Physicochemical and Pharmacokinetic Characterization of Highly Potent Recombinant l-Methionine γ-Lyase Conjugated with Polyethylene Glycol as an Antitumor Agent

Abstract

A highly potent recombinant L-methionine ;-lyase (METase) conjugated with polyethylene glycol (PEG) was characterized physicochemically and pharmacokinetically in vivo and in vitro. Pegylated METase (PEG-METase), which contains pyridoxal 5V-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 PEGMETase, 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

Methionine gamma‐lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers

“…The enzymatic activity of PEG-MGL was detected for 72 h, while that of unconjugated MGL was undetectable after 24 h, and the half life of PEG-MGL increased by 20 times (38 h), compared to unconjugated MGL (2 h) (72). Simultaneous coadministration of pyridoxal 5 0 -phosphate and oleic acid, or dithiothreitol treatment also strengthened effectiveness of PEG-MGL in the rodent model (72,73).…”

Isolation of a homocysteine γ-lyase-producing bacterium and study of its enzyme production conditions

“…This enzyme has been studied for decades, mainly for its l ‐methionine degradation activity. Based upon the fact that most tumours have an absolute requirement for l ‐methionine, whereas normal human cells are relatively resistant to depletion of l ‐methionine, this enzyme has been applied successfully in the treatment of cancer (Takakura et al. 2006; Tan et al.…”

L-Methionase: A Therapeutic Enzyme to Treat Malignancies

“…PEG-rMGL demonstrated a significant decrease in antigenicity [146]. The specific activity of PEG-MGL increased with DTT [147]. Although L -methionase from bacterial (prokaryotic) origin has immunogenic issues that can be overcome by PEGylation and by other methods such as deimmunization by combinational T-cell epitope removal using neutral drift [148].…”