Cancer Research Meeting Calendar Advances in Breast Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, Z.
Right arrow Articles by Hoffman, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, Z.
Right arrow Articles by Hoffman, R. M.
[Cancer Research 64, 5775-5778, August 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Circulating Half-Life of PEGylated Recombinant Methioninase Holoenzyme Is Highly Dose Dependent on Cofactor Pyridoxal-5'-Phosphate

Zhijian Yang1, Xinghua Sun1, Shukuan Li1, Yuying Tan1, Xiaoen Wang1, Nan Zhang1, Shigeo Yagi1, Tomoaki Takakura2, Yoshinao Kobayashi2, Akio Takimoto2, Takayuki Yoshioka2, Akinori Suginaka3, Eugene P. Frenkel4 and Robert M. Hoffman1

1 AntiCancer, Inc., San Diego, California; 2 Shionogi and Co., Ltd., Osaka, Japan; 3 NOF Corporation, Tokyo, Japan; and 4 Department of Internal Medicine, University of Texas at Dallas, Southwestern Medical School, Dallas, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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 µM, respectively, from the PLP baseline of 0.3 µM. 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 µm to ≤5 µM for 8, 24, 72, and 72 h, respectively. Thus, PLP infusion could extend the period of MET depletion by MEGC-PEG-rMETase by ~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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Methionine (MET) dependence, the elevated minimal MET requirement for cell growth relative to normal cells, has been observed in many human cancer cell lines and cancer xenografts in animal models (1, 2, 3) . MET dependence is a metabolic defect seen only in cancer cells and precludes the cells from growing in media in which methionine is depleted (4 , 5) .

Recombinant methioninase (rMETase), chemical name: L-methionine {alpha}-deamino-{gamma}-mercaptomethane lyase or EC 4.4.1.11, is a pyridoxal-L-phosphate (PLP)-dependent enzyme with four subunits of 43 KDa each. rMETase is found in Pseudomonas putida, Aeromonas, and Clostridium but not in yeast, plants, or mammals (1, 2, 3, 4, 5, 6, 7) . The enzyme has been cloned from Pseudomonas putida and produced in Escherichia coli (8 , 9) . rMETase has been demonstrated to be a powerful approach to methionine depletion in vivo (6 , 7) .

rMETase alone or in combination with chemotherapeutic agents such as cisplatin, 5-fluorouracil, and 1,3-bis(2-chloroethyl)-1-nitrosourea has shown efficacy and synergy, respectively, in mouse models of colon, lung, and brain cancer (10, 11, 12, 13) . A pilot Phase I clinical trial demonstrated that rMETase depleted plasma methionine levels without observed clinical toxicity over a period of ~24 h in patients with advanced cancer (14 , 15) . A recent study in primates, however, indicated that rMETase could cause anaphylactic reactions (16) .

Conjugation of protein therapeutics with polyethylene glycol (PEG) has been shown to confer important therapeutic benefits, most importantly reduced antigenicity (17) . The Food and Drug Administration has approved the PEGylated forms of several protein therapeutics for clinical use, including adenosine deaminase, asparaginase, {alpha}-IFN, and a growth hormone antagonist (18, 19, 20, 21, 22) .

rMETase was coupled to methoxypolyethylene glycol succinimidyl glutarate-5000 (MEGC-PEG; Ref. 23 ). Pharmacokinetic evaluation in mice showed that MEGC-PEG-rMETase had decreased antigenicity, increased serum half-life, and increased methionine depletion time compared with unmodified rMETase. However, the half-life of active MEGC-PEG-rMETase holoenzyme was shown to be limited because of in vivo dissociation of PLP (23) .

The present study demonstrates the dose dependence efficacy of infused PLP to markedly increase the serum half-life of the active holoenzyme form of PEGrMETase and the period of maintenance of serum MET depletion in mice, suggesting the clinical potential of the combination of MEGC-PEG-rMETase and PLP treatment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials.
rMETase was produced by Shionogi Co., Ltd. (Osaka, Japan) as described previously (23) . The rMETase was formulated in 50 mM sodium phosphate buffer (pH 7.2), containing 10 µM PLP, with protein concentration 31 mg/ml, and specific activity 50.7 units/mg. rMETase was >95% pure by high-performance liquid chromatography with a tetramer/oligomer ratio of 96.7/3.3 and endotoxin of 0.06 EU/mg. MEGC-PEG used for PEGylation of rMETase was provided by the NOF Corporation (Kawasaki-shi, Kanagawa, Japan). The average molecular mass was 5461 Da. PLP and other chemicals were purchased from Sigma (St. Louis, MO).

