [0126] R.sub.7 and R.sub.8 is each independently H, F,
Cl, Br, SO.sub.2Ph, CO.sub.2CH.sub.3, or SR.sub.12, [0127] where
R.sub.12 is H, aryl or a substituted or unsubstituted alkyl,
alkenyl or alkynyl, [0128] or a salt, enantiomer or zwitterion of
the compound.
[0129] In another embodiment of the above method, the cell is a
cancer cell that does not overexpress N--CoR. In another
embodiment, the cancer cell overexpresses TCTP.
DEFINITIONS
[0130] Certain embodiments of the disclosed compounds can contain a
basic functional group, such as amino or alkylamino, and are thus
capable of forming pharmaceutically acceptable salts with
pharmaceutically acceptable acids, or contain an acidic functional
group and are thus capable of forming pharmaceutically acceptable
salts with bases. The instant compounds therefore may be in a salt
form. As used herein, a "salt" is a salt of the instant compounds
which has been modified by making acid or base salts of the
compounds. The salt may be pharmaceutically acceptable. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as phenols. The
salts can be made using an organic or inorganic acid. Such acid
salts are chlorides, bromides, sulfates, nitrates, phosphates,
sulfonates, formates, tartrates, maleates, malates, citrates,
benzoates, salicylates, ascorbates, and the like. Phenolate salts
are the alkaline earth metal salts, sodium, potassium or lithium.
The term "pharmaceutically acceptable salt" in this respect, refers
to the relatively non-toxic, inorganic and organic acid or base
addition salts of compounds of the present invention. These salts
can be prepared in situ during the final isolation and purification
of the compounds of the invention, or by separately reacting a
purified compound of the invention in its free base or free acid
form with a suitable organic or inorganic acid or base, and
isolating the salt thus formed. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. For a
description of possible salts, see, e.g., Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19.
[0131] As used herein, "therapeutically effective amount" means an
amount sufficient to treat a subject afflicted with a disease (e.g.
cancer) or to alleviate a symptom or a complication associated with
the disease.
[0132] As used herein, "treating" means slowing, stopping or
reversing the progression of a disease, particularly cancer.
[0133] As used herein, "overexpressing TCTP" means that the level
of TCTP expressed in cells of the tissued tested are elevated in
comparison to the levels of TCTP as measure in normal healthy cells
of the same type of tissued under analgous conditions.
[0134] As used herein, "cancer cell" is a cell that is
characterized by uncontrolled growth and cell division and can
include tumor cells.
[0135] Cancer cells, which can include tumor cells, may or may not
overexpress N--CoR.
[0136] As used herein, "mitotic catastrophe" refers to a condition
of the cell characterized by abnormalities in the process of
mitosis that lead to cell death by any of the known cell death
pathways including apoptosis, necrosis, senescence, and
autophagy.
[0137] As used herein, "apoptosis" refers to programmed cell death
or any of a series morphological processes leading to controlled
cellular self-destruction.
[0138] As used herein, "proliferation" refers to a sustained
increase in the number of cells.
[0139] As used herein, "cell cycle progression" refers to the
advancement of a cell through a series of events that take place in
the cell leading to its division and replication.
[0140] As used herein, "cell cycle arrest" refers to the halting of
a series of events that take place in the cell leading to its
division and replication, which may be caused by a number of
factors, including, but not limited to, DNA damage, X-radiation,
ionizing radiation, and chemotherapeutic agents.
[0141] As used herein, anti-cancer agent means standard cancer
regimens which are currently known in the art. Examples include,
but are not limited to, x-radiation, ionizing radiation, DNA
damaging agents, DNA intercalating agents, microtubule stabilizing
agents, microtubule destabilizing agents, spindle toxins, and
chemotherapeutic agents. Further examples include cancer regimens
approved by the Food and Drug Administration, which include, but
are not limited to, abarelix, aldesleukin, alemtuzumab,
alitertinoin, allopurinol, altretamine, amifostin, anakinra,
anastrozole, arsenic trioxide, asparaginase, azacitidine,
bevacizumab, bexarotene, bleomycin, bortezomib, busulfan,
calusterone, capecitabine, carboplatin, carmustine, celecoxib,
cetuximab, chlorambucil, cisplatin, cladribine, clofarabine,
clyclophosphamide, cytarabine, dacarbazine, dactinomycin,
actinomycin D, dalteparin sodium, darbepoetin alfa, dasatinib,
daunorubicin, daunomycin, decitabine, denileukin, dexrazoxane,
docetaxel, doxorubicin, dromostanolone propionate, exulizumab,
epirubicin, epoetin alfa, erlotinib, estramustine, etoposide
phosphate, etoposide, VP-16, exemestane, fentanyl citrate,
filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant,
gefitinib, gemcitabine, gosereline acetate, histrelin acetate,
hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib
mesylate, interferon alfa 2a, interferon alfa 2b, irinotecan,
lapatinib ditosylate, lenalidomide, letrozole, leucovrin,
leuprolide acetate, levamisole, lomustine, meclorethamine,
megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone
phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin,
paclitaxel, palifermin, pamidronate, panitumumab, pegademase,
pegaspargase, pegfilgrastim, peginterferon alfa 2b, pemetrexed
disodium, pentostatin, pipobroman, plicamycin, mithramycin,
porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab,
sargrmostim, sorafenib, streptozocin, sunitinib, sunitinib maleate,
talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone,
thalidomide, thioguanine, G-TG, thiotepa, topotecan, toremifene,
tositumomab, trastuzumab, tretinoin ATRA, ruacil mustard,
valrunicin, vinblastine, vincristine, vinorelbine, vorinostat,
zoledronate, and zoledronic acid.
[0142] A complete list of all FDA approved cancer drugs can be
found at accessdata.fda.gov/scripts/cder/onctools/druglist.cfm
[0143] Examples of DNA intercalating agents include, but are not
limited to, doxorubicin, daunorubicin, dactinomycin. Examples of
Spindle Poisons include, but are note limited to vincristine,
vinblastine, taxol. DNA damaging agents include antracyclines,
bleomycin, cisplatin, etoposide, temozolomide, and
nitrosoureas.
[0144] As used herein, "alkyl" is intended to include both branched
and straight-chain saturated aliphatic hydrocarbon groups having
the specified number of carbon atoms. Thus, C.sub.1-C.sub.n as in
"C.sub.1-C.sub.n alkyl" is defined to include groups having 1, 2, .
. . , n-1 or n carbons in a linear or branched arrangement, and
specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl,
and so on. An embodiment can be C.sub.1-C.sub.12 alkyl. "Alkoxy"
represents an alkyl group as described above attached through an
oxygen bridge.
[0145] The term "alkenyl" refers to a non-aromatic hydrocarbon
radical, straight or branched, containing at least 1 carbon to
carbon double bond, and up to the maximum possible number of
non-aromatic carbon-carbon double bonds may be present. Thus,
C.sub.2-C.sub.n alkenyl is defined to include groups having 1, 2, .
. . , n-1 or n carbons. For example, "C.sub.2-C.sub.6 alkenyl"
means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and
at least 1 carbon-carbon double bond, and up to, for example, 3
carbon-carbon double bonds in the case of a C6 alkenyl,
respectively. Alkenyl groups include ethenyl, propenyl, butenyl and
cyclohexenyl. As described above with respect to alkyl, the
straight, branched or cyclic portion of the alkenyl group may
contain double bonds and may be substituted if a substituted
alkenyl group is indicated. An embodiment can be C.sub.2-C.sub.12
alkenyl.
