WO2006001954A2

WO2006001954A2 - Methods for promoting the formation of platelets and for treating blood and bone marrow disorders

Info

Publication number: WO2006001954A2
Application number: PCT/US2005/017735
Authority: WO
Inventors: Jonathan G. Drachman; Brian J. Lannutti; Manish Gandhi
Original assignee: Puget Sound Blood Center And Program
Priority date: 2004-05-20
Filing date: 2005-05-20
Publication date: 2006-01-05
Also published as: WO2006001954A3

Abstract

The invention provides methods for promoting the differentiation of megakaryocytes, methods for forming platelets, and methods for reducing the number of abnormal or malignant cells in bone marrow. In some embodiments, the methods comprise the step of contacting megakaryocyte progenitors with an amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective to promote their differentiation. In some embodiments, the 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound is 2-oxo-3-(4, 5, 6, 7-tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole-5-sulfonic acid dimethylamide (SU6656). The invention also provides methods for treating blood and bone marrow disorders.

Description

METHODS FOR PROMOTING THE FORMATION OF PLATELETS AND FOR TREATING BLOOD AND BONE MARROW DISORDERS

CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/573,447, filed May 20, 2004, and U.S. Provisional Application No. 60/620,402, filed October 19, 2004. STATEMENT OF GOVERNMENT LICENSE RIGHTS The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of HL065498 awarded by the National Institutes of Health. FIELD OF THE INVENTION The present invention relates to methods for promoting megakaryocyte differentiation and fragmentation, and methods for treating blood and bone marrow disorders. BACKGROUND OF THE INVENTION Thrombopoiesis is a complex process of megakaryocyte (MK) differentiation and fragmentation that can be divided into four distinct stages: (1) commitment of pluripotent stem cell to the MK lineage; (2) proliferation of MK progenitors (i.e., cell division without differentiation); (3) terminal differentiation of MKs, characterized by endomitosis and cellular expansion; and (4) platelet shedding through fragmentation (Vainchenker et al. (1995) Crit. Rev. Oncol. Hematol. 20:165-92; Italiano & Shivdasani (2003) Thromb. Haemost. 1:1174-82). The mainstay of therapy for clinically life-threatening thrombocytopenia is transfusion of allogeneic platelets. Platelet transfusions are associated with several problems, including viral and bacterial contamination, alloimunization, and a short shelf life (5 days). The infectious risk has been reduced but not eliminated through extensive screening, introduction of nucleic acid testing, and bacterial detection systems. Alloimmunization, which shortens survival of transfused platelets, can be reduced by eliminating unnecessary exposure and can be overcome through recruitment of HLA- compatible donors. There is a need for methods to induce thrombopoiesis in vitro to reduce both safety and inventory concerns. The present invention addresses this and other needs. SUMMARY OF THE INVENTION In one aspect, the invention provides methods for promoting the differentiation of megakaryocytes. In some embodiments, the methods comprise the step of contacting megakaryocyte progenitors with an amount of a 3-(cyclohexanoheteroarylidenyl)-2- indolinone compound effective for promoting the differentiation of megakaryocytes. The megakaryocyte progenitors used in the methods of the invention may be immature hematopoietic cells isolated from blood, umbilical cord blood, or bone marrow of a mammal, or cells from immortalized hematopoietic cell lines. The megakaryocyte progenitors may be contacted with a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound in vivo, ex vivo, or in vitro. In some embodiments, the 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound is 2-oxo-3-(4,5,6,7-tetrahydro- 1 H-indol-2-ylmethylene)-2,3 -dihydro- 1 H-indole-5-sulfonic acid dimethylamide (SU6656). In some embodiments, the megakaryocyte progenitors are promoted to differentiate into platelets or platelet-like fragments. Accordingly, in some embodiments the invention provides methods for forming platelets from megakaryocyte progenitors, comprising the step of contacting megakaryocyte progenitors with an amount of a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for promoting the formation of platelets or platelet-like fragments. In another aspect, the invention provides methods for reducing the number of abnormal or malignant cells in bone marrow, comprising the step of contacting bone marrow cells with an amount of a pharmaceutical composition comprising a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for reducing the number of abnormal or malignant cells. A further aspect of the invention provides methods for treating blood and bone marrow disorders. In some embodiments, the methods comprise the step of administering to a subject suffering from a blood or bone marrow disorder an amount of a pharmaceutical composition comprising a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound therapeutically effective for treating the blood or bone marrow disorder. In some embodiments or the methods, the 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound is SU6656. Exemplary blood or bone marrow disorders that may be treated using these methods include, but are not limited to, thrombocytopenias, myelodysplastic syndromes, leukemias, essential thrombocytoses, and myeloproliferative diseases. The subject to be treated is typically a mammalian subject, such as a human subject. Yet another aspect of the invention is directed to the use of a 3- (cyclohexanoheteroarylidenyl)-2-mdolinone compound in the manufacture of a medicament for the treatment of a blood or bone marrow disorder, including, but not limited to thrombocytopenias, myelodysplastic syndromes, leukemias, essential thrombocytoses, and