A combined pharmaceutical composition for treating glioblastoma

Abstract

The present invention discloses a combined pharmaceutical composition for treating glioblastoma. The pharmaceutical composition comprises temozolomide, MK-803, and / or MK-2206. Among them, the combination of temozolomide and MK-803 has a synergistic effect on the treatment of glioblastoma, which is significantly superior to the use of TMZ or MK-803 alone. The combination of temozolomide, MK-803, and / or MK-2206 can significantly inhibit the growth of glioblastoma cells and prolong the survival period of patients. The present invention provides a new strategy and idea for the treatment of glioblastoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a combined pharmaceutical composition for treating glioblastoma. Background Art

[0002] Glioblastoma (GBM) is the most common primary central nervous system tumor in the intracranial cavity, often showing infiltrative and destructive development and rapid progression. Due to the high invasiveness of tumor cells to surrounding tissues, glioblastoma is also considered the most aggressive and lethal brain tumor. According to the report of the American Brain Tumor Registry, the incidence rate of glioblastoma accounts for 80% of all intracranial malignant tumors. In China, the annual incidence rate is about 5 - 8 per 100,000. Glioblastoma is a tumor with abnormally high vascularization. Glioblastoma grows rapidly, and 70% - 80% of patients have a disease course of 3 - 6 months, and only 10% of patients have a disease course exceeding 1 year. Despite active treatment methods, most patients still have to face tumor recurrence. The progression of the tumor will be more rapid, the choice of treatment methods is very limited, and due to reasons such as the significant heterogeneity between internal cell populations of the tumor and the obstruction of the external blood - brain barrier, the possibility that drugs can slow down tumor progression in a short time is very low, the quality of life of patients is poor, and the prognosis is not ideal.

[0003] Currently, the main treatment strategy for glioblastoma is, on the premise of maximizing the resection of tumor tissue, to administer a standardized chemotherapy regimen containing alkylating agents plus radiotherapy after surgery (Sabo B. Primary malignant brain tumours, psychosocial distress and the intimate partner experience: what do we know[J]. Can J Neurosci Nurs, 2014, 36(3): 9 - 15.). However, most glioblastomas grow invasively and have no obvious boundary with brain tissue. Therefore, neither by naked eye nor by instruments can tumor tissue be completely distinguished from surrounding normal brain tissue during the operation. Therefore, it is very difficult to completely resect tumor tissue without damaging normal brain tissue. Moreover, glioblastoma is less sensitive to radiotherapy and chemotherapy compared to radiotherapy - and chemotherapy - sensitive tumors such as germ cell tumors, resulting in a very high recurrence rate after surgery for glioblastoma. Based on the above reasons, the median survival time of glioblastoma patients is only 12 - 15 months, only 30% of glioblastoma patients have a two - year survival period, less than 10% of glioblastoma patients can survive for more than 3 years, and very few can survive longer.

[0004] Therefore, there is an urgent need in the current field for researchers and clinicians to develop new treatment strategies and more effective therapeutic drugs for glioblastoma in order to extend the survival time of glioblastoma patients and improve the quality of life of glioblastoma patients. Summary of the Invention

[0005] The object of the present invention is to provide a combined pharmaceutical composition for treating glioblastoma, and the combined pharmaceutical composition is one or more of TMZ, MK-803, and MK-2206. Preferably, the combined pharmaceutical composition is TMZ and MK-803, TMZ and MK-803 and MK-2206. The present invention first discovers that the combination of TMZ and MK-803 has a synergistic effect on the treatment of glioblastoma, and the combination of TMZ, MK-803, and MK-2206 can significantly inhibit the growth of glioblastoma cells, showing a good therapeutic effect.

[0006] The above object of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a pharmaceutical composition for treating glioblastoma.

[0008] Furthermore, the pharmaceutical composition includes temozolomide and MK-803;

[0009] Furthermore, the pharmaceutical composition further includes MK-2206;

[0010] In a specific embodiment of the present invention, the pharmaceutical composition is temozolomide and MK-803.

[0011] In another specific embodiment of the present invention, the pharmaceutical composition is temozolomide, MK-803, and MK-2206.

[0012] Furthermore, the molar concentration ratio of temozolomide to MK-803 is 100-500:1-5.

[0013] Furthermore, the molar concentration ratio of temozolomide to MK-803 is 100:1.

[0014] Furthermore, the pharmaceutical composition further includes one or more of temozolomide, osimertinib, and atorvastatin.

[0015] In the present invention, TMZ refers to Temozolomide, which is a first-line clinical orphan drug with a broad anti-tumor spectrum, high activity, good oral absorption. Temozolomide can rapidly penetrate the blood-brain barrier, and the drug concentration in cerebrospinal fluid is 30% of that in plasma, and the concentration in tumor cells is much higher than that in normal cells. Therefore, it can kill tumor cells while maximizing the protection of normal tissues.

