WO2000038519A1 - 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy - Google Patents
3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy Download PDFInfo
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- WO2000038519A1 WO2000038519A1 PCT/US1999/031232 US9931232W WO0038519A1 WO 2000038519 A1 WO2000038519 A1 WO 2000038519A1 US 9931232 W US9931232 W US 9931232W WO 0038519 A1 WO0038519 A1 WO 0038519A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates generally to chemistry, biochemistry, pharmacology, medicine and cancer treatment. More particularly, it relates to 3-heteroarylidenyl-2- indolinone compounds that modulate the activity of protein kinases (PKs) and to methods for their use in treating disorders related to abnormal protein kinase activity including cancer wherein combinations of the compounds with other chemotherapeutic agents are used.
- PKs protein kinases
- PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins.
- the consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another depend on PK activity.
- abnormal PK activity has been related to a host of disorders, ranging from relatively non-life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer) .
- the PKs can conveniently be broken down into two classes, the protein tyrosine kinases (PTKs) and the serine- threonine kinases (STKs) .
- PTKs protein tyrosine kinases
- STKs serine- threonine kinases
- One of the prime aspects of PK activity is involvement with growth factor receptors.
- Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, growth factor receptors are converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor as well as other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules.
- RTKs receptor tyrosine kinases
- RTK subfamily consists of insulin receptor (IR) , insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR) .
- IR and IGF-1R interact with insulin, IGF-I and IGF-II to form a heterotetramer composed of two entirely extracellular glycosylated ⁇ subunits and two ⁇ subunits which cross the cell membrane and which contain the tyrosine kinase domain.
- a third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFR ⁇ , PDGFR ⁇ , CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences .
- PDGFR platelet derived growth factor receptor
- PDGFR subfamily is sometimes subsumed in the later group, is the fetus liver kinase ("flk”) receptor subfamily. This group is believed to be composed of kinase insert domain- receptor fetal liver kinase-1 (KDR/FLK-1) , flk-lR, flk-4 and fms-like tyrosine kinase 1 (flt-1) .
- FGF fibroblast growth factor
- This group consists of four receptors, FGFR1 - FGFR4, and seven ligands, FGF1 - FGF7. While not yet well characterized, it appears that the receptors also consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the PTK sequence is interrupted by regions of unrelated amino acid sequences.
- CTK non-receptor tyrosine kinases
- cellular tyrosine kinases a family of entirely intracellular PTKs. This latter designation, abbreviated “CTK”, will be used herein. CTKs do not contain extracellular and transmembrane domains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified.
- the Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
- Src Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
- STKs serine-threonine kinases
- STKs are the most common of the cytosolic kinases; i.e., kinases which perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton.
- the cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes .
- RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer.
- PTKs pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune disease and a variety of renal disorders.
- PK regulated functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto- oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs) , cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
- RTKs transmembrane growth factor PTK receptors
- CTKs cytoplasmic PTKs
- STKs cytosolic STKs
- Cancer continues to be one of the leading causes of death in human beings.
- the majority of cancers are solid tumor cancers such as, without limitation, ovarian cancer, colorectal cancer, brain cancer, liver cancer, kidney cancer, stomach cancer, prostate cancer, lung cancer, thyroid cancer, Kaposi ' s sarcoma and skin cancer.
- colorectal cancer is a particularly common malignancy; adenocarcinoma of the large bowel affects about one person in 20 in the United States and in most Westernized countries. In the United States, colorectal cancer represents about 15% of all newly diagnosed cancers. While colorectal cancer is the third leading cause of cancer-related death, prognosis and outcome is highly dependent on the stage the disease at diagnosis.
- colorectal cancer is highly curable using a multidisciplinary treatment regime. Nevertheless, 20 - 25% of patients diagnosed with the disease will present with metastases or will develop locally recurrent or metastatic disease; the majority of these patients will eventually die of the disease.
- the primary modes of treatment of solid tumor cancers are surgery, radiation therapy and chemotherapy, separately and in combination.
- any further growth does require neovascularization. That is, for tumors to grow beyond 3 to 4 mm 3 in volume, new blood vessel growth; i.e., angiogenesis, the sprouting of new capillaries from existing blood vessels, must occur.
- angiogenesis the sprouting of new capillaries from existing blood vessels.
- immunohistochemical analysis of tumor sections from the margins of growing tumors show a preponderance of blood vessels, irrespective of tumor type.
- angiogenic factors are released from hypoxic tumor cells and migrate to nearby blood vessel endothelial cells, activating these cells to undergo morphologic changes, to move and to divide. Tumors that lack adequate vasculature become necrotic (Brem, S., et al., Cancer Res., 1976, 36, 2807-12) and/or apoptotic
- VEGF vascular endothelial growth factor
- vascular permeability factor potency of VEGF is some 50,000 times higher than that of histamine which is a well-known vascular permeabilizing molecule (Dvorak, H. F., et al . , Am. J. Path., 1995, 146:1029-39).
- This increased permeability results in extravasion of macromolecules such as fibrogen from the circulation which provides a fibrin gel meshwork or substratum for the migration and organization of endothelial cells as well as tumor cells (Kumar, H., et al . , Clin. Cancer Res., 1998, 4:1279-85.
- VEGF expression has been demonstrated in vitro in a number of human cancer cells lines and surgically in resected tumors of the gastrointestinal tract, ovary, brain, breast and kidney (Thomas, K. A., J. Biol. Chem., 1996, 271:603-6).
- VEGF has also been closely associated with the development of colorectal cancer; i.e, increased levels of VEGF have been found in tumor tissue from patients with colorectal cancer. In fact, a strong correlation has been observed between the increases VEGF and the stage and depth of intestinal wall invasion (C. Barone, et al . , Brit. J. Cancer, 1998, 78 (6) : 765-70) . Consistent with this result is the finding that serum levels of VEGF correlate significantly with Dukes stage and carcinoembryotic antigen levels and that patients with hepatic and/or lymph node metastases tend to show higher serum VEGF levels than those patients without such metastases (Fujisaki, K. , et al . , Am.
- An additional advantage to such a method might be that the use of an angiogenic inhibitor that reduces the ability of the tumor to develop new blood vessels and thus would be cytostatic rather than cytotoxic may compliment standard cytotoxic chemotherapy; that is, utilize different mechanisms of action to increase the efficacy of the cytotoxic agent without additional toxicity.
- PKs protein kinases
- RTKs receptor tyrosine kinases
- CTKs cellular tyrosine kinases
- STKs serine-threonine tyrosine kinases
- Flk-1 is believed to play a critical role in angiogenesis and that that role is mediated by VEGF.
- 3-heteroarylidenyl-2- indolinones should be capable of inhibiting VEGF-mediated vascularization, and thereby the growth, of tumors during the period when no chemotherapeutic agent, such as, without limitation, a fluorinated pyrimidine, is being administered to a patient and thus should increase the efficacy of the chemotherapeutic agent.
- chemotherapeutic agent such as, without limitation, a fluorinated pyrimidine
- the present invention relates to a 3-heteroarylidenyl-2-indolinone compound that inhibits angiogenesis or vasculogenesis in a cell, the compound having the chemical structure:
- R x is H or alkyl
- R 2 is 0 or S
- R 3 is hydrogen
- R 4 , R 5 , R 6 , and R 7 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl , aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl , S(0)R, S0 2 NRR', S0 3 R, SR, N0 2 , NRR ⁇ OH, CN, C(0)R, OC(0)R, (CH 2 ) n C0 2 R, and CONRR ' ;
- A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3- triazole, 1 , 2 , 4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1 , 2 , 3 -oxadiazole, 1,2, 4 -oxadiazole, 1, 2 , 5-oxadiazole, 1, 3 , 4-oxadiazole, 1, 2, 3 , 4-oxatriazole, 1 , 2 , 3 , 5-oxatriazole, 1, 2 , 3 -thiadiazole, 1,2, 4-thiadiazole, 1 , 2 , 5-thiadiazole, 1, 3 , 4-thiadiazole, 1, 2 , 3 , 4-thiatriazole, 1, 2 , 3 , 5-thiatriazole, and tetrazol
- R and R 1 are independently selected from the group consisting of alkyl or aryl .
- Alkyl refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon.
- the alkyl group has 1 to 12 carbons. More preferably, it has from 1 to 7 carbons and most preferably, it is a lower alkyl having from
- Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl , hexyl, and the like.
- Alkenyl refers to an alkyl group containing at least one carbon-carbon double bond.
- Alkynyl refers to an alkyl group containing at least one carbon- carbon triple bond.
- Alkoxy refers to an "-Oalkyl” group wherein the alkyl group may be optionally substituted with one or more halo groups .
- Aryl refers to a group having at least one aromatic ring structure; that is, a one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups. The aryl group may be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SR, nitro, cyano, alkoxy, alkyl and NRR'.
- Alkaryl refers to an alkyl that is covalently joined to an aryl group. Preferably, the alkyl is an unsubstituted lower alkyl .
- Carbocyclic aryl refers to an aryl group wherein the ring atoms are carbon.
- Heterocyclic aryl refers to an aryl group having from 1 to 3 heteroatoms as ring atoms, the remainder of the ring atoms being carbon. Heteroatoms include oxygen, sulfur, and nitrogen. The ring may be five-membered or six-membered. Examples of heterocyclic aryl groups include furanyl , thienyl, pyridyl, pyrrolyl , N-alkylpyrrolyl , pyrimidyl, pyrazinyl, imidazolyl and the like.
- Amide refers to -C(0)NHR a , where R a is alkyl, aryl, alkylaryl or hydrogen.
- Thioamide refers to -C(S)NHR a
- Amino refers to an NRR 1 group in which both R and R' are hydrogen .
- Thioether refers to an -SR b group wherein R b is alkyl, aryl or alkylaryl .
- Halogen refers to fluorinem chlorine, bromine or iodine .
- Physiologically acceptable salts and prodrugs of the 3- heteroarylidenyl-2-indolinones are also within the scope of this invention.
- physiologically acceptable salt refers to a salt that is non-deleterious to the physical well-being of a patient to whom it is administered.
- the physiologically acceptable salts which the compounds of this invention may form include 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 wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid.
- Salts in which a compound of this invention 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 compound with an appropriate base (e.g. sodium hydroxide (NaOH) , potassium hydroxide (KOH) , Calcium hydroxide (Ca(OH)2) , etc.) .
- a “prodrug” refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
- prodrug a compound of the present invention which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial .
- prodrug might be a short polypeptide bonded to a carboxy group wherein metabolic removal of the polypeptide group releases the active compound .
- the 3 -heteroarylidenyl -2-indolinone compounds of this invention may exist as the E or the Z isomers of a combination thereof. All of these configurations are within the scope of this invention. In preferred embodiments of this invention, the 3-heteroarylidenyl-2-indolinones are predominantly (greater than 90%) the Z-isomer.
- inhibit is meant eliminate, reduce, contain, impede, prevent, slow, retard and/or restrict. In a presently preferred embodiment of this invention, inhibit refers to the inhibition of angiogenesis or vasculogenesis .
- angiogenesis activity is meant the formation of new blood vessels in a tissue.
- the 3 -heteroarylidenyl -2-indolinone compound of this invention is 3- [4- (2-carboxyethyl-3 , 5- dimethylpyrrol -2 -yl) methylidenyl] -2-indolinone (Structure 1) .
- the 3 -heteroarylidenyl -2-indolinone is 3- [(2,4- dimethylpyrrol -5-yl) methylidenyl] -2-indolinone (Structure 2) in yet another aspect of this invention.
