Method for assessing disorder of mucous membrane of gastrointestinal tract

Abstract

This method for assessing a disorder of the mucous membrane of the gastrointestinal tract comprises: a preparation step for preparing a biological sample collected from a subject to whom a dye compound for endoscopic examination has been administered; a measurement step for measuring the amount of the dye compound for endoscopic examination or a metabolite thereof in the biological sample; a comparison step for comparing the measured amount of the dye compound for endoscopic examination or the metabolite thereof with a reference value; and a determination step for determining that the subject is highly likely to have a mucosal disorder of the gastrointestinal tract if the measured amount of the dye compound for endoscopic examination or the metabolite thereof is equal to or greater than the reference value.

Description

Method for assessing damage to the mucosa of the digestive tract

[0001] The present invention relates to a method for assessing damage to the mucosa of the digestive tract.

[0002] The gastrointestinal tract (esophagus, stomach, small intestine, large intestine, etc.) prevents pathogens or allergens from entering the body through the barrier function of mucosal cells and the activity of immune cells. When the barrier function of mucosal cells is weakened, pathogens or allergens become more likely to enter the body. This condition is called "increased permeability of the gastrointestinal mucosa."

[0003] Increased permeability of the gastrointestinal mucosa occurs in various diseases, such as inflammatory bowel disease, functional gastrointestinal disorders, cancer, and lifestyle-related diseases. Objective evaluation of gastrointestinal mucosal permeability would be useful for determining the activity or therapeutic efficacy of various diseases accompanied by mucosal damage. Currently established methods for objectively evaluating gastrointestinal mucosal permeability and the degree of mucosal damage include measuring the sugar concentration excreted in urine after taking sugars with different absorbability (e.g., lactulose and mannitol) (Eur. Rev. Med. Pharmacol. Sci. 2019, 23, 795-810 (Non-Patent Document 1)).

[0004] Eur. Rev. Med. Pharmacol. Sci. 2019, 23, 795-810.

[0005] The evaluation method described in Non-Patent Document 1 requires hospitalization, prolonged fasting, or collection of a day's worth of urine, which places a heavy burden on patients. Furthermore, the evaluation method described in Non-Patent Document 1 tends to make it difficult to obtain stable evaluation results.

[0006] The present invention has been made in view of the above circumstances, and aims to provide a simple method for assessing damage to the mucosa of the digestive tract, which method enables evaluation of the degree of mucosal damage.

[0007] As a result of intensive research, the present inventors have found that the degree of mucosal damage can be evaluated by measuring the amount of the dye compound for endoscopic examination or its metabolites in a biological sample collected from a subject to which the dye compound for endoscopic examination has been administered, and have completed the present invention.

[0008] A first aspect of the present invention relates to a method for determining a gastrointestinal mucosal disorder, comprising: a preparation step of preparing a biological sample collected from a subject to which a dye compound for endoscopic examination has been administered; a measurement step of measuring the amount of the dye compound for endoscopic examination or a metabolite thereof in the biological sample; a comparison step of comparing the measured amount of the dye compound for endoscopic examination or a metabolite thereof with a reference value; and a determination step of determining that the subject is highly likely to have a gastrointestinal mucosal disorder when the measured amount of the dye compound for endoscopic examination or a metabolite thereof is equal to or greater than the reference value.

[0009] According to the present invention, it is possible to provide a simple method for assessing damage to the mucosa of the digestive tract, which method is capable of evaluating the degree of mucosal damage.

[0010] Figure 1 is a graph showing the concentration of indigo carmine in the blood of the control group (subject A), and Figure 2 is a graph showing the concentration of indigo carmine in the blood of the mucosal damage group (subject B).

