Novel treatment method of glioblastoma

Abstract

The invention provides a novel treatment method for gliocytoma. Specifically, the invention provides a novel application of a PFKFB4 inhibitor and / or a glutamine metabolism inhibitor, especially a combination of the PFKFB4 inhibitor and the glutamine metabolism inhibitor in treatment of glioblastoma. The new application comprises the steps of improving the drug resistance to chemotherapeutic agents, increasing the sensitivity to the chemotherapeutic agents and inhibiting the proliferation of glioblastoma.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a new method for treating glioblastoma. Background Art

[0002] Glioma is the most common malignant tumor of the central nervous system and one of the malignant tumors with the highest malignancy and mortality. It has the characteristics of infiltrative growth, poor differentiation, rapid proliferation, and strong invasiveness. WHO classifies gliomas into grades I to IV. The higher the grade, the higher the malignancy, the lower the degree of differentiation of tumor cells, the stronger the proliferation activity, and the more obvious the invasiveness.

[0003] Glioblastoma Multiforme (GBM) is the most common malignant primary brain tumor and one of the most deadly human cancers. It belongs to grade IV glioma, the most malignant type of glioma. It has the characteristics of rapid growth, strong invasiveness, and easy recurrence. It is at the "top" of the malignancy in the glioma family. At present, the etiology and pathogenesis of GBM are still not fully understood. Although progress has been made in molecular diagnosis, surgical treatment, chemotherapy and radiotherapy in recent years, the overall treatment effect is still unsatisfactory. Chemotherapy for GBM includes temozolomide (TMZ) and other alkylating agents such as carmustine, nimustine and lomustine. TMZ is still the only first-line anticancer drug. However, due to its intrinsic or acquired drug resistance, the clinical benefit of TMZ for patients with glioma is still limited. Drug resistance is often caused by a variety of reasons, including DNA damage repair pathways, heterogeneity of cancer cell populations, and cancer metabolic reprogramming, but it is still not thoroughly understood. This is why genetic testing results show multiple gene mutations but cannot effectively guide drug use. Therefore, on the basis of a clear study of the tumor resistance mechanism, it is crucial to select effective drug combination interventions and carry out combined therapies to improve treatment.

[0004] In summary, there is an urgent need in the art to develop new treatments for glioblastoma, especially new treatments that can address the problem of drug resistance. Summary of the invention

[0005] The purpose of the present invention is to provide a new treatment method for glioblastoma, especially a new treatment method that can solve the problem of drug resistance.

[0006] In a first aspect of the present invention, a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor is provided.

[0007] In another preferred embodiment, the PFKFB4 inhibitor and the glutamine metabolism inhibitor may be in separate preparations, or in the same preparation.

[0008] In another preferred embodiment, the PFKFB4 inhibitor and the glutamine metabolism inhibitor can be administered simultaneously, sequentially, or at intervals.

[0009] In another preferred embodiment, the molar ratio of the PFKFB4 inhibitor to the glutamine metabolism inhibitor is 0.001-1000:1000-0.001.

[0010] In another preferred embodiment, the PFKFB4 inhibitor and the glutamine metabolism inhibitor are as defined below.

[0011] In a second aspect of the present invention, there is provided a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor for use in the preparation of (a) a drug for treating or preventing glioblastoma, or (b) a sensitizer for anti-glioblastoma chemotherapeutic drugs, or (c) a drug for reducing resistance to anti-glioblastoma chemotherapeutic drugs.

[0012] In another preferred embodiment, the molar ratio of the PFKFB4 inhibitor to the glutamine metabolism inhibitor is 0.001-1000:1000-0.001.

[0013] In another preferred embodiment, the PFKFB4 inhibitor, glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined below.

[0014] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising

[0015] (a) PFKFB4 inhibitors;

[0016] (b) glutamine metabolism inhibitors; and

[0017] (c) a pharmaceutically acceptable carrier.

[0018] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the PFKFB4 inhibitor to the glutamine metabolism inhibitor is 0.001-1000:1000-0.001.

[0019] In another preferred embodiment, the total mass of the PFKFB4 inhibitor accounts for 0.1 to 99 wt % of the pharmaceutical composition.

[0020] In another preferred embodiment, the PFKFB4 inhibitor and the glutamine metabolism inhibitor are as defined below.

[0021] In a fourth aspect of the present invention, there is provided a use of a PFKFB4 inhibitor in the preparation of (a) a sensitizer for anti-glioblastoma chemotherapeutic drugs or (b) a drug for reducing drug resistance to anti-glioblastoma chemotherapeutic drugs.

[0022] In another preferred embodiment, the chemotherapy drug is an alkylating agent.

[0023] In another preferred embodiment, the alkylating agent includes: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof.

[0024] In another preferred embodiment, the chemotherapy drug is temozolomide (TMZ).

[0025] In another preferred embodiment, the PFKFB4 inhibitor is a selective inhibitor of PFKFB4.

[0026] In another preferred embodiment, the PFKFB4 inhibitor includes: a small molecule inhibitor, an antibody, a natural product, a lentiviral plasmid (such as a lentiviral plasmid transfected with sh-PFKFB4).

[0027] In another preferred embodiment, the PFKFB4 inhibitor includes: 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN), Bruceine A, or a combination thereof.

[0028] In another preferred embodiment, the glioblastoma is drug-resistant or non-drug-resistant glioblastoma.

[0029] In another preferred embodiment, the drug resistance refers to resistance to chemotherapeutic drugs, and the chemotherapeutic drugs are as defined above.

[0030] In another preferred embodiment, the glioblastoma is a PFKFB4-highly expressed glioblastoma.

[0031] In another preferred embodiment, in PFKFB4 high-expression glioblastoma, the ratio (A1 / A0) of the expression level of PFKFB4 in the glioblastoma (A1) to the expression level (A0) in normal tissues or cells is ≥1.5.

[0032] In another preferred embodiment, the expression level refers to the expression level of PFKFB4 protein.

[0033] In another preferred embodiment, the expression level refers to the mRNA level of PFKFB4.

[0034] In another preferred embodiment, the PFKFB4 inhibitor increases the sensitivity of chemotherapy drugs against glioblastoma by inhibiting glutamine metabolism.

[0035] In another preferred embodiment, glutamine metabolism is represented by the glutamine metabolic rate GLS / GLUL, wherein GLS represents kidney-type glutaminase GLS1 or liver-type glutaminase GLS2, and GLUL represents glutamine synthetase.

[0036] In another preferred embodiment, the glioblastoma is a glioblastoma with elevated glutamine metabolism.

[0037] In another preferred embodiment, the ratio (B1 / B0) of the metabolic rate of glutamine in glioblastoma with elevated glutamine metabolism to the metabolic rate of glutamine in normal tissues or cells (B0) is ≥2 (preferably, ≥3; more preferably, ≥8), wherein the metabolic rate refers to the ratio of GLS1 / GLUL.

[0038] In another preferred embodiment, the ratio (C1 / C0) of the metabolic rate of glutamine in glioblastoma with elevated glutamine metabolism to the metabolic rate of glutamine in normal tissues or cells is ≥60 (preferably ≥80; more preferably, ≥180), wherein the metabolic rate refers to the ratio of GLS2 / GLUL.

