Identification and planning of cancer treatment and kit used therefor

Abstract

Provided are a method for identifying a cancer patient suitable for the treatment with a compound of formula (I) and further planning an appropriate course of treatment for the patient, and a kit for performing the method. The method involves the detection of the expression of a biomarker in a biological sample obtained from an individual suffering from cancer, and the instruction of the individual, on the basis of the expression of the biomarker, to use a treatment regimen including the compound of formula (I), and / or the use of a pharmaceutically acceptable salt thereof in the treatment of cancer, wherein T1 is C6 cyclic hydrocarbon, and R1 is C1-C8 aliphatic hydrocarbon.

Description

Cancer treatment identification and planning and the kits used Technical Field

[0001] This invention relates to the field of cancer treatment, and more specifically, to the identification and planning of cancer treatment and the provision of kits for use in the identification and planning, so as to provide cancer patients with more precise and appropriate treatment, shorten the time of ineffective treatment, or even reduce the overall side effects during cancer treatment, thereby effectively prolonging the patient's life and improving the patient's quality of life during the extended life period. Background Technology

[0002] In medicine, a tumor refers to an abnormal cellular disease. This disease occurs when, under the influence of various carcinogenic factors, cells in a localized area of ​​the body lose control over their normal growth at the genetic level, leading to abnormal cell proliferation and aggregation into a mass, hence the name "tumor." Cancer is the most common type of tumor. The aggregation of abnormally proliferating cancer cells is called a "malignant tumor," which can spread or metastasize to other tissues or organs, causing severe and difficult-to-treat physiological dysfunctions and damage, and even death.

[0003] Current main methods for treating cancer include traditional surgical resection, chemotherapy, radiation therapy, targeted (gene) therapy, and immunotherapy. These methods remove the tumor or shrink or eliminate it, thereby prolonging the patient's life and increasing the chances of treatment or even a cure.

[0004] Chemotherapy is a systemic treatment that is effective against primary lesions, metastatic lesions, and subclinical metastatic lesions. However, because the chemical drugs administered in chemotherapy cannot distinguish between tumor cells and normal cells, they kill not only tumor cells but also normal and immune cells, thus causing harm to the patient's condition. Therefore, if the chemotherapy drugs are not administered effectively (i.e., they cannot effectively kill cancer cells), it will not only fail to improve the patient's condition but also subject the patient to unnecessary toxicity and delay treatment, which is extremely detrimental to cancer patients.

[0005] In addition, like other disease treatments, the chemotherapy drugs used in cancer treatment will more or less produce side effects on patients. Since cancer patients are usually in worse physical condition than healthy people and their recovery is slower, if they can understand in advance the possible side effects or diseases that may be caused by the medication, they can prepare in advance during chemotherapy or even before chemotherapy begins, reducing or even avoiding the potential harm of the chemotherapy drugs to the patient.

[0006] The above situation highlights the clinical need for methods to accurately assess and predict the treatment response and related side effects of chemotherapy drugs for specific cancers. This is because cancer, during its pathogenesis and development, significantly alters the expression of cellular molecules at various levels, including DNA, RNA, mRNA, miRNA, and proteins. These changes can influence disease progression and response to chemotherapy drugs. Previous studies have shown that the biomarker TOPO2A protease is frequently upregulated in breast cancer patients. Patients with upregulated TOPO2A, compared to those with congenital absence or different tumor types resulting in no upregulation, respond better to anthracyclines. Therefore, the upregulation of this biomarker can predict the therapeutic effect of anthracyclines, effectively assisting clinicians in determining the suitability of anthracyclines for breast cancer patients. This can be seen, for example: Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2 / neu-amplified breast cancer: Scandinavian Breast Group Trial 9401. J Clin Oncol. 2006; 24(16):2428-2436. doi:10.1200 / JCO.2005.02.9264, the full text of which is available here. However, the aforementioned techniques are limited to the detection of single or limited biomarkers, and cannot address the diverse heterogeneity of tumors, nor can they predict treatment efficacy based on individual patient differences or drug combinations; furthermore, these techniques also ignore situations where different states such as protein phosphorylation may lead to reduced treatment response rates and increased side effects.

[0007] Given the aforementioned unresolved issues, providing a medical strategy that allows for the screening of patients suitable for specific treatment plans based on multivariate molecular testing and the prediction of treatment responses and side effects would be a great boon to patients.

[0008] The inventors of this case discovered that specific protein groups can be used to identify whether cancer patients are suitable for treatment with specific anti-cancer drugs. They also found that based on changes in the expression of specific proteins, other anti-cancer drugs can be used as needed to reduce or eliminate the possible side effects of chemotherapy, and to develop treatment plans suitable for individual patients, thus achieving a precision medicine strategy. Summary of the Invention

[0009] One object of the present invention is to provide a method for identifying tumor patients suitable for treatment with a compound of formula (I), characterized by diagnosing individuals exhibiting specific biomarkers, wherein the active ingredient comprises a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof:

[0010] Wherein, T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group; and the biomarker is selected from the group consisting of: EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5, and combinations thereof. Preferably, R1 in formula (I) is a C1-C6 aliphatic hydrocarbon group, more preferably a C5 alkyl or alkenyl group, for example, Z-Butylidenephthalide (Z-BP) is used as the active ingredient.

[0011] In a preferred embodiment of the aforementioned identification method, the biomarker is PD-L1 or IDO1. 、 At least two of EGFR, Axl1, and cMet; in another preferred embodiment, the biomarker is at least one of TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5; in yet another preferred embodiment, the biomarker is at least one of cMet, VEGF, IDH1, IDH2, PARP1, and PARP14.

