Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine γ-lyase conjugated with polyethylene glycol as an antitumor agent

Tomoaki Takakura, Akio Takimoto, Yoshihide Notsu, Hiroshi Yoshida, Takaomi Ito, Hirofumi Nagatome, Masahiro Ohno, Yoshinao Kobayashi, Takayuki Yoshioka, Kenji Inagaki, Shigeo Yagi, Robert M. Hoffman, Nobuyoshi Esaki

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Abstract

A highly potent recombinant L-methionine γ-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.

Original languageEnglish
Pages (from-to)2807-2814
Number of pages8
JournalCancer Research
Volume66
Issue number5
DOIs
Publication statusPublished - Mar 1 2006

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Lyases
Methionine
Antineoplastic Agents
Pyridoxal Phosphate
Pharmacokinetics
Dithiothreitol
Holoenzymes
Serum Albumin
Half-Life
Therapeutic Uses
Oleic Acid
Cysteine

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine γ-lyase conjugated with polyethylene glycol as an antitumor agent. / Takakura, Tomoaki; Takimoto, Akio; Notsu, Yoshihide; Yoshida, Hiroshi; Ito, Takaomi; Nagatome, Hirofumi; Ohno, Masahiro; Kobayashi, Yoshinao; Yoshioka, Takayuki; Inagaki, Kenji; Yagi, Shigeo; Hoffman, Robert M.; Esaki, Nobuyoshi.

In: Cancer Research, Vol. 66, No. 5, 01.03.2006, p. 2807-2814.

Research output: Contribution to journalArticle

Takakura, T, Takimoto, A, Notsu, Y, Yoshida, H, Ito, T, Nagatome, H, Ohno, M, Kobayashi, Y, Yoshioka, T, Inagaki, K, Yagi, S, Hoffman, RM & Esaki, N 2006, 'Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine γ-lyase conjugated with polyethylene glycol as an antitumor agent', Cancer Research, vol. 66, no. 5, pp. 2807-2814. https://doi.org/10.1158/0008-5472.CAN-05-3910
Takakura, Tomoaki ; Takimoto, Akio ; Notsu, Yoshihide ; Yoshida, Hiroshi ; Ito, Takaomi ; Nagatome, Hirofumi ; Ohno, Masahiro ; Kobayashi, Yoshinao ; Yoshioka, Takayuki ; Inagaki, Kenji ; Yagi, Shigeo ; Hoffman, Robert M. ; Esaki, Nobuyoshi. / Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine γ-lyase conjugated with polyethylene glycol as an antitumor agent. In: Cancer Research. 2006 ; Vol. 66, No. 5. pp. 2807-2814.
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AU - Yoshida, Hiroshi

AU - Ito, Takaomi

AU - Nagatome, Hirofumi

AU - Ohno, Masahiro

AU - Kobayashi, Yoshinao

AU - Yoshioka, Takayuki

AU - Inagaki, Kenji

AU - Yagi, Shigeo

AU - Hoffman, Robert M.

AU - Esaki, Nobuyoshi

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N2 - A highly potent recombinant L-methionine γ-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.

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