关于一新型高效跨越血脑屏障的非嵌合药物输送载体K16ApoE的机制研究

The enzyme replacement therapy (ERT) has been proved to be the most effective way for lysosomal storage disorder (LSD) treatment. However, the blood-brain barrier (BBB) presents a major challenge to deliver therapeutics in central nervous system (CNS), which limits the application of ERT in CNS-involved LSD. Methods do exist to help recombinant lysosomal protein therapeutics cross the BBB in animal models but results have been modest in terms of low levels achievable in the brain and therapeutic value in terms of increasing the life-span of animal models remains to be demonstrated in a convincing way. We recently reported an efficacy study on a novel in-trans peptide carrier (K16ApoE) in late-infantile neuronal ceroid lipofuscinosis (LINCL) mouse model. Results demonstrated that supraphysiological levels of TPP1 were achieved in the brain after co-injection of TPP1 and K16ApoE mixture. Importantly, peptide-mediated delivery of TPP1 from the bloodstream into the brain translates into significantly increased lifespan. However, the mechanism of action of K16ApoE is yet to be definitively elucidated which greatly limits its clinical translational potential. Here, we propose this study to dissect the mechanism of K16ApoE mediated drug delivery across BBB. Specifically, we will explore the pharmacokinetics mechanism of K16ApoE, the route and distribution of K16ApoE from blood to brain, the specificity of the therapeutics delivered by K16ApoE and the interaction between K16ApoE and BBB. Based on the characterization of mechanism, we will further optimize the sequence of K16ApoE to screen for the candidate with optimal therapeutic index. Meanwhile, the computer simulation model for K16ApoE pharmacokinetics will be developed to compute the best injection parameters, which will then be testified in in vivo model. The study will lay the foundation for the long-term efficacy study in preclinical animal models. Overall, success in these studies will be a major breakthrough, changing the way we think about therapy for CNS disorders.

酶替代疗法（ERT）是治疗溶酶体疾病（LSD）最有效的方法。 血脑屏障(BBB)严重阻碍药物入脑， 限制了ERT在含中枢神经系统（CNS）病变的LSD中的应用。 目前已有的跨越BBB的方法其缺陷在于药物入脑量很低且无明显疗效如延长疾病小鼠平均寿命。 研究表明一新型多肽载体(K16ApoE)在一LSD疾病小鼠模型中， 通过与三肽基肽酶I非共价结合可大量将药物输送入脑且小鼠寿命显著延长。 然而K16ApoE的作用机制不清限制了其临床转化的可能性。因此，本项目将从K16ApoE的药代动力学机制及其入脑途径， K16ApoE与药物以及BBB的相互作用对该多肽的作用机理展开深入研究。 在阐明K16ApoE作用机制的基础上进一步对序列进行优化， 同时建立药代动力学生物仿真模型优化注射指数。为未来动物体内的长期功效研究提供重要支持。本项目的结果对LSD乃至CNS疾病的治疗具有变革性意义。

前期研究发现多肽K16ApoE可高效跨越血脑屏障(BBB)送药入脑。由于无需与被输送药物构建嵌合体，K16ApoE有望成为颅内药物的通用输送载体。然而其毒性导致治疗指数（有效剂量ED/最大承受剂量MTD）偏低，大大限制了该多肽的临床转化。本研究首先针对该多肽在BBB上的作用机制展开，随后对其毒性机制进行了探索。..对于K16ApoE的作用机制研究主要从以下三个方面进行：1）确定K16ApoE在血液及组织内的药代动力学曲线及其入脑途径；2）确定K16ApoE与携带药物之间是否具有特异性；3）揭示K16ApoE的重要功能序列，明确其在BBB上的作用机制。实验结果表明K16ApoE在携带药物进入脑实质后会快速降解，而颅内药物在深层神经元的分布多于表层；在K16ApoE的作用下，TPP1与血清蛋白均可入脑，然而脑内标准蛋白含量无明显改变，说明该多肽与被携带药物之间的作用非特异性，且输送药物过程不会破坏BBB造成泄露；对K16ApoE重要功能序列的研究发现带正电荷的K16序列对TPP1的入脑效力有着重要作用, 并同时与其毒性作用密切相关。..考虑到K16可能作为该多肽效力与毒性机制的共同靶点，我们从四个方面探索了毒性机制：1）小鼠毒性症状与病理观察；2）K16ApoE变体的细胞毒性；3）K16ApoE与乙酰胆碱酯酶（AChE）的相互作用；4）钙离子对K16ApoE毒性的影响。结果表明接受高剂量多肽注射后小鼠呼吸困难，肌肉痉挛，甚至死亡。尸检报告未发现明显器官病变，死亡前的脑电波无异常；当多肽中的K16序列被16个组氨酸取代后，该多肽细胞毒性消失，同时效力消失，再次证明K16是毒性与效力的共同关键序列；K16ApoE对AChE的酶活影响呈现复杂剂量性效应：抑制-激活-再抑制。改变K16ApoE中赖氨酸的数量虽然不影响抑制-激活-再抑制的效应，但对于激活期所需的多肽含量有显著改变，而且K12ApoE与K20ApoE在雌性和雄性小鼠中的剂量效应显著不同；钙离子在保证TPP1入脑效力的前提下可以显著降低K16ApoE的毒性，初步证实了K16ApoE与钙离子竞争结合肌钙蛋白导致其致死毒性的假说。..基于本研究，我们会进一步研究钙离子解毒机制，优化解毒方案，为未来K16ApoE临床转化提供重要理论依据。

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