Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE PROTECTION OF MITOCHONDRIA
BACKGROUND OF THE INVENTION
ivlitochondria are subcellular organelles present in all oxygen-utilizing
organisms in which
energy in the form of adenosine triphosphate (ATP) is generated, and oxygen in
reduced to
water. Ninety percent of the oxygen taken in is consumed in mitochondria. A
substantial
byproduct of this ATP generation is the formation of potentially toxic oxygen
radicals. For
example, it is estimated that 1-2% of all reduced oxygen yields superoxide (O~-
) and
hydrogen peroxide (H,O,). Other reactive oxygen species (ROS) that form are
singlet oxygen
(' O~) and hydroxyl radical (OH ~ ). Under stress conditions in the cell this
can rise to 10% of
all consumed oxygen. Mitochondrial membranes are sensitive to lipid
peroxidation and
depolarization resulting from these ROS. Mitochondrial damage is also a result
of exposure
to sunlight, which forms ROS as indicated above. Because damage to
mitochondria is
believed to be the cause or an important factor in some diseases, such as
cancer, diabetes,
cataract, neurodegenerative disease, porphyrias, cardiovascular disease, and
also a contributor
to the complications of aging, a method of protecting mitochondria from such
damage,
repairing such damage, is desired. Cellular damage from burns to the skin and
lungs from
contact with or exposure to fire and other sources of intense heat is mediated
through radical
damage. Furthermore, exposure to adverse environmental factors, including
industrial air
pollutants and petroleum and tobacco combustion products, may contribute to
oxidative
damage to pulmonary and other tissues of the body. In addition, various
therapeutic regimens
such as chernotherapeutic drugs and radiation therapy for the treatment of
dysproliferative
diseases induce significant oxidant-stress-related side effects, such as
cardiotoxicity. The
present invention relates to applied agents which protect the mitochondria
from such damage.
L-ergothioneine is a sulphur-containing amino acid which is found in many
mammalian
tissues but is not endogenously synthesized and must be consumed in the diet.
Although it
exists in some tissues in millimolar quantities, its exact role is uncertain
(see: Melville, 1959,
V itamins and Hormones 7:155-204). It is generally regarded as an antioxidant,
although
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results are conflicting. Some regard it as a scavenger of hydrogen peroxide
(see: Hartman,
1990, Methods in Enzymology 186:310-318), while others contend that it does
not readily
react with hydrogen peroxide but does scavenge hydroxyl radical (see: Akamnu
et al., 1991,
Arch. Biochem. Biophys. 298:10-16, 1991). Although previous in vitro studies
have
demonstrated its ability to protect DNA and proteins against phototoxic drug
binding induced
by UV radiation (e.g., van den Broeke et al., 1993, J. Photochem. Photobiol. B
17:279-286),
and to protect bacteriophage against gamma-irradiation ( Hartman et al., 1988.
Radiation
Research 114:319-330), in vivo results have not been as promising. Although L-
ergoth~oneine has been claimed as useful in topical formulations for
scavenging radicals and
UV light protectants for hair and skin damage (e.g., WO 9404129), Van den
Broeke et al.
(1993, Int. J. Radiat. Biol. 63:493-500) did not find topically-applied L-
ergothioneine
effective in an animal model of LTV-induced phototoxic drug binding to
epidermal
biomolecules. Other proposed in vivo uses have included lowering of
circulating lipoprotein
(a) levels (U. S. Patent 5,272,166), and inhibiting skin pigmentation, for
example, to remove
dark spots and freckles (JP 63008335 and JP 61155302).
As described above, numerous disease processes are attributed to the body's
adverse reaction
to the presence of elevated levels of reactive oxygen species (ROS) described
above. In the
eye, cataract, macular degeneration and degenerative retinal damage are
attributed to ROS.
