Title: Depletion method of
blood plasma ascorbate
United States Patent: 6,989,143
Issued: January 24, 2006
Park; Chan Hyung (611 S. 291st St., Federal Way, WA 98003);
Boo; Yong C. (Birmingham, AL)
Assignee: Jin Yang Pharm Co., Ltd. (Seoul,
KR); Park; Chan Hyung (Federal Way, WA)
Appl. No.: 275195
Filed: May 2, 2000
PCT Filed: May 2, 2000
PCT NO: PCT/KR00/00415
371 Date: October 31, 2002
102(e) Date: October 31, 2002
PCT PUB.NO.: WO01/82955
PCT PUB. Date: November 8, 2001
George Washington University's Healthcare MBA
Artificial modulation of ascorbate level
was investigated in mice capable of de novo synthesis of ascorbate. When
mice were given exogenous ascorbate or its physiological precursor, L-gulono-γ-lactone,
the plasma level of ascorbate was elevated substantially but immediately
returned to the basal levels. Comparably, the administration of ascorbate
oxidase caused a rapid disappearance of plasma ascorbate but followed by
an immediate restoration of ascorbate. These results indicate the
difficulties encountered in the modulation of ascorbate level in the
animal. However, the circulation life of the exogenous ascorbate oxidase
in the animal was successfully extended by chemical modification with
methoxypolyethylene glycol. The modified enzyme retained a full activity
and exerted a remarkably prolonged depletion of plasma ascorbate compared
with the native enzyme. This study suggests that the chemically modified
ascorbate oxidase should find many uses in the animal studies on ascorbate
since it was found to deplete plasma ascorbate even in the ascorbate-synthesizing
animal in the absence of dietary control. The enzyme should prove to be
useful in tumor control because there are tumor systems in mice and man
amenable to the manipulation of ascorbate level.
DISCLOSURE OF THE
It is clear that ascorbate has effects on
many physiological processes in humans . Insufficient intake of
ascorbate causes scurvy associated with decreased collagen synthesis.
Ascorbate influences on wound healing, gastric iron uptake, and many
immunological and biochemical reactions. Apparently, most, if not all, of
ascorbate's functions are related to the reducing property of ascorbate
keeping metal ions in reduced state. As a reductant, ascorbate can act
either as an antioxidant or as a prooxidant in aqueous environments .
However, ascorbate is generally designated as an important antioxidant for
humans based on many in vitro findings [3-5]. Further, it is widely held
that ascorbate may contribute to the prevention of pathological processes
associated with oxidative injury, but only circumstantial evidence is
available [6, 7].
Ascorbate has also been implicated to affect the growth of animal cells in
vitro [8-11]. The effect appears as either stimulatory or inhibitory
depending on the cells. Although the precise mechanism for the growth
modulation by ascorbate is not known, the effect has been proven to be
biological rather than physicochemical since optical isomers of ascorbate
or other redox compounds are without effect. Further, it is worthy to note
that malignant cells are more sensitive than normal counterparts to the
ascorbate effects. We have shown the unusual ascorbate sensitivity of
malignant cells from patients with acute myelogenous leukemia and
myelodysplastic syndromes [12, 13].
There are compelling evidences that this in vitro effect will translate to
in vivo situation ultimately resulting in tumor control. As explained
fully in the Description of the Prior Art, both extreme depletion and
supplementation will be beneficial to the control of cancer. Therefore the
depletion and supplementation periods will need to be cycled. One step
further, for depletion to be maximumly effective prior supplementation
period will be desirable and vice versa. In this Application one of these
2 extremes, depletion, is the subject.
Animal studies are critical for the demonstration of the physiological
relevance of many proposed functions of ascorbate. For the experimental
purpose, ascorbate level in animals needs to be modulated artificially but
dietary controls are not so promising . Ascorbate administered in
excess is readily excreted from the body and only minor fraction is
equilibrated with body stores of ascorbate. Furthermore, such laboratory
animals as mice are capable of endogenously synthesizing ascorbate, and by
nature they are insensitive to dietary intake of ascorbate. The importance
of animal studies on ascorbate modulation as related to tumor control, and
the usefulness of mice as laboratory animals prompted us to investigate
new methods of ascorbate modulation in mice. Two opposite methods are
investigated in the present study, one for the elevation of ascorbate
level and another for the selective depletion of ascorbate.
MODES FOR CARRYING
OUT THE INVENTION
Materials and Methods
Materials used in this invention are as follows.
Sodium ascorbate, L-gulono-γ-lactone, ascorbate oxidase (from Curcubita
species), methoxypolyethylene glycol (mPEG) activated with cyanuric
chloride (average molecular weight 5000), borax, sodium citrate,
mataphosphoric acid and dithiothreitol were purchased from SIGMA (St.
Louis, Mo.). All other chemicals are of reagent grade.
Chemical modification of ascorbate oxidase is as follows.
