U.S. patent application number 10/691915 was filed with the patent office on 2004-07-15 for method and composition for preventing and treating solid tumors.
Invention is credited to Gulati, Anil.
Application Number | 20040138121 10/691915 |
Document ID | / |
Family ID | 32176655 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138121 |
Kind Code |
A1 |
Gulati, Anil |
July 15, 2004 |
Method and composition for preventing and treating solid tumors
Abstract
Compositions and methods of preventing or treating a breast
cancer are disclosed. The composition and method can utilize an
endothelin B agonist and a chemotherapeutic agent as active
ingredients to treat a solid tumor in mammals, including humans.
Alternatively, the composition and method can utilize an endothelin
B antagonist and an optional angiogenesis inhibitor to treat a
solid tumor in mammals, including humans.
Inventors: |
Gulati, Anil; (Naperville,
IL) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Family ID: |
32176655 |
Appl. No.: |
10/691915 |
Filed: |
October 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60420960 |
Oct 24, 2002 |
|
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Current U.S.
Class: |
514/13.3 ;
514/19.4; 514/19.5; 514/267 |
Current CPC
Class: |
A61K 31/519 20130101;
A61K 38/21 20130101; A61P 43/00 20180101; A61K 31/4745 20130101;
A61K 31/337 20130101; A61K 38/208 20130101; A61K 38/2013 20130101;
A61K 33/243 20190101; A61K 45/06 20130101; A61K 38/2285 20130101;
A61N 2005/1098 20130101; A61K 41/0038 20130101; A61K 38/05
20130101; A61K 31/704 20130101; A61K 31/00 20130101; A61P 13/08
20180101; A61P 35/00 20180101; A61P 9/00 20180101; A61K 35/60
20130101; A61K 31/28 20130101; A61K 31/28 20130101; A61K 2300/00
20130101; A61K 31/337 20130101; A61K 2300/00 20130101; A61K 31/4745
20130101; A61K 2300/00 20130101; A61K 31/704 20130101; A61K 2300/00
20130101; A61K 33/24 20130101; A61K 2300/00 20130101; A61K 38/21
20130101; A61K 2300/00 20130101; A61K 38/2285 20130101; A61K
2300/00 20130101; A61K 38/2013 20130101; A61K 2300/00 20130101;
A61K 38/208 20130101; A61K 2300/00 20130101; A61K 38/05 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/012 ;
514/267 |
International
Class: |
A61K 038/17; A61K
031/519 |
Claims
What is claimed is:
1. A method of treating a solid tumor comprising administering to a
mammal in need thereof a therapeutically effective amount of an
endothelin B agonist and a therapeutically effective amount of a
chemotherapeutic agent.
2. The method of claim 1 wherein the solid tumor is selected from
the group consisting of an ovarian tumor, a colon tumor, Kaposi's
sarcoma, a breast tumor, a melanoma, a prostate tumor, a
meningioma, a liver tumor, and a breast phyllode tumor.
3. The method of claim 2 wherein the solid tumor is a breast
tumor.
4. The method of claim 1 wherein the endothelin agonist is selected
from the group consisting of ET-1, ET-2, ET-3, BQ3020, IRL1620,
sarafotoxin 56c, [Ala.sup.1, 3, 11, 15]ET-1, and mixtures
thereof.
5. The method of claim 4 wherein the endothelin B agonist comprises
IRL1620.
6. The method of claim 1 wherein the chemotherapeutic agent is
selected from the group consisting of adriamycin, camptothecin,
carboplatin, cisplatin, daunorubicin, doxorubicin, alpha, beta, or
gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel,
topotecan, and mixtures thereof.
7. The method of claim 1 wherein the endothelin B agonist and the
chemotherapeutic agent are administered simultaneously.
8. The method of claim 7 wherein the endothelin B agonist and the
chemotherapeutic agent are administered from a single
composition.
9. The method of claim 7 wherein the endothelin B agonist and the
chemotherapeutic agent are administered from separate
compositions.
10. The method of claim 1 wherein the endothelin B agonist and the
chemotherapeutic agent are administered sequentially.
11. The method of claim 10 wherein the chemotherapeutic agent is
administered prior to the endothelin B agonist.
12. The method of claim 10 wherein the endothelin B agonist is
administered prior to the chemotherapeutic agent.
13. The method of claim 1 wherein the mammal is a human.
14. A composition comprising a chemotherapeutic agent, an
endothelin B agonist, and an optional excipient.
15. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin B agonist, and; (b) an insert
providing instructions for administration of (a) to treat a solid
tumor in a mammal; and (c) a container for (a) and (b).
16. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin B agonist; (b) a packaged
composition comprising a chemotherapeutic agent; (c) an insert
providing instructions for a simultaneous or sequential
administration of (a) and (b) to treat a solid tumor in a mammal;
and (d) a container for (a), (b), and (c)
17. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin B agonist and a
chemotherapeutic agent; (b) an insert providing instructions for
administration of (a) to treat a solid tumor in a mammal; and (c) a
container for (a) and (b).
18. A method of treating a solid tumor comprising administering to
a mammal in need thereof a therapeutically effective amount of an
endothelin B antagonist.
19. The method of claim 18 wherein the solid tumor is selected from
the group consisting of an ovarian tumor, a colon tumor, Kaposi's
sarcoma, a breast tumor, a melanoma, a prostate tumor, a
meningioma; a liver tumor, and a breast phyllode tumor.
20. The method of claim 19 wherein the solid tumor is a breast
tumor.
21. The method of claim 18 wherein the endothelin B antagonist
comprises a specific endothelin B antagonist.
22. The method of claim 18 wherein the endothelin B antagonist
comprises a balanced endothelin B antagonist.
23. The method of claim 18 wherein the endothelin B antagonist is
selected from the group consisting of compounds 1 through 74 of
Appendices A, B, and C.
24. The method of claim 18 wherein the endothelin B antagonist is
selected from the group consisting of compounds 1 through 22 of
Appendix A.
25. The method of claim 18 wherein the endothelin B antagonist is
selected from the group consisting of compounds 23 through 32 of
Appendix B.
26. The method of claim 18 wherein the endothelin B antagonist is
selected from the group consisting of compounds 33 through 74 of
Appendix C.
27. The method of claim 18 wherein the endothelin B antagonist is
selected from the group consisting of atrasentan, tezosentan,
bosentan, sitaxsentan, enrasentan, Ro468443, TBC10950, TBC10894,
A192621, A308165, SB209670, SB17242, A182086, (s)-Lu302872,
J-104132, TAK-044, Sarafotoxin 56c, IRL2500, RES7011, Aselacins A,
B, and C, Ro470203, Ro462005, sulfamethoxazole, cochinmicin I, II,
and III, L749329, L571281, L754142, J104132, CGS27830, PD142893,
PD143296, PD145065, PD156252, PD159020, PD160672, PD160874,
TM-ET-1, IRL3630, Ro485695, L75037, LU224332, PD142893, LU302872,
PD145065, Ro610612, SB217242, BQ788, and mixtures thereof.
28. The method of claim 18 wherein the endothelin B antagonist
comprises BQ788.
29. The method of claim 18 further comprising administering a
therapeutically effective amount of an angiogenesis inhibitor.
30. The method of claim 29 wherein the angiogenesis inhibitor is
selected from the group consisting of thalidomide, marimastat,
COL-3, BMS275291, squalamine, 2-ME, SU6668, neovastat, Medi522,
EMD121974, CAI, celecoxib, interleukin-12, IM862, TNP470, avastin,
gleevac, herceptin, and mixtures thereof.
31. The method of claim 29 wherein the endothelin B antagonist and
the angiogenesis inhibitor are administered simultaneously.
32. The method of claim 31 wherein the endothelin B antagonist and
the angiogenesis inhibitor are administered from a single
composition.
33. The method of claim 29 wherein the endothelin B antagonist and
the angiogenesis inhibitor are administered from separate
compositions.
34. The method of claim 29 wherein the and endothelin B antagonist
and the angiogenesis inhibitor are administered sequentially.
35. The method of claim 34 wherein the angiogenesis inhibitor is
administered prior to the endothelin B antagonist.
36. The method of claim 34 wherein the endothelin B antagonist is
administered prior to the angiogenesis inhibitor.
37. The method of claim 18 further comprising treating the solid
tumor with radiation and an optional radiosensitizer.
38. The method of claim 37 wherein the radiosensitizer is selected
from the group consisting of metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole,
mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide,
5-bromodeoxyuridine, 5-iododeoxyuridine, bromodeoxycytidine,
fluorodeoxyuridine, hydroxyurea, cisplatin, therapeutically
effective analogs and derivatives thereof, and mixtures
thereof.
