Composition containing G4 stabilizer and DNA-PK inhibitor and application thereof

Abstract

The invention relates to the field of antitumor drugs, in particular to a composition containing a G4 stabilizer and a DNA-PK inhibitor and application of the composition. According to the present invention, the G4 stabilizer is combined with the core protein DNA-PK inhibitor in the NHEJ pathway to verify the synergistic lethal effect of the pharmaceutical composition on cancer cells and the safety of the pharmaceutical composition, and the pharmaceutical composition mode of the present invention provides a new effective treatment mode for the treatment of clinical cancer patients, especially highly invasive cancer patients.

Description

Technical Field

[0001] The present disclosure relates to the field of anti-tumor drugs, and in particular to a composition containing a G4 stabilizer and a DNA-PK inhibitor and use thereof in treating cancer. Background Art

[0002] G-quadruplex (G4) is a special nucleic acid secondary structure formed by guanine-rich nucleic acid sequences bound by Hoogsteen hydrogen bonds and stabilized by monovalent cations. More than 700,000 G4 structures can be formed in the human genome. These structurally diverse G4s are involved in regulating a variety of physiological and pathological functions, and play a vital role in cell proliferation, differentiation, aging, apoptosis, and the occurrence and development of tumors. Studies have shown that DNA G4s are mainly present in specific regions with important functions such as proto-oncogene promoters, 5′-untranslated regions, and telomeres. For example, the promoter regions of proto-oncogenes such as MYC, BCL-2, PDGFR-β, KRAS, HIF-1α, and VEGF all contain G4 structures. In addition to being associated with cancer, G4 has also been found to be associated with other diseases, including neurodegenerative diseases and viral and parasitic infections.

[0003] G4 provides a new target for anticancer drug research, and can solve the targeting problem of some "undruggable proteins", with great research and development potential. In addition, DNA G4 is significantly different from double-stranded DNA in structure, so it can achieve specific targeting, thereby avoiding the serious side effects caused by the lack of specificity of many double-stranded DNA targeted drugs.

[0004] CX5461 is a stabilizer of the G4 secondary structure of double-stranded DNA. G4 stabilization can lead to DNA double-strand breaks. In previous studies, the drug has little effect on normal tissue cells, but has a very obvious inhibitory effect on rapidly proliferating tumor cells and promotes apoptosis. However, an inevitable problem faced by all drug treatments is drug resistance. Tumor resistance is divided into primary resistance, secondary resistance and acquired resistance. Tumor heterogeneity plays an important role in drug resistance. Solutions to drug resistance include iteration of drugs with the same mechanism, development of drugs with new mechanisms of action, and combined use of two or more drugs with different mechanisms of action. Previous studies have shown the synergistic effect of CX5461 with ATM inhibitors, ATR inhibitors, PARP inhibitors and other drugs, but the side effects of the former two are large, and the applicable cancer types of PARP inhibitors are mainly limited to breast cancer and ovarian cancer. Summary of the invention

[0005] The purpose of the present disclosure is to provide a technical solution to improve the therapeutic effect of G4 stabilizer.

[0006] To achieve the above technical objectives, in one aspect, the present disclosure provides a composition, characterized in that it includes a G4 stabilizer and a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is AZD7648. In some embodiments, the G4 stabilizer is CX-5461 and / or PDS.

[0007] In another aspect, the present disclosure provides use of the above composition in preparing a medicament or a medicine kit for treating cancer.

[0008] In another aspect, the present disclosure provides use of a G4 stabilizer and a DNA-PK inhibitor in the preparation of a medicament or a drug kit for treating cancer.

[0009] According to any of the foregoing aspects, the cancer is a solid tumor and / or a hematological tumor; preferably, the cancer is selected from one or more of the following groups: non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small intestine cancer, appendix cancer, colorectal cancer, cancer of unknown primary site, endometrial cancer, mixed cancer types, pancreatic cancer, hepatobiliary cancer, small cell lung cancer, cervical cancer, germ cell cancer, breast cancer, ovarian cancer, gastrointestinal neuroendocrine cancer, bladder cancer, myeloma, myelodysplasia / myeloproliferative neoplasm, head and neck cancer, esophageal cancer, gastric cancer, soft tissue sarcoma, mesothelioma, thyroid cancer, lymphoma, leukemia and melanoma; more preferably, the cancer is non-small cell lung cancer.

[0010] In another aspect, the present disclosure provides use of a DNA-PK inhibitor in the preparation of a medicament or a drug kit for improving the therapeutic effect of a G4 stabilizer.

[0011] In another aspect, the present disclosure provides a drug or a drug kit for improving the therapeutic effect of a G4 stabilizer, characterized in that it comprises a DNA-PK inhibitor, wherein the DNA-PK inhibitor is administered at any time before, during, or after administration of the G4 stabilizer.

[0012] In another aspect, the present disclosure provides a method of treating cancer comprising administering to a subject a therapeutically effective amount of a G4 stabilizer and a therapeutically effective amount of a DNA-PK inhibitor.

[0013] In another aspect, the present disclosure provides a method for improving the therapeutic effect of a G4 stabilizer, comprising administering a DNA-PK inhibitor at any time before, during, or after administration of the G4 stabilizer.

[0014] According to any of the aforementioned aspects, the G4 stabilizer is CX5461 and / or PDS.

[0015] According to any of the preceding aspects, the DNA-PK inhibitor is AZD7648.

[0016] CX5461 shows specific killing ability against highly invasive tumors, but weak killing ability against normal cells, which shows its safety. CX5461 is administered orally, which shows its convenience. CX5461 has entered the clinical trial stage for the treatment of highly invasive acute myeloid leukemia, multiple myeloma and some solid tumors, showing greater convertibility. The present invention innovatively combines the G4 stabilizer with the core protein DNA-PK inhibitor in the NHEJ pathway, and verifies the synergistic lethal effect of the drug combination on cancer cells and the safety of the drug combination. The model of this drug combination will provide a new and effective treatment method for clinical tumor patients, especially those with highly invasive tumors. After the promotion of this patent, a large number of highly invasive tumor patients with poor prognosis will benefit from it. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments that conform to the specification, and are used together with the specification to explain the principles of the specification.

