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CA 02586420 2007-05-03
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ENHANCING TREATMENT OF CANCER AND HIF-i MEDIATED DISORDERS
WITH ADENOSINE A3 RECEPTOR ANTAGONISTS
1. CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of United States Provisional Application
No. 60/630,557, filed November 22, 2004, the disclosure of which is hereby
incorporated
by reference in its entirety.
2. FIELD OF THE INVENTION
The present invention relates to the use of adenosine receptor antagonists,
preferably A3 receptor antagonists, either alone or in combination with other
agents for the
treatment, prevention and/or management of diseases or disorders associated
with
overexpression of HIF-1 a and/or increased HIF-1 a activity (e.g., cancer,
respiratory
disease). The methods and compositions of the invention are particularly
usefuJ. for
preventing, treating, or ameliorating symptoms associated with a cancer,
disease or disorder
associated with hypoxia-inducible factor 1-a (HIF-l(X) using the A3 receptor
antagonists of
the invention. The present invention provides methods to inhibit the growth of
tumors,
particularly solid tumors, and more particularly hypoxic tumors.
3. BACKGROUND OF THE INVENTION
3.1 ADENOSINE
Adenosine, recently called a "primordial signalling molecule" (Linden, 2001,
Annu. Rev. Pharmacol. Toxicol, Unital 41: 775-87), has the potential of
influencing
development, is present in and modulates physiological responses in all
mammalian tissues.
The actions of adenosine are most prominent in tissues where oxygen demand is
high and
there is reduction in oxygen tension, i.e., within solid tumors, where cell
proliferation is
greater than the rate of blood vessel formation (Sitkovsky, 2004 Annu. Rev.
Immunol. 22,
657-82; Fredholm, 2001, Phar=macol. Rev. 53, 527-552). As a result, the tumor
has local
areas of hypoxia and adenosine accumulates to high levels (Hockel, 2001, J.
Natl. Cancer
Inst. 93, 266-76). In particular, it is recognized that significant levels of
adenosine are
present in the extracellular fluid of solid tumors (Blay, 1997, CancerRes.,
57, 2602-5),
suggesting a role for this nucleoside in tumor growth.
Adenosine has been linked to tumor development. Increased adenosine
concentration has been reported inside tumoral masses. It has been speculated
that it
represents the anti-tumor agent that prevents tumor growth in muscle tissue in
vivo and that
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impairs malignant cell growth and survival in vitro. However, it is known that
adenosine
acts as cyto-protective agent during ischemic damage in brain and heart.
Adenosine is
known to be released in hypoxia. Numerous studies have shown adenosine to
protect cells
in the heart from ischemic damage.
Adenosine has been shown to have protective roles in.numerous animal
models and in man (Am. J. Cardiol. 79(12A):44-48 (1997). For example, in the
heart, both
the Al and A3 receptors offer protection against ischemia (Am. J. Physiol.,
273(42)H501-
505 (1997)). However, it is the A3 receptor that offers sustained protection
against ischemia
(PNAS 95:6995-6999 (1998)). The ability of adenosine to protect tumor cells
against
hypoxia has not been recognized by others prior to the instant invention.
Adenosine interacts with cell surface receptors that are glycoproteins coupled
to different members of G protein family. By now four adenosine receptors have
been
cloned and characterised: Al, A2A, A2B and A3. Selective antagonists for the
A3 receptor
have been proposed for use as anti-inflammatory and antiischemic agents in the
brain.
Recently, A3 antagonists have been under development as antiasthmatic,
antidepressant,
anti-arrhythmic, renal protective, antiparkinson and cognitive enhancing
drugs. For
example, U.S. Pat. No. 5,646,156 to Marlene Jacobson et al. describes the
inhibition of
eosinophil activation by using selected A3 antagonists.
Recent studies in myocytes have shown the adenosine A3 receptors to be
responsible for long-term protection against ischemia (Liang and Jacobson,
PNAS, 1998,
95:6995-6999). While the present inventors have hypothesized that adenosine
plays a
protective role in other cell types, including tumor cells, in addition to
myocytes, no efforts
have been made to limit the protective effect of adenosine on tumor cells.
3.2 HIF-1 BIOLOGY
Hypoxia-inducible factor (HIF)-1 is a transcription factor that functions as a
master regulator of oxygen homeostasis (Semenza, 2001, Trends Mol. Med. 7, 345-
350.
HIF-1 is a heterodimer composed of an inducibly-expressed HIF-1a subunit
and a constitutively-expressed HIF-1 P subunit (Epstein, 2001,Cell, 107, 43-
54). HIF-1 a
and HIF-1(3 mRNAs are constantly expressed under normoxic and hypoxic
conditions
(Wiener, 1996 Biochem. Biophys. Res. Commun. 225, 485-488). The unique feature
of
HIF-1 is the regulation of HIF-l a expression: it increases as the cellular 02
concentration
is decreased (Cramer, 2003, Cell, 112, 645-657, Pugh, 2003, Nat. Med. 9, 677-
84). During
normoxia, HIF-1 a is rapidly degraded by the ubiquitin proteasome system,
whereas
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exposure to hypoxic conditions prevents its degradation (Minchenko, 2002 J.
Biol. Chem.,
277, 6183-6187; Semenza, 2000, J. Appl. Physiol., 88, 1474-1480).
A growing body of evidence indicates that HIF-1 contributes to tumor
progression and metastasis (Hopfl, 2004, Am. J. Physiol. Regul. Integr. Comp.
Physiol. 286,
R608-23; Welsh, 2003, Curr. Cancer Drug Targets. 3, 391-405).
Immunohistochemical
analyses have shown that HIF-la is present in higher levels in human tumors
than in
normal tissues (Zhong, 2000, Cancer Res. 60, 1541-5). Tumor progression is
associated
with adaptation to hypoxia, and there is an inverse correlation between tumor
oxygenation
and clinical outcome (Pugh, 2003, Ann. Med. 35, 380-90.; Semenza, 2000 J.
Appl. Physiol.,
88, 1474-1480). In particular, the levels of HIF-1 activity in cells are
correlated with
tumorigenicity and angiogenesis in nude mice (Chen, 2003, Am. J. Pathol.
162,1283-91).
Tumor cells lacking HIF-1 expression are markedly impaired in their growth and
vascularization (Carmeliet, 1998, Nature 394, 485-90; Jiang, 1997, Cancer
Res., 57, 5328-
5335 ; Maxwell, 1997, Proc. Natl. Acad. Sci. US.A., 94, 8104-8109; Ryan, 1998
EMBO J.
17, 3005-3015). Therefore, since HIF-la expression and activity appear central
to tumor
growth and progression, HIF-1 inhibition has become an appropriate anticancer
target
(Kung, 2000, Nat. Med. 6, 1335-40).
3.3 DISEASES OF RELEVANCE
3.3.1 CANCER
A neoplasm, or tumor, is a neoplastic mass resulting from abnormal
uncontrolled cell growth which can be benign or malignant. Benign tumors
generally
remain localized. Malignant tumors are collectively termed cancers. The term
"malignant"
generally means that the tumor can invade and destroy neighboring body
structures and
spread to distant sites to cause death (for review, see Robbins and Angell,
1976, Basic
Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can
arise in many
sites of the body and behave differently depending upon its origin. Cancerous
cells destroy
the part of the body in which they originate and then spread to other part(s)
of the body
where they start new growth and cause more destruction.
More than 1.2 million Americans develop cancer each year. Cancer is the
second leading case of death in the United States and if current trends
continue, cancer is
expected to be the leading cause of death by the year 2010. Lung and prostate
cancer are
the top cancer killers for men in the United States. Lung and breast cancer
are the top
cancer killers, for women in the United States, One in two men in the United
States will be
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diagnosed with cancer at some time during his lifetime. One in three women in
the United
States will be diagnosed with cancer at some time during her lifetime.
A cure for cancer has yet to be found. Current treatment options, such as
surgery, chemotherapy and radiation treatment, are oftentimes either
ineffective or present
serious side effects.
Currently, cancer therapy may involve surgery, chemotherapy, hormonal
therapy and/or radiation treatment to eradicate neoplastic cells in a patient
(See, for
example, Stockdale, 1998, "Principles of Cancer Patient Management", in
Scientific
American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section
IV).
Recently, cancer therapy could also involve biological therapy or
immunotherapy. All of
these approaches pose significant drawbacks for the patient. Surgery, for
example, may be
contraindicated due to the health of the patient or may be unacceptable to the
patient.
Additionally, surgery may not completely remove the neoplastic tissue.
Radiation therapy
is only effective when the neoplastic tissue exhibits a higher sensitivity to
radiation than
normal tissue, and radiation therapy can also often elicit serious side
effects. Hormonal
therapy is rarely given as a single agent and although can be effective, is
often used to
prevent or delay recurrence of cancer after other treatments have removed the
majority of
the cancer cells. Biological therapies/immunotherapies are limited in number
and may
produce side effects such as rashes or swellings, flu-like symptoms, including
fever, chills
and fatigue, digestive tract problems or allergic reactions.
With respect to chemotherapy, there are a variety of chemotherapeutic agents
available for treatment of cancer. A significant majority of cancer
chemotherapeutics act by
inhibiting DNA synthesis, either directly, or indirectly by inhibiting the
biosynthesis of the
deoxyribonucleotide triphosphate precursors, to prevent DNA replication and
concomitant
cell division (See, for example, Gilman et al., Goodman and Gilman's: The
Pharmacological Basis of Therapeutics, Eighth Ed. (Pergamom Press, New York,
2001, lOm
ed.)). These agents, which include alkylating agents, such as nitrosourea,
anti-metabolites,
such as methotrexate and hydroxyurea, and other agents, such as etoposides,
campathecins,
bleomycin, doxorubicin, daunorubicin, etc., although not necessarily cell
cycle specific, kill
cells during S phase because of their effect on DNA replication. Other agents,
specifically
colchicine and the vinca alkaloids, such as vinblastine and vincristine,
interfere with
microtubule assembly resulting in mitotic arrest. Chemotherapy protocols
generally involve
administration of a combination of chemotherapeutic agents to increase the
efficacy of
treatment.
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Despite the availability of a variety of chemotherapeutic agents,
chemotherapy has many drawbacks (See, for example, Stockdale, 1998,
"Principles Of
Cancer Patient Management" in Scientific American Medicine, vol. 3, Rubenstein
and
Federman, eds., ch. 12, sect. 10). Alinost all chemotherapeutic agents are
toxic, and
chemotherapy causes significant, and often dangerous, side effects, including
severe nausea,
bone marrow depression, immunosuppression, etc. Additionally, even with
administration
of combinations of chemotherapeutic agents, many tumor cells are resistant or
develop
resistance to the chemotherapeutic agents. In fact, those cells resistant to
the particular
chemotherapeutic agents used in the treatment protocol often prove to be
resistant to other
drugs, even those agents that act by mechanisms different from the mechanisms
of action of
the drugs used in the specific treatment; this phenomenon is termed
pleiotropic drug or
multidrug resistance. Thus, because of drug resistance, many cancers prove
refractory to
standard chemotherapeutic treatment protocols.
There is a significant need for alternative cancer treatments, particularly
for
treatment of cancer that has proved refractory to standard cancer treatments,
such as
surgery, radiation therapy, chemotherapy, and hormonal therapy. A promising
alternative is
immunotherapy, in which cancer cells are specifically targeted by cancer
antigen-specific
antibodies. Major efforts have been directed at harnessing the specificity of
the immune
response, for example, hybridoma technology has enabled the development of
tumor
selective monoclonal antibodies (See Green M.C. et al., 2000 Cancer Treat
Rev., 26: 269-
286; Weiner LM, 1999 Semin Oncol. 26(suppl. 14):43-51), and in the past few
years, the
Food and Drug Administration has approved the first MAbs for cancer therapy:
However,
there is still an unmet need for the treatment of cancers.
In the current chemotherapeutic treatment of human cancer, side effects
associated with the chemotherapeutic agent are often severe. The use of
paclitaxel or
docetaxel (both taxane family medicaments) for the treatment of neoplastic
diseases is
limited by acute hypersensitivity reactions experienced in many patients. For
example,
docetaxel administered at 100 mg/m2 causes acute hypersensitivity reaction in
13% of
patients, and severe hypersensitivity reaction in 1.2%. Due to these
reactions, patients are
normally premedicated with oral corticosteroids.
Similarly, side effects associated with the use of vinca alkaloids often limit
the useful dosages. For example, vincristine has been reported to be dose
limited due to
neurotoxicity. An enhancing agent providing a neuro-protective effect is
therefore
desirable. Chemotherapeutic agents are also costly to produce and provide to
patients. If
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the agents can be used at reduced dosages, both the cost and the extent of
undesirable side
effects can be similarly reduced. Another often-encountered challenge with
chemotherapeutic treatment is limitations in effectiveness due to the
cancerous growth or
cells developing multidrug-resistance (MDR). As most cancer cells are
genetically unstable
they are prone to mutations likely to produce drug resistant cells.
Multi-drug resistance is the name given to the circumstance when a disease
does not respond to a treatment drug or drugs. MDR can be either intrinsic,
which means
the disease has never been responsive to the drug or drugs, or it can be
acquired, which
means the disease ceases responding to a drug or drugs that the disease had
previously been
responsive to MDR is characterized by cross-resistance of a disease to more
than one
functionally and/or structurally unrelated drugs. MDR in the field of cancer,
is discussed in
greater detail in "Detoxification Mechanisms and Tumor Cell Resistance to
Anticancer
Drugs," by Kuzmich and Tew, particularly section VII "The Multidrug-Resistant
Phenotype
(MDR)," Medical Research Reviews, Vol. 11, No. 2, 185-217, (Section VII is at
pp. 208-
213) (1991); and in "Multidrug Resistance and Chemosensitization: Therapeutic
Implications for Cancer Chemotherapy," by Georges, Sharom and Ling, Advances
in
Pharmacology, Vol. 21, 185-220 (1990).
Different MDR mechanisms have been reported. One form of multi-drug
resistance (MDR) is mediated by a membrane bound 170-180 kD energy-dependent
efflux
pump designated as pleitotropic-glycoprotein or P-glycoprotein (P-gp) that is
codified by
MDR-1 gene (Endicott JA, Annu Rev Biochem 1989). P-glycoprotein has been shown
to
play a major role in the intrinsic and acquired resistance of a number of
human tumors.
Drugs that act as substrates for and are consequently detoxified by P-gp
include the vinca
alkaloids (vincristine and vinblastine), anthracyclines (Adriamycin), and
epipodophyllotoxins (etoposide).
Recently, MDR-1 gene has been identified as an additional risk factor in
advanced ovarian cancer. In the study by D.S. Alberts et al., patients with
phase III ovarian
cancer were screened for MDR-1. Ovarian cancer patients with high levels of
MDR-1
survived an average of 9.8 months. The patients having low or no MDR-1
expression
survived an average of 30 months or more.
While P-gp associated MDR is a major factor in tumor cell resistance to
chemotherapeutic agents, it is clear that the phenomenon of MDR is
multifactorial and
involves a number of different mechanisms. One such alternative pathway for
resistance to
anthracyclines involves the emergence of a 190 kD protein (p190) that is not P-
gp. See, T.
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McGrath, et al., Biochemical Pharmacolology, 38:3611 (1989). The protein p190
is not
found exclusively on the plasma membrane but rather appears to be localized
predominantly
in the endoplasmic reticulum. See, e.g., Marquardt, 1992 .Cancer Research,
52:3157.
The work of Cole and Deeley isolated a single open reading frame encoding
a protein of 1531 amino acids designated as multidrug resistance-associated
protein (MRP).
As reported by Fan et al., the MRP protein is thought to be the same as the
190 kD protein.
MRP has been observed in breast cancer, human leukemia, small cell lung
cancer, human
large cell lung cancer, fibrosarcoma, adenocarcinoma, thyroid cancer, and
cervical cancer.
Chen reports that MRP is associated with multi-drug resistance to camptothecin
and its
analogs.
Other MDR has been reported that is neither P-gp nor MRP related. (Kellen
editor, Alternative Mechanisms of Multidrug Resistance in Cancer, Birkhauser,
1995). The
results obtained by inventors are consistent for using adenosine A3
antagonists for
countering P-gp or MRP multi-drug resistance, but not other forms of MDR.
a-[3-[[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-
a-(1-methylethyl)benzeneacetonitrile (Verapamil) has been utilized as a
medicament to
counter the effect of P-gp associated MDR. Verapamil blocks L-type calcium
channels and
is used as a potent vasodilator of coronary and peripheral vessels and
decreases myocardial
oxygen consumption. Due to the Verapamil physiological effects, MDR use of
Verapamil
must be limited to patients not having low blood pressure, congestive heart
failure,
sinoatrial (SA) or atrioventricular (AV) node conduction disturbances,
digitalis toxicity,
Wolff-Parkinson-White syndrome, and further not being medicated with beta-
blockers or
Quinidine.
Recognizing that P-gp is also adenosine-5'-triphosphate (ATP) dependent,
another proposed method of countering MDR is to inhibit ATP synthesis in the
cancerous
cells. U.S. Patent No. 6,210,917 to Carson et al. discloses the use of L-
alanosine and other
adenosine kinase inhibitors for countering MDR. In addition U.S. Patent No.
6,391,884
identifies ATP-depleting agents including 2-deoxyglucose, cyanine, oligomycin,
valinomycin and azide, as well as salts and derivatives thereof. Approaches
relying upon
ATP depletion or inhibition in countering MDR have yet to receive clinical
success.
As a result, it is seen that there is a need for chemotherapeutic agent
enhancers and compounds that prevent or counter MDR either alone or with ATP
depleting
agents in cancer treatment but without the limitations imposed by Verapamil.
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The use of a combination of therapeutic agents is common in the treatment of
neoplastic diseases. For example, paclitaxel (TaxolTM) has been approved by
the U.S. FDA
for use with cisplatin in the treatment of ovarian carcinoma. U.S. Patent No.
5,908,835 to
Bissery et al. claims synergy of using paclitaxel or docetaxel in combination
with an
anthracycline antibiotic such as daunorubicin or doxorubicin. Similarly, U.S.
Patent No.
5,728,687 to Bissery et al. claims synergy of using paclitaxel or docetaxel in
combination
with an alkylating agent, epidophyllotoxin, antimetabolite, or vinca alkaloid.
However,
such combinations heretofore have not included the use of adenosine receptor
antagonists
and in particular A3 receptor antagonists.
It is therefore an object of the present invention to provide compositions and
methods of enhancing the treatment of neoplastic cells by minimizing or
eliminating the
protective effect of adenosine on cells with the use of adenosine A3 receptor
antagonists. It
is a further object of the present irivention to provide compositions and
methods suitable for
countering P-gp and/or MRP associated multi-drug resistance. It is further an
object of the
present invention to provide compositions and methods that reduce side effects
of other
currently used anti-cancer agents and/or therapies, in particular taxane
induced
hypersensitivity.
4. SUMMARY OF THE INVENTION
The present invention is based, in part, on the surprising discovery by the
inventors that adenosine, a purine nucleoside present within hypoxic regions
of solid
tumors, modulates hypoxia-inducible factor 1(HIF-1) expression. HIF-l, a
heterodimeric
transcription factor composed of HIF-la and HIF-1(3 subunits, is involved in
tumor growth
and angiogenesis (for reviews see, e.g., Semenza 2000, Nature, 3: 721-32). The
inventors
have found that in the human A375 melanoma cell line adenosine up-regulates
HIF-1a
protein expression in response to hypoxia in a dose- and time-dependent
manner. The
response to adenosine was not blocked by Al, A2A or A2B receptor antagonists,
while it was
abolished by A3 receptor antagonists. The inventors have found that Cl-IB-
MECA, an
adenosine analogue binding with high affinity to A3 receptors, mimicked
adenosine effect in
hypoxic cells. Furthermore, A3 receptor antagonists prevented HIF- l a protein
accumulation in response to A3 receptor stimulation. Although not intending to
be bound
by a particular mechanism of action, the response to adenosine is generated at
the cell
surface since the inhibition of A3 receptor expression, by using small
interfering RNA,
abolished the nucleoside effects. The main intracellular signaling pathways
sustained by A3
receptor stimulation in hypoxia involve p44/p42 mitogen-activated protein
kinase (MAPK)
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and p38 MAPK activation. The inventors have thus found for the first time that
adenosine
plays a critical role in HIF-1a regulation in hypoxia.
The inventors have found that compounds which are antagonists of the
adenosine receptors, preferably the A3 receptor, inhibit the protective effect
of adenosine on
growing tumor cells when such cells are starved of oxygen (i.e., before an
adequate
vasculature is developed, or when anti-angiogenesis agents are administered.
Although not
intending to be bound by a particular mechanism of action, the antagonists of
the invention
can bind in the same site as adenosine, or can be allosteric antagonists
(i.e., bind at a site
different from where adenosine binds, but adversely affect the ability of
adenosine to bind
to the site or adversely affect the ability of adenosine, once bound to the
adenosine
receptors, particularly A3 receptors, to protect growing tumor cells).
Accordingly, the present invention relates to methods for the treatment,
prevention, and/or management of diseases or disorders associated with
overexpression of
HIF-1a and/or increased HIF-1a activity (e.g., cancer, respiratory disorders
such as asthma
and obstructive pulmonary disorders) by using adenosine receptor antagonists,
particularly
A3 receptor antagonists alone or in combination with Al, A2A, A2B receptor
antagonists.
Although the Ai, A2A and A2B receptor antagonists may act through different
pathways than
the A3 receptor antagonist, the combination of these antagonists with A3
receptor antagonist
may be beneficial in the treatment, prevention, and/or management of diseases
or disorders,
e.g., cancer.
In most preferred embodiments, the methods of the invention relate to
treatment, prevention, and/or management of diseases or disorders associated
with
overexpression of HIF- 1 a and/or increased HIF-1 a activity by using
adenosine receptor
antagonists, particularly A3 receptor antagonists alone. Without being bound
to a particular
mechanism of action, administration of antagonists for the adenosine
receptors, particularly
A3 receptor antagonists, antagonizes the protective effects against hypoxia
and renders those
cells susceptible to destruction due to hypoxia. Since the adenosine
receptors, in particular,
the A3 receptor, are responsible for sustained cellular protection against
ischemia,
antagonists for the adenosine receptors, particularly A3 receptors are
particularly effective in
enhancing the activity of anti-tumor agents.
The methods and compositions of the invention comprising A3 receptor
antagonists are particularly useful when the levels of HIF-la expression
and/or activity are
elevated above the standard or background level, as determined using methods
known to
those skilled in the art and disclosed herein. As used herein, "elevation" of
a measured
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level of HIF-1 a relative to a standard level means that the amount or
concentration of HIF-
1a in a sample or subject is greater in a subject or sample relative to the
standard as
detected by any method now known in the art or to be developed in the future
for measuring
HIF-1 a levels. For example, elevation of the measured level relative to a
standard level
may be any statistically significant detectable elevation. Such an elevation
in HIF- l a
expression and/or activity may include, but is not limited to about a 10%,
about a 20%,
about a 40%, about an 80%, about a 2-fold, about a 4-fold, about an 10-fold,
about a 20-
fold, about a 50-fold, about a 100-fold, about a 2 to 20 fold, 2 to 50 fold, 2
to 100 fold, 20 to
50 fold, 20 to 100 fold, elevation, relative to the standard. The term "about"
as used herein,
refers to levels of elevation of the standard numerical value plus or minus
10% of the
numerical value.
The therapeutic methods of the invention comprising administering a
therapeutically effective amount of an A3 receptor antagonist of the invention
improve the
therapeutic efficacy of treatment for diseases or disorders associated with
overexpression of
HIF-1a and/or increased HIF-la activity (e.g., cancer, respiratory disorders
such as asthma
and obstructive pulmonary disorders) relative to the traditional modes of such
therapies.
Preferably the methods of the invention reduce the HIF-la level to the
background level
within one day, one week, 1 month, or 2 months, of the commencement of the
therapeutic
regime. In a most preferred embodiment, the methods of the invention result in
a reduction
of HIF-la level to the background level. The invention encompasses reduction
of the HIF-
l a level to a level which is within about 10%, about 20%, about 30%, about
40%, about
50% of the background level; about a 2-fold, about a 4-fold, about an 10-fold,
about a 20-
fold, about a 50-fold, about a 100-fold, about a 2 to 20 fold, 2 to 50 fold, 2
to 100 fold, 20 to
50 fold, 20 to 100 fold of the background level. Preferably, once the methods
of the
invention reduce the level of HIF- 1 a to a particular level, that level is
maintained during the
treatment regimen, such that the maintained level of HIF-1 a is sufficient and
effective to
result in regression of the disease, e.g., cancer.
In a preferred specific embodiment, the invention encompasses a method for
treatment, prevention and/or management of diseases or disorders associated
with
overexpression of HIF-la andlor increased HIF-la activity (e.g., cancer,
respiratory
disorders such as asthma and obstructive pulmonary disorders) comprising
administering a
therapeutically and/or prophylactically effective amount of an A3 receptor
antagonist
compound as disclosed herein alone or in combinationo with other therapeutic
or
prophylatic agents.
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The invention also encompasses a method for determining the prognosis of a
a disease or disorder associated with overexpression of HIF-1 a and/or
increased HIF-1 a
activity in a subject. Preferably, the subject is human. In yet another
embodiment, the
subject has been previously treated with a therapy regimen. The invention
encompasses
measuring a level of HIF-1 a in a subject to determine if the subject is in
need of the
therapeutic and or prophylactic methods of the inventon. The invention
encompasses
measuring a level of HIF-1 a in a sample obtained from the subject and
comparing the level
measured to a standard level, wherein elevation of the measured level of at
least one HIF-
1 a relative to the standard level indicates that the subject is at an
increased risk for
progression of the disease or disorder, e.g., metastasis of the cancer. The
HIF-1a level may
be measured using at least one of the methods for measuring and detecting HIF-
1 a such as
those known to those skilled in the art and exemplified herein.
The invention encompasses compounds which are A3 or Al receptor
antagonists for use in the methods of the invention. Examples of such
compounds are
disclosed in U.S. Patent Nos 6,326,390; 6,407,236; 6,448,253; 6,358,964; and
U.S.
Publication Nos. 2003/0144266 and 2004/0067932; all of which are incorporated
herein by
reference in their entireties. Furthermore; additional examples of such
compounds are
disclosed in the list of references in Table 1, the disclosures of which are
hereby
incorporated by reference in their entireties.
Table 1. A3 Receptor Antagonists
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US 6,288,070
US 5,573,772
WO 200350241 A2-A3
WO 200285308 A2-A3
WO 200283152 Al
WO 200270532 A2-A3
WO 200266020 A2-A3
WO 200257267 Al
WO 200139777 Al
WO 200051621 Al
WO 200003741 A2
WO 9962518 Al
WO 9920284 Al
WO 9421195 Al
JP 2004135657 A
The A3 receptor antagonists of the invention are particularly useful for
prevention, treatment, and/or management of cancer, for example, as a single
agent therapy
or in combination with other modes of therapy for cancer. In preferred
embodiments, the
invention encompasses methods of treatment, prevention, or management of
cancers which
are A3 receptor expressing cancers including, but not limited to, human
leukemia,
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WO 2007/040565 PCT/US2005/042551
melanoma, pancreatic carcinoma, ovarian carcinoma, breast carcinoma, prostrate
carcinoma, colon carcinoma, lung carcinoma, malignant melanomas, histiocytic
lymphoma,
and some fonns of astrocytoma cells. In other preferred embodiments, the
invention
encompasses methods of treatment, prevention, or management which have high
level of
expression of HIF-1 a including, but not limited to, cervical cancer (early
stage), lung cancer
(non-small cell lung carcinoma), breast cancer (including lymph node positive
breast cancer
and lymph node negative breast cancer), oligodendroglioma, orpharyngeal
squamous cell
carcinoma, ovarian cancer, oesophageal cancer, endometrial cancer, head and
neck cancer,
gastrointestinal stromal tumor of the stomach The methods of the invention are
particularly
useful in cancer therapy for providing tissue selectivity, such that the
biological effect is
observed in tumoral hypoxic cells, where high adenosine concentrations has
resulted in
increased HIF-la accumulation.
The methods and compositions of the invention comprising A3 receptor
antagonists (or Al receptor antagonists or a combination thereof) are
particularly useful for
the treatment, inhibition or regression of solid tumors. As used herein,
"solid tumors" refer
to a locus of tumor cells where the majority of the cells are tumor cells or
tumor-associated
cells, including but not limited to laryngeal tumors, brain tumors, and other
tumors of the
head and neck; colon, rectal and prostate tumors; breast and thoracic solid
tumors; ovarian
and uterine tumors; tumors of the esophagus; stomach, pancreas and liver;
bladder and gall
bladder tumors; skin tumors such as melanomas. Moreover, the tumors
encompassed
within the invention can be either primary or a secondary tumor resulting from
metastasis of
cancer cells elsewhere in the body to the chest.
The invention encompasses use of the A3 or Al receptor antagonist
compounds of the invention in inhibiting tumor growth in a mammal, including
humans.
The invention encompasses methods for administering an effective amount of
adenosine Al
or A3 antagonists, preferably, A3 antagonists sufficient to inhibit the
ability of adenosine to
protect tumor cells.
In other embodiments, the present invention encompasses methods for
treatment, prevention and/or management of cancer comprising administering a
compound
of the invention including A3 and/or Al receptor antagonists used in
combination with other
therapeutic and/or prophylactic agents, e.g., cytotoxic agents. The inventors
have
surprisingly found that adenosine A3 receptor antagonists synergistically
enhance cytotoxic
treatment and counter some forms of multi-drug resistance. Although not
intending to be
bound by a particular mechanism of action, the combination therapies of the
invention will
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attack the tumor cell directly, inhibit growth of new blood vessels around the
tumor cell,
and, by virtue of the adenosine A3 antagonists, inhibit the ability of the
cell to survive
without the growth of new blood vessels. Inventors have discovered that high
affinity
adenosine A3 receptor antagonists are useful as enhancers for many
chemotherapeutic
treatments of adenosine A3 receptor expressing cancers. Surprisingly, high
affinity
adenosine A3 receptor antagonists also counter P-glycoprotein (P-gp) effuse
pump multi-
drug resistance (MDR). Finally, high affinity adenosine A3 receptor
antagonists are helpful
in reducing or ameliorating side effects of cytoxic agents especially taxane
induced
hypersensitivity. For example, lower doses of taxane may be used once used in
combination with an adenosine A3 receptor antagonist of the invention.
In other embodiments, the present invention encompasses the use of high
affinity adenosine A3 receptor antagonists for enhancing chemotherapeixtic
treatment of
cancers expressing adenosine A3 receptors and countering multi-drug resistance
in cancers
expressing P-glycoprotein or MRP. In preferred embodiments, adenosine A3
receptor
antagonists are administered before or during administration of a taxane
family, vinca
alkaloid, camptothecin or antibiotic compound. Preferred high affinity A3
receptor
antagonists include compounds of the general formulas IIA, IIB, IIC and IID
described
herein, vide infi a.
The present invention encompasses therapies which involve administering
one or more of the compounds of the invention, to an animal, preferably a
mammal, and
most preferably a human, for preventing, treating, or ameliorating symptoms
associated
with a cancer, a disease or disorder associated with hypoxia-inducible factor
1-a (HIF-1a).
The invention further provides a pharmaceutical composition comprising a
therapeutically or prophylactically effective amount of a compound of the
invention that
specifically binds an Al or A3 receptor and a pharmaceutically acceptable
carrier.
Preferably the pharmaceutical formulation includes an adenosine A3 receptor
antagonist and
one or more excipients. The formulations can also include other anti-tumor
agents,
including cytotoxic agents and other anti-angiogenesis agents, including
adenosine A2A
antagonists, sucli as ZM241385 and SCH 5861, adenosine A2B antagonists, such
as MRE-
20290-F20 and AS-16, and anti-VEGF antibodies, including humanized and
chimeric
antibodies. In a preferred embodiment, the composition includes an effective
amount to
inhibit tumor growth of an adenosine A3 receptor antagonist, a cytotoxic
agent, and an anti-
angiogenesis agent.
4.1 DEFINITIONS
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As used herein, a compound is an "antagonist of an adenosine A3 receptor" if
it is able to prevent the action due to an agonist and is able to displace
[I21 I]-AB-MECA in
a competitive binding assay.
As used herein, a compound is selective for the A3 receptor if its affinity at
the A3 receptor is greater than its affinity at the Al, A2A and A2B receptors.
Preferably, the
ratio of Al /A3 and A2 /A3 activity is greater than about 50, preferably
between 50 and 100,
and more preferably, greater than about 100. Since the pharmacology at the A3
receptor
varies between species, especially between rodent A3 and human A3 receptors,
it is
important to determine the selectivity of the A3 compounds in human adenosine
receptors.
The same holds true for adenosine Al and A2A receptors in terms of whether
they are
selective.
As used herein "Adenosine Al Antagonists" carry their common ordinary
meaning. Adenosine Al antagonists are well known to those of skill in the art.
Some of the
more well known compounds include CPX, CVT 124, FK 352, and ILF 117, the
structures
of which are well known.
As used herein "Adenosine A3 Antagonists" carry their common ordinary
meaning. Adenosine A3 antagonists are well known to those of skill in the art.
Some of the
more well known compounds include MRS 1097, MRS 1191 and MRS 1220
(commercially
available from Research Biochemicals International, Natick Mass.). Other
suitable
antagonists include those disclosed in Jacobson et al., Neuropharmacology,
36:1157-1165
(1997); Yao et al., Biochem. Biophys. Res. Commun., 232:317-322 (1997); Kim et
al., J.
Med. Chem., 39(21):4142-4148 (1996); van Rhee et al., Drug Devel. Res., 37:131
(1996);
van Rhee et al., J. Med. Chem., 39:2980-2989 (1996); Siquidi et al.,
Nucleosides,
Nucleotides 15:693-718 (1996); van Rhee et al., J. Med. Chem., 39:398-406
(1996);
Jacobson et al., Drugs of the Future, 20:689-699 (1995); Jacobson et al., J.
Med. Chem.,
38:1720-1735 (1995); Karton et al., J. Med. Chem., 39:2293-2301 (1996); Kohno
et al.,
Blood, 88:3569-3574 (1996); Jiang et al., J Med. Chem., 39:4667-4675 (1996);
Yao et al.,
Biochem. Biophys. Res. Commun. 232:317-322 (1997); and Jiang et al., J. Med.
Chem.
40:2596-2608 (1996), the contents of which are hereby incorporated by
reference in their
entireties. In addition, other suitable adenosine A3 antagonists are disclosed
in the
references listed in Table 1, the disclosures of which are hereby incorporated
by reference in
their entireties.
As used herein, "elevation" of a measured level of HIF-1 a or A3 receptor
relative to a standard level means that the amount or concentration of HIF-1 a
or A3
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receptor in a sample or subject is sufficiently greater in a subject or sample
relative to the
standard as detected by any method now known in the art or to be developed in
the future
for measuring HIF-1a or A3 receptor levels. For example, elevation of the
measured level
relative to a standard level may be any statistically significant elevation
detectable. Such an
elevation in HIF-1 a expression and/or activity may include, but is not
limited to about a
10%, about a 20%, about a 40%, about an 80%, about a 2-fold, about a 4-fold,
about an 10-
fold, about a 20-fold, about a 50-fold, about a 100-fold, about a 2 to 20
fold, 2 to 50 fold, 2
to 100 fold, 20 to 50 fold, 20 to 100 fold, elevation, relative to the
standard. The term
"about" as used herein, refers to levels of elevation of the standard
numerical value plus or
minus 10% of the numerical value.
The term "standard level" or "background level" as used herein refers to a
baseline amount of an HIF-la level as determined in one or more normal
subjects, i.e., a
subject with no known history of past or current diseases, disorders or
cancer. For example,
a baseline may be obtained from at least one subject and preferably is
obtained from an
average of subjects (e.g., n=2 to 100 or more), wherein the subject or
subjects have no prior
history of diseases, disorders or cancer, especially no prior history of
diseases associated
with an aberrant level of expression and/or activity of HIF-1 a. In the
present invention, the
measurement of HIF-la level may be carried out using an HIF-1a probe or a HIF-
1 a activity assay (see Section 6).
As used herein, "enhancement" refers to a synergistic effect as determined
from measurement of the enhancement factor, as defined below. In general, an
enhancement factor of greater than one is considered synergistic. For example,
if one of the
compounds has little individual chemotherapeutic effect, an enhancement factor
greater than
one indicates a synergistic effect is occurring.
As used herein, the term "cancer" refers to a neoplasm or tumor resulting
from abnormal uncontrolled growth of cells. As used herein, cancer explicitly
includes,
leukemias and lymphomas. The term "cancer" refers to a disease involving cells
that have
the potential to metastasize to distal sites and exhibit phenotypic traits
that differ from those
of non-cancer cells, for example, formation of colonies in a three-dimensional
substrate
such as soft agar or the formation of tubular networks or weblike matrices in
a three-
dimensional basement membrane or extracellular matrix preparation. Non-cancer
cells do
not form colonies in soft agar and form distinct sphere-like structures in
three-dimensional
basement membrane or extracellular matrix preparations. Cancer cells acquire a
characteristic set of functional capabilities during their development, albeit
through various
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mechanisms. Such capabilities include evading apoptosis, self-sufficiency in
growth
signals, insensitivity to anti-growth signals, tissue invasion/metastasis,
limitless replicative
potential, and sustained angiogenesis. The term "cancer cell" is meant to
encompass both
pre-malignant and malignant cancer cells. In some embodiments, cancer refers
to a benign
tumor, which has remained localized. In other embodiments, cancer refers to a
malignant
tumor, which has invaded and destroyed neighboring body structures and spread
to distant
sites. In yet other embodiments, the cancer is associated with a specific
cancer antigen.
As used herein, the term "A3 expressing cancers" refers to human cancers
that express the adenosine A3 receptor or that otherwise comprise elevated
concentrations of
adenosine A3 receptors. Elevated concentration is determined by comparison to
normal,
non-cancerous tissues of a similar cell type. Examples of A3 expressing
cancers include,
without limitation, human leukemia, melanoma, pancreatic carcinoma, breast
carcinoma,
prostrate carcinoma, colon carcinoma, lung carcinomamalignant melanomas,
histiocytic
lymphoma, and some forms of astrocytoma cells.
As used herein the term HIF- 1 a expressing cancers refers to human cancer
that express HIF-1a or that otherwise comprise elevated concentrations of HIF-
la.
Elevated concentration is determined by comparison to normal, non-cancerous
tissues of a
similar cell type. Examples of HIF-1 a expressirig cancers include without
limitation
cervical cancer (early stage), melanoma, lung cancer (non-small cell lung
carcinoma), breast
cancer (including lymph node positive breast cancer and lymph node negative
breast
cancer), oligodendroglioma, orpharyngeal squamous cell carcinoma, ovarian
cancer,
oesophageal cancer, endometrial cancer, head and neck cancer, gastrointestinal
stromal
tumor of the stomach.
As used herein "hypoxia" or "ischemia" refers to any condition whereby the
physiology of the tissue is compromised and/or blood supply in one or more
tissues is
compromised. These two terms may be used interchangeably. The condition also
encompasses any reduction in partial pressure of 02 in one or more tissues.
