BRPI0608880A2 - anti-angiogenic agents with aldesleukin - Google Patents

Abstract

ANTI-ANGIOGENIC AGENTS WITH ALDESLEUCIN. The present invention relates to combined therapies with IL-2 compositions and anti-angiogenic agents for treating cancer. Further provided are methods of alleviating toxicities and increasing efficacy associated with administration of IL-2 compositions or anti-angiogenic compositions.

Description

AND ANGIOGENIC AGENTS WITH ALDESLEUCIN

REFERENCE TO RELATED APPLICATION

The present application claims benefit under 35 U.S.C. § 119 (e) of provisional application 60 / 654,341, filed February 18, 2005, which application is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods of therapy for diseases associated with abnormal cell proliferation. In some embodiments, recombinant IL-2 is combined with anti-angiogenic agents for use in cancer treatment.

BACKGROUND OF THE INVENTION

Capillaries reach almost all tissues of the human body and supply tissues with oxygen and nutrients, as well as removing waste products. Under typical conditions, endothelial cells that line the capillaries do not sedivate, and the number or size of capillaries, therefore, does not normally increase in a human adult. Under certain conditions, however, such as when tissue is damaged during certain parts of the menstrual cycle, capillaries begin to proliferate rapidly. This process of forming new capillaries from pre-existing blood vessels is known as angiogenesis or neovascularization. See Folkman, J. Scientific American 275, 150-154 (1996). Angiogenesis during wound healing is an example of pathophysiological neovascularization during adulthood. During wound healing, additional capillaries provide a supply of oxygen and nutrients, promote tissue granulation, and assist in the removal of waste material. Upon completion of the healing process, the capillaries usually regress. Lymboussaki, A. "Vascular Endothelial Growth Factors and their Receptors in Embryos, Adults and Tumors" Academic Dissertation, University of Helsinki, Molecular / Cancer Biology Laboratory and Department of Pathology, Haartman Institute, (1999).

Angiogenesis also plays an important role in cancer cell development. It is known that since a cluster of cancer cells reaches a certain size, approximately 1 to 2 mm in diameter, cancer cells must develop a blood supply so that the tumor grows, since diffusion may not be sufficient to supply cancer cells with enough oxygen and nutrients. Thus, inhibition of angiogenesis is expected to disrupt cancer cell growth. Of the angiogenic factors identified, vascular endothelial growth factor (VEGF) is the most potent and specific and has been identified as a crucial regulator of normal and pathological angiogenesis; although other factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), have been implicated in angiogenesis and cancer cell survival. Curr Opin Investig Drugs. February 2001; 2 (2): 280-6.

Specific anti-tumor immunity involves activation of multiple cell types in the immune system, the most efficient being cytolytic T lymphocytes. Required to induce the specific Tanti-tumor lymphocyte-mediated immune response, recognition not only of the tumor antigen of interest, but also of co-stimulatory interactions between specific ligands present on the tumor cell or antigen presenting cell and target T lymphocytes. This second costimulatory signal may also be provided by soluble factors, such as cytokines or other peptide molecules which select for specific receptors on the cell surface and initiate various signal transduction pathways, resulting in enhanced effector function.

Interleukin-2 (IL-2) is a T-derived lymphocyte cytokine which binds to specific receptors present on T cells and natural killer (NK) cells and will activate the same for tumor cytolysis, cytokine secretion and other effector functions. CD4 and CD8 T lymphocytes express the IL-2 receptor and develop increased decytocin and cytolytic effector synthetic function following exposure to the biological agent. The functional effects of IL-2 are mediated via the IL-2 receptor (IL-2R), which is structurally composed of three subunits: a, eg a common y chain that is shared by other decytocin receptors (Caligiuri et al., J Exp. Med 1990171 (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 consequent signaling are dependent on IL-2 receptor types on cells (Caligiuri et al., J ". Exp. Med.1990 171 (5): 1509-1526; Voss et al., J. Exp. Med.1992 176 (2): 531-541; Expinoza et al., J. "Leukoc.Biol. 1995 57 (1): 13-29; Theze et al., Immunol.Today 1996 17 (10): 481- 486). T cells express high affinity IL-2RaPY, whereas macrophage monocytosis NK cells predominantly express the intermediate-affinity form, IL-2R] 3y (Caligiuri ecolaboradores, J. Exp. Med. 1990 171 (5): 1509-1526; Your collaborators, J. Exp. Med. 1992 176 (2): 531-541; Theze collaborators, Iwmunol. Today 1996 17 (10): 481-486; Naglere collaborators, J. Exp. Med. 1990 171 (5): Central to its mechanism, IL-2 activates JAKs (JAK1, JAK3), which phosphorylate key tyrosines over IL-2RP, which serve as anchor sites for downstream de-signaling molecules, including Shc and Stat5 as well. which then catalyze the activation of two distinct pathways: Shc / Ras / Raf / MAPK and JAK / STAT5, which translocate to the nucleus and directly regulate a family of gene-regulated transcription factors - STATs (0'Shea, J. J., Immunity1997 7 (1): 1-11). Shc recruits the Grb-2 / Sos complex and activates it via Ras / Raf / MAPK and Grb-2 / Gab-2, which activates the phosphatidylinositol (PI3K) 3-kinase pathway. Together, these signaling proteins regulate degene transcription factors, ultimately controlling growth, division, cell differentiation, and immune activity. In addition, IL-2 induces apoptotic effects through regulation of pro-mitogenic genes leading to increased bcl-2, bcl-xl, c-myb, which affect cell cycle control through cdk activation 2,4,6 and inhibition of p27kipl or may be negatively regulated by SOCS (cytokine signaling suppressors) (Smith, KA, Science 1988 240 (4856): 1169-1176; Theze et al., Iwmunol. Today 1996 17 (10): 481-486). The interaction between IL-2 and IL-2R is reported to trigger downstream activation of the MAPK, PI-3K, and JAK / STAT signaling pathways, leading to cell survival and proliferation (Miyazaki et al., Science 1994 266 (5187): 1045- 1047; Beadling et al., Embo J. 1996 15 (8): 1902-1913; Nakajima et al., Iwmunity 1997 7 (5): 691-701; Moriggl et al., Iwmunity 1999 11 (2): 225-230).

Preclinical studies in various murine tumor models have shown that recombinant human IL-2, when administered to animals bearing tumors for periods of 10 to 14 days, may result in regression of tumor burden, long-term survival, and increased resistance to growth. tumor. Analysis of splenic lymphocytes obtained from these animals showed that anti-tumor effects due at least in part to increased cytolytic T-cell function. This effect includes direct decytolysis activation of tumor cells by CTL, as well as increased synthesis of other T-lymphocyte-derived cytokines, which may have other anti-tumor or indirect effects as well.

Metastatic melanoma and renal cell carcinoma (CCR) are largely incurable and resistant diseases of most types of systemic chemotherapy, so there remains a considerable need for the development of more effective therapies. Recent clinical data have supported the development of small molecule receptor kinase detirosin (RTK) inhibitors, particularly BAY 43-9006 or SU11248 in renal cell ecarcinoma melanoma. BAY 43-9006 and SU11248 inhibit multiple kinases that are involved in tumor growth, anti-angiogenesis proliferation. 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 ecolaborators, Cancer Res. 2004 64 (19): 7099-7109; Awada Eco-Workers, Br. J. Cancer 2005 92 (10): 1855-1861) inhibits the mutant B-Raf WT and B-Raf V599E. In addition to the effects of BAY 43-9006 on Raf, it is believed that most of its activity may be mediated via inhibition of other RTKs, particularly VEGFRs (VEGFR-2 and VEGFR-3), PDGFRp and other key FLT-3 and cKIT kinases. (Wilhelm et al., Cancer Res. 2004 64 (19): 7099-7109). Responses with BAY 43-9006 in preliminary solid tumor experiments have established that daily or twice daily administrations may result in stabilization of the disease, including some partial responses. To date, Phase I studies have indicated that BAY 43-9006 is generally well tolerated in patients. The most common toxicities associated with Bay43-9006 involved effects on the gastrointestinal tract (diarrhea, nausea, abdominal wind) or dermatological reactions, including itching or rashes (Richly et al., Int. J. Clin. Pharmacol. Ther.2003 41 (12 ): 620-621; Ahmad and Eisen, Cancer Clin Res 200410 (18 Pt 2): 6388S-92S; Awada et al., Br. J. Cancer 2005 92 (10): 1855-1861; Strumberg et al. Clin Oncol 2005 23 (5): 965-972).

In contrast, SU1124 8 is a highly potent, selective RTK inhibitor of VEGFR-1-3, PDGFRa, cKIT, FLT3 andPDGFRp (Abrams et al., Mol Cancer Ther. 2003 2 (5): 471-478; Mendel et al., Cancer Clin Res 20039 (1): 327-337; O'Farrell et al., Cancer Clin Res Res 2003 (15): 5465-5476). SU11248 showed antitumor activity in a series of advanced solid tumors (RCC, neuroendocrine, stromal and adenocarcinomas) (Faivre and co-workers, J. Clin. Oncol. 2006 24 (1): 23-35; Motzer and co-workers, J ". Clin. Oncol. 2006 24 (1): 16-24) Clinical data from SU1124 8 also indicate that the drug is generally tolerated with controllable toxicities, which include fatigue, lymphopenia, neutropenia, hyperlipasemia (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 form of clear-cell renal carcinoma, which occurs in von Hippel-Lindau syndrome, led to the identification of the von Hippel-Lindau tumor suppressor gene (VHL). The gene mutates into hereditary renal cell carcinoma (where one mutation is a germline mutation) and in most cases sporadic clear cell renal carcinoma (where both alleles acquired mutations or deletions). Gnarra JR, DuanDR, Weng Y et al., Molecular cloning of the vonHippel-Lindau gene suppressor gene and its role in renal carcinoma. Biochimica et Biophysica Acta. 1996; 1242 (3): 201-10. One consequence of these mutations is the overproduction of vascular endothelial growth factor through a mechanism involving hypoxia-inducible factor. Ilopoulous AL, C Jiang et al., Negative Regulation of Hypoxia-inducable genes by the Von-HippelLindau protein. Proc Natl Acad I know USA. 1996; 93: 10595-10599. Thus, by regulating vascular endothelial growth factor, von Hippel-Lindau protein is closely related to angiogenesis. Vascular endothelial growth factor stimulates endothelial cell growth and, as mentioned, appears to be a central factor in angiogenesis, particularly during embryogenesis, ovulation, wound healing, and tumor degrowth. Ferrara N. Molecular and biologicalproperties of vascular endothelial growth factor. J. Mol.Med. 1999; 77: 527-543.

Vascular endothelial growth factor is a good target for treatment of clear cell renal cancer because mutations in the von Hippel-Lindau tumor suppressor gene result in overproduction of this growth factor by tumors. A randomized, double-blind, placebo-controlled study of humanized monoclonal antibody (bevacizumab) in patients with metastatic clear cell cancer was conducted by Yange collaborators, A randomized trial of bevacizumab, ananti-vascular endothelial growth factor antibody. formetastatic renal cancer. New England Journal of Medicine. 349 (5): 427-34. Time to tumor progression was prolonged by a factor of 2.55 in patients who received bevacizumab per kilogram (10 mg / kg every two weeks) when compared to patients in the complacency group. Survival was not a primary goal in this experiment, which allowed patients to switch from place to bevacizumab therapy at the time of disease progression. Id. Treatments for kidney cancer that target angiogenic mechanisms may also be effective through other pathways other than those mediated by vascular endothelial growth factor. There are other proteins in the local microenvironment of some tumors that may promote angiogenesis. As such, there is a need for anti-angiogenic therapy that utilizes a rational combination of inhibitors directed at understanding renal cell carcinoma biology.

Recombinant human interleukin-2 (aldesleukin) has been approved by the FDA for the treatment of metastatic renal cancer based on the results of several multicenter experiments in 255 patients who received an intermittent high-dose debolus regimen. In these experiments, objective responses were observed in 15% of patients and the average survival was 16.3 months (Fisher and Rosenberg2000 Cancer J Sei Am 6S1: S55-57). Considering the significant toxicity associated with this regimen, a series of phase I and phase II experiments employing rIL-2 at different doses and using different routes of administration were conducted. A recent review of the efficacy of rIL-2 as a single agent indicated an overall response of 15% in more than 1,700 metastatic CCR patients who were treated in this series of studies. Complete responses were observed in 3 to 5% of patients. Bukowski RM. Natural history and therapy of metastatic renal cell carcinoma: therole of interleukin-2. Cancer. 1997; 80 (7): 1198-220. Astaxas do not seem to differ between these pathways. A randomized comparison between the high-dose intravenous bolus regimen and the low-dose intravenous bolus regimen and subcutaneous outpatient regimen showed no difference in mean survival between groups, but the low-dose ambulatory regimens were significantly less toxic. YangJC, Sherry RM, Steinberg SM et al., Randomized study of high-dose and low-dose interleukin-2 in patientswith metastatic renal cancer. Journal of Clinical Oncology.2003; 21 (16): 3127-32.

In addition to having direct immunomodulation effects, rIL-2 can also prevent tumor proliferation by causing secondary cytokine release (IFNγ) from activated NK cells. Saraya KLA, Balkwill FR. Temporal andcellular sequence originated from interleukin-2 stimulated cytokine geneexpression. British Journal of Cancer. 1993; 67 (3): 514-21.

Response rates and outcomes in patients with clear cell renal cancer may be improved. Clinical trials employing new drug combinations are needed. The combination of rIL-2 and anti-angiogenic agents, such as bevacizumab, may have additive effects that could translate into additional clinical benefit for patients with metastatic renal cell carcinoma as well as other cancers.

Another advantage to the complementary approach to cancer regression is that it provides a platform less easily overcome by resistance mutations. Therapeutic targets are thus polarized and specific (which may be necessary to avoid objectification of host cells), such as a particular substrate in a viral replicon or a kinase in a cancer cell line, a single point mutation in the diseased state can make it unchanged. by a drug, resulting in even more resistant insects of the disease in future generations.

New methods and mechanisms for the treatment of patients having abnormal proliferation-associated disorders that are resistant to or inadequately treated through conventional approaches using agents that target immune response mechanisms in the body and disease-free substrates are needed. The present invention provides such therapeutic agents and further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, on unique combined therapies using small molecule receptor tyrosinated kinase inhibitors and rIL-2 with non-overlapping toxicities. Since melanomas and CCR are generally responsive to immunotherapy, such as rIL-2, rational combinations of rIL-2 with potential receptor tyrosine kinase inhibitors based on the pharmacology of individual agents have been evaluated here. The invention provides clinically applicable drug schemes for circumventing potential drug interactions based on the toxicological profile of such agents. The potential for adverse pharmacokinetic / pharmacodynamic interactions and adverse pharmacological interactions is also elucidated. Effects of rIL-2 and small molecule tyrosine kinase inhibitors, such as BAY 43-9006, on T cells and the impact on IL-2-mediated signaling pathways (MAPK eJAK / STAT5) are described.

Thus, the present invention expands the indication of anti-tumor efficacy of IL-2 compounds, such as recombinant IL-2 (rIL-2, also known as aldesleukin) against cancer cell lines that respond poorly to conventional therapies, resulting in long-term survival. increased and enhanced tumor immunity. In addition, the immunomodulatory effects of rIL-2 aim to alleviate existing side effects through administration of anti-angiogenic decompositions for co-administration with asmesmas. Methods of treating an individual experiencing abnormal cellular deproliferation, particularly renal cell carcinoma, using a combination of an IL-2 compound, such as rIL-2, and at least one anti-angiogenic agent, are provided. In addition, methods of treating an individual suffering from abnormal cell proliferation, particularly renal cell carcinoma, using a combination of an IL-2 compound, such as rIL-2, and a small molecule are also provided. Preferred small molecules of the present 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-1H-pyrrole-3-carboxamide (also known as SU11248) and 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea (also known as Bay 43-9006).

Aldesleukin and anti-angiogenic agents may be administered together or separately as individual pharmaceutical compositions. If administered separately, aldesleukin may be administered prior to, concurrent with or subsequent to the anti-angiogenic agent. Particular regimens are provided comprising daily, weekly and monthly dosing schedules (or same interactions) for co-administration of aldesleukin with anti-angiogenic agent, such as 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolinecarboxylic acid. 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-one amine and 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

In one embodiment, the anti-angiogenic agents, preferably one of 6,7-bis (2-methoxyoxy) -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 -ethyl-1H-pyrrole-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine and 1- (4- (2- (methylcarbamoyl) pyridine) 4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea are anti-angiogenic comproteins such as monoclonal antibodies capable of VEGF inhibition co-administered. In a more particular embodiment, the anti-angiogenic proteins of the present invention are bevacizumab or VEGF-Trap. In another more particular embodiment, the protein is an EGF inhibitor such as ketuximab.

In one embodiment, administration of aldesleukin and anti-angiogenic agent (s) together in the manner presented herein potentiates the efficacy of the anti-angiogenic agent, resulting in a positive / synergistic therapeutic response that is improved over that observed with the inhibitor alone.

Other embodiments provide a commercially suitable therapeutic package for the treatment of a cancer patient comprising a container, a therapeutically effective amount of desleukin, and a therapeutically effective amount of an anti-angiogenic agent and / or small molecule, preferably listed in Tables 1-5, such. as 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-carboxamide, N- (4 -chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3 - (trifluoromethyl) phenyl) urea.

In still other embodiments, a kit is provided. Okit comprises a combination of drugs for the treatment of a cancer patient comprising: (a) aldesleukin and (b) a selected anti-angiogenic agent of 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolinomethane. 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-one amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea for simultaneous, sequential or separate use.

Methods of manufacture of the described combinations provided for and considered within the scope of the invention, as well as the use of the combinations in methods for the manufacture of medicaments for use in the invention methods.

Other embodiments of the invention include those described in the detailed description.