[0011] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0012] <Method for assessing damage to the mucosa of the digestive tract> The assessment method according to this embodiment is a method for assessing damage to the mucosa of the digestive tract, and includes the following steps: a preparation step of preparing a biological sample collected from a subject to which a dye compound for endoscopic examination has been administered; a measurement step of measuring the amount of the dye compound for endoscopic examination or a metabolite thereof in the biological sample; a comparison step of comparing the measured amount of the dye compound for endoscopic examination or a metabolite thereof with a reference value; and a assessment step of assessing that the subject is highly likely to have a mucosal disorder of the digestive tract, if the measured amount of the dye compound for endoscopic examination or a metabolite thereof is equal to or greater than the reference value.

[0013] <Preparation Step> In this step, a biological sample is prepared from a subject to which a dye compound for endoscopic examination has been administered. In this embodiment, a "dye compound for endoscopic examination" refers to a dye compound used in endoscopic examination that satisfies the following (A) and (B): (A) a dye, pigment, or compound made by a synthetic process or similar artificial technique, or a compound extracted, isolated, or derived from a plant, animal, mineral, or other source, regardless of whether intermediate or final changes in identity have occurred; (B) a compound that can impart a color (including black, white, and gray) when added to or applied to food, medicine, cosmetics, or the human body or a part thereof.

[0014] Examples of the dye compound for endoscopic examination include compounds listed in the U.S. Food and Drug Administration (FDA) list of "Dyes Used in Endoscopy" (https: / / www.ecfr.gov / current / title-21 / chapter-I / subchapter-A / part-74). In this embodiment, the dye compound for endoscopic examination preferably includes at least one dye selected from the group consisting of phthalocyanine blue, D&C Black No. 4, FD&C Blue No. 2 (indigo carmine), D&C Blue No. 6, D&C Green No. 6, D&C Red No. 17, D&C Violet No. 2, D&C Yellow No. 8, and D&C Yellow No. 10. In one aspect of this embodiment, the dye compound for endoscopic examination preferably includes indigo carmine. The IUPAC name of the above indigo carmine is disodium 3,3'-dioxo-2,2'-bis-indolidene-5,5'-disulfonate.

[0015] In one aspect of this embodiment, the dye compound for endoscopic examination is preferably a poorly absorbed compound. Indigo carmine is one type of poorly absorbed compound. In this embodiment, the term "poorly absorbed compound" refers to a compound that is poorly absorbed from the mucosa of the gastrointestinal tract when administered to a healthy subject without gastrointestinal mucosal disorders. One embodiment of the poorly absorbed compound is, for example, a compound that is used clinically and whose safety has been established.

[0016] In this embodiment, the "subject" is not particularly limited as long as it is a mammal to which the endoscopic dye compound is administered, and examples thereof include humans, chimpanzees, cows, sheep, pigs, goats, horses, dogs, cats, mice, rats, rabbits, and guinea pigs. In one aspect of this embodiment, the subject is preferably a human. The "subject" may also include adults, infants, and newborns.

[0017] The method of administering the dye compound for endoscopic examination to a subject can be any known method without particular limitation. In one aspect of this embodiment, the method of administering the dye compound for endoscopic examination to a subject may be a method of directly spraying the compound onto a target site in the gastrointestinal mucosa, or it may be oral administration. Here, the "target site" refers to a site in the gastrointestinal mucosa that is the subject of evaluation. Examples of the target site include a lesion site.

[0018] When the dye compound for endoscopic examination is administered orally, the dosage form of the drug containing the dye compound for endoscopic examination is not particularly limited as long as it can reach the target site in the gastrointestinal mucosa, and examples thereof include tablets, capsules, powders, etc.

[0019] The dose of the dye compound for endoscopic examination may vary depending on the body weight, age, etc. of the subject, but may be, for example, the amount used clinically in endoscopic examination (for example, 20 mg / 20 mL in the case of indigo carmine).

[0020] In this embodiment, the term "biological sample" refers to a biological sample collected from a subject, which may contain a dye compound for endoscopic examination administered to the subject or a metabolite thereof. Examples of the biological sample include blood, plasma, serum, and urine. In one aspect of this embodiment, the biological sample is preferably blood, serum, plasma, or urine.

