U.S. patent application number 17/372080 was filed with the patent office on 2022-04-07 for oral administration of melanin for protection against radiation.
The applicant listed for this patent is ALBERT EINSTEIN COLLEGE OF MEDICINE. Invention is credited to Arturo CASADEVALL, Ekaterina DADACHOVA.
Application Number | 20220105074 17/372080 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-07 |
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United States Patent
Application |
20220105074 |
Kind Code |
A1 |
DADACHOVA; Ekaterina ; et
al. |
April 7, 2022 |
ORAL ADMINISTRATION OF MELANIN FOR PROTECTION AGAINST RADIATION
Abstract
Methods and compositions are provided for alleviating and/or
preventing one or more side effects associated with exposure to
radiation in a subject exposed to radiation or at risk for exposure
to radiation comprising oral administration to the subject of an
amount of an edible source of melanin effective to alleviate a side
effect associated with radiation.
Inventors: |
DADACHOVA; Ekaterina;
(Mahopac, NY) ; CASADEVALL; Arturo; (Pelham,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT EINSTEIN COLLEGE OF MEDICINE |
Bronx |
NY |
US |
|
|
Appl. No.: |
17/372080 |
Filed: |
July 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16433169 |
Jun 6, 2019 |
11058666 |
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17372080 |
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15230617 |
Aug 8, 2016 |
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16433169 |
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14005601 |
Oct 15, 2013 |
9408882 |
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PCT/US2012/029213 |
Mar 15, 2012 |
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15230617 |
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61454242 |
Mar 18, 2011 |
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International
Class: |
A61K 31/404 20060101
A61K031/404; A61K 31/198 20060101 A61K031/198; A61K 31/136 20060101
A61K031/136; A61K 31/05 20060101 A61K031/05; A61K 36/07 20060101
A61K036/07; A61K 9/00 20060101 A61K009/00; A61K 36/06 20060101
A61K036/06 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers AI51519, AI087625, AI52733-07 and S10RR027308 awarded by
the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A method for alleviating and/or preventing one or more side
effects associated with exposure to radiation in a subject exposed
to radiation or at risk for exposure to radiation comprising oral
administration to the subject of an amount of an edible source of
melanin effective to alleviate a side effect associated with
radiation.
2. A method for increasing the survival rate of a plurality of
subjects exposed to an amount of radiation likely to kill the
plurality of subjects, comprising oral administration to each of
the plurality of subjects of an amount of an edible source of
melanin effective to increase the survival rate of the plurality of
subjects exposed to the amount of radiation likely to kill the
plurality of subjects.
3. The method of claim 1, wherein melanin is administered to the
subject, or to the subjects, in an amount equivalent to at least 8
mg of purified melanin per kg of body weight of the subject.
4. The method of claim 1, wherein the melanin is isolated or
derived from a melanin-containing biological source where melanin
constitutes at least 10% of the dry weight of the biological
source, or melanin is provided by administering a
melanin-containing biological source that comprises at least 10%
melanin per dry weight of the biological source.
5. The method of claim 4, wherein the biological source is a
melanin-containing plant, cell, fungus or microorganism.
6. The method of claim 4, wherein the biological source is grown in
the presence of a melanin precursor.
7. The method of claim 6, wherein the melanin precursor is one or
more of L-dopa (3,4-dihydroxyphenylalanin), D-dopa, catechol,
5-hydroxyindole, tyramine, dopamine, tyrosine, cysteine,
m-aminophenol, o-aminophenol, p-aminophenol, 4-aminocatechol,
2-hydroxyl-1,4-naphthaquinone, 4-metholcatechol, 3,4-dihydroxynaph
halene, gallic acid, resorcinol, 2-chloroaniline, p-chloroanisole,
2-amino-p-cresol, 4,5-dihydroxynaphthalene,
1,8-dihydroxynaphthalene, 2,7-disulfonic acid, o-cresol, m-cresol,
and p-cresol.
8. The method of claim 5, wherein the fungus is a
melanin-containing mushroom.
9. The method of claim 8, wherein the fungus is Auricularia
auricular-judae.
10. The method of claim 3, wherein melanin is administered in an
edible substance of at least 80 mg of edible substance per kg of
body weight of the subject.
11. The method of claim 10, wherein melanin is administered as at
least 80 mg of dry mushrooms per kg of body weight of the
subject.
12. The method of claim 1, wherein an internal organ of the subject
is protected from radiation.
13. The method of claim 12, wherein the organ that is protected is
one or more organ selected from the group consisting of bone
marrow, liver, spleen, kidneys, lungs, and gastrointestinal
tract.
14. The method of claim 1, wherein the side effect associated with
radiation is one or more of acute-nature nausea, vomiting,
abdominal pain, diarrhea, dizziness, headache, fever, cutaneous
radiation syndrome, low blood cell count, infection due to low
white blood cells, bleeding due to low platelets, anemia due to low
red blood cells, or death.
15-19. (canceled)
20. The method of claim 1, wherein the source of the radiation is
radiotherapy used for treatment of disease, radiation from a
medical imaging device, radiation used for radiation surgery, a
nuclear weapon, a nuclear reactor, high-altitude radiation.
21. The method of claim 20, wherein the source of radiation is from
a nuclear power plant.
22. The method of claim 1, wherein the edible source of melanin
comprises a drinkable suspension of melanin packaged for oral
administration to a subject for alleviating and/or preventing one
or more side effects associated with exposure of the subject to
radiation, wherein the drinkable suspension comprises at least 500
mg melanin in a volume of at least 10 mL.
23. The method of claim 1, further comprising administering one or
more antibiotics to the subject or to the subjects.
24. An edible source of melanin packaged for oral administration to
a subject for alleviating and/or preventing one or more side
effects associated with exposure of the subject to radiation,
wherein the edible source provides melanin in an amount equivalent
to at least 8 mg of purified melanin per kg of body weight of the
subject or a drinkable suspension of melanin packaged for oral
administration to a subject for alleviating and/or preventing one
or more side effects associated with exposure of the subject to
radiation, wherein the drinkable suspension comprises at least 500
mg melanin in a volume of at least 10 mL.
25. (canceled)
26. The drinkable suspension of claim 2, wherein the drinkable
suspension comprises at least 500 mg melanin in a volume of at
least 100 mL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 61/454,242 filed Mar. 18, 2011, the contents of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to use of melanin-containing
substances, such as black mushroom-based food supplements, for oral
administration for alleviating side effects associated with
exposure to radiation such as ionizing radiation.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various publications are
referred to in parenthesis. Full citations for these references may
be found at the end of the specification immediately preceding the
claims. The disclosures of these publications are hereby
incorporated by reference in their entireties into the subject
application to more fully describe the art to which the subject
application pertains.
[0005] Melanin is a high molecular weight pigment that is
ubiquitous in nature and has a variety of biological functions (5).
