U.S. patent application number 16/478421 was filed with the patent office on 2021-05-06 for diagnostic drug, diagnostic method and diagnostic device for permeability of intestinal mucosa.
The applicant listed for this patent is KOYO CHEMICAL CO., LTD., National University Corporation Shimane University. Invention is credited to Seiji KUROZUMI, Morihiko NAKAMURA, Yoshimori TAKAMORI, Haruki USUDA, Koichiro WADA.
Application Number | 20210132074 16/478421 |
Document ID | / |
Family ID | 1000005383708 |
Filed Date | 2021-05-06 |
![](/patent/app/20210132074/US20210132074A1-20210506\US20210132074A1-2021050)
United States Patent
Application |
20210132074 |
Kind Code |
A1 |
WADA; Koichiro ; et
al. |
May 6, 2021 |
DIAGNOSTIC DRUG, DIAGNOSTIC METHOD AND DIAGNOSTIC DEVICE FOR
PERMEABILITY OF INTESTINAL MUCOSA
Abstract
Provided is a diagnostic drug for evaluating permeability of
intestinal mucosa, including chitin and/or chitosan as a main
component. The chitin and/or chitosan to be used preferably has a
weight average molecular weight prepared to a range of from 1,000
to 11,600.
Inventors: |
WADA; Koichiro; (Shimane,
JP) ; USUDA; Haruki; (Shimane, JP) ; NAKAMURA;
Morihiko; (Shimane, JP) ; TAKAMORI; Yoshimori;
(Tottori, JP) ; KUROZUMI; Seiji; (Tottori,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National University Corporation Shimane University
KOYO CHEMICAL CO., LTD. |
Shimane
Osaka |
|
JP
JP |
|
|
Family ID: |
1000005383708 |
Appl. No.: |
16/478421 |
Filed: |
January 17, 2018 |
PCT Filed: |
January 17, 2018 |
PCT NO: |
PCT/JP2018/001266 |
371 Date: |
July 16, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0008 20130101;
A61K 9/0031 20130101; A61K 9/0053 20130101; G01N 33/66 20130101;
G01N 2800/06 20130101; G01N 33/5088 20130101 |
International
Class: |
G01N 33/66 20060101
G01N033/66; A61K 49/00 20060101 A61K049/00; A61K 9/00 20060101
A61K009/00; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 19, 2017 |
JP |
2017-007130 |
Claims
1. A diagnostic drug for evaluating permeability of intestinal
mucosa, comprising chitin and/or chitosan as a main component.
2. A diagnostic drug for evaluating permeability of intestinal
mucosa, comprising chitin and/or chitosan as a main component, the
diagnostic drug being used by orally administering or enema
administering the diagnostic drug to a test subject and measuring a
blood concentration thereof after a lapse of a predetermined period
of time.
3. A diagnostic drug according to claim 1 or 2, wherein the chitin
and/or chitosan has a weight average molecular weight prepared to a
range of from 1,000 to 11,600.
4. A diagnostic drug according to claim 2, wherein a dose of the
diagnostic drug is set to a range of from 8.33 mg to 20.83 mg per
kg of body weight.
5. A diagnostic method, comprising: orally administering or enema
administering chitin and/or chitosan to an animal, the animal being
other than a human; and measuring a concentration of the
administered substance in blood after a lapse of a predetermined
period of time, to thereby evaluate permeability of intestinal
mucosa of the animal.
6. A diagnostic method, comprising: orally administering or enema
administering chitin and/or chitosan to a test subject; and
measuring a concentration of the administered substance in blood
after a lapse of a predetermined period of time, to thereby
evaluate permeability of intestinal mucosa of the test subject.
7. A diagnostic method according to claim 5 or 6, wherein the
chitin and/or chitosan has a weight average molecular weight
prepared to a range of from 1,000 to 11,600.
8. A diagnostic method according to claim 6, wherein a dose of the
chitin and/or chitosan is set to a range of from 8.33 mg to 20.83
mg per kg of body weight.
9. A use of chitin and/or chitosan, for evaluation of permeability
of intestinal mucosa through oral administration and blood
concentration measurement after a lapse of a predetermined period
of time, or through enema administration and blood concentration
measurement after a lapse of a predetermined period of time.
10. A food and drink evaluation method, comprising: allowing a test
subject to eat and drink a single or a plurality of specific foods
and drinks; allowing the test subject to orally ingest chitin
and/or chitosan during the eating and drinking, or before or after
the eating and drinking; and measuring a concentration of the
ingested substance in blood after a lapse of a predetermined period
of time from the oral ingestion, to thereby determine whether the
foods and drinks have a potential to serve as a factor affecting
permeability of intestinal mucosa of the test subject.
11. A food and drink evaluation drug, comprising chitin and/or
chitosan as a main component, the food and drink evaluation drug
being used by: allowing a test subject to eat and drink a single or
a plurality of specific foods and drinks; allowing the test subject
to orally ingest chitin and/or chitosan during the eating and
drinking, or before or after the eating and drinking; and measuring
a concentration of the ingested substance in blood after a lapse of
a predetermined period of time from the oral ingestion, to thereby
determine whether the foods and drinks have a potential to serve as
a factor affecting permeability of intestinal mucosa of the test
subject.
12. An evaluation method, comprising: administering a given
substance; separately orally administering or enema administering
chitin and/or chitosan; and measuring blood concentrations of the
orally administered substance or the enema administered substance
before and after the administration of the given substance, to
thereby evaluate whether the given substance has a normalizing
action on permeability of intestinal mucosa, and how strong the
normalizing action, when present, is.
13. An evaluation agent, comprising chitin and/or chitosan as a
main component, to be orally administered or enema administered
separately from a given substance to be administered, the
evaluation agent being used for evaluating whether the given
substance has a normalizing action on permeability of intestinal
mucosa, and how strong the normalizing action, when present, is,
through measurement of blood concentrations of the evaluation agent
before and after the administration of the given substance.
