Combination application of G6PD inhibitors and inhibitors of new molecular points

Abstract

The present invention belongs to the field of medicine and biotechnology, and specifically relates to the combined use of a G6PD inhibitor and an inhibitor of a new molecular point. Specifically, a treatment scheme for the synergistic treatment of glioblastoma multiforme with a G6PD inhibitor and A922500, RSL-3, and Fatostatin is provided. At the same time, the application of G6PD in the diagnosis, grading, and prognosis of brain glioma is provided.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biotechnology, and specifically relates to the combined application of a G6PD inhibitor and an inhibitor of a new molecular point. Background Art

[0002] Glioblastoma Multiform (GBM) is a WHO grade IV glioma with a median survival of only 12-15 months after diagnosis. It is the malignant tumor with the highest mortality rate in the world. In addition, the prognosis of GBM patients has not improved significantly in the past few decades, mainly due to the lack of effective treatment strategies.

[0003] The standard treatment for GBM is to first perform maximal surgical resection, followed by simultaneous radiotherapy and temozolomide (TMZ) for 6 weeks, followed by continued TMZ treatment every 4 weeks for 6 cycles. However, this treatment is only effective for a few months, and almost all GBM tumors inevitably recur after treatment. In addition to TMZ, only bevacizumab is currently approved by the FDA for the clinical treatment of recurrent GBM. However, bevacizumab can only effectively relieve symptoms and improve quality of life in the short term, and bevacizumab has no combined effect with TMZ and radiotherapy; in addition, it reduces the absorption of TMZ, cannot improve overall survival, and has significant side effects such as hypertension, venous thrombosis, and infection.

[0004] Recent studies have shown that metabolic reprogramming is one of the core characteristics of tumors. Exploring the regulatory mechanism of GBM metabolism, analyzing its biological characteristics, and finding effective molecular targets to guide clinical treatment can provide new ideas and efficient targets for cutting off GBM energy supply. Summary of the invention

[0005] In order to provide an effective diagnosis and treatment approach for brain glioma, the present invention provides the following technical solutions:

[0006] Pharmaceutical composition

[0007] In a first aspect, the present invention provides a pharmaceutical composition for treating brain glioma, the pharmaceutical composition comprising a first pharmaceutical component and a second pharmaceutical component.

[0008] The first drug ingredient is a G6PD inhibitor,

[0009] The second drug ingredient is one or more of A922500, RSL-3, and Fatostatin.

[0010] As used herein, the term "G6PD inhibitor" refers to a substance that targets, decreases or inhibits at least one activity of G6PD.

[0011] Preferably, the G6PD inhibitor includes those synthesized artificially or those existing in nature.

[0012] Preferably, the G6PD inhibitor includes reagents used in gene editing technologies such as RNA interference technology, antisense oligonucleotide (ASO) technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology, and also includes compounds that can reduce the expression of G6PD.

[0013] Specifically, the G6PD inhibitor is G6PDi-1 or a pharmaceutically acceptable salt thereof. The G6PDi-1 is a reversible non-competitive 6-phosphate glucose dehydrogenase (G6PD) inhibitor with an IC50 of 0.07 μM.

[0014]

[0015] Its structural formula is:

[0016] Specifically, the A922500 includes a pharmaceutically acceptable salt thereof, the RSL-3 includes a pharmaceutically acceptable salt thereof, and the Fatostatin includes a pharmaceutically acceptable salt thereof.

[0017] Preferably, the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma.

[0018] Preferably, the brain glioma is glioblastoma multiforme (GBM, also known as glioblastoma), that is, grade IV brain glioma.

[0019] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients, which include any one or a combination of at least two of diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0020] In a specific embodiment, the dosage forms of the first drug component and the second drug component in the pharmaceutical composition are the same or different.

[0021] Preferably, the dosage form includes drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments or creams.

[0022] In a specific embodiment, the first pharmaceutical ingredient and the second pharmaceutical ingredient in the pharmaceutical composition are administered simultaneously or sequentially, specifically, they can be administered at an interval of 0, 1, 2, 3, 4, 5, 6, 7 or more days.