[0016] In the present invention, MK-803 refers to Lovastatin, which is an HMG-CoA reductase inhibitor with an IC50 of 3.4 nM in cell-free experiments and is used for lowering cholesterol (hypolipidemic drug). Lovastatin inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), blocking the enzyme-catalyzed conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonic acid. Lovastatin is a statin natural product isolated from various sources and exhibits cholesterol-lowering and anti-proliferative properties.

[0017] MK-2206 in the present invention is an AKT inhibitor. MK-2206 inhibits the autophosphorylation of AKT at threonine 308 and serine 473 sites. In addition, MK-2206 blocks the phosphorylation of downstream signaling molecules regulated by AKT (including TSC2, PRAS40, and ribosomal S6 protein).

[0018] In the specific embodiments of the present invention, it is found through experiments that the combination of Temozolomide and MK-803 can produce a synergistic effect on the treatment of glioblastoma, significantly inhibiting the growth of glioblastoma cells; the combination of Temozolomide, MK-803, and MK-2206 also shows a significant inhibitory effect on the growth of glioblastoma cells. In addition, the combination of the above drugs can significantly prolong the survival period of patients.

[0019] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0020] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes but is not limited to: diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, disintegrant.

[0021] Among them, the diluents include lactose, sodium chloride, glucose, urea, starch, water, etc.; the binders include starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropylcellulose, and hydroxypropylmethylcellulose, etc.; the surfactants include polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, cetyl alcohol, etc.; the humectants include glycerol, starch, etc.; the adsorption carriers include starch, lactose, bentonite, silica gel, kaolin, and saponite, etc.; the lubricants include zinc stearate, monoglyceride stearate, polyethylene glycol, talc powder, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, dodecyl magnesium sulfate, etc.; the fillers include mannitol (granular or powdery), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polydextrose, coupled sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminaran powder, agar powder, calcium carbonate, and sodium bicarbonate, etc.; the disintegrants include crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl cellulose, croscarmellose sodium, soy polysaccharide, etc.

[0022] Furthermore, the pharmaceutical composition described in the present invention may further include additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants.

[0023] Furthermore, the pharmaceutical composition described in the present invention can also be used in combination with other drugs for treating glioblastoma. Other therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition. Other therapeutic compounds can also be administered separately in a separate composition or in a different dosage form from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response to the treatment regimen.

[0024] Furthermore, the dosage form of the pharmaceutical composition described in the present invention is a dosage form conducive to drug administration prepared by conventional methods, including but not limited to: aqueous injection, powder for injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder inhaler, sustained-release agent, controlled-release agent, etc. The pharmaceutical excipients can be those conventionally used in various preparations, including but not limited to: isotonic agent, buffer, flavoring agent, excipient, filler, binder, disintegrant, lubricant, etc.; or those selected to adapt to the said substance, including but not limited to: emulsifier, solubilizer, bacteriostatic agent, analgesic agent, antioxidant, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, etc., thereby improving the metabolism of various compounds in vivo and further enhancing the drug administration effect of the composition. In addition, excipients can also be used to achieve specific drug administration purposes or methods, such as: sustained-release drug administration, controlled-release drug administration, pulsed drug administration, etc., including but not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials (for example: polyethylene glycol, polyurethane, polycarbonate and their copolymers, etc.). The main manifestations conducive to drug administration include: improving the therapeutic effect, improving bioavailability, reducing toxic and side effects, and improving patient compliance, etc.

[0025] The second aspect of the present invention provides the use of the pharmaceutical composition for treating glioblastoma described in the first aspect of the present invention in the preparation of products for treating and / or preventing glioblastoma.

[0026] Furthermore, the products include anti-glioblastoma drug preparations and anti-glioblastoma combination drugs.

[0027] Furthermore, the anti-glioblastoma drug preparation is a preparation prepared with the pharmaceutical composition for treating glioblastoma described in the first aspect of the present invention as the active ingredient, plus a pharmaceutically acceptable carrier and / or excipient.

[0028] Furthermore, the active ingredient is preferably a combination of temozolomide and MK-803, or a combination of temozolomide, MK-803 and MK-2206, more preferably a combination of temozolomide and MK-803.

[0029] Furthermore, the anti-glioblastoma combination drug contains one or more of temozolomide, MK-803, and MK-2206 administered simultaneously or separately, as well as a pharmaceutically acceptable carrier and / or excipient;

[0030] Preferably, the anti-glioblastoma combination drug contains temozolomide and MK-803 administered simultaneously or separately, as well as a pharmaceutically acceptable carrier and / or excipient;

[0031] Preferably, the combined drug for treating glioblastoma contains temozolomide, MK-803 and MK-2206 which are administered simultaneously or separately, as well as a pharmaceutically acceptable carrier and / or excipient.

[0032] In the present invention, the prevention refers to various means or measures for preventing the occurrence or development of a disease before it is recognized by clinical standards, including medical, physical or chemical methods, to prevent and reduce the occurrence or development of various symptoms of the disease.