[0012] Another object of the present invention is to provide a method for developing a treatment plan for an individual suffering from a tumor, comprising detecting a biomarker in a biological sample from the individual, and, when the detected biomarker is expressed, indicating that the individual's treatment plan includes the use of an active ingredient, wherein the biomarker is selected from the group consisting of EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5, and combinations thereof, and the active ingredient is a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof. Preferably, R1 in formula (I) is a C1-C6 aliphatic hydrocarbon group, more preferably a C5 alkyl or alkenyl group, for example, Z-BP is used as the active ingredient.

[0013] In a preferred embodiment of the above-described method for developing a treatment regimen, the biomarker is at least one of EGFR, PD-L1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5; in another preferred embodiment, the treatment regimen further includes the use of vaccines, gene therapy, GPCR inhibitors, LGR5 inhibitors, RNA interference agents, tyrosine kinase inhibitors, immunotherapy, anti-angiogenic drugs, metabolic enzyme inhibitors, and DNA repair inhibitors, or combinations thereof.

[0014] Another object of the present invention is to provide a kit comprising: a first component for detecting the performance of at least one of cMet, PLCE1, CCL2, PDGFRA, and LGR5; and a second component for detecting the performance of at least one of EGFR, Axl1, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2. Preferably, the kit of the present invention further comprises a third component containing a compound of formula (I) above, a pharmaceutically acceptable salt thereof, or a combination thereof. Preferably, the third component contains a compound of formula (I) wherein R1 is a C1-C6 aliphatic hydrocarbon group and / or a pharmaceutically acceptable salt thereof, more preferably containing a compound of formula (I) wherein R1 is a C5 alkyl or alkenyl group and / or a pharmaceutically acceptable salt thereof, for example, containing Z-BP and / or a pharmaceutically acceptable salt thereof.

[0015] Preferably, in the kit of the present invention, the first component and the second component are each stored in two independent spaces to facilitate the use of the first component and the second component in multiple uses, but each storage space may contain the first component and the second component at the same time.

[0016] The detailed technical content and some embodiments of the present invention will be described below to enable those skilled in the art to understand the features of the present invention. Attached Figure Description

[0017] Figure 1 is a photograph of tumor sections of biological samples from cancer patients analyzed by immunohistochemistry (IHC), showing the expression of EGFR protein, IDH1 protein, AXL protein, p-AXL protein, cMet protein, p-cMet protein, IDO1 protein, and PD-L1 protein, respectively.

[0018] Figure 2 shows the changes in mRNA expression levels in primary cells from extended samples of three brain tumor patients (T9, T14, and T21) analyzed by RT-PCR. Figures 2(A) to 2(F) are bar graphs showing the changes in mRNA expression levels of TP53, ANGPT2, CCL2, PLCE1, PDGFRA, and LGR5 after Z-BP treatment (untreated and after 24 hours). In the figures, "***" indicates a p-value < 0.001, "**" indicates a p-value < 0.01, and "*" indicates a p-value < 0.05. Detailed Implementation

[0019] The following describes some embodiments according to the present invention; however, the present invention can be practiced in many different forms without departing from the spirit of the present invention, and the scope of protection of the present invention should not be construed as limited to those stated in the specification.

[0020] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification shall be understood to include both singular and plural forms; “prediction” refers to the likelihood that an individual with a tumor will respond to treatment with an anticancer drug, which involves an individual assessment of the expected response of the tumor patient; “individual” refers to a mammal, which may be human or non-human.

[0021] In this article, "treatment" should not be interpreted as treating an individual until complete recovery, but rather includes therapeutic treatments that maintain an individual's disease progression or symptoms at a substantially static level, increase an individual's recovery rate, improve the severity of a specific condition, and improve a patient's quality of life, as well as prevention or intervention. The goal of treatment is to prevent or reduce undesirable physiological changes or diseases such as cancer. Beneficial treatment effects include, but are not limited to, relief of disease symptoms, shortening of disease duration, stabilization of disease state, and improvement of detectable and undetectable pathological conditions. "Treatment" can also refer to a longer survival compared to an untreated group. Individuals requiring treatment include those with cancer.

[0022] In this article, “biomarker performance” refers to at least one of the gene performance, protein performance, and protein phosphorylation performance of a biomarker in a biological sample taken from an individual with cancer.

[0023] Compounds of formula (I) and their pharmaceutically acceptable salts are known to have therapeutic benefits against a wide range of cancers, including colorectal cancer, rectal cancer, pancreatic cancer, bile duct cancer, liver cancer, gastric cancer, bladder cancer, lung cancer, pleural mesothelioma, breast cancer, gynecologic tumors, testicular cancer, brain cancers (including meningiomas and gliomas such as glioblastoma), and hematologic cancers (including leukemia, lymphoma, and multiple myeloma).

[0024] Wherein, T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group. Preferably, R1 is a C1-C6 aliphatic hydrocarbon group, and more preferably, R1 is a C5 alkyl or alkenyl group, such as Z-butylphthalide (Z-BP) or E-butylphthalide (E-BP).

[0025] The inventors of this case have discovered that for cancer patients whose lives can be effectively extended by administering the compound of formula (I) and / or its pharmaceutically acceptable salt, their biological samples will exhibit specific biomarkers, including EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5, especially at least one of EGFR, Axl1, cMet, PD-L1, and IDO1, or at least one of TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5, or at least one of cMet, VEGF, IDH1, IDH2, PARP1, and PARP14. Therefore, these biomarkers can be used to predict whether cancer patients are suitable for treatment with compounds of formula (I) and / or their pharmaceutically acceptable salts, avoiding the waste of treatment time and resources caused by ineffective treatment and improving the success rate of treatment.

