U.S. patent application number 14/640662 was filed with the patent office on 2015-12-10 for treatment of astrocytes-tumor cells inhibitors of endothelin receptors.
The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Isaiah FIDLER, Sun-jin KIM.
Application Number | 20150352113 14/640662 |
Document ID | / |
Family ID | 42938154 |
Filed Date | 2015-12-10 |
United States Patent
Application |
20150352113 |
Kind Code |
A1 |
FIDLER; Isaiah ; et
al. |
December 10, 2015 |
TREATMENT OF ASTROCYTES-TUMOR CELLS INHIBITORS OF ENDOTHELIN
RECEPTORS
Abstract
The disclosure relates to an endothelin receptor antagonist for
use in the prevention or treatment of brain metastases in
combination with a cytotoxic chemotherapy agent, radiotherapy or
both. The endothelin receptor antagonist may for example be
bosentan, macitentan or a mixture of bosentan and macitentan.
Inventors: |
FIDLER; Isaiah; (Houston,
TX) ; KIM; Sun-jin; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Family ID: |
42938154 |
Appl. No.: |
14/640662 |
Filed: |
March 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13390072 |
Feb 10, 2012 |
8999934 |
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PCT/US2010/044832 |
Aug 9, 2010 |
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14640662 |
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61232687 |
Aug 10, 2009 |
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Current U.S.
Class: |
600/1 ;
514/274 |
Current CPC
Class: |
A01K 67/027 20130101;
A61K 31/4188 20130101; A61K 31/506 20130101; A61K 31/513 20130101;
C12N 5/0693 20130101; C12N 5/0622 20130101; A01K 2227/105 20130101;
A61K 31/506 20130101; A61K 45/06 20130101; A61K 31/337 20130101;
A61P 35/00 20180101; A61K 31/495 20130101; A61K 31/495 20130101;
A01K 2267/0331 20130101; C12N 2502/08 20130101; A61K 31/4188
20130101; A61N 5/1084 20130101; A61P 43/00 20180101; C12N 2502/30
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61P 35/04 20180101; A61K
31/337 20130101; G01N 33/5008 20130101 |
International
Class: |
A61K 31/513 20060101
A61K031/513; A61N 5/10 20060101 A61N005/10; A61K 31/337 20060101
A61K031/337 |
Claims
1-26. (canceled)
27. A method of treating an existing brain metastasis tumor in a
subject comprising administering an endothelin receptor antagonist
and at least one cytotoxic chemotherapeutic agent, wherein the
endothelin receptor antagonist is macitentan and the at least one
cytotoxic chemotherapeutic agent is a mitotic inhibitor.
28. The method of claim 27, wherein the mitotic inhibitor is
paclitaxel.
29. A method for administering a cytotoxic chemotherapy to treat a
brain metastasis in a patient comprising: a. administering an
effective amount of endothelin receptor antagonist to desensitize
astrocyte mediated protection of a brain metastasis cell from a
cytotoxic chemotherapy induced cell death, wherein the endothelin
receptor antagonist is macitentan; and b. further administering a
cytotoxic chemotherapy agent to induce cell death, wherein the
cytotoxic chemotherapeutic agent is a mitotic inhibitor.
30. The method of claim 29, wherein the mitotic inhibitor is
paclitaxel.
31. The method of claim 29, wherein the brain metastasis cell is
located in an existing brain metastasis tumor in a subject and the
existing brain metastasis tumor is a visible tumor.
32. The method of claim 30, wherein the brain metastasis cell is
located in an existing brain metastasis tumor in a subject and the
existing brain metastasis tumor is a visible tumor.
33. A method of inhibiting in a human an astrocyte mediated
protection of a brain metastasis cell from a cytotoxic chemotherapy
induced cell death, wherein the brain metastasis cell is located in
an existing brain metastasis tumor in said human or is an isolated
cell, and said existing brain metastasis tumor is a
micrometastasis, said method comprising: a. administering an
effective amount of an endothelin receptor antagonist to the brain
metastasis cell and the astrocyte thereby inhibiting the astrocyte
mediated protection, wherein the endothelin receptor antagonist is
macitentan; and b. administering at least one cytotoxic
chemotherapeutic agent to the brain metastasis cell, wherein the
cytotoxic chemotherapeutic agent is a mitotic inhibitor; and c.
further performing whole brain radiotherapy, stereotactic
radiosurgery, or combinations thereof.
34. The method of claim 33, wherein the mitotic inhibitor is
paclitaxel.
35. A method of inhibiting in a human an astrocyte mediated
protection of a brain metastasis cell from a cytotoxic chemotherapy
induced cell death, wherein the brain metastasis cell is located in
an existing brain metastasis tumor in said human or is an isolated
cell, and said existing brain metastasis tumor is a visible tumor,
said method comprising: a. administering an effective amount of an
endothelin receptor antagonist to the brain metastasis cell and the
astrocyte thereby inhibiting the astrocyte mediated protection,
wherein the endothelin receptor antagonist is macitentan; and b.
administering at least one cytotoxic chemotherapeutic agent to the
brain metastasis cell, wherein the cytotoxic chemotherapeutic agent
is a mitotic inhibitor; and c. further performing whole brain
radiotherapy, stereotactic radiosurgery, or combinations
thereof.
36. The method of claim 35, wherein the mitotic inhibitor is
paclitaxel.
Description
BACKGROUND
[0001] Brain metastasis is one of the most difficult challenges
facing oncology. Metastatic tumors are resistant to most
chemotherapy agents. The treatments for brain metastasis are
primarily whole brain and focused radiotherapy, with surgical
resection of tumors in a minority of cases. Most chemotherapy
regimens involve 2-3 agents such as cisplatin, cyclophosphamide,
etoposide, teniposide, mitomycin, irinotecan, vinorelbine,
etoposide, ifosfamide, temozolomide and fluorouracil (5-FU). These
are administered in combination with radiotherapy. The effect of
these chemotherapies on prolonging survival is generally less than
a year. A fairly new chemotherapy for brain tumors is temozolomide
used with whole-brain irradiation. Results are preliminary but
temozolomide appears to have some limited effect on the response
rate compared to radiation alone and appears to have some clinical
activity in combination with radiation in phase II trials.
[0002] Despite intense efforts, the limited medical options
available for brain metastasis have remained poor and too often
more palliative than therapeutically effective. This state of
affairs has been long recognized but, to date, significant advances
have not materialized. Consequently, there is a great and present
medical need for new therapeutic approaches and pharmaceuticals
effective at treating brain metastasis.
[0003] The disclosure below discusses endothelin receptor
antagonists in relation to brain metastasis. Endothelin-1
(hereafter "ET-1"), a vasoactive peptide, is produced primarily in
endothelial, vascular smooth muscle, and epithelial cells. ET-1
exerts its physiological effect via two high-affinity
G-protein-coupled receptors, the endothelin-A (hereafter
"ET.sub.A") and the endothelin-B (hereafter "ET.sub.B") receptors.
Endothelin receptor antagonists (ERAs) are a well established class
of compounds capable of inhibiting these endothelin receptors
(hereafter "ETRs"). Within this class are subclasses of antagonists
specific to ET.sub.A or ET.sub.B and a subclass effective against
both (dual specificity). One member of the dual specificity
subclass, bosentan, is currently approved for use in treating
pulmonary arterial hypertension.
