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

Abstract

2',3'-Dideoxy-2',3'-didehydro-beta-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 beta-L(-) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 microM. As we previously reported for other beta-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 microM. 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.

FIG. 1

Chemical structures of anti-HIV nucleoside analogs.

FIG. 2

Antiviral isobolograms of drug combination data obtained in MT-2 cells with l(−)Fd4C plus D4T (A), l(−)Fd4C plus AZT (B), l(−)Fd4C plus ddC (C), and l(−)Fd4C plus ddI (D). Numbers along each axis are proportions of the EC₅₀ (taken as 1) for the drug indicated as a single agent. [EC₅₀s for single agents are 0.036 μM AZT, 1.8 μM D4T, 0.6 μM ddC, 12 μM ddI, and 0.5 μM l(−)Fd4C.] Each datum point represents a combination that produces an effect equivalent to that of the EC₅₀ for either drug used alone.

FIG. 3

HPLC analysis of l(−)Fd4C metabolites in CEM cells. (A) Methanol extracts prepared from CEM cells treated for 24 h with 2 μM [³H]L(−)Fd4C (20 mCi/mmol) were applied to a Partisil-SAX ion-exchange column as described in Materials and Methods. (B and C) Ion-exchange and reverse-phase chromatograms, respectively, of methanol-soluble extracts described above and digested with 25 U of alkaline phosphatase. Metabolites: I, l(−)Fd4C; II, l(−)Fd4C 5′-monophosphate; III, l(−)Fd4C 5′-diphosphate; IV, l(−)Fd4C 5′-triphosphate.

FIG. 4

β-l(−) Deoxycytidine analogs as substrates of HIV-RT and mitochondrial DNA pol γ. Shown is an autoradiograph of chain elongation assessed by using a ³²P-22-mer oligonucleotide primer annealed to M13mp19 phage DNA in the presence of enzyme and 0.5 μM nucleoside analog 5′-triphospate. The left lane shows the position of the primer in the incubation mixture without substrate. Reaction conditions were as described in Materials and Methods.