JP2008530239A - Anti-angiogenic agents containing aldesleukin - Google Patents

Abstract

The present invention relates to combination therapy using an IL-2 composition and an anti-angiogenic agent to treat cancer. The present invention further provides methods of alleviating the toxicity associated with administration of IL-2 compositions or anti-angiogenic compositions and increasing efficacy. Treat patients suffering from disorders associated with abnormal growth that are resistant to, or cannot be adequately treated by, traditional drugs using drugs that target the body's immune response mechanisms and disease state substrates New methods and mechanisms for doing so are needed. The present invention provides such therapeutic agents and provides other related advantages.

Description

(Citation of related application)
This application claims priority under 35 USC 119 (e) of US Provisional Patent Application No. 60 / 654,341 (filed 18 February 2005). This application is hereby incorporated by reference in its entirety.

(Field of Invention)
The present invention relates to a method for treating a disease associated with abnormal cell proliferation. In some embodiments, recombinant IL-2 is combined with an anti-angiogenic agent for use in the treatment of cancer.

(Background of the Invention)
Capillaries extend into almost every tissue in the human body, providing oxygen and nutrients to the tissue, and removing waste products. Under typical conditions, endothelial cells that lie along the inside of capillaries do not divide, and therefore capillaries usually do not increase in number or size in human adults. However, under certain conditions, such as when tissue is damaged or during certain periods of the menstrual cycle, capillaries begin to grow rapidly. This process of forming new capillaries from existing blood vessels is known as angiogenesis or angiogenesis. See Non-Patent Document 1. Angiogenesis during wound healing is an example of pathophysiological angiogenesis in adult life. During wound healing, additional capillaries provide a supply of oxygen and nutrients, promote granulation tissue and assist in the removal of waste products. After the healing process is complete, the capillaries usually disappear. (Non-patent document 2).

  Angiogenesis also plays an important role in the growth of cancer cells. When a cancer cell nest reaches a certain size, ie approximately 1-2 mm in diameter, the cancer cell grows larger because the diffusion is insufficient to provide sufficient oxygen and nutrients to the cancer cell It is known that a blood supply must be generated for this purpose. Therefore, inhibition of angiogenesis is expected to stop the growth of cancer cells. Of the angiogenic factors that have been identified, vascular endothelial growth factor (VEGF) is the most potent and specific and has been identified as an important regulator in both normal and pathological angiogenesis, Other factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet derived growth factor (PDGF) have also been associated with angiogenesis and cancer cell survival. Curr Opin Investig Drugs, February 2001; 2 (2): 280-6.

  Specific anti-tumor immunity involves the activation of multiple cell types in the immune system, the most efficient being cytolytic T lymphocytes. To elicit a specific, anti-tumor T lymphocyte-mediated immune response, not only the tumor antigen of interest, but also specific ligands and targets present on the surface of either tumor cells or antigen-presenting cells Recognition of costimulatory interactions with T lymphocytes is also necessary. This second costimulatory signal binds to specific cell surface receptors and initiates various signaling pathways, resulting in enhanced effector function, such as soluble cytokines or other peptide molecules It can also be provided by factors.

  Interleukin-2 (IL-2) binds to a specific receptor present on T cells and natural killer (NK) cells for tumor cell lysis, cytokine secretion, and other effector functions It is a cytokine derived from T lymphocytes that activates them. Both CD4 and CD8 T lymphocytes express the receptor for IL-2 and develop increased cytolytic effector and cytokine synthesis functions after exposure to biologic. The functional effect of IL-2 is mediated by binding to the IL-2 receptor (IL-2R), which is structurally shared with three subunits: α, β, and other cytokine receptors (Caligiuri et al., J. Exp. Med., 1990, 171 (5): 1509-1526; Bazan, J.F., Science, 1992, 257 (5068)): 410-413; Theze et al., Immunol. Today, 1996, 17 (10): 481-486). Activation of IL-2 receptor and subsequent signaling depends on the type of IL-2 receptor on the cell (Caligiuri et al., J. Exp. Med., 1990, 171 (5): 1509-1526; Voss J. Exp. Med., 1992, 176 (2): 531-541; Expinoza et al., J. Leukoc. Biol., 1995, 57 (1): 13-29; Theze et al., Immunol. Today, 1996, 17 (10): 481-486). T cells express high affinity IL-2Rαβγ, while NK cells, monocytes, and macrophages mainly express a moderate affinity form, IL-2Rβγ (Caligiuri et al., J. Exp). Med., 1990, 171 (5): 1509-1526; Voss et al., J. Exp. Med., 1992, 176 (2): 531-541; Theze et al., Immunol. Today, 1996, 17 (10): 481-486; Nagler et al., J. Exp. Med., 1990, 171 (5): 1527-1533). At the heart of its mechanism, IL-2 activates JAK (JAK1, JAK3), which phosphorylates key tyrosine on IL-2Rβ, which is the binding site for downstream signaling molecules including Shc and Stat5 It then catalyzes the activation of two different pathways, namely Shc / Ras / Raf / MAPK and JAK / STAT5, which translocate to the nucleus and activate STAT, a family of gene-regulated transcription factors Direct control (O'Shea, JJ, Immunity, 1997, 7 (1): 1-11). She calls the Grb-2 / Gos-2 to activate the Ras / Raf / MAPK pathway by activating the Grb-2 / Sos complex and also to activate the phosphatidylinositol 3-kinase (PI3K) pathway. Together, these signaling proteins control gene transcription factors and ultimately regulate cell growth, division, differentiation and immune activity. In addition, IL-2 also induces anti-apoptotic effects by regulating mitogenic genes, which leads to an increase in bcl-2, bcl-xl, c-myb, which is the cdk2,4,6 It affects cell cycle regulation by activation and inhibition of p27kip1, or is negatively controlled by SOCS (a suppressor of cytokine signaling) (Smith, KA, Science, 1988, 240 (4856). ): 1169-1176; Theze et al., Immunol. Today, 1996, 17 (10): 481-486). IL-2 and IL-2R interactions have been reported to trigger activation of downstream MAPK, PI-3K, and JAK / STAT signaling pathways leading to cell survival and proliferation (Miyazaki et al., Science, 1994, 266 (5187): 1045-1047; Beadling et al., Embo J., 1996, 15 (8): 1902-1913; Nakajima et al., Immunity, 1997, 7 (5 ): 691-701; Moriggl et al., Immunity, 1999, 11 (2): 225-230).

  Preclinical studies in various mouse tumor models have shown that recombinant human IL-2 has been shown to reduce tumor burden, long-term survival, and tumor resuscitation when administered to tumor-bearing animals for a period of 10-14 days. It has been demonstrated that it can lead to increased resistance to growth. Analysis of spleen lymphocytes obtained from these animals showed that the anti-tumor effect was due, at least in part, to the enhanced function of cytolytic T cells. This effect includes activation of both direct cytolysis of tumor cells by CTL and increased synthesis of other T lymphocyte-derived cytokines that may also have further direct or indirect anti-tumor effects. .

  Since metastatic melanoma and renal cell carcinoma (RCC) are very refractory diseases and are resistant to most types of systemic chemotherapy, there is still a considerable need to develop more effective treatments. Recent clinical data support the development of small molecule receptor tyrosine kinase (RTK) inhibitors in melanoma and renal cell carcinoma, particularly BAY 43-9006 or SU11248. Both BAY 43-9006 and SU11248 inhibit multiple kinases that are involved in tumor growth, proliferation and anti-angiogenesis. BAY 43-9006 is a potent inhibitor of Raf-1 which is a member of the Raf / MEK / ERK signaling pathway (Richly et al., Int. J. Clin. Pharmacol. Ther., 2003, 41 (12): Wilhelm et al., Cancer Res., 2004, 64 (19): 7099-7109; Awada et al., Br. J. Cancer, 2005, 92 (10): 1855-1861), WT B -Inhibits both Raf and mutant V599E B-Raf. In addition to the effects of BAY 43-9006 on Raf, most of its activity is attributed to other RTKs, particularly VEGFR (VEGFR-2 and VEGFR-3), PDGFRβ, and other major kinases FLT-3, and cKIT. (Wilhelm et al., Cancer Res., 2004, 64 (19): 7099-7109). The response with BAY 43-9006 in early solid tumor trials has established that once-daily or twice-daily administration can result in disease stabilization, including some partial response. To date, Phase I studies have shown that BAY 43-9006 is generally well tolerated by patients. The most common toxicities of Bay 43-9006 included gastrointestinal effects (diarrhea, nausea, abdominal cramps) or skin reactions including pruritus or rash (Richly et al., Int. J. Clin. Pharmacol. Ther., 2003, 41 (12): 620-621; Ahmad and Eisen, Clin. Cancer Res., 2004, 10 (18 part 2): 6388S-92S; Awada et al., Br. J. Cancer, 2005, 92 (10): 1855-1861; Strumberg et al., J. Clin. Oncol., 2005, 23 (5): 965-972).

  In contrast, SU11248 is a very potent selective RTK inhibitor of VEGFR-1 to 3, PDGFRα, cKIT, FLT3 and PDGFRβ (Abrams et al., Mol. Cancer Ther., 2003, 2 (5): 471-478; Mendel et al., Clin. Cancer Res., 2003, 9 (1): 327-337; O'Farrel et al., Clin. Cancer Res., 2003, 9 (15): 5465-5476. ). SU11248 has been shown to have anti-tumor activity in several advanced solid tumors (RCC, neuroendocrine tumors, stromal tumors and adenocarcinoma) (Faivre et al., J. Clin. Oncol., 2006, 24 (1 ): 23-35; Motzer et al., J. Clin. Oncol., 2006, 24 (1): 16-24). The clinical data of SU11248 also show that this drug has manageable toxicity including fatigue, lymphopenia, neutropenia, hyperlipaseemia and is generally tolerated (Faivre). Et al., J. Clin. Oncol., 2006, 24 (1): 23-35; Motzer et al., J. Clin. Oncol., 2006, 24 (1): 16-24).

  A genotypic study of clear cell renal cancer that occurs in von Hippel-Lindau syndrome has led to the identification of the von Hippel-Lindau tumor suppressor gene (VHL). This gene is hereditary renal cell carcinoma (one mutation is a germline mutation) and in most cases of sporadic clear cell renal cancer (both alleles gain mutations or deletions). In both cases). Gnarra JR, Duan DR, Weng Y, et al., “Molecular cloning of the von Hippel-Lindau tumour suppressor gene and it roll in real carcinoma.” Biochia. 1996; 1242 (3): 201-10. One consequence of these mutations is the overproduction of vascular endothelial growth factor by mechanisms involved in hypoxia-inducible factors. O Illiopoulos AL, C Jiang et al., “Negative Regulation of Hyperxia-inducible genes by the Von-Hippel Linda Protein.”, Proc Natl Acad Sc. 1996; 93: 10595-10599. Therefore, von Hippel-Lindau protein is closely related to angiogenesis by thereby controlling vascular endothelial growth factor. Vascular endothelial growth factor stimulates endothelial cell growth and, as mentioned, is considered to be a central factor in angiogenesis, especially in embryogenesis, ovulation, wound healing, and tumor growth. Ferrara N. "Molecular and biological properties of basal endothelial growth factor." Mol. Med. 1999; 77: 527-543.

  Because mutations in the von Hippel-Lindau tumor suppressor gene cause vascular endothelial growth factor to be overproduced by the tumor, this growth factor represents a good target in the treatment of clear cell kidney cancer. A randomized, double-blind, placebo-controlled study of a humanized monoclonal antibody (bevacizumab) in patients suffering from metastatic clear cell kidney cancer has been conducted by Yang et al. "A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for 3 years, 3rd, 3rd. Tumor progression time was extended 2.55 times in patients who received bevacizumab per kilogram (10 mg / kg every 2 weeks) compared to patients in the placebo group. Because survival was not the primary endpoint of the trial, patients could switch from placebo to bevacizumab treatment as the disease progressed. Same as above. Treatment of renal cancer targeting the mechanism of angiogenesis may be effective by routes other than those mediated by vascular endothelial growth factor. Within the local microenvironment of some tumors are other proteins that can promote angiogenesis. Thus, there is a need for anti-angiogenic treatments that utilize rational combinations of inhibitors, as directed by an understanding of renal cell carcinoma biology.

  Recombinant human interleukin-2 (Aldesleukin) is approved by the FDA for the treatment of metastatic kidney cancer based on the results of multiple multicenter trials in 255 patients who received intermittent high-dose bolus regimens Has been. In these trials, the desired response was seen in 15% of patients with a median survival of 16.3 months (Fisher and Rosenberg, 2000, Cancer J Sci Am, 6S1: S55-57). Due to the significant toxicity associated with this regimen, a series of Phase I and Phase II trials were attempted using different doses of rIL-2 and different routes of administration. A recent review of the efficacy of rIL-2 as a single agent suggests that in over 1700 patients suffering from metastatic RCC treated in this series of studies, the overall response may be 15% It is shown. A complete response was seen in 3-5% of patients. Bukowski RM. , “Natural history and therapeutic of metabolic ren cell carcinoma: the role of interleukin-2”, Cancer, 1997; 80 (7): 198-220. There appears to be no difference in speed between these paths. Randomized comparisons between the high-dose bolus IV regimen, the low-dose bolus IV regimen, and the subcutaneous outpatient regimen showed no difference in median survival between groups, but the low-dose and outpatient regimen It was significantly less toxic. Yang JC, Sherry RM, Steinberg SM, et al., “Randomized study of high-dose and low-dose interleukin-2 in covalent with the 21st.

  In addition to having a direct immunomodulatory effect, rIL-2 may also prevent tumor growth by causing secondary cytokine release (IFNγ) from activated NK cells. Saraya KA, Bulkwill FR, “Temporal sequence and cellular origin of interleukin-2 simulated cytogene gene expression.”, British Journal 3 (19);

  Response speed and outcome of clear cell renal cancer patients can be improved. Clinical trials using new drug combinations are needed. The combination of rIL2 and an anti-angiogenic agent such as bevacizumab may be an additional clinical benefit for patients suffering from metastatic renal cell carcinoma, and patients suffering from other cancers May have some synergistic effect.

Another advantage of the preferential approach to cancer regression is that it provides a platform that makes bypassing by resistance mutations more difficult. If the therapeutic target is very biased and specific, such as a specific substrate in a viral replicon or a kinase in a cancer cell line (this may be necessary to avoid targeting host cells ), A single point mutation in the pathology can be unaffected by the drug, resulting in more severe disease types in earlier generations.
Folkman, J. et al. , Scientific American, 275, 150-154, (1996). Lymbussaki, A .; , "Vascular Endothelial Growth Factors and their Receptors in Embryos, Adults, and in Tumors", Academic Dissertation, University of Helsinki, Molecular / Cancer Biology Laboratory and Department of Pathology, Haartman Institute, (1999 years)

  Treat patients suffering from disorders associated with abnormal growth that are resistant to, or cannot be adequately treated by, traditional drugs using drugs that target the body's immune response mechanisms and disease state substrates New methods and mechanisms for doing so are needed. The present invention provides such therapeutic agents and provides other related advantages.

(Concise Summary of Invention)
The present invention is based in part on a unique combination therapy with non-overlapping toxicity using a small molecule receptor tyrosine kinase inhibitor and rIL-2. Considering that melanoma and RCC are generally responsive to immunotherapy such as rIL-2, a reasonable combination of rIL-2 and a potential receptor tyrosine kinase inhibitor could be Based on the pharmacology of The present invention provides a clinically applicable drug schedule to avoid potential drug interactions based on the toxicity profiles of these agents. The potential for adverse pharmacokinetic / pharmacodynamic interactions and harmful pharmacological interactions was also elucidated. The effects of rIL-2 and small molecule receptor tyrosine kinase inhibitors such as BAY 43-9006 on T cells and the effects on IL-2 mediated signaling pathways (MAPK and JAK / STAT5) are described.

  Thus, the present invention provides an indication of the anti-tumor efficacy of IL-2 compounds such as recombinant IL-2 (rIL-2, also known as aldesleukin) against cancer cell lines that are poorly responsive to conventional therapies. Resulting in increased immunity against long-term survival and tumor reloading. Furthermore, the immunostimulatory effect of rIL-2 aims to alleviate existing side effects caused by administration of the anti-angiogenic composition to be administered simultaneously. Provided is a method of treating a subject suffering from abnormal cell proliferation, particularly renal cell carcinoma, using a combination of an IL-2 compound such as rIL-2 and at least one anti-angiogenic agent. Further provided is a method of treating a subject suffering from abnormal cell proliferation, particularly renal cell carcinoma, using a combination of an IL-2 compound such as rIL-2 and a small molecule. Preferred small molecules of the present invention are shown in Tables 1-5. A particularly preferred molecule is N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide. (Also known as SU11248) and 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea (Bay 43-9006 Also known as).

  The aldesleukin and the anti-angiogenic agent can be administered together or separately as separate pharmaceutical compositions. When administered separately, the aldesleukin can be administered prior to, simultaneously with, or subsequent to the anti-angiogenic agent. Aldesleukin and 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) ) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H -Pyrrol-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, and 1- (4- (2- (methylcarbamoyl) pyridine-4- Once a day for simultaneous administration of an anti-angiogenic agent such as (yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, Provide specific regimen comprising one, and monthly dosing schedules (or repetition).

  In one embodiment, an anti-angiogenic agent, preferably 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N— (3-Chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl ) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, and 1- (4- (2 One of-(methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea inhibits VEGF It is administered concurrently with the anti-angiogenic proteins such as monoclonal antibodies with force. In a more detailed embodiment, the anti-angiogenic protein of the invention is bevacizumab or VEGF-Trap. In another more detailed embodiment, the protein is an EGF inhibitor such as cetuximab.

  In another embodiment, the efficacy of the anti-angiogenic agent is achieved by administering the aldesleukin and the anti-angiogenic agent (or multiple anti-angiogenic agents) together in the manner described herein. Enhanced, resulting in an improved positive / synergistic therapeutic response over that observed with the inhibitor alone.

  Other embodiments include a container, a therapeutically effective amount of aldesleukin, a therapeutically effective amount of an anti-angiogenic agent and / or a small molecule, preferably 6,7-bis (2-methoxyethoxy) -N- (3 -Ethynylphenyl) quinazoline-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) Ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridine -4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro- - comprising a compound according to Tables 1-5, such as (trifluoromethyl) phenyl) urea, for treating a patient suffering from cancer provides a therapeutic package suitable for commercial.

  In a further embodiment, a kit is provided. The kit comprises (a) aldesleukin and (b) 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine for simultaneous, sequential or separate use. , 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5- Fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine- 1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl Comprising an anti-angiogenic agent selected from urea, comprising a combination of a medicament for treating a patient suffering from cancer.

  The methods of manufacturing combinations described herein are provided and contemplated to be within the scope of the present invention, as are the use of combinations in the manufacture of pharmaceuticals for use in the methods of the present invention.

  Further embodiments of the invention include those described in the best mode for carrying out the invention.

(Detailed description of the invention)
Using detection assays, several cancers have been characterized as being sensitive to modulation with anti-angiogenesis / VEGF inhibitors and cytokine administration or rIL-2. Such cancers include, for example, CML, AML, breast, stomach, endometrium, salivary gland, adrenal gland, non-small cell lung, pancreas, kidney, rectum, skin, melanoma, multiple myeloma, brain / CNS, Cervix, nasopharynx, malignant mesothelioma, hypopharynx, gastrointestinal carcinoid, peritoneum, omentum, mesentery, gallbladder, testis, lung, thyroid, ovary, peritoneum, prostate, head and neck, bladder, colon, Colorectal, lymphoma, and glioblastoma are included. The methods described herein are useful for the treatment of any such cancer.

  Treatment with a combination of aldesleukin and at least one anti-angiogenic agent in the manner described herein is for the treatment of cancers in which invariant proliferating cells are highly dependent on vascularization such as VEGF. Causes a beneficial physiological response.

  One embodiment of the present invention treats cancer patients suffering from hypotension from administration of aldesleukin comprising simultaneously administering to the patient a therapeutically effective amount of an anti-angiogenic agent to attenuate hypotension Provide a method.

  Another more detailed embodiment includes cancer remission in a patient.

  Another embodiment of the present invention is a method of treating a cancer patient suffering from hypertension from administration of an anti-angiogenic agent comprising simultaneously administering to the patient a therapeutically effective amount of aldesleukin to attenuate hypertension I will provide a.

  Another more detailed embodiment includes cancer remission in a patient. In certain embodiments, the cancer is sensitive to inhibition of angiogenesis and / or immunostimulation.

  In a more detailed embodiment of any of the preceding embodiments, the anti-angiogenic agent is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6 -(3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro- 2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine-1- From an amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Be-option.

  Another embodiment of the present invention relates to aldesleukin and 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy)- N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) ) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- A compound selected from (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Comprising administering, to provide a method of treating a patient suffering from cancer.

  Another embodiment of the present invention is that an anti-angiogenic agent is first administered at a dose capable of inducing hypoxia in a patient and then administered aldesleukin in a cancer patient. A method of increasing effectiveness is provided.

  Another embodiment provides a method of increasing the efficacy of an anti-angiogenic agent in a cancer patient, comprising reducing nitric oxide synthase by administering aldesleukin to the patient. Another embodiment provides a method of increasing anti-angiogenic agent efficacy in a cancer patient, comprising administering aldesleukin to a patient, wherein nitric oxide synthase is reduced by administering aldesleukin to the patient. provide.

  In a more detailed embodiment thereof, said anti-angiogenic agent is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy)- N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) ) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- Selected from (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

  Another embodiment of the present invention comprises first administering to said patient a therapeutically effective amount of aldesleukin, and then 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazoline-4 -Amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-(( 5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) Phthalazine-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) And a step of administering an anti-angiogenic agent such as those selected from) phenyl) urea, provides a method of treating a patient suffering from cancer.

  Another embodiment of the present invention provides the patient with 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- ( 3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- A therapeutically effective amount of an anti-angiogenic agent such as (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is first administered. A step of then includes the step of administering aldesleukin, provides a method of treating a patient suffering from cancer.

  Another embodiment of the invention comprises separately administering to the patient a therapeutically effective amount of an anti-angiogenic agent and aldesleukin according to a dosing schedule, wherein the aldesleukin is about 1 to 3 times a day, about 9 to about Provided is a method of treating a patient suffering from cancer that is administered at a dose of 130 MIU / day for a period of at least 3 consecutive days, optionally followed by a rest period of at least 3 consecutive days.