The term
"hypoxia" or "ischemia" encompasses any condition in which a cell, tissue or
organ
experiences a lack of oxygen or reduced blood flow.
As used herein, the terms "treat," "treating" and "treatment" refer to the
eradication, removal, modification, or control of primary, regional, or
metastatic cancer
tissue that results from the administration of one or more therapeutic agents.
In certain
embodiments, such terms refer to the minimizing or delaying the spread of
cancer resulting
from the administration of one or more therapeutic agents to a subject with
such a disease.
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The term also refers to the eradication, reduction or amelioration of a
symptom or disorder
related to aberrant levels of HIF-1 a level and/or activity.
As used herein, a "therapeutically effective amount" refers to that amount of
the therapeutic agent sufficient to delay or minimize the spread of cancer. A
therapeutically
effective amount may also refer to the amount of the therapeutic agent that
provides a
therapeutic benefit in the treatment or management of a disease or disorder.
Further, a
therapeutically effective amount with respect to a therapeutic agent of the
invention means
that amount of therapeutic agent alone, or in combination with other
therapies, that provides
a therapeutic benefit in the treatment or management of a disease or disorder.
Used in
connection with an amount of a compound of the invention, the term can
encompass an
amount that improves overall therapy, reduces or avoids unwanted effects, or
enhances the
therapeutic efficacy of or synergizes with another therapeutic agent.
As used herein, the terms "prophylactic agent" and "prophylactic agents"
refer to any agent(s) which can be used in the prevention of a disorder, or
prevention of
recurrence or spread of a disorder. A prophylactically effective amount may
refer to the
amount of prophylactic agent sufficient to prevent the recurrence or spread of
hyperproliferative disease, particularly cancer, or the occurrence of such in
a patient,
including but not limited to those predisposed to hyperproliferative disease,
for example
those genetically predisposed to cancer or previously exposed to carcinogens.
A
prophylactically effective amount may also refer to the amount of the
prophylactic agent
that provides a prophylactic benefit in the prevention of disease. Further, a
prophylactically
effective amount with respect to a prophylactic agent of the invention means
that amount of
prophylactic agent alone, or in combination with other agents, that provides a
prophylactic
benefit in the prevention of disease. Used in connection with an amount of a
compound of
the invention, the term can encompass an amount that improves overall
prophylaxis or
enhances the prophylactic efficacy of or synergizes with another prophylactic
agent, such as
but not limited to a therapeutic antibody.
As used herein, the terms "manage," "managing" and "management" refer to
the beneficial effects that a subject derives from administration of a
prophylactic or
therapeutic agent, which does not result in a cure of the disease or diseases.
In certain
embodiments, a subject is administered one or more prophylactic or therapeutic
agents to
"manage" a disease so as to prevent the progression or worsening of the
disease or diseases.
As used herein, the terms "prevent", "preventing" and "prevention" refer to
the methods to avert or avoid a disease or disorder or delay the recurrence or
onset of one or
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more symptoms of a disorder in a subject resulting from the administration of
a prophylactic
agent.
As used herein, the term "in combination" refers to the use of more than one
prophylactic and/or therapeutic agents. The use of the term "in combination"
does not
restrict the order in which prophylactic and/or therapeutic agents are
administered to a
subject with a disorder, e.g., hyperproliferative cell disorder, especially
cancer. A first
prophylactic or therapeutic agent can be administered prior to (e.g., 1
minute, 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8 weeks,
or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5
minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8 weeks,
or 12 weeks after) the administration of a second prophylactic or therapeutic
agent to a
subject which had, has, or is susceptible to a disorder. The prophylactic or
therapeutic
agents are administered to a subject in a sequence and within a time interval
such that the
agent of the invention can act together with the other agent to provide an
increased benefit
than if they were administered otherwise. Any additional prophylactic or
therapeutic agent
can be administered in any order with the other additional prophylactic or
therapeutic
agents.
As used herein, the term "alkyl" refers to a monovalent straight, branched or
cyclic saturated hydrocarbon group preferably having from 1 to 20 carbon
atoms, more
preferably 1 to 10 carbon atoms ("lower alkyl") and most preferably 1 to 6
carbon atoms.
This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-
propyl, -butyl, iso-
butyl, n-hexyl, and the like. The terms "alkylene" and "lower alkylene" refer
to divalent
radicals of the corresponding alkane. Further, as used herein, other moieties
having names
derived from alkanes, such as alkoxyl, alkanoyl, alkenyl, cycloalkenyl, etc.
when modified
by "lower," have carbon chains of ten or less carbon atoms. In those cases
where the
minimum number of carbons are greater than one, e.g., alkenyl (minimum of two
carbons)
and cycloalkyl, (minimum of three carbons), it is to be understood that
"lower" means at
least the minimum number of carbons.
As used herein, the term "substituted alkyl" refers to an alkyl group,
preferably of from 1 to 10 carbon atoms ("substituted lower alkyl"), having
from 1 to 5
substituents, and preferably 1 to 3 substituents, selected from the group
consisting of
alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
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cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino aminoacyl,
aminoacyloxy,
oxyacylamino, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,
carboxylalkyl, thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
heterocyclic,
hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -
SO-
heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-heteroaryl,
and mono-
and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-
arylamino, mono-
and di-heteroarylamino, mono- and di-heterocyclic amino, and unsymmetric di-
substituted
amines having different substituents selected from alkyl, aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic. As used
herein, other
moieties having the prefix "substituted" are intended to include one or more
of the
substituents listed above.
As used herein, "aralkyl" refers to an alkyl group with an aryl substituent.
Binding is through the alkyl group. "Alkaryl" refers to an aryl group with an
alkyl
substituent, where binding is through the aryl group.
As used herein, the term "alkoxy" refers to the group "alkyl-O-", where
alkyl is as defined above. Preferred alkoxy groups include, by way of example,
methoxy,
ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,
n-hexoxy,
1,2-dimethylbutoxy, and the like.
As used herein, the term "alkenyl" refers to an unsaturated, straight or
branched hydrocarbon group preferably having from 2 to 10 carbon atoms and
more
preferably 2 to 6 carbon atoms and having at least I and preferably from 1-2
double bonds.
Preferred alkenyl groups include ethenyl (-CH=CH2), n-propenyl (-CH2 CH=CH2),
iso-
propenyl (-C(CH3)=CH2), and the like.
As used herein, the term "alkynyl" refers to an unsaturated, straight or
branched hydrocarbon group preferably having from 2 to 10 carbon atoms and
more
preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2
triple bonds.
As used herein, the term "acyl" refers to the groups alkyl-C(O)-, substituted
alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-,
substituted
aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted
alkyl,
cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as
defined herein.
As used herein, the term "aminoacyl" refers to the group -C(O)NR33R33
where each R33 is independently hydrogen, alkyl, substituted alkyl, aryl,
substituted aryl,
heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl
and heterocyclic
are as defined herein.
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As used herein, the term "acylamino" refers to the group R34-C(O)-
N(R33)- where each R34 is independently alkyl, substituted alkyl, cycloalkyl,
substituted
cycloalkyl, aryl, substituted aryl, heteroaryl, or heterocyclic wherein alkyl,
substituted alkyl,
cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as
defined herein.
R33 is independently hydrogen, alkyl, substituted alkyl, aryl, substituted
aryl, heteroaryl, or
heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and
heterocyclic are as defined
herein.
As used herein, the term "acyloxy" refers to the group R34--C(O) - where
each R34 is independently alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, aryl,
substituted aryl, heteroaryl, or heterocyclic wherein alkyl, substituted
alkyl, cycloalkyl, aryl,
heteroaryl and heterocyclic are as defined herein.
As used herein the term "amino acid" means an alpha amino acid selected
from those amino acids which naturally occur in proteins but without regard
for specific
stereochemical properties. The term "protected amino acid" means an amino acid
of which
the alpha amino group has been converted to a less reactive moiety, but a
moiety which can
be converted back to the amino group with relative ease. The terms "amino acid
residue"
and "amino acid moiety" are used synonymously herein.
As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclic
group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or
multiple
condensed (fused) rings (e.g., naphthyl or anthryl). Preferred aryls include
phenyl, naphthyl
and the like. Unless otherwise constrained by the definition for the aryl
substituent, such
aryl groups can optionally be substituted with from 1 to 5 substituents and
preferably 1 to 3
substituents selected from the group consisting of hydroxy, acyl, alkyl,
alkoxy, alkenyl,
alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl,
substituted alkynyl,
amino, substituted amino, aminoacyl, acyloxy, acylamino, alkaryl, aralkyl,
aryl, aryloxy,
azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,
heterocyclic,
heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted
thioalkoxy,
thioaryloxy, thioheteroaryloxy, alkylthio, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-SO-heteroaryl, -S02-alkyl, -SO2 substituted alkyl, -SO2-aryl, -SO2 -
heteroaryl,
trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano,
nitro,
trihalomethyl, and thioalkoxy.
As used herein, the term "cycloalkyl" refers to cyclic alkyl groups of from 3
to 12 carbon atoms having a single cyclic ring or multiple condensed rings.
Such cycloalkyl
groups include, by way of example, single ring structures such as cyclopropyl,
cyclobutyl,
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cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as
adamantyl, and the
like.
As used herein, the terms "halo" or "halogen" refer to fluoro, chloro, bromo
and iodo and preferably is either fluoro or chloro.
As used herein, the term "heteroaryl" refers to an aromatic carbocyclic group
of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of
oxygen, nitrogen and sulfur within at least one ring (if there is more than
one ring).
Unless otherwise constrained by the defmition for the heteroaryl substituent,
such heteroaryl groups can be optionally substituted with from 1 to 5
substituents and
preferably 1 to 3 substituents selected from the group consisting of hydroxy,
acyl, alkyl,
alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted
alkenyl,
substituted alkynyl, amino, substituted amino, aminoacyl, acyloxy, acylamino,
alkaryl, aryl,
aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl,
heteroaryloxy,
heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,
substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-SO-heteroaryl, -S02-alkyl, -S02-substituted alkyl, -S02-aryl, -S02-
heteroaryl, and
trihalomethyl. Preferred substituents include alkyl, alkoxy, halo, cyano,
nitro,
trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a single ring
(e.g., pyridyl
or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or
unsaturated carbocyclic group having a single ring or multiple condensed
rings, from 1 to
15 carbon atoms and from 1 to 4 hetero atoms selected from the group
consisting of
nitrogen, sulfur and oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic
substituent, such heterocyclic groups can be optionally substituted with 1 to
5 substituents
selected from the group consisting of alkyl, substituted alkyl, alkoxy,
substituted alkoxy,
aryl, aryloxy, halo, nitro, heteroaryl, thiol, thioalkoxy, substituted
thioalkoxy, thioaryloxy,
trihalomethyl, and the like. Such heterocyclic groups can have a single ring
or multiple
condensed rings.
As to any of the above groups that contain one or more substituents, it is
understood, of course, that such groups do not contain any substitution or
substitution
patterns which are sterically impractical and/or synthetically non-feasible.
As used herein, "carboxylic acid derivatives" and "sulfonic acid derivatives"
refer to -C(X)R31, -C(X)-N(R31)z,, -C(X)OR31, -C(X)SR3', -SObR31, -SObOR31,
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-SObSR31, or SOb N(R31)Z, where X is 0, S or NR31, where R31 is hydrogen,
alkyl,
substituted alkyl or aryl, and activated derivatives thereof, such as
anhydrides, esters, and
halides such as chlorides, bromides and iodides.
"Pharmaceutically acceptable salts" refers to pharmaceutically acceptable
salts of a compound of Formulas IIA, IIB, IIC and IID which salts are derived
from a
variety of organic and inorganic counter ions well known in the art and
include, by way of
example only, sodium, potassium, calcium, magnesium, ammonium,
tetraalkylarnmonium,
and the like; and when the molecule contains a basic functionality, salts of
organic or
inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate,
acetate, maleate,
oxalate and the like can be used as the pharmaceutically acceptable salt.
5. BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A through 1H illustrate the saturation of [3H]-MRE 3008-F20
binding to A3 adenosine receptors in human cancers. KD and Bmax values are
reported in
Table 8. Values are the means and S.E. of the mean of three separate
experiments
performed in triplicate. In the inset the Scatchard plot of the same data is
shown.
Figure 2 illustrates colony formation assay of A375 cells. Cells were treated
with different drugs and colonies were counted after 7 days. The values
represent the mean
=L SEM of four independent experiments. D= DMSO (control); T= paclitaxel, 0.75
ng/ml;
M = MRE 3008F20, 10 M; C = CI-IB-MECA, 10 M; TM = paclitaxel plus MRE
3008F20; TC = paclitaxel plus CI-IB-MECA treated cells. *P<0.01 TM versus T.
Analysis
was by ANOVA followed by Dunnett's test.
Figure 3A through 3D illustrate typical dose response curves of A375 cells
exposed to increasing concentrations of cytotoxic agents vindesine (Figure 3A,
Figure 3B)
and Taxo1TM brand paclitaxel (Figure 3C, Figure 3D). The curves with open
symbols
represent the cytotoxic agent alone. The curves with closed symbols represent
the cytotoxic
agent in the presence of 10 gM MRE 3008F2010. Figure 3A and Figure 3C
illustrate
G2/M phases arrest calculated as percentage of untreated cells (control).
Figure 3B and
Figure 3D illustrate the accumulation (percentage of total living cells) of
the sub-Gl
(apoptosis) population. Cells were fixed in 70% ethanol, stained with PI, and
analysed by
flow cytometry.
Figure 4A through 4F illustrate induction of G2/M phases arrest by MRE
3008F20 on exponentially growing A375 cells treated with paclitaxel or with
vindesine.
A375 cells are treated for 24 hours with drug-vehicle as control (Figure 4A,
Figure 4D);
with 1 nM vindesine (Figure 4B), with 25 ng/ml paclitaxel (Figure 4E); with 1
nM
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vindesine plus 10 M MRE 3008F20 (Figure 4C); or with 25 ng/ml paclitaxel plus
10 M
MRE 3008F20 (Figure 4F). Cells were fixed in 70% ethanol, stained with PI, and
analysed
by flow cytometry. The percentage of cells at Gi, S and G2/M phases was
quantified.
Apoptotic cells (Apo) were also detected.
Figure 5A illustrates dose-response curve for G2/M phases arrest of A375
cells exposed to increasing concentrations of A3 adenosine receptor
antagonists in the
presence of 1 nM vindesine. Cells were fixed in 70% ethanol, stained with PI,
and analysed
by flow cytometry.
Figure 5B illustrates comparison between enhancing activity to vindesine by
adenosine receptor antagonists (SEC50) and binding affinity to A3 adenosine
receptors (Kj)
in A375 cells (r = 0.96; P<0.01).
Figure 6A through 6D illustrate flow chromatogram for Rhodamine 123
(Rh 123) accumulation by A375 cells and HeL023 cells. Figure 6A illustrates
the
accumulation by A375 cells. Figure 6B illustrates the accumulation by A375
cells in the
presence of 10 M MRE 3008F20. Figure 6C illustrates the accumulation by
HeL023
cells. Figure 6D illustrates the accumulation by HeL023 cells in the presence
of 10 gM
MRE 3008F20. In all cases, FM,9,t represents the maximum load of Rh 123 (gray
filled area).
FREs shows residual Rh 123 fluorescence after the P-gp mediated drug efflux
was allowed
for 3 hours (black filled area). Rh 123 unstained cell chromatogram is
reported as unfilled
area.
Figure 7A illustrates the effect of inhibitors of MEK- (PD98059), ERK 1/2-
(U0126) and p38"K- (SB203580) -signalling on the GZ/M phases arrest induced by
paclitaxel (filled bars) and by vindesine (empty bars). The residual G2/M
phases arrest is
reported as the percentage of control cells (cells treated with paclitaxel
plus MRE 3008F20
for TU, TS, TP or with vindesine plus MRE 3008F20, for VU, VS, VP.
Figure 7B illustrates the effect of inhibitors of MEK- (PD98059), ERK 1/2-
(U0126) and p38MAPK- (SB203580) -signalling on apoptosis induced by
paclitaxel. T=
paclitaxel (25 ng/ml), V= vindesine 1 M, U= U0126 30 M, P= PD98059 20 M, S
SB203580 1 M, M = MRE 3008F20 10 M, C = cells treated with metabolic
inhibitor
vehicle (DMSO) (control). Each bar represents the mean :L SE of four
independent
experiments performed on A375 cells. P<0.01 as follows: *TU versus internal
control
(paclitaxel plus MRE 3008F20); #VU versus internal control (vindesine plus MRE
3008F20); P<0.05: 2 versus 1, 4 versus 3, 6 versus 5 and 8 versus 7. Analysis
was by
ANOVA followed by Dunnett's test.
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Figure 8. Apoptosis and cell cycle analysis of A375 cells cultured in
normoxia or hypoxia for 24 hours. (A), Representative flow cytometric analysis
of cell
cycle using propidium iodide for DNA staining: shown is the pattern of A375
cells being in
apoptosis and in Go/Gl, S and G2/M phase during normoxia and hypoxia.
Apoptotic cells
(Apo) with sub-diploid DNA content are reported. (B), The quantitative
analysis of sub-
diploid and of cells in Go/Gl, S and G2/M phase is given in the graph. Plots
are mean ~-_ S.E.
values (n=3). *P<0.01 compared with hypoxia.
Figure 9. Induction of HIF-1 expression by hypoxia. A375 cells were
cultured in normoxia for 4 hours (lane normoxia) or under hypoxic conditions
for 2, 3, 4, 8,
16 and 24 hours (lanes 2-7). Whole cellular protein extracts were prepared and
subjected to
immunoblot assay using an anti-HIF-la monoclonal antibody. The blot was then
stripped
and used to determine HIF-1 P. expression using an anti-HIF-10 monoclonal
antibody.
Tubulin shows equal loading protein.
Figure 10. linduction of HIF-la expression by adenosine. (A), A375 cells
were cultured in normoxia for 4 hours (lane normoxia). A375 cells were treated
without
(lane 1) or with adenosine 10 nM (lane 2), 100 nM (lane 3), 1 M (lane 4), 10
M (lane 5)
and 100 M (lane 6) in hypoxia for 4 hours. Cellular extracts were prepared
and subjected
to immunoblot assay using an anti-HIF-la monoclonal antibody. The blot was
then
stripped and used to determine HIF-1(3 expression using an anti-HIF-1(3
monoclonal
antibody. Tubulin shows equal loading protein. (B), Typical dose response
curve of A375
cells exposed to adenosine in hypoxia is shown. The HIF-1a immunoblot signals
were
quantified using molecular analyst /PC densitometry software (Bio-Rad). The
mean
densitometry data from 12 independent experiments (one of which is shown in
Panel A)
were normalized to the result obtained in cells in the absence of adenosine
(control). Plots
are mean S.E. values (n=12). (C), Effect of Al, A2A, A2B and A3 adenosine
receptor
antagonists. A3 75 cells were treated without (lane 1, control) or with
adenosine 100 M
(lanes 2-6) and exposed to the Al receptor antagonist DPCPX 100 nM (lane 3),
or A2A
receptor antagonist SCH 58261 100 nM (lane 4), or A2B receptor antagonist MRE
2029F20
100 nM (lane 5), or A3 receptor antagonist MRE 3008F20 100 nM (lane 6) in
hypoxia for 4
hours. Cellular extracts were prepared and subjected to immunoblot assay using
an anti-
HIF-1 a monoclonal antibody. The blot was then stripped and used to determine
HIF-1(3
expression using an anti-HIF-1(3 monoclonal antibody. Tubulin shows equal
loading
protein. (D), The immunoblot signals were quantified using molecular
analyst/PC
densitometry software (Bio-Rad). The mean densitometry data from 5 independent
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experiments (one of which is shown in Panel C) were normalized to the result
obtained in
cells in the absence of adenosine (control). Plots are mean S.E. values
(n=5). *P<0.01
compared with the control.
Figure 11. Induction of HIF-1 expression by A3 receptor stimulation: time
course. (A), A375 cells were cultured in normoxia for 4 hours (lane normoxia)
or under
hypoxic conditions in the absence (-) or in the presence (+) of the A3
receptor agonist CI-IB-
MECA (100 nM) for 2, 4, 8, 16 and 24 hours. Whole cellular protein extracts
were
prepared and subjected to immunoblot assay using an anti-HIF-la monoclonal
antibody.
The blot was then stripped and used to determine HIF-1(3 expression using an
anti-HIF-
1(3 monoclonal antibody. Tubulin shows equal loading protein. (B), The
immunoblot
signals were quantified using molecular analyst/PC densitometry software (Bio-
Rad). The
mean densitometry data from 3 independent experiments (one of which is shown
in Panel
A) were normalized to the result obtained in cells in the absence of CI-IB-
MECA after 4
hours of hypoxia (control). Plots are mean S.E. values (n=3). *P<0.01
compared with the
control.
Figure 12. Induction of HIF-1 a expression by A3 receptor stimulation: dose
response. (A), A375 cells were treated without (lane 1) or with Cl-IB-MECA 0.1
nM (lane
2), 1 nM (lane 3), 10 nM (lane 4) 100 nM (lane 5) and 1 M (lane 6) in
normoxia and in
hypoxia for 4 hours. Cellular extracts were prepared and subjected to
immunoblot assay
using an anti-HIF-la monoclonal antibody. The blot was then stripped and used
to
determine HIF-1(3 expression using an anti-HIF-1(3 monoclonal antibody. (B),
Typical dose
response curve of A375 cells exposed to adenosine in hypoxia is shown. The
immunoblot
signals were quantified using molecular analyst /PC densitometry software (Bio-
Rad). The
mean densitometry data from 12 independent experiments (one of which is shown
in Panel
A) were normalized to the result obtained in cells in the absence of CI-IB-
MECA (control).
Plots are mean S.E. values (n=12).
Figure 13. Effect of A3 receptor antagonist MRE 3008F20. (A), A375 cells
were treated in hypoxia for 4 hours without (-) or with (+) Cl-IB-MECA 10 nM,
MRE
3008F20 10 nM (lanes 3, 4) and MRE 3008F20 100 nM (lanes 5, 6). Cellular
extracts were
prepared and subjected to immunoblot assay using an anti-HIF-la monoclonal
antibody.
The blot was then stripped and used to determine HIF-1(3 expression using an
anti-HIF-
1(3 monoclonal antibody. (B), The immunoblot signals were quantified using
molecular
analyst /PC densitometry software (Bio-Rad). The mean densitometry data from
independent experiments (one of which is shown in Panel A) were normalized to
the result
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obtained in hypoxic cells in the absence of CI-IB-MECA (control, lane 1).
Plots are mean
S.E. values (n=3); *P<0.01 compared with the control. (C), A375 cells were
treated in
hypoxia for 4 hours without (lane 1) or with CI-IB-MECA 10 nM (lanes 2-6) and
MRE
3008F20 0.3 nM (lane 3), 1 nM (lane 4), 3 nM (lane 5) and 10 nM (lane 6).
Cellular
extracts were prepared and subjected to immunoblot assay using an anti-HIF-Ia
monoclonal antibody. The blot was then stripped and used to determine HIF-1(3
expression
using an anti-HIF-1(3 monoclonal antibody. (D), Typical dose response curve of
A375 cells
exposed to MRE 3008F20 in hypoxia is shown. The immunoblot signals were
quantified
using molecular analyst /PC densitometry software (Bio-Rad). The mean
densitometry data
from 3 independent experiments (one of which is shown in Panel C) were
normalized to the
result obtained in cells in the absence of Cl-IB-MECA (control). Plots are
mean =L S.E.
values (n=3).
Figure 14. A3 receptor expression silencing by siRNA transfection. (A),
Analysis of siRNA transfection efficiency in A375 cells. Representative flow
chromatograms of siRNA-FITC accumulation (gray filled area) in A375 cells
transfected
with siRNA-FITC. Unfilled area shows A375 cells transfected with RNAiFectTM
Transfection reagent without siRNAA3. Fluorescence was quantified by flow
cytometry 5
hours post-transfection. (B), Relative A3 receptor mRNA quantification,
related to (3-actin
mRNA, by Real-Time RT-PCR. A375 cells were transfected by RNAiFectTM
Transfection
reagent or siRNAA3 and cultured for 24, 48 and 72 hours. Plots are mean S.E.
values
(n=3); *P<0.01 compared with the control. (C), Western blot analysis using an
anti-A3
receptor polyclonal antibody of protein extracts from A375 cells treated by
RNAiFectTM
Transfection reagent (control) or siRNAA3 and cultured for 24, 48 and 72 hours
in
normoxia. Tubulin shows equal loading protein. (D), Densitometric
quantification of A3
receptor Western blot; plots are mean S.E. values (n=5); *P<0.01 compared
with the
control. (E), Western blot analysis using an anti-HIF- 1 a monoclonal antibody
of protein
extracts from A375 cells treated by control-siRNA (-) or siRNAA3 (+) for 72
hours and
cultured with (+) or without (-) CI-IB-MECA 100 nM for 4 hours in hypoxia.
Tubulin
shows equal loading protein.
Figure 15. A3 receptor stimulation induces HIF-la accumulation in various
human cell lines under hypoxia. NCTC 2544 keratinocytes, U87MG glioblastoma,
U2OS
osteosarcoma human cells were treated without (-) or with (+) CI-IB-MECA 100
nM in
hypoxia for 4 hours. Cellular extracts were prepared and subjected to
immunoblot assay
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using an anti-HIF-la monoclonal antibody. The blot was then stripped and used
to
determine HIF-1(3 expression using an anti-HIF-1(3 monoclonal antibody.
Figure 16. A3 receptor stimulation induces HIF-1a accumulation through a
transcription-independent pathway. (A), A375 cells were pretreated with
actinomycin D (10
g/ml) for 30 minutes and then exposed to hypoxia. HIF-1 a accumulation was
induced by
the exposure of A375 cells to CI-IB-MECA 100 nM (+) for 4 hours in hypoxia in
the
absence (lane 2) or in the presence (lane 4) of actinomycin D. Cellular
extracts were
prepared and subjected to immunoblot assay using an anti-HIF-la monoclonal
antibody.
Tubulin shows equal loading protein. (B), The immunoblot signals were
quantified using
molecular analyst/PC densitometry software (Bio-Rad). The mean densitometry
data from
independent experiments (one of which is shown in Panel A) were normalized to
the result
obtained in cells in the absence of CI-IB-MECA after 4 hours of hypoxia
(control=lane 1).
Plots are mean S.E. values (n=3); *P<0.01 compared with the control.
Figure 17. Induction of HIF-la accumulation by A3 receptor stimulation in
normoxia. (A), A3 75 cells were exposed to 100 M CoC12 alone (lane 1) or in
the presence
of Cl-IB-MECA 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4), 1 M (lane 5)
and 10 M
(lane 6) in normoxia for 4 hours. Cellular extracts were prepared and
subjected to
immunoblot assay using an anti-HIF-Ia monoclonal antibody. The blot was then
stripped
and used to determine HIF-1(3 expression using an anti-HIF-1(3 monoclonal
antibody. (B),
A375 cells were exposed to 100 M CoC12 for 4 hours in normoxia. HIF-la
accumulation
was induced by the exposure of A375 cells to CI-IB-MECA 100 nM (+) for 4
(lanes 1 and
4) or 6 hours (lanes 5 and 6) in the absence (lanes 1 and 2) or in the
presence (lanes 3-6) of
cycloheximide (1 M). Cellular extracts were prepared and subjected to
immunoblot assay
using an anti-HIF- 1 a monoclonal antibody. Tubulin shows equal loading
protein.
Figure 18. A3 receptor activation does not affect HIF- 1 a degradation in
normoxia. (A), A375 cells were incubated in hypoxia in the absence (lanes 1 to
4) or in the
presence of Cl-IB-MECA 100 nM (lanes 5 to 8). After 4 hours, melanoma cells
were
exposed to normoxia and a time-course of HIF-la disappearance was performed at
0, 5, 10
and 15 minutes. Cellular extracts were prepared and subjected to immunoblot
assay using
an anti-HIF-la monoclonal antibody. Tubulin shows equal loading protein. (B),
The
immunoblot signals were quantified using molecular analyst /PC densitometry
software
(Bio-Rad). The mean densitometry data from 3 independent experiments (one of
which is
shown in Panel A) were normalized to the result obtained in cells at time 0
(control). The
fraction of HIF- 1 a remaining is indicated.
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Figure 19. Role of p38, p44 and p42 MAPKs in A3 signalling. (A), A375
cells were pretreated with or without SB202190 (1 and 10 M) or U0126 (10 and
30 M)
and then exposed to CI-IB-MECA 100 nM (+) or to drug vehicle (-) for 4 hours
in hypoxia.
Cellular extracts were prepared and subjected to immunoblot assay using an
anti-HIF- 1 a
monoclonal antibody. Tubulin shows equal loading protein. (B), A3 stimulation
via Cl-IB-
MECA induces p44/p42 activation after 4 hours hypoxia in A375 cells. Cl-IB-
MECA 0
(lane C), 10 (lane 1), 100 (lane 2), 500 (lane 3) and 1000 (lane 4) nM was
added to A375
cells. After 4 hours cells were harvested and subjected to immunoblot assay
using
antibodies specific for phosphorylated (Thrl83/Tyrl85) or total p44/p42 MAPKs.
(C), The
immunoblot signals were quantified using molecular analyst /PC densitometry
software
(Bio-Rad). Densitometric analysis of p44 and p42 phosphorylated isoforms is
reported. The
mean densitometry data from 3 independent experiments (one of which is shown
in Panel
B) were normalized to the results obtained in cells in the absence of Cl-IB-
MECA (lane C).
Plots are mean 1 S.E. values (n=3); *P<0.01 compared with the control (lane
C). (D), A3
stimulation via Cl-IB-MECA induces p38 activation after 4 hours hypoxia in
A375 cells.
Cl-IB-MECA 0 (lane C), 10 (lane 1), 100 (lane 2), 500 (lane 3) and 1000 (lane
4) nM was
added to A375 cells. After 4 hours cells were harvested and subjected to
immunoblot assay
using antibodies specific for phosphorylated (Thr180/Tyr182) or total p38
MAPKs. (E), The
immunoblot signals were quantified using molecular analyst /PC densitometry
software
(Bio-Rad). Densitometric analysis of p38 phosphorylated isoforms is reported.
The mean
densitometry data from 3 independent experiments (one of which is shown in
Panel D) were
normalized to the result obtained in cells in the absence of CI-IB-MECA (lane
C). Plots are
mean S.E. values (n=3); *P<0.01 compared with the control (lane C).
6. DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, in part, on the surprising discovery by the
inventors that adenosine, a purine nucleoside present within hypoxic regions
of solid
tumors, modulates hypoxia-inducible factor 1 (HIF- 1) expression. HIF- 1, a
heterodimeric
transcription factor composed of HIF-Ia and HIF-1(3 subunits, is involved in
tumor growth
and angiogenesis. The inventors have found that in the human A375 melanoma
cell line
adenosine up-regulates HIF-la protein expression in response to hypoxia in a
dose- and
time-dependent manner. The response to adenosine was not blocked by Al, A2A or
A2B
receptor antagonists, while it was abolished by A3 receptor antagonists. The
inventors have
found that Cl-IB-MECA, an adenosine analogue binding with high affinity to A3
receptors,
mimicked adenosine effect in hypoxic cells. Furthermore, A3 receptor
antagonists
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prevented HIF-la protein accumulation in response to A3 receptor stimulation.
Although
not intending to be bound by a particular mechanism of action, the response to
adenosine is
generated at the cell surface since the inhibition of A3 receptor expression,
by using small
interfering RNA, abolished the nucleoside effects. The main intracellular
signaling
pathways sustained by A3 receptor stimulation in hypoxia involve p44/p42
mitogen-
activated protein kinase (MAPK) and p38 MAPK activation. The inventors have
thus found
for the first time that adenosine plays a critical role in HIF- 1 a regulation
in hypoxia.
The inventors have found that compounds which are antagonists of the
adenosine receptors, preferably the A3 receptor, inhibit the protective effect
of adenosine on
growing tumor cells when such cells are starved of oxygen (i.e., before an
adequate
vasculature is developed, or when anti-angiogenesis agents are administered.
Although not
intending to be bound by a particular mechanism of action, the antagonists of
the invention
can bind in the same site as adenosine, or can be allosteric antagonists
(i.e., bind at a site
different from where adenosine binds, but adversely affect the ability of
adenosine to bind
to the site or adversely affect the ability of adenosine, once bound to the
adenosine receptors
particularly A3 receptors, to protect growing tumor cells).
Accordingly, the present invention relates to methods for the treatment,
prevention, and/or management of diseases or disorders associated with
overexpression of
HIF-la and/or increased HIF-la activity (e.g., cancer, respiratory disorders
such as asthma
and obstructive pulmonary disorders) by using adenosine receptor antagonists,
particularly
A3 receptor antagonists, alone or in combination with Al receptor antagonists.
Without
being bound to a particular mechanism of action, administration of antagonists
for the
adenosine receptors antagonizes the protective effects against hypoxia and
renders those
cells susceptible to destruction due to hypoxia. Since the adenosine
receptors, in particular,
the A3 receptors, are responsible for sustained cellular protection against
ischemia,
antagonists for the adenosine receptors, particularly A3 receptors are
particularly effective in
enhancing the activity of anti-tumor agents.
The methods and compositions of the invention comprising A3 receptor
antagonists are particularly useful when the levels of HIF- 1 a expression
and/or activity are
elevated above the standard or background level, as determined using methods
known to
those skilled in the art and dislosed herein. As used herein, "elevation" of a
measured level
of HIF-la relative to a standard level means that the amount or concentration
of HIF-la in
a sample or subject is sufficiently greater in a subject or sample relative to
the standard as
detected by any method now known in the art or to be developed in the future
for measuring
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HIF-la levels. For example, elevation of the measured level relative to a
standard level
may be any statistically significant elevation detectable. Such an elevation
in HIF-1 a
expression and/or activity may include, but is not limited to about a 10%,
about a 20%,
about a 40%, about an 80%, about a 2-fold, about a 4-fold, about an 10-fold,
about a 20-
fold, about a 50-fold, about a 100-fold, about a 2 to 20 fold, 2 to 50 fold, 2
to 100 fold, 20 to
50 fold, 20 to 100 fold, elevation, relative to the standard. The term "about"
as used herein,
refers to levels of elevation of the standard numerical value plus or minus
10% of the
numerical value.
The term "standard level" or "background level" as used herein refers to a
baseline amount of HIF-1a level as determined in one or more normal subjects,
i.e., a
subject with no known history of past or current diseases, disorders or
cancer. For example,
a baseline may be obtained from at least one subject and preferably is
obtained from an
average of subjects (e.g., n=2 to 100 or more), wherein the subject or
subjects have no prior
history of diseases, disorders or cancer, especially no prior history of
diseases associated
with an aberrant level of expression and/or activity of HIF-1a.
In the present invention, the measurement of HIF-1a level may be carried
out using an HIF-la probe or a HIF-la activity assay (see Section 5.4.4). As
used herein,
reference to measuring a level of HIF-la in a method of the invention relates
to any proxy
for HIF-la levels. For example, such levels may include, but are not limited
to, the
abundance of HIF-la nucleic acid or amino acid sequences in a sample from a
subject. A
level of HIF-1 a may correspond to the abundance of full-length HIF-1 a
protein.
Alternatively, a level of HIF- 1 a , may correspond to abundance of a
fragment, analog or
derivative of HIF- 1 a protein. A level of HIF- 1 a can be determined by
measuring the
abundance of nucleic acids (or sequences complementary thereto) that
corresponds to all or
fragments of HIF-1 a. In a preferred embodiment, the abundance of mRNA
encoding HIF-
1 a is measured.
As used herein, a probe with which the amount or concentration of HIF-
1 a can be determined, includes but is not limited to an antibody, an antigen,
a nucleic acid,
a protein, or a small molecule. In a specific embodiment, the probe is the HIF-
la protein or
a fragment thereof. In another embodiment, the probe is an antibody that
immunospecifically binds to HIF-la, such as e.g., a monoclonal antibody or a
binding
fragment thereof.
In a specific embodiment, measuring a level of HIF-1 a comprises testing at
least one aliquot of the sample, said step of testing comprising: (a)
contacting the aliquot
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with an antibody or a fragment thereof that is immunospecific for HIF-1 a, and
(b) detecting
whether and how much binding has occurred between the antibody or a fragment
thereof
and at least one species of HIF- 1 a in the aliquot. In yet another specific
embodiment,
measuring a level of HIF-la comprises testing at least one aliquot, said step
of testing
comprising: (a) contacting the aliquot with a nucleic acid probe that is
hybridizable to HIF-
la mRNA, and (b) detecting whether and how much hybridization has occurred
between
the nucleic acid probe and at least one species of HIF-1 a mRNA in the
aliquot. In both
embodiments measuring a level of HIF-1a involves quantitating the amount of
complex
formation. For example the amount of complex formation between the antibody or
a
fragment thereof and at least one species of HIF-Ia in the aliquot would
correlate with the
amount of at least one species of HIF-1 a in the aliquot of the sample.
In a further specific embodiment, the antibody or other probe is labeled with
a detectable marker. In yet another specific embodiment, the detectable marker
is a
chemiluminescent, enzymatic, fluorescent, or radioactive label.
The therapeutic methods of the invention comprising administering a
therapeutically effective amount of an A3 receptor antagonist of the invention
improve the
therapeutic efficacy of treatment for diseases or disorders associated with
overexpression of
HIF-la and/or increased HIF-la activity (e.g., cancer, respiratory disorders
such as asthma
and obstructive pulmonary disorders) relative to the traditional modes of such
therapies.
Preferably the methods of the invention reduce the HIF-1 a level to the
background level
within within one day, one week, 1 month, or 2 months of the commencement of
the
therapeutic regime. Preferably, once the methods of the invention reduce the
level of HIF-
la to a particular level, that level is maintained during the treatment
regimen, such that the
maintained level of HIF- 1 a is sufficient and effective to result in
regression of the disease,
e.g., cancer.
In a most preferred embodiment, the methods of the invention result in a
reduction of HIF-la level to the background level. The invention encompasses
reduction of
the HIF- 1 a level to a level which is within about 10%, about 20%, about 30%,
about 40%,
about 50% of the background level.
In a preferred specific embodiment, the invention encompasses a method for
treatment, prevention and/or management of diseases or disorders associated
with
overexpression of HIF-Ia and/or increased HIF-1a activity (e.g., cancer,
respiratory
disorders such as asthma and obstructive pulmonary disorders) comprising
administering a
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therapeutically and/or prophylactically effective amount of an A3 receptor
antagonist
compound as disclosed herein.
The invention also encompasses a method for determining the prognosis of a
a disease or disorder associated with overexpression of HIF-1a and/or
increased HIF-la
activity in a subject. Preferably, the subject is human. In some embodiments,
the subject
has been previously treated with a therapy regimen. The invention encompasses
measuring
at least a level of HIF-1 a in a subject to determine if the subject is in
need of the
therapeutic and or prophylcatic methods of the inventon. The invention
encompasses
measuring a level of at least one HIF-1 a in a sample obtained from the
subject and
comparing the level measured to a standard level, wherein elevation of the
measured level
of at least one HIF- 1 a relative to the standard level indicates that the
subject is at an
increased risk for progression of the disease or disorder, e.g., metastasis of
the cancer.
