U.S. patent application number 17/599211 was filed with the patent office on 2022-06-30 for use of cyclo(his-pro) (chp) for preventing, ameliorating, or treating fibrosis.
This patent application is currently assigned to NOVMETAPHARMA CO., LTD.. The applicant listed for this patent is NOVMETAPHARMA CO., LTD., SEOUL NATIONAL UNIVERSITY HOSPITAL, SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION. Invention is credited to Jong Su JEON, Hoe Yune JUNG, Ji Eun KIM, Yon Su KIM, Yong Chul KIM, Do Hyun LEE, Heon Jong LEE, Jong Joo MOON, Seung Hee YANG.
Application Number | 20220202897 17/599211 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220202897 |
Kind Code |
A1 |
JUNG; Hoe Yune ; et
al. |
June 30, 2022 |
USE OF CYCLO(HIS-PRO) (CHP) FOR PREVENTING, AMELIORATING, OR
TREATING FIBROSIS
Abstract
A Cyclo (his-pro) (CHP) is useful for preventing, ameliorating,
or treating fibrosis. A pharmaceutical composition containing CHP
may be used for preventing or treating fibrosis. A health
functional food composition containing CHP may be used for
preventing or ameliorating fibrosis. An antifibrotic composition, a
method of preventing, ameliorating, or treating fibrosis by using
CHP, and/or a use of CHP in the manufacture of the pharmaceutical
composition for preventing or treating fibrosis are disclosed.
Inventors: |
JUNG; Hoe Yune; (Pohang-si,
KR) ; LEE; Heon Jong; (Incheon, KR) ; JEON;
Jong Su; (Pohang-si, KR) ; LEE; Do Hyun;
(Pohang-si, KR) ; KIM; Yon Su; (Seoul, KR)
; YANG; Seung Hee; (Seoul, KR) ; KIM; Yong
Chul; (Seoul, KR) ; MOON; Jong Joo; (Seoul,
KR) ; KIM; Ji Eun; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVMETAPHARMA CO., LTD.
SEOUL NATIONAL UNIVERSITY HOSPITAL
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION |
Seoul
Seoul
Seoul |
|
KR
KR
KR |
|
|
Assignee: |
NOVMETAPHARMA CO., LTD.
Seoul
KR
SEOUL NATIONAL UNIVERSITY HOSPITAL
Seoul
KR
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION
Seoul
KR
|
Appl. No.: |
17/599211 |
Filed: |
March 30, 2020 |
PCT Filed: |
March 30, 2020 |
PCT NO: |
PCT/KR2020/004354 |
371 Date: |
September 28, 2021 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61P 19/04 20060101 A61P019/04; A61P 11/00 20060101
A61P011/00; A61P 1/16 20060101 A61P001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2019 |
KR |
10-2019-0036269 |
Mar 27, 2020 |
KR |
10-2020-0037859 |
Claims
1-7. (canceled)
8. A method for preventing or treating fibrosis in a subject in
need thereof, comprising administering to the subject an effective
amount of Cyclo-HisPro or a pharmaceutically acceptable salt
thereof.
9. The method of claim 8, wherein the fibrosis develops in any one
or more selected from the group consisting of: kidney, liver, lung,
skin, heart, pancreas, urinary system, genital system, sweat gland,
nerve, brain, bone marrow, muscle and joint.
10. The method of claim 8, wherein the fibrosis is any one or more
selected from the group consisting of: pulmonary fibrosis,
idiopathic pulmonary fibrosis, radiation-induced lung injury or
lung fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis,
endomyocardial fibrosis, myocardial infarction, artrial fibrosis,
glial scar, renal fibrosis, myelofibrosis, arthrofibrosis, fat
fibrosis, skin fibrosis, neurofibrosis and myofibrosis.
11. The method of claim 10, wherein the liver fibrosis comprises
non-alcoholic steatohepatitis.
12-13. (canceled)
14. A method for reducing or inhibiting fibrosis in a subject in
need thereof, comprising administering to the subject an effective
amount of Cyclo-HisPro or a pharmaceutically acceptable salt
thereof.
15. The method of claim 14, wherein the fibrosis develops in any
one or more selected from the group consisting of: kidney, liver,
lung, skin, heart, pancreas, urinary system, genital system, sweat
gland, nerve, brain, bone marrow, muscle and joint.
16. The method of claim 14, wherein the fibrosis is any one or more
selected from the group consisting of: pulmonary fibrosis,
idiopathic pulmonary fibrosis, radiation-induced lung injury or
lung fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis,
endomyocardial fibrosis, myocardial infarction, artrial fibrosis,
glial scar, renal fibrosis, myelofibrosis, arthrofibrosis, fat
fibrosis, skin fibrosis, neurofibrosis and myofibrosis.
17. The method of claim 16, wherein the liver fibrosis comprises
non-alcoholic steatohepatitis.
18. A method of inducing anti-fibrosis in a subject in need
thereof, comprising administering to the subject an effective
amount of Cyclo-HisPro or a pharmaceutically acceptable salt
thereof.
19. The method of claim 18, wherein the anti-fibrosis is induced in
any one or more selected from the group consisting of: kidney,
liver, lung, skin, heart, pancreas, urinary system, genital system,
sweat gland, nerve, brain, bone marrow, muscle and joint.
20. The method of claim 18, wherein the anti-fibrosis is induced in
any one or more selected from the group consisting of: pulmonary
fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung
injury or lung fibrosis, pulmonary edema, cystic fibrosis, liver
fibrosis, endomyocardial fibrosis, myocardial infarction, artrial
fibrosis, glial scar, renal fibrosis, myelofibrosis,
arthrofibrosis, fat fibrosis, skin fibrosis, neurofibrosis and
myofibrosis.
21. The method of claim 20, wherein the liver fibrosis comprises
non-alcoholic steatohepatitis.
Description
TECHNICAL FIELD
[0001] The present invention relates to a use of Cyclo-HisPro (CHP)
for preventing, ameliorating, or treating fibrosis and, more
particularly, to a pharmaceutical composition for preventing or
treating fibrosis comprising CHP, a health functional food
composition for preventing or ameliorating, an antifibrotic
composition, a method of preventing, or treating fibrosis by using
CHP, and/or a use of CHP in the manufacture of the pharmaceutical
composition for preventing or treating fibrosis.
BACKGROUND ART
[0002] Fibrosis is a disease in which abnormal production,
accumulation and deposition of extracellular matrix by fibroblasts
occurs, and is caused by fibrosis of an organ or tissue. Fibrosis
is a very fatal disease that causes long-term damage. For example,
idiopathic pulmonary fibrosis (IPF) appears as a result of
relapsing alveolar epithelial cell injury associated with
fibroblast accumulation and myofibroblast differentiation, and is a
chronic, progressive, and lethal disease that causes excessive
accumulation of extracellular matrix (ECM) with irreversible
destruction of lung parenchyma tissue.
[0003] Conventional treatment studies have largely targeted the
inflammatory process of fibrosis, using corticosteroids and
immunosuppressive drugs. However, these agents show little effect
in clinical trials, so there is a need for new drugs for treating
fibrosis.
[0004] Meanwhile, Korean Patent Laid-Open Publication No.
10-2013-0006170 discloses a composition for blood sugar regulation
comprising soybean hydrolysate with high CHP (CYCLO(His-Pro), but
the antifibrotic effect of CHP is not known.
DISCLOSURE OF INVENTION
Technical Problem
[0005] One aspect of the present invention is directed to providing
a pharmaceutical composition for preventing or treating fibrosis
comprising Cyclo-HisPro.
[0006] Another aspect of the present invention is directed to
providing a health functional food composition for preventing or
ameliorating fibrosis comprising Cyclo-HisPro.
[0007] Another aspect of the present invention is directed to
providing an antifibrotic composition comprising Cyclo-HisPro.
[0008] Another aspect of the present invention is directed to
providing a method of preventing, or treating fibrosis by using
Cyclo-HisPro.
[0009] Another aspect of the present invention is directed to
providing a use of Cyclo-HisPro in the manufacture of the
pharmaceutical composition for preventing or treating fibrosis.
Technical Solution
[0010] In order to solve the above problems, the present invention
provides a pharmaceutical composition for preventing or treating
fibrosis comprising Cyclo-HisPro or a pharmaceutically acceptable
salt thereof.
[0011] In addition, the present invention provides a health
functional food composition for preventing or ameliorating fibrosis
comprising Cyclo-HisPro or a sitologically acceptable salt
thereof.
[0012] Further, the present invention also provides a method for
preventing or treating fibrosis comprising administering to a
subject in need thereof an effective amount of Cyclo-HisPro.
[0013] The present invention also provides a use of Cyclo-HisPro in
the manufacture of a pharmaceutical composition for preventing or
treating fibrosis.
[0014] According to a preferred embodiment of the present
invention, the fibrosis may develop in any one or more selected
from the group consisting of: kidney, liver, lung, skin, heart,
pancreas, urinary system, genital system, sweat gland, nerve,
brain, bone marrow, muscle and joint.
[0015] According to another preferred embodiment of the present
invention, the fibrosis may be any one or more selected from the
group consisting of: pulmonary fibrosis, idiopathic pulmonary
fibrosis, radiation-induced lung injury or lung fibrosis, pulmonary
edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis,
myocardial infarction, artrial fibrosis, glial scar, renal
fibrosis, myelofibrosis, arthrofibrosis, fat fibrosis, skin
fibrosis, neurofibrosis and myofibrosis.
[0016] The present invention also provides an antifibrotic
composition comprising Cyclo-HisPro.
Advantageous Effects
[0017] The composition comprising Cyclo-HisPro of the present
invention is effective for preventing, ameliorating or treating
fibrosis by inhibiting fibrosis occurring in various tissues or
organs such as kidney, liver, lung, skin, heart, pancreas, urinary
system, genital system, sweat glands, nerves, brain, bone marrow,
muscles and joints.