BRIEF DESCRIPTION OF THE FIGURE Figure 1 shows the activity of a single agent, rlL-2, BAY 43-9006 or SU11248, in murine T-competent, IL-2-responsive tumor cell models. B16-F10 (2 x 10 6), CT26 colon (2 x 10 6) melanoma cells or RENCA (1 x 10 6) renal carcinoma were implanted s.c. into the right flank of C57BL6 or BALB / c female mice. Treatments were started when the tumors were set at an average size of 50 - 225 mm3, as outlined in the methods. The mice were randomly assigned to treatment groups (10 mice / group). rIL-2 was administered daily subcutaneously (0.2-3 mg / kg / d). BAY 43-9006 or SU11248 (1 - 100 mg / kg) was administered daily as a solution via forced oral ingestion. Figure 1 illustrates the mean inhibition of tumor growth (calculated as [1- (mean tumor volume of treated group / mean tumor volume of vehicle group) x 100]) of a single agent in BI 6- tumor models. FIO, CT26 and RENCA between the days 10-14. Data are compiled from multiple independent studies, with each 10 mouse / group study. * denotes statistical significance vs. vehicle treatment (p <0.05, ANOVA).

Figure 2 shows the effectiveness of concomitant therapy with rIL-2 and BAY 43-9006 in the murine B16-F10 melanoma model. B16-F10 cells (2 x 106 cells) were implanted subcutaneously in the right flank of female C57BL6 mice. Mice were randomly assigned to groups when tumors were -50 mm3 and treatments started on day 0 with vehicle, days 0-6 (♦), rIL-2 (3.3 mg / kg, sc, days 0-6 ) (A) or BAY 43-9006 (30 mg / kg, days 0-6) (x) or rIL-2 (3.3 mg / kg, sc, days 0-6) + BAY 43-9006 (30 mg / kg, po days 0-6) (■) combined. Figure 2 illustrates the mean tumor volume (mm3 + SE) vs. days after randomization (n = 10 mice / group).

Figures 3A and 3B show the effectiveness of sequential therapy with rIL-2 and BAY 43-9006 in the murine B16-F10 melanoma model. B16-F10 cells (2 x 10e cells) were implanted subcutaneously in the right flank of C57BL6 female mice. Mice were randomly assigned to groups when tumors were -50 mm3 and treatments started on day 0. Figure. 3A shows the results of a sequential regimen with rIL-2 administered first, then BAY 43-9006. The panel illustrates: vehicle (♦) days 0-6, 7-13; rIL-2 (3.3 mg / kg, sc, days 0-6) (A) or BAY 43-9006 (30 mg / kg, po days 7-13) (x) or rIL-2 (3.3 mg / kg, sc, days 0-6) + BAY 43-9006 (30 mg / kg, po days 7-13) (•) combined. Figure 3B shows sequential regimen results with first administered BAY 43-9006, followed by rIL-2. The panel illustrates: vehicle (♦) days 0-6, 7-13; BAY 43-9006 (30 mg / kg, p.o. days 0-6) (x); rIL-2 (3.3 mg / kg, sc, days 7-13) () or BAY 43-9006 (30 mg / kg, po days 0-6) + rIL-2 (3.3 mg / kg, sc , days 7-13) (▲) combined. Graphs show mean tumor volume (mm3 + SE) vs. days after randomization (n = 10 mice / group).

Figure 4 shows the efficacy of concomitant therapy with rIL-2 and SU11248 in the murine B16-F10 melanoma model. B16-F10 cells (2 x 106 cells) were implanted subcutaneously in the right flank of C57BL6 female mice. Mice were randomized to groups when tumors were -50 mm3 and treatments started on day 0 with vehicle, days 0-6 (O), rIL-2 (3.3 mg / kg, sc, days 0-6) () or SU11248 (40 mg / kg, days 0-6) (O) or rIL-2 (3.3 mg / kg, sc, days 0-6) + SU1124 8 (40 mg / kg, days 0-6) (A) combined. Figure 4 illustrates the mean tumor volume (mm3 ± SE) vs. days after randomization (n = 10 mice / group).

Figures 5A and 5B show the efficacy of sequential rIL-2 and SU11248 therapy in the murine B16-F10 melanoma model. B16-F10 cells (2 x 106 cells) were implanted subcutaneously in the right flow of female C57BL6 mice. The mice were randomly assigned to groups when the tumors were -50 mm3 and treatments started on day 0. Figure 5A shows the results of sequential regimen with rIL-2 administered first, then SU11248. The panel illustrates: vehicle (O) days 0-6, 7-13; rIL-2 (3.3 mg / kg, sc, days 0-6) (□) or SU11248 (40 mg / kg, po days 7-13) (x) or rIL-2 (3.3 mg / kg, sc, days 0-6) + SU11248 (40 mg / kg, po days 7-13) (•) combined. Figure 5B shows the results of sequential regimen with first administered SU11248, followed by rIL-2. The panel illustrates: vehicle (O) days 0-6, 7-13; SU11248 (40 mg / kg, p.o. days 0-6) (O); rlL-2 (3.3 mg / kg, sc, days 7-13) (A) or SU11248 (40 mg / kg, po days 0-6) + rIL-2 (3.3 mg / kg, sc, days 7-13) combined. Graphs show mean tumor volume (mm3 + SE) vs. days after randomization (n = 10 mice / group).

Figure 6 shows the efficacy of sequential therapy with rIL-2 and BAY 43-9006 in the murine TC26 colon adenocarcinoma model. CT26 cells (2 x 106 cells) were implanted subcutaneously in the right flank of BALB / c female mice. Mice were randomly assigned to groups when tumors were -225 mm3 and treatments started on day 0. The panel illustrates the sequential regimen with rIL-2 administered first, then BAY 43-9006: vehicle (♦) days 0- 6, 7-13; rIL-2 (1 mg / kg, sc, days 0-6) (□) or BAY 43-9006 (40 rog / kg, po days 7-13) (A) or rIL-2 (1 mg / kg, sc , days 0-6) + BAY 43-9006 (40 mg / kg, po days 7-13) (•) combined.

Figure 7 shows the efficacy of concomitant treatment with rIL-2 and SU11248 in the murine CT26 colon adenocarcinoma model. CT26 cells (2 x 106 cells) were implanted subcutaneously in the right flank of BALB / c female mice. Mice were randomly assigned to groups when tumors were -225 mm3 and treatments started on day 0. The panel illustrates: vehicle (♦) days 0-6; rIL-2 (1 mg / kg, sc, days 0-6) (□) or SU11248 (40 mg / kg, po days 0-6) (O) or rIL-2 (1 mg / kg, sc, days 0 -6) + SU11248 (40 mg / kg, days 0-6) (•) combined.

Figures 8A and 8B show the efficacy of sequential therapy with rIL-2 and SU11248 in the murine CT26 colon adenocarcinoma model. CT26 cells (2 x 106 cells) were implanted subcutaneously in the right flank of BALB / c female mice. The mice were randomly assigned to groups when the tumors were -225 mm3 and treatments started on day 0. The panel illustrates: Figure 8A shows the results of the sequential regimen with rIL-2 administered first, then SU11248: vehicle (♦ ) days 0-6, 7-13; rIL-2 (1 mg / kg, sc, days 0-6) (□) or SU11248 (40 mg / kg, po days 7-13) (O) or rIL-2 (1 mg / kg, sc, days 0 -6) + SU11248 (40 mg / kg, days 7-13) (■) combined. Figure 8B shows the results of the sequential regimen with SU1124 8 administered first, followed by rIL-2. The panel illustrates: vehicle (♦) days 0-6, 7-13; SU11248 (40 mg / kg, p.o. days 0-6) (O); rIL-2 (3.3 mg / kg, sc, days 7-13) (□) or SU11248 (40 mg / kg, po days 0-6) + rIL-2 (3.3 mg / kg, sc, days 7-13) (A) combined. Graphs show mean tumor volume (mm3 + SE) vs. days after randomization (n = 10 mice / group).

Figure 9 shows the efficacy of concomitant therapy with rIL-2 and BAY 43-9006 in the murine RENCA RCC tumor model. RENCA cells (1 x 106 cells) were implanted subcutaneously in the right flank of female BALB / c mice. Mice were randomly assigned to groups when tumors were -50 mm3 and treatments started on day 0 with vehicle, days 0-8 (♦), rIL-2 (1 mg / kg, sc, days 0-4, 7-11) (A) or BAY 43-9006 (30 mg / kg, po days 0-8) (x) or rIL-2 (1 mg / kg, sc, days 0-4, 7-11) + BAY 43-9006 (30 mg / kg, po days 0-8 (■) combined. Figure 9 illustrates the mean tumor volume (mm3 + SE) vs. days after randomization (n = 10 mouse / group).

Figure 10 shows the efficacy of concomitant treatment with rIL-2 and SU11248 in the murine RENCA RCC tumor model. RENCA cells (1 x 106 cells) were implanted subcutaneously in the right flank of female BALB / c mice. The mice were randomly assigned to groups when tumors were -50 mm3 and treatments started on day 0. The panel illustrates: vehicle (♦) days 0-6; rIL-2 (1 mg / kg, sc, days 0-6) or SU11248 (40 mg / kg, po days 0-6) (x) or rIL-2 (1 mg / kg, sc, days 0-6) + SU11248 (40 mg / kg, days 0-6) (A) combined.

DETAILED DESCRIPTION OF THE INVENTION

Using a detection assay, several types of cancers were characterized as susceptible to treatment with anti-angiogenesis / VEGF inhibitors and cytokine administration or rIL-2 modulation. Such cancers include, for example, CML, AML, breast, gastric, endometrial, salivary, adrenal, non-small cell lung, pancreatic, renal, rectal, skin, melanoma, multiple myeloma, brain / CNS , cervix, nasopharynx, malignant mesothelioma, hypopharynx, gastrointestinal carcinoid, peritoneum, omentum, mesentery, gallbladder, testis, esophageal, lung, thyroid, ovarian, peritoneal, prostate, head and neck, bladder, colon, colon, lymphomas and glioblastomas. The methods described herein are useful in treating any of such cancers.

Therapy with a combination of aldesleukin and at least one anti-angiogenic agent as presented herein causes a physiological response that is beneficial with respect to the treatment of cancers whose continuously proliferating cells are highly dependent on vascularization, such as VEGF.

One embodiment of the invention provides a method of treating a cancer patient suffering from hypotension from aldesleukin administration comprising: co-administering to the patient a therapeutically effective amount of an anti-angiogenic agent to attenuate hypotension.

Another more particular embodiment thereof comprises cancer relief in the patient.

Another embodiment of the invention provides a method of treating a cancer patient suffering from hypertension from administering an anti-angiogenic agent comprising: co-administering to the patient a therapeutically effective amount of aldesleukin to alleviate hypertension.

Another more particular embodiment thereof comprises cancer relief in the patient. In certain embodiments, cancer is susceptible to angiogenesis inhibition and / or immune stimulation.

In a more particular embodiment of any of the foregoing embodiments, said anti-angiogenic agent is selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2 - (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a method of treating a cancer patient comprising administering to said patient aldesleukin and a compound selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a method of increasing the efficacy of aldesleukin in a cancer patient comprising first administering an anti-angiogenic agent at a dose capable of inducing hypoxia in the patient, then administering aldesleukin.

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

In a more particular embodiment thereof, said anti-angiogenic agent is selected from 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-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl)) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a method for treating a cancer patient comprising the steps of first administering to said patient a therapeutically effective amount of aldesleukin, followed by administration of an anti-angiogenic agent (as) selected from 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-one amine, N- (2- (dimethylamino) ethyl) -5 - ((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrole-3-carboxamide, N- (4 -chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3 - (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a method for treating a cancer patient comprising the steps of first administering to said patient a therapeutically effective amount of an anti-angiogenic agent, such as 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - (( pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, followed by administration of aldesleukin.

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

In a more particular embodiment thereof, said anti-angiogenic agent is administered from 1 to 6 times every 2-3 weeks.

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

In a more particular embodiment thereof, the anti-angiogenic agent is selected from 6,7-bis (2-methoxyoxy) -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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin (4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

In a more particular embodiment thereof, aldesleukin is administered intravenously and said rest period is present.

In a more particular embodiment thereof, aldesleukin is administered subcutaneously and said rest period is absent.

In a more particular embodiment thereof, aldesleukin is administered 3 times daily at a dose of about 30-100 MIU / day.

In a more particular embodiment thereof, aldesleukin is administered for a period of 5 consecutive days, followed by a period of 9 days of rest. In a more particular embodiment thereof, said dosage schedule is repeated for at least two courses. In an even more particular embodiment, each course consists of 2-5 days of treatment, followed by a rest period of 9-15 days.

In another more particular embodiment thereof, aldesleukin is administered 3 times during the first day and once a day for the following days.

In a more particular embodiment thereof, said dosage schedule is repeated for at least two courses or 3 courses or 5 courses or 6 courses or 7 courses or 8 courses or 9 courses or 10 courses.

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

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

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

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and:

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

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and:

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

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and: a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt of the tautomer.

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and:

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

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and:

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

Another embodiment of the invention provides a method of treating a cancer patient comprising administering aldesleukin and a compound of formula I:

<formula> formula see original document page 28 </formula> where,

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

R2 is H or alkyl; or

Rx and R2 are joined together to form R1-2;

R 3 is H, -CN, -OH, halogen, alkyl, aryl, alkoxy, NR a R 1, -C (O) R c, -S (O) n R a or heterocyclyl;

R 4 is H, -CN, -OH, halogen, alkyl, aryl, -O- (CH 2) q R g, -O- (CH 2) q O -Re, -NRaRb, -S (O) nRd or -heterocyclyl-Rf ;

R 5 is H, -CN, -OH, halogen, alkyl, aryl, -O- (CH 2) q R g, -O- (CH 2) q O -Re, -NRaRb, -S (O) nRd 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 Ra and Rb is independently H, alkyl, -C (O) R 0, aryl, heterocyclyl or alkoxy; or

Ra and Rb are joined together to form Ri-2;

each R c is independently H, alkyl, alkoxy, C (O) alkyl, -C (O) aryl, -CHO, aryl or heterocyclyl;

each Rd is independently H, alkyl, alkenyl, aryl or -NRa R b;

each Re is independently H or alkyl;

Rf is H, halogen, -OH, -CN, - (CH2) q NRa Rh, alkoxy, -C (O) Rc, - (CH2) q CH3;

each Rg is independently H, halogen, -C (O) Rc, aryl, heterocyclyl or -NRaRb Rh is H or - (CH2) qS (O) nRd;

each R 1 is independently H, halo, alkyl, alkenyl, alkynyl or -O (CH 2) q -R 6;

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;

Rx-2 has the general structure as shown:

<formula> formula see original document page 30 </formula>

on what,

each R 8 is independently H, -OH, halogen, alkyl, alkoxy, -NR a R b or -S (O) n R a;

each R 9 is independently H, alkyl, -C (O) R c is absent if X is O, S or is absent,

each X is independently O, S, N, CH or is absent thereby forming a covalent bond; and

each m is independently 0, 1 or 2, or a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable dotautomer salt.

In a more particular embodiment thereof, said

a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt dotautomer.

In a more particular embodiment thereof, the referred compound is:

<formula> formula see original document page 30 </formula> a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt dotautomer.

Another embodiment of the invention provides a method for treating a cancer patient comprising administering aldesleukin and a compound of formula II:

on what:

R11 is alkyl, aryl or heterocyclyl;

R 12 / R 13 / R 14, and R 15 are independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NR a R b, -C (O) n R d / or heterocyclyl;

each Ra and Rb is independently H, alkyl, -C (O) Rz, aryl, heterocyclyl, or alkoxy;

each Rz is independently H, alkyl, alkoxy, -NH2, NH (alkyl), -N (alkyl) 2 / aryl, or heterocyclyl; each R d is independently H, alkyl, alkenyl, aryl, or -NRaRb;

each n is independently 0, 1, or 2; each q is independently 0, 1, or 2; or

a tautomer thereof, a pharmaceutically acceptable salt tautomer.

In a more particular embodiment thereof, RX1 is Rna:

R16f R17 and RX8 are independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NRaRb, -C (0) Rz, -S (0) nRd

<formula> formula see original document page 31 </formula>

wherein: or heterocyclyl.

In a more particular embodiment thereof, the referred compound is:.

a tautomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt dotautomer.

Another embodiment of the invention provides a method for treating a cancer patient comprising administering aldesleukin and a compound of formula III:

<formula> formula see original document page 32 </formula>

on what,

the dotted line represents an optional placement of an additional bond;

<formula> formula see original document page 32 </formula>

R-21, R22, R23 and R24 are each independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NRaRb, -C (O) R2, -S (O) nRd or heterocyclyl;

R25 is H, alkyl or -C (O) R2;

R 26 and R 27 are each independently H, -OH, halogen, alkyl, -NRaRb, -C (O) R 2, or -S (O) nRd; R 2s is H, -CH 3 or halogen;

each Ra and Rb is independently H, alkyl, -C (O) Rz, aryl, heterocyclyl or alkoxy;

each Rz is independently H, alkyl, alkoxy, -NH2 / -NH (alkyl), -N (alkyl) 2, aryl or heterocyclyl;

each Rd is independently H, alkyl, alkenyl, aryl or -NRa R b /;

each n is independently 0, 1 or 2; and

each q is independently 0, 1 or 2;

or a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable dotautomer salt.

In a more particular embodiment thereof, the referred compound is:

<formula> formula see original document page 33 </formula>

a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt dotautomer.

In a more particular embodiment thereof, the referred compound is:

<formula> formula see original document page 33 </formula>

a tautomer thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt dotautomer.

Another embodiment of the invention provides a method of decreasing the toxicity associated with administering dealdesleukin to a cancer patient comprising administering a quinolinone, preferably an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyoxy) -N- (3). -etinylphenyl) 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl ) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, the patient.

Another embodiment of the invention provides a method for reducing toxicity associated with administration of an anti-angiogenic agent, preferably selected from 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 N-(4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4-ylidene) methyl) -2,4-diethyl-1H-pyrrola-3-carboxamide - (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, to a cancer patient comprising dealdesleukin administration to the patient.

Another embodiment of the invention provides a method of decreasing IL-2 resistance in a decaying patient comprising administering an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyoxy) -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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin -1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea referred to above.

Another embodiment of the invention provides a method for treating a cancer patient, comprising the steps of first administering said patient a therapeutically effective amount of aldesleukin, followed by administering an anti-angiogenic agent, preferably selected from 6.7. -bis (2-methoxyoxy) -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-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl ) phenyl) urea.

Another embodiment of the invention provides a method for treating a cancer patient, comprising administering separately to said patient a therapeutically effective amount of an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxy) -N- (3-Ethinylphenyl) 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea and ealdesleucine according to a scheme Aldesleukin is administered from 1 to 3 times daily at a dose of from about 9 to about 130 MIU / day for a period of at least 3 consecutive days, optionally followed by a rest period of at least 3 consecutive days. More specifically, the dose is between about 30 and about 100 MIU / day. More specifically, the dose is between about 30 and about 60 MIU / day. More specifically, the dose is between about 30 and about 40 MIU / day. More specifically, the dose is between about 17 and about 30 MIU / day. More specifically, the dose is between about 9 and about 30 MIU / day.