[0146] The term "alkynyl" refers to a hydrocarbon radical straight
or branched, containing at least 1 carbon to carbon triple bond,
and up to the maximum possible number of non-aromatic carbon-carbon
triple bonds may be present. Thus, C.sub.2-C.sub.n alkynyl is
defined to include groups having 1, 2, . . . , n-1 or n carbons.
For example, "C.sub.2-C.sub.6 alkynyl" means an alkynyl radical
having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or
having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds,
or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds.
Alkynyl groups include ethynyl, propynyl and butynyl. As described
above with respect to alkyl, the straight or branched portion of
the alkynyl group may contain triple bonds and may be substituted
if a substituted alkynyl group is indicated. An embodiment can be a
C.sub.2-C.sub.n alkynyl.
[0147] As used herein, "aryl" is intended to mean any stable
monocyclic or bicyclic carbon ring of up to 10 atoms in each ring,
wherein at least one ring is aromatic. Examples of such aryl
elements include phenyl, naphthyl, tetrahydro-naphthyl, indanyl,
biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the
aryl substituent is bicyclic and one ring is non-aromatic, it is
understood that attachment is via the aromatic ring. The
substituted aryls included in this invention include substitution
at any suitable position with amines, substituted amines,
alkylamines, hydroxys and alkylhydroxys, wherein the "alkyl"
portion of the alkylamines and alkylhydroxys is a C.sub.2-C.sub.n
alkyl as defined hereinabove. The substituted amines may be
substituted with alkyl, alkenyl, alkynl, or aryl groups as
hereinabove defined.
[0148] The alkyl, alkenyl, alkynyl, and aryl substituents may be
unsubstituted or unsubstituted, unless specifically defined
otherwise. For example, a (C.sub.1-C.sub.6) alkyl may be
substituted with one or more substituents selected from OH, oxo,
halogen, alkoxy, dialkylamino, or heterocyclyl, such as
morpholinyl, piperidinyl, and so on.
[0149] In the compounds of the present invention, alkyl, alkenyl,
and alkynyl groups can be further substituted-by replacing one or
more hydrogen atoms by non-hydrogen groups described herein to the
extent possible. These include, but are not limited to, halo,
hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
[0150] The term "substituted" as used herein means that a given
structure has a substituent which can be an alkyl, alkenyl, or aryl
group as defined above. The term shall be deemed to include
multiple degrees of substitution by a named substitutent. Where
multiple substituent moieties are disclosed or claimed, the
substituted compound can be independently substituted by one or
more of the disclosed or claimed substituent moieties, singly or
plurally. By independently substituted, it is meant that the (two
or more) substituents can be the same or different.
[0151] As used herein, "zwitterion" means a compound that is
electrically neutral but carries formal positive and negative
charges on different atoms. Zwitterions are polar, have high
solubility in water and have poor solubility in most organic
solvents.
[0152] The compounds disclosed herein may also form zwitterions.
For example, a compound having the structure
##STR00026##
[0153] may also for the following zwitterionic structure
##STR00027##
[0154] where X is as defined throughout the disclosures herein.
[0155] As used herein, "administering" an agent may be performed
using any of the various methods or delivery systems well known to
those skilled in the art. The administering can be performed, for
example, orally, parenterally, intraperitoneally, intravenously,
intraarterially, transdermally, sublingually, intramuscularly,
rectally, transbuccally, intranasally, liposomally, via inhalation,
vaginally, intraoccularly, via local delivery, subcutaneously,
intraadiposally, intraarticularly, intrathecally, into a cerebral
ventricle, intraventicularly, intratumorally, into cerebral
parenchyma or intraparenchchymally.
[0156] The following delivery systems, which employ a number of
routinely used pharmaceutical carriers, may be used but are only
representative of the many possible systems envisioned for
administering compositions in accordance with the invention.
[0157] Injectable drug delivery systems include solutions,
suspensions, gels, microspheres and polymeric injectables, and can
comprise excipients such as solubility-altering agents (e.g.,
ethanol, propylene glycol and sucrose) and polymers (e.g.,
polycaprylactones and PLGA's).
[0158] Implantable systems include rods and discs, and can contain
excipients such as PLGA and polycaprylactone.
[0159] Oral delivery systems include tablets and capsules. These
can contain excipients such as binders (e.g.,
hydroxypropylmethylcellulose, polyvinyl pyrilodone, other
cellulosic materials and starch), diluents (e.g., lactose and other
sugars, starch, dicalcium phosphate and cellulosic materials),
disintegrating agents (e.g., starch polymers and cellulosic
materials) and lubricating agents (e.g., stearates and talc).
[0160] Transmucosal delivery systems include patches, tablets,
suppositories, pessaries, gels and creams, and can contain
excipients such as solubilizers and enhancers (e.g., propylene
glycol, bile salts and amino acids), and other vehicles (e.g.,
polyethylene glycol, fatty acid esters and derivatives, and
hydrophilic polymers such as hydroxypropylmethylcellulose and
hyaluronic acid).
[0161] Dermal delivery systems include, for example, aqueous and
nonaqueous gels, creams, multiple emulsions, microemulsions,
liposomes, ointments, aqueous and nonaqueous solutions, lotions,
aerosols, hydrocarbon bases and powders, and can contain excipients
such as solubilizers, permeation enhancers (e.g., fatty acids,
fatty acid esters, fatty alcohols and amino acids), and hydrophilic
polymers (e.g., polycarbophil and polyvinylpyrolidone). In one
embodiment, the pharmaceutically acceptable carrier is a liposome
or a transdermal enhancer.
[0162] Solutions, suspensions and powders for reconstitutable
delivery systems include vehicles such as suspending agents (e.g.,
gums, zanthans, cellulosics and sugars), humectants (e.g.,
sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene
glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens,
and cetyl pyridine), preservatives and antioxidants (e.g.,
parabens, vitamins E and C, and ascorbic acid), anti-caking agents,
coating agents, and chelating agents (e.g., EDTA).
[0163] It is understood that substituents and substitution patterns
on the compounds of the instant invention can be selected by one of
ordinary skill in the art to provide compounds that are chemically
stable and that can be readily synthesized by techniques known in
the art from readily available starting materials. If a substituent
is itself substituted with more than one group, it is understood
that these multiple groups may be on the same carbon or on
different carbons, so long as a stable structure results.
[0164] Compounds 100-108, as described herein, were obtained from
Lixte Biotechnology, Inc. 248 Route 25A, No. 2, East Setauket,
N.Y.
[0165] The present invention relates generally to compositions and
methods of inhibiting tumor genesis, tumor growth, and tumor
survival. The compositions comprise small molecule compounds that
reduce the amount of translational controlled tumor protein (TCTP)
in the cancer cell leading to its death.
[0166] Inhibitors of protein phosphatase 2A have been developed
that induce cancer cell death by induction of mitotic catastrophe
by a mechanism different from those mechanisms that underlie the
anti-cancer activity of these common chemotherapeutic agents.
Therefore, the compound 100 series of drugs have toxicities
different from most if not all commonly used chemotherapeutic
agents and thus, are combined with many active anti-cancer
therapeutic regimens to enhance therapeutic benefit.