PEGylation.
The protocol for preparation of PEGylated rMETase was previously described (23) : the activated MEGC-PEG derivative was used at a molar excess (2-fold) of MEGC-PEG to free lysines in rMETase (32 per rMETase tetramer), which corresponds to a molar ratio of PEG to rMETase of 60/1. A given amount of the activated MEGC-PEG was added to the rMETase solution (100 mg/ml) with three stepwise additions at 20-min intervals. The PEGylation reaction was carried out at 37°C (pH 9.0) under gentle stirring for 60 min. The PEGylation reaction was terminated by adjusting the pH to 7.2 with 0.1 M sodium diphosphate (pH 4.5). Diafiltration was followed by purification of MEGC-PEG-rMETase on DEAE-Sepharose and Sephacryl S200 columns. Purified MEGC-PEG-rMETase was concentrated and sterilized by filtration and stored at –80°C.

PLP Infusion.
Miniosmotic pumps with a reservoir volume of 200 µl, pumping rate of 1.0 µl/h and duration 7 days (Model 2001) were purchased from Durect Corporation (Cupertino, CA). Five-hundred mg of PLP and 150 mg of sodium hydroxide were dissolved in 1 ml of distilled water (pH 7.4). Other concentrations of PLP were prepared by dilution. The PLP solution was drawn into a 3-ml syringe, which was attached to the blunt-tipped, 27-gauge filling tube of the minipump. For s.c. placement of the pump, a small incision is made in the skin between the scapulae. Using a hemostat, a small pocket is formed by spreading the s.c. connective tissues apart. The pump is inserted into the pocket with the flow moderator pointing away from the incision. The skin incision is closed with sutures.

Determination of Protein Content.
Protein was measured colorimetrically using the Wako Protein Assay kit (Wako, Osaka, Japan) according to the instruction manual with slight modification (24) .

Determination of PEGylation Degree of rMETase.
The degree of modification of PEGylated rMETase was determined colorimetrically (25) and by matrix-assisted desorption ionization. For the colorimetric assay, various amounts of rMETase and MEGC-PEG-rMETase in 2 ml 0.1 M sodium phosphate buffer (pH 8.0) were mixed with 1 ml fluorescamine solution (0.3 mg/ml in acetone) and incubated for 5 min at room temperature. Samples were then assayed with a fluorescence spectrometer at 390 nm excitation and 475 nm emission. Results were plotted as fluorescence units versus concentration, with the slope of the line being determined by linear regression. The percentage of PEGylated primary amines was determined according to the following formula: 1 – (slope PEGylated rMETase/slope naked rMETase) x 100. Matrix-assisted desorption ionization analysis of naked and MEGC-PEG-rMETase was performed at the Scripps Research Institute using a PerSeptive Biosystems Voyager-Elite mass spectrometer (San Diego, CA).

