WO2017181934A1 - 唑来膦酸的用途 - Google Patents

唑来膦酸的用途 Download PDF

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WO2017181934A1
WO2017181934A1 PCT/CN2017/080879 CN2017080879W WO2017181934A1 WO 2017181934 A1 WO2017181934 A1 WO 2017181934A1 CN 2017080879 W CN2017080879 W CN 2017080879W WO 2017181934 A1 WO2017181934 A1 WO 2017181934A1
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zoledronic acid
rapamycin
pulmonary
group
cells
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薛斌
李朝军
赵丹丹
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Nanjing University
Nanjing Tech University
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Nanjing University
Nanjing Tech University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate

Definitions

  • the invention belongs to the field of use of zoledronic acid, and relates to the use of zoledronic acid in the preparation of a medicament for pulmonary lymphangioleiomyomatosis, in particular to the preparation of zoledronic acid in combination with rapamycin and/or simvastatin.
  • Lymphangiomyomatosis is a rare and multi-system disease that occurs in women of childbearing age and can occur in the lungs, thoracic duct, abdomen, retroperitoneal cavity, pelvis, uterus, ovary, etc. The lungs are the most susceptible.
  • Pulmonary lymphangioleiomyomatosis (PLAM) is a pulmonary lesion in lymphatic smooth muscle syndrome. The main clinical manifestations are progressive dyspnea, recurrent pneumothorax, and chylothorax effusion. Pulmonary lymphoperiodoma often occurs as an independent disorder, and a small number of secondary disorders occur as tuberous sclerosis (TSC).
  • the pathogenesis of pulmonary lymphangiomyomatosis is characterized by excessive proliferation, migration and differentiation of smooth muscle-like lung lymphatic cells, leading to destruction of the cystic structure of the lungs, obstructing the function of the trachea and lymphatic vessels, and gradually losing lung function.
  • the source of LAM cells in the lesion area of pulmonary lymphangioma is still unclear, and it is thought to be from lymphatic vessels or blood vessels.
  • Pathological diagnosis is difficult due to pulmonary lymphangioleiomyoma and asthma, chronic obstructive pulmonary disease, and clinical symptoms similar to emphysema.
  • HRCT is characterized by multiple thin-walled sac-like images with evenly distributed lungs. The lungs have clear circular cysts with uniform boundaries and even distribution.
  • Lung biopsy is the gold standard for the diagnosis of this disease.
  • pulmonary lymphangioleiomyoma there is no mature pathological diagnosis of pulmonary lymphangioleiomyoma, except for HMB 45 antibody-positive, estrogen receptor (ER) and progesterone receptors associated with melanoma.
  • ER estrogen receptor
  • PR progesterone receptors associated with melanoma.
  • Cathepsin-k which was newly discovered in recent years, is also one of the auxiliary molecular markers for its diagnosis.
  • there is no particularly effective method for the treatment of pulmonary lymphangioleiomyoma mainly drug control and lung transplantation.
  • TSC1/TSC2 tumor suppressor gene
  • TSC2/TSC1 hamartin and tuberin
  • mTOR mammalian target of rapamycin
  • the mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinase that integrates extracellular signals and phosphorylates downstream target eggs.
  • the ribosomal p70S6 kinases, such as S6K1 and 4E-BP1 affect gene transcription and protein translation, and thus participate in the regulation of cell growth and proliferation. According to research reports, most of the tumor development process will have excessive activation of the mTOR signaling pathway.
  • the macrolide antibiotic rapamycin is a drug that has been found to be effective in controlling tumor progression in recent years.
  • the point is the mTORC1 protein complex in the mTOR signaling pathway, but after the patient stopped using rapamycin, the tumor recurred in a short period of time and there was no therapeutic effect in some patients with sporadic pulmonary lymphangioma.
  • Lung lymphangioleiomyoma is the same as most stromal tumors, and a single therapeutic target is not conducive to the treatment of the disease.
  • Zoledronic acid is a diphosphate having the formula: C 5 H 10 N 2 O 7 P 2 ; the chemical name is 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1. 1-Diphosphoric acid; English name: zoledronic acid; chemical structural formula as shown in Formula I:
  • zoledronic acid is widely used in the treatment of bone metabolic diseases such as osteoporosis, osteoarthritis and cancerous bone metastasis, which has a significant inhibitory effect on bone resorption.
  • zoledronic acid can directly target Farnesyl pyrophosphate synthase, a key enzyme in the mevalonate/isoprene pathway of osteoclasts.
  • the activity of FPPS reduces the formation of isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), thereby blocking the transmission of multiple signaling pathways involved in GTPase.
