【作者】 张平；

【导师】 李刚； Jos H.Beijnen； Olaf van Tellingen；

【作者基本信息】 山东大学 ， 外科学（神经外科）， 2016， 博士

【摘要】 颅内肿瘤大致分为原发性肿瘤和转移瘤。胶质瘤是最常见的原发性脑肿瘤。胶质母细胞瘤(Glioblastoma, GBM)是发病率和恶性程度最高的胶质瘤。标准化治疗后,GBM患者的2年生存期仅有25%。其他系统的恶性肿瘤发生脑转移的几率也非常高,例如肺癌发生脑转移5年累积概率为16%,乳腺癌为7%,结肠癌为5%,而黑色素瘤脑转移率可高达40%以上。尽管人们对癌症发生、发展的分子机制了解有了巨大的进步,对治疗恶性肿瘤的靶向药物的研发也有了长足的发展,但是中枢神经系统(Central nervous system, CNS)就像肿瘤细胞的避难所,治疗颅内疾病(如原发颅内恶性肿瘤和转移瘤)的药物很难有效到达颅内病灶,这个问题一直困扰着所有CNS癌症研究者们。这一现象主要是由于血脑屏障(Blood-brain barrier, BBB)的存在而引起的。在正常状态下,BBB起到保护脑组织免受内源性和外源性有毒物质损伤。但是另一方面,这一屏障又可以阻挡大部分传统的或新型药物从血液循环系统进入脑实质或者脑内病灶。因此有很多实验在研究BBB形成的机制,以及如何规避BBB对治疗药物运输的阻碍作用。BBB主要是由血管内皮细胞构成。与身体其他部位的血管内皮细胞相比,BBB内皮细胞的胞膜缺少孔隙,胞饮作用活力低,紧密连接将它们紧紧的连接在一起。而且这些内皮细胞又同时被周细胞、星形细胞等紧密包绕,形成了第二层致密的脂质层。这些特性使得物质很难穿过BBB。一些必需的营养物质(如葡萄糖)进入脑内受一些摄取转运蛋白严格调控。其他物质通过BBB只能依靠被动扩散。被动扩散能力取决于分子脂溶性,分子质量,电离度,血浆蛋白结合力和组织结合力等。然而一些物质即使具备良好的被动扩散的分子特性,它们穿透BBB进入脑实质内的能力仍远远低于预期。经过多年努力,研究者发现BBB上存在一系列外排转运蛋白,它们可以进一步限制治疗药物进入脑内。在所有表达于BBB上的外排转运蛋白中,有两种主要负责将渗透入脑实质内的抗癌药物转运回循环血液,它们就是P-糖蛋白(P-glycoprotein, P-gp或ABCB1)和乳腺癌耐药蛋白(Breast cancer resistance protein, BCRP或ABCG2).因此,在研发有效抗癌药物的同时,如何提高药物渗透入脑也是决定治疗脑肿瘤成败的关键因素。目前规避BBB最有效的方法有两种,首先是研发或者筛选出可以直接规避BBB限制作用的药物。简单说就是寻找出既具有良好的被动扩散能力,又同时与外排转运蛋白,主要是ABCB1和ABCG2,亲和力较低的有效的抗癌靶向药物。第二个方法是,联合应用ABCB1和ABCG2的特异性抑制剂和有效的抗癌药物,从而抑制ABCB1和ABCG2外排作用,增加抗癌药物渗透入脑到达病灶能力。本课题包含两部分,第一部分我们研究了五种Zeste同源物2增强子(Enhancer of Zeste Homolog 2, EZH2)特异性抑制剂与ABCB1和ABCG2的亲和力,以及它们渗透入脑的能力。第二部分我们研究了特异性抑制ABCB1和ABCG2是否能增强BRAF抑制剂-威罗菲尼(Vemurafenib)治疗黑色素瘤脑转移灶的疗效。第一部分ABCB1和ABCG2限制多种EZH2抑制剂渗透入脑目前发现EZH2在GBM中表达上调,过表达的EZH2通过抑制分化来维持肿瘤细胞的干细胞性,这一作用提示EZH2是胶质瘤进展所必须的。另外,EZH2可以结合并甲基化信号转导与转录激活因子3(Signal Transducer and Activator of Transcription3, STAT3),从而增强胶质瘤肿瘤样干细胞(Glioma stem-like cell,GSC)中STAT3的活性,最终促进GSC自我更新及GBM恶化。而抑制EZH2功能,可以阻断GSC自我更新及存活相关的多条信号通路,提示EZH2是一个非常有前途的GBM治疗靶点。基于以上研究背景,我们探讨了5种结构非常相似的EZH2抑制剂(EPZ005687,EPZ-6438,UNC1999,GSK343和GSK126)与ABCB1和ABCG2的相互作用。我们先在体外利用Transwell实验对这些化合物进行了筛选,然后利用野生型(Wild-type, WT), Abcbl和／或Abcg2基因敲除鼠进行了体内实验,进一步研究了EPZ005687,EPZ-6438和GSK126体内药代动力学及BBB透过能力等。本实验发现,虽然GSK126非常低的膜穿透力导致体外实验难以检测其与转运蛋白的亲和力,但是结合体内实验结果,我们发现所有的EZH2抑制剂均可以被ABCB1和ABCG2转运。在体内,ABCB1和ABCG2都限制了EPZ005687和GSK126的BBB穿透力,而EPZ-6438在脑内的累积仅受ABCB1限制,并且ABCB1和ABCG2特异性抑制剂-依克利达可以完全抑制其针对EPZ-6438的外排作用。另外,在本实验所用的基因敲除小鼠体内存在一个未探明的因素,它明显延长了EPZ005687和EPZ-6438在敲除鼠血浆内停留时间,而这种现象并没有在GSK126相关的体内实验中发生。在WT小鼠中,所有组织内GSK126的组织-血浆比低于EPZ005687或EPZ-6438的组织-血浆比。此外,GSK126在WT小鼠的口服生物利用度仅为0.2%,在Abcbl;Abcg2基因敲除小鼠这一数值仅增加至14.4%。这些结果可能是由GSK126较差的膜渗透性导致的,因而本论文质疑GSK126的临床有效性。虽然本实验所检测的EZH2抑制剂都是ABCB1和ABCG2的转运底物,这一特性限制了这些化合物进入脑实质的能力和潜在的脑胶质瘤治疗效果,但是我们认为EPZ-6438是这一系列化合物中最适合用于治疗脑胶质瘤的EZH2抑制剂。第二部分抑制ABCBl和ABCG2可提高威罗菲尼治疗黑色素瘤脑转移瘤疗效在过去几十年里,黑色素瘤发病率逐年上升。Ⅳ级黑色素瘤患者发生脑转移的概率在40%以上,这是导致黑色素瘤患者死亡的主要因素之一。