Synthesis and biological characterization of protease-activated prodrugs of doxazolidine.

Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clinical impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common association with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (average IC(50) of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.

Preclinical efficacy of a carboxylesterase 2-activated prodrug of doxazolidine.

Doxazolidine (Doxaz) is a functionally distinct formaldehyde conjugate of doxorubicin (Dox) that induces cancer cell death in Dox-sensitive and resistant cells. Pentyl PABC-Doxaz (PPD) is a prodrug of Doxaz that is activated by carboxylesterase 2 (CES2), which is expressed by liver, non-small-cell lung, colon, pancreatic, renal, and thyroid cancer cells. Here, we demonstrate that in two murine models, PPD was effective at slowing tumor growth and demonstrated markedly reduced cardiotoxic and nephrotoxic effects, as well as better tolerance, relative to Dox. Hepatotoxicity, consistent with liver expression of the murine CES2 homologue, was induced by PPD. Unlike irinotecan, a clinical CES2-activated prodrug, PPD produced no visible gastrointestinal damage. Finally, we demonstrate that cellular response to PPD may be predicted with good accuracy using CES2 expression and Doxaz sensitivity, suggesting that these metrics may be useful as clinical biomarkers for sensitivity of a specific tumor to PPD treatment.

Anthracycline-formaldehyde conjugates and their targeted prodrugs.

The sequence of research leading to a proposal for anthracycline cross-linking of DNA is presented.The clinical anthracycline antitumor drugs are anthraquinones, and as such are redox active. Their redoxchemistry leads to induction of oxidative stress and drug metabolites. An intermediate in reductive glycosidiccleavage is a quinone methide, once proposed as an alkylating agent of DNA. Subsequent research nowimplicates formaldehyde as a mediator of anthracycline-DNA cross-linking. The cross-link at 5'-GC-3'sites consists of a covalent linkage from the amino group of the anthracycline to the 2-amino groupof the G-base through a methylene from formaldehyde, hydrogen bonding from the 9-OH to the G-base onthe opposing strand, and hydrophobic interactions through intercalation of the anthraquinone. The combinationof these interactions has been described as a virtual cross-linkof DNA. The origin of the formaldehyde in vivo remains a mystery. In vitro, doxorubicin reacts withformaldehyde to give firstly a monomeric oxazolidine, doxazolidine, and secondly a dimeric oxazolidine,doxoform. Doxorubicin reacts with formaldehyde in the presence of salicylamide to give the N-Mannich baseconjugate, doxsaliform. Doxsaliform is several fold more active in tumor cell growth inhibition than doxorubicin,but doxazolidine and doxoform are orders of magnitude more active than doxorubicin. Exploratory researchon the potential for doxsaliform and doxazolidine as targeted cytotoxins is presented. A promisinglead design is pentyl PABC-Doxaz, targeted to a carboxylesterase enzyme overexpressed in liver cancercells and/or colon cancer cells.