Pharmaceutical composition for treating glioma

Abstract

The invention provides a pharmaceutical composition for treating glioma, and relates to the technical field of medicines. The pharmaceutical composition for treating glioma is characterized by being composed of a small molecule compound and temozolomide. The small molecule compound H027-2175C disclosed by the invention can penetrate through a blood brain barrier, and is specifically combined with and inhibits milk acylation modification of a PDP2 K38 site. By inhibiting PDP2 K38 lactoacylation and H027-2175C, TCA circulation activation mediated by lactic acid is blocked, so that the drug resistance of glioma cells to temozolomide is reversed. The combined use of the small molecule compound H027-2175C and temozolomide can significantly enhance the antitumor activity, specifically, in an in-vitro experiment, the combined medication significantly inhibits the proliferation of glioma cells, and the IC50 value is reduced; in an in-vivo experiment, the lifetime of a glioma model mouse is remarkably prolonged and the tumor volume is reduced by virtue of drug combination. The small molecule compound H027-2175C and temozolomide are combined for use, so that the toxic and side effects of temozolomide can be reduced, and the treatment safety is improved.

Description

TECHNICAL FIELD

[0001] The application provides a pharmaceutical composition for treating glioma, and belongs to the technical field of medicine. BACKGROUND

[0002] Glioma is the most common primary malignant tumor of the central nervous system, with high invasiveness and high recurrence rate. Despite the current variety of treatment methods (such as surgical resection, radiotherapy and chemotherapy), due to the high heterogeneity of glioma and the drug resistance to traditional treatment, the prognosis of patients is still poor. Temozolomide (TMZ) as a first-line chemotherapy drug for glioma, induces tumor cell apoptosis by alkylating DNA. However, the problem of temozolomide resistance seriously limits its clinical efficacy, especially in patients with recurrent and refractory glioma.

[0003] In recent years, studies have found that temozolomide resistance is closely related to metabolic reprogramming in the tumor microenvironment. In particular, in temozolomide-resistant glioma patients, the level of lactic acid is significantly elevated. Lactic acid is not only a byproduct of tumor metabolism, but also regulates the function of various proteins through lactylation, thereby affecting the metabolism and survival of tumor cells. Studies have shown that lactic acid can modify the K38 site of pyruvate dehydrogenase phosphatase 2 (PDP2) through lactylation, activate the tricarboxylic acid cycle (TCA cycle), promote the energy metabolism of glioma cells, and thus enhance their resistance to temozolomide.

[0004] Although there have been studies attempting to overcome temozolomide resistance by targeting metabolic pathways, there is currently no effective drug that can specifically inhibit the lactylation modification of the K38 site of PDP2, thereby reversing temozolomide resistance. In addition, due to the presence of the blood-brain barrier, many potential therapeutic drugs are difficult to effectively reach the tumor site, further limiting the development of glioma treatment.

[0005] The generation of temozolomide resistance is closely related to the elevated level of lactic acid and its mediated PDP2 K38 lactylation modification, which provides a new target for glioma treatment. However, there is a lack of drugs in the existing technology that can specifically inhibit PDP2 K38 lactylation modification and penetrate the blood-brain barrier. Therefore, developing a treatment strategy that can effectively target this mechanism and overcome temozolomide resistance has important clinical significance. SUMMARY

[0006] Based on this, the application provides a pharmaceutical composition for treating glioma, in particular, provides a pharmaceutical composition of a new small molecule compound combined with temozolomide.

[0007] The application is implemented by adopting the following technical solutions:

[0008] A pharmaceutical composition for treating glioma is composed of a small molecule compound H027-2175C and temozolomide.

[0009] The structural formula of the small molecule compound (H027-2175C) is as follows:

[0010] ;

[0011] Temozolomide (TMZ), and the chemical structure is as follows:

[0012] .

[0013] Preferably, the weight ratio of the small molecule compound H027-2175C and temozolomide is 1:10-10:1.

[0014] Preferably, the weight ratio of the small molecule compound H027-2175C and temozolomide is 1:2-2:1.

