Benjamin L. Barthel

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.

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs.

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses.

Identification of Human Intestinal Carboxylesterase as the Primary Enzyme for Activation of a Doxazolidine Carbamate Prodrug

Doxazolidine (Doxaz), a formaldehyde-doxorubicin (Dox) conjugate, exhibits markedly increased tumor toxicity with respect to Dox without a concurrent increase in toxicity to cardiomyocytes. Pentyl PABC-Doxaz (PPD) is a Doxaz carbamate prodrug that is hydrolyzed by carboxylesterases. Here, we identify human intestinal carboxylesterase (hiCE) as the agent of activation for PPD. Upon prodrug treatment, cells that express higher levels of hiCE responded with lower IC50 values for growth inhibition. Exposing MCF-7 human breast cancer cells, which respond poorly and express little hiCE, to PPD together with hiCE resulted in a dramatic decrease in the IC50, a decrease that was absent when human carboxylesterase 1 was added to prodrug treatment. Finally, U373MG glioblastoma cells overexpressing hiCE displayed approximately 100-fold reduction in the IC50 for PPD compared to cells lacking the carboxylesterase. Overall, our studies indicate that PPD is selectively hydrolyzed to the active metabolite by hiCE.

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.

Correlation of in Situ Oxazolidine Formation with Highly Synergistic Cytotoxicity and DNA Cross-Linking in Cancer Cells from Combinations of Doxorubicin and Formaldehyde

Anthracyclines are a class of antitumor compounds that are successful and widely used but suffer from cardiotoxicity and acquired tumor resistance. Formaldehyde interacts with anthracyclines to enhance antitumor efficacy, bypass resistance mechanisms, improve the therapeutic profile, and change the mechanism of action from a topoisomerase II poison to a DNA cross-linker. Contrary to current dogma, we show that both efficient DNA cross-linking and potent synergy in combination with formaldehyde correlate with the anthracyclines ability to form cyclic formaldehyde conjugates as oxazolidine moieties and that the cyclic conjugates are better cross-linking agents and cytotoxins than acyclic conjugates. We also provide evidence that suggests that the oxazolidine forms in situ, since cotreatment with doxorubicin and formaldehyde is highly cytotoxic to dox-resistant tumor cell lines, and that this benefit is absent in combinations of formaldehyde and epirubicin, which cannot form stable oxazolidines. These results have potential clinical implications in the active field of anthracycline prodrug design and development.

Leveraging Colloidal Aggregation for Drug-Rich Nanoparticle Formulations

While limited drug loading continues to be problematic for chemotherapeutics formulated in nanoparticles, we found that we could take advantage of colloidal drug aggregation to achieve high loading when combined with polymeric excipients. We demonstrate this approach with two drugs, fulvestrant and pentyl-PABC doxazolidine (PPD; a prodrug of doxazolidine, which is a DNA cross-linking anthracycline), and two polymers, polysorbate 80 (UP80) and poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol) (PLAC-PEG; a custom-synthesized, self-assembling amphiphilic polymer). In both systems, drug-loaded nanoparticles had diameters < 200 nm and were stable for up to two days in buffered saline solution and for up to 24 h in serum-containing media at 37 °C. While colloidal drug aggregates alone are typically unstable in saline and serum-containing media, we attribute the colloid stability observed herein to the polymeric excipients and consequent decreased protein adsorption. We expect this strategy of polymer-stabilized colloidal drug aggregates to be broadly applicable in delivery formulations.