U.S. patent application number 09/823445 was filed with the patent office on 2001-12-20 for method for increasing the concentration of ascorbic acid in brain tissue of a subject.
This patent application is currently assigned to Sloan-Kettering Institute For Cancer Research. Invention is credited to Agus, David B., Golde, David W., Vera, Juan C..
Application Number | 20010053793 09/823445 |
Document ID | / |
Family ID | 26724830 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053793 |
Kind Code |
A1 |
Agus, David B. ; et
al. |
December 20, 2001 |
Method for increasing the concentration of ascorbic acid in brain
tissue of a subject
Abstract
This invention provides a method for increasing the ascorbic
acid concentration in brain tissues of a subject which comprises
administering to the subject an amount of dehydroascorbic acid
effective to increase the concentration of ascorbic acid in brain
tissues. This invention also provides that the dehydroascorbic acid
enters the tissues through the facilitative glucose
transporter.
Inventors: |
Agus, David B.; (Brooklyn,
NY) ; Vera, Juan C.; (New York, NY) ; Golde,
David W.; (New York, NY) |
Correspondence
Address: |
John P. White
Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Sloan-Kettering Institute For
Cancer Research
|
Family ID: |
26724830 |
Appl. No.: |
09/823445 |
Filed: |
March 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09823445 |
Mar 30, 2001 |
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09443785 |
Nov 19, 1999 |
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6221904 |
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09443785 |
Nov 19, 1999 |
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PCT/US98/10608 |
May 21, 1998 |
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60067185 |
Dec 1, 1997 |
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60047271 |
May 21, 1997 |
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Current U.S.
Class: |
514/474 |
Current CPC
Class: |
A61K 31/375 20130101;
A61P 43/00 20180101; A61K 31/34 20130101; A61P 25/16 20180101; A61K
31/34 20130101; A61P 25/28 20180101; A61K 45/06 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61P 25/00 20180101; A61K
31/375 20130101 |
Class at
Publication: |
514/474 |
International
Class: |
A61K 031/375 |
Goverment Interests
[0002] This invention was made with support under United States
Government Grant No. RO1 CA30388 and RO1 HL42107. Accordingly, the
United States Government has certain rights in the invention.
Claims
What is claimed is:
1. A method for increasing the ascorbic acid concentration in brain
tissues of a subject which comprises administering to the subject
an amount of dehydroascorbic acid effective to increase the
concentration of ascorbic acid in brain tissues.
2. The method of claim 1, wherein the dehydroascorbic acid enters
the brain tissues through the facilitative glucose transporter.
3. A method for increasing the ascorbic acid concentration in brain
tissues of a subject which comprises administering to the subject
an amount of dehydroascorbic acid effective to increase the
antioxidant potential of the brain tissues.
4. The method of claim 1, wherein the subject is a human.
5. The method of claim 1, wherein the human subject has
neurodegenerative disease.
6. The method of claim 5, wherein the neurodegenerative disease is
Alzheimer's Disease or Parkinson's Disease.
7. The method of claim 1, wherein the human subject has
neurovascular disease.
8. The method of claim 7, wherein the human subject has stroke.
9. The method of claim 1, wherein the human subject has diseases
which involve the oxidative modification of low-density lipoprotein
or lipid perioxidation.
10. The method of claim 9, wherein the human subject has stroke,
atherosclerosis or neurodegenerative disorders.
11. The method of claim 1, wherein the human subject has a
behavioral disorder.
12. The method of claim 11, wherein the behavioral disorder is
dysthymia, involution depression, aggressiveness via dominance,
hyperactivity, deprivation syndrome, separation anxiety,
intermittent anxiety, instrumental sociopathy, stereotypies, phobia
or socialization disorders.
13. The method of claim 1, wherein the dehydroascorbic acid is
administered orally, intravenously, subcutaneously or
intramuscularly.
14. A method for treating or preventing dementia of a subject
comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the concentration of
ascorbic acid in brain tissues.
15. A method for treating or preventing dementia of a subject
comprising administering to the subject an effective amount of
dehydroascorbic acid to increase the antioxidant potential of brain
tissues.
16. A method for treating or preventing neurodegenerative disease
of a subject comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the concentration of
ascorbic acid in brain tissues.
17. A method for treating or preventing neurodegenerative disease
of a subject comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the antioxidant
potential of the brain tissues.
18. The method of claim 16 or 17, wherein the neurodegenerative
disease is Alzheimer's Disease or Parkinson's Disease.
19. A method for treating or preventing stroke or neurovascular
disease of a subject comprising administering to the subject an
amount of dehydroascorbic acid effective to increase the
concentration of ascorbic acid in brain tissues.
20. A method for treating or preventing stroke or neurovascular
disease of a subject comprising administering to the subject an
amount of dehydroascorbic acid effective to increase the
antioxidant potential of the brain tissues.
21. A method for treating or preventing diseases which involve the
oxidative modification of low-density lipoprotein or lipid
perioxidation of a subject comprising administering to the subject
an amount of dehydroascorbic acid effective to increase the
concentration of ascorbic acid in brain tissues.