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • Zoledronic acid and alendronate and risedronate are third-generation bisphosphonates, which are distinguished from the first-generation nitrogen-free bisphosphonates, such as etidronate and chloroformate.
  • Sodium phosphonate and second-generation nitrogen-containing bisphosphonates, such as pamidronate and tiludronate have the strongest anti-bone resorption and less toxic side effects.
  • Its important molecular structure includes P-C-P group-active group and R1 and R2 side chain substituents. These important groups determine the biological activity and mechanism of action of zoledronic acid.
  • zoledronic acid mainly targets osteoclasts, such as blocking the destruction and absorption of bone by osteoclasts, interfering with the growth of osteoclasts and inducing apoptosis of osteoclasts.
  • osteoclasts such as blocking the destruction and absorption of bone by osteoclasts, interfering with the growth of osteoclasts and inducing apoptosis of osteoclasts.
  • zoledronic acid for the treatment of pulmonary lymphangioleiomyoma.
  • the present invention provides zoledronic acid in the preparation of pulmonary lymphangioleiomyomatosis the use of.
  • the medicament further comprises rapamycin and/or simvastatin.
  • the pulmonary lymphangioleiomyoma comprises lung tissue and smooth muscle nodular and diffuse hyperplasia in the hilar.
  • the dosage form of the zoledronic acid is an injection, a solution, a tablet, and a capsule.
  • the zoledronic acid is administered by intravenous drip, oral and nebulized administration.
  • the pulmonary lymphangioleiomyoma drug comprises zoledronic acid.
  • the concentration of zoledronic acid is from 200 ⁇ g/kg to 500 ⁇ g/kg.
  • the ratio of zoledronic acid to rapamycin is 1:5 to 1:1.
  • the ratio of zoledronic acid to compound X is from 1:10 to 1:1.
  • the ratio of zoledronic acid, rapamycin and simvastatin is 1:5:10 to 1:1:1.
  • the present invention proposes a novel use of zoledronic acid for the treatment of pulmonary lymphangioleiomyoma comprising pulmonary tissue and smooth muscle nodular and diffuse hyperplasia in the hilar.
  • the medicament of the invention can effectively treat pulmonary lymphangioleiomyoma in lymphangioleomostase, and can effectively reduce the recurrence of such diseases.
  • Figure 1 is a graph showing the toxicity of different zoledronic acid concentrations to TSC2-null cells
  • Figure 2 is a graph showing the toxicity of zoledronic acid and rapamycin on TSC2-null cytotoxicity
  • Figure 3 is a graph showing the apoptosis of TSC2-null cells induced by zoledronic acid, TUNEL assay and data statistics;
  • Figure 4 is a schematic diagram showing the construction of a subcutaneous tumor formation process in nude mice and a mouse model of pulmonary lymphangioleiomyoma;
  • Figure 5 shows the size of subcutaneous tumor and the immunofluorescence pattern of Ki67 and p-S6 in the cells after administration of zoledronic acid
  • Figure 6 is a pathological analysis of lung formation of lung lymphangioleiomyoma after administration of zoledronic acid and rapamycin (HE).
  • the experimental materials used in the present invention are as follows: the MTT test kit was purchased from Nanjing Jianjian Company, the TUNEL test kit was purchased from Promiga, and the DAPI, YAP, Ki67 antibodies were purchased from CST, 6-8 weeks old.
  • STOCK-Foxn1nu/Nju female nude mice were purchased from the Institute of Model Animals of Nanjing University.
  • the normal maintenance feed of mice was purchased from Jiangsu Synergy Bioengineering Co., Ltd. Zoledronic acid and rapamycin were purchased from Selleck Chemicals China.
  • TSC2- is selected when the zoledronic acid efficacy is verified at the cellular level of the following examples. Null mouse embryonic fibroblasts serve as cells for their detection.
  • control group zoledronic acid 10 ⁇ M, 25 ⁇ M, 50 ⁇ M group
  • control group was zoledronic acid solvent NaOH
  • the cell was TSC2-null mouse embryonic fibroblast
  • DMEM+10 % FBS was treated under normal culture conditions for 24 hours.
  • the experimental group was divided into seven groups, which were the control group, the zoledronic acid group, the rapamycin group, the simvastatin group, and the zoledronic acid + rapamycin.
  • the control group was a drug solvent, the concentration of zoledronic acid was 50 ⁇ M, the concentration of rapamycin was 20 nM, the concentration of simvastatin was 0.5 ⁇ M, the concentration of zoledronic acid + rapamycin was 50 ⁇ M + 20 nM, and zoledron.
