Use of chlorogenic acid in preparation of drug for treating diffuse midline gliomas

Abstract

The use of chlorogenic acid in the preparation of a drug for treating diffuse midline gliomas, which use belongs to the field of medicine. Cell experiments and animal experiments prove that chlorogenic acid can induce the apoptosis of diffuse midline brain stem glioma cells, and the proportion of apoptotic cells increases as the chlorogenic acid concentration increases. Compared with temozolomide, a conventional standard chemotherapeutic drug for glioblastoma, chlorogenic acid has a significantly improved inhibitory effect on diffuse brain stem glioma, and has low toxicity and few side effects. Chlorogenic acid significantly prolongs the survival time of a mouse model of diffuse brain stem glioma. Chlorogenic acid has an inhibitory effect on diffuse brain stem glioma, can inhibit the growth of diffuse brain stem glioma, provides a new, safer option for treating diffuse midline gliomas, and has good application prospects.

Description

Use of chlorogenic acid in preparing medicine for treating diffuse midline glioma Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to use of chlorogenic acid in preparing a medicine for treating diffuse midline glioma. Background Art

[0002] Diffuse midline gliomas (DMGs) are a type of primary brain tumor. They primarily occur in children and young adults, and are less common in older individuals. They are often located in midline structures such as the thalamus, brainstem, and spinal cord. The high incidence of histone H3K27M mutations is a key molecular biological basis for their occurrence. DMGs in the brainstem and thalamus are particularly aggressive, capable of diffusely invading surrounding normal brain tissue, making them one of the most lethal brain tumors.

[0003] The primary biological hallmark of DMG is the lysine 27 to methionine (K27M) mutation in histone 3 (H3), which induces widespread changes in the epigenome and gene expression and is considered a key driver of tumor initiation and maintenance. Due to the unique expression of H3K27 mutations in diffuse midline gliomas and their significant correlation with prognosis, the 2016 WHO classification of central nervous system tumors first classified them as WHO grade IV. Pathologically, these tumors are characterized by histone H3K27M mutations, diffuse infiltrative growth, and a high degree of malignancy. Treatment and prognosis are unsatisfactory, with a median survival of less than one year. Currently, there is no standard treatment regimen, and surgical resection and conventional chemoradiotherapy are unlikely to improve prognosis, particularly in cases located in the brainstem.

[0004] Diffuse midline brainstem glioma (DIPG) accounts for 10% to 15% of central nervous system tumors in children. Despite decades of research and development, the mortality rate remains 100% once diagnosed. Because the brainstem is densely packed with vital neural nuclei and fibers, surrounded by numerous blood vessels, and because brainstem gliomas grow infiltratingly, surgery offers little benefit and may often worsen neurological impairment. A growing number of chemotherapy options, including temozolomide, a standard chemotherapy drug routinely used for glioblastoma, have also been shown to be ineffective in improving the prognosis of DIPG. The current internationally recognized standard of care is conventional radiotherapy, but its effectiveness is limited to temporary symptom relief.

[0005] Therefore, in view of the dilemma that there is no effective treatment for diffuse midline glioma, exploring a drug for diffuse midline glioma has great value and significance in improving patients' prognosis and prolonging their survival.

[0006] Chlorogenic acid, a depsipeptide formed from caffeic acid and quinic acid, is a phenylpropanoid compound produced by plants via the shikimic acid pathway during aerobic respiration. Chlorogenic acid exhibits a wide range of biological activities, including anticancer, antibacterial, antiviral, hepatoprotective, choleretic, blood pressure-lowering, lipid-lowering, free radical scavenging, and central nervous system stimulating effects, demonstrating remarkable medicinal and social value.

[0007] It has been reported that chlorogenic acid can treat oligodendrogliomas and glioblastomas, but the "Guidelines for the Diagnosis and Treatment of Brain Glioma (2022 Edition)" clearly states that diffuse midline gliomas are fundamentally different from glioblastomas and oligodendrogliomas in terms of imaging characteristics: when MRI / CT performance is similar to astrocytoma or oligodendroglioma with enhancement, it indicates that anaplastic glioma is likely. Glioblastoma is characterized by irregular peripheral enhancement and massive central necrosis, and edema can be seen outside the enhancement. Diffuse midline gliomas often occur in midline structures such as the thalamus and brainstem. MRI shows long T1 and long T2 signals, and enhanced scans can have varying degrees of enhancement.