22. A method for treating or preventing a behavioral disorder of a
subject comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the concentration of
ascorbic acid in brain tissues.
23. A method for treating or preventing a behavioral disorder of a
subject comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the antioxidant
potential of the brain tissues.
24. The method of claim 22 or 23, wherein the behavioral disorder
is dysthymia, involution depression, aggressiveness via dominance,
hyperactivity, deprivation syndrome, separation anxiety,
intermittent anxiety, instrumental sociopathy, stereotypies, phobia
or socialization disorders.
25. The method of claim 14, 15, 16, 17, 19, 20, 21, 22, or 23
further comprising administering an effective amount of a
therapeutic agent.
Description
[0001] This application claims the benefit of U.S. Provisional
Applicaion No. 60/067,185, filed Dec. 1, 1997 and No. 60/047,271,
filed May 21, 1997, the contents of which are hereby incorporated
by reference.
[0003] Throughout this application, various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end the specification, preceding the
claims.
BACKGROUND OF THE INVENTION
[0004] Numerous connections have been made between the generation
and presence of oxidative free radicals in brain tissue and
neurological disorders. For example, 1) Jenner (26) links oxidative
stress to Parkinson's, Alzheimer's and Huntington's diseases. 2)
Recent clinical studies have demonstrated that alpha-tocopherol
(vitamin E) and selegiline (deprenyl), pharmacologic agents that
have antioxidant activity, can slow the progression of moderately
severe Alzheimer's disease (27). 3) Antioxidants such as vitamin C
and vitamin E may have an important role in the treatment of
diseases whose pathogenesis involves free radical formation and
impaired antioxidant defenses in the aging population. Oxidative
damage has been hypothesized as central to the neurodegenerative
processes such as Alzheimer's disease (28). According to the free
radical hypothesis, Alzheimer disease is an acceleration of the
normal aging process in affected brain regions which become
progressively more damaged by free radicals generated from
metabolism. In Alzheimer's disease, the cerebral cortex seems to
have increased antioxidant requirements, increased sensitivity to
free radicals, and levels of the free radical defense enzymes, such
as superoxide dismutase, that are reduced by 25-35% in the frontal
cortex and hippocampus. The loss of hippocampal cholinergic neurons
is a key feature of Alzheimer's disease and these neurons seem
particularly vulnerable to the deleterious effects of free radicals
on the muscarinic cholinergic receptor (29). 4) Antioxidants have
been tested as drugs for Parkinson's disease (30), and it was found
that selegiline, which may act as an antioxidant since it inhibits
oxidative deamination, delays the onset of the disability (31). 5)
Peyser et al. concluded that antioxidant therapy may slow the rate
of motor decline early in the course of Huntington's disease (35).
6) According to Challem (32) free radicals and oxidative stress may
be factors involved with the pathogenesis of Mad Cow disease. 7)
The oxidative modification of low-density lipoprotein (LDL), termed
lipid perioxidation has been shown to be an initiating event in
atherosclerosis. Probucol, an antioxidant, is effective in reducing
the rate of restenosis after balloon coronary angioplasty (36).
Oxidized LDL has several detrimental effects on cells including
brain cells such as cytotoxicity and vascular dysfunction.
[0005] Therefore, increasing the concentration of free-radical
scavengers or antioxidants in brain tissue may provide therapeutic
benefits to subjects suffering from neurodegenerative diseases.
Sano et al. conclude (27) that the use of the antioxidants,
selegiline or vitamin E may delay clinically important functional
deterioration in patients with Alzheimer's disease. Their results
are particularly significant because vitamin E does not cross the
blood-brain barrier in large amounts, and still it has a measurable
effect.
[0006] The enhancement of the antioxidant potential is useful in
treating of many diseases. For example, the increase of antioxidant
potential achieved by this invention will be able to treat stroke
and neurovascular diseases. It is known that ischemic stroke is the
most common neurologic disorder causing death or disability among
adults. Strokes of all types rank third as a cause of death,
surpassed only by heart disease and cancer. Ischemic stroke events
account for approximately 85% of all strokes. Because no medical or
surgical treatment has yet been established as reversing the
effects of acute ischemic stroke, early identification and
treatment of persons at the time they present with stroke is
compelling, if such a treatment is efficacious. Currently, there
are no approved treatments for stroke. The damage from stroke is
caused by occlusion of a vessel, thereby restricting the delivery
of oxygen in the blood to an area of the brain. Much of the damage
is caused by damage from oxygen free radicals in the area served by
the occluded vessel after reperfusion of the affected area (37).
Thus, increasing the antioxidant potential of the brain may have
beneficial effect on stroke and other neurovascular diseases.
[0007] Therefore, increasing vitamin C concentrations in the brain
by providing dehydroascorbic acid to the subject could enhance
antioxidant potential in the central nervous system and may be
therapeutic in stroke and neurovascular diseases as described.
[0008] Researchers have proposed that atherosclerosis, and its
deadly effects of heart attack and stroke, develops in relationship
to oxidation of low-density lipoproteins (LDL) carrying cholesterol
in the blood. The theory states that free radicals generated by the
body's own immune cells oxidize LDL which is taken up by cells of
the vascular intima initiating the atherosclerosis lesion.
