WO2025055599A1 - Use of chlorogenic acid in preparation of drug for treating diffuse midline gliomas - Google Patents

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 a drug for treating diffuse midline glioma Technical Field

The 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

Diffuse midline glioma (DMG) is a primary brain tumor. It is a type of tumor that mainly occurs in children and younger adults, and is rarely seen in the elderly. It is mostly located in midline structures such as the thalamus, brainstem, and spinal cord. The high incidence of histone H3K27M mutation is an important molecular biological basis. Among them, DMG in the brainstem and thalamus is extremely invasive and can diffusely invade the surrounding normal brain tissue. It is one of the most deadly brain tumors.

The main biological feature of DMG is the mutation of lysine 27 to methionine (K27M) in histone 3 (H3), which can cause extensive changes in epigenomics and gene expression and is considered to be an important driver of tumor initiation and maintenance. Due to the unique expression of H3K27 mutation in diffuse midline gliomas and its significant correlation with prognosis, the 2016 WHO Classification of Tumors of the Central Nervous System classified it as WHO grade IV for the first time. Its pathological characteristics are histone H3K27M mutation, diffuse infiltrative growth of lesions, and extremely high malignancy. The treatment and prognosis of this disease are not ideal, with a median survival of less than 1 year. There is currently no standard treatment regimen, and surgical resection and conventional chemoradiotherapy are difficult to improve prognosis, especially in cases located in the brainstem.

Diffuse midline brainstem glioma (DIPG) accounts for 10% to 15% of central nervous system tumors in children. Despite decades of continuous efforts by scholars at home and abroad, the mortality rate of this disease once diagnosed is still 100%. Because the brainstem is full of important nerve nuclei and fibers, there are many blood vessels around it, and brainstem glioma grows in an infiltrative manner, surgery cannot benefit patients and may often aggravate neurological damage. More and more chemotherapy treatments have also been proven to be of no help in improving the prognosis of DIPG, including temozolomide, a standard chemotherapy drug routinely used for glioblastoma. The current internationally recognized standard treatment is traditional radiotherapy, but the treatment effect is limited to temporary relief of symptoms.

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 for improving patients' prognosis and prolonging their survival.

Chlorogenic acid is a phenylpropanoid compound produced by caffeic acid and quinic acid during aerobic respiration in plants through the shikimic acid pathway. Chlorogenic acid has a wide range of biological activities, including anticancer, antibacterial, antiviral, liver-protecting and gallbladder-promoting, blood pressure-lowering, blood lipid-lowering, free radical scavenging and central nervous system excitation, reflecting extraordinary medicinal and social value.

It has been reported that chlorogenic acid can treat oligoastrocytoma and glioblastoma, 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 oligoastrocytoma in terms of imaging characteristics: when MRI/CT performance is similar to astrocytoma or oligodendroglioma with enhancement, it indicates that it is likely to be an anaplastic brain glioma. Glioblastoma is characterized by irregular peripheral enhancement and massive central necrosis, with enhanced peripheral gliomas and gliomas. Edema may be seen. 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 may show varying degrees of enhancement.

In the classification standard, glioblastoma, IDH wild type, belongs to adult diffuse glioma; diffuse midline glioma, H3K27 variant, belongs to pediatric diffuse high-grade glioma. Adult diffuse glioma and pediatric diffuse glioma: The new classification divides diffuse glioma into two major categories for the first time: adult diffuse glioma and pediatric diffuse glioma. It should be noted that this diagnostic classification is not entirely based on the age of tumor onset, but on clinical characteristics such as major molecular mutations and the distribution of such tumors in different age groups.

There is no report that chlorogenic acid has a therapeutic effect on diffuse midline glioma.

Summary of the invention

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

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

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

Furthermore, the diffuse midline glioma is a diffuse midline brainstem glioma.

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

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

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

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

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

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

Furthermore, the preparation is an oral preparation or an injection.

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

The present invention provides a use of chlorogenic acid in the preparation of a drug for treating diffuse midline gliomas, in particular, the use of chlorogenic acid 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 small toxic and side effects. Chlorogenic acid significantly prolongs the survival time of diffuse brainstem glioma model mice. 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, and has good application prospects.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described below by means of specific implementation methods in the form of embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies realized based on the above content of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the results of chlorogenic acid inducing apoptosis of diffuse midline brainstem glioma cells.

