G6pd inhibitor and rsl-3 combination therapy for brain glioma

Abstract

The application relates to a combined application of a G6PD inhibitor and RSL-3 in treating brain glioma and belongs to the fields of medicine and biotechnology. Specifically, a treatment scheme for the synergistic treatment of glioblastoma multiforme by the G6PD inhibitor and RSL-3 is provided. The application also provides the application of G6PD in the diagnosis, grading and prognosis of brain glioma.

Description

[0001] The present application is a divisional application, the parent application information is as follows: the invention name is the combination of G6PD inhibitors and new molecular point inhibitors; the original application date is October 20, 2023, and the original application number is 2023113640359. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine and biotechnology, and specifically relates to the combination of G6PD inhibitors and RSL-3 for treating brain glioma. BACKGROUND

[0003] Glioblastoma Multiform (GBM) is a WHO IV grade glioma, and the median survival time of patients after diagnosis is only 12-15 months. It is the malignant tumor with the highest mortality rate in the world, and the prognosis of GBM patients has not improved in the past few decades. The main reason is the lack of effective treatment strategies.

[0004] The standard treatment for GBM is first to perform maximum surgical resection, and then to perform radiotherapy and Temozolomide (TMZ) treatment simultaneously for 6 weeks, and then to continue TMZ treatment once every 4 weeks for 6 cycles. However, the effective period of this treatment method is only a few months, and almost all GBM tumors inevitably relapse after treatment. In addition to TMZ, only bevacizumab has been approved by FDA for clinical treatment of recurrent GBM. However, bevacizumab can only effectively alleviate symptoms and improve quality of life in the short term, and bevacizumab has no synergistic effect with TMZ and radiotherapy; in addition, it also reduces the absorption of TMZ, cannot improve the overall survival rate, and has obvious side effects such as hypertension, venous thrombosis and infection.

[0005] Recent studies have shown that metabolic reprogramming is one of the core characteristics of tumors, and 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 the energy supply of GBM. SUMMARY

[0006] In order to provide an effective diagnosis and treatment approach for brain glioma, the present application provides the following technical solutions:

[0007] Pharmaceutical composition

[0008] In a first aspect, the present application provides a pharmaceutical composition for treating brain glioma, the pharmaceutical composition comprising a first drug component and a second drug component,

[0009] The first drug component is a G6PD inhibitor,

[0010] The second pharmaceutical ingredient is one or more of A922500, RSL-3, Fatostatin.

[0011] The term “G6PD inhibitor” as used herein refers to a substance that targets, reduces or inhibits at least one activity of G6PD.

[0012] Preferably, the G6PD inhibitor comprises an artificially synthesized or naturally occurring one.

[0013] Preferably, the G6PD inhibitor comprises a reagent used in a gene editing technology such as an RNA interference technology, an antisense oligonucleotide (ASO) technology, a CRISPR technology, a TALEN technology, a ZFN technology, a Cre-loxP gene recombination technology, etc., and also comprises a compound that can reduce the expression of G6PD.

[0014] In particular, the G6PD inhibitor is G6PDi-1 or a pharmaceutically acceptable salt thereof. The G6PDi-1 is a reversible, non-competitive 6-phosphogluconate dehydrogenase (G6PD) inhibitor with an IC50 of 0.07 μM, and its structural formula is:

[0015] In particular, the A922500 comprises a pharmaceutically acceptable salt thereof, the RSL-3 comprises a pharmaceutically acceptable salt thereof, and the Fatostatin comprises a pharmaceutically acceptable salt thereof.

[0016] Preferably, the brain glioma comprises a grade I, grade II, grade III, or grade IV brain glioma.

[0017] Preferably, the brain glioma is Glioblastoma Multiform (GBM), also known as Glioblastoma, which is a grade IV brain glioma.

[0018] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, which comprises any one or a combination of at least two of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent, or a buffer.

[0019] In a particular embodiment, the first pharmaceutical ingredient and the second pharmaceutical ingredient in the pharmaceutical composition are in the same or different dosage forms.

[0020] Preferably, the dosage form comprises a drop, an oral liquid, a tablet, a capsule, a granule, a film, a gel, a powder, an emulsion, a dripping pill, a suppository, an aerosol, a spray, a powder spray, a patch, a solution, an ointment, or a cream.

[0021] In a specific embodiment, the first and second pharmaceutical components of the pharmaceutical composition are administered simultaneously or sequentially, in particular, administration can be separated by 0, 1, 2, 3, 4, 5, 6, 7 or more days.

[0022] In a specific embodiment, the dose ratio of the first and second pharmaceutical components of the pharmaceutical composition is 1:0.1-5; in particular, 5:1, 4:1, 3:1, 2.5:1, 2:1, 1:1, 1:2, 1:2.5, 1:3, 1:4, 1:5.

[0023] Certain compositions of the present application can further comprise a pharmaceutically acceptable carrier or excipient, by which is meant a nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0024] Preferably, the pharmaceutical composition can further comprise other cancer drugs, such as: Acivicin, Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium, Bropirimine, Busulfan, Cactinomycin, Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin Hydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, Cisplatin, Cladribine, etc.

[0025] In another aspect, the present application also provides a use of a G6PD inhibitor in the preparation of a medicament for enhancing the sensitivity of a patient to a new molecular inhibitor, wherein the new molecular inhibitor comprises one or more of A922500, RSL-3, Fatostatin.

[0026] Preferably, the patient is a brain glioma patient.

[0027] Preferably, the brain glioma comprises grade I, grade II, grade III, grade IV brain glioma.

[0028] Preferably, the brain glioma is Glioblastoma Multiform (GBM, also known as Glioblastoma), i.e. grade IV brain glioma.

[0029] On the other hand, the present invention provides a use of a pharmaceutical composition in the treatment of 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.

[0030] Preferably, the pharmaceutical composition can also be used in combination with other anticancer drugs and cancer treatment methods.

[0031] 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.

[0032] 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.

[0033] As used herein, the term "treating" refers to a method of reducing the symptoms / effects of a disease. As used herein, treating can refer to a decrease in severity or improvement in the symptoms / effects of an established disease by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. It is to be understood that treating does not necessarily mean curing or completely eliminating a disease, condition, or symptoms of a disease or condition.

[0034] 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, reptile, or amphibian. The term does not denote a particular age or sex. Thus, the term encompasses both adult and newborn subjects, regardless of sex.

[0035] As used herein, patient or subject are used interchangeably to 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, also known as glioblastoma multiforme (GBM).

[0036] Use as a marker

[0037] On the other hand, the present invention also provides the use of a reagent for detecting the expression level of G6PD in the preparation of a product for diagnosing glioblastoma.

[0038] Specifically, the expression level of G6PD is higher in GBM than in normal brain tissue.

[0039] In another aspect, the present application also provides use of a reagent for detecting the expression level of G6PD in the preparation of a product for grading brain glioma.

[0040] Specifically, the brain glioma includes grades 1-4, and the expression level of G6PD increases with the grade of brain glioma.

[0041] In another aspect, the present application also provides use of a reagent for detecting the expression level of G6PD in the preparation of a product for predicting the prognosis of brain glioma.

[0042] Preferably, the indicator of prognosis is the probability of survival.

[0043] Preferably, the reagent for detecting the expression level of G6PD includes a reagent for detecting the expression level of G6PD protein and / or G6PD mRNA.

[0044] Preferably, the reagent for detecting the expression level of G6PD protein includes a reagent used in the following methods: hematoxylin-eosin staining (HE staining), Ponceau O-Gallocyanin staining, Western Blot, ELISA, RIA, sandwich assay, immunohistochemical staining method, mass spectrometry method, immunoprecipitation analysis method, complement binding analysis method, flow cytometry fluorescence fractionation technology, and protein chip method.

[0045] Preferably, the reagent for detecting the expression level of G6PD mRNA includes a reagent 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, high-throughput sequencing platform method.

[0046] Preferably, the detection is performed on a sample from a subject.

[0047] Preferably, the sample includes tissue, cells (including cell culture), blood, urine, saliva, mucosal sample, feces, intestinal lavage, joint fluid, cerebrospinal fluid, bile sample, respiratory secretions, bronchoalveolar lavage fluid sample.

