CN111905102A

CN111905102A - Use of EZH2 inhibitors for the treatment of gliomas

Info

Publication number: CN111905102A
Application number: CN202010921634.6A
Authority: CN
Inventors: 黄菲; 洪金省; 伍兵; 王彩虹; 潘晓娴
Original assignee: First Affiliated Hospital of Fujian Medical University
Current assignee: First Affiliated Hospital of Fujian Medical University
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2020-11-10

Abstract

The invention relates to application of an EZH2 inhibitor in treating glioma, in particular to application in glioma radiotherapy sensitization. The inventor researches and discovers that the EZH2 inhibitor has synergistic effect with radiotherapy in the process of treating glioma, and can effectively assist in improving curative effect or reducing the dose of radiotherapy and reducing radiation injury without reducing curative effect.

Description

Use of EZH2 inhibitors for the treatment of gliomas

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of an EZH2 inhibitor in treatment of glioma, in particular to an application in sensitization of glioma radiotherapy.

Background

Brain glioma refers to tumor derived from neuroepithelia, accounts for 40% -50% of craniocerebral tumor, is the most common intracranial primary malignant tumor, has strong local invasion and migration capacity, has annual incidence of 3-8 people/10 million people, and has incidence increasing with age. Brain gliomas can be classified into grade I (least malignant, best prognosis) to grade IV (most malignant, worst prognosis) according to a grading system established by the World Health Organization (WHO). Among them, glioblastoma is classified as WHO grade IV. At present, the treatment means of glioma mainly comprises surgical excision, radiotherapy, drug therapy and the like.

Radiation therapy, which is an important component of clinical treatment of tumors as a method for locally treating malignant tumors, allows normal organs and tissues to be preserved, so that the number of patients treated by the method is rapidly increasing. The effectiveness of tumor radiotherapy is affected by a number of factors. Different types of tumors respond differently to radiation therapy. The extent of response to radiation therapy is also related to the type and size of the tumor. How to improve the effectiveness of radiation therapy is an important issue in clinical oncology.

Disclosure of Invention

The invention aims to provide a medicament capable of enhancing the curative effect of radiation therapy of glioma.

The invention provides an application of an EZH2 inhibitor in preparation of a glioma radiotherapy sensitizer.

In another preferred embodiment, the EZH2 inhibitor is selected from one or more of the following group: EPZ-6438, CPI-1205, GSK2816126 and GSK 343.

In another preferred embodiment, the EZH2 inhibitor inhibits tumor volume growth in glioma.

In another preferred embodiment, the EZH2 inhibitor can increase the survival rate of tumor-bearing mice.

In another preferred embodiment, the EZH2 inhibitor inhibits the growth of glioma cells.

In another preferred embodiment, the glioma comprises WHO grade I glioma, grade II glioma, grade III glioma, grade IV glioma.

In another preferred embodiment, the glioma is a glioblastoma.

In another preferred embodiment, the glioblastoma cell is a mouse glioblastoma cell line GL261, a human glioblastoma cell line U251 or a human glioblastoma cell line U87.

The invention also provides a glioma radiotherapy sensitizer, which takes an EZH2 inhibitor as an active ingredient.

In another preferred embodiment, the glioma radiosensitizer is an injection or an oral preparation.

In another preferred embodiment, the glioma is a glioblastoma.

In another aspect of the invention, there is provided a method of inhibiting growth of glioma cells in vitro by culturing the glioma cells with an EZH2 inhibitor prior to, simultaneously with, or after irradiation of the glioma cells.

In another preferred embodiment, the glioma is a glioblastoma.

In another preferred embodiment, the dose of irradiation is 2.5-4 Gy.

In another preferred embodiment, the concentration of the EZH2 inhibitor is 2.5-10 uM.

The invention also provides a method of increasing the efficacy of radiation therapy for a glioma patient characterized by administering to the patient an EZH2 inhibitor prior to, simultaneously with, or after radiation therapy to the patient.

In another preferred embodiment, the radiation therapy is administered in a dose of 2.5-4 Gy.

In another preferred embodiment, the EZH2 inhibitor is administered at a dose of 2.5-100 mg/kg.

