US20210132074A1 - Diagnostic drug, diagnostic method and diagnostic device for permeability of intestinal mucosa - Google Patents

Diagnostic drug, diagnostic method and diagnostic device for permeability of intestinal mucosa Download PDF

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US20210132074A1
US20210132074A1 US16/478,421 US201816478421A US2021132074A1 US 20210132074 A1 US20210132074 A1 US 20210132074A1 US 201816478421 A US201816478421 A US 201816478421A US 2021132074 A1 US2021132074 A1 US 2021132074A1
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chitin
chitosan
permeability
blood
concentration
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Koichiro Wada
Haruki USUDA
Morihiko Nakamura
Yoshimori TAKAMORI
Seiji Kurozumi
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Koyo Chemical Co Ltd
Shimane University NUC
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Koyo Chemical Co Ltd
Shimane University NUC
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Assigned to KOYO CHEMICAL CO., LTD., NATIONAL UNIVERSITY CORPORATION SHIMANE UNIVERSITY reassignment KOYO CHEMICAL CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KUROZUMI, SEIJI, NAKAMURA, MORIHIKO, TAKAMORI, YOSHIMORI, USUDA, Haruki, WADA, KOICHIRO
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
    • G01N2400/28—Chitin, chitosan
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • 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.
  • 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.
  • This method involves allowing a test subject to simultaneously take 10 g of lactulose (molecular weight ⁇ 340) and 5 g of mannitol (molecular weight ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a technology for evaluating leakiness of a substance having such large molecular weight does not exist in the first place.
  • 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.
  • the object is to provide a technology for diagnosing an increase in intestinal mucosal permeability, such as LGS.
  • Another object of the present invention is to provide a technology for determining a food and drink that affects permeability of intestinal mucosa.
  • the object is to provide a technology for determining a food and drink that induces LGS or a food and drink that inhibits LGS.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • (1) whether a substance having that molecular weight permeates the intestinal mucosa can be confirmed.
  • 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.
  • approximately how large the molecular weight of a substance that permeates the intestinal mucosa is can be confirmed by a single test.
  • 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.
  • the intake is smaller than that in a lactulose-mannitol test, and hence a burden on the test subject can be reduced.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the chitin and/or chitosan preferably has a weight average molecular weight prepared to a range of from 1,000 to 11,600.
  • 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.
  • 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.
  • the chitin and/or chitosan preferably has a weight average molecular weight prepared to a range of from 1,000 to 11,600.
  • the intake is preferably set to a range of from 8.33 mg to 20.83 mg per kg of body weight.
  • the term “affect” includes both increasing and reducing the permeability or leakiness of the intestinal mucosa.
  • increasing the leakiness means inducing or aggravating LGS, and reducing the leakiness means inhibiting, alleviating, ameliorating, or curing LGS.
  • examples of the specific single food and drink may include yogurt and a whey beverage.
  • an objective performance index for a food can also be provided.
  • examples of the plurality of specific foods and drinks may include: an oyster and wine; and a pork steak and beer.
  • screening of an inducer or an inhibitor can be performed for an individual.
  • 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.
  • 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.
  • the chitin and/or chitosan preferably has a weight average molecular weight prepared to a range of from 1,000 to 11,600.
  • the intake is preferably set to a range of from 8.33 mg to 20.83 mg per kg of body weight.
  • 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.
  • a subject to which the given substance is administered may be a human or may be an animal other than a human.
  • the chitin and/or chitosan preferably has a weight average molecular weight prepared to a range of from 1,000 to 11,600.
  • 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.
  • 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.
  • 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.
  • a subject to which the given substance is administered may be a human or may be an animal other than a human.
  • the chitin and/or chitosan preferably has a weight average molecular weight prepared to a range of from 1,000 to 11,600.
  • 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.
  • 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.
  • the diagnostic device may also be called an intestinal mucosal permeability evaluation device.
  • the degree of permeability of the intestinal mucosa can be directly evaluated with high reliability.
  • a food and drink that affects the permeability of the intestinal mucosa can be determined.
  • an objective evaluation of a pharmaceutical for normalizing the permeability of the intestinal mucosa or a candidate substance therefor can be performed.
  • FIG. 1 are test outlines of an AO model and an IR model.
  • FIG. 2 are photographs for showing a normal state of an intestinal tract and an LGS-induced state thereof.
  • FIG. 3 is a graph for showing the results of a lactulose-mannitol test for the AO model.
  • FIG. 4 are graphs for showing the results of an FITC-dextran test for the AO model.
  • FIG. 5 is a graph for showing the results of an FITC-dextran test for the IR model.
  • FIG. 6 are graphs for showing the measurement results of chitin-chitosan concentrations for the AO model and the IR model.
  • 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.
  • FIG. 8 is a graph for showing the blood concentrations of chitin-chitosan at various molecular weights for the IR(20) model.
  • FIG. 9 is a graph for showing the blood concentrations of chitin-chitosan at various molecular weights for the IR (10) model.
  • 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.
  • FIG. 11 are schematic diagrams of distribution modes of the molecular weight of a diagnostic drug and concentration measurement results.
  • FIG. 12 is a graph for showing the manner of molecular weight distribution of a purified sample.
  • 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.
  • 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.
  • 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.
  • FIG. 16 is an explanatory diagram for illustrating a protocol for inducing OVA allergy.
  • FIG. 17 is a graph for showing the blood concentration of chitin-chitosan using OVA-IgE mice.
  • FIG. 18 are graphs for showing the blood concentrations of chitin-chitosan for a high-fat diet model and a NASH-inducing diet model.
