ABCB1和ABCG2转运蛋白在脑肿瘤靶向药物治疗中的作用

发布时间：2018-07-06 10:03

本文选题：P-糖蛋白 + 乳腺癌耐药蛋白　；参考：《山东大学》2016年博士论文

【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。
[Abstract]:Intracranial tumors are roughly divided into primary tumors and metastatic tumors. Glioma is the most common primary brain tumor. Glioblastoma (GBM) is the most malignant and malignant glioma. After standardized treatment, the 2 year survival of patients with GBM is also very high in the incidence of brain metastases in other malignant tumors of 25%.. For example, the cumulative probability of 5 years of brain metastases in lung cancer is 16%, breast cancer is 7%, colon cancer is 5%, and the rate of melanoma brain metastases can be as high as 40%. Although people have great progress in the molecular mechanism of cancer development, the development of the molecular mechanism of cancer therapy has also developed greatly, but the central nervous system (Cent Ral nervous system, CNS) as a refuge of tumor cells, drugs for the treatment of intracranial diseases such as primary intracranial malignant tumors and metastases are difficult to reach intracranial lesions. This problem has been troubled by all CNS cancer researchers. This phenomenon is mainly caused by the presence of the blood brain barrier (Blood-brain barrier, BBB). In normal condition, BBB protects brain tissue from endogenous and exogenous toxic substances, but on the other hand, this barrier can block most traditional or new drugs from the blood circulation system into the brain parenchyma or brain lesions. So there are many experiments in the study of the mechanism of BBB formation and how to circumvent the treatment of BBB. .BBB is mainly composed of vascular endothelial cells. Compared with the vascular endothelial cells in other parts of the body, the membrane of the BBB endothelial cells is short of pore, and the activity of the cell is low, closely connected to connect them together. And these endothelial cells are closely wrapped around the pericytes and astrocytes. It is a dense layer of second layers of lipid. These properties make it difficult for the substance to pass through some of the essential nutrients (such as glucose) into the brain, which are strictly regulated by some uptake transporters in the brain. Other substances can only rely on passive diffusion through BBB. The passive diffusion ability depends on molecular fat solubility, molecular mass, ionization, plasma protein binding. However, even if some substances have good molecular properties of passive diffusion, their ability to penetrate BBB into the brain is still far lower than expected. After years of effort, researchers found a series of extracellular transporters on BBB, which can limit the entry of therapeutic drugs into the brain. In the outer row transporters on BBB, two kinds of drugs are responsible for transshipment of anticancer drugs into the brain parenchyma, which are P- glycoproteins (P-glycoprotein, P-gp or ABCB1) and breast cancer resistant proteins (Breast cancer resistance protein, BCRP, or ABCG2). Therefore, how to improve the effective anticancer drugs Drug penetration into the brain is also a key factor in the success of the treatment of brain tumors. At present, there are two most effective methods to avoid BBB. The first is to develop or screen out drugs that can directly circumvent the BBB restriction. It is simply to find out the good passive diffusion ability, and the same time and the external transporter, mainly ABCB1 and ABCG2, An effective anti-cancer targeting drug with low affinity. The second method is to combine the specific inhibitors of ABCB1 and ABCG2 and effective anticancer drugs to inhibit the ABCB1 and ABCG2 efflux and increase the ability of anti-cancer drugs to infiltrate into the brain to reach the focus. This topic includes two parts. In the first part, we studied the increase of five Zeste homology 2. The affinity between the Enhancer of Zeste Homolog 2, EZH2 (EZH2) specific inhibitors and ABCB1 and ABCG2, and their ability to infiltrate into the brain. Second we studied the efficacy of specific inhibition of ABCB1 and ABCG2 on the enhancement of BRAF inhibitor - Viv Lo Feeney (Vemurafenib) in the treatment of melanoma brain metastases. A variety of EZH2 inhibitors are infiltrated into the brain and the expression of EZH2 is up-regulated in GBM, and the overexpressed EZH2 maintains the stem cell nature of the tumor cells by inhibiting differentiation, which suggests that EZH2 is necessary for the progression of glioma. In addition, EZH2 can bind and methylation signal transduction and transfer activator 3 (Signal Transducer and Activator o) F Transcription3, STAT3), thereby enhancing the activity of STAT3 in glioma tumor like stem cells (Glioma stem-like cell, GSC), and ultimately promoting GSC self renewal and GBM deterioration. Inhibition of EZH2 function can block multiple signaling