Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMBINATIONS FOR THE TREATMENT OF
PROLIFERATIVE DISEASES
Background of the Invention
The present invention relates to the treatment of cancer and other
proliferative diseases.
Cancer is a disease marked by the uncontrolled growth of abnormal
cells. Cancer cells have overcome the barriers imposed in normal cells, which
have a finite lifespan, to grow indefinitely. As the growth of cancer cells
continue, genetic alterations may persist until the cancerous cell has
manifested
itself to pursue a more aggressive growth phenotype. If left untreated,
metastasis, the spread of cancer cells to distant areas of the body by way of
the
lymph system or bloodstream, may ensue, destroying healthy tissue.
The treatment of cancer has been hampered by the fact that there is
considerable heterogeneity even within one type of cancer. Some cancers, for
example, have the ability to invade tissues and display an aggressive course
of
growth characterized by metastases. These tumors generally are associated
with a poor outcome for the patient. Ultimately, tumor heterogeneity results
in
the phenomenon of multiple drug resistance, i.e., resistance to a wide range
of
structurally unrelated cytotoxic anticancer compounds, J. H. Gerlach et al.,
Cancer Surveys, 5:25-46, 1986. The underlying cause of progressive drug
resistance may be due to a small population of drug-resistant cells within the
tumor (e.g., mutant cells) at the time of diagnosis, as described, for
example, by
J. H. Goldie and Andrew J. Coldman, Cancer Research, 44:3643-3653, 1984.
Treating such a tumor with a single drug can result in remission, where the
tumor shrinks in size as a result of the killing of the predominant drug-
sensitive
cells. However, with the drug-sensitive cells gone, the remaining drug-
resistant cells can continue to multiply and eventually dominate the cell
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population of the tumor. Therefore, the problems of why metastatic cancers
develop pleiotropic resistance to all available therapies, and how this might
be
countered, are the most pressing in cancer chemotherapy.
Anticancer therapeutic approaches are needed that are reliable for a wide
variety of tumor types, and particularly suitable for invasive tumors.
Importantly, the treatment must be effective with minimal host toxicity. In
spite of the long history of using multiple drug combinations for the
treatment
of cancer and, in particular, the treatment of multiple drug resistant cancer,
positive results obtained using combination therapy are still frequently
unpredictable.
Summary of the Invention
The invention features compositions, methods, and kits for treating
proliferative diseases such as cancer.
In a first aspect, the invention features a composition that includes a first
agent that reduces the biological activity of a mitotic kinesin and a second
agent that reduces the biological activity of a protein tyrosine phosphatase.
The invention also features a method for treating a patient who has a
proliferative disease, or inhibiting the development of a proliferative
disease in
the patient by administering to the patient a first agent that reduces the
biological activity of a mitotic kinesin and a second agent that reduces the
biological activity of a protein tyrosine phosphatase. Desirably, the two
agents
are administered simultaneously or within 14 days of each other, within 7 days
of each other, within 1 day of each other, within 1 hour of each other in
amounts sufficient to treat the patient.
The invention also features a method of reducing cell proliferation by
contacting cells with a first agent that reduces the biological activity of a
mitotic kinesin and a second agent that reduces the biological activity of a
protein tyrosine phosphatase.
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The methods and compositions further include an additional
antiproliferative agent such as an anticancer agent.
In yet another aspect, the invention features a method for identifying a
combination that may be useful for the treatment of a proliferative disease.
In
this method, proliferating cells (e.g., cancer cells or a cancer cell line)
are
contacted in vitro with (i) an agent that reduces mitotic kinesin biological
activity and (ii) a candidate compound. Using any acceptable assay, it is then
determined whether the combination of the agent and the candidate compound
reduces cell proliferation, relative to proliferation of cells contacted with
the
agent but not contacted with the candidate compound. A reduction in cell
proliferation identifies the combination as a combination that may be useful
for
the treatment of a proliferative disease.
In another aspect, the invention features another method for identifying
a combination that may be useful for the treatment of a proliferative disease.
This method includes the steps of (a) identifying a compound that reduces
protein tyrosine phosphatase biological activity; (b) contacting proliferating
cells in vitro with an agent that reduces mitotic kinesin biological activity
and
the compound identified in step (a); and (c) determining whether the
combination of the agent and the compound identified in step (a) reduces cell
proliferation, relative to proliferation of cells contacted with the agent but
not
contacted with the compound identified in step (a) or contacted with the
compound identified in step (a) but not contacted with the agent. A reduction
in cell proliferation identifies the combination as a combination that may be
useful for the treatment of a proliferative disease.
In either of the foregoing aspects, the agent that reduces mitotic kinesin
biological activity may be, for example, a mitotic kinesin inhibitor, an
antisense
compound or RNAi compound that reduces the expression levels of a mitotic
kinesin, a dominant negative mitotic kinesin, an expression vector encoding
such a dominant negative mitotic kinesin, an antibody that binds a mitotic
kinesin and reduces mitotic kinesin biological activity, or an aurora kinase
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inhibitor. Desirably, the agent that reduces mitotic kinesin biological
activity
reduces the biological activity of HsEgS/KSP. Exemplary mitotic kinesin
biological activities are enzymatic activity, motor activity, and binding
activity.
In still another aspect, the invention features another method for
identifying a compound that may be useful for the treatment of a proliferative
disease. This method includes the steps of: (a) providing proliferating cells
engineered to have reduced mitotic kinesin biological activity; (b) contacting
the cells with a candidate compound; and (c) determining whether the
candidate compound reduces cell proliferation, relative to cells not contacted
with the candidate compound. A reduction in cell proliferation identifies the
compound as a compound that may be useful for the treatment of a
proliferative disease.
In another aspect, the invention features yet another method for
identifying a combination that may be useful for the treatment of a
proliferative
disease. This method includes the steps of: (a) contacting proliferating cells
in
vitro with an agent that reduces protein tyrosine phosphatase biological
activity
and a candidate compound; and (b) determining whether the combination of the
agent and the candidate compound reduces cell proliferation, relative to
proliferation of cells contacted with the agent but not contacted with the
candidate compound. A reduction in cell proliferation identifies the
combination as a combination that may be useful for the treatment of a
proliferative disease.
In a related aspect, the invention features a method for identifying a
combination that may be useful for the treatment of a proliferative disease.