14. A diagnostic device, comprising: concentration-measuring means
for measuring a concentration of chitin and/or chitosan in blood
collected from a test subject; and evaluation means for evaluating
permeability of intestinal mucosa of the test subject on the basis
of the concentration measured by the concentration-measuring means.
Description
TECHNICAL FIELD
[0001] The present invention relates to a technology for evaluating
permeability of intestinal mucosa, and more particularly, to a
technology for evaluating a degree of leaky gut syndrome and a
technology for identifying or evaluating an inducer or inhibitor
thereof.
BACKGROUND ART
[0002] There are various diseases based on permeability of
intestinal mucosa, and for example, leaky gut syndrome (hereinafter
referred to simply as "LGS" as appropriate) is known. LGS refers to
a condition in which a food molecule, foreign matter, or the like
penetrates into blood from the intestinal mucosa, and it is
considered that this leads to occurrence of diarrhea or occurrence
of allergic symptoms.
[0003] As a method of determining whether or not LGS is present,
there is known a lactulose-mannitol test.
[0004] This method involves allowing a test subject to
simultaneously take 10 g of lactulose (molecular
weight.apprxeq.340) and 5 g of mannitol (molecular
weight.apprxeq.180), and measuring a ratio between concentrations
of lactulose and mannitol (L/M ratio) in urine, to thereby evaluate
damage to an intestinal tract, i.e., a degree of leakage. The test
utilizes the fact that, when the test subject is healthy, mannitol
passes through the intestinal mucosa while lactulose hardly passes
through the intestinal mucosa from the viewpoint of a molecular
weight. A liquid chromatograph-mass spectrometer or the like is
used for the measurement.
[0005] In addition, as an animal experiment, there is also known an
FITC-dextran test.
[0006] This test involves orally administering FITC-dextran
(fluorescence-labeled dextran) having an average molecular weight
of about 4,000, and measuring its blood concentration. A
fluorometer or the like is used for the measurement.
[0007] However, hitherto, the following problems have been
known.
[0008] First, there has been a problem in that LGS does not have a
sufficient definition that is definite, and a detection method or
diagnostic method therefor has not been established. Conversely,
this may be expressed as follows: because the detection method or
diagnostic method has not been established, the definition is not
definite.
[0009] In actuality, even in the lactulose-mannitol test, because
urine is used, accurate measurement cannot be performed when a
renal disorder is present. In addition, sugars, such as lactulose
and mannitol, are absorbed through the intestinal mucosa by means
of a special transporter, and hence there is also a possibility of
a transport disorder due to the transporter itself.
[0010] In addition, there has also been a problem in that lactulose
has a relatively small molecular weight, and hence it cannot be
evaluated whether the test subject is in a state in which a
substance having a larger molecular weight is also absorbed by an
intestine (migrates to blood).
[0011] Meanwhile, the FITC-dextran has a somewhat large molecular
weight, but has had a problem in that FITC has toxicity, preventing
its use for a human.
[0012] Further, there are many reports that blood of an
enteritis-affected individual or an individual who excessively
ingests a high-fat diet contains a lipopolysaccharide having an
average molecular weight of from 5,000 to 8,000. There has also
been a problem in that a technology for evaluating leakiness of a
substance having such large molecular weight does not exist in the
first place.
[0013] That is, hitherto, there have been problems in that
evaluation of the permeability of the intestinal mucosa typified by
LGS is indirect, does not have high reliability, and has a narrow
evaluation range.
CITATION LIST
Patent Literature
[0014] [PTL 1] JP 2016-151559 A
SUMMARY OF INVENTION
Technical Problem
[0015] The present invention has been made in view of the
foregoing, and an object of the present invention is to provide a
technology capable of directly evaluating the degree of
permeability of intestinal mucosa with high reliability. As an
example, the object is to provide a technology for diagnosing an
increase in intestinal mucosal permeability, such as LGS.
[0016] Another object of the present invention is to provide a
technology for determining a food and drink that affects
permeability of intestinal mucosa. As an example, the object is to
provide a technology for determining a food and drink that induces
LGS or a food and drink that inhibits LGS.
[0017] Still another object of the present invention is to provide
a technology for giving an objective evaluation of a pharmaceutical
for normalizing permeability of intestinal mucosa or a candidate
substance therefor. As an example, the object is to provide a
technology for promoting the development of an LGS therapeutic
drug, an LGS alleviating drug, and an intestinal mucosal
permeability modulatory drug.
Solution to Problem
[0018] The invention according to the first aspect is directed to a
diagnostic drug for evaluating permeability of intestinal mucosa,
including chitin and/or chitosan as a main component.
[0019] The diagnostic drug may be used by oral administration or
may be used by enema administration. A subject to be diagnosed may
be other than a human.
[0020] The invention according to the second aspect is directed to
a diagnostic drug for evaluating permeability of intestinal mucosa,
including chitin and/or chitosan as a main component, the
diagnostic drug being used by orally administering or enema
administering the diagnostic drug to a test subject and measuring a
blood concentration thereof after a lapse of a predetermined period
of time.
[0021] The predetermined period of time only needs to be set as
appropriate, and may be set to 30 minutes in the case of the oral
administration and 5 minutes in the case of the enema
administration.
[0022] The invention according to the third aspect is directed to a
diagnostic drug according to the first or the second aspect of the
present invention, wherein the chitin and/or chitosan has a weight
average molecular weight prepared to a range of from 1,000 to
11,600.
[0023] Having a weight average molecular weight prepared to from
1,000 to 11,600 may be any of: (1) having an average molecular
weight at any one value of from 1,000 to 11,600; (2) having a
plurality of average molecular weight peaks between 1,000 and
11,600; and (3) containing molecules having molecular weights of
from 1,000 to 11,600 in an essentially uniform manner. In the case
of (1), whether a substance having that molecular weight permeates
the intestinal mucosa can be confirmed. In the case of (2),
approximately how large the molecular weight of a substance that
permeates the intestinal mucosa is can be accurately confirmed by a
single test using an agent having a small half-width of each peak.
