Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm

The compounds WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5 '-[ p-metboxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5 ,6-dihydro-3'-azidothymidine-5 '-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti -human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV a ctivation as well as to achieve spermicidal activity. We investigated the T K activity and intracellular metabolism of WHI-05 and WHI-07 in normal huma n vaginal and cervical epithelial cells as well as sperm. The time- and con centration-dependent intracellular formation of ZDV metabolites following a ddition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and end ocervical epithelial cells as well as motile sperm was studied by analytica l HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescen t nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert PH]thymidine to thymidine monophosphate in co mparison to NALM-6, a pre-B leukemia cell line. TK activity of genital trac t epithelial cells and sperm was found to be relatively low or lacking. Add ition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulte d in their concentration- and time-dependent conversion to alaninyl ZDV mon ophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites, Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP, These results demonstrate that human female geni tal tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.