39. The method of claim 18 wherein the mammal is a human.
40. A composition comprising (a) an endothelin B antagonist, (b) an
angiogenesis inhibitor, and (c) an optional excipient.
41. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin B antagonist; (b) a packaged
composition comprising an angiogenesis inhibitor; (c) an insert
providing instructions for a simultaneous or sequential
administration of (a) and (b) to treat a solid tumor in a mammal;
and (d) a container for (a), (b), and (c).
42. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin antagonist and an angiogenesis
inhibitor; (b) an insert providing instructions for administration
of (a) to treat a solid tumor in a mammal; and (c) a container for
(a) and (b).
43. An article of manufacture comprising: (a) a packaged
composition comprising an endothelin B antagonist; (b) an insert
providing instructions for administration of (a) to treat a solid
tumor in a mammal; and (c) a container for (a) and (b).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
patent application No. 60/420,960, filed Oct. 24, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to the prevention and
treatment of solid tumors, such as breast tumors, in a mammal,
either by administration of therapeutically effective amounts of an
endothelin agonist and a chemotherapeutic drug, or by
administration of a therapeutically effective amount of an
endothelin antagonist.
BACKGROUND OF THE INVENTION
[0003] Although the present specification is directed primarily to
breast tumors, the invention disclosed and claimed herein can be
used in the treatment and prevention of solid tumors in general, as
set forth hereafter.
[0004] Breast cancer incidence has increased substantially in the
last 10 years, and is the single leading cause of death for women
ages 40-49 years in the United States. In 2001, 192,000 cases and
40,000 deaths made breast cancer the most common cancer, after
superficial skin cancers, and the second leading cause of cancer
death (Lacey et al., Environ Mol Mutagen, 39(2-3):82-88
(2002)).
[0005] The development of a breast cancer is a complex process
involving a combination of factors, such as environmental and
genetic factors. One extensively studied breast tumor model is the
chemically induced rat mammary carcinogenesis model (Refs. 9, 18,
19, 39, 54). Chemically induced mammary tumorigenesis in rats is
the model most closely resembling a human cancer (40).
[0006] Chemically induced rat mammary carcinogenesis typically is
achieved by administration of 7,12-dimethylbenzene(a)anthracene
(DMBA) (37) or N-methylnitrosourea (MNU) (37). Tumors induced by
DMBA or MNU have different morphological characteristics. In
particular, tumors induced by MNU are more localized at the breast
and are less likely to metastasize (25). Therefore, MNU often is
chosen as the chemical agent for the specific induction of breast
tumors in rats. These breast tumors can be benign with
fibroadenomas and papillomas, or they can be malignant (54). Rats
have six pairs of mammary glands, one in the cervical region, two
in the thoracic region, one in the abdominal region, and two in the
ingual region (4, 54). Virgin rats treated with MNU develop more
tumors in the thoracic region than the abdominal region (41).
[0007] The development of tumor vasculature has been studied
extensively. Tumors greater than a few millimeters in size require
a constant nutrient supply, and, therefore, have their own vascular
bed and blood flow (10). Recruitment of new vasculature from
preexisting blood vessels is termed "angiogenesis." Without
constant nourishment from these developing blood vessels, the
tumors become hypoxic and subsequently die. Therefore, tumor
vasculature has been a target of cancer therapy for a considerable
time (10).
[0008] Tumor blood vessels develop substantially differently from
normal vasculature, and have different properties. Single layered
epithelial cells are the first hastily formed tumor blood vessels.
It has been suggested that these blood vessels are recruited when
the tumor secretes certain growth factors, like vascular
endothelial growth factor (VEGF), in response to hypoxic conditions
(23). These newly formed tumor blood vessels do not have a smooth
muscle layer or innervation (29, 36, 57).
[0009] Tumors also incorporate mature blood vessels that possess
all their autoregulatory functions (29). Normal tissue vascular
tone is governed by a host of endogenous factors like H.sup.+,
K.sup.+, Ca.sup.2+, pO.sub.2, pCO.sub.2, nitric oxide (NO), as well
as other regulatory substances like endothelin (ET-1) (24, 46).
[0010] ET-1 is a potent vasoconstrictor and contributes
significantly in regulating vascular tone (61). In breast cancer
tissue, ET.sub.B receptors are found on stromal fibroblast cells
(5, 34). Endothelins have been found to be mitogenic to fibroblasts
(53), melanocytes, vascular smooth muscle, and endothelium (3, 35,
52). Investigators have shown an increase in ET-1, ET-3, and
ET.sub.B receptor expression in breast carcinomas (1). It has been
shown that both ET-1 and ET-3 cause an increase in VEGF, which is
an important angiogenic factor (35). Thus, an increase in ET-1
promotes tumor growth. Several studies have reported an increase in
ET-1 levels in breast tumors (1, 21, 31, 33, 59, 60).
[0011] The present invention is directed to the effect of
endothelin antagonists and endothelin agonists on systemic
hemodynamics and blood circulation in solid tumor tissues. The
present invention also is directed to the use of endothelin
agonists and endothelin antagonists in the treatment of solid
tumors.
SUMMARY OF THE INVENTION
[0012] The present invention is directed to administration of
therapeutically effective amounts of an endothelin agonist and a
chemotherapeutic agent to an individual in need thereof in the
treatment of a solid tumor. The present invention also is directed
to administration of a therapeutically effective amount of an
endothelin antagonist to an individual in need thereof in the
prevention and treatment of a solid tumor, such as a breast
tumor.
[0013] In particular, tumors need a blood supply to grow. ET is a
powerful regulator of blood flow. ET.sub.A receptors have been
found to be vasoconstrictors, and ET.sub.B receptors have been
found to be vasodilators. In accordance with the present invention,
it has been demonstrated that breast tumor tissue has abundant
ET.sub.B receptors, and that an ET.sub.B receptor antagonist can
block the increased blood flow to breast tumor tissue induced by
ET-1. Accordingly, an endothelin antagonist, particularly an
ET.sub.B receptor antagonist, is useful to prevent the growth of
breast or other solid tumors having ET.sub.B receptors regulating
their blood flow.
[0014] In addition, because ET.sub.B receptors are vasodilators, it
has been found that an ET.sub.B receptor agonist, in combination
with a chemotherapeutic agent, is useful in the treatment of a
solid tumor, such as those found in breast cancer. In this
embodiment, the ET.sub.B receptor agonist more effectively delivers
the chemotherapeutic agent to the breast tumor resulting in an
enhanced treatment.
[0015] Accordingly, one aspect of the present invention is to
provide a method of treating solid tumors comprising administering
to a mammal in need thereof a therapeutically effective amount of
an endothelin agonist and a chemotherapeutic agent.
[0016] Another aspect of the present invention is to provide a
composition comprising an endothelin agonist, in particular an
ET.sub.B agonist. The composition is useful in the treatment of
solid tumors. The endothelin agonist is used in conjunction with a
chemotherapeutic agent. In particular, the present invention also
is directed to compositions containing an endothelin agonist, and
to methods of administering the endothelin agonist, in conjunction
with a chemotherapeutic agent, to treat solid tumors.
[0017] Still another aspect of the present invention is to provide
a composition comprising an endothelin agonist, a second
therapeutic agent useful in the treatment of a solid tumor, and an
excipient.
[0018] Still another aspect of the present invention is to provide
a method of preventing or treating solid tumors comprising
administering to a mammal in need thereof a therapeutically
effective amount of an endothelin antagonist. The endothelin
antagonist can be an endothelin B antagonist or a mixed endothelin
A/B antagonist. Preferably, the endothelin antagonist comprises a
specific endothelin B (ET.sub.B) antagonist. The endothelin
antagonist optionally is used in conjunction with an angiogenesis
inhibitor, radiation treatment, or both.
[0019] Another aspect of the present invention is to provide a
composition comprising an endothelin antagonist, in particular an
ET.sub.B antagonist, to an individual in need thereof. The
composition is useful in the prevention and treatment of solid
tumors.
[0020] Another aspect of the present invention is to provide a
composition comprising an endothelin antagonist, a second
therapeutic agent useful in the prevention or treatment of a solid
tumor, and an excipient.
