KR20070108909A - Antiangiogenic agents with aldesleukin - Google Patents

Abstract

The present invention relates to combination therapies with IL-2 compositions and antiangiogenic agents for the treatment of cancer. Further provided are methods of alleviating toxicities and increasing the efficacy associated with the administration of IL-2 compositions or antiangiogenic compositions.

Description

ANTIANGIOGENIC AGENTS WITH ALDESLEUKIN}

Cross Reference of Related Applications

This application claims 35 U.S.C. Claims the benefit of Provisional Application No. 60 / 654,341, filed February 18, 2005, under § 119 (e), which is incorporated herein by reference in its entirety.

Field of technology

The present invention relates to a method of treating diseases associated with abnormal cell proliferation. In some embodiments, recombinant IL-2 is combined with antiangiogenic agents used to treat cancer.

Capillaries touch almost all body tissues, providing oxygen and nutrients to tissues as well as removing waste. Under typical conditions, the endothelial cells inside the capillaries do not divide, so in adults the capillaries do not normally increase in number or size. However, under certain conditions, capillaries begin to multiply rapidly, such as when tissue is damaged or at any time during the menstrual cycle. This process of forming new capillaries from existing blood vessels is known as angiogenesis or angiogenesis. See Folkman, J. Scientific American 275, 150-154 (1996). Angiogenesis during wound healing is an example of pathophysiological angiogenesis in an adult's lifetime. Capillaries added during wound healing provide a supply of oxygen and nutrients, promote granulation tissue and help remove waste. After the healing process is finished, the capillary tube normally degenerates. Lymboussaki, A. "vascular endothelial growth factor and its receptor in embryos, adults and tumors", Thesis, Department of Pathology, Laboratory of Molecular / Cancer Biology, University of Helsinki, Hartman Institute (1999).

In addition, angiogenesis plays an important role in cancer cell growth. Once the cancerous site reaches a size of about 1 to 2 mm in diameter, the cancer cells must develop a wider blood supply in order to grow the tumor, and diffusion will not be sufficient to supply sufficient oxygen and nutrients to the cancer cells. Therefore, inhibition of angiogenesis is expected to stop the growth of cancer cells. Of the angiogenesis factors identified, vascular endothelial growth factor (VEGF) was found to be the most potent and specific and important regulator of both normal and pathological angiogenesis. However, other factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) have also been implicated in 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 effective being cytolytic T lymphocytes. Induction of specific anti-tumor T lymphocyte mediated immune responses requires recognition of co-stimulatory interactions between target tumor lymphocytes and specific ligands present in the tumor antigen as well as in the tumor cells or antigen transducing cells. . This second co-stimulatory signal may also be provided by soluble factors, such as other peptide molecules that enhance effector function by binding to cytokines or specific cell surface receptors to initiate various signal transduction pathways.

Interleukin-2 (IL-2) is a lymphocyte-derived cytokine that binds to specific receptors on T cells and natural cytotoxic (NK) cells, and these cells are responsible for tumor cytolysis, cytokine secretion, and other effector functions. Will be activated. Both CD4 and CD8 T lymphocytes express the receptor of IL-2 and show increased cytolysis effector and cytokine synthesis functions after exposure to biologics. The functional effect of IL-2 is mediated by the association with the IL-2 receptor (IL-2R), which is structurally composed of three subunits, in which a common γ chain shared with α, β, and other cytokine receptors (Caligiuri et al., J. Exp. Med. 1990 171 (5): 1509-1526; Bazan, JF, Science 1992 257 (5068): 410-413; Theze et al., Immunol. Today 1996 17 (10): 481 -486). IL-2 receptor activation and thus signaling depends on the type of IL-2 receptor on the cell (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; Expinoza et al., J. Leukoc. Biol. 1995 57 (1): 13-29). T cells express high-affinity IL-2Rαβγ, whereas NK cells, monocytes and macrophages preferentially express the medium-affinity form of 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). The central mechanism is that IL-2 activates JAK (JAK1, JAK3) and JAK phosphorylates key tyrosine on IL-2Rβ, which acts as a docking site for downstream signaling molecules, including Shc and Stat5. Then catalyzes the activation of two separate pathways, Shc / Ras / Raf / MAPK, and JAK / STAT5, where JAK / STAT5 is translocated into the nucleus to directly regulate the family of gene-regulated transcription factors-STAT ( O'Shea, JJ, Immunity 1997 7 (1): 1-11). She recruits the Grb-2 / Sos complex to activate the Ras / Raf / MAPK pathway, and Grb-2 / Gab-2 activates the phosphatidylinositol 3-kinase (PI3K) pathway. These signaling proteins, in addition to regulating gene transcription factors, ultimately control cell growth, division, differentiation and immune activity. In addition, IL-2 also induces anti-apoptotic effects by regulating pro-digestion genes to increase bcl-2, bcl-x1, c-myb, which activates cdk 2, 4, 6 and p27kip1. Inhibition may affect cell cycle control or may be negatively regulated by SOCS (cytokine signaling inhibitors) (Smith, KA, Science 1988 240 (4856): 1169-1176; Theze et al., Immunol.Today 1996 17 (10): 481-486). It has been reported that the interaction between IL-2 and IL-2R triggers downstream activation of MAPK, PI-3K, and JAK / STAT signaling pathways resulting in 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 murine tumor models may result in tumor regression, long-term survival, and increased resistance to tumor regrowth when recombinant human IL-2 is administered to tumor-bearing animals for 10 to 14 days. This has been proven. Analysis of splenic lymphocytes obtained from these animals revealed that the anti-tumor effect was at least in part due to increased cytolytic T cell function. This effect includes the activation of direct cytolysis of tumor cells by CTL, as well as increased synthesis of other T lymphocyte-derived cytokines, which may also have direct or indirect anti-tumor effects.

Metastatic melanoma and renal cell carcinoma (RCC) are extremely difficult to treat and resistant to most types of systemic chemotherapy, so there is a great need to develop more effective therapies. Recent clinical data support the development of small molecule receptor tyrosine kinase (RTK) inhibitors, particularly BAY 43-9006 or SU11248, in melanoma and renal cell carcinoma. BAY 43-9006 and SU11248 both inhibit a number of kinases involved in tumor growth, proliferation and anti-angiogenesis. BAY 43-9006 is a potent inhibitor of Raf-1, a member of the Raf / MEK / ERK signaling pathway (Richly et al., Int. J. Clin. Pharmacol. Ther. 2003 41 (12): 620-621; Wilhelm et al., Cancer Res. 2004 64 (19): 7099-7109; Awada et al., Br. J. Cancer 2005 92 (10): 1855-1861), inhibits both WT B-Raf and mutant V599E B-Raf. In addition to the effect of BAY 43-9006 on Raf, most of its activity is mediated by inhibition of other RTKs, particularly VEGFR (VEGFR-2 and VEGFR-3), PDGFRβ, and other key kinase FLT-3, and cKIT. (Wilhelm et al. Cancer Res. 2004 64 (19): 7099-7109). It was established that responses with BAY 43-9006 in early solid tumor trials could result in disease stabilization, including some partial responses, by dosing once or twice daily. To date, Phase I studies have shown patients generally tolerate BAY 43-9006 well. The most common toxicity in BAY 43-9006 included gastrointestinal effects (diarrhea, nausea, abdominal cramps) or dermatological reactions including skin itch or rash (Richly et al., Int. J. Clin. Pharmacol. Ther. 2003 41 (12 ): 620-621; Ahmad and Eisen, Clin.Cancer Res. 2004 10 (18 Pt 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 PTK inhibitor of VEGFR-1-3, 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'Farrell et al., Clin. Cancer Res. 2003 9 (15): 5465-5476). SU11248 has shown anti-tumor activity in many advanced solid tumors (RCC, neuroendocrine, stroma 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 indicate that patients generally withstand the drug and have manageable toxicity, including fatigue, lymphopenia, neutropenia, hyperlipemia (Faivre et al., J. Clin. Oncol. 2006 24 ( 1): 23-35; Motzer et al., J. Clin. Oncol. 2006 24 (1): 16-24).

Studies of the hereditary forms of clear cell kidney carcinoma of the von Hippel-Lindau syndrome have confirmed the presence of the present Hippel-lindo tumor suppressor gene (VHL). This gene is mutated in most cases of hereditary renal cell carcinoma (in which case a mutation is a point-line mutation) and sporadic clear cell kidney carcinoma (in which two alleles have acquired mutations or deletions). Gnarra J. R., Duan D. R., Weng Y et al. "Molecular Cloning and Role of Bonn Hippel-Rinde Tumor Suppressors in Renal Carcinoma", Biochimica et Biophysica Acta. 1996, 1242 (3): 201-10. One consequence of these mutations is the overproduction of vascular endothelial growth factors through mechanisms involving hypoxia-inducing factors. O. Iliopoulous A.L., C. Jiang et al., “Negative Regulation of Hypoxia-Induced Genes by Bon-Hipfellin Proteins”, Proc. Natl. Acad. Sci. USA. 1996, 93: 10595-10599. Thus, the Hippel-Rindo protein is closely linked to angiogenesis by regulation of vascular endothelial growth factor. Vascular endothelial growth factor stimulates endothelial cell growth and, as mentioned, has been shown to be a central factor in angiogenesis, in particular embryonic, ovulation, wound healing, and angiogenesis during tumor growth. Ferrara N. "Molecular and biological properties of vascular endothelial growth factor", J. Mol. Med. 1999, 77: 527-543.

Vascular endothelial growth factor is a good target for the treatment of clear cell kidney cancer because the growth factor is overproduced by mutation of the present hippel-lin tumor suppressor gene. A randomized double-blind placebo-controlled study of humanized monoclonal antibody (bevacizumab) in metastatic clear cell kidney cancer patients was performed by Yang et al. "Random Test of Anti-vascular Endothelial Growth Factor Antibody, Bevacizumab in Metastatic Renal Cancer," New England Journal of Medicine. 2003, 349 (5): 427-34. The time to tumor progression was extended by 2.55 times in patients receiving bevacizumab per kilogram (10 mg / kg every two weeks) compared to placebo groups. Survival was not the primary end point in this trial and patients were crossed from placebo to bevacizumab treatment at disease progression. Renal cancer treatment that targets angiogenesis mechanisms may also be effective through pathways other than mediated by vascular endothelial growth factors. There are other proteins in the local microenvironment of some tumors that can promote angiogenesis. As such, there is a need for anti-angiogenic therapies with rational combinations of inhibitors, which is related to an understanding of the ecology of renal cell carcinoma.

Recombinant human interleukin-2 (Aldeleukin) has been approved by the FDA as a treatment for metastatic kidney cancer based on the results of several multicenter trials in 255 patients who received intermittent high-dose involvement therapy. In these trials, 15% of patients had a target response and median survival was 16.3 months (Fisher and Rosenberg 2000 Cancer J. Sci. Am. 6S1: S55-57). Because of the significant toxicity associated with this therapy, a series of Phase I and II trials was undertaken using different doses of rIL-2 and using different routes of administration. Recent reviews of the efficacy of rIL-2 as a single agent show the overall response of 15% of the more than 1700 metastatic RCC patients treated in this series of studies. The complete response was recorded only in 3 to 5% of patients. Bukowski R. M., "Natural History and Treatment of Metastatic Renal Cell Carcinoma: The Role of Interleukin-2," 1997, 80 (7): 1198-220. There seemed to be no speed difference between these paths. Randomized comparisons between high-dose intravenous and low-dose intravenous therapy and outpatient subcutaneous therapy showed no difference in median survival between groups, but significantly lower toxicity in low-dose and outpatient therapies. Yang J. C., Sherry R. M., Steinberg S. M. et al., “Random Study of High Dose and Low Dose Interleukin-2 in Patients with Metastatic Renal Cancer,” Journal of Clinical Oncology, 2003, 21 (16): 3127-32.

In addition to having a direct immunomodulatory effect, rIL-2 can also prevent tumor growth by causing secondary cytokine release (IFNγ) from activated NK cells. Saraya K. A., Balkwill F. R., "Time Sequence and Cell Origin of Interleukin-2 Stimulating Cytokine Gene Expression", British Journal of Cancer. 1993, 67 (3): 514-21. Response rates and outcomes of clear cell kidney cancer patients may be improved. Clinical trials using new drug combinations are needed. Combinations of anti-angiogenic agents such as rIL-2 and bevacizumab may have the additional effect of modifying the added clinical benefit to patients with metastatic renal cell carcinoma as well as other cancers.

Another advantage of a complementary approach to cancer regression is that it provides a platform that resistant mutations are not easy to bypass. If the therapeutic target is too polar or specific (this may be necessary to avoid targeting the host cell), for example, a single disease state, such as a specific substrate in viral replicon or a kinase in cancer cell line, etc. Point mutations can make them unaffected by drugs, which can make the disease more severe in future generations.

New methods and mechanisms for treating patients with disorders associated with abnormal proliferation that are resistant to conventional approaches or that are insufficiently treated by conventional approaches, using agents that target the immune response mechanisms of body and disease-state substrates need. The present invention provides such therapeutics and further provides other related advantages.

Brief overview of the invention

The present invention is based in part on a unique combination therapy with rIL-2 and a small molecule receptor tyrosine kinase inhibitor with non-duplicate toxicity. Assuming that melanoma and RCC generally respond to immunotherapy such as rIL-2, a rational combination of rIL-2 and a potential receptor tyrosine kinase inhibitor was evaluated based on the pharmacology of the individual formulations. The present invention provides a clinically applicable drug schedule for avoiding potential drug interactions based on the toxicological profile of these agents. The possibility of adverse pharmacokinetic / pharmacodynamic interactions and adverse pharmacologic interactions is also described. The effect of small molecule receptor tyrosine kinase inhibitors such as rIL-2 and BAY 43-9006 on T cells and the effects on IL-2-mediated signaling pathways (MAPK and JAK / STAT5) are described.

Accordingly, the present invention provides for signs of anti-tumor efficacy of IL-2 compounds, such as recombinant IL-2 (rIL-2, also known as aldeleukin), on cancer cell lines that respond poorly to conventional therapies. Expand, resulting in increased long-term survival and immunity to tumor reinfection. Moreover, the immunostimulatory effect of rIL-2 aims to alleviate the current side effects caused by administration of the anti-angiogenic composition for co-administration with it. Methods of treating subjects suffering from abnormal cell proliferation, in particular renal cell carcinoma, are provided using a combination of an IL-2 compound, eg, rIL-2 and at least one antiangiogenic agent. Furthermore, there is also provided a method of treating a subject suffering from abnormal cell proliferation, in particular renal cell carcinoma, using an IL-2 compound, for example a combination of rIL-2 and a small molecule. Preferred small molecules of the invention are listed in Tables 1-5. Particularly preferred molecules are N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1 H-pyrrole- 3-carboxamide (also called SU11248) and 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl ) Phenyl) urea (also known as BAY 43-9006).

Aldeleukin and the anti-angiogenic agent may be administered together or separately as separate pharmaceutical compositions. If administered separately, the aldeleukin may be administered before, concurrently with, or after the antiangiogenic agent. Aldehydes and antiangiogenic agents, for example

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine-1-amine, and

1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea

Specific therapies are provided, including daily, weekly, and monthly dosing schedules (or repetitions thereof) for co-administration of.

In one embodiment, antiangiogenic agents, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

N- (4-chlorophenyl) -4-((pyridin-4-yl) methyl) phthalazine-1-amine, and

1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea

One of them is co-administered with anti-angiogenic proteins such as monoclonal antibodies capable of inhibiting VEGF. In a more particular embodiment, the anti-angiogenic protein of the invention is bevacizumab or VEGF-Trap. In another more specific embodiment, the protein is an EGF inhibitor, for example cetuximab.

In another embodiment, administering aldesleukin and antiangiogenic agent (s) together in the manner presented herein increases the effectiveness of the antiangiogenic agent, thus improving positive / relative to what is observed when using only inhibitors. Results in a synergistic therapeutic response.

Other embodiments provide a therapeutic package suitable for commercial sale for the treatment of cancer patients, which comprises a container, a therapeutically effective amount of aldesleukin, and a therapeutically effective amount of an angiogenesis agent and / or a small molecule, preferably Table 1 To those described in Examples 5 to 5

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-((fluoro-2-oxoindolin-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) urea

It includes.

In another embodiment, a kit is provided. The kit includes a combination of medications for the treatment of a cancer patient, which comprises (a) aldehydes, 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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Anti-angiogenic agents selected from, which are used simultaneously, continuously or separately.

Methods of making the combinations described herein are provided and are considered to fall within the scope of the present invention as well as the use of the combinations in the preparation of a medicament for use in the methods of the invention.

Further embodiments of the present invention include those described in the detailed description.

1 shows single agent activity of rIL-2, BAY 43-9006 or SU11248 in an IL-2-reactive T-cell competent murine tumor model. B16-F10 melanoma (2 × 10 ⁶ ), CT26 colon (2 × 10 ⁶ ) or RENCA kidney carcinoma (1 × 10 ⁶ ) cells were subcutaneously transplanted to the right flank of female C57BL6 or BALB / c mice. As outlined in the method, treatment was initiated when tumors were established with an average size of 50-225 mm 3. Mice were randomized into treatment cohorts (10 mice / group). rIL-2 was administered subcutaneously daily (0.2-3 mg / kg / d). BAY 43-9006 or SU11248 (1-100 mg / kg) was administered daily as a solution by oral gavage. Figure 1 shows the average tumor growth inhibition of single agents in B16-F10, CT26 and RENCA tumor models between 10-14 days (calculated as [1- (average tumor volume of the treatment group / excipient group) x 100]) To illustrate. These data were collected from multiple independent studies, each studying 10 mice / group. * Indicates statistical significance for excipient treatment (p <0.05, ANOVA).

Figure 2 shows the efficacy of the simultaneous treatment of rIL-2 and BAY 43-9006 in the B16-F10 murine melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female C57BL6 mice. When tumors reached about 50 mm 3 mice were randomized into treatment groups and treatment started on day 0 as follows: excipients, days 0-6 (◆), rIL-2 (3.3 mg / kg, subcutaneous, 0-6). (Day) (△) or BAY 43-9006 (30 mg / kg, intraperitoneal, 0-6 days) (×) or rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) + BAY 43-9006 (30 mg / kg, intraperitoneal, 0-6 days) combination (■). 2 illustrates mean tumor volume (부 ± SE) over days after randomization (n = 10 mice / group).

