U.S. patent application number 15/597588 was filed with the patent office on 2017-09-07 for use of opioid antagonists to attenuate endothelial cell proliferation and migration.
The applicant listed for this patent is The University of Chicago. Invention is credited to Joe G.N. Garcia, Mark Lingen, Jonathan Moss, Patrick A. Singleton, Chun-Su Yuan.
Application Number | 20170252336 15/597588 |
Document ID | / |
Family ID | 51789729 |
Filed Date | 2017-09-07 |
United States Patent
Application |
20170252336 |
Kind Code |
A1 |
Moss; Jonathan ; et
al. |
September 7, 2017 |
USE OF OPIOID ANTAGONISTS TO ATTENUATE ENDOTHELIAL CELL
PROLIFERATION AND MIGRATION
Abstract
The invention provides methods of attenuating, e.g., inhibiting
or reducing, cellular proliferation and migration, particularly
endothelial cell proliferation and migration, including that
associated with angiogenesis, using opioid antagonists, including,
but not limited to, those that are peripherally restricted
antagonists.
Inventors: |
Moss; Jonathan; (Chicago,
IL) ; Lingen; Mark; (Oak Park, IL) ;
Singleton; Patrick A.; (Chicago, IL) ; Garcia; Joe
G.N.; (Chicago, IL) ; Yuan; Chun-Su; (Chicago,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago |
Chicago |
IL |
US |
|
|
Family ID: |
51789729 |
Appl. No.: |
15/597588 |
Filed: |
May 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14292816 |
May 30, 2014 |
9662325 |
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15597588 |
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13972122 |
Aug 21, 2013 |
9675602 |
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14292816 |
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11379010 |
Apr 17, 2006 |
8524731 |
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13972122 |
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PCT/US2006/007892 |
Mar 7, 2006 |
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11379010 |
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60760851 |
Jan 20, 2006 |
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60731009 |
Oct 28, 2005 |
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60725703 |
Oct 12, 2005 |
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60659193 |
Mar 7, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/337 20130101;
A61K 31/475 20130101; A61K 31/337 20130101; A61K 31/517 20130101;
A61K 31/7068 20130101; A61K 31/7068 20130101; A61K 31/485 20130101;
A61K 45/06 20130101; A61K 31/475 20130101; A61K 39/39558 20130101;
A61K 31/485 20130101; A61K 2300/00 20130101; A61K 39/39558
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 31/517 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 31/485 20060101
A61K031/485; A61K 31/475 20060101 A61K031/475; A61K 31/337 20060101
A61K031/337; A61K 39/395 20060101 A61K039/395; A61K 31/7068
20060101 A61K031/7068; A61K 45/06 20060101 A61K045/06; A61K 31/517
20060101 A61K031/517 |
Goverment Interests
STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was supported in part by National Institutes
of Health grants: DE12322; DE00470; and DE015830. The United States
government has certain rights in this invention.
Claims
1. A method of inhibiting hyperproliferation of cells, comprising
administering to a subject in need thereof an effective amount of a
peripheral opioid antagonist and a chemotherapeutic agent, wherein
the peripheral opioid antagonist is not a quaternary derivative of
noroyxmorphone.
2. The method of claim 1, wherein a disorder/disease is treated by
the inhibitory effect of the peripheral opioid antagonist and the
chemotherapeutic agent on the hyperproliferation of the cells.
3. The method of claim 2, wherein the disorder/disease is a
cancer.
4. The method of claim 1, wherein the hyperproliferation is
agonist-induced hyperproliferation.
5. The method of claim 5, wherein the agonist-induced
hyperproliferation is VEGF-induced hyperproliferation
6. The method of claim 1, wherein the cells are endothelial
cells.
7. The method of claim 6, wherein the endothelial cells are
vascular endothelial cells and the unwanted migration and/or
proliferation of the vascular endothelial cells is
abnormal/unwanted angiogenesis.
8. The method of claim 1, wherein the subject is a human cancer
patient, and the combination of the peripheral opioid antagonist
and the chemotherapeutic agent is effective to inhibit or attenuate
the hyperproliferaton of endothelial cells.
9. The method of claim 1, wherein the peripheral opioid antagonist
is administered simultaneously or sequentially with the
chemotherapeutic agent.
10. The method of claim 1, wherein the subject is taking concurrent
opioid therapy.
11. The method of claim 1, wherein the subject is not taking
concurrent opioid therapy.
12. The method of claim 1, wherein the chemotherapeutic agent is
anti-metabolite, an anti-microtubule, an anti-neovascularization
agent, or a tyrosine kinase inhibitor.
13. The method of claim 12, wherein the anti-metabolite is
5-fluorouracil or gemcitabine.
14. The method of claim 12 wherein the tyrosine kinase inhibitor is
an EGFR inhibitor.
15. The method of claim 14, wherein the EGFR inhibitor is
erlotinib.
16. The method of claim 12, wherein the anti-neovascularization
agent is a VEGF monoclonal antibody.
17. The method of claim 16, wherein the anti-VEGF monoclonal
anti-body is bevacizumab.
18. The method of claim 12, wherein the anti-microtubule is
vinorelbine, paclitaxel, docetaxel, or a combination thereof.
19. The method of claim 1, wherein the peripheral opioid antagonist
is a tertiary ammonium derivative of morphinan, benzomorphan or
normorphinan.
20. The method of claim 1, wherein the peripheral opioid antagonist
is a N-substituted piperidine.
21. The method of claim 17, wherein the N-piperidine is a
piperidine-N-alkylcarbonylate.
22. The method of claim 18, wherein the
piperidine-N-alkylcarbonylate is a N-alkylamino-3,4,4 substituted
piperidine.
23. The method of claim 19, wherein N-alkylamino-3,4,4 substituted
piperidine is alvimopan.
24. The method of claim 20, wherein the
piperidine-N-alkylcarboxylate is represented by formula (III):
##STR00008## wherein R.sup.1 is hydrogen or alkyl; R.sup.2 is
hydrogen, alkyl, or alkenyl; R.sup.3 is hydrogen, alkyl, alkenyl,
aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R.sup.4
is hydrogen, alkyl, or alkenyl; A is OR.sup.5 or NR.sup.6R.sup.7;
wherein R.sup.5 is hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R.sup.6
is hydrogen or alkyl; R.sup.7 is hydrogen, alkyl, alkenyl, aryl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl or aryl-substituted alkyl, or
alkylene-substituted B or together with the nitrogen atom to which
they are attached, R.sup.6 and R.sup.7 form a heterocyclic ring
selected from pyrrole and piperidine; B is ##STR00009## wherein
R.sup.8 is hydrogen or alkyl; R.sup.9 is hydrogen, alkyl, alkenyl,
aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl or aryl-substituted alkyl or
together with the nitrogen atom to which they are attached, R.sup.8
and R.sup.9 form a heterocyclic ring selected from pyrrole and
piperidine; W is OR.sup.10, NR.sup.11R.sup.12, or OE; wherein
R.sup.10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,
cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkenyl, or
aryl-substituted alkyl; R.sup.11 is hydrogen or alkyl; R.sup.12 is
hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,
cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,
aryl-substituted alkyl, or alkylene-substituted C(.dbd.O)Y or,
together with the nitrogen atom to which they are attached,
R.sup.11 and R.sup.12 form a heterocyclic ring selected from
pyrrole and piperidine; E is ##STR00010## alkylene-substituted
(C.dbd.O)D, or --R.sup.13OC(.dbd.O)R.sup.14; wherein R.sup.13 is
alkyl-substituted alkylene; R.sup.14 is alkyl; D is OR.sup.15 or
NR.sup.16R.sup.17; wherein R.sup.15 is hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl substituted alkyl, or aryl-substituted alkyl; R.sup.16
is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl,
cycloalkyl, cycloalkenyl, cycloalkyl substituted alkyl, or
cycloalkenyl-substituted alkyl; R.sup.17 is hydrogen or alkyl or,
together with the nitrogen atom to which they are attached,
R.sup.16 and R.sup.17 form a heterocyclic ring selected from the
group consisting of pyrrole or piperidine; Y is OR.sup.18 or
NR.sup.19R.sup.20; wherein R.sup.18 is hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R.sup.19
is hydrogen or alkyl; R.sup.20 is hydrogen, alkyl, alkenyl, aryl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl or,
together with the nitrogen atom to which they are attached,
R.sup.19 and R.sup.20 form a heterocyclic ring selected from
pyrrole and piperidine; R.sup.21 is hydrogen or alkyl; and n is 0
to 4.
25. The method of claim 22, wherein the N-alkylamino-3,4,4
substituted piperidine represented by formula (IV):
##STR00011##
26. A method of enhancing the efficacy of a cancer chemotherapeutic
agent in a subject, the method comprising administering to a
subject having a cancer the cancer chemotherapeutic agent and a
peripheral mu-opioid antagonist, wherein the peripheral opioid
antagonist is not a quaternary derivative of noroxymorphone, and
wherein the chemotherapeutic agent is a tyrosine kinase inhibitor,
an anti-microtubule, an anti-neovascularization agent or an
antimetabolite.
27. The method of claim 26, wherein the subject is taking
concurrent opioid therapy.
28. A method of achieving an effect in endothelial cells,
comprising contacting the cells with an effective amount of a
peripheral opioid antagonist and a chemotherapeutic agent, wherein
the peripheral opioid antagonist is not a quaternary derivative of
noroxymorphone and wherein the effect is inhibiting VEGF activity
or Rho A activation.
29. A method of treating cancer, comprising administering an
effective amount of a peripheral opioid antagonist and a
chemotherapeutic agent to a subject in need thereof to inhibit the
hyperproliferation of cancer cells, wherein the peripheral opioid
antagonist is not a quaternary derivative of noroxymorphone.
30. The method of claim 29, wherein the cancer cells overexpress
mu-opioid receptors.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/292,816, filed May 30, 2014, now U.S. Pat. No. 9,662,325,
which is a continuation-in-part of U.S. application Ser. No.
13/972,122, filed Aug. 21, 2013, which is a continuation of U.S.
application Ser. No. 11/379,010, filed Apr. 17, 2006, now U.S. Pat.
No. 8,524,731, which is a continuation-in-part of PCT Application
No. PCT/US2006/07892 filed on Mar. 7, 2006, which claims benefit
under 35 U.S.C. 119(e) of the filing dates of U.S. Ser. Nos.
60/659,193 filed on Mar. 7, 2005, 60/725,703 filed Oct. 12, 2005,
60/731,009 filed on Oct. 28, 2005, and 60/760,851 filed Jan. 20,
2006, the entire disclosures of which are incorporated herein by
reference.
FIELD OF INVENTION
[0003] The invention relates to methods of attenuating migration
and/or proliferation of endothelial cells, especially associated
with tumors, utilizing opioid antagonists.
INTRODUCTION
[0004] Cellular proliferation is a normal ongoing process in all
living organisms that involves numerous factors and signals that
are delicately balanced to maintain regular cellular cycles.
Whether or not mammalian cells will grow and divide is determined
by a variety of feedback control mechanism, which includes the
availability of space in which a cell can grow, and the secretion
of specific stimulatory and inhibitory factors in the immediate
environment.
[0005] Angiogenesis and angiogenesis-related diseases are affected
by cellular proliferation. The process of angiogenesis results in
the formation of new blood vessels. Under normal physiological
conditions, animals, including humans, undergo angiogenesis only in
very specific restricted situations. For example, angiogenesis is
normally observed in wound healing, fetal and embryonic
development, and formation of the corpus luteum, endometrium and
placenta.
[0006] During the process of angiogenesis, endothelial cells, which
normally exist in a quiescent state as part of an existing blood
vessel, enter a migratory, proliferative state. This migratory,
proliferative state of endothelial cells is eventually resolved
when the cells return to the quiescent state as part of a
functional new blood vessel. The generation of new capillaries
involves a complex process that requires a number of cellular and
molecular events to occur in both a spatial and temporal pattern.
Some of these activities include the degradation of the surrounding
basement membrane of the originating vessel, the migration of the
endothelial cells through the connective tissue stroma, cell
proliferation, the formation of tube-like structures, and the
maturation of these endothelial-lined tubes into new blood vessels.
(Cliff, 1963; Schoefl, 1963; Ausprunck and Folkman, 1977). Some
essential angiogenic factors include fibroblast growth
factor-basic, vascular endothelial growth factor (VEGF),
angiopoietins, cytokines, extracellular matrix proteins, and matrix
metalloproteases. These factors are produced locally by stromal
cells and by activated leukocytes that are recruited to the area
(Risau, W. (1997) Nature 386(6626):671-674; Risau and Flamme (1995)
Ann. Rev. Cell Dev. Biol. 11:73-91). Unlike other angiogenic
factors, VEGF acts as an endothelial cell specific mitogen during
angiogenesis (Terman et al., 1992 and Ferrara, 1993).
[0007] Angiogenesis can be stimulated and harnessed by some
neoplasms (e.g., tumors) to increase nutrient uptake. It has been
found that angiogenesis is essential for the growth of solid tumors
beyond 2-3 mm in diameter and for tumor metastasis (Folkman, 1995;
reviewed in Bouck et al., 1996). In contrast to normal
angiogenesis, which leads to anastomoses and capillary maturation,
angiogenesis associated with neoplasia is a continuous process.
Endothelial cells are activated by nearby neoplastic cells to
secrete not only VEGF which stimulates angiogenesis, but also
matrix metalloproteases (MMP) which degrade the surrounding
extracellular matrix. The endothelial cells then invade the
extracellular matrix where they migrate, proliferate, and organize
to form new blood vessels, which support neoplasm growth and
survival. The newly vascularized neoplasm continues to grow,
leading to further nutrient deprivation and chronic pro-angiogenic
signaling. The vasculature of neoplasms is characterized by the
presence of lacunae and a low rate of anastomoses. This partially
dysfunctional vasculature fuels the permanent requirement for
angiogenesis. Additionally, this incomplete vasculature allows the
shedding of neoplastic cells into the systemic circulation. Hence,
the angiogenic potential of a neoplasm correlates with metastatic
potential. (Weidner et al. (1991) N. Engl. J. Med. 324(1):1-8;
Folkman and Shing (1992) J. Biol. Chern. 267(16): 10931-10934).
[0008] As a significant proportion of neoplasms are dependent on
continued angiogenesis, inhibition of angiogenesis blocks neoplasm
growth which often leads to complete necrosis of the neoplasm.
(Weidner et al. (1991) N. Engl. J. Med. 324(1):1-8; Folkman and
Shing (1992) J. Biol. Chern. 267(16):10931-10934). Suppression of
anyone of the steps of and/or factors involved in angiogenesis
could inhibit the formation of new vessels, and therefore, affect
tumor growth and generation of metastases. Indeed, it has been
estimated that the elimination of a single endothelial cell could
inhibit the growth of 100 tumor cells (Thorpe et al., 1995). It has
also been found that antibodies raised against the angiogenic
factor VEGF have been shown to suppress tumor growth in vivo (Kim
et al., 1993).
[0009] As part of treating and managing patients with cancer and
many medical conditions, opioid agonists, such as morphine, are
widely used for associated pain. For example, morphine is used in
the terminal phase of care of approximately one-half of the
patients that die of cancer each year in the United States. Opioid
agonists, such as morphine comprise a group of compounds that act
on a series of endogenous opioid receptors, such as mu-, kappa-,
and delta-receptors in biological systems. Normally, these
endogenous receptors bind endogenous opioids. Endogenous opioids
are natively produced by mammalian cells.
[0010] Endogenous opioids include beta-endorphins, enkephalins, and
dynorphins. Beta-endorphins show a preference for mu receptors,
enkephalins for delta receptors and dynorphins for kappa receptors.
Opioid agonists are classified by their preferential effects on the
endogenous opioid receptors. Generally, the mu-receptor is
associated with pain relief, and chemical dependence (e.g., drug
addiction and alcoholism). Morphine, for example, is a mu-opioid
agonist. Opioid receptors are not limited to the brain and central
nervous system (eNS), e.g., to central receptors. Peripheral opioid
receptors may be found in other tissues throughout the body, e.g.,
gastrointestinal tissue.
[0011] Despite wide use in pain management, morphine and other
opioid medications can have severe side effects that may be caused
by activation of the peripheral receptors. The side effects can be
difficult to manage and can result in the patient refusing
opioid-based pain management. Side effects of opioid treatment
include nausea, constipation, inhibition of gastrointestinal
motility, respiratory suppression and immunosuppression.
