U.S. patent application number 13/744815 was filed with the patent office on 2016-07-21 for cerium oxide nanoparticles for the treatment and prevention of stroke and cardiovascular disease.
This patent application is currently assigned to EDWARD Via COLLEGE OF OSTEOPATHIC MEDICINE. The applicant listed for this patent is Marc J. Billings, Jayce Cook, Justin Himler, Kevin Hockey, Landon M. Klein, Beverly A. Rzigalinski, Christopher A. Sholar. Invention is credited to Marc J. Billings, Jayce Cook, Justin Himler, Kevin Hockey, Landon M. Klein, Beverly A. Rzigalinski, Christopher A. Sholar.
Application Number | 20160206652 13/744815 |
Document ID | / |
Family ID | 49995121 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206652 |
Kind Code |
A9 |
Rzigalinski; Beverly A. ; et
al. |
July 21, 2016 |
CERIUM OXIDE NANOPARTICLES FOR THE TREATMENT AND PREVENTION OF
STROKE AND CARDIOVASCULAR DISEASE
Abstract
A method of treating or preventing neurological injury in a
subject who has suffered a stroke is described. The method includes
administering a therapeutically effective amount of cerium oxide
nanoparticles to the subject. Methods for prophylaxis against
neurological injury from stroke, and methods for treating or
preventing cardiovascular disease by administration of a
therapeutically effective amount of cerium oxide nanoparticles are
also described.
Inventors: |
Rzigalinski; Beverly A.;
(Radford, VA) ; Hockey; Kevin; (Radford, VA)
; Klein; Landon M.; (Blacksburg, VA) ; Sholar;
Christopher A.; (Rocky Point, NC) ; Himler;
Justin; (Uniontown, OH) ; Billings; Marc J.;
(Roanoke, VA) ; Cook; Jayce; (Yanceyville,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rzigalinski; Beverly A.
Hockey; Kevin
Klein; Landon M.
Sholar; Christopher A.
Himler; Justin
Billings; Marc J.
Cook; Jayce |
Radford
Radford
Blacksburg
Rocky Point
Uniontown
Roanoke
Yanceyville |
VA
VA
VA
NC
OH
VA
NC |
US
US
US
US
US
US
US |
|
|
Assignee: |
EDWARD Via COLLEGE OF OSTEOPATHIC
MEDICINE
Blacksburg
VA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140030335 A1 |
January 30, 2014 |
|
|
Family ID: |
49995121 |
Appl. No.: |
13/744815 |
Filed: |
January 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13539564 |
Jul 2, 2012 |
8747907 |
|
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13744815 |
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11993260 |
Mar 4, 2010 |
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PCT/US2006/024963 |
Jun 27, 2006 |
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13539564 |
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61587818 |
Jan 18, 2012 |
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60693930 |
Jun 27, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 33/24 20130101 |
International
Class: |
A61K 33/24 20060101
A61K033/24 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made partially with U.S. Government
support from the United States National Institutes of Health under
Contract No. NS40490 (National Institute of Neurological Disorders
& Stroke) and AG022617 (National Institute on Aging). The U.S.
Government has certain rights in the invention.
Claims
1. A method of treating or preventing neurological injury in a
subject who has suffered a stroke, comprising administering a
therapeutically effective amount of cerium oxide nanoparticles to
the subject.
2. The method of claim 1, wherein the cerium oxide nanoparticles
are administered within one hour after the stroke.
3. The method of claim 1, wherein the cerium oxide nanoparticles
are administered within 15 minutes after the stroke.
4. The method of claim 1, wherein the cerium oxide nanoparticles
are administered more than once to the subject.
5. The method of claim 1, wherein the subject is human.
6. The method of claim 1, wherein the nanoparticles have a size
from about 5 nm to about 25 nm.
7. The method of claim 1, wherein the nanoparticles have an average
size of about 10 nm.
8. The method of claim 1, wherein the cerium oxide nanoparticles
are delivered together with a pharmaceutically suitable
carrier.
9. The method of claim 1, wherein the subject is exhibiting
impaired motor function.
10. A method of providing prophylactic protection from neurological
injury in a subject comprising administering a therapeutically
effective amount of cerium oxide nanoparticles to the subject.
11. The method of claim 10, wherein the subject has one or more
risk factors associated with the occurrence of a stroke.
12. The method of claim 10, wherein the cerium oxide nanoparticles
are administered more than once to the subject.
13. The method of claim 10, wherein the subject is human.
14. The method of claim 10, wherein the nanoparticles have a size
from about 5 nm to about 25 nm.
15. The method of claim 10, wherein the nanoparticles have an
average size of about 10 nm.
16. The method of claim 10, wherein the cerium oxide nanoparticles
are delivered together with a pharmaceutically suitable
carrier.
17. A method of treating or preventing cardiovascular disease in a
subject, comprising administering a therapeutically effective
amount of cerium oxide nanoparticles to the subject.
18. The method of claim 17, wherein the cardiovascular disease is
ischemic heart disease.
19. The method of claim 18, wherein the subject is exhibiting acute
chest pain.
20. The method of claim 17, wherein the cerium oxide nanoparticles
are administered more than once to the subject.
21. The method of claim 17, wherein the subject is human.
22. The method of claim 17, wherein the nanoparticles have an
average size of about 10 nm.
23. The method of claim 17, wherein the cerium oxide nanoparticles
are delivered together with a pharmaceutically suitable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application 61/587,818 and US patent application Ser. No.
13/539,564; filed on Jul. 2, 2012, which is a continuation of U.S.
patent application Ser. No. 11/993,260, filed Dec. 20, 2007, which
is a U.S. National Stage Application of PCT/US2006/024963, filed
Jun. 27, 2006, which claims priority to U.S. Provisional Patent
Application Ser. No. 60/693,930, filed Jun. 27, 2005, all of which
are incorporated herein by reference in their entirety.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The present invention relates to the field of medicine. More
specifically, the invention relates to compositions containing
cerium oxide nanoparticles for the treatment and prevention of
stroke and cardiovascular disease.
[0005] 2. Description of the Related Art
[0006] Many approaches have been taken to treat, either
therapeutically or prophylactically, diseases, disorders, and other
medically important conditions that have, as a major component,
cell injury or death due to free radicals, such as oxygen radicals.
Among those approaches were the use of free radical scavengers,
such as Vitamin E and its related compounds, Vitamin C and its
related compounds, and melatonin, to name a few. While the
beneficial effects of these compounds have been noted, researchers
and clinicians continue to search for compounds with higher
activities and half-lives.
[0007] In early experiments performed by the present inventors and
their colleagues, cerium oxide nanoparticles prepared by a sol-gel
process were utilized to enhance cell longevity. The cerium oxide
nanoparticles were proposed to act as free radical scavengers to
bring about the observed results. However, the sol-gel process
posed several difficulties. For example, particle size was not
well-controlled within the reported 2-10 nm range, making
variability between batches high. That is, the process, while
satisfactory for producing nanoparticles with free radical
scavenging activity, did not reproducibly produce particles of a
specific size range. Thus, each batch of particles needed to be
tested to confirm the size range and the suitability of the batch
for use. In addition, the process resulted in tailing of
surfactants used in the process into the final product. The
presence of these surfactants produced biological difficulties when
used, primarily due to the toxicity of the surfactants in the
product. Furthermore, the inability to control the amount of
surfactant tailing posed problems with agglomeration when
nanoparticles were placed in biological media. These difficulties
reduced particle efficacy and biological deliverability. Removal of
surfactant after sol-gel synthesis produced particles that appeared
prone to agglomeration in biological media, and had a lack of
biological effects. Further, difficulties were encountered with
changes in valence state of cerium associated with these particles,
causing alterations in the ratio of valence states of cerium
(+3/+4) that occurred over time, particularly when particles were
placed in biological media. It is possible that the +3/+4 ratio of
valence states in the nanoparticles might alter free radical
scavenging and cellular delivery, including delivery in vivo.
[0008] Damage from ischemic stroke results from generation of free
radicals in neurons and other brain cells, which cause in cellular
demise and loss of function. Loss of energy production due to
damaged mitochondria is also evident. Depending on the size and
location of the stroke, functional deficits can range from mild
loss of coordination and limb movement to coma.
[0009] It has been shown that cerium oxide nanoparticles (CeONP)
are potent and effective regenerative free radical scavengers and
mitochondrial protectants (Bailey et al., Nature Biotechnology 14,
112 (2003); Rzigalinski et al., Nanomedicine, 1: 399-412 (2006);
Rzigalinski et al., Antioxidant Nanoparticles in Nanomedicine in
Health and Disease, Science Publishers, 2012). It has also been
shown that CeONP show promise in treatment of traumatic brain
injury (Whiting et al., J. Neurotrauma 26, 101 (2009)) and
Parkinson's Disease (Dillon et al., "Cerium oxide nanoparticles
protect against MPTP-induced dopaminergic neurodegeneration in a
mouse model for Parkinson's Disease" Proc. of International Conf.
on Nanotechnology, in press), and other neurodegenerative
disorders. However, the use of cerium oxide nanoparticles for the
treatment of stroke has not been previously demonstrated.
