Method for screening medicine for treating recurrent glioma

Abstract

The invention discloses a method for screening a medicine for treating recurrent glioma. The invention provides a method for screening a medicine for treating relapse brain glioma, which comprises the following steps: performing medicine treatment on in-vitro relapse brain glioma primary cells from a subject according to a candidate medication scheme, and detecting and comparing the activity of the relapse brain glioma primary cells under different medication schemes; and selecting a medicine with the strongest inhibition effect on the primary cells of the recurrent brain glioma as a treatment medicine for a corresponding subject. The invention proves that patients with brain glioma relapse can be screened by the method provided by the invention to obtain the most suitable drug treatment scheme.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for screening drugs for treating recurrent brain glioma. Background Art

[0002] Gliomas are the most common brain tumors, accounting for about 60% of brain tumor incidence. Gliomas are clinically recognized as intractable tumors with extremely high mortality rates. In my country, the annual incidence of gliomas is 50,000 to 100,000, and the 5-year overall survival rate does not exceed 35%. Among them, the median survival of glioblastoma, the most malignant type, is only 12-18 months, and the five-year survival rate does not exceed 5%. Gliomas are very prone to recurrence, and disease recurrence is also the main cause of death in glioma patients. The median survival of recurrent gliomas is only 6-12 months.

[0003] Primary brain malignant tumors have limited treatment options and poor prognosis for patients. They have become the number one lethal tumor for children and young adults. Among them, high-grade gliomas (WHO grade III-IV) are the most common primary brain malignant tumors. After current standard surgery, radiotherapy and chemotherapy, almost 100% of them recur, and there is currently no clear and effective treatment plan after recurrence. Taking WHO grade IV glioma - glioblastoma as an example, its overall median survival time is less than 15 months. Once it recurs, its median survival time is only 6 months. Glioma grows in the brain, an important functional organ, and its highly invasive characteristics. The tumor and its recurrence cause great damage to brain function and have a high disability rate. This brings great pain to patients and their families.

[0004] At present, the treatment of newly diagnosed gliomas is very simple, that is, temozolomide-based chemoradiotherapy after surgical treatment. There is no effective treatment for recurrent gliomas in clinical practice. The NCCN guidelines recommend that patients with recurrent gliomas participate in clinical trials in order to obtain potential benefits. This treatment model for brain gliomas has had almost no improvements and breakthroughs in the past 20 years. In recent years, research on new drug therapies in the field of gliomas has been hindered. Currently, no targeted drug / immune checkpoint drug / antibody-linked drug or cell therapy has achieved success in phase III clinical trials in the field of brain gliomas.

[0005] Although scientific research and medical institutions around the world have made great efforts to study the causes and development of brain tumors, humans still know very little about this disease. Brain tumors are a type of complex disease. Their occurrence and development is a dynamic process that involves the interaction of many signal molecules, forming a complex molecular regulatory network, and is also affected by external environmental factors. The causes and development of brain tumors have strong individual differences and cannot be generalized. In addition, some types of brain tumors lack cell lines that can be used for basic scientific research and drug development, which greatly restricts the progress of new diagnostic and treatment research and development and drug development for gliomas. Therefore, using primary cell cultures of solid brain tumors as models for individualized precision research is a trend in the field of brain tumor research and even in the field of brain tumor diagnosis and treatment. Summary of the invention

[0006] The purpose of the present invention is to provide a method for screening a drug (combination) for treating recurrent brain glioma, which can effectively improve the therapeutic effect on recurrent brain glioma.

[0007] In a first aspect, the present invention claims a method for screening a drug (combination) for treating recurrent brain glioma.

[0008] The method for screening drugs (combinations) for treating recurrent brain gliomas claimed in the present invention may include the following steps: treating recurrent brain glioma primary cells isolated from a subject with drugs according to candidate medication regimens, detecting and comparing the activities of the recurrent brain glioma primary cells under different medication regimens; selecting the drug (combination) with the strongest in vitro inhibitory effect on the recurrent brain glioma primary cells or the drug with a cell proliferation inhibition efficiency higher than 30% as or as a candidate for the treatment drug for the subject;

[0009] The medication regimen is selected from any of the following:

[0010] (a1) Combination therapy of temozolomide and lovastatin;

[0011] (a2) Combination therapy of temozolomide and everolimus,

[0012] (a3) Combination therapy of temozolomide and simvastatin,

[0013] (a4) combination of temozolomide and olaparib;

[0014] (a5) Combination of procarbazine, lomustine and vincristine.

[0015] The subject is a patient with recurrent brain glioma.

[0016] Furthermore, in the method, when the isolated recurrent glioma primary cells from the subject are treated with drugs, for the medication regimen (a1), the action concentration of temozolomide can be 100 μM, the action concentration of lovastatin can be 5 μM, and the action time can be 48 hours. The same applies below.

[0017] Furthermore, in the method, when the recurrent glioma primary cells isolated from the subject are treated with drugs, for the medication regimen (a2), the action concentration of temozolomide can be 100 μM, the action concentration of everolimus can be 1 μM, and the action time can be 48 hours. The same applies below.

[0018] Furthermore, in the method, when the isolated recurrent glioma primary cells from the subject are treated with drugs, for the medication regimen (a3), the action concentration of temozolomide can be 100 μM, the action concentration of simvastatin can be 3 μM, and the action time can be 48 hours. The same applies below.