myeloproliferative diseases. In some embodiments, the compound used in the manufacture of a medicament is 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2- ylmethylene)-2,3-dihydro-lH-indole-5-sulfonic acid dimethylamide (SU6656). In another aspect, the invention provides methods for inhibiting the activity of Aurora kinase B. The methods comprise the step of contacting cells with an amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective for inhibiting the activity of Aurora kinase B. In some embodiments, the compound is 2-oxo-3-(4,5,6,7- tetrahydro- 1 H-indol-2-ylmethylene)-2,3 -dihydro- 1 H-indole-5-sulfonic acid dimethylamide (SU6656). The inhibition of Aurora kinase B activity may be used for reducing the growth of cancer cells and for treating subjects suffering from cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT One aspect of the invention provides methods for promoting the differentiation of megakaryocytes. In some embodiments, the methods comprise the step of contacting megakaryocyte progenitors with an amount of a 3-(cyclohexanoheteroarylidenyl)-2- indolinone compound effective for promoting the differentiation of megakaryocytes. As used herein, "promoting differentiation of megakaryocytes" refers to the process of inducing or advancing the terminal differentiation of megakaryocytes from megakaryocyte progenitors, characterized by polyploidization through endomitosis, cellular expansion, and platelet shedding. The term "megakaryocyte progenitor" refers to a hematopoietic cell that is capable of being induced to differentiate into one or more megakaryocytes. Any suitable source of megakaryocyte progenitors may be used in the methods, such as immature hematopoietic cells isolated from blood, umbilical cord blood, or bone marrow of a mammal, and immortalized hematopoietic cell lines. Exemplary immortalized hematopoietic cells lines that may be used include, but are not limited to, megakaryoblastic cells lines such as UT7-TPO, HEL, and K562. The megakaryocyte progenitors are contacted with an amount of a 3-(cyclohexanoheteroarylidenyl)-2- indolinone compound effective for promoting the differentiation of megakaryocytes. As used herein, the term "3-(cyclohexanoheteroarylidenyl)-2-indolinone compound" refers to indolinone compounds having a cyclohexanoheteroarylidenyl substituent at the 2-position. It will be appreciated that the 3- (cyclohexanoheteroarylidenyl)-2-indolinone compounds may be further substituted. Representative substituted and unsubstituted 3-(cyclohexanoheteroarylidenyl)-2- indolinone compounds are described in U.S. Patent Nos. 6,051,593; 6,114,371; 6,130,238; 6,350,754; and 6,579,897, each incorporated herein by reference in its entirety. In certain embodiments, the cyclohexanoheteroaryl group of the 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound is a tetrahydroindole group. These compounds are referred to as 3-(tetrahydroindolidenyl)-2-indolinone compounds. Representative 3-(tetrahydroindolidenyl)-2-indolinone compounds include 2-oxo-3- (4,5,6,7-tetrahydro-lH-indol-2-yhnethylene)-2,3-dihydro-lH-indole compounds. The 2- oxo-3-(4,5,6,7-tetrahydro-lH-indol-2-yhnethylene)-2,3-dihydro-lH-indole compounds may be further substituted. In certain embodiments, the 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2- yhnethylene)-2,3-dihydro-lH-indole compounds include those compounds in which the indole group has a sulfonamide substituent (i.e., -SO₂NR₂, where each R is independently selected from hydrogen, C1-C6 alkyl, or C5-C10 aryl). Representative sulfonamido substituents include, for example, sulfonic acid amide (i.e., -SO₂NH₂), sulfonic acid methyl amide (i.e., -SO₂NHCH₃), and sulfonic acid diethylamide (i.e., -SO₂N(CH₃)₂), among others. In one embodiment, the 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2-ylmethylene)- 2,3-dihydro-lH-indole compound is a 5-sulfonamide 2-oxo-3-(4,5,6,7-tetrahydro-lH- indol-2-ylmethylene)-2,3-dihydro-lH-indole compound. Two representative 2-oxo-3- (4,5,6,7-tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole compounds having 5-sulfonamide substituents are described in Blake et al. (2000) MoI. Cell. Biol. 20(23):9018-27, incorporated herein by reference in its entirety. In one embodiment, the 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole compound is 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole-5- sulfonic acid dimethylamide, also known as SU6656. In another embodiment, the 2-oxo- 3-(4,5,6,7-tetrahydro-lH-indol-2-yhτiethylene)-2,3-dihydro-lH-indole compound is 2- oxo-3-(4,5,6,7-tetrahydro- 1 H-indol-2-yhnethylene)-2,3-dihydro- 1 H-indole-5-sulfonic acid amide, also known as SU6657. 2-Oxo-3-(4,5,6,7-tetrahydro- 1 H-indol-2-ylmethylene)-2,3 -dihydro- 1 H-indole-5- sulfonic acid dimethylamide (also referred to herein as "SU6656")_? a representative of the larger class of 3-(cyclohexanoheteroarylidenyl)-2-indolinone compounds, is described herein as useful in methods of the invention. The 3-(cyclohexanoheteroarylidenyl)-2- indolinone compound may be formulated as a pharmaceutical composition, as described below. The megakaryocyte progenitors may be contacted with a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound in vivo, ex vivo, or in vitro. The determination of an amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective to promote the differentiation of megakaryocytes is well within the competence of one of ordinary skill in the art. hi some embodiments, the amount of a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective to promote the differentiation of megakaryocytes is between about 0.05 μM and about 50 μM, such as between about 0.25 μM and about 10 μM or between about 1 μM and about 5 μM. Exemplary methods for contacting megakaryocyte progenitors with an effective amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound are provided in EXAMPLES 1-3. In some embodiments, the megakaryocyte