[0015] Preferably, the dosage form of the pharmaceutical composition is a tablet, a capsule or an injection.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The small molecule compound H027-2175C of the application can penetrate the blood-brain barrier, specifically bind to and inhibit the lactoylation modification of PDP2 K38. By inhibiting the lactoylation of PDP2 K38, H027-2175C blocks the lactic acid-mediated activation of the TCA cycle, thereby reversing the drug resistance of glioma cells to temozolomide. The small molecule compound H027-2175C of the application combined with temozolomide can significantly enhance the anti-tumor activity, which is specifically manifested as follows: in the in vitro experiment, the combination of drugs significantly inhibits the proliferation of glioma cells, and the IC 50The drug combination can reduce the side effects of temozolomide and improve the safety of treatment. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Comparison of the experimental effects of the drug composition of Example 1 and H027-2175C single drug and temozolomide single drug on tumor cell proliferation and apoptosis;

[0019] Figure 2 Comparison of the experimental effects of the drug composition of Example 1 and H027-2175C single drug and temozolomide single drug on mice;

[0020] Figure 3 Comparison of the effects of the drug composition of Example 1 and H027-2175C single drug and temozolomide single drug on each organ of mice. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present application clearer, the preferred embodiments of the present application will be further described in detail below in combination with examples. All other examples obtained by those skilled in the art without creative labor on the basis of the examples in the present application belong to the scope of protection of the present application. The small molecule compound H027-2175C described in the present application is provided by Antje Biotechnology Co., Ltd.

[0022] Example 1

[0023] A drug composition for treating glioma is composed of small molecule compound H027-2175C and temozolomide with a weight ratio of 1:2.

[0024] Example 2

[0025] A drug composition for treating glioma is composed of small molecule compound H027-2175C and temozolomide with a weight ratio of 1:1.

[0026] Example 3

[0027] A drug composition for treating glioma is composed of small molecule compound H027-2175C and temozolomide with a weight ratio of 2:1.

[0028] Example 4

[0029] A drug composition for treating glioma is composed of small molecule compound H027-2175C and temozolomide with a weight ratio of 5:2.

[0030] Example 5

[0031] A pharmaceutical composition for treating glioma consists of a small molecule compound H027-2175C and temozolomide in a weight ratio of 6:1.

[0032] Example 6

[0033] A pharmaceutical composition for treating glioma consists of a small molecule compound H027-2175C and temozolomide in a weight ratio of 10:1.

[0034] Example 7

[0035] A pharmaceutical composition for treating glioma consists of a small molecule compound H027-2175C and temozolomide in a weight ratio of 1:10.

[0036] A comprehensive drug evaluation was conducted on the pharmaceutical composition prepared in Example 1, temozolomide monotherapy, and H027-2175C monotherapy. The specific methods are as follows:

[0037] Blank control group: Contains no cells, only culture medium (for background fluorescence calibration).

[0038] Negative control group: Cells that have not been treated with any drugs.

[0039] H027 monotherapy group: using the small molecule compound H027-2175C.

[0040] TMZ monotherapy group: Temozolomide was used.

[0041] TMZ+H027 group: Pharmaceutical composition prepared in Example 1

[0042] Human glioblastoma U251 cell line was cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested, digested with 0.25% trypsin, resuspended, and counted. Cells were divided into groups of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 500 μL per well in 24-well plates. The plates were then incubated for 24 hours to allow for full adhesion.

[0043] Drug preparation: Both H027-2175C and TMZ were first prepared into high-concentration stock solutions using DMSO, then dispensed and stored at -20℃ protected from light. Before use, they were diluted with complete culture medium to the designed concentration.

[0044] Drug addition: After cell adhesion, discard the old culture medium. Add fresh complete culture medium containing the corresponding drug to each group as set up:

[0045] Negative control: complete medium containing 0.1% DMSO. H027-2175C single drug group: medium containing specific concentration of H027-2175C. TMZ single drug group: medium containing specific concentration of TMZ. Combination group: medium containing the same concentration of H027-2175C and TMZ. Put the cells back into the incubator for 48 hours of incubation.

