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US8147876
Medical agent for preventing or treating diseases resulting
from one of inflammation and remodeling,
and method for preventing or treating the
diseases
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a medical agent for preventing or
treating diseases resulting from inflammation or remodeling,
particularly diseases such as arteriosclerosis, heart failure,
cerebrovascular disorder, and hypertensive kidney disease; and to
a method for preventing or treating the diseases.
2. Description of the Related Art
In recent years, with the aging of the population and Westernized
eating habits, prevalence of high blood pressure, hyperlipemia,
and diabetes has increased. As a result, arteriosclerotic diseases
are increasing year by year. Arteriosclerotic diseases include
diseases such as stroke, an ischemic heart disease, hypertensive
nephropathy, ophthalmopathy, heart failure, aortic aneurysm,
arteriosclerosis obliterans, hypertensive emergency, and
cerebrovascular disorder. More than half of the elderly people
aged 75 or more has some sort of disease and reduced quality of
life (QOL). It is known that these diseases are caused by common
pathologic conditions, arteriolar and aortic damages.
Chronic injury in endothelial cells triggers the onset of
arteriosclerosis. Endothelial cells line the artery, forming a
layer, and play an important role such as regulation of vascular
permeability, production and/or secretion of antithrombotic
substances, smooth muscle cell-growth inhibition, and production
of vasoactive substances. In the injured vascular endothelium,
these functions are impaired. In addition, on the surface of
activated or injured endothelial cells, adhesion molecules such as
intercellular adhesion molecule-1 (ICAM-1) and vascular cell
adhesion molecule-1 (VCAM-1) are expressed. Monocyte recognizes
adhesion molecules, adheres to the surface of endothelial cells,
is recruited under the layer of endothelial cells, and then
matures into a macrophage. The macrophage beneath endothelial
cells is eventually converted into a foam cell, forming
atherosclerotic plaque. Inflammatory cytokine is involved in the
expression of adhesion molecules and migration of monocyte.
It is also known that in the pathologic condition such as high
blood pressure that is involved in the development of
arteriosclerosis, responding to various load applied on the blood
vessel, the cytoarchitecture of blood vessel wall changes, causing
remodeling of blood vessel. Remodeling of blood vessel refers to
the change of the structure of vascular tissue as a result of the
injury or abnormal proliferation of vascular tissue due to
mechanical load or change of humoral factor. Originally,
remodeling is a mechanism for responding to change in pathologic
condition; it is known that remodeling has an adverse effect on
blood vessel such as hardening of blood vessel wall and decrease
of inner diameter during the process. It is known that in the
remodeling of blood vessel, smooth muscle cells from medial layer
in blood vessel, become hypertrophied. Growth factors such as
angiotensin II and platelet derived growth factor (PDGF) are
involved in this hypertrophy of smooth muscle cells.
As described above, the mechanism of onset and/or development of
arteriosclerosis is complicated. Besides, many of the mechanisms
are yet to be elucidated. There is a need for efficient methods
for preventing and/or treating arteriosclerosis. Moreover, there
is a need for efficient methods for preventing and/or treating
diseases resulting from one of inflammation and remodeling
including arteriosclerosis.
In recent years, attention has been paid to various bioactive
effects of water that contains nanobubbles of oxygen in large
amount (oxygen-nanobubble water) on living organisms. For example,
oxygen-nanobubble water improves adaptability of fish and
shellfish to environmental change, or restores a debilitated
individual quickly. Nanobubbles are ultrafine bubbles with a
nano-order diameter and are typically generated in the process
where microbubbles (minute bubble with a diameter of 50 µm or
less) shrink. Since nanobubbles are self-pressurized by the action
of surface tension, they are completely dissolved rapidly. Thus,
the lifetime was considered to be short in general. However, it is
reported that in the case where nanobubbles are coated with shell
by a surfactant, or in the case where they are subjected to
electrostatic repulsion due to surface charging, even bubbles in
nano-order can exist for a certain period. Especially, nanobubbles
stabilized due to charging effect retain properties as bubble;
thus various applications, such as direct action to organisms at
cellular level, are expected (See, for example, Japanese Patent
Application Laid-Open (JP-A) No. 2005-245817).
BRIEF SUMMARY OF THE INVENTION
An object of the present invention is to solve the conventional
problems and to achieve the following objects. Specifically, an
object of the present invention is to provide a medical agent that
has an excellent preventive or therapeutic effect on the diseases
resulting from one of inflammation and remodeling and that can
prevent or treat them in response to various mechanisms of onset
and development of the diseases.
As a result of dedicated investigations conducted by the present
inventors to settle the above-mentioned problems, they have made
the following findings. Specifically, they have found that
nanobubble water which contains bubbles with a nano-order diameter
can exhibit an excellent preventive or therapeutic effect on the
diseases resulting from one of inflammation and remodeling.
As described above, it has been known that oxygen-nanobubble water
which contains bubbles of oxygen with a nano-order diameter has
various bioactive effects on living organisms. For example, it
improves adaptability of fish and shellfish to environmental
change, or restores a debilitated individual quickly. Thus, the
oxygen-nanobubble water is attracting attention. In addition,
recently a method for producing nanobubble water has been
established that can maintain nanobubbles of oxygen or ozone
stably for a long time (See, for example, JP-A No. 2005-245817).
Previously, however, it has not been known that the nanobubble
water can exhibit an excellent preventive or therapeutic effect on
the diseases resulting from one of inflammation and remodeling,
which was newly found by the present inventors.