This method includes the steps of: (a) identifying a compound that reduces
mitotic kinesin biological activity; (b) contacting proliferating cells in
vitro
with an agent that reduces protein tyrosine phosphatase biological activity
and
the compound identified in step (a); and (c) determining whether the
combination of the agent and the compound identified in step (a) reduces cell
proliferation, relative to proliferation of cells contacted with the agent but
not
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contacted with the compound identified in step (a) or contacted with the
compound identified in step (a) but not contacted with the agent. A reduction
in cell proliferation identifies the combination as a combination that may be
useful for the treatment of a proliferative disease.
In either of the foregoing aspects, the agent that reduces protein tyrosine
phosphatase biological activity is a protein tyrosine phosphatase inhibitor,
an
antisense compound or RNAi compound that reduces the expression levels of a
protein tyrosine phosphatase, a dominant negative protein tyrosine
phosphatase, an expression vector encoding said dominant negative protein
tyrosine phosphatase, an antibody that binds a protein tyrosine phosphatase
and
reduces protein tyrosine phosphatase biological activity, or a
farnesyltransferase inhibitor. Desirably, the agent reduces the biological
activity of a protein tyrosine phosphatase selected from PTP1B, PRL-1, PRL-2,
PRL-3, SHP-l, SHP-2, MKP-1, MKP-2, CDC14, CDC25A, CDC25B, and
CDC25C.
In another aspect, the invention features another method for identifying
a compound that may be useful for the treatment of a proliferative disease.
This method includes the steps of: (a) providing proliferating cells
engineered
to have reduced protein tyrosine phosphatase biological activity; (b)
contacting
the cells with a candidate compound; and (c) determining whether the
candidate compound reduces cell proliferation, relative to cells not contacted
with the candidate compound. A reduction in cell proliferation identifies the
compound as a compound that may be useful for the treatment of a
proliferative disease.
In any of the foregoing aspect, the cells are desirably cancer cells or
cells from a cancer cell line.
By "more effective" is meant that a method, composition, or kit exhibits
greater efficacy, is less toxic, safer, more convenient, better tolerated, or
less
expensive, or provides more treatment satisfaction than another method,
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composition, or kit with which it is being compared. Efficacy may be
measured by a skilled practitioner using any standard method that is
appropriate for a given indication.
By "mitotic kinesin inhibitor" is meant an agent that binds a mitotic
kinesin and reduces, by a significant amount (e.g., by at least 10%, 20% 30%
or more), the biological activity of that mitotic kinesin. Mitotic kinesin
biological activities include enzymatic activity (e.g., ATPase activity),
motor
activity (e.g., generation of force) and binding activity (e.g., binding of
the
motor to either microtubules or its cargo).
By "dominant negative" is meant a protein that contains at least one
mutation that inactivates its physiological activity such that the expression
of
this mutant in the presence of the normal or wild-type copy of the protein
results in inactivation of or reduction of the activity of the normal copy.
Thus,
the activity of the mutant "dominates" over the activity of the normal copy
such that even though the normal copy is present, biological function is
reduced. In one example, a dimer of two copies of the protein are required so
that even if one normal and one mutated copy are present there is no
activity; another example is when the mutant binds to or "soaks up" other
proteins that are critical for the function of the normal copy such that not
enough of these other proteins are present for activity of the normal copy.
By "protein tyrosine phosphatase" or "PTPase" is meant an enzyme that
dephosphorylates a tyrosine residue on a protein substrate.
By "protein tyrosine phosphatase inhibitor" is an agent that binds a
protein tyrosine phosphatase and inhibits (e.g. by at least 10%, 20%, or 30%
or
more) the biological activity of that protein tyrosine phosphatase.
By "dual specificity phosphatase" is meant a protein phosphatase that
can dephosphorylate both a tyrosine residue and either a serine or threonine
residue on the same protein substrate. Dual specificity phosphatases include
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MKP-1, MKP-2, and the cell division cycle phosphatase family (e.g., CDC14,
CDC25A, CDC25B, and CDC25C). Dual specificity phosphatases are
considered to be protein tyrosine phosphatases.
By "antiproliferative agent" is meant a compound that, individually,
inhibits cell proliferation. Antiproliferative agents of the invention include
alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors,
antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate
synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump
inhibitors, histone acetyltransferase inhibitors, metalloproteinase
inhibitors,
ribonucleoside reductase inhibitors, TNF alpha agonists and antagonists,
endothelin A receptor antagonists, retinoic acid receptor agonists,
immunomodulators, hormonal and antihormonal agents, photodynamic agents,
and tyrosine kinase inhibitors.
By "inhibits cell proliferation" is meant measurably slows, stops, or
reverses the growth rate of cells in vitro or in vivo. Desirably, a slowing of
the
growth rate is by at least 20%, 30%, 50%, 60%, 70%, 80%, or 90%, as
determined using a suitable assay for determination of cell growth rates
(e.g., a
cell growth assay described herein). Typically, a reversal of growth rate is
accomplished by initiating or accelerating necrotic or apoptotic mechanisms of
cell death in the neoplastic cells.
By "a sufficient amount" is meant the amount of a compound, in a
combination according to the invention, required to inhibit the growth of the
cells of a neoplasm in vivo. The effective amount of active compounds) used
to practice the present invention for therapeutic treatment of proliferative
diseases (i.e., cancer) varies depending upon the manner of administration,
the
ages race, gender, organ affected, body weight, and general health of the
subject. Ultimately, the attending physician or veterinarian will decide the
appropriate amount and dosage regimen.
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By a "low dosage" is meant at least 5% less (e.g., at least 10%, 20%,
50%, 80%, 90%, or even 95%) than the lowest standard recommended dosage
of a particular compound formulated for a given route of administration for
treatment of any human disease or condition.
By a "high dosage" is meant at least 5% (e.g., at least 10%, 20%, 50%,
100%, 200%, or even 300%) more than the highest standard recommended
dosage of a particular compound for treatment of any human disease or
condition.
The phrase "pharmaceutically acceptable" refers to molecular entities
and compositions that do not produce an adverse, allergic or other untoward
reaction when administered to patient.
As used herein, "pharmaceutically acceptable carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents and the like. The use of such media
and agents for pharmaceutical active substances is well known in the art.
By "patient" is meant any animal (e.g., a human). Non-human animals
that can be treated using the methods, compositions, and kits of the invention
include horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea
pigs,
rats, mice, lizards, snakes, sheep, cattle, fish, and birds.
Compounds useful in the invention include those described herein in any
of their pharmaceutically acceptable forms, including isomers such as
diastereomers and enantiomers, salts, solvates, and polymorphs, thereof, as
well as racemic mixtures of the compounds described herein.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
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Detailed Description
The invention features compositions, methods, and kits for treating
proliferative disorders.
