U.S. patent application number 13/646718 was filed with the patent office on 2013-02-14 for two-pore channels as regulators of proliferation in cancer.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. The applicant listed for this patent is Steven J. Feinmark, Richard B. Robinson. Invention is credited to Steven J. Feinmark, Richard B. Robinson.
Application Number | 20130040298 13/646718 |
Document ID | / |
Family ID | 38694482 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130040298 |
Kind Code |
A1 |
Feinmark; Steven J. ; et
al. |
February 14, 2013 |
Two-pore channels as regulators of proliferation in cancer
Abstract
The present invention relates to the discovery that two pore
K.sup.+ channel (2PK) gene expression is increased in tumors and
tumor cell lines, especially prostate tumor cells. The present
invention encompasses methods for disease diagnosis, drug screening
and the treatment of cancer.
Inventors: |
Feinmark; Steven J.;
(Haworth, NJ) ; Robinson; Richard B.; (Cresskill,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Feinmark; Steven J.
Robinson; Richard B. |
Haworth
Cresskill |
NJ
NJ |
US
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York City
NY
|
Family ID: |
38694482 |
Appl. No.: |
13/646718 |
Filed: |
October 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12299852 |
Jun 15, 2009 |
8309528 |
|
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PCT/US07/11339 |
May 9, 2007 |
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13646718 |
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60799486 |
May 10, 2006 |
|
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60835336 |
Aug 2, 2006 |
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Current U.S.
Class: |
435/6.11 ;
435/34; 435/6.12; 435/6.13; 435/7.21; 435/7.23 |
Current CPC
Class: |
C07K 14/705
20130101 |
Class at
Publication: |
435/6.11 ;
435/6.13; 435/7.21; 435/34; 435/7.23; 435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/04 20060101 C12Q001/04; G01N 33/574 20060101
G01N033/574; G01N 33/566 20060101 G01N033/566 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with Government support under
Contract No. W81XWH-06-1-0141 awarded by the Department of Defense.
The Government has certain rights in the invention.
Claims
1. A method for identifying a compound that activates TREK-1
activity comprising (i) contacting a cell expressing TREK-1 with a
test compound and measuring the level of TREK-1 activity; (ii) in a
separate experiment, contacting a cell expressing TREK-1 protein
with a vehicle control and measuring the level of TREK-1 activity
where the conditions are essentially the same as in part (i), and
then (iii) comparing the level of TREK-1 activity measured in part
(i) with the level of TREK-1 activity measured in part (ii),
wherein an increased level of TREK-1 activity measured in part (i)
compared to the level measured in part (ii) indicates that the test
compound is a TREK-1 activator.
2. A method for identifying a compound that inhibits TREK-1
activity comprising (i) contacting a cell expressing TREK-1 protein
with a test compound and measuring the level of TREK-1 activity,
(ii) in a separate experiment, contacting a cell expressing TREK-1
protein with a vehicle control and measuring the level of TREK-1
activity, where the conditions are essentially the same as in part
(i) and then (iii) comparing the level of TREK-1 activity measured
in part (i) with the level of TREK-1 activity measured in part
(ii), wherein a decreased level of TREK-1 activity in part (i)
compared to the level measured in part (ii) indicates that the test
compound is a TREK-1 inhibitor.
3. A method for identifying a compound that inhibits proliferation
of cells expressing TREK-1 activity comprising (i) contacting a
cell expressing TREK-1 protein with a test compound and measuring
the level of cell proliferation, (ii) in a separate experiment,
contacting a cell expressing TREK-1 protein with a vehicle control
and measuring the level of cell proliferation, where the conditions
are essentially the same as in part (i) and then (iii) comparing
the level of cell proliferation measured in part (i) with the level
of cell proliferation measured in part (ii), wherein a decreased
level of cell proliferation measured in part (i) compared to the
level measured in part (ii) indicates that the test compound is an
inhibitor of cell proliferation.
4. A method for diagnosis and/or prognosis of cancer in a subject
comprising: (a) detecting the level of TREK-1 expression in a
sample derived from a subject; and (b) comparing the level of
TREK-1 expression detected in the subject's sample to the level of
TREK-1 expression detected in a control sample, wherein an increase
in the level of TREK-1 expression detected in the subject's sample
as compared to a control sample is an indicator of a subject with
cancer or progression toward cancer.
5. The method of claim 1 wherein the level of TREK-1 RNA expression
is measured.
6. The method of claim 1 wherein the level of TREK-1 protein
expression is measured.
7. The method of claim 3 wherein the level of TREK-1 protein
expression is measured using an immunoassay.
8. The method of claim 1 wherein an increase in the level of TREK-1
expression detected in the subject's sample as compared to a
control sample is an indicator of a subject with cancer or
progression toward cancer.
9. A method for monitoring the therapeutic effect of a cancer
treatment on a subject comprising: measuring at time intervals
before, during, or after cancer treatment the amount of a TREK-1
expression wherein a change or absence of change in the amount of
the TREK-1 gene expression is correlated with the effect of the
cancer treatment on the subject.
10. The method of claim 5 wherein a decrease in the level of TREK-1
expression during or after cancer treatment as compared to the
level of TREK-1 expression before treatment is correlated with a
positive response to the treatment.
11. The method of claim 6 wherein the level of TREK-1 RNA
expression is measured.
12. The method of claim 6 wherein the level of TREK-1 protein
expression is measured
13. The method of claim 9 wherein the level of TREK-1 protein
expression is measured using an immunoassay.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority as a divisional
application to application Ser. No. 12/299,852, filed Nov. 6, 2008
under 35 U.S.C. .sctn.121, which is a national phase (371)
application of international application PCT/US2007/011339, filed
May 9, 2007 which claims benefit of priority to provisional
applications, 60/835,336 filed Aug. 2, 2006 and 60/799,486 filed
May 10, 2006 under 35 U.S.C. .sctn.119(e), the entire contents of
which are hereby incorporated by reference as if fully set forth
herein.
1. INTRODUCTION
[0003] The present invention relates to the discovery that two pore
K.sup.+ channel (2PK) gene expression is increased in tumor cell
lines, especially prostate tumor cells. The present invention
encompasses methods for disease diagnosis, drug screening and the
treatment of cancer.
2. BACKGROUND OF INVENTION
[0004] Cancer is a disease marked by the uncontrolled growth of
abnormal cells. Cancer cells have overcome the natural controls
imposed in normal cells, which have a finite lifespan. As the
growth of cancer cells continues the cancerous cell may develop a
more aggressive growth phenotype. If left untreated, metastasis,
the spread of cancer cells to distant areas of the body by way of
the lymph system or bloodstream, may ensue, destroying healthy
tissue. Carcinoma of the prostate (PCA) is the most frequently
diagnosed cancer in men in the United States, and is the second
leading cause of male cancer deaths (Karp et al., 1996, Cancer Res.
56:5547-5556).
[0005] It would therefore be beneficial to provide methods and
reagents for the diagnosis, staging, prognosis, monitoring, and
treatment of cancers.
3. SUMMARY OF THE INVENTION
[0006] The present invention relates to methods and compositions
for modulating the activity of the two-pore domain K.sup.+ channels
as a means for modulating the proliferation of cancer cells.
Specifically, the present invention relates to methods and
compositions for modulating the activity of the TREK-1 two-pore
domain K.sup.+ channels ("TREK-1") as a means for modulating the
proliferation of prostate cancer cells. Such modulation can be used
to reduce or inhibit the proliferation of prostate tumor cells. The
invention is based on the discovery that TREK-1 is over expressed
in human prostate cancer cells as compared to normal prostate
cells, as well as in breast, colon and bladder cancer cells.
Moreover, inhibition of TREK-1 activity was found to inhibit the
proliferation of tumor cells.
[0007] The present invention further relates to methods for the
diagnostic evaluation and prognosis of cancer, especially prostate
cancer. For example, TREK-1 nucleic acid molecules can be used as
diagnostic hybridization probes or as primers for diagnostic PCR
analysis for detection of abnormal levels of expression of the
TREK-1 gene. Antibodies to TREK-1 gene product can be used in a
diagnostic test to detect the level of TREK-1 gene product in
tissue samples. In specific embodiments, measurement of TREK-1 gene
product levels can be made to detect or stage cancer, especially
prostate cancer.
[0008] Still further, the present invention relates to screening
assays that utilize the TREK-1 gene and/or TREK-1 gene product for
the identification of compounds which modulate TREK-1 gene
expression and/or the activity of TREK-1 gene products. In a
preferred embodiment of the invention, the compound is one that is
capable of inhibiting the activity of TREK-1 and effectively
reducing or inhibiting the proliferation of cancer cells. Such
compounds can be used as agents to prevent and/or treat cancer.