Additionally, morphine and other opioid receptor agonists can
stimulate human microvascular endothelial cell proliferation and
angiogenesis in vitro and in vivo at typical morphine or
morphine-equivalent blood concentrations. This pro-angiogenesis
activity of the opioid agonists, while palliative for pain, may
hasten tumor progression.
[0012] Opioid antagonists are similarly classified by their effects
on the opioid receptors, e.g., by their ability to antagonize one
receptor more effectively than another receptor. For example, the
opioid antagonist naloxone acts as a competitive antagonist at all
opioid receptors, but is approximately ten times more effective at
mu-receptors than at kappa receptors, and is, therefore, classified
as a mu-opioid antagonist. Opioid antagonists may antagonize
central receptors, peripheral receptors or both. Opioid
antagonists, and in particular peripheral opioid antagonists, have
been used to lessen the side-effects of exogenously administered
opioids, as well as to lessen the unwanted effects of excessive
endogenous opioids.
[0013] Opioid antagonists also have been examined for their
potential use as anticancer agents for particular types of cancer,
as described in U.S. Pat. Nos. 6,384,044 and 6,136,780 and in the
scientific literature Gupta et al. Cancer Research, 62: 4491-98
(2002). The anticancer effects of opioid antagonists have been
controversial and not well understood, but it has been held that
the opioid antagonist anticancer effects, to the extent they have
been shown at all, are unrelated to angiogenesis (Poonawala T, et
al., Wound Repair Regen. 2005 March-April; 13(2): 165-74; Popov I.,
Acta Chirlugosl. 2004; 51(2): 117-21; Blebea J, et al., J Vasc
Surg. 2002 March; 35(3):532-8; Balasubramanian S, et al., J Mol
Cell Cardiol. 2001 December; 33(12):2179-87; Zagon I S, et al., Int
J Oncol. 2000 November; 17(5):1053-61; Blebea J et al., J Vasc
Surg. 2000 August; 32(2):364-73; Pasi A, et al., Gen Pharmacol.
(991; 22(6):1077-9.) In fact, it has been reported that in
xenograft tumor model in mice, the opioid antagonist naloxone did
not exhibit a significant effect on morphine induced angiogenesis
Gupta et al. Cancer Research, 62: 4491-98 (2002). Therefore, it is
surprising that it is now discovered that opioid antagonists can
inhibit endothelial proliferation and migration associated with
angiogenesis.
BRIEF DESCRIPTION OF THE INVENTION
[0014] The invention provides methods of attenuating, e.g.,
inhibiting or reducing, cellular proliferation and migration,
particularly endothelial cell proliferation and migration,
including that associated with angiogenesis, using opioid
antagonists, including, but not limited to, those that are
peripherally restricted antagonists.
[0015] According to one aspect of the invention, a method of
treatment is provided. The method involves administering to a
subject with a disorder characterized by unwanted migration or
proliferation of endothelial cells an effective amount of an opioid
antagonist. The treatment may inhibit one or both of migration and
proliferation. The unwanted migration or proliferation of
endothelial cells can be unwanted migration or proliferation of
vascular endothelial cells, including, but not limited to, unwanted
neovascularization or angiogenesis. Examples of unwanted
neovascularization include, but are not limited to,
neovascularization associated with cancer and ocular
neovascularization. The disorder can be any disorder characterized
by unwanted migration or proliferation of endothelial cells.
Important such disorders are cancer, sickle cell anemia, vascular
wounds, proliferative retinopathies, and unwanted endothelial cell
proliferation in the kidneys and the lung.
[0016] In important embodiments, the opioid antagonist is a
peripheral opioid antagonist. Peripheral opioid antagonists
include, but are not limited to, quaternary or tertiary morphinan
derivatives, piperidine-N-alkylcarboxylates, and quaternary
benzomorphans. One important such peripheral opioid antagonist is
methylnaltrexone. Another opioid antagonist is alvimopan. In
important embodiments, the effective amount is such that the
subject has effective circulating blood plasma levels of the opioid
antagonist continuously for at least 1 week, at least 2 weeks, at
least three weeks and, preferably, at least 4 weeks.
[0017] The invention also includes the coadministration of the
opioid antagonists with agents that are not opioid antagonists, but
which are nonetheless useful in treating disorders characterized by
unwanted migration or proliferation of endothelial cells. Examples
of such agents include anticancer agents, antineovascularization
agents (for example, anti-VEGF monoclonal antibody), antidiabetes
agents, anti-sickle cell agents, wound healing agents, and
antiendothelial cell proliferative agents.
[0018] It will be understood that the subjects may be, or may not
be, on concurrent opioid therapy, depending on the particular
disorder the subject has, the severity of the disorder, and the
need the subject has for pain management. In some embodiments, the
subject is taking concurrent opioid therapy. In some embodiments,
the subject is not taking concurrent opioid therapy. In some
embodiments, the subject is taking concurrent chronic opioid
therapy. In some embodiments, the subject is not taking concurrent
chronic opioid therapy.
[0019] According to another aspect of the invention, a method of
inhibiting VEGF activity in endothelial cells is provided. The
method involves contacting the cells with an effective amount of an
opioid antagonist.
[0020] According to another aspect of the invention, a method of
inhibiting exogenous opioid-induced cellular migration or
proliferation in endothelial cells is provided. The method involves
contacting the cells with an effective amount of an opioid
antagonist.
[0021] According to another aspect of the invention, a method of
inhibiting Rho A activation in endothelial cells is provided. The
method involves contacting the cells with an effective amount of an
opioid antagonist.
[0022] According to any of the foregoing embodiments, the opioid
antagonist preferably is a peripheral opioid antagonist, and most
preferably is methylnaltrexone.
[0023] The invention provides methods of attenuating migration
and/or proliferation of endothelial cells of a tumor or cancer,
comprising contacting the cells with an antimigratory or
antiproliferative amount of an opioid antagonist. In another
aspect, the invention provides methods of attenuating angiogenesis
associated with cancer. Thus, the invention contemplates treating a
human cancer patient, for example, by a method of attenuating
angiogenesis in a cancerous tissue of a patient, comprising
administering to the cancer tissue of the patient an effective
amount of an opioid antagonist.
[0024] The invention also provides a method of treating abnormal
neovascularization, comprising administering to a patient in need
of such treatment, an amount of an opioid antagonist effective to
inhibit the formation of blood vessels.
[0025] The invention also includes a method of attenuating tumor
progression and metastasis in animal tissues, comprising contacting
tumor cells or tissues with a growth-inhibiting amount of an opioid
antagonist, and a method of attenuating proliferation of
hyperproliferative cells in a subject, comprising administering to
the subject at least one opioid antagonist, in an amount which is
effective to attenuate proliferation of the hyperproliferative
cells. In one embodiment, the method involves administering a
peripheral opioid antagonist, and, in particular, a quaternary
derivative of noroxymorphone, to a subject with cancer, whether or
not the cancer involves angiogenesis, to treat or inhibit the
development or recurrence of the cancer. Cancers not involving
angiogenesis include those that do not involve the formation of a
solid tumor fed by neovasculature. Certain blood cell cancers fall
into this category, for example: leukemias (cancer of the
leukocytes or white cells), lymphomas (arising in the lymph nodes
or lymphocytes), and some cancers of the bone marrow elements.
Thus, in one aspect of the invention, a method of treatment is
provided. The method involves administering to a subject with a
disorder characterized by hyperproliferation of cells an effective
amount of a peripheral opioid antagonist. In one embodiment, the
cells are cancer cells. The cancer cells may be cancer cells
associated with angiogenesis or they may be unassociated with
angiogenesis. In one embodiment, the peripheral opioid antagonist
is methylnaltrexone.
[0026] In further embodiments, the invention provides methods of
treating cancer, wherein a peripheral opioid antagonist and at
least one other therapeutic agent that is not an opioid or opioid
antagonist are co-administered to the patient. Suitable therapeutic
agents include anticancer agents (including chemotherapeutic agents
and antineoplastic agents), as well as other antiangiogenesis
agents. It has been discovered that opioid antagonists
co-administered with various anticancer drugs, radiotherapy or
other antiangiogenic drugs can give rise to a significantly
enhanced antiproliferative effect on cancerous cells, thus
providing an increased therapeutic effect, e.g., employing
peripheral opioid antagonists to certain tumors can potentiate
their response to other therapeutic regimens. Specifically, a
significantly increased antiproliferative effect, including but not
limited to a significantly increased antiangiogenic effect, is
obtained with co-administered combinations as described in more
detail below. Not only can an existing regimen be enhanced, but new
regimens are possible, resulting, for example, in lower
concentrations of the anticancer compound, a lower dosing of
radiation, or lower concentration of other antiangiogenic drugs,
compared to the treatment regimes in which the drugs or radiation
are used alone. There is the potential, therefore, to provide
therapy wherein adverse side effects associated with the anticancer
or other antiangiogenic drugs or radiotherapy are considerably
reduced than normally observed with the anticancer or other
antiangiogenic drugs or radiotherapy when used alone. Thus, in one
aspect of the invention, a method of treatment is provided. The
method involves administering to a subject with a disorder
characterized by hyperproliferation of cells an effective amount of
an opioid antagonist and an anticancer agent, radiation, or an
antiangiogenic agent.
[0027] In one embodiment, the cells are cancer cells. In one
embodiment, the opioid antagonist is a peripheral opioid
antagonist. In one embodiment, the peripheral opioid antagonist is
methylnaltrexone. In another aspect of the invention, a method of
reducing the risk of recurrence of a cancer in a subject after
medical intervention is provided. The method involves administering
to the subject before, during or after the medical intervention an
effective amount of an opioid antagonist and an anticancer agent,
radiation, or an antiangiogenic agent. In one embodiment, the
opioid antagonist is a peripheral opioid antagonist. In one
embodiment, the peripheral opioid antagonist is
methylnaltrexone.
[0028] In another aspect of the invention, the opioid antagonists
are used peri-operatively. By "peri-operatively," it is meant
immediately before (e.g., in preparation for), during, and/or
immediately after a surgery or a surgical or endoscopic procedure,
e.g. colonoscopy, gastrolaparoscopy, and especially a surgery or
surgical procedure involving the removal of a tumor. The opioid
antagonists act to attenuate the recurrence of and/or the
metastasis of the tumor, especially that arising from angiogenesis
associated therewith. It is anticipated that the opioid antagonist
will preferably be given in a continuous dosing regimen, e.g., a
regimen that maintains a minimum, and even more preferably
relatively constant, blood level. It is further contemplated that
the methods of the present invention may have prophylactic value in
certain disorders associated with abnormal angiogenesis. Thus, the
invention provides a method of preventing the appearance or
re-appearance of a disorder in a mammal, the disorder being
characterized by unwanted endothelial cell migration or
proliferation, including abnormal angiogenesis, comprising
administering to a mammal in need of such treatment, an effective
amount of an opioid antagonist, wherein the disorder is a cancer,
sickle cell anemia, ocular neovascular diseases, diabetes, ocular
retinopathy, or other unwanted endothelial proliferation in
kidneys, eye or lung. It will therefore be understood that, as used
herein, treating a subject with a disorder characterized by
unwanted endothelial cell proliferation or migration includes
treating a subject with an active disorder to inhibit or cure the
disorder and treating a subject to inhibit a disorder from
reoccurring. For example, the subject may have had a solid tumor
removed, and the subject may receive the treatment to inhibit the
tumor from reoccurring.
[0029] In attenuating cell proliferation, the invention provides a
method for the treatment of abnormal cell proliferation of a cell
expressing vascular endothelial growth factor (VEGF) in a mammal
which comprises administering to the mammal a therapeutically
effective amount of an opioid antagonist. The invention also
includes a method of treating cancerous tissue in a subject
comprising, administering to the subject an amount of an opioid
antagonist sufficient to inhibit VEGF production in the cancerous
tissue, as well as a method of treating angiogenic disease, the
method comprising contacting a tissue or a population of
endothelial cells with a composition comprising an amount of at
least one of an opioid antagonist under conditions effective to
inhibit VEGF-induced angiogenesis and to treat angiogenic
disease.
[0030] In another aspect, the present invention provides a method
of inhibiting or reducing angiogenesis, particularly opioid-induced
angiogenesis, e.g., of tumor cells, by administrating or providing
an opioid antagonist, particularly a peripheral opioid antagonist,
to cells undergoing angiogenesis. In further aspect, the invention
provides methods of treating opioid-induced angiogenesis in
patients receiving opioid treatment or in patients where the
angiogenesis is induced by endogenous opioids. The former group is
typically cancer patients on opioid-based pain management. The
methods comprise administering an opioid antagonist to a patient in
an antiangiogenic amount, e.g., an amount sufficient to inhibit or
reduce the opioid-induced angiogenesis. In those patients receiving
opioid treatment, the opioid and the peripheral opioid antagonist
may be co-administered. Peripheral opioid antagonists can, thus, be
used to inhibit or reduce the angiogenic effects of opioids on
tumor cells, and attenuate the growth of a tumor.
[0031] Suitable opioid antagonists generally include heterocyclic
amine compounds that belong to several different classes of
compounds. For example, one class is suitably tertiary derivatives
of morphinan, and in particular, tertiary derivatives of
noroxymorphone. In one embodiment, the tertiary derivative of
noroxymorphone is, e.g. naloxone or naltrexone.
[0032] Suitable peripheral opioid antagonists are also generally
heterocyclic amine compounds that may belong to several different
classes of compounds. For example, one class is suitably quaternary
derivatives of morphinan, and in particular, quaternary derivatives
of noroxymorphone. In one embodiment, the quaternary derivative of
noroxymorphone is, e.g., N-methylnaltrexone (or simply
methylnaltrexone). Another class is N-substituted piperidines. In
one embodiment, the N-piperidine is a
piperidine-N-alkylcarbonylate, such as, e.g., alvimopan. Another
class of compounds which may be of value in the methods of the
present invention is quaternary derivatives of benzomorphans.
[0033] In some embodiments of the invention, the opioid antagonist
may be a mu opioid antagonist. In other embodiments, the opioid
antagonist may be a kappa opioid antagonist. The invention also
encompasses administration of more than one opioid antagonist,
including combinations of mu antagonists, combinations of kappa
antagonists and combinations of mu and kappa antagonists, for
example, a combination of methylnaltrexone and alvimopan, or a
combination of naltrexone and methyl naltrexone.
[0034] In further embodiments, the invention provides methods of
treating opioid-induced angiogenesis in patients receiving an
opioid, wherein a peripheral opioid antagonist and at least one
other therapeutic agent that is not an opioid or opioid antagonist
are co-administered to the patient. Suitable therapeutic agents
include anticancer agents (including chemotherapeutic agents and
antineoplastic agents), as well as other antiangiogenesis
agents.
[0035] In yet another aspect, the invention provides a method of
reducing the risk of recurrence of a cancer or tumor after medical
intervention (such intervention to include but not be limited to
surgery, e.g. pulmonary surgery, surgical and endoscopic
procedures, e.g. colonoscopy, gastrolaparoscopy, chemotherapy,
etc.), comprising co-administering to a cancer patient an opioid
antagonist. Thus, the invention contemplates, for example, a method
of minimizing the post-operative recurrence of, e.g., breast cancer
in a patient, comprising administering to the patient an effective
amount of an opioid antagonist. Peripheral opioid antagonists in
accordance with the present invention, e.g., MNTX, can also inhibit
VEGF, platelet-derived growth factor (PDGF), or sphingosine
I-phosphate (SIP)-stimulated or induced cell proliferation in the
endothelial cells.
[0036] Further provided herein is a method of enhancing the
efficacy of a cancer chemotherapeutic agent in a subject having a
cancer, wherein the method comprises administering the cancer
chemotherapeutic agent and an opioid antagonist to the subject.
[0037] In some embodiments, the chemotherapeutic agent is a
tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase
inhibitor is an EGFR inhibitor. In some embodiments, the tyrosine
kinase inhibitor is erlotinib.
[0038] In some embodiments, the chemotherapeutic agent is an
anti-microtubule agent. In some embodiments, the anti-microtubule
agent is selected from the group consisting of: vinorelbine,
paclitaxel, docetaxel, and a combination thereof.
[0039] In some embodiments, the chemotherapeutic agent is an
antimetabolite. In some embodiments, the antimetabolite is
gemcitabine.
[0040] Also provided herein is a method of inhibiting cancer
progression in a subject, comprising administering a
therapeutically effective amount of a chemotherapeutic agent with a
therapeutically effective amount of an opioid antagonist. In some
embodiments, the chemotherapeutic agent is a tyrosine kinase
inhibitor, an anti-microtubule agent or an antimetabolite.
