CN116392497A

CN116392497A - Anticancer medical application of glufosfamide

Info

Publication number: CN116392497A
Application number: CN202310049367.1A
Authority: CN
Inventors: 段建新; 李安蓉; 孟繁英; 荣唐纳德·T
Original assignee: Shenzhen Ascentawits Pharmaceutical Technology Co ltd
Current assignee: Shenzhen Ascentawits Pharmaceutical Technology Co ltd
Priority date: 2017-11-28
Filing date: 2018-10-22
Publication date: 2023-07-07
Also published as: CN110248664A; CN110248664B; WO2019105149A1

Abstract

The meglumine or analogue thereof has a specific inhibitory effect on cells of a specific genetic variation, in particular cells with impaired DNA repair, which cells or tissues have at least one or more of the genes BRCA1, BRCA2, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, RAD50, MER11, p53, NBS1, XRS2, XRCC3, ERCC1, ERCC2, ERCC3, ERCC4, XRCC1, ku80, MHS6, MGMT, PARP or ERCC 5. The analogues refer to: esters of one or more hydroxyl groups in the meglumine amide molecule with organic acids and inorganic oxyacids by esterification; esters of one or more hydroxyl groups in the phosphoramide molecule with amino acids by esterification; salts obtained by reacting a meglumine molecule with an acid. For this purpose, the pharmaceutical use of the glufosfamide or the analogues thereof in the treatment of tumors and cancer diseases in cancer patients with the above specific genetic variations is provided.

Description

Anticancer medical application of glufosfamide

The present application is a divisional application of a Chinese patent application of the invention with the application number of CN201880009940.3 and the invention name of 'anti-cancer medical application of the glufosfamide', which is filed on 10/22 2018.

Technical Field

The present invention relates to the treatment of cancer, tumors in patients with cancer or tumors in which a specific gene has been mutated by glufosfamide, in particular.

Background

Glufosfamide (glufosfamide), chemical name beta-D-Glucopyranosyl-N, N '-di (2-chloroethyl) phosphoramide, english name beta-D-Glucopyranosyl- [ N, N' -bis [ (2-chloroethyl) ] phosphoric acid diamide, is a new alkylating agent antineoplastic medicine, and is formed by connecting one molecule of isophosphamide nitrogen mustard with direct alkylation and one molecule of glucose through glycosidic bond. The glufosfamide is transported into tumor cells by sodium-dependent glucose transmembrane transporter SAAT1 and then active by the hydrolytic release of the isophosphamide mustard by glucosidase. .

The compound was first developed by the Asta medical (Degussa) located in Heidelberg, germany in cooperation with the cancer research center (DKFZ). The Baxter International, 10 th 2001, purchased a tumor portion of ASTAMIA and was renamed Baxter Oncology GmbH (Ion Niculiscu-Duvaz, current opinion in investigational drug,2002,3 (10): 1527-1532), and the post Baxter Oncology's meglumine project was picked up by Threshold, U.S. and used its metabolic localization technology to selectively target meglumine to tumor sites for commercial investigation of meglumine amide.

The U.S. Threshold corporation received rapid approval by the U.S. FDA in 2004 for the treatment of unresectable locally advanced or metastatic pancreatic cancer that had previously received gemcitabine (gemcitabine) treatment (w.steve am ons, jin-Wei Wang y, zhijian Yang y, george f. Tidmarshz and Robert m. Hoffmanny, neoplasia,2007.8,9 (8): 625-633), but the corporation announced no significant increase in overall survival rate as a two-wire treatment for the iii phase clinical trial of metastatic pancreatic cancer patients (Tudor e. Ciulema, alexander v. Pavlovsky b, gyorgy Bodokyc, avgustm. Garind, virginia k. Langmuire, stewart, george t. Tish, A randomised Phase III trial of glufosfamide compared with best supportive care in metastatic pancreatic adenocarcinoma previously treated with gemcitabine, european Journal Of Cancer,45 (2009): 1599-1599), as a final clinical trial of failure.

New drug application is also carried out by the Qilu pharmaceutical Co., ltd and Jiangsu Haoshen pharmaceutical industry Co., ltd in 2005 in China, and wholesale text of the Chinese food and drug administration is obtained, but the Chinese food and drug administration is not developed and marketed in the follow-up.

Disclosure of Invention

Experimental study shows that the glufosfamide has high sensitive growth inhibition effect on cancer cells with specific genetic variation, specifically cells with damaged DNA repair enzyme, the cells are at least BRCA1, BRCA2 and FANCA, FANCD1, FANCD2, 981, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2, ERCC3, ERCC4, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA Ligase I (Ligase I), MMS19, MNAT1, RAD23A, RAD, RPA2, IIH, XAB2, XPA, XPC, MBD, NEIL1, BAP1, CDK, 12, O1, FAO 20, FAN1, FAN 62N 1, F62N 51, DNA polymerase (DNA polymerase), DNA polymerase (DNA polymerase) or a more than one of the gene of the multiple gene (polymerase, the mutant gene) is used to make a direct DNA, the gene of the mutant gene (more than one of the gene) or more than the DNA polymerase, the gene (more than is Flap endonuclease).

For this purpose, based on the above experimental findings, the following technical solutions are provided:

technical scheme 1:

glufosfamide or an analog thereof is useful for treating a tumor or cancer patient with impaired DNA repair enzymes. Tumor (tumor) refers to a new growth (neogram) of a body formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new growth is often in the form of occupied massive protrusions, also called neoplasms (neoplasms). In medicine, cancer refers to malignant tumors originating from epithelial tissues, and is the most common type of malignant tumors. Correspondingly, malignant tumors originating from mesenchymal tissue are collectively referred to as sarcomas. There are a few malignant tumors which are not named according to the above principles, such as nephroblastoma, malignant teratoma, etc. The term "cancer" is generally used to refer to all malignant tumors. The tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 any one or more of ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, DNA ligase,

Preferably one or more of BRCA1, BRCA2, BARD1, FANCA, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, XRCC3, XRCC4/XPF, XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. Technical scheme 2:

the use according to claim 1, wherein the impaired DNA repair is

One or more of homologous recombination DNA repair enzyme (homologous recombination repair) damage, nucleotide excision repair enzyme (nucleotide excision repair) damage, non-homologous end joining enzyme damage (nonhomologous end joining), base excision repair enzyme (base excision repair) damage, mismatch repair enzyme (mismatch repair) damage, vanconi's anemia) pathway repair enzyme damage,

preferably any one or more of the homologous recombination DNA repair enzyme, nucleotide excision repair enzyme, base excision repair enzyme,

more preferably a patient with impaired homologous recombination DNA repair enzyme alone or with both homologous recombination DNA repair enzyme and nucleotide excision repair enzyme,

Preferably BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN and TMB of the gene corresponding to AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, DNA ligase are high,

more preferably a patient with impaired homologous recombination DNA repair enzymes.

Technical scheme 3:

the use according to any one of claims 1-2, wherein the medicament further comprises other anti-cancer agents, anti-tumor agents and agents for radiotherapy and surgery, including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxicity/cell growth inhibitors, antiproliferative agents, prenyl-proteinase inhibitors, HMG-CoA reductase inhibitors, HIV-proteinase inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

Technical scheme 4:

the use according to any one of claims 1-3, wherein the tumor, cancer comprises:

lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, hazy cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary cancer, papillary gland carcinoma, cystic gonadal carcinoma, cystic cancer, medullary cancer, bronchi cancer, bone cell carcinoma, epithelial cancer, cholangiocarcinoma, choriocarcinoma, embryo cancer, seminoma, wilms' cancer, glioblastoma, astrocytoma, medulloblastoma, craniopharyngeoma, ependymoma, pineal tumor, hemangioblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibromatoid tumor, fibroadenoma, fibromatoid osteoma, fibromatoid tumor, wilm fibrocyst, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocyst, myxochoma, myxochondrosarcoma, mucochondrofibrosarcoma, myxoadenoma, fibromatoma, fibroma, fibro myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondrioma, lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma myxoblastoma, liposarcoma, lipoadenoma, lipoblastoma, lipochoma lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma, vascular lipoma, vascular lymphangioma, vascular liposmooth myoma, vascular myoneuroma, vascular myxoma, vascular reticuloendothelioma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, ewing's tumor, smooth myoma, smooth myosarcoma, smooth myoma, smooth myofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lympholeukemia, acute myelogenous leukemia, chronic disease cells, erythrocytosis, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial cancer, multiple myeloma, preferably ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer.

Technical scheme 5:

the use according to any one of claims 1-4, wherein the analogue means: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

a. Esters of one or more hydroxyl groups in the meglumine amide molecule with organic acids and inorganic oxyacids by esterification;

b. esters of one or more hydroxyl groups in the phosphoramide molecule with amino acids by esterification;

c. salts obtained by reacting a meglumine molecule with an acid.

Technical scheme 6:

the use according to any one of claims 1-5, wherein the patient is a human (humanbeing) patient or a mammalian (mammal) patient other than a human.

Technical scheme 7:

a pharmaceutical composition for treating tumors and cancers of patients with damaged DNA repair enzymes containing glufosfamide or its analogues, the tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. Technical scheme 8:

the pharmaceutical composition according to claim 7, wherein the DNA repair enzyme is damaged as

One or more of homologous recombination DNA repair enzyme damage, nucleotide excision repair enzyme damage, non-homologous end joining enzyme damage, base excision repair enzyme damage, mismatch repair enzyme damage, vankini anemia pathway repair enzyme damage,

More preferably a patient with impaired homologous recombination DNA repair enzymes,

technical scheme 9:

the pharmaceutical composition according to any one of claims 7-8, wherein the pharmaceutical product further comprises other anti-cancer agents, anti-tumor agents and agents for radiotherapy and surgery, including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxicity/cell growth inhibitors, antiproliferative agents, prenyl-proteinase inhibitors, HMG-CoA reductase inhibitors, HIV-proteinase inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

The technical scheme 10 is as follows:

the pharmaceutical composition according to any one of claims 7-9, wherein the tumor, cancer comprises:

Technical scheme 11:

the pharmaceutical composition according to any one of claims 7-10, wherein the analogue means: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

Technical scheme 12:

the pharmaceutical composition according to any one of claims 7 to 11, wherein the patient is a human patient or a mammalian patient other than human.

Technical scheme 13:

glufosfamide or an analog thereof is useful for treating tumors, cancers in a patient with impaired DNA repair enzymes, tumors in the patient, the cancer tissue is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 any one or more of the genes corresponding to ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. Technical scheme 14:

the use of glufosfamide or an analog thereof according to claim 13 for the treatment of tumors, cancers with damaged DNA repair enzymes, wherein the damaged DNA repair enzymes are

more preferred are patients with impaired homologous recombination DNA repair enzymes alone or with both homologous recombination DNA repair enzymes and nucleotide excision repair enzymes.

Technical scheme 15:

the use of a meglumine or an analogue thereof according to any one of claims 13-14 for the treatment of tumors, cancers in which DNA repair enzymes are damaged, wherein the meglumine or an analogue thereof is also used in combination with other anti-cancer agents, anti-tumor agents including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

Technical scheme 16:

the use of glufosfamide or an analog thereof according to any one of claims 13-15 for treating a tumor, cancer in a patient with impaired DNA repair enzymes, wherein the tumor, cancer comprises:

Technical scheme 17:

the use of glufosfamide or an analogue thereof according to any one of claims 13-16 for the treatment of tumors, cancers in which DNA repair enzymes are damaged, wherein the analogue refers to: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

Technical scheme 18:

the use of glufosfamide or an analogue thereof according to any one of claims 13-17 for the treatment of tumors, cancers in which DNA repair enzymes are impaired, wherein tumors or cancers refer to tumors or cancers in humans and mammals other than humans.