  • the concentration of acid + simvastatin was 50 ⁇ M + 0.5 ⁇ M, and the concentration of simvastatin + rapamycin was 0.5 ⁇ M + 20 nM.
  • the experimental treatment time was 48 hours, and the same treatment method as in Example 1 was carried out.
  • Fig. 2 It can be seen from Fig. 2 that the cell viability decreased significantly after 48 hours of treatment with zoledronic acid, rapamycin and simvastatin. Among them, zoledronic acid has the most obvious effect, and the cell viability decreases by about 30%. The combination of zoledronic acid in combination with rapamycin and simvastatin was the most effective in the combination treatment group, with a decrease of about 35%. At the same time, as can be seen from Fig. 2, the combined effect of the combination on the cell viability was stronger than that of the single drug group and the zoledronic acid combination group was the strongest.
  • zoledronic acid at a concentration of 25 ⁇ M and 50 ⁇ M and rapamycin used clinically were examined by TUNEL kit to see if zoledronic acid caused apoptosis of the cells.
  • a NaOH solution of zoledronic acid was prepared as a control group, and the cells were treated for 24 hours.
  • apoptotic cells in the zoledronic acid-treated group were significantly increased compared with the rapamycin-treated group, and the apoptosis increased with increasing concentration, while zoledronic acid did not cause cell significance. Apoptosis, therefore apoptosis is concentration dependent on zoledronic acid.
  • zoledronic acid was continuously injected into the tail vein of a subcutaneous tumor-forming nude mouse twice a week at a dose of 1 mg/kg.
  • the growth rate of subcutaneous tumors in the zoledronic acid treatment group was significantly slowed down, and the tumor volume increased significantly after 15 days of drug treatment, but there was no significant difference in body weight between the two groups. . It can be concluded that zoledronic acid can effectively inhibit the growth of subcutaneous parenchyma of TSC2-null cells.
  • the present embodiment further detects the proliferation of subcutaneous tumor cells and the expression of p-S6 protein by immunofluorescence technique.
  • p-S6 is a signaling molecule downstream of the mTOR signaling pathway, and its expression is increased when the mTOR signaling pathway is activated. Ki67 protein expression is observed when cells enter the cycle for cell proliferation. As can be seen from Fig. 5C, the expression of Ki67 protein in the zoledronic acid-treated group was significantly reduced, indicating that zoledronic acid can effectively inhibit the proliferation of TSC2-null cells. The expression of p-S6 protein in the cells was examined. As shown in Fig.
  • this example constructs a model of pulmonary lymphangioleiomyoma.
  • Purchase 6- to 8-week-old female nude mice and wipe 75% of the TSC2-null cells with a 0.45 mm diameter needle on the bilaterally vascularized areas, and then inject 10 ⁇ 7 TSC2-null cells. The cells overflow.
  • subcutaneous parenchyma grew on both sides of the nude mice.
  • the nude mouse is sacrificed, and the subcutaneous tumor is taken out in a clean bench and cut.
  • the cut subcutaneous tumor was digested with collagenase and digested at 37 ° C for half an hour, then filtered through a 100-mesh sieve, and the primary tumor cells were centrifuged at 1000 rpm, the supernatant was removed, and a new medium was added to resuspend.
  • the cells were incubated in a Petri dish for two days.
  • Subcutaneously tumorigenic cells increase the tumorigenicity of the cells relative to the previous cells.
  • the well-adhered cells were re-digested and suspended, and 10 ⁇ 5 of the primary tumor cells were injected into the tail vein of each nude mouse, and the tail vein and intra-abdominal administration of the mice were performed three days later.
  • the specific construction flow of mouse pulmonary lymphangioleiomyoma is shown in Fig. 4B.
  • TSC2-null cells are not primary tumor cells.
  • TSC2-null cells In order to increase the tumorigenicity of TSC2-null cells, it is necessary to form tumors under the skin to increase cell formation. Tumorous, then injected into the nude mice in the tail vein will specifically form a lesion area in the lungs of nude mice, so the microenvironment of the lung may be more conducive to the growth of TSC2-null cells.
  • Figure 6 The specific results are shown in Figure 6.
  • pulmonary chyle appears in the general morphology of the lungs of nude mice, alveolar reduction in the HE staining of the lungs, increased spatial spacing of the lungs, and most of the TSC2-null cells accumulate in the arteries, veins , as well as near the trachea.
  • the drug-administered group was divided into five groups.
  • the control group was a placebo, a placebo, a zoledronic acid 200 ⁇ g/kg group, a zoledronic acid 500 mg/kg group, and a rapamycin group.