[0033] In the present invention, the treatment refers to various measures for preventing and reducing the occurrence or development of a disease, so as to inhibit, contain, alleviate, improve, slow down, stop, delay or reverse the development or aggravation of the disease course, and the various indicators of the disease, disorder or pathological state during treatment or maintenance include alleviating or reducing symptoms or complications, or curing or eliminating the disease, disorder or condition.

[0034] The inventors of the present invention used primary tumor cells derived from glioblastoma patients (with abnormal activation of the RTK pathway and fatty acid synthesis and metabolism pathways) for orthotopic inoculation in nude mice. Seven days after inoculation, treatment was administered (TMZ and / or MK-803 and / or MK-2206). The results showed that the treatment effects of the two inhibitors MK-803 and MK-2206 alone were not good for glioblastoma, but their combination with temozolomide (TMZ) and the triple combination could significantly inhibit the growth of tumors in vivo and prolong the survival time of tumor-bearing mice by more than twice. In addition, through experimental verification, the present invention first found that the combination of temozolomide and MK-803 had a synergistic effect on the treatment of glioblastoma. This result is a technical effect that could not be expected before experimental verification. This result also shows that the combination of temozolomide and MK-803, or the triple combination of temozolomide, MK-803 and MK-2206 can be used as a new anti-glioblastoma drug composition for the effective treatment of glioblastoma to prolong the survival time of glioblastoma patients and improve their quality of life.

[0035] Advantages and beneficial effects of the present invention compared with the prior art:

[0036] The present invention first found that the combined drug compositions TMZ and MK-803, TMZ and MK-803 and MK-2206 have a significant inhibitory effect on the growth of glioblastoma cells and can significantly prolong the survival period of patients. Among them, the combination of TMZ and MK-803 can play a synergistic effect, and the use effect is significantly better than that of using TMZ or MK-803 alone. Therefore, it has better clinical application prospects. Description of the Drawings

[0037] Figure 1Show the overall results of single-cell sequencing of clinical GBM patients. Among them, Figure A: 26 cell clusters were obtained after dimensionality reduction; Figure B: Heatmap of differentially expressed genes in 26 cell clusters; Figure C: Results of cell type identification; Figure D: Violin plot of marker genes for each cell type; Figure E: Cell distribution in tumor tissues of each patient; Figure F: inferCNV analysis of tumor cells;

[0038] Figure 2 Show the results of the correlation between the RTK pathway and the fatty acid synthesis and metabolism pathway. Among them, Figure A: Tumor cells can be divided into 4 groups according to 6 genes; Figure B: Expression distribution map of 6 genes; Figure C: Heatmap of differentially expressed genes in 4 groups of cells; Figure D: Survival curves of patients in the database divided into 3 groups according to the expression of 6 genes; Figure E: Correlation analysis between RTK pathway genes and fatty acid synthesis and metabolism pathway genes;

[0039] Figure 3 Show the results of the analysis of the prognostic discrimination effect of the RTK-fatty acid gene set scoring system in the TCGA database on GBM patients. Among them, Figure A: Scoring results of patients with different WHO grades; Figure B: Correlation analysis between this score and tumor purity; Figure C: Survival analysis of high- and low-scoring patients; Figure D: Univariate / multivariate COX regression analysis of various influencing factors in glioma patients;

[0040] Figure 4 Show the results of the analysis of the prognostic discrimination effect of the RTK-fatty acid gene set scoring system in the CGGA database on GBM patients. Among them, Figure A: Scoring results of patients with different WHO grades; Figure B: Correlation analysis between this score and tumor purity; Figure C: Survival analysis of high- and low-scoring patients; Figure D: Univariate / multivariate COX regression analysis of various influencing factors in glioma patients;

[0041] Figure 5 Show the results of the analysis of the prognostic discrimination effect of the RTK-fatty acid gene set scoring system in the Rembrandt database on GBM patients. Figure A: Scoring results of patients with different WHO grades; Figure B: Correlation analysis between this score and tumor purity; Figure C: Survival analysis of high- and low-scoring patients; Figure D: Univariate / multivariate COX regression analysis of various influencing factors in glioma patients;

[0042] Figure 6 Show the results of the combined analysis of RNA sequencing and metabolomics of clinical GBM tumor samples. Among them, Figure A: Heatmap of the expression of EGFR, ACSS3, ACSL3, ELOVL2 and the levels of various metabolites; Figure B: Heatmap of the correlation between the expression levels of 4 genes and metabolite levels; Figure C: Heatmap of the expression level of EGFR and cholesterol level;

[0043] Figure 7Results graphs showing the effects of inhibitors of EGFR-AKT and cholesterol synthesis on cell energy metabolism and proliferation. Among them, Figure A: Seahorse experiment exploring the effects of MK-2206 and MK-803 on the energy metabolism level and ATP production level of primary cell TBD0220 (EGFR pathway activation); Figure B: Seahorse experiment exploring the effects of EGFR-vIII overexpression and MK-2206 and MK-803 on the energy metabolism level and ATP production level of glioma cell line U87-MG cells; Figure C: ATP kit exploring the effects of MK-2206 and MK-803 on ATP production in TBD0220 and U87-MG; Figure D: Effects of MK-2206 and MK-803 on the cloning ability of TBD0220 and U87-MG cells; Figure E: Effects of MK-2206 and MK-803 on the proliferation ability of TBD0220 and U87-MG cells;

