WO2022000708A1 - 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 - Google Patents

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 Download PDF

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WO2022000708A1
WO2022000708A1 PCT/CN2020/108212 CN2020108212W WO2022000708A1 WO 2022000708 A1 WO2022000708 A1 WO 2022000708A1 CN 2020108212 W CN2020108212 W CN 2020108212W WO 2022000708 A1 WO2022000708 A1 WO 2022000708A1
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osteosarcoma
cells
proliferation
preparation
inhibitor
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赵子建
李芳红
许丽君
周素瑾
赵正刚
张馨丹
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广州华真医药科技有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

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  • the present application belongs to the field of anti-tumor drugs, and specifically, the present application provides the application of PDE4 inhibitor ZL-n-91 in the preparation of anti-osteosarcoma drugs.
  • Osteosarcoma is one of the most common malignant bone tumors, and its incidence ranks first among primary bone tumors. It is most common in adolescents aged 0-24, followed by the elderly. The incidence and incidence of adolescents are Years are relatively stable, while the incidence and age of onset in the elderly vary greatly. In patients with osteosarcoma without metastases at initial diagnosis, the 5-year survival rate ranges from 40% to 75%. Local recurrence and distant metastasis are the most important causes of death in patients with osteosarcoma. The local recurrence rate after osteosarcoma surgery is about 10%-20%. Once the tumor recurs, the survival rate is significantly reduced.
  • tumor cell metastasis and recurrence are still the main causes of death in patients with osteosarcoma. Therefore, metastasis and recurrence are difficult problems in the treatment of osteosarcoma.
  • One of the main reasons for tumor recurrence may be the increase in the number of circulating tumor cells (CTCs), especially some chemo-resistant CTCs, which may be the source of recurrence after treatment. Tumor self-seeding of CTCs is also considered to be the cause of tumor metastasis.
  • Phosphodiesterases have the function of hydrolyzing the intracellular second messenger cAMP or cGMP, thereby affecting the signaling pathways mediated by these second messengers and regulating cellular functions.
  • PDEs are divided into 11 subtypes, among which phosphodiesterase 4 (PDE4) specifically hydrolyzes cAMP.
  • PDE4 is mainly distributed in various inflammatory cells, including mast cells, macrophage lymphocytes, epithelial cells, etc., and is involved in the promotion of monocyte and macrophage activation, neutrophil infiltration, proliferation of vascular smooth muscle, and vasodilation.
  • PDE4 inhibitors 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 leukocytes (such as respiratory burst) and inhibiting leukocyte migration; 3 ) Inhibit the expression or up-regulation of cell adhesion factor (CAM); 4) Induce the production of cytosolic 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.
  • COPD chronic obstructive pulmonary disease
  • PDE4 inhibitors also have a significant inhibitory effect on malignant tumors.
  • Patricia Goldhoff xenografts human brain astrocytoma cells U87 into nude mice the use of PDE4 inhibitors can prolong the survival of mice.
  • MotoshiNarita found that PDE4i can inhibit the growth of human melanoma cells
  • PetrosX.E.Mouratidis found that adding PDE4 inhibitors CC-8075 and CC-8062 to pancreatic cancer cells can reduce cell proliferation and increase apoptosis.
  • PDE4 inhibitors mainly include Rolipram, Cilomilast, Roflumilast and the like. Because Rolipram and Cilomilast caused dizziness, headache, nausea, vomiting and other gastrointestinal adverse reactions, the clinical application of the drug was affected.
  • One of the possible reasons for gastrointestinal adverse reactions is the poor specificity of PDE4 inhibitors, which moderately selectively inhibit the entire PDE family.
  • the Ki of Cilomilast to PDE4 is 92 uM, which is only 500 to 1000 times that of PDE1, 2, 3, and 5. Therefore, the use of higher doses of Cilomilast can interact with other PDE family members and cause side effects. In fact, with most PDE4 inhibitors, the side effect of vomiting at high doses is common.
  • Roflumilast has been approved by the US FDA for the treatment of COPD, it reduces lung inflammation, resists oxidative stress, effectively relieves lung fibrosis, enhances mucosal clearance, and airway remodeling.