progenitors are promoted to differentiate into platelets or platelet-like fragments. Accordingly, in some embodiments the invention provides methods for forming platelets from megakaryocyte progenitors, comprising the step of contacting megakaryocyte progenitors with an amount of a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for promoting the formation of platelets or platelet-like fragments, hi some embodiments, the amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective to promote the formation of platelets are as described above for promoting the differentiation of megakaryocytes. An exemplary method for promoting the formation of platelets or platelet-like fragments is provided in EXAMPLE 3. Transfusion of allogeneic platelets is the mainstay of therapy for patients with life-threatening thrombocytopenia. However, currently in the United States donated platelets can only be stored for 5 days and are maintained at room temperature, increasing the risk of bacterial overgrowth. Therefore, methods for producing functional platelets in vitro is highly advantageous for transfusion therapy. As described in EXAMPLES 1 and 3, SU6656 induces cellular enlargement, polyploidization, and cytoplasmic fragmentation of megakaryocyte progenitors, such as hematopoietic cell lines and immature human primary megakaryocytes. After 6 days in the presence of thrombopoietin and SU6656, a majority of the cells became polyploid and started shedding platelet-like fragments (PFs). The PFs showed rapid and sustained aggregation in response to each of the standard agonists (collagen, arachidonic acid, adenosine diphosphate (ADP), and epinephrine). PFs generated in the presence of SU6656 had higher amplitude and more prolonged aggregation in each of three experiments. As seen by electron microscopy, primary progenitors developed demarcation-membranes within 72 hours and evidence of dense- granules, glycogen, and PFs after 6 days. These PFs demonstrated properties consistent with platelets, that is, aggregation in response to multiple agonists without spontaneous aggregation. These studies provide evidence that SU6656 promotes megakaryocytic differentiation and thrombopoiesis. In another aspect, the invention provides methods for reducing the number of abnormal or malignant cells in bone marrow, comprising the step of contacting bone marrow cells with an amount of a pharmaceutical composition comprising a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for reducing the number of abnormal or malignant cells. The bone marrow cells may be contacted with a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound in vivo, ex vivo, or in vitro. The determination of an amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound that is effective for reducing the number of abnormal or malignant cells in bone marrow is well within the competence of one of ordinary skill in the art. In some embodiments, the amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective for reducing the number of abnormal or malignant cells are as described above for promoting the differentiation of megakaryocytes. Exemplary 3- (cyclohexanoheteroarylidenyl)-2-indolinone compounds for use in these methods are as described above. In some embodiments, the compound is SU6656. As described in EXAMPLES 1 and 3, SU6656 is effective at inducing megakaryocyte differentiation of immortalized hematopoietic cells. By inducing differentiation of such cells, the methods of the invention effectively reduce the number of abnormal or malignant cells. A further aspect of the invention provides methods for treating blood and bone marrow disorders. In some embodiments, the methods comprise the step of administering to a subject suffering from a blood or bone marrow disorder an amount of a pharmaceutical composition comprising a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound therapeutically effective for treating the blood or bone marrow disorder. Exemplary 3-(cyclohexanoheteroarylidenyl)-2-indolinone compounds for use in these methods are as described above. In some embodiments, the compound is SU6656. Exemplary blood or bone marrow disorders that may be treated using these methods include, but are not limited to, thrombocytopenias, myelodysplastic syndromes, leukemias, essential thrombocytoses, and myeloproliferative diseases. The subject to be treated is typically a mammalian subject, such as a human subject. Suitable routes of administration of the pharmaceutical compositions may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal injections. The pharmaceutical composition may also be administered in a local rather than systemic manner, for example, in a sustained release formulation. In the pharmaceutical composition, the 3-(cyclohexanoheteroarylidenyl)-2- indolinone compound is typically mixed with suitable carriers or excipient(s). Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. The pharmaceutical compositions may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the compounds may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical compositions for oral use can be made with the use of a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions for oral administration may include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the compounds in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin and the like for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. The compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The pharmaceutical compositions may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the compounds for use in the methods of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate, etc. formed by the reaction of an amino group with the appropriate acid. Salts in which the compound forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the molecule with the appropriate base (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH^)). Pharmaceutical compositions suitable for use in the methods of the invention include compositions in which the active ingredients are contained in an amount effective to achieve its intended purpose. The term "therapeutically effective amount" as used herein refers to an amount of the compound being administered that will prevent, alleviate, ameliorate or reduce to some extent at least one of the symptoms of the blood or bone marrow disorder being treated, or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Moreover, animal models may be used to formulate a dose that achieves a circulating concentration range that includes the IC₅₀ as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal megakaryocyte- differentiation activity). Such information can be used to more accurately determine useful doses in human subjects. Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED_5Q (the dose therapeutically effective hi 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage may be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al. (1975) in "The Pharmacological Basis of Therapeutics", Ch. 1, p.l). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to achieve and maintain the megakaryocyte-differentiation activity, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% promotion of megakaryocyte differentiation using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10- 90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Yet another aspect of the invention is directed to the use of a 3- (cyclohexanoheteroarylidenyi)-2-indolinone compound in the manufacture of a medicament for the treatment of a blood or bone marrow disorder, including, but not limited to thrombocytopenias, myelodysplastic syndromes, leukemias, essential thrombocytoses, and myeloproliferative diseases. In some embodiments, the compound used in the manufacture of a medicament is 2-oxo-3-(4,5,6,7-tetrahydro-lH-indol-2- yhnethylene)-2,3-dihydro-lH-indole-5-sulfonic acid dimethylamide (SU6656). Exemplary methods for manufacturing a medicament are as described above for pharmaceutical compositions. In another aspect, the invention provides methods for inhibiting the activity of Aurora kinase B. The methods comprise the step of contacting cells with an amount of a 3-(cyclohexanoheteroarylidenyl)-2-indolinone compound effective for inhibiting the activity of Aurora kinase B. In some embodiments, the compound is 2-oxo-3-(4,5,6,7- tetrahydro- 1 H-indol-2-ylrnethylene)-2,3-dihydro- 1 H-indole-5-sulfonic acid dimethylamide (SU6656). Aurora kinases are a conserved family of three serine/threonine kinases that have essential functions in cell division (Carmena & Earnshaw (2003) Nat. Rev. MoI. Cell. Biol. 4:842-54). Recent studies have implicated all three Aurora kinases in cancer (see, e.g., Kayama et al (2003) Cancer Metastasis Rev. 22(4):451-64), and Aurora kinase inhibitors are being evaluated as anticancer agents (Doggrell (2004) Expert Opin. Investig. Drugs 13(9): 1199-201; Keen & Taylor (2004) Nat. Rev. Cancer 4(12):927-36). For example, expression levels of Aurora kinase B has been shown to be elevated in several cancer cell lines relative to normal cells (Ota et al. (2002) Cancer Res. 62:186-77). Moreover, overexpression of Aurora kinase B has been associated with thyroid carcinoma, and blocking Aurora kinase expression significantly reduced the growth of thyroid anaplastic carcinoma cells (Sorrentino et al. (2005) J. CHn. Endocrinol. Metab. 90(2):928-35). Accordingly, inhibiting the activity ofAurora kinase B activity may be useful for reducing the growth of cancer cells and for treating subjects suffering from cancer. The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. EXAMPLE 1 This Example describes the induction of polyploidization and differentiation by SU6656 in leukemic cell lines and primary bone marrow. Although polyploidization through endomitosis is seen in a variety of cell types, including salivary glands, trophoblast, and urinary bladder epithelium (Odell & Jackson (1968) Blood 32:102-10; Brodksy & Uryvaeva (1977) Int. Rev. Cytol. 50:275-332), it is unique to MKs among hematopoietic cells. The progression of cell cycle through chromosome duplication, assembly of nuclear spindles, dissolution of the nuclear envelope, and partial separation of homologous chromosomes has been well documented in MKs (Vitrat et al. (1998) Blood 91:3711-23; Nagata et al. (1997) J. Cell Biol. 139:449- 57). However, the process is interrupted prior to cell division, resulting in cells with twice the number of chromosomes. Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). This Example describes the effect of SU6656 on TPO-induced growth and differentiation. Materials and Methods Cells and cell culture'. Myelodysplastic bone marrow cells and cadaveric organ donor marrow were used. K562 and HEL cell lines were cultured in Iscove's modified Dulbecco's medium (IMDM, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD, USA) 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (BioWhittaker). UT-7/TPO (kindly provided by Kenneth Kaushansky) was maintained in IMDM with 10% fetal calf serum and 5 ng/ml human TPO (PeproTech, Rocky Hill, NJ). SU6656 (CalBiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) and added to cells at a final concentration of 2.5 μM and 0.1% DMSO (dosage was determined based on titration vs. phenotypic effect). Primary human CD34+/CD38¹⁰ were isolated and maintained as previously described (Lannutti et al. (2003) Exp. Hematol 31:1268-74). After 10 days in culture, cytokines were removed by washing the cells 3 times, and the pellet was resuspended in serum-free media containing rhTPO (35 ng/mL). Flow cytometry: Cells were labeled with propidium iodide and nuclear ploidy was determined by flow cytometry as previously described (Lannutti et al. (2004) Blood 103:3736-43). Flow cytometric analysis after immuno-staining for surface expression of CD41, CD61, or an isotype-matched control antibody was performed as previously described (Lannutti et al. (2003) Exp. Hematol. 