Determination of MEGC-PEG-rMETase Apoenzyme.
MEGC-PEG-rMETase apoenzyme was determined, in the presence of PLP, from {alpha}-ketobutyrate produced from L-methionine according to the method of Esaki and Soda (26) with slight modification. Fifty µl of sample, diluted in 100 mM potassium phosphate buffer (pH 8.0), containing 0.01% DTT, 1 mM EDTA2Na, 10 µM PLP, and 0.05% Tween 80, were mixed with 1 ml substrate solution [100 mM potassium phosphate buffer (pH 8.0), containing 25 mM L-methionine and 10 µM PLP] in a glass test tube. The reaction mixture was vortexed immediately and incubated at 37°C without shaking for precisely 10 min. The reaction was stopped by adding 100 µl 50% trichloroacetic acid. The suspension was centrifuged at 13,000 rpm for 2 min. The supernatant (0.8 ml) was collected in a glass tube containing 1.6 ml of 1 M acetate buffer (pH 5.0). Then, 0.6 ml N-methyl-2-benzothiazolone hydrazone hydrochloride solution containing 0.1% 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (Wako) was added to the tube, mixed well, and incubated at 50°C for 40 min. The absorbance of the reaction mixture was measured at 320 nm. The assay was carried out in triplicate. {Delta}E was calculated by subtracting the average absorbance of blanks from the average absorbance of the reaction mixture. The enzyme activity was calculated by the following formula: activity (units/ml) = 0.548 (1.07 + 2.2{Delta}E) {Delta}E. One unit of enzyme is defined as the amount of enzyme which produced 1 µM {alpha}-ketobutyrate/minute at an infinite concentration of MET.

Determination of MEGC-PEG-rMETase Holoenzyme Activity.
For determination of plasma MEGC-PEG-rMETase holoenzyme activity, no PLP was added in the substrate solution. All procedures were the same as for the determination of MEGBC-PEG-rMETase apoenzyme, except that 5 µl of the sample in 45 µl distilled water were mixed with 1 ml substrate solution in the first reaction step. Because of the change of sample volume, the enzyme activities are calculated from the following formula: activity (units/ml) = 27.4 (1.07 + 2.2{Delta}E) {Delta}E.

Plasma MET Determination.
The MET level in the plasma was measured by precolumn derivatization, followed by high-performance liquid chromatography separation (27) . Briefly, 10 µl plasma sample or MET standard were precipitated with 30 µl acetonitrile, followed by centrifugation at 10,000 rpm for 5 min. Ten µl of the supernatant were mixed with 5 µl of a fluoraldehyde derivative reagent, o-phthaldialdehyde, for 1 min at room temperature, followed by addition of 150 µl 0.1 M sodium acetate (pH 7.0). Twenty µl of the reaction mixture were loaded on a reversed-phase Supelcosil LC-18DB column (25 x 4.8-cm, particle size 5 µm; Supelco, Bellefonte, PA). The amino acid derivatives were separated by using a gradient elution of 40–60% solution B (methanol) in solution A [tetrahydrofuran/methanol/0.1 M sodium acetate (pH 7.2); 5/95/900] at a flow rate of 1.5 ml/min. A fluorescence spectrophotometer was used for detection with excitation at 350 nm and emission at 450 nm. The plasma methionine was identified by the retention time of a MET standard solution and quantitated according to a MET standard curve.

Determination of Plasma PLP.
PLP in plasma was determined based on the conversion of pyridoxal 5'-phosphate to 4-pyridoxic acid 5'-phosphate by cyanide in alkaline medium, followed by a high pressure liquid chromatographic separation (28) . All work was performed in a semi-darkened laboratory with low actinic lighting to avoid light-catalyzed destruction of the derivatives. Briefly, the diluted plasma sample and PLP standard solutions were mixed with an equal volume of trichloroacetic acid (100 g/liter) and incubated at 50°C in the dark for 5 min for removal of protein. After centrifugation at 10,000 rpm for 5 min, the supernates were taken and transferred to new vials. To 100 µl of the resulting supernatant, 35.5 µl dipotassium hydrogen phosphate (3.3 mM) were added, followed by 10 µl sodium cyanide (0.04 mM). The reaction mixture was kept at 50°C for 25 min in the dark, then 12.5 µl orthophosphoric acid (2.86 mM) were added. Fifty µl of the supernatant were loaded on a Waters Symmetry Shield RP8, 5µm, 4.6 x 250 mm column (Blackmoor Lane, Watford, United Kingdom). The column was eluted with water containing 75 mM semicarbazide and 50 mM potassium dihydrogen phosphate (pH 2.85) at a flow rate of 1.5 ml/min. A fluorescence spectrophotometer was used for detection with excitation at 325 nm and emission at 418 nm. The PLP peak was identified by the retention time of a PLP standard. The concentration of plasma PLP was calculated using a calibration curve.