[0019] Furthermore, in the method, when the isolated recurrent glioma primary cells from the subject are treated with drugs, for the medication regimen (a4), the concentration of procarbazine can be 150 nM, the concentration of lomustine can be 7.5 μM, the concentration of vincristine can be 100 nM, and the action time can be 48 h. The same applies below.

[0020] Furthermore, in the method, when the isolated recurrent glioma primary cells from the subject are treated with drugs, for the medication regimen (a5), the action concentration of temozolomide can be 100 μM, the action concentration of olaparib can be 10 μM, and the action time can be 48 hours. The same applies below.

[0021] In a second aspect, the present invention claims a method for screening sensitive drugs (combinations) for patients with recurrent brain glioma.

[0022] The method for screening sensitive drugs (combinations) for patients with recurrent brain gliomas claimed in the present invention may include the following steps:

[0023] (A1) Data input: inputting sample data, wherein the sample data is activity data of the recurrent glioma primary cells isolated from a subject under different drug regimens after the cells are treated with drugs according to a candidate drug regimen;

[0024] (A2) Data comparison and result output: Processing and comparing the sample data, thereby determining and outputting the medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%, and the corresponding drug (combination) is the screened sensitive drug for the subject;

[0025] The medication regimen is selected from any of the following:

[0026] (a1) Combination therapy of temozolomide and lovastatin;

[0027] (a2) Combination therapy of temozolomide and everolimus,

[0028] (a3) Combination therapy of temozolomide and simvastatin,

[0029] (a4) combination of temozolomide and olaparib;

[0030] (a5) Combination of procarbazine, lomustine and vincristine.

[0031] The subject is a patient with recurrent brain glioma.

[0032] In a third aspect, the present invention claims protection for any of the following applications:

[0033] (B1) Use of temozolomide and lovastatin in the preparation of a pharmaceutical composition 1 for treating recurrent brain glioma in a subject 1; the subject 1 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and lovastatin and is screened using the method described in the second aspect above;

[0034] (B2) Use of temozolomide and everolimus in the preparation of a pharmaceutical composition 2 for treating recurrent brain glioma in a subject 2; the subject 2 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and everolimus and is screened using the method described in the second aspect above;

[0035] (B3) Use of temozolomide and simvastatin in the preparation of a pharmaceutical composition 3 for treating recurrent brain glioma in a subject 3; the subject 3 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and simvastatin and is screened and determined by the method described in the second aspect above;

[0036] (B4) Use of temozolomide and olaparib in the preparation of a pharmaceutical composition 4 for treating recurrent brain glioma in a subject 4; the subject 4 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and olaparib and is screened using the method described in the second aspect above;

[0037] (B5) Use of procarbazine, lomustine and vincristine in the preparation of a pharmaceutical composition 5 for treating recurrent brain glioma in a subject 5; the subject 5 is a patient with recurrent brain glioma who is sensitive to the combination of procarbazine, lomustine and vincristine as determined by screening using the method described in the second aspect above.

[0038] Furthermore, in the pharmaceutical composition 1, the mass ratio of temozolomide to lovastatin is 50:28. The same applies below.

[0039] Furthermore, in the pharmaceutical composition 2, the ratio of temozolomide to everolimus is 100:28. The same applies below.

[0040] Furthermore, in the pharmaceutical composition 3, the mass ratio of temozolomide to simvastatin is 50:28. The same applies below.

[0041] Furthermore, in the pharmaceutical composition 4, the mass ratio of temozolomide to olaparib is 20:84. The same applies below.

[0042] Furthermore, in the pharmaceutical composition 5, the mass ratio of procarbazine, lomustine and vincristine is 1428:187:8. The same applies below.

[0043] In a fourth aspect, the present invention claims protection for a computer device.

[0044] The computer device claimed in the present invention comprises a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement the following steps:

[0045] (C1) Data input: inputting sample data, wherein the sample data is activity data of the recurrent glioma primary cells isolated from a subject (a recurrent glioma patient) under different medication regimens after the primary cells are treated with drugs according to a candidate medication regimen;

[0046] (C2) Data comparison and result output: Processing and comparing the sample data, thereby determining and outputting a medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%;

[0047] The medication regimen is selected from any of the following:

[0048] (a1) Combination therapy of temozolomide and lovastatin;

[0049] (a2) Combination therapy of temozolomide and everolimus,

[0050] (a3) Combination therapy of temozolomide and simvastatin,

[0051] (a4) combination of temozolomide and olaparib;

[0052] (a5) Combination of procarbazine, lomustine and vincristine.

[0053] In a fifth aspect, the present invention claims protection for a computer program product.

[0054] The computer program product claimed in the present invention comprises a computer program; when the computer program is executed by a processor, the steps described in the fourth aspect above are implemented.

[0055] The computer program product may be a software product that implements its solution mainly through a computer program.

[0056] In a sixth aspect, the present invention claims protection for a computer-readable storage medium.

[0057] The computer-readable storage medium claimed for protection by the present invention stores a computer program thereon, and when the computer program is executed by a processor, the steps described in the fourth aspect above are implemented.

[0058] Among them, the computer-readable storage medium refers to a carrier for storing data, which may be a tape, disk, floppy disk, CD, magneto-optical disk, ROM, PROM, VCD, DVD, hard disk, flash memory, U disk, CF card, SD card, MMC card, SM card, memory stick (MemoryStick) or xD card, etc.

[0059] In a seventh aspect, the present invention claims protection for a system (product) for screening sensitive drugs (combinations) for patients with recurrent brain glioma.

[0060] The system for screening sensitive drugs (combinations) for patients with recurrent brain gliomas claimed in the present invention may include:

[0061] (D1) Reagents or kits for detecting cell activity;

[0062] (D2) device; the device includes a data input module and a data comparison and result output module;

[0063] The data input module is configured to input sample data, wherein the sample data is activity data of the recurrent glioma primary cells under different medication regimens after the recurrent glioma primary cells isolated from the subject (recurrent glioma patient) are treated with drugs according to the candidate medication regimens;

[0064] The data comparison and result output is configured to process and compare the sample data, thereby determining and outputting a medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%, and the corresponding drug is the screened sensitive drug for the subject;

[0065] The medication regimen is selected from any of the following:

[0066] (a1) Combination therapy of temozolomide and lovastatin;

[0067] (a2) Combination therapy of temozolomide and everolimus,

[0068] (a3) Combination therapy of temozolomide and simvastatin,

[0069] (a4) combination of temozolomide and olaparib;

[0070] (a5) Combination of procarbazine, lomustine and vincristine.

[0071] In each of the above related aspects, the recurrent brain glioma primary cells are suspended and cultured using a brain tumor solid tumor primary cell culture medium;

[0072] The brain tumor solid tumor primary cell culture medium is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein VEGF, human recombinant protein GDNF, A83-01, N-acetyl-L-cysteine, N-2Supplement, SB431542, Y-27632, CHIR99021 and basal culture medium;

[0073] Wherein, the final concentration of HEPES is 8-12mM (such as 10mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%) by volume; the final concentration of human recombinant protein bFGF is 10-50ng / mL (such as 30ng / mL); the final concentration of human recombinant protein HGF is 5-25ng / mL (such as 20ng / mL); the final concentration of human recombinant protein MSP is 5-25ng / mL (such as 20ng / mL); the final concentration of human recombinant protein VEGF is 10-50ng / mL (such as 40ng / mL) ; The final concentration of the human recombinant protein GDNF is 50-200 ng / mL (such as 100 ng / mL); the final concentration of the A83-01 is 0.25-1.25 μM (such as 1 μM); the final concentration of the N-acetyl-L-cysteine ​​is 0.5-2 mM (such as 1 mM); the final concentration of the N-2Supplement is 1% volume percentage; the final concentration of the SB431542 is 1-5 μM (such as 2 μM); the final concentration of the Y-27632 is 5-20 μM (such as 4 μM); the final concentration of the CHIR99021 is 1-5 μM.

[0074] Furthermore, the basic culture medium may be composed of Advanced DMEM / F12 culture medium.

[0075] Further, in the brain tumor solid tumor primary cell culture medium, the final concentration of penicillin in the antibacterial and antifungal agent triple antibody is 100-200U / mL (such as 100U / mL); the final concentration of streptomycin in the antibacterial and antifungal agent triple antibody is 100-200μg / mL (such as 100μg / mL); the final concentration of amphotericin B in the antibacterial and antifungal agent triple antibody is 250ng / mL; the final concentration of HEPES is 8-12mM (such as 10mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%) volume percentage; the final concentration of human recombinant protein bFGF is 30ng / mL; the final concentration of human recombinant protein bFGF is 10 ... The final concentration of histone HGF is 20 ng / mL; the final concentration of the human recombinant protein MSP is 20 ng / mL; the final concentration of the human recombinant protein VEGF is 40 ng / mL; the final concentration of the human recombinant protein GDNF is 100 ng / mL; the final concentration of the A83-01 is 1 μM; the final concentration of the N-acetyl-L-cysteine ​​is 1 mM; the final concentration of the N-2Supplement is 1% by volume; the final concentration of the SB431542 is 2 μM; the final concentration of the Y-27632 is 10 μM; the final concentration of the CHIR99021 is 4 μM; and the remainder is Advanced DMEM / F12 culture medium.

[0076] In an eighth aspect, the present invention claims a pharmaceutical composition for treating recurrent brain glioma.

[0077] The pharmaceutical composition for treating recurrent brain glioma claimed in the present invention may be any of the following:

[0078] (D1) a pharmaceutical composition 1 for treating recurrent brain glioma in a subject 1, wherein the subject 1 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and lovastatin as determined by screening using the method described in the second aspect above; the pharmaceutical composition 1 consists of temozolomide and lovastatin;

[0079] (D2) a pharmaceutical composition 2 for treating recurrent brain glioma in subject 2, wherein subject 2 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and everolimus as determined by screening using the method described in the second aspect above; the pharmaceutical composition 2 consists of temozolomide and everolimus;

[0080] (D3) a pharmaceutical composition 3 for treating recurrent brain glioma in a subject 3, wherein the subject 3 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and simvastatin as determined by screening using the method described in the second aspect above; the pharmaceutical composition 3 consists of temozolomide and simvastatin;

[0081] (D4) A pharmaceutical composition 4 for treating recurrent brain glioma in subject 4, wherein subject 4 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and olaparib as determined by screening using the method described in the second aspect above; the pharmaceutical composition 4 consists of temozolomide and olaparib.

[0082] In one embodiment of the present invention, all of the above-mentioned recurrent brain gliomas may be grade III-IV gliomas.

[0083] The present invention proves that patients with recurrent brain glioma can obtain the most suitable drug treatment plan by screening through the method provided by the present invention, and can obtain significant clinical benefits by using temozolomide and statins in combination, or temozolomide and olaparib in combination during the treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 is the effective frequency of different drugs in 155 samples.