[0004] Certain ERAs have been investigated for use in cancer
therapy. [Nelson J B, et al., Phase 3, randomized, controlled trial
of atrasentan in patients with nonmetastatic, hormone-refractory
prostate cancer. Cancer, 2008 November 1; 113(9):2376-8.; Chiappori
A A, et al. Phase I/II study of atrasentan, an ET.sub.A receptor
antagonist, in combination with paclitaxel and carboplatin as
first-line therapy in advanced non-small cell lung cancer. Clin
Cancer Res, 2008 Mar 1; 14(5):1464-9.] These studies have largely
excluded patients with active brain metastasis. Ibid. This
exclusion is done on the general view that existing brain
metastases will not respond to treatment and, thus, morbidity and
symptoms due to these metastases would mask the effects of the test
treatment on the primary tumor. [Carden C P, et al., Eligibility of
patients with brain metastases for phase I trials: time for a
rethink? The Lancet Oncology, Vol 9, Issue 10, Pages 1012-1017,
October 2008 doi:10.1016/S1470-2045(08)70257-2.] This standard
clinical trial design strategy serves to emphasize the general
expectation that therapies effective against primary tumors and
even non-brain metastasis tumors will fail to effect brain
metastasis tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1: In vitro culture of MDA-MB-231 breast cancer cells
(T) and murine astrocytes (A) were evaluated by scanning electron
microscopy. Direct contact between the astrocytes (extending
pods-feet) and tumor cells is evident;
[0006] FIG. 2: The astrocyte-metastatic cancer cell co-cultures
showed dye transfer between co-cultured cells;
[0007] FIG. 3: Culturing of human MDA-MB-231 breast cancer cells or
human PC14Br4 lung cancer cells with astrocytes (but not 3T3
fibroblasts) reduced the relative apoptotic index (increased
resistance) of tumor cells incubated for 72 hours with paclitaxel
(5 ng/ml) by 58.3+8.9% (mean.+-.S.D., P<0.01) and 61.8.+-.6.7%
(mean.+-.S.D., P<0.05), respectively (the apoptotic index was
compared by the Student's t test);
[0008] FIG. 4: Human lung cancer PC14Br4 cells were cultured alone
or with astrocytes (direct cell to cell contact) in medium
containing P-glycoprotein-associated Adriamycin (200 ng/ml),
paclitaxel (5 ng/ml), vinblastine (3 ng/ml), vincristine (8 ng/ml),
and P-glycoprotein-dissociated 5-FU (500 ng/ml) or cisplatinum (2.4
.mu.g/ml);
[0009] FIG. 5: Astrocyte-mediated protection of brain metastasis
cells from cytotoxic chemotherapy-induced cell death does not last
longer than 72 hours after direct astrocyte-brain metastasis cell
contact is lost;
[0010] FIG. 6: Gene transcription profiling conditions
distinguished between murine and human mRNA;
[0011] FIG. 7: In the MDA-MB-231 cells, 1069 genes, and in the
PC14Br4 cells, 594 genes were differentially expressed. A two-gene
list comparison revealed increased expression of several genes well
known to be associated with anti-apoptosis and survival:
glutathione S transferase 5 (GSTA5), BCL2L1, TWIST;
[0012] FIG. 8: The expression of several anti-apoptosis and
survival genes was confirmed at the protein level by Western blot
analysis;
[0013] FIG. 9: Gene transcription data were collected from two
cycles of co-culture experiments;
[0014] FIG. 10: Increased expression of ETA-R in MDA-MB-231 human
breast cancer cells co-cultured with astrocytes but not with
fibroblasts (3T3);
[0015] FIG. 11: Expression of pAKT by MDA-MB-231 human breast
cancer cells co-cultured with astrocytes/Taxol;
[0016] FIG. 12: ACT-064992 added alone to MDA-MB-231 human breast
cancer cells or with astrocytes or with 3T3 fibroblasts did not
produce any measurable cytotoxic effects;
[0017] FIG. 13: ACT-064992 added alone to PC14 lung cancer cells or
with astrocytes or with 3T3 fibroblasts did not produce any
measurable cytotoxic effects;
[0018] FIG. 14A-D: Immunostaining for ET.sub.AR and ET.sub.BR in
several in vivo experimental models for metastatic brain cancer
shows relatively high expression associated specifically with
tumors and not normal brain tissues;
[0019] FIG. 15: Combination therapy of paclitaxel and ACT-064992 on
MDA-MB-231 human breast cancer cells co-cultured with astrocytes or
with 3T3 fibroblasts;
[0020] FIG. 16: Combination therapy of paclitaxel and ACT-064992 on
human lung cancer cells PC14Br4 co-cultured with astrocytes or with
3T3 fibroblasts;
[0021] FIG. 17: The addition of ACT-064992 to co-cultures of
astrocytes and tumor cells inhibited the expression of the survival
factors pAKT and pMAPK;
[0022] FIG. 18A-D: Brain sections were fixed and stained for brain
metastasis visualization. Control (vehicle) (FIG. 18A);
temozolomide ("TMA") 10 mg/kg p.o., daily (FIG. 18B); ACT-064992 50
mg/kg, p.o. daily (FIG. 18C); or combination TMZ+ACT-064992 (FIG.
18D); combination TMZ+ACT-064992 at higher magnification (FIG.
18E);
[0023] FIG. 19: Brain sections were fixed and stained for brain
metastasis visualization. A. Control (injected with vehicle
solution); B. Paclitaxel (8 mg/kg administered intraperitoneally
once per week); C. ACT-064992 (50 mg/kg administered orally once
per day); and D. Combination of ACT-064992 and paclitaxel;
[0024] FIG. 20: Representative brain slices from the four treatment
groups were stained for CD31 (endothelial cell marker) and Ki67
(cell proliferation marker): A. control mice; B. mice treated with
only paclitaxel; C. ACT-064992; D. paclitaxel and ACT-064992;
[0025] FIG. 21: Brain slices of mice from different treatment
groups were analyzed by in situ Terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL): A. control; B.
paclitaxel-treated; C. ACT-064992-treated; D. Combination of
ACT-064992 plus paclitaxel;
[0026] FIG. 22: Brain slices of mice from the four treatment groups
were immunostained for ET.sub.A (red) and phosphoserine (green)
colocalization produced orange-yellow color. A. Control brain; B.
brain from mice treated with paclitaxel; C. brain from mice treated
with ACT-064992 alone; D. brain from mice treated with
ACT-064992+paclitaxel.
DETAILED DESCRIPTION
Definitions
[0027] As used herein, the use of the word "a" or "an" when used in
conjunction with the term "comprising" in the claims and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one." Still further, the terms "having", "containing", "including"
and "comprising" are interchangeable and one of skill in the art is
cognizant that these terms are open ended terms.
[0028] The term "treating" and "treatment" as used herein refers to
administering to a subject a therapeutically effective medical
intervention, such as chemotherapy, so that the subject has an
improvement in the parameters relating to a cancer. The improvement
is any observable or measurable improvement including changes in
the size of a cancer tumor, reduction in the rate of growth of a
cancer tumor, or subjective or objective measures of pain
associated with a cancer tumor. Thus, one of skill in the art
realizes that a treatment may improve the patient's condition but
may not be a complete cure of the disease.
[0029] The term "effective amount" or "therapeutically effective
amount" as used herein refers to an amount that results in an
improvement or remediation of the symptoms of the disease or
condition.
[0030] The term "existing brain metastasis tumor" as used herein
refers to a multi-celled brain tumor and brain metastasis
surrounded by and infiltrated with GFAP-positive astrocytes.
Existing brain metastatic tumors are of two clinically distinct
types, micrometastases, which are too small to be visualized by
radiological means, and visible metastases, which are those tumors
large enough to be discernable by clinical radiological means, such
as magnetic resonance imaging, computerized tomography, or positron
emission tomography. These metastatic lesions are distinct from
metastatic cancer cells in the systemic circulation and single
cancer cells extravasating into brain tissue or quiescently
residing therein. [See generally Johanna A. Joyce & Jeffrey W.
Pollard, Microenvironmental regulation of metastasis, Nat Rev
Cancer 9, 239-252 (April 2009)1 doi:10.1038/nrc2618.]
[0031] "Micrometastasis" as used herein is preferably defined as a
group of confluent cancer cells measuring from greater than 0.2 mm
and/or having greater than 200 cells to 2 mm in maximum width. More
preferably "micrometastasis" is defined as a group of confluent
cancer cells from 0.2 mm to 2 mm in maximum width. See The AJCC
Cancer Staging Manual and Handbook, 7.sup.th ed. (2010), Edge, S.
B.; Byrd, D. R.; Compton, C. C.; Fritz, A. G.; Greene, F. L.;
Trotti, A. (Eds.), ISBN: 978-0-387-88440-0. An alternative
preferred definition of "micrometastasis" is a confluent group of
at least 1000 cancer cells and at least 0.1 mm in widest dimension
up to 1 mm in widest dimension. Micrometastasis are generally not
visible in standard contrast MRI imaging or other clinical imaging
techniques. However, in certain cancers, radioactive antibodies
directed to tumor selective antigens (e.g. Her2 for breast cancer
metastasis) will allow visualization of micrometastasis. Other
indirect detection methods include contrast media leakage at brain
micrometastasis sites due to VEGF induced vascular leakage. Yano S;
et al. (2000), Expression of vascular endothelial growth factor is
necessary but not sufficient for production and growth of brain
metastasis. Cancer research 2000; 60(17):4959-67. More sensitive
imaging techniques may also be applied to detect micrometastases.
For example, blood volume may be imaged by MRI using the
alternative contrast agent USPIO (Molday Iron, Biopal, Worcester,
Mass., sold as Molday ION.TM.) to detect micrometastasis. JuanYin
J, et al. Noninvasive imaging of the functional effects of
anti-VEGF therapy on tumor cell extravasation and regional blood
volume in an experimental brain metastasis model. Clin Exp
Metastasis. 2009; 26(5):403-14. Epub 2009 March 11.
[0032] The term "astrocyte mediated protection" as used herein
refers to the ability of an astrocyte to reduce the cytotoxicity of
a chemical for another cell type in direct physical contact with
the astrocyte. This physical contact includes astrocytes connected
to cancer cells, in particular via gap junction communication
(GJC).
[0033] The term "cytotoxic chemotherapy induced cell death" as used
herein refers to the induction of apoptosis or necrotic cell death
by a cytotoxic chemical. Most medically used chemotherapy agents
function to kill rapidly dividing tumor cells this way.
