Kinase inhibitor prodrugs for the treatment of cancer

Abstract

The present invention relates to kinase inhibitor prodrugs for treating cancer. Compositions containing TH-4000 and methods of administering TH-4000 can be used to treat cancer alone or in combination with other anticancer substances.

Description

[0001] This application is a divisional application of an invention patent with an application date of December 3, 2015, application number 201580075139.5, and invention name “Kinase inhibitor prodrug for treating cancer”. Technical Field

[0002] The present invention provides compositions containing kinase inhibitors in active form or in prodrug form, and methods of using the compositions alone or in combination with other anticancer substances or therapies for treating cancer or other hyperproliferative disease conditions. In addition, the present invention relates generally to analytical tests, and more specifically to gene expression analysis or hematological analysis as biological markers for predicting the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in treating cancer. In addition, the present invention relates to kits for identifying patients with cancers at risk for resistance to EGFR TKIs. Therefore, the present invention relates to the fields of medicine, pharmacology, chemistry and biology. Background Art

[0003] Kinases play an important role in regulating a variety of cellular processes, which has led to the development of kinase inhibitors for use as therapeutic agents in the treatment of a wide range of conditions, including cancer. Treating cancer is challenging because it is difficult to kill cancer cells while leaving normal cells unaffected or to a lesser extent. During cancer treatment, killing or otherwise deleteriously affecting normal cells can cause adverse side effects in patients. EGFR TKIs such as erlotinib, gefitinib or afatinib have been shown to be useful in the treatment of several cancers including non-small cell lung cancer (see, e.g., Felip et al. Clinical Cancer Research, 2008, 14:3867-3874; Smith et al., 2008, Br. J. Cancer, 98:1630-32; Hamilton et al., 2006, Clin. Can. Res. 12:2166-71), squamous cell carcinoma of the head and neck or skin (see, e.g., Calvo et al., Annals of Oncology, 2007, 18:761-767); ovarian cancer (see, e.g., Posada et al., Cancer, 2007, 109:1323-1330). In addition, new drugs designed to target mutant forms of EGFR while ignoring wild-type EGFR, such as AZD9291, CO-1686, etc., have been developed for the treatment of cancerous tumors (Cross et al., 2014, Can. Discov. 4; 1046).

[0004] However, cancer cells can differ from some normal cells in terms of oxidation levels and can be more hypoxic than normal cells. Hypoxia can induce adaptations in gene regulation and related cellular functions, especially epidermal growth factor receptor (EGFR; HER1) (Franovic et al. 2007, PNAS; 104: 13092; Wang et al., 2009, Nature Med., 15: 319; Wang et al., 2010, Carcinogenesis, 31: 1202; Minakata et al., 2012, Cancer Sci; 103(11): 1946-1954). Tumor hypoxia upregulates wild-type EGFR protein and its cognate ligand TGFα through several HIF-dependent mechanisms (Curr Pharm Des 2013; 19: 907). One consequence of upregulation of wild-type EGFR protein and cognate ligand in tumor cells is the induction of resistance to tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib or afatinib, which show preferential inhibitory activity against specific activated mutant forms of EGFR (Yun et al., 2008, Proc Natl Acad Sci; 105: 2070; Takezawa et al. Cancer Discover 2012; 2: 922-33; Camidge et al. Nat. Rev. Clin. Oncol. 2014; 11: 473-481; Murakami et al. 2014, Plos ONE 9(1): e86459). EGFR activating mutant-positive non-small cell lung cancer (NSCLC) is often heterozygous (Soh J, et al. 2009, PLoS ONE 4(10):e7464; Bai et al., 2013, PLoS ONE 8:e54170), and the presence of the wild-type allele is associated with limited response to TKI treatment regimens (Taniguchi et al., 2008, Cancer Sci;99:929-35).One possibility to overcome the TKI-resistant state is to silence wild-type EGFR signaling generated in hypoxic tumor areas, however, conventional EGFR TKIs lack the therapeutic index necessary to achieve this goal due to dose-limiting side effects associated with wild-type EGFR inhibition mechanisms in the skin and intestine (Hynes et al., 2005, Nat Rev Can 5:341; Sharma et al., Clin. Cancer Res 2006;12:4392s-4395s; Sharma et al., Genes Dev. 2007 21:3214-3231; Sharma et al., 2007 Nat Rev Can 7:169; Janjigian et al., Cancer Discovery 2014, 4:1-10).

[0005] Despite the progress made in this area, all available data indicate that significant non-responsiveness is undesirable in cancer patients due to EGFR genetic polymorphisms. Therefore, there is a need for new, safer and more effective methods for treating cancer that address EGFR resistance and non-responsive patients with EGFR genetic polymorphisms, especially for high-risk patients.

[0006] Some substances have been prepared for treating cancer by targeting hypoxic cancer cells (see, e.g., PCT Patent Publication Nos. WO 2010 / 104406 and WO 2011 / 028135, each of which is incorporated herein by reference), which disclose hypoxia-activated prodrugs (HAPs) of EGFR TKIs. An example of such a HAP EGFR TKI is TH-4000. Hypoxia-activated prodrugs (HAPs) of EGFR TKIs (e.g., TH-4000) can overcome such inherent resistance mechanisms.

[0007] There is a need for compositions and methods for treating cancer. The present invention meets this need by providing compositions and methods related to the use of TH-4000, a hypoxia-activated nitroimidazole prodrug of a kinase inhibitor, in treatment. The hypoxia-dependent mechanism provides tumor-selective release of EGFR TKIs in hypoxic tumor cells. The present invention addresses the problem of drug resistance and provides methods for identifying individuals at risk for resistance to EGFR treatment in patients with EGFR genetic polymorphisms. The present invention meets this and other needs. High local concentrations provide the therapeutic index required to silence wild-type EGFR signaling in hypoxic tumor compartments, as summarized below. Brief description of the invention

[0008] In one aspect, the present invention relates to a pharmaceutically acceptable composition comprising the known compound PR610, TH-4000 or Hypoxin as shown below:

[0009]

[0010] and a pharmaceutically acceptable carrier, excipient or diluent, such as 2-hydroxypropyl-β-cyclodextrin. In vivo, TH-4000 is converted into the tyrosine kinase inhibitor TH-4000E (or PR610E):

[0011]

[0012] In another aspect, the invention provides a pharmaceutically acceptable formulation comprising up to about 40 wt% 2-hydroxypropyl-β-cyclodextrin. The formulation may be diluted (eg, with an aqueous vehicle) prior to administration to a patient.

[0013] In another aspect, the present invention provides a unit dose of a pharmaceutically acceptable TH-4000 formulation. In one embodiment, a unit dose of a pharmaceutically acceptable formulation comprises about 5 mg - about 700 mg, or about 10 mg - about 180 mg, or about 20 mg - about 150 mg TH-4000. In one embodiment, the unit dose comprises about 100 mg TH-4000.

[0014] In another aspect, the present invention provides a method of treating cancer by administering to a patient in need of such treatment about 9 mg / m 2 – About 350 mg / m 2 , about 10mg / m 2 – About 200 mg / m 2 , about 15mg / m 2 – About 150 mg / m 2 , about 40mg / m 2 – About 100 mg / m 2 In another embodiment, the present invention provides for administration of about 80 mg / m 2 In another embodiment, the present invention provides a method of treating cancer with TH-4000 by administering about 32 mg / m 2 In one embodiment, the present invention provides a method of treating cancer with TH-4000 by administering about 20 mg / m 2Methods of treating cancer with TH-4000. In one embodiment, TH-4000 is administered intravenously (iv). In one embodiment, TH-4000 is administered intraperitoneally. In one embodiment, a therapeutically effective amount of TH-4000 is administered at a frequency of at least once a day, at least once a week, or at least once a month. In one embodiment, TH-4000 may be administered for a period of 1-3 weeks, or up to at least 30 weeks or more, at least 1 day, or up to at least 150 days or more. In some embodiments, longer administration times are used.

[0015] In one embodiment, the cancer treated is selected from non-small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, head and neck cancer, skin cancer, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer, prostate cancer, liver cancer, melanoma, brain cancer and mesothelioma.

[0016] In another aspect, the present invention provides pharmaceutically acceptable formulations comprising other tyrosine kinase and / or EGFR inhibitors, including but not limited to erlotinib, dacomitinib, AZD9291, CO-1686, afatinib, gefitinib, rociletinib, cetuximab, icotinib, AZD-8931, lapatinib, dacomitinib, neratinib, AP-26113, Poziotinib, ASP-8273, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab and patritumumab.

[0017] These and other aspects and embodiments are described in the drawings and detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Now, the present invention will be described in more detail with reference to the accompanying drawings, in which:

[0019] Figure 1 and 2 To show the comparison of erlotinib and TH-4000 in EGFR mutation-positive NSCLC in the clinically relevant HED;

[0020] Figure 3 Comparison of TH-4000–plasma vs tumor PK;

[0021] Figure 4It was shown that in nude mice bearing PC9 tumors, EGFR phosphorylation was inhibited for 7 days after a single administration of TH-4000 (15 mg / kg, ip).

[0022] Figure 5 Provides human plasma AUC0-inf of TH-4000 as a function of dose (mg / m2);

[0023] Figure 6 Western blot showing TKI-dependent target regulation in PC9 cells.

[0024] Figure 7 and 8 Western blot showing TKI-dependent target regulation in PC9 cells with erlotinib and AZD9291.

[0025] Fig. 9 Comparison of IC50 of TH-4000 in oxygen / anoxic conditions.

[0026] Fig.10 TH-4000 metabolism in selected NSCLC cell lines is shown.

[0027] Figure 11-13 Shown is a comparison of the efficacy of weekly administered TH-4000 vs daily administered erlotinib in PC-9 xenografts in individual mice.

[0028] Fig.14 Comparison was made from day 14, erlotinib (HED 150 mg) ± weekly TH-4000 (HED 32 mg / m2).

[0029] Fig.15 TH-4000 was shown to be effective against WT EGFR A431 xenografts at clinically relevant doses.

[0030] Fig.16 TH-4000 was shown to be effective against HER2-positive NCI-N87 gastric tumor xenografts.

[0031] Fig.17 The antiproliferative activity of cetuximab, erlotinib, TH-4000 and TH-4000E is shown in the human hypopharyngeal cell line FaDu after 5 consecutive days of exposure under aerobic conditions.

[0032] Fig.18 The antiproliferative activities of cetuximab, erlotinib, TH-4000 and TH-4000E in the human hypopharyngeal bone cell line FaDu were demonstrated.

[0033] Fig.19Shown is the expression of each member of the EGFR signaling axis after treatment with graded concentrations of TH-4000E.

[0034] Fig. 20 Shown is the expression of various components of the EGFR signaling axis after treatment with graded concentrations of cetuximab.

[0035] Fig.21 Shown is the expression of each member of the EGFR signaling axis after treatment with graded concentrations of TH-4000.

[0036] Fig. 22 Shown is EGFR expression in aerobic and hypoxia-pretreated (48 h) FaDu cells after exposure to various concentrations of TH-4000E.

[0037] Fig.23 Shown is EGFR expression after a period of hypoxic exposure with and without a period of aerobic recovery.

[0038] Fig.24 : Mean tumor volume (± SEM) of H125 tumors treated with vehicle control (qw x6), 48 mg / kg tarloxotinib (qw x6), 0.25 mg / mouse cetuximab (q3d x14), and the combination of tarloxotinib & cetuximab.

[0039] Fig.25 : Mean tumor volume (± SEM) of H125 tumors treated with vehicle control (qw x6), 6 mg / kg afatinib (qd x42), 0.25 mg / mouse cetuximab (q3d x14), and the combination of afatinib & cetuximab.

[0040] Fig.26 : Mean tumor volume (± SEM) of H125 tumors treated with vehicle control (qw x6), 48 mg / kg tasotinib (qw x6), 0.25 mg / mouse cetuximab (q3d x14), the combination of tasotinib & cetuximab, 6 mg / kg afatinib (qd x42), and the combination of afatinib & cetuximab.

[0041] Fig. 27 : Mean body weight loss (± SEM) of H125 tumor-bearing NIH-III mice treated with vehicle control (qw x6), 48 mg / kg tasotinib (qw x6), 0.25 mg / mouse cetuximab (q3d x14), the combination of tasotinib & cetuximab, 6 mg / kg afatinib (qd x42), and the combination of afatinib & cetuximab.

[0042] Fig.28 : Individual weight volumes of H125 tumors treated with vehicle control (qw x6), 48 mg / kg tasotinib (qw x6), 0.25 mg / mouse cetuximab (q3d x14), the combination of tasotinib & cetuximab, 6 mg / kg afatinib (qd x42), and the combination of afatinib & cetuximab. DETAILED DESCRIPTION OF THE INVENTION

[0043] The detailed description of aspects and embodiments of the present invention is organized into the following sections. Section I provides definitions of terms used herein. Section II describes pharmaceutically acceptable formulations of the present invention. Section III provides methods of treatment of the present invention. The detailed description is organized into sections for the convenience of the reader only, and the disclosure found in any section applies to disclosures elsewhere in this specification sheet.

[0044] Abbreviation and Definition

[0045] Abbreviations used herein are conventional unless otherwise defined. The following abbreviations are used: g = gram, H2O = water; h = hour, IC 50= concentration of inhibitor required for 50% inhibition of the enzyme in vitro, μM = micromolar, μL = microliter, mg = milligram, mm = millimeter, mM = millimolar, mmol = millimolar, mL = milliliter, mOD / min = milliomodensity units / minute, min = minute, M = molar, ACLS = Advanced Cardiovascular Life Support, ADL = activities of daily living, AE = adverse event, ALT = alanine aminotransferase, AST = aspartate aminotransferase, ATC = anatomic therapeutic chemical, ATP = adenosine triphosphate, AUC = area under the plasma concentration-time curve from 0 to infinity, AUClast = area under the plasma concentration-time curve from 0 to the time of the last quantifiable concentration, BP = blood pressure, BQL = below limit of quantification, Cmax = maximum plasma concentration, CFR = Code of Federal Regulations, CL = clearance, Cmax = peak plasma concentration, CNS = central nervous system, CR = complete response, CRO = clinical research organization, CT = computer tomography, CTC = circulating tumor cells, CTCAE = Common Toxicity Criteria for Adverse Events, D5W = dextrose in water 5%, DLT = dose-limiting toxicity, DOR = duration of response, ECG = electrocardiogram, ECOG = Eastern Cooperative Oncology Group, eCRF = electronic case report form, EGFR = epidermal growth factor receptor, GI = gastrointestinal, FDA = Food and Drug Administration, H = hour, HAP = hypoxia-activated prodrug, HED = human equivalent dose, HIV = human immunodeficiency virus, HR = heart rate, HRT = hormone replacement therapy, ICF = informed consent form, ICH = International Conference on Harmonization Conference on Harmonization), INR = international normalized ratio, IRB / EC = institutional review board / ethics committee, IV = intravenous, Kel = clearance constant, LLT = lowest level term, LLN = lower limit of normal, MedDRA = Medical Dictionary for Regulatory Affairs, MRI = magnetic resonance imaging, MTD = maximum tolerated dose, NSAID = nonsteroidal anti-inflammatory drug, NSCLC = non-small cell lung cancer, OR = overall response, ORR = overall response rate, OS = overall survival, PD = progressive disease, PET = positron emission tomography, PFS = progression-free survival, PK = pharmacokinetics, PR = partial response, PT = prothrombin time or preferred term, QT = QT interval, QTc = QT interval corrected for ECG heart rate, QTcB = QTc using Bazett formula, QTcF = QTc using Fridericia formula, RECIST 1.1 = Response Evaluation Criteria in Solid Tumors 1.1 version, RR = respiratory rate, SAE = serious adverse event, SD = stable disease, SOC = system organ class, T1 / 2 = terminal half-life, TdP = torsades de Pointes, TGFα = transforming growth factor-α, TH-4000E = TKI effector derived from TH-4000, TKI = tyrosine kinase inhibitor, Tmax = time to peak plasma concentration, ULN = upper limit of normal, Vβ = volume of distribution in the post-distribution phase, Vss = volume of distribution at steady state, WT = wild type. .