Such compounds can also be used to palliate the symptoms of the
disease, and control the metastatic potential of the cancer.
[0009] Specifically, the present invention provides a method for
identifying a compound that activates TREK-1 activity comprising
(i) contacting a cell expressing TREK-1 with a test compound and
measuring the level of TREK-1 activity; (ii) in a separate
experiment, contacting a cell expressing TREK-1 protein with a
vehicle control and measuring the level of TREK-1 activity where
the conditions are essentially the same as in part (i), and then
(iii) comparing the level of TREK-1 activity measured in part (i)
with the level of TREK-1 activity in part (ii), wherein an
increased level of TREK-1 activity in the presence of the test
compound indicates that the test compound is a TREK-1
activator.
[0010] The invention also provides a method for identifying a
compound that inhibits TREK-1 activity comprising (i) contacting a
cell expressing TREK-1 protein with a test compound and measuring
the level of TREK-1 activity, (ii) in a separate experiment,
contacting a cell expressing TREK-1 protein with a vehicle control
and measuring the level of TREK-1 activity, where the conditions
are essentially the same as in part (i) and then (iii) comparing
the level of TREK-1 activity measured in part (i) with the level of
TREK-1 activity in part (ii), wherein a decrease level of TREK-1
activity in the presence of the test compound indicates that the
test compound is a TREK-1 inhibitor.
[0011] In yet another embodiment of the invention, a method for
identifying a compound that inhibits proliferation of cells
expressing TREK-1 activity comprising (i) contacting a cell
expressing TREK-1 protein with a test compound and measuring the
level of cell proliferation, (ii) in a separate experiment,
contacting a cell expressing TREK-1 protein with a vehicle control
and measuring the level of cell proliferation, where the conditions
are essentially the same as in part (i) and then (iii) comparing
the level of cell proliferation measured in part (i) with the level
of cell proliferation in part (ii), wherein a decrease level of
cell proliferation in the presence of the test compound indicates
that the test compound is an inhibitor of cell proliferation.
[0012] The invention further provides pharmaceutical compositions
comprising a biologically active agent that modulates the activity
of TREK-1 in combination with a pharmaceutically acceptable
carrier.
4. BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. TREK-1 is expressed in prostate cancer cell lines
but not in normal prostate epithelium. Cell lysates were prepared
from LNCaP (lane 1), PC-3 (lane 2) and normal prostate epithelial
(lane 3) cell cultures and fractionated by SDS-PAGE. The proteins
were blotted to nitrocellulose and detected by ECL according to the
manufacturer's instructions with a polyclonal rabbit anti-TREK-1
primary antibody (Alomone). Beta-actin was measured as a loading
control on the same blots. These results are typical of two
experiments.
[0014] FIG. 2. PC-3 cells express TREK-1 current which can be
"knocked-out" by over-expression of a dominant-negative mutant
(dn-TREK). Panel A. TREK-1 current is defined as the
sipatrigine-sensitive current and is measured using a ramp
protocol. A series of 800 ms steps from -120 mV to +70 mV
(increasing 10 mV each step) from a holding potential of -20 mV are
applied. To eliminate contamination by Na.sup.+ current a prestep
to -90 mV (40 ms) is applied before each step. Sipatrigine (50
.mu.M) is applied by superfusion for 3 min while recordings are
taken every 30 s. The traces reported in the figure are the result
of the subtraction of the control current from the current in the
presence of sipatrigine at steady state. Panel B. PC-3 cells were
co-transfected with dn-TREK and pEGFP-C1 plasmids. 48 h later,
dn-TREK cells were identified by green fluorescence and recordings
were made as described above. Panel C. Data from numerous cells are
summarized in the bar graph.
[0015] FIG. 3. Dominant-negative TREK-1 reduces proliferation of
PC-3 cells. PC-3 cells were transfected with either dn-TREK (or a
control vector (pEGFP) using GeneJammer (Stratagene) under standard
conditions. Proliferation of these cells was measured using the MTT
assay after the cells were plated at 1.times.10.sup.5 cells/well in
96 well plates. Data are presented as the mean.+-.SEM of five
experiments. * p<0.05 vs control and EGFP FIG. 4. Normal
prostate epithelial cells (NPE) express no TREK-1 current but a
TREK-1 virus can be used to over-express the channel. Current was
measured in cultured NPE by patch-clamp recording as described in
FIG. 1. Panel A. TREK-1 current is not detectable in NPE. Panel B.
NPE were infected with a TREK-1 bearing adenovirus and current was
recorded. Panel C. Summary of numerous trials.
[0016] FIG. 5. Expression of TREK-1 increases proliferation of
normal prostate epithelial cells (NPE). Normal prostate epithelial
cell cultures were obtained from Clonetics and infected with a
TREK-1-containing adenovirus. Some virally infected cells were also
treated with sipatrigine (10 .mu.M), a TREK-1 blocker. Cell
proliferation was assessed using the MTT assay as described in FIG.
3. These data are presented as mean.+-.SEM from five paired
experiments. * p<0.05, the NPE/TREK-1 group differed
significantly from each of the other treatments and no other groups
were different.
[0017] FIG. 6 Immunohistochemical staining of human prostate tissue
reveals that TREK-1 is over-expressed in cancer. Human tissue
samples were stained with rabbit polyclonal anti-human TREK-1
antibody (Alomone). Panels A and B show a low power and high power
view of the same field. Under the low power, a normal gland is
visible (center right). The epithelial cells lining the luminal
surface are very lightly stained. An adjacent cancerous gland
(center left) shows a very small luminal space and the epithelial
cells lining it are very densely stained indicating an
overexpression of TREK-1. The same features are visible under
higher power in Panel B.
[0018] FIG. 7. Meclofenamate activates TREK-1 current in CHO cells
that heterologously express the channel. CHO cells were transfected
with a plasmid encoding human TREK-1 and the current was studied by
patch clamp. The current-voltage relation was determined using a
ramp protocol that went from -130 mV to +40 mV in 6 s (after
correction for the junction potential). Current was greater in the
presence of meclofenamate. This is typical of 7 cells.
[0019] FIG. 8. Trifluoperazine inhibits TREK-1 current in CHO cells
that heterologously express the channel. CHO cells were transfected
with a plasmid encoding human TREK-1 and the current was studied by
patch clamp. The current-voltage relation was determined using a
ramp protocol that went from -130 mV to +40 mV in 6 s (after
correction for the junction potential). Current was less in the
presence of trifluoperazine. This is typical of 3 cells.
[0020] FIG. 9. Structure of ONO-RS-082, BML263 and BLM 264.
[0021] FIG. 10. TREK-1 expression increases proliferation in CHO
cells which significantly correlates with current. CHO cells were
transfected with TREK-1 using standard methods. Transfected cells
were exposed to sipatrigine in doses from 0 to 100 uM and then
tested for proliferation and for TREK-1 current by methods
described elsewhere in this application. The percent of control
proliferation, as measured by the MTT assay, was plotted against
the inhibition of TREK-1 current, as measured by patch clamp
analysis. The data were then analyzed by linear regression which
shows a significant correlation between the rate of growth and the
expression of current in these cells (p<0.0001).
[0022] FIG. 11. TREK-1 stable over-expressor cell lines have a
significantly higher proliferation rate than control CHO cells.
Several clones of CHO cells that over-express TREK-1 have been
isolated as described elsewhere. Each clone was tested for the
expression of current (right panel) and for its proliferation rate
(left panel).
[0023] FIG. 12. TREK-1 over-expression in CHO cells significantly
increases the number of cells in S-phase. CHO cells were
transfected with TREK-1 by standard methods and then fixed for cell
cycle analysis as described elsewhere. Cells were analyzed by FACS
analysis and compared to control CHO cells, TREK-1 expressing cells
had a significant shift from G0/G1 to S and G2/M.
[0024] FIG. 13. TREK-1 over-expression in CHO cells promotes
anchorage independent growth. Stable TREK-1 over-expressors were
tested for growth in soft agar as described elsewhere. Top panel
shows the absence of colonies formed by control cells (left) and
the presence of many colonies (red spots) formed by clones 6
(middle) and 10 (right). A micrographic analysis of some of these
colonies is shown in the bottom panel. Control cells can be seen
(left) but these cells, while alive, are not dividing.
Over-expressors, however, continue to grow even when suspended in
agar and a few examples of colonies formed of many cells are shown
(middle and right).
5. DETAILED DESCRIPTION OF THE INVENTION
[0025] Described herein is the discovery that the expression of
two-pore K.sup.+ channels is increased in tumor cells as compared
to normal cells. The methods and compositions of the invention may
be used for disease diagnosis, drug screening and treatment of
cancer. The invention is described in detail in the subsections
below.