  • the zoledronic acid group 200 ⁇ g/kg was injected intravenously three times a week, the zoledronic acid group 500mg/kg twice a week, the rapamycin 1mg/kg three times a week, rapamycin combined with zoledronic acid
  • the acid group, zoledronic acid 200 ⁇ g/kg plus rapamycin 1 mg/kg was intraperitoneally injected three times a week.
  • the lungs of the mice were eroded and white, and after the group administration of the drug, the lungs of all the mice showed significant improvement, the blood vessels were clear, and the surface of the lungs was smooth.
  • the lungs of the mice were subjected to paraffin-embedded sections for pathological diagnosis. As a result, as shown in Fig.
  • mice 6A a large amount of tumor cells accumulated in the lungs of the experimental group, but After 6 weeks of administration of zoledronic acid 200 ⁇ g/kg and 500 ⁇ g/kg, the tumors of the lungs of mice at a concentration of 200 ⁇ g/kg were significantly reduced, and the mice of 500 ⁇ g/kg had almost no tumor in the lungs, while Repa The lungs of mice treated with mycin and rapamycin in combination with zoledronic acid showed no tumors after one month of administration.
  • one group after one month of withdrawal experimental group + rapamycin group, experimental group + zoledronic acid + rapamycin group, to observe whether there was recurrence in the lung lesion area of mice.
  • Fig. 6B The specific results are shown in Fig. 6B.
  • white lung parenchyma appeared on the surface of the lung of the mice.
  • the lungs of the mice showed obvious proliferation of tumor cells. Tumor cells in the vicinity of the arteries of the lungs are enriched.
  • the lung surface of the mice was smooth and no lesions, and the lungs of the mice were also tumor-free. No lesion area.
  • pathological examination of other organs also showed that no metastasis of tumor cells occurred, and the results are shown in Fig. 6C.
  • zoledronic acid in the mouse lymphangioleiomyoma model The treatment effect is even more.
  • Zoledronic acid was found to be effective in inhibiting tumor growth in the treatment of subcutaneous tumors, but did not shrink or disappear.
  • zoledronic acid can effectively inhibit the formation of tumors, and can effectively inhibit the recurrence of tumors in the treatment of rapamycin, so zoledronic acid on the lung lymph The treatment of leiomyomas is better.
  • zoledronic acid can be effective in lymphangioleiomyoma of lymphangioleiotrophic muscle disease, and the treatment with rapamycin can be more effective, and the tumor is less prone to recurrence after stopping the drug.
  • zoledronic acid has a certain effect on the accompanying disease of nodular sclerosis such as renal angiomyolipoma.

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Abstract

本发明公开了唑来膦酸在制备肺部淋巴管平滑肌瘤病药物中的用途。本发明提出了唑来膦酸用于治疗肺淋巴管平滑肌瘤药物的新用途，所述肺部淋巴管平滑肌瘤包括肺组织以及肺门内的平滑肌结节性及弥漫性增生。根据本文发明唑来膦酸可以有效的治疗肺部淋巴管平滑肌瘤，并且联合雷帕霉素可以有效的较少肺部淋巴管平滑肌瘤的复发。

Description

唑来膦酸的用途 技术领域
本发明属于唑来膦酸的用途领域，涉及唑来膦酸在制备肺淋巴管平滑肌瘤病药物中的用途，尤其涉及将唑来膦酸与雷帕霉素和/或辛伐他汀结合制备淋巴管平滑肌增多症中肺淋巴管平滑肌瘤病药物中的用途。
背景技术
淋巴管平滑肌增多症(lymphangiomyomatosis，LAM)是一种罕见多系统的疾病，此病多发病于育龄期的妇女，可发病于肺部，胸导管，腹部，腹膜后腔，盆腔，子宫，卵巢等，肺部最易受累。肺淋巴管平滑肌瘤(pulmonary lymphangioleiomyomatosis，PLAM)是淋巴管平滑肌增多症中肺部病变，临床主要表现为渐进性的呼吸困难，反复发作性气胸，乳糜性胸腔积液。肺淋巴平滑肌瘤大多作为独立的病症发生，也有少数作为结节性硬化症(TSC)的继发性病症发生。肺淋巴管肌瘤病的发病特征是平滑肌样的肺淋巴管细胞的过多的增殖，迁移和分化，导致肺囊性结构的破坏，阻碍气管和淋巴管的功能，使得肺功能逐渐丧失。对于肺淋巴管肌瘤病病灶区LAM细胞的来源现阶段还是不清楚，多认为其来自于淋巴管或是血管。由于肺淋巴管平滑肌瘤和哮喘，慢性阻塞性肺病，以及肺气肿相似的临床症状，因此其病理诊断比较困难。HRCT以肺部均匀分布的多个薄壁囊状影为特征，双肺出现边界清晰的圆形囊肿，分布均匀。肺组织活检是该病诊断的金标准，目前肺淋巴管平滑肌瘤病尚无成熟的病理诊断意见，除了与黑色素瘤相关的HMB 45抗体阳性，雌激素受体(ER)及孕激素受体(PR)阳性为LAM的标记性抗体，以及近几年新发现的Cathepsin-k也是作为其诊断的辅助分子标记之一。现阶段针对肺淋巴管平滑肌瘤的治疗无特别有效方法，主要是药物控制和肺移植。
研究表明抑癌基因TSC1/TSC2的基因突变是肺淋巴管肌瘤病发生的原因，其中TSC2基因的突变占大多数。TSC2/TSC1所编码的蛋白hamartin和tuberin会调控mTOR(mammalian target of rapamycin)信号通路的表达。哺乳动物的雷帕霉素靶(mammalian target of rapamycin,mTOR)是一种非典型丝氨酸/苏氨酸蛋白激酶,可整合细胞外信号,磷酸化下游靶蛋 白核糖体p70S6激酶,如S6K1及4E-BP1,影响基因转录与蛋白质翻译,从而参与调控细胞生长、增殖等过程。据研究报道，大部分的肿瘤发生发展过程中都会有mTOR信号通路的过度激活。