[0021] Yet another aspect of the present invention is to provide an
article of manufacture for human pharmaceutical use, comprising (a)
a container, and (b1) a packaged composition comprising an
endothelin agonist and, optionally, (b2) a packaged composition
comprising a second therapeutic agent useful in the treatment of a
solid tumor, and (c) a package insert containing directions for use
of the composition or compositions administered simultaneously or
sequentially, in the treatment of a solid tumor. In a preferred
embodiment, the endothelin agonist is an ET.sub.B receptor agonist
and the second therapeutic agent is a chemotherapeutic agent.
[0022] Another aspect of the present invention is to provide an
article of manufacture for human pharmaceutical use, comprising (a)
a container, (b1) a packaged composition comprising an endothelin
antagonist and, optionally, (b2) a packaged composition comprising
a second therapeutic agent useful in the treatment of a solid
tumor, and (c) a package insert containing directions for use of
the composition or compositions, administered simultaneously or
sequentially, in the prevention or treatment of a solid tumor. In a
preferred embodiment, the endothelin antagonist is an ET.sub.B
receptor antagonist, and the second therapeutic agent is an
angiogenesis inhibitor, radiation treatment, or both.
[0023] These and other novel aspects of the present invention will
become apparent from the following detailed description of the
preferred embodiments of the invention taken in conjunction with
the figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 contains bar graphs showing the effect of ET-1 on
systemic hemodynamics of saline-treated and MNU-treated,
tumor-bearing rats;
[0025] FIG. 2 contains bar graphs showing the effect of ET-1 on
blood flow and regional vascular resistance in the breast tissue of
saline-treated and MNU-treated rats;
[0026] FIG. 3 contains plots showing the effect of ET-1 on
perfusion, CMBC, and velocity of blood cells in breast tissue of
saline-treated and tumor tissue of MNU-treated rats;
[0027] FIG. 4 contains plots showing the effect of BQ788 on
ET-1-induced changes in blood perfusion, CMBC, and velocity of
blood cells in breast tissue of saline-treated and tumor tissue of
MNU-treated rats; and
[0028] FIG. 5 contains plots showing the effect of IRL1620 on
paclitaxel-induced changes in tumor perfusion.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] The present invention is directed to compositions and
methods of preventing and treating solid tumors, including breast
tumors. In particular, the present invention is directed to
pharmaceutical compositions comprising either (a) an endothelin
agonist and, optionally, a chemotherapeutic agent or (b) an
endothelin antagonist, and optionally, angiogenesis inhibitor.
[0030] The present invention also is directed to articles of
manufacture comprising an endothelin antagonist and an optional
angiogenesis inhibitor, packaged separately or together, and an
insert having instructions for using these active agents to prevent
or treat a solid tumor.
[0031] In addition, the present invention is directed articles of
manufacture comprising an endothelin agonist and a chemotherapeutic
agent, packaged separately or together, and an insert having
instructions for using these active agents to treat a solid
cancerous tumor.
[0032] One method disclosed herein utilizes an endothelin agonist
and a chemotherapeutic agent in the treatment of a solid tumor. The
agonist and chemotherapeutic agent can be administered in
sufficient amounts, simultaneously or sequentially, to achieve the
desired therapeutic effect.
[0033] Another method disclosed herein utilizes an endothelin
antagonist, optionally with an angiogenesis inhibitor, in the
treatment of solid tumors. The antagonist and angiogenesis
inhibitor can be administered in sufficient amounts, simultaneously
or sequentially, to achieve the desired effect.
[0034] For the purposes of the invention disclosed herein, the term
"treatment" includes preventing, retarding the progression of,
shrinking, or eliminating a solid tumor. As such, the term
"treatment" includes both medical therapeutic and/or prophylactic
administration, as appropriate.
[0035] The term "container" means any receptacle and closure
therefor suitable for storing, shipping, dispensing, and/or
handling a pharmaceutical product.
[0036] The term "insert" means information accompanying a
pharmaceutical product that provides a description of how to
administer the product, along with the safety and efficacy data
required to allow the physician, pharmacist, and patient to make an
informed decision regarding use of the product. The package insert
generally is regarded as the "label" for a pharmaceutical
product.
[0037] The term "prodrug" means compounds that transform rapidly in
vivo to a compound useful in the invention, for example, by
hydrolysis. A thorough discussion of prodrugs is provided in
Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, of the
A.C.S.D. Symposium Series, and in Roche (ed.), Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987.
[0038] Endothelin is a vasoactive substance known to modulate blood
flow and also has mitogenic properties. Endothelin is present in
large concentrations in breast carcinoma tissues compared to normal
breast tissue. In accordance with the present invention, it has
been shown that a subtype of endothelin receptor (ET.sub.B) also is
increased in breast cancer. Endothelin acts on ET.sub.B receptors
to produce vascular dilation and increase in blood flow to the
breast tumor tissue. Importantly, it also has been found that an
ET.sub.B receptor antagonist can block the increase in tumor blood
flow induced by endothelin.
[0039] Because endothelin and ET.sub.B receptors are overexpressed
in breast cancer, a selective ET.sub.B receptor antagonist, e.g.,
BQ788, can be used to block endothelin-induced vasodilation in the
breast tumor tissue, and cut off or reduce the blood supply and
nutrient supply needed for the breast tumor to grow. An ET.sub.B
antagonist can be used alone, or in combination with an
angiogenesis inhibitor, like thalidomide, that inhibits the
formation of new blood vessels in the tumor tissue. Once the blood
supply and nutrient supply to the tumor tissue are reduced, the
growth of the tumor also is reduced.
[0040] In addition, most chemotherapeutic agents have cytotoxic
properties that are targeted to destroy cancer cells, but in the
process inflict considerable damage to the body's normal
physiological systems. It would be of great advantage, therefore,
to selectively deliver chemotherapeutic agents to the tumor tissue.
Accordingly, an ET.sub.B receptor agonist that selectively
increases blood supply to the tumor can increase the delivery and
efficacy of the chemotherapeutic agent. Therefore, ET.sub.B
receptor agonists can selectively increase the delivery of
chemotherapeutic agents, like tamoxifen, to a breast tumor and
increase efficacy of the chemotherapeutic agent.
[0041] More particularly, tumor blood supply has become a target of
cancer therapy. Several vasoactive substances are known to modulate
blood flow including endothelin-1 (ET-1). ET-1 is present in large
concentrations in breast carcinoma tissues (i.e., 11.95 pg/mg
tissue) compared to normal breast tissue (i.e., 0.12 pg/mg tissue)
(Kojima et al., Surg. Oncol., 4(6):309-315 (1995); Kurbel et al.,
Med. Hypotheses, 52(4):329-333 (1999); Patel et al., Mol. Cell
Endocrinol., 126(2):143-151 (1997); Yamashita et al., Cancer Res.,
52(14):4046-4049 (1992); Yamashita et al., Res. Commun. Chem.
Pathol. Pharmacol., 74(3):363-369 (1991).
[0042] Studies have shown that ET-1, ET-3, and ET.sub.B receptor
expression is increased in breast cancer (grade III, strong
staining compared to negative staining in controls) (Alanen et al.,
Histopathology, 36(2):161-167 (2000)). It also has been found that
ET-1 produces an increase in blood flow to the breast tumor by
stimulating ET.sub.B receptors. BQ788, an ET.sub.B receptor
antagonist, completely blocked ET-1 induced increase in tumor blood
flow. Because breast tumor tissue has enhanced ET.sub.B receptor
expression, an ET.sub.B receptor antagonist can be used to
selectively decrease breast tumor blood supply, and an ET.sub.B
receptor agonist can be used to increase blood flow to the breast
tumor tissue.
[0043] Accordingly, an ET.sub.B receptor agonist in combination
with a chemotherapeutic agent decreases breast tumor growth. In
addition, an ET.sub.B receptor antagonist, either alone or in
combination with an angiogenesis inhibitor, significantly decreases
the breast tumor growth.
[0044] Administration of an ET.sub.B receptor agonist in
combination with a chemotherapeutic agent also can be used to treat
or prevent other solid tumors, including, but not limited to,
ovarian cancer, colon carcinoma, Kapoli's sarcoma, breast cancer,
and melanomas. An endothelin antagonist, alone or in combination
with an angiogenesis inhibitor, also can be used in the treatment
and prevention of solid tumors.
[0045] The following table lists the ET receptor expression for
various solid tumors.
