Note: Descriptions are shown in the official language in which they were submitted.
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NEUROPROTECTIVE COMPOSITION AND USES THEREOF
Background of the invention
1 ) Field of the invention
The present invention relates to the use of an amphiphilic antioxidant
composition as a neuroprotective agent and to methods for using and preparing
the same. More particularly, the present invention pertains to the use of a
formulation of pyruvate, antioxidant, and lipids) such as fatty acids for
protecting
neurons against oxidative stress.
2) Description of the prior art
Reactive oxygen species (ROS) have been implicated in the development
of many heart and brain dysfunctions. Ischemia/reperfusion insults to these
organs
are among the leading causes of mortality in America. These insults are caused
by
complete or partial local occlusions of heart and brain vasculature, by heart
stroke
or attack, and by cerebral attacks and trauma to the brain. In addition, ROS
are
involved in artherosclerotic lesions, in the evolution of various
neurodegenerative
diseases, and are also produced in association to epileptic episodes, in
inflammation, in the mechanisms of action of various neurotoxicants, or as
side-
effects of drugs.
Until now, no ideal therapeutic agent is known to protect neuronal cells
against oxidant species associated with various types of oxidative stress. It
would
therefore be highly desirable to have such neuroprotective agent.
TRIAD is combination of pyruvate, antioxidant and fatty acids. This
composition has been patented in 1997 in the U.S. as a therapeutic wound
healing
compositions (No 5,652,274). Many related U.S. patents have also been issued
for
covering the uses of TRIAD in antikeratolytic compositions (No 5,641,814); in
anti-
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fungal compositions (No 5,663,208); in acne healing compositions (No
5,646,190);
in anti-inflammatory compositions (No 5,648,380); in dermatological
compositions
(No 5,602,183); in sunscreen compositions (No 5,674,912); in antihistamine
compositions (No 5,614,561 ); in cytoprotective compositions (No 5,633,285);
in
wound healing composition affixed to razor cartridges (No 5,682,302); and in
regenerating compositions (EP 0 573 465 B1 ). However, none of these patents
discloses or suggests the use of TRIAD as neuroprotective agent.
In view of the above, it is clear that there is a need for an amphiphilic
antioxidant composition comprising pyruvate, antioxidant, and lipids) such as
fatty
acids to protect neuronal cells against oxidant species.
The purpose of this invention is to fulfil this need along with other needs
that
will be apparent to those skilled in the art upon reading the following
specification.
SUMMARY OF THE INVENTION
The present invention relates to a neuroprotective composition and more
particularly to an amphiphilic antioxidative composition and its uses.
According to an aspect of the invention, the neuroprotective composition
comprises a therapeutically effective amount of a mixture of pyruvate,
antioxidant(s), and lipids) such as fatty acids. These components are present
in
an amount that have a synergistic protective effect on neuronal cells.
In a preferred embodiment, lipids consist of a mixture of saturated and
unsaturated fatty acids selected from the group consisting of monogylcerides,
digylcerides, trigylcerides, free fatty acids, and mixtures thereof.
Preferably, pyruvate is selected from the group consisting of pyruvic acid,
pharmaceutically acceptable salts of pyruvic acid, prodrugs of pyruvic acid,
and
mixtures thereof.
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Preferably, also the antioxidant is selected from lipid-soluble antioxidants,
and more preferably the antioxidant is selected from the group consisting of
Vitamin A, carotene, Vitamin E, pharmaceutically acceptable salts thereof, and
mixtures thereof.
According to an other aspect of the invention, the neuroprotective
composition is used as such or as an active agent in the preparation of a
medication for the treatment of neuronal cells. Such treatments include the
treatment brain trauma, brain or cerebrovascular ischemia, neurodegenerative
diseases, poisoning of neuronal cells, the diminution of drugs side effects
and for
preservation of neuronal grafts.
According to an other aspect of the invention, the invention provides a
method for treating neuronal oxidative stress related condition, the method
comprising administrating to a patient in need thereof a therapeutically
effective
amount of an antioxidative composition comprising pyruvate, at least one
antioxidant and at least one lipid.
Alternatively, the invention also provides a method for treating neuronal
oxidative stress related condition comprising: a) administrating to a patient
in need
thereof, a therapeutically effective amount of an antioxidative composition
comprising pyruvate and at least one antioxidant; and b) providing, into the
blood
circulation of this patient, at least one lipid having a synergistic
therapeutic effect
on neuronal cells in combination with said antioxidative composition. The
lipids)
could be provided to the patient by increasing its lipidic blood level ratio
through its
diet. Examples of neuronal oxidative stress related condition include a
neurodegenerative disease, such as amyotrophic lateral sclerosis, Alzheimer's
disease, Parkinson's disease Huntington's disease, etc, brain trauma, brain or
cerebrovascular ischemia, neuronal cells poisoning, side effects caused by a
drug
and the preservation of neuronal grafts.
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According to an other aspect of the invention it is provided a method for
preparing a neuroprotective composition, the method comprising the steps of:
a) providing a therapeutically effective amount of: i) pyruvate, ii) at least
one
antioxidant; and iii) at least one lipid;
b) mixing together the components i), ii) and iii) of step a) in a
physiological
buffered saline solution to obtain a pharmaceutically acceptable homologous
suspension; and optionally
c) centrifuging or filtering the homologous suspension obtained in step b).
The buffered saline solution may comprises sodium, potassium, magnesium
and calcium ions at physiological concentrations and if necessary, an
emulsifier.
An advantage of the present invention is that it provides effective means for
preventing the loss of viability or functions of neuronal cells in conditions
of
oxidative stress. It can also protect a neuronal cell from a toxic substance,
stabilizes the cellular membrane of a neuronal cell and/or helps in the
normalization of neuronal cellular functions.
Other objects and advantages of the present invention will be apparent
upon reading the following non-restrictive descripti~~~~~ of several preferred
embodiments made with reference to the accompanying drawings.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the in vitro ROS production by peroxide-based
prooxidant systems used with P19 neurons.
FIG. 2 is a graph showing the protection provided by TRIAD to P19 neurons
exposed for different times to XA/XAO mediated oxidative stress.
FIG. 3 is a graph showing the protection provided by TRIAD components to
P19 neurons exposed for different times to XA/XAO mediated oxidative
stress.
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FIG. 4 is a graph showing the protection provided by TRIAD to P19 neurons
exposed to oxidative stress mediated by different concentrations of XAO.
FIG. 5 is a graph showing the protection provided by TRIAD to P19 neurons
exposed to hydrogen peroxide mediated oxidative stress.
FIG. 6 is a graph showing the protection provided by TRIAD components to
P19 neurons exposed to hydrogen peroxide mediated oxidative stress.
FIG. 7 is a graph showing the protection provided by TRIAD to P19 neurons
exposed to H202/Fe2+ prooxidant system.
FIG. 8 is a graph showing the protection provided by TRIAD components to
P19 neurons exposed to H202/Fe2+ prooxidant system.
FIG. 9 is a graph showing the in vitro antioxidant capacity of TRIAD in the
H202 prooxidant system used with P19 neurons.
FIG. 10 is a graph showing the in vitro antioxidant capacity of TRIAD in the
conditions of H202/Fe2+ prooxidant system used with P19 neurons.
FIG. 11 is a graph showing the in vitro antioxidant capacity of TRIAD
components in the conditions of H202 prooxidant system used with P19
neurons.