[0085] Figure 2 This is the clinical trial process for recurrent glioma. In the figure, PVC represents the combination of procarbazine, lomustine and vincristine. DETAILED DESCRIPTION

[0086] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0087] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0088] Example 1: Cultivation of primary cells of recurrent glioma

[0089] 1. Preparation of reagents for culturing primary brain tumor solid tumor cells

[0090] 1. Sample preservation solution (100mL)

[0091] The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0092] Table 1. Sample storage solution (100 mL)

[0093]

[0094] After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

[0095] 2. Sample cleaning solution (100mL)

[0096] The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0097] Table 2. Sample cleaning solution (100 mL)

[0098]

[0099] The sample cleaning solution must be prepared and used immediately.

[0100] 3. Sample dissociation solution (10mL)

[0101] The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0102] Table 3. Sample dissociation solution (10 mL)

[0103]

[0104]

[0105] Note: The sample dissociation solution should be prepared and used immediately.

[0106] In Table 3, the preparation of collagenase stock solution is shown in Table 4 and Table 5.

[0107] Table 4. 10× collagenase I stock solution (100 mL)

[0108]

[0109] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0110] Table 5. 10× Collagenase IV stock solution (100 mL)

[0111]

[0112] After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0113] In Tables 4 and 5, the unit U of collagenase (the collagenase I or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0114] 4. Cell digestion solution (10mL)

[0115] The specific formula of the cell digestion solution (10 mL) is shown in Table 6.

[0116] Table 6. Cell digestion solution (10 mL)

[0117]

[0118] The cell digestion solution is prepared and used immediately.

[0119] 5. Digestion stop solution (100 mL)

[0120] The specific formula of the digestion stop solution (100 mL) is shown in Table 7.

[0121] Table 7. Digestion stop solution (100 mL)

[0122]

[0123] After the digestion stop solution is prepared, it can be stored at 4°C for one month.

[0124] 6. Brain tumor solid tumor primary cell culture medium (100mL)

[0125] The specific formula of brain tumor solid tumor primary cell culture medium (100 mL) is shown in Table 8.

[0126] Table 8. Brain tumor solid tumor primary cell culture medium (100 mL)

[0127]

[0128] After the preparation of the brain tumor solid tumor primary cell culture medium is completed, it is filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0129] In Table 8, the preparation of human recombinant protein stock solutions is shown in Tables 10 to 14, the preparation of A83-01 stock solutions is shown in Table 15, the preparation of N-acetyl-L-cysteine ​​stock solutions is shown in Table 16, the preparation of SB431542 stock solutions is shown in Table 17, the preparation of Y-27632 stock solutions is shown in Table 18; and the preparation of CHIR99021 stock solutions is shown in Table 19. The preparation of 100×BSA solutions required for preparing these stock solutions is shown in Table 9.

[0130] Table 9, 100×BSA solution (1 mL)

[0131]

[0132] 100× BSA solution is prepared and used immediately.

[0133] Table 10, 1000× human recombinant protein bFGF stock solution (2.5 mL)

[0134]

[0135] After the 1000× human recombinant protein bFGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0136] Table 11, 1000× human recombinant protein HGF stock solution (5 mL)

[0137]

[0138] After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0139] Table 12, 1000× human recombinant protein MSP stock solution (2.5 mL)

[0140]

[0141] After the 1000× human recombinant protein MSP stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0142] Table 13, 1000× human recombinant protein VEGF stock solution (2.5 mL)

[0143]

[0144] After the 1000× human recombinant protein VEGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0145] Table 14. 1000× human recombinant protein GDNF stock solution (1 mL)

[0146]

[0147] After the 1000× human recombinant protein GDNF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0148] Table 15, 100000×A83-01 stock solution (1.05 mL)

[0149]

[0150] After the 1000×A83-01 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0151] Table 16, 1000×N-acetyl-L-cysteine ​​stock solution (5 mL)

[0152]

[0153] After the 1000× N-acetyl-L-cysteine ​​stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0154] Table 17, 1000×Y-27632 stock solution (3.125 mL)

[0155]

[0156] After the 1000×Y-27632 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0157] Table 18, 10000×SB431542 stock solution (1.30 mL)

[0158]

[0159] After the 10000×SB431542 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0160] Table 19, 10000×CHIR99021 stock solution (0.996 mL)

[0161]

[0162] After the 10000×CHIR99021 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0163] II. Obtaining postoperative specimens / biopsy specimens for recurrent glioma

[0164] 1. Cooperate with tertiary hospitals, and the cooperation has passed formal medical ethics review.

[0165] 2. The attending physician selects patients for inclusion in the study according to the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture based on the intraoperative clinical indications. The sample selection criteria are: recurrent brain glioma specimens weighing more than 20 mg.

[0166] 3. During the operation, the surgeon collects fresh specimens in the sterile environment of the operating room and places them in the sample preservation solution prepared in advance (see Example 1). After the specimen is removed from the body, it is temporarily stored on ice and transported to the laboratory within two hours for the next step of operation.

[0167] 3. Pre-dissociation treatment of recurrent glioma tissue samples

[0168] The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

[0169] The surgical instruments used in the following operations must be sterilized at high temperature and high pressure and dried before use.

[0170] 1. Weigh the sample.

[0171] 2. Clean the sample surface with 75% (volume percentage) ethanol for 10 to 30 seconds.

[0172] 3. Wash the sample 5 times with sample cleaning solution and 5 times with sterile PBS solution.