Melanins are found in all biological kingdoms. These pigments are
among the most stable, insoluble, and resistant of biological
materials (6). Melanins can have different structures depending on
the biosynthetic pathway and precursor molecules. Some definitions
of melanin have focused on chemical and physical properties of
melanins instead of defined structures (7). Melanins can be
synthesized in the laboratory by chemical means or by many living
organisms. Melanins formed by the oxidative polymerization of
phenolic compounds are usually dark brown or black (6). However,
melanins may have other colors as illustrated by the finding that
dopamine-derived melanin is reddish-brown. Fungi can make melanins
from at least two major biosynthetic pathways, employing the
precursor 1,8-dihydroxynapthalene (DHN melanin) or the oxidation of
suitable tyrosine derivatives like dihydroxyphenylalanine
(DOPA-melanin) (6). The fungus C. neoformans can make melanins from
a wide variety of phenolic compounds which are oxidized by a
laccase enzyme (8-10). Many fungi constitutively synthesize melanin
(11).
[0006] Every year 1.4 million people are diagnosed with cancer in
the U.S. and half of them will undergo some form of radiation
therapy in the course of their disease. The availability of
radioprotective compounds would alleviate the morbidity associated
with the radiation exposure. The doses received by millions of
patients during diagnostic radiological procedures are also very
high (the dose of a multi-slice cardiac CT scan is equal to the
dose from 300 chest X-rays) and are of great concern as well; thus
such patients would also benefit from the affordable and effective
radioprotectors. There is also importance for public safety to have
radioprotective agents readily available in the event of a nuclear
accident or terrorist attack.
[0007] Radioprotective agents that could be given prior to, or even
during, radiation exposure would be of significant value in
alleviating the side effects associated with exposure to ionizing
radiation. Currently there are no FDA-approved radioprotectors. It
would be extremely beneficial for hundreds of millions of people to
have access to food supplements that could fill the niche in the
absence of radioprotective drugs.
[0008] Fungal melanins can function as energy transducing molecules
capable of capturing high energy electromagnetic radiation and
converting it into an energy form that is useful to fungal cells
(1). Furthermore, fungal melanins can be effective shields against
radiation; the efficacy of radioprotection by melanins is dependent
on their chemical composition and spatial arrangement (2). In
addition to free reactive radical scavenging, radioprotection by
melanins involves prevention of free radical generation by Compton
recoil electrons through gradual recoil electron energy dissipation
by the .pi.-electron-rich melanin until the kinetic energy of
recoil electrons becomes low enough to be trapped by stable free
radicals present in the pigment (3). It has also been shown that
melanin-based nanoparticles protect bone marrow in mice subjected
to external whole body radiation or radioimmunotherapy (4).
[0009] The present invention addresses the need for
radioprotectants in humans at risk for radiation exposure using
melanin-based products.
SUMMARY OF THE INVENTION
[0010] The invention provides methods for alleviating and/or
preventing one or more side effects associated with exposure to
radiation in a subject exposed to radiation or at risk for exposure
to radiation comprising oral administration to the subject of an
amount of an edible source of melanin effective to alleviate a side
effect associated with radiation.
[0011] The invention also provides a method for increasing the
survival rate of a plurality of subjects exposed to an amount of
radiation likely to kill the plurality of subjects, comprising oral
administration to each of the plurality of subjects of an amount of
an edible source of melanin effective to increase the survival rate
of the plurality of subjects exposed to the amount of radiation
likely to kill the plurality of subjects.
[0012] The invention also provides edible sources of melanin
packaged for oral administration to a subject for alleviating
and/or preventing one or more side effects associated with exposure
of the subject to radiation, wherein the edible source provides
melanin in an amount equivalent to at least 8 mg of purified
melanin per kg of body weight of the subject.
[0013] The invention also provides a drinkable suspension of
melanin packaged for oral administration to a subject for
alleviating and/or preventing one or more side effects associated
with exposure of the subject to radiation, wherein the drinkable
suspension comprises at least 500 mg melanin in a volume of 500 mL
or less.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1. Survival of CD1 mice receiving synthetic pheomelanin
before 9 Gy whole body radiation dose. Three hours before receiving
the whole body dose of 9 Gy, the mice were given by oral gavage
either 100 mg/kg body weight synthetic pheomelanin followed by 5
days of antibiotic support, or PBS followed by 5 days of antibiotic
support, or PBS alone. AB--antibiotic support. 10 mice per group
were used. P values were 0.001 and 0.002 when the survival in
pheomelanin group was compared with PBS and PBS plus antibiotics
groups, respectively.
[0015] FIG. 2. Survival of CD1 mice after 9 Gy whole body radiation
dose. Three hours before receiving the whole body dose of 9 Gy the
mice were given by oral gavage either 1 g/kg body weight of
Auricolaria judae mushroom suspended in PBS, or 1 g/kg body weight
of Auricolaria judae mushroom followed by antibiotics support for 5
days, or PBS followed by antibiotics support for 5 days, or PBS
alone. There were 5 mice in PBS alone and in PBS plus antibiotic
support groups, and 6 mice in mushrooms and mushrooms plus
antibiotics groups. The P value was 0.015 for both mushrooms and
mushrooms plus antibiotics when compared to PBS alone and to PBS
plus antibiotics controls. Ab--antibiotics support,
Mum--mushrooms.
[0016] FIG. 3A-3E. Chemical composition of melanins and appearance
of melanins from various sources and mushrooms used in the study:
a) structure of eumelanin oligomer; b) structure of pheomelanin
oligomer; c) electron micrograph of purified microbial melanin
(melanin "ghosts"); d) synthetic melanin--eumelanin (black) on the
left and pheomelanin (brown) on the right; e) edible mushrooms used
in the study--Boletus edulis (white mushrooms) on the left and
Auricularia auricula-judae (black mushrooms) on the right.
[0017] FIG. 4A-4G. Physico-chemical characterization of black and
white mushrooms: a, b) EPR of dried mushrooms: a) black mushrooms;
b) white mushrooms; c-e) oxidative HPLC of melanin purified from
black mushrooms: c) background solution; d) PDCA standard eluting
at 8 min.; e) melanin from black mushrooms showing PDCA peak; f, g)
results of DPPH assay for antioxidant presence: f) butylated
hydroxyanisole (BHA) positive control; g) methanol extracts from
black and white mushrooms.
[0018] FIG. 5A-5H. Survival of irradiated CD-1 mice fed with black
edible mushrooms, blood counts in the surviving mice and histology
of the GI tract and bone marrow. Mice were divided into groups of
5-6 and fed 1 g/kg body weight black mushroom suspension in PBS, or
PBS alone, or 1 g/kg white mushroom suspension, or 1 g/kg white
mushroom suspension supplemented with 100 mg/kg synthetic melanin
via gavage needle. One hour after mushroom administration mice were
irradiated with 9 Gy dose of Cs-137 radiation at a dose rate of 2.5
Gy/min. a) Kaplan-Meyer survival curves. The experiment was
performed twice and was terminated at day 45; b) white blood cells
counts; c) platelet counts; d-h) H&E stained slides with
tissues from control and irradiated mice. Left, non-irradiated
controls; middle, black mushroom group; right, white mushroom
supplemented with melanin. d) stomach, magnification .times.400; e)
LI, magnification 400; f) SI, magnification .times.200; g) bone
marrow, magnification .times.400; h) spleen, magnification
.times.100.