[0026] The inventors of this case further discovered that after cancer patients were treated with compound (I) and / or its pharmaceutically acceptable salts, there were significant changes in the levels of EGFR, PDL1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5 in their bodies. However, the significant changes in the levels of the aforementioned biomarkers reflect that the administration of compound (I) and / or its pharmaceutically acceptable salts may have adverse effects on the patient's health. Therefore, when treating with compound (I) and / or its pharmaceutically acceptable salts, other treatments or other drugs can be administered in combination to alleviate or eliminate the adverse effects caused by the aforementioned significant changes and improve the overall treatment efficacy.

[0027] Therefore, the present invention relates to a method for identifying patients suitable for treatment with an active ingredient in cancer, characterized in that it diagnoses individuals exhibiting specific biomarkers, wherein the active ingredient comprises a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof:

[0028] Wherein, T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group; and the biomarker is selected from the group consisting of: EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5, and combinations thereof. Preferably, R1 in formula (I) is a C1-C6 aliphatic hydrocarbon group, more preferably a C5 alkyl or alkenyl group. In some specific embodiments of the identification method according to the present invention, Z-BP is contained in the active ingredient.

[0029] Preferably, the pharmaceutically acceptable salt of the compound of formula (I) refers to the lithium, sodium, potassium, magnesium, calcium and / or zinc salts of the compound.

[0030] In the identification method according to the present invention, an in vitro analysis is performed on a biological sample from an individual suffering from a tumor to determine whether the biological sample exhibits a desired biomarker. The biological sample can be any biological material obtained from the individual suffering from the tumor, and the biological sample contains gene or protein expression suitable for detection. In some embodiments, the sample contains the patient's genetic material, such as DNA, RNA, and mRNA. In some embodiments, the sample contains proteins.

[0031] For example, the biological sample can be an extended specimen of an individual suffering from a tumor, such as cellular and / or non-cellular materials, including tumor tissue, blood, plasma, serum, cerebrospinal fluid, extracellular vesicles (exosomes), exosomes, cell-free nucleic acid (cfDNA), primary cells isolated from the tumor, ascites, peritoneal lavage fluid, bone marrow fluid, pleural effusion, urine, saliva, or a combination thereof, wherein the exosomes contain deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein.

[0032] In some embodiments, the biological sample is tumor tissue. In some embodiments, the biological sample is primary cells isolated from a tumor. In some embodiments, the biological sample is blood, plasma, or serum, preferably plasma.

[0033] The desired biological sample can be obtained by any suitable method. For example, but not limited to, the desired biological sample can be obtained by blood draw, biopsy, surgical removal, or by simple collection.

[0034] The biomarker can be detected and analyzed using any suitable method. For example, but not limited to, it can be detected and analyzed using immunohistochemistry (IHC), immunofluorescence assay (IF), flow cytometry, Western ink dot assay, enzyme-linked immunosorbent assay (ELISA), RT-PCR, gene sequencing, and / or microarrays.

[0035] In some specific embodiments of the identification method according to the present invention, it is preferred to diagnose individuals exhibiting at least one of TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5. In another specific embodiment of the identification method according to the present invention, it is preferred to diagnose individuals exhibiting at least one of cMet, VEGF, IDH1, IDH2, PARP1, and PARP14.

[0036] In some specific embodiments of the identification method according to the present invention, it is preferred to diagnose an individual with a tumor exhibiting at least two of PD-L1, IDO1, EGFR, Axl1 and cMet, especially an individual exhibiting EGFR and Axl1, EGFR and cMet, Axl1 and cMet, or EGFR and all three of Axl1 and cMet.

[0037] In some embodiments of the present invention, the tumor patient has not received any anti-cancer treatment prior to the identification method of the present invention. In yet another embodiment of the present invention, the tumor patient is a brain cancer recurrence patient who has previously received first-line treatment such as surgery, chemotherapy, or radiation therapy.

[0038] The present invention further relates to a method for developing a treatment regimen for an individual suffering from a tumor, comprising detecting a biomarker in a biological sample from the individual, and when the detected biomarker is expressed, indicating that the individual's treatment regimen includes the use of an active ingredient, wherein the biomarker is selected from the group consisting of EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5 and combinations thereof, and the active ingredient is a compound of formula (I) above, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0039] In the method for developing a treatment regimen according to the present invention, suitable biological samples and detection methods are as described in the foregoing identification methods. Preferably, in the method for developing a treatment regimen according to the present invention, at least one of EGFR, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5 is detected.

[0040] In the method of developing a treatment plan according to the present invention, it is preferable to further compare the changes in the biomarker performance of the individual with tumor before and after administration of the active ingredient, and when there are significant changes, to further combine the developed treatment plan with other therapeutic drugs or methods corresponding to the biomarker, so as to avoid situations that are detrimental to the health of the individual with tumor related to changes in the performance of the biomarker.

[0041] For example, studies have found that some cancer patients who can achieve beneficial therapeutic effects by administering compound (I) and / or its pharmaceutically acceptable salts experience an increase in the levels of VEGF, IDH1, PARP14, TP53, and / or ANGPT2 after administration. This indicates that treating these cancer patients with compound (I) and / or its pharmaceutically acceptable salts may cause corresponding side effects, and therefore appropriate therapeutic drugs or methods should be used to alleviate these side effects. For example, increased expression of VEGF and ANGPT2 is known to be associated with angiogenesis, so anti-angiogenic drugs such as bevacizumab, ranibizumab, aflibercept, sunitinib, sorafenib, and faricimab can be used in combination; increased expression of IDH1 is known to be associated with cancer cell metabolism and tumor growth, so metabolic enzyme inhibitors such as enasidenib and ivonirb can be used in combination; increased expression of PARP14 is known to be associated with cancer cell gene repair and drug resistance, so it can be used in combination with... DNA repair inhibitors, such as olaparib, niraparib, tazolephosphonate, and rucaparib, can be used. Increased TP53 mutations are known to be associated with cancer cell growth, mutation, invasion, and metastasis; therefore, tyrosine kinase inhibitors, such as gefitinib, erlotinib, osimertinib, lapatinib, avapritinib, regorafenib, imatinib, and axitinib, can be used in combination.

