Classifications

• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Diaryl urea compounds e.g. 4 ⁇ 4-[3-(4-chloro-3-trifluoromethylphenyl)-ureido]-3-fluorophenoxy ⁇ - 5 pyridine-2-carboxylic acid methylamide as described e.g. in US 20050038080 are potent anticancer and anti-angiogenic agents that possess various activities, including inhibitory activity on the VEGFR, PDGFR, raf, p38, and/or flt-3 kinase signalling molecules.
• the RAS/RAF/MEK/ERK pathway is involved in cellular proliferation, differentiation, and transformation, and is implicated in many cancers.
• the PI3K/AKT signalling pathway is another important physiological pathway 10 in cells.
• AKT pathway appears to be active in many types of human cancer (Nicholson and Anderson, Cell Signal, 14: 381-395, 2002).
• the present invention provides drug combinations, compositions, and methods for treating diseases and conditions, including, but not limited to, cell proliferative disorders (such as cancer), inflammation, immunomodulatory disorders, and conditions associated with abnormal or undesirable angiogenesis.
• the drug combinations comprise a compound of formula I and at least one second compound that is an inhibitor of the PI3K/AKT signalling pathway.
• a diaryl urea compound as described below and a signalling pathway inhibitor, pharmaceutically-acceptable salts thereof, and derivatives thereof, etc.
• PI3K phosphatidylinositol-3-kinase
• AKT Protein Kinase B
• PI3K family members activate PI3K family members to specifically convert one lipid signalling molecule, PIP2, into another, PI(3,4,5)P3.
• the phosphorylated product recruits Akt family members to the inner plasma membrane, stimulating their protein kinase activity.
• Akt effectors involved in several biological processes have been identified.
• the Akt kinases mediate cell survival though phosphorylation and inactivation of apoptotic machinery components.
• the PI3K/AKT signalling pathway includes any members or components that participate in the signal transduction cascade.
• PI3- kinase Akt-kinase
• FKBP 12 FKBP 12
• mTOR mimmalian target of rapamycin; also known as FRAP, RAFTl, or RAPTl
• RAPTOR regulatory associated protein if mTOR
• TSC tuberous sclerosis complex
• PTEN phosphatase and tensin homolog
• Combinations of the present invention can be used to treat and/or prevent any condition and/or diseases associated with any of the aforementioned activities.
• An inhibitor of the PI3K/AKT signalling pathway is a compound that inhibits one or more members of the aforementioned signal transduction cascade. While such compounds may be referred to as pathway inhibitors, the present invention includes the use of these inhibitors to treat any of the mentioned diseases or conditions, regardless of the mechanism of action or how the therapeutic effect is achieved. Indeed, it is recognized that such compounds may have more than one target, and the initial activity recognized for a compound may not be the activity that it possesses in vivo when administered to a subject, or whereby it achieves its therapeutic efficacy.
• a compound as a pathway or protein target (e.g., Akt or mTOR) inhibitor indicates that a compound possesses such activity, but in no way restricts a compound to having that activity when used as a therapeutic or prophylactic agent.
• AKT family members include: Aktl, Akt2 (commonly over-expressed in tumors; Bellacosa et al., Int. J. Cancer, 64:280-285, 1995), and Akt3.
• PI3K family members include: pl lO-alpha, pl lO-beta, pl lO-delta, and pl lO-gamma (catalytic).
• PI3K/AKT signalling pathway inhibitors include, but are not limited to, e.g., FTY720 (e.g., Lee et al., Carcinogenesis, 25(12):2397-2405, 2004);
• PI3-kinase inhibitors examples include, but are not limited to, e.g., PI3-kinase
• celecoxib and analogs thereof such as OSU-03012 and OSU-03013 (e.g., Zhu et al., Cancer Res., 64(12):4309-18, 2004);
• 3-deoxy-D-myo-inositol analogs e.g., U.S. Application No. 20040192770; Meuillet et al., Oncol. Res., 14:513-27, 2004
• PX-316 2 '-substituted, 3'-deoxy-phosphatidyl-myo-inositol analogs (e.g., Tabellini et al., Br. J. Haematol., 126(4):574-82, 2004);
• Ly294002 (e.g., Vlahos, et al., J. Biol., Chem., 269(7) 5241-5248, 1994);
• quinazoline-4-one derivatives such as IC486068 (e.g., U.S. Application No. 20020161014; Geng et al., Cancer Res., 64:4893-99, 2004);
• viridins including semi-synthetic viridins such as such as PX-866 (acetic acid ( 1 S,4E, 1 OR, 11 R, 13 S, 14R)-[4-dially laminomethy lene-6-hydroxy- 1 -methoxymethy 1- 10,13 -dime- thy 1-3 ,7, 17-tr ioxo- 1, 3 ,4,7,10,11, 12,13, 14,15, 16,17-dodecahy dro-2-oxa-cyclopenta[a]phenanthren- 1 1-yl ester) (e.g., IhIe et al., MoI Cancer Ther., 3(7):763-72, 2004; U.S. Application No. 20020037276; U.S. Pat. 5,726,167); and
• wortmannin and derivatives thereof e.g., U.S. Pat. Nos. 5,504,103; 5,480,906, 5,468,773; 5,441,947; 5,378,725; 3,668,222).