Technical scheme 19:

the preparation containing the glufosfamide or the analogues thereof is used as a medicine for treating tumors and cancer patients with damaged DNA repair enzymes, the tumors of the patients, the cancer tissue is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 any one or more of the genes corresponding to ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. The technical scheme 20 is as follows:

the pharmaceutical preparation comprising glufosfamide or its analogues according to claim 19 for use as a tumor or cancer therapeutic in a patient suffering from DNA repair enzyme damage, wherein the DNA repair enzyme damage is

more preferably a patient with impaired homologous recombination DNA repair enzyme alone or with both homologous recombination DNA repair enzyme and nucleotide excision repair enzyme

technical scheme 21:

the use of a formulation comprising glufosfamide or an analogue thereof according to any one of claims 19-20 as a medicament for the treatment of tumors, cancers in patients with impaired DNA repair, wherein the formulation comprising glufosfamide or an analogue thereof further comprises other anti-cancer agents, anti-tumor agents and radiotherapy and surgical treatments, other anti-cancer agents, anti-tumor agents including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

Technical scheme 22:

the use of a formulation comprising glufosfamide or an analogue thereof according to any one of claims 19-21 as a medicament for treating a tumor, cancer in a patient suffering from impaired DNA repair enzymes, wherein the tumor, cancer comprises:

Technical scheme 23:

the use of a formulation comprising glufosfamide or an analogue thereof according to any one of claims 19-22 as a medicament for the treatment of DNA repair enzyme-damaged tumors, cancers, wherein the analogue is: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

Technical scheme 24:

the pharmaceutical product for treating tumors and cancers, which is characterized by comprising a preparation containing glufosfamide or an analogue thereof according to any one of the claims 19-23.

Technical scheme 25:

a method of treating tumors, cancers in a patient suffering from a DNA repair enzyme damage, which method comprises administering to said patient suffering from the disease a pharmaceutical formulation comprising glufosfamide or an analogue thereof, the tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. Technical scheme 26:

the method of claim 25, wherein the DNA repair enzyme is damaged as

technical scheme 27:

the method of any one of claims 25-26, wherein the pharmaceutical formulation further comprises additional anti-cancer agents, anti-tumor agents and agents for radiation therapy and surgery, including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxicity/cell growth inhibitors, antiproliferative agents, prenyl-proteinase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, and apoptosis inducers.

Technical scheme 28:

the method of treatment according to any one of claims 25-27, wherein the tumor, cancer comprises:

lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, hazy cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary cancer, papillary gland carcinoma, cystic gonadal carcinoma, cystic cancer, medullary cancer, bronchi cancer, bone cell carcinoma, epithelial cancer, cholangiocarcinoma, choriocarcinoma, embryo cancer, seminoma, wilms' cancer, glioblastoma, astrocytoma, medulloblastoma, craniopharyngeoma, ependymoma, pineal tumor, hemangioblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibromatoid tumor, fibroadenoma, fibromatoid osteoma, fibromatoid tumor, wilm fibrocyst, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocyst, myxochoma, myxochondrosarcoma, mucochondrofibrosarcoma, myxoadenoma, fibromatoma, fibroma, fibro myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondrioma, lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma myxoblastoma, liposarcoma, lipoadenoma, lipoblastoma, lipochoma lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma, vascular lipoma, vascular lymphangioma, vascular adipose leiomyoma, vascular myoma, vascular myoneuroma, vascular myxoma, vascular reticuloendothelioma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoma, leiomyomata, rhabdomyoma, rhabdomyomyxoma, acute lympholeukemia, acute myelogenous leukemia, chronic disease cells, erythrocytosis, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial cancer or multiple myeloma, preferably ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, breast cancer.

Technical scheme 29:

the method of treatment according to any one of claims 25-28, wherein the analogue means: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

The technical scheme 30 is as follows:

the method of any one of claims 25-29, wherein the patient is a human patient or a mammalian patient other than a human.

Technical scheme 31:

an immune combination therapeutic for treating tumors and cancers in a patient having an impaired DNA repair enzyme, the therapeutic comprising:

glufosfamide or an analog thereof; and

an immune checkpoint inhibitor,

wherein,, the tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK, CETN2, DDB 1' any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG. Technical scheme 32:

the therapeutic-free combination treatment drug of claim 31, wherein the DNA repair enzyme is damaged as

preferably BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, FAN1, FANCE, FANCM, MDC, NONO, POLQ, RAD B, RBBP, USP11, WRN and DNA endonucleases for patients, and DNA endonucleases of the patient's, and the endonucleases of the DNA to the DNA,

Technical scheme 33:

the therapeutic combination treatment drug according to claim 31, wherein the immune checkpoint inhibitor is a drug that interferes with a cell cycle checkpoint, and comprises inhibitors such as PD-1 (immunosuppressive receptor) inhibitor, PD-L1 (immunosuppressive receptor ligand) inhibitor, and CTL4 (soluble T lymphocyte 4) inhibitor.

Technical scheme 34:

the combination therapy of any one of claims 31-33, wherein the combination therapy further comprises other anti-cancer agents, anti-tumor agents and agents for radiation therapy and surgery, other anti-cancer agents, anti-tumor agents including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxicity/cell growth inhibitors, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

Technical scheme 35:

the combination therapy-free medicament of any one of claims 31-33, wherein the tumor, cancer comprises:

The technical scheme 36 is as follows:

the therapeutic-free combination treatment drug of any one of claims 31-33, wherein the analog is: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

Technical scheme 37:

the therapeutic-free combination treatment drug of any one of claims 31-33, wherein the patient is a human patient or a mammalian patient other than a human.

Technical scheme 38:

a method for treating a patient suffering from cancer or tumor, that is, the patient is first subjected to BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonucleases, end-processing enzymes, DNA polymerase, flap endonucleases, gene diagnostic reagents or gene diagnostic methods of DNA ligase or gene sequencing, to screen patients suffering from the above-mentioned gene mutation and to administer to these patients a drug containing glufosfamide or an analogue thereof.

Technical scheme 39:

the method of claim 38, further comprising administering to the subject a drug, formulation or combination of agents that enhance expression of glucose transporters or an analog thereof, wherein the subject is treated to enhance expression of glucose transporters, in order to enhance anticancer targeting of the meglumine or the analog thereof.

Furthermore, the compound preparation of the glucagons amide or the analogues thereof and the medicament for enhancing the expression of the glucose transport protein can be directly taken,

Technical scheme 40:

a compound formulation for treating cancer or tumor comprising:

glufosfamide or an analog thereof; and

a drug for enhancing the expression of glucose transporter,

wherein,, the tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease,

More preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG,

Technical scheme 41:

to detect whether or not the above BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS19, 12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC, 95, B, RBBP, WRN 5, USP11, DNA ligase, DNA polymerase, and the like should be mutated or not, the invention also provides a corresponding detection reagent, the reagent comprises a reagent selected from the group consisting of BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and inhibitors of the genes encoding the AP endonucleases, end-processing enzymes, DNA polymerases, flap endonucleases, proteins or enzymes encoded by the genes corresponding to the DNA ligases, and other suitable adjuvants.

Technical scheme 42:

an immunomodulatory compound preparation for treating cancer or tumor, comprising:

glufosfamide or an analog thereof; and

an immunomodulating agent, which comprises an immunomodulating agent,

The immunomodulating drugs include immunosuppressants and immunopotentiators.

Immunosuppressants include cyclosporin (Ciclosporin), tacrolimus (Tacrolimus), azathioprine (Azathioprine), and the like.

The immunopotentiator comprises: 1 non-specific active ingredients that enhance, modulate and restore the immune response of the body; 2 an interferon or an interferon inducer; 3 thymus hormone, thymus factor; 4 lymphokines, cytokines; 5 monoclonal antibodies and cross-links; 6 reactivating the immunocompetent cells; 7 tumor antigen and immune vaccine, and the specific medicine includes thymosin, metastasis factor, recombinant human interferon, pidotimod (Pidotimod), BCG polysaccharide nucleic acid preparation, ginseng, astragalus root, schisandra, wolfberry fruit, dangshen, aweto, glossy ganoderma, tremella polysaccharide and other natural medicine with immunity raising effect.

Technical scheme 43:

a method of treating cancer or tumor comprising

Administering to the patient a treatment with glufosfamide or an analog thereof; and

the immune adjustment treatment is carried out, and the immune adjustment treatment is carried out,

The immunomodulating drugs include immunosuppressants and immunopotentiators.

Immunosuppressants include cyclosporine, tacrolimus, azathioprine, and the like.

The immunopotentiator comprises: 1 non-specific active ingredients that enhance, modulate and restore the immune response of the body; 2 an interferon or an interferon inducer; 3 thymus hormone, thymus factor; 4 lymphokines, cytokines; 5 monoclonal antibodies and cross-links; 6 reactivating the immunocompetent cells; 7 tumor antigen and immune vaccine, and the specific medicine includes thymosin, metastasis factor, recombinant human interferon, pidotimod, BCG polysaccharide nucleic acid preparation, ginseng, astragalus root, schisandra, wolfberry fruit, dangshen, aweto, glossy ganoderma, tremella polysaccharide and other natural medicine with immunity raising effect.

Specifically, the administration of the glufosfamide or its analogue may be performed to the patient first and then the administration of the immunotherapy may be performed, and the administration of the glufosfamide or its analogue may be intravenous administration or may include intratumoral administration.

Due to the difference in TMB (Tumor mutation load (burden), i.e. tumor gene mutation load) levels between different tumor species: it is generally considered that: TMB is higher than 20 mutations/Mb (Mb represents millions of bases), i.e., high; below 10 mutations/Mb, i.e. low, in the middle, i.e. medium. In 2017, the world lung cancer conference, shi Guibao corporation published a clinical test result named CheckMate-032. This is a phase II clinical trial of 401 patients with advanced lung cancer who failed first line therapy who received PD-1 inhibitors alone or in combination with ipilimus. According to the TMB height, the patients are classified into TMB high, TMB middle and TMB low, so that the effective rate of the three groups in the group receiving the combined treatment is 62%, 20% and 23%, and the effective rate of the group with TMB high is 3 times higher; the median total survival of the three groups is respectively: 22.0 months, 3.6 months, 3.4 months-22.0 months differs from 3.4 months by a factor of 6. This experiment demonstrates that for different cancer therapeutic drugs, different TMB levels have a great impact on the efficacy of the drug.

For this reason, the present invention is preferably the following genes according to the conclusions of the literature of the prior study: BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK, CETN2, DDB1, DDB2, ERCC5/XPG ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, TMB of the gene for DNA ligase are high patients.

Term interpretation:

BRCA1 and BRCA2, namely Breast Cancer 1 and Breast 2 genes.

FANCA is Fanconi anemia complememtation groupA gene FANCD1 and FANCD2 is Fanconi anemia group D and 2protein genes.

ATM ataxia telangiectasia Mutated gene (Ataxia Telangiectasia-Mutated gene).

ATR, ATM-Rad3-Related gene.

CHEK1, CHEK2,

checkpoint kinase

1, 2 genes.

CTP, cytidine Triphosphate gene.

BARD1, BRCA1-associated RING domainprotein 1 gene.

BRIP1, BRCA1-interacting protein 1 gene.

RAD51D, RAD, 51C, RAD, RAD54, RAD55, RAD57, namely Restriction-site associated DNA, namely Restriction sites 51D, 51C, 52, 54, 55, 57.

FAM175, family with sequence similarity 175 membrane gene.

NBN, nijmegen breakage syndrome 1 gene.

Rad50, rad23A, RAD, 23B, RAD B, DNA repair protein RAD50, rad23A, RAD, 23B, RAD B genes.

MRE11, mismatch Repair Endonuclease 11 gene.

p53, tumor protein p53 gene.

NBS1, nijmegen breakage syndrome, no. 1 gene.

XRS2, a homologous gene to human Nbs 1.

XRCC1, XRCC2, XRCC3, DNA repair protein X-Ray Repair Cross Complementing1, 2, 3 genes.

