Methods of treating gefitinib resistant cancers

Abstract

The present invention relates to methods of treating gefitinib and / or erlotinib resistant cancers. A cancer patient is monitored for cancer progression following treatment with gefitinib and / or erlotinib. The progression of cancer indicates that the cancer is resistant to gefitinib and / or erlotinib. Once progression of cancer is indicated, a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor is administered to the patient. In a preferred embodiment, the irreversible EGFR inhibitor is EKB-569, HKI-272, and HKI-357.

Description

[0001] This application is a divisional application of the invention patent application with the application date of February 2, 2006, application number 202111229046.7, and name “Method for treating gefitinib-resistant cancer”. Background of the Invention

[0003] Epithelial cell cancer, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, spleen cancer, testicular cancer, thymic cancer etc., is a disease characterized by the abnormal accelerated growth of epithelial cells. This accelerated growth initially causes tumor formation. Finally, metastasis to different organ positions may also occur. Although progress has been made in the diagnosis and treatment of multiple cancers, these diseases still cause significant mortality.

[0004] Lung cancer is the leading cause of cancer death in industrialized countries. Cancers that begin in the lungs are divided into two main types, non-small cell lung cancer and small cell lung cancer, based on how the cells appear under a microscope. Non-small cell lung cancer (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) generally spread to other organs more slowly than small cell lung cancer. Approximately 75% of lung cancer cases are classified as non-small cell lung cancer (e.g., adenocarcinoma), while the other 25% are small cell lung cancer. Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the United States, Japan, and Western Europe. For patients with advanced disease, chemotherapy offers modest benefits in terms of survival, but at the cost of significant toxicity, and there is a strong need for therapeutic agents that specifically target key genetic lesions involved in tumor growth (Schiller JH et al., N Engl J Med, 346:92-98, 2002).

[0005] The epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) membrane-bound protein expressed on the surface of epithelial cells. EGFR is a member of a family of growth factor receptors that encodes numerous protein tyrosine kinases, a group of cell cycle regulatory molecules (WJ Gullick et al., 1986, Cancer Res., 46:285-292). When EGFR ligands (either EGF or TGF-α) bind to the extracellular domain, EGFR is activated, leading to autophosphorylation of the receptor's intracellular tyrosine kinase domain (S. Cohen et al., 1980, J. Biol. Chem., 255:4834-4842; A.S. Schreiber et al., 1983, J. Biol. Chem., 258:846-853).

[0006] EGFR is the protein product of the growth-promoting oncogene, erbB or ErbBl, which is a member of the ERBB family of protooncogenes and is believed to play a key role in the development and progression of many human cancers. In particular, enhanced expression of EGFR has been observed in breast cancer, bladder cancer, lung cancer, head cancer, neck cancer, and gastric cancer, as well as glioblastoma. The ERBB family of oncogenes encodes four structurally related transmembrane receptors, namely EGFR, HER-2 / neu (erbB2), HER-3 (erbB3), and HER-4 (erbB4). Clinically, ERBB oncogene amplification and / or receptor overexpression in tumors have been reported to be associated with disease recurrence and poor patient prognosis, as well as with response to therapy. (L. Harris et al., 1999, Int. J. Biol. Markers, 14: 8-15; and J. Mendelsohn and J. Baselga, 2000, Oncogene, 19: 6550-6565).

[0007] EGFR is composed of three basic domains: an extracellular domain (ECD, which is glycosylated and contains a ligand-binding pocket with two cysteine-rich regions; a short transmembrane domain; and an intracellular domain with intrinsic tyrosine kinase activity. The transmembrane region connects the ligand-binding domain to the intracellular domain. Amino acid and DNA sequence analysis and studies of the non-glycosylated form of EGFR have shown that the EGFR protein backbone has a mass of 132 kDa and has 1186 amino acid residues (AL Ullrich et al., 1984, Nature, 307: 418-425; J. Downward et al., 1984, Nature, 307: 521-527; C. R. Carlin et al., 1986, MoI. Cell. Biol., 6: 257-264; and FLV Mayes and MD Waterfield, 1984, The EMBO J., 3: 531-537).

[0008] Binding of EGF or TGF-α to EGFR activates signal transduction pathways and leads to cell proliferation. Dimerization, conformational changes, and internalization of EGFR molecules play a role in transmitting intracellular signals, leading to cell growth regulation (G. Carpenter and S. Cohen, 1979, Ann. Rev. Biochem., 48:193-216). Genetic alterations that affect the regulation of growth factor receptor function or lead to overexpression of receptors and / or ligands induce cell proliferation. In addition, EGFR has been identified as playing a role in cell differentiation, enhanced cell motility, protein secretion, neovascularization, cancer cell invasion, metastasis, and drug resistance to chemotherapeutic agents and radiation (M.-J. Oh et al., 2000, Clin. Cancer Res., 6:4760-4763).

[0009] A variety of EGFR inhibitors have been identified, including some that are currently undergoing clinical trials for the treatment of various cancers. For a recent overview, see de Bono, JS and Rowinsky, EK (2002), "The ErbB Receptor Family: A Therapeutic Target For Cancer", Trends in Molecular Medicine, 8, S 19-26.

[0010] A group of promising targets for therapeutic intervention in cancer treatment includes members of the HER-kinase axis. They are frequently upregulated in solid epithelial tumors such as prostate, lung, and breast tumors, and are also upregulated in glioblastomas. The epidermal growth factor receptor (EGFR) is a member of the HER-kinase axis and has been a target of choice for the development of a variety of different cancer therapies. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are among these therapies because reversible phosphorylation of tyrosine residues is required for EGFR pathway activation. In other words, EGFR-TKIs block cell surface receptors that are responsible for triggering and / or maintaining cellular signaling pathways that lead to tumor cell growth and differentiation. In particular, these inhibitors are believed to interfere with the EGFR kinase domain, known as HER-1. Among the more promising EGFR-TKIs are three series of compounds: quinazolines, pyridopyrimidines, and pyrrolopyrimidines.

[0011] Two more advanced compounds in clinical development include gefitinib (compound ZD 1839, developed by AstraZeneca UK Ltd.; available under the trade name IRESSA; hereinafter "IRESSA") and erlotinib (compound OSI-774, developed by Genentech, Inc. and OSI Pharmaceuticals, Inc., available under the trade name TARCEVA; hereinafter "TARCEVA"); both have produced encouraging clinical results. Conventional cancer treatment with both IRESSA and TARCEVA involves oral administration of no more than 500 mg of each compound daily. In May 2003, IRESSA was the first of these products to enter the U.S. market, when it was approved for the treatment of patients with advanced non-small cell lung cancer.

[0012] IRESSA is an orally active quinazoline that works by directly inhibiting tyrosine kinase phosphorylation on the EGFR molecule. It competes for adenosine triphosphate (ATP) binding sites, leading to inhibition of the HER-kinase axis. The exact mechanism of IRESSA's response is not fully understood; however, studies suggest that the presence of EGFR is a necessary prerequisite for its action.

[0013] A significant limitation in the use of these compounds is that recipients may develop resistance to their therapeutic effects after an initial response to therapy, or they may fail to respond to EGFR-TKIs at any detectable level. Response rates to EGFR-TKIs vary across different ethnic groups. Among low-end EGFR-TKI responders, in certain populations, only 10-15% of patients with advanced non-small cell lung cancer respond to EGFR kinase inhibitors. Therefore, a better understanding of the molecular mechanisms underlying the sensitivity of IRESSA and TARCEVA will greatly benefit targeted therapies for those patients most likely to benefit from such treatments.