SUMMARY
[0010] The present invention addresses the need for treatments to
improve recovery after stroke or cardiovascular disease by
providing compositions and methods for treating and/or preventing
stroke and cardiovascular disease, and for improving neuronal and
cardiovascular recovery after stroke and cardiovascular
disease.
[0011] The inventors have demonstrated that cerium oxide
nanoparticles can be formulated as nanopharmaceuticals that can be
used in the treatment of stroke and cardiovascular disease. The
data presented herein shows that, in a tissue culture model of
stroke (anoxia), treatment with cerium oxide nanoparticles improved
neuronal survival by 78% and maintained normal mitochondrial
membrane potential and calcium signaling. Further, treatment of
cells up to eight (8) hours post anoxia improved survival and
cellular function as compared to untreated cells. In a Drosophila
model of stroke, cerium oxide nanoparticles improved survival by
30% and allowed flies subjected to stroke to maintain better motor
function and climbing ability than untreated controls. The results
show that cerium oxide nanoparticles can be used for the treatment
and prevention of stroke and cardiovascular disease.
[0012] The invention thus provides compositions comprising cerium
oxide nanoparticles for the treatment of stroke, cardiovascular
disease, or both. It likewise provides compositions comprising
cerium oxide nanoparticles for the prevention of stroke,
cardiovascular disease, or both.
[0013] In one aspect, the present invention provides a method of
treating or preventing neurological injury in a subject who has
suffered a stroke that includes administering a therapeutically
effective amount of cerium oxide nanoparticles to the subject. In
another aspect, a method of providing prophylactic protection from
neurological injury in a subject is provided that includes
administering a therapeutically effective amount of cerium oxide
nanoparticles to the subject. In a further aspect, a method of
treating or preventing cardiovascular disease in a subject is
provided that includes administering a therapeutically effective
amount of cerium oxide nanoparticles to the subject. In some
embodiments, the cardiovascular disease is ischemic heart
disease.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 provides a bar graph showing that Cerium Oxide
Nanoparticles protect neurons of mixed organotypic cultures from
cell damage associated with oxygen/glucose deprivation. Mixed
organotypic brain cell cultures (15 days old) were treated with a
single dose of 100 nM cerium oxide nanoparticles on day 5 in vitro.
On day 15, cultures were exposed to oxygen/glucose deprivation for
30, 60, or 90 minutes as indicated in the figure, followed by
return to normal culture conditions for 24 hours. At this time,
cell death/damage was measured by uptake of Propidium iodide,
expressed on the Y axis as "injured cells per mg of protein".
*Significantly different from OGD, P<0.01.
[0015] FIG. 2 provides a bar graph showing Cerium Oxide
Nanoparticles promote cell survival when delivered 15 minutes after
oxygen/glucose deprivation. Mixed organotypic brain cell cultures
(15 days old) were exposed to oxygen/glucose deprivation for 30,
60, or 90 minutes as indicated in the figure. After oxygen/glucose
deprivation, cells were returned to normal culture conditions, in
medium containing 100 nM cerium oxide nanoparticles. Twenty four
hours later, cell death/damage was measured by uptake of Propidium
iodide, expressed on the Y axis as "injured cells per mg of
protein". *Significantly different from OGD, P<0.01.
[0016] FIG. 3 provides a bar graph showing Cerium Oxide
Nanoparticles promote cell survival when delivered 1 hour after
oxygen/glucose deprivation. Mixed organotypic brain cell cultures
(15 days old) were exposed to oxygen/glucose deprivation for 30,
60, or 90 minutes as indicated in the figure. After oxygen/glucose
deprivation, cells were returned to normal culture conditions. One
hour after oxygen/glucose deprivation, 100 nM cerium oxide
nanoparticles were added to the cultures. Twenty four hours later,
cell death/damage was measured by uptake of Propidium iodide,
expressed on the Y axis as "injured cells per mg of protein".
*Significantly different from OGD, P<0.01.
[0017] FIG. 4 provides a bar graph showing Cerium Oxide
Nanoparticles preserve basal [Ca.sup.2+]i when delivered before
oxygen/glucose deprivation. Mixed organotypic brain cell cultures
were treated with 100 nM cerium oxide nanoparticles on day 5 in
vitro. On day 15, cultures were exposed to oxygen/glucose
deprivation for 30, 60, or 90 minutes as indicated in the figure.
After oxygen/glucose deprivation, cells were returned to normal
culture conditions. Twenty four hours later, cells were loaded with
Fura-2 and basal [Ca.sup.2+]i was determined
microspectrophotometrically. *Significantly different from OGD,
P<0.01.
[0018] FIG. 5 provides a bar graph showing Cerium Oxide
Nanoparticles preserve basal [Ca.sup.2+]i when delivered after
oxygen/glucose deprivation. Mixed organotypic brain cell cultures
(15 days old) were exposed to oxygen/glucose deprivation for 30,
60, or 90 minutes as indicated in the figure. After oxygen/glucose
deprivation, cells were returned to normal culture conditions and
treated with 100 nM cerium oxide nanoparticles at either 15 minutes
post-deprivation, or 1 hour post-deprivation. Twenty four hours
later, cells were loaded with Fura-2 and basal [Ca.sup.2+]i was
determined microspectrophotometrically. *Significantly different
from OGD, P<0.01.
[0019] FIG. 6 provides a bar graph showing Cerium Oxide
Nanoparticles preserve near-normal glutamate signaling when
delivered prior to oxygen/glucose deprivation. Mixed organotypic
cultures were treated with 100 nM cerium oxide nanoparticles on day
5 in vitro. On day 15, cultures were exposed to oxygen/glucose
deprivation for 30, 60, or 90 minutes as indicated in the figure.
After oxygen/glucose deprivation, cells were returned to normal
culture conditions. Twenty four hours later, cell cells were loaded
with Fura-2. The change in [Ca.sup.2+]i in response to a 100 mM
glutamate stimulus was measured. *Significantly different from OGD,
P<0.01.
[0020] FIG. 7 provides a bar graph showing Cerium Oxide
Nanoparticles preserve glutamate-stimulated [Ca.sup.2+]i when
delivered after oxygen/glucose deprivation. Mixed organotypic
cultures (15 days old) were exposed to oxygen/glucose deprivation
for 30, 60, or 90 minutes as indicated in the figure. After
oxygen/glucose deprivation, cells were returned to normal culture
conditions and treated with 100 nM cerium oxide nanoparticles at
either 15 minutes post-deprivation, or 1 hour post-deprivation.
Twenty four hours later, cell cells were loaded with Fura-2. The
change in [Ca.sup.2+]i in response to a 100 mM glutamate stimulus
was measured. *Significantly different from OGD, P<0.01.
[0021] FIG. 8 provides a bar graph showing Motor Function in Normal
Drosophila. Flies were fed standard food (Jazz Mix) or food
containing the indicated concentrations of cerium oxide
nanoparticles for 14 days. On day 14, pre-stroke motor function was
assessed by measuring negative geotaxis, the ability of flies to
climb the walls of an empty vial to 3, 5.5, and 8 cm, in 10
seconds. Data is expressed as the percentage of flies achieving
each height goal in the required time.
[0022] FIG. 9 provides a bar graph showing CeONP-treated male flies
had normal motor function after stroke. Flies were fed as described
in FIG. 8. On day 15 flies were exposed to anoxia for 2.5 hrs,
followed by return to their respective food group. Two days after
stroke, motor function was assessed by negative geotaxis. Note that
flies fed standard food and exposed to stroke had significantly
decreased motor function as compared to normal flies. CeONP
preserved the negative geotactic response in stroked flies, to
levels equivalent to normal controls at the 3 and 5.5 cm goal
heights. At the 8 cm height, CeONP significantly improved
performance as compared to stroked flies. *Sig. from all control,
P<0.01; # Sig. from stroke, P<0.01.
[0023] FIG. 10 provides a bar graph showing CeONP-treated male
flies have improved motor function 6 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Six days after
stroke, motor function was assessed by negative geotaxis. Flies fed
standard food and exposed to stroke continued to have significantly
decreased motor function at all heights compared to normal
controls. However flies fed 100 and 200 .mu.M CeONP continued to
show improved motor function as compared to stroked flies. *Sig.
from control, P<0.01; g .sup.#Sig from stroke, P<0.01.
[0024] FIG. 11 provides a bar graph showing CeONP-treated male
flies have improved motor function 14 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Fourteen days
after stroke, motor function was assessed by negative geotaxis.
Flies fed standard food and exposed to stroke continued to have
significantly decreased motor function at all goal heights compared
to normal controls. CeONP preserved the negative geotactic response
in stroked flies, to levels equivalent to normal controls, with the
exception of the 200 .mu.M food group at the 8 cm height. *Sig.
from control, P<0.01; .sup.#Sig from stroke, P<0.01.
[0025] FIG. 12 provides a bar graph showing CeONP-treated male
flies have improved motor function 36 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Thirty six days
after stroke, motor function was assessed by negative geotaxis.
