Zebrafish model of blood-brain barrier to improve drug delivery to the brain

血脑屏障斑马鱼模型可改善药物向大脑的输送

• 批准号: 10702837
• 负责人: Michael Gottesman
• 金额: $ 60.75万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702837
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Astrocytes; Biological Assay; Biological Models; Blood; Blood - brain barrier anatomy; Brain; Caring; Cells; Collaborations; Consumption; Data; Drug Delivery Systems; Drug resistance; Embryo; Firefly Luciferases; Fishes; Glial Fibrillary Acidic Protein; Goals; Homologous Gene; Human; Incubated; Kidney; Knockout Mice; Light; Liver; Luciferases; Malignant Neoplasms; Measures; Modeling; Monitor; Mus; National Center for Advancing Translational Sciences; Nature; Permeability; Play; Property; Resistance; Role; Signal Transduction; Site; Study models; Substrate Specificity; System; Testing; Time; Transgenic Organisms; Variant; Work; Zebrafish; base; blood-brain barrier crossing; blood-brain barrier permeabilization; coelenterazine; high throughput analysis; high throughput screening; improved; inhibitor; luciferin; luminescence; mouse model; nanoluciferase; novel; prevent; promoter

Not only are ABC transporters responsible for drug resistance in cancer, but they are a major component of the blood-brain barrier (BBB) and blood-placental barrier. The three most prominent transporters at the blood-brain barrier are ABCB1, ABCC1, and ABCG2. We previously developed a murine model for analysis of ABCG2 expression at the blood-brain barrier based on the fact that luciferin is an ABCG2 substrate and its entry into the brain is prevented by transporter expression. In this model, firefly luciferase is under the expression of the GFAP promoter, leading to its expression in the astrocytes. When mice are injected with luciferin, no light signal from the brain is detected due to ABCG2 preventing luciferin from crossing the blood-brain barrier. However, when luciferin is coadministered with an ABCG2 inhibitor, it can cross the blood-brain barrier and react with luciferase expressed in the astrocytes to produce light which can be quantitatively measured. Because studies of the BBB in mice are time-consuming and expensive, we are developing homologous models in the zebrafish, as components of the zebrafish BBB appear to be very similar to those of the mammalian BBB. Two transgenic zebrafish lines have been developed with either firefly luciferase or nanoLuc under the control of the GFAP promoter. Luciferin is the substrate for firefly luciferase and is transported by ABCG2, while coelenterazine is one of the substrates for nanoLuc and is transported by both ABCB1 and ABCG2. Thus, either model could potentially be used to study the role of transporters at the blood-brain barrier, but they could also be used to screen compounds that might increase permeability of the barrier irregardless of the mechanism. If zebrafish are to be considered an appropriate model for study of transporters at the blood-brain barrier, the zebrafish homologs of human transporters must be carefully characterized. Zebrafish do not have a direct homolog of human ABCB1 but instead have 2 similar variants-Abcb4 and Abcb5. Expression of these transporters in heterologous systems has enabled their detailed characterization and inhibition properties. In collaboration with Matthew Hall at NCATS, we have found that zebrafish Abcb4 is nearly identical to human ABCB1 in conferring resistance to 90 known ABCB1 substrates. Abcb5 is also a functional transporter and confers resistance to many ABCB1 substrates but has a slightly narrower substrate specificity. While zebrafish Abcb4 is the only homolog that localizes to the BBB, Abcb4 and Abcb5 are expressed at other barrier and excretory sites in zebrafish, such as the gut, liver and kidneys. Zebrafish also have 4 homologs of human ABCG2-Abcg2a, Abcg2b, Abcg2c and Abcg2d. We have determined that Abcg2a is the only ABCG2 homolog expressed at the zebrafish BBB and a detailed characterization of the substrate specificity of the transporters is underway. Preliminary data in transfected cells suggest that Abcg2a has the most similar substrate specificity to human ABCG2, but they are not identical. As the light signal from nanoLuc is significantly brighter than that of firefly luciferase, we initially focused on the nanoLuc transgenic fish. Native coelenterazine and several of its derivatives are compatible with the nanoLuc system and we identified furimazine and coelenterazine h as the brightest. Both furimazine and coelenterazine h were found to be transported by zebrafish Abcg2a but not Abcb4. When embryonic fish were incubated with coelenterazine h in the presence of the ABCG2 inhibitor Ko143, we noted higher levels of luminescence compared to fish incubated with coelenterazine h alone, a proof-of-concept result. Further work will include testing other nanoLuc substrates as well as other known inhibitors.