[0018] Figure 1 The cell viability and IC50 values ​​of human non-small cell lung cancer cell lines A549 and H358 treated with different concentrations of CX5461 and AZD7648 alone or in combination are shown.

[0019] Figure 2 The clonogenicity of human non-small cell lung cancer cell lines A549 and H358 treated with CX5461 and AZD7648 alone or in combination is shown.

[0020] Figure 3 The figure shows the proportion of cells in each phase of the cell cycle after treatment with CX5461 and AZD7648 alone or in combination with AZD7648 in human non-small cell lung cancer cell lines A549 and H358.

[0021] Figure 4 The figure shows the proportion of apoptotic cells in human non-small cell lung cancer cell lines A549 and H358 after treatment with CX5461 and AZD7648 alone or in combination.

[0022] Figure 5 The cell viability and IC50 values ​​of human non-small cell lung cancer cell lines A549 and H358 treated with different concentrations of PDS and AZD7648 alone or in combination are shown. DETAILED DESCRIPTION

[0023] (I) Definition and Explanation

[0024] In order to make it easier to understand the present disclosure, certain technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present disclosure are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0025] As used in this specification and the appended claims, the singular forms "a," "an," "the," "said," and similar referents include plural referents unless the content clearly dictates otherwise.

[0026] As used herein, the conjunction term "and / or" between various elements is intended to include both the meanings of "and" and "or", for example, the phrase "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0027] As used herein, the terms "comprises," "comprising," "having," and "containing," and any variations thereof, are intended to cover a non-exclusive inclusion. The terms are intended to be open ended, to specify the presence of any stated features, elements, integers, steps, or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of."

[0028] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0029] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0030] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0031] As used herein, the terms "G quadruplex", "G4" and "G4s" can be used interchangeably. G-quadruplex is a special four-stranded nucleic acid secondary structure formed by guanine-rich sequences connected by Hoogsteen hydrogen bonds, including DNA G4, RNA G4, and i-motif. Studies have shown that G-rich sequences (G-quadruplex-forming sequences) are widely present in telomeres, promoter regions of pathogenic genes, mRNA 5'UTR regions, etc., and G quadruplexes can be formed in the promoter sequences of various genes under appropriate conditions. G4 is involved in many cellular functions, such as DNA replication, gene expression, telomere length and cell apoptosis, and is closely related to the occurrence and development of cancer, nervous system diseases, and viral diseases. Some genomic sequences include guanine-rich domain regions that are more thermodynamically stable than double-stranded DNA and can directly form G4. It is predicted that nearly 50% of human genes produce G4 near their promoter regions, and these G4s are usually located near oncogene promoters or regulatory genes, rather than near neighboring family genes. Since there is a dynamic balance between single-stranded, double-stranded and G-quadruplex promoters with transcriptional activity, gene transcription can be inhibited by stabilizing the structure of G-quadruplex. (Kosiol et al., Molecular Cancer, 2021, 20:40; Cai Jiongheng et al., Chinese Journal of Medicinal Chemistry, 2023, 33(10):736-751; Chen Zhiyuan et al., Chinese Journal of Traditional Chinese Medicine, 2024, 49(13):3421-3431, the contents of which are incorporated herein by reference.)

[0032] As used herein, the term "CX-5461" (Pidnarulex) includes CX5461 and pharmaceutically acceptable salts thereof. The chemical name of CX-5461 is 2-(4-methyl-1H-1,4-diazepan-1-yl)-N-[(5-methyl-2-pyrazinyl)methyl]-5-oxo-5H-benzothiazolo[3,2-a][1,8]naphthyridine-6-carboxamide, with CAS number 1138549-36-6. It is a small molecule drug that can stabilize the G-quadruplex structure of the DNA replication fork. During DNA replication, the G-quadruplex interacts with the polymerase on the replication fork, causing the replication fork to stagnate and the genome to be unstable, thereby causing DNA damage or breakage. CX5461 is also an rRNA synthesis inhibitor with oral activity, which can inhibit PolI-driven rRNA transcription. Because tumors proliferate rapidly and have high energy demands, the nucleolar structure enlarges and ribosome production increases. This is another mechanism of CX5461's anti-tumor effect. However, CX5461 requires a higher concentration to inhibit ribosome production, while DNA damage can occur at lower concentrations. The contribution values ​​of the above two anti-tumor mechanisms may not be the same in different types of tumors. As a first-in-class drug targeting G4s, CX-5461 has achieved good results in the treatment of BRCA1 / 2-deficient breast cancer and ovarian cancer patients, and can also play a tumor-suppressing role in lymphoma.

[0033] As used herein, the term "PDS" (pyridostatin) includes PDS and pharmaceutically acceptable salts thereof, which are a class of G4 ligands with a central pyridine ring. The chemical name of the PDS is 4-(2-aminoethoxy)-N2, N6-di[4-(2-aminoethoxy)-2-quinolyl]-2,6-pyridinedicarboxamide, CAS No. 1085412-37-8, which is a highly selective DNA G quadruplex binding small molecule designed to form a complex with the G quadruplex structure and stabilize its structure. In cultured primary neurons, the hydrochloride of PDS induces the formation of DNA DSBs, leading to neurite retraction, synaptic loss, and dose-dependent neuronal death.