[0044] Figure 8 Results graphs showing the effects of single-agent and combination drug administrations on tumor imaging results and survival results in tumor-bearing mice. Among them, Figure A: Imaging graph of the effects of single-agent and combination drug administrations on tumor cell growth in tumor-bearing mice; Figure B: Statistical result graph of the effects of single-agent and combination drug administrations on the survival time of tumor-bearing mice; Figure C: Statistical result graph of the effects of single-agent and combination drug administrations on the tumor bioluminescence level in tumor-bearing mice. Detailed implementation manners

[0045] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods without specific conditions noted in the following embodiments are usually implemented according to conventional conditions or according to the conditions recommended by the manufacturers.

[0046] Example 1 Acquisition of clinical glioblastoma (GBM) samples and single-cell sequencing

[0047] 1. Sample source

[0048] A total of 5 glioblastoma patients were included in this study. Before the surgery of inpatients with glioblastoma, the experimental situation was explained to the patients and their family members, and informed consent forms were signed with them. Multiple samples were taken from the postoperative samples of each patient, and a total of 12 samples were taken.

[0049] 2. Acquisition of clinical glioblastoma samples

[0050] (1) After the glioma of a glioblastoma patient is to be surgically removed, use a scalpel to obtain tumor samples from different positions of the tumor tissue. Each sample should be the size of a soybean, and pay attention to avoiding the necrotic area and the blood clot area;

[0051] (2) Use normal saline to wash the blood on the surface of the tissue, immediately put it into Miltenyi tissue preservation solution, seal it with sealing film, and store it at 4°C.

[0052] 3. Single-cell sequencing and analysis

[0053] (1) Place the obtained clinical glioblastoma sample in a 4°C transport box and send it to the company for single-cell suspension preparation and library construction and sequencing;

[0054] (2) Integrate and analyze the sequencing data off the machine, remove batch effects, and obtain a sparse matrix by comparing with the reference genome;

[0055] (3) After data filtering, dimensionality reduction and clustering using the Seurat v3.7.2 R package in R language, 26 cell clusters are obtained, and the two-dimensional UMAP plot is visualized. The resulting figure is shown in Figure 1 A;

[0056] (4) Compare the gene expression in each cell cluster, find the top 10 genes, and visualize them with a heat map. The resulting figure is shown in Figure 1 B;

[0057] (5) Use the data information in the cell type identification database CellMarker for comparison, obtain cell type information and visualize it with a UMAP plot. The resulting figure is shown in Figure 1 C;

[0058] (6) Visualize the expression of marker genes of different cell types with a violin plot. The resulting figure is shown in Figure 1 D;

[0059] (7) Visualize the cell distribution of 12 GBM samples with a UMAP plot. The resulting figure is shown in Figure 1 E;

[0060] (8) Using the gene expression of non-tumor cells as a reference, use the inferCNV R package to analyze the copy number variation of tumor cells and visualize it with a heat map. The resulting figure is shown in Figure 1 F;

[0061] (9) Isolate the tumor cell subtypes in the single-cell data for separate analysis, and visualize them with a UMAP plot after dimensionality reduction and clustering;

[0062] (10) The cell populations of the single-cell tumor data were divided into four groups based on the expression of GFAP, CHI3L1, OLIG2, SOX6, EGFR, and PDGFA, and visualized. The results are shown in Figure 2. Figure 2 A and Figure 2 B;

[0063] (11) Calculate the top 50 genes of each of the four cell groups and visualize them as heat maps. See the results in the figure. Figure 2 C.

[0064] 4. Experimental results

[0065] After single-cell sequencing of GBM patient tumor samples, 26 cell clusters were obtained (see Figure 1 A), after comparing the differentially expressed genes in each cluster and the marker genes of each cell, 7 cell types were obtained, of which tumor cells accounted for the majority (see Figure 1 C) with typical features of GBM including chromosome 7 amplification and chromosome 10 loss (see Figure 1 F). The expression levels of GBM classic marker genes GFAP (representing astrocyte origin), CHI3L1 (high expression indicates poor prognosis), OLIG2 and SOX6 (representing oligodendrocyte origin) are used to distinguish tumor cells. Tumor cells can be divided into three groups, namely, GFAP and CHI3L1 positive group (representing classic and mesenchymal GBM, with poor prognosis), OLIG2 and SOX6 positive group (representing proneuronal GBM, with relatively good prognosis), and all negative group. Among the GFAP and CHI3L1 positive group cells, there is a group of cells that are positive for EGFR and PDGFA (representing the activation state of the RTK pathway, usually positive expression indicates poor prognosis). Therefore, these two genes are used to further subdivide this group of cells into two groups. Comparing the differentially expressed genes of these four groups of cells, it was found that three genes (ACSS3, ACSL3, ELOVL2) of the fatty acid anabolic pathway were highly expressed in the GFAP, CHI3L1, EGFR, and PDGFA positive cell groups (see Figure 2 C) showed that astrocyte-derived tumor cells with activated RTK pathway had vigorous fatty acid anabolism. This result suggested that the activation state of EGFR signaling pathway and the vigorous degree of fatty acid anabolism were closely related to the prognosis of GBM patients, that is, the expression levels of genes GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 were closely related to the prognosis of GBM patients. GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 could be used as potential biomarkers for predicting the prognosis of GBM patients.