ABC转运蛋白不仅对癌症的耐药性负责，而且是血脑屏障(BBB)和血胎盘屏障的主要组成部分。血脑屏障上最突出的三种转运蛋白是ABCB1、ABCC1和ABCG2。我们之前开发了一个小鼠模型来分析ABCG2在血脑屏障的表达，这是基于荧光素是ABCG2的底物，它通过转运蛋白的表达阻止其进入大脑的事实。在这个模型中，萤火虫荧光素酶在GFAP启动子的表达下，导致其在星形胶质细胞中表达。当小鼠注射荧光素时，由于ABCG2阻止荧光素穿过血脑屏障，因此没有检测到来自大脑的光信号。然而，当荧光素与ABCG2抑制剂联合使用时，它可以穿过血脑屏障，与星形胶质细胞中表达的荧光素酶反应，产生可以定量测量的光。由于对小鼠血脑屏障的研究既耗时又昂贵，我们正在斑马鱼中开发类似的模型，因为斑马鱼血脑屏障的成分似乎与哺乳动物的血脑屏障非常相似。在GFAP启动子的控制下，已经培育出两个转基因斑马鱼系，分别含有萤火虫荧光素酶和纳米Luc。荧光素是萤火虫荧光素酶的底物，由ABCG2转运，而腔肠净是纳米LUC的底物之一，由ABCB1和ABCG2转运。因此，这两种模型都有可能被用来研究转运蛋白在血脑屏障中的作用，但它们也可以用来筛选可能增加屏障通透性的化合物，而不考虑其机制。如果斑马鱼被认为是研究血脑屏障转运蛋白的合适模型，必须仔细描述人类转运蛋白的斑马鱼同源物。斑马鱼没有人类ABCB1的直接同源物，而是有两个类似的变体-Abcb4和ABCB5。这些转运蛋白在异源系统中的表达使其具有详细的表征和抑制特性。在与NCATS的Matthew Hall合作中，我们发现斑马鱼Abcb4在对90种已知ABCB1底物产生抗性方面与人类ABCB1几乎相同。ABCB5也是一种功能转运蛋白，对许多ABCB1底物具有抗性，但底物特异性略低。虽然斑马鱼Abcb4是唯一定位于BBB的同源物，但Abcb4和ABCB5在斑马鱼的其他屏障和排泄部位表达，如肠道、肝脏和肾脏。斑马鱼也有人类ABCG2的4个同源物-Abcg2a、Abcg2b、Abcg2c和Abcg2d。我们已经确定Abcg2a是唯一在斑马鱼BBB中表达的ABCG2同源物，对转运蛋白底物特异性的详细表征正在进行中。在转基因细胞中的初步数据表明，Abcg2a与人ABCG2具有最相似的底物特异性，但它们并不完全相同。由于NanLuc的光信号明显强于萤火虫荧光素酶的光信号，我们最初将重点放在了NanLuc转基因鱼上。天然的中草药及其几个衍生物与纳米LUC系统兼容，我们确定呋喃西林和中草药H是最亮的。研究发现，呋喃西林和乙胺嘧啶均可通过斑马鱼Abcg2a转运，但不能通过Abcb4转运。当胚胎鱼在含有ABCG2抑制剂Ko143的情况下与coelenterazine h一起孵育时，我们注意到与单独使用coelenterazine h孵化的鱼相比，有更高水平的发光，这是一个概念验证结果。进一步的工作将包括测试其他纳米LUC底物以及其他已知的抑制剂。