pathways associated with GSC self renewal and survival. In the study background, we explored the interaction of 5 very similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with ABCB1 and ABCG2. We screened these compounds using Transwell experiments in vitro, and then used the wild type (Wild-type, WT), Abcbl and / or gene knockout mice. In this experiment, the pharmacokinetics and BBB transmittance of EPZ005687, EPZ-6438 and GSK126 were further studied. It was found that although the very low membrane penetration of GSK126 was difficult to detect its affinity with the transporter in vitro, we found that all of the EZH2 inhibitors were able to be ABCB1 and ABC in combination with the results of the experiment in vivo. G2 transport. In vivo, both ABCB1 and ABCG2 restrict the BBB penetration of EPZ005687 and GSK126, and the accumulation of EPZ-6438 in the brain is limited only by ABCB1, and ABCB1 and ABCG2 specific inhibitors, kkli, can completely inhibit the EPZ-6438 exocytosis. In addition, there is an unexplored gene knockout mouse in this experiment. It obviously prolongs the time of EPZ005687 and EPZ-6438 in the knockout rat plasma, and this phenomenon does not occur in GSK126 related in vivo experiments. In WT mice, the tissue plasma ratio of GSK126 in all tissues is lower than that of EPZ005687 or EPZ-6438. In addition, the oral bioavailability of GSK126 in WT mice is only the only oral bioavailability. For 0.2%, in Abcbl, Abcg2 gene knockout mice, which only increased to 14.4%., may be caused by the poor membrane permeability of GSK126, so this paper questions the clinical effectiveness of GSK126. Although the EZH2 inhibitors detected in this experiment are the transport bases of ABCB1 and ABCG2, this property restricts the entry of these compounds into the brain. Substantial ability and potential therapeutic effects of glioma, but we think that EPZ-6438 is the most suitable EZH2 inhibitor for the treatment of glioma in this series of compounds. The second part inhibition of ABCBl and ABCG2 can improve the efficacy of willfield in the treatment of melanoma brain metastases. The incidence of melanoma has increased year by year in the past few decades. The incidence of brain metastases in patients with grade IV melanoma is more than 40%, which is one of the major factors leading to the death of melanoma patients. As a BRAF protein inhibitor, willfield is one of the most effective chemotherapeutic agents for melanoma. Because Viv Lo Feeney is a transport substrate for ABCB1 and ABCG2, the oral utilization of willfield The accumulation of the two transporters in the brain is obviously limited by these transporters, which reduces the efficacy, especially for the brain metastases of melanoma. Based on the above research background, we used the ABCB1 and ABCG2 specific inhibitors, Cicia, to observe the mouse model of melanoma brain metastases. We first injected melanoma cells into WT mice, Abcb1 and Abcg2 gene knockout mouse brains to make a mouse model of melanoma brain metastases. Then we gave different doses of willfield or combined use of kkli to study its pharmacokinetics and efficacy. After 24 hours of willfield, the plasma concentration of WT mice was 1.4 times lower than the plasma concentration of Abcbla/1b and Abcg2-/- mice, but the concentration in the brain was more than 700 times lower than that of the gene knockout mice. 0 times. It shows that ABCB1 and ABCG2 significantly inhibit the infiltration of willoni into the brain. Although cideridis can significantly improve the ability of willfield to infiltrate the brain, it does not completely reverse the restriction of ABCB1 and ABCG2. Using the subcutaneous tumor model without BBB restriction, we present 10 mg/kg willfield to Abcbl a/1b; Abcg2-/- mice. The effect was almost the same as the effect of 25mg/kg willfield on WT mice. Using the brain metastasis model, we found that ABCBl and ABCG2 significantly inhibited the therapeutic effect of willfield on brain metastases, and the effect of willfield could be improved to a certain extent. In the beginning of the drug, the intracranial tumor of the gene knockout rat is more sensitive to the drug reaction, and the volume of the tumor no longer increases. But after a week or two weeks, the tumor no longer responds to willoni and the tumor grows rapidly. We try to explore the mechanism of the tumor's resistance to such a short time. Unfortunately, we use it. A variety of methods such as immunohistochemistry, Western and PCR were used to detect the related signaling pathways, and the mechanism of the drug resistance was not clearly identified. This study showed that ABCBl and ABCG2 significantly restricted the effect of willfield's treatment on melanoma brain metastases. Completely reversing the limitation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.4

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