[0034] As used herein, the term "DNA-PK" is a serine / threonine kinase, which is composed of a DNA-PK catalytic subunit (DNA-PKcs) and a Ku70 / 80 heterodimer, wherein DNA-PKcs is a polypeptide composed of 4128 amino acids, which includes an N-terminal structure, a "central cradle" structure formed by HEAT repeats, and a C-terminal kinase head structure, and has a protein phosphorylation function. DNA-PK is an important factor in the repair of DSBs mediated by NHEJ. After the cell generates DSBs, the cyclic Ku70 / Ku80 heterodimer quickly recognizes the damage signal and binds to the broken DNA end, while recruiting DNA-PKcs to the damaged DNA end to form a DNA-PK complex. The activated DNA-PKcs then recruits and phosphorylates a series of regulatory factors in NHEJ, such as the nuclear enzyme Artemis, XRCC4, and DNA ligase IV, and then connects the damaged DNA double strands together through DNA ligase IV, and then releases the repaired DNA from the complex to end the repair process. Studies have shown that abnormal expression of DNA-PK is closely related to the occurrence, development, metastasis and drug resistance of various tumors. (Lieber MR, Annual Review of Biochemistry, 2010, 79: 181-211; Cai Tian et al., Acta Pharmaceutica Sinica, 2023, 58(8): 2218-2225, the contents of which are incorporated herein by reference.)

[0035] As used herein, the term "AZD7648" includes AZD7648 and its pharmaceutically acceptable salts. The chemical name of AZD7648 is 7,9-dihydro-7-methyl-2-[(7-methyl[1,2,4]triazolo[1,5-A]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-8H-purin-8-one, CAS No. 2230820-11-6, which is a highly selective DNA-PK inhibitor with anti-tumor activity. There are currently two clinical trials on AZD7648: one is a phase I / IIa clinical trial for the treatment of advanced solid tumors as a monotherapy or in combination with cytotoxic drugs / new anticancer drugs (such as olaparib), and the other is a phase I clinical trial for the treatment of adult soft tissue sarcoma in combination with radiotherapy.

[0036] As used herein, the term "G4-mediated disease" refers to a disease or disease state in which G4 is involved in regulation, including but not limited to cancer, nervous system diseases, and viral diseases in which G4 is involved in regulation, and the expression of genes associated with the disease can be inhibited by stabilizing the structure of G-quadruplexes.

[0037] As used herein, the term "cancer" refers to a disease or disease state characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Examples of various cancers are described herein, including but not limited to non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, bladder cancer, myeloma, liver cancer, skin cancer, glioma, breast cancer, melanoma, malignant glioma, rhabdomyosarcoma, ovarian cancer, astroglioma, Ewing's sarcoma, retinoblastoma, epithelial cell cancer, colon cancer, renal cancer, gastrointestinal stromal tumor, esophageal cancer, gastric cancer, thyroid cancer, leukemia, lymphoma and nasopharyngeal carcinoma, etc. The terms "tumor" and "cancer" are used interchangeably in this article, for example, these two terms include solid and liquid, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include precancerous lesions and malignant cancers and tumors.

[0038] As used herein, the term "disease state" is or includes the likelihood of developing a clinically significant disease, such as the likelihood of developing a clinically significant disease within a particular time frame or natural life span, including the presence or likelihood of relapse in a subject, and / or whether a subject will benefit from treatment.

[0039] As used herein, the term "treatment" is intended to include prevention and treatment, for example, treatment of a G4-mediated disease includes prevention and / or treatment of a G4-mediated disease. Methods by which beneficial or desired results, including clinical results, can be obtained through treatment. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or improving the quality of life, increasing weight gain and / or prolonging survival. "Treatment" also includes a reduction in the pathological results of cancer (e.g., tumor volume). In the context of cancer, "treatment" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing overall tumor burden, and improving one or more symptoms associated with the disease.

[0040] As used herein, the term "effective amount" or "therapeutically effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The term "effective action amount" includes an amount in the in vivo environment or cellular microenvironment sufficient to improve or prevent the symptoms or symptoms of a medical condition. The effective amount or effective action amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. The effective amount or effective action amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0041] As used herein, the terms "subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, apes, humans, farm animals, competitive animals, and pets.

[0042] The amount of the drug administered may depend on the subject being treated, the subject's age, health, sex, and weight, the type of concurrent treatment (if any), the severity of the condition, the nature of the desired effect, the manner and frequency of treatment, and the judgment of the prescribing physician. The frequency of administration may also depend on the pharmacodynamic effect on arterial oxygen tension. However, the most preferred dosage may be adjusted for individual subjects, as will be understood by those skilled in the art and can be determined without undue experimentation. This generally includes adjusting the standard dosage (e.g., if the patient is of low weight, then reducing the dosage).

[0043] As used herein, the term "pharmaceutical composition" refers to a mixture of one or more components. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism. The pharmaceutical composition of the present disclosure may include one or more pharmaceutically acceptable salts, pharmaceutically acceptable carriers and / or adjuvants, such as antioxidants, preservatives, wetting agents, emulsifiers and dispersants, etc.

[0044] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation adjuvant or carrier conventional in the art for use with a therapeutic agent, which together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed. Suitable carriers are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymer micelles or inorganic nanocarriers, etc.

[0045] The "pharmaceutical composition" or "drug" of the present disclosure can also be administered to patients or subjects in need of such treatment in any suitable manner, such as oral, intravenous, parenteral, rectal, pulmonary or topical administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, such as oral solid preparations, such as tablets, capsules, pills, granules, etc.; or, oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When prepared into oral preparations, the pharmaceutical preparations may also contain suitable fillers, binders, disintegrants, lubricants, etc.

[0046] The "pharmaceutical composition" or "medication" of the present disclosure can be prepared into any pharmaceutically acceptable dosage form for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration, for example, it can be formulated into tablets, lozenges, capsules, pills, solutions, suspensions, syrups, injections, suppositories, inhalants or sprays.

[0047] As used herein, the term "kit" refers to one or more components of a package, which can be packaged separately or placed in a container, such as a tube, bottle, vial, bag, blister, syringe or other suitable container device. The kit described herein may also include instructions for implementing the subject method using the components of the kit (e.g., instructions for preparing a pharmaceutical composition described herein and / or instructions for using a pharmaceutical composition). The instructions for implementing the subject method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can be present in the kit in the form of a package insert, in the label of the container of the kit or its components (i.e., components associated with the package or small package). In some embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium (e.g., CD-ROM, disk, etc.). In other embodiments, the actual instructions are not present in the kit, but are provided for obtaining instructions from a remote source, such as through the Internet. An example of this embodiment is a kit including a website where the instructions can be viewed and / or from which the instructions can be downloaded.