FIG. 12 is a graph showing the in vitro antioxidant capacity of TRIAD
components in the conditions of H202/Fe2+ prooxidant system used with P19
neurons.
DETAILED DESCRIPTION OF THE INVENTION
As stated hereinbefore the present invention relates to the use of an
amphiphilic antioxidant compositions as neuroprotective agent. As disclosed
herein, a composition comprising sodium pyruvate, antioxidant and lipids) such
as
fatty acids have neuroprotective actions against oxidative stress.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one ordinary skilled in the
art
to which this invention belongs.
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As used herein, the term "neuroprotective agent" or "neuroprotective
composition" refers to any compound (or to any mixture of compounds) that
protects a neuronal cell from a toxic substance, stabilizes the cell membrane
of a
neuronal cell and/or helps in the normalization of neuronal cell functions. A
"neuroprotective agent" thereby prevents the loss of viability or functions of
neuronal cells in stressing conditions.
Therefore, the term "neuroprotection" as used herein refers to the capacity
of a neuroprotective agent to maintain or stimulate the capacity of neuronal
cells to
maintain or recover their neuronal functions even in pathological or harmful
conditions such as oxidative stress conditions.
As stated out above, the neuroprotective composition of the invention
comprises a mixture of (a) pyruvate, (b) at least one antioxidant, and (c) at
least
one lipid such as fatty acids, preferably a mixture of saturated and
unsaturated
fatty acids. According to the invention, these three components have a
synergistic
beneficial effect on neuronal cells, i.e. their combined effect is greater
than the
sum of their individual effects.
The pyruvate in the present invention may be selected from the group
consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid,
prodrugs of pyruvic acid, and mixtures thereof. In general, the
pharmaceutically
acceptable salts of pyruvic acid may be alkali salts and alkaline earth salts.
Preferably, the pyruvate is selected from the group consisting of pyruvic
acid,
lithium pyruvate, sodium pyruvate, potassium pyruvate, magnesium pyruvate,
calcium pyruvate, zinc pyruvate, manganese pyruvate, methyl pyruvate, a-
ketoglutaric acid, and mixtures thereof. More preferably, the pyruvate is
selected
from the group of salts consisting of sodium pyruvate, potassium pyruvate,
magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and
the like, and mixtures thereof. Most preferably, the pyruvate is sodium
pyruvate.
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The amount of pyruvate present in the neuroprotective composition of the
present invention is a therapeutically effective amount. A therapeutically
effective
amount of pyruvate is that amount of pyruvate necessary for the
neuroprotective
composition to prevent and/or reduce injury of a neuronal mammalian cell. The
exact amount of pyruvate will vary according to factors such as the type of
condition being treated as well as the other ingredients in the composition.
Typically, the amount of pyruvate should vary from about 0.01 mM to about 100
mM. In a preferred embodiment, pyruvate is present in the composition of the
neuroprotective extracellular medium in an amount from about 0.1 mM to about
30
mM, preferably from about 0.5 mM to about 10 mM. In the most preferred
embodiment, the neuroprotective composition comprises about 10 mM of sodium
pyruvate.
Antioxidants, including vitamin antioxidants, are substances which inhibit
oxidation or suppress reactions promoted by oxygen, oxygen free radicals
(OFR),
oxygen reactive species (ORS) including peroxides. Antioxidants, especially
lipid-
soluble antioxidants, can be absorbed into the cell membrane to neutralize
oxygen
radicals and thereby protect the membrane. The antioxidants useful in the
present
invention are preferably vitamin antioxidants that may be selected from the
group
consisting of all forms of Vitamin A including retinal and 3,4-
didehydroretinal, all
forms of carotene such as alpha-carotene, ~3-carotene, gamma-carotene, delta-
carotene, all forms of Vitamin C (D-ascorbic acid, L-ascorbic acid), all forms
of
tocopherol such as Vitamin E (Alpha-tocopherol, 3,4-dihydro-2,5,7,8-
tetramethyl-2-
(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol), ~3-tocopherol, gamma-
tocopherol, delta-tocopherol, tocoquinone, tocotrienol, and Vitamin E esters
which
readily undergo hydrolysis to Vitamin E such as Vitamin E acetate and Vitamin
E
succinate, and pharmaceutically acceptable Vitamin E salts such as Vitamin E
phosphate, prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,
pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, and
Vitamin
E, and the like, and mixtures thereof. Preferably, the antioxidant is selected
from
the group of lipid-soluble antioxidants consisting of Vitamin A, ~-carotene,
Vitamin
E, Vitamin E acetate, and mixtures thereof. More preferably, the antioxidant
is
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Vitamin E or Vitamin E acetate. Most preferably, the antioxidant is Vitamin E
acetate. Analogues of Vitamin E such as Trolox~, a compound which is more
hydrosoluble than natural forms of Vitamin E and which could reach
intracellular
sites more rapidly, could also be used according to the present invention.
The amount of antioxidant present in the neuroprotective composition of the
present invention is a therapeutically effective amount. A therapeutically
effective
amount of antioxidant is that amount of antioxidant necessary for the
neuroprotective composition to prevent and/or reduce injury of a neuronal
mammalian cell. The exact amount of antioxidant will vary according to factors
such as the type of condition being treated as well as the other ingredients
in the
composition. Typically, the amount of antioxidant should vary from about 0.01
unit/ml to about 10 unit/ml. In a preferred embodiment, vitamin E antioxidant
is
present in the composition of the neuroprotective extracellular medium in an
amount from about 0.01 unit/ml to about 10 unit/ml, preferably from about 0.05
to
about 5 unit/ml. In the most preferred embodiment, the neuroprotective
composition comprises about 1 unit of antioxidant (a-tocopherol type VI in
oil) per
ml of neuroprotective composition.
As it is well known, lipids are esters or carboxylic ~Gid compounds found in
animal and vegetable fats and oils. The composition may comprises a single
type
of lipid or various types of different lipids. Preferably lipids are in the
form of a
mixture of saturated and unsaturated fatty acids. However, other types of
lipids
could be used such as glycolipids and phospholipids (e.g. lecithin). Lipids)
or
mixture thereof are selected among those lipids required for the stabilization
and/or repair of the membrane of neuronal mammalian cells. These lipids may be
derived from animal or vegetables. In a preferred embodiment, selected lipids
are
in the form of mono-, di-, or triglycerides, or free fatty acids, or mixtures
thereof,
which are readily available for the stabilization or repair of the membrane of
neuronal mammalian cells. Artificial lipids which are soluble in organic
solvents
and are of a structural type which includes fatty acids and their esters,
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cholesterols, cholesteryls esters could also be used according to the present
invention.
In a more preferred embodiment, the saturated and unsaturated fatty acids
are those deriving from egg yolk. According to the use of the neuroprotective
compositions of the invention, replacing egg yolk as a source of fatty acids
by
chemical preparations of unsaturated, polyunsaturated and/or saturated fatty
acids
compatible with, and in proportions similar to those found in cell membranes
may
be advantageous or reveal necessary to insure a controllable quality of
preparations.
The amount of lipids) such as fatty acids present in the neuroprotective
composition of the present invention is a therapeutically effective amount. A
therapeutically effective amount of fatty acids for instance is that amount of
fatty
acids necessary for the neuroprotective composition to prevent and/or reduce
injury of a neuronal mammalian cells. The exact amount of lipids) or fatty
acids
will vary according to factors such as the type of condition being treated as
well as
the other ingredients in the composition. Typically, the amount of lipids) or
fatty
acids should vary from about 0.001 % v/v to about 1 % v/v. In a preferred
embodiment, fatty acids are present in the neuroprotective composition in an
amount from about 0.001 % v/v to about 0.3 % v/v, preferably from about 0.005
v/v to about 0.1 % v/v. In the most preferred embodiment, the neuroprotective
composition comprises about 0.1 % v/v of fresh egg yolk.