[0173] 4. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

[0174] 4. Dissociation of Recurrent Glioma Tissue Samples

[0175] The surgical instruments used in the following embodiments must be sterilized at high temperature and high pressure in advance and dried before use.

[0176] 1. Use ophthalmic scissors to cut the tissue into 1mm pieces. 3 Small pieces around.

[0177] 2. Treat the chopped tissue samples with sample dissociation solution preheated at 37°C at a dosage of 0.1 mL sample dissociation solution (see step 1) per mg of tissue. Dissociate the samples at 37°C for 15 minutes to 3 hours. Observe the dissociation of the samples under a microscope every 15 minutes until a large number of single cells are observed.

[0178] 3. Use 10 times the volume of digestion stop solution (see step 1) to terminate the dissociation reaction and collect the cell suspension.

[0179] 4. Filter the cell suspension with a 100 μm sterile cell strainer to remove tissue debris and adherent cells.

[0180] 5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

[0181] 6. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0182] 7. Resuspend the cell pellet with brain tumor solid tumor primary cell culture medium (see step 1), observe the cell status under a microscope, and count the cells.

[0183] The results showed that in addition to tumor cells, the dissociated single cell suspension was mixed with a large number of other types of cells, such as red blood cells, lymphocytes, fibroblasts, etc. One of the advantages of this method is that in the subsequent culture process, only cancer cells can be amplified in large quantities, while the proportion of other cells gradually decreases or even disappears, and finally a high-purity recurrent brain glioma primary cell is obtained.

[0184] 5. Primary cell culture of recurrent glioma

[0185] 1. Use a low-attachment-surface to culture the primary cells of recurrent glioma in suspension. The culture medium used is the primary cell culture medium of solid brain tumor in step 1 (wherein the final concentration of human recombinant protein bFGF is 30 ng / mL; the final concentration of human recombinant protein HGF is 20 ng / mL; the final concentration of human recombinant protein MSP is 20 ng / mL; the final concentration of human recombinant protein VEGF is 40 ng / mL; the final concentration of human recombinant protein GDNF is 100 ng / mL; the final concentration of A83-01 is 1 μM; the final concentration of N-acetyl-L-cysteine ​​is 1 mM; the final concentration of SB431542 is 2 μM; the final concentration of Y-27632 is 10 μM; the final concentration of CHIR99021 is 4 μM). Taking a six-well plate as an example, 10 cells / well are added to each well. 6 The cells were plated at a density of 100 cells / mL and cultured in a cell culture incubator at 37°C and 5% CO2.

[0186] 2. Observe the cell status every day and replace the culture medium every 3 days until the cells form clumps with a diameter of about 100 μm.

[0187] The results showed that after 3-10 days of culture, tumor cells proliferated to form cell clusters with a diameter of 100 μm, and the total number of tumor cells could exceed 10 7 , the number of other types of cells was significantly reduced or even disappeared.

[0188] Example 2: Method for drug screening using primary cell culture of recurrent glioma

[0189] In a 96-well cell culture plate, 2 × 10 4 The in vitro recurrent brain glioma primary cells cultured by the method described in Example 1 were inoculated at a density of 1 / well. 24 hours after inoculation, the drugs to be tested were added as shown in Table 20. The number of replicate wells for each drug group was 3.

[0190] Table 20. Brain tumor drug testing plan

[0191] Drug Screening Program Drug concentration Drug duration Procarbazine + lomustine + vincristine 150nM, 7.5μM, 100nM 48 hours Irinotecan (SN-38) 200nM 48 hours Carmustine 10μM 48 hours Etoposide 20μM 48 hours Temozolomide + procarbazine 100μM, 150nM 48 hours Temozolomide + lomustine 100μM, 7.5μM 48 hours Temozolomide + Olaparib 100μM, 10μM 48 hours Temozolomide + Hydroxyurea Sulfate 100μM, 20μM 48 hours Temozolomide + cisplatin 100μM, 10μM 48 hours Temozolomide + everolimus 100μM, 1μM 48 hours Temozolomide + Lovastatin 100μM, 5μM 48 hours Temozolomide + gemcitabine 100μM, 200nM 48 hours Temozolomide + simvastatin 100μM, 3μM 48 hours Dabrafenib + trametinib 10nM, 500nM 48 hours

[0192] After 48 hours of drug action, the cell activity in each well was detected using the Celltiter-Glo cell viability detection kit (Promega). The specific detection method is as follows:

[0193] 1. Equilibrate the Celltier-Glo detection reagent to room temperature;

[0194] 2. Remove the culture supernatant;

[0195] 3. Add Celltier-Glo detection reagent, shake and mix for 5 minutes, and incubate at room temperature for 25 minutes to fully lyse the cells;

[0196] 4. Use an enzyme-labeled instrument to detect the luminescence value of the chemiluminescence in each well;

[0197] 5. The inhibitory efficiency of each drug group on cell proliferation was calculated by comparing the luminescence value between each drug-treated group and the negative control group without any drug treatment (mean of 3 replicates).

[0198] Example 3: Results of drug sensitivity screening of primary glioma cells

[0199] Using the method of Example 1, surgical samples from 170 glioma patients were processed, and 155 primary glioma cells were successfully cultured, with a culture success rate of 91% (155 / 170). Drug screening was performed for each sample according to the method of Example 2, and the results are shown in Table 21.