[0039] In another preferred embodiment, the glioblastoma is a glioblastoma with high expression of PFKFB4 and elevated glutamine metabolism.

[0040] In a fifth aspect of the present invention, a combination of a PFKFB4 inhibitor and a chemotherapeutic drug is provided; wherein the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0041] In another preferred embodiment, the PFKFB4 inhibitor and the chemotherapeutic drug may be in separate preparations, or in the same preparation.

[0042] In another preferred embodiment, the PFKFB4 inhibitor and the chemotherapeutic drug can be administered simultaneously, sequentially, or at intervals.

[0043] In another preferred embodiment, the molar ratio of the PFKFB4 inhibitor to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0044] In another preferred embodiment, the PFKFB4 inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0045] In the sixth aspect of the present invention, there is provided a use of a combination of a PFKFB4 inhibitor and a chemotherapeutic drug in the preparation of a drug for treating or preventing glioblastoma; wherein the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0046] In another preferred embodiment, the PFKFB4 inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0047] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0048] (a) PFKFB4 inhibitors;

[0049] (b) anti-glioblastoma chemotherapeutic drugs; and

[0050] (c) a pharmaceutically acceptable carrier.

[0051] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for treating or preventing glioblastoma.

[0052] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the FKFB4 inhibitor to the chemotherapeutic drug is 0.01-1000:1000-0.001.

[0053] In another preferred embodiment, the total weight of the FKFB4 inhibitor and the chemotherapeutic drug accounts for 0.1 to 99 wt % of the pharmaceutical composition.

[0054] In another preferred embodiment, the PFKFB4 inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0055] In an eighth aspect of the present invention, there is provided a use of a glutamine metabolism inhibitor in the preparation of (a) a sensitizer for anti-glioblastoma chemotherapeutic drugs, or (b) a drug for reducing resistance to anti-glioblastoma chemotherapeutic drugs, or (c) for treating or preventing glioblastoma. In another preferred embodiment, the chemotherapeutic drug is an alkylating agent.

[0056] In another preferred embodiment, the alkylating agent includes: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof.

[0057] In another preferred embodiment, the chemotherapy drug is temozolomide (TMZ).

[0058] In another preferred embodiment, the glutamine metabolism inhibitor includes: antibodies, small molecule inhibitors, natural products, and lentiviral plasmids.

[0059] In another preferred embodiment, the glutamine metabolism inhibitor includes: a glutaminase inhibitor, a glutamine synthetase inhibitor, or a combination thereof.

[0060] In another preferred embodiment, the glutamine metabolism inhibitor is a glutaminase inhibitor (GLS inhibitor).

[0061] In another preferred embodiment, the glutaminase inhibitor is a renal glutaminase inhibitor (GLS1 inhibitor), a hepatic glutaminase inhibitor (GLS2 inhibitor), or a renal and hepatic glutaminase inhibitor (GLS1 / GLS2 inhibitor).

[0062] In another preferred embodiment, the glutaminase inhibitors include: CB-839 (Telaglenastat) (CAS: 1439399-58-2), BPTES (CAS: 314045-39-1), DON (CAS: 157-03-9), JHU-083, Compound 27 (CAS: 1832646-52-2), Glutaminase C-IN-1 (CAS: 311795-38-7)), or a combination thereof.

[0063] In another preferred embodiment, the glutamine synthetase inhibitor includes: L-Methionine-DL-sulfoximine (MSX) (CAS: 15985-39-4), JFD01307SC (CAS: 51070-56-5), or a combination thereof.

[0064] In another preferred embodiment, the glioblastoma is drug-resistant or non-drug-resistant glioblastoma.

[0065] In another preferred embodiment, the drug resistance refers to resistance to chemotherapeutic drugs, and the chemotherapeutic drugs are as defined above.

[0066] In another preferred embodiment, the glioblastoma is a PFKFB4 high-expression glioblastoma or a PFKFB4 low-expression or normal-expression glioblastoma.

[0067] In another preferred embodiment, in PFKFB4 low-expression or normal-type glioblastoma, the ratio (A1 / A0) of the expression level of PFKFB4 in the glioblastoma (A1) to the expression level (A0) in normal tissues or cells is ≤1.5.

[0068] In another preferred embodiment, in PFKFB4 high-expression glioblastoma, the ratio (A1 / A0) of the expression level of PFKFB4 in the glioblastoma (A1) to the expression level (A0) in normal tissues or cells is ≥1.5.

[0069] In another preferred embodiment, the expression level refers to the expression level of PFKFB4 protein.

[0070] In another preferred embodiment, the expression level refers to the mRNA level of PFKFB4.

[0071] In another preferred embodiment, the glioblastoma is a glioblastoma with elevated glutamine metabolism.

[0072] In another preferred embodiment, the ratio (B1 / B0) of the metabolic rate of glutamine in glioblastoma with elevated glutamine metabolism to the metabolic rate of glutamine in normal tissues or cells (B0) is ≥2 (preferably, ≥3; more preferably, ≥8), wherein the metabolic rate refers to the ratio of GLS1 / GLUL.

[0073] In another preferred embodiment, the ratio (C1 / C0) of the metabolic rate of glutamine in glioblastoma with elevated glutamine metabolism to the metabolic rate of glutamine in normal tissues or cells is ≥60 (preferably ≥80; more preferably, ≥180), wherein the metabolic rate refers to the ratio of GLS2 / GLUL.

[0074] In the ninth aspect of the present invention, a combination of a glutamine metabolism inhibitor and a chemotherapeutic drug is provided; wherein the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0075] In another preferred embodiment, the glutamine metabolism inhibitor and the chemotherapeutic drug can be in separate preparations, or in the same preparation.

[0076] In another preferred embodiment, the glutamine metabolism inhibitor and the chemotherapeutic drug can be administered simultaneously, sequentially, or at intervals.

[0077] In another preferred embodiment, the molar ratio of the glutamine metabolism inhibitor to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0078] In another preferred embodiment, the molar ratio of the glutamine metabolism inhibitor to the chemotherapeutic drug is 1:0.1-2000; preferably, 1:10-1000, more preferably, 1:500±200, and most preferably, 1:500±100.

[0079] In another preferred embodiment, the glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0080] In the tenth aspect of the present invention, there is provided a use of a combination of a glutamine metabolism inhibitor and a chemotherapeutic drug in the preparation of a drug for treating or preventing glioblastoma; wherein the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0081] In another preferred embodiment, the glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0082] In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0083] (a) Glutamine metabolism inhibitors;

[0084] (b) anti-glioblastoma chemotherapeutic drugs; and

[0085] (c) a pharmaceutically acceptable carrier.

[0086] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for treating or preventing glioblastoma.

[0087] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the glutamine metabolism inhibitor to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0088] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the glutamine metabolism inhibitor to the chemotherapeutic drug is 1:0.1-2000; preferably, 1:10-1000, more preferably, 1:500±200, and most preferably, 1:500±100.

[0089] In another preferred embodiment, the total weight of the glutamine metabolism inhibitor and the chemotherapeutic drug accounts for 0.1 to 99 wt % of the pharmaceutical composition.

[0090] In another preferred embodiment, the glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0091] In the twelfth aspect of the present invention, a combination of an active substance and a chemotherapeutic drug is provided; wherein the active substance is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; and the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0092] In another preferred embodiment, the active substance and the chemotherapeutic drug may be in separate preparations, or in the same preparation.

