Activity against human immunodeficiency virus type 1, intracellular metabolism, and effects on human DNA polymerases of 4'-ethynyl-2-fluoro-2'-deoxyadenosine

Abstract

We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 microM [(3)H]EFdA or [(3)H]3'-azido-2',3'-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/10(9) cells, while that of AZT was 396.5 pmol/10(9) cells. When CEM cells were exposed to 10 microM [(3)H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/10(9) cells), while the amount of [(3)H]AZT-TP increased only moderately by 2.4-fold (970 pmol/10(9) cells). The intracellular half-life values of EFdA-TP and AZT-TP were approximately 17 and approximately 3 h, respectively. When MT-4 cells were cultured with 0.01 microM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1(NL4-3), and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 microM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases alpha, beta, and gamma were >100 microM, >100 microM, and 10 microM, respectively, while those of ddA-TP were >100 microM, 0.2 microM, and 0.2 microM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.

FIG. 1.

Structure of EFdA.

FIG. 2.

Cellular uptake and intracellular phosphorylation profiles of EFdA. CEM cells (10⁶ cells/ml) were incubated in the presence of various concentrations (0.1, 1, and 10 μM) of [³H]EFdA or [³H]AZT for 6 h and washed, and intracellular nucleosides/nucleotides were extracted with 60% methanol. The extracts were subjected to HPLC. One-minute fractions were collected until 40 min, and the radioactivity of the collected samples was determined with a scintillation counter for EFdA (A) and AZT (C). The radioactivity of the sample showing each peak and its flanking samples' radioactivity (total of 3-min fractions) were summed and plotted for EFdA (B) and AZT (D). For the sake of clarity, the profiles of [³H]EFdA and [³H]AZT metabolites in the cells exposed to 1 μM of each agent have been omitted from panels A and C. Determination of intracellular phosphorylation profiles of EFdA and AZT was conducted on two independent occasions, and comparable profiles were obtained in those two experiments. Shown in panels A and C are representative data. The data shown in panels B and D are geometric means ± 1 standard deviation.

FIG. 3.

Intracellular stability of AZT and EFdA metabolites. CEM (A) or MT-4 (B) cells (10⁶ cells/ml) were incubated with 0.2 μM of [³H]EFdA or [³H]AZT for 6 h and thoroughly washed to remove the extracellular drug. After further incubation of the cells for 0, 2, 4, 8, 12, and 24 h, cell extracts were prepared and subjected to HPLC analysis. Results are shown as arithmetic means of two or three independently conducted experiments (± 1 standard deviation).

FIG. 4.

Persistence of anti-HIV-1 activity after removal of EFdA, AZT, and TDF from culture media. MT-4 (A), PBMC (B), or CCR5⁺-MAGI cells (C) were exposed to 0.01, 0.1, or 1 μM EFdA (or AZT) for 24 h, thoroughly washed to deplete extracellular drugs, cultured for various periods of time (0, 2, 6, 12, 24, and 48 h), exposed to HIV-1, and further cultured for an additional 5 days with MT-4 cells and PBMC or for an additional 48 h with CCR5⁺-MAGI cells. Anti-HIV-1 activity was monitored using p24 production or with an MTT assay or a MAGI assay.