U.S. patent application number 17/348279 was filed with the patent office on 2021-10-07 for composition for preventing or treating ischemic enteritis containing dna fragment mixture isolated from sperm or testis of fish.
This patent application is currently assigned to PharmaResearch Co., Ltd.. The applicant listed for this patent is PharmaResearch Co., Ltd.. Invention is credited to Seung Gul BAEK, Jung Won JEON, Chang Ju KIM, Ik Soo KIM.
Application Number | 20210308192 17/348279 |
Document ID | / |
Family ID | 1000005654801 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210308192 |
Kind Code |
A1 |
KIM; Ik Soo ; et
al. |
October 7, 2021 |
COMPOSITION FOR PREVENTING OR TREATING ISCHEMIC ENTERITIS
CONTAINING DNA FRAGMENT MIXTURE ISOLATED FROM SPERM OR TESTIS OF
FISH
Abstract
The present invention relates to a composition for preventing or
treating ischemic enteritis containing a DNA fragment mixture
isolated from sperm or testis of fish. The composition of the
present invention was verified to have excellent effects in the
prevention or treatment of ischemic enteritis. In addition, the
composition for preventing or treating ischemic enteritis of the
present invention was verified to be safe and have few side effects
even when administered for a long period of time. Therefore, a
medicine for ischemic enteritis, which is safe without side effects
and has an excellent treatment effect, is developed by using the
composition for preventing or treating ischemic enteritis of the
present invention, and thus the composition of the present
invention is expected to be a great help in the treatment of
ischemic enteritis.
Inventors: |
KIM; Ik Soo; (Seongnam-si,
KR) ; BAEK; Seung Gul; (Seongnam-si, KR) ;
KIM; Chang Ju; (Seoul, KR) ; JEON; Jung Won;
(Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PharmaResearch Co., Ltd. |
Gangneung-si |
|
KR |
|
|
Assignee: |
PharmaResearch Co., Ltd.
Gangneung-si
KR
|
Family ID: |
1000005654801 |
Appl. No.: |
17/348279 |
Filed: |
June 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15739646 |
Dec 22, 2017 |
11065281 |
|
|
PCT/KR2016/005528 |
May 25, 2016 |
|
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17348279 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/711 20130101;
A61K 9/08 20130101; A61K 9/0019 20130101; A61K 35/60 20130101; A61P
1/00 20180101; A61K 47/02 20130101 |
International
Class: |
A61K 35/60 20060101
A61K035/60; A61K 47/02 20060101 A61K047/02; A61K 9/08 20060101
A61K009/08; A61K 9/00 20060101 A61K009/00; A61P 1/00 20060101
A61P001/00; A61K 31/711 20060101 A61K031/711 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2015 |
KR |
10-2015-0090851 |
May 3, 2016 |
KR |
10-2016-0054666 |
Claims
1. A composition for treating ischemic colitis, the composition
comprising a DNA fragment mixture as an active ingredient and a
pharmaceutical excipient, wherein the DNA fragment mixture is
isolated from sperm or testis of fish.
2. The composition of claim 1, wherein the fish is Salmonidae
fish.
3. The composition of claim 1, wherein the DNA fragment mixture has
a molecular weight of 30-2,500 kDa.
4. The composition of claim 3, wherein the DNA fragment mixture has
a molecular weight of 40-2,000 kDa.
5. The composition of claim 4, wherein the DNA fragment mixture has
a molecular weight of 50-1,500 kDa.
6. The composition of claim 1, comprising the DNA fragment mixture
in an amount of 0.001-50 wt % to the total weight of the
composition.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional application of U.S.
application Ser. No. 15/739,646 filed on Dec. 22, 2017, which is a
35 U.S.C. 371 National Phase Entry Application from
PCT/KR2016/005528, filed May 25, 2016, which claims the benefit of
Korean Patent Application No. 10-2015-0090851 filed on Jun. 26,
2015; and Korean Patent Application No. 10-2016-0054666 filed on
May 3, 2016, the disclosures of which are herein incorporated by
reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a composition for
preventing or treating ischemic colitis, the composition containing
a DNA fragment mixture isolated from sperm or testis of fish.
BACKGROUND ART
[0003] Patients with digestive system diseases are on the rise due
to various types of stress and environmental factors resulting from
the development of the industrial society. Ischemia is a common
disease frequently occurring in the digestive system, and refers to
a state in which the supply of blood to body organs, tissues, or
sites is reduced due to the contraction or occlusion of blood
vessels. The reperfusion of blood occurs after ischemia and causes
various after effects such as nerve cell damage. Ischemia
ultimately results in irreversible damage, i.e., necrosis of cells
and tissues, and a disease such as ischemic colitis may occur.