The present invention is based on the above-mentioned findings by
the present inventors, and means for solving the above-mentioned
problems are as follows. Specifically,
<1> A medical agent for preventing or treating a disease
resulting from one of inflammation and remodeling in blood vessel,
including nanobubbles.
<2> The medical agent according to <1>, wherein the
disease is at least one selected from arteriosclerosis, heart
failure, cerebrovascular disorder, and hypertensive kidney
disease.
<3> A histone H3 acetylation-inhibitor including
nanobubbles.
<4> An ICAM-1 expression inhibitor including nanobubbles.
<5> A VCAM-1 expression inhibitor including nanobubbles.
<6> An inhibitor of adhesion of macrophage cells to vascular
endothelial cells, including nanobubbles.
<7> An ERK activation inhibitor including nanobubbles.
<8> An EGF receptor transactivation-inhibitor including
nanobubbles.
<9> A vascular smooth muscle cell hypertrophy-inhibitor
including nanobubbles.
<10> An inhibitor for hypertensive glomerular injury,
including nanobubbles.
<11> A method for preventing or treating a disease resulting
from one of inflammation and remodeling in blood vessel, including
administering the medical agent of <1> to a patient.
The present invention can solve the conventional problems and
provide a medical agent that has an excellent effect on the
diseases resulting from one of inflammation and remodeling and
that can prevent or treat them. Nanobubble water affects various
in vivo mechanisms associated with the diseases. Thus, the present
invention can further provide a reagent and medicine for
controlling these mechanisms.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 shows results of Western blotting in Example 1.
FIG. 2 shows results of Western blotting in Example 1.
FIG. 3 shows results of Northern blot analysis in
Example 1.
FIG. 4A is an optical microscope image showing a
result of functional analysis in Example 1.
FIG. 4B is an optical microscope image showing a
result of functional analysis in Example 1.
FIG. 4C is an optical microscope image showing a
result of functional analysis in Example 1.
FIG. 4D is an optical microscope image showing a
result of functional analysis in Example 1.
FIG. 5 shows results of signal transduction
experiments in Example 1.
FIG. 6A shows results of signal transduction
experiments in Example 1.
FIG. 6B shows results of signal transduction
experiments in Example 1.
FIG. 7 shows results of the evaluation of histone
acetyltransferase (HAT) activity in Example 1.
FIG. 8 shows results of signal transduction
experiments in Example 2.
FIG. 9A shows results of signal transduction
experiments in Example 2.
FIG. 9B shows results of signal transduction
experiments in Example 2.
FIG. 10A is a graph showing the results of the
evaluation of protein content and viable cell number in Example
2.
FIG. 10B is a graph showing the results of the
evaluation of protein content and viable cell number in Example
2.
FIG. 11 is a graph showing the results of the
evaluation of amount of water intake in Example 3.
FIG. 12 is a graph showing the results of the
evaluation of body weight in Example 3.
FIG. 13 is a graph showing the results of the
evaluation of systolic blood pressure and the evaluation of
pulse rate in Example 3.
FIG. 14 shows the results of hematoxylin-eosin
staining of glomerular apparatus from 40-week-old rats in
Example 3.
&c...
DETAILED DESCRIPTION OF THE INVENTION
Medical Agent for Prevention or Treatment
The medical agent of the present invention comprises nanobubbles.
<Nanobubble>
In the present invention, the “nanobubble” refers to a bubble
which has a nano-order diameter. The type of gas as the nanobubble
is not particularly limited and can be appropriately selected
according to the purpose. Examples thereof include oxygen, ozone,
hydrogen, nitrogen, natural gas (e.g. methane), and the like.
Among these, oxygen is preferable as the gas in that it has a
better ability to treat diseases resulting from one of
inflammation and remodeling in blood vessel. In the present
invention, nanobubble composed of oxygen is particularly referred
to as “oxygen nanobubble”, and an aqueous solution that comprises
the oxygen, part of which is in the form of the oxygen nanobubble,
is particularly referred to as “oxygen nanobubble water”.
The diameter of the nanobubble can be appropriately selected
according to the purpose. The bubble diameter is preferably 200 nm
or less, more preferably 100 nm or less and most preferably less
than 10 nm. Nanobubbles with a diameter of 100 nm or less are
advantageous in that there is less possibility for incorporation
of electrolytes or foreign substances. It is considered that the
smaller the diameter is, the more stable in long-term storage the
nanobubbles are, and the less impurity the nanobubbles contain.
Nanobubbles with a diameter less than 10 nm are further
advantageous in that incorporation of many foreign substances
including viruses can be prevented.
The bubble diameter of the nanobubble can be adjusted to a desired
size using, for example, a reverse osmosis membrane (for ozone
nanobubble, use of reverse osmosis membrane is not appropriate).
The bubble diameter of the nanobubble can be measured with, for
example, a dynamic light scattering equipment.
As long as at least part of bubble contained in the medical agent
exists as the nanobubble, the medical agent may comprise, in
addition to the nanobubble, bubble with a larger diameter (for
example, bubble which has a micro-order diameter (more than 1 µm,
and 1 mm or less)). The concentration of the nanobubble in the
tissue repair and/or regeneration solution is particularly
preferably saturated one. It is important that nanobubble be
dissolved in the solution sufficiently.