Among other organs and their ROS-related diseases include: lung cancer induced
by tobacco
combustion products and asbestos; accelerated aging and its manifestations,
including skin
damage; atherosclerosis; ischemia and reperfusion injury, diseases of the
nervous system such
as Parkinson disease, Alzheimer disease, muscular dystrophy, multiple
sclerosis; other lung
diseases including emphysema and bronchopulmonary dysphasia; iron overload
diseases such
as hemochromatosis and thalassemia; pancreatitis; diabetes; renal diseases
including
autoimmune nephrotic syndrome and heavy metal-induced nephrotoxicity; and
radiation
injuries. Certain anti-neoplastic drugs such as adriamycin and bleomycin
induce severe
oxidative damage, especially to the heart, limiting the patient's exposure to
the drug. Redox-
active metals such as iron induce oxidative damage to tissues; industrial
chemicals and
ethanol, by exposure and consumption, induce an array of oxidative damage-
related injuries,
such as cardiomyopathy and liver damage. Airborne industrial and petrochemical-
based
~ _ T
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_J_
pollutants, such as ozone, nitric oxide, radioactive particulates, and
halogenated
hydrocarbons, induce oxidative damage to the lungs, gastrointestinal tract.
and other organs.
Radiation poisoning from industrial sources, including leaks from nuclear
reactors and
exposure to nuclear weapons, are other sources of radiation and radical
damage. Other routes
of exposure may occur from living or working in proximity to sources of
electromagnetic
radiation, such as electric power plants and high-voltage power lines, x-ray
machines, particle
accelerators, radar antennas, radio antennas, and the like, as well as using
electronic products
and gadgets which emit electromagnetic radiation such as cellular telephones,
and television
and computer monitors. Protecting mitochondria from these many etiologic
agents is
desirable.
SUMMARY OF THE INVENTION
It is an objective of the present invention to provide a means for protecting
mitochondria
from damage by providing methods and compositions by which mammalian
mitochondria)
membranes are protected.
It is another objective of the invention to provide methods and compositions
by which
mammalian mitochondria can be protected from oxidative damage.
It is a further objective of the invention to provide methods and compositions
which protect
mammalian mitochondria from being damaged by the effects of incident sunlight
as well as
other damaging radiation.
It is yet a further objective of the invention to provide methods and
compositions which
protect mammalian mitochondria from being damaged by the effects of airborne
oxidative
toxins, such as are present in industrial pollutants and in petrochemical and
tobacco
combustion products.
It is as yet another objective of the invention to provide methods and
compositions which
protects mammalian mitochondria from being damaged by the effects of elevated
levels of
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oxidative compounds and reactive oxygen species which occur in various disease
processes,
as well as that induced by various therapeutic agents and regimens.
It is another object of the present invention to provide diagnostic tests for
determining in a
mammal the extent and susceptibility to mitochondrial damage from radiation,
radicals, and
reactive oxygen species.
Mitochondria are damaged by oxidative damage due to natural and disease
processes as well
as by the effects of exogenous factors such as incident sunlight, exposure via
inhalation to
oxidative environmental toxins, electromagnetic radiation, consumption of
dietary oxidants,
and oxidative-stress-inducing pharmaceuticals, among others. In the present
invention,
pretreatment or treatment of cells with L-ergothioneine protects mitochondria
from such
damage and reduces the damage caused to mitochondria by sunlight and that
caused by the
presence of oxygen radicals. In one non-limiting example. L-ergothioneine is
combined in a
1 S pharmaceutically-acceptable carrier to form an agent for topical
application to the skin. The
invention includes methods for treatment using L-ergothioneine administered
orally or
parenterally. Effective application and delivery of L-ergothioneine is
enhanced by
encapsulation in a liposome, a preferred embodiment. In one example, a
liposome composed
of phosphatidyl choline, phosphatidyl ethanolamine, oleic acid and cholesteryl
hemisuccinate
is used. The liposome-encapsulated L-ergothioneine is also combined with a
pharmaceutically-acceptable carrier for topical application.
DETAILED DESCRIPTION OF THE INVENTION
Inhibition of oxidative damage to mitochondria in various tissues of the
mammalian body is
of therapeutic benefit for the prophylaxis and treatment of many pathological
conditions
ranging from those responsible for significant morbidity and mortality, such
as
atherosclerosis and cancer, to those of a less pathological but significant
adverse
psychological component, such as unsightly changes to the skin as a result of
long-term
photoaging. In diverse diseases such as cancer, diabetes, atherosclerosis,
cataract, and certain
neurological diseases, among others, reactive oxygen species (ROS) are
implicated in the
.r.,_
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pathophysiology of the disease. Ischemia, in which tissues are deprived of
blood flow and
oxygen such as occurs during stroke and myocardial infarction, followed by
reperfusion of
the ischemic tissue, initiates significant ROS damage to the tissue which is
not directly killed
during the infarction. Cancer chemotherapeutic agents such as adriamyein and
bleomycin
induce oxidant damage, as does anti-cancer radiation (e.g., X-ray) therapy .