[0026] 5.1. Diagnostic Methods
[0027] In various embodiments, the present invention provides a
variety of methods for the diagnostic and prognostic evaluation of
cancer. Such methods may, for example, utilize reagents such as the
TREK-1 gene nucleotide sequences and antibodies directed against
TREK-1 gene products.
[0028] Specifically, such reagents may be used to detect the level
of TREK-1 expression, for example, for: (1) the detection of
over-expression of TREK-1 gene mRNA relative to normal cells;
and/or (2) the detection of an over-abundance of TREK-1 gene
product relative to normal cells, each of which correlates with
cancer or a progression toward cancer or metastasis.
[0029] Thus, the present invention provides a method for diagnosis
and/or prognosis of cancer in a subject comprising: (a) detecting
the level of TREK-1 expression in a sample derived from a subject;
and (b) comparing the level of TREK-1 expression detected in the
subject's sample to the level of TREK-1 expression detected in a
control sample, wherein an increase in the level of TREK-1
expression detected in the subject's sample as compared to a
control sample is an indicator of a subject with cancer or
progression toward cancer.
[0030] The methods described herein may be applied to samples of
cells or cellular materials taken directly from a patient. Any
method known in the art for collection or isolation of the desired
cells or materials can be used. In particular, for prostate, as
well as breast, colon and bladder cancer, samples for testing may
be obtained by techniques known in the art.
[0031] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic test kits comprising at least
one specific TREK-1 gene nucleic acid or anti-TREK-1 gene product
antibody reagent, which may be conveniently used, e.g., in clinical
settings or in home settings, to diagnose patients exhibiting
preneoplastic or neoplastic abnormalities, and to screen and
identify those individuals exhibiting a predisposition to such
neoplastic changes.
[0032] The present invention is useful for the diagnosis and
prognosis of malignant diseases in which the TREK-1 gene or gene
product is implicated or is suspected to be implicated. Such
malignancies include but are not limited to cancer of the prostate
gland, as well as cancer of the breast, colon and bladder. Nucleic
acid-based detection techniques are described, below. Peptide
detection techniques are described, below.
[0033] Expression levels of the TREK-1 gene can be detected by
utilizing a number of techniques. For the detection of TREK-1
transcripts, any cell type or tissue in which the TREK-1 gene is
expressed, such as, for example, prostate cancer cells, including
metastases, may be utilized.
[0034] In a preferred embodiment of the invention, quantitative
aspects of TREK-1 gene expression are assayed. For example, RNA
from a cell type or tissue known, or suspected, to express the
TREK-1 gene, such as prostate cancer cells, including metastases,
may be isolated and tested utilizing hybridization or PCR
techniques. Diagnostic methods for the detection of aberrant TREK-1
gene expression can include hybridization techniques which involve
for example, contacting and incubating nucleic acids, including RNA
molecules obtained from a sample, e.g., derived from a patient
sample, with one or more labeled TREK-1 nucleic acid reagents under
conditions favorable for the specific annealing of these reagents
to their complementary sequences within the TREK-1 RNA molecule.
Preferably, the lengths of these TREK-1 nucleic acid reagents are
at least 15 to 30 nucleotides. After incubation, all non-annealed
nucleic acids are removed from the nucleic acid:TREK-1 molecule
hybrid. The presence of nucleic acids which have hybridized, if any
such molecules exist, is then detected. Using such a detection
scheme, the nucleic acid from the cell type or tissue of interest
can be immobilized, for example, to a solid support such as a
membrane, or a plastic surface such as that on a microtitre plate
or polystyrene beads. In this case, after incubation, non-annealed,
labeled nucleic acid reagents are easily removed. Detection of the
remaining, annealed, labeled TREK-1 nucleic acid reagents is
accomplished using standard techniques well-known to those in the
art. The TREK-1 gene sequences to which the nucleic acid reagents
have annealed can be compared to the annealing pattern expected
from a normal TREK-1 gene expressing cell in order to determine
whether the TREK-1 gene is over-expressed.
[0035] In another embodiment of the invention, RT-PCR techniques
can be utilized to detect differences in levels of TREK-1
transcripts which may be due to normal or abnormal alternative
splicing. TREK-1 nucleic acid sequences may be derived by
performing PCR using two oligonucleotide primers designed on the
basis of the TREK-1 nucleotide sequences disclosed herein. The
template for the reaction may be cDNA obtained by reverse
transcription of mRNA prepared from cell lines or tissue known to
express TREK-1, i.e, prostate tumor cells.
[0036] Mammalian TREK-1 sequences that may be used in the design of
hybridization probes and/or PCR primers include, for example, those
disclosed in Genebank accession number is NM001017424, Fink et al.
(1996, EMBO Journal 15:6854-6862) or Meadows et al., (2000,
Pflugers Arch. 439:714-22). The disclosures of these publications
in their entireties are hereby incorporated by reference into this
application
[0037] Additionally, it is possible to perform such TREK-1 gene
expression assays "in situ", i.e., directly upon tissue sections
(fixed and/or frozen) of patient tissue obtained from biopsies or
resections, such that no nucleic acid purification is necessary.
Nucleic acid reagents such as those described above may be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols
And Applications", Raven Press, N.Y.).
[0038] Antibodies, and fragments thereof, directed against TREK-1
gene product may also be used as diagnostics and prognostics, as
described herein. Such diagnostic methods may be used to detect
abnormalities in the level of TREK-1 expression. Such antibodies
and fragments thereof include, but are not limited to, naturally
occurring antibodies, bivalent fragments such as (Fab').sub.2,
monovalent fragments such as Fab, single chain antibodies, single
chain Fv (scFv), single domain antibodies, multivalent single chain
antibodies, diabodies, triabodies, and the like that bind
specifically with antigens.
[0039] Antibodies, and fragments thereof, to be used in the
diagnostic and prognostic methods of the invention are those that
bind specifically to an epitope of a mammalian TREK-1 protein. Such
TREK-1 proteins include, for example, those having the amino acid
sequences disclosed in Genebank accession number is NM 001017424,
Fink et al. (1996, EMBO Journal 15:6854-6862) or Meadows et al.,
(2000, Pflugers Arch. 439:714-22). For the diagnostic and
prognostic methods of the invention described below, a directly
labeled anti-TREK-1 antibody may be utilized. Alternatively, an
unlabeled anti-TREK-1 antibody may be utilized followed by indirect
labeling of the antibody with an anti-Ig antibody.
[0040] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the TREK-1
gene, such as, for example, prostate, breast, colon or bladder
cancer cells or metastatic cells. The protein isolation methods
employed herein may, for example, be such as those described in
Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A
Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.), which is incorporated herein by reference in
its entirety.
[0041] Preferred diagnostic methods for the detection of TREK-1
gene product may involve, for example, immunoassays wherein the
TREK-1 gene products are detected by their interaction with an
anti-TREK-1 gene product-specific antibody. For example,
antibodies, or fragments of antibodies useful in the present
invention may be used to quantitatively detect the presence of
TREK-1 gene product. The antibodies (or fragments thereof) useful
in the present invention may, additionally, be employed
histologically, as in immunofluorescence or immunoelectron
microscopy, for in situ detection of TREK-1 gene products. In situ
detection may be accomplished by removing a histological specimen
from a patient, such as paraffin embedded sections of prostate
tissue and applying thereto a labeled antibody of the present
invention. The antibody (or fragment) is preferably applied by
overlaying the labeled antibody (or fragment) onto a biological
sample. It may also be desirable to introduce the antibody inside
the cell, for example, by making the cell membrane permeable.
Through the use of such a procedure, it is possible to determine
not only the presence of the TREK-1 gene product but also its
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0042] Immunoassays for TREK-1 gene product will typically comprise
incubating a sample, such as a biological fluid, a tissue extract,
freshly harvested cells, or lysates of cells, in the presence of an
antibody capable of identifying TREK-1 gene products and detecting
the bound antibody by any of a number of techniques well-known in
the art.
[0043] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled TREK-1 gene specific antibody. The solid
phase support may then be washed with the buffer a second time to
remove unbound antibody. The amount of bound label on solid support
may then be detected by conventional means.
[0044] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0045] The binding activity of anti-TREK-1 gene product antibody
may be determined according to well known methods. Those skilled in
the art will be able to determine operative and optimal assay
conditions for each determination by employing routine
experimentation.
[0046] One of the ways in which the TREK-1 peptide-specific
antibody can be detectably labeled is by linking the same to an
enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic
Horizons 2:1-7, Microbiological Associates Quarterly Publication,
Walkersville, Md.); Voller et al., 1978, J. Clin. Pathol.