[0019] FIG. 6A-6D. Survival and weight change in CD-1 mice ted with
different doses of synthetic pheomelanin and/or antibiotics and
irradiated with 9 Gy gamma radiation at 2.5 Gy/min: a) mice fed
with 0-100 mg/kg body weight pheomelanin; b) mice fed with 100
mg/kg pheomelanin followed by antibiotics for 5 days, or given PBS
only, or given PBS followed by antibiotics for 5 days; c) combined
results from a) and b); d) weight change in irradiated groups
modeled using linear regression. AB--antibiotics.
[0020] FIG. 7A-7C. Histological evaluation of the tissue in
surviving mice post-irradiation with 9 Gy gamma radiation at 2.5
Gy/min: a) stomach, small intestine, large intestine, liver and
bone marrow. Mice received 100 mg/kg pheomelanin plus antibiotics
(upper row); 75 mg/kg pheomelanin (middle row); 0 mg/kg plus
antibiotics (lower row); b) tissues from a mouse receiving 100
mg/kg pheomelanin plus antibiotics--focal microadenoma of the small
intestine (left panel) and bone marrow (right panel); c) cecum of a
single survivor in 0 mg/kg plus antibiotics group. The same region
of the cecum is shown with magnification .times.250 in the left
panel, .times.400 in the middle panel and .times.1,000 in the right
panel Each slide is a higher magnification of the same region.
Magnification .times.400 in a) and b).
[0021] FIG. 8A-8B. Toxicity evaluation of microbial and synthetic
eumelanin in non-irradiated CD-1 mice: a) body weight of mice fed
with 15 mg/kg microbial or synthetic eumelanin; b) histology of GI
organs from CD-1 mice fed with microbial eumelanin and sacrificed
24 hr later: left, stomach; middle, small intestine; right, colon.
Original magnification .times.400.
[0022] FIG. 9A-9H. Radiation effects in CD-1 mice fed with 15 mg/kg
body weight microbial or synthetic eumelanin and irradiated with 9
Gy gamma radiation at 2.5 Gy/min: (a-f) histology of GI tract
tissues obtained from irradiated CD-1 mice sacrificed at 4 hr (a-c)
and at 24 hr (d-f) post-irradiation: a) stomach, synthetic
eumelanin group; b) stomach, microbial eumelanin group; c) stomach,
PBS. Fewer apoptotic cells are seen in stomach tissue of microbial
melanin fed mice than in synthetic eumelanin or PBS groups; d)
colon, synthetic eumelanin group; e) colon, microbial eumelanin
group; f) colon, PBS control group; g) cumulative weight loss in
CD-1 mice; h) survival of the irradiated mice. Original
magnification .times.400.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides a method for alleviating one or more
side effects associated with exposure to radiation in a subject
exposed to radiation or at risk for exposure to radiation
comprising oral administration to the subject of an amount of an
edible source of melanin effective to alleviate a side effect
associated with radiation.
[0024] The invention also provides a method for increasing the
survival rate of a plurality of subjects exposed to an amount of
radiation likely to kill the plurality of subjects, comprising oral
administration to each of the plurality of subjects of an amount of
an edible source of melanin effective to increase the survival rate
of the plurality of subjects exposed to the amount of radiation
likely to kill the plurality of subjects. One skilled in the art
will know from then literature the amount of radiation likely to
kill the plurality of subjects. For example, for human beings the
LD.sub.50/60d (i.e. the dose that causes 50% mortality with 60 days
of exposure) in humans from acute, whole body radiation exposure is
in excess of 250 rad (2.5 Gy) and usually approximately 400 to 500
rads (4-5 Gy).
[0025] Preferably, melanin is administered to the subject in an
amount equivalent to at least 8 mg of purified melanin per kg of
body weight of the subject. For example, melanin can be
administered in an edible substance, containing at least 10%
melanin by dry weight, of at least 80 mg of edible substance per kg
of body weight of the subject. For example, melanin can be
administered as at least 80 mg of dry mushrooms per kg of body
weight of the subject, where the mushrooms contain at least 10%
melanin by dry weight. In an embodiment, the melanin is
administered in the form of a drinkable suspension. In an
embodiment, the edible source of melanin comprises a drinkable
suspension of melanin packaged for oral administration to a subject
for alleviating and/or preventing one or more side effects
associated with exposure of the subject to radiation, wherein the
drinkable suspension comprises at least 500 mg melanin in a volume
of at least 10 mL.
[0026] The invention also provides an edible source of melanin
packaged for oral administration to a subject for alleviating
and/or preventing one or more side effects associated with exposure
of the subject to radiation, wherein the edible source provides
melanin in an amount equivalent to at least 8 mg of purified
melanin per kg of body weight of the subject. In an embodiment, the
edible source provides melanin in an amount equivalent to at least
9, 10, 15 or 20 mg of purified melanin per kg of body weight of the
subject.
[0027] The invention also provides a drinkable suspension of
melanin packaged for oral administration to a subject for
alleviating and/or preventing one or more side effects associated
with exposure of the subject to radiation, wherein the drinkable
suspension comprises at least 250 mg melanin in a volume of at
least 10 mL. In an embodiment, the drinkable suspension comprises
at least 500 mg melanin in a volume of at least 10 mL. In an
embodiment, the drinkable suspension comprises at least 560 mg
melanin in a volume of at least 10 mL. In an embodiment, the
drinkable suspension comprises the melanin in at least 25 mL, 50
mL, 75 mL, 100 mL, 125 mL, 150 mL, 175 mL, 200 mL, 250, mL, 500 mL
or 750 mL. In an embodiment, substantially all the melanin is in
particulate form or smaller. The drinkable suspension can be
galenical.
[0028] The invention also provides a powderized form of melanin
packaged for making a drinkable suspension by dilution with a
drinkable liquid. The powderized form of melanin may be packaged,
for example in a sachet. In an embodiment, the powderized form of
melanin is formulated so as to permit, upon reconstitution with at
least 10 mL, 25 mL, 50 mL, 75 mL, 100 mL, 125 mL, 150 mL, 175 mL,
200 mL, 250, mL, 500 mL or 750 mL a drinkable suspension providing
at least 8 mg of purified melanin per kg of body weight of the
subject who will drink the drinkable suspension. In an embodiment,
the powderized form of melanin is formulated so as to permit, upon
reconstitution with at least 10 mL, 25 mL, 50 mL, 75 mL, 100 mL,
125 mL, 150 mL, 175 mL, 200 mL, 250, mL, 500 mL or 750 mL a
drinkable suspension providing at least 250 mg, 500 mg or 560 mg
melanin.