• Akt-kinase also known as protein kinase B
• examples of Akt-kinase (also known as protein kinase B) inhibitors include, but are not limited to, e.g.,
• Akt-1-1 (inhibits Aktl) (Barnett et al., Biochem. J., 385 (Pt.2):399-408, 2005);
• Akt-1-1,2 (inhibits AkI and 2) (Barnett et al., Biochem. J., 385 (Pt.2):399-408, 2005);
• API-59CJ-Ome e.g., Jin et al., Br. J. Cancer., 91 :1808-12, 2004;
• indole-3-carbinol and derivatives thereof e.g., U.S. Pat. Nos. 6,656,963; Sarkar and Li, J Nutr., 134(12 Suppl):3493S-3498S, 2004;
• perifosine e.g., interferes with Akt membrane localization; Dasmahapatra et al., Clin. Cancer Res., 10(15):5242-52, 2004); phosphatidylinositol ether lipid analogues (e.g., Gills and Dennis, Expert. Opin. Investig. Drugs, 13:787-97, 2004);
• triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al., Cancer Res., 64:4394-9, 2004).
• mTOR inhibitors include, but are not limited to, e.g.,
• rapamycins and derivatives thereof including: CCI-779 (temsirolimus), RADOOl (Everolimus; WO 9409010), TAFA93 and AP23573; rapalogs, e.g. as disclosed in WO 98/02441 and WO 01/14387, e.g.
• AP23573, AP23464, AP23675, or AP23841 40-(2-hydroxyethyl)rapamycin, 40-[3- hydroxy(hydroxymethyl) methylpropanoate]-rapamycin (also called CCl 779), 40-epi-(tetrazolyt)- rapamycin (also called ABT578), 32-deoxorapamycin, 16-pentynyloxy-32(S)-dihydrorapamycin, and other derivatives disclosed in WO 05005434; derivatives disclosed in USP 5,258,389, WO 94/090101, WO 92/05179, USP 5,118,677, USP 5,1 18,678, USP 5,100,883, USP 5,151,413, USP 5,120,842, WO 93/111130, WO 94/02136, WO 94/02485, WO 95/14023, WO 94/02136, WO - 95/16691 (e.
• phosphorus-containing rapamycin derivatives e.g., WO 05016252
• Examples of compounds in preclinical or clinical use include, e.g., AP23573, AP23841, CCI-779, and RADOOl.
• PI3-kinase inhibitors of interest are wortmannin and the derivatives or analogs thereof and the pharmaceutically acceptable salts of wortmannin and its derivatives and analogs. Consequently, methods of this invention include the use of the PI3-kinase inhibitors of formula W:
• R is H ( 11 -desacetoxywortmannin) or acetoxy and R' is Ci -C 6 alkyl, b) ⁇ 9,l 1- dehydrodesacetoxy wortmannin compounds of formula W2
• R' is C ,-C 6 alkyl
• R is H or acetoxy and R' is CpC 6 alkyl, and R" is H, CpC 6 alkyl,
• Ri is H, methyl or ethyl and R 2 is H or methyl or e) 1 1 -substituted and 17- substituted derivatives of wortmannin of formula W5
• R 4 0 or -0(CO)R 6
• R 3 0, -OH or -0(CO)R 6
• C]- 6 alkyl means straight, branched chain or cyclic alkyl groups having from one to six carbon atoms, which may be cyclic, linear or branched with single or multiple branching. Such groups include for example methyl, ethyl, w-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-buty ⁇ , cyclopropyl, cyclobutyl and the like.
• the present invention also relates to useful forms of the compounds as disclosed herein, such as pharmaceutically acceptable salts and metabolites.
• the present invention also relatesto prodrugs of the compound of formula (T).
• pharmaceutically acceptable salt refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention.
• Pharmaceutically acceptable salts include those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid and citric acid.
• Pharmaceutically acceptable salts also include those in which the main compound functions as an acid and is reacted with an appropriate base to form, e.g., sodium, potassium, calcium, mangnesium, ammonium, and choline salts.