[0200] Table 21. Results of drug screening in primary glioma cells (cell proliferation inhibition efficiency)

[0201]

[0202]

[0203]

[0204]

[0205] Note: N / A in the table means that the sample has not been tested for the corresponding drug

[0206] In the above screening experiment, we defined that if the tumor cell proliferation inhibition efficiency exceeded 30%, the corresponding drug regimen was a potentially effective regimen. Based on this, the analysis results were as follows: Among all 155 samples screened, 123 cases screened out at least one effective drug, and 32 cases did not screen out any effective drug.

[0207] Figure 1 is the effective frequency of different drugs in 155 samples, from which the four drug combinations with the highest effective frequency were obtained:

[0208] (1) Temozolomide + lovastatin, effective frequency 53.55% (83 / 155);

[0209] (2) temozolomide + everolimus, effective frequency 53.55% (83 / 155);

[0210] (3) temozolomide + simvastatin, effective frequency 44.74% (68 / 152);

[0211] (4) Temozolomide + olaparib, effective frequency 40% (62 / 155).

[0212] Example 4: Clinical safety and efficacy verification of potentially effective drug combinations

[0213] In order to verify the safety and effectiveness of the above-mentioned potentially effective drug regimen in clinical practice, this example selected patients with recurrent glioma and designed a clinical trial. The clinical trial process is shown in Figure 2 The basic process is as follows: After informed consent, the patient undergoes surgery for recurrent glioma and obtains surgical samples. The surgical samples are used for primary cell culture and drug screening to obtain potentially effective drug combinations. A multidisciplinary expert consultation is conducted to select the most suitable drug regimen for the patient from the effective drug combinations for treatment. The treatment process is tracked and followed up.

[0214] 1. Inclusion criteria:

[0215] (1) Aged 18-75 years old, regardless of gender;

[0216] (2) The first surgical pathology was WHO grade II-IV glioma;

[0217] (3) received radiotherapy and temozolomide chemotherapy in the past five years;

[0218] (4) Imaging showed recurrent glioma, with indications for surgical resection, and completed neurosurgery resection treatment;

[0219] (5) The histopathological diagnosis of the resected recurrent lesion was WHO grade III-IV glioma;

[0220] (6) At the time of enrollment, the neuro-oncologist determined that there was no standard treatment available after surgery;

[0221] (7) Those who can understand this trial and have signed the informed consent.

[0222] 2. Grouping standards:

[0223] (1) Accompanied by other uncontrolled malignant tumors;

[0224] (2) The amount of specimen removed during surgery is too small to complete gene sequencing or drug screening;

[0225] (3) had undergone carmustine sustained-release implantation surgery within 6 months;

[0226] (4) Active hepatitis B or C, HIV infection;

[0227] (5) Subjects with grade 2-3 hypertension or poorly controlled hypertension;

[0228] (6) Patients with severe cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, cerebral thrombosis, cerebral hemorrhage, etc.;

[0229] (7) Any other uncontrolled active diseases that prevent participation in the trial, such as hemolytic anemia, autoimmune skin diseases such as psoriasis and rheumatoid arthritis;

[0230] (8) Subjects with a history of uncontrolled psychiatric illness, or subjects whose medical or mental history, in the opinion of the investigator, may increase the risks associated with participation in the study or administration of study drugs, or may interfere with the results;

[0231] (9) Those who have been using immunosuppressants for a long time due to organ transplantation, etc.;

[0232] (10) Unstable pulmonary embolism, deep vein thrombosis, or other major arterial / venous thromboembolic events occurred within 30 days before enrollment, or patients were receiving anticoagulant therapy;

[0233] (11) Subjects who are pregnant or breastfeeding, or subjects who plan to become pregnant during treatment or within 2 months after the end of treatment;

[0234] (12) Active infection or uncontrollable infection requiring systemic treatment (except simple urinary tract infection or upper respiratory tract infection) within 14 days before enrollment;

[0235] (13) Patients who had received CAR-T therapy or other gene-modified cell therapy before enrollment;

[0236] (14) Those who are considered by the researchers to be unsuitable for participation in this trial;

[0237] (15) Subjects who suffer from illness that affects their ability to sign the written informed consent or comply with research procedures; or subjects who are unwilling or unable to comply with research requirements.

[0238] 3. Treatment plan:

[0239] (1) Temozolomide + lovastatin;

[0240] The treatment cycle is 28 days: oral temozolomide (200 mg / day) is taken on days 1 to 5, and oral lovastatin (20 mg / day, once a day) is taken on days 1 to 28.

[0241] (2) temozolomide + everolimus;

[0242] The treatment cycle is 28 days: temozolomide (200 mg / day) is taken orally on days 1-5, and everolimus (10 mg / day, once every night, before bedtime) is taken orally on days 1-28.

[0243] (3) temozolomide + simvastatin;

[0244] The treatment cycle is 28 days: oral temozolomide (200 mg / day) is taken on days 1 to 5, and oral simvastatin (20 mg / day, once a day) is taken on days 1 to 28.

[0245] (4) temozolomide + olaparib;

[0246] The treatment cycle is 28 days: temozolomide (200 mg / day) is taken orally on days 1-5, and olaparib (150 mg / day, once every night, before bedtime) is taken orally on days 1-28.

[0247] (5) procarbazine + lomustine + vincristine;

[0248] A treatment cycle of 29 days:

[0249] Lomustine: 110 mg / m 2 , oral, day 1. Vincristine: 4 mg, IV, day 8 and day 29. Procarbazine: 60 mg / m 2 , oral, day 8-21. Among them, m 2 Refers to the patient's body surface.

[0250] (6) For patients without sensitive treatment options, temozolomide alone can be used as maintenance therapy.