[0093] In another preferred embodiment, the active substance and the chemotherapeutic drug can be administered simultaneously, sequentially, or intermittently.

[0094] In another preferred embodiment, the molar ratio of the active substance to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0095] In another preferred embodiment, the active substance is a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor (such as the combination described in the first aspect).

[0096] In another preferred embodiment, the PFKFB4 inhibitor, the glutamine metabolism inhibitor, the chemotherapeutic drug and the glioblastoma are as defined above.

[0097] In the thirteenth aspect of the present invention, there is provided a use of a combination of an active substance and a chemotherapeutic drug in the preparation of a drug for treating or preventing glioblastoma; wherein the active substance is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; and the chemotherapeutic drug is an anti-glioblastoma chemotherapeutic drug.

[0098] In another preferred embodiment, the active substance is a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor (such as the combination described in the first aspect).

[0099] In another preferred embodiment, the PFKFB4 inhibitor, glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0100] In a fourteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0101] (a) an active ingredient; wherein the active ingredient is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor;

[0102] (b) anti-glioblastoma chemotherapeutic drugs; and

[0103] (c) a pharmaceutically acceptable carrier.

[0104] In another preferred embodiment, the active ingredient is a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor (such as the combination described in the first aspect).

[0105] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the active ingredient to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0106] In another preferred embodiment, the total weight of the active ingredient and the chemotherapeutic drug accounts for 0.1 to 99 wt % of the pharmaceutical composition.

[0107] In another preferred embodiment, the PFKFB4 inhibitor, glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0108] In a fifteenth aspect of the present invention, a kit is provided, comprising:

[0109] (a) a container containing an active ingredient; wherein the active ingredient is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; and

[0110] (b) Instructions or labels.

[0111] In another preferred embodiment, the instructions or label indicate that the kit is used for (a) treating or preventing glioblastoma, or (b) increasing the sensitivity of chemotherapeutic drugs against glioblastoma, or (c) reducing the resistance of chemotherapeutic drugs against glioblastoma.

[0112] In another preferred embodiment, the active ingredient is a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor (such as the combination described in the first aspect).

[0113] In another preferred embodiment, in the pharmaceutical composition, the molar ratio of the active ingredient to the chemotherapeutic drug is 0.001-1000:1000-0.001.

[0114] In another preferred embodiment, the kit further comprises: (c) a detection reagent, wherein the detection reagent is a detection reagent for detecting the expression of PFKFB4, a detection reagent for detecting the expression of PFKFB4, or a combination thereof.

[0115] In another preferred embodiment, the detection reagents for detecting the expression of PFKFB4 include: anti-PFKFB4 antibody, ELISA kit, fluorescent quantitative PCR, and immunohistochemistry.

[0116] In another preferred embodiment, the detection reagents for detecting glutamine metabolism include: detection reagents for magnetic resonance spectroscopy and ELISA.

[0117] In another preferred embodiment, the instructions or label further indicate: the detection reagent is used to detect and determine whether the glioblastoma meets the standard of using the first active ingredient to increase the sensitivity to or reduce the resistance to the chemotherapy drug.

[0118] In another preferred embodiment, the instructions or label further states: the detection reagent is used to detect and determine whether the glioblastoma meets the criteria for treatment or prevention using the first active ingredient.

[0119] In another preferred embodiment, the standard refers to one or more of the following:

[0120] (1) the ratio (A1 / A0) of the expression level of PFKFB4 in the glioblastoma (A1) to the expression level (A0) in normal tissues or cells is ≥1.5;

[0121] (2) the ratio of the metabolic rate of glutamine (B1) in the glioblastoma with elevated glutamine metabolism to the metabolic rate of glutamine (B0) in normal tissues or cells (B1 / B0) is ≥2 (preferably, ≥3; more preferably, ≥8), wherein the metabolic rate refers to the ratio of GLS1 / GLUL;

[0122] (3) The ratio (C1 / C0) of the glutamine metabolic rate (C1) in the glioblastoma with elevated glutamine metabolism to the glutamine metabolic rate (C0) in normal tissues or cells is ≥60 (preferably ≥80; more preferably ≥180), wherein the metabolic rate refers to the ratio of GLS2 / GLUL.

[0123] In another preferred embodiment, the kit further comprises: (d) anti-glioblastoma chemotherapy drugs.

[0124] In another preferred embodiment, the PFKFB4 inhibitor, glutamine metabolism inhibitor, chemotherapeutic drug and glioblastoma are as defined above.

[0125] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 The results show that PFKFB4 induces the proliferation of GBM cells and resistance to TMZ. Among them, 1A: the relative expression level of PFKFB4 in normal tissues and GBM patient tissues in the TCGA database; 1B: the expression level of PFKFB4 mRNA in HAC and U87, U251, and T98G cells; 1C: the expression level of PFKFB4 protein in HAC and U87, U251, and T98G cells; 1D-F: the construction of PFKFB4 knockdown and control U87 stable transgenic cell lines, and the knockdown effect was detected by QPCR and Western Blot. 1G: CCK-8 was used to detect the proliferation and sensitivity to TMZ of sh-control and sh-PFKFB4 U87 cells; 1H: Flow cytometry was used to detect the apoptosis and sensitivity to TMZ of sh-control and sh-PFKFB4 U87 cells. ***P<0.001,**P<0.01,*P<0.05,nosignificance(ns)P≥0.05.

[0127] Figure 2 It shows that PFKFB4 affects glutamine metabolism. Among them, 2A: neurotransmitter metabolomics heat map; 2B: six neurotransmitters with differences in neurotransmitter metabolomics; 2C: transcriptome sequencing Venn diagram; 2D: PFKFB4 resistance-related gene bubble map; 2E: glutamate synaptic gene heat map; ***P<0.001, **P<0.01, *P<0.05, no significance (ns) P≥0.05.

[0128] Figure 3PFKFB4 accelerates glutamine metabolism. 3A: Glutamine was supplemented, and CCK-8 was used to detect cell survival rate; 3B: Glutamine was supplemented, and EDU was used to detect cell proliferation rate; 2C: Glutamate was supplemented, and CCK-8 was used to detect cell survival rate; 3D: Glutamate was supplemented, and EDU was used to detect cell proliferation rate; 3E: Glutamine and glutamate metabolism flow chart; 3F and 3G: sh-control and sh-PFKFB4 U87 cells were cultured with DMSO or TMZ, and the expression levels of GLUL mRNA and protein were detected by QPCR and Western Blot; 3H: sh-control and sh-PFKFB4 U87 cells were cultured with DMSO or TMZ, and the level of GLS1 mRNA was detected by QPCR; 3I: sh-control and sh-PFKFB4 U87 cells were cultured with DMSO or TMZ, and the level of GLS2 mRNA was detected by QPCR; 3J: GLS1:GLUL mRNA ratio; 3K: GLS2:GLUL mRNA ratio. ***P<0.001, **P<0.01, *P<0.05, no significance(ns)P≥0.05.