[0008] According to the classification standards, glioblastomas with wild-type IDH are classified as adult-type diffuse gliomas, while diffuse midline gliomas with H3K27 mutations are classified as pediatric-type diffuse high-grade gliomas. The new classification for the first time divides diffuse gliomas into two categories: adult-type diffuse gliomas and pediatric-type diffuse gliomas. It is important to note that this diagnostic classification is not based solely on the age of tumor onset, but rather on clinical characteristics such as the primary molecular alterations and the distribution of these tumors across different age groups.

[0009] So far, there are no reports that chlorogenic acid has a therapeutic effect on diffuse midline glioma.

[0010] Summary of the Invention

[0011] The object of the present invention is to provide a use of chlorogenic acid in preparing a medicament for treating diffuse midline glioma.

[0012] The present invention provides a use of chlorogenic acid in preparing a medicine for treating childhood diffuse high-grade glioma.

[0013] Furthermore, the pediatric diffuse high-grade glioma is a diffuse midline glioma.

[0014] Furthermore, the diffuse midline glioma is a diffuse midline brainstem glioma.

[0015] Furthermore, the drug is a drug that inhibits the growth of diffuse midline brainstem glioma.

[0016] Furthermore, the drug is a drug that inhibits tumors caused by H3K27 mutations.

[0017] Furthermore, the drug is a preparation prepared with an effective amount of chlorogenic acid as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0018] Furthermore, each preparation unit of the medicine contains 1 to 2000 mg of chlorogenic acid.

[0019] Furthermore, each preparation unit of the medicine contains 10 to 200 mg of chlorogenic acid.

[0020] Furthermore, each preparation unit of the medicine contains 30 mg of chlorogenic acid.

[0021] Furthermore, the preparation is an oral preparation or an injection.

[0022] H3K27 mutation refers specifically to a lysine 27 to methionine (K27M) mutation in histone 3 (H3).

[0023] The present invention provides a use of chlorogenic acid in the preparation of a drug for treating diffuse midline gliomas, in particular, its use in the preparation of a drug for treating diffuse brainstem gliomas. Cell experiments and animal experiments of the present invention have shown that chlorogenic acid can induce apoptosis in diffuse midline brainstem glioma cells, and the proportion of apoptotic cells increases with the increase of chlorogenic acid concentration. Compared with temozolomide, a standard chemotherapy drug conventionally used for glioblastoma, chlorogenic acid has a significantly improved inhibitory effect on diffuse brainstem gliomas, and chlorogenic acid has low toxic and side effects. Chlorogenic acid significantly prolongs the survival time of mice with diffuse brainstem glioma models. The present invention is the first to discover that chlorogenic acid has an inhibitory effect on diffuse brainstem gliomas and can inhibit the growth of diffuse brainstem gliomas, providing a new and safe option for the treatment of diffuse midline gliomas, with good application prospects.

[0024] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0025] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a graph showing the results of chlorogenic acid-induced apoptosis of diffuse midline brainstem glioma cells.

[0027] FIG2 is a graph showing the tumor weights of mice in each group.

[0028] FIG3 is a graph showing the survival time of mice in each group. DETAILED DESCRIPTION

[0029] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0030] Chlorogenic acid, provided by Sichuan Jiuzhang Biotechnology Co., Ltd., has a content of 99.61%.

[0031] Patient tissues from diffuse midline brainstem gliomas were provided by Beijing Tiantan Hospital, Capital Medical University.

[0032] Example 1 Prescription of oral preparation of the pharmaceutical composition of the present invention

[0033] 1. Prescription 1

[0034] Chlorogenic acid 1000g.

[0035] Preparation method: Weigh chlorogenic acid according to the prescription and aseptically package it into powders.

[0036] 2. Prescription 2

[0037] Chlorogenic acid 1000g, filler 2000g, binder 10g.

[0038] Preparation method: Weigh chlorogenic acid, filler, and binder according to the prescription, granulate, shape the particles, and package them into granules.

[0039] 3. Prescription Three

[0040] Chlorogenic acid 1000g, filler 2000g, binder 10g, lubricant 5g.

[0041] Preparation method: Weigh chlorogenic acid, filler, and binder according to the prescription, granulate, shape the granules, add lubricant, and press into tablets to obtain tablets.

[0042] The filler is one or more of mannitol, lactose, pregelatinized starch, microcrystalline cellulose, and dextrin; the binder is sodium carboxymethyl cellulose and PVP; and the lubricant is magnesium stearate and micropowder silica gel.

