CN113521053B

CN113521053B - Application of PDE4 inhibitor in preparation of medicine for treating leukemia

Info

Publication number: CN113521053B
Application number: CN202010302022.9A
Authority: CN
Inventors: 赵子建; 毛萍; 李芳红; 赵正刚; 李玉玉; 周素瑾; 李美蓉; 张馨丹
Original assignee: Guangzhou Huazhen Pharmaceutical Co ltd
Current assignee: Shenzhen Hanhui Pharmaceutical Technology Co ltd
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2023-01-10
Anticipated expiration: 2040-04-16
Also published as: WO2021208295A1; CN113521053A

Abstract

The invention provides application of a phosphodiesterase 4 inhibitor ZL-n-91 in preparation of a medicament for treating leukemia. In vitro cytology experiments and animal experiments show that the phosphodiesterase 4 inhibitor ZL-n-91 can obviously inhibit the proliferation of leukemia cells, and the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target for anti-leukemia proliferation research and has good further development and application prospects.

Description

Application of PDE4 inhibitor in preparation of medicine for treating leukemia

Technical Field

The invention belongs to the field of antitumor drugs, in particular to leukemia drugs, and particularly provides application of a PDE4 inhibitor ZL-n-91 in preparation of drugs for treating leukemia.

Background

Leukemia is a malignant disease of the hematopoietic system characterized by extensive and uncontrolled proliferation of a large number of leukemia cells in the body and infiltration of normal bone marrow and other organs and tissues. The etiology and pathogenesis of the disease are complex and not clear at present. The mortality rate of leukemia in China is approximately 4.8/10 ten thousand population, and 6.2/10 ten thousand in European and American countries. It is one of ten high-incidence malignant tumors in China, and is also the malignant tumor with the highest morbidity and mortality in people under 35 years old. Acute leukemia is more common than chronic leukemia in China, and the situation is opposite in European and American countries. Acute myelocytic leukemia is common in adults, acute lymphocytic leukemia is common in children, and slightly more in males than in females. Leukemia has poor prognosis, and the treatment mainly comprises combination and support therapy. Because the non-solid blood tumor represented by leukemia has larger difference with solid tumor in drug metabolism, cancer cell distribution mode, heterogeneity and curative effect judgment, the type of the existing drugs for treating leukemia is far from that of solid tumor, the curative effect is generally limited, and the finding of efficient therapeutic drugs has important significance.

Phosphodiesterases (PDEs) have the function of hydrolyzing cAMP or cGMP, which are second messengers in cells, thereby influencing signal pathways mediated by the second messengers and regulating the functions of the cells. PDEs are divided into 11 subtypes, of which phosphodiesterase 4 (PDE 4) specifically hydrolyzes cAMP. PDE4 is mainly distributed in various inflammatory cells, including mast cells, macrophage lymphocytes, epithelial cells and the like, participates in the related physiological and pathological processes of promoting activation of monocytes and macrophages, neutrophil infiltration, proliferation of vascular smooth muscle, vasodilation, myocardial contraction and the like, and has influence on the functions of the central nervous system, cardiovascular functions, inflammation/immune system, cell adhesion and the like. The research shows that the PDE4 inhibitor (PDE 4 i) has the effects of resisting inflammation, allergy and platelet activation. The action mechanism mainly relates to: 1) Inhibit the release of various inflammatory mediators/cytokines, and can inhibit the expression of IL-4 and IL-5 genes; 2) Inhibiting activation of leukocytes (e.g., respiratory burst), inhibiting leukocyte migration; 3) Inhibiting expression or upregulation of cell adhesion factor (CAM); 4) Inducing the production of cells with inhibitory activity, such as IL-6; 5) Inducing apoptosis; 6) Stimulate the release of endogenous hormones and catecholamines. PDE4 inhibitors that have been developed or are under development are mainly directed to Chronic Obstructive Pulmonary Disease (COPD), asthma, inflammatory bowel disease, arthritis and the like. However, many studies have shown that PDE4 inhibitors also have a significant inhibitory effect on malignant tumors. After xenotransplantation of human brain astrocytoma cells U87 into nude mice by Patricia Goldhoff, the survival of the mice was prolonged by the use of PDE4 inhibitors. In 2006, motoshi Narita discovered that PDE4i can inhibit the growth of human melanoma cells, and Petros X.E. Moratidis discovered that after PDE4 inhibitors CC-8075 and CC-8062 are added into pancreatic cancer cells, the proliferation of the pancreatic cancer cells can be reduced and the apoptosis of the pancreatic cancer cells can be increased.