[0042] Furthermore, IDH2 is known to be associated with cancer cell oxidation and metabolism; therefore, if significant changes occur in IDH2 expression, metabolic enzyme inhibitors such as enasidenib and ivonib can be used in combination. PARP1 is known to be associated with cancer cell gene repair; therefore, if significant changes occur in PARP1 expression, DNA repair inhibitors such as olaparib, niraparib, tazolephosphonate, and rucaparib can be used in combination. PLCE1 is known to be associated with cancer cell metastasis and growth signal transduction; therefore, if significant changes occur in PLCE1 expression, gene therapy or RNA interference agents can be used in combination. Gene therapies include CAR-T cell therapy, TCR-T cell therapy, and / or administration of at least one of oncolytic viruses, gene-edited viruses, and gene-edited cells. RNA interference agents include siRNA, miRNA, shRNA, and antisense oligonucleotides. CCL2 is known to be associated with tumor immunity, growth, and metastasis. Therefore, if significant changes occur in CCL2 levels, gene therapy or RNA interference agents can be used in combination. Gene therapies include cell therapy such as CAR-T therapy, TCR-T therapy, and / or administration of at least one of oncolytic viruses, gene-edited viruses, and gene-edited cells. RNA interference agents include siRNA, miRNA, shRNA, and antisense oligonucleotides. PDGFRA is known to be associated with the role of tyrosine kinase in driving tumor growth, metastasis, and survival. Therefore, if significant changes occur in PDGFRA levels, tyrosine kinase inhibitors can be used in combination, such as gefitinib, erlotinib, osimertinib, lapatinib, avapritinib, regorafenib, imatinib, and axitinib.LGR5 is known to be associated with the regenerative capacity of cancer cell tissues. Therefore, if significant changes occur in LGR5 levels, LGR5 inhibitors can be used in combination, such as anti-LGR5 monoclonal antibodies, LGR5 antibody-drug conjugates (ADCs) like LGR5-MMAE ADC or LGR5-DM1 ADC, spike protein inhibitors, tanase inhibitors, β-catenin inhibitors, gene therapies like LGR5 CAR-T therapy, and RNA interference agents like LGR5 siRNA. shRNA, etc.; In addition, since LGR5 is a protein of the G protein-coupled receptor (GPCR) family, when LGR5 shows significant changes, GPCR inhibitors, such as mogamulizumab, plerixafor, and maraviroc, can also be used in combination. Similarly, IDO1 is known to be associated with immune escape from cancer cells, so if IDO1 shows significant changes, immunotherapy can be used in combination, such as pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0043] Therefore, the treatment regimen formulated according to the present invention may further include the use of at least one of a vaccine, gene therapy, GPCR inhibitor, LGR5 inhibitor, RNA interference agent, tyrosine kinase inhibitor, immunotherapy, anti-angiogenic drug, metabolic enzyme inhibitor, and DNA repair inhibitor.

[0044] In some specific embodiments of the invention, the treatment regimen includes the use of a vaccine comprising at least one of the following: Sipuleucel-T, Talimogene laherparepvec (T-VEC), CIMAvax-EGF, and Bacillus Calmette-Guérin (BCG) vaccine.

[0045] In some specific embodiments of the present invention, the formulated treatment regimen includes the use of gene therapy, wherein the gene therapy includes cell therapy and / or administration of at least one of oncolytic viruses, gene-editing viruses and gene-edited cells; wherein the cell therapy includes at least one of CAR-T therapy and TCR-T therapy.

[0046] In some specific embodiments of the invention, the treatment regimen includes the use of a GPCR inhibitor selected from at least one of the following: mogamulizumab, plerixafor, and maraviroc.

[0047] In some specific embodiments of the present invention, the formulated treatment regimen includes the use of an LGR5 inhibitor selected from at least one of the following: anti-LGR5 monoclonal antibodies, LGR5 antibody-drug conjugates (ADCs), spike protein inhibitors, tank enzyme inhibitors, β-catenin inhibitors, LGR5 CAR-T therapy, LGR5 siRNA, and LGR5 shRNA.

[0048] In some specific embodiments of the invention, the treatment regimen includes the use of an RNA interference agent selected from at least one of the following: siRNA, miRNA, shRNA, and antisense oligonucleotides.

[0049] In some specific embodiments of the present invention, the formulated treatment regimen includes the use of a tyrosine kinase inhibitor, which comprises at least one of the following: gefitinib, erlotinib, osimertinib, lapatinib, avapritinib, regorafenib, imatinib, and axitinib.

[0050] In some specific embodiments of the present invention, the formulated treatment regimen includes immunotherapy, which includes administration of at least one of the following: pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and ipilimumab.

[0051] In some specific embodiments of the present invention, the formulated treatment regimen includes the use of an anti-angiogenic drug, which includes at least one of the following: bevacizumab, ranibizumab, aflibercept, sunitinib, sorafenib, and faricimab.

[0052] In some specific embodiments of the invention, the treatment regimen includes the use of a metabolic enzyme inhibitor, which comprises at least one of the following: enasidenib and ivonib.

[0053] In some specific embodiments of the present invention, the formulated treatment regimen includes the use of a DNA repair inhibitor, which is a PARP inhibitor. The PARP inhibitor may be at least one of olaparib, niraparib, tazolephosphonate, and rucaparib.