[0043] Example 2 Prescription of the injection preparation of the pharmaceutical composition of the present invention

[0044] 1. Prescription 1

[0045] Chlorogenic acid 1000g.

[0046] Preparation method (1): Aseptically weigh chlorogenic acid according to the prescription, mix evenly, and aseptically package into powder injection. Preparation method (2): Aseptically weigh chlorogenic acid according to the prescription, dissolve it in water for injection, filter sterilize, and freeze-dry to obtain freeze-dried powder injection.

[0047] 2. Prescription 2

[0048] Chlorogenic acid 1000g, scaffolding agent 2667g, antioxidant 67g.

[0049] Preparation method: Weigh chlorogenic acid, scaffold agent, and antioxidant according to the prescription, dissolve in water for injection, filter and sterilize, and freeze-dry (freeze-drying conditions: pre-freezing: temperature ≤-40°C, normal pressure, drying time 2-4h, freezing; primary drying: temperature ≤-13°C, negative pressure, drying time ≥12h, sufficient drying; secondary drying, temperature 20-30°C, negative pressure, drying time ≥2h) to obtain freeze-dried powder injection.

[0050] The above-mentioned scaffolding agents are mannitol, lactose and glucose; the antioxidants are sodium bisulfite, vitamin C, glutathione and folic acid.

[0051] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0052] Experimental Example 1 In vitro study of chlorogenic acid in the treatment of diffuse midline glioma

[0053] 1. Experimental Methods

[0054] (1) Experimental materials

[0055] 1. Chlorogenic acid, provided by Sichuan Jiuzhang Biotechnology Co., Ltd.

[0056] 2. Patient tissues of diffuse midline brainstem glioma were provided by Beijing Tiantan Hospital affiliated to Capital Medical University.

[0057] 3. Main instruments: clean bench, CO2 constant temperature and humidity incubator, micropipette pipettes of various specifications, culture vessels, centrifuge, flow cytometer, electric pressure steam sterilizer, micro electronic balance, etc.

[0058] (2) Experimental steps

[0059] 1. Primary diffuse midline brainstem glioma cell line

[0060] After centrifugation of the tumor tissue, discard the hibernation buffer, add 2 ml of RPMI1640 cell culture medium, chop the tissue, add type IV collagenase and DNase I, and place it in a 37°C cell culture incubator for digestion for 30 minutes. Mix by blowing several times every 10 minutes until a single cell suspension is formed. Centrifuge at 1500 r / min for 5 minutes, remove the supernatant, resuspend the cells in RPMI1640, centrifuge at 1500 r / min for 5 minutes, remove the supernatant, and repeat the above steps twice. Resuspend the cells in RPMI1640 culture medium containing serum. After counting the cells, use 30,000-60,000 / cm 2 The primary cells were subcultured and the impurity cells were removed to finally obtain the primary diffuse midline brainstem glioma cell line.

[0061] 2. Tumor-infiltrating lymphocytes (TIL cells)

[0062] After centrifugation of the tumor tissue, the hibernation buffer was discarded, and the tissue was rinsed with RPMI1640 medium. DNAse, type IV collagenase, and hyaluronidase were added to the RPMI1640 medium, and the tissue was placed in a 37°C cell culture incubator for digestion for 30 minutes. The mixture was blown and mixed several times every 10 minutes until a single cell suspension was formed. After centrifugation at 1500r / min for 5 minutes, the supernatant was removed, and the cells were resuspended with RPMI1640. The cells were centrifuged at 1500r / min for 5 minutes, and the RPMI1640 was discarded. The above steps were repeated twice. 100% and 75% lymphocyte separation fluid were layered in a sterile centrifuge tube, and the cell suspension was gently added along the wall of the tube. After centrifugation at 2000r / min for 20 minutes, the cells on the 100% separation fluid interface were collected as a suspension rich in TIL cells. The TIL cells were washed twice with RPMI1640 culture fluid to remove the cell separation fluid. The cells were resuspended with RPMI1640 culture medium. The cell concentration was adjusted to 5×10 6 / ml, for future use.