As critical
subcellular organelles involved in aerobic energy metabolism and the oxidative
reactions
therein, mitochondria are sensitive to endogenous and exogenous influences and
may be
easily damaged or destroyed. Dysfunctional energy metabolism and, more
severely, damaged
mitochondria, may lead to cell senescence and death, and downstream tissue and
organ
dysfunction and damage. In the skin, increased oxidative damage as a
consequence of UV
light exposure can damage the cellular structure of the skin leading to
premature,
psychologically-debilitating changes related to aging, such as thinning of the
skin, wrinkling,
and abnormal pigmentation. Exposure of environmental oxidants to the lungs can
induce
mitochondria) and attendant cellular damage leading to chronic airways
obstructive disorders.
Exposure to electromagnetic and nuclear radiation also induce oxidative
damage.
In accordance with the present invention, protection is afforded to
mitochondria by the
application or administration of a composition comprising L-ergothioneine (L-
ET).
Administration to the target cells, tissue, or organ may be parenterally;
transmucosally, e. g. ,
orally, nasally, rectally; or transdermally. Parenteral administration is via
intravenous
injection, and also including, but is not limited to, intraarterial,
intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, intrathecal and intracranial
administration.
For example, the composition of the present invention may be infused directly
into a tissue or
organ that had undergone an infarct, such as the brain or heart following a
stroke or heart
attack, in order to protect mitochondria in the cells of the ischemic
penumbra, those outside
of the immediate infarct zone which are not killed during the cessation of
blood flow but
undergo extensive ROS-mediated damage when blood flow is restored. L-ET may be
prepared as a tablet or capsule formulation for oral administration. For
topical delivery, a
solution of L-ET in water, buffered aqueous solution or other pharmaceutically-
acceptable
carrier, or in a hydrogel lotion or cream, comprising an emulsion of an
aqueous and
hydrophobic phase, at a concentration of between 50 pM and 5 mM, is used. A
preferred
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concentration is about 1 mM. To this may be added ascorbic acid or its salts,
or other
ingredients, or a combination of these, to make a cosmetically-acceptable
formulation.
Metals should be kept to a minimum. It may be preferably formulated by
encapsulation into
a liposome for oral, parenteral, or, preferably, topical administration. As
will be seen below,
a composition of L-ET within a liposome improves the efficacy of protection of
mitochondria
from oxidative damage resulting from radiation damage.
It was found unexpectedly that the use of a liposome formulation for L-ET
enhances the
effectiveness of the compound for the protection of mitochondria. While
liposome delivery
has been utilized as a pharmaceutical delivery system for many other compounds
for a variety
of applications [see Langer, Science 249:1527-1533 (1990); Treat et al., in
Liposomes in
the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler
(eds.), Liss:
New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see
generally ibid.],
subcellular delivery of L-ET in an efficacious form was discovered by the
inventor herein and
is a preferred embodiment of the compositions and methods of the present
invention. The
function of the liposome is t~ increase the delivery of the L-ET to the
mitochondria, and
distinctly or additionally, to protect the L-ET until it reaches the target
cell or tissue. A non-
limiting example of a liposome formulation is that formed from
phosphatidylcholine,
phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio
of 2:2:1:5,
encapsulating 10 mM L-ET. A final concentration of 1 ~M to 10 mM L-ET is used,
preferably about 12 uM. This final concentration can be achieved by dilution
of the purified
liposomes in a pharmaceutically-acceptable carrier. Many other suitable
liposome
formulations ale known to the skilled artisan, and may be employed for the
purposes of the
present invention. For example, see: U.S. Patent No. 5,190,762; "Method of
Administering
Proteins to Living Skin Cells" to Yarosh which is incorporated herein by
reference. A
general discussion of liposomes and liposome technology can be found in a
three volume
work entitled "Liposome Technology" edited by G. Gregoriadis, 1993, published
by CRC
Press, Boca Raton, Florida. The pertinent portions of this reference are
incorporated herein
by reference.
