阅读说明：本技术 磷酸二酯酶4抑制剂ZL-n-91在制备抗骨肉瘤药物中的应用 (Application of phosphodiesterase 4 inhibitor ZL-n-91 in preparation of anti-osteosarcoma medicine ) 是由 赵子建 李芳红 许丽君 周素瑾 赵正刚 张馨丹 于 2020-07-01 设计创作，主要内容包括：本发明提供了PDE4抑制剂ZL-n-91在制备抗骨肉瘤药物中的应用。选择性PDE4抑制剂ZL-n-91对PDE4B和PDE4D的抑制剂强度是其他PDE家族成员的5000倍以上,特异性强且呕吐等副作用小；体外细胞学实验结果显示ZL-n-91可以显著抑制骨肉瘤细胞的增殖,具有良好的开发应用前景。(The invention provides application of a PDE4 inhibitor ZL-n-91 in preparing an anti-osteosarcoma medicament. The selective PDE4 inhibitor ZL-n-91 has the inhibitor strength of PDE4B and PDE4D which is more than 5000 times that of other PDE family members, strong specificity and small side effects such as vomit and the like; the results of in vitro cytology experiments show that ZL-n-91 can obviously inhibit the proliferation of osteosarcoma cells and has good development and application prospects.)

The use of the PDE4 inhibitor ZL-n-91 in the manufacture of a medicament for the treatment of osteosarcoma.

2. The use of claim 1, wherein the anti-osteosarcoma drug inhibits osteosarcoma cell proliferation.

3. The use of claim 1, wherein the anti-osteosarcoma drug induces apoptosis of osteosarcoma cells.

4. The use according to any one of claims 1 to 3, wherein the medicament is in an oral or injectable dosage form.

5. The use according to any one of claims 1-4, wherein the osteosarcoma is an orthotopic or metastatic osteosarcoma.

6. The use of claim 5, wherein the metastatic osteosarcoma is a pulmonary metastatic osteosarcoma.

7. An anti-osteosarcoma drug, which comprises ZL-n-91.

8. The medicament of claim 7, wherein ZL-n-91 is the only active ingredient in the anti-osteosarcoma medicament.

9. A method of non-therapeutically inhibiting osteosarcoma cell proliferation comprising administering ZL-n-91.

10. The method of claim 9, wherein the osteosarcoma cell is U2OS, SaoS-2, or MNNG/HOS.

Technical Field

The application belongs to the field of antitumor drugs, and particularly provides application of a PDE4 inhibitor ZL-n-91 in preparation of an anti-osteosarcoma drug.

Background

Osteosarcoma (OS) is one of the most common malignant bone tumors, the incidence rate of the osteosarcoma is the first of primary bone tumors, and is better to be found in teenagers of 0-24 years old, and the incidence rate and incidence age of the osteosarcoma are more stable, and the incidence rate and incidence age of the osteosarcoma are more variable in the elderly. The survival rate of 5 years of osteosarcoma patients without metastasis at the initial diagnosis is between 40% and 75%. Local recurrence and distant metastasis are the most important causes of death in osteosarcoma patients. The local recurrence rate after osteosarcoma surgery is about 10% -20%. Once the tumor recurs, survival rates significantly decrease. About 10% to 20% of patients have distant metastases at the first visit, of which 90% are lung metastases and about 50% of patients receiving conventional treatment have lung metastases. Once a patient's osteosarcoma has developed distant metastases, their 5-year survival rate drops to 20% -30%. In the past, the amputation operation mode is mostly adopted to radically remove the osteosarcoma affected limb, however, the amputation operation is not obvious to improve the long-term survival rate of the patient. Since the emergence of neoadjuvant chemotherapy in the 70 th 20 th century, combined with extensive tumor resection, the 5-year survival rate of osteosarcoma patients was improved from 20% to 70% and the limb protection rate was improved from 20% to 80%. However, metastasis and recurrence of tumor cells remain the major cause of death in osteosarcoma patients. Thus, metastasis and recurrence are difficult problems in osteosarcoma treatment. One major cause of tumor recurrence may be an increase in the number of Circulating Tumor Cells (CTCs), particularly some chemotherapy-resistant CTCs, which may be the source of post-treatment recurrence. Tumor self-seeding of CTCs is also thought to be responsible for tumor metastasis.