[0029] The melanin can be isolated or derived from a
melanin-containing biological source where melanin constitutes at
least 10% of the dry weight of the biological source. Melanin can
also be synthesized chemically. Melanin can also be provided by
administering a melanin-containing biological source that comprises
at least 10% melanin per dry weight of the biological source. In an
embodiment, the melanin is in a composition substantially free of
fungal material.
[0030] The biological source can be, for example, a
melanin-containing plant, cell, fungus or microorganism such as a
bacterium. Preferred fungi include melanin-containing edible
mushrooms, such as Auricolaria auricular-judae or Pleurotus
cystidiosus. A chemical source for melanin can be auto- or
catalytic-polymerization of certain phenolic compounds like
L-dopa.
[0031] The biological source can be grown in the presence of a
melanin precursor, such as, for example, one or more of L-dopa
(3,4-dihydroxyphenylalanin), D-dopa, catechol, 5-hydroxyindole,
tyramine, dopamine, tyrosine, cysteine, m-aminophenol,
o-aminophenol, p-aminophenol, 4-aminocatechol,
2-hydroxyl-1,4-naphthaquinone, 4-metholcatechol,
3,4-dihydroxynaphthalene, gallic acid, resorcinol, 2-chloroaniline,
p-chloroanisole, 2-amino-p-cresol, 4,5-dihydroxynaphthalene,
1,8-dihydroxynaphthalene, 2,7-disulfonic acid, o-cresol, m-cresol,
and p-cresol.
[0032] The melanin can comprise allomelanin, pheomelanin and/or
eumelanin. Eumelanins are derived from the precursor tyrosine.
Pheomelanin is derived from the precursors tyrosine and cysteine.
Allomelanins are formed from nitrogen-free precursors such as
catechol and l,8-dihydroxynaphthalenes. In one embodiment, the
ratio of pheomelanin to eumelanin is at least 1:1. Preferably, the
melanin contains divalent sulfur.
[0033] Preferably, one or more internal organs of the subject are
protected from radiation. Preferably, the organ that is protected
is one or more organ selected from the group consisting of bone
marrow, liver, spleen, kidneys, lungs, and gastrointestinal
tract.
[0034] The side effect associated with radiation can be one or
more. of nausea, vomiting, abdominal pain, diarrhea, dizziness,
headache, fever, cutaneous radiation syndrome, low blood cell
count, infection due to low white blood cells, bleeding due to low
platelets, anemia due to low red blood cells, or death. Preferably,
the subject's chance of survival is increased following exposure to
radiation. In one embodiment, the dose of radiation received by the
subject would be lethal to the subject in the absence of
radioprotection.
[0035] The subject can be any animal. Preferably the subject is a
mammal and more preferably a human.
[0036] The radiation can comprise ionizing radiation. Ionizing
radiation is of sufficiently high energy that it ionizes atoms. The
radiation can be, for example, one or more of gamma radiation,
x-ray radiation, bremsstrahlung radiation, ultraviolet radiation,
and particulate radiation (e.g., .alpha.-radiation and
.beta.-radiation). The source of the radiation can be a medical
isotope. In a preferred embodiment the ionizing radiation is gamma
radiation, .alpha.-radiation or .beta.-radiation. In an preferred
embodiment, the radiation is from a man-made source of radiation.
For example, the source of the radiation can be radiation therapy
used for treatment of disease (such as radiotherapy), radiation
from a medical imaging device (such as a CT scanner), radiation
used for radiation surgery (e.g. stereotactic radiation surgery), a
nuclear weapon, or a nuclear reactor, such as a nuclear reactor in
a power plant or submarine or high-altitude radiation, e.g. as
experienced in commercial or military flights or space flight. In
an embodiment, the high-altitude radiation is natural ionizing
radiation experienced at altitudes in excess of 20,000 ft. The
source of radiation can result from a terrorist attack. Thus, a
man-made source of radiation can include that resulting from
natural radioactive isotopes, but as applied in a man-made therapy,
power source or device.
[0037] Subjects expected to benefit from the present invention
include, but are not limited to, the following. Every second
patient in the U.S. who is diagnosed with cancer (1.4 million
people per year are diagnosed in the U.S.) will undergo some form
of radiation therapy during the course of their disease. Another
group of patients who will benefit are those who undergo CT
(computer tomography) scans. 72 million CT scans are performed in
the U.S. every year. There is growing concern about high doses of
radiation that many patients receive during those scans, which are
often recommended for them several times per year. The dose from
one high resolution cardiac multi-slice CT scan is equivalent to
approximately 100-600 chest X-rays or over 3-years' worth of
natural background radiation. Yet another group of patients who can
benefit from the present invention are people undergoing so-called
stereotactic radiosurgery (done with Particle beam (proton)), or
Cobalt-60 based (photon), or linear accelerator-based for
conditions such as arteriovenous malformations, benign brain
tumors, and functional disorders including trigeminal neuralgia,
essential tremor, and Parkinson's tremor/rigidity. Additional
subject who could benefit from the present invention are frequent
fliers and airline personnel whose doses are known to exceed the
annual limit for radiation occupational workers, nuclear medicine
and radiology professionals, personnel at the nuclear power plants
and nuclear reactors, and military personnel in nuclear submarines,
as well as victims of radiation accidents and terrorist
attacks.
[0038] In an embodiment of the methods, the treatment results in
reducing the likelihood that the exposed subject will develop a
cancer as a result of chronic radiation exposure over an extended
time period.
[0039] Melanin could be provided in the form of dry black mushrooms
suspended in palatable liquid ("melanin shakes"). The mushroom that
could be used include black edible mushrooms such as Auricularia
auricular-judae. Mushrooms such as Auricularia auricular-judae can
be grown as other edible mushrooms in a basement of a building when
provided with humidity and nutrients, dried, powderized and
formulated into "melanin shakes" by mixing it with flavored water
or fruit juice. Shakes with different flavors can be made. The
packaging can be standard individual juice cartons, e.g. 100 mL
volume. Melanin could also be provided in other edible forms, e.g.,
melanin brownies. Alternatively, naturally occurring or synthetic
melanins can be isolated or synthesized, respectively, and added to
foodstuffs to create products suitable for oral ingestion. In
non-limiting embodiments, the melanin from black mushrooms can be
processed so as to be particulate or powderized. In an embodiment,
the melanin is from an organism, such as a fungi, which has been
exposed itself to radiation in an amount effective to increase the
melanin production in the organism (radiosynthesis). In an
embodiment, the organism has been grown under conditions comprising
the presence of a melanin precursor. Methods for both
radiosynthesis and growing in the presence of a melanin precursor
are described in U.S. Patent Application Publication No. US
2009-0328258 A1, published Dec. 31, 2009, which is hereby
incorporated by reference.
[0040] Also provided is a drinkable suspension of melanin packaged
for oral administration to a subject for alleviating and/or
preventing one or more side effects associated with exposure of the
subject to radiation, wherein the drinkable suspension comprises at
least 500 mg melanin in a volume of at least 10 mL. In an
embodiment, the drinkable suspension comprises at least 500 mg
melanin in a volume of at least 100 mL.