[0014] There is a clear need in the art for satisfactory treatments for cancer, and particularly epithelial cell cancers, such as lung, ovarian, breast, brain, colon, and prostate cancers, that integrate the benefits of TKI therapy and overcome the non-response exhibited by patients. Such treatments can have a profound impact on the health of patients, particularly elderly patients, among whom cancer is particularly common. Summary of the Invention

[0016] The inventors of the present invention have surprisingly found that irreversible EGFR inhibitors are effective in treating cancer in patients who no longer respond to gefitinib and / or erlotinib therapy. Therefore, in one embodiment, the present invention provides a method for treating gefitinib and / or erlotinib-resistant cancer. In this embodiment, the development of the patient's cancer is monitored at a time point after the initial administration of gefitinib and / or erlotinib treatment. The development of cancer is an indication of a cancer that is resistant to gefitinib and / or erlotinib treatment, and the patient is given a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor.

[0017] In a preferred embodiment, the irreversible EGFR inhibitor is EKB-569, HKI-272 or HKI-357. Alternatively, the irreversible EGFR inhibitor may be any compound that binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0018] The progression of cancer can be monitored using methods well known to those skilled in the art. For example, the progression of cancer can be monitored by visual inspection of the cancer, such as by X-ray, CT scan, or MRI. Alternatively, the progression of cancer can be monitored by detecting tumor biomarkers.

[0019] In one embodiment, the patient is monitored at multiple time points throughout cancer treatment. For example, the progression of the cancer can be monitored by analyzing the progression of the cancer at a second time point and comparing this analysis with the analysis at a first time point. The first time point can be before or after initial treatment with gefitinib and / or erlotinib, while the second time point is after the first time point. Increased growth of the cancer indicates progression of the cancer.

[0020] In one embodiment, the growth of the cancer is monitored by analyzing the size of the cancer. In one embodiment, the size of the cancer is analyzed by visual inspection of the cancer using X-rays, CT scans, or MRI. In one embodiment, the size of the cancer is monitored by detecting tumor biomarkers.

[0021] In one embodiment, the cancer is an epithelial cell cancer. In one embodiment, the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

[0022] In one embodiment, the size of the cancer is monitored at additional time points, and the additional time points are subsequent to the second time point.

[0023] In one embodiment, the later time point is at least 2 months after the previous time point. In one embodiment, the later time point is at least 6 months after the previous time point. In one embodiment, the later time point is at least 10 months after the previous time point. In one embodiment, the later time point is at least one year after the previous time point.

[0024] In another embodiment, the present invention provides a method for treating cancer comprising administering a pharmaceutical composition comprising an irreversible EGFR inhibitor to a patient having a mutation in EGFR, namely a methionine substitution of threonine at position 790 (T790M) of SEQ ID. NO. 1. The T790M mutation confers resistance to treatment with gefitinib and / or erlotinib. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A-1B Shown are EGFR sequence analysis in recurrent metastatic lesions from two NSCLC patients with acquired gefitinib resistance. Figure 1A Sequence analysis of Case 1 is shown. The T790M mutation in EGFR was present in a recurrent liver lesion following the development of clinical gefitinib resistance. (Left) No mutation was detected in the primary lung lesion at diagnosis. (Right) Both the primary lung tumor and the recurrent liver lesion harbored the L858R gefitinib-sensitive mutation. Notably, the L858R mutation was present in both the primary and recurrent lesions at the expected heterozygous mutation ratio, whereas T790M was detectable at low levels compared to the wild-type allele. The polymorphism (G / A) shown in the same tracing demonstrates equivalent reproduction of both alleles in the non-clonal PCR product (SEQ ID NOS 3 & 4, respectively, are disclosed in order of appearance). Figure 1B Sequence analysis of Case 2 is shown. The T790M mutation was present in a minority of gefitinib-resistant cells. (Left) The T790M mutation was not detected by sequencing nonclonal PCR products in either the primary lung tumor or in eight recurrent liver lesions from this case. Heterozygosity at adjacent polymorphisms (G / A) confirmed amplification of both EGFR alleles from these samples. The heterozygous gefitinib-sensitive mutation, L861Q, was detected in the expected proportions in each of the primary lung tumor and eight recurrent liver lesions (SEQ ID NOS 3 & 5, respectively, are disclosed in order of appearance).

[0027] Figures 2A-2C We show that bronchoalveolar carcinoma cell lines acquire resistance to gefitinib and persistent sensitivity to irreversible ERBB family inhibitors. Figure 2AShown is the inhibition of proliferation of bronchoalveolar carcinoma cell lines harboring wild-type EGFR (NCI-H1 666) with an activating delE746-A750 mutation in EGFR (NCI-H1 650) or two representative gefitinib-resistant subclones of NCI-H1 650 (G7 and C11) by tyrosine kinase inhibitors. The effects of the reversible inhibitor gefitinib were compared with those of the irreversible inhibitor HKI-357. Comparable results were observed with other irreversible inhibitors. Cell number was determined by crystal violet staining after 72 h of exposure to the indicated drug concentrations and incubation in 5% FCS with 100 ng / ml EGFR. Each data point represents the mean of four samples. Figure 2B Shown are the chemical structures of the reversible EGFR inhibitor gefitinib, the irreversible EGFR inhibitor EKB-569, and two irreversible dual EGFR and ERBB2 inhibitors, HKI-272 and HKI-357. Figure 2C Shown are drug-resistant NCI-HI 650 cells developed after treatment with different concentrations of gefitinib or the irreversible ERBB inhibitor EKB-569. Colonies were stained after 12 days of culture in the presence of inhibitors.

[0028] Figures 3A-3D Shows sustained dependence on EGFR and ERBB2 signaling and altered receptor trafficking in gefitinib-resistant cells. Figure 3A Shown are cell viability after siRNA-mediated knockdown of EGFR and ERBB2 in a bronchoalveolar cell line with wild-type EGFR (NCI-H1 666) compared to the viability of cells harboring the activating delE746-A750 mutation in EGFR (NCI-H1 650) and two gefitinib-resistant derivatives (G7 and C11). Viable cells were counted after 72 h of double-stranded RNA treatment and are shown as the fraction relative to cells treated with nonspecific siRNA, with standard deviations calculated for triplicate samples. Figure 3B Shown is inhibition of EGFR autophosphorylation (Y1068) and phosphorylation of the downstream effectors AKT and MAPK (ERK) in cells treated with increasing concentrations of gefitinib or the irreversible inhibitor HKI-357, followed by a 2-h pulse with EGF. The parental cell line, NCI-H1650, was compared to the representative gefitinib-resistant line, G7. Total AKT and MAPK are shown as controls; tubulin was used as a loading control for the lower limit of total EGFR levels detected in these cells. Figure 3CShown is the EGFR internalization phenomenon of the change in gefitinib-resistant NCI-H 1650 (G7) cells compared with the sensitive NCI-H1 650 parental cell line.When adding ligand after 5 and 20min, use rhodamine-labeled EGF to mark EGFR.In NCI-H1 650 (G7) cells, the enhanced internalization phenomenon of EGFR is most obvious when 20min (Zeiss microscope, x63 magnification). Figure 3D Immunoblot showing EGFR internalized from NCI-H1 650 parental cells and the resistant derivative G7 after biotinylation and chase for more than 20 min pulse labeling of cell surface proteins. The enhanced intracellular EGFR in NCI-H1 650 (G7) cells was compared with the unchanged ferroportin receptor (TR) internalization phenomenon.