In a more particular embodiment, said anti-angiogenic agent, preferably selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin (4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is administered from 1 to 9 times every 2-3 weeks. In a more particular embodiment, said anti-angiogenic agent, preferably selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin (4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is a VEGF inhibitor.

In another embodiment, the anti-angiogenic agent, preferably selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4- yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea, combevacizumab or cetuximab or VEGF-Trap is also administered. More specifically, said bevacizumab is administered at a dose between 1 and 12 mg / kg once every 12-16 days.

In another embodiment, the anti-angiogenic agent is administered within 72 hours of rIL-2 administration, where "within" is intended to indicate before or after, as in: the anti-angiogenic agent is administered 72 hours before or 72 hours. hours after rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 14 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 7 days of rIL-2 administration. . In another embodiment, the anti-angiogenic agent is administered within 6 days of rIL-2 administration. Otherwise, the anti-angiogenic agent is administered within 5 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 4 days of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 48 hours of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 36 hours of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 24 hours of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 12 hours of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 6 hours of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered within 1 hour of rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered at the same time as rIL-2 administration. In another embodiment, the anti-angiogenic agent is administered in combination with rIL-2. In a more particular embodiment thereof, the anti-angiogenic agent is selected from 6,7-bis (2-methoxyoxy) -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 -ethyl-1H-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridine) 4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a method for treating a cancer patient comprising aldesleukin administration and an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolinomethane. 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-one amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment of the invention provides a commercially suitable therapeutic package for treating a cancer patient comprising a container, a therapeutically effective amount of desleukin and a therapeutically effective amount of an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyoxy). ) -N- (3-Ethinylphenyl) 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin -4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea. A therapeutic package thereof wherein said patient is suffering from renal cell carcinoma. A therapeutic package thereof, further comprising written instructions to guide the patient receiving treatment withldesleukin prior to treatment with the anti-angiogenic agent, preferably selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1 -amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

Another embodiment provides a pharmaceutical composition comprising an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyoxy) -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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin -4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea and aldesleucine.

Clear cell renal cell carcinoma (CCR) is associated with increased vascularization, particularly VEGF expression. VEGF is a protein that plays a crucial role in tumor angiogenesis (the formation of new tumor blood vessels) and maintenance of established tumor blood vessels. It binds to specific receptors on blood vessels to stimulate extensions to existing blood vessels. Some, but not all RCCa, have increased serum VEGF levels.

There is evidence that increased VEGF correlates with resistance to IL-2 (Lissoni ecolaboradores, 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) could represent a mechanism for VEGF-induced resistance to IL-2 treatment.

In addition, VEGF increases the permeability and it has been observed that anti-VEGF treatment leads to increased blood pressure in some patients. Therefore, it is likely that some of the major toxicities of anti-VEGF (eg, hypertension) and rIL-2 (eg, hypotension) would be counteracting and would lead to an improved overall therapeutic index if the combinations of the present invention were administered to patients suffering from the disease. cancer, such as RCCa.

Other combinations of the present invention, such as aldesleukin with bevacizumab or cetuximab, provide a greater proportion and / or better durability of responses compared to agents administered alone.

The particular aldesleukin dosage regimens disclosed herein provide for intermittent stimulation of natural killer cell (NK) reactivity and decreased risk of rIL-2 related side effects that may be associated with long-term exposure to rIL-2 dosing. Note, hypotension typically associated with dosage with rIL-2 is compensated for by administration of the anti-angiogenic compositions of the present invention since they are generally associated with hypertension. In addition, hypoxytypically associated with VEGF inhibitors would increase sensitivity to efficacy of rIL-2 treatment, while providing palliative benefits at the same time.

Another embodiment of the invention provides for the use of desleukin in the manufacture of a cancer treatment medicament, wherein said medicament is separate, simultaneous or sequential use with an anti-angiogenic agent, preferably selected from 6,7-bis (2-methoxyoxy). ) -N- (3-Ethinylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimelhylamino ) ethyl) -5 - ((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin -4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea. In a more particular embodiment thereof, said anti-angiogenic agent, preferably selected from 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-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl)) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea is a lactate salt. In another embodiment, said cancer is renal cell carcinoma.

Another embodiment of the invention provides the use of an anti-angiogenic agent, preferably selected from 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-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl)) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea for the manufacture of a cancer treatment medicament wherein said medicament is for separate, simultaneous or sequential use with aldesleukin.

Preferred angiogenes-associated molecules (such as inhibition) by the compositions of the present invention include: Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF), Interleukin-8 (IL-8), Angiogenin, Angiotropin, Epidermal Growth Factor (EGF), Platelet-Derived Endothelial Cell Growth Factor, Transformation Growth Factor a (THF-a), Transformation Growth Factor b (TGF-b) or Nitric Oxide. Also, modulation (such as potentiation) by the present compositions The invention of Thrombospondin, Angiostatin and Endostatin is considered within the present invention.

General Compositions For Co-administration with rIL-2: QUINAZOLINE

Compounds of Formula I have the following structure:

<formula> formula see original document page 43 </formula>

on what,

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

R2 is H or alkyl; or

R 1 and R 2 are linked together to form R 1-2;

R3 is H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NRaRb, -C (O) Rc, -S (O) nRa or heterocyclyl;

R4 is H, -CN, -OH, halogen, alkyl, aryl, -O- (CH2) q -Rg, -O- (CH2) qO-Re, -NRaRb, -S (O) nRd or -heterocyclyl-Rf ;

R5 is H, -CN, -OH, halogen, alkyl, aryl, -O- (CH2) q -Rg, -O- (CH2) q -O-Re, -NRaRb, -S (O) nRd 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 Ra and Rb is independently H, alkyl, -C (O) R 0, aryl, heterocyclyl or alkoxy; or

Ra and Rb are joined together to form Ri-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 -NRa R b, each Re is independently H or alkyl;

Rf is H, halogen, -OH, -CN, - (CH2) q NRa Rh, alkoxy, -C (O) RC1 - (CH2) q CH3;

each Rg is independently H, halogen, -C (O) Rc, aryl, heterocyclyl or -NRaRb;

Rh is H OR - (CH 2) q S (0) n Rd;

each R 1 is independently H, halo, alkyl, alkenyl, alkynyl or -O (CH 2) q -R 6;

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;

R1-2 has the general structure as shown:

each R 8 is independently H, -OH, halogen, alkyl, alkoxy, -NR a R b or -S (O) n R a;

each R 9 is independently H, alkyl, -C (O) R c is absent if X is O, S or is absent;

each X is independently O, S, N, CH or is absent thereby forming a covalent bond; and

each m is independently 0, 1 or 2.

In a more particular embodiment of the invention, R 2 is H. In an even more particular embodiment, R x is -aryl (R 1) p. In another embodiment wherein R 1 is -aryl (R 1) P; aryl within R 1 is phenyl, p within R 1 is 2 and both R 1 within R 1 are halo.

In another embodiment wherein R 1 is -aryl (R 1) p, R 1 aryladenyl is phenyl, p within R 1 is 1 and Ridentro R 1 groups are alkynyl, preferably ethinyl.

<formula> formula see original document page 44 </formula>

wherein, In another embodiment wherein R1 is -aryl (Ri) P, the aryladent of R1 is phenyl, p within R1 is 2, one group R1 within Rx is halo and the other group R1 within R1 is -0 (CH 2) q -Rg. In a more particular embodiment thereof, where R 1 is 1 and R g within R 1 is halophenyl.

In another embodiment wherein R 1 is -aryl (R 1) p, within R% is 1 and R 1 within R 1 is alkynyl.

Another more particular embodiment of the invention is provided, wherein R 1 and R 2 are linked together to form R 1-2:

<formula> formula see original document page 45 </formula>

wherein R8 is H, X is Ne R g -C (O) NHRb. In a more particular embodiment thereof, Rb within R 9 is -phenyl-O-CH 2 (CH 3) 2.

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

In a more particular embodiment of the invention, R 4 is -0- (CH 2) q -Rg. In a more particular embodiment thereof, where R4 is 1 and Rg is H.

In another embodiment wherein R4 is -O- (CH2) q -Rg, Rg within R4 is heterocyclyl.

In a more particular embodiment of the invention, R 4 and R 5 are each -O- (CH 2) q -O-Re. In a more particular embodiment thereof, q within R4 and R5 is 2 and Re within R4 and R5 is methyl.

In a more particular embodiment of the invention, R4 is -heterocyclyl-Rf and R5 is H. In a more particular embodiment thereof, said heterocyclyl within R4 is furanyl. In an even more particular embodiment, Rf within R4 is - (CH2 ) gNHRh. Still, Rh is - (CH2) qS (O) 2CH3.

In a more particular embodiment of the invention, R 5 is -0- (CH 2) q -R 6. In another embodiment thereof, Rg within R5 is heterocyclyl.

Another embodiment is provided, wherein R7 is H.

Another embodiment is provided, wherein R3, R6 and R7 are all H.INDOLINONA

Formula II compounds have the following structure:

<formula> formula see original document page 46 </formula>

R11 is alkyl, aryl or heterocyclyl;

R 12 R 13 / R 14 and R 8 are each independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NR a R b / -C (O) R 2, -S (O) n R d or heterocyclyl;

each Ra and Rb is independently H, alkyl, -C (O) Rz, aryl, heterocyclyl or alkoxy;

each Rz is independently H, alkyl, alkoxy, -NH2, -NH (alkyl), -N (alkyl) 2, aryl or heterocyclyl;

each Rd is independently H, alkyl, alkenyl, aryl or -NRa R b;

each n is independently 0, 1 or 2; and

each q is independently 0, 1 or 2.

In a more particular embodiment, Rn is heterocyclyl.

In an even more particular embodiment, R11 and R11a

<formula> formula see original document page 46 </formula>

in which in which

R16 and R17 are each independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NRaRb, -C (O) R2, S (O) nRd or heterocyclyl. In yet another more particular embodiment wherein R 1 is R 1, R 7 is -C (O) - (CH 2) PN (H) (2 r) (alkyl) r, wherein p is 0, 1, 2, 3, 4 or 5 and r is 0, 1 or 2. In yet another more particular embodiment wherein Rn is Rua / R13 is F. In yet another more particular embodiment wherein Rn is Rua, R17 is - (CH2) t C00H, where t is 1, 2, 3 or 4. Yet another more particular embodiment wherein Rn is R11a, Ree R8 are both methyl. In yet another more particular embodiment wherein Rn is Rlla, R17 is H.

In yet another more particular embodiment of the invention, Rn is aryl. Even more particularly, Rn is substituted or unsubstituted phenyl. In another embodiment, Ri 3 is diode.

ISOINDOLINONES

Formula III compounds have the following structure:

<formula> formula see original document page 47 </formula>

on what,

the dotted line represents an optional placement of an additional bond: R 20 is H or = 0;

R21 / R22, R23 and R24 are each independently H, -CN, -OH, halogen, alkyl, aryl, alkoxy, -NRaRb, -C (O) R2, -S (O) nRd or heterocyclyl;

R25 is H, alkyl or -C (O) R2;

R 26 and R 27 are each independently H, -OH, halogen, alkyl, -NR a R b, -C (O) R 2, or -S (O) n R c R 28 is H, -CH 3 or halogen;

each Ra and Rb is independently H, alkyl, -C (O) R2, aryl, heterocyclyl or alkoxy;

each Rz is independently H, alkyl, alkoxy, -NH2, -NH (alkyl), -N (alkyl) 2, aryl or heterocyclyl;

each Rd is independently H, alkyl, alkenyl, aryl or -NRa R b;

each n is independently 0, 1 or 2; and

each q is independently 0, 1 or 2.

DEFINITIONS:

AUC Area under the curve

CR Full Answer

DLT Dose-Limiting Toxicity

IL Interleukin

IL-2 Interleukin-2

UI International Units

IV Intravenous (as a mode of administration)

LAK Lymphokine-Activated Killer

MTD Maximum tolerated dosage

MIU International Units per million

NK Natural Killer

PK Pharmacokinetics

PR partial answer

RCC Renal Cell Carcinoma

RCCa Clear Cell Renal Carcinoma

rIL-2 Recombinant Interleukin-2

RTK receptor tyrosine kinase

TNF Tumor necrosis factor

VEGF Vascular Endothelial Growth Factor In general, reference to a particular element, such as hydrogen or H, should be understood to include all isotopes of that element. For example, if an R group is defined as including hydrogen or H, it also includes deuterium and tritium.

By "anti-angiogenic agent" is meant any small molecule, more specifically any having a molecular weight of less than 1,100 g / mol, which shows or will be shown to suppress angiogenesis in a system. Preferred molecules associated with angiogenesis modulated (such as inhibition) by 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), Endothelial Cell Growth Factor Platelets, Transforming Growth Factor a (THF-a), Transforming Growth Factor b (TGF-b) or Nitric Oxide. Also, modulation (such as potentiation) by the present invention compositions of Thrombospondin, Angiostatin and Endostatin is contemplated within the present invention. Preferred anti-angiogenic compositions of the present invention include: 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro-4 -fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5- ((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl-1H pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine and 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl ) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea.

The term "effective amount" is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a compound to treat renal cell carcinoma may be an amount necessary to cause tumor cell regression in renal cells. The effective amount may vary depending, for example, on the condition treated, weight of the individual and severity of the disease. Those skilled in the art can readily determine the effective amount empirically without undue experimentation.

As used herein, an "effective treatment amount" refers to an amount sufficient to calm, alleviate, stabilize, reverse, slow, or slow the progression of a condition, such as a sick state.

An "individual" or "patient" is intended to describe a human or vertebrate animal, including a dog, cat, pocket animal, tamarin, horse, cow, pig, sheep, goat, elephant, giraffe, chicken, lion, monkey, owl, rat, squirrel, lory and mouse.

A "pocket animal" refers to a group of vertebrate animals that can fit into a comfortable pouch such as hamsters, chinchillas, ferrets, rats, guinea pigs, gerbils, rabbits and sugar petauros.

As used herein, the term "pharmaceutically acceptable ester" refers to esters which hydrolyze in vivo and include those which readily decompose in the human body to leave the precursor compound or a salt thereof. Suitable ester groups include, for example, those derived from carboxylic acids pharmaceutically acceptable aliphatics, particularly alkanoic, alkenic, cycloalkanoic and alkanedioic acids, wherein each alkyl or alkenyl moiety advantageously has no more than 6 carbon atoms. Representative examples of esters include, but are not limited to, formats, acetates, propionates, butyrates, acrylates and ethyl succinates.

The compounds of the present invention may be used as salts, as in "pharmaceutically acceptable salts" derived from inorganic or organic acids. These salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzene -sulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate,

cyclopentanopropionate, dodecyl sulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, sulfate, 3-phenylpropionate, picrate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, groups containing basic nitrogen may be quaternized with agents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromide and iodide; dialkyl sulphates such as dimethyl, diethyl, dibutyl and diamyl sulphates, long halides such as chloride, bromide and iodide dedecyl, lauryl, myristyl and stearyl, dearalkyl halides such as benzyl and phenethyl bromides and others. Dispersible or oil-soluble products are thereby obtained. The term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of medical judgment, suitable for use in contact with lower human and animal tissues without toxicity, irritation, similar undesirable, commensurate undesirable allergy response with a proportion

reasonable and effective benefit / risk for their intended use, as well as zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to provide the precursor compound of the above formula, for example by hydrolysis in a blood. A full discussion is provided in T. Higuchi and V. Stella, Pro-drugs as NovelDelivery Systems, Vol. 14 of the A. CS series. Symposium and in Edward B. Roche, ed., Bioreversible Carriers in DrugDesign, American Pharmaceutical Association and PergamonPress, 1987. Prodrugs, as described in U.S. Patent. No. 6,284,772, for example, may be used.

Reference to "halo", "halide" or "halogen" refers to atoms of F, Cl, Br or I.

The word "alkyl" refers to unsubstituted and substituted alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The word also includes branched chain isomers of straight chain alkyl groups including, but not limited to, the following, which are provided by way of example: -CH (CH3) 2, -CH (CH3) (CH2CH3), -CH (CH2CH3) ) 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 ( CH2CH3) 3, - CH (CH3) CH (CH3) (CH2CH3), - CH2CH2CH (CH3) 2, CH2CH2CH (CH3) (CH2CH3), -CH2CH2CH (CH2CH3) S, -CH2CH2C (CH3) 3, CH2CH2C (CH2CH3) 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 others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl and rings substituted by straight and branched chain alkyl groups as defined above. The word also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl and bicyclo [2.2.2] octyl and such rings substituted by straight and branched chain alkyl groups as defined above. The word alkyl also includes groups in which one or more bonds to carbon (s) or hydrogen (s) are substituted by a bond to non-hydrogen and non-carbon atoms such as, but not limited to, a halogen atom in halides such as F Cl, Br and I; and a deoxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups and ester groups; a sulfur atom in groups such as thiol groups, alkyl and arylsulfide groups, sulfone groups, sulfonyl groups and group sulfoxide; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides and eenamines; a silicon atom in groups such as triaalkyl silyl, dialkylaryl silyl groups, alkyldiaryl silyl groups and triaryl silyl groups; and other heteroatoms in several other groups. Alkyl groups are those limited to having 1 to 20 carbon atoms and as much as 5 additional heteroatoms, as described above. More preferred alkyl groups have from 1 to 5 carbon atoms and as much as 2 heteroatoms.

The word "aryl" refers to substituted and unsubstituted aryl groups that do not contain heteroatoms. Thus, the word includes, but is not limited to, groups such as phenyl, biphenyl, anthracenyl, naphthenyl, by way of example. Aryl groups also include those in which one of the aromatic carbons is attached to a non-carbon or non-hydrogen atom described above (in the definition of alkyl) and also includes aryl groups in which one or more aromatic carbons of the aryl group are attached to a substituted and / or unsubstituted alkyl, alkenyl or alkynyl group as defined herein. This includes bonding arrangements in which two carbon atoms of an aryl group are attached to two atoms of an alkyl, alkenyl or alkynyl group to define a fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, the word "aryl" includes, but is not limited to, tolyl and hydroxyphenyl, among others.

