Development of Delivery Systems for the Control of Pharmacokinetics and Intracellular Trafficking of Antisense Drugs

反义寡核苷酸具有序列特异性抑制基因表达的潜能，有望成为治疗癌症、病毒感染和遗传性疾病的一类新型化疗药物。为了达到体内的治疗效果，有必要控制寡核苷酸在体内的分布特征以及在细胞内的药代动力学。该项目的目的是开发能够控制体内药代动力学和随后的反义寡核苷酸在细胞内转运的递送系统。我们使用20聚磷酸二酯(PO)及其硫代硫酸盐(PS)衍生物作为模型反义分子，补充了人c-myc mRNA。从整体、器官、细胞和亚细胞水平研究了模型寡核苷酸的基本药代动力学。体内研究和器官灌流实验表明，全身给药后，寡核苷酸在肝脏和肾脏迅速消除，并被核酸酶降解。在体外实验中，使用了各种培养的细胞，并结合激光共聚焦显微镜，阐明了寡核苷酸在细胞内的命运。在这些发现的基础上，我们评估了半乳糖化和甘露糖化的大分子载体L赖氨酸的可行性，它可以控制寡核苷酸的体内处置和细胞内的药代动力学。

Antisense oligonucleotides have been expected as a novel class of chemotherapeutic agents for the treatment of cancer, viral infections and genetic disorders because of their potentials to inhibit gene expression in a sequence-specific manner. To achieve therapeutic effect in vivo, it is necessary to control the in vivo disposition characteristics as well as intracellular pharmacokinetics of oligonucleotides. The purpose of this project was to develop delivery systems which can control in vivo pharmacokinetics and subsequent intracellular trafficking of antisense oligonucleotides. We used 20 mer phosphodiester (PO) and its phosphorothioate (PS) derivative, complimentary to the human c-myc mRNA,as model antisense molecules. The basic pharmacokinetics of the model oligonucleotides were studied at whole body, organ, cellular and subcellular levels. The in vivo studies and organ perfusion experiments have demonstrated that the oligonucleotides underwent rapid elimination by the liver and kidney as well as degradation by nucleases after systemic administration. In vitro experiments using a variety of cultured cells in combination with confocal laser microscopy have clarified the intracellular fate of oligonucleotides. On a basis of these findings, we assessed the feasibility of macromolecular carriers, galactosylated and mannosylated poly (L-lysine), which can control both in vivo disposition and intracellular pharmacokinetics of oligonucleotides.