[0054] The present invention further provides a kit for identifying tumor patients suitable for treatment with an active ingredient comprising a compound of formula (I) above and / or a pharmaceutically acceptable salt thereof. The kit comprises a first component and a second component, wherein the first component is used to detect the expression of at least one biomarker of cMet, PLCE1, CCL2, PDGFRA, and LGR5, and the second component is used to detect the expression of at least one biomarker of EGFR, Axl1, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2. For example, the first component comprises an antibody and / or detection gene capable of specifically binding to at least one of cMet, PLCE1, CCL2, PDGFRA, and LGR5, and the second component comprises an antibody and / or detection gene capable of specifically binding to at least one of EGFR, Axl1, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2.

[0055] Preferably, the second component of the kit according to the invention is for detecting the expression of at least one of EGFR, Axl1, PD-L1, and IDO1 (e.g., for detecting the expression of EGFR and Axl1), and therefore may contain antibodies and / or detection genes that specifically bind to at least one of EGFR, Axl1, PD-L1, and IDO1 (e.g., antibodies and / or detection genes that specifically bind to EGFR, and antibodies and / or detection genes that specifically bind to Axl1); or for detecting the expression of at least one of VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2, and therefore contains antibodies and / or detection genes that specifically bind to at least one of VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2.

[0056] The present invention also provides a kit for developing customized treatment plans for cancer patients, the kit comprising the first and second components described above, and further comprising a third component containing the compound of formula (I) and / or its pharmaceutically acceptable salt.

[0057] When using the kit of the present invention to develop a customized treatment plan, if there is already performance data of the target biomarker of the patient before treatment (i.e., the first set of data), the patient is first given the third set of data, and then the biological sample (e.g., blood sample) taken from the patient who received the third set of data is processed with the first set of data and the second set of data respectively, and the performance data of the target biomarker of the sample is analyzed (i.e., the second set of data). Subsequently, as described above for the method of developing a treatment plan for an individual with a tumor, the second set of data is compared with the first set of data to understand whether there is an improvement in the performance of the biomarker, so as to determine whether other therapeutic drugs and / or methods should be combined when treating the tumor patient with the active ingredient containing the compound of formula (I) and / or its pharmaceutically acceptable salt.

[0058] When using the kit of the present invention to develop a customized treatment plan, if lesion cells are obtained before treatment with the active ingredient, for example, the cell sample can be treated with a portion of the first component and a portion of the second component to obtain a first set of data. Then, another sample of the cells can be treated with the third component for a sufficient period of time. The remaining first and second components can then be used to treat the cells treated with the third component to obtain a second set of data. The second set of data is then compared with the first set of data to determine whether there is an improvement in the performance of a single or multiple biomarkers. This determines whether other therapeutic drugs and / or methods should be combined when treating cancer patients with an active ingredient containing a compound of formula (I) and / or its pharmaceutically acceptable salts, thereby developing a customized treatment plan. Therefore, when the kit of the present invention includes the third component, the first and second components can be stored in two separate spaces (e.g., two separate spaces, each containing the first and second components; or the first and second components can be stored separately as needed) for multiple uses.

[0059] In the kit according to the present invention, the first component and the second component can be packaged separately and stored in different independent spaces, or stored in the same space (i.e., the first component and the second component coexist in the same space), provided that the first component and the second component do not interfere with each other to obtain the desired detection results. When the kit of the present invention includes a third component, the third component is stored in a storage space independent of the first component and the second component. The kit of the present invention may also include an instruction manual to facilitate the user in processing and applying the reagents according to the prescribed procedures and processes, and to adjust the application method and dosage of each reagent as needed.

[0060] The present invention will now be further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the invention. The scope of protection of the invention is as described in the claims.

[0061] Example

[0062] Experimental materials

[0063] 1. "Cerebraca chip" is a chip containing Z-BP that can be implanted in the brain of patients (75 mg / chip; provided by Changhong Biotechnology Co., Ltd., product numbers: 040715070200, 040718040100, 040721100100).

[0064] 2. RNA extraction kit (RNeasy plus kit), purchased from Qiagen NV, Germany.

[0065] 3. Illumina kit (Illumina Stranded mRNA Prep), purchased from Illumina Inc., USA.

[0066] 4. RevertAid First Strand cDNA Synthesis Kit, purchased from Thermo Fisher Scientific Inc.

[0067] 5.2100 Bioanalyzer microfluidic electrophoresis analysis system (Agilent 2100 Bioanalyzer), purchased from Agilent Technologies, Inc.

[0068] 6. RNA sequencing kit (HiSeq Rapid PE Cluster Kit v2), purchased from Illumina Inc., USA.

[0069] 7. PGS (Partek Genomics Suite) software for gene alignment, purchased from Inmena Technologies, Inc., USA.

[0070] 8. The RefSeq database is a non-redundant sequence database created by NCBI by further reorganizing GenBank sequences. Its sequence format is almost identical to that of GenBank.

[0071] Experimental methods

[0072] 1. RNA Sequencing Method: The main steps include extracting RNA from the sample using TRIzol, reverse transcribing it into cDNA, cutting the cDNA into appropriately sized fragments, and ligating specific DNA sequence adapters to both ends of the cDNA fragments to facilitate subsequent sequencing analysis. The cDNA library is then fed into a high-throughput sequencer (such as the Inmena platform) for sequencing to obtain a large number of short DNA fragment data, allowing for the understanding of transcriptional activity at specific time points. Next, bioinformatics tools are used to compare the obtained short sequence data with a reference genome or transcriptome, quantifying the expression levels of each gene to obtain the differences in gene expression and their biological significance under different samples or conditions.