Normal cells have signaling mechanisms that regulate growth, mitosis,
differentiation, cell function, and cell death in a programmed fashion.
Defects
in the signaling pathways that regulate these functions can result in
uncontrolled growth and proliferation, which can manifest as cancer,
hyperplasias, restenosis, cardiac hypertrophy, immune disorders and
inflammatory disorders.
Mitotic kinesins are essential motors in mitosis. They control spindle
assembly and maintenance, attachment and proper positioning of the
chromosomes to the spindle, establish the bipolar spindle and maintain forces
in the spindle to allow movement of chromosomes toward opposite poles.
Perturbations of mitotic kinesin function cause malformation or dysfunction of
the mitotic spindle, frequently resulting in cell cycle arrest and cell death.
Protein tyrosine phosphatases (PTPases) are intracellular signaling
molecules that dephosphorylate a tyrosine residue on a protein substrate,
thereby modulating certain cellular functions. In normal cells, they typically
act in concert with protein tyrosine kinases to regulate signaling cascades
through the phosphorylation of protein tyrosine residues. Phosphorylation and
dephosphorylation of the tyrosine residues on proteins controls cell growth
and
proliferation, cell cycle progression, cytoskeletal integrity, differentiation
and
metabolism. In various metastatic and cancer cell lines, PTP1B and the family
of Phosphatases of Regenerating Liver (PRL-1, PRL-2, and PRL-3) have been
shown to be overexpressed. For example, PRL-3 (also known as PTP4A3) is
expressed in relatively high levels in metatstatic colorectal cancers (Saha et
al.,
Science 294: 1343-1346, 2001.). PRL-1 localizes to the mitotic spindle and is
required for mitotic progression and chromosome segregation. PRL
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phosphatases promote cell migration, invasion, and metastasis, and inhibition
of these PTPases has been shown to inhibit proliferation of cancer cells in
vitro
and tumors in animal models.
We previously demonstrated that the combination of chlorpromazine
and pentamidine work in concert to reduce cell proliferation (U.5. Patent No.
6,569,853). We now show that chlorpromazine acts as an inhibitor of mitotic
kinesin. Pentamidine has been demonstrated to be an inhibitor of the PRL
phosphatases (Pathak et al., Mol. Cancer Ther. 1:1255-1264, 2002).
Based on the foregoing observations, we conclude that combinations of
an agent that reduces the biological activity of a mitotic kinesin with an
agent
that reduces the activity of a protein tyrosine phosphatase are useful for
reducing cell proliferation and, hence, for treating proliferative diseases.
Mitotic kinesins
Mitotic kinesins include HsEgS/KSP, KIFC3, CH02, MKLP, MCAK,
Kin2, Kif4, MPP1, CENP-E, NYREN62, LOC8464, and KIFB. Other mitotic
kinesins are described in U.S. Patent Nos. 6,414,121, 6,582,958, 6,544,766,
6,492,158, 6,455,293, 6,440,731, 6,437,115, 6,420,162, 6,399,346, 6,395,540,
6,383,796, 6,379,941, and 6,248,594. The GenBank Accession Nos. of
representative mitotic kinesins are provided in Table 1.
Table 1
Human mitotic
kinesins
II
Protein name GenBank Accession No.
E 5/KSP AA857025, U37426, X85137
KIFC3 BC001211
MKLPI A1131325, AU133373, X67155
MCAK AL046197, U63743
KIN2 Y08319
KI F4 A F071592
MPP 1 AL 117496
CEN P-E Z 15005
CH02 AL021366
HsNYREN62 AF155117
HsLOC8464 NM 032559
KIF8 AB001436
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HsEgS/KSP has been cloned and characterized (see, e.g., Blangy et al.,
Cell, 83:1159-69 (1995); Galgio et al., J. Cell Biol., 135:399-414, 1996;
Whitehead et al., J. Cell Sci., 111:2551-2561, 1998; Kaiser, et al., J. Biol.
Chem., 274:18925-31, 1999; GenBank accession numbers: X85137, NM
004523). Drosophila (Heck et al., J. Cell Biol., 123:665-79, 1993) and Xenopus
(Le Guellec et al., Mol. Cell Biol., 11:3395-8, 1991) homologs of KSP have
been reported. Drosophila KLP61F/KRP130 has reportedly been purified in
native form (Cole, et al., J. Biol. Chem., 269:22913-22916, 1994), expressed
in
E. coli, (Barton, et al., Mol. Biol. Cell, 6:1563-74, 1995) and reported to
have
motility and ATPase activities (Cole, et al., supra; Barton, et al., supra).
Xenopus Eg5/KSP was expressed in E. coli and reported to possess motility
activity (Sawin, et al., Nature, 359:540-3, 1992; Lockhart and Cross,
Biochemistry, 35:2365-73, 1996; Crevel, et al, J. Mol. Biol., 273:160-170,
1997) and ATPase activity (Lockhart and Cross, supra; Crevel et al., supra).
Besides KSP, other members of the BimC family include BimC, CINB,
cut7, KIP1, KLP61F (Barton et al., Mol. Biol. Cell. 6:1563-1574, 1995;
Cottingham & Hoyt, J. Cell Biol. 138:1041-1053, 1997; DeZwaan et al., J. Cell
Biol. 138:1023-1040, 1997; Gaglio et al., J. Cell Biol. 135:399-414, 1996;
Geiser et al., Mol. Biol. Cell 8:1035-1050, 1997; Heck et al., J. Cell Biol.
123:665-679, 1993; Hoyt et al., J. Cell Biol. 118:109-120, 1992; Hoyt et al.,
Genetics 135:35-44, 1993; Huyett et al., J. Cell Sci. 111:295-301, 1998;
Miller
et al., Mol. Biol. Cell 9:2051-2068, 1998; Roof et al., J. Cell Biol. 118:95-
108,
1992; Sanders et al., J. Cell Biol. 137:417-431, 1997; Sanders et al., Mol.
Biol.
Cell 8:1025-0133, 1997; Sanders et al., J. Cell Biol. 128:617-624, 1995;
Sanders & Hoyt, Cell 70:451-458, 1992; Sharp et al., J. Cell Biol. 144:125-
138,
1999; Straight et al., J. Cell Biol. 143:687-694, 1998; Whitehead & Rattner,
J.
Cell Sci. 111:2551-2561, 1998; Wilson et al., J. Cell Sci. 110:451-464, 1997).