Ultraviolet and gamma radiation, cigarette smoke and other
environmental pollutants, also cause oxidative damage to cells and
vital compounds. The damage leads to the development of several
chronic diseases including cancer and coronary heart disease (CHD).
It was further proposed that antioxidants such as vitamin E and C
and the carotenoids could prevent damage and the ensuing diseases.
Many epidemiologic and animal studies have offered evidence to
support the theory (33, 34). Recent studies demonstrated that the
antioxidant proburol is effective in reducing the rate of
restenosis after balloon coronary angioplasty (36).
[0009] Evidence suggests that the neuropathology of Huntington's
disease, a neuropsychiatric disorder, results from excessive
activation of glutamate-gated ion channels, which kills neurons by
oxidative stress. It was reported that antioxidant therapy may slow
the rate of motor decline early in the course of Huntington's
disease (35).
[0010] Vitamin C enters cells, in vitro, through the facilitative
glucose transporter GLUT1 in the form of dehydroascorbic acid and
is retained intracellularly as ascorbic acid (1). In order to test
the hypothesis that GLUT1 transport of dehydroascorbic acid is a
primary physiological mechanism for tissue acquisition of vitamin
C, we investigated the transport of vitamin C across the
blood-brain barrier (BBB) in rodents. GLUT1 is expressed at the BBB
on endothelial cells and is responsible for glucose entry into the
brain. Ascorbic acid, the predominant form of vitamin C in blood,
was incapable of crossing the BBB while dehydroascorbic acid
readily entered the brain and was retained in the form of ascorbic
acid. The transport of dehydroascorbic acid into the brain was
competitively inhibited by D-glucose, but not by L-glucose. These
findings define the transport of dehydroascorbic acid by GLUT1 as
the mechanism by which the brain acquires vitamin C, and point to
the oxidation of vitamin C as the important regulatory step in the
accumulation of the vitamin by the brain.
[0011] Dehydroascorbic acid, the oxidized form of vitamin C, was
previously found to be transported through the facilitative glucose
transporters. Expression of GLUT1, GLUT2, and GLUT4 in Xenopus
oocytes conferred the ability to take up dehydroascorbic acid which
was retained intracellularly after it was reduced to ascorbic acid
(1). It was also established that facilitative glucose transporters
are involved in the transport and accumulation of vitamin C by
normal human neutrophils and the myeloid leukemia cell line, HL60
(1-3). In these cells dehydroascorbic acid is transported across
the cell membrane and accumulated in the reduced form, ascorbic
acid, which is not transportable through the bidirectional glucose
transporter (1-3). Ascorbic acid may be transported through a
Na.sup.+-ascorbate co-transporter that is reported to be present in
small intestine, kidney and adrenomedullary chromaffin cells (4).
The co-transporter has not been molecularly characterized and no
Na.sup.+-dependent ascorbic acid uptake in white blood cells has
been found (2,3).
[0012] GLUT1 is expressed on endothelial cells at the BBB and is
responsible for glucose transport into the brain (5, 6). In the
1880's, Ehrlich found that intravenously injected aniline dyes
colored all of the organs of experimental rabbits except the brain
and the spinal cord (7,8). This observation led to the eventual
discovery that the BBB is comprised of a wall of capillaries
forming an endothelial barrier between the blood and the brain,
functioning primarily to regulate the transport of nutrients and
waste products (9,10). Several nutrient transporters have been
identified at the BBB including GLUT1, a monocarboxylic acid
transporter, neutral amino acid transporter, amine transporter,
basis amino acid transporter, nucleoside transporter, and purine
base transporter (11). Here it is shown in rodents that vitamin C
cross the BBB through GLUT1 only in the oxidized form,
dehydroascorbic acid, and is retained in the brain in the reduced
form, ascorbic acid. The present invention allows for the
controlled introduction of the antioxidant vitamin C into brain
tissue, which should serve as an important therapeutic method to
treat and prevent various disorders associated with free radicals
and oxidative damage.
SUMMARY OF THE INVENTION
[0013] This invention provides a method for increasing the ascorbic
acid concentration in brain tissues of a subject which comprises
administering to the subject an amount of dehydroascorbic acid
effective to increase the concentration of ascorbic acid in brain
tissues. This invention also provides the above-described method
wherein the dehydroascorbic acid enters the tissues through the
facilitative glucose transporter.
[0014] This invention also provides a method for treating
neurodegenerative disease of a subject comprising administering to
the subject an amount of dehydroascorbic acid effective to increase
the antioxidant potential of brain tissues.