3A and 3B show the efficacy of continuous treatment of rIL-2 and BAY 43-9006 in the B16-F10 murine melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female C57BL6 mice. When the tumor was about 50 mm 3 mice were randomized into treatment groups and treatment started on day 0. 3A shows the results of continuous therapy with rIL-2 administered first followed by BAY 43-9006. Panels were excipient (◆) 0-6 days, 7-13 days; rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) (△) or BAY43-9006 (30 mg / kg, intraperitoneal, 7-13 days) (×) or rIL-2 (3.3 mg / kg, subcutaneous , 0-6 days) + BAY43-9006 (30 mg / kg, intraperitoneal, 7-13 days). 3B shows the results of continuous therapy with first administration of BAY 43-9006 followed by rIL-2. Panels were excipient (◆) 0-6 days, 7-13 days; BAY 43-9006 (30 mg / kg, intraperitoneal, 0-6 days) (×); rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days) (□) or BAY 43-9006 (30 mg / kg, intraperitoneal, 0-6 days) + rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days) The combination (▲) is displayed.

The graph shows mean tumor volume (㎣ ± SE) over days after randomization (n = 10 mice / group).

4 shows the efficacy of the simultaneous treatment of rIL-2 and SU11248 in the B16-F10 murine melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female C57BL6 mice. When tumors reached about 50 mm 3 mice were randomized into treatment groups and treatment started on day 0 as follows: excipients, days 0-6 (o), rIL-2 (3.3 mg / kg, subcutaneous, 0-6). Sun) (□) or SU11248 (40 mg / kg, intraperitoneal, 0-6 days) (○) or rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) + SU11248 (40 mg / kg, intraperitoneally, 0-6 days) combination (▲). 4 illustrates mean tumor volume (㎣ ± SE) over days after randomization (n = 10 mice / group).

5A and 5B show the efficacy of continuous treatment of rIL-2 and SU11248 in the B16-F10 murine melanoma model. B16-F10 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female C57BL6 mice. When the tumor was about 50 mm 3 mice were randomized into treatment groups and treatment started on day 0. 5A shows the results of continuous therapy with rIL-2 first followed by SU11248. Panels were excipient (◇) 0-6 days, 7-13 days; rIL-2 (3.3 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, intraperitoneal, 7-13 days) (×) or rIL-2 (3.3 mg / kg, subcutaneous, 0 -6 days) plus SU11248 (40 mg / kg, intraperitoneal, 7-13 days). 5B shows the results of continuous therapy with SU11248 first followed by rIL-2. Panels were excipient (◇) 0-6 days, 7-13 days; SU11248 (40 mg / kg, intraperitoneal, 0-6 days) (○); rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days) (△) or SU11248 (40 mg / kg, intraperitoneal, 0-6 days) + rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days ) Displays the combination (■). The graph shows mean tumor volume (㎣ ± SE) over days after randomization (n = 10 mice / group).

6 shows the efficacy of continuous treatment of rIL-2 and BAY 43-9006 in a CT26 murine colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female BALB / c mice. When the tumor was about 225 mm 3, mice were randomized into treatment groups and treatment started on day 0. The panel displays continuous therapy with rIL-2 administered first followed by BAY 43-9006: excipients (◆) days 0-6, 7-13 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (□) or BAY 43-9006 (40 mg / kg, intraperitoneal, 7-13 days) (△) or rIL-2 (1 mg / kg, subcutaneous, 0-6 days) + BAY 43-9006 (40 mg / kg, intraperitoneal, 7-13 days) combination (○).

7 shows the efficacy of the simultaneous treatment of rIL-2 and SU11248 in CT26 murine colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female BALB / c mice. When the tumor was about 225 mm 3, mice were randomized into treatment groups and treatment started on day 0. Panel was excipient (◆) 0-6 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, intraperitoneal, 0-6 days) (○) or rIL-2 (1 mg / kg, subcutaneous, 0-6 1) + SU11248 (40 mg / kg, intraperitoneal, 0-6 days).

8A and 8B show the efficacy of continuous treatment of rIL-2 and SU11248 in CT26 murine colon adenocarcinoma model. CT26 cells (2 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female BALB / c mice. When the tumor was about 225 mm 3, mice were randomized into treatment groups and treatment started on day 0. 8A shows the results of continuous therapy with rIL-2 administered first followed by SU11248. The panel displays the following: excipients (◆) 0-6 days, 7-13 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (□) or SU11248 (40 mg / kg, intraperitoneal, 7-13 days) (○) or rIL-2 (1 mg / kg, subcutaneous, 0-6 Day) + SU11248 (40 mg / kg, intraperitoneal, 7-13 days) combination (■). 8B shows the results of continuous therapy with SU11248 first followed by rIL-2. The panel displays the following: excipients (◆) 0-6 days, 7-13 days; SU11248 (40 mg / kg, intraperitoneal, 0-6 days) (○); rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days) (□) or SU11248 (40 mg / kg, intraperitoneal, 0-6 days) + rIL-2 (3.3 mg / kg, subcutaneous, 7-13 days ) Combination (▲). The graph shows mean tumor volume (㎣ ± SE) over days after randomization (n = 10 mice / group).

Figure 9 shows the efficacy of the simultaneous treatment of rIL-2 and BAY 43-9006 in a murine RCC RENCA tumor model. RENCA cells (1 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female BALB / c mice. When tumors reached about 50 mm 3, mice were randomized into treatment groups and treatment started on day 0 as follows: excipients (◆) 0-8 days; rIL-2 (1 mg / kg, subcutaneous, 0-4 days, 7-11 days) (△) or BAY 43-9006 (30 mg / kg, intraperitoneal, 0-8 days) (×) or rIL-2 (1 mg / kg, subcutaneous, 0-4 days, 7-11 days) + BAY 43-9006 (30 mg / kg, intraperitoneal, 0-8 days) combination (■). 9 illustrates mean tumor volume (㎣ ± SE) over days after randomization (n = 10 mice / group).

10 shows the efficacy of the simultaneous treatment of rIL-2 and SU11248 in a murine RCC RENCA tumor model. RENCA cells (1 × 10 ⁶ cells) were subcutaneously implanted into the right flank of female BALB / c mice. When the tumor was about 50 mm 3 mice were randomized into treatment groups and treatment started on day 0. Panel was excipient (◆) 0-6 days; rIL-2 (1 mg / kg, subcutaneous, 0-6 days) (■) or SU11248 (40 mg / kg, intraperitoneal, 0-6 days) (×) or rIL-2 (1 mg / kg, subcutaneous, 0-6 1) + SU11248 (40 mg / kg, intraperitoneal, 0-6 days) combination (▲).

Detection assays were used to characterize several types of cancer that could be treated or modulated with rIL-2 with anti-angiogenic / VEGF inhibitors and cytokine administration. Such cancers include, for example, CML, AML, breast cancer, gastric cancer, endometrial cancer, salivary gland cancer, adrenal cancer, non-small cell lung cancer, pancreatic cancer, kidney cancer, rectal cancer, skin cancer, melanoma, multiple myeloma, brain cancer / CNS, uterus Cervical cancer, nasopharyngeal cancer, malignant mesothelioma, hypopharyngeal cancer, gastrointestinal carcinoma, peritoneal cancer, adenocarcinoma, mesenteric cancer, gallbladder cancer, testicular cancer, esophageal cancer, lung cancer, thyroid cancer, ovarian cancer, peritoneal cancer, prostate cancer, head and neck cancer, bladder cancer, colon cancer, colorectal cancer , Lymphomas, and glioblastomas. The methods described herein are useful for the treatment of all such cancers.

Therapies using a combination of Aldesleukin and at least one antiangiogenic agent in the manner presented herein provide a beneficial physiological response with respect to the treatment of cancer in which the proliferating cells are not dependent on cancer and are highly dependent on angiogenesis such as VEGF. Cause.

One embodiment of the present invention provides a method for treating a cancer patient suffering from hypotension due to administration of Aldes Leukine, which comprises co-administering to a patient a therapeutically effective amount of an antiangiogenic agent that attenuates hypotension. .

Another more specific embodiment of the present invention includes improving cancer in a patient.

Another embodiment of the present invention provides a method for treating a cancer patient suffering from hypertension due to the administration of an antiangiogenic agent, which comprises co-administering to a patient a therapeutically effective amount of aldesleukin that attenuates hypertension. .

Another more specific embodiment of the present invention includes improving cancer in a patient. In some embodiments, the cancer is susceptible to inhibition of angiogenesis and / or immunostimulation.

In more specific embodiments of any of the preceding embodiments, the antiangiogenic agent

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is selected from.

Another embodiment of the present invention provides a method for treating a cancer patient, the method 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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Administering a compound selected from.

Another embodiment of the present invention provides a method of increasing the efficacy of aldesleukin in a cancer patient, which comprises first administering an antiangiogenic agent to a patient at a dose capable of causing hypoxia, and then administering aldesleukin. It includes.

Another embodiment provides a method of increasing the efficacy of an antiangiogenic agent in cancer patients, comprising reducing nitric oxide (NO) synthase by administering aldehydes to the patient. Another embodiment provides a method of increasing the efficacy of an antiangiogenic agent in cancer patients, the method comprising administering aldehydes to a patient, thereby administering nides leucine (NO) by administering aldehydes to the patient. Synthase is reduced.

In a more specific embodiment of the invention, the antiangiogenic agent

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is selected from.

Another embodiment of the invention provides a method of treating a cancer patient, which is first administered a therapeutically effective amount of aldesleukin to the patient, followed by (eg)

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Administering an antiangiogenic agent selected from

Another embodiment of the present invention provides a method of treating a cancer patient, wherein the therapeutically effective amount of anti-angiogenic agent,

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is administered first, followed by Aldeleukin.

Another embodiment of the present invention provides a method for treating a cancer patient, the method comprising administering to the patient a therapeutically effective amount of an angiogenesis agent and an aldeleukin separately according to a dosing schedule, wherein the aldeleukin May be administered 1 to 3 times per day at a dose of about 9 to about 130 MIU / day for at least 3 consecutive days, followed by an optional rest period of at least 3 consecutive days.

In a more specific embodiment of the invention, the antiangiogenic agent is administered 1 to 6 times every 2-3 weeks.

In a more specific embodiment of the invention, said antiangiogenic agent is a VEGF inhibitor.

In a more specific embodiment of the invention, the antiangiogenic agent

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is selected from.

In a more specific embodiment of the invention, the aldeleukin is administered intravenously and the resting period is present.

In a more specific embodiment of the invention, the aldoleukin is administered subcutaneously and the resting period is absent.

In a more particular embodiment of the invention, the aldoleukin is administered three times per day at a dose of about 30-100 MIU / day.

In a more specific embodiment of the invention, the aldoleukin is administered for 5 consecutive days, followed by a 9 day rest period.

In a more particular embodiment, the dosing schedule is repeated at least two courses. Also in more specific embodiments, each course consists of 9-15 days of rest after 2-5 days of treatment.

In another more specific embodiment of the present invention, the aldeleukin is administered three times on the first day and once per day on each progression day.

In a more specific embodiment of the invention, the dosing schedule is repeated at least 2 courses, or 3 courses, or 4 courses, or 5 courses, or 6 courses, or 7 courses, or 8 courses, or 9 courses, or 10 courses. .

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

In a more specific embodiment of any of the preceding embodiments, upon administration of the aldeleukin, acetaminophen, mereridine, indomethacin, ranitidine, nizatidine, diastop, loperamide, diphenhydramine, Or at least one compound selected from purosemide is also administered to the patient.

In a preferred embodiment of any of the preceding embodiments, the antiangiogenic agent

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

to be.

Another embodiment of the present invention provides a method for treating cancer patients, which comprises:

Administering tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method for treating cancer patients, which comprises:

Administering tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method for treating cancer patients, which comprises:

Administering tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method for treating cancer patients, which comprises:

Administering tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method for treating cancer patients, which comprises:

Administering tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method of treating a cancer patient, wherein the pharmaceutically acceptable salt of aldeleukin and a compound of formula (I), a tautomer thereof, a pharmaceutically acceptable salt thereof, or a tautomer Administering a salt comprising:

Formula I

Where

R ₁ is alkyl, -aryl (R _i ) _p , or heterocyclyl;

R ₂ is H or alkyl; or

R ₁ and R ₂ join 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) nR _d , or heterocyclyl;

R ₄ is H, —CN, —OH, halogen, alkyl, aryl, —O (CH ₂ ) _q -R _g , -O- (CH ₂ ) _q -OR _e , -NR _a R _b , -S (O ) _n R _d , or -heterocyclyl-R _f ;

R ₅ is H, —CN, —OH, halogen, alkyl, aryl, —0 (CH ₂ ) _q -R _g , -O- (CH ₂ ) _q -OR _e , -NR _a R _b , -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 combine together to form R _{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 —0 (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:

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, thereby forming a covalent bond;

Each m is independently 0, 1, or 2.

In a more particular embodiment of the invention, the compound is

Tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

In a more particular embodiment of the invention, the compound is

Tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method of treating a cancer patient, wherein the pharmaceutically acceptable salt of aldeleukin and a compound of formula II, a tautomer thereof, a pharmaceutically acceptable salt thereof, or a tautomer Administering acceptable salts includes:

Formula II

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 specific embodiment of the invention, R ₁₁ is R _11a :

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 particular embodiment of the invention, the compound is

Tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method of treating a cancer patient, wherein the pharmaceutically acceptable salt of aldeleukin and a compound of formula III, tautomer thereof, a pharmaceutically acceptable salt thereof, or tautomer Administering acceptable salts includes:

Formula III

In which the dashed line represents the selective placement of further bonds;

R ₂₀ is H or = 0;

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 O, 1, or 2;

Each q is independently O, 1, or 2.

In a more particular embodiment of the invention, the compound is

Tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

In a more particular embodiment of the invention, the compound is

Tautomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutically acceptable salts of tautomers.

Another embodiment of the present invention provides a method of reducing toxicity associated with administering Aldesleukin to a cancer patient, which comprises to the patient a quinolinone, preferably an antiangiogenic agent, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Administering the selected one.

Another embodiment of the invention is an anti-angiogenic agent, preferably in cancer patients

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Provided is a method of reducing toxicity associated with administering a selection from a subject, which comprises administering an aldeleukin to a patient.

Another embodiment of the present invention provides a method of reducing IL-2 fragility in cancer patients, which is directed to the patient with 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-methoxyquinazolin-4-amine,

N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Administering the selected one.

Another embodiment of the invention provides a method of treating a cancer patient, which is first administered to said patient a therapeutically effective amount of aldesleukin, followed by an antiangiogenic agent, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Administering the selected one.

Another embodiment of the present invention provides a method of treating cancer patients, wherein the therapeutically effective amount of an angiogenesis agent, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

And the separate administration of aldehydes according to the dosing schedule, wherein the aldehydes are administered 1-3 times daily at a dose of about 9 to about 130 MIU / day for at least 3 consecutive days, and then Optionally, a rest period of at least three consecutive days may be provided. 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 more specific embodiments, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

The antiangiogenic agent selected from is administered 1 to 9 times every 2-3 weeks. In more specific embodiments, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

The antiangiogenic agent selected from is a VEGF inhibitor.

In another embodiment, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Antiangiogenic agents selected from are also administered with bevacizumab, or cetuximab, or VEGF-Trap. More specifically, the bevacizumab is administered at a dose of 1 to 12 mg / kg once every 12 to 16 days.

In another embodiment, the anti-angiogenic agent is administered within 72 hours based on rIL-2 administration; "Within" herein means before or after, meaning that the anti-angiogenic agent is administered 72 hours before or 72 hours after rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 14 days based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 7 days based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 6 days based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 5 days based on rIL-2 administration. In another embodiment, the antiangiogenic agent is administered within 4 days based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 48 hours based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 36 hours based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 24 hours based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 12 hours based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 6 hours based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 1 hour based on rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered simultaneously with the administration of rIL-2. In another embodiment, the anti-angiogenic agent is administered in combination with rIL-2. In a more specific embodiment of the invention, the antiangiogenic agent

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is selected from.

Another embodiment of the present invention provides a method of treating cancer patients, which comprises aldehyde and antiangiogenic agents, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Co-administration of the one selected from.

Another embodiment of the invention provides a therapeutic package suitable for commercial sale for the treatment of cancer patients, which comprises a container, a therapeutically effective amount of aldesleukin, and a therapeutically effective amount of an angiogenic agent, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

It includes one selected from. In the therapeutic package of the present invention, the patient is a patient suffering from renal cell carcinoma. The therapeutic package of the present invention further comprises a written statement indicating that the patient is treated with an antiangiogenic agent after receiving Aldeleukin treatment, wherein the antiangiogenic agent is preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Is selected from.

Another embodiment is an antiangiogenic agent, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

It provides a pharmaceutical composition comprising one selected from Aldes Leukine.

Clear cell kidney cell carcinoma (RCC) is associated with increased angiogenesis, especially VEGF expression. VEGF is a protein that plays an important role in tumor angiogenesis (the formation of new blood vessels in the tumor) and in maintaining established tumor vessels. It binds to specific receptors on blood vessels and stimulates expansion to already existing vessels. In some but not all cases, RCCa showed elevated serum VEGF levels.

There is evidence that increased VEGF correlates with resistance to IL-2 (Lissoni et al., Anticancer Res. 2001, 21: 777-9). The immunosuppressive effects of VEGF on T cells and dendritic cells (Gabrilovich et al., J. Leukoc. Biol. 2002, 72: 285-96) may be a mechanism of VEGF induced resistance to IL-2 treatment.

In addition, VEGF has been reported to increase vascular permeability, such that treatment with anti-VEGF results in increased blood pressure in some patients. Thus, some of the main toxicities of anti-VEGF (e.g. hypertension) and rIL-2 (e.g. hypotension) may be counterbalanced and if the combination of the present invention has been administered to cancer patients, e.g. RCCa patients In this case, an improved treatment index will be obtained.

Furthermore, the combination of the present invention, such as the combination of aldehydes and bevacizumab or cetuximab, yields a higher proportion and / or better durability response compared to the formulation administered alone.