31:507-520; Butler 1981, Meth. Enzymol. 73:482-523; Maggio, E.
(ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.;
Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin,
Tokyo). The enzyme which is bound to the antibody will react with
an appropriate substrate, preferably a chromogenic substrate, in
such a manner as to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorimetric or by
visual means. Enzymes which can be used to detectably label the
antibody include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0047] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect TREK-1
protein through the use of a radioimmunoassay (RIA) (see, for
example, Weintraub, B., Principles of Radioimmunoassays, Seventh
Training Course on Radioligand Assay Techniques, The Endocrine
Society, March, 1986, which is incorporated by reference herein).
The radioactive isotope can be detected by such means as the use of
a gamma counter or a scintillation counter or by
autoradiography.
[0048] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0049] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0050] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0051] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0052] In various embodiments, the present invention provides the
measurement of TREK-1 gene product, and the uses of such
measurements in clinical applications. The measurement of TREK-1
gene product can be valuable in detecting and/or staging cancer in
a subject, in screening of cancer in a population, in differential
diagnosis of the physiological condition of a subject, and in
monitoring the effect of a therapeutic treatment on a subject
[0053] In an embodiment of the present invention, measurements of
TREK-1 gene expression can be used to stage the cancer in a
subject. Staging refers to the grouping of patients according to
the extent of their disease. Staging is useful in choosing
treatment for individual patients, estimating prognosis, and
comparing the results of different treatment programs. Staging of
cancer is performed initially on a clinical basis, according to the
physical examination and laboratory radiologic evaluation.
[0054] Any immunoassay, such as those described above, can be used
to measure the amount of TREK-1 gene expression which is compared
to a baseline level. This baseline level can be the amount which is
established to be normally present in the tissue or body fluid of
subjects with various degrees of the disease or disorder. An amount
present in the tissue or body fluid of the subject which is similar
for detection of the amplified product, the nucleic acid
amplification may be performed using radioactively or
non-radioactively labeled nucleotides. Alternatively, enough
amplified product may be made such that the product may be
visualized by standard ethidium bromide staining or by utilizing
any other suitable nucleic acid staining method. So a standard
amount, established to be normally present in the tissue or body
fluid of the subject during a specific stage of cancer, is
indicative of the stage of the disease in the subject. The baseline
level could also be the level present in the subject prior to the
onset of disease or the amount present during remission of the
disease.
[0055] In specific embodiments of this aspect of the invention,
measurements of levels of the TREK-1 gene product can be used in
the detection of prostate cancer or the presence of metastases or
both. In yet another embodiment of the invention, measurements of
levels of the TREK-1 gene product can be used in the detection
breast, colon and bladder cancer.
[0056] The present invention also provides a method for monitoring
the effect of a therapeutic treatment on a subject who has
undergone the therapeutic treatment. TREK-1 gene product can be
identified and detected in cancer patients with different
manifestations of disease, providing a sensitive assay to monitor
therapy. The therapeutic treatments which may be evaluated
according to the present invention include but are not limited to
radiotherapy, surgery, chemotherapy, vaccine administration,
endocrine therapy, immunotherapy, and gene therapy, etc.
[0057] The method of the invention comprises measuring at suitable
time intervals before, during, or after therapy, the amount of a
TREK-1 gene expression. Any change or absence of change in the
amount of the TREK-1 gene expression can be identified and
correlated with the effect of the treatment on the subject, such
as, for example, a reduction of the transformed phenotype in cancer
cells.
[0058] In a preferred aspect, the approach that can be taken is to
determine the levels of TREK-1 gene expression levels at different
time points and to compare these values with a baseline level. The
baseline level can be either the level of the marker present in
normal, disease free individuals; and/or the levels present prior
to treatment, or during remission of disease, or during periods of
stability. These levels can then be correlated with the disease
course or treatment outcome. Elevated levels of TREK-1 gene
expression relative to the baseline level indicates a poor response
to treatment.
[0059] 5.2. Screening Assays for Compounds that Modulate TREK-1
Activity
[0060] The present invention further provides methods for the
identification of compounds that may, through their interaction
with the TREK-1 gene or TREK-1 gene product, affect the onset,
progression and metastatic spread of cancer; especially prostate
cancer.
[0061] The following assays are designed to identify: (i) compounds
that bind to TREK-1 gene products; (ii) compounds that bind to
other intracellular proteins that interact with a TREK-1 gene
product; and (iii) compounds that modulate the activity of TREK-1
gene (i.e., modulate the level of TREK-1 gene expression and/or
modulate the level of TREK-1 gene product activity). Compounds
identified via assays such as those described herein may be useful,
for example, in elaborating the biological functions of the TREK-1
gene product, and for ameliorating symptoms of cancer. It is to be
noted that the compositions of the invention include pharmaceutical
compositions comprising one or more of the compounds identified via
such methods. Such pharmaceutical compositions can be formulated,
for example, as discussed, below.
[0062] Assays may be utilized which identify compounds which bind
to TREK-1 gene regulatory sequences (e.g., promoter sequences) and
which may modulate the level of TREK-1 gene expression. Such
methods for identifying compounds that modulate TREK-1 gene
expression, comprise, for example: (a) contacting a test compound
with a cell or cell lysate containing a reporter gene operatively
associated with a TREK-1 gene regulatory element; and (b) detecting
expression of the reporter gene product. Also provided is another
method for identifying compounds that modulate TREK-1 gene
expression comprising: (a) contacting a test compound with a cell
containing TREK-1 transcripts; and (b) detecting the translation of
the TREK-1 transcript. Any reporter gene known in the art can be
used, such as but not limited to, green fluorescent protein,
.beta.-galactosidase, alkaline phosphatase, chloramphenicol
acetyltransferase, etc.
[0063] In yet another embodiment of the invention, in vitro systems
may be designed to identify compounds capable of interacting with,
e.g., binding to, the TREK-1 gene product. Such compounds may be
useful, for example, in modulating the activity of TREK-1 gene
product, in elaborating the biological function of the TREK-1 gene
product, or may be utilized in screens for identifying compounds
that disrupt normal TREK-1 gene product interactions, or may in
themselves disrupt such interactions.
[0064] The principle of the assays used to identify compounds that
interact with the TREK-1 gene product involves preparing a reaction
mixture of the TREK-1 gene product, or fragments thereof and the
test compound under conditions and for a time sufficient to allow
the two components to interact with, e.g., bind to, thus forming a
complex, which can represent a transient complex, which--can be
removed and/or detected in the reaction mixture. These assays can
be conducted in a variety of ways. For example, one method to
conduct such an assay would involve anchoring TREK-1 gene product
or the test substance onto a solid phase and detecting TREK-1 gene
product/test compound complexes anchored on the solid phase at the
end of the reaction. In one embodiment of such a method, the TREK-1
gene product or fragment thereof may be anchored onto a solid
surface, and the test compound, which is not anchored, may be
labeled, either directly or indirectly.
[0065] In yet another embodiment of the invention, displacement
assays may be used to identify compounds that interact with the
TREK-1 gene product, or fragments thereof. The assay is based on
the ability of such compounds to displace or preventing binding of
compounds known to interact with the TREK-1 gene product or
fragments thereof.
[0066] The basic principle of the displacement assay system used to
identify compounds that interact with the TREK-1 gene product or
fragments thereof involves preparing a reaction mixture containing
the TREK-1 gene product, or fragments thereof, and the compound
know to bind to TREK-1 under conditions and for a time sufficient
to allow the two to interact and bind, thus forming a complex. In
order to test a compound for inhibitory activity, the reaction
mixture is prepared in the presence and absence of the test
compound. The test compound may be initially included in the
reaction mixture, or may be added at a time subsequent to the
addition of TREK-1 gene product and its intracellular interacting
partner. Control reaction mixtures are incubated without the test
compound or with a placebo. The formation of any complexes between
the TREK-1 gene product or fragments thereof and the compound known
to bind to TREK-1 is then detected. The formation of a complex in
the control reaction, but not in the reaction mixture containing
the test compound, indicates that the compound interferes with the
interaction of the TREK-1 gene product and the compound known to
bind to TREK-1.
[0067] The assay for compounds that interfere with the interaction
of the TREK-1 gene product and compounds known to bind to TREK-1
can be conducted in a heterogeneous or homogeneous format.
Heterogeneous assays involve anchoring either the TREK-1 gene
product or the compound known to bind to TREK-1 onto a solid phase
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the TREK-1 gene products and the
compounds known to bind to TREK-1, e.g., by competition, can be
identified by conducting the reaction in the presence of the test
substance; i.e., by adding the test substance to the reaction
mixture prior to or simultaneously with the TREK-1 gene protein and
compound known t bind to TREK-1. Alternatively, test compounds that
disrupt preformed complexes, e.g. compounds with higher binding
constants that displace one of the components from the complex, can
be tested by adding the test compound to the reaction mixture after
complexes have been formed.