[0004] Ischemic colitis (IC) is the most common form of vascular
disease invading the colon, and accounts for approximately 50-60%
of all cases of ischemic gastrointestinal disease (Suh D. C. et
al., 2007). Moderate ischemic colitis is characterized by
nonspecific symptoms, such as abdominal pain and diarrhea, which
are often improved before patients visit a hospital, and most
patients with main symptoms, such as hemorrhagic diarrhea and
abdominal pain, visit a hospital, but such symptoms are often
improved by conservative therapy.
[0005] The most causes of ischemic colitis are difficult to
identify, but ischemic colitis occurs mainly in the elderly when
the mesenteric artery is occluded by thrombosis or an embolism or
when the splanchnic artery is contracted by myocardial infarction
or heart failure. Ischemic colitis may be caused by mesenteric
venous thrombosis, drugs causing blood hyper-coagulation or
hypotension, or vasculitis (Reinus J. F. et al., 1981; Hunter G. C.
et al., 1988). Ischemic colitis is mainly classified into three
types in view of pathology. The first is the transient reversible
type in which ischemia occurs only in mucosa and submucosa and
recovery is possible through conservative treatment alone; the
second is the chronic type in which ischemia occurs in inner
circular muscles; and the third is the fulminant type in which
acute ischemia occurs in the anterior layer (Toursarkissian B et
al., 1997; Bower T. C., 1993; Deana D. G. et al., 1995). About half
of ischemic colitis cases are a transient reversible type which can
be spontaneously cured, so only conservative therapy is sufficient.
However, the other half may reach necrosis of the enteric
full-layer or intestinal perforation, and in these cases,
aggressive therapy including the consideration of surgery is needed
due to a high mortality rate (Green B. T. et al., 2005; Jung S. H.
et al., 2008).
[0006] The present inventors, while having been advancing studies
using a DNA fragment mixture extracted from sperm or testis of
fish, confirmed that the DNA fragment mixture inhibits cell
necrosis in ischemic colitis rat models, and therefore completed
the present invention.
[0007] Korean Patent Registration No. 0818752 as a prior art
discloses a composition containing, as an active ingredient, siRNA
inhibiting human FAF1 protein for treating ischemic disease, but
does not disclose a DNA fragment mixture. Korean Patent Publication
No. 2003-0001370 discloses that splanchnic ischemia can be treated
using a peptide having EGF activity and a growth hormone secretion
promoting hexapeptide, but is different from the present invention
with respect to the DNA fragment mixture.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0008] An aspect of the present invention is to provide a
composition for preventing or treating ischemic colitis, the
composition containing a DNA fragment mixture isolated from sperm
or testis of fish.
Technical Solution
[0009] In accordance with an aspect of the present invention, there
is provided a composition containing a DNA fragment mixture for
preventing or treating ischemic colitis.
[0010] The DNA fragment mixture may be isolated from sperm or
testis of fish.
[0011] The fish may be Salmonidae fish.
[0012] The DNA fragment mixture may have a molecular weight of
30-2,500 kDa.
[0013] Hereinafter, the present invention will be described in
detail.
[0014] The DNA fragment mixture refers to a DNA molecule
corresponding to a biological polymer composed of phosphate, four
types of bases, and deoxyribose, wherein the DNA fragment mixture
is present in a form in which fragments with a specific molecular
weight are mixed.
[0015] The fish may be Salmonidae fish, preferably salmon or trout,
and most preferably salmon.
[0016] The DNA fragment mixture may have a molecular weight of
30-2,500 kDa, preferably 40-2,000 kDa, and most preferably 50-1,500
kDa.
[0017] In addition, the present invention provides a pharmaceutical
composition for preventing or treating ischemic colitis, the
composition containing a DNA fragment mixture and a pharmaceutical
excipient. The DNA fragment mixture may be added in a content of
preferably 0.001-50 wt %, more preferably, 0.001-40 wt %, and most
preferably, 0.001-30 wt %, relative to the total weight of the
entire composition.