Controls (unstroked) flies showed decreased motor function as
compared to their motor function on days 2-14. This is typical, as
the flies are now reaching the end of their life span (flies are
now 54 days old with an average lifespan of 58-60 days). Flies fed
standard food and exposed to stroke continued to have significantly
decreased motor function at all goal heights compared to normal
controls. CeONP preserved the negative geotactic response in
stroked flies at all goal heights. Additionally, flies fed CeONP
had improved motor function, as compared to unstroked controls
(with the exception of the 200 mM food group at 8 cm. *Sig. from
control, P<0.01; .sup.#Sig from stroke, P<0.01.
[0026] FIG. 13 provides a bar graph showing Motor Function in
Normal Female Drosophila. Flies were fed standard food (Jazz Mix)
or food containing the indicated concentrations of cerium oxide
nanoparticles for 14 days. On day 14, motor function was assessed
by measuring negative geotaxis, the ability of flies to climb to 3,
5.5, and 8 cm in a 10 second minute period. Data is expressed as
the percentage of flies achieving each height goal in the required
time.
[0027] FIG. 14 provides a bar graph showing CeONP-treated female
flies had increased motor function after stroke. Flies were fed as
described in FIG. 8. On day 15 flies were exposed to anoxia for 2.5
hrs, followed by return to their respective food group. Two days
after stroke, motor function was assessed by negative geotaxis.
Note that flies fed standard food and exposed to stroke had
significantly decreased motor function as compared to normal flies.
CeONP significantly improved the negative geotactic response in
stroked female flies, to levels equivalent to normal controls at
the 3 cm climbing height. *Sig. from control, P<0.01; # Sig.
from stroke, P<0.01.
[0028] FIG. 15 provides a bar graph showing CeONP-treated female
flies have improved motor function 6 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Six days after
stroke, motor function was assessed by negative geotaxis. Females
fed standard food and exposed to stroke continued to have
significantly decreased motor function at all heights. Females
treated with 100 and 200 .mu.M CeONP showed improved motor function
at all goal heights, as compared to stroked flies, with climbing to
the 3 cm height equivalent to unstroked controls. *Sig. from
control, P<0.01; .sup.#Sig from stroke, P<0.01.
[0029] FIG. 16 provides a bar graph showing CeONP-treated female
flies have improved motor function 14 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Fourteen days
after stroke, motor function was assessed by negative geotaxis.
Flies fed standard food and exposed to stroke continued to have
significantly decreased motor function at all goal heights compared
to normal controls. CeONP preserved the negative geotactic response
in stroked flies at the 1 and 100 .mu.M doses, to levels equivalent
to normal controls. *Sig. from control, P<0.01; .sup.#Sig from
stroke, P<0.01.
[0030] FIG. 17 provides a bar graph showing CeONP-treated female
flies have improved motor function 36 days after stroke. Flies were
fed and exposed to anoxia as described in FIG. 9. Thirty six days
after stroke, motor function was assessed by negative geotaxis.
Controls (unstroked) flies continued to show decreased motor
function as compared to their motor function on days 2-14. This is
typical, as the flies are now reaching the end of their life span
(flies are now 54 days old with an average lifespan of 58-60 days).
Flies fed standard food and exposed to stroke continued to have
significantly decreased motor function at all goal heights compared
to normal controls. All doses of CeONP preserved the negative
geotactic response in stroked flies at all goal heights, to levels
that were greater than control (unstroked) flies. *Sig. from
control, P<0.01; .sup.#Sig from stroke, P<0.01.
DETAILED DESCRIPTION
[0031] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this application pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
exemplary embodiments, suitable methods and materials are described
below. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
Definitions
[0032] The terminology as set forth herein is for description of
the embodiments only and should not be construed as limiting the
application as a whole. Unless otherwise specified, "a," "an,"
"the," and "at least one" are used interchangeably. Furthermore, as
used in the description of the application and the appended claims,
the singular forms "a", "an", and "the" are inclusive of their
plural forms, unless contraindicated by the context surrounding
such.
[0033] The recitations of numerical ranges by endpoints include all
numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5,
2, 2.75, 3, 3.80, 4, 5, etc.).
[0034] The expression "therapeutically effective amount" as used
herein, refers to a sufficient amount of agent to exhibit a
therapeutic effect. The exact amount required will vary from
subject to subject, depending on the species, age, and general
condition of the subject, the particular therapeutic agent, its
mode and/or route of administration, and the like. It will be
understood, however, that the total daily usage of the compounds
and compositions of the present invention can be decided by an
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
subject or organism will depend upon a variety of factors including
the disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the subject; the time of administration, route of
administration, and rate of excretion of the specific composition
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific composition employed; and like
factors well known in the medical arts.
[0035] Treatment, as used herein, encompasses the administration of
cerium oxide nanoparticles to a subject that has already suffered
symptoms of a disease. In some embodiments, treatment is effective
to eliminate the disease and/or its symptoms; in another
embodiment, administration of the cerium oxide nanoparticles is
effective to decrease the severity of the disease and/or its
symptoms. Treatment includes improvement in the condition through
lessening or suppression of at least one symptom and/or delaying in
progression of the disease. Preventing, as used herein, refers to
avoiding the development of additional symptoms or the development
of a more severe case of a disease.
[0036] To address the shortcomings of prior attempts to develop
cerium oxide nanoparticles for use in treating damage caused by
free radicals, different methods of synthesizing nanoparticles, and
thus different nanoparticles, were investigated. Efforts were
directed toward examining the biological efficacy of commercially
available cerium oxide nanoparticles prepared by existing
manufacturing processes. These included cerium oxide nanoparticles
available from Nanophase Technologies Corporation (Romeoville,
Ill.), Advanced Powder Technology Pty Ltd. (Welshpool, Western
Australia), and NanoScale Materials Inc. (Manhattan, Kans.). In
summary, in a series of experiments, it was found that cerium oxide
nanoparticles produced by Nanophase Technology Corporation, using
specific, patented mechanisms of synthesis, provided consistently
reproducibly sized nanoparticles that consistently showed high
levels of biological activity. With sizes of 20 nm and below,
particles readily entered cells and reduced free-radical mediated
damage. Synthesis for these particles has been described in the
following patents, the disclosures of the entireties of all of
which are incorporated herein by reference: U.S. Pat. No.
6,669,823, U.S. Pat. No. 5,460,701, U.S. Pat. No. 5,514,349, U.S.
Pat. No. 5,874,684, Japanese Patents JP2980987 and JP3383608,
European Patent EP0711217B1, German Patent DE69426886, French
Patent FR94922757, Great Britain Patent GB94922757, and Australian
Patent AU068582882.
[0037] It was surprisingly found that the new source of cerium
oxide nanoparticles provided superior reproducibility of activity
from batch to batch, and showed lower toxicity to mammalian cells.
It was determined that the cerium oxide nanoparticles used in the
present invention were different from the prior nanoparticles in
quality and size distribution, factors that significantly
contribute to their improved characteristics in treating subjects
according to the methods of the invention. In developing the
invention, it was determined that, regardless of source, cerium
oxide particles having a small size, narrow size distribution, and
low agglomeration rate are most advantageous. Also, for delivery,
the nanoparticles are advantageously in a non-agglomerated form. To
accomplish this, stock solutions of about 10% by weight can be
sonicated in ultra-high purity water (16 megaohms). These
nanoparticles are superior to previously developed cerium oxide
nanoparticles for treatment of and protection against, damage
caused by free radicals. This new and useful improvement allows
cerium oxide nanoparticles to be used in extending the life of a
cell in vivo as well as in vitro. In particular, it is shown herein
the novel finding that cerium oxide nanoparticles of a defined size
range and distribution and made by a method other than sol-gel
synthesis increase the lifespan of cells, such as cells of an
organism in vivo. Also shown is that cerium oxide nanoparticles
enhance the lifespan of mammalian cells in culture and in vivo, act
as potent free radical scavengers, and possess significant
anti-inflammatory and radioprotective properties in vivo.
[0038] While not wishing to be limited to any single method of
action, it is thought that cerium oxide nanoparticles have a unique
oxide lattice and valence structure that might confer them with the
ability to scavenge (detoxify) intracellular free radicals, and
might thus convey their anti-inflammatory, radioprotective, and
longevity-enhancing properties. Further, the data obtained by the
inventors, and provided herein, suggests that the valence and
oxygen lattice structure conveys the ability of cerium oxide
nanoparticles to regenerate a biologically active matrix after a
free radical scavenging event. This allows small, single doses of
nanoparticles to remain active within the cell for long periods of
time, conveying regenerative biological effects. In contrast, most
commonly available free radical scavengers, such as vitamin E,
nitrosone compounds, and vitamin C are inactivated by alteration of
their chemical structure after scavenging a single free radical.
This loss of structure limits their pharmacological efficacy and
requires high dosing regimens.
[0039] It appears that the regenerative activity of the cerium
oxide nanoparticles may be dependent on a well-known oscillating
chemical phenomenon, known as the Belousov-Zhabotinsky (B-Z)
reaction, in which cerium oxide serves to facilitate oscillation of
electrons (or free radicals) from one compound to another. Cerium
in the nanoparticles exists in two valence states, +3 and +4.