The solution that makes up the medical agent is preferably an
aqueous solution, but other liquids can be appropriately selected
according to the purpose.
—Other Component—
The nanobubble water may comprise other components than the
nanobubbles as necessary. The other component is not particularly
limited and can be appropriately selected according to the
purpose, including, for example, iron, manganese, and salts.
The salt concentration, pH, and hardness of the nanobubble water
are not particularly limited and can be appropriately selected
according to the purpose. For example, each of them can be
adjusted to a desired degree during the production process of
nanobubble water described below or after the production of
nanobubble water.
—Production—
The nanobubble water can be produced by any method without
limitation and the method can be appropriately selected depending
on the application. For example, the nanobubble water can be
produced by the production method disclosed in JP-A Nos.
2005-245817, 2005-246294, etc. The production method disclosed in
the gazette is preferable since it can produce nanobubble water in
which nanobubbles exist stably and do not disappear from the
aqueous solution over a long period of several months or more.
In the production process of the nanobubble water, iron,
manganese, and/or salts can be preferably added.
The salt concentration of the aqueous solution used in the
production process of the nanobubble water is preferably 0.2% by
mass to 3.0% by mass, and more preferably 0.8% by mass to 1.2% by
mass. When the salt concentration is within the range of from 0.8%
by mass to 1.2% by mass, nanobubbles (core of gas) can be produced
easily; thus it is advantageous in that production efficiency of
the nanobubble water is excellent. The salt concentration can be
measured using, for example, a known instrument for measuring salt
concentration.
It is considered that the pH and hardness of the aqueous solution
used in the production process of the nanobubble water does not
affect the production efficiency of nanobubbles as greatly as does
the salt concentration. Typically, the pH is preferably 7 to 8,
and the hardness is preferably 20 to 30. The pH and hardness can
be measured using, for example, a known instrument for measuring
pH and a known instrument for measuring hardness, respectively.
More specifically, microbubbles of 50 µm or less are produced
using hard water (groundwater) with a salt concentration of 1.0%
by mass as a raw material. Then, by rapidly collapsing or crushing
the microbubbles, nanobubble water can be produced. Further,
nanobubble water with a salt concentration of 0% by mass can be
prepared by passing the resulting nanobubble water through a
reverse osmosis membrane of 10 angstrom twice (oxygen-nanobubble
water with a salt concentration of 0% by mass is “Naga no shizuku”
(drips of Naga) (manufactured by NAGA Co., Ltd.), which is
drinking water approved by the Ministry of Health, Labour and
Welfare.). Meanwhile, nanobubble water with a salt concentration
of 1.0% by mass is nanobubble water that is not passed through a
reverse osmosis membrane of 10 angstrom. Varying the mixing ratio
of both of the nanobubble waters can provide nanobubble waters
with a salt concentration of from 0% by mass to 1.0% by mass. As
mentioned above, in the case of ozone nanobubble, use of reverse
osmosis membrane is not preferable because when ozone nanobubble
water is passed through a reverse osmosis membrane, device may
melt and get damaged.
The nanobubble water, prepared as described above, may be used
itself as the medical agent, or may be used as the medical agent
by combining with other components. For example, it is expected
that the ability to prevent or treat diseases resulting from one
of inflammation and remodeling in blood vessel can be further
improved by adding to the nanobubble water an existing medical
agent, etc. that can be used for the purpose of prevention or
treatment of diseases resulting from one of inflammation and
remodeling in blood vessel, or by using the nanobubble water for
preparing an existing medical agent, etc. that can be used for the
purpose of prevention or treatment of diseases resulting from one
of inflammation and remodeling in blood vessel. Thus, such medical
agents that utilize the nanobubble water in part are also included
within the scope of the medical agent of the present invention.
<Aspect of Medical Agent>
Since the medical agent has excellent capability of prevention or
treatment of diseases resulting from one of inflammation and
remodeling in blood vessel, it is suitable for animals, tissues,
and the prevention or treatment of diseases.
—Target Animal—
The animal, to which the medical agent is applied, is not
particularly limited, including those from mammals, birds,
reptiles, amphibian, and the like. Among these, mammals are
preferable. The mammal is not particularly limited, and examples
thereof include human, monkey, cattle, horses, pigs, mice, rats,
and the like. Among these, human is preferable.
—Target Tissue—
The tissue, to which the medical agent is applied, is not
particularly limited and can be appropriately selected according
to the purpose; examples thereof include epithelial tissue,
connective tissue, muscular tissue, nerve tissue, and the like.
The word “tissue” also includes “cells” and “organs” below. The
“cell” is not particularly limited and can be appropriately
selected according to the purpose; examples thereof include
epidermal cells, pancreatic parenchymal cells, pancreatic ductal
cells, hepatic cells, blood cells, cardiac muscle cells, skeletal
muscle cells, osteoblasts, skeletal myoblasts, nerve cells,
endothelial cells, pigment cells, smooth muscle cells, fat cells,
bone cells, cartilage cells, and the like. The “organ” is not
particularly limited and can be appropriately selected according
to the purpose; examples thereof include skin, blood vessel,
cornea, kidney, heart, liver, umbilical cord, intestine, nerve,
lung, placenta, pancreas, brain, peripheral extremities, retina,
and the like.
The organism from which the tissue is derived is not particularly
limited and can be appropriately selected according to the
purpose. Mammals are preferable, and human is more preferable.
The tissue may be those present inside of the body, or may be
those present outside of the body (for example, cultured tissue).
