Metabolism of 2',3'-dideoxy-2',3'-didehydro-β-L(-)-5-fluorocytidine and its activity in combination with clinically approved anti-human immunodeficiency virus β-D(+) nucleoside analogs in vitro

Abstract

2'3'-Dideoxy2',3'-didehydro-β-L(-)-5-fluorocytidine [L(-)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination of L(-)Fd4C plus 2',3'-didehydro-2',3'-dideoxythymidine (D4T, or stavudine) or 3'-azido-3'deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive antiviral activity was observed with L(-)Fd4C in combination with 2',3'- dideoxycytidine (ddC, or zalcitabine) or 2',3'-dideoxyinosine (ddI, or didanosine). This β-L(-) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 μM. As we previously reported for other β-L(-) nucleoside analogs, [(-)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified as cytoplasmic deoxycytidine kinase, and the Km is 100 μM. L(-)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine nucleoside kinase. Also, L(-)Fd4C was not a substrate for deoxycytidine deaminase. L(-)Fd4C 5'-triphosphate served as an alternative substrate to dCTP for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by L(-)Fd4C in two-drug combinations favors the further development of L(-)Fd4C as an anti- HIV agent.