  In more detailed embodiments thereof, the anti-angiogenic agent is administered 1-6 times every 2-3 weeks.

  In a more detailed embodiment thereof, said anti-angiogenic agent is a VEGF inhibitor.

  In its more detailed embodiment, the anti-angiogenic agent is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy)- N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) ) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- Selected from (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

  In a more detailed embodiment thereof, aldesleukin is administered intravenously and there is a rest period.

  In a more detailed embodiment thereof, aldesleukin is administered subcutaneously and there is no such rest period.

  In more detailed embodiments thereof, aldesleukin is administered three times daily at a dose of about 30-100 MIU / day.

  In its more detailed embodiment, aldesleukin is administered continuously for a period of 5 days, followed by a 9-day rest period.

  In a more detailed embodiment, the dosing schedule is repeated at least 2 cools. In an even more detailed embodiment, each course consists of 2-5 days of treatment followed by a 9-15 day rest period.

  In another more detailed embodiment, aldesleukin is administered three times on the first day and once a day each subsequent day.

  In a more detailed embodiment, the dosing schedule is repeated at least 2 cools, or 3 cools, or 4 cools, or 5 cools, or 6 cools, or 7 cools, or 8 cools, or 9 cools, or 10 cools.

  In more detailed embodiments of any of the foregoing embodiments, the cancer is colon cancer, renal cell carcinoma or malignant melanoma.

  In a more detailed embodiment of any of the preceding embodiments, at least one selected from acetaminophen, meperidine, indomethacin, ranitidine, nizatidine, diastop, loperamide, diphenhydramine, or furosemide when administering aldesleukin. One compound is also administered to the patient.

  In a preferred embodiment of any of the preceding embodiments, the anti-angiogenic agent is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3- Morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline) -3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1 -(4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

  Another embodiment of the present invention relates to an aldesleukin:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

, A tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer, and a method for treating a patient suffering from cancer.

  Another embodiment of the present invention relates to an aldesleukin:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

, A tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer, and a method for treating a patient suffering from cancer.

  Another embodiment of the present invention relates to an aldesleukin:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

, A tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer, and a method for treating a patient suffering from cancer.

  Another embodiment of the present invention relates to an aldesleukin:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

, A tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer, and a method for treating a patient suffering from cancer.

  Another embodiment of the present invention relates to an aldesleukin:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

, A tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer, and a method for treating a patient suffering from cancer.

  Another embodiment of the invention is a compound of formula I with aldesleukin:

［式中、
Ｒ_１は、アルキル、−アリール（Ｒ_ｉ）_ｐ、もしくはヘテロシクリルであり；
Ｒ_２は、Ｈもしくはアルキルであるか；または
Ｒ_１およびＲ_２は、一緒に結合してＲ_１−２を形成し；
Ｒ_３は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｃ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_４は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、−Ｏ−（ＣＨ_２）_ｑ−Ｒ_ｇ、−Ｏ−（ＣＨ_２）_ｑ−Ｏ−Ｒ_ｅ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、または−ヘテロシクリル−Ｒ_ｆであり；
Ｒ_５は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、−Ｏ−（ＣＨ_２）_ｑ−Ｒ_ｇ、−Ｏ−（ＣＨ_２）_ｑ−Ｏ−Ｒ_ｅ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_６は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｃ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_７は、Ｈ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｃ、アリール、ヘテロシクリル、もしくはアルコキシであるか；または
Ｒ_ａおよびＲ_ｂは、一緒に結合してＲ_１−２を形成し；
それぞれのＲ_ｃは、独立してＨ、アルキル、アルコキシ、−Ｃ（Ｏ）アルキル、−Ｃ（Ｏ）アリール、−ＣＨＯ、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのＲ_ｅは、独立してＨまたはアルキルであり；
Ｒ_ｆは、Ｈ、ハロゲン、−ＯＨ、−ＣＮ、−（ＣＨ_２）_ｑＮＲ_ａＲ_ｈ、アルコキシ、−Ｃ（Ｏ）Ｒ_ｃ、−（ＣＨ_２）_ｑＣＨ_３であり
それぞれのＲ_ｇは、独立してＨ、ハロゲン、−Ｃ（Ｏ）Ｒ_ｃ、アリール、ヘテロシクリル、または−ＮＲ_ａＲ_ｂであり；
Ｒ_ｈは、Ｈ、または−（ＣＨ_２）_ｑＳ（Ｏ）_ｎＲ_ｄであり；
それぞれのＲ_ｉは、独立してＨ、ハロ、アルキル、アルケニル、アルキニル、または−Ｏ（ＣＨ_２）_ｑ−Ｒ_ｇであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｐは、独立して０、１、２、または３であり；
それぞれのｑは、独立して０、１、または２であり；
Ｒ_１−２は、以下に示す一般構造を有し：

[Where:
R ₁ is alkyl, -aryl (R _i ) _p , or heterocyclyl;
R ₂ is H or alkyl; or R ₁ and R ₂ are joined together to form R _1-2 ;
R ₃ is, H, -CN, -OH, halogen, alkyl, aryl, _{alkoxy, -NR a R b, -C (O} ) R c, -S (O) n R d , or heterocyclyl;
R ₄ is H, —CN, —OH, halogen, alkyl, aryl, —O— (CH ₂ ) _q —R _g , —O— (CH ₂ ) _q —O—R _e , —NR _a R _b , -S _(O) n _{R d,} or - heterocyclyl -R _f;
R ₅ is H, —CN, —OH, halogen, alkyl, aryl, —O— (CH ₂ ) _q —R _g , —O— (CH ₂ ) _q —O—R _e , —NR _a R _b , It is -S _(O) n _{R d,} or heterocyclyl;
R ₆ is H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _c , —S (O) _n R _d , or heterocyclyl;
R ₇ is H, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —S (O) _n R _d , or heterocyclyl;
Each R _a and R _b is independently H, alkyl, —C (O) R _c , aryl, heterocyclyl, or alkoxy; or R _a and R _b are joined together to form R _{1 1- 2} ;
Each R _c is independently H, alkyl, alkoxy, —C (O) alkyl, —C (O) aryl, —CHO, aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each R _e is independently H or alkyl;
R _f is H, halogen, —OH, —CN, — (CH ₂ ) _q NR _a R _h , alkoxy, —C (O) R _c , — (CH ₂ ) _q CH ₃ , and each R _g is Independently H, halogen, —C (O) R _c , aryl, heterocyclyl, or —NR _a R _b ;
R _h is H or — (CH ₂ ) _q S (O) _n R _d ;
Each R _i is independently H, halo, alkyl, alkenyl, alkynyl, or —O (CH ₂ ) _q —R _g ;
Each n is independently 0, 1, or 2;
Each p is independently 0, 1, 2, or 3;
Each q is independently 0, 1, or 2;
R _1-2 has the general structure shown below:

［式中、
それぞれのＲ_８は、独立してＨ、−ＯＨ、ハロゲン、アルキル、アルコキシ、−ＮＲ_ａＲ_ｂ、または−Ｓ（Ｏ）_ｎＲ_ｄであり；
それぞれのＲ_９は、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｃであるか、またはＸがＯ、Ｓ、もしくは存在しない場合は存在せず；
それぞれのＸは、独立してＯ、Ｓ、Ｎ、ＣＨであるか、または存在せず、したがって共有結合を形成し；
それぞれのｍは、独立して０、１、または２である］］；または
その互変異性体、製薬上許容されるその塩、もしくは互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

[Where:
Each R ₈ is independently H, —OH, halogen, alkyl, alkoxy, —NR _a R _b , or —S (O) _n R _d ;
Each R ₉ is independently H, alkyl, —C (O) R _c , or absent when X is O, S, or absent;
Each X is independently O, S, N, CH or absent, thus forming a covalent bond;
Each m is independently 0, 1, or 2]]; or a tautomer, pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer. A method for treating a patient suffering from cancer.

  In a more detailed embodiment thereof, said compound is:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩である。

A tautomer, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a tautomer.

  In a more detailed embodiment thereof, said compound is:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩である。

A tautomer, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a tautomer.

  Another embodiment of the present invention provides a compound of formula II with aldesleukin:

［式中、
Ｒ_１１は、アルキル、アリールまたはヘテロシクリルであり；
Ｒ_１２、Ｒ_１３、Ｒ_１４、およびＲ_１５は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｚ、アリール、ヘテロシクリル、またはアルコキシであり；
それぞれのＲ_ｚは、独立してＨ、アルキル、アルコキシ、−ＮＨ_２、−ＮＨ（アルキル）、−Ｎ（アルキル）_２、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｑは、独立して０、１、または２である］；または
その互変異性体、製薬上許容されるその塩、もしくは互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

[Where:
R ₁₁ is alkyl, aryl or heterocyclyl;
R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S. (O) _n R _d , or heterocyclyl;
Each R _a and R _b is independently H, alkyl, —C (O) R _z , aryl, heterocyclyl, or alkoxy;
Each R _z is independently H, alkyl, alkoxy, —NH ₂ , —NH (alkyl), —N (alkyl) ₂ , aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each n is independently 0, 1, or 2;
Each q is independently 0, 1, or 2]; or a tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer. A method for treating a patient suffering from cancer is provided.

In a more detailed embodiment thereof, R ₁₁ is R _11a :

［式中、
Ｒ_１６、Ｒ_１７およびＲ_１８は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルである］。

[Where:
R ₁₆ , R ₁₇ and R ₁₈ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S (O) _n. R _d , or heterocyclyl].

  In a more detailed embodiment thereof, said compound is:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩である。

A tautomer, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a tautomer.

  Another embodiment of the present invention is a compound of formula III with aldesleukin:

［式中、
点線は、追加の結合の任意選択の配置を表し；
Ｒ_２０は、Ｈまたは＝Ｏであり；
Ｒ_２１、Ｒ_２２、Ｒ_２３、およびＲ_２４は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_２５は、Ｈ、アルキル、または−Ｃ（Ｏ）Ｒ_ｚであり；
Ｒ_２６およびＲ_２７は、それぞれ独立してＨ、−ＯＨ、ハロゲン、アルキル、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、または−Ｓ（Ｏ）_ｎＲ_ｄであり；
Ｒ_２８は、Ｈ、−ＣＨ_３、またはハロゲンであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｚ、アリール、ヘテロシクリル、またはアルコキシであり； それぞれのＲ_ｚは、独立してＨ、アルキル、アルコキシ、−ＮＨ_２、−ＮＨ（アルキル）、−Ｎ（アルキル）_２、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｑは、独立して０、１、または２である］；または
その互変異性体、製薬上許容されるその塩、もしくは互変異性体の製薬上許容される塩とを投与することを含む、癌に罹患している患者の治療方法を提供する。

[Where:
The dotted line represents an optional arrangement of additional bonds;
R ₂₀ is H or ═O;
R ₂₁ , R ₂₂ , R ₂₃ , and R ₂₄ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S. (O) _n R _d , or heterocyclyl;
R ₂₅ is H, alkyl, or —C (O) R _z ;
R ₂₆ and R ₂₇ are each independently H, —OH, halogen, alkyl, —NR _a R _b , —C (O) R _z , or —S (O) _n R _d ;
R ₂₈ is H, —CH ₃ , or halogen;
Each R _a and R _b is independently H, alkyl, —C (O) R _z , aryl, heterocyclyl, or alkoxy; each R _z is independently H, alkyl, alkoxy, —NH ₂ , —NH (alkyl), —N (alkyl) ₂ , aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each n is independently 0, 1, or 2;
Each q is independently 0, 1, or 2]; or a tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of the tautomer. A method for treating a patient suffering from cancer is provided.

  In a more detailed embodiment thereof, said compound is:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩である。

A tautomer, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a tautomer.

  In a more detailed embodiment thereof, said compound is:

、その互変異性体、製薬上許容されるその塩、または互変異性体の製薬上許容される塩である。

A tautomer, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of a tautomer.

  Another embodiment of the present invention is a quinolinone, preferably 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N -(3-Chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- ( Preferably selected from 2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Angiogenic agents comprising administering to a patient, the provides a method of reducing the toxicity associated with the administration to a cancer patient aldesleukin.

  Another embodiment of the present invention comprises 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3), comprising administering aldesleukin to a patient. -Morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxo Indoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or Selected from 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Be it a preferably anti-angiogenic agent to provide a method for reducing the toxicity associated with the administration to a cancer patient.

  Another embodiment of the present invention is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro -4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2, 4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) Said patient with an anti-angiogenic agent, preferably selected from:) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Comprising administering, to provide a method of reducing IL-2 resistance in a patient suffering from cancer.

  Another embodiment of the present invention comprises first administering to said patient a therapeutically effective amount of aldesleukin, and then 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazoline-4 -Amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-(( 5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) Phthalazine-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) And a step is selected from) phenyl) urea is administered preferred antiangiogenic agent, provides a method of treating a patient suffering from cancer.

  Another embodiment of the present invention provides the patient with a therapeutically effective amount of 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholino Propoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline- 3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- Preferably selected from (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea. Comprising separately administering an anti-angiogenic agent and aldesleukin according to a dosing schedule, wherein aldesleukin is administered at a dose of about 9 to about 130 MIU / day for at least 3 consecutive days Provided is a method of treating a patient suffering from cancer that is administered for a period of time, optionally followed by a rest period of at least 3 consecutive days. More specifically, the dose is about 30 to about 100 MIU / day. More specifically, the dose is about 30 to about 60 MIU / day. More specifically, the dose is about 30 to about 40 MIU / day. More specifically, the dose is about 17 to about 30 MIU / day. More specifically, the dose is about 9 to about 30 MIU / day.

  In a more detailed embodiment, 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4 -Fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4- Diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridine Preferably, the anti-angiogenic agent selected from -4-yloxy) phenyl) -3- (4-chloro-3-trifluoromethyl) phenyl) urea is 2 to Administered 1-9 times every week. In a more detailed embodiment, 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4 -Fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4- Diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridine Preferably, the anti-angiogenic agent selected from -4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is VE An F-inhibitor.

  In another embodiment, 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4- Fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl 1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridine- An anti-angiogenic agent, preferably selected from 4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, is bevacizumab Or cetuximab, or administered with VEGF-Trap. More specifically, the bevacizumab is administered at a dose of 1-12 mg / kg every 12-16 days.

  In another embodiment, the anti-angiogenic agent is administered within 72 hours of administration of rIL-2. Here, “within” means to indicate before or after, such as “administering an anti-angiogenic agent 72 hours before or 72 hours after administration of rIL-2”. In another embodiment, the anti-angiogenic agent is administered within 14 days of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 7 days of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 6 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 5 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 4 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 48 hours of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 36 hours of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 24 hours of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 12 hours of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 6 hours of administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered within 1 hour of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered concurrently with the administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered in combination with rIL-2. In its more detailed embodiment, the anti-angiogenic agent is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy)- N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) ) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- Selected from (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

  Another embodiment of the present invention relates to aldesleukin and 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-Chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl ) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2 Anti-blood preferably selected from-(methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea And forming agent simultaneously comprising administering, to provide a method of treating a patient suffering from cancer.

  Another embodiment of the present invention comprises a container, a therapeutically effective amount of aldesleukin, and a therapeutically effective amount of 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine. , 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5- Fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine- 1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urine It is selected from and a preferably antiangiogenic agents, for treating a patient suffering from cancer provides a therapeutic package suitable for commercial. In that treatment package, the patient suffers from renal cell carcinoma. Patient is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H- Pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) ) Phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea before treatment with an anti-angiogenic agent, preferably selected from alde Further comprising that therapeutic package documents indicating that treated with an interleukin.

  Another embodiment is 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4- Fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl 1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridine- Anti-angiogenic agents and aldesleukins, preferably selected from 4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea It provides a pharmaceutical composition comprising a.

  Clear cell renal cell carcinoma (RCC) is associated with increased vascularization, particularly VEGF expression. VEGF is a protein that plays an important role in tumor angiogenesis (formation of new blood vessels to the tumor) and maintenance of established tumor blood vessels. This binds to specific receptors on the blood vessels and stimulates extension to existing blood vessels. Some, but not all, cases of RCCa have elevated serum VEGF levels.

  There is evidence that increased VEGF is correlated with resistance to IL-2 (Lissoni et al., AntiCancer Res., 2001; 21: 777-9). The immunosuppressive effect of VEGF on T cells and dendritic cells (Gabrilovich et al., J Leukoc Biol., 2002; 72: 285-96) represents a mechanism for the treatment of VEGF-induced resistance to IL-2. There is a possibility.

  Furthermore, it has been noted that VEGF increased vascular permeability and that treatment with anti-VEGF resulted in increased blood pressure in some patients. Thus, when the combination of the present invention is administered to patients suffering from cancers such as RCCa, some of the major toxicities of anti-VEGF (eg hypertension) and rIL-2 (eg hypotension) cancel each other out. And may result in an overall improved therapeutic index.

  Further combinations of the present invention, such as aldesleukin and bevacizumab or cetuximab, provide a higher rate and / or better response durability than when the drug is administered alone.

  Certain aldesleukin dosing regimens disclosed herein have side effects associated with rIL-2 that can be associated with intermittent stimulation of natural killer (NK) cell activity and long-term exposure to rIL-2 dosing. Provides a reduced risk of. It should be noted that the hypotension typically associated with rIL-2 dosing is offset by administration of the anti-angiogenic compositions of the present invention, which is the anti-angiogenic properties of the present invention. This is because the composition is generally associated with hypertension. Furthermore, hypoxia, typically associated with VEGF inhibitors, increases sensitivity to the effectiveness of rIL-2 treatment, while also providing symptomatic benefits.

  Another embodiment of the present invention provides the use of aldesleukin in the manufacture of a medicament for treating cancer, said medicament comprising 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) ) Quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl)- 5-((5-Fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridine-4- Yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- ( Separately and it is preferred antiangiogenic agent selected from trifluoromethyl) phenyl) urea, is for simultaneous or sequential, use. In more detailed embodiments thereof, 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro- 4-Fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4 -Diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl)) Preferably said anti-angiogenic agent selected from pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea It is an acid salt. In another embodiment, the cancer is renal cell carcinoma.

  Another embodiment of the present invention relates to 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3) in the manufacture of a medicament for treating cancer. -Morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxo Indoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea It provides the preferred use of antiangiogenic agents, the medicament is separate and aldesleukin is for simultaneous or sequential, use.

Preferred molecules associated with angiogenesis that are modulated (such as inhibited) by the compositions of the present invention include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukin-8 (IL-8). , Angiogenin, angiotropin, epidermal growth factor (EGF), platelet-derived endothelial cell growth factor, transforming growth factor a (TGF-a), transforming growth factor b (TGF-b), or nitric oxide. Also contemplated by the present invention is the modulation (such as enhancement) of thrombospondin, angiostatin, and endostatin by the compositions of the present invention.
General composition for co-administration with rIL-2:
Quinazoline The compound of formula I has the following structure:

［式中、
Ｒ_１は、アルキル、−アリール（Ｒ_ｉ）_ｐ、もしくはヘテロシクリルであり；
Ｒ_２は、Ｈもしくはアルキルであるか；または
Ｒ_１およびＲ_２は、一緒に結合してＲ_１−２を形成し；
Ｒ_３は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｃ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_４は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、−Ｏ−（ＣＨ_２）_ｑ−Ｒ_ｇ、−Ｏ−（ＣＨ_２）_ｑ−Ｏ−Ｒ_ｅ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、または−ヘテロシクリル−Ｒ_ｆであり；
Ｒ_５は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、−Ｏ−（ＣＨ_２）_ｑ−Ｒ_ｇ、−Ｏ−（ＣＨ_２）_ｑ−Ｏ−Ｒ_ｅ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_６は、Ｈ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｃ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_７は、Ｈ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｃ、アリール、ヘテロシクリル、もしくはアルコキシであるか；または
Ｒ_ａおよびＲ_ｂは、一緒に結合してＲ_１−２を形成し；
それぞれのＲ_ｃは、独立してＨ、アルキル、アルコキシ、−Ｃ（Ｏ）アルキル、−Ｃ（Ｏ）アリール、−ＣＨＯ、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのＲ_ｅは、独立してＨまたはアルキルであり；
Ｒ_ｆは、Ｈ、ハロゲン、−ＯＨ、−ＣＮ、−（ＣＨ_２）_ｑＮＲ_ａＲ_ｈ、アルコキシ、−Ｃ（Ｏ）Ｒ_ｃ、−（ＣＨ２）_ｑＣＨ_３であり、
それぞれのＲ_ｇは、独立してＨ、ハロゲン、−Ｃ（Ｏ）Ｒ_ｃ、アリール、ヘテロシクリル、または−ＮＲ_ａＲ_ｂであり；
Ｒ_ｈは、Ｈ、または−（ＣＨ_２）_ｑＳ（Ｏ）_ｎＲ_ｄであり；
それぞれのＲ_ｉは、独立してＨ、ハロ、アルキル、アルケニル、アルキニル、または−Ｏ（ＣＨ_２）_ｑ−Ｒ_ｇであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｐは、独立して０、１、２、または３であり；
それぞれのｑは、独立して０、１、または２であり；
Ｒ_１−２は、以下に示す一般構造を有し：

[Where:
R ₁ is alkyl, -aryl (R _i ) _p , or heterocyclyl;
R ₂ is H or alkyl; or R ₁ and R ₂ are joined together to form R _1-2 ;
R ₃ is, H, -CN, -OH, halogen, alkyl, aryl, _{alkoxy, -NR a R b, -C (O} ) R c, -S (O) n R d , or heterocyclyl;
R ₄ is H, —CN, —OH, halogen, alkyl, aryl, —O— (CH ₂ ) _q —R _g , —O— (CH ₂ ) _q —O—R _e , —NR _a R _b , -S _(O) n _{R d,} or - heterocyclyl -R _f;
R ₅ is H, —CN, —OH, halogen, alkyl, aryl, —O— (CH ₂ ) _q —R _g , —O— (CH ₂ ) _q —O—R _e , —NR _a R _b , It is -S _(O) n _{R d,} or heterocyclyl;
R ₆ is H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _c , —S (O) _n R _d , or heterocyclyl;
R ₇ is H, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —S (O) _n R _d , or heterocyclyl;
Each R _a and R _b is independently H, alkyl, —C (O) R _c , aryl, heterocyclyl, or alkoxy; or R _a and R _b are joined together to form R _{1 1- 2} ;
Each R _c is independently H, alkyl, alkoxy, —C (O) alkyl, —C (O) aryl, —CHO, aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each R _e is independently H or alkyl;
R _f is H, halogen, —OH, —CN, — (CH ₂ ) _q NR _a R _h , alkoxy, —C (O) R _c , — (CH 2) _q CH ₃ ,
Each R _g is independently H, halogen, —C (O) R _c , aryl, heterocyclyl, or —NR _a R _b ;
R _h is H or — (CH ₂ ) _q S (O) _n R _d ;
Each R _i is independently H, halo, alkyl, alkenyl, alkynyl, or —O (CH ₂ ) _q —R _g ;
Each n is independently 0, 1, or 2;
Each p is independently 0, 1, 2, or 3;
Each q is independently 0, 1, or 2;
R _1-2 has the general structure shown below:

［式中、
それぞれのＲ_８は、独立してＨ、−ＯＨ、ハロゲン、アルキル、アルコキシ、−ＮＲ_ａＲ_ｂ、または−Ｓ（Ｏ）_ｎＲ_ｄであり；
それぞれのＲ_９は、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｃであるか、またはＸがＯ、Ｓ、もしくは存在しない場合は存在せず；
それぞれのＸは、独立してＯ、Ｓ、Ｎ、ＣＨであるか、または存在せず、したがって共有結合を形成し；
それぞれのｍは、独立して０、１、または２である］］。

[Where:
Each R ₈ is independently H, —OH, halogen, alkyl, alkoxy, —NR _a R _b , or —S (O) _n R _d ;
Each R ₉ is independently H, alkyl, —C (O) R _c , or absent when X is O, S, or absent;
Each X is independently O, S, N, CH or absent, thus forming a covalent bond;
Each m is independently 0, 1, or 2]].