[0046] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols and other scientific or medical terms or expressions used herein are intended to have the meanings generally understood by those skilled in the art of chemistry and medicine. In some cases, terms with generally understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such terms herein should not be construed as representing essential differences beyond the definition of terms generally understood in the art.

[0047] It is noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0048] "About" refers to ±20% of an amount, and includes, but is not limited to, ±15%, ±10%, and ±5% of an amount.

[0049] "Administering" a drug to a patient (and grammatical equivalents of that term) refers to direct administration, which may be administered to a patient by a medical professional or may be self-administered, and / or indirect administration, which may be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer the drug and / or provides a prescription for the drug to a patient administers the drug to the patient.

[0050] "Antagonist" or "inhibitor" refers to a substance or molecule that inhibits or binds to a receptor of the present invention, partially or completely blocks the stimulation or activity of a receptor of the present invention, reduces, shuts down, prevents, delays the activation or enzymatic activity of a receptor of the present invention, inactivates, desensitizes, or downregulates the activity of a receptor of the present invention. As used herein, "antagonist" also includes inverse agonists.

[0051] "Anti-cancer effects" include, but are not limited to, anti-cancer effects, response rates, time to disease progression, and survival rates. "Anti-tumor" effects include, but are not limited to, inhibition of tumor growth, tumor growth delay, tumor regression, tumor shrinkage, prolonged tumor regrowth after treatment cessation, and delayed disease progression.

[0052] "Cancer" refers to a potentially indefinitely growing malignant tumor that can spread locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, non-small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, head and neck cancer, skin cancer, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer, prostate cancer, liver cancer, melanoma, brain cancer, and mesothelioma.

[0053] "Combination therapy" refers to the use of two or more drugs for treatment, i.e., the use of a hypoxia-activated prodrug as described herein together with a conventional drug for the treatment of blood cancer is a combination therapy. "Combination" administration refers to the administration of two substances (e.g., a hypoxia-activated prodrug and a substance known to be used to treat blood cancer) in any manner in which the pharmacological effects of the two drugs are simultaneously manifested in the patient. Thus, combined administration does not require a single pharmaceutical composition, the same dosage form, or even the same route of administration to be used for the administration of the two substances or that the two substances be administered at exactly the same time. For example, and without limitation, it is contemplated that one or more of the following substances may be administered in combination with a hypoxia-activated prodrug according to the present invention.

[0054] As used herein, the term "disorder" refers to the disease state against which the compounds, compositions and methods of the present invention are used.

[0055] As used herein, the term "clinical response" refers to any or all of the following: a quantitative measure of response, non-response, and adverse responses (ie, side effects).

[0056] As used herein, the term "clinical trial" refers to any study designed to collect clinical data on response to a specific treatment, and includes, but is not limited to, Phase I, Phase II, and Phase III clinical trials. Standard methods are used to define patient populations and for recruiting subjects.

[0057] As used herein, "expression" includes, but is not limited to, one or more of the following: transcription of a gene into a pre-mRNA; splicing and other processing of the pre-mRNA to produce a mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modification of the translation product, if necessary for proper expression and function.

[0058] As used herein, the term "gene" shall mean a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns and other untranslated regions that control expression.

[0059] As used herein, the term "genotype" shall mean the unphased 5' to 3' sequence of nucleotide pairs found at one or more polymorphic sites in a locus of a pair of homologous chromosomes of an individual. As used herein, genotype includes full genotype and / or subgenotype.

[0060] As used herein, the term "locus" shall mean a location on a chromosome or DNA molecule that corresponds to a gene or a physical or phenotypic trait.

[0061] "Patient" refers to a mammal in need of cancer treatment. Generally, the patient is a human. The patient can also be a "warm-blooded animal".

[0062] "Pharmaceutically acceptable" means useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable and includes those that are acceptable for veterinary as well as human pharmaceutical uses.

[0063] "Pharmaceutically acceptable carrier, excipient or diluent" refers to a carrier, excipient or diluent that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers, excipients or diluents that are acceptable for human pharmaceutical use as well as veterinary use. "Pharmaceutically acceptable carrier, excipient or diluent" includes one or more than one carrier, excipient or diluent.

[0064] "Pharmaceutically acceptable salts" are meant to include salts of the active compounds, which are prepared with relatively nontoxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, for example, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts may be obtained by contacting the neutral form of the compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and those derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid, and the like (see, e.g., Berge, SM et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain basic and acidic functional groups that can convert the compounds into base addition salts or acid addition salts. By contacting the salt with an alkali or acid and isolating the parent compound in a conventional manner, the neutral form of the compound can be regenerated. The parent form of the compound differs from the various salt forms in some physical properties, such as solubility in polar solvents, but otherwise, for the purposes of the present invention, the salt is equivalent to the parent form of the compound.

[0065] As used herein, the term "polymorphism" will mean any sequence variant that is present at a frequency of >1% in a population. Sequence variants may be present at a frequency significantly greater than 1%, such as 5% or 10% or greater. Also, the term may be used to refer to sequence changes observed in individuals at polymorphic sites. Polymorphisms include nucleotide substitutions, insertions, deletions, and microsatellites, and may, but need not, produce detectable differences in gene expression or protein function.

[0066] As used herein, the term "polymorphic site" shall mean a position within a locus at which at least two alternative sequences are found in a population, the most frequently occurring sequence having a frequency of no more than 99%.

[0067] As used herein, the term "polynucleotide" will mean any RNA or DNA, which can be unmodified or modified RNA or DNA. Polynucleotides include but are not limited to single-stranded and double-stranded DNA, DNA, single-stranded and double-stranded RNA, and RNA, hybrid molecules containing DNA and RNA, which are mixtures of single-stranded and double-stranded regions, and which can be single-stranded or more typically, double-stranded or mixtures of single-stranded and double-stranded regions. In addition, polynucleotides refer to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a skeleton modified for stability or other reasons.

[0068] As used herein, the term "polypeptide" shall mean any polypeptide containing two or more amino acids interconnected by peptide bonds or modified peptide bonds (i.e., isosteres of peptides). Polypeptides refer to short chains commonly referred to as peptides, glycopeptides or oligomers, as well as longer chains commonly referred to as proteins. Polypeptides may contain amino acids other than the 20 genetically encoded amino acids. Polypeptides include amino acid sequences modified by natural methods such as post-translational processing or by chemical modification techniques well known in the art. The modifications are described in detail in basic texts and more detailed monographs, as well as in most research literature.

[0069] "Prodrug" refers to a compound that is metabolized or otherwise converted into a biologically active or more active compound (or drug) after administration with respect to at least one property. Relative to the drug, the prodrug is chemically modified in a form that renders it less active or inactive relative to the drug, but the chemical modification is such that after the prodrug is administered, the corresponding drug is produced through metabolic processes or other biological processes. Relative to the active drug, the prodrug may have altered metabolic stability or transport characteristics, fewer side effects or lower toxicity or improved taste (e.g., see reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392, incorporated herein by reference). Prodrugs may be synthesized using reactants other than the corresponding drug.

[0070] "Relief" of a symptom (and grammatical equivalents of this phrase) refers to a reduction / decrease in severity or frequency of the symptom, or the elimination of the symptom.

[0071] "Relapsed or refractory" refers to a type of blood cancer that is resistant to treatment with a substance or that responded to treatment with a substance but relapsed and is not resistant to the substance, or that responded to treatment with a substance but relapsed and is resistant to the substance.

[0072] "Single substance therapy" or "monotherapy" refers to the use of a single drug to treat a disease, i.e., the use of a hypoxia-activated prodrug, such as TH-4000, as the only chemical substance to treat blood cancer. The administration of palliatives and / or vitamins and / or other substances administered for purposes other than direct treatment of the disease may be administered in a single substance therapy. Patients undergoing single substance therapy may also undergo radiation therapy and / or surgery.

[0073] "Standard chemotherapy" refers to treatment with a drug in accordance with FDA label instructions and / or good clinical practice. Standard chemotherapy is well known to those skilled in the medical arts.

[0074] As used herein, "SNP nucleic acid" is a nucleic acid sequence that contains nucleotides that are variable (and therefore exist as alleles) within an otherwise identical nucleotide sequence between individuals or between groups of individuals. The SNP nucleic acid is preferably about 15 to about 500 nucleotides in length. The SNP nucleic acid can be a part of a chromosome, or it can be an exact copy of a part of a chromosome, for example, by PCR or by amplification of a cloned part of the chromosome. Hereinafter, the SNP nucleic acid is simply referred to as "SNP". The SNP probe according to the present invention is an oligonucleotide complementary to the SNP nucleic acid.

[0075] A "therapeutically effective amount" of a drug refers to an amount of the drug that, when administered to a patient suffering from cancer or another hyperproliferative disease, will have the desired therapeutic effect, such as alleviating, ameliorating, reducing or eliminating one or more clinical manifestations of the patient's cancer or another hyperproliferative disease. The therapeutic effect does not necessarily occur through the administration of a single dose, and may occur only after a series of doses are administered. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0076] "Treatment" of a disease or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. For purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviation or improvement of one or more symptoms of cancer or another hyperproliferative disease; reduction in the severity of the disease; delay or slowing of the progression of the disease; improvement, alleviation or stabilization of the disease state; or other beneficial results.

[0077] A "unit dose" of a drug is a single dose of a therapeutically effective amount of the drug at a specified time.

[0078] "Warm-blooded animal" means any member of the class Mammalia, which includes, but is not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats and pigs; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents such as rats, mice and guinea pigs, etc.

[0079] Pharmaceutically acceptable formulations

[0080] In one aspect, the present invention relates to a pharmaceutically acceptable formulation comprising a compound called TH-4000 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent. TH-4000 is described in PCT Patent Application Publication No. WO 2011 / 028135. Aqueous suspensions contain the active substance mixed with an excipient suitable for preparing an aqueous suspension. The excipient is a suspending agent such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; the dispersant or wetting agent can be a naturally occurring phosphatide such as lecithin, or a condensation product of an alkylene oxide with a fatty acid such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long-chain fatty alcohol such as heptadecaethylene oxycetanol, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride such as polyoxyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

[0081] Dispersible powders and granules suitable for preparing aqueous suspensions are provided by adding water to the active ingredient mixed with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are, for example, those mentioned above. Other excipients such as sweeteners, flavoring agents and coloring agents may also be present.

[0082] In one embodiment, a pharmaceutically acceptable formulation comprises TH-4000 and 2-hydroxypropyl-β-cyclodextrin as an excipient. The TH-4000 formulation is described in Example 1 below.

[0083] In one embodiment, a unit dose of a pharmaceutically acceptable formulation comprises about 5 mg - about 700 mg, or about 10 mg - about 180 mg, or about 20 mg - about 150 mg TH-4000. In one embodiment, a unit dose comprises about 100 mg TH-4000.

[0084] In another aspect, the present invention provides a method of treating a patient in need of such treatment by administering a therapeutically effective amount of about 9 mg / m 2 – About 350 mg / m 2 , about 10mg / m 2 – About 200 mg / m 2 , about 15mg / m 2 – About 150 mg / m 2 , about 40mg / m 2 – About 100 mg / m 2 In another embodiment, the present invention provides a method of treating cancer by administering about 80 mg / m 2 In another embodiment, the present invention provides a method of treating cancer by administering about 32 mg / m 2 Methods of treating cancer with TH-4000. In one embodiment, the present invention provides for administering about 20 mg / m 2 Methods of treating cancer with TH-4000. Pharmaceutically acceptable formulations of suitable unit doses of TH-4000 are described in the following examples.

[0085] Treatment

[0086] In one embodiment, the cancer treated is selected from non-small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, head and neck squamous cell carcinoma, skin squamous cell carcinoma, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer and mesothelioma.

[0087] The therapeutic dose of TH-4000 will likely be 1 mg to 3000 mg / day. The specific dose level selected for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration and rate of excretion, drug combination, and severity of the condition being treated.

[0088] In another aspect, the present invention provides a method of treating a patient in need of said treatment by administering about 9 mg / m 2 – About 350 mg / m 2 , about 10mg / m 2 – About 200 mg / m 2 , about 15mg / m 2 – About 150 mg / m 2 , about 40mg / m 2 – About 100 mg / m 2 In another embodiment, the present invention provides a method of treating cancer by administering about 80 mg / m 2 In another embodiment, the present invention provides a method of treating cancer by administering about 32 mg / m 2 Methods of treating cancer with TH-4000. In one embodiment, the present invention provides for administering about 20 mg / m 2 Methods of treating cancer with TH-4000. Animal studies of TH-4000 showing therapeutically effective administration of TH-4000 with anti-tumor effects are described in the Examples below (eg, Examples 1-4). A human clinical trial protocol is described in Example 5 below.

[0089] Application

[0090] As used herein, administration of a substance or drug to a subject or patient includes self-administration and administration by another person.

[0091] In one embodiment, the TH-4000 compound can be administered by injection in a dosage unit formulation. The term "administered by injection" includes intravenous, intramuscular, subcutaneous, and parenteral injections, as well as the use of infusion techniques. Effective intravenous (iv) administration of TH-4000 for the treatment of cancer is described in the Examples. The bioavailability of effective intravenous injection of TH-4000 is described in Example 3. The typical injection period is 1 to about 6 hours.

