KR20130121122A - Methods of treating tumors - Google Patents

Abstract

The present invention relates to a method of treating tumors in which PAK1 is overexpressed or amplified by co-administration of a PAK1 inhibitor and a second anti-hyperproliferative agent.

Description

Method of treatment of tumors {METHODS OF TREATING TUMORS}

The present invention discloses a method of treating cancerous tumors which amplify or overexpress PAK1 by contacting the cancerous tumor with a PAK1 inhibitor with a second anti-proliferative agent.

Protein kinases are a family of enzymes that catalyze the phosphorylation of hydroxyl groups of certain tyrosine, serine or threonine residues in proteins. Typically, such phosphorylation can drastically change the function of a protein, so protein kinases can be pivotal in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation and cell survival. The mechanisms of these cellular processes provide the basis for targeting protein kinases to treat disease conditions arising out of or involving the impairment of these cellular processes. Examples of such diseases include, but are not limited to, cancer and diabetes.

Protein kinases can be classified into two types, protein tyrosine kinase (PTK) and serine-threonine kinase (STK). Both PTK and STK can be receptor protein kinases or non-receptor protein kinases. PAKs are a family of non-receptor STKs. The p21-activated protein kinase (PAK) family of serine / threonine protein kinases plays an important role in cytoskeletal organization, cell morphogenesis, cellular processes and cell survival (Daniels et al., Trends Biochem. Sci. 1999 24: 350-355; Sells et al., Trends Cell. Biol. 1997 7: 162-167]. The PAK family consists of six members subdivided into two groups: PAK1 to PAK3 (group I) and PAK4 to PAK6 (group II), identified based on sequence homology and the presence of self-suppressive regions within group I PAK. . p21-activated kinases (PAKs) serve as important mediators of Rac- and Cdc42 GTPase functions as well as pathways required for Ras-induced tumorigenesis (Manser et al., Nature 1994 367: 40-46; B Dummler et. al., Cancer Metathesis Rev. 2009 28: 51-63; R. Kumar et al., Nature Rev. Cancer 2006 6: 459-473].

The present invention treats a patient with a PAK1 inhibitor and a second anti-hyperproliferative or anti-tumor agent selected from inhibitors of the EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptosis protein inhibitors or the tumor is treated with a PAK1 inhibitor And a method for treating a tumor or hyperproliferative condition by contacting with said second anti-hyperproliferative agent or anti-tumor agent, wherein the tumor cell or hyperproliferative cell overexpresses or amplifies PAK1.