威罗菲尼作为BRAF蛋白抑制剂,是目前治疗黑色素瘤最有效的化疗药物之一。由于威罗菲尼是ABCB1和ABCG2的转运底物,因此威罗菲尼口服利用率和脑内累积量明显受到这两个转运蛋白限制,从而降低了其疗效,尤其是对黑色素瘤脑转移灶的疗效。基于以上研究背景,我们在应用威罗菲尼治疗黑色素瘤脑转移小鼠模型的时候,同时应用ABCB1和ABCG2特异性抑制剂-依克利达,以观察该联合用药方法能否增加威罗菲尼的疗效。我们首先将黑色素瘤细胞注射入WT小鼠,Abcb1和Abcg2基因敲除鼠脑内,制成黑色素瘤脑转移小鼠模型。然后给予不同剂量的威罗菲尼或联合应用依克利达,研究其药代动力学及疗效。本研究发现,口服威罗菲尼24小时后,尽管WT小鼠血浆内浓度比Abcbla/1b;Abcg2-/-小鼠血浆浓度仅低了1.4倍,但脑内浓度却比基因敲除鼠低了700多倍。依克利达可以提高威罗菲尼的口服利用率和脑通透能力,但是WT小鼠脑内浓度仍低于基因敲除鼠脑内浓度20多倍。表明ABCB1和ABCG2显著抑制威罗菲尼渗透入脑,依克利达虽然可以显著提高威罗菲尼渗透入脑的能力,但是并不能完全逆转ABCB1和ABCG2对其限制作用。利用没有BBB限制的皮下肿瘤模型时,我们发现10 mg/kg威罗菲尼对Abcbl a/1b;Abcg2-/-小鼠的疗效与25mg/kg威罗菲尼对WT小鼠的疗效几乎是一致的。利用脑转移模型我们发现,ABCBl和ABCG2显著抑制了威罗菲尼对脑转移黑色素瘤的治疗作用,而依克利达可以一定程度地提高威罗菲尼的疗效。另外我们还发现一个有趣的现象：在开始用药的时候,基因敲除鼠颅内肿瘤对药物反应比较敏感,肿瘤体积不再增大。但是用药一周或两周之后,肿瘤不再对威罗菲尼有反应,肿瘤迅速增长。我们试图去探讨该肿瘤在如此短的时间内获得耐药性的机制。但很不幸的是我们利用免疫组织化学、Western和PCR等多种方法检测相关信号通路,均未明确找出该耐药性的产生机制。本实验研究表明ABCBl和ABCG2明显的限制了威罗菲尼对黑色素瘤脑转移瘤的治疗效果,联合应用其抑制剂依克利达可以增强其疗效,但仍不足以完全逆转该限制作用。更多还原

【Abstract】 Brain tumors can be devided into two groups:the primary and metastatic tumors. Gliomas are the most common primary brain tumors. Glioblastomas are by far the most common and most malignant type. After standard treatment, the 2-year overall survival of glioblastoma patients is approximately only 25%. Brain metastases occur in a significant percentage of patients with common malignancies, with 5-year cumulative incidence rates of 16% in lung cancer patients,7% of breast cancer patients, and 5% of patients with colon cancer. In diseases such as melanoma, the incidence of brain metastatic disease is reported to be as high as 40%.Despite the dramatic advances in understanding the molecular basis for carcinogenesis and the development of new targeting agents to treat malignancies, a critical challenge that continues to face cancer researchers is overcoming the sanctuary for primary and metastatic disease found within the central nervous system (CNS). This sanctuary is formed by the blood-brain barrier (BBB), a mechanism that protects the brain from exposure to toxins, both endogenous and exogenous. This barrier prevents many of our traditional and new drugs from crossing from the circulation into the brain parenchyma.The BBB is formed by endothelial cells that are closely linked by tight junctions. In contrast to most endothelial cells in the rest of the body, endothelial cells in the BBB lack fenestra and have low endocytic activity. Moreover, the pericytes and astrocytes intimately surrounding the endothelial cells form a secondary lipid layer. Consequently, the BBB is almost impermeable. Entry of essential nutrients (e.g., glucose) is strictly