WO2021208295A1 - Use of pde4 inhibitor in preparation of drug for treating leukemia - Google Patents

Abstract

Disclosed is the use of phosphodiesterase 4 inhibitor ZL-n-91 in preparation of a drug for treating leukemia. In vitro cytology experiments and animal experiments show that the phosphodiesterase 4 inhibitor ZL-n-91 can significantly inhibit the proliferation of leukemia cells, showing that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target in studies concerning anti-leukemia proliferation, and has a good prospect for further development and application.

Description

Application of PDE4 inhibitor in preparing medicine for treating leukemia Technical field

The present invention belongs to the field of anti-tumor drugs, especially leukemia drugs. Specifically, the present invention provides the application of PDE4 inhibitor ZL-n-91 in the preparation of drugs for treating leukemia.

Background technique

Leukemia is a malignant disease of the hematopoietic system, which is characterized by the extensive and uncontrolled proliferation of a large number of leukemia cells in the body and the infiltration of normal bone marrow and other organs and tissues. Its etiology and pathogenesis are complex, and it is still not very clear. The mortality rate of leukemia in China is roughly 4.8 per 100,000 population, and that in European and American countries is 6.2 per 100,000. It is one of the ten high-incidence malignant tumors in China, and it is also the malignant tumor with the highest morbidity and mortality among people under the age of 35. Acute leukemia is more common than chronic leukemia in China, while the situation is the opposite in European and American countries. Acute myeloid leukemia is more common in adults, and acute lymphoblastic leukemia is more common in children. It is slightly more common in men than in women. The prognosis of leukemia is poor, and the treatment is mainly combination and supportive therapy. Because non-solid hematological tumors represented by leukemia are quite different from solid tumors in drug metabolism, cancer cell distribution, heterogeneity, and efficacy judgments, the current types of drugs for treating leukemia are far less than solid tumors, and their efficacy is generally limited. Seeking high efficiency The therapeutic drugs are of great significance.

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, especially non-solid tumors, such as blood/lymphoid tumors, has not been verified by any research.

Summary of the invention

Based on the early treatment of lung diseases and malignant solid tumors by the PDE4 inhibitor ZL-n-91, the inventors tried to use it for the treatment of non-solid tumors, especially leukemia.

In one aspect, the present invention provides the application of the PDE4 inhibitor ZL-n-91 in the preparation of drugs for the treatment of non-solid tumors.

Further, the non-solid tumor is leukemia.

Further, the leukemia is lymphocytic leukemia.

Further, the drug inhibits the proliferation of leukemia.

Further, the drug is administered at a dose of 10 mg·kg-1.

Further, the medicine is an oral dosage form.

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

Further, the leukemia is lymphocytic leukemia.

Further, the drug is administered at a dose of 10 mg·kg-1.

Further, the pharmaceutical composition is an oral dosage form.

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

Further, the non-solid tumor is leukemia.

Further, the leukemia is lymphocytic leukemia.

Further, the ZL-n-91 inhibits the proliferation of leukemia.

Further, the ZL-n-91 is administered at a dose of 10 mg·kg-1.

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 leukemia described in this application includes various specific types of leukemia, including, but not limited to, acute/chronic leukemia, myeloid/granulocyte/lymphocytic leukemia, and childhood/adult leukemia.

The dosage forms that can be used in the present invention include but are not limited to tablets, capsules, oral liquids, injections, powder injections, etc. In addition to commonly used oral and injection dosage forms, those skilled in the art can also design or select according to needs and common knowledge in the pharmaceutical field Other dosage forms.

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 by the present invention does not exclude the use of other known Chinese and Western medicines/therapies for the treatment of leukemia. 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 tumor cells, indicating that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target for anti-leukemia proliferation research. The preparation of anti-leukemia proliferation drugs provides a basis and has good development and application prospects. 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, less side effects such as vomiting, and has good clinical adaptability and safety when it is extended to the treatment of leukemia.

Description of the drawings

Figure 1: The inhibitory effect of ZL-n-91 on the proliferation of leukemia cells (a) The proliferation results of L1210, HL-60, K562 and JurKat cells treated with ZL-n-91 at different concentrations for 48 hours; (b) Different concentrations The proliferation inhibition rate of L1210, HL-60, K562 and JurKat corresponding to 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 group B.