The word "alkenyl" refers to straight and branched chain cyclic groups, such as those described with respect to alkyl groups, as defined above, except that at least one double bond exists between two carbon atoms. Examples include, but are not limited to, vinyl, -CH = C (H) (CH 3), -CH = C (CH 3) 2, -C (CH 3) = C (H) 2, C (CH 2) = C ( H) (CH 3), -C (CH 2 CH 2) = CH 2, cyclohexenyl,

cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl and hexadienyl, among others. Included are also groups in which a non-carbon or non-hydrogen atom is bonded to a carbon double bonded to another carbon and to those in which one of the non-carbon or non-hydrogen atom is bonded to a carbon not involved in a bond. double to another carbon. Alkenyl groups are those limited to having 2 to 15 carbon atoms and as much as 4 additional heteroatoms, as described above. More preferred alkenyl groups have from 2 to 5 carbon atoms and as much as 2 heteroatoms.

The word "alkoxy" refers to substituted or unsubstituted alkoxy groups of the formula -O-alkyl, wherein the fixing point is the oxy group and the alkyl group is as defined above. Alkoxy groups are those limited to having 1 to 20 carbon atoms and as much as 5 additional hetero atoms, including the oxygen atom. More preferred alkoxy groups have from 1 to 5 carbon atoms and as much as 2 hetero atoms, including the oxygen atom.

The word "alkynyl" refers to straight and branched chain groups, such as those described with respect to alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Examples include, but are not limited to, C = C (H), -C 3 C (CH 3), -C = C (CH 2 CH 3), -C (H 2) C = C (H), -C (H) 2 C = C (CH 3) and -C (H) 2 C = C (CH 2 CH 3), among others. Included are also alkynyl groups in which a non-carbon or non-hydrogen atom is included in a triple carbon attached to another carbon and those in which a non-carbon or non-hydrogen atom is attached to a carbon not involved in a carbon. triple bond to another carbon. Alkynyl groups are those limited to having 2 to 15 carbon atoms and as much as 4 additional heteroatoms, as described above. More preferred alkynyl groups have from 2 to 5 carbon atoms and as much as 2 heteroatoms.

The word "heterocyclyl" refers to aromatic and non-aromatic ring compounds, including monocyclic, bicyclic and polycyclic ring compounds such as, but not limited to, quinuclidyl containing 3 or more elements in ring 1, of which one is a but not limited to, N, O and S. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3- to 8-membered rings containing 1 to 4 nitrogen atoms such as but not limited to, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g. 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1 2,3-triazolyl, etc.), tetrazolyl, (e.g. 1H-tetrazolyl, 2H-tetrazolyl, etc.); saturated 3- to 8-membered rings containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; unsaturated condensed heterocyclic groups containing 1 to 4 nitrogen atoms such as, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl; unsaturated 3- to 8-membered rings containing 1 to 2 oxygen atoms such as, but not limited to, furanyl; unsaturated 3- to 8-membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (e.g. 1,2,4-oxadiazolyl, 1,3, 4-oxadiazolyl, 1,2,5-oxadiazolyl, etc); saturated rings of 3 to 8 elements containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, 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- to 8-membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolyl, isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3- to 8-membered rings containing 1 to 2 sulfur atoms and 3 nitrogen atoms such as, but not limited to, thiazolodinyl; saturated and unsaturated 3- to 8-membered rings containing 1 to 2 sulfur atoms such as, but not limited to, thienyl), dihydrodithynyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; condensed unsaturated heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g. 2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g. 2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated condensed heterocyclic rings containing 1 to 2 oxygen atoms, such as benzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3- to 8-membered rings containing one oxygen atom and 1 to 2 sulfur atoms such as, but not limited to, dihydroxyatiinyl; saturated 3- to 8-membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms, such as 1,4-oxatian; condensed unsaturated rings containing 1 to 2 sulfur atoms, such as benzothienyl, benzodithynyl; and heterocyclic condensed unsaturated rings containing one oxygen atom 1 to 2 oxygen atoms, such as benzoxathiinyl. The heterocyclyl group also includes those described above, wherein one or more ring S atoms are double bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include

tetrahydrothiophene, tetrahydrothiophene oxide and tetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5 or 6 ring elements. More preferred heterocyclyl groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine in which the thiomorpholine S atom is attached to a or more O, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan and tetrahydrofuran atoms. "Heterocyclyl" also refers to those groups as defined above wherein one of the elements in the ring is attached to a non-hydrogen atom as described above with respect to substituted alkyl groups and substituted aryl groups. Examples include, but are not limited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1-methyl piperazinyl and 2-chloropyridyl, among others. Heterocyclyl groups are those limited to having 2 to 15 carbon atoms and as much as 6 additional heteroatoms, as described above. More preferred heterocyclyl groups have from 3 to 5 carbon atoms and as much as 2 heteroatoms.

The term "substituted", when applied to an indefinite group, still well known in the art, such as phenyl, will have the same meaning with respect to optional attachments as described in the definition of alkyl.

Within the present invention, it should be understood that compounds described herein may exhibit the phenomenon of tautomerism and that formulas drawn within the present specification may represent only one of the possible tautomeric forms. It should be understood that the invention encompasses any tautomeric form which has anti-angiogenic activity and is not intended to be limited merely to any of the tautomeric forms used within the formula drawings.

It should also be understood that certain compounds and embodiments of the invention may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It should be understood that the invention encompasses all of such solvated forms which have anti-angiogenic activity.

The invention also includes isotopically labeled compounds which are structurally identical to those disclosed above except that one or more atoms are replaced by an atom having a different atomic mass or mass number than the atomic mass or mass number usually found in nature. Examples of isotopes which may be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F and 36Cl, respectively. Compounds of the present invention, prodrugs thereof and pharmaceutically acceptable salts of said compounds and said prodrugs containing the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, for example, those in which radioactive isotopes, such as He C, are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated isotopes, i.e. 3H and carbon-14, i.e. 14C, are particularly preferred for their ease of preparation and detectability. In addition, substitution by heavier isotopes, such as deuterium, i.e. 2H, may provide certain therapeutic advantages resulting from increased metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and may therefore be preferred in some circumstances. Isotopically labeled compounds of the present invention and prodrugs thereof may generally be prepared by performing known or mentioned procedures and by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

Reference to "IL-2" or "interleukin-2" indicates a lymphocyte that is produced by normal peripheral blood lymphocytes and is present in the body at low concentrations. IL-2 was first described by Morgan et al. (1976) Science 193: 1007-1008 and originally called T cell growth factor because of its ability to induce proliferation of stimulated T lymphocytes. It is a protein with a reported molecular weight in the range of 13,000 to 17,000 (Gillis and Watson (1980) J. Exp. Med. 159: 1709) and has an isoelectric point in the range of 6-8.5. For purposes of the present invention, the term "IL-2" is intended to encompass any source of IL-2, including mammalian sources such as, for example, mouse, rat, rabbit, primate, pig, pocket animals and humans. and may be native or recombinantly obtained, such as recombinant IL-2 polypeptides produced by various microbial hosts. IL-2 may be the native polypeptide sequence or may be a variant of the native IL-2 polypeptide as described herein below, as long as the IL-2 variant polypeptide retains the biological activity of IL-2 of interest. , as defined here. Preferably, the IL-2 polypeptide or variant thereof is derived from a human source and includes recombinantly produced human IL-2, such as recombinant human IL-2 polypeptides produced by microbial hosts and variants thereof that retain the same. IL-2 biological activity of interest. Any pharmaceutical composition comprising IL-2 as a therapeutically active component may be used to practice the present invention.

Anti-cancer agents:

Compositions of the present invention may be administered in conjunction with other anti-cancer agents. In particular, compositions will be formulated together as a combined therapeutic product or administered separately. Anti-cancer agents for use with the invention include, but are not limited to, one or more of the following presented below. Kinase Inhibitors

Kinase inhibitors for use as anticancer agents in conjunction with the compositions of the present invention include Epidermal Growth Factor Receptor (EGFR) kinase inhibitors such as small molecule quinazolines, for example gefitinib (US 5457105, US 5616582 and US 5,770,599), 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, including 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), vatalanib or PTK-787 (US 6,258,812), VEGF-Trap (WO 02/57423), B43 Genistein (WO-09606116), fenretinide (retinoic acid p-hydroxyphenylamine) (US 4,323,581), IM-862 (WO 02/62826), bevacizumab or Avastin® (WO 94/10202), KRN-951, 3- [5- (methylsulfonylpiperadine methyl) -indolyl] quinolone, AG-13736 and AG-13925, pyrrolo [2,1-f] [1,2,4] triazines, ZK-304709, Veglin®, VMDA-3601, EG-004, CEP- 701 (US 5,621,100), Cand5 (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 C kinase (PKC) inhibitors such as LY-317615 (WO 95/17182) and periphosine (US 2003171303); Phosphoinositide 3-Kinase (PI3K) inhibitors, including SF-1126 and PI-103, PI-509, PI-516 and PI-540 (produced by PIramed); Raf / Map / MEK / Ras kinase inhibitors, including sorafenib (BAY 43-9006), ARQ-350RP, LErafAON, BMS-3 54 825 AMG-548 and others disclosed in WO 03/82272; Fibroblast Growth Factor Receptor (FGFR) kinase inhibitors; Cell Dependent Kinase (CDK) inhibitors, including CIC-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®.

B. Antiestrogens

Estrogen targeting agents for use in anticancer therapy in conjunction with the compositions of the present invention include Selective Estrogen Receptor Modulators (SERMs), including tamoxifen, toremifene, raloxifene; aromatase inhibitors, including Arimidex® or anastrozole; Estrogen Receptor Upregulators (ERDs), including Faslodex® or fulvestrant.

C. Anti-Androgens

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

D. Other Inhibitors

Other inhibitors for use as anticancer agents in conjunction with the compositions of the present invention include farnesyl protein transferase inhibitors, including tipifarnib or R-115777 (US 2003134846 and WO 97/21701), BMS-214662, AZD-3409 and FTI. -277; topoisomerase inhibitors including merbarone and diflomotecan (BN-80915); mitotic kinesin spindle protein (KSP) inhibitors, including SB-743921 and MKI-833; protease modulators such as bortezomib or Velcade® (US 5,780,454), XL-784; and cyclooxygenase 2 (COX-2) inhibitors, including non-steroidal anti-inflammatory drugs I (NSAIDs).

E. Cancer Chemotherapeutic Drugs

Cancer chemotherapeutic agents in particular for use as anticancer agents in conjunction with the compositions of the present invention include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), injection of busulfan (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-S-deoxy-S-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine ( Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan) , daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®, US 2004073044), hydrochloride doxorubicin (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacytibine, Gemcitabine (difluorodeoxyhydhoxy) ( Hidrea®), Idarubicin (Idamycin®), Ifosfamide (IFEX®), Irinotecan (Camptosar®), L-Asparaginase (ELSPAR®), Leucovorin Calcium, Melphalan (Alqueran®), 6-Mercaptopurine (Purinethol®), Methotrexate (Folex ®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine (Gliadel®) implant, tamoxifen citrate (Nolvadex®), teniposide ( Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®).

F. Alkylating Agents

Alkylating agents for use in conjunction with the compositions of the present invention for anti-cancer therapeutic products include VNP-40101M or chloretizine, oxaliplatin (US 4,169,846, WO 03/24978 and WO 03/04505), glufosfamide, mafosfamide, etopophos ( 5,041,424), prednimustine; treosulfan; busulfan; irofluven (acylfulveno); penclomedine; pyrazoloacridine (PD-115934); Benzylguanine; decitabine (5-aza-2-deoxycytidine); brostalicin; mitomycin C (MitoExtra); TLK-286 (Telcyta®); temozolomide; trabectedin (US 5,478,932); AP-5280 (Cisplatin Platinate formula); porphyromycin; and clearazide (mechlorethamine).

G. Chelating Agents

Chelating agents for use in conjunction with the compositions of the present invention for anti-cancer therapeutic products include tetrathiomolybdate (WO 01/60814); RP-697; Chimeric T84.66 (cT84.66); gadofosveset (Vasovist®); deferoxamine; and bleomycin optionally in combination with electroporation (EPT).

H. Biological Response Modifiers

Biological response modifiers, such as immune modulators, for use in conjunction with the compositions of the present invention for anti-cancer therapeutic products include staurosporine and macrocyclic analogs thereof, including UCN-01, CEP-701 and midostaurin (see WO 02/3 0941 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 and WO 88/07045); squalamine (WO 01/79255); DA-9601 (WO 98/04541 and US 6,025,387); alemtuzumab; interferons (e.g., 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 biological variants thereof having amino acid sequences greater than 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, 5,238 944 and 5,525,612); and SMIPs, including benzazoles, anthraquinones, thiosemicarbazones and triptantrins (WO 04/87153, WO 04/64759 and WO 04/60308). I. Cancer Vaccines

Anti-cancer vaccines for use in conjunction with the compositions of the present invention include Avicine® (Tetrahedron Letters 26, 1974 2269-70); oregovomab (OvaRex®); Theratope® (STn-KLH); Melanoma Vaccines; the GI-4000 series (GI-4014, GI-4015 and GI-4016), which targets five mutations in the Ras protein; GlioVax-1; MelaVax; Advexin® or INGN-201 (WO 95/12660); Sig / E7 / LAMP-1, encoding HPV-16 E7; MAGE-3 or M3TK vaccine (WO 94/05304); HER-2VAX; ACTIVE, which stimulates tumor-specific T cells; GM-CSF cancer vaccine; evacins based on Listeria monocytogenes. J. Antisense Therapy

Anti-cancer agents for use in conjunction 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; oblimersen (Genasense®); JFS2; aprinocarsen (WO 97/29780); GTI-2040 (ribonucleotide reductase mRNA oligo antisense R2) (WO 98/05769); GTI-2501 (WO 98/05769); liposome-encapsulated c-Raf antisense oligodeoxynucleotides (LErafAON) (WO 98/43095); and Sirna-027 (RNAi-based therapeutic product targeting VEGFR-1 mRNA). K. IL-2

A preferred IL-2 compound is "aldesleukin" or "Proleucine®" manufactured by Chiron Corporation of Emeryville, California. IL-2 in this formulation is a recombinantly produced unglycosylated human IL-2 mutein which differs from the amino acid sequence of native human IL-2 by having the initial alanine residue deleted and the cysteine residue at position 125 replaced by a serine residue (referred to as des-alanyl-1 human interleukin-2, serine-125). Such IL-2 mutein may be expressed in E. coli and subsequently purified by diafiltration and cation exchange chromatography as described in U.S. Patent No. 4,931,543.

Aldesleukin was compared to native IL-2 (Jurkat) in vitro. No significant differences were observed and in vivo induction of cytolytic cells in mice and serum life after IV administration are equivalent for aldesleukin and native IL-2 (Jurkat); although there are beneficial attributes of the aldesleukin-encompassed form of IL-2 and therefore reference to "aldesleukin" encompasses only that composition and not all possible forms of the IL-2 protein.

In 1983, when the Cetus lymphocyte proliferation bioassay was developed, no official IL-2 reference preparation was available. One unit of Cetus was defined for this preparation as the amount of IL-2 in 1 ml that induced IL-2-dependent murine T cells to incorporate tritiated thymidine at 50% of their maximum level after 24 hours of incubation. The IL-2 containing product was assigned a specific activity of 3 x 106 Cetus units per mg. In 1988, the National Institute of Biological Standards and Controls (NIBSC), which is a WHO laboratory for Biological Standards in England, established an International Standard of IL-2. Also in 1988, the assay procedure for the Cetus lymphocyte proliferation bioassay was changed. The in-laboratory Cetus standard was calibrated against the IL-2 International Standard in the new bioassay procedure. The following correction factor was established:

International Units = Cetus Units x 6

Amounts of aldesleukin will be designated as mass units based on 1 mg aldesleukin having a nominal specific activity of 18 x 106 International Units (18 MIU). The protocol incorporates International Units.

Pharmaceutical compositions useful in the invention methods may comprise biologically active variants of IL-2. Such variants will retain the desired biological activity of the native polypeptide, so that the pharmaceutical composition comprising the variant polypeptide will have the same therapeutic effect as the pharmaceutical composition comprising the native polypeptide when administered to an individual. That is, the variant polypeptide will serve as a therapeutically active component in the pharmaceutical composition in a manner similar to that observed for native polypeptide. Methods are available in the art to determine whether a variant polypeptide retains the desired biological activity and therefore serves as a therapeutically active component in the pharmaceutical composition. Biological activity can be measured using assays specifically designed for measuring activity of native polypeptide or protein, including assays described in the present invention. Additionally, antibodies stimulated against a biologically active native polypeptide can be tested for their ability to bind to the variant polypeptide, where effective binding is indicative of a polypeptide having a conformation similar to that of the native polypeptide.

For purposes of the present invention, the IL-2 biological activity of interest is IL-2's ability to activate and / or expand natural killer (NK) cells to mediate lymphokine-activated killer (LAK) activity and antibody-dependent cellular cytotoxicity. (ADCC). Thus, an IL-2 variant (e.g., a human IL-2 mutein) for use in the methods of the present invention will activate and / or expand NK cells to mediate LAK and ADCC activity. Assays to determine NK cell IL-2 activation or expansion and LAC or ADCC activity mediation are well known in the art.

Suitable biologically active variants of native or naturally occurring IL-2 may be fragments, analogs and derivatives of such polypeptide. By "fragment" is meant a polypeptide consisting of only part of the sequence and structure of the intact polypeptide and may be a C-terminal deletion or N-terminal deletion of the native polypeptide. By "analog" is meant an analog of the native polypeptide or a fragment of the native polypeptide, wherein the analog comprises a native polypeptide sequence and structure having one or more amino acid substitutions, insertions or deletions. "Muteins" such as those described herein, and peptides having one or more peptides (peptide mimics) are also encompassed by the term analog (see International Publication No. WO 91/04282). By "derivative" is meant any suitable modification of the native polypeptide of interest, a fragment of the native polypeptide or analogs thereof, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol) or other addition of foreign moieties. as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs and derivatives are generally available in the art.