[0092] In one embodiment, a therapeutically effective amount of TH-4000 is administered at least once a day, at least once a week, at least once every two weeks, or up to once a month. In one embodiment, TH-4000 is administered for a period of 1-40 weeks, at least 1 week, at least 2 weeks, or up to at least 40 weeks. In another embodiment, it is administered for a longer period of time.

[0093] For example, in Examples 2-5 below, various frequencies and durations of TH-4000 administration for effective treatment of cancer according to the present methods are described.

[0094] In one embodiment, TH-4000 is administered in combination with another antiproliferative substance, such as those that regulate tyrosine kinases. Optional antiproliferative substances that may also be administered include, but are not limited to, erlotinib, dacomitinib, AZD9291, CO-1686, afatinib, gefitinib, rociletinib, cetuximab, icotinib, AZD-8931, lapatinib, dacomitinib, neratinib, AP-26113, Poziotinib, ASP-8273, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab, and patritumumab.

[0095] In a specific embodiment, the present invention provides for the administration of about 10 mg / m 2 Up to about 200 mg / m 2 A method of treating a patient suffering from non-small cell lung cancer (NSCLC) by administering to a patient an amount of TH-4000. In one embodiment, the patient is also treated with erlotinib. These and other aspects and embodiments are described in the drawings and detailed description of the invention.

[0096] In one embodiment, the present invention provides for the administration of about 10 mg / m 2 Up to about 200 mg / m 2 In one embodiment, the patient is also treated with AZD9291.

[0097] In one embodiment, the invention provides a method of treating cancer by administering a pharmaceutically acceptable formulation of the invention comprising a hypoxia-activated nitroimidazole prodrug other than TH-4000, and those described in WO 2011 / 028135.

[0098] Diagnostic methods and kits

[0099] Hypoxia markers

[0100] The present invention advantageously provides a method for determining whether a patient will experience drug resistance during drug therapy before actually taking the drug. Thus, by helping clinicians (1) provide additional or alternative concomitant medications, (2) change the dosage of a drug, or (3) choose not to prescribe the drug to the patient, the present invention provides a safer treatment regimen for the patient.

[0101] In various embodiments of the present invention, markers of hypoxia are used to select patients for treatment and / or identify patients who respond (or do not respond) to treatment. During the study, hypoxia markers have been developed, which show that hypoxia promotes a more aggressive solid tumor phenotype and is associated with resistance to radiotherapy and many chemotherapies, as well as the possibility of tumor invasion and low patient survival. Specifically, cells with pO2<10 mm Hg resist the ionizing effects of radiotherapy and the cytotoxic effects of chemotherapy. Hypoxic necrotic lesions of pseudopalisading cells are one of the characteristics that define, for example, glioblastoma (GBM). Therefore, various methods have been designed to evaluate the degree of hypoxia in xenografts and patient tumors, and according to the present invention, these methods, appropriately modified and practiced as described herein, are used in some embodiments of the methods of the present invention to select patients and evaluate responses to treatment. In general, the present invention provides methods for identifying patients suitable for treatment with a hypoxia-activated prodrug, wherein a hypoxia marker is used to determine that the patient's cancer is hypoxic, and then the patient is treated with a hypoxia-activated prodrug, i.e., the higher the degree of hypoxia, the greater the likelihood that the patient will respond to treatment with the hypoxia-activated prodrug. Those skilled in the art will appreciate that, based on this disclosure, the methods can be used for all cancers, not just blood cancers.

[0102] Traditionally, the gold standard for measuring hypoxia has always been the use of polar oxygen sensitive probes, which provide direct measurements of tissue oxygen tension. However, the method has limitations, such as its inability to distinguish between viable and necrotic lesions, the inaccessibility of many tumor tissues including those associated with myeloid malignancies, and the lack of practical means for large-scale application of the technology. Two 2-nitroimidazole compounds, pimonidazole and EF5, are hypoxia markers, and immunohistochemical identification of pimonidazole or EF5 protein adducts can provide reliable evaluation of radio-biologically relevant hypoxia. Molecular oxygen competes with reducing equivalents, thereby effectively inhibiting pimonidazole (and EF5) from combining at oxygen concentrations exceeding 14 micromolar. The method reliably and specifically identifies viable hypoxic cells (necrotic cells cannot metabolize pimonidazole or EF5).

[0103] Other hypoxia markers suitable for use according to the methods of the present invention have been identified in preclinical studies, including GLUT-1, HIF-1a, CA-IX, LDH-A, osteopontin, microRNA markers, including but not limited to miR-210 and VEGF. Each of these proteins or RNAs is upregulated under hypoxia, and they can be detected by tumor biopsy. However, more conveniently, some of the markers, namely CA-IX LDH-A, osteopontin, microRNAs including but not limited to miR-210 and VEGF, are detectable in the patient's blood, serum or plasma, so that a simple blood test, rather than a tumor biopsy, can be used to select patients for hypoxia-activated prodrug treatment.

[0104] In addition, studies have validated the spatial relationship between tumor hypoxia assessed by immunohistochemistry and [18F]-FDG and [18F]-FMISO autoradiography and PET imaging, and these compounds and similar PET tracers such as [18F]-EF5, [18F]-FAZA and [18F]-HX4 can be used in accordance with the methods of the present invention. In addition to autoradiography and PET imaging, MRI imaging of hypoxia, particularly dynamic contrast-enhanced MRI (DCE-MRI), can be used to identify hypoxic cancers and thereby identify ideal patients for treatment with hypoxia-activated prodrugs.

[0105] When administered IV or orally, Hypoxyprobe TM -1 (pimonidazole hydrochloride, marketed by Hypoxyprobe, Inc.) is distributed to all tissues in the body, including the brain, but forms adducts with proteins only in those cells where the oxygen concentration is below 14 micromolar (equivalent to 10 mm Hg pO2 at 37 degrees Celsius). Hypoxyprobe-1 MAb1 is a mouse IgG1 monoclonal antibody that detects protein adducts of Hypoxyprobe-1 in hypoxic cells. Typically, this reagent is added to each tissue sample. Then, according to the present invention, a chromogenic or fluorescent secondary antibody reagent is used to show where Hypoxyprobe-1 adducts are formed in hypoxic tissues.

[0106] In addition to these hypoxia markers, there are other markers that can be used to select patients for hypoxia-activated prodrug treatment. The hypoxia-activated prodrugs of the present invention are activated by reductases, so a biopsy or blood test showing that the patient has a higher level of activated reductases such as POR (P450 oxidoreductase), MTRR (methionine synthase reductase) and / or NOS (nitric oxide synthase) indicates that the patient is more likely to respond to hypoxia-activated prodrug treatment. In addition, the HDR (also known as HR) system repairs DNA damage induced by the hypoxia-activated prodrug, and in the patient's blood or tumor biopsy, the lower the level of the proteins in the system, including but not limited to BRCA, FANG, XPF (also known as ERCC4), XRCC2 and / or XRCC3, the more likely the patient is to respond to hypoxia-activated prodrug treatment.

[0107] A relevant population with wild-type EGFR can be determined in the TH-4000 clinical trial. Subjects with and without EGFR resistance were identified and genotyped as described below.

[0108] The maximum tolerated dose (MTD) of a compound is determined using methods and materials known in the field of medicine and pharmacology, for example, by a dose expansion test. First, one or more patients are treated with a low dose of a compound, typically based on the results of an in vitro cell culture test, 10% of the expected therapeutic dose. Patients are observed for a period of time to determine toxicity. Typically, toxicity is shown as observing one or more of the following symptoms: vomiting, diarrhea, peripheral neuropathy, ataxia, neutropaenia, or elevated liver enzymes. If toxicity is not observed, the dosage is increased by 2-times, and the patient's toxicity evidence is observed again. The cycle is repeated until the dosage for producing toxicity evidence is reached. The dosage immediately before the onset of unacceptable toxicity is used as MID. The determination of MTD is provided above.

[0109] Using various techniques well known in the art, individuals carrying wild-type alleles can be detected at the DNA, RNA or protein level. Identification and detection strategies are described in EP 730,663, EP717,113 and PCT US97 / 02102. The identification of alleles containing wild-type can involve the amplification of DNA in the target sample. This can be achieved by, for example, PCR. Generally, see PCR Technology: Principles and Applications for DNA Amplification (Erlich compiled, Freeman Press, New York, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Innis et al. compiled, Academic Press, San Diego, Calif., 1990). Detection of the wild type in a particular DNA sequence can be accomplished by a variety of methods, including, but not limited to, restriction fragment length wild-type detection based on allele-specific restriction-endonuclease cleavage (Kan & Dozy, Lancet II:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al., Nucl. Acids Res. 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1969)) or oligonucleotide arrays (Maskos & Southern, Nucl. Acids Res. 21:2269-2270 (1993)), allele-specific PCR (Newton et al., Nucl. Acids Res. 21:2269-2270 (1993)), allele-specific PCR (Newton et al., Nucl. Acids Res. 21:2269-2270 (1993)), allele-specific PCR (Newton et al., Nucl. Acids Res. 21:2269-2270 (1993)), allele-specific PCR (Wallace ... Res. 17: 2503-2516 (1989)), mismatch repair assay (MRD) (Faham & Cox, Genome Res. 5: 474-482 (1995)), MutS protein binding (Wagner et al., Nucl. Acids Res. 23: 3944-3948 (1995), denaturing-gradient gel electrophoresis (DGGE) (Fisher & Lerman, Proc. Natl. Acad. Sci. USA 80: 1579-1583 (1983)), single-stranded conformation-wild-type assay (Orita et al., Genomics 5: 874-879 (1983)), RNAse cleavage of mismatched base pairs (Myers et al., Science 230:1242 (1985)), the chemistry of heteroduplex DNA (Cotton et al., Proc. Natl. Acad. Sci. USA, 8Z: 4397-4401 (1988)) or enzyme (Youil et al., Proc. Natl. Acad. Sci. USA 92: 87-91 (1995)) cleavage, allele-specific primer extension-based methods (Syvanen et al., Genomics 8: 684-692 (1990)), genetic bit analysis (GBA) (Nikiforov et al., Nucl. Acids Res. 22: 4167-4175 (1994)), oligonucleotide ligation analysis (OLA) (Landegren et al., Science 241:1077 (1988)), allele-specific ligation chain reaction (LCR) (Barrany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), gap-LCR (Abravaya et al., Nucl. Acids Res. 23:675-682 (1995)), radioactive and / or fluorescent DNA sequencing using standard methods well known in the art, and peptide nucleotide (PNA) analysis (Orum et al., Nucl. Acids Res. 21:5332-5356 (1993); Thiede et al., Nucl. Acids Res. 24:983-984 (1996)). Sambrook J et al., Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Press, Cold Spring Harbor, NY, 2000) provides additional guidance. In one embodiment, the technique for detecting gene expression includes the use of gene chips. The structure and use of gene chips are well known in the art. See, U.S. Patent Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795,716 and 5,800,992. See also Johnston, M. Curr Biol 8: R171-174 (1998); Iyer VR et al., Science 283: 83-87 (1999) and Elias P, "New human genome `chip` is a revolution in the offing" Los Angeles Daily News (October 3, 2003). .

[0110] In a particularly preferred embodiment, detection of the wild-type gene can be performed using the so-called SNP genotyping analysis (available from Applied Biosystems, Foster City, CA) was performed.

[0111] It is preferred that the individuals included in the clinical population have been graded according to the target medical condition present. The classification of potential patients can utilize standard physical examination or one or more laboratory tests. Alternatively, for situations where there is a strong correlation between gene expression pattern and disease susceptibility or severity, the classification of patients can utilize gene expression pattern.

[0112] The therapeutic treatment of interest is administered to each individual in the trial population, and the response of each individual to the treatment is measured using one or more predetermined criteria. It is contemplated that in many cases the trial population will show a range of responses, and the investigator will select the number of responder groups consisting of the various responses (e.g., low, medium, high).

[0113] After clinical and wild-type data have been obtained, the correlation between individual response and genotype or haplotype capacity is established. Correlation can be generated in various ways. In one method, individuals are grouped according to their genotype or haplotype (or haplotype pairing) (also referred to as wild-type group), and the mean and standard deviation of the clinical response displayed by each wild-type group member is then calculated.

[0114] The results are analyzed to determine whether any changes observed in clinical response between the wild-type groups are statistically significant. Statistical analysis methods that can be used are described in LD Fisher & G. van Belle, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, New York, 1993).

[0115] A second approach to finding correlations between gene expression patterns and clinical responses utilizes predictive models based on optimization algorithms that minimize errors. One of some possible optimization algorithms is a genetic algorithm (R. Judson, "Genetic Algorithms and Their Uses in Chemistry" in Reviews in Computational Chemistry, Vol. 10, pp. 1-73, KB Lipkowitz and DB Boyd, ed. (VCH Publishers, New York, 1997). Simulated annealing (Press et al., "Numerical Recipes in C: The Art of Scientific Computing", Cambridge University Press (Cambridge) 1992, Chapter 10), neural networks (E. Rich and K. Knight, "Artificial Intelligence", 2nd ed., McGraw-Hill, New York, 1991, Chapter 18), standard gradient descent (Press et al., supra, Ch. 10) or other global or local optimization methods (see discussion in Judson, supra).

[0116] ANOVA is used to verify the hypothesis that the response variable is caused by or is related to one or more measurable characteristics or variables (Fisher & van Belle, see above, Ch. 10). For the statistical methods used in the methods of the present invention, see: Statistical Methods in Biology, 3rd Edition, Bailey NTJ, (Cambridge Univ. Press, 1997); Introduction to Computational Biology, Waterman MS (CRC Press, 2000) and Bioinformatics, Baxevanis A D & Ouellette BFF (John Wiley & Sons, Inc., 2001).

[0117] Based on the above analysis, a mathematical model can be easily constructed by a skilled artisan to predict clinical response as a function of gene expression pattern.

[0118] The confirmation of the association between the genotype or haplotype (or haplotype pair) of clinical response and gene can be the basis of designing diagnostic method, to determine the individuality that will respond to treatment or not respond, or alternatively, will respond with lower level and need more treatment thus, i.e. those individuals of larger dosage medicine. Diagnostic method can adopt one of several forms: for example direct DNA test (i.e. gene expression pattern), serological test or physical examination measurement. Only requirement is that there is good correlation between diagnostic test result and potential genotype or haplotype, and described genotype or haplotype are then relevant to clinical response. In preferred embodiments, described diagnostic method uses predictive haplotype analysis method described above.