31:1268-74). Analysis was performed using a FACScan analyzer using CELLQuest software (Becton Dickinson, San Diego, CA). Cell viability and histological analysis: Cells were observed by inverted light microscopy, aliquots were stained with trypan blue and counted for total viable cells by hemocytometer. Results from each cell count was performed four times and were counted in triplicate. Cytospins were prepared and stained as previously described (Lannutti et al. (2003) Exp. Hematol. 31:1268-74). Western blotting and kinase assays: Whole cell lysates were analyzed on 10% polyacrylamide gels. Transfer to nitrocellulose, blocking, probing with antibodies, and chemiluminescence were performed as previously described (Lannutti et al. (2004) Blood 103:3736-43). Kinase reactions were performed at 30⁰C for 10 min in kinase buffer (20 mM HEPES, pH 7.6, 5 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 7.5 mM magnesium chloride, 200 μM ATP, 1 μg Histone H3 (Upstate Biotechnology, Lake Placid, NY)), and 1 μg of active Aurora B kinases (Upstate Biotechnology) in a total volume of 15 μl. Kinase reactions were terminated by the addition of 7 μL of 3x SDSPAGE loading buffer, separated by SDS-PAGE, blotted to nitrocellulose, and probed with anti-phosphohistone H3 (Upstate Biotechnology). Results and Discussion SU6656 induces polyploidization of transformed human MK cell lines: UT- 7/TPO is a human cell line, derived from the leukemic UT-7 line, that has characteristics of both megakaryocyte and erythroid cells. These cells proliferate but do not differentiate significantly in response to exogenous rhTPO (Komatsu et al. (1996) Blood 87:4552-60). It was found that the addition of SU6656 at a concentration of 2.5 μM, results in rapid terminal differentiation of UT-7/TPO cells. As early as 12 hours a dramatic shift in cells cycle was apparent with the majority of the cells shifted to 4N. After 24 hours 8N cells appeared, and by 48 hours, 16N were readily seen. Continued growth in the presence of SU6656 yielded higher ploidy states (32 and 64N). In contrast, cells grown in the presence of 0.1% DMSO only showed no change in ploidy. A similar effect on polyploidization was found when other cells with megakaryocytic potential (i.e. HEL and K562) were cultured in the presence of added SU6656. Of note, HEL and K562 cells do not require exogenous cytokines for proliferation. UT-7 /TPO cells undergo terminal differentiation in the presence of SU6656: In addition to polyploidization, morphological changes indicative of megakaryocytic maturation after prolonged exposure to TPO and SU6656 were found. Cells cultured longer than 48 hours displayed cellular enlargement and the appearance of proplatelet- like processes and platelet-sized particles. Interestingly, cell division ceased in SU6656, but there was no significant change in the apoptotic rate as measured by annexin V staining. The expression of MK-specifϊc markers was then evaluated using flow cytometry. The percentage of cells expressing glycoprotein lib (CD41) and glycoprotein HIa (CD61) increased significantly (by at least 25%) after 48 hrs in the presence of SU6656. These results suggest that SU6656 induces MK differentiation, as assessed by cell morphology and expression of specific differentiation markers. Furthermore, this represents a powerful in vitro model system to study polyploidization and cell cycle regulation. SU6656 induces polyploidization of expanded human bone marrow progenitors: To test the effects of SU6656 on primary cells, human undifferentiated hematopoietic progenitors (CD34⁺/CD38^l0) were isolated by flow cytometry from whole marrow. Purified CD34⁺/CD38^l0 cells were cultured in serum-free media supplemented with IL-3 (50 pg/mL), IL-6 (10 ng/mL), SCF (10 ng/mL), and TPO (50 ng/mL) (PeproTech, Rocky, NJ) to expand MKs as previously described (Lannutti et al. (2003) Exp. Hematol. 31:1268-74). After 10 days in culture, cytokines were removed by washing the cells 3 times, and cells were then resuspended in serum-free media. Half of the cells received TPO (35 ng/mL) + 0.1% DMSO and half were treated with TPO (35 ng/mL) + SU6656 (2.5 μM). Similar to the previous studies using cell lines, primary cells demonstrated an increase in the number of higher ploidy megakaryocytes as well as an increase in cell size when SU6656 was present. After 72 hours in culture in the presence of SU6656 and TPO, primary cells showed 8N and 16N cells, whereas in the presence of TPO alone, primary cells cycled between 2N and 4N only. Myelodysplastic syndrome (MDS) and SU6656: MDS is a relatively common cause of acquired thrombocytopenia and increases in incidence with advanced age. In many cases, thrombocytopenia results from ineffective thrombopoiesis and abnormal (i.e., dysplastic) MKs. Therefore, to test the hypothesis that SU6656 could improve MK differentiation, bone marrow cells were obtained from two individuals with confirmed MDS and were cultured under serum-free conditions with rhTPO (35 ng/mL) +/- SU6656 (2.5 μM). After 72 hrs incubation, polyploid cells (8N and 16N) and cells with increased size and MK morphology were substantially increased in the presence of SU6656. Of interest, Western blots of cultured MDS cell Iy sates using Lyn kinase-specific antibodies demonstrated constitutive activation of Lyn kinase, which was substantially decreased in the presence of SU6656. Finally, there were fewer small cells in the presence of SU6656, suggesting that SU6656 diminishes the growth rate of clonal malignant cells. In each of the above experiments the level of Lyn kinase activation was decreased in the presence of SU6656. A similar decrease in intracellular tyrosine phosphorylation was observed in cells grown in the presence of SU6656, as compared to previous results using the Src inhibitor PP2 (Lannutti et al. (2004) Blood 103:3736-43). Although SU6656 is clearly a potent inhibitor of the Src family kinases (Blake et al. 2000) MoI. Cell. Biol. 20:9018-27), studies were undertaken to determine if additional targets can be identified. No effect was found on Jak2, STAT3, and STAT5 tyrosine phosphorylation. However, the activity of Aurora kinase B was inhibited in vitro by as little as 50 nM SU6656. It is recognized that SU6656 may, in fact, have other actions beside SFK inhibition. Prior studies demonstrated that activation of the Src family kinases (SFKs) Lyn and Fyn may partially block MK development (Lannutti et al. (2003) Exp. Hematol. 