Mitotic kinesin biological activities include its ability to affect ATP
hydrolysis; microtubule binding; gliding and polymerization/depolymerization
(effects on microtubule dynamics); binding to other proteins of the spindle;
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binding to proteins involved in cell-cycle control; serving as a substrate to
other
enzymes, such as kinases or proteases; and specific kinesin cellular
activities
such as spindle pole separation.
Methods for assaying biological activity of a mitotic kinesin are well
known in the art. For example, methods of performing motility assays are
described, e.g., in Hall, et al., 1996, Biophys. J., 71:3467-3476, Turner et
al.,
1996, Anal. Biochem. 242:20-25; Gittes et al., 1996, Biophys. J. 70:418-429;
Shirakawa et al., 1995, J. Exp. Biol. 198: 1809-1815; Winkelmann et al., 1995,
Biophys. J. 68: 2444-2453; and Winkelmann et al., 1995, Biophys. J. 68:725.
Methods known in the art for determining ATPase hydrolysis activity also can
be used. U.S. application Ser. No. 09/314,464, filed May 18, 1999, hereby
incorporated by reference in its entirety, describes such assays. Other
methods
can also be used. For example, P; release from kinesin can be quantified. In
one embodiment, the ATP hydrolysis activity assay utilizes 0.3 M perchloric
acid (PCA) and malachite green reagent (8:27 mM sodium molybdate II, 0.33
mM malachite green oxalate, and 0.8 mM Triton X-100). To perform the
assay, 10 ~L of reaction is quenched in 90 ~L of cold 0.3 M PCA. Phosphate
standards are used so data can be converted to nM inorganic phosphate
released. When all reactions and standards have been quenched in PCA, 100
~.L of malachite green reagent is added to the relevant wells in e.g., a
microtiter
plate. The mixture is developed for 10-15 minutes and the plate is read at an
absorbance of 650 nm. If phosphate standards were used, absorbance readings
can be converted to nM P; and plotted over time. Additionally, ATPase assays
known in the art include the luciferase assay.
ATPase activity of kinesin motor domains also can be used to monitor
the effects of modulating agents. In one embodiment ATPase assays of kinesin
are performed in the absence of microtubules. In another embodiment, the
ATPase assays are performed in the presence of microtubules. Different types
of modulating agents can be detected in the above assays. In one embodiment,
the effect of a modulating agent is independent of the concentration of
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microtubules and ATP. In another embodiment, the effect of the agents on
kinesin ATPase may be decreased by increasing the concentrations of ATP,
microtubules, or both. In yet another embodiment, the effect of the modulating
agent is increased by increasing concentrations of ATP, microtubules, or both.
Agents that reduce the biological activity of a mitotic kinesin in vitro
may then be screened in vivo. Methods for in vivo screening include assays of
cell cycle distribution, cell viability, or the presence, morphology,
activity,
distribution, or amount of mitotic spindles. Methods for monitoring cell cycle
distribution of a cell population, for example, by flow cytometry, are well
known to those skilled in the art, as are methods for determining cell
viability
(see, e.g., U.S. Patent No. 6,617,115).
Mitotic kinesin inhibitors
Mitotic kinesin inhibitors include chlorpromazine, monasterol,
terpendole E, HR22C 16, and SB715992. Other mitotic kinesin inhibitors are
those compounds disclosed in Hopkins et al., Biochemistry 39:2805, 2000,
Hotha et al., Angew Chem. Inst. Ed. 42:2379, 2003, PCT Publication Nos.
W001/98278, W002/057244, W002/079169, W002/057244, W002/056880,
W003/050122, W003/050064, W003/049679, W003/049678,
W003/049527, W003/079973, and W003/039460, and U.S. Patent
Application Publication Nos. 2002/0165240, 2003/0008888, 2003/0127621,
and 2002/0143026; and U.S. Patent Nos., 6,437,115, 6,545,004, 6,562,831,
6,569,853, and 6,630,479, and the chlorpromazine analogs described in U.S.
Patent Application No. 10/617,424 (see, e.g., Formula (I)).
Protein tyrosine phosphatases
Protein tyrosine phosphatases include the PRL family (PRL-1, PRL-2,
and PRL-3), PTP1B, SHP-1, SHP-2, MKP-1, MKP-2, CDC14, CDC25A,
CDC25B, CDC25C, PTPa, and PTP-BL. Protein tyrosine phosphatase
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biological activities include dephosphorylation of tyrosine residues on
substrates. The GenBank Accession Nos. of representative tyrosine
phosphatases are provided in Table 2.
Table 2
Human
rotein
t rosine
hos hatases
Protein GenBank Accession No.
name
PRL-1 AJ420505, BI222469, U48296
PRL-2 AF208850, B1552091, L48723
PRL-3 AF041434, BC003105
PTP1B AUl 17677, M33689
SHP-1 BC002523, BG754792, M77273, BM742181,
AF178946
SHP-2 AU123593, BF515187, BX537632, D13540
MKP-1 U01669, X68277
MKP-2 BC014565, U21108, U48807, AL137704
CDC 14A AF000367, AF064102, AF064103
CDC14B AF023158, AF064104
CDC25A M81933
CDC25B M81934, 268092, AF036233
CDC25C M34065, 229077, AJ304504, M34065
PTPaI M36033
ha
PTP-BL D21210, D21209, D21211, U12128
Protein tyrosine phosphatase inhibitors
Inhibitors of protein tyrosine phosphatases include pentamidine,
levamisole, ketoconazole, bisperoxovanadium compounds (e.g., those
described in Scrivens et al., Mol. Cancer Ther. 2:1053-1059, 2003, and U.S.
Patent No. 6,642,221), vanadate salts and complexes (e.g., sodium
orthovanadate), dephosphatin, dnacin A1, dnacin A2, STI-571, suramin,
gallium nitrate, sodium stibogluconate, meglumine antimonate, 2-(2-
mercaptoethanol)-3-methyl-1,4-naphthoquinone, 2,5-bis(4-
amidinophenyl)furan-bis-O-methylamidoxime, known as DB289 (Immtech),
2,5-bis(4-amidinophenyl)furan (DB75, Immtech), disclosed in U.S. 5,843,980,
and compounds described in Pestell et al., Oncogene 19:6607-6612, 2000,
Lyon et al., Nat. Rev. Drug Discov. 1:961-976, 2002, Ducruet et al., Bioorg.
Med. Chem. 8:1451-1466, 2000, U.S. Patent Application Publication Nos.