Adequate propagation of B-Z requires a specific ratio of Ce+3 to +4
in the nanoparticles. If the composition changes to have too much
+3 cerium, the reaction will not propagate. Research has shown that
as the cerium oxide nanoparticle size is reduced from 30 nm to 3
nm, lattice strain in the nanoparticles causes more cerium to be in
the +3 state. Although this mechanism has only been studied in
vitro up to now, this mechanism of action may also be true in vivo
and would provide a significant advantage to using larger sizes of
cerium oxide nanoparticles.
[0040] Further research has also shown that cerium oxide
nanoparticles have a beneficial effect on mitochondrial dysfunction
which may also contribute to their beneficial effects in treating
stroke and cardiovascular disease. The inventors have shown that
cerium oxide nanoparticles enter the mitochondria, and can
substitute for damaged elements of the electron transport change,
thereby improving ATP synthesis and mitochondrial membrane
potential in diseases, such as stroke, in which mitochondrial
damage is evident. As such, cerium oxide nanoparticles replace
damaged semiconductor elements of cellular mitochondria and improve
energy production in disease and damaged tissue. Further discussion
of the role of cerium oxide nanoparticles in treating mitochondrial
dysfunction can be found in U.S. patent pplication Ser. No.
12/252,905 (Rzigalinski et al.), the disclosure of which is
incorporated herein by reference.
[0041] Broadly speaking, the present invention provides a method of
treating at least one cell with cerium oxide particles. The method
generally comprises contacting at least one cell with an amount of
cerium oxide nanoparticles that reduces or eliminates damage caused
by free radicals, which are unstable, highly reactive molecules
such as nitric oxide, superoxide, hydroxyl radicals, peroxynitrite,
and other unstable reactive compound formed from the above. They
cause aging and various diseases by taking electrons from other
molecules in the body, a process that causes cell or oxidative
damage. As used herein, cell or oxidative damage has the same
meaning as oxidative stress.
[0042] "Contacting" means any action that results in at least one
cerium oxide nanoparticle physically contacting at least one cell.
It thus may comprise exposing the cell(s) to cerium oxide
nanoparticles in an amount sufficient to result in contact of at
least one cerium oxide nanoparticle with at least one cell. The
method can be practiced in vivo, in which case contacting means
exposing at least one cell in a subject to at least one cerium
oxide nanoparticle. According to the invention, contacting thus may
comprise exposing at least one cell to at least one cerium oxide
particles, such as, for example by administering cerium oxide
particles to a subject via any suitable route. It also may comprise
exposing cells in vitro or ex vivo by introducing, and preferably
mixing, cerium oxide particles and cells in a controlled
environment, such as a culture dish or tube. Optionally, where
practiced in vitro or ex vivo, some or all of the cerium oxide
particles that are not taken up or adsorbed by cells are removed,
for example by washing the cells in suitable media, buffer, water,
etc. According to the invention, contacting may comprise
introducing, exposing, etc. the cerium oxide particles at a site
distant to the cells to be contacted, and allowing the bodily
functions of the subject, or natural (e.g., diffusion) or
man-induced (e.g., swirling) movements of fluids to result in
contact of the nanoparticle(s) and cell(s). Where practiced ex
vivo, the cells may also be re-introduced into a subject,
preferably the subject from which they were originally obtained. In
one embodiment, this includes putting the particles into a gel or
other packet that limits diffusion, followed by implanting it into
a body area such as a knee joint.
[0043] According to the method of the invention, the subject,
individual, or patient can be any organism to whom the cerium oxide
nanoparticles are administered. Thus, the subject may be a human or
a non-human animal, such as another mammal, including, but not
limited to a rodent (e.g., mouse, rat, rabbit), a canine (e.g., a
dog), a feline (e.g., a cat), an equine (e.g., a horse), an ovine
(e.g., a sheep), an orcine (e.g., a pig), or a bovine (e.g., a cow
or steer). The subject can be any other animal such as a bird,
reptile, amphibian, or any other companion or agricultural
animal.
[0044] The method can be practiced in vivo as either a therapeutic
method of treating a disease or disorder involving free radicals or
as a prophylactic method to prevent free radical damage. In
embodiments where the method is a method of treating (i.e., a
therapeutic method), the amount is an amount that is effective for
reducing or eliminating cell death or dysfunction or tissue or
organ damage due to free radicals that are being produce, or were
produced previously, in the subject, or mitochondrial damage
produced by stroke or cardiovascular disease. The subject,
individual, or patient may be one who is in immediate or apparent
need of, or suspected of being in need of, treatment for a disease
or disorder associated with free radicals, or it may be one who is
in immediate or apparent need of, or suspected of being in need of,
treatment for an injury or other trauma resulting from or known to
result in production of free radicals. In such situations, where a
pre-existing condition related to cell, tissue, or organ damage due
to free radicals is evident or suspected, the method is a
therapeutic method. For example, if a subject has had a stroke, it
may be beneficial to treat the subject with cerium oxide
nanoparticles to reduce the effects of the stroke.
[0045] In addition, according to the methods of the invention, the
subject, individual, or patient may be one who is not in or
suspected of being in need of treatment of a pre-existing disease,
disorder, or injury or trauma. In such situations, the method is a
prophylactic method. Prophylactic methods are useful in situations
where the subject is currently engaged in, or soon to be engaged
in, one or more activities that might result in an injury or
trauma. They are also useful in situations where the patient has a
likelihood of developing a disease or disorder associated with
cell, tissue, or organ damage due to free radicals. Thus, the
present methods are useful not only for treating patients with a
disease or disorder, but for treating patients who are suspected of
having a predisposition to a disease or disorder. For example, if
the family of a subject has been shown to be prone to a certain
neurodegenerative disease, the subject may be given cerium oxide
nanoparticles to avoid or reduce the effects of that disease.
Likewise, if a subject exhibits one or more risk factors associated
with stroke, it may be beneficial to prophylactically administer
cerium oxide nanoparticles to decrease the amount of neurological
damage that may result should a stroke occur.
[0046] As another example to compare prophylactic and therapeutic
methods, in embodiments where the method is a prophylactic method,
the amount is an amount that is effective in reducing or blocking
cell death or dysfunction or tissue or organ damage due to free
radicals that might be produced in the subject in the future. For
example, in a therapeutic method, the cerium oxide nanoparticles
may be administered to a patient following a head injury to reduce
the amount of damage to the brain as a result of the injury. In
contrast, in a prophylactic method, the cerium oxide nanoparticles
may be administered to a subject prior to engaging in an activity
that has a likelihood of head injury, such as a car race or other
high-speed activity.
[0047] The act of administering cerium oxide nanoparticles can be
any act that provides the cerium oxide nanoparticles to a subject
such that the particles can function for their intended purpose.
For example, administering can be by injection or infusion. It can
thus be an intramuscular, intraperitoneal, subcutaneous, or
intrathecal injection, or a slow-drip or bolus infusion. Other
non-limiting examples of methods of administration include topical
administration, such as by way of lotions, salves, or bandages,
often on intact skin but also through open wounds, lesions, or
sores. Yet other non-limiting examples include administration
through mucous membranes, such as by way of intranasal
administration through inhalation of dry particles or a mist
comprising the particles, oral ingestion, sublingual absorption, by
subcutaneous means, and rectal or vaginal delivery. The vehicle of
delivery may be in any suitable form, such as the form of an oral
solution, gel, tablet, capsule, powder, suppository, infusible,
lozenge, cream, lotion, salve, inhalant, or injection.
[0048] According to embodiments of the method, the method can
comprise repeating the act of contacting (e.g., administering) the
cerium oxide nanoparticles. In embodiments relating to
administering the cerium oxide to subjects, repeating the
administration can include one or more administrations in addition
to the original administration. The amount to be administered to
each subject will vary depending on usual factors taken into
consideration for dosing of pharmaceuticals, such as weight,
general health, and metabolic activities of the patient. Likewise,
the mode of administration (e.g., injection, oral administration)
will be taken into account when determining the proper amount of
nanoparticles to administer per dose.
[0049] In general, a dosing of about 0.005 to about 500 micrograms
per gram of body weight, with doses in the range of about 0.05
micrograms to about 50 micrograms per gram of body weight of 10-20
nm cerium oxide nanoparticles being more preferred. Specific
embodiments may use about 50 ng, 100 ng, 500 ng, 1 .mu.g, 5 .mu.g,
10 .mu.g, or 50 .mu.g per administration or per gram body mass per
administration should be effective in providing the desired
therapeutic or prophylactic result. Of course, injection or
infusion amounts will tend to be on the lower end of the range
while oral administration amounts will tend to be on the upper end.
Current results suggest that the optimal dose for 10-20 nm cerium
oxide nanoparticles is 10 nM to 1 uM for blood and intracellular
fluid levels. However, the action of the particles is highly
dependent on other variables and so these amounts will vary
depending on the surface area, the species of the subject, the
reason for administration, etc. Amounts may be higher when the
method is practiced in vitro or ex vivo because excess particles
may be easily removed at any time by washing, etc.