—Target Diseases—
The disease, to which the medical agent is applied, is not
particularly limited as long as the disease is diseases resulting
from one of inflammation and remodeling in blood vessel that can
occur in the target tissue which the target animal has, and the
disease can be appropriately selected according to the purpose.
Specific examples of the disease resulting from inflammation in
blood vessel include acute coronary syndrome resulting from
rupture of coronary atheroma (acute myocardial infarction and
unstable angina), aortitis syndrome, Buerger's disease, Kawasaki
disease, and the like. Specific examples of the disease resulting
from remodeling in blood vessel include hypertension,
arteriosclerosis, diabetic macro- and microangiopathy, restenosis
after angioplasty, arteriovenous shunt stenosis, graft
angiostenosis, aortic aneurysm, arteriovenous fistula,
arteriosclerosis after heart or kidney transplantation,
nephrosclerosis, pulmonary hypertension, and the like. Examples of
the disease, with which arteriosclerosis is deeply associated and
which is caused or developed by inflammation or remodeling,
include stroke, an ischemic heart disease, hypertensive
nephropathy, ophthalmopathy, heart failure, aneurysm,
arteriosclerosis obliterans, hypertensive emergency,
cerebrovascular disorder, and the like.
—Usage—
The medical agent can be used in any manner without limitation as
long as the effect of the present invention is achieved, and the
manner can be appropriately selected according to the purpose. For
example, the medical agent can be used by contacting with the
tissue in blood vessel where inflammation and/or remodeling is/are
caused by any method.
The medical agent can be stored in any way without limitation and
the way can be appropriately selected according to the purpose.
When ozone-nanobubble is used, it is preferable that the medical
agent be shielded from ultraviolet light and refrigerated under
dark.
<Other Use>
The present inventors revealed that nanobubble water is involved
in various mechanisms related to the diseases resulting from one
of inflammation and remodeling in blood vessel.
Thus, nanobubble water not only can be used as a medical agent for
the prevention or treatment of diseases resulting from one of
inflammation and remodeling in blood vessel, but also can be put
to various uses shown below.
—Histone H3 Acetylation-Inhibitor—
The histone H3 acetylation-inhibitor of the present invention
comprises nanobubbles. Of course, if necessary, other components
can be appropriately selected. The histone H3
acetylation-inhibitor includes both an inhibitor used for reagent
and an inhibitor used for prevention and/or treatment of diseases.
The histone H3 acetylation-inhibitor has an effect to inhibit
acetylation of histone H3, particularly has an effect to inhibit
acetylation of histone H3, which is induced by tumor necrosis
factor a (TNF a) stimulation. Such effect allows us to utilize the
histone H3 acetylation-inhibitor for the studies of acetylation
mechanism of histone H3, chromatin structure controlled by the
acetylation of histone H3, transcriptional mechanism of a specific
gene, which is controlled by the chromatin structure, relative
increase in histone acetylation due to reduced histone
deacetylation, and diseases exacerbated by the acetylation of
histone H3; and for the prevention or treatment of diseases
exacerbated by the acetylation of histone H3. The disease
exacerbated by the acetylation of histone H3 also includes the
target diseases listed in the section of (medical agent for
prevention or treatment).
The histone H3 acetylation-inhibitor may be used in any manner
without limitation as long as the effect of the present invention
is achieved, and the manner can be appropriately selected
according to the purpose. For example, the histone H3
acetylation-inhibitor can be used by contacting with the tissue in
blood vessel where inflammation and/or remodeling is/are caused by
any method.
—Intercellular Adhesion Molecule-1 (ICAM-1) Expression
Inhibitor—
The ICAM-1 expression inhibitor of the present invention comprises
nanobubbles. Of course, if necessary, other components can be
appropriately selected. The ICAM-1 expression inhibitor includes
both an inhibitor used for reagent and an inhibitor used for
prevention and/or treatment of diseases.
The ICAM-1 expression inhibitor has an effect to inhibit
expression of ICAM-1, particularly has an effect to inhibit
expression of ICAM-1, which is induced by interleukin-1ß (IL-1ß)
stimulation and TNF a stimulation. Such effect allows us to
utilize the ICAM-1 expression inhibitor for the studies of
expression mechanism of ICAM-1, factors adhering to ICAM-1,
abnormality in ICAM-1 function, and diseases exacerbated by the
expression of ICAM-1; and for the prevention or treatment of
diseases exacerbated by the expression of ICAM-1. The disease
exacerbated by the expression of ICAM-1 also includes the target
diseases listed in the section of (medical agent for prevention or
treatment).
The ICAM-1 expression inhibitor can be used in any manner without
limitation as long as the effect of the present invention is
achieved, and the manner can be appropriately selected according
to the purpose. For example, the ICAM-1 expression inhibitor can
be used by contacting with the tissue in blood vessel where
inflammation and/or remodeling is/are caused by any method.
—Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression
Inhibitor—
The VCAM-1 expression inhibitor of the present invention comprises
nanobubbles. Of course, if necessary, other components can be
appropriately selected. The VCAM-1 expression inhibitor includes
both an inhibitor used for reagent and an inhibitor used for
prevention and/or treatment of diseases.