[0068] In a specific embodiment of the invention, compounds know to
bind to TREK-1, that may be used in the practice of the invention
include, for example, ONO-RS-082, BML263 and BLM 264 (FIG. 9). To
facilitate the detection of such compounds, the compounds may be
radioactively or fluorescently labeled.
[0069] In a specific embodiment of the invention, membrane
preparations derived from cells known to express TREK-1, or
genetically engineered to express TREK-1, may be used in the
displacement assays of the invention. In yet another embodiment of
the invention, membrane preparations may be derived from tissues
derived from transgenic animals engineered to express TREK-1.
[0070] In practice, microtitre plates may conveniently be utilized
as the solid phase. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0071] In order to conduct the assay, the nonimmobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously nonimmobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously nonimmobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0072] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for TREK-1 gene product or the test compound to anchor any
complexes formed in solution, and a labeled antibody specific for
the other component of the possible complex to detect anchored
complexes.
[0073] In another embodiment of the invention, assays may be
utilized to identify intracellular proteins that interact with the
TREK-1 gene product. Any method suitable for detecting
protein-protein interactions may be employed for identifying TREK-1
protein-intracellular protein interactions. Among the traditional
methods which may be employed are co-immunoprecipitation,
crosslinking and co-purification through gradients or
chromatographic columns. Utilizing procedures such as these allows
for the isolation of intracellular proteins which interact with
TREK-1 gene product. Once isolated, such an intracellular protein
can be identified and can, in turn, be used, in conjunction with
standard techniques, to identify additional proteins with which it
interacts.
[0074] Assays may also be utilized to identify compounds that
interfere with TREK-1 gene product/intracellular macromolecular
interactions. TREK-1 gene product may, in vivo, interact with one
or more intracellular macromolecules, such as proteins and nucleic
acid molecules. For purposes of this discussion, such intracellular
macromolecules are referred to herein as "interacting partners."
Compounds that disrupt TREK-1 interactions in this way may be
useful in regulating the activity of the TREK-1 gene product.
[0075] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between the TREK-1
gene product and its intracellular interacting partner or partners
involves preparing a reaction mixture containing the TREK-1 gene
product, or fragments thereof, and the interacting partner under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence and absence of the test compound. The test compound may be
initially included in the reaction mixture, or may be added at a
time subsequent to the addition of TREK-1 gene product and its
intracellular interacting partner. Control reaction mixtures are
incubated without the test compound or with a placebo. The
formation of any complexes between the TREK-1 gene product or
fragments thereof and the intracellular interacting partner is then
detected. The formation of a complex in the control reaction, but
not in the reaction mixture containing the test compound, indicates
that the compound interferes with the interaction of the TREK-1
gene product and the interacting partner.
[0076] The assay for compounds that interfere with the interaction
of the TREK-1 gene product and interacting partners can be
conducted in a heterogeneous or homogeneous format. Heterogeneous
assays involve anchoring either the TREK-1 gene product or the
binding partner onto a solid phase and detecting complexes anchored
on the solid phase at the end of the reaction. In homogeneous
assays, the entire reaction is carried out in a liquid phase. In
either approach, the order of addition of reactants can be varied
to obtain different information about the compounds being tested.
For example, test compounds that interfere with the interaction
between the TREK-1 gene products and the interacting partners,
e.g., by competition, can be identified by conducting the reaction
in the presence of the test substance; i.e., by adding the test
substance to the reaction mixture prior to or simultaneously with
the TREK-1 gene protein and intracellular interacting partner.
Alternatively, test compounds that disrupt preformed complexes,
e.g. compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been
formed.
[0077] In yet another embodiment of the invention, cell-based
assays may be used for identification of compounds which modulate
TREK-1 activity and which may be used in treating cancer by
modulating TREK-1 activity. Specifically, such assays identify
compounds which affect TREK-1-dependent processes, such as but not
limited to the manifestation of a transformed phenotype, i.e,
changes in cell morphology, cell division, differentiation,
adhesion, motility, or phosphorylation, dephosphorylation of
cellular proteins. Other TREK-1-dependent processes which may be
affected include but are not limited to stimulation of K+ channel
activity. For example, changes in channel activity may be measured
by changes in net current by patch clamp recording or changes in
resting membrane potential. Compounds identified via such methods
can, for example, be utilized in methods for treating cancer and
metastasis.
[0078] In an embodiment, cell-based assays are based on expression
of the TREK-1 gene product in a mammalian cell and measuring the
TREK-1-dependent process. Any mammalian cell that can express the
TREK-1 gene and allow the functioning of the TREK-1 gene product
can be used, in particular, cancer cells derived from the prostate,
such as PC-3 and LNCaP may be used. Other cancer cell lines such as
those derived from breast, lung, colon, or other epithelial-derived
cancers, may also be used provided that a detectable TREK-1 gene
product is produced. Additionally, cells may be recombinantly
engineered to express the TREK-1 gene using methods well known to
those of skill in the art. In these assays, cells producing
functional TREK-1 gene products are exposed to a test compound for
an interval sufficient for the compound to modulate the activity of
the TREK-1 gene product. The activity of TREK-1 gene product can be
measured directly or indirectly through the detection or
measurement of TREK-1-dependent cellular processes such as, for
example, the manifestation of a transformed phenotype. As a
control, a cell not producing the TREK-1 gene product may be used
for comparisons. Depending on the cellular process, any techniques
known in the art may be applied to detect or measure it.
[0079] As disclosed above, over-expression of TREK-1 can result in
an increase in cell proliferation. In contrast, decreasing the
TREK-1 mediated current may slow proliferation. Accordingly, the
present invention provides methods for identifying modulators of
TREK-1 activity based on cell proliferation assays. For example,
TREK-1 expressing cells may be grown in a 96-well plate and exposed
to varying concentrations of a test substance for 4-24 h followed
by measurement of cell proliferation.
[0080] Cells that may be utilized in the proliferation assays of
the invention include cells over-expressing TREK-1 wherein said
over-expression results in an increase in cell proliferation. Such
cells include cells that naturally over-express TREK-1 as well as
cells genetically engineered to overexpress TREK-1. Cells include,
for example, PC-3 cells as well as CHO cells transfected with the
TREK-1 gene. As demonstrated herein, CHO cells over-expressing
TREK-1 have increased proliferation rates and acquire a tumorigenic
phenotype, i.e., they exhibit anchorage independent growth. Thus,
in a preferred embodiment of the invention genetically engineered
CHO cells may be used in the proliferation assays of the
invention.
[0081] Methods of measuring cell proliferation are well known in
the art and most commonly include determining DNA synthesis
characteristic of cell replication. There are numerous methods in
the art for measuring DNA synthesis, any of which may be used
according to the invention. For example, DNA synthesis may be
determined using a radioactive label ([.sup.3H]thymidine) or
labeled nucleotide analogues (BrdU) for detection by
immunofluorescence. Alternatively, the rate of proliferation can be
measured using any of a number of commercial colorimetric kits,
such as the MTT assay. Additionally, the cells may be assayed to
determine whether there are changes in levels, or modification, of
proteins known to be associated with cell proliferation. Such
proteins include, for example, cyclin D1, CDK4 or p107. The
efficacy of the test compound can be assessed by generating dose
response curves from data obtained using various concentrations of
the test compound. A control assay can also be performed to provide
a baseline for comparison. Compounds which are found to alter cell
proliferation may then be screened in an electrophysiological assay
to confirm that the effect is due to modulation of TREK-1.
[0082] Using such proliferation assays, non-narcotic
analgesics/non-steroidal anti-inflammatory drugs (NSAIDs) were
identified as a class of compounds capable of positive modulation
of TREK-1 activity. Accordingly, NSAIDs having the following
general structure:
##STR00001##
where R.sub.1-R.sub.9 may be the same or different and are selected
from the group consisting of hydrogen, halogen, alkyl, or
haloalkyl, may be used to modulate the proliferation of cells. The
free --COOH group may also be in the form of a pharmaceutically
acceptable salt or ester.
[0083] In a specific embodiment of the invention, Meclofenamic acid
having the molecular formula C.sub.14H.sub.11Cl.sub.2O.sub.2N and
the following chemical structure:
##STR00002##
may be used to modulate the activity of TREK-1.