The existing PDE4 inhibitors mainly comprise Rolipram (Rolipram), cilomilast (Cilomilast), roflumilast (Roflumilast) and the like. The adverse reactions of gastrointestinal tracts, such as dizziness, headache, nausea, vomiting and the like, caused by Rolipram and Cilomilast influence the popularization and application of the medicine in clinic. One of the possible causes of adverse gastrointestinal effects is the poor specificity of PDE4 inhibitors, which inhibit the entire PDE family with moderate selectivity. For example, the Ki =92nM for Cilomilast for PDE4, is only 500 to 1000 times the Ki for PDE1, 2, 3, 5. Therefore, cilomilast can interact with other PDE family members at higher doses and produce side effects. In fact, the side effects of emesis at high doses are common in most PDE4 inhibitors. Roflumilast has been approved by the FDA in the united states for marketing for the treatment of COPD, reduction of inflammation in the lungs, resistance to oxidative stress, effective alleviation of fibrosis in the lungs, enhancement of mucosal clearance and airway remodeling, among others. But also has adverse reactions, mainly manifested by diarrhea, weight loss, nausea, atrial fibrillation, and aggravation of mental diseases (such as insomnia, anxiety and depression).

Several novel PDE4 selective inhibitors have been developed to address the above problems, such as ZL-n-91, developed by Kyogming, university of North Carolina:

IC thereof ₅₀ Reaching 18nM, it has been tried to treat lung diseases such as COPD and even lung cancer and prostate cancer at home and abroad with good effect. In further expanding the course of this PDE4 inhibitor for the treatment of PDE 4-related diseases, it has not been validated in any study as to whether it is effective against other cancers, particularly non-solid tumors, such as hematologic/lymphoid tumors.

Disclosure of Invention

The inventors have attempted to use the PDE4 inhibitor ZL-n-91 in the treatment of non-solid tumours, particularly leukaemia, based on the treatment of pulmonary disease and malignant solid tumours.

In one aspect, the invention provides the use of the PDE4 inhibitor ZL-n-91 in the manufacture of a medicament for the treatment of a non-solid tumour.

Further, the non-solid tumor is leukemia.

Further, the leukemia is lymphocytic leukemia.

Further, the medicament inhibits leukemia proliferation.

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

Further, the medicament is an oral dosage form.

In another aspect, the present invention provides a pharmaceutical composition for treating leukemia, which comprises ZL-n-91 as a sole active ingredient.

Further, the leukemia is lymphocytic leukemia.

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

Further, the pharmaceutical composition is an oral dosage form.

The chemical structure of ZL-n-91 described herein is disclosed and those skilled in the art can obtain ZL-n-91 of suitable purity using synthesis, purchase, application gifting, etc. using techniques common in the art of organic chemistry, such as synthesis, purchase, application gifting, etc., in reference to the prior art literature (e.g., ruihong Ma, bin-yan Yang, chang-you Wu. A selective phosphor assay 4 (PDE 4) inhibitor ZL-n-91 precursors IL-17production by human memory. International immunopharmacology,2008,8 (10): 1408-1417).

The leukemias described in this application include various specific classes of leukemia, including, but not limited to, acute/chronic leukemia, myeloid/granulocytic/lymphoid leukemia, childhood/adult leukemia.

The dosage forms adopted by the invention include but are not limited to tablets, capsules, oral liquid, injection, powder injection and the like, and besides the common oral and injection dosage forms, other dosage forms can be designed or selected by persons skilled in the art according to the needs and the common knowledge in the field of medicaments.

According to the requirement of dosage form and general knowledge of pharmacy, the prepared medicine can contain various pharmaceutically acceptable auxiliary materials and excipients, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators, flavoring agents and the like.

The technical scheme claimed by the invention does not exclude the use of other known traditional Chinese and western medicines/therapies for treating leukemia, such as chemotherapeutics, biological targeting medicines, radiotherapy, immunotherapy and stem cell therapy, and ZL-n-91 can be prepared in the same pharmaceutical composition with the medicines or used in combination in the form of separate medicines.

The selective PDE4 inhibitor ZL-n-91 can obviously inhibit the proliferation of tumor cells, indicates that the phosphodiesterase 4 inhibitor ZL-n-91 is expected to become an important target for anti-leukemia proliferation research, provides a basis for preparing anti-leukemia proliferation medicines, and has good development and application prospects. ZL-n-91 is more than 5000-fold more potent than other PDE family members as inhibitors of PDE4B and PDE 4D. Compared with other PDE4 inhibitors, the compound has higher selectivity on PDE4B and PDE4D, strong specificity and small side effects such as generated vomit, and has good clinical adaptability and safety when being expanded to be used for treating leukemia.