As the lipidic blood level of an individual is normally about 0.5-0.6% of the
total serum volume, the lipidic portion could be omitted from the
neuroprotective
composition of the invention. It could be possible to provide into the blood
circulation of this individual at least one lipid having a synergistic
therapeutic effect
on neuronal cells with the others component of the antioxidative
neuroprotective
composition of the invention. For instance, selected lipids) could be provided
by
increasing the lipidic blood level ratio of this individual through the diet.
Lipids
which could have a synergistic therapeutic effect without being harmful to a
patient
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could be selected from the group consisting of phospholipids, glycolipids,
fatty
acids, and mixture thereof.
Further agents can be joint to the neuroprotective composition of the
invention. For examples various antioxidants may complete the action of
neuroprotective composition such as
-ceruloplasmin or its analogues since it can scavenge '02 radicals and has a
ferroxidase activity which oxidizes Fe2+ to Fe3+ ;
-metal chelators/scavengers (e.g. desferrioxamine [Desferal~], a small
substance capable to scavenge Fe3+ and other metal ions);
-proteins or their fragments that can bind metal ions such as ferritin or
transferrin which both bind Fe3+;
-scavengers of'OH (hydroxyl) or NO (nitric oxide) radicals (e.g. mannitol).
-small scavengers of '02- (superoxide), 'OH (hydroxyl) or NO (nitric oxide)
radicals (e.g. acetyl salicylic acid, scavenger of 'OZ ; mannitol or
captopril, scavengers of 'OH) or molecules that inhibit the generation of
these radicals (e.g. arginine derivatives, inhibitors of nitric oxide
synthase which produce NO);
-proteins or their fragments that scavenge oxygen free radicals and can
assist the protective action of ceruloplasmin (e.g. superoxide dismutase
which dismutate '02-; hemoglobin which traps NO); and
-proteins or their fragments that can scavenge H202 (hydrogen peroxide) in
cases where they may exert a more potent or durable protective action
than pyruvate (e.g. catalase, glutathion peroxidase).
The composition of the invention may also comprises modulators of brain
functions such as neurotransmitters, neuropeptides, hormones, trophic factors,
or
analogs of these substances that act by binding to brain receptors (e.g. DOPA
in
Parkinson's disease).
Further to the therapeutic agents, the neuroprotective composition of the
invention may also contain preserving agents, solubilizing agents, stabilizing
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agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts,
buffers,
or coating agents. For preparing the neuroprotective composition, methods well
known in the art may be used.
The method of preparation of the neuroprotective compositions of the
invention is very simple as it consists simply in the mixing of components in
a
buffered saline solution in order to get a homogenous suspension. Suitable
saline
solution comprises sodium, potassium, magnesium and calcium ions at
physiological concentrations, has an osmotic pressure varying from 280 to 340
mosmol, and a pH varying from 7.2 to 7.4. Depending of the amount and of type
of
lipids) which is used, the saline may also comprises an emulsifier.
Preferably, the
buffered saline solution is selected from the group consisting of modified
Krebs-
Henseleit buffer (KH) and phosphate buffer saline (PBS), both at pH 7.4. The
homogenous suspension obtained can further be centrifuged and/or filtered to
reduce its viscosity and/or eliminated non-soluble particles.
Obviously, this simple method can be modified according to the use of the
neuroprotective composition. In the example found hereunder, a genuine
preparation was used. Centrifuged and/or filtered preparations could also have
been used. However, it is important to note that modifications in the modality
of
preparation can influence the resulting effects of the neuroprotective
composition.
For example, varying the pH of the composition (or buffer) can slightly modify
the
ionization state of carboxylic functions of pyruvate and thus alter its
solubility
and/or reaction with H202, while the dialysis of the composition would reduce
the
amount of pyruvate in the final preparation, unless it is done before addition
of
pyruvate. A person skilled in the art will know how to adapt the preparation
of the
neuroprotective composition of the invention according to its desired use in
specific conditions in order to obtain positive desired effects.
The neuroprotective composition of the invention could be suitable to treat
diseases and pathological conditions such as brain trauma and diseases which
were shown to involve oxidative stress conditions such as amyotrophic lateral
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sclerosis and neurodegenerative Parkinson's, Alzheimer's and Huntington's
diseases. These neuroprotective compositions could also be involved in the
treatment of poisoning or diminution of side effects of various drugs (such as
chemotherapeutic and immunosuppressive drugs) to the brain and/or to neuronal
cells. Indeed, deleterious action of various toxicants and drugs is exerted
via
production of ROS.
The neuroprotective composition of the invention has potential applications
in both fast (in minutes; especially due to the pyruvate) and long term
treatments
(hours and days; due to the antioxidant and lipids) such as fatty acids). The
amount to be administered is a therapeutically effective amount. A
therapeutically
effective amount of a neuroprotective composition is that amount necessary for
protecting a neuronal cell from the loss of viability or function induced by a
toxic
substance, stabilizing the cell membrane of neuronal cells and/or helping in
the
normalization of neuronal cell functions. Suitable dosages will vary,
depending
upon factors such as the type and the amount of each of the components in the
composition, the desired effect (fast or long term), the disease or disorder
to be
treated, the route of administration and the age and weight of the individual
to be
treated.
The neuroprotective composition of the invention and/or more complex
pharmaceutical compositions comprising the same may be given orally (per os)
in
the form of tablets, capsules, powders, syrups, etc. since all their
components are
absorbable by the gastrointestinal tract. Others administration ways can also
be
considered (rectal and vaginal capsules or nasally by means of a spray). They
may also be formulated as creams or ointments for topical administration. They
may also be given parenterally, for example intravenously, intramuscularly or
sub-
cutaneously by injection or by infusion. Intravenous administration can be a
way
for fast answer in various clinical conditions (e.g. ischemic brain, brain
trauma,
stroke and heart attacks, post-surgery treatments, etc). Obviously, the
neuroprotective compositions of the invention may be administered alone or as
part of a more complex pharmaceutical composition according to the desired use
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and route of administration. Anyhow, for preparing such compositions, methods
well known in the art may be used.
As it will now be demonstrated by way of an example hereinafter, the
composition of the invention possesses a strong neuroprotective activity i.e.
the
capacity to maintain the viability and functions of neurons at their normal
level or to
induce a fast recovery to the normal level, even in pathological or harmful
conditions such as oxidative stress conditions. These conditions can occur at
post-
ischemia reperfusion of the brain associated with an attack to brain
vasculature,
cerebral trauma or a heart stroke/attack, in various neurodegenerative
diseases, in
epilepsy, following an exposure to neurotoxicants, or as side-effects of drugs
and
inflammation. Although any methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention,
the preferred methods and materials are described.