[0021] The method for collecting the biological sample is not particularly limited, and any known method can be used. For example, blood can be collected by drawing blood using a syringe or the like.

[0022] In one aspect of this embodiment, the biological sample is preferably collected from a subject to whom the dye compound for endoscopic examination has been administered by direct spraying onto a target site of the gastrointestinal mucosa. Direct spraying onto a target site of the gastrointestinal mucosa can be achieved, for example, by spraying using an endoscope. By administering the dye compound for endoscopic examination in this manner, it becomes possible to determine the presence or absence of mucosal damage and the degree of mucosal damage for each target site.

[0023] The timing of collecting the biological sample is not particularly limited, as long as it is any time between "after the endoscopic dye compound or its metabolites have been transferred to the biological sample" and "before they have been released from the biological sample" via the bloodstream. In one aspect of this embodiment, the timing of collecting the sample is preferably any time between 30 and 120 minutes, and more preferably any time between 30 and 60 minutes, after the endoscopic dye compound has been administered to the subject. In one aspect of this embodiment, the biological sample may be collected once, or multiple times (e.g., two to five times) at different times. When the biological sample is collected multiple times at different times, it becomes possible to examine changes over time in the amount of the endoscopic dye compound or its metabolites in the biological sample.

[0024] In one aspect of this embodiment, prior to carrying out the measurement step described below, a biological sample collected from a subject may be pretreated by a method known to those skilled in the art. For example, when the biological sample is blood, peripheral blood from the subject is first collected in a blood collection tube, allowed to clot, and then centrifuged to obtain a supernatant. The obtained supernatant fraction can be subjected to pretreatment such as solid-phase extraction to prepare a measurement sample.

[0025] <Measurement Step> In this step, the amount of the endoscopic dye compound or its metabolite in the biological sample is measured. In this embodiment, a metabolite of the endoscopic dye compound refers to a compound produced by conversion from the endoscopic dye compound due to the biological functions of the subject to which the dye compound is administered. For example, metabolites of indigo carmine include 2-amino-5-sulfobenzoic acid (5-sulfoanthranilic acid) and 2,3-dihydro-2,3-dioxo-1H-indole-5-sulfonic acid (isatin-5-sulfonic acid). In other words, the metabolite of indigo carmine is preferably isatin-5-sulfonic acid or 5-sulfoanthranilic acid.

[0026] In the present embodiment, the method for measuring the amount of the dye compound for endoscopic examination or its metabolite is not particularly limited, and examples thereof include ELISA (Enzyme-Linked Immunosorbent Assay), mass spectrometry, liquid chromatography, immunochromatography, fluorescence detection, spectrophotometry, and the like.

[0027] Examples of columns for liquid chromatography include reverse phase columns and HILIC (Hydrophilic Interaction Liquid Chromatography) columns.

[0028] The mobile phase for liquid chromatography can be appropriately selected depending on the column used. For example, when a reverse phase column is used, methanol, acetonitrile, ammonium acetate solution, etc. are preferably used.

[0029] Examples of the mass spectrometry include liquid chromatography-mass spectrometry (LC-MS), laser desorption ionization mass spectrometry (LDI-MS), etc. There are no particular limitations on the equipment used for mass spectrometry as long as it is commercially available, and examples include LCMS-8060 and AXIMA Performance manufactured by Shimadzu Corporation.

[0030] In one aspect of this embodiment, the amount of the dye compound for endoscopic examination or its metabolites is preferably measured using LC-MS or LDI-MS in the measuring step.

[0031] In this embodiment, the "amount of the dye compound for endoscopic examination or its metabolites" is not particularly limited as long as it is a physical quantity that indicates the presence of the dye compound for endoscopic examination or its metabolites in a biological sample, and examples include the density in the biological sample, the concentration in the biological sample, etc. When the biological sample is blood, serum, etc., the "amount of the dye compound for endoscopic examination or its metabolites" is usually expressed as the blood concentration.