ERCC1, ERCC2, ERCC3, ERCC4, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, excision

Repair Cross-Complementation group

1, 2, 3, 4, 5, 6, 8 genes.

Ku80, a gene encoding Ku80 protein.

MHS6, mutS homolog 6 gene.

MGMT, methylguanine Methyltransferase gene.

PARP, poly ADP-ribose polymerase polyadenylation-ribose polymerase gene.

CCNH, cyclin H gene.

CDK7, 12,Cyclin Dependent Kinase 7, 12 genes.

CETN2, centrin 2 gene.

DDB1, DDB2, damage Specific DNA Binding Protein 1/2 genes.

LIG1/DNA Ligase I, DNA Ligase 1 gene.

MMS19，MMS19 Homolog,Cytosolic Iron-Sulfur Assembly Component。

MNAT1, NAT1, CDK Activating Kinase Assembly Factor genes.

RPA1, RPA2, replication Protein A1, A2 genes.

TFIIH, transcription Factor IIH gene.

XAB2, XPA Binding Protein 2 gene.

XPA, DNA Damage Recognition And Repair Factor gene.

XPC, PC Complex subnit, DNA Damage Recognition And Repair Factor gene.

MBD4, methyl-CpG Binding Domain 4,DNA Glycosylase gene.

NEIL1, nei Like DNA Glycosylase 1 gene.

BAP1，BRCA1 Associated Protein 1。

EXO1，Exonuclease 1。

FAAP20，Fanconi Anemia Core Complex Associated Protein 20。

FAN1，FANCD2 And FANCI Associated Nuclease 1。

FANCE，Fanconi Anemia Complementation Group E。

FANCM，Fanconi Anemia Complementation Group M。

MDC1，Mediator Of DNA Damage Checkpoint 1。

NONO，Non-POU Domain Containing Octamer Binding。

POLQ，DNA Polymerase Theta。

RBBP8，RB Binding Protein 8,Endonuclease。

SMC5，Structural Maintenance Of Chromosomes 5。

USP11，Ubiquitin Specific Peptidase 11。

WRN，Werner Syndrome RecQ Like Helicase。

AP endonucleases, AP endonucleases.

End processing enzymes, end-processing enzyme.

DNA polymerase.

Flap endonuclease, flap endonuclease.

Obviously, the specific treatment method of the disease provided by the invention is to firstly carry out the gene diagnosis test of the genes on the subject or patient, even if the corresponding BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, gene diagnostic reagent or gene diagnostic method or gene sequencing of the gene corresponding to DNA ligase, to screen out patients suffering from the above gene mutation, and to administer or treat.

Further, in order to enhance the anticancer targeting effect of the glufosfamide or the analogues thereof, a drug or a preparation for enhancing the expression of the glucose transporter or a treatment for enhancing the expression of the glucose transporter can be used in combination with the patient, and further, a compound preparation of the glufosfamide or the analogues thereof and the drug for enhancing the expression of the glucose transporter can be directly taken.

To detect BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 whether or not the gene corresponding to ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, DNA ligase is mutated or the like, the invention also provides a corresponding detection reagent, the reagent comprises a reagent selected from the group consisting of BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/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, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonucleases, end-processing enzymes, DNA polymerases, flap endonucleases, inhibitors of proteins or enzymes encoded by genes corresponding to DNA ligases, and other suitable adjuvants, in the test, whether the corresponding BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, or not is judged by observing the effect of the corresponding inhibitor on the cells MGMT, PARP, ERCC5/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonucleases, end-processing enzymes, DNA polymerases, flap endonucleases, DNA ligase.

Clearly, as a broad spectrum anticancer agent, the tumors, cancers that can be treated are quite broad, including but not limited to the above: lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, hazy cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary cancer, papillary gland carcinoma, cystic gonadal carcinoma, cystic cancer, medullary cancer, bronchi cancer, bone cell carcinoma, epithelial cancer, cholangiocarcinoma, choriocarcinoma, embryo cancer, seminoma, wilms' cancer, glioblastoma, astrocytoma, medulloblastoma, craniopharyngeoma, ependymoma, pineal tumor, hemangioblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibromatoid tumor, fibroadenoma, fibromatoid osteoma, fibromatoid tumor, wilm fibrocyst, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocyst, myxochoma, myxochondrosarcoma, mucochondrofibrosarcoma, myxoadenoma, fibromatoma, fibroma, fibro myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondrioma, lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma myxoblastoma, liposarcoma, lipoadenoma, lipoblastoma, lipochoma lipofibroma, lipohemangioma, myxoma, chondrosarcoma, chondrioma, chondromyoma, chordoma, vascular lipoma, vascular lymphangioma, vascular liposmooth myoma, vascular myoneuroma, vascular myxoma, vascular reticuloendothelioma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphangioepithelial tumors, lymphoblastoma, endothelioma, endothelial cell tumors, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, ewing's tumor, smooth myoma, smooth myosarcoma, smooth myofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lympholeukemia, acute myelogenous leukemia, chronic disease cells, erythrocytosis, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial cancer, and multiple myeloma.

As a preference for the above 41 technical solutions, the analogues are:

a. esters of one or more hydroxyl groups in the molecule of the meglumine amide with organic acid and inorganic oxyacid by esterification, wherein the organic acid can be carboxylic acid (-COOH) or sulfonic acid (-SO) ₃ H) Sulfinic acid (RSOOH), thiocarboxylic acid (RCOSH);

b. esters of one or more hydroxyl groups in the phosphoramide molecule with amino acids, which may be naturally occurring α -amino acids or other β -, γ..w-amino acids obtained by proteolysis, more preferably α -amino acids;

c. the acid can be organic acid or inorganic acid, and the organic acid can be carboxylic acid (-COOH) or sulfonic acid (-SO) ₃ H) Sulfinic acid (RSOOH), thiocarboxylic acid (RCOSH), and inorganic acid such as sulfuric acid, hydrochloric acid, phosphoric acid, hydrobromic acid, etc.

The combination of the meglumine or analogues thereof according to the invention with other anti-cancer or chemotherapeutic agents, immunotherapeutic agents, obviously should also be able to achieve the objects of the invention, and should also be protected. Non-limiting examples of such agents can be found in oncology for cancer principles and practices, v.t. devita and s.hellman (editors), 6 th edition (month 15 of 2001), lippincottWilliams & Wilkins press. One skilled in the art can identify what combination of agents may be used based on the particular characteristics of the drug and the cancer (or other indication) involved. Anticancer agents incorporated for use as a compound include, but are not limited to, the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, microtubule inhibitors/stabilizers, topoisomerase inhibitors, antisense RNA and DNA oligonucleotides, antimetabolites, antibodies or radioisoforms conjugated to cytotoxic agents, HMG-CoA reductase inhibitors, prenyl transferase inhibitors, farnesyl protein transferase inhibitors, angiogenesis inhibitors, kinase inhibitors, COX2 inhibitors, integrin blockers, PPAR agonists, MDR inhibitors, and immunotherapeutic agents. Other anticancer agents also include hypoxia activatable agents, proteasome inhibitors, ubiquitin inhibitors, HDM2 inhibitors, TNF activators, BUB-R inhibitors, CENP-E inhibitors, and interferons (e.g., interferon-alpha). Such anti-cancer agents may be small molecules or biological agents (e.g., RNA antisense agents and antibodies).

HDAC inhibitors are histone deacetylase inhibitors comprising:

a. fatty acids such as butyrate, phenyl butyrate and valproic acid;

b. hydroxamates, such as TSA, are found as the 1 st natural hydroxamates that inhibit HDACs, SAHA is structurally similar to TSA and is approved as the 1 st clinically useful agent;

c. cyclic peptides such as the natural products depsipeptides FK-228, apicidin and epoxyoxime acid;

d. benzamide such as MS-275, MGCD0103, etc.

By "estrogen receptor modulator" is meant herein a compound that interferes with or inhibits estrogen binding to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, anastrozole, and letrozole.

"androgen receptor modulator" refers to a compound that interferes with or inhibits androgen binding to a receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride (finasteride) and other 5 alpha-reductase inhibitors, nilutamide (nilutamide), flutamide, bicalutamide (bicalutamide), liazol (liarozole), and abiraterone (abiraterone) acetate.

"retinoid receptor modulator" refers to a compound that interferes with or inhibits the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, difluoromethylornithine, ILX23-7553, trans-N- (4' -hydroxyphenyl) retinoamide and N-4-carboxyphenyl retinoamide.

Examples of cytotoxic agents include, but are not limited to, servanef, cachexin, isophosphamide (ifosfamide), tasonermin, lonidamine (lonidamine), carboplatin, hexamethylenemine (altramine), prednisolide (prednimustine), dibromodulcitol, ranimustine (raniustine), fostidine TM (fotemustine), nedaplatin (nedaplatin), platinum oxalate, temozolomide (temozolomide), cyclophosphamide, heptylplatinamine (hepaplatin), estramustine (estramustine), promethamine (improsulfan) tosylate, chlorocyclophosphamide, nimustine (nimustine), dibromo Shi Pi pyridine (dibrospidium chloride), pumitepa (pudapamide), luo Babo amine (lobaplatin), opamine (satraplatin), proformycin (proformine), cisplatin, oxamine (promethamine), oxamide (gplatin), oxamide (6-hydroxy-1, 6-dichloro-6-acetyl-1-dichloro-1-peripheral-1-diamine, 6-dichloro-1-acetyl-1-dichloro-amide (6-dichloro-oxamide), oxamide (6-dichloro-1, dichloro-hydroxy-1-acetyl-1, 6-dichloro-acetyl-1-amine (oxamide), oxamide (oxamide) and (oxamide) bis-hydroxy-1-bis-methyl-1, oxamide (oxamide) Rhodocin (zorubicin), idarubicin, daunorubicin, biscantrene (biscantrene), mitoxantrone (mitoxantrone), pi Lagong bacteriocin (pirrubicin), pinacolin Huai De (pinafide), rhodocin, azarhodocin (amrubicin), anti-new plastron (antineoplastin), 3 '-deammon (deansino) -3' -morpholino-13-deoxy-10-hydroxycarminomycin, anamycin (anamycin), garamycin (galubecin), also lina Huai De (elinafide), MEN10755, 4-desmethoxy-3-deamino-3-aziridinyl-4-methylsulfonyl-daunorubicin (see WO 00/50032), methotrexate, gemcitabine, and mixtures thereof.

Examples of microtubule inhibitors/microtubule stabilizing agents include paclitaxel (paclitaxel), vinca-sulfate, 3',4' -didehydro-4 '-deoxy-8' -N-vinca-leukomycin, docetaxel, vincristine, vinblastine, vinorelbine (vinorelbine), sitagliptin (rhizoxin), dolastatin, mi Wo protein (mivobulin), isethionate, auristatin (auristatin), cerocin Ma Duoting (cemadetin), RPR109881, BMS 184740, wen Fulu-ning (vinflunine), cryptophycin, 2,3,4,5, 6-pentafluoro-N- (3-fluoro-4-methoxyphenyl) benzenesulfonamide, dehydrated vinca-alkaloid, N-dimethyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-t-butyl-proline, x258, 62, and (e.g., see U.S. patent No. 62,284,786).

Some examples of topoisomerase inhibitors are topotecan (topotecan), sea catamide (hycaptamine), irinotecan (irinotecan), lu Biti kang (rubitecan), and the like.