[0041] In some embodiments, the opioid antagonist is a peripheral
opioid antagonist. In some embodiments, the peripheral opioid
antagonist comprises a compound of formula (I). In some
embodiments, the peripheral opioid antagonist comprises
methylnaltrexone.
BRIEF DESCRIPTION OF DRAWINGS
[0042] The invention may be better understood and appreciated by
reference to the detailed description of specific embodiments
presented herein in conjunction with the accompanying drawings of
which:
[0043] FIG. 1 is a bar graph of dose-dependent inhibition of human
microvascular endothelial cell (HMVEC) migration, depicting the
results from Example 1.
[0044] FIG. 2 is a bar graph of dose-dependent inhibition of human
microvascular endothelial cell migration, depicting the results
from Example 2.
[0045] FIG. 3 is a bar graph of dose-dependent inhibition of HMVEC
migration using MNTX and MNTX+DAMGO.
[0046] FIG. 4 is a bar graph of dose-dependent inhibition of HMVEC
migration 15 using naloxone and naloxone+DAMGO.
[0047] FIG. 5 is a bar graph of dose-dependent effect of M3G and
M6G on HMVEC migration.
[0048] FIG. 6 is a photomicrograph that shows morphine induced
endothelial cell migration in the presence and absence of MNTX.
Panel A=Control, Panel B=20 MS (morphine sulfate), Panel C=MNTX,
and Panel D=MS+MNTX. Arrows are shown in Panel A to highlight
several cells that have successfully migrated across the
membrane.
[0049] FIG. 7 is a bar graph of percent proliferation (A) and
migration (B) of human pulmonary microvascular endothelial cells in
the presence of VEGF, 25 morphine and DAMGO with or without
MNTX.
[0050] FIG. 8 is a panel of immunoblots indicating the tyrosine
phosphorylation (activation) of (A) of anti-VEGF R.1 (Flt-1) and 2
(Flk-1) using immunoprecipitated VEGF R.1 or 2 and
anti-phospho-tyrosine in human pulmonary microvascular endothelial
cells in the presence of VEGF, morphine and DAMGO with or without
MNTX and a bar graph (B) of percent proliferation and migration of
human pulmonary microvascular endothelial cells in the presence of
VEGF, morphine and DAMGO with or without VEGF R. inhibitor.
[0051] FIG. 9 is a panel of immunoblots indicating RhoA activation
using anti-RhoA in human pulmonary microvascular endothelial cells
in the presence of VEGF, morphine and DAMGO with or without MNTX
(A) or VEGF R. Inhibitor (B).
[0052] FIG. 10 is a panel of immunoblots (A) of anti-RhoA of human
pulmonary microvascular endothelial cells in the presence of
scramble siRNA (targeting no known human mRNA sequence) or RhoA
siRNA and a bar graph of percent proliferation (B) and migration
(C) of human pulmonary microvascular endothelial cells in the
presence of VEGF, morphine and DAMGO with or without scramble siRNA
(targeting no known human mRNA sequence) or RhoA siRNA.
[0053] FIG. 11 is a schematic diagram summarizing the mechanism of
MNTX effects on angiogenesis.
[0054] FIG. 12 is a bar graph of percent proliferation above
control of pulmonary microvascular endothelial cells in the
presence of SIP, VEGF, PDGF, morphine and DAMGO with or without
MNTX.
[0055] FIG. 13 is a bar graph of percent migration above control of
pulmonary microvascular endothelial cells in the presence of SIP,
VEGF, PDGF, morphine and DAMGO with or without MNTX.
[0056] FIG. 14 is a bar graph of percent proliferation above
control of pulmonary microvascular endothelial cells in the
presence of SIP, VEGF, PDGF, morphine and DAMGO with scramble
(control) siRNA or with mu opioid receptor siRNA.
[0057] FIG. 15 is a bar graph of percent migration above control of
pulmonary 25 microvascular endothelial cells in the presence of S
IP, VEGF, PDGF, morphine and DAMGO with scramble (control) siRNA or
with mu opioid receptor siRNA.
[0058] FIG. 16 is a panel of immunoblots indicating phosphorylation
(activation) of the mu opioid receptor using immunoprecipitated mu
opioid receptor and (A, C) anti-phospho-serine, (B, D)
anti-phospho-threonine of human pulmonary microvascular endothelial
cells in the presence of morphine, DAMGO, SIP, VEGF, PDGF with MNTX
(C, D) or without MNTX (A, B); (E) is an immunoblot of anti-mu
opioid receptor.
[0059] FIG. 17 is an anti-RhoA immunoblot of (A, B) activated RhoA
and (C) total RhoA of human pulmonary microvascular endothelial
cells in the presence of morphine, DAMGO, SIP, VEGF, PDGF with MNTX
(B) and without MNTX (A).
[0060] FIG. 18 is a panel of immunoblots of top panel: (A, B)
anti-phospho-tyrosine, (C) anti-VEGF R and bottom panel: (A, B)
anti-phospho-tyrosine, (C) anti-PDGF R, of human pulmonary
microvascular endothelial cells in the presence of morphine, DAMGO,
VEGF (top panel) or PDGF (bottom panel) with MNTX (B in each panel)
or without MNTX (A in each panel).
[0061] FIG. 19 is a panel of immunoblots indicating tyrosine
phosphorylation (activation) of the S1P.sub.3 receptor using
immunoprecipitated S1P.sub.3 receptor and (A, B)
anti-phospho-tyrosine, (C) anti-S1P.sub.3 R, of human pulmonary
microvascular endothelial cells in the presence of morphine, DAMGO,
and S1P with MNTX (B) or without MNTX (A).
[0062] FIG. 20 is a bar graph of percent proliferation above
control of pulmonary microvascular endothelial cells in the
presence of S1P, VEGF, PDGF, morphine and DAMGO with scramble
(control) siRNA or with RhoA siRNA.
[0063] FIG. 21 is a bar graph of percent migration above control of
pulmonary 20 microvascular endothelial cells in the presence of
S1P, VEGF, PDGF, morphine and DAMGO with scramble (control) siRNA
or with RhoA siRNA.
[0064] FIG. 22 is an schematic diagram summarizing the mechanism of
MNTX effects on RhoA activation and angiogenesis.
[0065] FIG. 23 is a graph of percent proliferation above control of
microvascular 25 endothelial cells in the presence of VEGF with
MNTX, with 5-FU and with a combination of MNTX and 5-FU.
[0066] FIG. 24 is a graph of percent migration above control of
microvascular endothelial cells in the presence of VEGF with MNTX,
with Bevacizumab and with a combination of MNTX and
Bevacizumab.
[0067] FIG. 25 is a bar graph of the effects of MNTX, 5-FU, and a
combination of both on SW480 human colorectal cancer cell line.
[0068] FIG. 26 is a bar graph of the effects of MNTX, 5-FU, and a
combination of both on HCT116 human colorectal cancer cell
line.
[0069] FIG. 27 is a bar graph of the effects of MNTX, 5-FU, and a
combination of both on MCF-7 human breast cancer cell line.
[0070] FIG. 28 is a bar graph of the effects of MNTX, 5-FU, and a
combination of both on non-small lung cancer cell (NSLCC) line.
[0071] FIG. 29 is a graph of percent inhibition of NSCLC cells in
the presence of (A) erlotinib, (B) paclitaxel, (C) gemcitabine, (D)
vironrelbine, and (E) docetaxel with morphine and combinations of
morphine and MNTX.
DETAILED DESCRIPTION OF THE INVENTION
[0072] The present invention provides methods of attenuating
abnormal or undesirable migration and/or proliferation of
endothelial cells. As such, the invention provides methods for
attenuating angiogenesis in a tissue or an organ of a subject by
the use of opioid antagonists, and a novel approach for treating
angiogenic related diseases and other hyperproliferative diseases
in mammals.
[0073] For example, as described above, solid tumors rely on the
generation of new blood vessels for nutrients to reach the cells
within the tumor. The growth factors required for angiogenesis can
be produced by the tumor cells or alternatively, exogenous factors,
such as opioids can stimulate new blood vessel growth. The present
invention by the use of opioid antagonists provides a novel
therapeutic approach to the treatment of such tumors, wherein the
generation of new blood vessels within the tumor, rather than the
tumor cells themselves, is the target. This treatment is not likely
to lead to the development of resistant tumor cells.
[0074] Described herein are opioid antagonists inhibit
proliferation and migration induced by opioids, endogenous or
exogenous, and growth factors, such as VEGF, PDGF, S1P etc.
Peripheral opioid antagonists, in particular, showed a substantial
efficacy in inhibiting opioid and growth factor induced
proliferation and migration of endothelial cells. The peripheral
opioid antagonist methylnaltrexone (MNTX) inhibited both opioid and
growth factor induced proliferation and migration in a
concentration dependent manner. In addition, naloxone also
inhibited opioid-induced endothelial migration. It should be noted,
however, that the naloxone inhibition of DAMGO induced migration of
endothelial cells occurred at a relatively high, micromolar,
concentration of naloxone. Furthermore, it has now been discovered
that opioid antagonists, and the peripheral opioid antagonist MNTX
in particular, inhibit agonist induced endothelial cell (EC)
proliferation and migration via inhibition of receptor
phosphorylation and/or transactivation and subsequent inhibition of
Rho A activation. The agonists can be opioids, exogenous and/or
endogenous, angiogenic factors (VEGF), and other proliferation
and/or migration stimulating factors (PDGF, S1P, S1P.sub.3
receptor, RhoA, etc). These results suggest that inhibition of
angiogenesis by opioid antagonists can be a useful therapeutic
intervention for, among other disorders, cancer.
[0075] The present invention also provides methods of attenuating
abnormal or undesirable proliferation of cancer cells per se. This
aspect of the invention is useful in situations involving the
presence or absence of angiogenic activity. The absence of
angiogenic activity is evidenced by one or more of the following
characteristics: nonsolid tumors or tumors where there is repulsion
of existing blood vessels and/or absence of micro vessels within
the tumor, limited growth, for example up to about 1 mm in diameter
in vivo, at which time further expansion is stopped, harmless to
the host until it switches to an angiogenic phenotype, etc.
Nonangiogenic tumors can be completely avascular or they can
contain empty lumen without red blood cells. The gross difference
between the nonangiogenic and angiogenic tumors (i.e. white vs. red
tumors) is most likely due to the reactive hyperemia that
accompanies the onset of blood flow after the angiogenic switch is
completed in a previously hypoxic tumor. Examples of nonangiogenic
tumor lineages include but are not limited to breast
adenocarcinoma, osteosarcoma, glioblastoma, embryonic kidney tumors
etc. There are many factors that could play a role in tumor
dormancy and the rate-determining step for tumor expansion of
nonangiogenic tumors could be governed by lack of angiogenesis
and/or differentiation programs, tumor cell survival, immune
response to the host etc. Although some nonagiogenic tumors never
switch to an angiogenic phenotype, many undergo spontaneous
transformation into an angiogenic and harmful phenotype. Therefore,
treatment of nonangiogenic tumors is of therapeutic
significance.
[0076] Cancers not involving angiogenesis include those that do not
involve the formation of a solid tumor fed by neovasculature.
Certain blood cell cancers can fall into this category, for
example: leukemias, including acute lymphocytic leukemia (ALL),
acute myelogenous leukemia (AML), chronic lymphocytic leukemia
(CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia;
lymphomas (arising in the lymph nodes or lymphocytes) including
Hodgkin lymphoma, Burkitt's lymphoma, cutaneous lymphoma, cutaneous
T-cell lymphoma, follicular lymphoma, lymphoblastic lymphoma, MALT
lymphoma, mantle cell lymphoma, Waldenstrom's macroglobulinemia,
primary central nervous system lymphoma; and some cancers of the
bone marrow elements including Ewing's sarcoma and
osteosarcoma.
[0077] Before any embodiments of the invention are explained in
detail, it is to be understood that the invention is not limited in
its application to the details of the structure and function of the
invention set forth in the following description or illustrated in
the appended FIGS. of the drawing. The invention is capable of
other embodiments and of being practiced or carried out in various
ways. Also, it is to be understood that the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of terms such as
"including," "comprising," or "having" and variations thereof
herein is meant to encompass the item listed thereafter and
equivalents thereof as well as additional items.
[0078] Unless otherwise noted, technical terms are used according
to conventional usage. As used herein, however, the following
definitions may be useful in aiding the skilled practitioner in
understanding the invention:
[0079] "Subject" refers to humans, dogs, cats, and horses.
[0080] "Chronic opioid use" refers to and is characterized by the
need for substantially higher levels of opioid to produce the
therapeutic benefit as a result of prior opioid use, as is well
known in the art. Chronic opioid use as used herein includes daily
opioid treatment for a week or more or intermittent opioid use for
at least two weeks.
[0081] "Alkyl" refers to an aliphatic hydrocarbon group which is
saturated and which may be straight, branched or cyclic having from
1 to about 10 carbon atoms in the chain, and all combinations and
subcombinations of chains therein. Exemplary alkyl groups include
methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, see-butyl,
tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.
[0082] "Lower alkyl" refers to an alkyl group having 1 to about 6
carbon atoms.
[0083] "Alkenyl" refers to an aliphatic hydrocarbon group
containing at least one carbon-carbon double bond and having from 2
to about I 0 carbon atoms in the chain, and all combinations and
sub combinations of chains therein. Exemplary alkenyl groups
include vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,
octenyl, nonenyl and decenyl groups.
[0084] "Alkynyl" refers to an aliphatic hydrocarbon group
containing at least one carbon-carbon triple bond and having from 2
to about 10 carbon atoms in the chain, and combinations and sub
combinations of chains therein. Exemplary alkynyl groups include
ethynyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,
nonenyl and decenyl groups.
[0085] "Alkylene" refers to a bivalent aliphatic hydrocarbon group
having from 1 to about 6 carbon atoms, and all combinations and
subcombinations of chains therein. The alkylene group may be
straight, branched or cyclic. There may be optionally inserted
along the alkylene group one or more ox)"gen, sulfur or optionally
substituted nitrogen atoms, wherein the nitrogen substituent is
alkyl as described previously.
[0086] "Alkenylene" refers to an alkylene group containing at least
one carbon-carbon double bond. Exemplary alkenylene groups include
ethenylene (--CH.dbd.CH--) and propenylene
(CH.dbd.CHCH.sub.2--).
[0087] "Cycloalkyl" refers to any stable monocyclic or bicyclic
ring having from about 3 to about 10 carbons, and all combinations
and subcombinations of rings therein. The cycloalkyl group may be
optionally substituted with one or more cyloalkyl-group
substituents. Exemplary cycloalkyl groups include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl
groups.
[0088] "Cycloalkyl-substituted alkyl" refers to a linear alkyl
group, preferably a lower alkyl group, substituted at a terminal
carbon with a cycloalkyl group, preferably a C3-C8 cycloalkyl
group. Exemplary cycloalkyl-substituted alkyl groups include
cyclohexylmethyl, cyclohexylethyl, cyclopentylethyl,
cyclopentylpropyl, cyclopropylmethyl and the like.
[0089] "Cycloalkenyl" refers to an olefinically unsaturated
cycloaliphatic group having from about 4 to about 10 carbons, and
all combinations and subcombinations of rings therein.
[0090] "Alkoxy" refers to an alkyl-O-group where alkyl is as
previously described. Exemplary alkoxy groups include, for example,
methoxy, ethoxy, propoxy, butoxy and heptoxy.
[0091] "Alkoxy-alkyl" refers to an alkyl-O-alkyl group where alkyl
is as previously described.
[0092] "Acyl" means an alkyl-CO group wherein alkyl is as
previously described. Exemplary acyl groups include acetyl,
propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
[0093] "Aryl" refers to an aromatic carbocyclic radical containing
from about 6 to about 10 carbons, and all combinations and
subcombinations of rings therein. The aryl group may be optionally
substituted with one or two or more aryl group substituents.
Exemplary aryl groups include phenyl and naphthyl.
[0094] "Aryl-substituted alkyl" refers to a linear alkyl group,
preferably a lower alkyl group, substituted at a terminal carbon
with an optionally substituted aryl group, preferably an optionally
substituted phenyl ring. Exemplary aryl-substituted alkyl groups
include, for example, phenylmethyl, phenyl ethyl and
3-(4-methylphenyl)propyl.