在结节性硬化症相关的肺淋巴管肌瘤病的临床治疗方面，大环内酯类抗生素雷帕霉素(rapamycin)是近几年发现的可以有效控制肿瘤恶化的药物，其作用的靶点就是mTOR信号通路中的mTORC1蛋白复合物，但是病人停止使用雷帕霉素后，短期内肿瘤出现复发，并且在某些散发性的肺淋巴管肌瘤病人身上并无治疗效果。肺淋巴管平滑肌瘤与大部分的恶质瘤一样，单个的治疗靶点不利于该病的治疗。
唑来膦酸是一种二磷酸盐，其的分子式：C5H10N2O7P2；化学名称为：1-羟基-2-(咪唑-1-基)-亚乙基-1,1-二磷酸；英文名称为：zoledronic acid；化学结构式如式I所示：
Figure PCTCN2017080879-appb-000001
目前，唑来膦酸在临床上被广泛用于治疗骨代谢性疾病，如骨质疏松、变形性骨炎以及癌性骨转移等骨病，其对骨吸收有明显的抑制作用。唑来膦酸作为甲羟戊酸代谢途径下游的抑制剂，可以直接靶向破骨细胞的甲羟戊酸/异戊二烯通路的关键酶—法尼基焦磷酸合成酶(farnesyl pyrophosphate synthase，FPPS)的活性，进而减少异戊二烯类化合物如法尼基焦磷酸(FPP)和香叶基香叶基焦磷酸(GGPP)的形成，从而阻断GTPase参与的多种信号通路的传递。唑来膦酸与阿仑膦酸钠和利塞膦酸钠等是第三代二膦酸盐药物，其区别于第一代不含氮二膦酸盐，如依替膦酸钠与氯屈膦酸钠和第二代含氮二膦酸盐，如帕米膦酸钠与替鲁膦酸钠，其抗骨吸收作用最强而毒副作用更小。其重要分子结构包括P-C-P基团—活性基团和R1与R2侧链取代基，这些重要基团决定了唑来膦酸的生物活性与作用机理。临床上的唑来膦酸主要靶向破骨细胞，如阻挡破骨细胞对骨的破坏与吸收、干扰破骨细胞的生长和诱导破骨细胞的凋亡等。但目前尚无唑来膦酸用于治疗肺淋巴管平滑肌瘤的报道。
发明内容
解决的技术问题：为了开发一种对肺淋巴管平滑肌瘤病治疗效果好，且能够有效防止肺部肿瘤复发的药物，本发明提供了唑来膦酸在制备肺淋巴管平滑肌瘤病中的用途。
技术方案：
唑来膦酸在制备肺淋巴管平滑肌瘤病药物中的用途。
优选的，所述药物还包括雷帕霉素和/或辛伐他汀。
优选的，所述肺淋巴管平滑肌瘤包括肺组织以及肺门内的平滑肌结节性及弥漫性增生。
优选的，所述唑来膦酸的剂型为针剂、溶液剂、片剂和胶囊剂。
优选的，所述唑来膦酸的给药方式为静脉滴注、口服和雾化吸入式给药。
所述的肺淋巴管平滑肌瘤病药物，包含唑来膦酸。
优选的，唑来膦酸的用药浓度为200μg/kg～500μg/kg。
优选的，唑来膦酸与雷帕霉素联合使用时，唑来膦酸与雷帕霉素的用药比例为1:5～1:1。
优选的，唑来膦酸与辛伐他汀联合使用时，唑来膦酸与化合物X的用药比例为1:10～1:1。
优选的，唑来膦酸、雷帕霉素及辛伐他汀联合使用时，唑来膦酸、雷帕霉素及辛伐他汀的用药比例为1:5:10～1:1:1。
有益效果：本发明提出了唑来膦酸用于治疗肺淋巴管平滑肌瘤的新用途，所述肺淋巴管平滑肌瘤包括肺组织以及肺门内的平滑肌结节性及弥漫性增生。本发明所述的药物可以有效的治疗淋巴管平滑肌增多症中的肺淋巴管平滑肌瘤，并且可以有效的较少该类疾病的复发。
附图说明
图1是不同唑来膦酸浓度对TSC2-null细胞毒性的检测图；
图2是唑来膦酸联和雷帕霉素对TSC2-null细胞毒性的检测图；
图3是唑来膦酸引起TSC2-null细胞的凋亡，TUNEL检测以及数据统计图；
图4是裸鼠皮下成瘤过程图和肺淋巴管平滑肌瘤的小鼠模型构建示意图；
图5唑来膦酸用药后检测皮下肿瘤大小以及细胞Ki67和p-S6免疫荧光图；
图6是唑来膦酸以及联合雷帕霉素用药后肺淋巴管平滑肌瘤肺部成瘤病理分析图(HE)。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
本发明采用的实验材料来源如下：MTT检测试剂盒购买于南京建成公司，TUNEL检测试剂盒购买于Promiga公司,DAPI、YAP、Ki67抗体购买于CST公司，6～8周龄的 STOCK-Foxn1nu/Nju雌性裸鼠购于南京大学模式动物研究所。小鼠正常维持饲料购于江苏省协同生物工程有限公司。唑来膦酸以及雷帕霉素购于Selleck Chemicals中国分公司。
由于大部分散发肺淋巴管平滑肌瘤和结节性硬化伴随症大部分的基因诊断都是依赖TSC2基因的突变，因此在以下实施例的细胞水平验证唑来膦酸药效时，选择TSC2-null小鼠胚胎成纤维细胞作为其检测的细胞。
