Cellular delivery of nucleoside diphosphates: a prodrug approach.
OBJECTIVE
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.
Kang
S H
SH
Division of Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill 27599-7360, USA.
Sinhababu
A K
AK
Cory
J G
JG
Mitchell
B S
BS
Thakker
D R
DR
Cho
M J
MJ
D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003565:Cytidine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000494:pharmacology; D054306:Dideoxynucleotides; D006868:Hydrolysis; D010539:Permeability; D010766:Phosphorylation; D011355:Prodrugs / Q000493:pharmacokinetics / Q000494:pharmacology*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology
D000970:Antineoplastic Agents; D001386:Azides; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D011355:Prodrugs; D003841:Deoxycytidine; C073515:bis(pivaloyloxymethyl) 2',3'dideoxyuridine 5'-monophosphate; C053847:2'-azido-2'-deoxyuridine 5'-diphosphate; C038474:2'-azido-2'-deoxycytidine 5'-diphosphate; D003565:Cytidine Diphosphate; D014542:Uridine Monophosphate; D012264:Ribonucleotide Reductases; D003842:Deoxycytidine Kinase; D003857:Deoxyuridine