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

Division Of Basic Sciences - Nci

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Abstract

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.