Also in the case of (3), approximately how large the molecular
weight of a substance that permeates the intestinal mucosa is can
be confirmed by a single test.
[0024] The invention according to the fourth aspect is directed to
a diagnostic drug according to the second aspect of the present
invention, wherein a dose of the diagnostic drug is set to a range
of from 8.33 mg to 20.83 mg per kg of body weight.
[0025] The intake is smaller than that in a lactulose-mannitol
test, and hence a burden on the test subject can be reduced.
[0026] The invention according to the fifth aspect is directed to a
diagnostic method, including: orally administering or enema
administering chitin and/or chitosan to an animal, the animal being
other than a human; and measuring a concentration of the
administered substance in blood after a lapse of a predetermined
period of time, to thereby evaluate permeability of intestinal
mucosa of the animal.
[0027] The concentration of the administered substance means: a
chitin concentration when only chitin is administered; a chitosan
concentration when only chitosan is administered; and the
concentration of a mixture of chitin and chitosan when the mixture
of chitin and chitosan is administered.
[0028] The invention according to the sixth aspect is directed to a
diagnostic method, including: orally administering or enema
administering chitin and/or chitosan to a test subject; and
measuring a concentration of the administered substance in blood
after a lapse of a predetermined period of time, to thereby
evaluate permeability of intestinal mucosa of the test subject.
[0029] The invention according to the seventh aspect is directed to
a diagnostic method according to the 5th or the 6th aspect of the
present invention, wherein the chitin and/or chitosan has a weight
average molecular weight prepared to a range of from 1,000 to
11,600.
[0030] The invention according to the eighth aspect is directed to
a diagnostic method according to the 6th aspect of the present
invention, wherein an oral dose of the chitin and/or chitosan is
set to a range of from 8.33 mg to 20.83 mg per kg of body
weight.
[0031] The invention according to the ninth is directed to a use of
chitin and/or chitosan, for evaluation of permeability of
intestinal mucosa through oral administration and blood
concentration measurement after a lapse of a predetermined period
of time, or through enema administration and blood concentration
measurement after a lapse of a predetermined period of time.
[0032] The chitin and/or chitosan preferably has a weight average
molecular weight prepared to a range of from 1,000 to 11,600.
[0033] The dose is preferably set to a range of from 8.33 mg to
20.83 mg per kg of body weight in the case of a human.
[0034] The invention according to the 10th aspect is directed to a
food and drink evaluation method, including: allowing a test
subject to eat and drink a single or a plurality of specific foods
and drinks; allowing the test subject to orally ingest chitin
and/or chitosan during the eating and drinking, or before or after
the eating and drinking; and measuring a concentration of the
ingested substance in blood after a lapse of a predetermined period
of time from the oral ingestion, to thereby determine whether the
foods and drinks have a potential to serve as a factor affecting
permeability of intestinal mucosa of the test subject.
[0035] The chitin and/or chitosan preferably has a weight average
molecular weight prepared to a range of from 1,000 to 11,600.
[0036] The intake is preferably set to a range of from 8.33 mg to
20.83 mg per kg of body weight.
[0037] The term "affect" includes both increasing and reducing the
permeability or leakiness of the intestinal mucosa. With regard to
LGS, increasing the leakiness means inducing or aggravating LGS,
and reducing the leakiness means inhibiting, alleviating,
ameliorating, or curing LGS.
[0038] From the viewpoint of the inhibition of LGS, examples of the
specific single food and drink may include yogurt and a whey
beverage. Along with this, an objective performance index for a
food touted as a conditioner for gut flora can also be
provided.
[0039] From the viewpoint of the induction of LGS, examples of the
plurality of specific foods and drinks may include: an oyster and
wine; and a pork steak and beer.
[0040] According to the present invention, screening of an inducer
or an inhibitor can be performed for an individual.
[0041] The term "before or after the eating and drinking" may mean,
for example, a time point 20 minutes before the start of the eating
and drinking or a time point 15 minutes after the end of the eating
and drinking.
[0042] The invention according to the 11th is directed to a food
and drink evaluation drug, including chitin and/or chitosan as a
main component, the food and drink evaluation drug being used by:
allowing a test subject to eat and drink a single or a plurality of
specific foods and drinks; allowing the test subject to orally
ingest chitin and/or chitosan during the eating and drinking, or
before or after the eating and drinking; and measuring a
concentration of the ingested substance in blood after a lapse of a
predetermined period of time from the oral ingestion, to thereby
determine whether the foods and drinks have a potential to serve as
a factor affecting permeability of intestinal mucosa of the test
subject.
[0043] The chitin and/or chitosan preferably has a weight average
molecular weight prepared to a range of from 1,000 to 11,600.
[0044] The intake is preferably set to a range of from 8.33 mg to
20.83 mg per kg of body weight.
[0045] The invention according to the 12th aspect is directed to an
evaluation method, including: administering a given substance;
separately orally administering or enema administering chitin
and/or chitosan; and measuring blood concentrations of the orally
administered substance or the enema administered substance before
and after the administration of the given substance, to thereby
evaluate whether the given substance has a normalizing action on
permeability of intestinal mucosa, and how strong the normalizing
action, when present, is.
[0046] A subject to which the given substance is administered may
be a human or may be an animal other than a human.
[0047] The chitin and/or chitosan preferably has a weight average
molecular weight prepared to a range of from 1,000 to 11,600.
[0048] An oral dose or enema dose is preferably set to a range of
from 8.33 mg to 20.83 mg per kg of body weight in the case of a
human.
[0049] The invention according to the 13th aspect is directed to an
evaluation agent, including chitin and/or chitosan as a main
component, to be orally administered or enema administered
separately from a given substance to be administered, the
evaluation agent being used for evaluating whether the given
substance has a normalizing action on permeability of intestinal
mucosa, and how strong the normalizing action, when present, is,
through measurement of blood concentrations of the evaluation agent
before and after the administration of the given substance.
[0050] A subject to which the given substance is administered may
be a human or may be an animal other than a human.