1 Tumor ET receptor expression References Ovarian cancer ET.sub.A
and ET.sub.B receptors Bagnato et al., Cancer Res, 1999, 59,
720-727 Colon carcinoma ET.sub.A receptors are present Egidy et
al., Am J in stroma Pathology, 2000, 157, ET.sub.B receptors in
1863-1874 endothelium and myofibroblasts Kaposi's sarcoma ET.sub.A
and ET.sub.B receptors in Bagnato et al., Am J tumor and
intratumoral Pathol, 2001, 158, vessels 841-847 Breast cancer
ET.sub.B receptors Alanen et al., Histopathology: 2000: 36(2): 161
Melanoma ET.sub.B receptors Kikuchi et al., Biochem Biophys Res
Comm, 1996, 219, 734-739
[0046] In one embodiment of the present invention, a solid tumor is
treated using an endothelin agonist in conjunction with a
chemotherapeutic agent. In this method, the endothelin agonist,
notably an ET.sub.B agonist, increases blood flow in the breast
tumor, which is rich in ET.sub.B receptors. The ET.sub.B agonist,
therefore, provides a more selective target for the
chemotherapeutic agent and improves the chemotherapeutic effect of
the agent.
[0047] ET.sub.B agonists useful in the present invention include,
but are not limited, to, ET-1, ET-2, ET-3, BQ3020, IRL1620,
sarafotoxin S6c, [Ala .sup.1, 3, 11, 15]ET-1, and mixtures
thereof.
[0048] It is theorized, but not relied upon herein, that endothelin
agonists stimulate ET.sub.B receptors and dilate tumor blood
vessels, thereby increasing delivery of the chemotherapeutic agent
to the tumor. Endothelin agonists also increase blood perfusion of
the solid tumor, and thereby increase oxygenation of the tissue.
Improved oxygenation is known to enhance the therapeutic action of
chemotherapeutic agents. The mitogenic action of endothelin also
can help increase the action of chemotherapeutic agents, when
administered together. The mitogenic action of an endothelin
agonist can improve incorporation of chemotherapeutic agents in the
dividing cells, and increase the efficacy of the chemotherapeutic
agents.
[0049] In this embodiment, the ET.sub.B agonist is used in
conjunction with a chemotherapeutic agent. The ET.sub.B agonist
enhances the therapeutic benefit of chemotherapy treatment,
including induction chemotherapy and primary (neoadjuvant)
chemotherapy. In addition, chemotherapy is frequently indicated as
an adjuvant to surgery in the treatment of a cancer. The goal of
chemotherapy in the adjuvant setting is to reduce the risk of
recurrence and enhance disease-free survival when the primary tumor
has been controlled. Chemotherapy is utilized as a treatment
adjuvant for a cancer, frequently when the disease is metastatic.
An ET.sub.B agonist, therefore, is particularly useful following
surgery in the treatment of a solid tumor in combination with
chemotherapy.
[0050] Chemotherapeutic agents that can be used in the present
method include, but are not limited to, alkylating agents,
antimetabolites, hormones and antagonists thereof, radioisotopes,
antibodies, as well as natural products, and mixtures thereof. For
example, an ET.sub.B agonist can be administered with antibiotics,
such as doxorubicin and other anthracycline analogs, nitrogen
mustards, such as cyclophosphamide, pyrimidine analogs such as
5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and
synthetic derivatives, and the like. As another example, in the
case of mixed tumors, such as adenocarcinoma of the breast, where
the tumors include gonadotropin-dependent and
gonadotropin-independent cells, the ET.sub.B agonist can be
administered in conjunction with leuprolide or goserelin (synthetic
peptide analogs of LH-RH). Examples of chemotherapeutic agents
useful in the method of the present invention are listed in the
following table.
2 Alkylating agents Nitrogen mustards mechlorethamine
cyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureas
carmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)
Ethylenimine/Methylmelamine thriethylenemelamine (TEM) triethylene
thiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine)
Alkyl sulfonates busulfan Triazines dacarbazine (DTIC)
Antimetabolites Folic Acid analogs methotrexate trimetrexate
Pyrimidine analogs 5-fluorouracil fluorodeoxyuridine gemcitabine
cytosine arabinoside (AraC, cytarabine) 5-azacytidine
2,2'-difluorodeoxycytidine Purine analogs 6-mercaptopurine
6-thioguanine azathioprine 2'-deoxycoformycin (pentostatin)
erythrohydroxynonyladeni- ne (EHNA) fludarabine phosphate
2-chlorodeoxyadenosine (cladribine, 2-CdA) Type I Topoisomerase
Inhibitors camptothecin topotecan irinotecan Natural products
Antimitotic drugs paclitaxel Vinca alkaloids vinblastine (VLB)
vincristine vinorelbine Taxotere .RTM. (docetaxel) estramustine
estramustine phosphate Epipodophylotoxins etoposide teniposide
Antibiotics actimomycin D daunomycin (rubidomycin) doxorubicin
(adriamycin) mitoxantroneidarubicin bleomycinsplicamycin
(mithramycin) mitomycinC dactinomycin Enzymes L-asparaginase
Biological response modifiers interferon-alpha IL-2 G-CSF GM-CSF
Differentiation Agents retinoic acid derivatives Radiosensitizers
metronidazole misonidazole desmethylmisonidazole pimonidazole
etanidazole nimorazole RSU 1069 EO9 RB 6145 SR4233 nicotinamide
5-bromodeozyuridine 5-iododeoxyuridine bromodeoxycytidine
Miscellaneous agents Platinium coordination complexes cisplatin
carboplatin Anthracenedione mitoxantrone Substituted urea
hydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)
procarbazine Adrenocortical suppressant mitotane (o,p'-DDD)
ainoglutethimide Cytokines interferon (.alpha., .beta., .gamma.)
interleukin-2 Hormones and antagonists Adrenocorticosteroids/
antagonists prednisone and equivalents dexamethasone
ainoglutethimide Progestins hydroxyprogesterone caproate
medroxyprogesterone acetate megestrol acetate Estrogens
diethylstilbestrol ethynyl estradiol/ equivalents Antiestrogen
tamoxifen Androgens testosterone propionate
fluoxymesterone/equivalents Antiandrogens flutamide
gonadotropin-releasing hormone analogs leuprolide Nonsteroidal
antiandrogens flutamide Photosensitizers hematoporphyrin
derivatives Photofrin .RTM. benzoporphyrin derivatives Npe6 tin
etioporphyrin (SnET2) pheoboride-a bacteriochlorophyll-a
naphthalocyanines phthalocyanines zinc phthalocyanines
[0051] Examples of chemotherapeutic agents that are particularly
useful in conjunction with an ET.sub.B agonist include, for
example, adriamycin, camptothecin, carboplatin, cisplatin,
daunorubicin, doxorubicin, interferon (alpha, beta, gamma),
interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and
therapeutically effective analogs and derivatives of the same.
[0052] In another embodiment of the present invention, an
endothelin antagonist utilized in the method and composition can be
any ET.sub.B receptor antagonist known in the art. ET.sub.B
receptors are potent vasodilators. ET.sub.B antagonists inhibit the
activity of ET.sub.B, and are used to restrict blood flow.
[0053] ET.sub.B antagonists useful in the present invention can be
selective ET.sub.B antagonists or balanced ET.sub.A/ET.sub.B
antagonists. ET.sub.B receptor antagonists, and balanced
ET.sub.A/ET.sub.B antagonists, useful in the treatment and/or
prevention of solid tumors are set forth in Appendices A through C
herein. Additional useful endothelin antagonists can be found in
U.S. Patent Application Publication No. US 2002/0082285 A1,
incorporated herein by reference.
[0054] Examples of ET.sub.B antagonists useful in the present
invention include, but are not limited to, atrasentan, tezosentan,
bosentan, sitaxsentan, enrasentan, Ro468443, TBC10950, TBC10894,
A192621, A308165, SB209670, SB217242, A182086, (s)-Lu302872,
J-104132, TAK-044, Sarafotoxin 56c, IRL2500, RES7011, Aselacins A,
B, and C, Ro470203, Ro462005, sulfamethoxazole, cochinmicin I, II,
and III, L749329, L571281, L754142, J104132, CGS27830, A182086,
PD142893, PD143296, PD145065, PD156252, PD159020, PD160672,
PD160874, TM-ET-1, IRL3630, Ro485695, L753037, LU224332, PD142893,
LU302872, PD145065, Ro610612, SB217242, BQ788, and mixtures
thereof. BQ-788 is a preferred specific endothelin B antagonist,
and is the sodium salt of
N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-me-
thylleucyl-D-1-methoxycarbonyl triptophanyl-DNIe (see Proc. Natl.
Acad. Sci. USA, 91:4892-4896 (1994)).