[0050] It should be noted that this method shows low toxicity in
mammalian cells, fruit flies, rats, and mice, and thus is expected
to show low toxicity in other animal cells. This new and useful
improvement allows the method of the present invention to be used
in subjects with lower toxicity than in previous inventions. This
important feature of the present invention means that the cerium
oxide nanoparticles can be used in a broad range of applications.
In preferred embodiments, the cerium oxide nanoparticles do not
contain docusate sodium, which has been shown to produce toxicity
in tissue culture. Also, in preferred embodiments, there are less
than 0.01% (w/w or w/v) of any other contaminating ions, metals, or
other substances, which can also cause toxicity to cells.
[0051] Although the cerium oxide nanoparticles show very low
toxicity, in some instances it might be desirable to provide
multiple, low doses of particles to an individual. In such cases,
the method may comprise two or more administrations of less than
the total effective amount, where the amount ultimately
administered is an effective amount. Likewise, multiple
administrations of an effective dose may be desirable where the
second or subsequent administration is performed at a time well
separated from the first administration. That is, because the
cerium oxide nanoparticles are highly stable, even after being
administered, repeated administrations of effective doses are
envisioned as occurring at widely spaced intervals, such as months
or years apart.
[0052] The invention thus includes a method of providing
prophylactic protection from neurological injury in a subject. This
aspect involves administering a therapeutically effective amount of
cerium oxide nanoparticles to the subject before the occurrence of
stroke. Because of the high stability of cerium oxide
nanoparticles, the nanoparticles can be administered well in
advance of the occurrence of a stroke, with repeated administration
being provided in some embodiments at widely spaced intervals. In
some embodiments, the cerium oxide nanoparticles are
prophylactically administered to subjects who have one or more risk
factors associated with the occurrence of stroke.
[0053] Furthermore, where multiple administrations are performed,
different modes of administration may be used. For example, if two
doses are administered, one can be an injection whereas the other
can be oral. In addition, if three or more doses are administered,
two or more may be by the same mode, while the remaining may be
from one or more different mode, in any combination, number, and
order. Of course, where multiple administrations are used, each
administration may be by a different mode. The mode of
administration, the number of times it is repeated, and the
sequence of modes of administration may be selected by those of
skill in the art based on numerous considerations, and such
selection is well within the abilities of those of skill in the
art.
[0054] The method can also be practiced in vitro which means that
contacting at least one cell with at least one cerium oxide
nanoparticle can occur in a petri dish, a test tube, an IV tube, or
any other container applicable for contacting. When practiced in
vitro, it may be a method for identifying parameters that are
useful in in vivo treatment regimens. The method can be practiced
to study the effects of combinations of nanoparticles with drugs on
cells. For example, the cerium oxide nanoparticles can be combined
with other known antioxidants such as vitamin E, n-acetyl cysteine,
or melatonin. The cerium oxide nanoparticles could also be combined
with disease specific drugs. The in vitro methods can also comprise
using the cerium oxide nanoparticles as a research tool to observe
the effects of free radicals on cells or observe the cells for
changes in protein expression, cell morphology, or any other
characteristic of interest.
[0055] In preferred embodiments, the method is practiced with
size-limited cerium oxide nanoparticles made by a method other than
a sol-gel method. The nanoparticles useful in the present invention
have pre-defined sizes clustered tightly within a range. In
general, the particles have a size of about 1 nm or less to about
500 nm. In embodiments, the particles are 11 nm or more. In
embodiments where particles are taken into the interior of cells,
the preferable range of particles that are taken into the cell are
from about 11 nm to about 50 nm, such as about 20 nm. In
embodiments where particles exert their effects on cells from
outside of the cells, the preferable range of particles that are
extracellular are from about 11 nm to about 500 nm. In embodiments,
the particles are from about 40 nm to about 500 nm. In other
embodiments, the particles are from about 11 nm to about 40 nm,
such as from about 11 nm to about 20 nm, about 15 nm to about 20
nm, about 11 nm to about 15 nm, or about 30 nm to 40 nm. Of course,
any specific size range within these general sizes can be provided,
the size being selected by the practitioner based on any number of
parameters. According to the invention, the term "about" is used to
indicate a margin of error for a statistically significant portion
of the particles of 10%. Thus, particles of a size of 20 nm include
those in which a majority of the particles fall within the range of
18 nm to 22 nm. In embodiments, 95% of the cerium oxide
nanoparticles have a size of between about 15 nm and about 25 nm.
In embodiments, 95% of the cerium oxide nanoparticles are within 5%
of 20 nm. In other embodiments, 90% of the cerium oxide
nanoparticles have a size of between about 18 nm and about 22
nm.
[0056] In certain embodiments, the invention provides compositions
comprising cerium oxide nanoparticles for improving neuronal
recovery after stroke, or for treating or preventing cardiovascular
disease. Cerium oxide nanoparticles having a size below 10 nm
results in decreased ability to scavenge multiple types of free
radicals. As size decrease, only superoxide radicals were
scavenged, with less scavenging of hydroxyl and nitroxyl radicals.
In stroke, as in other neurodegenerative diseases, superoxide
radicals represent only a small fraction of the radicals actually
produced, with hydroxyl and nitroxyl radicals being more abundant.
For further discussion of the effect of particle size on the
scavenging of different types of free radicals, see Rzigalinski et
al., "Antioxidant Nanoparticles," Nanomedicine in Health and
Disease, Hunter R. J. & Preedy, V. R. (eds.), CRC press, NY,
2011, the disclosure of which is incorporated herein by reference.
Accordingly, in some embodiments, the cerium oxide nanoparticles
have a size range of from about 5 nm to about 25 nm, such as from 7
nm to 20 nm, 7 nm to 12 nm, 13 nm to 20 nm, 14 nm to 20 nm, 15 nm
to 20 nm. In embodiments, a majority of the particles have a size
within the range of 18 nm to 22 nm. In other embodiments, the
nanoparticles have an average size of about 10 nm.
[0057] The present invention provides methods of treating
individuals suffering from, or suspected of suffering from, a
disease or disorder involving free radicals, such as oxygen
radicals, or a disease involving mitochondrial dysfunction. It
likewise provides methods of treating individuals suffering from,
or suspected of suffering from a complication of an injury that
results from free radicals, such as oxygen radicals, or results in
the production of free radicals, such as oxygen radicals. In
general, the methods of the invention comprise administering to an
individual (used interchangeably herein with "subject" and
"patient") an amount of cerium oxide nanoparticles sufficient to
reduce or eliminate cell, tissue, or organ damage in the individual
that is caused by free radicals. Thus, the invention encompasses
the use of cerium oxide nanoparticles in enhancement of cell and
organism longevity, reduction of inflammation and inflammatory
disorders, reduction in tissue damage due to inflammatory
disorders, and reduction in radiation injury.
[0058] While the above disclosure discusses administration in vivo,
it is important to recognize that the present invention also
encompasses administering ex vivo. Thus, a method according to the
invention can comprise removing at least one cell from an organism,
administering cerium oxide nanoparticles to that cell, then
returning the cell to its natural environment (e.g., into the body
of the patient). In such situations, the act of administering can
be simply exposing the nanoparticles to the cell, for example in a
culture dish or a tube. In one particular embodiment, the method of
ex vivo administration comprises obtaining blood from a patient,
exposing the blood to cerium oxide nanoparticles, and returning the
treated blood to the patient. The method can comprise separating
cerium oxide nanoparticles from the blood prior to returning the
blood to the patient.
[0059] In another embodiment, the present invention is used to
affect, either prophylactically or therapeutically, cell longevity
in organisms. The methods treat or affect, either prophylactically
or therapeutically, diseases or disorders associated with free
radicals, or cell death or tissue or organ damage due to free
radicals. In general, the methods comprise administering to a
subject an amount of cerium oxide nanoparticles sufficient to
reduce, eliminate, or block cell, tissue, or organ damage caused by
free radicals in the subject.
[0060] In one embodiment, the cerium oxide nanoparticles can be
taken up by the cell. In this case, they can act to reduce or
eliminate free radicals within the cell. This method can be used
for the prevention or treatment of brain disease, spinal cord
disease, or other neurological trauma. This method can also be used
for the treatment or prevention of neurodegenerative disorders such
as Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis (ALS), multiple sclerosis,
toxin-mediated damage, or stroke. This method may be used in the
treatment or prevention of cardiovascular disease, diabetes,
diseases of the retina, asthma, respiratory dysfunctions, and
allergic or autoimmune diseases, such as chronic obstructive
pulmonary disease and lupus. It is to be understood that the
diseases stated above are only examples and are not to be
understood as limiting the invention in anyway.
[0061] A stroke is a medical condition that can cause permanent
neurological damage and death. Strokes can be classified into two
major categories: ischemic and hemorrhagic. Ischemic strokes are
those that are caused by interruption of the blood supply, while
hemorrhagic strokes are the ones which result from rupture of a
blood vessel or an abnormal vascular structure In an ischemic
stroke, blood supply to part of the brain is decreased as a result
of thrombosis (obstruction of a blood vessel by a blood clot
forming locally), embolism (obstruction due to an embolus from
elsewhere in the body, see below), systemic hypoperfusion (general
decrease in blood supply, e.g., in shock), or venous thrombosis.