For example, amino acid sequence variants of the polypeptide may be prepared by mutations in the cloned DNA sequence encoding the native polypeptide of interest. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Walquer and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Know. USA 82: 488-492; Kunkel et al. (1987) Methods Enzymol. 154: 367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, New York); U.S. Patent No. 4,873,192; and the references cited therein; incorporated herein by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the Diahoff et al. (1978) model in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D. C), incorporated herein by reference. Conservative substitutions, such as exchanging one amino acid for another having similar properties, may be preferred. 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 so that the variants continue to possess the desired activity. Any mutations made in the DNA encoding the variant polypeptide should not place the sequence outside the reading frame and preferably will not create complementary regions that could produce a secondary mRNA structure.

Biologically active variants of IL-2 will generally have at least 70%, preferably at least 80%, more preferably about 90% to 95% or more, and even more preferably about 98% or more amino acid sequence identity to the protein. amino acid sequence of the reference polypeptide molecule, which serves as the basis for comparison. Thus, where the IL-2 reference molecule is human IL-2, a biologically active variant thereof will be at least 70%, preferably at least 80%, more preferably about 90% to 95% or more and even more. more preferably about 98% or more sequence identity with the human IL-2 amino acid sequence. A biologically active variant of a native polypeptide of interest may differ from the native polypeptide by as little as 1-15 amino acids, as little as 1-10, such as 6-10, as little as 5, as little as 4, 3, 2 or even 1 amino acid residue. By 'sequence identity' is meant that the same amino acid residues are found within the variant polypeptide and polypeptide molecule which serves as a reference when a specified continuous segment of the variant amino acid sequence is aligned and compared with the amino acid of the reference molecule. The percent sequence identity between the two amino acid sequences is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to provide the number of equivalent positions by dividing the number of equivalent positions by the number of positions. total positions in the segment being compared to the reference molecule and multiplying the result by 100 to provide the percent sequence identity.

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

With respect to the optimal alignment of two amino acid sequences, the continuous segment of the variant amino acid sequence may have additional amino acid residues or amino acid residues deleted relative to the reference amino acid sequence. The continuous segment used for comparison to the reference amino acid sequence will include at least twenty (20) continuous amino acid residues and may have 30, 40, 50 or more amino acid residues. Corrections to sequence identity associated with conservative residue substitutions or gaps can be made (see Smith-Waterman homology search algorithm).

The precise chemical structure of a polypeptide having IL-2 activity depends on a number of factors. Since ionizable amino and carboxyl groups are present in the molecule, a particular polypeptide may be obtained as an acidic or basic or neutral salt form. All of such preparations which retain their biological activity when placed under appropriate environmental conditions are included in the definition of polypeptides having IL-2 activity as used herein.

In addition, the primary amino acid sequence of the polypeptide may be increased by derivatization using sugar moieties (glycosylation) or by other supplementary molecules such as lipids, phosphate, acetyl groups and the like. It can also be increased by conjugation with saccharides. Certain aspects of such an increase are obtained through post-translational processing systems of the production host; other such modifications may be introduced in vitro. In any case, such modifications are included in the definition of an IL-2 polypeptide used herein insofar as the IL-2 activity of the polypeptide is not destroyed. Such modifications are expected to affect, quantitatively or qualitatively, activity, either by enhancing or decreasing polypeptide activity, in the various assays. In addition, individual amino acid residues in the chain may be modified by oxidation, reduction or other derivatization and the polypeptide may be cleaved to retain activity-retaining fragments. Such non-activity-destroying changes do not remove the polypeptide sequence from the definition of polypeptides. IL-2 of interest as used herein.

The technique provides substantial guidance regarding the preparation and use of polypeptide variants. In the preparation of IL-2 variants, those skilled in the art can readily determine which modifications to the native protein nucleotide or amino acid sequence will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition used in the methods of the present invention. invention.

IL-2 or variants thereof for use in the methods of the present invention may be from any source but preferably is recombinant IL-2. By "recombinant IL-2" is meant interleukin-2 which has comparable biological activity to native sequence IL-2 and which has been prepared by recombinant DNA techniques as described, for example, by Taniguchi et al. (1983). Nature 302: 305-310 and Devos (1983) Nucleic Acids Research 11: 4307-4323 or mutationally altered IL-2, as described by Wang et al. (1984) Science 224: 1431-1433. In general, the IL-2 coding gene is cloned and then expressed in transformed organisms, preferably a microorganism and more preferably E. coli, as described herein. The host organism expresses the foreign gene to produce IL-2 under expression conditions. Synthetic recombinant IL-2 may also be made in eukaryotes, such as yeast or human cells. Processes for growing, harvesting, disrupting or extracting IL-2 from cells are substantially described, for example, in U.S. Pat. 4,604,377; 4,738,927; 4,656,132; 4,569. 790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543.

For examples of variant IL-2 proteins see European Patent Publication (EP) No. EP 136,489 (which discloses one or more of the following changes to the naturally occurring IL-2 amino acid sequence: Asn26 for Gln26; Trpl21 for Phel21 Cys58 to Ser58 or Ala58, Cys105 to Ser105 or Alal05; Cys125 to Ser15 or Alal25; deletion of all residues after Argl20; and Met-1 forms thereof); and the recombinant IL-2 muteins described in European Patent Application No. 83306221.9, filed October 13, 1983 (published May 30, 1984 under Publication No. EP 109,748), which is equivalent to Belgian Patent No. 893,016 and commonly assigned US Patent No. 4,518,584 (which discloses recombinant human IL-2 mutein wherein the cysteine at position 125, numbered according to native human IL-2, is deleted or replaced by a neutral amino acid; alanyl-ser-25-IL-2; and des-alanyl-ser-25-IL-2). See also US Patent No. 4,752,585 (which discloses the following variant IL-2 proteins: alal04 serl25 IL-2, alal04 seral25 IL-2, vall04 serl25 IL-2, vall04 IL-2, vall04 alal25 IL-2, desalal alal04 serl25 IL-2, desalalal alal04 IL-2, 10 desalal alal04 alal25 IL-2, desalal vall04 serl25 IL-2, desalal vall04 IL-2, desalal vall04 alal25 IL-2, desalal des-pro2 alal04 serl25 IL-2, desalal des-pro2 alal04 IL-2, desalal des-pro2 alal04 aial25 IL-2, desalal des-pro2 vall04 serl25 IL-2, desalal des pro2 vall04 IL-2, desalal des-pro2 vall04 seral25 IL-2, desalal des-pro2 desal3 alal04serl25 IL-2, desalal des-pro2 des- thr3 alal04 IL-2, desalal des-pro2 des-thr3 alal04 15 alal25 IL-2, desalal des-pro2 des-thr3 vall04 serl25 IL-2, desalal des-pro2 des-thr3 vall04 IL-2 , desalal des-pro2 des-thr3 vall04 alal25 IL-2, desalal des-pro2 des-thr3 des ser4 alal04 serl25 IL-2, desalal des-pro2 des-thr3 des-ser4 alal04 IL-2, desalal des-pro2 des-thr3 des-ser4 alal04 alal25 IL-2, desalal des-pro2 des- thr3 des-ser4 vall04 serl25 IL-2, desalal des-pro2 des-thr3 des-ser4 vall04 IL-2, desalal des-pro2 des-ser3 vall0420 alal25 IL-2, desalal des-al2 pro2 deser3 des-ser4 deser5 alal04 serl25 IL-2, desalal des pro2 des-thr3 des-ser4 des-ser5 alal04 IL-2, desalal des-pro2 des-thr3 des-ser5 des-ser5 alal04 alal25 IL-2, desalal des-pro2 des-thr3 des-ser4 des-ser5 vall04 serl25 IL-2, desalal des-pro2 des-thr3 des-ser4 des-ser5 goes 104 IL-2, desalal alal des-pro2 des-thr3 des-ser4 des-ser5 vall04 ala! 25 IL-2, desalal des-pro2 des-thr3 des-ser4 des-ser6 alal04 25 alal25 IL-2, desalal des -pro2, des-thr3 des-ser4 des-ser5 des-ser6 alal04 IL-2, desalal des pro2 des-thr3 des-ser4 des-ser5 des-ser6 alal04 serl25 IL-2, desalal des-ser2 des -t hr3 des ser4 des-ser5 des-ser6 vall04 serl25 IL-2, desalal des-pro2 des-thr3 des-ser5 des serore vall04 IL-2 and desalal des-pro2 des-thr3 des-ser4 des -ser5 des-ser6 vall04 alal25 IL-2) and US Patent No. 4,931.54 3 (which discloses the des-alanyl-1 IL-2 mutein, human serine 30 125 IL-2 used in the examples herein, as well as other IL-2 muteins).

See also European Patent Publication No. EP 200,280 (published December 10, 1986), which discloses recombinant IL-2 muteins, in which methioninan position 104 has been replaced by a conservative amino acid.

Examples include the following muteins: ser4 des-ser5 alal04 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 alal04 alal25 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 glul04 serl25 IL-2; desalal des-pro2 des-thr3 des ser4 des-ser5 glul04 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 glul04 alal25 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 alal04 alal25 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 alal04 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 5 des-ser6 alal04 serl25 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 glul04 serl25 IL-2; desalal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 glul04 IL-2; and desalal des-pro2 des-thr3 des-ser4 des-ser5 des-ser6 glul04 alal25 IL-2. See also European Patent Publication No. EP 118,617 and U.S. Patent No. 5,700,913, which discloses unglycosylated human IL-2 variants bearing alanine rather than methionine in native IL-2 as the N-terminal amino acid; an unglycosylated human IL-2 with the deleted initial methionine, so proline is the N-terminal amino acid; and an 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 substitutions at position 20 for histidine or isoleucine, asparagine at position 8 for arginine, glycine or isoleucine or glutamine at position 126 for leucine or glutamic acid) which are reported to have selective activity by high affinity IL-2 receptors expressed by T-cell receptors, preferably NK cells, and reduced IL-2 toxicity; the muteins disclosed in US Patent No. 5,229,109 (alanine 38 position arginine substitutions or lysine position 42 phenylalanine substitutions) which exhibit reduced binding to the high affinity IL-2 receptor as compared to IL -2 native while maintaining the ability to stimulate LAK cells; muteins disclosed in International Publication No. WO 00/58456 (alteration or deletion of a naturally occurring (x) D (y) sequence in native IL-2, where D is aspartic acid, (x) is leucine, isoleucine, glycine or valine and (y) is valine, leucine or serine), which are claimed to reduce vascular leakage syndrome; IL-2 pl-30 peptide disclosed in International Publication No. WO 00/04 04 8 (corresponding to the first 30 amino acids of IL-2, which contains the entire IL-2 α-helix A and interacts with the chain IL-2 receptor P) which, according to information, stimulates NK cells and induces LAK cells; and a mutant form of peptide IL-2 pl-30 also disclosed in WO 00/04048 (substitution of aspartic acid at position 20 for lysine) which is reportedly unable to induce vascular bleeding but remains capable of generating cells. LAK Additionally, IL-2 may be modified with polyethylene glycol to provide enhanced solubility and an altered pharmacokinetic profile (see U.S. Patent No. 4,766,106).

The term IL-2 as used herein is also intended to include fusions of IL-2 or conjugates comprising IL-2 fused to a second protein or covalently conjugated to polyproline or a water-soluble polymer to reduce dosing frequencies or improve tolerability to IL-2. For example, IL-2 (or a variant thereof as defined herein) may be fused to human albumin or an albumin fragment using methods known in the art (see WO 01/79258). Alternatively, IL-2 may be covalently conjugated to polyproline or polyethylene glycol homopolymers and polyoxyethylated polyols, wherein the homopolymer is unsubstituted or substituted at one end by an alkyl group and the polyol is unsubstituted using methods known in the art ( see, for example, U.S. Patent Nos. 4,766,106, 5,206,344 and 4,894,226). Any pharmaceutical composition comprising IL-2 as the therapeutically active component may be used in the methods of the invention. Such pharmaceutical compositions are known in the art and include, but are not limited to, those disclosed in U.S. Patent Nos. 4,745,180; 4,766,106; 4,816,440; 4,894,226; 4,931,544; and 5,078,997. Thus, lyophilized or spray-dried liquid compositions comprising IL-2 or variants thereof which are known in the art may be prepared as an aqueous or non-aqueous solution or suspension for subsequent administration to an individual according to the methods of the invention. Each of these compositions will comprise IL-2 or variants thereof as a therapeutic or prophylactically active component. By "therapeutically or prophylactically active component" is meant that IL-2 or variants thereof is specifically incorporated into the composition to achieve a desired therapeutic or prophylactic response with respect to the treatment, prevention or diagnosis of a disease or condition within an individual when the pharmaceutical composition is administered to that individual. Preferably, the pharmaceutical composition comprises appropriate stabilizing agents, bulking agents or both to minimize problems associated with loss of protein stability and biological activity during storage preparation. In preferred embodiments of the invention, IL-2-containing pharmaceutical compositions useful in the methods of the invention are compositions comprising stabilized monomeric IL-2 or variants thereof, compositions comprising multimeric IL-2 or variants thereof and compositions comprising lyophilized or dried IL-2. spray or stabilized variants thereof.

Pharmaceutical compositions comprising stabilized monomeric IL-2 or variants thereof are disclosed in International Publication No. WO 01/24814, entitled "Stabilized Liquid Polypeptide-Pharmaceutical Compositions". By "monomeric" IL-2 is meant that protein molecules are present substantially in their monomeric form, not in an aggregate form, in the pharmaceutical compositions described herein. Consequently, covalent or hydrophobic oligomers or aggregates of IL-2 are not present. Briefly, IL-2 in such liquid compositions is formulated with an amount of an amino acid base sufficient to decrease the formation of IL-2 aggregates during storage. Amino acid base is an amino acid or combination of amino acids, where any given amino acid is present in its free base form or in its salt form. Preferred amino acids are selected from the group consisting of arginine, lysine, aspartic acid and glutamic acid. Such compositions further comprise a buffering agent for maintaining the pH of liquid compositions within an acceptable range for IL-2 stability, where the buffering agent is an acid substantially free of its salt form, an acid in its salt form. or a mixture of an acid and its salt 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 serves to stabilize IL-2 against aggregate formation during storage of the liquid pharmaceutical composition, whereas use of an acid substantially free of its salt form, an acid in its salt form or a mixture of a Acid and its salt form as a buffering agent results in a liquid composition having an osmolarity that is approximately isotonic. The liquid pharmaceutical composition may additionally incorporate other stabilizing agents, more particularly methionine, a nonionic surfactant such as polysorbate 80, and EDTA, to further enhance the stability of the polypeptide. Such liquid pharmaceutical compositions are mentioned as being stabilized, since the addition of the amino acid base in combination with an acid substantially free of its salt form, an acid in its salt form or a mixture of an acid and its salt form, results in compositions having increased storage stability over liquid pharmaceutical compositions formulated in the absence of a combination of these two components. Such liquid pharmaceutical compositions comprising stabilized monomeric IL-2 may be used in an aqueous liquid form or stored for later use in a frozen or dry form for further reconstitution into a liquid form or other form suitable for administration to a subject. according to the methods of the present invention. By "dry form" is meant that the liquid pharmaceutical composition or formulation is dried by freeze drying (i.e. lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38: 48 59). ), spray drying (see Masters (1991) in Spray-Drying Handbook (8th ed; Longman Scientific and Technical, Essez, UK), pages 491-676; Broadhead et al. (1992) Drug Devi. Ind. Pharm. 18 : 1169-1206; and Mumenthaler et al. (1994) Pharm Res. 11: 12-20 or air drying (Carpenter and Crowe (1988) Cryobiology 25: 459-470; and Roser (1991) Biopharm. 4: 47- 53).

Other examples of IL-2 formulations comprising IL-2 in their unaggregated monomeric state include those described in Whittington and Faulds (1993) Drugs 46 (3): 446-514. Such formulations include the recombinant IL-2 product in which the recombinant IL-2 mutein Teceleucine (unglycosylated human IL-2 with a methionine residue added to the amino terminus) is formulated with 0.25% human serum albumin. in a lyophilized powder which is reconstituted with isotonic saline and recombinant IL-2 mutein Bioleucine (human IL-2 with a methionine residue added to the amino terminus and a replacement of the cysteine residue at position 125 of the IL-2 sequence. Alanine) formulated such that 0.1 to 1.0 mg / ml IL-2 mutein is combined with acid, wherein the formulation has a pH of 3.0 to 4.0, advantageously without buffer and a conductivity of less than 1,000 mS / cm (mmhos / cm) (advantageously less than 500 mS / cm (mmhos / cm)). See EP 373,679; Xhang et al. (1996) Pharmaceut. Res. 13 (4): 643-644 and Prestrelski et al. (1995) Pharmaceut. Res. 12 (9): 1250-1258.

Examples of pharmaceutical compositions comprising multimeric IL-2 are disclosed in commonly assigned U.S. Patent No. 4,604,377. By "multimeric" is meant that protein molecules are present in the pharmaceutical composition in a microaggregated form having an average molecular association of 10-50 molecules. These multimers are present as loosely bound, physically associated IL-2 molecules.

A lyophilized form of such compositions is commercially available under the trademark Proleucina® IL-2 (Chiron Corporation, Emeryville, California). The lyophilized formulations disclosed in that reference comprise selectively oxidized microbially produced recombinant IL-2 in which the recombinant IL-2 is mixed with a water-soluble carrier such as mannitol which provides volume and a sufficient amount of sodium dodecyl sulfate to ensure solubility of recombinant IL-2 in water. These compositions are suitable for reconstitution into aqueous injections for parenteral administration and are stable and well tolerated in human patients. When reconstituted, the IL-2 maintains 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.

