Use of hypoxia-activated compounds for preparation of medicaments for treatment of cancer patients

Abstract

The invention provides an application of a hypoxia-activated compound single drug or a combination of the hypoxia-activated compound and a PARP inhibitor in preparation of drugs for treating cancer patients, particularly, the patients are detected to have mutation of treatment genes such as RAD51D, and meanwhile, a method for treating cancer, a drug combination and a drug preparation are provided. The method, the drug combination and the pharmaceutical preparation have a good tumor inhibition effect on patients with mutation of pathogenic genes such as RAD51D and the like through single drug administration, and the good tumor inhibition effect is still achieved even if the patients are resistant to PARP inhibitors. The medicine is combined with a PARP inhibitor, and compared with a single medicine, the medicine has a certain synergistic effect.

Description

Use of hypoxia-activated compounds in the preparation of medicaments for treating cancer patients Technical Field

[0001] The present invention relates to the use of hypoxia-activated compounds alone or in combination with PARP inhibitors (PARPi) in treating diseases caused by homologous recombination pathogenic gene mutations, in particular in treating RAD51 pathogenic mutations, and provides corresponding treatment methods, drug combinations and pharmaceutical preparations, belonging to the field of biomedicine. Background Art

[0002] Reactive oxygen species produced by cellular metabolism, as well as various endogenous and exogenous factors such as ultraviolet light and genotoxic chemicals, can continuously induce DNA damage. Types of DNA damage can be broadly categorized as base damage, DNA crosslinks, single-strand breaks (SSBs), and double-strand breaks (DSBs). Double-strand breaks are the most lethal form of DNA damage. If not promptly and correctly repaired, they can cause chromosome deletions, fusions, and translocations, ultimately leading to cell death and even the development and progression of tumors. Homologous recombination (HR) is the primary mechanism for double-strand DNA repair.

[0003] DNA damage repair is a complex, tightly controlled signaling network that monitors damage, regulates transient cycles, and ultimately completes the entire process. Multiple proteins, including PARP, ATR, ATM, Wee1, DNA-PK, BRCA, RAD51, and PRMT5, play crucial roles in this process.

[0004] The RAD51 gene encodes a 339-amino acid sequence with a relative molecular mass of 4.3×10 4 The polypeptide has five homologs: XRCC2, XRCC3, RAD51B (RAD51L1), RAD51C (RAD51L2), and RAD51D (RAD551L3), which participate in the HR process together with RAD51.

[0005] PARP inhibitors can inhibit PARP's DNA single-strand damage repair function, resulting in a large amount of single-strand DNA damage in cells that cannot be repaired in a timely manner. Unrepaired single-strand DNA damage triggers replication fork collapse and generates double-strand DNA damage. Double-strand DNA damage with strong cytotoxicity can be repaired in normal cells through the homologous recombination (HR) repair pathway mediated by proteins such as BRCA and RAD51.

[0006] The initial study of PARPi in ovarian cancer enrolled people who had previously received platinum chemotherapy, and the study found that platinum sensitivity was directly associated with PARPi response (platinum chemotherapy drugs are DNA damaging agents that cause DNA cross-links, some of which can be repaired by the HR pathway, so DNA repair-deficient tumors are expected to be sensitive to platinum chemotherapy). Two PARPi have been approved for ovarian cancer: Niraparib and Rucaparib: The FDA and EMA have approved the maintenance treatment of Niraparib (regardless of BRCA1 / 2 status); Rucaparib has also been registered by the FDA and EMA as an optional treatment option for patients with BRCA1 / 2 mutation-related ovarian cancer who have previously received two lines of chemotherapy. And talazoparib has also been approved by the FDA for the treatment of BRCA mutation / HER-2 negative metastatic breast cancer (Mateo, J., Lord, CJ, Serra, V., Tutt, A., J., Castroviejo-Bermejo, M., Cruz, C., Oaknin, A., Kaye, SB, & de Bono, JS (2019). A decade of clinical development of PARP inhibitors in perspective. Annals of oncology: official journal of the European Society for Medical Oncology, 30(9), 1437-1447. https: / / doi.org / 10.1093 / annonc / mdz192).

[0007] In the prior art WO2019133697A1 (corresponding to the Chinese application CN111801117A), researchers used the PARP inhibitor Niraparib to benefit patients with homologous recombination repair (HRR) deficiencies (not BRCA1 or BRCA2). With the clinical application of PARPi, PARPi resistance has become an inevitable problem in its clinical application. Existing studies have shown that homologous recombination repair restoration (HRR), DNA replication fork protection, PARPi pharmacokinetics changes, etc. are the main causes of PARPi resistance. In order to overcome PARPi resistance and increase PARPi drug sensitivity, a variety of combined treatments are being developed, many of which have entered the clinical stage. Mainly including: PARPi-DNA alkylating agent combination; PARPi-monosporalytic herpesvirus (oHSVs) combination; PARPi-ionizing radiation combination; PARPi-immunotherapy combination; PARPi-HSP90 inhibitor combination; PARPi-WEE1 / ATR inhibitor combination; PARPi-DNMTi inhibitor combination; PARPi-CDK inhibitor combination, etc. (He Li, Zhao-Yi Liu, Nayiyuan Wu, Yong-Chang Chen, Quan Cheng and Jing Wang. PARP inhibitor resistance: the underlying mechanisms and clinical implications. Mol Cancer, 2020 Jun 20; 19(1): 107.2020. https: / / doi.org / 10.1186 / s12943-020-01227-0; Rose, M., Burgess, JT, O'Byrne, K., Richard, DJ, & Bolderson, E. (2020). PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance. Frontiers in cell and developmental biology, 8, 564601. https: / / doi.org / 10.3389 / fcell.2020.564601).

[0008] TH-302 (Evofosfamide, CAS No. 918633-87-1) and its analogs are 2-nitroimidazole-triggered hypoxia-activated prodrugs (HAPs) of bromoisophosphamide, developed by Threshold Corporation in the United States. Under hypoxic conditions, the inactive TH-302 prodrug releases the highly toxic Br-IPM. TH-302 exhibits broad in vitro and in vivo biological activity, as well as specific hypoxia-selective activation activity, inducing γH2AX phosphorylation and DNA cross-linking, leading to cell cycle arrest and cell death. Consequently, the compound is being developed as an anticancer drug by numerous pharmaceutical companies and research institutes.