Risk factors associated with an increased likelihood of having a
stroke include old age, high blood pressure, previous stroke or
transient ischemic attack, diabetes, high cholesterol, tobacco
smoking and atrial fibrillation.
[0062] The neurological damage caused by a stroke can result in
various symptoms, such as motor function disorders and various
sensory and cognitive disorders. More specifically, symptoms
include numbness, altered smell, taste, hearing, or vision,
drooping of eyelid, decreased reflexes (e.g., gag, swallow, pupil
reactivity to light), balance problems, altered breathing and heart
rate, various speech disorders such as aphasia and dysarthria,
memory deficits, confusion, altered walking gait, and lack of
movement coordination.
[0063] One embodiment of the invention provides a method of
treating or preventing neurological injury in a subject who has
suffered a stroke by administering a therapeutically effective
amount of cerium oxide nanoparticles to the subject. As described,
the neurological injury from a stroke results from a disruption in
the blood supply, resulting in cell death due in part to free
radical formation. Accordingly, the neurological injury can be
treated or prevented by scavenging the free radicals. To scavenge
free radicals, the cerium oxide nanoparticles can be administered
either before or after the occurrence of stroke. While it would be
preferable to administer the cerium oxide nanoparticles before the
occurrence of stroke in order to minimize free radical formation,
it is also beneficial to administer the cerium oxide nanoparticles
once a stroke has occurred. For example, the cerium oxide
nanoparticles can be administered immediately after a stroke,
within 5, 10, or 15 minutes of a stroke, within a half hour of a
stroke, within one hour after the stroke, or within 24 hours of the
stroke. Even after the acute phase of neurological injury,
administration of cerium oxide nanoparticles have been shown to
have a beneficial effect in mitigating neurological injury
resulting from stroke.
[0064] Cerium oxide nanoparticles can also be used to treat or
prevent cardiovascular disease. Examples of cardiovascular disease
include coronary heart disease (e.g., ischemic heart disease),
cardiomyopathy, heart failure, cardiac dysrhythmias, inflammatory
heart disease, and peripheral arterial disease. In particular,
cerium oxide nanoparticles can be used to treat or prevent
cardiovascular disease involving ischemia such as ischemic heart
disease. Ischemic heart disease is characterized by a reduced blood
supply of heart muscle, usually due to atherosclerosis. Signs and
symptoms of ischemic heart disease include angina pectoris (chest
pain on exertion, in cold weather or emotional situations), acute
chest pain (i.e., heart attack) such as acute coronary syndrome,
unstable angina or myocardial infarction, heart failure with
associated difficulty in breathing or swelling of the extremities,
and heartburn. Risk factors for ischemic heart disease include age,
smoking, hypercholesterolaemia, diabetes, and hypertension.
[0065] In another embodiment, the cerium oxide nanoparticles are
not taken up in any significant amount by the cells, but go into
intravascular or interstitial spaces. In this embodiment, the
nanoparticles can act to reduce or eliminate free radicals outside
the cell. This can result in reduction of inflammation and
inflammatory disorders. The cerium oxide nanoparticles can reduce
inflammation systemically (throughout a subject's body) or locally
(at the site of the inflammatory cells). The nanoparticles can
reduce or eliminate inflammation that leads to preeclampsia or
inflammation caused by wounding. This can also reduce or eliminate
inflammation caused by the insertion of a medical prosthesis into
the subject. Nanoparticles may be retained at particular sites, at
least substantially retained for periods of time, by inclusion of
the nanoparticles into compositions, such as dissolvable or porous
matrices and the like.
[0066] In a further aspect, cerium oxide nanoparticles and
compositions comprising cerium oxide nanoparticles are provided.
The cerium oxide nanoparticles are size-limited and provided in an
amount sufficient to provide one or more doses to a subject in need
of, or suspected of being in need of, treatment for a disease or
disorder involving free radicals. Compositions may comprise cerium
oxide particles of the invention along with one or more other
substances, which are typically substances that are biologically
tolerable in that they may be exposed to living cells without
killing the cells. In embodiments, the other substances are
pharmaceutically acceptable substances. As used herein,
"pharmaceutically acceptable substance" is intended to include
solvents, coatings, antibacterial and antifungal agents, and any
other ingredient that is biologically tolerable. Examples of such
carriers include, but are not limited to, water, buffered saline,
dextrose solution, human serum albumin, liposomes, and hydrogels.
The use of such media and agents for pharmaceutically active
substances is well known in the art, and thus further examples and
methods of incorporating each into compositions at effective levels
need not be discussed here.
[0067] Certain aspects of the invention provide for the use of
cerium oxide nanoparticles in the treatment of diseases and
disorders associated with free radicals, such as oxygen free
radicals, or mitochondrial dysfunction. The use is in particular
for in vivo therapeutic or prophylactic methods of protecting cells
from free radical damage. Certain other aspects of the invention
provide for the use of cerium oxide nanoparticles in the
preparation of compositions for medical use, such as pharmaceutical
or therapeutic compositions. In general, use of the particles is in
combining them with other substances to make medicinal
compositions.
[0068] Another aspect of the invention provides a container
containing cerium oxide nanoparticles. In general, a container
according to the invention contains a sufficient amount of
size-limited cerium oxide nanoparticles made by a method other than
a sol-gel method to provide at least one dose of cerium oxide to a
subject suffering from, or at risk of suffering from, a disease or
disorder involving free radicals, such as oxygen radicals. For
example, the container may contain sufficient cerium oxide
nanoparticles and, optionally, one or more other biologically
tolerable substance, for one dose to a human or non-human animal
subject. In certain embodiments, the container is provided in a
package with one or more other containers and/or with one or more
articles of manufacture or devices having use in delivery of
substances to subjects (e.g., syringes, needles, antiseptic swabs,
sterile saline solution). In some embodiments, kits comprising one
or more containers are provided.
[0069] Regardless of whether provided alone, as part of a
composition, or as part of a kit, the cerium oxide nanoparticles
may be provided in any suitable physical form. Thus, they may be
provided as dry particles or as part of a liquid composition. When
part of a liquid composition, the composition typically will
comprise water or an aqueous buffer, such as phosphate buffered
saline (PBS) or other salt buffers. In general, it is preferred
that the liquid composition be suitable for introduction into a
living organism or for contact with a living cell without causing
deleterious effects, such as cell toxicity. It is to be understood
that this general preference permits inclusion of toxic components
in the liquid composition as long as those components, when exposed
to a living cell upon exposure to the cell, are present in a
non-toxic form or at non-toxic levels. In embodiments where dry
nanoparticles are administered, the nanoparticles may be in a
purified state or may be in a composition comprising one or more
other component. It is preferred that the other component(s) be
non-toxic or, if toxic, present in an amount that, when
administered, is not toxic to the cell or subject as a whole.
Examples of non-toxic components include, but are not limited to,
salts (e.g., sodium salts such as sodium phosphate or sodium
chloride); sugars (e.g., glucose, sucrose); preservatives; and
antibiotics, anti-inflammatories, albumin, lipids, or other drugs.
The vehicle of delivery may be in the form of an oral solution,
gel, tablet, capsule, powder, suppository, infusible, lozenge,
cream, salve, inhalant, or injection.
[0070] Typically, the particles or composition comprising the
particles will be sterile or will have been sterilized prior to
administration to a subject or other use. The particles may be
sterilized using any suitable technique known in the art,
including, but not limited to, heat sterilization, filtration, and
irradiation. Thus, in embodiments, the method of the invention
further comprises providing sterile or sterilized cerium oxide
nanoparticles, or further comprises sterilizing the nanoparticles
prior to administering them to a subject.
[0071] The invention provides compositions comprising cerium oxide
nanoparticles. The compositions can comprise a pharmaceutically
suitable carrier, a nutritional supplement, or a dietary
supplement. While not being so limited, typically the compositions
comprise one or more other substances other than the nanoparticles,
where the other substances are biologically tolerable (i.e.,
non-toxic or present in an amount that is non-toxic). Examples of
such substances are well known to those of skill in the art and
include, without limitation, sugars, salts, lipids, drugs,
excipients, carriers, flavorants, fillers, binders, gums,
colorants, water, buffers, detergents, biologically active
compounds, and the like.
[0072] The present invention also provides kits. In general, the
kits comprise cerium oxide nanoparticles in an amount sufficient to
treat at least one patient at least one time to reduce or eliminate
free radicals that can cause cell, tissue, or organ damage.
Typically, the nanoparticles of the kit will be supplied in one or
more container, each container containing a sufficient amount of
nanoparticles for at least one dosing of the patient. The kits can
comprise other components, such as some or all of the components
necessary to practice a method of the invention. For example, in
embodiments of the kit, albumin is included, either as a separate
component or as part of a composition comprising the nanoparticles.