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2003/0114703, 2003/0144338, and 2003/0161893, and PCT Patent Publication
Nos. W099/46237, W003/06788 and W003/070158. Still other analogs are
those that fall within a formula provided in any of U.S. Patent Nos.
5,428,051;
5,521,189; 5,602,172; 5,643,935; 5,723,495; 5,843,980; 6,008,247; 6,025,398;
6,172,104; 6,214,883; and 6,326,395, and U.S. Patent Application Publication
Nos. US 2001/0044468 and US 2002/0019437, and the pentamidine analogs
described in U.S. Patent Application No. 10/617,424 (see, e.g., Formula (II)).
Other protein tyrosine phosphatase inhibitors can be identified, for example,
using the methods described in Lazo et al. (Oncol. Res. 13:347-352, 2003),
PCT Publication Nos. W097/40379, W003/003001, and W003/035621, and
U.S. Patent Nos. 5,443,962 and 5,958,719.
Other biological activity inhibitors
In addition to reducing biological activity through the use of compounds
that bind a mitotic kinesin or protein tyrosine phosphatase, other inhibitors
of
mitotic kinesin and protein tyrosine phosphatase biological activity can be
employed. Such inhibitors include compounds that reduce the amount of target
protein or RNA levels (e.g., antisense compounds, dsRNA, ribozymes) and
compounds that compete with endogenous mitotic kinesins or protein tyrosine
phosphatases for binding partners (e.g., dominant negative proteins or
polynucleotides encoding the same).
Antisense compounds
The biological activity of a mitotic kinesin and/or protein tyrosine
phosphatase can be reduced through the use of an antisense compound directed
to RNA encoding the target protein. Mitotic kinesin antisense compounds
suitable for this use are known in the art (see, e.g., U.S. Patent No.
6,472,521,
W003/030832, and Maney et al., J. Cell Biol., 1998, 142:787-801), as are
antisense compounds against protein tyrosine phosphatases (see, e.g., U.S.
Patent Publication No. 2003/0083285 and Weil et al., Biotechniques 33:1244,
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WO 2005/046607 PCT/US2004/037527
2002). Other antisense compounds that reduce mitotic kinesins can be
identified using standard techniques. For example, accessible regions of the
target mitotic kinesin or protein tyrosine phosphatase mRNA can be predicted
using an RNA secondary structure folding program such as MFOLD (M.
Zuker, D. H. Mathews & D. H. Turner, Algorithms and Thermodynamics for
RNA Secondary Structure Prediction: A Practical Guide. In: RNA
Biochemistry and Biotechnology, J. Barciszewski & B. F. C. Clark, eds.,
NATO ASI Series, Kluwer Academic Publishers, (1999)). Sub-optimal folds
with a free energy value within 5% of the predicted most stable fold of the
mRNA are predicted using a window of 200 bases within which a residue can
find a complimentary base to form a base pair bond. Open regions that do not
form a base pair are summed together with each suboptimal fold and areas that
are predicted as open are considered more accessible to the binding to
antisense
nucleobase oligomers. Other methods for antisense design are described, for
example, in U.S. Patent No. 6,472,521, Antisense Nucleic Acid Drug Dev.
1997 7:439-444, Nucleic Acids Research 28:2597-2604, 2000, and Nucleic
Acids Research 31:4989-4994, 2003.
RNA interference
The biological activity of a mitotic kinesin and/or protein tyrosine
phosphatase can be reduced through the use of RNA interference (RNAi),
employing, e.g., a double stranded RNA (dsRNA) or small interfering RNA
(siRNA) directed to the mitotic kinesin or protein tyrosine phosphatase in
question (see, e.g., Miyamoto et al., Prog. Cell Cycle Res. 5:349-360, 2003;
U.S. Patent Application Publication. No. 2003/0157030). Methods for
designing such interfering RNAs are known in the art. For example, software
for designing interfering RNA is available from Oligoengine (Seattle, WA).
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Dominant negative proteins
One skilled in the art would know how to make dominant negative
mitotic kinesins and protein tyrosine phosphatases. Such dominant negative
proteins are described, for example, in Gupta et al., J. Exp. Med., 186:473-
478,
1997; Maegawa et al., J. Biol. Chem. 274:30236-30243, 1999; Woodford-
Thomas et al., J. Cell Biol. 117:401-414, 1992;
Aurora kinase inhibitors
Aurora kinases have been shown to be protein kinases of a new family
that regulate the structure and function of the mitotic spindle. One target of
Aurora kinases include mitotic kinesins. Aurora kinase inhibitors thus can be
used in combination with a compound that reduces protein tyrosine
phosphatase biological activity according to a method, composition, or kit of
the invention.
There are three classes of aurora kinases: aurora-A, aurora-B and aurora-
C. Aurora-A includes AIRK1, DmAurora, HsAurora-2, HsAIK, HsSTKIS,
CeAIR-l, MmARKl, MmAYKl, MmIAKI and XIEg2. Aurora-B includes
AIRK-2, DmIAL-1, HsAurora-1, HsAIK2, HsAIM-1, HsSTKI2, CeAIR-2,
MmARK2 and XAIRK2. Aurora-C includes HsAIK3 (Adams, et al., Trends
Cell Biol. 11:49-54, 2001 ).
Aurora kinase inhibitors include VX-528 and ZM447439; others are
described, e.g., in U.S. Patent Application Publication No. 2003/0105090 and
U.S. Patent Nos. 6,610,677, 6,593,357, and 6,528,509.
Farnesyltransferase inhibitors
Farnesyltransferase inhibitors alter the biological activity of PRL
phosphatases and thus can be used in combination with a compound that
reduces mitotic kinesin activity in a method, composition, or kit of the
invention. Farnesyltransferase inhibitors include arglabin, lonafarnib, BAY-
43-9006, tipifarnib, perillyl alcohol, FTI-277 and BMS-214662, as well as
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those compounds described, e.g., in Kohl, Ann. NY Acad. Sci. 886:91-102,
1999, U.S. Patent Application Publication Nos. 2003/0199544, 2003/0199542,
2003/0087940, 2002/0086884, 2002/0049327, and 2002/0019527, U.S. Patent
Nos. 6,586,461 and 6,500,841, and W003/004489.