[0029] Figures 4A-4B Demonstrating the efficacy of irreversible ERBB inhibitors in inhibiting the T790M EGFR mutant. Figure 4A Shown is a comparison of the ability of gefitinib and two irreversible inhibitors (HKI-357 and HKI-272) to inhibit EGFR autophosphorylation (Y1068) and phosphorylation of the downstream effectors AKT and MAPK (ERK) in the NCI-H1975 bronchoalveolar cell line (containing both a sensitizing mutation (L858R) and a mutation associated with resistance (T790M). Total EGFR, AKT, and MAPK are shown as loading controls. Figure 4B Shown is the inhibition of proliferation of NCI-H1975 cells harboring L858R and T790M mutations using three irreversible ERBB family inhibitors compared to gefitinib.

[0030] Figure 5 The nucleotide sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 1) of EGFR are shown.

[0031] Figure 6 It was shown that like gefitinib, HKI 357 and EKB 569 (labeled "Wyeth") demonstrated enhanced cell killing ability against NSCLC cells harboring EGFR mutations, but unlike gefitinib, they were less prone to generating clones resistant to these drugs in vitro and they retained their efficacy against gefitinib-resistant clones. Detailed Description of the Invention

[0033] Cancers resistant to gefitinib and erlotinib

[0034] Gefitinib (compound ZD 1839, developed by AstraZeneca UK Ltd.; available under the trade name IRESSA) and erlotinib (compound OSI-774, developed by Genentech, Inc. and OSI Pharmaceuticals, Inc., available under the trade name TARCEVA) have produced surprising clinical responses in non-small cell lung cancers (NSCLCs) that harbor activating mutations in the EGF receptor (EGFR) (1-3), the target of these competitive inhibitors of ATP binding (4, 5). The efficacy of these tyrosine kinase inhibitors may result from both alterations in ATP cleavage (cleft) associated with these mutations, which result in enhanced inhibition of the mutant kinase by these drugs, and from the biological dependence of these cancer cells on the enhanced survival signals transduced by the mutant receptors, a phenomenon described as "oncogene addiction" (6, 7).

[0035] Although responses to both gefitinib and erlotinib can last up to 2-3 years, the average duration of response in most NSCLC cases is only 6-8 months (8-10). The underlying mechanisms of acquired drug resistance are not well understood. By analogy, with imatinib (GLEEVEC), which inhibits the BCR-ABL kinase involved in chronic myeloid leukemias (CMLs), the C-KIT kinase involved in gastrointestinal stromal tumors (GISTs), and the FIP1L1-PDGFR-α kinase involved in idiopathic hypereosinophilic syndrome (HES), secondary kinase domain mutations can effectively inhibit drug binding (11-16). However, recurrent NSCLC is not easily biopsied; therefore, only limited clinical specimens are available for analysis. Recently, a single secondary mutation, T790M, in the EGFR kinase domain was reported in three of six cases of recurrent disease after gefitinib or erlotinib therapy (17, 18). Codon 315 of BCR-ABL, similar to EGFR codon 790, is frequently mutated in imatinib-resistant CML (11, 12), and mutations of the corresponding residues in C-XJr (codon 670) and FIP1L1-PDGFR-a (codon 674) are associated with imatinib-resistant GIST and HES, respectively (15, 16). Early in vitro models of resistance to EGFR inhibitors suggested that codon 790 mutations in wild-type receptors would similarly suppress inhibition by EGFR tyrosine kinase inhibitors (19). More recently, transfected EGFR proteins containing activating mutations, along with the T790M substitution, were shown to reduce inhibition by gefitinib and erlotinib (17, 18). Although the T790M mutation appears to contribute to acquired resistance in some cases of NSCLC, its potential mechanism of treatment failure in the absence of secondary EGFR mutations remains unclear.

[0036] In contrast to the cytoplasmic kinase BCR-ABL, signal transduction by membrane-bound EGFR involves a complex ligand binding pathway, homodimerization and heterodimerization of the receptor with ERBB2 and other family members, followed by internalization and recycling of the ligand-bound receptor or ubiquitin-mediated receptor degradation (20). Important EGF-dependent signal transduction is believed to occur during the internalization process, which is also associated with the dissociation of the EGFR complex at low pH in intracellular vesicles. Thus, multiple factors regulate the intensity and quality of signal transduction by the receptor, and changes in EGFR trafficking are closely linked to the regulation of EGF-dependent cellular responses (20).

[0037] The present invention is based on the discovery that gefitinib-resistant cancers can include those in which the T790M EGFR mutation is present only in a subset of resistant tumor cells and those in which no T790M mutation is observed, but enhanced EGFR internalization is observed. The present invention is also based on the discovery that irreversible EGFR inhibitors that covalently crosslink the receptor are effective in inhibiting cancers with the T790M mutation, as well as in cancers with altered EGFR trafficking that render such cancers resistant to treatment with gefitinib and / or erlotinib. Thus, the present invention provides methods for treating gefitinib- and / or erlotinib-resistant cancers comprising administering an irreversible EGFR inhibitor.

[0038] Methods of treating patients

[0039] In one embodiment, the present invention provides a method for treating gefitinib / erlotinib-resistant cancer. The method comprises administering to a patient in need of such treatment an effective amount of certain irreversible EGFR inhibitors, including EKB-569 (4-anilinoquinoline-3-carbonitrile; Greenberger et al., 1996). th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, November 7-10, 2000, Abstract 388; Wyeth), HKI-357 (a derivative of 4-anilinoquinoline-3-carbonitrile; Tsou et al., J. Med. Chem. 2005, 48: 1107-1131; Wyeth) and / or HKI-272 (a derivative of 4-anilinoquinoline-3-carbonitrile; Rabindran et al., Cancer Res. 2004, 64, 3958-3965; Wyeth). In a preferred embodiment, the present invention provides a method comprising administering an effective amount of EKB-569 to a patient in need of such treatment. In a preferred embodiment, the present invention provides a method comprising administering an effective amount of HKI-357 to a patient in need of such treatment.

[0040] The treatment may also include a combination of therapies, including, but not limited to, a tyrosine kinase inhibitor in combination with other tyrosine kinase inhibitors, chemotherapy, radiation therapy, and the like.

[0041] Cancers can be initially diagnosed as gefitinib / erlotinib sensitive or predicted to be gefitinib / erlotinib sensitive using the methods described in Lynch et al., 2004;350:2129-2139. Gefitinib / erlotinib sensitivity can be predicted by the presence of EGFR mutations in the tumor, including, for example, deletion of residues 747 (lysine) to 749 (glutamic acid) in combination with mutations in residue 750 (alanine), deletion of residues 747 (lysine) to 750 (alanine), substitution of arginine for leucine at residue 858, and substitution of glutamine for leucine at residue 861.