实施例1
96孔板每孔10^4个TSC2-null细胞，贴壁12-24小时。按实验分组加入不同浓度的药物，每组都有至少3个重复孔，作用24小时。药物处理结束后每孔100μL的无血清培养基加入10μL的MTT溶液，37℃培养箱培养4小时，小心吸出培养基，每孔加入100μL的DMSO，使结晶物充分的溶解，用酶标仪在570nm波长检测其吸光值。MTT结晶的形成量与细胞增殖成正比，增殖更好的细胞其吸光值更高。
本实验共分为三组分别是对照组，唑来膦酸10μM，25μM，50μM组，其中对照组为唑来膦酸的溶剂NaOH，细胞为TSC2-null小鼠胚胎成纤维细胞，DMEM+10％FBS正常培养条件下处理24小时。
由图1可知，随着唑来膦酸的浓度越来越高，细胞的增殖活力越来越低，并且相对于25μM的浓度，50μM的唑来膦酸细胞增殖效率的下降更加明显，出现显著性的差异。因此唑来膦酸的不同浓度对细胞的增殖有很大的影响，存在唑来膦酸药物的浓度依赖性。
实施例2
与实施例1相同的细胞实验方法，本次实验组共分为七组，分别是对照组，唑来膦酸组，雷帕霉素组，辛伐他汀组，唑来膦酸+雷帕霉素组，唑来膦酸+辛伐他汀组，辛伐他汀+雷帕霉素组。其中对照组为药物溶剂，唑来膦酸浓度为50μM，雷帕霉素浓度为20nM，辛伐他汀浓度为0.5μM，唑来膦酸+雷帕霉素的浓度为50μM+20nM，唑来膦酸+辛伐他汀的浓度为50μM+0.5μM，辛伐他汀+雷帕霉素的浓度为0.5μM+20nM。实验处理时间为48小时，与实施例1同样的处理方法。
由图2可知，唑来膦酸，雷帕霉素，辛伐他汀处理细胞48小时后，细胞活力明显下降。其中唑来膦酸的单独作用效果最为明显，细胞的活力下降约30％。联合用药处理组中唑来膦酸联合雷帕霉素组和辛伐他汀组作用效果最为明显，下降约35％。同时，由图2可知，联合用药对细胞活力的抑制效果强于单独用药组并且唑来膦酸联合用药组最强。
实施例3
进一步，通过TUNEL试剂盒检测25μM和50μM浓度的唑来膦酸以及临床上使用的雷帕霉素，观察唑来膦酸是否引起细胞的凋亡。其中配制唑来膦酸的NaOH溶液作为对照组，细胞处理24小时。
结果如图3所示，唑来膦酸处理组相对于雷帕霉素处理组凋亡细胞明显增加，并且随着浓度的增加细胞凋亡增多，而唑来膦酸并未引起细胞的显著性的凋亡，因此细胞凋亡对唑来膦酸有浓度依赖性。
实施例4
购买6～8周的雌性裸鼠，在其背部擦拭酒精后，0.45mm的针头注射10^7个细胞，拔出针头时注意无细胞溢出。在小鼠背部肿瘤长至直径约为3mm时，进行尾静脉唑来膦酸给药。裸鼠尾静脉给药的剂量为1mg/kg，每周两次，共给药六周12次。裸鼠皮下实质瘤的具体构建流程如图4A所示，调查给药后小鼠的肿瘤的生长速度，并以只注射PBS作为对照组，结果如图5所示。
由图5A可知，在皮下成瘤的裸鼠尾静脉连续注射唑来膦酸，一周两次，剂量为1mg/kg。相对于打PBS的对照组，唑来膦酸治疗组裸鼠皮下的肿瘤的生长速度明显减缓，肿瘤体积的增长从药物治疗15天后出现明显的差异，但两组鼠之间没有明显的体重差异。可以得出结论唑来膦酸能有效的抑制TSC2-null细胞皮下实质瘤的生长。
为了进一步检测唑来膦酸对裸鼠皮下肿瘤生长的抑制作用，本实施例进一步利用免疫荧光技术检测了皮下肿瘤细胞的增殖，以及p-S6蛋白的表达。p-S6是mTOR信号通路下游的信号分子，mTOR信号通路激活时，其表达的增加。细胞进入周期进行细胞增殖会有Ki67蛋白表达。从图5C中可见，唑来膦酸处理组Ki67蛋白的表达明显减少，说明唑来膦酸能够有效的抑制TSC2-null细胞的增殖。检测p-S6蛋白在细胞的表达，结果如图5B所示，经过唑来膦酸治疗的裸鼠，其皮下肿瘤细胞中p-S6的表达量明显减少，表明唑来膦酸有效的抑制了mTOR信号通路的表达。因此从动物模型的唑来膦酸治疗中进一步得出结论：唑来膦酸在动物体内可以有效的控制皮下肿瘤的生长。
实施例5
为了进一步说明唑来膦酸可以更加有效的治疗肺淋巴管平滑肌瘤，本实施例构建了肺淋巴管平滑肌瘤的模型。购买6～8周龄的雌性裸鼠，在其两侧腋下血管丰富处擦拭75％的酒精后用孔径0.45mm的注射针头注射10^7个TSC2-null细胞，同时注意针头拔出时无细胞溢出。 1～1.5个月以后裸鼠两侧长出皮下实质瘤。待实质瘤长到直径为1cm时，牺牲裸鼠，在超净台中取出其皮下瘤，将其剪碎。利用胶原酶消化剪碎的皮下瘤，并放在37℃摇床消化半个小时，之后用100目的筛子过滤后，1000rpm离心原代肿瘤细胞，去除上清，加入新的培养基重新悬浮。将细胞接入培养皿中重新贴壁培养两天。皮下成瘤的细胞相对于之前的细胞增加了细胞的成瘤性。将贴壁较好的细胞重新消化悬浮，每只裸鼠尾静脉注射10^5个分出的原代肿瘤细胞，三天以后可以进行小鼠尾静脉和腹腔给药。小鼠肺淋巴管平滑肌瘤的具体构建流程如图4B所示。