EXAMPLE:
Neuroprotective actions of TRIAD against oxidative stress
Abstract
This work shows that TRIAD, a combination of sodium pyruvate, vitamin E
and egg yolk fatty acids, has an antioxidant protective action on cultured P19
neurons exposed to oxidative stress. Oxidative stress was induced by
incubation
with prooxidant systems that generate major reactive oxygen species produced
by
ischemia-reperfusion of the brain in vivo, namely 1) xanthine/xanthine oxidase
system to produce ~02 superoxide radicals and H202, 2) H202 it self, and 3)
H202
in the presence of Fe2+ to produce ~0H hydroxyl radicals. TRIAD-induced
resistance to injury caused by oxidative stress was assessed by measurement of
cell viability. TRIAD concentrations less than 3X permitted to achieve
complete
protection of neurons. Optimal concentrations of TRIAD with neurons exposed to
peroxide-based systems were directly related to the oxidant power of the
systems
as measured by oxidation of N,N-diethyl-p-phenylenediamine. However, higher
concentrations of TRIAD than those predicted from in vitro analyses were
required
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to protect neurons against oxidative stress. In addition, the results also
show that
the respective contribution of pyruvate and of fatty acids + vitamin E
combination
may differ between prooxidant systems and between in vitro or cell culture
situations. These results indicate that TRIAD components have different
mechanisms of action and that these mechanisms are further modulated by cell
metabolism. Generally, in our experimental models, pyruvate was a major
contributor of the antioxidant action of TRIAD and its effect was increased by
fatty
acids and vitamin E in some cases in an additive manner and in other cases
synergistically.
Abbreviations DPD : N,N-diethyl-p-phenylenediamine; KH : Krebs-Henseleit;
LDSp : lethal dose 50 or dose that causes 50% mortality; PBS : phosphate
buffer
saline; OFR : oxygen free radical; ROS : reactive oxygen species; XA :
xanthine;
XAO: xanthine oxidase, SOD: superoxide dismutase; CAT: catalase; GP:
glutathion (GSH)-peroxidase.
1. Introduction
1.1 Oxidative stress and antioxidant defenses in normal and pathophysiological
heart and brain
Reactive oxygen species (ROS) including hydrogen peroxide, oxygen free
radicals (OFR) such as superoxide and hydroxyl radicals, and their derivatives
are
generated by normal cellular metabolism but are potent cellular toxicants when
they are produced in excess and thus cause an oxidative stress to cells (LeBel
and Bondy, 1991; Gutteridge, 1994; Chan, 1996). The organism has several
strategies to maintain ROS-induced damage at low levels : a) to eliminate ROS
(e.g. SOD, CAT and GP enzymes shown in Fig.1 ), b) to scavenge ROS by
trapping them (e.g. ascorbic acid) or by breaking their propagation (e.g.
vitamin E),
c) to sequester iron or other metals in non- or low reactive forms, and d) to
repair
molecular damages (Gutteridge, 1994).
ROS have been implicated in the development of many heart and brain
dysfunctions (Takemura et al., 1994; Chan, 1996; Maiese, 1998) and
ischemiaireperfusion insults to these organs are among the leading causes of
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mortality in America (Takemura et al., 1994; Chan, 1996; Maiese, 1998). These
insults are caused by complete or partial local occlusions of vasculature and
by
trauma to heart and brain. ROS as those found in ischemia-reperfusion events
are also involved in the evolution of several neurodegenerative diseases or
produced in brain following an exaggerated activity of this organ (e.g.
epilepsy).
Various pathways generating superoxide radical (~Oz-) and other ROS - also
known as reactive oxygen intermediates (R01) - have been identified, such as:
activation of polymorphonuclear leukocytes, autoxidation of catecholamines,
reactions of xanthine oxidase and NADPH oxidase, or metabolism of arachidonic
acid. The harmful effects of superoxide radical and its by-products are
dramatically
increased in the presence of transition metals. The ferrous (Fe2+) ion
generated by
the Haber-Weiss reaction catalyses the formation of the highly aggressive
hydroxyl (~OH) radical, via Fenton reaction. The OFR concentration at
reperfusion
is higher than during ischemia. OFR may contribute to reperfusion injury by
interacting with membrane polyunsaturated fatty acids (PUFA) and generating
lipid
peroxides which increase membrane permeability and alter ionic homeostasis.
Inhibition of free radical accumulation with OFR scavengers, antioxidant
enzymes,
and spin-trap agents was shown to reduce the severity of damages to brain.
However, the benefits of these treatments gradually vanish with time,
especially
during long-term utilization in neurodegenerative diseases, or are lessen by
the
apparition of adverse secondary effects. Therefore, the identification of
other
therapeutics agents still remains highly desirable.
1.2 Aspects on TRIAD and its therapeutic role
As stated herein before, TRIAD is a combination of pyruvate, antioxidant and
fatty acids for which many uses have been patented. Preferably, TRIAD
comprises
sodium pyruvate, vitamin E and egg yolk. Although this combination is also
known
under the name of CRT (Cellular Resuscitation Therapy), the current
denomination of TRIAD is use throughout this document.
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These three agents were shown to act synergistically to ameliorate wound
healing (Martin, 1996; Sheridan et al., 1997) and to reduce oxidative damage
to
keratinocytes and monocytes exposed to ultraviolet light (Martin, 1996) or to
hepatocytes treated with doxorubicin (Gokhale et al., 1997). The presumed
respective role of each agent of the antioxidant combination is a) for
pyruvate, to
bind stochiometrically to H202, b) for vitamin E, to interrupt the propagation
of lipid
peroxidation, and c) for egg yolk, to provide a balanced mix of fresh
unsaturated
and saturated fatty acids which will help in membrane repair (Martin, 1996).
1.3 Presentation of the study
The goal of this study was to determine if TRIAD has an antioxidant
protective action on cultured P19 neurons exposed to oxidative stress. The
choice
of this model is related to the fact that the P19 cell line is establishing
itself as a
flexible model of neurons of central nervous system. Oxidative stress was
induced
by incubation with prooxidant systems that generate major ROS produced by
ischemia-reperfusion in vivo. Prooxidant systems used are: i) XA/XAO system to
produce ~02 and H202 ii) H202 it self, and iii) H202 in the presence of Fe2+
to
produce ~OH. Resistance of neurons to injury induced by oxidative stress was
assessed by measurement of cell viability. In all cases, different
concentrations of
TRIAD were tested in order to determine those that permitted to achieve a
complete protection and also tested the contribution of TrIAD components to
the
overall protection. In addition, when applicable, the antioxidant properties
of
TRIAD in vitro were measured in order to understand some aspects of the
protection afforded by this mix in live models.
2. Materials and Methods
Materials
Vitamin E (a-tocopherol type VI in oil), sodium pyruvate, ethylenediamine
tetraacetic acid (EDTA), N,N-diethyl-p-phenylenediamine (DPD), and xanthine
(XA) were purchased from Sigma Chem. Co. Xanthine oxidase (XAO) was from
Boehringer Mannheim. Neurobasal~ , L-glutamine and B27 supplement were from
Gibco-BRL. Alamar Blue was purchased from Medicorp (Montreal, Quebec). Fresh
egg yolk was used as the source of fatty acids. The other current chemicals
were
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reagent grade (from Sigma Chem. Co., St-Louis) and were used without further
purification.