[0034] The term "endothelin receptor antagonist" as used herein
refers to the class of compounds recognized in the art as such, and
in particular to a compound that, when submitted to the "Test for
the determination of ET.sub.A or ET.sub.B IC.sub.50" described in
the present patent application, has an IC.sub.50 equal or lower
than 1 .mu.M against ET.sub.A, against ET.sub.B or against both
ET.sub.A and ET.sub.B. An ERA is a drug that blocks endothelin
receptors from interaction with ET-1 or prevents an ETR from
responding to bound ET-1. Two main kinds of ERAs exist: selective
ERAs, such as sitaxentan, ambrisentan and atrasentan, which affect
ET.sub.A receptors, and dual ERAs, such as bosentan, which affect
both ET.sub.A and ET.sub.B receptors. Exemplary members of the ERA
class of compounds may be found in the patent literature cited in
[HAM Mucke "Small-molecule endothelin receptor antagonists: A
review of patenting activity across therapeutic areas" IDrugs 2009
12:366-375.] Representative ERAs which have already been
investigated in human clinical trials or approved for medical use
include sitaxentan, tezosentan, clazosentan, abbrisentan, bosentan,
macitentan (also known as ACT-064992) and/or atrasentan.
[0035] The term "ET.sub.A antagonist" as used herein refers a
compound that, when submitted to the "Test for the determination of
ET.sub.A or ET.sub.B IC.sub.50" described in the present patent
application, has an IC.sub.50 equal or lower than 1 .mu.M against
ET.sub.A.
[0036] The term "ET.sub.B antagonist" as used herein refers a
compound that, when submitted to the "Test for the determination of
ET.sub.A or ET.sub.B IC.sub.50" described in the present patent
application, has an IC.sub.50 equal or lower than 1 .mu.M against
ET.sub.B.
[0037] The term "dual endothelin receptor antagonist" or "dual
"ERAs" as used herein refers a compound that, when submitted to the
"Test for the determination of ET.sub.A or ET.sub.B IC.sub.50"
described in the present patent application, has an IC.sub.50 equal
or lower than 1 .mu.m against ET.sub.A and an IC.sub.50 equal or
lower than 1 .mu.M against ET.sub.B. Dual ERAs include bosentan and
macitentan.
[0038] The term "cytotoxic chemotherapy agent" as used herein
refers to a substance inducing apoptosis or necrotic cell death.
Examples of cytotoxic chemotherapy agents which may be used in
combination with ERAs in accordance to the present invention
include: [0039] alkylating agents (for example mechlorethamine,
chlorambucil, cyclophosphamide, ifosfamide, streptozocin,
carmustine, lomustine, melphalan, busulfan, dacarbazine,
temozolomide, thiotepa or altretamine); [0040] platinum drugs (for
example cisplatin, carboplatin or oxaliplatin); [0041]
antimetabolite drugs (for example 5-fluorouracil, capecitabine,
6-mercaptopurine, methotrexate, gemcitabine, cytarabine,
fludarabine or pemetrexed); [0042] anti-tumor antibiotics (for
example daunorubicin, doxorubicin, epirubicin, idarubicin,
actinomycin-D, bleomycin, mitomycin-C or mitoxantrone); and [0043]
mitotic inhibitors (for example paclitaxel, docetaxel, ixabepilone,
vinblastine, vincristine, vinorelbine, vindesine or estramustine);
and [0044] topoisomerase inhibitors (for example etoposide,
teniposide, topotecan, irinotecan, diflomotecan or elomotecan).
[0045] "Super-sensitization" or "Super-sensitize" is defined as a
relative increase in cell death caused by cytotoxic chemotherapy
agent(s) in physical contact with astrocytes over that seen in
cells either in the absence of astrocytes or with no direct
physical contact with astrocytes.
[0046] "Simultaneously" or "simultaneous", when referring to a
therapeutic use, means in the present application that the
therapeutic use concerned consists in the administration of two or
more active ingredients by the same route and at the same time.
[0047] "Separately" or "separate", when referring to a therapeutic
use, means in the present application that the therapeutic use
concerned consists in the administration of two or more active
ingredients at approximately the same time by at least two
different routes.
[0048] By therapeutic administration "over a period of time" is
meant in the present application the administration of two or more
ingredients at different times, and in particular an administration
method according to which the entire administration of one of the
active ingredients is completed before the administration of the
other or others begins. In this way it is possible to administer
one of the active ingredients for several days, weeks or months
before administering the other active ingredient or ingredients. In
this case, no simultaneous administration occurs.
DISCLOSURE
[0049] In the brain parenchyma, the role of astrocytes in
maintaining homeostasis is well recognized. Astrocytes enwrap every
blood vessel with specialized end-feet and communicate with other
brain cells, such as neurons. This unique structure allows
astrocytes to transport essential nutrients, such as glucose and
amino acids, from the circulation to dependent neurons, and
glycolysis in astrocytes has been recently shown to regulate
neuronal activity, the so called "neuron-astrocyte metabolic
coupling." Under pathological conditions, such as hypoxia,
ischemia, and degenerative conditions, astrocytes will become
activated and express a protein designated GFAP. GFAP reactive
astrocytes have been shown to protect neurons from various
challenges and to rescue neurons from excitotoxicity produced by
accumulation of glutamate. Activated astrocytes can also protect
neurons from apoptosis produced by ethanol, hydrogen peroxide, and
copper-catalyzed cysteine cytotoxicity.
[0050] Clinical brain metastases are commonly surrounded by and
infiltrated by activated (GFAP-positive) astrocytes. [Yoshimine T,
et al. (1985) Immunohistochemical study of metastatic brain tumors
with astroprotein (GFAP), a glia-specific protein. Tissue
architecture and the origin of blood vessels. J Neurosurg 62:
414-418.] The Inventors have confirmed this phenomenon is
reproduced in a xenotransplantation model below.
[0051] Various embodiments of the present invention are presented
thereafter:
1) The invention firstly relates to an endothelin receptor
antagonist for use in the prevention or treatment of brain
metastases in combination with a cytotoxic chemotherapy agent,
radiotherapy or both. 2) According to one main variant of
embodiment 1), the endothelin receptor antagonist will be for use
in combination with a cytotoxic chemotherapy agent. 3) According to
one sub-embodiment of embodiment 2), the cytotoxic chemotherapy
agent will comprise (and in particular be) an alkylating agent. 4)
In particular, the alkylating agent of embodiment 3) will be
selected from the group consisting of mechlorethamine,
chlorambucil, cyclophosphamide, iphosfamide, streptozocin,
carmustine, lomustine, melphalan, busulfan, dacarbazine,
temozolomide, thiotepa and altretamine. 5) According to another
sub-embodiment of embodiment 2), the cytotoxic chemotherapy agent
will comprise (and in particular be) a platinum drug. 6) In
particular, the platinum drug of embodiment 5) will be selected
from the group consisting of cisplatin, carboplatin and
oxaliplatin. 7) According to yet another sub-embodiment of
embodiment 2), the cytotoxic chemotherapy agent will comprise (and
in particular be) an antimetabolite drug. 8) In particular, the
antimetabolite drug of embodiment 7) will be selected from the
group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine,
methotrexate, gemcitabine, cytarabine, fludarabine and pemetrexed.
9) According to a further sub-embodiment of embodiment 2), the
cytotoxic chemotherapy agent will comprise (and in particular be)
an anti-tumor antibiotic. 10) In particular, the anti-tumor
antibiotic of embodiment 9) will be selected from the group
consisting of daunorubicin, doxorubicin, epirubicin, idarubicin,
actinomycin-D, bleomycin, mitomycin-C and mitoxantrone. 11)
According to another sub-embodiment of embodiment 2), the cytotoxic
chemotherapy agent will comprise (and in particular be) a mitotic
inhibitor. 12) In particular, the mitotic inhibitor of embodiment
11) will be selected from the group consisting of paclitaxel,
docetaxel, ixabepilone, vinblastine, vincristine, vinorelbine,
vindesine and estramustine. 13) According to yet another
sub-embodiment of embodiment 2), the cytotoxic chemotherapy agent
will comprise (and in particular be) a topoisomerase II inhibitor.