31:1268-74; Lannutti et al. (2004) Blood 103:3736-43). It has been shown that SFK inhibitors PPl and PP2 as well as a dominant negative form of Lyn lead to increased proliferation, higher ploidy classes, and increased Erkl/2 activity (Lannutti et al. (2004) Blood 103:3736-43). Remarkably, when SU6656 (2.5 μM) was added to a megakaryocyte cell line, UT-7/TPO, the cells ceased cell division but continued to accumulate DNA by endomitosis. During this interval, CD41 and CD61 expression on the cell surface increased. Similar effects on polyploidization and MK differentiation were seen with expanded primary MKs, bone marrow from two patients with myelodysplastic syndrome, and other cell lines with MK potential. These data indicate that SU6656 may be used as a differentiation-inducing agent for MKs and is an important tool for understanding the molecular basis of MK endomitosis. The results presented above add to the growing application of protein kinase inhibitors as tools in treating cancer, inflammatory, neurodegenerative and cardiovascular diseases (Roginskaya et al. (1999) Leukemia 13:855-61; Luskova & Draper (2004) Curr. Pharm. Des. 10:1727-37; Wolfrum et al. (2004) Arterioscler. Thromb. Vase. Biol. 10:1842-7; Zhang et al. (2004) Am. J. Pathol. 165:843-53). Recently, it was demonstrated that Src family kinase (SFK) inhibitors are capable of blocking growth of leukemic cells, suggesting that targeted inhibition of SFKs may have a therapeutic role in human disease (Roginskaya et al. (1999) Leukemia 13:855-61). Currently, a number of studies are utilizing SU6656 to examine molecular aspects of signal transduction pathways (Luskova & Draper (2004) Curr. Pharm. Des. 10:1727-37; Talmor-Cohen et al. (2004) Reproduction 127:455-63; Tapia et al. (2003) J. Biol. Chem. 278:35220-30). This Example demonstrates that SU6656 may also be used to induce polyploidization and maturation of human leukemic cell lines and primary human bone marrow progenitors. These results demonstrate that SU6656 is an important tool for understanding the molecular basis of MK endomitosis and identifies SU6656 as candidate therapeutic agent for the treatment of blood and bone marrow disorders. EXAMPLE 2 This Example describes changes in gene expression in UT7/TPO cells treated with SU6656. To examine which genes are significantly up- and down-regulated in response to SU6656, UT7/TPO cells were grown in the presence of 2.5 μM of SU6656. Samples of the culture were withdrawn at 0, 6, 12, and 24 hours, total RNA was extracted, converted to double-stranded cDNA, biotin-labeled cRNA was transcribed, and hybridized to Affymetrix U133a human gene chips for analysis. Comparisons between the SU6656(+) and SU6656(-) cells revealed gene expression differences over the full range of signal intensity. The expression of GP II-a, cyclin Al, myosin heavy chain 13, PDGF-beta, DUSP4, tousled-like kinase, and CBFA1/Runx2 were more highly expressed in response to SU6656, whereas CDC25C, and the IL-6 receptor-alpha expression was reduced. Interestingly, many genes with increased expression were related to platelets and MK development, whereas genes involved in proliferation (CDC25C, cyclin B phosphatase) were down-regulated. This experimental approach helps to assess the overall function of SU6656 in MK development, as well as to identify a subset of signaling events that are essential for MK terminal differentiation and platelet production. In an effort to clarify the mechanism of the SU6656-induced megakaryocytic differentiation, changes in the level of cell cycle regulating molecules, including cyclins, Cdk, and Aurora kinase, were examined by Western blot analysis. These experiments demonstrated that cyclin D3 expression was unchanged. However, cyclin B levels began to gradually decrease after 12 hours, whereas Aurora B (a kinase involved in the regulation mitosis and cytokinesis) levels appear to slightly increase after 24 hours. EXAMPLE 3 This Example describes the generation of platelet-like fragments from megakaryocytic cell lines and human progenitor cells. As described in EXAMPLE 1, SU6656, results in polyploidization and differentiation of hematopoietic cell lines and primary bone marrow progenitors (Lannutti et al. (2004) Blood 103:3736-43; Lannutti et al. (2005) Blood 105(10):3875-8). This Example provides evidence that SU6656 also induces thrombopoiesis, including the formation of demarcation membranes and shedding of functional platelet-like fragments (PFs). Materials and Methods Cells and Cell Culture: Bone marrow cells were harvested from cadaveric organ donors. UT-7/TPO (kindly provided by N. Komatsu, Japan) cells were cultured and maintained as described in EXAMPLE 1. SU6656 (CalBiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) and added to cells at a final concentration of 2.5 μM and 0.1% DMSO (dosage based on titration vs. phenotypic effect). CD34⁺ CD38¹⁰ cells were selected from bone marrow and cultured in serum free conditions with a four-cytokine cocktail (IL-3, IL-6, SCF and TPO) as previously described (Shim et al. (2004) Exp. Hematol. 