The VCAM-1 expression inhibitor has an effect to inhibit
expression of VCAM-1, particularly has an effect to inhibit
expression of VCAM-1, which is induced by IL-1ß stimulation and
TNF a stimulation. Such effect allows us to utilize the VCAM-1
expression inhibitor for the studies of expression mechanism of
VCAM-1, factors adhering to VCAM-1, abnormality in VCAM-1
function, and diseases exacerbated by the expression of VCAM-1;
and for the prevention or treatment of diseases exacerbated by the
expression of VCAM-1. The disease exacerbated by the expression of
VCAM-1 also includes the target diseases listed in the section of
(medical agent for prevention or treatment).
The VCAM-1 expression inhibitor can be used in any manner without
limitation as long as the effect of the present invention is
achieved, and the manner can be appropriately selected according
to the purpose. For example, the VCAM-1 expression inhibitor can
be used by contacting with the tissue in blood vessel where
inflammation and/or remodeling is/are caused by any method.
—Inhibitor of Adhesion of Macrophage Cells to Vascular
Endothelial Cells—
The inhibitor of adhesion of macrophage cells to vascular
endothelial cells comprises nanobubbles. Of course, if necessary,
other components can be appropriately selected. The inhibitor of
adhesion of macrophage cells to vascular endothelial cells
includes both an inhibitor used for reagent and an inhibitor used
for prevention and/or treatment of diseases.
The inhibitor of adhesion of macrophage cells to vascular
endothelial cells has an effect to inhibit adhesion of macrophage
cells to vascular endothelial cells, particularly has an effect to
inhibit adhesion through ICAM-1 and VCAM-1 expressed on the
surface of vascular endothelial cell. Such effect allows us to
utilize the inhibitor of adhesion of macrophage cells to vascular
endothelial cells for the studies of adhesion mechanism of
macrophage cells to vascular endothelial cells, factors adhering
to macrophage cell, diseases caused by abnormal macrophage
migration, and diseases exacerbated by the adhesion of macrophage
cells to vascular endothelial cells; and for the prevention or
treatment of diseases caused by abnormal macrophage migration and
diseases exacerbated by the adhesion of macrophage cells to
vascular endothelial cells. The disease caused by abnormal
macrophage migration and disease exacerbated by the adhesion of
macrophage cells to vascular endothelial cells also include the
target diseases listed in the section of (medical agent for
prevention or treatment).
The inhibitor of adhesion of macrophage cells to vascular
endothelial cells can be used in any manner without limitation as
long as the effect of the present invention is achieved, and the
manner can be appropriately selected according to the purpose. For
example, the inhibitor of adhesion of macrophage cells to vascular
endothelial cells can be used by contacting with the tissue in
blood vessel where inflammation and/or remodeling is/are caused by
any method.
—Extracellular Signal Regulated Kinase (ERK) Activation
Inhibitor—
The ERK activation inhibitor of the present invention comprises
nanobubbles. Of course, if necessary, other components can be
appropriately selected. The ERK activation inhibitor includes both
an inhibitor used for reagent and an inhibitor used for prevention
and/or treatment of diseases.
The ERK activation inhibitor has an effect to inhibit activation
of ERK, particularly has an effect to inhibit activation of ERK
due to angiotensin II (A II) stimulation, or activation of ERK via
tyrosine phosphorylation of EGF receptor. Such effect allows us to
utilize the ERK activation inhibitor for the studies of mechanism
of ERK activation, factors controlled by ERK activation, abnormal
cellular proliferation and/or differentiation through ERK
activation, and diseases exacerbated by the ERK activation; and
for the prevention or treatment of diseases exacerbated by the ERK
activation. The disease exacerbated by the ERK activation also
includes the target diseases listed in the section of (medical
agent for prevention or treatment).
The ERK activation inhibitor can be used in any manner without
limitation as long as the effect of the present invention is
achieved, and the manner can be appropriately selected according
to the purpose. For example, the ERK activation inhibitor can be
used by contacting with the tissue in blood vessel where
inflammation and/or remodeling is/are caused by any method.
—Epidermal Growth Factor (EGF) Receptor
Transactivation-Inhibitor—
The EGF receptor transactivation-inhibitor of the present
invention comprises nanobubbles. Of course, if necessary, other
components can be appropriately selected. The EGF receptor
transactivation-inhibitor includes both inhibitors used for
reagent and inhibitors used for prevention and/or treatment of
diseases.
The “EGF receptor transactivation” means that EGF receptor is not
activated directly by its ligand, EGF, but is activated
secondarily by the activation of G protein-coupled receptor such
as angiotensin II. It is considered that transactivation has an
important role on the mechanism of hypertrophy by angiotensin II.
The EGF receptor transactivation-inhibitor has an effect to
inhibit transactivation of EGF receptor, particularly has an
effect to inhibit transactivation of EGF receptor due to
angiotensin II stimulation. Such effect allows us to utilize the
EGF receptor transactivation-inhibitor for the studies of
mechanism of EGF receptor transactivation, factors controlled by
EGF receptor transactivation, elucidation of mechanism of EGF
receptor transactivation, and diseases exacerbated by the EGF
receptor transactivation; and for the prevention or treatment of
diseases exacerbated by the EGF receptor transactivation. The
disease exacerbated by the EGF receptor transactivation also
includes the target diseases listed in the section of (medical
agent for prevention or treatment).
The EGF receptor transactivation-inhibitor can be used in any
manner without limitation as long as the effect of the present
invention is achieved, and the manner can be appropriately
selected according to the purpose. For example, the EGF receptor
transactivation-inhibitor can be used by contacting with the
tissue in blood vessel where inflammation and/or remodeling is/are
caused by any method.