  • adverse reactions mainly manifested as diarrhea, weight loss, nausea, atrial fibrillation and aggravation of mental diseases (such as insomnia, anxiety, depression) and so on.
  • IC50 value for PDE4D2 is 18nM, and it has a strong inhibitory effect on lipopolysaccharide-induced release of tumor necrosis factor alpha (TNF ⁇ ) from human peripheral blood mononuclear cells.
  • TNF ⁇ tumor necrosis factor alpha
  • Zln-91 has a high selectivity for PDE4D, and produces little side effects, which can effectively avoid adverse reactions such as dizziness, nausea, and vomiting. It has been tried at home and abroad for the treatment of lung diseases, such as COPD and even lung cancer and prostate cancer, with good results. Its efficacy in other cancers has not been verified by any actual studies.
  • the invention utilizes tumor cell culture in vitro to study the pathophysiological effect of ZL-n-91 through cell proliferation experiment, cell cycle experiment and cell apoptosis experiment.
  • the inhibitor can significantly inhibit the proliferation of human osteosarcoma cells U2OS, SaoS-2, MNNG/HOS; can significantly block the cell cycle of U2OS, SaoS-2, MNNG/HOS; can significantly induce U2OS, SaoS-2 2.
  • Apoptosis of MNNG/HOS osteosarcoma cells It lays a foundation for the preparation of anti-osteosarcoma proliferation drugs.
  • the present invention provides the application of PDE4 inhibitor ZL-n-91 in the preparation of anti-osteosarcoma drugs.
  • the anti-osteosarcoma drug inhibits the proliferation of osteosarcoma cells.
  • the anti-osteosarcoma drug induces apoptosis of osteosarcoma cells.
  • the medicine is in the form of oral or injection.
  • osteosarcoma is in situ or metastatic osteosarcoma.
  • metastatic osteosarcoma is lung metastatic osteosarcoma.
  • the present application provides an anti-osteosarcoma drug comprising ZL-n-91.
  • ZL-n-91 is the only active ingredient in the anti-osteosarcoma drug.
  • the present application provides methods of non-therapeutic inhibition of osteosarcoma cell proliferation comprising administering ZL-n-91.
  • osteosarcoma cells are U2OS, SaoS-2 or MNNG/HOS.
  • Osteosarcoma described in this application includes various types and characteristics of osteosarcoma, including but not limited to traditional osteosarcoma, intramedullary well-differentiated osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, telangiectasia osteosarcoma , Small cell osteosarcoma.
  • oral and injectable dosage forms in this application include, but are not limited to, tablets, capsules, oral liquid preparations, pills, granules, powders, water injections, oil injections, emulsion injections, powder injections, and the like.
  • transdermal administration inhalation administration
  • targeted carrier administration can also be routinely selected and designed by those skilled in the pharmaceutical field according to the situation.
  • ZL-n-91 can be used in combination with other known or researched drugs for the treatment of osteosarcoma or prepared into the same preparation.
  • Other drugs for the treatment of osteosarcoma include but are not limited to chemotherapy drugs, such as doxorubicin , platinum, cyclophosphamide, methotrexate and other chemotherapeutic agents, targeted drugs such as apatinib, anlotinib, crizotinib, imatinib, nilotinib, cedirnib step and so on.
  • the phosphodiesterase 4 inhibitor ZL-n-91 of the present invention can be purchased directly, and can be synthesized by itself or by entrustment with reference to existing literature. For example, you can refer to articles 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. Synthetic preparation.
  • 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 anti-osteosarcoma proliferation research
  • the target which provides a basis for the preparation of anti-osteosarcoma proliferation drugs, has a good development and application prospect.
  • ZL-n-91 is more than 5000 times more potent than other PDE family members against PDE4B and PDE4D.
  • the compound has higher selectivity for PDE4B and PDE4D, strong specificity, and fewer side effects, and can effectively reduce or even avoid adverse reactions such as vomiting.