[0119] Computer can perform any or all analysis and mathematical operations relevant to practice the inventive method.In addition, computer can perform the program of the view (or screen) shown on display device, and utilize it, user can interact, to see and analyze a large amount of information relevant to gene and genome change, comprise chromosome location, gene structure and gene family, gene expression data, wild type data, gene sequence data and clinical data, group data (for example based on the data of geographic population (ethnogeographic) source, clinical response, genotype and the haplotype of one or more colonies).Wild type data described in the text can be stored as a part of relational database (for example, example of Oracle database or a group of ASCII flat files).Described wild type data can be stored in computer hard drive or can for example be stored in CD-ROM or computer readable one or more other storage devices.For example, data can be stored in one or more databases contacted with computer by network.

[0120] In other embodiments, the present invention provides methods, compositions and test kits for individual gene haplotype analysis and / or genotyping. Compositions include oligonucleotide probes and primers that specifically hybridize with one or more target regions of design, and the regions include wild-type sites or are adjacent to wild-type sites. The methods and compositions for setting up individual genotypes or haplotypes at wild sites described herein can be used for studying the effects of wild types in the etiology of diseases affected by protein expression and function, studying the effectiveness of drug targeting, predicting the susceptibility of individuals to diseases affected by the protein expression and function, and predicting the responsiveness of individuals to drugs of targeted gene products.

[0121] Still in another embodiment, the invention provides methods for determining the association between a gene expression pattern and a trait. In a preferred embodiment, the trait is a susceptibility to disease, the severity of disease, the staging of disease, or the response to a drug. The method is applicable to the development of diagnostic tests and therapeutic treatments for all pharmacogenetic applications, where there is the possibility that genotype and treatment outcome are associated, including efficacy measurements, PK measurements, and side effect measurements.

[0122] The present invention also provides a computer system for storing and displaying the measured wild-type data of genes. The computer system comprises a computer processing unit; a display; and a database comprising gene expression pattern data. The gene expression pattern data may include gene expression patterns in a reference population. In a preferred embodiment, the computer system can generate an image that displays gene expression patterns organized according to their evolutionary relationships.

[0123] In practicing the present invention, many other conventional techniques in molecular biology, microbiology and recombinant DNA are used. The techniques are well known and explained in, for example, the following references: "Current Protocols in Molecular Biology", Volumes I-III, Ausubel, ed. (1997); Sambrook et al., "Molecular Cloning: A Laboratory Manual", Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); "DNA Cloning: A Practical Approach", Volumes I and II, Glover, ed. (1985); "Oligonucleotide Synthesis", Gait, ed. (1984); "Nucleic Acid Hybridization", Hames & Higgins, ed. (1985); "Transcription and Translation", Hames & Higgins, ed. (1984); "Animal Cell Culture", Freshney, ed. (1986); "Immobilized Cells and Enzymes", IRL Press (1986); Perbal, "A Practical Guide to Molecular Cloning"; the series, Methods in Enzymol., Academic Press, Inc. (1984); "Gene Transfer Vectors for Mammalian Cells", Miller and Calos, eds.; Cold Spring Harbor Laboratory, NY (1987); and Methods in Enzymology, Vols. 154 and 155, Wu & Grossman and Wu, eds.

[0124] Then, the standard control level of the gene expression product measured in different control groups can be compared with the level of measurement of the gene expression product in a given patient.Described gene expression product can be the characteristic mRNA relevant to the polypeptide gene expression product of described specific genotype group or described genotype group.Then, can be based on how similar the measured level of given group is compared with the control level, patient classification or be assigned to concrete genotype group.

[0125] As those skilled in the art should understand, there is a certain degree of uncertainty associated with carrying out the described determination.Therefore, the standard deviation of the control group level will be used to carry out probability determination, and the method of the present invention will be applicable to the genotype group determination based on probability widely.Therefore, for example and not limited to, in one embodiment, if the level of the measurement of gene expression product falls within 2.5 standard deviations of any control group mean, then the individual can be assigned to the genotype group.In another embodiment, if the level of the measurement of gene expression product falls within 2.0 standard deviations of any control group mean, then the individual can be assigned to the genotype group.Also in another embodiment, if the level of the measurement of gene expression product falls within 1.5 standard deviations of any control group mean, then the individual can be assigned to the genotype group.Also in another embodiment, if the level of the measurement of gene expression product is 1.0 of any control group mean or lower standard deviation, then the individual can be assigned to the genotype group.

[0126] Thus, the method will enable determination (with varying degrees of probability) of which group a particular patient should be placed in, and then said assignment to genotype groups will determine into which risk category the individual should be placed.

[0127] Methods for detecting and measuring mRNA levels and polypeptide gene expression product levels are well known in the art and include nucleotide microarray and polypeptide assay methods involving mass spectrometers and / or antibody assays and quantitation techniques. See also Human Molecular Genetics, 2nd Edition. Tom Strachan & Andrew, Read (John Wiley and Sons, Inc. Publication, NY, 1999).

[0128] The target therapeutic treatment is applied to each individual in the colony, and one or more predetermined criteria are used to measure the response of each individual to the treatment. It is considered that in many cases, the colony will show a series of responses, and the researcher will select the numbering (e.g., low, medium, high) of the responder group consisting of various responses. In addition, the gene of each individual in the colony is genotypic and / or haplotype, which can be performed before or after the treatment is applied.

[0129] It should be understood that the methods of the invention described herein may also generally comprise the use of a kit according to the invention. Generally, the diagnostic methods of the invention may be performed ex vivo, and such ex vivo methods are specifically contemplated by the invention. Also, where the methods of the invention may comprise steps that may be performed on the human or animal body, methods comprising only those steps that are not performed on the human or animal body are specifically contemplated by the invention. In a preferred embodiment, the kit may comprise a DNA sample collection tool.

[0130] Therefore, the present invention also includes a kit for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the present invention in a biological sample, such as any body fluid including but not limited to serum, plasma, lymph, cystic fluid, urine, feces, CSF, acitic fluid or blood, and including a biopsy sample of body tissue. For example, the kit may include a marker compound or substance that can detect a polypeptide corresponding to a marker of the present invention or an mRNA encoding a polypeptide in a biological sample, and a tool for determining the amount of the polypeptide or mRNA in the sample, such as an antibody or oligonucleotide probe that binds to the polypeptide, and the probe is bound to the DNA or mRNA encoding the polypeptide. The kit also includes instructions for explaining the results obtained using the kit.

[0131] For oligonucleotide-based kits, the kit can include, for example, 1) an oligonucleotide, such as a detectably labeled oligonucleotide, that hybridizes to a nucleotide sequence encoding a polypeptide corresponding to a marker of the invention or 2) a pair of primers that can be used to amplify a nucleotide molecule corresponding to a marker of the invention.

[0132] The kit may also include, for example, a buffer, a preservative or a protein stabilizer. The kit may also include components such as enzymes or substrates required for detecting detectable labels. The kit may also contain a control sample or a series of control samples, which may be analyzed and compared with the test sample. The components of the kit may be contained in individual containers, and all of the containers may be in a single package, and there are instructions for explaining the analysis results completed using the kit.

[0133] The kits of the present invention may contain written products on or within the kit container. The written products describe how to use the reagents contained within the kit to determine whether a patient will experience hepatotoxicity during drug therapy. In several embodiments, the use of the reagents can be in accordance with the methods of the present invention. In one embodiment, the reagents are primer pairs for performing genetic wild-type PCR analysis.

[0134] The invention, which has been generally and specifically described, is illustrated but not limited by the following examples which provide pharmaceutically acceptable formulations of TH-4000 of the present invention and demonstrate the efficacy of TH-4000 administered to treat cancer according to the present method. Example

[0135] General

[0136] Material:

[0137] HCC827 cell line was obtained from ATCC (CRL-2868). PC9 cell line was obtained from RIKEN Bio Resource Center, Japan. TH-4000 can be synthesized and separated from unwanted reaction materials as described in PCT patent application publication number WO2011 / 028135. Erlotinib (SN 31254) was provided by Auckland Cancer Society Research Centre, Medicinal Chemistry Department. (2-Hydroxypropyl)-β-cyclodextrin was obtained from Sigma Aldrich (Cat#778966-500G). Poly(ethylene glycol) 400 (PEG-400) can be obtained from Sigma Aldrich (Cat#P3265-1KG). Dimethyl sulfoxide (DMSO) was obtained from ECP Ltd, New Zealand (Cat#2200.1-500ml). Water for injection (WFI) was obtained from DEMO SA Pharmaceutical Industry, Greece.

[0138] Example 1. Unit Dose Formulation of TH-4000

[0139] TH-4000 is formulated in ultrapure water purified by distillation or reverse osmosis "WFI" and 20% 2-hydroxypropyl-β-cyclodextrin to produce a unit dosage form of TH-4000.

[0140] Example 2. Demonstration of the efficacy of TH-4000 in killing hypoxic tumor cells, particularly EGFR mutation-positive non-small cell lung cancer, at clinically relevant human equivalent doses

[0141] HCC827 and PC9 cells are NSCLC cell lines that contain exon 19 deletion mutations in the tyrosine kinase domain of EGFR. HCC827 is homozygous for mutant EGFR (100% mutant alleles), while a portion of the alleles in PC9 cells contain wild-type EGFR (87% mutant and 13% wild type) and are thus heterozygous at this point. Using an in vivo xenograft model of lung cancer driven by mutant EGFR, this example evaluates the relative efficacy of TH-4000 and erlotinib at clinically achievable dose levels in two genetic backgrounds.

[0142] HCC827 and PC9 human non-small cell lung cancer (NSCLC) cells were prepared in culture. Then, 10 million cells / inoculum were injected subcutaneously into 5-8 week old female NIH-III nude mice (18-24 g). 3 HCC827 tumors were recruited and at ∼400 mm3 PC9 tumor recruitment. When tumors reached the desired volume, mice were randomly assigned to groups A–E and treated as detailed in Table 1 below:

[0143] Table 1:

[0144] Group Treatment A Program / Approach N A Solvent (β-cyclodextrin) Q3dx8,ip 5–6 B Erlotinib (10 mg / kg) Qdx21,po 5–6 C TH-4000 (30 mg / kg) Q3dx8,ip 5–6 D TH-4000 (15 mg / kg) Q3dx8,ip 5–6 E TH-4000 (7.5 mg / kg) Q3dx8,ip 5–6

[0145] TH-4000 was formulated according to Example 1. Erlotinib was prepared in a 5% DMSO solution, 40% PEG-400 in a 40% 2-hydroxypropyl-β-cyclodextrin solution. Body weight was recorded every 3 days or before administration, and the length and width of the subcutaneous tumor were measured every 3 days with an electronic caliper, and the volume was calculated according to the prolate ellipsoid formula (π / 6xlength x width x width). All other clinical signs were recorded.

[0146] The results are described below. When mice bearing established subcutaneous tumors were treated with erlotinib (10 mg / kg, po daily), it provided a mean blood exposure (AUC inf 20,000ng*h / ml), only tumors containing homozygous EGFR mutants were successfully controlled ( Figure 1-2 , Table 2-4).

[0147] Table 2 (corresponding to Figure 1 HCC827 tumors in

[0148]

[0149] Table 3 (corresponding to Figure 2 PC9 tumors in

[0150]

[0151] Table 4 (corresponding to Figure 2 PC9 tumors in

[0152]

[0153] TH-4000 is highly active in both homozygous and heterozygous tumor settings, compared to plasma concentrations of only 10%-20% of those achieved at the maximum safe dose of TH-4000 (150 mg / m2) in Phase I clinical trials. TH-4000 was evaluated for growth delay in HCC827 xenografts with 3 doses of 30, 15 and 7.5 mg / kg (q3d X8). At both dose levels, all HCC827 tumors regressed; 100% CR. Within 90 days, 92% of tumors failed to grow. Only mild weight loss (1.1%-2.5%) was observed in both TH-4000-treated groups.

[0154] The results provide evidence that in hypoxic tumor cells, hypoxia-dependent metabolism provides tumor-selective release of EGFR TKI. High local concentrations provide the necessary therapeutic index to silence wild-type EGFR signal transduction in hypoxic tumor compartments. The results also establish the efficacy of TH-4000 in NSCLC models containing exon 19 deletion mutations. Therefore, under plasma exposures that are easily achieved by human subjects, EGFR NSCLC can overcome hypoxia-induced resistance to erlotinib. In the xenograft model driven by WT EGFR, TH-4000 shows greater activity compared to erlotinib and afatinib, and under plasma exposures that are easily achieved by human subjects, TH-4000 has good activity in HER2-amplified xenograft models.

[0155] Example 3. Confirmation of the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of TH-4000 and TH-4000E in PC-9 tumors

[0156] Pharmacokinetic and pharmacodynamic studies were performed in NIH-III mice bearing PC-9 tumors when 15 mg / kg TH-4000 was administered ip. 6 Cells / mouse, a total of 42 mice). TH-4000 was prepared as above, and mice were euthanized and 3 tumors were excised at the following time points after TH-4000 administration: 0.5hrs., 3hrs., 12hrs., 24hrs., 48hrs., 72hrs., 96hrs., 120hrs., 144hrs and 168hrs. The intact tumors were snap frozen (with liquid nitrogen) and bio-crushed. Tumors were measured daily until collection to correlate growth kinetics with target shutdown. After bio-crushing, half of the bio-crushed tumors were processed to measure TH-4000 and TH-4000E concentrations using LC-MS / MS. The other half of the tumors were processed for protein extraction and Western blotting to determine pEGFR and total protein (EGFR, α-tubulin) levels.

[0157] For LC-MS / MS, 4 volumes of ice-cold acetonitrile (containing 0.5 μM D6-TH-4000 & TH-4000E) were added to the tumor sample. The sample was rotated at 13,000 rpm for 5 min, and 40 μL of supernatant was taken. To the supernatant, 80 μL of 0.01% FA-water was added, and the sample was mixed. 10 μL of the sample was injected for analysis. Mass spectrometry detection was completed using an Agilent 6410 triple quadrupole mass spectrometer equipped with a polymorphic ion (MMI) source. The mass spectrometer was operated in electrospray positive ion mode using multiple reaction detection (MRM). Agilent MassHunter software (v.4.04.00) was used for data acquisition and chromatographic peak integration.

[0158] For Western blot of tumor samples, collect the crushed tissue and quickly transfer it to an Eppendorf tube buried in dry ice. Once all samples are biologically crushed, the sample is weighed immediately, the tissue weight is recorded, and the tube is kept in dry ice. In a fume hood, add a volume of cold 1xLaemmli buffer + protease inhibitor (1:100 dilution), which corresponds to 10x sample weight, and the sample is transferred to wet ice. The sample is vortexed for 1 minute, and the dissociated tissue is visually dissolved. The sample is incubated for 20 minutes at 70°C, in an Eppendorf Thermomixer Compact at 300RPM shaking. By centrifugation at room temperature for 5 minutes at 13,000xg, any residual undissolved tissue is precipitated. 20ml aliquots are set aside for measuring protein concentration using Bio-Rad protein (Bradford) Assay. The remaining samples are frozen at -20°C. The results are listed in the table below.