—Vascular Smooth Muscle Cell Hypertrophy-Inhibitor—
The vascular smooth muscle cell hypertrophy-inhibitor of the
present invention comprises nanobubbles. Of course, if necessary,
other components can be appropriately selected. The vascular
smooth muscle cell hypertrophy-inhibitor includes both inhibitors
used for reagent and inhibitors used for prevention and/or
treatment of diseases.
The vascular smooth muscle cell hypertrophy-inhibitor has an
effect to inhibit hypertrophy of vascular smooth muscle cell,
particularly has an effect to inhibit hypertrophy of vascular
smooth muscle cell due to angiotensin II stimulation and platelet
derived growth factor (PDGF) stimulation. Such effect allows us to
utilize the vascular smooth muscle cell hypertrophy-inhibitor for
the studies of mechanism of vascular smooth muscle cell
hypertrophy, factors controlled by vascular smooth muscle cell
hypertrophy, elucidation of mechanism of vascular smooth muscle
hypertrophy, and diseases exacerbated by the vascular smooth
muscle cell hypertrophy; and for the prevention or treatment of
diseases exacerbated by the vascular smooth muscle cell
hypertrophy. The disease exacerbated by the vascular smooth muscle
cell hypertrophy also includes the target diseases listed in the
section of (medical agent for prevention or treatment).
The vascular smooth muscle cell hypertrophy-inhibitor can be used
in any manner without limitation as long as the effect of the
present invention is achieved, and the manner can be appropriately
selected according to the purpose. For example, the vascular
smooth muscle cell hypertrophy-inhibitor can be used by contacting
with the tissue in blood vessel where inflammation and/or
remodeling is/are caused by any method.
—Inhibitor for Hypertensive Glomerular Injury—
The inhibitor for hypertensive glomerular injury of the present
invention comprises nanobubbles. Of course, if necessary, other
components can be appropriately selected. The inhibitor for
hypertensive glomerular injury includes both inhibitors used for
reagent and inhibitors used for prevention and/or treatment of
diseases.
The inhibitor for hypertensive glomerular injury has an effect to
prevent or treat hypertensive glomerular injury, particularly has
an effect on hypertensive glomerular injury exacerbated by the
intake of salts. Such effect allows us to utilize the inhibitor
for hypertensive glomerular injury for the studies of mechanism of
hypertensive glomerular injury, factors controlled by hypertensive
glomerular injury, and diseases exacerbated by the hypertensive
glomerular injury; and for the prevention or treatment of diseases
exacerbated by the hypertensive glomerular injury. The disease
exacerbated by the hypertensive glomerular injury also includes
the target diseases listed in the section of (medical agent for
prevention or treatment).
The inhibitor for hypertensive glomerular injury can be used in
any manner without limitation as long as the effect of the present
invention is achieved, and the manner can be appropriately
selected according to the purpose. For example, the inhibitor for
hypertensive glomerular injury can be used by contacting with the
tissue in blood vessel where inflammation and/or remodeling is/are
caused by any method.
Due to the effect of nanobubble, the medical agent for prevention
or treatment of the present invention can be suitably used for
prevention or treatment of diseases resulting from one of
inflammation and remodeling in blood vessel. In addition, the
effect of nanobubble has an influence on various in vivo
mechanisms associated with the diseases. For example, the medical
agent can be used as a histone H3 acetylation-inhibitor, ICAM-1
expression inhibitor, VCAM-1 expression inhibitor, inhibitor of
adhesion of macrophage cells to vascular endothelial cells, ERK
activation inhibitor, EGF receptor transactivation-inhibitor,
vascular smooth muscle cell hypertrophy-inhibitor, and inhibitor
for hypertensive glomerular injury.
EXAMPLES
Hereinafter, Examples of the present invention will be described,
which however shall not be construed as limiting the present
invention thereto.
In Examples, OXNB means oxygen nanobubble, and OZNB means ozone
nanobubble. Both are available from REO Institute.
Example 1
Objective:
To investigate the effect of nano-bubbles on inflammatory
responses in the cultured endothelial cells induced by cytokines.
Methods:
(1) Medium preparation: Although accurate assay for OXNB density
is not established, the activity of OXNB is increased with
increasing the concentration of NaCl in the solution. REO
Institute provided us OXNB solution including 1.0% by mass of
NaCl. We prepared NaCl-free MCDB medium powder and dissolved the
powder by using OXNB solution as vehicle. Because ordinary MCDB
media contains 0.64% by mass of NaCl, we diluted the nano-bubble
solution by distilled water to make OXNB with 0.64% by mass of
NaCl. Then we dissolved the sodium free-MCDB medium powder with
0.64% by mass of NaCl OXNB solution to adjust NaCl concentration
to ordinary MCDB media and defined the media 100%-OXNB MCDB. To
examine the dose-response relationship between inflammatory
response and concentration of OXNB, we diluted 100%-OXNB MCDB by
ordinary MCDB media. The “50%-OXNB” and “75%-OXNB” described below
mean those prepared by diluting 100%-OXNB MCDB by ordinary MCDB
media (based on mass).
(2) Cell culture: Rat aortic endothelial cells (RAOECs, passage 7
to 10) were incubated in the MCDB culture media (containing fetal
bovine serum, endothelial cell growth factor and antibiotics) with
or without OXNB for 24 hours and were stimulated with inflammatory
cytokines (10 ng/mL of IL-1ß or 20 ng/mL of TNFa). Expression of
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), endothelial NO synthase (eNOS) and a-tubulin
was examined by Western blotting.