Methods
2.1 Preparation of TRIAD
The 1X TRIAD concentration was prepared as per Gokhale et al. (1997) and
contained 0.1 % v/v fresh egg yolk, 1 unit/ml vitamin E (a-tocopherol type VI
in oil)
and 10 mM sodium pyruvate. Stock 5X (5 fold) or 10X (10 fold) concentration of
TRIAD was freshly prepared before each experiment by carefully mixing the
three
agents to get a homogenous suspension. TRIAD mixtures were made in
phosphate buffer saline (PBS; 136 mM NaCI, 2.7 mM KCI, 1.5 mM KH2P04 and
8 mM Na2HP04, pH 7.4). Pyruvate was soluble in and egg yolk miscible with this
saline physiological buffer. Aseptically drawn egg yolk and vitamin E
suspension
(vitamin E in oil combined to 70% ethanol in a 2.5:1 ratio) were added at the
desired final concentrations to a 100 mM stock pyruvate solution prepared in
PBS
and filter-sterilized on 0.22 Nm.
Although not tested with neuronal cells, a modified preparation of TRIAD was
shown to be effective to protect isolated hearts. Modification of TRIAD
preparations was as follows: 5X or 10X genuine preparations were centrifuged
at
15 000 x g for 20 min, at 4°C, and the resulting supernatants (S1 )
filtered on
Whatman paper filter #54. The final filtered supernatant was named TRIAD (S2)
and used to perfuse hearts. The different concentrations of TRIAD (S2)
preparation were obtained by subsequent dilution with Krebs-Henseleit
physiological saline buffer (i.e. TRIAD (S2) 1X was obtained by 10 fold
dilution of
stock TRIAD (S2) 10X preparation).
2.2 Culture and neuronal differentiation of P19 cells
Culture and neuronal differentiation of P19 embryonal carcinoma cells were
done according to the procedures of Jeannotte et al. (1997) with the following
modifications for microscale adaptation of cultures to 96-well plates: cell
aggregates obtained at day 4 of the treatment of P19 cells with retinoic acid
were
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trypsinized with 0.025% trypsin-1 mM EDTA in PBS and subjected to mechanical
passages to obtain individual cells which were seeded in gelatin-precoated
microwells at a density of 0.7-1 x 105 cells per well. The newly seeded cells
(neurons) were cultured in supplemented Neurobasal medium (Neurobasal~
containing 0.5 mM L-glutamine and 0.5% B27 supplement) until exposure to
oxidative conditions at day 7. Because this defined serum-free medium sustains
growth of P19 neurons (Yao et al., 1995) but discourages the proliferation of
fibroblasts, another cell derivative of the differentiation of P19 cells with
retinoic
acid (McBurney, 1993; Jeannotte et al., 1997), the cell populations in
microwells
were composed mostly of neurons (>_ 95%).
2.3 Exposure of P19 neurons to prooxidant systems
The prooxidant systems tested with P19 neurons were XA/XAO, H202, and
H202/Fe+2. Before neurons were exposed to either one of these systems, they
were carefully washed with PBS and then incubated at 37°C, in an
atmosphere of
95% ambient air and 5% C02, and in the specific conditions of each system, as
follows: i) from 0 to 90 min in the presence of PBS containing 500 pM XA and
50 mU/ml XAO, with the enzyme added last to start the reaction; ii) for 30 min
in
the presence of PBS containing 0 to 10 mM H202 with peroxide added last; and
iii)
for 30 min in the presence of PBS containing 0 to 10 mM H202 plus 50 pM FeCl2,
with peroxide added last. Conditions were initially taken from Cini et al.
(1994) for
XA/XAO, from Desagher et al. (1996) for H202, and from Takemura et al. (1994)
for H202/Fe+2 systems respectively, and adapted to produce dose- or time-
dependent cell mortality in P19 neurons. The first studies done with the
H202/Fe2+
system included the addition of 500 NM ascorbic acid; however, since responses
of P19 neurons were similar whether or not ascorbic acid was present, this
vitamin
was omitted in subsequent experiments.
When TRIAD or its components were tested for their antioxidant action,
they were administered to cells just prior the addition of XAO or H202. After
incubation of cells under oxidative conditions, the prooxidant medium was
removed and replaced with 200 p1 of supplemented Neurobasal-minus AO
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(Neurobasal~ containing L-glutamine and the B27-minus AO supplement). B27-
minus AO is a version of B27 supplement sold by Gibco-BRL from which normally
present antioxidants (AO = vitamin E, catalase, SOD and GSH) have been
removed. Cells were incubated in this Neurobasal-minus AO medium for 16 h, at
37 ~C, 5 % C02, and for a further 7 h in the same medium but in the presence
of
Alamar Blue for viability measurement.
Another protocol was also tested for XA/XAO system to generate a milder
oxidative stress. In this new protocol, P19 neurons were exposed during 40 min
to
250 NM XA and varying concentrations of XAO (0 to 20 mU/mL) in PBS. At end of
stress, PBS was changed for Neurobasal-minus AO medium and cells were
incubated for 7 h, readily in the presence of Alamar Blue, for viability
measurement.
2.4 Cell viability assay
Fifteen (15) p1 Alamar Blue was added to the culture medium of each well
(200 p1) and incubation resumed for 7h at 37°C, 5% C02. A 180-NI
aliquot of each
culture medium was read by fluorescence using a wavelength of 544 nm for
excitation and of 590 nm for emission; fluorescence increases upon reduction
of
the dye by metabolic activity of viable cells. Fluorescence determinations
were
done with a fluorimeter adapted to read microplates. Viability is reported as
%,
comparing the fluorescence units obtained for cells exposed to oxidative
conditions to those of control (non-exposed) cells.
2.5 In vitro antioxidant capacity
Oxidation of N,N-diethyl-p-phenylenediamine (DPD) by a prooxidant system
was used as a general reporter of the amount of ROS generated by that system
(Anonymous, 1985; Chahine et al., 1991 ). Antioxidant capacity of preparations
of
TRIAD (or of its components) was defined as the ability to inhibit the
oxidation of
DPD by prooxidants.
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To estimate the antioxidant capacity of TRIAD preparations in the prooxidant
conditions used with P19 neurons, DPD was added to a final concentration of
32 mM to 200 p1 of each prooxidant system described above (see section 2.4)
and
incubated for the times tested with the cells. At the end of incubation, the
amount
of oxidized DPD was determined at 560 nm using a spectrophotometer adapted to
microscale measurement.
3. Results
The P19 embryonal carcinoma cell line is establishing its place as a versatile
cell model for neurons of central nervous system (McBurney, 1993; Yao et al.,
1994; Finley et al., 1996; Parnas and Linial, 1997; Jeannotte et al., 1997).
These
cells differentiate into neurons, astrocytes and fibroblast-like cells
following
induction with retinoic acid, and their neuronal derivatives mature into
functional
neurons. Indeed, P19-derived neurons express a variety of neuron-specific
proteins, acquire cell polarity of neurons, form synapses, synthesize and
release
neurotransmitters and neuropeptides, and their membranes respond to
electrophysiological stimuli (McBurney, 1993; Finley et al., 1996; Parnas and
Linial, 1997; Jeannotte et al., 1997). The P19 system presents several
advantages
over other neuronal models for screening tests using cultured cells: i) P19
neurons
like primary neurons are highly differentiated (in contrast, neuroblastoma
cells
often used as neuronal models are poorly differentiatc:~~), ii) acquisition of
P19
neurons does not depend on the sacrifice of animals, and iii) although P19
neurons are post-mitotic and therefore do not divide as do neuroblastoma
cells,
they can be obtained in large quantities since P19 stem cells propagate at
high
rates. Considering that they resemble primary neurons and can be easily and
reproductively obtained, we thus used P19 neurons to study the neuroprotective
action of TRIAD.
