CN115531546B

CN115531546B - Combined antimetabolite for treating high-grade glioma and preparation method thereof

Info

Publication number: CN115531546B
Application number: CN202211036071.8A
Authority: CN
Inventors: 李腾飞
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-08-27
Filing date: 2022-08-27
Publication date: 2024-02-27
Anticipated expiration: 2042-08-27
Also published as: CN115531546A

Abstract

The invention relates to a combined antimetabolite for treating high-grade glioma, which comprises the following raw material components of glycolytic inhibitor, lactic acid metabolism inhibitor, mitochondrial oxidative phosphorylation key enzyme inhibitor and mitochondrial scavenger, wherein four different mechanisms of the combined glycolytic inhibitor, lactic acid metabolism inhibitor, mitochondrial oxidative phosphorylation inhibitor and mitochondrial scavenger are used for acting, so that the comprehensive inhibition of the main energy supply mode glycometabolism pathway of the high-grade glioma tumor cells is realized, and better effect of killing the tumor cells is achieved in a synergistic way. The final combined antimetabolite scheme can be used for treating high-grade glioma, and effectively kills tumor cells and reduces drug resistance through multiple inhibition of tumor specific cell glycometabolism targets, so that the problems that the antimetabolite in the prior art is easy to cause obvious metabolic reprogramming, compensation and the like in the tumor treatment process are effectively solved.

Description

Combined antimetabolite for treating high-grade glioma and preparation method thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a combined antimetabolite capable of targeting tumor glycolysis and mitochondrial metabolism and treating high-grade glioma and a preparation method thereof.

Background

Glioblastoma (GBM) is the most common incurable, highly invasive malignancy of the central nervous system, accounting for 14.3% of all primary CNS tumors and 49.2% of all CNS primary malignancies. Although some progress has been made in the treatment modality, and comprehensive treatment specifications such as a Stupp scheme and electric field treatment are established, the heterogeneity of tumors limits the effectiveness of various treatment modalities, and the resistance of tumors to chemotherapy and radiotherapy makes recurrence unavoidable, resulting in poor prognosis of tumor patients, with annual survival rates of about 40.9% and five-year survival rates of about 6.6%. The current treatment of high-grade glioma comprises safe excision of the maximum range of the tumor and temozolomide synchronous chemoradiotherapy.

Genomics research brings about revolutionary changes in the pathogenesis of tumors and targeted therapies, however only a few tumor patients with certain specific mutations may benefit from targeted therapies. The current knowledge of glioma heterogeneity has made significant progress, but no new drug has been demonstrated that is clearly effective against gliomas, other than temozolomide. Glioma genomics studies have found that several molecular markers with diagnostic and prognostic evaluations can best explain the complexity of the tumor formation process. Recent studies of glioma heterogeneity using single cell RNA sequencing have yielded many new insights, including genetic heterogeneity, epigenetic heterogeneity, tumor microenvironment heterogeneity. Among them, metabolic reprogramming is an important part of glioma development, so that tumor cells survive severe changes in microenvironment, and show remarkable plasticity in metabolic adaptation.

Isocitrate Dehydrogenase (IDH) mutation is an important early event of glioma occurrence, and has diagnostic and prognostic evaluation values. Currently, inhibitors targeting IDH1 mutations have been approved for the treatment of acute myeloid leukemia, chondrosarcoma, and cholangiocarcinoma, but their clinical efficacy in IDH mutant gliomas is not established. The IDH1-R132H inhibitor AGI-5198 blocks the mutant enzyme from producing 2-hydroxyglutarate (2-HG) in a dose-dependent manner, but when used for glioma treatment, produces contradictory effects with radiotherapy and reverses the killing effect of radiation on tumor cells. 2-deoxyglucose, a glucose analog that can be phosphorylated but cannot be further processed, competitively inhibits hexokinase and exerts a negative feedback effect, has been clinically tested in GBM and prostate cancer, but is dose-limited in GBM due to neurotoxicity. Studies have found that various drugs target mitochondrial metabolism to inhibit GBM, such as nigericin, metformin, phenformin, ivermectin, etc., but none play an expected role in killing tumor cells, where metabolic reprogramming plays an important role. Bevacizumab treatment targeting human Vascular Endothelial Growth Factor (VEGF) can promote glucose uptake and conversion into lactic acid, reduce glucose inflow into tricarboxylic acid (TCA) circulation, and substantial prolongation of total survival time of patients is not achieved in clinical research, and metabolic reprogramming plays an important role in bevacizumab failure in glioma treatment. GBM antimetabolite therapy faces a number of difficulties, and reprogramming of tumor metabolism and metabolic regulation after chemoradiotherapy also present additional challenges for antimetabolite therapy of tumors.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a combined antimetabolite capable of targeting glycolysis of tumors and metabolism of mitochondria and treating high-grade glioma and a preparation method thereof. The combined antimetabolite can be used for treating high-grade glioma, and effectively kills tumor cells and reduces drug resistance through multiple inhibition of tumor specific cell carbohydrate metabolism targets, so that the problems that the antimetabolite in the prior art is easy to cause obvious metabolic reprogramming, compensation and the like in the tumor treatment process are effectively solved.