The main advantages of the invention are: the inventor unexpectedly finds that the EZH2 inhibitor can effectively improve the curative effect of radiation therapy of glioma, and has a remarkable synergistic effect on tumor growth inhibition and survival rate extension of tumor-bearing mice together with radiation therapy.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows the fold change in tumor volume before and after treatment (P < 0.05) for each experimental group of mice. Among these, Con (control, solvent treatment); IR (radiotherapy group, radiotherapy + solvent treatment), GSK (drug treatment group, GSK343 treatment), IR-GSK (radiotherapy + drug treatment, radiotherapy + GSK343 treatment).

Figure 2 shows the survival curves of mice in each experimental group after treatment (P < 0.01). Among these, Con (control, solvent treatment); IR (radiotherapy group, radiotherapy + solvent treatment), GSK (drug treatment group, GSK343 treatment), IR-GSK (radiotherapy + drug treatment, radiotherapy + GSK343 treatment).

FIG. 3 shows the cell clonogenic profiles of mouse glioblastoma cell line GL261, and human glioblastoma cell lines U251 and U87 in each experimental group. Among them, Control (Control, solvent treatment); IR (radiation treatment group, radiation treatment + solvent treatment), GSK434 (drug treatment group, GSK343 treatment), IR + GSK434 (radiation treatment + drug treatment, radiation treatment + GSK343 treatment).

FIG. 4 shows the cell clonogenic profiles of the mouse glioblastoma cell line GL261 and the human glioblastoma cell line U87 in the respective experimental groups. Among them, Control (Control, solvent treatment); IR (radiation treatment group, radiation treatment + solvent treatment), EPZ-6438 (drug treatment group, Tazemetostat treatment), IR + EPZ-6438 (radiation treatment + drug treatment, radiation treatment + Tazemetostat treatment).

Detailed Description

The inventor screens and proves that the EZH2 inhibitor has a remarkable effect on improving the curative effect of radiation therapy of glioma through long-term experiments, so that the EZH2 inhibitor becomes an ideal glioma radiotherapy sensitizer.

The EZH2 is an important epigenetic regulation gene, has Histone Methyltransferase (HMT) activity, and can catalyze 27 th lysine trimethyl (H3K27me3) of histone H3 so as to inhibit the expression of a target gene. Tazemetostat (also known as EPZ-6438) is an inhibitor of EZH2 that is already on the market. Tazemetostat is accelerated to market by FDA in the early years due to its significant phase II clinical effect in interstitial sarcoma, but is commonly expressed in leukemia and lymphoma, urogenital tumors. Other EZH2 inhibitors that have entered clinical trials include CPI-1205 and GSK 2816126. These three drugs are currently in phase I/II clinical studies. GSK343 is an unpmercurified EZH2 inhibitor. GSK343 has not yet been developed for any clinical trials. Although GSK343 has also been reported in recent years to be useful for inhibiting the growth of glioblastoma, its inhibitory effect is very limited. The invention provides an application of an EZH2 inhibitor in preparing a glioma radiotherapy sensitizer.

The invention also provides a glioma radiotherapy sensitizer, which takes an EZH2 inhibitor as an active component.

The glioma radiosensitizer can be administered orally or non-orally, and can be administered according to the administration of the EZH2 inhibitor per se. When the EZH2 inhibitor is administered orally, the glioma radiosensitizer can be administered in any conventional form, such as powder, granule, tablet, capsule, pill, drop pill, soft capsule, floating agent, oral liquid, suspension, syrup, buccal tablet, spray or aerosol, etc.; when the EZH2 inhibitor is administered non-orally, the glioma radiosensitizer can be administered in any conventional form, such as suppositories, injections (intravenous, intramuscular), ointments, inhalants and the like.

The glioma radiosensitizer can also comprise a pharmaceutically acceptable carrier or excipient besides the active ingredient. Pharmaceutically acceptable carriers or excipients for use herein are well known in the art and may be solid or liquid. As specific examples, there may be mentioned lactose, starch, dextrin, calcium carbonate, synthetic or natural aluminum sulfate, magnesium chloride, magnesium stearate, sodium bicarbonate, dried yeast, etc.; liquid carriers or excipients include water, glycerol, propylene glycol, simple syrup, ethanol, ethylene glycol, polyethylene glycol, sorbitol, etc.; the excipient of ointment can be fatty oil, hydrophobic agent or hydrophilic agent composed of aqueous lanolin, vaseline, glycerol, beeswax, wood wax, liquid paraffin, resin, and high grade wax.

