Treatment of patients resistant to PARP inhibitors with TH-302

Abstract

The present invention provides a method of treatment for patients resistant to PARP inhibitors with TH-302, either alone or in combination, a drug, and pharmaceutical use thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for treating cancer, and in particular to a method for treating cancer patients resistant to PARP inhibitors (PARPi). [Background technology]

[0002] The first human clinical trial of the PARPi drug olaparib has demonstrated for the first time that PARPi drugs can inhibit the growth of tumor cells harboring BRCA1 / 2 mutations. This is primarily based on the synthetic lethality theory (Ashworth, A., & Lord, CJ. (2018). Synthetic lethal therapies for cancer: what's next after PARP inhibitors?. Nature reviews. Clinical oncology, 15(9), 564-576. https: / / doi.org / 10.1038 / s41571-018-0055-6), which states that PARP inhibitors can inhibit the DNA single-strand damage repair function of PARP, resulting in the inability to timely repair large amounts of single-strand DNA damage in cells. Unrepaired single-strand DNA damage leads to replication fork collapse, resulting in double-strand DNA damage. While these highly cytotoxic double-strand DNA damages can be repaired in normal cells through the homologous recombination repair (HR) pathway, mediated jointly by proteins such as BRCA1 and BRCA2, in BRCA1 / 2-deficient tumor cells, these double-strand DNA damages cannot be repaired, leading to the eventual death of the tumor cells. PARPi was originally developed for radiotherapy and chemotherapy sensitization, and preclinical studies support the potential for PARPi to be developed as a single drug to treat BRCA1 / 2-deficient cancers. Therefore, the initial target population to test the PARPi-BRCA hypothesis was selected as carriers of BRCA1 / 2 germline mutations (gBRCA1 / 2). All of the initial study cohort for PARPi in ovarian cancer had previously received platinum-based chemotherapy, and the study found that platinum sensitivity was directly related to PARPi response (since platinum-based chemotherapy agents are DNA-damaging agents that result in DNA crosslinks, some of which can be repaired by the HR pathway, DNA repair-deficient tumors are expected to be sensitive to platinum-based chemotherapy).Two other PARPi drugs, niraparib and rucaparib, are approved for the treatment of ovarian cancer; the FDA and EMA have approved maintenance therapy with niraparib (regardless of BRCA1 / 2 status); rucaparib is also registered by the FDA and EMA as an alternative treatment protocol for patients with BRCA1 / 2 mutation-associated ovarian cancer after two prior chemotherapy protocols; and talazoparib is also approved by the FDA for the treatment of BRCA mutation / HER-2 negative metastatic breast cancer. (Mateo, J., Lord, C.J., Serra, V., Tutt, A., Balmana, J., Castroviejo-Bermejo, M., Cruz, C., Oaknin, A., Kaye, S.B., & de Bono, J.S. (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).

[0003] As PARPi is used clinically, PARPi resistance has become an inevitable problem, with over 40% of BRCAm (BRCA-mutated) ovarian cancer patients failing to benefit from PARPi. Existing studies have shown that alterations in homologous recombination repair restoration (HRR), DNA replication fork protection, and PARPi pharmacokinetics are major causes of PARPi resistance. To overcome PARPi resistance and enhance PARPi sensitivity, various combination therapies have been developed, many of which have entered the clinical stage. These mainly include PARPi-DNA alkylating agent combinations, PARPi-oncolytic herpes simplex virus (oHSVs) combinations, PARPi-ionizing radiation combinations, PARPi-immunotherapy combinations, PARPi-HSP90 inhibitor combinations, PARPi-WEE1 / ATR inhibitor combinations, PARPi-DNMTi inhibitor combinations, and PARPi-CDK inhibitor combinations (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 Jun20;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 Tumor Resistance. Frontiers in cell and developmental biology, 8,564601. https: / / doi.org / 10.3389 / fcell.2020.564601).

[0004] TH-302 (Evofosfamide, CAS number 918633-87-1) is a 2-nitroimidazole-derived hypoxia-activated prodrug (HAP) of bromoisophosphamide, developed by Threshold, Inc., USA. The inactive TH-302 prodrug may release the highly toxic Br-IPM in the presence of hypoxia. TH-302 possesses broad biological activity in vitro and in vivo and specific hypoxia-selective activation activity, inducing H2AX phosphorylation, DNA crosslinking activity, and cell cycle arrest. Therefore, this compound is being developed as an anticancer drug by numerous pharmaceutical companies and scientific research institutes.

[0005] A research paper by Meng FY et al. demonstrated that TH-302 has broad activity against various tumors and exhibits enhanced activity due to its excellent hypoxia selectivity. Studies have shown that the in vitro cytotoxicity of TH-302 in 32 human cancer cell lines under hypoxic conditions is significantly stronger than that under normoxic conditions, demonstrating that this compound has selective cytotoxicity against cancer cells under hypoxic conditions. Using human cells overexpressing one-electron reductase (POR), the principle of one-electron reductase-dependent enhanced activity of TH-302 under hypoxic conditions was demonstrated. This is shown in Reaction Scheme 1 below. JPEG0007762796000001.jpg51170

[0006] Cytochrome P450 oxidoreductase reduces the prodrug TH-302 to generate an intermediate radical anion, which then decomposes into the unstable, cytotoxic cytotoxin Br-IPM. The key step in this process is the one-electron reduction process. Studies have demonstrated that the presence of oxygen reverses this process, i.e., inhibits it. Therefore, TH-302 can only be reduced under oxygen-deficient conditions, resulting in stronger cytotoxicity. We further used Chinese hamster ovary cell-based DNA repair mutant cell lines (which lack homology-dependent repair), including cell lines deficient in base excision repair, nucleotide excision repair, nonhomologous end joining repair, or homologous end joining repair. Studies have shown that cell lines deficient in homologous end joining repair individually or in both homologous end joining repair and nucleotide excision repair exhibit significantly enhanced TH-302 hypoxia sensitivity. However, cell lines individually deficient in base excision repair, nucleotide excision repair, or non-homologous end joining repair have no effect on TH-302 sensitivity. Consistent with this finding, enhanced TH-302 sensitivity was also observed in in vitro cell experiments lacking BRCA1, BRCA2, and FANCA.In clinical trials, TH-302 has been shown to have a better therapeutic effect on patients with BRCA gene mutations (Meng F, Evans JW, Bhupathi D, et al. Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302. [J]. Molecular Cancer Therapeutics, 2012, 11(3):740; Conroy, M., Borad, MJ, & Bryce, AH (2017). Hypoxia-Activated Alkylating Agents in BRCA1-Mutant Ovarian Serous Carcinoma. Cureus, 9(7), e1517. https: / / doi.org / 10.7759 / cureus.1517; WO2015013448A1, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and A taxane; WO2020007106A1, Anticancer pharmaceutical use of evofosfamide).

[0007] These studies on the mechanism of action of TH-302, especially the fact that TH-302's particular sensitivity to BRCA mutations was revealed, suggest that the drug TH-302 may be able to overcome the drawback of drug resistance to PARPi drugs when used in combination.

[0008] However, in application PCT / US2012 / 031677 (Publication No. WO2012135757A2, Methods for treating cancer, Applicant: Threshold, Inc., USA), Threshold researchers conducted in vitro combination studies using TH-302 and the PARPi candidate drug ABT-888 (i.e., Veliparib, CAS:912444-00-9).

