title: Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.
abstract: Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has 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-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic 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 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition 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 constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.
authors.affiliation: Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, New Jersey 08854-3969, USA.
authors.forename: Maoliang
authors.initials: M
authors.lastname: Wang
authors.forename: Amartya
authors.initials: A
authors.lastname: Basu
authors.forename: Thomas
authors.initials: T
authors.lastname: Palm
authors.forename: Jack
authors.initials: J
authors.lastname: Hua
authors.forename: Stephen
authors.initials: S
authors.lastname: Youngster
authors.forename: Lisa
authors.initials: L
authors.lastname: Hwang
authors.forename: Hsien-Ching
authors.initials: HC
authors.lastname: Liu
authors.forename: Xiguang
authors.initials: X
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Peng
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Zhang
authors.forename: Clifford
authors.initials: C
authors.lastname: Longley
authors.forename: Mary
authors.initials: M
authors.lastname: Mehlig
authors.forename: Virna
authors.initials: V
authors.lastname: Borowski
authors.forename: Prakash
authors.initials: P
authors.lastname: Sai
authors.forename: Manickam
authors.initials: M
authors.lastname: Viswanathan
authors.forename: Eun
authors.initials: E
authors.lastname: Jang
authors.forename: Gerald
authors.initials: G
authors.lastname: Petti
authors.forename: Sam
authors.initials: S
authors.lastname: Liu
authors.forename: Karen
authors.initials: K
authors.lastname: Yang
authors.forename: David
authors.initials: D
authors.lastname: Filpula
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D045645:Mycoplasma arthritidis / Q000201:enzymology* / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry*; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase