In vitro and in vivo study of an albumin-binding prodrug of doxorubicin that is cleaved by cathepsin B

Purpose: This study was designed to evaluate the in vitro and in vivo antitumor activity of an albumin-binding prodrug of doxorubicin 1 which incorporates a maleimide moiety and a para-aminobenzyloxycarbonyl (PABC) spacer coupled to the dipeptide Phe-Lys that is cleaved by Cathepsin B.

Methods: Cleavage of the albumin conjugate was studied with Cathepsin B and in homogenates of MDA-MB 435 tumors. For in vivo studies, nude mice were injected with (a) glucose buffer, (b) doxorubicin (2 x 8 mg/kg, i.v, on days 10 and 17), or (c) compound 1 (3 x 24 mg/kg doxorubicin equivalent, on days 10, 17 and 24).

Results: Prodrug 1 once bound to albumin was effectively cleaved by Cathepsin B and in tumor homogenates releasing doxorubicin. A cytotoxicity assay of the albumin conjugate of 1 in two human tumor cell lines showed that doxorubicin was ~6 times more active than the conjugate. In contrast, in an in vivo study, the prodrug exhibited superior antitumor activity (T/C 15%) compared to doxorubicin (T/C 49%) in an equitoxic comparison.

Conclusions: The Cathepsin B cleavable spacer Phe-Lys-PABC incorporated in an albumin-binding prodrug is an effective way to increase the therapeutic index of doxorubicin.