  • Cell proliferation inhibition rate (%) (1-average OD value of administration group/average OD value of control group) ⁇ 100;
  • the cells were harvested, washed twice with cold PBS, and prepared into a cell suspension of 1 ⁇ 106 cells/mL with 1*BindingBuffer. Take 100 ⁇ l into a flow tube, and add 5 ⁇ l 7AA-D and 5 ⁇ L PE according to the kit instructions. For staining, vortex the cells gently, incubate at room temperature for 15 minutes in the dark, add 400u1* BindingBuffer to the tube, perform flow cytometry within 1 hour, and analyze the results with FlowJoV10 analysis software.
  • ZL-n-91 can significantly induce the apoptosis of U2OS, SaoS-2, MNNG/HOS osteosarcoma cells.

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Abstract

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。

Description

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 技术领域
本申请属于抗肿瘤药物领域，具体地，本申请提供了PDE4抑制剂ZL-n-91在制备抗骨肉瘤药物中的应用。
背景技术
骨肉瘤(OS)是最常见的恶性骨肿瘤之一，其发病率居原发性骨肿瘤的第一位，好发于0～24岁青少年，次为老年人，其中青少年的发病率及发病年比较稳定，而老年人发病率及发病年龄变异较大。初诊时无转移的骨肉瘤患者，5年生存率介于40％-75％之间。局部复发和远处转移是骨肉瘤患者死亡的最重要原因。骨肉瘤术后局部复发率约为10％-20％。一旦肿瘤复发，生存率显著降低。约10％-20％的患者第一次就诊时有远处转移，其中90％是肺转移，大约50％接受常规治疗的患者有肺转移。一旦患者骨肉瘤发展为远处转移，其5年生存率下降到20％-30％。以往多采取截肢的手术方式对骨肉瘤患肢进行根治性切除，然而，截肢手术对于提高患者的长期生存率改善并不明显。自从20世纪70年代新辅助化疗的出现，联合肿瘤广泛性切除术，使得骨肉瘤患者的5年生存率由20％提高到70％，保肢率从20％提高到80％。然而，肿瘤细胞的转移和复发仍然是骨肉瘤患者死亡的主要原因。因此，转移和复发是骨肉瘤治疗的难题。肿瘤复发的一个主要原因可能是循环肿瘤细胞(CTC)数量的增加，特别是一些化疗耐药的CTC，这些CTC可能成为治疗后复发的根源。CTC的肿瘤自播种也被认为是肿瘤转移的原因。
近年来，发展出了一些骨肉瘤的基因靶向治疗及免疫治疗方法，但疗效并不理想，多仅用于化疗无效、复发等晚期骨肉瘤的辅助治疗，例如使用阿帕替尼能够维持转移性骨肉瘤患者平均无进展生存期约6-12个月；在近30年的临床研究统计中发现，尽管使用了增加化疗时间和剂量，应用靶向或免疫治疗等手段，OS患者化疗联合保肢手术治疗后5年生存率约为70％，并未发生显著变化。故新型抗骨肉瘤药物的研发仍由很大的需求。
