STUDIES OF PHOSPHORAMIDATE PRONUCLEOTIDES

描述(逐字摘自申请者的摘要)：克服许多 阻碍使用核苷酸作为治疗药物的障碍，发展一种 体内给药的前药物方法学(即原核苷酸) 核苷酸已经被提出作为一种解决方案。这样的方法应该允许 核苷酸为：A)静脉注射。和/或口服剂量，b)无限期稳定在 血液，以及c)目标在细胞内转化为活性物种 组织。拟议研究的目标是制定一套原则，以 将原核苷酸转化为相应核苷的设计 单磷酸在体内的靶组织中的作用，并利用我们对 AZT的抗乳腺癌活性。最近，我们已经证明了氨基 抗病毒和抗肿瘤核苷的酸性磷酸酯单酯 水溶性、无毒、高度稳定、有效的抗病毒和抗肿瘤 探员们。试图刻画这些活动的机械论研究 独特的化合物提供了支持细胞内直接P-N的证据 一种未知的酶活性导致的键断裂。另外，我们发现， 氨基酸磷酸盐在血浆中是稳定的，并且有显著的 体内血浆半衰期更长，分布体积更大 母体核苷。因此，它们有可能对 体内的核苷酸递送。因此，我们建议定义 关于氨基酸磷酰酯有用性的原则 原核苷酸方法，通过使用模型实现以下特定目标 具有抗病毒和抗癌活性的AZT的氨基酸磷酸酯。 1)直接测定D和L细胞内P-N键断裂程度 人外周血中AZT的甲胺氨基酸磷酸酯 单个核细胞(PBMC)、人T淋巴细胞白血病细胞株(CEM)和 人乳腺癌细胞株(MCF-7)。2)分子表征 推测为人磷酸水解酶，负责转换氨基酸 AZT磷酰化为AZT单磷酸盐。3)确定是否 AZT-D-和L-氨基酸磷酸酯被淋巴细胞和 乳腺癌细胞主要是扩散、促进或内吞的。 受驱动的流程。4)测定D-和L-色氨酸的能力 AZT的磷酸酯盐对化学诱导的乳房生长的抑制作用 大鼠肿瘤和SCIC小鼠人乳腺肿瘤。

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.