Transdermal delivery of L-ET, either as a liposome formulation or free L-ET,
is also
T..
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contemplated. Various and numerous methods are known in the art for
transdermal
administration of a drug, e. g. , via a transdermal patch. It can be readily
appreciated that a
transdermal route of administration may be enhanced by use of a dermal
penetration
enhancer.
In yet another aspect of the present invention, provided are pharmaceutical
compositions of
L-ET. Such pharmaceutical compositions may be for administration for
injection, or for oral,
pulmonary, nasal or other forms of administration. In general, comprehended by
the
invention are pharmaceutical compositions comprising effective amounts of L-ET
together
with pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants
and/or carriers. Such compositions include diluents of various buffer content
(e.g., Tris-HC1,
acetate, phosphate), pH and ionic strength; additives such as detergents and
solubilizing
agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,
sodium
metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol) and bulking
substances (e.g.,
I 5 lactose, mannitol); incorporation of the material into particulate
preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes
(infra).
Hylauronic acid may also be used. Such compositions may influence the physical
state,
stability, rate of in vivo release, and rate of in vivo clearance of L-ET. The
compositions may
be prepared in liquid form, or may be in dried powder, such as lyophilized
form.
Controlled release oral formulation may be desirable. The drug may be
incorporated into an
inert matrix which permits release by either diffusion or leaching mechanisms,
e.g., gums.
Slowly degenerating matrices may also be incorporated into the formulation.
Some enteric
coatings also have a delayed release effect. Another form of a controlled
release of this
therapeutic is by a method based on the Oros therapeutic system (Alza Corp.),
i.e. the drug is
enclosed in a semipermeable membrane which allows water to enter and push drug
out
through a single small opening due to osmotic effects.
Also contemplated herein is pulmonary delivery of the pharmaceutical
compositions of the
present invention, for the treatment or protection of mitochondria from
oxidative damage.
Pulmonary delivery may be used to treat the lung tissue itself, or serve as a
delivery route
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to the blood stream and thus other locations within the body. A pharmaceutical
composition of the present invention is delivered to the lungs of a mammal
while inhaling
and traverses across the lung epithelial lining to the blood stream.
Contemplated for use in
the practice of this invention are a wide range of mechanical devices designed
for
pulmonary delivery of therapeutic products, including but not limited to
nebulizers, metered
dose inhalers, and powder inhalers, all of which are familiar to those skilled
in the art.
With regard to construction of the delivery device, any form of aerosolization
known in the
art, including but not limited to spray bottles, nebulization, atomization or
pump
aerosolization of a liquid formulation, and aerosolization of a dry powder
formulation, can
be used in the practice of the invention.
Ophthalmic delivery of the compositions of the present invention are also
contemplated for
the protection and treatment of mitochondria, for example, in the lens of the
eye, in which
oxidative damage is believed to account for a high incidence of cataracts.
Other ophthalmic
uses include treatment or prophylaxis of macular degeneration and degenerative
retinal
damage.
Nasal delivery of a pharmaceutical composition of the present invention is
also
contemplated. Nasal delivery allows the passage of a pharmaceutical
composition of the
present invention to the blood stream directly after administering the
therapeutic product to
the nose, without the necessity for deposition of the product in the lung.
Formulations for
nasal delivery include those with dextran or cyclodextran. For nasal
administration, a
useful device is a small, hard bottle to which a metered dose sprayer is
attached. In one
embodiment, the metered dose is delivered by drawing the pharmaceutical
composition of
the present invention solution into a chamber of defined volume, which chamber
has an
aperture dimensioned to aerosolize and aerosol formulation by forming a spray
when a
liquid in the chamber is compressed. The chamber is compressed to administer
the
pharmaceutical composition of the present invention. In a specific embodiment,
the
chamber is a piston arrangement. Such devices are commercially available.
In a further aspect, the L-ET liposomes can cross the blood-brain barrier,
which would
t.
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allow for intravenous or oral administration. Many strategies are available
for crossing the
blood-brain barrier, including but not limited to, increasing the hydrophobic
nature of a
molecule; introducing the molecule as a conjugate to a carrier, such as
transferrin, targeted
to a receptor in the blood-brain barrier; and the like. In another embodiment,
the molecule
can be administered intracranially or, more preferably, intraventricularly. In
yet another
embodiment, L-ET can be administered in a liposome targeted to the blood-brain
barrier.