[0095] "Heterocyclic" refers to a monocyclic or multicyclic ring
system carbocyclic radical containing from about 4 to about 10
members, and all combinations and subcombinations of rings therein,
wherein one or more of the members of the ring is an element other
than carbon, for example, nitrogen, oxygen or sulfur. The
heterocyclic group may be aromatic or nonaromatic. Exemplary
heterocyclic groups include, for example, pyrrole and piperidine
groups.
[0096] "Halo" refers to fluoro, chloro, bromo or iodo.
[0097] "Peripheral," in reference to opioid antagonists, designates
opioid antagonists that act primarily on physiological systems and
components external to the central nervous system, e.g., they do
not readily cross the blood-brain barrier in an amount effective to
inhibit the central effects of opioids. In other words, peripheral
opioid antagonists do not effectively inhibit the analgesic effects
of opioids when administered peripherally, e.g., they do not reduce
the analgesic effect of the opioids. For example, the peripheral
opioid antagonist compounds employed in the methods of the present
invention exhibit high levels of activity with respect to
gastrointestinal tissue, while exhibiting reduced or substantially
no central nervous system (CNS) activity. The peripheral opioid
antagonist compounds employed in the present methods suitably
exhibit less than about 15% of their pharmacological activity in
the CNS, with about 0% (e.g., no CNS activity) being most suitable.
The non-central acting characteristic of a peripheral opioid
antagonist is often related to charge, polarity and/or size of the
molecule. For example, peripherally-acting quaternary amine opioid
antagonists are positively charged while the central-acting
tertiary amine opioid antagonists are neutral molecules. The
peripheral opioid antagonists useful in the present invention are
typically mu and/or kappa opioid antagonists.
[0098] As used herein, "antiangiogenesis" or "antiangiogenic" is
meant to refer to the capability of a molecule/compound to
attenuate, e.g., inhibit, reduce or modulate, proliferation of new
blood vessels, in general, and for example, to reduce or inhibit
migration and proliferation of human microvascular endothelial
cells in culture in the presence of certain growth factors. As
described above, the formation of new blood vessels by endothelial
cells involves migration, proliferation and differentiation of the
cells.
[0099] In the following description of the methods of the
invention, process steps are carried out at room temperature and
atmospheric pressure unless otherwise specified. It also is
specifically understood that any numerical range recited herein
includes all values from the lower value to the upper value, e.g.,
all possible combinations of numerical values between the lowest
value and the highest value enumerated are to be considered to be
expressly stated in this application. For example, if a
concentration range or beneficial effect range is stated as 1% to
50%, it is intended that values such as 2% to 40%, 10% to 30%, or
1% to 3%, etc., are expressly enumerated in this specification.
These are only examples of what is specifically intended.
[0100] In one aspect, the present invention relates to methods of
attenuating abnormal or undesirable cellular, particularly
endothelial cell migration and/or proliferation, and angiogenesis
in tissue or an organ of a subject. The methods comprise providing
or administering one or more opioid antagonists in an effective
amount to endothelial cells of the tissue or organ of a patient to
inhibit endothelial cell migration and proliferation, and
angiogenesis. The angiogenesis may, in part, be the result of
receiving opioid treatment, particularly for pain management in
cancer patients, or having high levels of endogenous opioids.
[0101] It was observed that morphine and the mu agonist enkephalin
DAMGO ([D-Ala.sup.2, N-McPhe.sup.4 Gly.sup.5-ol] enkephalin), each
cause a dose-dependent increase in migration of endothelial cells
similar to that of vascular endothelial growth factor (VEGF) as
measured by, e.g., a chemotaxis assay (as detailed in the examples
below) or other similar assays used to identify factors in tumor
angiogenesis and the drugs that affect it. At clinically relevant
concentrations of morphine, the magnitude of the effect is
approximately 70% of that which is achieved by VEGF. This
morphine-based endothelial cell migration is attenuated by the mu
opioid antagonist methylnaltrexone (MNTX) in a dose-dependent
fashion. For example, endothelial cell migration induced by
morphine, in concentrations as low as 10.sup.-7M, is significantly
blocked by 10.sup.-7M MNTX (FIG. 2). This attenuation strongly
suggesting that endothelial cell migration is mediated by morphine
action on the mu opioid receptor (MOR). As described in the
examples below, the effect via the MOR rather than other opioid
receptors is confirmed by experiments that show the highly
selective synthetic enkephalin mu agonist DAMGO also induces
migration. The migratory effect induced by DAMGO is also blocked by
MNTX (FIG. 3).
[0102] In one comprehensive review (Neumann et al. Pain 1982;
13:247-52), analgesia in cancer patients was associated with a
range of steady state concentrations of morphine and plasma ranging
from 6 to 364 ng/mL. It was observed an effect of morphine that
causes endothelial cell migration at 100 ng/mL well within the
clinical dose range. It therefore is believed by the inventors
herein that a dose of MNTX which will maintain plasma levels of
MNTX at minimum levels of plasma MNTX between about 25 and 150
ng/mL would be suitable. Such doses are attainable and are well
tolerated (Yuan et al., J Clin Pharmacol 2005; 45:538-46).
[0103] Alvimopan, another selective peripheral opioid antagonist
given orally, is in late stage development for prophylaxis of
postoperative ileus and the management of opioid induced
constipation (Moss et al., Pain relief without side effects:
peripheral opioid antagonists. In Schwartz, A J., editor. 33rd ASA
Refresher Course in Anesthesiology. Philadelphia: Lippincott
Williams & Wilkins (in press).) There is some transpassage of
alvimopan across the membrane (J. Foss, et al., Clin. Pharm. &
Ther. 2005, PII-90, p. 74) and it may, therefore, possess the
ability to reverse some of the systemic effects of opioids without
affecting analgesia even when given orally.
[0104] Without being bound by any particular theory, it may be that
the mechanism of mu opioid effect on endothelial cell migration
occurs at the membrane level as MNTX, unlike naloxone, is a charged
molecule at physiological pH. Morphine acts via G-protein coupled
receptors, while VEGF acts by receptor tyrosine kinases. While the
actions of mu agonists and VEGF may be independent, there is
growing evidence of receptor transactivation as a mechanism. A
prior investigation demonstrated that pertussis toxin dependent
GPCRs transactivate VEGF receptor-2/F1 K1 (Zeng, H. et al., J.
Biol. Chem. 2003; 278:20738-45). By this manner morphine could
transactivate F11 c-1 and promote an environment where endothelial
cell proliferation and tumor growth could occur. A recent study of
MOR knockout mice infected with T241 fibrosarcoma cells
demonstrated significant differences in the incidences of tumor
growth and a 10-fold increase in F11 c-1 expression in morphine
treated mice versus controls, versus no increase in morphine
treated KO mice (K. Gupta, personal communication). This provides
further evidence that morphine stimulates endothelial cell
proliferation and promotes tumor growth probably by trans
activating FLK1 phosphorylation. As such, the present invention
provides potential clinical strategies using MNTX as well as other
peripheral opioid antagonist in conjunction with current therapies
targeting VEGF. Although a direct effect by receptor
transactivation is possible, a potential additional factor involved
in the proliferation of tumors may well be the role of chemokines
as integrators of pain and inflammation. A recent review on this
subject (White et al., Nature Rev. Drug Discovery 2005; 4:834-44)
also suggests a possible role for leukocytes in activating opioid
receptors.
[0105] Furthermore, it was observed that morphine, DAMGO and VEGF
stimulate RhoA activation which is inhibited by opioid antagonists,
such as MNTX. RhoA is an important signaling molecule involved in
angiogenesis (Aepfelbacher et al., 1997; Cascone et al., 2003;
Hoang et al., 2004; Liu and Sanger, 2004.) VEGF receptor
transactivation is important for opiate-induced RhoA activation.
Silencing RhoA expression blocked opioid and VEGF induced EC
proliferation and migration, demonstrating a role for RhoA
activation in agonist-induced EC angiogenic activity. The MNTX
mediated attenuation of RhoA activation may be important for the
inhibitory role of MNTX on opioid and VEGF induced
angiogenesis.
[0106] Because morphine and other opioids at clinical doses enhance
endothelial cell migration, the present invention may be of
therapeutic value in opioid antagonist treatment for patients on
significant and sustained doses of opioids that have tumors relying
on the angiogenic process. Further, while the inventor's clinical
observations have focused on morphine, which is exogenously
administered, endogenous opioids, which are released by stress or
pain, may also play a role in endothelial cell migration. Based on
endothelial cell migration experiments detailed below in the
examples, MNTX and opioid antagonists generally are of therapeutic
value as an antiangiogenic therapy even absent exogenous opioid
administration (as detailed herein). It is envisioned that the
methods of the present invention will inhibit or reduce the growth
of blood vessels within and to a tumor. Inhibiting the growth of
blood vessels within tumors prevents nutrients and oxygen from
being supplied to the tumor to support growth beyond a certain
size. Minimizing the number of blood vessels or other tumors also
lessens the probability that the tumor will metastasize.
[0107] Multidrug resistance, an important mechanism by which many
cancers develop resistance to chemotherapeutic drugs, is a major
contributing factor to the failure of cancer drug therapy. It
affects patients with a variety of cancers including lung, breast,
ovarian and lower gastrointestinal tract cancers. Resistance to
therapy is regulated by at least two classes of molecular pumps in
tumor-cell plasma membranes that actively expel chemotherapeutic
drugs from inside the cell. This allows tumor cells to avoid the
toxic effects of the drugs and promotes multidrug resistance. The
two pumps commonly found in cancer are P-glycoprotein and the
multidrug resistance-associated proteins (MRP) that belong to the
ATP-binding cassette (ABC) transporters. A major MRP expressed in
NSCLC is ABCC3. Morphine may promote drug resistance to a variety
of chemotherapeutic agents with different mechanisms of action
including tyrosine kinase inhibitor, microtubule modifying agents
and DNA synthesis inhibitor. Among other things, the present
invention may be useful to reverse the multidrug resistance effects
of morphine.
[0108] The present invention may be of therapeutic value in opioid
antagonist treatment for patients who have tumors relying on the
angiogenic process. Tumors that rely on angiogenic processes are
solid tumors, leukemias and myelomas. Solid tumors include, but are
not limited to adrenal cortical carcinoma, tumors of the bladder:
squamous cell carcinoma, urothelial carcinomas; tumors of the bone:
adamantinoma, aneurysmal bone cysts, chondroblastoma, chondroma,
chondromyxoid fibroma, chondrosarcoma, fibrous dysplasia of the
bone, giant cell tumour, osteochondroma, osteosarcoma; breast
tumors: secretory ductal carcinoma, chordoma; colon tumors:
colorectal adenocarcinoma; eye tumors: posterior uveal melanoma,
fibrogenesis imperfecta ossium, head and neck squamous cell
carcinoma; kidney tumors: chromophobe renal cell carcinoma, clear
cell renal cell carcinoma, nephroblastoma (Wilms tumor), kidney:
papillary renal cell carcinoma, primary renal ASPS CR 1-TFE3 tumor,
renal cell carcinoma; liver tumors: hepatoblastoma, hepatocellular
carcinoma; lung tumors: non-small cell carcinoma, small cell
cancer; malignant melanoma of soft parts; nervous system tumors:
medulloblastoma, meningioma, neuroblastoma, astrocytic tumors,
ependymomas, peripheral nerve sheath tumors, phaeochromocytoma;
ovarian tumors: epithelial tumors, germ cell tumors, sex
cord-stromal tumors, pericytoma; pituitary adenomas; rhabdoid
tumor; skin tumors: cutaneous benign fibrous histiocytomas; smooth
muscle tumors: intravenous leiomyomatosis; soft tissue tumors:
liposarcoma, myxoid liposarcoma, low grade fibromyxoid sarcoma,
leiomyosarcoma, alveolar soft part sarcoma, angiomatoid fibrous
histiocytoma (AFH), clear cell sarcoma, desmoplastic small round
cell tumor, elastofibroma, Ewing's tumors, extraskeletal myxoid
chondrosarcoma, inflammatory myofibroblastic tumor, lipoblastoma,
lipoma/benign lipomatous tumors, liposarcoma/malignant lipomatous
tumors, malignant myoepithelioma, rhabdomyosarcoma, synovial
sarcoma, squamous cell cancer; tumors of the testis: germ cell
tumors, spermatocytic seminoma; thyroid tumors: anaplastic
(undifferentiated) carcinoma, oncocytic tumors, papillary
carcinoma; uterus tumors: carcinoma of the cervix, endometrial
carcinoma, leiomyoma etc.
[0109] In one embodiment of the invention the tumors are prostate
cancer, gastrointestinal tumors such as colon or pancreatic cancer
and the compounds of the invention are co-administered with other
anticancer agents as described herein.
[0110] The opioid antagonists in accordance with the present
invention include both centrally and peripherally acting opioid
antagonists. It is contemplated that those antagonists of
particular value are suitably the peripheral opioid antagonists.
Especially suitable may be a mu opioid antagonist, especially a mu
peripheral opioid antagonist. Opioid antagonists form a class of
compounds that can vary in structure while maintaining the
peripheral restrictive property. These compounds include tertiary
and quaternary morphinans, in particular noroxymorphone
derivatives, N-substituted piperidines, and in particular,
piperidine-N-alkylcarboxylates, and tertiary and quaternary
benzomorphans. Peripherally restricted antagonists, while varied in
structure, are typically charged, polar and/or of high molecular
weight, each of which impedes their crossing the blood-brain
barrier.
[0111] Examples of opioid antagonists, which cross the blood-brain
barrier and are centrally (and peripherally) active, include, e.g.,
naloxone, naltrexone (each of which is commercially available from
Baxter Pharmaceutical Products, Inc.) and nalmefene (available,
e.g., from DuPont Pharma). These may be of value in attenuating
angiogenesis in the central nervous system or in patients not being
treated for pain management or other opioid treatment.
[0112] A peripheral opioid antagonist useful for the present
invention may be a compound which is a quaternary morphinan
derivative, and in particular, a quaternary noroxymorphone of
formula (I):
##STR00001##
wherein R is alkyl, alkenyl, alkynyl, aryl, cycloalkyl-substituted
alkyl or aryl-substituted alkyl, and X' is the anion, especially a
chloride, bromide, iodide or methylsulfate anion. The
noroxymorphone derivatives of formula (I) can be prepared, for
example, according to the procedure in U.S. Pat. No. 4,176,186,
which is incorporated herein by reference; see also, U.S. Pat. Nos.
4,719,215; 4,861,781; 5,102,887; 5,972,954 and 6,274,591, U.S.
Patent Application Nos. 2002/0028825 and 2003/0022909; and PCT
publication Nos. WO 99/22737 and WO 98/25613, all of which are
hereby incorporated by reference.
[0113] A compound of formula (I) of particular value is
N-methylnaltrexone (or simply methylnaltrexone), wherein R is
cyclopropylmethyl as represented in formula (II):
##STR00002##
wherein X-- is as described above. Methylnaltrexone is a quaternary
derivative of the opioid antagonist naltrexone. Methylnaltrexone
exists as a salt, and "methyl naltrexone" or "MNTX", as used
herein, therefore embraces salts. "Methylnaltrexone" or "MNTX"
specifically includes, but is not limited to, bromide salts,
chloride salts, iodide salts, carbonate salts, and sulfate salts of
methylnaltrexone. Names used for the bromide salt of MNTX in the
literature include: methylnaltrexone bromide; N-methylnaltrexone
bromide; naltrexone methobromide; naltrexone methyl bromide;
SC-37359, MRZ-2663-BR, and
N-cyclopropylmethylnoroxy-morphine-methobromide. Methylnaltrexone
is commercially available from, e.g., Mallinclaodt Pharmaceuticals,
St. Louis, Mo. Methylnaltrexone is provided as a white crystalline
powder, freely soluble in water, typically as the bromide salt. The
compound as provided is 99.4% pure by reverse phase HPLC, and
contains less than 0.011% unquaternized naltrexone by the same
method. Methylnaltrexone can be prepared as a sterile solution at a
concentration of, e.g., about 5 mg/mL.