- a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of another chemotherapeutic agent and a therapeutically effective amount of a 3 -heteroarylidenyl -2- indolinone wherein the 3-heteroarylidenyl-2-indolinone has the chemical structure:
- R l t R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and A are the same as set forth above .
- the chemotherapeutic agent is a fluorinated pyrimidine.
- physiologically acceptable salts or prodrugs of the 3 -heteroarylidenyl -2 -indolines are within the scope of this combination chemotherapy aspect of the present invention.
- method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
- treating simply means that the life expectancy of an individual affected with a cancer will be increased, that one or more of the symptoms of the disease will be reduced and/or that quality of life will be enhanced.
- administer refers to the delivery of a compound, salt or prodrug of the present invention or of a pharmaceutical composition containing a compound, salt or prodrug of this invention to a patient for the purpose of treatment of cancer or the prevention or treatment of a PK-related disorder.
- “Comprising” as used herein in connection with “administering” is intended to mean that drugs being administered pursuant to the present invention may be administered as simply a combination of a 3 -heteroarylidenyl-2- indolinone compound and a chemotherapeutic agent alone or may be expanded to include additional drugs, such as, when the chemotherapeutic agent is a fluorinated pyrimidine, leucovorin, which are known or expected to offer additional beneficial characteristics to the combination.
- a “therapeutically effective amount” refers to that amount of a drug or its metabolite which is effective to prevent, alleviate, reduce or ameliorate symptoms of disease or prolong the survival of the patient being treated.
- a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of (or preferably eliminating) the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or preferably eliminating) one or more symptoms associated with the cancer.
- a "therapeutically effective amount" of a chemotherapeutic agent is meant any amount administered in any manner and in any treatment regime as may be currently recognized in the medical arts or as may come about as the result of future developments regarding the use of these agents .
- the chemotherapeutic agent is a fluorinated pyrimidine, in particular, fluorouracil, and the treatment regimes are those known in the chemotherapeutic art for the administation of fluorouracil .
- a “treatment regime” refers to specific quantities of selected chemotherapeutic agents (and, optionally, other agent such as the 3 -heteroarylidenly-2 -indolinone compound of this invention) administered at set times in a set manner over an established time period.
- a common treatment regime for treating colorectal cancer with fluorouracil/leucovorin comprises administering 425 mg/m 2 (milligrams per square meter of body surface area, a manner of measuring chemotherapeutic agent dosage well known to those skilled in the art) flourouracil plus 20 mg/m 2 leucovorin (specific quantities of selected agents) daily for 5 days (set times) by intravenous push (set manner) repeated at 4 to 5 week intervals (established time period) .
- Consecutive days means consectutive calendar days; i.e., Monday, Tuesday, Wednesday, etc.
- “Staggered” days means calendar days with other calendar days between them, e.g., without limitation, Monday, Wednesday, Saturday, etc.
- a “therapeutically effective amount of a 3-heteroarylidenyl-2-indolinone” refers to an amount of the compound sufficient to inhibit the growth, size and vascularization; i.e., angiogenesis and/or vasculogenesis, of tumors during the "recovery" periods, i.e., the periods in a treatment regime when no other chemotherapeuic agent is being administered to a patient.
- a “patient” refers to any higher organism which is susceptible to a PK related disorder including in particular cancer.
- patient refers to a mammal, especially a human being.
- Fluorinated pyrimidine chemotherapeutic agents are well known to those skilled in chemotherapeutic art; examples, without limitation, of fluorinated pyrimidines which may be used with the compounds of this invention include, without limitation, carmofur, doxifluridine, fluorouracil, floxuridine, tegafur, capecitabine and uracil - ftorafur (UFT) .
- the fluorinated pyrimidine chemotherapeutic agent is fluorouracil .
- the above method for the treatment of cancer also comprises leucovorin.
- the 3-heteroarylidenyl-2-indolinone used to treat cancer incombination with other chemotherapeutic agents is selected from the group consisting of 5-hydroxy-3 - [ (2 , 4- dimethylpyrrol-5-yl) methylidenyl] -2-indolinone (Structure 3), 4 -methyl -5- (2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -1H- pyrrole-2 -carboxylic acid (Structure 4), 4 -methyl -5- (2-oxo- 1, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-2-carboxylic acid methyl ester (Structure 5), 3- (5-hydroxymethyl-3 -methyl - lH-pyrrol-2-ylmethylene) -1, 3-dihydroindole-2-one (Structure 6) and 4-methyl-5- (2-oxo-l , 2-d
- the 3- heteroarylidenly-2 -indolinone compound used to treat cancer in combination with other chemotherapeutic agents is 3- [4- (2- carboxyethyl-3 , 5-dimethylpyrrol-2-yl) methylidenyl] -2 - indolinone (Structure 1) .
- the 3- heteroarylidenyl-2 -indolinone compound used to treat cancer in combination with other chemotherpeutic agents is 3- [(2,4- dimethylpyrrol -5-yl) methylidenyl] -2-indolinone (Structure 2).
- the cancer which may be treated using the above- described method may be selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, renal cancer, hepatic cancer, pancreatic cancer, bladder cancer, thyroid cancer, prostate cancer and colorectal cancer.
- Yet another aspect of this invention is a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of fluorouracil and a therapeutically effective amount of a compound selected from the group consisting of 3- [4- (2- carboxyethyl-3 , 5-dimethylpyrrol-2-yl) methylidenyl] -2- indolinone and 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2- indolinone .
- the cancer is colorectal cancer.
- the above method for the treatment of cancer includes the use of leucovorin.
- the therapeutically effective amount of fluorouracil comprises from about 300 to about 800 mg/m 2 , preferably from about 400 to about 500 mg/m 2 of the compound.
- the therapeutically effective amount of fluoruracil may be administered as an intravenous bolus injection or as a continuous intravenous infusion in yet another aspect of this invention.
- the therapeutically effective amount of 3- [(2,4- dimethylpyrrol-5-yl) -methylidenyl] -2-indolinone comprises from about 4 to about 190 mg/m 2 , preferrably from about 72 to 145 mg/m 2 of the compound.
- the therapeutically effective amount of leucovorin comprises from about 20 to about 500 mg/m 2 , preferrably from about 20 to about 200 mg/m 2 of the compound.
- a still further aspect of this invention is a treatment regime comprising the administration of from about 400 to about 500 mg/m 2 fluorouracil on one or more days, which may be consecutive or staggered, after which from about 72 to about 145 mg/m 2 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2- indolinone are administered on one or more days, which days likewise may be consecutive or staggered.
- 20 mg/m 2 leucovorin may also be adminstered on the days on which fluorouracil is administered.
- the above treatment regime is a four week treatment regime, fluorouracil (and, optionally, leucovorin) being administered as an intravenous bolus injection on days 1, 2, 3, 4 and 5 of the first week of the treatment regime while the 3- [(2,4- dimethylpyrrol -5 -yl) methylidenyl] -2-indolinone being administered as an intravenous bolus injection twice a week during weeks 2 , 3 and 4 of the treatment regime .
- Another aspect of this invention is a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of 3- [ (2 , 4 -dimethylpyrrol-5-yl) methylidenyl] -2-indolinone and a therapeutically effective amount of gemcitabine, another fluoropyrimidine compound.
- Gemcitabine has shown particular effectiveness in the treatment of advanced pancreatic cancer.
- chemotherapeutic agents e.g., paclitaxel, carboplatin, doxorubicin (in particular, liposomal doxorubicin) and topotecan
- gemcitabine has shown substantial activity against other refractory solid tumor cancers including advanced ovarian cancer, small cell lung cancer and kidney cancer.
- Another pyrimidine analog which should benefit from combination with 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2- indolinone is capecitabine which has shown effectiveness against metastatic breast cancer; such a combination is another aspect of this invention.
- the chemotherapeutic combination of 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2-indolinone with either of the pyrimidine chemotherapeutic agents 5-FU or UFT or derivatives, analogs or agents related thereto is an aspect of this invention.
- a further aspect of this invention is the combination of 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2-indolinone with carboplatin, oxaliplatin, cisplatin or related chemotherapeutic agents.
- Carboplatin and cisplatin are presently the pre-eminent drugs for the treatment of advanced ovarian cancer while oxaliplatin is a first-line chemotherapeutic agent in metastatic colorectal cancer.
- 3- [ (2, 4 -dimethylpyrrol-5-yl) methylidenyl] -2-indolinone in combination with carboplatin or cisplantin may permit a reduction in the amount of these two very toxic chemotherapeutic agents necessary to treat the cancer.
- combination of carboplatin or cisplatin with paclitaxel has shown promise in the treatment of ovarian cancer.
- 3- [ (2 , 4-dimethylpyrrol-S- yl) methylidenyl] -2-indolinone to this combination of chemotherapeutic agents could result in the same advantages discussed with regard to the above combinations.
- a presently preferred chemotherapeutic combination is comprised of 3- [ (2,4-dimethylpyrrol-5-yl)methylidenyl] -2-indolinone, cisplatin and gemcitabine.
- a further aspect of this invention is the combination of 3- [ (2 , 4-dimethylpyrrol-5-yl) methylidenyl] -2-indolinone with paclitaxel (taxol) , its synthetic analog docetaxel or polyglutamated taxanes.
- Paclitaxel has been approved by the FDA for the treatment of ovarian, breast, lung and AIDS- related cancers.
- Paclitaxel/docetaxel work by a different mechanism than the compounds of this invention, that is, they block a cell's ability to break down the mitotic spindle during mitosis.
- a still further aspect of this invention is the combination of 3- [ (2 , 4 -dimethylpyrrol -5 -yl) methylidenyl] -2- indolinone with thalidomide which is showing substantial chemotherapeutic utility particularly against refractory myelomas but also against glioblasoma multiforma, an extremely virulent brain cancer.
- Other cancer which may be responsive to this combination include prostate, breast and skin (Kaposi's sarcoma) cancers.
- An aspect of this invention is a chemotherapeutic combination of 3- [ (2 , 4 -dimethylpyrrol-5 -yl) methylidenyl] -2- indolinone with COX-2 inhibitors.
- the inhibition of cyclooxygenase-2 prevents production of factors that prompt angiogenesis.
- the combination would provide a two way attack on the vascularization essential to the vitality of cancer cells .
- a combination therapy consisting of 3- [(2,4- dimethylpyrrol-5-yl) methylidenyl] -2-indolinone and tamoxifen or derivatives thereof is as aspect of this invention.
- Tamoxifen interferes with the activity of estrogen which has been shown to promote the growth of breast cancer cells.
- the combination of 3- [ (2 , 4 -dimethylpyrrol-5-yl) methylidenyl] -2- indolinone, an anti-angiogenesis compound, with this "anti- estrogen” compound could provide a potent additional treatment for breast cancer.
- Another aspect of this invention is the combination of 3- [ (2 , 4 -dimethylpyrrol-5-yl) methylidenyl] -2-indolinone with leuprolide, a synthetic nonapeptide analog of naturally ocurring gonadotropin-releasing hormone that has demonstrated effectiveness particularly aginst testicular cancer but also against ovarian and breast cancer.
- Combination therapy using agents related to leuprolide is also contemplated by this invention. Again, a substantial benefit could be gained by combining the two different mode of action compounds.
- the chemotherapeutic combination of 3- [(2,4- dimethylpyrrol -5 -yl) methylidenyl] -2-indolinone with angiostatin, endostatin or similar chemotherapeutic agents, which inhibit angiogenesis by apoptosis, is likewise an aspect of this invention.