[0042] Cancer can be diagnosed as resistant to gefitinib and / or erlotinib after initiation of treatment with gefitinib and / or erlotinib. Alternatively, cancer can be diagnosed as resistant to gefitinib and / or erlotinib before initial treatment with these compounds. Gefitinib and / or erlotinib resistance in the tumor can occur after gefitinib and / or erlotinib treatment, e.g., 6 months or more. Alternatively, gefitinib and / or erlotinib resistance in the tumor can be diagnosed less than 6 months after initiation of treatment with gefitinib and / or erlotinib. Diagnosis of gefitinib and / or erlotinib resistance can be accomplished by monitoring the progression of the tumor during gefitinib and / or erlotinib treatment. Tumor progression can be determined by comparing the status of the tumor between various time points after initiation of treatment, or by comparing the status of the tumor between time points after initiation of treatment and time points before initiation of gefitinib and / or erlotinib treatment. During treatment with gefitinib and / or erlotinib, the development of the tumor can be monitored by visual inspection, for example, using radiography, for example, X-rays, CT scans or other monitoring methods known to the skilled person, including methods of monitoring cancer biomarker levels or monitoring tumor biomarker levels. The development of cancer during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. An increase in tumor biomarker levels indicates tumor development. Thus, an increase in tumor biomarker levels during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. Detection of new tumors or detection of metastasis indicates tumor development. Cessation of tumor shrinkage indicates tumor development. The growth of cancer is represented by, for example, an increase in tumor size, metastasis or detection of new cancer and / or an increase in tumor biomarker levels.

[0043] The development of gefitinib and / or erlotinib resistance can be monitored by testing circulating tumor cells from the patient's circulation or other body fluids for the presence of mutations associated with gefitinib and / or erlotinib resistance. The presence of mutations associated with gefitinib and / or erlotinib resistance in tumor cells from the patient is indicative of a gefitinib and / or erlotinib-resistant tumor.

[0044] In one embodiment, the patient's tumor comprises a mutation indicating sensitivity to gefitinib and / or erlotinib, yet it is resistant to treatment with gefitinib and / or erlotinib. In one embodiment, the patient's tumor comprises a mutation indicating sensitivity to gefitinib and / or erlotinib, and comprises a mutation indicating resistance to gefitinib and / or erlotinib, such as a T790M mutation, i.e., a methionine residue replaces a natural threonine residue in EGFR, such as enhanced EGFR internalization. In one embodiment, the patient's tumor does not comprise a mutation indicating sensitivity to gefitinib and / or erlotinib and does not comprise a mutation indicating resistance to gefitinib and / or erlotinib, such as a T790M mutation in EGFR, such as enhanced EGFR internalization.

[0045] When used in connection with the administration of a drug, an "effective amount" refers to an amount that results in a beneficial effect in at least a statistically significant portion of patients, such as improvement of symptoms, cure, reduction in disease burden, reduction in tumor or cell number, prolongation of life, improvement in quality of life, or other effect normally recognized as positive by a physician familiar with treating a particular type of disease or condition.

[0046] The effective dose of the active ingredient used may vary depending on the specific compound used, the mode of administration, and the severity of the condition being treated. It is noted by the skilled artisan that the effective dose for each patient may vary depending on the severity of the disease, individual genetic variation, or metabolic rate. However, generally, satisfactory results are obtained when the compounds of the present invention are administered at a daily dose of about 0.5 to about 1000 mg / kg body weight, optionally in divided doses 2 to 4 times a day, or in a sustained-release form. The total daily dose is planned to be about 1 to 1000 mg, preferably about 2 to 500 mg. Dosage forms suitable for oral administration include about 0.5 to 1000 mg of the active compound intimately mixed with a pharmaceutically acceptable solid or liquid carrier. The dosage regimen can be adjusted to provide the optimal therapeutic effect. For example, multiple divided doses may be administered daily or the dose may be proportionally reduced as indicated by the urgency of the treatment situation.

[0047] The route of administration may be intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal (ID), intraperitoneal (IP), intrathecal (IT), intrapleural, intrauterine, rectal, vaginal, topical, intratumoral, etc. The compounds of the present invention may be administered parenterally by injection or gradual infusion over time and may be transported by peristaltic means.

[0048] Administration can be through mucosal or transdermal means. For transmucosal or transdermal administration, penetrants with appropriate permeability barriers are used in the formulation. Such penetrants are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to promote penetration. Transmucosal administration can be through nasal sprays, for example, or using suppositories. For oral administration, the compounds of the present invention are formulated into conventional oral administration forms such as capsules, tablets, and tonics.

[0049] For topical administration, the pharmaceutical compositions (kinase activity inhibitors) are formulated into ointments, salves, gels, or creams generally known in the art.

[0050] The therapeutic compositions of the present invention, such as irreversible EGFR inhibitors, are, for example, administered intravenously by unit dose injection. The term "unit dose" when used to refer to the therapeutic compositions of the present invention refers to physically discrete units suitable as unitary dosages for administration to a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required diluent, i.e., carrier or vehicle.

[0051] The composition is administered in a manner consistent with the dosage form and in a therapeutically effective amount. The amount and frequency of administration will depend on the patient being treated, the patient's systemic capacity for utilizing the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required to be administered depends on the judgment of the practitioner and will vary from individual to individual.

[0052] The therapeutic compositions for implementing the method of the present invention are described herein, such as, irreversible EGFR inhibitors. Any formulation or drug delivery system generally known to those skilled in the art can be used, which contains an active ingredient suitable for the intended use. Suitable pharmaceutically acceptable carriers for oral, rectal, topical or parenteral (including inhalation, subcutaneous, abdominal, intramuscular and intravenous) administration are known to those skilled in the art. The carrier must be pharmaceutically acceptable, meaning that it is compatible with the other ingredients in the preparation and is harmless to its recipient.

[0053] As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, as they refer to compositions, carriers, diluents, and reagents, are used interchangeably and represent that the substance is capable of being administered to, or does not produce undesirable physiological effects in, a mammal.

[0054] Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparations of the active compound, which are preferably isotonic with the blood of the recipient. Thus, such preparations may conveniently contain distilled water, 5% dextrose in distilled water or saline. Useful formulations also include concentrated solutions or solids containing the compound, which are diluted with a suitable solvent to provide the above-mentioned solutions suitable for parenteral administration.

[0055] For enteral administration, the compound may be mixed with an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or as an aqueous or non-aqueous liquid suspension or solution, such as a syrup, elixir, emulsion or draught. Suitable carriers may be starch or powdered sugar and may include lubricants, flavorings, binders and other materials of the same nature.

[0056] Tablets may be prepared by compression or molding, optionally with one or more adjuvants. Compressed tablets may be prepared by compressing the active compound in a free-flowing form such as a powder or granules, optionally mixed with an adjuvant such as a binder, lubricant, inert diluent, surfactant or dispersant, in a suitable machine. Molded tablets may be prepared by molding a mixture of the powdered active compound and any suitable carrier in a suitable machine.

[0057] Syrups or suspensions may be prepared by adding the active compound to a concentrated sugar solution, such as sucrose, in water, with any adjuvants added. Such adjuvants may include flavorings, agents to prevent sugar crystallization, or agents to increase the solubility of any other ingredients, such as polyols, for example, glycerol or sorbitol.

[0058] Formulations for rectal administration may be presented as suppositories with a conventional suppository base such as cocoa butter or Witepsol S55 (a trademark of Dynamite Nobel Chemical, Germany).

[0059] Formulations for oral administration may contain enhancers. Orally acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoyl carnitine, polyethylene glycol monolaurate-9, phosphatidylcholine, cyclodextrins and their derivatives; bile salts such as sodium deoxycholate, sodium taurocholate, glycocholate and sodium fusidate; chelating agents including EDTA, citric acid and salicylates; and fatty acids (e.g., oleic acid, lauric acid, acylcarnitines, mono- and diglycerides). Other oral absorption enhancers include benzalkonium chloride, benzethonium chloride, CHAPS (3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate), Big-CHAPS (N,N-bis(3-D-glucosamidopropyl)-choleamine), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols and alkyl alcohols. A particularly preferred oral absorption enhancer for use in the present invention is sodium lauryl sulfate.