A subject in whom administration of L-ET is an effective therapeutic regiment
for
mitochondrial protection is preferably a human, but can be any animal. Thus,
as can be
readily appreciated by one of ordinary skill in the art, the methods and
pharmaceutical
compositions of the present invention are particularly suited to
administration to any
animal, particularly a mammal, and including, but by no means limited to,
domestic
animals, such as feline or canine subjects, farm animals, such as but not
limited to bovine,
equine, caprine, ovine, and porcine subjects, wild animals (whether in the
wild or in a
zoological garden), research animals, such as mice, rats, rabbits, goats,
sheep, pigs, dogs,
cats, etc., avian species, such as chickens, turkeys, songbirds, etc. , i. e.
, for veterinary
medical use.
The protection of mitochondria from oxidative damage may be used for the
prevention and
treatment of a number of disorders, including effects of solar,
electromagnetic and nuclear
radiation to the body, disease processes, exposure to pollutants including
tobacco combustion
products, and protection against the damaging effects of certain
pharmaceuticals whose
mechanisms of action involve generation of reactive oxygen species and other
radicals. For
example, certain anti-neoplastic agents induce oxidative radicals as their
mechanism of
action, but a significant and limiting side effect in patients is
cardiotoxicity; higher doses and
thus increased anti-cancer efficacy is achievable by protecting the
mitochondria of the heart
and other tissues with the compositions and methods of the present invention.
In addition,
various types of radiation used for anti-cancer therapy, as an alternative or
adjunct to surgery,
induces significant damage to tissues; prior administration of L-ET may be
used to reduce or
prevent the toxicity of radiation therapy to the body. It may further be used
in combination
with fibrinolytic therapy, such as tissue plasminogen activator or
streptokinase, wherein clots
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in obstructed blood vessels causing stroke and heart attack are dissolved.
Protection of
tissues from ROS arising as a result of reperfusion is an object of the
present invention.
Furthermore, numerous disease processes involve reactive oxygen species. In
the eye,
cataract, macular degeneration and degenerative retinal damage are attributed
to ROS and
may be treated with topical, oral or parenterally-administered L-ET. A
liposome formulation
is preferred. ROS-related diseases of the lungs such as emphysema and
bronchopulmonary
dysphasia and including pathology induced by inhalation of tobacco combustion
products and
asbestos may be treated by an aerosolized form of L-ET as described above.
Various diseases
of the nervous system such as Parkinson disease, Alzheimer disease, muscular
dystrophy, and
multiple sclerosis may be treatable by oral or parenteral formulations or
direct delivery to the
central nervous system via intrathecal, intraventricular and intracranial
administration. Iron
overload diseases such as hemochromatosis and thalassemia may also be treated
by the
compositions and methods of the present invention. Other diseases include
pancreatitis;
diabetes; renal diseases including autoimmune nephrotic syndrome and heavy
metal-induced
nephrotoxicity; and radiation injuries. Local and system injury as a result of
burns involve
ROS damage.
In addition to the aforementioned therapeutic and prophylactic uses of the
compositions of
the present invention, various diagnostic utilities are also contemplated. The
potential of L-
ergothioneine to protect a mammal from mitochondria) damage and the level of L-
ET
necessary to afford protection may be assessed in vitro exposing aliquots of a
cellular sample
from said mammal to the damaging agent or condition, said aliquots containing
various
concentrations of L-ET. The damage to mitochondria of the various aliquots is
determined,
a well as the lowest concentration, if any, of L-ET providing sufficient
protection from
damage. To determine the degree of therapeutic benefit of L-ET to a mammal
after exposure
to a mitochondria) damaging agent, a similar diagnostic test as described
above may be
employed, with a variation in that the various concentrations of L-ET are
applied to the
cellular sample aliquots after exposure to the mitochondria) damaging agent.
In another
embodiment, the extent of exposure of a mammal to ROS may be assessed by
determining
the effect of L-ET on a sample of cells taken from the mammal. These
diagnostic utilities
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further offer assistance in selecting a effective therapeutic dose of L-ET.