[0009] Different cancer cells were pretreated with ABT-888 for 1 hour under normoxia and then incubated with TH-302 for an additional 2 hours under normoxia or hypoxia. After 3 days of incubation in the presence of ABT-888, cell viability was measured using Alamar Blue. The results are shown in the table below. Results for H460 cell line (human large cell lung cancer cells): JPEG0007762796000002.jpg30170HCT116 cell line (human colon cancer cells) results: JPEG0007762796000003.jpg32170A375 cell line (human melanoma cells) results: JPEG0007762796000004.jpg29170

[0010] The above results demonstrate that in vitro cell experiments, the combination of TH-302 and ABT-888 does not have an additive effect, i.e., TH-302 activity is not substantially affected by the presence of ABT-888.

[0011] However, PCT / US2019 / 065065 (published WO2020118251A2, title: Hypoxia targeting compositions and combinations thereof with a parp inhibitor and methods of use It has been disclosed that the combination of a hypoxia-activated drug or a prodrug thereof (e.g., apaziquone, AQ4N, etanidazole, evofosfamide (TH-302), nimorazole, pimonidazole, porfiromycin, PR-104, tarloxotinib, tirapazamine) with a PARPi has an additive effect, and in particular, a combined administration test of tirapazamine and olaparib in an animal body has also been disclosed, and as a result, compared with a single administration protocol using tirapazamine or a PARPi, a combined administration protocol of tirapazamine, which is a hypoxia-activated anticancer prodrug, with the PARPi olaparib significantly slows the tumor growth rate in a PDX animal model, indicating that the combination of a hypoxia-activated anticancer drug and a PARPi has an additive effect.

[0012] In other words, whether the combination of hypoxia-activated anticancer prodrugs and PARPi has an additive effect in various research studies remains controversial, indicating the complexity of additive effects. Summary of the Invention [Problem to be solved by the invention]

[0013] In efficacy studies, the applicant's researchers found that the combination of TH-302 and PARPi showed additive effects in some in vivo tumor growth inhibition tests, which was completely different from the results of in vitro cell experiments conducted by Threshold in 2012. Therefore, the applicant conducted further research and obtained the unexpected result that TH-302 alone has excellent therapeutic effects on PARPi-resistant cancer models. [Means for solving the problem]

[0014] Based on experimental results, the present application provides the following cancer treatment methods. The present invention provides a therapeutic method for treating patients with PARPi-resistant cancers and tumors by using a single or combined drug containing an anoxia-activated compound of formula (I). JPEG0007762796000005.jpg26170In the formula, each R is independently selected from H, -CH3, and -CH2CH3, and each X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO. The present invention provides a method for treating patients with cancer or tumor resistant to PARP inhibitors by using a drug containing an anoxia-activated compound of formula (I) in combination with a PARP inhibitor. JPEG0007762796000006.jpg26170In the formula, each R is independently selected from H, -CH3, and -CH2CH3, and each X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO.

[0015] A drug as described herein refers to a medicine or formulation, wherein the prepared drug contains an anoxic activated compound of formula (I), a salt or solvate thereof as an active ingredient in a particular dosage range, and / or the prepared drug is administered in a particular dosage form, in a particular mode of administration.

[0016] The prepared medicine, drug, or formulation may further contain a pharmaceutically acceptable adjuvant or excipient. The drug may be in any clinically administered dosage form, such as a tablet, suppository, dispersible tablet, enteric-coated tablet, chewable tablet, orally disintegrating tablet, capsule, sugar-coated tablet, granule, dry powder, oral liquid, small-volume injection, lyophilized powder injection, or large-volume parenteral preparation. Depending on the specific dosage form and administration mode, the pharmaceutically acceptable adjuvant or excipient in the drug may include one or more of diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and dyes.

[0017] JPEG0007762796000007.jpg26170 includes oral formulations, lyophilized formulations, and concentrated injectable formulations, and related formulations, preparation methods, and clinical dosing and administration methods are described and disclosed in detail in Threshold's related patents WO2010048330A1, WO2012142520A2, and WO2008083101A1, the entire contents of which are incorporated herein by reference.

[0018] JPEG0007762796000008.jpg26170-class anti-cancer agents, which have the potential to treat a wide range of cancers. Related cancer indication experiments and clinical trials are described in patent applications of Threshold and other pharmaceutical companies (e.g., WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106 A1,WO2020118251A1,WO2014169035A1,WO2013116385A1,WO2019173799A2,WO2016081547A1,WO2014062856A1,WO2015069489A1,WO2012006032A2, WO2018026606A2, WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A2, WO20 16014390A2, 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), wherein the present invention incorporates all of the above related application and clinical trial information.

[0019] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) capable of potentially unlimited growth that can spread locally by attack and systemically by metastasis.

[0020] JPEG0007762796000009.jpg25170 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 gland. Other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, dysdifferentiation of cervical epithelium and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforme, hairy cell tumor, intestinal ganglioneuroma, proliferative corneal nerve tumor, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumor, malignant melanoma, malignant hypercalcemia, equine tumor, myeloid epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, granuloma mycosis, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, squamous cell carcinoma, both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilms' tumor.

[0021] PARP is an enzyme collectively known as poly ADP-ribose polymerase (PARP). PARP is a DNA repair enzyme that plays an important role in the DNA repair pathway. When DNA is damaged or broken, PARP is activated, which acts as a molecular receptor for DNA damage, recognizing and ligating the DNA break site, and further activating and catalyzing poly ADP-ribosylation of receptor proteins, thereby participating in the DNA repair process.

[0022] PARP inhibitors block the activity of PARP enzymes, preventing these "repairmen" from functioning normally, resulting in DNA damage not being repaired and cell death.

[0023] Cells have more than just one "repairman" called PARP. Even if PARP becomes a problem, the cell's DNA damage is carried over to the next step, and another "repairman" is waiting to repair the DNA. The protein produced by the BRCA gene is an important member of this other "repairman." Normal cells have this double insurance mechanism; even if a PARP inhibitor destroys one insurance, the other insurance still works, so the cell does not die.

[0024] However, in ovarian or breast cancer cells that have a BRCA gene mutation, the BRCA "repairman" can no longer function properly, and the PARP group, of course, continues to function, preventing the cancer cells from dying.

[0025] When PARP inhibitors specifically enter cancer cells, they inhibit the activity of the PARP enzyme, preventing it from functioning normally and preventing the DNA of cancer cells from being repaired. This allows PARP inhibitors to kill only cancer cells without killing normal cells.

[0026] When PARP inhibitors and BRCA gene mutations occur simultaneously, a condition known as "synthetic lethality" occurs, in which cell death occurs when two different genes (BRCA) or proteins (PRAP) are altered at the same time. However, if only one of these two genes / proteins is abnormal, cell death does not occur. A PARP inhibitor is a compound that has an inhibitory effect on the PARP enzyme, and any substance that can inhibit PARP enzyme activity is a PARP inhibitor.

[0027] It is clear that the PARP inhibitor is selected from the group consisting of five marketed drugs, Olaparib, Rucaparib, Niraparib, Talazoparib, Fluzoparib, and the drug Pamiparib which has entered Phase 3 clinical trials, and in this context it means a drug which substantially contains the active ingredient of a PARP inhibitor. JPEG0007762796000010.jpg42170JPEG0007762796000011.jpg40170JPEG0007762796000012.jpg43170

[0028] Talazoparib is indicated for adults with harmful or suspected harmful germline BRCA-mutated (gBRCAm) HER2-negative locally advanced or metastatic breast cancer. The commercially available dosage form is 0.25 mg / 1 mg talazoparib tosylate capsules, administered orally at 1 mg once daily. If adverse reactions occur, treatment interruption or dosage reduction should be considered. At the first occurrence of adverse reactions, reduce the oral dose to 0.75 mg (three 0.25 mg capsules) once daily. If adverse reactions occur a second time, reduce the oral dose to 0.5 mg (two 0.25 mg capsules) once daily. If adverse reactions occur after the third dose, reduce the oral dose to 0.25 mg (one 0.25 mg capsule) once daily.