[0009] A research article published by Meng FY et al. indicates that TH-302 has broad-spectrum activity against various tumors and exhibits excellent hypoxia-selective activity enhancement. The study demonstrated that the in vitro cytotoxicity of TH-302 in 32 human cancer cell lines under hypoxia was significantly stronger than that under normoxic conditions, indicating that the compound exhibits hypoxia-selective cytotoxicity against cancer cells in hypoxic environments. The single-electron reductase (POR)-dependent activity enhancement mechanism of TH-302 under hypoxia was confirmed using human cells overexpressing POR, as shown in the following reaction formula 1:

[0010] Cytochrome P450 oxidoreductase reduces the prodrug TH-302 to produce an intermediate free radical anion. The unstable free radical anion then decomposes into the cytotoxic cytotoxin Br-IPM. The key step in this process is the single-electron reduction process. Studies have shown that the presence of oxygen reverses this process, impeding it. Therefore, TH-302 can only be reduced to produce a more cytotoxic substance in an oxygen-deficient / hypoxic environment.

[0011] The applicant modified TH-302 by modifying the aminophosphate part into an aziridine structure and the nitroimidazole part into a fused ring structure containing a nitrobenzene ring (patent application PCT / CN2020 / 114519, publication number WO2021120717A1), and also obtained a broad-spectrum anticancer prodrug with hypoxic activity.

[0012] Summary of the Invention

[0013] In the efficacy test, the applicant's researchers found that hypoxia-activated TH-302 and its structurally modified aziridine analogs (patent application PCT / CN2020 / 114519, publication number WO2021120717A1) have a good tumor inhibitory effect when administered as a single drug to patients with RAD51D pathogenic gene mutations, and even if the patient is resistant to PARP inhibitors (PARPi), it still has a good tumor inhibitory effect. Further studies have found that the above-mentioned hypoxia-activated TH-302 and its structurally modified aziridine analogs, respectively, combined with PARPi, have a certain synergistic effect compared to the efficacy of a single drug.

[0014] Based on this, the present application provides the following methods for treating cancer, pharmaceutical uses, drug combinations and related pharmaceutical preparations.

[0015] A method of treating a patient with cancer or tumor whose DNA repair enzyme is damaged by using a hypoxia-activated compound or its salt, ester, solvate, isotopic variant, or isomer alone or in combination with other drugs.

[0016] In particular, the DNA repair enzyme is damaged by one or more gene defects selected from the group consisting of BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, Rad50, MRE11, p53, NBS1, XRS2, XRCC4 / XPF, ERCC1, ERCC2 / XPD, ERCC3 / XPB, ERCC4 / XPF, XRCC1, Ku80, MHS6, MGMT, PARP, ERCC5 / XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5 / XPG, ERCC6 / CSB, ERCC8 / CSA, LIG1 / DNA Ligase I, MMS19, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RBBP8, SMC5, USP11, WRN; the RAD51 is selected from XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, preferably RAD51D.

[0017] In one embodiment, the DNA repair enzyme is impaired and selected from the group consisting of:

[0018] Damage to homologous recombination DNA repair enzymes,

[0019] Damage to nucleotide excision repair enzymes,

[0020] Impaired nonhomologous end joining enzymes

[0021] Damage to base excision repair enzymes,

[0022] Damage to mismatch repair enzymes,

[0023] One or more of the Fanconi's anemia pathway repair enzymes are impaired.

[0024] More preferably, any one or more of the following are impaired homologous recombination DNA repair enzymes, impaired nucleotide excision repair enzymes, and impaired base excision repair enzymes. Even more preferably, only the homologous recombination DNA repair enzyme is impaired, or both the homologous recombination DNA repair enzyme and the nucleotide excision repair enzyme are impaired.

[0025] Particularly, the above-mentioned other drugs include PARP inhibitors, and the PARP inhibitors are selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

[0026] Particularly, the above cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

[0027] In particular, the patient is resistant or insensitive to the above-mentioned PARP inhibitors.

[0028] Sensitivity and drug resistance are qualitative judgments that evaluate whether a patient is resistant to a drug, and are commonly seen in drug sensitivity tests of microbial pathogens and antibacterial (bacterial and fungal) drugs.

[0029] Sensitivity and drug resistance are two different results that appear during clinical drug sensitivity tests. Sensitivity means that during the drug sensitivity test, the pathogen was found to be very sensitive to the drug, which means that the drug can kill all pathogens; while drug resistance means that during the test, the pathogen was found to be unable to kill the drug, which means that the drug is ineffective. Obtaining data on drug resistance in the clinic can be very meaningful for the treatment of the disease, as it can be used to select more sensitive drugs to kill the pathogen. In addition to sensitive and resistant, there is also moderate sensitivity, which means that the pathogen is moderately sensitive and can easily develop drug resistance, but it may also be useful, but the efficacy will not be very good and will not be as effective as sensitive treatment.

[0030] In chemotherapy for cancer treatment (compared to surgery and radiotherapy, chemotherapy here is broadly defined, referring to the use of drugs to treat cancer), according to clinical treatment guidelines, a series of chemotherapy drugs are usually used in sequence: when the first-administered drug has no therapeutic effect on the patient after a period of administration or the therapeutic effect is very weak, it is considered that the cancer patient has developed drug resistance, resistance or insensitivity to this drug, and other drugs (second-line drugs) are needed. Correspondingly, second-line drugs may also develop drug resistance, resistance or insensitivity after a period of administration, and third-line drugs are needed. Sensitivity and resistance are diagnosed and judged by medical professionals or clinicians.

[0031] Obviously, the specific meaning of cancer or tumor patients being sensitive to PARP inhibitors is that they have an effect after being administered with PARP inhibitors or are judged to have an effect after being diagnosed and treated by a doctor.

[0032] In one embodiment, the method of treating a patient with a cancer or tumor caused by a RAD51D gene defect is treated with a hypoxia-activated compound or a salt, ester, solvate, isotopic variant, or isomer thereof as a monotherapy, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer.

[0033] In one embodiment, the treatment method uses a hypoxia-activated compound or its salt, ester, solvate, isotopic variant, or isomer in combination with a PARP inhibitor to treat patients with cancer or tumors caused by RAD51D gene defect, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer; and the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib.