[0159] Table 5 (corresponding to Figure 3 ).

[0160] PK parameters (TH-4000)±SE TH-4000-TKI±SE <![CDATA[C max obs (ng / mL)]]> 11717±1048 47±3.6 <![CDATA[C max pred (ng / mL)]]> 29797±74 50 <![CDATA[AUC 0-∞ (ng*h / mL)]]> 15960±13 106 <![CDATA[T 1 / 2 (h)]]> 0.37±0.00 1.28

[0161] Western blot was also used to detect the inhibition of EGFR autophosphorylation by erlotinib (0.1–10 uM). HCC827 and PC9 cell cultures were established from logarithmic phase passage flasks. Cells were seeded into 6-well plates at a density of 1 million cells / well and maintained under normoxic conditions or hypoxic conditions (1% O2) for 48 hours. Erlotinib drug stock was diluted with culture medium to achieve the final concentrations in each well (as indicated in the figure). After 3 hours of drug exposure, cells were rinsed with ice-cold PBS and lysed on ice for 30 minutes with radioimmunoprecipitation assay (RIPA) buffer containing sodium orthovanadate and sodium fluoride at a final concentration of 1 mM each. Cell debris was pelleted by centrifugation (13,000 rpm for 2 minutes). Protein concentration in each sample was determined using bicinchoninic acid (BCA) analysis. Equal amounts of protein (10 μg) were denatured (98°C for 5 minutes) and loaded onto 4-20% Tris-glycine protein gels for protein separation. After transfer to nitrocellulose membranes, each membrane was blocked with 5% bovine serum albumin (in Tris-buffered saline) for 1 hour. Anti-pEGFR (Y1068) primary antibody (rabbit, polyclonal) was added at a dilution of 1:2000 in 5% bovine serum albumin (containing Tris-buffered saline) overnight. Excess primary antibody in each membrane was washed off with Tris-buffered saline. Secondary antibody (goat anti-rabbit) was added to each membrane at a dilution of 1:5000 for 2 hours. For protein detection, the membrane was rinsed with Tris-buffered saline for 5 minutes to remove excess secondary antibody and added chemiluminescent substrate (Pierce Supersignal West Pico chemiluminescent substrate). The membrane was observed using a Fujifilm LAS 4000 imager. The in vitro evidence suggests a role for hypoxic exposure (1% O2) in resistance to erlotinib in a heterozygous PC-9 cell line (see 6 and 7).

[0162] A single dose of 15 mg / kg TH-4000 in nude mice achieved an efficacy equivalent to 32 mg / m in human subjects. 2 The maximum safe dose defined in the Phase I trial (MTD = 150 mg / m 2 / week; NCT01631279). TH-4000 is rapidly cleared from mouse plasma (T1 / 2=0.37h), but has a persistent residence in PC9 tumors (T1 / 2=39h), releasing TKI above effective levels for 7 days (T1 / 2β=84h). Consistent with the PK properties, the total shutdown of EGFR signaling was profound and persistent, with no recovery by day 7. To confirm the mechanism of action, we prepared chemical-biological tools (CBTs) that reflect the physicochemical properties of TH-4000, but are designed not to release TKI after 1e-reduction. Using a panel of human NSCLC cell lines, TH-4000 was efficiently metabolized under hypoxic conditions (TKI release rate 0.4-2.1nmol / hr / 10 6 cells), an O2-inhibited process (TKI < 0.002 nmol / hr / 10 6 cells). CBT was metabolically inert under all conditions. Cell antiproliferation and receptor phosphorylation assays showed that TH-4000 was 14-80-fold inactivated relative to the parent TKI, while CBT was inactivated regardless of oxygen status. In vivo, hyperbaric O2 breathing inhibited >80% of PC9 tumor TKI production derived from TH-4000 (538vs 99nmol / kg; p<0.01), while CBT did not release significant TKI (<1%). In summary, the data suggest that TH-4000 has a unique hypoxia activation mechanism, is active in a heterozygous model of mutant EGFR NSCLC, and can overcome hypoxia-induced resistance to erlotinib at plasma exposures readily achievable in human subjects.

[0163] Example 4. Demonstration of the efficacy of TH-4000 in combination with erlotinib for cancer treatment in PC-9 xenografts

[0164] To further test the therapeutic advantage of TH-4000 in a heterozygous (mutant / wild-type) tumor setting, PC9 xenografts were treated with erlotinib (10 mg / kg, oral daily) for 14 days before the introduction of weekly TH-4000 (15 mg / kg). In addition, PC9 xenografts were treated with erlotinib (10 mg / kg, oral daily) as a single agent or TH-4000 (3, 7.5 and 15 mg / kg, ip) as a single agent or with a combination of erlotinib and TH-4000 at the above doses and routes of administration.

[0165] TH-4000 and erlotinib were formulated as follows: TH-4000 in WFI containing 20% ​​2-hydroxypropyl-β-cyclodextrin; erlotinib in 40% 2-hydroxypropyl-β-cyclodextrin solution in 5% DMSO, 40% PEG-400. PC9 tumor cells were inoculated subcutaneously in 5-8wk female NIH-III nude mice (17-22g). Tumors grew to ~400mm 3 When the tumor is 400mm 3 At 4 hr, mice were randomly assigned to groups A–J and treated as follows:

[0166]

[0167]

[0168] The length and width of the subcutaneous tumor were measured with an electronic caliper every 3 days, and the volume was calculated according to the prolate ellipsoid formula (π / 6 x length x width x width). Body weight was recorded every 3 days or before administration. All other clinical signs were recorded.

[0169] Administration of TH-4000 provided immediate tumor shrinkage. In PC9 cells, TH-4000 efficacy was demonstrated at 3 doses (3, 7.5 & 15 mg / kg; qw X4). Tumor regression was observed for all mice administered 7.5 & 15 mg / kg TH-4000 ( Figure 11-12 For mice administered 3 mg / kg TH-4000, stable disease was achieved ( Fig.13 The reduction in PC9 tumor volume starting at day 14 following administration of 15 mg / kg TH-4000 was not associated with any signs of toxicity, as indicated by the absence of weight loss in the murine hosts ( Fig.14 ). However, over a period of dosing (21 days), erlotinib only achieved stable disease. While not being bound by theory, the observations are consistent with the necessity of silencing wild-type EGFR signaling in the hypoxic compartment of the tumor and mutant EGFR signaling in the tumor. They also show that in the setting of heterozygous EGFR tumors, TH-4000 provides a significant therapeutic index.

[0170] In addition, Western blot data comparing AZD9291 activity in homozygous or heterozygous cell lines under aerobic or hypoxic conditions ( Figure 6-8 ) is consistent with resistance to AZD9291 in the heterozygous EGFR setting in the presence of tumor hypoxia, as seen with erlotinib.

[0171] As shown above, TH-4000 provided significant shrinkage in erlotinib-resistant preclinical NSCLC models at doses that are readily achievable in human trials.

[0172] Example 5. Demonstration of TH-4000 efficacy in a HER2-amplified gastric cancer transplant model

[0173] The antitumor activities of erlotinib, afatinib, and TH-4000 were compared in A431 xenografts expressing WT EGFR at clinically relevant dose levels and schedules. NIH-III mice were administered 30 mg / kg TH-4000 (ip) to achieve an AUC of 60.2 ug*hr / mL, a dose equivalent to 104 mg / m in human subjects. 2 , for non-binding TH-4000. Daily oral administration of erlotinib or afatinib failed to prevent tumor progression. Only TH-4000 (30 mg / kg, ip) once a week (Qw x4) or twice a week (Q3d x8) was able to control WT EGFR-driven A431 tumor xenografts with 100% tumor regression. All treatments were well tolerated with <5% body weight change. Fig.15 and 16 TH-4000 was found to be active against HER2-positive NCI-N87 gastric tumor xenografts with minimal (<5%) body weight loss.

[0174] Example 6. Determination of the antiproliferative activity of various EGFR-targeting substances in FaDu cells

[0175] The goal of this study was to determine the sensitivity of FaDu cells to various EGFR-targeting agents after 5 days of exposure.

[0176] Evaluated substances

[0177] The substances evaluated in this study are as follows:

[0178]

[0179] Cell lines

[0180] This study focused on the human hypopharyngeal bone cell line FaDu (ATCC #HTB-43). FaDu cells were grown in αMEM medium supplemented with 5% FBS without routine use of antibiotics (P / S). Cultures were reestablished from validated frozen stocks in liquid nitrogen at >24 passages or >90 days in culture, whichever came first. Frozen cell stocks were confirmed to be mycoplasma-free using the PCR-ELIZA kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0181] Antiproliferation assay

[0182] FaDu cells were seeded (1500 cells / well in αMEM medium with 5% FCS) in 96-well plates and placed overnight under 21% O2. The cells were then treated with 10 serial dilutions (3-fold) of each test compound. The cells were grown in a humidified incubator (5% CO2, 37°C) for 5 days. The cells were then fixed with 40% trichloroacetic acid at 4°C for 1 h. The cells were then stained for Sulforhodamine B (SRB) expression. Cell density was determined using a Biotek ELx808 Absorbance Microplate Reader. The IC was derived by extrapolating the value required to reduce the cell density by 50%. 50 value.

[0183] In a 5-day antiproliferative assay, TH-4000E was 150-fold more potent than cetuximab. Thus, cetuximab-resistant head / neck cancers may retain sensitivity to TH-4000E. The antiproliferative activity of erlotinib was slightly superior to that of cetuximab (IC 50 The values ​​differed by 2-fold. Therefore, FaDu cells showed relative resistance to cetuximab and erlotinib. TH-4000E showed an 80-fold lower IC than erlotinib. 50 Thus, erlotinib-resistant cells can maintain sensitivity to TH-4000E. In FaDu cells exposed for 5 days, the antiproliferative activity of the prodrug TH-4000 was 13-fold lower than that of TH-4000E (effector), indicating inactivation of the prodrug relative to the effector.

[0184] Fig.17 In IC 50 Values ​​represent the concentration of substance required to reduce proliferation by 50% relative to untreated controls. 50 Values ​​are means ± standard errors of ≥3 independent experiments. FaDu cells are relatively resistant to the clinically approved EGFR inhibitors cetuximab and erlotinib. Despite resistance to cetuximab and erlotinib, FaDu cells showed significant sensitivity to TH-4000E. The prodrug TH-4000 is inactivated relative to TH-4000E.

[0185] Example 7. Antiproliferation assay of various EGFR-targeting substances in Fadu cells

[0186] The goal of this study was to determine how hypoxic exposure alters the sensitivity of FaDu cells to various EGFR-targeting agents.

[0187] Evaluated substances

[0188] The substances evaluated in this study are as follows:

[0189]

[0190] Cell lines

[0191] This study focused on the human hypopharyngeal bone cell line FaDu (ATCC #HTB-43). FaDu cells were grown in αMEM medium supplemented with 5% FBS without routine use of antibiotics (P / S). Cultures were reestablished from validated frozen stocks in liquid nitrogen at >24 passages or >90 days in culture (whichever came first). Frozen cell stocks were confirmed to be mycoplasma-free using the PCR-ELIZA kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0192] Antiproliferation assay

[0193] Monolayers of FaDu cells were harvested by trypsinization and counted using a hemocytometer. For aerobic assays, cells were seeded (1500 cells / well) in 96-well plates and placed in a humidified incubator (37°C / 5% CO2) for 2 hours. Cells were then treated with 10 serial dilutions (3-fold) of each test compound for 24 hours. For the anoxic plates, all supplies were pre-equilibrated for at least 72 hours in a H2 / palladium-catalyst anaerobic chamber (Bactron, Shellab) to remove any residual oxygen. On the day of the experiment, all cells were centrifuged (1000 rpm, 5 min) to pellet the cells. The culture medium was aspirated. The pellet was then moved to an anaerobic chamber and diluted with hypoxia-equilibrated culture medium (αMEM medium, 5% FCS, 0.2 mmol / L 2'-deoxycytidine, 10 mmol / L glucose) to achieve the appropriate cell density. Cells were seeded under conditions consistent with the aerobic plates. Once seeded, the cells were placed in a humidified incubator (37°C) in an anaerobic chamber for 2 hours. The anoxic plates were similarly treated with 10 serial dilutions (3-fold) of each test compound. 4 hours after drug addition, the anoxic plates were removed from the anaerobic chamber and placed in a humidified incubator (37°C / 5% CO2) for an additional 20 hours. After a total of 24 hours of drug exposure, the aerobic and anoxic plates were rinsed 3 times with fresh medium (αMEM+5% FCS+P / S). The cells were allowed to grow for a total of 5 days. Thereafter, the cells were fixed with 40% trichloroacetic acid for 1 hour at 4°C. The plates were then stained for Sulforhodamine B (SRB) expression. Cell density was determined using a Biotek ELx808 Absorbance Microplate Reader. The IC was derived by extrapolating the value required to reduce the cell density by 50%. 50 value.

[0194] Hypoxia reduces the sensitivity of FaDu cells to cetuximab. 50 The values ​​were 4-fold higher than those under aerobic conditions. Thus, hypoxic exposure appears to promote cetuximab resistance in FaDu cells, possibly due to increased intracellular retention of EGF receptors (membrane internalization). Under aerobic exposure conditions, the antiproliferative activity of erlotinib was slightly superior to that of cetuximab (IC 50 The erlotinib sensitivity of FaDu cells was not affected by hypoxia. After a period of hypoxia exposure, the erlotinib IC 50 The value is 13-fold lower than that of cetuximab, reflecting the change in sensitivity of cetuximab. Under hypoxic exposure conditions, TH-4000 is 3000 times more dose-effective than cetuximab (relative IC 50 TH-4000E is an irreversible HER1 / 2 / 4 inhibitor that is released from its homologous prodrug TH-4000 under hypoxic conditions. TH-4000E exhibits 1400-fold greater dose potency than erlotinib (based on IC 50 =values). Therefore, erlotinib-resistant cells can maintain sensitivity to TH-4000E. After a period of hypoxic exposure, TH-4000 was 244 times more dose-effective than erlotinib, based on IC 50 The results were determined by the change in the values ​​of TH-4000. Therefore, erlotinib-resistant head / neck cells can maintain sensitivity to TH-4000. TH-4000 is a hypoxia-activated prodrug of the irreversible HER1 / 2 / 4 inhibitor TH-4000E. In air, the antiproliferative activity of the effector (TH-4000E) is superior to that of the prodrug (TH-4000). Thus, the prodrug TH-4000 is inactivated 62-fold relative to its homologous effector TH-4000E. In FaDu cells, TH-4000 showed hypoxia-selective antiproliferative activity, with a 15-fold greater activity after 4 h of hypoxia.

