title: Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine gamma-lyase conjugated with polyethylene glycol as an antitumor agent.
abstract: A highly potent recombinant L-methionine gamma-lyase (METase) conjugated with polyethylene glycol (PEG) was characterized physicochemically and pharmacokinetically in vivo and in vitro. Pegylated METase (PEG-METase), which contains pyridoxal 5'-phosphate (PLP) as a cofactor in the molecule, is a potent anticancer agent that can deplete L-methionine from plasma. Although pegylation decreased its specific activity, dithiothreitol (DTT) treatment increased it over three times with the detachment of one PEG moiety modified with a cysteine residue. We can produce DTT-treated PEG-METase on a large scale in sufficient quality for therapeutic use. The superiority of DTT-treated PEG-METase was confirmed by the enhancement of L-methionine depletion and amelioration of pharmacokinetics in mice. The holoenzyme of DTT-treated PEG-METase gave a several times larger area under the plasma concentration curve than that of DTT-untreated PEG-METase, not because of an increase of the half-life but because of high specific activity. Conversely, simultaneous PLP infusion led to a greatly increased half-life of the holoenzyme. DTT-treated PEG-METase administration with PLP infusion was the most useful combination for maximizing the potency of the enzyme. We showed that serum albumin interfered with holoenzyme activity in vitro. The decrease of holoenzyme activity was dependent on the type of serum albumin. We concluded that PLP was released from PEG-METase by serum albumin in vivo and in vitro. The deleterious effect of PLP dissociation from PEG-METase could be improved by supplementing PLP and oleic acid. Their synergistic effect in preventing a decrease of the holoenzyme activity was also observed.
authors.affiliation: Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan. tomoaki.takakura@shionogi.co.jp
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Takakura
authors.forename: Akio
authors.initials: A
authors.lastname: Takimoto
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Notsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takaomi
authors.initials: T
authors.lastname: Ito
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nagatome
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ohno
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshioka
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yagi
authors.forename: Robert M
authors.initials: RM
authors.lastname: Hoffman
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006207:Half-Life; D006801:Humans; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011092:Polyethylene Glycols / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D012709:Serum Albumin / Q000378:metabolism / Q000494:pharmacology; D005719:gamma-Globulins / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D012709:Serum Albumin; D005719:gamma-Globulins; D011092:Polyethylene Glycols; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D004229:Dithiothreitol