[0073] 2. Reverse Transcription Polymerase Chain Reaction (RT-PCR): A method that uses reverse transcriptase to convert RNA samples into cDNA, and then uses PCR technology to exponentially amplify and detect the cDNA. Its core steps include: reverse transcription of the RNA template into cDNA by reverse transcriptase; then, using primers and DNA polymerase, repeating denaturation (approximately 95°C to dissociate double-stranded DNA into single strands), annealing (lowering the temperature to allow the primers to bind to the target DNA sequence), and extension (raising the temperature to approximately 72°C to allow the DNA polymerase to extend the new DNA strand from the primers) for approximately 25-35 cycles to massively replicate the cDNA fragment; finally, real-time monitoring is performed by binding a fluorescent substance, detecting changes in the fluorescence signal during PCR to determine the presence and content of the target RNA.

[0074] 3. Immunohistochemistry (IHC): IHC is a method for detecting the presence of specific biomarkers in cells or tissues. It utilizes the specific binding between antibodies and antigens to detect the expression level and location of target proteins in tissues. The procedure involves preparing a slide of the tissue to be studied; applying a primary antibody to the slide to bind to the antibody-antigen complex generated by the specific antigen; then adding a secondary antibody; in the presence of the antigen and chromogen, enzymes form colored deposits at the antibody-antigen binding sites, which can be imaged under a microscope to confirm the presence and degree of staining of the deposits.

[0075] Example 1

[0076] Brain tumors from glioblastoma patients were prepared into pathological tissue sections, which were then subjected to immunohistochemical staining (IHC) with antibodies against EGFR, IDH1, AXL1, p-AXL1, cMET, p-cMET, IDO-1, and PD-L1. The tumor area was then observed under a microscope, and the results are shown in Figure 1.

[0077] Figure 1 shows that the biomarkers EGFR, Axl1, p-Axl1, cMET, p-cMET, IDH1, IDO1, and PD-L1 proteins in the tumor region of glioblastoma patients all showed significant signal expression. This indicates that these specific proteins are significantly expressed in tumor sections of cancer patients and can be used as a combination of potential biomarkers for identifying cancer patients.

[0078] Example 2

[0079] In this study, the biomarker expression of tumor tissues in seven glioblastoma patients treated with a Cerebraca wafer containing Z-BP, a representative example of the compound of formula (I) of the present invention, was detected. The median overall survival (mOS) of glioblastoma patients who expressed at least one of the above biomarker groups, namely EGFR, IDH1, Axl1, p-Axl1, cMET, p-cMET, IDO-1, and PD-L1, was compared with that of glioblastoma patients who did not express any of the biomarkers.

[0080] The median overall survival was compared by using Kaplan-Meier curves to estimate and compare the survival probabilities of individuals in different groups at specific time points to assess the impact of biomarker performance on patient survival. The results are shown in Table 1, where n is the number of patients. Furthermore, the median overall overall survival for glioblastoma patients in this embodiment was 477 days, or 15.7 months.

[0081] Table 1

[0082] As shown in Table 1, cancer patients exhibiting specific biomarkers showed significantly higher median overall survival after Z-BP treatment compared to patients without biomarkers. Furthermore, cancer patients exhibiting the biomarkers cMET and / or the combination of EGFR and Axl1 had significantly higher median overall survival than cancer patients exhibiting other single biomarkers.

[0083] The results of this embodiment show that cancer patients exhibiting specific biomarkers or combinations thereof demonstrate significantly higher median overall survival after treatment with compound (I) than patients without biomarkers. This indicates that the performance of these biomarkers can be used to predict the clinical response of glioblastoma patients treated with compound (I), and thus can help identify cancer patients suitable for treatment regimens containing compound (I).

[0084] Example 3

[0085] Three primary cells were cultured from extended specimens of patients with recurrent glioblastoma, and the changes in the expression of various genes in the cells before and after 24 hours of Z-BP treatment were examined.

[0086] First, the cultured primary cells were divided into an experimental group and a control group (Ctl group). The former was treated with Z-BP (400 μM concentration, 400 μmol) for 24 hours, while the latter was not treated with Z-BP.

[0087] RNA extraction was performed using an RNA extraction kit, followed by reverse transcription of total RNA into cDNA using an Illumina kit (Inmena Biosciences, USA). The specific steps were as follows: 2 μg of total RNA was incubated at 37°C for 1 hour with RNase-free DNase I (1 unit / µL); 2 μL of EDTA (25 mmol / L) was added and incubated at 65°C for 10 minutes to inactivate DNase I; then, the mixture was incubated at 56°C for 5 minutes with 3 μL of random primers and 1 μL of dNTPs (10 mmol / L); the temperature was lowered to 4°C, and then incubated at room temperature with RNaseOUT reagent for 2 minutes; next, 3 μL of reverse transcriptase (200 units) was added and incubated at room temperature for 10 minutes, followed by incubation at 42°C for 1 hour; finally, the mixture was incubated at 70°C for 15 minutes. The final cDNA (50 nmol / µL) was obtained for subsequent RNA sequencing.

[0088] Table 2 below shows the initiation sequences used in this embodiment.

[0089] Table 2

[0090] Next, RNA sequencing was performed to determine the gene changes induced by Z-BP treatment. First, samples with an RNA integrity number (RIN) greater than 8 were screened using a 2100 Bioanalyzer microfluidic electrophoresis system. Then, 2 micrograms of total RNA were collected, and an RNA sequence library was prepared using RNA sequencing kits. The library was read using single-end sequencing, with a minimum read depth of 400 million reads per sample. Subsequently, the read sequences were aligned with the human genome GRCh37 / Hg19 using PGS software. Based on mRNA and long non-coding RNA (lncRNA) annotations from the RefSeq database, the transcriptional level was quantified as Reads Per Kilobase Million (RPKM). The sequenced data underwent alignment, differential expression analysis, fold change calculation, and p-value analysis. The results are shown in Table 3. ENSG is the Human Gene Identification ID used in the Ensembl Genome Browser, which is the gene number.

