STUDIES OF PHOSPHORAMIDATE PRONUCLEOTIDES

描述（申请人摘要中的逐字记录）：为了克服许多 阻碍了核苷酸作为治疗药物的使用， 前药方法学（即，原核苷酸）用于体内递送 已经提出了核苷酸作为解决方案。这种方法应允许 a）静脉内和/或口服给药，B）在体内无限期稳定， 血液，和c）在细胞内被靶转化为活性物质 组织.拟议研究的目标是制定一套原则， 原核苷酸的设计转化为相应的核苷 在体内靶组织的单磷酸，并利用我们的发现， AZT的抗乳腺癌活性。最近，我们证明了氨基 抗病毒和抗肿瘤核苷的酸性氨基磷酸酯单酯， 水溶性、无毒、高稳定性和有效的抗病毒和抗肿瘤 剂.机制研究试图表征这些活性 独特的化合物提供了细胞内直接P-N的支持证据， 通过未知的酶活性进行键断裂。此外，我们发现， 氨基酸氨基磷酸酯在血浆中稳定，并且具有显著的 体内血浆半衰期更长，分布容积更大， 母体核苷。因此，它们有可能用于 核苷酸的体内递送。因此，我们建议界定 氨基酸氨基磷酸酯的有效性原则 原核苷酸的方法，通过执行以下具体目标与模型 具有抗病毒和抗癌活性的AZT的氨基酸氨基磷酸酯。 1)直接测定D和L的胞内P-N键裂解程度 人外周血中AZT的甲酰胺氨基酸磷酸酯 单个核细胞（PBMC）、人T淋巴母细胞白血病细胞系（CEM）和 人乳腺癌细胞系（MCF-7）。2)分子表征 负责将氨基酸 AZT氨基磷酸酯化为AZT单磷酸盐。3)确定是否 AZT D-和L-氨基酸氨基磷酸酯被淋巴细胞内化， 乳腺癌细胞主要是扩散，促进或内吞 驱动过程。4)测定D-和L-色氨酸 AZT的氨基磷酸酯抑制化学诱导的乳腺癌生长 大鼠中的肿瘤和SCIC小鼠中的人乳腺肿瘤。

DESCRIPTION (verbatim from the applicant's abstract): To overcome the many hurdles preventing the use of nucleotides as therapeutics, the development of a prodrug methodology (i.e., pronucleotide) for the in vivo delivery of nucleotides has been proposed as a solution. Such an approach should allow nucleotides to be: a) i.v. and/or orally dosed, b) indefinitely stable in blood, and c) converted intracellularly to the active species by the target tissue. The goal of the proposed study is to develop a set of principles for the design of pronucleotides that are converted to the corresponding nucleoside monophosphate by the target tissue in vivo and to exploit our discovery of the anti-breast cancer activity of AZT. Recently, we have demonstrated that amino acid phosphoramidate monoesters of antiviral and antitumor nucleosides are water soluble, non-toxic, highly stable and potent antiviral and antitumor agents. Mechanistic studies attempting to characterize the activity of these unique compounds have provided supporting evidence of direct intracellular P-N bond cleavage by an unknown enzymatic activity. In addition, we have found that amino acid phosphoramidates are stable in plasma, and have a significantly longer in vivo plasma half-life and larger volume of distribution than their parent nucleoside. Consequently, they have the potential to be useful for the in vivo delivery of nucleotides. Therefore, we propose to define the principles governing the usefulness of an amino acid phosphoramidate pronucleotide approach, by carrying out the following specific aims with model amino acid phosphoramidates of AZT that have antiviral and anticancer activity. 1) Directly determine the extent of intracellular P-N bond cleavage of D and L methyl amide amino acid phosphoramidates of AZT by human peripheral blood mononuclear cells (PBMCs) a human T-Lymphoblast leuckemia cell-line (CEM) and a human breast cancer cell line (MCF-7). 2) Molecular characterization of the putative human phosphoramidate hydrolase responsible for converting amino acid AZT phosphoramidates to AZT monophosphate. 3) Determine whether the internalization of AZT D-and L-amino acid phosphoramidates by lymphocytic and breast cancer cells are primarily a diffusion, facilitated or endocytotic driven process. 4) Determine the ability of the D- and L- tryptophan phosphoramidates of AZT to inhibit the growth of chemically induced breast tumors in rats and human breast tumors in SCIC mice.