Certain Aldesleukin dosing regimens disclosed herein provide intermittent stimulation of natural cytotoxic (NK) cell activity and reduce the risk of rIL-2-related side effects that may be associated with long term exposure to rIL-2 dosing. Of course, hypotension typically associated with rIL-2 dosing is also counteracted by administration of the anti-angiogenic compositions of the present invention, since anti-angiogenic compositions are generally associated with hypertension. Furthermore, hypoxia typically associated with VEGF inhibitors will also increase the sensitivity to rIL-2 therapeutic efficacy while also providing the benefits of remission.

Another embodiment of the invention provides the use of aldesleukin in the manufacture of a medicament for the treatment of cancer, wherein the medicament is 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-methoxyquinazolin-4-amine,

N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

It is used separately or simultaneously with the one selected from. In a more specific embodiment of the invention, preferably

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

The anti-angiogenic agent selected from is a lactate salt. In another embodiment, the cancer is renal cell carcinoma.

Another embodiment of the invention is an anti-angiogenic agent, preferably in the manufacture of a medicament for the treatment of cancer

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

Provided for the use of the one selected from, wherein the medicament is used separately or simultaneously or sequentially with the aldeleukin.

Preferred molecules associated with angiogenesis modulated (eg 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 growth factor, conversion growth factor a (TGF-a), conversion growth factor b (TGF-b), or nitric oxide. Also contemplated by the composition of the invention of thrombospodine, angiostatin, and endostatin (eg, elevation) is contemplated within the scope of the invention.

rIL General compositions co-administered with -2:

Quinazoline

The compound of formula I has the structure

Formula I

Where

R ₁ is alkyl, -aryl (R _i ) _p , or heterocyclyl;

R ₂ is H or alkyl; or

R ₁ and R ₂ join 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 -OR _e , -NR _a R _b , -S (O ) _n R _d , or -heterocyclyl-R _f ;

R ₅ is H, —CN, —OH, halogen, alkyl, aryl, —0 (CH ₂ ) _q -R _g , -O- (CH ₂ ) _q -OR _e , -NR _a R _b , -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 combine together to form R _{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 —0 (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:

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, thereby forming a covalent bond;

Each m is independently 0, 1, or 2.

In a more particular embodiment of the invention R ₂ is H. In a more particular embodiment of the invention R ₁ is -aryl (R _i ) _p . In further embodiments, R ₁ is -aryl, and (R _{i) p,} in R _1, and aryl is phenyl, in R ₁ p is 2, the two R _i groups are halo in the R _1.

In further embodiments, R ₁ is-aryl (R _{i) p,} in R _1, and aryl is phenyl, in R ₁ p is 1, and the ethynyl from R ₁ R _i groups are alkynyl, preferably.

In further embodiments, R ₁ is -aryl (R _{i) p,} and in R ₁ and aryl is phenyl, in R ₁ p is 2 and one R _i in the R ₁ is halo, in R ₁ other 1 The dog R _i is -O (CH ₂ ) _q -R _g . In a more particular embodiment of the invention, q in R ₁ is 1 and R _g in R ₁ is halophenyl.

In further embodiments, R ₁ is -aryl (R _i ) _p , _p of R ₁ is 1 and R _i of R ₁ is alkynyl.

Another more specific embodiment of the invention is provided wherein R ₁ and R ₂ are joined together to form the following R _{1 -2} :

Where

R ₈ is H, X is N and R ₉ is —C (O) NHR _b . In a more particular embodiment of the invention, R _b of R ₉ is -phenyl-O-CH ₂ (CH ₃ ) ₂ .

In more specific embodiments of the invention R ₃ and R ₆ are H.

In a more particular embodiment of the invention R ₄ is -O- (CH ₂ ) _q -R _g . In a more specific embodiment of the invention, q in R ₄ is 1 and R _g is H.

In other embodiments, R ₄ is —O— (CH ₂ ) _q —R _g and R _g of R ₄ is heterocyclyl.

In a more particular embodiment of the invention R ₄ and R ₅ are each -O- (CH ₂ ) _q -OR _e . In a more particular embodiment of the invention, q of R ₄ and R ₅ is 2 and R _e of R ₄ and R ₅ is methyl.

In a more particular embodiment of the invention R ₄ is -heterocyclyl-R _f and R ₅ is H. In a more particular embodiment of the invention said heterocyclyl of R ₄ is furanyl. In more particular embodiments R _f of R ₄ is — (CH ₂ ) _q NHR _h . Furthermore, R _h is — (CH ₂ ) _q S (O) ₂ CH ₃ .

In a more particular embodiment of the invention R ₅ is -O- (CH ₂ ) _q -R _g . In another embodiment of the invention R _g of 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 structure

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 more specific embodiments R ₁₁ is heterocyclyl. In more specific embodiments R ₁₁ is R _11a :

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 another more specific embodiment, R ₁ is R _1a and R ₇ is —C (O) — (CH ₂ ) _p -N (H) _(2-r) (alkyl) _r , wherein p is O, 1, 2, 3, 4, or 5 and r is 0, 1, or 2. In another more specific embodiment, R ₁₁ is R _11a and R ₁₃ is F. In another more specific embodiment, R ₁₁ is R _11a and R ₁₇ is-(CH ₂ ) _t COOH, wherein t is 1, 2, 3, or 4. In another more specific embodiment, R ₁₁ is R _11a and R ₆ and R ₈ are both methyl. In another more specific embodiment, R ₁₁ is R _11a and R ₁₇ is H.

In another more specific embodiment of the invention R ₁₁ is aryl. More specifically, R ₁₁ is substituted or non-substituted phenyl. In another embodiment R ₁₃ is iodide.

Isoindolinone

The compound of formula III has the structure

In which the dashed line represents the selective placement of further bonds;

R ₂₀ is H or = 0;

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 O, 1, or 2;

Each q is independently O, 1, or 2.

Justice:

Area under the AUC curve

CR complete response

DLT toxicity limit

IL interleukin

IL-2 Interleukin-2

IU International Unit

IV Intravenous (dose)

LAK lymphokine-activated cytotoxicity

MTD Maximum Allowable Dosage

MIU Million International Unit

NK Natural Cytotoxicity

PK Pharmacokinetics

PR partial reaction

RCC Renal Cell Carcinoma

RCCa clear cell kidney cell carcinoma

rIL-2 recombinant interleukin-2

RTK Receptor Tyrosine Kinase

TNF tumor necrosis factor

VEGF Endothelial Growth Factor

In general, reference to an element, such as hydrogen or H, is meant to include all isotopes of that element. For example, if an R group is defined to contain hydrogen or H, it also includes deuterium and tritium.

"Anti-angiogenic agent" means any small molecule, and more specifically any compound with a molecular weight of less than 1,100 g / mol that has been or has been shown to inhibit angiogenesis in the system. Preferred molecules associated with angiogenesis modulated (eg inhibited) by the compositions of the invention include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukin-8 (IL-8), angiogenin, Angiotropin, epidermal growth factor (EGF), platelet-derived endothelial growth factor, conversion growth factor a (TGF-a), conversion growth factor b (TGF-b), or nitric oxide. Also contemplated by the composition of the invention of thrombospodine, angiostatin, and endostatin (eg, elevation) is contemplated within the scope of the invention. Preferred anti-angiogenic compositions of the invention

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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid,

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) urea

It includes.

The term "effective amount" is an amount necessary or sufficient to realize the desired biological effect. For example, an effective amount of a compound for the treatment of renal cell carcinoma may be that amount necessary to cause regression of tumor growth in renal cells. The effective amount can vary depending, for example, on the condition to be treated, the weight of the subject, and the severity of the disease. One skilled in the art can easily determine the effective amount experimentally without undue experimentation.

As used herein, “effective amount for treatment” refers to an amount sufficient to alleviate, ameliorate, stabilize, reverse, slow or slow the progression of a condition, such as a disease state.

A "subject" or "patient" is a person or dog, cat, pouch pet, silk monkey, horse, cow, pig, sheep, goat, elephant, giraffe, chicken, lion, monkey, owl, rat, squirrel, slender It is meant to describe vertebrates, including Lawless, and mice.

A "pocket pet" refers to a group of vertebrates that fit snugly in a wide coat pocket, such as hamsters, chinchillas, ferrets, mice, guinea pigs, gerbils, rabbits, and marsupials.

As used herein, the term "pharmaceutically acceptable ester" refers to an ester that hydrolyzes in vivo and includes those that are readily degraded in the human body, leaving the parent compound or salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, in particular alkanic acid, alkenic acid, cycloalkanoic acid and alkanedioic acid, wherein each alkyl or alkenyl moiety is 6 or less It is advantageous to have carbon atoms. 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 are acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsul Fate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrogen bromide, hydrogen iodide, 2-hydroxyethanesulfonate, lactate , Maleate, methanesulfonate, nicotinate, 2-naphth-allensulfonate, oxalate, pamoate, pectinate, sulfate, 3-phenylpropionate, picrate, pivalate, propionate, Succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate, but are not limited to these. Basic nitrogen-containing groups also include lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; Dialkylsulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromide, and iodides, arals May be quaternized with agents such as chel halides such as benzyl and phenethyl bromide and the like. This gives a water soluble or oil soluble or dispersible product.

As used herein, the term “pharmaceutically acceptable prodrugs”, within the scope of sufficient medical judgment, is suitable for use in contact with human tissues and tissues of lower animals without undue toxicity, irritation, allergic reactions, etc. Prodrugs of the compounds of the present invention that correspond to a risk ratio and are effective for the intended use, where possible include amphoteric ionic forms of the compounds of the present invention. The term “prodrug” refers to a compound which is rapidly modified in vivo to yield the parent compound of the above formulas, for example by hydrolysis in blood. T. Higuchi and V. Stella, "Prodrugs as New Delivery Systems", A.C.S. Symposium series 14, and edited by Edward B. Roche, "Bioreversible Carriers in Drug Design," American Pharmaceutical Association, Pergamon Press, 1987. For example, prodrugs such as those described in US Pat. No. 6,284,772 can be used.

"Halo", "halide", or "halogen" refers to an F, Cl, Br, or I atom.

The term "alkyl" refers to substituted and non-substituted alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The term also encompasses branched chain isomers of straight chain alkyl groups, for example

-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 ₂ 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, but are not limited to these. The term also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, which rings may be substituted with straight and branched chain alkyl groups as defined above. The term also includes, but is not limited to, polycyclic alkyl groups, for example adamantyl, norbornyl, and bicyclo [2.2.2] octyl, which rings may be substituted with straight and branched chain alkyl groups as defined above. Can be. The term "alkyl" also includes groups in which one or more bonds with carbon (s) or hydrogen (s) have been replaced by bonds with non-hydrogen and non-carbon atoms, with non-hydrogen and non-carbon atoms Are, but are not limited to, halogen atoms of halides such as, for example, F, Cl, Br and I; Oxygen atoms of groups such as hydroxyl groups, alkoxy groups, aryloxy groups and ester groups; Sulfur atoms of groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups and sulfoxide groups; Nitrogen atoms of groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, amides and enamines; Silicon atoms of groups such as trialkylsilyl group, dialkylarylsilyl group, alkyldiarylsilyl group and triarylsilyl group; And other hetero atoms of various other groups. Alkyl groups are limited to those having from 1 to 20 carbon atoms and up to 5 additional hetero atoms as described above. More preferred alkyl groups have 1 to 5 carbon atoms and up to 2 hetero atoms.

The term "aryl" refers to substituted and non-substituted allyl groups that do not contain heteroatoms. Thus, the term includes, but is not limited to, for example, groups such as phenyl, biphenyl, anthracenyl, naphthenyl. Also, aryl groups include those in which one of the aromatic carbons is bonded to a non-carbon or non-hydrogen atom described above (in the definition of alkyl), wherein one or more aromatic carbons of the aryl group is defined herein and And / or aryl groups bonded to non-substituted alkyl, alkenyl, or alkynyl groups. This includes a bonding arrangement in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, or alkenyl group to form a fused ring system (eg, dihydronaphthyl or terterhydronaphthyl). . Thus, the term "aryl" includes, but is not limited to, tolyl and hydroxyphenyl in particular.

The term "alkenyl" refers to straight, branched and cyclic groups, such as those described in connection with alkyl groups as defined above, wherein at least one double bond is present between two carbon atoms. In particular, 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, pentadiethyl, and hexadienyl include, but are not limited to these. In addition, groups in which a non-carbon or non-hydrogen atom is bonded to a carbon double-bonded with another carbon, and those in which one of the non-carbon or non-hydrogen atoms is bonded to a carbon not included in a double bond with another carbon Included. Alkenyl groups are limited to those having from 2 to 15 carbon atoms and up to 4 additional hetero atoms as described above. More preferred alkenyl groups have from 2 to 5 carbon atoms and up to 2 hetero atoms.

The term "alkoxy" refers to a substituted or non-substituted alkoxy group represented by '-O-alkyl', wherein the point of attachment is oxy group and the alkyl group is as defined above. Alkoxy groups are limited to those having up to 5 additional hetero atoms, including 1 to 20 carbon atoms and oxygen atoms. More preferred alkoxy groups have up to 2 hetero atoms, including 1 to 5 carbon atoms and oxygen atoms.

The term "alkynyl" refers to straight and branched chain groups, such as those described with respect to alkyl groups as defined above, wherein at least one triple bond is present between two carbon atoms. Specifically, -C≡C (H), -C≡C (CH3), -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 ) may be exemplified, but is not limited thereto. Also included are alkynyl groups in which non-carbon or non-hydrogen atoms are bonded to carbon triple-bonded with other carbon, and those in which non-carbon or non-hydrogen atoms are bonded to carbon not included in double bonds with other carbons. . Alkynyl groups are limited to those having from 2 to 15 carbon atoms and up to 4 additional hetero atoms as described above. More preferred alkynyl groups have 2 to 5 carbon atoms and up to 2 hetero atoms.

The term “heterocyclyl” refers to both aromatic and non-aromatic ring compounds, and includes, but is not limited to, monocyclic, bicyclic and polycyclic ring compounds, and contains three or more ring members, one or more of which But quinuclidyl, which is a hetero atom such as N, O, and S. Examples of heterocyclyl groups include, but are not limited to, unsaturated 3-8 membered rings containing 1-4 nitrogen atoms, including but not limited to pyrrole, 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 and the like), tetrazolyl (eg, 1H-tetrazolyl, 2H-tetrazolyl and the like); Saturated 3 to 8-membered rings containing 1 to 4 nitrogen atoms, including but not limited to pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; Condensed unsaturated heterocyclic groups containing 1 to 4 nitrogen atoms, including but not limited to indolyl, isoindoleyl, indolinyl, indolinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotria Jolyl; Unsaturated 3-8 membered rings containing 1 to 2 oxygen atoms, including but not limited to furanyl; Unsaturated 3-8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, including but not limited to, for example, oxazolyl, isoxazolyl, oxdiazolyl (eg, 1,2,4 Oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, and the like); Saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, including but not limited to morpholinyl; Unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example benzoxazolyl, benzoxadiazolyl, benzoxazinyl (e.g., 2H-1,4-benzoxazinyl Etc); Unsaturated 3-8 membered rings containing 1 to 3 sulfur 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 and the like); Saturated 3 to 8-membered rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, including but not limited to thiazolodinyl; Saturated and unsaturated 3-8 membered rings containing 1 to 2 sulfur atoms, including but not limited to thienyl, dihydrodithiyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; Unsaturated condensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, including but not limited to benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g., 2H-1,4 -Benzothiazinyl and the like), dihydrobenzothiazinyl (eg, 2H-3,4-dihydrobenzothiazinyl and the like); Unsaturated condensed heterocyclic rings containing 1 to 2 oxygen atoms, such as benzodioxolyl (eg, 1,3-benzodioxoyl, etc.); 3 to 8-membered rings containing oxygen atoms and 1 to 2 sulfur atoms, including but not limited to dihydrooxathiinyl; Saturated 3 to 8-membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as 1,4-oxatian; Unsaturated condensed rings containing 1 to 2 sulfur atoms such as benzothienyl, benzodithiinyl; And unsaturated condensed heterocyclic rings containing oxygen atoms and 1 to 2 oxygen atoms, for example benzoxathynyl. Heterocyclyl groups also include those described above wherein at least one sulfur atom in the ring is double bonded with one or two oxygen atoms (sulfoxide and sulfone). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide and tetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5 or 6 ring members. More preferred heterocyclyl groups are morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine , Thiomorpholine, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidin, thiazole, in which the sulfur atom of thiomorpholine is bonded with one or more oxygen atoms , Isoxazole, furan, and tetrahydrofuran. "Heterocyclyl" also refers to the groups described above wherein one of the ring members is bonded to a non-hydrogen atom as described above in connection with a substituted alkyl group and a substituted aryl group. In particular, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1-methylpiperazinyl, and 2-chloropyridyl are exemplified, but are not limited to these. Heterocyclyl groups are limited to those having from 2 to 15 carbon atoms and up to 6 additional hetero atoms as described above. More preferred heterocyclyl groups have 3 to 5 carbon atoms and up to 2 hetero atoms.

The term "substitution", although not defined, will have the same meaning with respect to optional additions as described in the definition of alkyl when applied to groups well known in the art, for example groups such as phenyl.

It is to be understood that within the scope of the present invention, the compounds described herein may exhibit tautomerism and that the chemical schemes herein may represent only one of the possible tautomeric forms. It is to be understood that the present invention includes all tautomeric forms with anti-angiogenic activity and is not limited to any one tautomeric form used in chemical schemes.

In addition, it is to be understood that certain compounds and embodiments of the present invention may exist in solvated forms as well as in non-solvated forms such as, for example, hydrated forms. It should be understood that the present invention includes all such solvated forms having anti-angiogenic activity.

In addition, the present invention includes isotopically-labelled compounds, which are structurally identical to those described above, except that at least one atom has an atomic mass or mass value that is different from the atomic mass or mass value normally found in nature. Only the replacement is different. Examples of isotopes that may be bound to the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, each of which is ² H, ³ H, ¹³ C, ¹⁴ C , ¹⁵ N, ¹⁸ 0, ¹⁷ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. Compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds and the prodrugs that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, such as those in which radioisotopes such as H and C are bound, are useful in drug and / or substrate tissue distribution analysis. Tritium, i.e. ³ H and carbon-14, i.e. ¹⁴ C isotopes, is particularly preferred because of its ease of preparation and detectability. Furthermore, substitution with heavier isotopes such as deuterium, i.e. ² H, may also have certain therapeutic advantages due to greater metabolic stability, for example increased in vivo half-life or reduced dosage required, and therefore in some circumstances It may be desirable. Isotopically-labelled compounds and prodrugs thereof of the present invention can generally be prepared by carrying out known or mentioned procedures and substituting non-isotopically labeled reagents with readily available isotopically-labeled reagents.