The dose of the active ingredient in the glioma radiosensitizing agent may vary depending on the mode of administration, the age and weight of the patient, the severity of the condition of the patient, and other similar factors. For example, the oral dose can be 100-500 mg/time/person, 1-3 times daily; the dosage of the injection can be 100-1000 mg/time/person and 1 time per day. Also for example, the dose may be 2.5-500mg/kg body weight, or 10-200mg/kg body weight, or 20-100mg/kg body weight, or 30-60mg/kg body weight, and the like. The pharmaceutical composition of the present invention may be administered continuously, for example, for 3 to 10 days, 3 to 7 days, 3 to 5 days, or the like. The pharmaceutical compositions of the invention may also be administered at intervals, such as every other day, two days, or more, and the like.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

Construction of a nude mouse transplantation tumor:

mouse glioblastoma cell GL261 (purchased from ATCC) was suspended in PBS and adjusted to 5X 10⁷mL, inoculated into C57BL/6 mice. A4-6 week old C57BL/6 mouse was placed in a mouse brain positioning instrument and a small hole was drilled 1.5mm forward and 1mm to the left of the bregma orientation of the skull. The cell suspension was aspirated by a 10uL syringe, a needle was inserted into the well to a depth of 2.5mm, and 2uL (1X 10) of cells were injected⁵One), the needle insertion rate is 1mm/min (1 uL/min); standing for 20min after injection, withdrawing the needle at a needle withdrawal rate of 1mm/min (1 uL/min). 7 days after inoculation, the cells were observed using a mouse NMR spectrometerThe tumor can grow to 0.5-1 mm³。

The treatment modalities for each group were as follows:

one week (day 7) after tumor bearing in mice, the average tumor volume grows to 0.5mm³At the moment, the treatment is started,

control group (no radiation treatment, no drug treatment): 10% ethanol (solvent for preparing GSK343 medicinal liquid) was injected 3 times a week at a dose of about 380 uL/injection from day 7 for two weeks.

Radiation treatment group (IR group, no drug treatment): the mouse head was treated with radiation starting on day 7 using a total of 2.5Gy at a dose rate of 500cGy with a 12MeV electron beam. Irradiation was stopped after 3 consecutive days. At the same time, injection of 10% ethanol (solvent for preparation of GSK343 medicinal liquid) was started simultaneously with radiotherapy, 3 times per week, about 380uL each time, for two weeks.

Drug treatment group (GSK group, no radiotherapy): the injection of GSK343 medicinal liquid (GSK343 medicinal liquid dissolved in absolute ethanol, stock solution concentration 125ug/mL) was started to the mice at a dose of 2.5mg/kg in synchronization with the radiotherapy, and the injection was performed 3 times per week at a dose of about 380 uL/mouse (125ug/mL) per body weight per time for two weeks.

Radiotherapy + drug treatment group (IR + GSK group): the mouse head was treated with radiation starting on day 7 using a total of 2.5Gy at a dose rate of 500cGy with a 12MeV electron beam. Irradiation was stopped after 3 consecutive days. Meanwhile, GSK343 liquid medicine was injected into mice at a dose of 2.5mg/kg in synchronization with the radiotherapy, 3 times per week, about 380 uL/mouse (125ug/mL) per time in terms of body weight, for a total of two weeks.

After the two-week treatment (day 21), fold changes in tumor volume before and after treatment were compared for each experimental group. The tumor volume was calculated according to the small animal nuclear magnetic imaging results using the mimics 16.0 software. Fold change is the ratio of tumor volume size after treatment (day 21) to before treatment (day 7). The results are shown in FIG. 1. The results show that the tumor volume increase rate is obviously reduced after the radiotherapy and the GSK343 medicament are combined for treatment.

Example 2

The construction procedure and treatment mode of the nude mouse transplanted tumor were the same as those of example 1.

After the two-week treatment (day 21), the survival curves of the mice after the treatment of each experimental group were compared. The mouse survival curve is a Kaplan-Meier curve, and the Log Rank method is adopted to compare whether the survival curves are different.