SUMMARY
[0167] Compound 100 Preferentially Inhibits Cancer Cells Compared
to Normal Cells and May be Combined with Standard Anti-Cancer
Chemotherapy and/or Radiotherapy Regimens to Improved Therapeutic
Effect
[0168] Compound 100 and homologs inhibit many human cancer cell
types growing in cell culture and growing in vivo as xenografts
(PCT application on bicycloheptanes etc). Exposure of cancer cells
to Compound 100 is associated with a rapid and marked decrease in
translationally controlled tumor protein, TCTP, one of the most
highly conserved and most abundant proteins in eukaryotic cells
(Bommer and Thiele, 2004). TCTP is essential to cancer cell growth
but is not critical to the survival of normal (untransformed) cells
(Chen et al, 2007B). Targeting TCTP with Compound 100 (and its
homologs) is an effective means for disrupting cancer cell division
and therefore for treating cancers in general.
[0169] Reduction in TCTP by Compound 100 leads to disordered cell
replication and division. The addition of Compound 100 to standard
cancer regimens enhances the effectiveness of other cancer
treatments that inhibit cell growth and/or division. Compound 100
exerts its anti-cancer activity by a mechanism of action that is
not toxic to normal cells, at least in non-embryonic cells. Since
reduction of TCTP is not toxic to normal adult cells such as the
bone marrow, GI tract, peripheral nerves, or auditory nerves,
normal tissue often damaged by most cancer chemotherapeutic agents,
compound 100 can be combined with standard anti-cancer regimens to
enhance anti-cancer activity while avoiding increased toxicity.
[0170] In the instance of compound 100 (and its homologs), the
likelihood of a particular cell type being vulnerable to treatment
with a drug and the extent of potency of a drug can be simply and
rapidly estimated by the extent to which exposure to Compound 100
reduces TCTP. Assays that measure the ability of compounds to
decrease the abundance of TCTP in cancer cell lines are useful for
the identification of compounds that may be effective anti-cancer
drugs.
[0171] Assay of TCTP is a Tool for Screening Compounds for Activity
Likely to be Useful in Cancer Treatment and for Determining Cell
Types Likely to be Inhibited by Compound 100.
[0172] Assays that measure the ability of compounds to decrease the
abundance of TCTP in cancer cell lines are useful for the
identification of compounds that may be effective anti-cancer
drugs. In the instance of compound 100 (and its homologs), the
likelihood of a particular cell type being vulnerable to treatment
with a drug and the extent of potency of a drug can be simply and
rapidly estimated by the extent to which exposure to compound 100
reduces TCTP.
[0173] Introduction
[0174] We have discovered that inhibition of the serine/threonine
protein phosphatase inhibitor PP2A leads to a reduction in the
amount of TCTP in multiple human cancer cell lines including lines
derived from glioblastoma multiforme, medulloblastoma,
neuroblastoma, central nervous system lymphoma, and breast cancer.
We synthesized a series of small molecule inhibitors of PP2A with
varying degrees of lipophilicity and showed that both the water
soluble lead compound compound 100 and the lipid soluble lead
compound, compound 102, lead to increased phosphorylation and a
decrease in the amount of TCTP in cell lines in vitro and growing
as xenografts of human glioblastomas and neuroblastomas.
[0175] Because these molecular changes, i.e. increased
phosphorylation and reduction of TCTP, are likely not to affect the
integrity of normal adult cells, we believe the inhibition of TCTP
via small molecule inhibitors of the pathway regulating the
integrity of the interaction of TCTP with the anti-apoptic
machinery is an effective means of treating cancer. In addition,
this pathway is also exploitable for the inhibition of other cancer
cell types undergoing excessive replication and white blood cell
proliferation in the inflammatory response.
[0176] Disclosed herein is a method for the treatment of human and
animal cancers based on inducing alterations of multiple components
of processes responsible for cell growth and replication with a
single pharmacologic intervention. In the adult, most normal cells
are not prepared for cell replication and cannot be forced into
cell replication by a pharmacologic intervention. Many types of
cancers, however, are characterized by a state of activation of
multiple enzymes that initiate and carry out cell replication. This
abnormal state of heightened activation can be further intensified
by inhibition of serine/threonine protein phosphatase 2A (PP2A),
causing increased activation of the mitotic process to a level at
which chaotic cell division results in cell death. The critical and
final step in activation of this pathway by inhibition of PP2A is a
reduction in TCTP and concomitant reduction in mcl-1. In the
absence of sufficient amounts TCTP, cell death rapidly occurs in
transformed cells.
[0177] Our claim is for a novel method for the treatment of cancer
based on pharmacologic induction of conditions that lead to
diminution in the amount of TCTP in the cancer cell. The structural
hallmarks of the induction of this process are referred to as
mitotic catastrophe (MC). MC refers to a condition of the cell
characterized by abnormalities in the process of mitosis that lead
to cell death by any of the known cell death pathways including
apoptosis, necrosis, senescence, and autophagy (Gullizzi et al
2007). We present an example of pharmacologic induction of MC by
pan-modification of the extent of phosphorylation of serine and/or
threonine regulatory sites in proteins controlling orderly cell
replication and division and, finally cell death by reduction in
TCTP. Pan-deregulation of serine/threonine phosphorylation is
achieved in one instance by the inhibition of protein phosphatase
2A (PP2A) by a small molecule, Compound 100 and/or several of its
homologs.
[0178] We further show that the opposite modulations of DNA-damage
response pathways result paradoxically in enhancement of the
effectiveness of cytotoxic chemotherapy. We demonstrate that a
small molecule inhibitor (Compound 102) of protein phosphatase 2A
(PP2A) (Westermark and Hahn, 2008) activates Plk-1 and Akt-1 and
decreases p53 abundance in tumor cells. Combined with temozolomide
(TMZ; a DNA-methylating chemotherapeutic drug), compound 102 causes
complete regression of glioblastoma multiforme (GBM) (Prados et al,
2008) xenografts without recurrence in 50% of animals (greater than
28 weeks) and complete inhibition of growth of neuroblastoma (NB)
(Rubie et al, 2006) xenografts (for at least 7 weeks). Treatment
with either drug alone results in only short-term
inhibition/regression, with all xenografts resuming rapid growth.
Compound 102-inhibition of PP2A increases entry of cancer cells
into disordered mitosis with accumulation of cells in the G2M phase
and blocks cell cycle arrest in the presence of TMZ.
[0179] Previously, it was demonstrated that a shellfish toxin
(okadaic acid), which inhibits serine/threonine protein
phosphatases PP2A and PP1, inhibits the growth and promotes cell
differentiation of primary GBM cells (Park et al, 2007; Lu et al,
2008). Small molecules derived from cantharidin (a vesicant
originally extracted from beetles) or its demethylated homolog
(nor-cantharidin) mimic the effects of okadaic acid and have
anti-cancer activity in vitro and in vivo (Hart et al, 2004;
Bonness et al, 2006). Reported clinical benefit of cantharidin is
modest and constrained by urologic toxicity and nor-cantharidin,
while less toxic, has limited effectiveness (Hart et al, 2004). A
series of nor-cantharidin derivatives have been synthesized and
their anti-phosphatase and anticancer activity characterized in
vitro (Kovach and Johnson, 2008).