[0091] As shown in Table 3, compared with the control group without Z-BP treatment, biomarkers such as VEGF, IDH1, IDH2, PARP1, and PARP14 showed significant changes in gene expression levels in extended sample cells from Z-BP-treated patients. Therefore, they can be used as biomarkers to identify whether cancer patients are suitable for treatment with compound (I). Table 3 also shows that VEGF, IDH1, and PARP14 all showed significant upregulation of gene expression levels after Z-BP treatment, indicating the side effects that treatment with compound (I) may cause, further providing information on appropriate treatment options.

[0092] Table 3

[0093] Example 4

[0094] Based on the fold change in the expression of other genes in RNA sequencing and in accordance with the RNA extraction and sequencing method in Example 3, the expression of other biomarkers in glioblastoma patients was further compared, and the results are shown in Table 4.

[0095] As shown in Table 4, compared with the control group without Z-BP treatment, the extended specimen cells of the patients showed significant changes in gene expression levels of biomarkers such as TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5 after Z-BP treatment. Therefore, these can be used as biomarkers to identify whether cancer patients are suitable for treatment with compound (I). Table 4 also shows that TP53 and ANGPT2 showed significant upregulation of gene expression levels after Z-BP treatment, indicating the side effects that may be caused by treatment with compound (I), further providing relevant information on appropriate treatment options.

[0096] Table 4

[0097] In addition, changes in the mRNA expression levels of specific biomarkers TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5 were confirmed after Z-BP treatment.

[0098] Three primary cell lines obtained from relapsed glioblastoma patients were cultured using the aforementioned method, and were divided into an experimental group treated with Z-BP (400 μmol concentration) for 24 hours and a control group not treated with Z-BP. Total RNA was then extracted using the RNeasy Plus kit (Kagem GmbH, Germany) according to the manufacturer's instructions. Subsequently, the total RNA (2 μg) was reverse transcribed into cDNA using the RevertAid kit (Thermo Fisher Scientific, USA). The procedure for using this kit is as follows: RNA is mixed with nuclease-free water and reacted at 60°C for 10 minutes. Then, 1 μL of oligomeric (dT) primer is added at 65°C and reacted for 5 minutes. Next, 2 μL of dNTPs (10 mmol / L), 4 μL of 5X reaction buffer, 1 μL of RevertAid reverse transcriptase, and 1 μL of RiboLock RNase inhibitor are added. The mixture is then reacted at 45°C for 60 minutes, followed by a final reaction at 70°C for 5 minutes. The resulting cDNA (50 nmol / μL) is the sample used for RT-PCR. The results are shown in Figure 2.

[0099] As shown in Figure 2, compared to the control group without Z-BP treatment, the experimental group showed significant changes in the mRNA expression levels of specific biomarkers TP53, ANGPT2, CCL2, PLCE1, PDGFRA, and LGR5. These results can be used to predict the clinical response of glioblastoma patients treated with compound (I). Furthermore, changes in specific biomarker expression can also help develop treatment regimens for individuals with tumors that include compound (I) and related adjuvant anticancer drugs, thereby reducing resistance to compound (I) and improving overall efficacy.

Claims

1. A method for identifying patients suitable for treatment of tumors with an active ingredient, characterized in that, Diagnosing individuals exhibiting specific biomarkers, among whom, The active ingredient comprises a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof: Wherein, T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group; and The biomarker was selected from the following groups: EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5, and combinations thereof.

2. The method as described in claim 1, characterized in that, The biomarker is at least two of PD-L1, IDO1, EGFR, Axl1, and cMet.

3. The method as described in claim 1, characterized in that, The biomarker is at least one of TP53, PLCE1, CCL2, ANGPT2, PDGFRA, or LGR5.

4. The method as described in claim 1, characterized in that, The biomarker is at least one of cMet, VEGF, IDH1, IDH2, PARP1, and PARP14.

5. A method for developing a treatment plan for an individual suffering from a tumor, characterized in that, It includes detecting a biomarker in a biological sample from the individual, and when the detected biomarker is present, indicating that the individual's treatment regimen includes the use of an active ingredient, wherein the biomarker is selected from the group consisting of: EGFR, Axl1, cMet, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, LGR5, and combinations thereof, and the active ingredient is a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof. Wherein, T1 is a C6 cyclic hydrocarbon and R1 is a C1-C8 aliphatic hydrocarbon group.

6. The method as described in claim 5, characterized in that, The biomarker is at least one of EGFR, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53, PLCE1, CCL2, ANGPT2, PDGFRA, and LGR5.

7. The method as described in claim 6, characterized in that, The treatment regimen further includes at least one of the following: vaccine, gene therapy, GPCR inhibitor, RNA interference agent, tyrosine kinase inhibitor, immunotherapy, anti-angiogenic drug, metabolic enzyme inhibitor, and DNA repair inhibitor.

8. The method as described in claim 6, characterized in that, The treatment regimen further includes at least one of the following: vaccine, gene therapy, LGR5 inhibitor, RNA interference agent, tyrosine kinase inhibitor, immunotherapy, anti-angiogenic drug, metabolic enzyme inhibitor, and DNA repair inhibitor.

9. The method as described in claim 7, characterized in that, The treatment regimen includes the use of a tyrosine kinase inhibitor, which includes at least one of the following: gefitinib, erlotinib, osimertinib, lapatinib, avatinib, regorafenib, imatinib, and axitinib.

10. The method as described in claim 8, characterized in that, The treatment regimen includes the use of a tyrosine kinase inhibitor, which includes at least one of the following: gefitinib, erlotinib, osimertinib, lapatinib, avatinib, regorafenib, imatinib, and axitinib.