- Apoptosis is programmed cell death.
- the combination of cell-killing anti -angiogenesis with cell stasis anti-angiogenesis could be a powerful chemotherapeutic combination.
- MMPs having been shown to be involved in many disease states including cancer.
- MMP inhibitors such as, without limitation, AG3340, are showing tumoristatic efficacy againt solid tumor cancers such as non- small cell lung cancer and hormone-refractory prostate cancer.
- an angiogenesis inhibitor could provide a synergistic combination.
- Interferon alpha and its various subtypes (e.g., without limitation, interferons alpha A/2a, alpha/2b, alpha B2/alpha 8) are well-established chemotherapeutic agents against such cancers as hairy-cell leukemia, chronic myeloid leukemia, kidney cancer, melanoma, low grade lymphomas, multiple myeloma and Kaposi's sarcoma.
- a further aspect of this invention is the chemotherapeutic combination of 3- [ (2 , 4 -dimethylpyrrol -5- yl) methylidenyl] -2-indolinone with doxorubicin, daunorubicin and other anthracycline antineoplastic antibiotic, and derivatives and formulations thereof such as, without limitation, liposomal doxorubicin.
- Doxorubicin is widely used in the treatment of malignant lymphomas, leukemias, squamous cell cancer of the head and neck, breast cancer and thyroid cancer. Liposomal doxorubicin has been approved for the treatment of Kaposi's sarcoma.
- Tumor cells weakened by the anti-angiogenesis activity of 3 - [ (2 , 4-dimethylpyrrol-S- yl) methylidenyl] -2-indolinone could be much more susceptible to doxorubicin.
- Combination therapy using 3- [(2,4- dimethylpyrrol-5 -yl) methylidenyl] -2-indolinone and metoxantrone, a related chemotherapeutic agent, is specifically contemplated by this invention.
- chemotherapeutic combination which is an aspect of this invention is the combination of 3- [(2,4- dimethylpyrrol- 5 -yl) methylidenyl] -2-indolinone with estramustine and chemotherapeutic agents related thereto, which has shown particular utility in the treatment of refractory prostate cancer.
- Estramustine causes cell death by interferring with DNA synthesis.
- DNA synthesis disruption and anti- angiogenesis could provide a useful chemotherapeutic combination.
- Combination therapy using 3- [ (2 , 4-dimethylpyrrol-S- yl) methylidenyl] -2-indolinone and the vinca alkaloids including, without limitation, vincristine and vinblastine is also contemplated by the present invention.
- a further aspect of this invention is a 3- heteroarylidenyl-2-indolinone selected from the group consisting of 5 -hydroxy-3 -[ (2 , 4 -dimethylpyrrol -5- yl) methylidenyl] -2-indolinone (Structure 3), 4-methyl-5- (2- oxo-1 , 2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-2-carboxylic acid (Structure 4), 4-methyl-5- (2-oxo-l , 2-dihydroindol-3- ylidenemethyl) -lH-pyrrole-2 -carboxylic acid methyl ester (Structure 5), 3- (5-hydroxymethyl-3-methyl-lH-pyrrol-2- ylmethylene) -1, 3-dihydroindole-2-one (Structure 6) and 4- methyl-5- (2-oxo-l, 2 -dihydroindo
- this present invention relates to a method of modulating the catalytic activity of PKs comprising contacting the PK with a compound having one of the structures shown above.
- modulation refers to the alteration of the catalytic activity of RTKs, CTKs and STKs.
- modulating refers to the activation of the catalytic activity of RTKs, CTKs and STKs, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs.
- catalytic activity refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs.
- contacting refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent.
- Such "contacting” can be accomplished “in vitro,” i.e., in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with a compound in that environment.
- the ability of a particular compound to affect a PK related disorder i.e., the IC 50 of the compound, defined below, can be determined before use of the compounds in vivo with more complex living organisms is attempted.
- a PK related disorder i.e., the IC 50 of the compound, defined below.
- the above-referenced PK is selected from the group consisting of an RTK, a CTK or an STK in another aspect of this invention.
- the receptor protein kinase whose catalytic activity is modulated by a compound of this invention is selected from the group consisting of EGF, HER2 , HER3 , HER4 , IR, IGF-1R, IRR, PDGFR ⁇ , PDGFR ⁇ , CSFIR, C-Kit, C-fms, Flk-IR, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
- the cellular tyrosine kinase whose catalytic activity is modulated by a compound of this invention is selected from the group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
- this invention relates to a method for treating or preventing a PK-related disorder in a patient in need of such treatment comprising administering to the patient a therapeutically effective amount of one or more of the compounds described above .
- PK related disorder As used herein, "PK related disorder, " "PK driven disorder, “ and “abnormal PK activity” all refer to a condition characterized by inappropriate; i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs; (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth.
- Over- activity of a PK refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases) .
- Under-activity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK activity decreases.
- Treating refers to alleviating or abrogating the cause and/or the effects of a PK-related disorder.
- the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from acquiring a PK related disorder in the first place.
- the PK related disorder may be selected from the group consisting of an RTK, a CTK and an STK related disorder in a further aspect of this invention.
- the above referenced PK related disorder may be selected from the group consisting of an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder and a flk related disorder.
- the above referenced protein kinase related disorder is a cancer selected from the group consisting of squamous cell carcinoma, astrocytoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small -cell lung cancer, colorectal cancer, gastrointestinal cancer and glioma in a further aspect of this invention.
- the above referenced protein kinase related disorder is selected from the group consisting of diabetes, an autoimmune disorder, a hyperproliferation disorder, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, an inflammatory disorder and angiogenesis in yet another aspect of this invention.
- disorders which might be treated with compounds of this invention include, without limitation, immunological and cardiolovascular disorders such as, for instance aetherosclerosis .
- compositions of the above compounds are a further aspect of this invention.
- a “pharmaceutical composition” refers to a mixture of one or more of the compounds or drugs described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients.
- the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
- a “physiologically acceptable carrier” refers to a carrier or diluent that does not abrogate the biological activity and properties of the administered compound while facilitating administration by, for example, stabilizing or solubilizing the compound. Preferably, the carrier does not cause significant irritation to the organism.
- An “excipient” refers to a substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, surfactants and polyethylene glycols .
- Yet another aspect of this invention is a method for inhibiting tumorigenic activity in a cell comprising contacting the cell with a 3-heteroarylidenyl-2 -indolinone of this invention.
- Tumorgenic activity refers to both intracellular and extracellular biochemical activity which contributes to the formation of a neoplasm.
- a "neoplasm” is an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow even after the stimuli that initiated the new growth cease.
- a neoplasm partially or completely lacks structural organization and functional coordination with the normal tissue and usually forms a distinct mass of tissue. Such masses may be benign (benign tumors) or malignant (solid tumor cancer) .
- Malignant neoplasms are locally invasive and destructive and in many cases metastasize (spread to and invade and destroy tissues in areas of the affected organism remote from the site of origin) .
- the process of neoplasm formation is generally referred to as "neoplasia" ; i.e. neoplasia is the biochemical process by which a neoplasm forms and grows .
- malignant neoplasm neoplasm
- cancer tumor necrosis neoplasm
- tumor tumor necrosis neoplasm
- solid tumor cancer solid tumor cancer
- inhibitor refers to eliminating, reducing, containing, impeding, preventing, slowing, retarding and/or restricting neoplasia.
- chemotherapeutic agent refers to a chemical substance or drug used to treat a disease; the term is most often applied to such substances or drugs which are used primarily for the treatment of cancer.
- the PKs whose catalytic activity is modulated by the compounds of this invention include protein tyrosine kinases of which there are two types, receptor tyrosine kinases
- RTKs cellular tyrosine kinases
- CTKs cellular tyrosine kinases
- STKs serine- threonine kinases
- each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor.
- phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
- STKs being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation.
- PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism.
- Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis .
- PKs typically possess a bi- lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs.
- Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs .
- the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP. Specificity of a particular molecule for a particular PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs. Thus, different indolinone substituents may contribute to preferential binding to particular PKs.
- the ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site.
- the compounds disclosed herein may thus have utility as in vitro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins.
- the protein kinase is a protein tyrosine kinase, more particularly, a receptor protein tyrosine kinase.
- receptor protein tyrosine kinases whose catalytic activity can be modulated with a compound of this invention, or salt thereof, are, without limitation, EGF, HER2 , HER3 , HER4 , IR, IGF-1R, IRR, PDGFR ⁇ , PDGFR ⁇ , CSFIR, C-Kit, C-fms, Flk-IR, Flk4, KDR/Flk-1, Flt-1, FGFR-IR, FGFR-2R, FGFR-3R and FGFR-4R.
- the protein tyrosine kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or a prodrug thereof, can also be a non-receptor or cellular protein tyrosine kinase (CTK) .
- CTKs such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compound or salt of this invention.
- Still another group of PKs which may have their catalytic activity modulated by contact with a compound of this invention are the serine-threonine protein kinases such as, without limitation, CDK2 and Raf .
- this invention relates to a method for treating or preventing a PK related disorder by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism.
- a pharmaceutical composition containing a compound of this invention or a salt or prodrug thereof is administered to an organism for the purpose of preventing or treating a PK related disorder.
- This invention is therefore directed to compounds which modulate PK signal transduction by affecting the enzymatic activity of RTKs, CTKs and/or STKs, thereby interfering with the signals transduced by such proteins. More particularly, the present invention is directed to compounds which modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinomas, sarcomas including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreatic cancers, colon cancers, blood cancers, lung cancers and bone cancers .
- cell proliferative disorders which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders.
- Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels) .
- vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive.
- Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.
- disorders related to the shrinkage, contraction or closing of blood vessels are also implicated and may be treated or prevented by the methods of this invention.
- Fibrotic disorders refer to the abnormal formation of extracellular matrices.
- fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders.
- Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar.
- An increased extracellular matrix resulting in a hepatic scar can also be caused by a viral infection such as hepatitis.
- Lipocytes appear to play a major role in hepatic cirrhosis.
- Other fibrotic disorders implicated include atherosclerosis.
- Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells.
- Mesangial proliferative disorders include various human renal diseases such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well as such disorders as thro botic microangiopathy syndromes, transplant rejection, and glomerulopathies.
- the RTK PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al . , 1993, Kidney International, 43:47S-54S.
- PKs have been associated with cell proliferative disorders.
- PKs such as, for example, members of the RTK family have been associated with the development of cancer.
- Some of these receptors like EGFR (Tuzi et al.,Br. J. Cancer, 1992, 63:227-233; Torp et al . , 1992, APMIS 100:713-719)
- HER2/neu Slamon et al . , Science, 1989, 244:707-712
- PDGF-R Kerabe et al .
- EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer.
- HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.
- PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer.
- the RTK c-met has also been associated with malignant tumor formation.
- c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas . Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease.
- Flk has likewise been associated with a broad spectrum of tumors including, without limitation, mammary, ovarian and lung tumors as well as gliomas such as glioblastoma.
- IGF-IR in addition to being implicated in nutritional support and in type- II diabetes, has also been associated with several types of cancers.
- IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al . , J. Clin. Invest., 1989, 84:1418-1423) and small lung tumor cells (Macauley et al . , Cancer Res., 1989, 50:2511-2517).
- IGF- I while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas.
- Sandberg-Nordqvist et al is an autocrine stimulator of human gliomas.
- IGF-IR insulin growth factor-IR
- fibroblasts epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stem cells of the bone marrow)
- IGF-I insulin growth factor-I
- Goldring and Goldring Eukaryotic Gene Expression, 1991, 1:301-326.
- Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies.
- RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration and hemangiomas .
- diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration and hemangiomas .
- EGFR has been indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-IR are indicated in type-II diabetes mellitus.
- a more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., DN&P, 1994, 7:334-339.
- CTKs including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al . , FASEB J. , 1993, 6:3403- 3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed.
- mutated src v-src
- pp60 v"src oncoprotein
- pp60 c ⁇ src transmits oncogenic signals of many receptors.
- Over- expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60 c"src , which is characteristic of malignant cells but absent in normal cells.
- mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c- src in osteoclast function and a possible involvement in related disorders.
- Zap70 has been implicated in T-cell signaling which may relate to autoimmune disorders.
- STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis .
- PKs have also been implicated in embryo implantation.
- the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents.
- a method for identifying a chemical compound that modulates the catalytic activity of one or more of the above discussed protein kinases is another aspect of this invention.
- the method involves contacting cells expressing the desired protein kinase with a compound of this invention
- VEGF vascular endothelial growth factor
- Flk-1/KDR angiogenesis and Colorectal Cancer
- Tumor cells stimulate quiescent endothelial cells to divide and form new blood vessels by releasing growth factors, which bind to nearby endothelial cells (a paracrine mode of action) . Binding of vascular endothelial growth factor ("VEGF”) to one of its receptors begins the signaling cascade that regulates cellular events involved in new blood vessel formation.
- VEGF vascular endothelial growth factor
- VEGF vascular endothelial growth factor
- Flk-1 have also been implicated in pathological angiogenesis to support the growth of many solid tumors, including gliomas, breast cancer, bladder cancer, colon carcinoma and other gastrointestinal tract cancers.
- a correlation has been observed between VEGF expression and vessel density in breast tumors, renal cell carcinoma and colon cancer.
- transcripts for VEGF and its receptors were identified by in situ hybridization; transcripts were not detected in the less vascular, low grade gliomas or in normal brain tissue.
- Flk-1 receptors were detected in the endothelial cells of the vessels while VEGF localized to the tumor cells. Expression of VEGF and its receptors has been shown for hematopoietic tumor cell lines including multiple myeloma.
- VEGF is mitogenic for endothelial cells in vitro.
- neutralizing antibodies against Flk-1 inhibit mitogenesis.
- ribozymes that cleave flk-1 or flt-1 mRNAs reduce the growth of human microvasculature endothelial cells, presumably by decreasing the number of receptors on the cells.
- a variety of in vivo techniques have been used to investigate the role of VEGF signaling in tumor angiogenesis.
- Flk-1 receptors which lack the intracellular kinase domain block the activation of the endogenous Flk-1 receptor activity in cultured cells, inhibiting the growth of tumors implanted subcutaneously into nude mice. Any tumors that did form in this animal model contained significantly reduced vessel density. Also, reduction in VEGF expression with antisense constructs inhibits the growth of C6 rat glioma cells in nude mice with concurrent reduced blood vessel density in these tumors and inhibits the growth of human melanoma cells in nude/SCID mice.
- VEGF vascular endothelial growth factor
- 3- [ (2, 4 -dimethylpyrrol- 5 -yl) methylidenyl] -2-indolinone inhibits in vitro proliferation of endothelial cells induced by VEGF with IC 50 values of approximately 0.07 ⁇ M.
- 3- [ (2, 4-dimethylpyrrol-5-yl)methylidenyl] -2-indolinone exerts a time-dependent increase in potency, with detectable activity first observed after a 5-minute exposure to drug.
- One-hour exposure to 3- [ (2 , 4 -dimethylpyrrol -5 -yl) methylidenyl] -2- indolinone results in in vitro antiproliferative activity for 3 to 4 days thereafter.
- 3- [ (2 , 4-dimethylpyrrol-S- yl) methylidenyl] -2-indolinone has no direct inhibitory effects on a variety of tumor cell lines at concentrations up to 50 ⁇ M.
- 3- [ (2 , 4 -dimethylpyrrol -5- yl) methylidenyl] -2-indolinone in which a variety of tumor cell lines were subcutaneously implanted into immunocompromised mice, 3- [ (2 , 4-dimethylpyrrol-S- yl) methylidenyl] -2-indolinone demonstrates a significant suppression of tumor growth against a broad spectrum of tumor types whose growth are driven by various growth factors such as PDGF, EGF and Her2.
- Daily intraperitoneal dosing (ranging from 12.5 - 25 mg/kg/day for 28 days) resulted in 30-80% inhibition of tumor growth.
- Preliminary pharmacokinetic data from a Phase 1 study in patients with advanced malignancies in which patients were treated at doses between 4.4 - 190 mg/m 2 indicates that the drug has a half-life in humans of approximately 60 minutes.
- the alpha half-life is rapid, with a mean 5.8 ⁇ 1.9 minutes.
- the beta half-life or elimination phase has a mean value 43.4 ⁇ 21.9 minutes with a range from 10-111 minutes.
- AUC and C, ⁇ increase linearly with dose .
- the primary pathway for metabolism of 3- [(2,4- dimethylpyrrol-5-yl) methylidenyl] -2-indolinone is through sequential oxidation reactions of the 5 -methyl group on the pyrrol ring. Four metabolites are measurable in serum, all of which involve serial oxidations of this methyl group on the pyrrol ring.
- Fluorouracil and Fluorouracil/Leucovorin - The chemical structure of fluorouracil is 5-fluoro-2,4 (IH, 3H) -pyrimidinedione . While the precise mode of action of fluorouracil is not clear, the drug is thought to function as an antimetabolite in at least three ways. In one aspect, as its deoxyribonucleotide derivative, 5-fluoro-2 ' -deoxyuridine- 5 '-phosphate (F-dUMP) , the drug inhibits thymidylate synthetase which results in inhibition of methylation of deoxyuridylic acid to thymidylic acid. This, in turn, interferes with the synthesis of DNA.
- 5-fluoro-2 ' -deoxyuridine- 5 '-phosphate F-dUMP
- fluorouracil is found to be incorporated into RNA to a an extent which, although small, is sufficient to have a major effect on both the processing and functions of the RNA.
- fluorouracil has been shown to block uracil phosphatase thus inhibiting utilization of preformed uracil in RNA synthesis (Goodman and Gilman's, "The Pharmacological Basis of Therapeutics", 1985, pages 1268-1271) .
- Fluorouracil can be administered alone or in combination with other drugs.
- the most common combination involves the use of leucovorin (folinic acid) .
- Leucovorin potentiates the cytotoxic effect of fluorouracil by, it is thought, increasing the extracellular concentration of reduced folates which in turn appears to stabilize the covalent ternary complex formed by (F-dUMP) , 5 , 10 -methylenetetrahydrofolate and thymidine synthetase. The stabilization of this complex enhances inhibition of the synthetase, thereby increasing the efficacy of fluorouracil.
- chemotherapeutic combinations with fluorouracil for the treatment of advanced stage colorectal cancer include, without limitation, combination of fluorouracil with: methotrexate, alone (Blijham, G., et al . , J. Clin Oncol., 1996, 14 (8) : 2266-73) and in combination with leucovorin (Romero, A. 0., et al . , Am. J. Clin. Onocol . , 1998, 21(1): 94-8); interferon alfa-2a (Greco, F. A., et al . , J. Clin.
- Fluorouracil may be adminstered by either intravenous bolus injection or continuous infusion.
- the volume of distribution is slightly larger than the extracellular space.
- Intravenous bolus doses of 370 to 720 mg/m 2 produce an elimination half-life of 8 to 14 minutes with plasma levels below 1 ⁇ M within 2 hours, an approximate threshold for cytotoxic effects. Less than 10% of the drug is excreted in urine, while the balance is cleared through metabolic pathways.
- Frequently used administration schedules include short- bolus injections over three to five days every 3-4 weeks, continuous intravenous infusions of 96 - 120 hour duration every 4 weeks, and weekly infusions for six weeks out of every eight weeks.
- the incidence of serious clinical toxicity tends to increase with higher systemic exposure (for example, with higher steady- state plasma concentrations during constant infusions and higher AUC with bolus administration) .
- each of the above schedules of treatment includes substantial intervals during which no fluorouracil is administered. This is due primarily to the inherent toxicity of fluorouracil, which is exacerbated by the addition of leucovorin. Unfortunately, this time interval substantially reduces the efficacy of fluorouracil. That is, initial treatment of a patient with fluorouracil or fluorouracil/leucovorin produces about a three log unit (three orders of magnitude or 1000-fold) reduction in tumor number and size. However, during the no-treatment "recovery" period, tumor number and size rebound to the extent of about two log units (100 -fold) .
- the overall effect of a course of treatment with fluorouracil is only about one log unit (an approximately 10-fold decrease in tumor number and size) per administration of fluorouracil.
- prolonged treatment with fluorouracil cause a problem with regard to cost of treatment, patient quality of life, etc., it can result in secondary resistance to the drug.
- the methods of this invention are directed to maintaining a more substantial portion of the effect of each administration of fluorouracil during the recovery period. Subsequent administrations in the full course of treatment will thus be confronted with tumors of reduced size and number, thus improving the overall effectiveness of fluorouracil.
- intensive-course fluorouracil plus low-dose leucovorin fluorouracil 425 mg/m 2 plus leucovorin 20 mg/m 2 intravenous [IV] push daily for 5 days with courses repeated at 4- to 5- week intervals
- a compound of the present invention, a prodrug thereof or a physiologically acceptable salt of either the compound or its prodrug can be administered as such to a human patient or it can be administered in pharmaceutical compositions in which the foregoing materials are mixed with suitable carriers or excipient (s) .
- suitable carriers or excipient s
- Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
- the preferred routes of administration are oral and parenteral .
- one may administer the compound in a local rather than systemic manner for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
- one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
- compositions of the present invention 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.
- compositions for use in accordance with the present invention 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.
- the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
- physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- the compounds can be formulated 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, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient.
- compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores.
- Useful 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 and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
- disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.
- Dragee cores are provided with suitable coatings.
- suitable coatings may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene giycol, 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 .
- compositions which can be used orally 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 can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers.
- the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also.
- the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane or carbon dioxide.
- a suitable propellant e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane or carbon dioxide.
- the dosage unit may be controlled by providing a valve to deliver a metered amount.
- Capsules and cartridges of, for example, gelatin 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 also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g., 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 formulating materials such as suspending, stabilizing and/or dispersing agents.
- Pharmaceutical compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle.
- Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as 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 and/or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
- a suitable vehicle e.g., sterile, pyrogen-free water
- the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
- the compounds may also be formulated as depot preparations.
- Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
- a compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil) , with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.
- a non-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer and an aqueous phase such as the VPD co-solvent system.
- VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene giycol 300, made up to volume in absolute ethanol.
- the VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.
- This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
- the proportions of such a co- solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
- identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene giycol may be varied; other biocompatible polymers may replace polyethylene giycol, e . g. , polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
- hydrophobic pharmaceutical compounds may be employed.
- Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
- certain organic solvents such as dimethylsulfoxide also may be employed, although often at the cost of greater toxicity.
- the compounds may be delivered using a sustained-release system, such as semi -permeable matrices of solid hydrophobic polymers containing the therapeutic agent.
- sustained-release materials have been established and are well known by those skilled in the art.
- Sustained- release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
- additional strategies for protein stabilization may be employed.
- the pharmaceutical compositions herein also 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.
- This compound may be formulated as any of the compositions and formulations described above.