In a further embodiment, the ability of L-ET to protect a cellular sample from
the damaging
effects of a therapeutic regimen that causes oxidative damage, such as an anti-
neoplastic
agent or radiation therapy to be administered to a mammal with cancer, can be
performed in
vitro by combining the anti-neoplastic agent with various concentrations of L-
ET, applying
the combination to identical aliquots of a cellular sample from a mammal, and
determining
the extent of mitochondrial damage in said series of samples. These data may
be used to
determine an effective dose of L-ET to prevent mitochondriai damage in the non-
diseased
cells of said mammal. In a parallel manner using a sample of diseased or
cancerous cells
from said mammal, it may be determined whether L-ET will effect any diminution
of the
anti-cancer activity of said anti-cancer agent; based on these two tests, a
level of L-ET for co-
administration with the anti-cancer agent may be selected to provide optimal
protection of the
non-diseased cells of the mammal from the anti-cancer agent while providing
maximum anti-
cancer therapy. These are non-limiting examples of useful diagnostic tests
assessing the
prophylactic and therapeutic benefits of the compositions and methods of the
present
invention.
Application of L-ET and its effect on mitochondrial damage is demonstrated by
the following
experimental examples in which mouse keratinocytes are treated with
unencapsulated or
liposome-encapsulated L-ET. The mitochondria are then subjected to the
potentially
damaging effects of UV-B light and to alloxan (which is known to induce oxygen
radicals)
and the results measured. Damage to mitochondria were detected by two methods:
1 ) the
MTT assay and 2) the JC-1 assay.
EXAMPLE I
UV-B Light: Mouse keratinocytes were pretreated with different concentrations
of L-
ergothioneine (unencapsulated) and then exposed to ultraviolet B radiation (UV-
B), the
shorter wavelength range of UV light present in sunlight which is responsible
for significant
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photodamage to the skin. The light was generated by a FS20 sunlamp filtered
with 2 sheets
of Kodicell to eliminate light having a wavelength less than 280 nm.
MTT Assav: This assay measures the specific activity of mitochondria to cleave
the
tetrazolium ring of the soluble dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl
tetrazolium bromide] to form the insoluble blue formazan form. Living
mitochondria
metabolize MTT and make the blue formazan; dead mitochondria immediately stop
forming
blue formazan. In the MTT assay, mammalian cells are pretreated with L-
ergothioneine and
then treated with the mitochondrial damaging agent, in this example UV-B. MTT
is then
added and the formation of the blue dye is measured spectrophotometrically.
Results: Table I provides the percent optical density of the formazan blue
present relative to
unexposed (control) mitochondria in cells for various UV-B levels, expressed
as joules per
square meter, and L-ergothioneine concentrations.
TABLEI
UV-B L-ergothioneine
concentration
(J/m'-) 0 mM 0.1 0.~ 1 mM
mM mM
0 100.0 100.0 100.0 100.0
100 84.2 95.7 97.2 97.1
200 80.7 97.0 99.4 103.9
500 79.9 9I.0 90.8 96.0
These data show that in the absence of L-ET, increasing UV-B irradiation
intensity results in
a decreasing numbers of living mitochondria in the lceratinocytes, as shown by
the dose-
responsive decreasing level of conversion of MTT to formazan. Ultraviolet-
irradiated
keratinocytes are protected by L-ergothioneine: at 100 and 200 J/m2, all three
levels of L-ET
have maintained greater than 95% mitochondrial viability; at the highest UV-B
dose, L-ET
still protected the mitochondria.
EXAMPLE 2
i r ,
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Alloxan: Mouse keratinocytes were pretreated with various concentrations of
L-ergothioneine (unencapsulated) and then exposed to alloxan at various
concentrations.
MTT Assav: The effect of alloxan on the pretreated mitochondria was determined
by the
MTT assay as in Example 1.
Results: Table 2 provides the percent optical density of the formazan blue
present relative to
unexposed (control) mitochondria in cells for various alloxan concentrations
and
L-ergothioneine concentrations.