[0048] As used herein, the term "synergistic effect" refers to a phenomenon in which the effects of two drugs used in combination are more effective than their individual effects, as opposed to antagonistic effects.

[0049] The "combination" or "combination use" described in the present disclosure is a mode of administration, which includes various situations in which two drugs are administered sequentially or simultaneously. The so-called "simultaneously" here refers to the administration of G4 stabilizer and DNA-PK inhibitor in the same administration cycle, for example, the two drugs are administered within 2 days, or within 1 day. The so-called "sequential or sequential" administration includes the situation in which G4 stabilizer and DNA-PK inhibitor are administered separately in different administration cycles. The "combined" mode of administration includes simultaneous administration, independent preparation and co-administration, or independent preparation and sequential administration. These modes of administration all belong to the combined administration described in the present disclosure.

[0050] (II) Detailed technical solution

[0051] The inventors found that CX5461 alone can not only inhibit the repair of single-strand DNA damage such as nucleic acid excision repair, base excision repair, and mismatch repair, but also inhibit the damage to the HR pathway that causes double-strand break repair. This is repeated with PARPi, ATRi, and ATMi. At this time, tumor cells may be more dependent on the NHEJ repair pathway for DNA damage repair. Therefore, NHEJ inhibitors such as DNA-PK inhibitors can play a better role in combination with CX5461.

[0052] One aspect of the present disclosure provides a composition, characterized in that it includes a G4 stabilizer and a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor includes AZD7648. In some embodiments, the G4 stabilizer includes CX-5461 and / or PDS.

[0053] In some embodiments, the composition includes 7.8-1000nM of CX-5461, for example, it can be 7.8nM, 10nM, 15.6nM, 20nM, 31.2nM, 40nM, 50nM, 62.5nM, 80nM, 100nM, 125nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM (1μM), preferably 15.6-500nM, and more preferably 31.2-250nM.

[0054] In some embodiments, the composition includes 0.3125-10 μM of PDS, for example, it can be 0.3125 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.625 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.25 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, preferably 0.625-10 μM, more preferably 1.25-5 μM, and further preferably 2.5-5 μM.

[0055] In some embodiments, the composition comprises 0.3125-10 μM of AZD7648, for example, it can be 0.3125 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.625 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.25 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, preferably 0.625-10 μM, more preferably 0.625-5 μM, and further preferably 1.25-5 μM.

[0056] In some embodiments, the molar ratio of CX-5461 to AZD7648 is 0.0008-3.2:1, for example, 0.0008:1, 0.0009:1, 0.001:1, 0.002:1, 0.003:1, 0.005:1, 0.008:1, 0.010:1, 0.015:1, 0.020:1, 0.025:1, 0.030:1, 0.035:1, 0.04:1, 0.0 5:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 2.3:1, 2.5:1, 2.8:1, 3.0:1, 3.2:1, preferably 0.025-0.1:1.

[0057] In some embodiments, the molar ratio of PDS to AZD7648 is 0.03-32:1, for example, 0.03:1, 0.035:1, 0.04:1, 0.05:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0. 6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 2.5:1, 3.0:1, 4.0:1, 5.0:1, 6.0:1, 7.5:1, 10:1, 15:1, 20:1, 25:1, 28:1, 30:1, 31:1, 32:1, preferably 0.25-1:1.

[0058] In some embodiments, the composition includes CX5461 and AZD7648. In some embodiments, CX5461 and AZD7648 may be present in the same preparation, or may be separately prepared in the form of a combined package. When the above two components are prepared separately, the dosage forms may be the same or different. The dosage form may be any clinically acceptable dosage form, such as an injection dosage form, an oral dosage form, etc. The injection dosage form includes liquid injection, powder for injection, tablets for injection, etc.; the oral dosage form includes tablets, capsules, oral liquid preparations, etc.

[0059] In some embodiments, the composition includes PDS and AZD7648. In some embodiments, PDS and AZD7648 may be present in the same preparation, or may be separately prepared in the form of a combined package. When the above two components are prepared separately, the dosage forms may be the same or different. The dosage form may be any clinically acceptable dosage form, such as an injection dosage form, an oral dosage form, etc. The injection dosage form includes liquid injection, powder for injection, tablets for injection, etc.; the oral dosage form includes tablets, capsules, oral liquid preparations, etc.

[0060] In some embodiments, the composition comprises 7.8 nM CX5461 and 10 μM AZD7648.

[0061] In some embodiments, the composition comprises 10 nM CX5461 and 9 μM AZD7648.

[0062] In some embodiments, the composition comprises 20 nM CX5461 and 7 μM AZD7648.

[0063] In some embodiments, the composition comprises 50 nM CX5461 and 5 μM AZD7648.

[0064] In some embodiments, the composition comprises 80 nM CX5461 and 3 μM AZD7648.

[0065] In some embodiments, the composition comprises 62.5 nM CX5461 and 2.5 μM AZD7648.

[0066] In some embodiments, the composition comprises 150 nM CX5461 and 2.5 μM AZD7648.

[0067] In some embodiments, the composition comprises 200 nM CX5461 and 2 μM AZD7648.

[0068] In some embodiments, the composition comprises 250 nM CX5461 and 1 μM AZD7648.

[0069] In some embodiments, the composition comprises 400 nM CX5461 and 0.8 μM AZD7648.

[0070] In some embodiments, the composition comprises 500 nM CX5461 and 0.6 μM AZD7648.

[0071] In some embodiments, the composition comprises 800 nM CX5461 and 0.4 μM AZD7648.

[0072] In some embodiments, the composition comprises 1000 nM CX5461 and 0.3125 μM AZD7648.