In a more detailed embodiment of the invention, R ₂ is H. In an even more detailed embodiment, R ₁ is -aryl (R _i ) _p . In a further embodiment where R ₁ is -aryl (R _i ) _p , the aryl in R ₁ is phenyl, p in R ₁ is 2, and both R _i groups in R ₁ are halo. .

In a further embodiment where R ₁ is -aryl (R _i ) _p , the aryl in R ₁ is phenyl, p in R ₁ is 1, and the R _i group in R ₁ is alkynyl, preferably ethynyl. It is.

In a further embodiment where R ₁ is -aryl (R _i ) _p , the aryl in R ₁ is phenyl, p in R ₁ is 2, and one R _i group in R ₁ is halo , The other R _i group in R ₁ is —O (CH ₂ ) _q —R _g . In more detailed embodiments thereof, q in R ₁ is 1 and R _{g in} R ₁ is halophenyl.

In a further embodiment where R ₁ is -aryl (R _i ) _p , _p in R ₁ is 1 and R _{i in} R ₁ is alkynyl.

Provided is another more detailed embodiment of the present invention wherein R ₁ and R ₂ are joined together to form R _1-2 :

［式中、Ｒ_８はＨであり、ＸはＮであり、Ｒ_９は−Ｃ（Ｏ）ＮＨＲ_ｂである］。そのより詳細な実施形態では、Ｒ_９中のＲ_ｂは−フェニル−Ｏ−ＣＨ_２（ＣＨ_３）_２である。

[Wherein R ₈ is H, X is N, and R ₉ is —C (O) NHR _b ]. In a more detailed embodiment, the _{R b} in _{R 9} - phenyl _-O-CH 2 (CH _{3) 2.}

In a more detailed embodiment of the invention, R ₃ and R ₆ are H.

In a more detailed embodiment of the invention, R ₄ is —O— (CH ₂ ) _q —R _g . In more detailed embodiments thereof, q in R ₄ is 1 and R _g is H.

In another embodiment R ₄ is _{-O- (CH 2) q -R g} , R g in _{R 4} is heterocyclyl.

In a more detailed embodiment of the invention, R ₄ and R ₅ are each —O— (CH ₂ ) _q —O—R _e . In more detailed embodiments thereof, q in both R ₄ and R ₅ is 2, and R _{e in} both R ₄ and R ₅ is methyl.

In a more detailed embodiment of the invention, R ₄ is -heterocyclyl-R _f and R ₅ is H. In a more detailed embodiment thereof, said heterocyclyl in R ₄ is furanyl. In an even more detailed embodiment, R _{f in} R ₄ is — (CH ₂ ) _q NHR _h . Furthermore, R _h is — (CH ₂ ) _q S (O) ₂ CH ₃ .

In a more detailed embodiment of the invention, R ₅ is —O— (CH ₂ ) _q —R _g . In another embodiment thereof, R _{g in} R ₅ is heterocyclyl.

Another embodiment is provided wherein R ₇ is H.

Another embodiment is provided wherein R ₃ , R ₆ , and R ₇ are all H.
Indolinone The compound of formula II has the following structure:

［式中、
Ｒ_１１は、アルキル、アリールまたはヘテロシクリルであり；
Ｒ_１２、Ｒ_１３、Ｒ_１４、およびＲ_１５は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｚ、アリール、ヘテロシクリル、またはアルコキシであり；
それぞれのＲ_ｚは、独立してＨ、アルキル、アルコキシ、−ＮＨ_２、−ＮＨ（アルキル）、−Ｎ（アルキル）_２、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｑは、独立して０、１、または２である］。

[Where:
R ₁₁ is alkyl, aryl or heterocyclyl;
R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S. (O) _n R _d , or heterocyclyl;
Each R _a and R _b is independently H, alkyl, —C (O) R _z , aryl, heterocyclyl, or alkoxy;
Each R _z is independently H, alkyl, alkoxy, —NH ₂ , —NH (alkyl), —N (alkyl) ₂ , aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each n is independently 0, 1, or 2;
Each q is independently 0, 1, or 2.]

In a more detailed embodiment, R ₁₁ is heterocyclyl. In an even more detailed embodiment, R ₁₁ is R _11a :

［式中、
Ｒ_１６、Ｒ_１７およびＲ_１８は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルである］。Ｒ_１がＲ_１ａであるさらに別のより詳細な実施形態では、Ｒ_７は−Ｃ（Ｏ）−（ＣＨ_２）_ｐ−Ｎ（Ｈ）_{（２−ｒ）}（アルキル）_ｒであり、ｐは０、１、２、３、４、または５であり、ｒは０、１、または２である。Ｒ_１１がＲ_１１ａであるさらに別のより詳細な実施形態では、Ｒ_１３はＦである。Ｒ_１１がＲ_１１ａであるさらに別のより詳細な実施形態では、Ｒ_１７は−（ＣＨ_２）_ｔＣＯＯＨであり、ｔは１、２、３、または４である。Ｒ_１１がＲ_１１ａであるさらに別のより詳細な実施形態では、Ｒ_６およびＲ_８はどちらもメチルである。Ｒ_１１がＲ_１１ａであるさらに別のより詳細な実施形態では、Ｒ_１７はＨである。

[Where:
R ₁₆ , R ₁₇ and R ₁₈ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S (O) _n. R _d , or heterocyclyl]. In yet another more detailed embodiment wherein R ₁ is R _1a , R ₇ is —C (O) — (CH ₂ ) _p —N (H) _(2-r) (alkyl) _r , p is 0, 1, 2, 3, 4, or 5 and r is 0, 1, or 2. In yet another more detailed embodiment wherein R ₁₁ is R _11a , R ₁₃ is F. In yet another more detailed embodiment wherein R ₁₁ is R _11a , R ₁₇ is — (CH ₂ ) _t COOH and t is 1, 2, 3, or 4. In yet another more detailed embodiment, where R ₁₁ is R _11a , R ₆ and R ₈ are both methyl. In yet another more detailed embodiment wherein R ₁₁ is R _11a , R ₁₇ is H.

In another more detailed embodiment of the present invention, R ₁₁ is aryl. Even more particularly, R ₁₁ is substituted or unsubstituted phenyl. In another embodiment, R ₁₃ is iodide.
Isoindolinone The compound of formula III has the following structure:

［式中、
点線は、追加の結合の任意選択の配置を表し；
Ｒ_２０は、Ｈまたは＝Ｏであり；
Ｒ_２１、Ｒ_２２、Ｒ_２３、およびＲ_２４は、それぞれ独立してＨ、−ＣＮ、−ＯＨ、ハロゲン、アルキル、アリール、アルコキシ、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、−Ｓ（Ｏ）_ｎＲ_ｄ、またはヘテロシクリルであり；
Ｒ_２５は、Ｈ、アルキル、または−Ｃ（Ｏ）Ｒ_ｚであり；
Ｒ_２６およびＲ_２７は、それぞれ独立してＨ、−ＯＨ、ハロゲン、アルキル、−ＮＲ_ａＲ_ｂ、−Ｃ（Ｏ）Ｒ_ｚ、または−Ｓ（Ｏ）_ｎＲ_ｄであり；
Ｒ_２８は、Ｈ、−ＣＨ_３、またはハロゲンであり；
それぞれのＲ_ａおよびＲ_ｂは、独立してＨ、アルキル、−Ｃ（Ｏ）Ｒ_ｚ、アリール、ヘテロシクリル、またはアルコキシであり；
それぞれのＲ_ｚは、独立してＨ、アルキル、アルコキシ、−ＮＨ_２、−ＮＨ（アルキル）、−Ｎ（アルキル）_２、アリール、またはヘテロシクリルであり；
それぞれのＲ_ｄは、独立してＨ、アルキル、アルケニル、アリール、または−ＮＲ_ａＲ_ｂであり；
それぞれのｎは、独立して０、１、または２であり；
それぞれのｑは、独立して０、１、または２である］。
定義：
ＡＵＣ 曲線下面積
ＣＲ 完全応答
ＤＬＴ 用量規制毒性
ＩＬ インターロイキン
ＩＬ−２ インターロイキン−２
ＩＵ 国際単位
ＩＶ 静脈内（投与様式として）
ＬＡＫ リンホカイン活性化キラー
ＭＴＤ 最大耐用量
ＭＩＵ 百万国際単位
ＮＫ ナチュラルキラー
ＰＫ 薬物動態学
ＰＲ 部分応答
ＲＣＣ 腎細胞癌
ＲＣＣａ 明細胞腎細胞癌
ｒＩＬ−２ 組換えインターロイキン−２
ＲＴＫ 受容体チロシンキナーゼ
ＴＮＦ 腫瘍壊死因子
ＶＥＧＦ 血管内皮成長因子
一般に、水素またはＨなどの特定の元素の言及には、その元素のすべての同位体が含まれることを意味する。例えば、Ｒ基が、水素またはＨが含まれると定義されている場合は、これには重水素およびトリチウムも含まれる。

[Where:
The dotted line represents an optional arrangement of additional bonds;
R ₂₀ is H or ═O;
R ₂₁ , R ₂₂ , R ₂₃ , and R ₂₄ are each independently H, —CN, —OH, halogen, alkyl, aryl, alkoxy, —NR _a R _b , —C (O) R _z , —S. (O) _n R _d , or heterocyclyl;
R ₂₅ is H, alkyl, or —C (O) R _z ;
R ₂₆ and R ₂₇ are each independently H, —OH, halogen, alkyl, —NR _a R _b , —C (O) R _z , or —S (O) _n R _d ;
R ₂₈ is H, —CH ₃ , or halogen;
Each R _a and R _b is independently H, alkyl, —C (O) R _z , aryl, heterocyclyl, or alkoxy;
Each R _z is independently H, alkyl, alkoxy, —NH ₂ , —NH (alkyl), —N (alkyl) ₂ , aryl, or heterocyclyl;
Each R _d is independently H, alkyl, alkenyl, aryl, or —NR _a R _b ;
Each n is independently 0, 1, or 2;
Each q is independently 0, 1, or 2.]
Definition:
AUC Area under the curve CR Complete response DLT Dose-limiting toxicity IL Interleukin IL-2 Interleukin-2
IU International Unit IV Intravenous (as mode of administration)
LAK lymphokine-activated killer MTD maximum tolerated dose MIU million international unit NK natural killer PK pharmacokinetic PR partial response RCC renal cell carcinoma RCCa clear cell renal cell carcinoma rIL-2 recombinant interleukin-2
RTK receptor tyrosine kinase TNF tumor necrosis factor VEGF vascular endothelial growth factor In general, reference to a particular element such as hydrogen or H means that all isotopes of that element are included. For example, if the R group is defined to include hydrogen or H, this includes deuterium and tritium.

  An “anti-angiogenic agent” is any small molecule that has been shown or will be shown to inhibit angiogenesis in the system, and more specifically has a molecular weight of less than 1,100 g / mol. Means any compound. Preferred molecules associated with angiogenesis that are modulated (such as inhibited) by the compositions of the present invention include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukin-8 (IL-8). , Angiogenin, angiotropin, epidermal growth factor (EGF), platelet-derived endothelial cell growth factor, transforming growth factor a (TGF-a), transforming growth factor b (TGF-b), or nitric oxide. Also contemplated by the present invention is the modulation (such as enhancement) of thrombospondin, angiostatin, and endostatin by the compositions of the present invention. Preferred anti-angiogenic compositions of the present invention include 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-Chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl ) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, and 1- (4- (2 -(Methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is included.

  The term “effective amount” is that amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of a compound for treating renal cell carcinoma can be that amount necessary to cause regression of tumor growth in renal cells. The effective amount can vary depending on, for example, the condition being treated, the weight of the subject and the severity of the disease. One skilled in the art can readily determine the effective amount empirically without undue experimentation.

  As used herein, “therapeutically effective amount” refers to an amount sufficient to alleviate, ameliorate, stabilize, reverse, slow down or delay the progression of a condition such as a medical condition.

  “Subject” or “patient” refers to dogs, cats, pocket pets, marmosets, horses, cattle, pigs, sheep, goats, elephants, giraffes, chickens, lions, monkeys, owls, rats, squirrels, slender loris, and mice Is meant to describe humans or vertebrates, including

  “Pocket pet” refers to a group of vertebrates that can enter a relaxed coat pocket, such as, for example, hamsters, chinchillas, ferrets, rats, guinea pigs, gerbils, rabbits, and owl moths.

  As used herein, the term “pharmaceutically acceptable ester” refers to an ester that hydrolyzes in vivo and includes those that readily decompose in the human body to leave the parent compound or salt thereof. Suitable ester groups include, for example, pharmaceutically acceptable aliphatic carboxylic acids, especially alkanoic acids, alkenoic acids, cycloalkanoic acids and alkanes, where each alkyl or alkenyl moiety advantageously has 6 or fewer carbon atoms. Those derived from diacids are included. Representative examples of specific esters include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, and ethyl succinate.

  The compounds of the present invention can be used in the form of salts, such as “pharmaceutically acceptable salts” derived from inorganic or organic acids. These salts include but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, hydrogensulfate, butyrate, camphor salt, camphor Sulfonate, digluconate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride , Hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, shu Acid salt, pamoate, pectate, sulfate, 3-phenylpropionate, picrate, piva Emissions, propionate, succinate, tartrate, thiocyanate, p- toluenesulfonate and undecanoate. In addition, basic nitrogen-containing groups include methyl, ethyl, propyl, and lower alkyl halides such as butyl chloride, butyl bromide, and butyl iodide; dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfate. Can be quaternized with agents such as decyl, lauryl, myristyl and long chain halides such as stearyl chloride, stearyl bromide and stearyl iodide, or alkyl halides such as benzyl bromide and phenethyl bromide . Thereby a product is obtained which is soluble or dispersible in water or oil.

  As used herein, the term “pharmaceutically acceptable prodrug” is within the scope of sound medical judgment and has a reasonable benefit / risk ratio with excessive toxicity, irritation, allergic response, etc. Prodrugs of the compounds of the present invention suitable for use in contact with human and lower animal tissues that are balanced and effective for their intended use, and, where possible, of the present invention The zwitterionic form of the compound. The term “prodrug” refers to a compound that is rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. For a thorough discussion, see T.W. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems”, A.M. C. S. Symposium Series Vol. 14, and Edward B. Edited by Roche, “Bioreversible Carriers in Drug Design”, American Pharmaceutical Association and Pergamon Press, 1987. For example, prodrugs described in US Pat. No. 6,284,772 can be used.

  Reference to “halo”, “halide”, or “halogen” refers to an F, Cl, Br, or I atom.

The phrase “alkyl” refers to substituted and unsubstituted alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. This term also includes, but is not limited to, branched chain isomers of straight chain alkyl groups, provided as examples -CH (CH ₃ ) ₂ , -CH (CH ₃ ) (CH ₂ CH _{3), - CH (CH 2} CH 3) 2, -C (CH 3) 3, -C (CH 2 CH 3) 3, -CH 2 CH (CH 3) 2, -CH 2 CH (CH 3) ( _{CH 2 CH 3), - CH} 2 CH (CH 2 CH 3) 2, -CH 2 C (CH 3) 3, -CH 2 C (CH 2 CH 3) 3, -CH (CH 3) CH (CH 3 _{) (CH 2 CH 3),} - CH 2 CH 2 CH (CH 3) 2, -CH 2 CH 2 CH (CH 3) (CH 2 CH 3), - CH 2 CH 2 CH (CH 2 CH 3) 2 _{, -CH 2 CH 2 C (CH} 3) 3, -CH 2 CH 2 C (CH _{CH 3) 3, -CH (CH} 3) CH 2 CH (CH 3) 2, -CH (CH 3) CH (CH 3) CH (CH 3) 2, -CH (CH 2 CH 3) CH (CH 3 ) CH (CH ₃ ) (CH ₂ CH ₃ ) and the like. The phrase also includes cyclic rings such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, and such rings substituted with straight and branched chain alkyl groups as defined above. It is. This phrase is substituted with polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl and bicyclo [2.2.2] octyl, as well as linear and branched alkyl groups as defined above. Such rings are also included. The phrase “alkyl” includes, but is not limited to, one or more bonds to carbon or hydrogen, halogen atoms in halides such as F, Cl, Br, and I; hydroxyl groups, alkoxy groups, aryls Oxygen atoms in groups such as oxy groups and ester groups; sulfur atoms in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; amines, amides, alkylamines, dialkylamines, Nitrogen atoms in groups such as arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups Silicon atoms in various other groups Groups are replaced by a bond with no atom carbon in hydrogen such as other heteroatoms are also included. Alkyl groups are limited to those having 1-20 carbon atoms and as many as 5 additional heteroatoms as described above. More preferred alkyl groups have 1 to 5 carbon atoms and as many as 2 heteroatoms.

  The phrase “aryl” refers to substituted and unsubstituted aryl groups that do not contain heteroatoms. Thus, this phrase includes, but is not limited to, groups such as phenyl, biphenyl, anthracenyl, naphthenyl, and the like. Aryl groups also include those in which one of the aromatic carbons is bonded to an atom (in the definition of alkyl) that is not a carbon or hydrogen as described above, and one or more aromatic carbons of the aryl group are Also included are aryl groups attached to a substituted and / or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bond configurations in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system (eg, dihydronaphthyl or tetrahydronaphthyl). Thus, the phrase “aryl” includes, but is not limited to, tolyl and hydroxyphenyl, among others.

The phrase “alkenyl” refers to straight and branched chain and cyclic groups such as those described for alkyl groups as defined above, provided that there is at least one double bond between two carbon atoms. To do. Examples include, but are not limited to, vinyl, —CH═C (H) (CH ₃ ), —CH═C (CH ₃ ) ₂ , —C (CH ₃ ) ═C (H) ₂ , — _{C (CH 3) = C (} H) (CH 3), - C (CH 2 CH 3) = CH 2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl. In addition, a group in which an atom that is neither carbon nor hydrogen is bonded to a carbon that is double-bonded to another carbon, and one of atoms that are not carbon or hydrogen are involved in a double bond to another carbon. Also included are groups that are bonded to carbons that are not. Alkenyl groups are limited to those having 2 to 15 carbon atoms and as many as four additional heteroatoms as described above. More preferred alkenyl groups have 2 to 5 carbon atoms and as many as 2 heteroatoms.

  The phrase “alkoxy” refers to a substituted or unsubstituted alkoxy group of formula —O-alkyl, where the point of attachment is an oxy group, and the alkyl group is as defined above. Alkoxy groups are limited to those having additional heteroatoms including 1-20 carbon atoms and as many as 5 oxygen atoms. More preferred alkoxy groups have 1-5 carbon atoms and heteroatoms including as many as 2 oxygen atoms.

The phrase “alkynyl” refers to straight and branched groups, such as those described for alkyl groups as defined above, provided that there is at least one triple bond between the two carbon atoms. Examples include, but are not limited to, —C≡C (H), —C≡C (CH ₃ ), —C≡C (CH ₂ CH ₃ ), —C (H ₂ ) C≡C ( H), —C (H) ₂ C≡C (CH ₃ ), and —C (H) ₂ C≡C (CH ₂ CH ₃ ). In addition, an alkynyl group in which an atom that is neither carbon nor hydrogen is bonded to a carbon atom that is triple-bonded to another carbon, and an atom that is not carbon or hydrogen to a carbon that is not involved in a triple bond to another carbon. Bound alkynyl groups are also included. Alkynyl groups are limited to those having 2-15 carbon atoms and as many as four additional heteroatoms as described above. More preferred alkynyl groups have 2 to 5 carbon atoms and as many as 2 heteroatoms.