"IL-2" or "Interleukin-2" refers to lymphocytes produced by normal peripheral blood lymphocytes and present in low concentrations in the body. IL-2 was first described by Morgan et al. (Science 193: 1007-1008, 1976) and was originally called a T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. It is reportedly a protein with a molecular weight ranging from 13,000 to 17,000 (Gillis and Watson (1980) J. Exp. Med. 159: 1709) and has an isoelectric point ranging from 6 to 8.5. For the purposes of the present invention, the term "IL-2" includes any source of IL-2, including, for example, mammalian sources such as horses, mice, rabbits, primates, pigs, pocket pets, and humans. It is intended to be native and may be obtained by recombinant techniques, such as recombinant IL-2 polypeptides produced by microbial hosts. IL-2 may be a native polypeptide sequence or may be a variant of a native IL-2 polypeptide as described below, provided that variant IL-2 polypeptides possess the corresponding IL-2 biological activity as defined herein. Preferably, the IL-2 polypeptide or variant thereof is derived from a human source and recombinantly produced human IL-2, such as a recombinant human IL-2 polypeptide produced by a microbial host, and that possesses the corresponding IL-2 biological activity. Variants of. In the practice of the present invention, any pharmaceutical composition comprising IL-2 as a therapeutically active ingredient can be used.

Anticancer drugs:

The composition of the present invention can be administered with other anticancer agents. In particular, the compositions may be formulated together as a combination therapeutic or administered separately. Anticancer agents for use with the present invention include, but are not limited to, one or more of those set forth below:

A. Kinase Inhibitors

, US 5,747,498 및 WO 96/30347), 및 라파티닙(US 6,727,256 및 WO 02/02552); 혈관내피 성장인자 수용체(VEGFR) 키나제 억제제, 예를 들어 SU-11248(WO 01/60814), SU 5416(US 5,883,113 및 WO 99/61422), SU 6668(US 5,883,113 및 WO 99/61422), CHIR-258(US 6,605,617 및 US 6,774,237), 바탈라닙 또는 PTK-787(US 6,258,812), VEGF-Trap(WO 02/57423), B43-제니스테인(WO-09606116), 펜레티니드(레티노산 p-히드록시페닐아민)(US 4,323,581), IM-862(WO 02/62826), 베바시쥬맵 또는 Avastin

(WO 94/10202), KRN-951, 3-[5-(메틸술포닐피페라딘메틸)인돌릴]-퀴놀론, AG-13736 및 AG-13925, 피롤로[2,1f][1,2,4]트리아진, ZK-304709, Veglin

, VMDA-3601, EG-004, CEP-701(US 5,621,100), Cand5(WO 04/09769); Erb2 티로신 키나제 억제제, 예를 들어 퍼투쥬맵(WO 01/00245), 트라스투쥬맵 및 리툭시맵; AKT 단백질 키나제 억제제, 예를 들어 RX-0201; 단백질 키나제 C(PKC) 억제제, 예를 들어 LY-317615(WO 95/17182) 및 페리포신(US 2003171303); 포스포이노시티드 3-키나제(PI3K) 억제제, 예를 들어 SF-1126 및 PI-103, PI-509, PI-516 및 PI-540(PIramed에 의해서 제조); Raf/Map/MEK/Ras 키나제 억제제, 예를 들어 소라페니브(BAY43-9006), ARQ-350RP, LErafAON, BMS-354825, AMG-548, 및 WO 03/82272에 개시된 다른 것들; 섬유모세포 성장인자 수용체(FGFR) 키나제 억제제; 세포의존성 키나제(CDK) 억제제, 예를 들어 CYC-202 또는 로스코비틴(WO 97/20842 및 WO 99/02162); 혈소판-유래 성장인자 수용체(PGFR) 키나제 억제제, 예를 들어 CHIR-258, 3G3 mAb, AG-13736, SU-11248 및 SU6668; 그리고 Bcr-Abl 키나제 억제제 및 융합 단백질, 예를 들어 STI-571 또는 Gleevec

을 포함한다.Kinase inhibitors used as anticancer agents with the compositions of the present invention include epidermal growth factor receptor (EGFR) kinase inhibitors such as small molecule quinazolines, such as zephytinib (US 5457105, US 5616582 and US 5770599), ZD-6474. (WO 01/32651), erlotinib (Tarceva

, US 5,747,498 and WO 96/30347), and lapatinib (US 6,727,256 and WO 02/02552); Vascular endothelial growth factor receptor (VEGFR) kinase inhibitors, for example SU-11248 (WO 01/60814), SU 5416 (US 5,883,113 and WO 99/61422), SU 6668 (US 5,883,113 and WO 99/61422), CHIR- 258 (US 6,605,617 and US 6,774,237), batalanib or PTK-787 (US 6,258,812), VEGF-Trap (WO 02/57423), B43-Geninistein (WO-09606116), fenretinide (retinoic acid p-hydroxy Phenylamine) (US 4,323,581), IM-862 (WO 02/62826), bevacizumab or Avastin

(WO 94/10202), KRN-951, 3- [5- (methylsulfonylpiperadinemethyl) indolyl] -quinolone, AG-13736 and AG-13925, pyrrolo [2,1f] [1,2, 4] triazine, ZK-304709, Veglin

VMDA-3601, EG-004, CEP-701 (US 5,621, 100), Cand 5 (WO 04/09769); Erb2 tyrosine kinase inhibitors such as pertuzumab (WO 01/00245), trastuzumab and rituximab; AKT protein kinase inhibitors such as RX-0201; Protein kinase C (PKC) inhibitors such as LY-317615 (WO 95/17182) and perifosine (US 2003171303); Phosphoinositide 3-kinase (PI3K) inhibitors such as SF-1126 and PI-103, PI-509, PI-516 and PI-540 (manufactured by PIramed); Raf / Map / MEK / Ras kinase inhibitors such as sorafenib (BAY43-9006), ARQ-350RP, LErafAON, BMS-354825, AMG-548, and others disclosed in WO 03/82272; Fibroblast growth factor receptor (FGFR) kinase inhibitors; Cell dependent kinase (CDK) inhibitors such as CYC-202 or roscovitine (WO 97/20842 and WO 99/02162); Platelet-derived growth factor receptor (PGFR) kinase inhibitors such as CHIR-258, 3G3 mAb, AG-13736, SU-11248 and SU6668; And Bcr-Abl kinase inhibitors and fusion proteins such as STI-571 or Gleevec

It includes.

B. Anti-estrogens

또는 아나스트로졸; 에스트로겐 수용체 하향조절제(ERD), 예를 들어 Faslodex

또는 풀베스트란트를 포함한다.Estrogen-targeting agents used in anticancer therapy with the compositions of the present invention include selective estrogen receptor modulators (SERMs) such as tamoxifen, toremifene, raloxifene; Aromatase inhibitors, for example Arimidex

Or anastrozole; Estrogen Receptor Downregulators (ERDs), for example Faslodex

Or fulvestrant.

C. Anti-Androgens

Androgen-targeting agents used in anticancer therapy with the compositions of the present invention include flutamide, bicalutamide, finasteride, aminoglutetamide, ketoconazole, and corticosteroids.

D. Other Inhibitors

(US 5,780,454), XL-784; 및 시클로옥시게나제 2(COX-2) 억제제, 예를 들어 비-스테로이드계 항염제 I(NSAID)를 포함한다. Other inhibitors used as anticancer agents in combination with the compositions of the present invention include protein panesyl transferase inhibitors, such as Tipifarnib or R-115777 (US 2003134846 and WO 97/21701), BMS-214662, AZD-3409, and FTI. -277; Isomerase inhibitors such as mebarone and diflomotecan (BN-80915); Mitotic kinesin spindle protein (KSP) inhibitors such as SB-743921 and MKI-833; Protease modulators, for example botezomib or Velcade

(US 5,780,454), XL-784; And cyclooxygenase 2 (COX-2) inhibitors, such as non-steroidal anti-inflammatory agents I (NSAIDs).

E. Cancer Chemotherapy

), 비칼루타미드(Casodex

), 블레오마이신술페이트(Blenoxane

), 부술판(Myleran

), 부술판주사액(Busulfex

), N4-펜톡시카르보닐-5-데옥시-5-플루오로시티딘, 카보플라틴(Paraplatin

), 카뮤스틴(BiCNU

), 카페시타빈(Xeloda

), 클로람부실(Leukeran

), 시스플라틴(Platinol

), 클라드리빈(Leustatin

), 시클로포스파미드(Cytoxan

또는 Neosar

), 시타라빈, 시토신아라비노시드(Cytosar-U

), 시타라빈 리포솜주사액(DepoCyt

), 다카바진(DTIC-Dome

), 닥티노마이신(액티노마이신D, 코스메간), 다우노루비신염산염(Cerubidine

), 다우노루비신시트레이트 리포솜주사액(DaunoXome

), 덱사메타손, 독소루비신염산염(Adriamycin

, Rubex

), 도세탁셀(Taxotere

, US 2004073044), 에토포시드(Vepesid

), 플루다라빈포스페이트(Fludara

), 플루타미드(Eulexin

), 5-플루오로우라실(Adrucil

, Efudex

), 테자시티빈, 젬시타빈(디플루오로데옥시시티딘), 히드록시유레아(Hydrea

), 이다루비신(Idamycin

), 이포스파미드(IFEX

), 이리노테칸(Camptosar

), L-아스파라기나제(ELSPAR

), 멜팔란(Alkeran

), 류코보린칼슘, 6-메르캅토퓨린(Purinethol

), 미톡산트론(Novantrone

), 메토트렉세이트(Folex

), 미로타그, 파클리탁셀(Taxol

), 포에닉스(이트륨90/MX-DTPA), 펜토스타틴, 카뮤스틴 임플란트 폴리페프로산 20(Gliadel

), 테니포시드(Vumon

), 타목시펜시트레이트(Nolvadex

), 티라파자민(Tirazone

), 6-티오구아닌, 티오테파, 빈블라스틴(Velban

), 토포테칸염산염 주사액(Hycamptin

), 빈크리스틴(Oncovin

), 및 비노렐빈(Navelbine

)을 포함한다.Certain cancer chemotherapeutic agents used as anticancer agents with the compositions of the present invention include: Anastrozole (Arimidex

), Bicalutamide (Casodex

), Bleomycin sulfate (Blenoxane

), Smashpan (Myleran

), Busulex injection

), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin

), Camustine (BiCNU)

), Capecitabine (Xeloda)

), Chlorambucil (Leukeran

), Cisplatin (Platinol)

), Cladribine (Leustatin

), Cyclophosphamide (Cytoxan

Or Neosar

), Cytarabine, cytosine arabinoside (Cytosar-U

), Cytarabine liposome injection (DepoCyt)

), Takabazine (DTIC-Dome

), Dactinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride (Cerubidine

Daunorubicin citrate liposome injection (DaunoXome)

), Dexamethasone, doxorubicin hydrochloride (Adriamycin

, Rubex

), Docetaxel (Taxotere

, US 2004073044), etoposide (Vepesid

Fludarabine phosphate

), Flutamide (Eulexin

), 5-fluorouracil (Adrucil

, Efudex

), Tezacitin, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea)

), Idarubicin (Idamycin

), Phosphamide (IFEX

), Irinotecan (Camptosar)

), L-asparaginase (ELSPAR

, Melphalan (Alkeran)

), Leucovorin calcium, 6-mercaptopurine (Purinethol

), Mitoxantrone

), Methotrexate (Folex

), Mirotag, paclitaxel (Taxol

), Phonics (yttrium 90 / MX-DTPA), pentostatin, chamustine implant polypeproic acid 20 (Gliadel)

), Teniposide (Vumon

Tamoxifen Citrate (Nolvadex)

), Tirazone (Tirazone)

), 6-thioguanine, thiotepa, vinblastine (Velban

, Topotecan Hydrochloride Injection (Hycamptin)

), Vincristine (Oncovin)

), And vinorelbine (Navelbine

).

F. Alkylating Agent

); 테모졸로마이드; 트라벡테딘(US 5,478,932); AP-5280 (시스플라틴 플라티네이트 제제); 포피로마이신; 및 클레아라지드(메클로레타민)을 포함한다.Alkylating agents used with the compositions of the present invention as anticancer agents include VNP-40101M or chloretizin, oxaliplatin (US 4,169,846, WO 03/24978 and WO 03/04505), glufosamide, maphosphamide, etopo Force (US 5,041,424), prednisostin; Treosulfan; Busulfan; Irofluben (acylpulben); Fenclomedine; Pyrazoloacridine (PD115934); O6-benzylguanine; Decitabine (5-aza-2-deoxycytidine); Brostallysin; Mitomycin C (Mito Extra); TLK-286 (Telcyta

); Temozolomide; Trabectedin (US 5,478,932); AP-5280 (cisplatin platinum formulation); Porphyromycin; And clairazide (mechloretamine).

 G. Chelating Agents

); 데페록사민; 및 일렉트로포레이션(EPT)와 선택적으로 조합되는 블레오마이신을 포함한다.Chelating agents used with the compositions of the present invention as anticancer agents include tetrathiomolybdate (WO 01/60814); RP-697; Chimeric T84.66 (cT84.66); Gadophossetet (Vasovist

); Deferoxamine; And bleomycin optionally in combination with electroporation (EPT).

H. Biological Response Modifiers

); SU 101 또는 레플루노마이드(WO 04/06834 및 US 6,331,555); 이미다조퀴놀린류, 예를 들어 레시퀴모드 및 이미퀴모드(US 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, 및 5,525,612); 및 SMIP류, 예를 들어 벤자졸류, 안트라퀴논류, 티오세미카르바존류, 및 트립탄트린류(WO 04/87153, WO 04/64759, 및 WO 04/ 60308).Biological response modifiers, such as immunomodulators, used in combination with the compositions of the present invention as anticancer agents include: staurosphrine and its macrocyclic analogues such as UCN-01, CEP-701 and midostauline (see : WO 02/30941, WO 97/07081, WO 89/07105, US 5,621,100, WO 93/07153, WO 01/04125, WO 02/30941, WO 93/08809, WO 94/06799, WO 00/27422, WO 96/13506, WO 88/07045); Squalane (WO 01/79255); DA-9601 (WO 98/04541 and US 6,025,387); Alemtuzumab; Interferons (eg, IFN-a, IFN-b, etc.); Interleukins, specifically IL-2 or Aldesleukin, as well as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12, and active biological variants thereof having an amino acid sequence of at least 70% of the native human sequence; Altretamine (Hexalen

); SU 101 or leflunomide (WO 04/06834 and US 6,331,555); Imidazoquinolines such as resiquimod and imiquimod (US 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, 5,238,944612) and 5,525 And SMIPs such as benzazoles, anthraquinones, thiosemicarbazones, and tryptanthrins (WO 04/87153, WO 04/64759, and WO 04/60308).

I. Cancer vaccine

(1974, Tetrahedron Letters 26, 2269-70); 오레고보맵(OvaRex

); Theratope

(STn-KLH]); 흑색종 백신류; GI-4000 시리즈(GI-4014, GI-4015, 및 GI-4016), 이것들은 Ras 단백질에 있는 다섯 돌연변이에 관한 것이다; GlioVax-1; MelaVax; Advexin

또는 INGN-201 (WO 95/12660); Sig/E7/LAMP-1, HPV-16 E7 암호화; MAGE-3 백신 또는 M3TK(WO 94/ 05304); HER-2VAX; ACTIVE, 이것은 종양 특이적 T 세포를 자극한다; GM-CSF 암백신; 및 리스테리아 모노시토게네스-기반 백신을 포함한다.The anticancer vaccine used with the composition of the present invention is Avicine

(1974, Tetrahedron Letters 26, 2269-70); Oregovo map (OvaRex

); Theratope

(STn-KLH]); Melanoma vaccines; GI-4000 series (GI-4014, GI-4015, and GI-4016), these relate to five mutations in the Ras protein; GlioVax-1; MelaVax; Advexin

Or INGN-201 (WO 95/12660); Sig / E7 / LAMP-1, HPV-16 E7 Encryption; MAGE-3 vaccine or M3TK (WO 94/05304); HER-2VAX; ACTIVE, which stimulates tumor specific T cells; GM-CSF cancer vaccine; And Listeria monocytogenes-based vaccines.

J. Antisense Therapy

); JFS2; 아프리노카센(WO 97/29780); GTI-2040(R2 리보뉴클레오티드 환원효소 mRNA 안티센스 올리고)(WO 98/05769); GTI-2501(WO 98/05769); 리포솜-캡슐화 c-Raf 안티센스 올리고데옥시뉴클레오티드류(LErafAON)(WO 98/43095); 및 Sirna-027(VEGFR-I mRNA을 표적으로 하는 RNAi-기반 치료제)를 포함한다.Anticancer agents for use with the compositions of the present invention also include antisense compositions such as AEG-35156 (GEM-640); AP-12009 and AP-11014 (TGF-beta2-specific antisense oligonucleotides); AVI-4126; AVI-4557; AVI-4472; Oblimesen

); JFS2; Aprinocesen (WO 97/29780); GTI-2040 (R2 ribonucleotide reductase mRNA antisense oligo) (WO 98/05769); GTI-2501 (WO 98/05769); Liposome-encapsulated c-Raf antisense oligodeoxynucleotides (LErafAON) (WO 98/43095); And Sirna-027 (RNAi-based therapeutics targeting VEGFR-I mRNA).

K. IL-2

"이며, 캘리포니아 에머리빌 소재 카이론 코포레이션에서 제조된다. 이 제제에서 IL-2는 재조합적으로 생산되며, 초기 알라닌 잔기가 제거되고, 125번 위치의 시스테인 잔기가 세린 잔기로 치환되었다는 점에서 네이티브 사람 IL-2 아미노산 서열과는 다른 글리코실화되지 않은 사람 IL-2 뮤테인이다(데스-알라닐-1, 세린-125 사람 인터류킨-2라고 한다). 이 IL-2 뮤테인은 E. coli에서 발현된 다음, 정용여과 및 양이온 교환 크로마토그래피에 의해 정제될 수 있으며, 이에 대해서는 미국특허 제4,931,543호에 설명된다.One preferred IL-2 compound is "Aldesleukin" or "Proleukin".