- a presently preferred formulation is comprised of 3- [(2,4- dimethylpyrrol- 5-yl) methylidenyl] -2-indolinone in sufficient sterile parenteral solution to afford a 4.5 mg/ml final concentration.
- Additional components of the formulation include polyethylene giycol 400; polyoxyl 35 castor oil (Cremophor ® ) ; benzyl alcohol and dehydrated alcohol. It should be noted that this formulation, since it contains Cremophor ® , is not compatible with standard PVC-lined syringes, intravenous bags and administration sets. Fluorouracil/Leucovorin composition
- Fluorouracil is commercially available in compositions and formulations which are known to those skilled in the chemotherapeutic art and may be administered in the methods of this invention as those compositions/formulations. Examples of such compositions/formulations are shown in the Package Insert which accompanies commercial fluorouracil and which is incorporated by reference as if fully set forth herein. The use of any other or different composition/formulation as such may be developed or become available in the future is also within the scope of this invention.
- leucovorin is also commercially available in compositions/formulations known to those in the chemotherapeutic art and may also be administered in the methods of this invention as those compositions/formulations. Examples of such compositions/formulations are shown in the Package Insert that accompanies commercial leucovorin and which is incorporated as if fully set forth herein. As above, any other or different composition/formulation as such may be developed or become available in the future is also within the scope of this invention. DOSAGE
- compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose; i.e., the modulation of PK activity or the treatment or prevention of a PK-related disorder.
- a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
- the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half -maximal inhibition of the PK activity) . Such information can then be used to more accurately determine useful doses in humans.
- Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC 50 and the LD 50 (both of which are discussed elsewhere herein) for a subject compound.
- the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can 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 species, which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs) .
- MECs minimal effective concentrations
- the MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. 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 that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
- the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval .
- the amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. 3- [ (2 , 4-dimethylpyrrol-5-yl)methylidenyl] -2-indolinone dosage.
- the compound may be administered in doses ranging from about 4 mg/m 2 to about 195 mg/m 2 .
- the dosage is between about 72.5 mg/m 2 and about 145 mg/m 2 .
- the dilution described in the above composition section may be administered to a patient at a rate of from about 50 to about 350 cc/hour.
- the rate is from about 150 to about 250 cc/hour. Most preferably, it is from about 175 to about 225 cc/hour.
- the 3- [(2,4- dimethylpyrrol- 5-yl) methylidenyl] -2-indolinone dose is administered during rest periods when no fluorouracil or fluorouracil/leucovorin is being administered to a patient.
- fluorouracil or fluorouracil/leucovorin may be administered in numerous treatment regimes, the choice of which is within the knowledge and expertise of the treating physician. Fluorouracil and Fluorouracil/Leucovorin dosage
- fluoruracil may be administered in doses ranging from about 300 mg/m 2 to about 800 mg/m 2
- schedules of fluorouracil which provide a dose intensity of approximately 400 -500 mg/m 2 /week are presently considered to be optimal therapy.
- leucovorin is included in the treatment, differences in clinical outcome for low and high dose leucovorin are minimal which, given the additional toxicity of the high dose regimen, the low dose regimen presently appears most appropriate.
- fluorouracil or fluorouracil/leucovorin may, within the scope of this invention, be administered in any presently approved manner or in any manner found in the future to be efficacious, given the above data
- a presently preferred embodiment of this invention is to administer fluorouracil at a dose of about 400 to 500 mg/m 2 as a bolus intravenous injection on day 1-5 of a 4 week cycle.
- the 4- week cycle may be repeated as necessary or until adverse side effects as recognized by the physician conducting the treatment are encountered.
- Leucovorin may be administered with the fluorouracil.
- Leucovorin may be administered in doses of from about 20 to about 500 mg/m 2 , preferably from about 20 to about 200 mg/m 2 and in a presently preferred embodiment of this invention as a low-dose administration of about 20 mg/m 2 , also as a bolus injection, with each administration of fluorouracil.
- the compounds when 3- [(2,4- dimethylpyrrol-5-yl) methylidenyl] -2-indolinone is administered in combination with fluorouracil or fluorouracil/leucovorin, the compounds may be administered simultaneously, sequentially, continuously, intermittantly, etc. in accordance with a treatment regime calculated to take maximum advantage of the characteristics of each of the components.
- 3- [(2,4- dimethylpyrrol- 5-yl) methylidenyl] -2-indolinone is administered on days when no fluorouracil or fluorouracil/leucovorin is administered.
- the above-described dose of 3- [ (2 , 4 -dimethylpyrrol -5-yl) methylidenyl] -2-indolinone is administered in any pattern desired; e.g., without limitation, on each day, every other day, every third day, etc. of a treatment regime selected for fluorouracil or fluorouracil/leucovorin on which fluorouracil or fluorouracil/leucovorin is not administered.
- the 3- [(2,4- dimethylpyrrol-5-yl) methylidenyl] -2-indolinone may be administered as a bolus intravenous injection or as a continuous intravenous infusion.
- 3- [ (2 , 4 -dimethylpyrrol -5yl) methylidenyl] -2- indolinone may be administered over a relatively short time period (5 to 30 minutes) and exert antiproliferative activity on the endothelial cells for 3 to 4 days thereafter.
- the in vivo data demonstrate that dosing with 3- [ (2 , 4 -dimethylpyrrol -5-yl) methylidenyl] -2-indolinone at 3 to 4 day intervals was sufficient to inhibit tumor growth without toxicity.
- no cumulative toxicity was observed in Phase I dose escalation studies in patients treated with up to 52 weeks of treatment.
- the indicated dose of 3- [ (2 , 4 -dimethylpyrrol -5-yl) methylidenyl] - 2-indolinone is administered twice weekly in weeks 2-4 of each four week treatment regime.
- 3- [ (2 , 4 -Dimethylpyrrol - 5-yl) methylene] -2-indolinone might be expected to work in combination with other chemotherapeutic agents as well.
- the combination of 3- [ (2 , 4-Dimethylpyrrol-5-yl) methylene] -2-indolinone with other alkylating agents might afford synergistic activity without concomitant increased toxicity.
- alkylating agents could include, without limitation, the alkyl sulfonates; e.g., busulfan (used for treatment of chronic granulocytic leukemia) , improsulfan and piposulfan; the aziridines; e.g., benzodepa, carboquone, meturedepa, and uredepa; the ethyleneimines and methylmelamines ; e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine and the nitrogen mustards; e.g., chlorambucil (used in treatment of chronic lymphocytic leukemia, primary macroglobulinemia and non-Hodgkin ' s lymphoma), cyclophosphamide (used in treatment of Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, Wilm's tumor and rhabdom
- 3 -[ (2 , 4 -Dimethylpyrrol -5-yl) methylene] -2- indolinone could have a beneficial effect in combination with other antimetabolite chemotherapeutic agents such as, without limitation, folic acid analogs (e.g., methotrexate (used in treating acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, neck and head and lung cancer, osteogenic sarcoma) and pteropterin) the purine analogs such as mercaptopurine and thioguanine which find use in the treatment of acute granulocytic, acute lymphocytic and chronic granulocytic leukemias) .
- folic acid analogs e.g., methotrexate (used in treating acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast, neck and head and lung cancer, osteogenic sarcoma) and
- 3- [ (2 , 4 -Dimethylpyrrol -5-yl) methylene] -2-indolinone could also prove effective in combination with natural product chemotherapeutic agents such as, without limitation, the vinca alkaloids (vinblastine (used for breast and testicular cancer) , vincristine, vindesine) , the epipodophylotoxins (etoposide, teniposide (both used in the treatment of testicular cancer and Kaposi ' s sarcoma)), the antibiotic chemotherapeutic agents (daunorubicin, doxorubicin, bleomycin, mitomycin (used for stomach, cervix, colon, breast, bladder and pancreatic cancer), dactinomycin, plicamycin, bleomycin (used for skin, esophagus and genitourinary tract cancer) and the enzymatic chemotherapeutic agents such as L-Asparaginase .
- the vinca alkaloids vinblastine (
- 3- [(2,4- Dimethylpyrrol-5-yl) methylene] -2-indolinone might also benefit the activity of chemotherapeutic agents such as platinum coordination complexes (cisplatin, etc.), substituted ureas (hyroxyurea) , methylhydrazine derivatives (procarbazine) , adrenocortical suppressants (mitotane, aminoglutethimide) as well as hormones and antagonists such as adrenocorticosteroids (prednisone) , progestins (hydroxyprogesterone caproate) , estrogens
- chemotherapeutic agents such as platinum coordination complexes (cisplatin, etc.), substituted ureas (hyroxyurea) , methylhydrazine derivatives (procarbazine) , adrenocortical suppressants (mitotane, aminoglutethimide) as well as hormones and antagonists such as adre
- compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
- the pack may for example comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration.
- Such notice for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
- compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like. 4. SYNTHESIS
- the compounds of this invention, as well as the precursor 2-oxindoles and aldehydes, may be readily synthesized using techniques well known in the chemical arts. It will be appreciated by those skilled in the art that other synthetic pathways for forming the compounds of the invention are available and that the following is offered by way of example and not limitation.
- Phosphorus oxychloride (0.186 mL, 1.44 mmol) was added dropwise to a solution of dimethyformamide (0.15 mL, 1.44 mmol) in dichloromethane (4mL) at 0 °C. The mixture was warmed to room temperature and stirred for 30 minutes and then cooled to 0° C. 4-Methyl-2-pyrrolecarboxylate methyl ester (100 mg, 0.72 mmol) was added portion-wise and the mixture was then stirred at 40-50° C for 4 hours. Sodium hydroxide (10% aqueous solution, 2 ml) was added and the reaction mixture was stirred for 30 minutes.
- Phosphorus oxychloride (0.66 mL, 7.2 mmol) was added dropwise to an ice-cold solution of dimethylforamide (0.6 mL, 7.2 mmol) in dichloromethane (30 mL) . The mixture was stirred at room temperature for 30 minutes and then cooled in an ice-bath. 4-methyl-2-pyrrolecarboxylate ethyl ester (919 mg, 6 mmol) was added slowly to the reaction mixture. The resulting reaction mixture was then stirred at room temperature for 2.4 hours. The mixture was then cooled in an ice-bath and 2N sodium hydroxide was added and the mixture stirred for 30 minutes.
- this invention relates to novel 3-heteroarylidenyl-2-indolinones demonstrating the ability to modulate RTK, CTK, and STK activity.
- the following assays are employed to select those compounds demonstrating the optimal degree of the desired activity.
- Assay Procedures The following in vitro assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the PKs. Similar assays can be designed along the same lines for any PK using techniques Well known in the art.
- the cellular/catalytic assays described herein are performed in an ELISA format.
- the general procedure is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. The cells are lysed and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody recognizing the substrate of the enzymatic phosphorylation reaction. Non-substrate components of the cell lysate are washed away and the amount of phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine compared with control cells that were not contacted with a test compound.
- the cellular/biologic assays described herein measure the amount of DNA made in response to activation of a test kinase, which is a general measure of a proliferative response.
- the general procedure for this assay is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of " time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine is added. The amount of labeled DNA is detected with either an anti -BrdU antibody or by measuring radioactivity and is compared to control cells not contacted with a test compound.
- Enzyme linked immunosorbent assays may be used to detect and measure the presence of PK activity.