(3) Analysis of mRNA expression: Northern blot analysis was
performed to examine mRNA expression of ICAM-1 and VCAM-1 induced
by inflammatory cytokines 6 hours after the stimulation.
(4) Functional analysis: RAOECs were incubated either with or
without OXNB. Rat alveolar macrophage cells (NR8383) were
stimulated with 0.1 µM of lipopolysaccharide (LPS). Then RAOECs
and NR8383 were co-cultured for 1 hour and adhesion of NR8383 to
RAOECs was evaluated.
(5) Signal transduction experiments: RAOECs were incubated in the
culture media with or without OXNB for 24 hours and were
stimulated with 20 ng/mL of TNFa. Intracellular signal
transduction was evaluated by Western blot analysis. Activation of
NFkB was measured by NFKB p50/65 transcription factor assay kit
(Chemicon International Inc.) (DiDonato J A, Mercurio F, Karin M.:
Phosphorylation of IKBa precedes but is not sufficient for its
dissociation from NF-kB. Mol. Cell. Biol. 1995; 15: 1302-1311;
Huang T, Miyamoto S.: Postrepression activation of NF-kB requires
the amino-terminal nuclear export signal of IKBa. Mol. Cell. Biol.
2001; 21: 4737-1311).
(6) Histone acetyltransferase (HAT) activity: HAT activity was
measured by commercially available kit (Upstate Biotechnologies
Inc.) (Dignam J D, Lebovits R M, Roeder R G.: Accurate
transcription initiation by RNA polymerase II in a soluble extract
from isolated mammalian nuclei. Nucleic Acid Res. 1983; 11:
1475-1489; Nakatani F, Tanaka K, Sakimura R et al.: Identification
of p21WAF/CIP1 as a direct target of EWS-Fli1 oncogenic fusion
protein. J Biol Chem. 2003; 278: 15105-15115).
Results:
IL-1ß and TNFa induced ICAM-1 and VCAM-1 expression in RAOECs with
a peak at 24 hours after stimulation. Incubation of RAOECs with
culture media containing OXNB inhibited induction of ICAM-1 by
TNFa (FIG. 1). ICAM-1 expression was perfectly abolished over
50%-OXNB media and VCAM-1 expression was perfectly abolished over
75%-OXNB media (FIG. 2). In contrast, expression of eNOS or
a-tublin was not changed. Northern blot analysis revealed that
OXNB inhibited induction of mRNA expression of ICAM-1 and VCAM-1
by TNFa (FIG. 3). When RAOECs were treated with OXNB, adhesion of
NR8383 to RAOECs was significantly decreased (FIG. 4). Similar
results were obtained in the experiments using IL-1ß instead of
TNFa. As shown in FIG. 5, OXNB did not inhibit activation of c-Jun
NH2 terminal kinase (JNK). OXNB did not affect degradation of IKBa
that is an internal inhibitor for NFkB (FIG. 6A). Consequently,
activation of NFkB was not decreased by treatment of OXNB (FIG.
6B). These results suggest that intracellular signal transduction
was not changed by OXNB. These results indicate that OXNB might
affect transcriptional regulation of VCAM-1 by TNFa. Curcumin, a
specific HAT inhibitor is reported to inhibit TNFa-induced VCAM-1
expression (Lee C W, Lin W N, Lin C C, Luo S F, Wang J S,
Pouyssegur J, Yang C M: Transcriptional regulation of VCAM-1
expression by tumor necrosis factor-a in human tracheal smooth
muscle cells: Involvement of MAPKs, NF-kB, p300 and histone
acetylation. J Cell Physiol. 2006; 207: 174-186). Thus it is
possible that OXNB attenuates VCAM-1 expression via an inhibition
of HAT in endothelial cells. We investigated the effect of OXNB on
HAT activity in RAOECs. We observed that OXNB inhibited the
acetylation of histone H3 (FIG. 7). Acetylation of histone H4 was
not changed by OXNB. Similar results were obtained when ozone
nano-bubbles are used instead of OXNB.
Conclusion:
These results indicate that nano-bubbles (OXNB and OZNB) could be
a novel therapeutic measure to attenuate atherosclerosis via a
selective inhibition of histone H3 acetylation of in vitro
cultured endothelial cells.
Example 2
Objective:
To investigate the effect of nano-bubbles on hypertrophic response
of vascular smooth muscle cells.
Methods:
(1) Medium preparation: DMEM media for vascular smooth muscle
cells were prepared with the same procedure of MCDB media in
Example 1.
(2) Cell culture: Rat aortic vascular smooth muscle cells (RASMCs,
passage 7 to 10) were used. RASMCs were incubated in the serum
free DMEM media with or without OXNB (75%) for 48 hours and were
stimulated with A II (A II, 10<-8 >M), epidermal growth
factor (EGF, 100 ng/mL) or platelet-derived growth factor (PDGF,
50 ng/mL).
(3) Signal transduction analysis: Activation of extracellular
signal-regulated kinase (ERK) was evaluated by Western blotting
analysis. Tyrosine phosphorylation of EGF receptor was analyzed by
immunoprecipitation (IP) of EGF receptor with immunoblotting with
anti-phosphotyrosine (clone 4G10).