Treatment of Mice with MEGC-PEG-rMETase.
BALB-C NCR nude mice ages 4 weeks (20–25g) were used for the study with five mice/group. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH National Research Council’s "Guide for the Care and Use of Laboratory Animals"5 under assurance number A3873-1. Osmotic pumps filled with 250 µl PLP at different concentrations ranging from 5 to 500 mg/ml were implanted s.c. as described above. Twelve h after pump implantation, MEGC-PEG-rMETase at a dose of 4000 or 8000 units/kg was i.v. injected via the tail vein into each mouse, respectively. Blood (200 µl) was collected from the retrorbital plexus of each animal using heparinized capillary tubes. Blood was collected before injection and 1, 8, 12, 24, 48, 72, and 96 h after injection. The plasma was separated and stored in small aliquots at –80°C. MEGC-PEG-rMETase activity, methionine concentration and PLP level in the plasma were measured as described above.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Characterization of MEGC-PEG-rMETase.
rMETase was PEGylated at a ratio of MEGC-PEG to rMETase of 60:1, which resulted in MEGC-PEG-rMETase molecules with a range of three to seven PEG chains/monomer of rMETase, specific enzyme activity, 44 units/mg (23) .

Dose Response of PLP Infusion on Plasma PLP Levels.
The activity of rMETase depends on stable binding of PLP to the protein to form the holoenzyme (29) . We have previously shown that PLP readily dissociates from MEGC-PEG-rMETase in vivo (23) . To increase the time of maintenance of the holoenzyme form of PEG-rMETase and MET-depletion time in vivo, PLP was continuously infused to MEGC-PEG-rMETase-treated mice using implanted miniosmotic pumps containing various concentrations of PLP. Plasma PLP levels were found to increase in a dose-dependent manner (Fig. 1)Citation . Peak plasma PLP concentrations in mice implanted with 5, 50, 100, 200, and 500 mg/ml PLP-containing pumps resulted in 7, 24, 34, 60, and 95 µM plasma concentrations, respectively, compared with a baseline of 0.3 µM. Pumps with 200 mg/ml PLP could sustain plasma PLP at ~20 µM for 24 h and 10 µM for 72 h. Pumps with 500 mg/ml PLP could sustain plasma PLP levels at ~40 µM for 48 h and 10 µM for 96 h (Fig. 1)Citation .



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Dose-dependent efficacy of PLP on plasma PLP concentration. Osmotic pumps filled with PLP ranging from 5 to 500 mg/ml were implanted s.c. 12 h before MEGC-PEG-rMETase was i.v. injected in mice. Blood samples were collected at different time points and measured for plasma PLP concentration as described in "Materials and Methods."

 
Dose Response of PLP Infusion on MEGC-PEG-rMETase Holoenzyme Half-Life.
MEGC-PEG-rMETase holoenzyme activity is measured in vitro without addition of PLP in the enzyme assay solution. PEG-rMETase holoenzyme activity was markedly dependent on the dose of PLP infused in vivo (Table 1Citation , Fig. 2ACitation ). MEGC-PEG-rMETase holoenzyme half-life was 1.5 h without PLP infusion, 2.5 h with 5 mg/ml PLP pumps, and ranged to 7 h with 500 mg/ml PLP pumps (Table 1)Citation , a 4.5-fold increase compared with the PEG-rMETase half-life without PLP supplementation. A high degree of correlation of PLP concentration in the osmotic pumps and MEGC-PEG-rMETase holoenzyme half-life was found up to 200 µm PLP (r2 = 0.96; Table 1Citation , Fig. 2ACitation ). In contrast, the half-life of PEG-rMETase apoenzyme, measured in the presence of PLP in vitro, was ~18 h in animals with or without PLP infusion (Table 1Citation , Fig. 2BCitation ). Thus, MEGC-PEG-rMETase holoenzyme half-life was highly dependent on the dose of PLP in contrast to apoenzyme half-life (Fig. 2A)Citation .