[0034] In particular, the patient is resistant or insensitive to the PARP inhibitor.

[0035] The drug described herein refers to a medicine or preparation, wherein the prepared medicine comprises a hypoxia-activated compound or a salt or solvate thereof as an active ingredient within a specific dosage range, and / or the prepared medicine is in a specific dosage form and is administered by a specific administration method.

[0036] The prepared medicine, drug, preparation may also include pharmaceutically acceptable adjuvants or excipients. The medicine may be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder for injection, or large infusions. Depending on the specific dosage form and mode of administration, the pharmaceutically acceptable adjuvants or excipients in the medicine may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavorings, sweeteners, antioxidants, surfactants, preservatives, wrappers, and pigments.

[0037] In particular, in the above-mentioned treatment method, the hypoxia-activated compound is selected from the compounds of the following formula I or II:

[0038] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO;

[0039] Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1, and the entire text is hereby incorporated into this application.

[0040] The modified TH-302 compound, Formula II, retains its hypoxia-activated mechanism of action: it exhibits enhanced cytotoxicity against cancer cells under hypoxic conditions compared to normoxic conditions. Formula II is not a P-gp substrate and effectively enters the brain and other central nervous systems, accumulating to sufficient concentrations to exert its effects. It holds promise as a treatment for primary brain cancer, tumors, or metastatic cancers or tumors that have metastasized to the brain. Formula II is relatively stable in human plasma and liver microsomes, suggesting potential for drug development. In vivo animal studies have further demonstrated that Formula II exhibits a higher mean mean (MTD) tolerance, meaning the new compound possesses a higher safety profile and clinically effective dose than previously reported compounds, potentially leading to improved efficacy against cancer or tumors. In contrast, its tumor growth inhibition efficacy is significantly superior to that of the classic chemotherapy agent ifosfamide (also a DNA alkylating agent).

[0041] Specifically, R1, R2, R3, and Cx are defined as follows:

[0042] Cx is a 5-10 membered aromatic ring or aromatic heterocycle, aliphatic heterocycle or cycloalkane, which shares two carbon atoms with the nitrobenzene ring to form a fused ring structure;

[0043] R1 is connected to any backbone atom of the Cx ring and is selected from hydrogen, halogen atoms, cyano or isocyano, hydroxyl, thiol, amino, OTs, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, alkoxy of 1-6 carbon atoms or Z-substituted alkoxy of 1-6 carbon atoms, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 、-OCOR 6 、-NR 6 SO2R 7 、-NR 6 SO2NR 6 R 7 ,

[0044] R2 and R3 are each independently hydrogen, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, or R2, R3 and the benzylic carbon atom to which they are bonded together form a 3-6 membered ring;

[0045] The group can replace the hydrogen atom at any position on the condensed ring carbon atom, and the number of substitutions is 1;

[0046] The substituent Z is a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, a C1-C3 alkyl group or a substituted alkyl group, a C1-C3 alkoxy group or a substituted alkoxy group, a C2-C3 alkenyl group or a substituted alkenyl group, a C2-C3 alkynyl group or a substituted alkynyl group, or a C3-C8 cycloalkyl group or a substituted cycloalkyl group;

[0047] R 6 、R 7 are each independently hydrogen, C1-C6 alkyl or Z-substituted C1-C6 alkyl, C2-C6 alkenyl or Z-substituted C2-C6 alkenyl, C2-C6 alkynyl or Z-substituted C2-C6 alkynyl, C3-C8 cycloalkyl or Z-substituted C3-C8 cycloalkyl, C6-C 10 Aryl or Z-substituted C6-C 10 aryl, 4-15 membered heterocyclic group or Z-substituted 4-15 membered heterocyclic group, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 6 、R 7 Together with the atoms to which it is bonded, it forms a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group.

[0048] In particular, the compound of formula I is selected from the group consisting of compounds of the following structures:

[0049] In particular, the compound of formula II is selected from the group consisting of compounds of the following structures:

[0050] Preparations related to TH-302 or its similar compounds of Formula I include oral preparations, lyophilized preparations, and concentrated injections. The relevant prescriptions, preparation methods, clinical compatibility, and administration methods are described and disclosed in detail in Threshold's relevant patents: WO2010048330A1, WO2012142520A2, and WO2008083101A1. The present invention hereby incorporates the full text of the above application texts.

[0051] TH-302 or a compound similar to Formula I is a DNA alkylating anticancer drug with a wide range of cancer therapeutic potential. TH-302-related cancer indication experiments and clinical trials are disclosed in patent applications filed by Threshold and other pharmaceutical companies (e.g., WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO20 ... 106A1, WO2020118251A1, WO2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO201200603 2A2, WO2018026606A2, WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A 2. WO2016014390A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2) and FDA-registered clinical trials (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, NCT02712567). The present invention hereby introduces all the above-mentioned related application texts and clinical trial information.

[0052] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) with potentially unrestricted growth that can spread locally by invasion and systemically by metastasis.

[0053] Examples of cancers that can be treated with TH-302 or a compound of formula I include, but are not limited to, cancers of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid. Some other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinomas, adenomas, basal cell carcinomas, dysplastic epithelial cells and carcinoma in situ of the cervix, Ewing's sarcoma, epidermoid carcinoma, giant cell tumors, glioblastoma multiforme, hair cell tumors, intestinal ganglioneuromas, proliferative corneal nerve tumors, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumors, malignant melanoma, malignant hypercalcemia, Marfanoid body type tumors, medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, squamous cell carcinoma of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilm's tumor.

[0054] PARP is an enzyme known as poly (ADP-ribose) polymerase (PARP). PARP is a DNA repair enzyme that plays a key role in the DNA repair pathway. When single-strand DNA breaks, PARP is activated. As a molecular sensor of DNA damage, it recognizes and binds to the DNA break site, activating and catalyzing the poly (ADP-ribosyl)ation of the receptor protein, thus participating in the DNA repair process.

[0055] PARP inhibitors inhibit the work of PARP enzymes, preventing these "repairmen" PARP enzymes from working properly. DNA damage cannot be repaired, and cells may die. PARP inhibitors are compounds that inhibit PARP enzymes. In other words, any substance that can inhibit PARP enzyme activity is a PARP inhibitor.