[0129] Figure 4 The results show that PFKFB4 promotes the proliferation of GBM cells and resistance to TMZ by accelerating glutamine metabolism. 4A, B, and C: GLUL was overexpressed in 293T cells, and its expression was detected by QPCR and Western Blot; 4D and E: GLUL was overexpressed in sh-control and sh-PFKFB4 U87 cells, and its expression was detected by Western Blot; 4F: CCK-8 was used to detect the survival rate of cells after overexpression of GLUL; 4G: CCK-8 was used to detect the survival rate of cells using the GLUL inhibitor MSX; 4H and 4I: EDU was used to detect the cell proliferation rate using the GLUL inhibitor MSX; 4J and 4K: QPCR was used to detect the inhibitory effect of compound 968 on GLS1 and GLS2; 4L: CCK-8 was used to detect the cell survival rate using the GLS1 / GLS2 inhibitor compound 968; 4M and 4N: EDU was used to detect the cell proliferation rate using the GLS1 / GLS2 inhibitor compound 968. ***P<0.001, **P<0.01, *P<0.05, nosignificance(ns)P≥0.05.

[0130] Figure 5It shows that the increase in glutamine metabolism promotes asparagine consumption-induced GBM cell proliferation and TMZ resistance. 5A: Arginine replenishment, CCK-8 detection of cell survival rate and sensitivity to TMZ; 5B: Asparagine replenishment, CCK-8 detection of cell survival rate and sensitivity to TMZ; 5C: QPCR detection of ASNS mRNA level after overexpression of GLUL; 5D: QPCR detection of ASNS mRNA level after compound 968 treatment. ***P<0.001,**P<0.01,*P<0.05,ns P≥0.05

[0131] Figure 6 Schematic diagram showing that PFKFB4 promotes GBM cell proliferation and TMZ resistance by accelerating glutamine metabolism.

[0132] Figure 7 It was shown that knockdown of PFKFB4 combined with compound 968 treatment significantly inhibited GBM growth and TMZ resistance in vivo. 6A: Flowchart of the establishment of transplanted tumor model. 6B: Images of nude mouse tumor tissues in different treatment groups after treatment; 6C: Tumor growth curve of nude mice; 6D: Tumor volume of nude mice after treatment; 6E: Tumor weight of nude mice after treatment. 6F: Immunohistochemical staining of paraffin sections of tumor tissues was performed to detect the expression of Ki67 in tumor tissues. ***P<0.001,**P<0.01,*P<0.05,no significance (ns)P≥0.05.

[0133] Figure 8 The results showed that knockdown of PFKFB4 and combined treatment with compound 968 significantly reduced glutamine metabolism in tumors in vivo. 7A: Glutamine content in tumor tissues was detected by colorimetry. 7B: Glutamate content in tumor tissues was detected by colorimetry. 7C: GLUL protein expression in tumor tissues after TMZ or TMZ combined with compound 968 treatment. 7D: GLS1 mRNA expression in tumor tissues. 7E: GLS2 mRNA expression in tumor tissues. 7F: GLUL mRNA expression in tumor tissues. 7G: GLS1: GLUL mRNA ratio in tumor tissues. 7H: GLS2: GLUL mRNA ratio in tumor tissues. 7I: ASNS mRNA expression in tumor tissues. 7J: HE staining results of nude mouse kidneys. 7K: HE staining results of nude mouse livers. ***P<0.001,**P<0.01,*P<0.05,no significance (ns)P≥0.05. DETAILED DESCRIPTION

[0134] After extensive and in-depth research, the inventor unexpectedly found that the inhibition of PFKFB4 can significantly improve the sensitivity of glioblastoma to chemotherapy drugs such as temozolomide, reduce drug resistance, and significantly improve the therapeutic effect of chemotherapy drugs on glioblastoma through the pathway of glutamine metabolism. In addition, it is also found that the pathway of directly inhibiting glutamine metabolism can also play a similar role. Based on this, the inventor has completed the present invention.

[0135] the term

[0136] Unless otherwise defined, each abbreviation herein has a meaning well known to those skilled in the art.

[0137] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0138] As used herein, a±b refers to the range from ab to a+b (including endpoints), for example, 500±100 refers to the range of ≥400 and ≤600.

[0139] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0140] PFKFB4 pathway and its inhibitors

[0141] 6-phosphofructokinase / fructose-2,6-bisphosphatase (PFK-2 / FBPase-2, PFKFB for short) is widely present in various biological cells. It has bifunctional enzyme activity of kinase and phosphatase, catalyzing the formation and degradation of fructose 2,6-bisphosphate (F-2,6-BP). F-2,6-BP is an allosteric activator of phosphofructokinase-1 and the most effective stimulus for glycolysis. The PFKFB family regulates the level of glycolysis in cells by regulating the expression of F-2,6-BP. So far, four different PFK-2 / FBPase-2 isozymes (PFKFB1, PFKFB2, PFKFB3 and PFKFB4) have been identified.

[0142] The associated kinase and phosphatase activities of PFKFB4 can adapt to the changing needs of the tumor microenvironment. Glucose in tumor cells is mainly utilized through glycolysis or the pentose phosphate pathway, so shunting it into these two pathways to balance anabolism and redox homeostasis is key to ensure unlimited tumor proliferation and escape from apoptosis. Selective inhibition of PFKFB4 expression can reduce F-2,6-BP, glucose uptake and tumor growth in vivo.

[0143] To date, only one study has focused on the design of PFKFB4 inhibitors, which reported that 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN) significantly inhibited PFKFB4 activity and was a selective inhibitor of PFKFB4, inhibiting glycolysis and proliferation of multiple human cancer cell lines in vitro. Subsequent studies have demonstrated that 5MPN can inhibit breast cancer development and myeloma cell growth. Importantly, 5MPN has high oral bioavailability, and oral administration of non-toxic doses of 5MPN to mice can inhibit glucose metabolism and tumor growth.

[0144] Glioma proliferation and drug resistance and tumor metabolic reprogramming

[0145] Any kind of life activity is inseparable from the corresponding material and energy basis. During the rapid development of tumors, in order to meet the energy and nutrient requirements of malignant proliferation, tumor cells will undergo adaptive metabolic changes, so that the flow and flux of nutrients in the metabolic network are redefined. This process is called metabolic reprogramming of tumors. Metabolic reprogramming is a common feature of many tumors, enabling cancer cells to meet their specific growth needs by changing glucose, lipid and amino acid metabolism. In addition to the well-known phenomenon of aerobic glycolysis, there are also cancer-specific metabolic changes, including increased lactate production, increased glutamine metabolism, and reduced fatty acid synthesis and fatty acid oxidation. At the same time, some metabolites inside and outside the cells change, which can have a profound impact on gene expression, cell differentiation and tumor microenvironment. GBM cells have a high metabolic rate, which enables them to adapt to the harsh tumor microenvironment and survive. Therefore, if there is a way to selectively control certain metabolic pathways of GBM cells, it is possible to block their corresponding malignant phenotypes.

[0146] Pharmaceutical compositions and methods of administration

[0147] Since PFKFB4 inhibitors, glutamine metabolism inhibitors or a combination thereof have the effects of significantly increasing the sensitivity of glioblastoma to chemotherapy drugs for treating glioblastoma, reducing the drug resistance of glioblastoma to chemotherapy drugs for treating glioblastoma, and reducing the proliferation of glioblastoma cells, PFKFB4 inhibitors, glutamine metabolism inhibitors or a combination thereof, and pharmaceutical compositions or drugs having PFKFB4 inhibitors, glutamine metabolism inhibitors or a combination thereof as main active ingredients can be used to treat, prevent and alleviate glioblastoma, as well as for adjuvant treatment of glioblastoma (such as in combination with existing chemotherapy drugs such as alkylating agents).

[0148] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0149] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0150] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0151] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0152] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0153] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0154] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0155] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0156] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0157] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0158] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0159] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0160] Combination therapy

[0161] In some aspects, the active substances provided herein (such as PFKFB4 inhibitors, glutamine metabolism inhibitors or combinations thereof) are used to treat or adjuvantly treat glioblastoma in subjects in need thereof. When used to adjuvantly treat glioblastoma, the method for treating glioblastoma further comprises administering an anti-glioblastoma chemotherapeutic drug.

[0162] In some embodiments, the active substances described herein are administered in combination with the chemotherapeutic drug.

[0163] The active substances provided herein can be administered sequentially or alternately with chemotherapeutic drugs. In alternating or sequential step treatments, the effective dose of each agent is administered continuously or sequentially. The dose administered depends on the absorption, inactivation and excretion rate of the drug and other factors known to those skilled in the art. For any particular object, a specific dosage regimen and schedule should be adjusted over time according to individual needs and the professional judgment of the person who manages or supervises the administration of the composition.

[0164] In some embodiments, provided herein is a dose of a chemotherapeutic agent used in a combination therapy. In some embodiments, the dose of a chemotherapeutic agent used in a combination therapy provided herein is lower than the dose that has been or is currently used for glioblastoma. The recommended dose of the second agent can be obtained from the knowledge of those skilled in the art.

[0165] In some embodiments, the active substances provided herein and the chemotherapeutic drugs are administered simultaneously. In some embodiments, the active substances provided herein and the chemotherapeutic drugs are administered at intervals of about 2 to 4 days, about 4 to 6 days, about 1 week, about 1 to 2 weeks, or more than 2 weeks.

[0166] In some embodiments, the active substance provided herein and the chemotherapeutic drug are administered to the patient in sequence and at intervals, and in some embodiments, are administered to mammals, such as humans, so that the active substance provided herein can act together with the chemotherapeutic drug to provide an increased benefit than when administered in other ways. In some embodiments, the chemotherapeutic drug can be administered simultaneously or in any order in sequence at different times in a timely manner; however, if not administered simultaneously, they should be administered close enough in time to provide the desired treatment or preventive effect. In some embodiments, the active substance provided herein and the chemotherapeutic drug play their role at overlapping times. Each active substance and chemotherapeutic drug can be administered separately in any appropriate form and by any appropriate route. In some embodiments, the active substance provided herein is administered before, at the same time, or after the administration of the chemotherapeutic drug.

[0167] The main advantages of the present invention include:

[0168] (a) The present application found that inhibition of PFKFB4 can significantly increase the sensitivity of glioblastoma to chemotherapeutic drugs such as temozolomide and reduce drug resistance.

[0169] (b) The present application found that inhibition of PFKFB4 can significantly reduce the ratio of GLS1 / GLS2 to GLUL and reduce glutamine metabolism, thereby reducing the proliferation and drug resistance of glioblastoma and increasing the sensitivity of the compound to chemotherapeutic drugs.

[0170] (c) The present application discovered that inhibition of glutamine metabolism may also significantly reduce the proliferation and drug resistance of glioblastoma and increase the sensitivity of the compound to chemotherapeutic drugs.

[0171] (d) In vivo studies in the present application found that simultaneous inhibition of PFKFB4 and glutamine metabolism can significantly enhance sensitivity to chemotherapeutic drugs such as alkylating agents, significantly inhibit tumors, and even cause tumor regression.

[0172] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0173] I. Experimental Methods

[0174] Method 1. CCK-8 assay for cytotoxicity or cell viability

[0175] The experiment selected sh-control U87 and sh-PFKFB4 U87 cell lines obtained by stable lentiviral transfection in the early stage (the lentiviral plasmid used was pGMLV-SC5, commercially available), and used 89% MEM basal medium with 10% fetal bovine serum and 1% penicillin-streptomycin, and 0.25μg / ml puromycin to select stable transfected cells. The main steps are as follows: 2000 cells / well were planted in a 96-well plate, and cell culture medium containing 1mM DMSO or 1mM TMZ was added for culture, and 100μl cell culture medium was used as a blank control. In the amino acid replenishment experiment, glutamine (1mM), glutamic acid (1mM), asparagine (1mM), and arginine (1mM) were added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ; GLUL inhibitor MSX (1mM) was added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ; GLS1 / GLS2 inhibitor compound 968 (2μM) was added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ. After 48h, the original culture medium was removed, and 100μl of basal culture medium containing CCK-8 detection solution was added. After culturing in the incubator for 2h, the absorbance value of each well was detected by an enzyme reader (wavelength of 450nm), and the value was recorded. Cell survival rate % = (OD value of drug-added cells-OD value of blank wells) / (OD value of control cells-OD value of blank wells) × 100%.

[0176] Compound 968 (also known as Glutaminase C-IN-1) is an allosteric inhibitor of GLS. Its molecular formula is: 27 H 27 BrN2O, the structure is as follows:

[0177]

[0178] Method 2. Flow cytometry to detect cell apoptosis

[0179] AnnexinⅤ-PE / 7-AAD cell apoptosis detection kit (Vazyme) was used for flow cytometry apoptosis experiment. sh-control U87 and sh-PFKFB4 U87 cells were selected for the experiment. The main steps are as follows: 1×10 cells were seeded in a 6-well plate 51mM DMSO or 1mM TMZ was added to each well and cultured. After 48 hours, the cell culture medium was collected into a clean centrifuge tube, the cells were washed with PBS, the cells were digested with trypsin, and then the cells were resuspended in PBS and counted. 100μl 1×BindingBuffer was added to the cells, and then 5μl Annexin V-PE and 5μl 7-AAD Staining Solution were added and mixed thoroughly. Incubate at room temperature for 10 minutes in the dark. Detect the samples as soon as possible using a flow cytometer, and select the software FlowJo to analyze the experimental results.

[0180] Method 3. EdU detection of cell proliferation

[0181] According to the instructions of the Bio-Tech EDU-555 Cell Proliferation Detection Kit, sh-control U87 and sh-PFKFB4 U87 cells were selected for the experiment. The cell seeding density in the 6-well plate was 1×10 5 / well, amino acid replenishment experiment, glutamine (1mM) and glutamate (1mM) were added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ; GLUL inhibitor MSX (1mM) was added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ; GLS1 / GLS2 inhibitor compound968 (10μM) was added to the basal medium without glutamine containing 1mM DMSO or 1mM TMZ. After 48h, the EDU working solution of the corresponding concentration was prepared and added according to the instructions and incubated for three hours. After EDU labeling, 4% paraformaldehyde was fixed, permeabilized with 1ml permeabilization solution, and the corresponding EDU detection solution was prepared and added according to the instructions. After incubation in the dark for 30 minutes, Hoechst 33342 was used to stain the cell nucleus, and EDU stained cells were detected by fluorescence microscopy.