[0066] Example 2 Analysis using the glioblastoma database

[0067] In this example, the glioblastoma data in the TCGA, CGGA, and Rembrandt databases were used for bioinformatics analysis. The analysis software used was the R language to verify the performance of the evaluation criteria.

[0068] 1. Experimental method

[0069] (1) Data download: TCGA data was downloaded from the UCSC XENA website (https: / / xenabrowser.net / ), and CGGA and Rembrandt data were downloaded from the official CGGA website (http: / / www.cgga.org.cn / ). Both included gene expression data and clinical data;

[0070] (2) Using the expression values of GFAP, CHI3L1, OLIG2, and SOX6, calculate according to the following formula

[0071] Calculate. Patients with values less than the median were in group G3, and patients with values greater than the median were in groups G1+G2;

[0072] (3) In groups G1+G2, further calculate using the expression values of EGFR and PDGFA. Patients with values greater than the median were in group G1, and patients with values less than the median were in group G2;

[0073] (4) Calculate the survival times of patients in groups G1, G2, and G3, and draw survival curves. The resulting figure is shown in Figure 2 D;

[0074]

[0075] (5) Calculate the expression correlations of the genes EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 in the database. The resulting figure for the correlation analysis is shown in Figure 2 E;

[0076] (6) Patient scoring: Use the expression values of the genes GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 and the survival times of the corresponding patients to perform univariate COX regression analysis to obtain the β values of each gene. Add the products of the β values of these 7 genes and the corresponding gene expression values to obtain the comprehensive score of the patient, which is the RTK-fatty acid gene set score;

[0077] (7) Group the patients according to the WHO grade, and use a bar chart to show the comprehensive scores of each group of patients. The resulting figures corresponding to the GBM sample sequencing / microarray data in the TCGA, CGGA, and Rembrandt databases are shown inFigure 3 A, Figure 4 A, Figure 5 A;

[0078] (8) Calculate the tumor purity of the patient using the ESTIMATE method, and use a scatter plot to show the relationship between the comprehensive score of the patient and the tumor purity. The result graphs corresponding to the sequencing / microarray data of GBM samples in the TCGA, CGGA, and Rembrandt databases are shown in Figure 3 B, Figure 4 B, Figure 5 B;

[0079] (9) Group the patients according to the median of the comprehensive score and draw the survival curve. The result graphs corresponding to the sequencing / microarray data of GBM samples in the TCGA, CGGA, and Rembrandt databases are shown in Figure 3 C, Figure 4 C, Figure 5 C;

[0080] (10) Select parameters such as gender, age, WHO grade, IDH mutation status, 1p / 19q co-deletion status, MGMT promoter methylation level, the expression levels of genes GFAP, CHI3L1, OLIG2, SOX6, EGFR, PDGFA, ACSS3, ACSL3, ELOVL2, as well as the Group grouping and the comprehensive score grouping, and perform univariate / multivariate COX regression analysis. The result graphs corresponding to the sequencing / microarray data of GBM samples in the TCGA, CGGA, and Rembrandt databases are shown in Figure 3 D, Figure 4 D, Figure 5 D.

[0081] 2. Experimental results

[0082] Through the analysis of the sequencing / microarray data of GBM samples and the corresponding clinical data in the TCGA, CGGA, and Rembrandt databases, it is found that EGFR, PDGFA have a good correlation with ACSS3, ACSL3, ELOVL2 (see Figure 2E); Patients in the database were grouped according to the expression level distributions of GFAP, CHI3L1, OLIG2, SOX6, EGFR, and PDGFA. Survival analysis found that patients positive for GFAP, CHI3L1, EGFR, and PDGFA had the shortest survival, while patients positive for OLIG2 and SOX6 had the relatively longest survival. Then, the expression levels of GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 in each patient's tumor were integrated to create a scoring system (scoring model), and it was found that this score could significantly indicate the GBM grade of the patients. A high score predicted a high WHO grade, and the median survival of patients with a high score was significantly shortened by 4 times compared to those with a low score, which could be used for prognostic prediction of GBM patients. Subsequently, univariate and multivariate COX regression analyses were performed (see Figure 3 D, Figure 4 D, Figure 5 D), and the above results showed that WHO grade (a high grade predicts poor prognosis of patients), IDH mutation status (an indicator recognized in neuro-oncology research, and the wild type usually predicts poor prognosis of patients) both had good predictive ability for the prognosis of patients (P < 0.05); the integrated scoring system also had good predictive ability compared with them (P < 0.05); the 1p / 19q co-deletion status had acceptable results in univariate COX analysis (P < 0.05), but there was no significant difference in the 1p / 19q co-deletion status in multivariate COX analysis (P > 0.05), indicating that the scoring system provided by the present invention can have the same ability to distinguish the prognosis of glioma patients as classical methods such as WHO grade and IDH mutation status, and is stronger than the 1p / 19q co-deletion method, further indicating that this scoring system can be used as another powerful method for independently distinguishing the survival prognosis of glioma patients;