FIG2 is a graph showing the tumor weight of mice in each group.

FIG. 3 is a graph showing the survival time of mice in each group.

DETAILED DESCRIPTION

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

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

Patient tissues of diffuse midline brainstem glioma were provided by Beijing Tiantan Hospital, Capital Medical University.

Example 1 Prescription of oral preparation of the pharmaceutical composition of the present invention

1. Prescription 1

Chlorogenic acid 1000g.

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

2. Prescription 2

Chlorogenic acid 1000g, filler 2000g, binder 10g.

Preparation method: Weigh chlorogenic acid, filler and binder according to the prescription, granulate, granulate and package into granules.

3. Prescription 3

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

Preparation method: Weigh chlorogenic acid, filler, and binder according to the prescription, granulate, resize, add lubricant, and press to obtain tablets.

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 micro-powder silica gel.

Example 2 Prescription of the injection preparation of the pharmaceutical composition of the present invention

1. Prescription 1

Chlorogenic acid 1000g.

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 and sterilize, freeze-dry, and obtain freeze-dried powder injection.

2. Prescription 2

Chlorogenic acid 1000g, scaffold agent 2667g, antioxidant 67g.

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.

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

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

Experimental Example 1 In vitro test of chlorogenic acid of the present invention in treating diffuse midline glioma

1. Experimental Methods

1. Experimental Materials

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

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

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.

(II) Experimental steps

1. Primary diffuse midline brainstem glioma cell line

After centrifugation of the tumor tissue, the hibernation buffer was discarded, 2 ml of RPMI1640 cell culture medium was added, the tissue was cut into pieces, type IV collagenase and DNase I were added, and then digested in a 37°C cell culture incubator for 30 minutes. The mixture was blown and mixed several times every 10 minutes until a single cell suspension was formed. Centrifuge at 1500r/min for 5 minutes, remove the supernatant, resuspend the cells with RPMI1640, centrifuge at 1500r/min for 5 minutes, remove the supernatant, and repeat the above steps twice. Resuspend the cells with RPMI1640 culture medium containing serum. After counting the cells, inoculate them at a density of 30,000-60,000/ ^cm2 in a culture plate pre-coated overnight. The cultured primary cells were subcultured to remove impurity cells, and finally screened to obtain the primary diffuse midline brainstem glioma cell line.

2. Tumor-infiltrating lymphocytes (TIL cells)

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 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. After centrifugation at 1500r/min for 5 minutes, RPMI1640 was discarded, and the above steps were repeated twice. 100% and 75% lymphocyte separation fluid were placed in a sterile centrifuge tube, and the cell suspension was gently added along the tube wall. After centrifugation at 2000r/min for 20 minutes, the cells on the interface of 100% separation fluid were collected as a suspension rich in TIL cells, and 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 and set aside.

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

Take 5 μL of 10 mM CFDA-SE stock solution and add 5 mL of PBS to prepare 10 μM CFDA-SE solution; centrifuge at 1500 r/min for 5 minutes, wash the cells 3 times with PBS, resuspend and wash once with PBS, then add 10 μM CFDA-SE solution, incubate at room temperature in the dark for 8 minutes with slight shaking; after the incubation, add 10 mL of RPMI 1640 complete medium and gently blow evenly to terminate CFDA-SE staining; centrifuge at 1500 r/min for 5 minutes, remove the supernatant, and wash the cells twice with PBS; resuspend and count with RPMI 1640 complete medium; adjust the cell concentration to 1×10 ⁵ /mL with RPMI 1640 complete medium and set aside.