[0055] In addition to a conventional endothelin antagonist, a
compound that inhibits the formation of endogenous endothelin also
can be used as the endothelin antagonist in the present invention.
Such compounds are useful because they prevent endothelin formation
and, therefore, decrease the activity of endothelin receptors. One
class of such compounds is the endothelin converting enzyme (ECE)
inhibitors. Useful ECE inhibitors include, but are not limited to,
CGS34225 (i.e., N-((1-((2-(S)(acetylthio-
)-1-oxopentyl)-amino)-1-cyclopentyl)-carbonyl-S-4-phenylphenyl-alanine
methyl ester) and phosphoramidon (i.e.,
N-(1-rhamnopyranosyloxyhydroxypho- sphinyl)-Leu-Trp).
[0056] As discussed more fully hereafter, the ET.sub.B receptor
antagonist can be used in conjunction with an angiogenesis
inhibitor. As previously stated, angiogenesis is the generation of
new vasculature from preexisting blood vessels. An angiogenesis
inhibitor retards or eliminates the generation of new
vasculature.
[0057] Any angiogenesis inhibitor known in the art can be used with
an ET.sub.B antagonist in the present method. Examples of
angiogenesis inhibitors include, but are not limited to,
thalidomide, marimastat, COL-3, BMS-275291, squalamine, 2-ME,
SU6668, neovastat, Medi-522, EMD121974, CAI, celecoxib,
interleukin-12, IM862, TNP470, avastin, gleevac, herceptin, and
mixtures thereof.
[0058] In one method of the present invention, wherein an ET.sub.B
antagonist and an optional angiogenesis inhibitor are administered
to an individual in need thereof to treat a solid tumor by
restricting blood flow and inhibiting the formation of new
vasculature, the individual also can be treated using radiation
therapy and/or a radiosensitizer.
[0059] The term "radiosensitizer," as used herein, is defined as a
compound administered to a human or other animal in a
therapeutically effective amount to increase the sensitivity of
cells to electromagnetic radiation and/or to promote the treatment
of diseases that are treatable with electromagnetic radiation.
Radiosensitizers can be administered in conjunction with an
ET.sub.B antagonist and optional angiogenesis inhibitor.
[0060] The terms "electromagnetic radiation" and "radiation" as
used herein include, but are not limited to, radiation having the
wavelength of 10-20 to 100 meters. Preferred embodiments of the
present invention employ the electromagnetic radiation of
gamma-radiation (10-20 to 10-13 m), X-ray radiation (10-12 to 10-9
m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to
700 nm), infrared radiation (700 nm to 1.0 mm), and microwave
radiation (1 mm to 30 cm).
[0061] Many cancer treatment protocols currently employ
radiosensitizers activated by electromagnetic radiation, e.g.,
X-rays. Examples of X-ray-activated radiosensitizers include, but
are not limited to, the following: metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole,
mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide,
5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),
bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea,
cisplatin, and therapeutically effective analogs and derivatives of
the same.
[0062] Photodynamic therapy (PDT) of cancers employs visible light
as the radiation activator of the sensitizing agent. Examples of
photodynamic radiosensitizers include, but are not limited to,
hematoporphyrin derivatives, PHOTOFRIN.RTM., benzoporphyrin
derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide-a,
bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc
phthalocyanine, and therapeutically effective analogs and
derivatives of the same.
[0063] In summary, the structure, growth, and function of the blood
vessels in breast tumors are markedly different from that of normal
breast tissue due to changes in the production of growth factors,
like vascular endothelial growth factor (VEGF), vasoactive
substances like endothelin-1 (ET-1), and cytokines. The role of
ET-1 in breast tumor angiogenesis is not adequately understood.
Studies have shown that the expression of proET-1, proET-3, and
ET.sub.B receptors is increased in breast tumor. However, it is
unclear whether there is any change in ET-1 induced vascular
responses in the breast tumor. Hence, the systemic hemodynamics and
regional circulatory effects of ET-1 in rats with breast tumors was
investigated.
[0064] For the first time, it has been demonstrated that ET-1
produces an increase in blood flow to the breast tumor by
stimulating ET.sub.B receptors. BQ788, an ET.sub.B receptor
antagonist, completely blocked an ET-1 induced increase in tumor
blood flow. Because breast tumor tissue has enhanced ET.sub.B
receptor expression, an ET.sub.B receptor antagonist can be used to
decrease blood supply selectively to tumor tissue.
[0065] Similarly, an ET.sub.B receptor agonist increases blood
supply to tumor tissue, thereby facilitating administration of a
chemotherapeutic drug to the tumor. Accordingly, an ET.sub.B
receptor agonist can be used in combination with a chemotherapeutic
agent in the treatment of a solid tumor, like a breast tissue. In
addition, most chemotherapeutic agents have cytotoxic properties
and are targeted to destroying cancer cells. However, in the
process, chemotherapeutic agents inflict considerable damage to the
body's normal physiological systems. ET.sub.B receptor agonists
that selectively increase blood supply to the tumor therefore can
increase the delivery and efficacy of chemotherapeutic agents.
[0066] ET.sub.B receptor antagonists can be used in the treatment
of a breast cancer either alone or in combination with an
angiogenesis inhibitor. Angiogenesis inhibitors prevent the
formation of new blood vessels needed for the growth of the tumor.
Therefore, a combination of an angiogenesis inhibitor with an
ET.sub.B receptor antagonist, which selectively decreases the blood
supply to breast tumor tissue, significantly decreases tumor
growth.
[0067] Therefore, an ET.sub.B receptor agonist in combination with
a chemotherapeutic agent decreases solid tumor growth. In addition,
an ET.sub.B receptor antagonist, either alone or in combination
with an angiogenesis inhibitor, significantly decreases solid tumor
growth.
[0068] Materials and Methods
[0069] Animals
[0070] Female Sprague Dawley rats (Harlan Co., Madison, Wis.)
weighing 180-200 grams (g) were used. All animals were housed,
three to a cage, in a temperature controlled room (23.+-.1.degree.
C.), humidity (50.+-.10%), and artificial light (0600-1800 hr). The
animals were given food and water ad libitum. The experiments were
conducted after the animals had been acclimatized to the
environment for at least four days.
[0071] Drugs
[0072] N-methylnitrosourea (MNU) was purchased from Ash Stevens
Inc., Detroit, Mich. BQ788
(N-cis2,6-dimethylpiperidinocarbonyl-L-gamma-methyll-
eucyl-D-1-methoxycarbonyltrptophanyl-D-Nle), IRL1620, and
Endothelin-1 (ET-1) were obtained from American Peptide Company
Inc., Sunnyvale, Calif. BQ788 was dissolved in saline and ET-1 was
dissolved in 0.1% albumin.
[0073] Methods for Effect of IRL1620 and Taxol on Breast Tumor
Perfusion
[0074] MNU (50 mg/kg, i.p.) or saline (1 ml/kg, i.p.) was
administered to female Sprague Dawley rats. After the tumors
reached 2-4 cm in diameter, the blood flow experiments were
performed. The animals were divided into the following groups:
[0075] (i) Saline injection followed by taxol (3 mg/kg) after 15
minutes in normal rats (N=4);
[0076] (ii) IRL 1620 (3 nmol/kg) injection followed by taxol (3
mg/kg) after 15 minutes in normal rats (N=4);
[0077] (iii) Saline injection followed by taxol (3 mg/kg) after 15
minutes in tumor bearing rats (N=4); and
[0078] (iv) IRL 1620 (3 nmol/kg) injection followed by taxol (3
mg/kg) after 15 minutes in tumor bearing rats (N=4).
[0079] Surgical Preparations
[0080] Rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma
Chemicals, St. Louis, Mo.). The left femoral vein was cannulated
(PE 50 tubing, Clay Adams, Parsipanny, N.J.) for drug
administration. The left femoral artery was cannulated, and was
used for withdrawal of reference blood samples. The right femoral
artery was cannulated and connected to a Gould P23 ID pressure
transducer for recording the blood pressure on a Grass P7D
polygraph (Grass Instrument Co., Quincy, Mass., USA) through a 7PI
preamplifier. The heart rate (HR) was recorded through a 7P4B Grass
tachograph (Grass Instrument Co., Quincy, Mass.) triggered from
blood pressure signals.
[0081] Breast Blood Perfusion Measurement by Laser Doppler
Flowmetry (LDF)
[0082] The blood perfusion to the mammary gland of the rats was
measured using laser Doppler flowmetry. The animals were shaved
around the nipples and the skin surrounding the mammary glands was
dissected out. A standard model fiber optic probe was secured to
the mammary artery and connected to a Periflux PF2b 4000 Laser
Doppler Flowmetry (Perimed KB, Stockholm, Sweden). The time
constant was set to 1.5 seconds, and the band width was set to 4
KHz.