Drawings

FIG. 1: the proliferation inhibition effect of ZL-n-91 on leukemia cells (a) a cell proliferation result graph is obtained after ZL-n-91 with different concentrations is used for treating L1210, HL-60, K562 and JurKat cells for 48 hours; (b) The proliferation inhibition rates of ZL-n-91 with different concentrations on L1210, HL-60, K562 and JurKat are respectively improved. All data are expressed as mean ± standard deviation. (n = 3), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to solvent control B.

FIG. 2: after L1210, HL-60, K562 and JurKat cells were treated for 48h by ZL-n-91 with different concentrations, the morphology of the cells changed.

FIG. 3: influence of ZL-n-91 on leukemia cell cycle distribution (a) cell cycle flow assay of ZL-n-91 at different concentrations after treatment of L1210, HL-60, K562 and JurKat cells for 48 h; (b-c) percentage of each phase of the cycle in L1210, HL-60, K562 and JurKat after treatment with ZL-n-91 at different concentrations. All data are expressed as mean ± standard deviation. (n = 3), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to the solvent control group.

FIG. 4: ZL-n-91 induces leukemia cell apoptosis (a) apoptosis flow detection maps after ZL-n-91 with different concentrations treats L1210, HL-60, K562 and JurKat cells for 48 h; (b) Total apoptosis rates in different groups of L1210, HL-60, K562 and JurKat. All data are expressed as mean ± standard deviation. (n = 3), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to the solvent control.

FIG. 5: ZL-n-91 profile of body weight over time in mice of different groups following inoculation in the nude mice L1210 leukaemia subcutaneous model tumour suppression test (a); (b) Graph of tumor volume versus time for different groups of mice after inoculation; (c) histograms of tumor weights of different groups of mice; (d) different groups of tumor pictures. All data are expressed as mean ± standard deviation. (n = 7-8), P <0.05, P <0.01, P <0.001and P <0.0001, all compared to the solvent control group.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: the CCK8 method is used for detecting the influence of ZL-n-91 on the proliferation of leukemia cells.

1) Taking the cells L1210, HL-60, K562 and JurKat in the logarithmic growth phase to prepare single cell suspension. Inoculating 100 μ L of cell suspension (K562, L1210 containing 8 × 103 cells; HL-60, jurKa4t containing 1 × 104 cells) per well in 96-well plate, and dividing into 9 groups, including complete control group A, solvent control group B,10 μ M,20 μ M,40 μ M,80 μ M,100 μ M,160 μ M,320 μ M, and 3 auxiliary wells per group;

2) After the plates are paved, respectively adding ZL-n-91 with different concentrations into each component, and continuously culturing the cells for 48h (adding the medicine again when culturing for 24 h);

3) Adding 10ul of CCK-8 solution into each hole to avoid generating bubbles;

4) The cells were incubated for an additional 1-2 hours, the plates were removed and absorbance at 450nm was measured using a microplate reader. And calculating the cell proliferation rate and the cell inhibition rate, and calculating the IC50 result by adopting Graphpad software. Cell proliferation rate (%) = (OD 450 experimental group-OD 450 background)/(OD 450 solvent control group-OD 450 background) × 100%; cell inhibition (%) = 1-cell proliferation (%)

The results are shown in FIG. 1: along with the increase of the concentration of ZL-n-91, the proliferation capacities of leukemia cells L1210, HL-60, K562 and JurKat are obviously reduced.

Example 2: leukemia cell morphology change 48h after ZL-n-91 treatment

1) Taking cells in logarithmic growth phase L1210, HL-60, K562 and JurKat to prepare single cell suspension. Inoculating 100 μ L cell suspension (K562, L1210 containing 8 × 103 cells; HL-60, jurKa4t containing 1 × 104 cells) per well into 96-well plate, and dividing into 3 groups, i.e. solvent control group of 100 μ M and 160 μ M, each group having 3 auxiliary wells;

2) After the plates are paved, adding ZL-n-91 with different concentrations into each group respectively, and continuously culturing the cells for 48h (adding the medicine when culturing for 24 h);

3) After 48h of cell culture, morphological changes of L1210, HL-60, K562 and JurKat were observed and recorded with a 40-fold microscope.

The results are shown in FIG. 2: compared with the control group, the dosage group has morphological changes such as intracellular vacuoles, abundant cytoplasm, cell shrinkage, and cracking of a plurality of cells into fragments.