11. The method as described in claim 7, characterized in that, The treatment regimen involves the use of an RNA interference agent, wherein the RNA interference agent comprises at least one of the following: siRNA, miRNA, shRNA, and antisense oligonucleotides.

12. The method as described in claim 8, characterized in that, The treatment regimen involves the use of an RNA interference agent, wherein the RNA interference agent comprises at least one of the following: siRNA, miRNA, shRNA, and antisense oligonucleotides.

13. The method as described in claim 7, characterized in that, The treatment regimen includes the use of a GPCR inhibitor, wherein the GPCR inhibitor comprises at least one of the following: movamutumab, pelesafur, or maravirox.

14. The method as described in claim 8, characterized in that, The treatment regimen includes the use of an LGR5 inhibitor, wherein the LGR5 inhibitor comprises at least one of the following: an anti-LGR5 monoclonal antibody, an LGR5 antibody-drug conjugate, a spike protein inhibitor, a tank enzyme inhibitor, a β-catenin inhibitor, LGR5 CAR-T therapy, LGR5 siRNA, and LGR5 shRNA.

15. The method as described in claim 7, characterized in that, The treatment regimen includes the use of gene therapy, which includes cell therapy and / or administration of at least one of the following: oncolytic viruses, gene-editing viruses, and gene-edited cells.

16. The method as described in claim 8, characterized in that, The treatment regimen includes the use of gene therapy, which includes cell therapy and / or administration of at least one of the following: oncolytic viruses, gene-editing viruses, and gene-edited cells.

17. The method as described in claim 15, characterized in that, This cell therapy includes at least one of the following: CAR-T therapy and TCR-T therapy.

18. The method as described in claim 16, characterized in that, This cell therapy includes at least one of the following: CAR-T therapy and TCR-T therapy.

19. The method as described in claim 7, characterized in that, The treatment regimen includes the use of anti-angiogenic drugs, which include at least one of the following: bevacizumab, ranibizumab, aflibercept, sunitinib, sorafenib, and fareximab.

20. The method as described in claim 8, characterized in that, The treatment regimen includes the use of anti-angiogenic drugs, which include at least one of the following: bevacizumab, ranibizumab, aflibercept, sunitinib, sorafenib, and fareximab.

21. The method as described in claim 7, characterized in that, The treatment regimen includes the use of a metabolic enzyme inhibitor, which includes at least one of the following: ensidipin and evanixib.

22. The method as described in claim 8, characterized in that, The treatment regimen includes the use of a metabolic enzyme inhibitor, which includes at least one of the following: ensidipin and evanixib.

23. The method as described in claim 7, characterized in that, The treatment regimen includes the use of immunotherapy, which includes administration of at least one of the following: pembrolizumab, nivolumab, atezolizumab, acitumab, devastatin, and ivevolumab.

24. The method as described in claim 8, characterized in that, The treatment regimen includes the use of immunotherapy, which includes administration of at least one of the following: pembrolizumab, nivolumab, atezolizumab, acitumab, devastatin, and ivevolumab.

25. The method as described in claim 7, characterized in that, The treatment regimen includes the use of a DNA repair inhibitor, specifically a PARP inhibitor.

26. The method as described in claim 8, characterized in that, The treatment regimen includes the use of a DNA repair inhibitor, specifically a PARP inhibitor.

27. The method as described in claim 25, characterized in that, The PARP inhibitor is at least one of the following: olaparib, niraparib, tazoledrone, and rucaparib.

28. The method as described in claim 26, characterized in that, The PARP inhibitor is at least one of the following: olaparib, niraparib, tazoledrone, and rucaparib.

29. The method according to any one of claims 1 to 28, characterized in that, The tumor is colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, bile duct cancer, liver cancer, stomach cancer, bladder cancer, lung cancer, pleural mesothelioma, breast cancer, gynecological tumor, testicular cancer, brain cancer, blood cancer, or a combination of the foregoing.

30. The method according to any one of claims 1 to 28, characterized in that, The biological sample may be tumor tissue, blood, plasma, serum, cerebrospinal fluid, extracellular vesicles, exosomes, free nucleic acids, primary cells isolated from the tumor, ascites, peritoneal lavage fluid, bone marrow fluid, pleural effusion, urine, saliva, or a combination thereof.

31. The method according to any one of claims 1 to 28, characterized in that, The biomarker is expressed through at least one of its gene expression, protein expression, and protein phosphorylation expression.

32. A set, characterized in that, It contains The first component is used to detect the performance of at least one of cMet, PLCE1, CCL2, PDGFRA and LGR5; as well as The second component is used to detect the expression of at least one of EGFR, Axl1, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53 and ANGPT2.

33. The set as described in claim 32, characterized in that, The second component is used to detect the performance of EGFR and Axl1.

34. The set as described in claim 32, characterized in that, The second component is used to detect the performance of at least one of EGFR, Axl1, PD-L1, and IDO1.

35. The set as described in claim 32, characterized in that, The second component is used to detect the activity of at least one of VEGF, IDH1, IDH2, PARP1, PARP14, TP53, and ANGPT2.

36. The kit of claim 32, further comprising a third component, the third component comprising a compound of formula (I), a pharmaceutically acceptable salt thereof, or a combination thereof. in, T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group.

37. The set as described in any one of claims 32 to 36, characterized in that, The first component contains antibodies and / or detection genes that can specifically bind to at least one of the following: cMet, PLCE1, CCL2, PDGFRA and LGR5, and the second component contains antibodies and / or detection genes that can specifically bind to at least one of the following: EGFR, Axl1, PD-L1, IDO1, VEGF, IDH1, IDH2, PARP1, PARP14, TP53 and ANGPT2.

38. The set as described in claim 36, characterized in that, The first component and the second component are each stored in two or more independent spaces.

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