[0051] The chitin and/or chitosan preferably has a weight average
molecular weight prepared to a range of from 1,000 to 11,600.
[0052] The dose is preferably set to a range of from 8.33 mg to
20.83 mg per kg of body weight in the case of a human.
[0053] The invention according to the 14th aspect is directed to a
diagnostic device, including: concentration-measuring means for
measuring a concentration of chitin and/or chitosan in blood
collected from a test subject; and evaluation means for evaluating
permeability of intestinal mucosa of the test subject on the basis
of the concentration measured by the concentration-measuring
means.
[0054] The diagnostic device may also be called an intestinal
mucosal permeability evaluation device.
Advantageous Effects of Invention
[0055] According to the present invention, the degree of
permeability of the intestinal mucosa can be directly evaluated
with high reliability.
[0056] In addition, according to the present invention, a food and
drink that affects the permeability of the intestinal mucosa can be
determined.
[0057] In addition, according to the present invention, an
objective evaluation of a pharmaceutical for normalizing the
permeability of the intestinal mucosa or a candidate substance
therefor can be performed.
BRIEF DESCRIPTION OF DRAWINGS
[0058] FIG. 1 are test outlines of an AO model and an IR model.
[0059] FIG. 2 are photographs for showing a normal state of an
intestinal tract and an LGS-induced state thereof.
[0060] FIG. 3 is a graph for showing the results of a
lactulose-mannitol test for the AO model.
[0061] FIG. 4 are graphs for showing the results of an FITC-dextran
test for the AO model.
[0062] FIG. 5 is a graph for showing the results of an FITC-dextran
test for the IR model.
[0063] FIG. 6 are graphs for showing the measurement results of
chitin-chitosan concentrations for the AO model and the IR
model.
[0064] FIG. 7 is a graph for showing the manner of distribution of
samples containing chitin-chitosan having weight average molecular
weights of 1,000, 3,000, and 11,600 as main components.
[0065] FIG. 8 is a graph for showing the blood concentrations of
chitin-chitosan at various molecular weights for the IR(20)
model.
[0066] FIG. 9 is a graph for showing the blood concentrations of
chitin-chitosan at various molecular weights for the IR (10)
model.
[0067] FIG. 10 is a graph for showing results for the IR(20) model
obtained by orally administering a diagnostic agent having a weight
average molecular weight of 1,000 at each of 1.25 mg/mouse and
0.625 mg/mouse, and measuring its blood concentration.
[0068] FIG. 11 are schematic diagrams of distribution modes of the
molecular weight of a diagnostic drug and concentration measurement
results.
[0069] FIG. 12 is a graph for showing the manner of molecular
weight distribution of a purified sample.
[0070] FIG. 13 are a graph for showing the measurement results of a
chitin-chitosan concentration using the purified sample, and the
external appearance of an intestinal tract and an HE-stained image,
for the IR (20) model.
[0071] FIG. 14 is a graph of the measurement of a temporal change
in chitin-chitosan amount in circulating blood in the IR (10) model
in which the purified sample was orally administered to mice.
[0072] FIG. 15 are graphs of the measurement of a temporal change
in blood concentration of chitin-chitosan in the case where the
purified sample was intravenously administered to mice not
subjected to ischemia-reperfusion treatment.
[0073] FIG. 16 is an explanatory diagram for illustrating a
protocol for inducing OVA allergy.
[0074] FIG. 17 is a graph for showing the blood concentration of
chitin-chitosan using OVA-IgE mice.
[0075] FIG. 18 are graphs for showing the blood concentrations of
chitin-chitosan for a high-fat diet model and a NASH-inducing diet
model.
[0076] FIG. 19 are photographs for showing a whole intestine image
and HE-stained image before onset in a DSS-induced ulcerative
colitis model. Comparative images without the administration of DSS
are also shown.
[0077] FIG. 20 is a graph for showing the blood concentrations of
chitin-chitosan before onset in the DSS-induced ulcerative colitis
model.
[0078] FIG. 21 is a conceptual diagram for illustrating a time lag
between an increase in permeability of intestinal mucosa and the
occurrence of inflammation or disorder.
DESCRIPTION OF EMBODIMENTS
[0079] <Construction of LGS-Induced Models>
[0080] In this embodiment, LGS was assumed as an example in which
the leakiness of an intestinal tract was evaluated, and first,
induction tests therefor were performed.
[0081] One model uses aspirin and omeprazole (hereinafter referred
to as "AO model" as appropriate). Conditions were modified on the
basis of the literature (Innate Immun. 2015 July;
21(5):537-45).
[0082] An outline of the test is as follows: per kg of body weight
of mice, 100 mg of aspirin (100 mg/kg) is orally administered twice
a day for 6 days and 10 mg of omeprazole (10 mg/kg) is
intraperitoneally administered twice a day for 6 days, and the
degree of LGS is measured on the 7th day.
[0083] Another model is an intestinal tract ischemia-reperfusion
model (hereinafter referred to as "IR model" as appropriate).
Conditions were modified on the basis of the literature
(Gastroenterology. 2001 February; 120 (2):460-9.) and the like.
[0084] An outline of the test is as follows: an intestinal tract is
clipped continuously for 30 minutes to be brought into an ischemia
state, and then unclipped to achieve reperfusion, and 30 minutes
later, the degree of LGS is measured. As an expression specifying
an ischemia time of 30 minutes, this model is referred to as "IR
(30) model" as appropriate.
[0085] Outlines of both models are illustrated in FIG. 1. In FIG.
1b, an IR(20) model and an IR(10) model, which are described later,
are also illustrated.
[0086] In FIGS. 2, external appearance photographs, HE-stained
images, and electron micrographs of the intestinal tract are shown.
As compared to a normal tissue, in the AO model, cracks are found
in intestinal villi, and hence the occurrence of mild LGS is
recognized. In addition, in the IR (30) model, intestinal villi are
significantly damaged, and hence the occurrence of severe LGS is
recognized.