Ascorbate oxidase (EC 126.96.36.199)) was modified with MPEG activated with
cyanuric chloride, as in Jackson et al. . Briefly describing, 4 mg of
ascorbate oxidase was dissolved in 10 ml of ice-cold 0.1 M borax buffer
(pH 9.2) and then 200 mg of activated mPEG was directly added to the
enzyme solution with stirring on ice. The reaction mixture was kept on ice
for 60 min and dialyzed against normal saline at 4° C. across a
Spectrapor-2 membrane (SPECTRUM. Houston, Tex.). The dialyzed protein,
i.e. pegylated ascorbate oxidase, was used immediately or stored at -60°
C. until used.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed on 6% gel prepared by Laemmli's formulation  and proteins
were stained with Coomassie Blue. Ascorbate oxidase activity was assayed
spectrophotometrically, as in Kim et al. .
Animals used in this invention are as follows.
Specific pathogen-free female BALB/c mice (body weight, ca. 25 g) were
used in the present study. Animals were obtained from Dae-Han Laboratory
Animal Center (Daejeon, Korea) and cared for according to the ILAR
guidelines in the animal care facility of Samsung Biomedical Research
Center (Seoul, Korea). They were fed a diet from Jell Laboratory Chow
(Seoul, Korea). Feed and water were offered ad libitum.
Extraction and assay of ascorbate are as follows.
The animal was anesthetized with diethyl ether vapour and blood was
obtained by choroidal venous plexus. About one ml of blood was collected
into a microcentrifuge tube that contained 50 μl of 130 mM sodium citrate
as an anticoagulant. The blood was centrifuged at 1000×g for 10 min to
obtain plasma, which was rapidly cooled on ice. Equal volume of ice-cold
10% metaphosphoric acid was added to the plasma and the mixture was
centrifuged at 10000×g for 10 min. The ascorbate content in the
supernatant was measured directly by HPLC method. An aliquot of the sample
was treated with 100 mM dithiothreitol at neutral pH to reduce
dehydroascorbate to ascorbate, and then total ascorbate content was
measured. The content of dehydroascorbate was estimated from the
difference between the two measurements. In some cases, blood plasma was
directly ultrafiltrated by using a Microcon YM-10 centrifugal filter
device (MILLIPORE, Bedford, Mass.), as described by Mendiratta et al. .
The ultrafiltrate was injected to HPLC either directly for the assay of
ascorbate, or after incubation with dithiothreitol for the assay of total
ascorbate (ascorbate plus dehydroascorbate).
Ascorbate was assayed by HPLC with the electrochemical detection, as
previously described . HPLC system (SHIMADZU, Tokyo, Japan) consisted
of CBM-10A communication module, LC-10AD pump, SIL-10A auto injector,
CTO-10A column oven, and L-ECD-6A electrochemical detector. Separation was
carried out on a Shim-pack CLC-ODS column (6 mm×15 cm) with a guard column
of the same material (4 mm×1 cm). Mobile phase was 50 mM potassium
phosphate (pH 2.5) containing 1 mM EDTA, eluted at a flow rate of 1.0
ml/min. The potential of the glassy carbon working electrode was set at
+0.6 V versus an Ag/AgCl reference electrode.
Data analysis is as follows. Statistical software package StatView 4.0
(Abacus concepts, Berkeley, Calif.) was used for the data analysis.
Results and Discussion
Ascorbate is synthesized from glucose via the hexuronic acid pathway in
ascorbate synthesizing animals . L-Gulono-γ-lactone is an immediate
precursor of ascorbate and its conversion into ascorbate is catalyzed by
L-gulono-γ-lactone oxidase . Human's inability to synthesize ascorbate
is due to the lack of this enzyme . As a physiological precursor of
ascorbate, L-gulono-γ-lactone might be useful for the elevation of
ascorbate level in ascorbate-synthesizing animals including mice. This
notion was subjected to experimental scrutiny in the present study (FIG.
1). When mice were given 5 mmole/kg of L-gulono-γ-lactone, there was a
substantial increase in plasma level of ascorbate, in agreement with the
capacity of the animal to synthesize ascorbate. However, the accumulated
ascorbate rapidly diminished to normal levels within a few hours,
similarly to the directly administered ascorbate (as sodium salt, 5 mmole/kg),
implicating that the animal has the capacity to keep ascorbate level from
abnormal elevation. Plausible mechanisms may include the renal excretion
and metabolic consumption of excessive ascorbate [14, 21], and inhibition
of L-gulono-γ-lactone oxidase by ascorbate . All these mechanisms
would be beneficial for the life of mice, but due to these mechanisms, we
have hardly a chance to investigate the physiological consequences of
ascorbate elevation in the laboratory animal.
Ascorbate oxidase is an enzyme that catalyzes the oxygen-dependent
oxidation of ascorbate to dehydroascorbate . Due to the high substrate
specificity and catalytic efficiency, the enzyme has widely been used in
numerous in vitro studies on ascorbate, including selective determination
of ascorbate . However, its compatibility with animal systems is
unknown. The enzyme was tested as a drug for the selective depletion of
ascorbate in the present study. As shown in FIG. 2, the administration of
ascorbate oxidase into mice lowered plasma level of ascorbate in a
dose-dependent manner. The ascorbate depletion was not observed when the
enzyme was heat-inactivated (data not shown). These results implicate a
competition in the blood between the activity of exogenous ascorbate
oxidase and the ascorbate maintenance mechanisms, such as ascorbate
regeneration from dehydroascorbate by red blood cells [5, 25] and
ascorbate release from the liver where de novo synthesis of ascorbate
takes place . The minimal dose of ascorbate oxidase inducing a total
clearance of plasma ascorbate appeared to be about 100 units/kg equivalent
to 1 μg protein/animal, indicating the effectiveness of ascorbate oxidase
in the blood. Time-sequential monitoring of the ascorbate level in mice
given 400 units/kg of ascorbate oxidase, however, revealed a rapid
restoration of plasma ascorbate, probably associated with short
circulation life of the exogenous enzyme.
Enzymes can be chemically modified with retention of activities by
attaching biologically inert polymers at sites other than the active site.
Such chemical modifications have been shown to prolong the circulation
life of the enzymes in animals, by eliminating immunogenicity of native
proteins and/or reducing the renal excretion [26, 27]. mPEGs are very
useful polymers in this respect and readily can be attached to proteins by
use of cyanuric chloride as a coupling agent . Ascorbate oxidase could
be chemically modified with cyanuric chloride-activated mPEG in the
present study. A far excessive amount of the activated mPEG (1000
molecules per one enzyme molecule) was used to ensure the reaction. The
attachment of the polymers to the enzyme was verified through SDS-PAGE
(FIG. 3). The modified ascorbate oxidase exhibited a very slow mobility on
6% gel compared with the unmodified. The former appeared as a broad band
close to the stacking gel, whereas the latter occurred as a sharp band
with apparent molecular weight of 70 kDa corresponding to the subunit
. Enzyme activity loss by the modification was very slight (less than
To assess the influence of chemical modification, mice were given either
native ascorbate oxidase or the modified enzyme, both at 400 unit/kg, and
plasma level of ascorbate was monitored time-sequentially. Drugs were
administered via intraperitoneal injections that were found effective as
intravenous routes. As shown in FIG. 4, the modified enzyme exerted a
rapid clearance of plasma ascorbate as the native enzyme, in line with its
retention of enzyme activity. Furthermore, the duration of ascorbate
depletion was remarkably prolonged; the modified enzyme depleted for about
120 h compared with 3-4 h for the native enzyme. These results clearly
demonstrate the benefit of chemical modification to extend the circulation
life and the action time of the exogenous enzyme in animals.
Ascorbate is just oxidized to dehydroascorbate by the activity of
ascorbate oxidase. Nonetheless, any trace amount of dehydroascorbate was
not detected in plasma during the ascorbate depletion (data not shown). At
least two different methods of sample preparation employed in the present
study gave the same results. The results are not surprising, however,
since dehydroascorbate is known to be very unstable in plasma .
Although further studies are required, it is tentatively inferred that the
modified ascorbate oxidase may lead a rather comprehensive depletion of
plasma ascorbate including its oxidized form, by steadily oxidizing
available ascorbate to dehydroascorbate which decomposes subsequently.
In summary, the present paper describes two different approaches to attain
the purpose of ascorbate modulation in mice capable of de novo synthesis
of ascorbate. In the first trial, we tested the administration of L-gulono-γ-lactone
as a physiological precursor of ascorbate to elevate ascorbate level, but
no significant merits over direct ascorbate supplementation were observed.
However, in the following trial, the exogenous ascorbate oxidase was found
very effective for the depletion of plasma ascorbate. The catalytic
efficiency of the enzyme appeared to compete readily with the ascorbate
synthesis and/or recycling mechanisms in animals. Employing a biologically
inert polymer to the enzyme, we could surmount the problem associated with
the short circulation life of the exogenous enzyme. The chemical
modification of enzyme did not require additional risks such as severe
loss of enzyme activity or toxicity to animals. The modified ascorbate
oxidase should find numerous uses in vivo researches on ascorbate.
Notably, it seems likely to be used in animal studies to demonstrate
physiological relevancy of many in vitro findings such as antioxidant
actions of ascorbate [3-5]. The most straight-forward application will be
the growth retardation of malignant cells sensitive to ascorbate[8,
11-13], ultimately leading to the control of malignancies.
Claim 1 of 3 Claims
1. A method for depleting
ascorbate in blood plasma extracted from a living body, by using ascorbate
oxidase modified with biologically inert polymers.
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