[0041] In an embodiment, the subject has been, is being, or will be
exposed to a single radiation exposure of 10 mGy, 20 mGy, 50 mGy,
100 mGy, 500 mGy, 1Gy, 1.5 Gy, 2Gy or greater, 5 Gy or greater, 7.5
Gy or greater, 10 Gy or greater or greater than 10 Gy. In humans, a
whole-body exposure to 5 or more Gy of high-energy radiation at one
time usually leads to death within 14 days.
[0042] In embodiments of the methods and compositions, including
suspensions, the melanin is not in the form of melanized
nanoparticles.
[0043] In an embodiment, the methods further comprise administering
one or more antibiotics to the subject.
[0044] All combinations of the various elements described herein
are within the scope of the invention unless otherwise indicated
herein or otherwise clearly contradicted by context.
[0045] This invention will be better understood from the
Experimental Details, which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
Experimental Details
EXAMPLE 1
[0046] Radiation protection with synthetic melanin: The
radioprotective properties of fungal and synthetic melanins were
tested by oral administration of synthetic pheomelanin to mice
before whole body exposure to. 1.5 lethal dose (9 Gy) of gamma
radiation. The whole body dose of 9 Gy is also 2.5 times the lethal
dose for a human. The resulting survival of mice protected with
melanins (FIG. 1) provided encouragement for the use and
development of melanin-based products as radioprotectants in humans
at risk for radiation exposure.
[0047] Radiation protection with black edible mushrooms: In a
follow-up experiment, it was investigated whether the result with
synthetic melanin would apply to natural melanins. The edible
fungus Auricularia auricular-judae was selected since it is heavily
melanized. Three hours before receiving the whole body dose of 9
Gy, groups of 5-6 CD1 mice were given by oral gavage 1 g/kg body
weight of Auricularia auricular-judae mushroom suspended in PBS, or
1 g/kg body weight of Auricularia auricular-judae mushroom followed
by antibiotics support for 5 days after irradiation, or PBS
followed by antibiotics support for 5 days after irradiation, or
PBS alone. Mice were monitored for their survival for 30 days since
in radioprotection experiments mice are considered to be surviving
indefinitely beyond that point. The results of the experiment are
shown in FIG. 2. Black mushroom Auricularia auricular-judae
significantly prolonged the survival of lethally irradiated mice,
with 30% of mice given mushrooms alone or mushrooms with antibiotic
support surviving for 30 days. There were 5 mice in PBS alone and
in PBS plus antibiotic support groups, and 6 mice in mushrooms and
mushrooms plus antibiotics groups. The P value was 0.015 for both
mushrooms and mushrooms plus antibiotics when compared to PBS alone
and to PBS plus antibiotics controls.
[0048] Given that synthetic melanins and melanin in microscopic
fungi have a similar structure as the melanin found in edible
mushrooms, a food supplement could be used to supply melanin in the
form, for example, of dry black mushrooms suspended in palatable
liquid ("melanin shakes") to individuals to be subjected to
radiation exposure. In studies with oral administration of melanin
the protective dose of purified melanin was 100 mg/kg in a mouse,
which will be 8 mg/kg purified melanin in a human taking into
consideration the different weight to body surface area ratios in
mice and humans. Provided that melanin constitutes at least 10% of
a dry mushroom weight--in a mouse experiment described above, mice
received 1 g/kg of Auricularia auricular-judae which in a human
will be equal to 80 mg/kg of dry mushrooms, or 5.6 g per 70 kg
person. Auricularia auricular-judae can be grown as other edible
mushrooms in a basement of a building when provided with humidity
and nutrients, dried, powderized and formulated into "melanin
shakes" by mixing it with flavored water or fruit juice. Shakes
with different flavors can be made. The packaging can be standard
individual juice cartons, e.g. 100 mL volume.
EXAMPLE 2
[0049] Melanin-containing edible mushrooms offered the highest
degree of radioprotection without antibiotic support. The
radioprotective efficacy of melanin delivered as a natural food
source was evaluated. The black edible mushroom Auricularia
auricula-judae (common names Jelly Ear or Judas Ear) was selected
as a source of edible melanin and the white mushroom Boletus edulis
(common names porcino or bun bun) as a melanin-devoid control (FIG.
3E). Both types of mushroom are basidiomycetes that are used in
Western and Asian cuisines and are available commercially in dried
form. The presence of melanin in Auricularia auricula-judae (black
mushrooms) and its absence in Boletus edulis (white mushrooms) was
demonstrated by electron paramagnetic resonance (EPR) with
characteristic melanin "signature" signal in black mushrooms (FIG.
4A) and background only--in white mushrooms (FIG. 4B). Melanin
purified from black mushrooms using the protocol developed in our
laboratories (19) constituted approximately 10% of black mushrooms
dry weight and was further characterized by elemental analysis and
oxidative high performance liquid chromatography (HPLC). Eumelanins
are composed of 5,6-dihydroxyindole (DHI) and
5,6-dihydroxyindole-2-carboxylic acid (DHICA) monomer units with
6-9% nitrogen (20, 21). In parallel, fungi also synthesize
eumelanin from 1,8-dihydroxynaphthalene (DHN) via pentaketide
synthetic pathway and such melanin does not contain nitrogen in its
structure (22). The elemental analysis determined that there was
44% carbon, 5% hydrogen and 2% nitrogen in black mushroom melanin.
The low percentage of nitrogen suggested that the pigment was
primarily DHN-melanin, while the HPLC of oxidized melanin gave
additional information about its structure (FIG. 4C-E). The
presence of pyrrole-2,3-dicarboxylic acid (PDCA), which is an
oxidation product of DHI-derived units in oxidized melanin allowed
a conclusion that melanin in black mushrooms was a mixture of DHN
and DHI melanins. In addition, it was considered whether
mushroom-associated antioxidants could contribute to the
radioprotective effect and compared the antioxidant contents of
black and white mushrooms using 2,2-diphenyl-1-picrylhydrazyl
(DPPH) assay. The DPPH is a stable free radical having a deep
violet color in solution. The radical scavenging activity of a
sample can be measured as a decolorizing effect following the
trapping of the unpaired electron of DPPH12. There was no
difference in soluble antioxidant content between black and white
mushrooms (FIG. 4F, 4G), thus excluding differences in antioxidants
as the basis of black mushroom-mediated radiation protection in
vivo. It is important to note, that only soluble antioxidants are
measured in this assay as they .are extracted into methanol.
Melanin which also possesses powerful free radical scavenging
properties by virtue of being a stable free radical (24) cannot
contribute to the results of this assay as it is not soluble in
methanol or any other common solvents.