[0029] Niraparib is indicated for the maintenance treatment of adult patients with platinum-sensitive recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer after achieving complete or partial remission with platinum-based chemotherapy. The commercially available dosage is 100 mg niraparib tosylate capsules, administered orally at a dose of 300 mg once daily until disease progression or intolerable side effects occur, with treatment interruption or dose reduction considered if side effects occur. The dose is initially reduced from 3 capsules (300 mg) per day to 2 capsules (200 mg) per day. If further dose reduction is necessary, the second dose may be reduced from two capsules (200 mg) daily to one capsule (100 mg) daily. If drug discontinuation or dose reduction fails to control adverse reactions, drug discontinuation is recommended.

[0030] Rucaparib is used for women with advanced ovarian cancer whose tumors have a specific genetic mutation (detrimental BRCA) and who have been treated with two or several 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. Treatment is continued until disease progression or toxicity is unacceptable. Adverse reactions may lead to treatment interruption or dose reduction.

[0031] Olaparib is indicated for the maintenance treatment of adult patients with initially treated, germline or somatically mutated (gBRCAm or sBRCAm) advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer after achieving complete or partial remission with platinum-based chemotherapy, and for the maintenance treatment of adult patients with platinum-sensitive, recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer after achieving complete or partial remission with platinum-based chemotherapy. It is commercially available in two tablets, 150 mg and 100 mg. The recommended dose is 300 mg (two 150 mg tablets) twice daily, corresponding to a total daily dose of 600 mg. The 100 mg tablet is used for dose reduction. Treatment discontinuation or dose reduction may be considered to address adverse events such as nausea, vomiting, diarrhea, and anemia. If a dose reduction is necessary, reduce the recommended dose to 250 mg (one 150 mg tablet and one 100 mg tablet) taken twice daily (equivalent to a total daily dose of 500 mg). If further 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).

[0032] Fluzoparib is indicated for the treatment of patients with germline BRCA-mutated (gBRCAm) platinum-sensitive recurrent ovarian, fallopian tube, or primary peritoneal cancer who have received at least second-line chemotherapy. It is commercially available in 50 mg capsules.

[0033] For other investigational PARPi drug candidates entering clinical trials, please see the web link https: / / www.selleckchem.com / PARP.html and related academic review literature.

[0034] JPEG0007762796000013.jpg26170 Recommended dosage forms are disclosed in patent applications of Threshold and other pharmaceutical companies (e.g., WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118251A1, WO 2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO2012006032A2, WO2018026606 A2, 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, NCT0186 4538, NCT01149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, NCT02712567). 120 mg / m per day 2 ~460mg / m 2 It is administered intravenously at a dose of 480 mg / m 2 ~about 670mg / m 2 , or, for example, 575 mg / m 2 Administered intravenously at a weekly dose of

[0035] TH-302 (concentrate for use in solution) used in clinical trials is a sterile liquid formulation of TH-302. TH-302 was prepared using 70% absolute ethanol, 25% dimethylacetamide, and 5% polysorbate 80. It was provided by the sponsor in 10 mL glass vials with rubber stoppers and flip-off seals. TH-302 drug product is a clear, colorless to pale yellow solution, substantially free of visible particles. For a nominal total dose of 650 mg of TH-302, each single-use vial contains a nominal fill volume of 6.5 mL (equivalent to 100 mg / mL) of TH-302 drug product and is clearly labeled to disclose the lot number, route of administration, required storage conditions, name of the sponsor, and appropriate warning markings required by applicable regulations. It must be diluted according to the pharmacy manual before administration.

[0036] Prior to administration, the desired final concentration was obtained by diluting the solution with commercially available 5% glucose in water to a total volume of 500 mL (1000 mL for a total dose of ≥1000 mg). Each dose of TH-302 was prepared in di(2-ethylhexyl) phthalate-free (DEHP-free) 5% glucose in water and infused intravenously using a DEHP-free intravenous infusion administration device. Of course, the lyophilized formulation developed by Threshold Corporation can also be used.

[0037] A solution (20 mL) of TH-302 (100 mg) and sucrose (1 g) was added to a lyophilization vial and lyophilized to give a concentration of 5 mg / cm 3 A lyophilized unit dosage form of TH-302 is obtained with a drug loading of less than 100 mg / kg. For human administration, the unit dosage form is dissolved in 5% glucose injection and an appropriate volume of the solution is administered to the patient.

[0038] The administration protocol for the subsequent Phase I clinical trial of TH-302 in human patients involves preparing a lyophilized formulation for injection in a 100mL glass vial with a drug loading of 100mg / 100mL and storing it under controlled conditions at 2-8°C. At the time of use, 250mL of 5% glucose injection solution is injected into the lyophilized formulation vial, which is then administered intravenously via an infusion pump within 30 minutes.

[0039] A single drug is a monotherapy. A combination is a combination therapy. Monotherapy refers to the use of only one anticancer drug in one treatment course. Combination therapy refers to the use of two or more anticancer drugs simultaneously or sequentially in one treatment course.

[0040] In general, in combination therapy, different dosages and administration cycles must be considered depending on the characteristics of the disease state and the type of combined drug. Only based on the above circumstances can the combination drug therapy protocol obtained through consideration achieve a higher therapeutic effect than monotherapy.

[0041] The drug dosage and administration cycle for both monotherapy and combination therapy protocols should be investigated through clinical trials, taking into account the dosage and administration protocols for TH-302, its analogues, and PARPi mentioned above. Furthermore, the patient's DNA repair enzymes are impaired. According to relevant research literature, damage to DNA repair enzymes: Damage to homologous recombination DNA repair enzymes Damage to nucleotide excision repair enzymes, damage to nonhomologous end joining; Damage to base excision repair enzymes, Damage to mismatch repair enzymes, Impairment of Fanconi's anemia pathway repair enzymes The compound is one or more selected from the group consisting of:

[0042] Preferably, the damage is one or more of damage to a homologous recombination DNA repair enzyme, damage to a nucleotide excision repair enzyme, or damage to a base excision repair enzyme, and more preferably, damage to a single homologous recombination DNA repair enzyme or damage to both a homologous recombination DNA repair enzyme and a nucleotide excision repair enzyme.

[0043] Furthermore, the patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2, or the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2. Genetic mutations in either or both of the genes corresponding to BRCA1 and BRCA2 can be detected by commercially available (associated) diagnostic kits.

[0044] OlaparibMate detection kit BRACAnalysisCDx gene detection is used to detect BRCA gene mutations in blood samples from ovarian cancer patients.

[0045] The BRCA1 / 2 Gene Mutation Detection Kit (Multiplex Probe Anchor Sequencing Method) is used to qualitatively detect germline mutations in the exon regions and adjacent intron regions of the BRCA1 / 2 gene in patients clinically diagnosed with ovarian cancer and breast cancer.

[0046] The human BRCA1 and BRCA2 gene detection kit (reversible end-termination sequencing method) is used in the drug guide for the PARP inhibitor olaparib. BRCA1 and BRCA2 mutations include germline mutations (gBRCAm) and somatic mutations (sBRCAm) of BRCA1 and BRCA2. The anoxic activating compound of formula (I) is selected from compounds of the following structure: JPEG0007762796000014.jpg56170

[0047] Furthermore, the cancer or tumor is selected from ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, lung cancer, liver cancer, colon cancer, rectal cancer, bladder cancer, etc., and the lung cancer is preferably non-small cell lung cancer or small cell lung cancer.