[0114] Other suitable peripheral opioid antagonists may include
N-substituted piperidines, and in particular,
piperidine-N-alkylcarboxylates as represented by formula (III):
##STR00003##
wherein R.sup.1 is hydrogen or alkyl; R.sup.2 is hydrogen, alkyl,
or alkenyl; R.sup.3 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl,
cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl or aryl-substituted alkyl; R.sup.4
is hydrogen, alkyl, or alkenyl; A is OR.sup.5 or NR.sup.6R.sup.7;
wherein R.sup.5 is hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl or aryl-substituted alkyl; R.sup.6
is hydrogen or alkyl; R.sup.7 is hydrogen, alkyl, alkenyl, aryl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl or aryl-substituted alkyl, or
alkylene-substituted B or together with the nitrogen atom to which
they are attached, R.sup.6 and R.sup.7 form a heterocyclic ring
selected from pyrrole and piperidine; B is
##STR00004##
wherein R.sup.8 is hydrogen or alkyl; R.sup.9 is hydrogen, alkyl,
alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted
alkyl, cycloalkenyl-substituted alkyl or aryl-substituted alkyl or
together with the nitrogen atom to which they are attached, R.sup.8
and R.sup.9 form a heterocyclic ring selected from pyrrole and
piperidine; W is OR.sup.10, NR.sup.11R.sup.12, or OE; wherein
R.sup.10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl,
cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkenyl, or
aryl-substituted alkyl; R.sup.11 is hydrogen or alkyl; R.sup.12 is
hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl,
cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl,
aryl-substituted alkyl or alkylene-substituted C(.dbd.O)Y or,
together with the nitrogen atom to which they are attached,
R.sup.11 and R.sup.l2 form a heterocyclic ring selected from
pyrrole and piperidine;
E is
##STR00005##
[0115] alkylene-substituted (C.dbd.O)D, or
--R.sup.l30C(.dbd.O)R.sup.14; wherein R.sup.l3 is alkyl-substituted
alkylene; R.sup.l4 is alkyl; D is OR.sup.l5 or NR.sup.16R.sup.17;
wherein R.sup.l5 is hydrogen, alkyl, alkenyl, cycloalkyl,
cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl
substituted alkyl, or aryl-substituted alkyl; R.sup.l6 is hydrogen,
alkyl, alkenyl, aryl, aryl-substituted alkyl, cycloalkyl,
cycloalkenyl, cycloalkyl substituted alkyl or
cycloalkenyl-substituted alkyl; R.sup.17 is hydrogen or alkyl or,
together with the nitrogen atom to which they are attached,
R.sup.16 and R.sup.17 form a heterocyclic ring selected from the
group consisting of pyrrole or piperidine; Y is OR.sup.l8 or
NR.sup.l9R.sup.20; wherein R.sup.18 is hydrogen, alkyl, alkenyl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl; R.sup.l9
is hydrogen or alkyl; R.sup.20 is hydrogen, alkyl, alkenyl, aryl,
cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl,
cycloalkenyl-substituted alkyl, or aryl-substituted alkyl or,
together with the nitrogen atom to which they are attached,
R.sup.19 and R.sup.20 form a heterocyclic ring selected from
pyrrole and piperidine; R.sup.21 is hydrogen or alkyl; and n is 0
to 4.
[0116] Particular piperidine-N-alkylcarbonylates which may be of
value are N-alkylamino-3, 4, 4 substituted piperidines, such as
alvimopan represented below as formula (IV):
##STR00006##
[0117] Suitable N-substituted piperidines may be prepared as
disclosed in U.S. Pat. Nos. 5,270,328; 6,451,806; 6,469,030, all of
which are hereby incorporated by reference. Alvimopan is available
from Adolor Corp., Exton, Pa. Such compounds have moderately high
molecular weights, a zwitterion form and a polarity which prevent
penetration of the blood-brain barrier.
[0118] Still other suitable peripheral opioid antagonist compounds
may include quaternary benzomorphan compounds. The quaternary
benzomorphan compounds employed in the methods of the present
invention exhibit high levels of morphine antagonism, while
exhibiting reduced, and preferably substantially no, agonist
activity.
[0119] The quaternary benzomorphan compounds which may be employed
in the methods of the present invention have the following formula
(V):
##STR00007##
wherein; R.sup.1 is hydrogen, acyl or acetoxy; and R.sup.2 is alkyl
or a1kenyl; R is alkyl, alkenyl or alkynyl and X.sup.- is an anion,
especially a chloride, bromide, iodide or methylsulfate anion.
[0120] Specific quaternary derivatives of benzomorphan compounds
that may be employed in the methods of the present invention
include the following compounds of formula (V): 2'-hydroxy
5,9-dimethyl-2, 2-diallyl-6,7-benzomorphanium-bromide;
2'-hydroxy-5,9-dimethyl-2-n-propyl-2 ally 1-6,
7-benzomorphanium-bromide; 2'-hydroxy-5,9-dimethy
1-2-n-propyl-2-propargyl-6, 7 benzomorphanium-bromide; and
2'-acetoxy-5,9-dimethyl-2-n-propyl-2-ally)-6,7
benzomorphanium-bromide.
[0121] Other quaternary benzomorphan compounds that may be employed
in the methods of the present invention are described, for example,
in U.S. Pat. No. 3,723,440, the entire disclosure of which is
incorporated herein by reference. The compounds employed in the
methods of the present invention may exist in prodrug form. As used
herein, "prodrug" is intended to include any covalently bonded
carriers which release the active parent drug according to formulas
(I) to (V) or other formulas or compounds employed in the methods
of the present invention in vivo when such prodrug is administered
to a mammalian subject. Since prodrugs are known to enhance
numerous desirable qualities of pharmaceuticals (e.g., solubility,
bioavailability, manufacturing, etc.), the compounds employed in
the present methods may, if desired, be delivered in prodrug form.
Thus, the present invention contemplates methods of delivering
prodrugs. Prodrugs of the compounds employed in the present
invention may be prepared by modifying functional groups present in
the compound in such a way that the modifications are cleaved,
either in routine manipulation or in vivo, to the parent
compound.
[0122] Accordingly, prodrugs include, for example, compounds
described herein in which a hydroxy, amino, or carboxy group is
bonded to any group that, when the prodrug is administered to a
mammalian subject, cleaves to form a free hydroxyl, free amino, or
carboxylic acid, respectively.
[0123] Examples include, but are not limited to, acetate, formate
and benzoate derivatives of alcohol and amine functional groups;
and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl,
ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl,
cyclopropyl, phenyl, benzyl, and phenethyl esters, and the
like.
[0124] As noted, the compounds employed in the methods of the
present invention may be prepared in a number of ways well known to
those skilled in the art. All preparations disclosed in association
with the present invention are contemplated to be practiced on any
scale, including milligram, gram, multigram, kilogram,
multikilogram or commercial pharmaceutical scale.
[0125] Compounds employed in the present methods may contain one or
more asymmetrically substituted carbon atom, and may be isolated in
optically active or racemic form. Thus, all chiral, diastereomeric,
racemic form, epimeric form and all geometric isomeric form of a
structure are intended, unless the specific stereochemistry or
isomeric form is specifically indicated. It is well known in the
art how to prepare and isolate such optically active form. For
example, mixtures of stereo isomers may be separated by standard
techniques including, but not limited to, resolution of racemic
form, normal, reverse-phase, and chiral chromatography,
preferential salt formation, recrystallization, and the like, or by
chiral synthesis either from chiral starting materials or by
deliberate synthesis of target chiral centers.
[0126] In some embodiments of the invention, the opioid antagonist
may be a mu opioid antagonist. In other embodiments, the opioid
antagonist may be a kappa opioid antagonist. The invention also
encompasses administration of more than one opioid antagonist,
including combinations of mu antagonists, combinations of kappa
antagonists and combinations of mu and kappa antagonists, for
example, a combination of methylnaltrexone and alvimopan.
[0127] The methods of the present invention encompass providing a
therapeutic or prophylactic role in other endothelial-based
diseases, e.g., in a variety of angiogenesis and/or
proliferation-related neoplastic and non-neoplastic diseases, e.g.,
sickle cell disease, neovascular disease of the eye (such as
diabetic retinopathy, neovascular glaucoma, retinopathy of
prematurity, age-related macular degeneration), endothelial
proliferation in the kidneys or lung and psoriasis. Non-neoplastic
conditions that are amenable to treatment include rheumatoid
arthritis, psoriasis, atherosclerosis, diabetic and other
proliferative retinopathies including retinopathy of prematurity,
retrolental fibroplasia, neovascular glaucoma, age-related macular
degeneration, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, chronic inflammation,
lung inflammation, nephrotic syndrome, preeclampsia, ascites,
pericardial effusion (such as that associated with pericarditis),
and pleural effusion. For example, it has been shown that morphine
induced proliferative retinopathy in sickle cell disease (Gupta et
al., personal communication). It is anticipated that treatment with
an opioid antagonist may significantly inhibit the retinopathy,
particularly with opioid-induced retinopathy in sickle cell
patients that are in active opioid therapy, and receive opioids for
long periods of time, including chronic therapy for weeks, months
or even years.
[0128] The methods of the present invention are also envisioned to
be of value in reducing the risk of recurrence of a malignancy or
neoplasm after treatment with other therapeutic modalities, e.g.,
after surgical intervention. For example, the present invention
provides a method for reducing the risk of recurrence of
postoperative cancer. The cancers may include, for example, breast
cancer or prostate cancer, and reduced risk may be achieved by
providing to the patient suffering from such cancer an effective
amount of an opioid antagonist, particularly a peripheral opioid
antagonist. For example, as described above, patients undergoing
breast cancer surgery had a significant difference (fourfold) in
the incidence of recurrence at 2-4 years depending on whether the
patients received regional or general anesthesia (with morphine
during their initial surgery. Co-administration of the opioid
antagonists, especially peripheral antagonist, in accordance with
the present invention with surgical treatment may be of value to
reduce the incidence of recurrence of the cancer.
[0129] It is also contemplated that the invention provides a method
of inhibiting the activity of VEGF by providing to the affected
cells or subject an effective amount of an opioid antagonist under
conditions sufficient to inhibit VEGF-induced angiogenesis. In
other words, the compounds of the present invention have
VEGF-inhibitory or antagonist activity.
[0130] As also shown in the examples below, it has further been
found that a peripheral opioid antagonist, MNTX, attenuates not
only VEGF-induced endothelial cell migration, but also induction of
endothelial migration and/or proliferation by other
pro-migration/pro-proliferative factors such as platelet derived
growth factor (PDGF), or sphingosine 1-phosphate (S1P). Such
attenuation ranges from about 10% to 60%, and provides further
evidence that the methods of the present invention have value in
inhibiting pro-migration, pro-angiogenic factors.
[0131] The methods of the invention also encompass treating
patients, e.g., cancer patients, who are undergoing treatment with
opioid agonists. Opioid agonists include, but are not limited to,
morphine, methadone, codeine, meperidine, fentidine, fentanil,
sufentanil, alfentanil and the like. As described above, opioid
agonists are classified by their ability to agonize one type of
receptor an order of magnitude more effectively than another. For
example, the relative affinity of morphine for the mu receptor is
200 times greater than for the kappa receptor, and is therefore
classified as a mu opioid agonist. Some opioid agonists may act as
agonists towards one receptor and antagonists toward another
receptor and are classified as agonist/antagonists, (also known as
mixed or partial agonists). "Agonist/antagonist," "partial
agonist," and "mixed agonist" are used interchangeably herein.
These opioids include, but are not limited to, pentazocine,
butorphanol, nalorphine, nalbufine, buprenorphine, bremazocine, and
bezocine. Many of the agonist/antagonist group of opioids are
agonists at the kappa receptors and antagonists at the mu
receptors. Further, it is envisioned the active metabolites of
opioid agonists may also be active as angiogenesis inducers. For
example, the metabolites of morphine, morphine 3-glucuronide (M3G)
and morphine 6-glucuronide (M6G) may be active proangiogenic
factors.
[0132] Generally, the peripheral opioid antagonists in accordance
with the present invention may be administered in an effective
amount such that the patient's plasma level of the peripheral
opioid antagonist is in the range from 10.sup.-6 M to 10.sup.-9M.
Patient drug plasma levels may be measured using routine HPLC
methods known to those of skill in the art.
[0133] As described in the examples below, the enkephalin analog
DAMGO induces endothelial migration. Thus, the methods of the
present invention may be of value to patient suffering from
angiogenic-related or hyperproliferative diseases, e.g., cancer,
quite apart from treatment with opioid agonists.
[0134] The particular mode of administration of the opioid
antagonist selected will depend, of course, upon the particular
combination of drugs selected, the severity of the tumor
progression being treated, in the cancer patient, the general
health condition of the patient, and the dosage required for
therapeutic efficacy. The methods of this invention, generally
speaking, may be practiced using any mode of administration that is
medically acceptable, e.g., any mode that produces effective levels
of the active compounds without causing clinically unacceptable
adverse effects. Such modes of administration include oral, rectal,
topical (as by powder, ointment, drops, transdermal patch or
iontophoretic devise), trans dermal, sublingual, intramuscular,
infusion, intravenous, pulmonary, intramuscular, intracavity, as an
aerosol, aural (e.g., via eardrops), intranasal, inhalation,
intraocular or subcutaneous. Direct injection could also be used
for local delivery. Oral or subcutaneous administration may be
suitable for prophylactic or long term treatment because of the
convenience of the patient as well as the dosing schedule. For
ocular diseases, ophthalmic formulations may be injected or
instilled directly.
[0135] Additionally, the opioid antagonists may be administered as
an enterically coated tablet or capsule. In some embodiments, the
opioid antagonist is administered by a slow infusion method or by a
time-release or controlled-release method or as a lyophilized
powder.
[0136] When administered, the compounds of the invention are given
in pharmaceutically acceptable amounts and in pharmaceutically
acceptable compositions or preparations. Such preparations may
routinely contain salts, buffering agents, preservatives, and
optionally other therapeutic ingredients. When used in medicine,
the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic,
formic, succinic, naphthalene-2-sulfonic, pamoic,
3-hydroxy-2-naphthalenecarboxylic, and benzene sulfonic. Suitable
buffering agents include, but are not limited to, acetic acid and
salts thereof (1-2% WN); citric acid and salts thereof (1-3% WN);
boric acid and salts thereof (0.5-2.5% WN); and phosphoric acid and
salts thereof (0.8-2% WN).
[0137] Suitable preservatives include, but are not limited to,
benzalkonium chloride (0.003-0.03% WN); chlorobutanol (0.3-0.9%
WIN); parabens (0.01-0.25% WN) and thimerosal (0.004-0.02% WN).
[0138] For ease of administration, a pharmaceutical composition of
the peripheral opioid antagonist may also contain one or more
pharmaceutically acceptable excipients, such as lubricants,
diluents, binders, carriers, and disintegrants. Other auxiliary
agents may include, e.g., stabilizers, wetting agents, emulsifiers,
salts for influencing osmotic pressure, coloring, flavoring and/or
aromatic active compounds.
[0139] A pharmaceutically acceptable carrier or excipient refers to
a non-toxic solid, semi-solid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. For
example, suitable pharmaceutically acceptable carriers, diluents,
solvents or vehicles include, but are not limited to, water, salt
(buffer) solutions, alcohols, gum arabic, mineral and vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, magnesium stearate, talc,
silicic acid, viscous paraffin, vegetable oils, fatty acid
monoglycerides and diglycerides, pentaerythritol fatty acid esters,
hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Proper
fluidity may be maintained, for example, by the use of coating
materials such as lecithin, by the maintenance of the required
particle size in the case of dispersions and by the use of
surfactants. Prevention of the action of microorganisms may be
ensured by the inclusion of various antibacterial and antifungal
agents such as paraben, chlorobutanol, phenol, sorbic acid and the
like.
[0140] If a pharmaceutically acceptable solid carrier is used, the
dosage form of the analogs may be tablets, capsules, powders,
suppositories, or lozenges. If a liquid carrier is used, soft
gelatin capsules, transdermal patches, aerosol sprays, topical
cream, syrups or liquid suspensions, emulsions or solutions may be
the dosage form.
[0141] For parental application, particularly suitable are
injectable, sterile solutions, preferably nonaqueous or aqueous
solutions, as well as dispersions, suspensions, emulsions, or
implants, including suppositories. Ampoules are often convenient
unit dosages. Injectable depot form may also be suitable and may be
made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide,
poly(orthoesters) and poly(anhydrides). Depending upon the ratio of
drug to polymer and the nature of the particular polymer employed,
the rate of drug release can be controlled.
[0142] Depot injectable formulations are also prepared by
entrapping the drug in liposomes or microemulsions which are
compatible with body tissues. The injectable formulations may be
sterilized, for example, by filtration through a
bacterial-retaining filter or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable media just
prior to use.
[0143] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules such as soft
gelatin capsules. A syrup, elixir, or the like can be used wherein
a sweetened vehicle is employed.