  The phrase “heterocyclyl” refers to both aromatic and non-aromatic cyclic compounds, one or more of which are heteroatoms such as, but not limited to, N, O, and S Monocyclic, bicyclic, and polycyclic cyclic compounds, such as quinuclidyl, containing three or more ring members are included. Examples of heterocyclyl groups include, but are not limited to: pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (eg 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), tetrazolyl, (e.g., 1H-tetrazolyl, 2H tetrazolyl, etc.), etc. A saturated 3- to 8-membered ring containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; but not limited to indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quino Fused unsaturated heterocyclic groups containing 1 to 4 nitrogen atoms such as ru, isoquinolyl, indazolyl, benzotriazolyl; unsaturated 3 containing 1 to 2 oxygen atoms such as but not limited to furanyl 8-membered ring; 1-2 oxygen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (eg 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.) And an unsaturated 3-8 membered ring containing 1-3 nitrogen atoms; a saturated 3-8 membered ring containing 1-2 oxygen atoms and 1-3 nitrogen atoms such as, but not limited to, morpholinyl; For example, 1 such as benzoxazolyl, benzooxadiazolyl, benzoxazinyl (such as 2H-1,4-benzoxazinyl) An unsaturated fused heterocyclic group containing 2 oxygen atoms and 1 to 3 nitrogen atoms; including but not limited to thiazolyl, isothiazolyl, thiadiazolyl (eg, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl) , 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.) and the like, an unsaturated 3-8 membered ring containing, but not limited to, 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms; Saturated 3-8 membered rings containing 1-2 sulfur atoms such as thiazorodinyl and 1-3 nitrogen atoms; including but not limited to thienyl, dihydrodithinyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran, etc. Of saturated and unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atoms of benzothiazolyl, 1 to 2 sulfur atoms such as Nzothiadiazolyl, benzothiazinyl (such as 2H-1,4-benzothiazinyl), dihydrobenzothiazinyl (such as 2H-3,4-dihydrobenzothiazinyl) and 1-3 Unsaturated fused heterocycles containing 1 nitrogen atom, unsaturated fused heterocycles containing 1 to 2 oxygen atoms such as benzodioxolyl (eg, 1,3-benzodioxoyl, etc.); Unsaturated 3 to 8 membered rings containing 1 oxygen atom and 1 to 2 sulfur atoms such as dihydrooxathiinyl; 1 to 2 oxygen atoms and 1 to 2 sulfur such as 1,4-oxathiane Saturated 3- to 8-membered rings containing atoms; unsaturated condensed rings containing 1-2 sulfur atoms such as benzothienyl, benzodithiynyl; and one oxygen atom such as benzooxathinyl They include unsaturated condensed heterocyclic ring containing preliminary 1-2 oxygen atoms. Heterocyclyl groups also include those groups described above in which one or more S atoms in the ring are double bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5 to 6 ring members. More preferred heterocyclyl groups include thiomorpholine with one or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, and Included are morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine bound to tetrahydrofuran. “Heterocyclyl” also refers to a group as defined above in which one of the ring members is bonded to a non-hydrogen atom such as those described for substituted alkyl and substituted aryl groups. Examples include, but are not limited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1-methylpiperazinyl, and 2-chloropyridyl, among others. Heterocyclyl groups are limited to those having 2 to 15 carbon atoms and as many as 6 additional heteroatoms as described above. More preferred heterocyclyl groups have 3 to 5 carbon atoms and as many as 2 heteroatoms.

  When applied to an undefined but well-known group such as phenyl, the term “substituted” has the same meaning as described in the definition of alkyl for the optional appendages.

  Within the present invention, it is understood that the compounds described herein may exhibit tautomeric phenomena and that the formula illustrations herein represent only one of the possible tautomers. I want to be. It is to be understood that the present invention encompasses any tautomer with anti-angiogenic activity and is not limited to any one tautomer used in the formula diagram.

  It is to be understood that certain compounds and embodiments of the present invention can exist in unsolvated forms, such as solvated forms, eg, hydrate forms. It should be understood that the present invention encompasses all such solvated forms having anti-angiogenic activity.

Also, the present invention is structurally identical to those disclosed above, but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Also included are isotopically labeled compounds. Examples of isotopes that can be incorporated into the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes, eg, ² H, ³ H, ¹³ C, ^{14 respectively.} C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl are included. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds and said prodrugs that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. . Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as ³ H and ¹⁴ C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritium, ie ³ H, and carbon-14, ie ¹⁴ C-labeled isotopes are particularly preferred due to their ease of preparation and detection capability. In addition, substitution with a heavier isotope such as deuterium, ie ² H, may provide certain therapeutic benefits resulting from better metabolic stability, eg, increased in vivo half-life or reduced required dose Therefore, this may be preferable in some situations. The isotopically labeled compounds of the present invention and their prodrugs are generally not isotopically labeled by performing known or reference procedures and with readily available isotopically labeled reagents. It can be prepared by replacing the reagent.

  Reference to “IL-2” or “interleukin-2” refers to lymphocytes produced by normal peripheral blood lymphocytes and present in the body at low concentrations. IL-2 was first described by Morgan et al., (1976), Science, 193: 1007-1008, and originally because of its ability to induce proliferation of stimulated T lymphocytes, T cell growth factor and It was called. This is a protein reported to have a molecular weight ranging from 13,000 to 17,000 (Gillis and Watson, (1980), J. Exp. Med., 159: 1709), 6-8.5. With an isoelectric point in the range For the purposes of the present invention, the term “IL-2” refers to any IL-2 source, including mammalian sources such as mice, rats, rabbits, primates, pigs, pocket pets, and humans. It is intended to be encompassed and may be native or obtained by recombinant techniques, such as recombinant IL-2 polypeptides produced by a microbial host. Although IL-2 is of the native polypeptide sequence, as long as the variant IL-2 polypeptide retains the desired IL-2 biological activity as defined herein, It can also be a variant of a native IL-2 polypeptide as described below. Preferably, the IL-2 polypeptide or variant thereof is derived from a human source, recombinantly produced human IL-2, such as a recombinant human IL-2 polypeptide produced by a microbial host, and the desired IL- 2 variants thereof retaining biological activity are included. The present invention can be practiced with any pharmaceutical composition comprising IL-2 as a therapeutically active ingredient.

Anticancer drugs:
The composition of the present invention may be administered in conjunction with other anticancer agents. Specifically, the compositions are formulated together as a combination therapy or administered separately. Anti-cancer agents used in the present invention include, but are not limited to, one or more of the following:
A. Kinase inhibitors Kinase inhibitors for use as anti-cancer agents in conjunction with the compositions of the present invention include inhibitors of epidermal growth factor receptor (EGFR) kinases such as small molecule quinazoline, such as gefitinib (US Pat. No. 5,457,105, US). Patent US5616582, and US Pat. No. 5,770,599), ZD-6474 (International Publication WO01 / 32651), Erlotinib (Tarceva®, US Pat. No. 5,747,498 and International Publication WO96 / 30347), And lapatinib (US Pat. No. 6,727,256 and International Publication No. WO 02/02552); SU-11248 (International Publication WO 01/60814), SU 5416 (US Pat. No. 5,883,113 and International Publication WO 99 / 61422), SU6668 (US Pat. No. 5,883,113 and International Publication No. WO99 / 61422), CHIR-258 (US Pat. No. 6,605,617 and US Pat. No. 6,774,237), batalanib or PTK -787 (US Pat. No. 6,258,812), VEGF-Trap (International Publication WO02 / 57423), B43-Genistein (International Publication WO-09606116), Fenretinide (P-hydroxyphenylamine retinoic acid) ( US Pat. No. 4,323,581), IM-862 (International Publication WO02 / 62826), Bevacizumab or Avastin (registered trademark) (International Publication WO94 / 10202), KRN-951, 3- [5- ( Methylsulfonyl pipe Ginmethyl) -indolyl] -quinolone, AG-13736 and AG-13925, pyrrolo [2,1-f] [1,2,4] triazine, ZK-304709, Veglin®, VMDA-3601, EG-004 Vascular endothelial growth factor receptor (VEGFR) kinase inhibitors, including CEP-701 (US Pat. No. 5,621,100), Cand5 (International Publication WO04 / 09769); Pertuzumab (International Publication WO01 / 00245) ), An Erb2 tyrosine kinase inhibitor such as trastuzumab, and rituximab; an AKT protein kinase inhibitor such as RX-0201; LY-317615 (International Publication No. WO 95/17182), and perifosine (US Pat. Protein kinase C (PKC) inhibitors such as No. 1303); phosphoinositide 3-kinase (PI3K) inhibitors including SF-1126 and PI-103, PI-509, PI-516 and PI-540 (produced by PIramed) Raf / Map / MEK / Ras kinase inhibitors, including sorafenib (BAY 43-9006), ARQ-350RP, LErafAON, BMS-354825 AMG-548, and other compounds disclosed in International Publication No. WO 03/82272; Blast growth factor receptor (FGFR) kinase inhibitors; cell-dependent kinase (CDK) inhibitors including CYC-202 or roscovitine (WO 97/20842 and WO 99/02162); CHIR- 258, Includes platelet derived growth factor receptor (PGFR) kinase inhibitors such as G3 mAb, AG-13736, SU-11248 and SU6668; and fusion proteins such as Bcr-Abl kinase inhibitors and STI-571 or Gleevec® .

B. Antiestrogens Estrogen targeting agents for use in anticancer therapy in conjunction with the compositions of the present invention include selective estrogen receptor modulators (SERM) including Tamoxifen, Toremifene, Raloxifene; Arimidex® or Anastro Aromatase inhibitors including sols; estrogen receptor down-regulators (ERDs) including Faslodex® or fulvestrant.

C. Antiandrogens Androgen targeting agents for use in anticancer therapy in conjunction with the compositions of the present invention include flutamide, bicalutamide, finasteride, aminoglutetamide, ketoconazole, and corticosteroids.

D. Other inhibitors Other inhibitors for use as anticancer agents in conjunction with the compositions of the present invention include tipifarnib or R-115777 (US Patent US2003134848 and International Publication No. WO97 / 21701), BMS-214662, AZD- Protein farnesyltransferase inhibitors including 3409, and FTI-277; topoisomerase inhibitors including melvalon and difurotecan (BN-80915); mitotic kinesin spindle proteins including SB-743921 and MKI-833 ( KSP) inhibitors; including bortezomib or Velcade® (US Pat. No. 5,780,454), protease modulators such as XL-784; and non-steroidal anti-inflammatory drug I (NSAID) Cyclooxygenase 2 (COX-2) inhibitors are included.

E. Cancer Chemotherapeutic Agents Specific cancer chemotherapeutic agents for use as anti-cancer agents in conjunction with the compositions of the present invention include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate ( Blenoxane (registered trademark), busulfan (Myleran (registered trademark)), busulfan injection (Busulex (registered trademark)), capecitabine (Xeloda (registered trademark)), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine , Carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®) , Cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC) -Dome (registered trademark)), dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine (registered trademark)), daunorubicin citrate liposome injection (DanoXome (registered trademark)), dexamethasone, docetaxel (registered trademark) Trademark), US Patent No. US2004073044), doxorubicin hydrochloride (Adriamycin (registered trademark), Rubex (registered trademark)), etoposide (Vepesid (registered trademark)), Fludarabine acid (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibin, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, mel Faran (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxant (Novantrone (R)), Mirotag, Paclitaxel (Taxol (R)), Phoenix (Yttrium 90 / MX-DTPA), Pentostatin, Polyfeprosan 20 and carmustine graft (Gliadel (R)), Quen Tamoxifen acid (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban (Velban)) Registered trademark)), vincristine (Oncovin®), and vinorelbine (Navelbine®).

F. Alkylating agents Alkylating agents for use in conjunction with the compositions of the present invention for anti-cancer treatment include VNP-40101M or chloretidine, oxaliplatin (US Pat. No. 4,169,846, International Publication). WO 03/24978 and International Publication No. WO 03/04505), glufosfamide, mafosfamide, etopophos (US Pat. No. 5,041,424), predonimustine; treosulfan; busulfan; ilofulvene (acylfulvene); Acridine (PD-115934); O6-benzylguanine; decitabine (5-aza-2-deoxycytidine); brostallicin; mitomycin C (MitoExtra); TLK- 286 (Telcyta®); temozamide; trabectedin (US Pat. No. 5,478,932); AP-5280 (platinate formulation of cisplatin); porphyromycin; and clerazide (mechloretamine) It is.

G. Chelating agents Chelating agents for use in conjunction with the compositions of the present invention for anticancer therapy include tetrathiomolybdate (International Publication No. WO01 / 60814); RP-697; Chimeric T84.66 ( cT84.66); gadofosveset (Vasovist®); deferoxamine; and optionally bleomycin in combination with electroporation (EPT).

H. Biological Response Modifiers Biological response modifiers such as immunomodulators for use in conjunction with the compositions of the present invention for anti-cancer therapy include UCN-01, CEP-701 and midostaurin. Taurospurin and macrocyclic analogs thereof (International Publication WO02 / 30941, International Publication WO97 / 07081, International Publication WO89 / 07105, US Pat. No. 5,621,100, International Publication WO93 / 07153, International Publication No. WO01 / 04125, International Publication No. WO02 / 30941, International Publication No. WO93 / 08809, International Publication No. WO94 / 06799, International Publication No. WO00 / 27422, International Publication No. WO96 / 13506 and International Publication Gazette WO88 / 07045); squalamine (international public) DA-9601 (International Publication WO 98/04541 and US Pat. No. 6,025,387); Alemtuzumab; Interferon (eg IFN-a, IFN-b, etc.); Interleukins, especially IL -2 or Aldesleukin and IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, And an active biological variant thereof having an amino acid sequence that is more than 70% of the native human sequence; Artretamine (Hexalen®); SU101 or Leflunomide (International Publication No. WO 04/06834 and US Pat. No. 6) 331, 555); imidazoquinolines such as resiquimod and imiquimod US Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612); and SMIP (International Publication No. WO 04/87153, International Publication No. WO 04/64759, and International Publication No. WO 04/60308) including benzazole, anthraquinone, thiosemicarbazone, and tryptanthrin.

I. Cancer vaccine:
Anti-cancer vaccines for use in conjunction with the compositions of the present invention include Avicine® (Tetrahedron Letters, 26, 1974, pages 2269-70); Oregovomab (OvaRex®); Therapope® ) (STn-KLH); melanoma vaccine; GI-4000 series (GI-4014, GI-4015, and GI-4016) directed to five mutations in Ras protein; GlioVax-1; MelaVax; (Registered trademark) or INGN-201 (International Publication WO95 / 12660); Sig / E7 / LAMP-1 encoding HPV-16E7; MAGE-3 vaccine or M3TK (International Publication WO94 / 05304); HER-2VA X; ACTIVE stimulating T cells specific for tumors; GM-CSF cancer vaccine; and vaccines based on Listeria monocytogenes.

J. et al. Antisense treatment:
Anticancer agents for use in conjunction with the compositions of the present invention include AEG-35156 (GEM-640); AP-12009 and AP-11014 (antisense oligonucleotides specific for TGF-β2); AVI-4126; AVI AVI-4472; Oblimersen (Genasense (registered trademark)); JFS2; Aprinocarsen (International Publication No. WO 97/29780); GTI-2040 (R2 ribonucleotide reductase mRNA antisense oligo) (International Publication) GTI-2501 (International Publication No. WO 98/05769); c-Raf antisense oligodeoxynucleotide (LErafAON) encapsulated in liposome (International Publication No. WO) 8/43095 No.); antisense compositions, such as and Sirna-027 (VEGFR-1mRNA therapeutic targeting based on RNAi) is also included.

K. IL-2:
One preferred IL-2 compound is “Aldesleukin” or “Proleukin®”, which is manufactured by Chiron Corporation of Emeryville, California. IL-2 in this formulation is produced recombinantly and the non-glycosylated human IL-2 mutein eliminates the first alanine residue and replaces the cysteine residue at position 125 with a serine residue. Thus differing from the native human IL-2 amino acid sequence (referred to as des-alanyl-1, serine-125 human interleukin-2). This IL-2 mutein can be expressed in E. coli and then purified by diafiltration and cation exchange chromatography as described in US Pat. No. 4,931,543.

  Aldesleukin was compared with native (Jurkat) IL-2 in vitro. There were no significant differences, and induction of cytolytic cells in vivo in mice and serum half-life after IV administration were similar for aldesleukin and native (Jurkat) IL-2. However, the form of IL-2 encompassed by aldesleukin has beneficial attributes, and therefore references to “aldesleukin” encompass only its composition and all possible forms of IL-2 protein. Is not included.

In 1983, when the Cetus lymphocyte proliferation bioassay was developed, there was no official IL-2 standard preparation available. For this preparation, 1 Cetus unit induced IL-2 dependent mouse T cells to take up tritium-labeled thymidine at its maximum level of 50% after 24 hours of incubation, in 1 mL of IL-2. Defined as quantity. The product containing IL-2 was assigned a specific activity of 3 × 10 ⁶ cetas units per mg. In 1988, the National Institute for Biologics (NIBSC), a WHO laboratory for biological standards in the United Kingdom, established an international standard for IL-2. In 1988, the assay procedure for the Setas lymphocyte proliferation bioassay was also changed. A new bioassay procedure calibrated the Cetas laboratory standard to the IL-2 international standard. The following correction factors were established:
International unit = Setas unit x 6
The amount of aldesleukin is specified using mass units based on 1 mg aldesleukin with a nominal specific activity of 18 × 10 ⁶ international units (18 MIU). The protocol incorporates international units.

  Pharmaceutical compositions useful in the methods of the invention can include biologically active variants of IL-2. Such variants retain the desired biological activity of the native polypeptide such that when administered to a subject, the pharmaceutical composition comprising the variant polypeptide has the same therapeutic effect as the pharmaceutical composition comprising the native polypeptide. Should be. That is, the variant polypeptide serves as a therapeutically active ingredient in the pharmaceutical composition in a manner similar to that observed in the native polypeptide. There exist methods in the art to determine whether a variant polypeptide retains a desired biological activity and thus serves as a therapeutically active ingredient in a pharmaceutical composition. Biological activity can be measured using assays specifically designed to measure the activity of native polypeptides or proteins, including those described in the present invention. In addition, antibodies raised against biologically active native polypeptides can be tested for their ability to bind to variant polypeptides. Here, effective binding is an indication that the polypeptide has a conformation similar to the native polypeptide.

  For the purposes of the present invention, the desired IL-2 biological activity is that IL-2 activates and / or expands natural killer (NK) cells, resulting in lymphokine activated killer (LAK) activity and antibody dependence. The ability to mediate cellular cytotoxicity (ADCC). Thus, IL-2 variants (eg, human IL-2 muteins) for use in the methods of the invention activate and / or expand NK cells to mediate LAK activity and ADCC. Assays to determine NK cell activation or expansion by IL-2 and mediation of 1AC or ADCC activity are well known in the art.

  Suitable biologically active variants of native or naturally occurring IL-2 can be fragments, analogs, and derivatives of the polypeptide. By “fragment” is intended a polypeptide consisting of only a portion of an intact polypeptide sequence and structure, and can be a C-terminal deletion or an N-terminal deletion of the native polypeptide. By “analog” is intended an analog of either a native polypeptide or a fragment of a native polypeptide, where the analog is of a native polypeptide having one or more amino acid substitutions, insertions, or deletions. Has sequence and structure. Also included within the term “analog” are “muteins” such as those described herein, and peptides having one or more peptoids (peptidomimetics) (International Publication No. WO 91/04282). reference). A “derivative” refers to any suitable modification of a native polypeptide of interest, a fragment of a native polypeptide, or a respective analog thereof, such as glycosylation, so long as the desired biological activity of the native polypeptide is retained. Intended for phosphorylation, polymer conjugate formation (eg with polyethylene glycol), or the addition of other exogenous moieties. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

  For example, amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native polypeptide of interest. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Walker and Gaastra, (1983), “Techniques in Molecular Biology”, (MacMillan Publishing Company, New York); Kunkel, (1985), incorporated herein by reference. Natl. Acad. Sci. USA, 82: 488-492; Kunkel et al. (1987), Methods Enzymol. 154: 367-382; Sambrook et al. (1989), “Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, Plainview, New York 3, U.S. Pat. No. 4, and U.S. Pat. No. 4, 87); See the references cited in. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the polypeptide of interest is provided by Dayhoff et al. (1978), “Atlas of Protein Sequence and Structure (Natl. Biomed.), Incorporated herein by reference. (Res. Found., Washington, D. C.) Conservative substitutions such as exchanging one amino acid for another with similar properties may be preferred. Examples of substitutions include, but are not limited to, Gly: Ala, Val: Ile: Leu, Asp: Glu, Lys: Arg, Asn: Gln, and Phe: Trp: Tyr.

  In constructing a variant of the target IL-2 polypeptide, modification is performed so that the variant continues to have the desired activity. Any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of the reading frame and result in complementary regions that may result in secondary mRNA structure. Preferably not.

  A biologically active variant of IL-2 is generally at least 70%, preferably at least 80%, more preferably about 90% to 95% relative to the amino acid sequence of a reference polypeptide molecule that serves as a basis for comparison. As described above, the amino acid sequence identity is most preferably about 98% or more. Thus, when the IL-2 reference molecule is human IL-2, its biologically active variant is at least 70%, preferably at least 80%, more preferably about 90% relative to the amino acid sequence of human IL-2. % To 95% or more, most preferably about 98% or more of sequence identity. A biologically active variant of the native polypeptide of interest can be as little as 1-15 amino acids, no more than 1-10, such as 6-10, no more than 5, no more than 4, 3, 2, or even May differ from the native polypeptide by one amino acid residue. “Sequence identity” refers to the alignment between a variant polypeptide and a polypeptide molecule that serves as a reference when aligned with the amino acid sequence of a reference molecule by aligning consecutive segments of the variant with the specified amino acid sequence. It is intended that the same amino acid residue be found therein. The% sequence identity between two amino acid sequences determines the number of positions where the same amino acid residue appears in both sequences to obtain the number of matching positions, the number of matching positions is the reference molecule Calculated by dividing by the total number of positions in the segment being compared to and multiplying the result by 100 to obtain% sequence identity.

  Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferably, naturally occurring and non-naturally occurring variants of IL-2 are at least relative to a reference molecule, such as the amino acid sequence of native human IL-2, or a shorter portion of a reference IL-2 molecule. It has an amino acid sequence that is 70%, preferably 80%, more preferably 85%, 90%, 91%, 92%, 93%, 94% or 95% identical. More preferably, the molecules are 96%, 97%, 98% or 99% identical. The% sequence identity is determined using a Smith-Waterman homology search algorithm with an affine gap search using gap open penalty 12, gap extension penalty 2, BLOSUM matrix 62. The Smith-Waterman homology search algorithm is described by Smith and Waterman, Adv. Appl. Math. (1981) 2: 482-489. Variants can differ, for example, by as few as 1-10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even one amino auxiliary residue .

  For optimal alignment of two amino acid sequences, a contiguous segment of the variant amino acid sequence may have added or deleted amino acid residues compared to the reference amino acid sequence. A continuous segment used for comparison with a reference amino acid sequence includes at least 20 consecutive amino acid residues, and may be 30, 40, 50 or more amino acid residues. Corrections for sequence identity associated with conservative residue substitutions or gaps can be made (see Smith-Waterman homology search algorithm).