[0060] Alternatively, the compound can be administered in the form of liposomes or microspheres (or microparticles). Methods for preparing liposomes or microspheres for administration to patients are well known to those skilled in the art. U.S. Patent No. 4,789,734 describes a method for encapsulating biological raw materials in liposomes, the contents of which are incorporated herein by reference. Importantly, the raw material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, and if necessary, a surfactant is added, and the raw material is dialyzed or sonicated when necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

[0061] Microspheres formed from polymers or proteins are familiar to those skilled in the art and can be tailored for direct entry into the bloodstream via the gastrointestinal tract. Alternatively, the compound can be incorporated and the microspheres or microsphere composites implanted for slow release over a period of days to months. See, for example, U.S. Patent Nos. 4,906,474, 4,925,673, and 3,625,214 and Jein, TIPS 19:155-157 (1998), the contents of which are incorporated herein by reference.

[0062] In one embodiment, the tyrosine kinase inhibitors of the present invention can be formulated into liposomes or microparticles of a suitable size to accumulate in the capillary bed after intravenous administration. When the liposomes or microparticles are placed in the capillary bed surrounding the ischemic tissue, the drug can be administered locally to the site where it is most effective. Suitable liposomes for targeting ischemic tissue are generally less than about 200 nanometers and are also typically unilamellar vesicles, as disclosed in, for example, U.S. Patent No. 5,593,688 to Baldescliweiler, entitled "Liposomal targeting of ischemic tissue," the contents of which are incorporated herein by reference.

[0063] Preferred microparticles are those prepared from biodegradable polymers such as polyglycolide, polylactic acid and copolymers thereof. One skilled in the art can readily determine an appropriate carrier system based on various factors including the desired drug release rate and the desired dosage.

[0064] In one embodiment, the preparation is administered directly into the interior of a blood vessel via a catheter. For example, administration can be performed through the hole of a catheter. In those embodiments where the active compound has a relatively long half-life (approximately 1 day to 1 week or longer), the preparation can be incorporated into a biodegradable polymeric hydrogel, such as those disclosed in U.S. Patent No. 5,410,016 to Hubbell et al. These polymeric hydrogels can be delivered to the lumen of the tissue and the active compound can be released over time as the polymer degrades. If desired, the polymeric hydrogel can have microparticles or liposomes containing the active compound dispersed therein, unless there is another mechanism to provide controlled release of the active compound.

[0065] The formulations are conveniently presented in unit dosage form and can be prepared by any method familiar to the pharmaceutical art. All methods include the step of mixing the active compound with a carrier which comprises one or more adjuvants. Typically, the formulations are prepared by uniformly and intimately mixing the active compound with a liquid carrier or a finely divided solid carrier and then, if desired, shaping the product into the desired unit dosage form.

[0066] The preparation may also contain one or more optional adjuvants used in the field of pharmaceutical preparations, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, suspending agents, preservatives (including antioxidants), etc.

[0067] The compound (i.e., irreversible EGFR inhibitor) in the present method can be administered to the respiratory tract in the form of an inhaler or aerosol or a solution for a nebulizer or a fine powder for insufflation, alone or mixed with an inert carrier such as lactose. In such cases, the particles of the active compound preferably have a diameter of less than 50 microns, preferably less than 10 microns, and more preferably 2-5 microns.

[0068] Generally, when used for nasal administration, a weak acid pH will be preferred. Preferably, the pH of the composition of the present invention is about 3-5, more preferably about 3.5 to about 3.9 and most preferably 3.7. Adjustment of pH is achieved by adding an appropriate acid such as hydrochloric acid.

[0069] The formulation of pharmaceutical compositions containing active ingredients dissolved or dispersed therein is well known in the art and is not necessarily limited to such formulations. Typically, such compositions are prepared as injectable or liquid solutions or suspensions, however, solid forms suitable for solutions or suspensions can also be prepared and dissolved in a liquid prior to use. The formulation can also be emulsified.

[0070] The active ingredient can be mixed with a pharmaceutically acceptable excipient compatible with the active ingredient in an amount suitable for use in the methods of treatment described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Furthermore, if desired, the composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers, and the like that enhance the efficacy of the active ingredient.

[0071] The irreversible kinase inhibitors of the present invention may include pharmaceutically acceptable salts of their components. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. (formed with the free amino groups of the polypeptide). Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0072] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no substances other than the active ingredient and water, or containing a buffer such as sodium phosphate, saline, or both at physiological pH, such as phosphate-buffered saline. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.

[0073] Liquid compositions may also contain liquid phases other than water and in addition to water. Examples of such other liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions.

[0074] definition:

[0075] The terms "ErbBl," "epidermal growth factor receptor," and "EGFR" are used interchangeably herein and refer to native sequence EGFR as disclosed, for example, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987), including variants thereof (e.g., deletion mutant EGFR in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbBl refers to the gene encoding the EGFR protein product. As used herein, the EGFR protein has been disclosed as GenBank Accession No. NP_005219 (SEQ ID NO.: 1), encoded by the erbBl gene (GenBank Accession No. NM_005228 (SEQ ID NO: 2)). The nucleotide and amino acid sequences of erbBl / EGFR can be found at Figure 5 Found in.

[0076] As used herein, the term "nucleic acid variant that increases kinase activity" refers to a variation (i.e., a mutation) in the nucleotide sequence of a gene that results in increased kinase activity. The increased kinase activity is a direct result of the variation in the nucleic acid and is associated with the protein encoded by the gene.

[0077] As used herein, the term "drug" or "compound" refers to a chemical entity or biological product, or a combination of chemical entities or biological products, that is administered to a patient to treat, prevent, or manage a disease or condition. The chemical entity or biological product is preferably, but not necessarily, a low molecular weight compound, but may also be a larger compound, for example, a nucleic acid, an oligomer of amino acids, or a carbohydrate, including but not limited to proteins, oligonucleotides, ribozymes, DNA enzymes, glycoproteins, siRNAs, lipoproteins, aptamers, modifications thereof, and combinations thereof.

[0078] As used herein, the terms "effective" and "efficacy" include both pharmacological effects and physiological safety. Pharmacological effect refers to the ability of a treatment to produce a desired biological effect in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (usually side effects) at the cellular, organ, and / or biological level resulting from the administration of the treatment. "Poor effect" means that the treatment results in a therapeutically significantly lower level of pharmacological effect and / or a therapeutically higher level of adverse physiological effect.

[0079] Nucleic acid molecules can be isolated from a particular biological sample using any of a variety of methods known in the art, with the specific isolation process selected being appropriate for the particular biological sample. For example, nucleic acid molecules can be obtained from solid materials using freeze-thaw and alkaline lysis; nucleic acid molecules can be obtained from urine using heat and alkaline lysis; and nucleic acids can be obtained from blood using proteinase K extraction (Rolff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).