[0073] In some embodiments, the composition comprises 0.3125 μM PDS and 10 μM AZD7648.

[0074] In some embodiments, the composition comprises 0.4 μM PDS and 9 μM AZD7648.

[0075] In some embodiments, the composition comprises 0.8 μM PDS and 7 μM AZD7648.

[0076] In some embodiments, the composition comprises 1.25 μM PDS and 5 μM AZD7648.

[0077] In some embodiments, the composition comprises 2 μM PDS and 3 μM AZD7648.

[0078] In some embodiments, the composition comprises 2.5 μM PDS and 2.5 μM AZD7648.

[0079] In some embodiments, the composition comprises 3 μM PDS and 2 μM AZD7648.

[0080] In some embodiments, the composition comprises 4 μM PDS and 1 μM AZD7648.

[0081] In some embodiments, the composition comprises 5 μM PDS and 0.8 μM AZD7648.

[0082] In some embodiments, the composition comprises 7 μM PDS and 0.6 μM AZD7648.

[0083] In some embodiments, the composition comprises 9 μM PDS and 0.4 μM AZD7648.

[0084] In some embodiments, the composition comprises 10 μM PDS and 0.3125 μM AZD7648.

[0085] Another aspect of the present disclosure provides use of the above composition in preparing a medicament or a medicine kit for treating cancer.

[0086] Another aspect of the present disclosure provides use of a G4 stabilizer and a DNA-PK inhibitor in the preparation of a medicament or a drug kit for treating cancer.

[0087] In some embodiments, the cancer is a solid tumor and / or a hematological tumor. In some embodiments, the cancer is selected from one or more of the following groups: non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small intestine cancer, appendix cancer, colorectal cancer, cancer of unknown primary focus, endometrial cancer, mixed cancer types, pancreatic cancer, hepatobiliary cancer, small cell lung cancer, cervical cancer, germ cell cancer, breast cancer, ovarian cancer, gastrointestinal neuroendocrine cancer, bladder cancer, myeloma, myelodysplasia / myeloproliferative neoplasm, head and neck cancer, esophageal cancer, gastric cancer, soft tissue sarcoma, mesothelioma, thyroid cancer, lymphoma, leukemia or melanoma. In some embodiments, the cancer is non-small cell lung cancer.

[0088] Another aspect of the present disclosure provides use of a DNA-PK inhibitor in the preparation of a medicament or a drug kit for improving the therapeutic effect of a G4 stabilizer.

[0089] Another aspect of the present disclosure provides a drug or a drug kit for improving the therapeutic effect of a G4 stabilizer, characterized in that it comprises a DNA-PK inhibitor, wherein the DNA-PK inhibitor is administered at any time before, during, or after administration of the G4 stabilizer.

[0090] According to any of the preceding aspects, in some embodiments, the DNA-PK inhibitor is AZD7648.

[0091] According to any of the preceding aspects, in some embodiments, the G4 stabilizer comprises CX5461 and / or PDS.

[0092] According to any of the foregoing aspects, the effective amount of CX-5461 in the drug or kit is 7.8-1000nM, for example, it can be 7.8nM, 10nM, 15.6nM, 20nM, 31.2nM, 40nM, 50nM, 62.5nM, 80nM, 100nM, 125nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM (1μM), preferably 15.6-500nM, and more preferably 31.2-250nM.

[0093] According to any of the preceding aspects, the effective amount of the PDS in the drug or kit is 0.3125-10 μM, for example, 0.3125 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.625 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.25 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, preferably 0.625-10 μM, more preferably 1.25-5 μM, and further preferably 2.5-5 μM.

[0094] According to any of the foregoing aspects, the effective amount of AZD7648 in the drug or kit is 0.3125-10 μM of the DNA-PK inhibitor, for example, it can be 0.3125 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.625 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.25 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, preferably 0.625-10 μM, more preferably 0.625-5 μM, and further preferably 1.25-5 μM.

[0095] According to any of the foregoing aspects, the molar ratio of the effective amount of CX-5461 to the effective amount of AZD7648 in the drug or kit is 0.0008-3.2:1, for example, 0.0008:1, 0.0009:1, 0.001:1, 0.002:1, 0.003:1, 0.005:1, 0.008:1, 0.010:1, 0.015:1, 0.020:1, 0.025:1, 0.030:1, 0.035:1, 0.0 4:1, 0.05:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 2.3:1, 2.5:1, 2.8:1, 3.0:1, 3.2:1, preferably 0.025-0.1:1.

[0096] According to any of the foregoing aspects, the molar ratio of the effective amount of PDS to the AZD7648 in the drug or kit is 0.03-32:1, for example, 0.03:1, 0.035:1, 0.04:1, 0.05:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1 .5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 2.5:1, 3.0:1, 4.0:1, 5.0:1, 6.0:1, 7.5:1, 10:1, 15:1, 20:1, 25:1, 28:1, 30:1, 31:1, 32:1, preferably 0.25-1:1.

[0097] In some embodiments, the drug or kit includes a therapeutically effective amount of CX5461 and a therapeutically effective amount of AZD7648. In some embodiments, CX5461 and AZD7648 may be present in the same preparation, or may be separately prepared in the form of a combined package. When the above two components are prepared separately, the dosage forms may be the same or different. The dosage form may be any clinically acceptable dosage form, such as an injection dosage form, an oral dosage form, etc. The injection dosage form includes a liquid injection, an injection powder, an injection tablet, etc.; the oral dosage form includes a tablet, a capsule, an oral liquid preparation, etc.

[0098] In some embodiments, the drug or kit includes a therapeutically effective amount of PDS and a therapeutically effective amount of AZD7648. In some embodiments, PDS and AZD7648 may be present in the same preparation, or may be separately prepared in the form of a combined package. When the above two components are prepared separately, the dosage forms may be the same or different. The dosage form may be any clinically acceptable dosage form, such as an injection dosage form, an oral dosage form, etc. The injection dosage form includes liquid injection, powder for injection, tablets for injection, etc.; the oral dosage form includes tablets, capsules, oral liquid preparations, etc.