  The exact chemical structure of a polypeptide having IL-2 activity depends on several factors. Because ionizable amino and carboxyl groups are present in the molecule, certain polypeptides can be obtained as acidic or basic salts or in neutral form. All such preparations that retain their biological activity when placed under appropriate environmental conditions are included in the definition of a polypeptide having IL-2 activity as used herein.

  Furthermore, the primary amino acid sequence of a polypeptide can be enhanced by derivatization with sugar moieties (glycosylation) or other supplemental molecules such as lipids, phosphates, acetyl groups. Alternatively, it may be enhanced by forming a conjugate with a saccharide. A specific aspect of such enhancement is achieved by the production host's post-translational processing system. Other such modifications can be introduced in vitro. In any event, such modifications are included in the definition of IL-2 polypeptide as used herein as long as the IL-2 activity of the polypeptide is not destroyed. Such modifications are expected to affect the activity quantitatively or qualitatively in various assays by enhancing or decreasing the activity of the polypeptide. In addition, individual amino acid residues in the chain may be modified by oxidation, reduction, or other derivatization, and the polypeptide may be cleaved to obtain fragments that retain activity.

  Such alterations that do not destroy activity do not remove the polypeptide sequence from the definition of an IL-2 polypeptide of interest as used herein.

  There is considerable guidance in the art regarding the preparation and use of polypeptide variants. In preparing an IL-2 variant, those skilled in the art can make any modification to the nucleotide or amino acid sequence of the native protein for use as a therapeutically active component of the pharmaceutical composition used in the methods of the invention. It can easily be determined if a suitable variant is produced.

  Although IL-2 or a variant thereof for use in the methods of the invention can be derived from any source, it is preferably recombinant IL-2. “Recombinant IL-2” has biological activity comparable to that of native sequence IL-2, for example, Taniguchi et al. (1983), Nature, 302: 305-310 and Devos, (1983). ), Nucleic Acids Research, 11: 4307-4323, interleukin-2 prepared by recombinant DNA technology, or Wang et al. (1984) Science 224: 1431-1433. IL-2 modified by In general, a gene encoding IL-2 is cloned and then expressed in a transformed organism, preferably a microorganism, most preferably E. coli, as described herein. The host organism expresses the foreign gene under expression conditions to produce IL-2. Synthetic recombinant IL-2 can also be produced in eukaryotic organisms such as yeast or in human cells. Methods for growing, collecting, disrupting or extracting IL-2 from cells are described, for example, in U.S. Pat. Nos. 4,604,377; 4,738,927; 4,656,132; 4,569. 790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543. ing.

  Examples of variant IL-2 proteins include European Patent (EP) Publication EP 136,489 (which discloses one or more of the following changes in the amino acid sequence of naturally occurring IL-2) : Asn26 to Gln26; Trp121 to Phe121; Cys58 to Ser58 or Ala58, Cys105 to Ser105 or Ala105; Cys125 to Ser125 or Ala125; deletion of all residues following Arg120; and its Met-1 form); and Belgium Equivalent to Patent No. 893,016 and co-owned with US Pat. No. 4,518,584, European Patent Application No. 83306221.9 filed October 13, 1983 (published on May 30, 1984) Published under EP109,748) The recombinant IL-2 mutein (which is deleted at position 125 [numbered according to native human IL-2], or has been replaced by a neutral amino acid). 2 muteins; alanyl-ser125-IL-2; and des alanyl-ser125-IL-2 are disclosed). U.S. Pat. No. 4,752,585 (which discloses the following variant IL-2 proteins: ala104 ser125 IL-2, ala104 IL-2, ala104 ala125 IL-2, val104 ser125 IL-2, val104 IL-2, val104 ala125 IL-2, des-ala1 ala104 ser125 IL-2, des-ala1 ala104 IL-2, 10 des-ala1 ala104 ala125 IL-2, des-ala1 val104 ser125 IL-2, des-ala1 val104 IL-2, des-ala1 val104 ala125 IL-2, des-ala1 des-pro2 ala104 ser125 IL-2, des-al 1 des-pro2 ala104 IL-2, des-ala1 des-pro2 ala104 aia125 IL-2, des-ala1 des-pro2 val104 ser125 IL-2, des-ala1 des-pro2 val104 IL-2, des-ala1 des2 val104 ala125 IL-2, des-ala1 des-pro2 des-thr3 ala104 ser125 IL-2, des-ala1 des-pro2 des-thr3 ala104 IL-2, des-ala1 des-pro2 des-thr3 ala125 ala151515 , Des-ala1 des-pro2 des-thr3 val104 ser125 IL-2, d s-ala1 des-pro2 des-thr3 val104 IL-2, des-ala1 des-pro2 des-thr3 val104 ala125 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 ala125 sel125 IL-2 ser125 sel125 IL-2 des-pro2 des-thr3 des-ser4 ala104 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 ala104 ala125 IL-2, des-alal des-pro2 des-thr3 des104-ser4 125 des-ala1 des-pro2 des-thr3 des-ser4 val104 IL-2, d s-ala1 des-pro2 des-thr3 des-ser4 val104 20 ala125 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 ala104 ser125 des- IL2 des-ala1 des-thr3 des-thr3 des-2 -Ser4 des-ser5 ala104 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 ala104 ala125 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser4 des104 2, des ala1 des-pro2 des-thr3 des-ser4 des- er5 val104 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 val104 ala125 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser6 des-ser5 des104 2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 ala104 IL-2, des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 ser104 serIL2 104 -Ala1 des-pro2 des-thr3 des ser4 des-ser5 des-se r6 val104 ser125 IL-2, des-alal des-pro2 des-thr3 des-ser4 des-ser5 des ser6 val104 IL-2, and des-alal des-pro2 des-thr6 des-ser6 des-ser5 des104 IL-2) and U.S. Pat. No. 4,931,543 (which includes the IL-2 mutein des-alanyl-1, serine 30 125 human IL-2 used in the examples herein, and other ILs) -2 muteins are also disclosed).

  See also European Patent Publication EP200,280 (10, published 10 December 1986), which discloses a recombinant IL-2 mutein in which the methionine at position 104 is replaced by a conservative amino acid. I want.

  Examples include the following muteins: ser4 des-ser5 ala104 IL-2; des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 ala104 ala125 IL-2; des-ala1 des-pro2 des -Thr3 des-ser4 des-ser5 glu104 ser125 IL-2; des-alal des-pro2 des-thr3 des ser4 des-ser5 glu104 IL-2; IL-2; des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 de -Ser6 ala104 ala125 IL-2; des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 ala104 IL-2; ala104 ser125 IL-2; des-ala1 des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 glu104 ser125 IL-2; des-ala1 des-pro2 des-thr6 des-ser6 des6 ser4 des-ser6 des4 -2; and des-ala1 des-pro2 des-thr3 des-ser4 d s-ser5 des-ser6 glu104 ala125 IL-2. Also, a non-glycosylated human IL-2 variant having alanine instead of methionine of native IL-2 as the N-terminal amino acid; glycosylation wherein the first methionine is deleted such that proline is the N-terminal amino acid Unpublished human IL-2; and European Patent Publication EP 118,617 and US Patents disclosing unglycosylated human IL-2 with an alanine inserted between the N-terminal methionine and proline amino acids See also 5,700,913.

  Other IL-2 muteins have preferential activity over high affinity IL-2 receptors expressed by cells expressing T cell receptors over NK cells, and IL- 2 as reported in International Publication No. WO 99/60128, where toxicity is reported (substitution of aspartic acid at position 20 with histidine or isoleucine, asparagine at position 88 with arginine, glycine, or Substituted with isoleucine, or glutamine at position 126 with leucine or glutamate); LAK has been shown to have reduced binding to the high affinity IL-2 receptor compared to native IL-2 Mutein (position) disclosed in US Pat. No. 5,229,109, which maintains the ability to stimulate cells Arginine of 8 is replaced by alanine or phenylalanine at position 42 is replaced by lysine); a mutant protein disclosed in WO 00/58456, which is alleged to reduce blood leak syndrome (native IL- Naturally occurring (x) D (y) sequence alterations or deletions in 2; D is aspartic acid, (x) is leucine, isoleucine, glycine, or valine; (y) is valine, leucine Or IL-2pl-30 peptide (IL-2 a-helix A) disclosed in WO 00/04048, which has been reported to stimulate the induction of NK and LAK cells; Corresponding to the first 30 amino acids of IL-2, including the whole and interacting with the b chain of the IL-2 receptor); and blood A mutant of the IL-2pl-30 peptide, also disclosed in WO 00/04048, which has been reported to be unable to induce hemorrhage but still be able to generate LAK cells (position) 20 aspartic acids replaced with lysine). In addition, IL-2 can be modified with polyethylene glycol to provide increased solubility and altered pharmacokinetic profile (see US Pat. No. 4,766,106).

  As used herein, the term “IL-2” includes IL-2 or polyproline fused to a second protein or water soluble to reduce dosing frequency or to improve the tolerance of IL-2. It is also intended to include IL-2 fusions or conjugates comprising IL-2 covalently conjugated to a sex polymer. For example, IL-2 (or a variant thereof as defined herein) can be fused to human albumin or an albumin fragment using methods known in the art (WO 01/79258). reference). Alternatively, IL-2 can be covalently conjugated with polyproline or polyethylene glycol homopolymer and polyoxyethylated polyol using methods known in the art, where the homopolymer is unsubstituted. Or one end is substituted with an alkyl group and the popriol is unsubstituted (eg, US Pat. Nos. 4,766,106, 5,206,344, and 4,894). No. 226). Any pharmaceutical composition comprising IL-2 as a therapeutically active ingredient can be used in the methods of the invention. Such pharmaceutical compositions are known in the art and include, but are not limited to, U.S. Pat. Nos. 4,745,180; 4,766,106; 4,816,440; 894,226; 4,931,544; and 5,078,997. Accordingly, a liquid composition, lyophilized composition, or spray-dried composition comprising IL-2 or a variant thereof known in the art is prepared as an aqueous or non-aqueous solution or suspension; It can be administered to a subject according to the methods of the invention. Each of these compositions includes IL-2 or a variant thereof as a therapeutically or prophylactically active ingredient. A “therapeutically or prophylactically active ingredient” means that when a pharmaceutical composition is administered to a subject, it provides the desired therapeutic or prophylactic response for the treatment, prevention, or diagnosis of the disease or condition in the subject. Thus, it is intended that IL-2 or a variant thereof is specifically incorporated into the composition. Preferably, the pharmaceutical composition includes appropriate stabilizers, fillers, or both to minimize problems associated with loss of protein stability and biological activity during preparation and storage. In a preferred embodiment of the invention, the pharmaceutical composition comprising IL-2 in the method of the invention comprises a composition comprising stabilized monomeric IL-2 or a variant thereof, multimeric IL-2 or a variant thereof. And a composition comprising stabilized lyophilized or spray dried IL-2 or a variant thereof.

  A pharmaceutical composition comprising stabilized monomeric IL-2 or a variant thereof is disclosed in International Publication No. WO01 / 24814 entitled “Stabilized Liquid Polypeptide-Containing Pharmaceutical Compositions.”. “Monomer” IL-2 is intended to mean that the protein molecule is present in its substantially monomeric form, rather than in aggregated form, in the pharmaceutical compositions described herein. Thus, there are no IL-2 covalent or hydrophobic oligomers or aggregates. Briefly, IL-2 in these liquid compositions is formulated with a sufficient amount of amino acid base to reduce IL-2 aggregate formation during storage. An amino acid base is an amino acid or combination of amino acids in which any given amino acid is present in its free base form or in its salt form. Preferred amino acids are selected from the group consisting of arginine, lysine, aspartic acid, and glutamic acid. These compositions further comprise a buffering agent for maintaining the phi of the liquid composition within an acceptable range for IL-2 stability, wherein the buffering agent is an acid substantially free of its salt form, An acid in its salt form, or a mixture of an acid and its salt fonde. Preferably, the acid is selected from the group consisting of succinic acid, citric acid, phosphoric acid, and glutamic acid.

  Such compositions are referred to herein as stabilized monomeric IL-2 drug compositions. The amino acid bases in these compositions serve to stabilize IL-2 against aggregate formation during storage of the liquid drug composition, while the acid, its salt, substantially free of its salt form Using an acid in form or a mixture of an acid and its salt form as a buffer results in a liquid composition having a nearly isotonic osmolarity. The liquid drug composition may further incorporate other stabilizers, more specifically nonionic surfactants such as methionine, polysorbate 80, and EDTA to further increase polypeptide stability. . Such liquid pharmaceutical compositions are said to be stable, an amino acid base in combination with an acid substantially free of salt fonde, an acid in the salt fond, or a mixture of an acid and its salt form Adding a composition results in an increased storage stability compared to a liquid pharmaceutical composition formulated without the combination of these two ingredients. These liquid pharmaceutical compositions comprising stabilized monomeric IL-2 can be used in aqueous liquid form or stored frozen for later use, or to a liquid fond or subject according to the methods of the present invention. It may be in any dry form for subsequent reconstitution into other forms suitable for administration. “Dry form” refers to freeze-drying a liquid drug composition or formulation (ie, lyophilization; see, for example, Williams and Polli, (1984), J. Parental Sci. Technol., 38 48:59), spray drying (Masters, (1991), "Spray-Drying Handbook, (S edition; Longman Scientific and Technical, Essez, UK), 491-676; Broadhead et al., ( 1992), Drug Devl. Ind. Pharm., 18: 169-1206; and Mummentaler et al., (1994), Pharm. See page 20, or intended to dry by air drying (Carpenter and Crowe, (1988), Cryobiology, 25: 459-470; and Roser (1991), Biopharm., 4: 47-53). .

  Other examples of IL-2 formulations comprising IL-2 in its non-aggregated monomer state include those described in Whittington and Faulds, (1993) Drugs 46 (3): 446-514. Is included. These formulations include recombinant IL-2 mutein teseleukin (methionine at the amino terminus) formulated with 0.25% human serum albumin in lyophilized powder, reconstituted with isotonic saline. Non-glycosylated human IL-2 with added residues), and the formulation has a pH of 3.0-4.0, 10 advantageously no buffer is present, and the conductivity is 1000 mmhos / Recombinant IL-2 mutein bioleukin, formulated so that 0.1 to 1.0 mg / ml IL-2 mutein is combined with acid, less than cm (preferably less than 500 mmhos / cm) Recombinant IL-2 products, such as (human IL-2 with a methionine residue added to the amino terminus and a cysteine residue at position 125 of the human IL-2 sequence replaced with alanine) It is. European Patent Publication EP 373,679; Xhang et al. (1996), Pharmaceut. Res. 13 (4): 643-644 and Prestrelski et al. (1995) Pharmaceut. Res. 12 (9): 1250-1258.

  Examples of pharmaceutical compositions comprising multimeric IL-2 are generally disclosed in US Pat. No. 4,604,377. By “multimer” is intended that protein molecules are present in the pharmaceutical composition in a micro-aggregated form with an average molecular association of 10-50 molecules. These multimers exist as loosely bound, physically associated IL-2 molecules. Lyophilized forms of these compositions are commercially available under the trade name Proleukin® IL-2 (Chiron Corporation, Emeryville, CA). The lyophilized formulation disclosed in this reference comprises selectively oxidized, microorganism-produced recombinant IL-2, which is a soluble carrier that provides a bulk such as mannitol , And mixed with a sufficient amount of sodium dodecyl sulfate to ensure that the recombinant IL-2 dissolves in water.

  These compositions are suitable for reconstitution with aqueous injections for parenteral administration, are stable in human patients and are well tolerated. When reconstituted, IL-2 maintains its multimeric state. Such lyophilized or liquid compositions comprising multimeric IL-2 are encompassed by the methods of the invention. Such a composition is referred to herein as a multimeric IL-2 drug composition.

  The method of the present invention may employ a stabilized lyophilized or spray-dried drug composition comprising IL-2, which is a liquid or other suitable form for administration according to the method of the present invention. Can be reconstructed Such a pharmaceutical composition is disclosed in International Publication No. WO01 / 49274 entitled “Methods for Pulmonary Del bef of of Inter1euk1Tz-2.”. These compositions may further comprise at least one filler, an amount of at least one agent sufficient to stabilize the protein during the drying process, or both. “Stabilized” means that the IL-2 protein or variant thereof is in the form of monomer or multimer as well as quality, purity after lyophilization or spray drying to obtain a solid or dry powder form of the composition. , And its other main properties of efficacy are intended to be retained. In these compositions, preferred carrier materials for use as fillers include glycine, mannitol, alanine, valine, or any combination thereof, most preferably glycine. Fillers are present in the formulation in the range of 0% to about 10% (w / v) depending on the drug used. Preferred carrier materials for use as stabilizers include any sugar or sugar alcohol or any amino acid. Preferred sugars include sucrose, trehalose, raffinose, stachyose, sorbitol, glucose, lactose, dextrose or any combination thereof, preferably sucrose. When the stabilizer is a sugar, it is about 0% to about 9.0% (w / v), preferably about 0.5% to about 5.0%, more preferably about 1.0% to about It is present in the range of 3.0%, most preferably about 1.0%. When the stabilizer is an amino acid, it ranges from about 0% to about 1.0% (w / v), preferably from about 0.3% to about 0.7%, most preferably about 0.5%. Exists. These stabilized lyophilized or spray-dried compositions optionally comprise methionine, one of its salts such as ethylenediaminetetraacetic acid (EDTA) or disodium EDTA, or IL-2 or a variant thereof. Other chelating agents that protect against methionine oxidation may be included. The use of such agents in this manner is described in US application 09 / 677,643, which is incorporated herein by reference. The stabilized lyophilized composition or spray-dried composition is within the acceptable range of the pH of the pharmaceutical composition in the liquid phase, such as during the compounding process or after reconstitution of the dried form of the composition, preferably about pH 4. It may be formulated with a buffering agent that maintains from 0 to about pH 8.5. The buffer is selected so that it is compatible with the drying process and does not affect the quality, purity, potency, and stability of the protein during processing and storage.

  The previously described stabilized monomers, multimers, and stabilized lyophilized or spray dried IL-2 drug compositions represent suitable compositions for use in the methods of the present invention. . However, any pharmaceutical composition comprising an IL-2 compound as a therapeutically active ingredient is encompassed by the methods of the present invention.

  Also provided herein is a kit or package comprising at least one inventive combination composition and accompanying instructions for use. For example, if each drug itself is administered as a separate or separate dosage form, the kit includes each drug with instructions for use. The drug components can be packaged in any manner suitable for administration as long as the packaging clearly indicates the mode of administration of each drug component when considered along with the instructions for administration. Alternatively, each of the drug components of the combination may be combined as a single administrable dosage form such as a single composition.

  For example, in an exemplary kit comprising rIL-2 and an anti-angiogenic agent, the kit may be arranged at any suitable time period, such as daily. By way of example, on day 1, a representative kit may contain respective unit doses of rIL-2 and an anti-angiogenic agent. If each drug is administered twice a day, the kit may include two rows of unit dosage forms of rIL-2 and an anti-angiogenic agent, as well as instructions for when to administer, corresponding to day 1. . Alternatively, if the time or amount of one or more drugs administered is different compared to the other drug species in the combination, that is reflected in the package and instructions. For example, if rIL-2 is administered twice a day and the anti-angiogenic agent is taken only once a day, an exemplary day 1 package would have a unit dosage form of rIL-2 of “1 day Corresponding to "Eye, Dose 1" and the dosage form of the anti-angiogenic agent may correspond to "Day 1, Dose 2".

  Various embodiments according to the above can easily be envisioned, but of course will depend on the specific drug combination used for treatment, its corresponding dosage form, the recommended dose, the intended patient population, etc. . The package may be in any form commonly used for pharmaceutical packaging, utilizing any of several features such as various colors, packaging materials, tamper-evident packaging, blister packs, desiccants, etc. Can do.

  The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any manner.

  Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

Example 1
Compositions for co-administration with rIL-2 Compound Table 1: Examples of quinazoline

Ｂ．合成
キナゾリン
スキーム１：

B. Synthesis Quinazoline Scheme 1:

スキーム１は、無数の置換キナゾリン化合物を合成するためのモジュール方法を記載している。ＡＧへの言及は、例えばハロゲン化物、トリフレート、またはケトンなどの活性化基を示し、４個ものＡＧ基が存在し得る。第３ステップに示す４−クロロの位置はベンジル環状の位置５〜８よりも活性があるため、ＮＨＲＲ’を用いた置換は、位置５〜８のいずれかのＡＧの同時置換があまり多く起こらずに進行し得る。次いで、最終ステップにおけるＡＧ基のＲ’’による置換は、弱〜中等度の塩基の存在下で進行し得る。あるいは、所望の生成物を得るためにＡＧ基（または複数のＡＧ基）を修飾してもよく、例えば、ＮＯ_２基をＥｔＯＨおよびＨ_２Ｏ中のＦｅおよびＡｃＯＨで還元してアミノ置換基を得て、さらにそれを、例えばパラホルムアルデヒドを用いた還元性アミノ化によって置換し得る。

Scheme 1 describes a modular method for synthesizing a myriad of substituted quinazoline compounds. Reference to AG refers to an activating group, such as a halide, triflate, or ketone, and there can be as many as four AG groups. Since the 4-chloro position shown in the third step is more active than the benzylic cyclic positions 5-8, substitution with NHRR 'does not result in much simultaneous substitution of any of the AGs at positions 5-8. Can progress to. The replacement of the AG group with R ″ in the final step can then proceed in the presence of a weak to moderate base. Alternatively, the AG group (or multiple AG groups) may be modified to obtain the desired product, for example by reducing the NO ₂ group with Fe and AcOH in EtOH and H ₂ O to reduce the amino substituent. It can then be further replaced by reductive amination, for example using paraformaldehyde.

As will be apparent to those skilled in the art, a large number of functionalized 2-aminobenzamide starting materials are either commercially available or readily synthesized by known procedures. Subsequently, the AG group in the starting material may be a desirable substituent in the final product (such as R ″), such as an alkoxy group or a substituted alkyl group. Further, available are a number of functionalized 2-nitrobenzamide starting materials, they are converted into readily aminobenzamide starting material in the presence of a reducing agent such as H _{2 /} Pd _/ C in EtOH The

  Alternative methods for making the quinazolines of the present invention include WO 04/24703, WO 01/32651, US Pat. No. 5,457,105, US Pat. No. 5,616,582, US Pat. No. 5,770. , 599, International Publication No. WO02 / 16351, US Patent No. 6,727,256, International Publication No. WO02 / 02552, US Patent No. 5,747,498, and International Publication No. WO96 / 30347. Yes.