14) In particular, the topoisomerase II inhibitor of embodiment 13)
will be selected from the group consisting of etoposide,
teniposide, topotecan, irinotecan, diflomotecan and elomotecan. 15)
In a preferred sub-embodiment of embodiment 2), the cytotoxic
chemotherapy agent will be selected from the group consisting of
paclitaxel, doxorubicin, vinblastine, vincristine, 5-fluorouracil,
cisplatin, cyclophosphamide, etoposide, teniposide, mitomycin-C,
irinotecan, vinorelbine, ifosfamide and temozolomide (and in
particular from paclitaxel and temozolomide). 16) In particular,
the cytotoxic chemotherapy agent of embodiment 15) will be selected
from paclitaxel, temozolomide and a mixture of paclitaxel and
temozolomide. 17) According to a preferred variant of embodiment
16), the endothelin receptor antagonist of embodiment 16) will be
selected from the group consisting of bosentan, macitentan and a
mixture of macitentan and bosentan (and will notably be
macitentan). 18) The invention also relates to an endothelin
receptor antagonist for use in combination with at least one of the
cytotoxic chemotherapeutic agents mentioned in one of embodiments
2) to 17) and with radiotherapy. 19) According to a preferred
embodiment of this invention, the endothelin receptor antagonist
used in embodiments 1) to 18) will comprise (and in particular be)
a dual endothelin receptor antagonist. 20) In particular, the dual
endothelin receptor antagonist of embodiment 19) will be selected
from the group consisting of bosentan, macitentan and a mixture of
bosentan and macitentan. 21) In a particularly preferred
embodiment, the dual endothelin receptor antagonist of embodiment
20) will be macitentan. 22) According to one variant of embodiments
1) to 21), the endothelin receptor antagonist and the cytotoxic
chemotherapeutic agent will be administered separately. 23)
According to another variant of embodiments 1) to 21), the
endothelin receptor antagonist and the cytotoxic chemotherapeutic
agent will be administered simultaneously. 24) According to yet
another variant of embodiments 1) to 21), the endothelin receptor
antagonist and the cytotoxic chemotherapeutic agent will be
administered over a period of time. 25) According to yet another
main variant of embodiment 1), the endothelin receptor antagonist
will be for use in combination with radiotherapy (whereby this
radiotherapy is preferably whole brain radiotherapy or stereotactic
radiosurgery). 26) According to a preferred embodiment of this
invention, the endothelin receptor antagonist used in embodiment
25) will comprise (and in particular be) a dual endothelin receptor
antagonist. 27) In particular, the dual endothelin receptor
antagonist of embodiment 26) will be selected from the group
consisting of bosentan, macitentan and a mixture of bosentan and
macitentan. 28) In a particularly preferred embodiment, the dual
endothelin receptor antagonist of embodiment 27) will be
macitentan. 29) In another main variant of this invention, the
endothelin receptor antagonist for use with a cytotoxic
chemotherapy agent according to one of embodiments 2) to 23) will
be for use together with radiotherapy (whereby this radiotherapy is
preferably whole brain radiotherapy or stereotactic radiosurgery).
30) Another main variant of this invention, combinable with any one
or more of the foregoing embodiments 1) to 29), is an endothelin
receptor antagonist for use in the treatment of an existing brain
metastasis tumor in a subject wherein the existing brain metastasis
tumor is a micrometastasis tumor such as micrometastasis tumor
selected from the group consisting of a lung cancer, breast cancer,
colon cancer, melanoma or renal carcinoma brain micrometastasis
tumor. 31) The invention also relates to a method of inhibiting an
astrocyte mediated protection of a brain metastasis cell, which
method comprises administering an effective amount of an endothelin
receptor antagonist to the brain metastasis cell and the astrocyte
to inhibit the astrocyte mediated protection. 32) The invention
further relates to the method of embodiment 31), which further
comprises administering an effective amount of at least one
cytotoxic chemotherapeutic agent to the brain metastasis cell. 33)
The invention furthermore relates to the method of embodiment 31),
which further comprises submitting the brain metastasis cell to
radiotherapy (whereby this radiotherapy is preferably whole brain
radiotherapy or stereotactic radiosurgery). 34) The invention
moreover relates to the method of embodiment 31), which further
comprises administering an effective amount of at least one
cytotoxic chemotherapeutic agent to the brain metastasis cell and
submitting the brain metastasis cell to radiotherapy (whereby this
radiotherapy is preferably whole brain radiotherapy or stereotactic
radiosurgery). 35) The invention furthermore relates to a method of
manufacturing a medicament for use according to any of the
foregoing 1) to 34) and any combinations thereof. Preferably, the
medicament produced by the foregoing is further packaged in a
commercial package with instruction for carrying out one or more of
1)-34), and any combinations thereof. 36) The invention further
relates to an endothelin receptor antagonist for use in the
reduction of the risk of and/or reducing the rate of expansion of
brain metastases, including brain micrometastasis, in combination
with a cytotoxic chemotherapy agent, radiotherapy or both,
according to one or more of 1)-34), and any combinations
thereof.
EXPERIMENTAL SECTION
Experiment 1--Immunofluorescent Analysis of Brain Metastasis
Materials and Methods
[0052] Experimental brain metastases were produced by the injection
of human lung adenocarcinoma cells PC14Br4 into the internal
carotid artery of nude mice (S1). Mice were killed 5 weeks later
and tissue samples were processed in OCT compound for frozen
section as previously described (S2). Tissues were sectioned (8-10
.mu.m), mounted on positively charged slides, and air-dried for 30
minutes. Tissue fixation was performed using a protocol consisting
of three sequential immersions in ice-cold solutions of acetone,
50:50 (v/v) acetone:chloroform, and acetone (5 minutes each).
Samples were then washed three times with PBS, incubated with
protein blocking solution containing 5% normal horse serum and 1%
normal goat serum in PBS for 20 minutes at room temperature, and
then incubated with a 1:400 dilution of rabbit anti-GFAP polyclonal
antibody (Biocare Medical, Concord, Calif.) for 18 hours at
4.degree. C. The samples were rinsed four times with PBS for 3
minutes each and then incubated for 1 hour with a 1:1500 dilution
of goat anti-rabbit Cy5 antibody (Jackson ImmunoResearch
Laboratories, West Grove, Pa.). Control samples were labeled with
an identical concentration of isotype control antibody and goat
anti-rabbit Cy5 antibody. All samples were rinsed and then briefly
incubated with sytox green nucleic acid stain (Eugene, Oreg.). The
slides were mounted with a glycerol/PBS solution containing 0.1
mol/L propyl gallate (Sigma) to minimize fluorescent bleaching.
Confocal images were collected on a Zeiss LSM 510 laser scanning
microscope system (Carl Zeiss, Inc., Thornwood, N.Y.) equipped with
a motorized Axioplan microscope, argon laser, HeNe laser, LSM 510
control and image acquisition software, and appropriate filters
(Chroma Technology Corp., Brattleboro, Vt.).
[0053] 18 Composite images were constructed with Photoshop software
(Adobe Systems, Inc., Mountain View, Calif.).
Results
[0054] The results were determined from color images (not shown) of
immunohistochemically reacted samples. Tumor cells were surrounded
by and infiltrated with GFAP-positive (red) astrocytes. This
experiment confirmed prior observations of astrocyte infiltration
of clinical brain metastasis samples. The same infiltration by
GFAP-positive astrocytes were seen with a syngenic murine mouse
model. Immunohistochemical analysis of mouse Lewis lung carcinoma
(3LL) in the brain of a C57 mouse. Dividing 3LL cells
(PCNA-positive, blue) were infiltrated and surrounded by activated
astrocytes (GFAP-positive, brown). Together, these experiments
validated the murine-human xenograft model which reproduced the
phenomenon seen in clinical human samples and in the syngenic mouse
model. One aspect of the present invention is therefore an in vivo
mouse-human brain metastasis cell model which is useful, for
example, in studying the phenomenon of human metastatic brain
cancer.
Experiment 2--Scanning Electron Microscopy Studies
[0055] Having established that murine astrocytes interact in vivo
with human tumor cells in the formation of brain metastasis in the
same way as is seen in primary human clinical specimens, the
Inventors examined the interactions of these two cell types in
vitro in a simplified co-culture system. It was uncertain whether
such a system would result in any cell-to-cell interaction, much
less having direct physiological relevance to the in vivo situation
described above. It was therefore a surprising discovery that
intercellular interactions between human metastatic cancer cell
lines and murine astrocytes were achieved in vitro.
Materials and Methods
[0056] Cell Lines and Culture Conditions.
[0057] Human breast cancer cell line, MDA-231 (S3), a brain
metastatic variant of human lung adenocarcinoma cell line PC14Br4
(Si) and murine NIH 3T3 fibroblasts were maintained as monolayer
cultures in a complete Eagle's minimum essential medium (CMEM)
supplemented with 10% fetal bovine serum (FBS; HyClone, Logan,
Utah), L-glutamine pyruvate, nonessential amino acids, two-fold
vitamin solution, and penicillin streptomycin (GIBCO/Invitrogen,
Carlsbad, Calif.). All reagents used for tissue culture were free
of endotoxin as determined by the limulus amebocyte lysate assay
(Associate of Cape Cod, Woodshole, Mass.), and the cell lines were
free of the following murine pathogens: Mycoplasma spp, Hantan
virus, hepatitis virus, minute virus, adenovirus (MAD1, MAD2),
cytomegalovirus, ectromelia virus, lactate dehydrogenase-elevating
virus, polyma virus, and Sendai virus (assayed by the Research
Animal Diagnostic Laboratory, University of Missouri, Columbia,
Mo.). Cells used in this study were from frozen stock and all
experiments were carried out within 10 in vitro passages after
thawing.
[0058] Isolation and Maintenance of Murine Astrocytes.