[0056] In particular, the PARP inhibitor of the present application is selected from the following group: ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR2313, E7016, E7449, fluzoparib, IMP 4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP 124, niraparib, NU 1025, NU1064, NU 1076, NU1085, olaparib, ONO2231, PD 128763, R 503, R554, rucaparib, SBP 101, SC 101914, ximinperi, pamiparib, talazoparib, veliparib, WW 46. ​​2-(4-(Trifluoromethyl)phenyl)-7,8-dihydro-5H-thiofuro[4,3-d]pyrimidin-4-ol and its salts or derivatives.

[0057] Preferably, the PARP inhibitor is selected from five drugs that have been marketed, namely Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib, a drug that has entered clinical phase III. Obviously, the PARP inhibitor here actually refers to a drug containing the active ingredient of the PARP inhibitor.

[0058] Talazoparib is indicated for adults with deleterious or suspected deleterious germline BRCA-mutated (gBRCAm) HER2-negative locally advanced or metastatic breast cancer. Talazoparib is available in 0.25 mg / 1 mg talazoparib tosylate capsules, taken orally once daily. Treatment interruption or dose reduction should be considered in the event of adverse reactions:

[0059] At the first occurrence of an adverse reaction, the oral dose was reduced to 0.75 mg (three 0.25 mg capsules) once daily;

[0060] If the adverse reaction occurs a second time, the oral dose is reduced to 0.5 mg (two 0.25 mg capsules) once daily;

[0061] The third time an adverse reaction occurred, the oral dose was reduced to 0.25 mg (one 0.25 mg capsule) once daily.

[0062] Niraparib is used for the maintenance treatment of adult patients with platinum-sensitive recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who have achieved complete or partial remission after platinum-containing chemotherapy. The commercial dosage form is 100 mg of niraparib tosylate capsules, taken orally once a day at 300 mg until disease progression or intolerable adverse reactions occur. In the event of adverse reactions, treatment interruption or dose reduction should be considered:

[0063] Dose reduction was first from 3 capsules (300 mg) per day to 2 capsules (200 mg) per day;

[0064] If further dose reduction is required, the dose can be reduced a second time from 2 capsules (200 mg) per day to 1 capsule (100 mg) per day;

[0065] If suspending the drug and reducing the dose cannot control the adverse reaction, discontinuation of the drug is recommended.

[0066] Rucaparib is used for women with advanced ovarian cancer whose tumors carry a specific gene mutation (harmful BRCA) and who have been treated with two or more chemotherapy drugs. It is available in three tablet forms: 200 mg, 250 mg, and 300 mg. The recommended dose is 600 mg, taken orally twice daily, with or without food. Continue treatment until disease progression or unacceptable toxicity. For adverse reactions, consider interrupting treatment or reducing the dose.

[0067] Olaparib is used for maintenance treatment of adult patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer who have germline or somatic BRCA mutations (gBRCAm or sBRCAm) after achieving complete remission or partial remission with platinum-containing chemotherapy; and for maintenance treatment of adult patients with platinum-sensitive recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer after achieving complete remission or partial remission with platinum-containing chemotherapy. The marketed dosage form is tablets: 150 mg and 100 mg, with two specifications. The recommended dose is 300 mg (2 150 mg tablets) twice a day, equivalent to a total daily dose of 600 mg. The 100 mg tablet is used for dose reduction:

[0068] To manage adverse events, such as nausea, vomiting, diarrhea, and anemia, interruption of treatment or dose reduction may be considered;

[0069] If dose reduction is necessary, the recommended dose is reduced to 250 mg (one 150 mg tablet, one 100 mg tablet) taken twice daily (equivalent to a total daily dose of 500 mg);

[0070] If further dose reduction is necessary, the recommended dose is reduced to 200 mg (two 100 mg tablets) taken twice daily (equivalent to a total daily dose of 400 mg).

[0071] Fluzoparib is used to treat patients with platinum-sensitive recurrent ovarian, fallopian tube, or primary peritoneal cancer who have undergone two or more lines of chemotherapy and harbor germline BRCA mutations (gBRCAm). The commercially available dosage form is a 50mg capsule.

[0072] For other PARPi candidate drugs under development and entering clinical practice, please refer to the web link https: / / www.selleckchem.com / PARP.html and related academic review literature.

[0073] The recommended dosage of TH-302 or its similar formula I compounds for treating cancer can refer to the patent application texts of Threshold and other pharmaceutical companies (such as WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118251A1, WO20 14169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO2012006032A2, WO2018026606A 2. WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A2, WO20160 14390A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2) and FDA-registered clinical trials (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01 Dosage in:

[0074] 120 mg / m 2 Up to 460 mg / m 2 The daily dose is administered intravenously;

[0075] 480 mg / m 2 Up to approximately 670 mg / m 2 Or for example 575 mg / m 2 The weekly dose is administered intravenously.

[0076] TH-302 (concentrate for administration solution) used in clinical trials is a sterile liquid formulation of TH-302. TH-302 is formulated with 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80. It is provided by the sponsor in 10 mL glass vials with rubber stoppers and flip-off closures. The TH-302 drug product is a clear, colorless to pale yellow solution that is essentially free of visible particulates. For a nominal total amount of 650 mg of TH-302, each single-use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product (equivalent to 100 mg / mL) and is clearly labeled, disclosing the batch number, route of administration, required storage conditions, the sponsor's name, and appropriate precautionary labeling as required by applicable regulations. Dilution is required according to the pharmacy manual prior to administration.

[0077] Prior to administration, dilute to a total volume of 500 mL (1000 mL for a total dose of ≥1000 mg) with a commercially available 5% dextrose aqueous solution to achieve the desired final concentration. Each dose of TH-302 was prepared with a 5% dextrose aqueous solution that was free of di(2-ethylhexyl) phthalate (DEHP) and administered intravenously using a DEHP-free intravenous infusion device.

[0078] Of course, you can also use the freeze-dried preparation developed by Threshold:

[0079] A solution (20 mL) of TH-302 (100 mg) and sucrose (1 g) was added to lyophilization vials and lyophilized to produce a lyophilized unit dosage form of TH-302 with a drug loading of less than 5 mg / cm 3 For human administration, the unit dosage form is dissolved in 5% dextrose injection and an appropriate amount of this solution is administered to the patient;

[0080] The subsequent Phase I clinical trial dosing regimen for TH-302 in human patients used a lyophilized preparation. The TH-302 lyophilized preparation for injection was prepared in a 100mL glass vial with a drug load of 100mg / 100ml and stored under controlled conditions at 2-8°C. When used, the preparation was injected into 250mL of 5% glucose injection in the lyophilized preparation bottle and administered intravenously within 30 minutes via an infusion pump.