regulated by a range of uptake transporters. Other substances can only enter the brain by passive diffusion across the BBB, and the ability to do so is determined by a series of molecular parameters such as sufficient lipid solubility, molecular weight, degree of ionization, plasma protein binding and tissue binding. Nonetheless, even compounds that have molecular characteristics in favor of passive diffusion demonstrate much lower brain penetration than expected due to the activity of drug efflux transporters. ABC drug transporters expressed at the BBB have well-known roles in the restriction of therapeutic agents into the brain. Of all the efflux transporters present in the BBB, two transporters are mainly responsible for the efflux of anti-cancer agents back into the blood capillaries. These proteins are ABCB1 and ABCG2Thereby, the key factors for anti-brain tummors are exploring new targeting agents and increasing their brain penetration. There are two efficient ways:firstly, exploring or selecting agents showing good membrane penetration and low affinity for ABCB1 and ABCG2; secondly, combination of ABCB1/ABCG2 specific inhibitors and anti-tumor agents.In this study, we first eveluated the affinity of five EZH2 inhibitors for ABCB1 and ABCG2, and their brain penetraion. Then we investigated the ability of ABCB1/ABCG2 inhibitor-elacridar enhancing the efficacy of vemurafenib anti-brain metastases of melanoma.Part I ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-inhibitorsEZH2 up-regulation in glioblastoma (GBM), a group of highly malignant and lethal tumors of the brain, can maintain sternness of tumor cells by inhibiting differentiation, suggesting EZH2 is necessary for glioma progression. Moreover, functional inactivation of EZH2 might attenuate multiple key signals involved in glioma stem cells self-renewal and survival, pinpointing EZH2 as a potential therapeutic target for GBM.We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcbl and/or Abcg2 knockout mice.We found that all EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by its low membrane permeability. Both P-gp and Bcrpl restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcbl;Abcg2 knockout mice. These results are likely due to its poor membrane permeability and question the clinical usefulness of GSK126.Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.Part II Inhibiting ABCB1 and ABCG2 enhances the efficacy of vemurafenib on melanoma brain metastasesThe incidence of melanoma has been steadily increasing worldwide over the past few decades. Brain metastases happens to more than 40% of patients with stage IV melanoma, which is a major cause of death in melanoma cases. Vemurafenib is a specific and potent inhibitor of mutated BRAF. Vemurafenib is a good substrate of ABCB1 and ABCG2, which limits the efficacy of vemurafenib on melanoma especially on brain metastases.Based on this, we performed the present study to evaluate the roles of ABCB1 and ABCG2 in treatment of brain metastases of melanoma with vemurafenib, in hope that these preclinical data would help in further advance of a durable response or less side effects in patients with melanoma brain metastases.We employed WT and Abcbla/1b;Abcg2-/- mice for intracranial injection of melanoma cells. Then we investigated the efficacy of different doses of vemurafenib combined with or without elacridar.The plasma AUCo-24h of Abcbla/1b;Abcg2-/- mice was only 1.4-fold higher than that of WT mice treated with 25 mg/kg vemurafenib, while the brain concentration in Abcb1a/1b;Abcg2-/- mice was 700-fold higher than in WT mice. Albeit elacridar enhanced the brain entry profoundly, the brain concentration in knockout mice dosed with 10 mg/kg vemurafenib was still 24 times higher than in WT mice with elacridar and same dose of vemurafenib. The bioavailability and brain penetration of vemurafenib is restricted by Abcbla/lb and Abcg2, what is more elacridar can enhance them considerably. We employed the subcutaneous tumor models where the penetration of vemurafenib to tumors was not limited by BBB, finding that the tumors were notably and similarly reduced by vemurafenib in the two treated groups even with different doses. With the intracranial melanoma models, the Abcbl and Abcg2 hindered the efficacy of vemurafenib against the brain metastases of melanoma, and elacridar is not powerful enough to reverse the limitation. Interestingly, in the first week, the growth of tumor in knockout mice was arrested by vemurafenib. But after the seventh treatment tumors regrew to the same size as that at the start of treatment, and did not respond to the therapeutics any more. Several methods were tried to explore the mechanism for the acquired resistance in our experiment. Unluckily, no clue was found.The current study showed the ABCB1 and ABCG2 transporters attenuated the initial response of the brain metastases of melanoma to vemurafenib. Coadministration with the ABC inhibitors (elacridar) is a strategy to improve the potency of vemurafenib, although it could not completely reverse the limitation by ABC transporters. Further studies are needed to explore the quickly resistant mechanism in our study.更多还原