[0050] To test the radioprotective properties of edible mushrooms
in mice we first need to determine the time between feeding mice
with mushrooms and irradiation to ascertain the presence of
mushrooms in GI tract during irradiation. The fluorescent imaging
was performed by utilizing the natural autofluorescence of white
mushrooms. Mice were given mushroom suspension with a gavage needle
and imaged on IVIS Spectrum Imaging System at 15, 30 and 60 min
post-feeding. 675/30 nm and 840/20 nm filters were used for
excitation and emission, respectively. Mice were given 1 g/kg body
weight white mushrooms suspension in water via gavage needle and
imaged in supine position under Isoflurane anesthesia. The
mushrooms were in the stomach at 15 and 30 min post-feeding and
moved into the intestines to the large extent at 60 min (data not
shown). As intestines are more sensitive to ionizing radiation than
stomach, we selected 60 min as time to administer radiation in
order to ensure maximum protection for the most sensitive part of
GI tract. Armed with these results, we conducted a radioprotection
study in mice given lethal whole body dose of 9 Gy. Groups of 5-6
CD-1 mice were used in the experiment which was then repeated with
the similar results. Black mushrooms were administered as
suspension in sterile PBS via gavage as 1 g/kg body weight. The
control groups included mice given only sterile PBS, or white
mushrooms as 1 g/kg body weight dose. To establish that the melanin
pigment was indeed the radioprotective substance in black mushrooms
an additional group of mice was given white mushrooms as 1 g/kg
body weight dose supplemented with 100 mg/kg of synthetic melanin
dosed to match its contents in black mushrooms. All mice in the PBS
group and 80% in the white mushrooms--fed group died by day 14 post
irradiation (FIG. 5A). The remaining 20% of mice in the white
mushroom-treated group died by day 25. This trend toward
prolongation in survival in comparison with PBS alone group which
was not statistically significant (P=0.07) and might be explained
by the presence of antioxidants in white mushrooms which could have
produced local protective effects in the gut. Strikingly, in black
mushrooms and in white mushrooms supplemented with synthetic
melanin groups, 60 and 75% of mice survived (P=0.002 and 0.001),
respectively, up to day 45 when the experiment was terminated to
perform the histological examination of their tissues (FIG. 5A). At
the same time the white blood cell counts in black mushroom and in
melanin-supplemented groups were not different from the
non-irradiated controls (P=0.06) (FIG. 5B), while platelet counts
were lower in both irradiated groups (P=0.03) (FIG. 5C), however,
at the levels which ensure recovery in mice receiving radiation
treatment (25). In lethally irradiated mice mortality results from
damage to rapidly dividing tissues such as GI mucosa (26) and bone
marrow suppression (27, 28). There were no signs of radiation
damage in the stomachs, large and small intestines (LI and SI,
respectively) in the surviving mice in black mushroom and
melanin-supplemented groups (FIG. 5D-F). Bone marrow of irradiated
mice had slight myeloid hyperplasia (FIG. 5G) and the spleens
architecture was normal with some extramedullary hematopoesis (FIG.
5).
[0051] Synthetic pheomelanin in combination with antibiotics
protected the majority of mice against lethal dose of radiation. It
was hypothesized that high linear attenuation coefficient and
stable radical contents (18) of the synthetic pheomelanins would
translate into radioprotection in mice. The dose response
experiments demonstrated that orally administrated synthetic
pheomelanin protected mice irradiated with 9 Gy at 2.5 Gy/min in a
dose-dependent manner. Over a 40 day survival study, no protection
was identified in mice receiving 25 mg/kg pheomelanin. Mice fed
with 50 mg/kg had a 6.6% survival rate (P=0.02), while those
receiving 75 mg/kg had a 20% survival rate (P=0.01) (FIG. 6A).
However, no protection was observed when the pheomelanin dose was
increased to 100 mg/kg (P=0.06). The possibility that the lack of
protection at the higher melanin dose was a consequence of
bowel-related effects that predisposed animals to bacterial sepsis
was considered. Hence, in a follow-up study, the effect of
antimicrobial therapy on survival after pheomelanin administration
and lethal irradiation was evaluated. Mice were given either PBS
alone, or PBS in combination with antibiotics or 100 mg/kg
pheomelanin in combination with antibiotics which resulted in 40
day survival of 0%, 16.7%, and 80% (P=0.005), respectively (FIG.
6B, 6C).
[0052] The rate of weight loss in the different treatments groups
was analyzed by linear regression (FIG. 6D). The highest weight
loss was observed in the groups given PBS alone or PBS in
combination with antibiotics: a 1.6 and 1.1% decrease in total body
weight per day, respectively. Mice given pheomelanin displayed
significantly less weight loss when compared to PBS alone group:
the group treated with 50 mg/kg of pheomelanin lost 1.0% of their
body weight per day (P=0.02), while the group treated with 75 mg/kg
had a 0.7% loss in body weight per day (P=0.015). Most importantly,
mice receiving 100 mg/kg pheomelanin plus antibiotics actually
gained weight at a rate of 1.0% per day after irradiation, a
difference that was significant in comparison with all other groups
(P<0.05).
[0053] Histological evaluation of the tissue in surviving mice
confirmed the body weight data by revealing no damage to the
stomach, small intestine, large intestine, liver and bone marrow in
surviving mice treated with 100 mg/kg pheomelanin plus antibiotics
(FIG. 7A, upper row) or 75 mg/kg pheomelanin (FIG. 7A, middle row).
Among the surviving mice in the 100 mg/kg pheomelanin plus
antibiotics group (80% survival) only one mouse had a focal
microadenoma in the small intestine (FIG. 7B, left panel) and
moderately depleted bone marrow cellularity (FIG. 7B, right panel).
The single survivor in PBS plus antibiotics group exhibited
multifocal lymphohistiocytic and plasmacytic periportal infiltrates
in the liver (FIG. 7A, tower row) and a focal perforation in the
cecum with chronic active peritonitis (FIG. 7C).
[0054] Microbial and synthetic eumelanins prolonged survival of
lethally irradiated mice. Before carrying out irradiation studies
we evaluated whether there was any toxicity associated with oral
administration of microbial and synthetic melanins to CD-1 mice.
Measures of toxicity were the body weight over 10 days and
histological evaluation of gut tissue. Microbial and synthetic eu-
and pheomelanins proved to be non-toxic with mice steadily gaining
weight during the observation period which was confirmed by the
normal histology of the gut (FIG. 8).
[0055] Encouraged by the lack of toxicity of microbial and
synthetic melanins, the efficacy of orally administered synthetic
and microbial eumelanins in protecting CD-1 mice against lethal
irradiation was evaluated. Histological evaluation of GI tissues
obtained from mice 4 hr post-irradiation with 9 Gy at 2.5 Gy/min
from 137Cs source revealed that mice fed with microbial eumelanin
had approximately 40% fewer apoptotic cells in stomach tissue than
mice fed synthetic eumelanin or PBS (FIG. 10A-C). At 24 hr this
trend continued with glandular cells being less attenuated in
stomachs of mice fed with microbial eumelanin than in mice fed with
synthetic eumelanin. Simultaneously, there were approximately 25%
more mitotic figures and less apoptotic cells in both eumelanins
groups in comparison with control PBS fed mice. In the small
intestine there was no apparent difference between treatment
groups. At 24 hr in the colonic glands of mice fed microbial
eumelanin there was 30% less cellular reaction and apoptosis
compared to the other colon samples (FIG. 9D-F).