Figure 2: The morphological changes of L1210, HL-60, K562 and JurKat cells treated with different concentrations of ZL-n-91 for 48 hours.

Figure 3: The effect of ZL-n-91 on the cell cycle distribution of leukemia (a) Cell cycle flow cytometry diagram of L1210, HL-60, K562 and JurKat cells treated with different concentrations of ZL-n-91 for 48 hours; (bc) After treatment with different concentrations of ZL-n-91, the percentage of each stage of the cycle in L1210, HL-60, K562 and JurKat. 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 4: ZL-n-91 induces apoptosis of leukemia cells (a) Flow cytometric detection of apoptosis in L1210, HL-60, K562 and JurKat cells treated with different concentrations of ZL-n-91 for 48 hours; (b) L1210 , HL-60, K562 and JurKat, 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.

Figure 5: ZL-n-91 on the nude mouse L1210 leukemia subcutaneous model tumor inhibition test (a) after inoculation, the curve of the weight change of different groups of mice with time; (b) after the inoculation, the tumor volume of different groups of mice Curve graph over time; (c) histogram of tumor weights of mice in different groups; (d) pictures of tumors in different groups. All data are expressed as mean ± standard deviation. (n=7-8), *P<0.05, **P<0.01, ***P<0.001 and****P<0.0001 are all 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 of leukemia cells.

1) Take the cells L1210, HL-60, K562 and JurKat in the logarithmic growth phase to prepare a single cell suspension. 100μL of cell suspension per well (K562, L1210 containing 8×103 cells; HL-60, JurKa4t containing 1×104 cells) was inoculated into 96-well plates, divided into 9 groups: complete control group A, solvent control Group B, 10μM, 20μM, 40μM, 80μM, 100μM, 160μM, 320μM, 3 sub-holes in each group;

2) After plating, each group added different concentrations of ZL-n-91, and the cells were cultured for 48 hours (the drug was added again after 24 hours of culture);

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

4) Continue to incubate the cells for 1-2 hours, take out the culture plate, and measure the absorbance at 450 nm with a microplate reader. And calculate the cell proliferation rate and cell inhibition rate, using Graphpad 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 increased, the proliferation ability of leukemia cells L1210, HL-60, K562 and JurKat decreased significantly.

Example 2: Changes in the morphology of leukemia cells after 48 hours of treatment with ZL-n-91

1) Take the cells L1210, HL-60, K562 and JurKat in the logarithmic growth phase to prepare a single cell suspension. 100μL of cell suspension per well (K562, L1210 containing 8×103 cells; HL-60, JurKa4t containing 1×104 cells) was seeded in 96-well plates, divided into 3 groups: solvent control group 100μM, 160μM, 3 auxiliary holes per group;

2) After plating, each group added different concentrations of ZL-n-91, and the cells were cultured for 48 hours (the drug was added again after 24 hours of culture);

3) After the cells are cultured for 48 hours, observe and record the morphological changes of L1210, HL-60, K562 and JurKat with a 40-fold lens.

The results are shown in Figure 2: Compared with the control group, vacuoles were seen in the cells of the dose group, with abundant cytoplasm, cell shrinkage, and morphological changes such as lysis of many cells into fragments.

Example 3: Flow cytometry to detect the influence of Zl-n-91 on cell cycle distribution of leukemia

1) Take the L1210, HL-60, K562 and JurKat cells in the logarithmic growth phase, resuspend them in serum-free basal medium, inoculate 1*105 cells/ml in a 6-well culture plate, 2ml per well, and place in culture Box culture, starvation treatment for 24h;

2) After 24h, add serum and experimental concentration ZL-n-91 (100μM, 150μM) respectively, and set up a solvent control group at the same time, and continue to culture the cells for 48h (add the drug again when cultured for 24h);

3) Harvest the cells after 48h, wash twice with cold PBS, prepare a cell suspension of 1×10^6 cells/mL with PBS, add 1ml of 70% absolute ethanol, and fix at 4℃ or -20℃ for more than 24 hours;

4) Centrifuge, wash with cold PBS twice, add 500μl PE stain according to the kit instructions, gently vortex the cells, incubate at room temperature and dark for 15min, ModiFit LT5.0 software for cell cycle analysis.

