AD-143

N-Trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a DNA nonbinding derivative of Adriamycin, was studied for its effect on the uptake of labeled nucleosides into human leukemia (ML-1) and lymphoma (P3HR-1) cells in culture. After preincubation with AD 143 at concentrations as low as 5.2 microM (ML-1) or 13 microM (P3HR-1), the ability of the cells to take up extracellular labeled nucleosides was decreased by more than 50%. Similar experiments with Escherichia coli cells showed that AD 143 at the same concentrations did not have any effect. Influx of [3H]thymidine or [3H]uridine was studied by centrifugation of the cells through phthalate oil mixture, and it was found that the influx of the labeled nucleosides was decreased after treatment of the cells with AD 143. An increase in the membrane fluidity was observed after treatment of the cells with AD 143, as revealed by electron paramagnetic resonance spectroscopy studies. These observations suggest that the decreased incorporation of [3H]thymidine and [3H]uridine into acid-precipitable material that we observed earlier in the AD 143-treated cells may in part be the result of the AD 143-induced alteration of cell membrane activities, which in turn causes an inhibition of nucleoside uptake.

The N-acylanthracyclines AD32 (N-trifluoroacetyladriamycin-14-valerate) and AD143 (N-trifluoroacetyladriamycin-14-O-hemiadipate) are analogs of Adriamycin (ADR) undergoing clinical or advanced pre-clinical screening. Their principal metabolites, following the cleavage of the 14-acyl side-chain, are N-trifluoroacetyladriamycin (AD41) and its reduced form N-trifluoroacetyladriamycinol (AD92). Both these compounds are biologically active and detectable in treated patients, laboratory animals, and in tissue culture cells. Unlike ADR, AD32, as well as AD143 and metabolites, show no detectable binding to double-strand DNA. Their effects on DNA have been previously investigated in vivo and in vitro using the alkaline filter-elution assay. It has been shown that all of the compounds cause approximately equivalent amounts of protein-associated DNA breaks (PAB) and DNA-protein crosslinks in a mouse lymphoma and in tissue-culture leukemia cells. In order to establish whether the induction of PAB by the drugs requires DNA topoisomerase II mediation, cleavage mapping analysis was done with tested compounds using purified human topoisomerase II. DNA fragmentation was significantly enhanced in the presence of the enzyme and either AD41 or AD92. In contrast, no fragmentation enhancement was observed in the presence of the parental drugs AD32 or AD143. The results strongly suggest that metabolic activation of N-acylanthracyclines by nonspecific esterases is a prerequisite for their interaction with DNA topoisomerase II and for stabilization of the cleavable complex.