[0083] The diagnostic efficacy of the scoring model for predicting the prognosis of glioblastoma patients composed of GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 in combination is good, and the AUC value is 0.7516. The diagnostic efficacy results of any 3 - 7 genes of GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 in combination for predicting the prognosis of glioblastoma patients are shown in Table 1. The results show that the AUC value of the combination of CHI3L1 + EGFR + ACSL3 is as high as 0.7947, indicating that one or more of GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, and ELOVL2 can be applied to the prognostic prediction of glioblastoma patients and have good diagnostic efficacy.

[0084] Table 1 Diagnostic efficacy of genes for prognostic prediction of glioblastoma

[0085]

[0086] Example 3 RNA Sequencing and Untargeted Metabolomics Analysis of Clinical GBM Samples

[0087] 1. Experimental Methods

[0088] (1) Twelve clinical GBM patients were recruited. After obtaining the consent of the patients and their families, fresh glioma tissue samples the size of a soybean were taken after surgery, avoiding the necrotic area and blood clot area. Multiple samples were taken from each patient's postoperative sample, and a total of 63 samples were collected;

[0089] (2) The blood on the tissue surface was washed off with normal saline, placed in a cryotube, immediately frozen with liquid nitrogen, and sent to the company by dry ice for RNA extraction, library construction and sequencing, as well as metabolite extraction and detection;

[0090] (3) The sequencing data and metabolomics data were integrated and analyzed;

[0091] (4) The expression levels of genes EGFR, ACSS3, ACSL3, and ELOVL2, as well as the metabolite levels of fatty acid synthetic metabolites phosphatidylcholine (PC), lysophosphatidylcholine (LysoPC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LysoPE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) were extracted and visualized using a heatmap. The resulting figure is shown in Figure 6 A;

[0092] (5) The correlation between the gene expression level and the fatty acid synthetic metabolite level was calculated and visualized using a heatmap. The resulting figure is shown in Figure 6 B;

[0093] (6) The expression level of EGFR and the cholesterol synthetic metabolite level were extracted and visualized using a heatmap. The resulting figure is shown in Figure 6 C.

[0094] 2. Experimental Results

[0095] Since GBM patients with poor prognosis have high expression of fatty acid synthetic metabolism genes, fresh postoperative tumor samples of clinical GBM patients were collected for RNA sequencing and untargeted metabolomics analysis. The results showed that the expression levels of genes EGFR, ACSS3, ACSL3, and ELOVL2 were significantly positively correlated. As the end products of fatty acid synthetic metabolism, untargeted metabolomics analysis found that the intracellular levels of phosphatidylcholine and cholesterol were positively correlated with the EGFR expression level, while lysophosphatidylcholine was negatively correlated with the EGFR expression level (see Figure 6)。The above results further confirmed that the fatty acid synthesis and metabolism pathway is also abnormally activated in GBM tumor cells derived from astrocytes and with RTK pathway activation.

[0096] Example 4 Seahorse Mitochondrial Stress Test and Cell ATP Level Detection

[0097] 1. Seahorse Mitochondrial Stress Test

[0098] (1) Cultivate glioma cells U87-MG and primary glioma cells TBD0220 (EGFR pathway activated) routinely;

[0099] (2) Seed the cells into a Seahorse cell plate, with 15,000 cells per well;

[0100] (3) After the cells adhered, add drugs for treatment, adding 5 μM MK-2206, 5 μM MK-803, and the corresponding volume of DMSO respectively;

[0101] (4) One night before the detection, hydrate the probe plate with the hydration solution and place it in a 37°C CO2-free incubator overnight;

[0102] (5) Prepare the detection medium 1 hour in advance. After the cells are treated for 24 h, discard the cell medium completely. Wash the cells twice with the detection medium, then add 500 μL of the detection solution to each well and place it in a 37°C CO2-free incubator for 1 h;

[0103] (6) During this period, add the corresponding concentrations of Oligomycin, FCCP, Retenone and Antimycin A drugs provided in the kit to the respective drug addition wells in the probe plate and put it into the machine for quality inspection;

[0104] (7) After the quality inspection is completed and the cell plate has been static for the required time, place the cell plate into the machine for detection, with a duration of about 2 h;

[0105] (8) Detect the oxygen consumption rate (OCR) after adding different drugs. After the detection, export the data for analysis and graphing.