BRIEF DESCRIPTION OF DRAWINGS
[0018] The above and other aspects, features, and advantages of the
present invention will become more apparent to those of ordinary
skill in the art by describing embodiments thereof in detail with
reference to the accompanying drawings, in which:
[0019] FIG. 1 shows the process of cell isolation and primary
culture from human glomeruli and renal tubules (left: glomerulus,
right: tubule);
[0020] FIG. 2 shows the morphological changes of cells according to
CHP treatment (4, 20 and 100 .mu.g/ml, respectively) by
concentration in a human-derived proximal tubule epithelial cell
fibrosis model;
[0021] FIG. 3 shows a result of confirming changes in the
expression level of E-cadherin, fibronectin and pSTAT3 proteins by
Western blot according to CHP treatment (4, 20 and 100 .mu.g/ml,
respectively) by concentration in a human-derived proximal tubule
epithelial cell fibrosis model;
[0022] FIG. 4 shows the morphological changes of cells according to
CHP treatment (40 and 100 .mu.g/ml, respectively) by concentration
in a human-derived glomerular endothelial cell fibrosis model;
[0023] FIG. 5A shows a band result of confirming changes in the
expression level of fibronectin protein by Western blot according
to CHP treatment (62.5, 125 and 250 ng/ml, respectively) by
concentration in a human-derived liver cell fibrosis model, and
FIG. 5B is a graph of quantifying the size of the band;
[0024] FIG. 6A shows a band result of confirming changes in the
expression level of fibronectin protein by Western blot according
to CHP treatment (5 and 35 mg/kg, respectively) by concentration in
an animal model of liver fibrosis, and FIG. 6B is a graph of
quantifying the size of the band;
[0025] FIG. 7A shows a band result of confirming changes in the
expression level of fibronectin protein by Western blot according
to CHP treatment (62.5 and 125 ng/ml, respectively) by
concentration in a human-derived lung cell fibrosis model, and FIG.
7B is a graph of quantifying the size of the band;
[0026] FIG. 8 is a graph showing changes in the expression level of
TGF.beta. and Collagen 3 genes according to CHP administration (5
and 35 mg/kg, respectively) by concentration in an animal model of
lung fibrosis;
[0027] FIG. 9A shows a band result of confirming changes in the
expression level of fibronectin protein by Western blot according
to CHP treatment (62.5, 125, 250 and 500 ng/ml, respectively) by
concentration in a human-derived skin cell fibrosis model, and FIG.
9B is a graph of quantifying the size of the band;
[0028] FIG. 10 is a graph showing changes in the expression level
of fibrosis markers TGF.beta., Fibronectin, Collagen 1, Collagen 2,
Collagen 3 and Collagen 4 genes according to CHP administration (5
and 35 mg/kg, respectively) by concentration in an animal model of
cardiac fibrosis;
[0029] FIG. 11 is a graph showing changes in the expression level
of fibrosis markers TGF.beta., Fibronectin, Collagen 3, CTGF genes
according to CHP administration (5 and 35 mg/kg, respectively) by
concentration in an animal model of fat fibrosis.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] Hereinafter, the present invention will be described in more
detail.
[0031] As described above, conventional fibrosis-related treatment
studies have largely targeted the inflammatory process of fibrosis,
using corticosteroids and immunosuppressive drugs, but these agents
show little effect in clinical trials, so there is a need for new
drugs for treating fibrosis.
[0032] Accordingly, the present inventors confirmed that
Cyclo-HisPro is effective in preventing, ameliorating, or treating
fibrosis by inhibiting fibrosis occurring in various tissues or
organs, and completed the present invention.
[0033] Accordingly, the present invention provides a pharmaceutical
composition for preventing or treating fibrosis comprising
Cyclo-HisPro or a pharmaceutically acceptable salt thereof and/or a
health functional food composition for preventing or ameliorating
fibrosis comprising Cyclo-HisPro or a sitologically acceptable salt
thereof.
[0034] The present invention also provides an antifibrotic
composition comprising Cyclo-HisPro.
[0035] In the present invention, "Cyclo-HisPro (CHP)" is a
naturally occurring circular dipeptide composed of
histidine-proline which is a metabolite of thyrotropin-releasing
hormone (TRH) or a physiologically active dipeptide that is also
synthesized in the body through the TRH metabolic process and de
novo, and refers to a substance widely distributed throughout the
brain and in spinal cord and gastrointestinal tract.
[0036] In the composition of the present invention, the CHP may be
synthesized or commercially available. In addition, it can be used
after purification from substances containing CHP, for example,
prostate extract and soybean hydrolysate.
[0037] By use of the term "purified", it is intended to mean that
CHP is in a concentrated form compared to a form obtainable from a
natural origin, such as a prostate extract. Purified ingredients
can be concentrated from their natural sources or obtained through
chemical synthesis methods.
[0038] As used herein, the term "fibrosis" is used interchangeably
with "fibrotic condition", "fibroproliferative condition",
"fibrotic disease", "fibroproliferative disease", "fibrotic
disorder" and "fibroproliferative disorder", and refers to a
condition, disease or disorder characterized by dysregulated
proliferation or activity of fibroblasts, abnormal accumulation of
fibronectin and/or pathological or excessive accumulation of
collagenous tissue. Typically, such conditions, diseases or
disorders are treatable by administration of a compound having
antifibrotic activity.
[0039] The fibrosis may develop, for example, in any one or more
selected from the group consisting of: kidney, liver, lung, skin,
heart, pancreas, urinary system, genital system, sweat gland,
nerve, brain, bone marrow, muscle and joint.
[0040] For example, the fibrosis of the present invention may be
any one or more selected from the group consisting of: pulmonary
fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung
injury or lung fibrosis, pulmonary edema, cystic fibrosis, liver
fibrosis, endomyocardial fibrosis, myocardial infarction, artrial
fibrosis, glial scar, renal fibrosis, myelofibrosis,
arthrofibrosis, fat fibrosis, skin fibrosis, neurofibrosis and
myofibrosis, but is not limited thereto.
[0041] Specifically, the CHP according to the present invention has
therapeutic effects on the various fibrosis described herein.
[0042] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a first aspect
relates to renal fibrosis, which is fibrosis occurring in the
kidney.
[0043] Kidney disease is classified into acute renal failure or
chronic renal failure depending on the progression status, or
depending on the cause of disease, divided into glomerulonephritis
due to deposition of vascular complexes, diabetic kidney disease
accompanying diabetes or hypertensive renal disease accompanying
hypertension, toxic nephropathy caused by drug administration such
as antibiotics or anticancer agents, bacterial infection, and the
like. Regardless of the causative kidney disease, if the glomerular
filtration rate is reduced to 50% or less due to chronic renal
dysfunction, in most cases, the glomerular filtration rate
continues to decrease, ultimately leading to end-stage renal
failure, developing complications such as hematologic
abnormalities, nervous system complications, gastrointestinal
complications, immunological complications, infections, or
osteodystrophy, and in severe cases, leading to death.
[0044] Chronic renal failure is a state in which the renal function
gradually decreases in one direction (irreversibly), and the
homeostasis of the living body cannot be maintained. All kidney
diseases involve fibrosis of the kidneys, eventually leading to
end-stage renal failure. In particular, since chronic renal
function decline is deeply related to the progression of renal
fibrosis, inhibition of the progression of fibrosis may lead to
inhibition of the progression of chronic renal failure.
[0045] As used herein, the term "renal fibrosis" includes all
diseases in which fibrosis occurs in the kidney due to various
causes, and the fibrosis may include, but is not limited to, those
caused by any one or more selected from the group consisting of:
catheter installation, glomerulosclerosis, glomerulonephritis,
nephritis, acute renal failure, chronic renal failure, end-stage
renal disease, and metabolic disease.
[0046] The type of nephritis may include, but is not limited to,
for example, any interstitial nephritis, for example, lensacoccal
(Streptococcus sp.) nephritis, staphylococcal nephritis; viral
nephritis accompanying alastrim, hepatitis B, hepatitis C, HIV,
etc.; nephritis caused by parasitic infections such as malaria;
fungal nephritis, infectious interstitial nephritis accompanying
mycoplasma nephritis, systemic erythematosus (lupus nephritis),
systemic scleroderma (collagen disease kidney), interstitial
nephritis accompanying collagen diseases such as Sjgren's syndrome,
purpura nephritis, polyarteritis, nephritis accompanying vascular
immune diseases such as rapidly progressive glomerulonephritis,
interstitial nephritis accompanying radiation exposure;
drug-induced interstitial nephritis caused by anticancer drugs such
as gold agents, NSAID, penicillamine, and bleomycin, antibiotics,
paraquat, etc.; allergic nephritis caused by insect bites, pollen,
or rhus plants, etc.; nephritis accompanying amyloidosis nephritis,
diabetic renal failure, chronic glomerulonephritis, malignant
nephrosclerosis, polycystic kidney failure, etc.;
tubulointerstitial nephritis, nephritis accompanying pregnancy
toxaemia or cancer; idiopathic interstitial nephritis such as
membranous proliferative glomerulonephritis, IgA renal failure,
mixed cryoglobulinemia nephritis, Goodpasture's-syndrome nephritis,
beguena granulomatous nephritis, acute interstitial nephritis.
[0047] As demonstrated in FIGS. 2 to 4, CHP has a fundamental
antifibrotic effect by restoring the expression of the splicing
marker E-cadherin protein and reducing the expression of
fibronectin and pSTAT3 protein in the proximal tubular epithelial
cells induced by fibrosis, and therefore, regardless of the cause
that induces fibrosis, it can be applied to treat various renal
fibrosis.
[0048] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a second aspect
relates to liver fibrosis, which is fibrosis occurring in the
liver.
[0049] As used herein, the term "liver fibrosis" refers to a
symptom in which fibrous tissue proliferates due to chronic damage
to the liver, and may include, but is not limited to, those caused
by any one or more selected from the group consisting of: chronic
liver disease, hepatitis B virus infection, hepatitis C virus
infection, hepatitis D virus infection, schistosomiasis, alcoholic
liver disease or non-alcoholic steatohepatitis, metabolic disease,
protein deficiency, coronary artery disease, autoimmune hepatitis,
cystic fibrosis, alpha-1 antitrypsin deficiency, primary biliary
cirrhosis, drug reactions and toxins.
[0050] Liver fibrosis is a precursor to cirrhosis and is initiated
by the action of various cytokines and growth factors as a result
of severe liver damage, leading to chronic liver disease. In
general, liver fibrosis consists of reversible and thin fibrils,
and when there is no nodule formation and the cause of liver damage
is temporary, the extracellular matrix (ECM) increased by the
process of apoptosis and matrix metalloproteinases (MMP) is
decomposed, and normal recovery is possible, but if the liver
fibrosis process continues repeatedly, it forms thick fibrils and
progresses to nodular cirrhosis. In addition, liver cirrhosis is
induced through the process of liver fibrosis, in which hepatocytes
are damaged by various inflammatory factors and so abnormal
extracellular matrix proteins including collagen are accumulated,
and it is important to control the accumulation of extracellular
matrix to control the expression of cirrhosis. Inflammatory
response when hepatocytes are damaged activates resting hepatic
stellate cells to secrete extracellular matrix and various
cytokines and chemokines, among which TGF-.beta.1 acts as a strong
growth inhibitory agent. TGF-.beta.1 is a 25 kD substance, which
binds to the latent TGF-.beta.1 binding protein and is secreted in
the form of an inactive latent, and it exists in a state combined
with extracellular matrix such as type 1 and 4 collagen, laminin
and decorin, and is activated by various stimuli. TGF-.beta.1
regulates collagen expression by decreasing the production of
collagenase or increasing the production of collagenase inhibitors,
increases the production of TNF-.alpha., IL-1 and PDGF in
macrophages, and play an important role in the fibrosis process.