在肺淋巴管平滑肌瘤的裸鼠模型的构建过程中，TSC2-null细胞并非是原发性的肿瘤细胞，为增加TSC2-null细胞的成瘤性，需要在皮下成瘤以增加细胞的成瘤性，之后再尾静脉注入到裸鼠体内会在裸鼠肺部特异的形成病灶区，因此可能肺部的微环境更加有利于TSC2-null细胞的生长。具体结果如图6所示。如图所示，在裸鼠的肺部的大体形态上出现肺部乳糜，在肺部HE染色片上肺泡减小，肺空间间距增大，并且大部分的TSC2-null细胞聚集在动脉血管，静脉，以及气管附近。
本实施例中给药组共分为五组，对照组即小鼠成瘤后注射安慰剂，唑来膦酸200μg/kg组，唑来膦酸500mg/kg组，雷帕霉素组，雷帕霉素联合唑来膦酸组。唑来膦酸组200μg/kg每周三次尾静脉注射，唑来膦酸组500mg/kg每周两次尾静脉注射，雷帕霉素1mg/kg每周三次，雷帕霉素联合唑来膦酸组，唑来膦酸200μg/kg加上雷帕霉素1mg/kg腹腔注射每周三次。实验组的小鼠肺部糜烂泛白，在分组给予药物治疗后，全部小鼠的肺部出现明显的改善，血管清晰，肺部表面光滑。为了进一步观察药物治疗效果，对小鼠的肺部进行了石蜡的包埋切片，进行病理症断，结果如图6A所示，实验组的小鼠肺部出现大量的肿瘤细胞的聚集，但是在唑来膦酸200μg/kg，500μg/kg给药六周后，200μg/kg给药浓度的小鼠肺部的肿瘤明显减少，500μg/kg的小鼠的肺部几乎未出现肿瘤，同时雷帕霉素以及雷帕霉素联合唑来膦酸治疗的小鼠的肺部在给药一个月后均未出现肿瘤。为了进一步观察药物治疗的作用，停药一个月后两组：实验组+雷帕霉素组，实验组+唑来膦酸+雷帕霉素组，观察小鼠肺部病灶区有没有复发。具体结果如图6B所示，雷帕霉素停药后小鼠的肺部表面出现白色的实质瘤，雷帕霉素治疗组停药后小鼠的肺部出现明显的肿瘤细胞的快速增殖，肺部的动脉附近肿瘤细胞出现富集。但是，唑来膦酸联合雷帕霉素治疗组，小鼠的肺部未出现肿瘤的复发，小鼠肺部表面光滑无病灶点，并且病理症断方面小鼠的肺部也是未出现肿瘤，无病灶区。另外其他器官的病理检测也显示未出现肿瘤细胞的转移，结果如图6C所示。
综上所述，相比较皮下肿瘤的治疗效果，唑来膦酸在小鼠的肺淋巴管平滑肌瘤模型中的 治疗效果更加。唑来膦酸在干预皮下肿瘤的治疗中发现，其能有效的抑制肿瘤的生长，但是并未会使肿瘤缩小或是消失。而在肺部的淋巴管平滑肌瘤中，唑来膦酸能够有效的抑制肿瘤的形成，并且在联合雷帕霉素的治疗中能够有效的抑制肿瘤的复发，因此唑来膦酸对肺淋巴管平滑肌瘤的治疗效果更佳。
根据本发明唑来膦酸能够有效的淋巴管平滑肌增多症中的肺淋巴管平滑肌瘤，在联合雷帕霉素治疗可以效果更佳，并且在停药后肿瘤不易出现复发。根据对小鼠皮下瘤的治疗效果，唑来膦酸对于肾血管平滑肌脂肪瘤等结节性硬化症的伴随症也是有一定的疗效。

Claims (10)

  1. 唑来膦酸在制备肺淋巴管平滑肌瘤病药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述药物还包括雷帕霉素和/或辛伐他汀。
  3. 根据权利要求1所述的用途，其特征在于，所述肺淋巴管平滑肌瘤包括肺组织以及肺门内的平滑肌结节性及弥漫性增生。
  4. 根据权利要求1所述的用途，其特征在于，所述唑来膦酸的剂型为针剂、溶液剂、片剂和胶囊剂。
  5. 根据权利要求1所述的用途，其特征在于，所述唑来膦酸的给药方式为静脉滴注、口服和雾化吸入式给药。
  6. 权利要求1所述的肺淋巴管平滑肌瘤病药物，其特征在于，包含唑来膦酸。
  7. 根据权利要求6所述的药物，其特征在于，唑来膦酸的用药浓度为200μg/kg～500μg/kg。
  8. 根据权利要求6所述的药物，其特征在于，唑来膦酸与雷帕霉素联合使用时，唑来膦酸与雷帕霉素的用药比例为1:5～1:1。
  9. 根据权利要求6所述的药物，其特征在于，唑来膦酸与辛伐他汀联合使用时，唑来膦酸与化合物X的用药比例为1:10～1:1。
  10. 根据权利要求6所述的药物，其特征在于，唑来膦酸、雷帕霉素及辛伐他汀联合使用时，唑来膦酸、雷帕霉素及辛伐他汀的用药比例为1:5:10～1:1:1。
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