[0195] for Fig.18 For the anoxic test, FaDu cells were inoculated under anaerobic conditions. Four hours after drug addition, the anoxic plates were removed from the anaerobic chamber and placed in a humidified incubator (37°C / 5% CO2) for another 20 hours. For the aerobic test, FaDu cells were inoculated and treated with drugs for 24 hours in a humidified incubator (37°C / 5% CO2). 50 The values ​​represent the concentration of substance required to reduce proliferation by 50% relative to the untreated control. 50 Values ​​are means ± SEM of ≥3 independent experiments.

[0196] Hypoxic conditions significantly reduced cetuximab sensitivity of FaDu cells. Hypoxic conditions did not alter the sensitivity of FaDu cells to erlotinib exposure. Hypoxic conditions significantly increased TH-4000 sensitivity in FaDu cells. TH-4000E was significantly more dose-effective than cetuximab or erlotinib in FaDu cells. TH-4000 showed a 62-fold inactivation ratio relative to TH-4000E. TH-4000 was 15-fold more dose-effective against FaDu cells after 4 hours in hypoxia vs aerobic conditions.

[0197] Example 8. Target Regulation of Various EGFR-Targeting Substances in FaDu Cells

[0198] The aim of this study was to determine the concentration dependence of various EGFR-targeting agents on the expression of EGFR and its downstream signal transducers pAkt and pERK.

[0199] Cell lines

[0200] The human hypopharyngeal bone cell line FaDu (ATCC #HTB-43) was used in this study. FaDu cells were grown in αMEM medium supplemented with 5% FBS without routine use of antibiotics (P / S). Cultures were reestablished from validated frozen stocks frozen in liquid nitrogen at >24 passages or >90 days in culture (whichever came first). Frozen cell stocks were confirmed to be mycoplasma-free using the PCR-ELIZA kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0201] Culture conditions and lysate preparation

[0202] FaDu cells (700k / well) were seeded on 6-well plates and left overnight. Then, the cells were incubated with gradient concentrations of TH-4000, TH-4000E or cetuximab (3-fold dilution) for 1 h. Then, the cells were stimulated with 50 ng / mL EGF (Sigma #E9644) for 15 mins.

[0203] Lysate preparation for western blotting

[0204] The cells were rinsed once with ice-cold PBS. Lysates were prepared by incubating the cells with modified RIPA buffer (RIPA [50mmol / L Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mmol / L NaCl, 1.0mmol / L EDTA] + 1.0mmol / L Na3VO4 + 1.0mmol / L NaF + 1x protease inhibitor cocktail (Sigma # P8340)) on ice for 30min. After clarification, BCA analysis was performed to determine the protein concentration of the sample. Absorbance readings were performed at 550nm.

[0205] Western blotting

[0206] Cell lysates were resolved by gel electrophoresis and immunoblotted for various members of the EGFR signaling axis. Each loading volume included 10 μg of protein and 25% SDS sample buffer (Life Technologies # NP0007). Samples were loaded onto NuPAGE 4-12% Bis-Tris 15well gels (Life Technologies # NP0336) and run at 120V in MES running buffer (Life Technologies # NP0002) for approximately 2 hours or until the sample had run off the gel (whichever came first). Samples were then transferred at 100V (transfer buffer: 25mM Tris, 192mM glycine, 20% methanol) for 80mins onto nitrocellulose membranes. Blots were blocked with 5% BSA or 5% phosphoblocker (CellBiolabs # AKR-104) in TBS-T for at least 1 hour. The primary and secondary antibodies used are summarized in Tables 6 & 7. Briefly, blots were incubated with primary antibodies overnight at 4°C. After 3 washes with TBS-T for 5 min each, blots were incubated with appropriate HRP-conjugated secondary antibodies for at least 2 h at room temperature. In all cases, blots were imaged on Fijifilm LAS 4000 using Supersignal West PicoChemiluminescent substrate (Life Technologies, #34080). Because the target proteins (e.g., pERK and pAkt) had similar band sizes to β-actin (control), bound antibodies were removed by incubating the blots with Restore protein blot regeneration buffer (Thermofisher Scientific #21059) for 15 mins. Then, after a single wash in TBS-T, blots were probed for β-actin or α-tubulin.

[0207] Table 6: Blocking conditions and primary antibodies

[0208]

[0209]

[0210] Table 7: Secondary Antibodies

[0211] Secondary Antibodies Source (Catalog Number) dilution Goat anti-mouse IgG-HRP Santa Cruz (#J2212) 1:5000 Goat anti-rabbit IgG-HRP Santa Cruz (#H2112) 1:5000

[0212] TH-4000E is superior to cetuximab in inhibiting EGFR signal transduction. At concentrations of 0.12 μM or greater, TH-4000E completely silences pEGFR (Y1068) and pERK expression. Similarly, at concentrations exceeding 0.12 μM, pAkt expression is largely silenced. At concentrations of 1.1 μM or greater, cetuximab partially silences pEGFR expression, but this has minimal effect on downstream signal transduction through pAkt and pERK. Exposure to up to 10 μM cetuximab cannot silence pAkt and pERK activity. Judging from the inhibition of EGFR pathway signal transduction, TH-4000E is at least 250 times more effective than cetuximab. In terms of inhibiting EGFR signal transduction, TH-4000E (the effector of TH-4000) is superior to TH-4000 (the prodrug). TH-4000 completely silenced pEGFR expression up to 1.1 μM (equivalent to 0.04 μM TH-4000E).

[0213] At 0.37 μM, pAkt expression was completely silenced. TH-4000E was approximately 28 times more dose-effective than TH-4000, as judged by inhibition of EGFR pathway signaling.

[0214] TH-4000E is at least 250-fold more dose-effective than cetuximab. TH-4000 is less potent than TH-400E in inhibiting EGFR signaling, consistent with its mechanism of action.

[0215] Example 9. Effect of hypoxia on TH-4000E sensitivity in FaDu cells

[0216] The goal of this study was to determine how hypoxic preconditioning alters TH-4000E sensitivity in FaDu cells.

[0217] Cell lines

[0218] The human hypopharyngeal bone cell line FaDu (ATCC #HTB-43) was utilized in this study. FaDu cells were grown in αMEM medium supplemented with 5% FBS without routine use of antibiotics (P / S). Cultures were reestablished from validated frozen stocks in liquid nitrogen at >24 passages or >90 days in culture (whichever came first). Frozen cell stocks were confirmed to be mycoplasma-free using the PCR-ELIZA kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0219] Culture conditions and lysate preparation

[0220] FaDu cells were seeded (500k / well) on 6-well plates for 48 hours under 0.1% O2 and 21% O2. After 48h hypoxic pretreatment, cells were incubated with various concentrations of TH-4000E for 1h. Then, cells were stimulated with 50ng / mL EGF (Sigma #E9644) for 15mins.

[0221] Lysate preparation for western blotting

[0222] After the specified period of hypoxia exposure, the cells were rinsed once with ice-cold PBS. Lysates were prepared by incubating the cells with modified RIPA buffer (RIPA [50mmol / L Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mmol / L NaCl, 1.0mmol / L EDTA] + 1.0mmol / L Na3VO4 + 1.0mmol / L NaF + 1x protease inhibitor cocktail (Sigma # P8340)) on ice for 30min. After clarification, BCA analysis was performed to determine the protein concentration of the samples. Absorbance readings were performed at 550nm.

[0223] Western blotting

[0224] Cell lysates were resolved by gel electrophoresis and immunoblotted for various members of the EGFR signaling axis. Each loading volume included 10 μg of protein and 25% LDS sample buffer (Life Technologies # NP0007). Samples were loaded onto NuPAGE 4-12% Bis-Tris 15well gels (Life Technologies # NP0336) and run at 120V in MES running buffer (Life Technologies # NP0002) for approximately 2 hours or until the sample had run off the gel (whichever came first). Samples were then transferred at 100V (transfer buffer: 25mM Tris, 192mM glycine, 20% methanol) for 80mins onto nitrocellulose membranes. Blots were blocked with 5% BSA or 5% phosphoblocker (CellBiolabs # AKR-104) in TBS-T for at least 1 hour. The primary and secondary antibodies used have been summarized in Tables 8 & 9. Briefly, blots were incubated with primary antibodies overnight at 4°C. After 3 washes with TBS-T for 5 min each, blots were incubated with appropriate HRP-conjugated secondary antibodies for at least 2 h at room temperature. In all cases, blots were imaged on Fijifilm LAS 4000 using Supersignal West PicoChemiluminescent substrate (Life Technologies, #34080). Because the target proteins (e.g., pERK and pAkt) had similar band sizes to β-actin (control), bound antibodies were removed by incubating the blots with Restore protein blot regeneration buffer (Thermofisher Scientific #21059) for 15 mins. Then, after a single wash in TBS-T, the blots were probed for β-actin or α-tubulin.

[0225] Table 8: Blocking conditions and primary antibodies

[0226]

[0227] Table 9: Secondary Antibodies

[0228] Secondary Antibodies Source (Catalog Number) dilution Goat anti-mouse IgG-HRP Santa Cruz (#J2212) 1:5000 Goat anti-rabbit IgG-HRP Santa Cruz (#H2112) 1:5000

[0229] TH-4000E sensitivity was comparable for aerobic and hypoxic pre-treated cells. Almost complete silencing of EGFR signaling was observed over the range of TH-4000E concentrations used (10.0-0.37 umol / L).

[0230] Hypoxic pretreatment did not alter TH-4000E sensitivity at the dilutions of TH-4000E used.

[0231] Example 10. Kinetics of EGFR Induction

[0232] The goal of this study was to determine how hypoxia alters EGFR expression in the human hypopharyngeal bone cell line FaDu.

[0233] Cell lines

[0234] The human hypopharyngeal bone cell line FaDu (ATCC #HTB-43) was utilized in this study. FaDu cells were grown in αMEM medium supplemented with 5% FBS without routine use of antibiotics (P / S). Cultures were reestablished from validated frozen stocks in liquid nitrogen at >24 passages or >90 days in culture (whichever came first). Frozen cell stocks were confirmed to be mycoplasma-free using the PCR-ELIZA kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0235] Culture conditions

[0236] FaDu was inoculated (700 k / ) in T25 bottles and placed overnight at 21% O2. The culture medium (αMEM + 5% FCS) was aspirated and the bottles were placed in a H2 / palladium-catalyst hypoxic culture chamber (Bactron, Shellab). Fresh hypoxia-equilibrated culture medium (αMEM + 5% FCS + P / S + 1% additive) was added to each bottle. Cells were exposed to hypoxia for different periods with and without aerobic recovery (Table 10).

[0237] Table 10: FaDu culture conditions

[0238]

[0239]

[0240] Lysate preparation for western blotting

[0241] After the specified period of hypoxia exposure, the cells were rinsed once with ice-cold PBS. Lysates were prepared by incubating the cells with modified RIPA buffer (RIPA [50mmol / L Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mmol / L NaCl, 1.0mmol / L EDTA] + 1.0mmol / L Na3VO4 + 1.0mmol / L NaF + 1x protease inhibitor cocktail (Sigma # P8340)) on ice for 30min. After clarification, BCA analysis was performed to determine the protein concentration of the samples. Absorbance readings were performed at 550nm.

[0242] Western blotting

[0243] Cell lysates were resolved by gel electrophoresis and immunoblotted for various members of the EGFR signaling axis. Each loading volume included 10 μg of protein and 25% LDS sample buffer (Life Technologies # NP0007). Samples were loaded onto NuPAGE 4-12% Bis-Tris 15well gels (Life Technologies # NP0336) and run at 120V in MES running buffer (Life Technologies # NP0002) for approximately 2 hours or until the sample had run off the gel (whichever came first). Samples were then transferred at 100V (transfer buffer: 25mM Tris, 192mM glycine, 20% methanol) for 80mins onto nitrocellulose membranes. Blots were blocked with 5% phosphoblocker or 5% BSA (CellBiolabs # AKR-104) in TBS-T for at least 1 hour. The primary and secondary antibodies used have been summarized in Tables 11 & 12. Briefly, the blots were incubated with primary antibodies overnight at 4°C. After rinsing with TBS-T 3 times for 5 min each, the blots were incubated with the appropriate HRP-conjugated secondary antibodies for at least 2 h at room temperature. In all cases, the blots were imaged on Fijifilm LAS 4000 using Supersignal West PicoChemiluminescent substrate (Life Technologies, #34080). Because the target proteins (e.g., pERK and pAkt) have similar band sizes to actin (control), bound antibodies were removed by incubating the blots with 0.2M NaOH for 5 mins. Then, after blocking with 5% BSA in TBS-T for at least 1 h, the blots were probed for actin.

[0244] Table 11: Blocking conditions and primary antibodies

[0245]

[0246] Table 12: Secondary Antibodies

[0247] Secondary Antibodies Source (Catalog Number) dilution Goat anti-mouse IgG-HRP Santa Cruz (#J2212) 1:5000 Goat anti-rabbit IgG-HRP Santa Cruz (#H2112) 1:5000

[0248] Table 13: Gel running order

[0249]

[0250]

[0251] Total EGFR (tEGFR) expression was unchanged by 14 hours of hypoxia. Phospho-EGFR (Y1092) levels increased in FaDu cells during hypoxia. Phospho-EGFR (Y1092) levels remained elevated in FaDu cells 5 hours after re-exposure to normoxia.

[0252] Under hypoxic culture conditions, transient induction of pEGFR(Y1092) was observed in FaDu cells. After re-exposure to normoxia, pEGFR(Y1092) levels were sustainable for at least 5 hours.

[0253] Example 11: TH-4000 in combination with cetuximab vs afatinib in combination with cetuximab in wild-type EGFR-driven H125 non-small cell lung cancer xenografts

[0254] The primary objectives of this example were to compare the efficacy of weekly single agent tasotinib with the combination of tasotinib / cetuximab in H125 xenografts, and to compare the efficacy of tasotinib / cetuximab with afatinib / cetuximab in H125 xenografts, and to evaluate the toxicity of the combination of tasotinib / cetuximab compared with afatinib / cetuximab.

[0255] All substances were administered at human PK equivalents. In WT EGFR-driven H125 xenografts, the combination of tasotinib + cetuximab was significantly more effective than the combination of afatinib + cetuximab (ORR was 67% and 37%, respectively). For tasotinib, cetuximab and afatinib, the ORRs of single substances were 29%, 17% and 0%, respectively (Table 3). Tumor growth inhibition (TGI%; 27 days) analysis showed that the combination of tasotinib / cetuximab was the most effective (TGI = 81%), followed by tasotinib alone (TGI = 69%). The combination of afatinib / cetuximab showed moderate activity (TGI = 67%). The single substance treatment groups of afatinib and cetuximab had poor results (Table 1). For all treatment groups, no significant weight loss was recorded.

