WO2021208294A1 - Application of pde4 inhibitor in preparation of drug for inhibiting proliferation in glioma - Google Patents

Abstract

An application of phosphodiesterase-4 inhibitor ZL-n-91 in preparation of a drug for treating a glioma, especially glioblastoma. In vitro cytological experiments show that phosphodiesterase-4 inhibitor ZL-n-91 can significantly inhibit proliferation of glioma cell lines in a dose-dependent manner, block the cell cycle, and induce apoptosis; in vivo animal models show that ZL-n-91 can significantly inhibit the growth of glioma subcutaneous tumors, and therefore has good development and application prospects.

Description

Application of PDE4 inhibitor in the preparation of drugs for inhibiting the proliferation of glioma Technical field

This application belongs to the field of anti-tumor drugs, especially glioma drugs. Specifically, the application provides the application of the PDE4 inhibitor ZL-n-91 in the preparation of drugs for the treatment of glioma proliferation.

Background technique

Central nervous system cancer (CNS) has always been one of the top ten malignant tumors with high morbidity and high mortality in China. In 2016, China had 106,207 new cases of central nervous system (CNS) cancer and 59,120 deaths. It is the country with the largest number of new cases and deaths from CNS cancer in the world. The prevention and treatment of CNS cancer in China has a long way to go. The most common primary CNS cancer is glioma, a type of glial cell malignant tumor, which is divided into 4 grades (WHO grade Ⅰ to Ⅳ) according to its malignancy. Glioblastoma (GBM, grade Ⅳ) ) Is the most common, most malignant, and most aggressive tumor in gliomas, accounting for the majority of gliomas (56.6%), with an incidence rate of 3.21 per 100,000. Although progress has been made in its diagnosis and standard treatments such as surgery, radiotherapy, and chemotherapy, the prognosis of GBM is still very poor due to the complexity of the onset of GBM, tumor resistance to radiation and chemotherapy, and the patient's survival after diagnosis is only 12 -15 months. Since the current types of drugs used to treat gliomas are far less than those of other solid tumors, and the efficacy and drug resistance properties are generally limited, it is of great significance to seek efficient treatment drugs.

Phosphodiesterase (abbreviated as PDEs) has the function of hydrolyzing the second messenger cAMP or cGMP in the cell, thereby affecting the signal pathway mediated by these second messengers and regulating cell functions. PDEs are divided into 11 subtypes, in which phosphodiesterase 4 (PDE4) specifically hydrolyzes cAMP. PDE4 is mainly distributed in various inflammatory cells, including mast cells, macrophages, lymphocytes, epithelial cells, etc. It participates in the promotion of monocyte and macrophage activation, neutrophil infiltration, vascular smooth muscle proliferation, and vasodilation As well as related physiological and pathological processes such as myocardial contraction, it has an impact on central nervous system function, cardiovascular function, inflammation/immune system, cell adhesion and so on. Studies have shown that PDE4 inhibitors (PDE4i) have anti-inflammatory, anti-allergic, and anti-platelet activation effects. Its mechanism of action mainly involves: 1) inhibiting the release of various inflammatory mediators/cytokines, which can inhibit the expression of IL-4 and IL-5 genes; 2) inhibiting the activation of white blood cells (such as respiratory burst) and inhibiting the migration of white blood cells; 3 ) Inhibit the expression or up-regulation of cell adhesion factor (CAM); 4) induce the production of cells with inhibitory activity, such as IL-6; 5) induce apoptosis; 6) stimulate the release of endogenous hormones and catecholamines. The diseases targeted by PDE4 inhibitors that have been developed or are being developed are mainly chronic obstructive pulmonary disease (COPD), asthma, inflammatory bowel disease, arthritis and so on. However, many studies have shown that PDE4 inhibitors also have a significant inhibitory effect on malignant tumors. After Patricia Goldhoff xenotransplanted nude mice into human brain astroblastoma cells U87, the use of PDE4 inhibitors can prolong the survival of the mice. In 2006, Motoshi Narita discovered that PDE4i can inhibit the growth of human melanoma cells. Petros X.E. Mouratidis discovered that the addition of PDE4 inhibitors CC-8075 and CC-8062 to pancreatic cancer cells can reduce cell proliferation and increase cell apoptosis.