The invention encompasses compounds which are adenosine receptor
antagonists particularly A3 antagonists for use in the methods of the
invention. Examples of
such compounds are disclosed in U.S. Patent Nos 6,326,390; 6,407,236;
6,448,253;
6,358,964; and U.S. Publication Nos. 2003/0144266 and 2004/0067932; all of
which are
incorporated herein by reference in their entireties. In addition, other
suitable adenosine A3
antagonists are disclosed in the references listed in Table 1, supra, the
disclosures of which
are hereby incorporated by reference in their entireties.
The A3 receptor antagonists of the invention are particularly useful for
prevention, treatment, and/or management of cancer, for example, as a single
agent therapy
or in combination with other modes of therapy for cancer. In preferred
embodiments, the
invention encompasses methods of treatment, prevention, or management of
cancers which
are A3 expressing cancers including, but not limited to, human leukemia,
melanoma,
pancreatic carcinoma, ovarian carcinoma, breast carcinoma, prostrate
carcinoma, colon
carcinoma, lung carcinoma, malignant melanomas, histiocytic lymphoma, and some
forms
of astrocytoma cells. In other preferred embodiments, the invention
encompasses methods
of treatment, prevention, or management which have high level of expression of
HIF-1 a
including, but not limited to, cervical cancer (early stage), lung cancer (non-
small cell lung
carcinoma), breast cancer (including lymph node positive breast cancer and
lymph node
negative breast cancer), oligodendroglioma, orpharyngeal squamous cell
carcinoma, ovarian
cancer, oesophageal cancer, endometrial cancer, head and neck cancer,
gastrointestinal
stromal tumor of the stomach The methods of the invention are particularly
useful in
cancer therapy for providing tissue selectivity, such that the biological
effect is observed in
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tumoral hypoxic cells, where high adenosine concentrations has resulted in
increased HIF-
1 a accumulation.
The methods and compositions of the invention comprising A3 receptor
antagonists (or Al receptor antagonists or a combination thereof) are
particularly useful for
the treatment, inhibition or regression of solid tumors. As used herein,
"solid tumors" refer
to a locus of tumor cells where the majority of the cells are tumor cells or
tumor-associated
cells, including but not limited to laryngeal tumors, brain tamors, and other
tumors of the
head and neck; colon, rectal and prostate tumors; breast and thoracic solid
tumors; ovarian
and uterine tumors; tumors of the esophagus, stomach, pancreas and liver;
bladder and gall
bladder tumors; testicular cancer; skin tumors such as melanomas. Moreover,
the tumors
encompassed within the invention can be either primary or a secondary tumor
resulting
from metastasis of cancer cells elsewhere in the body to the chest.
The invention encompasses use of the adenosine receptor antagonists of the
invention, particulalry A3 receptor antagonists in inhibiting tumor growth in
a mammal,
including humans. The invention encompasses methods for administering an
effective
amount of an adenosine receptor antagonists, preferably A3 receptor
antagonists to inhibit
the ability of adenosine to protect tumor cells.
In other embodiments, the present invention encompasses methods for
treatment, prevention and/or management of cancer comprising administering an
adenosine
receptor antagonist, particularly an A3 receptor antagonist combination with
other
therapeutic and/or prophylactic agents, e.g., cytotoxic agents. The inventors
have
surprisingly found that adenosine A3 receptor antagonists synergistically
enhance cytotoxic
treatment and counter some forms of multi-drug resistance. Although not
intending to be
bound by a particular mechanism of action, the combination therapies of the
invention will
attack the tumor cell directly, inhibit growth of new blood vessels around the
tumor cell,
and, by virtue of the adenosine A3 antagonists, inhibit the ability of the
cell to survive
without the growth of new blood vessels. Inventors have discovered that high
affinity
adenosine A3 receptor antagonists are useful as enhancers for many
chemotherapeutic
treatments of adenosine A3 receptor expressing cancers. Surprisingly, high
affinity
adenosine A3 receptor antagonists also counter P-glycoprotein (P-gp) effuse
pump multi-
drug resistance (MDR). Finally, high affinity adenosine A3 receptor
antagonists are helpful
in reducing or ameliorating side effects of cytotoxic agents, e.g., taxane
In other embodiments, the present invention encompasses the use of high
affinity adenosine A3 receptor antagonists for enhancing chemotherapeutic
treatment of
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cancers expressing adenosine A3 receptors and countering multi-drug resistance
in cancers
expressing P-glycoprotein or MRP. In preferred embodiments, adenosine A3
receptor
antagonists are administered before or during administration of a taxane
family, vinca
alkaloid, camptothecin or antibiotic compound. Preferred high affinity A3
receptor
antagonists include compounds of the general formulas IIA, IIB, IIC and IID
described
herein, vide infi a.
The present invention encompasses therapies which involve administering
one or more of the compounds of the invention, to an animal, preferably a
mammal, and
most preferably a human, for preventing, treating, or ameliorating symptoms
associated
with a cancer, a disease or disorder associated with hypoxia-inducible factor
1-a (HIF-1 a).
The invention further provides a pharmaceutical composition comprising a
therapeutically or prophylactically effective amount of a compound of the
invention that
specifically binds an Al or A3 receptor and a pharmaceutically acceptable
carrier.
Preferably the pharmaceutical formulation includes an A3 receptor antagonist
and one or
more excipients. The formulations can also include other anti-tumor agents,
including
cytotoxic agents and other anti-angiogenesis agents, including adenosine A2A
antagonists,
such as ZM241385 and SCH 5861, adenosine A2B antagonists, such as MRE-20290-
F20
and AS-16, and anti-VEGF antibodies, including humanized and chimeric
antibodies. In a
preferred embodiment, the composition includes an effective amount to inhibit
tumor
growth of an adenosine A3 receptor antagonist, a cytotoxic agent, and an anti-
angiogenesis
agent.
6.1 COMPOUNDS OF THE INVENTION
6.1.1 COMPOUNDS OF THE GENERAL FORMULA IIA, IIB AND
IIC
In a specific embodiment, the invention provides methods for treating a
disease or disorder associated with HIF-ta in a patient, comprising
administering to a
patient in need thereof an effective amount of a compound or a
pharmaceutically acceptable
salt of the compound having the following general formulas IIA and IIB:
R3
N--~,
R2 A I N
N~NHR IIA
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R3
R2 G N, R2
N~R2
n(R2CH)-N
R IIB
wherein:
A is imidazole, pyrazole, or triazole;
R is -C(X)Rl, -C(X)-N(Rl)2, -C(X)OR', -C(X)SRI, -SObRI, -SObORI, -
SObSRI, or SOIN(Rl)2 ;
R' is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl,
substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic,
substituted heterocyclic, lower alkenyl, lower alkanoyl, or, if linked to a
nitrogen atom, then
taken together with the nitrogen atom, forms an azetidine ring or a 5-6
membered
heterocyclic ring containing one or more heteroatoms such as N, 0, S;
R2 is hydrogen, alkyl, alkenyl, alkynyl, substituted alkyl, substituted
alkenyl,
substituted alkynyl, aralkyl, substituted aralkyl, aryl, substituted aryl,
heteroaryl, or
substituted heteroaryl;
R3 is furan, pyrrole, thiophene, benzofuran, indole, benzothiophene,
optionally
substituted with one or more substituents as described herein for substituted
heteroaryl
rings;
XisO,S,orNRl
bis 1 or2.
Preferably, R' is hydrogen; Ci to C8 alkyl; C2 to C7 alkenyl, C2 to C7
alkynyl;
C3 to C7 cycloalkyl; Cl to C5 alkyl substituted with one or more halogen
atoms, hydroxy
groups, C1 to C4 alkoxy, C3 to C7 cycloalkyl or groups of formula NR32 R32, -
C(O)NR32R32 ; aryl, substituted aryl wherein the substitution is selected from
the group
consisting of C1 to C4 alkoxy, C1 to C4 alkyl, nitro, amino, cyano, C1 to C4
haloalkyl, CI to
C4 haloalkoxy, carboxy, carboxyamido; C7 to Clp aralkyl in which the aryl
moiety can be
substituted with one or more of the substituents indicated above for the aryl
group; a group
of formula -(CH2)rõ-Het, wherein Het is a 5-6 membered aromatic or non
aromatic
heterocyclic ring containing one or more heteroatoms selected from the group
consisting of
N, 0, and S and m is an integer from 0 to 5, and in some embodiments from 1-5.
Preferred CI to C8 alkyl groups are methyl, ethyl, propyl, butyl and
isopentyl.
Examples of C3 to C7 cycloalkyl groups include cyclopropyl, cyclopentyl, and
cyclohexyl.
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Examples of C1 to C5 alkyl groups substituted with C3 to C7 cycloalkyl groups
include
cyclohexylmethyl, cyclopentylmethyl, and 2-cyclopentylethyl. Examples of
substituted C1
to CS alkyl groups include 2-hydroxyethyl, 2-methoxyethyl, trifluoromethyl, 2-
fluoroethyl,
2-chloroethyl, 3-aminopropyl, 2-(4-methyl-l-piperazine)ethyl, 2-(4-
morpholinyl)ethyl, 2-
aminocarbonylethyl, 2-dimethylaminoethyl, 3-dimethylaminopropyl. Aryl is
preferably
phenyl, optionally substituted with Br, Cl, F, methoxy, nitro, cyano, methyl,
trifluoromethyl, difluoromethoxy groups. Examples of 5 to 6 membered ring
heterocyclic
groups containing N, 0 and/or S include piperazinyl, morpholinyl, thiazolyl,
pyrazolyl,
pyridyl, furyl, thienyl, pyrrolyl, triazolyl, tetrazolyl. Examples of C7 to
Clo aralkyl groups
comprise benzyl or phenethyl optionally substituted by one or more
substituents selected
from Cl, F, methoxy, nitro, cyano, methyl, trifluoromethyl, and
difluoromethoxy.
Preferably, Rl is hydrogen, C1 to C8 alkyl, aryl or C7 to ClO aralkyl,
optionally substituted, preferably with halogen atoms. Preferably, X is 0, RZ
is C2-C3 alkyl
or substituted alkyl and R3 is furan.
In a specific embodiment R2 is alkyl.
Particularly preferred compounds are those in which R is a phenethyl group
in which the phenyl ring is substituted with one or more substituents selected
from the
group consisting of chlorine, fluorine atoms, methoxy, nitro, cyano, methyl,
trifluoromethyl,
and difluoromethoxy groups.
The possible meanings of A canbe represented by the following structural
formulae:
</N Q NV R2-N z N N ~
N N N N
R2 R2 R2
R2, N N
. , ~
N, I R2_N, ~
N N
In a particular embodiment, the invention provides methods for treating a
disease or disorder associated with HIF- 1 a in a patient, comprising
administering to a
patient in need thereof an effective amount of a compound or a
pharmaceutically acceptable
salt of the compound having the general formula IIA:
wherein:
A is imidazole, pyrazole, or triazole;
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R is -C(X)R', -C(X)-N(Rl)Z, -C(X)OR', -C(X)SRI, -SOõRl, -SOSR', or
-SOõN(Rl)Z;
R' is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl,
substituted alkynyl, aryl, substituted aryl, heteroaryl, heterocyclic, lower
alkenyl, lower
alkanoyl, or, if linked to a nitrogen atom, then taken together with the
nitrogen atom, forms
an azetidine ring or a 5-6 membered heterocyclic ring containing one or more
heteroatoms;
R2 is hydrogen, alkyl, substituted alkyl, alkenyl, aralkyl, substituted
aralkyl,
heteroaryl, substituted heteroaryl or aryl;
R3 is furan, pyrrole, thiophene, benzofuran, benzypyrrole, benzothiophene,
optionally substituted with one or more substituents selected from the group
consisting of
hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted
alkoxy,
substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl,
acyloxy,
acylamino, alkaryl, aryl, substituted aryl, aryloxy, azido, carboxyl,
carboxylalkyl, cyano,
halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
aminoacyloxy,
thioalkoxy, substituted thioalkoxy, -SO-alkyl, -SO-substituted alkyl, -SO-
aryl, -SO-
heteroaryl, -S02-alkyl, -SO2-substituted alkyl, -S02-aryl, -S02-heteroaryl,
and
trihalomethyl;
X is 0, S, or NR1; and
nis 1 or2.
In a specific embodiment of the invention the method is conducted using the
compound of the general formula IIA, wherein RZ is selected from the group
consisting of
hydrogen, alkyl, alkenyl and aryl.
In another specific embodiment of the invention the method is conducted
using the compound of the general formula IIA, wherein A is a triazolo ring.
In another specific embodiment of the invention the method is conducted
using the compound of the general formula IIA, wherein A is a pyrazolo ring.
In a specific embodiment, the method uses as high affinity adenosine A3
receptor antagonist the "phenyl-carbamoyl-amino" compounds of the general
formula IIC
and pharmaceutical salts thereof:
R3
N-~
N
Rz A N O
N~N N' R 6
R H IIC
wherein:
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A is imidazole, pyrazole, or triazole;
R2 is hydrogen, alkyl, substituted alkyl, alkenyl, aralkyl, substituted
aralkyl,
heteroaryl, substituted heteroaryl or aryl;
R3 is furan; and
R6 is aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle
or
substituted heterocycle.
Non-limiting examples of the compounds of the general formula IIA and IIC
include compounds listed below in Tables 2 and 3.
Table 2
# Compound
lc 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-
e]-
1,2,4-triazolo [ 1, 5-c]pyrimidine,
2c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3
-
e]-1,2,4-triazolo[1,5-c]pyrimidine,
3c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-ethyl-2-(2-furyl)-pyrazolo[4,3-e
]-
1,2,4-triazolo [ 1, 5-c]pyrimidine,
4c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-ethyl-2-(2-furyl)-pyrazolo[4,3-
e]-
1,2,4-triazolo [1,5-c]pyrimidine,
5c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-propyl-2-(2-furyl)-pyrazolo[4,3-
e]-
1,2,4-triazolo [ 1,5-c]pyrimidine;
6c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-propyl-2-(2-furyl)-pyrazolo[4,3
-e]-
1,2,4-triazolo [ 1,5-c] pyrimidine,
7c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-butyl-2-(2-furyl)-pyrazolo[4,3-e
]-
1 ,2,4-triazolo [ 1, 5-c] pyrimidine,
8c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-butyl-2-(2-furyl)-pyrazolo[4,3-
e]-
1,2,4-triazolo [ 1,5-c]pyrimidine,
9c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-isopentyl-2-(2-furyl)-pyrazolo[4
,3-
e] -1,2,4-triazolo [ 1, 5-c] pyrimidine,
10c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-isopentyl-2-(2-furyl)-
pyrazolo[ 4,3-
e] -1, 2,4-triazolo [ l, 5-c] pyrimidine,
llc 5-[[(3-Chlorophenyl)amino]]carbonyl]amino-8-(2-isopentenyl)-2-(2-
furyl)pyrazolo [4,3 -e] 1,2,4-triazolo [ 1,5-c]pyrimidine,
12c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-(2-isopentenyl)-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine,
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13c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-(2-phenylethyl)-2 (2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine,
14c 5-[[(4-Methoxyphenyl)amino]carbonylamino-8-(2-phenylethyl)-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine,
15c 5-[[(3-Chlorophenyl)amino]carbonyl]amino-8-(3-phenylpropyl)-2-(2-fiuyl)-
pyrazolo[4,3-e]-1,2,4-triazolo [ 1,5-c]pyrimidine,
16c 5-[[(4-Methoxyphenyl)amino]carbonyl]amino-8-(3-phenylpropyl)-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo [ 1,5-c]pyrimidine,
17c 5-[(Benzyl)carbonyl]amino-8-isopentyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine,
18c 5-[(Benzyl)carbonyl]amino-8-(3-phenylpropyl)-2-(2-furyl)-pyrazolo[4,3-e]-
1, 2,4-
triazolo [ 1, 5-c] pyrimidine.
101d N-[4-(diethylamino)phenyl]-N'-[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-
e] [ 1,2, 4] tri azo l o[ 1, 5- c] pyrimidin- 5-yl] ure a
102d N-[8-methyl-2-(2-furyl)-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-yl]-N'-
[4-(diethylamino)phenyl]urea hydrochloride
103d N-[8-methyl-2-(2-furyl)-8H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-
c]pyrimidin-5-yl]-N'-
[4-(dimethylamino)phenyl]urea hydrochloride
104d N-(2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]
pyrimidin-5-yl)-
N'-[4-(morpholin-4-ylsulfonyl)phenyl]urea
105d N-[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]
pyrimidin-5-yl]-
N'- { 4- [(4-methylpiperazin-l-yl) sulfonyl] -phenyl } urea
106d N-[2-(2-furyl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-yl]-N'-
pyridin-4-yl urea
107d N-[2-(2-furyl)-8-methyl=8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-
5-yl]-N'-
pyridin-4-ylurea hydrochloride
Table 3
NHR
N)-~N'N~~
i ~N O
N I
N
RZ
Compound No RZ R
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61d H 4-MeO-Ph-NHCO
62d H 3-Cl-Ph-NHCO
64d t-C4H9 4-MeO-Ph-NHCO
65d t-C4H9 3-Cl-Ph-NHCO
66d CH3 Ph-NHCO
67d CH3 4-SO3H-Ph-NHCO
68d CH3 3,4-C12-Ph-NHCO
69d CH3 3,4-(OCH2-O)-Ph-NHCO
70d CH3 4-(N02)-Ph-NHCO
71d CH3 4-(CH3)-Ph-NHCO
72d CH3 Ph-(CH2)-CO
73d C2H5 Ph-NHCO
74d C2H5 4-SO3H-Ph-NHCO
75d C2H5 3,4-C12-Ph-NHCO
76d C2H5 3,4-(OCH2-O)-Ph-NHCO
77d C2H5 4-(N02)-Ph-NHCO
78d C2H5 4-(CH3)-Ph-NHCO
79d C2H5 Ph-(CH2)-CO
80d n-C3H7 Ph-NHCO
81d n-C3H7 4-SO3H-Ph-NHCO
82d n-C3H7 3,4-C12 -Ph-NHCO
83d n-C3H7 3,4-(OCH2-O)-Ph-NHCO
84d n-C3H7 4-(NO2)-Ph-NHCO
85d n-C3H7 4-(CH3)-Ph-NHCO
86d n-C3H7 Ph-(CH2)-CO
87d n-C4H9 Ph-NHCO
88d n-C4H9 4-SO3H-Ph-NHCO
89d n-C4H9 3,4-C12-Ph-NHCO
90d n-C4H9 3,4-(OCH2-O)-Ph-NHCO
91d n-C4H9 4-(NO2)-Ph-NHCO
92d n-C4H9 4-(CH3)-Ph-NHCO
93d 2-(a-napthyl)ethyl Ph-(CH2)-CO
95d 2-(a-napthyl)ethyl 4-MeO-Ph-NHCO
96d 2-(a-napthyl)ethyl 3-Cl-Ph-NHCO
98d 2-(2,4,5-tribromo- 4-MeO-Ph-NHCO
phenyl)ethyl
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99d 2-(2,4,5-tribromo- 3-C1-Ph-NHCO
phenyl)ethyl
100d 2-propen-1-yl 4-MeO-Ph-NHCO
108d n-C3H7 4-MeO-Ph-NHCO
109d C2H5 4-MeO-Ph-NHCO
6.1.2 COMPOUNDS OF THE GENERAL FORMULA IID
In another specific embodiment, the invention provides methods for treating
a disease or disorder associated with HIF-1a in a patient, comprising
administering to a
patient in need thereof an effective amount of a compound or a
pharmaceutically acceptable
salt of the compound having the following general formula IID:
R3
N-{
~
R ~ ,N
~ N
~
N~NHR IID
wherein:
R is -C(X)Rl, -C(X)-N(R')2, -C(X)OR', -C(X)SR', -SObRI, -SObORI, -
SObSR', or -SObN(R')2;
Rl is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl,
substituted alkynyl, aryl, heteroaryl, heterocyclic, lower alkenyl, lower
alkanoyl, or, if
linked to a nitrogen atom, then taken together with the nitrogen atom, forms
an azetidine
ring or a 5-6 membered heterocyclic ring containing one or more heteroatoms
such as N, 0,
S;
Rz is hydrogen, halogen, preferably chloro, alkyl, alkenyl, alkynyl,
substituted alkyl,
substituted alkenyl, substituted alkynyl, aralkyl, substituted aralkyl, aryl,
substituted aryl,
heteroaryl or substituted heteroaryl;
R3 is furan, pyrrole, thiophene, benzofuran, indole, benzothiophene,
optionally
substituted with one or more substituents as described herein for substituted
heteroaryl
rings;
XisO,S,orNRl;and
bislor2.
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In a specific embodiment the compound is of the formula IID as described
above,
with the proviso that R2 is not halogen when R is -C(X)Rl. A preferred R group
is -
C(X)-N(R')2, where X is O.
For compounds of the general formula IID, preferably, Rl is hydrogen; C1.8
alkyl; C2.7 alkenyl, C2-7 alkynyl; C3_7 cycloalkyl; C1.5 alkyl substituted
with one or more
halogen atoms, hydroxy groups, C1-4 alkoxy, C3.7 cycloalkyl or groups of
formula NR32,
-C(O)NR32 ; aryl, substituted aryl wherein the substitution is selected from
the group
consisting of Cl-4 alkoxy, Cl-4 alkyl, nitro, amino, cyano, C1-4 haloalkyl,
C1.4 haloalkoxy,
carboxy, carboxyamido; C7.I0 aralkyl in which the aryl moiety can be
substituted with one
or more of the substituents indicated above for the aryl group; a group of
formula -
(CH2)õ7--Het, wherein Het is a 5-6 membered aromatic or non aromatic
heterocyclic ring
containing one or more heteroatoms selected from the group consisting of N, 0,
and S and
m is an integer from 1 to 5; For compounds of the general formula IID,
preferred C1_$ alkyl groups are
methyl, ethyl, propyl, butyl and isopentyl. Examples of C3.7 cycloalkyl groups
include
cyclopropyl, cyclopentyl, and cyclohexyl. Examples of C1_5 alkyl groups
substituted with
C3_7 cycloalkyl groups include cyclohexylmethyl, cyclopentylmethyl, and 2-
cyclopentylethyl. Examples of substituted C1.5 alkyl groups include 2-
hydroxyethyl, 2-
methoxyethyl, trifluoromethyl, 2-fluoroethyl, 2-chloroethyl, 3-aminopropyl, 2-
(4methyl-l-
piperazine)ethyl, 2-(4-morpholinyl)ethyl, 2-aminocarbonylethyl, 2-
dimethylaminoethyl, 3-
dimethylaminopropyl. Aryl is preferably phenyl, optionally substituted with
Cl, F, methoxy,
nitro, cyano, methyl, trifluoromethyl, difluoromethoxy groups. Examples of 5
to 6
membered ring heterocyclic groups containing N, 0 and/or S include
piperazinyl,
morpholinyl, thiazolyl, pyrazolyl, pyridyl, furyl, thienyl, pyrrolyl,
triazolyl, tetrazolyl.
Examples of C7_10 aralkyl groups comprise benzyl or phenethyl optionally
substituted by
one or more substituents selected from Cl, F, methoxy, nitro, cyano, methyl,
trifluoromethyl, and difluoromethoxy. Preferably, Rl is hydrogen, C1_8 alkyl,
aryl or C7_10
aralkyl, optionally substituted, preferably with halogen atoms. Preferably, X
is 0, RZ is
chloro, C2_3 alkyl or substituted alkyl and R3 is furan.
Particularly preferred compounds are those in which R is a phenethyl group
in which the phenyl ring is substituted with one or more substituents selected
from the
group consisting of chlorine, fluorine atoms, methoxy, nitro, cyano, methyl,
trifluoromethyl,
and difluoromethoxy groups.
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Non-limiting examples of compounds of the formula IID include compounds
listed below in Table.
Table 4
# Compound
lb 5-[[4-methoxyphenyl)amino]carbonyl]amino-9-chloro-2-(2-furyl)-1,2,4-
triazolo[1,5-
bc]quinazoline
2b 5-[[3-chlorophenyl)amino]carbonyl]amino-9-chloro-2-(2-furyl)-1,2,4-
triazolo[l,5-
c]quinazoline
6.2 METHODS OF PREPARING THE COMPOUNDS OF THE
INVENTION
Compounds of the general formulas IIA, IIB and IIC can be prepared, for
example, as described in U.S. Patent No.'s 6,407,236; 6,448,253 and U.S.
Patent
Application Serial No. 10/134,219, filed Apri126, 2002; the disclosures of
which are hereby
incorporated by reference in their entireties. Compounds of the general
formula IID can be
prepared, for example, as described in U.S. Patent No. 6,358,964; the
disclosure of which is
hereby incorporated by reference in its entirety.
Where necessary, certain synthetic intermediates used to prepare compounds
used in the invention may contain reactive moeities. Those skilled in the art
of organic
chemistry will appreciate that reactive and fragile functional groups often
must be protected
prior to a particular reaction, or sequence of reactions, and then restored to
their original
forms after the last reaction is completed. Usually groups are protected by
converting them
to a relatively stable derivative. For example, a hydroxyl group may be
converted to an
ether group and an amine group converted to an amide or carbamate. Methods of
protecting
and de-protecting, also known as "blocking" and "de-blocking," are well known
and widely
practiced in the art, e.g., see T. Green, Protective Groups in Organic
Synthesis, John Wiley,
New York (1981) or Protective Groups in Organic ChemistNy, Ed. J. F. W.
McOmie,
Plenum Press, London (1973).
6.3 PROPHYLACTIC AND THERAPEUTIC METHODS
The present invention relates to methods for the treatment, prevention, and/or
management of diseases or disorders associated with overexpression of HIF-1 a
and/or
increased HIF-1 a activity (e.g., cancer, respiratory disorders such as asthma
and obstructive
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pulmonary disorders) by using A3 receptor antagonists alone or in combination
with Al
receptor antagonists.
The methods and compositions of the invention comprising A3 receptor
antagonists are particularly useful when the levels of HIF- 1 a expression
and/or activity are
elevated above the standard or background level, as determined using methods
known to
those skilled in the art and dislosed herein. For example, elevation of the
measured level
relative to a standard level may be any statistically significant detectable
elevation. Such an
elevation of HIF-1 a expression and/or activity may include, but is not
limited to about a
10%, about a 20%, about a 40%, about an 80%, about a 2-fold, about a 4-fold,
about a 10-
fold, about a 20-fold, about a 50-fold, about a 100-fold, about a 2 to 20
fold, 2 to 50 fold, 2
to 100 fold, 20 to 50 fold, 20 to 100 fold, elevation, relative to the
standard. The term
"about" as used herein, refers to levels of elevation of the standard
numerical value plus or
minus 10% of the numerical value.
The therapeutic methods of the invention comprising administering a
therapeutically effective amount of an A3 receptor antagonist of the invention
improve the
therapeutic efficacy of treatment for diseases or disorders associated with
overexpression of
HIF-la and/or increased HIF-1a activity (e.g., cancer, respiratory disorders
such as asthma
and obstructive pulmonary disorders) relative to the traditional modes of such
therapies.
Preferably the methods of the invention reduce the HIF-1 a level to the
background level
within 1 day, 1 week, I month, at least 2 months of the commencement of the
therapeutic
regime. In a most preferred embodiment, the methods of the invention result in
a reduction
of an HIF-l a level to the background level. The invention encompasses
reduction of the
HIF-1a level to a level which is within about 10%, about 20%, about 30%, about
40%,
about 50% of the background level.
In a preferred specific embodiment, the invention encompasses a method for
treatment, prevention and/or management of diseases or disorders associated
with
overexpression of HIF-la and/or increased HIF-la activity (e.g., cancer,
respiratory
disorders such as asthma and obstructive pulmonary disorders) comprising
administering a
therapeutically and/or prophylactically effective amount of an A3 receptor
antagonist
compound as disclosed herein.
Compounds of the present invention that function as prophylactic and/or
therapeutic agents of a disease or disorder can be administered to an animal,
preferably a
mammal, and most preferably a human, to treat, prevent or ameliorate one or
more
symptoms associated with the disease or disorder. The subject is preferably a
mammal such
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as non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a
primate (e.g., monkey,
such as a cynomolgous monkey and a human). In a preferred embodiment, the
subject is a
human. Compounds of the invention can be administered in combination with one
or more
other prophylactic and/or therapeutic agents useful in the treatment,
prevention or
management of a HIF-1 a-mediated disorders, which are improved or ameliorated
by
modulation of HIF-1 expression or activities.
The amount of the compounds of the invention (including adenosine Al
and/or A3 antagonists) required to be effective will, of course, vary with the
individual
mammal being treated and is ultimately at the discretion of the medical or
veterinary
practitioner. The factors to be considered include the condition being
treated, the route of
administration, the nature of the formulation, the mammal's body weight,
surface area, age
and general condition, and the particular compound to be administered.
However, a suitable
effective dose is in the range of about 0.1 g/kg to about 100 mg/kg, about
0.1 g/kg to
about 500 mg/kg, about 0.1 g/kg to about lg/kg, about 100 ug/kg to about 500
mg/kg,
about 100 ug/kg to aboutlg/kg, about 1 mg/kg to about 100 mg/kg, about 1 mg/kg
to about
500 mg/kg, about 1 mg/kg to about lg/kg of the patient's body weight.
The total daily dose may be given as a single dose, multiple doses, e.g=, two
to six times per day, or by intravenous infusion for a selected duration.
Dosages above or
below the range cited above are within the scope of the present invention and
may be
administered to the individual patient if desired and necessary.
The methods and compositions of the invention comprise the administration
of one or more compounds of the invention to subjects/patients suffering from
or expected
to suffer from a disease or disorder.
6.3.1 COMBINATION THERAPY
The invention encompasses combination therapies by administration of one
or more compounds of the invention in combination with administration of one
or more
other therapies that are traditionally used for the treatment and/or
prevention of the
particular disease or disorder being treated or prevented. For example in the
case of cancer,
the A3 and/or AI receptor antagonists of the invention may be administered in
combination
with one or more cancer therapies such as, but not limited to, chemotherapies,
radiation
therapies, hormonal therapies, and/or biological therapies/immunotherapies.
Prophylactic and therapeutic compounds that may be used in the methods
and compositions of the invention include, but are not limited to,
proteinaceous molecules,
including, but not limited to, peptides, polypeptides, proteins, including
post-translationally
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modified proteins, antibodies, etc.; small molecules (less than 1000 daltons),
inorganic or
organic compounds; nucleic acid molecules including, but not limited to,
double-stranded or
single-stranded DNA, double-stranded or single-stranded RNA, as well as triple
helix
nucleic acid molecules. Prophylactic and therapeutic compounds can be derived
from any
known organism (including, but not limited to, animals, plants, bacteria,
fungi, and protista,
or viruses) or from a library of synthetic molecules. In certain embodiments,
one or more
compounds of the invention are administered to a mammal, preferably a human,
concurrently with one or more other therapeutic agents useful for the
treatment of cancer or
a disorder. The term "concurrently" is not limited to the administration of
prophylactic or
therapeutic agents at exactly the same time, but rather it is meant that
compounds of the
invention and the other agent are administered to a subject in a sequence and
within a time
interval such that the compounds of the invention can act together with the
other agent to
provide an increased benefit than if they were administered otherwise. For
example, each
prophylactic or therapeutic agent may be administered at the same time or
sequentially in
any order at different points in tiine; however, if not administered at the
same time, they
should be administered sufficiently close in time so as to provide the desired
therapeutic or
prophylactic effect. Each therapeutic agent can be administered separately, in
any
appropriate form and by any suitable route.
In various embodiments, the prophylactic or therapeutic agents are
administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to
about 2 hours
apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4
hours apart, at
about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart,
at about 6
hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at
about 8 hours to
about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10
hours to about 11
hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours
apart or no
more than 48 hours apart. In preferred embodiments, two or more components are
administered within the same patient visit.
The dosage amounts and frequencies of administration provided herein are
encompassed by the terms therapeutically effective and prophylactically
effective. The
dosage and frequency further will typically vary according to factors specific
for each
patient depending on the specific therapeutic or prophylactic agents
administered, the
severity and type of cancer, the route of administration, as well as age, body
weight,
response, and the past medical history of the patient. Suitable regimens can
be selected by
one skilled in the art by considering such factors and by following, for
example, dosages
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reported in the literature and recommended in the Physician's Desk Reference
(58th ed.,
2004).
6.3.2 ANTAGONIST-BASED THERAPY/PROPHYLAXIS
6.3.2.1 CANCERS
Compounds of the invention, particularly the Al and A3 receptor antagonists,
can be used alone or in combination with other anti-cancer agents, e.g.,
therapeutic
antibodies known in the art to prevent, inhibit or reduce the growth of
primary tumors or
metastasis of cancerous cells. In a specific embodiment, a compound of the
invention,
when administered alone or in combination with an anti-cancer agent, inhibits
or reduces
the growth of primary tumor or metastasis of cancerous cells by at least 99%,
at least 95%,
at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least
60%, at least
50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at
least 25%, at
least 20%, or at least 10% relative to the growth of primary tumor or
metastasis in absence
of said compound of the invention. In a preferred embodiment, compounds of the
invention
particularly the Al and A3 receptor antagonists in combination with an anti-
cancer agent
inhibit or reduce the growth of primary tumor or metastasis of cancer by at
least 99%, at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least 60%,
at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least
30%, at least
25%, at least 20%, or at least 10% relative to the growth or metastasis in
absence of said
compounds. The methods of the invention are particularly useful for the
treatment of solid
tumors, hypoxic tumors, A3 expressing tumors and HIF-la expressing tumors.
The compounds of the invention particularly the Al and A3 receptor
antagonists can be used to treat solid tumors. Solid tumors which can be
treated in
accordance with the methods of the invention include without limitation
fibrodysplasia
ossificans progressiva, prostate cancer, benign prostatic hyperplasia,
recessive dystrophic
epidermolysis bullosa (RDEB), Lewis lung carcinoma, breast cancer, and brain
tumors.
As used herein, the term "A3 expressing cancers" refers to human cancers
that express the adenosine A3 receptor or that otherwise comprise elevated
concentrations of
adenosine A3 receptors. Elevated concentration is determined by comparison to
normal,
non-cancerous tissues of a similar cell type. Examples of A3 expressing
cancers include,
without limitation, human leukemia, melanoma, pancreatic carcinoma, breast
carcinoma,
prostrate carcinoma, colon carcinoma, lung carcinomamalignant melanomas,
histiocytic
lymphoma, and some forms of astrocytoma cells.
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As used herein the term "HIF-1 a expressing cancers" refers to human cancer
that express HIF- 1 a or that otherwise comprise elevated concentrations of
HIF-1 a.
Elevated concentration is determined by comparison to normal, non-cancerous
tissues of a
similar cell type. Examples of HIF-1 a expressing cancers include without
limitation
cervical cancer (early stage), lung cancer (non-small cell lung carcinoma),
breast cancer
(including lymph node positive breast cancer and lymph node negative breast
cancer),
oligodendroglioma, orpharyngeal squamous cell carcinoma, ovarian cancer,
oesophageal
cancer, endometrial cancer, head and neck cancer, gastrointestinal stromal
tumor of the
stomach.
In particular embodiments, the methods and compositions of the invention
comprise the administration of one or more A3 receptor antagonist compounds of
the
invention (alone or in combination with other anti-cancer agents) to
subjects/patients
suffering from or expected to suffer from cancer, e.g., have a genetic
predisposition for a
particular type of cancer, have been exposed to a carcinogen, or are in
remission from a
particular cancer. The methods and compositions of the invention may be used
as a first
line or second line cancer treatment. Included in the invention is also the
treatment of
patients undergoing other cancer therapies and the methods and compositions of
the
invention can be used before any adverse effects or intolerance of these other
cancer
therapies occurs. The invention also encompasses methods for administering one
or more
A3 receptor antagonist compounds of the invention to treat or ameliorate
symptoms in
refractory patients. In a certain embodiment, that a cancer is refractory to a
therapy means
that at least some significant portion of the cancer cells are not killed or
their cell division
arrested. The determination of whether the cancer cells are refractory can be
made either in
vivo or in vitro by any method known in the art for assaying the effectiveness
of treatment
on cancer cells, using the art-accepted meanings of "refractory" in such a
context. In
various embodiments, a cancer is refractory where the number of cancer cells
has not been
significantly reduced, or has increased. The invention also encompasses
methods for
administering one or more A3 receptor antagonist compounds of the invention to
prevent the
onset or recurrence of cancer in patients predisposed to having cancer.
In particular embodiments, the A3 receptor antagonists of the invention are
administered to reverse resistance or reduced sensitivity of cancer cells to
certain hormonal,
radiation and chemotherapeutic agents thereby resensitizing the cancer cells
to one or more
of these agents, which can then be administered (or continue to be
administered) to treat or
manage cancer, including to prevent metastasis.
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In alternate embodiments, the invention provides methods for treating cancer
in a subject by administering one or more A3 receptor antagonists of the
invention in
combination with any other treatment or to patients who have proven refractory
to other
treatments but are no longer on these treatments. In certain embodiments, the
patients being
treated by the methods of the invention are patients already being treated
with
chemotherapy, radiation therapy, hormonal therapy, or biological
therapy/immunotherapy.
Among these patients are refractory patients and those with cancer despite
treatment with
existing cancer therapies. In other embodiments, the patients have been
treated and have no
disease activity and one or more A3 receptor antagonist compounds of the
invention are
administered to prevent the recurrence of cancer.
Additionally, the invention also provides methods of treatment of cancer as
an alternative to chemotherapy, radiation therapy, hormonal therapy, and/or
biological
therapy/immunotherapy where the therapy has proven or may prove too toxic,
i.e., results in
unacceptable or unbearable side effects, for the subject being treated. The
subject being
treated with the methods of the invention may, optionally, be treated with
other cancer
treatments such as surgery, chemotherapy, radiation therapy, hormonal therapy
or biological
therapy, depending on which treatment was found to be unacceptable or
unbearable.
In other embodiments, the invention provides administration of one or more
A3 receptor antagonist compounds of the invention without any other cancer
therapies for
the treatment of cancer, but who have proved refractory to such treatments. In
specific
embodiments, patients refractory to other cancer therapies are administered
one or more A3
receptor antagonist compounds in the absence of cancer therapies.
In a preferred embodiment when treating A3 expressing cancers, a high
affinity adenosine A3 receptor antagonist and a chemotherapeutic cancer agent
are
administered to the patient. The combination therapy enhances the effect of
the
chemotherapeutic cancer agent and prevents multi-drug resistance from
developing.
Although not intending to be bound to a particular mechanism of action, the
chemotherapeutic cancer agents showing desirable response to the present
invention are
typical of agents noted for developing P-gp or MRP class multi-drug
resistance, including,
without limitation, taxane compounds, vinca alkaloids, camptothecins and
antibiotics useful
as chemotherapeutic agents.
The invention encompasses combination therapies for treating cancers that
have already developed multi-drug resistance. In this case, the high affinity
adenosine A3
receptor antagonist counters the existing MDR while further enhancing the
effect of the
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chemotherapeutic cancer agent. As exemplified in the working examples, the
present
invention is not effective for all forms of MDR cancers. The MDR cancers
showing
desirable response to the present invention are in the P-gp and MRP classes.