[0144] As noted, other delivery system may include time-release,
delayed-release or sustained-release delivery system. Such system
can avoid repeated administrations of the compounds of the
invention, increasing convenience to the patient and the physician
and maintain sustained plasma levels of compounds. Many types of
controlled-release delivery system are available and known to those
of ordinary skill in the art. Sustained- or controlled-release
compositions can be formulated, e.g., as liposomes or those wherein
the active compound is protected with differentially degradable
coatings, such as by microencapsulation, multiple coatings,
etc.
[0145] For example, compounds of this invention may be combined
with pharmaceutically acceptable sustained-release matrices, such
as biodegradable polymers, to form therapeutic compositions. A
sustained-release matrix, as used herein, is a matrix made of
materials, usually polymers, which are degradable by enzymatic or
acid-base hydrolysis or by dissolution. Once inserted into the
body, the matrix is acted upon by enzymes and body fluids. A
sustained-release matrix may be desirably chosen from biocompatible
materials such as liposomes, polymer-based system such as
polylactides (polylactic acid), polyglycolide (polymer of glycolic
acid), polylactide co-glycolide (copolymers of lactic acid and
glycolic acid), polyanhydrides, poly (ortho)esters,
polysaccharides, polyamino acids, hyaluronic acid, collagen,
chondroitin sulfate, polynucleotides, polyvinyl propylene,
polyvinyl pyrrolidone, and silicone; nonpolymer system such as
carboxylic acids, fatty acids, phospholipids, amino acids, lipids
such as sterols, hydrogel release system; silastic system;
peptide-based system; implants and the like. Specific examples
include, but are not limited to: (a) erosional system in which the
polysaccharide is contained in a form within a matrix, found in
U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 (herein
incorporated by reference in their entireties), and (b) diffusional
system in which an active component permeates at a controlled rate
from a polymer such as described in U.S. Pat. Nos. 3,854,480,
5,133,974 and 5,407,686 (herein incorporated by reference in their
entireties). In addition, pump-based hard-wired delivery system can
be used, some of which are adapted for implantation. Suitable
enteric coatings are described in PCT publication No. WO 98/25613
and U.S. Pat. No. 6,274,591, both incorporated herein by
reference.
[0146] Use of a long-term sustained-release implant may be
particularly suitable for treatment of chronic conditions.
"Long-term" release, as used herein, means that the implant is
constructed and arranged to deliver therapeutic levels of the
active ingredient for at least 7 days, and suitably 30 to 60 days.
Long-term sustained-release implants are well-known to those of
ordinary skill in the art and include some of the release system
described above.
[0147] For topical application, there are employed as nonsprayable
form, viscous to semi-solid or solid form comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include, but
are not limited to, solutions, suspensions, emulsions, cream,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, etc.
[0148] Transdermal or iontophoretic delivery of pharmaceutical
compositions of the peripheral opioid antagonists is also
possible.
[0149] Respecting MNTX specifically, aqueous formulations may
include chelating agent, a buffering agent, an anti-oxidant and,
optionally, an isotonicity agent, preferably pH adjusted to between
3.0 and 3.5. Preferred such formulations that are stable to
autoclaving and long term storage are described in application Ser.
No. 10/821,811, now published as US 2004/0266806, entitled
"Pharmaceutical Formulation," the disclosure of which is
incorporated herein by reference.
[0150] In one embodiment, compounds of the invention are
administered in a dosing regimen which provides a continuous dosing
regimen of the compound to a subject, e.g., a regimen that
maintains minimum plasma levels of the opioid antagonist, and
preferably eliminates the spikes and troughs of a drug level with
conventional regimens. Suitably, a continuous dose may be achieved
by administering the compound to a subject on a daily basis using
any of the delivery methods disclosed herein. In one embodiment,
the continuous dose may be achieved using continuous infusion to
the subject, or via a mechanism that facilitates the release of the
compound over time, for example, a transdermal patch, or a
sustained release formulation. Suitably, compounds of the invention
are continuously released to the subject in amounts sufficient to
maintain a concentration of the compound in the plasma of the
subject effective to inhibit or reduce opioid induced angiogenesis;
or in cancer patients, to attenuate growth of a tumor. Compounds in
accordance with the present invention, whether provided alone or in
combination with other therapeutic agents, are provided in an
antiangiogenic effective amount. It will be understood, however,
that the total daily usage of the compounds and compositions of the
present invention will be decided by the attending physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular patient will depend upon a
variety of factors including the disorder being treated and the
severity of the disorder; activity of the specific compound
employed; the specific composition employed; the age, body weight,
general health, sex and diet of the patient; the time of
administration; the route of administration; the rate of excretion
of the specific compound employed; the duration of the treatment;
drugs used in combination or coincidental with the specific
compound employed and like factors well known in the medical arts.
For example, it is well within the level of ordinary skill in the
art to start doses of the compound at levels lower than those
required to achieve the desired therapeutic effect and to gradually
increase the dosage until the desired effect is achieved.
[0151] If desired, the effective daily dose may be divided into
multiple doses for purposes of administration. Consequently, single
dose compositions may contain such amounts or submultiples thereof
to make up the daily dose. As noted, those of ordinary skill in the
art will readily optimize effective doses and co-administration
regimens (as described herein) as determined by good medical
practice and the clinical condition of the individual patient.
[0152] Generally, oral doses of the opioid antagonists,
particularly peripheral antagonists, will range from about 0.01 to
about 80 mg/kg body weight per day. It is expected that oral doses
in the range from 1 to 20 mg/kg body weight will yield the desired
results. Generally, parenteral administration, including
intravenous and subcutaneous administration, will range from about
0.001 to 5 mg/kg body weight. It is expected that doses ranging
from 0.05 to 0.5 mg/kg body weight will yield the desired results.
Dosage may be adjusted appropriately to achieve desired drug
levels, local or systemic, depending on the mode of administration.
For example, it is expected that the dosage for oral administration
of the opioid antagonists in an enteric ally coated formulation
would be from 10 to 30% of the non-coated oral dose. In the event
that the response in a patient is insufficient of such doses, even
higher doses (or effectively higher 30 dosage by a different, more
localized delivery route) may be employed to the extent that the
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compounds. Appropriate
system levels can be determined by, for example, measurement of the
patient's plasma level of the drug using routine HPLC methods known
to these of skill in the art.
[0153] In some embodiments of the invention, the opioid antagonists
are co-administered with the opioid. The term "co-administration"
is meant to refer to a combination therapy by any administration
route in which two or more agents are administered to a patient or
subject. Co-administration of agents may also be referred to as
combination therapy or combination treatment. The agents may be in
the same dosage formulations or separate formulations. For
combination treatment with more than one active agent, where the
active agents are in separate dosage formulations, the active
agents can be administered concurrently, or they each can be
administered at separately staggered times. The agents may be
administered simultaneously or sequentially (e.g., one agent may
directly follow administration of the other or the agents may be
give episodically, e.g., one can be given at one time followed by
the other at a later time, e.g., within a week), as long as they
are given in a manner sufficient to allow both agents to achieve
effective concentrations in the body. The agents may also be
administered by different routes, e.g., one agent may be
administered intravenously while a second agent is administered
intramuscularly, intravenously or orally. In other words, the
co-administration of the opioid antagonist compound in accordance
with the present invention with an opioid is suitably considered a
combined pharmaceutical preparation which contains an opioid
antagonist and a opioid agent, the preparation being adapted for
the administration of the peripheral opioid antagonist on a daily
or intermittent basis, and the administration of opioid agent on a
daily or intermittent basis. Thus, the opioid antagonists may be
administered prior to, concomitant with, or after administration of
the opioids. Co-administrable agents also may be formulated as an
admixture, as, for example, in a single formulation or single
tablet. These formulations may be parenteral or oral, such as the
formulations described, e.g., in U.S. Pat. Nos. 6,277,384;
6,261,599; 5,958,452 and PCT publication No. WO 98/25613, each
hereby incorporated by reference.
[0154] It is further contemplated that the present method can be
used alone or in conjunction with other treatments to control the
growth or migration of endothelial cells in connection with the
various conditions described above. The peripheral opioid
antagonist may be co-administered with another therapeutic agent
that is not an opioid or opioid antagonist. Suitable such
therapeutic agents include anticancer agents, e.g.,
chemotherapeutic agents, radiotherapy, or other antiangiogenic
agents such as suramin, or anti-VEGF mab, an endostatin or
radiotherapy. It is envisioned that the opioid antagonists in
accordance with the present invention are of particular value when
co-administered with those agents that inhibit VEGF activity, e.g.,
anti-VEGF mab. The anti-VEGF antibodies are useful in the treatment
of various neoplastic and non-neoplastic diseases and disorders,
including endometrial hyperplasia, endometriosis, abnormal vascular
proliferation associated with phakomatoses, edema (such as that
associated with brain tumors and Meigs' syndrome. One example of an
anti-VEGF mab is bevacizumab (Avastin, Genentech) described in U.S.
Pat. No. 6,884,879 and WO94/10202 hereby incorporated in their
entirety. In a certain embodiments of the invention, MNTX is
co-administered with Avastin.
[0155] In other words, the compounds of the present invention may
also be useful for the treatment of cancer in patients, as
described above, either when used alone or in combination with one
or more other anticancer agents, e.g., radiotherapy and/or other
chemotherapeutic, including antiangiogenic, treatments
conventionally administered to patients for treating cancer. The
main categories and examples of such drugs are listed herein and
include, but are not limited to metalloproatease inhibitors,
inhibitors of endothelial cell proliferation/migration, antagonists
of angiogenic growth factors, inhibitors of Integrin/Survival
signaling, and chelators of copper.
[0156] In certain embodiments the compounds of the invention can be
combined with known combinations of anticancer agents. The
compounds of the invention can be combined with an antiangiogenic
agent and a chemotherapeutic agent and administered to a cancer
patient. For example, MNTX can be administered to cancer patients
in combination with Avastin and 5-fluorouracil. It is anticipated
that the opioid antagonists co-administered with various anticancer
drugs, radiotherapy or other anti angiogenic drugs can give rise to
a significantly enhanced antiproliferative effect on cancerous
cells, and thus providing an increased therapeutic effect, e.g.,
employing peripheral opioid antagonists to certain tumors can
potentiate their response to other therapeutic regimens.
Specifically, a significantly increased anti angiogenic or
antihyperproliferative effect is obtained with the above disclosed
co-administered combinations, even if utilizing lower
concentrations of the anticancer, a lower dosing of radiation, or
other antiangiogenic drugs compared to the treatment regimes in
which the drugs or radiation are used alone. Therefore there is the
potential to provide therapy wherein adverse side effects
associated with the anticancer or other antiangiogenic drugs or
radiotherapy are considerably reduced than normally observed with
the anticancer or other antiangiogenic drugs or radiotherapy used
alone in larger doses. For example, co-administration of an opioid
antagonist in accordance with the present invention with an
anti-VEGF agent, e.g., anti-VEGF mab, may reduce the dose of the
anti-VEGF agent or increase potency or efficacy or both. Further,
as detailed herein, the co-administration of an opioid antagonist
in accordance with the present invention with other anticancer
modalities may have prophylactic value.
[0157] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
metalloprotease inhibitors such as for example: Marimastat,
synthetic matrix metalloprotease inhibitor (MMPI), British Biotech;
Bay 12-9566, synthetic MMPI and inhibitor tumor growth, Bayer;
AG3340, synthetic MMPI, Agouron/Wamer-Lambert; CGS 27023A,
synthetic MMPI, Novartis; CGS 27023A, Synthetic MMPI; COL-3,
synthetic MMPI, tetracycline derivative, Collagenex; AE-941
(Neovastat), naturally occurring MMPI, AEterna, BMS-275291,
synthetic MMPI, Bristol-Myers Squibb; Penicillamine, urokinase
inhibitor, NCI-NABTT.
[0158] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
direct inhibitors of endothelial cell proliferation/migration such
as: TNP-470 (fumagillin derivative), inhibits endothelial cell
growth, TAP Pharmaceuticals; Squalamine, inhibits sodium-hydrogen
exchanger, NIHE3, Magainin; Combretastatin, induction of apoptosis
in proliferating endothelial cells, Oxigene; Endostatin, inhibition
of endothelial cells, EntreMed; Penicillamine, blocks endothelial
cell migration and proliferation, NCI-NABTT; Farnesyl Transferase
Inhibitor (FTI), blocks endothelial cell migration and
proliferation, NCI-NABTT, -L-778,123 Merck, -SCH66336
Schering-Plough, -RI15777 Janssen.
[0159] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
antagonists of angiogenic growth factors such as: anti-VEGF
antibody, monoclonal antibody that inactivates VEGF, Genentech;
thalidomide, blocks activity of angiogenic growth factors (bFGF,
VEGF, TNF-alpha), Celgene; SU5416 (sexaminib;
3[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one),
blocks VEGF receptor (Flk-1/KDR) signaling (tyrosine kinase),
Sugen-NCI; ribozyme (Angiozyme), attenuates mRNA of VEGF receptors,
Ribozyme Pharmaceuticals, Inc; SU6668
(5-[1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole--
3-propanoic acid), blocks VEGF, bFGF, and PDGF receptor signaling,
Sugen; PTK787/ZK22584 (vatalnib), blocks VEGF receptor signaling,
Novartis; Interferon-alpha, inhibition of bFGF and VEGF production;
Suramin, blocks binding of growth factor to its receptor,
NCI-NABTT.
[0160] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
drugs that inhibit endothelial-specific Integrin/Survival
signaling: Vitaxin, antibody to alpha-v-beta3 integrin present on
endothelial cell surface, lxsys; EMD121974, small molecule blocker
of integrin present on endothelial cell surface, Merck KGaA.
[0161] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
chelators of copper, such as: penicillamine, sulfhydryl group binds
copper; clears copper through urinary excretion, NCI-NABTT;
tetrathiomolybdate, thiol groups tightly bind copper, inactivate
copper available to tumor, University of Michigan Cancer Center;
captopril, chelates copper and zinc; also, inhibitor of MMP and
angiotensin converting enzyme, Northwestern University.
[0162] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
angiogenesis antagonists with distinct mechanisms: CAI
(carboxyamidotriazole), inhibitor of calcium influx, NCI; ABT-627
(atrasentan), endothelin receptor antagonist, Abbott/NCI;
CM101/ZDO101, group B streptococcus toxin that selectively disrupts
proliferating endothelium by interaction with the (CM201) receptor,
CarboMed/Zeneca; Interleukin-12, induction of interferon-gamma,
down-regulation of IL-10, induction of IP-10, M.D. Anderson Cancer
Center/Temple University, Temple University, Genetics Institute,
Hoffman LaRoche; IM862 (small anti-VEGF dipeptide; L-glu-L-trp),
blocks production of VEGF and bFGF; increases production of the
inhibitor IL-12, Cytran; PNU-145156E
(7,7-(carbony-bis-[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-p-
yrrole]-carbonylimino)-bis(1,30-naphthaline disulfonate), blocks
angiogenesis induced by Tat protein, Pharmacia and Upjohn.
[0163] When used in the treatment of hyperproliferative diseases,
compounds of the present invention may be co-administered with
chemotherapeutic agents such as, for example, alpha interferon,
COMP (cyclophosphamide, vincristine, methotrexate and prednisone),
etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,
cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP
(prednisone, methotrexate (w/leucovin rescue), doxorubicin;
cyclophosphamide, paclitaxol, docetaxol, etoposide/mechlorethamine,
vincristine, prednisone and procarbazine), vincristine,
vinblastine, angioinhibins, TNP-470, pentosan polysulfate, platelet
factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan,
thalidomide, SP-PG and the like.
[0164] Anticancer agents which may be co-administered with
compounds of the present invention also suitably include
antimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine),
antimicrotubule agents (e.g., vincristine, vinblastine, taxanes
such as paclitaxel, docetaxel), an alkylating agent (e.g.,
cyclophosphamide, melphalan, biochoroethylnitrosurea, hydroxyurea),
nitrogen mustards, (e.g., mechloethamine, melphan, chlorambucil,
cyclophosphamide and Ifosfamide); nitrosoureas (e.g., carmustine,
lomustine, semustine and streptozocin), platinum agents (e.g.,
cisplatin, carboplatin, oxaliplatin, JM-2I6, C 1-973),
anthracyclines (e.g., doxrubicin, daunorubicin), antibiotics (e.g.,
mitomycin, idarubicin, adriamycin, daunomycin), topoisomerase
inhibitors (e.g., etoposide, camptothecins), alkyl sulfonates
including busulfan; triazines (e.g., dacarbazine); ethyenimines
(e.g., thiotepa and hexamethylmelamine); folic acid analogs (e.g.,
methotrexate); pyrimidine analogues (e.g., 5 fluorouracil, cytosine
arabinoside); purine analogs (e.g., 6-mercaptopurine,
6-thioguanine); antitumor antibiotics (e.g., actinomycin D;
bleomycin, mitomycin C and methramycin); hormones and hormone
antagonists (e.g., tamoxifen, cortiosteroids) and any other
cytotoxic agents, (e.g., estramustine phosphate,
prednimustine).