Currently, it is known that TGF-.beta.1 plays an important role in
liver fibrosis, since TGF-.beta.1 is expressed only in areas where
fibrosis has progressed and is not expressed in normal liver
tissues or inactive areas.
[0051] Meanwhile, non-alcoholic fatty liver can be caused by causes
such as obesity, diabetes, hyperlipidemia, drugs, etc. regardless
of drinking alcoholic, and depending on the progress, refers to a
wide range of diseases including from simple steatosis without
inflammatory response to non-alcoholic steatohepatitis (NASH)
showing hepatocellular inflammatory response, advanced fibrosis and
liver cirrhosis.
[0052] In the case of nonalcoholic fatty liver disease (NAFLD), it
has been reported that due to the increase in adult diseases caused
by high-fat and high-calorie diets in modern society, 20-30% of the
adult population in developed countries develop nonalcoholic fatty
liver disease (NAFLD), and 2-3% of them transition to nonalcoholic
steatohepatitis (NASH) patients, and in particular, they show
histological findings of steatohepatitis accompanied by fibrosis
and inflammation, highly increasing the risk of developing
cirrhosis, liver failure, and liver cancer.
[0053] Accordingly, in an embodiment of the present invention, an
animal model of liver fibrosis induced by a high-fat diet was
established, and it was confirmed that CHP exhibits the effect of
reducing fibronectin protein expression in the model.
[0054] Fatty liver is not a pathological condition in itself, and
is a reversible symptom that naturally recovers when the causative
substance is removed. However, if the state of excessive fat
accumulation in the liver tissue is continuously maintained,
steatohepatitis occurs, and as a result, hepatocyte necrosis and
regeneration occur repeatedly, during which the fibrous
extracellular matrix (ECM) increases, leading to liver fibrosis.
When liver damage reaches a certain stage, the accumulation of ECM
increases regardless of the type of causative agent, and as a
result of continuous destruction and regeneration of hepatocytes,
regenerative nodules are formed, which worsens into irreversible
liver cirrhosis.
[0055] Therefore, in the present invention, liver fibrosis is a
disease clearly distinguished from fatty liver, which is a
reversible symptom, and refers to any disease in which liver
function is reduced by changing liver tissue to fibrotic tissue
such as regenerative nodules.
[0056] As demonstrated in FIG. 5, CHP reduces the expression level
of fibronectin protein, a fibrotic protein, in fibrosis-induced
liver cells, and as demonstrated in FIG. 6, CHP reduces the
expression of fibronectin protein in an animal model in which liver
fibrosis is induced, thereby exhibiting a fundamental antifibrotic
effect, so it can be applied to the treatment of liver failure and
liver cancer that have progressed from cirrhosis, as well as
various liver fibrosis, regardless of the cause of fibrosis.
[0057] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a third aspect
relates to pulmonary fibrosis, which is fibrosis occurring in the
lung.
[0058] Especially among fibrosis, pulmonary fibrosis refers to a
disease in which chronic inflammatory cells infiltrate into the
alveolar wall of the lung tissue and induce tissue fibrosis to
cause serious structural changes in the lung tissue. Once fibrosis
is progressed due to any cause, the lung tissue hardens and the
alveolar wall thickens, thereby reducing the amount of oxygen
supplied by the blood, which makes breathing difficult. Currently,
there is no treatment method that can completely restore the lung
tissue that has already progressed to fibrosis, so if it is
detected at an early stage of fibrosis or except for lung
transplantation, usually 3 to 5 years after the onset of symptoms,
the patient will eventually die.
[0059] Specifically, as used herein, the term "pulmonary fibrosis"
refers to the development of scarred (fibrous) tissue due to the
formation or development (fibrosis) of excessive fibrous connective
tissue in the lung. Specifically, pulmonary fibrosis is a chronic
disease that causes swelling and scarring of the alveoli and
interstitial tissues of the lungs. Such scar tissue replaces
healthy tissue and causes inflammation, and chronic inflammation
can be identified as a precursor of fibrosis. Due to such damage to
the lung tissue, the lungs may become stiff, and it may become
difficult for the individual to breathe on their own.
[0060] Pulmonary fibrosis in the present invention may include, but
is not limited to, any one or more selected from the group
consisting of: idiopathic pulmonary fibrosis, radiation-induced
lung injury, nonspecific interstitial pneumonia, acute interstitial
pneumonia, respiratory bronchiolitis associated interstitial lung,
desquamative interstitial pneumonia, lymphoid interstitial
pneumonia, interstitial pulmonary fibrosis, diffuse pulmonary
fibrosis, pulmonary edema, cystic fibrosis, and pulmonary fibrosis
due to metabolic disease.
[0061] The pulmonary fibrosis may be caused by various causes, for
example, microscopic damage to the lungs induced by inhalation of
fine particles (asbestos, stone dust, metal dust, particles present
in cigarette smoke, silica dust, etc.). In addition, pulmonary
fibrosis may also occur as a secondary effect of other diseases
(such as autoimmune diseases, viral or bacterial infections) and
may be caused by certain drugs such as cytotoxic agents (such as
bleomycin, busulfan and methotrexate); antibiotics (such as
nitrofurantoin and sulfasalazine); antiarrhythmic drugs (such as
amiodarone and tocainide); anti-inflammatory drugs (such as gold
and penicylamine); illegal drugs (such as narcotics, cocaine and
heroin), and the idiopathic pulmonary fibrosis may be caused by
other unknown causes other than causes above. In addition,
long-term intake of high-fat diet induces pulmonary fibrosis, which
appears to be due to increased inflammatory levels following
high-fat intake.
[0062] As demonstrated in FIG. 7, CHP reduces the expression level
of fibronectin protein, a fibrotic protein, in fibrosis-induced
lung cells, and as demonstrated in FIG. 8, CHP reduces the
expression of TGF.beta. and Collagen 3 genes, which are major
fibrosis markers, in a pulmonary fibrosis-induced animal model,
thereby exhibiting a fundamental antifibrotic effect, and
therefore, regardless of the cause that induces fibrosis, it can be
applied to treat various pulmonary fibrosis.
[0063] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a fourth aspect
relates to skin fibrosis.
[0064] As used herein, the term "skin fibrosis" is excessive
scarring on the skin and is a result of a pathological wound
healing response. There is a wide range of fibrotic skin diseases:
scleroderma, renal fibrosing dermatosis, mixed connective tissue
disease, scleromyxedema, sclerosing edema, and eosinophilic
fasciitis. Exposure to chemicals or physical agents (for mechanical
trauma, burn wounds) is also a potential cause of fibrotic skin
disease. Skin fibrosis may be driven by immune, autoimmune and
inflammatory mechanisms. The balance of collagen production and
degradation in fibroblasts plays an important role in the
pathophysiological process of skin fibrosis. Certain cytokines,
such as transforming growth factor-.beta. (TGF-.beta.) and
interleukin-4 (IL-4) promote wound healing and fibrosis. Normal
skin fibroblasts are stationary. They control the amount of
connective tissue protein and have low proliferative activity.
After skin injury, these cells are activated, that is, they express
.alpha.-smooth muscle actin (.alpha.-SMA) and synthesize large
amounts of connective tissue protein. Such activated cells are
often called myofibroblasts.
[0065] As used herein, the term "skin fibrosis" is also meant to
include "scleroderma".
[0066] Scleroderma is a chronic autoimmune connective tissue
disease of unknown cause characterized by sclerotic changes and
vascular abnormalities in which the skin of a part or the whole
body becomes hard and thickened due to excessive accumulation of
collagen in the dermis. Collagen forms connective tissue and serves
to support and connect the tissues of the body. There are several
forms of scleroderma, some showing symptoms only in a specific part
of the body, while others appearing all over the body including
internal organs. That is, it is divided into localized scleroderma,
in which only a part of the skin becomes hard, and systemic
scleroderma, which exhibits fibrosis symptoms due to the increase
in collagen not only in the skin but also in internal organs such
as the lungs, digestive organs, kidneys and heart. Systemic
scleroderma is further classified into limited type and diffuse
type according to the degree of invasion of the skin and internal
organs, prognosis, and immunological examination findings.
[0067] In the use of the present invention for preventing,
ameliorating or treating skin fibrosis, skin fibrosis may include,
but is not limited to, any one or more selected from the group
consisting of: scars, hypertrophic scars, keloid scars, localized
scleroderma and systemic scleroderma.
[0068] Although the cause of scleroderma has not yet been clearly
identified, tissue fibrosis is thought to play an important
role.
[0069] The early symptoms of scleroderma are very diverse,
especially in the latter stage, the skin symptoms become clear.
Common symptoms of scleroderma include joint pain, morning
stiffness, fatigue, and weight loss. In addition, exposure to cold
also temporarily limits the blood supply to fingers, toes, nose and
ears. These symptoms are early symptoms of scleroderma patients and
are one of the frequent symptoms. The skin of patients with
scleroderma becomes hard. This hardening of the skin spreads widely
and typically occurs on both sides of the body. Eventually, the
tissue is damaged, and the skin is pigmented.
[0070] "Localized scleroderma" is 2.6 times more common in women
than in men. Localized scleroderma occurs in 75% of patients
between 20 and 50 years of age, and linear scleroderma tends to
occur at an earlier age. Localized scleroderma is classified into
localized scleroderma, systemic localized scleroderma, linear
localized scleroderma, or subcutaneous localized scleroderma.
[0071] Localized scleroderma often starts with erythema or
reddish-purple patches, and the border with the surrounding normal
skin becomes clear, and the skin in a certain area loses elasticity
and becomes hard. The area will be brown or, in many cases,
discolored, giving it a white color. Most of the time it occurs in
one place, but sometimes there are several, and they vary in size
and can range from the size of a coin to the size of an adult palm.
It causes cosmetic problems but does not progress to systemic
scleroderma. In some patients, even without treatment, the hard
skin softens and recovers on its own.