[0048] A method of treatment is provided for treating patients with olaparib-resistant ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, colon cancer, rectal cancer, or bladder cancer with a single drug containing an anoxia-activated compound of the following formula: JPEG0007762796000015.jpg30170

[0049] The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2, or the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2.

[0050] A method of treatment is provided in which olaparib is used in combination with a drug containing an anoxia-activated compound of the following formula to treat patients with olaparib-resistant ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, colon cancer, rectal cancer, or bladder cancer. JPEG0007762796000016.jpg30170

[0051] The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2, or the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2.

[0052] 1. A method of treatment comprising: detecting the BRCA1 and BRCA2 gene mutation status of patients with PARP inhibitor-resistant cancers and tumors; If the patient has a BRCA1 or BRCA2 gene mutation, treating the patient with a drug containing an anoxia-activated compound of formula (I) alone or in combination with a PARP inhibitor; Includes. JPEG0007762796000017.jpg26170 wherein each R is independently selected from H, -CH3, and -CH2CH3; each X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Preferably, the TMB (tumor mutation burden) of the genetic mutation is at a moderate level, 20. Use of an anoxia-activated compound of formula (I) in the preparation of a medicament for treating cancer in a patient, either alone or in combination with a PARP inhibitor. JPEG0007762796000018.jpg26170The patient is a patient resistant to a PARP inhibitor, Each R is independently selected from H, -CH3, and -CH2CH3, and each X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO.

[0053] In the above pharmaceutical use, the patient's DNA repair enzymes are impaired or The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1, BRCA2, or The patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2. BRCA1 and BRCA2 mutations include germline (gBRCAm) and sBRCAm BRCA1 and BRCA2 mutations.

[0054] In the above pharmaceutical use, the anoxic activating compound of formula (I) is selected from compounds having the following structure: JPEG0007762796000019.jpg31170The PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, fluzoparib, and pamiparib; or The cancer or tumor is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, colon cancer, rectal cancer, and bladder cancer; or The TMB (tumor mutation burden) of the gene mutations is at a moderate level.

[0055] Because tumor mutation load (TMB) levels vary among different tumor types, a TMB greater than 20 mutations / Mb (Mb stands for per million bases) is generally considered high, less than 10 mutations / Mb is considered low, and intermediate levels are considered moderate. At the 2017 World Lung Cancer Conference, Bristol-Myers Squibb (BMS) announced the results of a clinical trial called CheckMate-032. This was a phase II clinical trial involving 401 patients with advanced lung cancer who had failed phase I treatments and were treated with a PD-1 inhibitor alone or in combination with epilin. Patients were divided into three groups according to their TMB levels: high, medium, and low. In the group receiving combination therapy, the efficacy rates for the three groups were 62%, 20%, and 23%, respectively. The high TMB group had a three-fold higher efficacy rate, and the median overall survival times for the three groups were 22.0 months, 3.6 months, and 3.4 months, respectively, a six-fold difference between 22.0 months and 3.4 months. This study demonstrated that different TMB levels have a significant impact on the efficacy of different cancer therapeutic drugs.

[0056] The present invention further provides a drug containing an anoxia-activated compound of formula (I) for treating patients with PARP inhibitor-resistant cancer or tumor, which can be used as a single agent or in combination to treat patients with PARP inhibitor-resistant cancer or tumor. JPEG0007762796000020.jpg26170In the formula, each R is independently selected from H, -CH3, and -CH2CH3, and each X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO.

[0057] Preferably, the patient's DNA repair enzymes are impaired or The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1, BRCA2, or The patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2.

[0058] Preferably, the BRCA1 and BRCA2 mutations include germline mutations (gBRCAm) and somatic mutations (sBRCAm) of BRCA1 and BRCA2.

[0059] In particular, in the above medicament, the hypoxic activating compound of formula (I) is selected from compounds of the following structure: JPEG0007762796000021.jpg31170The PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, fluzoparib, and pamiparib; or The cancer or tumor is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, fallopian tube cancer, primary peritoneal cancer, gastric cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, colon cancer, rectal cancer, and bladder cancer; or The TMB (tumor mutation burden) of the gene mutations is at a moderate level.

[0060] The above-mentioned drug contains the hypoxic activating compound of formula (I), and pharmaceutically acceptable adjuvants or excipients are added according to the characteristics of the medicine, drug, and formulation. The drug can be in any clinically administered dosage form, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral liquids, small-volume injections, lyophilized powder injections, or large-volume parenteral preparations. Depending on the specific dosage form and administration method, the pharmaceutically acceptable adjuvants or excipients in the drug may include one or more of diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and dyes. When these drugs are used, they can be used alone or in combination with PARPi treatment. [Brief explanation of the drawings]

[0061] [Figure 1] Figure 1 shows the inhibition curves of compounds TH-302 and tirapazamine against Capan-1 cell line under normoxic and hypoxic conditions, where con.Log(nM) represents the base 10 logarithm of the concentration value in nmol / L, and inhibition represents the inhibition rate. [Figure 2] Figure 2 shows the inhibition curves of compounds TH-302 and tirapazamine against Capan-1 cell line under normoxic and hypoxic conditions, where con.Log(nM) represents the base 10 logarithm of the concentration value in nmol / L, and inhibition represents the inhibition rate. [Figure 3] FIG. 3 is a graph showing the growth of tumor volume in mice of each group in a subcutaneous model of human-derived pancreatic cancer, Capan-1. [Figure 4] FIG. 4 is a graph showing the relative tumor inhibition rate of each group of mice in a subcutaneous model of human-derived pancreatic cancer, Capan-1. [Figure 5] FIG. 5 is a graph showing the body weight of mice in each group in the Capan-1 subcutaneous model of human-derived pancreatic cancer. [Figure 6] FIG. 6 is a graph showing the percentage change in body weight of mice in each group in the Capan-1 subcutaneous model of human-derived pancreatic cancer. [Figure 7] FIG. 7 is a graph showing the growth of tumor volume in each group of mice in the pancreatic cancer Capan-1CDX model. [Figure 8] FIG. 8 is a graph showing the change in body weight over time for each treatment group in the Capan-1CDX pancreatic cancer model. [Figure 9] FIG. 9 is a graph showing the growth of tumor volume in each group of mice in the lung cancer LU6429 PDX model. [Figure 10] FIG. 10 is a graph showing the change in body weight over time for each treatment group in the LU6429 PDX lung cancer model. [Figure 11] FIG. 11 is a graph showing the growth of tumor volume in each group of mice in the BL3325PDX bladder cancer model. [Figure 12] FIG. 12 is a graph showing the change in body weight over time for each treatment group in the BL3325PDX bladder cancer model. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present invention and not limiting the scope of the present invention.

[0063] Unless otherwise specified, all experimental methods in the following examples are conventional. The medicinal materials, reagents, etc. used are commercially available unless otherwise specified.

[0064] "Patient" and "individual" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, a patient is a human. Typically, a patient is a human who has been diagnosed with cancer. In certain examples, a "patient" or "individual" can refer to a non-human mammal, e.g., a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used in screening, characterizing, and evaluating drugs and treatments.

[0065] A "prodrug" refers to a compound that, after administration or application, is converted metabolically or otherwise into a compound (or drug) that is more biologically active or active in at least one property. A prodrug is chemically modified to be less active or inactive relative to a drug, but the chemical modification allows the corresponding drug to be produced by metabolism or other biological processes after administration of the prodrug. A prodrug can have altered metabolic stability or delivery characteristics, fewer side effects, or lower toxicity, or improved flavor compared to an active drug. Prodrugs can be synthesized using reactants other than the corresponding drug. "Treatment" or "treating a patient" means the administration, use, or administration of a therapeutically effective amount of a drug in accordance with the present invention to a patient.