And manufactured at Kyron Corporation, Emeryville, California. In this formulation, IL-2 is produced recombinantly, native human IL in that the initial alanine residue was removed and the cysteine residue at position 125 was replaced with a serine residue. Is a non-glycosylated human IL-2 mutein other than the -2 amino acid sequence (referred to as des-alanyl-1, serine-125 human interleukin-2), which is expressed in E. coli It can then be purified by diafiltration and cation exchange chromatography, which is described in US Pat. No. 4,931,543.

Aldes-leukin was compared in vitro with native (Jurkat) IL-2. There was no significant difference, and the serum half-life after in vivo introduction and intravenous administration of cytolytic cells to mice is equivalent to that of Aldeleukin and native (Jurkat) IL-2. Nevertheless, since the form of IL-2 contained in Aldeleukin has advantageously contributed, "Aldeleukin" does not include all possible forms of IL-2 protein, but only the composition thereof.

There was no official IL-2 reference preparation available until 1983 when Cetus' lymphocyte proliferation biological assay was developed. One cetus unit for this preparation was defined as the amount of IL-2 in 1 mL that induced IL-2 dependent murine T cells capable of binding tritium-thymidine to 50% of the maximum level after 24 hours of incubation. . This IL-2 containing product was assigned a specific activity of 3 x 10 ⁶ cetus units per mg. In 1988, the National Institute of Biopharmaceutical Standardization, a WHO-based biopharmaceutical standardization laboratory, established the international standard for IL-2. Also, in 1988, the analysis process of Setus lymphocyte proliferation biological assay was changed. In this new biological assay, Setus's in-house standard was tested against the international standard for IL-2. The following correlation coefficients have been established:

International Units = Setus Units x 6

The amount of aldesleukin will be determined in mass units based on 1 mg of aldesleukin with nominal inactivation of 18 x 10 ⁶ international units (18 MIU). This protocol is integrated with international units.

Pharmaceutical compositions useful in the methods of the invention may include biologically active variants of IL-2. Such variants must retain the desired biological activity of the native polypeptide such that when a pharmaceutical composition comprising a variant polypeptide is administered to a subject, it has the same therapeutic effect as a pharmaceutical composition comprising the native polypeptide. In other words, variant polypeptides will act as therapeutically active ingredients in pharmaceutical compositions in a manner similar to that observed for native polypeptides. There are methods available in the art for determining whether a variant polypeptide maintains its desired biological activity, thereby acting as a therapeutically active ingredient in a pharmaceutical composition. Biological activity can be measured using assays specifically designed for measuring the activity of a native polypeptide or protein, including the assays described herein. In addition, antibodies raised against biologically active native polypeptides can be tested for their ability to bind variant polypeptides, where effective binding is an indication of a polypeptide having a conformation similar to a native polypeptide having.

For the purposes of the present invention, the IL-2 biological activity of interest activates natural cytotoxic (NK) cells to mediate lymphokine-activated cytotoxic (LAK) activity and cytotoxicity (ADCC) of antibody-dependent cells. And / or the ability of IL-2 to expand. Thus, IL-2 variants (eg, muteins of human IL-2) used in the methods of the invention will activate and / or expand NK cells to mediate LAK activity and ADCC. Assays to determine IL-2 activation or expansion of NK cells and the mediation of LAC 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 this polypeptide. "Fragment" means a polypeptide consisting solely of parts of an intact polypeptide sequence and structure, and may be a C-terminal deletion or an N-terminal deletion of a native polypeptide. An “analog” means an analog of a native polypeptide or native polypeptide fragment, wherein the analog includes a native polypeptide sequence and structure having one or more amino acid substitutions, insertions, or deletions. Peptides having “muteins” and one or more peptoids (peptide mimetics) such as those described herein are also included in the term analogues (see WO 91/04282). A "derivative" is any suitable modification of a native polypeptide, a fragment of a naked polypeptide, or an analog thereof, such as glycosylation, phosphorylation, polymer conjugation (eg with polyethylene glycol), or other foreign moiety. Means the addition of these compounds, provided that the desired biological activity of the native polypeptide is retained. Methods of making polypeptide fragments, analogs and derivatives are generally available in the art.

For example, amino acid sequence variants of a polypeptide can be prepared by mutagenesis of the cloned DNA sequence encoding the native polypeptide. Mutagenesis and nucleotide sequencing methods are well known in the art. See, eg, Walker and Gaastra, "Molecular Biology" (MacMillan Publishing Company, New York, 1983); Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); Kunkel et al., Methods Enzymol. 154: 367-382 (1987); Sambrook et al., “Molecular Cloning: Laboratory Manual” (Cold Spring Harbor Laboratory Press, Plainview, New York, 1989); US Patent No. 4,873, 192; And the references cited herein, which are incorporated herein by reference. Guidance on suitable amino acid substitutions that do not affect the biological activity of the polypeptide in question can be found in Dayhoff et al., "Illustrations of Protein Sequences and Structures" (Natl. Biomed. Res. Found., Washington, 1978), incorporated herein by reference. ). Conservative substitutions may be desirable, such as when one amino acid is exchanged for another amino acid of similar nature. Examples of conservative substitutions include, but are not limited to, Gly: Ala, Val: Ile: Leu, Asp: Glu, Lys: Arg, Asn: Gln, and Phe: Trp: Tyr.

In constructing variants of the IL-2 polypeptide of interest, modifications are made such that the variants continue to possess the desired activity. All mutations made in the DNA encoding the variant polypeptide should not be positioned outside the reading frame, and preferably do not create a complementarity region capable of producing a secondary mRNA structure.

The biologically active variant of IL-2 is generally at least 70%, preferably at least 80%, more preferably at least about 90% to 95%, and most preferably relative to the amino acid sequence of the reference polypeptide molecule to which the reference is being compared. Will have at least about 98% amino acid sequence identity. 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% to 95% of the amino acid sequence of human IL-2. Will have at least%, and most preferably at least about 98% sequence identity. Biologically active variants of the native polypeptide may be on the order of 1-15 amino acids, 1-10 amino acids, for example 6-10, 5, 4, 3, 2, or even 1 amino acid residues. May differ from the native polypeptide. "Sequence identity" means that the same amino acid residue is found in the reference polypeptide molecule and the variant polypeptide when certain consecutive segments of the amino acid sequence of the variant are aligned and compared to the amino acid sequence of the reference molecule. Percent sequence identity between two amino acid sequences measures the number of positions where identical amino acid residues are found in two sequences, yielding the number of matched positions, and dividing the number of matched positions by the total number of positions in the segment compared to the reference molecule. The result is then multiplied by 100 to yield the percent sequence identity.

Thus, determination of percent identity between any two sequences can be accomplished using mathematical algorithms. Preferably, the naturally occurring or non-naturally occurring IL-2 variant is at least 70%, preferably 80%, with the amino acid sequence of the reference molecule, eg native human IL-2, or a shorter portion of the reference IL-2 molecule. , More preferably 85%, 90%, 91%, 92%, 93%, 94% or 95% identical amino acid sequences. More preferably, this molecule is 96%, 97%, 98% or 99% identical. Percent sequence identity is determined using the Smith-Waterman homology investigation algorithm, using the gap gap search, gap open penalty 12, gap extension penalty 2, BLOSUM matrix 62. Smith-Waterman homology investigation algorithms are described in Smith and Waterman, Adv. Appl. Math. (1981) 2: 482-489. Variants may vary, for example, from 1 to 10 amino acid residues, for example 6-10 amino acid residues, 5, 4, 3, 2 amino acid residues or even 1 amino acid residue.

With regard to optimal alignment of two amino acid sequences, contiguous segments of variant amino acid sequences may have added amino acid residues or deleted amino acid residues relative to the reference amino acid sequence. Consecutive segments used for comparison with a reference amino acid sequence will include at least 20 contiguous amino acid residues and may be 30, 40, 50, or more amino acid residues. Correction of sequence identity associated with conservative residue substitutions or gaps can be made (see Smith-Waterman homology investigation algorithm).

The exact chemical structure of a polypeptide having IL-2 activity depends on many 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 biological activity when subjected to suitable environmental conditions are included in the definition of polypeptide having IL-2 activity as used herein.

Furthermore, the primary amino acid sequence of a polypeptide can be expanded by derivatization (glycosylation) with sugar moieties or by other supplemental molecules such as lipids, phosphates, acetyl groups and the like. Conjugation with sugars can also be expanded. Some aspect of this expansion is achieved through the post-translational processing system of the generating subject. Other such modifications can be introduced in vitro. In any case, such modifications are included in the definition of an IL-2 polypeptide as used herein, provided that the IL-2 activity of the polypeptide is not disrupted. It is anticipated that such modifications may affect the activity quantitatively or qualitatively by enhancing or decreasing the activity of the polypeptide in various assays. Furthermore, each amino acid residue in the chain can be modified by oxidation, reduction, or other derivatization, and the polypeptide can be cleaved to obtain fragments that retain activity.

Such alterations that do not destroy activity do not delete this polypeptide sequence from the definition of the corresponding IL-2 polypeptide as used herein.

The art provides practical guidance regarding the preparation and use of polypeptide variants. In the preparation of IL-2 variants, one of skill in the art can readily determine whether modifications to native protein nucleotide or amino acid sequences will result in variants suitable for use as therapeutically active ingredients of pharmaceutical compositions used in the methods of the present invention.

The IL-2 or variant thereof used in the methods of the invention may be from any source, but is preferably recombinant IL-2. "Recombinant IL-2" is prepared by recombinant DNA techniques described in Taniguchi et al. (1983) Nature 302: 305-310 and Devos (1983) Nucleic Acids Research 11: 4307-4323, or Wang et al. (1984) Science 224 Mutant described by: 1431-1433 means interleukin-2 with biological activity similar to the altered native-sequence IL-2. Generally, the gene encoding IL-2 is cloned and then expressed in the transformed organism, preferably the microorganism, most preferably E. coli , as described herein. The host organism expresses a foreign gene that produces IL-2 under expression conditions. Synthetic recombinant IL-2 can also be produced in eukaryotes such as yeast or human cells. Growth, collection, rupture, or extraction processes of IL-2 from cells are described, for example, in US Pat. No. 4,604,377; 4,738,927; 4,738,927; No. 4,656,132; No. 4,569,790; 4,748,234; 4,748,234; 4,530,787; 4,530,787; 4,572,798; 4,572,798; 4, 748,234; And 4,931,543.

As an example of variant IL-2 protein, 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; Cys1O5 to Ser1O5 or Ala1O5; Cys125 to Ser125 or Ala125; deletion of all residues after Arg120; and their Met-1 forms), and the recombinant IL-2 mutein Described in European Patent Application No. 83306221.9 filed March 13 (published EP 109,748, published May 30, 1984), which is Belgian Patent 893,016 and co-owned US Patent No. 4,518,584 (this is a native human IL- And recombinant human IL-2 muteins deleted from the cysteine at position 125 numbered according to 2 or neutral amino acid substituted; alanyl-ser125-IL-2; and desalanyl-ser125-IL-2); It is the same patent. In addition, U. S. Patent No. 4,752, 585, which discloses the following variant IL-2 proteins:

And US Pat. No. 4,931,543, which discloses IL-2 mutein des-alanyl-1, serine 30 125 human IL-2 used in the examples herein, as well as the remaining IL-2 muteins.

European Patent Publication EP 200,280 (published December 10, 1986) also discloses a recombinant IL-2 mutein, wherein methionine at position 104 has been replaced with a conservative amino acid.

Examples include the following muteins:

See also EP 118,617 and US Pat. No. 5,700,913, which are non-glycosylated human IL-2 variants with alanine instead of methionine of native IL-2 as the N-terminal amino acid; Unglycosylated human IL-2 in which the starting methionine is deleted so that proline becomes the N-terminal amino acid; And unglycosylated human IL-2 with an alanine inserted between the N-terminal methionine and proline amino acids.

Other IL-2 muteins include those disclosed in WO 99/60128 (aspartate at position 20 replaced with histidine or isoleucine, asparagine at position 88 replaced with arginine, glycine or isoleucine, or 126 The glutamine in position is replaced by leucine or glutamic acid), these reportedly show selective activity and reduced IL on high-affinity IL-2 receptors expressed by cells expressing T cell receptors in preference to NK cells -2 has toxicity; Muteins disclosed in US Pat. No. 5,229,109 (arginine at position 38 replaced with alanine or phenylalanine at position 42 replaced with lysine), while maintaining the ability to stimulate LAK cells while maintaining native IL Reduced binding to high-affinity IL-2 receptor when compared to -2; Muteins disclosed in International Publication WO 00/58456 (change or deletion of naturally occurring (x) D (y) in native IL-2, where D is aspartic acid, (x) is leucine, isoleucine, glycine or valine, and (y) is valine, leucine or serine), these are claimed to reduce vascular leakage syndrome; IL-2 pl-30 peptide disclosed in International Publication WO 00/04048 (corresponds to the first 30 amino acids of IL-2, which contains the entire a-helix A of IL-2, and the b chain of IL-2 receptor Interactions), which, according to records, stimulates NK cell and LAK cell induction; And a mutant form of the IL-2 pl-30 peptide, also disclosed in WO 00/04048, wherein aspartic acid at position 20 is substituted with lysine, which is reported to not produce vascular hemorrhage but can produce LAK cells To maintain; In addition, IL-2 can be modified with polyethylene glycol to enhance solubility and alter pharmacokinetic profiles (see US Pat. No. 4,766,106).

The term IL-2, as used herein, also refers to IL-2 fused with a second protein or covalently conjugated with polyproline or a water soluble polymer for the purpose of reducing the frequency of administration or improving IL-2 tolerance. It is meant to include an IL-2 fusion or conjugate comprising a. For example, IL-2 (or a variant thereof as defined herein) can be fused with human albumin or albumin fragments using methods known in the art (see WO 01/79258). Alternatively, IL-2 may be covalently conjugated with polyproline or polyethyleneglycol homopolymers and polyoxyethylated polyols using methods known in the art, wherein the homopolymers are unsubstituted or It may be substituted at the end with an alkyl group and the polyol is unsubstituted (see, eg, US Pat. Nos. 4,766,106, 5,206,344, and 4,894,226). 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, US Pat. Nos. 4,745, 180; No. 4,766,106; 4,816,440; 4,816,440; 4,894,226; 4,931,544; And 5,07 8,997. Thus, liquid, lyophilized, or spray dried compositions comprising IL-2 or variants thereof known in the art can be prepared as aqueous or non-aqueous solutions or suspensions for continuous administration to a subject in accordance with the present invention. . Each of these compositions will include IL-2 or a variant thereof as a therapeutic or prophylactic active ingredient. A "therapeutically or prophylactically active ingredient" is specifically included in a composition such that IL, when administered to a subject, results in a desired therapeutic or prophylactic response associated with the treatment, prevention, or diagnosis of a disease or condition in the subject. -2 or a variant thereof. Preferably, the pharmaceutical composition comprises a stabilizer, bulking agent, or both suitable for the purpose of minimizing problems associated with loss of protein stability and biological activity during manufacture and storage. In a preferred embodiment of the invention, an IL-2 containing pharmaceutical composition useful in the method of the invention comprises a composition comprising a stabilized monomer IL-2 or a variant thereof, a composition comprising a stabilized multimer IL-2 or a variant thereof, And stabilized lyophilized or spray dried IL-2 or variants thereof.

Pharmaceutical compositions comprising stabilized monomers IL-2 or variants thereof are disclosed in International Publication WO 01/24814, entitled “Stabilized Liquid Polypeptide-Containing Pharmaceutical Compositions”. "Monomer" IL-2 means that in the pharmaceutical compositions described herein, the protein molecules are substantially in monomeric form and are not in aggregated form. Thus, no covalent or hydrophobic oligomers or aggregates of IL-2 are present. In brief, IL-2 in these liquid compositions is formulated with an amount of amino acid base sufficient to reduce aggregate formation of IL-2 during storage. Amino acid bases are amino acids or combinations of amino acids, wherein any given amino acid is present in free base form or in salt form. Preferred amino acids are selected from the group consisting of arginine, lysine, aspartic acid, and glutamic acid. These compositions further comprise a buffer for the purpose of maintaining the pH of the liquid composition within the tolerance of the stability of IL-2, wherein the buffer is an acid substantially free of salt form, an acid in salt form, or a mixture of acid and salt form. . 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 pharmaceutical compositions. The amino acid base in these compositions acts to stabilize IL-2 against aggregate formation during storage of the liquid pharmaceutical composition, and the use of an acid substantially free of salt form, acid in salt form, or mixture of acid and salt form as a buffer It makes the osmolarity of the liquid composition nearly isotonic. The liquid pharmaceutical composition may further comprise other stabilizers, more specifically methionine, nonionic surfactants such as polysorbate 80, and EDTA for the purpose of further increasing the stability of the polypeptide. Such liquid pharmaceutical compositions may be prepared in comparison with liquid pharmaceutical compositions prepared without a combination of these two components due to the addition of an acid substantially free of salt form, an acid in salt form, or an amino acid base combined with a mixture of acid and salt form. It is said to be stable because of increased storage stability. These liquid pharmaceutical compositions comprising stabilized monomers IL-2 are suitable for use in aqueous liquid form, or stored frozen for later use, or for later administration to a subject in liquid form or in accordance with the methods of the present invention. It may be in a dried form for restoring to another form. “Dried form” means that a liquid pharmaceutical composition or formulation is lyophilized (ie, lyophilized, eg see Williams and Polli, (1984) J. Parenteral Sci. Technol. 38: 48-59), spray drying (Masters, (1991) "Spray Dry Handbook", Sth ed., Longman Scientific and Technical, British Ezez, pp. 491-676; Broadhead et al., (1992) Drug Devel.Ind. Pharm. 18: 1169-1206; and Mumenthaler et al., (1994) Pharm. Res. 11: 12-20, or air drying (Carpenter and Crowe, (1988) "cold biology", 25: 459-470; and Roser, (1991) Biopharm. 4: 47-53). Means that.

