Recently we have described an
HPMA copolymer conjugate carrying both the
aromatase inhibitor aminoglutethimide (AGM) and
doxorubicin (Dox) as combination
therapy. This showed markedly enhanced in vitro cytotoxicity compared to the
HPMA copolymer-Dox (
FCE28068), a conjugate that demonstrated activity in
chemotherapy refractory
breast cancer patients during early clinical trials. To better understand the superior activity of
HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (
aromatase-transfected)
breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of
HPMA copolymer-Dox-AGM and
HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal
thiol-dependant
proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same
polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a
cholesterol-dependent pathway), however, the pattern of
enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from
HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the
anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for
HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the
anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of
HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity.