Methoxypoly(ethylene glycol)-conjugated carboxypeptidase A for solid tumor targeting: part I: synthesis and...

In vivo efficacy of novel anticancer agents has been hindered by the inability to deliver effective concentrations of drugs to tumors. The use of macromolecules such as antibodies and polymers for enzyme delivery to tumors has revealed that catalyzing the conversion of a nontoxic prodrug into its cytotoxic form can generate an effective level of cytotoxic agents at tumor sites. This study primarily focuses on the synthesis and characterization of methoxypoly(ethylene glycol)-modified carboxypeptidase A (CPA) for solid tumor targeting. The molecular weight of CPA has been successfully altered from 35 to 40-50 kDa via attachment of a defined number of mPEG moieties. Relatively pure mPEG-CPA conjugates containing one, two, and three mPEG chains were obtained at preparative scale quantities through controlled PEGylation followed by fractionation that involved size-exclusion chromatography. An enhancement in kinetic properties including k(cat) and k(cat)/K(m) towards hippuryl-L-phenylalanine (hipp-L-phe) was observed in mPEG-CPA conjugates. An increase in the V(m) appeared to be responsible for this enhancement. The attachment of mPEG to CPA substantially improved the stability of the enzyme with respect to the specific peptidase activity toward the model substrate. This finding is particularly important in the development of a novel CPA/methotrexate-alpha-peptide system in solid tumor chemotherapy.