[0046] (I) EdU detection after drug treatment, as follows:

[0047] 1) After drug treatment, according to the kit (such as BeyoClick™ EdU Cell Proliferation Kit) instructions.

[0048] 2) Preparation of EdU working solution: dilute the EdU stock solution with pre-warmed medium according to the recommended proportion (usually 10 μM).

[0049] 3) Absorb the drug-containing medium in each well, add 500 μL EdU working solution to each well.

[0050] 4) Put the cell plate back into the incubator for 2 hours of incubation, so that the cells in proliferation incorporate EdU into newly synthesized DNA.

[0051] 5) After EdU incubation, carefully aspirate the EdU working solution.

[0052] 6) Collect cells to 105mL EP tube by 0.25% trypsin digestion.

[0053] 7) Cell fixation: add 500 μL 4% paraformaldehyde (PFA) or formaldehyde fixing solution to each group, fix at room temperature for 15-30 minutes.

[0054] 8) Cell permeabilization: aspirate the fixing solution, wash each well with PBS for 5 minutes, 3 times. Aspirate the PBS, add 500 μL 0.5% Triton X-100 permeabilization solution to each group, incubate at room temperature for 15-20 minutes.

[0055] 9) Preparation of Click reaction solution: mix Azide dye, buffer, CuSO4 and additives according to the kit instructions in proportion. This step needs to be operated in the dark.

[0056] 10) Aspirate the permeabilization solution, wash the cells with PBS 2 times.

[0057] 11) Add enough freshly prepared Click reaction solution to each well (make sure to completely cover the sample), incubate at room temperature for 30 minutes in the dark.

[0058] 12) Flow cytometry detection, use Flowjo for plottingFigure 1 - A)

[0059] 13) Repeat three times to obtain statistical graph Figure 1 - B)

[0060] 14) Use software such as GraphPad Prism to perform One-way ANOVA and post-hoc test (such as Tukey's test) to assess the statistical significance of differences between groups (p < 0.05 is generally considered a significant difference).

[0061] (B) Perform apoptosis detection, specifically: 1) Collect cells: After treatment, carefully collect the culture supernatant into a flow tube with a pipette. Digest the adherent cells with trypsin without EDTA (digestion time should not be too long to avoid false positives), until the cells can be blown down gently. Use the collected supernatant to terminate digestion, and transfer the entire cell suspension to the corresponding flow tube. 2) Centrifugation: centrifuge at 1000 rpm for 5 minutes at 4°C, discard the supernatant. 3) Washing: resuspend the cell pellet with pre-cooled PBS, centrifuge again, and discard the supernatant. Repeat this step once. 4) Annexin V-APC / PI staining, follow the instructions of the Annexin V-FITC Apoptosis Detection Kit. 5) Flow cytometry detection 6) Data analysis and result interpretation: use FlowJo software to analyze the data and plot as shown in Figure (1-C); 7) Repeat three times to obtain statistical graph Figure 1 - D)

[0062] (C) Cell clone detection

[0063] 1) Take U251 cells in logarithmic growth phase, digest to prepare a single cell suspension, and count the cells. Seed 500 cells per well in a 6-well plate with 2 mL complete medium. Gently shake the cross to distribute the cells evenly. Place the cell plate in a 37°C, 5% CO2 incubator. After the cells adhere: culture for 24 hours, and after the cells completely adhere, discard the original culture medium. According to the experimental grouping, add fresh complete medium containing the corresponding concentration of drug. Each concentration group has at least 3 replicate wells.

[0064] 2) After 48 hours of drug treatment, carefully aspirate the drug-containing culture medium, and gently wash the cells with pre-warmed PBS twice to completely remove the drug. Replace each well with 2 mL of fresh complete medium, and then replace the fresh medium every 3 days.

[0065] 3) Clone culture and fixation: continue to culture for 10-14 days, or until visible cell clones (usually > 50 cells) form in the blank control group. At the end of the culture, discard the culture medium and wash the cells with PBS twice.

[0066] 4) Cell fixation: Add 1-2 mL 4% paraformaldehyde or methanol per well, and fix at room temperature for 15-30 minutes. Discard the fixative, and gently wash once with PBS.