Figure 1-Analysis of PAK1 genome amplification and functional role in human breast tumors. (A) GISTIC (Genome Identification of Significant Targets in Cancer) Analysis of 11q13 Replication Number Increase. The points are proportionally spaced and arranged in genomic order. The vertical line represents the chromosomal location of the PAK1 gene. GISTIC Q-values for DNA increase are defined as the frequency of increase, expressed as negative log ₁₀ of Q-values for each SNP array probe set, and the multi-test-corrected probability of accidental mean replication increase. (B) PAK1 DNA Replication and mRNA Expression The viscosity table shows the relationship between DNA replication number and 226507_at Affymetrix MAS 5.0 signal for 51 tumor samples. Pearson correlation statistics (0.75) for the plot are shown. The straight line represents the best fit line through these points. (C) The increasing proportion of Annexin V positive cells after knockdown of PAK1 expression was found in three breast cancer cell lines with local PAK1 genomic amplification, namely MDA-MB-175, HCC1500 and MDA-MB-134. Marked for IV. Cells were harvested three to five days after transient transfection of pooled siRNA oligonucleotides. (D) Fluorescence-activated cell sorting assay for Annexin V / PI. MDA-MB-175 cells were incubated for 48 hours in the presence or absence of IPA-3. Annexin V / PI staining was then performed to assess apoptosis / necrosis. Annexin V markers (lower right quadrant) indicate populations undergoing early apoptosis. Annexin V and PI double labels (upper right quadrant) indicate cells that have already been killed by apoptosis. Surviving cells are indicated in the lower left quadrant. The percentage of cells for each quadrant is indicated.
2-PAK1 is highly expressed in human lung tumors and plays a key role in the proliferation of squamous NSCLC cell lines. (A) Analysis of PAK1 mRNA expression in laser-capture microdissection lung tissue. Data for Affymetrix probe 226507_at is plotted as mean (horizontal line), median 50% (box) and 95% confidence interval (line) of the data. Pairwise comparisons were performed with the Student's t-test. PAK1 expression was significantly higher in squamous NSCLC (n = 16; **, p = 0.0005) and adenocarcinoma NSCLC (n = 29; *, p = 0.008) relative to normal tissue (n = 9). The difference in PAK1 mRNA expression between squamous NSCLC and adenocarcinoma NSCLC was not significant in this lung tumor panel (p = 0.1). (B) Proliferation of squamous NSCLC cell line panels was measured by [ ³ H] thymidine uptake assay. Non-targeting control siRNA oligonucleotides (black bars) for EBC-1, NCI-H520, KNS-62, SK-MES-1 and NCI-H441 cells (black bars), or pools of siRNA oligonucleotides for PAK1 and PAK2 (white bars) Transfection. The extent of proliferation under each condition was plotted as a percentage of normalized non-targeting control values for each cell line, and data are expressed as mean ± SD.
3-(A) Accumulation of cells at G ₁ phase of the cell cycle after PAK1 knockdown is evident. NCI-H520.X1 cells were treated with 200 ng / ml Dox for 4 days and analyzed by propidium iodide staining and flow cytometry. (B) NCI-H520.X1 cells were not serum fed for 24 hours, and cell cycle reintroduction was monitored by growing in 10% serum containing medium and collecting cell lysates at the indicated time points. Cell lysates were analyzed by immunoblotting using antibodies against PAK1, p27 ^Kip1 , E2F1 and actin. (C) The percentage of cells with nuclear accumulation of p27 ^Kip1 is indicated (total 2000 cells per condition). Bars represent mean ± SD (*, p <0.05. **, p <0.0001).
4-PAK1 is required for the growth of established NCI-H520.X1 and EBC-1 squamous NSCLC tumors. (A) NCI-H520.X1 cells expressing inducible shRNA against LacZ, PAK1, PAK2 or PAK1 + PAK2 were transplanted into the flanks of athymic mice as described in Materials and Methods. Treatment was started when the tumor size was between 200 mm and 250 mm in each experiment. Administration of 1 mg / ml doxycycline through drinking water inhibited tumor growth for mice bearing shPAK1 and shPAK1 + 2 NCI-H520.X1 cells. Induction of PAK2 or LacZ specific shRNA did not affect tumor growth rate. No animal weight loss was observed. The data consist of 10 mice per treatment group and error bars represent standard error. Individual mice were removed from the data plot when tumors reached a volume end point of 2000 mm 3: shLacZ control n = 5; shLacZ + Dox n = 3; shPAK1 control n = 2; shPAK2 control n = 5; shPAK2 + Dox n = 2; shPAK1 + 2 control n = 5. (B) EBC-1 tumors expressing shLacZ or shPAK1 were grown from 200 kPa to 250 kPa prior to doxycycline administration to groups of mice with equally sized tumors to inhibit PAK1. The data consist of 10 mice per treatment group and error bars represent standard error.
5-PAK1 inhibition reduces NF-κB pathway activation and promotes apoptosis of NSCLC cells in combination with IAP antagonists. (A) EBC-1-shPAK1 and -shLacZ cells were treated with BV6 IAP antagonist and 300 ng / ml doxycycline (Dox). The highest concentration of BV6 was 20 μΜ and 2-fold serial dilutions were evaluated on a 10 point dilution curve. Cells were preincubated for 3 days in the presence of Dox before adding BV6 for an additional 3 days. Cell cultures were then analyzed by CellTiterGlo viability assay. Data points were performed in triplicate. (B) Fluorescence-activated cell sorting analysis for Annexin V and propidium iodide (PI) staining. EBC-1-shPAK1 cells were cultured for 72 hours in the presence or absence of 300 ng / ml doxycycline (Dox) and for an additional 24 hours after addition of 5 μM BV6. Annexin V / PI staining and fluorescence-activated cell sorting (FACS) analysis were then performed to assess apoptosis / necrosis.
6-(A) Downregulation of XIAP expression enhances the proapoptotic activity of PF-3758309 PAK small molecule inhibitor (PAK SMI; p <0.0001, Dunnett's t-test). Cells were transfected for 48 hours with non-targeting control (NTC) or XIAP specific siRNA oligonucleotides prior to further 72 hours of treatment with DMSO or PAK SMI as indicated. Cell viability was measured via CellTiterGro analysis, and the results represent mean ± standard deviation from three experiments. (B) Combination antagonism of XIAP with PAK1 promotes efficient cleavage of PARP and caspase-3. XIAP siRNA oligonucleotides were transfected for 72 hours before treatment with DMSO or 5 mM PAK SMI.
7-(A) Combination accumulation of cleaved PARP and caspase-3 by PAK1 and IAP antagonism. Cells were incubated and lysed with Dox and 5 μM BV6 for the indicated times and used for western blot analysis. (B) Dual PAK1 and IAP inhibition synergistically reduces the viability of SK-MES-1 (squamous subtype) and NCI-H441 (adenocarcinoma subtype) NSCLC cells. As indicated, transient siRNA-mediated inhibition of PAK1 and cellular ATP consumption after 5 μM BV6 treatment were measured via CellTiterGlo analysis. Inhibition of cell viability was significantly higher with the combination of PAK1 siRNA and BV6 than with the single agent (p <0.0001, Student's t-test).
8-PAK1 inhibition leads to cleavage of caspase and poly ADP ribose polymerase (PARP) in breast cancer cells with local genome amplification of PAK1. (A) HCC-1500 cells were transiently transfected with individual or pooled siRNA oligonucleotides (100 nM) to induce PAK1 knockdown. Cell lysates were harvested after 48 hours and monitored for induction of apoptosis by immunoblotting with antibodies to cleaved caspase-3, cleaved caspase-7 and cleaved PARP. (B) Removal of PAK1 protein expression in MDA-MB-134 IV cells was also associated with a decrease in MEK1 (Ser298) and ERK1 / 2 (Thr202 / Tyr204) phosphorylation. Total protein and β-actin were used as controls.
9-Combined PAK1 inhibition and IAP inhibition result in apoptosis of squamous NSCLC cells. (A) The percentage of Annexin V positive cells for each treatment condition is indicated. (B) Cellular apoptosis markers increased after genetic clearance of PAK1 and IAP antagonist treatment for the indicated time. Cell lysates were analyzed by immunoblotting. PARP cleavage and caspase-3 / 6/7/9 activation were sharply elevated by the combined Dox and BV6 treatment.
FIG. 10-Combination of ATP competition pan-PAK inhibitor PF-3758309 (BW Murray et al. Proc. Nat. Acad. Sci. USA 2010 107 (20): 9446-9471) with IAP small molecule antagonists Apoptosis of squamous NSCLC cells is generated. (A) Catalytic inhibition of PAK1 via PF-3758309 treatment was tested with BV6 to determine in vitro combination efficacy in EBC-1 cells using a 4-day CellTiterGlo viability assay. Chou and Talalay for calculating synergy (Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984; 22: 27-55]) The level of synergy was measured by calculating the concomitant index using Calcusyn, a program using the method. Strong synergy, indicated by a combination index (CI) value of 0.3 or less, was observed (CI = 0.113). (B) Combination of 5 μM PAK inhibitor (PAKi) and 5 μM BV6 for the indicated time drastically induced cellular apoptosis markers.
Figure 11-Combination effect of PAK1, PAK2, MEK and PI3K inhibition on tumor cell viability. As indicated, CellTiterGlo® (CTG) analysis of cell viability was performed after PAK1 and PAK2 siRNA transfection and compound treatment for 3 days. GDC-0623 is a potent highly selective inhibitor of MEK1 and MEK2. GDC-0941 is a potent inhibitor of class I PI3K isoforms with biochemical IC ₅₀ values of 3 nM to 75 nM for the four class I isoforms of PI3K. (A) Viability of SKMES-1 (KRAS ^N85K mutant) lung cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 0.2 μM GDC-0623 and 0.5 μM GDC-0941. (B) Viability of Calu-6 (KRAS ^Q61K mutant) lung cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 0.2 μM GDC-0623 and 0.4 μM GDC-0941. (C) Viability of Cal-120 (basal subtype) breast cancer cells treated with PAK1 and PAK2 siRNA oligonucleotides, 2 μM GDC-0623 and 2.5 μM GDC-0941.
12-Combination regulation of apoptosis and proliferative biomarkers after PAK1, PAK2, MEK and PI3K inhibition in combination in NSCLC cells. SKMES-1 (KRAS ^N85K mutant) NSCLC cells were ^{treated with PAK1} and PAK2 siRNA oligonucleotides, 0.4 μM GDC-0623 and 1 μM GDC-0941 for 24 hours. Accumulation of cleaved caspase-3 and poly ADP ribose polymerase (PARP), and reduction of cyclin D1 protein were enhanced by the combination of PAK knockdown and inhibitors of the MEK and PI3K pathways.

As used herein, the phrase “one” or “one” (ie, singular) material means one or more of the above materials, for example, one compound means one or more compounds or at least one compound. Thus, the terms "one" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

As used in this specification and claims, the terms “consisting”, “consisting”, “comprises”, “comprising” and “comprising” are intended to specify the presence of the stated feature, integer, component or step. But does not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

The terms “treat” and “treatment” both mean therapeutic treatments, wherein the purpose is to prevent or slow down (reduce) the growth, development or spread of unwanted physiological changes or disorders, such as cancer. For the purposes of the present invention, favorable or desired clinical outcomes are alleviating symptoms, reducing the extent of disease, stabilizing (ie, not worsening) the condition, delaying or slowing disease progression, whether detectable or undetectable. , Alleviation or sedation of disease state, and (partial or total) remission. "Treatment" can also mean prolonging survival compared to expected survival if treatment is not provided. Subjects in need of treatment include those already suffering from the condition or disorder, as well as subjects susceptible to the condition or disorder or subjects to which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means (i) treating a particular disease, condition or disorder, (ii) attenuating, alleviating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) By an amount of a compound of the present invention is meant to prevent or delay the onset of one or more symptoms of a particular disease, condition or disorder. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; Reduce tumor size; May inhibit (i.e., slow to some extent, preferably stop and / or inhibit) cancer cell invasion into a peripheral organ; May inhibit (i.e., slow to some extent, preferably stop and / or inhibit) tumor metastasis; Inhibit tumor growth to some extent; It may relieve to some extent one or more symptoms associated with cancer. The drug may prevent the growth of existing cancer cells and / or may exhibit cell proliferation inhibition and / or cytotoxic effects to the extent that existing cancer cells can be killed. In the case of cancer treatment, efficacy can be measured, for example, by evaluating time to disease progression (TTP) and / or measuring response rate (RR).

As used herein, the term "synergistic" means a therapeutic combination that is more effective than the additive effect of two or more single substances. The determination of the synergistic interaction between the PAK1 inhibitor and the second anti-proliferative agent can be based on the results obtained from the assays described herein. The combination provided by the present invention has been evaluated in several assay systems, and data can be analyzed through the use of standard programs to quantify synergism, addition and antagonism between anticancer agents. The program used preferably is the program described by Chow and Talaray in "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2. Combination index values below 0.8 indicate synergy, values above 1.2 indicate antagonism, and values between 0.8 and 1.2 indicate additional effects. Combination treatment may provide a "synergy" and demonstrate a "synergistic" effect (ie, the effect achieved when the active ingredients are used together is greater than the sum of the effects generated when the compounds are used separately) ). The synergistic effect is when the active ingredients are administered or delivered simultaneously in (1) co-formulated and combined unit dosage form; (2) delivered alternately or in parallel as separate formulations; Or (3) when delivered by some other therapy. When delivered in alternation therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, eg, by different injections through separate syringes. In general, each active ingredient of the effective dose is administered sequentially, ie sequentially, during the alternation treatment, while in combination therapy two or more active ingredients of the effective dose are administered together.