• an appropriate base e.g., sodium, potassium, calcium, mangnesium, ammonium, and choline salts.
• acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.
• alkali and alkaline earth metal salts are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.
• Representative salts of the compounds of this invention include the conventional non-toxic salts and the quaternary ammonium salts which are formed, for example, from inorganic or organic acids or bases by means well known in the art.
• acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cinnamate, - cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate, mandelate, methanesulfonate
• Base salts include alkali metal salts such as potassium and sodium salts, alkaline earth metal salts such as calcium and magnesium salts, and ammonium salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine. Additionally, basic nitrogen containing groups may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aryl or aralkyl halides like benzyl and phenethyl bromides and others monosubstituted aralkyl halides or polysubstituted aralkyl
• Solvates for the purposes of the invention are those forms of the compounds where solvent molecules form a complex in the solid state and include, but are not limited to for example ethanol and methanol. Hydrates are a specific form of solvates, where the solvent molecule is water.
• Certain pharmacologically active agents can be further modified with labile functional groups that are cleaved after in vivo administration to furnish the parent active agent and the pharmacologically inactive derivatizing group.
• These derivatives commonly referred to as prodrugs, can be used, for example, to alter the physicochemical properties of the active agent, to target the active agent to a specific tissue, to alter the pharmacokinetic and pharmacodynamic properties of the active agent, and to reduce undesirable side effects.
• Prodrugs of the invention include, e.g., the esters of appropriate compounds of this invention that are well-tolerated, pharmaceutically acceptable esters such as alkyl esters including methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters. Additional esters such as phenyl-C r C 5 alkyl may be used, although methyl ester is preferred.
• the metabolites of the compounds of this invention include oxidized derivatives of the compounds of formula I, wherein one or more of the nitrogens are substituted with a hydroxy group; which includes derivatives where the nitrogen atom of the pyridine group is in the oxide form, referred to in the art as 1 -oxo-pyridine or has a hydroxy substituent, referred to in the art as 1-hydroxy- pyridine.
• the compounds of the invention may be prepared by use of known chemical reactions and procedures as described e.g. in the following published international application WO 2005/009961.
• the compounds of formula I have been previously characterized as having various activities, including for inhibiting the Raf/MEK/ERK pathway, raf kinase, p38 kinase, VEGFR kinase, PDGFR kinase. These activities and their use in treating various diseases and conditions are disclosed in, e.g., WO 2005/009961, which are hereby incorporated by reference in their entirety.
• Drug combinations of the present invention can be utilized to treat any diseases or conditions that
• ⁇ are associated with, or mediated by, the cellular pathways modulated by the compounds comprising the combinations.
• These pathways include, but are not limited to signalling pathways which comprise, e.g., VEGFR, VEGFR2, Raf/Mek/Erk, Akt/PI3K, MTOR, PTEN, etc. (see also above).
• the drug combinations can be useful to treat diseases that are associated with, or mediated by, mutations in one of more genes present in these pathways, including cancer- associated mutations in PTEN, ras, Raf, Akt, PDK, etc.
• the compounds may be known as specific inhibitors, the present invention includes any ameliorative or therapeutic effect, regardless of the mechanism of action or how it is achieved.
• the drug combination can have one or more of the following activities, including, antiproliferative; anti-tumor; anti-angiogenic; inhibiting the proliferation of endothelial or tumor cells; anti-neoplastic; immunosuppressive; immunomodulatory; apoptosis-promoting, etc.
• Conditions or diseases that can be treated in accordance with the present invention include proliferative disorders (such as cancer), inflammatory disorders, immuno-modulatory disorders, allergy, autoimmune diseases, (such as rheumatoid arthritis, or multiple sclerosis), abnormal or excessive angiogenesis, etc.
• Any tumor or cancer can be treated, including, but not limited to, cancers having one or more mutations in raf, VEGFR-2, VEGFR-3, PDGFR-beta, Flt-3, ras, PTEN, Akt, PI3K, mTOR, as well as any upstream or downstream member of the signalling pathways of which they are a part.
• a tumor or cancer can be treated with a drug combination of the present invention irrespective of the mechanism that is responsible for it.
• cancers of any organ can be treated, including cancers of, but are not limited to, e.g., colon, pancreas, breast, prostate, bone, liver, kidney, lung, testes, skin, pancreas, stomach, prostate, ovary, uterus, head and neck, blood cell, lymph, etc.