[0067] 5) Clone staining and imaging: Cell staining: Add appropriate amount of crystal violet staining solution (0.1% or 0.5% w / v) or Giemsa staining solution per well, and stain at room temperature for 15-30 minutes. Carefully discard the staining solution, and rinse the plates very slowly and gently with running water until the background is transparent and no excess dye remains. Invert the plates in a well-ventilated area, and air-dry at room temperature.

[0068] 6) Clone counting and analysis: Take a photo of each well using the naked eye or a microscope scanner (e.g. Figure 1 -E). Clone counting criteria: A cell mass with more than 50 cells is usually defined as a countable clone. Count the number of clones in each well manually.

[0069] 7) Calculate the clone formation rate by repeating the experiment three times (e.g. Figure 1 -F)

[0070] Statistical analysis was performed using software such as GraphPad Prism. Data are expressed as mean ± standard deviation (Mean ± SD), and one-way ANOVA was used to compare differences between groups, with *p < 0.05, **p < 0.01, and ***p < 0.001 considered statistically significant.

[0071] Research plan for constructing a nude mouse orthotopic glioma model using the U251-Luc cell line and evaluating the combined treatment effect of H027-2175C and TMZ

[0072] 1. Purpose of the experiment

[0073] A stable and monitorable orthotopic model of human U251-Luc glioma in nude mice was established to evaluate the effects of H027-2175C monotherapy, temozolomide (TMZ) monotherapy, and their combination on tumor growth and mouse survival, and to perform final verification through immunohistochemistry.

[0074] 2. Experimental materials and animals

[0075] Cell line: U251-Luc cells (a human glioblastoma cell line stably expressing luciferase)

[0076] Animals: BALB / c nude mice (female, 4 weeks old), weighing 18-22 g. All animals were raised in a SPF-level environment with free access to food and water. The experiment was approved by the animal ethics committee.

[0077] 3. Experimental grouping and protocol

[0078] Grouping: 20 nude mice were randomly divided into 4 groups (n=5), as follows:

[0079] Control group: After injection of tumor cells, the corresponding volume of drug solvent was given.

[0080] H027 monotherapy group: After injection of tumor cells, H027-2175C 10 mg / kg was given.

[0081] TMZ monotherapy group: After injection of tumor cells, TMZ 5 mg / kg was given.

[0082] TMZ+H027 combination group (Example 1 drug composition): After injection of tumor cells, H027-2175C and TMZ were given simultaneously (doses same as monotherapy group).

[0083] Among them, the administration route and frequency are shown in the schematic diagram Figure 2 -A).

[0084] 4. Construction of orthotopic glioma model (stereotactic injection)

[0085] Cell preparation: Take U251-Luc cells in logarithmic growth phase, digest and resuspend with PBS or serum-free medium, adjust the cell concentration to 5×10 7 cells / mL, and place on ice for standby.

[0086] Animal anesthesia and fixation: Anesthetize the mice with isoflurane (3% induction, 1.5-2% maintenance) or pentobarbital sodium (50 mg / kg ip). Fix the head of the mouse on the stereotaxic instrument.

[0087] Surgical preparation: Disinfect the skin with iodophor and 75% alcohol alternately.

[0088] Positioning and injection: Position at the Bregma position. Injection coordinates: 1.0 mm in front of the bregma, 2.0 mm right of the midline, depth 3.0 mm (can be fine-tuned according to the reference). Use a skull drill to drill a micro-hole at the target coordinates. Use a Hamilton microsyringe (with a 26G needle) to suck 5 μL of cell suspension (about 2.5×10 5Cell injection: The needle is inserted slowly to the target depth, and wait for 1 minute for tissue stabilization. Inject the cell suspension slowly at a rate of 1 μL / min. After injection, leave the needle for 5 minutes, then slowly withdraw the needle at a rate of 0.5 mm per minute to prevent backflow of cells. Post-operative care: Suture the skin, place the mouse on a heating pad until it wakes up, and return it to the cage for continued feeding.

