LIGAND DIRECTED UPTAKE ON NONIONIC ANTICODERS

非离子抗编码剂的配体定向摄取

• 批准号: 2392243
• 负责人: PAUL O. P. TS'O
• 金额: $ 23.46万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392243
• 关键词: analog; antisense nucleic acid; antiviral agents; athymic mouse; biotransformation; chemical association; chemical conjugate; chemical stability; covalent bond; fluorescence microscopy; fluorescent dye /probe; folate; glycopeptides; herpes simplex virus 1; ligands; nuclear matrix; nucleic acid metabolism; nucleotide metabolism; oligonucleotides; pharmacokinetics; phosphonate; prodrugs; radiotracer; single photon emission computed tomography; tissue /cell culture

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.

非离子抗编码剂的配体定向摄取 核酸及其类似物、寡肽的细胞摄取过程 而分子量在5-10,000之间的低聚糖则不太好 明白了。 然而，这些化合物可能具有巨大的潜力 有效的治疗剂。 这些分子是为它们的 与其细胞内靶标的相互作用。 因此，彻底 对这些物质的 ht 细胞摄取机制的了解是 开发这些化合物作为分子生物学工具所必需的 并有可能成为有效的药物。 我们关于寡核苷的论文 甲基膦酸酯（ONMP，8 聚体）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 但最有可能通过吸附内吞过程和液相 胞饮作用。 我们最近的手稿报告说，新糖肽 [YEE(ah-GalNAc)3] 定向、受体介导的内吞作用高度 有效（高出 50 倍）诱导细胞摄取 ONMP 培养中的人类肝细胞。 吸收的增强是组织- 特异性的、配体特异性的并且依赖于之间的共价连接 ONMP 和新糖肽。 ONMP的生物活性 目前正在检测肝细胞的抗病毒作用 在细胞核（抗剪接）和细胞核中对抗 HSV-1 的活性 细胞质（翻译中的抗 mRNA）。 初步结果表明 ONMP 在细胞核中的抗剪接效果 与新糖肽连接后增加 20-25 倍。 这 ONMP 缀合物的阳性生物活性表明 组织特异性 ONMP 输送系统的有效系统现在可以 为人类肝细胞构建。 同样，我们的初步研究 表明叶酸可以作为配体，增强细胞的摄取 ONMP 细胞系（如 Igrov-1 和 kB 细胞）具有高 叶酸受体的表达。 因此，修订后的研究计划 应用程序是专门为各种 组织和器官； “递送组件”由配体-支架-组成 前药。 “交付”结构的以下变化 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 - ONMP、寡核苷酸、多肽/蛋白质和 化疗剂。 细胞有效性测定系统 构建的“交付组件”将基于以下测量 放射性标记或荧光标记的前药用于吸收 率和退出率，细胞内位置的显微镜检查，以及 抗HSV活性，特别是ONMP或ONM{P-寡核苷酸嵌合体的抗HSV活性 具有已知的抗 HSV 活性。 我们将评估和开发 用于细胞进入淋巴细胞的有效“递送组件”， 除肝细胞外，还有肾细胞和前列腺细胞。 随后， 动物和肿瘤异种移植物中的生物分布和药代动力学 杜克大学的 Michael Colvin 博士将作为合作者对小鼠进行研究。 这 动物体内的实时生物分布和药代动力学也将 通过单光子发射通过 123I 标记的传输组件进行研究 计算机断层扫描 (SPECT) 与 J. James Frost 博士合作 约翰·霍普金斯大学放射学系。 各种共价键 将评估配体-支架-前药之间的连接 效力。 这些研究将提供关于如何 前药可能从内涵体中逸出并变得具有生物活性，以及​​如何 膜受体可能会回收以发挥更多作用。 该研究将 制定组织特异性药物输送系统的原则和实践 从基础科学到临床应用。