• Cancers that can be treated in accordance with the present invention include, especially, but not limited to, brain tumors, breast cancer, bone sarcoma (e.g., osteosarcoma and Ewings sarcoma), bronchial premalignancy, endometrial cancer, glioblastoma, hematologic malignancies, hepatocellular carcinoma, Hodgkin's disease, kidney neoplasms, leukemia, leimyosarcoma, liposarcoma, lymphoma, Lhermitte-Duclose disease, malignant glioma, melanoma, malignant melanoma, metastases, multiple myeloma, myeloid metaplasia, myeloplastic syndromes, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell carcinoma (e.g., advanced, advanced refractory), rhabdomyosarcoma, soft tissue sarcoma, squamous epithelial
• breast cancer examples include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
• cancers of the respiratory tract include, but are not limited to, small-cell, non-small- cell lung carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
• brain cancers include, but are not limited to, brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumor.
• Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer.
• Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
• Tumors of the digestive tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland cancers.
• Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.
• Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.
• liver cancers include, but are not limited to, hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
• Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
• Head-and-neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, and/or oropharyngeal cancers, and lip and oral cavity cancer.
• Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.
• Sarcomas include, but are not limited to, sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
• Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
• drug combinations of the present invention can also cause tumor regression, e.g., a decrease in the size of a tumor, or in the extent of cancer in the body.
• Angiogenesis-related conditions and disorders can also be treated with drug combinations of the present invention. Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism.
• a number of pathological conditions are associated with the growth of extraneous blood vessels. These include, e.g., diabetic retinopathy, neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, restenosis, etc.
• the increased blood supply associated with cancerous and neoplastic tissue encourages growth, leading to rapid tumor enlargement and metastasis.
• the growth of new blood vessels in a tumor provides an escape route for renegade cells, encouraging metastasis and the consequence spread of the cancer.
• Useful systems for modulating angiogenesis include, e.g., neovascularization of tumor explants (e.g., U.S. Pat. Nos. 5,192,744; 6,024,688), chicken chorioallantoic membrane (CAM) assay (e.g., Taylor and Folkman, Nature, 297:307-312, 1982; Eliceiri et al., J. Cell Biol., 140, 1255-1263, 1998), bovine capillary endothelial (BCE) cell assay (e.g., U.S. Pat. No. 6,024,688; Polverini, P. J.
• CAM chicken chorioallantoic membrane
• BCE bovine capillary endothelial
• useful systems for modulating lymphangiogenesis include, e.g., rabbit ear model (e.g., Szuba et al., FASEB J., 16(14): 1985-7, 2002).
• Modulation of angiogenesis can be determined by any suitable method.
• the degree of tissue vascularity is typically determined by assessing the number and density of vesssels present in a given sample.
• microvessel density MMD
• MMD microvessel density
• CD31 also known as platelet-endothelial cell adhesion molecule or PECAM.
• a CD31 antibody can be employed in conventional immunohistological methods to immunostain tissue sections as described by, e.g., Penfold et al., Br. J. Oral and Maxill.
• Vezfl e.g., Xiang et al., Dev. Bio., 206: 123-141, 1999
• angiopoietin e.g., Tie-1, and Tie-2 (e.g., Sato et al., Nature, 376:70-74, 1995).
• the drug combinations of this invention also have a broad therapeutic activity to treat or prevent the progression of a broad array of diseases, such as inflammatory conditions, coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain kidney diseases associated with proliferation of glomerular or mesangial cells, and ocular diseases associated with retinal vessel proliferation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, restenosis, vascular graft restenosis, in-stent stenosis, angiogenesis, ocurlar diseases, pulmonary fibrosis, obliterative bronchiolitis, glomerular nephritis, rheumatoid arthritis.
• diseases such as inflammatory conditions, coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, inflammation, certain kidney diseases associated with proliferation of glomerular or mesangial cells, and ocular diseases associated with retinal vessel proliferation, psorias
• the present invention also provides for treating, preventing, modulating, etc., one or more of the following conditions in humans and/or other mammals: retinopathy, including diabetic retinopathy, ischemic retinal-vein occlusion, retinopathy of prematurity and age related macular degeneration; rheumatoid arthritis, psoriasis, or bullous disorder associated with subepidermal blister formation, including bullous pemphigoid, erythema multiforme, or dermatitis herpetiformis, rheumatic fever, bone resorption, postmenopausal osteoperosis, sepsis, gram negative sepsis, septic shock, endotoxic shock, toxic shock syndrome, systemic inflammatory response syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), Jarisch-Herxheimer reaction, asthma, adult respiratory distress syndrome, acute pulmonary fibrotic disease, pulmonary sarcoidosis, allergic respiratory disease, silicos
• the present invention provides methods of treating any of the aforementioned diseases and/or conditions (including those mentioned in any of the cited references), comprising administering effective amounts of a compound of formula I and at least one second compound that is an inhibitor of the PI3K/AKT signalling pathway (e.g. rapamycin or a derivative or analog of rapamycin, or wortmannin or a derivative or analog of wortmannin).