[0018] The pharmaceutical composition may be formulated into an
oral dosage form, an external applicable dosage form, a suppository
dosage form, and a sterile injection solution dosage form, such as
a powder, a granule, a tablet, a capsule, a suspension, an
emulsion, syrup, and an aerosol, by using conventional methods,
respectively. Examples of a carrier, an excipient, and a diluent
that may be contained in the pharmaceutical composition may be
lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl
hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,
and mineral oil. A preparation is formulated by using a diluent or
an excipient, such as a filler, an extender, a binder, a wetting
agent, a disintegrant, or a surfactant, which is conventionally
used.
[0019] The composition containing a DNA fragment mixture for
preventing or treating ischemic colitis according to the present
invention is preferably administered orally or through an
injection.
[0020] Examples of a solid preparation for oral administration
include a tablet, a pill, a powder, a granule, a capsule, and the
like, and these solid preparations are formulated by mixing the
extract or compound of the present invention with at least one
excipient, for example, starch, calcium carbonate, sucrose,
lactose, or gelatin. Also, in addition to a simple excipient, a
lubricant, such as magnesium stearate or talc, may be used.
Examples of a liquid preparation for oral administration may
include a suspension, a liquid for an internal use, an emulsion, a
syrup, and the like. In addition to commonly used simple diluents,
such as water and liquid paraffin, various excipients, such as a
wetting agent, a sweetener, a flavoring agent, a preservative, and
the like may be contained in the liquid preparation.
[0021] An injection may be formulated in a dosage form of a sterile
injection solution by a conventional method. Preferably, the
composition of the present invention may be used as a sterile
injection solution by being dissolved in water for injection
(Korean Pharmacopoeia).
[0022] The dosage of the composition for treatment of the present
invention may vary according to the age, sex, body weight of the
subject to be treated, the pathological condition, the severity of
the pathological condition, the administration route, and the
judgment of the prescriber.
[0023] The DNA fragment mixture may be administered in a
concentration of 2-25 mg/kg--preferably 2-16 mg/kg. If the dose
concentration is less than 2 mg/kg, treatment effects are difficult
to obtain, and if the dose concentration exceeds 25 mg/kg, the
increase in treatment effect relative to the dosage is not
large.
[0024] The DNA fragment mixture may be administered once a day or
divided into multiple doses.
[0025] The composition for treatment of the present invention may
be administered to mammals, such as mouse, livestock, and human,
through various routes to prevent or treat ischemic colitis. All
modes of administration may be expected, for example, oral, rectal,
intravenous, muscular, subcutaneous, intrauterine dural, or
intracerebral injection.
Advantageous Effects
[0026] The present invention relates to a composition for
preventing or treating ischemic colitis, the composition containing
a DNA fragment mixture isolated from sperm or testis of fish, and
the composition of the present invention was confirmed to have an
excellent prevention or treatment effect on ischemic colitis. In
addition, the composition for preventing or treating ischemic
colitis of the present invention was confirmed to be safe and have
few side effects even during long-term administration.
[0027] Therefore, the composition for preventing or treating
ischemic colitis of the present invention is used to develop
medicines for ischemic colitis, which are safe and have no side
effects and have excellent treatment effects. As a result, the
composition is expected to be helpful in treating ischemic
colitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the change of the temperature of the affected
part (body temperature) caused by the occurrence of ischemic
colitis.
[0029] FIG. 2 shows the results of naked eye observation of the
colonic tissue due to the administration of a DNA fragment mixture
in ischemic colitis-induced rats.
[0030] FIG. 3 shows H&E staining results of the colonic
histological change due to the administration of a DNA fragment
mixture in ischemic colitis-induced rats.
[0031] FIG. 4 shows Masson's trichrome staining results of clefts
of colonic mucosa due to the administration of a DNA fragment
mixture in ischemic colitis-induced rats.
[0032] FIG. 5 shows the protein expression of apoptotic factors due
to the administration of a DNA fragment mixture in ischemic
colitis-induced rats.
[0033] In each of the drawings, (A) a normal group without ischemic
colitis induction, (B) an ischemic colitis-induced group, (C) a DNA
fragment mixture administration (2 mg/kg) group, (D) a DNA fragment
mixture administration (4 mg/kg) group, (E) a DNA fragment mixture
administration (8 mg/kg) group, and (F) a DNA fragment mixture
administration (16 mg/kg) group
BEST MODE FOR CARRYING OUT THE INVENTION
[0034] Hereinafter, preferable embodiments of the present invention
will be described in detail. However, the present invention is not
limited to the embodiments described herein but may be embodied in
other forms. Rather, the embodiments disclosed herein are provided
so that these disclosures will be thorough and complete, and those
skilled in the art will fully understand the concept of the present
invention.