Antimetabolite drugs include: 5-fluorouracil, enocitabine (enocitidine), azoxystrobin (carmofur), tegafur (tegafur), penstatin, doxifluridine, trimethazine (trimetrexate), fludarabine (fludarabine), capecitabine (capecitabine), garostabine (galocitabine), cytarabine (ocfosfote), sodium hydrate of frieabine (fosteabine), raltitrexed (raltitrexed) Balti Cui Xide (pattrexid), ethirimofluoride (emitefur), thizofurin (tizofurin), thizofurin (decetabine), nolatrexed (nolaterexed), pemetrexed (pemetrexed), nizhalabine (nelzarabine), 2' -deoxy-2 ' -methylenecytidine, 2' -fluoromethylene-2 ' -deoxycytidine, N- [5- (2, 3-dihydro-benzofuranyl) sulfonyl ] -N ' - (3, 4-dichlorophenyl) urea, N6- [ 4-deoxy-4- [ N2 (E), 4 (E) -tetradecadienoyl ] glycinamido ] -L-glyceryl-B-L-mannopyranosyl ] adenine, april (aplidine), also tenacitin (ectoin), troxacitabine (troxacitabine), 4- [ 2-amino-4-oxo-4, 6,7, 8-tetrahydro-3H-pyrimido [5,4-B ] [1,4] thiazin-6-yl- (S) -ethyl ] -2, 5-thiophenoyl-L-glutamic acid, aminopterin, 5-fluorouracil, nitrosohydroxyalanine, 11-acetyl-8- (carbamoyloxymethyl) -4-formyl-6-methoxy-14-oxo-1, 11-diazabicyclo (7.4.1.0.0) -tetradec-2, 4, 6-trien-9-yl acetate, swainsonine, lometrexol, dexrazoxane, methioninase, 2 '-cyano-2' -deoxy-N4-palmitoyl-1-B-D-arabinofuranosyl cytosine, and 3-aminopyridine-2-carboxyaldehyde thiosemicarbazone.

"HMG-CoA reductase inhibitor" refers to an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include, but are not limited to, lovastatin (lovastatin) (see U.S. Pat. nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (simvastatin) (see U.S. Pat. nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (pravastatin) (see U.S. Pat. nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (fluvastatin) (see U.S. Pat. nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), and atorvastatin (atorvastatin) (see U.S. Pat. nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The term HMG-CoA reductase inhibitors as used herein is intended to include all pharmaceutically acceptable lactone and ring-opened acid forms (i.e., wherein the lactone ring is opened to form the free acid), as well as salt and ester forms of compounds having HMG-CoA reductase inhibitory activity, and thus, the use of such salt, ester, open acid and lactone forms is included within the scope of the present invention.

"prenyl-protein transferase inhibitor" refers to a compound that inhibits any one or any combination of prenyl-protein transferases, including farnesyl protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type II (GGPTase-II, also known as Rab GGPTase).

Examples of farnesyl protein transferase inhibitors include SARASARTM (4- [2- [4- [ (11R) -3, 10-dibromo-8-chloro-6, 11-dihydro-5H-benzo [5,6] cyclohepta [1,2-b ] pyridin-11-yl- ] -1-piperidinyl ] -2-oxoethyl ] -1-piperidinecarboxamide, tipifarnib, and the like.

An "angiogenesis inhibitor" refers to a compound that inhibits neovascularization, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosinase inhibitors, such as inhibitors of the tyrosinase receptors Flt-1 (VEGFR 1) and Flk-1/KDR (VEGFR 2), inhibitors of epidermal-, fibroblast-, or platelet-derived growth factors, MMP (interstitial Metalloprotease) inhibitors, integrin blockers, metaleukocyte-12, pentosan polysulfate, cyclooxygenase inhibitors, including non-steroidal anti-inflammatory agents (NSAIDs), such as aspirin and ibuprofen (ibuprofen), and selective cyclooxygenase-2 inhibitors, such as celecoxib and Luo Feiku west ratio (rofecoxib)

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, 5-methoxy-4- [ 2-methyl-3- (3-methyl-2-butenyl) oxiranyl ] -1-oxospiro [2,5] oct-6-yl (chloroacetyl) ester, acetyldinaline (dinanaline), 5-amino-1- [ [3, 5-dichloro-4- (4-chlorobenzoyl) phenyl ] methyl ] -1H-1,2, 3-triazole-4-carboxamide, CM101, squalamine, pinostatin, RPI4610, NX31838, sulfated mannosaccharose phosphate, 7- (carbonyl-bis [ imino-N-methyl-4, 2-pyrrolocarbonyimino [ N-methyl-4, 2-pyrrole ] -carbonylimino ] -bis- (1, 3-naphthalenedisulfonate), and 3- [ (2, 4-dimethylpyrrolyl-5-methylene2-indoline) SU (5416).

Other therapeutic agents that modulate or inhibit angiogenesis and which may also be used in combination with glufosfamide or an analog thereof include agents that modulate or inhibit the coagulation and plasmin action systems. Examples of such agents that modulate or inhibit the coagulation and plasmin action pathways include, but are not limited to, heparin (see Thromb. Haemost.80:10-23 (1998)), low molecular weight heparin, and inhibitors of carboxypeptidase U (also known as inhibitors of active thrombin-activatable plasmin action inhibitor [ TAFIa ]). Examples of TAFIa inhibitors have been described in PCT patent WO03/013,526.

Examples of kinase inhibitors include: agents that inhibit cell surface receptors and downstream information transduction cascade of these surface receptors. Such agents inhibit cell proliferation and survival. It includes inhibitors of EGFR (e.g., ji Feiting Nib and supple and graceful Luo Dini primary (erlotinib)), antibodies to EGFR (e.g., C225), inhibitors of ERB-2 (e.g., trastuzumab)), inhibitors of IGFR, inhibitors of cytokine receptors, inhibitors of MET, inhibitors of PI3K (e.g., LY 294002), inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt, such as those described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138), inhibitors of Raf enzymes (e.g., BAY-43-9006), inhibitors of MEK (e.g., CI-1040 and PD-098059), inhibitors of mTOR (e.g., wyeth CCI-779), and inhibitors of C-ab1 kinase. Other kinase inhibitors include those that inhibit proteins involved in the cell cycle. Including aurora kinase inhibitors, CDK inhibitors (e.g., flavonopyridines, CYC202, BMS387032, and polar kinase inhibitors). These also include agents that interfere with the cell cycle checkpoint and pass through to sensitize cancer cells to DNA damaging agents. Such agents include, for example, inhibitors of ART, ATM, chk1 and Chk 2.

By "integrin blocker" is meant a compound that selectively antagonizes, inhibits, or neutralizes the binding of a physiological ligand to the αvβ3 integrin, a compound that selectively antagonizes, inhibits, or neutralizes the binding of a physiological ligand to the αvβ5 integrin, a compound that antagonizes, inhibits, or neutralizes the binding of a physiological ligand to both the αvβ3 integrin and the αvβ5 integrin, and a compound that antagonizes, inhibits, or neutralizes the activity of a particular integrin expressed on microvascular endothelial cells. The term also refers to antagonists of αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1, and α6β4 integrins. This term also refers to antagonists of any combination of αvβ3, αvβ5, αvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1, and α6β4 integrins.

Combinations with compounds other than anticancer compounds are also contemplated in the methods of the invention. For example, a combination of glufosfamide or an analog thereof with a PPAR-gamma (i.e., PPAR-gamma) agonist and a PPAR-delta (i.e., PPAR-delta) agonist (collectively, "PPAR agonists") may be useful for treating certain malignant conditions. PPAR-gamma and PPAR-delta are the nuclear peroxisome proliferator-activated receptors gamma and delta, respectively. The expression of PPAR-gamma on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol.1998;31:909-913; J. Biol. Chem.1999;274:9116-9121;Invest.Ophthalmol Vis.Sci.2000;41:2309-2317). Recently, PPAR-gamma agonists have been shown to inhibit the angiogenic response to VEGF in vitro; troglitazone and rosiglitazone maleate inhibit the progression of retinal neovascularization in mice (Arch. Ophthamol.2001; 119:709-717). Examples of PPAR-gamma agonists and PPAR-gamma/alpha agonists include, but are not limited to, thiazolidinediones (e.g., DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfirozil, antomine (clofiirate).

Glufosfamide or an analog thereof may also be administered in combination with one or more inhibitors of inherent multi-drug resistance (MDR), particularly MDR associated with high levels of delivered sub-protein expression. Such MDR inhibitors include inhibitors of P-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).

Other anticancer agents also include hypoxia activatable agents (e.g., tirapazamine), proteasome inhibitors (e.g., lactacystine and bortezomib), ubiquitin inhibitors, HDM2 inhibitors, TNF activators, BUB-R inhibitors, CENP-E inhibitors, and interferon alpha.

There are currently over 200 clinical trials evaluating the clinical efficacy of combination therapy of DNA damaging agents (chemotherapy, abbreviated as chemotherapy) and immune checkpoint inhibitors (immunotherapy, abbreviated as immune therapy). Especially in the last two years, the number of chemo/non-therapeutic clinical trials has grown considerably. There have been reports describing significant clinical benefits associated with the use of DNA damaging agents having immunoup-regulating activity in combination with immune checkpoint inhibitors. At the same time, combination therapy has proven to be very safe due to the different toxicity profile of the two drugs.

The design principle of chemotherapy/immunotherapy is that the DNA damaging agent (chemotherapeutic drug) is used for enhancing the immunocompetence of cancer cells, and then the immunotherapy is used, so that the anticancer effect of the immunotherapy is greatly increased. When considering this superior combination therapy, it is most critical that a DNA damaging agent with immune up-regulating function must be selected.

DNA damaging agents up-regulate the immune activity of cancer cells by the following mechanisms:

1) Induction of Immunocompetent Cell Death (ICD);

2) Creating an inflammatory Tumor Microenvironment (TME);

3) Increasing the number and variety of new antigen production by increasing the mutation load (open reading frame mutation and DNA repair mutation or defect);

4) Increasing antigen expression.

For this reason, in order to greatly increase the combined therapeutic effect, conventional strategies are adopted:

1) Selecting a DNA damaging agent capable of inducing immune-active cell death (ICE);

2) Selecting a dose of an optimal DNA damaging agent that protects the population of immunoreactive T cells;

3) Selecting an optimal administration time and order;

because glufosfamide increases the immune activity of cancer cells by inducing DNA damage, thereby up-regulating the sensitivity of cancer cells to immune checkpoint inhibitors and the effectiveness of anti-cancer therapies. In cancer cases where homologous recombination DNA repair is defective, this combination therapy is expected to produce a safe and more effective anti-tumor therapeutic effect. The reasons are as follows:

1) The ability of glufosinate to induce more DNA damage (experimental evidence has shown that cells with damaged homologous recombination DNA repair enzymes, nucleotide excision repair enzymes, non-homologous end joining enzymes, base excision repair enzymes, mismatch repair enzymes, van kanni anemia pathway repair enzymes, and repair defects due to damage are sensitive to glufosinate, and cells with homologous recombination DNA repair defects are more sensitive to glufosinate), resulting in a heavier mutational load;

2) The defect of homologous recombination DNA repair itself has greatly increased the tumor mutation load. Clinical data has shown that tumor mutational burden is positively correlated with immunotherapy effectiveness. The heavier the mutation load, the more effective the immunotherapy;

3) Increased Tumor Infiltrating Lymphocytes (TILs);

4) An increased lymphocyte attractant;

5) Affecting PD-L1 expression.

In conclusion, the combination of glufosfamide and immunotherapy can more effectively treat cancer patients with damaged homologous recombinant DNA repair enzymes, damaged nucleotide excision repair enzymes, damaged non-homologous end joining enzymes, damaged base excision repair enzymes, damaged mismatch repair enzymes, and damaged van conney anemia pathway repair enzymes.

Immunotherapeutic agents include immune checkpoint inhibitors, such as PD-1 inhibitors (e.g., keytruda), PD-L1 inhibitors (e.g., tecantriq, imfinzi) and CTLA4 inhibitors (e.g., yervoy).

Effects and effects of the invention

Experiments prove that the meglumine or the analogues thereof have specific inhibition effect on cells with specific genetic variation, in particular to cells with damaged DNA repair enzyme, the cell is one of the genes BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, IL1, EXP 1, XNEP 1, FAP 20, FAAP20, DNA endonuclease, flex-5, and the DNA polymerase, and the like, and the DNA endonucleases of which process the end of the cell.