[0080] As used herein, "cancer" in a subject or patient refers to the presence of cells that have characteristic features typical of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In certain instances, cancer cells will be present in the form of a tumor, or such cells may be localized in the animal's body or circulate in the bloodstream as independent cells. Example

[0081] Compounds used herein include EKB-569, HKI-357, and HKI-272 as described in the following documents: U.S. Patent No. 6,002,008; Greenberger et al., Proc. th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Clinical.Cancer Res.Vol.6 Supplement, Nov.2000, ISSN 1078-0432; Rabindran et al., Cancer Res. 64: 3958-3965 (2004); Holbro and Hynes, Ann. Rev. Pharm. Tox. 44: 195-217 (2004); and Tejpar et al., J. Clin. Oncol. ASCO Annual Meeting Proc. Vol. 22, No. 14S: 3579 (2004).

[0082] Relapsed NSCLC Analysis and Generation of Gefitinib-Resistant NCI-H1650 Cells. Relapsed NSCLC clinical samples were obtained by autopsy after obtaining appropriate consent. Following analysis of nonclonal PCR products, the entire kinase domain of EGFR was sequenced. Multiple clones of exon 20 were sequenced to detect codon 790. Mutation analysis of EGFR (exons 1-28), ERBB2 (exons 1-24), PTEN (exons 1-9), Kras (codons 12, 13, and 61), and p53 (exons 5-8) in gefitinib-resistant clones, as well as the parental NCI-H1650 cell line, was performed by automated sequencing of individual exons using dye-terminator chemistry (BIGDYE version 1.1, Applied Biosystems) and flanking intronic sequences using bidirectional sequencing (PCR conditions available upon request). Sequencing reactions were performed on an ABI3100 sequencer (Applied Biosystems) and electropherograms were analyzed by using SEQUENCE NAVIGATOR and FACTURA software (Applied Biosystems).

[0083] To generate drug-resistant subclones of NCI-H1650 cells, these cells were treated with ethyl methanesulfonate (EMS; 600 μg / ml), allowed to recover for 72 h, and then seeded with 20 μM gefitinib at a density of 10 cm per 10-cm 2 Tray 6x10 4 Compared with the irreversible inhibitor, these cells are relatively resistant to gefitinib, which can be determined by incubating 5x10 4 Cells were seeded in six-well plates in 5% FCS and 100 ng / ml EGF (Sigma). After 72 h of fixation with 4% formaldehyde, the cells were stained with 0.1% crystal violet and the cell population was quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). For micro-intervention RNA (siRNA) knockdown experiments, cells were transfected with double-stranded RNA oligonucleotides targeting EGFR and ERBB2 (two SMART pools from Dharmacon, Lafayette, CO), or non-specific controls (LRT1B) were performed using X-treme gene transfection reagent (Roche Applied Science). After 72 h, cells were stained with crystal violet and analyzed using an Odyssey infrared scanner.

[0084] Immunoblotting and signal transduction studies. Inhibition of EGFR signaling by increasing concentrations of gefitinib or irreversible inhibitors was determined by the following assay: 9x10 4 Cells were seeded in 24-well plates, and drugs were added to medium containing 5% FCS for 15 minutes. Lysates were then pulsed with 100 ng / ml EGF for 2 hours, and lysates were harvested. Lysates were prepared in 2x gel loading buffer, sonicated, boiled, and separated by 10% SDS / PAGE. The membranes were then electrotransferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted. Antibodies used were phospho-EGFR Y1068 and phospho-mitogen-activated protein kinase (MAPK) (Cell Signaling Technology, Beverly, MA), phospho-AKT (BioSource International, Camarillo, CA), and total EGFR, MAPK, AKT, and tubulin (Santa Cruz Biotechnology).

[0085] EGFR internalization analysis. To demonstrate EGFR internalization by fluorescence microscopy, cells were grown on coverslips and incubated with 1 ng / ml recombinant human (rh) EGF (Molecular Probes, Eugene, OR) for various time intervals before being fixed in 4% paraformaldehyde for 10 min. The coverslips were washed with PBS and mounted with ProLong Gold antifade reagent (Molecular Probes). To quantify EGFR internalization by cell surface biotinylation, cells were grown to confluence, pretreated with cyclohexamide, incubated on ice with 1.5 mg / ml of sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce) for 1 h, washed with blocking buffer (50 nM NH4CL / 1 mM MgCl2A) 1 mM CaCl2 in PBS to quench free sulfo-NHS-SS-biotin, and then washed several times with PBS. The cells were then incubated at 37°C in culture medium for various time intervals to allow internalization of the biotinylated molecules, washed twice on ice with glutathione solution (50 mM glutathione / 75 mM NaCl / 75 mM NaOH / 1% BSA) for 20 min to strip all biotin groups from the cell surface, and then scraped and lysed in 500 μM radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.4, with 150 mM NaCl / 0.1% SDS / 1% Triton X-100) supplemented with NaF, sodium orthovanadate, and protease inhibitors. The cell extract was centrifuged, and the supernatant was incubated with streptavidin beads (Sigma) to collect the biotinylated proteins, which were then analyzed by SDS / PAGE and immunoblotting using anti-EGFR antibody (SC-03, Santa Cruz Biotechnology) or anti-transferrin receptor antibody (Santa Cruz Biotechnology).

[0086] Results and discussion

[0087] Analysis of Recurrent Lung Cancer with Acquired Resistance to Gefitinib. Recurrent gefitinib-resistant NSCLC developed in two patients whose tumors already contained activating mutations in the EGFR kinase at diagnosis and who had shown a dramatic initial clinical response to the drug (1). In both cases, metastatic disease developed in the liver 1-2 years after initial treatment, leading to the patients' death. In case 1, analysis of the majority of liver metastases obtained at autopsy showed persistence of the sensitizing EGFR mutation (L858R) and the presence of a newly acquired T790M mutation ( Figure 1A Interestingly, analysis of nonclonal PCR products revealed that the initial L858R mutation was present at an abundance consistent with a heterozygous mutation present in all tumor cells, whereas the secondary T790M mutation was seen at approximately one-fifth the abundance of the corresponding wild-type allele. Thus, this resistance-associated mutation appears to be present only in a subset of cells in recurrent tumors.

[0088] Case 2 relates to eight obvious recurrence metastases in liver after gefitinib therapy failure.In all these independent damages, the L861Q EGFR mutation of sensitization exists with the ratio of expected heterozygous mutation.By the analysis of the non-clone PCR product from any of these transfers, no secondary EGFR mutation is detected.However, after PCR product subclone, find in two of four metastatic tumor analyses (T790M, 2 in 50 clones of self-injury 1 order-checking and 1 in 56 clones of self-injury 2 order-checking), T790M mutation exists with very low frequency, but from two other recurrence metastases (0 in 55 clones of self-injury 3 order-checking and 0 in 59 clones of self-injury 4 order-checking) or primary tumor (0 in 75 clones), fail to find ( Figure 1B and Table 1). Taken together, these results are consistent with previous reports that the T790M mutation is present in some, but not all, cases of acquired gefitinib resistance (3 of 7 tumors; see references 17, 18, and 21). Furthermore, as previously noted (18), even in some cases where this resistance-associated mutation was present, it appeared to be present in only a small fraction of tumor cells in recurrent lesions. These observations suggest that additional mechanisms of resistance are involved in cases without secondary EGFR mutations and that such mechanisms coexist with the T790M mutation in other cases.