[0072] Systemic localized scleroderma is a severe form of localized
scleroderma, in which the skin in a large area becomes hard and
hyperpigmentation is observed. Extensive hardening of the skin of
the trunk, buttocks, and legs. However, it is distinguished from
systemic sclerosis because there is no Raynaud's phenomenon or
internal organ involvement.
[0073] Linear localized scleroderma is a case in which the skin
becomes long and hard in a straight line, and it occurs most often
in the order of the legs, arms, forehead, and chest, in particular,
if it is depressed in a vertical line on the forehead, it is called
en coup de sabre and causes cosmetic problems. Unlike localized
scleroderma, linear scleroderma involves not only the skin but also
the underlying muscle and periosteum tissue and is fixed in the
underlying tissue. It occurs more frequently in children and can
sometimes cause severe skeletal developmental anomaly in the limbs
and face.
[0074] Subcutaneous localized scleroderma is characterized by
sclerosis of the fat layer, fascia, muscle and sometimes bone, and
joint movement may be limited. Because the lesion appears in the
deep part, the characteristic skin pigment change of scleroderma is
not observed.
[0075] Systemic scleroderma occurs four times more often in women
than in men, and can occur at any age, but usually occurs between
the ages of 30 and 50. The diffuse type is known to occur at a
younger age than the limited type. Raynaud's phenomenon is the
first symptom of systemic scleroderma, followed by sclerosis of the
skin and internal organs. Systemic scleroderma is divided into skin
symptoms and internal organ symptoms.
[0076] In systemic sclerosis, the first skin symptoms begin on the
fingers and hands. At first, only the Raynaud's phenomenon is seen,
but the fingers and hands become swollen, stiff, and turn red.
Gradually, the skin becomes hard, and it spreads to the arms and
face, and in the case of the diffuse type, the skin of the whole
body including the trunk is continuously hardened. When sclerosis
occurs on the face, the overall wrinkles disappear, and it becomes
difficult to make expressions. The nose looks pointed, it is
difficult to open the mouth, and the lips become thin, and radial
wrinkles around the mouth appear, making it look like an elderly
person's mouth. The movement of the joint is limited, making it
impossible to hold the hand all the way, and sclerodactyly appears
in which the fingers are bent and pointed. Painful and
difficult-to-heal ulcers develop on the fingertips and joints.
[0077] Extensive hyperpigmentation and local hypopigmentation may
occur, and hair loss and sweat secretion reduction occur in the
involved area. Capillary dilatation appears in the form of round
spots on the face and upper body, or is observed around the nail
folds. If there is an abnormality in the capillaries of the nail
folds in patients with Raynaud's disease, systemic scleroderma may
occur later, so changes in capillary are important in determining
the prognosis. Skin calcification can be observed and is relatively
common around the finger joints.
[0078] Internal organ symptoms occur in the gastrointestinal tract,
lungs, heart, or kidneys. In the gastrointestinal tract, the
esophagus is the most frequently involved part and is involved in
more than 90% of cases. In the esophagus, peristaltic dysfunction,
dysphagia, and reflux esophagitis are common. Symptoms such as
constipation, diarrhea, and malabsorption may appear due to
decreased intestinal motility. The most important cause of death in
patients with systemic scleroderma is due to pulmonary symptoms,
occurring in about 70% of patients. Due to fibrosis of the lungs,
people complain of difficulty in breathing and cough during
exercise. Alveolitis is a common occurrence, and pulmonary function
continues to decline. Cardiac symptoms may include cardiac
conduction disorder, heart failure, and pericarditis, and
myocardial sclerosis may occur in 50-70% of patients. Kidney
symptoms occur in approximately 45% of patients with systemic
scleroderma. It appears as sudden onset of acute renal failure,
hypertension, etc., and uremia that progresses slowly may
occur.
[0079] Accordingly, in the use of the present invention for
preventing, ameliorating or treating skin fibrosis, skin fibrosis
encompasses, without limitation, fibrosis occurring in any part of:
internal cavities of organs or glands such as blood vessels and
veins, ducts of submandibular, gallbladder, thyroid follicles,
sudoriferous ducts, ovaries, kidneys; epithelial cells of the gums,
tongue, palate, nose, larynx, esophagus, stomach, intestines,
rectum, anus and vagina; and any skin tissue and epithelial cells,
including the dermis, scars, skin and scalp.
[0080] As confirmed in FIG. 9, CHP exhibits a fundamental
antifibrotic effect by reducing the expression level of fibronectin
protein, a fibrotic protein, in fibrosis-induced skin cells, and
therefore, regardless of the cause that induces fibrosis, it can be
applied to treat various skin fibrosis (scleroderma).
[0081] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a fifth aspect
relates to fibrosis, which is cardiac fibrosis occurring in the
heart.
[0082] As used herein, the term "cardiac fibrosis" refers to a
phenomenon in which the heart hardens due to excessive deposition
of matrix proteins between heart cells and also refers to a
phenomenon that occurs mainly in the heart of patients with
myocardial infarction and is the main cause of decreased heart
function, and may include, but not limited to, any one or more
selected from the group consisting of: endomyocardial fibrosis,
atrial fibrosis, heart failure, myocardial infarction, and cardiac
fibrosis due to metabolic disease.
[0083] Cardiac fibrosis is characterized by a disproportionate
accumulation of fibrosing collagen that occurs after cardiomyocyte
death, inflammation, increased workload, hypertrophy and
stimulation by a number of hormones, cytokines and growth
factors.
[0084] Cardiac fibrosis may also refer to abnormal thickening of
heart valves due to disproportionate proliferation of cardiac
fibroblasts, but more generally refers to proliferation of
fibroblasts in the heart muscle. Fibrocyte cells normally act to
secrete collagen and provide structural support for the heart. When
overactivated, this process leads to valve thickening and
fibrosis.
[0085] Since fibrosis of the heart is a major cause of heart
failure and myocardial infarction, the term "cardiac fibrosis" may
be interpreted to encompass heart failure and/or myocardial
infarction caused by cardiac fibrosis.
[0086] As confirmed in FIG. 10, CHP exhibits a fundamental
antifibrotic effect by reducing the expression of Fibronectin,
Collagen 1, Collagen 2, Collagen 3 and Collagen 4 genes, which are
major fibrosis markers, in a fibrosis-induced animal model, and
therefore, regardless of the cause that induces fibrosis, it is
useful for the treatment of various cardiac fibrosis.
[0087] In the use of the composition of the present invention for
preventing, ameliorating or treating fibrosis, a sixth aspect
relates to fat fibrosis.
[0088] As used herein, the term "fat fibrosis" is meant to
encompass all diseases in which fibrosis occurs in adipose
tissue.
[0089] It has been reported that fibrosis of adipose tissue occurs
when normal mice are fed a high-fat diet containing 60% fat for 16
to 24 weeks (Hu, M et al., Evidence-Based Complementary and
Alternative Medicine, 1-12, 2018; Kwon, E. Y., & Choi, M. S.,
Nutrients, 10(10), 1415, 2018; Nakazeki, F et al., Scientific
Reports, 8(1), 2018; Muniappan, L et al. Scientific Reports, 7(1),
2017; Lancha, A et al., PLoS ONE, 9(5), e98398, 2014;
Vel.alpha.zquez, K. T et al., Physiological Reports, 5(18), e13412,
2017; Wang, L., Ye, X., Hua, Y., & Song, Y, Biomedicine &
Pharmacotherapy, 105, 121-129, 2018).
[0090] Accordingly, the present inventors established an animal
model of fat fibrosis induced by a high-fat diet and checked the
expression changes of the fibrosis markers TGF.beta., Fibronectin,
Collagen 3, and CTGF genes according to CHP administration. As a
result, as confirmed in FIG. 11, CHP exhibits an antifibrotic
effect by remarkably reducing the expression of the fibrosis
markers, and thus is useful for the treatment of fat fibrosis.
[0091] As described above, in the present invention, it was
confirmed that CHP exhibits antifibrotic activity by reducing the
expression of various fibrosis marker genes or proteins in various
fibrosis-induced tissue cells and animal models, therefore, it can
be expected that it can be effectively applied to prevent,
ameliorate, or treat fibrosis occurring in various tissues and/or
organs in the body, including kidney, liver, lung, skin, heart,
pancreas, urinary system, genital system, sweat glands, nerves,
brain, bone marrow, muscles and joints.
[0092] In the composition for preventing, ameliorating or treating
fibrosis of the present invention, the terms "prevent",
"ameliorate" and/or "treat" refer to any act that inhibits or
delays the onset of a disease or disease symptom, any act that
ameliorates or beneficially alters the state of a disease or
disease symptom, and any act that delays, stops or reverses the
progression of a disease or disease symptom.
[0093] As used herein, the term "pharmaceutically acceptable"
refers to those that are physiologically acceptable and do not
normally cause allergic reactions or similar reactions when
administered to humans, and the salt is preferably an acid addition
salt formed by a pharmaceutically acceptable free acid.
[0094] The pharmaceutically acceptable salt may be an acid addition
salt formed using an organic acid or an inorganic acid, and the
organic acid include, for example, formic acid, acetic acid,
propionic acid, lactic acid, butyric acid, isobutyric acid,
trifluoroacetic acid, malic acid, maleic acid, malonic acid,
fumaric acid, succinic acid, succinic acid monoamide, glutamic
acid, tartaric acid, oxalic acid, citric acid, glycolic acid,
glucuronic acid, ascorbic acid, benzoic acid, phthalic acid,
salicylic acid, anthranilic acid, dichloroacetic acid, aminooxy
acetic acid, benzenesulfonic acid, p-toluenesulfonic acid or
methanesulfonic acid. The inorganic acid includes, for example,
hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid,
nitric acid, carbonic acid or boric acid. The acid addition salt
may preferably be in the form of hydrochloride salt or acetate
salt, and more preferably in the form of hydrochloride salt.
[0095] In addition, additional salts are available in the form of
GABA salt, gabapentin salt, pregabalin salt, nicotinate salt,
adipate salt, hemimalonate salt, cysteine salt, acetylcysteine
salt, methionine salt, arginine salt, lysine salt, ornithine salt
or aspartate salt etc.
[0096] In addition, the pharmaceutical composition of the present
invention may further include a pharmaceutically acceptable
carrier. The pharmaceutically acceptable carrier may further
include, for example, a carrier for oral administration or a
carrier for parenteral administration. Carriers for oral
administration may include lactose, starch, cellulose derivatives,
magnesium stearate, stearic acid, and the like. Carriers for
parenteral administration may include water, suitable oils, saline,
aqueous glucose and glycols, and the like. In addition, it may
further include a stabilizer and a preservative. Suitable
stabilizers include antioxidants such as sodium bisulfite, sodium
sulfite or ascorbic acid. Suitable preservatives include
benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
As to other pharmaceutically acceptable carriers, reference may be
made to those described in the following literature (Remington's
Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton,
Pa., 1995).