The existing PDE4 inhibitors mainly include Rolipram, Cilomilast, and Roflumilast. Rolipram and Cilomilast caused dizziness, headache, nausea, vomiting and other gastrointestinal adverse reactions, which affected the clinical promotion and application of the drug. One of the possible reasons for gastrointestinal adverse reactions is poor specificity of PDE4 inhibitors, thereby moderately and selectively inhibiting the entire PDE family. For example, the Ki of Cilomilast to PDE4=92nM, which is only 500 to 1000 times the Ki of PDE1, 2, 3, and 5. Therefore, the use of higher doses of Cilomilast will interact with other members of the PDE family and cause side effects. In fact, for most PDE4 inhibitors, the side effect of vomiting at high doses is common. Although Roflumilast has been approved by the US FDA for the treatment of COPD, it can reduce lung inflammation, resist oxidative stress, effectively alleviate lung fibrosis, enhance mucosal clearance, and airway remodeling. But there are also adverse reactions, mainly manifested as diarrhea, weight loss, nausea, atrial fibrillation, and aggravation of mental illness (such as insomnia, anxiety, depression) and so on.

In response to the above problems, a variety of new PDE4 selective inhibitors have been developed, such as ZL-n-91 developed by Ke Hengming of the University of North Carolina:

Its IC ₅₀ reaches 18nM. It has been tried at home and abroad to treat lung diseases, such as COPD and even lung cancer and prostate cancer, with good results. In the process of further expanding the PDE4 inhibitor to treat PDE4-related diseases, whether it is effective for other cancers, such as glioma, has not been verified by any research.

Summary of the invention

The inventors tried to treat gliomas based on the early PDE4 inhibitor ZL-n-91 in the treatment of lung diseases and malignant solid tumors. In vitro cytological experiments show that the phosphodiesterase 4 inhibitor ZL-n-91 of the present invention can significantly inhibit the proliferation of glioma cells, block the cycle and induce cell apoptosis. The subcutaneous tumor model in vivo shows that Zl-n-91 can significantly inhibit tumor growth, indicating that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target for anti-glioma proliferation research, and is for the preparation of anti-glioma proliferation The medicine provides the foundation and has good prospects for development and application.

In one aspect, the present invention provides the application of ZL-n-91 in the preparation of drugs for treating solid tumors.

Further, the solid tumor is glioma.

Further, the glioma includes glioblastoma.

Further, the drug inhibits the proliferation of gliomas.

Further, the medicine is in the form of oral, injection or atomization.

On the other hand, the present invention provides a pharmaceutical composition for treating glioma, which contains ZL-n-91 as the sole active ingredient.

Further, the glioma includes glioblastoma.

In another aspect, the present invention provides a method of using ZL-n-91 to inhibit the proliferation of gliomas.

Further, the glioma includes glioblastoma.

Further, the glioma cells are U87, snb19 and U251 cells.

Further, the in vivo animal experiment uses a subcutaneous tumor-bearing model of glioma.

In another aspect, the present invention provides a method of treating solid tumors, which includes administering a PDE4 inhibitor ZL-n-91 to a subject in need.

Further, the solid tumor is glioma.

Further, the glioma includes glioblastoma.

The chemical structure of ZL-n-91 described in this application has been disclosed, and those skilled in the art can refer to prior art documents (such as Ruihong Ma, Bin-yan Yang, Chang-you Wu. A selective phosphodiesterase 4(PDE4) inhibitor Zl-n-91 suppresses IL-17 production by human memory Th17 cells. International Immunopharmacology, 2008, 8(10): 1408-1417.) and conventional techniques in the field of organic chemistry use synthesis, purchase, and application for gifts to obtain suitable purity The ZL-n-91.

The gliomas mentioned in this application include those derived from astrocytes, oligodendrocytes, mixed, ependymal, choroid plexus, other neuroepithelial sources, neurons and mixed sources, and various malignant degrees/stages Glioma.

The dosage forms that can be used in the present invention include, but are not limited to, tablets, capsules, oral liquids, injections, powder injections, atomized liquids, etc. In addition to the commonly used oral, injection, and atomized dosage forms, those skilled in the art can also according to needs Design or select other dosage forms based on common sense in the field of pharmacy.

According to the needs of the dosage form and the common knowledge of pharmacy, the prepared medicine may contain various pharmaceutically acceptable excipients and excipients, including but not limited to coating materials, solvents, solubilizers, adhesives, stabilizers, and antioxidants. , PH adjusters, flavoring agents, etc.

The technical solution claimed in the present invention does not exclude the use of other known Chinese and Western medicines/therapies for the treatment of gliomas. These medicines include, but are not limited to, chemotherapeutic drugs, biological targeted drugs, radiotherapy, immunotherapy, stem cell therapy, ZL -n-91 can be prepared in the same pharmaceutical composition with these drugs or used in combination as a single drug.