[0087] <Evaluation of Degree of LGS>
[0088] The above-mentioned models were used to determine leakiness
based on a difference in molecular weight.
[0089] First, a lactulose-mannitol test was performed for the AO
model. Lactulose and mannitol were both orally administered at 500
mg/kg, urine was collected for 4 hours, and an L/M ratio was
measured. The results are shown in FIG. 3. As compared to mice
administered with none of aspirin and omeprazole and not having LGS
induced, i.e., a control, the AO model had an about 2-fold increase
in L/M ratio. It was able to be confirmed from the foregoing that a
state in which the leakiness of a substance having a molecular
weight of more than 300, such as mannitol, was raised was
found.
[0090] Next, an FITC-dextran test was performed for the AO model.
FITC-dextran was orally administered at 600 mg/kg, blood was
collected 60 minutes later, and the concentration in plasma thereof
was measured. The results are shown in FIG. 4. It was able to be
confirmed that the concentration of dextran in the plasma was
increased to some degree as compared to the control (FIG. 4b).
[0091] In addition, an FITC-dextran test was performed for the IR
(30) model. FITC-dextran was orally administered at 600 mg/kg,
ischemia was started 30 minutes later, blood was collected 30
minutes after reperfusion, and the concentration in plasma thereof
was measured. The results are shown in FIG. 5. The dextran
concentration in plasma had an increase as high as about 15-fold as
compared to the control.
[0092] As apparent from a comparison between FIG. 4a and FIG. 5,
the IR (30) model may be said to be a model capable of inspecting a
state in which a substance having a large molecular weight is
allowed to migrate to blood more easily, as compared to the AO
model. In other words, the IR (30) model may be said to be a model
capable of evaluating severe LGS.
[0093] On the other hand, on the basis of a comparison between FIG.
3 and FIG. 4b, the AO model may be said to be a model capable of
inspecting a state that is not so severe as to allow a substance
having a large molecular weight to easily migrate to blood. In
other words, the AO model may be said to be a model capable of
evaluating mild LGS.
[0094] <Evaluation of LGS using Chitin-Chitosan>
[0095] Next, evaluation of LGS using chitin-chitosan was
performed.
[0096] First, the shell of a crab was deproteinized, decalcified,
and deacetylated to provide a mixture of chitin and chitosan.
Subsequently, the mixture of chitin and chitosan was decomposed
into small molecules to provide a chitin-chitosan sample having a
weight average molecular weight of 7,900. As a method for the
decomposition into small molecules, which is not particularly
limited, there are given a method involving hydrolyzing the mixture
of chitin and chitosan with concentrated hydrochloric acid (JP
5714963 B2), and a method involving dissolving the mixture with
hydrochloric acid or an organic acid, such as acetic acid, citric
acid, or lactic acid, and then decomposing the mixture into small
molecules using a chitosanase enzyme (JP 2013-79217 A).
[0097] Next, for the AO model, 2.50 mg of the above-mentioned
sample was orally administered on the 7th day, blood was collected
60 minutes later, and the concentration of chitin-chitosan in
plasma was measured (see FIG. 1a).
[0098] In addition, for the IR(30) model, 2.50 mg of the
above-mentioned sample was orally administered 30 minutes before
the start of ischemia, blood was collected 30 minutes after
reperfusion, and the concentration of chitin-chitosan in plasma was
measured (see FIG. 1b).
[0099] For the measurement, first, the plasma was extracted from
the blood by a conventional method, and chitin-chitosan in the
plasma was decomposed into chitose (2,5-anhydro-D-mannose) serving
as a constituent monosaccharide by a nitrous acid decomposition
method. Next, its aldehyde group was allowed to react with
3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and
iron (III) chloride to develop a blue color. Finally, the degree of
the blue color was measured with an absorbance meter, and the
concentration was calculated on the basis of the dye amount.
[0100] The results are shown in FIG. 6. As shown in FIG. 6, it can
be confirmed that chitin-chitosan is allowed to migrate into the
blood more remarkably in the IR (30) model than in the AO model.
This means a larger molecular weight of the sample used, and agrees
with the relationship between FIG. 4b and FIG. 5.
[0101] In view of the foregoing, the inventors of the present
invention have decided to investigate the applicability of the
chitin-chitosan as a diagnostic drug for the degree of LGS, and by
extension, a diagnostic drug for diagnosing permeability of
intestinal mucosa (evaluation drug for evaluating permeability of
intestinal mucosa). The concentration evaluation has been performed
with the absorbance in the foregoing, but is not limited thereto,
and chitin and chitosan do not particularly need to be
distinguished from each other as long as the blood concentration
can be detected. Therefore, in the present invention, the
expression "chitin-chitosan" is used, and means chitin and/or
chitosan.
[0102] First, a solution of hydrochloric acid or an organic acid
was prepared, and the chitin-chitosan sample having a weight
average molecular weight of 7, 900 was fractionated using UF
membranes (having molecular weight cut-offs of 3,000, 6,000,
10,000, and the like) into chitin-chitosan having weight average
molecular weights of 1,000, 3,000, 7,900, and 11,600. In FIG. 7,
the manner of distribution of the prepared samples is shown. In
addition, a measurement method for the weight average molecular
weight is also described.
[0103] Next, for the IR model, 2.5 mg each of the above-mentioned
samples was orally administered, and the blood concentration of the
chitin-chitosan was measured. It was considered that an ischemia
time of 30 minutes caused excessively large damage to an intestinal
tract, and hence, in this case, a test was performed with the
ischemia time shortened to 20 minutes. This model is referred to as
"IR(20) model".
[0104] For the IR(20) model, the measurement results of the blood
concentrations of the chitin-chitosan at various molecular weights
are shown in FIG. 8. In FIG. 8, the results of a control, i.e.,
without ischemia-reperfusion are also shown. As apparent from FIG.
8, although the concentration at a weight average molecular weight
of 11,600 is slightly small, in general, the sample having any
molecular weight has migrated into blood.