[0180] Recent work led to the discovery that treatment with the
compound, compound 100, and several homologs inhibit the
serine/threonine protein phosphatases PP2A (FIG. 1). Compound 102
inhibits PP2A (IC.sub.50=.about.0.4 .mu.M) more potently than PP1
(IC.sub.50=.about.80 .mu.M) (FIG. 2). Associated with their
inhibition of PP2A, Compound 100 and homologs inhibit a variety of
human cancer cell types growing in cell culture and growing in vivo
as xenografts implanted subcutaneously in SCID mice (FIG. 3 and
FIG. 4). Given intraperitoneally (i.p.), a single dose of compound
102 at 1.5 mg/kg inhibits PP2A activity in subcutaneous (s.c.)
xenografts of the human GBM cell line, U87 MG, and in normal brain
tissue (FIG. 5). In vitro, compound 102 showed dose-dependant
inhibition of GBM cell growth (IC.sub.50=.about.4 uM) (FIG. 6).
Death of these cancer cells is associated with profound disruption
of microtubular structures. Such patterns of disordered
microtubules during mitosis have been noted after exposure of
cancer cells to spindle toxins include such vincristine,
vinblastine, taxol, taxotere, ionizing radiation, and DNA damaging
agents including anthracyclines and the platinum based compounds.
The morphologic appearance of cells displaying these
characteristics has been called the mitotic catastrophe phenotype
(Castedo et al 2004).
[0181] Compound 100 Reduces TCTP Leading to Cancer Cell Death
[0182] As shown in the examples that follow, exposure of cancer
cells to Compound 100 is associated with increased phosphorylation
of several regulatory proteins involved in cell growth and division
including Akt-1, Aurora A, N--CoR, Plk-1 and TCTP. In particular,
the phosphorylation of the serine/threonine kinase, Plk-1, is
associated with the disruption of the homogeneous cytoplasmic
distribution of alpha-tubulin (FIG. 7). Surprisingly, we also found
that phosphorylation of Plk-1 is associated with a rapid and marked
decrease in the amount of TCTP (FIG. 8 and FIG. 9).
[0183] TCTP is associated with many functions in the cell and is
essential for fetal development (Bommer and Thiele, 2004; Chen et
al, 2007B). TCTP is also essential to cancer cell growth but is not
critical to the survival of normal adult (untransformed) cells
(Chen et al, 2007B). For this reason, TCTP is an attractive target
for anti-cancer treatments. Compound 100 and its homologs
consistently reduce cellular concentrations of TCTP in cancer cells
as early as 4 hours after exposure to the drugs. Even at this early
time, loss of TCTP is associated with disruption of microtubular
morphology and mitotic disruption (FIG. 7), accompanied
subsequently by apoptosis, necrosis, and autophagy. Thus, targeting
TCTP with compound 100 is an effective means for inhibiting cancer
cell growth and division and therefore for treating cancers.
[0184] Compound 100 Preferentially Inhibits Cancer Cells Compared
to Normal Cells
[0185] The therapeutic benefit of reducing TCTP by treatment with
Compound 100 and its homologs is further enhanced by combining
treatment with Compound 100 with other anti-cancer treatments
including ionizing radiation and agents used for the treatment of
cancer that induce abnormalities in DNA and/or that interfere with
one or more constituents of the mitotic process. In particular, the
anti-cancer activity of X-ray, DNA alkylating agents, DNA
intercalating agents, and microtubule stabilizing and disrupting
agents is enhanced by treatment with Compound 100. For example,
compound 100 enhances cancer cell inhibition by the standard
chemotherapeutic agents cisplatin, doxyrubicin and taxol (FIGS.
10A, 10B and 10C).
[0186] Most current strategies for pharmacologic treatment of
cancers are based on developing drugs or biologicals, primarily
antibodies and anti-sense RNAs, that specifically inhibit the
activity of an enzyme in a signaling pathway or a gene(s) encoding
an enzyme upon which the cancer cell is dependent for growth and
survival (Shoshan and Linder 2008). Dependence of a particular type
of cancer on excessive activity of a specific signaling pathway has
been termed "oncogene addiction" (Lim et al 2008). Interference
with the function or abundance of an addicting oncogene may inhibit
growth and, in some cases, result in the death of cancer cells that
are dependent upon this pathway. Inhibition of a single oncogene,
however, is usually insufficient for complete inhibition of a
cancer and inhibition is overcome by mutation leading to drug
resistance. Older approaches to cancer treatment have involved
primarily the use of non-specific agents alone and in combinations
of drugs with non-overlapping toxicities to normal tissues to
damage DNA or to interfere with cell metabolic pathways including
modulation of microtubule stability.
[0187] We provide evidence that a more effective means of
inhibiting the growth of many, if not all, cancers, is to target
master regulatory molecules that affect the function of multiple
other regulatory molecules simultaneously. We developed a method to
preferentially target cancer cells compared to normal cells by
taking advantage of the fact that cancer cells are preparing for or
are engaged in active growth and replication.
[0188] Coordination and inhibition of molecular events necessary
for the survival of the normal cell and the cancer cell are
accomplished by counterbalancing chemical activities. Among the
most important of these regulatory activities are phosphorylation
and de-phosphorylation and acetylation and de-acetylation of
proteins controlling many cell functions. By altering the activity
of one or a few enzymes controlling phosphorylation and/or
acetylation, the activity of complex processes essential to a
variety of cell functions can be altered (Johnson et al 2008).
Deregulation of systems essential to cell replication should have
general applicability for the treatment of multiple types of human
cancers, particularly those with a high proportion of cells in
active growth and cell division.
[0189] PP2A is one of the most abundant and most highly conserved
of all proteins, playing a critical role in the life of the cell,
primarily during development of the fetus and at times of cell
replication in the adult. PP2A modulates the state of
phosphorylation of multiple enzymes, some of which are necessary
for proper assembly and disassembly of the mitotic machinery
(Andrabi et al 2007, van de Weerdt 2005, Westermarck and Hahn 2008,
Juntilla et al 2007). When DNA damage occurs during mitosis, PP2A
is activated and dephosphorylates the serine/threonine kinase,
Plk1. Dephosphorylation of Plk1 in turn halts mitosis providing
time for DNA repair before replication is completed. Plk1 has
several other activities affecting cell growth and division. It
regulates spindle formation and dissolution. An increase in
phosphorylation of Plk1 leads to its activation and its
phosphorylation of the transcriptionally controlled protein (TCTP),
another serine/threonine kinase. Phosphorylation of TCTP leads to a
reduction in its abundance and cell death. In the normal cell, upon
conclusion of mitosis, Plk1 undergoes dephosphorylation by PP2A
that allows the spindle to be disassembled with tubulin undergoing
re-polymerization. (Yarm 2002, van Vugt and Medema 2005, Johnson et
al 2008). As normal adult cells are not subject to regulation of
cell death by the function of TCTP, inhibition of PP2A and
destruction of TCTP would preferentially lead to cancer cell death.
We developed small molecule inhibitors of PP2A and demonstrated
that exposure of cancer cells in vitro and in vivo to a lead
compound, compound 100, results in abnormal spindle formation,
alteration in cell shape, and incomplete cell division of a variety
of human cancer cell types, associated with a decrease in TCTP.