[0165] In addition to the co-administration of anti-VEGF agent with
the compounds in accordance with the present invention, other
chemotherapeutics, such as anti-microtubules, antimetabolites, and
other tyrosine kinase inhibitors, may be combined with the
compounds of the present invention. Tyrosine kinases are an
especially important target because they play an important role in
the modulation of growth factor signaling. Tyrosine kinase
inhibitors compete with the ATP binding site of the catalytic
domain of several oncogenic tyrosine kinases. Several tyrosine
kinase inhibitors have been found to have effective antitumor
activity; these include imatinib mesylate (STI571; Gleevec),
gefitinib (Iressa), erlotinib (OSI-1774; Tarceva), lapatinib
(GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib
(PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and
leflunomide (SU101).
[0166] As demonstrated in the Examples below, methylnaltrexone
enhances the ability of erlotinib (an EGF receptor inhibitor),
gemcitabine (an antimetabolite), vironrelbine (an
anti-microtubule), paclitaxel (an anti-microtubule), and docetaxel
(an anti-microtubule) to inhibit NSCLC cell proliferation. Morphine
on the other hand, leads to relative chemotherapeutic resistance
(e.g., see FIG. 29). Thus, an opioid antagonist can enhance the
efficacy of a chemotherapeutic agent.
[0167] It will be understood that agents which can be combined with
the compounds of the present invention for the inhibition,
treatment or prophylaxis of angiogenesis and/or cancers are not
limited to those listed above, but include, in principle, any
agents useful for the treatment opioid induced angiogenic diseases
and tumor growth.
[0168] The present invention is further explained by the following
examples, which should not be construed by way of limiting the
scope of the present invention.
Examples
Example 1: Endothelial Cell Migration Assay
[0169] The antiangiogenic activity of the peripheral opioid
antagonists in accordance with the present invention was evaluated
in experiments testing the ability of the antagonist to inhibit or
modulation capillary endothelial cell migration using a modified
Boyden chamber.
[0170] The endothelial cell migration assay was performed as
described by Lingen, M. W., Methods in Molecular Medicine, 78:
337-347 (2003), the disclosure of which is incorporated by
reference. Briefly, Human Microvascular Endothelial Cells (HMVEC)
(Cell Systems, Kirkland, W A.) were starved overnight in
Endothelial Growth Medium (EGM) containing 0.1% bovine serum
albumin (BSA). Cells were then trypsinized and resuspended in
Dulbecco's Modified Eagle Medium (DME) with 0.1% BSA at a
concentration of 1.times.10.sup.6 cells/mL. Cells were added to the
bottom of a 48-well modified Boyden chamber (NeuroPore Corporation,
Pleasanton, Calif.). The chamber was assembled and inverted, and
cells were allowed to attach for 2 hours at 37.degree. C. to
polycarbonate chemotaxis membranes (5 .mu.m pore size) (NeuroProbe)
that had been soaked in 0.1% gelatin overnight and dried. The
chamber was then reinverted and the compound to be tested at
varying concentrations in quadruple, vascular endothelial growth
factor (VEGF) (as a positive control) or vehicle were added to the
wells of the upper chamber (to a total volume of 50 mL); the
apparatus was then incubated for 4 hours at 37.degree. C. Membranes
were recovered, fixed and stained (DiffQuick, Fisher Scientific,
Pittsburgh, Pa.) and the number of cells that had migrated to the
upper chamber per 10 high power fields were counted. Background
migration to DME+O.l % BSA was subtracted and the data reported as
the number of cells migrated per 10 high power fields (400 times).
Each substance was tested in quadruplicate in each experiment, and
all experiments were repeated to least twice. VEGF (R&D System,
Minneapolis, Minn.) was used as a positive control at a
concentration of 200 pg/mL. The optimal concentration for VEGF was
determined previously by dose-response experiments (data not
shown). The compounds tested as described above were morphine,
naloxone, methylnaltrexone, and the combination of methylnaltrexone
and morphine. The concentrations of each tested substance ranged
for 0.001 to 10.0 .mu.M. The concentration of the morphine was
constant at 0.1 .mu.M. The results are shown in FIG. 1.
[0171] FIG. 1 shows that morphine increased migration in a
concentrationdependent manner. The co-addition of methylnaltrexone
and morphine, however, decreased migration in a
concentration-dependent manner. Neither methylnaltrexone or
naloxone alone affected migration.
Example 2: Endothelial Cell Migration Assay
[0172] Another set of experiments was conducted in accordance with
the procedure described in Example 1. In this set of experiments,
methylnaltrexone and the combination of methylnaltrexone and
morphine was again tested for ability to inhibit endothelial cell
migration. The methylnaltrexone concentrations when tested alone
varied from 0.001 to 10.0 .mu.M. In combination, the concentrations
of methylnaltrexone varied from 0.001 to 10.0 .mu.M, while the
morphine concentration remained constant at 0.1 .mu.M as described
in Example 1. The results are shown in FIG. 2.
[0173] FIG. 2 shows the combination of methylnaltrexone and
morphine decreased migration in a concentration-dependent manner,
while methylnaltrexone alone did not affect migration.
Example 3: Endothelial Cell Migration Induced by DAMGO
[0174] The drugs used in this study were [D-Ala 2, N-McPhe4,
Gly5-ol] enkephalin or DAMGO (Sigma, St. Louis, Mo.); naloxone
(Sigma, St. Louis, Mo.); N-methylnaltrexone bromide or
methylnaltrexone (Mallinckrodt Specialty Chemicals, Phillipsburg,
N.J.). The endothelial cell migration assay was performed as
previously described (9). Human dermal microvascular endothelial
cells (Cell Systems, Kirkland, W A) were starved overnight in media
containing 0.1% bovine serum albumin (BSA), harvested, resuspended
into Dulbecco's Modified Eagle's media (DME) with 0.1% BSA, and
plated on a semi-porous gelatinized membrane in a modified Boyden
chamber (Nucleopore Corporation, Pleasanton, Calif.). Test
substances were then added to the wells of the upper chamber and
cells were allowed to migrate for four hours at 37.degree. C.
[0175] Membranes were recovered, fixed, and stained and the number
of cells that had migrated to the upper chamber per ten high power
fields counted by a blinded observer. Background migration to
DME+0.1% BSA was subtracted and the data reported as the number of
cells migrated per 10 high power fields (400.times.). Each
substance was tested in quadruplicate in each experiment and all
experiments were repeated at least twice. The concentration of DAM
GO was 1 .mu.M, VEGF (R&D Systems, Minneapolis, Minn.) was used
as a positive control at a concentration of 200 pg/mL. The optimal
concentration for VEGF was determined previously by dose-response
experiments (data not shown).
[0176] The results are shown in FIG. 3 which shows that
methylnaltrexone and DAMGO decreased migration in a
concentration-dependent manner. FIG. 4 illustrates similar results
with naloxone and DAMGO. The inactive morphine metabolite M3G
exerts no angiogenic activity while M6G known to act at the mu
receptor exhibited a concentration dependent effect on angiogenesis
(FIG. 5).
Example 4: Treatment of Human and Mammalian Subjects with
Methylnaltrexone
[0177] In a first set of experiments, mice are induced to develop
tumors by transformation, inbreeding or transplantation of tumor
cells. Thirty-six mice, each bearing tumors having a volume of at
least 60 mm3, are randomly divided into three groups. The first
group receives a control substance comprising neither an opioid nor
an opioid antagonist. The second group receives an opioid, e.g.
morphine administered orally at a dose of 0.5 mg/kg/day. The third
group receives an opioid, e.g. morphine administered orally at a
dose of 0.5 mg/kg/day, and the peripheral opioid antagonist
methylnaltrexone, administered orally at a dose of 5 mg/kg/day.
[0178] The compounds are administered daily for a period of eight
weeks. Differences in the rate of tumor growth, tumor size, a
reduction in angiogenesis in the tumor and mortality of the mice
between each of the groups are recorded. The results demonstrate a
reduction in tumor growth and angiogenesis compared to controls or
morphine alone.
[0179] In a second set of experiments, human cancer patients are
enrolled in a study. Enrollees in the study are controlled for age,
stage of disease, treatment types and genetic and familial factors.
Participants are divided into two groups according to whether they
are receiving opioids, e.g. morphine. The group receiving opioids
is further randomly divided into two subgroups. One of the two
subgroups receiving opioids receives a peripheral opioid
antagonist, e.g., methylnaltrexone administered orally at a dose of
5 mg/kg/day for a period of eight weeks. The other of the two
subgroups receives placebo for the same period. Differences in the
rate of tumor growth, tumor size, a reduction in angiogenesis in
the tumor and mortality of the participants in each of the groups
are recorded.
Example 5: Treatment of Human and Mammalian Subjects with
Alvimopan
[0180] Mice that have been induced to develop tumors are subjected
to the protocol as described in Example 3, except that the
peripheral opioid antagonist is alvimopan. The results demonstrate
a reduction in tumor growth and angiogenesis compared to controls
or opioid alone.
[0181] Human cancer patients are enrolled in a study conducted as
described in Example 4, except that the peripheral opioid
antagonist is alvimopan.
Example 6: Therapies Comprising Co-Administration of the Peripheral
Opioid Antagonist Methyl Naltrexone and Second Therapeutic
Agent
[0182] In a first set of experiments, mice are induced to develop
tumors by transformation, inbreeding or transplantation of tumor
cells. Forty-eight mice, each bearing tumors having a volume of at
least 60 mm.sup.3, are randomly divided into six groups. The first
group receives a control substance which does not comprise an
opioid, an opioid antagonist, or an anticancer agent. The second
group receives an opioid, e.g. morphine administered orally at a
dose of 0.5 mg/kg/day. The third group receives an opioid, e.g.
morphine administered orally at a dose of 0.5 mg/kg/day, and the
peripheral opioid antagonist methylnaltrexone, administered orally
at a dose of 5 mglkg/day. The fourth group receives an opioid, e.g.
morphine administered orally at a dose of 0.5 mglkg/day, and the
peripheral opioid antagonist methylnaltrexone administered orally
at a dose of 5 mg/kg/day with an anticancer therapeutic agent, e.g.
bevacizumab (Avastin) at a dose of 5 mglkg every 14 days. The sixth
group receives an opioid, e.g. morphine, at a dose of 0.5 mglkg/day
and an anticancer therapeutic agent, e.g. bevacizumab (Avastin) at
a dose of 5 mg/kg every 14 days.
[0183] The compounds are administered daily for a period of eight
weeks. Differences in the rate of tumor growth, tumor size, a
reduction in angiogenesis in the tumor and mortality of the mice in
each of the groups are recorded. The results demonstrate an
enhanced result (e.g., reduction in angiogenesis and tumor growth)
for the groups administered the combination of opioid, opioid
antagonist, and anticancer agent compared to the other groups.
[0184] In a second set of experiments, human cancer patients
receiving an opioid, e.g. morphine, an anticancer therapeutic
agent, e.g. bevacizumab (Avastin) or both are enrolled in a study.
Enrollees in the study are controlled for age, stage and type of
disease, treatment types and genetic and familial factors.
Participants receiving an opioid are randomly divided into first
and second groups; participants receiving an anticancer therapeutic
agent, e.g. bevacizumab (Avastin) are randomly divided into third
and fourth groups; participants receiving an opioid plus an
anticancer agent, e.g. bevacizumab (Avastin) are randomly divided
into fifth and sixth groups. The first, third and fifth groups each
receive a peripheral opioid antagonist, e.g., methylnaltrexone
administered orally at a dose of 5 mg/kg/day for a period of eight
weeks. The second, fourth and sixth groups receive placebo for the
same period. Differences in the rate of tumor growth, tumor size, a
reduction in angiogenesis in the tumor and mortality of the
participants in each of the groups are recorded. The results
demonstrate an enhanced result (e.g., reduction in angiogenesis and
tumor growth) for the groups administered the combination of
opioid, opioid antagonist, and anticancer agent compared to the
other groups.
Example 7: Therapies Comprising Co-Administration of the Peripheral
Opioid Antagonist Alvimopan and Second Therapeutic Agent
[0185] Mice that have been induced to develop tumors are subjected
to the protocol as described in Example 5, except that the
peripheral opioid antagonist is alvimopan. The results demonstrate
an enhanced result (e.g., reduction in angiogenesis and tumor
growth) for the groups administered the combination of opioid,
opioid antagonist, and anticancer agent compared to the other
groups.
[0186] Human cancer patients are enrolled in a study conducted as
described in Example 6, except that the peripheral opioid
antagonist is alvimopan. The results demonstrate an enhanced result
(e.g., reduction in angiogenesis and tumor growth) for the groups
administered the combination of opioid, opioid antagonist, and
anticancer agent compared to the other groups.
Example 8: Effect of Opioid Antagonists on Endothelial Cell
MigrationfProliferation
[0187] Cell culture and reagents-Human dermal microvascular
endothelial cells (Cell Systems, Kirkland, W A) and human pulmonary
microvascular endothelial cells (Clonetics, Walkersville, Md.) were
cultured as previously described in EBM-2 complete medium
(Clonetics) at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2, 95% air, with passages 6-10 used for experimentation
(Garcia et al. 2001). Unless otherwise specified, reagents were
obtained from Sigma (St. Louis, Mo.). Reagents for SDS-P AGE
electrophoresis were purchased from Bio-Rad (Richmond, Calif.),
Immobilon-P transfer membrane from Millipore (Millipore Corp.,
Bedford, Mass.). The drugs used in this study were [D-Ala.sup.2,
N-MePhe.sup.4, Gly.sup.5-ol] enkephalin or DAMGO (Sigma, St. Louis,
Mo.); naloxone, morphine-3-glucuronide (M3G) and
morphine-6-glucuronide (M6G) (Sigma, St. Louis, Mo.);
N-methylnaltrexone bromide or methylnaltrexone (Mallinckrodt
Specialty Chemicals, Phillipsburg, N.J.), morphine (Baxter,
Deerfield, Ill.). VEGF Receptor Tyrosine Kinase Inhibitor was
purchased from Calbiochem (San Diego, Calif.). Mouse anti-RhoA
antibody, mouse anti-phosphotyrosine antibody and rho binding
domain (RBD)-conjugated beads were purchased from Upstate
Biotechnology (Lake Placid, N.Y.). Rabbit anti-VEGF receptor 1
(Flt-I) and antiVEGF receptor 2 (Flk-I) antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, Calif.). Mouse
anti-.about.-actin antibody was purchased from Sigma (St. Louis,
Mo.). Secondary horseradish peroxidase (HRP)-labeled antibodies
were purchased from Amersham Biosciences (Piscataway, N.J.).
[0188] Immunoprecipitation and immunoblotting--Cellular materials
were incubated with IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl,
20 mM MgC12, 1% TritonX-100, 0.1% SDS, 0.4 mM Na3V04, 40 mM NaF,
50/IM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1:250
dilution of Calbiochem protease inhibitor mixture 3). The samples
were then immunoprecipitated with anti-VEGF receptor 1 or anti-VEGF
receptor 2 IgG followed by SDS-PAGE in 4-15% polyacrylamide gels,
transfer onto Immobilon.TM. membranes, and developed with specific
primary and secondary antibodies. Visualization of immunoreactive
bands was achieved using enhanced chemiluminescence (Amersham
Biosciences).
[0189] Determination of tyrosine phosphorylation of VEGF Receptors
1 and 2--Solubilized proteins in IP buffer (see above) were
immunoprecipitated with either rabbit anti-VEGF receptor 1 or
rabbit anti-VEGF receptor 2 antibody followed by SDS-PAGE in 4-15%
polyacrylamide gels and transfer onto Immobilon.TM. membranes
(Millipore Corp., Bedford, Mass.). After blocking nonspecific sites
with 5% bovine serum albumin, the blots were incubated with either
rabbit anti-VEGF receptor 1 antibody, rabbit anti-VEGF receptor 2
antibody or mouse anti-phosphotyrosine antibody followed by
incubation with horseradish peroxidase (HRP)-labeled goat
anti-rabbit or goat anti-mouse IgG. Visualization of immunoreactive
bands was achieved using enhanced chemiluminescence (Amersham
Biosciences).