[0059] Neonatal mice homozygous for a temperature-sensitive SV40
large T antigen (H-2Kb-tsA58 mice; CBA/ca.times.C57BL/10 hybrid;
Charles River Laboratories, Wilmington, Mass.) were euthanized in a
carbon dioxide chamber, and the skin was prepared for surgery in
standard fashion (S4). Sterile micro forceps (Roboz Surgical
Instrument Co., Gaithersburg, Md.) were used to remove the skin
from the skull, and microscissors were used to create a circular
posterior incision from the opening of the left ear to the opening
of the right ear. Another incision was made along the brain
midline, and the skull was divided to allow access to the cranial
cavity. The optic nerves were clipped and the brain removed with
blunt forceps and placed into 100-mm ice-cold phosphate buffered
saline (PBS). Whole neocortices were dissected, and the hippocampus
and internal structures were removed to leave only the cortical
sheets. The meninges were stripped away, and the cortical sheets
were minced with a scalpel and digested for 30 minutes at
37.degree. C. in Dulbecco's modified essential medium (DMEM)
containing 0.1% collagenase (150 U/ml; Worthington Biochemical
Corp., Lakewood, N.J.) and 40 pg/ml deoxyribonuclease (Sigma
Chemical Co., St. Louis, Mo.). The cortical tissue was then
triturated in DMEM containing 10% FBS and filtered through a 50-pm
sterile mesh. The resulting single-cell suspension was plated onto
75-cm 2 tissue culture flasks that had been previously coated with
5 .mu.g/ml mouse laminin (Sigma). The cells were allowed to grow
for 7 days in DMEM containing 10% FBS and supplements (see above)
in an atmosphere of 8% carbon dioxide (to achieve a proper
buffering of pH at 33.degree. C.). At this time, astroglial cells
formed a confluent monolayer with neurons, oligodendrocytes, and
fibroblasts growing on top. These contaminating cells were removed
by rotary shaking the flasks overnight at 250 revolutions per
minute in a warm room. The resulting cultures were composed of more
than 95% astrocytes as determined by immunoreactivity with
antibodies directed against GFAP. These cultures were expanded, the
procedure was repeated, and the percentage of astrocytes in these
cultures was determined to exceed 99%.
[0060] Scanning Electron Microscope Imaging of Cultured Tumor Cells
and Astrocytes.
[0061] Human breast cancer MDA-MB-231 cells and murine astrocytes
were plated in DMEM containing 5% FBS onto sterilized glass
coverslips in 24 well plates at a density of 2.4.times.10.sup.4
cells. After 48 hours, the coverslips were removed and fixed for 1
hour at room temperature in a solution containing 3%
glutaraldehyde/2% parpformaldehyde/7.5% sucrose in 0.1 M cacodylate
buffer (pH 7.3). The samples were then washed with 0.1 m cacodylate
buffer and post-fixed for 1 hour with 1% cacodylate buffered osmium
tetroxide containing 7.5% sucrose. The samples were washed with 0.1
M cacodylate buffer followed by distilled water and sequentially
treated for 30 minutes in the dark with Millipore-filtered aqueous
1% tannic acid, washed in distilled water and Millipore-filtered 1%
aqueous uranyl acetate for 1 hour in the dark. The samples were
rinsed thoroughly with distilled water, dehydrated through an
ascending series of ethanols, and then transferred for 5 minutes
each to a graded series of increasing concentrations of
hexamethyldisilazane and allowed to air dry overnight. Samples were
mounted onto double-thick carbon tabs (Ted Pella, Inc., Redding,
Calif.) that had previously been mounted onto aluminum specimen
mounts (Electron Microscopy Sciences, Ft. Washington, Pa.). The
samples were then coated under vacuum using a Balzer MED 010
evaporator (Technotrade International, Manchester, N.H.) with
platinum alloy for a thickness of 25 nm and then immediately flash
carbon coated under vacuum. The samples were transferred to a
desiccator for examination at a later date. Samples were examined
using a JSM-5910 scanning electron microscope (JEOL, Inc., Peabody,
Mass.) at an accelerating voltage of 5 kV.
Results
[0062] The results are shown in FIG. 1. In vitro culture of
MDA-MB-231 breast cancer cells (T) and murine astrocytes (A) were
evaluated by scanning electron microscopy. Direct contact between
the astrocytes (extending pods-feet) and tumor cells is evident. A
single astrocyte can contact multiple tumor cells. These appear to
be fully formed gap junctions of the kind seen between astrocytes
and neurons of the central nervous system. Similar results are seen
with melanoma, breast cancer and lung cancer cell lines (not
shown).
Experiment 3--Gap Junction Assays
[0063] To further validate the functional nature of the gap
junctions shown in FIG. 1, The Inventors performed dye transfer
experiments to ascertain whether the gap junction like structures
seen in Experiment 2 were functional.
[0064] Materials and Methods
[0065] Gap Junction Communication.
[0066] Gap junction communication between recipient tumor cells
(MDA-MB-231) and donor cells (astrocytes, 3T3 cells, MDA-MB-231)
was analyzed by flow cytometry measuring the transfer of dye.
Briefly, recipient cells (300,000 cells/well) were plated into a
6-well plate and cultured overnight. At that time, donor cells were
labeled for 45 minutes with 1 .mu.g/ml green calcein-AM (Molecular
Probes) followed by extensive washing. Donor cells (60,000
cells/well) were co-cultured for 5 hours with recipient cells
either directly or in a transwell chamber (Transwell-Boyden
Chamber, 0.4 p.m pore size; Costar, Corning, N.Y.). Cells were
harvested, washed, fixed in ethanol, and analyzed by flow
cytometry. Gap junction formation was calculated as the percent of
shifted FITC peak (S9-S11).
Results
[0067] As shown in FIG. 2, the astrocyte-metastatic cancer cell
co-cultures showed dye transfer between co-cultured cells. The
Inventors' co-culture system thus results in active gap junctions
forming between murine astrocytes and human metastatic cancer cell
lines. The control transwell experiments demonstrate this to be a
genuine cell-cell interaction. A second aspect of the present
invention is therefore an in vitro mouse astrocyte-human metastasis
cell co-culture system which is useful, for example, in studying
the phenomenon of human metastatic brain cancer interaction with
astrocytes in a manipulable ex vivo setting.
Experiment 4--Chemoprotection Assays
[0068] Modeling of the chemoresistance of brain metastasis is and
will be a major use for both the foregoing in vivo model system and
the in vitro cell co-culture system. Because the cell based
co-culture system is more amenable to experimental manipulation, it
was further assessed to determine if the chemoresistance of brain
metastases seen in vivo was replicated by the cell based culture
system.
Material and Methods
[0069] In Vitro Co-Culture Chemoprotection Assay.
[0070] Astrocytes and NIH 3T3 fibroblasts were transfected with GFP
genes as previously described (S5, S6). Target tumor cells,
astrocytes, or 3T3 fibroblasts were harvested from a 60-70%
confluent culture by a brief (2-minute) exposure to 0.25% trypsin
in a 0.1% EDTA/PBS solution. The cells were dislodged by tapping
the culture flasks briskly and resuspended in CMEM. The murine
astrocytes, 3T3 fibroblasts, and tumor cells were plated alone or
as co-culture at a tumor cell/astrocyte/3T3 cell ratio of 1:2 onto
each of the 35-mm diameter well of the sterile 6-well tissue
culture multi-well dish. The cells were allowed to adhere overnight
in a humidified 37.degree. C. incubator in an atmosphere of 6.4%
carbon dioxide plus air. The cultures were then washed and
incubated with fresh CMEM (negative control) or medium containing
various concentrations of TAXOL.RTM. (Paclitaxel; NDC 0015-3476-30,
Bristol-Myers Squibb, Princeton, N.J.) and other chemotherapeutic
drugs (see below). After 72 hours, the GFP labeled astrocytes or
NIH 3T3 cells were sorted out and the apoptotic fraction of tumor
cells was determined by propidium iodide staining and FACS analysis
(see below). To determine whether direct contact between tumor
cells and astrocytes (or fibroblasts serving as control) was a
prerequisite to produce induction of resistance to chemotherapy, we
performed the co-culture assay using a Transwell-Boyden chamber,
i.e., plating the tumor cells in the chamber and the
ImmortoAstrocytes (or fibroblasts) in the well. After 72 hours of
incubation, the relative apoptotic index of the tumor cells was
determined as described below.
[0071] In the second set of in vitro studies, we determined whether
astrocyte-mediated induction of tumor cell resistance to
chemotherapeutic drugs is transient or permanent. The human lung
cancer PC14Br4 cells were co-cultured with either astrocytes or 3T3
fibroblasts in medium alone or medium containing 5 ng/ml
paclitaxel. After 72 hours, the astrocytes or 3T3 cells were
separated from tumor cells by FACS, and the relative apoptotic
index of the tumor cells was determined in multiple wells by
propidium iodide staining as described below. From parallel wells,
we harvested surviving tumor cells and re-plated them on different
monolayers of astrocytes or 3T3 cells. These co-cultures were of
tumor cells first co-cultured with astrocytes and then with either
astrocytes or 3T3 cells, or of tumor cells first cultured with 3T3
cells and then with either 3T3 cells or astrocytes. The second
round of co-cultures then received media containing 5 ng/ml of
paclitaxel. After 72 hours, the relative apoptotic index of tumor
cells was determined by propidium iodide staining and FACS
analysis.
[0072] Preparation for Propidium Iodide Staining and FACS
Analysis.
