Engineering an Arginine Catabolizing Bioconjugate: Biochemical and Pharmacological Characterization of PEGylated Derivatives of Arginine Deiminase from Mycoplasma arthritidis

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• 作者：Maoliang Wang ; Amartya Basu ; Thomas Palm ; Jack Hua ; Stephen Youngster ; Lisa Hwang ; Hsien-Ching Liu ; Xiguang Li ; Ping Peng ; Yue Zhang ; Hong Zhao ; Zhihua Zhang ; Clifford Longley ; Mary Mehlig ; Virna Boro
• 刊名：Bioconjugate Chemistry
• 出版年：2006
• 出版时间：November 2006
• 年：2006
• 卷：17
• 期：6
• 页码：1447 - 1459
• 全文大小：776K
• 年卷期：v.17,no.6(November 2006)
• ISSN：1520-4812

文摘

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivationhas been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth,angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate,a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylatedderivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic andpharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous,intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 ± 2 PEGstrands per protein dimer, providing an additional molecular mass of about 0.2-0.5 × 10⁶ Da and prolonging theplasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation wasdemonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallelmeasurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with thepharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginineto undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneousand intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growthinhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase.Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constrainingthe PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of theexperimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of theprimary amines retained about 60% or 48% of enzyme activity, respectively; the K_m and pH profiles were nearlyunchanged; IC₅₀ values were diminished by less than 30%; while stability studies demonstrated full retention ofactivity at 4 C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonasputida illustrated the superior characteristics of the M. arthritidis ADI enzyme.