View this table:
[in this window]
[in a new window]

  		Table 1 Dose-dependent efficacy of PLP on plasma half-life of MEGC-PEG-rMETase

 


View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Dose-dependent efficacy of PLP on plasma half-life of MEGC-PEG-rMETase holoenzyme (A) and apoenzyme (B) half-life. Osmotic pumps filled with PLP ranging from 5 to 500 mg/ml were implanted s.c. MEGC-PEG-rMETase at a dose of 4000 units/kg was i.v. injected into each mouse 12 h after pump implantation. Blood samples were collected at different time points and measured for PEG-rMETase holoenzyme (A) and apoenzyme (B) activity as described in "Materials and Methods."

 
Dose Response of PLP Infusion on Time of MET Depletion by PEG-rMETase.
MEGC-PEG-rMETase holoenzyme half-life highly correlated with time of MET depletion, both of which depend on the dose of infused PLP (Table 2Citation , Fig. 3, A and BCitation ). PLP-supplemented mice were injected with a single i.v. injection of 4000 or 8000 units/kg PEG-rMETase. Blood samples were collected at various time points and measured for plasma MET concentration. Four-thousand units/kg MEGC-PEG-rMETase administration in the mice infused with 5, 50, 200, and 500 mg/ml PLP-containing pumps resulted in plasma MET depletion to ≤5 µM for 8, 24, 72, and 72 h, respectively (Table 2Citation , Fig. 3ACitation ), demonstrating a strong PLP dose dependence of MET depletion. The mice receiving 8000 units/kg PEG-rMETase and the same varying amounts of PLP showed a similar plasma MET depletion time course (Table 2Citation , Fig. 3BCitation ). However, mice given 8000 units/kg MEGC-PEG-rMETase supplemented with 500 mg/ml PLP had no detectable levels of MET for 72 h (Fig. 3B)Citation . The efficacy of PLP to extend the time of MEGC-PEG-rMETase-mediated MET depletion to ≤ 5 µM was linearly dose dependent on PLP up to 200 mg/ml in the osmotic pump (r2 = 0.99; Fig. 4Citation ). Five-hundred mg/ml PLP-containing pumps did not further extend the time of MET depletion.


View this table:
[in this window]
[in a new window]

  		Table 2 Dose-dependent efficacy of PLP on time of plasma MET depletion by MEGC-PEG- rMETase

 


View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Time and extent of plasma MET depletion by MEGC-PEG-rMETase at various doses of PLP infusion. Osmotic pumps filled with PLP ranging from 5 to 500 mg/ml were implanted s.c. PEG-rMETase at a dose of 4000 units/kg (A) or 8000 units/kg (B) was i.v. injected into each mouse 12 h after pump implantation. Blood samples were collected at different time points and measured for plasma MET level as described in "Materials and Methods."

 


View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Dose-dependent efficacy of PLP on time of plasma MET depletion by MEGC-PEGrMETase. Osmotic pumps filled with PLP ranging from 5 to 500 mg/ml were implanted s.c. MEGC-PEG-rMETase at a dose of 4000 units/kg was i.v. injected in each mouse 12 h after pump implantation. Blood samples were collected at different time points and measured for plasma MET level as described in "Materials and Methods."

 
This study indicated that PLP supplementation in vivo greatly extended the half-life of MEGC-PEG-rMETase holoenzyme, which in turn greatly extended the time of MET depletion by MEGC-PEG-rMETase in a dose-dependent manner. PLP extended the half-life of MEGC-PEG-rMETase holoenzyme ~4–5-fold in a dose-dependent manner, which resulted in a ~10-fold increase in time of plasma MET depletion to ≤5 µm. PLP appeared to either associate with the MEGC-PEG-rMETase apoenzyme or prevent PLP dissociation from the holoenzyme in the plasma. Although PLP remains tightly bound to the enzyme in vitro, it readily dissociates from the enzyme in vivo. In a recent study with macaque monkeys, MEGC-PEG-rMETase had an extended apoenzyme half-life of 90-143 h,6 but holoenzyme half-life was only 1.5 h. The results of the present study in mice suggest that PLP would greatly extend holoenzyme half-life in primates as well as humans. The combination of MEGC-PEG-rMETase treatment with PLP supplementation suggests an effective clinical strategy for long-term MET depletion to arrest cancer cell growth.