The albumin is provided to lessen the amount or use of disruption
of the nanoparticles, for example by sonication at 5-20 Hz for 2
minutes, that can sometimes be needed to provide certain
formulations for delivery. The kits may contain a syringe for
administering a dose of the nanoparticles. The kits may also
comprise filters for sterilization of the particles prior to
delivery; however, it is preferred that the particles be sterilized
prior to packaging in the kits, or the entire kit be sterilized
after all components are packaged. It may likewise contain sterile
water or buffer for rehydration or reconstitution of dry
nanoparticles, prior to administration of the particles to a
patient. In embodiments, multiple doses of nanoparticles are
provided in the kit, either all in a single container (e.g., a
vial) or distributed among two or more containers. As the invention
contemplates administering or delivering (used synonymously herein)
of nanoparticles in liposomes, kits according to the invention may
comprise liposomes, particularly liposomes loaded with the
nanoparticles.
[0073] The following examples are included for purposes of
illustration and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Evaluation of CeONP Treatment of Stroke Using a Tissue Culture
Model
[0074] Given that cellular damage in human ischemic stroke arises
from oxidative stress and free radical production, as well as
mitochondrial dysfunction, we propose that CeONP may be used to
prevent and treat neuronal dysfunction and motor deficits
associated with stroke.
In Vitro Studies
[0075] We first investigated the efficacy of CeONP in treatment of
stroke using a tissue culture model. In human ischemic stroke,
blockade of an artery in the brain deprives a specific area of the
brain of blood flow. This results in deprivation of oxygen for
respiration and glucose for energy. Hence, tissue culture models
for ischemic stroke involve subjecting cultured brain cells to an
anoxic environment devoid of glucose and other sugars utilized to
produce energy.
Methods:
[0076] We utilized a well-established in vitro tissue culture model
for stroke in which cells grown in culture are deprived of oxygen
and glucose (oxygen/glucose deprivation, OGD). (S M Jones et al.,
J. Neurosci. Methods, 199, 241-248, 2011). For these studies, mixed
organotypic brain cell cultures containing neurons, astrocytes and
microglia were prepared as we have previously described (Zhang
& Rzigalinski, Science 274, 1921-1923, 1997). Cultures were
either pretreated with CeONP or treated with CeONP 15 minutes or 1
hr after OGD, as indicated in the figures. For pretreatment
studies, cultures were treated with 100 nM CeONP on day 5 in vitro.
Organotypic cultures were allowed 48 hrs to take up the
nanoparticles, and the medium was changed after 48 hrs. We have
previously shown that cells readily take up CeONP, and retain it in
the cytoplasm or mitochondria for up to 2 months, possibly longer
(Rzigalinski et al., Biological Nanoparticles for Cell
Engineering--A Radical Concept. In Nanotechnologies for Life
Sciences, C. Kumar, editor, Wiley & Sons, 2006). For post-OGD
treatment, cultures were treated with CeONP either 15 minutes or 1
hour after OGD.
[0077] On day 15 in vitro, cultures were subjected to OGD using an
anaerobic chamber. Prior to use, the chamber was purged with
nitrogen and filled with 90%N.sub.2/10%CO.sub.2. To assure lack of
oxygen in the chamber, oxygen levels were monitored with a gas
sensor, and were maintained at 0% for the duration of OGD. Just
prior to exposure of cell cultures to OGD, cells were washed and
placed in OGD medium without glucose, glutamine, or antibiotics.
The OGD medium used was previously bubbled with N.sub.2 for 30
minutes to remove any dissolved O.sub.2, and equilibrated in the
anaerobic chamber overnight prior to use.
[0078] After placement of cells in OGD medium, the cultures were
sealed in the anaerobic chamber and maintained at 37.degree. C. for
30, 60 or 90 minutes. After OGD, cultures were removed and placed
in their normal culture medium (Dulbecco's Minimal Essential Medium
with fetal calf serum) and cultured at 37.degree. C. for 24 hrs.
Controls (shams) were manipulated in the same manner, but were not
exposed to OGD.
[0079] Assessment of Neuronal Damage: Propidium iodide (PrI) was
used to assess neuronal damage. PrI is a dye that is excluded from
healthy cells with intact membranes. As cells are damaged or begin
to die, holes appear in the cell membrane that allow entry of PrI
to the intracellular space, where the dye stains the nuclei a
bright orange. PrI stained nuclei are then counted under a
fluorescent microscope. PrI uptake in the neuronal layer of cells
is determined by adjusting focal plane (neurons are the upper layer
of cells, growing on top of astrocytes) and cell morphology. PrI
data are expressed and the number of injured cells per mg of
protein. To assure that cell loss did not occur during the post-OGD
period, total protein in the medium and in the cellular layer was
assessed. There was no increase in medium protein during the 24 hr
post-OGD period, and no decrease in total protein in the attached
cellular layer, indicating that cell loss through detachment had
not occurred.
[0080] Intracellular Free Calcium ([Ca.sup.2+]i). [Ca.sup.2+].sub.i
was measured as we have previously described, using Fura-2
microspectrophotometery (Rzigalinski et al, J. Biol. Chem. 274,
175-182, 1999) and selective labeling of neurons (Weber,
Rzigalinski, et al, J. Biol. Chem. 276, 1800-1807, 2001). Normal
uninjured neurons maintain basal levels of [Ca.sup.2+].sub.i within
a very tight range, from 80-105 nM. As neurons are damaged and
mitochondrial function destroyed, ion gradients are dismantled and
basal [Ca.sup.2+].sub.i rises, activating many cellular
autodestructive functions that ultimately result in cell demise
over time. Therefore, basal [Ca.sup.2+].sub.i was determined at 24
hrs post-OGD. Additionally, since our cultures are prepared from
the cortex, glutamate is a major neurotransmitter in this area of
the brain. After OGD and other forms of brain injury,
excitotoxicity is often observed. Excitotoxicity is characterized
by and excessive and aberrant rise in [Ca.sup.2+].sub.i in response
to a neurotransmitter stimulus. This excessive rise in
[Ca.sup.2+].sub.i is hypothesized to result from excessive
glutamate release by neurons, as well as the inability of the
neuron to maintain ionic gradients within the normal range. To
assess excitotoxicity and neuronal signaling, we exposed cultures
to a 100 .mu.M glutamate stimulus, and recorded the change in
[Ca.sup.2+].sub.i.
Results
[0081] CeONP protect neurons from cell damage associated with OGD.
As shown in FIG. 1, sham controls had very little uptake of PrI,
indicative of healthy neurons in a normoxic environment (first set
of bars). After OGD (second set of bars) we see a dramatic increase
in injured neurons after 30, 60, and 90minutes of OGD. Pretreatment
of cultures with CeONP on day 5 in vitro significantly reduced
neuronal damage at all levels of OGD. These results suggest that
CeONP may be an effective pretreatment for prevention of neuronal
death associated with OGD and/or stroke.
[0082] CeONP promote cell survival when delivered after OGD. In
FIGS. 2 and 3, we see the same low cellular damage rate in our sham
controls. As expected, there is a dramatic increase in cell damage
at all time points of OGD. In FIG. 2, third set of bars, cultures
received 100 nM CeONP 15 minutes after the end of OGD. A single
dose of CeONP significantly decreased cell damage after all levels
of OGD, from 52-46%.
[0083] Similar results were observed when CeONP were delivered 1
hour after OGD (FIG. 3), with significant levels of neuroprotection
observed at all levels of OGD, even when CeONP were delivered 1 hr
after OGD. Taken together, these results suggest that CeONP may be
an effective post-stroke treatment to reduce neuronal damage.
[0084] CeONP preserve basal [Ca.sup.2+].sub.i when delivered before
OGD. As discussed earlier, basal Cai in neurons is maintained
within tight control, to keep normal cellular systems functioning
optimally and to promote neuronal signaling and communication. As
shown in FIG. 4, sham controls had basal [Ca.sup.2+].sub.i levels
of between 90-105 nM, consistent with our prior observations
(leftmost set of bars). Twenty four hrs after various 30, 60, and
90 min OGD, basal [Ca.sup.2+].sub.i was dramatically and
significantly elevated (middle set of bars). Elevation of
[Ca.sup.2+].sub.i to these levels is known to activate cellular
autodestructive functions, damage mitochondria, and blunt neuronal
signaling; often resulting in cell death. However in cultures
pretreated with CeONP, the rise in basal [Ca.sup.2+].sub.i after
OGD was significantly blunted, with near-normal basal
[Ca.sup.2+].sub.i levels being maintained. These results suggest
that CeONP pretreatment may preserve normal basal calcium levels in
neurons after stroke.
[0085] CeONP preserve basal [Ca.sup.2+]i when delivered after OGD.
Next, we determined whether delivery of CeONP after OGD would still
preserve basal [Ca.sup.2+].sub.i levels. As shown in FIG. 5,
delivery of 100 nM CeONP either 15 minutes or 1 hr after OGD still
resulted in a significant decrease in basal [Ca.sup.2+].sub.i to
levels much closer to that observed in normal cells (FIG. 5). Take
together, these findings indicate that CeONP may be an effective
pharmaceutical to block the elevations in basal [Ca.sup.2+].sub.i
that may induced neuronal damage after stroke.