The terms " cancer "and" cancerous "typically mean or describe the physiological condition of a mammal characterized by unregulated cell growth. A "tumor" includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma); Lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and squamous cell carcinoma of the lung; Peritoneal cancer; Hepatocellular carcinoma; Stomach cancer or gastrointestinal cancer including gastrointestinal cancer; Pancreatic cancer; Glioblastoma; Cervical cancer; Ovarian cancer; Liver cancer; Bladder cancer; Hepatocellular carcinoma; Breast cancer; Colon cancer; Rectal cancer; Colon cancer; Endometrial carcinoma or uterine carcinoma; Salivary gland carcinoma; Renal or neoplastic; Prostate cancer; Vulvar cancer; Thyroid cancer; Liver cancer; Anal carcinoma; Penis carcinoma; And head and neck cancer.

The term "carcinoma" refers to an invasive malignant tumor consisting of transformed epithelial cells. The term "squamous cell carcinoma" (SCC) is a subset of carcinomas affecting squamous cells that can occur in many different organs, including the skin, lips, mouth, esophagus, bladder, prostate, lungs, vagina and cervix. Means. This is a malignant tumor of the squamous epithelium.

A "chemotherapeutic agent" is a biological (molecule) or chemical (small molecule) compound useful for the treatment of cancer regardless of the mechanism of action. Classes of chemotherapeutic agents include alkylating agents, anti-metabolites, spindle poison plant alkaloids, cytotoxic / anti-tumor antibiotics, tooisomerase inhibitors, proteins, antibodies, photosensitizers, and kinase inhibitors It is not limited to these. Chemotherapeutic agents include compounds used in "targeted therapy" and non-targeted conventional chemotherapy.

Examples of chemotherapeutic agents include the following materials: erlotinib (TARCEVA®), Genentech / OSI Pharm., Bortezomib (belcade) (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), sunitib ( SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate ( Gleevec®, Novartis, finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®), Sanofi Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, Rapamune®), Wyeth), Lapatinib (TYKERB®), GSK572016, Glaxo Smith Kline, Lonafamib (SCH 66336), Sorafenib (Sorafenib) NEXAVAR®, Bayer Labs, gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and cy CYTOXAN® cyclophosphamide; Alkyl sulfonates such as busulfan, impprosulfan and piposulfan; Aziridine such as benzodopa, carboquone, meturedopa and uredopa; Ethyleneimine and methylamelamine including althretamine, triethylene melamine, triethylenephosphoramide, triethylene thiophosphoramide and trimethylo-melamine; Acetogenin (especially bullatacin and bullatacinone); Camptothecin (including the synthetic analog topotecan); Bryostatin; Calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); Cryptophycins (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); Eleutherobin; Pancratistatin; Sarcodictyin; Spongistatin; Nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine Chlorethamine oxide hydrochloride, melphalan, nomobichin, phenesterine, prednimustine, trofosfamide, uracil mustard; Nitrosourea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; Antibiotics such as enediyne antibiotics (eg, calicheamicin, in particular calicheamicin γ1I and calicheamicin ωII (Angew Chem. Intl. Ed. Engl. 1994 33: 183- 186)); dynemycin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocazinostatin chromophores and related chromophores Phosphorus Antibiotics Chromophore), alaccinomysin, actinomycin, atrumycin, azaserrine, bleomycin, cactinomycin, carabicin ), Caminomycin (caminomycin), carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5 -Oxo-L-norleucine, adriamycin (ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin ), Idarubicin, marcellomycin, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin ), Porfiromycin (porfiromycin), puromycin (puromycin), quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, bovine Benimex, zinostatin, zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azaciitidine, 6-azauridine, camofur, cytarabine, dideoxyuridine, Doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Anti-adrenals, such as aminoglutethimide, mitotane, trilostane; Folic acid supplements such as frolinic acid; Aceglatone; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfomithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamnol; Nitrarine; Pentostatin; Phennamet; Pyrarubicin; Losoxantrone; Podophyllinic acid; 2-ethyl hydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products); Razoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2 ', 2 "-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitobronitol; mitolactol; pipobroman; pipobroman; gacytosine; Arabinoside (“Ara-C”); cyclophosphamide; thiotepa; toxoid, for example Taxol (TAXOL) (paclitaxel; Bristol-Myers Squibb Oncology) , Princeton, NJ), ABRAXANE® (without Cremophor), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Shaum, Illinois, USA) Berg) and TAXOTERE® (docetaxel, poison wash) (doxetaxel); sanopi-aventis; chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carbople Latin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine® (vinoline®) vinorelbine); novantron; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (xELODA) (XELODA) Trademark)); ibandronate; CPT-11; Topoisomerase inhibitors RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; And pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

The definition of “chemotherapeutic agent” also includes the following substances: (i) anti-hormonal agents that act to modulate or inhibit hormonal action on tumors, such as tamoxifen (NOLVADEX®) ); Tamoxifen citrate), raloxifene, drroloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and Anti-estrogen and selective estrogen receptor modulators (SERM), including FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase that modulates estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglutetidemide, MEGASE® Megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (registered trademark) ( Borozole), FEMARA® (Retrosol; Novartis) and Arimidex® (Anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; And troxacitabine (1,3-dioxolane nucleoside cytosine analogue); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides that inhibit the expression of genes, such as PKC-alpha, Ralf and H-Ras, in the signal transduction pathways involved in antisense oligonucleotides, particularly abnormal cell proliferation; (vii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME (TM)) and HER2 expression inhibitors; (viii) Vaccines, such as gene therapy vaccines, such as ALLOVECTIN, LEUVECTIN and VAXID (registered trademark); PROLEUKIN &lt; (R) &gt;,rIL-2; Topoisomerase 1 inhibitors such as LURTOTECAN (R); ABARELIX rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®), Genentech); And (x) pharmaceutically acceptable salts, acids and derivatives of any of the above materials.

Definitions of “chemotherapeutic agents” also include the following substances: therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (Avastin®, Genentech); Cetuximab (ERBITUX®, Imclone); Panitumumab (VECTIBIX (registered trademark), Amgen), rituximab (RITUXAN (registered trademark), Genentech / Biogen Idec) , Pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech) and tositumomab (Bexxar, Corixia).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents for use with the PI3K inhibitors of the present invention include the following antibodies: alemtuzumab, apolilizumab, aslizumab, atelizumab ( atlizumab), bapineuzumab, bevacizumab, vivauzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cerfulizumab pegol Tuzumab, cidtuzumab, daclizumab, eculizumab, epalizumab, epratuzumab, erlizumab, erlizumab, fellizumab Felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab ), Matuzumab, mepolizumab (mepolizu mab), motavizumab, motovizumab, motizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab , Omalizumab, palivizumab, pascolizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, pexelizumab, pexelizumab Ralivizumab, ranibizumab, resliizumab, reslizizumab, reslizumab, resizizumab, rovelizumab, rovelizumab, ruplizumab, sibrotuzumab (sibrotuzumab), siplizumab, sontuzumab, tautuzumab, tetracatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, and tocili Tocilizumab, toralizumab, trastuzumab, tucotuzumab celmol eukin), tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

The following abbreviations are used herein: DCIS (endothelial ductal carcinoma), SCLC (small cell lung cancer), NSCLC (non-small cell lung cancer); SCC (squamous cell carcinoma).