Other examples of IL-2 preparations comprising IL-2 in non-aggregated monomeric state include those described in Whittington and Faulds (1993) "Drugs", 46 (3): 446-514. These formulations comprise a recombinant IL-2 product, wherein the recombinant IL-2 mutein teseryukkin (unglycosylated human IL-2 with methionine residues added at the amino-terminus) is 0.25% human serum albumin and Lyophilized powder, restored to isotonic saline, recombinant IL-2 mutein bioleukin (methionine residue added at amino-terminus and cysteine residue at position 125 of human IL-2 sequence replaced with alanine Human IL-2) is formulated to combine 0.1-1.0 mg / ml IL-2 mutein with acid, wherein the pH of the formulation is advantageously 3.0-4.0, with no 10 buffers, and the conductivity is less than 1000 mmhos / cm ( Advantageously less than 500 mmhos / cm). EP 373,679; Xhang et al., (1996) Pharmaceut. Res. 13 (4): 643-644, and Prestrelski et al., (1995) Pharmaceut. Res. See 12 (9): 1250-1258.

IL-2(카이론 코포레이션, 캘리포니아 에머리빌)로 상업적으로 입수가능하다. 이와 관련하여 개시된 동결건조 제제는 선택적으로 산화된, 미생물에 의해 생산된 재조합 IL-2이며, 여기서 재조합 IL-2는 벌크를 제공하는 만니톨과 같은 수용성 담체 및 재조합 IL-2의 수용해도를 확보할 만큼 충분한 양의 나트륨도데실술페이트와 함께 혼합된다.Examples of pharmaceutical compositions comprising multimeric IL-2 are disclosed in co-owned US Pat. No. 4,604,377. "Multimer" means that the protein molecules are present in the pharmaceutical composition in microaggregated 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 sold under the trade name Proleukin

Commercially available as IL-2 (Cairon Corporation, Emeryville, CA). The lyophilized formulations disclosed in this regard are recombinant IL-2 produced by microorganisms, optionally oxidized, wherein the recombinant IL-2 will ensure water solubility of the recombinant IL-2 and an aqueous carrier such as mannitol to provide bulk. Mix with sufficient amount of sodium dodecyl sulfate.

These compositions are suitable for restoration in aqueous injections for parenteral administration and are stable and well tolerated in human patients. When restored IL-2 retains its multimeric state. Such lyophilized or liquid compositions comprising multimeric IL-2 are encompassed by the methods of the present invention. Such compositions are referred to herein as multimeric IL-2 pharmaceutical compositions.

In addition, the methods of the present invention may employ stabilized, lyophilized or spray dried pharmaceutical compositions comprising IL-2, which may be restored to liquid or other suitable form for administration according to the methods of the present invention. Such pharmaceutical compositions are disclosed in International Publication WO 01/49274, titled “Method of Delivering Lung Pathways of Interleukin-2”. These compositions may further comprise at least one bulking agent, at least one agent in an amount sufficient to stabilize the protein during the drying process, or both. “Stable” means that after lyophilization or spray drying to obtain a composition in solid or dry powder form, the IL-2 protein or variant thereof, as well as its monomeric or multimeric forms, as well as other important properties of quality, purity, and titer It means to hold. Preferred carrier materials used as bulking agents in these compositions include glycine, mannitol, alanine, valine, or any combination thereof, most preferably glycine. The bulking agent is present in the formulation in the range of 0% to about 10% (w / v), depending on the formulation used. Preferred carrier materials used as stabilizers include any sugars or sugar alcohols or any amino acids. Preferred sugars include sucrose, trehalose, raffinose, stachiose, sorbitol, glucose, lactose, dex or any combination thereof, preferably sucrose. If the stabilizer is sugar, it is from about 0% to about 9.0% (w / v), preferably from about 0.5% to about 5.0%, more preferably from about 1.0% to about 3.0%, most preferably from about 1.0% Exists in scope. If the stabilizer is an amino acid, it is present in the range of about 0% to about 1.0% (w / v), preferably about 0.3% to about 0.7%, most preferably about 0.5%. These stabilized, lyophilized or spray dried compositions may optionally comprise one or other chelating agents of those salts such as methionine, ethylenediaminetetraacetic acid (EDTA) or disodium EDTA, which may contain IL- for methionine oxidation. 2 or a variant thereof. The use of these agents in this manner is described in US Application 09 / 677,643, which is incorporated herein by reference. The stabilized, lyophilized or spray dried composition maintains the pH of the pharmaceutical composition at an acceptable range when in the liquid phase, preferably from about pH 4.0 to about pH 8.5, such as during preparation or after restoration of the dry form composition. It can be prepared using a buffer. The buffer is compatible with the drying process and is chosen so as not to affect the quality, purity, titer, and stability of the protein during processing and storage.

The stabilized monomers, multimers, and stabilized lyophilized or spray dried IL-2 pharmaceutical compositions described above are 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 included by the methods of the present invention.

Also provided herein are kits or packages containing at least one combination composition of the present invention with instructions for use. For example, in an example where the drugs are each administered in separate or separate formulations, the kit includes each of the drugs along with instructions for use. The drug ingredients may be packaged in any way suitable for administration, provided that the package, when considered together with the dosing instructions, should clearly indicate the manner in which each drug ingredient is administered. Alternatively, each drug component of the combination may be combined into a single administrable formulation such as a single composition.

For example, in the example of a kit comprising rIL-2 and an angiogenesis agent, the kit can be organized in any suitable time period, such as by 'day'. By way of example, for 'Daily', a representative kit can include each unit dose of rIL-2 and an angiogenesis agent. If each drug is to be administered twice daily, the kit may contain a second row of each unit dosage form of rIL-2 and antiangiogenic agent along with the dosing instructions, corresponding to the day. Alternatively, if the timing of administration of one or more of the drugs or the amount of drug to be administered differs compared to the rest of the drug members of the combination, it may be reflected in the packaging and instructions. For example, if rIL-2 is administered twice daily and the anti-angiogenic agent should only be taken once daily, the unit dosage form of rIL-2 in a typical 'daily' package would be “1 day, 1 dose”. Correspondingly, the formulation of the anti-angiogenic agent may correspond to "1 day, 2 doses".

Various embodiments according to the above can be readily envisioned, which of course will depend on the particular drug combination used in the treatment, the corresponding formulation, the recommended dosage, the intended patient population, and the like. The packaging may be in any form commonly used in pharmaceutical packaging and may utilize any of a number of features such as different colors, skins, anti-damage packaging, blister packs, desiccants and the like.

The following are examples of specific embodiments to carry out the invention. This embodiment is provided for illustrative purposes only and does not limit the scope of the invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be tolerated.

Example  One

rIL Composition for co-administration with -2

A. Compound

B. Synthesis

Quinazoline

Scheme 1 illustrates the basic method of synthesizing many substituted quinazoline compounds. AG denotes an activation group such as, for example, a halide, triflate, or ketone, and up to four AG groups can be present. Since the 4-chloro position shown in the third step is more active than positions 5-8 on the benzyl ring, the substitution with NHRR 'can proceed without multiple simultaneous substitutions of AG at any of positions 5-8. Subsequent replacement of the AG group with R ″ in the final step can then proceed in the presence of weak or intermediate bases. Alternatively, the AG group (s) can be modified to obtain the desired product, for example NO ₂ groups are selected from EtOH and Fe and AcOH in H ₂ O can be reduced to yield amino substituents, which can be further substituted, for example, by reductive amination with paraformaldehyde.

As will be apparent to those skilled in the art, excess functionalized 2-aminobenzamide starting materials are commercially available or readily synthesized by known procedures. Subsequently, the AG group (s) of the starting material may be the desired substituent (such as R ″) in the final product, for example an alkoxy group (s) or substituted alkyl group (s). Furthermore, many functionalized 2- Nitrobenzamide starting materials are available, which are readily converted to 2-aminobenzamide starting materials in the presence of a reducing agent such as H ₂ / Pd / C in EtOH.

Alternative methods of preparing the quinazolines of the present invention are described in WO 04/24703, WO 01/32651, US 5,457,105, US 5,616,582, US 5,770,599, WO 02/16351, US 6,727,256, WO 02/02552, US 5,747,498, and WO 96/30347.

Indolinone

Scheme 2a is a one-pot process and the reagents in step a are NH ₄ OH, CuCl in H ₂ O. Subsequently, aqueous HCl is added in step b. R ₂ -R ₅ is as defined herein.

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

Reagents in Scheme 2c are 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 described herein. The structure may be substituted for Formula II to allow substitution at R ₉ , where all other substituents are as defined herein.

(Synthesis of Compound 6)

Phthalazine

The preparation of substituted phthalazines, such as compound 10, is described as follows, as shown in US Pat. No. 6,258,812, which also includes other schemes that may aid in the synthesis of the compounds of the present invention.

Reaction 3a: 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine dihydrochloride

15.22 g (59.52 mmol) 1-chloro-4- (4-pyridylmethyl) phthalazine (see German Application No. 061 788 for the preparation, published July 23, 1959), 7.73 g (60.59 mmol) The mixture of 4-chloroaniline and 200 ml 1-butanol is heated at reflux for 2 hours. The mixture is slowly cooled to 5 ° C., then the crystals obtained are filtered off and washed with 1-butanol and ether. The filter residue is dissolved in about 200 ml of hot methanol, the solution is treated with 0.75 g activated carbon, filtered with Hyflo Super Cel, and the pH of the filtrate is adjusted to about 2.5 with 7 ml 3N methanolic HCl. The filtrate is evaporated to about half its original volume, ether is added until a slight turbidity is produced, and then cooled to precipitate the crystals. The crystals were filtered off, washed with a mixture of methanol / ether (1: 2) and ether, dried at 110 ° C. for 8 hours under HV, and allowed to equilibrate for 72 hours in a 20 ° C. room temperature atmosphere. In this way the title compound is obtained with a water content of 8.6%.

mp> 270 ° C .; ¹ H NMR (DMSO-d6) 11.05-12.20 (br), 9.18-9.23 (m, 1H), 8.88 (d, 2H), 8.35-8.40 (m, 1H), 8.18-8.29 (m, 2H), 8.02 (d, 2H), 7.73 (d, 2H), 7.61 (d, 2H), 5.02 (s, 2H); ESI-MS: (M + H) ⁺ = 347.

Reaction 3b: 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride

A mixture of 0.972 g (3.8 mmol) 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.656 g (4 mmol) 4-chloroaniline hydrochloride (Research Organics, Inc., Cleveland, Ohio) and 20 ml ethanol Heat at reflux for 2 hours. The reaction mixture is cooled in an ice bath and filtered, then the crystals are washed with some ethanol and ether. After drying at 110 ° C. for 8 hours and H 150 ° C. under HV, the title compound is obtained as a result of heat removal of HCl.

mp> 270 ° C .; ¹ H NMR (DMSO-d6) 9.80-11.40 (br), 8.89-8.94 (m, 1H), 8.67 (d, 2H), 8.25-8.30 (m, 1H), 8.06-8.17 (m, 2H), 7.87 (d, 2H), 7.69 (d, 2H), 7.49 (d, 2H), 4.81 (s, 2H); ESI-MS: (M + H) ⁺ = 347.

Reaction 3c: 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride

A mixture of 1.28 g (5 mmol) 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.67 g (5.25 mmol) 4-chloroaniline and 15 ml 1-butanol was stirred at 100 ° C. for 0.5 hour Heat. The mixture is cooled to room temperature and filtered, then the filtrate is washed with 1-butanol and ether. The crystals are dissolved in 40 ml hot methanol for purification, the solution is treated with activated charcoal and filtered through Hyflo Super Cel, then the filtrate is evaporated to about half its original volume to form crystalline precipitate. After cooling to 0 ° C., it is filtered, the filter residue is washed with ether and dried at 130 ° C. for 8 h under HV to give the title compound.

mp> 270 ° C .; ¹ H NMR (DMSO-d6) 9.80-11.40 (br), 8.89-8.94 (m, 1H), 8.67 (d, 2H), 8.25-8.30 (m, 1H), 8.06-8.17 (m, 2H), 7.87 (d, 2H), 7.69 (d, 2H), 7.49 (d, 2H), 4.81 (s, 2H); ESI-MS: (M + H) ⁺ = 347.

Example  2

rIL Manufacture of -2:

, 카이론)의 제조:Aldes-leukin, rIL-2 (NSC3773364) (Proleukin

Manufacture of Chiron):

의 분자량은 대략 15,600 달톤이다. 아미노산 조성 및 N-말단 서열화에 의한 분석에 의해 알데스류킨이 예상된 단백질 서열을 가진다는 것을 확인했다.Double stranded cDNA is made using the messenger RNA of the human Jurkat cell line and hybridized with the pBR322 plasmid. IL-2 gene containing clones are identified using ³² P-labeled oligonucleotide probes corresponding to the short IL-2 base sequence. The gene is inserted into the pBR322 plasmid region with convenient restriction sites. Inserting the appropriate promoter and ribosomal-binding site in front of the IL-2 gene, the resulting expression clone encodes the modified recombinant rIL-2. In vitro mutagenesis of cloned IL-2 is used to replace serine with cysteine at position 125. The resulting molecule is indistinguishable from native IL-2 in in vitro biological activity. Production strains of E. coli carrying the Aldesleukin gene are grown in fermentors. The cultures are collected to extract aldeleukin. A series of chromatographic steps is performed to purify the aldesleukin. The pH of the prepared product is adjusted to 7.2-7.8. Proleukin

Has a molecular weight of approximately 15,600 Daltons. Analysis by amino acid composition and N-terminal sequencing confirmed that the aldeleukin had the expected protein sequence.

Restoration and Dilution Process:

은 1.3mg 단백질을 함유하는 5cc 바이알에 동결건조 케이크 상태로 들어있다. Proleukin

바이알은 주사를 위해 1.2ml 주사용 멸균수(미국약전) 로 복원된다. 희석액은 과도한 거품이 생기는 것을 피하기 위해 바이알의 측면에서 흘려 넣고, 흔들지 말고 부드럽게 빙빙 돌려 내용물을 완전히 용해한다. 복원되었을 때 ml 당 1.1mg(18백만 IU) Proleukin

이 함유된다. 복원된 Proleukin

은 직접 정맥내 주사하는데 적합하거나, 또는 필요에 따라 0.1% 사람 알부민(미국약전)을 갖는 5% 덱스트로스 주사액(미국약전)을 사용, 50mL 내지 500mL로 희석될 수 있다. 희석하는 경우 사람 알부민(미국약전)은 복원된 Proleukin

을 첨가하기 전에 5% 덱스트로스 주사액(미국약전)에 첨가한다.Proleukin

5 cc vials containing 1.3 mg protein are lyophilized cakes. Proleukin

The vial is restored with 1.2 ml sterile water for injection (US Pharmacopoeia) for injection. The diluent is poured on the side of the vial to avoid excessive foaming, and do not shake but gently whirl to dissolve the contents completely. 1.1 mg (18 million IU) Proleukin per ml when restored

It contains. Restored Proleukin

Is suitable for direct intravenous injection or can be diluted to 50 mL to 500 mL using 5% dextrose injection (US Pharmacopoeia) with 0.1% human albumin (US Pharmacopoeia) as needed. When diluted, human albumin (US Pharmacopeia) was restored to Proleukin

Add to 5% dextrose injection (US Pharmacopoeia) before adding.

Example  3

Single agent rIL -2 treatment with the composition rIL -2 composition Antiangiogenic agents  Combination Therapy Used

The results of the high-dose rIL-2 are summarized in Table 6.

Many factors promote combination therapy with anti-angiogenic compositions. Significant morbidity associated with high-dose rIL-2 requires careful patient selection, and the number of potential patients who can benefit from therapies is dramatically reduced. Unfortunately, the accompanying disease is most often found in patients with the highest RCC prevalence. Single agent high-dose rIL-2 administration is expensive and limited to use in large medical institutions and requires careful patient monitoring and occasional intensive medical care. The actual effect of this is of limited use only in a small number of patients.

Low-dose rIL-2 was importantly evaluated, which is shown in Table 7.

The most commonly used subcutaneous therapies have been published by Buter et al. In Buter's literature, rIL-2 is given once a day and lasts six weeks, five days a week. During the first five days of the cycle, 18 x 10 ⁶ IU (MIU) was given once daily, and after the first two days in the next cycle the capacity was reduced to 9 MIU. Response rate and survival data may be similar to those published for high-dose intravenous mass administration of rIL-2. Buter / Sleijfer therapy was recently compared with high-dose rIL-2 in a planned randomized trial. 96 patients received randomized high-dose intravenous rIL-2 and 92 patients received subcutaneous rIL-2 (Buter / Sleijfer dose schedule). Previously, the response to high-dose rIL-2 was 20% and the response to subcutaneous rIL-2 was 10%. However, overall survival was not different from high-dose (p = 0.34). As such, catalysts for combination with anti-angiogenic compositions are present to increase efficacy and low overall patient responsiveness to low-dose therapies.

Therapies for the combination of aldehyde leukine and antiangiogenic agents were importantly evaluated and are shown in Table 8.

Treatment with rIL-2 is an 8-week treatment cycle with 6 consecutive weeks (1-42 days, Monday through Friday each week), followed by a 2 week rest period.