In recent years, gene-targeted therapy and immunotherapy methods for osteosarcoma have been developed, but the curative effect is not ideal, and the method is mostly only used for adjuvant therapy of chemotherapy-ineffective, recurrent and other advanced osteosarcoma, for example, apatinib can maintain the average progression-free survival period of metastatic osteosarcoma patients for about 6-12 months; in clinical study statistics of recent 30 years, it was found that the survival rate of OS patients 5 years after chemotherapy combined with limb protection surgery is about 70% and does not change significantly despite the use of measures such as increasing chemotherapy time and dose, applying targeting or immunotherapy, and the like. Therefore, the development of new anti-osteosarcoma drugs is still in great demand.

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(PDE4) specifically hydrolyzes cAMP. PDE4 is mainly distributed in various inflammatory cells, including mast cells, macrophage lymphocytes, epithelial cells, and the like, and participates in the related physiological and pathological processes of promoting activation of monocytes and macrophages, neutrophil infiltration, proliferation of vascular smooth muscle, vasodilatation, myocardial contraction, and the like, and has effects on central nervous system function, cardiovascular function, inflammation/immune system, cell adhesion, and the like. The research shows that the PDE4 inhibitor (PDE4i) 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. Although PDE4 inhibitors have been or are being developed for diseases primarily Chronic Obstructive Pulmonary Disease (COPD), asthma, inflammatory bowel disease, arthritis, and the like. However, many studies have shown that PDE4 inhibitors also have significant inhibitory effects on malignant tumors. After xenotransplantation of human brain astrocytoma cells U87 into nude mice by Patricia Goldhoff, the survival of mice was prolonged by the use of PDE4 inhibitors. In 2006, MotoshinNarita found that PDE4i can inhibit the growth of human melanoma cells, and PetrosX.E. Moratidis found that after PDE4 inhibitors CC-8075 and CC-8062 are added to 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 include 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 reactions is the poor specificity of PDE4 inhibitors, which results in a moderately selective inhibition of the entire PDE family. For example, the Ki of Cilomilast to PDE4 is 92uM, which is only 500 to 1000 times that of PDE1, 2, 3 and 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 with 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:

the compound has an IC50 value of 18nM to PDE4D2, and has strong inhibition effect on tumor necrosis factor alpha (TNF alpha) released by human peripheral blood mononuclear cells induced by lipopolysaccharide. The research shows that the inhibitor has more than 5000 times of the inhibition effect on PDE4D compared with other PDE family members. Zln-91 has high selectivity to PDE4D, produces little side effect, and can effectively avoid adverse reactions such as dizziness, nausea, emesis, etc. The application of the traditional Chinese medicine composition for treating lung diseases such as COPD and lung cancer and prostatic cancer has been tried at home and abroad, and good effects are achieved. The curative effect of the traditional Chinese medicine composition on other cancers is not verified by any actual research.

Disclosure of Invention

In the process of further expanding ZL-n-91 to treat various solid tumors and non-solid tumors, the applicant finds that ZL-n-91 has a good inhibition effect on osteosarcoma which is clinically difficult to treat at present. The invention researches the pathophysiology effect of ZL-n-91 by using in vitro tumor cell culture and through cell proliferation experiments, cell cycle experiments and apoptosis experiments. The experiment proves that: the inhibitor can remarkably inhibit proliferation of human osteosarcoma cells U2OS, SaoS-2 and MNNG/HOS; can obviously block the cell cycle of U2OS, SaoS-2 and MNNG/HOS; can obviously induce the apoptosis of U2OS, SaoS-2 and MNNG/HOS osteosarcoma cells. Lays a foundation for the research of preparing anti-osteosarcoma proliferation medicines.