[0066] "Administration" or "application" (use) of a drug to a patient means direct administration or administration (which may be administered or administered to a patient by a health care professional or 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 administers or administers a drug to a patient.

[0067] A "therapeutically effective amount" of a drug refers to the amount of drug that, when administered or applied to a cancer patient, will have the desired therapeutic effect (e.g., alleviation, improvement, remission, or elimination of one or more clinical symptoms of cancer in the patient). The therapeutic effect does not necessarily occur by administration or application of a single dose, but may occur only after administration or application of a series of doses. Thus, a therapeutically effective amount can be administered or applied once or multiple times.

[0068] "Treatment" of a disease state or patient refers to taking measures to obtain beneficial or desired results (including clinical results). For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more cancer symptoms, reduction in disease severity, delay or slowing of disease progression, improvement, remission, or stabilization of the disease state, or other beneficial results. In some cases, treating cancer may result in a partial response or stabilization of the disease. "Tumor cell" refers to a tumor cell of any appropriate species (eg, mammalian, eg, murine, canine, feline, equine, or human).

[0069] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications based on the present invention without departing from the spirit of the present invention, all of which fall within the scope of the appended claims of the present invention. Specific experiments of the present invention will be described below.

[0070] The study protocol, any modifications, animal welfare, and application of the animal experiments during the study process disclosed herein have all been reviewed and approved by the local CRO (Joint Research Organization) IACUC committee. All animal welfare and experimental procedures during the study process meet AAALAC requirements.

[0071] (1) Comparison of in vitro cytotoxicity of compound TH-302 in BRCA knockout and wild-type tumor cells

[0072] TH-302 is a small molecule prodrug that is activated under hypoxic conditions, releasing cytotoxins and killing tumor cells and tumor tissues. To evaluate the association between TH-302 and BRCA pathogenic mutations at the cellular level in vitro, we used human colon cancer cell lines (DLD1 and BRCA2) in which the DLD1 and BRCA2 proteins were knocked out. - / - Tumor cell lines were selected to detect whether there was a difference in the killing ability of TH-302 against these two types of tumor cell lines under hypoxic conditions depending on whether they expressed BRCA2 protein.

[0073] This clonogenesis experiment was performed using IC 90 The (90% inhibitory concentration) value was used to evaluate the killing ability of compound TH-302 on cells. The specific experimental method is as follows.

[0074] (1)Cell culture a) DLD1 and DLD1-BRCA2- / - cells were cultured in RPMI medium supplemented with 10% FBS and 1% diabody and cultured at 37°C and 5% CO2.

[0075] (2) Cell plating a) Cells were cultured until cell saturation reached 80% to 90%, and once the required amount was reached, the cells were harvested. b) The cells were suspended in the corresponding medium, counted, and prepared into a cell suspension of appropriate density. c) Add the cell suspension to a 10 cm glass dish and incubate with DLD1-BRCA2 - / - The cell density was 3.0 × 10 5 The cell density was 300 / dish and the DLD1 cell density was 300 / dish. d) The cells were cultured in an incubator at 37°C and 5% CO2 for 2 days.

[0076] (3) Preparation of compounds The compounds were prepared according to the experimental requirements.

[0077] (4) Compound-treated cells a) Cells were treated with the compounds for 3 hours under conditions of oxygen content <0.01% (anoxia). b) The cells were washed once with 1x PBS, digested with trypsin, and then counted. c) Cells were suspended in 3 mL of medium and plated at 2000 / well with DLD1-BRCA2 - / - Cells were seeded into 6-well plates at a density of 300 DLD1 cells / well. d) The cell plates were placed in an incubator for 10 days. e) The medium was discarded, the cells were fixed, and then stained with crystal violet for 40 minutes. f) Perform cell clone counting using Colony Counter (VWR) and IC according to CalcuSyn software (http: / / www.biosoft.com / w / calcusyn.htm) 90 The value was calculated. The IC value of the test compound TH-302 in two types of cells obtained by the above experimental method 90 The values ​​are shown in Table 1 below.

[0078] JPEG0007762796000022.jpg23170

[0079] Experimental results: Compound TH-302 was activated by hypoxia and then inhibited the growth of DLD1 wild-type and BRCA2-deficient DLD1-BRCA2 - / - Significant differences in in vitro cytotoxicity of wild-type DLD1 cells were observed. 90 Values ​​are DLD1-BRCA2 - / - The sensitivity of DLD1 cells to TH-302 was increased by 70-fold compared to that of DLD1 cells. These results indicate that under hypoxic conditions, the BRCA2 protein deficiency significantly increased the sensitivity of DLD1 cells to TH-302.

[0080] (2) In vitro cell proliferation study of compound TH-302 / tirapazamine on Capan-1 / BxPc-3 cells The present applicant specifically investigated the in vitro cell growth inhibition of the Capan-1 and BxPc-3 cell lines under normoxic and hypoxic conditions using the hypoxic-activated anticancer prodrugs TH-302 and tirapazamine. The Capan-1 cell line is a BRCA-mutated cell line, while the BxPc-3 cell line is a BRCA-wild-type, i.e., non-BRCA-mutated, cell line. This experiment verified the difference in the sensitivity of TH-302, which is also a hypoxia-activated anticancer prodrug, and tirapazamine to BRCA mutations. The experimental method, data and results are as follows: Experimental Method

[0081] 1) Add 495 μL of Capan-1 / BxPc-3 cell suspension to each well of two 24-well plates at a cell density of 6 × 10 4 24-well plates with glass inserts were used for hypoxia experiments, and regular plastic 24-well plates were used for normoxia experiments. 2) The cells were cultured overnight in an incubator at 37°C and 5% CO2. 3) Compound treatment Hypoxic conditions: The hypoxic workstation was adjusted to an oxygen-deficient environment (O2<0.01%), and the oxygen-deficient state of the workstation was confirmed using an oxygen indicator. After plating the cells for 24 hours, the 24-well plates with glass inserts were transferred to a hypoxia station. The 24-well plate was placed on a screw shaker, and the well plate cover was opened and shaken to allow gas exchange for 5 minutes. For the single agent groups, compound solutions at 100 times the test concentration were added directly to each well. Normoxic conditions: After plating the cells for 24 hours, 5 μL of compound solution at 100x the test concentration was added to each well, with 3 wells in each experimental group. For the single agent groups, compound solutions at 100 times the test concentration were added directly to each well.

[0082] 4) After 3 hours of compound treatment, all 24-well plates were washed twice with complete medium, 500 μL per well. 5) 1000 μL of complete medium was added per well to Capan-1 cell culture plates, and 500 μL of complete medium was added per well to BxPc-3 cell culture plates. 6) The cells were left in a 37°C, 5% CO2 incubator for 72 hours.

[0083] 7) For Capan-1 cells, 800 μL of medium was discarded per well, and for BxPc-3 cells, 300 μL of medium was discarded per well, 50 μL of CTG was added, and the plate was shaken evenly for 2 minutes and then left at room temperature in the dark for 15 minutes. 8) The medium was transferred from the 24-well plate to a 96-well plate at 100 μL per well. 9) The chemiluminescence signal value was read using a multifunctional microplate reader, and the reading time was 1000 ms. 10) IC with GraphPad Prism5 software 50 Calculate the IC of the compound 50 values ​​(half maximal inhibitory concentrations) were obtained.

[0084] Experimental data Experimental data on cell growth inhibition of TH-302 and tirapazamine in the BRCA-mutated Capan-1 cell line under normoxic and hypoxic conditions are shown in Tables 2 and 3, and the IC 50 The curve is shown in FIG.