磷酸二酯酶(简称PDEs)具有水解细胞内第二信使cAMP或cGMP的功能，从而影响这些第二信使所介导的信号通路，调节细胞功能。PDEs共分为11个亚型，其中磷酸二酯酶4(PDE4)特异性水解cAMP。PDE4主要分布于各种炎性细胞内，包括肥大细胞、巨噬细胞淋巴细胞、上皮细胞等，参与了促进单核细胞与巨噬细胞活化、中性粒细胞浸润、 血管平滑肌的增殖、血管扩张以及心肌收缩等相关生理病理过程，对中枢神经系统功能、心血管功能、炎症/免疫系统、细胞黏附等都有影响。研究显示，PDE4抑制剂(PDE4i)具有抗炎、抗过敏、抗血小板活化作用。其作用机制主要涉及：1)抑制多种炎症介质/细胞因子的释放，能够抑制IL-4、IL-5基因的表达；2)抑制白细胞的激活(如呼吸爆发)，抑制白细胞游走；3)抑制细胞粘附因子(CAM)的表达或上调；4)诱导产生具有抑制活性的细胞子，如IL-6；5)诱导细胞凋亡；6)刺激内源性激素和儿茶酚胺类物质的释放。虽然已经开发或正在开发的PDE4抑制剂针对的疾病主要是慢性阻塞性肺疾病(COPD)、哮喘、炎症性肠疾病、关节炎等。但是，很多研究表明PDE4抑制剂对于恶性肿瘤也有显著的抑制作用。PatriciaGoldhoff给裸鼠异种移植入人脑星形胶质母细胞瘤细胞U87后，使用PDE4抑制剂可以延长小鼠生存期。2006年MotoshiNarita发现PDE4i可以抑制人黑色素瘤细胞的生长，PetrosX.E.Mouratidis发现在胰腺癌细胞中加入PDE4抑制剂CC-8075和CC-8062后，可以使其细胞增殖降低和细胞凋亡增加。
现有的PDE4抑制剂主要有咯利普兰(Rolipram)、西洛司特(Cilomilast)、罗氟司特(Roflumilast)等。由于Rolipram和Cilomilast引发头晕、头痛和恶心、呕吐等胃肠道不良反应，影响了药物在临床中推广应用。导致胃肠道不良反应的可能原因之一是PDE4抑制剂特异性差，从而中度选择性地抑制了整个PDE家族。如Cilomilast对PDE4的Ki＝92uM，仅是PDE1、2、3、5的Ki的500到1000倍。因此使用较高剂量Cilomilast会与其他PDE家族成员发生作用从而产生副作用。事实上，大多数PDE4抑制剂，在高剂量时产生呕吐的副作用是普遍现象。Roflumilast虽然已经通过美国FDA批准上市用于治疗COPD，降低肺部的炎症、抵抗氧化应激、有效的缓解肺部的纤维化、增强粘膜的清除能力以及气道的重塑等等。但是也有不良反应，主要表现为腹泻、体重下降、恶心、心房纤颤及精神方面疾病的加重(如失眠、焦虑、抑郁)等。
针对上述问题已经开发多种新型PDE4选择性抑制剂，如北卡罗来纳大学柯衡明开发的ZL-n-91：
Figure PCTCN2020108212-appb-000001
其对PDE4D2的IC50值为18nM，对脂多糖诱导的人外周血单个核细胞释放肿瘤坏死因子α(TNFα)有很强的抑制作用。研究表明其对PDE4D的抑制作用是其他PDE家族成员的5000倍以上。Zln-91对PDE4D具有较高的选择性，产生的副作用小，能够有效避免头晕、恶心、呕吐等不良反应。国内外已经尝试其用于治疗肺部疾病，如COPD乃至肺癌和前列腺癌，取得了良好的效果。其对于其他癌症的疗效，尚无任何实际研究验证。
发明内容
申请人在进一步扩展ZL-n-91治疗多种实体瘤和非实体瘤的过程中，发现ZL-n-91对目前临床上处置较为困难的骨肉瘤有着良好的抑制效果。本发明利用体外肿瘤细胞培养，通过细胞增殖实验，细胞周期实验，细胞凋亡实验，研究ZL-n-91的病理生理学作用。实验证实：该抑制剂能够显著抑制人源骨肉瘤细胞U2OS、SaoS-2、MNNG/HOS的增殖；能显著阻滞U2OS、SaoS-2、MNNG/HOS的细胞周期；能显著诱导U2OS、SaoS-2、MNNG/HOS骨肉瘤细胞的凋亡。为制备抗骨肉瘤增殖药物的研究奠定了基础。
一方面，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。
进一步地，所述抗骨肉瘤药物抑制骨肉瘤细胞增殖。
进一步地，所述抗骨肉瘤药物诱导骨肉瘤细胞凋亡。
进一步地，所述药物为口服或注射剂型。
进一步地，所述骨肉瘤为原位或转移骨肉瘤。
进一步地，所述转移骨肉瘤为肺转移骨肉瘤。
另一方面，本申请提供了抗骨肉瘤药物，其包含ZL-n-91。
进一步地，ZL-n-91为所述抗骨肉瘤药物中的唯一有效成分。
另一方面，本申请提供了非治疗性抑制骨肉瘤细胞增殖的方法，包括施用ZL-n-91。
进一步地，所述骨肉瘤细胞为U2OS、SaoS-2或MNNG/HOS。
本申请中所述的骨肉瘤包括各种类型，各种特征的骨肉瘤，包括但不限于传统型骨肉瘤、髓内高分化骨肉瘤、骨旁骨肉瘤、骨膜骨肉瘤、毛细血管扩展骨肉瘤、小细胞型骨肉瘤。
本申请中的口服和注射剂型包括但不限于片剂、胶囊剂、口服液体制剂、丸剂、颗粒剂、散剂、水针剂、油针剂、乳针剂、粉针剂等。
除口服或注射剂型外，其他已知或研究中的剂型如透皮给药、吸雾给药、靶向载体给药剂型也可以根据情况由药剂领域技术人员常规选用并设计剂型。
治疗骨肉瘤时ZL-n-91可以与其他已知或研究中的治疗骨肉瘤的药物联合使用或者制备入同一制剂中，其他治疗骨肉瘤的药物包括但不限于化疗药物，如阿霉素类、铂类、环磷酰胺类、甲氨蝶呤类等化疗剂，靶向药物如阿帕替尼、安罗替尼、克唑替尼、伊马替尼、尼罗替尼、西地尼步等。
本发明所述的磷酸二酯酶4抑制剂ZL-n-91可直接购买，参考现有文献自行合成或委托合成，例如可以参考文如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.合成制备。
有益效果：本发明所述的选择性PDE4抑制剂ZL-n-91，可以显著抑制肿瘤细胞的增殖，预示该磷酸二酯酶4抑制剂ZL-n-91有望成为抗骨肉瘤增殖研究的重要靶点，为制备抗骨肉瘤增殖药物提供了基础，具有良好的开发应用前景。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。相比其他PDE4抑制剂，该化合物对PDE4B和PDE4D具有更高的选择性，特异性强，产生的副作用小，能有效减弱甚至避免呕吐等不良反应。
附图说明
图1是ZL-n-91对骨肉瘤细胞的增殖抑制作用效果图：(a)不同浓度的ZL-n-91处理U2OS、SaoS-2、MNNG/HOS细胞48h后，细胞增殖结果图；(b)以200uM作为实验浓度，分别检测了U2OS、SaoS-2、MNNG/HOS细胞在0h、24h、48h、72h、96h的OD值；所有数据均用平均值±标准偏差表示。(n＝3)，*P<0.05,**P<0.01，***P<0.001and****P<0.0001均与溶剂对照组相比；
图2是ZL-n-91对骨肉瘤细胞周期分布的影响：200uM的ZL-n-91处理U2OS、SaoS-2、MNNG/HOS细胞48h后的细胞周期流式检测图；200uM的ZL-n-91处理后，U2OS、SaoS-2、MNNG/HOS中周期各个阶段的百分比；所有数据均用平均值±标准偏差表示；(n＝3)，*P<0.05,**P<0.01，***P<0.001and****P<0.0001均与溶剂对照组相比；
图3是ZL-n-91诱导骨肉瘤细胞凋亡图：不同浓度的ZL-n-91处理U2OS、SaoS-2、 MNNG/HOS细胞48h后的细胞凋亡流式检测图；不同浓度的ZL-n-91处理后，U2OS、SaoS-2、MNNG/HOS细胞中细胞不同状态的百分比；所有数据均用平均值±标准偏差表示。(n＝3)，*P<0.05,**P<0.01，***P<0.001and****P<0.0001均与溶剂对照组相比。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明，下面将结合附图，对本发明作进一步的说明。
实施例1
CCK-8法检测ZL-n-91对骨肉瘤细胞增殖的影响
1)取对数生长期的细胞U2OS、SaoS-2、MNNG/HOS，制备单细胞悬液。按每孔100μL细胞悬液接种于96孔板中，共分为9组：完全对照组(不添加任何物质)，溶剂对照组(添加同等体积的溶剂EtOH)，实验组(分别添加20uM、40uM、80uM、160uM、320uM、400uM、480uM的EtOH溶剂溶解的ZL-n-91)，每组3个副孔；