When the chemotherapeutic cancer agents is a compound of the taxane
family, the high affinity adenosine A3 receptor antagonist is preferably
administered either
before or during administration of the taxane compound. Although not intending
to be
bound to a particular theory, this is done to reduce the incidence of
hypersensitivity to the
taxane agent. Commercially available compounds of the taxane family are
paclitaxel
(commercially available under the tradename TAXOL from Bristol-Myers Squibb
Company, Princeton, NY and as a generic drug from IVAX Corp, Miami, FL) and
docetaxel (commercially available under the tradename TAXOTERE from Aventis
Pharmaceuticals, Collegeville, PA), which are encompassed in the instant
invention. It is
noted that additional taxane family chemotherapeutic cancer agents are
presently in
development and testing and encompassed within the invention.
In another preferred embodiment for treating existing tumors, the
composition comprises an effective amount of an adenosine A3 receptor
antagonist and a
chemotherapeutic agent that is a taxane family compound, for example
paclitaxel or
docetaxel to inhibit tumor growth. Paclitaxel is commercially available under
the
tradename TAXOL from Bristol-Myers Squibb Company, Princeton, NY and as a
generic
drug from IVAX Corp, Miami, FL. Docetaxel is commercially available under the
tradename TAXOTERE from Aventis Pharmaceuticals, Collegeville, PA. In this
embodiment the A3 antagonist may reduce the growth rate of cancerous cells,
interfere with
adenosine protective effects against hypoxia, enhance the chemotherapeutic
effect of the
taxane, reduce hypersensitivity reactions in the patient and counter
development of multi-
drug resistance.
Cancers and related disorders that can be treated or prevented by methods
and compositions comprising Al and/or A3 receptor antagonists of the present
invention
include, but are not limited to, the following: Leukemias including, but not
limited to, acute
leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as
myeloblastic,
promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and
myelodysplastic syndrome, chronic leukemias such as but not limited to,
chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia;
polycythemia
vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's
disease;
multiple myelomas such as but not limited to smoldering multiple myeloma,
nonsecretory
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myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma
and
extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal
gammopathy of undetermined significance; benign monoclonal gammopathy; heavy
chain
disease; bone and connective tissue sarcomas such as but not limited to bone
sarcoma,
osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,
fibrosarcoma
of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma
(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,
liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain
tumors
including but not limited to, glioma, astrocytoma, brain stem glioma,
ependymoma,
oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain
lymphoma;
breast cancer including, but not limited to, adenocarcinoma, lobular (small
cell) carcinoma,
intraductal carcinoma, medullary breast cancer, mucinous breast cancer,
tubular breast
cancer, papillary breast cancer, Paget's disease, and inflammatory breast
cancer; adrenal
cancer, including but not limited to, pheochromocytom and adrenocortical
carcinoma;
thyroid cancer such as but not limited to papillary or follicular thyroid
cancer, medullary
thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but
not limited to,
insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and
careinoid
or islet cell tumor; pituitary cancers including but not limited to, Cushing's
disease,
prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers
including but not
limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and
cilliary body
melanoma, and retinoblastoma; vaginal cancers, including but not limited to,
squamous cell
carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not
limited to,
squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma,
sarcoma, and
Paget's disease; cervical cancers including but not limited to, squamous cell
carcinoma, and
adenocarcinoma; uterine cancers including but not limited to, endometrial
carcinoma and
uterine sarcoma; ovarian cancers including but not limited to, ovarian
epithelial carcinoma,
borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers
including but not
limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,
mucoepidermoid
carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers including but
not limited to,
adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading,
diffusely
spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma;
colon
cancers; rectal cancers; liver cancers including but not limited to
hepatocellular carcinoma
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and hepatoblastoma, gallbladder cancers including but not limited to,
adenocarcinoma;
cholangiocarcinomas including but not limited to, pappillary, nodular, and
diffuse; lung
cancers including but not limited to, non-small cell lung cancer, squamous
cell carcinoma
(epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell
lung cancer;
testicular cancers including but not limited to, gerninal tumor, seminoma,
anaplastic,
classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma
carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers including but not limited
to,
adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral
cancers
including but not limited to, squamous cell carcinoma; basal cancers; salivary
gland cancers
including but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and
adenoidcystic carcinoma; pharynx cancers including but not limited to,
squamous cell
cancer, and verrucous; skin cancers including but not limited to, basal cell
carcinoma,
squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular
melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney
cancers
including but not limited to, renal cell cancer, adenocarcinoma, hypemephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms'
tumor; bladder
cancers including but not limited to, transitional cell carcinoma, squamous
cell cancer,
adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic
sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,
synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic
carcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and
papillary
adenocarcinomas (for a review of such disorders, see Fishman et al., 1985,
Medicine, 2d
Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed
Decisions: The
Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,
Penguin
Books U.S.A., Inc., United States of America).
Accordingly, the methods and compositions of the invention are also useful
in the treatment or prevention of a variety of cancers or other abnormal
proliferative
diseases, including (but not limited to) the following: carcinoma, including
that of the
bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix,
thyroid and
skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid
lineage,
including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell
lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid
lineage, including acute and chronic myelogenous leukernias and promyelocytic
leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
other
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tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and
glioma;
tumors of the central and peripheral nervous system, including astrocytoma,
neuroblastoma,
glioma, and schwannomas; tumors of mesenchymal origin, including fibrosafcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma,
xenoderma
pegmeritosum, keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma.
It is also contemplated that cancers caused by aberrations in apoptosis would
also be treated
by the methods and compositions of the invention. Such cancers may include but
not be
limited to follicular lymphomas, carcinomas with p53 mutations, hormone
dependent
tumors of the breast, prostate and ovary, and precancerous lesions such as
familial
adenomatous polyposis, and myelodysplastic syndromes. In specific embodiments,
malignancy or dysproliferative changes (such as metaplasias and dysplasias),
or
hyperproliferative disorders, are treated or prevented by the methods and
compositions of
the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or
uterus. In other
specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented
by the
methods and compositions of the invention.
Cancers associated with the cancer antigens may be treated or prevented by
administration of the A3 receptor antagonists of the invention in combination
with an
antibody that binds the cancer antigen and is cytotoxic. In one particular
embodiment, the
A3 receptor antagonists of the invention enhance the antibody mediated
cytotoxic effect of
the antibody directed at the particular cancer antigen. For example, but not
by way of
limitation, cancers associated with the following cancer antigen may be
treated or prevented
by the methods and compositions of the invention. KS 1/4 pan-carcinoma antigen
(Perez
and Walker, 1990, J Immunol. 142:32-37; Bumal, 1988, Hybridoma 7(4):407-415),
ovarian
carcinoma antigen (CA125) (Yu et al., 1991, Cancer Res. 51(2):48-475),
prostatic acid
phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(l):4928), prostate
specific antigen
(Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 10(2):903-910; Israeli
et al.,
1993, Cancer Res. 53:227-230), melanoma-associated antigen p97 (Estin et al.,
1989, J.
Natl. Cancer Instit. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et
al., 1990, J.
Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA)
(Natali et al., 1987, Cancer 59:55-3; Mittelman et al., 1990, J. Clin. Invest.
86:2136-2144)),
prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et
al., 1994,
Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen,
human milk fat
globule antigen, Colorectal tumor-associated antigens such as: CEA, TAG-72
(Yokata et
al., 1992, Cancer Res. 52:3402-3408), C017-1A (Ragnhammar et al., 1993, Int.
J. Cancer
68
CA 02586420 2007-05-03
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53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2:135), CTA-1
and LEA,
Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-
1336),
human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445), CD33
(Sgouros
et al., 1993, J. Nucl. Med. 34:422-430), melanoma specific antigens such as
ganglioside
GD2 (Saleh et al., 1993, J.Immunol., 151, 3390-3398), ganglioside GD3 (Shitara
et al.,
1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et
al.,
1994, J Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer
Res.
53:5244-5250), tumor-specific transplantation type of cell-surface antigen
(TSTA) such as
virally-induced tumor antigens including T-antigen DNA tumor viruses and
envelope
antigens of RNA tumor viruses, oncofetal antigen-alpha-fetoprotein such as CEA
of colon,
bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-
2188),
differentiation antigen such as human lung carcinoma antigen L6, L20
(Hellstrom et al.,
1986, Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia T
cell
antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. oflmmun. 141:1398-
1403),
neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal
growth
factor receptor), HER2 antigen (p185HEP2), polymorphic epithelial mucin (PEM)
(Hilkens et
al., 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte
antigen-APO-1
(Bernhard et al., 1989, Science 245:301-304), differentiation antigen (Feizi,
1985, Nature
314:53-57) such as I antigen found in fetal erthrocytes and primary endoderm,
I(Ma) found
in gastric adencarcinomas, M18 and M3 9 found in breast epithelium, SSEA-1
found in
myeloid cells, VEP8, VEP9, Myl, VIM-D5,and D156-22 found in colorectal cancer,
TRA-1-
85 (blood group H), C 14 found in colonic adenocarcinoma, F3 found in lung
adenocarcinoma, AH6 found in gastric cancer, Y hapten, LeY found in embryonal
carcinoma
cells, TL5 (blood group A), EGF receptor found in A43 1 cells, El series
(blood group B)
found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric
adenocarcinoma, CO-514 (blood group Lea) found in adenocarcinoma, NS-10 found
in
adenocarcinomas, CO-43 (blood group Leb), G49, EGF receptor, (blood group
ALeb/Ley)
found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer
mucins, T5A7
found in myeloid cells, R24 found in melanoma, 4.2, GD3, D1.1, OFA- 1, GM2,
OFA-2, GD2,
M1:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-
cell
stage embryos. In another embodiment, the antigen is a T cell receptor derived
peptide
from a cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62).
6.3.2.1.1 OTHER AGENTS
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The high affinity adenosine A3 receptor antagonists of the invention can be
administered alone or in combination with other therapeutic and/or
prophylactic agents.
The methods of the invention encompass the administration of one or more
chemotherapeutic cancer agents. Chemotherapeutic cancer agents are defined as
agents that
attack and kill cancer cells. Chernotherapeutic agents that may be used
in.=the methods of
the invention include taxane compounds and derivatives thereof, paclitaxel and
its
derivatives or docetaxel and its derivatives. Additional taxane derivatives
and methods of
synthesis are disclosed in U.S. Patent No. 6,191,287 to Holton et al., U.S.
Patent No.
5,705,508 to Ojima et al., U.S. Patent Nos. 5,688,977 and 5,750,737 to Sisti
et al., U.S.
Patent No. 5,248,796 to Chen et al., U.S. Patent No. 6,020,507 to Gibson et
al., U.S. Patent
No. 5,908,835 to Bissery, all of which are incorporated herein by reference in
their
entireties.
Other chemotherapeutic cancer agents that may be used in the methods of the
invention include mitotic inhibitors (vinca alkaloids). These include
vincristine, vinblastine,
vindesine and NavelbineTM (vinorelbine,5'-noranhydroblastine). In yet other
embodiments,
chemotherapeutic cancer agents include topoisomerase I inhibitors, such as
camptothecin
compounds. As used herein, "camptothecin compounds" include CamptosarTM
(irinotecan
HCL), HycamtinTM (topotecan HCL) and other compounds derived from camptothecin
and
its analogues. Another category of chemotherapeutic cancer agents that may be
used in the
methods and compositions of the invention are podophyllotoxin derivatives,
such as
etoposide, teniposide and mitopodozide. The invention further encompasses
other
chemotherapeutic cancer agents known as alkylating agents, which alkylate the
genetic
material in tumor cells. These include without limitation cisplatin,
cyclophosphamide,
nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan,
chlorambucil,
belustine, uracil mustard, chlornaphazin, and dacarbazine. The invention
encompasses
antimetabolites as chemotherapeutic agents. Examples of these types of agents
include
cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine,
azathioprime, and
procarbazine. An additional category of chemotherapeutic cancer agents that
may be used
in the methods and compositions of the invention include antibiotics. Examples
include
without limitation doxorubicin, bleomycin, dactinomycin, daunorubicin,
mithramycin,
mitomycin, mytomycin C, and daunomycin. There are numerous liposomal
formulations
commercially available for these compounds. The invention further encompasses
other
chemotherapeutic cancer agents including without limitation anti-tumor
antibodies,
dacarbazine, azacytidine, amsacrine, melphalan, ifosfamide and mitoxantrone.
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The A3 antagonists can be administered alone or in combination with other
anti-tumor agents, including cytotoxic/antineoplastic agents and anti-
angiogenic agents.
Cytotoxic/anti-neoplastic agents are defined as agents which attack and kill
cancer cells.
Some cytotoxic/anti-neoplastic agents are alkylating agents, which alkylate
the genetic
material in tumor cells, e.g., cis-platin, cyclophospharnide, nitrogen
mustard, trimethylene
thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil
mustard,
chlornaphazin, and dacabazine. Other cytotoxic/anti-neoplastic agents are
antimetabolites
for tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate,
mercaptopuirine,
azathioprime, and procarbazine. Other cytotoxic/anti-neoplastic agents are
antibiotics, e.g.,
doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin,
mytomycin
C, and daunomycin. There are numerous liposomal formulations commercially
available
for these compounds. Still other cytotoxic/anti-neoplastic agents are mitotic
inhibitors
(vinca alkaloids). These include vincristine, vinblastine and etoposide.
Miscellaneous
cytotoxic/anti-neoplastic agents include taxol and its derivatives, L-
asparaginase, anti-tumor
antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide,
mitoxantrone, and vindesine.
Anti-angiogenic agents are well known to those of skill in the art. Suitable
anti-angiogenic agents for use in the methods and compositions of the
invention include
anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF
aptamers
and antisense oligonucleotides. Other known inhibitors of angiogenesis include
angiostatin,
endostatin, interferons, interleukin 1 (including a and (3) interleukin 12,
retinoic acid, and
tissue inhibitors of inetalloproteinase-1 and -2. (TIMP- 1 and -2). Small
molecules,
including topoisomerases such as razoxane, a topoisomerase II inhibitor with
anti-
angiogenic activity, can also be used.
In one embodiment, the anti-angiogenesis compound is an adenosine A2a
antagonist. A2a antagonists are well known to those of skill in the art.
Examples of these
compounds include SCH 58261 from Schering Plough, ZM241385 (Palmer et al.,
Mol.
Pharmacol., 48:970-974 (1995), the tricyclic non-xanthine antagonists and the
triazoloquinazolines, including CP 66,713, disclosed in Nikodijevic et al., J.
Pharmacol.
Exp. Ther., 259:286-294 (1991), as well as the compounds disclosed in von
Lubitz et al.,
Eur. J. Pharmacol., 287:295-302 (1995). The contents of each of the references
above are
hereby incorporated by reference.
Anti-cancer agents that can be used in combination with A3 receptor
antagonists of the invention in the various embodiments of the invention,
including
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pharmaceutical conzpositions and dosage forms and kits of the invention,
include, but are
not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine;
adozelesin;
aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide;
amsacrine;
anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;
azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide
dimesylate;
bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;
cactinomycin;
calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride;
carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine;
crisnatol mesylate;
cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin
hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone;
docetaxel;
doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanolone
propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin;
enloplatin;
enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin
hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide
phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;
fludarabine
phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;
gemcitabine;
gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine;
interleukin II (including recombinant interleukin II, or rIL2), interferon
alfa-2a; interferon
alfa-2b; interferon alfa-nl ; interferon alfa-n3; interferon beta-I a;
interferon gamma-I b;
iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;
leuprolide acetate;
liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate=,
melengestrol
acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate
sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin;
mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;
nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase;
peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone
hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin;
prednimustine;
procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine;
rogletimide; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium;
sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;
streptonigrin;
streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafiar; teloxantrone
hydrochloride;
temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa;
tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine
phosphate;
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trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride;
uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;
vindesine;
vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine
tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin
hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-
1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene;
adecypenol;
adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;
amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;
antarelix;
anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis
gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine
deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin
3; azasetron;
azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin
B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene;
bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine;
calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;
capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700;
cartilage
derived inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B;
cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine;
clomifene analogues; clotrimazole; collismycin A; collismycin B;
combretastatin A4;
combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin
8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam;
cypemycin;
cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
dehydrodidemnin
B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone;
didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-
;
dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron;
doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;
edrecolomab;
eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine
analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;
fluasterone;
fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin;
fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase
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inhibitors; gemeitabine; glutathione inhibitors; hepsulfam; heregulin;
hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone;
ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like
growth
factor-1 receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane;
iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;
isohomohalicondrin B;
itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;
leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte
alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole;
linear polyamine analogue; lipophilic disaccharide peptide; lipophilic
platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;
maitansine;
mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopiamide; MIF inhibitor; mifepristone; miltefosine; mirimostim;
mismatched double
stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide;
mitotoxin
fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal
antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium
cell
wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor
suppressor
1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell
wall
extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;
nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;
nemorubicin;
neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators;
nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;
oligonucleotides;
onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;
ormaplatin;
osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives;
palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;
parabactin;
pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium;
pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
phosphatase
inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim;
placetin A; placetin
B; plasminogen activator inhibitor; platinum complex; platinum compounds;
platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl
bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune
modulator;
protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein
tyrosine
phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins;
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pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists;
raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras
inhibitors; ras-GAP
inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII
retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1;
raboxyl;
safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;
semustine;
senescence derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal
transduction modulators; single chain antigen binding protein; sizofiran;
sobuzoxane;
sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding
protein;
sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1;
squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide;
stromelysin
inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen
methiodide;
tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;
telomerase inhibitors;
temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine;
thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;
thymopoietin receptor
agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell factor;
translation
inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron;
turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;
ubenimex; urogenital
sinus-derived growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin
B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin;
vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;
and zinostatin
stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil and
leucovorin.
Examples of therapeutic antibodies that can be used in methods of the
invention include but are not limited to HERCEPTIN (Trastuzumab) (Genentech,
CA)
which is a humanized anti-HER2 monoclonal antibody for the treatment of
patients with
metastatic breast cancer; REOPRO (abciximab) (Centocor) which is an anti-
glycoprotein
IIb/IIIa receptor on the platelets for the prevention of clot formation;
ZENAPAX
(daclizumab) (Roche Pharmaceuticals, Switzerland) which is an
immunosuppressive,
humanized anti-CD25 monoclonal antibody for the prevention of acute renal
allograft
rejection; PANOREXTM which is a murine anti-17-IA cell surface antigen IgG2a
antibody
(Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)
IgG
antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody
(ImClone System); VITAXINTM which is a humanized anti-aV03 integrin antibody
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(Applied Molecular Evolution/Medlmmune); Campath 1H/LDP-03 which is a
humanized
anti CD52 IgGl antibody (Leukosite); Smart M195 which is a humanized anti-CD33
IgG
antibody (Protein Design Lab/Kanebo); RITUXANTM which is a chimeric anti-CD20
IgGl
antibody (IDEC Phaml/Genentech, Roche/Zettyaku); LYMPHOCIDETM which is a
humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-
ICAM3
antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDEC
Pharm/Mitsubishi); ZEVALINTM is a radiolabelled murine anti-CD20 antibody
(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai);
IDEC-
151 is a primatized anti-CD4 antibody (IDEC); IDEC-1 52 is a primatized anti-
CD23
antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein
Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody
(Alexion
Pharm); D2E7 is a humanized anti-TNF-a antibody (CAT/BASF); CDP870 is a
humanized
anti-TNF-a Fab fragment (Celltech); IDEC- 151 is a primatized anti-CD4 IgGl
antibody
(IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody
(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-a IgG4 antibody
(Celltech);
LDP-02 is a humanized anti-a4(37 antibody (LeukoSite/Genentech); OrthoClone
OKT4A is
a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVATM is a humanized
anti-
CD40L IgG antibody (Biogen); ANTEGR.ENTM is a humanized anti-VLA-4 IgG
antibody
(Elan); and CAT-152 is a human anti-TGF-(32 antibody (Cambridge Ab Tech).
Other examples of therapeutic antibodies that can be used in combination
with the A3 receptor antagonists of the invention are presented in Table 5
below:
TABLE 5. MONOCLONAL ANTIBODIES FOR CANCER THERAPY THAT CAN BE USED IN
COMBINATION WITH THE A3 RECEPTOR ANTAGONISTS OF THE INVENTION.
Company Product Disease Target
Abgenix ABX-EGF Cancer EGF receptor
AltaRex OvaRex ovarian cancer tumor antigen CA125
BravaRex metastatic cancers tumor antigen MUC I
Antisoma Theragyn (pemtumomabytrrium-90) ovarian cancer PEM antigen
Therex breast cancer PEM antigen
Boehringer Ingelheim blvatuzumab head & neck cancer CD44
Centocor/J&J Panorex Colorectal cancer 17-1A
ReoPro PTCA gp IIIb/IIIa
ReoPro Acute MI gp IIIb/IIIa
ReoPro Ischemic stroke gp IIIb/IIIa
Corixa Bexocar NHL CD20
CRC Technology MAb, idiotypio 105AD7 colorectal cancer vaccine gp72
Crucell Anti-EpCAM cancer Ep-CAM
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Company Product Disease Target
Cytoclonal MAb, lung cancer non-small cell lung NA
cancer
Genentech Herceptin metastatic breast cancer HER-2
Herceptin early stage breast cancer HER-2
Rituxan Relapsed/refractory low- CD20
grade or follicular NHL
Rituxan intermediate & high- CD20
grade NHL
MAb-VEGF NSCLC, metastatic VEGF
MAb-VEGF Colorectal cancer, VEGF
metastatic
AMD Fab age-related macular CD18
degeneration
E-26 (2" gen. IgE) allergic asthma & rhinitis IgE
IDEC Zevalin (Rituxan + yttrium-90) low grade of follicular, CD20
relapsed or refractory,
CD20-positive, B-cell
NHL and Rituximab-
refractory NHL
ImClone Cetuximab + innotecan refractory colorectal EGF receptor
carcinoma
Cetuximab + cisplatin & radiation newly diagnosed or ' EGF receptor
recurrent head & neck
cancer
Cetuximab + gemcitabine newly diagnosed EGF receptor
metastatic pancreatic
carcinoma
Cetuximab + cisplatin + 5FU or recurrent or metastatic EGF receptor
Taxol head & neck cancer
Cetuximab + carboplatin + newly diagnosed non- EGF receptor
paclitaxel small cell lung carcinoma
Cetuximab + cisplatin head & neck cancer EGF receptor
(extensive incurable
local-regional disease &
distant metasteses)
Cetuximab + radiation locally advanced head & EGF receptor
neck carcinoma
BEC2 + Bacillus Calmette Guerin small cell lung carcinoma mimics ganglioside
GD3
BEC2 + Bacillus Calmette Guerin melanoma mimics ganglioside GD3
IMC-1C11 colorectal cancer with VEGF-receptor
liver metasteses
ImmonoGen nuC242-DM1 Colorectal, gastric, and nuC242
pancreatic cancer
ImmunoMedics LymphoCide Non-Hodgkins CD22
lymphoma
LymphoCide Y-90 Non-Hodgkins CD22
lymphoma
CEA-Cide metastatic solid tumors CEA
CEA-Cide Y-90 metastatic solid tumors CEA
CEA-Scan (Tc-99m-labeled colorectal cancer CEA
arcitumomab) (radioimaging)
CEA-Scan (Tc-99m-labeled Breast cancer CEA
arcitumomab) (radioimaging)
CEA-Scan (Tc-99m-labeled lung cancer CEA
arcitumomab) (radioimaging)
CEA-Scan (Tc-99m-labeled intraoperative tumors CEA
arcitumomab) (radio imaging)
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Company Product Disease Target
LeukoScan (Tc-99m-labeled soft tissue infection CEA
sulesomab) (radioimaging)
LymphoScan (Tc-99m-labeled) lymphomas CD22
(radioimaging)
AFP-Scan (Tc-99m-labeled) liver 7 gem-cell cancers AFP
(radioimaging)
Intracel HumaRAD-HN (+ yttrium-90) head & neck cancer NA
HumaSPECT colorectal imaging NA
Medarex MDX-101 (CTLA-4) Prostate and other CTLA-4
cancers
MDX-210 (her-2 overexpression) Prostate cancer HER-2
MDX-210/MAK Cancer HER-2
Medlmmune Vitaxin Cancer q,yO3
Merck KGaA MAb 425 Various cancers EGF receptor
IS-IL-2 Various cancers Ep-CAM
Millennium Campath (alemtuzumab) chronic lymphocytic CD52
leukemia
NeoRx CD20-streptavidin (+ biotin-yttrium Non-Hodgkins CD20
90) lymphoma
Avidicin (albumin+ NRLU 13) metastatic cancer NA
Peregrine Oncolym (+ iodine-131) Non-Hodgkins HLA-DR 10 beta
lymphoma
Cotara (+ iodine-131) unresectable malignant DNA-associated proteins
glioma
Pharmacia Corporation C215 (+ staphylococcal enterotoxin) pancreatic cancer NA
MAb, lung/kidney cancer lung & kidney cancer NA
nacolomab tafenatox (C242 + colon & pancreatic NA
staphylococcal enterotoxin) cancer
Protein Design Labs Nuvion T cell malignancies CD3
SMART M195 AML CD33
SMART 1D10 NHL HLA-DR antigen
Titan CEAVac colorectal cancer, CEA
advanced
TriGem metastatic melanoma & GD2-ganglioside
small cell lung cancer
TriAb metastatic breast cancer MUC-1
Trilex CEAVac colorectal cancer, CEA
advanced
TriGem metastatic melanoma & GD2-ganglioside
small cell lung cancer
TriAb metastatic breast cancer MUC-1
Viventia Biotech NovoMAb-G2 radiolabeled Non-Hodgkins NA
lymphoma
Monopharm C colorectal & pancreatic SK-1 antigen
carcinoma
GlioMAb-H (+ gelonin toxin) glioma, melanoma & NA
neuroblastoma
Xoma Rituxan Relapsed/refractory low- CD20
grade or follicular NHL
Rituxan intermediate & high- CD20
grade NHL
ING-1 adenomcarcinoma Ep-CAM
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The A3 receptor antagonists of the invention may be used in combination with
one or more
ATP depleting agents for enhancing the therapeutic efficacy of treating a
disease or disorder
as disclosed herein, i.e., cancer. Alternatively, the A3 receptor antagonists
of the invention
may be used with verapamil for enhancing the therapeutic efficacy of
conventional modes
of treating a disease or disorder as discussed herein, i.e., cancer.
6.3.2.2 RESPIRATORY DISORDERS
Compounds of the invention, particularly the Al and A3 receptor antagonists,
can be used alone or in combination with other therapeutic and/or prophylactic
agents to
prevent, treat or manage respiratory disorders such as asthma and obstructive
pulmonary
disorders (COPD).
The methods and compositions comprising Al and/or A3 receptor antagonists
of the invention are effective for treatment, prevention, and/or management of
asthma. The
therapeutic and prophylactic methods of the invention for asthma may be used
in
combination with other methods known in the art for the treatmerit, prevention
and/or
management of asthma including but not limited to inhaled beta 2 agonists,
inhaled
corticosteroids, retinoic acid, anti-IgE antibodies, phosphodiesterase
inhibitors, leukotriene
antagonists, anti IL-9 antibody, and/or anti-mucin therapies (e.g., anti
hCLCA1 therapy
such as LomucinTM). (3-adrenergic drugs (e.g. epinephrine and isoproterenol),
theophylline,
anticholinergic drugs (e.g., atropine and ipratorpium bromide), and
corticosteroids,
adrenergic stimulants (e.g., catecholamines (e.g., epinephrine, isoproterenol,
and
isoetharine), resorcinols (e.g., metaproterenol, terbutaline, and fenoterol),
and saligenins
(e.g., salbutamol), other steroids, immunosuppressant agents (e.g.,
methotrexate and gold
salts), mast cell modulators (e.g., cromolyn sodium (INTALTM) and nedocromil
sodium
(TILADETM)), and mucolytic agents (e.g., acetylcysteine)).
The methods and compositions comprising Al and/or A3 receptor antagonists
of the invention are effective for treatment, prevention, and/or management of
COPD
including fixed airways disorders, chronic bronchitis and emphysema.
The therapeutic and prophylactic methods of the invention may be used in
combination with other therapeutic methods known in the art for the treatment,
prevention
and/or management of COPD including but not limited tiotropium and/or
ipratropium
Other respiratory disorders that may be treated, prevented or managed using
the methods and compositions of the invention include without limitation lung
fibrosis,
bronchial hyper responsiveness.
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6.4 COMPOSITIONS AND METHODS OF ADMINISTERING
The invention provides methods and pharmaceutical compositions
comprising compounds of the invention. The invention also provides methods of
treatment,
prophylaxis, and amelioration of one or more symptoms associated with a
disease, disorder
or cancer by administering to a subject an effective amount of compound of the
invention.
In a specific embodiment, the subject is an animal, preferably a ma.mmal such
as non-
primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g.,
monkey such as, a
cynomolgous monkey and a human). In a preferred embodiment, the subject is a
human.
The compositions of the invention include bulk drug compositions useful in
the manufacture of pharmaceutical compositions (e.g., impure or non-sterile
compositions)
and pharmaceutical compositions (i.e., compositions that are suitable for
administration to a
subject or patient) which can be used in the preparation of unit dosage forms.
Such
compositions comprise a prophylactically or therapeutically effective amount
of a
prophylactic and/or therapeutic agent disclosed herein or a combination of
those agents and
a pharmaceutically acceptable carrier. Preferably, compositions of the
invention comprise a
prophylactically or therapeutically effective amount of compounds of the
invention and a
pharmaceutically acceptable carrier.
In one particular embodiment, the pharmaceutical composition comprises a
therapeutically or prophylactically effective amount of an A3 or Al receptor
antagonist and a
pharmaceutically acceptable carrier. In another embodiment, said
pharmaceutical
composition further comprises one or more anti-cancer agents.
In a specific embodiment, the tenn "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state government or listed
in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in animals,
and more
particularly in humans. The term "carrier" refers to a diluent, adjuvant
(e.g., Freund's
adjuvant (complete and incomplete), excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and oils,
including those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the
pharmaceutical composition is administered intravenously. Saline solutions and
aqueous
dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the
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like. The composition, if desired, can also contain minor amounts of wetting
or emulsifying
agents, or pH buffering agents. These compositions can take the form of
solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained-release
formulations and
the like.
Generally, the ingredients of compositions of the invention are supplied
either separately or mixed together in unit dosage form, for example, as a dry
lyophilized
powder or water free concentrate in a hermetically sealed container such as an
ampoule or
sachette indicating the quantity of active agent. Where the composition is to
be
administered by infusion, it can be dispensed with an infusion bottle
containing sterile
pharmaceutical grade water or saline. Where the composition is administered by
injection,
an ampoule of sterile water for injection or saline can be provided so that
the ingredients
may be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include, but are not limited to those formed
with anions
such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and
those formed with captions such as those derived from sodium, potassium,
ammonium,
calcium, ferric hydroxides, isopropylamine, triethylarnine, 2-ethylamino
ethanol, histidine,
procaine, etc.
The compounds described above are preferably administered in formulations
including an active compound, i.e., an adenosine Al and/or A3 receptor
antagonist, together
with an acceptable carrier for the mode of administration. Suitable
pharmaceutically
acceptable carriers are known to those of skill in the art. The compositions
can optionally
include other therapeutically active agents, including anti-angiogenic and
anti-cancer
agents. Other optional ingredients include antivirals, antibacterials, anti-
inflammatories,
analgesics, and immunosuppresants. The carrier must be pharmaceutically
acceptable in the
sense of being compatible with the other ingredients of the formulation and
not deleterious
to the recipient thereof.
The compounds of the invention, e.g., high affinity adenosine A3 receptor
antagonists, can be administered in a physiologically acceptable diluent in a
pharmaceutical
carrier, such as a sterile liquid or mixture of liquids, including water,
saline, aqueous
dextrose and related sugar solutions, an alcohol (e.g., ethanol, isopropanol,
or hexadecyl
alcohol), glycols (e.g., propylene glycol or polyethylene glycol), glycerol
ketals, (e.g., 2,2-
dimethy11,3-dioxolane-4-methanol), ethers, (e.g., poly(ethyleneglycol) 400),
an oil, a fatty
acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride
with or without the
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addition of a pharmaceutically acceptable surfactant (e.g., a soap or a
detergent), a
suspending agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxyrnethylcellulose, or emulsifying
agents and other
pharmaceutical excipients and adjuvants.
The formulations can include carriers suitable for oral, rectal, topical or
parenteral (including subcutaneous, intramuscular and intravenous)
administration.
Preferred carriers are those suitable for oral or parenteral administration.
Formulations suitable for parenteral administration conveniently include
sterile aqueous preparation of the active compound which is preferably
isotonic with the
blood of the recipient. Thus, such formulations may conveniently contain
distilled water,
5% dextrose in distilled water or saline. Useful formulations also include
concentrated
solutions or solids containing the compounds which upon dilution with an
appropriate
solvent give a solution suitable for parental administration above.
Formulations suitable for parenteral administration include but are not
limited to aqueous and non-aqueous solutions, isotonic sterile injection
solutions, which
may comprise anti-oxidants, buffers, bacteriostats, and solutes that render
the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that may comprise suspending agents, solubilizers, thickening
agents,
stabilizers, and preservatives.
The parenteral formulations will typically contain from about 0.5 to about
25% by weight of the active ingredient in solution. Suitable preservatives and
buffers can
be used in such formulations. In order to minimize or eliminate irritation at
the site of
injection, such compositions may contain one or more nonionic surfactants
having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity
of
surfactant in such formulations ranges from about 5 to about 15% by weight.
Suitable
surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and
the high molecular weight adducts of ethylene oxide with a hydrophobic base,
formed by
the condensation of propylene oxide with propylene glycol. The parenteral
formulations
can be presented in unit-dose or multi-dose sealed containers, such as ampules
and vials,
and can be stored in a freeze-dried (lyophilized) condition requiring only the
addition of the
sterile liquid carrier, for example, water, for injections, immediately prior
to use.
Extemporaneous injection solutions and suspensions can be prepared from
sterile powders,
granules, and tablets of the kind previously described.
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The compounds of the inventione, e.g., high affinity adenosine A3 receptor
antagonists may be made into injectable formulations. The requirements for
effective
pharmaceutical carriers for injectable compositions are well known to those of
ordinary skill
in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,
Philadelphia,
Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on
Injectable
Drugs, Toissel, 4th ed., pages 622-630 (1986); which are incorporated herein
by reference
in their entireties.
For enteral administration, the compound can be incorporated into an inert
carrier in discrete units such as capsules, cachets, tablets or lozenges, each
containing a
predetermined amount of the active compound; as a powder or granules; or a
suspension or
solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir,
an emulsion or a
draught. Suitable carriers may be starches or sugars and include lubricants,
flavorings,
binders, and other materials of the same nature.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the compound dissolved in diluents,
such as water,
saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and
troches, each containing
a predetermined amount of the active ingredient, as solids or granules; (c)
powders; (d)
suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid
formulations may
include diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the
polyethylene alcohols, either with or without the addition of a
pharmaceutically acceptable
surfactant, suspending agent, or emulsifying agent. Capsule forms can be of
the ordinary
hard- or soft-shelled gelatin type containing, for example, surfactants,
lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and cornstarch.
Tablet forms can include one or more of lactose, sucrose, mannitol, corn
starch, potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
calcium stearate,
zinc stearate, stearic acid, and other excipients, colorants, diluents,
buffering agents,
disintegrating agents, moistening agents, preservatives, flavoring agents, and
pharmacologically compatible carriers. Lozenge forms can comprise the active
ingredient
in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles
comprising the
active ingredient in an inert base, such as gelatin and glycerin, or sucrose
and acacia,
emulsions, gels, and the like containing, in addition to the active
ingredient, such carriers as
are known in the art.
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A tablet may be made by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared by compressing
in a
suitable in a machine the active compound in a free-flowing form, e.g., a
powder or
granules, optionally mixed with accessory ingredients, e.g., binders,
lubricants, inert
diluents, surface active or dispersing agents. Molded tablets may be made by
molding in a
suitable machine, a mixture of the powdered active compound with any suitable
carrier.
A syrup or suspension may be made by adding the active compound to a
concentrated, aqueous solution of a sugar, e.g., sucrose, to which may also be
added any
accessory ingredients. Such accessory ingredients may include flavoring, an
agent to retard
crystallization of the sugar or an agent to increase the solubility of any
other ingredient, e.g.,
as a polyhydric alcohol, for example, glycerol or sorbitol.
The compounds can also be administered locally by topical application of a
solution, ointment, cream, gel, lotion or polymeric material (for example, a
PluronicTM,
BASF), which may be prepared by conventional methods known in the art of
pharmacy. In
addition to the solution, ointment, cream, gel, lotion or polymeric base and
the active
ingredient, such topical formulations may also contain preservatives,
perfumes, and
additional active pharmaceutical agents. Topical formulations for high
affinity adenosine
A3 receptor antagonists include ointments, creams, gels and lotions that may
be prepared by
conventional methods known in the art of pharmacy. Such topical formulation
may also
furhter comprise preservatives, perfumes, and additional active pharmaceutical
agents.
Preferred additional pharmaceutical agents include the chemotherapeutic agents
for cancer
treatments noted to be enhanced or benefited by the A3 receptor antagonist
(for example by
preventing MDR). An example of a preferred topical formulation includes a high
affinity
adenosine A3 receptor antagonist and a taxane family compound.
Oils, which can be used in parenteral formulations include petroleum,
animal, vegetable, and synthetic oils. Specific examples of oils include
peanut, soybean,
sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids
for use in
parenteral formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and
isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps
for use in
parenteral formulations include fatty alkali metal, ammonium, and
triethanolamine salts,
and suitable detergents include (a) cationic detergents such as, for example,
dimethyl
dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents
such as, for
example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates,
and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine
oxides, fatty
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acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d)
amphoteric
detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-
imidazoline
quatemary ammonium salts, and (e) mixtures thereof.
Additionally, the compounds of the invention, e.g., high affinity adenosine
A3 receptor antagonist may be made into suppositories by mixing with a variety
of bases,
such as emulsifying bases or water-soluble bases. Formulations suitable for
vaginal
administration may be presented as pessaries, tampons, creams, gels, pastes,
foams, or spray
formulas containing, in addition to the active ingredient, such carriers as
are known in the
art to be appropriate.
Formulations for rectal administration may be presented as a suppository
with a conventional carrier, e.g., cocoa butter or Witepsol S55 (trademark of
Dynamite
Nobel Chemical, Germany), for a suppository base.
The formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of pharmacy. All
methods
include the step of bringing the active compound into association with a
carrier which
constitutes one or more accessory ingredients. In general, the formulations
are prepared by
uniformly and intimately bringing the active compound into association with a
liquid carrier
or a finely divided solid carrier and then, if necessary, shaping the product
into desired unit
dosage form.