[0085] JPEG0007762796000023.jpg20170

[0086] JPEG0007762796000024.jpg20170

[0087] The experimental data of TH-302 and tyrapamine on the cell growth inhibition of BRCA wild-type BxPc-3 cell line under hypoxic and normoxic conditions are shown in Tables 4 and 5. 50 The curve is shown in FIG.

[0088] JPEG0007762796000025.jpg20170

[0089] JPEG0007762796000026.jpg20170

[0090] Experimental results The above experimental data show that TH-302 inhibits the IC50 expression in the BRCA mutant Capan-1 cell line under hypoxic conditions. 50 was 0.82 μM, and the IC in the BRCA wild-type BxPc-3 cell line 50 was 3.07 μM, a 3.7-fold difference between the two, indicating that BRCA mutations give TH-302 stronger growth inhibitory activity against tumor cell lines, i.e., BRCA mutations increase the sensitivity of tumor cells to the drug TH-302.

[0091] Tirapamine, another hypoxia-activated anticancer prodrug, exhibited IC β -reducing activity in BRCA-mutated Capan-1 cell lines under hypoxic conditions. 50 was 29.06 μM, and the IC in wild-type BRxPc-3 cell line 50 was 33.23 μM, a difference of 1.1-fold, which is not a significant difference, indicating that tyrapamine is not associated with BRCA mutations, i.e., BRCA mutations do not significantly affect the growth inhibitory activity of tyrapamine against tumor cell lines, i.e., BRCA mutations cannot increase the sensitivity of tumor cells to tyrapamine drugs.

[0092] (3) Efficacy and safety studies of TH-302 in olaparib-resistant animal CDX and PDX models 3.1 Efficacy and safety evaluation of TH-302 in the Capan-1CDX model of pancreatic cancer resistant to olaparib The Capan-1CDX model is an Olaparib-resistant model with a pathogenic BRCA2 mutation. Each BALB / c female nude mouse was implanted with 5 × 10 5Capan-1 cells were subcutaneously inoculated and resuspended in a 1:1 ratio of PBS and Matrigel (0.1 ml / mouse). A total of 64 female mice were inoculated. The inoculation date was June 23, 2021, and the average tumor volume was 140 mm. 3 Once tumor size reached 100 mg / kg, mice were randomly grouped according to tumor size. Seven groups were assigned to the study: Olaparib 100 mg / kg monotherapy (Group 2); TH-302 75 mg / kg in combination with Olaparib 100 mg / kg (Group 5); TH-302 75 mg / kg monotherapy (Group 7); and a vehicle control group containing 10% absolute ethanol, 10% polyoxyethylene (35) castor oil, and 80% glucose injection solution, D5W (pH 7.4). The vehicle control group, TH-302 monotherapy, and combination therapy groups were all administered intravenously via the tail vein once weekly for a total of three weeks. Olaparib was administered orally via intragastric administration once daily for a total of 30 days. Treatment efficacy was assessed by the relative tumor inhibition rate (TGI) (%), and safety was assessed by body weight change and mortality.

[0093] The group treated with the test drug olaparib 100 mg / kg (Group 2) showed no tumor inhibition effect at 35 days after tumor cell inoculation, with a relative tumor inhibition rate (TGI) of -7.1%, which was not statistically significant compared to the control group (p>0.05). The group treated with the combination of olaparib 100 mg / kg and TH-302 75 mg / kg (Group 5) showed significant tumor inhibition at 35 days after tumor cell inoculation, with a statistically significant difference compared to the control group (p<0.001), with a relative tumor inhibition rate (TGI) of 84.47%. The group treated with TH-302 75 mg / kg alone (Group 7) showed significant tumor inhibition at 35 days after tumor cell inoculation, with a statistically significant difference compared to the control group (p<0.001), with a relative tumor inhibition rate (TGI) of 87.66%. The TH-302 monotherapy group showed no significant difference in tumor inhibition effect compared with the Olaparib and TH-302 combination treatment group (p>0.05). The Olaparib 100 mg / kg, TH-302 75 mg / kg monotherapy groups, and the Olaparib 100 mg / kg and TH-302 75 mg / kg combination group did not significantly decrease mouse body weight, and the treatment was well tolerated. The specific dosing protocol for each group is shown in Table 6 below.

[0094] JPEG0007762796000027.jpg51170 The tumor volume of each group of mice was measured on different days, and the average values ​​obtained are shown in Table 7 below.

[0095] The tumor growth status of the treatment group and the control group is shown in Table 7 and Figure 3. The efficacy evaluation is shown in Table 8.

[0096] JPEG0007762796000029.jpg90170

[0097] Table 8: Relative tumor growth rate, T / C%, is the percentage value of the relative tumor volume or tumor weight of the treatment and control groups at a specific time point. The calculation formula is as follows:

[0098] T / C%=T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV : Mean RTV of the vehicle control group; RTV=Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of this animal after treatment).

[0099] The relative tumor inhibition rate (TGI) (%) was calculated as follows: TGI% = (1-T / C) × 100%, where T and C are the mean relative tumor volumes (RTV) of the treatment and control groups, respectively, at a particular time point.

[0100] JPEG0007762796000030.jpg34170Graphing the above table gives Figure 4. The body weights of the mice in the different groups were measured on different days, and the average results are shown in Table 10 below.

[0101] JPEG0007762796000031.jpg25170The above table can be plotted as a graph to obtain Figure 5, which shows the mouse weight curves for each group in the Capan-1 subcutaneous model of human-derived pancreatic cancer. Processing the data in Table 10 in the same way gives Table 11 below.

[0102] JPEG0007762796000032.jpg27170By graphing the above table, we obtain Figure 6.

[0103] Analysis of experimental data reveals the effectiveness of treatment.

[0104] 1. The Capan-1CDX model is indeed an Olaparib-resistant model, and Olaparib has no inhibitory effect on tumor growth in this model, i.e., it is resistant to Olaparib. 2. TH-302 monotherapy has a good therapeutic effect on olaparib-resistant pancreatic cancer (TGI 82.67%). 3. The combination of TH-302 and olaparib has a good therapeutic effect on olaparib-resistant pancreatic cancer (TGI was 87.43%). 4. The tumor inhibition effect of the TH-302 monotherapy group was slightly higher than that of the olaparib and TH-302 combination treatment group, but the difference was not significant (p>0.05).

[0105] Analysis of the experimental data showed that there was no significant decrease in body weight in any of the mice in the test drug Olaparib 100 mg / kg, TH-302 75 mg / kg group, or the combination of Olaparib 100 mg / kg and TH-302 75 mg / kg group, and that the treatment was well tolerated.

[0106] The inventors further investigated TH-302 in the Olaparib-resistant Capan-1CDX pancreatic cancer model and found that (1) the TH-302 monotherapy group showed dose-dependent tumor-inhibitory effects, and (2) specific dose combinations of Olaparib and TH-302 could exhibit synergistic tumor-inhibitory effects.

[0107] The present inventors simultaneously investigated the efficacy and safety of TH-302 in the olaparib-resistant lung cancer LU6429 PDX model and bladder cancer BL3325 PDX model. Further study protocols and experimental data are described below.

