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

2′,3′-Dideoxy-2′,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, l(−)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 K(m) 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.