Drawings

FIG. 1 is a graph of cell viability of AA8 cells after application of various concentrations of glufosfamide during the first experiment in an example of the invention;

FIG. 2 is a graph of cell viability of UV41 cells after application of varying concentrations of glufosfamide during the first experiment in an example of the invention;

FIG. 3 is a graph of cell viability of AA8 cells after application of various concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 4 is a graph of cell viability of EM9 cells after application of various concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 5 is a graph of cell viability of UV5 cells after application of different concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 6 is a graph of cell viability of UV20 cells after application of different concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 7 is a graph of cell viability of UV24 cells after application of different concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 8 is a graph of cell viability of UV135 cells after application of different concentrations of glufosfamide during a second experiment in an example of the invention;

FIG. 9 is a graph of cell viability of AA8 cells after application of different concentrations of cisplatin during the first experiment in an example of the present invention;

FIG. 10 is a graph showing the cell viability of UV41 cells after different concentrations of cisplatin applied during the first experiment in an example of the present invention

FIG. 11 is a graph of cell viability of AA8 cells after the application of different concentrations of cisplatin during a second experiment in an example of the present invention;

FIG. 12 is a graph of cell viability of EM9 cells after application of different concentrations of cisplatin in a second experiment in an embodiment of the present invention;

FIG. 13 is a graph of cell viability of UV5 cells after application of different concentrations of cisplatin in a second experiment in an embodiment of the present invention;

FIG. 14 is a graph of cell viability of UV20 cells after application of different concentrations of cisplatin in a second experiment in an embodiment of the present invention;

FIG. 15 is a graph of cell viability of UV24 cells after application of different concentrations of cisplatin in a second experiment in an embodiment of the present invention;

FIG. 16 is a graph of cell viability of UV135 cells after application of different concentrations of cisplatin in a second experiment in an embodiment of the present invention;

FIG. 17 is a graph comparing cell viability curves of UV41 cells and AA8 cells after application of different concentrations of glufosfamide in the first experiment in the example of the present invention;

FIG. 18 is a graph comparing the cell viability of EM9 cells and AA8 cells after application of different concentrations of glufosfamide in a second experiment in an example of the invention;

FIG. 19 is a graph comparing cell viability curves of UV5 cells and AA8 cells after application of different concentrations of glufosfamide in a second experiment in an example of the invention;

FIG. 20 is a graph comparing cell viability curves of UV20 cells and AA8 cells after application of different concentrations of glufosfamide in a second experiment in an example of the invention;

FIG. 21 is a graph comparing cell viability curves of UV24 cells and AA8 cells after application of different concentrations of glufosfamide in a second experiment in an example of the invention;

FIG. 22 is a graph comparing cell viability curves of UV135 cells and AA8 cells after application of different concentrations of glufosfamide in a second experiment in an example of the invention;

FIG. 23 is a graph comparing cell viability curves of UV41 cells and AA8 cells after application of different concentrations of cisplatin in the first experiment in an example of the present invention;

FIG. 24 is a graph comparing cell viability curves of EM9 cells and AA8 cells after application of different concentrations of cisplatin in a second experiment in an example of the present invention;

FIG. 25 is a graph comparing cell viability curves of UV5 cells and AA8 cells after application of different concentrations of cisplatin in a second experiment in an example of the present invention;

FIG. 26 is a graph comparing cell viability curves of UV20 cells and AA8 cells after application of different concentrations of cisplatin in a second experiment in an example of the present invention;

FIG. 27 is a graph comparing cell viability curves of UV24 cells and AA8 cells after application of different concentrations of cisplatin in a second experiment in an example of the present invention;

FIG. 28 is a graph comparing cell viability curves of UV135 cells and AA8 cells after application of different concentrations of cisplatin in a second experiment in an example of the present invention;

FIG. 29 is a graph showing the weight of tumor-bearing mice in the MCF-7 xenograft model as a function of time following GLUF administration. Data points represent average body weights for each group, error bars represent Standard Error (SEM);

FIG. 30 is a graph showing relative body weight over time calculated based on body weight of animals at the time of starting administration after MCF-7 cells were transplanted into BALB/c nude mice. Data points represent percent mean weight change for each group, error bars represent Standard Error (SEM);

FIG. 31 is a graph showing tumor growth in tumor-bearing mice of MCF-7 xenograft model following GLUF administration. Data points represent average tumor volumes for each group, error bars represent Standard Error (SEM);

FIG. 32 is a graph showing the weight of tumor-bearing mice in the RPMI-8226 xenograft model as a function of time following GLUF administration. Data points represent average body weights for each group, error bars represent Standard Error (SEM). And (3) injection: day28 or tumor mean volume up to 2000mm was observed 14 days after dosing ³ ；

FIG. 33 is a graph of relative body weight over time calculated based on body weight of animals at the time of initiation of dosing after RPMI-8226 cells were transplanted into CB-17 SCID mice. Data points represent the percent mean weight change for each group, error bars represent Standard Error (SEM). And (3) injection: day28 or tumor mean volume up to 2000mm was observed 14 days after dosing ³ ；

FIG. 34 is a graph showing tumor growth curve of tumor-bearing mice in the RPMI-8226 xenograft model following GLUF administration. Data points represent mean tumor volumes for each group, error bars represent Standard Error (SEM). And (3) injection: day28 or tumor mean volume up to 2000mm was observed 14 days after dosing ³ ；

FIG. 35 shows the body weight change of SK-MEL-28 xenograft model tumor-bearing mice after GLUF administration. Data points represent average body weights for each group, error bars represent Standard Error (SEM);

FIG. 36 is a graph of relative body weight over time calculated based on body weight of animals at the time of initiation of dosing after RPMI-8226 cell transplantation into BALB/c nude mice. Data points represent percent mean weight change for each group, error bars represent Standard Error (SEM);

FIG. 37 is a graph showing tumor growth curves of SK-MEL-28 xenograft model tumor-bearing mice after GLUF administration. Data points represent mean tumor volumes for each group, error bars represent Standard Error (SEM).

Detailed Description

In vitro cell experiments

1. Materials and methods

1.1 materials and instruments

AA8 cell line, purchased from american type culture collection (American type culture collection) (ATCC #crl-1859);

UV41 cell line, purchased from American type culture Collection (ATCC #CRL-1860);

EM9 cell line, purchased from American type culture Collection (ATCC #CRL-1861);

UV5 cell lines were purchased from American type culture Collection (ATCC #CRL-1865);

UV20 cell line, purchased from American type culture Collection (ATCC #CRL-1862);

UV24 cell line, purchased from American type culture Collection (ATCC #CRL-1866);

UV135 cell lines were purchased from American type culture Collection (ATCC #CRL-1867);

MEM-alpha medium, available from Fisher reagent company (Fisher # 12-561-056);

fetal bovine serum (Fetal bovine serum, abbreviated as FBS), available from ThermoFisher company (ThermoFisher # 26140-079);

penicillin streptomycin solution-dual anti Penn-step, available from Hyclone company (hyclone#sv30010);

AlamarBlue reagent, available from ThermoFisher corporation (ThermoFisher#DAL1100).

1.2 test Compounds

Compounds of formula (I)	Solvent for dissolution	Testing concentration (M/L)	Diluting solution
				Glufosfamide	DMSO	0.015-1000	Culture medium solution containing 0.5% DMSO
Cisplatin (cisplatin)	DMSO	0.003-200	Culture medium solution containing 0.5% DMSO

1.3 test conditions

1.3.1 cell culture

AA8 and UV41 cells were cultured in MEM-alpha medium with 10% FBS and 1% Penn-strep.

1.3.2 experimental conditions

For proliferation assays AA8 and EM9, UV5, UV20, UV24, UV41, UV135 cells were seeded at 4000 cells/well in 96-well, black, transparent bottom tissue culture plates. Cells at 37℃and 5% CO ₂ Incubate overnight. Cells were then treated with the compounds in section 1.2 for 72 hours. The total volume of medium per well was 200ul. After the treatment, the cell proliferation rate was measured using AlamarBlue fluorescent quantitation method. The AlamarBlue assay is based on detecting fluorescent and colorimetric increases to detect metabolic activity. Specifically, the active ingredient resazurin of AlamarBlue reagent is blue and hardly fluoresces. After entry into the cell, resazurin is reduced to resorufin, a red and highly fluorescent compound. Sustained cell growth maintains a reducing environment, thus increasing the medium surrounding the cells Is added to the total fluorescence and color of the light source. Due to laboratory limitations, this experiment was performed in total in two batches, the first batch being experiments with two AA8 and UV41 cells, respectively, and the second batch being experiments with six AA8 and EM9, UV5, UV20, UV24, UV135 cells, respectively, a total of 8 sets of data.

Experiments have shown that the fluorescence intensity of AlamarBlue reagent is proportional to the number of cells. For AlamarBlue assay, 20ul of AlamarBlue reagent was added to each well and incubated for 4 hours in a 37℃incubator. Using BioTek Synergy ^TM The 2-enzyme-labeled instrument measures fluorescence intensity under 530nm excitation and 590nm emission.

2. Experimental results. The experimental data obtained are shown in tables 1 to 8 below.

Table 1, experimental data on proliferation of glufosfamide on AA8 cells

* Annotating the data as experimental data of the first batch

Table 2, proliferation assay data of glufosfamide on UV41 cells

* Annotating the data as experimental data of the first batch

TABLE 3 proliferation assay data of Glufosfamide on AA8 cells ^#

Annotation, # data was the experimental data of the second batch

Table 4 proliferation assay data of Glufosfamide on EM9 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 5 proliferation assay data of Glufosfamide on UV5 cells ^#

Annotation # the data is the experimental data of the second batch,

* The data is culled as bad values.

TABLE 6 proliferation assay data of glufosfamide on UV20 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 7 proliferation assay data of glufosfamide on UV24 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 8 proliferation assay data of glufosfamide on UV135 cells ^#

Annotation, # data was the experimental data of the second batch

Table 9, cisplatin versus AA8 cell proliferation assay data

* Note that the data is experimental data for the first lot, the set of data is bad, and no statistical calculation is performed by kicking.

Table 10, cisplatin versus UV41 cell proliferation experimental data

* Annotating the data as experimental data of the first batch

TABLE 11 proliferation assay data of cisplatin on AA8 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 12 proliferation assay data of cisplatin on EM9 cells ^#

Annotation, # the data was experimental data for the second batch.

TABLE 13 proliferation assay data of cisplatin on UV5 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 14 proliferation assay data of Glufosfamide on UV20 cells ^#

Annotation, # data was the experimental data of the second batch

TABLE 15 proliferation assay data of glufosfamide on UV24 cells ^#

Annotation, # data was the experimental data of the second batch

Table 16, proliferation assay data of Glufosfamide on UV135 cells ^#

Annotation, # data was the experimental data of the second batch

3. Data analysis

Each concentration of cell proliferation assay was performed in triplicate. Experimental data were analyzed using GraphPadPrism software. In the absence of compound, the fluorescence intensity (Ft) was defined as 100%. In the absence of cells, the fluorescence intensity (Fb) was defined as 0%. The percent of cells surviving at each compound = (F-Fb)/(Ft-Fb) was calculated according to the following formula, where F = fluorescence intensity in the presence of the compound.

The experimental data of concentration of the compound versus percentage of surviving cells, i.e., the response curve, is plotted using nonlinear regression analysis with cell survival on the Y-axis, concentration of the compound on the X-axis, and concentration corresponding to 50% of maximum activity, designated IC ₅₀ As shown in fig. 1 to 16.

Finally, IC of the glufosfamide and cisplatin to AA8 and UV41 cells is obtained ₅₀ The values are shown in Table 17 below.