[0063] 3. CFDA-SE Labeling of Primary Cultured Diffuse Midline Brainstem Glioma Cells

[0064] 5 μL of 10 mM CFDA-SE stock solution was added to 5 mL of PBS to prepare a 10 μM CFDA-SE solution; centrifuged at 1500 r / min for 5 minutes, washed the cells three times with PBS, resuspended and washed once with PBS, and then added with 10 μM CFDA-SE solution. The cells were incubated at room temperature in the dark for 8 minutes with slight shaking; after the incubation, 10 mL of RPMI 1640 complete medium was added and gently pipetted to terminate CFDA-SE staining; centrifuged at 1500 r / min for 5 minutes, removed the supernatant, and washed the cells twice with PBS; resuspended in RPMI 1640 complete medium and counted; the cell concentration was adjusted to 1 × 10 5 / mL, for future use.

[0065] Add 0.5 mL of 1×10 5 / mL of CFDA-SE-labeled diffuse midline brainstem glioma cells and 0.5mL of 5.0×10 6 / mL of TIL cells were shaken evenly and placed in a cell culture incubator for co-culture for 4 hours; after the culture was completed, chlorogenic acid solution prepared with PBS was added to treat them, and the final concentrations of chlorogenic acid solution were 0, 100 μM, and 300 μM, respectively (3 replicates were set up in each group), and they were placed in a cell culture incubator for incubation for 24 hours; after the incubation was completed, the culture medium in the well was collected into a centrifuge tube, and the adherent diffuse midline brainstem glioma cells were digested with trypsin and the cells were collected into a centrifuge tube; the cells were washed once with the buffer solution in the cell apoptosis PI staining kit (centrifugation at 1500 r / min, 5 min), and the cells were resuspended in an appropriate amount of buffer solution; the cells were stained with the PI staining solution in the cell apoptosis PI staining kit and stained at room temperature in the dark for 10 min; flow cytometric analysis was performed using a flow cytometer.

[0066] 2. Experimental Results

[0067] The results of the cell apoptosis assay are shown in Figure 1. Chlorogenic acid can induce apoptosis in diffuse midline brainstem glioma cells. Furthermore, when the chlorogenic acid concentration was 300 μM, the proportion of apoptotic cells was higher than when the chlorogenic acid concentration was 100 μM.

[0068] Experimental Example 2 Animal Test on the Inhibitory Effect of Chlorogenic Acid of the Present Invention on Diffuse Midline Glioma

[0069] 1. Experimental Methods

[0070] (1) Construction of a mouse diffuse brainstem glioma model

[0071] Fresh surgical specimens of diffuse midline brainstem glioma patients were collected and cut into small tissue pieces. The obtained tumor tissue was ground into single cell suspension using a tissue grinder in phosphate buffer saline. After counting, the cell suspension was adjusted to a cell concentration of 2.5×10 6 pieces / mL.

[0072] Seventy female SPF-grade ICR mice weighing 18-22 g were selected. After wiping the skin at the inoculation site with an alcohol cotton ball, 0.2 mL of tumor fluid was inoculated into the right scapula of the mice. Three days after the tumor inoculation, the tumor was transplanted subcutaneously into the mice. The mice were randomly divided into three groups, and the survival and growth characteristics of the transplanted tumors were observed.

[0073] (2) Grouping

[0074] 70 ICR mice were successfully modeled and randomly divided into the following groups:

[0075] 1. Tumor inhibition effect observation group

[0076] The blank control group consisted of 10 mice per group, and no drug was given.

[0077] Temozolomide group (50 mg / kg): 10 rats in each group. The temozolomide group was given 10 g / 0.2 mL of body weight by oral gavage once a day for 5 consecutive days.

[0078] Chlorogenic acid group (40 mg / kg): 10 rats per group, 10 g / 0.1 mL of chlorogenic acid was intraperitoneally injected once a day until the end of the experiment. The chlorogenic acid injection preparation of Experimental Example 2, Formulation 1, was used here.

[0079] The weight and health of the mice were observed twice a week. The tumor size was measured every 3 days using a vernier caliper. The tumor volume was expressed as V (mm 3 ) = length × width 2 ×0.5 to estimate.

[0080] The tumor size of the blank test group was 1500 mm 3 The experiment was terminated at approximately 1:10 p.m. Animals in the blank control group, temozolomide group, and chlorogenic acid group were treated simultaneously. Tumors were dissected subcutaneously from the animals. Tumor inhibition rate (%) was calculated based on tumor weight. Tumor inhibition rate = (1-T / C) × 100%, where T represents the tumor weight of the experimental group and C represents the tumor weight of the blank control group.