In addition to the aforementioned ingredients, the formulations of this
invention may further include one or more cytotoxic agent as well as one or
more optional
accessory ingredient(s) utilized in the art of pharmaceutical formulations,
e.g., diluents,
buffers, flavoring agents, binders, surface active agents, thickeners,
lubricants, suspending
agents, preservatives (including antioxidants) and the like.
Various delivery systems are known and can be used to administer a
composition comprising compounds of the invention, e.g., encapsulation in
liposomes,
microparticles, microcapsules.
In some embodiments, the compounds of the invention are formulated in
liposomes for targeted delivery of the compounds of the invention. Liposomes
are vesicles
comprised of concentrically ordered phopsholipid bilayers which encapsulate an
aqueous
phase. Liposomes typically comprise various types of lipids, phospholipids,
and/or
surfactants. The components of liposomes are arranged in a bilayer
configuration, similar to
the lipid arrangement of biological membranes. Liposomes are particularly
preferred
delivery vehicles due, in part, to their biocompatibility, low immunogenicity,
and low
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toxicity. Methods for preparation of liposomes are known in the art and are
encompassed
within the invention, see, e.g., Epstein et al., 1985, Proc. Nati. Acad. Sci.
USA, 82: 3688;
Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patent No.'s
4,485,045 and
4,544,545; all of which are incorporated herein by reference in their
entirety.
The invention also encompasses methods of preparing liposomes with a
prolonged serum half-life, i.e., enhanced circulation time, such as those
disclosed in U.S.
Patent No. 5,013,556. Preferred liposomes used in the methods of the invention
are not
rapidly cleared from circulation, i.e., are not taken up into the mononuclear
phagocyte
system (MPS). The invention encompasses sterically stabilized liposomes which
are
prepared using common methods known to one skilled in the art. Although not
intending to
be bound by a particular mechanism of action, sterically stabilized liposomes
contain lipid
components with bulky and highly flexible hydrophilic moieties, which reduces
the
unwanted reaction of liposomes with serum proteins, reduces oposonization with
serum
components and reduces recognition by MPS. Sterically stabilized liposomes are
preferably
prepared using polyethylene glycol. For preparation of liposomes and
sterically stabilized
liposome see, e.g., Bendas et al., 2001 BioDrugs, 15(4): 215-224; Allen et
al., 1987 FEBS
Lett. 223: 42-6; Klibanov et al., 1990 FEBS Lett., 268: 235-7; Blum et al.,
1990, Biochim.
Biophys. Acta., 1029: 91-7; Torchilin.et al., 1996, J. Liposome Res. 6: 99-
116; Litzinger et
al., 1994, Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al., 1991, Chem.
Pharm.
Bull., 39: 1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8;
Allen et al.,
1994, Adv. Drug Deliv. Rev, 13: 285-309; all of which are incorporated herein
by reference
in their entirety. The invention also encompasses liposomes that are adapted
for specific
organ targeting, see, e.g., U.S. Patent No. 4,544,545. Particularly useful
liposomes for use
in the compositions and methods of the invention can be generated by reverse
phase
evaporation method with a lipid composition comprising phosphatidylcholine,
cholesterol,
and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded
through filters of defined pore size to yield liposomes with the desired
diameter. In some
embodiments, a fragment of an antibody of the invention, e.g., F(ab'), may be
conjugated to
the liposomes using previously described methods, see, e.g., Martin et al.,
1982, J Biol.
Chem. 257: 286-288, which is incorporated herein by reference in its entirety.
Methods for preparing liposomes and microspheres for administration to a
patient are well known to those of skill in the art. U.S. Pat. No. 4,789,734,
the contents of
which are hereby incorporated by reference, describes methods for
encapsulating biological
materials in liposomes. essentially, the material is dissolved in an aqueous
solution, the
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appropriate phospholipids and lipids added, along with surfactants if
required, and the
material dialyzed or sonicated, as necessary. A review of known methods is
provided by G.
Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine,
pp. 287-341
(Academic Press, 1979), which is incorporated herein by reference in its
entirety.
Microspheres formed of polymers or proteins are well known to those skilled
in the art, and can be tailored for passage through the gastrointestinal tract
directly into the
blood stream. Alternatively, the compound can be incorporated and the
microspheres, or
composite of microspheres, implanted for slow release over a period of time
ranging from
days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and
3,625,214, the
contents of which are hereby incorporated by reference.
Preferred microparticles are those prepared from biodegradable polymers,
such as polyglycolide, polylactide and copolymers thereof. Those of skill in
the art can
readily determine an appropriate carrier system depending on various factors,
including the
desired rate of drug release and the desired dosage.
In another embodiment, the compositions can be delivered in a vesicle, in
particular a liposome (See Langer, Science 249:1527-1533 (1990); Treat et al.,
in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 3 17-
327; see
generally ibid.).
In yet another embodiment, the compositions can be delivered in a controlled
release or sustained release system. Any technique known to one of skill in
the art can be
used to produce sustained release formulations comprising one or more A3
receptor
antagonists of the invention. See, e.g., U.S. Patent No. 4,526,938; PCT
publication WO
91/05548; PCT publication WO 96/20698; Ning et al., 1996, "Intraturnoral
Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-
Release
Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody
Mediated Lung
Targeting of Long-Circulating Emulsions," PDA Journal of PhaNmaceutical
Science &
Technology 50:372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers
for a bFGF
Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel.
Bioact. Mater.
24:853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized
Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel.
Bioact. Mater.
24:759-760, each of which is incorporated herein by reference in its entirety.
In one
embodiment, a pump may be used in a controlled release system (See Langer,
supra; Sefton,
1987, CRC Crit. Ref Biorned. Eng. 14:20; Buchwald et al., 1980, Surgery
88:507; and
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Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,
polymeric
materials can be used to achieve controlled release of A3 receptor antagonists
(see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres.,
Boca
Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design
and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas, 1983,
J., Macromol. Sci. Rev. Macrornol. Chem. 23:61; See also Levy et al., 1985,
Science
228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 7
1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No.
5,912,015;
U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO
99/15154;
and PCT Publication No. WO 99/20253). Examples of polymers used in sustained
release
formulations include, but are not limited to, poly(2-hydroxy ethyl
methacrylate),
poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl
acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl
pyrrolidone),
poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides
(PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet another
embodiment, a
controlled release system can be placed in proximity of the therapeutic target
(e.g., the
lungs), thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-13 8 (1984)). In
another
embodiment, polymeric compositions useful as controlled release implants are
used
according to Dunn et al. (See U.S. 5,945,155). This particular method is based
upon the
therapeutic effect of the in situ controlled release of the bioactive material
from the polymer
system. The implantation can generally occur anywhere within the body of the
patient in
need of therapeutic treatment. In another embodiment, a non-polymeric
sustained delivery
system is used, whereby a non-polymeric implant in the body of the subject is
used as a
drug delivery system. Upon implantation in the body, the organic solvent of
the implant
will dissipate, disperse, or leach from the composition into surrounding
tissue fluid, and the
non-polymeric material will gradually coagulate or precipitate to form a
solid, microporous
matrix (See U.S. 5,888,533). Controlled release systems are discussed in the
review by
Langer (1990, Science 249:1527-1533). Any technique known to one of skill in
the art can
be used to produce sustained release formulations comprising one or more
therapeutic
agents of the invention. See, e.g., U.S. Patent No. 4,526,938; International
Publication Nos.
WO 91/05548 and WO 96/20698; Ning et al., 1996, Radiotherapy & Oncology 39:179-
189;
Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50:372-
397;
Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bloact. Mater. 24:853-854;
and Lam et
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al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is
incorporated herein by reference in its entirety.
Methods of administering a compound of the invention include, but are not
limited to, parenteral administration (e.g., intradermal, intramuscular,
intraperitoneal,
intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and
oral routes). In a
specific embodiment, the compounds of the invention are administered
intramuscularly,
intravenously, or subcutaneously. The compositions may be administered by any
convenient route, for example, by infusion or bolus injection, by absorption
through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.)
and may be administered together with other biologically active agents.
Administration can
be systemic or local. In addition, pulmonary administration can also be
employed, e.g., by
use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S.
Patent Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064; 5,855,913;
5,290,540;
and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013;
WO 98/31346; and WO 99/66903, each of which is incorporated herein by
reference in its
entirety.
In one embodiment, the compounds are administered intravenously in a
liposome or microparticle with a size such that the particle can be delivered
intraveneously,
but gets trapped in a capillary bed around a growing tumor. Suitable particle
sizes for this
embodiment are those currently used, for example, in liposomes sold under the
name
DaunoXome.TM., which are believed to be between about 200 and 500 um. The
compounds are then released locally, over time, at the location of the tumor.
This can be,
effective in limiting systemic effects of the administration of cytotoxic
agents, and also
targets the adenosine antagonist and any other anti-angiogenic agents directly
to the site at
which blood vessel growth is to be inhibited.
In another embodiment, the compounds are administered in a tissue coating,
preferably a polymeric tissue coating, more preferably, a biodegradable tissue
coating,
which is applied to the site at which a tumor is surgically removed. Suitable
polymeric
materials are disclosed, for example, in U.S. Pat. No. 5,410,016 to Hubbell et
al., the
contents of which are hereby incorporated by reference.
The polymeric barrier, in combination with the adenosine A3 antagonists,
and optionally in combination with other anti-angiogenic agents and/or
cytotoxic agents,
provides a physical barrier as well as a chemical barrier to further tumor
growth and
vasculation around the tumor.
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The two embodiments described above can be particularly useful when
cytotoxic agents with pronounced systemic side effects are used. By targeting
the
administration of these compounds to a specific site, and locally delivering
the compounds,
systemic effects can be minimized.
In yet another embodiment, anti-VEGF antibodies can be covalently coupled
to microparticles or liposomes including the compounds. The antibodies can be
used to not
only target the delivery of the compounds, but also to assist in inhibiting
tumor growth by
inhibiting angiogenesis.
The amount of the composition of the invention which will be effective in
the treatment, prevention or amelioration of one or more symptoms associated
with a
disorder can be determined by standard clinical techniques. The precise dose
to be
employed in the formulation will also depend on the route of administration,
and the
seriousness of the condition, and should be decided according to the judgment
of the
practitioner and each patient's circumstances. Effective doses may be
extrapolated from
dose-response curves derived from in vitro or animal model test systems.
In a specific embodiment, it may be desirable to administer the
pharmaceutical compositions of the invention locally to the area in need of
treatment; this
may be achieved by, for example, and not by way of limitation, local infusion,
by injection,
or by means of an implant, said implant being of a porous, non-porous, or
gelatinous
material, including membranes, such as sialastic membranes, or fibers.
Treatment of a subject with a therapeutically or prophylactically effective
amount of compounds of the invention can include a single treatment or,
preferably, can
include a series of treatments. In a preferred example, a subject is treated
with compounds
of the invention in the range of between about 0.1 g/kg to about 100 mg/kg,
about 0.1
gg/kg to about 500 mg/kg, about 0.1 g/kg to about lg/kg, about 100 ug/kg to
about 500
mg/kg, about 100 ug/kg to aboutlg/kg, about 1 mg/kg to about 100 mg/kg, about
1 mg/kg to
about 500 mg/kg, about 1 mg/kg to about 1 g/kg of the patient's body weight.
one time per
week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably
between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. In other
embodiments, the pharmaceutical compositions of the invention are administered
once a
day, twice a day, or three times a day. In other embodiments, the
pharmaceutical
compositions are administered once a week, twice a week, once every two weeks,
once a
month, once every six weeks, once every two months, twice a year or once per
year. It will
also be appreciated that the effective dosage of the compounds used for
treatment may
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increase or decrease over the course of a particular treatment. The amount of
a compound
required to be effective as an antagonist of adenosine A3 receptors will, of
course, vary with
the active moiety selected, the individual mammal being treated and is
ultimately at the
discretion of the medical or veterinary practitioner. The factors to be
considered include the
binding affinity of the active, the route of administration, the nature of the
formulation, the
mammal's body weight, surface area, age and general condition, and the
particular
compound to be administered.
The total daily dose may be given as a single dose, multiple doses, e.g., two
to six times per day, or by intravenous infusion for a selected duration.
Dosages above or
below the range cited above are within the scope of the present invention and
may be
administered to the individual patient if desired and necessary. For example,
for a 75 kg
mammal, a dose range would be about 75 ug to about 50 mg per day, and a
typical dose
would be about 50 mg per day. If discrete multiple doses are indicated,
treatment might
typically be 50 mg of a compound of the present invention given 3 times per
day.
The invention also provides that the compounds of the invention are
packaged in a hermetically sealed container such as an ampoule or sachette
indicating the
quantity of antibody. In one embodiment, the compounds of the invention are
supplied as a
dry sterilized lyophilized powder or water free concentrate in a hermetically
sealed
container and can be reconstituted, e.g., with water or saline to the
appropriate concentration
for administration to a subject. Preferably, the compounds of the invention
are supplied as a
dry sterile lyophilized powder in a hermetically sealed container at a unit
dosage of at least
5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least
35 mg, at least
45 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 200 mg, at
least 500 mg or at
least 1 gram.. The lyophilized compounds of the invention should be stored at
between 2
and 8 C in their original container and the compounds should be administered
within 12
hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1
hour after being
reconstituted. In an alternative embodiment, compounds of the invention are
supplied in
liquid form in a hermetically sealed container indicating the quantity and
concentration of
the compound. Preferably, the liquid form of the compounds are supplied in a
hermetically
sealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml, at
least 5 mg/ml, at
least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at
least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of the compounds.
The formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of pharmacy. All
methods
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include the step of bringing the active compound into association with a
carrier which
constitutes one or more accessory ingredients. In general, the formulations
are prepared by
uniformly and intimately bringing the active compound into association with a
liquid carrier
or a finely divided solid carrier and then, if necessary, shaping the product
into desired unit
dosage form.
In addition to the aforementioned ingredients, the formulations may further
include one or more optional accessory ingredient(s) utilized in the art of
pharmaceutical
formulations, e.g., diluents, buffers, flavoring agents, binders, surface
active agents,
thickeners, lubricants, suspending agents, preservatives (including
antioxidants) and the
like.
The formulations include, but are not limited to, those suitable for oral,
rectal, topical or parenteral (including subcutaneous, intramuscular and
intravenous)
administration. Preferred are those suitable for oral or parenteral
administration.
The pharmaceutically acceptable carriers described herein, for example,
vehicles, adjuvants, excipients, or diluents, are well known to those who are
skilled in the
art and are readily available to the public. It is preferred that the
pharmaceutically
acceptable carrier be one which is chemically inert to the active compounds
and one which
has no detrimental side effects or toxicity under the conditions of use.
The choice of carrier will be determined in part by the particular active
agent, as well as by the particular method used to administer the composition.
Accordingly,
there are a wide variety of suitable formulations of the pharmaceutical
composition of the
present invention. The following formulations for oral, aerosol, parenteral,
subcutaneous,
intravenous, intraarterial, intramuscular, interperitoneal, intrathecal,
rectal, and vaginal
administration are merely exemplary and are in no way limiting.
The compounds of the invention, e.g., high affinity adenosine A3 receptor
antagonist, alone or in combination with other suitable components, can be
made into
aerosol formulations to be administered via inhalation. These aerosol
formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane,
nitrogen, and the like. They also may be formulated as pharmaceuticals for non-
pressured
preparations, such as in a nebulizer or an atomizer.
6.5 CHARACTERIZATION AND DEMONSTRATION OF
THERAPEUTIC/PROPHYLACTIC UTILITY
Several aspects of the pharmaceutical compositions, prophylactic or
therapeutic agents of the invention are preferably tested in vitro, e.g., in a
cell culture
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system, and then in vivo, e.g., in an animal model organism, such as a rodent
animal model
system, for the desired therapeutic activity prior to use in humans. For
example, assays
which can be used to determine whether administration of a specific
pharmaceutical
composition is indicated, include cell culture assays in which a patient
tissue sample is
grown in culture, and exposed to or otherwise contacted with a pharmaceutical
composition,
and the effect of such composition upon the tissue sample is observed, e.g.,
inhibition of or
decrease in growth and/or colony formation in soft agar or tubular network
formation in
three-dimensional basement membrane or extracellular matrix preparation. The
tissue
sample can be obtained by biopsy from the patient. This test allows the
identification of the
therapeutically most effective prophylactic or therapeutic molecule(s) for
each individual
patient. Alternatively, instead of culturing cells from a patient, therapeutic
agents and
methods may be screened using cells of a tumor or malignant cell line. In
various specific
embodiments, in vitro assays can be carried out with representative cells of
cell types
involved in an autoimmune or inflammatory disorder (e.g., T cells), to
determine if a
pharmaceutical composition of the invention has a desired effect upon such
cell types.
Many assays standard in the art can be used to assess such survival and/or
growth; for
example, cell proliferation can be assayed by measuring 3H-thymidine
incorporation, by
direct cell count, by detecting changes in transcriptional activity of known
genes such as
proto-oncogenes (e.g., fos, myc) or cell cycle markers; cell viability can be
assessed by
trypan blue staining, differentiation can be assessed visually based on
changes in
morphology, decreased growth and/or colony formation in soft agar or tubular
network
formation in three-dimensional basement membrane or extracellular matrix
preparation.
Combinations of prophylactic and/or therapeutic agents can be tested in
suitable animal model systems prior to use in humans. Such animal model
systems include,
but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs,
rabbits, etc. Any
animal system well-known in the art may be used. In a specific embodiment of
the
invention, combinations of prophylactic and/or therapeutic agents are tested
in a mouse
model system. Such model systems are widely used and well-known to the skilled
artisan. --
Prophylactic and/or therapeutic agents can be administered repeatedly. Several
aspects of
the procedure may vary such as the temporal regime of administering the
prophylactic
and/or therapeutic agents, and whether such agents are administered separately
or as an
admixture.
Once the prophylactic and/or therapeutic agents of the invention have been
tested in an animal model they can be tested in clinical trials to establish
their efficacy.
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Establishing clinical trials will be done in accordance with common
methodologies known
to one skilled in the art, and the optimal dosages and routes of
administration as well as
toxicity profiles of the compositions of the invention can be established
using routine
experimentation.
Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the
instant invention can be determined by standard pharmaceutical procedures in
cell cultures
or experimental animals, e.g., for determining the LD50 (the dose lethal to
50% of the
population) and the EDso (the dose therapeutically effective in 50% of the
population). The
dose ratio between toxic and therapeutic effects is the therapeutic index and
it can be
expressed as the ratio LD5O/ED50. Prophylactic and/or therapeutic agents that
exhibit large
therapeutic indices are preferred. While prophylactic aiid/or therapeutic
agents that exhibit
toxic side effects may be used, care should be taken to design a delivery
system that targets
such agents to the site of affected tissue in order to minimize potential
damage to uninfected
cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used
in formulating a range of dosage of the prophylactic and/or therapeutic agents
for use in
humans. The dosage of such agents lies preferably within a range of
circulating
concentrations that include the ED50 with little or no toxicity. The dosage
may vary within
this range depending upon the dosage form employed and the route of
administration
utilized. For any agent used in the method of the invention, the
therapeutically effective
dose can be estimated initially from cell culture assays. A dose may be
formulated in
animal models to achieve a circulating plasma concentration range that
includes the IC50
(i.e., the concentration of the test compound that achieves a half-maximal
inhibition of
symptoms) as determined in cell culture. Such information can be used to more
accurately
determine useful doses in humans. Levels in plasma may be measured, for
example, by
high performance liquid chromatography.
The anti-cancer activity of the therapies used in accordance with the present
invention also can be determined by using various experimental animal models
for the study
of cancer such as the SCID mouse model or transgenic mice or nude mice with
human
xenografts, animal models, such as hamsters, rabbits, etc. known in the art
and described in
Relevance of Tumor Models for Anticancer Drug Development (1999, eds. Fiebig
and
Burger); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology
Research
(1991, eds. Boven and Winograd); and Anticancer Dr=ug Development Guide (1997
ed.
Teicher), herein incorporated by reference in their entireties.
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The protocols and compositions of the invention are preferably tested in
vitro, and then in vivo, for the desired therapeutic or prophylactic activity,
prior to use iri
humans. Therapeutic agents and methods may be screened using cells of a tumor
or
malignant cell line. Many assays standard in the art can be used to assess
such survival
and/or growth; for example, cell proliferation can be assayed by measuring 3H-
thymidine
incorporation, by direct cell count, by detecting changes in transcriptional
activity of known
genes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers; cell
viability can be
assessed by trypan blue staining, differentiation can be assessed visually
based on changes
in morphology, decreased growth and/or colony formation in soft agar or
tubular network
formation in three-dimensional basement membrane or extracellular matrix
preparation, etc.
Compounds for use in therapy can be tested in suitable animal model
systems prior to testing in humans, including but not limited to in rats,
mice, chicken, cows,
monkeys, rabbits, hamsters, etc., for example, the animal models described
above. The
compounds can then be used in the appropriate clinical trials.
6.5.1 ANTI-PROLIFERATIVE ASSAYS
The anti-proliferative activity of compounds of the invention can be readily
determined using no more than routine experimentation using cell growth
inhibitory assays.
Selection of cell lines for such assays is based upon desired future
pharmaceutical use.
Numerous cell lines are available for study from American Type Culture
Collection,
Manassas, VA. Cell lines suitable for assays include, but are not limited to,
HL-60 human
leukemia, A375 human melanoma, SKMES human lung carcinoma, HT29 human colon
carcinoma and Panc-1 human pancreatic carcinoma. Cell lines can be maintained
in RPMI-
1640 medium supplemented with 100 units/ml penicillin G sodium, 100 g/mi
streptomycin
sulfate, 0.25 g/ml amphotericin B (fungizone), 2 mM glutamine, 10 mM HEPES,
25 g/ml
gentamycin, and 10% heat-inactivated fetal bovine serum. Such maintained cells
can be
cultured in a T25 Falcon Tissue Culture flask in a humidified incubator at 37
C with 5%
C02-95% air. For example, such maintained cells can be sub-cultured twice a
week with
approximate doubling times of 22 hours for A375 human melanoma, 46 hours for
SKMES
human lung carcinoma, 48 hours for HT29 human colon carcinoma and 60 hours for
Panc-1
human pancreatic carcinoma.
One of the growth inhibitory assays that can be used in accordance with the
methods of the invention is the MTT assay. An exemplary assay may comprise the
following steps: Cells (1000-1500 cells/well) are seeded in a 96-well micro
culture plate in
a total volume of 100 1/we11. After overnight incubation in a humidified
incubator at 37 C
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with 5% CO2 - 95% air, chemotherapeutic drug solutions diluted with culture
medium at
various concentrations are added in the amount of 100 l to each well. The
plates are
placed in a humidified incubator at 37 C with 5% CO2 - 95% air for 7-10 days.
The plates
are then centrifuged briefly and 100 gl of the growth medium is removed. Cell
cultures are
incubated with 50 gl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium
bromide] reagent (1 mg/ml in Dulbecco's phosphate-buffered saline) for 4 hr at
37 C. The
resultant purple formazan precipitate is solubilized with 200 gl of 0.04 N HCl
in
isopropanol. Absorbance is measured at a wavelength of 595 nm and at a
reference
wavelength of 655 nm using a Bio-Rad Mode13550 Microplate Reader. Preferably,
all tests
are run in duplicate for each dose level. Bio-Rad brands Microplate Readers,
when properly
equipped, transmit measured test results to a personal computer for
interpretation via
computer programs such as the EZED50 program. The EZED50 computer program
estimates the concentration of agent that inhibits cell growth by 50% as
compared to the
control cells. This is termed the IC50 and is determined by curve fitting test
data using the
following four logistic equation.
Y Amax-Amin +Amin
1 + (X / IC5o)n
where A,,,. is the absorbance of the control cells, A;,, is the absorbance of
the cells in the
presence of the highest agent concentration, Y is the observed absorbance, X
is the agent
concentration, IC50 is the concentration of agent that inhibits the cell
growth by 50%
compared to the control cells, and n is the slope of the curve.
If testing concentrations are properly selected (i.e., no or little growth
inhibition at low concentrations and complete inhibition at high
concentrations), EZED50
program fits the data extremely well and estimates the IC50 value accurately.
If the testing
agent was too potent and inhibits cell growth by more than 50% at all of the
concentrations
tested, EZED50 cannot estimate the IC50 value accurately (as indicated by an
erroneously
fitted Amax value). In these instances, the data could be re-analyzed by
fixing the Amax
value.
On the other hand, if the compound being tested produces incomplete
inhibition at the highest agent concentration, EZED50 will overestimate the
potency of the
testing agent (the fitted IC50value is lower than the "actual" IC50 value). In
this case, the
IC50 could be estimated more accurately if both the A,,,,,X and Am;n values
are fixed.
Although it may be feasible to estimate IC50 value by fixing Amax and/or An,~x
and Ain
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without repeating the experiment, the best way to determine the IC50
accurately is to
decrease or increase the concentrations of the test agent and repeat the test.
Inhibitory assay testing is also used to determine enhanced therapeutic
effects from combining A3 receptor antagonists with other tumor inhibiting
agents. The
inhibitory cell growth assays are run with and without selected concentrations
of A3
receptor antagonists. An enhanced therapeutic effect is present when the IC50
of the tumor
inhibiting agent is lower with the A3 receptor antagonist.
Quantitatively, the enhancement factor (EF) is calculated by dividing the
IC50 value of the tumor inhibiting agent for a tumor cell line by the IC50
value of the agent
with A3 receptor antagonist for the same cell line:
IC50 of anti-tumor agent
Enhanced Factor (EF)= ICSO of anti-tumor agent with A3 receptor
antagonist
Interpretation of the EF is dependent upon the concentrations of A3 receptor
antagonist tested. For example, when using very low concentrations of A3
receptor
antagonist, any EF above 1.0 is synergistic if the A3 receptor antagonist
concentration is
below the threshold of cell growth inhibition.
6.5.2 ADENOSINE RECEPTOR BASED ASSAYS
The activity and selectivity of the compounds of the invention as adenosine
A3 antagonists can be readily determined using no more than routine
experimentation using
any of the assays disclosed herein or known to one skilled in the art. Since
the Al and A2a
receptors express similar pharmacology between humans and rodents, endogenous
receptors
from the rat can be used for the Al and A2a binding assays.
An exemplary rat Al and A2A Adenosine receptor binding assay may
comprise the following steps. Membrane preparations: Male Wistar rats (200-250
g) can be
decapitated and the whole brain (minug brainstem, striatum and cerebellum)
dissected on
ice. The brain tissues can be disrupted in a Polytron (setting 5) in 20 vols
of 50 mM Tris
HCI, pH 7.4. The homogenate can then be centrifuged at 48,000 g for 10 min and
the pellet
resuspended in Tris-HCL containing 2 IU/ml adenosine deaminase, type VI (Sigma
Chemical Company, St. Louis, Mo., USA). After 30 min incubation at 37 C, the
membranes can be centrifuged and the pellets stored at -70 C. Striatal
tissues can be
homogenized with a Polytron in 25 vol of 50 mM Tris HCl buffer containing 10
mM MgCl2
pH 7.4. The homogenate can then be centrifuged at 48,000 g for 10 min at 4 C.
and
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resuspended in Tris HCl buffer containing 2 IU/ml adenosine deaminase. After
30 min
incubation at 37 C, membranes can be centrifuged and the pellet stored at -70
C. The
radioligand binding assays may comprise the following: Binding of [3H]-DPCPX
(1,3-
dipropyl-8-cyclopentylxanthine) to rat brain membranes can be performed
essentially
according to the method previously described by Bruns et al., 1980, Proc.
Natl. Acad. Sci.
77, 5547-5551, which is incorporated herein by reference in its entirety.
Displacement
experiments can be performed in 0.25 ml of buffer containing 1 nM [3H]-DPCPX,
100 ul of
diluted membranes of rat brain (100 g of protein/assay) arid at least 6-8
different
concentrations of examined compounds. Non specific binding can be determined
in the
presence of 10 uM of CHA (N6 cyclohexyladenosine) and this is always <10% of
the total
binding. Incubation times are typically 120 min at 25 C.
Radioligand binding assays may comprise the following: Binding of [3H]-
SCH 58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-
c]pyrimidine) to rat striatal membranes (100 ug of protein/assay) can be
performed
according to methods described in Zocchi et al., 1996, J. Pharm. and Exper.
Ther. 276:398-
404, which is incorporated herein by reference in its entirety. In competition
studies, at
least 6-8 different concentrations of examined compounds should be used. Non
specific
binding can be determined in the presence of 50 uM of NECA (5'-(N-
ethylcarboxamido)adenosine). -Incubation time is typically 60 min at 25 C.
Bound and
free radioactivity can be separated by filtering the assay mixture through
Whatman GF/B
glass-fiber filters using a Brandel cell harvester (Gaithersburg, Md., USA).
The incubation
mixture can be diluted with 3 ml of ice-cold incubation buffer, rapidly vacuum
filtered and
the filter can be washed three times with 3 ml of incubation buffer. The
filter bound
radioactivity can be measured, for example, by liquid scintillation
spectrometry. The
protein concentration can be determined, for example, according to a Bio-Rad
method
(Bradford, 1976, Anal. Biochem. 72:248, which is incorporated herein by
reference in its
entirety) with bovine albumin as reference standard.
An exemplary assay for human cloned A3 Adenosine Receptor Binding
Assay may comprise the following: Binding assays can be carried out according
to methods
described in Salvatore et al., 1993, Proc. Natl. Acad. Sci. 90:10365-10369;
which is
incorporated herein by reference in its entirety. In saturation studies, an
aliquot of
membranes (8 mg protein/ml) from HEK-293 cells transfected with the human
recombinant
A3 adenosine receptor (Research Biochemical International, Natick, Mass., USA)
can be
incubated with 10-12 different concentrations of [125 I]AB-MECA ranging from
0.1 to 5
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nM. Competition experiments can be carried out in duplicate in a final volume
of 100 ul in
test tubes containing 0.3 nM [125 I]AB-MECA, 50 mM Tris HC1 buffer, 10 mM
MgC12, pH
7.4 and 20 ul of diluted membranes (12.4 mg protein/ml) and at least 6-8
different
concentrations of examined ligands. Incubation time was 60 min at 37 C,
according to the
results of previous time-course experiments. Bound and free radioactivity were
separated
by filtering the assay mixture through Whatman GF/B glass-fiber filters using
a Brandel cell
harvester. Non-specific binding was defined as binding in the presence of 50
uM R-PIA
and was about 30% of total binding. The incubation mixture was diluted with 3
ml of ice-
cold incubation buffer, rapidly vacuum filtered and the filter was washed
three times with 3
ml of incubation buffer. The filter bound radioactivity was counted in a
Beckman gamma
5500B gamma counter. The protein concentration can be determined according to
a Bio-
Rad method with bovine albumin as reference standard.
Data Analysis may be carried out as follows: Inhibitory binding constant,
Ki, values can be calculated from those of IC50 according to the Cheng &
Prusoff equation
(Cheng and Prusoff, 1973, Biochem. Pharnaacol. 22:3099-3108), Ki.
=IC50/(1+[C*]/KD*),
where [C*] is the concentration of the radioligand and KD * its dissociation
constant. A
weighted non linear least-squares curve fitting program LIGAND (Munson and
Rodbard,
1990, Anal. Biochem. 107:220-239) can be used for computer analysis of
saturation and
inhibition experiments. Data are typically expressed as geometric mean, with
95% or 99%
confidence limits in parentheses.
In some embodiments, the efficacy of the compounds can be tested in animal
models. For example, the efficacy of the adenosine A3 antagonists alone or in
combination
with conventional anti-tumor agents such as cytotoxic/anti-neoplastic agents
and anti-
angiogenic agents can be compared to the conventional agents alone. Typically,
a tumor of
a given size is present in a rat or mouse. The mouse is treated with the agent
and the size of
the tumor is measured over time. The mean survival time of the animals can
also be
measured. The adenosine A3 antagonists which are used must actively bind the
adenosine
A3 receptor in the animal which is to be tested. Xenografts can be implanted
into the animal
to test the ability of species specific cytotoxic compounds. However, the
effect of the anti-
angiogenic therapy depends on how well the anti-angiogenetic compounds work on
inhibiting the specific animal's vasculation.
Assay methods that have demonstrated the efficacy of endostatin and
angiostatin in suppressing tumor growth can be applied to Al, A2a, and A3
antagonists. For
example, Lewis lung carcinoma, T241 fibrosarcoma or B 16F 10 melanoma can be
grafted to
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mice (Boehm et al., 1977, Nature, 390:404-407; which is incorporated herein by
reference
in its entirety). Other tumors, such as PC-3 human prostate carcinoma, the CCL
188 human
colon carcinoma and the UBC urinary bladder carcinoma have been shown to
release
angiogenic agents (Chen et al., 1996, Cancer Res., 55:4230-4322; which is
incorporated
herein by reference in its entirety). these tumors can also be grafted in mice
for the
evaluation of anti-tumor activities of adenosine antagonists. Adenosine
antagonists can be
administered to mice by the methods disclosed herein. This therapy can be used
alone or in
conjunction with anti-angiogenic therapy or conventional chemotherapy.
6.5.3 METHODS FOR DETERMINING AND MEASURING HIF-I
ALPHA LEVELS
The invention encompasses methods to assess quantitative and qualitative
aspects of HIF-la expression. In one example, the increased expression of an
HIF-la gene
or gene product indicates a predisposition for the development of cancer or a
disease or
disorder associated with enhanced HIF-la expression. Alternatively, enhanced
expression
levels of an HIF-la gene or gene product can indicate the presence of cancer
in a subject or
the risk of metastasis of said cancer in said subject. Techniques well known
in the art, e.g.,
quantitative or semi-quantitative RT PCR or Northern blot, can be used to
measure
expression levels of HIF-la. Methods that describe both qualitative and
quantitative
aspects of HIF-1 a gene or gene product expression are described in detail in
the examples
infra. The measurement of HIF-la gene expression levels can include measuring
naturally
occurring HIF-1a transcripts and variants thereof as well as non-naturally
occurring
variants thereof, however for the diagnosis and/or prognosis of diseases or
disorders in a
subject the HIF-la gene product is preferably a naturally occurring HIF-la
gene product
or variant thereof. Thus, the invention relates to methods of measuring the
expression of the
HIF-la gene or gene product in a subject.
Any method known in the art for detecting and/or quantitating an HIF-1 a
level may be used in the methods and kits of the invention, a number of which
are
exemplified herein. Particularly preferred are methods known in the art for
detecting and/or
quantitating an HIF- 1 a activity or an HIF-la related activity, e.g.,
phosphorylation of
downstream effector molecules in the HIF-la pathway. In some embodiments, the
invention encompasses measuring an HIF-la activity or an HIF-la related
activity
including but not limited to, measuring an activity of one or more downstream
effectors of
anHIF-la signaling cascade. Measuring an HIF-la activity or anHIF-1a related
activity
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can be done using any of the methods disclosed herein or any standard method
known to
one skilled in the art.
In other embodiments, the invention encompasses quantitation of a nucleic
acid encoding HIF-la in a sample obtained from a subject using methods
disclosed herein
or any standard method known in the art.
In yet other embodiments, the invention encompasses quantitation of HIF-la
protein in a sample obtained from a subject with a disease or disorder, e.g.,
cancer. Any
method known in the art for the detection and quantitation of a HIF-la protein
is
encompassed within the present invention.
6.5.3.1 DETECTION OF NUCLEIC ACID MOLECULES
The methods and kits of the invention encompass detection and/or
quantitation of a nucleic acid sequence encoding HIF-1 a in a sample obtained
from a
subject. In certain embodiments, the invention provides methods for amplifying
a specific
HIF-1 a nucleic acid sequence in a sample obtained from a subject with cancer,
and
detecting and/or quantitating the same. Nucleic acids encoding HIF-la are well
known in
the art. See, for example, Wang et aL, 1995, Proc. Natl. Acad. Sci. USA, 92:
5510-4; and
WO 96/39426 each of which is incorporated herein by reference in their
entireties.
The methods and kits of the invention may use any nucleic acid
amplification or detection method known to one skilled in the art, such as
those described in
U.S. Patent No.'s 5,525,462; 6,528,632; 6,344,317; 6,114,117; 6,127,120;
6,448,001; all of
which are incorporated herein by reference in their entirety.
In some embodiments, the nucleic acid encoding an HIF-l a is amplified by
PCR amplification using methodologies known to one skilled in the art. One of
skill in the
art will recognize, however, that amplification of target sequences (i.e.,
nucleic acid
sequences encoding HIF-la) in a sample obtained from a subject with cancer can
be
accomplished by any known method, such as ligase chain reaction (LCR), QP-
replicase
amplification, transcription amplification, and self-sustained sequence
replication, each of
which provides sufficient amplification. The PCR process is well known in the
art and is
thus not described in detail herein. For a review of PCR methods and
protocols, see, e.g.,
Innis et al., eds., PCR Protocols, A Guide to Methods and Application,
Academic Press,
Inc., San Diego, Calif. 1990; which is incorporated herein by reference in its
entirety). Also
see U.S. Patent No. 4,683,202; which is incorporated herein by reference in
its entirety.
PCR reagents and protocols are also available from commercial vendors, such as
Roche
Molecular Systems.
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The invention encompasses methods to determine quantitative and/or
qualitative levels of expression of HIF- 1 a. Any technique known in the art
for measuring
the expression of an HIF-1a is within the scope of the invention, including
but not limited,
to quantitative and/or semi-quantitative RT PCR and Northern blot analysis.
In some embodiments, the invention encompasses detecting and/or
quantitating an HIF- 1 a nucleic acid using fluorescence in situ hybridization
(FISH) in a
sample, preferably a tissue sample, obtained from a subject with cancer in
accordance with
the methods of the invention. FISH is a common methodology used in the art,
especially in
the detection of specific chromosomal aberrations in tumor cells, for example,
to aid in
diagnosis and tumor staging. As applied in the methods of the invention, it
can also be used
as a method for detection and/or quantitation of an HIF- 1 a nucleic acid. For
a review of
FISH methodology, see, e.g., Weier et al., 2002, Expert Rev. Mol. Diagn. 2(2):
109-119;
Trask et al., 1991, Trends Genet. 7(5): 149-154; and Tkachuk et al., 1991,
Genet. Anal.
Tech. Appl. 8: 676-74; all of which are incorporated herein by reference in
their entirety.
The invention encompasses measuring naturally occurring HIF-la
transcripts and variants thereof as well as non-naturally occurring variants
thereof. For the
prognosis of cancer in a subject using the methods of the invention, the HIF-1
a transcript is
preferably a naturally occurring HIF-1a transcript.
In some embodiments, the invention relates to methods of prognosis of a
cancer in a subject by measuring the expression of an HIF-la transcript in a
subject. For
example, the increased level of mRNA encoding an HIF- 1 a, as compared to a
standard,
e.g., a non-cancerous sample, would indicate the increased risk of developing
cancer in said
subject. In another embodiment, the increased level of mRNA encoding an HIF-1
a as
compared to a standard would indicate the risk of metastasis of the cancer in
said subject or
the likelihood of a poor prognosis in said subject.