[0056] For the first four days post-irradiation, mice fed with
microbial eumelanin lost slightly less weight than mice fed either
PBS or synthetic eumelanin (FIG. 9G). By day 5, the cumulative
weight loss in all groups had equalized and for the rest of the
observation period the weight loss was the least pronounced in mice
fed with synthetic eumelanin. The overall survival on day II
post-irradiation was 100% in synthetic eumelanin group, 66%--in the
microbial eumelanin group and 33%--in control mice fed with PBS,
with the last mouse in this group dying on day 16 (FIG. 9J). For
the duration of study, the mean survival for mice fed with
microbial eumelanin was 13 d, for control PBS fed mice--12.7 days
and for synthetic eumelanin group--19 days (P=0.01, Mandel-Cox
test).
Discussion
[0057] There is an ongoing and urgent need for oral radioprotectors
that are inexpensive, do not require refrigeration for storage and
transportation ("cold chain"), and are suitable for distribution to
large numbers of people in the event of radiation emergencies such
as the recent nuclear accident at Fukushima-Daiichi nuclear plants.
This need is enhanced by the fact that many developing nations are
considering increased reliance on nuclear power as an alternative
to fossil fuels and that a major expansion in nuclear programs
carries significant risks as evidenced by two major accidents at
Chernobyl and Fukushima-Daiichi in the space of one generation. One
potential radioprotector that has been studied extensively is
amifostine (28-30). It belongs to the class of free radical
scavengers that includes aminothiols and phosphorothioates, and is
administered as a prodrug that must be metabolized to an active
form to be effective. While this drug has some radioprotective
efficacy, it also has several undesirable properties, including a
relatively low radioprotective capacity, potentially serious side
effects such as anaphylaxis and the need for intravenous
administration. In a study by Burdelya et al. (31), a different
approach to radioprotection was taken by pharmacologically
suppressing apoptosis in the irradiated cells. This was done by
pre-treating experimental animals with flagellin-derived
polypeptide which binds to Toll-like receptor 5 and activates
nuclear factor-.kappa.B signaling. While this method showed some
promise, the drug also has to be given parenterally and might have
carcinogenic side effects by virtue of interfering with the process
of apoptosis.
[0058] Herein, in vivo studies were conducted to evaluate
protective effect of different types of orally administered
melanins on the GI tract in mice receiving a lethal dose of 9 Gy at
a high dose rate. The radioprotective effects of melanin are
proposed to be based on controlled dissipation of Compton electron
energy by melanin which results in a decreased number of
interactions between Compton electrons and cellular milieu and the
scavenging of free reactive radicals by melanin (18). The
protective effects of two eumelanins--microbial eumelanin purified
from C. neoformans and commercially available synthetic eumelanin
were compared. Histological examination of the stomachs and colons
of the irradiated mice revealed that the mice given microbial
eumelanin were better protected than those given synthetic
eumelanin or controls. However, this early protective effect of
microbial eumelanin did not extend into the long-term protection
while synthetic eumelanin administration resulted in statistically
significant prolongation in survival. This surprising observation
may be explained by the inflammation which microbial eumelanin can
cause in the mucosa due to the persistent presence of immunogenic
proteins and polysaccharides intertwined with its structure even
after rigorous multi-step purification (32). Melanin `ghosts`
derived from melanized fungal cells contain cell wall components
which are known to be highly immunogenic. For example, zymosan
particles prepared from yeast cell wall are notoriously
pro-inflammatory (33) and C. neoformans-derived melanin have been
shown to trigger direct inflammation (34). Such inflammation might
increase the damage sustained from radiation and be accompanied by
edema which could explain the less significant weight loss in the
microbial eumelanin group in comparison with the synthetic
eumelanin and control groups during the first four days after
irradiation. The synthetic eumelanin afforded statistically
significant prolongation in survival for the overall duration of
experiment, which provided impetus for further investigation of its
role in radioprotection by orally administering to mice synthetic
pheomelanin which has higher number of stable free radicals than
eumelanin and was more radioprotective in vitro (18).
[0059] Pheomelanin protected mice in a dose-dependent manner in the
dose range of 25-75 mg/kg body weight. Since the 100 mg/kg dose did
not protect mice, it was hypothesized that the higher dose of
melanin may have had unanticipated adverse effects in damaged
tissue. To explore the contribution of associated bacteremia to the
mortality antibiotics were administered to mice post-irradiation.
Antibiotic administration resulted in 80% survival of irradiated
mice treated with 100 mg/kg pheomelanin. When compared to published
data--pheomelanin plus antibiotics was more protective then
amifostine (60% survival after 9 Gy delivered at 1 Gy/min (17)),
and equal to flagellin-derived polypeptide (80% survival after 9 Gy
delivered at 2.3 Gy/min (20)). The increase in radiation dose rate
is known to make the cellular repair mechanisms less efficient
(35). The histological evaluation of the surviving mice in groups
protected with pheomelanin alone or with pheomelanin and
antibiotics revealed no obvious radiation damage to the major
organs. Among the survivors in the group receiving 100 mg/kg
pheomelanin plus antibiotics only one mouse had any abnormality in
its major organs, which consisted of a moderate depletion of the
bone marrow and an isolated microadenoma of the small intestine.
These abnormalities may or may not reflect the effect of
irradiation. In contrast, the single survivor in the antibiotics
only group had focal typhlitis and perforation associated with
peritonitis. Ionizing radiation induces disruption of the mucosal
integrity which is often complicated by ulceration (26, 36). Focal
ulcerations are common; these vary from simple loss of epithelial
layer with acute inflammation of the lamina propria to ulcers that
may penetrate to varying depths of the intestinal wall, even to the
serosa. A perforated appendix and associated peritonitis is a
frequent clinical consequence of exposure to ionizing radiation in
patients (26). It was concluded that the cecal perforation was a
result of radiation injury, and the mouse survived until the end of
the study due to antibiotic administration, which prevented fatal
peritonitis.
[0060] The ideal radioprotective agent would both be protective and
cost-effective. Black edible mushrooms, in their native form,
provide a natural radioprotector that is readily available. The
equal survival of mice protected with either black mushrooms or
white mushrooms supplemented with melanin establishes the causality
between the presence of melanin in black mushrooms and their
radioprotective properties. Interestingly, approximately the same
percentage of mice survived in experiments with mushrooms when no
antibiotics were given as in the experiment with the synthetic
pheomelanin where the supplementation with antibiotics was required
for the protection. This effect is most likely due to the
combination of melanin and soluble antioxidants which are present
in mushrooms (FIG. 2). Given that a significant proportion of black
mushroom- or white mushroom-supplemented with melanin-fed mice
became long term survivors it must follow that the presence of
melanin in the GI tract provided local protection that allowed
these mice to recover. Protection of GI mucosa would prevent death
by a GI syndrome and sepsis. Hence, local GI protection appears to
translate into systemic protection and this observation establishes
a new concept in the approach to protecting against radiation
sickness. Black edible mushrooms could be prepared as a suspension
in a palatable liquid and distributed as food supplement to
affected populations. This radioprotection may also benefit cancer
patients undergoing radiation treatment, as radiation-induced
injury to the GI tract is common in patients undergoing external
radiation beam therapy (EBRT).