[0084] Using proliferation assays, tricyclic antipsychotics were
identified as a class of compounds capable of negative modulation
of TREK-activity. Accordingly, in an embodiment of the present
invention, a TREK-1 inhibitor may be a compound selected from the
group consisting of one or more tricyclic antipsychotics, tricyclic
antidepressants, thiazines, dibenzoxazepines. In preferred
embodiments, the compound is selected from the group consisting of
tricyclic thiazines, phenothiazines, thioxanthenes, more preferable
from the group consisting of phenothiazines. In one of the more
preferred embodiments the TREK-1 inhibitor is the tricyclic
antipsychotic phenothiazine Trifluoperazine.
[0085] In instances where specific drug use is associated with
behavioral effects, the drug may be modified so that it maintains
the ability to modulate TREK-1 activity while minimized the CNS
effect.
[0086] Compounds which may be screened in accordance with the
invention include, but are not limited to, small organic or
inorganic compounds, peptides, antibodies and fragments thereof,
and other organic compounds e.g., peptidomimetics) that modulate
TREK-1 activity. Compounds may include, but are not limited to,
peptides such as, for example, soluble peptides, including but not
limited to members of random peptide libraries (see, e.g., Lam, K.
S. et al., 1991, Nature 354:82-84; Houghten, R. et al., 1991,
Nature 354:84-86); and combinatorial chemistry-derived molecular
library made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to, members of random
or partially degenerate, directed phosphopeptide libraries; (see,
e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies
(including, but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression library fragments, and epitope
binding fragments thereof), and small organic or inorganic
molecules.
[0087] 5.3. Methods of Treating Proliferative Disorders
[0088] Described below are methods and compositions for treating
cancer wherein the TREK-1 gene or gene product is used as a
therapeutic target. Such compositions include, but are not limited
to, peptides, including soluble peptides, small organic or
inorganic molecules, therapeutic nucleic acid molecules, including
antisense, ribozymes and siNA, all of which function as TREK-1
inhibitors. Additionally, anti-TREK-1 antibodies, or fragments
thereof, may be used to treat cancer. Such antibodies and fragments
thereof include, but are not limited to, naturally occurring
antibodies, bivalent fragments such as (Fab').sub.2, monovalent
fragments such as Fab, single chain antibodies, single chain Fv
(scFv), single domain antibodies, multivalent single chain
antibodies, diabodies, triabodies, and the like that bind
specifically with antigens.
[0089] The outcome of a treatment is designed to produce in a
treated subject a healthful benefit, which in the case of cancer,
includes but is not limited to remission of the cancer, palliation
of the symptoms of the cancer, control of metastatic spread of the
cancer. All such methods involve modulating TREK-1 gene activity
and/or expression which in turn modulate the phenotype of the
treated cell.
[0090] As discussed, above, successful treatment of cancer can be
brought about by techniques which serve to decrease TREK-1
activity. Activity can be decreased by, for example, directly
decreasing TREK-1 gene product activity and/or by decreasing the
level of TREK-1 gene expression. Compounds that may be used to
decrease TREK-1 activity include, but are not limited to,
oleylamine, sipatrigine or trifluoperazine. Additionally, compounds
that inhibit the activity of phospholipase A2 or 15-lipoxygenase
(15-LOX) isozymes may be utilized to inhibit the proliferation of
cancer cells.
[0091] For example, compounds such as those identified through
assays described above, which decrease TREK-1 activity, can be used
in accordance with the invention to treat cancer. Such compounds
can include, but are not limited to peptides, including soluble
peptides, and small organic or inorganic compounds, and can be
referred to as TREK-1 inhibitors.
[0092] It should be understood that compounds capable of modulating
TREK-1 activity, as disclosed herein, include functional
derivatives and analogs, including pharmaceutically acceptable
salts, esters, or hydrates thereof.
[0093] In the context of the invention, preference is given to
pharmaceutically acceptable salts. "Pharmaceutically acceptable
salts" refers to an acid addition salt or a basic addition salt of
a compound of the invention in which the resulting counter ion is
understood in the art to be generally acceptable for pharmaceutical
uses. Pharmaceutically acceptable salts can be salts of the
compounds according to the invention with inorganic or organic
acids. Preference is given to salts with inorganic acids, such as,
for example, hydrochloric acid, hydrobromic acid, phosphoric acid
or sulfuric acid, or to salts with organic carboxylic or sulfonic
acids, such as, for example, acetic acid, maleic acid, fumaric
acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic
acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfonic
acid, toluenesulfonic acid or naphthalenedisulfonic acid.
Pharmaceutically acceptable salts can also be metal or ammonium
salts of the compounds according to the invention. Particular
preference is given to, for example, sodium, potassium, magnesium
or calcium salts, and also to ammonium salts which are derived from
ammonia or organic amines, such as, for example, ethylamine, di- or
triethylamine, di- or triethanolamine, dicyclohexylamine,
dimethylaminoethanol, arginine, lysine, ethylenediamine or
2-phenylethylamine. (see, Berge et al. J. Pharm. Sci. 1977, 66,
1-19.)
[0094] The substances according to the invention may also be
present as pharmaceutically acceptable ester, such as the methyl
ester, ethyl ester and the like.
[0095] When one or more chiral centers are present in the compounds
of the present invention, the individual isomers and mixtures
thereof (e.g., racemates, etc.) are intended to be encompassed by
the formulae depicted herein. In certain embodiments, compounds of
the invention may exist in several tautomeric forms. Accordingly,
the chemical structures depicted herein encompass all possible
tautomeric forms of the illustrated compounds. Compounds of the
invention may exist in various hydrated forms.
[0096] In an embodiment of the invention, the level of TREK-1
expression can be modulated using TREK-1 based oligonucleotide
molecules including but not limited to antisense, ribozyme, or RNAi
approaches to inhibit or prevent translation of TREK-1 mRNA
transcripts or triple helix approaches to inhibit transcription of
the TREK-1 gene (herein after referred to as "therapeutic nucleic
acid molecules"). Antisense, RNAi and ribozyme approaches involve
the design of oligonucleotides (either DNA or RNA) that are
complementary to TREK-1 mRNA. The antisense, siNA or ribozyme
oligonucleotides will be targeted to complementary TREK-1 mRNA
transcripts and prevent translation. Absolute complementarity,
although preferred, is not required. One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex. Mammalian TREK-1 sequences that may be used in the design
of antisense, RNAi and ribozymes include those disclosed in
Genebank accession number is NM001017424, Fink et al. (1996, EMBO
Journal 15:6854-6862) or Meadows et al., (2000, Pflugers Arch.
439:714-22).
[0097] In a preferred embodiment of the invention, double-stranded
short interfering nucleic acid (siNA) molecules may be designed to
inhibit TREK-1 expression. In one embodiment, the invention
features a double-stranded siNA molecule that down-regulates
expression of the TREK-1 gene product, wherein said siNA molecule
comprises about 15 to about 28 base pairs.
[0098] In one embodiment, the invention features a double stranded
short interfering nucleic acid (siNA) molecule that directs
cleavage of a TREK-1 RNA via RNA interference (RNAi), wherein the
double stranded siNA molecule comprises a first and a second
strand, each strand of the siNA molecule is about 18 to about 28
nucleotides in length, the first strand of the siNA molecule
comprises nucleotide sequence having sufficient complementarity to
the TREK-1 RNA for the siNA molecule to direct cleavage of the
TREK-1 RNA via RNA interference, and the second strand of said siNA
molecule comprises nucleotide sequence that is complementary to the
first strand.
[0099] The use of antisense molecules as inhibitors of gene
expression is a specific, genetically based therapeutic approach
(for a review, see Stein, in Ch. 69, Section 5 "Cancer: Principle
and Practice of Oncology", 4th ed., ed. by DeVita et al., J. B.
Lippincott, Philadelphia 1993). The present invention provides the
therapeutic use of nucleic acids of at least six nucleotides that
are antisense to the TREK-1 gene or a portion thereof. An
"antisense" TREK-1 nucleic acid as used herein refers to a nucleic
acid capable of hybridizing to a portion of a TREK-1 RNA
(preferably mRNA) by virtue of some sequence complementarity.
[0100] The antisense nucleic acid of the invention may be
complementary to a coding and/or noncoding region of a TREK-1 mRNA.
The antisense molecules will bind to the complementary TREK-1 gene
mRNA transcripts and reduce or prevent translation. Absolute
complementarity, although preferred, is not required. A sequence
"complementary" to a portion of an RNA, as referred to herein,
means a sequence having sufficient complementarity to be able to
hybridize with the RNA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0101] In yet another embodiment of the invention, ribozyme
molecules designed to catalytically cleave TREK-1 mRNA transcripts
can also be used to prevent translation of TREK-1 mRNA and
expression of TREK-1. (See, e.g., PCT International Publication
WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science
247:1222-1225).