[0015] This invention finally provides a method for preventing
neurodegenerative disease of a subject comprising administering to
the subject an amount of dehydroascorbic acid effective to increase
the antioxidant potential of brain tissues.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 Dehydroascorbic acid is transported across the BBB
and accumulates in the brain as ascorbic acid. (A) Balb/c mice (age
6-8 weeks) and (B) Fischer F344 rats (70-80 gram body weight) were
injected into the tail vein with 5 .mu.Ci(mouse) or 10 uCi(rat)
1.sup.4C-ascorbic acid (L-[.sup.41-C]-ascorbic acid, specific
activity, 6.6 mCi/mmol, Dupont NEN), .sup.14C-dehydroascorbic acid
or H-sucrose ([fructose-1-.sup.3H]-sucrose, specific activity 20.0
Ci/mmol, Dupont NEN). Each group consists of 12 animals and the
values are expressed as mean .+-.SEM. (C)HPLC analysis of the
methanol soluble fraction of the brain and (H) serum of a mouse
injected with 20 .mu.Ci .sup.14C-dehydroascorbic acid and
sacrificed at 5 min (injected material, hashed line). (C)
Accumulation of vitamin C in the brain is in the form of ascorbic
acid (.about.90& retention time.apprxeq.11.80 min, solid line).
(H) Radioactivity present in serum is in the form of ascorbic acid
(>98%; retention time.apprxeq.11.80 min, solid line). (D) The
initial kinetics and (E) 2 hr kinetics of accumulation of
radioactivity in the brain of mice injected intravenously with
.sup.14C-ascorbic acid (.circle-solid.), .sup.14C-dehydroascorbic
acid (.box-solid.) or.sup.3 H-sucrose (.smallcircle.). (F) The
initial kinetics and (G) 2 hr kinetics of radioactivity in the
serum of mice injected intravenously with .sup.14C-ascorbic
acid(.circle-solid.),.sup.14 C-dehydroascorbic acid (.box-solid.)
or .sup.3H-sucrose(.smallcircle.). Each data set in (D) through (G)
represents 4 mice.+-.SEM.
[0017] FIG. 2 Specificity of the transport of dehydroascorbic acid
through GLUT1 at the Balb/c mouse BBB. (A) .sup.14C-Dehydroascorbic
acid (.box-solid.) entered the brain and its accumulation was
blocked by increasing amounts of D-deoxyglucose which is
transported through GLUT1. Transport of .sup.3H-leucine
(.smallcircle.) or .sup.14C-ascorbic acid (.circle-solid.) across
the BBB was not affected by D-deoxyglucose. (B) L-glucose, which is
not transported through GLUT1, had no effect on the transport of
.sup.14C-dehydroascorbic acid. Transport of .sup.3H-leucine
(.smallcircle.) or .sup.14C-ascorbic acid (.circle-solid.) across
the BBB was not affected by L-glucose. All experiments were carried
out over a 30-second time course. Each data set included 4 mice and
the data were expressed as mean .+-.SEM. A mouse has a baseline
serum glucose concentration of approximately 12 mM, which
calculates to 2.67 mg glucose in the entire mouse based on the
average plasma volume of the mouse. The amount of exogenous glucose
administered in this experiment was based on this number and
subsequent multiples to a maximum tolerable level.
[0018] FIG. 3 Brain digital autoradiography of rat with
.sup.14C-labelled ascorbic acid, dehydroascorbic acid,
D-deoxyglucose and sucrose. (A) Digital autoradiography was
performed on a Fisher F344 rat (8 wks of age) 3 min after
intravenous injection with 40 pCi of .sup.14C-dehydroascorbic acid,
(B) 40 .mu.Ci .sup.14C-ascorbic acid and (C)40 .sup.14 .mu.Ci
C-sucrose ([glucose-.sup.14C(U)]-sucrose, specific activity, 310
mCi/mmol, Dupont NEN). The area of the brain is denoted with an *
in the figure. The photo-stimulated luminescence (PSL) /mm.sup.2
ratio of brain/background counts for the dehydroascorbic
acid-injected rat was 8.6.+-.0.3 (mean of 3 sections.+-.SEM). The
PSL/mm.sup.2 ratio in the ascorbic acid-injected rat was 1.5.+-.0.1
and 1.4.+-.0.1 in the sucrose-injected rat.
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention provides a method for increasing the ascorbic
acid concentration in brain tissues of a subject which comprises
administering to the subject an amount of dehydroascorbic acid
effective to increase the concentration of ascorbic acid in brain
tissues. This invention also provides the above-described method
wherein the dehydroascorbic acid enters the tissues through the
facilitative glucose transporter.
[0020] This invention also provides a method for increasing the
ascorbic acid concentration in brain tissues of a subject which
comprises administering to the subject an amount of dehydroascorbic
acid effective to increase the antioxidant potential of the brain
tissues.
[0021] In an embodiment of this invention, the subject is a human.
In a separate embodiment, the human subject has a neurodegenerative
disease. Such neurodegenerative disease includes but is not limited
to Alzheimer's Disease, Parkinson's Disease or other forms of
presenile dementia.
[0022] In another embodiment, the subject has neurovascular
disease. This invention is useful for treating or preventing stroke
or neurovascular diseases.
[0023] The subject may carry genetic diseases with central nervous
system manifestations. In an embodiment, the genetic disease is the
Huntington's disease.