[0256] Test parameters

[0257]

[0258]

[0259] method

[0260] NIH-III mice bearing H125 tumors were randomly assigned to one of the following treatment groups:

[0261] Group A: vehicle (WFI containing 20% ​​2-hydroxypropyl-β-cyclodextrin), ip, qw x6

[0262] Group B: Tasotinib, 48 mg / kg, qw x6

[0263] Group C: Cetuximab, 0.25 mg / mouse, q3d x14

[0264] Group D: Tasotinib, 48 mg / kg, qw x6, combined with cetuximab, 0.25 mg / mouse, q3d x14

[0265] Group E: Afatinib, 6 mg / kg, qd x42

[0266] Group F: Afatinib, 6 mg / kg, qd x42, combined with cetuximab, 0.25 mg / mouse, q3d x14

[0267] All doses and treatment regimens were based on the best available information for clinically relevant doses and regimens for each substance. The human equivalent doses of tasotinib and afatinib were determined in-house in NIH-III nude mice after adjustment for plasma protein binding in both species. A dose of 48 mg / kg tasotinib in mice (NIH-III) produced a human equivalent of 150 mg / m 2 The free drug plasma exposure (Investigator's Manual TH-CR-601, Threshold Pharmaceuticals, INC) was determined and administered once a week. The 6 mg / kg afatinib dose in mice was found to be equivalent to 40 mg / kg in humans (Wind et al., 2013, Clin Pharmacokinet; 52: 1101–1109), and was administered orally daily. The cetuximab dose used was based on the findings of Luo et al. (2005, Cancer Chemother Pharmacol; 56[5]: 455), in which a dose of 0.25 mg per mouse was equivalent to a clinically available dose of cetuximab (with observed target saturation). Administration in mice was q3d to correct for the shorter elimination half-life of mice relative to human subjects (40-42 vs 114 hr, respectively).

[0268] The combination of tasotinib and cetuximab was more effective than single agent treatment. The combination produced the strongest antitumor activity compared to all other treatment groups, with 4 / 6 (67%) evaluable tumors achieving PR and 2 / 6 SD.

[0269] The combination of afatinib / cetuximab was more effective than either afatinib or cetuximab alone. 3 / 8 tumors (38%) treated with the combination of afatinib / cetuximab achieved PR, whereas with single agent afatinib, none achieved SD or PR.

[0270] The combination of tasotinib / cetuximab produced 81% tumor growth inhibition (TGI %, Day 27) relative to vehicle-treated controls, while the combination of afatinib / cetuximab produced 67% TGI.

[0271] Body weight loss was <5% in all treatment groups.Apart from one case of diarrhea in one mouse treated with the combination of afatinib / cetuximab (day 13), which resolved within 48 hours, no other signs of toxicity were observed.

[0272] In patients with EGFR-mutation-positive NSCLC with acquired resistance to first-line TKIs, a phase 1b evaluation of afatinib and cetuximab showed that the dose intensity achieved with dual inhibition of EGFR with afatinib / cetuximab was associated with mechanistically 3 / 4 toxicities such as diarrhea and rash (Janjigian et al., 2014, Can Discov, 4: 10). It is assumed that in the described setting, the combination of tasotinib and cetuximab may provide the dose intensity required for adequate inhibition of ErbB signaling and maintaining the therapeutic window. This study was conducted to evaluate the therapeutic potential of tasotinib + cetuximab relative to the combination of afatinib and cetuximab in a wild-type (WT) EGFR-driven NSCLC model.

[0273] In WT EGFR driven H125 xenografts, the combination of tasotinib / cetuximab was significantly more effective than the combination of afatinib / cetuximab. The combination of tasotinib and cetuximab was well tolerated and was not associated with any significant toxicity.

[0274] Table 14: Tumor growth inhibition of H125 tumors (compared to control) on day 27 after treatment with 48 mg / kg tasotinib (qw), 0.25 mg / mouse cetuximab (q3d), the combination of tasotinib & cetuximab, 6 mg / kg afatinib (qd), and the combination of afatinib & cetuximab

[0275]

[0276]

[0277] Table 15: Research results

[0278]

[0279] Note / Abbreviation:

[0280] Solvent: 20% 2-hydroxypropyl-β-cyclodextrin (Sigma Aldrich, lot MKBG9088V), dissolved in water for injection (DEMO SA Pharmaceutical Industry, lot 105467).

[0281] Deaths not related to drug toxicity were excluded before RTV was reached 4 And selected mice (for example, mice selected early because of grade II ulcers).

[0282] Cause of death: Drug toxicity related: a – death found, b – evidence of morbidity, c – evidence of treatment-related adverse effects, d – excessive weight loss (>20% from starting size).

[0283] Unrelated to drug toxicity: e – irregular tumor growth, f – tumor ulceration grade III, g – tumor metastasis, h – dose abnormality, i – treatment-unrelated disease, j – animal husbandry problem

[0284] Table 16: Tumor Growth Response Measured by RECIST Criteria

[0285]

[0286] Note / Abbreviation:

[0287] PD – progressive disease: <50% regression from starting volume throughout the study period and >25% increase in starting volume by the end of dosing

[0288] SD – Stable Disease: <50% regression from starting volume throughout the study period and ≤25% increase in starting volume by the end of dosing

[0289] PR – partial response: ≥50% regression from starting volume at any time point throughout the study period, but with measurable tumor (≥25 mm 3 )

[0290] CR – Complete response: disappearance of measurable tumor volume (<25 mm) at any time point during the study period. 3 ).

[0291] R% - Response rate: percentage of evaluable tumors with a partial (PR) or complete (CR) response.

[0292] *The sample size used to evaluate tumor response includes all mice that completed the dosing regimen or reached the tumor endpoint before dosing was completed. All mice culled before completion of dosing due to drug toxicity, tumor ulceration, or dosing aberrations were excluded from the tumor response analysis.

[0293] Example 12. Phase I Clinical Trial of TH-4000 in Human Patients

[0294] This example describes an open-label, multicenter Phase I trial administering TH-4000 to cancer patients.

[0295] Preparation and administration of TH-4000

[0296] TH-4000 was prepared for infusion in glass bottles and administered intravenously over 1 hour via an infusion pump. The starting dose (Level 1) was 10 mg / m 2 x8. A modified accelerated titration design (Simon et al., JNCI, 89: 1138-47, incorporated herein by reference) was used. At the starting dose level, 27 patients were enrolled. The dose was increased by 100% across the continuous levels until the first dose-limiting toxicity (DLT) or grade 2 toxicity clearly unrelated to disease progression, intercurrent illness, concurrent medication, or other non-drug intervention occurred during cycle 1. DLT was defined as:

[0297] Level 4 at 200 mg / m 2 Patients with QTc prolongation (DLT)

[0298] Level 3 at 200 mg / m 2 Patient facial pain (DLT)

[0299] Level 3 at 200 mg / m 2 Patients with vestibular disorders

[0300] The medical monitor and the principal investigator reviewed and discussed available relevant safety and PK data before proceeding to the next dose level.

[0301] Study Group

[0302] This study enrolled 27 patients (all comers) with advanced solid tumors. The primary objective of this study was to determine the MTD and DLTs. Secondary objectives included PK, safety, and efficacy studies. The MTD was established at 150 mg / m 2 / w.

[0303] Treatment period:

[0304] For treatment, patients were given an initial dose of 10 mg / m by IV over 1 hour.2 , once a week. Disease assessment was performed at week 6 and then every 8 weeks. PK analysis of TH-4000 and TH-4000E was evaluated in cycles 1 and 2.

[0305] Gradually increase the dose

[0306] A traditional 3+3 dose escalation design (≤100%) was implemented. Each of the consecutive groups of participants (4-6 participants / group) was started at a fixed dose. For the first two groups, real-time PK was studied.

[0307] result

[0308] In 15 patients, ≤80 mg / m 2 Administration of TH-4000 (1h infusion) did not produce acneiform dermatitis or pruritus (0 / 15); and resulted in low incidences of grade 1 / 2 maculopapular rash (4 / 15 patients) and diarrhea (1 / 15 patient). Compared with afatinib administered at 40mg / d, 80mg / m 2 Plasma exposures at doses produced AUC0-inf≈daily for 5 consecutive weeks. Systemic TH-4000-E exposures were <1% of TH-4000 (median 0.88%; range 0.29%-2.4%). The toxicokinetic relationship is consistent with systemic inactivation of TH-4000 in humans.

[0309] TH-4000 showed tumor-selective dose enhancement of TH-4000E (e.g., >8-fold increase in AUC), with a tumor half-life of ≈2.5 days and p-EGFR shutdown of greater than 4 days, which is better than afatinib, which had p-EGFR shutdown of less than 6 h. Thus, TH-4000 showed an advantage over other treatments in having irreversible inhibition of HIF2α-driven wild-type EGFR and wild-type EGFR hidden in endocytic vesicles in a hypoxic environment.

[0310] Example 13: TH-4000 in patients with EGFR-mutant, T790M-negative, advanced non-small cell lung cancer who have worsened on EGFR tyrosine kinase inhibitors

[0311] This example describes a single-arm, open-label, multicenter, two-stage, Phase II trial in which TH-4000 was administered to patients with GFR-mutant, T790M-negative, advanced non-small cell lung cancer.

[0312] Preparation and administration of TH-4000

[0313] TH-4000 is filled frozen (-20 ± 5 ° C) into 20 mL glass bottles containing a minimum of 120 mg TH-4000 in at least 10 mL of a sterile, preservative-free solution at pH 3-4 containing 40% 2-hydroxypropyl-β-cyclodextrin, 20 mM citrate. The concentration of TH-4000 is 12 mg / mL. Before use, TH-4000 vials are thawed on the shelf at room temperature. Once thawed, dilute the TH-4000 vials into 5% dextrose water (D5W) within 12 hours. D5W used to dilute TH-4000 can be obtained from commercial sources and stored at room temperature. The final TH-4000 concentration of the prepared TH-4000 / D5W solution is approximately 1 mg / mL.

[0314] TH-4000 infusion was prepared in a glass bottle and administered intravenously via an infusion pump over 1 hour. The starting dose was 150 mg / m2 on Days 1, 8, 15, and 22 of each 28-day cycle. 2 , until progressive disease (PD) or unacceptable toxicity. This study utilized the optimal Simon two-phase design (Simon et al., JNCI, 89: 1138-47, incorporated herein by reference). Dose adjustment rules were based on toxicities that were considered to be potentially related to TH-4000. Prior to serial dose administration of TH-4000, patients who had experienced one of the following treatment-related toxicities with the previous dose must meet the following criteria:

[0315] Grade 1 or lower QTcF interval prolongation;

[0316] Rash of Grade 2 or lower; and

[0317] Gastrointestinal (GI) toxicity of grade 2 or less.

[0318] Any other clinically significant TH-4000-related toxicity must have resolved to at least Grade 1.

[0319] In the event that TH-4000 administration was delayed because of TH-4000-related toxicity, patients could receive subsequent TH-4000 administrations at a dose reduced by 25% to 50% from the previous dose.

[0320] Study Group

[0321] In this study, up to 37 patients with epidermal growth factor (EGFR)-mutant, T790M-negative, advanced non-small cell lung cancer (NSCLC) (all comers) will be enrolled. The primary objective of this study shall be to evaluate the antitumor activity of TH-4000, as determined by the response rate in patients with EGFR-mutant, T790M-negative, advanced NSCLC that have progressed on EGFR TKIs. Secondary objectives of this study include evaluating the safety and tolerability of TH-4000 in the study population; evaluating further measures of TH-4000 antitumor activity, including duration of response (DOR), progression-free survival (PFS), and overall survival (OS); studying the pharmacokinetics (PK) of TH-4000 (prodrug) and TH-4000E (TKI effector) in the study population, and exploring potential PK / pharmacodynamic relationships; and evaluating whether there is a relationship between the plasma exposure of TH-4000 and its active metabolite TH-4000E, and the effects on cardiac repolarization.

[0322] Study duration:

[0323] This trial is defined as follows: 3 phases: screening, treatment, and follow-up. The estimated total duration of the active portion of the study for each patient is approximately 30 weeks, as follows:

[0324] Screening period - 28 days

[0325] Screening and baseline procedures may be up to 28 days prior to the first scheduled dose.

[0326] Treatment period: Week 1–26 (26 weeks)

[0327] After the last dose of study drug, an average of six 28-day treatment periods (24 weeks) were followed by a 2-week end-of-study treatment visit. Patients continued study treatment until they had significant treatment-related toxicity and evidence of progressive disease. The accrual period was approximately 12 months.

[0328] Treatment period:

[0329] For treatment, patients were given an initial dose of 150 mg / m by IV over 1 hour. 2, administered on days 1, 8, 15, and 22 of each 28-day cycle until progressive disease (PD) or unacceptable toxicity. The potential prediction of TH-4000 clinical activity was evaluated based on hypoxic PET imaging (e.g., hypoxic tumor volume and hypoxic fraction of available locations only) and analysis from tumor tissue, circulating tumor cells, or plasma (e.g., WT: mutant EGFR allele ratio; EGFR / pEGFR and HER2 / pHER2 protein expression; EGFR and HER2 copy number acquisition; Veristrat plasma analysis for likelihood of response to EGFR TKIs; and / or identification of other known molecular abnormalities associated with TKI resistance (e.g., MET amplification or KRAS mutations).

[0330] Plasma TH-4000 and TH-4000E pharmacokinetic samples were collected from patients before dosing, 30 minutes after the start of infusion, at the end of infusion, 30 minutes after the end of infusion, and 1h, 2h, 3h, 5h, and 24h after the end of infusion on day 1 of cycle 1. The concentrations of TH-4000 and TH-4000E, as well as the following parameters, were reported: the maximum observed time to peak plasma concentration (Tmax); plasma concentration (Cmax), the time to peak); the magnitude of the linear regression slope of logarithmic concentration (Tmax), the terminal clearance constant versus time (Kel),); terminal half-life (T1 / 2); area under the concentration-time curve (AUC), the area under the curve from Hr0 to the time of the final measurable concentration (AUClast),); area under the curve (AUC); clearance (CL), the volume of distribution in the post-distribution phase (cl); the volume of distribution at steady state (Vss); the apparent volume of distribution in the post-distribution phase (Vβ) exploration.

[0331] Follow-up: Up to 1 year after first dose

[0332] Patients will be contacted approximately every 3 months regarding survival and follow-up information after the first dose, for up to 12 months, until death, loss to follow-up, or study discontinuation.