Materials and Methods
[0061] Melanin sources and physico-chemical analyses. Commercial
synthetic eumelanin made from tyrosine was obtained from
Sigma-Aldrich. The microbial eumelanin from C. neoformans strain
24067 in form of "ghosts" (hollow melanin spheres from which all
cellular contents has been removed via multi-step purification
procedure) was purified as previously described8. Synthetic
pheomelanin using 5-S-cysteinyldopa was produced by incubating 0.5
mmol 5-S-cysteinyldopa with 0.025 mmol L-DOPA, added as a catalyst,
in 0.05 M sodium phosphate buffer, pH 6.8 and with mushroom
tyrosinase (Sigma) in the amount of 8300 units (773 .mu.L of 2
mg/mL solution) with constant agitation overnight at 37.degree. C.
After the overnight incubation, the oxidation reaction was halted
by the addition of 250 .mu.L 6 M HCl to lower the pH to
approximately 3.0. This acidified mixture was kept at 2.degree. C.
for 1 hour. The precipitate was collected by centrifugation, washed
three times with 15 mL 1% acetic acid, washed twice with 15 mL
acetone, once more with 15 mL 1% acetic acid, and re-suspended in
de-ionized water. The resulting pheomelanin was then lyophilized
and suspended in PBS at a concentration of 12.5 mg/mL to create the
stock suspension.
[0062] Dried Auricularia auricula-judae (black mushrooms) and
Boletus edulis (white mushrooms) were purchased from Trader Joe's
(Monrovia, Calif.). Melanin from black mushrooms was purified as
described previously (19). Elemental analysis of melanin was
carried out by QTI (Whitehouse, N.J.). EPR of dried mushrooms and
oxidative HPLC of melanin using permanganate oxidation were
performed as in (18). The antioxidant capacity of methanol extracts
from black and white mushrooms in DPPH assay was measured as in
(23).
[0063] Evaluation of potential toxicity of melanins. All animal
studies were carried out in accordance with the guidelines of the
Albert Einstein College of Medicine Animal Care and Use Committee.
Six-eight weeks old CD-1 female mice (Charles River Breeding
Laboratories, Portage, Mich.) were used in all experiments. Mice
were divided into groups of five and fed 15 mg/kg body weight
either synthetic or microbial eumelanins, or 100 mg/kg synthetic
pheomelanin or PBS via gavage needle. Mice were evaluated daily for
body weight and their physical condition. Two mice per group were
humanely sacrificed at 24 hr post-feeding with melanin, and the
remaining mice were sacrificed at day 14. The stomach and small and
large intestines were fixed in 10% neutral buffered formalin and
routinely processed for paraffin embedding. Samples were sectioned
to 5 .mu.m and stained with hematoxylin and eosin (H&E) for
histological evaluation.
[0064] Imaging. The in vivo imaging was performed with IVIS
Spectrum Imaging System (Caliper Life Sciences, Hopton, Mass.) in
epifluorescence mode equipped with 675/30 nm and 840/20 nm filters
for excitation and emission, respectively. Mice were fed with
non-fluorescent chow for 5 days and then fasted overnight before
the imaging experiment to exclude the interference from the remnant
autofluorescence of the chow. They were given 1 g/kg body weight
white mushrooms suspension in water via gavage needle and imaged in
supine position under Isoflurane anesthesia at 15, 30 and 60 min
post-feeding.
[0065] In vivo radioprotection with various melanins. Microbial and
synthetic eumelanins. CD-1 mice (13 mice per group) were fed either
synthetic eumelanin or microbial eumelanin or PBS via gavage needle
at a dose of 15 mg/kg body weight. One hour post-eumelanin feeding
the mice were subjected to whole body irradiation in a 137-Cs
irradiator with a total body dose of 9 Gy delivered at 2.5 Gy/min.
At 4 and 24 hr, 2 mice per group were humanely sacrificed and their
stomachs, small intestine and colon were removed and processed as
previously described. The remaining animals were monitored until
death with daily measurements of body weight. Moribund animals were
humanely euthanized.
[0066] Synthetic pheomelanin. CD-1 mice were divided into 5 groups
of fifteen mice. The groups were treated with 0, 25, 50, 75, 100
mg/kg melanin suspension in PBS via gavage. One hr post feeding the
mice were subjected to whole body irradiation in a 137-Cs
irradiator with a total body dose of 9 Gy delivered at 2.5 Gy/min
and their body weight and survival were monitored for 40 days. The
rate of weight change was quantified by using a linear regression
analysis (Prism, GraphPad, San Diego, Calif.). In a follow-up study
mice were divided into three groups. Group 1 was treated by oral
gavage with 100 mg/kg melanin suspension in PBS and group 2 and 3
were treated by oral gavage with only PBS followed by irradiation
as above of all three groups. Starting at 2 days after irradiation
groups 1 and 2 were dosed subcutaneously with penicillin (10,000
units/mL) and streptomycin (10 mg/mL) (Sigma, St. Louis, Mo.) at
120 .mu.L twice a day for 5 days. At the completion of the study on
day 40, all surviving mice were sacrificed and their stomachs,
small intestine, large intestine, liver, sternum and femur were
removed and processed as previously described for histological
evaluation.
[0067] Black mushrooms. Since dried black mushroom contain 10%
melanin, black mushrooms were administered as suspension in sterile
PBS via gavage as 1 g/kg body weight dose to match the melanin
concentrations in the described above experiments with pure
synthetic melanins. CD-1 mice were divided into groups of 5-6 and
fed 1 g/kg body weight black mushroom suspension in PBS, or PBS
alone, or 1 g/kg white mushroom suspension, or 1 g/kg white
mushroom suspension supplemented with 100 mg/kg synthetic melanin
via gavage needle. One hour after mushroom administration mice were
irradiated with 9 Gy dose of Cs-137 radiation at a dose rate of 2.5
Gy/min. Mice were evaluated daily for body weight and their
physical condition for 45 days. The experiment was performed twice.
At the conclusion of the experiment the surviving mice were
humanely sacrificed, their blood chemistry was analyzed for white
blood cells and platelet count, gross pathology was performed and
the stomach, small and large intestines, spleen and bone marrow
were subjected to histological evaluation. Survival of mice was
analyzed using log-rank test, the WBC and platelet counts--by one
tail Student's test. The differences in results were considered
statistically significant when P was <0.05.
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