[0023] In a specific embodiment, the dosage ratio of the first drug ingredient to the second drug ingredient in the pharmaceutical composition is 1:0.1-5; specifically including 5:1, 4:1, 3:1, 2.5:1, 2:1, 1:1, 1:2, 1:2.5, 1:3, 1:4, and 1:5.

[0024] Certain compositions of the present invention may further comprise a pharmaceutically acceptable carrier or excipient, wherein the “pharmaceutically acceptable carrier or excipient” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary.

[0025] Preferably, the pharmaceutical composition may further comprise other cancer drugs, for example: Acivicin, Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium Sodium), Bropirimine, Busulfan, Cactinomycin, Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin Hydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, Cisplatin, Cladribine, etc.

[0026] On the other hand, the present invention also provides the use of a G6PD inhibitor in the preparation of a drug for enhancing the sensitivity of a patient to a new molecule inhibitor, wherein the new molecule inhibitor comprises one or more of A922500, RSL-3, and Fatostatin.

[0027] Preferably, the patient is a glioma patient.

[0028] Preferably, the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma.

[0029] Preferably, the brain glioma is glioblastoma multiforme (GBM, also known as glioblastoma), that is, grade IV brain glioma.

[0030] On the other hand, the present invention provides use of a pharmaceutical composition in treating brain glioma, wherein the pharmaceutical composition comprises a G6PD inhibitor and an inhibitor of a new molecular point, wherein the inhibitor of the new molecular point is one or more of A922500, RSL-3, and Fatostatin.

[0031] Preferably, the pharmaceutical composition can also be used in combination with other anticancer drugs and cancer treatment methods.

[0032] Specifically, the other anticancer drugs include adriamycin, vincristine, vinorelbine, paclitaxel, cisplatin, dactinomycin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, epirubicin, etoposide, etoposide, epipodophyllotoxin, fluoroarabinoside, etc. Other cancer treatment methods include chemotherapy, radiotherapy, immunotherapy, gene therapy, surgery, etc.

[0033] On the other hand, the present invention also provides a method for treating brain glioma, comprising administering the aforementioned pharmaceutical composition to a cancer patient.

[0034] As used herein, the term "treat" refers to a method of reducing the symptoms / effects of a disease. In this context, treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in severity or improvement in the symptoms / effects of an established disease. It is to be understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptoms of a disease or condition.

[0035] As used herein, the term subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), fish, bird, or reptile or amphibian. The term does not indicate a particular age or sex. Thus, the subject encompasses both adult and newborn subjects, regardless of sex.

[0036] As used herein, patient or subject are used interchangeably and refer to a subject suffering from / suspected of suffering from a disease. The disease targeted by the present invention is a patient with a glioma, preferably a grade IV glioma, i.e., glioblastoma multiforme (GBM, also known as glioblastoma).

[0037] Application as a marker

[0038] On the other hand, the present invention also provides the use of a reagent for detecting the expression amount of G6PD in the preparation of a product for diagnosing glioblastoma.

[0039] Specifically, the G6PD expression level in GBM is higher than that in normal brain tissue.

[0040] On the other hand, the present invention also provides the use of a reagent for detecting the expression amount of G6PD in the preparation of a product for grading brain gliomas.

[0041] Specifically, the brain glioma includes grades 1 to 4, and the expression level of G6PD increases with the increase of the grade of the brain glioma.

[0042] On the other hand, the present invention also provides the use of a reagent for detecting the expression amount of G6PD in the preparation of a product for predicting the prognosis of brain glioma.

[0043] Preferably, the prognostic indicator is probability of survival.

[0044] Preferably, the reagent for detecting the expression level of G6PD includes a reagent for detecting the expression level of G6PD protein and / or the expression level of G6PD mRNA.

[0045] Preferably, the reagents for detecting the expression amount of G6PD protein include reagents used in the following methods: hematoxylin-eosin staining (HE staining), safranin O-fast green staining, protein blotting (Western Blot method), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemistry (Immunohistochemistry) staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence separation technology and protein chip method.

[0046] Preferably, the reagents for detecting the expression of G6PD mRNA include reagents used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, and high-throughput sequencing platform method.

[0047] Preferably, the detection is performed on a sample from a subject.

[0048] Preferably, the sample comprises tissue, cell (including cell culture), blood, urine, saliva, mucosal sample, feces, intestinal lavage, joint fluid, cerebrospinal fluid, bile sample, respiratory secretion, bronchoalveolar lavage fluid sample.

[0049] Preferably, the sample is tissue or cell.

[0050] Preferably, the product includes a kit, a chip, a test strip, or a device, an apparatus, or a computer-readable storage medium.

[0051] It is well known to those skilled in the art that the contents disclosed in the present invention can be specifically implemented as complete hardware, complete software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the comparison result of G6PD expression in GBM and normal brain tissue.

[0053] Figure 2 This is the comparison result of the expression level of G6PD in gliomas of different grades.

[0054] Figure 3 This is the survival curve of G6PD in GBM.

[0055] Figure 4 This is the result of using tissue chips to analyze the expression of G6PD in gliomas of different grades.

[0056] Figure 5 It is a survival curve drawn based on the results of tissue chip detection.

[0057] Figure 6 This is the result of the correlation analysis between G6PD and other genes.

[0058] Figure 7 It is the expression result of SREBP-1 and G6PD in tumor tissues of GBM patients.

[0059] Figure 8 This is a graph showing the cell number statistics of U87 cells treated with the SREBP1 inhibitor Fatostatin in combination with G6PDi-1.

[0060] Fig. 9 This is a microscopic image of U87 cells treated with the SREBP1 inhibitor Fatostatin in combination with G6PDi-1.

[0061] Fig.10 This is a graph showing the cell number statistics of U251 cells treated with the DGAT1 inhibitor A922500 in combination with G6PDi-1.

[0062] Fig.11 These are microscope images of U251 cells treated with the DGAT1 inhibitor A922500 in combination with G6PDi-1.

[0063] Fig.12This is a graph showing the colony formation results of DGAT1 inhibitor A922500 and G6PDi-1.

[0064] Fig.13 This is a graph showing the cell number statistics of U251 cells treated with the GPX4 inhibitor RSL-3 in combination with G6PDi-1.

[0065] Fig.14 These are microscope images of U251 cells treated with the GPX4 inhibitor RSL-3 and G6PDi-1. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0067] Example 1. Role of G6PD in the diagnosis, grading, and prognosis of GBM

[0068] By analyzing RNAseq data of 32 solid tumors from The Cancer Genome Atlas (TCGA), it was found that the expression of G6PD in GBM was significantly higher than that in normal brain tissue ( Figure 1 ); the expression in GBM was also significantly higher than that in low-grade glioma (LGG) ( Figure 2 ). High expression of G6PD was significantly associated with poor survival in the GBM cohort ( Figure 3 ).

[0069] Accordingly, we further detected the G6PD protein level in tumor tissues of patients with grade I-IV gliomas using tissue microarray (TMA) (n=145). IHC staining results showed that the G6PD protein content in grade IV GBM tissues was the highest compared with grade III gliomas, grade II gliomas, and grade I gliomas ( Figure 4 ); In addition, survival analysis showed that high levels of G6PD protein in these 145 glioma tissues were closely associated with low patient survival rates ( Figure 5 ). These results suggest that G6PD may be a new molecular marker for GBM and may play an important role in the occurrence and development of GBM.

[0070] Example 2: G6PDi enhances the sensitivity of cancer cells to lipid synthesis inhibitors

[0071] The RNAseq data of 153 primary tumor patients in the GBM cohort in TCGA were analyzed. The results showed that the expression of G6PD was significantly positively correlated with the expression of lipid synthesis-related genes such as SREBP1, FASN, ACC, etc., and was also significantly positively correlated with the expression of genes related to maintaining intracellular redox homeostasis such as GPX4, TXN, B166, etc. ( Figure 6 ).