(4) Protein content and the viable cell number: Protein content of
cell lysate from RASMCs was measured by chromomeric assay using
bicinchoninic acid solution. Cell viability was measured by
dehydrogenase enzyme activity found in metabolically active cells
using
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt; MTS.
Results:
FIG. 8 shows the time course of ERK activation by A II. DMEM
containing 100%-OXNB inhibited ERK activation by A II over 10
minutes. As shown in FIG. 9A, A II and EGF activated ERK in RASMCs
5 minutes after the stimulation. Treatment of RASMCs with OXNB
inhibited ERK activation by A II, however ERK activation by EGF
was not changed suggesting the specific inhibition of A
II-mediated ERK activation. It has been reported that
transactivation of EGF receptor has an important role in ERK
activation by A II (H Daub, C Wallasch, A Lankenau, A Herrlich, A
Ullrich: Signal characteristics of G protein-transactivated EGF
receptor. EMBO J. 1997; 16: 7032-7044). We checked the effect of
OXNB on transactivation of EGF receptor by A II. As shown in FIG.
9B, OXNB inhibited transactivation of EGF receptor, suggesting
that decreased ERK activation is at least, partially mediated by
an inhibition of EGF receptor transactivation. As we found the
inhibitory effect of OXNB on ERK activation, we have investigated
the effect of OXNB on protein synthesis of RASMCs. As shown in
FIG. 10A, A II increased protein content of RASMCs under
inhibition of A II type-2 receptor (10<-6 >M of PD123319).
No significant change was observed in cell viability by A II,
suggesting that A II promotes hypertrophy of RASMCs. OXNB
inhibited this hypertrophic response of RASMCs by A II. Similarly
PDGF increased protein content of RASMCs without changing the
viable cell number. OXNB significantly inhibited the increases in
protein content in RASMCs without affecting the viable cell number
(FIG. 10B). Similar results were obtained in the experiments using
OZNB instead of OXNB.
Conclusion:
These results suggest that nano-bubbles inhibit hypertrophy of
vascular smooth muscle cells. It is possible that nano-bubbles
attenuate vascular remodeling induced by atherogenic stimuli.
Example 3
Objective:
To investigate the in vivo effect of OXNB using the animal model
of genetic hypertension.
Methods:
Spontaneously hypertensive rats (SHR) at 4 week-old were used. SHR
were divided into control (n=4) and OXNB group (n=4). They were
kept in the separated cages and allowed to drink either water with
0.1% by mass of NaCl or water with 10-fold diluted OXNB with 1% by
mass of NaCl (final concentration of 0.1% by mass of NaCl). The
amount of water intake, body weight, systolic blood pressure (SBP)
and pulse rate (PR) were checked once a week. They were sacrificed
at 40 week-old to examine histological changes in
hypertension-target organs (heart and kidney).
Results:
As shown in FIG. 11, the amount of water did not differ between
two groups. Body weight in OXNB group was higher than that in
control group (FIG. 12). Blood pressure and pulse rate were not
significantly different between two groups (FIG. 13). The organ
weight ratio of heart and kidney did not differ between two groups
(Table 1). As shown in FIG. 14, hematoxylin-eosin staining of
glomerular apparatus in SHR kidney showed marked hyaline
degeneration. In contrast, OXNB group did not show any
degenerative changes in the kidney. The quantitative analysis of
histological specimen revealed that the administration of OXNB to
SHR decreased the degeneration rate of glomerular apparatus
significantly (control: 92.1+/-0.49%, OXNB: 24.4+/-5.8%,
p<0.001).
We also did the same experiments using OZNB and found protective
effect of OZNB in SHR kidney. No significant histological changes
were found in the heart.
TABLE 1
The organ to body weight ratio in SHR
Control OXNB p value
Heart (g/kg) 4.88 ± 0.38 4.72 ± 0.42 0.79
Right kidney (g/kg) 5.03 ± 0.36 5.50 ±
0.53 0.53
We checked the effect of OXNB on the redox state in SHR because
the excess administration of oxygen might results in the increase
in oxidative stress. We have measured the plasma levels of low
density lipoprotein cholesterol (LDL-cholesterol), a marker of
oxidative stress, in SHR. We did not find significant changes in
plasma LDL cholesterol levels in the two groups (control:
59.8+/-9.6 mg/dL, OXNB: 71.0+/-2.1 mg/dL, p=0.30).
Conclusion:
These results suggest that OXNB prevented glomerular injury in the
hypertension model animals. This protective effect of glomerular
apparatus is exerted without reducing blood pressure, suggesting
that nano-bubbles can protect kidneys even the blood pressure is
elevated. The anti-inflammatory and anti-proliferative property of
nano-bubbles is supposed to be involved in this unique biological
activity such HAT inhibition.
(Conclusions)
OXNB and OZNB prevented glomerular injury in the animal models of
hypertension. The renal protective effect might be exerted by
attenuation of inflammation and remodeling of blood vessels.
Inhibition of HAT is at least, partially involved in the
mechanism. Precise mechanism how nano-bubbles inhibit HAT is still
not clear, however this property of nano-bubbles might be applied
to other diseases such as atherosclerosis, heart failure and
cerebrovascular diseases. Application of nano-bubbles could be a
novel treatment for renal diseases caused by hypertension. This
technology might be applied in immunological, diabetic and
drug-induced glomerular injury that shares common pathogenic
features with hypertensive renal disease.