In one aspect, the invention provides the use of the PDE4 inhibitor ZL-n-91 in the preparation of an anti-osteosarcoma medicament.

Further, the anti-osteosarcoma drug inhibits cell proliferation of osteosarcoma.

Further, the anti-osteosarcoma drug induces apoptosis of osteosarcoma cells.

Further, the medicine is in an oral or injection dosage form.

Further, the osteosarcoma is an in situ or metastatic osteosarcoma.

Further, the metastatic osteosarcoma is a pulmonary metastatic osteosarcoma.

In another aspect, the present application provides an anti-osteosarcoma agent comprising ZL-n-91.

Furthermore, ZL-n-91 is the only effective component in the anti-osteosarcoma medicine.

In another aspect, the present application provides a method of non-therapeutically inhibiting osteosarcoma cell proliferation comprising administering ZL-n-91.

Further, the osteosarcoma cell is U2OS, SaoS-2 or MNNG/HOS.

Osteosarcomas described herein include various types of osteosarcomas of various characteristics, including, but not limited to, traditional osteosarcoma, intramedullary well-differentiated osteosarcoma, paraosteosarcoma, periosteal osteosarcoma, capillary-expanded osteosarcoma, and small cell osteosarcoma.

Oral and injectable dosage forms in the present application include, but are not limited to, tablets, capsules, oral liquid preparations, pills, granules, powders, water injections, oil injections, milk injections, powder injections and the like.

In addition to oral or injectable dosage forms, other known or under-developed dosage forms such as transdermal administration, inhalation administration, targeted carrier administration, may be routinely selected and designed by those skilled in the pharmaceutical arts as appropriate.

In the treatment of osteosarcoma, ZL-n-91 may be used in combination with or formulated into other known or studied osteosarcoma-treating agents, including, but not limited to, chemotherapeutic agents such as doxorubicin, platinum, cyclophosphamide, methotrexate, etc., and targeted agents such as apatinib, aritinib, crizotinib, imatinib, nilotinib, cediranib, etc.

The phosphodiesterase 4 inhibitor ZL-n-91 of the present invention can be purchased directly, synthesized by itself or by itself in reference to the existing literature, and can be prepared synthetically, for example, in reference to Ruihong Ma, Bin-yan Yang, Chang-you Wu.A selective phosphodiesterase 4(PDE4) inhibitor ZL-n-91 supresses IL-17production by human memory Th17 cells.

Has the advantages that: 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-osteosarcoma proliferation research, provides a foundation for preparing anti-osteosarcoma proliferation medicines, and has good development and application prospects. ZL-n-91 has 5000-fold greater potency as inhibitors of PDE4B and PDE4D than other PDE family members. Compared with other PDE4 inhibitors, the compound has higher selectivity on PDE4B and PDE4D, strong specificity and small side effect, and can effectively reduce or even avoid adverse reactions such as vomit and the like.

Drawings

FIG. 1 is a graph showing the effect of ZL-n-91 on the inhibition of osteosarcoma cell proliferation: (a) after U2OS, SaoS-2 and MNNG/HOS cells are treated for 48 hours by ZL-n-91 with different concentrations, a cell proliferation result graph is obtained; (b) OD values of U2OS, SaoS-2 and MNNG/HOS cells at 0h, 24h, 48h, 72h and 96h are respectively detected by taking 200uM as an experimental concentration; 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. 2 is a graph showing the effect of ZL-n-91 on cell cycle distribution in osteosarcoma: cell cycle flow assay of U2OS, SaoS-2, MNNG/HOS cells treated with 200uM ZL-n-91 for 48 h; percentage of each phase of the cycle in U2OS, SaoS-2, MNNG/HOS after ZL-n-91 treatment at 200 uM; 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. 3 is a graph of ZL-n-91 induction of apoptosis in osteosarcoma cells: flow detection of apoptosis of ZL-n-91 cells treated with U2OS, SaoS-2 and MNNG/HOS cells for 48h at different concentrations; percentage of different states of cells in U2OS, SaoS-2, MNNG/HOS cells after treatment with different concentrations of ZL-n-91; 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.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

