CA2545423A1 - Combinations for the treatment of proliferative diseases - Google Patents

Abstract

The invention features combinations of drugs for the treatment of proliferative diseases (e.g., cancer). The invention also features methods for identifying new combination therapies for the treatment of cancer and other proliferative diseases.

Description

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 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.

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 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 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, 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 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.

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.

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

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 MKLPI A1131325, AU133373, X67155 MCAK AL046197, U63743 HsNYREN62 AF155117 HsLOC8464 NM 032559 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;

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 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 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, SHP-2 AU123593, BF515187, BX537632, D13540 MKP-1 U01669, X68277 MKP-2 BC014565, U21108, U48807, AL137704 CDC 14A AF000367, AF064102, AF064103 CDC14B AF023158, AF064104 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.

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, 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).

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 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, 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.

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 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 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 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 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 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]

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

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 a 12 9.9 13 15 16 18 27 46 69 82 87 ~ 19 22 I 25 36 40 49 70 82 94 94 ~ ~ ~ ~ ~

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:

Claims (75)

1. A composition comprising a first agent that reduces mitotic kinesin biological activity and a second agent that reduces protein tyrosine phosphatase biological activity, wherein said first and second agents are present in amounts that, when administered to a patient having a proliferative disease, are sufficient to treat said disease.

2. The composition of claim 1, wherein said first agent is a mitotic kinesin inhibitor.

3. The composition of claim 1, wherein said first agent is an antisense compound or RNAi compound that reduces the expression levels of said mitotic kinesin.

4. The composition of claim 1, wherein said first agent is a dominant negative mitotic kinesin or an expression vector encoding said dominant negative mitotic kinesin.

5. The composition of claim 1, wherein said first agent is an antibody that binds said mitotic kinesin and reduces mitotic kinesin biological activity.

6. The composition of any one of claims 1-5, wherein said mitotic kinesin is HsEg5/KSP.

7. The composition of claim 1, wherein said first agent in an aurora kinase inhibitor.

8. The composition of claim 1, wherein said mitotic kinesin biological activity is enzymatic activity, motor activity, or binding activity.

9. The composition of claim 1, wherein said second agent is a protein tyrosine phosphatase inhibitor.

10. The composition of claim 1, wherein said second agent is an antisense compound or RNAi compound that reduces the expression levels of said protein tyrosine phosphatase.

11. The composition of claim 1, wherein said second agent is a dominant negative protein tyrosine phosphatase or an expression vector encoding said dominant negative protein tyrosine phosphatase.

12. The composition of claim 1, wherein said second agent is an antibody that binds said protein tyrosine phosphatase and reduces protein tyrosine phosphatase biological activity.

13. The composition of any one of claims 9-12, wherein said protein tyrosine phosphatase is PTP1B, PRL-1, PRL-2, PRL-3, SHP-1, SHP-2, MKP-1, MKP-2, CDC14, CDC25A, CDC25B, or CDC25C.

14. The composition of claim 1, wherein said second agent is a farnesyltransferase inhibitor.

15. The composition of any one of claims 1-14, wherein said first or second agent is present in said composition in a low dosage.

16. The composition of any one of claims 1-14, wherein said first or second agent is present in said composition in a high dosage.

17. The composition of any one of claims 1-16, wherein said composition is formulated for topical administration.

18. The composition of any one of claims 1-16, wherein said composition is formulated for systemic administration.

19. A method for treating a patient who has a proliferative disease, said method comprising administering to said patient a combination of:
a) a first agent that reduces mitotic kinesin biological activity; and b) a second agent that reduces protein tyrosine phosphatase biological activity, wherein the first and second agents are administered simultaneously or within 28 days of each other, in amounts that together are sufficient to treat said patient.

20. The method of claim 19, wherein said first agent is a mitotic kinesin inhibitor.

21. The method of claim 19, wherein said first agent is an antisense compound or RNAi compound that reduces the expression levels of said mitotic kinesin.

22. The method of claim 19, wherein said first agent is a dominant negative mitotic kinesin or an expression vector encoding said dominant negative mitotic kinesin.

23. The method of claim 19, wherein said first agent is an antibody that binds said mitotic kinesin and reduces mitotic kinesin biological activity.

24. The method of any one of claims 19-23, wherein said mitotic kinesin is HsEg5/KSP.

25. The method of claim 19, wherein said first agent in an aurora kinase inhibitor.

26. The method of claim 19, wherein said second agent is a protein tyrosine phosphatase inhibitor.

27. The method of claim 19, wherein said second agent is an antisense compound or RNAi compound that reduces the expression levels of said protein tyrosine phosphatase.

28. The method of claim 19, wherein said second agent is a dominant negative protein tyrosine phosphatase or an expression vector encoding said dominant negative protein tyrosine phosphatase.

29. The method of claim 19, wherein said second agent is an antibody that binds said protein tyrosine phosphatase and reduces protein tyrosine phosphatase biological activity.

30. The method of any one of claims 21-25, wherein said protein tyrosine phosphatase is PTP1B, PRL-1, PRL-2, PRL-3, SHP-1, SHP-2, MKP-1, MKP-2, CDC14, CDC25A, CDC26B, or CDC25C.

31. The method of claim 19, wherein said second agent is a farnesyltransferase inhibitor.

32. The method of any one of claims 19-31, wherein said first and second agents are administered within 14 days of each other.