The results are shown in Figure 3: 100μM ZL-n-91 blocked L1210 cell cycle in G2-M phase, 150μM ZL-n-91 partially blocked L1210 cell cycle in G2-M phase, and partly blocked in G0- G1 phase; block HL-60 cell cycle in G0-G1 phase; ZL-n-91 block K562 cell cycle in G0-G1 phase; 150μM ZL-n-91 block JurKat cell cycle in G0-G1 Expect.

Experimental Example 4: Detection of Zl-n-91 inducing apoptosis of leukemia cells by flow cytometry

1) Take the L1210, HL-60, K562 and JurKat cells in the logarithmic growth phase and inoculate them in a 6-well culture plate at 1*105 cells/ml, 2ml per well;

2) After plating the plate, add experimental concentration ZL-n-91 (100μM, 150μM), and set a solvent control group at the same time, and continue to culture the cells for 48h (add the drug again when cultured for 24h);

3) Harvest the cells after 48h, wash twice with cold PBS, prepare 1×10^6 cells/mL cell suspension with 1*Binding Buffer, take 100μl into the flow tube, add 5μl 7AA-D and 5μl PE stain, gently vortex the cells, incubate at room temperature for 15min in the dark, add 400μl 1*Binding Buffer to the tube, perform flow cytometry within 1h, and analyze the results by FlowJo V10 analysis software.

The results are shown in Figure 4: ZL-n-91 can significantly induce apoptosis of L1210, HL-60, K562 and JurKat leukemia cells.

Experimental Example 5: In vivo tumor inhibition test in nude mice

1) Cell preparation: Culture the leukemia cell L1210 to the logarithmic growth phase, wash twice with PBS and resuspend, and count the cells. The final injection volume is about 5*10 ⁴ /100μl/cell.

2) Divide the nude mice into two groups: a solvent control group and an administration group (10mg·kg-1). Gavage drugs every day, and measure the volume of the nude mice’s subcutaneous tumors every day, and calculate the tumor volume (V=0.52×long diameter) ×Short diameter ² ), draw the tumor volume growth curve, when the mouse tumor volume reaches about 1500mm ³ , take out the mouse tumor and compare the tumor size,

3) The results are shown in Figure 5: The tumor of the L1210 nude mouse transplantation model grows rapidly, and the intragastric administration of drugs did not affect the weight of the mice (Figure 5a). The tumor volume in the control group was significantly larger than that on the 10th day after the administration. In the drug treatment group, the tumor volume was about 1/3 of the control group (Figure 5b), and the tumor weight was about 1/2 of the control group (Figure 5c). It shows that ZL-n-91 has a significant inhibitory effect on the growth of L1210 subcutaneous implantation tumor, which is statistically significant.

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

Claims (15)

1. Application of ZL-n-91 in the preparation of drugs for treating non-solid tumors.
2. The use according to claim 1, wherein the non-solid tumor is leukemia.
3. The use according to claim 2, wherein the leukemia is lymphocytic leukemia.
4. The use according to claim 2 or 3, wherein the drug inhibits the proliferation of leukemia.
5. The use according to any one of claims 1 to 4, wherein the drug is administered at a dose of 10 mg·kg-1.
6. The use according to any one of claims 1-5, wherein the drug is in an oral dosage form.
7. A pharmaceutical composition for treating leukemia, which contains ZL-n-91 as the sole active ingredient.
8. The pharmaceutical composition according to claim 7, wherein said leukemia is lymphocytic leukemia.
9. The pharmaceutical composition according to claim 7 or 8, wherein the pharmaceutical composition is administered at a dose of ZL-n-91 10 mg·kg-1.
10. The pharmaceutical composition according to any one of claims 7-9, wherein the pharmaceutical composition is an oral dosage form.
11. A method of treating non-solid tumors, characterized by comprising administering ZL-n-91 to a subject in need.
12. The method according to claim 11, wherein said non-solid tumor is leukemia.
13. The method according to claim 12, wherein said leukemia is lymphocytic leukemia.
14. The method according to claim 12 or 13, wherein said ZL-n-91 inhibits the proliferation of leukemia.
15. The method according to any one of claims 11-14, wherein said ZL-n-91 is administered at a dose of 10 mg·kg-1.

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