[0083] Statistical Analysis
[0084] All data are presented as mean.+-.SEM. Data were analyzed
using analysis of variance followed by Duncan's test. A level of
p<0.05 was considered significant.
[0085] RESULTS
[0086] Effect of IRL1620 and Taxol on Breast Tumor Perfusion
[0087] No change in blood flow to the breast tissue of normal rats
was observed following the administration of saline or IRL1620 and
taxol. Significant differences were observed between the blood flow
in the tumor tissue after IRL1620 injection (36.3%, p<0.05) and
after taxol administration (51.9%, p<0.0-5) from baseline (see
FIG. 5).
[0088] Effect of IRL 1620 and Taxol on Blood Pressure
[0089] No change in blood pressure was observed following the
administration of saline or IRL 1620 and taxol in normal and tumor
bearing rats.
[0090] Experimental Protocol for ET-1 Infusion Into Rats
[0091] The following groups of animals were studied to evaluate the
effect of ET-1 infusion on systemic hemodynamics and blood flow to
the mammary tissue of normal and tumor-bearing rats.
[0092] (i) ET-1 (50 ng/kg/min) infusion for 30 minutes in rats
treated with saline (N=6); and
[0093] (ii) ET-1 (50 ng/kg/min) infusion for 30 minutes in treated
with MNU (50 mg/kg, i.p.) (N=6).
[0094] The following groups were studied to evaluate the role of
ET.sub.B receptors on the changes induced by ET-1 infusion on the
systemic hemodynamics and blood flow to the mammary tissue of
normal rats and rats with breast tumors:
[0095] (i) BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by
ET-1 (50 ng/kg/min) infusion for 30 minutes in rats treated with
saline (N=5);
[0096] (ii) BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by
ET-1 (50 ng/kg/min) infusion for 30 minutes in rats treated with
MNU (50 mg/kg, i.p.) (N=5).
[0097] MNU and saline treatments were performed as intraperitoneal
(i.p.) injections three months prior to the study. Rats were
palpated regularly starting four weeks after the treatments. Once
tumors reached an optimal size (i.e., 4-8 mm in diameter), the
experiments were initiated. Systemic hemodynamic and regional
circulation parameters were determined at baseline, 30, 60, and 120
minutes after starting ET-1 (50 ng/kg/min) infusion. Because ET-1
infusion was performed for 30 minutes, the 30-minute data shows the
effect of ET-1, and the 60- and 120-minute data indicates duration
of the ET-1 effect.
[0098] Surgical Preparations
[0099] Rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma
Chemicals, St. Louis, Mo.). All surgical areas were shaved and
cleaned with alcohol swabs. The left femoral vein was cannulated
(PE 50 tubing, Clay Adams, Parsipanny, N.J.) for drug
administration. The left femoral artery was cannulated (PE 50
tubing) and was used for withdrawal of reference blood sample in
microsphere studies using a withdrawal pump (Model 22, Harvard
Apparatus, South Natick, Mass.). The right femoral artery was
cannulated (PE 50 tubing) and connected to a Gould P23 ID pressure
transducer for recording the blood pressure on a Grass P7D
polygraph (Grass Instrument Co., Quincy, Mass., USA) through a 7PI
preamplifier. The heart rate (HR) was recorded through a 7P4B Grass
tachograph (Grass Instrument Co., Quincy, Mass.) triggered from
blood pressure signals. The right carotid artery was exposed and a
PE 50 tubing was guided through the common carotid artery into the
left ventricle. The presence of the cannula in the left ventricle
was confirmed by recording the pressure on the Grass polygraph
using the Statham P23 DC pressure transducer (Grass Instrument Co.,
Quincy, Mass.). When the cannula reached the left ventricle; the
diastolic pressure dropped to zero. In order to maintain the blood
pO.sub.2, pCO.sub.2, and pH constant, and to avoid the effect of
respiration on blood pressure and HR, animals were kept on constant
rate artificial respiration by inserting an endotracheal cannula
connected to a rodent ventilator (Model 683, Harvard Apparatus
Inc., South Natick, Mass.).
[0100] Determination of Systemic Hemodynamics and Regional
Circulation
[0101] Systemic hemodynamics and regional blood circulation were
determined using a literature described procedure (13, 16, 47). At
each measurement, a thoroughly mixed suspension of approximately
100,000 microspheres (15.+-.1 .mu.m diameter) labeled with
.sup.46Sc (scandium), .sup.113Sn (tin), .sup.141Ce (cerium), or
.sup.95Nb (niobium) (New England Nuclear Corporation, Boston,
Mass., USA) in 0.2 ml saline were injected into the left ventricle
and flushed with 0.3 ml saline over a 15 second period. In order to
calculate blood flow, arterial blood was withdrawn at a rate of 0.5
ml/min through the right femoral artery. Blood was withdrawn for 90
seconds starting about 5-10 seconds before microsphere injection.
At the end of the experiment, the animals were sacrificed with an
overdose of pentobarbital sodium. All tissues and organs were
dissected out, weighed, and placed in vials. The radioactivity in
the standards, the blood samples, and the tissue samples were
counted in a Packard Minaxi Auto-Gamma 5000 series gamma counter
(Packard Instruments Co., Downers Grove, Ill.) with preset windows
discriminating the isotope energies. The following parameters were
calculated: (1) cardiac output (CO) ((radioactivity injected x
withdrawal rate of arterial blood)/radioactivity in sampled
arterial blood), (2) stroke volume (SV) (CO/HR), (3) total
peripheral resistance (TPR) (mean arterial pressure (MAP)/CO), (4)
regional blood flow ((radioactivity in tissue x withdrawal rate of
arterial blood)/radioactivity in sampled arterial blood), and (5)
regional vascular resistance (MAP/regional blood flow). The data
were calculated using computer programs described in the literature
(45).
[0102] Breast Blood Perfusion Measurement by Laser Doppler
Flowmetry (LDF)
[0103] Blood perfusion to the mammary gland of the rats was
measured using laser Doppler flowmetry as described in literature
procedures (50, 51). The animals were shaved around the nipples.
The skin surrounding the mammary glands was dissected out as a
lambeau about 6 cm wide and 4 cm long. A standard model fiber optic
probe was applied to the surface of the lambeau, and secured to the
tissue by double stick tape. The lambeau was placed in a metal
holder and taped down to prevent movement, then connected to a
Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed KB, Stockholm,
Sweden). The time constant was set at 1.5 seconds and the bandwidth
was set at 4 KHz.
[0104] Statistical Analysis
[0105] All data are presented as mean.+-.SEM. Data were analyzed
using analysis of variance followed by Duncan's test. A level of
p<0.05 was considered significant.
[0106] RESULTS
[0107] Effect of ET-1 on Systemic Hemodynamics in Normal and
Tumor-Bearing Rats
[0108] The baseline systemic hemodynamic parameters in normal
(saline treated) rats were MAP: 111.1.+-.4.8 mmHg; CO:268.6.+-.17.6
ml/min; SV:0.87.+-.0.06 ml; TPR:419.6.+-.24.37 mmHg.min/ml; and
HR:312.5.+-.20.2 beats/min. In normal rats, a significant increase
in MAP was observed at 30 minutes (14.5%; p<0.05), and a
decrease at 120 minutes (17.8%; p<0.05) following ET-1 infusion.
TPR increased at 120 minutes (49.2%; p<0.05). CO decreased at 60
and 120 minutes (22.9% and 42.5% respectively; p<0.05) after
ET-1 infusion. SV decreased at 60 and 120 minutes (20.9% and 36%
respectively; p<0.05). No significant change in HR was observed
(FIG. 1).
[0109] The baseline systemic hemodynamic parameters in
tumor-bearing (MNU treated) rats were similar to that in normal
rats. A significant increase in MAP was observed at 30 minutes
(19.1%; p<0.05) and at 60 minutes (15.3%; p<0.05) following
ET-1 infusion in tumor-bearing rats. TPR increased at 30 minutes
(73.9%; p<0.05), 60 minutes (39.7%; p<0.05), and 120 minutes
(71.4%; p<0.05) following administration of ET-1. CO decreased
at 30, 60 and 120 minutes (29.4%, 16.7% and 36.1% respectively;
p<0.05). SV decreased significantly at 30, 60 and 120 minutes
(31.1%, 17.9% and 32.1% respectively; p<0.05). No change in HR
was observed (FIG. 1).
