CELLULAR DELIVERY OF NUCLEOSIDE DIPHOSPHATES - A PRODRUG APPROACH

Purpose. This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CD P), potent inhibitors of ribonucleotide reductase. It characterizes th e phosphorylation steps involved in the conversion of 2'-azido-2'-deox yuridine (N(3)Urd) and 2'-azido-2'-deoxycytidine (N(3)Cyd) to the corr esponding diphosphates and explores a prodrug approach in cellular del ivery of the inhibitor which circumvents the requirement of deoxynucle oside kinases. Methods. Cell growth of CHO and 3T6 cells of known deox ycytidine kinase level was determined in the presence of N(3)Urd and N (3)Cyd. Activity of ribonucleotide reductase was determined in the pre sence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monoph osphate of N(3)Urd was prepared and its biological activity was evalua ted with CHO cells as well as with cells transfected with deoxycytidin e kinase. Results. N(3)Urd failed to inhibit the growth of both cell l ines, while N(3)Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kin ase levels found in the cells. Importance of the kinase was further es tablished with the finding that the nucleoside analogs were inactive a s reductase inhibitors in a permeabilized cell assay system while thei r 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 re ductase by two sequential phosphorylation steps. The present study ind icates that the first step to monophosphate is rate-limiting, justifyi ng a prodrug approach with the monophosphate.