32:638-48; Guerriro et al. (1995) Blood 86:3725-36). After 10 days in culture, cytokines were removed by washing the cells 3 times, and the pellet was resuspended hi serum-free media (X Vivo- 10, BioWhittaker) containing either recombinant human thrombopoietrn (TPO 35 ng/mL) or replacement of the four-cytokine cocktail. Each of these cultures was further divided into two wells, one of which received SU6656 (2.5 μM final concentration) and the other received 0.1% DMSO. The cells were subsequently cultured for an additional six days and then analyzed. Isolation of platelet-like fragments (PFs): Cell morphology was monitored daily by inverted light microscopy to determine maximal PF formation. Cells were harvested after six days by differential centrifugation (20Og x 10 minutes to sediment the intact cells; supernatant, containing PFs, was then centrifuged at 150Og x 10 minutes to concentrate the fragments). The concentrated PFs were suspended in 2-5ml of IX PBS and subjected to a discontinuous gradient albumin column (2% and 4%) as previously described (Drachman et al. (1997) Blood 89:483-92; Drachman et al. (1999) Methods 17:238-49). After 90 minutes at room temperature, cell fractions were collected, examined under a light microscope, and pooled based on cell size and morphology. The fractions were concentrated by centrifugation and were resuspended in human AB plasma (60% plasma, 40% IXPBS). Aggregation studies: Aggregation analysis was performed on PACKS4 aggregometer (Helena Labs, Beaumont, TX). Aggregation was initiated in vitro by adding ADP (20.6 μM), collagen (10 μg/mL), epinephrine (55 μM), arachidonic acid (0.5 mg/mL), or normal saline (control) to cell fractions that were stirred continuously. Aggregation was measured spectrophotometrically as a function of light transmission, which was plotted vs. time. Electron Microscopy: PFs, including the remaining large cells generated from human CD34⁺ CD38¹⁰ cells, were harvested after three and six days in culture in the presence of SU6656 or DMSO. These cells were examined by electron microscopy as previously described (Luthi et al. (2003) Exp. Hematol. 31:150-8). Results and Discussion UT-7/TPO cells retain characteristics of both MK and erythroid cells but fail to differentiate into recognizable cells of either lineage under normal growth conditions. UT-7/TPO is a sub-line developed from UT-7 based on the ability to proliferate in response to TPO as well as IL3 and GM-CSF (Komatsu et al. (1996) Blood 87:4552-60). As described in EXAMPLE 1, when SU6656 was added to MK cell lines (UT-7/TPO cells in TPO, HEL cells, and K562 cells) or CD34+ CD38¹⁰ human primary cells, a dramatic shift in cell cycle progression occurs, resulting in higher ploidy states as early as 12 hours after culture. In addition, there was increased cytoplasmic fragmentation and increased surface expression of CD41 and CD61. UT-7/TPO derived PFs aggregate in the presence of platelet specific aggregating agents: Cellular fractions, including PFs isolated from UT-7/TPO cells, were studied as described above. It was found that the PFs in the presence of SU6656 do not undergo spontaneous activation but have rapid (within 15-30 seconds) and sustained (for five to more than 10 minutes) aggregation in response to each of the standard agonists. Furthermore, they demonstrate higher amplitude and more prolonged aggregation in each of three experiments compared to the PFs generated under control conditions (0.1% DMSO). Although PFs can be isolated from UT-7/TPO cells with aggregation properties similar to platelets, electron microscopy demonstrates that these fragments do not have the usual structure of normal platelets (i.e., dense granules and circumferential tubulin ring). Nonetheless, making PFs from cell lines is worth pursuing for several reasons. First, cells are readily available and can be maintained and expanded in culture indefinitely. Second, differentiation is inducible in a single-step synchronous manner upon addition of SU6656. However, PFs derived from a leukemic cell line may not have similar survival, biologic function, and immunogenicity compared to normal platelets. Furthermore, gamma-irradiation would be required to avoid inadvertent transmission of intact leukemic cells. SU6656 induces terminal differentiation of expanded MKs fi'om human bone marrow: Culture-derived MKs obtained from human CD34⁺ CD38^1D cells were studied as described above. Reduced proliferative rate and dramatic increases in nuclear ploidy and cell size were seen in the presence of the SU6656 but not with TPO alone or the four- cytokine cocktail. Furthermore, these cells gave rise to abundant PFs that undergo agonist-induced aggregation under physiological conditions. Electron microscopic examination of CD34⁺ CD38^lσ cells after 10-day culture in the four-cytokine cocktail demonstrated that most cells have characteristics of immature MKs. After 3 days in growth factor alone there was marked apoptosis and cell death compared to the cells with cytokine plus SU6656. In addition, there was increased cytoplasmic maturation (demarcation membranes) after 3 days exposure to SU6656. These changes were more pronounced in combination with TPO rather than the four- cytokine cocktail. By day six, electron microscopy identified abundant dense storage vesicles, glycogen, and platelet-like particles. Once again, these changes were better developed in the presence of TPO than the four-cytokine cocktail. In contrast, cells in the presence of vehicle (DMSO) were smaller in size with smaller nuclei and absence of demarcation membranes. These results show that SU6656 induces terminal differentiation and thrombopoiesis of primary hematopoietic precursor cells as well as a MK cell line (UT- 7/TPO). The PFs released by these cells aggregate like platelets, indicating that thrombocytes may be produced in vitro. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for promoting the differentiation of megakaryocytes, comprising the step of contacting megakaryocyte precursors with an amount of a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for promoting the differentiation of megakaryocytes.

