Cellular pharmacology of PNU-159682, a liver microsomal metabolite of methoxymorpholinyl doxorubicin (Nemorubicin; MMDX)

PNU-159682 is a major liver microsomal metabolite of MMDX, a doxorubicin (DX) derivative currently undergoing a phase II/111clinical trial. The purposes of this work were to obtain preliminary information about the cytotoxicity, effects on cell cycle progression, and DNA-binding properties of PNU-159682. In a first set of experiments we compared the in vitro cytotoxicity of PNU-159682 with that of MMDX and DX toward a panel of eight tumor cell lines including three human leukemias (EM-2, Jurkat and CEM), a human colon adenocarcinoma (HT-29), a human ovarian carci- noma (A2780), a human prostatic carcinoma (DU145), a murine melanoma (B16F10) and a murine leukemia (L1210). Each tumor cell line was ex- posed to the drugs for 1 h and then cultured in drug-free medium for 72 h. Drug concentrations that decreased cell growth by 70% (IC70) were calculated from dose-response curves by linear interpolation. The IC70 values of PNU-159682 ranged between 0.07 and 0.58 nM; these values were considerably lower than that recorded for both MMDX (67.6-577.9 nM) and DX (181.0-1,717.3 nM). Further experiments analyzed the cell cycle perturbations induced in LoVo colon adenocarcinoma cells by exposure to equitoxic concentrations of each drug. Cells were continuously exposed to the IC70 value of each compound recorded after 72 h of drug treatment, and the cell cycle distribution profiles analyzed using flow cytometry. Exposure to PNU- 159682 determined an accumulation of the cells in the S or G2/M phase at 24 h; in contrast, MMDX and DX induced a G1 and a G2/M arrest, respec- tively. Between 24 and 48 h of treatment a small number of cells started to undergo apoptosis as revealed by the presence of a sub-G1 peak at 48 h. This phenomenon became more evident at 72 h of drug exposure. Prelimi- nary DNA unwinding studies showed that PNU-159682 is able to intercalate into DNA with a higher affinity than MMDX and DX. In conclusion, the results described indicate that PNU-159682 is a compound endowed with a much higher cytotoxicity than MMDX and DX. Furthermore, the different ef- fect of PNU-159682 on cell cycle progression and its higher DNA binding affinity, compared with that of MMDX and DX, suggest that it might possess a different molecular mechanism of action