The technical scheme adopted by the invention is as follows:

a combined antimetabolite for the treatment of high-grade glioma comprises the following raw materials: glycolytic inhibitors, lactate metabolism inhibitors, mitochondrial oxidative phosphorylation key enzyme inhibitors, and mitochondrial scavengers.

Further preferably, the molar ratio of glycolytic inhibitor, lactic acid metabolism inhibitor, mitochondrial oxidative phosphorylation key enzyme inhibitor and mitochondrial scavenger in the raw material components of the combined antimetabolite for treating high-grade glioma is 150:60:10000:15.

the glycolysis inhibitor is one or more of 3-bromopyruvate, 2-deoxy-D-glucose (2-DG) and lonidamine.

The lactic acid metabolism inhibitor is one or more of malonic acid derivative, sodium oxalate and pekinetin.

The key enzyme inhibitor for mitochondrial oxidative phosphorylation is one or more of dichloroacetic acid, TM-1, AZD7545 and PDHK-IN.

The mitochondrial scavenger is one or more of flunarizine.

The preparation method of the combined antimetabolite for treating the high-grade glioma comprises the following steps:

dispersing glycolytic inhibitor and mitochondrial scavenger in organic solvent respectively, dispersing lactic acid metabolism inhibitor and mitochondrial oxidative phosphorylation key enzyme inhibitor in water respectively, and mixing the four solutions uniformly to obtain the combined antimetabolite for treating high-grade glioma.

Dispersing the lactic acid metabolism inhibitor in DMSO to obtain 5-20mM lactic acid metabolism inhibitor stock solution.

Dispersing the mitochondrial scavenger in DMSO to prepare 10-30mM of mitochondrial scavenger stock solution.

Dispersing glycolysis inhibitor in water to obtain 25-100mM glycolysis inhibitor stock solution;

dispersing the mitochondrial oxidative phosphorylation key enzyme inhibitor in water to prepare 100-400mM mitochondrial oxidative phosphorylation key enzyme inhibitor stock solution.

The inventors of the present application have found in long-term studies that 3-bromopyruvate is capable of inhibiting a variety of key glycolytic or related metabolic enzymes, such as inhibiting glycolysis (e.g., HK2, GAPDH, and 3-PGK), mitochondrial OXPHOs (e.g., PDH, SDH, IDH and αKD), PPP (e.g., G6 PDH), and the like, and also selectively inducing cell death by apoptosis or necrosis. Malonic acid derivative AZ-33 can reverse the Warburg effect, leading to an increase in tumor OXPHOS and a mitochondrial mediated reactivation of apoptosis. Dichloroacetic acid (DCA) can activate mitochondria in cancer cells, inhibit glycolysis, restore the functions of mitochondria OXPHOS, reduce the production of intracellular lactic acid and increase a large amount of active oxygen, and promote apoptosis of cancer cells, thereby showing anti-tumor effect on various cancers and having good tolerance to patients. Flunarizine has the effects of inhibiting growth and promoting apoptosis on glioblastoma, and plays an anti-tumor effect in cooperation with temozolomide, 3-bromopyruvate and dichloroacetic acid.

The beneficial effects of the invention are as follows:

the raw material components of the combined antimetabolite for treating the high-grade glioma comprise glycolysis inhibitor, lactic acid metabolism inhibitor, mitochondrial oxidative phosphorylation key enzyme inhibitor and mitochondrial scavenger, and four different mechanisms of combined glycolysis inhibitor, lactic acid metabolism inhibitor, mitochondrial oxidative phosphorylation inhibitor and mitochondrial scavenger are used for acting, so that the main energy supply mode of the high-grade glioma tumor cells is comprehensively inhibited, and better effect of killing the tumor cells is achieved in a synergistic way. The final combined antimetabolite scheme can be used for treating high-grade glioma, and effectively kills tumor cells and reduces drug resistance through multiple inhibition of tumor specific cell glycometabolism targets, so that the problems that the antimetabolite in the prior art is easy to cause obvious metabolic reprogramming, compensation and the like in the tumor treatment process are effectively solved.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of toxicity detection of a monoclonal antibody metabolizing drug to a U87MG cell line;