TABLE 17 IC of glufosfamide AST1001 and cisplatin (cispratin) against AA8, UV41 cells ₅₀ Value of

4. Conclusion of the experiment

4.1, AA8 cells and mutant UV41 cells

The UV41 cell line is a derivative of a CHO-AA8 cell line and is derived from an AA8 ultraviolet sensitive line, and the cell line is a cell line with damaged homologous recombination DNA repair enzyme and damaged nucleotide excision repair enzyme caused by DNAERCC4/XPF gene mutation.

UV41 Cell lines are literature-deficient cells with respect to AA8, which are sensitive to large volumes of adduct-induced mutations, belonging to excision repair supplement group 4, UV41 is extremely sensitive to DNA crosslinkers (see Thompson LH, et al Repair of DNA adducts in asynchronous CHO cells and the role of repair in Cell killing and mutation induction in synchronous cells treated with-bromoxybenz [ a ] anti-foam Cell mol. Genet.10:183-194,1984.PubMed:6584989;Thompson LH,et al.Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 78:3734-3737,1981.PubMed:6943579;Hoy CA,et al.Defective DNA cross-link removal in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents. Cancer rRs. 45:7-1743,1985.PubMed:3919945;Busch D,et al.Summary of complementation groups of UV-sensitive CHO Cell mutants isolated by large-scale screening. Mutagens 4:349-354,1989.PubMed:2687628;Bessho T,et al.Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions "to the cross-linked base and removes a 22-to 28-nucleic acid-free-strand and. Mol. Biol.17:6822-6830,1997.PubMed:9372913;Thompson LH,et al.Hypersensitivity to mutation and sister-link removal in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents 6:954-753:753).

DNA repair enzyme damage in UV41 cells can result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ The value shows that the glufosfamide has selective inhibition effect on normal AA8 cells and UV41 cells after the AA8 cells are subjected to gene mutation, and has inhibition activity IC on normal cells ₅₀ At 23. Mu.M, the mutant cell UV41 (cancer or tumor) has an inhibitory activity of up to 32.8-fold, i.e., 0.7. Mu.M. As shown in FIG. 17, it is found that the glucamide has a selective inhibitory effect on cancer cells having the above-mentioned gene mutation, and further shows the relative safety and less toxicity of glucamide, as compared with cisplatin as a cytotoxic drug, as shown in FIG. 23.

4.2, AA8 cells and mutated EM9 cells

EM9 is a repair-deficient mutant derived from AA8 (see ATCC CRL-1859). The cell line was selected to enhance sensitivity to Ethyl Methanesulfonate (EMS). The cell line has defects in DNA single-strand break repair, the baseline frequency of sister chromatid exchange is 10 times higher than that of AA8 before mutation, and the sensitivity to X-ray killing is 2 times higher than that of AA8 cells. This defect was corrected by the human XRCC1 gene.

It has been shown that this Cell line is a Cell line in which the base excision repair enzyme has been impaired by DNAXRCC1/RCC gene mutation (1.Thompson LH,et al.ACHO-Cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency ofsister-chromaid exchange. Mutat. Res.95:427-440,1982;2.Thompson LH,et al.A screening method for isolating DNA repair-deficient mutants of CHO cells. Systemic Cell Genet.6:391-405, 1980). The DNA repair enzyme damage of EM9 cells may result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK, CETN2, DDB 1' any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ As can be seen from FIG. 18, glufosfamide has a selective inhibitory effect on normal AA8 cells and EM9 cells after the AA8 cells have undergone gene mutation, and has inhibitory activity IC on normal cells ₅₀ The inhibition activity was 16. Mu.M, but was similar and slightly smaller for the mutated cells EM9 (cancer or tumor cells) and was further found to be stronger for the mutated EM9 cells at lower concentrations. As shown in fig. 24, the relative safety of the glufosfamide was further demonstrated, along with less toxicity.

4.3, AA8 cells and mutated UV5 cells

The UV5 cell line is a derivative of a CHO-AA8 cell line and is derived from an AA8 ultraviolet sensitive line, and the cell line is a cell line with damaged nucleotide excision repair enzyme caused by DNA ERCC1/RAD10 gene mutation. It has been shown that the UV5 Cell line is a Cell defective for the nucleotide excision repair enzyme relative to AA8, which is sensitive to bulky adduct-induced mutations, belonging to excision repair complement group 1 (see Thompson LH, et al reply of DNA adducts in asynchronous CHO cells andthe role of repair in Cell killing andmutation induction in synchronous cells treated with-bromoxybenz [ a ] anthrene. Systemic Cell mol. Genet.10:183-194,1984.PubMed:6584989;Thompson LH,et al.Genetic diversity ofUV-sensitive DNArepair mutants of Chinese hamster ovary cells Proc.Natl.Acad.Sci.USA 78:3734-3737,1981.PubMed:6943579;Hoy CA,et al.Defective DNA cross-link removal in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents.cancer rRs.45:1737-1743,1985.PubMed:3919945;Busch D,et al.Summary of complementation groups of UV-sensitive CHO Cell mutants isolated by large-scale screening.Mutagenesis 4:349-354,1989.PubMed:2687628;Bessho T,et al.Initiation of DNA interstrand cross-linkrepair in humans: the nucleotide excision repair system makes dual incisions "to the cross-linkedbase andremoves a 22-to 28-nucleic acid-long damage-free strand. Mol.cell.biol.17:6822-6830,1997.PubMed:9372913;Reardon JT,et al.Isolation and characterization of two human transcription factor IIH (TFIIH) -related complexes:ERCC 2/candTFH.Proc.Natl.Acad.Sci.USA 93:6482-6487,1996.PubMed:8692841;Thompson LH,et al.Hypersensitivity to mutation and sister-chromatography-exchange induction in CHO Cell mutants defective in incising DNA containing UV scales.Somatic Cell et.8:759-773,1982.PubMed: 63954; cellular responses to DNA damage. New York: lists; 1983; hay, R.J., caputo, J.L., and Macy, M.L., eds. (1992), ATCC Quality Control Methods for Cell lines.2nd edition, publishendbyATCC; caputo, J.L., biosafety procedures in cell culture.J.tissue Culture Methods 11:223-227,1988; fleming, D.O., richardson, J.H., tulis, J.J., and Vesley, D., (1995) Laboratory Safety: principles and practice.

DNA repair enzyme damage in UV5 cells can result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ The value shows that the glufosfamide has selective inhibition effect on normal AA8 cells and UV5 cells after the AA8 cells are subjected to gene mutation, and has inhibition activity IC on normal cells ₅₀ At 16 μm, the mutant cell UV20 (cancer or tumor) had similar inhibitory activity, as shown in fig. 19, compared to cisplatin as a cytotoxic drug, as shown in fig. 25, further demonstrating the relative safety of glufosfamide, and less toxicity.

4.4, AA8 cells and mutated UV20 cells

The UV20 cell line is a derivative of a CHO-AA8 cell line and is derived from an AA8 ultraviolet sensitive line, and the cell line is a cell line with damaged homologous recombination DNA repair enzyme and damaged nucleotide excision repair enzyme caused by DNA ERCC2/XPD gene mutation.

It has been shown that the UV20 Cell line is a Cell defective in nucleotide excision repair enzymes relative to AA8, which is sensitive to bulky adduct-induced mutations, belonging to excision repair supplementation group 2, UV20 is extremely sensitive to DNA cross-linking agents (see Thompson LH, et al repai ofDNAadducts in asynchronous CHO cells andthe role of repair in Cell killing and mutation induction in synchronous cells treated with-bromoxybenz [ a ] anthracenene.genetic Cell mol. Genet.10:183-194,1984.PubMed:6584989;Thompson LH,et al.Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells Proc.Natl.Acad.Sci.USA 78:3734-3737,1981.PubMed:6943579;Hoy CA,et al.Defective DNA cross-link removal in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents.cancer Res.45:1737-1743,1985.PubMed:3919945;Busch D,et al.Summary of complementation groups of UV-sensitive CHO Cell mutants isolated by large-scale screening.Mutagenesis 4:349-354,1989.PubMed:2687628;Bessho T,et al.Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions "to the cross-linked base and removes a-to 28-nucleic acid-long damage-free strand. Mol.cell.biol.17:6822-6830,1997.PubMed:9372913;Thompson LH,et al.Hypersensitivity to mutation and sister-chromatography-exchange induction in CHO Cell mutants defective in incising DNA containing UV crystals.soy Cell Genet.8:759-773,1982.PubMed:7163954;Thompson LH,et al.Ascreening method for isolating DNA repair-deficient mutants of CHO cells.soy Cell Genet.6:391-405,1980.PubMed:7404270; hay, R.J., caputo, J.L., macand M.L., eds (19956), published by ATCC; caputo, J.L., biosafety procedures in cell culture.J.tissue Culture Methods 11:223-227,1988; fleming, D.O., richardson, J.H., tulis, J.J., and Vesley, D., (1995) Laboratory Safety: principles and practice, second edition, ASM press, washington, DC; biosafety in Microbiological and Biomedical Laboratories,5th ed.HHS.U.S.Department of Health and Human Services,Centers for Disease Control and Prevention.Washington DC:U.S.Government Printing Office; 2007.).

DNA repair enzyme damage in UV20 cells can result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ The value shows that the glufosfamide has selective inhibition effect on normal AA8 cells and UV20 cells after the AA8 cells are subjected to gene mutation, and has inhibition activity IC on normal cells ₅₀ UV20 (cancer or tumor cells) has an inhibitory activity of up to 16-fold, i.e.1. Mu.M, against the mutated cells at 16. Mu.M. As shown in FIG. 20, it is evident that the glucamide has a selective inhibitory effect on cancer cells having the above-mentioned gene mutation, and further shows the relative safety and less toxicity of glucamide, compared with cisplatin as a cytotoxic drug, as shown in FIG. 26.

4.5, AA8 cells and mutant UV24 cells

The UV24 cell line is a derivative of a CHO-AA8 cell line and is derived from an AA8 ultraviolet sensitive line, and the cell line is a cell line with damaged nucleotide excision repair enzyme caused by DNA ERCC3/XPB gene mutation.

UV24 Cell lines are literature-deficient cells with respect to AA8, which are sensitive to large-volume adduct-induced mutations, belonging to excision repair complement group 3 (see Thompson LH, et al, repair of DNA adducts in asynchronous CHO cells and the role of repair in Cell killing andmutation induction in synchronous cells treated with-bromoxynil [ a ] anthracene. Somatic Cell mol. Gene. 10:183-194,1984.PubMed:6584989;Thompson LH,et al.Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells Proc. Natl. Acad. Sci. USA 78:3734-3737,1981.PubMed:6943579;Hoy CA,et al.Defective DNA cross-link removal in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents.cancer Res. 45:7-1743,1985.PubMed:3919945;Busch D,et al.Summary of complementation groups of UV-sensitive CHO Cell mutants isolated by large-scale screening. Mutagene. 349-354,1989.PubMed:2687628;Reardon JT,et al.Isolation and characterization of two human transcription factor IIH (IIH) -related complexes:ERCC2/CAKand TFIIH. Proc. Natl. Acad. Sci. USA 93:3982-6487,1996.PubMed:8692841;Thompson LH,et al.Hypersensitivity to mutation and sister-chmatoid-exchange induction in CHO Cell mutants defective in incising DNA containingUV les. Soc. 759-1982:954 b.De.De.De.3); cellular responses to DNA damage. New York: lists; 1983; bessaho T, et al initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions "to the cross-linked base and removes a-to 28-nucleic-long damage-free strand and. Mol. Cell. Biol.17:6822-6830,1997.PubMed:9372913; hay, R.J., caputo, J.L., and Macy, M.L., eds. (1992), ATCC Quality Control Methods for Cell lines.2nd edition, published by ATCC; caputo, J.L., biosafety procedures in cell culture.J.tissue Culture Methods 11:223-227,1988; fleming, D.O., richardson, J.H., tulis, J.J., and Vesley, D., (1995) Laboratory Safety: principles and practice, second edition, ASM press, washington, DC.).