Legends to the Figures
Fig. 1. In vitro ROS production by peroxide-based prooxidant systems used with
P19 neurons. The relative amount of ROS produced by H202 and H202/Fe2+
systems in the conditions used with P19 neurons were measured
spectrophotometrically, in absence of cells, by following oxidation of N,N-
diethyl-p-
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phenylenediamine (DPD). The experiment was done twice, in triplicate
determinations, and results are expressed as means ~ errors to the means. The
H202/Fe2+ system contained 50 pM FeCl2; the addition of 500 NM ascorbic acid
to
this system did not change the amount of oxidized DPD (not shown), indicating
that iron ions were in a concentration sufficient to increase the oxidative
stress
induced by H202 alone.
Fig. 2. Protection provided by TRIAD to P19 neurons exposed for different
times
to XA/XAO mediated oxidative stress. P19 neurons were exposed from 0 to 90
min to 500 ~M XA and 50 mU/mL XAO in the absence (No protection) or presence
of different concentrations of TRIAD. At end of stress, cells were incubated
for 16
h in a fresh provision of culture medium lacking XA, XAO and TRIAD. Afterward,
Alamar Blue was added and cells were further incubated for 7 h for viability
determination. Viability values are reported as percentages, with 100
corresponding to the response of P19 neurons not exposed to XA/XAO. The
experiment was done once, in duplicate determinations, and results are
expressed
as means (~ errors to the means). Error bars are similar for all curves
although
they are shown for two curves only, for the purpose of clarity.
Fig. 3. Protection provided by TRIAD components to P19 neurons exposed for
different times to XA/XAO mediated oxidative stress. Exposure to XA/XAO and
viability measurement were done as indicated in the legend to Fig. 2. The
experiment was done twice, in duplicate determinations, and results are
expressed
as means ~ errors to the means.Vit.E, vitamin E; F.A., fatty acids.
Fig. 4. Protection provided by TRIAD to P19 neurons exposed to oxidative
stress
mediated by different concentrations of XAO. P19 neurons were exposed for 40
min to 250 pM XA and different concentrations of XAO in the absence (No
protection) or presence of 0.5X TRIAD. At end of stress, cells were incubated
for 7
h in a fresh provision of culture medium lacking XA, XAO and TRIAD, but
containing Alamar Blue for viability determination. Viability values are
reported as
percentages, with 100 % corresponding to the response of P19 neurons not
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exposed to XA/XAO. The experiment was done twice, in triplicate
determinations,
and results are expressed as means ~ errors to the means.
Fig. 5. Protection provided by TRIAD to P19 neurons exposed to hydrogen
peroxide mediated oxidative stress. P19 neurons were exposed during 30 min to
different concentrations of H202 in the absence (control = no protection) or
presence of different concentrations of TRIAD. At end of stress, cells were
incubated for 16 h in a fresh provision of culture medium lacking H202 and
TRIAD.
Alamar Blue was then added and cells were further incubated for 7 h for
viability
determination. Viability values are reported as percentages, with 100
corresponding to the response of P19 neurons not exposed to H202. The
experiment was done 5 times, in duplicate determinations, and results are
expressed as means ~ S.D. Omission of the 16 h incubation period did not
change
the relative responses.
Fig. 6. Protection provided by TRIAD components to P19 neurons exposed to
hydrogen peroxide mediated oxidative stress. Exposure to H202 and viability
measurement were done as indicated in the legend to Fig. 5. The experiment was
done four times, in triplicate determinations, and results are expressed as
means ~
S.D. Vit.E, vitamin E; F.A., fatty acids.
Fig. 7. Protection provided by TRIAD to P19 neurons exposed to H202/Fe2+
prooxidant system. Cell treatments were as described in the legend to fig. 5
except that iron (50 NM FeCl2) was present in the stress medium to generate
hydroxyl radicals. Viability values are reported as percentages, with 100%
corresponding to the response of P19 neurons not exposed to H202/Fe2+. The
experiment was done 3 times, in duplicate determinations, and results are
expressed as means ~ S.D.
Fig. 8. Protection provided by TRIAD components to P19 neurons exposed to
H202/Fe2+ prooxidant system. Exposure to H202/Fe2+ and viability measurement
were done as indicated in the legend to Fig. 7. The experiment was done twice,
in
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duplicate determinations, and results are expressed as means ~ errors to the
means. Vitamin E + fatty acids did not protect in that system (not shown).
Fig. 9. In vitro antioxidant capacity of TRIAD in the conditions of H202
prooxidant
system used with P19 neurons. Effect of different concentrations of TRIAD on
the
oxidation of DPD induced by H202. The experiment was done 5 times, in
duplicate
determinations, and results are expressed as means ~ S.D.
Fig. 10. In vitro antioxidant capacity of TRIAD in the conditions of H202/Fe2+
prooxidant system used with P19 neurons. Effect of different concentrations of
TRIAD on the oxidation of DPD induced by H202/Fe2+. The experiment was done 7
times, in duplicate determinations, and results are expressed as means ~ S.D.
Fig. 11. In vitro antioxidant capacity of TRIAD components in the conditions
of
H202 prooxidant system used with P19 neurons. Effect of TRIAD components on
the oxidation of DPD induced by H202. The experiment was done 4 times, in
duplicate determinations, and results are expressed as means ~ S.D. Vit. E,
vitamin E; F.A., fatty acids.
Fig. 12. In vitro antioxidant capacity of TRIAD components in the conditions
of
H202/Fe2+ prooxidant system used with P19 neurons. Effect of TRIAD
components on the oxidation of DPD induced by H202/Fe2+. The experiment was
done 3 times, in duplicate determinations, and results are expressed as means
~
S.D. Vit. E, vitamin E; F.A., fatty acids.
3.1 In vitro oxidant capacity of peroxide-based prooxidant systems used with
P19
neurons
Three prooxidant systems were used to induce oxidative stress in P19
neurons, namely XA/XAO, H202, and H202/Fe+2. The relative oxidant capacity of
the two peroxide-based system was determined by following oxidation of DPD in
vitro. Fig. 1 shows that addition of Fe+2 increased the oxidant power of H202.
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Comparison with the oxidant power of XA/XAO system could not be done since
this system did not directly oxidize DPD. It is believed that addition of SOD
enzyme to XA/XAO system in vitro would convert ~02- radicals produced by the
system to measurable proportionate amounts of H202 molecules.
3.2 Neuroprotection afforded by TRIAD against oxidative stress induced by
XAlXAO
When exposed during 90 min to TRIAD in the absence of an oxidative stress,
P19 neurons remained completely viable even for concentration 9X of the
antioxidant mix (results not shown). Since genuine preparations of TRIAD were
not toxic to these cells, they were used without further treatment (i.e. TRIAD
and
not TRIAD (S2) was used with P19 neurons). However, as explained hereinbefore
(see section 2.1 ), the genuine preparations could have been centrifuged and
the
resulting supernatants filtered.
XA/XAO system was used to generate superoxide radicals (~OZ ) in addition
to H202. When exposed to that system for different times, a large proportion
of
P19 neurons died after 5 min (Fig. 2, No protection). When TRIAD was present
in
the culture medium, it decreased cell mortality cai.~s~;d by the prooxidant
conditions. Protection was concentration dependent and reached almost
completion at concentration 9X of TRIAD (Fig. 2). The individual contribution
of
fatty acids, vitamin E and pyruvate to the protection provided by TRIAD
against
neuronal death caused by XA/XAO was also determined. A 1X concentration for
TRIAD and its components was used instead of an optimal value of 9X in order
to
reveal the eventual synergistic effects, if any. Fig. 3 shows that pyruvate 1X
protected only slightly less than TRIAD 1X. Fatty acids and vitamin E together
protected similarly to pyruvate or TRIAD during the 30 first min of incubation
with
XA/XAO (Fig. 3). However, the protective effect fell off rapidly from 30 to 90
min
while that of pyruvate remained stable (Fig.3). At 90 min of exposure, the
summation of the protective effect separately shown by pyruvate and by fatty
acids
+ vitamin E reproduced the protection provided by TRIAD itself (Fig. 3),
indicating
that fatty acids and vitamin E had an additive effect on pyruvate action.