[0097] The pharmaceutical composition of the present invention may
be administered to mammals, including humans by any method. For
example, it may be administered orally or parenterally, and
parenteral administration methods include, but are not limited to,
intravenous, intramuscular, intraarterial, intramedullary,
intradural, intracardiac, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral, topical, sublingual or rectal
administration.
[0098] The pharmaceutical composition of the present invention may
be formulated as a formulation for oral administration or
parenteral administration according to the administration route as
described above. When formulated, it may be prepared using one or
more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose,
mannose, sucrose, or dextran, etc.), antioxidants, bacteriostatic
agents, chelating agents (e.g., EDTA or glutathione), fillers,
extending agents, binding agents, adjuvants (e.g. aluminum
hydroxide), suspending agents, thickening agents, wetting agents,
disintegrating agents or surfactants, diluents or excipients.
[0099] Solid formulations for oral administration include tablets,
pills, powders, granules, liquids, gels, syrups, slurries,
suspensions or capsules, etc., and these solid formulations may be
prepared by mixing the pharmaceutical composition of the present
invention with at least one or more excipients, for example, starch
(including corn starch, wheat starch, rice starch, potato starch,
etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol,
mannitol, xylitol, erythritol maltitol, cellulose, methyl
cellulose, sodium carboxymethyl cellulose and
hydroxypropylmethyl-cellulose or gelatin, etc. For example, tablets
or sugar-coated tablets may be obtained by blending the active
ingredient with solid excipients, grinding them, adding suitable
adjuvants, and processing them into a granular mixture.
[0100] In addition to simple excipients, lubricants such as
magnesium stearate talc are also used. Liquid formulations for oral
use may include suspensions, internal liquid formulations,
emulsions, or syrups, and various excipients, such as wetting
agents, sweetening agents, air fresheners, or preservatives in
addition to water or liquid paraffin, which are commonly used
simple diluents.
[0101] In addition, in some cases, cross-linked
polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be
added as a disintegrating agent, and an anti-aggregating agent, a
lubricant, a wetting agent, a flavoring agent, an emulsifier, and a
preservative may be additionally included.
[0102] When administered parenterally, the pharmaceutical
composition of the present invention may be formulated according to
methods known in the art in the form of injections, transdermal
administration agents and nasal inhalants together with suitable
parenteral carriers. In the case of the injection, it must be
sterilized and protected from contamination of microorganisms such
as bacteria and fungi. Examples of suitable carriers for injection
may include, but are not limited to, water, ethanol, polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol, etc.),
mixtures thereof and/or a solvent or dispersion medium containing
vegetable oil. More preferably, suitable carriers may include
Hanks' solution, Ringer solution, phosphate buffered saline (PBS)
with triethanolamine or isotonic solutions such as sterile water
for injection, 10% ethanol, 40% propylene glycol and 5% dextrose,
etc. In order to protect the injection from microbial
contamination, it may further include various antibacterial and
antifungal agents such as parabens, chlorobutanol, phenol, sorbic
acid, thimerosal, and the like. In addition, in most cases, the
injection may further include an isotonic agent such as sugar or
sodium chloride.
[0103] In the case of transdermal administration agent, forms such
as ointment, cream, lotion, gel, external solution, pasta,
liniment, and air roll are included. In the above, `transdermal
administration` means that an effective amount of the active
ingredient contained in the pharmaceutical composition is delivered
into the skin by topically administering the pharmaceutical
composition to the skin.
[0104] In the case of inhalant, the compounds for use according to
the present invention may be conveniently delivered in the form of
an aerosol spray from pressurized packs or nebulizers using a
suitable propellant, for example, dichlorofluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. For example, gelatin capsules and cartridges for
use in inhalers or insufflators may be formulated to contain a
compound and a suitable powder base powder mixture such as lactose
or starch. Formulations for parenteral administration are described
in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack
Publishing Company, Easton, Pa. 18042, Chapter 87: Blaug, Seymour,
a formulary commonly known to all pharmaceutical chemistry.
[0105] When the pharmaceutical composition of the present invention
contains Cyclo-HisPro in an effective amount, it is possible to
provide a desirable effect of preventing, ameliorating or treating
fibrosis. As used herein, the term "effective amount" refers to an
amount that exhibits a response greater than that of a negative
control group, and preferably refers to an amount sufficient to
prevent, ameliorate or treat fibrosis. The pharmaceutical
composition of the present invention may contain 0.01 to 99.9% of
Cyclo-Hispro, and the remaining amount may be occupied by a
pharmaceutically acceptable carrier. The effective amount of
Cyclo-HisPro included in the pharmaceutical composition of the
present invention will vary depending on the form in which the
composition is commercialized.
[0106] The total effective amount of the pharmaceutical composition
of the present invention may be administered to a patient as a
single dose, and may be administered by a fractionated treatment
protocol in which multiple doses are administered for a long period
of time. The pharmaceutical composition of the present invention
may vary the content of active ingredients according to the
severity of the disease. For example, it may be administered in one
to several divided doses to be administered in an amount of
preferably 0.001 to 100 mg, more preferably 0.01 to 10 mg per 1 kg
of body weight per day based on Cyclo-HisPro. However, in the case
of the dose of Cyclo-HisPro, since the effective dosage for a
patient is determined in consideration of various factors such as
administration route of the pharmaceutical composition and number
of treatments, as well as the patient's age, weight, health status,
sex, disease severity, diet, and excretion rate, in consideration
of this point, one of ordinary skill in the art will be able to
determine an appropriate effective dosage of the Cyclo-HisPro
according to a specific use for preventing, treating or
ameliorating fibrosis. The pharmaceutical composition according to
the present invention is not particularly limited in its
formulation, administration route and administration method as long
as the effect of the present invention is exhibited.
[0107] The pharmaceutical composition for preventing or treating
fibrosis of the present invention may be used alone or in
combination with methods using surgery, radiation therapy, hormone
therapy, chemotherapy, or biological response modifiers.
[0108] The pharmaceutical composition for preventing or treating
fibrosis of the present invention may also be provided in the
formulation of an external application containing Cyclo-HisPro. In
this aspect, the composition of the present invention may be a
quasi-drug composition for preventing or ameliorating fibrosis and
a quasi-drug comprising the composition.
[0109] The external application may be directly applied to the skin
or oral cavity. When the pharmaceutical composition for preventing
or treating fibrosis of the present invention is used for external
application, it may further contain adjuvants commonly used in the
field of dermatology, such as any other ingredients commonly used
for external applications for skin including fatty substance,
organic solvent, solubilizer, thickening and gelling agent,
emollient, antioxidant, suspending agent, stabilizer, foaming
agent, air freshener, surfactant, water, ionic emulsifier, nonionic
emulsifier, filler, sequestering agent, chelating agent,
preservative, vitamin, blocking agent, wetting agent, essential
oil, dye, pigment, hydrophilic active agent, lipophilic active
agent or lipid vesicle. In addition, the above ingredients may be
introduced in an amount generally used in the field of
dermatology.
[0110] When provided for external application, the composition of
the present invention may be in the formulation of a liquid,
ointment, patch, gel, cream or spray, but is not limited thereto.
According to an embodiment of the present invention, the
quasi-drugs of the present invention may include toothpaste, oral
care products including mouthwash and mouth spray, ointments,
masks, poultices, plasters, and transdermal absorbents.
[0111] When the composition of the present invention is used as a
quasi-drug composition, Cyclo-HisPro may be added as it is or may
be appropriately used in combination with other quasi-drug
ingredients according to a conventional method. The mixing amount
of the active ingredients may be appropriately determined according
to the purpose of use (prevention, health or therapeutic
treatment).
[0112] The contents of the pharmaceutical composition and health
functional food composition of the present invention may be applied
mutatis mutandis to the quasi-drug composition and quasi-drug of
the present invention.
[0113] As used herein, the term "health functional food" includes
both the meanings of "functional food" and "health food".
[0114] As used herein, the term "functional food" is the same term
as food for special health use (FoSHU), and refers to food with
high medical effects processed to efficiently exhibit bioregulatory
functions in addition to nutritional supply.
[0115] As used herein, the term "health food" refers to food having
an active health maintenance or promotion effect compared to
general food, and health supplement food refers to food for the
purpose of health supplementation. In some cases, the terms
functional food, health food, and health supplement food are used
interchangeably. The above food may be prepared in various forms
such as tablets, capsules, powders, granules, liquids, pills, etc.
in order to obtain a useful effect in improving and recovering
liver function.
[0116] As a specific example of such a functional food, by using
the composition, it is possible to produce a processed food with
good storage properties while at the same time being transformed by
taking advantage of the characteristics of agricultural products,
livestock products or aquatic products.
[0117] The health functional food composition of the present
invention may also be prepared in the form of nutritional
supplements, food additives and feeds, and is intended for
consumption by animals, including humans or livestock.
[0118] Food compositions of this type can be prepared in various
forms according to conventional methods known in the art. It may be
prepared as regular food by adding Cyclo-HisPro to the following
food including, but not limited to, beverages (including alcoholic
beverages), fruits and their processed foods (e.g., canned fruit,
bottled, jam, marmalade, etc.), fish, meat and their processed
foods (e.g., ham, sausages, corn beef, etc.), breads and noodles
(e.g., udon noodles, soba noodles, ramen, spaghetti, macaroni,
etc.), fruit juice, various drinks, cookies, yeot (Korean hard
taffy), dairy products (e.g., butter, cheese, etc.), edible
vegetable oils and fats, margarine, vegetable protein, retort food,
frozen food, various seasonings (e.g., soybean paste, soy sauce,
sauce, etc.).
[0119] In addition, it may be prepared as the nutritional
supplement by adding Cyclo-HisPro to capsules, tablets, pills, and
the like, but it is not limited thereto.
[0120] For health functional food, the Cyclo-HisPro may be prepared
in the form of tea, juice, and drink and ingested by liquefaction,
granulation, encapsulation, and powder so that it can be drinkable
(as health drink), but it is not limited thereto. In addition, in
order to use the Cyclo-HisPro in the form of a food additive, it
may be prepared and used in the form of a powder or a concentrate.
In addition, it may be prepared in the form of a composition by
mixing the Cyclo-HisPro with an active ingredient known to be
effective in preventing or ameliorating fibrosis.