[0251] 4. Main observation indicators

[0252] The feasibility of clinical treatment of recurrent glioma through drug screening and multidisciplinary consultation, that is, the proportion of patients who complete treatment according to the recommended medication regimen.

[0253] Completion of treatment: means completing at least one cycle of treatment according to the above regimen.

[0254] If treatment is not carried out or the drug is used for less than one cycle, the treatment is not completed.

[0255] After completing one cycle of treatment, maintenance treatment will continue until the patient's disease progresses, or side effects occur that indicate discontinuation of treatment, or the patient is unwilling to continue treatment.

[0256] 5. Secondary observation index

[0257] Patients who have received CAR-T therapy or other gene-modified cell therapy before enrollment;

[0258] (1) Safety indicators: Investigators are required to monitor the safety of all subjects who are enrolled and receive at least one recommended medication and record any adverse reactions from the start of treatment to 4 weeks after the last treatment.

[0259] (2) Efficacy indicators: 6-month overall survival rate, 12-month overall survival rate, progression-free survival (PFS), and overall survival (OS).

[0260] 6. Results and Analysis

[0261] A total of 35 patients with recurrent glioma were enrolled in this clinical trial, of whom 5 were excluded due to abandonment of treatment or meeting the exclusion criteria. Primary cell culture and drug screening were performed on 30 patients, and the screening results are shown in Table 22.

[0262] Table 22. Drug screening results and optimal medication regimen for patients with recurrent glioma

[0263]

[0264]

[0265] Primary tumor cells were successfully cultured for 29 patients and drug screening was performed, and treatment plans were determined through multidisciplinary consultation:

[0266] The best treatment option for 9 patients was temozolomide combined with statins, and 7 of them completed the treatment according to the established regimen;

[0267] The best treatment for 6 patients was temozolomide combined with everolimus, and 5 of them completed the treatment according to the established regimen;

[0268] The best treatment option for 6 patients was temozolomide combined with olaparib, and 6 of them completed the treatment according to the established plan;

[0269] The best regimen for 5 patients was procarbazine, lomustine, and vincristine, and 4 of them completed the treatment according to the established regimen;

[0270] Three patients were not screened for sensitive regimens and all completed temozolomide monotherapy maintenance treatment.

[0271] The 29 patients were followed up every 2 months for one year after the start of drug treatment. The follow-up results are shown in Table 23.

[0272] Table 23. Follow-up results of patients with recurrent glioma

[0273]

[0274]

[0275]

[0276] The best treatment was procarbazine, lomustine combined with vincristine, and the four patients who completed the treatment had an overall survival of 100% at 6 months and 50% at 12 months. The overall survival of all patients exceeded the median survival of patients with recurrent glioma.

[0277] The best treatment was temozolomide combined with olaparib, and the six patients who completed the treatment had an overall survival of 100% at 6 months and 83.3% at 12 months. The overall survival of all patients exceeded the median survival of patients with recurrent glioma.

[0278] The best treatment was temozolomide combined with statin (lovastatin or simvastatin). The 7 patients who completed the treatment had an overall survival of 100% at 6 months and 71.4% at 12 months. The overall survival of all patients exceeded the median survival of patients with recurrent glioma.

[0279] The best treatment was temozolomide combined with everolimus, and the five patients who completed the treatment had an overall survival of 100% at 6 months and 40% at 12 months. The overall survival of all patients exceeded the median survival of patients with recurrent glioma.

[0280] For the 7 patients who did not complete the treatment according to the established plan or had no optimal treatment option, the overall survival at 6 months was 28.6% and the overall survival at 12 months was 0%.

[0281] It can be seen that through the methods of primary cell culture and drug screening, effective drug treatment options can be screened for patients, significantly improving the patient's survival period.

[0282] The drug combinations of temozolomide combined with statins (lovastatin or simvastatin), temozolomide combined with olaparib, and temozolomide combined with everolimus can all bring significant survival benefits to patients with recurrent glioma.

[0283] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for screening drugs for treating recurrent brain glioma, comprising the following steps: treating recurrent brain glioma primary cells isolated from a subject with drugs according to candidate drug regimens, detecting and comparing the activity of the recurrent brain glioma primary cells under different drug regimens; selecting the drug with the strongest inhibitory effect on the recurrent brain glioma primary cells or the drug with a cell proliferation inhibition efficiency higher than 30% as or as a candidate for the treatment drug for the subject; The medication regimen is selected from any of the following: (a1) Combination therapy of temozolomide and lovastatin; (a2) Combination therapy of temozolomide and everolimus, (a3) Combination therapy of temozolomide and simvastatin, (a4) combination of temozolomide and olaparib; (a5) Combination of procarbazine, lomustine and vincristine.

2. A method for screening sensitive drugs for patients with recurrent brain glioma, comprising the following steps: (A1) Data input: inputting sample data, wherein the sample data is activity data of the recurrent glioma primary cells isolated from a subject under different drug regimens after the cells are treated with drugs according to a candidate drug regimen; (A2) Data comparison and result output: Processing and comparing the sample data, thereby determining and outputting a medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%, and the corresponding drug is the screened sensitive drug for the subject; The medication regimen is selected from any of the following: (a1) Combination therapy of temozolomide and lovastatin; (a2) Combination therapy of temozolomide and everolimus, (a3) Combination therapy of temozolomide and simvastatin, (a4) combination of temozolomide and olaparib; (a5) Combination of procarbazine, lomustine and vincristine.