[0048] Preferably, the sample is tissue, cells.

[0049] Preferably, the product comprises a kit, a chip, a test strip, or, a device, an apparatus, a computer readable storage medium.

[0050] Those skilled in the art of the relevant technical field understand that the disclosure can be implemented as complete hardware, complete software (including firmware, resident software, microcode, etc.), or a combination of hardware and software. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 is the comparison result of G6PD expression in GBM and normal brain tissue.

[0052] Figure 2 is the comparison result of G6PD expression in different grades of glioma.

[0053] Figure 3 is the survival curve of G6PD in GBM.

[0054] Figure 4 is the result of analyzing G6PD expression in different grades of glioma using tissue chips.

[0055] Figure 5 is the survival curve drawn according to the detection result of the tissue chip.

[0056] Figure 6 is the correlation analysis result graph of G6PD and other genes.

[0057] Figure 7 is the expression result of SREBP-1 and G6PD in the tumor tissue of GBM patients.

[0058] Figure 8 is the cell number statistical result graph of U87 cells treated with the SREBP1 inhibitor Fatostatin and G6PDi-1 in combination.

[0059] Figure 9 is the microscopic image of U87 cells treated with the SREBP1 inhibitor Fatostatin and G6PDi-1 in combination.

[0060] Figure 10 is the cell number statistical result graph of U251 cells treated with the DGAT1 inhibitor A922500 and G6PDi-1 in combination.

[0061] Figure 11 is the microscopic image of U251 cells treated with the DGAT1 inhibitor A922500 and G6PDi-1 in combination.

[0062] Figure 12A922500, an inhibitor of DGAT1, and G6PDi-1. The results of colony formation are shown in the graph.

[0063] Figure 13 RSL-3, an inhibitor of GPX4, and G6PDi-1. The results of cell counting are shown in the graph.

[0064] Figure 14 RSL-3, an inhibitor of GPX4, and G6PDi-1. The microscopic images of U251 cells treated with the combination are shown. DETAILED DESCRIPTION

[0065] The application will be further described below in conjunction with the embodiments. The following description is only the preferred embodiments of the application and does not limit the application in other forms. Any skilled person in the art can modify the above disclosed technical content into equivalent embodiments with equivalent changes. Any simple modification or equivalent change made to the following embodiments according to the technical essence of the application without departing from the scheme of the application falls within the protection scope of the application.

[0066] Example 1, role of G6PD in GBM diagnosis, grading, prognosis

[0067] Through analysis of 32 kinds of solid tumor RNAseq data of The Cancer Genome Atlas (TCGA), it was found that the expression of G6PD in GBM was significantly higher than that in normal brain tissue (P<0.001) Figure 1 ); the expression in GBM was also significantly higher than that in low-grade glioma (LGG) (P<0.001) Figure 2 ). The high expression of G6PD was significantly related to the poor survival rate of patients in the GBM cohort (P<0.001) Figure 3 ).

[0068] Accordingly, we further detected the G6PD protein level in tumor tissues of patients with I-IV grade glioma (n=145) using tissue microarray (TMA). The results of IHC staining showed that the G6PD protein content in IV grade GBM tissue was the highest compared with III grade glioma, II grade glioma and I grade glioma (P<0.001) Figure 4 ); in addition, survival analysis showed that the high G6PD protein level in the 145 glioma tissues was closely related to the low survival rate of patients (P<0.001) Figure 5 ). These results suggest that G6PD may be a new molecular marker of GBM and may play an important role in the occurrence and development of GBM.

[0069] Example 2, G6PDi enhances sensitivity of cancer cells to lipogenesis inhibitors

[0070] The analysis of RNAseq data of 153 patients with primary tumors in the GBM cohort of TCGA showed that the expression of G6PD was significantly positively correlated with the expression of lipid synthesis-related genes such as SREBP1, FASN, ACC, and other genes related to maintaining the redox state of the cell such as GPX4, TXN, B166, etc. Figure 6 ).