FIG. 2 shows the results of toxicity detection of a monoclonal antibody metabolism drug against the U251 cell line;

FIG. 3 shows the results of toxicity assays of the combination antimetabolite on U87MG cell lines;

FIG. 4 shows the results of toxicity assays of the combination antimetabolite on the U251 cell line.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

Example 1

The embodiment provides a combined antimetabolite for treating high-grade glioma, which comprises the following raw material components: 3-bromopyruvate (3-BrPA), malonic acid derivative (AZ-33), dichloroacetic acid (DCA) and Flunarizine (FNZ). Wherein, the mol ratio of the 3-bromopyruvate, malonic acid derivative, dichloroacetic acid and flunarizine is 150:60:10000:15.

further, the preparation method of the combined antimetabolite for treating high-grade glioma according to the embodiment comprises the following steps:

3-bromopyruvate 3-BrPA is dispersed in water to prepare a glycolysis inhibitor stock solution with the concentration of 50 mM; dispersing malonic acid derivative AZ-33 in DMSO to prepare 10mM lactic acid metabolism inhibitor stock solution; dispersing dichloroacetic acid DCA in water to prepare 200mM mitochondrial oxidative phosphorylation key enzyme inhibitor stock solution; dispersing flunarizine FNZ in DMSO to prepare 15mM mitochondrial scavenger stock solution;

and finally, uniformly mixing the four stock solutions to obtain the combined antimetabolite for treating the high-grade glioma.

Example 2

This example provides a combination antimetabolite for the treatment of high-grade gliomas, which differs from example 1 only in that: in this example, the stock solution concentrations of 3-bromopyruvate, malonic acid derivative, dichloroacetic acid and flunarizine were 25mM, malonic acid derivative was 5mM, dichloroacetic acid was 100mM and flunarizine was 10mM.

Example 3

This example provides a combination antimetabolite for the treatment of high-grade gliomas, which differs from example 1 only in that: in this example, the stock solution concentrations of 3-bromopyruvate, malonic acid derivative, dichloroacetic acid and flunarizine were 100mM, malonic acid derivative was 20mM, dichloroacetic acid was 400mM, and flunarizine was 30mM.

Comparative example 1

The difference between this comparative example and example 1 is that: the flunarizine is not added, and the method specifically comprises the following steps: 3-bromopyruvate, malonic acid derivative and dichloroacetic acid according to the mole ratio of 150:60: 10000.

Experimental example

The test protocol for in vitro treatment of gliomas with the combination antimetabolites obtained in example 1 and comparative example is as follows:

(1) In vitro culture of glioma cell lines

The invention adopts glioma cell line U87 and U251 as experimental objects. Glioma cell lines were purchased from Shanghai cell banks from the national academy of sciences typical culture Collection and stored in liquid nitrogen. During cell recovery, quickly taking out frozen cell tube from liquid nitrogen, placing into a constant-temperature water bath kettle at 37deg.C, continuously shaking the frozen cell tube to accelerate cell dissolution, quickly transferring the frozen cell tube into high-sugar DMEM medium containing 10% Fetal Bovine Serum (FBS) under aseptic condition after the frozen cell tube is completely melted, centrifuging (800 rpm/4 min) after blowing, discarding supernatant, taking DMEM complete medium to resuspend cells, transferring cell suspension into a culture dish, shaking, transferring cell suspension into 5% CO at 37deg.C ₂ Is cultured in an incubator of (a). The medium was changed every 1/2 day according to the growth conditions, and the growth conditions of the cells, such as state, density, morphology, etc., were observed and recorded daily.

(2) Spreading and adding medicine

a. Respectively digesting and collecting U87 or U251 cells in logarithmic growth phase, diluting with complete culture medium, accurately counting, mixing, and regulating living cell concentration to 3×10 ⁴ Individual/ml;

b. cells were plated in 96-well plates at a cell plating density of about 3000 (100 μl) per well, no cells were added to the edge wells, PBS was added to avoid the effect of edge effects, and incubated in an incubator at 37deg.C. The following day, CCK8 cell viability detection and drug experiments were performed after cell attachment.