Exposure to compound 100 caused dose dependent inhibition of human
cancer cell lines derived from the breast, colon, stomach, liver,
ovary, prostate, brain, lung, and of leukemias of myeloid and
lymphoid lineage and of lymphomas.
[0190] The compound 100 series of drugs was developed to target
serine threonine protein phosphatase 2A. PP2A regulates the
activity of a multitude of cell signaling proteins especially those
essential for cell growth, mitosis, and division (Janssens and
Goris, 2001). We reasoned that, although PP2A is important to many
cell functions (Forester et al, 2007; Westermarck and Hahn 2008),
its activity may be particularly important to the cancer cell.
Cancer cells (transformed cells) are characterized by alterations
in at least some signaling (enzyme) systems that are regulated by
phosphorylation and dephosphorylation. Inhibition of PP2A, the
major serine threonine phosphatase in the mammalian cell, might
disrupt several pathways important to cancer cell survival. The
targeting of a multifunctional enzyme such as PP2A that disrupts
the function of several (many) pathways important to cancer cell
growth and division should be more effective than targeting a
single pathway. Thus, inhibition of PP2A will alter many pathways
simultaneously rendering the cancer cell less likely to overcome
inhibition by bypassing the activity of any one regulatory
molecule. Mutational alteration of PP2A itself that bypasses
inhibition by compound 100 while maintaining its multiple
regulatory capabilities may not be easily accomplished, thereby
minimizing the chances of acquired compound 100 resistance.
[0191] We found that exposure of cancer cells in vitro and in vivo
to a compound 100 results in abnormal spindle formation, alteration
in cell shape, and incomplete cell division of a variety of human
cancer cell types. This induced deregulation of cell division led
to cancer cell death accompanied in some cancers by cell
differentiation. Exposure to Compound 100 caused dose dependent
inhibition of human cancer cell lines derived from the breast,
colon, stomach, liver, ovary, prostate, brain, lung, and of
leukemias of myeloid and lymphoid lineage and of lymphomas. We
showed that compound 100 treatment of cancer cells induces
abnormalities in mitotic spindle structures in a large proportion
of the cell mass, and leads to cell death. Thus, the use of
compound 100 to decrease TCTP should be effective for the treatment
of cancers in general.
[0192] In all cell types studied, exposure to compound 100 lead to
prompt and marked reduction in TCTP. The mechanism(s) by which
compound 100 induces a reduction in TCTP and leads to death of the
cancer cell is not known (Gachet et al, 1999: Bommer and Thiele,
2004; Chen et al 2007A). TCTP appears to be critical to the proper
functioning of proteins with apoptotic regulatory activity. One
such protein is mcl-1, a member of the bcl-2 family (Craig 2002,
Warr and Shore, 2008). Mcl-1 is a highly labile molecule important
to many developmental processes and is essential for fetal
development (Rinkenberger et al 2000; Craig, 2002; Liu et al,
2005). The presence of mcl-1 is also required for the growth and
development of T and B lymphocytes (Opfermann et al 2003).The
mechanism by which diminished or absent mcl-1 leads to cell death
of the embryo is not firmly established. In the absence of mcl-1,
however, fetal death occurs at an early stage of development and a
variety of cancer cell types undergo apoptosis. Stimulation of
cells by cell growth factors is associated with rapid synthesis of
mcl-1 and leads to increases in cell survival and/or
differentiation. Withdrawal of growth stimuli results in cessation
of synthesis and rapid degradation of mcl-1 and cell death (Liu et
al 2005).
[0193] TCTP binds to mcl-1 and to Bc1-xL, another anti-apoptotic
protein (Yang et al 2005). Susini et al (2008) reported that loss
of TCTP expression results in a marked increase in cell death
during embryogenesis. They suggested that TCTP exerts its
anti-apoptotic effect by interfering with Bax dimerization in the
mitochondrial membrane. Of crucial importance to TCTP as a target
for cancer therapy is the fact that survival of cells of the adult
is independent of the abundance of mcl-1 (Liu et al 2005). Thus,
loss of TCTP activity induced by compound 100 may be one mechanism
by which compound 100 differentially inhibits the cancer cell while
sparing damage to the adult (differentiated) normal cell.
[0194] Thus, we demonstrated the effectiveness of reducing
pharmacologically the abundance of TCTP as a method of cancer
treatment. Exposure to compound 100 of glioblastoma,
medulloblastoma, B-cell lymphoma, and breast cancer cell lines
induced the mitotic phenotype in a large proportion of the cell
mass as well as inducing apoptosis and differentiation of other
cells. Inhibition of cell growth and cell death by compound 100 was
associated with increased phosphorylation of several kinases
including Akt and Plk1 as well as reduction of TCTP and mcl-1. The
effects of PP2A inhibition on several components of the mitotic
machinery have been characterized.
[0195] Akt is a kinase target of PI3 kinase that regulates multiple
cell functions including the activity of proteins involved in cell
cycle progression (Andrabi et al, 2007). Plk1 has multiple
activities in cell growth and division and it is critically
important for regulating spindle formation and dissolution by
regulating the phosphorylation of TCTP (Yarm et al, 2002; van Vugt
and Medema 2005). Increased phosphorylation of two specific sites
in the Plk1 molecule leads to activation of its serine/threonine
kinase activity, causing increased phosphorylation of the kinase,
TCTP. These molecular changes are associated with depolymerization
of microtubules, a process necessary for rendering tubulin
available for spindle formation and the orderly distribution of DNA
during mitosis. Upon conclusion of mitosis, Plk1 undergoes
dephosphorylation permitting in turn the spindle to be disassembled
with tubulin undergoing polymerization. TCTP is associated with
microtubule function and has been shown to affect cell-cycle
progression among other aspects of cell growth and transformation
(Johnson et al, 2008). The Aurora kinases are regulatory proteins
demonstrated to have roles in mitosis, affecting centrosome
function and bipolar spindle formation (Anand et al, 2003; Jiang et
al, 2003; Gautschi et al. 2008). The activity of Cdk1 (Cdc2), a
cyclin dependent kinase, is required for cells to exit mitosis
(Forester et al 2007). Activation of Cdc2 requires
dephosphorylation by the phosphatase Cdc25C, whose activity is
dependent upon dephosphorylation by PP2A. Thus, inhibition of PP2A
prevents the dephosphorylation of Cdc2, which in turn prevents exit
from mitosis (Forester et al 2007).
[0196] These and other proteins regulated by serine/threonine
phosphorylation mediated by PP2A play critical roles in cell
replication, a process essential for development of the fetus and
child and maintenance of normal tissue structure and function in
the adult but also a process, that when unregulated, underlies the
virulent hallmark of the cancer cell, unregulated proliferation
(Westermarck and Hahn 2008). Interference with the orderly
formation and dissolution of spindle structures by excessive
activity of any or all of these molecules results in deregulation
of the mitotic process and failure of quantitative apportionment of
DNA between daughter cells during cell division.