[0108] 3.2 Further investigation of the efficacy and safety of TH-302 in the Capan-1CDX model of pancreatic cancer resistant to olaparib Protocol: BALB / c nude mice were subcutaneously inoculated with human pancreatic cancer Capan-1 cells to establish a subcutaneous pancreatic cancer model. They were divided into seven groups (6 mice per group): olaparib 100 mg / kg (Group 2), TH-302 50 mg / kg (Group 3, QD), TH-302 100 mg / kg (Group 4), TH-302 50 mg / kg (Group 5, QW), TH-302 25 mg / kg (Group 6), TH-302 25 mg / kg plus olaparib 100 mg / kg (Group 7), and a control group (Group 1) containing 10% absolute ethanol, 10% polyoxyethylene (35) castor oil, and 80% glucose injection solution, D5W (pH 7.4). Both the vehicle control group and TH-302 were administered via tail vein injection. The 50 mg / kg TH-302 monotherapy group (Group 3, QD) received TH-302 once daily for three consecutive days, followed by a four-day break, a two-week break, and then another three consecutive days of daily administration. TH-302 was administered once weekly for three weeks in the 100 mg / kg (Group 4, QW), 50 mg / kg (Group 5, QW), and 25 mg / kg (Group 6, QW) TH-302 monotherapy groups, as well as the 25 mg / kg TH-302 and 100 mg / kg olaparib combination group (Group 7). Olaparib was administered orally and intragastrically once daily for a total of 30 days. The specific administration route, dose, and protocol for the Capan-1 human pancreatic cancer animal model are shown in Table 12.

[0109] JPEG0007762796000033.jpg66170Note: The administration volume is 10 μL / g.

[0110] As shown in Table 13, tumor growth in each treatment group and control group was recorded on different test days. Figure 7 shows the corresponding mouse tumor volume growth graph for each group. Efficacy was evaluated based on the relative tumor growth rate and relative tumor inhibition rate, and the efficacy analysis for each group is shown in Table 14. Post-administration weight changes in the treatment and control groups were recorded, and safety of each group was examined in the Capan-1 subcutaneous model of human pancreatic cancer. The results of the mouse weight change rate on day 43 are shown in Table 15, and a graph of weight change over time for each treatment group is shown in Figure 8.

[0111] Relative tumor growth rate, T / C%, is the percentage value of the relative tumor volume or tumor weight of the treatment and control groups at a specific time point. The calculation formula is as follows:

[0112] T / C%=T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV : Mean RTV of the solvent control group; RTV = V t / V0, V0 is the tumor volume of the animals in the group, and V t is the tumor volume of the animal after treatment). or T / C%=T TW / C TW ×100%(T TW : Mean tumor weight at the end of the experiment for the treatment group, C TW : Mean tumor weight at the end of the experiment in the vehicle control group).

[0113] The relative tumor inhibition rate (TGI) (%) was calculated as follows: TGI% = (1-T / C) × 100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups, respectively, at a specific time point.

[0114] JPEG0007762796000034.jpg99170

[0115] JPEG0007762796000035.jpg85170Notes: 1. Data are expressed as "mean ± standard error." 2. T / C% = T RTV / C RTV ×100% 3.CR: The tumor was completely remitted and the tumor regressed to 0.

[0116] JPEG0007762796000036.jpg75170

[0117] The above data shows the following: Mice in the vehicle control group had a mean tumor volume of 1301.38 mm on Day 43 after tumor cell inoculation. 3 In mice treated with the test drug Olaparib 100 mg / kg (Group 2), the mean tumor volume on Day 43 after tumor cell inoculation was 846.86 mm 3 The relative tumor inhibition rate (TGI) (%) was 37.43%, which was not statistically significant compared with the control group (p>0.05).

[0118] In the test drug TH-302 50 mg / kg treatment group (Group 3, QD), the mean tumor volume on Day 43 after tumor cell inoculation was 146.99 mm 3 There was a statistically significant difference compared to the control group (p<0.001), and the relative tumor inhibition rate (TGI) (%) was 89.17%. The test drug TH-302 100 mg / kg treatment group (Group 4) had a mean tumor volume of 124.68 mm on Day 43 after tumor cell inoculation. 3 The difference was statistically significant compared to the control group (p<0.001), with a relative tumor inhibition rate (TGI) of 90.89%. The test drug TH-302 50 mg / kg treatment group (Group 5, QW) had a mean tumor volume of 263.45 mm on Day 43 after tumor cell inoculation. 3There was a statistically significant difference compared to the control group (p<0.001), with a relative tumor inhibition rate (TGI) of 80.18% and a complete tumor inhibition rate of 33.3%. The test drug TH-302 25 mg / kg treatment group (Group 6) had a mean tumor volume of 1521.33 mm on Day 43 after tumor cell inoculation. 3 There was no statistically significant difference compared to the control group (p>0.05), and the relative tumor inhibition rate (TGI) (%) was -21.59%. The Olaparib 100 mg / kg and TH-302 25 mg / kg combination treatment group (Group 7) had a mean tumor volume of 378.56 mm on Day 43 after tumor cell inoculation. 3 This showed a statistically significant difference compared to the control group (p<0.001), and the relative tumor inhibition rate (TGI) (%) was 74.36%.

[0119] The TH-302 monotherapy groups, 100 mg / kg (Group 4), 50 mg / kg (Group 5, QW), and 25 mg / kg (Group 6), demonstrated a dose-dependent tumor-inhibitory effect. The TH-302 monotherapy groups, 100 mg / kg (Group 4, QW) and 50 mg / kg (Group 5, QW), showed statistically significant differences compared with the TH-302 monotherapy group, 25 mg / kg (Group 6, QW) (p<0.001 for all groups).

[0120] The combination treatment group of Olaparib 100 mg / kg and TH-302 25 mg / kg (Group 7) showed a superior antitumor effect than the Olaparib 100 mg / kg (Group 2) and TH-302 25 mg / kg single-agent group (Group 6), with statistically significant differences (p<0.05 and p<0.001), suggesting that the specific dose combination of Olaparib and TH-302 may exhibit synergistic tumor-inhibitory effects.

[0121] None of the mice in the test drug group (Olaparib 100 mg / kg (Group 2), TH-302 25 mg / kg (Group 6), TH-302 50 mg / kg (Group 3, QD), TH-302 50 mg / kg (Group 5, QW), TH-302 100 mg / kg (Group 4), the combination group of Olaparib 100 mg / kg and TH-302 25 mg / kg (Group 7), and the vehicle control group (Group 1) showed significant weight loss and were well tolerated.

[0122] 3.3 Efficacy and safety evaluation of TH-302 in the olaparib-resistant lung cancer LU6429 PDX model The LU6429 PDX model is an Olaparib-resistant model with a pathogenic BRCA2 mutation. Protocol: A human lung cancer subcutaneous tumor xenograft model was established by subcutaneously inoculating HuPrimeR lung cancer LU6429 tumor masses into Balb / c nude female mice. The study included six groups (6 mice per group) receiving the test drug Olaparib 50 mg / kg monotherapy (Group 02), TH-302 80 mg / kg monotherapy (Group 03), TH-302 40 mg / kg monotherapy (Group 04), TH-302 20 mg / kg monotherapy (Group 05), TH-302 40 mg / kg in combination with Olaparib 50 mg / kg (Group 06), and a glucose injection vehicle control group (Group 01). The study included six groups, each containing six mice. Both the vehicle control group and TH-302 were administered via the tail vein once weekly for three weeks. Olaparib was administered via the stomach once daily for a total of 28 days. Specific administration routes, doses, and protocols for HuPrime® lung cancer LU6429 in animal models are shown in Table 16.

[0123] JPEG0007762796000037.jpg55170Notes: 1. Dosage volume 10μL / g, 2. QD×28: Administered once daily for 28 consecutive days, 3. QW×3: Administered once weekly for 3 consecutive weeks.

