LIGAND DIRECTED UPTAKE ON NONIONIC ANTICODERS

配基导向摄取非离子反编码子 核酸及其类似物寡肽的细胞摄取过程 而相对分子质量在5-10,000之间的低聚糖不是细胞 明白了。然而，这些化合物可能具有很大的潜力 有效的治疗剂。这些分子是为他们的 与细胞内目标的相互作用。因此，一次彻底的 对这些物质的细胞摄取机制的理解(S)是 这些化合物作为分子生物学工具发展所必需的 并有可能成为有效的药物。我们关于寡核苷的论文 甲基膦酸盐(ONMP，8-mer)表明ONMP进入细胞 不太可能通过被动扩散或受体介导的内吞作用发生 但很可能是通过吸附的内吞过程和液体相 胞饮增多症。我们最近的手稿报告说，新糖肽 [Yee(ah-GalNAc)3]导向的、受体介导的内吞作用高度 有效地(高出50倍)诱导细胞摄取ONMP 培养中的人肝细胞。摄取的增强是组织- 特定的，配体特定的，并依赖于 ONMP和新糖肽。ONMP的生物活性研究 目前正在对其抗病毒作用进行检测。 抗HSV-1核内活性(反剪接)和抗HSV-1活性 细胞质(翻译中的抗mRNA)。初步结果表明， ONMP在细胞核内的反剪接效果一直是 与新糖肽连接后增加20-25倍。这个 ONMP结合物的阳性生物活性表明 用于组织特异性ONMP递送系统的有效系统现在可以 是为人类肝细胞构建的。同样，我们的初步研究 表明叶酸可以作为一种配体，增强细胞的摄取 ONMP对高表达的细胞系(如Igrov-1和kB细胞)的作用 叶酸受体的表达。因此，修订后的研究方案 应用程序是专门为各种不同的应用程序构建“交付组件” 组织和器官；“传递组件”由配体-支架- 支持毒品。在“交付”一词的结构中有以下变化 组装“是：1)配体--糖和叶酸；2)支架-- 新糖肽、牛血清白蛋白/人血清白蛋白寡肽； 3)前药物-ONMP、寡核苷酸、多肽/蛋白质和 化疗药物。检测药物有效性的细胞检测系统 要建造的“交付组件”的尺寸将基于 放射性标记或荧光标记前药物用于两种摄取 比率和退出比率，细胞内位置的显微镜检查，以及 抗HSV活性，特别是ONMP或ONM(P-寡核苷酸嵌合体 具有已知的抗单纯疱疹病毒活性。我们将评估和发展 为进入淋巴细胞的细胞提供有效的“传递组件”， 除了肝脏细胞外，还有肾脏和前列腺细胞。随后， 动物和裸鼠移植瘤的生物分布和药代动力学 杜克大学的迈克尔·科尔文博士将作为合作者对小鼠进行研究。这个 在动物体内的实时生物分布和药代动力学也将 ~(123)I标记的单光子发射递送组件的研究 计算机断层扫描(SPECT)与The 约翰霍普金斯大学放射科。各种共价键 将对配体-支架-前药物之间的联系进行评估 有效性。这些研究将为人们提供新的理解，了解 前药物可能从内吞体内逃逸成为生物活性以及如何 膜受体可能会再循环，发挥更大的作用。这项研究将 制定组织特异性给药系统的原理和实践 从基础科学到临床应用。

Ligand-Directed Uptake of Nonionic Anticoders The cellular uptake process of nucleic acid and its analogs, oligopeptides and oligosaccharides with molecular weights between 5- 10,000 is not ell understood. However, these compounds may possess great potential a effective therapeutic agents. These molecules are designed for their interactions with their intracellular targets. Therefore, a thorough understanding of ht cellular uptake mechanism(s) for these substances is necessary for development of these compounds as molecular biological tools and potentially as effective drugs. Our paper on oligonucleoside methylphosphonates (ONMP, 8-mer) indicates that cellular entrance of ONMPs is unlikely to occur via passive diffusion or receptor-mediated endocytosis but most likely through an adsorptive endocytotic process and fluid-phase pinocytosis. Our recent manuscript reports that the neoglycopeptide [YEE(ah-GalNAc)3]-directed, receptor-mediated endocytosis is highly effective (50 times higher) in inducing the cellular uptake of ONMP into human liver cells in culture. The enhancement of uptake is tissue- specific, ligand-specific and is dependent on a covalent linkage between the ONMP and the neoglycopeptide. The biological activities of ONMP taken up by liver cells are currently being assayed for their antiviral activities against HSV-1 in the nucleus (anti-splicing) and in the cytoplasm (anti-mRNA in translation). The preliminary results indicated that the anti-splicing effectiveness of ONMP in the nucleus has been increased by 20-25 fold upon linking with the neoglycopeptide. The positive biological activity of the ONMP conjugate indicates that an effective system for a tissue-specific ONMP delivery system can now be constructed for human liver cells. Similarly, our preliminary study indicates that folate can serve as a ligand, enhancing the cellular uptake of ONMP to cell lines (such as Igrov-1 and kB cells) possessing a high expression of folate receptors. Thus, the research program of the revised application is to build a "delivery assembly" specifically for various tissues and organs; the "delivery assembly" consists of ligand-Scaffold- Pro-drug. The following variations in the construction of a "delivery assembly" are: 1) Ligands - sugars and folate; 2) Scaffold - neoglycopeptides, bovine serum albumin/human serum albumin oligopeptides; 3) Pro-drug - ONMP, oligonucleotides, polypeptides/protein and chemotherapeutic agents. The cellular assay system for the effectiveness of the "delivery assembly" to constructed will be based on measurement of radioactively labeled or fluorescently labeled pro-drug for both uptake rate and exit rate, microscopic examination of intracellular location, and anti-HSV activity, particularly of ONMP or ONM{P-oligonucleotides chimera possessing known anti-HSV activities. We will evaluate and develop effective "delivery assemblies' for the cellular entrance to lymphocytes, kidney and prostate cells in addition to liver cells. Subsequently, the biodistribution and pharmacokinetics in animals and tumor xenograft-nude mice will be studied by Dr. Michael Colvin in Duke as collaborator. The real time biodistribution and pharmacokinetics in animals will also be investigated by 123I-labeled delivery assembly via Single-Photon Emission Computer Tomography (SPECT) in collaboration with Dr. J. James Frost of the Radiology Department of The Johns Hopkins University. Various covalent linkages among the ligand-scaffold-pro-drug will be evaluated for their effectiveness. The studies will provide new understanding about how the pro-drug may escape from endosomes to become biologically active and how membrane receptors may recycle back for more action. The research will develop principles and practices of tissue-specific drug delivery systems from basic science to clinical application.