In one embodiment, the invention encompasses isolating RNA from a
sample obtained from a subject with cancer, and testing the RNA utilizing
hybridization or
PCR techniques as described above for determining the level of an HIF- 1 a. In
another
embodiment, the invention encompasses synthesizing cDNA from the isolated RNA
by
reverse transcription. All or part of the resulting cDNA is then used as the
template for a
nucleic acid amplification reaction, such as a PCR or the like. The nucleic
acid reagents
used as synthesis initiation reagents (e.g., primers) in the reverse
transcription and nucleic
acid amplification steps of this method are chosen from among the HIF-1 a
nucleic acid
reagents described below. The preferred lengths of such nucleic acid reagents
are at least 9-
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30 nucleotides. For detection of the amplified product, the nucleic acid
amplification may
be performed using radioactively or non-radioactively labeled nucleotides.
Alternatively,
enough amplified product may be made such that the product may be visualized
by standard
ethidium bromide staining or by utilizing any other suitable nucleic acid
staining method.
In alternative embodiments, standard Northern analysis techniques known to
one skilled in the art can be performed on a sample obtained from a subject
with cancer.
The preferred length of a probe used in Northern analysis is 9-50 nucleotides.
Utilizing
such techniques, quantitative as well as size related differences among HIF-1a
transcripts
can also be detected.
In alternative embodiments, the invention encompasses gene expression
assays in situ, i.e., directly upon tissue sections (fixed and/or frozen) of
patient tissue
obtained from biopsies or resections, such that no nucleic acid purification
is necessary.
Nucleic acid reagents such as those described below may be used as probes
and/or primers
for such in situ procedures (see, e.g., Nuovo, G.J., 1992, PCR In Situ
Hybridization:
Protocols And Applications, Raven Press, NY, which is incorporated herein by
reference in
its entirety).
The target HIF-la nucleic acids of the invention can also be detected using
other standard techniques well known to those of skill in the art. Although
the detection
step is typically preceded by an amplification step, amplification is not
necessarily required
in the methods of the invention. For instance, the HIF-1 a nucleic acids can
be identified by
size fractionation (e.g., gel electrophoresis). The presence of different or
additional bands
in the sample as compared to the control is an indication of the presence of
target nucleic
acids of the invention. Alternatively, the target HIF-1 a nucleic acids can be
identified by
sequencing according to well known techniques. In alternative embodiments,
oligonucleotide probes specific to the target HIF- 1 a nucleic acids can be
used to detect the
presence of specific fragments.
Sequence-specific probe hybridization is a well known method of detecting
desired nucleic acids in a sample comprising a biological fluid or tissue
sample and is
within the scope of the present invention. Briefly, under sufficiently
stringent hybridization
conditions, the probes hybridize specifically only to substantially
complementary
sequences. The stringency of the hybridization conditions can be relaxed to
tolerate varying
amounts of sequence mismatch. If the target is first amplified, detection of
the amplified
product utilizes this sequence-specific hybridization to insure detection of
only the correct
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amplified target, thereby decreasing the chance of a false positive caused by
the presence of
homologous sequences from related organisms or other contaminating sequences.
A number of hybridization formats well known in the art, including but not
limited to solution phase, solid phase, mixed phase, or in situ hybridization
assays are
encompassed within the nucleic acid detection methods of the invention. In
solution (or
liquid) phase hybridizations, both the target nucleic acid and the probe or
primer are free to
interact in the reaction mixture. In solid phase hybridization assays, either
the target or
probes are linked to a solid support where they are available for
hybridization with
complementary nucleic acids in solution. Exemplary solid phase formats include
Southern
hybridizations, dot blots, and the like. The following articles provide an
overview of the
various hybridization assay formats, all of which are incorporated herein by
reference in
their entirety: Singer et al., 1986 Biotechniques 4: 230; Haase et al., 1984,
Methods in
Virology, Vol. VII, pp. 189-226; Wilkinson, In Situ Hybridization, D. G.
Wilkinson ed.,
IRL Press, Oxford University Press, Oxford; and Nucleic Acid Hybridization: A
Practical
Approach, Hames, B. D. and Higgins, S. J., eds., IRL Press (1987).
The invention encompasses homogenous based hybridization assays as well
as heterogeneous based assays for detection and/or quantitation of HIF-1 a
nucleic acid
sequences in accordance with the methods of the invention. Heterogeneous based
assays
depend on the ability to separate hybridized from non-hybridized nucleic
acids. One such
assay involves immobilization of either the target or probe nucleic acid on a
solid support so
that non-hybridized nucleic acids which remain in the liquid phase can be
easily separated
after completion of the hybridization reaction (see, e.g., Southern, 1975, J.
Mol. Biol. 98:
503-517; which is incorporated herein by reference in its entirety). In
comparison,
homogeneous assays depend on other means for distinguishing between hybridized
and
non-hybridized nucleic acids. Because homogeneous assays do not require a
separation
step, they are generally considered to be more desirable. One such homogeneous
assay
relies on the use of a label attached to a probe nucleic acid that is only
capable of generating
a signal when the target is hybridized to the probe (see, e.g., Nelson, et
al., 1992,
Nonisotopic DNA Probe Techniques, Academic Press, New York, N.Y., pages 274-3
10;
which is incorporated herein by reference in its entirety).
The invention encompasses any method known in the art for enhancing the
sensitivity of the detectable signal in such assays, including but not limited
to the use of
cyclic probe technology (Bakkaoui et al., 1996, BioTechniques 20: 240-8, which
is
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incorporated herein by reference in its entirety); and the use of branched
probes (Urdea et
al., 1993, Clin. Chem. 39: 725-6; which is incorporated herein by reference in
its entirety).
The hybridization complexes are detected according to well known
techniques in the art. Nucleic acid probes capable of specifically hybridizing
to a target can
be labeled by any one of several methods typically used to detect the presence
of hybridized
nucleic acids. One common method of detection is the use of autoradiography,
using probes
labeled with 3H, 125I335S, 14C, or 32P, or the like. The choice of radioactive
isotope depends
on research preferences due to ease of synthesis, stability, and half lives of
the selected
isotopes. Other labels include compounds (e.g., biotin and digoxigenin), that
bind to anti-
ligands or antibodies labeled with fluorophores, chemiluminescent agents, or
enzymes.
Alternatively, probes can be conjugated directly to labels such as
fluorophores,
chemiluminescent agents or enzymes. The choice of label depends on sensitivity
required,
ease of conjugation with the probe, stability requirements, and available
instrumentation.
The probes and primers of the invention can be synthesized and labeled
using techniques known to one skilled in the art. Oligonucleotides for use as
probes and
primers may be chemically synthesized according to the solid phase
phosphoramidite
triester method described by Beaucage, S. L. and Caruthers, M. H., 1981,
Tetrahedron Lett.
22(20): 1859-1862, using an automated synthesizer, as described in Needham-
VanDevanter,
D. R., et al. 1984, Nucleic Acids Res. 12: 6159-6168. Purification of
oligonucleotides can
be by either native acrylamide gel electrophoresis or by anion-exchange HPLC,
as described
in Pearson, J. D. and Regnier, F. E., 1983, ,I. Chrom. 255:137-149. All of the
references
cited supra are incorporated herein by reference in their entirety.
6.5.4 DETECTION OF PROTEINS
The methods and kits of the invention encompass detection and/or
quantitation of HIF-la in a sample obtained from a subject. Any method known
to one
skilled in the art for the detection and quantitation of an HIF- 1 a protein
is encompassed
within the present invention. HIF-1 a protein sequences useful in the methods
and kits of
the invention are well known in the art. See, for example, Wang et al., 1995,
Proc. Natl.
Acad. Sci. USA, 92: 5510-4; Merighi et al., HIF-la Expression in Cancer Cells
in up-
regulated by adeerrozine A3 receptors, paper submitted to Nature, 2004; and WO
96/39426
each of which is incorporated herein by reference in its entireties.
HIF-la proteins and anti- HIF-la antibodies and immunospecific fragments
thereof are suitable in the assays of the invention. Detection and
quantitation of an HIF-1 a
gene product encompasses the detection of proteins exemplified herein.
Detection of
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elevated levels of an HIF-la gene product in a sample obtained from a subject
in
accordance with the methods of the invention is generally compared to a
standard sample.
In some embodiments, antibodies directed against naturally occurring HIF-
la proteins may be used in the methods of the invention. The invention
encompasses the
use of any standard immunoassay method known to one skilled in the art,
including but not
limited to Western blot, ELISA, and FACS.
In one embodiment, the invention encompasses use of an immunoassay
comprising contacting a sample from a subject with an anti- HIF-la antibody or
an
immunospecific fragment thereof under conditions such that immunospecific
binding to the
HIF- 1 a receptor in the sample can occur, thereby forming an immune complex,
and
detecting and/or measuring the amount of complex formed. In a specific
embodiment, an
antibody to an HIF-la is used to assay a sample for the presence of the HIF-
la, wherein an
increased level of the HIF-1 a is detected relative to a standard sample.
In some embodiments, the biological sample may be brought in contact with
and immobilized onto a solid phase support or a carrier such as nitrocellulose
or other solid
support capable of immobilizing cells, cell particles or soluble proteins. The
support can be
washed with suitable buffers followed by treatment with the antibody that
selectively or
specifically binds to an HIF-la protein. The solid phase support can then be
washed with
buffer to remove unbound antibody. The amount of antibody bound to the solid
support can
then be detected by ponveintional means.
"Solid phase support or carrier" as used herein refers to any support capable
of binding an antigen or an antibody. Well-known supports or carriers include
glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural
and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier
can be either
soluble to some extent or insoluble for the purposes of the present invention.
The support
material may have virtually any possible structural configuration so long as
the coupled
molecule is capable of binding to an antigen or antibody. Thus, the support
configuration
may be spherical, as in a bead, or cylindrical, as in the inside surface of a
test tube, or the
external surface of a rod. Alternatively, the surface may be flat such as a
sheet, test strip,
etc. Preferred supports include polystyrene beads. Those skilled in the art
will know many
other suitable carriers for binding antibody or antigen, or will be able to
ascertain the same
by use of routine experimentation.
In some embodiments, the anti- HIF-1 a antibody or an immunospecific
fragment thereof can be detectably labeled by linking the same to an enzyme
and using the
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labeled antibody in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme
Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1, Microbiological
Associates Quarterly Publication, Walkersville, MD; Voller, A. et al., 1978,
J. Clin. Pathol.
31:507-520; Butler, J.E., 1981, Meth. Enzymol. 73:482; Maggio, E. ed., 1980,
Enzyme
Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., eds., 1981,
Enzyme
Immunoassay, Kgaku Shoin, Tokyo, all of which are incorporated herein by
reference in
their entirety). The enzyme bound to the antibody will react with an
appropriate substrate,
preferably a chromogenic substrate, in such a manner as to produce a chemical
moiety
which can be detected, for example, by spectrophotometric, fluorimetric or
visual means.
Enzymes that can be used to detectably label the antibody include but are not
limited to
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate
isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-
galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase,
glucoamylase and acetylcholinesterase, among others. The detection can be
accomplished
by colorimetric methods that employ a chromogenic substrate for the enzyme.
Detection
can also be accomplished by visual comparison of the extent of enzymatic
reaction of a
substrate in comparison with similarly prepared standards.
Detection can also be accomplished using any other method known to one
skilled in the art. For example, by radioactively labeling the antibodies or
antibody
fragments, it is possible to detect HIF-la protein through the use of a
radioimmunoassay
(RIA) (see, for example, Weintraub, B., Principles of Radioimrnunoassays,
Seventh
Training Course on Radioligand Assay Techniques, The Endocrine Society, March,
1986).
The radioactive isotope can be detected by such means as the use of a gamma
counter or a
scintillation counter, or by autoradiography.
In other embodiments, the invention encompasses labeling the antibody with
a fluorescent compound. When the fluorescently labeled antibody is exposed to
light of the
proper wave length, its presence can then be detected due to fluorescence.
Among the most
commonly used fluorescent labeling compounds are fluorescein isothiocyanate,
rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
In yet
other embodiments, the antibody can also be detectably labeled using
fluorescence emitting
metals such as 152Eu, or others of the lanthanide series. These metals can be
attached to the
antibody using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
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The invention further encompasses detectably labeling the antibody by
coupling it to a chemiluminescent compound. The presence of the
chemiluminescent-
tagged antibody is then determined by detecting the presence of luminescence
that arises
during the course of a chemical reaction. Examples of particularly useful
chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium ester,
imidazole,
acridinium salt and oxalate ester. Likewise, a bioluminescent compound can be
used to
label the antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic protein
increases the
efficiency of the chemiluminescent reaction. The presence of a bioluminescent
protein is
determined by detecting the presence of luminescence. Bioluminescent compounds
for
purposes of labeling include, e.g., luciferin, luciferase and aequorin.
The invention also encompasses methods for indirect detection of HIF-la.
In a specific embodiment, the invention encompasses use of an immunoassay
comprising
contacting a sample derived from a subject with cancer with an anti- HIF-la
antibody
(primary antibody) or an immunospecific fragment thereof under conditions such
that
immunospecific binding to the HIF-1 a protein in the sample can occur, thereby
forming an
immune complex, adding a secondary antibody that is labeled under conditions
such that
immunospecific binding to the primary antibody occurs and detecting and/or
quantitating
the amount of complex formed indirectly.
Anti- HIF- 1 a antibodies or immunospecific fragments thereof may be used
quantitatively or qualitatively to detect an HIF-1a in a sample. In some
embodiments,
when the sample is a tissue, the anti- HIF-l a antibodies or immunospecific
fragments
thereof may be used histologically, e.g., immunofluorescence or microscopic
studies, using
common techniques known to one skilled in the art, for in situ detection of an
HIF- 1 a
receptor. In situ detection may be accomplished by preparing a histological
specimen from
a subject, such as a paraffin embedded section of tissue, e.g., breast
tissues, and applying
thereto a labeled antibody of the present invention. The antibody (or
fragment) is preferably
applied by overlaying the labeled antibody (or fragment) onto the biological
sample.
Through the use of such a procedure, it is possible to determine not only the
presence of an
HIF-1 a protein but also its distribution in the examined tissue. Using the
methods of the
present invention, those of ordinary skill will readily perceive that any of a
wide variety of
histological methods (such as staining procedures) can be modified in order to
achieve such
in situ detection.
6.6 NUCLEIC ACIDS ENCODING HIF-1 ALPHA
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The methods of the invention may use any nucleic acid encoding HIF-1 a, an
analog, fragment or derivative thereof, as a proxy for determining a HIF-1 a
level. A
nucleic acid is intended to include DNA molecules (e.g., cDNA, genomic DNA),
RNA
molecules (e.g., h.nRNA, pre-mRNA, mRNA) and DNA or RNA analogs (e.g., peptide
nucleic acids) generated using techniques known to one skilled in the art. The
nucleic acid
measured as a proxy for a HIF-1 a level can be single-stranded or double
stranded.
For example, but not by way of limitation, nucleotide sequences for use in
the methods and kits of the invention may include all or a portion of any of
the following:
nucleotide sequences disclosed in U.S. Patent Nos. 6,455,674 (issued to Einat
et al);
6,652,799 (issued to Semenza); 6,222,018(issued to Semenza); Wang et al., 1995
PNAS
USA, 92: 5510-4; and WO 96/39426. The invention encompasses all or a portion
of the
nucleotide sequence of human HIF-la with GENBANK Accession Nos. NM 001530 and
N1VI l 81054. All nucleotide sequences of the references cited supra are
incorporated herein
by reference in their entirety.
Generally, any HIF-la nucleic acid known in the art may be useful in the
methods and kits of the invention. Such nucleic acids generally encode at
least a portion of
HIF-la, or have a sequence that hybridizes to a HIF-la, -encoding nucleic acid
under
hybridizing conditions, as described herein.
In one embodiment, the methods of the invention may use a coding sequence
or a 5' or 3' untranslated region of a nucleic acid encoding HIF-1a or a
fragment thereof as
a probe, including naturally occurring and non-naturally occurring variants. A
non-
naturally occurring variant is one that is engineered by man (e.g., a peptide
nucleic acid
probe). In the methods of the invention wherein HIF-1 a, or an mRNA encoding
HIF- 1 a, in
a sample from a subject is detected or measured, naturally occurring gene
products are
detected, including but not limited to wild-type gene products as well as
mutants, allelic
variants, splice variants, polymorphic variants, etc. In general, variants
will be highly
homologous to the wild-type gene product encoding HIF-la, e.g., having at
least 90%,
95%, 98% or 99% amino acid sequence identity (as determined by standard
algorithms
known in the art, see, e.g., Altschul, 1990 Proc. Natl. Acad. Sci. U.S.A. 87:
2264-2268;
Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5877; Altschul et al.,
1990 J. Mol.
Biol. 215: 403-410).
HIF-1 a variants to be used as probes may be encoded by a nucleic acid
which is hybridizable under stringent conditions to a nucleic acid encoding
HIF-Ia.
Nucleic acid hybridization methods are well known in the art (see, e.g.,
Sambrook et al.,
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2001 Molecular Cloning, A Laboratory Manual, 3'd ed., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, New York; Ausubel et al., eds., 1994-1997, in the
Current
Protocols in Molecular Biology: Series of laborato try echnigue manuals, John
Wiley and
Sons, Inc.; Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-
92; Dyson,
1991 Essential Molecular Biology: A Practical Approach, vol. 2, T.A. Brown,
ed., 111-156,
Press at Oxford University Press, Oxford, UK). The term "stringent conditions"
refers to
the ability of a first polynucleotide molecule to hybridize, and remain bound
to a second
filter-bound polynucleotide molecule in 0.5 M NaHPO4, 7% sodium dodecyl
sulfate (SDS),
1 mM EDTA at 65 C, followed by washing in 0.2X SSC/0.1% SDS at 42 C (see
Ausubel et
al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). In
specific
embodiments, the variants being detected or measured comprise (or, if nucleic
acids,
encode) not more than 1, 2, 3, 4, 5, 10, 15 or 20 point mutations
(substitutions) relative the
wild-type sequence.
An isolated nucleic acid probe encoding HIF-1a or a portion thereof, can be
obtained by any method known in the art, e.g., from a deposited plasmid, by
PCR
amplification using synthetic primers hybridizable to the 3' and 5' ends of
the sequence,
and/or by cloning from a eDNA or genomic library using standard screening
techniques, or
by polynucleotide synthesis. Use of such probes for detection and quantitation
of specific
sequences is well known in the art. See e.g., Erlich, e.d, 1989, PCR
Technology Principles
and Applications for DNA Amplification, Macmillan Publishers Ltd., England;
Sambrook
et al, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, NY, 2001.
In some embodiments, the methods of the invention may use a gene coding
sequence, e.g., cDNA, of HIF-la, which preferably hybridizes under stringent
conditions as
described above to at least about 6, preferably about 12, most preferably
about 18 or more
consecutive nucleotides of the gene coding sequence of HIF-la protein, useful
for the
detection of an HIF-la protein
Using all or a portion of a nucleic acid sequence encoding HIF-1 cc protein,
such as those exemplified herein as a hybridization probe, full length nucleic
acid molecules
encoding HIF-1 a protein can be quantitated using standard hybridization
techniques (see,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold
Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, 2001)
for use in the methods of the invention, i.e., as a proxy for HIF-la level.
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The HIF-1 a sequences used in the methods of the invention are preferably
humansequences. However, homologs of the human HIF-la isolated from other
animals
can also be used in the methods of the invention as a proxy for HIF- 1 a
level, particularly
where the subject is a non-human animal. Thus, the invention also includes the
use of HIF-
1 a homologs identified from non-human animals such as non-human primates,
rats, mice,
farm animals including but not limited to cattle, horses, goats, sheep, pigs,
etc., household
pets including but not limited to cats, dogs, etc., in the methods of the
invention.
The methods of the invention may use fragments of any of the nucleic acids
disclosed herein in any of the methods of the invention. A fragment preferably
comprises at
least 10, 20, 50, 100, or 200 contiguous nucleotides of a sequence described
herein.
Standard recombinant DNA techniques known in the art may be used to
provide a HIF-la protein or a nucleic acid encoding an HIF-1 a protein, or a
fragment
thereof, for use in the methods and kits of the invention. In some
embodiments, in order to
provide a HIF-la or nucleic acid as a standard, the corresponding nucleotide
sequence
encoding HIF-la protein of interest can be cloned. For a review of PCR
technology and
cloning strategies which may be used in accordance with the invention, see,
e.g., PCR
Primer, 1995, Dieffenbach et al., ed., Cold Spring Harbor Laboratory Press;
Sambrook et
al., 2001, supra which are incorporated herein by reference in their
entireties.
6.7 HIF-1 ALPHA PROTEINS
The present invention provides for the use of HIF-1a proteins, or fragments
thereof, for the generation of antibodies for methods of the invention. HIF- I
a polypeptides
and fragments can also be used as protein abundance or activity standards in
the methods of
the invention.
For example, but not by way of limitation, the invention encompasses amino
acid sequences of HIF-1 a as dislcosed in U.S. Patent Nos. 6,455,674 (issued
to Einat et al);
6,652,799 (issued to Semenza); 6,222,018(issued to Semenza); Wang et al., 1995
PNAS
USA, 92: 5510-4; 6,436,654 (issued to Berkenstam); WO 96/39426. The amino acid
sequences cited in the above-identified references are incorporated herein by
reference in
their entirety. The invention encompasses the amino acid sequences of human
HIF-1 a with
GENBANK Accession No.s NP_001521 and NP_851397 each of which is incorporated
herein by reference in its entirety.
In some embodiments, the HIF-1 a protein comprises an amino acid
sequence that exhibits at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
99%
sequence similarity to the amino acid sequence of any of HIF-la proteins known
in the art.
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Algorithms for determining percent identity between two protein sequences are
well known
in the art, see, e.g., Altschul, 1990 Proc. Natl. Acad. Sci. U.S.A. 87: 2264-
2268; Altschul,
1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5877; Altschul et al., 1990 J.
Mol. Biol. 215:
403-410; each of which is incorporated herein by reference.
In a specific embodiment, proteins are provided consisting of or comprising
a fragment of HIF- 1 a protein consisting of at least ten contiguous amino
acids. In another
embodiment, the fragment consists of or comprises at least 20, 30, 40, or 50
contiguous
amino acids from a HIF-la protein for use, for example, in raising antibodies.
Such
fragments can also be useful, for example, as standards or controls in the
methods and kits
of the invention.
A variety of host-expression vector systems may be utilized to express HIF-
1 a proteins or fragments for use in the methods of the invention. Such host-
expression
systems are well known and provide the necessary means by which a protein of
interest may
be produced and subsequently purified. Examples of host-expression vector
systems that
may be used in accordance with the invention are: bacterial cells (e.g., E.
coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors containing a HIF-la nucleic acid coding sequence, yeast
cells (e.g.,
Saccharomyces, Pichia) transformed with a recombinant yeast expression vector
containing
the HIF-la coding sequence; insect cells infected with a recombinant virus
expression
vector (e.g., baculovirus) containing the HIF-la coding sequence; plant cells
infected with
a recombinant virus expression vector (e.g., cauliflower mosaic virus, CaMV;
tobacco
mosaic virus, TMV) or transformed with a recombinant plasmid expression vector
(e.g., Ti
plasmid) containing the HIF-la coding sequence; or mammalian cells (e.g., COS,
CHO,
BHK, 293, 3T3) harboring recombinant expression constructs containing
promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K
promoter).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the HIF-la being expressed. For
example,
when a large quantity of such a protein is to be produced for raising
antibodies, vectors that
direct the expression of high levels of protein products that are readily
purified may be
desirable. Such vectors include but are not limited to the E, coli expression
vector pUR278
(Ruther et al., 1983, EMBO J. 2:1791), in which the HIF-la coding sequence can
be
ligated into the vector in-frame with the lac Z coding region so that a fusion
protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101; Van
Heeke &
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Schuster, 1989, J. Biol. Chem. 264:5503); and the like. pGEX vectors can also
be used to
express foreign polypeptides as fusion proteins with glutathione S-transferase
(GST). In
general, such fusion proteins are soluble and can easily be purified from
lysed cells by
adsorption and binding to a column comprising of glutathione-agarose beads
followed by
elution in the presence of free glutathione. The pGEX vectors are designed to
include, e.g.,
thrombin or factor Xa protease cleavage sites so that the cloned target gene
product can be
released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) can be used as a vector to express foreign genes. The virus grows in
Spodoptera
fi ugiperda cells. The HIF-1 a coding sequence can be cloned individually into
non-
essential regions (for example the polyhedrin gene) of the virus and placed
under control of
an AcNPV promoter (for example the polyhedrin promoter). Successful insertion
of a HIF-
1 a coding sequence will resiilt in inactivation of the polyhedrin gene and
production of
non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat
coded for by the
polyhedrin gene). These recombinant viruses can be used to infect
Spodopterafrugiperda
cells in which the inserted gene is expressed (e.g., see Smith et al., 1983,
J. Virol. 46:584;
Smith, U.S. Patent No. 4,215,051).
In mammalian host cells, a number of viral-based expression systems can be
utilized. In cases where an adenovirus is used as an expression vector, the
HIF-1 a coding
sequence of interest may be ligated to an adenovirus transcription/translation
control
complex, e.g., the late promoter and tripartite leader sequence. This chimeric
gene may
then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in
a non-essential region of the viral genome (e.g., region El or E3) will result
in a
recombinant virus that is viable and capable of expressing HIF-la in infected
hosts (see,
e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655). Specific
initiation signals
may also be required for efficient translation of inserted HIF-la coding
sequences. These
signals include the ATG initiation codon and adjacent sequences. In cases
where an entire
HIF-1a gene, including its own initiation codon and adjacent sequences, is
inserted into the
appropriate expression vector, no additional translational control signals may
be needed.
However, in cases where only a portion of the HIF-la coding sequence is
inserted,
exogenous translational control signals, including, if necessary, the ATG
initiation codon,
must be provided. These exogenous translational control signals and initiation
codons can
be of a variety of origins, both natural and synthetic. Furthermore, the
initiation codon must
be in phase with the reading frame of the desired coding sequence to ensure
correct
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translation of the entire insert. The efficiency of expression may be enhanced
by the
inclusion of appropriate transcription enhancer elements, transcription
terminators, etc. (see
Bittner et al., 1987, Methods in Enz niol. 153: 516).
In addition, a host cell strain may be chosen that modulates the expression of
the inserted sequences, or modifies and processes the gene product in the
specific fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have
characteristic and specific mechanisms for the post-translational processing
and
modification of proteins and gene products. Appropriate cell lines or host
systems can be
chosen to ensure the correct modification and processing of the foreign
protein expressed.
To this end, eukaryotic host cells that possess the cellular machinery for
proper processing
of the primary transcript, glycosylation, and phosphorylation of the gene
product can be
used. Such mammalian host cells include but are not limited to CHO, VERO, BHK,
HeLa,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such
as, for
example, BT483, Hs578T, HTB26, BT20 and T47D, and normal mammary gland cell
lines
such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines that stably express the HIF-
la gene
product can be engineered. Rather than using expression vectors that contain
viral origins
of replication, host cells can be transformed with DNA controlled by
appropriate expression
control elements (e.g., promoter, enhancer, sequences, transcription
terininators,
polyadenylation sites, etc.), and a selectable marker.
Following introduction of the foreign DNA, engineered cells can be allowed
to grow for 1-2 days in an enriched media, and then can be switched to a
selective media. A
selectable marker in a recombinant construct, such as a plasmid, can confer
resistance to the
selective media, allow cells to stably integrate the plasmid into their
chromosomes, and
grow to form foci which, in turn, can be cloned and expanded into cell lines.
This method
can advantageously be used to engineer cell lines that stably express the HIF-
1 oc gene
product. Such engineered cell lines can be particularly useful in screening
and evaluating
compounds that affect the endogenous activity of the HIF-1a gene product.
A number of selection systems including but not limited to the herpes
simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci.
USA 48:
2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817)
genes can
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be employed in tk', hgprt- or aprt' cells, respectively. Also, anti-metabolite
resistance can be
used as the basis of selection for the following genes: dhfr, which confers
resistance to
methotrexate (Wigler et al., 1980, Proc Natl. Acad. Sci. USA 77: 3567; O'Hare
et al., 1981,
Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which
confers
resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol.
Biol. 150:
1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984,
Gene 30: 147).
6.8 ANTIBODIES TO HIF-1 ALPHA
The methods and kits of the invention encompass use of anti- HIF-1 a
antibodies or fragments thereof that specifically recognize one or more
epitopes of a HIF-
1a protein. Accordingly, any HIF-1a protein, derivative, or fragment can be
used as an
immunogen to generate antibodies that irimmunospecifally bind a HIF- 1 a
protein. Such
antibodies and fragments can be used in the detection and quantitation of a
HIF-1 a in a
sample to carry out any of the methods of the invention as disclosed herein.
Such antibodies can include but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain
antibodies,
Fab fragments, F(ab')2 fragments, Fv fragments, fragments produced by a Fab
expression
library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of
any of the
above. In a specific embodiment, antibodies to human HIF- 1 a protein are
used.
Described herein are general methods for the production of antibodies or
immunospecific fragments thereof. Any of such antibodies or fragments can be
produced
by standard immunological methods or by recombinant expression of nucleic acid
molecules encoding the antibody or an immunospecific fragment thereof in an
appropriate
host organism.
For the production of antibodies against HIF-la , any of various host
animals can be immunized by injection with a HIF-la gene product, or a portion
thereof.
Such host animals can include but are not limited to rabbits, mice, and rats.
Various
adjuvants can be used to increase the immunological response depending on the
host
species, including but not limited to Freund's (complete or incomplete),
mineral gels such
as aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol
or
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
CoNynebacteriutn paNvutn.
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Anti- HIF- 1 a monoclonal antibodies are preferred for use in the methods
and kits of the invention. Monoclonal antibodies can be obtained by any
technique that
provides for the production of antibody molecules by continuous cell lines in
culture. These
include but are not limited to the hybridoma technique of Kohler and Milstein,
(1975,
Nature 256: 495; and U.S. Patent No. 4,376,110), the human B-cell hybridoma
technique
(Kosbor et al., 1983, Immunology Today 4: 72; Cole et al., 1983, Proc. Natl.
Acad. Sci. USA
80: 2026), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies
And Cancer Therapy, Alan R. Liss, Inc., pp. 77). Such antibodies can be of any
immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass
thereof. The
hybridoma producing the mAb of this invention can be cultivated in vitro or in
vivo.
Techniques developed for the production of "chimeric antibodies" (Morrison
et al., 1984, Proc. Natl. Acad. Sci. 81, 6851-6855; Neuberger et al., 1984,
Nature 312, 604-
608; Takeda et al., 1985, Nature 314, 452-454) by splicing the genes from a
mouse
antibody molecule of appropriate antigen specificity together with genes from
a human
antibody molecule of appropriate biological activity can be used in preparing
antibodies
useful in the present invention. A chimeric antibody is a molecule in which
different
portions are derived from different animal species, such as those having a
variable region
derived from a murine mAb and a human immunoglobulin constant region. (See,
e.g.,
Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No.
5,816,397). The
invention thus contemplates chimeric antibodies that are specific or selective
for a HIF-la
protein. While often designed to be therapeutic, such chimeric antibodies can
be useful to
quantitate a HIF-1 a level according to the methods of the invention.
Further, humanized antibodies can be used in the methods and kits of the
invention. Briefly, humanized antibodies are antibody molecules from non-human
species
having one or more hypervariable regions or complementarity determining
regions (CDRs)
from the non-human species and framework regions from a human immunoglobulin
molecule. The extent of the framework region and Cars have been precisely
defined (see,
"Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.S.
Department of
Health and Human Services (1983)). Examples of techniques that have been
developed for
the production of humanized antibodies are known in the art and useful within
the scope of
the present invention. (See, e.g., Queen, U.S. Patent No. 5,585,089 and
Winter, U.S. Patent
No. 5,225,539). Humanized antibodies are typically developed as therapeutic
agents.
However, such antibodies can also be used in the methods and kits of the
present invention,
as they can be used to quantitate a HIF-la level in accordance with the
instant invention.
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Phage display technology can be used to increase the affinity of an antibody
to a HIF-1 a gene product. This technique can be useful in obtaining higher
affinity
antibodies to an HIF-1 a gene product, which could be used for the diagnosis
and prognosis
of a subject with cancer according to the present invention. The technology,
referred to as
affinity maturation, employs mutagenesis or CDR walking and re-selection using
the HIF-
1 a gene product antigen to identify antibodies that bind with higher affinity
to the antigen
when compared with the initial or parental antibody (see, e.g., Glaser et al.,
1992, J.
Immunology 149:3903). Mutagenizing entire codons rather than single
nucleotides results
in a semi-randomized repertoire of amino acid mutations. Libraries can be
constructed
consisting of a pool of variant clones, each of which differs by a single
amino acid alteration
in a single CDR, and contain variants representing each possible amino acid
substitution for
each CDR residue. Mutants with increased binding affinity for the antigen can
be screened
by contacting the immobilized mutants with labeled antigen. Any screening
method known
in the art can be used to identify mutant antibodies having increased avidity
to the antigen
(e.g., ELISA) (see Wu et al., 1998, Proc Natl. Acad Sci. USA 95:6037; Yelton
et al., 1995,
J. Immunology 155:1994). CDR walking that randomizes the light chain may also
be useful
(see Schier et al., 1996, J Mol. Blo. 263:551).
Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et
al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879; and Ward et al., 1989, Nature 334: 544)
can be
adapted to produce single chain antibodies against HIF-1a gene products.
Single chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region via an
amino acid bridge, resulting in a single chain polypeptide. Techniques for the
assembly of
functional Fv fragments in E. coli can also be used (Skerra et al., 1988,
Science 242:1038).
Antibody fragments that recognize specific epitopes can be generated by
known techniques. Such fragments can be used for quantitating a HIF-la gene
product
according to any available method known in the art. For example, such
fragments include
but are not limited to: F(ab')2 fragments, which can be produced by pepsin
digestion of the
antibody molecule; and Fab fragments, which can be generated by reducing the
disulfide
bridges of the F(ab')2 fragments; Fab fragments, which can be generated by
treating the
antibody molecule with papain and a reducing agent; and Fv fragments.
Alternatively, Fab
expression libraries can be constructed (Huse et al., 1989, Science 246:1275-
1281) to allow
rapid and easy identification of monoclonal Fab fragments having the desired
specificity.
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A molecular clone of an antibody to an antigen of interest can be prepared by
techniques known to one skilled in the art. Recombinant DNA methodology (see
e.g.,
Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor
Laboratory, Cold Spring Harbor, New York) can be used to construct nucleic
acid
sequences that encode a monoclonal antibody molecule, or an immunospecific
fragment
thereof.
Antibody molecules can be purified by well-known techniques, e.g.,
immunoabsorption or immunoaffinity chromatography, chromatographic methods
such as
HPLC (high performance liquid chromatography), or a combination thereof.
In the production of antibodies, screening for the desired antibody can be
accomplished by techniques known in the art, e.g., ELISA (enzyme-linked
immunosorbent
assay). For example, to select antibodies that recognize a specific domain of
an HIF-la ,
generated hybridomas can be assayed for a product that binds to a HIF- 1 a
fragment
containing such domain.
The foregoing antibodies can be used to quantify a HIF-la protein, e.g., to
measure levels thereof in appropriate samples, in the methods and kits of the
invention.
The methods of antibody production employed herein include those
described in Harlow and Lane (Harlow, E. and Lane, D., 11988, and later
editions,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, New York), which is incorporated herein by reference in its entirety.
Any antibody directed to one or more epitopes of an HIF-1 a can be used in
the methods and kits of the invention. Commercially available HIF-la
antibodies can be
used in accordance with the instant invention, for example those available
from Novus
Biologicals, Inc. (Littleton, CO); Affinity BioReagents, (Golden, CO).
6.9 KITS
The invention provides a pharmaceutical pack or kit comprising one or more
containers filled with compounds of the invention. Additionally, one or more
other
prophylactic or therapeutic agents useful for the treatment of a disease can
also be included
in the pharmaceutical pack or kit. The invention also provides a
pharmaceutical pack or kit
comprising one or more containers filled with one or more of the ingredients
of the
pharmaceutical compositions of the invention. Optionally associated with such
container(s)
can be a notice in the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products, which
notice reflects
approval by the agency of manufacture, use or sale for human administration.
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The present invention provides kits that can be used in the above methods.
In one embodiment, a kit comprises one or more compounds of the invention. In
another
embodiment, a kit further comprises one or more other prophylactic or
therapeutic agents
useful for the treatment of cancer, in one or more containers. In another
embodiment, a kit
further coinprises one or more anti-cancer agents that bind one or more cancer
antigens
associated with cancer. In certain embodiments, the other prophylactic or
therapeutic agent
is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic
agent is a
biological or hormonal therapeutic.
7. EXAMPLES
The following examples illustrate aspects of this invention but should not be
construed as limitations. The symbols and conventions used in these examples
are intended
to be consistent with those used in the contemporary, international, chemical
literature, for
example, the Journal of the American Chemical Society (J.Am.Chem.Soc.) and
Tetrahedron.
MATERIALS AND METHODS--Chemicals and Reagents
Human A375, SKMES, HT29, Panc-1, Jurkat, HeL023 and NCTC2544 cells
were obtained from American Tissue Culture Collection (ATCC). Tissue culture
media and
growth supplements were obtained from BioWhittaker. Unless otherwise noted,
all other
chemicals were purchased from Sigma. MRE 3046F20, 5-N-(4-methylphenyl-
carbamoyl)amino-8-methyl-2(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5-
c]pyrimidine;
IL-10 salt, 5-N-(4-diethylamino-phenyl-carbamoyl)amino-8-methyl-(2(2furyl)-
pyrazolo-
[4,3e]1,2,4-triazolo[1,5-c]pyrimidine); MRE 3008F20, 5-N-(4-methoxyphenyl-
carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]1,2,4-triazolo[1,5-
c]pyrimidine, MRE
3055F20, 5-N- (4-phenylcarbamoyl)amino- 8-propyl- 2 (2furyl) -pyrazolo[4,3e]-
1,2,4-
triazolo-[1,5c]pyrimidine and MRE 3062F20, 5-N-(4-phenyl-carbumoyl)amino-8-
butyl-
2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5-c]pyrimidine were synthesized by
Prof. P.G.
Baraldi, University of Ferrara, Italy. CGS15943, 5-amino-9-chloro-2-
(furyl)1,2,4-
triazolo[1,5-c] quinazoline and ZM 241385, 4-(2-[7-amino-2-(2-furyl)-
[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol were obtained from
RBI [Zeneca
Pharmaceuticals, Cheshire, UK]. PD 98059, 2-Amino-3-methoxyflavone, (a
selective
inhibitor of MAP kinase (MEK)) and rhodamine 123 were obtained from
Calbiochem.
U0126 (an inhibitor of MEK-1 and MEK-2) and SB 203580 (an inhibitor of
p381V1AP
kinase) were from Promega. RNAse was purchased from Boehringer.
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Cell Culture: For chemotherapeutic enhancement testing, the human
melanoma A375, human lung carcinoma SKMES, colon carcinoma HT29, and
pancreatic
cancer Panc-1 cell lines were originally obtained from the ATCC (American Type
Culture
Collection) and have been maintained at PRC. Cell lines are maintained in RPMI-
1640
medium supplemented with 100 units/ml penicillin G sodium, 100 g/mi
streptomycin
sulfate, 0.25 g/ml amphotericin B(fungizone), 2 mM glutamine, 10 mM HEPES, 25
g/ml
gentamycin, and 10% heat-inactivated fetal bovine serum. The cells are
cultured in a T25
Falcon Tissue Culture flask in a humidified incubator at 37 C with 5% C02-95%
air. The
cells are sub-cultured twice a week. The doubling times of A375, SKMES, HT29,
and
Panc-1 cultures are approximately 22, 45, 48, and 60 hr, respectively.