- the ELISA may be conducted according to known protocols which are described in, for example, Voller, et al . , 1980, "Enzyme- Linked Immunosorbent Assay," In: Manual of Clinical
- the disclosed protocol may be adapted for determining activity with respect to a specific PK. That is, the preferred protocols for conducting the ELISA experiments for specific PKs is provided below. However, adaptation of these protocols for determining a compound's activity for other members of the RTK family, as well as for CTKs and STKs, is well within the scope of knowledge of those skilled in the art.
- An ELISA assay is conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1. Materials and Reagents. a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96) ; b. Cappel goat anti-rabbit IgG (catalog no. 55641) ; c. PBS (Gibco Catalog No. 450-1300EB) ; d.
- TBSW Buffer 50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20) ; e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4°C) ; f. HNTG buffer (20mM HEPES buffer (pH 7.5), 150mM
- L-glutamine Gibco (catalog no. 25030-016); o. VEGF, PeproTech, Inc. (catalog no. 100-20) (kept as 1 ⁇ g/100 ⁇ l stock in Milli-Q dH 2 0 and stored at -20° C; p. Affinity purified anti -FLK-1 antiserum; q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et al . , 1990, Cancer Research 50:1550-1558) ; r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011) ; s.
- media should be removed from the cells and washed one time with 200 ⁇ l/well PBS.
- HNTG formulation includes sodium ortho vanadate, sodium pyrophosphate and EDTA.
- EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells.
- HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described below: Materials and Reagents.
- EGF stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.
- b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain) .
- Anti-phosphotyrosine antibody (anti-Ptyr) polyclonal) (see, Fendley, et al . , supra) .
- Detection antibody Goat anti -rabbit IgG horseradish peroxidase conjugate, TAGO, Inc., Burlingame, CA. e . TBST buf fer :
- Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 ⁇ g per well in PBS, 100 ⁇ l final volume/well, and store overnight at 4°C. Coated plates are good for up to 10 days when stored at 4°C.
- An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and intracellular HER2 kinase domain can be used for this assay.
- 2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, at room temperature for 5 minutes . 3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable.
- EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 ⁇ l dilute EGF (1:12 dilution), 100 nM final concentration is attained.
- the maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.
- All cell culture media, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37°C.
- cells (U1242, obtained from Joseph Schlessinger, NYU) are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium, cells are changed to serum- free medium and treated with test compound for 2 hr in a 5%
- Cells are then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 V0 4 , 0.2% Triton X- 100, and 2 mM NaPyr) .
- HNTG 20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 V0 4 , 0.2% Triton X- 100, and 2 mM NaPyr
- Cell lysates (0.5 mg/well in PBS) are transferred to ELISA plates previously coated with receptor- specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X- 100) at room temperature for 30 min. Lysates are incubated with shaking for 1 hour at room temperature.
- the plates are washed with TBST four times and then incubated with polyclonal anti -phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody is removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody is added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times.
- the following protocol may be used to measure phosphotyrosine level on IGF-1 receptor, which indicates IGF- 1 receptor tyrosine kinase activity.
- Materials and Reagents a.
- the cell line used in this assay is 3T3/IGF-1R, a cell line genetically engineered to overexpresses IGF-1 receptor.
- b. NIH3T3/IGF-1R is grown in an incubator with 5% C0 2 at 37 °C. The growth media is DMEM + 10% FBS (heat inactivated) + 2mM L-glutamine.
- D-PBS D-PBS:
- Blocking Buffer TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk) . f . TBST buffer:
- ABTS solution should be kept in dark and 4°C. The solution should be discarded when it turns green.
- Hydrogen Peroxide 30% solution is kept in the dark and at 4°C.
- ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. Pat plate dry with paper towels.
- the cells grown in tissue culture dish (Corning 25020-100) to 80-90% confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO). 2. Resuspend the cells in fresh DMEM + 10% FBS +
- the drugs are tested under serum-free condition.
- Cone is 20 nM) , and incubate at 5% C0 2 at 37°C for 10 minutes . 5. Remove media and add 100 ⁇ l/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed.
- EGF Receptor kinase activity in cells genetically engineered to express human EGF-R can be measured as described below:
- UBI 05-101
- Anti-phosphotyosine antibody anti-Ptyr
- Detection antibody Goat anti-rabbit IgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, CA.
- TBST buf fer
- Coat ELISA plates (Corning, 96 well, Cat. #25805- 96) with 05-101 antibody at 0.5 ⁇ g per well in PBS, 150 ⁇ l final volume/well, and store overnight at 4°C. Coated plates are good for up to 10 days when stored at 4°C.
- NIH 3T3/C7 cell line (Honegger, et al . , Cell 51:199-209, 1987) can be use for this assay.
- EGF ligand dilute stock EGF in DMEM so that upon transfer of 10 ⁇ l dilute EGF (1:12 dilution), 25 nM final concentration is attained.
- HNTG* comprises: HNTG stock (2.0 ml), milli-Q
- Met Autophosphorylation Assay This assay determines Met tyrosine kinase activity by analyzing Met protein tyrosine kinase levels on the Met receptor. Reagents a. HNTG (5X stock solution): Dissolve 23.83 g " HEPES and 43.83 g NaCl in about 350 ml dH 2 0.
- This procedure can be performed the night before or immediately prior to the start of receptor capture.
- This assay is used to determine src protein kinase activity measuring phosphorylation of a biotinylated peptide as the readout .
- Yeast transformed with src (Sugen, Inc., Redwood City, California) .
- Cell lysates Yeast cells expressing src are pelleted, washed once with water, re-pelleted and stored at - 80°C until use.
- N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standard procedures well known to those skilled in the art .
- DMSO Sigma, St. Louis, MO.
- e. 96 Well ELISA Plate Corning 96 Well Easy Wash, Modified flat Bottom Plate, Corning Cat. #25805-96. f .
- Vecastain ELITE ABC reagent Vector, Burlingame, CA.
- Anti-src (327) mab Schizosaccharomyces Pombe is used to express recombinant Src (Superti-Furga, et al . , EMBO J., 12:2625-2634; Superti-Furga, et al . , Nature Biochem. , 14:600-605).
- Src Superti-Furga, et al . , EMBO J., 12:2625-2634; Superti-Furga, et al . , Nature Biochem. , 14:600-605
- Pombe strain SP200 (h-s leul.32 ura4 ade210) is grown as described and transformations are pRSP expression plasmids are done by the lithium acetate method (Superti- Furga, supra) . Cells are grown in the presence of 1 ⁇ M thiamine to repress expression from the nmtl promoter or in the absence of thiamine to induce expression.
- i Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be used instead) .
- Turbo TMB-ELISA peroxidase substrate Pierce Chemical .
- Buffer Solutions a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. # 450-1300EB. b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS. c. Carbonate Buffer: Na 2 C0 4 from Fischer, Cat. # S495, make up 100 mM stock solution. d.
- PBS Dulbecco's Phosphate-Buffered Saline
- Blocking Buffer 5% Non-fat milk (Carnation) in PBS.
- Carbonate Buffer Na 2 C0 4 from Fischer, Cat. # S495, make up 100 mM stock solution. d.
- Kinase Buffer 1.0 ml (from 1M stock solution) MgCl 2 ; 0.2 ml (from a 1M stock solution) MnCl 2 ; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ H 2 0. e.
- Lysis Buffer 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na 3 V0 4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H 2 0. f . ATP: Sigma Cat. # A- 7699, make up 10 mM stock solution (5.51 mg/ml).
- g TRIS-HC1 Fischer Cat.
- MgCl 2 Fischer Cat. # M33-500, make up 1M stock solution with MilliQ H 2 0. k.
- HEPES Fischer Cat. # BP 310-500; to 200 ml MilliQ H 2 0, add 59.6 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ H 2 0, sterile filter (1M stock solution) .
- MnCl 2 Fischer Cat. # M87-100, make up 1M stock solution with MilliQ H 2 0. n.
- DTT Fischer Cat. # BP172-5.
- EEEYEEYEEEYEEEYEEEY make peptide stock solution (ImM, 2.98 mg/ml) in water fresh just before use.
- Vectastain ELITE ABC reagent To prepare 14 ml of working reagent, add 1 drop of reagent A to 15 ml TBST and invert tube several times to mix. Then add 1 drop of reagent B. Put tube on orbital shaker at room temperature and mix for 30 minutes. Procedures Preparation of src coated ELISA plate.
- This assay is used to determine lck protein kinase activities measuring phosphorylation of GST- ⁇ as the readout.
- Materials and Reagents a. Yeast transformed with lck. Schizosaccharomyces Pombe is used to express recombinant Lck (Superti-Furga, et al., EMBO J, 12:2625-2634; Superti-Furga, et al . , Nature Biotech. , 14:600-605). S.
- Pombe strain SP200 (h-s leul.32 ura4 ade210) is grown as described and transformations with pRSP expression plasmids are done by the lithium acetate method (Superti-Furga, supra) . Cells are grown in the presence of 1 ⁇ M thiamine to induce expression.
- Cell lysates Yeast cells expressing lck are pelleted, washed once in water, re-pelleted and stored frozen at -80°C until use.
- GST- ⁇ DNA encoding for GST- ⁇ fusion protein for expression in bacteria obtained from Arthur Weiss of the Howard Hughes Medical Institute at the University of California, San Francisco. Transformed bacteria are grown overnight while shaking at 25°C.
- GST- ⁇ is purified by glutathione affinity chromatography, Pharmacia, Alameda, CA. d. DMSO: Sigma, St. Louis, MO. e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat Bottom Plate, Corning Cat. #25805-96. f . NUNC 96-well V-bottom polypropylene plates for dilution of compounds: Applied Scientific Cat. # AS-72092. g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia) Cat. #90001605. h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301. i. Sheep ant -mouse IgG (H+L) : Jackson Labs Cat. #
- Buffer solutions a. PBS (Dulbecco's Phosphate-Buffered Saline) IX solution: GIBCO PBS, GIBCO Cat. # 450-1300EB. b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS (pH7.5), 58.44 g NaCl, 10 ml Tween-20, bring up to 1 L total volume with MilliQ H 2 0. c. Carbonate Buffer: Na 2 C0 4 from Fischer, Cat.
- HEPES HEPES; 0.1 ml TX-100; bring to 10 ml total volume with MilliQ H 2 0.
- Lysis Buffer 5.0 HEPES (from IM stock solution.); 2.74 ml NaCl (from 5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml Na 3 V0 4 (from a 0.1 M stock solution); bring to 100 ml total volume with MilliQ H 2 0. f .
- ATP Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml).
- TRIS-HC1 Fischer Cat. # BP 152-5, to 600 ml MilliQ H 2 0 add 121.14 g material, adjust pH to 7.5 with HCI, bring to 1 L total volume with MilliQ H 2 0. h.
- NaCl Fischer Cat. # S271-10, Make up 5M stock solution with MilliQ H 2 0. i Na 3 V0 4 : Fischer Cat.
- UB40 plate 1.0 ⁇ g/well UB40 in 100 ⁇ l of PBS overnight at 4°C and block with 150 ⁇ l of Blocking Buffer for at least 1 hour.
- Assay measuring phosphorylating function of RAF The following assay reports the amount of RAF-catalyzed phosphorylation of its target protein MEK as well as MEK' s target MAPK.
- the RAF gene sequence is described in Bonner et al., 1985, Molec. Cell. Biol. , 5:1400-1407, and is readily accessible in multiple gene sequence data banks. Construction of the nucleic acid vector and cell lines utilized for this portion of the invention are fully described in Morrison et al . , 1988, Proc. Natl. Acad. Sci. USA, 85:8855-8859.
- Sf9 Spodoptera frugiperda cells
- GIBCO-BRL Gaithersburg, MD.