[0105] In view of the foregoing, the ischemia time was changed to
10 minutes to further reduce the degree of LGS, inducing
medium-degree LGS, and sample-screening performance was
investigated. This test is referred to as "IR(10) model".
[0106] For the IR(10) model, the measurement results of the blood
concentrations of the chitin-chitosan at various molecular weights
are shown in FIG. 9. As can be seen in FIG. 9, it can be confirmed
that a sample having a smaller weight average molecular weight is
more liable to leak, and a sample having a larger weight average
molecular weight is less liable to leak.
[0107] As apparent from the above-mentioned experiments, through
the use of the chitin-chitosan, evaluation can be performed
regarding the following: how large the molecular weight of a
substance that leaks through the intestinal tract is; and a state
in which a substance having how large a molecular weight leaks
through the intestinal tract to what degree is found.
[0108] That is, the chitin and/or chitosan can be used for
evaluation of permeability of intestinal mucosa through oral
administration and blood concentration measurement after a lapse of
a predetermined period of time.
[0109] In other words, it may be said that a diagnostic drug for
evaluating permeability of intestinal mucosa, containing chitin
and/or chitosan as a main component, was able to be obtained.
[0110] In addition, it may also be said that a diagnostic drug for
evaluating permeability of intestinal mucosa, containing chitin
and/or chitosan as a main component, the diagnostic drug being used
by allowing a test subject to orally ingest the diagnostic drug and
measuring a blood concentration thereof after a lapse of a
predetermined period of time, was able to be obtained.
[0111] It may also be said that an evaluation technology for an
increase in intestinal mucosal permeability was able to be
obtained.
[0112] As shown in FIG. 8 and FIG. 9, the IR model can induce LGS
of any degree from mild to severe through the adjustment of the
ischemia time, and may be said to be a model capable of
constructing a versatile and objective evaluation system.
[0113] For the IR (20) model, a diagnostic drug having a weight
average molecular weight of 1,000 was orally administered at each
of 1.25 mg/mouse and 0.625 mg/mouse, and a blood concentration in
the case where the dose was reduced was measured. The results are
shown in FIG. 10. As apparent from FIG. 10, it was found that
significant concentration measurement was possible even at 0.625
mg/mouse. When this value is converted for a human having a body
weight of 60 kg, the dose of the diagnostic drug is 1.25 g. In
addition, in terms of measurement limit taking also a control into
consideration, significant concentration measurement is in theory
possible even at 0.500 g of oral ingestion for a 60 kg human. That
is, it is appropriate that the dose or the intake be set to a range
of from 8.33 mg to 20.83 mg per kg of body weight. In consideration
of individuals ranging from a child having a body weight of 5 kg to
an adult having a body weight of 200 kg, it may be said that the
oral intake may be set to a range of from 0.04 g to 4.20 g. In any
case, an oral intake of as much as 15 g required in the
lactulose-mannitol test is not required, and hence the diagnostic
drug may be said to relieve a burden on the test subject.
[0114] It is considered that the same applies to the case of enema
administration.
[0115] In addition, although depending on a detection system, it is
preferred that the diagnostic drug have a weight average molecular
weight prepared to a range of from 200 to 20,000, more preferably
from 1,000 to 11,600. FIG. 11 are schematic diagrams of
distribution modes of the molecular weight of the diagnostic drug
and concentration measurement results. In FIG. 11a, a single-peak
diagnostic drug is shown. Whether a state in which a substance
having at least up to this molecular weight leaks is found can be
determined. In FIG. 11b, a multi-peak diagnostic drug is shown. How
large the maximum molecular weight of a substance that is liable to
leak is can be determined. In FIG. 11c, a diagnostic drug having a
uniform spread of molecular weights is shown. Also in this case,
how large the maximum molecular weight of a substance that is
liable to leak is can be determined.
[0116] The diagnostic drug or diagnostic method described above has
the following advantages. [0117] Having no toxicity (Usable for a
human. Allowing an animal experiment as well.). [0118] Orally
administrable. [0119] Not produced in a living body including an
intestine (Allowing direct concentration measurement.). [0120] Not
easily decomposed in a living body (Allowing direct concentration
measurement.). [0121] Hardly absorbed from an intestinal tract in a
normal state. [0122] Allowing the adjustment of a molecular
weight.
[0123] <Test Using Highly Purified Chitin-Chitosan>
[0124] Next, a test was performed using chitin-chitosan having a
sharper molecular weight distribution. FIG. 12 is a graph for
showing the manner of molecular weight distribution of a
chitin-chitosan sample used in the following test. The sample is a
highly purified sample which has a sharp distribution as compared
to FIG. 7, and in which, according to the results of separate
analysis, molecules having molecular weights of from 823 to 1,984
(having molecular weights of from about 1,000 to about 1,200 as
hydrochlorides) account for 98 wt % of the entirety. This sample is
referred to as "purified sample" as appropriate.
[0125] For the IR(20) model, a chitin-chitosan concentration in
serum was measured using the purified sample. The measurement
results, and the external appearance of an intestinal tract and an
HE-stained image thereof are shown in FIG. 13. It is found from the
HE-stained image that a mucosal disorder has only slightly
advanced, but as with FIG. 8, it can be confirmed that a state in
which chitin-chitosan having a molecular weight of about 1,000
leaks into blood is found.
[0126] <Safety Confirmation>
[0127] In the IR(10) model in which the purified sample was orally
administered to mice, a temporal change in chitin-chitosan amount
in circulating blood was measured. A temporal change from 30
minutes after reperfusion is shown in FIG. 14. An oral dose was set
to 2.5 mg/mouse.
[0128] The circulating blood amount of a mouse is estimated to be
an amount corresponding to 1/13 of its body weight, and hence the
amount is 1.93 ml when the body weight is 25 g. Meanwhile, the Area
Under the Curve (AUC) shown in FIG. 14 was 162.16 .mu.g/ml between
0 h and 8 h. Therefore, the amount of the chitin-chitosan that
leaked into circulating blood in 8 hours is 162.16*1.93=312.97
.mu.g. It may be said from the foregoing that 12.5% of the oral
dose of the purified sample leaked into circulating blood (312.97
.mu.g/2.5 mg=0.125). The mice remained alive even after 8
hours.