[0182] Method 4. Transcriptome Sequencing

[0183] The sh-control U87 and sh-PFKFB4 U87 cells were selected for transcriptome sequencing. The number of cells in each sample was kept at 5×10 6 The above cells were cultured in cell culture medium containing 1mM DMSO or 1mM TMZ, and divided into four groups: sh-control U87+DMSO, sh-control U87+TMZ, sh-PFKFB4U87+DMSO, sh-PFKFB4 U87+TMZ, with five replicates in each group. After 48h of culture, the cells were washed with PBS and an appropriate amount of Trizol lysis buffer was added and mixed. The subsequent high-throughput sequencing work of this experiment was completed by Biomarker Biotechnology Co., Ltd.

[0184] Method 5. Neurotransmitter metabolome analysis

[0185] The sh-control U87 and sh-PFKFB4 U87 cells were selected for neurotransmitter metabolome analysis. The number of cells in each sample was ensured to be 5×10 6 The above was divided into two groups: sh-control U87 and sh-PFKFB4U87, with three replicates in each group. After the cells reached the appropriate number, they were washed with PBS and an appropriate amount of Trizol lysis buffer was added and mixed. The subsequent high-throughput sequencing work of this experiment was completed by Shanghai Aqu Biotechnology Co., Ltd.

[0186] Method 6: Animal Experiment Methods

[0187] This study used Balb / c nude five-week-old male mice (16-20 g), which were purchased from Beijing Weitonglihua Technology Co., Ltd. This animal experiment has passed the review standards of the Experimental Animal Welfare Ethics Review Committee of the Second Affiliated Hospital of Kunming Medical University.

[0188] (1) Thirty 5-6-week-old Balb / c nude mice weighing 16-18 g were randomly divided into 6 groups, with 5 mice in each group;

[0189] (2) Take stably transfected cell lines (sh-control and sh-PFKFB4 U87 cell lines) in the logarithmic growth phase, preferably with a confluence of 90%. Digest the cells with trypsin containing 0.25% EDTA and then wash them twice with pre-cooled PBS to remove residual serum in the cells.

[0190] (3) PBS was blown to the cell pellet to an appropriate concentration. The number of cells inoculated into the subcutaneous tumor was about 3×10 6 cells / mouse, the inoculation volume was 100 μl.

[0191] (4) After the cells are digested, they should be inoculated subcutaneously into nude mice as soon as possible. The implantation site should be the middle and posterior region of the axilla, which has a rich blood supply. During the process, the cell suspension should be placed on ice to reduce cell metabolism and thus maintain cell activity as much as possible.

[0192] (5) Raise nude mice in an SPF animal room and monitor their growth status and the growth of implanted tumors. When the transplanted tumor grows to be visible to the naked eye, measure it with a vernier caliper every other day according to the formula V = 1 / 2 × a × b 2 Calculate the volume of transplanted tumor (a is the long diameter of transplanted tumor, b is the short diameter of transplanted tumor). 3Afterwards, 30 nude mice were divided into six treatment groups, 5 mice in each group, and intraperitoneally injected with PBS+DMSO (control), TMZ (40 mg / kg, dissolved in PBS), and TMZ+compound 968 (100 μg / mouse, dissolved in DMSO). The specific groups were: sh-control U87+PBS+DMSO, sh-PFKFB4 U87+PBS+DMSO, sh-control U87+TMZ+DMSO, sh-PFKFB4 U87+TMZ+DMSO, sh-control U87+TMZ+compound 968, and sh-PFKFB4 U87+TMZ+compound 968. The injections were continued for 10 days, and the mental and physical conditions of the mice were observed at any time. The tumor size and weight of the mice were measured every 2 days. After the treatment, the nude mice were killed after anesthesia and the tumors were collected.

[0193] II. Embodiment

[0194] Example 1: PFKFB4 induces GBM cell proliferation and TMZ resistance.

[0195] In the TCGA database, the expression of PFKFB4 in GBM patient tissues was significantly increased compared with normal tissues ( Figure 1 A); In this application, the expression of PFKFB4 mRNA and protein in normal human astrocytes HAC and human glioma cell lines U87, U251, and T98G was compared, and it was found that the expression of PFKFB4 in glioma cell lines was significantly higher than that in HAC ( Figure 1 B and C).

[0196] Next, sh-control U87 and sh-PFKFB4 U87 cell lines were obtained by stable transfection with lentivirus ( Figure 1 DF); CCK-8 and flow cytometry results showed that after knocking down PFKFB4, cell proliferation and resistance to TMZ were significantly reduced ( Figure 1 G and H).

[0197] The above results indicate that PFKFB4 induces the proliferation of GBM cells and resistance to TMZ, and knocking down PFKFB4 can reduce resistance to alkylating agents (such as TMZ).

[0198] Example 2: PFKFB4 affects glutamine metabolism.

[0199] In recent years, studies have found that neurotransmitters have become important microenvironmental components that regulate tissue homeostasis and affect various malignant phenotypes of human cancer. Many neurotransmitters and / or their analogs or antagonists / agonists of their receptors have medicinal properties and can be used as drugs for various diseases including cancer. PFKFB4, as an important regulator in the glycolysis pathway, can affect amino acid metabolism by affecting glycolysis. Among them, certain amino acids, as neurotransmitters, are important microenvironmental components that can affect the malignancy of human cancer. Next, this application investigated whether PFKFB4 would affect the proliferation and sensitivity to TMZ of GBM by affecting the content of neurotransmitters. In this application, neurotransmitter metabolomics was performed on sh-control and sh-PFKFB4 U87 cells, and the heat map is shown in the figure. Figure 2 A. Among them, six neurotransmitters showed significant differences, including glutamine (Gln), asparagine (Asn), arginine (Arg), ornithine (Orn), kynurenine (Kyn), methionine (Met) ( Figure 2 B). To further explore which neurotransmitter plays a major role, sh-control and sh-PFKFB4 U87 cells were treated with DMSO or TMZ, respectively, and transcriptome sequencing was performed. The results showed that glutamate synapse-related genes were highly correlated with TMZ resistance ( Figure 3 D and 3E).

[0200] Example 3: PFKFB4 accelerates glutamine metabolism.

[0201] Glutamine is the major precursor for the synthesis of glutamate, a key excitatory neurotransmitter and the most abundant amino acid in human plasma.

[0202] In order to further clarify the effect of PFKFB4 on glutamine and glutamate, we first supplemented glutamine and glutamate in tumor cells. The results showed that after supplementing glutamine, the cell proliferation rate and resistance to TMZ increased significantly, even higher than the control group ( Figure 3 A and B). After glutamate replenishment, cell proliferation rate and resistance to TMZ also increased significantly ( Figure 3 C and D). Therefore, the inventors believe that glutamine and glutamate may be one of the important reasons why PFKFB4 causes the malignant proliferation and drug resistance of glioma.

[0203] This application also studies how glutamine and glutamate affect the proliferation and drug resistance of GBM. Glutamine is decomposed into glutamate and ammonium ions under the catalysis of glutaminase (GLS). Conversely, glutamate can be resynthesized into glutamine under the catalysis of glutamine synthetase (GLUL). Figure 3 E).

[0204] Next, the inventors detected the changes in GLUL and GLS mRNA and found that compared with the control group sh-controlU87, the expression of GLUL in sh-PFKFB4 U87 cells was significantly increased, but there was no significant difference under TMZ culture conditions ( Figure 3 F and G).