(3) CCK8 cell viability assay

The first CCK8 assay time was recorded as 0h and was dosed according to Table 1 (combination group wherein A represents AZ-33, B represents 3-BrPA, D represents DCA, F represents FLN), table 2 (single drug group), followed by 24h to 72h row CCK8 cell viability assay and liquid change dosing every interval.

TABLE 1 composition of the Combined antimetabolite, pharmaceutical use, CCK8 detection time Point

Note that: "-" applies not "+" applies.

TABLE 2 pharmaceutical compositions of the single drug groups and CCK8 detection time points

The specific operation of CCK8 cell viability detection is as follows:

(S1) light-shielding pressing CCK8:10% complete DMEM broth = 1:9, preparing premix (for on-site preparation), sucking the original culture solution, adding 100 μl of premix into each detection hole under the condition of light shielding, taking 3 holes without CCK8 as blank control groups, mixing with a light shaking culture plate, and placing into an incubator;

(S2) after incubation for 90min, the medium added to CCK8 wells was seen to turn yellow in color, the bubbles in the wells were removed, and absorbance values (OD values) were detected using a microplate reader set at a wavelength of 450 nm. The detection process is to avoid pollution, and 75% alcohol is used for wiping until the detection process is dry;

and (S3) preparing a dosing premix, and continuously culturing in an incubator after the liquid change treatment until the detection is completed in 72 h.

(S4) data processing: the measurement minus the blank measurement was used as a measure of cell viability for this group. The experiment was repeated three times, the data was normalized with the first detection value as 1, and proliferation line graphs of each group of cells were plotted with the detection time point as x-axis and the normalized OD value as y-axis (fig. 1, 2). Statistical tests used analysis of variance, with P < 0.05 considered statistical differences.

(4) Description of experimental data

The CCK8 cytotoxicity detection result shows that the single drug antimetabolite treatment has poor therapeutic effect on glioma cell lines. FIGS. 1 and 2 show the toxicity test results of the monoclonal antibody metabolism drugs on the U87MG cell line and the U251 cell line, respectively, and a larger dose is required to exert the effect of killing cells.

The combined antimetabolite therapy has significant toxic effects on glioma cell lines. FIGS. 3 and 4 show the toxicity test results of the combination antimetabolite on the U87MG cell line and the U251 cell line, respectively.

In the U87MG cell line, the ABD and ABDF groups showed clear tumor cytotoxicity at 48h of treatment, cell viability was significantly reduced compared to blank 0 and control DMSO, and at 72h both the ABD and ABDF groups showed clear cytotoxicity (n=3). The ABD group still has obvious proliferation after 24 hours, which indicates that the cells have metabolic reprogramming or function recovery, and the ABDF combination drug administration scheme group has cell activity inhibition, especially almost all cells in the ABDF72 group die.

In the U251 cell line, at 72h of dosing treatment, significant cytotoxicity occurred in the ABDF24 and ABDF72 groups (n=3), indicating that the combined use of ABDF had a more satisfactory toxic effect on U251 cells.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A combined antimetabolite for treating high-grade glioma, which is characterized by comprising the following raw material components: glycolytic inhibitors, lactate metabolism inhibitors, mitochondrial oxidative phosphorylation key enzyme inhibitors and mitochondrial scavengers;

in the raw material components, the molar ratio of the glycolysis inhibitor, the lactic acid metabolism inhibitor, the mitochondrial oxidative phosphorylation key enzyme inhibitor and the mitochondrial scavenger is 150:60:10000:15;

the glycolytic inhibitor is 3-bromopyruvate, the lactic acid metabolism inhibitor is malonic acid derivative AZ-33, the mitochondrial oxidative phosphorylation key enzyme inhibitor is dichloroacetic acid, and the mitochondrial scavenger is flunarizine.

2. The method for preparing the combined antimetabolite for the treatment of high grade glioma according to claim 1 comprising the steps of:

dispersing lactic acid metabolism inhibitor and mitochondrial scavenger in organic solvent respectively, dispersing glycolysis inhibitor and mitochondrial oxidative phosphorylation key enzyme inhibitor in water respectively, and mixing the four solutions uniformly to obtain the combined antimetabolite for treating high-grade glioma.

3. The method according to claim 2, wherein the lactic acid metabolism inhibitor is dispersed in DMSO to prepare a lactic acid metabolism inhibitor stock solution of 5-20 mM.

4. The method of claim 2, wherein the mitochondrial scavenger is dispersed in DMSO to provide a 10-30mM stock solution of mitochondrial scavenger.

5. The preparation method according to claim 2, wherein the glycolysis inhibitor is dispersed in water to prepare 25-100mM glycolysis inhibitor stock solution;