   FOOTNOTES
 
Grant support: United States National Cancer Institute Grant 1 R43 CA86166.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Robert M. Hoffman, AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111. Phone: (858) 654-2555; Fax: (858) 268-4175; E-mail: all{at}anticancer.com

5 Internet address: http://oacu.od.nih.gov/regs/guide/guidex.htm. Back

6 Z. Yang, J. Wang, Q. Lu, J. Xu, Y. Kobayashi, T. Takura, A. Takimoto, T. Yoshioka, C. Lian, C. Chen, D. Zhang, Y. Zhang, S. Li, X. Sun, Y. Tan, S. Yagi, E. P. Frenkel, and R. M. Hoffman, unpublished data. Back

Received 4/23/04. Accepted 5/17/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Mecham JO, Rowitch D, Wallace CD, Stern PH, Hoffman RM. The metabolic defect of methionine dependence occurs frequently in human tumor cell lines. Biochem Biophys Res Commun, 117: 429-34, 1983.[CrossRef][Medline]
  2. Hoffman RM, Erbe RW. High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine. Proc Natl Acad Sci USA, 73: 1523-7, 1976.[Abstract/Free Full Text]
  3. Hoffman RM. Altered methionine metabolism, DNA methylation, and oncogene expression in carcinogenesis: a review and synthesis. Biochim Biophys Acta, 738: 49-87, 1984.[Medline]
  4. Guo H, Herrera H, Groce A, Hoffman RM. Expression of the biochemical defect of methionine dependence in fresh patient tumors in primary histoculture. Cancer Res, 53: 2479-83, 1993.[Abstract/Free Full Text]
  5. Tisdale M, Eridani S. Methionine requirement of normal and leukaemic haemopoietic cells in short-term cultures. Leuk Res, 5: 385-94, 1981.[CrossRef][Medline]
  6. Lishko VK, Lishko OV, Hoffman RM. The preparation of endotoxin-free-L-methionine alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Pseudomonas putida. Protein Expr Purif, 4: 529-33, 1993.[CrossRef][Medline]
  7. Lishko VK, Lishko OV, Hoffman RM. Depletion of serum methionine by methioninase in mice. Anticancer Res, 13: 1465-8, 1993.[Medline]
  8. Hori H, Takabayashi K, Orvis L, Carson DA, Nobori T. Gene cloning and characterization of Pseudomonas putida L-methionine alpha-deamino-gamma-mercaptomethane-lyase. Cancer Res, 56: 2116-22, 1996.[Abstract/Free Full Text]
  9. Tan Y, Xu M, Tan XZ, et al Overexpression and large-scale production of recombinant L-methionine-{alpha}-deamine-{gamma}-mercaptomethane-lyase for novel anticancer therapy. Protein Expr Purif, 9: 233-45, 1997.[CrossRef][Medline]
  10. Tan Y, Sun X, Xu M, et al Efficacy of recombinant methioninase in combination with cisplatin on human colon tumors in nude mice. Clin Cancer Res, 5: 2157-63, 1999.[Abstract/Free Full Text]
  11. Yoshioka T, Wada T, Uchida N, et al Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil and safety of recombinant methioninase. Cancer Res, 58: 2583-7, 1998.[Abstract/Free Full Text]
  12. Kokkinakis DM, Schold SC, Jr, Hori H, Nobori T. Effect of long-term depletion of plasma methionine on the growth and survival of human brain tumor xenografts in athymic mice. Nutr Cancer, 29: 195-204, 1997.[Medline]
  13. Kokkinakis DM, Wick JB, Zhou Q-X. Metabolic response of normal and malignant tissue to acute and chronic methionine stress in athymic mice bearing human glial tumor xenografts. Chem Res Toxicol, 15: 1472-9, 2002.[CrossRef][Medline]
  14. Tan Y, Zavala J, Sr, Xu M, Zavala J, Jr, Hoffman RM. Serum methionine depletion without side effects by methioninase in metastatic breast cancer patients. Anticancer Res, 16: 3937-42, 1996.[Medline]
  15. Tan Y, Zavala J, Sr, Han Q, et al Recombinant methioninase infusion reduces the biochemical endpoint of serum methionine with minimal toxicity in high-stage cancer patients. Anticancer Res, 17: 3857-60, 1997.[Medline]