[0032] In one aspect of this embodiment, the above measurement may be carried out in the presence of an internal standard substance, such as tar-based dyes such as Orange G and Sunset Yellow FCF.

[0033] <Comparison Step> In this step, the measured amount of the dye compound for endoscopic examination or its metabolite is compared with a reference value.

[0034] In this embodiment, the "reference value" refers to the "amount of the dye compound for endoscopic examination or its metabolites" that serves as a reference for determining whether a subject is likely to have a gastrointestinal mucosal disorder. The reference value can be set arbitrarily. For example, the reference value may be the "amount of the dye compound for endoscopic examination or its metabolites" in a subject without a gastrointestinal mucosal disorder (e.g., a healthy subject), or may be a preset amount. The preset amount may be an appropriate value that does not include the patient group among the measurements of healthy subjects (healthy subject group) and patients with a gastrointestinal mucosal disorder (patient group), or a value that is not included in either the healthy subject group or the patient group, but is somewhere between the healthy subject group and the patient group.

[0035] <Determination step> In this step, if the measured amount of the dye compound for endoscopic examination or its metabolite is equal to or greater than the reference value, it is determined that the subject is highly likely to have a mucosal disorder of the digestive tract.

[0036] In one aspect of this embodiment, the phrase "when the measured amount of the dye compound for endoscopic examination or a metabolite thereof is equal to or greater than the reference value" encompasses both a case where the measured amount of the dye compound for endoscopic examination or a metabolite thereof is equivalent to the reference value and a case where the measured amount of the dye compound for endoscopic examination or a metabolite thereof exceeds the reference value.

[0037] In another aspect of this embodiment, it may be determined that the greater the measured amount of the dye compound for endoscopic examination or its metabolite, the greater the degree of mucosal damage in the digestive tract.

[0038] In one aspect of this embodiment, the gastrointestinal mucosal disorder preferably includes irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), such as ulcerative colitis.

[0039] Conventionally, the presence or absence of mucosal damage has been evaluated by spraying an endoscopic dye compound such as indigo carmine on the inner wall of the large intestine and observing it with an endoscope (contrast method). This is because areas with mucosal damage have a rough (uneven) surface, which allows indigo carmine to accumulate, resulting in a deep blue color in those areas. However, the contrast method has not been able to quantitatively evaluate the degree of mucosal damage. The assessment method of this embodiment focuses on the fact that endoscopic dye compounds such as indigo carmine are more easily absorbed in areas with mucosal damage than in normal mucosa, and quantitatively evaluates the permeability of endoscopic dye compounds such as indigo carmine by measuring their blood concentration. Therefore, the assessment method of this embodiment is capable of quantitatively evaluating the degree of mucosal damage.

[0040] Furthermore, according to the determination method of this embodiment, by quantitatively evaluating gastrointestinal mucosal damage at a target site where a dye compound for endoscopic examination has been sprayed, it becomes possible to evaluate the pathology of diseases that have symptoms of intestinal mucosal damage, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).It is also expected that the method will be applicable to "diseases that have symptoms of gastrointestinal mucosal damage," such as cancer, metabolic diseases, diabetes, and neuropsychiatric disorders, and will be useful in treatment plans such as confirming the effectiveness of therapeutic drugs and changing or adjusting therapeutic drugs according to the pathology.

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0042] Indigo carmine (a poorly absorbed compound) was administered to subjects (subjects) in the control group and mucosal damage group shown below, and the amount (concentration) of indigo carmine in the blood was evaluated. The specific procedure is shown below. For both the control group and the mucosal damage group, subjects aged 20 years or older and with no history of gastrointestinal surgery were selected. Control group: Subjects without severe diarrhea or constipation, and in whom endoscopic examination revealed no colonic inflammation, ulcers, cancer, or other diseases (Mayo classification: Grade 0). Mucosal damage group: Subjects clinically diagnosed with inflammatory bowel disease (IBD) (Mayo classification: Grade 2 or 3).