[0073] The supernatant medium containing floating cells were
collected from each dish into a 15-ml conical centrifuge tube. The
attached cells were harvested by briefly exposing the cells to
0.25% trypsin in a solution containing 0.1% EDTA/PBS. Cells were
combined with the corresponding supernatant medium. The samples
were pelleted by centrifugation at 100 g for 5 minutes. The pellets
were resuspended in 10 ml of HBSS and further pelleted at 100 g for
5 minutes. The samples were resuspended by vortex and the cells
fixed in 1 ml of 1% paraformaldehyde for 10 minutes at room
temperature. The samples were then transferred into polypropylene
microcentrifuge tubes and the fixed cells were washed in 1 ml of
PBS and then pelleted at 10,000 g for 1 minute. The pellets were
resuspended by vortex and the cells fixed overnight in 1 ml of
ethanol at -20.degree. C. The cells were subsequently vortexed and
pelleted by a microcentrifuge at 10,000 g for 1 minute. The samples
were then vortexed and the pellets resuspended and stained in 300
of propidium iodide (50 .mu.g/ml; Cat. P4864, Sigma) containing
RNAse (15 .mu.g/ml; Cat. A7973, Promega, Madison, Wis.) for 20-30
minutes at 37.degree. C. The samples were finally transferred to
5-ml plastic culture tubes for FACS analysis using a Coulter EPICS
Cytometer (Beckman Coulter, Inc., Fullerton, Calif.). Relative
apoptotic index was determined by comparing the apoptotic index of
tumor cells/apoptotic index of tumor cells and ImmortoAstrocytes or
tumor cells and NIH 3T3 fibroblasts.times.100 (%) (S7).
Results
[0074] Culturing of human MDA-MB-231 breast cancer cells or human
PC14Br4 lung cancer cells with astrocytes (but not 3T3 fibroblasts)
reduced the relative apoptotic index (i.e. increased
chemoresistance) of tumor cells incubated for 72 hours with
paclitaxel (5 ng/ml) by 58.3+8.9% (mean.+-.S.D., P<0.01) and
61.8.+-.6.7% (mean.+-.S.D., P<0.05), respectively (the apoptotic
index was compared by the Student's t test) (FIG. 3). This
reduction was dependent on direct contact between tumor cells and
astrocytes. The Inventors base this conclusion on the data showing
that when tumor cells and astrocytes were separated by a
semi-permeable membrane (Transwell-Boyden Chamber, 0.4 pm pore size
membrane; Costar, Corning, N.Y.), the chemoprotective effect of
astrocytes was not observed. Co-culture of tumor cells with 3T3
fibroblasts did not protect tumor cells from chemotherapy (FIG. 3).
Co-culture of human tumor cells with an alternative control using
fibroblasts isolated from the H-2k.sup.b-tsA58 mouse also did not
protect the tumor cells from chemotherapeutic agents (data not
shown). Analogous results were seen using standard MTT assays to
determine the degree of cytotoxicity (data not shown). [Cory A H,
Owen T C, Barltrop J A, Cory J G (July, 1991) "Use of an aqueous
soluble tetrazolium/formazan assay for cell growth assays in
culture." Cancer Communications 3 (7): 207-212.]
[0075] These data validate the cell based co-culture system
disclosed herein as reproducing the phenomenon of brain metastasis
chemoresistance. The unexpected ability of the co-culture system to
replicate chemoresistance thus renders it well suited for use in
studying the mechanism of chemoresistance and potential therapeutic
interventions for abrogating chemoresistance in existing brain
metastasis in vivo.
[0076] To expand upon these initial experiments, the Inventors have
tested several additional chemotherapy agents, representative of
the major classes of drugs in use to day. Human lung cancer PC14Br4
cells were cultured alone or with astrocytes (direct cell to cell
contact) in medium containing P-glycoprotein-associated Adriamycin
(200 ng/ml), paclitaxel (5 ng/ml), vinblastine (3 ng/ml),
vincristine (8 ng/ml), and P-glycoprotein-dissociated 5-FU (500
ng/ml) or cisplatinum (2.4 .mu.g/ml). Co-culture with astrocytes
induced significant (P<0.01) protection against all drugs (FIG.
4). This unexpected finding further validates the cell based
co-culture system. The cell culture system demonstrates the
robustness of the chemoresistance induced by astrocytes in a manner
which is directly comparable to brain metastasis chemoresistance
seen in vivo in the clinical setting. A third aspect of the present
invention is therefore an in vitro cell based chemoresistance assay
which is useful, for example, in studying the phenomenon of
astrocyte mediated protection of brain metastasis cells from
cytotoxic chemotherapy induced cell death. In particular
embodiments the in vitro cell based chemoresistance assay may be
used to screen one or more candidate chemotherapy agents to assess
the degree of astrocyte mediated protection against the cytotoxic
effects of the chemotherapy agents.
[0077] Additional experiments summarized in FIG. 5 demonstrate that
astrocyte-mediated protection of brain metastasis cells from
cytotoxic chemotherapy-induced cell death does not last longer than
72 hours after direct astrocyte-brain metastasis cell contact is
lost. Further, cells having lost the protective effect afforded by
prior astrocyte contact can be re-protected by a second
co-culturing with astrocytes to reacquire astrocyte-mediated
protection against the cytotoxic effects of the chemotherapy
agents. This reflects the clinical observations of primary tumors
and even other non-brain metastasis being chemoresponsive while
their derived brain metastasis are chemoresistant.
Experiment 5--Mechanism of Astrocyte-Mediated Chemoresistance
[0078] The Inventors applied the above cell-based chemoresistance
assay to investigate potential mechanisms underlying the
phenomenon. Gene expression profiling plus Western blot
confirmation of protein production were used to investigate
astrocyte-mediated protection of brain metastasis cells from
cytotoxic chemotherapy-induced cell death.
Materials and Methods
Gene Expression Profiles by RNA Microarray Analysis.
[0079] In the first set of experiments, MDA-MB-231 or PC14Br4 cells
were incubated alone, with murine astrocytes, or with NIH 3T3
fibroblasts in a 35-mm diameter 6-well plate (Cat. 353046, BD
Falcon.TM., San Jose, Calif.). To ascertain cell-to-cell contact,
the ratio of tumor cells to murine astrocytes or NIH 3T3 cells was
1:2. After 72 hours, GFP-labeled murine astrocytes or fibroblasts
were sorted out by FACS, and the tumor cells were processed for
microarray analyses. In the second set of experiments, we
determined whether the expression of genes associated with tumor
cell resistance to chemotherapeutic drugs was dependent on a
continuous contact with astrocytes. MDA-231 or PC14Br4 cells were
co-cultured with either murine astrocytes or NIH 3T3 cells for 72
hours. The murine astrocytes or NIH 3T3 cells were sorted out, and
tumor cells were either processed to determine gene expression
profiles by microarray analyses or plated for a second round of
co-culture with either murine astrocytes or fibroblasts. Thus,
tumor cells first cultured with murine astrocytes were co-cultured
again with murine astrocytes or with fibroblasts and, conversely,
tumor cells initially cultured with fibroblasts were co-cultured
again with fibroblasts or astrocytes. After a 72-hour incubation,
murine astrocytes or fibroblasts were sorted out and the tumor
cells were processed for microarray analyses.
[0080] Microarray Analyses.
[0081] Total RNAs (500 ng) were used for labeling and hybridization
according to the manufacturer's protocols (Illumina, Inc., San
Diego, Calif.). Briefly, cDNA was generated from total RNA using
IlluminaR Total Prep RNA Amplification Kit (Applied Biosystem,
Austin, Tex.). Next, in vitro transcription was carried out to
incorporate biotin-labeled nucleotides into cRNA for 4 hours at
37.degree. C. A total of 1500 ng of biotin-labeled cRNA was
hybridized to Illumina's SentrixR human 6-v2 Expression BeadChips
at 58.degree. C. overnight (16 hours) according to the
manufacturer's instructions. The hybridized biotinylated cRNA was
detected with 1 .mu.g/ml cyanine 3-streptavidine (GE Healthcare,
Piscataway, N.J.), and the BeadChips were scanned with Illumina
BeadArray Reader (Illumina, Inc.). The results of microarray data
were extracted with Bead Studio 3.7 (Illumina, Inc.) without any
normalization or background subtraction. Gene expression data were
normalized using quantile normalization method in LIMMA package in
R (www.r-project.org) (S12). The expression level of each gene was
transformed into a log 2 before further analysis. To select genes
that are differentially expressed in two groups of tissues, we used
a class comparison tool in BRB Array Tools (v 3.6; Biometrics
Research Branch, National Cancer Institute, Bethesda, Md.) as a
method for two sample t-tests with the estimation of FDR.
[0082] Western Blot Analysis.