[0024] For a separate embodiment, the subject has schizophrenia. In
a still another embodiment, the human subject has a behavioral
disorder. Such behavioral disorder includes, but is not limited to
dysthymia, involution depression, aggressiveness via dominance,
hyperactivity, deprivation syndrome, separation anxiety,
intermittent anxiety, instrumental sociopathy, stereotypies, phobia
or socialization disorders.
[0025] As it will be easily appreciated by persons of skills in the
art, this invention is applicable to both human and animal diseases
which could be treated by antioxidants. This invention is intended
to be used in husbandry and veterinary medicine.
[0026] In this invention, the dehydroascorbic acid may be
administered orally, intravenously, subcutaneously, intramuscularly
or by other routes or circumstances of administration by which the
dehydroascorbic acid will not be hydrolyzed. Dehydroascorbic acid
hydrolyses easily in aqueous solution. It is the intention of this
invention to administer the dehydroascorbic acid in a stabilized
form. It is known that dehydroascorbic acid is stable under low pH
conditions. Accordingly, dehydroascorbic acid may be stored in low
pH and then administered directly to a large vein of a subject.
Alternatively, dehydroascorbic acid may be stored in powdered form
and hydrated before administering to a subject.
[0027] Moreover, dehydroascorbic acid may be encapsulated in
liposomes at low pH. The encapsulated dehydroascorbic acid will
then be administered to a subject. In a preferred embodiment, the
encapsulated dehydroascorbic acid is administered orally.
[0028] U.S. Pat. No. 4,822,816 describes uses of aldono-lactones
and salts of L-threonic, L-xylonic and L-lyxonic to stabilize the
dehydroascorbic acid. The content of U.S. Pat. No. 4,822,816 is
hereby incorporated into this application by reference.
Accordingly, this method provides another means for stabilization
of the dehydroascorbic acid.
[0029] Finally, appropriate amounts of ascorbic acid and ascorbate
oxidase may be administered together to a subject to produce an
amount of dehydroascorbic acid effective to increase the
concentration of ascorbic acid in the brain tissues of the subject.
Ascorbate oxidase catalyzes oxidation of L-ascorbic acid, and it is
commercially available. U.S. Pat. No. 5,612,208 describes a new
ascorbate oxidase and its gene, the content of which is hereby
incorporated into this application by reference. Accordingly,
ascorbate oxidase may be produced by the recombinant DNA
technology.
[0030] Using this invention, the brain tissues of a subject may be
loaded with the maximum amount of ascorbic acid.
[0031] Dehydroascorbic acids may exist in various salt forms. It is
the intention of this invention to encompass these forms. The salts
upon hydration will generate dehydroascorbic acid. This invention
provides a method for treating or preventing dementia of a subject
comprising administering to the subject an amount of
dehydroascorbic acid effective to increase the concentration of
ascorbic acid in brain tissues.
[0032] This invention also provides a method for treating or
preventing neurodegenerative disease of a subject comprising
administering to the subject an amount of dehydroascorbic acid
effective to increase the antioxidant potential of the brain
tissues.
[0033] This invention also provides a combination therapy wherein
an effective amount of dehydroascorbic acid is administered with
therapeutic agents for the neurodegenerative disease. The
administration may be performed concomitantly or at different time
points. When treating the Alzheimer's disease, the therapeutic
agents include, but are not limited to, Estrogen, Vitamin E
(alpha-tocopherol), Tacrine (Tetrahydroacridinamine), Selegiline
(Deprenyl), and Aracept (Donepezil). With respect to the
Parkinson's disease, the therapeutic agents include, but are not
limited to, the anticholinergic class of drugs, clozapine, levodopa
with carbidopa or benserazide, Selegiline (Deprenyl), and dopamine
agonist class of drugs.
[0034] This invention provides a method for treating or preventing
stroke or neurovascular disease or other diseases which can be
caused by lipid perioxidation of a subject comprising administering
to the subject an amount of dehydroascorbic acid effective to
increase the concentration of ascorbic acid in brain tissues.
[0035] This invention also provides a method for treating or
preventing stroke or neurovascular disease or other diseases which
can be caused by lipid perioxidation of a subject comprising
administering to the subject an amount of dehydroascorbic acid
effective to increase the antioxidant potential of the brain
tissues.
[0036] These diseases include, but are not limited to stroke,
atherosclerosis and neurodegenerative disorders.
[0037] Moreover, this invention provide a method for treating or
preventing central nervous system manifestations of genetic
diseases. The conditions of the disease will be improved by
increasing the antioxidant potential of the brain. Prevention of
such central nervous system manifestations of genetic disease may
even be prevented if the antioxidant potential of the brain
maintain to be a high level. This genetic disease includes, but not
limited to, Huntington's disease.
[0038] This invention provides a method for preventing or treating
behavioral disorders of a subject comprising administering to the
subject an amount of dehydroascorbic acid effective to increase the
concentration of ascorbic acid in brain tissues. This invention
finally provides a method for preventing or treating behavioral
disorders of a subject comprising administering to the subject an
amount of dehydroascorbic acid effective to increase the
antioxidant potential of the brain tissues. Such behavioral
disorder includes, but is not limited to dysthymia, involution
depression, aggressiveness via dominance, hyperactivity,
deprivation syndrome, separation anxiety, intermittent anxiety,
instrumental sociopathy, stereotypies, phobia or socialization
disorders.