For multi-drug resistance testing, A375 and NCTC2544 cells are grown
adherently and maintained in DMEM and EMEM medium, respectively, containing
10%
fetal calf serum, penicillin (100 U/ml), streptomycin (100 g/ml), L-glutamine
(2 mM) at
37 C in 5% C02/95% air. HeL023 and Jurkat were grown in RPMI-1640 medium,
containing 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100
g/ml), L-
glutamine (2 mM) at 37 C in 5% C02/95% air. Cells are passaged two or three
times
weekly at a ratio between 1:5 and 1:10. Lymphocytes were isolated from buffy
coats kindly
provided by the Blood Bank of the University Hospital of Ferrara. Blood
donated by
healthy volunteers, after informed consent for research was obtained.
Lymphocytes were
isolated by density gradient centrifugation (Ficoll/Histopaque 1.077 g/ml).
The cells are
stimulated with purified phytohemoagglutinin (1 g/ml) and expanded in RPMI
medium
added of interleukin 2 (20 Units/ml) and 10% fetal calf serum.
Colony Formation Assay: Exponentially growing A375 cells were seeded at
300 cells per well in six-well plates with 2 ml of fresh medium and treated
with paclitaxel,
vindesine and adenosine receptor agonists and antagonists dissolved in DMSO
solution.
Control plates received the same volume of DMSO alone. After 7 days of growth
at 37 C
in humidified atmosphere containing 5% C02, the cells were fixed with absolute
methanol
for 5' and stained with 1/10 Giemsa/phosphate-buffered saline (PBS) staining
solution for
10 minutes. Staining solution was removed and colonies of greater than 30
cells were
scored as survivors. For each treatment, six individual wells were scored.
Flow CXtometry Analysis: A375 and NCTC2544 adherent cells were
trypsinized, mixed with floating cells, washed with PBS and permeabilised in
70% (vol/vol)
ethanol/PBS solution at 4 C for at least 24 hours. Jurkat, HeL023 and PBMC
cells were
centrifuged for 10 minutes at 1000 x g. The cell pellet was then resuspended
and
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permeabllised in 70% (vol/vol) ethanol/PBS solution at 4 C for at least 24
hours. The cells
were washed with PBS and the DNA was stained with a PBS solution, containing
20 g/ml
of propidium iodide and 100 g/ml of RNAse, at room temperature for 30
minutes. Cells
were analysed by FACScan (Becton-Dickinson) and the content of DNA was
evaluated by
the Cell-LISYS program (Becton-Dickinson). Cell distribution among cell cycle
phases and
apoptotic cells was evaluated as previously described (Secchiero et al.,
2001). Briefly, the
cell cycle distribution is shown as the percentage of cells containing 2n (Gl
phase), 4n (G2
and M phases), 4n>x>2n. DNA amount (S phase) judged by propidium iodide
staining.
The apoptotic population is the percentage of cells with DNA content lower
than 2n.
Rhodamine 123 E~'flux Assay: 5x105 cells from each subline were loaded
with 50 ng/ml of rhodamine 123 for 30 minutes at 37C in medium. The cells were
washed
and resuspended in dye-staining agent free medium for 3 hours at 37 C. to
allow rhodamine
123 efflux. The cells were then washed twice and the fluorescence of
intracellular
rhodamine 123 were analysed with Flow cytometer (residual fluorescence FRES).
The
fluorescence was compared with loaded cells maintained at 4 C. to prevent drug
export
(maximal fluorescence FMAX). The cells were treated with adenosine receptor
antagonists
to evaluate the ability of adenosine receptors to interfere with P-gp drug
efflux activity.
Cells from each subline that were not being exposed to rhodamine 123 were used
as
negative controls.
Metabolic Inhibitors: Cells were treated for 30 minutes with metabolic
inhibitors or with drug vehicle (DMSO) prior to being challenged with
adenosine receptor
antagonists and paclitaxel or vindesine. After 24 hours, cells were collected
for flow
cytometry analysis. PD 98059 was used at 20 uM as an inhibitor of MEK to
prevent MEK-
1 activation. U01 26 was used at 10 uM as inhibitor of MEK-1 and MEK-2 to
prevent
extracellular signal-regulated kinase ERK-1 and ERK-2 activation. SB 203580
was used at
1 uM as an inhibitor of p38MAP kinase (p38MAPK).
Statistical Analysis: All values in the figures and text are expressed as mean
+.standard deviation (SD) of n observation (with n>3). Data sets were examined
by
analysis of variance (ANOVA) and Dunnett's test (when required). A P value
less than 0.05
was considered statistically significant. Representative images obtained by
FACscan are
reported, with similar results having been obtained in at least three
different experiments.
Inventors have verified the concentration of adenosine A3 receptors in human
cancer cells that is elevated in the cancerous tumors compared to non-
cancerous tumors or
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normal tissue. Table 8 and FIG. 1 indicate elevated levels of adenosine A3
receptors found
by the inventors in human A375 (human melanoma), Panc-1 (human pancreatic
carcinoma),
MX-1 (human breast carcinoma), PC-1 (human prostate carcinoma), HT29 (human
colon
carcinoma), and SKMES (human lung carcinoma) and A2780AD 10 (human ovarian
carcinoma).
Three A3 receptor antagonists have been tested for their'Degree of Growth
Inhibitory activity and enhancement of inhibitory growth function of known
anti-neoplastic
agents. Table 6 indicates receptor binding assay results for three A3 receptor
antagonist
compounds. Structures of the three compounds are as follow:
0 OCH3
\ I
HN J~ N
H
N/ N'N
i N O
N
H3C
MRE3008F20: 5-[[(4-Methoxyphenyl)amino] carbonyl] amino-8-propyl-2-(2-furyl)-
pyrazolo[4,3-e] 1,2,4-triazolo[1,5-c]pyrimidine
r CH3
O NvCH3
~
HN J N
~ H
N N N 6 ~ I
~ N N I
N
H3C
IL- 10: N- 1-(4-diethylamino-phenyl)-N'-S-[8-methyl-2-(2-furyl)-pyrazolo[4,3-
e]
1,2,4-triazolo [ 1 , 5-c] pyrimidine] -urea
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H3C
OII ~ I N, CH3
Jl
HN N
H
N N'N
Ni1 N O
N
H3C
IL- 11: N-1-(4-dimethylamino-phenyl)-N'-5-[8-methyl-2-(2-furyl)-pyrazolo[4,3-
e]
1,2,4 -triazolo[1,5-c]pyrimidine]-urea
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Table 6. Binding Affinity at rA1, rA2 and hA3 Adenosine Receptors
rA1(Kõ nM) rA2A (Kõ nM) hA3 (K;, nM) rA1/hA3 rA2A/hA3
MItE3008F20 >10,000 1,993 0.29 >34,482 6,872
(1,658-2,397) (0.27-0.32)
IL-10 580 523 27 21.5 19.4
IL-11 >10,000 >10,000 15 >6700 6700
Table 6 shows MRE3008F20, IL-IO and IL-11 to be potent, selective
antagonists for the human adenosine A3 receptor.
In addition to binding affinity studies of these three compounds, growth
inhibitory studies were also performed. Results of growth studies of the
compounds,
uncombined with other compounds, are shown in Table 7. Table 7 also indicates
the growth
inhibitory activity of common anti-neoplastic agents.
Table 7: Growth Inhibitory Activity of A3 Antagonists and Anti-Neoplastic
Agents
Used Separately
IC50 Values (gg/ml) ~ Mean
Agent Human Human Colon Human Human Lung
Melanoma Carcinoma HT29 Pancreatic Cancer Carcinoma
A375 Panc-1 SKMES
MRE3008F20 91.3 ~ 1.5 39.4 ~= 7.7 15.8 =1: 13.5 19 =t= 1.2
IL-10 11.9~0.4 10t0.7 3.9 3.3 5.9t0.3
IL-11 49.3~40.7 56.5 8.3 8~= 5.7 36.3~31.5
irinotecan HCL 0.88 ~ 0.19 1.05 0.07 1.12 ~ 0.29 1.23 f 0.36
paclitaxel 0.004 f 0.0002 0.00112 =L 0.0003 0.0027 ~= 0.0003 0.0026 =L 0.0007
docetaxel 0.0002 f 0.00007 0.00024 0.00005 0.00037 0.00031 0.00084
0.00037
vinblastine 0.0016 0.0019 f 0.0004 0.0018 f 0.0008 0.0019 f 0.0009
Compounds MRE3008F20, IL-10 and IL-11 were obtained from King
Pharmaceutical, Inc. Irinotecan HC1(Camptosar ; 20 mg/ml) was obtained from
Pharmacia & Upjohn Co. Paclitaxel (Taxol , 6 mg/ml) was obtained from Bristol-
Myers
Squibb, Co. Docetaxel (Taxotere ) was obtained from Rhone-Poulenc Rorer.
Vinblastine
sulfate salt was obtained from Sigma Chemical Co., (V 1377). Irinotecan was
diluted with
culture medium. All other agents were dissolved in 100% DMSO at appropriate
concentrations. The DMSO stock solutions were diluted 100-fold with growth
medium so
that the final DMSO concentration was 1%. We have previously shown that DMSO
has no
effect on the growth of culture cells at concentrations up to 1%. MTT (3-[4,5-
Dimethylthiazol-2-y1]2,5-diphenyltetrazolium bromide) was obtained from Sigma
Chemical
Co. RPMI- 1640 medium, antibiotic antimycotic 100 X consisting of 10,000
units/ml
penicillin G sodium, 10,000 g/mi streptomycin sulfate, 25 g/ml Amphotericin
B
(fungizone), glutamine (200mM), HEPES buffer (1 M), gentamicin (50 mg/ml),
sodium
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bicarbonate (7.5%), and fetal bovine serum were obtained from GibcoBRL. The
complement in fetal bovine serum was inactivated at 56 C for 30 min.
The human melanoma A375, human lung carcinoma SKMES, colon
carcinoma HT29, and pancreatic cancer Panc-1 cell lines were originally
obtained from the
ATCC (American Type Culture Collection) and have been maintained in RPMI-1640
medium supplemented with 100 units/ml penicillin G sodium, 100 gg/mi
streptomycin
sulfate, 0.25 g/ml amphotericin B(fungizone), 2 mM glutamine, 10 mM HEPES, 25
g/ml
gentamycin, and 10% heat-inactivated fetal bovine serum.
Most of the agents showed a broad spectrum and equally potent growth
inhibitory activity against these four histologically distinct human tumor
cell lines (A375
melanoma, HT29 colon carcinoma, Panc-1 pancreatic carcinoma, and SKMES lung
carcinoma). The IC50 values were approximately the same against all four cell
lines.
Interestingly, HT29 is approximately 30-fold more refractory than the A375
cell line to
doxorubicin (IC50 value of 0.211 vs. 0.0064 g/ml) and mitoxantrone (ICSO
value of 0.1 vs.
0.0032 g/ml), respectively. It is possible that the HT29 cell line has an
altered form of
topoisomerase II, and is therefore more refractory to doxorubicin and
mitoxantrone, which
mediate their growth inhibitory activity by trapping topoisomerase II, DNA,
and drug in
ternary complexes.
7.1 ABUNDANCE OF A3 RECEPTORS IN HUMAN SOLID TUMORS
AND MELANOMA.
Inventors verified the concentration of adenosine A3 receptors in human
cancer cells that is elevated in the cancerous tumors compared to non-
cancerous tumors or
normal tissue. Table 8 and Figure 1 indicate elevated levels of adenosine A3
receptors
found by the inventors in human A375 (human melanoma), Panc-1 (human
pancreatic
carcinoma), MX-1 (human breast carcinoma), PC-1 (human prostate carcinoma),
HT29
(human colon carcinoma), and SKMES (human lung carcinoma).
Table 8 - Abundance of A3 receptors in human solid tumors and melanoma.
Data shown are equilibrium binding parameters at 4 C expressed as
dissociation constant, KD (nM), and BMAX (finol/mg protein) for [3H]MRE
3008F20 derived
from saturation experiments to human A3 adenosine receptors expressed in
tumour tissues.
Tumor types KD BMAX Figure
Human malignant 1.9 0.2 256 31 lA
melanoma A375
Human colon DLDl 2.1 0.3 434 40 1B
Human pancreatic 2.6 f 0.3 249 34 1C
MiaPaCa
Human pancreatic 2.7 0.1 441 46 1D
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Panel
Human breast tumor 1.9 0.2 435 50 1E
MXl
Human lung squamour 2.4 0.6 169 20 1F
carcinoma SKMES
Human pancreatic PC3 3.1 0.1 379 ::L 40 1G
Human colon HT29 3.3 0.3 213 23 IH
7.2 HUMAN MELANOMA A375
The growth inhibitory activity of paclitaxel against the human melanoma
A375 cell line was determined in the absence or in the presence of 10 gg/ml of
MRE3008F20 or 5 g/ml each of IL- 10 and IL-11 (Table 9). At these sub-
cytotoxic
concentrations, MRE3008F20, IL-10, and IL-11 (approximately 3 0-45% growth
inhibition
in the presence of A3 antagonists alone), enhanced the growth inhibitory
activity of
paclitaxel by 8-12-fold; IC50 values decreased from 0.0046gg/ml (paclitaxel
alone) to
0.0004-0.00054 g/ml (paclitaxel plus MRE3008F20, IL-10, and IL-11).
Table 9: Growth Inhibitory Activity of A3 Antagonists and Anti-Neoplastic
Agents Used Jointly With A375 Cells
Anti-Neoplastic Agent Antagonist (Concentration) IC50 ( g/ml) Enhancement
(Concentration Range) Factor
paclitaxel (0.0002-0.1 g/m1) None 0.0046
paclitaxel (0.0002-0.1 gg/m1) MRE3008F20 (10 gg/ml) 0.00054 8.5
paclitaxel (0.0002-0.1 g/ml) IL-10 (5 gg/ml) 0.0005 9.2
paclitaxel (0.0002-0.1 g/m1) IL-11 (5 g/m1) 0.0004 11.5
It was also detennined that IL- 10 and IL-11 enhance the growth inhibitory
activity of paclitaxel in a concentration-dependent manner. Results of further
testing of the
enhancement effects with taxane family compounds is given in Table 10. Both of
the
common taxane compounds paclitaxel (taxolTM) and docetaxel (taxotereTM) are
included.
Table 10 illustrates concentration-dependencies for concentrations of 1, 3,
and 10 gg/ml for MRE3008F20 and for concentrations of 0.5, 1.5, and 5 g/ml
for
compounds IL-10 and IL-1 l. MRE3008F20 enhanced the growth inhibitory activity
of
paclitaxel in a concentration-independent manner (enhancement factor = 6.4-7.1
at all three
concentrations). On the other hand, IL-10 and IL-11 enhanced the growth
inhibitory
activity of paclitaxel in a concentration-dependent manner. This testing also
indicated that
compounds MRE3008F20, IL-10 and IL-11 have enhancement factors with docetaxel
that
are indicative of a synergistic effect.
Table 10: Growth Inhibitory Activity of Antagonists and Taxane Compounds used
Jointly with A375 Cells
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Anti-Neoplastic Agent A3 Antagonist IC50 ( g/ml) Enhancement
(Concentration Rainge) (Concentration) Factor
aclitaxel (0.0001-0.05 gg/ml) None 0.0042
paclitaxel (0.0001-0.05 g/ml) MRE3008F20 (1 ml) 0.00059 7.1
paclitaxel (0.0001-0.05 g/ml) MRE3008F20 (3 ml) 0.00066 6.4
paclitaxel (0.0001-0.05 g/ml) MRE3008F20 (10 g/ml) 0.00059 7.1
paclitaxel (0.0001-0.05 g/ml) none 0.0044
paclitaxel (0.0001-0.05 g/ml) IL-10 (0.5 gg/ml) 0.0012 3.7
paclitaxel (0.0001-0.05 gg/ml) IL-10 (1.5 g/ml) 0.00061 7.2
paclitaxel (0.0001-0.05 gg/ml) IL-10 (5 g/ml) 0.0005 8.8
paclitaxel (0.0001-0.05 gg/ml) none 0.0044
paclitaxel (0.0001-0.05 g/ml) IL-11 (0.5 g/ml) 0.0014 3.1
paclitaxel (0.0001-0.05 ml) IL-11 (1.5 jig/mi) 0.00067 6.6
paclitaxel (0.0001-0.05 g/ml) IL-11 (5 g/ml) 0.00046 9.6
docetaxel (0.00001-0.005 ml) none 0.000038
docetaxel (0.00001-0.005 /ml) MRE3008F20 (1 gg/ml) 0.0000006 63.3
docetaxel (0.00001-0.005 ml) MRE3008F20 (3 ml) 0.0000055 6.9
docetaxel (0.00001-0.005 g/ml) MRE3008F20 (10 g/ml) 0.0000072 5.3
docetaxel (0.00001-0.005 g/ml) none 0.000021
docetaxel (0.00001-0.005 g/ml) IL-10 (0.5 g/ml) 0.0000025 8.4
docetaxel (0.00001-0.005 g/ml) IL-10 (1.5 /ml) 0.0000023 9.1
docetaxel (0.00001-0.005 g/ml) IL-10 (5 g/ml) 0.0000072 2.9
docetaxel (0.00001-0.005 gg/ml) none 0.000046
docetaxel (0.00001-0.005 g/ml) IL-11 (0.5 gg/ml) 0.0000087 5.3
docetaxel (0.00001-0.005 g/ml) IL-11 (1.5 gg/ml) 0.000005 9.2
docetaxel (0.00001-0.005 g/ml) IL-11 (5 g/ml) 0.0000071 6.5
7.3 HUMAN LUNG CARCINOMA SKMES
In human lung carcinoma SKMES, all three A3 antagonists enhance the
growth inhibitory activity of paclitaxel, docetaxel, irinotecan and vindesine
(Table 11).
Table 11: Growth Inhibitory Activity of A3 Antagonists and Anti-Neoplastic
Agents
used Jointly with SKMES Cells
Anti-Neoplastic Agent A3 Antagonist ICso ( g/ml) Enhancement
(Concentration Range) (Concentration) Factor
paclitaxel (0.0001-0.05 gg/ml) None 0.0033
paclitaxel (0.0001-0.05 gg/ml) MRE3008F20 (1 gg/ml) 0.0008 4.1
paclitaxel (0.0001-0.05 g/ml) IL-10 (0.5 gg/ml) 0.0012 2.8
paclitaxel (0.0001-0.05 gg/ml) IL-11 (0.5 g/mI) 0.00078 4.2
docetaxel (0.00001-0.005 /ml) none 0.00047
docetaxel (0.00001-0.005 g/ml) MRE3008F20 (1 g/ml) 0.00013 3.6
docetaxel (0.00001-0.005 /ml) IL-10 (0.5 g/ml) 0.00017 2.8
docetaxel (0.00001-0.005 gg/ml) IL-11 (0.5 gg/ml) 0.00025 1.9
docetaxel (0.00001-0.005 g/ml) none 1.17
irinotecan HCL (0.04-20 g/ml) MRE3008F20 (1 g/ml) 0.57 2.1
irinotecan HCL (0.04-20 g/ml) IL-10 (0.5 gg/ml) 0.67 1.7
irinotecan HCL (0.04-20 g/ml) IL-11 (0.5 g/ml) .061 1.9
vinblastine (0.00001-0.005 gg/ml) none 0.0015
vinblastine (0.00001-0.005 gg/ml) MRE3008F20 (1 g/ml) 0.0013 1.2
vinblastine (0.00001-0.005 g/ml) IL-10 (0.5 g/ml) 0.0011 1.4
vinblastine (0.00001-0.005 gg/ml) IL-11 (0.5 gg/ml) 0.00094 1.6
In comparing Table 11 with the results obtained with A375 human
melanoma, the magnitude of enhancement for the taxane compounds in SKMES cells
is less
than that observed in A375 melanoma. These differences in absolute magnitude
of
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synergism may be related to the activity of P-gp in the different tumors under
the test
conditions employed. However, the enhancement between A3 antagonists and
taxane
compounds is consistently observed. Enhancement in SKMES cells is also
consistent with
a mechanism of inhibiting P-gp, in the case of vinblastine, and inhibiting
MRP, as in the
case of irinotecan HCL.
7.4 HUMAN COLON CARCINOMA HT29
The growth inhibitory activity of paclitaxel and docetaxel against the human
colon carcinoma HT29 cell line was determined in the absence or in the
presence of 1 g/ml
of MRE3 008F20 or 0.5 g/ml each of IL- 10 and IL-11 (Table 12). It was found
that A3
antagonists enhance the growth inhibitory activity of paclitaxel, docetaxel,
doxorubicin and
irinotecan.
Table 12: Growth Inhibitory Activity of A3 Antagonists and Taxan Compounds
Used
Jointly With HT29 Cells
Anti-Neoplastic (Concentration A3 Antagonist IC50 ( g/ml) Enhancement
Range) Agent (Concentration) Factor
paclitaxel (0.001-0.05 g/ml) none 0.0025
paclitaxel (0.001-0.05 g/ml) MRE3008F20 (I ml) 0.00085 2.9
paclitaxel (0.001-0.05 g/ml) IL-10 (0.5 g/ml) 0.00100 2.5
paclitaxel (0.001-0.05 g/ml) IL-11 (0.5 g/ml) 0.00099 2.5
docetaxel (0.0001-0.005 ~tg/ral) none 0.000018
docetaxel (0.0001-0.005 g/ml) MRE3008F20 (1 g/ml) 0.0000012 15.0
docetaxel (0.0001-0.005 g/ml) IL-10 (0.5 g/ml) 0.0000033 5.5
docetaxel (0.0001-0.05 g/ml) IL-11 (0.05 g/ml) 0.0000028 6.4
doxorubicin (0.0002-0.1 g/ml) none 0.46
doxorubicin (0.0002-0.1 g/ml) MRE3008F20 (1 g/ml) 0.28 1.6
doxorubicin (0.0002-0.1 g/ml) IL-10 (0.5 g/ml) 0.31 1.5
doxorubicin (0.0021-0.1 g/ml) IL-I1 (0.5 g/ml) 0.38 1.2
irinotecan HC1 (0.04-20 g/ml) none 0.96
irinotecan HC1 (0.04-20 /ml) 1VIRE3008F20 (1 ml) 0.53 1.8
irinotecan HC1 (0.04-20 /ml) IL-10 (0.5 /ml)) 0.77 1.2
irinotecan HC1 (0.04-20 /ml) IL-11 (0.5 /ml) 0.85 1.1
7.5 HUMAN PANCREATIC CANCER PANC-1
In human pancreatic cancer Panc-1, A3 antagonists potentiated the growth
inhibitory activity of taxane family compounds paclitaxel and docetaxel (Table
13).
However, the potentiation observed is of a smaller magnitude compared to that
observed in
A375 melanoma.
Table 13: Growth Inhibitory Activity of A3 Antagonists and Taxane Compounds
Used
Jointly With Panc-1 Cells
Anti-Neoplastic (Concentration A3 Antagonist IC50 ( g/ml) Enhancement
Range) Agent (Concentration) Factor
aclitaxel (0.0001-0.005 g/ml) None 0.0029
aclitaxel (0.0001-0.05 ml) MRE3008F20 (1 g/ml) 0.0015 1.9
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paclitaxel (0.0001-0.005 gg/ml) IL-10 (0.5 gg/ml) 0.0017 1.7
aclitaxel (0.0001-0.05 gg/ml) IL-11 (0.5 g/ml) 0.0016 1.8
docetaxel (0.00001-0.005 g/ml) none 0.00068
docetaxel (0.00001-0.005 gg/ml) MRE3008F20 (1 g/ml) 0.00038 1.8
docetaxel (0.00001-0.005 g/ml) IL-10 (0.5 gg/ml) 0.00047 1.4
docetaxel (0.00001-0.005 gg/ml) IL-11 (0.5 g/ml) 0.00050 1.4
It is seen from the above data and tables that the use of high affinity
adenosine A3 receptor antagonists in conjunction with chemotherapeutic cancer
agents
result in a notable enhancement effect for many of the agents.
7.6 MDR CANCER CELL TREATMENTS
Having noted that chemotherapeutic cancer agents can be selected for A3
antagonist enhancement from the form of MDR they are associated with, the
inventors have
further established that high affinity A3 adenosine receptor antagonists can
be used to
counter P-gp and MRP associated multi-drug resistance.
At first, inventors evaluated whether A3 adenosine receptor could protect the
cell by the toxic effect of conventional chemotherapeutic drug. Colony
formation assay
experiments were performed on A375 melanoma cells treated with increasing
concentration
of paclitaxel (0.25-75 g/ml). A3 stimulation is achieved with the selective
agonist CI-IB-
MECA while A3 blockade is obtained with the selective antagonist MRE 3008F20.
Colony
formation of A375 cells is abolished when both paclitaxel (0.75 g/ml) and MRE
3008F20
(10 M) are applied whereas colony formation is only partly decreased when
paclitaxel
alone or MRE 3008F20 alone are applied. (Figure 2)
Colony formation of A375 cells is increased of about 30% when the
adenosine A3 agonist CI-IB-MECA (10 M) is applied. This is seen in the "C"
bar of
Figure 2 being 130% of the DMSO control bar "D" Figure 2 further shows that
increased
colony formation occurs when CI-IB-MECA is combined with the taxane family
compound
paclitaxel (0.75 g/ml). When paclitaxel alone is added ("T" bar of Figure 2),
colony
formation is 64% of the control. Colony formation is increased 32% to 85% of
control
when the CI-IB-MECA is combined ("TC" bar of Figure 2).
Using the adenosine A3 antagonist MRE 3008F20 (10 M) alone decreases
colony formation to 59% of control ("M" bar of Figure 2). Surprisingly, when
the A3
antagonist is combined with the taxane compound, virtually all colony
formation ceases
("TM" bar of Figure 2). This clearly identifies the synergistic nature of
combining A3
antagonists with chemotherapeutic agents. In comparison to the surprising
result of 0.2%
colony formation, a geometrical combination predicts a result of 38%.
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One explanation for these results is that A3 receptors trigger a pro-survival
signal, not able to restore colony formation ability of A375 cells treated
with the taxane
paclitaxel, while the blockage of As increased paclitaxel mediated deleterious
effects
(p<0.01, "TM" bar versus "T" bar of Figure 2).
Subsequent to colony formation experiments, the inventors also analysed the
ability of A3 adenosine receptor antagonists to enhance chemotherapeutic
effects by
performing an acute treatment of A375 cells with the taxane family compound
paclitaxel
and the vinca alkaloid vindesine. Cell proliferation and apoptosis are
quantified by flow
cytometer analysis after propidium-iodide (PI) DNA staining. At 24 hours post-
exposure,
paclitaxel and vindesine induced a dose dependent cell accumulation into G2/M
cell cycle
phases and a parallel decrease of the Gl population.
To quantify the effect of paclitaxel and vindesine to alter cell proliferation
the inventors determined the concentration exerting the 50% of the GZ/M
accumulation
(ECSO). When exposed to increasing concentrations of chemotherapeutic drugs,
EC50 are
16.60+2.00 ngJml and 1.90+0.20 nM for paclitaxel and vindesine, respectively
(means of
four experiments). Analyses further show that EC5o of paclitaxel and vindesine
for decrease
of Gl population are not significantly changed respect to EC50 of G2/M arrest.
The S-phase
population, representative of replicating DNA, is not appreciably changed.
Additional experiments quantified the sub-G, population, representative of
cells undergoing apoptosis. In A375 cells, the percentage of apoptotic cells
increases
progressively with paclitaxel concentration, reaching the maximum value
(ranging from 35
to 53% on different experiments) at 5 ng/ml. An increase of the paclitaxel
concentration
results in a decrease of A375 cells at sub-Gl. The concentration exerting the
maximal
apoptosis (ECmAx) is 6.00 0.63 ng/ml (mean of four experiments). Similarly,
experiments
performed with vindesine obtain an EC mAx value of 3.54 + 0.42 nM (mean of
four
experiments).
A375 cells treated with paclitaxel or with vindesine with or without the A3
adenosine receptor selective A3 antagonist MRE 3008F20 demonstrate
enhancement.
Figures 3A and 3C show that MRE 3008F20 (10 M) improved vindesine and
paclitaxel
ability to alter cell proliferation: MRE 3008F20 reduced paclitaxel and
vindesine EC50 of
1.9 and 4.0 fold, respectively. Similar results were obtained analysing EC50
values
calculated for the Gl population.
Furthermore, MRE 3008F20 (10 M) reduced ECmAx of 2.0 and 2.1 fold, for
paclitaxel and vindesine, respectively (Figure 3B and 3D).
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Representative flow cytometry profiles of DNA content in A375 cells (Apo
(sub diploid cells), Gl, S and G2/M phases) are shown in Figure 4. Figure 4C
shows that,
under both treatments (A3 antagonist MRE3008F20 plus vinca alkaloid
vindesine),
vindesine response increased as cells progressed from Gl to G2/M phases
respect to
vindesine-treated cells alone (Figure 4B). Similar results were obtained with
the taxane
compound paclitaxel (Figure 4E-F).
To verify whether this enhancement activity was not related to toxic
contaminants present in the MRE3008F20 solution but due to A3 adenosine
receptor
specific blockade, inventors quantified the ability of other adenosine
receptor antagonists to
enhance A375 cell responses to vindesine and paclitaxel.
A375 cells were treated with vindesine (1 nM) with increasing
concentrations of adenosine receptor antagonists (MRE3055F20, MRE3062F20,
MRE3046F20, MRE3008F20, IL-10, CGS 15943, ZM 241385). As illustrated in Figure
5A, the order of potency of adenosine antagonists to enhance Vindesine effect
was:
MRE3055F20 (highest) > MRE3062F20 > MRE 3046F20 > MRE3008F20 > IL-10 >
CGS15943 > ZM241385 (lowest). The concentrations exerting the 50% of the
enhancing
activity (SEC50) are reported in Table 14 as the mean of four experiments.
SEC50 values are
in good agreement with inhibitory equilibrium binding constants (Ki) observed
in binding
experiments for the adenosine A3 receptor (Figure 5B).
Table 14 Adenosine receptor antagonist parameters of enhancing activity to
vindesine
and binding affmity to A3 adenosine receptor in A375 cells.
SEC50 Ki
(p.M) (nM)
MRE 3055F20 0.31 + 0.03 1.4 + 0.2
MRE 3062F20 0.43 + 0.04 2.3 0.2
MRE 3046F20 0.57 0.06 3.0 0.3
MRE 3008F20 0.55 :~ 0.06 3.1 :L 0.3
IL-10 0.97 :1:0.10 30.0 =L 2.5
CGS 15943 12.60 :L 1.41 118 :L 12
ZM 241385 25.00 2.23 270 25
SEC50: adenosine receptor antagonist dose that induces 50% of the
enhancing activity to vindesine (1 nM), calculated on G2/M accumulation dose
response
curve. Ki equilibrium constant of binding affinity at human A3 adenosine
receptor. Data
represents the mean of four independent experiments.
To verify the role of A3 receptor antagonists on paclitaxel and vindesine
mediated alteration of cell proliferation and apoptosis, testing was performed
with
additional cell lines. Cell lines were selected with a pattern of surface
adenosine receptor
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expression similar to A375 cell line, namely HeL023, Jurkat, NCTC2544 and PBMC
blasts.
Experimental conditions are similar to those for the A375 cell testing. The
effect upon ECsn
values of vindesine and paclitaxel mediated cell cycle distribution alteration
(G2/M
accumulation) under treatment with the A3 antagonist MRE 3008F20 is given in
Table 15.
The variation in the level of apoptosis as reflected in EC50 values are also
indicated.
In Table 15, the HeLa23 cell line has the lowest sensitivity to paclitaxel and
vindesine alone. Interestingly, HeLa23 cells also show the greatest amount of
enhancement
by MRE3008F20 co-treatment.
Table 15: Induction of Ga/M accumulation and apoptis of different cells by
paclitaxel
and vindesine with or without A3 receptor antagonist MRE3008F20 treatment
A375 HeLa23 Jurkat
paclitaxel vindesine paclitaxel vindesine paclitaxel vindesine
ECso ECso EC50 ECso EC50 ECso
DMSO 16.60:~2.00 1.9W:0.20 50.6015.30 25.8013.25 2.964:0.31 0.9310.08
MRE 3008F20 8.60 0.81* 0.48 0.05* 4.10 0.40* 0.71d:0.08* 2.52 0.30 0.37 0.04*
ECnA, ECMAX ECMpX ECMpX ECMAX ECMpX
DMSO 6.00 0.63 3.54t0.422 33.42 3.55 33.11 4.02 3.32 0.52 nd
MRE3008F20 3.04f0.32* 1.6610.20 3.19 0.30* 1.0510.09* 2.29f0.31* nd
NCTC 2544 PBMC
paclitaxel vindesine paclitaxel vindesine
ECgo ECso ECso ECso
DMSO 2.69f0.28 0.89 0.08 5.30 0.60 1.10 0.90
MRE 3008F20 2.41 0.30 0.51 0.06* 5.30 0.60 0.86 0.09
ECMax ECmAX ECMAX ECmax
DMSO 2.69 0.23 2.50 0.27 nd nd
MRE 3008F20 2.55=L0.24 1.34~W.15* nd nd
Data represent the mean SE of four independent experiments. 1VIRE3008F20 is
10 M.
EC50 values were obtained by analysing G2/M accumulation dose response
curve. ECMAx values were obtained by analysing sub-Gl accumulation dose
response
curve. Paclitaxel values have units of ng/ml. Vindesine values have units of
nM. nd: not
done. * P<0.01 vs DMSO; analysis was by ANOVA followed by Dunnett's test.
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The greater relative enhancement in the HeL023 cell line may be related to a
factor absent in other cell lines or related to the intracellular
concentration of actives that is
modulated by P-glycoprotein drug expulsion activity.
Rhodamine 123 (Rh123) retention, a P-gp functional assay, was studied in
all cell lines. This assay was performed by incubating cells with Rh123 and
determining
Rh123 accumulation by measuring its fluorescence. The maximum load of Rh123
(FmAx)
was quantified at the end of treatment harvesting the cells and storing them
at 4 C to
prevent any active Rh123 efflux. The P-gp drug efflux was allowed incubating
Rh123-
loaded cells in fresh new medium Rh123-free for 3 hours at 37 C. After this
incubation,
residual fluorescence (FREs) was measured and compared to FMAX.
Results of the Rh123 retention in A375 cells is shown in Figure 6A and
Figure 6B. Figure 6A shows the flow chromatogram for Rh123 accumulation by
A375
cells when the adenosine A3 antagonist is not present. Two cell populations
are found,
characterised by an Fus having a mean fluorescence intensity lower than FMAX.
The
population with the lowest fluorescence, accounting for 26+5% of total cells,
represents the
cells expressing functional P-gp and having low intracellular level of Rh 123.
In contrast, Figure 6B shows the A375 cellular accumulation of Rh123 in
the presence of MRE3008F20 (10 M). With the high affinity A3 antagonist
present, the
response yielded a FREs chromatogram comparable to FMAX, consistent with a
completely
blockade of P-gp mediated Rh123 transport.
Results of the Rh123 retention in human herithro-lcukemia HeL023 cells is
shown in Figure 6C and Figure 6D. Figure 6C shows that HeL023 cells have
higher P-gp
activity than A375 cells. FREs (dark shaded area) is similar to the
chromatogram obtained
with cells not treated with Rh123 (autofluorescence). This is consistent with
nearly all
(100+1 %) the cells expressing high level of functional P-gp.
With the addition of the A3 antagonist MRE3008F20, HeL023 cells showed
a P-gp inhibitor behaviour (Figure 6D). Fp~Es in the presence of MRE 3008F20,
is similar
to FmAx. This is indicative of near total P-gp inhibition.
The Jurkat and NCTC2544 cells studied did not appear to have a significant
change of Rh123 fluorescence after 3 hours of incubation at 37 C (Fus was
similar to
FMAx) consistent with the absence or not detectable P-pg drug pumping activity
in these
cells.
To verify that the anti-P-gp activity of A3 antagonists is not related to
toxic
contaminants present in the MRE3008F20 solution, the ability of other
adenosine receptor
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antagonists to interfere with P-gp mediated drug-efflux was quantified. HeL023
cells were
treated with adenosine receptor antagonists (IL-10, MRE3008F20, MRE3055F20,
MRE3062F20, MRE3046F20, CGS15943 and ZM241385). Results are given in Table 16.
Table 16 shows the percentage of cells expressing P-gp activity (% of Rh123
negative cells) in the presence of adenosine receptor antagonists. The high
affinity
adenosine A3 antagonists IL-I0, MRE3008F20, MRE3055F20, MRE3062F20 and
MRE3046F20 are strong inhibitors of P-gp activity. In contrast the low
affinity antagonists,
ZM241385 and CGS 15943 had much lower effect on P-gp activity. This may be due
to the
higher A3 affinity or due to the existence of a structure-activity
relationship for the
inhibition of P-gp drug expulsion activity mediated by adenosine receptor
antagonists.
TABLE 16
Treatment Concentration % of Rh123 Negative
( M) Cells
DMSO 99.7 1.2
IL10
10 0.410.2
CH3
O N~CH3
HN~NJ('\~~~/
H HCI
N~N\N
N 0
N
H3d
MRE 3008F20
10 0.5 0.3
0 OCH3
HNNJr\~~
H
N VN
N
N
H3C
MRE3055F20
10 0.8d:0.2
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~I
HN~N \
H
N N'N
N~I ~N O
N
H3C
MRE 3062F20
1.0--1:0.2
HNN
H
N'~'N\N
Ni N O
N
CH3
MRE 3046F20
10 10 22.0~1.0
O ~CH3
HN~N I
~ H
N N'N / I
N~~ N
N
H3C
CGS 15943
70 87.1:L1.0
NH2
N- N'N
I ~ N O
~/
ZM 241385
70 93.0 1.1
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NHz
~ I N~NN 0 I
H
Table 16 data represents the mean of four independent experiments.
To underline the molecular mechanisms sustaining the A3 antagonist
mediated response to vindesine and paclitaxel anti-proliferative effects,
signalling studies
were performed.
Previous research has related paclitaxel and Vinca alkaloid derivatives
lethality to activation of mitogen-activated protein kinase (MAPK) family
(Lieu in
Sequence-dependent potentiation mol. Pharmacol. 2001). Three MAPK family
members
have been characterised thus far: ERKs (or p42/44M~K) JNK (or SAPK) and
p38"~K.
Signalling studies investigated the effect of ERKs and p38"K on MRE 3008F20
mediated
enhancement to vindesine and to paclitaxel. The JNK pathway was not studied
due to the
lack of commercially available JNK inhibitor. Results are shown in Figure 7A
and
Figure 7B.