[0083] The Western blot was used to confirm the results of the
microarray. Briefly, whole-cell lysates of FACS-sorted tumor cells
were prepared using 1 ml of lysis buffer (10 mM Tris [pH 8.0], 1 mM
EDTA, 0.1% SDS, 1% deoxycholate, 1% NP40, 0.14 M NaCl, 1 .mu.g/ml
leupeptin, 1 .mu.g/ml aprotinin, and 1 .mu.g/ml pepstatin)
containing a protease inhibitor mixture (Roche, Indianapolis,
Ind.). Samples containing equal amounts of protein (30 fag) were
separated by electrophoresis on 4-12% Nu-PAGE gels (Invitrogen) and
transferred to nitrocellulose membranes. After blocking with TTBS
(TBS+0.1% Tween 20) containing 5% non-fat milk, the membranes were
incubated at 4.degree. C. overnight with mouse monoclonal antibody
against BCL2 (1:1,000, BD PharMingen, San Diego, Calif.), rabbit
polyclonal antibody against BCL2L1 (1:1,000, Cell Signaling,
Beverly, Mass.), rabbit polyclonal antibody against TWIST (1:1000,
Cell Signaling), mouse monoclonal antibody against glutathione
S-transferase (1:1,000, BD PharMingen), and mouse monoclonal
antibody against I3-actin (Sigma). Blots were then exposed to
horseradish peroxidase-conjugated secondary antibodies (1:3000) and
visualized by the enhanced chemiluminescence system from Amersham
(Piscataway, N.J.). Equal protein loading was confirmed by
stripping the blots and re-probing them with an anti-13-actin
antibody.
[0084] Statistical Analysis.
[0085] For statistical analysis of gene expression profiles, the
expression level of each gene was transformed into a log 2 before
further analysis. Class comparison tool in BRB Array Tools (v3.6;
Biometrics Research Branch, National Cancer Institute, Baltimore,
Md.) for a two-sample t test with the estimation of FDR was the
method used to determine the statistical significance of
differentially expressed genes between tumor cells co-cultured with
different host cells. Genes for Venn diagram were selected by
univariate test (two-sample t test) with multivariate permutation
test (10,000 random permutations). We applied a cut-off P-value of
less than 0.001 to retain genes whose expression is significantly
different between two groups of tissues examined.
Results
[0086] The Inventors identified tumor cell genes whose expression
is commonly altered subsequent to co-culture with astrocytes by
applying two-sample t tests (P<0.001). Using this procedure, the
Inventors identified in the MDA-MB-231 cells, 1069 genes, and in
the PC14Br4 cells, 594 genes that were differentially expressed
(FIG. 7). A two-gene list comparison revealed increased expression
of several genes well known to be associated with anti-apoptosis
and survival: glutathione S transferase 5 (GSTA5), BCL2L1, TWIST
(FIG. 7). The expression of these genes was confirmed at the
protein level by Western blot analysis (FIG. 8). The Inventors then
determined whether the altered gene expression pattern in tumor
cells co-cultured with astrocytes (but not fibroblasts) was
permanent or transient. The Inventors co-cultured tumor cells for
one cycle with astrocytes or fibroblasts, and then harvested the
tumor cells and plated them in a second round on astrocytes or
fibroblasts. When the gene expression data were collected from the
two cycles of co-culture experiments, the influence of the second
co-culture was dominant in gene expression patterns of the cancer
cells. Regardless of the first co-culture condition, cancer cells
co-cultured with astrocytes in the second cycle exhibited a
distinctive gene expression signature that was detected in the
first cycle culture experiments (high expression of GSTA5, BCL2L1,
and TWIST), whereas cancer cells co-cultured with astrocytes in the
first round lost the specific gene expression signatures when they
were co-cultured with fibroblasts in the second round (FIG. 9).
This result parallels that of the in vitro chemoprotection assay
results summarized in FIG. 5 and proves that the gene expression
pattern in the tumor cells depends on constant contact with the
astrocytes. Tumor cells co-cultured in the second round with
astrocytes also expressed a high level of TCF4, CD24, CARD14, and
EFNB2 genes (data not shown). Clinical studies have shown that
tumor cell expression of these genes is correlated with a poor
prognosis. A fourth aspect of the present invention is therefore an
in vitro cell based chemoresistance assay having a molecular
diagnostic component which is useful, for example, in studying the
phenomenon of astrocyte mediated protection of brain metastasis
cells from cytotoxic chemotherapy induced cell death in an ex vivo
setting. In certain embodiments, the molecular diagnostic component
is one or more of a gene expression profiling step and an analysis
of cellular protein concentrations. In specific embodiments, a
predetermined gene expression signature is used to evaluate the
effects of experimental interventions to, e.g., abrogate astrocyte
mediated protection. A fifth aspect of the invention is a gene
expression signature or a combination of protein level profiles
indicative of astrocyte mediated protection of brain metastasis
cells from cytotoxic chemotherapy induced cell death. A sixth
aspect is the process of producing said gene signature or protein
level profiles as described and exemplified above.
Experiment 6--Expression of Endothelin Receptors by Tumor Cells and
Astrocytes
[0087] 300,000 MDA-MB-231 human breast cancer cells were cultured
for 24 hours with 600,000 GFP-labeled astrocytes or with
GFP-labeled 3T3 fibroblasts. The cells were then collected and
sorted to isolate the MDA-MB-231 cells. Proteins were extracted,
analyzed by Western blots, and hybridized with anti-ETAR antibody.
The data shown in FIG. 10 clearly demonstrate that tumor cells
co-cultured with astrocytes express a higher level of ETAR.
[0088] In the next set of studies, 10,000 GFP-labeled MDA-MB-231
cells were co-cultured with 20,000 astrocytes in chamber slides.
Twenty-four hours later, the cultures were treated for 24 hours
with Taxol (15 ng/ml) and then stained for phosphorylated pAKT (4%
PFA fixation). As shown in FIG. 11, tumor cells co-cultured with
astrocytes (and Taxol) expressed high levels of pAKT. Hence,
co-culture with astrocytes produces increased expression of ETR and
survival factors by tumor cells which are correlated with tumor
cell increased resistance to chemotherapeutic drugs. Similar
studies with an anti-ETBR antibody revealed ETBR expression.
Experiment 7--Endothelin Receptor Antagonists do not Produce
Cytotoxic Effects Against Tumor Cells
[0089] Using the in vitro cell based chemoresistance assay scheme
described above, the Inventors tested two endothelin receptor
antagonists having dual ETAR and ETBR affinity to assess the degree
of astrocyte-mediated protection against the cytotoxic effects of
the chemotherapy agent. One ETR antagonist tested was Bosentan
which is approved by the EMEA for use in the treatment of pulmonary
artery hypertension (PAH). The second drug tested was designated
ACT-064992 and is a derivative of Bosentan also having dual
ETAR/ETBR affinity. ACT-064992 is formally designated Macitentan,
and has the structure
[N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4--
yl]-N'-propylaminosulfonamide]:
##STR00001##
[0090] Iglarz M, et al., Pharmacology of macitentan, an orally
active tissue-targeting dual endothelin receptor antagonist, J
Pharmacol Exp Ther. 2008 December; 327(3):736-45. Epub 2008
September 9. The original disclose of the ACT-064992 molecule, its
synthesis and its pharmacological activity may be found in
WO02/053557. ACT-064992 is roughly three times more
pharmaceutically active than Bosentan (i.e. it requires 1/3 the
dose). The detailed data herein refer to the ACT-064992
experiments, however analogous results are achieved by higher dose
Bosentan experiments.
[0091] ACT-064992 (100 nM) was added to the cell-based co-culture
assay described above with a tumor cell:astrocyte cell ratio of 1:2
or tumor cell:3T3 fibroblasts for 48 hours, and the degree of
apoptosis was measured as described above. As shown in FIG. 12
(MDA-231 breast cancer) and FIG. 13 (PC14 lung cancer), ACT-064992
added alone or with astrocytes or with 3T3 fibroblasts did not
produce any measurable cytotoxic effects.
Experiment 8--Endothelin Receptor Antagonists in Combination with
Chemotherapeutic Agents
[0092] ETR antagonists as shown above were ineffective as a single
agent chemotherapy. This negative result made the dramatic effect
seen with ETR antagonists as a component of combination therapies
highly unexpected. In co-culture experiments, cell culture ratios
were 1:2 tumor cell:astrocyte cell (50,000:100,000), and treatments
were using paclitaxel (TAXOL.RTM.) (6 ng/ml) and/or ACT-064992 (100
nM) for 48 hours (FIG. 15; *p<0.01). For the control experiments
using MDA-MB-231 cells, the same astrocyte-mediated protection from
paclitaxel occurred as in prior experiments. There were two
surprising results from these combination experiments. First, the
combination of paclitaxel and ACT-064992 in the controls lacking
astrocytes did not yield a significant increase in cell death over
paclitaxel alone. This was seen in at least three independent
experiments (FIG. 15). These results were reproduced using human
lung cancer cells PC14Br4 (FIG. 16). Thus, under the conditions
tested, ETR antagonists were also ineffective in combination as was
observed in Experiment 6 as a single agent. In the tumor
cell-astrocyte co-cultures, ACT-064992 showed the unexpected
ability to negate the astrocyte-mediated protection from
paclitaxel. Even more surprising, ACT-064992 actually enhanced the
efficacy of paclitaxel, as compared to control experiments without
astrocytes, to super-sensitize the metastatic tumor cells to
paclitaxel. The addition of ACT-064992 to co-cultures of astrocytes
and tumor cells inhibited the expression of the survival factors
pAKT and pMAPK (FIG. 17). This inhibition was directly correlated
with the increased tumor cell death mediated by the
chemotherapeutic drug. This synergism is made even more surprising
because the experiments lacking astrocytes did not demonstrate even
additive effects. These dramatic results demonstrate the importance
of the new co-culture screening methods disclosed herein because
the effects of ETR antagonists in combination therapy would not
have been seen in a standard tumor cell line assay scheme lacking
co-cultured astrocytes. A seventh aspect of the present invention
is thus the use of endothelin receptor antagonists in combination
with one or more other cytotoxic chemotherapy agents and/or
radiation therapy to treat an existing brain metastasis tumor in a
subject. In particular embodiments, this combination therapy
super-sensitizes the existing brain metastasis to the cytotoxic
chemotherapy agents and/or radiation therapy co-administered with
the ETR antagonist.