[0129] In addition, 2.5 mg of the purified sample was intravenously
administered to nontreated mice, i.e., mice not subjected to
ischemia-reperfusion treatment, and a temporal change in blood
concentration of the chitin-chitosan was measured. The measurement
results are shown in FIG. 15. It was confirmed that the blood
concentration was about 70 .mu.g/ml even when the intravenous
administration was performed (the blood concentration estimated by
extrapolation was about 150 .mu.g/ml even immediately after the
administration), and the chitin-chitosan rapidly disappeared from
the blood in about 2 hours (the concentration decrease was linear
when replotted on a logarithmic scale). This is considered to be
due to renal excretion. No shock symptom due to the intravenous
administration was found, and the mice remained alive even after 2
hours.
[0130] The following may be said in consideration of the two
tests.
1) The mice continued to live even after the tests, and hence it
was able to be confirmed again that the chitin-chitosan was safe
for a living body (at least the possibility of immediately
seriously affecting the living body is extremely low). 2) The
chitin-chitosan can be said to be safe to a living body also
because of rapid disappearance thereof from the blood. 3) Under the
above-mentioned conditions, the chitin-chitosan in the blood was
detected from the blood even after 8 hours. Accordingly, the
chitin-chitosan allows concentration measurement even after a lapse
of some time from administration, and hence is useful as a
diagnostic drug/evaluation drug. In addition, a temporal change can
also be grasped. 4) The chitin-chitosan rapidly disappears after
leaking into blood, and hence, through oral administration or enema
administration thereof, a real-time state of the leakiness or
permeability of an intestine can be grasped. More simply, it may be
said that the state of the intestine can be grasped.
[0131] <Permeability Evaluation with Model Other than IR
Model>
[0132] Next, evaluation of permeability or leakiness was performed
for models other than the above-mentioned evaluation test involving
directly applying a load to the intestine like
ischemia-reperfusion.
[0133] <Permeability Evaluation with Food Allergy Model>
[0134] First, mucosal permeability evaluation of the small
intestine was performed for mice having egg allergy, i.e., OVA-IgE
mice. A protocol for inducing OVA allergy is as illustrated in FIG.
16. First, mice were sensitized by being intraperitoneally
administered with ovalbumin (OVA), followed continuously by oral
administration therewith. 28 Days after the sensitization, the mice
were orally administered with OVA and the purified sample and
evaluated. In the OVA-IgE mice, severe diarrhea, a typical symptom
of food allergy, was found.
[0135] The blood concentration measurement results of the
chitin-chitosan are shown in FIG. 17. For comparison, the results
of a lactulose-mannitol test are also shown. As shown in FIG. 17,
it was able to be found that, when diarrhea resulting from food
allergy occurred, a state in which the permeability of the
intestine was raised was found. It was also found that the raising
of permeability was able to be more clearly judged by the
evaluation than by the lactulose-mannitol test.
[0136] <Permeability Evaluation with High-fat Diet Model and
NASH-inducing Diet Model>
[0137] Next, permeability evaluation was performed for mice kept
fed with a high-fat diet and a nonalcoholic steatohepatitis
(NASH)-inducing diet, respectively.
[0138] The components of the high-fat diet and the NASH-inducing
diet are as shown below.
TABLE-US-00001 TABLE 1 High-fat diet NASH-inducing diet (g/100 g of
feed) Carbohydrate 26 45 Protein 26 22 Lipid 35 20
Cholesterol amounts in the lipid and fructose amounts in the
carbohydrate are as shown below.
TABLE-US-00002 High-fat diet NASH-inducing diet (g) Cholesterol in
lipid 0.028 2 Fructose in carbohydrate 0 22
[0139] High-fat diet model: 6-Week-old male C57BL/6 mice were
allowed to ingest the high-fat diet ad libitum for 5 weeks, and
then given neither water nor feed for 21 hours. After that, 2.5 mg
of the purified sample was orally administered to the mice, and 1
hour after that, blood was collected from the inferior vena cava,
and a chitin-chitosan amount was measured.
[0140] NASH-inducing diet model: 6-Week-old male C57BL/6 mice were
allowed to ingest the NASH-inducing diet ad libitum for 4 weeks,
and then given neither water nor feed for 21 hours. After that, 2.5
mg of the purified sample was orally administered to the mice, and
1 hour after that, blood was collected from the inferior vena cava,
and a chitin-chitosan amount was measured.
[0141] The results are shown in FIG. 18. Although the permeability
is not raised for the high-fat diet, the permeability is clearly
raised in the case of the NASH-inducing diet. Thus, first, it has
been able to be confirmed that it may be said that nonalcoholic
steatohepatitis induces a state in which the permeability of the
intestine is raised. Next, the leakiness of the intestine was not
raised with the ingestion of the high-fat diet of the composition
for the period of time. Thus, it has been found, conversely, that
the use of the chitin-chitosan enables screening on how the
intestine is affected by what diet.
[0142] <Permeability Evaluation with DSS-Induced Ulcerative
Colitis Model>
[0143] Next, permeability evaluation was performed for mice having
ulcerative colitis induced with dextran sodium sulfate (DSS).
[0144] Ulcerative colitis model: First, mice were allowed to drink
water having dissolved therein 2.5 wt % of DSS ad libitum. After 72
hours from the start of the ad libitum water drinking, the mice
were enema administered or orally administered with the purified
sample, and the blood concentrations of the chitin-chitosan were
measured 1 hour after the administration for the mice subjected to
the enema administration, and 4 hours after the administration for
the mice subjected to the oral administration.
[0145] FIG. 19 are whole intestine images and HE-stained images of
the case of allowing the drinking of water with DSS for 72 hours (3
days) and the case of not allowing water drinking. As shown in FIG.