[0099] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 7.8 nM, and the effective amount of AZD7648 is and 10 μM.

[0100] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 10 nM, and the effective amount of AZD7648 is and 9 μM.

[0101] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 20 nM, and the effective amount of AZD7648 is and 7 μM.

[0102] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 50 nM, and the effective amount of AZD7648 is and 5 μM.

[0103] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 80 nM, and the effective amount of AZD7648 is and 3 μM.

[0104] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 62.5 nM, and the effective amount of AZD7648 is and 2.5 μM.

[0105] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 150 nM, and the effective amount of AZD7648 is and 2.5 μM.

[0106] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 200 nM, and the effective amount of AZD7648 is and 2 μM.

[0107] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 250 nM, and the effective amount of AZD7648 is and 1 μM.

[0108] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 400 nM, and the effective amount of AZD7648 is and 0.8 μM.

[0109] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 500 nM, and the effective amount of AZD7648 is and 0.6 μM.

[0110] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 800 nM, and the effective amount of AZD7648 is and 0.4 μM.

[0111] In some embodiments, in the medicament or kit, the effective amount of CX5461 is 1000 nM, and the effective amount of AZD7648 is and 0.3125 μM.

[0112] In some embodiments, in the medicament or kit, the effective amount of PDS is 0.3125 μM, and the effective amount of AZD7648 is 10 μM.

[0113] In some embodiments, in the medicament or kit, the effective amount of PDS is 0.4 μM and the effective amount of AZD7648 is 9 μM.

[0114] In some embodiments, in the medicament or kit, the effective amount of PDS is 0.8 μM and the effective amount of AZD7648 is 7 μM.

[0115] In some embodiments, in the medicament or kit, the effective amount of PDS is 1.25 μM and the effective amount of AZD7648 is 5 μM.

[0116] In some embodiments, in the medicament or kit, the effective amount of PDS is 2 μM and the effective amount of AZD7648 is 3 μM.

[0117] In some embodiments, in the medicament or kit, the effective amount of PDS is 2.5 μM, and the effective amount of AZD7648 is 2.5 μM.

[0118] In some embodiments, in the medicament or kit, the effective amount of PDS is 3 μM and the effective amount of AZD7648 is 2 μM.

[0119] In some embodiments, in the medicament or kit, the effective amount of PDS is 4 μM and the effective amount of AZD7648 is 1 μM.

[0120] In some embodiments, in the medicament or kit, the effective amount of PDS is 5 μM and the effective amount of AZD7648 is 0.8 μM.

[0121] In some embodiments, in the medicament or kit, the effective amount of PDS is 7 μM and the effective amount of AZD7648 is 0.6 μM.

[0122] In some embodiments, in the medicament or kit, the effective amount of PDS is 9 μM and the effective amount of AZD7648 is 0.4 μM.

[0123] In some embodiments, in the medicament or kit, the effective amount of PDS is 10 μM and the effective amount of AZD7648 is 0.3125 μM.

[0124] Another aspect of the present disclosure provides a method for treating cancer, comprising administering to an individual a therapeutically effective amount of a G4 stabilizer and a therapeutically effective amount of a DNA-PK inhibitor, or the pharmaceutical composition, medicament or kit of the aforementioned aspects.

[0125] Another aspect of the present disclosure provides a method for improving the therapeutic effect of a G4 stabilizer, comprising administering a DNA-PK inhibitor at any time before, during, or after administration of the G4 stabilizer.

[0126] In some embodiments, the G4 stabilizer includes CX5461 and / or PDS. In some embodiments, the DNA-PK inhibitor is AZD7648. In some embodiments, the DNA-PK inhibitor and the G4 stabilizer are administered to the individual simultaneously. In some embodiments, the DNA-PK inhibitor and the G4 stabilizer are administered to the individual in any order. In some embodiments, the DNA-PK inhibitor and the G4 stabilizer are administered to the individual in any suitable manner, such as oral, intravenous, parenteral, rectal, pulmonary or topical administration. The administration of the DNA-PK inhibitor and the G4 stabilizer may be the same or different. The administration cycle of the DNA-PK inhibitor and the G4 stabilizer may be the same or different.

[0127] Example

[0128] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. In the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. The following examples are used to further illustrate the present disclosure, but should not be construed as limitations on the present disclosure. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present disclosure should all be equivalent replacement methods and are included in the protection scope of the present disclosure.

[0129] Lung cancer is one of the malignant tumors with the heaviest disease burden in the world. In 2020, the estimated new cases of lung cancer worldwide accounted for about 11.4% of all cancer cases, ranking second in the most common malignant tumors. In 2020, the estimated deaths from lung cancer worldwide accounted for about 18% of all cancer deaths, ranking first in the death ranking of all malignant tumors. Non-small cell lung cancers (NSCLC) account for about 85% of the total number of lung cancers, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc., of which adenocarcinoma and squamous cell carcinoma are the most common subtypes. The following examples verify the effects of G4 stabilizer and DNA-PK inhibitor alone and in combination on non-small cell lung cancer.

[0130] Example 1. CX5461 and AZD7648 synergistically inhibit cancer cell growth

[0131] Human non-small cell lung cancer cell lines A549 (purchased from Wuhan Pronocell, catalog number CL0016) and H358 (purchased from Wuhan Pronocell, catalog number CL0400) were selected and inoculated in RPMI1640 cell culture medium containing 10% fetal bovine serum (supplemented with 100u / mL of penicillin and streptomycin) and placed at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator, the medium was changed every 2-3 days, and the cells were digested with 0.25% trypsin, passaged and collected.