PAKs participate in a number of pathways that are typically deregulated in human cancer cells. PAK1 is a component of mitogen-activated protein kinase (MAPK), JUN N-terminal kinase (JNK), steroid hormone receptors and nuclear factor κβ (NF κβ) signaling pathways involved in tumorigenesis. PAK activates MEK and RAF1 by phosphorylating MEK and RAF1 on serine 298 and serine 338, respectively. The increase in Ras-induced transformation by PAK1 correlates with its effect on signaling through the extracellular signal-regulated kinase (ERK) -MAPK pathway and is isolated from the effect on the JNK or p38-MAPK pathway. (R. Kumar et al. Nature Rev. Cancer 2006 6: 459). Persistent activation of the ERK / MEK pathway is involved in tumor formation, progression and survival, and furthermore associated with aggressive phenotypes characterized by unregulated proliferation, loss of regulation of apoptosis and weak prognosis (JA Spicer, Expert Opin.Drug Discov. 2008 3: 7].

Tumor formation and progression requires the inactivation of proapoptotic signals in cancer cells. PAK activity has been shown to downregulate several important proapoptotic pathways. PAK1 phosphorylation of RAF1 induces RAF1 translocation to mitochondria, where RAF1 phosphorylates the proapoptotic protein BCL2-antagonist (BAD) of cell death. In addition, PAK1, PAK2, PAK4 and PAK5 are selected from several cell types selected, such as CV-1 (COS) kidney cells derived from monkeys and carrying SV40, Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) 293T. It has been reported to directly phosphorylate and inactivate BAD in cells (R. Kumar et al., Supra). However, the relevant pathways downstream of PAK1 in human tumor cells remain only partially understood. have.

PAK1 is widely expressed in a variety of normal tissues, but expression is significantly increased in ovarian cancer, breast cancer and bladder cancer (S. Balasenthil et al., J. Biol. Chem. 2004 279: 4743; M. Ito et. al., J. Urol. 2007 178: 1073; P. Schraml et al., Am. J. Pathol. 2003 163: 985]. In luminal breast cancer, genomic amplification of PAK1 is associated with resistance to tamoxifen treatment, which is likely to occur as a result of direct phosphorylation and ligand-independent transcriptional activation of estrogen receptors by PAK1 (SK Rayala et al. al., Cancer Res. 2006. 66: 1694-1701]. PAK1 is an interesting target for the development of therapeutic agents that are effective for use in the treatment of hyperproliferative disorders (R. Kumar et al., Supra).

Amplification Experiments -PAK1 genome replication numbers and gene expression for large panels of breast, lung and head and neck tumors were measured. PAK1 genomic amplification was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node invasion and metastasis.

Several genomic regions with an increased number of copies have been identified in breast cancer through comparative genome hybridization methods (J. Climent et al., Biochem. Cell Biol. 2007 85: 497-508; EH van Beers and PM Nederlof, Breast Cancer Res. 2006 8: 210]. 51 breast tumors were analyzed for DNA replication number changes using high resolution single nucleotide polymorphism (SNP) arrays and data were analyzed using GISTIC (genomic identification of significant targets in cancer) (PM [PM] Haverty et al., Genes Chromosomes Cancer 2008 47: 530-542. 21; R. Beroukhim et al., Proc. Natl. Acad. Sci. USA 2007 104: 20007-20012]. Chromosome region 11 of the amplification is shown in A of FIG. Two different GISTIC peaks were observed at 69 Mb and 76 Mb, suggesting that the 11q13.5 region contains two independent amplicons. 69 Mb peak corresponds to amplification of CCND1 , a very well known genomic alteration in breast cancer (C. Dickson, et al., Cancer Lett. 1995 90: 43-50). The peak of the 76 Mb peak contains the PAK1 gene (indicated by dashed lines). The frequency of PAK1 amplification was 17% (number of replicates> 2.5) in this tumor panel, and the increase in the number of copies correlated well with mRNA expression (Pearson correlation = 0.75; FIG. 1B). Similar results were obtained with a larger panel of breast tumors (n = 165) analyzed for genomic amplification by high resolution SNP arrays. PAK1 gene amplification was predominant, mean DNA replication was highest in luminal hormone receptor positive tumors (average number of 7.7) and lowest in basal breast tumors (average number of 2.8) (Z. Kan et al. , Nature 2010 466: 869-873]. These experiments suggest that PAK1 may be a tumor promoting "promoter" gene in 76 Mb amplicon of chromosome 11. PAK1 expression was not present in normal breast epithelial cells but was detected in 39% malignant cells of primary mammary adenocarcinoma.

PAK1 protein expression levels and subcellular localization were confirmed through immunohistochemical (IHC) staining of tissue microarrays. Potent and selective IHC reactivity of PAK1 antibodies was confirmed in cancer cell lines by immunoblot analysis of protein extracts from these cells in parallel. PAK1 protein expression data for 226 primary breast cancers, 15 DCIS, 32 breast cancer lymph node metastases, 97 NSCLC, 27 SCLC and 130 head and neck squamous cell carcinoma are summarized in Table 1. PAK1 staining intensity varied between tumor tissues from the absence of staining in the cytoplasm and / or nucleus or from weak staining to very strong staining.

RNA was purified from 88 primary breast cancer samples and cytoplasmic PAK1 IHC staining was correlated with increased mRNA expression. These data show that PAK1 expression is widely upregulated in breast cancer and that high expression is correlated with disease aggressiveness.

Strong nuclear and cytoplasmic PAK1 expression was prevalent in squamous non-small cell lungs, and selective PAK1 inhibition was correlated with delayed cell cycle progression in vitro and in vivo.

Expression of PAK1 protein was analyzed on tissue microarrays of 27 SCLCs and 97 NSCLCs, which consisted of 30 adenocarcinomas and 67 SCCs. 43 of 64 (64%) squamous NSCLC samples were positive for PAK1 expression, and 52% of all cases showed medium (2+) or strong (3+) intensity staining in malignant cells. Nuclear localization of PAK1 was also evident in significant proportions (17 of 67; 25%) in squamous NSCLC tumors. In contrast to squamous cell carcinoma, NSCLC adenocarcinoma (p = 0.0008) and SCLC (p = 0.003) tumors expressed only mild to moderate levels of PAK1 only in the cytoplasm. Adjacent normal lung tissue did not express appreciable levels of PAK1. Elevated PAK1 expression was also prevalent in head and neck tumors, an additional sign of squamous cell carcinoma (79 of 130; 61%).

In head and neck tumors, elevated PAK1 expression was also prevalent in head and neck tumors, another sign of squamous cell carcinoma (79 of 130; 61%) (Table 1).

Anti-tumor efficacy of PAK1 inhibition in preclinical tumor models of squamous NSCLC-inhibition of PAK1 expression with shRNA in NCI-H520.X1 and EBC-1 xenograft models significantly inhibited tumor growth (FIGS. 4A and 4B). ). After tumor establishment (200 mm to 250 mm), Dox in sucrose drinking water was administered to the animals and tumor growth was monitored for 21 to 24 days. In NCI-H520.X1 tumor bearing animals, inhibition of PAK1 significantly impaired tumor growth relative to control shLacZ mice as measured on the last day of dosing, whereas inhibition of PAK2 was not so (dunnet t-test, p <0.0001; A) in FIG. 4. Combined knockdown of PAK1 and PAK2 resulted in inhibition of tumor growth comparable to PAK1 inhibition alone (63.7% and 59.7%, respectively). EBC-1 tumor xenografts were used to obtain equivalent results, with 66.8% inhibition of tumor growth following in vivo knockdown of PAK1 (FIG. 4B). Finally, tumor progression data for both xenograft models were analyzed for the time required for tumor size to double the tumor size at the start of treatment. By this measure, PAK1 inhibition also produced significant anti-tumor effects compared to the other populations (p <0.0001). Dox treatment was well tolerated and none of the animals showed any appreciable weight loss. Analysis of xenograft tumors by immunohistochemical method showed a substantial reduction of Ki-67 positive tumor cells in Dox-treated tumors expressing shPAK1 compared to shLacZ controls. The proportion of Ki-67 positive nuclei was quantified and the anti-proliferative effect of PAK1 knockdown in vivo was found to be statistically significant (p <0.01; NCI-H520.X1 shLacZ 69 ± 6%; NCI-H520.X1 shPAK1 50 ± 4%; EBC-1 shLacZ 91 ± 3%; EBC-1 shPAK1 75 ± 11%). PAK1 and PAK2 levels were reduced by more than 80% in Dox-treated tumors, and as implied, PAK1 knockdown was not associated with reduced AKT activation (TC Hallstrom and JR Nevins, Cell Cycle 2009 8: 532-535].