Indolinone Scheme 2a:

スキーム２ａはワンポット手順として行い、ステップａの試薬はＨ_２Ｏ中のＮＨ_４ＯＨ、ＣｕＣｌである。次いで、ステップｂでＨＣｌ水溶液を加える。Ｒ_２〜Ｒ_５は本明細書中で定義したとおりである。

Scheme 2a is performed as a one-pot procedure, where the reagent in step a is NH ₄ OH in H ₂ O, CuCl. Then, in step b, an aqueous HCl solution is added. R _{2 to} R ₅ are as defined in this specification.

  Scheme 2b:

スキーム２ｂでは、試薬をＥｔＯＨ中、ピペリジン（ａ）の存在下で攪拌して最終生成物を得る。当業者には明らかなように、具体的な出発物質の反応性に応じて、反応を加熱して収率を増大させ得る。

In Scheme 2b, the reagent is stirred in EtOH in the presence of piperidine (a) to give the final product. As will be apparent to those skilled in the art, depending on the reactivity of the particular starting material, the reaction can be heated to increase the yield.

  Scheme 2c:

スキーム２ｃでは、試薬をＥｔＯＨ中、ＮａＯＢｕ−ｔ（ａ）の存在下で還流させて最終生成物を得る。スキーム２に示すＲ_９は、本明細書中で定義したとおりＨ、−ＯＨ、−ＣＮ、アルキル、アリール、ヘテロシクリル、アルコキシ、または−ＮＲ_ａＲ_ｂである。Ｒ_９での置換を可能にするために上記構造で式ＩＩを置き換え得ることが企図され、他の置換基は本明細書中で定義したとおりである。

In Scheme 2c, the reagent is refluxed in EtOH in the presence of NaOBu-t (a) to give the final product. R ₉ shown in Scheme 2 is H, —OH, —CN, alkyl, aryl, heterocyclyl, alkoxy, or —NR _a R _{b as} defined herein. It is contemplated that Formula II can be replaced with the above structure to allow substitution at R ₉ , and other substituents are as defined herein.

  Scheme 2d (Synthesis of Compound 6):

フタラジン
化合物１０と同様の置換フタラジンの調製を以下のとおり、米国特許ＵＳ６，２５８，８１２号から描写したように説明し、これには本発明の化合物の合成に役立ち得る他の反応スキームも含まれる。

Phthalazine The preparation of substituted phthalazines similar to compound 10 is illustrated as described from US Pat. No. 6,258,812, as follows, including other reaction schemes that may be useful for the synthesis of the compounds of the present invention. .

Scheme 3a:
1-Chloro-4- (4-pyridylmethyl) phthalazine dihydrochloride 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine 15.22 g (59.52 mmol) (prepared in July 1959 A mixture of 7.73 g (60.59 mmol) of 4-chloroaniline and 200 ml of 1-butanol was heated to reflux for 2 hours. Thereafter, the crystals obtained when the mixture was slowly cooled to 5 ° C. were collected by filtration and washed with 1-butanol and ether. Dissolve the filter residue in about 200 ml of hot methanol, treat the solution with 0.75 g of activated carbon, filter through Hyflo Super Cel, and adjust the pH of the filtrate to about 2.5 using 7 ml of 3N methanolic HCl. did. The filtrate was evaporated to about half of the initial volume and ether was added until a slight turbidity occurred. Subsequent cooling resulted in the precipitation of crystals. The crystals were filtered off, washed with a mixture of methanol / ether (1: 2) and ether, dried for 8 hours at 110 ° C. under HV and equilibrated for 72 hours at 20 ° C. and room temperature. In this way, the title compound having a water content of 8.6% was obtained. Melting point> 270 ° C .;

スキーム３ｂ：塩酸１−（４−クロロアニリノ）−４−（４−ピリジルメチル）フタラジン
０．９７２ｇ（３．８ｍｍｏｌ）の１−クロロ−４−（４−ピリジルメチル）フタラジン、０．６５６ｇ（４ｍｍｏｌ）の塩酸４−クロロアニリン（Ｒｅｓｅａｒｃｈ Ｏｒｇａｎｉｃｓ， Ｉｎｃ．、米国オハイオ州Ｃｌｅｖｅｌａｎｄ）および２０ｍｌのエタノールの混合物を、２時間、加熱還流した。反応混合物を氷浴中で冷却し、濾過し、結晶を少量のエタノールおよびエーテルで洗浄した。ＨＶ下、１１０℃で８時間、および１５０℃で１０時間乾燥させたのち、ＨＣｌの熱除去の結果、表題化合物を得た。融点＞２７０℃。

Scheme 3b: 1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride 0.972 g (3.8 mmol) 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.656 g (4 mmol) A mixture of 4-chloroaniline hydrochloride (Research Organics, Inc., Cleveland, Ohio, USA) and 20 ml of ethanol was heated to reflux for 2 hours. The reaction mixture was cooled in an ice bath, filtered and the crystals were washed with a small amount of ethanol and ether. After drying under HV at 110 ° C. for 8 hours and 150 ° C. for 10 hours, HCl was removed by heat to obtain the title compound. Melting point> 270 ° C.

スキーム３ｃ：
塩酸１−（４−クロロアニリノ）−４−（４−ピリジルメチル）フタラジン
１．２８ｇ（５ｍｍｏｌ）の１−クロロ−４−（４−ピリジルメチル）フタラジン、０．６７ｇ（５．２５ｍｍｏｌ）の４−クロロアニリンおよび１５ｍｌの１−ブタノールの混合物を、窒素雰囲気下で攪拌しながら０．５時間、１００Ｃで加熱した。その後、混合物を室温まで冷却し、濾過し、濾液を１−ブタノールおよびエーテルで洗浄した。精製には、結晶を４０ｍｌの熱メタノールに溶かし、溶液を活性炭で処理し、Ｈｙｆｌｏ Ｓｕｐｅｒ Ｃｅｌで濾過し、濾液を最初の体積の約半分まで蒸発させて、結晶沈殿物の形成がもたらされた。０℃まで冷却し、濾過し、フィルターの残渣をエーテルで洗浄し、ＨＶ下、１３０℃で８時間乾燥させたのち、表題化合物を得た。融点＞２７０℃；

Scheme 3c:
1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride 1.28 g (5 mmol) of 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.67 g (5.25 mmol) of 4- A mixture of chloroaniline and 15 ml 1-butanol was heated at 100 C for 0.5 h with stirring under a nitrogen atmosphere. The mixture was then cooled to room temperature, filtered and the filtrate was washed with 1-butanol and ether. For purification, the crystals were dissolved in 40 ml of hot methanol, the solution was treated with activated charcoal, filtered through Hyflo Super Cel, and the filtrate was evaporated to about half of the original volume, resulting in the formation of a crystalline precipitate. . After cooling to 0 ° C. and filtration, the filter residue was washed with ether and dried at 130 ° C. under HV for 8 hours to give the title compound. Melting point> 270 ° C .;

（実施例２）
ｒＩＬ−２の調製
アルデスロイキン、ｒＩＬ−２（ＮＳＣ３７７３３６４）（Ｐｒｏｌｅｕｋｉｎ（登録商標）、Ｃｈｉｒｏｎ）の調製：
ヒトジャーカット細胞系からのメッセンジャーＲＮＡを用いて二本鎖ｃＤＮＡを作製し、これをｐＢＲ３２２プラスミド内にハイブリダイズさせた。短いＩＬ−２塩基配列に対応する^３２Ｐ標識したオリゴヌクレオチドプローブを用いてＩＬ−２遺伝子を含むクローンを同定した。遺伝子を、好都合の制限部位を有するｐＢＲ３２２プラスミドの領域内に挿入した。適切なプロモーターおよびリボソーム結合部位をＩＬ−２遺伝子の前に挿入することで、生じた発現クローンは改変された組換えｒＩＬ−２をコードする。クローニングしたＩＬ−２のｉｎ ｖｉｔｒｏ突然変異誘発を用いて、位置１２５でのセリンからシステインへの保存的置換を行った。生じた分子は、そのｉｎ ｖｉｔｒｏ生物活性がネイティブＩＬ−２と区別不可能であった。アルデスロイキン遺伝子を保有する大腸菌の産生株を発酵槽内で増殖させた。培養物を収集し、アルデスロイキンを抽出した。アルデスロイキンを精製するために一連のクロマトグラフィーステップを行った。配合された生成物をｐＨ７．２〜７．８に調節した。Ｐｒｏｌｅｕｋｉｎ（登録商標）の分子量は約１５，６００ダルトンである。アミノ酸組成およびＮ末端配列決定による分析により、アルデスロイキンが予測されたタンパク質配列を有することが確認された。

(Example 2)
Preparation of rIL-2 Preparation of aldesleukin, rIL-2 (NSC3773364) (Proleukin®, Chiron):
Double stranded cDNA was made using messenger RNA from a human Jurkat cell line and hybridized into the pBR322 plasmid. A clone containing the IL-2 gene was identified using a ³² P-labeled oligonucleotide probe corresponding to the short IL-2 nucleotide sequence. The gene was inserted into a region of the pBR322 plasmid with convenient restriction sites. By inserting an appropriate promoter and ribosome binding site in front of the IL-2 gene, the resulting expression clone encodes a modified recombinant rIL-2. A conservative substitution of serine to cysteine at position 125 was made using in vitro mutagenesis of cloned IL-2. The resulting molecule was indistinguishable from native IL-2 in its in vitro biological activity. An Escherichia coli production strain carrying the aldesleukin gene was grown in a fermenter. The culture was collected and Aldesleukin was extracted. A series of chromatographic steps were performed to purify aldesleukin. The blended product was adjusted to pH 7.2-7.8. Proleukin® has a molecular weight of about 15,600 daltons. Analysis by amino acid composition and N-terminal sequencing confirmed that Aldesleukin has the predicted protein sequence.

Reconstitution and dilution procedures:
Proleukin® exists as a lyophilized cake in a 5 cc vial containing 1.3 mg protein. Proleukin® vials for injection are reconstituted with 1.2 mL of sterile water for injection, USP. The diluent is directed to the side of the vial to avoid excessive foaming and gently rotated until the contents are completely dissolved while avoiding shaking. After reconstitution, each mL contains 1.1 mg (18 million IU) Proleukin®. The reconstituted Proleukin® is suitable for direct intravenous injection or, if necessary, 50 mL-500 mL using 0.1% human albumin, 5% dextrose injection containing USP, USP Can be diluted to In the case of dilution, human albumin and USP are added to 5% dextrose injection and USP, and then reconstituted Proleukin (registered trademark) is added.

(Example 3)
Treatment with single agent rIL-2 composition versus combination treatment with rIL-2 composition and anti-angiogenic agent The results of high dose rIL-2 are summarized in Table 6.

いくつかの要素が抗血管形成性組成物を用いた組合せ療法の起動力を生じさせている。高用量ｒＩＬ−２に関連する顕著な罹患率により、患者の注意深い選択が必要となり、治療から利益を得る可能性のある潜在的な患者の数が劇的に減少した。残念ながら、最も高いＲＣＣの発生率を有する患者で随伴疾患が最も頻繁に見つかっている。患者の注意深い監視および不定期の医療的集中治療の必要性により、単独薬剤高用量ｒＩＬ−２の投与が高価となり、その使用が大きな医療センターに限定されている。その実務上の効果は、利用可能性が患者の招集に限定されてしまうことである。

Several factors have created the driving force for combination therapy with anti-angiogenic compositions. The significant morbidity associated with high-dose rIL-2 required careful patient selection and dramatically reduced the number of potential patients that could benefit from treatment. Unfortunately, concomitant disease is most frequently found in patients with the highest incidence of RCC. The need for careful patient monitoring and occasional medical intensive care makes expensive administration of a single drug high dose rIL-2 limited to large medical centers. Its practical effect is that availability is limited to patient calls.

  Lower doses of rIL-2 were strictly evaluated as shown in Table 7.

最も一般的に利用されている皮下レジメンは、Ｂｕｔｅｒらによって公開されている。Ｂｕｔｅｒでは、１日１回、１週間に５日間、６週間の間、ｒＩＬ−２を与えた。最初の５日間サイクルの間、１８×１０^６ＩＵ（ＭＩＵ）を１日１回与えた。その後のサイクルでは、最初の２日間より後の用量を９ＭＩＵに減らした。応答速度および生存データはｒＩＬ−２の高用量ＩＶボーラス投与について公開されたものと類似であり得る。Ｂｕｔｅｒ／Ｓｌｅｉｊｆｅｒレジメンは、最近、前向きのランダム化治験において高用量ｒＩＬ−２と比較されている。９６人の患者を高用量ＩＶ ｒＩＬ−２にランダム化し、９２人の患者をＳＱ ｒＩＬ−２（Ｂｕｔｅｒ ／Ｓｌｅｉｊｆｅｒ用量スケジュール）にランダム化した。以前の高用量ｒＩＬ−２に対する応答は２０％であり、ＳＱ ｒＩＬ−２に対する応答は１０％であった。しかし、全体的な生存は高用量と差異がなかった（ｐ＝０．３４）。したがって、有効性およびより低い用量レジメンに対する患者の全体的な応答性を増大させるために、抗血管形成組成物と組み合わせるための触媒が存在する。

The most commonly used subcutaneous regimen is published by Buter et al. In Buter, rIL-2 was given once a day for 5 days a week for 6 weeks. During the first 5 day cycle, 18 × 10 ⁶ IU (MIU) was given once a day. In subsequent cycles, the dose after the first 2 days was reduced to 9 MIU. Response rates and survival data may be similar to those published for high dose IV bolus administration of rIL-2. The Butter / Sliejfer regimen has recently been compared to high dose rIL-2 in a prospective randomized trial. Ninety-six patients were randomized to high dose IV rIL-2 and 92 patients were randomized to SQ rIL-2 (Buter / Sleijfer dose schedule). The previous response to high dose rIL-2 was 20% and the response to SQ rIL-2 was 10%. However, overall survival did not differ from the high dose (p = 0.34). Thus, there are catalysts to combine with anti-angiogenic compositions to increase efficacy and overall patient responsiveness to lower dose regimens.

  As shown in Table 8, the combination regimen of aldesleukin and anti-angiogenic agent was evaluated strictly.

・１用量の抗血管形成性剤（用量レベルに従った平面化用量に等価）を−７日目に与える。
・抗血管形成性剤を１日目に再度与え、その後、８週間の治療サイクルで２週間毎に連続的に与える。

• Give one dose of anti-angiogenic agent (equivalent to a flattened dose according to dose level) on day -7.
• Anti-angiogenic agent is given again on day 1 and then continuously every 2 weeks for an 8 week treatment cycle.

Treatment with rIL-2 was continued for 6 weeks in a row (1-42 days, Monday to Friday of each week), followed by a 2-week rest period resulting in an 8-week treatment cycle.
Example 4
Combination therapy with rIL-2 and small molecule receptor tyrosine kinase inhibitors BAY 43-9006 and SU11248 Materials and Methods Drug Recombinant human interleukin-2 (Proleukin®, Aldesleukin / rIL-2); 18 MIU / ml, Chiron Corporation, Emeryville, Calif.) Was reconstituted with sterile water for injection prior to administration, Formulated with 5% dextrose. Vincristine (Vincristine sulfate) was from Mayne Pharma Ltd (Mulgrave, Australia). CHIR-258 is 4-amino-5-fluoro-3- [5- (4-methylpiperazin-1-yl) -1H-benzimidazol-2-yl] quinolin-2 (1H) -one (Chiron). is there. According to published procedures and patents, BAY 43-9006 (Sorafanib / Nexavar®) (Riedl et al., Proc. Am. Assoc. Cancer Res., 2001, 42 (Abstract 4956); Lowinger et al., Curr. Pharm. Des., 2002, 8 (25): 2269-2278; International Publication No. WO9932455) and SU11248 (Sunitinib / Sutent®) (Sun et al., J. Med. Chem., 2003, 46 ( 7): 1116-1119; International Publication No. WO0160814) synthesized and purified in-house. Stock solutions of BAY 43-9006 or SU11248 (20 mM) were prepared in DMSO and aliquots were stored at −20 ° C. before use. For in vitro assays, all drugs were diluted in optimal media. For in vivo administration, BAY 43-9006 was formulated in 100% PEG400 vehicle while SU11248 dosing solution was prepared in 5 mM citrate buffer. All other chemicals were research grade.

Cell Lines All mouse cell lines, namely CTLL-2 (IL-2-dependent T cell line), B16-F10 melanoma, CT26 colon, and renal cancer RENCA are obtained from the American Tissue Culture Collection (Rockville, MD). It was. CTLL-2 is 10% FBS (Fetal Bovine Serum, Gibco Life Technologies, Gaithersburg, MD), 2 mM L-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 0.5 nM rIL-2, 2 mM Grow in RPMI 1640 supplemented with β-mercaptoethanol. RENCA cells were cultured in medium containing EMEM containing 10% FBS, 2% 100 × vitamin, 1% 200 mM glutamine, 1% 100 mM NaPy, 1% non-essential amino acids. For the growth of CT26 cells, the medium contained EMEM, 10% FBS, 2% vitamins, 1% 200 mM glutamine, 1% 100 mM sodium pyruvate, 1% non-essential amino acids. B16-F10 cells are in RPMI 1640 with 10% FBS, 1% non-essential amino acids, 1% 100 mM sodium pyruvate, 2% vitamins; 2 mM 1-glutamine; 2% sodium bicarbonate. Allowed to grow. Yac-1 cells (ATCC) were cultured in RPMI + 10% FBS and subcultured 1-2 days before the assay to ensure log phase growth. Cells were maintained as suspensions or adherent cultures in a humidified atmosphere at 37 ° C., 5% CO ₂ . Cells were used in the logarithmic growth phase (not exceeding passage 6-8) with viability> 98% (assessed using trypan blue staining) and determined without mycoplasma.

In vivo efficacy studies Female BALB / c or C57BL6 mice (4-6 weeks old, 18-22 g) were obtained from Charles River (Wilmington, Mass.) in a pathogen free enclosure prior to the start of the study. Acclimatized for one week. Animals were given sterilized rodent feed and water ad libitum and were housed in sterile filter-capped cages using a 12 hour light / dark cycle. All experiments were under the guidance of the International Association for Laboratory Animal Care and Recognition.

For tumor implantation, B16-F10 (2 × 10 ⁶ ), CT26 (2 × 10 ⁶ ) or RENCA (1 × 10 ⁶ ) cells were collected, washed 3 times and resuspended in PBS. The hair on the flank of the mouse was shaved and subcutaneously implanted (sc) into the right flank of the mouse (0.2 ml). C57BL6 mice were used for the B16-F10 tumor model, while CT26 and RENCA tumors were transplanted into BALB / c mice. As outlined in the specific study design, treatment began after the average tumor size was established to 50-250 mm ³ (day 0). Mice were randomized into treatment cohorts (typically 10 mice / group). RIL-2 was administered subcutaneously once a day (0.2-3 mg / kg / day) on days 0-6 or 7-13 or days 0-4, 7-11. BAY 43-9006 or SU11248 (1-100 mg / kg) was administered as a liquid once daily by oral gavage starting on day 0 or 7 (5-12 days). All monotherapy and drug combinations at selected doses outlined in individual studies were well tolerated.

Tumor inhibition and response assessment Tumor volume and body weight were assessed 2-3 times per week. Using the formula: 1/2 (length (mm) × [width (mm)] ² ), the measured value of the tumor with caliper was converted to the average tumor volume (mm ³ ). Tumor growth inhibition (TGI) was calculated as [1- (mean tumor volume of treatment group / mean tumor volume of control group) × 100]. Response is defined as either a complete response (CR, no measurable tumor) or a partial response (PR, 50-99% tumor volume reduction) compared to the tumor volume of each animal at the start of treatment. did. Tumor growth delay analysis was calculated as [(days until treatment group reaches average tumor volume 1000 mm ³ ) − (days until control group reaches average tumor volume 1000 mm ³ )].

  Synergy refers to the predicted% tumor growth inhibition ratio of the combination therapy (% T / C prediction =% T / C treatment 1 ×% T / C treatment 2) and the observed% T / C of the combination therapy ( It was defined when the value divided by (% T / C actual measurement)> 1. Additive effects were defined when% T / C prediction /% T / C actual measurement = 1, and antagonism was defined when% T / C prediction /% T / C actual measurement <1 (Yokoyama et al., Cancer Res. , 2000, 60 (80): 2190-2196).

Pharmacokinetics For the evaluation of drug pharmacokinetics, mice were treated with a single subcutaneous dose of rIL-2 (6 mg / kg, 0.2 ml) or BAY 43-9006 (20 mg / kg, oral, 0.2 ml). Blood was collected at various times after drug administration. Plasma levels of BAY 43-9006 and rIL-2 were determined using HPLC or ELISA bioassay.

Western Blot Analysis After drug incubation under the indicated conditions, cells were harvested and washed with ice-cold PBS, protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN) and phosphatase inhibitors (Sigma, St. Louis, MT). ) In RIPA buffer (1 × phosphate buffered saline, 1% Nonidet P-40, pH 7.2, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) did. The protein content of the lysates was determined using the BCA assay (Bio-Rad, Hercules, CA). For Western blot analysis, 60 μg of protein was electrophoresed, pERK was detected using a mouse antibody against pERK (1: 1000, Cell Signaling, Beverly, Mass.), And incubated at 4 ° C. overnight. Detection of pSTAT5 antibody (1: 1000, Upstate) and pAKT (1: 1000, Cell Signaling) was performed with the same amount of protein by probing with an appropriate anti-phosphotyrosine antibody for 2 hours at room temperature. The membrane was then incubated with anti-rabbit IgG conjugated with 1: 5000 horseradish peroxidase (Jackson Immunoresearch, West Grove, Pa.) For 1 hour at room temperature. To verify equivalent loading, the blots are stripped and reprobed with anti-ERK (Cell Signaling), anti-STAT5 (BD Biosciences), and anti-AKT (Cell Signaling) antibodies to obtain total ERK, STAT5 and Each AKT protein was measured. Proteins were detected using sensitive chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) and visualized after exposure to Kodak film. Scanning densitometry was performed to quantify the intensity of the bands. The amount of pERK, pSTAT5 or pAKT was normalized to total ERK, STAT5 or AKT protein levels and compared to vehicle or untreated controls.