[0197] Compound 100 and its homologs inhibit the action of PP2A
allowing excessive phosphorylation of Plk1 and in turn of TCTP
leading the formation of spindle structures at inappropriate times
with respect to the cell cycle. Interference with the orderly
formation and dissolution of spindle structures by excessive
activity of any or all of these molecules results in deregulation
of the mitotic process and failure of quantitative DNA
apportionment between daughter cells during cell division. This
deregulation results in an unusual histologic appearance of cancer
cells called the mitotic phenotype that is characterized
histologically by micronuclei and lobulated nuclei and bizarre
abnormal spindle shapes and arrest of cell division. Extreme
activation of the mitotic process leads to MC, a state of
replicative disorder that has been associated with the death of
such affected cells either in mitosis or subsequently in the first
or second interphase (Galluzzi et al, 2007). What has not been
appreciated is that inhibition of PP2A results in marked diminution
of TCTP. It is this event that provides for preferential killing of
the cancer cell compared to the normal adult cell.
[0198] We also showed that the extent of pharmacological inhibition
of PP2A in cancer cells has paradoxical effects of cell growth. At
doses of Compound 100 that do not inhibit cancer cell proliferation
in cell culture (submicromolar to very low micromolar
concentrations), there is slight but clear-cut stimulation of the
growth of tumor cells whereas higher doses lead to mitotic
catastrophe and cell death. One possible explanation for this
phenomenon is that slight inhibition of PP2A increases
phosphorylation of molecules regulating entry into mitosis such as
Plk1 (Strebhardt and Ullrich, 2007), resulting in an increase in
the rate of cells of cells entering mitosis without significantly
decreasing the amount of TCTP. Activated Plk1 phosphorylates TCTP
leading to a decrease in microtubule stabilization, which normally
promotes microtubule reorganization after metaphase (Yarm, 2002;
Johnson et al, 2008). At higher doses of Compound 100, however,
there is a sharp reduction in TCTP, leading cell death.
[0199] Our data are compatible with the idea that many aspects of
cell division and cell cycle regulation are not so different
between the normal cell and the cancer cell. An intervention such
as inhibition of a key regulator of the phosphorylation of multiple
enzymes involved in cell division in cell types already prepared
for rapid proliferation, such as cancer cells, results in a chaotic
mitotic state. Inhibition of PP2A by compound 100 does not force
normal cells into excessive replication and, therefore, the normal
cell survives compound 100.
[0200] Compound 100 Enhances the Activity of Other Anti-Cancer
Agents
[0201] Mitotic enhancement by treatment with compound 100 not only
inhibits the growth and kills cancer cells in and of itself but
also renders cancer cells more vulnerable to inhibition and killing
by standard modalities of cancer treatment. Abnormal mitotic
structures are induced by exposure of cells to X-radiation, to
drugs that either interfere with tubulin polymerization or cause
hyperpolymerization, and to DNA damaging agents (Ianzini and
Mackey, 1998; Morse et al, 2005; Ngan et al, 2008). Despite their
significant toxicities, X-ray, spindle poisons, and DNA alkylating
agents are among the most widely used and most effective, if not
curative, anti-cancer modalities available.
[0202] The addition of spindle poisons and/or x-ray during or
following exposure of cancers to compound 100 will enhance the
extent of cancer cell killing without increasing toxicity to normal
cells. Specifically, the combinations of LB-1 combined with
ionizing radiation (X-ray therapy), spindle poisons including
taxol, vincristine (VCR), vinblastine (VBL), and to DNA damaging
agents including anthracyclines, bleomycin, cis-platin, etoposide,
temozolomide, and nitrosoureas are more effective anti-cancer
regimens than standard regimens of single anti-cancer agents or
combinations of agents in the absence of treatment with compound
100. This list of anti-cancer drugs is not meant to be inclusive of
all drugs that may be combined to advantage with compound 100.
Because the mechanism of action of LB-1 on TCTP and other
regulatory molecules is distinct from all other approved
anti-cancer regimens, compound 100 may be use to advantage in
combination with any of all FDA approved cancer regimens (for list
of FDA-approved anti-cancer drugs see:
www.accessdata.fda.gov.gov.scripts/cder/onctools/druglist.cfm)
[0203] Recently, several investigators have proposed to exploit
activation rather than inhibition of PP2A activity as a therapeutic
approach to cancers that have impaired PP2A function. Mutationally
reduced PP2A activity has been reported in melanomas, cancers of
the colon, lung, and breast and certain leukemias (Neviani et al
2007; Perrotti and Neviani 2008). In these cancers, functional
inactivation of various subunits of PP2A reduces its phosphatase
activity. Certain immunosuppressive drugs, including forskolin and
FTY720 enhance PP2A phosphatase activity resulting in inhibition of
the growth of these tumors in vitro and in vivo (Perrotti and
Neviani 2008). The most striking effect of activation of PP2A
phosphatase activity is reported against human blast crisis chronic
myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive
(phi-positive) acute lymphocytic leukemia (ALL). In a mouse model
of disseminated lymphoma-leukemia, Liu et al (2008 showed that
treatment with FTY720 daily for two weeks increased survival time.
This seemingly paradoxical effect, inhibition of cancer cell growth
by enhancement of PP2A activity rather than inhibition of PP2A,
underscores the complex concentration dependent effects of
modulating the state of phosphorylation of PP2A to reduce oncogenic
activity.
[0204] Functional impairment of PP2A increases activation of the
PKC, PI3 kinase-Akt, and ERK pathway, a mechanism known to
contribute to the cancer phenotype through enhanced signaling via
this pathway. Partial restoration of PP2A activity in such cells
reduces the extent of aberrant signaling leading to inhibition of
cell proliferation. In the case of FTY720 there is enhancement of
dephosphorylation (reduction of activation) of activated oncogenes
and, presumably a reduction of cells entering mitosis. In the case
of compound 100 inhibition of PP2A, there is increased
phosphorylation (increased activation) of oncogenes driving cells
into mitotic chaos and loss of TCTP, leading to cell death.
[0205] Tuynder et al (2004) noted that some anti-histaminic and
psychoactive drugs are associated with a reduction of TCTP in
certain leukemia cell lines and increase the life-span of mice
bearing these leukemias. This effect of the anti-histaminic
compounds was noted several days after drug exposure. To our
knowledge, except for these anti-histaminic and psychoactive drugs
and compound 100, no agents have been reported to reduce TCTP
activity.
[0206] Contrary to the conventional wisdom that inhibition of
certain regulatory proteins controlling cell proliferation and
division and restoration of acquired defects in DNA-damage defenses
are promising approaches to improved cancer treatments, quite the
opposite is the case. Namely, accentuation rather than inhibition
of cell cycle progression and of defense against DNA-damage enhance
the effectiveness of cancer chemotherapy. Global alteration of
signal transduction by inhibition of ubitquitous, highly conserved
regulatory protein phosphatase, PP2A, accelerates cell cycle
progression in cancer cells and blocks defenses against DNA damage
imparting curative activity to Temozolomide, a drug with
non-curative activity used alone. We also show that the mechanism
underlying potentiation of Temozolomide cytotoxicity is a general
effect as it applies to the cellular response to one of the most
commonly used anti-cancer drugs, doxorubicin.
[0207] Because the novel compounds used to inhibit signal
transduction pathways affecting cell growth and DNA damage response
mechanisms is of a class of pharmacologic agents, PP2A inhibitors,
which have been given safely to humans in the past, our approach is
successfully applied to the treatment of cancers in humans. The
following results demonstrate that, contrary to conventional
thinking in drug discovery, simultaneous perturbation of multiple
regulatory pathways already disordered in the cancer phenotype
differentially affects cancer cells compared to normal cells,
preventing recurrence of the cancer after treatment with standard
chemotherapeutic agents that are otherwise only partially effective
in reducing cancer cell burden.