The results are shown in FIG. 2. The results show that the survival rate of the mice is remarkably improved after the combination of radiotherapy and GSK343 medicament treatment.

Therefore, the EZH2 inhibitor GSK343 intraperitoneal injection mouse can remarkably improve the radiotherapy curative effect of mouse glioblastoma multiforme, and has the effects of reducing brain tumor focus and prolonging the life cycle of tumor-bearing mice.

Example 3

Mouse glioblastoma cell line GL261, and human glioblastoma cell lines U251 and U87, all from ATCC, were cultured in DMEM + 10% fetal calf serum at 37 deg.C and 5% CO₂。

Control group: without irradiation, the culture broth was treated with 0.1% DMSO for 14 days.

Radiation treatment group (IR group): after 1 radiation treatment, the cells were cultured for 14 days at a dose rate of 200cGy, 6MeV X-ray, total dose of 4 Gy.

Drug treatment group (GSK343 group): GSK343(2.5uM in DMSO) was added to the culture at a concentration of 0.1% and at a concentration of 2.5mM, and the culture was incubated for 14 days.

Radiation treatment + drug treatment group (IR + GSK343 group): after 1 treatment at a dose rate of 200cGy, 6MeV X-ray, total dose 4Gy, GSK343 was added to the culture medium at 0.1% 2.5mM GSK343(2.5uM in DMSO), and the culture was incubated for 14 days.

After 14 days of cell culture, the cells were stained with crystal violet and each group was observed for colony formation.

The results are shown in FIG. 3. The results show that: at the cellular level, we found that radiation therapy and treatment with the EZH2 inhibitor GSK343 synergistically inhibited clonogenic of glioblastoma cells GL261 (mouse) and U251 (human), U87 (human).

Example 4

Experiments were performed using another EZH2 inhibitor, Tazemetostat (also known as EPZ-6438), instead of GSK343 to observe the colony formation of each group of cells.

Mouse glioblastoma cell line GL261 and human glioblastoma cell line U87 were obtained from ATCC and cultured in DMEM + 10% fetal calf serum at 37 deg.C and 5% CO₂。

Radiation treatment group (IR group): after 1 radiation treatment, the cells were cultured for 14 days at a dose rate of 500cGy, 6MeV X-ray, total dose of 4 Gy.

Drug treatment group (EPZ-6438 group): cells in culture were treated for 14 days by adding 0.1% of 10mM Tazemetostat (10uM in DMSO);

radiation treatment + drug treatment group (IR + EPZ-6438 group): after 1 treatment at a dose rate of 500cGy, 6MeV X-ray, total dose 4Gy, cells were cultured by adding 0.1% of 10mM Tazemetostat (10uM in DMSO) to the culture medium for 14 days.

The results are shown in FIG. 4. The results show that: at the cellular level, Tazemetostat also inhibited clonogenic of glioblastoma cells GL261 (mouse) and U87 (human) in concert with radiation therapy. Its function is similar to GSK 343.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

The application of an EZH2 inhibitor in preparing a glioma radiotherapy sensitizer.
2. The use of claim 1, wherein the EZH2 inhibitor is selected from one or more of the group consisting of: EPZ-6438, CPI-1205, GSK2816126 and GSK 343.
3. The use of claim 1, wherein the EZH2 inhibitor inhibits tumor volume growth of glioma.
4. The use of claim 1, wherein the EZH2 inhibitor increases survival of tumor-bearing mice.
5. The use of claim 1, wherein the EZH2 inhibitor inhibits the growth of glioma cells.
6. The use of claim 1, wherein the glioma comprises a WHO grade I glioma, a grade II glioma, a grade III glioma, a grade IV glioma.
7. A glioma radiotherapy sensitizer is characterized in that the sensitizer takes an EZH2 inhibitor as an active component.
8. The glioma radiosensitizer of claim 7 wherein the glioma radiosensitizer is an injectable or oral formulation.
9. The glioma radiosensitizer of claim 7 wherein the EZH2 inhibitor is selected from one or more of the group consisting of: EPZ-6438, CPI-1205, GSK2816126 and GSK 343.
10. A method of inhibiting the growth of glioma cells in vitro comprising culturing the glioma cells with an EZH2 inhibitor prior to, simultaneously with, or after irradiation of the glioma cells.