3. Any of the following applications: (B1) Use of temozolomide and lovastatin in the preparation of a pharmaceutical composition 1 for treating recurrent brain glioma in a subject 1; the subject 1 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and lovastatin and is screened and determined by the method of claim 2; (B2) Use of temozolomide and everolimus in the preparation of a pharmaceutical composition 2 for treating recurrent brain glioma in a subject 2; the subject 2 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and everolimus and is screened and determined by the method of claim 2; (B3) Use of temozolomide and simvastatin in the preparation of a pharmaceutical composition 3 for treating recurrent brain glioma in a subject 3; the subject 3 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and simvastatin and is screened and determined by the method of claim 2; (B4) Use of temozolomide and olaparib in the preparation of a pharmaceutical composition 4 for treating recurrent brain glioma in a subject 4; the subject 4 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and olaparib and is screened and determined by the method of claim 2; (B5) Use of procarbazine, lomustine and vincristine in the preparation of a pharmaceutical composition 5 for treating recurrent brain glioma in a subject 5; the subject 5 is a patient with recurrent brain glioma who is sensitive to the combined use of procarbazine, lomustine and vincristine as determined by screening using the method of claim 2.

4. A computer device comprising a memory, a processor and a computer program stored in the memory, characterized in that: The processor executes the computer program to implement the following steps: (C1) Data input: inputting sample data, wherein the sample data is activity data of the recurrent glioma primary cells isolated from a subject under different drug regimens after the cells are treated with drugs according to a candidate drug regimen; (C2) Data comparison and result output: Processing and comparing the sample data, thereby determining and outputting a medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%; The medication regimen is selected from any of the following: (a1) Combination therapy of temozolomide and lovastatin; (a2) Combination therapy of temozolomide and everolimus, (a3) Combination therapy of temozolomide and simvastatin, (a4) combination of temozolomide and olaparib; (a5) Combination of procarbazine, lomustine and vincristine.

5. A computer program product, comprising a computer program, characterized in that: The computer program implements the steps recited in claim 4 when executed by a processor.

6. A computer-readable storage medium having a computer program stored thereon, characterized in that: The computer program implements the steps recited in claim 4 when executed by a processor.

7. A system for screening sensitive drugs for patients with recurrent brain glioma, comprising: (D1) Reagents or kits for detecting cell activity; (D2) device; The device comprises a data input module and a data comparison and result output module; The data input module is configured to input sample data, wherein the sample data is activity data of the recurrent glioma primary cells in vitro derived from the subject under different medication regimens after the recurrent glioma primary cells are treated with drugs according to the candidate medication regimens; The data comparison and result output is configured to process and compare the sample data, thereby determining and outputting a medication regimen with the strongest inhibitory effect on the recurrent brain glioma primary cells or a cell proliferation inhibition efficiency higher than 30%, and the corresponding drug is the screened sensitive drug for the subject; The medication regimen is selected from any of the following: (a1) Combination therapy of temozolomide and lovastatin; (a2) Combination therapy of temozolomide and everolimus, (a3) Combination therapy of temozolomide and simvastatin, (a4) combination of temozolomide and olaparib; (a5) Combination of procarbazine, lomustine and vincristine.

8. The method or application or computer device or computer program product or computer readable storage medium or system according to any one of claims 1 to 7, characterized in that: The recurrent brain glioma primary cells are suspended and cultured using a brain tumor solid tumor primary cell culture medium; The brain tumor solid tumor primary cell culture medium is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein VEGF, human recombinant protein GDNF, A83-01, N-acetyl-L-cysteine, N-2Supplement, SB431542, Y-27632, CHIR99021 and basal culture medium; The final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of human recombinant protein bFGF is 10-50 ng / mL; the final concentration of human recombinant protein HGF is 5-25 ng / mL; the final concentration of human recombinant protein MSP is 5-25 ng / mL; the final concentration of human recombinant protein VEGF is 10-50 ng / mL; the final concentration of human recombinant protein G The final concentration of DNF is 50-200 ng / mL; the final concentration of A83-01 is 0.25-1.25 μM; the final concentration of N-acetyl-L-cysteine ​​is 0.5-2 mM; the final concentration of N-2Supplement is 1% by volume; the final concentration of SB431542 is 1-5 μM; the final concentration of Y-27632 is 5-20 μM; and the final concentration of CHIR99021 is 1-5 μM.

9. A pharmaceutical composition for treating recurrent brain glioma, which is any of the following: (E1) A pharmaceutical composition 1 for treating recurrent brain glioma in a subject 1, wherein the subject 1 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and lovastatin as determined by the method of claim 2, characterized in that: The pharmaceutical composition 1 consists of temozolomide and lovastatin; (E2) a pharmaceutical composition 2 for treating recurrent brain glioma in a subject 2, wherein the subject 2 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and everolimus as determined by screening using the method of claim 2, characterized in that the pharmaceutical composition 2 consists of temozolomide and everolimus; (E3) a pharmaceutical composition 3 for treating recurrent brain glioma in a subject 3, wherein the subject 3 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and simvastatin as determined by the method of claim 2, characterized in that the pharmaceutical composition 3 consists of temozolomide and simvastatin; (E4) A pharmaceutical composition 4 for treating recurrent brain glioma in a subject 4, wherein the subject 4 is a patient with recurrent brain glioma who is sensitive to the combination of temozolomide and olaparib as determined by the method of claim 2, characterized in that the pharmaceutical composition 4 consists of temozolomide and olaparib.

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