PD98059, a selective inhibitor of MEK1/2 (dudley dt, PNAS 92:7686, 1995),
was used to inhibit the MEK pathways. U0126, an agent approximately 100-fold
more
potent than PD98059, was used as inhibitor of ERK activation. SB203580
selectively
inhibits p38MAPK activity (Young PR JBC 272:12116 1997). A375 cells were
pretreated
with PD98059 (20 M), U0126 (30 M), SB203580 (1 M] or with DMSO (as control)
and
then challenged with vindesine (1 nM) or with paclitaxel (15 ng/ml). At 24
hours post
treatment, cells were harvested and apoptosis and cell cycle were analysed.
The combination of PD98059, U0126 and SB203580 plus paclitaxel or
vindesine did not alter the G2/M accumulation rate respect to paclitaxel or
vindesine alone.
PD98059 and SB203580 failed to inhibit MRE3008F20 (10 M) improved
susceptibility of
A375 cells to vindesine and paclitaxel. Results for PD98059 with paclitaxel is
shown as bar
"TP" of Figure 7A and with vindesine is bar "VP". The results for SB203580
plus
paclitaxel is shown as bar "TS" of Figure 7A and with vindesine is bar "VS".
U0126 prevented MRE3008F20-induced accumulation in G2/M by 23 5%
and by 4815% in presence of paclitaxel (bar "TU" of Figure 7A) and vindesine
(bar "VU"
of Figure 7A), respectively. This infers that the molecular mediator of
enhancement
activity was ERK.
The effects on selectively blocking the pathways is shown in Figure 7B.
Noted is a significant reduction of apoptosis induced by paclitaxe125 ng/ml in
cells treated
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with PD98059 (35 6%, Figure 7B, lane 5) and U0126 (73::L10%, Figure 7B, lane
3),
whereas SB203580 did not exert any effect Figure 7B, lane 7). However, PD98059
and
SB203580 (Figure 7B, lanes 6 and 8, respectively) failed to enhance the
protective effect of
MRE3008F20 in apoptosis induced by paclitaxel, as observed in presence of
U0126
(Figure 7B, lane 4).
These results provide confirmation that the MRE3008F20-induced reduction
of apoptosis of paclitaxel is dependent on ERK activation. Additional testing
demonstrated
similar trends in HeL023, NCTC2544 and Jurkat cell lines (data not shown).
Further testing indicates that P-gp interference and ERK engagement can
occur independently of each other. A375 cells were first treated with U0126
(30 M) for 30
minutes and subsequently incubated with MRE3008F20 (10 M). The P-gp activity
is
compared which those of cells treated with MRE3008F20 alone (for example,
cells of
Figure 6B). U0126 failed to prevent MRE 3008F20 blockade of P-gp. This
confinns that
P-gp interference and ERK engagement can occur independently of each other.
Discussion: Results presented show that several adenosine receptor antagonists
exert
enhancing activity to chemotherapeutic agents. Noteworthy, this enhancing
activity is A3
adenosine receptor dependent. This pharmacological specificity was determined
by an
accurate Spermean's rank correlation between the dose exerting physiological
effect (SEC50
quantified on GZ/M accumulation) and the binding ability to A3 adenosine
receptors (Ki) of
different adenosine receptor antagonists (r=0.96, Figure 5B). A known and
clear
explanation is not yet available as to the mechanism of this enhancement.
However, the
high statistically significant Spermean's rank correlation coefficient between
SEC50 values
in the G2/M accumulation and receptor affinity values showed a highly
significant positive
correlation.
Results show that A3 adenosine receptor antagonists enhance
chemotherapeutic agent anti-proliferative and apoptotic effects. The A3
adenosine receptor
antagonists reduce EC50 doses of chemotherapeutic drugs (quantified by
analysing G2/M
accumulation rate) to 12.3, 1.9, 1.2-fold for paclitaxel and 36.3, 4.0, 2.5-
fold for vindesine
when challenged on HeL023, A375 and Jurkat cell lines, respectively. This
enhancement
activity was confirmed also when the apoptotic degree was evaluated: ECMAx
dose of
chemotherapeutic drugs are reduced to 10.5, 2.0, 1.5-fold for paclitaxel on
HeL023, A375
and Jurkat cells, respectively, and 31.5, 2.0-fold for vindesine on HeL023 and
A375 cells,
respectively.
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The variable degree of EC50 (and ECMAx) value observed in different cell
types suggest a cell type specific participation in drug-induced enhancement.
Of note, the
prerequisite of the drug activity is its delivery to the target site. However,
efficiency of drug
is limited by appearing resistance, i.e., lack of cell sensitivity to the
administered drug. The
tumor cells with multidrug resistance (MDR) phenotype are characterised by
lowered
intracellular accumulation of the compounds they are resistant to.
Moreover, most cell lines with MDR phenotype show the over-expression of
170 KDa membrane associated P-glycoprotein (P-gp) that acts as an energy-
dependent
efflux pump. It has been demonstrated that this protein plays an important
role in the
transport of toxic endogenous metabolites and it seems to be responsible for
the decreased
intracellular drug accumulation observed in resistant cells (Fojo A, Cancer
Res 45:3002-7,
1985). Previous studies reported that melanoma and HeL heamopoietic cell line
expressed
functional P-gp
On the other hand, MRP and not P-gp transporter is expressed in Jurkat
leukemia cells (T lymphocytic cell line). This is consistent with the results
as HeL023 and
A375 cells produced a P-gp efflux-activity whereas in Jurkat cells had low
rhodamine 123
efflux. It is shown that adenosine A3 antagonists are useful for enhancement
in both P-gp
expressing and MRP expressing cell lines.
The much lower ability of CGS15943 and ZM241385 to inhibit P-pg and
MRP correlates with the lower adenosine A3 affinity of these compounds. The
high affinity
A3 antagonist compounds tested have greater potency in both enhancement and
inhibiting P-
gp and MRP drug resistance. This may be due to the higher affinity or to the
molecular
structure of such compounds. For example, the tested high affinity compounds
have a
phenyl-carbarnoyl-amino derivative in the N5 position of a 2-furylpyrazolo-
triazolo-
pyrimidine structure. CGS 15943 and ZM241385 in addition to having much lower
A3
affinity, also have very different molecular structures.
7.7 EXAMPLE FORMULATIONS
The following examples illustrate aspects of this invention but should not be
construed as limitations. The symbols and conventions used in these examples
are indented
to be consistent with those used in the contemporary, international, chemical
literature, for
example, the Journal of the American Chemical Society and Tetrahedron.
Example - Pharmaceutical Formulations
(A) Transdermal System - for 1000 patches
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Ingredients Amount
Active compound l00g
Silicone fluid 450g
Colloidal silicon dioxide 2g
The silicone fluid and active compound are mixed together and the colloidal
silicone dioxide is added to increase viscosity. The material is then dosed
into a subsequent
heat sealed polymeric laminate comprised of the following: polyester release
liner, skin
contact adhesive composed of silicone or acrylic polymers, a control membrane
which is a
polyolefin, and an impermeable backing membrane made of a polyester
multilaminate. The
resulting laminated sheet is than cut into 10 sq. cm patches.
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(B) Oral Tablet - For 1000 Tablets
Ingredients Amount
Active compound 50g
Starch 50g
Magnesium Stearate 5g
The active compound and the starch are granulated with water and dried.
Magnesium stearate is added to the dried granules and the mixture is
thoroughly blended.
The blended mixture is compressed into tablets.
(C) Injection - for 1000, 1 mL Ampules
Ingredients Amount
Active compound lOg
Buffering Agents q.s.
Propylene glycol 400mg
Water for injection q.s. 1000mL
The active compound and buffering agents are dissolved in the propylene
glycol at about 50 )C. The water for injection is then added with stirring and
the resulting
solution is filtered, filled into ampules, sealed and sterilized by
autoclaving.
(D) Continuous Injection - for 1000 mL
Ingredients Amount
Active compound lOg
Buffering Agents q.s.
Water for injection q.s. 1000mL
The active compound and buffering agents are dissolved in water at about
50 C. The resulting solution is filtered, filled into appropriate
administration container,
sealed and sterilized.
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(E) Topical Ointment - for 1000, 1 g packs
Ingredients Amount
Active compound 10g
White petroleum base q.s. 990g
The active compound is blended into the petrolatum base under sterile
conditions and filled into 1 gram packs.
Although the present invention has been described in terms of specific
embodiments, various substitutions of materials and conditions can be made as
will be
known to those skilled in the art. For example, other excipients may be
utilized in preparing
the pharmaceutical formulations. In addition, many of the active adenosine A3
receptor
antagonists contain one or more asymmetric centers and may therefore give rise
to
enantiomers and diastereomers as well as their racemic and resolved,
enantiomerically pure
or diastereomerically pure forms, and pharmaceutically acceptable salts
thereof. It is often
desirable that the adenosine A3 receptor antagonists be given simultaneously
with the
cytotoxic agent. When this is the case, users of this invention may find it
advantageous to
combine the A3 receptor antagonists with the cytotoxin into a single dosage
form. These
and other variations will be apparent to those skilled in the art and are
meant to be included
herein. The scope of the invention is only to be limited by the following
claims:
7.8 REGULATION OF HIF-la BY ADENOSINE
Chemicals and Reagents: A375 melanoma, NCTC 2544 keratinocytes,
U2OS osteosarcoma, U87MG glioblastoma human cells were obtained from American
Tissue Culture Collection (ATCC). Tissue culture media and growth supplements
were
obtained from BioWhittaker. GasPak PouchTM System was obtained from Becton
Dickinson. Unless otherwise noted, all other chemicals were purchased from
Sigma. Anti-
HIF-1 a and anti-HIF 1(3 antibodies (mAb) were obtained from Transduction
Laboratories
(BD, Milano, Italy). U0126 (inhibitor of MEK- 1 and MEK-2), SB202190
(inhibitor of p38
MAP kinase), Anti-ACTIVE MAPK and anti-ERK 1/2 (pAb) were from Promega.
Phospho-p38 and p38 MAP Kinase antibodies were from Cell Signaling Technology.
Anti-
Adenosine A3 receptor (polyAb) was from Aviva Antibody Corporation.
Cell culture and hypoxia treatment: Cells were maintained in DMEM
(A375), EMEM (NCTC 2544) or RPMI 1640 (U87MG, U2OS) medium containing 10%
fetal calf serum, penicillin (100 U/ml), streptomycin (100 ug/ml), and L-
glutamine (2 mM)
at 37 C in 5% CO2/95% air. Cells were passaged two or tliree times weekly at a
ratio
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between 1:5 and 1:10. Hypoxic exposure was in BBLTM GasPak pouchTM System
(Becton
Dickinson) that reduce oxygen concentration of less than 2% within 2 hours of
incubation at
35 C.
[3HJ-Thymidine incorporation: cell proliferation test: Cells were seeded in
fresh medium with 1 uCi/ml of [3H]-Thymidine in DMEM containing 10% fetal calf
serum,
penicillin (100 U/ml), streptomycin (100 ug/ml), L-glutamine (2 mM). After 24
hours of
labelling, cells were trypsinised, dispensed in 4 wells of a 96 well plate,
and filtered through
Whatman GF/C glass-fiber filters using a Micro-Mate 196 cell harvester
(Packard
Instrument Company). The filter bound radioactivity was counted on Top Count
Microplate
Scintillation Counter (efficiency 57%) with Micro-Scint 20.
Flow C tometry analysis: A375 adherent cells were trypsinized, mixed with
floating cells, washed with PBS and permeabilized in 70% (vol/vol) ethanol/PBS
solution at
4 C for at least 24 hours. The cells were washed with PBS and the DNA was
stained with a
PBS solution, containing 20 ug/ml of propidium iodide and 100 ug/ml of RNAse,
at room
temperature for 30 minutes. Cells were analysed with an EPICS XL flow
cytometer
(Beckman Coulter, Miami, FL) and the content of DNA was evaluated by the Cell-
LISYS
program (Becton-Dickinson). Cell distribution among cell cycle phases and the
percentage
of apoptotic cells were evaluated as previously described (Merighi 2002).
Briefly, the cell
cycle distribution is shown as the percentage of cells containing 2n (G0/Gl
phases), 4n (G2
and M phases), 4n>x>2n DNA amount (S phase) judged by propidium iodide
staining. The
apoptotic population is the percentage of cells with DNA content lower than
2n.
Small interferinjz RNA (siRNA) desi~n: To generate a small interfering RNA
that targets A3 receptor mRNA (siRNAA3), eight oligonucleotides consisting of
ribonucleosides, except for the presence of 2'-deoxyribonucleosides at the 3'
end, were
synthesized and annealed, according to the recommendations of Elbashir (ref),
to the
manufacturer's instructions (SilenceNTM siRNA Construction Kit, Ambion) and as
previously described (Mirandola, 2004). For oligo-1, sense sequence: 5'-GCU
UAC CGU
CAG AUA CAA GUU-3' (SEQ ID NO:1) and antisense 5'-CUU GUA UCU GAC GGU
AAG CUU-3' (SEQ ID NO:2). For oligo-2, sense sequence: 5'-GAC GGC UAA GUC
CUU GUU UUU-3' (SEQ ID NO:3) and antisense 5'-AAA CAA GGA CUU AGC CGU
CUU-3' (SEQ ID NO:4). For oligo-3, sense sequence: 5'-ACA CUU GAG GGC CUG
UAU GUU-3' (SEQ ID NO:5) and antisense 5'-CAU ACA GGC CCU CAA GUG UUU-3'
(SEQ ID NO:6). For oligo-4, sense sequence 5'-CCU GCU CUC GGA GGA UGC CUU-
3' (SEQ ID NO:7) and antisense 5'-GGC AUC CUC CGA GAG CAG GUU-3' (SEQ ID
NO: 8). Target sequences were aligned to the human genome database in a BLAST
search
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to ensure sequences without significant homology to other genes. The target
sequences for
oligo-1, oligo-2, oligo-3 and oligo-4 are localized at position 337, 679, 1009
and 1356 bases
downstream of the start codon of A3 receptor mRNA sequence (L20463),
respectively.
Treatment of cells with siRNA: A375 cells were plated in six-well plates and
grown to 50-70% confluence before transfection. Transfection of siRNA was
performed at
a concentration of 100 nM using RNAiFectTM Transfection Kit (Qiagen). Cells
were
cultured in complete media and at 24, 48 and 72 hours total RNA was isolated
for Real-
Time RT-PCR analysis of A3 receptor mRNA and for Western blot analysis of A3
receptor
protein. At 48 hours from the transfection, A375 cells were serum starved for
another 24
hours and then exposed to increasing concentrations of the A3 adenosine
receptor agonist
CI-IB-MECA for 4 hours in hypoxia. Total protein were then harvested for
Western blot
analysis. As control, cells were exposed to RNAiFectTM Transfection reagent
without
siRNAA3. To quantify cell transfection efficiency we used siRNA-FITC labelled
(Qiagen).
After 24 hours of transfection, cells were tripsinized and resuspended in PBS
for flow
cytometry analysis. Fluorescence obtained from FITC-siRNA transfected cells
was
compared to autofluorescence generated by untransfected control.
Real-Time RT-PCR experiments: Total cytoplasmic RNA was extracted by
the acid guanidinium thiocyanate phenol method (Chomczynski & Sacchi, 1987).
Quantitative real-time RT-PCR assay (Higuchi, 1993) of HIF-la and A3 mRNA
transcripts
was carried out using gene-specific double fluorescently labelled TaqMan MGB
probe
(minor groove binder) in a ABI Prism 7700 Sequence Detection System (Applied
Biosystems, Warrington Cheshire, UK). The following primer and probe sequences
were
used for real-time RT-PCR: A3 forward primer, 5'-ATG CCT TTG GCC ATT GTT G-3'
(SEQ ID NO:9); A3 reverse primer, 5'-ACA ATC CAC TTC TAC AGC TGC CT-3' (SEQ
ID NO:10); A3 MGB probe, 5'-FAM-TCA GCC TGG GCA TC-TAMRA-3' (SEQ ID
NO:11); for the real-time RT-PCR of the HIF-1 a gene the assays-on-demandTM
Gene
expression Product Accession No. NM 019058 was used (Applied Biosystems,
Monza,
Italy). The fluorescent reporter FAM and the quencher TAMRA are 6-carboxy
fluorescein
and 6-carboxy-N,N,N',N'-tetramethylrhodamine, respectively. For the real-time
RT-PCR of
the reference gene the endogenous control human (3-actin kit was used, and the
probe was
fluorescent-labeled with VICTM (Applied Biosystems, Monza, Italy).
WesteNn blotting: A375, NCTC 2544, U2OS and U87MG cells were treated
with adenosine or adenosine analogues and exposed to normoxia and hypoxia for
different
times (2-24 hours). Cells were harvested and washed with ice-cold PBS
containing 1 mM
sodium orthovanadate, AEBSF 104 mM, aprotinin 0.08 mM, leupeptin 2 mM,
bestatin 4
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mM, pepstatin A 1.5 mM, E-64 1.4 mM. Cells were then lysed in Triton lysis
buffer. The
protein concentration was determined using BCA protein assay kit (Pierce).
Equivalent
amounts of protein (35 ug) were subjected to electrophoresis on 7.5% sodium
dodecyl
sulfate-acrylamide gel. The gel was then electroblotted onto a nitrocellulose
membrane.
Membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween-20
and
incubated with antibodies against HIF-la (1:250 dilution) and HIF-1 P (1:1000
dilution) in
5% nonfat dry milk in PBS/0.1% Tween-20 overnight at 4 C. Aliquots of total
protein
sample (50 ug) were analyzed using antibodies specific for phosphorylated
(Thrl 83/Tyrl 85) or total p44/p42 MAPK (1:5000 dilution), phosphorylated
(Thrl80/Tyrl82) or total p381VIAPK (1:1000 dilution) and for A3 receptor (1 ~
g/ml
dilution). Filters were washed and incubated for 1 hour at room temperature
with
peroxidase-conjugated secondary antibodies against mouse and rabbit IgG
(1:2000
dilution). Specific reactions were revealed with the Enhanced
Chemiluminescence Western
blotting detection reagent (Amersham Corp., Arlington Heights, Ill.). The
membranes were
then stripped and reprobed with tubulin (1:250) to ensure equal protein
loading.
Metabolic inhibitors: Cells were treated for 30 minutes with metabolic
inhibitors or with drug vehicle (DMSO) prior to being challenged with
adenosine or
adenosine analogues. U0126 was used at 10 and 30 uM as inhibitor of MEK-1 and
MEK-2
to prevent p44 and p42 MAPK activation. SB202190 was used at 1 and 10 uM as
inhibitor
of p38 MAPK.
Densitometry analysis: The intensity of each band in immunoblot assay was
quantified using molecular analyst/PC densitometry software (Bio-Rad). Mean
densitometry data from independent experiments were normalized to result in
cells in the
control. The data were presented as the mean + S.E., and analyzed by the
Student's test.
Statistical analysis: All values in the figures and text are expressed as mean
standard error (S.E.) of n observation (with n?3). Data sets were examined by
analysis of
variance (ANOVA) and Dunnett's test (when required). A P value less than 0.05
was
considered statistically significant.
Results: Adenosine induces HIF-la protein accumulation in hypoxia.We
have evaluated the biological effect produced by a prolonged oxygen
deprivation in the
human A375 melanoma cell line. Viability and proliferation of A375 cells
exposed to
hypoxia for 24 hours were assessed analyzing the percentage of apoptotic cells
and the
distribution among the different phases of the cell cycle. We employed flow
cytometry and
DNA staining by propidium iodide for discrimination of cells in apoptosis, in
Go/Gl, S and
G2/M phases. The results indicate that hypoxia did not promote significant
cell death while
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interfered with proliferation arresting melanoma cells in Go/Gl and S phases
and reducing
the number of cells in G2/M (Fig. 8). These data were confirmed by using
trypan blue
exclusion, cell counts and [3H]-thymidine incorporation assay (data not
shown).
Exposure of A375 cells to hypoxia induced HIF-la protein expression (Fig.
9). The hypoxic induction of HIF-1 a was rapid, and an increase could be seen
over the first
2 to 3 hours following hypoxic incubation. Maximal stimulation was obtained
between 4-8
hours of incubation in oxygen-deprived conditions, while the levels of HIF-la
protein were
somewhat lower with prolonged hypoxia. On the contrary, the levels of HIF-1(3
were not
altered.
To study the effect of adenosine on the transcription factor HIF- 1, A375
melanoma cells were treated for 4 hours with increasing concentrations of the
nucleoside in
hypoxic conditions. As observed in Fig. 10A, adenosine up-regulated HIF-la
protein
expression in hypoxic melanoma cells. In particular, adenosine induced HIF-1a
protein
accumulation in a dose-dependent manner, with an EC50 = 2.1 0.2 M and a
maximal
increase of 2.6 0.2 fold at 100 M (Fig. lOB). We did not observe any
modulation of HIF-
10 protein.
The family of adenosine receptors consists of four subtypes of G protein-
coupled receptors, designed Al, A2A, A2B and A3. We have previously
demonstrated that all
four adenosine receptors are expressed in human melanoma A375 cells. To
evaluate the
functional role of adenosine receptor subtypes on HIF-1a protein expression
under hypoxic
conditions, we tested the effect of adenosine in combination with DPCPX (an Al
receptor
antagonist), SCH 58261 (a selective A2A receptor antagonist), MRE 2029F20 (a
selective
A2B receptor antagonist), and MRE 3008F20 (a selective A3 receptor antagonist)
(Baraldi
2004; Merighi 2001; Varani 2000). While the Al, A2A and A2B receptor
antagonists were
not able to prevent adenosine-in.duced HIF-la protein expression, the A3
receptor
antagonist MRE 3008F20 abrogated the adenosine-induced increase of HIF-la
protein
expression (Fig. 1OC-D). Furthermore, HIF-1(3 expression was unaffected by
adenosine or
by synthetic adenosine receptor antagonists. These results indicate that
adenosine may
increase HIF-la protein expression via A3 receptors.
A3 adenosine receptor induces HIF-1a protein accumulation in hypoxia.
To investigate the involvement of A3 receptors in the modulation of HIF-1 a
protein
expression, we treated A375 cells with the selective A3 receptor agonist Cl-IB-
MECA. We
performed a time-course experiment in which A375 cells were exposed to CI-IB-
MECA
100 nM for 2-24 hours. A3 adenosine receptor stimulation did not promote HIF-
l a protein
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accumulation in normoxia, while under hypoxic conditions HIF-la protein
expression was
increased in a time-dependent manner (Fig. 11). In particular, HIF-1 a
increased from 2
hours and maximum peak levels were observed 4 hours after the addition of CI-
IB-MECA
to the culture media. The prolonged A3 receptor stimulation in hypoxia
resulted in a minor
effect in HIF=1a protein level modulation. As already observed with adenosine,
also Cl-IB-
MECA did not modify HIF-1(3 expression in normoxia and in hypoxia.
To characterize in more detail the induction of HIF-1a expression by A3
receptor stimulation, A375 cells were treated with various concentrations of
A3 agonist for 4
hours. As expected, in normoxia the activation of A3 receptors did not induce
detectable
levels of HIF-la. On the contrary, in hypoxia CI-IB-MECA induced HIF-la
protein
accumulation in a dose-dependent manner (Fig. 12A), reproducing the effect
produced by
adenosine (Fig. 10). The maximum expression of HIF-la protein was induced by
CI-IB-
MECA at a concentration of 100 nM, with an EC50= 10.6:L 1.2 nM (Fig. 12B). In
contrast,
A3 receptor stimulation did not affect the expression of HIF-1(3 protein,
either in normoxia
or in hypoxia.
To better characterize the ability of A3 receptor to significantly increase
HIF-
1 a protein expression in hypoxia, we performed a series of experiments to
evaluate the
ability of A3 selective receptor antagonists (MRE 3008F20 and MRE 3005F20)
(Baraldi
2004) to prevent this effect. A375 cells were treated with increasing
concentrations of MRE
3008F20 and MRE 3005F20 for 30 minutes with or without CI-IB-MECA (10 and 100
nM)
treatment. Both MRE 3008F20 and MRE 3005F20 (10 and 100 nM) were able to
abrogate
the effect of Cl-IB-MECA in HIF-la modulation. As expected, neither MRE
3008F20 nor
MRE 3005F20 had any effect on HIF-1(3 expression. Figure 13A-B shows the
results
obtained with MRE 3008F20. Similar results were obtained with the antagonist
MRE
3005F20 (data not shown).
We next investigated the effect of increasing concentrations of MRE
300SF20 on HIF-la protein increase induced by a submaximal dose of CI-IB-MECA.
HIF-
1 a protein increase, induced by 10 nM CI-IB-MECA, was inhibited by increasing
concentrations of MRE 3008F20 (0.3-30 nM) with an IC50 of 0.90-+0.08 nM (Fig.
13C-D).
Finally, to further demonstrate that A3 receptor is required for HIF-la
protein accumulation in response to adenosine, A375 cells were mock
transfected or
transfected with small interfering RNAs that target A3 receptor mRNA (siRNAA3)
for
degradation. To evaluate transfection efficiency A375 cells were also
transfected with a
siRNA control labeled with fluorescein. By flow cytometry we oberved a
transfection
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efficiency of 854-5% (Fig. 7A). After transfection, the cells were cultured in
complete
media and at 24, 48 and 72 hours total RNA was isolated for Real-Time RT-PCR
analysis
of A3 receptor mRNA and for Western blot analysis of A3 receptor protein. As
expected, A3
receptor mRNA levels were significantly reduced in cells transfected with
siRNAA3 (Fig.
14B). Furthermore, A3 receptor protein expression was strongly reduced in
siRNAA3-treated
cells (Fig. 14C-D). Neither mock transfection nor transfection with an siRNA
targeted to
an irrelevant mRNA inhibited A3 receptor mRNA or protein expression.
Therefore, at 72
hours from the siRNAA3 transfection, A375 cells were exposed to increasing
concentrations
of the A3 adenosine receptor agonist Cl-IB-MECA (1-100 nM) for 4 hours in
hypoxia.
Total protein were then harvested for Western blot analysis. As control, A375
cells were
exposed to RNAiFectTM Transfection reagent without siRNAA3. We found that the
inhibition of A3 receptor expression is sufficient to block CI-IB-MECA-induced
HIF-la
accumulation (Fig. 14E).
To determine whether the effect of A3 receptor stimulation on HIF-1 a
expression was a general phenomenon, we assessed the ability of Cl-IB-MECA to
induce
HIF-la levels in a variety of cell lines expressing A3 adenosine receptors .
After 4 hours of
hypoxia under Cl-IB-MECA treatment, we were able to detect a significant
increase in HIF-
la protein expression, both in human keratinocytes NCTC 2544 and in different
human
tumor U87MG glioblastoma and U2OS osteosarcoma cells (Figure 15).
A3 receptor mediates HIF-la accumulation through a transcription-
independent and translation-dependent pathway. To obtain a better
understanding of
the processes involved in HIF- 1 a accumulation in response to A3 receptor
stimulation in
hypoxia, we investigated the effect of CI-IB-MECA on the HIF-1 a mRNA
accumulation.
After a treatment of A375 cells for 4 hours in hypoxia, RNA was extracted, and
Real-Time
RT-PCR analysis was performed. Activation of melanoma cells with 10 nM, 100 nM
and 1
M Cl-IB-MECA produced, respectively, a 1.13 0.10, 1.25~=0.15 and 1.19f0.13
fold
increase of HIF-1a mRNA accumulation with respect to the corresponding
untreated cells,
suggesting that A3 receptor stimulation does not regulate HIF-1a mRNA
transcription. To
confirm this hypothesis, A375 cells were pretreated with 10 g/ml actinomycin
D (Act-D)
to inhibit de novo gene transcription. Then, A375 cells were cultured for 4
hours in hypoxia
in the presence of increasing concentrations of Cl-IB-MECA (100 nM). We found
that A3
receptor stimulation was able to increase HIF-la protein expression also in
the presence of
Act-D (Fig.16).
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HIF-la has been shown to be degraded through the proteasome pathway
during normoxia. The enzymatic hydroxylation of proline 564 of HIF-la controls
the
turnover of the protein by tagging it for interaction with the von Hippel
Lindau protein (Ivan
2001; Jaakkola, 2001; Yu 2001; Maxwell 1999). When cells are hypoxic, the
proline
residue is not hydroxylated and HIF-la protein accumulates. The effect of
hypoxia on Pro-
564 hydroxylation can be mimicked by transition metals like cobalt, iron
chelators and by
inhibitors of the prolyl hydroxylase enzymes (Ivan et al., 2001; Jaakkola et
al., 2001).
We tested the ability of A3 adenosine receptor to modulate HIF-1a
accumulation in the presence of the prolyl hydroxylase enzymes inhibitor,
cobalt chloride
(CoC12). We observed that A3 receptor stimulation was able to increase the
levels of HIF-
la protein also in CoC12-treated cells (Fig. 17A). To determine if A3 receptor
induces HIF-
1 a expression through a translation-dependent pathway, we determined HIF-1 a
protein
modulation in the presence of the protein translation inhibitor, cycloheximide
(CHX). To
do this, A375 cells were cultured in normoxia for 4 hours in the presence of
100 M CoC12,
preventing oxygen-dependent HIF-1 a protein degradation, and then A375 cells
were treated
with 100 nM CI-IB-MECA in the presence or absence of CHX (1 M). In cells
exposed to
CHX, Cl-IB-MECA failed to increase HIF-1 a levels within 6 hours, as observed
in the
absence of CHX (Fig. 17B). Together, these results suggest that A3 receptor
activation
increases HIF-1 a protein levels through a translation-dependent pathway.
After return of hypoxic A375 cultures to normoxia the levels of HIF-1 a
protein decreased very rapidly and were abrogated after 15 minutes (Fig. 18A).
Therefore,
to study the effect of A3 receptor activation on HIF-1a degradation, A375
cells were
incubated in hypoxia in the absence and in the presence of Cl-IB-MECA 100 nM.
After 4
hours, melanoma cells were exposed to normoxia and a time-course of HIF-1 a
disappearance was performed. Within 15 minutes after the removal of hypoxic
conditions,
a decrease in HIF-1a protein could be seen, in the absence and in the presence
of CI-IB-
MECA with unchanged degradation rate (Fig. 18B). These results indicate that
A3 receptor
activation is not able to prevent HIF- 1 a degradation in normoxic conditions.
The main intracellular signaling pathways sustained by A3 receptors
during HIF-la accumulation in hypoxia. It has been demonstrated that MAPK are
involved in HIF-1a activation. To determine whether MAPK pathway was required
for
HIF-la protein increase induced by A3 receptor activation, A375 cells were
pretreated with
U0126, which is a potent inhibitor of MEK1/2, an upstream regulator of the
phosphorylation of p44/p42 (Favata 1998), or with the inhibitor of p38 MAPK,
SB202190
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(Kramer 1996). Cells were then exposed to CI-IB-MECA 100 nM for 4 hours in
hypoxia,
and total cellular protein extracts were prepared for immunoblot assay of HIF-
1 a and
tubulin protein levels. As shown in Fig. 19A, both MEK inhibitor, U0126 (10
and 30 M),
and p38 MAPK inhibitor, SB202190 (1 and 10 M), were able to inhibit CI-IB-
MECA-
induced increase of HIF-la protein expression. These results suggest that
p44/p42 and p38
MAPK activity were required for the HIF- 1 a expression increase induced by A3
receptor
activation.
Furthermore, to confirm that p44/p42 and p38 MAPK belong to the signaling
pathways fired by A3 receptor stimulation, we also investigated endogenous
p44/p42 and
p38 MAPK activation levels in response to A3 receptor agonist treatment. A375
cells were
incubated with increasing concentrations of CI-IB-MECA (1-1000 nM) for 4 hours
in
hypoxia, and the total cellular protein extracts were used to determine levels
of phospho-
p44, phospho-p42, and phospho-p3 8.
As shown in Fig. 19B, phosphorylation of p44 and p42 was induced in
response to nanomolar concentrations of Cl-IB-MECA and the induction of
p44/p42 kinases
phosphorylation status was maximum by the treatment of A3 receptor agonist 100
nM (Fig.
19C). Furthermore, we have monitored the activation levels of p38 MAPK upon A3
receptor stimulation by the detection of its phosphorylated form on Western
blot. As can be
seen in Fig. 19D, a strong increase in the phosphorylation of p38 MAPK was
observed after
4 hours of A3 receptor stimulation in hypoxia. In particular, exposure of A375
cells to
various concentrations of Cl-IB-MECA increased the phosphorylation of p38 MAPK
in a
dose-dependent manner (Fig. 19E). Phospho-p44, phospho-p42 and phospho-p38
blots
were then stripped and reblotted with an antibody that equally recognizes
total p44, p42 and
p38 MAPKs. We found that the observed changes in phosphorylation level of p44,
p42 and
p38 MAPKs were not accompanied by a significative modulation in the expression
levels of
total proteins (Fig. 19 B-D).
DISCUSSION: To our knowledge, this is the first report which describes the
role of adenosine in modulating the cellular response during hypoxia in an 02-
sensitive cell.
Hypoxia represents one of the first events in the growth of the cancer; this
process creates conditions that, on one hand, are conducive to the
accumulation of
extracellular adenosine and, on the other hand, stabilize hypoxia-inducible
factors, such as
HIF-la (Winn, 1981; Decking 1997; Ledoux 2003).
The results of the present study indicate a new way by which hypoxia may
contribute to cancer development, based on the natural pathways of adenosine
receptor-
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mediated signaling. For the first time, here we demonstrate that adenosine is
able to
increase HIF- 1 a protein expression in response to hypoxia in a dose- and
time-dependent
manner in A375 human melanoma cells, whereas HIF-1(3 protein levels are not
affected.
We have previously demonstrated that all four adenosine receptors are
expressed in human melanoma A375 cells (Merighi, 2001). Here, we report that
A3
receptor subtype mediates the observed adenosine effects on HIF-1a regulation
in this cell
line.
The effects of adenosine on HIF- 1 a protein accumulation are not mediated
by Al, A2A or A2B receptors. In support of this conclusion, DPCPX, SCH 58261
and MRE
2029F20, adenosine receptor antagonists highly selective for Al, A2A and A2B
receptors,
respectively, did not block the stimulatory effect of adenosine on HIF- 1 a
protein increase.
The conclusion that the effects of adenosine on HIF-la accumulation are
mediated via A3 receptors is supported by the observation that the stimulatory
effects of this
nucleoside on HIF-la protein are mimicked by the A3 receptor agonist Cl-IB-
MECA and
inhibited by A3 receptor antagonists, MRE 3008F20 and MRE 3005F20. In
particular, the
potencies of these drugs are in agreement with their inhibitory equilibrium
binding constant
(Ki) observed in binding experiments for the adenosine A3 receptor (Merighi
2001).
Furthermore, the inhibition of A3 receptor expression at the mRNA and
protein level is sufficient to block A3 receptor-induced HIF-1a protein
accumulation.
Therefore, our results indicate that the cell surface A3 adenosine receptor
transduces
extracellular hypoxic signals into the cell interior. A3 receptors are present
in melanoma
cells and their expression appears to be a bridge between the hypoxic insult
and HIF- 1 a
accumulation, regulating the cellular response to hypoxia, like an oxygen-
sensing receptor.
The extent to which A3 receptor influences the ability of tumor cells to
respond to hypoxia
will require further investigation.
Similar results obtained in different cells (keratinocytes, melanoma,
osteosarcoma, glioblastoma) raised the concern that the A3 receptor
stimulation effect on
HIF- 1 a protein expression in hypoxia may be indiscriminate between normal
and cancer
cells, thereby demonstrating that this may be a general signaling pathway
shared by many,
if not all, cell types.
A3 adenosine receptor stimulation had no effect on HIF-la mRNA
accumulation, as observed by Real-Time RT-PCR experiments. Accordingly, Act-D
experiments indicate that A3 receptor does not regulate HIF-1a protein
expression through a
transcription-dependent mechanism. The lack of adenosine effect on HIF-1 a at
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transcriptional level is not surprising in view of the fact that hypoxic
regulation of HIF-la
is primarily determined by stabilization of HIF-la protein (Huang, 1998). In
addition, we
have obtained evidences that A3 adenosine receptors modulate HIF-1a protein
levels
through a translation-dependent pathway while did not affect HIF-la oxygen-
dependent
degradation. Our data suggest that A3 adenosine receptors does not increase
the half-life of
HIF-la protein while may increase the rate of HIF-1a protein synthesis, in a
manner similar
to the effect of various growth factors (Zhong 2000; Fukuda, 2002).
Nevertheless, we
cannot exclude the possibility that A3 adenosine receptor regulates the
translation of a
protein, which inhibits HIF-1 a degradation.
Phosphorylation and dephosphorylation activities have been suggested to be
critical in the signaling pathway leading to HIF-1 activation. Several reports
demonstrated
that hypoxia induces the phosphorylation of HIF-la by p44/p42 and p38 MAPKs,
which
increases both HIF-la nuclear localization and transcriptional activity
(Semenza
200 1 CurrOpCB; Richard 1999BBRC; Berra 2000; Richard 1999JBC; Conrad 1999;
Sodhi
2000; Mottet D, 2003; Semenza 2002). In addition, adenosine has been shown to
directly
enhance MAPK activity in A375 human melanoma cells (Merighi et al., 2002) but
also in
non human cell lines stably transfected with the human A3 receptor (Hammarberg
2004;
Schulte 2000-2002-2003). In the present study, we observed that p44/p42 and
p38 MAPKs
are necessary to increase HIF-1 a levels but also that these kinases are
included in the
molecular signaling pathways generated by A3 receptor engagement. In
conclusion, the
present study demonstrates that adenosine, via A3 receptors, is able to
increase the levels of
HIF- 1 a through p44/p42 and p38 MAPK pathways. Actually, further studies are
needed to
evaluate the role of p44/p42 and p38 MAPK in the reduced turnover, increased
life and
transduction of HIF-la protein in hypoxia.
HIF-1a is overexpressed in tumors as a result of hypoxia and is involved in
key aspects of tumor biology, such as angiogenesis, invasion and altered
energy metabolism
(Ratcliffe 2000). It is recognized that the inhibition of HIF-1 activity
represents a novel
therapeutic approach to cancer therapy, especially in combination with
angiogenesis
inhibitors, which would fiu ther increase intratumoral hypoxia and thus
provide an even
greater therapeutic window for use of an HIF-inhibitor. Recent studies
indicate that
pharmacologic inhibition of HIF-1 a and particularly of HIF-regulated genes
that are
important for cancer cell survival may be more advantageous than HIF-gene
inactivation
therapeutic approaches (Mabjeesh et al., 2003). Many normal tissues function
at p02 values
sufficient to activate HIF, and the system has important functions under
normal
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physiological conditions (Hopfl, 2004). This will need to be considered in the
development
of pharmacological inhibitors for clinical use.
Given the ability of A3 adenosine receptor antagonists to block HIF-1a
protein expression accumulation induced by adenosine, our data imply that A3
adenosine
receptor antagonists may be useful in cancer therapy. In particular, we remark
that in in
vivo system the extracellular fluid of solid tumors contains increased levels
of adenosine
(Blay ct al., 1997), the endogenous agonist responsible for adenosine receptor
functions.
Therefore, with A3 receptor antagonists in cancer therapy it may be possible
to achieve
tissue selectivity, such that a biological effect would only be observed in
tumoral hypoxic
cells, where high adenosine concentration increases HIF-la accumulation.
Additional studies are warranted to determine whether A3 receptor
antagonists can block the survival of hypoxic solid tumors.
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