[0205] At the same time, the changes of GLS were detected in this application. GLS is divided into two types: renal GLS (GLS1) and hepatic GLS (GLS2). GLS1 is expressed in a variety of tissue cells, especially in cells with active metabolism and high glutamine demand, while GLS2 is mainly expressed in liver tissues and has changes in expression under certain pathological conditions (such as some neurodegenerative diseases). After knocking down PFKFB4, there was no significant difference between GLS1 and GLS2; however, when cultured with TMZ, the expression of GLS1 and GLS2 in sh-PFKFB4 U87 cells was significantly increased, especially the GLS2 difference, which was as high as more than 200 times ( Figure 3 H and I). At the same time, the ratio analysis of glutamine metabolism rate, namely GLS1 / GLUL and GLS2 / GLUL, was performed. The results showed that the ratios of GLS1:GLUL and GLS2:GLUL were significantly reduced after knocking down PFKFB4, regardless of the presence or absence of TMZ ( Figure 3 J and K), suggesting that increased glutamine metabolism may mediate PFKFB4 regulation of glioblastoma malignant proliferation and TMZ resistance.

[0206] Example 4: PFKFB4 promotes GBM cell proliferation and TMZ resistance by accelerating glutamine metabolism.

[0207] To further verify the correlation between glutamine metabolism and its related genes and PFKFB4-mediated GBM proliferation and TMZ resistance, we constructed a GLUL overexpression plasmid. First, the overexpression of the plasmid was verified in 293T cells ( Figure 4 AC). Next, it was transfected into sh-control and sh-PFKFB4 U87 cells, and it was verified that after overexpression (OE) of GLUL, the levels of GLUL mRNA and protein increased significantly ( Figure 4 D and E).

[0208] In addition, it was found that overexpression of GLUL significantly reduced cell survival, and overexpression of GLUL while knocking down PFKFB4 resulted in the highest sensitivity of cells to TMZ ( Figure 4 F).

[0209] The present application further used the GLUL inhibitor MSX, and the results showed that compared with the control group, inhibition of GLUL reversed the decrease in cell proliferation and drug resistance caused by knockdown of PFKFB4 ( Figure 4 GI).

[0210] Next, the present application used compound 968, an inhibitor of GLS1 and GLS2, and QPCR detected its inhibitory effect on GLS1 / GLS2 ( Figure 4 J and K). Compound 968 significantly enhanced the inhibitory effect of TMZ on cells after treatment. Figure 4 LN). Therefore, high expression of PFKFB4 promoted the proliferation of GBM cells and resistance to TMZ by accelerating glutamine metabolism.

[0211] Example 5: Increased glutamine metabolism induces GBM cell proliferation and resistance to TMZ by promoting asparagine consumption.

[0212] The carbon in glutamine, in the form of α-ketoglutarate, is an important substrate for supporting the biosynthetic function of the tricarboxylic acid cycle, while the nitrogen derived from glutamine is required for the biosynthesis of molecules such as hexosamine, nucleotides, and other non-essential amino acids. Figure 2 In neurotransmitter metabolomics, the inventors found significant differences between asparagine and arginine ( Figure 2 B). Glutamate is converted to ornithine through transamination, and then to arginine through the urea cycle; glutamine and aspartate are synthesized into asparagine and glutamate under the catalysis of asparagine synthetase (ASNS). In order to further verify which amino acid downstream of the changes in glutamine and glutamate mediated by PFKFB4 affects the malignant proliferation and drug resistance of GBM, the inventors first supplemented arginine, and the results showed that there was no significant change in cell proliferation and resistance to TMZ ( Figure 5 A). Next, asparagine was supplemented, and it was found that similar to glutamine supplementation, asparagine supplementation significantly reversed the reduction in cell proliferation and TMZ resistance caused by knockdown of PFKFB4 ( Figure 5 B). The expression of ASNS was significantly increased after overexpression of GLUL in cells, and the expression of ASNS was significantly reduced after compound 968 inhibited GLS1 and GLS2 ( Figure 5 C and D).

[0213] The above results indicate that the increase in glutamine metabolism further promotes the utilization of asparagine, thereby inducing the malignant proliferation of GBM cells and resistance to TMZ.

[0214] Example 6: Knockdown of PFKFB4 combined with compound 968 treatment significantly inhibited GBM growth and resistance to TMZ in vivo.

[0215] In order to further study the effect of PFKFB4 on GBM in vivo, the present application conducted a subcutaneous tumor formation experiment in nude mice. In this study, sh-control and sh-PFKFB4 U87 cells were injected into 15 nude mice subcutaneously. When the subcutaneous tumors of nude mice injected with sh-control U87 cells reached about 500mm 3 Afterwards (day 12), the nude mice were treated with control (PBS+DMSO), TMZ, and combined treatment (TMZ+compound 968). They were divided into six groups: sh-control U87+PBS+DMSO, sh-PFKFB4 U87+PBS+DMSO, sh-control U87+TMZ+DMSO, sh-PFKFB4 U87+TMZ+DMSO, sh-controlU87+TMZ+compound 968, and sh-PFKFB4 U87+TMZ+compound 968, with five mice in each group. They were treated with intraperitoneal injection for a total of ten days. After the treatment, the tumor tissues were collected ( Figure 6 A). Weigh, measure and photograph the tissue ( Figure 6 B), Tumor tissues and visceral tissues were fixed or snap-frozen according to subsequent experiments.

[0216] By drawing the tumor growth curve, the applicant found that the subcutaneous tumor growth of the sh-PFKFB4 U87 group was significantly lower than that of the sh-control U87 group. After about 4 days of treatment with TMZ or TMZ combined with compound 968, the tumor growth of both groups was significantly inhibited, and the tumor growth of the sh-PFKFB4 U87+TMZ+compound 968 group was the slowest, and some mice showed tumor regression ( Figure 6 C). The tumors were measured after sampling, and the results showed that the tumor volume in the sh-control U87 group was significantly larger than that in the sh-PFKFB4 U87 group. After TMZ treatment, the tumor size was significantly reduced, and the combination of the two drugs further significantly reduced the tumor volume. After knocking down PFKFB4, the tumor volume was the smallest when TMZ and compound 968 were combined for treatment ( Figure 6 D). In addition, we also measured the tumor weight and obtained similar results as the tumor volume ( Figure 6 E).

[0217] To further explore the effects of TMZ alone and in combination with compound 968 on tumor proliferation in mice, immunohistochemical staining was performed on nude mouse tumor tissues to evaluate the Ki67 staining positivity in tumor sections. The results showed that tumor sections with high PFKFB4 expression showed significantly higher Ki67 expression than those with low PFKFB4 expression, indicating that the increase in glutamine metabolism induced by PFKFB4 promoted tumor proliferation. Importantly, the Ki67 positivity rate was lowest after combined treatment with TMZ and compound 968 in tumors with low PFKFB4 expression, which further illustrates that simultaneous inhibition of PFKFB4 and glutamine metabolism increases the sensitivity of tumor cells to TMZ ( Figure 6 F). Therefore, the above results suggest that knockdown of PFKFB4 combined with compound968 treatment significantly inhibits GBM growth and resistance to TMZ in vivo.