Analysis of cell lines with an increase in the number of PAK1 genome copies showed a dependency on PAK1 expression and activity for cell survival. Inhibition of PAK1 catalytic activity with IPA-3, an allosteric inhibitor that interferes with PAK1 to PAK3 activation by Rho family GTPase (J. Viaud, J. and JR Peterson, Mol. Cancer Ther. 2009 8 : 2559-2565) significantly induced apoptosis as measured by fluorescence-activated cell sorting analysis for Annexin-V / propidium iodide staining of dead cells (7-fold increase; FIG. 1D ). This phenotype was evident within 24 to 48 hours and was confirmed through the use of selective siRNA-mediated knockdown of PAK1 expression (two to six fold increase of Annexin-V introduction; FIG. 1C). Cell death induced by PAK1 inhibition was also associated with caspase activation, PARP cleavage, and attenuated phosphorylation of MEK1-S298 and ERK1 / 2 (FIG. 8). Thus, strong induction of cell death resulting from PAK1 inhibition in breast cancer cells with PAK1 amplification suggests that this kinase contributes to the oncogenic phenotype by at least partially inhibiting tumor cell apoptosis.

Dependence on PAK1, suggesting that PAK1 may be an "Achilles' heel" to a subpopulation of breast cancer, is evidence of oncogene addiction (IB Weinstein and A. Joe, Cancer Res. 2008 68: 3077). -3080) and provide a reasonable basis for PAK1-directed treatment in this disease indication.

Abnormal cytoplasmic expression of PAK1 in more than 50% squamous non-small cell lung cancer and head and neck squamous cell carcinoma further suggests that they may also depend on PAK1 expression for continued growth and survival.

Currently, further known genetic abnormalities in squamous NSCLC include loss of p53, p16 ^Ink4a , PTEN and LKB1 function through mutation or methylation, and activation mutations or amplification of protein kinases such as EGFR, MET, HER2 and PIK3CA. (RS Herbst et al., N. Engl. J. Med. 2008 359: 1367-1380). Thus, inhibition of PAK1 enzyme activity or scaffold function is synergistically combined with therapeutic agents that target these key growth and survival pathways, leading to anti-tumor efficacy and tumor cell death in tumor cells that over-amplify or overexpress PAK1. Will increase. Such tumor cells include, but are not limited to, DCIS, squamous NSCLC, and head and neck SCC.

Inhibitors of PAK kinases are described (D. Bouzida et al., International Patent Application Publication No. WO2006072831 published July 13, 2006; C. Guo et al., March 1, 2007). International Patent Application Publication No. WO2007023382 published as date; L. Dong et al., International Patent Application Publication No. WO2007072153 published June 30, 2007; D. Campbell, 6 2010 International Patent Application Publication No. WO2010 / 071846 published 24 May; K. Daly et al., US Patent Application Publication No. 20090275570, published November 5, 2009).

Combination of PAK1 knockdown induced by shRNA with molecularly targeted therapeutics induces apoptosis of NSCLC cells-screening small molecule libraries confirmed potent synergistic interactions between PAK1 antagonists and other anti-hyperproliferative agents (Table 1).

A cell viability screen was constructed using a panel of 200 small molecule compounds, including oncology drugs, signaling pathway inhibitors, and DNA damaging agents approved by the Food and Drug Administration (FDA), and EBC-1-shPAK1 isogenic cells. Was performed. Apoptotic protein inhibitors (IAP; 12 and 57 fold), epidermal growth factor receptor (EGFR; 2.9, 7.4, 12.8 and 15 fold), MAPK / ERK kinase-1 / 2 (MEK1 / 2) among the tested compounds 8.5 fold) and Src family kinase (5.4 fold) showed a sharply improved potency with PF-3758309 (Table 1). None of these substances showed a strong single substance effect (EC ₅₀ > 6 μM) on the growth and survival of EBC-1 cells in the absence of PAK1 inhibition. Thus, PAK1 inhibition can greatly enhance the efficacy of several classes of well characterized molecularly targeted therapeutics.

MEK kinase (S. Price, Expert Opin. Ther. Patents 2008 18 (6): 603-626; EM Wallace et al., Curr. Topics Med. Chem. 2005 5 (2): 215), Akt ( C. Lindsley, Curr. Top. Med. Chem. 2010 10: 458-477; SE Ghayad and PA Cohen, Rec. Pat.Anti-Cancer Drug Discov. 2010 5: 29-57], Src (Ref. X. Cao et al., Mini-Rev. Med. Chem. 2008 8: 1053-1063) and apoptosis protein inhibitors (IAP) (D. Vucic and WJ Fairbrother, Clin. Cancer Res. 2007 13 (20) 5995; AD Schimmer and S. Dalili, Hematology 2005 215).

The probiotic activity of the IAP protein is antagonized by caspase's second mitochondrial activator (SMAC) (C. Du et al., Cell 2000 102: 33-42; AM Verhagen, et al., Cell 2000 102: 43-53), similar to the SMAC amino-terminal peptide, a number of antagonists have been described that disrupt the binding of IAP to SMAC and activated caspase-9 (K. Zobel et al., ACS Chem Biol. 2006 1: 525-533; E. Varfolomeev et al., Cell 2007 131: 669-681]. In particular, BV6 (C. Ndubaku et al., Future Med. Chem. 2009 1 (8): 1509) binds to the baculovirus IAP repeat (BIR) domains to speed up c-IAP1 and cIAP-2. One such class of small molecule antagonists that promote self-ubiquitination and proteasome degradation (Zobel, supra). Consistent with the small molecule screening data, strong combined activity against double inhibition of PAK1 and IAP was confirmed in EBC-1 cells (FIG. 5A). Relative to the control (EC ₅₀ = 3.0 x 10 ^-3 μM), this rapid increase in BV6 efficacy for EBC-1-shPAK1 cells (EC ₅₀ = 4.1 x 10 ^-7 μM) when co-treated with Dox is As measured by Annexin-V flow cytometry analysis (FIG. 5B) and immunoblotting for cleaved caspase-3, caspase-6, caspase-7 and caspase-9 (FIG. 9). Switched to strong induction of cell apoptosis. Importantly, evidence of improved cell death was also observed when using pharmacological inhibitors of PAK (IPA-3, PF-3758309) or Rac1 (NSC23766) catalytic activity (FIG. 7A). To further investigate this apparent synergy, we investigated the possible molecular mechanisms of PAK1 and IAP inhibition on the induction of apoptosis. Combined PAK1 inhibition and IAP inhibition did not involve altered rates of IAP proteolysis induced by BV6 or increased autosecretory signaling by TNFα. Finally, additional NSCLC cell lines, including NSCLC cell lines that respond only minimally to the activity of a single substance, were investigated for anti-tumor efficacy resulting from the combined inhibition of PAK1 and IAP. BV6 treatment after transient siRNA-mediated knockdown of PAK1 expression significantly reduced SK-MES-1 and NCI-H441 cell viability (FIG. 7B).