[0072] To evaluate the potential clinical significance of our observations, we performed a correlation analysis of the immunohistochemical staining patterns of SREBP-1 and G6PD in tumor tissues of 16 GBM patients on tissue microarray (TMA). The results showed that the expression levels of SREBP-1 and G6PD in tumor tissues of GBM patients were significantly positively correlated ( Figure 7 , some cases), among which G6PD expression was detected in 15 GBM tumor samples.

[0073] Example 3: G6PDi combined with inhibitors of new molecular points for the treatment of GBM

[0074] GBM cells were treated with G6PDi-1 and a novel molecular point inhibitor to test their synergistic therapeutic effect. The drug was purchased from MedChemExpress.

[0075] Treatment of GBM cells U87 with the SREBP1 inhibitor Fatostatin (2 μM) combined with the G6PD inhibitor G6PDi-1 (5 μg / ml, 10 μg / ml) for 3 days produced a dose-dependent growth inhibition and promoted U87 cell death. However, treatment of U87 with Fatostatin (2 μM) or G6PDi-1 (5 μg / ml, 10 μg / ml) alone for 3 days did not produce or only produced a weak growth inhibition effect ( Figure 8-9 Fatostatin).

[0076] Treatment of GBM cells U251 with the DGAT1 inhibitor A922500 (DGATi, 20 μg / ml) combined with the G6PD inhibitor G6PDi-1 (5 μg / ml, 10 μg / ml) for 4 days produced a dose-dependent growth inhibition and promoted U251 cell death. However, treatment of U251 with A922500 (20 μg / ml) or G6PDi-1 (5 μg / ml, 10 μg / ml) alone for 4 days did not produce or only produced a weak growth inhibition effect ( Figure 10-11A922500). After 2 weeks of treatment of GBM cells U251 with DGAT1 inhibitor A922500 (DGATi, 2μg / ml, 5μg / ml) combined with G6PD inhibitor G6PDi-1 (5μg / ml, 10μg / ml), a dose-dependent growth inhibition and cell death promotion of U251 cells were observed. ( Fig.12 A922500 colony formation assay)

[0077] Treatment of GBM cells U251 with the GPX4 inhibitor RSL-3 (2 μM) combined with the G6PD inhibitor G6PDi-1 (5 μg / ml, 10 μg / ml) for 4 days produced a dose-dependent growth inhibition and promoted U251 cell death. However, treatment of U251 with RSL-3 (2 μM) or G6PDi-1 (5 μg / ml, 10 μg / ml) alone for 4 days only produced a weak growth inhibition effect ( Figure 13-14 RSL-3).

Claims

1. A pharmaceutical composition for treating brain glioma, comprising a first pharmaceutical component and a second pharmaceutical component, The dosage ratio of the first drug component to the second drug component in the pharmaceutical composition is 1:0.1-2; The first pharmaceutical ingredient is G6PDi-1 or a pharmaceutically acceptable salt thereof, The second pharmaceutical ingredient is A922500 or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable excipient, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient selected from one or more of the following: a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizing agent, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant or an antibacterial agent.

3. The pharmaceutical composition according to claim 1, wherein the dosage forms of the first pharmaceutical ingredient and the second pharmaceutical ingredient in the pharmaceutical composition are the same or different.

4. The pharmaceutical composition of claim 3, wherein the dosage form comprises drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, solutions, ointments or creams.

5. The pharmaceutical composition according to claim 1, wherein the first pharmaceutical ingredient and the second pharmaceutical ingredient in the pharmaceutical composition are administered simultaneously or sequentially.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a product for treating brain glioma.

7. The use according to claim 6, wherein the brain glioma includes grade I, grade II, grade III, and grade IV brain glioma.

8. The use according to claim 6, wherein the brain glioma is glioblastoma multiforme.

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