Preparation of Enzymatically Highly Active Pegylated-D-Amino Acid Oxidase and Its Application to Antitumor Therapy

Abstract

Background: D-Amino acid oxidase (DAO) is an H2O2-generating enzyme, and tumor growth suppression by selective delivery of porcine DAO in tumors via the cytotoxic action of H2O2 has been reported. DAO isolated from Fusariumspp. (fDAO) shows much higher enzyme activity than porcine DAO, although the application of fDAO for antitumor treatment has not yet been determined.

Objective: The purpose of this study was to prepare enzymatically highly active pegylated-fDAO, and to determine whether it accumulates in tumors and exerts a potent antitumor effect in tumor- bearing mice.

Methods: Polyethylene glycol (PEG; Mw. 2000) was conjugated to fDAO to form PEGylated fDAO (PEG-fDAO). PEG-fDAO was intravenously administered into S180 tumor-bearing mice, and the body distribution and antitumor activity of PEG-fDAO was determined.

Results: The enzyme activity of PEG-fDAO was 26.1 U/mg, which was comparable to that of fDAO. Intravenously administered PEG-fDAO accumulated in tumors with less distribution in normal tissue except in the plasma. Enzyme activity in the tumor was 60-120 mU/g-tissue over 7-20 h after i.v. injection of 0.1 mg of PEG-fDAO. To generate the H2O2 in the tumor tissue, PEG-fDAO was intravenously administered, and then, D-phenylalanine was intraperitoneally administered after a lag time. No remarkable tumor suppression effect was observed under conditions used in this study, compared to the non-treated group.

Conclusion: The results suggest that PEG-fDAO maintained high enzymatic activity after pegylation. Treatment with PEG-fDAO conferred high enzyme activity on tumor tissue; 3-6 fold higher than that of previously reported pDAO; however, high enzyme activity in the plasma limited repeated treatment owing to lethal toxicity, which seemingly led to poor therapeutic outcome. Overall, the use of PEG-fDAO is promising for antitumor therapy, although the suppression of DAO activity in the plasma would also be required rather than only the increase in DAO activity in the tumor for an antitumor effect.

Keywords: D-amino Acid Oxidase (DAO); D-amino acid; H2O2; antitumor therapy; enhanced permeability and retention (EPR) effect; polyethylene glycol..