[0102] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA (For a review see, for example Rossi,
J., 1994, Current Biology 4:469-471). The mechanism of ribozyme
action involves sequence specific hybridization of the ribozyme
molecule to complementary target RNA, followed by an
endonucleolytic cleavage. The composition of ribozyme molecules
must include one or more sequences complementary to the target gene
mRNA, and must include the well known catalytic sequence
responsible for mRNA cleavage. For this sequence, see U.S. Pat. No.
5,093,246, which is incorporated by reference herein in its
entirety. As such, within the scope of the invention are engineered
hammerhead motif ribozyme molecules that specifically and
efficiently catalyze endonucleolytic cleavage of RNA sequences
encoding target gene proteins.
[0103] Ribozyme molecules designed to catalytically cleave TREK-1
gene mRNA transcripts can also be used to prevent translation of
TREK-1 gene mRNA and expression of target or pathway gene. (See,
e.g., PCT International Publication WO90/11364, published Oct. 4,
1990; Sarver et al., 1990, Science 247:1222-1225). While ribozymes
that cleave mRNA at site specific recognition sequences can be used
to destroy TREK-1 gene mRNAs, the use of hammerhead ribozymes is
preferred Hammerhead ribozymes cleave mRNAs at locations dictated
by flanking regions that form complementary base pairs with the
target mRNA. The sole requirement is that the target mRNA have the
following sequence of two bases: 5'-UG-3'. The construction and
production of hammerhead ribozymes is well known in the art and is
described more fully in Haseloff and Gerlach, 1988, Nature,
334:585-591. Preferably the ribozyme is engineered so that the
cleavage recognition site is located near the 5' end of the TREK-1
gene mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts.
[0104] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and which has been extensively described by
Thomas Cech and collaborators (Zaug, et al., 1984, Science,
224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et
al., 1986, Nature, 324:429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been and
Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an
eight base pair active site which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes which target eight
base-pair active site sequences that are present in a TREK-1
gene.
[0105] Alternatively, endogenous TREK-1 gene expression can be
reduced by targeting deoxyribonucleotide sequences complementary to
the regulatory region of the TREK-1 gene (i.e., the TREK-1 promoter
and or enhancer) to form triple helical structures that prevent
transcription of the TREK-1 gene in targeted tumor cells in the
body. (See generally, Helene, C. et al., 1991, Anticancer Drug Des.
6:569-584 and Maher, I J, 1992, Bioassays 14:807-815).
[0106] Therapeutic nucleic acid molecules such as RNAi, antisense
and ribozyme molecules which inhibit TREK-1 gene expression can be
used in accordance with the invention to reduce the level of TREK-1
gene expression, thereby effectively reducing the level of TREK-1
activity. Still further, triple helix molecules can be utilized in
reducing the level of TREK-1 gene activity.
[0107] Such therapeutic nucleic acid molecules, i.e., RNAi,
antisense, ribozyme and triple helix forming oligonucleotides, may
be synthesized using standard methods known in the art for the
synthesis of DNA and RNA molecules. These include techniques for
chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides, such as for example, solid phase
phosphoramidite chemical synthesis. The nucleic acid molecule can
be DNA or RNA or chimeric mixtures or derivatives or modified
versions thereof, single-stranded or double-stranded. The nucleic
acid molecule can be modified at the base moiety, sugar moiety, or
phosphate backbone, for example, to improve stability of the
molecule, hybridization, etc. The nucleic acid molecule may include
other appended groups such as peptides (e.g., for targeting host
cell receptors in vivo), or agents facilitating transport across
the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl.
Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl.
Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published
Dec. 15, 1988), hybridization-triggered cleavage agents. (See,
e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating
agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end,
the nucleic acid molecules may be conjugated to another molecule,
e.g., a peptide, hybridization triggered cross-linking agent,
transport agent, hybridization-triggered cleavage agent, etc.
[0108] Alternatively, the therapeutic nucleic acid molecules can be
generated by in vitro and in vivo transcription of DNA sequences
encoding the therapeutic nucleic acid molecules. Such DNA sequences
can be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters.
[0109] Any technique which serves to selectively administer nucleic
acid molecules to a cell population of interest can be used, for
example, by using a delivery complex. Such a delivery complex can
comprise an appropriate nucleic acid molecule and a targeting
means. Such targeting means can comprise, for example, sterols,
lipids, viruses or target cell specific binding agents. In a
specific embodiment, pharmaceutical compositions comprising a
therapeutic nucleic acid molecule are administered via biopolymers,
liposomes, microparticles, or microcapsules. In various embodiments
of the invention, it may be useful to use such compositions to
achieve sustained release of the therapeutic nucleic acids. In a
specific embodiment, it may be desirable to utilize liposomes
targeted via antibodies to specific identifiable tumor antigens
(Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451;
Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).
[0110] It is often difficult to achieve intracellular
concentrations of the therapeutic nucleic acid molecule sufficient
to suppress translation of endogenous mRNAs. Therefore, a preferred
approach utilizes a recombinant DNA construct in which expression
of the therapeutic nucleic acid molecule is placed under the
control of a strong pol III or pol II promoter. For general reviews
of the methods of gene therapy, see Goldspiel et al., 1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,
Ann Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215).
Methods commonly known in the art of recombinant DNA technology
which can be used are described in Ausubel et al. (eds.), 1993,
Current Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.
(eds.), 1994, Current Protocols in Human Genetics, John Wiley &
Sons, NY.
[0111] The use of recombinant DNA constructs to transfect target
cells in the patient will result in the transcription of sufficient
amounts of the therapeutic nucleic acid molecule that will form
complementary base pairs with the endogenous TREK-1 gene
transcripts and thereby prevent translation of the TREK-1 gene
mRNA. For example, a vector can be introduced in vivo such that it
is taken up by a cell and directs the transcription of an antisense
RNA. Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA.
[0112] Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
a therapeutic nucleic acid can be by any promoter known in the art
to act in mammalian, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include but are not
limited to: the SV40 early promoter region (Bernoist and Chambon,
1981, Nature 290:304-310), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell
22:787-797), the herpes thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory
sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC or viral
vector can be used to prepare the recombinant DNA construct which
can be introduced either directly into the tissue site, or via a
delivery complex. Alternatively, viral vectors can be used which
selectively infect the desired tissue.
[0113] In a specific embodiment, a viral vector that contains the
nucleic acid of interest, i.e., a therapeutic nucleic acid molecule
is used. For example, a retroviral vector can be used (see Miller
et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors
have been modified to delete retroviral sequences that are not
necessary for packaging of the viral genome and integration into
host cell DNA. The TREK-1 nucleic acid to be used in gene therapy
is cloned into the vector, which facilitates delivery of the gene
into a patient. More detail about retroviral vectors can be found
in Boesen et al., 1994, Biotherapy 6:291-302, which describes the
use of a retroviral vector to deliver the mdr1 gene to
hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., 1994, J.
Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0114] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.
Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300.
[0115] Alternatively, mesenchymal stem cells which express gap
junctional protein may be genetically engineered to express the
therapeutic nucleic acid of interest followed by transplantation
into tumor tissue, i.e, prostate tumor tissue. It has been
demonstrated that siRNA, for example, can cross gap junctional
channels to affect the function of target cells (Valiunas et al.,
2005, J. Physiol. (Lond) 568:459-468).
[0116] In another embodiment of the invention, nucleic acid
molecules comprising a sequence encoding a dominant negative mutant
TREK-1 protein or non-functional fragment or derivative thereof,
are administered to inhibit TREK-1 function. The TREK-1 channel is
normally found in the cell as a homodimer. When a dominant-negative
mutant TREK-1 protein is overexpressed it forms a heterodimer with
the endogenous wild-type monomers resulting in a non-functional
channel. Thus, dominant negative TREK-1 mutants are those mutants
that are defective in function but effective in binding to form
heterodimers with the wild type TREK-1. Specifically, the nucleic
acid comprises a TREK-1 nucleic acid that is part of an expression
vector that expresses a dominant non-functional TREK-1 protein or
fragment or chimeric protein thereof in cancer cells. In a specific
embodiment of the invention, a dominant negative TREK-1 can be
created by mutating a key residue in the ion selectivity filter in
both pore-forming loops (G161E and G268E). Dominant non-functional
TREK-1 can be engineered for expression in cancer cells that
inappropriately overexpress TREK-1.
[0117] The present invention is directed to a method of modulating
cell proliferation comprising contacting a cell with a composition
comprising a nucleic acid sequence, wherein the nucleotide sequence
encodes a variant TREK-1 that has dominant negative activity. In a
specific embodiment, the nucleic acid encoding the dominant
negative mutant TREK-1 is directly administered in vivo, where it
is expressed to produce the non-functional TREK-1 gene product.
