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Cellular Delivery of Nucleoside Diphosphates: A Prodrug Approach

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Abstract

Purpose. This study is concerned with cellular delivery/generation of 2′-azido-2′-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2′-azido-2′-deoxyuridine (N3Urd) and 2′-azido-2′-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases.

Methods. Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5′-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase.

Results. N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level.

Conclusions. The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.

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Author notes

  1. Dhiren R. Thakker

    Present address: Division of Pharmaceutics, School of Pharmacy, CB # 7360, University of North Carolina, Chapel Hill, North Carolina, 27599-7360

Authors and Affiliations

  1. Division of Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina

    Shin Hong Kang & Moo J. Cho

  2. Department of BioMet, Glaxo Wellcome Research Institute, Research Triangle Park, North Carolina, 27709

    Achintya K. Sinhababu & Dhiren R. Thakker

  3. Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina

    Joseph G. Cory

  4. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina

    Beverly S. Mitchell

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  1. Shin Hong Kang
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  3. Joseph G. Cory
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  4. Beverly S. Mitchell
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Kang, S.H., Sinhababu, A.K., Cory, J.G. et al. Cellular Delivery of Nucleoside Diphosphates: A Prodrug Approach. Pharm Res 14, 706–712 (1997). https://doi.org/10.1023/A:1012133902314

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