[0089] 5. In vivo pharmacodynamic evaluation indicators

[0090] Survival observation: Observe the survival state of the mice every day, and record the survival time. Take euthanasia when neurological symptoms (such as head tilt, circling, lethargy, cachexia) or weight loss of more than 25% appear, and record the time as the death time. Draw the Kaplan-Meier survival curve**, and use the Log-rank test for intergroup comparison. The plot is shown in (2-B).

[0091] Body weight monitoring: Weigh and record the body weight of the mice every 2-3 days as an indicator for evaluating the systemic toxicity of the drug. More than 20% weight loss requires consideration of the humane endpoint. The plot is shown in (2-C).

[0092] In vivo imaging (IVIS) is used to monitor tumor growth, with the following steps:

[0093] a. Intraperitoneal injection of D-luciferin potassium salt (150 mg / kg).

[0094] b. 10-15 minutes after injection, anesthetize the mice with isoflurane.

[0095] c. Place the mice in the imaging chamber and collect photon signals (exposure time is usually 1-60 seconds, automatic or manual adjustment).

[0096] Data analysis: Use Living Image and other software to draw the region of interest (ROI) in the tumor area, and quantify the total photon flux (Total Flux, photons / s) as an indicator of tumor size. Draw the tumor growth curve. The plot is shown in (2-D, E).

[0097] 6. End-point sample collection and immunohistochemistry (IHC) analysis

[0098] At the end of the experiment (when the mice are near death or at the predetermined time point), euthanize all mice and collect brain tissue. Fix the brain tissue in 4% paraformaldehyde for 24-48 hours. Perform paraffin embedding and coronal sectioning along the needle tract (thickness 4-5 μm). Hematoxylin-eosin (H&E) staining: observe the tumor morphology, boundary, necrosis, etc.

[0099] Evaluation of organ toxicity of H027 in mice by histopathology and immunohistochemistry

[0100] 1. Purpose of the experiment

[0101] To systematically evaluate the potential organ toxicity of H027 by observing the histomorphological changes and expression changes of specific biomarkers in the major organs (liver, kidney, heart, lung, brain) of nude mice treated with H027, and to provide a basis for subsequent preclinical safety evaluation.

[0102] 2. Materials and samples

[0103] Tissue samples: liver, kidney, heart, lung, and brain organs from all groups (control group, H027 monotherapy group, TMZ monotherapy group, combination therapy group) of mice in the "in situ glioma model efficacy experiment".

[0104] 3. Experimental procedure

[0105] 3.1. Tissue sample collection and processing

[0106] Immediately after euthanasia of the mice at the end of the experiment, the complete liver, kidney, heart, lung, and brain tissues were carefully removed. The blood was gently washed with pre-cooled PBS, and the surface liquid was absorbed with filter paper. The tissue block was immediately immersed in sufficient 4% paraformaldehyde fixing solution (at least 10 times the volume of the tissue block), and fixed at room temperature for 24-48 hours. After fixation, the tissue was dehydrated, transparentized, and paraffin-embedded according to the routine procedure.

[0107] 3.2. Paraffin sectioning and H&E staining

[0108] Each tissue block was serially sectioned using a paraffin microtome with a thickness of 4-5 μm. The sections were mounted on glass slides and placed in a 60°C oven for 2 hours to tightly adhere the tissue. Hematoxylin-eosin (H&E) staining was performed.

[0109] H&E stained sections were used to observe basic tissue morphological structure, cell degeneration, necrosis, inflammatory cell infiltration, fibrosis, and other pathological changes. The results are shown in Figure 3 .

[0110] The above-described embodiments are part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

Claims

1. A pharmaceutical composition for treating glioma, characterized in that, consists of a small molecule compound and temozolomide; The small molecule compound has the following structural formula: 。 2. The pharmaceutical composition for treating glioma according to claim 1, wherein The weight ratio of the small molecule compound and temozolomide is 1:10-10:

1.

3. The pharmaceutical composition for treating glioma according to claim 2, wherein The weight ratio of the small molecule compound and temozolomide is 1:2-2:

1.

4. The pharmaceutical composition for treating glioma according to claim 1, wherein The dosage form of the pharmaceutical composition is a tablet, a capsule or an injection.

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