The combined inhibition of PAK1 and PAK2 by inhibitors of the MEK (GDC-0623) and PI3K (GDC-0941; AJ Folkes et al., J. Med. Chem. 2008 57: 5522-5532) pathways was also investigated. . Combination efficacy measured by reduced cell viability (FIG. 11) and induction of apoptosis biomarkers (FIG. 12) was observed after inhibition of PAK, MEK and PI3K signaling. In addition, these studies provide strong preclinical evidence that breast and NSCLC may offer many opportunities for rational combination treatment with PAK1 inhibitors. In one embodiment of the present invention there is provided a method of treating a tumor comprising contacting the tumor with a PAK1 inhibitor and a second anti-hyperproliferative compound.

In another embodiment of the invention, a method of treating a tumor, wherein the tumor is contacted with a PAK1 inhibitor and an inhibitor or antagonist of the EGFR, Raf / MEK / ERK pathway, Src, PI3K / AKT / mTOR pathway or apoptosis protein inhibitor. A method is provided that includes a step.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits a high level of PAK1, comprising contacting the tumor with a PAK1 inhibitor and a second anti-proliferative compound.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the tumor comprising a PAK1 inhibitor and an EGFR, Raf / MEK / ERK pathway, Src, PI3K / AKT / mTOR pathway or apoptosis protein inhibitor. A method is provided that includes contacting an inhibitor or antagonist.

In one embodiment of the present invention there is provided a method of treating a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor, the method comprising contacting the tumor with a PAK1 inhibitor and a second anti-hyperproliferative compound. .

In another embodiment of the invention, a method of treating a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor, wherein the tumor is a PAK1 inhibitor, and an EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptotic protein inhibitor. Provided is a method comprising contacting with an inhibitor or antagonist.

In another embodiment of the invention, there is provided a method of treating a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor exhibiting high levels of PAK1, the step of contacting the tumor with a PAK1 inhibitor and a second anti-proliferative compound There is provided a method comprising a.

In another embodiment of the invention, there is provided a method of treating breast tumors, squamous non-small cell lung tumors or squamous head and neck tumors exhibiting high levels of PAK1, said tumors comprising PAK1 inhibitors, and EGFR, Raf / MEK / ERK pathways, Src A method is provided that includes contacting an inhibitor or antagonist of an Akt or apoptosis protein inhibitor.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of Formula I (PF-3758309) and a second anti-proliferative compound:

(I)

In another embodiment of the invention, a method of treating a tumor comprising contacting the tumor with a compound of formula (I) and an inhibitor or antagonist of an EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptotic protein inhibitor A method is provided.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the method comprising contacting the tumor with a compound of Formula I and a second anti-proliferative compound.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, wherein the tumor is a compound of formula (I) and an inhibitor or antagonist of an EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptotic protein inhibitor. A method is provided that includes contacting with a.

In another embodiment of the invention, there is provided a method of treating a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor exhibiting high levels of PAK1, wherein said tumor is contacted with a compound of formula I and a second anti-proliferative compound There is provided a method comprising the step of making.

In another embodiment of the invention, there is provided a method of treating breast tumors, squamous non-small cell lung tumors or squamous head and neck tumors exhibiting high levels of PAK1, said tumors comprising the compounds of formula (I), and the EGFR, Raf / MEK / ERK pathway Provided is a method comprising contacting with an inhibitor or antagonist of a Src, Akt or apoptosis protein inhibitor.

In one embodiment of the present invention there is provided a method of treating a tumor comprising contacting the tumor with an inhibitor of a compound of Formula I and an apoptosis protein inhibitor.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits a high level of PAK1, the method comprising contacting the tumor with an inhibitor of a compound of Formula I and an apoptosis protein inhibitor.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of Formula (I) and BV6 or G24416.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits a high level of PAK1, the method comprising contacting the tumor with a compound of Formula I and BV6 or G24416.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula (I) and an EGFR inhibitor or antagonist.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the method comprising contacting the tumor with a compound of Formula I and an EGFR inhibitor or antagonist.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula (I) and erlotinib, gefitinib, or lapatinib.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits a high level of PAK1, the method comprising contacting the tumor with a compound of Formula I and erlotinib, zefitinib, or lapatinib.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of Formula I and an inhibitor of the Ras / Raf / MEK / Erk signaling cascade.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the method comprising contacting the tumor with a compound of Formula I and an inhibitor of the Ras / Raf / MEK / Erk signaling cascade Is provided.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of Formula I and an inhibitor of Akt kinase.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the method comprising contacting the tumor with a compound of Formula I and an inhibitor of Akt kinase.

In one embodiment of the invention, there is provided a method of treating a tumor comprising contacting the tumor with a compound of formula I and an inhibitor of Src kinase.

In another embodiment of the invention, there is provided a method of treating a tumor that exhibits high levels of PAK1, the method comprising contacting the tumor with a compound of Formula I and an inhibitor of Src kinase.

In another embodiment of the invention, a method of treating a patient suffering from cancer or a hyperproliferative disorder, comprising co-administering a PAK1 inhibitor and a second anti-proliferative agent to a patient in need thereof There is provided a method comprising a.

In another embodiment of the invention, a method of treating a patient suffering from cancer or a hyperproliferative disorder, the PAK1 inhibitor and an inhibitor or antagonist of an EGFR, Raf / MEK / ERK pathway, Src, Akt or apoptotic protein inhibitor. Provided is a method comprising co-administration to a patient in need thereof.

In another embodiment, the present invention provides a combination of a PAK1 inhibitor and a second anti-proliferative compound for the treatment of a tumor.

In another embodiment, the present invention provides co-administration of a PAK1 inhibitor and a second anti-hyperproliferative agent for the treatment of a cancer or hyperproliferative disorder.

In another embodiment, the present invention provides the use of a combination of a PAK1 inhibitor and a second anti-proliferative compound for the manufacture of a medicament for the treatment of tumors.

In another embodiment, the present invention provides the use of a PAK1 inhibitor and a second anti-proliferative agent for the manufacture of a medicament for the treatment of cancer or a hyperproliferative disorder.

The following examples illustrate the biological evaluation of compounds within the scope of the present invention. These examples are provided below to enable those skilled in the art to more clearly understand the present invention and to practice the present invention. These examples should not be regarded as limiting the scope of the present invention, but merely as illustrative and representative of the present invention.

Example  One

Tissue samples

Formalin-fixed paraffin-embedded tissue blocks and corresponding pathological reports were obtained for 97 sequential NSCLCs and 27 sequential SCLCs, 130 head and neck SCCs, 15 DCIS, 226 primary breast cancers and 32 breast cancer lymph node metastases. (John Radcliffe Hospital, Oxford, UK). Tissue microarrays (TMA) were assembled as previously described (L. Bubendorf, et al., J. Pathol. 2001 195: 72-79).

For sequential patients with breast adenocarcinoma, surgery was performed between 1989 and 1998, and patients were treated with extensive local resection and postoperative radiotherapy or with mastectomy with or without postoperative radiotherapy. I was treated. Patients received or did not receive adjuvant chemotherapy and / or adjuvant hormonal therapy. Tamoxifen has been used as an endocrine treatment for 5 years in estrogen receptor (ER) positive patients. Patients under 50 years of age with lymph node-positive tumors or ER- and / or primary tumors with diameters greater than 3 cm are treated with adjuvant cyclophosphamide, methotrexate and 5-fluorouracil (CMF) with 3-week intravenous therapy. Was provided for 6 cycles. In addition, patients over 50 years of age with ER- lymph node positive tumors received CMF. Estrogen receptor (ER) content was measured using an enzyme-linked immunosorbent assay technique (Abbott Laboratories, Abbott Park, Ill.). Tumors were considered positive when cell fluid ER levels exceeded 10 fmol / mg of total cell fluid protein. HER status was assessed by HercepTest (DAKO, Calif., California, USA). Receptor values were monitored by participation in the EORTC quality control scheme.