[0208] Experimental Details
EXAMPLE 1
Reduction of TCTP After Treatment with Compound 100 in U87 and DAOY
Cells
[0209] Administration compound 100 in U87 glioblastoma multiforme
cells grown as subcutaneous xenografts in SCID mice resulted in
reduction of TCTP concentration, as detected by 2-dimensional gel
electrophoresis. SCID mice were implanted with 5 million U87 cells
subcutaneously. On day 26, the mice were administered 1.5mg/kg of
compound 100 by intraperitoneal injection. The animals were
sacrificed after 4 hours of treatment and the subcutaneous mass of
tumor cells were removed for 2-dimensional gel electrophoretic
analysis. A comparable group of mice were exposed to vehicle alone.
As shown in FIG. 4, TCTP, subsequently identified by LC-MS-MS,
compound 100 treated cells resulted in a diminution in TCTP.
[0210] Administration of compound 100 in DAOY medulloblastoma cells
in cultures resulted in a reduction in concentration of TCTP and
activation of Plk-1, as detected by western blot analysis of cell
lystates. DAOY cells in culture were exposed to compound 100 for 4
hours and for 24 hours, and stained for TCTP, p-plk (phosphorylated
plk), and total plk on western blots. As early as 4 hours, there is
a decrease in the TCTP and an increase of plk-1 phosphorylation, as
shown in FIG. 9. In addition, at 24 hours, no TCTP is detectable at
loading of comparable concentrations of total cell protein.
EXAMPLE 2
Inhibition of PP2A Diminishes a Major Defense Against DNA Damage,
Cell-Cycle Arrest by p53
[0211] Exposure of U87MG cells in culture to compound 102 resulted
in the appearance of disordered microtubules and abnormal mitotic
figures that are characteristic of mitotic catastrophe, a form of
cell death distinct from apoptosis and cell senescence (Castedo et
al, 2004; d'Adda di Fagagna, 2008) (FIGS. 11A, 11B). Induction of
mitotic catastrophe by compound 102 was associated with increased
phosphorylated Akt-1 (pAkt-1, FIG. 11C), increased phosphorylated
Plk-1 (pPlk-1) and a marked decrease in translationally controlled
tumor protein (TCTP; FIG. 11D). TCTP is an abundant, highly
conserved, multifunctional protein that binds to and stabilizes
microtubules before and after mitosis and also exerts potent
anti-apoptotic activity (Sommer and Thiele, 2004; Yarm, 2002;
Susini et al, 2008) (FIG. 11E). Decreasing TCTP with anti-sense
eTCTP has been shown by others to enhance tumor reversion of
v-src-transformed NIH 3T3 cells and reduction of TCTP is suggested
to be the mechanism by which high concentrations of certain
anti-histaminics and psychoactive drugs inhibit growth of a human
lymphoma cell line (Tuynder et al, 2004).
[0212] pAkt-1 phosphorylation at Ser308 indicates downstream
activation of the phosphatidylinositol-3-kinase (PI3K) pathway, an
event generally considered to be growth-promoting (Brazil et al,
2004). Akt-1 activation, however, may be anti- or proapoptotic
depending on the context of cell signaling (Andrabi et al, 2007).
Compound 102 inhibition of PP2A increased pAkt-1 and activated
Plk-1, a regulator of a mitotic checkpoint and of the activity of
TCTP. Compound 102 exposure also increased phosphorylated MDM2, the
primary regulator of p53 activity (Vogelstein et al, 2000; Vazquez
et al, 2008) and decreased the abundance of p53 (FIGS. 11F, 11G.).
pAkt-1 can directly phosphorylate MDM2, increasing its stability,
and can phosphorylate MDMX, which binds to and further stabilizes
MDM2 (Olivier et al, 2008). Thus inhibition of PP2A diminishes a
major defense against DNA damage, cell-cycle arrest by p53.
EXAMPLE 3
Compound 100 Enhances the Cytotoxic Activity of Standard Cytotoxic
Chemotherapeutic Drugs
[0213] Exposure to compound 100 enhanced the inhibition of the
human glioblastoma cell line, U373, by cisplatin (FIG. 10A),
doxorubicin (FIG. 10B) and Taxol (FIG. 10C), as shown in FIGS. 10A,
10B, and 10C, respectively. Cells were exposed to vehicle alone
(control); compound 100 at 2.5 .mu.M, cisplatin at 0.1 .mu.M;
doxorubicin at 0.01 .parallel.M; or taxol at 0.3 nM alone or to the
combination of compound 100 plus each of the standard agents at the
same concentrations. In each case, the addition of compound 100
enhanced the effect of the cytotoxic agent at 7 days to an exten
greater than that expected from the activity of each agent used
alone. The expected percent inhibition from a combination of drugs
is calculated by multiplying the actual percent inhibition by each
drug alone and comparing that product to the actual percent
inhibition caused by the combination of the two drugs (Valeriote,
1975). The expected percent inhibition at 7 days is the product of
the inhibition by each agent alone.
[0214] For Cisplatin and Compound 100, expected inhibition at 7
days was 66% (93.5 for cisplatin alone.times.71% for compound 100
alone) versus the actual extent of inhibition by the combination of
50% (FIG. 10A).
[0215] For doxorubicin and compound 100, expected inhibition at 7
days was 53% (75.7 5 for doxorubicin alone.times.71% for compound
100 alone) versus the actual extent of inhibition by the
combination of 42.3%. (FIG. 10B)
[0216] For taxol and compound 100 expected inhibition at 7 days 80%
(114% for Taxol alone.times.71% for Compound 100 alone) versus the
actual extent of inhibition by the combination of 61% (C). (FIG.
10C)
EXAMPLE 4
The Effects of Compound 102 Combined with Temozolimide (TMZ), a
Non-Specific DNA-Methylating Drug
[0217] To determine the impact of altering DNA-damage defense
mechanisms by inhibiting PP2A on the efficacy of cytotoxic
chemotherapy, the effects of compound 102 combined with TMZ, a
non-specific DNA-methylating drug, routinely used for the
palliative treatment of GBM patients (Prados et al, 2008), were
studied. SCID mice bearing s.c. xenografts of either the GBM line
U87MG or the neuroblastoma line SH-SY5Y were treated with vehicle
alone, compound 102 alone, TMZ alone, or both drugs at the same
doses and schedules as when given alone. GBM xenografts (one in
each flank of five mice) grew rapidly in control animals, requiring
sacrifice at 3 weeks. Compound 102 alone minimally delayed growth.
TMZ alone caused complete regression for 5 weeks but with regrowth
of all xenografts requiring sacrifice of all animals by week 9. The
combination of compound 102 and TMZ also caused complete regression
of all xenografts but with delayed recurrence and regrowth in 3
animals requiring sacrifice of one mouse at 13 weeks and the other
two, at 15 weeks. Two mice, however, had no recurrence in either
flank after 7 months, suggesting their cancers had been eliminated.
A repeat study confirmed that the 2-drug combination can cause
complete regression without recurrence; in this study, three of
five animals each implanted with 2 s.c. xenografts remained disease
free for over 4 months. No evidence of drug toxicity was noted in
either experiment.