Therapy
The compounds of the invention are useful for the treatment of cancers
and other disorders characterized by hyperproliferative cells. Therapy may be
performed alone or in conjunction with another therapy (e.g., surgery,
radiation
therapy, chemotherapy, immunotherapy, anti-angiogenesis therapy, or gene
therapy). Additionally, a person having a greater risk of developing a
neoplasm or other proliferative disease (e.g., one who is genetically
predisposed or one who previously had such a disorder) may receive
prophylactic treatment to inhibit or delay hyperproliferation. The duration of
the combination therapy depends on the type of disease or disorder being
treated, the age and condition of the patient, the stage and type of the
patient's
disease, and how the patient responds to the treatment. Therapy may be given
in on-and-off cycles that include rest periods so that the patient's body has
a
chance to recovery from any as yet unforeseen side-effects. Desirably, the
methods, compositions, and kits of the invention are more effective than other
methods, compositions, and kits. By "more effective" is meant that a method,
composition, or kit exhibits greater efficacy, is less toxic, safer, more
convenient, better tolerated, or less expensive, or provides more treatment
satisfaction than another method, composition, or kit with which it is being
compared.
Cancers include, without limitation, leukemias (e.g., acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic
leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,
acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic
myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera,
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lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas
and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell
lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,
meningioma, melanoma, neuroblastoma, and retinoblastoma).
Other proliferative disease that can be treated with the combinations and
methods of the invention include lymphoproliferative disorders and psoriasis.
By "lymphoproliferative disorder" is meant a disorder in which there is
abnormal proliferation of cells of the lymphatic system (e.g., T-cells and B-
cells).
Additionally therapy can include the use of other antiproliferative agents
with the combinations of the invention. For example, when treatment is for
cancer, the combination may be administered with an anticancer agent, such as
the agents in Table 3, below.
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Table 3
Busulfan procarbazine
dacarbazine altretamine
ifosfamide estramustine phosphate
Alkylating hexamethylmelamine mechlorethamine
agents
thiotepa streptozocin
dacarbazine temozolomide
lomustine Semustine
cyclophosphamide cisplatin
chlorambucil
Platinum spiroplatin lobaplatin (Aeterna)
agents
tetraplatin satraplatin (Johnson Matthey)
ormaplatin BBR-3464 (Hoffmann-La
Roche)
iproplatin SM-11355 (Sumitomo)
ZD-0473 (AnorMED) AP-5280 (Access)
oxaliplatin
carbo Latin
Antimetabolitesazacytidine trimetrexate
Floxuridine deoxycoformycin
2-chlorodeoxyadenosinepentostatin
6-mercaptopurine hydroxyurea
6-thioguanine decitabine (SuperGen)
cytarabine clofarabine (Bioenvision)
2-fluorodeoxy cytidineirofulven (MGI Pharma)
methotrexate DMDC (Hoffmann-La Roche)
tomudex ethynylcytidine (Taiho)
fludarabine gemcitabine
raltitrexed ca ecitabine
Topoisomeraseamsacrine exatecan mesylate (Daiichi)
inhibitors epirubicin quinamed (ChemGenex)
etoposide gimatecan (Sigma-Tau)
teniposide or mitoxantronediflomotecan (Beaufour-Ipsen)
7-ethyl-10-hydroxy-camptothecinTAS-103 (Taiho)
dexrazoxanet (TopoTarget)elsamitrucin (Spectrum)
pixantrone (Novuspharma)J-107088 (Merck & Co)
rebeccamycin analogue BNP-1350 (BioNumerik)
(Exclixis)
BBR-3576 (Novuspharma)CKD-602 (Chong Kun Dang)
rubitecan (SuperGen) KW-2170 (Kyowa Hakko)
irinotecan (CPT-11) hydroxycamptothecin (SN-38)
to otecan
Antitumor dactinomycin (actinomycinazonafide
D)
antibiotics valrubicin anthrapyrazole
daunorubicin (daunomycin)oxantrazole
therarubicin losoxantrone
idarubicin bleomycinic acid
rubidazone MEN-10755 (Menarini)
plicamycin GPX-100 (Gem Pharmaceuticals)
porfiromycin epirubicin
mitoxantrone (novantrone)mitoxantrone
amonafide
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Table 3 (cont.)
Antimitotic colchicine E7010 (Abbott)
agents vinblastine PG-TXL (Cell Therapeutics)
vindesine IDN 5109 (Bayer)
dolastatin 10 (NCI) A 105972 (Abbott)
rhizoxin (Fujisawa) A 204197 (Abbott)
mivobulin (Warner-Lambert)LU 223651 (BASF)
cemadotin (BASF) D 24851 (ASTAMedica)
RPR 109881A (Aventis) ER-86526 (Eisai)
TXD 258 (Aventis) combretastatin A4 (BMS)
epothilone B (Novartis)isohomohalichondrin-B
(PharmaMar)
T 900607 (Tularik) ZD 6126 (AstraZeneca)
T 138067 (Tularik) AZ10992 (Asahi)
cryptophycin 52 (Eli IDN-5109 (Indena)
Lilly)
vinflunine (Fabre) AVLB (Prescient NeuroPharma)
auristatin PE (Teikokuazaepothilone B (BMS)
Hormone)
BMS 247550 (BMS) BNP-7787 (BioNumerik)
BMS 184476 (BMS) CA-4 prodrug (OXiGENE)
BMS 188797 (BMS) dolastatin-10 (N1H)
taxoprexin (Protarga) CA-4 (OXiGENE)
SB 408075 (GlaxoSmithKline)docetaxel
vinorelbine vincristine
aclitaxel
Aromatase aminoglutethimide YM-511 (Yamanouchi)
inhibitors atamestane (BioMedicines)formestane
letrozole exemestane
anastrazole
Thymidylate pemetrexed (Eli Lilly)nolatrexed (Eximias)
s nthase ZD-9331 (BTG) CoFactorTM (BioKe s
inhibitors
DNA antagoniststrabectedin (PhannaMar)edotreotide (Novartis)
glufosfamide (Baxter mafosfamide (Baxter International)
International)
albumin + 32P (Isotopeapaziquone (Spectrum
Solutions)
thymectacin (NewBiotics)Pharmaceuticals)
06 benz 1 uanine Pali
ent)
Farnesyltransferasearglabin (NuOncology tipifarnib (Johnson &
Labs) Johnson)
inhibitors lonafarnib (Schering-Plough)perillyl alcohol (DOR
BioPharma)
BAY-43-9006 (Ba er)
.