The selective PDE4 inhibitor ZL-n-91 of the present invention can significantly inhibit the proliferation of gliomas, indicating that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target for anti-glioma research It provides a basis for the preparation of anti-glioma proliferation drugs and has a good development and application prospect. The inhibitory strength of ZL-n-91 on PDE4B and PDE4D is more than 5000 times that of other PDE family members. Compared with other PDE4 inhibitors, the compound has higher selectivity for PDE4B and PDE4D, strong specificity, and less side effects such as vomiting, and has good clinical adaptability and safety when it is extended to the treatment of glioma.

Description of the drawings

Figure 1 is the proliferation inhibitory effect of ZL-n-91 on glioma cells. (A) Different concentrations of ZL-n-91 treated U87, snb19, U251 cells for 48 hours, the cell proliferation results; (B) different concentrations of ZL -The effect of n-91 treatment on the inhibition rate of U87, snb19, and U251 cells; (C) The inhibition rate of U87, snb19, and U251 corresponding to different concentrations of ZL-n-91. All data are expressed as mean ± standard deviation. (n=3), *P<0.05, **P<0.01, ***P<0.001 and****P<0.0001 are all compared with the solvent control 0μM group.

Figure 2 is the effect of ZL-n-91 on the cell cycle distribution of glioma cells (A) Cell cycle flow cytometry diagram of U87, snb19, and U251 cells treated with different concentrations of ZL-n-91 for 24 hours; (B) different concentrations After treatment with ZL-n-91, U87, snb19, U251, the percentage of each stage of the cycle. All data are expressed as mean ± standard deviation. (n=3), *P<0.05, **P<0.01, ***P<0.001 and****P<0.0001 are all compared with the solvent control group.

Figure 3 shows the apoptosis of glioma cells induced by ZL-n-91 (A) Flow cytometric detection of cell apoptosis in U87, snb19, and U251 cells treated with different concentrations of ZL-n-91 for 48 hours; (B) different concentrations After ZL-n-91 treatment, U87, snb19, U251 cells, the total apoptosis rate of different groups. All data are expressed as mean ± standard deviation. (n=3), *P<0.05, **P<0.01, ***P<0.001 and****P<0.0001 are all compared with the solvent control group.

Fig. 4 is Zl-n-91 inhibiting the growth of glioma subcutaneous tumors in nude mice (A) graph of weight change of nude mice after administration treatment; (B) graph of tumor volume change in nude mice after administration treatment; (C) tumor Weight map; (D) Tumor volume size map. All data are expressed as mean ± standard deviation. (n=5), *P<0.05, **P<0.01, both are compared with the solvent control group.

Detailed ways

According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

Example 1: CCK8 method to detect the effect of ZL-n-91 on the proliferation inhibition of glioma cells.

1) Take the glioma cells U87, snb19, and U251 in the logarithmic growth phase to prepare a single cell suspension. Inoculate 100μL of cell suspension (containing 2～3×10 ³ cells) per well in 96-well plates, divided into 9 groups: complete cell control group, solvent control group 0μM, 25μM, 50μM, 100μM, 200μM, 250μM , 300μM, 400μM, 500μM, 600μM, 800μM, 3 sub-holes in each group;

2) 24 hours after plating, after the cells are fully attached, different concentrations of ZL-n-91 are added to each group, and the cells are cultured for 48 hours;

3) Add 10ul CCK-8 solution to each hole to avoid bubbles;

4) Continue to incubate the cells for 1-2h, take out the culture plate, and measure the absorbance of the cells at 450nm with a microplate reader. And calculate the cell proliferation rate and cell inhibition rate, using Graphpad 7.0 software to calculate the IC50 results. Cell proliferation rate (%) = (OD450 experimental group-OD450 background) / (OD450 solvent control group-OD450 background) × 100%; cell inhibition rate (%) = 1-cell proliferation rate (%)

The results are shown in Figure 1: As the concentration of ZL-n-91 increases, the inhibitory ability of glioma cells U87, snb19, and U251 is significantly enhanced.