[0071] 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 from 16 GBM patients on a tissue microarray (TMA). The results showed that the expression of SREBP-1 and G6PD in tumor tissues from GBM patients was significantly positively correlated (Fig. 6B, part of cases), and G6PD expression was detected in 15 GBM tumor samples. Figure 7

[0072] Example 3, G6PDi combined with new molecular point inhibitors for GBM treatment

[0073] To test the synergistic therapeutic effect of G6PDi-1 and a new molecular point inhibitor, we purchased G6PDi-1 from MedChemExpress company

[0074] Treatment of GBM cells U87 with SREBP1 inhibitor Fatostatin (2 μM) in combination with G6PD inhibitor G6PDi-1 (5 μg / ml, 10 μg / ml) for 3 days resulted in dose-dependent inhibition of growth and promotion of U87 cell death. However, treatment of U87 with Fatostatin (2 μM) alone or G6PDi-1 (5 μg / ml, 10 μg / ml) alone for 3 days did not produce or only weakly produced an inhibitory effect on growth (Fig. 6C, Fatostatin). Figure 8-9

[0075] Treatment of GBM cells U251 with DGAT1 inhibitor A922500 (DGATi, 20 μg / ml) in combination with G6PD inhibitor G6PDi-1 (5 μg / ml, 10 μg / ml) for 4 days resulted in dose-dependent inhibition of growth and promotion of U251 cell death. However, treatment of U251 with A922500 (20 μg / ml) alone or G6PDi-1 (5 μg / ml, 10 μg / ml) alone for 4 days did not produce or only weakly produced an inhibitory effect on growth (Fig. 6D, DGATi). Figure 10-11 ​​A922500). Treatment of GBM cells U251 with the DGAT1 inhibitor A922500 (DGATi, 2 pg / ml, 5 pg / ml) in combination with the G6PD inhibitor G6PD i-1 (5 pg / ml, 10 pg / ml) for 2 weeks resulted in a dose-dependent inhibition of growth and promotion of U251 cell death (p<0.05, p<0.01, p<0.001, respectively) (Figure 6A). Figure 12 A922500 colony formation assay).

[0076] Treatment of GBM cells U251 with the GPX4 inhibitor RSL-3 (2 mM) in combination with the G6PD inhibitor G6PD i-1 (5 pg / ml, 10 pg / ml) for 4 days resulted in a dose-dependent inhibition of growth and promotion of U251 cell death. However, treatment of U251 with RSL-3 (2 mM) alone or G6PD i-1 (5 pg / ml, 10 pg / ml) alone for 4 days resulted in only weak inhibition of growth (p<0.05, p<0.01, respectively) (Figure 6B). Figure 13-14 RSL-3).

Claims

1. A pharmaceutical composition for treating brain glioma, characterized in that: The active ingredients of the pharmaceutical composition are a first component and a second component; The first component is a G6PD inhibitor; The second component is a GPX4 inhibitor; The G6PD inhibitor is G6PDi-1 or a pharmaceutically acceptable salt thereof; The GPX4 inhibitor is RSL-3 or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, which is selected from one or more of the following: Fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants or bacteriostats.

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

4. The pharmaceutical composition according to claim 3, characterized in that The dosage forms include 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, characterized in that The first component and the second component are administered simultaneously or sequentially.

6. The pharmaceutical composition according to claim 5, characterized in that The intervals between successive administrations are 0, 1, 2, 3, 4, 5, 6, 7 or more days.

7. The pharmaceutical composition according to claim 1, characterized in that The dosage ratio of the first component to the second component is 1:0.1-2.

8. The pharmaceutical composition according to claim 1, characterized in that The brain glioma includes grade I, grade II, grade III and grade IV brain glioma.

9. The pharmaceutical composition according to claim 1, characterized in that The brain glioma is glioblastoma multiforme.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a product for treating brain glioma.

11. The use according to claim 10, characterized in that The brain glioma includes grade I, grade II, grade III and grade IV brain glioma.

12. The use according to claim 10, characterized in that The brain glioma is glioblastoma multiforme.

13. The use according to claim 10, characterized in that The product includes a kit.

Images