[0039] When treating or preventing the behavioral disorders,
dehydroascorbic acid may be used in combination with other drugs.
They may be administered concomitantly or at different time
points.
[0040] In another embodiment, the behavioral disorder is
schizophrenia.
[0041] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
[0042] Experimental Details
[0043] Experimental Methods
[0044] Blood-brain Barrier Transport Studies.
[0045] .sup.14C-dehydroascorbic acid was generated in all
experiments by incubating the .sup.14C-ascorbic acid with ascorbate
oxidase, 1 unit/1.0 mmol L-ascorbate (derived from Cucurbita
species, Sigma). Dithiothreitol (0.1 mmol/liter) was added to the
vitamin C preparations as a reducing agent. Animals were sacrificed
at various time points after injection by cervical dislocation of
CO.sub.2 inhalation. The brain was then dissected out and
homogenized in 70% methanol. Samples were processed for
scintillation spectrometry or HPLC as described (2,3). HPLC was
performed on the methanol fraction with 1 mmol/L EDTA added (2,3).
Samples were stored at -70.degree. C. until analysis. HPLC samples
were separated on a Whatman strong anion exchange Partisil 10 SAX
(4.6-.times.25-cm) column (Whatman, Hillsboro, Oreg.). A
Whatman-type WCS solvent-conditioning column was used and the
eluates monitored with a Beckman System Gold liquid chromatograph
(Beckman Instruments, Irvine, Calif.) with a diode array detector
and radioisotope detector arranged in series. Ascorbic acid was
monitored by absorbance at 265 nm and by radioactivity.
Dehydroascorbic acid shows no absorbance at 265 nm and was
monitored by radioactivity.
[0046] Digital Autoradiography.
[0047] Animals were sacrificed, frozen in a dry ice/hexane mixture
and then embedded in -5% carboxymethylcellulose (Sigma Aldrich).
The animal blocks were allowed to equilibrate for -12 hours at
-20.degree. C. and the animals were sectioned in coronal cuts with
a slice thickness of .about.40-45 .mu.m in a cryo-microtome (PMV),
and tape lifted for direct exposure onto digital plates (23). The
exposure time was approximately 72 hours. All digital plates were
scanned on a Fuji Bas 5000 digital autoradiographic system (Fuji,
Inc.) At 25 .mu.m resolution.
[0048] Calculation of the BBB Permeability-surface Area
Product.
[0049] The amount of compound which crosses the BBB is dependent on
two parameters defined by the following equation: 1 PS = V D - V o
t
[0050] where PS is the BBB permeability-surface area product and
AUC is the plasma area under the concentration time-activity curve
at a given time (t) after injection. A variant of the single
intravenous injection technique termed the external organ technique
was used to quantify the BBB PS product in anesthetized animals.
The plasma and brain radioactivity was measured as decays per min
(DPM)/.mu.l of serum (after the ascorbic acid or sucrose was
solubilized from the cells in the presence of 70% methanol) which
was equivalent to the integral of the plasma radioactivity. The BBB
PS product is calculated:
% injected dose/.mu.m of brain tissue=PS.times.AUC
[0051] where the variables are defined, as follows:
[0052] t=time 2 VD = [ 14 C - AA or DHA ] dpm gm brain tissue
(brain) [ 14 C - AA or DHA ] dpm 1 serum (external organ) V o = [ 3
H - Sucrose ] dpm gm brain tissue (brain) [ 3 H - Sucrose ] dpm 1
serum (external organ)
[0053] The rats were anesthetized with a mixture of ketamine 90
mg/kg and xylazine 10 mg/kg anesthesia during the procedure. The
xylazine causes a hyperglycemia and hypoinsulinemia in the animals
with the serum glucose measured at approximately 280 mg/dl 30 min
after induction of anesthesia (24,25). This is almost three-fold
higher than baseline glucose concentrations in the rats and affects
transport through GLUT1 and therefore the PS calculations.
Radiolabeled test compound (.sup.3H-sucrose, .sup.14C-ascorbic
acid, .sup.14C-dehydroascorbic acid) was injected into a cannulated
femoral vein in groups of 3 rats. Sucrose was used as a V.sub.0
marker (plasma volume marker). For 30 seconds (t) after injection
arterial blood was collected by gravity from a catheter cannulated
in the abdominal aorta and then the animal was sacrificed and the
brain harvested.
[0054] Results and Discussions
[0055] Mice and rats were injected into the tail vein with
.sup.14C-ascorbic acid, .sup.14C-dehydroascorbic acid or
.sup.3H-sucrose. Three min after intravenous injection the animals
were sacrificed, the brains harvested and the methanol soluble
fraction counted by liquid scintillation. Approximately 4% of the
dehydroascorbic acid (expressed as percent of injected dose (ID)
per gram of brain tissue) was found in the brain after 3 min (FIGS.