[0132] The cells in logarithmic growth phase were cultured in RPMI1640 cell culture medium containing 10% fetal bovine serum (supplemented with 100u / mL of penicillin and streptomycin, respectively) to a concentration of 2.5×10 4 / mL concentration of cell suspension, according to each well 1000 A549 cells or each well 2000 H358 cells, added to the 96-well cell culture plate, cultured for 24 hours, each well added with 100μL of culture solution containing different concentrations of drugs, wherein A549 cell line and H358 cell line were respectively set up with CX5461 treatment group, AZD7648 treatment group, and combined treatment group, each treatment group was set up with 6 concentrations (see Table 1), and a blank control was set up, and each concentration was set up with 6 parallel wells. Before the drug was used, it was dissolved and diluted to the target concentration with RPMI1640 cell culture solution containing 10% fetal bovine serum (supplemented with 100u / mL of penicillin and streptomycin).

[0133] Table 1. Drug concentrations of CX5461 and AZD7648 alone or in combination in A549 and H358 cells

[0134]

[0135] After 72 hours of culture, the supernatant was discarded. 100 μL of freshly prepared CCK-8 culture medium was added to each well, and after incubation at 37°C for 1 hour, the absorbance (OD value) was measured using an enzyme reader, with the detection wavelength at 570 nm and the reference wavelength at 450 nm.

[0136] Data processing: Inhibition rate = (OD value of control group - OD value of drug administration group) / OD value of control group × 100%; drug effect index is expressed as half inhibition concentration (IC50). Graphpad Prism software was used to draw a graph and fit the tumor cell growth curve in the software to calculate the half inhibition concentration (IC50, nM). The specific results are as follows Figure 1 As shown, CX5461 and AZD7648 have significant synergistic inhibitory effects on human non-small cell lung cancer cell lines A549 and H358, and the IC50 of the combination group is significantly lower than that of the CX5461 and AZD7648 single-agent groups.

[0137] Example 2. CX5461 and AZD7648 synergistically inhibit cancer cell colony formation

[0138] (1) Cell preparation: A549 and H358 cells in the logarithmic growth phase were completely digested with trypsin, resuspended in culture medium to prepare cell suspension, and counted.

[0139] (2) Cell seeding: 500 A549 cells were seeded per well, and 1000 H358 cells were seeded per well in a 6-well culture plate.

[0140] (3) After 24 hours of cell culture, 100 μL of culture medium containing different concentrations of drugs was added, and CX5461 and AZD7648 were added separately or simultaneously. CX5461 was set at two concentrations of 50 nM and 100 nM (H358) or 15 nM and 30 nM (A549), and the concentration of AZD7648 was 1 μM. A DMSO blank control was set. The treatment was performed for 72 hours, and 3 replicates were set for each group of treatment. Before the drug was used, it was diluted to the target concentration with cell culture medium. The culture medium (culture medium) used in this example was the same as that in Example 1.

[0141] (4) Replace the culture medium with fresh one and continue culturing in the incubator for 14 days. Replace the culture medium every 3 days and observe the cell status.

[0142] (5) Before terminating the experiment, observe the cell clones under a microscope and wash the cells once with PBS.

[0143] (6) Add 1 mL of 4% paraformaldehyde to each well and fix the cells for 30 min. Wash the cells once with PBS.

[0144] (7) Add 500 μL of clean, impurity-free crystal violet stain to each well and stain the cells for 30 min.

[0145] (8) Wash the cells several times with ddH2O, dry them, and take pictures with a camera. The results are as follows: Figure 2 As shown, there was no obvious clone formation in the CX5461 and AZD764 combination groups.

[0146] Example 3. Synergistic effect of CX5461 and AZD7648 on the cell cycle of cancer cells

[0147] Referring to the method of Example 2, A549 and H358 cell lines were treated with DMSO, CX5461 100 nM, CX5461 1 μM, AZD7648 1 μM, CX5461 100 nM combined with AZD7648 1 μM, and CX5461 1 μM combined with AZD7648 1 μM for 36 h, respectively, with three replicate wells in each group, of which DMSO treatment was a blank control (NC); the cells were trypsinized, the culture medium was resuspended into a cell suspension, and the cells were collected in a 2 ml EP tube.

[0148] (1) Preparation of cell samples: The number of cells is controlled at 1×10 5 ~1×10 6 Carefully remove the cell culture medium, digest the cells with trypsin, and prepare a single-cell suspension. Centrifuge at 1000g for 5 minutes to precipitate the cells, discard the supernatant, rinse the cells once with 1mL of pre-cooled PBS, and collect the cells by centrifugation.

[0149] (2) Cell fixation:

[0150] The cell pellet was gently mixed with 1 mL of pre-cooled 70% ethanol and fixed overnight at 4°C. After centrifugation at 1000 g for 5 min to precipitate the cells, the supernatant was carefully removed, leaving about 50 μl of 70% ethanol to avoid aspirating the cells.

[0151] Add 1 mL of pre-cooled PBS to resuspend. Then centrifuge again at 1000g for 5 min to precipitate the cells. Carefully remove the supernatant, leaving about 50 μl of PBS to avoid sucking away the cells. Gently tap the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.

[0152] (3) Preparation of propidium iodide staining solution:

[0153] For one sample, add 25 μl of propidium iodide staining solution (20×) and 10 μl of RNase A (50×) to 0.5 mL of staining buffer, mix well and set aside.

[0154] (4) Dyeing:

[0155] Add 0.5 ml of propidium iodide staining solution to each tube of cell sample, gently mix to resuspend the cell pellet, and incubate at 37°C in the dark for 30 minutes.

[0156] (5) Flow cytometry and analysis:

[0157] The red fluorescence was detected by flow cytometry at an excitation wavelength of 488 nm, and the light scattering was detected at the same time. The cell DNA content and light scattering analysis were performed using appropriate Flowjo software.

[0158] The test and analysis results are as follows Figure 3 As shown, CX5461 alone can inhibit the proliferation of non-small cell lung cancer cell lines and produce G2 / M cycle arrest, and the combination of CX5461 and AZD7648 shows obvious synergistic effect.

[0159] Example 4. Synergistic effect of CX5461 and AZD7648 on cancer cell apoptosis

[0160] A549 and H358 cell lines were treated with CX5461 and AZD7648 alone or in combination, with the drug concentrations and treatment methods being the same as in Example 3.