- RIPA buffer 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5 % Triton X- 100;
- T-MEK Thioredoxin-MEK fusion protein
- His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cells transformed with pUC18 vector encoding His- MAPK. His-MAPK is purified by Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, CA, as described herein. 5. Sheep anti mouse IgG: Jackson laboratories, West Grove, PA. Catalog, # 515-006-008, Lot# 28563
- RAF-1 protein kinase specific antibody URP2653 from UBI .
- Coating buffer PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg, MD.
- wash buffer 50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1 % Triton X-100).
- Block buffer TBST, 0.1 % ethanolamine pH 7.4 10.
- DMSO fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, fetal sulfate, 0.1 % ethanolamine pH 7.4 10.
- DMSO Sigma, St. Louis, MO
- Kinase buffer 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1 % Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium orthovanadate, 0.5 MM DTT and 10 mM MgCl 2 .
- ATP mix 100 mM MgCl 2 , 300 mM ATP, 10 mCi ⁇ 33 P ATP (Dupont-NEN) /mL.
- ELISA plate coating ELISA wells are coated with
- Lysates from RAS/RAF infected Sf9 insect cells are prepared after cells are infected with recombinant baculoviruses at a MOI of 5 for each virus, and harvested 48 hours later. The cells are washed once with PBS and lysed in RIPA buffer. Insoluble material is removed by centrifugation (5 min at 10,000 x g) . Aliquots of lysates are frozen in dry ice/ethanol and stored at -80 °C until use.
- Pre-dilute compounds stock solution 10 mg/ml DMSO
- extracts 20 fold in TBST plus 1% DMSO.
- Start the kinase reaction by addition of 5 ml ATP mix; Shake the plates on an ELISA plate shaker during incubation.
- 40 mL aliquots from individual wells of the assay plate can be transferred to the corresponding positions on the phosphocellulose filter mat.
- After air drying the filters put the filters in a tray. Gently rock the tray, changing the wash solution at 15 min intervals for 1 hour. Air-dry the filter mats. Seal the filter mats and place them in a holder suitable for measuring the radioactive phosphorous in the samples. Insert the holder into a detection device and quantify the radioactive phosphorous on the filter mats.
- This assay analyzes the protein kinase activity of CDK2 in exogenous substrate.
- B Adjust pH to 7.2 with HCI.
- ATP solution 60 ⁇ M ATP, 300 ⁇ g/ml BSA, 3 mM DTT
- 120 ⁇ l 10 mM ATP 120 ⁇ l 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at -80° C.
- CDK2 solution cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, o .5 mM DTT, 10% glycerol, stored in 9 ⁇ l aliquots at
- PDGF human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.
- FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- Anti -BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- PBS Washing Solution IX PBS, pH 7.4 (Sugen, Inc., Redwood City, California) .
- 3T3 cell line genetically engineered to express human PDGF-R. Protocol (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gin in a 96 well plate. Cells are incubated overnight at 37°C in 5% C0 2 .
- ligand PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA
- test compounds are added to the cells simultaneously.
- the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound.
- Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
- FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel .
- Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.
- the blocking solution is removed by decanting and the wells are washed once with PBS.
- Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
- the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
- TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
- the absorbance of the samples are measured at 410 nm (in "dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
- EGF mouse EGF, 201; Toyobo, Co., Ltd, Japan.
- BrdU Labeling Reagent 10 mM, in PBS (pH7.4), Cat.
- FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
- TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- PBS Washing Solution IX PBS, pH 7.4 (Sugen, Inc.,
- Cells are seeded at 8000 cells/well in 10% CS, 2mM Gin in DMEM, in a 96 well plate. Cells are incubated overnight at 37°C in 5% C0 2 . (2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
- ligand EGF, 2 nM, prepared in DMEM with 0.1% BSA
- test compounds are added to the cells simultaneously.
- the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
- Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
- Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.
- the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
- TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
- the absorbance of the samples are measured at 410 nm (in "dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
- EGF mouse EGF, 201; Toyobo, Co., Ltd. Japan
- FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany .
- TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer Mannheim,
- PBS Washing Solution IX PBS, pH 7.4, made in house .
- the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.
- ligand (EGF 2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously.
- the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound.
- Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
- the FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
- the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
- TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
- the absorbance of the samples are measured at 410 nm (in "dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
- IGF1 Ligand human, recombinant; G511, Promega Corp, USA .
- FixDenat fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- TMB Substrate Solution tetramethylbenzidine (TMB) , ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.
- PBS Washing Solution IX PBS, pH 7.4 (Sugen, Inc., Redwood City, California) .
- Albumin, Bovine (BSA) fraction V powder; A-8551, Sigma Chemical Co., USA.
- Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2mM Gin in a 96- well plate. Cells are incubated overnight at 37°C in 5% C0 2 .
- test compounds are added to the cells simultaneously.
- the negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound.
- Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.
- the FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel . Milk is added (5% dehydrated milk in PBS, 200 ⁇ l/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 ⁇ l/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.
- the antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.
- TMB substrate solution is added (100 ⁇ l/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.
- the absorbance of the samples are measured at 410 nm (in "dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
- FGF-Induced BrdU incorporation Assay measures FGF-induced DNA synthesis in 3Tc7/EGFr cells that express endogenous FGF receptors. Materials and Reagents
- FGF human FGF2/bFGF (Gibco BRL, No. 13256-029).
- BrdU Labeling reagent (10 mM PBS (pH 7.4), Boehringer Mannheim Cat No. 1 647 229) .
- Fixdenat fixation solution (Boehringer Mannheim Cat No. 1 647 229) .
- Anti-BrdU-POD mouse monoclonal antibody conjugated with peroxidase, Boehringer Mannheim Cat. No. 1 647 229).
- TMB tetramethylbenzidine, Boehringer Mannheim Cat. No. 1 647 229) .
- PBS washing solution pH 7.4 (Sugen, Inc.) .
- 3T3 engineered cell line 3T3c7/EGFr.
- Cells are seeded at 8,000 cells/well in DMEM, 10% CS and 2 mM Gin in a 96-well plate. Incubate 24 hours at 37° C in 5% C0 2 .
- test compounds Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA) and test compound simultaneously. Negative control wells receive serum free DMEM with 0.1% BSA only; positive control wells receive FGF2 ligand but no test compound. Test compounds are prepared in serum- free DMEM with ligand in a 96-well plate and serially diluted to make seven (7) test concentrations.
- This assay measures the in vitro kinase activity of EGFR using ELISA.
- PBS Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No. 450-1300EB
- Blocking Buffer Reagent M.W. Working Amount per Concentration 100 ml
- A431 cell lysate (Screening Lab, SUGEN, Inc.)
- Adenosine-5 ' -triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394) .
- This reagent should be prepared immediately before use and kept on ice
- Ethylenediaminetetraacetic acid Prepare 200 mM working solution in dH 2 0. Adjust to pH 8.0 with 10 N NaOH.
- This assay measures the in vitro kinase activity of PDGFR using ELISA.
- PBS Dulbecco's Phosphate-Buffered Saline, Gibco Catalog No. 450-1300EB
- Blocking Buffer 6. PDGFR- ⁇ expressing NIH 3T3 cell lysate (Screening Lab, SUGEN, Inc.) .
- Adenosine-5 ' -triphosphate (ATP, from Equine muscle, Sigma Cat. No. A-5394).
- Ethylenediaminetetraacetic acid EDTA
- This assay measures in vitro kinase activity of the Myc-
- GyrB-FGFR fusion protein using ELISA GyrB-FGFR fusion protein using ELISA.
- HEPES 238.3 100 mM 23.83 g 20 mM NaCl 58.44 750 mM 43.83 g 150 mM Glycerol NA 50% 500 ml 10%
- HEPES pH 7.2 238.3 500 mM 50 mM MnCl 2 20 mM 2 mM
- Triton-X-100 1 % 0.1%
- ATP Bacillerial source, Sigma Cat. No. A-7699
- Biotin conjugated anti -phosphotyrosine mab (clone 4G10, Upstate Biotechnology Inc. Cat. No. 16-103, Ser. No. 14495) .
- Vectastain Elite ABC reagent (Avidin peroxidase conjugate, Vector Laboratories Cat. No. PK-6 100).
- HEPES Fisher Cat. No. BP310-500
- Blocking Buffer 5% Low Fat Milk in PBS
- This assay evaluates flk-1 autophosphorylation activity in vitro using ELISA.
- Flk-1/NIH cells NIH fibroblast line over- expressing human flk-1 clone 3 (SUGEN, Inc., obtained from MPI, Martinsried, Germany). 3. Growth medium: DMEM plus heat inactivated 10% FBS and 2 mM Glutamine (Gibco-BRL) .
- Starvation medium DMEM plus 0.5% heat -inactivated FBS, 2 mM Glutamine (Gibco-BRL) .
- PBS Dulbecco's Phosphate-Buffered Saline
- Gibco Cat. No. 450-1300EB Gibco Cat. No. 450-1300EB
- test compounds (normally 10-fold) into wells of a polypropylene plate containing 20% DMSO in TBS.
- step 4. Wash plates as in step 4. 17. Add 100 ⁇ l of the preformed A & B complex to all wells. Incubate 30 min with shaking at room temperature.
- HUV-EC-C cells human umbilical vein endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL) . Wash with Dulbecco ' s phosphate- buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm 2 of tissue culture flask.
- D-PBS Dulbecco ' s phosphate- buffered saline
- Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution Sigma Chemical Company; catalogue no. C-1544
- the 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco; catalogue no. 25200-049) in the cell dissociation solution. Trypsinize with about 1 ml/25-30 cm 2 of tissue culture flask for about 5 minutes at 37°C.
- test compound titrations make up two-fold test compound titrations in separate 96-well plates, generally 50 ⁇ M on down to 0 ⁇ M. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 ⁇ l/well of test compound at 200 ⁇ M (4X the final well concentration) to the top well of a particular plate column. Since the stock test compound is usually 20 mM in DMSO, the 200 ⁇ M drug concentration contains 2% DMSO.
- a diluent made up to 2% DMSO in assay medium (F12K + 0.5% fetal bovine serum) is used as diluent for the test compound titrations in order to dilute the test compound but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 ⁇ l/well. Take 60 ⁇ l from the 120 ⁇ l of 200 ⁇ M test compound dilution in the top well of the column and mix with the 60 ⁇ l in the second well of the column. Take 60 ⁇ l from this well and mix with the 60 ⁇ l in the third well of the column, and so on until two- fold titrations are completed.
- VEGF vascular endothelial cell growth factor
- aFGF acidic fibroblast growth factor
- test compound dilution 50 ⁇ l growth factor or media
- 100 ⁇ l cells which calculates to 200 ⁇ l/well total.
- 4X concentrations of test compound and growth factors become IX once everything has been added to the wells.
- human tumors to grow as xenografts in athymic mice (e . g. , Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol . Microbial. Scand. 77:758-760), many different human tumor cell lines ⁇ e . g. , mammary, lung, genitourinary, gastro- intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice.
- the following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention.
- Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and _____ vivo.
- the object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell.
- the object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell.
- the object of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder such as cancer.
- Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107) , A375 cells (melanoma, ATCC # CRL 1619) , A431 cells (epidermoid carcinoma, ATCC # CRL 1555) , Calu 6 cells (lung, ATCC # HTB 56) , PC3 cells (prostate, ATCC # CRL 1435) , SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-IR or any other test kinase.
- the following protocol can be used to perform xenograft experiments :
- mice Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories (Gilroy, CA) . All animals are maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They receive sterile rodent chow and water ad libitum.
- Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM glutamine (GLN) ) . All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% C0 2 at 37°C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco) .
- appropriate medium for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)
- All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY) unless otherwise specified. All cells are grown in
- 8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg Xylazine) are administered intraperitoneally . A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10 7 tumor cells in a volume of 100 ⁇ l medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin is closed by using wound clips. Animals are observed daily. Analysis
- mice After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry, in situ hybridization determination, etc . ) .
- Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect.
- a measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index; i.e., IC 50 /LD 50 .
- IC 50 the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein.
- LD 50 ⁇ the dosage which results in 50% toxicity, can also be measured by standard techniques as well (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.
- 3-heteroarylidenyl-2- indolinones are expected to have a beneficial effect on the chemotherapeutic efficacy of various chemotherapeutic agents, in particular fluorinated pyrimidine compounds.
- 3- [ (2, 4 -Dimethylpyrrol- 5-yl) methylene] -2-indolinone together with fluorouracil or fluorouracil/leucovorin is expected to be an effective chemotherapeutic combination for the treatment of colorectal cancer.
- compositions of the present invention are expected to modulate RTK and CTK activity and therefore to be effective as therapeutic agents against RTK- and CTK-related disorders .
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Priority Applications (15)
Application Number | Priority Date | Filing Date | Title |
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JP2000590484A JP2002533360A (en) | 1998-12-31 | 1999-12-30 | 3-Heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
MXPA01006742A MXPA01006742A (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy. |
IL14392099A IL143920A0 (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone derivatives and pharmaceutical compositions containing the same |
EP99966725A EP1139754A4 (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
AU22215/00A AU760964B2 (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activityand for use in cancer chemotherapy |
KR1020017008453A KR20010108063A (en) | 1998-12-31 | 1999-12-30 | 3-Heteroarylidenyl-2-Indolinone Compounds for Modulating Protein Kinase Activity and for Use in Cancer Chemotherapy |
BR9916735-2A BR9916735A (en) | 1998-12-31 | 1999-12-30 | 3-Heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
CA002357042A CA2357042A1 (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
DE60030164T DE60030164T2 (en) | 1999-12-30 | 2000-06-30 | 3-heteroarylidenyl-2-indolinone derivatives for modulating the activity of a protein kinase and for use in the chemotherapy of cancer |
PCT/US2000/018058 WO2001049287A1 (en) | 1999-12-30 | 2000-06-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
AU57819/00A AU5781900A (en) | 1999-12-30 | 2000-06-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activityand for use in cancer chemotherapy |
ES00943334T ES2269156T3 (en) | 1999-12-30 | 2000-06-30 | HETEROARILIDENIL-3-INDOLINONE COMPOUNDS TO MODULATE PROTEIN-KINASE ACTIVITY AND FOR USE IN CANCER CHEMOTHERAPY. |
JP2001549655A JP2003535038A (en) | 1999-12-30 | 2000-06-30 | 3-Heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
EP00943334A EP1259234B9 (en) | 1999-12-30 | 2000-06-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
AT00943334T ATE336245T1 (en) | 1999-12-30 | 2000-06-30 | 3-HETEROARYLIDENYL-2-INDOLINONE DERIVATIVES WITH PROTEIN KINASE MODULATING ACTIVITY AND FOR USE IN CANCER SEMOTHERAPY |
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US11431398P | 1998-12-31 | 1998-12-31 | |
US60/114,313 | 1998-12-31 |
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WO2000038519A1 true WO2000038519A1 (en) | 2000-07-06 |
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PCT/US1999/031232 WO2000038519A1 (en) | 1998-12-31 | 1999-12-30 | 3-heteroarylidenyl-2-indolinone compounds for modulating protein kinase activity and for use in cancer chemotherapy |
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EP (1) | EP1139754A4 (en) |
JP (1) | JP2002533360A (en) |
KR (1) | KR20010108063A (en) |
CN (1) | CN1356872A (en) |
AU (1) | AU760964B2 (en) |
BR (1) | BR9916735A (en) |
CA (1) | CA2357042A1 (en) |
IL (1) | IL143920A0 (en) |
MX (1) | MXPA01006742A (en) |
WO (1) | WO2000038519A1 (en) |
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WO2001060814A2 (en) * | 2000-02-15 | 2001-08-23 | Sugen, Inc. | Pyrrole substituted 2-indolinone protein kinase inhibitors |
WO2001062235A2 (en) * | 2000-02-28 | 2001-08-30 | Aventis Pharma S.A. | A composition comprising camptothecin and a pyrimidine derivative for the treatment of cancer |
WO2001037820A3 (en) * | 1999-11-24 | 2001-12-13 | Sugen Inc | Ionizable indolinone derivatives and their use as ptk ligands |
US6451838B1 (en) | 2000-05-24 | 2002-09-17 | Pharmacia & Upjohn Company | 1-(pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives |
WO2003035619A1 (en) * | 2001-10-25 | 2003-05-01 | Merck & Co., Inc. | Tyrosine kinase inhibitors |
WO2003035616A2 (en) * | 2001-10-25 | 2003-05-01 | Merck & Co., Inc. | Tyrosine kinase inhibitors |
WO2003035614A2 (en) * | 2001-10-25 | 2003-05-01 | Merck & Co., Inc. | Tyrosine kinase inhibitors |
US6569868B2 (en) | 1998-04-16 | 2003-05-27 | Sugen, Inc. | 2-indolinone derivatives as modulators of protein kinase activity |
US6599902B2 (en) | 2001-05-30 | 2003-07-29 | Sugen, Inc. | 5-aralkysufonyl-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives as kinase inhibitors |
US6686362B2 (en) | 2001-12-27 | 2004-02-03 | Theravance, Inc. | Indolinone derivatives |
US6706709B2 (en) | 2000-06-02 | 2004-03-16 | Sugen, Inc. | Indolinone derivatives as protein kinase/phosphatase inhibitors |
WO2004045617A1 (en) * | 2002-11-15 | 2004-06-03 | Warner-Lambert Company Llc | Combination chemotherapy comprising a mek inhibitor and capecitabine for treating cancer |
EP1427326A2 (en) * | 2001-08-15 | 2004-06-16 | Sugen, Inc. | Combination therapy for the treatment of cancer |
EP1453502A2 (en) * | 2001-11-21 | 2004-09-08 | Sugen, Inc. | Pharmaceutical formulations comprising indolinone derivatives |
US6797825B2 (en) | 2001-12-13 | 2004-09-28 | Abbott Laboratories | Protein kinase inhibitors |
US6831175B2 (en) | 2001-12-13 | 2004-12-14 | Abbott Laboratories | Kinase inhibitors |
EP1515680A2 (en) * | 2002-06-25 | 2005-03-23 | Wyeth | Use of thio-oxindole derivatives in treatment of skin disorders |
US6878733B1 (en) | 1999-11-24 | 2005-04-12 | Sugen, Inc. | Formulations for pharmaceutical agents ionizable as free acids or free bases |
EP1531806A2 (en) * | 2002-06-25 | 2005-05-25 | Wyeth | Use of thio-oxindole derivatives in treatment of hormone-related conditions |
US7312237B2 (en) | 2001-03-14 | 2007-12-25 | Bristol-Myers Squibb Co. | Combination of epothilone analogs and chemotherapeutic agents for the treatment of prolilferative diseases |
US8110591B2 (en) | 2003-10-23 | 2012-02-07 | Ab Science | 2-aminoaryloxazole compounds as tyrosine kinase inhibitors |
US8246966B2 (en) | 2006-08-07 | 2012-08-21 | University Of Georgia Research Foundation, Inc. | Trypanosome microsome system and uses thereof |
US8338433B2 (en) | 2006-11-22 | 2012-12-25 | University Of Georgia Research Foundation, Inc. | Tyrosine kinase inhibitors as anti-kinetoplastid agents |
US8450302B2 (en) | 2002-08-02 | 2013-05-28 | Ab Science | 2-(3-aminoaryl) amino-4-aryl-thiazoles and their use as c-kit inhibitors |
US8658659B2 (en) | 2005-04-04 | 2014-02-25 | Ab Science | Substituted oxazole derivatives and their use as tyrosine kinase inhibitors |
US8835435B2 (en) | 2002-08-02 | 2014-09-16 | Ab Science | 2-(3-aminoaryl) amino-4-aryl-thiazoles and their use as c-kit inhibitors |
WO2016112111A1 (en) | 2015-01-08 | 2016-07-14 | The Board Of Trustees Of The Leland Stanford Junior University | Factors and cells that provide for induction of bone, bone marrow, and cartilage |
US9844529B2 (en) | 2011-11-11 | 2017-12-19 | Eli Lilly And Company | Combination therapy for ovarian cancer |
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CN100432071C (en) * | 2004-11-05 | 2008-11-12 | 中国科学院上海药物研究所 | Substituted 1H-indole-2-ketone compound and its preparation method and uses |
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WO1998007695A1 (en) * | 1996-08-23 | 1998-02-26 | Sugen, Inc. | Indolinone combinatorial libraries and related products and methods for the treatment of disease |
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- 1999-12-30 WO PCT/US1999/031232 patent/WO2000038519A1/en not_active Application Discontinuation
- 1999-12-30 CA CA002357042A patent/CA2357042A1/en not_active Abandoned
- 1999-12-30 IL IL14392099A patent/IL143920A0/en unknown
- 1999-12-30 CN CN99816376A patent/CN1356872A/en active Pending
- 1999-12-30 AU AU22215/00A patent/AU760964B2/en not_active Ceased
- 1999-12-30 MX MXPA01006742A patent/MXPA01006742A/en unknown
- 1999-12-30 BR BR9916735-2A patent/BR9916735A/en not_active IP Right Cessation
- 1999-12-30 EP EP99966725A patent/EP1139754A4/en not_active Withdrawn
- 1999-12-30 KR KR1020017008453A patent/KR20010108063A/en not_active Application Discontinuation
- 1999-12-30 JP JP2000590484A patent/JP2002533360A/en not_active Withdrawn
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US6451838B1 (en) | 2000-05-24 | 2002-09-17 | Pharmacia & Upjohn Company | 1-(pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives |
US7008943B2 (en) | 2000-05-24 | 2006-03-07 | Pharmacia & Upjohn Company | 1-(Pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives |
US7053113B2 (en) | 2000-05-24 | 2006-05-30 | Sugen, Inc. | Mannich base prodrugs of 3-(pyrrol-2-yl-methylidene)-2-indolinone derivatives |
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US6482848B2 (en) | 2000-05-24 | 2002-11-19 | Sugen Incorporated | Prodrugs of 3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives |
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US6797825B2 (en) | 2001-12-13 | 2004-09-28 | Abbott Laboratories | Protein kinase inhibitors |
US6686362B2 (en) | 2001-12-27 | 2004-02-03 | Theravance, Inc. | Indolinone derivatives |
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Also Published As
Publication number | Publication date |
---|---|
AU760964B2 (en) | 2003-05-22 |
CA2357042A1 (en) | 2000-07-06 |
EP1139754A1 (en) | 2001-10-10 |
CN1356872A (en) | 2002-07-03 |
IL143920A0 (en) | 2002-04-21 |
BR9916735A (en) | 2001-09-25 |
KR20010108063A (en) | 2001-12-07 |
EP1139754A4 (en) | 2002-12-18 |
MXPA01006742A (en) | 2004-04-21 |
JP2002533360A (en) | 2002-10-08 |
AU2221500A (en) | 2000-07-31 |
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