Example 1: Preparation of Animals
[0035] 7 weeks old white male Sprague Dawley (S/D) rats weighing
180.+-.5 g were used.
[0036] All rats were given free access to solid feed and water,
maintained at 22-24.degree. C. with a humidity of 60%, and housed
in a laboratory environment with 12-hour day and night cycles. The
rats were divided into experimental groups shown in table 1 below,
and ten rats were used per experimental group.
TABLE-US-00001 TABLE 1 Ischemic colitis DNA fragment mixture
induction administration (mg/kg) A X 0 B 0 C 2 D 4 E 8 F 16
Example 2: Establishment of Ischemic Colitis Animal Models
[0037] The rats reared in example 1 were anesthetized by
intraperitoneal injection of Zoletil 50.RTM.. The abdomen of the
anesthetized rats was open by abdominal midline incision, and then
the peripheral vessels in the 4 cm-range from the descending colon
were ligated. After the operation, the rats were subjected to
incision suture, followed by disinfection with 1% iodine solution,
and then the rats were recovered in a constant temperature and
humidity device.
Example 3: Drug Administration
[0038] The ischemic colitis animal models in example 2 were
administered with the DNA fragment mixture of the present invention
to investigate the treatment effect thereof on ischemic
colitis.
[0039] The DNA fragment mixture (molecular weight: 50-1500 kDa) was
administered from 48 hours after ischemic colitis-induced surgery.
The DNA fragment mixture was intraperitoneally injected at
concentrations of 2, 4, 8, and 16 mg/kg with 500 .mu.l each once a
day for a total of 21 days. Here, as control groups, a normal group
without ischemic colitis induction and a DNA fragment mixture
non-administration ischemic colitis model group were used.
Example 4: Measurement of Temperature of the Affected Part
[0040] On the basis of the fact that heat increases in an affected
part, the ischemic colitis induction was investigated using a laser
thermometer (MT6, Raytek Co., CA. US) a total of four times
including once before ischemic colitis induction and three times on
a weekly basis after ischemic colitis induction. The results are
shown in FIG. 1.
[0041] Referring to FIG. 1, the temperature of the affected part
(body temperature) after ischemic colitis induction increased in
all the experimental groups excluding the normal group (A), and at
the second check, the temperature of the affected part was reduced
in a manner dependent on the concentration of the DNA fragment
mixture. Especially, the DNA fragment mixture (16 mg/kg)
administration group (F) showed the largest reduction effect. It
was confirmed that the reduction in the temperature of the affected
part in the DNA fragment mixture administration groups was also
statistically significant at the measurement three weeks after
administration of the DNA fragment mixture.
Example 5: Enteroscopy
[0042] Each rat was anesthetized by intraperitoneal injection of 10
mg/kg of Zoletil 50.RTM. 21 days after administration of the DNA
fragment mixture, and then the abdomen was opened to expose the
ischemic colitis-induced enteric tissue, which was then
photographed at the same angle and distance. The results are shown
in FIG. 2.
[0043] As shown in FIG. 2, in the ischemic colitis-induced group
(B), the necrosis of the colon part was increased and bleeding was
observed outside the colon. Whereas in the DNA fragment mixture
administration groups (C, D, E, and F), the necrosis due to
ischemia was alleviated and thus the colon morphology similar to
that of the normal group (A) was maintained.
[0044] It can be seen from these results that the administration of
the DNA fragment mixture can inhibit or treat the ischemic
colitis-induced necrosis of the colon part.
Example 6: Histological Staining
Example 6-1: Tissue Treatment
[0045] Tissues were extracted from each rat photographed for
colonic tissue in example 5. Of these, some tissues were fixed for
24 hours in a 4% paraformaldehyde fixative dissolved in 100 mM
phosphate buffer. Thereafter, the tissues were infiltrated with
paraffin through a three-step procedure of dehydration through
sequential introduction into 70%, 80%, 90%, and 100% ethanol,
immersion in xylene, and then using a liquid for hard paraffin
(Leica, USA). The tissue after infiltration was made into blocks in
order to prepare sections, and the paraffin tissue was cut into 5
.mu.m thick sections using a paraffin microtome (Shandon Finesse
325, Thermo Electron Co., England). The tissue section was attached
on a coating slide, and left in a 37.degree. C. slide oven for
16-18 hours.