  16. Yang Z, Wang J, Yoshioka T, et al Pharmacokinetics, methionine depletion, and antigenicity of recombinant methioninase in primates. Clin Cancer Res, 10: 2131-8, 2004.[Abstract/Free Full Text]
  17. Kozlowski A, Harris JM. Improvements in protein PEGylation: pegylated interferons for treatment of hepatitis C. J Control Release, 72: 217-24, 2001.[CrossRef][Medline]
  18. Maeda H Kabanov A Kataska K Okano T eds. . Advances in experimental medicine and biology: polymer drugs in the clinical stage, Vol. 519: Kluwer Academic/Plenum Publishers Dordrecht, the Netherlands 2003.
  19. Gilbert CW, Park C, Myung-ok, inventors (Enzon, Inc., assignee). Interferon polymer conjugates. United States patent US 5951974. 1999 Sept 14.
  20. Aguayo A, Cortes J, Thomas D, et al Combination therapy with methotrexate, vincristine, polyethylene-glycol conjugated-asparaginase, and prednisone in the treatment of patients with refractory or recurrent acute lymphoblastic leukemia. Cancer, 86: 1203-9, 1999.[CrossRef][Medline]
  21. Pool R. "Hairy enzymes" stay in the blood. Science (Wash. DC), 248: 305 1990.[Free Full Text]
  22. Hershfield MS. PEG-ADA replacement therapy for adenosine deaminase deficiency: an update after 8.5 years. Clin Immunol Immunopathol, 76: S228-32, 1995.[CrossRef][Medline]
  23. Sun X, Yang Z, Li S, et al In vivo efficacy of recombinant methioninase is enhanced by the combination of polyethylene glycol conjugation and pyridoxal 5'-phosphate supplementation. Cancer Res, 63: 8377-83, 2003.[Abstract/Free Full Text]
  24. Watanabe N, Kamei S, Ohkubo A, et al Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer. Clin Chem, 32: 1551-4, 1986.[Abstract/Free Full Text]
  25. Li S, Yang Z, Sun X, Tan Y, Yagi S, Hoffman RM. A simultaneous colorimetric assay of free and protein-coupled polyethylene glycol. Anal Biochem, 313: 335-7, 2003.[CrossRef][Medline]
  26. Esaki N, Soda K. L-Methionine gamma-lyase from Pseudomonas putida and Aeromonas. Methods Enzymol, 143: 459-65, 1987.[Medline]
  27. Jones BN, Gilligan JP. o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J Chromatogr, 266: 471-82, 1983.[CrossRef][Medline]
  28. Deitrick CL, Katholi RE, Huddleston DJ, Hardiek K, Burrus L. Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5'-phosphate in human plasma. J Chromatogr B Biomed Sci Appl, 751: 383-7, 2001.[CrossRef][Medline]
  29. Han Q, Xu M, Tang L, et al Homogeneous nonradioactive enzymatic assay for plasma pyridoxal 5-phosphate. Clin Chem, 48: 1560-4, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J BiochemHome page
D. Kudou, S. Misaki, M. Yamashita, T. Tamura, T. Takakura, T. Yoshioka, S. Yagi, R. M Hoffman, A. Takimoto, N. Esaki, et al.
Structure of the Antitumour Enzyme L-Methionine {gamma}-Lyase from Pseudomonas putida at 1.8 A Resolution
J. Biochem., April 1, 2007; 141(4): 535 - 544.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Takakura, A. Takimoto, Y. Notsu, H. Yoshida, T. Ito, H. Nagatome, M. Ohno, Y. Kobayashi, T. Yoshioka, K. Inagaki, et al.
Physicochemical and Pharmacokinetic Characterization of Highly Potent Recombinant L-Methionine {gamma}-Lyase Conjugated with Polyethylene Glycol as an Antitumor Agent.
Cancer Res., March 1, 2006; 66(5): 2807 - 2814.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, Z.
Right arrow Articles by Hoffman, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, Z.
Right arrow Articles by Hoffman, R. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online