Example 1

CCK-8 method for detecting influence of ZL-n-91 on osteosarcoma cell proliferation

1) Single cell suspensions were prepared from cells in logarithmic growth phase U2OS, SaoS-2, MNNG/HOS. The cells were seeded in 96-well plates at 100. mu.L per well, and divided into 9 groups: complete control (no addition of any substance), solvent control (addition of equal volume of solvent EtOH), experimental (20 uM, 40uM, 80uM, 160uM, 320uM, 400uM, 480uM of EtOH in solvent dissolved ZL-n-91), 3 secondary wells per group;

2) after the plates are paved, respectively adding ZL-n-91 with different concentrations into each group, and continuously culturing the cells for 48 hours;

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

4) continuously incubating the cells for 1-2 hours, taking out the culture plate, and measuring the absorbance at 450uM by using an enzyme-labeling instrument; and calculating the cell inhibition rate, and calculating the IC50 result by using Graphpad software. Cell growth inhibition (%) × 100 (1-mean OD value of administration group/mean OD value of control group);

the results are shown in FIG. 1: with the increase of ZL-n-91 concentration, the proliferation capacity of osteosarcoma cells U2OS, SaoS-2 and MNNG/HOS is remarkably reduced, and the inhibition effect is time-dependent.

Example 2

Flow cytometry for detecting influence of ZL-n-91 on cell cycle distribution of osteosarcoma

1) Taking U2OS, SaoS-2 and MNNG/HOS cells in logarithmic growth phase, re-suspending with a serum-free basic culture medium, inoculating 2 x 105 cells/ml into a 6-well culture plate, culturing in an incubator with 2ml of each well, and starving for 24 hours;

2) after 24h, adding 200uM ZL-n-91, setting a solvent control group (adding EtOH with the same volume), and continuously culturing the cells for 48 h;

3) harvesting cells after 48h, washing with cold PBS for 2 times, preparing 1 × 106 cell suspensions/mL with PBS, adding 1mL of 70% absolute ethanol, and fixing at 4 deg.C or-20 deg.C for more than 24 h;

centrifugation, cold PBS wash 2 times, add 500. mu.L PE stain according to kit instructions, gently vortex cells, incubate 15min at room temperature in the dark, ModiFitLT5.0 software for cell cycle analysis.

The results are shown in FIG. 2: 200uM ZL-n-91 blocks the cell cycle of U2OS and SaoS-2 in G0-G1; MNNG/HOS cells were arrested at G2-M phase.

Experimental example 3

Flow cytometry detection of induction effect of ZL-n-91 on osteosarcoma apoptosis

Taking U2OS, SaoS-2 and MNNG/HOS cells in logarithmic growth phase, inoculating 2 x 105 cells/ml into a 6-well culture plate, wherein each well is 2 ml;

2) after the plates are paved, adding experimental concentration ZL-n-91(100uM, 200uM and 300uM) respectively, setting a solvent control group (adding EtOH with the same volume), and continuously culturing the cells for 48 h;

3) after 48h cells were harvested, washed 2 times with cold PBS, prepared into 1X 106/mL cell suspensions with 1 BindingBuffer, 100. mu.l in flow tubes, 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, added 400u1 BindingBuffer in tubes, flow cytometric assay was performed within 1h, and the results were analyzed by FlowJoV10 analysis software.

The results are shown in FIG. 3: ZL-n-91 can remarkably induce apoptosis of U2OS, SaoS-2 and MNNG/HOS osteosarcoma cells.

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

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.