In Vivo Endothelin Receptor Antagonist Therapy for Existing Brain
Metastasis
[0093] In vivo delivery of endothelin receptor antagonist(s) and
co-administered chemotherapy agents may be achieved by the same
oral and or systemic delivery already developed and used in prior
clinical trials for various members of this class of compounds. The
development and optimization of such therapeutic regimens is
routine in the art. [Clinical trials: a practical guide to design,
analysis and reporting Edited by Ameet Bakhai and Duolao Wang.
Remedica, London 2005.] One issue specific to brain cancers is the
impact of the blood brain barrier. While this has been long cited
as a technical problem precluding systemic treatments for brain
metastasis, the art has now recognized that even in
micrometastasis, the blood brain barrier is partially disrupted.
[Cavaliere R. and Schiff D., Chemotherapy and cerebral metastases:
misperception or reality? Neurosurg Focus. 2007 March 15;
22(3):E6.] Thus it is reasonable to expect that systemic delivery
of endothelin receptor antagonist(s) will penetrate and have
therapeutic effect in brain metastasis tumors. In addition, certain
members of the endothelin receptor antagonist(s) family have the
ability to cross even intact blood brain barrier, thus rendering
them particularly suited for use in brain metastasis tumor therapy
in vivo. [Vatter H, et al., Cerebrovascular characterization of
clazosentan, the first nonpeptide endothelin receptor antagonist
shown to be clinically effective for the treatment of cerebral
vasospasm. Part II: effect on endothelin(B) receptor-mediated
relaxation. J Neurosurg. 2005 June; 102(6): 1108-14.] Finally,
direct tumor infusion or injection of endothelin receptor
antagonist(s) may be suitable where the size of metastatic tumors
render such an approach feasible.
In Vivo Therapy for Brain Metastasis in Mice
[0094] To further validate the efficacy of endothelin receptor
antagonist therapy for existing brain metastasis, the Inventors
performed exemplary in vivo experiments using mice. Nude mice were
injected with 10,000 viable MDA231 cells mice by way of the
internal carotid artery. Preliminary pathology work revealed that
visible established metastases formed two weeks, post injection
(data not shown). The Inventors thus started treatments at this
time point.
Experiment 9--ACT-064992 and Temozolomide Effects on In Vivo Tumor
Growth
[0095] Nude mice were injected into the internal carotid artery
with 10,000 viable MDA231 cells. The treatment groups at two weeks
post injection were: Control (vehicle) (FIG. 18A); temozolomide
("TMZ") 10 mg/kg p.o., daily (FIG. 18B); ACT-064992 50 mg/kg, p.o.
daily (FIG. 18C); or combination TMZ+ACT-064992 (FIG. 18D).
[0096] All mice were killed on day 28 of treatment. Brain sections
were fixed and stained. Brain metastases were evaluated visually.
Exemplary specimens are shown in FIG. 18A-D at the same
magnifications. As can be seen in more detail in FIG. 18E, the
combination TMZ and ACT-064992 dramatically reduced the size and
density of metastasis tumors compared to TMZ alone. This
demonstrates that the above described cell culture system results
correlate directly with the observed effects in vivo.
Experiment 10--ACT-064992 and Paclitaxel Effects on In Vivo Tumor
Growth
[0097] Female nude mice (10-12 weeks old) were injected in the
internal carotid artery with 10,000 viable MDA231 cells. Two weeks
after the injection when brain metastases were established, the
mice were randomized into 4 treatment groups (n=10): 1) Control
(injected with vehicle solution); 2) Paclitaxel (8 mg/kg
administered intraperitoneally once per week); 3) ACT-064992 (50
mg/kg administered orally once per day); and 4) Combination of
ACT-064992 and Paclitaxel. All mice were euthanized after 28 days
of treatment and autopsied. The brains were collected for
histologic study and immunohistochemistry. Exemplary results are
shown in FIG. 19. A. Control (19A), ACT-064992 (19B) and Paclitaxel
(19C) all have well defined metastatic tumors. The
ACT-064992+Paclitaxel group (19D) in contrast had much smaller
colonies of tumor cells.
[0098] The results with temozolomide and paclitaxel demonstrate
that endothelin receptor antagonist therapy sensitizes brain
metastasis to chemotherapy agents generally.
Experiment 11--ACT-064992 and Paclitaxel Effects on In Vivo Tumor
Cell Proliferation
[0099] Representative brain slices from the four treatment groups
were stained for CD31 (endothelial cell marker) and Ki67 (cell
proliferation marker). The brains of control mice, mice treated
with only paclitaxel, or only ACT-064992 contained a large number
of proliferating cells. In sharp contrast, the brains of mice
treated with both paclitaxel and ACT-064992 contained only a few
dividing cells. FIG. 20A, B, C and D, respectively.
Experiment 12--ACT-064992 and Paclitaxel Effects on In Vivo Tumor
Cell Apoptosis
[0100] Brain slices of mice from different treatment groups were
analyzed by in situ Terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL). Negoescu A, et al., J Histochem Cytochem.
1996 September; 44(9):959-68. The brains of control, Taxol-treated,
and ACT-064992-treated mice had few to no apoptotic cells. FIG. 21
A-C. In sharp contrast, the brains of mice treated with the
combination of ACT-064992 plus Taxol had a large number of
apoptotic tumor cells (green) and endothelial cells (yellow) (FIG.
21 D).
Experiment 13
[0101] Brain slices of mice from the four treatment groups were
immunostained for ET.sub.AR (red) and phosphoserine (green)
colocalization produced orange-yellow color. Control brains
expressed phosphorylated ET.sub.AR as did brains from mice treated
with paclitaxel (FIG. 22A-B). Treatment with ACT-064992 alone, and
with ACT-064992+paclitaxel, prevented phosphorylation of the
ET.sub.AR. (FIG. 22C-D). This result confirms the correlation of
endothelin receptor antagonist activity and the sensitization
metastatic tumors to chemotherapy agents.
Experiment 14
[0102] Survival Study for Treatment of Experimental Human MDA231
Breast Cancer Brain Metastasis with ACT064992 and Taxol
Experimental Details
[0103] 5.times.10.sup.3 MDA231 cells were injected according to the
protocol in Experiment 10. Treatment began on day 14 after
injection according to the protocol in Experiment 10. A Survival
curve is drawn and the p-value derived to compare the statistical
significance among treatment groups.
Treatment Groups
[0104] Control (vehicle): daily oral administrations and once
weekly i.p. injections. Paclitaxel (5 mg/kg): daily oral vehicle
administrations and once weekly i.p. injection of Paclitaxel.
ACT064992 (10 mg/kg): daily oral ACT064992 administrations and once
weekly i.p. injections of Paclitaxel.
Results
[0105] Day of death (after treatment)
Control: 42, 43, 53, 60, 64, 68, 68, 69, 69
Paclitaxel: 49, 53, 60, 63, 74, 74, 78
ACT064992: 51, 63, 68, 75, 78, 82
[0106] Paclitaxel+ACT: 48
Test for the Determination of ET.sub.A or ET.sub.B IC.sub.50:
[0107] For competition binding studies, membranes of CHO cells
expressing human recombinant ET.sub.A or ET.sub.B receptors are
used. Microsomal membranes from recombinant CHO cells are prepared
and the binding assay made as previously described (Breu V., et al,
FEBS Lett. (1993), 334, 210).
[0108] The assay is performed in 200 .mu.L 50 mM Tris/HCl buffer,
pH 7.4, including 25 mM MnCl.sub.2, 1 mM EDTA and 0.5% (w/v) BSA in
polypropylene microtiter plates. Membranes containing 0.5 ug
protein were incubated for 2 h at 20.degree. C. with 8 pM
[.sup.125I]ET-1 (4000 cpm) and increasing concentrations of
unlabelled antagonists. Maximum and minimum binding are estimated
in samples without and with 100 nM ET-1, respectively. After 2 h,
the membranes are filtered on filterplates containing GF/C filters
(Unifilterplates from Canberra Packard S.A. Zurich, Switzerland).
To each well, 50 .mu.L of scintillation cocktail is added
(MicroScint 20, Canberra Packard S.A. Zurich, Switzerland) and the
filter plates counted in a microplate counter (TopCount, Canberra
Packard S.A. Zurich, Switzerland).
[0109] All the test compounds are dissolved, diluted and added in
DMSO. The assay is run in the presence of 2.5% DMSO which is found
not to interfere significantly with the binding. IC.sub.50 is
calculated as the concentration of antagonist inhibiting 50% of the
specific binding of ET-1.
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[0135] All References cited or otherwise identified herein are
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cited.
* * * * *