The surgical NSCLC series included surgical resection specimens from 30 adenocarcinomas and 67 squamous cell carcinomas (operations performed from 1984 to 2000). Clinical and pathological data for 75 cancers were available. 35 cases (47%) were stage T1 and 40 cases (53%) were stage T2. 53 (71%) cases were stage N0 and 22 (29%) cases were stage N1. Patients did not receive adjuvant chemotherapy and no information on radiation therapy was available.

The head and neck squamous cell carcinoma series included surgical resection samples from 11 oropharyngeal cancers, 27 cancers occurring in the oral cavity, 17 laryngeal cancers and 75 hypopharyngeal cancers (final surgery was performed from 1995 to 2005). Nine cancers were UICC stage 1, 16 cancers were stage 2, 29 cancers were stage 3, and 76 cancers were stage 4. 108 patients (83%) were treated with radiation after surgery.

Example 2

Genome Copy Number and Expression Analysis

For Affymetrix 500K SNP array analysis, genomic DNA preparation, chip processing and data analysis were performed as previously published (P.M. Haverty et al., Genes Chromosomes Cancer 2008 47: 530-542). Regions of significant increase or loss were identified using the GISTIC (Genome Identification of Significant Targets in Cancer) algorithm (R. Beroukhim, et al., Proc Natl Acad Sci USA 2007 104: 20007-20012). . To collect expression array data for matched tumor samples, RNA was extracted from frozen tissue obtained from 88 cases of the primary breast cancer series to form Affymetrix (Santa Clara, CA, USA) HGU133 gene. It was applied to the expression microarray. Tumors were subclassified into basal, luminal-A, luminal-B, Her2 and normal types according to published criteria using gene probe intensity data (CM Perou et al., 2000 Nature 2000 406: 747-). 752]). The 226507_at probeset was selected to indicate PAK1 mRNA expression.

Example 3

Cell Culture and Viability Analysis

Cell lines were obtained from the Health Science Research Resources Bank (HSRRB, Japan) or the American Type Culture Collection (ATCC, Manassas, Va.) And were obtained at 37 ° C and 5%. Maintained in Dulbecos modified Eagle's medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI 1640) medium with 10% fetal bovine serum and 4 mM L-glutamine in CO ₂ . For cell proliferation analysis by thymidine incorporation into DNA, cells were incubated with 1 μCi / well [ ³ H] thymidine for 18 hours and Filtermate® 196 harvester (Perkin Elmer). Perkin Elmer, Waltham, Mass., USA, was used to collect on Unifilter® GF / C plates. MicroScint® 20 Liquid Scintillation Cocktail was added to the dried filter plate, then the filter plate was sealed and counted at Topcount® (Perkin Elmer, Waltham, Mass.). For cell cycle analysis via flow cytometry, cells were fixed in 70% ice-cold ethanol for 1 hour at a density of 1 × 10 ⁶ , then washed with PBS and propidium iodide (PI) solution (0.05 in PBS) at 4 ° C. Incubated in mg / ml RNase (Sigma, St. Louis, MO) and 0.05 mg / ml PI (Sigma, St. Louis, MO) solution for 3 hours. Cells were immediately analyzed with a FacScan flow cytometer (Becton Dickinson, San Jose, CA, USA).

To confirm the role of PAK1 in cell survival, the amount of cytoplasmic histone-bound DNA fragments was quantified using a cell death detection ELISA plus kit purchased from Roche (Mannheim, Germany). Alternatively, for cell death analysis via flow cytometry, cells are collected by centrifugation and subjected to annexin V-FITC and PI solutions (BD Biosciences, San Jose, USA) as directed by the manufacturer. Stained.

For palmarray viability screens with 200 compound libraries, EBC1-shPAK1 cells were cultured in complete growth medium and treated with or without 300 ng / ml doxycycline for 3 days prior to compound addition. Cells were then replated at appropriate density in 384-well plates and treated with each compound at 6 concentrations (4 fold serial dilution from 10 μM) for 72 hours of treatment. Cell viability was assessed via ATP content using CellTiterGro® luminescence assay (Promega, Madison, WI). Cell growth inhibition and EC ₅₀ differences for PAK1 knockdown and wild type cells were measured.

Example 4

Generation of RNA Interference and Inducible shRNA Cell Pools

Short interfering RNA (siRNA) oligonucleotides for PAK1 and PAK2 were obtained from Dharmacon RNAi technologies (Chicago, Ill.). The short hairpin RNA oligonucleotides used in this study were: LacZ shRNA (sense) 5'-CTT ATA AGT TCC CTA TCA GTG ATA GAG ATC CCC AAT AAG CGT TGG CAA TTT ATT CAA GAG ATA AAT TGC CAA CGC TTA TTT TTT TTG GAA-3 ', LacZ shRNA (antisense) 5'-TTC CAA AAA AAA TAA GCG TTG GCA ATT TAT CTC TTG AAT AAA TTG CCA ACG CTT ATT GGG GAT CTC TAT CAC TGA TAG GGA ACT TAT AAG-3', PAK1 shRNA-1 (sense) 5'-GAT CCC CGA AGA GAG GTT CAG CTA AAT TCA AGA GAT TTA GCT GAA CCT CTC TTC TTT TTT GGA AA-3 ', PAK1 shRNA-1 (antisense) 5'-AGC TTT TCC AAA AAA GAA GAG AGG TTC AGC TAA ATC TCT TGA ATT TAG CTG AAC CTC TCT TCG GG-3 ', PAK2 shRNA-3 (Sense) 5'-GAT CCC CCT GCA TAA CCT GAA TGA AAT TCA AGA GAT TTC ATT CAG GTT ATG CAG TTT TTT GGA AA-3 ', PAK2 shRNA-3 (antisense) 5'-AGC TTT TCC AAA AAA CTG CAT AAC CTG AAT GAA ATC TCT TGA ATT TCA TTC AGG TTA TGC AGG GG-3'. The vesicular stomatitis virus (VSV-G) envelope glycoprotein and the HIV-1 packaging protein (GAG-POL) were prepared using Lipofectamine (trademark) (Invitrogen, Carlsbad, CA). Methods previously described by co-transfecting pHUSH-Lenti-GFP and / or pHUSH-Lenti-dsRed constructs containing the desired shRNA with expressing plasmids in HEK293T cells (KP Hoeflich et al., Cancer Res. 2006 66: 999-1006; J. Climent et al., Biochem. Cell Biol. 2007 85: 497-508]. Target cells were transduced with these viruses and sterile sorted by flow cytometry for the presence of dsRed or GFP, or both (top 2% to 5%). Cells were characterized for doxycycline inducible protein knockdown by Western blot analysis.

Example  5

Immunoblotting and Immunofluorescence

Frozen tumors were crushed on dry ice using a small Bessman tissue grinder (Spectrum Laboratories, Rancho Dominguez, Calif.), And cell extraction buffer (Invitrogen, CA, USA) Carlsbad), 1 mM phenylmethylsulfonyl fluoride (PMSF), Phosphatease Inhibitor Cocktail 1/2 (Sigma-Aldrich, St. Louis, MO) and fully EDTA-free mini ( Protein extracts were prepared at 4 ° C. using 1 tablet of Mini) Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, Indiana, USA). Lysates were centrifuged at 16,100 g for 15 minutes and protein concentrations were measured using BCA protein analysis (Pierce Biotechnology, Rockford, Ill.). For Western blot analysis, proteins were separated by 4% to 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, Mass.). Immunble using primary antibodies against PAK1, p27, E2F1 (Cell Signaling Technology, Danvers, Mass.) And anti-β-actin (Sigma-Aldrich, St. Louis, MO) Lotting was performed. Secondary antibodies were obtained from Pierce Biotechnology (Rockford, Ill.).