[0086] Cerium Oxide Nanoparticles preserve near-normal glutamate
signaling when delivered prior to oxygen/glucose deprivation. We
next examined how CeONP might improve neuronal calcium signaling in
response to neurotransmitters after OGD, using the primary
excitatory neurotransmitter, glutamate. As shown in FIG. 6, sham
controls responded to 100 mM glutamate with a change in
[Ca.sup.2+].sub.i (above basal) from between 80-100 nM, similar to
our previously published reports (first set of bars). Twenty four
hrs after OGD, the response to glutamate was significantly
enhanced, achieving levels of 130-240 nM, consistent with what we
have previously observed with excitotoxicity. However in neurons
pretreated with CeONP, the glutamate stimulated [Ca.sup.2+].sub.i
elevations were significantly blunted, from 110-138 nM (right set
of bars). These results demonstrate that pretreatment with CeONP
may preserve neuronal signaling after stroke, and have the
potential to decrease stroke-associated dysfunction. In FIG. 7,
CeONP were delivered 15 min or 1 hr after OGD. Once again, we see
that the dramatic rise in glutamate-stimulated [Ca.sup.2+].sub.i
elevation was blunted in neurons treated with CeONP.
[0087] Taken together with observations of basal [Ca.sup.2+].sub.i
our results suggest that CeONP may be used to prevent neuronal
damage observed in stroke, may be used to prevent [Ca.sup.2+].sub.i
dysregulation induced by stroke, and may be used to treat neurons
after stroke, to block neuronal damage/death, calcium
dysregulation, and excitotoxicity
Example 2
Evaluation of CeONP Treatment of Stroke Using a Drosophila Animal
Model
[0088] The tissue culture studies described in Example 1 strongly
suggest that CeONP may be utilized as prevention and treatment for
neurological deficits produced by stroke. Next, we tested our
hypothesis in an animal model, Drosophila melanogaster, the fruit
fly. See Rodriquez et al., J. Exptl. Biol. 215, 4157-4165,
2012.
[0089] Drosophila is used as a model for many human diseases and
neurodegenerative disorders, including Alzheimer's disease,
Parkinson's disease, Huntington's disease, stroke, and numerous
others. Although it is an insect, utility of Drosophila models
arises from the fact that large numbers can be easily obtained and
culture is relatively inexpensive, compared to mammalian models.
The entire Drosophila genenome has been sequenced, making genetic
studies readily available. Further, animal care and use laws
consider Drosophila a viable alternative to immediate use of
mammalian models.
[0090] For this stroke study, we utilized a well-characterized
model for stroke, oxygen-glucose deprivation. Reports in the
literature demonstrate that Drosophila undergo neuronal damage and
loss via OGD and have histological neuronal damage similar to that
observed in humans. Additionally, functional deficits are also
similar to those observed in humans, including loss of learning,
memory and motor function.
Methods
[0091] One day old male and female flies were collected upon
enclosure from the pupa and cultured on commercial fly food (Jazz
mix) supplemented with CeONP at 1, 100 and 200 .mu.M doses. Control
group food was supplemented with vehicle (0.01% docusate sodium) as
we have previously described. Groups were separated into male and
female, and there were 100 male and female flies per group.
[0092] Flies were cultured for 14 days in standard vials at
25.degree. C. with 50% humidity, and were turned over into new food
vials every 2 days. On day 15, flies were placed into empty vials
and subjected to OGD. OGD was produced by placing the flies in a
tightly sealed gas tent. Flies were placed in the tent which was
then purged of all air and filled with nitrogen. Lack of oxygen in
the tent was measured with a sensor placed inside the tent. Within
15 minutes of beginning the oxygen removal and nitrogen
replacement, flies ceased activity and were immobilized on the
bottom of the vial. Flies remained in this environment for 2.5
hrs.
[0093] After the stroke period, flies were removed from the tent
and placed in standard vials containing their respective food
groups, and allowed to recover. In the male cohort, there was a 10%
loss due to death, with a 20% loss in the female cohort, within 3
days of stroke. No significant differences in immediate death were
noted between the groups.
[0094] Since loss of motor function is a common problem associated
with stroke, motor function in Drosophila was measured by assessing
the negative geotactic response. Negative geotaxis is the ability
of the flies to climb the walls of the vial to various heights. For
these experiments, flies were placed in an empty vial and gently
tapped to the bottom of the vial. Climbing to 3 heights was then
determined, 3 cm, 5.5 cm, and 8 cm. Flies were given 10 seconds to
achieve each respective height goal. The percentage of flies
achieving each height goal was determined. Negative geotaxis was
measured in all groups on day 14 (1 day prior to stroke) and at 2,
6, 14, and 36 days after stroke.
Results
Male Fly Data
[0095] Negative geotaxis data for all male fly groups prior to
delivery of stroke is shown in FIG. 8. There was no significant
difference between food groups (i.e. normal vs CeONP).
[0096] In FIG. 9, flies were assessed for motor function using
negative geotaxis, at 2 days after stroke. We see that stroke
(black bar) decreased the negative geotactic response at all levels
of climbing (3, 5.5, and 8 cm). However in flies treated with 1-200
.mu.M CeONP, the negative geotactic response was preserved to the
level seen in normal controls for the 3 and 5.5 cm goals. For the 8
cm goal, all CeONP groups showed significantly better performance
than stroked flies, with the 100 mM dose showing performance
equivalent to untreated controls.
[0097] At 6 days post-stroke (FIG. 10), untreated, stroked flies
continued to show depressed geotactic responses at all height
goals. Flies fed the lowest (1 .mu.M) dose of CeONP (white bars)
also showed a decline in motor function, scoring similar to
untreated stroked flies. However flies fed 100 and 200 .mu.M CeONP
continued to show improved motor function which was not different
from controls at 3 cm goal heights.
[0098] At 14 days post-stroke (FIG. 11), motor function remained
depressed in untreated stroked flies at all height goals. However
in flies treated with 1-200 .mu.M CeONP, motor function returned to
levels that were equal to that of non-stroked control male flies,
with the exception of the highest goal (8 cm) for the flies fed the
highest dose (200 .mu.M) CeNOP.
[0099] At 36 days after stroke, motor function was assessed again.
Controls (unstroked) flies showed decreased motor function as
compared to their motor function on days 2-14. This is typical, as
the flies are now reaching the end of their life span (flies are
now 54 days old with and have an average lifespan of 58-60 days).
Flies fed standard food and exposed to stroke continued to have
significantly decreased motor function at all goal heights compared
to normal controls. CeONP preserved the negative geotactic response
in stroked flies at all goal heights. Interestingly, flies fed
CeONP had improved motor function, as compared to unstroked
controls (with the exception of the 200 .mu.M food group at 8 cm),
suggesting that CeONP may also improve motor function with
aging.
Female Fly Data
[0100] Negative geotaxis data for all female fly groups prior to
delivery of stroke is shown in FIG. 13. There was no significant
difference between food groups (i.e. normal vs CeONP).
[0101] Two days after stroke, females showed a decline in motor
function, as shown in FIG. 14. Compared to males, the female
decline in motor function was somewhat more severe for all climbing
goal heights. Flies treated with CeONP showed significant
improvement in motor function for all climbing goal heights.
Climbing heights for the 3 and 5.5 cm goals in stroked flies
treated with 100 and 200 .mu.M CeONP were similar to unstroked
controls.
[0102] Six days after stroke females fed standard food and exposed
to stroke continued to have significantly decreased motor function
at all goal heights (FIG. 15). Females treated with 100 and 200
.mu.M CeONP showed significantly improved motor function at all
goal heights. Climbing to the 3 cm height was equivalent to that
observed in unstroked controls. The 1 mm dose, again showing a
trend toward improved motor function, did not show significantly
greater improvement as compared to stroked flies.
[0103] Fourteen days after stroke, motor function was again
assessed by negative geotaxis, as shown in FIG. 16. Note that
controls show a small decline in motor function as compared to that
observed on day 14 (just prior to stroke). Again, this is typical
with aging flies, as these groups are now 29 days old
(approximately midlife). Flies fed standard food and exposed to
stroke continued to have significantly decreased motor function at
all goal heights compared to normal controls. CeONP preserved motor
function in stroked flies at the 1 and 100 .mu.M doses, to levels
equivalent to normal controls.
[0104] Thirty six days after stroke (FIG. 17), controls (unstroked)
flies continued to show decreased motor function as compared to
their motor function on days 2-14. Again, this is typical, as the
flies are now reaching the end of their life span (flies are now 54
days old with an average lifespan of 58-60 days). Flies fed
standard food and exposed to stroke continued to have significantly
decreased motor function at all goal heights compared to normal
controls. All doses of CeONP preserved the negative geotactic
response in stroked flies at all goal heights. Interestingly, the
geotactic response was preserved to levels that significantly
exceeded normal, unstroked flies. This again suggests that CeONP
may blunt the normal decline in motor function seen with aging.
CONCLUSIONS
[0105] Female Drosophila are more susceptible to deleterious
effects of stroke/OGD. Male and female flies fed with CeONP (1-200
mM) prior to and after stroke had dramatically improved motor
function, equivalent to controls (unstroked) in some cases.
[0106] The complete disclosure of all patents, patent applications,
and publications, and electronically available material cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
* * * * *