Add 0.5 mL of 1×10 ⁵ /mL CFDA-SE-labeled diffuse midline brainstem into a 12-well plate. Glioma cells and 0.5mL 5.0×10 ⁶ /mL TIL cells were shaken evenly and placed in a cell culture incubator for co-culture for 4 hours; after the culture, chlorogenic acid solution prepared with PBS was added to treat, and the final concentrations of chlorogenic acid solution were 0, 100μM, and 300μM, respectively (three replicates were set for each group), and then placed in a cell culture incubator for incubation for 24 hours; after the incubation, 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 collected into a centrifuge tube; the cells were washed once with the buffer solution in the cell apoptosis PI staining kit (centrifugation 1500r/min, 5min), and the cells were resuspended with an appropriate amount of buffer solution; the cells were stained with the PI staining solution in the cell apoptosis PI staining kit, and stained for 10min at room temperature in the dark; flow cytometric analysis was performed using a flow cytometer.

2. Experimental Results

The results of the cell apoptosis test are shown in Figure 1, which shows that chlorogenic acid can induce apoptosis in diffuse midline brainstem glioma cells. When the concentration of chlorogenic acid is 300 μM, the proportion of apoptotic cells is higher than when the concentration of chlorogenic acid is 100 μM.

Experimental Example 2 Animal test on the inhibitory effect of chlorogenic acid on diffuse midline glioma

1. Experimental Methods

(I) Construction of a mouse diffuse brainstem glioma model

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

70 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 to observe the survival and growth characteristics of the transplanted tumors.

(II) Grouping

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

1. Tumor inhibition effect observation group

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

Temozolomide group (50 mg/kg): 10 mice in each group. The temozolomide group was intragastrically administered with 10 g/0.2 mL of body weight, once a day for 5 consecutive days.

Chlorogenic acid group (40 mg/kg): 10 mice 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 Prescription 1 was used here.

The body weight and health of the mice were observed twice a week. The tumor size was measured with a vernier caliper every 3 days, and the tumor volume was estimated according to V (mm ³ ) = length × width ² × 0.5.

The experiment was terminated when the tumor size of the blank test group was about 1500 ^mm3 , and the animals in the blank control group, temozolomide group, and chlorogenic acid group were treated at the same time. The tumor was dissected out from the subcutaneous tissue of the animal. The tumor inhibition rate (%) was calculated based on the 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.

2. Survival group

Temozolomide survival group (50 mg/kg): 20 mice in each group. The temozolomide group was intragastrically administered with 10 g/0.2 mL of body weight, once a day for 5 consecutive days.

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

The survival period of the temozolomide group and the survival period of the chlorogenic acid group were observed until all animals died, and the median survival time of the two groups was calculated.

2. Experimental Results

1. Chlorogenic acid inhibits diffuse brainstem glioma results

The results of chlorogenic acid's inhibition of 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 tumors significantly better than the positive control drug temozolomide group (P<0.05).

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.

Table 1 The 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.

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

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

The median survival time of mice in the temozolomide group was 36 days, while that in 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).

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.

The present invention provides a use of chlorogenic acid in the preparation of a drug for treating diffuse midline gliomas, in particular, the use of chlorogenic acid in the preparation of a drug for treating diffuse brainstem gliomas. The 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 little toxic and side effects. Chlorogenic acid significantly prolongs the survival time of diffuse brainstem glioma model mice. 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, A highly secure option with good application prospects.

Claims (10)

Use of chlorogenic acid in preparing a medicine for treating childhood diffuse high-grade glioma. The use according to claim 1, characterized in that the pediatric diffuse high-grade glioma is a diffuse midline glioma. The use according to claim 2, characterized in that the diffuse midline glioma is a diffuse midline brainstem glioma. The use according to claim 3 is characterized in that the drug is a drug for inhibiting the growth of diffuse midline brainstem glioma. The use according to claim 1 is characterized in that the drug is a drug for inhibiting tumors caused by H3K27 mutations. The use according to any one of claims 1 to 5, characterized in that the drug is a preparation prepared with an effective amount of chlorogenic acid as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients. The use according to claim 6 is characterized in that each preparation unit of the medicine contains 1 to 2000 mg of chlorogenic acid. The use according to claim 7 is characterized in that each preparation unit of the medicine contains 10 to 200 mg of chlorogenic acid. The use according to claim 8 is characterized in that each preparation unit of the medicine contains 30 mg of chlorogenic acid. The use according to any one of claims 6 to 9, characterized in that the preparation is an oral preparation or an injection.