[0218] NB xenografts in control animals also grew rapidly,
requiring sacrifice at 3 weeks. Compound 102 alone completely
suppressed growth for 2 weeks with tumors subsequently growing more
slowly than controls, not reaching a size requiring sacrifice by 7
weeks. TMZ alone was less inhibitory than compound 102. The
two-drug combination, however, completely inhibited growth, with
all xenografts remaining the same size as at the start of treatment
for 7 weeks (FIG. 12C). In the three drug treatment arms, some NB
xenografts ulcerated by week 4 and all xenografts ulcerated by 7
weeks requiring sacrifice per animal care protocol. None of the
xenografts in control animals ulcerated suggesting that tissue
breakdown at the xenograft site is an effect of treatment The
mechanism responsible for the necrosis, is not known. Histologic
examination of NB xenografts 24 hours after exposure to a single
i.p. injection of vehicle or drug showed a homogeneous field of
healthy appearing tumor cells in vehicle treated animals, whereas
compound 102 alone resulted in decreased cell size and pyknotic
nuclei in .about.50% of cells; TMZ alone produced cytoplasmic
swelling and vacuolization interspersed with a few (potentially
viable) pleomorphic cells in .about.50% of cells; and compound 102
plus TMZ resulted in small pyknotic nuclei in more than 90% of
cells but without the overt necrosis present after TMZ alone (FIG.
12D.). Thus, the two-drug combination prevented the growth and
induced ulceration of the NB xenografts but did not cause complete
regression, again without apparent toxicity.
EXAMPLE 5
Effects of Compound 102 are Not Specific to the Type of DNA Damage
Caused by TMZ
[0219] The increase in tumor cell killing by compound 102 plus TMZ
raised the possibilities that inhibition of PP2A renders cells more
vulnerable to TMZ and/or less efficient in repairing DNA damage
because of impaired mitotic and/or DNA damage arrest. The effects
of compound 102, TMZ, doxorubicin (DOX), a widely used anti-cancer
drug that disrupts DNA replication, compound 102 plus TMZ, and
compound 102 plus DOX on the amount of pAkt, p53 and MDM2 in U87MG,
a cell line with wild-type p53, and in U373, a cell line with
mutant p53 (Short et al, 2007) were assessed by Western blots.
Exposure of U87MG cells to compound 102 alone for 24 hours
increased both pAkt-1 and MDM2 and eliminated p53; TMZ alone and
DOX alone decreased pAkt-1, increased p53, and had little effect on
MDM2. Adding compound 102 prevented the decrease in pAkt-1 caused
by TMZ alone or DOX alone and increased MDM2 in the face of
continued increased expression of p53 (FIG. 12E), indicating that
the effects of compound 102 are not specific to the type of DNA
damage caused by TMZ.
[0220] In vivo, SCID mice implanted with 5 million U87 cells
divided into four groups of 10 were treated starting at time 0 when
average tumor volume was approximately 60 cubic millimeters by i.p.
injection of vehicle alone (100 uL of 50% DMSO in PBS), compound
102 alone, doxorubicin alone, or compound 102 and doxorubicin at
various concentrations. Compound 102 in combination with
doxorubicin effected the same molecular changes on regulation of
cell replication as with TMZ (FIG. 13).
EXAMPLE 6
Effects of PP2A Inhibition are Not Dependent Upon the Presence of
Functional p53
[0221] The same molecular changes in pAkt-1, p53, and MDM2 induced
by compound 102, TMZ, and compound 102 plus TMZ occurred in U373
cells (FIG. 12F), indicating that the effects of PP2A inhibition
are not dependent upon the presence of functional p53. Okadaic
acid, at a concentration (2 nM) that is expected to inhibit PP2A
and not PP1 (Hart et al, 2004), mimicked the effects of compound
102 on pAkt-1 and on mutant p53 in U373 cells (FIG. 12G),
supporting the hypothesis that the effects of compound 102 result
from inhibition of PP2A. The reduction of intracellular levels of
p53 by exposure to compound 102 alone and in combination with TMZ
was confirmed by immunofluorescence staining of U87 cells (FIG.
12H).
EXAMPLE 7
Changes in Cell Cycle are Not Dependent on the Specific Action of
the DNA Damaging Agent and/or on the Presence of Functional p53
[0222] We analyzed cell cycle patterns of U87MG and U373 cells 48
hours after exposure to TMZ or DOX alone and in combination with
compound 102. In U87MG cells, exposure to TMZ alone decreased the
number of G1 phase cells, markedly increased S phase cells, and had
little effect on G2/M phase cells. Exposure to compound 102 alone
also decreased G1, modestly increased S, but prominently increased
G2/M. Exposure to either of the two-drug combinations resulted in
patterns comparable to compound 102 alone, namely decreased G1 with
greatly increased S and G2/M (FIG. 14A). Compared to U87MG cells,
control U373 cells had slightly greater G1 and smaller G2/M
compartments and a comparable S component. Compound 102 alone had
no effect on this profile. Exposure to TMZ or DOX alone reduced G1
and G2/M and greatly increased S. Exposure to either of the
two-drug combinations markedly decreased G1 and increased G2/M.
(FIG. 14B). There were some quantitative differences but the
primary effects of compound 102 combined with TMZ or with DOX were
similar in both cells lines, indicating that the changes in cell
cycle are not dependent on the specific action of the DNA damaging
agent and/or on the presence of functional p53.
[0223] Inhibition of PP2A by compound 102 triggers a chain of
alterations in cancer cell signaling that accelerates inappropriate
entry of cells into mitosis and, at the same time, impairs arrest
of cell cycle at G1 and G2M (FIG. 14C.). In the face of
chemotherapy-induced DNA damage and disordered cell replication,
compound 102 up-regulates Akt-1, which has the potential to
stimulate cell growth, and, at the same time, interferes with
p53-mediated cell cycle arrest by stabilizing MDM2 (Lopez-Pajares
et al, 2008). An increase in pAkt-1 activates Plk-1, interfering
with activation of a checkpoint at G2/M (Lei and Erikson, 2008;
Garcia-Echeverria and Sellers, 2008) and activating TCTP by
phosphorylation (Bommer and Thiele, 2004). Phosphorylation of TCTP
decreases the stabilization of microtubules (Bommer and Thiele,
2004; Yarm, 2002), which may contribute to the development of
mitotic catastrophe after exposure of cancer cells to compound 102.
It has been found, however, that in the cancer cell lines and
xenografts studied, pPlk-1 phosphorylation of TCTP results in a
marked reduction in TCTP abundance. Loss of TCTP expression during
embryogenesis increases cell death (Chen et al, 2007), presumably
by reduction of TCTP anti-apoptotic activity that is mediated by
interference with Bax dimerization in the mitochondrial membrane
(Susini et al, 2008). Loss of TCTP induced by inhibition of PP2A
may enhance cancer cell killing by the same mechanism.
[0224] The foregoing results indicate that inhibition of PP2A
increases the anti-cancer activity of TMZ to the level of cure in
up to 50% of animals implanted with GBM xenografts and completely
suppresses the growth of NB xenografts. When toxicity is not
limiting in humans, inhibition of PP2A in cancers is a general
method for improving the effectiveness of anti-cancer regimens that
target DNA and/or components of the mitotic process. The forgoing
results indicate that pharmacologic inhibition of PP2A enhances the
effectiveness of cancer treatments that damage DNA or disrupt
components of cell replication by interfering with multiple
DNA-damage defense mechanisms.
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