[0110] Effect of ET-10n Regional Blood Flow and Vascular Resistance
in the Breast Tissue of Normal and Tumor-Bearing Rats
[0111] No change in blood flow to the breast tissue of normal
saline-treated rats was observed following the administration of
ET-1. A significant decrease (18.61%; p<0.05) in vascular
resistance at 60 minutes was observed, which is 30 minutes post
ET-1 infusion, in the breast tissue of normal rats (FIG. 2).
[0112] Significant differences were observed between the blood-flow
and the regional vascular resistance in the breast tissue of
tumor-bearing (MNU treated) and normal (saline treated) rats. A
significant increase (153%; p<0.05) in blood flow to the breast
tissue of tumor-bearing rats as compared to normal rats was
observed at 60 minutes following administration of ET-1. The
vascular resistance in the tumor-bearing rats was significantly
different at baseline (102%; p<0.05) and at 60 minutes (147%;
p<0,05) following ET-1 administration compared to normal
rats.
[0113] Effect of ET-1 on Blood Perfusion in the Breast Tissue of
Normal and TumorBearing Rats as Measured by LDF
[0114] FIG. 3 shows the changes in perfusion, concentration of
moving blood cells (CMBC), and velocity of red blood cells (RBC) in
the breast tissue of tumor-bearing and normal rats. Blood perfusion
in the breast tissue of normal rats did not change after ET-1
administration. Perfusion in the breast tissue of tumor-bearing
rats at 30 minutes following ET-1 administration increased
significantly (176%; p<0.05) compared to normal rats. This
increase in perfusion returned to baseline at 60 and 120 minutes
following ET-1 administration in tumor-bearing rats.
[0115] The CMBC in tumor-bearing rats increased significantly (54%;
p<0.05) at 60 minutes post ET-1 administration as compared to
normal rats. CMBC returned to baseline at 120 minutes after ET-1
administration. The velocity of RBC increased significantly (252%;
p<0.05) at 30 minutes post ET-1 administration compared to
normal rats. Two hours (120 minutes) after ET-1 administration, the
velocity of RBC in tumor-bearing rats returned to baseline.
[0116] Effect of BQ7880n ET-1 Induced Changes in Blood Perfusion in
the Breast Tissue of Normal and Tumor-Bearing Rats as Measured by
LDF
[0117] FIG. 4 shows the effect of BQ788 on changes induced by ET-1
in blood perfusion, CMBC, and velocity of RBC in tumor-bearing and
normal rats, respectively. Blood perfusion in the breast tissue of
normal rats did not change significantly after BQ788 administration
or ET-1 infusion. However, perfusion in the breast tumor tissue of
tumor-bearing rats decreased significantly at 30 (25.25.+-.5.7%;
P<0.05) and 60 minutes (25.17.+-.2.8%; P<0.05) following ET-1
infusion in BQ788 pretreated rats. Pretreatment with BQ788
attenuated the increase in perfusion induced by ET-1 in
tumor-bearing rats. No difference between the perfusion in breast
tissue of tumor-bearing rats and normal rats was observed following
ET-1 administration in BQ788 pretreated rats.
[0118] The baseline CMBC in tumor-bearing rats was significantly
higher than the baseline CMBC of breast tissue of normal rats
(42.4%; P<0.05). However, after BQ788 infusion, no difference
between CMBC of tumor-bearing and normal rats was observed. In
addition, no difference in velocity of RBC between the two groups
was observed.
[0119] The above tests show the effect of ET-1 on systemic
hemodynamics and blood flow to the breast tissue of saline-treated
and MNU-treated tumor-bearing rats. It is known that ET-1
stimulates angiogenesis by promoting production of VEGF. Studies
have shown that ET-1 is increased in many cancer tissues like
breast carcinoma (60), breast phyllode tumor (59), prostate
carcinoma (31), liver carcinoma (21), and some meningiomas (33).
The above tests demonstrate changes in ET-1-induced vascular
responses in the breast tumor. The method used in these tests was a
well-established radioactive microsphere technique to study the
systemic hemodynamics and regional blood circulation (12-15).
[0120] ET-1 is a powerful vasoconstrictor (61). ET-1 belongs to a
family of peptides approximately 21 amino acids long. At least
three forms of ET receptors exist, and are known as ET.sub.A,
ET.sub.B, and ET.sub.C. ET.sub.A has a higher affinity for ET-1,
but ET.sub.B has equal affinity for both ET-1 and ET-3 (2, 17, 42).
ET-1 has complex cardiovascular effects. When administered to
anesthetized and ventilated rats, an immediate decrease followed by
a sustained increase in blood pressure is observed (22). It has
been found that ET.sub.A receptors are responsible for the
vasoconstrictor responses, and ET.sub.B receptors are responsible
for the vasodilatory actions of ET-1. ET-1 administration resulted
in an increase in blood flow to the skin tumors possibly due to the
vasodilatory actions of ET.sub.B (6). Similar results in blood flow
to the breast tumor of rats are expected because of an increase in
ET-1 and ET.sub.B in breast tumors.
[0121] Infusion of 50 ng/kg/min of ET-1 caused a biphasic response
in blood pressure, i.e., an immediate but short lasting decrease
followed by a sustained increase. These results are in accordance
with previous studies (20, 30, 38, 56). ET-1 produced a marked
pressor response in both normal and tumor-bearing rats, which was
accompanied by a significant decrease in SV and CO. TPR
significantly increased in both normal and tumor-bearing rats and
may explain the observed pressor response.
[0122] Baseline blood flow to the breast tumor tissue of
tumor-bearing rats was higher than blood flow in normal animals.
This was observed in an earlier study and is theorized, but not
relied upon, as being attributed to the recruitment of new blood
vessels in the tumor (55). Blood flow to the breast tumor following
ET-1 administration was significantly increased as compared to that
observed in the breast tissue of normal rats. Laser Doppler
flowmetry showing an increase in blood perfusion to the breast
tumor confirmed an increase in blood flow observed in the breast
tumor tissue following ET-1 administration. The increase in blood
perfusion is theorized, but not relied upon, as being attributed to
an increase in either velocity of RBC velocity or CMBC, or both. At
the end of ET-1 infusion an increase in velocity of RBC was
observed, whereas an increase in CMBC was observed 30 minutes after
ET-1 infusion.
[0123] Further, the observed increase in blood flow in response to
ET-1 is theorized, but not relied upon, as being attributed to
ET.sub.B mediated vasodilation. Studies have shown that ET-1 and
ET.sub.B receptor expression is augmented in the breast cancer
tissue (1, 60). In accordance with the present invention, it was
found that administration of BQ788 blocked the ET-1-induced
increase in blood flow to the tumor tissue. BQ788 (i.e.,
N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyll-
eucyl-D-1-methoxycarbonyltrptophanyl-D-Nle) is a specific ET.sub.B
receptor antagonist. BQ788 inhibits binding to ET.sub.B receptors
with an IC.sub.50 value of 1.2 nM.
[0124] BQ788 was used to determine the role of ET.sub.B receptors
in ET-1 induced vasodilation in the breast tumor. This result
suggests that ET-1-induced vasodilatory responses are mediated
through ET.sub.B receptors. Expression of ET.sub.B receptors is
significantly higher in the endothelial cells than in the smooth
muscle cells, and is regulated by various growth factors and
cytokines (49). Normal breast tissue has a higher level of ET.sub.B
than ET.sub.A receptors (1), and it is theorized, but not relied
upon, that during breast cancer, ET.sub.B receptors are
overexpressed and contribute to maintaining blood flow to the tumor
tissue.
[0125] As tumors grow, new blood vessels are recruited to supply
nutrients. This recruitment can be incorporation of existing
vessels into the tumor or creation of new blood vessels (7).
Studies have shown that new vessels have different physical
properties than normal vasculature. Unlike normal vessels, these
vessels do not have any smooth muscle layers or any innervation,
but consist only of single layers of endothelial cells.
[0126] In summary, the present tests clearly demonstrate that the
infusion of ET-1 produced an increase in blood flow and a decrease
in vascular resistance of the breast tumor tissue, and that this
increase in blood flow can be blocked by an ET.sub.B receptor
antagonist, e.g., BQ788.
[0127] The increased blood flow observed in the rat breast tumor is
attributed to increased ET.sub.B receptors. Therefore, blocking
these receptors can reduce blood flow to the tumor. The clinical
significance of these findings is that ET.sub.B receptor
antagonists play a role in reducing blood supply to the breast
tumor tissue, and thereby prevent and/or reduce growth of the
breast tumor, and solid tumors in general.