Example 6-2: Hematoxylin and Eosin (H&E) Staining
[0046] The tissue section prepared in example 6-1 was reacted with
Mayer's hematoxyline (Sigma, USA) for 30 seconds, and then washed
with flowing water for 10 minutes. Thereafter, the tissue section
was reacted with eosin (Sigma, USA) solution for 3 seconds. The
tissue on the completion of staining was dehydrated and sealed with
Permount.RTM. (Fischer Scientific, USA). The H&E stained tissue
section was observed using a microscope to check the tissue change,
and the results are shown in FIG. 3.
[0047] As shown in FIG. 3, the tissue damage including condensation
and clefts in the colonic mucosa was observed in the ischemic
colitis-induced group (B) compared with the normal group (A).
However, the ischemia-caused condensation and clefts in colonic
mucosa were observed to be reduced in the DNA fragment mixture
administration groups (C, D, E, F), and the reduction was dependent
on the concentration of the DNA fragment mixture administered.
Example 6-3: Masson's Trichrome Staining
[0048] In order to investigate clefts of colonic mucosa due to the
administration of a DNA fragment mixture after ischemic colitis
induction, the expression of the total collagen fiber was checked.
Therefore, Masson's trichrome staining was conducted.
[0049] The tissue embedded in paraffin in example 6-1 was immersed
in xylene to remove paraffin, hydrated, and treated with the
Bouin's solution (Sigma, USA) for 1 hour. Thereafter, the tissue
was washed with Weigner's iron hematoxylin solution (Sigma, USA)
for 10 minutes, and washed with Biebrich scarlet-acid fuchsin
solution (Sigma, USA) for 2 minutes. The washed tissue was treated
with Phosphomolybdic-Phosphotungstic acid solution (Sigma, USA) for
10-15 minutes, treated with aniline blue solution (Sigma, USA) for
5 minutes, treated with the light green solution for 1 minute,
treated with acetic solution for 5 minutes, treated with 95%
ethanol and xylene, and then sealed with Permount.RTM. (Fischer
Scientific, USA). The tissue stained with Masson's trichrome was
observed using a microscope, and the results are shown in FIG.
4.
[0050] As shown in FIG. 4, the clefts of colonic mucosa due to
ischemia were increased in the ischemic colitis induction group (B)
compared with the normal group (A), but the clefts of colonic
mucosa due to ischemia were reduced in a manner dependent on the
concentration of the DNA fragment mixture in the DNA fragment
mixture administration groups (C, D, E, and F).
Example 7: Investigation of Protein Expression of Apoptotic
Factors
[0051] The expression of apoptotic factors in association with cell
necrosis due to ischemic colitis was investigated.
[0052] The colonic tissue extracted from each rat photographed for
colonic tissue in example 5 was subjected to protection extraction
with a lysis buffer, and the resultant product was centrifuged to
obtain supernatant. Thereafter, the protein concentration was
quantified using the Bio-Rad protein quantifying reagent, thereby
obtaining 50 .mu.l of proteins.
[0053] Then, 50 .mu.l of the obtained proteins were subjected to
electrophoresis using 10% SDS-PAGE, and then proteins were
transferred to the nitrocellulose membrane. The protein-transferred
membrane was blocked with 5% skim milk, and then treated with a
primary antibody against Bax or Bcl-2 at 4.degree. C. for 16-18
hours. Here, GAPDH antibody was used as a quantitative control. The
membrane treated with the primary antibody was washed with TBST
solution (Tris-buffered saline with 0.05% tween 20) three times for
10 minutes for each time. A secondary antibody against each primary
antibody was added to the washed membrane, followed by reaction at
room temperature for 1 hour, and the membrane was again washed with
TBST solution three times for 15 minutes for each time. The washed
membrane was treated with the enhanced chemiluminescence (ECL)
solution to check protein bands. The results are shown in FIG.
5.
[0054] As shown in FIG. 5, the expression of apoptotic factor Bax
was increased in the ischemic colitis-induced group (B) compared
with the normal group (A). The expression of Bax was increased in
the DNA fragment mixture administration (2 mg/kg and 4 mg/kg)
groups, but the expression of Bax was reduced in a
concentration-dependent manner in the DNA fragment mixture
administration (8 mg/kg and 16 mg/kg) groups. Whereas, the
expression of anti-apoptotic factor Bcl-2 was increased in the DNA
fragment mixture administration groups (C, D, E, F) compared with
the ischemic colitis induction group (B).
[0055] It can be predicted from the above results that the DNA
fragment mixture can treat ischemic colitis by inhibiting cell
necrosis on the sites of occurrence of ischemic colitis.
* * * * *