This can be accomplished using any of the gene therapy methods
described above for in vivo expression of therapeutic nucleic acid
molecules.
[0118] The form and amount of therapeutic nucleic acid envisioned
for use depends on the cancer, desired effect, patient state, etc.,
and can be determined by one skilled in the art.
[0119] Antibodies exhibiting capability to down regulate TREK-1
gene product activity can also be utilized to treat cancer. Such
antibodies can be generated using standard techniques. Such
antibodies include but are not limited to polyclonal, monoclonal,
Fab fragments, single chain antibodies, chimeric antibodies, and
the like.
[0120] 5.4. Pharmaceutical Preparations and Methods of
Administration
[0121] The compounds and nucleic acid sequences described herein
can be administered to a patient at therapeutically effective doses
to treat or prevent cancer. A therapeutically effective dose refers
to that amount of a compound sufficient to result in a healthful
benefit in the treated subject.
[0122] Toxicity and therapeutic efficacy of compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects can be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0123] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that inhibits TREK-1 by 50% as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma can be measured, for example, by high
performance liquid chromatography.
[0124] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvents can be formulated for administration by inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral or rectal administration. In a specific
embodiment of the invention, the pharmaceutical compositions of the
invention may be implanted directly into, or in close proximity, to
the tumor tissue. For example, the pharmaceutical compositions may
be implanted into the prostate, thereby, allowing the
administration of higher doses of drug to a more limited tissue
area. Such compositions may be formulated into a form that allows
for sustained release of the drug in the area to be treated.
[0125] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0126] Preparations for oral administration can be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions can take the form of tablets
or lozenges formulated in conventional manner.
[0127] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator can
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0128] The compounds can be formulated for parenteral
administration (i.e., intravenous or intramuscular) by injection,
via, for example, bolus injection or continuous infusion.
Formulations for injection can be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient can be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0129] The compounds can also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0130] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
6. EXAMPLE
Two-Pore Domain K+ Channels Are Over-Expressed in Cancer
[0131] Initial studies of prostate epithelium have identified
expression of the TREK-1 two pore channel in two prostate cancer
cell lines, PC-3 and LNCaP but not in normal prostate epithelial
cells (NPE; FIG. 1). Expression of functional channels in both PC-3
(FIG. 2, Panel A) and LNCaP (data not shown) cells has been
confirmed by whole-cell patch-clamp recording in the voltage-clamp
configuration. TREK-1 was identified in these recordings as the
sipatrigine-sensitive current in the presence of CsCl (5 mM), TEA
(10 mM) and nifedipine (5 .mu.M) which were are added to block
other K.sup.+ and the I.sub.CaL currents. Normal prostate
epithelial cells do not express any TREK-1 current under these
conditions (see FIG. 4, below). In addition, a TREK-1 channel
mutant has been created which is disrupted in the selectivity
filter of the pore region (G161E/G268E). This mutant channel
carries no current when expressed in CHO cells and functions as a
dominant-negative (dn-TREK) when co-expressed with wild-type
channel. Transfection of PC-3 cells with do-TREK (FIG. 2, Panel B)
leads to complete ablation of endogenous TREK-1 current.
[0132] Additional studies have identified the presence of TASK-1
(identified as a methanandamide-sensitive current) (Maingret et
al., 2001) and TASK-3 (identified as a ruthenium red-sensitive
current) (Czirjak and Enyedi, 2003) in both LNCaP and PC-3
cells.
[0133] Since TREK-1 is expressed in the prostate cancer cells but
not in NPE, such data indicates that TREK-1 might contribute to the
rapid proliferation rate of prostate cancer cells. To confirm this
hypothesis, the proliferation of untransfected PC-3 cells was
compared with cells that were transfected either with the
dominant-negative TREK-1 or an empty vector (pEGFP). The cells
which over-express the dn-TREK carry no TREK-1 current (see FIG. 2,
Panel B). Proliferation rate was determined using a standard
colorimetric assay (Cell Proliferation Kit I [MTT]; Roche) and
normalized to the proliferation rate of untransfected PC-3. pEGFP
transfection does not affect the proliferation rate of PC-3 cells.
However, over-expression of dn-TREK significantly slows the
proliferation rate by 44.+-.6.6% (FIG. 3; n=6). It should be noted
that the transfection efficiency in these experiments is
approximately 30%. While this is not an issue for the
electrophysiology experiments where cells expressing the
dominant-negative channel can be individually selected due to the
co-expression of EGFP, the efficiency should be expected to reduce
the effect observed in the proliferation assay which measures the
rate of all the cells in the microplate. Therefore, the 47%
inhibition is believed to be an underestimate of the effect.
[0134] To test whether over-expression of TREK-1 in normal prostate
epithelial cells would increase the proliferative rate experiments
were conducted with a TREK-1 bearing adenovirus. Normal prostate
epithelial cells do not exhibit TREK-1 current (FIG. 4, Panel A).
However, when NPE are infected with the TREK-1 virus a large
current is readily detected (FIG. 4, Panel B). The effect of this
current on the proliferation rate in NPE was tested and it was
determined that expression of TREK-1 significantly increased the
proliferation rate of NPE by 270.+-.27.7% (FIG. 5, n=5,
p<0.05).
[0135] As demonstrated in FIG. 5, expression of TREK-1 increases
proliferation of normal prostate epithelial cells (NPE). Normal
prostate epithelial cell cultures were obtained from Clonetics and
infected with a TREK-1-containing adenovirus. Some virally infected
cells were also treated with sipatrigine (10 .mu.M), a TREK-1
blocker. Cell proliferation was assessed using the MTT assay as
described in FIG. 3. These data are presented as mean.+-.SEM from
five paired experiments. * p<0.05, the NPE/TREK-1 group differed
significantly from each of the other treatments and no other groups
were different.
[0136] FIG. 6 demonstrates, by immunohistochemical staining of
human prostate tissue, that TREK-1 is over-expressed in cancer.
Human tissue samples were stained with rabbit polyclonal anti-human
TREK-1 antibody (Alomone). Panels A and B show a low power and high
power view of the same field. Under the low power, a normal gland
is visible (center right). The epithelial cells lining the luminal
surface are very lightly stained. An adjacent cancerous gland
(center left) shows a very small luminal space and the epithelial
cells lining it are very densely stained indicating an
overexpression of TREK-1. The same features are visible under
higher power in Panel B.
[0137] FIG. 7 reveals that Meclofenamate activates TREK-1 current
in CHO cells that heterologously express the channel. CHO cells
were transfected with a plasmid encoding human TREK-1 and the
current was studied by patch clamp. The current-voltage relation
was determined using a ramp protocol that went from -130 mV to +40
mV in 6 s (after correction for the junction potential). Current
was greater in the presence of meclofenamate. This is typical of 7
cells.
[0138] Further, FIG. 8 reveals that Trifluoperazine inhibits TREK-1
current in CHO cells that heterologously express the channel. CHO
cells were transfected with a plasmid encoding human TREK-1 and the
current was studied by patch clamp. The current-voltage relation
was determined using a ramp protocol that went from -130 mV to +40
mV in 6 s (after correction for the junction potential). Current
was less in the presence of trifluoperazine. This is typical of 3
cells.
[0139] Transiently transfected CHO cells (obtained through ATCC)
were fixed with 1 ml ice-cold 70% ethanol 48 h after transfection
with TREK-1. Fixation was followed by RNase treatment and staining
with propidium iodide (50 .mu.g/ml). Stained samples were analyzed
on a FACScan flow cytometer and data were analyzed using Cell Quest
software to assess the cell-cycle distribution patterns. FIGS.
10-12 demonstrate that TREK-1 CHO stable over-expressor cell lines
have significantly higher proliferation rates which correlate with
the TREK-1 current.
[0140] Anchorage-independent growth of stably transfected CHO cells
was also measured. Creation of the TREK-1 stable over-expressing
lines was done in CHO cells using zeocin selection. CHO cells that
stably over-expressed TREK-1 (1.times.10.sup.6 cells/well) were
suspended in 0.4% agar with complete RPMI medium in six well
plates. Twenty days later the plates were stained with
iodonitrotetrazolium chloride for 6 h. Colonies were scored using a
light microscope at .times.10 magnification. As demonstrated in
FIG. 13, TREK-1 over-expression promotes anchorage-independent
growth.
[0141] The present invention is not to be limited in scope by the
specific embodiments described herein which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the claims. Throughout this application various
publications are referenced. The disclosures of these publications
in the entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
those skilled therein as of the date of the invention described and
claimed herein.
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