[0161] Recover the cell culture medium, wash the cells once with PBS, digest the cells with trypsin, add the collected cell culture medium and gently blow, collect the cells in a 2ml EP tube. Centrifuge at 1500rpm for 5min, carefully wash off the supernatant, add 1ml 4℃ pre-cooled PBS, resuspend the cells, centrifuge again to precipitate the cells, and carefully remove the supernatant.

[0162] Dilute Binding Buffer with deionized water at a ratio of 1:9 (2 ml 10X Binding Buffer + 18 ml deionized water).

[0163] Resuspend cells in 1X Binding Buffer.

[0164] Take 100 μl of cell suspension into a 2 ml EP tube, add 5 μl of Annexin V / FITC, mix well, and incubate at room temperature in the dark for 5 min.

[0165] 5 μl of propidium iodide solution (PI) was added, and 400 μl of PBS was added for immediate flow cytometry detection.

[0166] Experimental design:

[0167] Blank tube: Negative control group cells, without AnnexinV / FITC, propidium iodide solution (PI). Used to adjust voltage.

[0168] Single staining tube: positive control group cells, only add AnnexinV / FITC. Used for compensation adjustment.

[0169] Detection tube: Treated cells, add Annexin V / FITC, propidium iodide solution (PI). Use blank tube and single-stained tube to adjust voltage compensation and obtain the required flow data.

[0170] Test results such as Figure 4 As shown, CX5461 alone can induce cell death (or apoptosis), and the combination of CX5461 and AZD7648 showed obvious synergistic effect. The cell mortality rate of the combination group was higher than that of CX5461 and AZD7648 alone, and the cell mortality rate of the combination group exceeded the sum of the mortality rates of the two single drug groups.

[0171] Example 5. PDS and AZD7648 synergistically inhibit cancer cell growth

[0172] According to the method of Example 1, the inhibitory effects of PDS and AZD7648 alone and in combination on human non-small cell lung cancer cell lines A549 and H358 were detected. A549 cell line and H358 cell line were set up with PDS treatment group, AZD7648 treatment group, and combination treatment group, respectively. Each treatment group was set up with 6 concentrations (0.3125μM, 0.625μM, 1.25μM, 2.5μM, 5μM, 10μM), and a blank control was set up. Six parallel wells were set up for each concentration. The results are shown in Figure 1. Figure 5 As shown, the combination of PDS and AZD7648 also exhibited a strong synergistic effect.

[0173] Due to the availability of materials, the above examples illustrate the synergistic effect of available G4 stabilizer drugs, CX5461 and PDS, and available DNA-PK inhibitor drugs, AZD7648, but those skilled in the art can understand that other G4 stabilizer drugs can have similar synergistic effects with other DNA-PK inhibitor drugs based on the same principle of action on the same target. The purpose of selecting and describing exemplary embodiments in the present disclosure is to explain the specific principles of the present disclosure and its practical application. Those of ordinary skill in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A composition, characterized in that Includes G4 stabilizers and DNA-PK inhibitors.

2. The composition according to claim 1, wherein The DNA-PK inhibitor is AZD7648; Preferably, the G4 stabilizer is CX-5461 and / or PDS.

3. The composition according to claim 1 or 2, wherein The content of CX-5461 is 7.8-1000nM, preferably 15.6-500nM, more preferably 31.2-250nM; or The content of PDS is 0.3125-10 μM, preferably 0.625-10 μM, more preferably 1.25-5 μM; Preferably, the content of AZD7648 is 0.3125-10 μM, preferably 0.625-10 μM, more preferably 0.625-5 μM.

4. The composition according to claim 2 or 3, wherein The molar ratio of the CX-5461 to the AZD7648 is 0.0008-3.2:1, preferably 0.025-0.1:1; or The molar ratio of the PDS to the AZD7648 is 0.03-32:1, preferably 0.25-1:

1.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a drug or a medicine kit for treating cancer.

6. Use of G4 stabilizer and DNA-PK inhibitor in the preparation of drugs or drug kits for treating cancer.

7. The use according to claim 5 or 6, wherein the cancer is a solid tumor or a hematological tumor; Preferably, the cancer is selected from one or more of the following groups: non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small intestine cancer, appendix cancer, colorectal cancer, cancer of unknown primary, endometrial cancer, mixed cancer types, pancreatic cancer, hepatobiliary cancer, small cell lung cancer, cervical cancer, germ cell cancer, breast cancer, ovarian cancer, gastrointestinal neuroendocrine cancer, bladder cancer, myeloma, myelodysplasia / myeloproliferative neoplasm, head and neck cancer, esophageal cancer, gastric cancer, soft tissue sarcoma, mesothelioma, thyroid cancer, lymphoma, leukemia and melanoma; More preferably, the cancer is non-small cell lung cancer.

8. Use of a DNA-PK inhibitor in the preparation of a drug or a drug kit for improving the therapeutic effect of a G4 stabilizer.

9. The use according to any one of claims 6 to 8, wherein the DNA-PK inhibitor is AZD7648; Preferably, the G4 stabilizer is CX5461 and / or PDS.

10. The use according to any one of claims 6, 7 and 9, wherein: The effective amount of CX-5461 in the drug or kit is 7.8-1000 nM, preferably 15.6-500 nM, more preferably 31.2-250 nM; or The effective amount of PDS is 0.3125-10 μM, preferably 0.625-10 μM, more preferably 1.25-5 μM; Preferably, the effective amount of AZD7648 is 0.3125-10 μM, preferably 0.625-10 μM, more preferably 0.625-5 μM.

11. The use according to any one of claims 6 to 10, wherein: The molar ratio of the effective amount of CX-5461 to the effective amount of AZD7648 is 0.0008-3.2:1, preferably 0.025-0.1:1; or The molar ratio of the PDS to the effective amount of AZD7648 is 0.03-32:1, preferably 0.25-1:1.

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