DNA repair enzyme damage in UV24 cells can result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ The value shows that the glufosfamide has selective inhibition effect on normal AA8 cells and UV24 cells after the AA8 cells are subjected to gene mutation, and has inhibition activity IC on normal cells ₅₀ At 16. Mu.M, there was a high inhibitory activity against the mutant cell UV24 (cancer or tumor cells). As shown in FIG. 21, it is clear that the glucamide has a selective inhibitory effect on cancer cells having the above-mentioned gene mutation, and further shows the relative safety and less toxicity of glucamide, as compared with cisplatin as a cytotoxic drug, as shown in FIG. 27.

4.6, AA8 cells and mutated UV135 cells

The UV135 cell line is a derivative of a CHO-AA8 cell line and is derived from an AA8 ultraviolet sensitive line, and the cell line is a cell line with damaged nucleotide excision repair enzyme caused by DNA ERCC5/XPG gene mutation.

It is well documented that UV135 Cell lines are cells with nucleotide excision repair enzyme defects relative to AA8, which are sensitive to bulky adduct-induced mutations, belonging to excision repair supplement group 5 (see Thompson LH, et al Repair of DNA adducts in asynchronous CHO cells and the role of repair in Cell killing and mutation induction in synchronous cells treatedwith 7-bromoxybenz [ a ] anthracene. Systemic Cell mol. Genet.10:183-194,1984.PubMed:6584989;Thompson LH,et al.Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells Proc. Natl. Acad. Sci. USA78:3734-3737,1981.PubMed:6943579;Hoy CA,et al.Defective DNA cross-linkremoval in Chinese hamster Cell mutants hypersensitive to bifunctional alkylating agents Res.45:1737-1743,1985.PubMed:3919945;BuschD,et al.Summary ofcomplementation groups of UV-sensitive CHO Cell mutants isolatedby large-scale screening. Mutagens 4:349-354,1989.PubMed:2687628;Cellular responses to DNA damage.New York:Liss;1983.; bessho T, et al initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair systemmakes dual incisions 5"to the cross-linkedbase and removes a 22-to 28-nucleic Cell Gene-free strand. Mol. Cell. Biol.17:6822-6830,1997.PubMed:9372913;Reardon JT,et al.Isolation and characterization of two human transcription factor IIH (TFIIH) -related complexes: ERCC2/CAKand TFIIH.Proc. Natl. Acad. Sci. USA 93:6482-6487,1996.PubMed:8692841;Thompson LH,et al.Hypersensitivity to mutation and sister-chromated-exchange induction in CHO Cell mutants defective in incising DNA containing UV versions.Somatic Cell Gene 8:759-773,1982.PubMed: 7163954.).

DNA repair enzyme damage in UV135 cells can result in BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1 any one or more of the genes for DDB2, ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease.

IC by comparison of Table 17 ₅₀ The value shows that the glufosfamide has selective inhibition effect on normal AA8 cells and UV135 cells after the AA8 cells are subjected to gene mutation, and has inhibition activity IC on normal cells ₅₀ At 16. Mu.M, the mutant cell UV135 (cancer or tumor) has similar inhibitory activity, as shown in FIG. 22, which further demonstrates the relative safety of glufosfamide, and less toxicity, as shown in FIG. 28.

4.7 summary

That is, the meglumine has a specific inhibitory effect on cells having a specific genetic variation, specifically cells having a damaged DNA repair enzyme, the cell is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD, C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHC 6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5/XPG ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN and AP endonuclease, terminal processor, DNA polymerase, flap endonuclease, the invention discloses a pharmaceutical composition containing the glucuromide or the analogues thereof for treating tumors and cancers of patients with damaged DNA repair enzymes, application of the glucuromide or the analogues thereof in preparing medicines for treating the tumors and cancers of the patients with damaged DNA repair enzymes, and an immune combination treatment medicine and a treatment method containing the glucuromide or the analogues thereof and an immune checkpoint inhibitor.

Further, the meglumine is formed by linking one molecule of the directly alkylated isophosphamide mustard with one molecule of glucose through a glycosidic bond, which is transported into tumor cells under the action of sodium-dependent glucose transmembrane transporter SAAT1, and then released by hydrolysis to act as an active agent, that is, the meglumine is a prodrug of isophosphamide. Since cancer cells grow and proliferate more strongly than normal cells, their use and demand for glucose and the like is also more abundant, that is, tumor cells have unique characteristics of extremely high use of glucose (high expression of glucose transporter gene), which results in enrichment of local concentrations of glufosfamide in the tumor condition in the patient, i.e., selectively targeting glufosfamide to tumor sites to enhance tumor killing effects.

Therefore, as a targeting prodrug, more of the killing toxin (ifosfamide nitrogen mustard) can be released only in cancer cells with high glucose transport and glucosidase expression, whereas in normal cells, the prodrug cannot release the toxin due to low or no glucose transport, so that it is inactive or less toxic.

II, animal experiment

This test uses the MCF-7 cell BALB/c nude mice subcutaneous engraftment model to evaluate the in vivo antitumor effect of test compound GLUF.

This test uses the RPMI-8226 cell CB-17 SCID mouse subcutaneous transplantation tumor model to evaluate the in vivo anti-tumor effect of test compound GLUF.

The test uses SK-MEL-28 cell BALB/c nude mice subcutaneously transplanted tumor model to evaluate the in vivo anti-tumor effect of test compound GLUF.

Test article

Name: AST-1001 (Glufosfamide, abbreviated as GLUF)

Providing units: bellen Chemistry Co.Ltd (Beijing Liuhningyun technology Co., ltd.)

Lot number: AE-050402

Description of the Properties powder

Molecular weight 383.16

Packaging 5 g/bottle

Preservation conditions: -20 DEG C

1. Experimental animal

BALB/c nude mice, 6-8 week old, females, 18-22 grams in weight, 24 (excluding the remaining mice in groups), suppliers: shanghai Ling Biotechnology Co., ltd;

CB-17 SCID mice, 6-8 weeks old, females, 18-22 g in weight, 24 (excluding the remaining mice in groups), suppliers: shanghai Ling Biotechnology Co., ltd;

2. experimental methods and procedures

2.1 cell culture

The MCF-7 cell culturing method is in vitro adherent culture, and the culturing condition is that 10% fetal bovine serum, 100U/ml penicillin and 100 mug/ml streptomycin are added into EMEM culture medium, and 5% CO2 is cultured at 37 ℃. Passaging is routinely performed 2 times a week. Cells were harvested, counted and inoculated while the cells remained in the exponentially growing phase.

The RPMI-8226 cell culture method is in vitro adherence culture, and the culture conditions are that 10% fetal bovine serum, 100U/ml penicillin and 100 μg/ml streptomycin are added into RPMI 1640 culture medium, and 5% CO2 is cultured at 37 ℃. Passaging is routinely performed 2 times a week. Cells were harvested, counted and inoculated while the cells remained in the exponentially growing phase.

The SK-MEL-28 cell culturing method is in vitro adherent culture, and the culturing condition is that 10% fetal calf serum, 100U/ml penicillin and 100 μg/ml streptomycin are added into EMEM culture medium, and 5% CO2 is cultured at 37 ℃. Passaging is routinely performed 2 times a week. Cells were harvested, counted and inoculated while the cells remained in the exponentially growing phase.

2.2 inoculation of tumor cells

0.2ml of 10X 106 MCF-7 cells were inoculated subcutaneously into the right back of each BALB/c nude mouse (PBS: matrigel=1:1). The average tumor volume reaches 161mm ³ The administration of the packets was started at that time (see table 18 below).

Table 18, case of sub-dermal administration of MCF-7 cells to BALB/c nude mice

Note that: number of mice per group N

0.2ml of 10X 106 RPMI-8226 cells were inoculated subcutaneously into the right back of each CB-17 SCID mouse nude mouse (PBS: matrigel=1:1). The average tumor volume reaches 153mm ³ The administration of the packets was started at that time (see table 19 below).

Table 19, case of sub-dermal administration of RPMI-8226 cells to CB-17 SCID mouse nude mice

Group of	Na	Compound treatment	Dosage (mg/kg)	Dosing volume parameter (μl/g)	Route of administration	Frequency b of administration
							1	8	Solvent control	--	10	Abdominal cavity	Once daily for 14 days
2	8	GLUF	30	10	Abdominal cavity	Once daily for 14 days
							3	8	GLUF	100	10	Abdominal cavity	Twice weekly for 2 weeks

0.2ml of 10X 106 SK-MEL-28 cells were inoculated subcutaneously into the right back of each BALB/c nude mouse (PBS: matrigel=1:1). Tumor average volume up to 181mm ³ The administration of the packets was started at that time (see table 20 below).

TABLE 20 grouping dosing of SK-MEL-28 cells subcutaneously vaccinated into BALB/c nude mice

Group of	Na	Compound treatment	Dosage (mg/kg)	Dosing volume parameter (μl/g)	Route of administration	Frequency of administration
							1	8	Solvent control	--	10	Abdominal cavity	Once daily for 14 days
2	8	GLUF	30	10	Abdominal cavity	Once daily for 14 days
							3	8	GLUF	100	10	Abdominal cavity	Twice weekly for 2 weeks

2.3 preparation of test substances

The formulation conditions of the test drugs are shown in table 21 below.

Table 21, method for preparing different test drugs

Note that: the drug should be gently mixed well before administration.

2.4 tumor measurement and Experimental indicators

The experimental index is to examine whether tumor growth is inhibited, retarded or cured. Tumor diameters were measured with a vernier caliper three times a week. The calculation formula of the tumor volume is: v=0.5a×b2, a and b represent the long and short diameters of the tumor, respectively.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects the tumor growth inhibition rate. Calculation of TGI (%): TGI (%) = [ (1- (average tumor volume at the end of the administration of a certain treatment group-average tumor volume at the beginning of the administration of the treatment group)/(average tumor volume at the end of the treatment of a solvent control group-average tumor volume at the beginning of the treatment of a solvent control group) ]x100% ].

Animals were euthanized after the end of the experiment and tumor weights were measured.

3. Results and statistical analysis

3.1 test for evaluating the in vivo antitumor effect of test compound GLUF using MCF-7 cell BALB/c nude mice subcutaneous transplantation tumor model.

3.1.1 statistical analysis

Statistical analysis, including mean and Standard Error (SEM) of tumor volumes at each time point for each group (see table 22 for specific data). The experiment ended 14 days after the start of dosing, so statistical analysis was performed to evaluate the differences between groups based on the day's data. Three or more comparisons were statistically analyzed using one-way ANOVA. All data analyses were performed with GraphPad Prism 5.0. p <0.05 was considered a significant difference.

Tumor volume

The change in tumor volume of each group of MCF-7 cell xenograft tumor BALB/c nude mice after GLUF treatment is shown in Table 22.

Table 22, tumor volume data at various time points for each group after MCF-7 cell xenograft tumor BALB/c nude mice

A, mean ± SEM; b. a total of 2 mice died from the dosing group (100 mg/kg) before day 7 of the post-dosing day

3.1.2 experimental results

Mortality, morbidity and weight changes

During the experiment, 4 mice in the GLUF 100mg/kg dose group showed a more pronounced weight loss on all days 6 to 11 of dosing, 2 of which died on day 7 of dosing. The GLUF 30mg/kg dose group had 1 mouse with a more pronounced weight loss on day 14 of dosing.

The effect of GLUF on the body weight of tumor-bearing BALB/c nude mice is shown in FIGS. 29 and 30.

Tumor growth curve

The tumor growth curve is shown in FIG. 31.