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It was realized that the pro-oxidant conditions used just above were
probably very drastic since most neurons died after 5 min (Figs 2 and 3, No
protection). According to the protocol used, cells were exposed to 500 pM XA
and
50 mU/mL XAO for up to 90 min, then incubated in fresh culture medium - not
containing XA/XAO - for 16 h hours in the absence of AlamarBlue, and for a
further 7 h in the presence of the dye. It was believed that any residual
amount of
XA/XAO left in the culture medium had time to continue to attack neurons,
explaining why viability was lost abruptly. Therefore, milder conditions were
applied in order to observe a gradual loss of viability. In the new protocol,
neurons
were first exposed for 40 min to 250 NM XAN in the presence of various
concentrations of XAO (0-20 mU/mL), and then readily incubated in fresh
culture
medium in the presence of AlamarBlue for 7 h. If there was residual amount of
XAO left in the fresh medium, it increased mortality but in a manner
proportional to
the initial concentration of the enzyme. With the new protocol, the loss of
viability
caused by XA/XAO was indeed gradual and depended on the concentration of
XAO in the solution (Fig. 4). Interestingly, 0.5X TRIAD was sufficient to
provide
almost complete protection in those conditions.
3.3 Neuroprotection afforded by TRIAD against oxidative stress induced by
H202
Fig. 5 shows that P19 neurons died in a concentration dependent manner
when exposed to hydrogen peroxide. The LD5p value was 0.3 mM hydrogen
peroxide. TRIAD protected cells against death caused by H202 and a complete
protection was achieved with 1X concentration of the antioxidant mix. TRIAD 3X
and 5X also provided complete protection (not shown). When assayed at 0.5X
suboptimal concentration of TRIAD, fatty acids or vitamin E alone did not
provide
protection against oxidative stress caused by hydrogen peroxide (not shown).
Combination of these agents was protective up to 0.5 mM H202 but fell off
rapidly
at higher concentrations of the prooxidant (Fig. 6). In contrast, pyruvate
provided
a substantial and stable protection up to 10 mM H202 (Fig. 6). Comparison of
the
protection separately provided by 0.5X pyruvate and 0.5X TRIAD shows that 0.5X
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TRIAD was more efficient by about 2-fold (Fig. 6), indicating that fatty acids
and
vitamin E increased the protective action of pyruvate in a synergistic manner.
3.4 Neuroprotection afforded by TRIAD against oxidative stress induced by H202
in the presence of iron
Addition of Fe2+ to generate hydroxyl radicals was slightly more deleterious
to
neurons than hydrogen peroxide alone. As an example, 1 mM H202 caused
approximately 70% cell mortality (Fig. 5) whereas the same concentration of
peroxide in the presence of iron caused more than 80% cell mortality (Fig. 7).
This
is in agreement with the relative oxidant power of each system (Fig. 1 and
section 3.1). Increased stress required 3X TRIAD instead of 1X to provide
complete neuroprotection (Fig.7). Pyruvate contributed for most of TRIAD
protective effect in this prooxidant system (Fig. 8).
3.5 In vitro antioxidant capacity of TRIAD with peroxide-based prooxidant
systems used with cultured neurons
Antioxidant capacity of TRIAD and of its components were evaluated in vitro
by following oxidation of DPD by the two peroxide-based prooxidant systems.
Figs. 9 and 10 show that optimal antioxidant concentrations of TRIAD for
peroxide
systems are smaller in vitro than in cell culture situations. Indeed, 0.5X and
1X
TRIAD respectively abolished DPD oxidation (Fig. 9) and neuron mortality (Fig.
5)
induced by oxidative stress in the H202 system, and the counterpart values
were
1X (Fig. 7) and 3X (Fig. 10) for the H202/Fe2+ system. These observations
suggest that even low levels of oxidative stress could exert irreversible
detrimental
effects on cells, requiring higher concentrations of TRIAD to be prevented.
Results of DPD oxidation measurement show that there are resemblance and
also differences regarding the relative protection afforded by each component
of
TRIAD in vitro, compared to cell culture situations, giving clues on the
possible
mechanism of action of TRIAD. Differences were obvious with the H202 system.
In
cultured neurons, pyruvate mostly (60%) contributed to the neuroprotective
action
of TRIAD against H202-induced injury whereas fatty acids and vitamin E did not
by
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themselves provide much protection to cells (less than 10% for peroxide
concentration higher than 1 mM) although they increased pyruvate action
synergistically (section 3.3 and Fig. 6). In contrast, in vitro, fatty acids +
vitamin E
completely inhibited the oxidation of DPD by H202 while pyruvate also provided
a
substantial although not total antioxidant effect (Fig. 11 ). Discrepancy
between
cultured cells and in vitro situations could be explained by the presence or
absence of a cell membrane barrier which distinguishes inside and outside
protection. In vitro, fatty acids + vitamin E combination and pyruvate can
separately inhibit DPD oxidation by H202 because they are all in the same
compartment. In contrast, there are at least two compartments in cell cultures
(inside and outside cells). Because pyruvate can be uptaken by neurons, it can
protect them from both exterior and interior damages induced by an excess of
H202 which is known to diffuse easily through cell membranes. Fatty acids
(including lecithin present in egg yolk) and vitamin E which do not pass
easily
through membranes during the 30 min of treatment, could not afford important
intracellular protection but rather helped pyruvate by providing extracellular
defense. Fatty acids + vitamin E combination were only as powerful as pyruvate
to
inhibit DPD oxidation when iron was added to hydrogen peroxide (Fig. 12). It
is
possible that this combination lost part of its antioxidant properties because
egg
yolk fatty acids were deteriorated by iron-catalyzed formation of hydroxyl
radicals
which are known to initiate lipid peroxidation (Gutteridge, 1994; Chan, 1996).
Neurons would thus count more on pyruvate for their protection in the
H202/Fe+2
than in the H202 system. Unfortunately, the XA/XAO system could not oxidize
DPD
by itself. Therefore direct comparison between this system and the peroxide-
based
systems cannot readily be made.
4. Discussion
This study showed that TRIAD has an antioxidant capacity in vitro and a
protective action on cultured P19 neurons exposed to oxidative stress. The
results
are summarized in Table I below and indicate that association of pyruvate,
vitamin
E and fatty acids can protect cells against extracellular and intracellular
oxidative
damages, by different mechanisms. Since oxidative damage in vivo can be caused
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by extracellular or intracellular (or both) ROS sources, association of the
three
components of TRIAD appears very useful.
Table I. Minimal concentration of TRIAD (X-fold) for complete antioxidant
protection.