1A and 1B). Injected ascorbic acid and sucrose yielded only trace
radioactivity in the brain homogenate at 3 min, indicating that
ascorbic acid could not pass the BBB. Because sucrose is not
metabolized or transported it is used as a marker of plasma volume
(12). The small amount of radioactivity present in the brain of the
sucrose and ascorbic acid-injected animals was consistent with the
radioactivity being present within the brain blood vessels.
High-performance liquid chromatography (HPLC) analysis of the
methanol (70%) fraction of the brain homogenate showed that the
form of the vitamin C accumulated in the brain of dehydroascorbic
acid-injected animal was >85% ascorbic acid (FIG. 1C). This
result indicated that dehydroascorbic acid was transported across
the BBB and retained as ascorbic acid in the brain.
[0056] Brain radioactivity, after dehydroascorbic acid injection,
reached a maximum of 4.3% of ID/gram brain tissue at 3 min,
decreased to 3.3% at 25 min, and remained at that level for up to 2
hours after injection (FIG. 1D, 1E). Injection of sucrose and
ascorbic acid resulted in a maximum brain accumulation of 0.4%
ID/gram brain tissue at 15 to 30 seconds after injection (FIG. 1D).
Brain radioactivity in the sucrose-injected animals decreased to
<0.1% after 15 min, concomitant with the fall in serum
radioactivity in these mice (FIG. 1E, 1G). In ascorbic
acid-injected mice there was an increase in brain radioactivity to
1.1% ID/gram brain tissue 2 hours after injection, a time period
during which there was a decreasing amount of radioactivity in the
serum (FIGS. 1E, IG). The serum radioactivity concentration at 15
seconds after dehydroascorbic acid injection was 8% ID/gram serum,
whereas the corresponding figure in mice injected with ascorbic
acid was 27%. Thus dehydroascorbic acid was cleared from the
circulation substantially faster than ascorbic acid (FIG. 1F). At
the 3-min time point the radioactivity in the serum of the ascorbic
acid and dehydroascorbic acid-injected animals was equivalent (FIG.
1G). Radioactivity remaining in the serum of the dehydroascorbic
acid-injected animals at 5 min was associated with ascorbic acid
(FIG. 1H).
[0057] Injected .sup.14C-ascorbic acid showed no measurable
transport into the brain over the first 30-min, but some
radioactivity accumulated in the brain at longer time periods.
There are at least three potential explanations for this result.
The first is that the ascorbic acid was metabolized in the interval
time period and the counts in the brain represented transported
radiolabeled metabolic breakdown products of ascorbic acid. Such an
explanation is unlikely as the HPLC results demonstrated that the
majority of the radioactivity in the dehydroascorbic acid-injected
brain was eluted in radioactive peaks consistent with intact
ascorbic acid. A second possibility is the presence of a small
number of Na.sup.+-ascorbate cotransporters at the BBB or choroid
plexus, which is unlikely since the accumulation of ascorbic acid
did not occur linearly with time, as it would in this case, but
only occurred after 30 min (13). The interpretation is that
oxidation of ascorbic acid in the microenvironment occurred in vivo
leading to the production of dehydroascorbic acid which was then
transported across the BBB and retained in the brain as ascorbic
acid.
[0058] The serum concentration of injected dehydroascorbic acid
reached only 20 to 25% of the serum concentration of ascorbic acid
or sucrose during the initial several minutes after injection.
Sucrose has no transport mechanism, therefore its clearance from
the serum was slow. Part of the clearance mechanisms for ascorbic
acid and dehydroascorbic acid are through transport, the GLUTs in
the case of dehydroascorbic acid and potentially a
Na.sup.+-ascorbate cotransporter in the case of ascorbic acid (4).
The rapid clearance of dehydroascorbic acid from the serum likely
reflected the large number of glucose transporters available for
transport.
[0059] The glucose transporter GLUT1 selectively transports
D-glucose but not L-glucose. In order to confirm that
dehydroascorbic acid passed the BBB through GLUTs, inhibition
experiments were conducted with D- and L-glucose. 2-Deoxy-D-glucose
(D-deoxyglucose) and D-glucose (data not shown) inhibited uptake of
dehydroascorbic acid in the brain in a dose-dependent fashion up to
70%, whereas L-glucose and leucine had no effect (FIG. 2A). The
uptake of leucine, which is not transported by GLUTs, but crosses
the BBB largely through L system transporters and to a minor extent
by the ASC system transporter (14), was not affected by increasing
concentrations of L-glucose of D-deoxyglucose (FIG. 2B) nor were
the serum concentrations of ascorbic acid, dehydroascorbic acid and
leucine affected by increasing concentrations of D-deoxyglucose or
L-glucose (data not shown). These results established that D-
deoxyglucose inhibits dehydroascorbic acid from entering the brain
through the glucose transporters but does not affect certain other
transport systems or alter general BBB permeability by osmotic
effects.
