title: In vivo efficacy of recombinant methioninase is enhanced by the combination of polyethylene glycol conjugation and pyridoxal 5'-phosphate supplementation.
abstract: Recombinant methioninase (rMETase) is an enzyme active in preclinical mouse models of human cancer. The efficacy of rMETase is due to depletion of plasma methionine, an amino acid for which tumors generally have an abnormally high methionine requirement. Furthermore, transient methionine depletion results in a markedly increased sensitivity of the tumors to several chemotherapeutic agents. This study characterized methods to prolong the half-life of rMETase to extend the in vivo period of depletion of plasma and tumor methionine. In the present study, rMETase was coupled to methoxypolyethylene glycol succinimidyl glutarate-5000 in order to prolong the half-life of rMETase and thus extend the in vivo period of depletion of plasma and tumor methionine. Matrix-assisted laser desorption ionization mass spectrometry indicated that one sub-unit of rMETase was modified by approximately 4, 6 and 8 PEG molecules when rMETase was PEGylated at molar ratios of PEG/rMETase of 30/1, 60/1, and 120/1, respectively. PEG-rMETase (120/1) had a serum half-life increase of 20-fold, and methionine depletion time increased 12-fold compared to unmodified rMETase. The increase in in vivo half-life depended on the extent of PEGylation of rMETase. In addition, a remarkable prolongation of in vivo activity and effective methionine depletion by the PEG-rMETase was achieved by the simultaneous administration of pyridoxal 5'-phosphate. PEGylation also reduced the immunogenicity of rMETase. The extent of reduction in immunogenicity depended on the number of residues PEGylated. PEG-rMETase 30/1 had a 10-fold decrease in IgG titer while PEG-rMETase 120/1 had a 10(4)-fold decreased titer compared to naked rMETase. Thus, the molecular modification of PEGylation confers critical new properties to rMETase for development as a cancer therapeutic.
authors.affiliation: AntiCancer, Inc, San Diego, California 92111, USA.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Sun
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Yang
authors.forename: Shukuan
authors.initials: S
authors.lastname: Li
authors.forename: Yuying
authors.initials: Y
authors.lastname: Tan
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.forename: Xiaoen
authors.initials: X
authors.lastname: Wang
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yagi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshioka
authors.forename: Akio
authors.initials: A
authors.lastname: Takimoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Mitsushima
authors.forename: Akinori
authors.initials: A
authors.lastname: Suginaka
authors.forename: Eugene P
authors.initials: EP
authors.lastname: Frenkel
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood; D000918:Antibody Specificity; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D002626:Chemistry, Pharmaceutical; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D005450:Fluorescamine / Q000378:metabolism; D006207:Half-Life; D008715:Methionine / Q000097:blood / Q000172:deficiency; D051379:Mice; D008819:Mice, Nude; D011092:Polyethylene Glycols / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D013388:Succinimides; D005450:Fluorescamine; D011092:Polyethylene Glycols; D011732:Pyridoxal Phosphate; C028210:monomethoxypolyethylene glycol; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase