Association of DNA cross-linking with potentiation of the morpholino derivative of doxorubicin by human liver microsomes.

The morpholino analog of doxorubicin (DOX), 3'-deamino-3'-(4"-morpholinyl)-doxorubicin (MRA), is 0.5- to 10-fold more potent than DOX in vitro but 100- to 200-fold more potent in vivo, which indicated that biotransformation in vivo may generate a highly potent metabolite(s). A likely mechanism for such biotransformation is hepatic mixed-function oxidation. At a concentration of 5 microM, MRA was incubated for 30 minutes at 37 degrees C with 1 mg of human liver microsomes/mL and 0.45 mM of NADPH. The cytotoxicity of the microsome- and NADPH-treated MRA was 44-fold higher than that of the untreated MRA in the human ovarian carcinoma cell line ES-2. This potentiation did not occur for MRA treated with boiled microsomes and NADPH, active microsomes in the absence of NADPH, or Tris buffer plus NADPH. No potentiation was observed with DOX or the highly potent cyanomorpholino derivative of DOX, MRA-CN, under any of the above conditions. After 2 hours of exposure of the ES-2 cells to microsome- and NADPH-treated MRA, dose-dependent DNA cross-links were observed with 5 nM or more of MRA, whereas only DNA strand breaks were detected in cells exposed to 500 nM of untreated MRA or MRA incubated under other conditions. These data indicate that MRA is biotransformed by the hepatic mixed-function oxidases to a potent DNA-alkylating metabolite(s), which may be important in the determination of the pharmacologic and toxicologic profile of MRA. The active metabolite(s) of MRA may be analogous to MRA-CN, which cross-links DNA without requiring bioactivation.