[0190] Construction and transfection of siRNA against RhoA--The
siRNA sequence targeting human against RhoA was generated using
mRNA sequences from Genbank.TM. (gi:33876092). For each mRNA (or
scramble), two targets were identified. Specifically, RhoA target
sequence 1 (5'-AAGAAACTGGTGATTGTTGGT-3') (SEQ ID NO:I), RhoA target
sequence 2 (5'-AAAGACATGCTTGCTCATAGT-3') (SEQ ID NO:2), scrambled
sequence I (5'-AAGAGAAA TCGAAACCGAAAA-3') (SEQ ID NO:3), and
scramble sequence 2 (5'-AAGAACCCAATTAAGCGCAAG-3') (SEQ ID NO:4),
were utilized. Sense and antisense oligonucleotides were purchased
from Integrated DNA Technologies (Coralville, Iowa). For
construction of the siRNA, a transcription-based kit from Ambion
was used (Silencer.TM. siRNA construction kit). Human lung
microvascular EC were then transfected with siRNA using
siPORTamine.TM. as the transfection reagent (Ambion, TX) according
to the protocol provided by Ambion. Cells (.about.40% confluent)
were serum-starved for 1 hour followed by incubated with 3 .mu.M
(1.5 .mu.M of each siRNA) of target siRNA (or scramble siRNA or no
siRNA) for 6 hours in serum-free media. The serum-containing media
was then added (1% serum final concentration) for 42 h before
biochemical experiments and/or functional assays were
conducted.
[0191] RhoA activation assay--After agonist and/or inhibitor
treatment, EC are solubilized in solubilization buffer and
incubated with rho bonding domain (RBD)-conjugated beads for 30
minutes at 4.degree. C. The supernatant is removed and the
RBD-beads with the GTP-bound form of Rho A bound are washed
extensively. The RBD beads are boiled in SDS-P AGE sample buffer
and the bound RhoA material is run on SDS-P AGE, transferred to
Immobilon.TM. and immunoblotted with anti-RhoA antibody (Garcia et
al 2001).
[0192] Human dermal microvascular EC migration assay--The
endothelial cell migration assay was performed as previously
described (Lingen 2002). Human dermal microvascular endothelial
cells (Cell Systems, Kirkland, W A) were starved overnight in media
containing 0.1% bovine serum albumin (BSA), harvested, resuspended
into Dulbecco's Modified Eagle's media (DME) with 0.1% BSA, and
plated on a semi-porous gelatinized membrane in a modified Boyden
chamber (Nucleopore Corporation, Pleasanton, Calif.). Test
substances were then added to the wells of the upper chamber, and
cells were allowed to migrate for 4 hr at 37.degree. C. Membranes
were recovered, fixed, and stained and the number of cells that had
migrated to the upper chamber per 10 high-power fields was counted
by a blinded observer. Background migration to DME+0.1% BSA was
subtracted, and the data were reported as the number of cells
migrated per 10 high-power fields (400.times.). Each substance was
tested in quadruplicate in each experiment and all experiments were
repeated at least twice. Vascular endothelial growth factor (VEGF,
R&D Systems, Minneapolis, Minn.) was used as a positive control
at a concentration of 200 pg/mL. The optimal concentration for VEGF
was determined previously by dose-response experiments (data not
shown).
[0193] Human pulmonary microvascular EC migration
assay--Twenty-four TranswelfM units with 8 M pore size were used
for monitoring in vitro cell migration. HPMVEC
(.about.I.times.10.sup.4 cells/well) were plated with various
treatments 30 (100 nM MNTX, 10 .mu.M VEGF Receptor Tyrosine Kinase
Inhibitor or siRNA) to the upper chamber and various agonists were
added to the lower chamber (100 nM MS, DAMGO or VEGF). Cells were
allowed to migrate for 18 hours. Cells from the upper and lower
chamber were quantitated using the CellTiter96.TM. MTS assay
(Promega, San Luis Obispo, Calif.) and read at 492 nm. % migration
was defined as the # of cells in the lower chamber % the number of
cells in both the upper and lower chamber. Each assay was set up in
triplicate, repeated at least five times and analyzed statistically
by Student's t test (with statistical significance set at
P<0.05).
[0194] Human pulmonary microvascular EC proliferation assay--For
measuring cell growth, HPMVEC [5.times.10.sup.3 cells/well
pretreated with various agents (100 nM MNTX, 10 .mu.M VEGF Receptor
Tyrosine Kinase Inhibitor or siRNA) were incubated with 0.2 mL of
serum-free media containing various agonists (100 nM MS, DAMGO or
VEGF) for 24 h at 37.degree. C. in 5% C02/95% air in 96-well
culture plates. The in vitro cell proliferation assay was analyzed
by measuring increases in cell number using the CellTiter96.TM. MTS
assay (Promega, San Luis Obispo, Calif.) and read at 492 nm. Each
assay was set up in triplicate, repeated at least five times and
analyzed statistically by Student's t test (with statistical
significance set at P<0.05).
[0195] Using the endothelial cell migration assay, it was found
that MS caused a concentration-dependent increase in endothelial
migration. Naloxone and MNTX alone had no effect on endothelial
cell migration over a wide range of concentrations. This is
demonstrated in representative photomicrographs and quantitatively
(FIGS. 6 and 1, respectively). At clinically relevant
concentrations of morphine, the magnitude of the effect was
approximately 70% of that achieved by VEGF. Endothelial cell
migration induced by morphine in concentrations as low as
10.sup.-7M (FIG. 2). Morphine-based endothelial cell migration was
attenuated by the mu opioid antagonists naloxone and MNTX (in doses
as low as 10.sup.-8 .mu.M) in a concentration-dependent fasmon,
strongly suggesting that endothelial cell migration is mediated by
morphine's action on the mu opioid receptor (MOR). That the effect
is via the MOR rather than other opioid receptors was confirmed by
our observations that the highly selective synthetic enkephalin mu
agonist DAMGO also induced migration in a concentration dependent
fashion. The effect of DAMGO was also blocked by MNTX (FIG. 3).
That the inactive morphine metabolite M6G exerts no angiogenic
activity, while M6G, known to act at the mu receptor, exhibits a
concentration-dependent effect on angiogenesis, confirms our
hypothesis that morphine's effect on the endothelium is mediated by
mu receptors (McQuay et al. 1997) (FIG. 5).
[0196] In order to assess the mechanisms of opioid and MNTX-induced
effects on angiogenesis, a well-characterized EC line was used,
human pulmonary microvascular endothelial cells (HPMVEC). In
agreement with the effects on human dermal microvascular EC, it was
observed that MS, DAMGO and VEGF induce HPMVEC migration which is
inhibited by MNTX (FIG. 7B). It was shown that MS, DAMGO and VEGF
also stimulate HPMVEC proliferation which is attenuated by MNTX
(FIG. 7A).
[0197] Considering the inhibitory effects of MNTX, a mu opioid
receptor antagonist, on VEGF-induced EC proliferation and
migration, the role of opioids on VEGF receptor transactivation was
examined. FIG. 8A shows that MS and DAMGO induce tyrosine
phosphorylation of both VEGF receptor 1 (Flt-1) and 2 (Flk-1) which
is blocked by MNTX. Further, MNTX attenuates the tyrosine
phosphorylation of VEGF receptors 1 and 2 induced by VEGF. These
results indicate that opioids induce VEGF receptor
transactivation.
[0198] In order to address if VEGF receptor tyrosine kinase
activity is required for opioid-induced angiogenesis, EC were
pre-treated with a VEGF receptor 1 and 2 tyrosine kinase inhibitor
and measured opioid-induced EC proliferation and migration (FIG.
8B). The results indicate that the tyrosine kinase activity of VEGF
receptors is important in opioid-induced EC angiogenic
functions.
[0199] One important signaling molecule involved in angiogenesis is
the small G-protein, RhoA (Aepfelbacher et al. 1997; Cascone et al.
2003; Hoang et al. 2004; Liu and Senger 2004). It was observed that
MS, DAMGO and VEGF stimulate RhoA activation which is inhibited by
MNTX (FIG. 9A). Further, VEGF receptor transactivation is important
for opioid-induced RhoA activation (FIG. 9B). Silencing RhoA
expression blocks opioid and VEGF-induced EC proliferation and
migration (FIG. 10). These results indicate the pivotal role of
RhoA activation on agonist-induced EC angiogenic activity.
[0200] Taken as a whole these findings suggest a model in which the
peripheral mu opioid receptor antagonist, MNTX, attenuates opioid
and VEGF-induced VEGF receptor and RhoA activation. This
attenuation is important for the inhibitory role of MNTX on opioid
and VEGF-mediated angiogenesis (FIG. 11).
Example 9: Methylna Itrexone Inhibits SIP, VEGF and PDGF-Induced
Angiogenesis: Role of Receptor Transactivation
[0201] Assays were conducted according to the procedure similar to
that described in Examples 1-3. It was observed that SIP, VEGF,
PDGF, morphine and DAM GO induced proliferation (FIG. 12) (as
measured by the colorimetric CellTiter.TM. (Promega) MTS assay) and
migration (FIG. 13) (as measured by the Transwell.TM. (Costar)
permeable membrane filter assay (8 J.Im pore diameter)) of EC which
were inhibited by pretreatment with MNTX (0.1 .mu.M, 1 hour).
Silencing mu opioid receptor expression (siRNA) blocks morphine and
DAMGO-induced EC proliferation (FIG. 14) and migration (FIG. 15)
while also significantly inhibiting SIP, VEGF and PDGF-induced EC
proliferation (FIG. 14) and migration (FIG. 15).
Immunoprecipitation followed by immunoblot analyses indicate that
SIP, VEGF and PDGF treatment of EC induced serine/threonine
phosphorylation of the mu opioid receptor (FIG. 16) (indicating
receptor transactivation) and activation of the cytoskeletal
regulatory small G-protein, RhoA (FIG. 17). Further, morphine and
DAMGO treatment of EC induced tyrosine phosphorylation of the VEGF
receptor (FIG. 18), PDGF receptor (FIG. 18) and SIP 3 receptor
(FIG. 19) along with RhoA activation. MNTX pretreatment of EC
attenuated morphine, DAMGO, SIP, VEGF and PDGF induced receptor
phosphorylation events and RhoA activation. Finally, silencing RhoA
expression (siRNA) blocked agonist-induced EC proliferation (FIG.
20) and migration (FIG. 21). Taken together, these results indicate
that MNTX inhibits agonist induced EC proliferation and migration
via inhibition of receptor phosphorylation/transactivation and
subsequent inhibition of Rho A activation (FIG. 22). These results
suggest that MNTX inhibition of angiogenesis can be a useful
therapeutic intervention for cancer treatment.
Example 10: Methylnaltrexone and Antiproloferative Compounds
Synergistically Inhibit VEGF-Induced Proliferation and
Migration
[0202] Assays were conducted according to the procedure similar to
that described in Examples 1-3. It was observed that
methylnaltrexone and 5-FU synergistically inhibit VEGF induced
proliferation of endothelial cells. (FIG. 23). It was likewise
observed that methylnaltrexone and Bevacizumab synergistically
inhibit VEGF induced migration of endothelial cells. (FIG. 24).
Example 11: Effects of MNTX on Various Cancer Cell Lines
[0203] The effects of MNTX on SW 480 human colorectal cancer cell
line were evaluated. As shown in FIG. 25, it was observed that MNTX
itself possesses antiproliferation activity in SW 480 cells (**,
P<0.01 compared to control). In addition, MNTX enhanced 5-FU's
tumoricidal effect (*, P<0.05 compared to 5-FU 10 uM only,
approx. 1050 for this cell line). As shown in FIGS. 26, 27, and 28,
respectively, similar results were obtained in HCT116 human
colorectal cancer cell line, MCF-7 human breast cancer cell line,
and non-small lung cancer cell (NSLCC) line.
[0204] In summary, the present invention provides methods of
attenuating endothelial cell migration and/or proliferation
associated with angiogenesis and/or enhancing endothelial cell
barrier function in tissue or an organ of a subject in need
therefor by administering one or more opioid antagonists,
especially peripheral opioid antagonists, in an effective amount to
the patient to inhibit the migration and/or proliferation and
angiogenesis, and/or improve barrier function. The methods of the
present invention may also involve administering a peripheral
opioid antagonist to a patent receiving opioid treatment.
Especially suitable may be a mu peripheral opioid antagonist. The
present invention also provides methods of co-administering an
opioid and a peripheral opioid antagonist to a subject in need
therefore. The peripheral opioid antagonist may also be
co-administered with an anticancer agent, as may the combination of
the opioid and peripheral opioid antagonist be co-administered with
an anticancer agent.
Example 12: MNTX Enhances the Ability of Chemotherapeutics to
Inhibit Cell Proliferation in Human Lung Cancer Cells
[0205] Cell Culture and Reagents--
[0206] The human Non-Small Cell Lung Cancer (NSCLC) cell line H1993
was obtained from ATCC (Walkersville, Md.) and cultured in Roswell
Park Memorial Institute complete medium (Cambrex, East Rutherford,
N.J.) at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2,
95% air, with passages 6-10 used for experimentation. Unless
otherwise specified, reagents were obtained from Sigma (St. Louis,
Mo.). Morphine was purchased from Baxter (Deerfield, Ill.).
N-methylnaltrexone bromide or methylnaltrexone (MNTX) was purchased
from Mallinckrodt Specialty Chemicals (Phillipsburg, N.J.).
Erlotinib (epidermal growth factor receptor tyrosine kinase
inhibitor), Vinorelbine Tartrate (inhibits mitosis through
interaction with tubulin), Paclitaxel (microtubule polymer
stabilizer), Docetaxel (inhibitor of depolymerisation of
microtubules) and Gemcitabine HCl (DNA synthesis inhibitor) were
purchased from Selleck Chemicals (Houston, Tex.).
[0207] Human NSCLC Cell Proliferation Assay--
[0208] H1993 cells (5.times.10.sup.3 cells/well) were incubated
with 0.2 ml of serum-free media containing 0, 0.01, 0.1, 0.5, 1, 5
or 10 uM of either Erlotinib (epidermal growth factor receptor
tyrosine kinase inhibitor), Vinorelbine Tartrate (inhibits mitosis
through interaction with tubulin), Paclitaxel (microtubule polymer
stabilizer), Docetaxel (inhibitor of depolymerisation of
microtubules) or Gemcitabine HCl (DNA synthesis inhibitor). These
are indicated as Control. In some cases, morphine (100 nM) or
morphine (100 uM) plus methylnaltrexone (MNTX, 10 nM or 100 nM)
were added in addition to the chemotherapeutic agents. Treated
cells were grown for 24 h at 37.degree. C. in 5% CO.sub.2/95% air
in 96-well culture plates. The in vitro cell proliferation assay
was analyzed by measuring the change in cell number using the
CellTiter96.TM. MTS assay (Promega, Madison, Wis.) and read at 492
nm.
[0209] Results are represented graphically (FIG. 29) with the
y-axis indicating Percent Inhibition of Cell Proliferation and the
x-axis indicating drug concentration. The results shown in FIG. 29
indicate that Erlotinib, Paclitaxel, Gemcitabine HCl, Vinorelbine
Tartrate and Docetaxel inhibit human NSCLC proliferation in a
dose-dependent manner. Morphine decreases the ability of these
chemotherapeutic agents to inhibit H1993 cell proliferation thus
promoting multidrug resistance. In contrast, MNTX reversed the
effects of morphine on multidrug resistance in a dose-dependent
manner and enhanced the effects of the chemotherapeutic agents.
[0210] While the present invention has now been described and
exemplified with some specificity, those skilled in the art will
appreciate the various modifications, including variations,
additions, and omissions that may be made in what has been
described. Accordingly, it is intended that these modifications
also be encompassed by the present invention and that the scope of
the present invention be limited solely by the broadest
interpretation that lawfully can be accorded the appended
claims.
[0211] All patents, publications and references cited herein are
hereby fully incorporated by reference. In case of conflict between
the present disclosure and incorporated patents, publications and
references, the present disclosure should control.
Sequence CWU 1
1
4121DNAHomo sapiens 1aagaaactgg tgattgttgg t 21221DNAHomo sapiens
2aaagacatgc ttgctcatag t 21321DNAHomo sapiens 3aagagaaatc
gaaaccgaaa a 21421DNAHomo sapiens 4aagaacccaa ttaagcgcaa g 21
* * * * *