• a compound of formula I e.g. rapamycin or a derivative or analog of rapamycin, or wortmannin or a derivative or analog of wortmannin.
• An "effective amount” is the quantity of the compound that is useful to achieve the desired result, e.g., to treat the disease or condition.
• the present invention also relates to methods of inhibiting angiogenesis in a system comprising cells, comprising administering to the system a combination of effective amounts of compounds described herein.
• a system comprising cells can be an in vivo system, such as a tumor in a patient, isolated organs, tissues, or cells, in vitro assays systems (CAM, BCE, etc), animal models (e.g., in vivo, subcutaneous, cancer models), hosts in need of treatment (e.g., hosts suffering from diseases having an angiogenic component, such as cancer; experiencing restenosis), etc.
• the drug combinations can be administered to modulate one or more the following processes, cell growth (e.g., proliferation), tumor cell growth (including, e.g., differentiation, cell survival, and/or proliferation), tumor regression, endothelial cell growth (including, e.g., differentiation, cell survival, and/or proliferation), angiogenesis (blood vessel growth), angiogenesis, and/or hematopoiesis (e.g., proliferation, T-cell development, etc.).
• cell growth e.g., proliferation
• tumor cell growth including, e.g., differentiation, cell survival, and/or proliferation
• tumor regression including, e.g., endothelial cell growth (including, e.g., differentiation, cell survival, and/or proliferation)
• angiogenesis blood vessel growth
• angiogenesis hematopoiesis
• Compounds or drug combinations of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosal, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active or inactive. They can be administered in any effective dosage, e.g., from about 0.1 to about 200 mg/kg of total body weight.
• the combinations of the present invention can be administered at any time and in any effective form.
• the compounds can be administered simultaneously, e.g., as a single composition or dosage unit (e.g., a pill or liquid containing both compositions), or they can be administered as separate compositions, but at the same time (e.g., where one drug is administered intravenously and the other is administered orally or intramuscularly.
• the drugs can also be administered sequentially at different times.
• Agents can be formulated conventionally to achieve the desired rates of release over extended period of times, e.g., 12-hours, 24-hours. This can be achieved by using agents and/or their derivatives which have suitable metabolic half-lives, and/or by using controlled release formulations.
• the drug combinations can be synergistic, e.g., where the joint action of the drugs is such that the combined effect is greater than the algebraic sum of their individual effects.
• reduced amounts of the drugs can be administered, e.g., reducing toxicity or other deleterious or unwanted effects, and/or using the same amounts as used when the agents are administered alone, but achieving greater efficacy, e.g., in having more potent antiproliferative and pro-apoptotic action.
• additives include any of the substances already mentioned, as well as any of those used conventionally, such as those described in Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition,
• pharmaceutically acceptable carriers are combined with the active drug and can be administered safely to a subject for therapeutic purposes.
• compounds or drug combinations of the present invention can be administered with other active agents or therapies (e.g., radiation) that are utilized to treat any of the above- mentioned diseases and/or conditions.
• active agents or therapies e.g., radiation
• the present invention provides combinations of at least one compound of Formula I and at least one second compound which is a PI3K/AKT signalling pathway inhibitor useful in treating a disease or disorder.
• “Combinations” for the purposes of the invention include:
• compositions or dosage forms which contain at least one compound of Formula I and at least one second compound which is an PI3K/AKT signalling pathway inhibitor;
• -kits which comprise at least one compound of Formula I and at least one second compound which is an PI3 K/ AKT signalling pathway inhibitor packaged separate from one another as unit dosages or as independent unit dosages, with or without instructions that they be administered concurrently or sequentially; and -separate independent dosage forms of at least one compound of Formula I and at least one second compound which is an PI3K/AKT signalling pathway inhibitor which cooperate to achieve a therapeutic effect, e.g., prophylaxis or treatment of the same disease, when administered concurrently or sequentially.
• a therapeutic effect e.g., prophylaxis or treatment of the same disease
• each agent of the combination can be selected with reference to the other and/or the type of disease and/or the disease status in order to provide the desired therapeutic activity.
• the active agents in the combination can be present and administered in a fixed combination.
• "Fixed combination” is intended here to mean pharmaceutical forms in which the components are present in a fixed ratio that provides the desired efficacy. These amounts can be determined routinely for a particular patient, where various parameters are utilized to select the appropriate dosage (e.g., type of cancer, age of patient, disease status, patient health, weight, etc.), or the amounts can be relatively standard.