19, at 72 hours from the start of the ad libitum water drinking,
inflammation was not found in intestinal mucosa, and no clear
change was observed anywhere across even the whole intestine.
[0146] However, as shown in FIG. 20, in both the oral
administration and the enema administration, leakage of the
chitin-chitosan into blood was observed. In addition, as a result
of continued observation, it was separately confirmed that the mice
that had drunk water with DSS ad libitum developed inflammation 96
hours (4 days) after the start of the water drinking.
[0147] That is, surprisingly, it was able to be confirmed that the
use of the chitin-chitosan enabled the detection of an abnormal
increase in permeability of intestinal mucosa before the onset of
ulcerative colitis.
[0148] <Chitin-Chitosan as Evaluation Agent for Pharmacological
Action>
[0149] As illustrated in FIG. 21, an increase in permeability of
intestinal mucosa in general, not limited to that before the onset
of ulcerative colitis, is considered to occur long before the
occurrence of inflammation or disorder. Therefore, grasping of
permeability or leakiness through the use of the chitin-chitosan
enables pre-onset diagnosis, onset prevention, onset prediction,
and evaluation of the pharmacological action of a therapeutic drug
or the like.
[0150] First, in the case where inflammation is caused by food
ingestion or the like, whereas it has hitherto been impossible to
judge what is a causative food or a causative food group without
continuing the ingestion until onset, the ingestion can be stopped
before onset to relieve a burden and effective screening can be
performed.
[0151] Next, the chitin-chitosan can also be used for screening for
drug discovery of an LGS therapeutic drug, an LGS alleviating drug,
an intestinal mucosal permeability modulatory drug, and the like.
That is, by: administering a given substance (candidate substance);
separately orally administering or enema administering chitin
and/or chitosan; and measuring blood concentrations of chitin
and/or chitosan before and after the administration of the
candidate substance, it is possible to evaluate whether the
candidate substance has a normalizing action on permeability of
intestinal mucosa, and how strong the normalizing action, when
present, is. The chitin-chitosan may also be provided as an
evaluation agent containing chitin and/or chitosan as a main
component, the evaluation agent being used as described above.
[0152] In addition, it also becomes possible to accumulate findings
on inflammatory bowel diseases (IBDs), such as ulcerative colitis
and Crohn's disease, and eosinophilic gastroenteritis. Findings on
irritable bowel syndrome can also be accumulated. That is, it
becomes possible to determine the active period or remission of
pathology, determine a therapeutic effect and a drug efficacy
evaluation, and predict pathology.
[0153] <Application of Diagnostic Drug>
[0154] In addition, when the results shown in FIG. 18 are also
taken into consideration, the chitin-chitosan can also be used as
an evaluation drug for a food and drink.
[0155] That is, by: allowing a test subject to eat and drink a
single or a plurality of specific foods and drinks; allowing the
test subject to orally ingest an evaluation drug containing the
chitin-chitosan as a main component during the eating and drinking,
or before or after the eating and drinking; and measuring a
concentration of the chitin-chitosan in blood after a lapse of a
predetermined period of time from the oral ingestion, it is
possible to determine whether the foods and drinks affect
permeability of intestinal mucosa of the test subject.
[0156] In the case of LGS, it can be determined whether LGS is
induced or LGS is inhibited. The evaluation drug may be orally
ingested before the eating and drinking, during the eating and
drinking, or after the eating and drinking as appropriate in
accordance with, for example, the kind of the food and drink.
[0157] Through the use of the evaluation drug, not only screening
of a general inducer or inhibitor can be performed, but also
screening of an inducer or inhibitor for an individual can be
performed. That is, a risk factor for an individual can be
identified.
[0158] For example, a test subject is allowed to first take the
evaluation drug, and then eat meat while drinking beer.
Concurrently, blood is collected every 10 minutes, and thereby, a
temporal transition of LGS can be monitored. As a result, when that
combination of food and drink causes the onset of medium-degree LGS
in about 20 minutes for that person, the person can be advised to
avoid such combination.
[0159] Further, through the use of the food and drink evaluation
drug, there can be provided, for example, an objective performance
index for a food touted as a conditioner for gut flora or an
intestinal environment, i.e., a probiotic food. Moreover, a
prebiotic food can also be evaluated.
[0160] A diagnostic device having applied thereto the technology
described above can also be constructed.
[0161] That is, a diagnostic device can be obtained by including:
concentration-measuring means for measuring a concentration of
chitin and/or chitosan in blood collected from a test subject; and
evaluation means for evaluating permeability of intestinal mucosa
of the test subject on the basis of the concentration measured by
the concentration-measuring means.
[0162] As a component technology of the concentration-measuring
means, for example, chromatography may be used. A process from
sample introduction to concentration calculation may be automated
as appropriate through the use of a general technology.
[0163] The evaluation means may be configured to, for example,
determine the degree of leakiness through analysis of the position
and height of a peak in an obtained chromatogram, the calculation
of a peak area, and the like. Not only a mere determination as
severe LGS or mild LGS, but also such a diagnosis as the following
can be made on the basis of the distribution and unevenness of
peaks in consideration of past data as well: being predisposed to
constantly having mild LGS though not predisposed to having
medium-degree LGS or severe LGS; predisposed to having
medium-degree LGS by taking exercise; or having an intestinal
disease other than LGS.
[0164] Besides, a specific detection antibody may be generated, and
an ELISA kit using the antibody may be adopted. With this, a large
number of samples can be evaluated at once.
[0165] The blood used for measurement and diagnosis is disposed of
without being returned to a human body.
INDUSTRIAL APPLICABILITY
[0166] According to the present invention, an objective index and
diagnosis system for LGS can be constructed. In addition,
involvement of LGS in various diseases can be explored. For
example, its relationship with chronic renal disorder, bronchitic
asthma, type I diabetes, food allergy, alcoholic hepatitis,
nonalcoholic steatohepatitis, or the like can be investigated. A
contribution can also be made to the development of a therapeutic
drug for an intestinal disease.
* * * * *