2)铺完板后，各组分别加入不同浓度的ZL-n-91，将细胞继续培养48h；
3)向每孔加入10ulCCK-8溶液，避免产生气泡；
4)将细胞继续孵育1-2小时，将培养板取出，用酶标仪测定在450uM处的吸光度；并计算细胞抑制率，采用Graphpad软件计算IC50结果。细胞增殖抑制率(％)＝(1-给药组平均OD值/对照组平均OD值)×100；
结果如图1所示：随着ZL-n-91浓度的升高，骨肉瘤细胞U2OS、SaoS-2、MNNG/HOS的增殖能力显著性降低，并且抑制作用具有时间依赖性。
实施例2
流式细胞术检测Zl-n-91对骨肉瘤细胞周期分布的影响
1)取对数生长期的U2OS、SaoS-2、MNNG/HOS细胞，用无血清的基础培养基重悬，以2*105个/ml接种于6孔培养板，每孔2ml，放在培养箱培养，饥饿处理24h；
2)24h后，加入200uM ZL-n-91，同时设溶剂对照组(添加同等体积的EtOH)，将细胞继续培养48h；
3)48h后收获细胞，冷PBS洗涤2次，用PBS制备成1×106个/mL的细胞悬液，加入1ml70％的无水乙醇，置于4℃或者-20℃固定24h以上；
离心，冷PBS洗涤2次，按试剂盒说明书加入500μL PE染色，轻柔的涡旋细胞，室温避光孵育15min，ModiFitLT5.0软件进行细胞周期分析。
结果如图2所示：200uM ZL-n-91将U2OS、SaoS-2细胞周期阻滞在G0-G1期；将MNNG/HOS细胞阻滞在G2-M期。
实验例3
流式细胞术检测Zl-n-91对骨肉瘤细胞凋亡的诱导作用
取对数生长期的U2OS、SaoS-2、MNNG/HOS细胞，以2*105个/ml接种于6孔培养板，每孔2ml；
2)铺完板后，分别加入实验浓度ZL-n-91(100uM，200uM，300uM)，同时设溶剂对照组(添加同等体积的EtOH)，将细胞继续培养48h；
3)48h后收获细胞，冷PBS洗涤2次，用1*BindingBuffer制备成1×106个/mL的细胞悬液，取100μl于流式管中，按试剂盒说明书加入5μl 7AA-D和5μL PE染色，轻柔的涡旋细胞，室温避光孵育15min，再加400u1*BindingBuffer于管中，1h内进行流式细胞检测，FlowJoV10分析软件分析结果。
结果如图3所示：ZL-n-91能显著诱导U2OS、SaoS-2、MNNG/HOS骨肉瘤细胞凋亡。
以上研究结果表明，本发明所采用的磷酸二酯酶4抑制剂ZL-n-91能抑制骨肉瘤细胞增殖，具有良好的抗肿瘤效果。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于这里的实施例，本领域技术人员根据本发明的揭示，对于本发明做出的改进和修改都应该在本发明的保护范围之内。

Claims (10)

  1. PDE4抑制剂ZL-n-91在制备抗骨肉瘤药物中的应用。
  2. 根据权利要求1所述的应用，其中所述抗骨肉瘤药物抑制骨肉瘤细胞增殖。
  3. 根据权利要求1所述的应用，其中所述抗骨肉瘤药物诱导骨肉瘤细胞凋亡。
  4. 根据权利要求1-3任一项所述的应用，其中所述药物为口服或注射剂型。
  5. 根据权利要求1-4任一项所述的应用，其中所述骨肉瘤为原位或转移骨肉瘤。
  6. 根据权利要求5所述的应用，其中所述转移骨肉瘤为肺转移骨肉瘤。
  7. 一种抗骨肉瘤药物，其包含ZL-n-91。
  8. 根据权利要求7所述的药物，其中ZL-n-91为所述抗骨肉瘤药物中的唯一有效成分。
  9. 一种非治疗性抑制骨肉瘤细胞增殖的方法，包括施用ZL-n-91。
  10. 根据权利要求9所述的方法，其中所述骨肉瘤细胞为U2OS、SaoS-2或MNNG/HOS。
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