The methods of the present invention may also use stabilized spray-dried or lyophilized pharmaceutical compositions comprising IL-2, which may be reconstituted in a liquid or other form suitable for administration according to the methods of the invention. Such pharmaceutical compositions are disclosed in International Publication No. WO 01/4 9274 entitled "Methods for Pulmonary Delivery of Interleukin-2". Such compositions may further comprise at least one bulk compounding agent, at least one agent in an amount sufficient to stabilize the protein during the drying process or both. By "stabilized" is meant that IL-2 protein or variants thereof retain their monomeric or multimeric form, as well as their other key properties of quality, purity and potency after freeze drying or spray-drying to obtain the dry powder form. or solid of the composition. In such compositions, preferred carrier materials for use as a bulking agent include glycine, mannitol, alanine, valine or any combination thereof, more preferably glycine. The bulking agent is present in the formulation in the range of 0% to about 10% (w / v), depending on the agent used. Preferred carrier materials for use as a stabilizing agent include any sugar or sugar alcohol or any amino acid. Preferred sugars include sucrose, trehalose, raffinose, stachyose, sorbitol, glucose, lactose, dextrose or any combination thereof, preferably sucrose. When the stabilizing agent is a sugar, it is present in the range from about 0% to about 9.0% (weight / v), preferably about 0.5% to about 5.0%, more preferably. about 1.0% to about 3.0%, even more preferably about 1.0%. When the stabilizing agent is an amino acid, it is present in the range from about 0% to about 1.0% (weight / v), preferably about 0.3% to about 0.7%, even more. preferably about 0.5%. Such stabilized lyophilized or spray-dried compositions may optionally comprise methionine, ethylenediaminetetraacetic acid (EDTA) or one of its salts, such as disodium EDTA or other chelating agent, which protects IL-2 or variants thereof against methionine oxidation. . The use of such agents in this manner is described in U.S. Application Serial No. 09 / 677,643, incorporated herein by reference. The stabilized lyophilized or spray-dried compositions may be formulated using a buffering agent which maintains the pH of the pharmaceutical composition within an acceptable range, preferably from a pH of about 4.0 to a pH of about 8%. 5 when in a liquid phase, such as during the formulation process or after reconstitution of the dry form of the composition. Buffers are chosen so that they are compatible with the drying process and do not affect protein quality, purity, potency and stability during processing and storage.

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

Also provided herein is a kit or package containing at least one combined composition of the invention, accompanied by instructions for use. For example, in cases where each of the drugs itself is administered as individual or separate dosage forms, the kit comprises each of the drugs, along with instructions for use. The drug components may be packaged in any manner suitable for administration, as the package, when considered together with the instructions for administration, clearly indicates the manner in which each of the drug components must be administered. Alternatively, each of the drug components of the combination may be combined into a single administrable dosage form, such as a single composition.

For example, for an illustrative kit comprising rIL-2 and an anti-angiogenic agent, the kit may be arranged for any appropriate period of time, such as per day. As an example, for Day 1, a representative kit may comprise unit dosages of each of rIL-2 and anti-angiogenic agent. If each drug must be administered twice daily, then the kit may contain, corresponding to Day 1, two rows of unit dosage forms of each of IL-2 and anti-angiogenic agent, with instructions for the time of administration. Alternatively, if one or more of the drugs differ in timing or amount of drug to be administered compared to the other drug elements of the combination, then this will be reflected in the package and instructions. For example, if rIL-2 has to be administered twice a day and the anti-angiogenic agent has to be taken once a day, the exemplary Day 1 pack would correspond to rIL-2 unit dosage forms as " Day 1, Dose 1 ", together with dosage forms for the anti-angiogenic agent corresponding to" Day 1, Dose 2 ".

Various embodiments according to the above may be readily considered and, of course, will be dependent upon the combination of particular drugs employed for treatment, their corresponding dosage forms, recommended dosages, intended patient population and the like. The package may be in any form commonly employed for pharmaceutical packaging and may utilize any of a number of features such as different colors, wrap, tamper resistant packaging, blister packs, dissectors and the like.

EXAMPLES

Below are examples of specific embodiments of the present invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperatures, etc.), but some error and experimental deviation may of course be allowed.

Example 1

Compositions for co-administration with rIL-2

A. COMPOUNDS

Table 1: Examples of Quinazoline

<table> table see original document page 89 </column> </row> <table> <table> table see original document page 90 </column> </row> <table> <table> table see original document page 91 < / column> </row> <table> <table> table see original document page 92 </column> </row> <table> <table> table see original document page 93 </column> </row> <table> <formula> formula see original document page 94 </formula>

Scheme 1 describes a modular method of synthesis of a myriad of substituted quinazoline compounds. Reference to AG indicates an activation group such as, for example, a halide, triflate or ketone, wherein as many as four AG groups may be present. Since the 4-chloro position, shown in the third step, is more active than positions 5-8 on the benzyl ring, NHRR 'displacement can be processed without much concomitant AG displacement at any of the 5- positions. 8 Subsequent displacement of the AG group to R "in the final step may be processed in the presence of a weak to moderate base. Alternatively, the AG group (s) may be modified to provide the desired product by For example, a NO2 group may be reduced with Fe and AcOH in EtOH and H2 O to provide an amino substituent which may be further substituted, for example, by reductive paraformaldehyde amination.

As will be apparent to those skilled in the art, a plethora of functionalized 2-aminobenzamide initiation materials is commercially available or readily synthesized by known procedures, subsequently the AG group (s) in the initiation material may (m) ) is a desired substituent on the final product (as in R ") such as, for example, (one) alkoxy group (s) or (one) substituted alkyl group (s). Functionalized 2-nitrobenzamide is available which are easily converted to the 2-aminobenzamide material in the presence of a reducing agent such as H2 / Pd / C in EtOH.

Alternative methods of manufacturing quinazolines of the 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.

INDOLINONES Scheme 2a:

<formula> formula see original document page 95 </formula>

Scheme 2a is performed as a procedure in uvass, with reagents in the step being NH 4 OH, CuCl in H 2 O, followed by the addition of aq. in step b. R2-R5 are as defined herein.

Scheme 2b:

<formula> formula see original document page 95 </formula>

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

Scheme 2c: <formula> formula see original document page 96 </formula>

In Scheme 2c, the reagents are refluxed in EtOH in the presence of NaOBu-t (a) to provide the final product. R9, as shown in Scheme 2, is H, -OH, -CN, alkyl, aryl, heterocyclyl, alkoxy or -NRaRb as defined herein. It is contemplated that the above structure may substitute for Formula II to allow substitution at R 9, so all other substituents are as defined herein.

Scheme 2d (Synthesis of Compound 6):

<formula> formula see original document page 96 </formula>

FTAAZINES

Preparation of substituted phthalazines, as in Compound 10, is described as follows, an outline of US 6,258,812, which also includes other reaction schemes that may be useful in the synthesis of the compounds of the present invention.

Scheme 3a:

1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine dihydrochloride

A mixture of 15.22 (59.52 mmol) of 1-chloro-4- (4-pyridylmethyl) phthalazine (for preparation seeGerman Patent Application No. 1,061,788, published July 23, 1959]), 7, 73 g (60.59 mmol) of 4-chloroaniline and 200 ml of 1-butanol is heated for 2 h under reflux. Crystallization, which is obtained when the mixture slowly cools to 5 ° C, is then filtered and washed with 1-butanol and ether. The residue on the filter is dissolved in about 200 ml of hot methanol, the solution is treated with 0.75 g of activated charcoal and filtered via a Hyflo Super Cel and the pH of the filtrate adjusted to about 2.5 with 7 ml. 3N methanolic HCl. The filtrate is evaporated to about half of the original volume and ether added until slight turbidity occurs; Cooling then leads to the precipitation of the crystals. The crystallize is filtered off, washed with a methanol / ether mixture (1: 2) as well as ether, dried for 8 hours at 110 ° C under HV and equilibrated for 72 hours at 20 ° C and at ambient atmosphere. Thus, the title compound is obtained with a water content of 8.6%; m.p. > 270 ° C. ; 1H 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.

Scheme 3b:

1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride

A mixture of 0.972 g (3.8 mmol) of 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.656 g (4 mmol) of 4-chloroaniline hydrochloride (Research Organics, Inc., Cleveland, Ohio, USA) and 20 ml of ethanol is heated for 2 h under reflux. The reaction mixture is cooled in an ice bath, filtered and the crystallize washed with a little ethanol and ether. After drying under HV for 8 h at 110 ° C and 10 h at 150 ° C, the title compound is obtained as a result of thermal removal of HCl; m.p. > 270 ° C; 1 H NMR (DMSO-d 6) 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.

Scheme 3c:

1- (4-Chloroanilino) -4- (4-pyridylmethyl) phthalazine hydrochloride

A mixture of 1.28 g (5 mmol) of 1-chloro-4- (4-pyridylmethyl) phthalazine, 0.67 g (5.25 mmol) of 4-chloroaniline and 15 mL of 1-butanol is heated for 0 0.5 h at 100 ° C while stirring in a nitrogen atmosphere. The mixture is then cooled to RT, filtered and the filtrate washed with 1-butanol and ether. For purification, the crystallize is dissolved in 40 ml of hot methanol, the activated charcoal treated solution filtered via Hyflo Super Cel and the filtrate evaporated to about half its original volume, resulting in the formation of a crystalline precipitate. After cooling to 0 ° C, filtration, washing the residue on the filter with ether and drying under HV for 8 h at 130 ° C, the title compound is obtained; m.p. > 270 ° C; 1H NMR (DMS0-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

Preparation of rIL-2

Aldesleukin Preparation, rIL-2 (NSC3773364) ("Proleucine", "Chiron): Messenger RNA from the human Jurkat cell line is used to create double stranded cDNA, which is hybridized to pBR322 plasmids. A clone containing the IL-2 gene is identified using a 32P-labeled oligonucleotide probe corresponding to a short portion of the IL-2 base sequence. The gene is inserted into a region of plasmid pBR322 that has a convenient restriction site. The appropriate promoter and ribosomal binding site are inserted in front of the IL-2 gene and the resulting expression clone encodes a modified recombinant rIL-2. In vitro mutagenesis of cloned IL-2 is used to make a conservative substitution of serine for cysteine at position 125. The resulting molecule is indistinguishable from native IL-2 for its in vitro biological activity. An E. coli producing strain bearing the aldesleukin gene is grown in fermenters. The culture is collected and aldesleukin is extracted. A series of chromatographic steps were performed to purify aldesleukin. The formulated product is adjusted to a pH of 7.2-7.8. The molecular weight of Proleucine * is approximately 15,600 daltons. Amino acid composition analysis and N-terminal sequencing confirmed that aldesleukin has the predicted protein sequence.

Reconstitution and Dilution Procedure:

Proleucina® is a lyophilized cake in 5 cc flasks containing 1.3 mg protein. Proleucina® vials for injection are reconstituted with 1.2 mL Sterile Water for Injection, USP. The diluent is directed against the side of the bottle to avoid excess foam, shaking the contents gently until completely dissolved while avoiding agitation. When reconstituted, each mL contains 1.1 mg (18 million IU) of Proleucine '. Reconstituted Proleucine0 is suitable for intravenous injection directly or may be diluted as needed in 50 mL to 500 mL volumes with 5% Dextrose Injection, USP, with 0.1% Human Albumin, USP When diluted, Human Albumin, USP is added to the 5% Dextrose Injection, USP, prior to the addition of reconstituted Proleucin *.

Example 3

Therapy with a single agent rIL-2 Composition versus combination therapy with rIL-2 composition and anti-angiogenic agents High-dose rIL-2 results were summarized in Table 6.

Table 6: Cake Infusion Scheme Experiments

<table> table see original document page 100 </column> </row> <table> <table> table see original document page 101 </column> </row> <table> International Units; CR: complete answer; PR: Partial Response; q: each; NS: not established

Complete response + partial response = 16.5% (95% confidence interval, 13.8% - 19.2%).

Adapted in part from Bukowski (39).

A number of factors have given rise to the momentum for combination therapies with anti-angiogenic compositions. Significant morbidity associated with high dose rIL-2 required careful patient selection, dramatically decreasing the number of potential patients who could benefit from therapy. Unfortunately, concomitant illness is most often found in those with the highest incidence of CCR. The requirement for careful patient monitoring and occasional intensive care has made high-dose rIL-2 administration a single expensive agent and limiting its use in large medical facilities. The practical effect of which is to restrict availability to only a minority of patients.

Lower doses of rIL-2 were critically evaluated, as observed in Table 7.

Table 7: rIL-2 Phase II Experiments as a Single Agent: Subcutaneous Schemes1

<table> table see original document page 102 </column> </row> <table> <table> table see original document page 103 </column> </row> <table> <table> table see original document page 104 < / column> </row> <table>

rIL-2: Recombinant Interleukin-2; MIU: Million International Units; CR: complete answer, - PR: partial response; q: each; NS: not established; i.v.:intravenous

Complete response + partial response = 18.5% (95% confidence interval, 15.8% - 21.5%).

bSpecified Unit Type, partly adapted from Bukowski (39).

Most commonly used subcutaneous regimens have been published by Buter et al. In Buter, rIL-2 was provided once a day, 5 days a week for 6 weeks. During the first 5-day cycle, 18 x 106 IU (MIU) was provided once a day; in the following cycles, the doses after the first 2 days were reduced to 9 MIU. Response rates and survival data may be similar to those published for rIL-2 high-dose bolus IV administration. The Butcher / Sleijfer regimen was recently compared to the high rIL-2 dose in a prospective randomized trial. Ninety six patients were randomly assigned to rIL-2 high IV and 92 patients to rIL-2 SQ (Buter / Sleijfer dosing schedule). Previously, the response to high rIL-2 dose was 20% and rIL-2 SQ was 10%. However, overall survival was no different (p = 0.34) from highs. As such, a catalyst for combination with anti-angiogenic compositions exists to increase the patient's overall efficacy and responsiveness to the lower dose regimen.

Regimens for combinations of aldesleukin with anti-angiogenic agents are critically evaluated, as observed in Table 8.

Table 8: Treatment, Dose and Duration

<table> table see original document page 105 </column> </row> <table>

"A dose of anti-angiogenic agent (equal to the dose planned according to dose level) is provided on Day-7.

"Anti-angiogenic agent is provided again on day1 and then every 2 weeks continuously in an 8-week detox cycle.

RIL-2 treatment is continued for 6 consecutive weeks (days 1-42, Monday-Friday of each week) followed by a 2-week rest period resulting in an 8-week treatment cycle.

Example 4

RIL-2 Combination Therapy and BAY 43-9006 Small Molecule Receptor Tyrosine Kinase Inhibitors and

SU11248

A. Material and Methods

Pharmaceuticals

Recombinant human interleukin-2 (Proleucine 8 ", Aldesleucine / rIL-2); 18 MIU / ml, Chiron Corporation, Emeryville, CA) was reconstituted with sterile water for injection and formulated in 5% dextrose prior to administration. Vincristine (vincristine sulfate) was from Mayne Pharma Ltda (Mulgrave, Australia) CHIR-258 is 4-amino-5-fluoro-3- [5- (4-methylpiperazin-1-yl) -1H-benzimidazol-2-yl] quinolin-2 (1H ) -ona (Chiron) BAY 43-9006 (Sorafanib / Nexavar®) (Riedl et al., Proc. Am. Assoc. Cancer Res. 2001 42 (Abs 4956); Lowinger and collaborators, Curr. Pharm. Des. 2002 8 ( 25): 2269-2278; WO9932455) and SU11248 (Sunitinib / Sutent®) (Sun and collaborators, J ". Med. Chem. 2003 46 (7): 1116-1119; WO0160814) were synthesized and purified in the laboratory according to procedures. and published patents. BAY 43-9006 or SU11248 (20 mM) stock solutions were prepared in DMSO and aliquots will be stored at -20 ° C prior to use. For in vitro assays, all drugs were diluted in optimal culture medium. For inventive administration, BAY 43-9006 was formulated in 100% PEG 400 vehicle, while SU11248 dosing solutions were prepared in 5 mM citrate buffer. All other chemicals used were research grade.

Cell Lineages

All murine cell line CTLL-2 (IL-2 dependent T cell line), B16-F10 melanoma, CT26 colon carcinoma and RENCA renal were obtained from the American Tissue Culture Collection (Rockville, MD). CTLL-2 were grown in RPMI1640 supplemented with 10% FBS (fetal bovine serum, Gibco Life Technologies, Gaithersburg, MD), 2 mM L-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, 0 rIL-2, 5 nM, 2 mM β-mercaptoethanol. RENCA cells were cultured in medium containing EMEM with 10% FBS, 100X 2% Vitamin, 1% 200 mM Glutamine, 1% 100 mM NaPy, 1% nonessential amino acids. For CT26 cell growth, the medium contained EMEM, 10% FBS, 2% vitamins, 1% 200 mM glutamine, 1% 100 mM sodium pyruvate, 1% nonessential amino acids. B16-F10 cells were grown in RPMI 1640 with 10% FBS, 1% nonessential amino acids, 1% 100mM sodium pyruvate, 2% vitamins; 2 mM 1-glutamine; 2% sodium bicarbonate. Yac-1 cells (ATCC) were cultured in RPMI + 10% FBS and subcultured 1-2 days prior to assay to ensure log-phase growth. Cells were maintained as suspension or adherent cultures in a humidified atmosphere at 37 ° C and 5% CO2. Cells were used in the exponential growth phase (not exceeding 6-8 passages) with> 98% viability (assessed using staining trypan blue) and determined free of mycoplasma.

In vivo efficacy studies

Female BALB / c or C57BL6 mice (4-6 weeks deity, 18-22 g) were obtained from Charles River (Wilmington, MA) and acclimated for 1 week in depatogen-free cages prior to commencing the study. The animals received sterile rodent chow and water ad libitum and were housed in sterile overhead filter cages with 12 hour light / dark cycles. All experiments were under the guidelines of the Association for Assessment and

Accreditation of Laboratory Animal Care International.

For tumor implantation, B16-F10 (2 x 106), CT26 (2 x 106) or RENCA (1 x 106) cells were collected, washed three times and resuspended in PBS. The mice were scraped on the flank and implanted (0.2 ml) subcutaneously (s.c.) into the right flank of the mouse. For the BI6-FIO tumor model, C57BL6 mice were used, while CT26 and RENCA tumors were implanted in BALB / c mice. Treatments were started when the tumors had an established mean size of 50-250 mm3 (day 0), as outlined in specific study designs. The mice were randomly assigned to groups of (typically 10 mice / group). rIL-2 was administered daily sc (0.2-3 mg / kg / day) on days 0-6 or 7-13 or days 0-4, 7-11.BAY 43-9006 or SU11248 (1-100 mg / day). kg) were administered daily (for 5-12 days) as a solution via forced oral ingestion beginning on day 0 or day 7. All monotherapy and dosed drug combinations outlined in individual studies were well tolerated.

Evaluation of Tumor Inhibition and ResponsesTumor volumes and body weights were evaluated 2-3 times a week. Tumor gauge measurements were converted to mean tumor volume (mm3) using the formula: 1/2 (length (mm) x [width (mm)] 2). Tumor growth inhibition (TGI) was calculated as [1- (mean tumor volume of treated group / mean tumor volume of control group) x 100]. Responses were defined as a complete response (CR, no measurable tumor) or partial response (PR, tumor volume reduction of 50-99%) compared with tumor volume for each animal at initiation of treatment. Tumor growth retardation analysis was calculated as: [(number of days for a treated group to reach an average tumor volume of 1000 mm3) - (number of days for the control group to reach an average tumor volume of 1000 mm3)] .