  • 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.
  • FIG. 20 is a graph for showing the blood concentrations of chitin-chitosan before onset in the DSS-induced ulcerative colitis model.
  • 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.
  • LGS was assumed as an example in which the leakiness of an intestinal tract was evaluated, and first, induction tests therefor were performed.
  • AO 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).
  • 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.
  • IR model 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.
  • 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.
  • FIG. 1 Outlines of both models are illustrated in FIG. 1 .
  • FIG. 1 b an IR(20) model and an IR(10) model, which are described later, are also illustrated.
  • FIGS. 2 external appearance photographs, HE-stained images, and electron micrographs of the intestinal tract are shown.
  • the AO model cracks are found in intestinal villi, and hence the occurrence of mild LGS is recognized.
  • the IR (30) model intestinal villi are significantly damaged, and hence the occurrence of severe LGS is recognized.
  • 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 .
  • 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.
  • 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. 4 b ).
  • 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.
  • 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.
  • the IR (30) model may be said to be a model capable of evaluating severe LGS.
  • 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.
  • the AO model may be said to be a model capable of evaluating mild LGS.
  • the shell of a crab was deproteinized, decalcified, and deacetylated to provide a mixture of chitin and chitosan.
  • 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.
  • 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).
  • 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.
  • chitose (2,5-anhydro-D-mannose) serving as a constituent monosaccharide by a nitrous acid decomposition method.
  • its aldehyde group was allowed to react with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and iron (III) chloride to develop a blue color.
  • MBTH 3-methyl-2-benzothiazolinone hydrazone hydrochloride
  • iron (III) chloride iron (III) chloride
  • 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.
  • 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.
  • UF membranes having molecular weight cut-offs of 3,000, 6,000, 10,000, and the like
  • FIG. 7 the manner of distribution of the prepared samples is shown.
  • a measurement method for the weight average molecular weight is also described.
  • IR(20) 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”.
  • the measurement results of the blood concentrations of the chitin-chitosan at various molecular weights are shown in FIG. 8 .
  • the results of a control, i.e., without ischemia-reperfusion are also shown.
  • 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.
  • 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”.
  • the measurement results of the blood concentrations of the chitin-chitosan at various molecular weights are shown in FIG. 9 .
  • 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.
  • 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.
  • 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.
  • a diagnostic drug for evaluating permeability of intestinal mucosa containing chitin and/or chitosan as a main component, was able to be obtained.
  • 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.
  • 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.
  • 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 .
  • FIG. 10 it was found that significant concentration measurement was possible even at 0.625 mg/mouse.
  • the dose of the diagnostic drug is 1.25 g.
  • significant concentration measurement is in theory possible even at 0.500 g of oral ingestion for a 60 kg human.
  • the dose or the intake be set to a range of from 8.33 mg to 20.83 mg per kg of body weight.
  • the oral intake may be set to a range of from 0.04 g to 4.20 g.
  • 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.
  • FIG. 11 are schematic diagrams of distribution modes of the molecular weight of the diagnostic drug and concentration measurement results.
  • 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.
  • 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.
  • 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.
  • the diagnostic drug or diagnostic method described above has the following advantages.
  • 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.
  • 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.
  • 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.
  • the chitin-chitosan can be said to be safe to a living body also because of rapid disappearance thereof from the blood.
  • 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.
  • 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.
  • mice having egg allergy i.e., OVA-IgE mice.
  • a protocol for inducing OVA allergy is as illustrated in FIG. 16 .
  • OVA-IgE mice severe diarrhea, a typical symptom of food allergy
  • the blood concentration measurement results of the chitin-chitosan are shown in FIG. 17 .
  • 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.
  • permeability evaluation was performed for mice kept fed with a high-fat diet and a nonalcoholic steatohepatitis (NASH)-inducing diet, respectively.
  • NASH nonalcoholic steatohepatitis
  • the components of the high-fat diet and the NASH-inducing diet are as shown below.
  • 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.
  • 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.
  • 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.
  • 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.
  • an increase in permeability of intestinal mucosa in general 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.
  • 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.
  • IBDs inflammatory bowel diseases
  • 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.
  • the chitin-chitosan can also be used as an evaluation drug for a food and drink.
  • 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.
  • 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.
  • 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.
  • an objective performance index for a food can be provided, for example, an objective performance index for a food advocated as a conditioner for gut flora or an intestinal environment, i.e., a probiotic food.
  • a prebiotic food can also be evaluated.
  • a diagnostic device having applied thereto the technology described above can also be constructed.
  • 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.
  • 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.
  • 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.
  • 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.
  • the blood used for measurement and diagnosis is disposed of without being returned to a human body.
  • an objective index and diagnosis system for LGS can be constructed.
  • 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.

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