Evaluation index of antitumor drug effect

Table 23, GLUF results of evaluation of tumor-inhibiting efficacy of MCF-7 subcutaneous transplantation tumor model (Day 14)

Group of	Tumor volume (mm) ³ )a	T/C％	TGI％	p-value (vs Vehicle)
					Vehicle	926±60	--	--	--
GLUF(30mg/kg)	847±65	91.4	10.4	＞0.05
					GLUF(100mg/kg)	768±51	82.9	20.7	＞0.05

Note that: a. mean ± SEM; b.p values were calculated by one-wayANOVA

3.2 test for evaluating in vivo anti-tumor Effect of test Compound GLUF Using the RPMI-8226 cell CB-17 SCID mouse subcutaneous transplantation tumor model

3.2.1 statistical analysis

Statistical analysis, including mean and Standard Error (SEM) of tumor volumes at each time point for each group (see table 24 for specific data). The experiment was stopped after 14 days from the start of the administration, and the end of day 28 was observed (the solvent control group had a tumor mean volume of more than 2000mm on day 23) ³ Euthanized according to IACUC animal welfare regulations). Statistical analysis was therefore performed to evaluate group-to-group differences based on day 14 data. Three or more comparisons were statistically analyzed using one-way ANOVA. All data analyses were performed with GraphPad Prism 5.0. P is p<0.05 was considered to be a significant difference.

Tumor volume

The change in tumor volume of each group of RPMI-8226 cell xenograft CB-17 SCID mice after GLUF treatment is shown in Table 24.

Table 24 tumor volumes at various time points for each group following RPMI-8226 cell xenograft tumor CB-17 SCID mice

Mean ± SEM

3.2.2 experimental results

Mortality, morbidity and weight changes

During the experiment, no significant weight loss occurred during the administration of GLUF 30mg/kg and 100mg/kg dose groups.

The effect of GLUF on body weight of tumor-bearing CB-17 SCID mice is shown in FIGS. 32 and 33.

Tumor growth curve

The tumor growth curve is shown in fig. 34.

Evaluation index of antitumor drug effect

Table 25, GLUF evaluation of tumor-inhibiting efficacy of RPMI-8226 subcutaneous graft tumor model (Day 14)

Group of	Tumor volume (mm) ³ )a	T/C％	TGI％	p value b (vs Vehicle)
					Vehicle	1085±134	--	--	--
GLUF(30mg/kg)	412±53	38.0	72.4	＜0.001
					GLUF(100mg/kg)	338±26	31.1	80.1	＜0.001

Note that: a. mean ± SEM; b.p values were calculated using one-way ANOVA

3.3 experiments to evaluate the in vivo anti-tumor effect of test compounds GLUF using SK-MEL-28 cell BALB/c nude mice subcutaneous transplantation tumor model.

3.3.1 statistical analysis

Statistical analysis, including mean and Standard Error (SEM) of tumor volumes at each time point for each group (see table 26 for specific data). The experiment ended 14 days after the start of dosing, so statistical analysis was performed to evaluate the differences between groups based on the day's data. Three or more comparisons were statistically analyzed using one-way ANOVA. All data analyses were performed with GraphPad Prism 5.0. p <0.05 was considered a significant difference.

Evaluation index of antitumor drug effect

Table 26, GLUF evaluation of tumor-inhibiting efficacy of SK-MEL-28 subcutaneous transplantation tumor model (Day 14)

Group of	Tumor volume (mm) ³ )a	T/C％	TGI％	p value b (vs Vehicle)
					Vehicle	715±68	--	--	--
GLUF(30mg/kg)	603±56	84.2	21.0	＞0.05
					GLUF(100mg/kg)	563±45	78.7	28.5	＞0.05

Note that: a. mean ± SEM; b.p values were calculated using one-way ANOVA

3.3.2 experimental results

Mortality, morbidity and weight changes

During the experiment, 2 mice in the GLUF 30mg/kg dose group showed more obvious weight loss on the 12 th day of administration, and no obvious weight loss occurred during the GLUF 100mg/kg dose group administration.

The effect of GLUF on body weight of tumor-bearing BALB/c nude mice is shown in FIGS. 35 and 36.

Tumor volume

The change in tumor volume of each group of SK-MEL-28 cell xenograft tumor BALB/c nude mice after GLUF treatment is shown in Table 27.

Table 27 tumor volumes at various time points for each group after SK-MEL-28 cell xenograft tumor BALB/c nude mice

Mean ± SEM

Tumor growth curve

The tumor growth curve is shown in FIG. 37.

4. Experimental results and discussion

4.1 test for evaluating in vivo anti-tumor Effect of test Compound GLUF Using MCF-7 cell BALB/c nude mice subcutaneous transplantation tumor model

In this experiment, we evaluated the in vivo efficacy of GLUF in a model of MCF-7 subcutaneous transplantation tumor. The tumor volumes at various time points for each experimental group are shown in table 22, table 23 and fig. 31.

Tumor average volume of solvent control tumor-bearing mice reached 926mm 14 days after administration ³ . The average tumor volumes of GLUF 30mg/kg group and 100mg/kg group after 14 days of administration were 847mm, respectively ³ (T/C＝91.4％,TGI＝10.4％,p>0.05 768 mm) ³ (T/C＝82.9％,TGI＝20.7％,p>0.05). GLUF showed no significant tumor inhibition at doses of 30mg/kg and 100 mg/kg.

The effect of GLUF on weight change in tumor-bearing mice is shown in FIG. 29, FIG. 30. Part of the tumor-bearing mice in the GLUF 100mg/kg dose group showed a more pronounced weight loss during the experiment, with 2 mice dying during the treatment period (day 7).

4.2 test for evaluating in vivo anti-tumor Effect of test Compound GLUF Using the RPMI-8226 cell CB-17 SCID mouse subcutaneous transplantation tumor model

In this experiment, we evaluated the in vivo efficacy of GLUF in the RPMI-8226 subcutaneous tumor model. The tumor volumes at various time points for each experimental group are shown in table 24, table 25 and fig. 34.

On day 14 of the observation period, the average tumor volume of the tumor-bearing mice in the solvent control group reached 1085mm ³ . GLUF 30mg/kg group and 100mg/kg group had average tumor volumes of 412mm on day 14 of observation period, respectively ³ (T/C＝38.0％,TGI＝72.4％,p<0.001 And 338mm ³ (T/C＝31.1％,TGI＝80.1％,p<0.001). GLUF showed significant tumor growth inhibition at 30mg/kg,100mg/kg, and at a dose-dependent Dependency. At the end of day 14 dosing, the GLUF group tumors showed a faster growth rate.

The effect of GLUF on weight change in tumor-bearing mice is shown in FIG. 32, FIG. 33. No significant weight loss occurred in the GLUF 30mg/kg,100mg/kg dose group at the frequency of dosing for this experiment.

4.3 test for evaluating in vivo anti-tumor Effect of test Compound GLUF Using SK-MEL-28 cell BALB/c nude mice subcutaneously transplanted tumor model

In this experiment, we evaluated the in vivo efficacy of GLUF in SK-MEL-28 subcutaneous engraftment tumor model. The tumor volumes at various time points for each experimental group are shown in table 26, table 27 and fig. 37.

Tumor average volume of solvent control tumor-bearing mice reached 715mm 14 days after administration ³ . The average tumor volumes of GLUF 30mg/kg group and 100mg/kg group after 14 days of administration were 603mm, respectively ³ (T/C＝84.2％,TGI＝21.0％,p>0.05 563 mm) ³ (T/C＝78.7％,TGI＝28.5％,p>0.05). GLUF showed no significant tumor inhibition at doses of 30mg/kg and 100 mg/kg.

The effect of GLUF on weight change in tumor-bearing mice is shown in FIG. 35, FIG. 36. 2 tumor-bearing mice in the GLUF 30mg/kg dose group showed about 10% weight loss during the experiment.

In the above animal experiments, the tumor proliferation was started by transplanting human skin malignant melanoma cells SK-MEL-28, human breast cancer cells MCF-7 and multiple myeloma cells RPMI-8226 onto BALB/c nude mice and CB-17SCID mice, respectively, and then administration of glufosfamide, and it was found that the tumor inhibition results after administration were remarkably effective only for tumors in which multiple myeloma cells RPMI-8226 were transplanted into CB-17SCID mice.

CB-17SCID mice: BALB/c mice were inbred homologously to generate C.B-17 mice, CB-17SCID mice were the SCID mutant (autosomal recessive mutation) of C.B-17, and Bosma was found in 1980 from the group of C.B-17/lcr mice raised by Fox Chase cancer center. The line showed severe combined immunodeficiency symptoms, B cell and T lymphocyte dysfunction. At the same time, the strain has normal NK cells, macrophages and granulocytes. The B-17 mice carry the immunoglobulin heavy chain lg-1b allele from C57BL/Ka, except that it is identical to the BALB/C mouse gene, so that the genetic background of the CB-17SCID mice is essentially identical to that of BALB/C.

Since CB-17SCID mice are mice having SCID mutant lines (autosomal recessive mutations) relative to BALB/c mice, the above animal experiments demonstrate that GULF has a selective inhibitory effect on cancer cells of animals (mice) having undergone a specific gene mutation.

Claims

1. The use of glufosfamide or an analogue thereof for the manufacture of a medicament for the treatment of a patient suffering from a tumor or cancer in which DNA repair enzymes are impaired, the tumor or cancer tissue of the patient is at least BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, rad50, MRE11, p53, NBS1, XRS2, XRCC3, XRCC4/XPF, ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XRCC1, ku80, MHS6, MGMT, PARP, ERCC/XPG, CCNH, CDK7, CETN2, DDB1, DDB2 any one or more of ERCC5/XPG, ERCC6/CSB, ERCC8/CSA, LIG1/DNA ligase I, MMS, MNAT1, RAD23A, RAD B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonuclease, end-processing enzyme, DNA polymerase, flap endonuclease, DNA ligase,

more preferably XRCC1/RCC, ERCC1/RAD10, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG.

2. The use according to claim 1, wherein the impaired DNA repair is

preferably any one or more of the homologous recombination DNA repair enzyme damage, nucleotide excision repair enzyme damage, base excision repair enzyme damage, more preferably a patient with the homologous recombination DNA repair enzyme damage alone or with both the homologous recombination DNA repair enzyme damage and the nucleotide excision repair enzyme damage,

3. The use according to any one of claims 1-2, wherein the medicament further comprises other anti-cancer agents, anti-tumor agents and agents for radiotherapy and surgery, including HDAC inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxicity/cell growth inhibitors, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducers.

4. The use according to any one of claims 1-3, wherein the tumor, cancer comprises:

5. Use according to any one of claims 1-4, wherein the analogue means: pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates or prodrugs (such as esters) of glufosfamide, particularly preferred are

c. salts obtained by reacting a meglumine molecule with an acid.

6. The use according to any one of claims 1-5, wherein the patient is a human patient and a mammalian patient other than a human.

7. An immune combination therapeutic for treating tumors and cancers in a patient having an impaired DNA repair enzyme, the therapeutic comprising:

glufosfamide or an analog thereof; and

an immune checkpoint inhibitor,

8. The therapeutic combination treatment drug according to claim 7, wherein the immune checkpoint inhibitor is a drug that interferes with a cell cycle checkpoint, and the drug includes inhibitors such as PD-1 (immunosuppressive receptor) inhibitor, PD-L1 (immunosuppressive receptor ligand) inhibitor, and CTL4 (soluble T lymphocyte 4) inhibitor.

9. A compound formulation for treating cancer or tumor comprising:

glufosfamide or an analog thereof; and

a drug for enhancing the expression of glucose transporter,

10. An immunomodulatory compound preparation for treating cancer or tumor, comprising:

glufosfamide or an analog thereof; and

an immunomodulating agent, which comprises an immunomodulating agent,

11. The immunomodulatory compound preparation for treating cancer or tumor according to claim 10, wherein:

the immunomodulating drugs include immunosuppressants and immunopotentiators,

The immunosuppressant comprises cyclosporine, tacrolimus and azathioprine.