Example  4

rIL -2 and Small molecule  Receptor tyrosine Kinase  Inhibitor BAY 43-9006 and SU11248 Combination therapy

A. Materials and Methods

drug

, 알데스류킨/rIL-2); 18 MIU/ml(카이론 코포레이션, 캐나다 에머리빌)을 투여 전에 주사용 멸균수로 복원하고 5% 덱스트로스 중에 조제했다. 빈크리스틴(빈크리스틴 술페이트)는 Mayne Pharma Ltd(호주 멀그레이브)로부터 구입했다. CHIR-258은 4-아미노-5-플루오로-3-[5-(4-메틸피페라진-1-일)-1H-벤지미다졸-2-일]퀴놀린-2(1H)-온(카이론)이다. BAY 43-9006(소라파닙/Nexavar

)(Riedl 등, Proc. Am. Assoc. Cancer Res. 2001 42(Abs4956); Lowinger 등, Curr. Pharm. Des. 2002 8(25):2269-2278; WO 9932455) 및 SU11248(수니티닙/Sutent

)(Sun 등 J. Med. Chem. 2003 46(7):1116-1119; WO 0160814)은 공개된 과정 및 특허에 따라 직접 합성하여 정제했다. BAY 43-9006 또는 SU11248의 스톡 용액(20mM)을 DMSO 중에 제조하고, 알리쿼트로 나누어 사용하기 전까지 -20℃에 보관한다. 시험관내 분석을 위해 모든 약물은 최적 배양배지로 희석했다. 생체내 투여를 위해, BAY 43-9006은 100% PEG 400 부형제 중에 조제했고, SU11248 투약 용액은 5mM 시트레이트 버퍼 중에 제조했다. 사용된 모든 다른 화학물질은 연구등급이었다.Recombinant Human Interleukin-2

, Aldesleukin / rIL-2); 18 MIU / ml (Cairon Corporation, Emeryville, Canada) was restored to sterile water for injection prior to administration and formulated in 5% dextrose. Vincristine (Vincristine Sulfate) was purchased from Mayne Pharma Ltd (Mulgrav, Australia). CHIR-258 is 4-amino-5-fluoro-3- [5- (4-methylpiperazin-1-yl) -1H-benzimidazol-2-yl] quinolin-2 (1H) -one (kyron )to be. BAY 43-9006 (Sorapanib / Nexavar

Riedl et al., Proc. Am. Assoc. Cancer Res. 2001 42 (Abs4956); Lowinger et al., Curr. Pharm. Des. 2002 8 (25): 2269-2278; WO 9932455) and SU11248 (sunitinib / Sutent)

(Sun et al. J. Med. Chem. 2003 46 (7): 1116-1119; WO 0160814) were synthesized and purified directly according to published procedures and patents. Stock solutions (20 mM) of BAY 43-9006 or SU11248 are prepared in DMSO, divided into aliquots and stored at -20 ° C until use. All drugs were diluted to optimal culture medium for in vitro analysis. For in vivo administration, BAY 43-9006 was formulated in 100% PEG 400 excipients and SU11248 dosage solution was prepared in 5 mM citrate buffer. All other chemicals used were research grade.

Cell line

AU murine cell lines, CTLL-2 (IL-2 dependent T cell lines), B16-F10 melanoma, CT26 colon and kidney carcinoma RENCA were obtained from the American Tissue Culture Collection (Rockville, Maryland). CTLL-2 is RPMI 1640 supplemented with 10% FBS (fetal bovine serum, Gibco Life Technologies, Gettysburg, MD), 2 mM L-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 0.5 nM rIL-2, 2 mM β-mercaptoethanol Grown in. RENCA cells were cultured in medium containing EMEM with 10% FBS, 2% 100X vitamin, 1% 200mM glutamine, 1% 100mM NaPy, 1% non-essential amino acids. The medium for the growth of CT26 cells contained EMEM, 10% FBS, 2% vitamin, 1% 200 mM glutamine, 1% 100 mM sodium pyruvate, 1% non-essential amino acids. B16-F10 cells contain 10% FBS, 1% non-essential amino acids, 1% 10 mM sodium pyruvate, 2% vitamin; 2 mM 1-glutamine; Grows in RPMI 1640 with 2% sodium bicarbonate. Yac-1 cells (ATCC) were cultured in RPMI + 10% FBS and subcultured 1-2 days prior to analysis to ensure long phase. The cells were maintained as a suspension or sticky culture in a humid atmosphere of 37 ° C. and 5% carbon dioxide. Exponential growth phase (not exceeding 6-8 passages) cells were used, with viability> 98% (evaluated using trypan blue staining) and no mycoplasma detected.

In vivo  Efficacy Study

Female BALB / c or C57BL6 mice (4-6 weeks old, 18-22 g) were obtained from the Charles River (Wilmington, Mass.) And were acclimated for one week in pathogen free cages before starting the study. Animals were given sterile rodent food and water randomly and were housed in a sterile filter-top cage with a 12 hour light / dark cycle. All experiments were in accordance with the guidelines of the International Association of Laboratory Animal Care Evaluation and Certification.

For tumor transplantation, B16-F10 (2 × 10 ⁶ ), CT26 (2 × 10 ⁶ ) or RENCA (1 × 10 ⁶ ) cells were collected, washed three times and suspended in PBS. The sides of the mice were shaved and subcutaneous (sc) implanted (0.2 ml) into the right sides of the mice. The B16-F10 tumor model used C57BL6 mice, and CT26 and RENCA tumors were transplanted into BALB / c mice. When an average tumor size of 50-250 mm 3 was established (day 0) treatment was initiated as outlined in the specified study plan. Mice were randomized to treatment cohorts (typically 10 per group). RIL-2 was administered subcutaneously daily on days 0-6 or 7-13 or days 0-4, 7-11 (0.2-3 mg / kg / day). BAY 43-9006 or SU11248 (1-100 mg / kg) was administered daily (5-12 days) as a solution by oral gavage starting on day 0 or day 7. All monotherapy and drug combinations of selected doses outlined in each study were well tolerated.

Tumor Suppression and Assessment of Response

Tumor volume and body weight were evaluated 203 times per week. Caliper measurements of tumors were converted to mean tumor volumes, using the formula of 1/2 (length (mm) x [width (mm)] ² ). Tumor growth inhibition (TGI) was calculated as [1- (average tumor volume of treated group / average tumor volume of control group) x 100]. Responses were defined as complete response (CR, no measurable tumor), or partial response (PR. 50-99% tumor volume reduction) compared to the tumor volume of each animal at the start of treatment. Tumor growth delay analysis was calculated as follows: [(days until treatment group reached average tumor volume 1000㎣)-(days until control group reached average tumor volume 1000㎣)].

Expected% tumor growth inhibition of combination therapy (% T / C Expected =% T / C Treatment 1 x% T / C Treatment 2) divided by the observed% T / C of the combination treatment (% T / C observed) 1 It was defined as a synergistic effect when exceeded. It was defined as an additive action when% T / C expected /% T / C observed = 1 and as an antagonist when% T / C expected /% T / C observed <1.

Pharmacokinetics

To assess 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, intraperitoneal, 0.2 ml) and at several hours after drug administration. Collected blood. Plasma levels of BAY 43-9006 and rIL-2 were measured using HPLC or ELISA biological assays.

Western Blot  analysis

After incubating the drug under the indicated conditions, cells were collected, washed with ice-cold PBS, and then RIPA buffer containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis) and phosphatase inhibitors (Sigma, St. Louis, MO) (1 x phosphate buffered saline, 1% Nonidet P-40 in pH 7.2, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Protein content of the cell lysate was measured using BCA analysis (Bio-Rad, Hercules, California). For Western blot analysis, 60 μg of protein was electrophoresed and pERK was detected using a mouse antibody against pERK (1: 1000, Cell Signaling, Mass.), And incubated at 4 ° C. overnight. Detection of pSTAT5 antibody (1: 1000 Upstate) and pAKT (1: 1000 Cell Signaling) was performed by probing with a suitable anti-phosphotyrosine antibody at room temperature for 2 hours using the same amount of protein. The membrane was then incubated with 1: 5000 horseradish peroxidase-conjugated anti-rabbit IgG (Jackson Immunoresearch, Pennsylvania Westgrove) for 1 hour at room temperature. To demonstrate the same loading, the blots were stripped and reprobed with anti-ERK (Cell Signaling), anti-STAT5 (BD Biosciences), and anti-AKT (Cell Signaling) antibodies to separate the total ERK, STAT5 and AKT proteins, respectively. Measured. Proteins were detected using enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) and visualized by exposure to Kodak film. Scanning density measurements were performed to quantify band intensities. The amount of pERK, pSTAT5 or pAKT was normalized to total ERK, STAT5 or AKT protein levels and compared with excipients or untreated controls.

After medication BALB / c hairless In mice  Cellular immunophenotype

Blood samples were collected at several hours after the indicated treatment. Whole blood (100 μl) was transferred to FACS / TruCount tubes (BD BioSciences) and stored on ice. Samples were treated with 0.5 μg mouse Fc blocks (anti-mouse CD16 / CD32; BD BioSciences) and incubated for 20 minutes on ice. Fluorescent dye-conjugated antibodies as shown below were added to the samples and incubated on ice for 20 minutes blocking the light. 2 ml 1 x FACS lysis solution (BD BioSciences) was added while turning the blood sample, incubated at room temperature for 10 minutes, and then centrifuged at 1250 rpm. All samples were washed twice, suspended in PBS and 2% FBS, stored at 4 ° C. and subsequently obtained from BD FACScalibur and analyzed by CellQuest Pro software. Absolute values of cells were measured against TruCount bead criteria. Cell populations were identified and controlled based on FSC and SSC characteristics as well as markers for total lymphocytes (CD45, BD BioSciences). T cell lymphocyte populations were identified based on CD3 staining and subpopulations were identified by CD4 or CD8 staining (BD Biosciences). Each cell population was identified by appropriately controlled events.

Histopathology and Immunohistochemistry

Mouse tumors were fixed in 10% neutral buffered formalin and then transferred to 70% ethanol and paraffin embedded using Excelsior Tissue Processor (Thermo Electron Corporation, Pittsburgh, PA). Tissue sections (4 μm) were cut out on a rotating microtome (RM2125, Leica Microsystems, Nussroach, Germany), and then hemotoxiline and eosin (H & E) stained sections were prepared. Immunostaining was then performed using the Discovery XT automated slide staining system (Ventana Medical Systems, Tuxan, Arizona). Murine T cells were detected using a rabbit anti-CD3 antibody (1:60 dilution, Dako Norden A / S, Danish Glossrup), and monocytes / macrophages were detected using F4 / 80 (Serotec). Mice tumors were stained for Ki-67 using monoclonal murine anti-mouse antibody (1:15 dilution, DAKO) for cell proliferation. Heat-induced epitope regeneration was performed using Ventana Medical Systems (CC1). The samples were then incubated with a suitable secondary antibody (goat anti-rabbit IgG biotinylated antibody, 1: 100 dilution, Jackson ImmunoReseach Laboratories). Antibodies were localized using horseradish peroxidase-labeled streptoavidin with a 3-3′-diaminobenzidine pigment source (Ventana Medical Systems). Sections were counterstained with intranuclear dyes to better reveal tissue morphology. For B16-F10 tumors, melanin deposition was observed in H & E tissue, which used the Veneta bluemap kit-alternative NBT / BCIP staining method because it aided in the visualization of T cells in tumors.

CTLL -2 T cell proliferation assay

CTLL-2 T cells were preincubated for 2 hours at 37 ° C. with and without BAY 43-9006 (3 μM). The cells were then washed and plated in 96-well microtiter plates at 5,000 cells / well in culture medium with rhIL-2 serial dilutions (1 pM to 100 nM). At the end of the incubation period (37 ° C., 72 hours), cell viability was measured by tetrazolium dye analysis using the cell proliferation reagent WST-1 (Roche Applied Science, Indianapolis, Indianapolis).

In vitro  Cytotoxicity Assay

Cells are plated in 96-well microtiter plates (CTLL-2: 5000 cells / well; RENCA: 1500 / well; B16-F10: 1000 / well; MV4; 11: 5000 / well) and BAY 43-9006 / Treated with serial dilutions of SU11248 or vincristine. Since CTLL-2 is an IL-2 dependent cell line, cytotoxicity assays were performed on these cells in medium containing 5 nM rIL-2. At the end of the incubation period (37 ° C., 72 hours), cell viability was measured by tetrazolium dye (WST-1) analysis or chemiluminescence (BrdU) analysis (Roche Applied Science, Indianapolis, Indianapolis). EC ₅₀ values were defined as the concentration required for a 50% reduction in absorbance of treated cells relative to untreated control cells.

In vitro Splenocytes  Cytotoxicity Assay

Splenocytes were obtained from drug-treated BALB / c mice (n = 3-5 / group) under sterile conditions and homogenized in cold PBS. After passing through a 70 μm nylon cell separator, the cells were briefly centrifuged at 4 ° C. Red blood cells were lysed using RBC cytolysis buffer (Sigma, St. Louis, MO). Cells were washed and ⁵¹ Cr-labeled Yac- at different E: T ratios (100: 1, 50: 1, 25: 1, 12/5: 1, 6.25: 1, 3: 1) in a 4 hour cytotoxicity assay. Plated with 1 target cell. A wallac plate reader was used to obtain data expressed in parts per minute (cpm). Amounts are expressed as percent specific cytolysis and calculated as% specific cytolysis = 100 x ((average experiment-mean spontaneous release) / (average maximum release-mean spontaneous release)). Spontaneous release was measured from wells containing labeled target cells but no effector cells, and maximal release was measured from wells containing target cells labeled in 1% Triton X-100.

Statistical analysis

Multiple comparisons were performed using one-way ANOVA, and post-tests were performed using the Student-Newman-Keuls test (SigmaStat) to compare different treatment means. The difference was considered statistically significant at p <0.05.

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

CTLL In -2 cells JAK / STAT and MAPK Signaling rIL By -2 In vitro  Activation

To account for the initiation, time course, and duration of the IL-2 / IL-2R signal transduction pathway in T cells, CTLL-2 cells (1 × 10 ⁷ cells) were fasted in serum for 24 hours, followed by various concentrations of rIL. Treatment with -2 (1 pM to 100 nM) was performed for 2 hours, and important signaling pathways: MAPK, STAT5, AKT were evaluated using Western blot analysis. Serum-fasting CTLL-2 cells were treated in vitro with a wide range of concentrations of rIL-2 (1 pM to 100 nM) to give two dominant IL-2R complexes, namely high-affinity (IL-2RαβγK _D = 10 ⁻¹¹ M) and medium. The interaction of the affinity (IL-2βγK _D = 10 ⁻⁹ M) receptor was outlined. In some experiments cells were exposed to free anti-IL-2α antibody (> 1000 fold excess; 10 nM) incubated 1 hour prior to rIL-2 addition. To assess the time course of IL-2 / IL-2R activation, serum-fasting CTLL-2 cells were activated with 10 nM rIL-2 and IL-2 signaling was tested at various times from 1 minute to 48 hours. IL-2R downstream phosphorylation of ERK1 / 2, STAT5 and AKT was assessed in rIL-2-treated cells using Western blot analysis. Relative levels of pERK, pSTAT5 or pAKT were compared with total protein levels of ERK, STAT5 or AKT, respectively.

CTLL-2 cells were exposed to rIL-2 followed by activation of pERK1 / 2. Phospho ERK was slightly activated at 1 pM at 2 hours and maximal activation was seen at> 100 pM concentration. The JAK / STAT5 pathway was maximally activated at 1 pM concentration, and increasing concentrations of rIL-2 did not change pSTAT5 levels (up to 100 nM). The basic pAKT pathway in CTLL-2 seems to be activated in T-cells under serum-fasting conditions. Moreover, the level of pAKT remained unchanged at the rIL-2 concentration tested (1 pM-100 nM; using pAKT antibody at the 483 phosphorylation site).

Blocking antibodies against IL-2Rα were used to determine whether the IL-2 signaling pathway was specifically mediated by binding to IL-2R. PSTAT5 levels of CTLL-2 cells were analyzed by Western blot. CTLL cells were serum-fasted 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). PSTAT5 was activated at 1 pM after rIL-2 treatment in the absence of blocking IL-2Rα antibody, but pSTAT5 signaling was eliminated (> 95% inhibition) when IL-2Rα inhibition was present, indicating that IL-2Rα was not found in STAT5 signaling. To confirm that it is essential. Interestingly, pSTAT5 levels are restored in CTLL-2 cells with increasing concentrations of rIL-2 (> 10 pM), which rIL-2 competitively replaces anti-IL-2Rα antibodies and / or is low- It suggests that pSTAT5 signaling is activated by binding to the affinity IL-2βγ chain (K _D = 10 ⁻⁹ M).

CTLL In -2 cells pSTAT5 Swift rIL -2 active and persistent

To assess the initiation time and duration of the IL-2R signaling response in CTLL-2 cells, serum-fasting cells were treated in vitro with rIL-2 (10 nM) and for phosphorylation of ERK1 / 2, STAT5 and AKT. The effect was evaluated using Western blot analysis. Phosphorylation of STAT5 was activated within minutes after addition of rIL-2 to CTLL-2 cells (<10 minutes) and the duration of the pSTAT5 response was maintained up to 48 hours. Activation of the MAPK pathway (pERK) pathway by rIL-2 seemed to be delayed slightly and was activated by 1 hour. The intensity of pERK was lower than the maximal response obtained when serum-fasting CTLL-2 cells were stimulated with PMA (50 ng / ml) + inomycin (0.4 μg / ml) for 15 minutes. In addition, pERK levels lasted up to 24 hours and returned to baseline levels by 48 hours. No apparent effect was observed for pAKT, confirming that signaling by the PI-3K / AKT pathway is still active in CTLL-2 cells.

CTLL BAY 43-9006 treatment in -2 cells was pERK But suppress pSTAT5 Does not suppress

BAY-43-9006 is a potent inhibitor of Raf-1 and also inhibits both wild type and mutant BRAF. In addition, BAY-43-9006 has been shown to contain several kinases, in particular VEGF2, 3; PDGFRβ; Inhibits FLT3 and cKIT (Wilhelm, S. M. et al., Cancer Res 2004; and kinase profiling data from Chiron) and to some extent inhibits two kinases involved in T cell functional responses, Lck and Fyn. Given the inhibitory kinase profile of BAY 43-9006, and its potential impact on MAPK signaling / T cell signaling, BAY 43-9006 could potentially interfere with IL-2 / IL-2R signaling in T cells. It was supposed to be. Thus, possible interactions of BAY 43-9006 with IL-2 signaling were assessed 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-fasting 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 inomycin (0.4 μg / ml) for 15 minutes. To test the effect of the simultaneous and continuous treatment of rIL-2 and BAY 43-9006 in CTLL-2 cells, serum-fasting CTLL-2 cells were treated for 2 hours with or without BAY 43-9006 (3 μM). treated with rIL-2 (10 nM). In the continuous treatment, BAY 43-9006 was treated for 2 hours. Cells were then washed and treated with rIL-2 and vice versa. In addition, the inhibitory effect of BAY 43-9006 (3 μM) stimulated or unstimulated with PMA (50 ng / ml) and inomycin (0.4 μg / ml) was tested for 15 minutes.

Since the concentration of BAY 43-9006 required to inhibit the MAPK pathway in different cell types is very variable, the effects of different concentrations of BAY 43-9006 (range of 0-20 μM) may or may not be stimulated with or without PMA + inomycin. Evaluation was made on serum-fasting CTLL-2 cells for 2 hours. At the end of the incubation period, treated CTLL-2 cells were lysed and protein lysates were analyzed by Western blot to determine pERK levels. BAY 43-9006 substantially inhibited pERK at concentrations of> 1 μM in murine CTLL-2 cells and human Jurkat T cell lines. The effect of BAY 43-9006 treatment on CTLL-2 cells did not alter the levels of pSTAT5 and pAKT in cells, suggesting that JAK / STAT5 pathway and PI-3K / AKT did not significantly affect T cells. do.