• the combination can comprise effective amounts of at least one compound of Formula I and at least one second compound which is a PI3K/AKT signalling pathway inhibitor, which achieves a greater therapeutic efficacy than when either compound is used alone.
• the combination can be
• the relative ratios of each compound in the combination can also be selected based on their respective mechanisms of action and the disease biology. For example, activating mutations of the B-RAF gene are observed in more than 60% of human melanomas and a composition for treatment of melanoma may advantageously comprise a formula I compound in a more potent amount than the compound which is a P13K/AKT signalling pathway inhibitor.
• a cancer is associated with a mutation in the PI3K/AKT signalling pathway (e.g., ovarian and breast cancers)
• an agent which has activity in this signalling pathway can be present in more potent amounts relative to the Ref/MEK/ERK pathway inhibitor.
• the relative ratios of each compound can vary widely and this invention includes combinations for treating cancer where the amounts of the formula I compound and the second active agent can be adjusted routinely such that either is present in higher amounts.
• the release of one or more agents of the combination can also be controlled, where appropriate, to provide the desired therapeutic activity when in a single dosage form, combination pack, kit or when in separate independent dosage forms.
• Activity of combinations of the present invention can be determined according to any effective in vitro or in vivo method.
• Kinase activity can be determined routinely using conventional assay methods.
• Kinase assays typically comprise the kinase enzyme, substrates, buffers, and components of a detection system.
• a typical kinase assay involves the reaction of a protein kinase with a peptide substrate and an ATP, such as 32 P-ATP, to produce a phosphorylated end-product (for instance, a phosphoprotein when a peptide substrate is used).
• the resulting end-product can be detected using any suitable method.
• a radioactively labeled phosphoprotein can be separated from the unreacted gamma- 32 P-ATP using an affinity membrane or gel electrophoresis, and then visualized on the gel using autoradiography or detected with a scintillation counter.
• Nonradioactive methods can also be used. Methods can utilize an antibody which recognizes the " " phosphorylated substrate, e.g., an anti-phosphotyrosine antibody.
• kinase enzyme can be incubated with a substrate in the presence of ATP and kinase buffer under conditions which are effective for the enzyme to phosphorylate the substrate..
• the reaction mixture can be separated, e.g., electrophoretically, and then phosphorylation of the substrate can be measured, e.g., by Western blotting using an anti-phosphotyrosine antibody.
• the antibody can be labeled with a detectable label, e.g., an enzyme, such as HRP, avidin or biotin, chemiluminescent reagents, etc.
• detectable label e.g., an enzyme, such as HRP, avidin or biotin, chemiluminescent reagents, etc.
• Other methods can utilize ELISA formats, affinity membrane separation, fluorescence polarization assays, luminescent assays, etc.
• TR- FRET time-resolved fluorescence resonance energy transfer
• a c-Raf kinase assay can be performed with a c-Raf enzyme activated (phosphorylated) by Lck kinase.
• Lck-activated c-Raf (Lck/c-Raf) is produced in Sf9 insect cells by co-infecting cells with baculoviruses expressing, under the control of the polyhedrin promoter, GST-c-Raf (from amino acid 302 to amino acid 648) and Lck (full-length). Both baculoviruses are used at the multiplicity of infection of 2.5 and the cells are harvested 48 hours post infection.
• MEK-I protein is produced in Sf9 insect cells by infecting cells with the baculovirus expressing GST-MEK-I (full-length) fusion protein at the multiplicity of infection of 5 and harvesting the cells 48 hours post infection. Similar purification procedure is used for GST-c-Raf 302-648 and GST-MEK-I .
• Transfected cells are suspended at 100 mg of wet cell biomass per mL in a buffer containing 10 mM sodium phosphate, 140 mM sodium chloride pH 7.3, 0.5% Triton X-100 and the protease inhibitor cocktail.
• the cells are disrupted with a Polytron homogenizer and centrifuged 30,00Og for 30 minutes. The 30,000g supernatant is applied applied onto GSH-Sepharose.
• the resin is washed with a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100.
• the GST-tagged proteins are eluted with a solution containing 100 mM Glutathione, 50 mM Tris, pH 8.0, 150 mM NaCl and 0.01% Triton X-100.
• the purified proteins are dialyzed into a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl and 20% Glycerol.
• Test compounds are serially diluted in DMSO using three-fold dilutions to stock concentrations ranging typically from 50 ⁇ M to 20 nM (e.g., final concentrations in the assay can range from 1 ⁇ M to 0.4 nM).
• the c-Raf biochemical assay is performed as a radioactive filtermat assay in 96- well Costar polypropylene plates (Costar 3365). The plates are loaded with 75 ⁇ L solution containing 50 mM HEPES pH 7.5, 70 mM NaCl, 80 ng of Lck/c-Raf and 1 ⁇ g MEK-I .
• Raf activity can also be monitored by its ability to initiate the cascade leading to ERK phosphorylation (i.e., raf/MEK/ERK), resulting in phospho-ERK.
• a Bio-Plex Phospho-ERKl/2 immunoassay can be performed as follows:
• MDA-MB-231 cells are plated at 50,000 cells per well in 96-well microtitre plates in complete growth media. For effects of test compounds on basal pERKl/2 inhibition, the next day after plating, MDA-MB-231 cells are transferred to DMEM with 0.1% BSA and incubated with test compounds diluted 1 :3 to a final concentration of 3 mM to 12 nM in 0.1% DMSO. Cells are incubated with test compounds for 2 h, washed, and lysed in Bio-Plex whole cell lysis buffer A.
• Samples are diluted with buffer B 1 :1 (v/v) and directly transferred to assay plate or frozen at -80 C degrees until processed.
• 50 mL of diluted MDA-MB-231 cell lysates are incubated with about 2000 of 5 micron Bio-Plex beads conjugated with an anti-ERKl/2 antibody overnight on a shaker at room temperature.
• biotinylated phospho-ERK 1/2 sandwich immunoassay is performed, beads are washed 3 times during each incubation and then 50 mL of PE-strepavidin is used as a developing reagent.
• the relative fluorescence units of pERKl/2 is detected by counting 25 beads with Bio-Plex flow cell
• the IC50 is calculated by taking untreated cells as maximum and no cells (beads only) as background.
• PKI3 activity can be determined routinely, e.g., using commercially available kits (e.g., Perkin- Elmer, FlashPlate Platform), Frew et al., Anticancer Res., 14(6B):2425-8, 1994. See also, publications listed under PKI3 inhibitors.
• AKT can be isolated from insect cells expressing His-tagged AKTl (aa 136-480) as described in WO 0501 1700. Expressing cells are lysed in 25 mM HEPES, 100 mM NaCl, 20 mM imidazole; pH 7.5 using a polytron (5 mis lysis buffer/g cells). Cell debris is removed by centrifuging at 28,000 x g for 30 minutes. The supernatant is filtered through a 4.5 micron filter then loaded onto a nickel-chelating column pre-equilibrated with lysis buffer.
• the column is washed with 5 column volumes (CV) of lysis buffer then with 5 CV of 20% buffer B, where buffer B is 25 mM HEPES, 100 mM NaCl, 300 mM imidazole; pH 7.
• His-tagged AKTl (aa 136-480) is eluted with a 20-100% linear gradient of buffer B over 10 CV.
• His-tagged AKTI (136-480) eluting fractions are pooled and diluted three-fold with buffer C, where buffer C is 25 mM HEPES, pH 7.
• the sample is then chromatographed over a Q-Sepharose HP column pre-equilibrated with buffer C.
• the column is washed with 5 CV buffer C, then step eluted with 5 CV 10 %D, 5 CV 20%D, 5 CV 30% D, 5 CV 50% D. and 5 CV of 100% D; where buffer D is 25 mM HEPES, 1000 mM NaCl; pH 7.5.
• His-tagged AKTI (aa 136-480) containing fractions are pooled and concentrated in a 10-kDa molecular weight cutoff concentrator. His-tagged AKTl (aa 136-480) is chromatographed over a Superdex 75 gel filtration column pre-equilibrated with 25 mM HEPES, 200 mM NaCl, 1 mM DTT; pH 7.5. His-tagged AKTl (aa 136-480) fractions are examined using SDS-PAGE and mass spec. The protein is pooled, concentrated, and stored at 80 0 C.
• His-tagged AKT2 (aa 138-481) and His-tagged AKT3 (aa 135-479) can be isolated and purified in a similar fashion.
• AKT Enzyme Assay Compounds can be tested for AKT protein serine kinase inhibitory activity in substrate phosphorylation assays. This assay examines the ability of small molecule organic compounds to inhibit the serine phosphorylation of a peptide substrate.
• the substrate phosphorylation assays use the catalytic domains of AKT 1, 2, or 3.
• AKT 17 2 and 3 are also commercially available from Upstate USA, Inc.
• the method measures the ability of the isolated enzyme to catalyze the transfer of the gamma-phosphate from ATP onto the serine - 72 residue of a biotinylated synthetic peptide (Biotin-ahx-ARKRERAYSFGHHA-amide).