[0218] Example 7: The combined treatment of PFKFB4 knockdown and compound 968 significantly reduced glutamine metabolism in tumors in vivo.

[0219] In order to further verify whether the changes in glutamine metabolism in tumor tissues in vivo are consistent with in vitro experiments. This study used amino acid colorimetry to detect glutamine and glutamate in tumor tissues. The results showed that the glutamine content in tumor tissues with PFKFB4 knockdown was significantly increased, indicating that high expression of PFKFB4 promotes glutamine metabolism ( Figure 7 A). After TMZ and compound 968 treatment of mice transplanted with PFKFB4 knockdown U87 cells, glutamine content was shown to be the highest, further proving the inhibitory effect of compound 968 on glutamine metabolism and preventing the metabolism of glutamine to glutamate. Next, the detection of glutamate found that the glutamate content in the tumor tissue with PFKFB4 knockdown was significantly reduced in the untreated group and the TMZ alone treatment group ( Figure 7 B), but the expression level of PFKFB4 did not affect the glutamate content in the combined treatment group.

[0220] Next, this study took tumor tissues to detect the protein and mRNA levels of glutamine metabolism genes. Western blot was used to detect the changes in GLUL protein in tumor tissues after TMZ alone or combined with compound 968 treatment. The results showed that the expression of GLUL was significantly increased after compound 968 was given, regardless of whether PFKFB4 was highly expressed or lowly expressed in tumors ( Figure 7C). After RNA was extracted from tumor tissues, RT-qPCR was used to detect changes in GLS1, GLS2, and GLUL mRNA. It was found that there was no significant difference in the expression of GLS1 and GLS2 in the untreated group. After TMZ treatment, the expression of GLS1 and GLS2 was significantly reduced. The combined treatment of the two drugs further reduced the expression of GLS1, while there was no significant difference in the expression of GLS2. This further proves the inhibitory effect of compound 968 on GLS1 and GLS2 in tumor tissues in vivo, and the inhibitory effect on GLS1 is stronger ( Figure 7 D and E). Similar to the protein level, the GLUL mRNA level was significantly increased after compound 968 administration, indicating that compound 968 not only changed glutamine metabolism by inhibiting GLS, but also further caused changes in the expression of GLUL ( Figure 7 F).

[0221] Finally, we analyzed the GLS1:GLUL and GLS2:GLUL ratios and found that knockdown of PFKFB4 significantly reduced the glutamine metabolism rate and treatment with compound 968 effectively inhibited glutamine metabolism, which is consistent with our previous cell experiment results ( Figure 7 G and H).

[0222] To further verify the effect of glutamine changes on asparagine synthesis in vivo, the expression level of ASNS mRNA in tumor tissues was detected. The results showed that the expression level of ASNS mRNA was similar to the level of GLS2:GLUL ratio ( Figure 7 I). This result suggests that, in vivo, glutamine metabolism further affects asparagine metabolism to promote GBM proliferation and drug resistance.

[0223] Compound 968 is an allosteric inhibitor of GLS. GLS1 is mainly expressed in the kidney, while GLS2 is mainly expressed in the liver. In order to explore whether the compound 968 treatment regimen would have toxic side effects on nude mice, this study used HE staining to detect the kidney and liver tissues of nude mice. The HE staining results showed that the kidneys and livers of nude mice did not show obvious toxic reactions regardless of whether they received compound 968 treatment or not ( Figure 7 J and K). Therefore, the present invention infers that intraperitoneal injection of compound 968 (100 mg / kg) can increase the sensitivity of nude mouse GBM to TMZ treatment, and the drug combination has no toxic side effects on normal tissues.

[0224] Therefore, it can be seen that GBM cells with high expression of PFKFB4 can increase glutamine metabolism by increasing the ratio of GLS1 / GLS2 to GLUL. The increase in glutamine metabolism further accelerates the consumption of asparagine and promotes the malignant proliferation of GBM cells and resistance to TMZ (e.g. Figure 8 shown).

[0225] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor.

2. Use of a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor in the preparation of (a) a drug for treating or preventing glioblastoma, or (b) a sensitizer for anti-glioblastoma chemotherapeutic drugs, or (c) a drug for reducing resistance to anti-glioblastoma chemotherapeutic drugs (preferably, the chemotherapeutic drug is an alkylating agent; more preferably, the alkylating agent is selected from the group consisting of temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; most preferably, the chemotherapeutic drug is temozolomide (TMZ)).

3. Use of a PFKFB4 inhibitor in the preparation of (a) a sensitizer for anti-glioblastoma chemotherapeutic drugs or (b) a drug for reducing drug resistance to anti-glioblastoma chemotherapeutic drugs.

4. The use according to claim 3, characterized in that The chemotherapy drug is an alkylating agent; preferably, the alkylating agent is selected from the following group: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; more preferably, the chemotherapy drug is temozolomide (TMZ).

5. Use of a glutamine metabolism inhibitor in the preparation of (a) a sensitizer for anti-glioblastoma chemotherapeutic drugs, or (b) a drug for reducing the drug resistance of anti-glioblastoma chemotherapeutic drugs, or (c) a drug for treating or preventing glioblastoma.

6. The use according to claim 5, characterized in that The chemotherapy drug is an alkylating agent; preferably, the alkylating agent is selected from the following group: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; more preferably, the chemotherapy drug is temozolomide (TMZ).

7. A combination of an active substance and a chemotherapeutic drug; wherein: The active substance is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; the chemotherapy drug is an anti-glioblastoma chemotherapy drug (preferably, the chemotherapy drug is an alkylating agent; more preferably, the alkylating agent is selected from the following group: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; most preferably, the chemotherapy drug is temozolomide (TMZ)).

8. Use of a combination of an active substance and a chemotherapeutic drug in the preparation of a drug for treating or preventing glioblastoma; wherein: The active substance is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; the chemotherapy drug is an anti-glioblastoma chemotherapy drug (preferably, the chemotherapy drug is an alkylating agent; more preferably, the alkylating agent is selected from the following group: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; most preferably, the chemotherapy drug is temozolomide (TMZ)).

9. A pharmaceutical composition comprising: (a) an active ingredient; wherein the active ingredient is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; (b) anti-glioblastoma chemotherapeutic drugs (preferably, the chemotherapeutic drug is an alkylating agent; more preferably, the alkylating agent is selected from the following group: temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; most preferably, the chemotherapeutic drug is temozolomide (TMZ)). ; and (c) a pharmaceutically acceptable carrier.

10. A kit comprising: (a) a container containing an active ingredient; wherein the active ingredient is a PFKFB4 inhibitor, a glutamine metabolism inhibitor, or a combination of a PFKFB4 inhibitor and a glutamine metabolism inhibitor; (b) instructions or labels; (c) an optional detection reagent, which is a detection reagent for detecting expression of PFKFB4, a detection reagent for detecting expression of PFKFB4, or a combination thereof; and (d) Optional anti-glioblastoma chemotherapeutic drug (preferably, the chemotherapeutic drug is an alkylating agent; more preferably, the alkylating agent is selected from the group consisting of temozolomide (TMZ), carmustine, nimustine, lomustine or a combination thereof; most preferably, the chemotherapeutic drug is temozolomide (TMZ)).

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