Cellular immunophenotyping of BALB / c nude mice after drug treatment Blood samples were collected at various times after the indicated treatment. Whole blood (100 μl) was transferred to a FACS / TruCount tube (BD BioSciences) and kept on ice. Samples were treated with 0.5 μg mouse Fc blocker (anti-mouse CD16 / CD32; BD BioSciences) and incubated on ice for 20 minutes. The following antibody conjugated with fluorochrome was added to the sample and incubated on ice for 20 minutes protected from light. The blood sample was vortexed while adding 2 ml of 1 × FACS lysis solution (BD BioSciences), then incubated at room temperature for 10 minutes, and then centrifuged at 1250 rpm. All samples were washed twice and suspended in PBS and 2% FBS and stored at 4 ° C. until sample acquisition with BD FACScalibur and subsequent analysis with CellQuest Pro software. The absolute number of cells was determined relative to the TruCount bead reference. A population of cells was identified and gated based on FSC and SSC characteristics and markers of total lymphocytes (CD45, BD BioSciences). T cell lymphocyte populations were identified based on CD3 staining and subpopulations were identified based on CD4 or CD8 staining (BD BioSciences). Individual cell populations were identified by appropriately gated events.

Histopathology and immunohistochemistry Mouse tumors were fixed in 10% neutral buffered formalin, then transferred to 70% ethanol, then paraffin embedded using Excelsior tissue processor (Thermo Electron Corporation, Pittsburgh, PA). Processed for. Tissue sections (4 μm) were cut with a rotary microtome (RM2125, Leica Microsystems, Nussloch, Germany). Sections prepared with hematoxylin and eosin (H & E) were prepared. Subsequently, immunostaining was performed using a Discovery XT automated slide staining system (Ventana Medical Systems, Tucson, Ariz.). Mouse T cells were detected using rabbit anti-CD3 antibody (1:60 dilution, Dako Norden A / S, Danish Glostrup) and monocytes / macrophages were detected using F4 / 80 (Serotec). For cell proliferation, mouse tumors were stained for Ki-67 using monoclonal rat anti-mouse antibody (1:15 dilution, DAKO). Recovery of heat-induced epitopes was performed using CC1 (Ventana Medical Systems). Samples were then incubated with the appropriate secondary antibody (goat anti-rabbit IgG biotin labeled antibody, 1: 100 dilution, Jackson ImmunoResearch Laboratories). Antibody localization was determined using a horseradish peroxidase labeled streptavidin biotin system and 3-3'-diaminobenzidine chromogen (Ventana Medical Systems). To enhance visualization of histomorphology, sections were counterstained with Nuclea First Red. For B16-F10 tumors, melanin deposition was observed in H & E tissues, so the Ventana Bluemap kit, an alternative method of NBT / BCIP staining, was used to help visualize T cells in the tumor.

CTLL-2 T Cell Proliferation Assay CTLL-2 T cells were preincubated with or without BAY 43-9006 (3 μM) for 2 hours at 37 ° C. Cells were then washed and seeded in 96-well microtiter plates at 5,000 cells / well in culture medium containing serial dilutions of rhIL-2 (1 pM-100 nM). At the end of the incubation period (72 hours at 37 ° C.), cell viability was determined by a tetrazolium dye assay using the cell growth reagent WST-1 (Roche Applied Science, Indianapolis, Ind.).

In Vitro Cytotoxicity Assay Cells are seeded in 96 well microtiter plates (CTLL-2: 5000 cells / well; RENCA: 1500 cells / well; B16-F10: 1000 cells / well; MV4; 11: 5000 cells / well ), BAY 43-9006 / SU11248 or vincristine serial dilutions. Since CTLL-2 is an IL-2 dependent cell line, cytotoxicity assays were performed in these cells in media containing 5 nM rIL-2. At the end of the incubation period (72 hours at 37 ° C.), cell viability was determined by either tetrazolium dye (WST-1) assay or chemiluminescence (BrdU) assay (Roche Applied Science, Indianapolis, Ind.). The EC ₅₀ value was defined as the concentration required for a 50% decrease in the absorbance of the treated cells relative to untreated control cells.

Ex vivo splenocyte cytotoxicity assay Splenocytes were obtained from drug-treated BALB / c mice (n = 3-5 / group) under aseptic conditions and homogenized in cold PBS. After passing through a 70 μm nylon cell strainer, the cells were briefly centrifuged at 4 ° C. Red blood cells were lysed using RBC lysis buffer (Sigma, St. Louis, MT). Cells were washed and with ⁵¹ Cr labeled Yac-1 target cells at various E: T ratios (100: 1, ⁵⁰ : 1, 25: 1, 12/5: 1, 6.25: 1, 3: 1) Planting, a 4 hour cytotoxicity assay was performed. Data was obtained using a Wallac plate reader and expressed as counts per minute (cpm). Quantitation was expressed as% specific lysis and was calculated as% specific lysis = 100 x ((average experimental release-average spontaneous release) / (average maximum release-average spontaneous release)). Spontaneous release was determined from wells containing labeled target cells and no effector cells, and maximal release was determined from wells containing target cells labeled in 1% Triton X-100.

Statistical analysis Multiple comparisons were made using one-way analysis of variance (ANOVA), and a post-hoc test to compare different treatment measures was performed using the Student-Newman Cools test (SigmaStat). The difference was p <0.05 and was considered statistically significant.

  B. In vitro studies with rIL-2 and BAY 43-9006 on T cell and tumor cell signaling pathways.

rIL-2 activates JAK / STAT and MAPK signaling in vitro in CTLL-2 cells To elucidate the onset, time course and duration of the IL-2 / IL-2R signaling pathway in T cells CTLL-2 cells (1 × 10 ⁷ cells) were serum starved for 24 hours before being treated with various concentrations of rIL-2 (1 pM-100 nM) for 2 hours and were subjected to major signaling using Western blot analysis. Routes, ie MAPK, STAT5, AKT were evaluated. Serum starved CTLL-2 cells have two major IL-2R complexes: a high affinity (K _D = 10 ⁻¹¹ M for IL-2Rαβγ) receptor and a moderate affinity (K for IL-2βγ). _D = 10 ⁻⁹ M) In order to delineate receptor interactions, treatments were performed in vitro with a wide range of concentrations of rIL-2 (1 pM-100 nM). In some experiments, cells were exposed to free anti-IL-2α antibody (> 1000 fold excess; 10 nM) and incubated for 1 hour before rIL-2 was added. To assess the time course of IL-2 / IL-2R activation, serum starved CTLL-2 cells were activated with 10 nM rIL-2 and IL-2 signaling was increased from 10 minutes to 48 hours. Examined at various times. IL-2R downstream phosphorylation of ERK1 / 2, STAT5 and AKT was assessed in rIL-2 treated cells using Western blot analysis. The relative level of pERK, pSTAT5 or pAKT was compared to the total protein level of ERK, STAT5 or AKT, respectively.

  Activation of pERK1 / 2 was observed after exposing CTLL-2 cells to rIL-2. Phospho ERK was slightly activated at 1 pM for 2 hours, while maximal activation was seen at concentrations ≧ 100 pM. The JAK / STAT5 pathway was maximally activated at a concentration of 1 pM, and increasing the concentration of rIL-2 did not change the level of pSTAT5 (up to 100 nM). The basal pAKT pathway in CTLL-2 appears to be activated in T cells under serum starved conditions. Furthermore, pAKT levels remained largely unchanged at the tested rIL-2 concentrations (1 pM-100 nM; using pAKT antibody against phosphorylated site 483).

A blocking antibody against IL-2Rα was used to confirm that the IL-2 signaling pathway was specifically mediated by binding to IL-2R. PSTAT5 levels in CTLL-2 cells were analyzed by Western blot. CTLL cells were serum starved and treated with excess free anti-IL-2Rα antibody (10 nM,> 1000 fold) for 1 hour prior to treatment with rIL-2 (0.1 pM to 10 nM). In the absence of blocking IL-2Rα antibody, pSTAT5 was activated after rIL-2 treatment at 1 pM, but in the presence of IL-2Rα inhibition, pSTAT5 signaling was suppressed (> 95% inhibition) This confirms that IL-2Rα is required for STAT5 signaling. Interestingly, pSTAT5 levels are repaired in CTLL-2 cells with increasing concentrations of rIL-2 (≧ 10 pM), which is associated with a low affinity IL-2Rβγ chain (K _D = 10 ⁻⁹ M). It suggests that by binding rIL-2 competitively replaces anti-IL-2Rα antibody and / or activates pSTAT5 signaling.

rIL-2 activation of pSTAT5 is rapid and persistent in CTLL-2 cells To assess the onset and duration of IL-2R signaling responses in CTLL-2 cells, serum-starved cells were Treatment with rIL-2 (10 nM) in vitro and the effect on phosphorylation of ERK1 / 2, STAT5 and AKT was assessed using Western blot analysis. STAT5 phosphorylation was activated within minutes (<10 minutes) of rIL-2 added to CTLL-2 cells, and the duration of the pSTAT5 response was maintained up to 48 hours. Activation of the MAPK pathway (pERK) pathway by rIL-2 appeared to be slightly delayed and was activated by 1 hour. The intensity of pERK was lower than the maximal response obtained by stimulating serum starved CTLL-2 cells with PMA (50 ng / ml) + ionomycin (0.4 μg / ml) for 15 minutes. Furthermore, pERK levels lasted up to 24 hours and returned to background levels by 48 hours. No discernable effect on pAKT was observed, confirming that signaling through the PI-3K / AKT pathway is continuously active in CTLL-2 cells.

BAY 43-9006 treatment inhibits pERK but does not inhibit pSTAT5 in CTLL-2 cells BAY-43-9006 is a potent inhibitor of Raf-1 and also inhibits BRAF wild type and mutants. Furthermore, BAY-43-9006 inhibits multiple kinases, in particular VEGF2,3; PDGFRβ; FLT3 and cKIT (Wilhelm, SM et al., Cancer Res, 2004; and Chiron's kinase profiling data), and T cell functional It inhibits to some extent the two kinases involved in the response, Lck and Fyn. Given the inhibitory kinase profile of BAY43-9006 and its potential impact on MAPK signaling / T cell signaling, BAY43-9006 can potentially interfere with IL-2 / IL-2R signaling in T cells It was thought. Therefore, the potential interaction of BAY 43-9006 on IL-2 signaling was evaluated in vitro in CTLL-2, B16-F10, or RENCA cells.

  To test the effect of BAY 43-9006 on CTLL-2, B16-F10 or RENCA model cells, serum starved cells were treated with various concentrations of BAY 43-9006 (0.01-20 μM). As a suitable control, cells were stimulated with PMA (50 ng / ml) and ionomycin (0.4 μg / ml) for 15 minutes. To examine the effect of rIL-2 and BAY 43-9006 in CTLL-2 cells in vitro and the effect of sequential treatment, serum-starved CTLL-2 cells were treated with rIL-2 (10 nM) for 2 hours. BAY 43-9006 (3 μM) was treated in the presence or absence. For sequential treatment, BAY 43-9006 was treated for 2 hours. The cells are then washed and then treated with rIL-2, and vice versa. The inhibitory effect of BAY 43-9006 (3 μM) was also examined with or without stimulation with PMA (50 ng / ml) and ionomycin (0.4 μg / ml).

  Since the concentration of BAY 43-9006 required for inhibition of the MAPK pathway in various cell types is very variable, the effects of various concentrations of BAY 43-9006 (ranging from 0-20 μM) can be compared with serum-starved CTLL- Two cells were evaluated for 2 hours with or without PMA + ionomycin stimulation. At the end of the incubation period, treated CTLL-2 cells were lysed and the protein lysates were subjected to Western blot analysis to determine pERK levels. BAY 43-9006 significantly inhibited pERK at concentrations ≧ 1 μM in mouse CTLL-2 cells and human Jurkat T cell lines. PSTAT5 and pAKT levels in cells were not altered by the effects of BAY 43-9006 treatment on CTLL-2 cells, indicating that the JAK / STAT5 pathway and PI-3K / AKT in T cells were largely unaffected. Show.

  To investigate the therapeutic basis of combining rIL-2 and BAY43-9006 in vitro, the effects of rIL-2 and BAY43-9006 on T cells and two IL-2 mediated in CTLL-2 cells Studies on the effects of signaling pathways (MAPK and JAK / STAT5) were conducted. IL-2 mediated T cell signaling effects using simultaneous or sequential regimens of the two drugs were investigated in CTLL-2 cells. Serum starved CTLL-2 cells were treated with BAY 43-9006 (3 μM) for 2 hours followed by either vehicle, rIL-2 (10 nM) or PMA + ionomycin. Alternatively, treatment with rIL-2 (10 nM, 2 hours) followed by BAY 43-9006 (3 μM, 2 hours) was also investigated. After drug exposure and / or PMA + ionomycin stimulation, Western blot analysis of pERK and pSTAT5 in cell lysates was performed (as previously described). While BAY 43-9006 (3 μM) treatment inhibited pERK levels, rIL-2 activated pERK levels (relative to baseline pERK levels) in serum-starved CTLL-2 cells, which resulted in MAPK The conflicting effects of the two drugs on the pathway were confirmed. All treatments with the combination of BAY 43-9006 (simultaneous and sequential drug exposure) significantly inhibited pERK in CTLL-2 cells. No effect was observed on the STAT5 or AKT pathway in all combination treatments tested (as outlined in the method).

BAY 43-9006 at high concentrations inhibits pERK levels in tumor cell lines The effect of BAY 43-9006 treatment on mouse tumor cell lines, namely B16-F10 melanoma, CT26 colon and RCC RENCA models was determined. A mouse cell line was selected based on its responsiveness to in vivo rIL-2 treatment in an immunocompatible model (a mouse compatible with T / NK / monocytes / macrophages), where rIL- The combined treatment effects of 2 and BAY 43-9006 could be investigated. Serum starved B16-F10 melanoma and RENCA cells were exposed to BAY 43-9006 at concentrations ranging from 0-20 μM. Phospho ERK levels in cell lysates after drug exposure were determined by Western blot analysis. BAY 43-9006 inhibited pERK levels (B16-F10 and RENCA) in both cell lines tested at concentrations as high as ≧ 5 μM, with almost complete disappearance of pERK at 20 μM.

C. In vitro effect of BAY 43-9006 on IL-2 mediated cell proliferation To examine whether pERK inhibition by BAY 43-9006 affected IL-2 mediated proliferation response in CTLL-2 cells. Proliferation assays were performed by preincubating CTLL-2 cells (5000 cells / well) with a concentration of BAY 43-9006 (3 μM, 2 hours) that inhibits pERK. After incubating the cells with BAY 43-9006 (3 μM) for 2 hours, the cells were seeded in 96-well plates and exposed to various concentrations of rIL-2 (0-100 nM) for 72 hours. Untreated cells received sham treatment prior to incubation with rIL-2 (same concentration). The proliferative response of CTLL-2 cells treated and untreated with BAY 43-9006 was evaluated using the WST-1 assay. Preincubation of cells with BAY 43-9006 (3 μM) inhibited pERK signaling in the CTLL-2 cell line, but did not affect the IL-2-induced proliferative response.

D. Anti-proliferative activity of BAY 43-9006 or SU11248 against CTLL-2 and tumor cells in vitro Cytotoxicity of BAY43-9006 or SU11248 in each of these cell types (CTLL-2, B16-F10, RENCA, CT26), respectively To assess activity, cells were seeded in 96-well microtiter plates and treated with serial dilutions of BAY 43-9006 (0-50 μM) at 37 ° C. for 72 hours. Since the CTLL-2 cell line is dependent on IL-2 for growth, cytotoxicity against these cells was performed in media containing 5 nM rIL-2. At the end of the 72 hour incubation period, cell viability was determined by either tetrazolium dye (WST-1) assay or chemiluminescence (BrdU) assay. As controls, MV4; 11 (human FLT3 ITD AML cell line) was treated with BAY 43-9006 (0-50 μM) or B16-F10 cells were treated with vincristine (0-1 μM) to determine drug cytotoxicity and assay. The validity of was confirmed. The inhibitory concentration of the drug was expressed as an EC ₅₀ value, which was defined as the concentration required for a 50% reduction in proliferative response measured as the absorbance of cells treated with the drug relative to the untreated / vehicle control.

The relative EC ₅₀ of BAY 43-9006 or SU11248 is shown in Table 9.

細胞を９６ウェルマイクロタイタープレートに植え（ＣＴＬＬ−２：５０００個の細胞／ウェル；ＲＥＮＣＡ：１５００個／ウェル；Ｂ１６−Ｆ１０：１０００個／ウェル；ＣＴ２６：１０００個の細胞／ウェル；ＭＶ４；１１：５０００個／ウェル）、ＢＡＹ４３−９００６、ＳＵ１１２４８またはビンクリスチンの段階希釈液で処置した。ＣＴＬＬ−２はＩＬ−２依存性細胞系であるので、細胞毒性アッセイは５ｎＭのｒＩＬ−２を含む培地中で実施した。インキュベーション期間（３７℃で７２時間）の終わりに、テトラゾリウム色素（ＷＳＴ−１）アッセイのどちらかによって細胞生存度を決定した。

Cells are seeded in 96-well microtiter plates (CTLL-2: 5000 cells / well; RENCA: 1500 cells / well; B16-F10: 1000 cells / well; CT26: 1000 cells / well; MV4; 11: 5000 / well), BAY 43-9006, SU11248 or vincristine serial dilutions. Since CTLL-2 is an IL-2 dependent cell line, cytotoxicity assays were performed in media containing 5 nM rIL-2. At the end of the incubation period (72 hours at 37 ° C.), cell viability was determined by either tetrazolium dye (WST-1) assay.

^a EC50 = concentration required for 50% reduction in proliferative response measured as absorbance of cells treated with drug versus untreated / vehicle control.

^b MV4; 11 is a human acute myeloid leukemia (AML) cell line that expresses FLT3 internal tandem duplication (ITD). BAY 43-9006 (a potent FLT3 kinase inhibitor; IC50 = 58 nM) demonstrates potent in vitro antiproliferative activity against MV4; 11 cells.

^d Cell viability was assessed using the Promega Cell-Titer Glo ™ assay, which measured the ATP content of cells.

Generally, to inhibit the growth of CTLL-2 cells and various cell lines (B16-F10, RENCA, CT26), a relatively high concentration of BAY 43-9006 (or SU11248) compared to the antimitotic agent vincristine. ) Was required (defined by relative EC50; see Table 9). The cytotoxicity of the effect of BAY 43-9006 on RENCA cells was also examined using the BrdU assay to assess its effect on DNA synthesis (as opposed to antiproliferative activity using the mitochondrial tetrazolium dye assay). The EC ₅₀ of BAY 43-9006 (˜5 μM) on RENCA cells obtained using the BrdU method was similar to that observed in the WST-1 assay. When RENCA tumor cells are exposed to a range of rIL-2 at a concentration (0-1 μM), no direct proliferative response (or cytotoxicity) is observed, thereby causing the mechanism of rIL-2 to be immune effector cells. It was confirmed that it was dependent on the activation of the components.

E. In Vivo Efficacy Study Pharmacokinetics of rIL-2 and BAY 43-9006 in Mice In the published Phase I report of BAY 43-9006 (400 mg dose), a high C _{max of} 10-20 μM and about 24 hours A long drug half-life was achieved in patients (Strumberg et al., J. Clin. Oncol., 2005, 23 (5): 965-972). To determine whether plasma drug exposure can affect T cell viability and proliferative responses in vivo, single dose pharmacokinetics of rIL-2 and BAY 43-9006 were measured in mice.

A single oral dose of 30 mg / kg BAY 43-9006 in mice achieved a C _max of about 5500-8000 ng / ml (-10 μM) at t _max 2 hours. The plasma elimination rate of BAY 43-9006 was relatively slow, resulting in a half-life of about 4 hours. In contrast, the PK profile of rIL-2 after subcutaneous administration of 6 mg / kg demonstrated a C _max of about 550-850 ng / ml at a t _{max of} about 30 minutes. The t _1/2 of rIL-2 in mice when exposed to ≧ 50 ng / ml for 4 hours was about 1 hour. Interestingly, both treatment after a single dose and each route of administration showed a non-overlapping C _max (at t _max ), as supported by findings from PK, either simultaneous or sequential treatment It was also avoided that it could be feasible. Given the possibility that BAY 43-9006 is mostly bound to proteins in the blood, the effects of exposure of these drugs on T cell viability in vivo must still be addressed.

Treatment of rIL-2 and BAY 43-9006 reduces immune effector function ex vivo Inhibition of one or more T lymphoid kinases (Lck, Fyn, Syk, Btk, Src, Tck2, MAPK, JAK) is a T cell The effects of rIL-2 and BAY 43-9006 on T cell proliferative and functional responses in vivo were investigated.

In these experiments, BALB / c mice bearing RENCA tumors were treated with rIL-2 (1 mg / kg / day, subcutaneous, days 6-10), BAY 43-9006 (30 mg / kg / day, oral, 6-10 Day) or simultaneously or sequentially (rIL-2, 1 mg / kg / day, subcutaneous, days 1-5 + BAY 43-9006, 30 mg / kg / day, oral, days 6-10; or BAY 43-9006, 30 mg / kg / day, oral, day 1-5 + rIL-2, 1 mg / kg / day, subcutaneous, day 6-10) treated with a combination of rIL-2 and BAY 43-9006 . Spleen cells isolated from treated mice were then subjected to ex vivo killing assays against Yac-1 target cells at various effector: target (E: T) ratios and% specific of ⁵¹ Cr-labeled Yac-1 cells Dissolution was determined. Mice treated with rIL-2 (1 mg / kg) significantly increased splenocyte-mediated killing of the Yac-1 target compared to vehicle treatment (23% with rIL-2 vs. 0 with vehicle treatment). %). The specific killing effect (1%) observed with BAY 43-9006 treatment was negligible and was not different from vehicle treatment. All treatments with rf and BAY 43-9006 resulted in reduced lytic activity mediated by splenocytes (co-treatment = 16%; sequential treatment ≦ 9% vs. rIL-2 monotherapy = 23%).

