STUDIES OF PHOSPHORAMIDATE PRONUCLEOTIDES

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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.