Pump inhibitorsCBT-1 (CBA Pharma) zosuquidar trihydrochloride
(Eli Lilly)
tariquidar (Xenova) biricodar dicitrate (Vertex)
MS-209 (Schering AG)
Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate
(Titan)
acetyltransferaseSAHA (Aton Pharma) depsipeptide (Fujisawa)
inhibitors MS-275 Scherin AG)
MetalloproteinaseNeovastat (Aeterna CMT-3 (CollaGenex)
Laboratories)
inhibitors marimastat (British BMS-275291 (Celltech)
Biotech)
Ribonucleosidegallium maltolate (Titan)tezacitabine (Aventis)
reductase tria ine (Vion didox Molecules for Health)
inhibitors
21
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Table 3 (cont.)
TNF alpha virulizin (Lorus Therapeutics)revimid (Celgene)
a onists/antagonistsCDC-394 (Cel ene)
Endothelin atrasentan (Abbott) YM-598 (Yamanouchi)
A
rece for ZD-4054 (AstraZeneca)
ants onist
Retinoic fenretinide (Johnson alitretinoin (Ligand)
acid & Johnson)
rece for LGD-1550 (Ligand
a onists
Immuno- interferon dexosome therapy (Anosys)
modulators oncophage (Antigenics) pentrix (Australian Cancer
GMK (Progenies) Technology)
adenocarcinoma vaccine ISF-154 (Tragen)
(Biomira)
CTP-37 (AVI BioPharma) cancer vaccine (Intercell)
IRX-2 (Immuno-Rx) norelin (Biostar)
PEP-005 (Peplin Biotech)BLP-25 (Biomira)
synchrovax vaccines MGV (Progenies)
(CTL Immuno)
melanoma vaccine (CTL f3-alethine (Dovetail)
Immuno)
21 RAS vaccine (GemVax CLL thera y (Vasogen)
Hormonal estrogens dexamethasone
and
antihormonalconjugated estrogens prednisone
agents ethinyl estradiol methylprednisolone
chlortrianisen prednisolone
idenestrol aminoglutethimide
hydroxyprogesterone leuprolide
caproate
medroxyprogesterone octreotide
testosterone mitotane
testosterone propionateP-04 (Novogen)
fluoxymesterone 2-methoxyestradiol (EntreMed)
methyltestosterone arzoxifene (Eli Lilly)
diethylstilbestrol tamoxifen
megestrol toremofine
bicalutamide goserelin
flutamide leuporelin
nilutamide bicalutamide
Photodynamictalaporfin (Light Sciences)Pd-bacteriopheophorbide
(Yeda)
agents Theralux (Theratechnologies)lutetium texaphyrin (Pharmacyclics)
motexafin adolinium h ericin
(Pharmac clics
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Table 3 (cont.
Kinase Inhibitorsimatinib (Novartis)EKB-569 (Wyeth)
leflunomide (Sugen/Pharmacia)
kahalide F (PharmaMar)
ZD1839 (AstraZeneca)CEP-701 (Cephalon)
erlotinib (OncogeneCEP-751 (Cephalon)
Science)
canertinib (Pfizer)MLN518 (Millenium)
squalamine (Genaera)PKC412 (Novartis)
SU5416 (Phannacia)Phenoxodiol (Novogen)
SU6668 (Pharmacia)C225 (ImClone)
ZD4190 (AstraZeneca)rhu-Mab (Genentech)
ZD6474 (AstraZeneca)MDX-H210 (Medarex)
vatalanib (Novartis)2C4 (Genentech)
PKI166 (Novartis)MDX-447 (Medarex)
GW2016 (GlaxoSmithKline)ABX-EGF (Abgenix)
EKB-509 (Wyeth) IMC-1C11 (ImClone)
trastuzumab (Genentech)Tyrphostins
Gefitinib (Iressa)
Miscellaneous
agents
SR-27897 ceflatonin (apoptosis promotor,
(CCK A inhibitor, ChemGenex)
Sanofi-Synthelabo)
tocladesine BCX-1777 (PNP inhibitor, BioCryst)
(cyclic
AMP agonist,
Ribapharm)
alvocidib ranpirnase (ribonuclease stimulant,
(CDK inhibitor, Alfacell)
Aventis)
CV-247 (COX-2 galarubicin (RNA synthesis
inhibitor, inhibitor, Dong-A)
Ivy Medical)
P54 (COX-2 tirapazamine (reducing agent,
inhibitor, SRI International)
Phytopharm)
CapCellTM N-acetylcysteine (reducing
(CYP450 agent, Zambon)
stimulant,
Bavarian
Nordic)
GCS-100 (gala R-flurbiprofen (NF-kappaB inhibitor,
antagonist, Encore)
GlycoGenesys)
G17DT immunogen 3CPA (NF-kappaB inhibitor,
(gastrin Active Biotech)
inhibitor,
Aphton)
efaproxiral seocalcitol (vitamin D receptor
(oxygenator, agonist, Leo)
Allos Therapeutics)
PI-88 (heparanase 131-I-TM-601 (DNA antagonist,
inhibitor, TransMolecular)
Progeny
tesmilifene eflornithine (ODC inhibitor
(histamine , ILEX Oncology)
antagonist,
YM
BioSciences) minodronic acid (osteoclast
inhibitor,
histamine Yamanouchi)
(histamine
H2 receptor
agonist,
Maxim) indisulam (p53 stimulant, Eisai)
tiazofurin aplidine (PPT inhibitor, PharmaMar)
(IMPDH inhibitor,
Ribapharm)
cilengitide gemtuzumab (CD33 antibody,
(integrin Wyeth Ayerst)
antagonist,
Merck KGaA)
SR-31747 PG2 (hematopoiesis enhancer,
(IL-1 antagonist, Pharmagenesis)
Sanofi-Synthelabo)
CCI-779 (mTOR lmmunolT"' (triclosan oral
kinase inhibitor, rinse, Endo)
Wyeth)
exisulind triacetyluridine (uridine prodrug
(PDE V inhibitor, , Wellstat)
Cell Pathways)
CP-461 (PDE SN-4071 (sarcoma agent, Signature
V inhibitor, BioScience)
Cell Pathways)
AG-2037 (GART TransMID-107TM (immunotoxin,
inhibitor, KS Biomedix)
Pfizer)
WX-UK1 (plasminogen PCK-3145 (apoptosis promotor,
activator Procyon)
inhibitor,
Wilex) doranidazole (apoptosis promotor,
Pola)
PBI-1402 CHS-828 (cytotoxic agent, Leo)
(PMN stimulant,
ProMetic
LifeSciences) trans-retinoic acid (differentiator,
NIH)
bortezomib MX6 (apoptosis promotor, MARIA)
(proteasomc
inhibitor,
Millennium)
SRL-172 (T apomine (apoptosis promotor,
cell stimulant, ILEX Oncology)
SR Pharma)
TLK-286 (glutathione urocidin (apoptosis promotor,
S transferase Bioniche)
inhibitor,
Telik) Ro-31-7453 (apoptosis promotor,
La Roche)
PT-100 (growth brostallicin (apoptosis promotor,
factor agonist, Pharmacia)
Point
Therapeutics)
midostaurin
(PKC inhibitor,
Novartis)
bryostatin-1
(PKC stimulant,
GPC Biotech)
CDA-Il (apoptosis
promotor,
Everlife)
SDX-101 (apoptosis
promotor,
Salmedix)
rituximab
(CD20 antibod
, Genentech
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Examples
The following examples are to illustrate the invention. They are not
meant to limit the invention in any way.