To investigate the therapeutic basis of the combination of rIL-2 and BAY 43-9006, the effects of rIL-2 and BAY 43-9006 on T cells and the two IL-2-mediated signaling pathways in CTLL-2 cells The effects on (MAPK and JAK / STAT5) were studied. The effect of IL-2-mediated T cell signaling on the simultaneous or continuous therapy of the two drugs was investigated in CTLL-2 cells. Serum-fasting CTLL-2 cells were treated with BAY 43-9006 (3 μM) for 2 hours and then with excipients, rIL-2 (10 nM) or PMA + inomycin. Alternatively, the treatment with BAY 43-9006 (3 μM, 2 hours) after treatment with rIL-2 (10 nM, 2 hours) was also investigated. After drug exposure and / or PMA + inomycin stimulation, Western blot analysis of pERK and pSTAT5 in cell lysates was performed (as previously described). Treatment with BAY 43-9006 (3 μM) inhibited pERK levels, whereas rIL-2 activated pERK levels in serum-fasting CTLL-2 cells (for baseline pERK levels), indicating that the two drugs for the MAPK pathway It is to confirm the opposite effect. AU treatment (simultaneous and continuous drug exposure) with the BAY 43-9006 combination substantially inhibited pERK in CTLL-2 cells. No effects on the STAT5 pathway or AKT pathway were observed in all combination treatments tested (as outlined in the method).

High concentrations of BAY 43-9006 are tumors In the cell line pERK  Suppress the level

The effect of BAY 43-9006 treatment on the murine tumor cell line -B16-F10 melanoma, CT26 colon and RCC RENCA model was measured. Murine cell lines were selected based on the response to in vivo rIL-2 therapy in the immunocompetent model (T- / NK- / monocyte- / macrophage-competent mice), rIL-2 and BAY 43 The effects of -9006 combination therapy may be investigated. Serum-fasted B16-F10 melanoma and RENCA cells were exposed to BAY 43-9006 in the concentration range of 0-20 μM. Phospho-ERK levels in cell lysates after drug exposure were measured by Western blot analysis. BAY 43-9006 inhibited pERK levels in two cell lines (B16-F10 and RENCA) tested at very high concentrations> 5 μM and showed nearly complete removal of pERK at 20 μM.

C. In Vitro Effects of BAY 43-9006 on IL-2-Mediated Cell Proliferation

To test whether pERK inhibition by BAY 43-9006 in CTLL-2 cells affected the IL-2-mediated proliferative response, CTLL-2 at a concentration of BAY 43-9006 (3 μM, 2 hours) that inhibits pERK Proliferation assays were performed by pre-incubating the cells (5000 cells / well). After incubating the cells with BAY 43-9006 (3 μM) for 2 hours, the cells were plated in 96-well plates and exposed to various concentrations of rIL-2 (0-100 nM) for 72 hours. Untreated cells were given mock treatment prior to incubation with rIL-2 (at the same concentration). Proliferative responses of BAY 43-9006-treated and untreated CTLL-2 cells were evaluated using the WST-1 assay. Pre-incubation of cells with BAY 43-9006 (3 μM) inhibited pERK signaling, but did not affect IL-2 induced proliferative responses in CTLL-2 cell lines.

D. In Vitro Antiproliferative Activity of BAY 43-9006 or SU11248 on CTLL-2 and Tumor Cells

To assess the in vitro cytotoxic activity of BAY 43-9006 or SU11248 in each of these cell types (CTLL-2, B16-F10, RENCA, CT26), cells were plated in 96-well microtiter plates and plated at 37 ° C. Treated with serial dilutions of BAY 43-9006 (0-50 μM) for 72 h. Since CTLL-2 cell lines are dependent on IL-2 for proliferation, cytotoxicity against these cells was performed in medium containing 5 nM rIL-2. At the end of the 72 hour incubation period, cell viability was measured by tetrazolium dye (WST-1) analysis or chemiluminescence (BrdU) analysis. As a control, 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 the cytotoxicity of the formulation and Analysis validity was confirmed. The inhibitory concentration of the drug is expressed as an EC ₅₀ value, which is defined as the concentration required for a 50% reduction in the proliferative response measured as the absorbance of the drug-treated cells versus the untreated / excipient control.

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

In general, relatively high concentrations of BAY 43-9006 (or SU11248) are used to inhibit the proliferation of CTLL-2 cells as well as several cell lines (B16-F1O, RENCA, CT26) when compared to vincristine, an antimitotic agent. Required (defined by relative EC50; see Table 9). In addition, the cytotoxicity of the BAY 43-9006 effect on RENCA cells was tested using BrdU assay (for anti-proliferative activity using mitochondrial tetrazolium dye assay) for the purpose of evaluating the effect on DNA synthesis. The EC50 for BAY 43-9006 (about 5 μM) for RENCa cells obtained using the BrdU method was similar to that observed in the WST-1 assay. No direct proliferative response (or cytotoxicity) was observed when RENCA tumor cells were exposed to the rIL-2 concentration range (0-1 μM), confirming that the mechanism of rIL-2 is dependent on immune effector cell components.

E. In vivo efficacy studies

In mice rIL -2 and BAY 43-9006 Pharmacokinetics

A published Phase I report of BAY 43-9006 (400 mg dose) achieved a high Cmax of 10-20 μM and a long drug half-life of 24 hours in patients (Strumberg et al., J. Clin. Oncol. 2005 23 (5): 965 -972). Single dose pharmacokinetics of rIL-2 and BAY 43-9006 were measured in mice to determine if plasma drug exposure could affect T cell viability and proliferative responses in vivo.

A single oral dose of 30 mg / kg BAY 43-9006 in mice achieved a Cmax of about 5500-8000 ng / ml (about 10 μΜ) at a tmax of 2 hours. Plasma removal rate of BAY 43-9006 was quite slow, and the half-life of the result was about 4 hours. In contrast, the PK profile of rIL-2 after subcutaneous administration of 6 mg / kg showed a Cmax of about 550-850 ng / ml at a Tmax of about 30 minutes. RIL-2 t1 / 2 was approximately 1 hour in mice when exposed to> 50ng / ml for 4 hours. Interestingly, it is not easy to understand that after a single dose, non-overlapping Cmax (at tmax) was seen in both treatment and each route of administration, and both simultaneous and continuous treatment could be supported by PK discovery. BAY 43-9006 can be very large proteins bound in the blood and the effect of these drug exposures on T cell viability is still a problem to be addressed in vivo.

rIL -2 and BAY 43-9006 treatment In vitro  immune Effector  Reduces function

RIL-2 and BAY 43 on T cell proliferation because inhibition of one or multiple T lymphocyte kinases (Lck, Fyn, Syk, Btk, Src, Tck2, MAPK, JAK) abolishes T cell expansion and immune effector function. The effect of -9006 and its functional response in vivo were investigated.

In these experiments, BALB / c mice with RENCA tumors were treated as follows: rIL-2 (lmg / kg / day, subcutaneous, 6-10 days), BAY 43-9006 (30 mg / kg / day, intraperitoneally, 6-10 days) or a combination of rIL-2 and BAY 43-9006 administered simultaneously or sequentially (rIL-2, 1 mg / kg / day, subcutaneous, 1-5 days + BAY 43-9006, 30 mg / kg / day , Intraperitoneal, 6-10 days; or BAY 43-9006, 30 mg / kg / day, intraperitoneal, 1-5 days + rIL-2 1 mg / kg / day, subcutaneous, 6-10 days). Isolated splenocytes from isolated mice were subjected to in vitro killing assays for Yac-1 target cells at various effector: target (E: T) ratios and the specific percent cell lysis of ⁵¹ Cr-labeled Yac-1 cells was measured. Mice treated with rIL-2 (1 mg / kg) significantly increased splenocyte-mediated killing of Yac-1 targets compared to excipient treatment (23% rIL-2 vs 0% excipient treatment). The specific killing effect observed in 9006 treatment (1%) was negligible and did not differ from excipient treatment. All treatments with 43-9006 provided reduced splenocyte-mediated cytolytic activity (simultaneous treatment = 16%, continuous treatment <9%, rIL-2 monotherapy = 23%).

Circulating immunity Effector  For cell populations rIL -2 and the effect of BAY 43-9006 therapy

The pharmacodynamic effects of rIL-2 and / or BAY-43-9006 therapy on circulating lymphocyte and monocyte population tumor-bearing BALB / c mice were tested. In these studies, blood was collected after a single agent or a combination therapy with rIL-2 and BAY 43-9006 (simultaneous or continuous treatment as described above). The absolute number of lymphocytes and T cell sub-populations (CD4 + or CD8 +) in whole blood was quantified using TruCount ™ tubes and appropriate immunostaining.

rIL-2 treatment was used for the treatment of circulating lymphocytes and monocytes (CD45 + cells: 2387 cells / μl vs 1575 cells / μl, excipient), and T cells (1550 cells / μl vs 664 cells / μl, excipient) in mice with tumors. Reduced number. Significantly increased CD4: CD8 cell ratios were observed in rIL-2 treatment compared to excipient treatment (5: 3; rIL-2: excipient, ie 1.7-fold), indicating the effect of the rIL-2 mechanism of expanding T cell numbers. Suggest. Relative numbers of non-T and monocyte cells (CD45 + CD3-) after rIL-2 treatment were similar to excipient treatment (837 cells / μl versus 911 cells / μl, excipient).

In contrast, BAY 43-9006 in combination with single agent BAY 43-9006 or rIL-2 had little effect or increased the absolute number of lymphocytes and monocytes (2417-3577 cells / μl range versus 2387 cells / μL). Μl, excipient), non-T cell and mononuclear cell populations were also increased (1241-1563 cells / μl versus 837 cells / μl, excipient). The effect of BAY 43-9006 treatment on the total number of T cells was similar to excipient treatment (1827 cells / μl versus 1550 cells / μl, excipient). A slight increase in the total number of T cells (including both the CD4 + and CD8 + populations) was observed when initiating with rIL-2 in a continuous regime of rIL-2 and BAY 43-9006 (2081 T cells / μl versus 1550 cells / μl , Excipients). When rIL-2 and BAY 43-9006 were given at the same time, or BAY 43-9006 was given first and rIL-2 was given subsequently, the total number of T cells decreased slightly compared to excipient treatment (about 1170-1244). T cells / μl to 1550 cells / μl, excipient). Similar trends were observed for each CD4 + and CD8 + T cell population.

 In addition, the histology of tumors was measured after treatment with rIL-2 and BAY 43-9006 or SU11248 therapy (as described above). To test the pharmacokinetics of T cells in vivo, tumor infiltrating T cells were detected using mouse anti-CD3 antibodies. Anti-proliferative effects in tumors after drug treatment were assessed using Ki67 staining. In comparison to excipient treatment, an increase in T cell numbers was seen in both invasive RENCA tumors (and B16-F10 tumors) in rIL-2 treatment. In general, fewer T cells were observed in the BAY 43-9006-treated group in the RENCA model. Since some T cells were detected in all treatment groups, the effects of rIL-2 and BAY 43-9006 or SU11248 on invasive T cells in B16-F10 tumors were equivalent. Tumors with increased necrosis (tumor suppression) generally had higher numbers of T cells interspersed between tumor cells. Overall, these data indicate that rIL-2 activates T cells in the circulatory system, migrates cells to extravascular sites, including tumors, and BAY43-9006 or SU11248 treatment can partially eliminate T cell proliferation and migration. Imply that there is.

rIL -2 and BAY 43-9006 Therapies Are IL-2 Reactive Rat  Increase the efficacy of tumor models

Drug interactions of rIL-2 with BAY 43-9006 or SU11248 were tested (see FIGS. 1-10 and Tables 10-12). This combination therapy was evaluated in three experimental T cell competents, a murine IL-2-reactive tumor model (B16-F10 melanoma, CT26 colon, RCC RENCA model) (FIGS. 1-10). In these studies, mice were randomized when a tumor size of 50-225 mm 3 was established, and the tumor size after daily oral administration of BAY 43-9006 or SU11248 or daily subcutaneous administration of rIL-2 (as indicated in the method). Was monitored by measuring with a caliper. Single agent efficacy and tolerability of rIL-2, BAY 43-9006 and SU11248 were investigated in the dose and treatment schedule ranges (FIG. 1). In all models, rIL-2, which showed potent efficacy and tumor suppression at the effective dose, was generally in the range of 40-60% (for excipient treatment) (FIG. 1). The minimum effective dose of BAY 43-9006 was> 30 mg / kg / day (for excipient treatment). SU11248 was generally effective at the> 40 mg / kg / day dose, with the exception of the B16-F10 tumor model only (FIG. 1).

Based on single agent efficacy and tolerability, rIL-2 was combined with a dose of BAY 43-9006 or SU11248 that did not show any deleterious effects on the body weight or any adverse clinical signs. Both continuous and concurrent treatment schedules were tested in the B16-F10 and CT26 tumor models (FIGS. 2-8, Tables 10 and 11). Almost all combination therapies (simultaneous or continuous) investigated with rIL-2 and BAY 43-9006 or rIL-2 and SU12248 in B16-F10 and CT26 tumor models showed anti-tumor activity as compared to monotherapy or excipient therapy. Increased (Figures 2-5). Combination drug interactions of the various combination therapies are briefly summarized in Tables 10 and 11.

In one case in the CT26 model, when BAY 43-9006 (40 mg / kg / day, intraperitoneal, 1-7 days) was administered before rIL-2 (1 mg / kg / day, subcutaneous, 8-13 days), the combination The effect of treatment was below the optimal effect.

Only simultaneous schedules were evaluated in the RENCA model (FIGS. 9-10; Table 12), and the combination of rIL-2 and BAY 43-9006 or SU11248 showed greater tumor suppression compared to single agent therapy.

Continuous treatment in the RENCA model was not easy due to the short model duration and mouse ataxia (animal consumption / weight loss). Taken together, these data indicate that rIL-2 is enhanced in combination with targeted small molecule therapies such as BAY 43-9006 and SU11248 in a preclinical model, suggesting that this treatment strategy may be adapted to clinical practice.

While the present invention has been described with reference to specific embodiments, including presently preferred ways of practicing the invention, those skilled in the art will understand that many variations and permutations of the systems and techniques described above fall within the spirit and scope of the invention. will be.

Inclusion of References

All contents of the above cited patents, patent applications and journal articles are incorporated by reference as if fully set forth herein.

Claims (32)

With a therapeutically effective amount of Aldes Leukine 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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid, 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) urea A method of treating cancer patients comprising administering to the patient an antiangiogenic agent selected from the group. 10. The method of claim 1, wherein the aldeleukin is administered prior to the antiangiogenic agent. The method of claim 1, wherein the aldehyde leukine is administered followed by an antiangiogenic agent. 2. The method of claim 1, wherein the aldeleukin is administered simultaneously with the antiangiogenic agent. 5. The method of claim 4, wherein the aldeleukin is administered as a composition separate from the antiangiogenic agent. 6. The method of any one of claims 1 to 5, comprising the step of separately administering to the patient a therapeutically effective amount of aldesleukin and an angiogenesis agent according to the dosing schedule, wherein at least continuous Administering one to three times per day at a dose of about 9 to about 130 MIU / day for 3 days, followed by an optionally at least 3 consecutive days of rest. 7. The method of claim 6, wherein the antiangiogenic agent is administered 1 to 6 times every 2-3 weeks. 7. The method of claim 6, wherein the aldosekin is administered intravenously and the resting period is present. 7. A method according to claim 6, wherein aldehydes are administered subcutaneously and the resting period is absent. 10. The method of claim 9, wherein the aldeleukin is administered 1-3 times a day at a dose of about 9-30 MIU / day. 7. The method of claim 6, wherein the aldeleukin is administered three times a day at a dose of about 30-130 MIU / day. 7. The method of claim 6, wherein the aldose-leukin is administered continuously for 5 days followed by a 9 day rest period. 7. The method of claim 6, wherein the dosing schedule is repeated at least two courses. 7. The method of claim 6, wherein the dosing schedule is repeated at least three courses. 7. The method of claim 6, wherein the dosing schedule is repeated at least four courses. 7. The method of claim 6, wherein the aldehyde leukine is administered three times on the first day and then once daily for each progression day. 17. The method of any one of claims 1-16, wherein the cancer is renal cell carcinoma, melanoma or colon cancer. 18. The compound of claim 17, wherein at least one compound selected from acetaminophene, meperidine, indomethacin, ranitidine, nizatidine, diastop, loperamide, diphenylhydramine, or furosemide The method further comprises administering to the patient following or concurrently with the administration. 19. The method of any one of claims 1 to 18, wherein cancer is improved in the patient as a result of the method. 19. The method of any one of claims 1 to 18, wherein hypotension is attenuated in the patient as a result of the method. 19. The method of any one of claims 1 to 18, wherein hypertension is attenuated in the patient as a result of the method. 19. The method of any one of claims 1-18, wherein nitric oxide synthase is reduced in the patient as a result of the method. The method of claim 1, wherein the cancer is sensitive to angiogenesis and / or inhibition of immunostimulation. The method of claim 1, wherein the angiogenic agent is N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindolin-3-ylidene ) Methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide. The method of claim 1, wherein the angiogenic agent is 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro- 3- (trifluoromethyl) phenyl) urea. (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-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5-((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid, 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) urea Therapeutically effective amount of an angiogenesis agent selected from; And (c) pharmaceutically acceptable excipients Composition comprising a. (a) aldehydes; 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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid, 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) urea A kit comprising a pharmaceutical combination for the treatment of cancer patients, characterized in that it comprises anti-angiogenic agents selected from, which are used simultaneously, consecutively or separately. 27. The kit of claim 26, wherein each medicament is individually packaged. In the manufacture of one or more medicaments for the treatment of cancer patients, Aldeleukin 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-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carbox mid, 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) urea Use of antiangiogenic agents selected from. 30. The use according to claim 29, wherein the aldeleukin is formulated to be administered prior to the antiangiogenic agent. 30. The use according to claim 29, wherein the aldeleukin is formulated for subsequent administration to an antiangiogenic agent. 30. The use according to claim 29, wherein the aldeleukin is formulated to be administered simultaneously with the antiangiogenic agent.

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