Toshihiro Noguchi

Cytotoxicity of Amrubicin, a Novel 9‐Aminoanthracycline, and Its Active Metabolite Amrubicinol on Human Tumor Cells

Amrubicin, a completely synthetic 9‐aminoanthracycline derivative, was previously shown to have potent antitumor activities against various human tumor xenografts. In this study, the in vitro activities of amrubicin and its major metabolite, amrubicinol, were examined using 17 human tumor cell lines. Amrubicinol was 5 to 54 times more potent than amrubicin, and as potent as doxorubicin, in inhibiting the growth of the cells following 3‐day continuous drug exposure. Amrubicinol closely resembled doxorubicin in its profile of activities on the 17 human tumor cell lines. Cells were incubated with the drugs for 1 h, and the intracellular drug concentration and cell growth inhibition after 3 days were determined. Amrubicinol attained similar intracellular concentrations at lower medium concentrations compared to amrubicin, and the intracellular concentration of amrubicinol necessary to produce 50% cell growth inhibition was 3 to 8 times lower than that of amrubicin in 4 cell lines tested. Amrubicinol has a higher activity level inside the cells than does amrubicin. When cells were incubated with amrubicin for 5 h, a substantial amount of amrubicinol, more than 9% of that of amrubicin, was found in cells in 4 of the 8 cell lines tested. Amrubicinol may contribute to the in vitro growth‐inhibitory effect of amrubicin on these cells. The results suggest that amrubicinol plays an important role in the in vivo antitumor effect of amrubicin as an active metabolite.

A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9‐aminoanthracycline derivative. Amrubicin and its C‐13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA‐protein complex formation and double‐strand DNA breaks in CCRF‐CEM cells and KU‐2 cells. In this study, we found that ICRF‐193, a topo II catalytic inhibitor, antagonized both DNA‐protein complex formation and double‐strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF‐193. Taken together, these findings indicate that the cell growth‐inhibitory effects of amrubicin and amrubicinol are due to DNA‐protein complex formation followed by double‐strand DNA breaks, which are mediated by topo II.

In vivo efficacy and tumor-selective metabolism of amrubicin to its active metabolite

The tissue distribution of a novel antitumor anthracycline antibiotic, amrubicin, was studied using seven human tumor xenografts implanted into nude mice, in order to identify the principal factors determining its therapeutic efficacy. We found a good correlation between the level of the metabolite amrubicinol in the tumor and the in vivo efficacy. High metabolic activity of amrubicin to amrubicinol was detected in tumor tissue homogenates, especially in cell lines highly sensitive to amrubicin in vivo. In contrast to amrubicin, the administration of amrubicinol showed less tumor‐selective toxicity in these human tumor xenograft models. These data indicate that the tumor‐selective metabolism of amrubicin to amrubicinol resulted in a tumor‐selective disposition of amrubicinol, leading to good efficacy in in vivo experimental therapeutic models.

Tumor-selective Distribution of an Active Metabolite of the 9-Aminoanthracycline Amrubicin

It has been reported that the 9‐aminoanthracycline amrubicin shows good efficacy in human tumor xenograft models. We studied the disposition and metabolism of amrubicin in mice, in comparison with those of doxorubicin. Amrubicinol, a 13‐hydroxy metabolite of amrubicin, which is 10 to 100 times more cytotoxic than amrubicin, was detected as a major metabolite in blood and tissues, and aglycones of amrubicin were also detected. A pharmacokinetic study revealed that amrubicin had a smaller distribution volume and a shorter half‐life than doxorubicin. In several normal tissues, the levels of amrubicin and amrubicinol were lower than those of doxorubicin. In contrast, the tumor levels of amrubicinol in the mice treated with amrubicin were higher than those of doxorubicin in the mice treated with that drug, in tumors that are sensitive to amrubicin. These results suggest that the potent therapeutic activity of amrubicin is caused by the selective distribution of its highly active metabolite, amrubicinol, in tumors.

Uptake and intracellular distribution of amrubicin, a novel 9-amino-anthracycline, and its active metabolite amrubicinol in P388 murine leukemia cells.

Amrubicin, a 9‐aminoanthracycline anti‐cancer drug, and its C‐13 hydroxyl metabolite amrubicinol, were examined for growth‐inhibitory activity as well as cellular uptake and distribution in P388 murine leukemia cells and doxorubicin‐resistant P388 cells. Also discussed are the differences in the mechanisms of action among amrubicin, amrubicinol and doxorubicin in terms of their cellular pharmacokinetic character. In P388 cells, amrubicinol was about 80 times as potent as amrubicin, and about 2 times more potent than doxorubicin in a 1‐h drug exposure growth‐inhibition test. A clear cross‐resistance was observed to both amrubicin and amrubicinol in doxorubicin‐resistant P388 cells, though the resistance index was lower for amrubicin. The intracellular concentration of amrubicinol was about 6 times and 2 times higher than those of amrubicin and doxorubicin, respectively. Compared to doxorubicin, amrubicin and amrubicinol were released rapidly after removal of the drugs from the medium. A clear correlation was found between the growth‐inhibiting activity and the cellular level of amrubicin and amrubicinol in P388 cells. About 10 to 20% of amrubicin or amrubicinol taken up by the cells was detected in the cell nuclear fraction, whereas 70 to 80% of doxorubicin was localized in this fraction. These results suggest that amrubicin and amrubicinol exert cytotoxic activity via a different mechanism from that of doxorubicin.

Amrubicin induces apoptosis in human tumor cells mediated by the activation of caspase-3/7 preceding a loss of mitochondrial membrane potential

Amrubicin, a completely synthetic 9‐aminoanthracycline derivative, inhibits cell growth by stabilizing a topoisomerase II–DNA complex. This study was designed to examine the apoptosis induced in human leukemia U937 cells by amrubicin and its active metabolite amrubicinol. Amrubicin, amrubicinol and other antitumor agents, such as daunorubicin and etoposide, induced typical apoptosis with characteristic nuclear morphological change and DNA fragmentation. Measuring the population of sub‐G1 phase cells, it was found that under conditions where cell growth was inhibited by either amrubicin or amrubicinol, U937 cells underwent apoptotic cell death in a dose‐dependent manner accompanied by an arrest of the cell cycle at G2/M. Furthermore, amrubicin‐ and amrubicinol‐induced apoptosis was mediated by the activation of caspase‐3/7, but not caspase‐1, preceding a loss of mitochondrial membrane potential. These results indicate that both a reduction in mitochondrial membrane potential and the activation of caspase‐3/7 are key events in the apoptosis induced by amrubicin and amrubicinol as well as the other antitumor agents. In addition, studies with oligomycin suggested that the apoptosis induced by amrubicin and amrubicinol involved substantially different pathways from that triggered by daunorubicin and etoposide. Oligomycin blocked the etoposide‐induced increase in the number of sub‐G1 phase cells without preventing the activation of caspase‐3/7, and had no inhibitory effect on the expansion of the sub‐G1 population in daunorubicin‐treated cells, whereas apoptosis‐related changes caused by amrubicin and amrubicinol were suppressed in the presence of oligomycin. (Cancer Sci 2006; 97: 1396–1403)