[0121] When the food composition of the present invention is used
as a health drink composition, the health drink composition may
contain various flavoring agents or natural carbohydrates as
additional ingredients, like a conventional drink. The
above-mentioned natural carbohydrates may be monosaccharides such
as glucose and fructose; disaccharides such as maltose and sucrose;
polysaccharides such as dextrin and cyclodextrin; or sugar alcohols
such as xylitol, sorbitol, and erythritol. As sweetening agents,
natural sweetening agents such as taumatine and stevia extract,
synthetic sweetening agents such as saccharin and aspartame, or the
like may be used. The proportion of the natural carbohydrate is
generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per
100 mL of the composition of the present invention.
[0122] Cyclo-HisPro may be contained as an active ingredient in a
food composition for preventing or ameliorating fibrosis, and the
amount may be an effective amount to obtain the preventing or
ameliorating effect, for example, preferably 0.01 to 100% by weight
based on the total weight of the total composition, but not
particularly limited thereto. The food composition of the present
invention may be prepared by mixing Cyclo-HisPro with other active
ingredients known to be effective in preventing or ameliorating
fibrosis.
[0123] In addition to the above, the health functional food of the
present invention may include various nutritional supplements,
vitamins, electrolytes, flavoring agents, coloring agents, pectic
acid, pectic acid salts, alginic acid, alginic acid salts, organic
acids, protective colloidal thickeners, pH regulators, stabilizers,
preservatives, glycerin, alcohols, or carbonating agents, and the
like. In addition, the health food of the present invention may
contain fruit flesh for the production of natural fruit juice,
fruit juice beverage, or vegetable beverage. These ingredients may
be used independently or in combination. The proportion of these
additives is not particularly important, but is generally selected
in the range of 0.01 to 0.1 parts by weight per 100 parts by weight
of the composition of the present invention.
[0124] The present invention also relates to a method for
preventing or treating fibrosis comprising administering to a
subject in need thereof an effective amount of Cyclo-HisPro.
[0125] In the method of the present invention, the term "subject"
includes any animal (e.g., human, horse, pig, rabbit, dog, sheep,
goat, non-human primate, cow, cat, guinea pig or rodent), but is
not limited thereto. These terms do not denote a specific age or
sex. Accordingly, whether female or male, adult and neonatal
subjects as well as fetuses are intended to be included. A patient
refers to a subject with a disease or disorder. The term patient
includes human and veterinary subjects.
[0126] In the method of the present invention, the description of
the constitution including the effect of CHP, its administration
route, the number of administrations, the dosage, etc. is the same
as described above, and thus description thereof will be
omitted.
[0127] The present invention also provides a use of Cyclo-HisPro in
the manufacture of a pharmaceutical composition for preventing or
treating fibrosis.
[0128] Hereinafter, the present invention will be described in more
detail through examples. However, since the present invention may
have various changes and may have various forms, the specific
examples and descriptions described below are only for helping the
understanding of the present invention, and it is not intended to
limit the invention to any particular form disclosed. It should be
understood that the scope of the present invention includes all
modifications, equivalents and substitutes included in the spirit
and scope of the present invention.
MODE FOR CARRYING OUT THE INVENTION
Preparation Example
[0129] CHP (Cyclo-HisPro) used in the following examples was
purchased from Bachem and used.
Example 1
[0130] Confirmation of Antifibrotic Effect in Human-Derived
Proximal Tubular Epithelial Cells According to CHP Treatment P 1-1.
Isolation and Culture of Human-Derived Proximal Tubular Epithelial
Cells
[0131] As shown in FIG. 1, human-derived glomerular endothelial
cells and human-derived proximal tubular epithelial cells, which
can be used for cell experiments, were purely isolated and cultured
by isolating glomeruli and tubular interstitial tissue from normal
adult kidney tissue and primarily culturing them.
[0132] 1-2. Observation of Cytological Morphology after CHP
Treatment in a Human-Derived Proximal Tubular Epithelial Cell
Fibrosis Model
[0133] By inducing cell fibrosis in human-derived proximal tubular
epithelial cells that were isolated and primarily cultured in
Example 1-1 by treating recombinant TGF.beta. (2 ng/ml) to the
cells, cytoskeletal remodeling and morphological changes (became
thin, elongated and structure disappeared) were occurred, which
confirming that fibrosis was properly induced (FIG. 2).
[0134] Thereafter, it was confirmed cytomorphologically that
fibrosis was improved when various concentrations of CHP (4, 20 and
100 .mu.g/ml) were treated (FIG. 2).
[0135] 1-3. Confirmation of Expression of E-Cadherin, Fibronectin
and pSTAT3 Proteins after CHP Treatment in a Human-Derived Proximal
Tubular Epithelial Cell Fibrosis Model
[0136] Under the same conditions as in Example 1-2, the expression
levels of proteins of E-cadherin, a junction marker, fibronectin, a
fibrosis marker, and pSTAT3, a fibrosis transcription factor, after
CHP administration were checked by Western blot.
[0137] As a result, as shown in FIG. 3, it was confirmed that the
protein expression of E-cadherin was restored and the protein
expression of fibronectin and pSTAT3 decreased according to the CHP
administration.
Example 2
[0138] Confirmation of Antifibrotic Effect in Human-Derived
Glomerular Endothelial Cells According to CHP
[0139] Observation of Cytological Morphology after CHP Treatment in
a Human-Derived Glomerular Endothelial Cell Fibrosis Model
[0140] As in the human-derived proximal tubular epithelial cell
fibrosis model of Example 1-1, by inducing cell fibrosis in
glomerular endothelial cells by treatment with rTGF.beta. (2
ng/ml), cytoskeletal remodeling and morphological changes were
occurred, which confirming that fibrosis was properly induced (FIG.
4).
[0141] Thereafter, it was confirmed cytomorphologically that
fibrosis was improved when various concentrations of CHP (40 and
100 .mu.g/ml) were treated (FIG. 4).
[0142] Through the above results, it was possible to confirm the
excellent therapeutic effect of CHP on renal fibrosis.
Example 3
[0143] Confirmation of Antifibrotic Effect in Human-Derived
Hepatocytes According to CHP Treatment
[0144] 3-1. CHP Treatment after Culturing Human-Derived Hepatocytes
and Inducing Fibrosis Thereof
[0145] DMEM medium and FBS were purchased from Hyclone for
culturing the Huh7 liver cell line, and TGF.beta. for inducing
fibrosis was purchased from R&D Systems. The cells were
cultured in a DMEM medium supplemented with 10% FBS in a 5%
CO.sub.2 incubator at 37.degree. C., treated with TGF.beta. at a
concentration of 2 ng/ml and treated with CHP at 0, 62.5, 125, or
250 ng/ml, and cells were harvested at 48 hours of treatment.
[0146] 3-2. Confirmation of Fibronectin Protein Expression after
CHP Treatment in a Human-Derived Hepatocyte Fibrosis Model
[0147] RIPA lysis buffer and Halt.TM. protease and phosphatase
inhibitor cocktails was purchased from Thermo to analyze the
expression of fibronectin protein, a fibrotic protein, and for
protein quantification, BCA protein quantification kit was
purchased from Thermo. For Western blot, Thermo's Bolt.TM. protein
gel electrophoresis system and Bio-rad's wet transfer system were
prepared. The antibody of fibronectin protein, a marker of liver
fibrosis, and the GAPDH antibody used as a loading control were
purchased from Abcam. 100 ul of RIPA buffer containing protease and
phosphatase inhibitors was added to the harvested Huh7 cells, and
the cells were lysed by pipetting. After leaving on ice for 10
minutes, centrifugation was performed at 15,000 rpm at 4.degree. C.
for 10 minutes. The supernatant was collected, and the protein
concentration was measured by BCA quantitation, and the protein was
separated from the same amount of samples using the Bolt.TM.
protein gel electrophoresis system, and transferred to a
nitrocellulose membrane. The membrane was blocked with 5% skim milk
at room temperature for 1 hour, and then reacted with fibronectin
and GAPDH antibodies, which are primary antibodies, overnight at
4.degree. C. After washing 3 times for 10 minutes with TBST, the
reaction was performed with the secondary antibody at room
temperature for 1 hour. After washing 3 times for 10 minutes with
TBST, the expression level was measured by reacting with ECL. The
size of the appearing band was quantified using the ImageJ program
and corrected by dividing the fibronectin band size value by the
GAPDH band size value. Statistical significance was analyzed using
Student's t-test statistical method with the control group.
#p<0.05 (negative control group: TGF.beta. treated control
group).
[0148] As a result of the experiment, as shown in FIG. 5a-b, it was
confirmed that the expression level of fibronectin protein, a
fibrotic protein, which increases according to TGF.beta. treatment,
showed a tendency to decrease when treated with CHP 250 ng/ml
together with TGF.beta.. Through this, it was confirmed that CHP
can ameliorate fibrosis in the liver cell line Huh7 cells.
Example 4
[0149] Confirmation of Antifibrotic Effect in an Animal Model of
Liver Fibrosis According to CHP Administration
[0150] Long-term high-fat diet intake is known to be a risk factor
for liver fibrosis. An inflammatory environment is induced in the
liver tissue due to insulin resistance and increased blood fatty
acids, adipokines, and cytokines that appear when a fatty diet is
consumed for a long period of time, and the interaction of immune
cells accumulated in the liver tissue proceeds with fibrosis, in
particular, IL-13 cytokine secreted by Th2 CD4+ T cells causes
macrophages to produce large amounts of TGF.beta., a key inducing
factor for fibrosis. Signal transduction by TGF.beta. causes
transformation of cells in liver tissue and promotes fibrosis
progression (Rosselli, M et al., Current Pharmaceutical Design,
20(31), 5010-5024, 2014; Wu. D et al., Science, 332:243-7, 2011; J
Investig Med, 60(8): 1147-1150, 2012; Fichtner-Feigl S et al., Nat
Med, 12:99-106, 2006; Lee C G et al., J Exp Med, 194:809-21,
2001).
[0151] When normal mice were fed a high-fat diet containing 60% fat
for 10 to 16 weeks, liver fibrosis was observed, which can be
confirmed through the expression of fibronectin, a representative
fibrosis marker protein (Kim, I. H et al., AGE, 38(4), 291-302,
2016; Chen, H. J., & Liu, J., Biomedicine &
Pharmacotherapy, 97, 1386-1396, 2018.1-2).