33. The method of claim 32, wherein said first and second agents are administered within 7 days of each other.

34. The method claim 33, wherein said first and second agents are administered within 1 day of each other.

35. The method of any one of claims 19-34, wherein said first or second agent is administered in a low dosage.

36. The method of any one of claims 19-34, wherein said first or second agent is administered in a high dosage.

37. The method of any one of claims 19-36, wherein said first or second agents is administered topically or systemically.

38. The method of any one of claims 19-37, wherein said proliferative disease is cancer.

39. The method of claim 38, wherein said cancer is selected from 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, Hodgkin's disease, non-Hodgkin's disease, Waldenstrom's macroglobulinemia, heavy chain disease, 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.

40. The method of claim 38, further comprising administering to said patient an antiproliferative agent listed in Table 3.

41. A method of inducing cell cycle arrest in a cell, comprising contacting the cell with a first agent that reduces mitotic kinesin biological activity and a second agent that reduces protein tyrosine phosphatase biological activity.

42. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising the steps of:
(a) contacting proliferating cells in vitro with an agent that reduces mitotic kinesin 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

43. The method of claim 42, wherein the cells are cancer cells or cells from a cancer cell line.

44. A method for identifying a compound that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the compound as a compound that may be useful for the treatment of a proliferative disease.

45. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

46. The method of claim 45, wherein the cells are cancer cells or cells from a cancer cell line.

47. A method for identifying a compound that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the compound as a compound that may be useful for the treatment of a proliferative disease.

48. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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 contacted with the compound identified in step (a) or contacted with the compound identified in step (a) but not contacted with the agent, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

49. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

50. A kit, comprising:
(i) a composition comprising a first agent that reduces mitotic kinesin biological activity and a second agent that reduces protein tyrosine phosphatase biological activity; and (ii) instructions for administering said composition to a patient diagnosed with a proliferative disease.

51. A kit, comprising:
(i) a first agent that reduces mitotic kinesin biological activity;
(ii) a second agent that reduces protein tyrosine phosphatase biological activity; and (iii) instructions for administering said first and second agents to a patient diagnosed with a proliferative disease.

52. A kit comprising (i) a first agent that reduces mitotic kinesin biological activity and (ii) instructions for administering said first agent and a second agent that reduces protein tyrosine phosphatase biological activity to a patient diagnosed with a proliferative disease

53. A kit comprising (i) a first agent that reduces protein tyrosine phosphatase biological activity and (ii) instructions for administering said first agent and a second agent that reduces mitotic kinesin biological activity to a patient diagnosed with a proliferative disease.

54. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising the steps of:
(a) contacting proliferating cells in vitro with an agent that reduces mitotic kinesin 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

55. The method of claim 54, wherein said agent that reduces mitotic kinesin biological activity is a mitotic kinesin inhibitor.

56. The method of claim 54, wherein said agent that reduces mitotic kinesin biological activity is an antisense compound or RNAi compound that reduces the expression levels of said mitotic kinesin.

57. The method of claim 54, wherein said agent that reduces mitotic kinesin biological activity is a dominant negative mitotic kinesin or an expression vector encoding said dominant negative mitotic kinesin.

58. The method of claim 54, wherein said agent that reduces mitotic kinesin biological activity is an antibody that binds said mitotic kinesin and reduces mitotic kinesin biological activity.

59. The method of claim 54, wherein said mitotic kinesin is HsEg5/KSP.

60. The method of claim 54, wherein said agent that reduces mitotic kinesin biological activity in an aurora kinase inhibitor.

61. The method of claim 54, wherein said mitotic kinesin biological activity is enzymatic activity, motor activity, or binding activity.

62. The method of claim 54, wherein the cells are cancer cells or cells from a cancer cell line.

63. A method for identifying a compound that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the compound as a compound that may be useful for the treatment of a proliferative disease.

64. The method of claim 63, wherein the cells are cancer cells or cells from a cancer cell line.

65. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

66. The method of claim 65, wherein said agent that reduces protein tyrosine phosphatase biological activity is a protein tyrosine phosphatase inhibitor.

67. The method of claim 65, wherein said agent that reduces protein tyrosine phosphatase biological activity is an antisense compound or RNAi compound that reduces the expression levels of said protein tyrosine phosphatase.

68. The method of claim 65, wherein said agent that reduces protein tyrosine phosphatase biological activity is a dominant negative protein tyrosine phosphatase or an expression vector encoding said dominant negative protein tyrosine phosphatase.

69. The method of claim 65, wherein said agent that reduces protein tyrosine phosphatase biological activity is an antibody that binds said protein tyrosine phosphatase and reduces protein tyrosine phosphatase biological activity.

70. The method of claim 64, wherein said protein tyrosine phosphatase is PTP1B, PRL-1, PRL-2, PRL-3, SHP-1, SHP-2, MKP-1, MKP-2, CDC14, CDC25A, CDC25B, or CDC25C.

71. The method of claim 65, wherein said second agent is a farnesyltransferase inhibitor.

72. The method of claim 65, wherein the cells are cancer cells or cells from a cancer cell line.

73. A method for identifying a compound that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the compound as a compound that may be useful for the treatment of a proliferative disease.

74. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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 contacted with the compound identified in step (a) or contacted with the compound identified in step (a) but not contacted with the agent, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.

75. A method for identifying a combination that may be useful for the treatment of a proliferative disease, the method comprising 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, wherein a reduction in cell proliferation identifies the combination as a combination that may be useful for the treatment of a proliferative disease.