Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine gamma-lyase conjugated with polyethylene glycol as an antitumor agent.
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.
Takakura
Tomoaki
T
Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan. tomoaki.takakura@shionogi.co.jp
Takimoto
Akio
A
Notsu
Yoshihide
Y
Yoshida
Hiroshi
H
Ito
Takaomi
T
Nagatome
Hirofumi
H
Ohno
Masahiro
M
Kobayashi
Yoshinao
Y
Yoshioka
Takayuki
T
Inagaki
Kenji
K
Yagi
Shigeo
S
Hoffman
Robert M
RM
Esaki
Nobuyoshi
N
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
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