[0081] Monotherapy refers to single-drug therapy. Combination therapy refers to combined drug therapy. Monotherapy refers to the use of only one anticancer drug in a single course of treatment. Combination therapy refers to the use of two or more anticancer drugs simultaneously or sequentially in a single course of treatment.

[0082] Generally speaking, combination therapy requires exploring different dosages and dosing cycles based on the characteristics of the disease and the types of combined drugs. Only based on the above conditions can the combination drug treatment plan explored achieve better therapeutic effects than single drug treatment.

[0083] The drug dosage and administration cycle of monotherapy and combination therapy need to be explored through clinical trials with reference to the dosage and administration regimen of the above-mentioned TH-302 and its similar compounds and PARPi.

[0084] The present application further provides the steps of the above-mentioned treatment method, including:

[0085] Detection of RAD51D gene mutations in cancer or tumor patients;

[0086] If the patient or the patient's tumor tissue has a RAD51D pathogenic gene mutation, treatment is performed using a drug containing a hypoxia-activated compound of the following formula I or II, alone or in combination with a PARP inhibitor:

[0087] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO;

[0088] Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1, and the entire text is hereby incorporated into this application.

[0089] Specifically, R1, R2, R3, and Cx are defined as follows:

[0090] Cx is a 5-10 membered aromatic ring or aromatic heterocycle, aliphatic heterocycle or cycloalkane, which shares two carbon atoms with the nitrobenzene ring to form a fused ring structure;

[0091] R1 is connected to any backbone atom of the Cx ring and is selected from hydrogen, halogen atoms, cyano or isocyano, hydroxyl, thiol, amino, OTs, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, alkoxy of 1-6 carbon atoms or Z-substituted alkoxy of 1-6 carbon atoms, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 、-OCOR 6 、-NR 6 SO2R 7 、-NR 6 SO2NR 6 R 7 ,

[0092] R2 and R3 are each independently hydrogen, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, or R2, R3 and the benzylic carbon atom to which they are bonded together form a 3-6 membered ring;

[0093] The group can replace the hydrogen atom at any position on the condensed ring carbon atom, and the number of substitutions is 1;

[0094] The substituent Z is a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, a C1-C3 alkyl group or a substituted alkyl group, a C1-C3 alkoxy group or a substituted alkoxy group, a C2-C3 alkenyl group or a substituted alkenyl group, a C2-C3 alkynyl group or a substituted alkynyl group, or a C3-C8 cycloalkyl group or a substituted cycloalkyl group;

[0095] R 6 、R 7 are each independently hydrogen, C1-C6 alkyl or Z-substituted C1-C6 alkyl, C2-C6 alkenyl or Z-substituted C2-C6 alkenyl, C2-C6 alkynyl or Z-substituted C2-C6 alkynyl, C3-C8 cycloalkyl or Z-substituted C3-C8 cycloalkyl, C6-C 10 Aryl or Z-substituted C6-C 10 aryl, 4-15 membered heterocyclic group or Z-substituted 4-15 membered heterocyclic group, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 6 、R 7 Together with the atoms to which it is bonded, it forms a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group.

[0096] In particular, the TMB (tumor mutation burden) level of the above-mentioned RAD51D gene mutation is medium.

[0097] Because TMB (tumor mutation burden) varies among different tumor types, it's generally considered high if it exceeds 20 mutations / Mb (Mb stands for bases per million), low if it's below 10 mutations / Mb, and moderate if it's in between. At the 2017 World Lung Cancer Conference, Bristol-Myers Squibb (BMS) announced the results of the CheckMate-032 clinical trial, a Phase II study that enrolled 401 patients with advanced lung cancer who had failed first-line therapy. The patients were treated with a PD-1 inhibitor alone or in combination with ipilimumab. Patients were divided into three groups based on TMB levels: high, intermediate, and low. Among those receiving the combination therapy, the response rate was 62%, 20%, and 23%, respectively, with the response rate three times higher in those with high TMB. The median overall survival was 22.0 months, 3.6 months, and 3.4 months, respectively—a six-fold difference! This study demonstrated that different TMB levels have a significant impact on the efficacy of different cancer treatment drugs.

[0098] Particularly, the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

[0099] In particular, the patient is resistant or insensitive to the above-mentioned PARP inhibitors.

[0100] The present application also provides pharmaceutical uses of the hypoxia-activated compound of the following formula I or II or its salts, esters, solvates, isotopic variants, and isomers, which are used to prepare drugs for treating cancer and tumor patients with RAD51D pathogenic gene mutations, either alone or in combination with other therapeutic drugs:

[0101] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO;

[0102] Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1, and the entire text is hereby incorporated into this application.

[0103] In particular, for the above-mentioned pharmaceutical use, the TMB (tumor mutation burden) level of the RAD51D gene mutation is medium.

[0104] In particular, for the above-mentioned pharmaceutical use, the other therapeutic drugs include PARP inhibitors, and the PARP inhibitors are selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib;

[0105] In particular, for the above pharmaceutical use, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer.

[0106] In particular, patients are insensitive or resistant to the above-mentioned PARP inhibitors.

[0107] The present application also provides a drug for treating cancer caused by RAD51D pathogenic gene mutation, which contains a hypoxia-activated compound of the following formula I or II or a salt, ester, solvate, isotopic variant, or isomer thereof:

[0108] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO;

[0109] Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1, and the entire text is hereby incorporated into this application.

[0110] In particular, the above-mentioned drugs can be used alone or in combination with a PARP inhibitor.

[0111] Preferably, the PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

[0112] Preferably, the TMB (tumor mutation burden) level of the gene mutation is medium.

[0113] Preferably, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, bladder cancer.

[0114] Particularly, among the above drugs, the patient is resistant or insensitive to the PARP inhibitor.

[0115] The present application also provides a pharmaceutical combination comprising the following substances as active ingredients:

[0116] Hypoxia-activated compounds or salts, esters, solvates, isotopic variants, isomers thereof, and

[0117] PARP inhibitors;

[0118] The active ingredients are formulated together or separately for combined use, simultaneous use or separate use.