TABLE 2
Alloxan (mM) L-ergothioneine
concentration
0 mM 0.1 mM 0.5 mM 1 mM
0 100.0 I 00.0 100.0 100.0
0.625 81.6 86.3 88.3 91.2
1.25 76.5 80.8 81.0 86.5
2.5 77.1 81.1 81.0 82.9
5 69.2 73.3 73.3 67.8
10 70.7 74.9 74.1 68.5
20 74.5 79.1 78.4 72.7
40 69.2 73.3 72.3 66.6
Alloxan induces oxidative damage to mitochondria; a dose-responsive reduction
in
mitochondrial viability can be seen up to a level of 5 mM, above which the
damage has
plateaued. In the dose-responsive portion of the curve, L-ergothioneine
afforded protection.
from oxidative damage.
EXAMPLE 3
Mouse keratinocytes were pretreated with L-ergothioneine, both unencapsulated
and
encapsulated in liposomes (prepared as described below), at a final
concentration of 12.5 ~M.
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A control group was not treated with L-ergothioneine. The mouse keratinocytes
were then
exposed to UV-B as in Example 1.
Li~osome Encapsulation: The L-ergothioneine was encapsulated into liposomes
composed of
phosphatidyl choline, phosphatidyl ethanolamine, oleic acid and cholesteryl
hemisuccinate in
a ratio of 2:2:1:5. To calculate the concentration of entrapped L-
ergothioneine, the liposomes
are extracted with chloroform, and the ODZ58 is measured in the aqueous layer.
The
concentration of L-ergothioneine is calculated using the E~5$ of L-
ergothioneine of 14,500.
The final concentration of L-ET in the purified liposome was about I .1 mM.
This
concentration. was reduced by diluting the liposomes with cell culture media
to a final
concentration of 12.5 ~M in the media. Unencapsulated L-ergothioneine was
adjusted to the
same concentration by dilution.
The effect of the UV-B on the pretreated and untreated mitochondria was
determined by the
MTT assay as in Example 1.
Results: Table 3 provides the percent optical density of the formazan blue
present in each
case relative to unexposed (control) mitochondria in cells for various UV-B
levels.
TABLE 3
UV-B (J/m2) L-ergothioneine Without L-ET
at 12.5 qM
Encapsulated Llnencapsulated
L-ET L-ET
0 100.0 100.0 100.0
100 I 06.8 89.9 75.4
500 110.3 89.9 76.8
In the absence of L-ergothioneine, keratinocytes showed significant
mitochondria) damage at
both UV-B doses. Unencapsulated L-ET afforded significant but not complete
protection
under these conditions, but complete protection was afforded by the
encapsulated formulation
of L-ET, at the same concentration. Thus, the liposome formulation of L-ET
provides
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superior protection.
EXAMPLE 4
Alloxan: Mouse keratinocytes were pretreated with L-ergothioneine
(unencapsulated) at 1
mM and then treated with 8 mM alloxan. One day after treatment with L-
ergothioneine, the
cells were treated for 10 minutes with JC-1 and the cells examined by
fluorescence
microscopy.
JC-I Assav: The JC-1 assay makes use of the fluorescent dye JC-1 (5,5',6,6'-
tetrachloro-
l,1',3,3'-tetraethylbenzimidazoylcarbocyanine iodide) (Molecular Probes, Inc.,
Eugene, OR).
This dye immediately and specifically intercalates into mitochondria)
membranes. In living,
c>,arged membranes the JC-1 dye is maintained in the membrane as a monomer,
and
fluoresces green. When the mitochondria) membrane is damaged, aggregation of
the JC-1
dye into J-aggregates occurs and the fluorescence changes to orange. Orange
coloration is
then characteristic of mitachondrial membrane damage.
Results: Untreated control cells were predominantly green. Cells treated with
alloxan alone
showed significant patches of orange. Cells treated with alloxan and L-
ergothioneine showed
much less orange than cells treated with alloxan alone. Cells treated with L-
ergothioneine
alone showed green fluorescence.
Thus, using a different means of determining mitochondria) viability, the
protection afforded
mitochondria by L-ET is confirmed.
This invention may be embodied in other forms or carried out in other ways
without
departing from the spirit or essential characteristics thereof. The present
disclosure is
therefore to be considered as in all respects illustrative and not
restrictive, the scope of the
invention being indicated by the appended Claims, and all changes which come
within the
meaning and range of equivalency are intended to be embraced therein.
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WO 98/36748 PCT/US98/03352
-16-
Various citation to the literature present above are incorporated herein by
reference.