Synergistic effects were defined when the expected tumor growth inhibition proportion of combined therapy (% T / Cexp =% T / C treatment 1 x% T / Treatment 2) divided by the observed% T / C (% T / Cobs) combined treatment was> 1. Additive effects were defined when% T / Cexp /% T / Cobs = 1 and antagonism when% T / Cexp /% T / Cobs <1 (Yokoyama et al., Cancer Res.2000 60 (80): 2190-2196).

Pharmacokinetics

To evaluate the pharmacokinetics of the drug, mice were treated with a single sc dose of rIL-2 (6 mg / kg, 0.2 ml) or BAY 43-9006 (20 mg / kg, po, 0.2 ml) and blood was collected at various times after drug administration. Plasma BAY 43-9006 and rIL-2 levels were determined using HPLC or an ELISA bioassay.

Western blot analysis

After incubations of the drug under indicated conditions, cells were collected, washed with ice cold PBS and lysed with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate in IX phosphate buffered saline, pH 7.2) containing protease inhibitors (Roche MolecularBiochemicals, Indianapolis, IN) and phosphatase inhibitors (Sigma, St. Louis, MO). Protein content in lysates was determined using the BCA assay (Bio-Rad, Hercules, CA). For western blot analysis, 60 µg of protein was electrophoresed and pERK detection was performed with a common pERK mouse antibody (1: 1000). , CellSignaling, Beverly, MA) and incubated at 4 ° C overnight. Antibody Detection pSTAT5 (1: 1000, Upstate) and pAKT (1: 1000, Cell Signaling) were performed with the same amount of protein by anti-comanbody hybridization. appropriate phosphotyrosine for 2 hours at room temperature. The membranes were then incubated for 1 hour at room temperature with 1: 5000 armoraceous-conjugated peroxidase buttock IgG (Jackson Immunoresearch, West Grove, PA). To verify equal loading, the blots were extracted and re-hybridized with anti-ERK (CellSignaling), anti-STAT5 (BD Biosciences) and anti-AKT (CellSignaling) antibodies to measure total ERK, STAT5 and AKT proteins, respectively. Proteins were detected using enhanced chemiluminescence (ECL; Amersham

Biosciences, Buckinghamshire, England) and viewed after exposure to a Kodak film. Scanning densitometry was performed to quantify band intensities. The amount of pERK, pSTAT5 or pAKT was normalized to total ERK, STAT5or AKT protein levels and compared to vehicle or untreated controls. Cellular Immunophenotyping in BALB / c NusAfter Drug Treatment

Blood samples were collected at various times after the indicated treatments. Whole blood (100 µl) was transferred to FACS / TruCount tubes (BD BioSciences) and kept on ice. The samples were treated with 0.5 µg mouse Fc block (CD16 / CD32 anti-mouse; BDBioSciences) and incubated on gel 20 minutes. Fluorochrome-conjugated antibodies, as indicated below, were added to the samples and incubated for 20 minutes on ice protected from light. The blood samples were vortexed while adding 2 ml of 1x FACS lysis solution (BD BioSciences), followed by incubation at room temperature for 10 minutes, then centrifuged at 1250 rpm. All samples were washed twice, suspended in PBS and 2% FBS and stored at 4 ° C prior to sample acquisition on a BDFACScalibur and subsequent analysis using CellQuest Pro software. Absolute cell numbers were determined relative to the TruCount reference globule. Cell populations were identified and separated 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 identified by CD4 staining. or CD8 (BD Biosciences). Individual cell populations were identified through appropriately separated events.

Histopathology and Immunohistochemistry Mouse tumors were fixed in a 10% neutral buffered form and then transferred to 70% ethanol and subsequently processed for paraffin inlay using an Excelsior tissue processor (ThermoElectron Corporation, Pittsburgh, PA). Tissue sections (4um) were cut over a rotating microtome (RM2125, Leica Microsystems, Nussloch, Germany). Haemotoxylin and eosin (H&E) stained sections were prepared. Immunostaining was then performed using a Discovery XT automated slide staining system (VentanaMedical Systems, Tucson, AZ). Murine T cells were detected with a murine anti-CD3 antibody (1:60 dilution, Dako Norden S / A, Glostrup Denmark) and monocytes / macrophages were detected using F4 / 80 (Serotec). For cell proliferation, mouse tumors were stained for Ki-67 using a mouse anti-mouse monoclonal antibody (1: 15 dilution, DAKO). Heat-induced epitope recovery was performed using CC 1 (Ventana Medical Systems). Samples were then incubated with appropriate secondary antibodies (goat anti-rabbit IgG biotinylated antibody, 1: 100 dilution, Jackson ImmunoReseach Laboratories). A 3-3'-diaminobenzidine chromogen peroxidase labeled biotin / streptavidin system (VentanaMedical Systems) was used for antibody localization. Sections were counterstained with Fast Red Nuclear to enhance visualization of tissue morphology. For B16-F10, the Ventana Bluemap kit - an alternative NBT / BCIP decolorization method - was used in virtues of melanin deposits observed in H&E tissues, which queauxiliou in visualizing T cells in tumors.

CTLL-2 T-cell Proliferation Assay

CTLL-2 T cells were preincubated with or without BAY43-9006 (3 µM) at 37 ° C for 2 hours. The cells were then washed and placed in 96-well microtiter slides, 5,000 cells / well in culture medium with serial rhIL-2 dilutions (1 pM to 100 nM). At the end of the incubation period (72 hours at 37 ° C), cell viability was determined by a corantetetrazolium assay using WST-I cell proliferation reagent (Roche Applied Science, Indianapolis, IN).

In vitro cytotoxicity assays

Cells were plated in 96-well microtiter slides (CTLL-2: 5000 cells / well; RENCA: 1500 / well; B16-F10: 100O / well; MV4.11: 5000 / well) and treated with serial dilutions of BAY 43- 9006 / SU11248 or vincristine. CTLL-2 is an IL-2-dependent cell line and, therefore, for these cells, the cytotoxicity assay was conducted on 5 nM rIL-2. At the end of the incubation period (72 hours at 37 ° C), cell viability was determined by a tetrazolium dye (WST-I) or chemiluminescent dye (BrdU) assay (Roche Applied Science, Indianapolis, IN). EC50 values were defined as the concentration required for a 50% reduction in treated cells vs. absorbance. untreated control

Ex Vivo Splenocyte Cytotoxicity Assays

Splenocytes were obtained from drug-treated BALB / mice (n = 3-5 / group) under aseptic conditions homogenized in ice cold PBS. After passage through a 70 µm nylon cell retainer, the cells were rapidly centrifuged at 4 ° C. Red blood cells were lysed using RBC lysis buffer (Sigma, St. Louis, MO). Cells were washed and plated on Yac-1 51 Cr-labeled target cells in various E: T ratios (100: 1, 50: 1, 25: 1, 12/5: 1,6,25: 1, 3: 1 ) in a 4 hour cytotoxicity assay. Data were obtained using a Wallac slide reader and expressed as counts per minute (cpm). Quantification is expressed as percent specific lysis and was calculated as:% specific lysis = 100 x ((experimental mean-spontaneous mean release) / (mean spontaneous mean maximum release)). Spontaneous release was determined from a well containing no dawn cells and no effector cells, and maximum release was determined from wells containing 1% Triton X-100 dawn cells.

Statistical analysis

Multiple comparisons were made using one-way variance analysis (ANOVA) and posttest to compare different means of treatment using the Student-Newman Keuls test (SigmaStat). Differences were considered statistically significant at p <0.05.

B. In vitro Studies with rIL-2 and BAY 43-9006 on T-Cell Signaling Pathways and Tumor Cells

rIL-2 Activates JAK / STAT and MAPK Signaling in In vitro CTLL-2 Cells

To elucidate the onset, time course and duration of IL-2 / IL-2R signal transduction pathways in T cells, CTLL-2 cells (1x107 cells) were deprived of serodurant 24 hours prior to treatment with various rIL-2 concentrations. (1 pM to 100 nM) over 2 hours of MAPK, STAT5, AKT key signaling were evaluated using Western blot analysis. Serum-deprived CTLL-2 cells were treated in vitro with a wide range of rIL-2 concentrations (from 1 pM to 100 nM) to delineate the interactions of two predominant IL-2R complexes: high affinity receptor (IL-2 KD). 2Rapy = 10-11M) and intermediate affinity (IL-2py KD = 10 "9M). In some experiments, cells were exposed to free anti-IL-2a antibody (excess> 1000-fold; 10 nM) incubated for 1 hour before rIL-2 addition.To evaluate IL-2 / IL-2R activation course time, serum-deprived TCLL-2 cells were activated with 10 nM rIL-2 and IL-2 signaling was examined at various time points. 10 minutes to 48 hours Downstream IL-2R phosphorylation of ERK1 / 2, STAT5 and AKT was evaluated in rlL-2-treated cells using Western blot analysis .The relative levels of depERK, pSTAT5 or pAKT were compared with s total protein levels for ERK, STAT5 or AKT, respectively.

Activation of pERK1 / 2 was observed after exposure of CTLL-2 cells to rIL-2. Phospho ERK was slightly activated at 1 pM within 2 hours, however, maximum activation was observed at concentrations> 100 pM. The JAK / STAT5 pathway was optimally activated at a concentration of 1 pM; and increased rIL-2 concentrations did not alter depSTAT5 levels (up to 100 nM). The basal pAKT pathway in CTLL-2 appears to be activated on T cells under serum deprivation conditions. In addition, pAKT levels remained largely unchanged at the tested rIL-2 concentrations (1 pM to 100nM; using a pAKT antibody to the phosphorylation 483).

An IL-2Ra blocking antibody was used to confirm whether IL-2 signal pathways are specifically mediated by binding to IL-2R. PSTAT5 levels in CTLL-2 cells were analyzed by Western blot. CTLL cells were serum-deprived and treated with excess free anti-IL-2Ra antibody (10 nM,> 1000-fold) for 1 hour prior to rIL- 2 (0.1 pM to 10nM). In the absence of blocking IL-2Ra antibody, pSTAT5 was activated at 1 pM after rIL-2 treatment, however, in the presence of IL-2Ra inhibition, pSTAT5 signaling was aborted (inhibition> 95%), confirming the requirement of IL-2Ra. in STAT5 signaling. Interestingly, pSTAT5 levels were restored in CTLL-2 cells with increasing concentrations of rIL-2 (> 10 pM), suggesting that rIL-2 competitively displaces anti-IL-2Rae antibody or activates pSTAT5 signaling by binding to the low affinity IL-2RJ3Y chain (KD = 10-9 M).

PSTAT5 Activation by rIL-2 is Fast and Sustained on CTLL-2 Cells

To assess the time of onset and duration of IL-2R signaling responses in in vitro treated seroforam deprived CTLL-2 cells with rIL-2 (10 nM) and the effects on ERK1 / 2, STAT5 and AKT phosphorylation were evaluated using analyzes. by Western blot. ST AT 5 phosphorylation was activated in minutes (<10 minutes) after the addition of rLL-2 to CTLL-2 cells and the response duration of pSTAT5 was maintained for up to 48 hours. Activation of the pelarIL-2 MAPK (pERK) pathway appeared to be slightly delayed and was activated within 1 hour. The intensity of pERK was lower than the maximum response obtained by stimulation of serum-deprived CTLL-2 cells with PMA (50 ng / ml) + ionomycin (0.4ug / ml) for 15 minutes. Additionally, depERK levels were sustained for up to 24 hours and returned to baseline levels at 48 hours. No discernible effects were observed on pAKT, confirming that the PI-3K / AKT pathway is continually active in CTLL-2 cells.

BAY 43-9006 Treatment Inhibits pERK and not pSTAT5 in CTLL-2 Cells

BAY-43-9006 is a potent inhibitor of Raf-1, which also inhibits mutant and wild type BRAF. In addition, BAY-43-9006 inhibits multiple kinases, particularly VEGF2,3; PDGFRJ3; FLT3 and cKIT (Wilhelm, SMe et al., Cancer Res 2004; and Chiron kinase profile definition data) and to some extent inhibit aLck and Fyn, two kinases that are involved in T-cell functional responses. 43-9006 and the potential impact on MAPK signaling / T-cell signaling, it is believed that BAY 43-9006 can potentially interfere with IL-2 / IL-2R signaling in T cells. Therefore, potential BAY interactions 43-9006 on IL-2 signaling were evaluated in CTLL-2, B16-F10 or RENCA invitro cells.

To examine the effects of BAY 43-9006 on CTLL-2, B16-F10, or RENCA model cells, sputum deprived cells were treated with various concentrations of BAY 43-9006 (0.01-20 µM). As an appropriate control, cells were stimulated with PMA (50 ng / ml) and ionomycin (0.4 µg / ml) for 15 minutes. To examine the effects of concurrent and sequential treatments with rIL-2 and BAY 43-9006 on CTLL-2 cells in vitro, rIL-2 (10 nM) -treated seroforan CTLL-2 cells in the presence or absence of BAY 43-9006 (3 µM) for 2 hours. For sequential treatments, BAY 43-9006 was treated for 2 hours. Ascells were then washed and then treated with rIL-2 and vice versa. The inhibitory effects of BAY 43-9006 (3 µM) were also examined without or with stimulation with PMA (50ng / ml) and ionomycin (0.4 µg / ml) for 15 minutes.

Since the BAY 43-9006 concentration required to inhibit the MAPK pathway in different cell types is very variable, the effects of various BAY43-9006 concentrations (ranging from 0 to 20 µM) were evaluated in serum deprived TCLL-2 cells. for 2 hours with or without stimulation with PMA + Ionomicin. At the end of the incubation period, treated CTLL-2 cells were then lysed and protein lysates were subjected to Western blot analysis to determine pERK levels. BAY43-9006 substantially inhibited pERK at concentrations> 1uM in murine CTLL-2 cells and the Thumana Jurkat cell line. The effects of treatment with BAY 43-9006 on CTLL-2 cells did not alter pSTAT5 and pAKTem cell levels, indicating that the JAK / STAT5 and PI-3K / AKT pathway in T cells remained largely unaffected.

To investigate the therapeutic basis of combination derIL-2 and BAY 43-9006 in vitro, the effects of rIL-2 and BAY 43-9006 on T cells and the impact on the two IL-2-mediated deinalization pathways (MAPK and JAK / STAT5) in TCLL-2 cells were studied. The effects of IL-2-mediated T-cell signaling with concomitant or sequential two-drug regimens were investigated in CTLL-2 cells. Serum deprived TCLL-2 cells were treated with BAY 43-9006 (3uM) for 2 hours and then treated with vehicle, rIL-2 (10 nM) or PMA + Ionomicin. Alternatively, treatment with rIL-2 (10 nM, 2 hours), followed by BAY 43-9006 (3 µM, 2 hours) was also investigated. Following drug exposure and / or stimulation with PMA + Ionomicin, Western blot analyzes of pERK and pSTAT5 on cell lysates were performed (as described above). Treatment with BAY 43-9006 (3 pM) inhibited pERK levels, whereas rIL-2 activated pERK levels in serum-deprived CTLL-2 cells (vs. baseline pERK levels), confirming the opposite effects of both. MAPK aviation drugs. All treatments with combinations of BAY43-9006 (concomitant and sequential drug exposures) substantially inhibited pERK in CTLL-2 cells. No effects were observed on the STAT5 pathway or the AKT pathway, with all combined treatments tested (as outlined in methods).

BAY 43-9006 at High Concentrations Inhibits DepERK Levels in Tumor Cell Lines

The effects of treatment with BAY 43-9006 on murine tumor cell lines - B16-F10 melanoma, colon CT26 and RCC RENCA model were determined. Murine cell lines were selected based on their responsiveness to rIL-2 in vitro therapy. live in immunocompetent models (T- / NK- / monocyte- / macrophage-competent mice), where the effects of rIL-2 and BAY 43-9006 combination therapy could be investigated. Serum-deprived BI6-FIO and RENCA melanoma cells were exposed to BAY 43-9006 concentration range from 0 to 20 µM. Fosfo-ERK levels in cell lysates following drug exposure were determined by Western blot analysis. BAY 43-9006 inhibited pERK levels in all cell lines tested (B16-F10 and RENCA) at very high concentrations of> 5uM, with almost complete elimination of pERK observed at 20 µM.

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

To examine whether inhibition of pERK by BAY 43-9006 affected IL-2-mediated proliferative responses in CTLL-2 cells, proliferation assays were conducted by CTLL-2 cell pre-incubation (5000 cells / wells) at BAY 43-9006 concentrations. (3 µM, 2 hours) inhibiting ERK. After a 2 hour incubation of the cells with BAY 43-9006 (3 µM), the cells were placed in 96 well slides and exposed to various concentrations of rIL-2 (0-100nM) for 72 hours. Untreated cells were given sham treatments prior to rIL-2 incubation (at the same concentrations). Proliferative responses of BAY 43-9006 treated and untreated CTLL-2 cells were evaluated using the WST-I assay. Preincubation of the cell with BAY 43-9006 (3 µM) inhibited pERK signaling, but did not affect IL-2-induced proliferative responses in the CTLL-2 cell line.

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

To evaluate the in vitro cytotoxic activity of BAY43-9006 or SU11248 in each of these cell types (CTLL-2, B16-F10, RENCA, CT26), cells were placed in 96-well microtiter slides and treated with serial BAY dilutions. 43-9006 (from 0 to 50 µM) for 72 hours at 37 ° C. Since the CTLL-2 cell line is IL-2 dependent for proliferation, cytotoxicity against these cells was conducted in medium containing 5 nM rIL-2. At the end of the 72 hour incubation period, cell viability was determined by the tetrazolium dye assay (WST-I) or a chemiluminescent assay (BrdU). As controls, MV4.11 (AMLhuman FLT3 ITD cell line) was treated with BAY 43-9006 (0 to 50 µM) or B16-F10 cells were treated with vincristine (0 to 1 pM) to confirm agent cytotoxicity and validity. Assays. Inhibitory drug concentrations were expressed as the EC50 value, which was defined as the concentration required for a 50% reduction in proliferative response measured as the absorbance of drug-treated cells vs. vehicle control / untreated.