Example 3: flow cytometry for detecting influence of ZL-n-91 on cell cycle distribution of leukemia

1) Taking L1210, HL-60, K562 and JurKat cells in the logarithmic growth phase, re-suspending the cells by using a serum-free basic culture medium, inoculating 1 x 105 cells/ml to a 6-well culture plate with 2ml per well, culturing the cells in a culture box, and performing starvation treatment for 24 hours;

2) After 24h, respectively adding serum and experimental concentration ZL-n-91 (100 muM, 150 muM), setting solvent control group, and continuously culturing cells for 48h (adding medicine again when culturing for 24 h);

3) Harvesting cells after 48h, washing with cold PBS for 2 times, preparing 1 × 10^ 6/mL cell suspension with PBS, adding 1mL of 70% absolute ethyl alcohol, and fixing at 4 ℃ or-20 ℃ for more than 24h;

4) Centrifugation, cold PBS wash 2 times, according to kit instructions add 500 u lPE staining, gentle vortex cells, room temperature light-shielded incubation for 15min, modiFit LT5.0 software for cell cycle analysis.

The results are shown in FIG. 3: 100 μ M ZL-n-91 blocks the L1210 cell cycle in the G2-M phase, 150 μ M ZL-n-91 blocks part of the L1210 cell cycle in the G2-M phase and part in the G0-G1 phase; stopping HL-60 cell cycle at a G0-G1 stage; ZL-n-91 blocks the K562 cell cycle in the G0-G1 phase; 150 μ M ZL-n-91 arrested JurKat cell cycle in G0-G1.

Experimental example 4: flow cytometry detection of induction effect of ZL-n-91 on leukemia cell apoptosis

1) Taking L1210, HL-60, K562 and JurKat cells in the logarithmic growth phase, inoculating 1 × 105 cells/ml in a 6-well culture plate, wherein each well contains 2ml;

2) After the plates are paved, the experimental concentration ZL-n-91 (100 mu M and 150 mu M) is respectively added, meanwhile, a solvent control group is arranged, and the cells are continuously cultured for 48h (the medicine is added again when the cells are cultured for 24 h);

3) After 48h, cells were harvested, washed 2 times with cold PBS, prepared into 1 × 10^ 6/mL cell suspension with 1 binding Buffer, 100. Mu.l in Flow tube, stained with 5. Mu.l 7AA-D and 5. Mu.l PE according to kit instructions, vortexed gently, incubated 15min at room temperature in the dark, 400. Mu.l of 1 binding Buffer in tube, flow cytometric assay was performed within 1h, and results were analyzed by Flow Jo V10 analysis software.

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

Experimental example 5: tumor inhibition test in nude mice

1) Preparation of cells: leukemia cells were cultured to log phase L1210, washed twice with PBS and resuspended, and cell counted, with a final injection of approximately 5 x 10 ⁴ The volume of the solution is 100 mu l/piece,

2) Nude mice were divided into two groups: the solvent control group and the administration group (10 mg. Kg-1) were treated with the gastric lavage drug daily, and the subcutaneous tumor volume of the nude mice was measured daily to calculate the tumor volume (V = 0.52X major axis X minor axis) ² ) Drawing a tumor volume growth curve, when the tumor volume of the mouse reaches 1500mm ³ At the right and left times, the mice were taken outTumors, comparing the size of the tumors,

3) The results are shown in FIG. 5: the tumor growth of the L1210 nude mouse transplantation model was rapid, and the weight of the mice was not affected by the gavage administration of the drug (FIG. 5 a), and the tumor volume of the control group was significantly larger than that of the drug-treated group at the 10 th day after the administration, the tumor volume was about 1/3 of that of the control group (FIG. 5 b), and the tumor weight was about 1/2 of that of the control group (FIG. 5 c). The results show that ZL-n-91 has a remarkable inhibition effect on the growth of L1210 subcutaneous implantation tumors and has statistical significance.

The research results show that the phosphodiesterase 4 inhibitor ZL-n-91 adopted by the invention can inhibit the proliferation of leukemia cells and has good anti-tumor effect.

Claims

Application of ZL-n-91 in preparing medicine for treating lymphocytic leukemia.
2. The use of claim 1, wherein the medicament inhibits lymphoblastic leukemia proliferation.
3. The use according to claim 1, wherein the medicament is administered at a dose of 10 mg-kg "1.
4. The use according to any one of claims 1 to 3, wherein the medicament is in an oral dosage form.