[0060] The external organ approach, utilizing serum as the external
organ, was used to calculate the BBB permeability-surface areas
product (PS) in the Fischer F344 rat (15). The calculated PS of
.sup.14C-dehydroascorbic acid was 136.+-.12 (SEM).mu.l/min/gm brain
tissue, .sup.14C-ascorbic acid was -0.44.+-.0.24 .mu.l/min/gm brain
tissue, and .sup.3H-D-deoxyglucose was 44.+-.3.2 .mu.l/min/gm brain
tissue. The difference in the BBB permeability-surface area
products (PS) between ascorbic acid and dehydroascorbic acid
illustrated the marked differences in the BBB transport between the
redox states of vitamin C. The calculated PS of ascorbic acid was
approximately Opl/min/gm brain tissue at 30 seconds, similar to
sucrose, which indicates no transport across the BBB. The PS of
dehydroascorbic acid was 3-fold greater than D-deoxyglucose which
corresponds with the difference in the Km values between the two
compounds. The apparent Km of D-deoxyglucose for transport was 2.5
mM in HL60 cells compared with an apparent K.sub.m of 0.85 mM for
dehydroascorbic acid in HL60 cells (2,3).
[0061] Digital autoradiography of the brain of a rat injected with
.sup.14C-dehydroascorbic acid and a rat injected with
.sup.14C-ascorbic acid was performed to confirm the anatomical
distribution of the injected compounds (FIG. 3). Autoradiographic
evidence of activity accumulation in the brain was seen only in
animals injected with dehydroascorbic acid. .sup.14C-sucrose was
used as a marker of intravascular volume.
[0062] The results of this study established that the transport of
vitamin C into the brain is mediated by GLUTs at the BBB which
transport dehydroascorbic acid. Ascorbic acid itself is not
transportable across the BBB. The glucose transport in vivo
therefore was found to function comparably to in vitro models in
that only the oxidized form of vitamin C, dehydroascorbic acid, was
transportable (1-3). Dehydroascorbic acid was reduced to ascorbic
acid after passing the BBB and was retained in the brain as
ascorbic acid. This trapping mechanism allows for the accumulation
of higher concentrations of vitamin C in the brain than in the
blood. Overall, the findings point to the oxidation of ascorbic
acid as being the critical step in the regulation of the
accumulation of vitamin C in the brain.
[0063] The current recommended daily allowance of vitamin C is 60
mg daily and yields a steady-state plasma concentration of
approximately 24 .mu.M in human volunteers (16). Only ascorbic acid
is detected in the serum, with dehydroascorbic acid at trace serum
levels or not measurable (17). The vitamin C injected in this study
was approximately 500 .mu.M, which is 5-fold greater than the
physiologic serum concentration of vitamin C in rodents (18). In
this study, at physiologic glucose concentrations, dehydroascorbic
acid transport through GLUTI did occur. The serum concentration of
glucose in normal rodents is approximately 10 mM yet there is still
dehydroascorbic acid transport to the brain indicating that both
dehydroascorbic acid and glucose are substrates of the GLUTs under
physiologic conditions. This result is consistent with in vitro
data demonstrating that a deoxyglucose concentration greater than
50 mM is necessary to block the transport of dehydroascorbic acid
through GLUT1 (2,3).
[0064] James Lind detailed the clinical description of scurvy in A
Treatise of the Scurvy in 1772. He concluded his report of the
autopsy results of scorbutic patients' "ravaged bodies" as follows,
"What was very surprising, the brains of those poor creatures were
always sound and entire . . . " (19). There thus appeared to be a
mechanism for the accumulation and storage of ascorbic acid in the
brain such that the brain would be the last organ depleted of
vitamin C. The normal human brain has a vitamin C concentration of
approximately lmM, 10 times the normal serum concentration (20).
The precise role of vitamin C in the brain is uncertain, but
ascorbic acid may be a cofactor of dopamine .beta.-hydroxylase and
is thus involved in the biosynthesis of catecholamines. Vitamin C
can also inhibit the peroxidation of membrane phospholipids and act
as a scavenger of free radicals in the brain (21,22). The results
of this study demonstrate the physiological importance of vitamin C
transport through GLUT1 in the form of dehydroascorbic acid and
define the mechanism by which the brain obtains and retains vitamin
C.
[0065] Recent data show that large quantities of vitamin C can be
loaded into the brain. An experiment was done in which the carotid
artery of a subject rat was cannulated with a catheter and 24 mg of
dehydroascorbic acid was injected into the artery. The injected
dehydroascorbic acid was spiked with a tracer amount of radioactive
(.sup.14C-labeled) dehydroascorbic acid. The dehydroascorbic acid
was infused over forty minutes and the brain was harvested. The
amount of radioactive vitamin C was quantitated in the brain and
total amount of injected vitamin C that accumulated in the brain
was thus extrapolated. The experiment demonstrated that 2.6 mg of
vitamin C accumulated in the brain of the subject rat during the
forty minute injection period, which was approximately 11% of the
injected dose. This shows that it is possible to achieve
pharmacologic concentrations of vitamin C in the brains of subject
animals. It is of note that the total vitamin C concentration in
the normal adult rat brain is approximately 150 .mu.g. A log-fold
greater Vitamin C than baseline normal concentration of Vitamin C
was thus achieved.
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