The relative EC50 of BAY 43-9006 or SU11248 is shown in Table 9.

<table> table see original document page 121 </column> </row> <table> <table> table see original document page 122 </column> </row> <table>

The cells were placed in 96-well microtiter slides (CTLL-2: 5000 cells / well; RENCA: 150O / well; B16-F10:

1000 / well; CT26: 1000 cells / well; MV4; 11: 50 / well) and treated with serial dilutions of BAY 43-9006, SU11248 or Vincristine. CTLL-2 is an IL-2-dependent cell line and, consequently, the decitotoxicity assay was conducted on medium containing 5 nM rIL-2. At the end of the incubation period (72 hours at 37 ° C), cell viability was determined. through a tetrazolium dye assay (WST-I).

aEC50 = concentration required for 50% reduction in proliferative response, measured as absorbance of drug-treated cells vs. vehicle / untreated controls.

bMV4.11 is a human acute myelogenous leukemia (AML) cell line that expresses FLT3 Internal Random Duplication (ITD). BAY 43-9006 (potent FLT3 kinase inhibitor; IC50 = 58 nM) demonstrates potent anti-proliferative activity in vitro against MV4.11 cells. Cell viability was evaluated using the PromegaCell-Titer Glo ™ assay which measures APT content of cells.

In general, relatively high concentrations of BAY 43-9006 (or SU11248) were required to inhibit CTLL-2 cell proliferation as well as several cell lines (B16-F10, RENCA, CT26) compared with vincristine antimitotic agent (defined by EC50's). see Table 9). The cytotoxicity of the effects of BAY 43-9006 on RENCA cells was also examined using the BrrdU assay to evaluate the effect on DNA synthesis (vs. antiproliferative activity using the mitochondrial corantetetrazolium assay). The EC50 for BAY 43-9006 (~ 5 µM) on RENCA cells obtained using the BrdU method was similar to that observed with the WST-I assay. No direct proliferative response (or cytotoxicity) was observed when RENCA tumor cells were exposed to the rIL-2 concentration range (from 0 to 1 µM), confirming that the rIL-2 mechanism relies on activation of the immune effector component.

E. In vivo Effectiveness Studies

Pharmacokinetics of rIL-2 and BAY 43-9006 in Mice In published Phase I reports of BAY 43-9006 (at a dose of 400 mg), high Cmax of 10-20 µM and long drug half-lives of about 24 hours. were obtained from patients (Strumberg et al., J. Clin. Oncol. 200523 (5): 965-972). To determine whether exposure to noplasma drug may have an impact on the viability of proliferative T cell responses in vivo, the pharmacokinetics of a single dose of rIL-2 and BAY 43-9006 in mice were measured.

A single oral dose of 30 mg / kg BAY 43-9006 in mice achieved a Cmax of about 5500 to 8000 ng / ml (-10 µM) at a tmax of 2 hours. The BAY43-9006 plasma clearance rate was reasonably slow and the resulting half-life was about 4 hours. In contrast, the PKda rIL-2 profile following subcutaneous administration of 6 mg / kg showed a Cmax of about 550 - 850 ng / ml at a tmax of about 30 minutes. The t1 / 2 of rIL-2 in mice was approximately 1 hour, with exposures of> 50 ng / ml obtained for 4 hours. Interestingly, treatments after a single dose and their amino acid pathways demonstrated non-overlapping Cmax (at tmax), concluding that concomitant and sequential treatments may be feasible, as supported by PK findings. Because BAY 43-9006 may be highly protein bound in the blood, the impact of these drug exposures on T cell viability in vivo remains to be considered.

Treatment with rIL-2 and BAY 43-9006 decreases ex vivo immune effector function

Since inhibition of one or multiple T-lymphocytic kinases (Lck, Fyn, Syk, Btk, Src, Tck2, MAPK, JAKs) can eliminate T cell expansion and effectorimmune function, the effects of rIL-2 and BAY 43-9006 on proliferative and functional T cell responses in vivo were investigated.

In these experiments, BALB / c mice bearing RENCA tumors were treated with rIL-2 (1 mg / kg / day, days 6-10), BAY 43-9006 (30 mg / kg / day, po, days 6-10) or combinations. rIL-2 and BAY 43-9006 administered concomitantly or sequentially (rIL-2, 1 mg / kg / day, sc, days 1-5 + BAY 43-9006, 30 mg / kg / day, po, days 6-10; or BAY 43-9006, 30 mg / kg / day, po, days 1-5 + rIL-2, 1 mg / kg / day, sc, days 6-10). Isolated splenocytes from treated mice were then subjected to ex vivo death assays against target Yac-1 cells at various effector: target (E: T) ratios and specific percent lysis of labeled 51 Cr-labeled Yac-1 cells was determined. -2 (1 mg / kg) significantly increased splenocyte-mediated death of Yac-1 targets compared with vehicle treatment (23% with rIL-2vs. 0% with vehicle treatment). The specific death effects observed with BAY 43-9006 treatment (1%) were negligible and did not differ from vehicle treatment. All treatments with rf and BAY 43-9006 provided reduced splenocyte-mediatic lytic activity (concomitant treatment = 16%; subsequent treatments <9% vs. rIL-2 monotherapy = 23%).

Effect of rIL-2 and BAY 43-9006 Therapy on Circulating Immune Effector Cells Populations

The pharmacodynamic effects of rIL-2 and / or BAY-43-9006 therapy on circulating lymphocyte and monocyte populations in BALB / c mice bearing tumor and tumor were evaluated. In these studies, blood was collected after a single agent or combination therapy of rIL-2 and BAY 43-9006 (concomitant or sequential treatments as previously described). Absolute T-cell delymphocyte counts and subpopulations (CD4 + or CD8 +) were quantified in whole blood using TruCount ™ tubes and appropriate immunostaining.

Treatment with rIL-2 decreased the absolute numbers of circulating lymphocytes and monocytes (CD45 + cells: 2387 cells / pl vs. 1575 cells / pl with vehicle) and T cells (1550 cell / ul vs. 664 cell / ul with vehicle) in mice. A significant proportion of CD4: CD8 cells was observed with rIL-2 therapy compared with vehicle treatment (5: 3; rIL-2: Vehicle, ie 1.7-fold) as indicative of the mechanism of action of rIL-2 on T-cell number expansion. Relative numbers of non-T and monocytic (CD45 + CD3-) cells after treatment with rIL-2 were similar to vehicle treatment (83 7 cells / ul vs. 911 cells / ul with vehicle). .

In contrast, the single agent BAY 43-9006 or BAY 43-9006 combined with rIL-2 had little impact or increased absolute lymphocyte and monocyte numbers (range 2417-3577 cells / pl vs. 2387 cells / pl with vehicle) and also increased non-T cell and monocyte populations (1241-1563 cells / pl vs. 837 cells / pl with vehicle). The effect of BAY 43-9006 therapy on total T cell numbers was similar to vehicle treatment (1827 cells / p1 vs. 1550 cells / p1 with vehicle). Total T cell numbers increased slightly (including CD4 + and CD8 + populations) with the sequential rIL-2and BAY 43-9006 regimen when started with rIL-2 (2081 T cells / plvs. 1550 cells / pl with vehicle). When rIL-2 and BAY 43-9006 were given concomitantly or sequentially as BAY 43-9006, then, rIL-2, total T cell numbers decreased slightly compared to vehicle treatment (-1170-1244 T cells / pl vs. 1550 cells / pl comvehicle). Similar trends were observed in individual CD4 + and CD8 + T cell populations. In addition, tumor histology following rIL-2 and BAY 43-9006 or SU11248 treatment therapy (as previously described) was determined. To examine T cell pharmacodynamics in vivo, tumor infiltrating T cells were detected with a mouse anti-CD3 antibody. Antiproliferative effects in tumors following drug treatments were evaluated using KI67 staining. With rIL-2 treatment, increased T-cell numbers were observed seeping into RENCA (and B16-F10) tumors compared with vehicle treatment. Generally, fewer T cells were detected in the Bayer 43-9006 treated group named RENCA. The effects of rIL-2 and BAY 43-9006or SU1124 8 therapy on T cell infiltration in B16-F10 tumors were equivocal since some T cells were detected in all treated groups. Tumors that demonstrated increased necrosis (tumor inhibition) generally showed higher numbers of Tinterdispersed cells among tumor cells. Collectively, the data suggest that rIL-2 activates circulating T cells and cells travel to extravascular sites, including tumors, and that treatment with BAY43-9006 or SU11248 may partially eliminate proliferative responses and T cell traffic.

RIL-2 and BAY 43-9006 Therapy Increases Effectiveness of IL-2-Responsive Murine Tumor Models

Drug interactions of rIL-2 with BAY 43-9006 or SU11248 were examined (see Figures 1-10 and Table 10-12). Combined treatments were evaluated on three experimental IL-2-responsive, T-competent cell tumor models (B16-F10 melanoma, colon CT26, RCCRENCA model) (Figures 1-10). In these studies, mice were randomly distributed when tumors were commonly established at a size of 50-252 mm3 and growth of tumors was monitored by gauge measurements following daily oral dosing of BAY 43-9006 or SU11248 or subcutaneous daily administration of rIL-2 (as indicated). methods). The efficacy and tolerability of a single rIL-2 agent, BAY 43-9006 and SU11248 were investigated over a range of doses and treatment regimens (Figure 1). In all models, rIL-2 demonstrated potent efficacy and effective dose inhibitions were generally within the range of 40-60% (vs. vehicle treatment) (Figure 1). Minimum effective dose for BAY 43-9006 was> 30 mg / kg / day (vs. Vehicle treatment). SU11248 was generally effective at doses> 40 mg / kg / day, except for tumor model B16-F10 (Figure 1).

Based on the efficacy and tolerability of a single agent, rIL-2 was then combined with BAY 43-9006 or SU1124 8 at doses that were tolerated and exhibited no detrimental effects on body weight or any adverse clinical symptoms. Sequential and concurrent treatment regimens were examined in the B16-F10e CT26 tumor models (Figures 2-8, Tables 10 and 11). In the B16-F10 and CT26 tumor models, almost all combined (regimeconcomitant or sequential) therapies investigated with rIL-2 and BAY43-9006 or rIL-2 and SU12248 increased anti-tumor activity compared with monotherapy or vehicle treatments (Figures 2 -5). A summary of the combined drug interactions of the various combination therapies is summarized in the <table> table see original document page 129 </column> </row> <table> <table> table see original document page 130 </column> </row> <table> <table> table see original document page 131 </column> </row> <table>

an = 10 C57BL6 mice / group in each study. Mice bearing BI6-FIO tumor were treated when ostumors had an established mean size of -50mm3.

b Observed T / C (O) =% T / C

c T / C (E) =% T / C treatment 1 x% T / C treatment 2

d Synergistic effects were defined when the expected% tumor growth inhibition ratio of the combined therapy (% T / Cesp =% T / C treatment 1 x% T / C treatment 2) divided by the observed% T / C (% T / Cobs) of the combined treatment was> 1.

and Drug interactions were defined as additive when% T / Cesp /% T / Cobs = 1 and antagonism when% T / Cesp /% T / Cobs <! • N / A, not applicable.

Table 11. Efficacy of rIL-2, BAY 43-9006, SU11248 and rIL-2 and BAY 43-9006 / SU11248 e-combinations in the <table> table model see original document page 132 </column> </row> <table> <table> table see original document page 133 </column> </row> <table> <table> table see original document page 134 </column> </row> <table>

an = 10 BALB-c mice / group in each study. Mice bearing CT26 tumors were treated when ostumors had an established mean size of ~ 225mm3.

b Observed T / C (O) =% T / C

c T / C (E) =% T / C treatment 1 x% T / C treatment 2d Synergistic effects were defined when the expected% tumor growth inhibition ratio of combined therapy (% T / CesP =% T / C treatment 1 x % T / C treatment 2) divided by the observed% T / C (% T / C0bS) of the combined treatment was> 1.

and Drug interactions were defined as additive when% T / Cesp /% T / Cobs = 1 and antagonism when% T / Cesp /% T / C0bs <1-N / A, not applicable.

In only one case in model CT26, when BAY 43-9006 (40 mg / kg / day, days 1-7) was administered before derIL-2 (1 mg / kg / day, days 8-13), the effects The combined treatment were sub-optimal.

In the RENCA model, only concomitant schemata evaluated (Figures 9-10; Table 12) and combinations of rIL-2 with BAY 43-9006 or SU11248 demonstrated greater tumor inhibition compared to single agent therapy.

<table> table see original document page 135 </column> </row> <table> <table> table see original document page 136 </column> </row> <table> b T / Observed (O) =% T /Ç

c T / C (E) =% T / C treatment 1 x% T / C treatment 2

d Synergistic effects were defined when the expected% tumor growth inhibition ratio of the combined therapy (% T / Cesp =% T / C treatment 1 x% T / C treatment 2) divided by the observed% T / C (% T / Cobs) of the combined treatment was> 1.

and Drug interactions were defined as additive when% T / CeSp /% T / Cobs = 1 and antagonism when% T / Cesp /% T / Cobs <1-N / A, not applicable.

Sequential treatments in the RENCA model are not possible due to the short duration of the mouse model and caquexed mouse (weakening / weight loss). Collectively, the data demonstrate intensified activity when rIL-2 was combined with small molecule therapies such as BAY 43-9006. and SU11248, in preclinical models, indicating that such therapeutic strategies could be translated to the clinician.

While the present invention has been described with respect to specific examples, including presently preferred embodiments of the invention, those skilled in the art will appreciate that there are various variations and exchanges of the above described systems and techniques which fall within the inventive concept and scope of the invention.

INCORPORATION BY REFERENCE

The contents of all patents, patent applications and newspaper articles mentioned above are incorporated by reference as fully set forth herein.

Claims (32)

A method of treating a patient suffering from cancer comprising administering to said patient a therapeutically effective amount of aldesleukin and a selected anti-angiogenic agent of 6,7-bis (2-methoxyoxy) -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-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl ) phenyl) urea. Method according to claim 1, characterized in that aldesleukin is administered before the anti-angiogenic agent. Method according to claim 1, characterized in that aldesleukin is subsequently administered to the anti-angiogenic agent. Method according to claim 1, characterized in that aldesleukin is administered concurrently with the anti-angiogenic agent. Method according to Claim 4, characterized in that the aldesleukin is administered in a separate composition of the anti-angiogenic agent. A method according to any one of claims 1, 2, 3, 4 or 5, comprising separately administering to said patient a therapeutically effective amount of desleukin and an anti-angiogenic agent according to a dosage schedule wherein aldesleukin is administered. 1-3 times daily at a dose of from about 9 to about 130 MIU / day over a period of at least 3 consecutive days, optionally followed by a pause period of at least 3 consecutive days. Method according to claim 6, characterized in that said anti-angiogenic agent is administered from 1 to 6 times every 2-3 weeks. Method according to claim 6, characterized in that the aldesleukin is administered intravenously and said withdrawal period is present. Method according to Claim 6, characterized in that the aldesleukin is administered subcutaneously and said rest period is absent. Method according to claim 9, characterized in that aldesleukin is administered 1-3 times daily at a dose of about 9-30 MIU / day. Method according to claim 6, characterized in that aldesleukin is administered 3 times daily at a dose of about 30-130 MIU / day. Method according to claim 6, characterized in that aldesleukin is administered for a period of about 5 consecutive days followed by a 9-day break period. Method according to claim 6, characterized in that said dosage schedule is repeated for at least two courses. Method according to claim 6, characterized in that said dosing schedule is repeated for 3 courses. Method according to claim 6, characterized in that said dosage schedule is repeated for 4 strokes. Method according to claim 6, characterized in that aldesleukin is administered 3 times during the first day and once a day thereafter. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15 or 16, characterized in that the sercarcinoma cancer of renal cells, melanoma and colon cancer. A method according to claim 17 further comprising administering to the patient at least one selected compound deacetaminophen, meperidine, indomethacin, ranitidine, nizatidine, diastop, loperamide, diphenhydramine or furosemide subsequent to or concurrently with aldesleukin co-administration. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17 or 18, characterized in that of this method results in cancer relief in the patient. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17 or 18, characterized in that such method results in attenuation of hypotension in the patient. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17 or 18, characterized in that this method results in attenuation of hypertension in the patient. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17 or 18, characterized in that of this method results in the reduction of nitric oxide synthase in the patient. Method according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17 or 18, characterized in that from susceptible cancer to inhibition of angiogenesis and / or immune stimulation. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17, 18, 19, 20 21, 22 or 23, characterized in that said anti-angiogenic agent is N- (2- (dimethylamino) ethyl) -5 - ((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4- diethyl-1H-pyrrole-3-carboxamide. Method according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15, 16, 17, 18, 19, 20 , 21, 22, 23 or 24, characterized in that the anti-angiogenic agent is 1- (4- (2-methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea. A composition comprising: (a) a therapeutically effective amount of aldesleukin; (b) a therapeutically effective amount of an anti-angiogenic agent selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1- (trifluoromethyl) phenyl) urea; and (c) a pharmaceutically acceptable excipient. A kit comprising a combination of drugs for the treatment of a cancer patient comprising: (a) aldesleukin and (b) an anti-angiogenic agent selected from 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-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin -1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4-yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea for simultaneous, sequential or separate use. A kit according to claim 26, characterized in that each of said drugs is separately packaged. 29. Use of aldesleukin and a selected anti-angiogenic agent of 6,7-bis (2-methoxyethoxy) -N- (3-ethynylphenyl) quinazolin-4-amine, 6- (3-morpholinopropoxy) -N- (3-chloro -4-fluorophenyl) -7-methoxyquinazolin-4-amine, N- (2- (dimethylamino) ethyl) -5 - ((5-fluoro-2-oxoindolin-3-ylidene) methyl) -2,4-diethyl 1H-pyrrola-3-carboxamide, N- (4-chlorophenyl) -4 - ((pyridin-4-yl) methyl) phthalazin-1-amine, or 1- (4- (2- (methylcarbamoyl) pyridin-4- yloxy) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea characterized in that it is for the manufacture of one or more drugs for the treatment of a cancer patient. Use according to claim 29, characterized in that aldesleukin is formulated for administration prior to the anti-angiogenic agent. Use according to claim 29, characterized in that aldesleukin is formulated for subsequent administration to the anti-angiogenic agent. Use according to claim 29, characterized in that aldesleukin is formulated for concurrent administration with the anti-angiogenic agent.