[0081] 2. Survival group

[0082] Temozolomide survival group (50 mg / kg): 20 mice in each group. Temozolomide group was given oral administration at 10 g / 0.2 mL of body weight, once a day for 5 consecutive days.

[0083] Chlorogenic acid survival group (40 mg / kg): 20 mice per group, 10 g / 0.1 mL body weight administered by intraperitoneal injection once a day for 28 consecutive days. The chlorogenic acid injection preparation of Experimental Example 2, Formulation 1, was used here.

[0084] The survival time of the animals in the temozolomide group and the chlorogenic acid group was observed until all animals died, and the median survival time of the two groups was calculated.

[0085] 2. Experimental Results

[0086] 1. Chlorogenic acid inhibits diffuse brainstem glioma results

[0087] The results of chlorogenic acid's inhibition on diffuse brainstem glioma in mice are shown in Figure 2 and Table 1. As can be seen from Figure 2 and Table 1, both the temozolomide group and the chlorogenic acid group can significantly inhibit the growth of ectopic diffuse brainstem glioma in mice, with tumor inhibition rates of 31% and 49%, respectively; and the chlorogenic acid group inhibited the growth of diffuse midline brainstem glioma significantly better than the positive control drug temozolomide group (P<0.05).

[0088] It can also be seen from Table 1 that, compared with the blank control group, chlorogenic acid had no significant effect on the body weight of animals, but the body weight of animals in the temozolomide group was significantly reduced.

[0089] Table 1 Tumor inhibition results of each group in the mouse diffuse brainstem glioma model Note: Compared with the blank control group, * indicates P < 0.05, ** indicates P < 0.01; compared with the temozolomide group, # indicates P < 0.05, ## indicates P < 0.01.

[0090] 2. Chlorogenic acid prolongs the survival time of tumor-bearing mice

[0091] The results of the survival time experiment in the mouse diffuse brainstem glioma model are shown in Figure 3.

[0092] The median survival time of mice in the temozolomide group was 36 days, while that of the chlorogenic acid group increased to 74 days. It can be seen that compared with the temozolomide group, the chlorogenic acid group can significantly prolong the survival time of tumor-bearing mice (Figure 3).

[0093] The above experimental results show that compared with the administration of temozolomide, the administration of chlorogenic acid has a significantly improved inhibitory effect on diffuse brainstem glioma, with less toxic side effects, and can significantly prolong the survival time of diffuse brainstem glioma model mice.

[0094] The present invention provides a use of chlorogenic acid in the preparation of a medicament for treating diffuse midline gliomas, particularly diffuse brainstem gliomas. Cell and animal experiments of the present invention demonstrate that chlorogenic acid can induce apoptosis in diffuse midline brainstem glioma cells, and the proportion of apoptotic cells increases with increasing chlorogenic acid concentration. Compared with temozolomide, a standard chemotherapy drug commonly used for glioblastoma, chlorogenic acid has a significantly improved inhibitory effect on diffuse brainstem gliomas, with minimal toxic side effects. Chlorogenic acid significantly prolongs the survival time of mice with a diffuse brainstem glioma model. The present invention is the first to discover that chlorogenic acid has inhibitory effects on diffuse brainstem gliomas and can inhibit the growth of diffuse brainstem gliomas, providing a new, safer option for the treatment of diffuse midline gliomas with promising application prospects.

Claims

1. Use of chlorogenic acid in the preparation of a drug for treating childhood diffuse high-grade glioma.

2. The use according to claim 1, characterized in that The pediatric diffuse high-grade glioma is a diffuse midline glioma.

3. The use according to claim 2, characterized in that: The diffuse midline glioma is a diffuse midline brainstem glioma.

4. The use according to claim 3, characterized in that: The drug is a drug that inhibits the growth of diffuse midline brainstem glioma.

5. The use according to claim 1, characterized in that: The drug is a drug for inhibiting tumors caused by H3K27 mutation.

6. The use according to any one of claims 1 to 5, characterized in that: The drug is a preparation prepared by taking an effective amount of chlorogenic acid as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

7. The use according to claim 6, characterized in that: Each preparation unit of the medicine contains 1 to 2000 mg of chlorogenic acid.

8. The use according to claim 7, characterized in that: Each preparation unit of the medicine contains 10 to 200 mg of chlorogenic acid.

9. The use according to claim 8, characterized in that Each preparation unit of the medicine contains 30 mg of chlorogenic acid.

10. The use according to any one of claims 6 to 9, characterized in that: The preparation is an oral preparation or an injection.

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