We have recently investigated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin after intravenous administration, and (b) acid-sensitive or enzymatic release of the albumin-bound drug at the tumor site (1,2). In the present work we set out to develop albumin-binding prodrugs of doxorubicin that are cleaved by prostate-specific antigen (PSA). PSA is a serine protease that is over-expressed in prostate carcinoma and represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. Based on our synthetic experience, we developed albumin-binding prodrugs with the structures MT-Ser-Ser-Tyr-Tyr-Ser-Gly-DOXO; MT-Asn-Ser-Ser-Tyr-–Phe-Gln-DOXO [MT = maleimidotriethyleneglycol acid; DOXO = doxorubicin), EMC-(Arg)-Arg-Ser-Ser-Tyr-Tyr-Ser-X-DOXO [EMC: ε-Maleimidocaproic acid; X = amino acid]. All of the maleimide derivatives bound rapidly to the cysteine-34 position of endogenous and exogenous albumin and were efficiently cleaved by PSA at the P1-P′1scissile bond releasing a respective doxorubicin dipeptide. The derivatives containing arginine residues (EMC-(Arg)-Arg-Ser-Ser-Tyr-Tyr-Ser-X-DOXO) exhibited excellent water-solubility for intravenous administration. Subsequent biological evaluation was focused on a PSA-negative xenograft model (PC 3) and PSA-positive xenograft model (CWR22) in order to assess the selectivity of our therapeutic approach. EMC-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-DOXO showed no in vivo activity in the PSA-negative PC-3 model, but good activity in the CWR22 PSA-positive model that was comparable to doxorubicin. Incubation studies with CWR22 tumor homogenates revealed, however, that the full potential of EMC-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-DOXO had not been exploited considering that a less active doxorubicin dipeptide DOXO-Gly-Ser and not doxorubicin was released in PSA-positive prostate carcinoma tissues. We thus optimized the structure of this prodrug by substituting Arg for Gly in the C-terminal position. In incubation studies with CWR22 tumor homogenates, the resulting albumin conjugate of EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-DOXO initially released Ser-Arg-DOXO which was further degraded to doxorubicin as the final cleavage product. In vivo studies with this optimized PSA specific prodrug are underway. Kratz et al., Journal of Medicinal Chemistry. 45, 5523-33, 2002 Mansour et al., Cancer Res. 63, 4062-4066, 2003

[Proc Amer Assoc Cancer Res, Volume 46, 2005]