[0089] Generation of gefitinib-resistant cell lines sensitive to irreversible inhibitors. Given the strong correlation between clinical responses of EGFR-mutant NSCLC and the enhanced gefitinib sensitivity of NSCLC cell lines harboring these mutations (2, 6, 22, 23), and the limited availability of clinical samples from relapsed patients, the applicants established an in vitro model of gefitinib resistance. The applicants cultured the bronchoalveolar carcinoma cell line NCI-H1650, which has an in-frame deletion of the EGFR kinase (delE746-A750), in 20 μM gefitinib, with or without prior exposure to the mutagen ethyl methanesulfonate. This cell line exhibited a 100-fold enhanced sensitivity to gefitinib compared to certain NSCLC lines expressing wild-type EGFR (6). While the vast majority of these cells were effectively killed by 20 μM gefitinib, at ~10 ～5Resistant clones were readily observed with a frequency of 50-fold (95% CI, 1.0-2.0) with or without mutagen treatment. Forty-nine independent resistant clones were isolated, showing an average 50-fold reduction in sensitivity to gefitinib ( Figure 2A All of these showed no persistence of sensitizing mutations with altered EGFR expression, and none had acquired secondary EGFR mutations or new mutations in ERBB2, p53, Kras, or PTEN. Gefitinib-resistant clones demonstrated considerable resistance to related anilinoquinazoline inhibitors. However, strikingly, they exhibited resistance to ERBB family ( Figure 2A ) of the three inhibitors: EGFR and ERBB2 (for EGFR, IC 50 values ​​of 92 and 34 nM, respectively, and for ERBB2, 59 and 33 nM, respectively) for the dual inhibitors HKI-272 (24) and HKI-357 (compound 7f in reference 25), and EGFR (for EGFR, IC 50 The value was 39 nM and for ERBB2, IC 50 Value is 1.3 μM) selective inhibitor EKB-569 (26) (Wyeth) ( Figure 2B All three drugs are irreversible inhibitors, most likely through covalent binding to Cys773 residue in the catalytic domain of EGFR or Cys805 of ERBB2. Like gefitinib, these compounds demonstrated enhanced killing of NSCLC cells harboring EGFR mutations compared to cells expressing wild-type receptors ( Figure 2A However, in contrast to gefitinib, where drug-resistant clones are readily generated even at high drug concentrations, the applicants were unable to establish cell clones resistant to the irreversible inhibitor at concentrations of approximately 10 μM, even after mutagenesis with ethyl methanesulfonate ( Figure 2C ).

[0090] Gefitinib-resistant cells rely on EGFR and ERBB2 expression. In order to gain insight into the mechanism of gefitinib resistance and sustained sensitivity to irreversible inhibitors, the applicant first determined whether the resistant cell lines still rely on EGFR for their viability. The applicant has previously shown that siRNA-mediated knockout of EGFR triggers apoptosis in cells containing mutant EGFRs, but not in cells with wild-type alleles (6). Significantly, after transfection with siRNA-targeted EGFR, parental NCI-H1 650 cells and their gefitinib-resistant derivatives showed comparable reductions in cell viability ( Figure 3A). Thus, the acquisition of gefitinib-resistance does not involve EGFR-independent activation of downstream effectors. Because HKI-272 and HKI-357 target both EGFR and ERBB2, the applicants also tested inhibition of these related receptors. Knockout of ERBB2 in NCI-H1650 and its gefitinib-resistant derivatives also caused loss of viability ( Figure 3A ), suggesting a role for EGFR-ERBB2 heterodimers in transducing essential survival signals in tumor cells harboring EGFR mutations. Inhibition of EGFR by irreversible inhibitors alone appears to be sufficient to induce apoptosis in gefitinib-resistant cells, as demonstrated by the efficacy of EKB-569, which primarily targets EGFR (26). However, given the potential complementary effects of targeting both EGFR and ERBB2 using siRNA and the availability of irreversible inhibitors targeting these family members, the potential benefits of dual inhibition are being considered.

[0091] The applicant compared the ability of gefitinib and irreversible ERBB family inhibitors to inhibit signal transduction through downstream effectors of EGFR that mediate its proliferation and survival pathways. HKI-357 was 10-fold more potent than gefitinib in inhibiting EGFR autophosphorylation (detected at residue Y1068) and AKT and MAPK phosphorylation in parental NCI-H1650 cells containing the delE746-A750 EGFR mutation. Figure 3B In the gefitinib-resistant derivative, NCI-H1650 (G7), gefitinib exhibited a considerably reduced inhibitory effect on AKT phosphorylation, a key EGFR signaling effector associated with gefitinib response (6), whereas HKI-357 was shown to have sustained activity ( Figure 3B ).

[0092] Altered EGFR internalization in gefitinib-resistant clones. Given the lack of secondary mutations in EGFR and the continued susceptibility of gefitinib-resistant cells to siRNA-mediated inhibition of EGFR, the applicants tested whether the mechanism underlying the differential inhibition of EGFR signaling by reversible and irreversible inhibitors in gefitinib-resistant cells might be related to alterations in receptor trafficking, a well-documented regulator of EGFR-dependent signaling (20). Indeed, as measured by fluorescein-labeled EGF internalization ( Figure 3C Analysis of EGFR trafficking in NCI-H1650-derived resistant cells demonstrated a consistent enhancement of EGFR internalization, as measured by both quantification of cytoplasmic biotinylated EGFR ( Figure 3DSuch an effect was not observed in the transferrin receptor, suggesting that this is not the result of a general alteration in all receptor processing. Although further work is needed to elucidate the precise mechanism of this alteration in EGFR trafficking, many regulatory proteins have been implicated in this complex process, and these results suggest that the ability of gefitinib to inhibit EGFR activation compromises these cells, while the effects of irreversible inhibitors are undetectably affected.

[0093] Inhibition of T790M EGFR signaling and enhanced cell killing by irreversible inhibitors. The enhanced inhibition of EGFR signaling by irreversible ERBB inhibitors creates the possibility that these drugs may also show sustained activity in cells containing T790M secondary mutations in EGFR. Therefore, the applicant tested the effects of these inhibitors on the NCI-H1 975 bronchioalveolar carcinoma cell line containing L858R and T790M mutations in EGFR (18). Significantly, the cell line was derived from patients who were not treated with EGFR inhibitors, indicating that the mutation is not uniquely associated with acquired drug resistance. In inhibiting ligand-induced EGFR autophosphorylation and its downstream signaling, both HKI-357 and HKI-272 were significantly more effective than gefitinib when measured by AKT and MAPK phosphorylation ( Figure 4A Similarly, all three irreversible inhibitors inhibited proliferation in this cell line under conditions of gefitinib resistance ( Figure 4B ). Thus, irreversible ERBB inhibitors appear to be effective in cells containing T790 MEGFR as well as in cells with altered transmission of the wild-type receptor.

[0094] The applicant's results confirm reports of the T790M mutation in EGFR as a secondary mutation that has previously occurred in sensitive NSCLCs containing activating mutations and is associated with the emergence of acquired resistance (17, 18). However, this mutation is only present in a subset of cases, and even tumors containing the T790M mutation may contain only a small fraction of cells with this mutation. These observations mean that multiple resistance mechanisms may coexist in recurrent tumors after an initial response to gefitinib or similar reversible EGFR inhibitors. In addition, these findings suggest that resistance mechanisms independent of T790M may be the same, if not more effective than the T790M substitution itself in conferring drug resistance, and may explain why recurrent tumors rarely show clonality to T790M (17, 18). In vitro mechanisms of acquired gefitinib resistance do not significantly and frequently involve secondary EGFR mutations, but are instead associated with altered receptor trafficking. However, it should be noted that the applicants did not test EGFR trafficking in all of the resistant clones established in vitro by the applicants, and that there may be other mechanisms that may contribute to gefitinib resistance in some clones. Nevertheless, in fact, all gefitinib-resistant clones showed comparable sensitivity to irreversible ERBB inhibitors.

[0095] Applicants' results demonstrate a significant difference between competitive EGFR inhibitors such as gefitinib, whose efficacy is limited by the rapid development of resistance in vitro, and irreversible inhibitors, to which acquired resistance appears to be rare ( Figure 2C ). The applicant speculates that the enhanced internalization phenomenon of ligand-bound EGFR in drug-resistant cells may be associated with the dissociation of gefitinib-EGFR complexes at low pH in intracellular vesicles. In contrast, the irreversible cross-linking of receptors will not be affected by changes in such receptor transport. In the absence of drugs, cells are passaged up to 20 generations later, and acquired resistance to gefitinib remains stable, suggesting that genetic or epigenetic changes in genes regulating EGFR conversion may explain this phenomenon. Because receptor transport is not easy to study using available clinical samples, it may be necessary to identify such genetic changes before possible clinical relevance. Nevertheless, such a mechanism may work on acquired gefitinib-resistance in patients with recurrent disease who do not have secondary mutations in EGFR.

[0096] Irreversible ERBB inhibitors also appear to be effective in overcoming gefitinib resistance mediated by the T790M mutation, which is presumed to maintain the effect of inhibitor binding regardless of the alteration of this important residue. While this work was underway, another irreversible EGFR inhibitor [CL-387,785, Calbiochem (27)] was shown to inhibit the kinase activity of the T790M EGFR mutant (17). The efficacy of CL-387,785 in the presence of T790M is thought to be a result of the lack of a chloride at position 3 of the anilino group, which is present in gefitinib and presumably sterically hinders binding to the mutant methionine at codon 790. However, EKB-569, HKI-272, and HKI-357 all have a chloride moiety at this position on the anilino ring, suggesting that their shared ability to irreversibly bind to EGFR may explain their efficacy, rather than a lack of specific steric interaction with T790M (24-26). Thus, these irreversible inhibitors could prove broadly effective across multiple resistance mechanisms, in addition to the T790M mutation.

[0097] Table 1. EGFR T790M mutation is present at a very low frequency in the recurrent tumor of case 2.

[0098]

[0099] Sequencing of a large number of cloned PCR products revealed that the minority allele in two of the four cases of liver injury contained the T790M mutation.

[0100] References cited throughout this specification are hereby incorporated by reference in their entirety. Specific implementation method:

[0102] 1. A method for treating gefitinib and / or erlotinib-resistant cancer, comprising the following steps:

[0103] a. monitoring the patient for the development of cancer at a time point after the patient is initially treated with gefitinib and / or erlotinib, wherein the development of the cancer is an indication of a cancer that is resistant to treatment with gefitinib and / or erlotinib; and

[0104] b. administering a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor to a cancer patient resistant to gefitinib and / or erlotinib treatment.

[0105] 2. The method of embodiment 1, wherein the irreversible EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0106] 3. The method of embodiment 1, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0107] 4. The method of embodiment 1, wherein the progression of the cancer is monitored by visual inspection of the cancer.

[0108] 5. The method of embodiment 4, wherein the visual detection of cancer is performed by X-ray, CT scan or MRI.

[0109] 6. The method of embodiment 1, wherein the progression of the cancer is monitored by detecting tumor biomarkers.

[0110] 7. The method of embodiment 1, wherein monitoring the progression of the cancer comprises comparing the cancer at a second time point to the cancer at a first time point, wherein the second time point is subsequent to the first time point, wherein the first time point is before or after initial treatment with gefitinib and / or erlotinib, wherein increased growth of the cancer indicates progression of the cancer.

[0111] 8. The method of embodiment 7, wherein the cancer at the additional time point is compared to the cancer at the previous time point.

[0112] 9. The method of embodiment 1, wherein the cancer is epithelial cell cancer.

[0113] 10. The method of embodiment 1, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

[0114] 11. A method for treating cancer, comprising the steps of:

[0115] a. Giving cancer patients gefitinib and / or erlotinib;

[0116] b. monitoring the patient's cancer progression; and

[0117] c. Once cancer progression is confirmed, the patient is given an irreversible EGFR inhibitor.

[0118] 12. The method of embodiment 11, wherein the irreversible EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0119] 13. The method of embodiment 11, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0120] 14. The method of embodiment 11, wherein the progression of the cancer is monitored by visual inspection of the cancer.

[0121] 15. The method of embodiment 14, wherein visual detection of cancer is performed using X-ray, CT scan, or MRI.

[0122] 16. The method of embodiment 11, wherein the progression of the cancer is monitored by detecting tumor biomarkers.

[0123] 17. The method of embodiment 11, wherein monitoring the progression of the cancer comprises comparing the cancer at a second time point to the cancer at a first time point, wherein the second time point is subsequent to the first time point, wherein the first time point is before or after initial treatment with gefitinib and / or erlotinib, wherein increased growth of the cancer indicates progression of the cancer.

[0124] 18. The method of embodiment 17, wherein the cancer at the additional time point is compared to the cancer at the previous time point.

[0125] 19. The method of embodiment 11, wherein the cancer is epithelial cell cancer.

[0126] 20. The method of embodiment 11, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

[0127] 21. The method of embodiment 11, wherein the irreversible EGFR inhibitor is administered concurrently with a reversible EGFR inhibitor.

[0128] 22. The method of embodiment 21, wherein the reversible EGFR inhibitor is gefitinib or erlotinib.

[0129] 23. A method for treating cancer, comprising administering a pharmaceutical composition comprising an irreversible EGFR inhibitor to a cancer patient, wherein the patient has a mutation in EGFR (SEQ ID NO: 1), wherein the mutation is a substitution of threonine at position 790 with methionine.

[0130] 24. The method of embodiment 23, wherein the EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0131] 25. The method of embodiment 23, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0132] 26. The method of embodiment 23, wherein the cancer is epithelial cell cancer.

[0133] 27. The method of embodiment 23, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

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Claims

1. A method for treating gefitinib and / or erlotinib-resistant cancer, the method The following steps are involved: a. monitoring the patient for the development of cancer at a time point after the patient is initially treated with gefitinib and / or erlotinib, wherein the development of the cancer is an indication of a cancer that is resistant to treatment with gefitinib and / or erlotinib; and b. administering a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor to a cancer patient resistant to treatment with gefitinib and / or erlotinib.

2. The method of claim 1, wherein the irreversible EGFR inhibitor is selected from the group consisting of EKB-569, HKI-272 and HKI-357.

3. The method of claim 1, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 (SEQ ID NO: 1) of EGFR.

4. The method of claim 1, wherein the progression of the cancer is monitored by visual inspection of the cancer.

5. The method of claim 4, wherein the visual detection of cancer is performed by X-ray, CT scan or MRI.

6. The method of claim 1, wherein the progression of the cancer is monitored by tumor biomarker detection.

7. The method of claim 1, wherein monitoring the progression of the cancer comprises comparing the cancer at a second time point to the cancer at a first time point, wherein the second time point is subsequent to the first time point, wherein the first time point is before or after initial treatment with gefitinib and / or erlotinib, wherein increased growth of the cancer indicates progression of the cancer.

8. The method of claim 7, wherein the cancer at the additional time point is compared to the cancer at the previous time point.

9. The method of claim 1, wherein the cancer is an epithelial cell cancer.

10. The method of claim 1, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

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