[0106] 2. Cell ATP Level Detection

[0107] (1) Seed the cells into a 6-well plate and add drugs for stimulation according to the stimulation conditions described in Example 4;

[0108] (2) After 24 h, wash the cells twice with PBS, add the cell lysate in the kit, pipette the cells down and transfer them to a 1.5 mL tube;

[0109] (3) According to the operation steps of the kit, use the ATP standard product to prepare and plot the standard curve, and add samples to the sample wells for detection;

[0110] (4) Calculate the ATP level in the sample according to the standard curve.

[0111] 3. Experimental results

[0112] As a key receptor of the RTK pathway, after receiving external stimuli, EGFR activates the PI3K-AKT signaling pathway downward, causing cleavage of SREBP1, thereby upregulating the key enzymes of the fatty acid synthesis and metabolism pathway. Therefore, the specific AKT phosphorylation inhibitor MK-2206 was selected to block the EGFR-AKT signaling pathway. The key step in cholesterol synthesis is the generation of mevalonic acid from acyl coenzyme A catalyzed by HMG-CoA reductase. Therefore, the HMG-CoA reductase inhibitor MK-803 was selected to block this pathway. Through the Seahorse mitochondrial stress experiment, it was found that EGFR activation could enhance the cell energy metabolism level. Adding the two inhibitors MK-2206 and MK-803 respectively could lead to abnormal cell energy metabolism and a significant decrease in ATP production (see Figure 7 A, Figure 7 B, Figure 7 C).

[0113] Example 5 Colony formation assay

[0114] 1. Experimental method

[0115] (1) Cultivate glioma cells U87-MG and primary glioma cells TBD0220 routinely;

[0116] (2) Digest the cells into single-cell suspensions and count them, and seed the cells into 6-well plates at a density of 500 cells / well;

[0117] (3) After the cells adhered, discard the original culture medium and perform drug stimulation. The concentration of MK-2206 was 1 μM, and the concentration of MK-803 was 1 μM;

[0118] (4) After 10 - 12 days, cell colonies could be seen with the naked eye. At this time, wash the cells 2 times with PBS, and add 4% paraformaldehyde to fix at room temperature for 10 min;

[0119] (5) After washing 2 times with PBS, add 1 mL of 0.2% crystal violet staining solution to each well for staining, and gently shake at room temperature for 2 h;

[0120] (6) After staining, rinse thoroughly with PBS, air dry, and take pictures for recording.

[0121] 2. Experimental results

[0122] The results of the colony formation assay showed that the addition of two inhibitors, MK-2206 and MK-803, respectively, could lead to abnormal cell energy metabolism, significantly reduce ATP production, and ultimately result in slow cell growth and a sharp decrease in the number of cell colonies (see Figure 7 D).

[0123] Example 6 Cell Proliferation Assay

[0124] 1. Experimental Method

[0125] (1) Cultivate glioma cells U87-MG and primary glioma cells TBD0220 routinely;

[0126] (2) Digest the cells into single-cell suspensions and count them, then seed the cells into 6 96-well plates at a density of 2000 cells / well;

[0127] (3) After the cells adhered, discard the original culture medium and perform drug stimulation. The concentration of MK-2206 was 5 μM, and the concentration of MK-803 was 5 μM;

[0128] (4) Take out 1 96-well plate every day, add CCK-8 reagent, and react at 37 °C for 1 h;

[0129] (5) Take out the cell plate and use a microplate reader to read the absorbance value of each well at a wavelength of 450 nm;

[0130] (6) Detect for 6 days in total, perform integrated analysis, and draw a proliferation curve.

[0131] 2. Experimental Results

[0132] The results showed that the addition of two inhibitors, MK-2206 and MK-803, respectively, could lead to better inhibition of tumor cell growth by blocking cholesterol synthesis for a long time (see Figure 7 E).

[0133] Example 7 In Vivo Animal Experiment

[0134] 1. Experimental Method

[0135] (1) Select 6-week-old female nude mice weighing 16 - 18 g, with 10 mice in each group;

[0136] (2) Select primary glioma cells TBD0220 for tumorigenesis;

[0137] (3) Anesthetize the mice with chloral hydrate and fix them on a stereotaxic apparatus;

[0138] (4) Use a scalpel to cut open the scalp and use a syringe needle to drill a hole in the skull at a position 2 mm to the right of the midline between the anterior fontanelle and the posterior fontanelle;

[0139] (5) Insert the needle, with a depth of 3 mm, then retract the needle by 1 mm, start the injection at a speed of 1 μL / min, inject 3 μL for each mouse, and the total cell number is 300,000;

[0140] (6) After injection, wait for 1 min and then retract the needle, and suture the scalp with a thin thread;

[0141] (7) Seven days after modeling, start drug treatment. MK-2206 and MK-803 are administered by oral gavage, and TMZ is administered intraperitoneally for a total of 14 days;

[0142] (8) Perform in vivo imaging of the mice every 7 days and record the tumor signals;

[0143] (9) Observe the status of the mice every day and record the survival time.