[0124] As shown in Table 17, tumor growth in each treatment group and control group was recorded on different days. Figure 9 shows the corresponding mouse tumor volume growth graph for each group. Efficacy was evaluated based on the relative tumor growth rate and relative tumor inhibition rate, and the efficacy analysis for each group is shown in Table 18. Post-administration weight changes in the treatment and control groups were recorded, and the safety of each group in the HuPrime® lung cancer LU6429 subcutaneous model was examined. Mouse weight changes on day 25 are shown in Table 19, and a graph of weight changes over time for each treatment group is shown in Figure 10.

[0125] JPEG0007762796000038.jpg83170

[0126] JPEG0007762796000039.jpg75170Note: Data are expressed as "mean value ± standard error."

[0127] JPEG0007762796000040.jpg50170

[0128] The above data shows the following: The olaparib monotherapy group showed no tumor inhibitory effect, and the LU6429 PDX lung cancer model was resistant to olaparib. In this study, the 80 mg / kg, 40 mg / kg, and 20 mg / kg doses of the test drug TH-302 monotherapy and the 40 mg / kg TH-302 / 50 mg / kg olaparib combination therapy groups all had significant anti-tumor growth effects in the HuPrime® LU6429 subcutaneous lung cancer model. One mouse in the 80 mg / kg TH-302 (Group 03) treatment group experienced complete tumor elimination, representing a tumor elimination rate of 16.7%. There was a statistically significant difference (p <0.05) between the 80 mg / kg, 40 mg / kg, and 20 mg / kg TH-302 treatment groups, demonstrating dose-dependent effects. The combined therapeutic effect of TH-302 40 mg / kg and Olaparib 50 mg / kg was significantly superior to that of the Olaparib 50 mg / kg monotherapy group, but was slightly superior to that of the TH-302 40 mg / kg monotherapy group, the difference being small and not significant. Mice in each test drug treatment group did not lose weight during the treatment period and were well tolerated.

[0129] 3.4 Efficacy and safety evaluation of TH-302 in the BL3325 PDX model of bladder cancer resistant to olaparib The BL3325PDX model is an Olaparib-resistant model with a pathogenic mutation in BRCA2. Protocol: A human bladder cancer subcutaneous tumor xenograft model was established by subcutaneously inoculating HuPrime® BL3325 bladder cancer tumor masses into Balb / c nude female mice. The study included the following groups: Olaparib 50 mg / kg monotherapy group (Group 02), TH-302 80 mg / kg monotherapy group (Group 03), TH-302 40 mg / kg monotherapy group (Group 04), TH-302 20 mg / kg monotherapy group (Group 05), TH-302 40 mg / kg in combination with Olaparib 50 mg / kg (Group 06), and a glucose injection vehicle control group (Group 01). This study included six groups, each consisting of six mice. The vehicle control group and TH-302 were both administered via the tail vein once weekly for three weeks, while Olaparib was administered via the stomach once daily for 30 days. Specific administration routes, doses and protocols in the HuPrime® bladder cancer BL3325 animal model are shown in Table 20.

[0130] JPEG0007762796000041.jpg64170

[0131] Notes: 1. Dosage volume 10μL / g, 2. QD×30: once daily for 30 consecutive days, 3. QW×3: once weekly for 3 consecutive weeks, 4. Iv: tail vein administration, po: stomach administration.

[0132] Tumor growth in each treatment and control group was recorded on different test days, as shown in Table 21. A graph of the tumor volume growth in each group is shown in Figure 11. Efficacy was evaluated based on the relative tumor growth rate and relative tumor inhibition rate, and the efficacy analysis for each group is shown in Table 22. Post-administration weight changes in the treatment and control groups were recorded, and the safety of each group in the HuPrime® BL3325 subcutaneous bladder cancer model was evaluated. The results of mouse weight changes on day 35 are shown in Table 23, and a graph of the weight changes over time for each treatment group is shown in Figure 12.

[0133] JPEG0007762796000042.jpg89170

[0134] JPEG0007762796000043.jpg70170Notes: 1. Data are expressed as "mean ± standard error." 2. T / C% = T RTV / RTV *100%

[0135] JPEG0007762796000044.jpg129170Notes: 1. Data are expressed as "mean value ± standard error."

[0136] The above data shows the following: The TH-302 monotherapy groups at doses of 80 mg / kg, 40 mg / kg, and 20 mg / kg, as well as the TH-302 40 mg / kg and Olaparib 50 mg / kg combination treatment groups, all showed significant anti-tumor growth effects in the HuPrime® BL3325 subcutaneous bladder cancer model. Olaparib 50 mg / kg monotherapy did not show significant tumor inhibition. The TH-302 80 mg / kg group showed a statistically significant difference in tumor inhibition compared with the 20 mg / kg group, indicating that TH-302-mediated inhibition of BL3325 tumor growth was dose-dependent. One mouse in the TH-302 80 mg / kg treatment group (Group 03) completely eliminated the tumor, representing a tumor elimination rate of 16.7%. The combined therapeutic effect of TH-302 40 mg / kg and Olaparib 50 mg / kg was significantly superior to that of the Olaparib 50 mg / kg monotherapy group, and was also superior to that of the TH-302 40 mg / kg monotherapy group, with the difference being significant. Mice in each test drug treatment group did not lose weight during the treatment period and were well tolerated.

[0137] The PARP inhibitor selected in the examples of the present application is Olaparib, but Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, etc. are also PARP inhibitors with mechanisms of action similar to that of Olaparib, and all act by blocking enzymes involved in the repair of damaged DNA. Therefore, it can be assumed that PARPi such as Rucaparib, Niraparib, Talazoparib, Fluzoparib, Pamiparib, etc. will have tumor-inhibiting effects similar to those of Olaparib in the above experiments. TH-302 is an anoxia-activated DNA alkylating agent and is a compound of the general formula set forth in claim 1. JPEG0007762796000045.jpg26170

[0138] Since a similar mechanism to that of TH-302 has been demonstrated in related patent applications, it is entirely predictable that such compounds will have similar effects to TH-302.

Claims

1. 1. Use of an anoxic activated compound of formula (I) in the preparation of a medicament for treating cancer in a patient, either alone or in combination with a PARP inhibitor, comprising: the patient is a patient resistant to a PARP inhibitor; the PARP inhibitor is olaparib; The cancer or tumor is selected from pancreatic cancer, lung cancer, and bladder cancer.

2. The use according to claim 1, wherein a PARP inhibitor is used in combination with a drug containing an anoxia-activated compound of formula (I) to treat a patient with cancer or tumor resistant to a PARP inhibitor.

3. the patient's DNA repair enzymes are impaired, or The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1, BRCA2, or The use according to claim 1 or 2, wherein the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2.

4. The BRCA1 and BRCA2 mutations include germline mutations (gBRCAm) and somatic mutations (sBRCAm) of BRCA1 and BRCA2; The use according to claim 3, wherein the TMB (tumor mutation burden) of the genetic mutations is 10-20 mutations / Mb.

5. A method for treating patients with pancreatic cancer, lung cancer, or bladder cancer who are resistant to olaparib with a single drug containing an anoxia-activated compound of the following formula: The use described in claim 1, wherein the patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2, or the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2.

6. A drug containing an anoxia-activated compound of formula (I) for treating PARP inhibitor-resistant cancer, tumor patients, comprising: the PARP inhibitor is olaparib; The cancer or tumor is selected from pancreatic cancer, lung cancer, and bladder cancer. Drugs.

7. the patient's DNA repair enzymes are impaired, or The patient's tumor or cancer tissue is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1, BRCA2, or The drug described in claim 6, wherein the patient is detected to have a genetic mutation in one or both of the genes corresponding to BRCA1 and BRCA2, and the BRCA1 and BRCA2 mutations include germline mutations (gBRCAm) and somatic mutations (sBRCAm) of BRCA1 and BRCA2.

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