655. Development of a Human Carboxylesterase That Can Efficiently Metabolize CPT-11
Authors: Monika Wierdl, RandyM. Wadkins, MatthewR. Redinbo, Lyudmila Tsurkan, PhilipM. Potter
Journal: Molecular Therapy (2006)

CPT-11 is a water-soluble camptothecin analogue that has shown remarkable antitumor activity against a variety of tumors in xenograft models and in clinical trials. It is currently approved for the treatment of advanced colorectal cancers, and is likely to be approved for use against a variety of other solid tumors. CPT-11 is a prodrug that is metabolized in vivo by carboxylesterases (CEs) to generate the potent topoisomeraseI inhibitor, SN-38. Among the highly homologous mammalian CEs, a rabbit liver carboxylesterase (rCE) is the most efficient at CPT-11 activation. The human intestinal CE (hiCE), and the human liver CE (hCE1), however, are 5-fold, and 1000-fold less efficient at drug conversion, respectively. As a result, in humans, less than 5% of the administered CPT-11 is converted to SN-38. Furthermore, CPT-11 dose escalation is limited by severe delayed diarrhea due in part to CPT-11 conversion in the small intestine. Thus, development of an enzyme-prodrug therapy approach that would allow selective drug activation at the tumor site, without the need for CPT-11 dose escalation, could increase the clinical efficacy of this drug. For such an approach, development of a human CE that can activate CPT-11 with high efficiency is required. This CE will likely be less immunogenic than rCE in humans, and may allow development of protocols for improved enzyme-prodrug therapy approaches with CPT-11.Based on the differences in the crystal structures of rCE, and hCE1, we generated multiple mutations in hCE1, and evaluated these mutants for their ability to metabolize CPT-11. One mutant (hCE1mut6) metabolized CPT-11 with similar efficiency as rCE. Furthermore, we demonstrated that Cos7 cells expressing this mutant protein were sensitized to CPT-11. Molecular modeling studies of the mutant protein revealed that there is an alteration in the shape of the active site gorge opening compared to the wild-type protein. These results indicate that the mutations allow better access of the drug to the catalytic amino acids that are buried at the bottom of a long active site gorge. We are currently evaluating the ability of adenovirus expressing hCE1mut6 to sensitize human tumor cell lines to CPT-11 after viral transduction. If successful, this CE may be suitable for enzyme-prodrug therapy in combination with CPT-11 in vivo.This work was supported by the NIH and by the American Lebanese Syrian Associated charities. CPT-11 is a water-soluble camptothecin analogue that has shown remarkable antitumor activity against a variety of tumors in xenograft models and in clinical trials. It is currently approved for the treatment of advanced colorectal cancers, and is likely to be approved for use against a variety of other solid tumors. CPT-11 is a prodrug that is metabolized in vivo by carboxylesterases (CEs) to generate the potent topoisomeraseI inhibitor, SN-38. Among the highly homologous mammalian CEs, a rabbit liver carboxylesterase (rCE) is the most efficient at CPT-11 activation. The human intestinal CE (hiCE), and the human liver CE (hCE1), however, are 5-fold, and 1000-fold less efficient at drug conversion, respectively. As a result, in humans, less than 5% of the administered CPT-11 is converted to SN-38. Furthermore, CPT-11 dose escalation is limited by severe delayed diarrhea due in part to CPT-11 conversion in the small intestine. Thus, development of an enzyme-prodrug therapy approach that would allow selective drug activation at the tumor site, without the need for CPT-11 dose escalation, could increase the clinical efficacy of this drug. For such an approach, development of a human CE that can activate CPT-11 with high efficiency is required. This CE will likely be less immunogenic than rCE in humans, and may allow development of protocols for improved enzyme-prodrug therapy approaches with CPT-11. Based on the differences in the crystal structures of rCE, and hCE1, we generated multiple mutations in hCE1, and evaluated these mutants for their ability to metabolize CPT-11. One mutant (hCE1mut6) metabolized CPT-11 with similar efficiency as rCE. Furthermore, we demonstrated that Cos7 cells expressing this mutant protein were sensitized to CPT-11. Molecular modeling studies of the mutant protein revealed that there is an alteration in the shape of the active site gorge opening compared to the wild-type protein. These results indicate that the mutations allow better access of the drug to the catalytic amino acids that are buried at the bottom of a long active site gorge. We are currently evaluating the ability of adenovirus expressing hCE1mut6 to sensitize human tumor cell lines to CPT-11 after viral transduction. If successful, this CE may be suitable for enzyme-prodrug therapy in combination with CPT-11 in vivo. This work was supported by the NIH and by the American Lebanese Syrian Associated charities.