• Substrate phosphory- lation can be detected by the following procedure described in WO 05011700.
• Assays are performed in 384 well U-bottom white plates. 10 nM activated AKT enzyme is incubated for 40 minutes at room temperature in an assay volume of 20ul containing 50 mM MOPS, pH 7.5, 20 mM MgCl 2 , 4uM ATP, 8uM peptide, 0.04 uCi [g- 33 P] ATP/well, " l mM CHAPS, 2 mM DTT, and 1 ⁇ l of test compound in 100% DMSO.
• the reaction is stopped by the addition of 50 ⁇ l SPA bead mix (Dulbecco's PBS without Mg2+ and Ca2+, 0.1 % Triton X-100, 5 mM EDTA, 50 ⁇ M ATP, 2.5mg/ml Streptavidin-coated SPA beads).
• 50 ⁇ l SPA bead mix (Dulbecco's PBS without Mg2+ and Ca2+, 0.1 % Triton X-100, 5 mM EDTA, 50 ⁇ M ATP, 2.5mg/ml Streptavidin-coated SPA beads).
• the plate is sealed, the beads are allowed to settle overnight, and then the plate was counted in a Packard Topcount Microplate Scintillation Counter (Packard Instrument Co., Meriden, CT).
• proliferation assays can be performed by any suitable method.
• a breast carcinoma cell proliferation assay can be performed as follows.
• Other cell types can be substituted for the MDA-MB-231 cell line.
• Human breast carcinoma cells (MDA MB-231, NCI) are cultured in standard growth medium (DMEM) supplemented with 10% heat-inactivated FBS at 37 0 C in 5% CO 2 (vol/ vol) in a humidified incubator. Cells are plated at a density of 3000 cells per well in 90 ⁇ L growth medium in a 96 well culture dish. In order to determine T Oh CTG values, 24 hours after plating, 100 ⁇ L of CellTiter-Glo Luminescent Reagent (Promega) is added to each well and incubated at room temperature for 30 minutes. Luminescence is recorded on a Wallac Victor II instrument. The CellTiter-Glo reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present, which, in turn is directly proportional to the number of cells present.
• DMEM standard growth medium
• FBS heat-inactivated FBS
• Test compounds are dissolved in 100% DMSO to prepare 10 mM stocks. Stocks are further diluted 1 :400 in growth medium to yield working stocks of 25 ⁇ M test compound in 0.25% DMSO. Test compounds are serially diluted in growth medium containing 0.25% DMSO to maintain constant - DMSO concentrations for all wells. 60 ⁇ L of diluted test compound are added to each culture well to give a final volume of 180 ⁇ L. The cells with and without individual test compounds are incubated for 72 hours at which time ATP dependent luminescence was measured, as described previously, to yield T 72h values. Optionally, the IC 50 values can be determined with a least squares analysis program using compound concentration versus percent inhibition.
• T 72h t est ATP dependent luminescence at 72 hours in the presence of test compound
• One useful model to study angiogenesis is based on the observation that, when a reconstituted basement membrane matrix, such as Matrigel, supplemented with growth factor (e.g., FGF-I), is injected subcutaneously into a host animal, endothelial cells are recruited into the matrix, forming new blood vessels over a period of several days. See, e.g., Passaniti et al., Lab. Invest., 67:519- 528, 1992.
• growth factor e.g., FGF-I
• angiogenesis can be temporally dissected, permitting the identification of genes involved in all stages of angiogenesis, including, e.g., migration of endothelial cells into the matrix, commitment of endothelial cells to angiogenesis pathway, cell elongation and formation of sac-like spaces, and establishment of functional capillaries comprising connected, and linear structures containing red blood cells.
• the growth factor can be bound to heparin or another stabilizing agent.
• the matrix can also be periodically re-infused with growth factor to enhance and extend the angiogenic process.
• neovascularization of tumor explants e.g., U.S. Pat. Nos. 5,192,744; 6,024,688
• CAM chicken chorioallantoic membrane
• BCE bovine capillary endothelial
• HUVEC human umbilical cord vascular endothelial cell growth inhibition assay
• the present invention provides one or more of the following features.
• a method of treating any of the aforementioned diseases and/or conditions comprising administering effective amounts of a compound of formula I and a second compound which is an PI3K/AKT signalling pathway inhibitor.
• a method of modulating (e.g., inhibiting) one or more aforementioned activities comprising administering effective amounts of a compound of formula I and a second compound which is an PI3K/ AKT signalling pathway inhibitor.
• Combinations comprising a compound of formula I and a second compound which- is an PI3K/AKT signalling pathway inhibitor.

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