[0119] Typically, the active ingredients that characterize the pharmaceutical combinations described herein may be formulated together (single dosage unit) or separately (kit).

[0120] The two active ingredients, formulated together or separately, may generally be administered simultaneously; or, given the nature of their respective formulations, separately with a time interval which allows for the optimization of the synergistic effect of the two active ingredients.

[0121] The drug combination can be a compound drug, which is a pharmaceutical composition comprising one active ingredient being the above-mentioned hypoxia-activated compound or a salt, ester, solvate, isotopic variant, or isomer thereof, and another active ingredient being a PARP inhibitor. In this case, the above active ingredients are co-formulated and administered simultaneously.

[0122] A drug combination can also be a packaged product, similar to Pfizer's COVID-19 drug Paxlovid, that can be sold together or separately. In this case, the active ingredients are formulated separately, but the two or more separately formulated drugs can be administered simultaneously or at intervals, depending on the properties of their respective formulations.

[0123] As a specific embodiment of the present application, when formulated separately, one drug comprises the above-mentioned hypoxia-activated compound or its salt, ester, solvate, isotopic variant, isomer, and pharmaceutically acceptable excipients, and the other drug comprises a PARP inhibitor and pharmaceutically acceptable excipients. The drug combination consisting of the above two drugs can be administered simultaneously or separately at a certain time interval depending on the properties of the respective formulations.

[0124] The drug combination provided herein is used to treat cancer or tumor patients with impaired DNA repair enzymes. In other words, the drug combination may have a better therapeutic effect on cancer or tumor patients with impaired DNA repair enzymes.

[0125] In particular, the DNA repair enzyme is damaged by one or more gene defects selected from the group consisting of BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, Rad50, MRE11, p53, NBS1, XRS2, XRCC4 / XPF, ERCC1, ERCC2 / XPD, ERCC3 / XPB, ERCC4 / XPF, XRCC1, Ku80, MHS6, MGMT, PARP, ERCC5 / XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5 / XPG, ERCC6 / CSB, ERCC8 / CSA, LIG1 / DNA Ligase I, MMS19, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RBBP8, SMC5, USP11, WRN; the RAD51 is selected from XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, preferably RAD51D.

[0126] Particularly, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

[0127] As a preferred embodiment of the present application, the active ingredient PARP inhibitor in the drug combination is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib; the active ingredient hypoxia-activated compound in the drug combination is selected from the compound of the following formula I or II:

[0128] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO;

[0129] Wherein, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

[0130] As a preferred embodiment, the hypoxia activated compound of formula I is selected from the compounds of the following structures:

[0131] As a preferred embodiment, the hypoxia activated compound of formula II is selected from the compounds of the following structures:

[0132] The present application also provides a pharmaceutical preparation, which is prepared from the active ingredients of the above-mentioned drug combination and pharmaceutically acceptable excipients.

[0133] The dosage form of the above-mentioned pharmaceutical preparation is selected from granules, tablets, pills, capsules, injections or freeze-dried powder injections.

[0134] The present application also provides the use of the above-mentioned drug combination and / or the above-mentioned drug preparation in the preparation of anti-tumor drugs.

[0135] As a preferred use of the above-mentioned drug combination and / or the above-mentioned drug preparation in the preparation of anti-tumor drugs, the tumor is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

[0136] In addition to containing the hypoxia-activated compound of Formula I or II, or its salts, esters, solvates, isotopic variants, or isomers, the above-mentioned drugs should also contain pharmaceutically acceptable excipients or vehicles, depending on the characteristics of the drug, medicament, or formulation. The drug can be in any dosage form suitable for clinical administration, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small injections, lyophilized powders for injection, or infusions. Depending on the specific dosage form and route of administration, the pharmaceutically acceptable excipients or vehicles in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and pigments. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG1 is a graph showing the growth curve of tumor volume in each treatment group of mice in the LD1-2032-361588 human ovarian cancer transplanted tumor PDX model;

[0138] Figure 2 is a graph showing the percentage change in body weight of mice in each treatment group over time in the LD1-2032-361588 human ovarian cancer transplanted tumor PDX model. DETAILED DESCRIPTION

[0139] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0140] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used are all commercially available products unless otherwise specified.

[0141] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, the patient is a human. Typically, the patient is a human diagnosed with cancer. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used to screen, characterize, and evaluate drugs and therapies.

[0142] "Prodrug" refers to a compound that, after administration or administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug is chemically modified relative to a drug in a manner that renders it less active or inactive relative to the drug, but the chemical modification allows for the production of the corresponding drug by metabolism or other biological processes following administration of the prodrug. A prodrug may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or an improved flavor relative to the active drug. Prodrugs may be synthesized using reactants other than the corresponding drug.

[0143] "Treatment" or "treating a patient" refers to administering, using or applying to a patient a therapeutically effective amount of a drug related to the present invention.

[0144] "Administering" or "applying" a drug to a patient refers to direct administration or administration (which may be administered or administered to a patient by a medical professional or may be self-administered or administered) and / or indirect administration or administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer or administer a drug and / or provides a prescription for a drug to a patient is administering or administering a drug to a patient.

[0145] A "therapeutically effective amount" of a drug refers to an amount of the drug that, when administered or used to a patient suffering from cancer, will have the desired therapeutic effect (e.g., alleviation, amelioration, remission, or elimination of the clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur by administering or applying a single dose and may only occur after administering or applying a series of doses. Thus, a therapeutically effective amount can be administered or applied in one or more doses.

[0146] "Treatment" of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results). For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, remission, or stabilization of the disease state; or other beneficial results. In some instances, treatment of cancer may result in a partial response or stabilization of the disease.

[0147] "Tumor cell" refers to a tumor cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).

[0148] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

[0149] The following provides specific experiments of the present invention.

[0150] The experimental protocols, any modifications, animal welfare, and use of animals in the experiments disclosed herein were reviewed and approved by the IACUC committee of the contract research organization (CRO). Animal welfare and experimental procedures met AAALAC requirements.

[0151] Compound AST is the S isomer of compound 01 in Table 1 of patent application PCT / CN2020 / 114519, publication number WO2021120717A1, or compound B in patent application PCT / CN2021 / 101870, publication number WO2022052564A1, and its structural formula is shown below:

[0152] 1. Antitumor Effects and Safety Evaluation of TH-302, AST, and Olaparib as Single Agents and Their Combination in the LD1-2032-361588 Human Ovarian Cancer Xenograft PDX Model

[0153] This model was established from human ovarian cancer tissue from LD1-2032-361588 and passaged to FP3+3 for this efficacy study. The tumor originated from a 62-year-old female patient with ovarian cancer liver metastasis. The pathological diagnosis was poorly differentiated adenocarcinoma, which had developed resistance to PARPi during treatment. Genetic testing confirmed that this model harbors the pathogenic RAD51D gene mutation, p.Lys91Ilefs*13.

[0154] The LD1-2032-361588 human ovarian cancer xenografts were cut into approximately 3mm x 3mm x 3mm (approximately 45-60mg) pieces and inoculated subcutaneously into NU / NU mice. The mice were observed and tumor growth was monitored. On day 27 of inoculation, the average tumor volume of the tumor-bearing mice was 183.67mm. 3 The day of group administration was defined as day 0. The specific administration route, dosage and regimen information are shown in Table 1.

[0155] Table 1: Administration routes, dosages, and regimens Note: N: number of animals; Dosage volume: adjusted according to the weight of tumor-bearing mice (0.2 mL / 20 g); po: oral administration; iv: tail vein injection; QW: once a week; QD: once a day.

[0156] Tumor growth in each treatment group and control group was recorded on different days of the experiment, as shown in Table 2. The corresponding tumor volume growth curves of mice in each group are shown in Figure 1. The efficacy was evaluated based on the relative tumor growth rate and relative tumor inhibition rate. The efficacy analysis of each group is shown in Table 3, and the statistical significance analysis is shown in Table 4. The body weight changes of the treatment and control groups after administration were recorded to study the safety of each group in the human ovarian cancer transplant tumor PDX model. The results of mouse body weight changes are shown in Table 5. The percentage change of body weight of each treatment group over time is shown in Figure 2 (percentage change of body weight: RCBW (%) = (BW i -BW0) / BW0×100,BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first administration).

[0157] Table 2: Changes in tumor volume of mice in each group with treatment time (mm 3 )

[0158] Tumor volume and tumor-bearing mouse weight measurement: Use vernier calipers to measure twice a week. The tumor volume is calculated as V = 0.5a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively.

[0159] Table 3: Summary of the antitumor efficacy evaluation of the test compounds in the LD1-2032-361588 human ovarian cancer subcutaneous xenograft tumor model

[0160] The relative tumor growth rate T / C (%) is calculated as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; CRTV : RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., d0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0161] Tumor growth inhibition rate TGI (%) = [1-(T i -T0) / (V i -V0)]×100,T i is the average tumor volume of the compound group after the start of administration, T0 is the average tumor volume of the compound group at the first administration, V0 is the average tumor volume of the vehicle control group at the first administration, and V i The mean tumor volume of the vehicle control group after the start of drug administration.

[0162] Table 4: Statistical analysis

[0163] Table 5: Changes in mouse body weight (g) over treatment time

[0164] On the 32nd day after the start of administration, the average tumor volume of the tumor-bearing mice in the control group was 1709.32±271.80mm 3 The average tumor volumes of the tumor-bearing mice in the test compound Olaparib (50 mg / kg, PO, QD*32 days) monotherapy group, TH-302 (80 mg / kg, IV, QW×3) monotherapy group, Olaparib combined with TH-302 administration group, AST (20 mg / kg, IV, QD×5, 2 days off; 2 weeks off; QD×5) monotherapy group, and Olaparib combined with AST administration group were 1261.91±136.43 mm, respectively. 3 、239.28±101.70mm 3 、118.07±25.29mm 3 、823.26±100.46mm 3 and 476.89±73.20mm 3 ; The relative tumor proliferation rates T / C (%) were 73.82%, 14.04%, 6.91%, 42.28% and 27.96% respectively; the tumor volume growth inhibition rates TGI (%) were 29.33%, 96.33%, 104.32%, 58.06% and 80.77% respectively.

[0165] The experimental results showed that in a human ovarian cancer tumor model with a RAD51D pathogenic gene mutation, the test compound Olaparib alone had a poor tumor inhibitory effect, proving that the tumor model was indeed resistant to Olaparib; the test compound TH-302 alone, Olaparib combined with TH-302, AST alone, and Olaparib combined with AST all showed statistically significant tumor growth inhibition compared with the control group (p<0.05 and T / C%<40%); there was no statistically significant difference between the Olaparib combined with TH-302 group and the TH-302 alone group, and there was a statistically significant difference between the Olaparib combined with AST and the AST alone group, indicating that the combination of AST and Olaparib can enhance its tumor growth inhibition effect to a certain extent.

[0166] The body weight of experimental animals is used as an indirect indicator to monitor drug toxicity. No significant weight loss was observed in the mice in each drug group after drug administration, indicating that the tumor-bearing mice tolerated the treatment of each drug at the experimental dose well.

[0167] In clinical studies, researchers used PARP inhibitors to treat patients with homologous recombination repair deficiency. However, with the clinical application of PARPi, PARPi resistance has become an inevitable problem in its clinical application. Existing studies have shown that homologous recombination repair, DNA replication fork protection, and changes in PARPi pharmacokinetics are the main causes of PARPi resistance. The mechanism of action of TH-302 and AST is completely different from that of PARPi, but they are equally effective for patients with homologous recombination repair deficiency (RAD51D pathogenic gene mutation is one of the homologous recombination repair deficiency mutations). This provides a new treatment option for patients who develop PARPi resistance clinically and have homologous recombination repair deficiency mutations.

[0168] Our research also showed that both TH-302 and AST exhibited strong tumor suppression in an olaparib-resistant mouse ovarian cancer PDX model, and that the combination of AST and olaparib exhibited a significant synergistic effect compared to AST alone. This provides new hope for the clinical treatment of patients with pathogenic HRR gene mutations who are resistant to PARPi, and provides important theoretical and experimental evidence.

[0169] TH-302 and AST are small molecule prodrugs that can be activated under hypoxic / anoxic conditions and release cytotoxins, thereby specifically killing tumor cells and tumor tissues. TH-302 and AST are hypoxia-activated DNA alkylating agents.

[0170] The following general compound of formula I:

[0171] It has been demonstrated in relevant patent applications that the compound has a mechanism similar to that of TH-302, so it is entirely predictable that the compound of this general formula has an effect similar to that of TH-302.

[0172] Similarly, the general compound of formula II below:

[0173] It has also been proven in the relevant patent application PCT / CN2020 / 114519 (publication number WO2021120717A1) to have a mechanism similar to AST, so it is completely predictable that this type of general formula compound has an effect similar to AST.

[0174] Although the PARP inhibitor selected in the examples of the present application is Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, etc. are also PARP inhibitors, and their mechanism of action is similar to that of Olaparib, which is to block the enzymes involved in repairing damaged DNA. Therefore, it can be inferred that PARPi such as Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, etc. have similar tumor inhibitory effects as Olaparib in the above experiments.

Claims

1. A method of treating a patient with cancer or tumor whose DNA repair enzyme is damaged by using a hypoxia-activated compound or its salt, ester, solvate, isotopic variant, or isomer alone or in combination with other drugs.

2. The method of claim 1, wherein Impaired DNA repair enzymes selected from one or more gene defects selected from the group consisting of BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, Rad50, MRE11, p53, NBS1, XRS2, XRCC4 / XPF, ERCC1, ERCC2 / XPD, ERCC3 / XPB, ERCC4 / XPF, XRCC1, Ku80, MHS6, MGMT, PARP, ERCC5 / XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5 / XPG, ERCC6 / CSB, ERCC8 / CSA, LIG1 / DNA Ligase I, MMS19, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RBBP8, SMC5, USP11, WRN; the RAD51 is selected from XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, preferably RAD51D.

3. The method of claim 1, wherein: The other drugs include PARP inhibitors, which are selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib; The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

4. The method of claim 3, wherein: The patient is resistant or insensitive to the PARP inhibitor.

5. A method of treating a patient with cancer or tumor caused by RAD51D gene defect using a hypoxia-activated compound or its salt, ester, solvate, isotopic variant, or isomer as a single agent, wherein: The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer.

6. A method of treating a patient with cancer or tumor caused by RAD51D gene defect using a hypoxia-activated compound or its salt, ester, solvate, isotopic variant, or isomer in combination with a PARP inhibitor, wherein: The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer; The PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

7. The method of claim 6, wherein: The patient is resistant or insensitive to the PARP inhibitor.

8. The method of any one of claims 1 to 7, wherein the hypoxia-activated compound is selected from the group consisting of compounds of Formula I or II below: in, R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

9. The method of claim 8, wherein: The hypoxia activated compound of formula I is selected from the group consisting of compounds of the following structures: The hypoxia activated compound of formula II is selected from the compounds of the following structures:

10. A method of treatment comprising the following steps: Detection of RAD51D gene mutations in cancer or tumor patients; If the patient or the patient's tumor tissue has a RAD51D pathogenic gene mutation, treatment is performed using a drug containing a hypoxia-activated compound of the following formula I or II, alone or in combination with a PARP inhibitor: wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

11. The method of claim 10, wherein: The PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib and Pamiparib.

12. The method of claim 10 or 11, wherein: The patient is resistant or insensitive to the PARP inhibitor.

13. Pharmaceutical use of the hypoxia-activated compound of Formula I or II below, or its salts, esters, solvates, isotopic variants, or isomers, for preparing a medicament for treating cancer or tumor patients with RAD51D pathogenic gene mutations, either alone or in combination with other therapeutic agents: in, R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

14. The pharmaceutical use according to claim 13, wherein The other therapeutic drugs include PARP inhibitors, which are selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib; The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer.

15. The pharmaceutical use according to claim 13 or 14, wherein The patient is resistant or insensitive to the PARP inhibitor.

16. A drug for treating cancer caused by a RAD51D pathogenic gene mutation, the drug comprising a hypoxia-activated compound of the following formula I or II or a salt, ester, solvate, isotopic variant, or isomer thereof: in, R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

17. The drug according to claim 16, which is used in combination with a PARP inhibitor, wherein PARP inhibitors selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib; The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer.

18. The drug according to claim 17, wherein The patient is resistant or insensitive to the PARP inhibitor.

19. A drug combination, characterized in that: Contains the following substances as active ingredients: Hypoxia-activated compounds or salts, esters, solvates, isotopic variants, isomers thereof, and PARP inhibitors; The active ingredients are formulated together or separately for combined use, simultaneous use or separate use.

20. The drug combination according to claim 19, characterized in that The drug combination is used to treat cancer or tumor patients with damaged DNA repair enzymes.

21. The method of claim 20, wherein: The DNA repair enzyme is damaged and is selected from one or more gene defects of the following: BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, Rad50, MRE11, p53, NBS1, XRS2, XRCC4 / XPF, ERCC1, ERCC2 / XPD, ERCC3 / XPB, ERCC4 / XPF, XRCC1, Ku80, MHS6, MGMT, PARP, ERCC5 / XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5 / XPG, ERCC6 / CSB, ERCC8 / CSA, LIG1 / DNA Ligase I, MMS19, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RBBP8, SMC5, USP11, WRN; the RAD51 is selected from XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, preferably RAD51D; The cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

22. The drug combination according to claim 19, wherein The PARP inhibitor is selected from Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and Pamiparib; The hypoxia-activated compound is selected from the compounds of the following formula I or II: wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Among them, the definitions of R1, R2, R3, and Cx are as recorded in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1.

23. The drug combination according to claim 22, wherein The hypoxia activated compound of formula I is selected from the group consisting of compounds of the following structures: The hypoxia activated compound of formula II is selected from the compounds of the following structures:

24. A pharmaceutical preparation, characterized in that It is prepared from the active ingredients of the drug combination according to claim 19 and pharmaceutically acceptable excipients.

25. The pharmaceutical preparation according to claim 24, characterized in that The dosage form of the pharmaceutical preparation is selected from granules, tablets, pills, capsules, injections or freeze-dried powder injections.

26. Use of the drug combination according to claim 19 and / or the pharmaceutical preparation according to claim 24 in the preparation of an anti-tumor drug.

27. The use according to claim 26, wherein The tumor is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, liver cancer, colon cancer, rectal cancer, lung cancer, and bladder cancer, and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

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