2. The method of Claim 1, wherein the compound is 2-oxo-3-(4,5,6,7- tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole-5-sulfonic acid dimethylamide.

3. The method of Claim 1, wherein the megakaryocyte progenitors are immortalized hematopoietic cells.

4. The method of Claim 1, wherein the megakaryocyte progenitors are immature hematopoietic cells isolated from a mammal.

5. The methods of Claim 1, wherein the megakaryocyte progenitors are promoted to differentiate into platelets or platelet-like fragments.

6. A method for reducing the number of abnormal or malignant cells in bone marrow, comprising the step of contacting bone marrow cells with an amount of a 3- (cyclohexanoheteroarylidenyl)-2-indolinone compound effective for reducing the number of abnormal or malignant cells.

7. The method of Claim 6, wherein the compound is 2-oxo-3-(4,5,6,7- tetrahydro-lH-indol-2-ylmethylene)-2,3-dihydro-lH-indole-5-sulfonic acid dimethylamide.

8. A method for treating blood and bone marrow disorders, comprising the step of administering to a subject suffering from a blood or bone marrow disorder an amount of a pharmaceutical composition comprising a 3-(cyclohexanoheteroarylidenyl)- 2-indolinone compound therapeutically effective for treating the blood or bone marrow disorder.

9. The method of Claim 8, wherein the compound is 2-oxo-3-(4,5,6,7- tetrahydro- 1 H-indol-2-ylmethylene)-2,3 -dihydro- 1 H-indole-5-sulfonic acid dimethylamide.

10. The method of Claim 8, wherein the blood or bone marrow disorder is one of thrombocytopenia, myelodysplastic syndrome, leukemia, essential thrombocytosis, and myeloproliferative disease.

11. The use of a 3-(cyclohexanoheteroarylidenyl)-2-indolmone compound in the manufacture of a medicament for the treatment of a blood or bone marrow disorder.

12. The use of Claim 11, wherein the compound is 2-oxo-3-(4,5.,6,7- tetrahydro- 1 H-indol-2-ylmethylene)-2,3-dihydro- 1 H-indole-5-sulfonic acid dimethylamide.