Metabolism and accumulation of the lipophilic deoxynucleoside analogs elacytarabine and CP-4126

Abstract

Cytarabine (ara-C) and gemcitabine (dFdC) are commonly used anticancer drugs, which depend on the equilibrative (ENT) and concentrative-nucleoside-transporters to enter the cell. To bypass transport-related drug resistance, lipophilic derivatives elacytarabine (CP-4055), ara-C-5'elaidic-acid-ester, and CP-4126, (CO 1.01) gemcitabine-5'elaidic-acid-ester, were investigated for the entry into the cell, distribution, metabolism and retention. The leukemic CEM-cell-line and its deoxycytidine-kinase deficient variant (CEM/dCK-) were exposed for 30 and 60 min to the radiolabeled drugs; followed by culture in drug-free medium in order to determine drug retention in the cell. The cellular fractions were analyzed with thin-layer-chromatography and HPLC. Elacytarabine and CP-4126 were converted to the parent compounds both inside and outside the cell (35-45%). The ENT-inhibitor dipyridamole did not affect their uptake or retention. Inside the cell Elacytarabine and CP-4126 predominantly localized in the membrane and cytosolic fraction, leading to a long retention after removal of the medium. In contrast, in cells exposed to the parent drugs ara-C and dFdC, intracellular drug concentration increased during exposure but decreased to undetectable levels after drug removal. In the dCK- cell line, no metabolism was observed. The concentrations of ara-CTP and dFdCTP reached a peak at the end of the incubation with the drugs, and decreased after drug removal; peak levels of dFdCTP were 35 times higher than ara-CTP and was retained better. In contrast, after exposure to elacytarabine or CP-4126, ara-CTP and dFdCTP levels continued to increase not only during exposure but also during 120 min after removal of the elacytarabine and CP-4126. Levels of ara-CTP and dFdCTP were higher than after exposure to the parent drugs. In conclusion, the lipophilic derivatives elacytarabine and CP-4126 showed a nucleoside-transporter independent uptake, with long retention of the active nucleotides. These lipophilic nucleoside analogues are new chemical entities suitable for novel clinical applications.

Fig. 1

Extracellular concentration of ara-C and dFdC after incubation for 30 or 60 min (marked as 30 and 60) of the CEM or CEM/dCK- cell lines with radioactive drugs. The concentrations of the prodrug and parent drug were measured after 60 min exposure to the drugs and washing with drug-free medium and culture for 60 min in drug free medium (marked at 60 + 60). a Ara-C after incubation with 9.7 μM [³H]-ara-C or [³H]-elacytarabine (CP-4055), b dFdC after incubation with 8.9 μM [³H]-dFdC or [³H]-CP-4126. Values represent means ± SEM of at least three separate experiments. Ara-C levels in medium of CEM-dCK- cells were significantly higher than in medium of CEM cells (P < 0.01)

Fig. 2

Intracellular accumulation of ara-C and dFdC after incubation for 30 or 60 min of the CEM or CEM/dCK- cell line with radioactive drugs (marked as 30 and 60). Retention of the prodrug and the parent drug was measured after exposure for 60 min and washing with drug-free medium and culture for 60 min in drug free medium (marked as 60 + 60). a Ara-C after incubation with 9.7 μM [³H]-ara-C or [³H]-elacytarabine (CP-4055), b dFdC after incubation with 8.9 μM [³H]-dFdC or [³H]-CP-4126. Values represent means ± SEM of at least three separate experiments. Accumulation of ara-C from CP-4055 after 60 min and 60 min retention period was significantly higher in CEM-cells than in CEM-dCK- cells, similarly accumulation of dFdC from CP-4126 after 30 min was significantly higher in CEM compared to CEM-dCK- cells (P < 0.05)

Fig. 3

Nuclear accumulation and retention of radiolabeled ara-C (a), elacytarabine (CP-4055) (b), dFdC (c) and CP-4126 (d). The nuclear pellets of CEM and CEM/dCK- cells were analyzed for radiolabeled metabolites after incubation for 30 or 60 min (marked as 30 and 60) with 9.7 μM [³H]-ara-C (a) or [³H]-elacytarabine (CP-4055) (b), 8.9 μM [³H]-dFdC (c) or [³H]-CP-4126 (d). Retention was studied after 60 min exposure followed by washing the cells and resuspension in drug-free medium for 60 min (marked as 60 + 60). Values represent means ± SEM of at least three separate experiments. Accumulation of radiolabeled CP-4126 was significantly lower than that of CP-4055 (P < 0.01)

Fig. 4

Effect of dipyridamole on (a) Extracellular concentration and (b) intracellular accumulation of ara-C and elacytarabine (CP-4055) in CEM cells (marked as 30 and 60). Cells were incubated for 30 and 60 min with 9.7 μM [³H]-ara-C or [³H]-elacytarabine (CP-4055), after which elacytarabine and ara-C were measured. Concentrations of the prodrug and parent drug were measured after 60 min exposure and washing the cells with drug free medium and incubation for 60 min in drug free medium (marked as 60 + 60). Values represent means ± SEM of at least three separate experiments

Fig. 5

Ara-CTP (a) and dFdCTP (b) accumulation in the CEM cell line after 60 min incubation with 10 μM ara-C, 100 μM elacytarabine (CP-4055), 1 μM dFdC or 10 μM CP-4126 with and without inhibition of hENT by dipyridamole. Intracellular concentrations of the triphosphates were measured after 60 min exposure and washing the cells with drug-free medium and subsequent culture for 60 and 120 min in drug-free medium (marked as 60 + 60 and 60 + 120). Values represent means ± SEM of at least three separate experiments. Accumulation (60 min) of ara-CTP and dFdCTP was not different when comparing ara-C incubation with elacytarabine or dFdC with CP-4126. However, the retention (60 + 120) of ara-CTP and of dFdCTP was significantly higher in cells incubated with elacytarabine (p < 0.01) or CP-4126 (p < 0.05), respectively, compared to incubation with ara-C or dFdC, as well as the effect of dipyridamole on dFdCTP retention (P < 0.01) of ara-CTP retention (P < 0.05)

Fig. 6

Subcellular localization of radioactivity from 9.7 μM Elacytarabine (CP-4055) (a,b) and ara-C (a,b), or 8.9 μM CP-4126 (c,d) and dFdC (c,d) in the CEM (a,c) and CEM/dCK- (b,d) cell lines. Relative amounts after 60 min incubation with the radioactive drugs; total activity was calculated by setting the sum of radioactivity in all fractions at 100%, and subsequently calculating the percentage in each fraction. Accumulation of elacytarabine label and CP-4126 label was significantly higher in the membrane fraction compared to accumulation of ara-C (p < 0.05) or dFdC (p < 0.02) label, respectively