Circulating half-life of PEGylated recombinant methioninase holoenzyme is highly dose dependent on cofactor pyridoxal-5'-phosphate.
Recombinant methioninase (rMETase) has been shown to target the elevated methionine (MET) dependence of tumor cells and arrest their growth as well as make tumors more sensitive to standard chemotherapy agents. Polyethylene glycol (PEG)-modified rMETase (PEG-rMETase) has reduced antigenicity compared with unmodified rMETase. However, PEG-rMETase has a limited active circulating half-life due to rapid in vivo dissociation of its cofactor pyridoxal-5'-phosphate (PLP), a surprising finding, because PLP is tightly bound to PEG-rMETase in buffer. The question asked in the current study was on the effect of increasing doses of PLP to extend the circulating half-life of active PEG-rMETase holoenzyme in vivo. rMETase was conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/ml PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 microm, respectively, from the PLP baseline of 0.3 microm. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. Pumps containing 500 mg/ml PLP increased the half-life of MEGC-PEG-rMETase holoenzyme 4.5-fold from 1.5 to 7 h. Infused PLP did not extend the half-life of MEGC-PEG-rMETase apoenzyme. With a dose of 4000 units/kg MEGC-PEG-rMETase in the mice infused with 5, 50, 200, and 500 mg/ml PLP, plasma MET was depleted from 50 microm to < or = 5 microm for 8, 24, 72, and 72 h, respectively. Thus, PLP infusion could extend the period of MET depletion by MEGC-PEG-rMETase by approximately 10-fold in a dose-dependent manner. The mice given 8000 units/kg MEGC-PEG-rMETase showed a similar plasma MET depletion time course, indicating that the limiting factor for MEGC-PEG-rMETase-mediated MET depletion in vivo was PLP. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP. The infused PLP either bound to apo-MEGC-PEG-rMETase and/or inhibited dissociation of PLP from holo-PEG-rMETase, thereby maintaining the holoenzyme form of MEGC-PEG-rMETase in vivo. The combination of MEGC-PEG-rMETase treatment with PLP infusion suggests an effective clinical strategy for long-term MET depletion to arrest cancer growth.
Yang
Zhijian
Z
AntiCancer, Inc., San Diego, California 92111, USA.
Sun
Xinghua
X
Li
Shukuan
S
Tan
Yuying
Y
Wang
Xiaoen
X
Zhang
Nan
N
Yagi
Shigeo
S
Takakura
Tomoaki
T
Kobayashi
Yoshinao
Y
Takimoto
Akio
A
Yoshioka
Takayuki
T
Suginaka
Akinori
A
Frenkel
Eugene P
EP
Hoffman
Robert M
RM
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D013437:Carbon-Sulfur Lyases / Q000097:blood* / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D006207:Half-Life; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011092:Polyethylene Glycols / Q000737:chemistry / Q000493:pharmacokinetics*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics
D000964:Antimetabolites, Antineoplastic; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D011732:Pyridoxal Phosphate; C028210:monomethoxypolyethylene glycol; D013437:Carbon-Sulfur Lyases; C474516:PEG-methioninase