[0043] <Preparation Step> Indigo carmine was sprayed onto the upper part of the large intestine (cecum) using an endoscope for each of subject A in the control group and subjects B to D in the mucosal damage group. Blood was collected immediately before spraying, 30 minutes after spraying, and 60 minutes after spraying. The collected blood was left to stand without the addition of an anticoagulant to allow the blood cell components to coagulate. The blood was then centrifuged to separate it into serum and blood clots, and the serum was collected as a sample for measurement. This serum corresponds to a "biological sample collected from a subject administered with a dye compound for endoscopic examination."

[0044] A methanol solution (100 μl) containing Orange G (Fujifilm Wako Pure Chemical Industries, Ltd., final concentration 100 ng / ml) was added to the serum (20 μl) to obtain a mixture. The resulting mixture was centrifuged (12,000 × g, 10 minutes) to recover the supernatant (80 μl). 20 μl of 5% formic acid in methanol was added to the recovered supernatant to obtain a mixture. The mixture was purified by passing it through a pre-conditioned affinity column (MonoSpin Phospholipid, GL Sciences Inc.), and the filtrate was recovered. The recovered filtrate was vacuum dried and then redissolved in 150 μl of ultrapure water to obtain a sample for analysis.

[0045] <Measurement Step> The above analytical samples were analyzed under the following conditions using a high-performance liquid chromatograph mass spectrometer (LC-MS / MS) (Shimadzu Corporation, product name: LCMS-8060) (Table 1). The amount of indigo carmine was calculated from the obtained analytical results using the following method. The results are shown in Figure 1 (control group, subject A) and Figure 2 (mucosal disorder group, subject B), as well as Table 2.

[0046] (LC-MS / MS analysis conditions) <LC conditions> Column: Shim-pack XR-ODS II (3.9 mm ID x 75 mm, 2.2 μm) Column oven: 40°C Mobile phase: A: 10 mM ammonium acetate solution, B: acetonitrile Gradient: 3% (0 min) → 3% (0.25 min) → 95% (5 min) → 95% (7 min) → 3% (7.1 min) → 3% (10 min) Flow rate: 0.4 ml / min Sample cooler: 4°C Injection volume: 5 μl

[0047] Nebulizer gas flow rate: 3 L / min Heating gas flow rate: 10 L / min Interface temperature: 300°C Desolvation temperature: 526°C DL temperature: 250°C Heat block temperature: 400°C Drying gas flow rate: 10 L / min

[0048]

[0049] <Comparison step, judgment step> From the results of Figure 1 and Table 2, in the control group (subject A), the presence of indigo carmine was confirmed in the blood 30 minutes after spraying indigo carmine (1.269 ng / ml), but its presence was not confirmed 60 minutes after spraying. On the other hand, from the results of Figure 2 and Table 2, in the mucosal disorder group (subjects B to D), the presence of indigo carmine was confirmed at high concentrations in the blood 30 minutes after spraying indigo carmine (2.781 to 9.807 ng / ml), and its presence was also confirmed 60 minutes after spraying (1.192 to 4.878 ng / ml). Furthermore, in the mucosal disorder group, the blood concentration of indigo carmine increased as the Mayo classification grade progressed, and a correlation was observed between the two.

[0050] These results indicate that the blood indigo carmine concentration in the mucosal damage group was higher than that in the control group. This result is thought to be due to increased absorption of indigo carmine from the digestive tract due to mucosal damage in the digestive tract. It also suggests that measuring the blood indigo carmine concentration after endoscopic examination can quantitatively evaluate the degree of mucosal damage. Since indigo carmine in the blood is thought to be transferred to urine, similar results were expected when urine was used as a biological sample.

[0051] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0052] (Item 1) A method for assessing damage to the mucosal membrane of the gastrointestinal tract, according to one embodiment, includes the following steps: preparing a biological sample collected from a subject to which a dye compound for endoscopic examination has been administered; measuring the amount of the dye compound for endoscopic examination or a metabolite thereof in the biological sample; comparing the measured amount of the dye compound for endoscopic examination or a metabolite thereof with a reference value; and assessing that the subject is highly likely to have a mucosal membrane disorder of the gastrointestinal tract if the measured amount of the dye compound for endoscopic examination or a metabolite thereof is equal to or greater than the reference value. The method for assessing damage to the mucosal membrane of the gastrointestinal tract according to item 1 can provide a simple method for assessing damage to the mucosal membrane of the gastrointestinal tract, which is capable of evaluating the degree of mucosal membrane disorder.

[0053] (Item 2) In the determination method according to item 1, the dye compound for endoscopic examination includes at least one selected from the group consisting of phthalocyanine blue, D&C Black No. 4, indigo carmine, D&C Blue No. 6, D&C Green No. 6, D&C Red No. 17, D&C Violet No. 2, D&C Yellow No. 8, and D&C Yellow No. 10. According to the determination method according to item 2, damage to the mucosa of the digestive tract can be determined more simply.

[0054] (Item 3) In the determination method according to item 1 or 2, the biological sample is blood, serum, plasma, or urine. According to the determination method according to item 3, damage to the mucosa of the digestive tract can be determined more simply.

[0055] (Item 4) In the determination method according to any one of Items 1 to 3, the amount of the dye compound for endoscopic examination or its metabolite is measured using LC-MS or LDI-MS in the measuring step. According to the determination method according to Item 4, the degree of mucosal damage can be evaluated more quantitatively.

[0056] (Item 5) In the method of any one of Items 1 to 4, the biological sample is collected from a subject to whom the dye compound for endoscopic examination has been administered by directly spraying the dye compound onto a target site of the gastrointestinal mucosa. According to the method of Item 5, the degree of mucosal damage can be evaluated for each target site.

[0057] (Item 6) In the method for determining a gastrointestinal mucosal disorder according to any one of Items 1 to 5, the gastrointestinal mucosal disorder includes irritable bowel syndrome and inflammatory bowel disease. The method for determining a gastrointestinal mucosal disorder according to Item 6 can suitably evaluate the severity of irritable bowel syndrome or inflammatory bowel disease.

Claims

1. A method for determining damage to the mucous membrane of the digestive tract, Preparation steps include preparing biological samples collected from subjects who have been administered endoscopic dye compounds, and A measurement step of measuring the amount of the endoscopic dye compound or its metabolite in the biological sample, A comparison step involves comparing the measured amount of the endoscopic dye compound or its metabolite with a reference value. A determination step in which, if the measured amount of the endoscopic dye compound or its metabolite is equal to or greater than the reference value, it is determined that the subject is highly likely to have a gastrointestinal mucosal disorder. A determination method that includes this.

2. The determination method according to claim 1, wherein the biological sample is a biological sample collected from a subject to which the endoscopic dye compound has been administered by directly spraying it on a target site of the gastrointestinal mucosa.

3. The determination method according to claim 1, wherein the endoscopic dye compound comprises at least one selected from the group consisting of phthalocyanine blue, D&C Black No. 4, indigo carmine, D&C Blue No. 6, D&C Green No. 6, D&C Red No. 17, D&C Violet No. 2, D&C Yellow No. 8, and D&C Yellow No.

10.

4. The determination method according to claim 1, wherein the biological sample is blood, serum, plasma, or urine.

5. The determination method according to claim 1, wherein in the measurement step, the amount of the endoscopic dye compound or its metabolite is measured using LC-MS or LDI-MS.

6. The method for determining the gastrointestinal mucosal disorder according to claim 1, wherein the gastrointestinal mucosal disorder includes irritable bowel syndrome and inflammatory bowel disease.