Effect of rIL-2 and BAY 43-9006 treatment on circulating immune effector cell population Pharmacodynamics of rIL-2 and / or BAY-43-9006 treatment on circulating lymphocytes and monocyte populations of tumor bearing BALB / c mice Effects were examined. In these studies, blood was collected after a single agent or a combination of rIL-2 and BAY 43-9006 treatment (simultaneous or sequential treatment as described above). Absolute counts of lymphocytes and T cell subpopulations (CD4 + or CD8 +) were quantified in whole blood using TruCount ™ tubes and appropriate immunostaining.

  With rIL-2 treatment, the absolute number of circulating lymphocytes and monocytes (CD45 + cells: 2387 cells / μl vs. 1575 cells / μl in vehicle) and the absolute number of T cells (1550) in tumor-bearing mice. Cells / μl vs. 664 cells / μl) in the vehicle. Compared to vehicle treatment, a significant increase in the CD4: CD8 cell ratio was observed with rIL-2 therapy (5: 3; rIL-2: vehicle, ie, 1.7 fold), which is in an increasing number of T cells It is an index of the mechanism of action of rIL-2. The relative number of non-T cells and monocytes (CD45 + CD3-) after rIL-2 treatment was similar to vehicle treatment (837 cells / μl vs. 911 cells / μl in vehicle).

  In contrast, the single agents BAY 43-9006 or BAY 43-9006 combined with rIL-2 were less affected or increased the absolute number of lymphocytes and monocytes (cells / μl ranging from 2417 to 3577) 2387 cells / μl versus vehicle), and non-T cell and monocyte populations were also increased (1241-1563 cells / μl vs 837 cells / μl vehicle). The effect of BAY 43-9006 treatment on the total number of T cells was similar to vehicle treatment (1827 cells / μl vs. 1550 cells / μl in vehicle). Slightly increased total T cells observed with sequential regimens of rIL-2 and BAY 43-9006 when starting with rIL-2 (2081 T cells / μl vs. 1550 cells / μl in vehicle) Number (including both CD4 + and CD8 + populations). When rIL-2 and BAY 43-9006 were given simultaneously or sequentially, such as BAY 43-9006, followed by rIL-2, the total number of T cells was slightly reduced compared to vehicle treatment (approximately 1170-1244). T cells / μl vs. 1550 cells / μl in vehicle). Similar trends were observed in individual CD4 + and CD8 + T cell populations.

  In addition, tumor histology after treatment with rIL-2 and BAY 43-9006 or SU11248 treatment (described previously) was determined. In order to examine the pharmacodynamics of T cells in vivo, T cells infiltrating the tumor were detected with a mouse anti-CD3 antibody. Anti-proliferative effects in tumors after drug treatment were evaluated using Ki67 staining. rIL-2 therapy showed an increase in the number of T cells infiltrating both RENCA tumors (and B16-F10 tumors) compared to vehicle treatment. In general, fewer T cells were detected in the Bayer 43-9006 treatment group in the RENCA model. Since some T cells were detected in all treatment groups, the effect of rIL-2 and BAY 43-9006 or SU11248 treatment on infiltrating T cells in B16-F10 tumors was uncertain. Tumors that showed increased necrosis (tumor inhibition) were generally shown to have more T cells dispersed among the tumor cells. Taken together, the data indicate that rIL-2 activates circulating T cells to transport cells to the extravascular space, including tumors, and that BAY 43-9006 or SU11248 treatments promote T cell proliferative responses and transport. This suggests that it can be partially suppressed.

rIL-2 and BAY 43-9006 treatment enhances the efficacy of IL-2-responsive mouse tumor models The drug interaction between rIL-2 and either BAY 43-9006 or SU11248 was examined (Figures 1-10 and Table 10-12 reference). Combination treatment was evaluated in a mouse IL-2-responsive tumor model compatible with three experimental T cells (B16-F10 melanoma, CT26 colon, RCC RENCA model) (FIGS. 1-10). In these studies, mice were randomized after tumor size was established to 50-225 mm ³ , with daily oral dosing of BAY 43-9006 or SU11248 or once daily subcutaneous administration of rIL-2. Later (as indicated in the method), tumor growth was monitored by caliper measurements. The efficacy and tolerability of rIL-2, BAY 43-9006 and SU11248 alone were investigated over a range of doses and treatment schedules (FIG. 1). RIL-2 showed potent efficacy in all models, and tumor inhibition at effective doses was generally in the range of 40-60% (vs. vehicle treatment) (FIG. 1). The minimum effective dose of BAY 43-9006 was ≧ 30 mg / kg / day (vs. vehicle treatment). SU11248 was generally effective at doses ≧ 40 mg / kg / day, with the exception of the B16-F10 tumor model (FIG. 1).

  Based on the efficacy and tolerability of the single agent, rIL-2 was then combined with BAY 43-9006 or SU11248 at a dose that was tolerated and did not show any adverse effects on body weight or any adverse clinical symptoms. Both sequential and concurrent treatment schedules were examined with B16-F10 and CT26 tumor models (Figures 2-8, Tables 10 and 11). In the B16-F10 and CT26 tumor models, almost all combination therapies (simultaneous or sequential regimens) investigated using rIL-2 and BAY 43-9006 or rIL-2 and SU12248 compared to monotherapy or vehicle treatment. Thus, the antitumor activity was enhanced (FIGS. 2 to 5). A summary of combination drug interactions for various combination therapies is summarized in Tables 10 and 11.

^ａｎ＝各研究で１０匹のＣ５７ＢＬ６マウス／群。Ｂ１６−Ｆ１０腫瘍を有するマウスは、腫瘍の平均の大きさが〜５０ｍｍ^３まで確立された後に治療した。

^a n = 10 C57BL6 mice / group in each study. Mice bearing B16-F10 tumors were treated after the average tumor size was established to ˜50 mm ³ .

^b T / C _{actual measurement} (O) =% T / C
^c T / C _prediction (E) =% T / C treatment 1 ×% T / C treatment 2
^d Synergy is the predicted% tumor growth inhibition of the combination therapy (% T / C prediction =% T / C treatment 1 x% T / C treatment 2) and the observed% T / C (% T / C actual measurement) was defined when the ratio was> 1.

^e Drug interaction is defined as additive when% T / C prediction /% T / C measurement = 1 and antagonized when% T / C prediction /% T / C measurement <1 Defined.

  N / A, not applicable

^ａｎ＝各研究で１０匹のＢＡＬＢ／ｃマウス／群。ＣＴ２６腫瘍を保有するマウスは、腫瘍の平均の大きさが〜２２５ｍｍ^３まで確立された後に治療した。

^an = 10 BALB / c mice / group in each study. Mice bearing CT26 tumors were treated after the average tumor size was established to ˜225 mm ³ .

^b T / C _{actual measurement} (O) =% T / C
^c T / C _prediction (E) =% T / C treatment 1 ×% T / C treatment 2
^d Synergy is the predicted% tumor growth inhibition of the combination therapy (% T / C prediction =% T / C treatment 1 x% T / C treatment 2) and the observed% T / C (% T / C actual measurement) was defined when the ratio was> 1.

^e Drug interaction is defined as additive when% T / C prediction /% T / C measurement = 1 and antagonized when% T / C prediction /% T / C measurement <1 Defined.

N / A, not applicable BAY 43-9006 (40 mg / kg / day, oral, days 1-7) was administered before rIL-2 (1 mg / kg / day, subcutaneous, days 8-13), In only one example of the CT26 model, the effect of the combination treatment was not optimal.

  The RENCA model only evaluated the concurrent schedule (Figures 9-10; Table 12), and the combination of rIL-2 with either BAY 43-9006 or SU11248 showed greater tumor inhibition compared to single agent treatment. It was done.

^ａｎ＝各研究で１０匹のＢＡＬＢ／ｃマウス／群。ＲＥＮＣＡモデルの腫瘍を有するマウスは、腫瘍の平均の大きさが〜５０〜７０ｍｍ^３まで確立された後に治療した。ＲＥＮＣＡ腫瘍モデルは重篤な悪液質（動物のるいそう）を示し、薬剤の逐次的な投与が不可能となり、評価が不可能であったので、同時レジメンを示した。

^an = 10 BALB / c mice / group in each study. Mice with RENCA model tumors were treated after the average tumor size was established to ˜50-70 mm ³ . The RENCA tumor model showed severe cachexia (animal suspicion), making sequential administration of drugs impossible and impossible to evaluate, indicating a concurrent regimen.

^b T / C _{actual measurement} (O) =% T / C
^c T / C _prediction (E) =% T / C treatment 1 ×% T / C treatment 2
^d Synergy is the predicted% tumor growth inhibition of the combination therapy (% T / C prediction =% T / C treatment 1 x% T / C treatment 2) and the observed% T / C (% T / C actual measurement) was defined when the ratio was> 1.

^e Drug interaction is defined as additive when% T / C prediction /% T / C measurement = 1 and antagonized when% T / C prediction /% T / C measurement <1 Defined.

N / A, not applicable Sequential treatment in the RENCA model was not feasible due to the short duration of the model and mouse cachexia (animal leopard / weight loss). Taken together, the data indicate that activity is enhanced when rIL-2 is combined with targeted small molecule treatments such as BAY 43-9006 and SU11248 in preclinical models, indicating that such a therapeutic strategy. It can be transferred to the clinic.

  Although the present invention has been described with reference to specific examples, including preferred modes of carrying out the invention, those skilled in the art will recognize that the above systems and techniques have many variations and modifications that are within the spirit and scope of the invention. It will be understood that a substitution exists.

INCORPORATION BY REFERENCE The entire contents of the above-mentioned patents, patent applications and journal articles are incorporated by reference as if fully set forth herein.

FIG. 1 shows the activity of rIL-2, BAY 43-9006 or SU11248 alone in a mouse tumor model compatible with IL-2-responsive T cells. B16-F10 melanoma (2 × 10 ⁶ ), CT26 colon (2 × 10 ⁶ ) or RENCA kidney cancer (1 × 10 ⁶ ) cells were implanted subcutaneously into the right flank of female C57BL6 or BALB / c mice. . Treatment was initiated as outlined in the method after the average tumor size was established to 50-225 mm ³ . Mice were randomized into treatment cohorts (10 mice / group). rIL-2 was administered subcutaneously once a day (0.2-3 mg / kg / day). BAY 43-9006 or SU11248 (1-100 mg / kg) was administered as a solution once daily by oral gavage. FIG. 1 shows 10-14 day mean tumor growth inhibition ([1− (mean tumor volume in treatment group / mean tumor volume in vehicle group) × 100] of single agent in B16-F10, CT26 and RENCA tumor models. Calculated). Data were compiled from multiple independent studies, each study conducted in 10 mice / group. ^* Represents statistical significance vs. vehicle treatment (p <0.05, ANOVA). FIG. 2 shows the effectiveness of simultaneous treatment with rIL-2 and BAY 43-9006 in a B16-F10 mouse melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female C57BL6 mice. Mice were randomized into groups after tumors were ˜50 mm ³ , treatment started on day 0, vehicle, days 0-6 (♦), rIL-2 (3.3 mg / kg, subcutaneous, 0 Day 6) (Δ) or BAY 43-9006 (30 mg / kg, oral, days 0-6) (x) or combined rIL-2 (3.3 mg / kg, subcutaneous, days 0-6) + BAY43 -9006 (30 mg / kg, oral, 0 to 6 days) (■) was used. FIG. 2 illustrates mean tumor volume (mm ³ ± SE) vs. number of days after randomization (n = 10 mice / group). FIGS. 3A and 3B show the efficacy of sequential treatment with rIL-2 and BAY 43-9006 in a B16-F10 mouse melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female C57BL6 mice. Mice were randomized into groups after tumors were ˜50 mm ³ and treatment started on day 0. FIG. 3A shows the results from sequential regimens in which rIL-2 is administered first, followed by BAY 43-9006. Panels illustrate: vehicle (♦) 0-6, 7-13 days; rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) (Δ) or BAY 43-9006 (30 mg / kg) , Oral, days 7-13) (x) or combined rIL-2 (3.3 mg / kg, subcutaneous, days 0-6) + BAY 43-9006 (30 mg / kg, oral, days 7-13) ( ●). FIG. 3B shows the results from sequential regimens in which BAY 43-9006 is administered first, followed by rIL-2. The panel illustrates: vehicle (♦) 0-6, 7-13 days; BAY 43-9006 (30 mg / kg, oral, 0-6 days) (x); rIL-2 (3.3 mg / kg) , Subcutaneous, days 7-13) (□) or combined BAY 43-9006 (30 mg / kg, oral, days 0-6) + rIL-2 (3.3 mg / kg, subcutaneous, days 7-13) ( ▲). The graph shows mean tumor volume (mm ³ ± SE) vs. number of days after randomization (n = 10 mice / group). FIG. 4 shows the effectiveness of simultaneous treatment with rIL-2 and SU11248 in a B16-F10 mouse melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female C57BL6 mice. Mice were randomized into groups after tumors were ˜50 mm ³ , treatment started on day 0, vehicle, days 0-6 (◇), rIL-2 (3.3 mg / kg, subcutaneous, 0 ˜6) (□) or SU11248 (40 mg / kg, oral, 0-6 days) (◯) or combined rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) + SU11248 (40 mg / Kg, oral, 0 to 6 days) (▲). FIG. 4 illustrates mean tumor volume (mm ³ ± SE) vs. number of days after randomization (n = 10 mice / group). Figures 5A and 5B show the effectiveness of sequential treatment with rIL-2 and SU11248 in a B16-F10 mouse melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female C57BL6 mice. Mice were randomized into groups after tumors were ˜50 mm ³ and treatment started on day 0. FIG. 5A shows the results from sequential regimens in which rIL-2 is administered first, followed by SU11248. Panels illustrate: vehicle (ビ) 0-6, 7-13 days; rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, oral , Day 7-13) (x) or combined rIL-2 (3.3 mg / kg, subcutaneous, days 0-6) + SU11248 (40 mg / kg, oral, days 7-13) (●). FIG. 5B shows results from sequential regimens in which SU11248 is administered first, followed by rIL-2. Panels illustrate: vehicle (ビ) 0-6, 7-13 days; SU11248 (40 mg / kg, oral, 0-6 days) (）); rIL-2 (3.3 mg / kg, subcutaneous) , 7-13 days) (Δ) or combined SU11248 (40 mg / kg, oral, 0-6 days) + rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days) (■). The graph shows mean tumor volume (mm ³ ± SE) vs. number of days after randomization (n = 10 mice / group). FIG. 6 shows the effectiveness of sequential treatment with rIL-2 and BAY 43-9006 in a CT26 mouse colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female BALB / c mice. Mice were randomized into groups after tumors were ˜225 mm ³ and treatment started on day 0. The panel illustrates a sequential regimen in which rIL-2 is administered first followed by BAY 43-9006: vehicle (♦) 0-6, 7-13 days; rIL-2 (1 mg / kg, subcutaneous) , 0-6 days) (□) or BAY 43-9006 (40 mg / kg, oral, 7-13 days) (Δ) or combined rIL-2 (1 mg / kg, subcutaneous, 0-6 days) + BAY43 -9006 (40 mg / kg, oral, day 7-13) (●). FIG. 7 shows the effectiveness of simultaneous treatment with rIL-2 and SU11248 in the CT26 mouse colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female BALB / c mice. Mice were randomized into groups after tumors were ˜225 mm ³ and treatment started on day 0. Panels illustrate: vehicle (♦) 0-6 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, oral, 0-6 days) ) (◯) or combined rIL-2 (1 mg / kg, subcutaneous, days 0-6) + SU11248 (40 mg / kg, oral, days 0-6) (●). Figures 8A and 8B show the efficacy of sequential treatment with rIL-2 and SU11248 in the CT26 mouse colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female BALB / c mice. Mice were randomized into groups after tumors were ˜225 mm ³ and treatment started on day 0. The panel illustrates the following: FIG. 8A shows the results from sequential regimens in which rIL-2 is administered first, followed by SU11248: vehicle (♦) 0-6, 7-13 days; rIL -2 (1 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, oral, 7-13 days) (◯) or combined rIL-2 (1 mg / kg, subcutaneous, 0 -Day 6) + SU11248 (40 mg / kg, oral, days 7-13) (■). FIG. 8B shows the results from sequential regimens in which SU11248 is administered first and then rIL-2. Panels illustrate: vehicle (♦) 0-6, 7-13 days; SU11248 (40 mg / kg, oral, days 0-6) (O); rIL-2 (3.3 mg / kg, subcutaneous) , 7-13 days) (□) or combined SU11248 (40 mg / kg, oral, days 0-6) + rIL-2 (3.3 mg / kg, subcutaneous, days 7-13) (▲). The graph shows mean tumor volume (mm ³ ± SE) vs. number of days after randomization (n = 10 mice / group). FIG. 9 shows the effectiveness of simultaneous treatment with rIL-2 and BAY 43-9006 in a mouse RCC RENCA tumor model. RENCA cells (1 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female BALB / c mice. Mice were randomized into groups after tumors were ˜50 mm ³ , treatment started on day 0, vehicle, days 0-8 (♦), rIL-2 (1 mg / kg, subcutaneous, 0-4 , 7-11 days) (Δ) or BAY 43-9006 (30 mg / kg, oral, days 0-8) (x) or combined rIL-2 (1 mg / kg, subcutaneous, 0-4, 7-11) Day) + BAY 43-9006 (30 mg / kg, oral, any of days 0-8 (■)) Figure 9 shows the mean tumor volume (mm ³ ± SE) versus the number of days after randomization (n = 10 mice / group). FIG. 10 shows the effectiveness of co-treatment with rIL-2 and SU11248 in the treatment of the mouse RCC RENCA tumor model. RENCA cells (1 × 10 ⁶ cells) were implanted subcutaneously into the right flank of female BALB / c mice. Mice were randomized into groups after tumors were ˜50 mm ³ and treatment started on day 0. Panels illustrate: vehicle (♦) 0-6 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (■) or SU11248 (40 mg / kg, oral, 0-6 days) ) (X) or combined rIL-2 (1 mg / kg, subcutaneous, days 0-6) + SU11248 (40 mg / kg, oral, days 0-6) (▲).

Claims (32)

  Patients receive therapeutically effective amounts of aldesleukin and 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro -4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2, 4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) ) Administering an anti-angiogenic agent selected from:) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Including the door, a method of treating a patient suffering from cancer.   2. The method of claim 1, wherein the aldesleukin is administered prior to the anti-angiogenic agent.   The method of claim 1, wherein the aldesleukin is administered next to the anti-angiogenic agent.   2. The method of claim 1, wherein the aldesleukin is administered concurrently with the anti-angiogenic agent.   5. The method of claim 4, wherein the aldesleukin is administered in a composition separate from the anti-angiogenic agent.   Administering to the patient a therapeutically effective amount of aldesleukin and an anti-angiogenic agent separately according to a dosing schedule, wherein aldesleukin is administered at a dose of about 9 to about 130 MIU / day, 1 to 3 times a day, 6. The method of any one of claims 1-5, wherein the administration is for a period of at least 3 consecutive days, optionally followed by a rest period of at least 3 consecutive days.   The method according to claim 6, wherein the anti-angiogenic agent is administered 1 to 6 times every 2 to 3 weeks.   7. The method of claim 6, wherein aldesleukin is administered intravenously and the rest period is present.   7. The method of claim 6, wherein aldesleukin is administered subcutaneously and the rest period is absent.   10. The method of claim 9, wherein the aldesleukin is administered 1-3 times daily at a dose of about 9-30 MIU / day.   7. The method of claim 6, wherein aldesleukin is administered at a dose of about 30-130 MIU / day, 3 times a day.   7. The method of claim 6, wherein aldesleukin is administered continuously for a period of 5 days, followed by a 9-day rest period.   7. The method of claim 6, wherein the dosing schedule is repeated at least 2 cools.   7. The method of claim 6, wherein the dosing schedule is repeated 3 cools.   The method of claim 6, wherein the dosing schedule is repeated 4 cools.   7. The method according to claim 6, wherein aldesleukin is administered three times on the first day and once a day for each subsequent day.   The method according to any one of claims 1 to 16, wherein the cancer is renal cell carcinoma, melanoma or colon cancer.   Further comprising administering to the patient at least one compound selected from acetaminophen, meperidine, indomethacin, ranitidine, nizatidine, diastop, loperamide, diphenhydramine, or furosemide following or simultaneously with administration of aldesleukin. The method of claim 17 comprising.   19. A method according to any one of claims 1 to 18, wherein a cancer remission is provided in the patient.   19. A method according to any one of claims 1 to 18, wherein hypotension is attenuated in the patient.   19. A method according to any one of claims 1 to 18, wherein a reduction in hypertension is provided in the patient.   19. A method according to any one of claims 1 to 18, wherein a reduction in nitric oxide synthase is effected in the patient.   19. A method according to any one of claims 1 to 18, wherein the cancer is sensitive to angiogenesis inhibition and / or immunostimulation.   The anti-angiogenic agent is N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3 24. A method according to any one of claims 1 to 23, which is a carboxamide.   The anti-angiogenic agent is 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea. 24. The method according to any one of 1 to 23.   (A) a therapeutically effective amount of aldesleukin; (b) 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N -(3-Chloro-4-fluorophenyl) -7-methoxyquinazoline-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindoline-3-ylidene) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- ( A therapeutically effective amount of an antivascular agent selected from 2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Forming agent and; (c) a composition comprising a pharmaceutically acceptable excipient.   (A) aldesleukin for simultaneous, sequential or separate use, and (b) 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- ( 3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2- Oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazin-1-amine, Or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Anti-vascular and a forming agent, a kit comprising a combination of a medicament for treating a patient suffering from a cancer that is to be.   27. The kit of claim 26, wherein each of the medicaments is packaged separately.   Aldesleukin and 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazoline-4-in the manufacture of one or more medicaments for the treatment of patients suffering from cancer Amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5 -Fluoro-2-oxoindoline-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine -1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) ) Phenyl) use of anti-angiogenic agent selected from urea.   30. Use according to claim 29, wherein the aldesleukin is formulated for administration prior to the anti-angiogenic agent.   30. Use according to claim 29, wherein the aldesleukin is formulated for subsequent administration of the anti-angiogenic agent.   30. Use according to claim 29, wherein the aldesleukin is formulated for co-administration with an anti-angiogenic agent.

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