[0152] 4-1. Design of Experimental Animals and Induction of Liver
Fibrosis
[0153] For liver fibrosis experiments in animals, 12-week-old
C57BL/6 mice fed a high-fat diet containing 60% fat for 6 weeks
were purchased from Jackson, USA, and Research diets D12492, a 60%
fat-containing high-fat dietary feed purchased from Saeron Bio,
were ingested for 17 weeks. Mice were bred to allow free access to
feed and water in an environment of 24.+-.3.degree. C. C57BL/6 mice
fed a high-fat diet for 6 weeks were acclimatized to the breeding
environment for 1 week, and their weights were measured and evenly
divided into 3 groups as shown in Table 1. CHP was orally
administered to each group at a concentration of 5 mg/kg or 35
mg/kg once a day for 16 weeks, and the same amount of distilled
water was orally administered to the control group in the same
manner.
TABLE-US-00001 TABLE 1 Group HFD CHP5 CHP35 Administration Vehicle
CHP 5 mg/kg CHP 35 mg/kg (water) Number of mice 10 8 8
[0154] 4-2. Confirmation of the Expression of Fibronectin Protein,
a Marker of Fibrosis, in an Animal Model of Liver Fibrosis
Administered with CHP
[0155] Isoflurane required for anesthesia and dissection of mice
was purchased from Hana Pharmaceutical, and Vetequip's RC2 Rodent
Circuit Controller Anesthesia System was prepared. Phosphate
buffered saline (PBS) was purchased from Hyclone. To isolate the
mouse liver tissue, the mice were anesthetized by respiratory
anesthesia with 3-3.5% isoflurane. After blood was drawn from the
heart of anesthetized mice, the liver tissue was immediately
excised and washed with PBS. Then, 50 mg of the liver tissue was
cut out and placed in 500 ul of RIPA buffer containing protease and
phosphatase inhibitors, and was pulverized using IKA's T10
homogenizer. After leaving on ice for 15 minutes, centrifugation
was performed at 15,000 rpm at 4.degree. C. Western blotting was
performed in the same manner as in Example 3-2 to analyze the
expression of fibronectin protein, a fibrotic protein. Statistical
significance was analyzed using the Student's t-test statistical
method with HFD control group (*p<0.05, **p<0.01).
[0156] As a result of the experiment, as shown in FIG. 6a-b, it was
confirmed that the expression level of fibronectin protein, a
representative fibrosis marker, was significantly decreased in the
CHP-administered group, and the effect was confirmed to be stronger
according to the concentration of CHP. This means that CHP
ameliorates liver fibrosis, which is a clear evidence that it can
be applied to the treatment of liver fibrosis.
Example 5
[0157] Confirmation of Antifibrotic Effect in Human-Derived Lung
Cells According to CHP Treatment
[0158] 5-1. CHP Treatment after Culturing Human-Derived Lung Cells
and Inducing Fibrosis Thereof
[0159] MEM medium and FBS were purchased from Hyclone for culturing
the L-132 lung cell line, and TGF.beta. for inducing fibrosis was
purchased from R&D Systems. The cells were cultured in a MEM
medium supplemented with 10% FBS in a 5% CO.sub.2 incubator at
37.degree. C., treated with TGF.beta. at a concentration of 2 ng/ml
and treated with CHP at 0, 62.5, or 125 ng/ml, and cells were
harvested at 48 hours of treatment.
[0160] 5-2. Confirmation of Fibronectin Protein Expression after
CHP Treatment in a Human-Derived Lung Cell Fibrosis Model
[0161] Western blotting was performed in the same manner as in
Example 3-2 to analyze the expression of fibronectin protein, a
fibrotic protein. Statistical significance was analyzed using
Student's t-test statistical method with the control (#p<0.05
(negative control group: TGF.beta.-treated control group),
**p<0.01 (TGF.beta.-treated control group: CHP 125 ng/ml-treated
group)).
[0162] As a result of the experiment, as shown in FIG. 7a-7b, it
was confirmed that the expression level of fibronectin protein, a
fibrotic protein, which increases according to TGF.beta. treatment,
was significantly decreased when treated with CHP 125 ng/ml
together with TGF.beta.. Through this result that CHP ameliorated
fibrosis in L-132 cells, a lung cell line, it was confirmed that
CHP exhibited an excellent therapeutic effect on lung fibrosis.
Example 6
[0163] Confirmation of Antifibrotic Effect in an Animal Model of
Pulmonary Fibrosis According to CHP Administration
[0164] Pulmonary fibrosis is caused by prolonged high-fat diet
intake. This appears to be due to an increase in the level of
inflammation caused by high fat intake, and it has been reported
that pulmonary fibrosis appeared when normal mice were fed a
high-fat diet containing 60% fat for 15 weeks (Ge, X. N et al.,
Experimental Lung Research, 39(9), 365-378, 2016). Specifically,
long-term intake of high-fat diet induces obesity, and adipokines
and cytokines secreted by hypertrophic adipose tissue and immune
cells gathered here disrupt the migration of immune cells from bone
marrow to lungs and cause chronic inflammatory response (de Vries A
et al., Clin Exp Allergy, 39:731-739, 2009). In addition, IL-13
cytokine secreted by Th2 CD4+ T cells causes large amounts of
TGF.beta., a key fibrosis-inducing factor, to be produced, and
signal transduction by TGF.beta. promotes lung tissue
transformation and fibrosis (Fichtner-Feigl S et al., Nat Med,
12:99-106, 2006; Lee C G et al., J Exp Med, 194:809-21, 2001).
Pulmonary fibrosis can be confirmed through the expression level of
the TGF.beta. gene increased compared to normal (Ge, X. N et al.,
Experimental Lung Research, 39(9), 365-378, 2016).
[0165] 6-1. Design of Experimental Animals and Induction of
Pulmonary Fibrosis
[0166] For pulmonary fibrosis experiments in animals, an animal
model of pulmonary fibrosis was established in the same manner as
in Example 4-1.
[0167] 6-2. Confirmation of the Expression of Fibrosis Markers
TGF.beta. and Collagen 3 Genes in an Animal Model of Pulmonary
Fibrosis Administered with CHP
[0168] Isoflurane required for anesthesia and dissection of mice
was purchased from Hana Pharmaceutical, and Vetequip's RC2 Rodent
Circuit Controller Anesthesia System was prepared. Phosphate
buffered saline (PBS) was purchased from Hyclone. To isolate the
mouse lung tissue, the mice were anesthetized by respiratory
anesthesia with 3-3.5% isoflurane. After blood was drawn from the
heart of the anesthetized mice, the lung tissue was immediately
excised, 50 mg of the lung was cut out, and 500 .mu.L of NucleoZOL
was added thereto, followed by pulverization using a T10
homogenizer from IKA. Then, RNA was extracted according to
NucleoZOL's Total RNA Isolation protocol, and cDNA was synthesized
from 1 .mu.g of RNA by reverse transcription polymerase chain
reaction using the iScript cDNA synthesis kit. The synthesized cDNA
was analyzed by real-time PCR with iQ SYBR Green Supermix using
forward/reverse primer sets corresponding to each gene. The
expression value of each gene was corrected by dividing by the
expression value of GAPDH, a housekeeping gene. Primers for
real-time PCR were synthesized and used by Bioneer with the
sequence shown in Table 2.
TABLE-US-00002 TABLE 2 Gene SEQ ID SEQ ID name Forward primer NO:
Reverse primer NO: TGF.beta. 5'-cctgagtggctgtcttttga-3' 1
5'-aatcgaaagccctgtattccg-3' 2 Collagen 3 5'-agtcaaggagaaagtggtcg-3'
3 5'-ccagggaaacccatgacac-3' 4 GAPDH 5'-acactgagcaagagagaggc-3' 5
5'-tgggggtctgggatggaaat-3' 6
[0169] Statistical significance was analyzed using one-way ANOVA
statistical analysis, and the Dunnett post hoc test was used to
compare the significance with the control group (*p<0.05,
***p<0.001).
[0170] As a result of the experiment, as shown in FIG. 8, it was
confirmed that the expression of TGF.beta. and Collagen 3 genes,
which are major fibrosis markers shown in the lungs, decreased more
significantly according to the CHP administration concentration.
This means that CHP ameliorates pulmonary fibrosis, confirming an
excellent therapeutic effect in the treatment of pulmonary
fibrosis.
Example 7
[0171] Confirmation of Antifibrotic Effect in Human-Derived Skin
Cells According to CHP Treatment
[0172] 7-1. CHP Treatment after Culturing Human-Derived Skin Cells
and Inducing Fibrosis Thereof
[0173] DMEM medium and FBS were purchased from Hyclone for
culturing the HS68 skin fibroblast-derived cell line, and TGF.beta.
for inducing fibrosis was purchased from R&D Systems. The cells
were cultured in a DMEM medium supplemented with 10% FBS in a 5%
CO.sub.2 incubator at 37.degree. C., treated with TGF.beta. at a
concentration of 2 ng/ml and treated with CHP at 0, 62.5, 125, 250,
or 500 ng/ml, and cells were harvested at 48 hours of
treatment.
[0174] 7-2. Confirmation of Fibronectin Protein Expression after
CHP Treatment in a Human-Derived Skin Cell Fibrosis Model
[0175] Western blotting was performed in the same manner as in
Example 3-2 to analyze the expression of fibronectin protein, a
fibrotic protein. Statistical significance was analyzed using
Student's t-test statistical method with the control (#p<0.05
(negative control group: TGF.beta.-treated control group),
*p<0.05 (TGF.beta.-treated control group: CHP 500 ng/ml-treated
group)).
[0176] As a result of the experiment, as shown in FIG. 9a-9b, it
was confirmed that the expression level of fibronectin protein, a
fibrotic protein, which increases according to TGF.beta. treatment,
was significantly decreased when treated with CHP 500 ng/ml
together with TGF.beta.. Through this result that CHP ameliorated
fibrosis in HS68 cells, a skin cell line, it was confirmed that CHP
can be applied to the treatment of skin fibrosis.
Example 8
[0177] Confirmation of Antifibrotic Effect in an Animal Model of
Cardiac Fibrosis According to CHP Administration
[0178] Risk factors for heart fibrosis are well known, including
poor diet, obesity, and high cholesterol. When a high-fat diet is
consumed for a long period of time, hypervolemia due to obesity
occurs, overloading the heart, and causing deformation of the
contractile and diastolic functions of the heart. In addition,
symptoms such as hyperglycemia, insulin resistance, hyperlipidemia,
and chronic inflammation are known to play a decisive role in
cardiac fibrosis (Kaltman A J, Goldring R M., Am J Med, 60:645-653,
1976; Xia Y et al., Histochem Cell Biol, 131:471-481, 2009; Ulasova
E et al., J Mol Cell Cardiol., 50:147-156, 2011; Lo C S et al., J
Cell Biochem., 103:1999-2009, 2008; Cavalera, M et al.,
Translational Research, 164(4), 323-335, 2014). In particular, it
is known that the increase in cardiac mast cells and their
degranulation due to obesity provide fibrosis-inducing factors such
as TGF.beta. to the heart tissue and further promote cardiac
fibrosis (Kong P, Christia P, Frangogiannis N G, Cell Mol Life
Sci., 71:549-574, 2014).