[0128] The test results, therefore, clearly demonstrate that
ET.sub.B antagonists, like BQ788, can prevent or treat solid
tumors. ET.sub.B antagonists optionally can be combined with an
angiogenesis inhibitor to potentiate the effects of the ET.sub.B
antagonist.
[0129] The ET.sub.B antagonist, optional angiogenesis inhibitor,
ET.sub.B agonist, and chemotherapeutic agent (hereafter
collectively "active ingredients") can be formulated in suitable
excipients for oral administration or for parenteral
administration. Such excipients are well known in the art. The
active ingredients typically are present in such a composition in
an amount of about 0.1% to about 75% by weight.
[0130] Pharmaceutical compositions containing the active
ingredients are suitable for administration to humans or other
mammals. Typically, the pharmaceutical compositions are sterile,
and contain no toxic, carcinogenic, or mutagenic compounds that
would cause an adverse reaction when administered. Administration
of the pharmaceutical composition can be performed before, during,
or after the onset of solid tumor growth.
[0131] A method of the present invention can be accomplished using
active ingredients as described above, or as a physiologically
acceptable salt, derivative, prodrug, or solvate thereof. The
active ingredients can be administered as the neat compound, or as
a pharmaceutical composition containing either or both
entities.
[0132] The active ingredients can be administered by any suitable
route, for example by oral, buccal, inhalation, sublingual, rectal,
vaginal, intracisternal through lumbar puncture, transurethral,
nasal, percutaneous, i.e., transdermal, or parenteral (including
intravenous, intramuscular, subcutaneous, and intracoronary)
administration. Parenteral administration can be accomplished using
a needle and syringe, or using a high pressure technique, like
POWDERJECT.TM..
[0133] The pharmaceutical compositions include those wherein the
active ingredients are administered in an effective amount to
achieve their intended purpose. More specifically, a
"therapeutically effective amount" means an amount effective to
prevent development of, to eliminate, to retard the progression of,
or to reduce the size of a solid tumor. Determination of a
therapeutically effective amount is well within the capability of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0134] A "therapeutically effective dose" refers to that amount of
the active ingredients that results in achieving the desired
effect. Toxicity and therapeutic efficacy of such active
ingredients can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., 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, which is expressed as the ratio between
LD.sub.50 and ED.sub.50. A high therapeutic index is preferred. The
data obtained can be used in formulating a range of dosage for use
in humans. The dosage of the active ingredients preferably lies
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.
[0135] The exact formulation, route of administration, and dosage
is determined by an individual physician in view of the patient's
condition. Dosage amount and interval can be adjusted individually
to provide levels of the active ingredients that are sufficient to
maintain therapeutic or prophylactic effects.
[0136] The amount of pharmaceutical composition administered is
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration, and
the judgment of the prescribing physician.
[0137] Specifically, for administration to a human in the curative
or prophylactic treatment of a breast tumor, oral dosages of active
ingredients, individually generally are about 10 to about 200 mg
daily for an average adult patient (70 kg), typically divided into
two to three doses per day. Thus, for a typical adult patient,
individual tablets or capsules contain about 0.1 to about 50 mg
active ingredients, in a suitable pharmaceutically acceptable
vehicle or carrier, for administration in single or multiple doses,
once or several times per day. Dosages for intravenous, buccal, or
sublingual administration typically are about 0.1 to about 10 mg/kg
per single dose as required. In practice, the physician determines
the actual dosing regimen that is most suitable for an individual
patient, and the dosage varies with the age, weight, and response
of the particular patient. The above dosages are exemplary of the
average case, but there can be individual instances in which higher
or lower dosages are merited, and such are within the scope of this
invention.
[0138] The active ingredients can be administered alone, or in
admixture with a pharmaceutical carrier selected with regard to the
intended route of administration and standard pharmaceutical
practice. Pharmaceutical compositions for use in accordance with
the present invention thus can be formulated in a conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries that facilitate processing of
the active ingredients into preparations which can be used
pharmaceutically.
[0139] These pharmaceutical compositions can be manufactured in a
conventional manner, e.g., by conventional mixing, dissolving,
granulating, dragee-making, emulsifying, encapsulating, entrapping,
or lyophilizing processes. Proper formulation is dependent upon the
route of administration chosen. When a therapeutically effective
amount of the active ingredients are administered orally, the
composition typically is in the form of a tablet, capsule, powder,
solution, or elixir. When administered in tablet form, the
composition can additionally contain a solid carrier, such as a
gelatin or an adjuvant. The tablet, capsule, and powder contain
about 5% to about 95% of an active ingredients, and preferably from
about 25% to about 90% active ingredients. When administered in
liquid form, a liquid carrier, such as water, petroleum, or oils of
animal or plant origin, can be added. The liquid form of the
composition can further contain physiological saline solution,
dextrose or other saccharide solutions, or glycols. When
administered in liquid form, the composition contains about 0.5% to
about 90% by weight of active ingredients, and preferably about 1%
to about 50% of active ingredients.
[0140] When a therapeutically effective amount of the active
ingredients is administered by intravenous, cutaneous, or
subcutaneous injection, the composition is in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable solutions, having due
regard to pH, isotonicity, stability, and the like, is within the
skill in the art. A preferred composition for intravenous,
cutaneous, or subcutaneous injection typically contains, in
addition to an isotonic vehicle.
[0141] Suitable active ingredients can be readily combined with
pharmaceutically acceptable carriers well-known in the art. Such
carriers enable the active agents to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can be obtained
by adding the active ingredients with a solid excipient, optionally
grinding the resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients include, for example,
fillers and cellulose preparations. If desired, disintegrating
agents can be added.
[0142] The active ingredients can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampules or in multidose 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.
[0143] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active agent in water-soluble
form. Additionally, suspensions of the active ingredients can be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils or synthetic
fatty acid esters. Aqueous injection suspensions can contain
substances which increase the viscosity of the suspension.
Optionally, the suspension also can contain suitable stabilizers or
agents that increase the solubility of the compounds and allow for
the preparation of highly concentrated solutions. Alternatively, a
present composition can be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0144] The active ingredients also can be formulated in rectal
compositions, such as suppositories or retention enemas, e.g.,
containing conventional suppository bases. In addition to the
formulations described previously, the active ingredients also can
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 active ingredients 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.
[0145] In particular, the active ingredients can be administered
orally, buccally, or sublingually in the form of tablets containing
excipients, such as starch or lactose, or in capsules or ovules,
either alone or in admixture with excipients, or in the form of
elixirs or suspensions containing flavoring or coloring agents.
Such liquid preparations can be prepared with pharmaceutically
acceptable additives, such as suspending agents. The active
ingredients also can be injected parenterally, for example,
intravenously, intramuscularly, subcutaneously, or intracoronarily.
For parenteral administration, the active ingredients are best used
in the form of a sterile aqueous solution which can contain other
substances, for example, salts, or monosaccharides, such as
mannitol or glucose, to make the solution isotonic with blood.
[0146] For veterinary use, the active ingredients are administered
as a suitably acceptable formulation in accordance with normal
veterinary practice. The veterinarian can readily determine the
dosing regimen and route of administration that is most appropriate
for a particular animal.
[0147] As stated above, it has been discovered that using an
ET.sub.B antagonist, alone or together with an angiogenesis
inhibitor, is useful in the treatment and prevention of solid
tumors.
[0148] The angiogenesis inhibitor, like the ET.sub.B antagonist, is
administered in an effective amount to perform its intended
function. The angiogenesis inhibitor can be administered by any
suitable means, typically using a composition containing the
angiogenesis inhibitor.
[0149] The angiogenesis inhibitor can be administered
simultaneously with the ET.sub.B antagonist, or prior to or after
ET.sub.B antagonist administration. The ET.sub.B antagonist and
optional angiogenesis inhibitor also can be administered in
conjunction with radiation treatment of the solid tumor and an
optional radiosensitizer.
[0150] In another embodiment, the solid tumor can be treated by
administration of therapeutically effective amounts of an ET.sub.B
agonist and a chemotherapeutic agent. Administration of the
ET.sub.B agonist and chemotherapeutic agent can be performed as
described above for the ET.sub.B antagonist and angiogenesis
inhibitor.
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hereinbefore set forth can be made without departing from the
spirit and scope thereof, and, therefore, only such limitations
should be imposed as are indicated by the appended claims. 123456
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