Prooxidant system
Model XA/XAO H202 H2021Fe2+
Neurons - 0.5X 1X 3X
(Pyruvate > F.A. + Vit.E) (Pyruvate > F.A. + Vit.E) (Pyruvate > F.A. + Vit.E)
T or 1'1' 1'1' or TT1' 1' or TT
In vitro Not determined* 0.5X 1X
(F.A. + Vit.E > Pyruvate) (pyruvate - F.A. + Vit.E)
T TT
The results are presented for cultured P19 neurons and their in vitro
counterpart (i.e.
prooxidant conditions tested on DPD, in absence of cells). The relative
contribution of
pyruvate and of F.A. + Vit.E is given between parentheses. The accompanying
arrows
indicate that pyruvate action was apparently increased in les:~ tP~an additive
(T), additive
(TT) or synergistic (1'TT) manner by F.A. + Vit.E. *: not determined because
the
prooxidant system cannot directly oxidize DPD. F.A.: fatty acids; Vit.E:
vitamin E.
Although not shown in this study, the Inventors have demonstrated that
TRIAD protected hearts against oxidative stress generated via several
important
ROS (~02-, H202 and ~OH), physiologically produced in ischemia-reperfusion
conditions. Smaller concentrations of TRIAD was needed to protect isolated
heart
from ischemia-reperfusion induced damages than to protect isolated neurons.
There are several explanations to this difference. First, concentrations of
ROS
used in this study with neurons were high and likely more important than those
encountered naturally. In addition, ROS produced exogenously have conceivably
an easier access to cells grown as monolayers than to cells tightly organized
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within an organ, and organ being formed of different types of cells, it likely
possesses a larger spectrum of antioxidant defenses than have monotypic cells
in
cultures. However, 1X and 3X were very effective concentrations of TRIAD in
cultured neurons. In peroxide-based prooxidant systems, concentration
dependency of the protective effect of TRIAD in cultured cells matched that of
its
antioxidant capacity in vitro. However, higher concentrations than those
predicted
from in vitro analyses were systematically needed with neurons. These results
suggest that cellular damages can accumulate before TRIAD entirely exerts its
protective action and/or that cellular metabolism can trigger ROS
transformation
from one type to a more reactive one.
Pyruvate was the most important component of TRIAD with cultured
neurons, accounting for 60 to 90% of the protective action of TRIAD. Fatty
acids
and vitamin E by themselves did not provide much protection but they increased
the protective effect of TRIAD, most often in an additive but sometimes in a
synergistic manner. Pyruvate is considered as an important scavenger of H202
and compared to the other agents of TRIAD, provides important intracellular
neuroprotection due to the capacity of neurons to import pyruvate from
extracellular sources. As an exception to the important contribution of
pyruvate,
antioxidant capacities of TRIAD in vitro' with H202 prooxidant system was
mainly
contributed by fatty acids and vitamin E. Addition of Fe+2 to H202 diminished
the
antioxidant power of fatty acids + vitamin E in vitro, an effect which could
be
related to a possible peroxidation of egg yolk lipids by newly formed hydroxyl
radicals.
The Applicant is aware of the apparent contradiction between the results
obtained with cultured neurons (Fig. 6, where pyruvate is the major protector)
and
those obtained with DPD (Fig. 11, where pyruvate is not the major protector)
for
H202 system, as discussed in the precedent paragraph. A possible explanation
for
this observation is the existence of at least two compartments in the cell
situation
(intracellular and extracellular compartments) compared to only one
compartment
in the test tube assay.
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In vitro, either pyruvate alone or the combination of vitamin E + fatty acids
is
in concentration sufficient to decrease or prevent DPD oxidation by hydrogen
peroxide (Fig. 11 ). However, damages caused to cells by hydrogen peroxide are
intra- as well as extracellular, since this ROS can pass through cell
membrane. In
vivo, pyruvate would provide intracellular protection because it is uptaken by
cells,
while vitamin E and fatty acids which do not pass cell membranes provide
extracellular protection only. One could imagine that if cells are
permeabilized,
then vitamin E + fatty acids and pyruvate would perhaps provide an overall
protection resembling that seen in vitro. However, the formulation of TRIAD
tested,
has the advantage of both extracellular (membrane) and intracellular effects.
The neuroprotective action of TRIAD is likely related to its three
components. Pyruvate, able to enter the cell, will enhance intracellular
defense,
while vitamin E and fatty acids will improve membrane functionality,
eventually
limiting the leakage of cellular Fe2+ ion (easily generated by reduction of
Fe3+ ~
Fe2+, induced by superoxide anion which is a reductive agent), preventing thus
the
production of hydroxyl radical (~OH) via the Fenton and Haber-Weiss reactions,
Fenton reaction : Fe2+ + H202 ~ Fe3+ + ~OH + OH-
Haber-Weiss reaction : Fe3+ + ~02- -~ Fe2+ + 02
Mechanisms of iron involvement are not fully elucidated, but there is a
growing consensus that oxidative tissue damage is related to non-heme cellular
iron mobilized from cytosolic metal-containing sites: e.g. ferritin stores
within cells.
In this work, the protective effect of TRIAD was studied during co-exposure of
neurons to both prooxidant conditions and TRIAD. In a therapeutic point of
view,
this antioxidant TRIAD mix could conceivably be also used to prevent damages
caused to tissues by acute or chronic exposure to oxidative stress or to
recover
from such injuries. In this aspect, the potential of egg yolk to serve as a
source of
fatty acids to repair membrane damages and that of pyruvate to serve as fuel
for
cells could confer important neurotrophic properties to TRIAD and extent
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31
application of TRIAD to neurodegenerative diseases. This is particularly
relevant
since oxidative stress is considered as an etiologic or at least an
aggravating
factor in several of these diseases. TRIAD thus has a high therapeutic
potential in
preventive or reparative strategies.
5. Conclusive remarks
This study shows that TRIAD has an antioxidant neuroprotective action on
cultured P19 neurons exposed to oxidative stress. Optimal concentrations vary
with the type and prooxidant power of ROS generating systems. Pyruvate is a
major contributor of antioxidant properties of TRIAD ex vivo (heart, not
shown) and
in neuronal cultures, especially when TRIAD is administered just prior
induction of
an oxidative stress and remains present for short time of treatment (30-40 min
for
neurons). The contribution of vitamin E and egg yolk fatty acids may appear
even
more important in antioxidant defense when TRIAD is administered for longer
periods (before, during and after oxidative stress).
This study also yields in the development of an essential concept which
comprises two aspects:
i) combinations of antioxidants having different mechanism of action provide
higher protection to oxidative stress than any single antioxidant; and
ii) synergistic protection is a "latent" property of antioxidant combinations
and
does not necessarily manifest itself in all prooxidant conditions.
Aspect ii) is best illustrated by the results of Fig. 6 which showed that
while
pyruvate and vitamin E + fatty acids each provides half of the protective
effect of
TRIAD at low hydrogen peroxide concentrations, at higher concentrations of the
prooxidant, TRIAD remained almost as protective even though vitamin E + fatty
acids were no longer active by themselves. Synergistic neuroprotection was
thus
seen under more pronounced oxidative stress conditions.
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Finally, although the term "TRIAD" used herein refers to a composition
comprising sodium pyruvate, vitamin E and egg yolk fatty acids, a person
skilled in
the art will understand that the compositions of the present invention are not
restricted to these sole specific components as explained previously in the
first
part of the section "DETAILED DESCRIPTION OF THE INVENTION".
6. References
Throughout this paper, reference is made to a number of articles of
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Of course, numerous modifications and improvements could be made to the
embodiments that have been disclosed herein above. These modifications and
improvements should, therefore, be considered a part of the invention.