Immunofluorescence imaging was performed using a primary antibody against p27 ^Kip1 (Becton Dickinson, San Jose, CA, USA). Secondary antibodies were obtained from Millipore Corporation, Billerica, Massachusetts. Images were analyzed in Metamorph (version 7.5.3.0, MDS Analytical; Sunnyvale, Calif.) Using an automated analysis routine. In summary, a smoothing filter was applied to the DAPI channel to flatten the nuclear staining pattern. The MWCS application in Metamorph was then used to identify and count DAPI stained nuclei and classify them as positive or negative for p27 in the Cy3 channel.

Example 6

Tumor Xenograft Model

Cultured NCI-H520.X1 and EBC1 cells were removed from the culture, suspended in Hank's buffered saline solution (HBSS), mixed 1: 1 with Matrigel (BD Biosciences, USA) and naive Subcutaneously implanted into the right flank of female NCR nude mice (Taconic Farms, Hudson, NY, USA). Mice with tumors with an average volume of about 250 mm 3 were divided into treatment groups of 10 mice each. Mice received only 5% sucrose or 5% sucrose plus 1 mg / ml doxycycline (Clontech, Mountain View, CA) for each of the control and knockdown groups. All water bottles were replaced three times per week. Body weight and tumor volume measurements (obtained by length and width measurements with calipers) were obtained twice per week during the study. All experimental procedures followed the guidelines of the American Physiological Society and were approved by Genentech's Animal Care and Use Organizing Committee. Tumor volume was calculated by the following formula: Tumor volume = 0.5 * (a * b ² ), where “a” is the maximum tumor diameter and “b” is the vertical tumor diameter. Tumor volume results are presented as mean tumor volume ± standard error of the mean (SEM). At the end of the study (EOS),% growth inhibition (% INH) was calculated as% INH = 100 [(EOS vehicle-EOS treatment) / (EOS vehicle)]. Data analysis and the generation of p values using Dunnett's t-test were performed using JMP software (SAS Institute, Cary, NC).

Xenografts were fixed in 10% neutral buffered formalin for 24 hours before processing and paraffin embedding. Sections were cut to a thickness of 3 μm and specimens with sufficient viable tumors (as assessed on H & E-stained slides) were further evaluated by immunohistochemical method. Anti-Ki-67 (clone MIB-1, mouse anti-human) was used with the Dako ARK kit for detection. Tissues were counterstained with hematoxylin, dehydrated and fixed on slides. Antigen recovery was performed using the Daco Target Recovery Kit according to the manufacturer's instructions. To immunohistochemically quantify Ki-67 positive cells, images were acquired with an Ariol SL-50 automated slide scanning platform (Genetix Ltd.) at 100-fold final magnification. Tumor specific regions were manually identified for analysis in Ariol software. Staining area was quantified by specifying the 3,3'-diaminobenzidine specific color range using hue, saturation and intensity color space, and the result was total Ki-67 positive cells relative to the total cell count.

The features disclosed in the above description or in the following claims, which are expressed in a particular form or expressed in terms of a means for performing a disclosed function, or a method or process for achieving a disclosed result, are intended to embody the invention in its various forms, where appropriate. To be realized separately or in any combination of these features.

The invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. It will be apparent to those skilled in the art that changes and modifications can be made within the scope of the appended claims. Accordingly, it is to be understood that the above description is intended to be illustrative, and not restrictive. Accordingly, the scope of the invention should not be determined by reference to the above description, but instead should be determined by reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. .

The patents, published applications, and scientific literature cited herein are hereby incorporated by reference in their entirety to the same extent as those specifically and individually described, which establish the knowledge of those skilled in the art and are each incorporated by reference. Any discrepancy between any reference cited herein and the specific teachings herein should be interpreted in favor of the latter. Likewise, any discrepancy between the definitions in the art of the term or phrase and the definition of the term or phrase specifically taught herein should be interpreted to favor the latter.

Claims (36)

A combination of a PAK1 inhibitor and a second anti-proliferative compound for the treatment of a tumor. The method of claim 1,
Combination, where tumors exhibit high levels of PAK1 protein. 3. The method according to claim 1 or 2,
The combination is a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor. 4. The method according to any one of claims 1 to 3,
Combination wherein PAK1 inhibitor is a compound of Formula I:
Formula I

5. The method according to any one of claims 1 to 4,
The combination of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of apoptosis protein inhibitor. The method of claim 5,
The combination of the inhibitor of apoptosis protein inhibitor is BV6 or G24416. 7. The method according to any one of claims 1 to 6,
The combination of claim 2, wherein the anti-hyperproliferative compound is an EGFR inhibitor or antagonist. The method of claim 7, wherein
The EGFR inhibitor is erlotinib, gefitinib or lapatinib. 5. The method according to any one of claims 1 to 4,
The combination of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade. 5. The method according to any one of claims 1 to 4,
The combination of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade. 5. The method according to any one of claims 1 to 4,
The combination of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of Src kinase. Co-administration of a PAK1 inhibitor and a second anti-hyperproliferative agent for the treatment of cancer or a hyperproliferative disorder. Use of a combination of a PAK1 inhibitor and a second anti-hyperproliferative compound for the manufacture of a medicament for the treatment of tumors. The method of claim 13,
Use wherein the tumor exhibits high levels of PAK1 protein. The method according to claim 13 or 14,
The tumor is a breast tumor, a squamous non-small cell lung tumor or a squamous head and neck tumor. 16. The method according to any one of claims 13 to 15,
Use where the PAK1 inhibitor is a compound of Formula I:
Formula I

17. The method according to any one of claims 13 to 16,
The second anti-hyperproliferative compound is an inhibitor of an apoptosis protein inhibitor. 18. The method of claim 17,
The inhibitor of apoptosis protein inhibitor is BV6 or G24416. 17. The method according to any one of claims 13 to 16,
The second anti-hyperproliferative compound is an EGFR inhibitor or antagonist. 20. The method of claim 19,
The EGFR inhibitor is erlotinib, zefitinib or lapatinib. 17. The method according to any one of claims 13 to 16,
The second anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade. 17. The method according to any one of claims 13 to 16,
The second anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade. 17. The method according to any one of claims 13 to 16,
The second anti-hyperproliferative compound is an inhibitor of Src kinase. Use of a PAK1 inhibitor and a second anti-hyperproliferative agent for the manufacture of a medicament for the treatment of cancer or a hyperproliferative disorder. Contacting the tumor with a PAK1 inhibitor with a second anti-proliferative compound. 26. The method of claim 25,
The method of treatment, wherein the tumor exhibits high levels of PAK1 protein. 27. The method of claim 25 or 26,
The method of treatment, wherein the tumor is a breast tumor, a squamous non-small cell lung tumor, or a squamous head and neck tumor. 28. The method of claim 27,
The method of treatment, wherein the PAK1 inhibitor is a compound of Formula I:
Formula I

29. The method according to any one of claims 25 to 28,
The method of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of apoptosis protein inhibitor. 30. The method of claim 29,
The inhibitor of apoptosis protein inhibitor is BV6 or G24416. 29. The method according to any one of claims 25 to 28,
The method of claim 2, wherein the anti-hyperproliferative compound is an EGFR inhibitor or antagonist. 32. The method of claim 31,
The method of treatment, wherein the EGFR inhibitor is erlotinib, zefitinib, or lapatinib. 29. The method according to any one of claims 25 to 28,
The method of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of the Ras / Raf / MEK / Erk signaling cascade. 29. The method according to any one of claims 25 to 28,
The method of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of the PI3K / AKT / mTOR signaling cascade. 29. The method according to any one of claims 25 to 28,
The method of claim 2, wherein the anti-hyperproliferative compound is an inhibitor of Src kinase. A method of treating a patient suffering from cancer or a hyperproliferative disorder, comprising co-administering a PAK1 inhibitor and a second anti-hyperproliferative agent to a patient in need thereof.

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