[0144] 2. Experimental results

[0145] The results showed that when using the patient-derived primary tumor cells TBD0220 (with abnormal activation of the RTK pathway and fatty acid synthesis and metabolism pathways) for orthotopic inoculation in nude mice and starting drug treatment 7 days after inoculation, it was found that the efficacy of the two inhibitors MK-2206 and MK-803 administered alone was not very ideal, but co-administration with temozolomide (TMZ) could significantly inhibit the growth of tumors in glioblastoma mice and extend the survival time of tumor-bearing mice by more than twice (see Figure 8 A, Figure 8 B and Figure 8 C);

[0146] The above results confirmed that the evaluation criteria established using the above 7 genes (GFAP, CHI3L1, EGFR, PDGFA, ACSS3, ACSL3, ELOVL2) can not only independently and accurately distinguish the survival prognosis of glioblastoma patients, but also guide the treatment strategy of combining RTK pathway inhibitors and lipid-lowering drugs with temozolomide for high-score patients;

[0147] To verify whether there is a synergistic effect between the combination of TMZ and MK-2206 and the combination of TMZ and MK-803, their inhibition rates were calculated, and the inhibition rates of the combination of TMZ and MK-2206 and the combination of TMZ and MK-803 on glioblastoma were obtained respectively. Among them, the inhibition rates of the combination of TMZ and MK-2206 at different concentrations on glioblastoma are shown in Table 2, and the inhibition rates of the combination of TMZ and MK-803 at different concentrations on glioblastoma are shown in Table 3;

[0148] Table 2 Inhibition rates of the combination of TMZ and MK-2206 at different concentrations on glioblastoma

[0149]

[0150] Table 3 Inhibition rate of TMZ and MK-803 at different concentrations in combination against glioblastoma

[0151]

[0152] Using the King's formula expression q = E A+B / (E A + E B - E A × E B ), determine whether the combination of the two (TMZ and MK-2206, TMZ and MK-803) has a synergistic effect on the treatment of glioblastoma. Among them, E A+B is the inhibition rate of TMZ and MK-2206, TMZ and MK-803 against glioblastoma, E A is the inhibition rate of TMZ against glioblastoma, E B is the inhibition rate of MK-2206 or MK-803 against glioblastoma; if q = 0.85 - 1.15, it is simple addition, 1.15 < q < 20 is synergy, q > 20 is significant synergy, q < 0.85 is antagonism, that is, when q > 1.15, it can be determined that the combination of the two (TMZ and MK-2206, TMZ and MK-803) has a synergistic effect on the treatment of glioblastoma, rather than simple addition;

[0153] According to the results of Table 2 and Table 3, use the above formula to judge whether the combination of TMZ and MK-2206, TMZ and MK-803 has a synergistic effect on the treatment of glioblastoma. The calculated q values are shown in Table 4 and Table 5. The results show that the combination of TMZ and MK-2206 does not have a synergistic effect on the treatment of glioblastoma, while the combination of TMZ and MK-803 has a synergistic effect on the treatment of glioblastoma. Among them, the molar concentration ratio of TMZ and MK-803 is 100 - 500:1 - 5. Within this range of molar concentration ratio, the combination of TMZ and MK-803 has a synergistic effect on the treatment of glioblastoma.

[0154] Table 4 q value and its effect of the combination of TMZ and MK-2206

[0155]

[0156] Table 5 q value of the combination of TMZ and MK-803

[0157]

[0158] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for the in vivo synergistic treatment of glioblastoma, characterized in that, The active ingredients in the pharmaceutical composition consist of temozolomide and lovastatin MK-803; The molar concentration ratio of temozolomide to lovastatin MK-803 is 200 - 500:

1.

2. The pharmaceutical composition for in vivo combined treatment of glioblastoma according to claim 1, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. Use of the pharmaceutical composition according to claim 1 or 2 for in vivo synergistic treatment of glioblastoma in the preparation of a product for treating and / or preventing glioblastoma.

4. Use of the pharmaceutical composition for the in vivo combined treatment of glioblastoma according to claim 3 in the preparation of a product for the treatment and / or prevention of glioblastoma, characterized in that, The product includes an anti-glioblastoma drug preparation and a combined anti-glioblastoma drug.

5. Use of the pharmaceutical composition for in vivo combined treatment of glioblastoma according to claim 4 in the preparation of a product for treating and / or preventing glioblastoma, characterized in that, The anti-glioblastoma drug preparation is a preparation prepared with the pharmaceutical composition according to claim 1 or 2 for in vivo synergistic treatment of glioblastoma as the active ingredient and a pharmaceutically acceptable carrier.

6. Use of the pharmaceutical composition for the in vivo combined treatment of glioblastoma according to claim 4 in the preparation of a product for the treatment and / or prevention of glioblastoma, characterized in that, The combined anti-glioblastoma drug is temozolomide and lovastatin MK-803 administered simultaneously or separately, and a pharmaceutically acceptable carrier.

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