[0179] In addition, there is a report that cardiac fibrosis occurs
when normal mice are fed a high-fat diet for a long period of time.
It has been reported that when normal mice are fed a high-fat diet
containing 60% fat for 14 to 22 weeks, cardiac fibrosis is induced,
and changes in the expression levels of TGF.beta. and Collagen
genes, which are increased compared to normal, can be confirmed
(Li, W et al., Nutrition & Metabolism, 14(1);68, 2017;
Ternacle, J et al., European Heart Journal, 18(11), 1283-1291,
2017; Zhao, Y et al., Biochimica et Biophysica Acta
(BBA)--Molecular Basis of Disease, 1863(8), 1991-2000, 2017; Xu,
Z., & Kong, X. Q., Biomedicine & Pharmacotherapy, 89,
991-1004, 2017).
[0180] 8-1. Design of Experimental Animals and Induction of Cardiac
Fibrosis
[0181] For cardiac fibrosis experiments in animals, an animal model
of cardiac fibrosis was established in the same manner as in
Example 4-1.
[0182] 8-2. Confirmation of the Expression of Fibrosis Markers
TGF.beta., Fibronectin, Collagen 1, Collagen 2, Collagen 3 Collagen
4 Genes in an Animal Model of Cardiac Fibrosis Administered with
CHP
[0183] Isoflurane required for anesthesia and dissection of mice
was purchased from Hana Pharmaceutical, and Vetequip's RC2 Rodent
Circuit Controller Anesthesia System was prepared. Phosphate
buffered saline (PBS) was purchased from Hyclone. To isolate the
mouse heart tissue, the mice were anesthetized by respiratory
anesthesia with 3-3.5% isoflurane. After blood was drawn from the
heart of the anesthetized mice, the heart tissue was immediately
excised, 50 mg of the heart was cut out, and RNA extraction and
gene expression analysis were performed in the same manner as in
Example 6-2. Primers for real-time PCR were synthesized and used by
Bioneer with the sequence shown in Table 3. The expression value of
each gene was corrected by dividing by the expression value of
(.beta.-actin, a housekeeping gene.
TABLE-US-00003 TABLE 3 Gene SEQ ID SEQ ID name Forward primer NO:
Reverse primer NO: Fibronectin 5'-ttggagaggagtgggagc-3' 7
5'-gaaatgaccactgccaaagc-3' 8 TGF.beta. 5'-cctgagtggctgtcttttga-3' 1
5'-aatcgaaagccctgtattccg-3' 2 Collagen 1 5'-ctaacgtggttcgtgaccg-3'
9 5'-atccatcggtcatgctctct-3' 10 Collagen 3
5'-agtcaaggagaaagtggtcg-3' 3 5'-ccagggaaacccatgacac-3' 4 Collagen 4
5'-cggtacacagtcagaccatt-3' 11 5'-catcacgaaggaatagccga-3' 12
.beta.-actin 5'-gggaaggtgacagcattg-3' 13
5'-atgaagtattaaggcggaagatt-3' 14
[0184] Statistical significance was analyzed using one-way ANOVA
statistical analysis, and the Dunnett post hoc test was used to
compare the significance with the HFD control group (*p<0.05,
**p<0.01, ****p<0.0001).
[0185] As a result of the experiment, as shown in FIG. 10, it was
confirmed that the expression of Fibronectin, TGF.beta., Collagen
1, 3, 4 genes, which are major fibrosis markers shown in the heart,
was significantly reduced by CHP administration. However, a further
decreased effect according to the concentration of CHP was not
confirmed, which can be considered because a sufficient effect has
already occurred with only a concentration of 5 mg/kg of CHP. These
results that CHP inhibited the progression of cardiac fibrosis and
ameliorated cardiac fibrosis suggest that CHP can be applied to the
treatment of cardiac fibrosis.
Example 9
[0186] Confirmation of Antifibrotic Effect in an Animal Model of
Fat Fibrosis According to CHP Administration
[0187] It is known that the best way to prevent fibrosis is to
block the inflammatory response. In particular, adipose tissue
secretes inflammatory substances, causing a vicious cycle of fat
fibrosis due to inflammation. It has been reported that fibrosis of
adipose tissue occurs when normal mice are fed a high-fat diet
containing 60% fat for 16 to 24 weeks (Hu, M et al., Evidence-Based
Complementary and Alternative Medicine, 1-12, 2018; Kwon, E. Y,
& Choi, M. S., Nutrients, 10(10), 1415, 2018; Nakazeki, F et
al., Scientific Reports, 8(1), 2018; Muniappan, L et al. Scientific
Reports, 7(1), 2017; Lancha, A et al., PLoS ONE, 9(5), e98398,
2014; Vel.alpha.zquez, K. T et al., Physiological Reports, 5(18),
e13412, 2017; Wang, L., Ye, X., Hua, Y, & Song, Y, Biomedicine
& Pharmacotherapy, 105, 121-129, 2018). When a high-fat diet is
consumed for a long period of time, adipocytes expand due to
excessive nutrition, causing triglyceride accumulation, adipocyte
death, production of adipokines and cytokines, endoplasmic
reticulum stress, adipose tissue hypoxia, etc., resulting in
infiltration of immune cells into adipose tissue and occurrence of
chronic inflammation (Schenk S et al., J Clin Invest,
118:2992-3002, 2008; Sun K, Kusminski C M, Scherer P E, J Clin
Invest, 121:2094-101, 2011). The interaction between various
inflammatory immune cells accumulated in adipose tissue and
macrophages-adipocytes produces TGF.beta., a key inducing factor
for fibrosis, which causes adipose tissue to be deformed, leading
to fat fibrosis (Lee C G et al., J Exp Med, 194:809-21, 2001;
Fichtner-Feigl S et al., Nat Med, 12:99-106, 2006; Pessin, J. E.,
& Kwon, H, Journal of Investigative Medicine, 60(8), 1147-1150,
2012). Fibrosis of adipose tissue can be confirmed through changes
in the expression of representative fibrosis gene markers that are
increased compared to normal adipose tissue.
[0188] 9-1. Design of Experimental Animals and Induction of Fat
Fibrosis
[0189] For fat fibrosis experiments in animals, an animal model of
fat fibrosis was established in the same manner as in Example
4-1.
[0190] 9-2. Confirmation of the Expression of Fibrosis Markers
TGF.beta., Fibronectin, Collagen 3 and CTGF Genes in an Animal
Model of Fat Fibrosis Administered with CHP
[0191] Isoflurane required for anesthesia and dissection of mice
was purchased from Hana Pharmaceutical, and Vetequip's RC2 Rodent
Circuit Controller Anesthesia System was prepared. Phosphate
buffered saline (PBS) was purchased from Hyclone. To isolate the
mouse adipose tissue, the mice were anesthetized by respiratory
anesthesia with 3-3.5% isoflurane. After blood was drawn from the
heart of the anesthetized mice, the epididymal adipose tissue (EAT)
was immediately excised, 100 mg of the fat was cut out, and RNA
extraction and gene expression analysis were performed in the same
manner as in Example 6-2. Primers for real-time PCR were
synthesized and used by Bioneer with the sequence shown in Table 4.
The expression value of each gene was corrected by dividing by the
expression value of GAPDH, a housekeeping gene.
TABLE-US-00004 TABLE 4 Gene SEQ ID SEQ ID name Forward primer NO:
Reverse primer NO: Fibronectin 5'-ttggagaggagtgggagc-3' 7
5'-gaaatgaccactgccaaagc-3' 8 TGF.beta. 5'-cctgagtggctgtcttttga-3' 1
5'-aatcgaaagccctgtattccg-3' 2 Collagen 3 5'-agtcaaggagaaagtggtcg-3'
3 5'-ccagggaaacccatgacac-3' 4 CTGF 5'-ctgtgcctgccattacaact-3' 15
5'-ccatgtctccgtacatcttcc-3' 16 GAPDH 5'-acactgagcaagagagaggc-3' 5
5'-tgggggtctgggatggaaat-3' 6
Statistical significance was analyzed using one-way ANOVA
statistical analysis, and the Dunnett post hoc test was used to
compare the significance with the control group (*p<0.05,
**p<0.01).
[0192] As a result of the experiment, as shown in FIG. 11, it was
confirmed that the expression of Fibronectin, TGF.beta., Collagen
3, CTGF genes, which are major fibrosis markers shown in the fat,
was markedly reduced by CHP administration. This means that CHP
ameliorates fat fibrosis, confirming an excellent therapeutic
effect in the treatment of fat fibrosis.
Sequence CWU 1
1
16120DNAArtificialTGFbeta forward primer 1cctgagtggc tgtcttttga
20221DNAArtificialTGFbeta reverse primer 2aatcgaaagc cctgtattcc g
21320DNAArtificialCollagen 3 forward primer 3agtcaaggag aaagtggtcg
20419DNAArtificialCollagen 3 reverse primer 4ccagggaaac ccatgacac
19520DNAArtificialGAPDH forward primer 5acactgagca agagagaggc
20620DNAArtificialGAPDH reverse primer 6tgggggtctg ggatggaaat
20718DNAArtificialFibronectin forward primer 7ttggagagga gtgggagc
18820DNAArtificialFibronectin reverse primer 8gaaatgacca ctgccaaagc
20919DNAArtificialCollagen 1 forward primer 9ctaacgtggt tcgtgaccg
191020DNAArtificialCollagen 1 reverse primer 10atccatcggt
catgctctct 201120DNAArtificialCollagen 4 forward primer
11cggtacacag tcagaccatt 201220DNAArtificialCollagen 4 reverse
primer 12catcacgaag gaatagccga 201318DNAArtificialBeta-actin
forward primer 13gggaaggtga cagcattg 181423DNAArtificialBeta-actin
reverse primer 14atgaagtatt aaggcggaag att 231520DNAArtificialCTGF
forward primer 15ctgtgcctgc cattacaact 201621DNAArtificialCTGF
reverse primer 16ccatgtctcc gtacatcttc c 21
* * * * *