Example 2: Detection of the influence of Zl-n-91 on the cell cycle distribution of glioma by flow cytometry

1) Take the U87, snb19, and U251 cells in the logarithmic growth phase, resuspend them in serum-free basal medium, ^{inoculate them on a 6-well culture plate at 2*10 5} cells/ml, culture them in an incubator, and starve them for 24 hours;

2) After 24h, add serum and experimental drug concentration ZL-n-91 (150μM, 200μM) respectively, and set a solvent control group at the same time, and continue to culture the cells for 24h;

3) Collect the cells after 24 hours, wash twice with pre-cooled PBS, prepare a ^{cell suspension of 1×10 6} cells/mL with PBS, add 1 ml of 70% absolute ethanol, and fix at 4°C or -20°C for more than 24 hours ；

4) Centrifuge, wash twice with cold PBS, add 500μL PE stain according to the kit instructions, gently vortex the cells, incubate at room temperature for 15min in the dark, test on the machine within 1h, use ModiFit LT5.0 software for cell cycle analysis.

The results are shown in Figure 2: The growth cycle of the drug-treated cells was blocked in the G0/G1 period, and the higher the drug concentration, the more significant the blockade. The cells that were dividing in the S period decreased to varying degrees.

Experimental example 3: Flow cytometry to detect the inducing effect of Zl-n-91 on glioma cell apoptosis

1) Take the U87, snb19, and U251 glioma cells in the logarithmic growth phase and inoculate them in a 6-well culture plate ^{at 1.5-2*10 5 cells/ml;}

2) 24h after plating, after the cells are fully attached, add experimental concentrations of ZL-n-91 (150μM, 200μM, 300μM), and set up a solvent control group, and continue to culture the cells for 48h;

3) Harvest the cells after 48h, wash twice with pre-cooled PBS, prepare 1×10 ⁶ cells/mL cell suspension with 1×Binding Buffer, take 100μL into the flow tube, add 5uL 7-AAD according to the kit instructions Stain with 5uL PE, vortex the cells gently, incubate at room temperature for 15min in the dark, then put 200uL 1×Binding Buffer in the tube, perform flow cytometry within 1h, and analyze the results with Flow Jo V10 analysis software.

The results are shown in Figure 3: ZL-n-91 induced apoptosis of U87, snb19, and U251 glioma cells in a dose-dependent manner.

Experimental example 4: Zl-n-91 inhibits the growth of glioma subcutaneous tumors in nude mice

In order to study the inhibitory effect of Zl-n-91 on the proliferation of glioma U87 cells in vivo, we planted U87 cells under the skin of 6-week-old nude mice. When the tumor volume grows to ^{about 100mm 3} , the tumor-bearing mice are randomly divided into two groups: a solvent control group and an administration group (5mg/kg). The mice are given intragastric administration every day. The mice are weighed and tumor volume is measured every two days. .

The results are shown in Figure 4: There is no significant difference in body weight between the administration group and the control group. The tumor volume of the mice in the administration group was significantly smaller than that of the control group after 24 days of administration. The tumor was stripped and weighed. The weight of the tumor in the administration group (182.6±109.4 mg) was significantly smaller than the weight of the tumor in the control group (813.6±511.6 mg). These results show that Zl-n-91 can not only significantly inhibit the growth of gliomas, but also has less side effects on mice.

The above research results show that the phosphodiesterase 4 inhibitor ZL-n-91 used in the present invention can inhibit the proliferation of glioma cells and has a good anti-tumor effect.

Claims (14)

1. Application of PDE4 inhibitor ZL-n-91 in the preparation of drugs for treating solid tumors.
2. The use according to claim 1, wherein the solid tumor is a glioma.
3. The use according to claim 2, wherein the glioma comprises glioblastoma.
4. The use according to any one of claims 1 to 3, wherein the drug inhibits the proliferation of gliomas.
5. The application according to any one of claims 1 to 4, wherein the medicine is in the form of oral, injection or atomization.
6. A pharmaceutical composition for treating glioma, which contains ZL-n-91 as the sole active ingredient.
7. The pharmaceutical composition according to claim 6, wherein said glioma includes glioblastoma.
8. Use Zl-n-91 in vitro cytology and in vivo animal experiments to inhibit the proliferation of gliomas.
9. The method according to claim 8, wherein said glioma cells comprise astroblastoma.
10. The method according to claim 9, wherein the glioma cells are U87, snb19, U251 cells.
11. The method according to claim 8, wherein the in vivo animal experiment is a subcutaneous tumor-bearing model of glioma.
12. A method for treating solid tumors, which is characterized in that it comprises applying a PDE4 inhibitor ZL-n-91 to a subject in need.
13. The method of claim 12, wherein the solid tumor is a glioma.
14. The method of claim 13, wherein the glioma comprises glioblastoma.

Images