[0333] Example 14: TH-4000 in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or skin

[0334] This example describes an open-label, multicenter, two-stage, Phase II trial administering TH-4000 to patients with recurrent or metastatic squamous cell carcinoma of the head and neck or skin.

[0335] TH-4000 Preparation and Administration

[0336] TH-4000 is filled frozen (-20 ± 5 ° C) into 20 mL glass bottles, which contain a minimum of 120-150 mg TH-4000 in at least 10 mL of a sterile, preservative-free solution at pH 3-4 containing 40% 2-hydroxypropyl-β-cyclodextrin, 20 mM citrate. The concentration of TH-4000 is 12 mg / mL. Before use, TH-4000 vials are thawed on the shelf at room temperature. Once thawed, dilute the TH-4000 vials into 5% dextrose water (D5W) within 12 hours. D5W used to dilute TH-4000 can be obtained from commercial sources and stored at room temperature. The final TH-4000 concentration of the prepared TH-4000 / D5W solution is approximately 1 mg / mL.

[0337] TH-4000 infusion was prepared in a glass bottle and administered intravenously via an infusion pump over 1 hour. The starting dose was 150 mg / m2 on Days 1, 8, 15, and 22 of each 28-day cycle. 2 , until progressive disease (PD) or unacceptable toxicity. This study utilized the optimal Simon two-phase design (Simon et al., JNCI, 89: 1138-47, incorporated herein by reference). Dose adjustment rules were based on toxicities that were considered to be potentially related to TH-4000. Prior to serial dose administration of TH-4000, patients who had experienced one of the following treatment-related toxicities with the previous dose must meet the following criteria:

[0338] Grade 1 or lower QTcF interval prolongation;

[0339] Rash of Grade 2 or lower; and

[0340] Gastrointestinal (GI) toxicity of grade 2 or less.

[0341] Any other clinically significant TH-4000-related toxicity must have resolved to at least Grade 1.

[0342] In the event that TH-4000 administration was delayed because of TH-4000-related toxicity, patients could receive subsequent TH-4000 administrations at a dose reduction of 25% to 50% from the previous dose.

[0343] Study Group

[0344] In this study, 30 patients with recurrent or metastatic squamous cell carcinoma of the head and neck or skin will be enrolled. The primary objective of this study shall be to evaluate the antitumor activity of TH-4000, as measured by response rate in patients with recurrent and / or metastatic squamous cell carcinoma of the head and neck or skin. Secondary objectives include evaluating the safety and tolerability of TH-4000 in the study population; evaluating further measures of TH-4000 antitumor activity, including duration of response (DOR), progression-free survival (PFS), and overall survival (OS); studying the pharmacokinetics (PK) of TH-4000 (prodrug) and TH-4000E (TKI effector) in this study population, and exploring potential PK / pharmacodynamic relationships; and evaluating whether there is a relationship between the plasma exposure of TH-4000 and its active metabolite TH-4000E, and the effect on cardiac repolarization.

[0345] Study duration:

[0346] This trial is defined as follows: 3 phases: screening, treatment, and follow-up. The estimated total duration of the active portion of the study for each patient is approximately 30 weeks, as follows:

[0347] Screening period - 28 days

[0348] Screening and baseline procedures may be up to 28 days prior to the first scheduled dose.

[0349] Treatment period: Week 1–26 (26 weeks)

[0350] After the last dose of study drug, an average of six 28-day treatment periods (24 weeks) were followed by a 2-week end-of-study treatment visit. Patients continued study treatment until they had significant treatment-related toxicity and evidence of progressive disease. The accrual period was approximately 12 months.

[0351] Treatment period:

[0352] For treatment, patients were given an initial dose of 150 mg / m by IV over 1 hour. 2 , administered on days 1, 8, 15, and 22 of each 28-day cycle until progressive disease (PD) or unacceptable toxicity. The potential prediction of TH-4000 clinical activity was evaluated based on hypoxic PET imaging (e.g., hypoxic tumor volume and hypoxic fraction of available locations only) and analysis from tumor tissue, circulating tumor cells, or plasma (e.g., WT: mutant EGFR allele ratio; EGFR / pEGFR and HER2 / pHER2 protein expression; EGFR and HER2 copy number acquisition; Veristrat plasma analysis for likelihood of response to EGFR TKIs; and / or identification of other known molecular abnormalities associated with TKI resistance (e.g., MET amplification or KRAS mutations).

[0353] Plasma pharmacokinetic samples of TH-4000 and TH-4000E were collected from patients before dosing, 30 minutes after the start of infusion, at the end of infusion, 30 minutes after the end of infusion, and 1h, 2h, 3h, 5h, and 24h after the end of infusion on day 1 of cycle 1. The concentrations of TH-4000 and TH-4000E, as well as the following parameters, were reported: the maximum observed time to peak plasma concentration (Tmax); plasma concentration (Cmax), the time to peak); the magnitude of the linear regression slope of logarithmic concentration (Tmax), the terminal clearance constant vs time (Kel),); terminal half-life (T1 / 2); area under the concentration-time curve (AUC), the area under the curve from Hr0 to the time of the final measurable concentration (AUClast),); area under the curve (AUC); clearance (CL), the volume of distribution in the post-distribution phase (cl); the volume of distribution at steady state (Vss); the apparent volume of distribution in the post-distribution phase (Vβ) exploration.

[0354] Follow-up: Up to 1 year after first dose

[0355] Patients will be contacted approximately every 3 months regarding survival and follow-up information after the first dose, for up to 12 months, until death, loss to follow-up, or study discontinuation.

[0356] Although the present invention has been described with reference to its specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present invention. In addition, some modifications may be made to adapt to specific circumstances, materials, material compositions, methods, method steps, to obtain the benefits provided by the present invention without departing from the scope of the present invention. All such modifications are contemplated within the scope of the claims appended hereto.

[0357] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended to indicate that any such document is relevant prior art, nor does it constitute any acceptance of the contents or date of such documents.

[0358] Additional embodiments

[0359] 1. A pharmaceutically acceptable preparation comprising TH-4000 or a pharmaceutically acceptable salt or solvate thereof and 2-hydroxypropyl-β-cyclodextrin.

[0360] 2. A unit dose of the formulation of embodiment 1.

[0361] 3. A unit dose of a pharmaceutically acceptable formulation comprising about 75 mg - about 150 mg TH-4000.

[0362] 4. A method for treating cancer, the method comprising administering to a patient in need of such treatment a therapeutically effective dose of about 5 mg / m 2 – About 350 mg / m 2 TH-4000.

[0363] 5. The method of embodiment 4, wherein the amount of TH-4000 administered is about 75 mg / m 2 – About 150 mg / m 2 .

[0364] 6. The method of embodiment 4 or 5, wherein TH-4000 is administered as a single substance therapy.

[0365] 7. The method according to any one of embodiments 4-6, wherein TH-4000 is administered intravenously.

[0366] 8. The method of any one of embodiments 4-7, wherein TH-4000 is administered at a frequency of at least once a day to once a month.

[0367] 9. The method according to any one of embodiments 4-8, wherein TH-4000 is administered at a frequency of at least once a week.

[0368] 10. The method of any one of embodiments 4-9, wherein TH-4000 is administered for a period of about 1 week to about 60 weeks.

[0369] 11. The method of any one of embodiments 4-10, wherein TH-4000 is administered for one or more three-week cycles, each cycle comprising administering TH-4000 once a week for 4 consecutive weeks.

[0370] 12. The method of any one of embodiments 4-10, wherein TH-4000 is administered over an infusion period of about 1 hour to about 6 hours.

[0371] 13. The method of any one of embodiments 4-12, wherein the cancer treated is selected from non-small cell lung cancer, esophageal cancer, pancreatic cancer, rectal cancer, head and neck squamous cell carcinoma, skin squamous cell carcinoma, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer and mesothelioma.

[0372] 14. The method of any one of embodiments 4-13, wherein the cancer is relapsed or refractory or the patient is not a candidate for standard chemotherapy.

[0373] 15. The method of any one of embodiments 4-14, wherein TH-4000 is administered in combination with another tyrosine kinase selected from the group consisting of erlotinib, dacomitinib, AZD9291, CO-1686, afatinib, gefitinib, rociletinib, cetuximab, icotinib, AZD-8931, lapatinib, dacomitinib, neratinib, AP-26113, poziotinib, ASP-8273, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab, and patritumumab.

[0374] 16. The method according to any one of embodiments 4-15, wherein the patient is also treated with erlotinib.

[0375] 17. The method according to any one of embodiments 4-15, wherein the patient is also treated with cetuximab.

[0376] 18. A method for diagnosing an increased risk of tyrosine kinase drug resistance in a subject suffering from a condition or disorder mediated at least in part by EGFR, comprising (a) obtaining the genotype of a subject to be treated for the EGFR-mediated condition or disorder or (b) determining the presence of hypoxia; and (c) determining whether the subject is at risk for tyrosine kinase drug resistance.

[0377] 19. A method for identifying a subject susceptible to developing resistance during treatment with a tyrosine kinase inhibitor, comprising (a) determining the presence of hypoxia; and (b) determining whether the subject is at risk for developing resistance to the tyrosine kinase drug.

[0378] 20. A method for treating cancer in a subject, comprising the steps of: (i) identifying whether the subject is susceptible to tyrosine kinase drug resistance; and (ii) administering a therapeutically effective amount of TH-4000 to the subject.

[0379] 21. A method for treating cancer in a patient who is resistant or unresponsive to a dose of a tyrosine kinase inhibitor, comprising: providing a therapeutically effective amount of a tyrosine kinase inhibitor; and administering a resistant-surpassing amount of TH-4000 to the patient.

[0380] 22. A method for treating cancer in a patient who is resistant or unresponsive to doses of a tyrosine kinase inhibitor, comprising administering a therapeutic amount of TH-4000 to the patient.

[0381] 23. The method of any one of embodiments 4-17 and 20-22, wherein the cancer treated is non-small cell lung cancer.

[0382] 24. The method of any one of embodiments 4-17 and 20-22, wherein the cancer treated is head and neck or skin squamous cell carcinoma.

[0383] 25. The method of any one of embodiments 4-17 and 20-22, wherein the patient is treated prophylactically with a topical substance to reduce or prevent mucosal and / or skin damage.

[0384] 26. Use of TH-4000 in the preparation of a medicament for treating cancer in a selected patient population that is unresponsive to a tyrosine kinase inhibitor, wherein the patient population is selected based on the patient's genotype or the presence of hypoxia.

[0385] 27. A kit for diagnosing an increased risk of tyrosine kinase drug resistance in a subject suffering from cancer, comprising: (a) a reagent for detecting wild-type EGFR; or (b) a substance for detecting hypoxia; and (c) a container for the reagent or substance; and (d) a written product on or in the container describing the use of the reagent or substance in predicting tyrosine kinase resistance in a subject.

Claims

1. Use of the compound [(E)-4-[[4-(3-bromo-4-chloroanilino)]pyrido[3,4-d]pyrimidin-6-yl]amino]-4-oxobut-2-enyl]-dimethyl-[(3-methyl-5-nitroimidazol-4-yl)methyl]ammonium bromide in the preparation of a pharmaceutical composition for treating cancer in a patient in need of treatment, wherein the cancer is characterized by wild-type activity of EGFR protein, wherein the pharmaceutical composition comprises the compound in an amount of 10 mg-180 mg.

2. The use of claim 1, wherein the pharmaceutical composition is prepared for administration as a single agent therapy.

3. The use of claim 1, wherein the pharmaceutical composition is prepared for intravenous administration.

4. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition is prepared for administration at a frequency of once a day to once a month.

5. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition is prepared for administration at a frequency of once every three days.

6. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition is prepared for administration over a period of time ranging from 1 week to 60 weeks.

7. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered once every three days for 24 days.

8. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition is prepared for administration over an infusion period of 1 hour to 6 hours.

9. The method of any one of claims 1 to 3, wherein the cancer treated is selected from the group consisting of non-small cell lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, head or neck squamous cell carcinoma, skin squamous cell carcinoma, glioblastoma, colon cancer, cervical cancer, bladder cancer, breast cancer, gastrointestinal stromal cancer (GIST), ovarian cancer, gastric cancer, endometrial cancer, uterine cancer and mesothelioma.

10. The use of any one of claims 1 to 3, wherein the cancer is relapsed or refractory or the patient is not suitable for standard chemotherapy.

11. The use of any one of claims 1 to 3, wherein the pharmaceutical composition is prepared for administration in combination with another tyrosine kinase inhibitor selected from the group consisting of erlotinib, dacomitinib, AZD9291, CO-1686, afatinib, gefitinib, rociletinib, cetuximab, icotinib, AZD-8931, lapatinib, dacomitinib, neratinib, AP-26113, poziotinib, ASP-8273, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab and patritumumab.

12. The use according to any one of claims 1 to 3, wherein the patient is also treated with erlotinib.

13. The use according to any one of claims 1 to 3, wherein the patient is also treated with cetuximab.

14. The method of any one of claims 1 to 3, wherein the patient is resistant or unresponsive to a dose of a tyrosine kinase inhibitor selected from the group consisting of erlotinib, dacomitinib, AZD9291, CO-1686, afatinib, gefitinib, rociletinib, cetuximab, icotinib, AZD-8931, lapatinib, dacomitinib, neratinib, AP-26113, poziotinib, ASP-8273, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab and patritumumab.

15. The use according to any one of claims 1 to 3, wherein the cancer treated is non-small cell lung cancer.

16. The use according to any one of claims 1 to 3, wherein the cancer treated is squamous cell carcinoma of the head and neck or skin.

17. The use according to any one of claims 1 to 3, wherein the patient is treated prophylactically with a topical agent to reduce or prevent mucosal and / or skin damage.

18. The use according to any one of claims 1 to 3, wherein the composition comprises the compound or a pharmaceutically acceptable salt thereof and 2-hydroxypropyl-β-cyclodextrin.

19. The use of any one of claims 1-3, wherein the cancer is heterozygous for EGFR protein.

20. The use of any one of claims 1-3, wherein the cancer is characterized by a genotype comprising one mutant EGFR allele and one wild-type EGFR allele.

21. The use of any one of claims 1-3, wherein the cancer is homozygous for wild-type activity of EGFR protein.

22. The use according to any one of claims 1 to 3, wherein the activity of wild-type EGFR protein is overexpressed or upregulated.

23. The use according to any one of claims 1 to 3, wherein the expressed EGFR protein has a wild-type active site.

24. The use of any one of claims 1-3, wherein the cancer is characterized by at least one copy of exon 19 of the EGFR gene.

25. The use according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises the compound in an amount of 100 mg.

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