Chlorpromazine is a mitotic kinesin inhibitor
We determined that chlorpromazine is a mitotic kinesin inhibitor using a
cell free motor assay. This assay measures organic phosphate (P;) generated
during microtubule activated ATPase activity of kinesin motor proteins.
Recombinant HsEgS/KSP kinesin motor protein activity was assayed using the
Kinesin ATPase End Point Biochem Kit (Cytoskeleton, catalog # BK053)
following the manufacturer's instructions for amounts of reaction buffer, ATP
and microtubules. The amount of HsEgS/KSP kinesin protein was optimized to
0.8 ~,g per reaction and included where indicated. Each assay was performed
in a total reaction volume of 30 ~,L in a clear 96 well '/2 area plate
(Corning
Inc., Costar and cat # 3697) and included the following conditions:
1. a reaction blank consisting of reaction buffer and ATP only;
2. negative control reactions containing:
a. microtubules and ATP without kinesin protein or
b. kinesin HsEgS/KSP and ATP without microtubules; and
3. experimental reactions containing ATP, kinesin, and microtubules with
or without compound at the indicated final concentrations.
Reactions were pre-incubated for 15 minutes at room temperature prior
to the addition of ATP. After ATP addition, reactions were allowed to proceed
for 10 minutes at room temperature prior to termination by the addition of 70
~.L of CytoPhos Reagent. Following a last 10-minute incubation at room
temperature, reactions were quantitated by reading the absorbance at 650 nm
on a spectrophotometer (Beckman Instruments, Inc., Model DU 530). Raw
absorbance values were corrected by subtracting the absorbance of the blank.
Absorbance was converted into Pi concentration by comparison with a standard
Pi curve. Percent inhibition was calculated from Pi concentration according to
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the following formula: %Inhibition = (untreated-treated)/untreated x 100. The
arithmetic mean was generated from percent inhibition of experimental
replicates. The results are shown in Table 4.
Table 4
Percent inhibition of kinesin motor activity (n=4)
Chlorpromazine [pM]
1 2 4 8 16 32 64
Mean -5.51 -11.18 17.42 52.91 85.82 97.79 104.54
STDEV 11.87 25.94 17.54 6.99 10.84 6.40 10.96
Other phenothiazines capable of reducing mitotic kinesin biological activity
include promethazine, thioridazine, trifluoperazine, perphenazine,
fluphenazine, clozapine, and prochlorperazine.
The combination of chlorpromazine and pentamidine reduce cell
proliferation in vitro
The ability of pentamidine (a protein tyrosine phosphatase inhibitor) and
chlorpromazine (a mitotic kinesin inhibitor), in combination, to reduce cell
proliferation in vitro was determined. Human colon adenocarcinoma cell line
HCT116 (ATCC#CCL-247) were grown at 37°~ 5°C and 5% C02in
DMEM
supplemented with 10% FBS, 2 mM glutamine, 1% penicillin and 1%
streptomycin. The anti-proliferation assays were performed in 384-well plates.
l OX stock solutions (6.6 ~L) from the combination matrices were added to 40
~,L of culture media in assay wells. The tumor cells were liberated from the
culture flask using a solution of 0.25% trypsin. Cells were diluted in culture
media such that 3000 cells were delivered in 20 ~L of media into each assay
well. Assay plates were incubated for 72-80 hours at 37°C X0.5°C
with 5%
C02. Twenty microliters of 20% Alamar Blue warmed to 37°C
X0.5°C was
added to each assay well following the incubation period. Alamar Blue
metabolism was quantified by the amount of fluorescence intensity 3.5 - 5.0
hours after addition. Quantification, using an LJL Analyst AD reader (LJL
CA 02545423 2006-05-10
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Biosystems), was taken in the middle of the well with high attenuation, a 100
msec read time, an excitation filter at 530 nm, and an emission filter at 575
nm.
For some experiments, quantification was performed using a Wallac Victor2
reader. Measurements were taken at the top of the well with stabilized energy
lamp control; a 100 msec read time, an excitation filter at 530 nm, and an
emission filter at 590 nm. No significant differences between plate readers
were measured.
The percent inhibition (%I) for each well was calculated using the
following formula:
%I = [(avg. untreated wells - treated well)/(avg. untreated wells)] x 100
The average untreated well value (avg. untreated wells) is the arithmetic mean
of 40 wells from the same assay plate treated with vehicle alone. Negative
inhibition values result from local variations in treated wells as compared to
untreated wells.
The data, expressed as percent inhibition, are shown in Table 5.
Table 5
Chlor
romazine
M
0 4 6 7.5 9 10 12 16 20 22
0 0.63 2.9 0.115.4 4.1 16 22 39 56 59
0.5 1.2 -0.13 6.1 4.3 7.9 16 31 45 64 65
1 1.9 2.2 9.1 5.5 16 21 25 56 57 68
2 3.1 3.1 5.8 5.1 9.7 18 30 57 70 73
4 -0.77 4.0 2.7 12 10 20 26 59 69 74
6 5 7.1 15 9.9 16 22 38 58 74 78
9 9 13 13 22 16 37 41 68 79 88
a 12 9.9 13 15 16 18 27 46 69 82 87
15 16 20 22 35 26 40 52 78 84 92
~ 19 22 I 25 36 40 49 70 82 94 94
~ ~ ~ ~ ~
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Other Embodiments
All publications and patents mentioned in the above specification are
herein incorporated by reference. Various modifications and variations of the
described method and system of the invention will be apparent to those skilled
in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with specific
preferred embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention that are
obvious to those skilled in oncology or related fields are intended to be
within
the scope of the invention.
We claim:
27
