AGENT AND COMPOSITION FOR IMPROVING INTRAUTERINE BACTERIAL FLORA, AND METHOD FOR DETERMINING WHETHER INTRAUTERINE BACTERIAL FLORA HAS BEEN IMPROVED OR NORMALIZED

Abstract

The purpose of the present invention is to provide an agent for improving intrauterine bacterial flora, and a method for determining whether intrauterine bacterial flora has been improved or normalized. An aspect of the present invention is an agent for improving intrauterine bacterial flora that contains lactoferrin or a salt thereof as an active ingredient. Additionally provided are: an agent or composition for improving intrauterine flora or treating or preventing diseases caused by the imbalance of intrauterine bacterial flora, the agent or composition containing lactoferrin or a salt thereof; a method for treating or preventing diseases caused by imbalance of intrauterine bacterial flora, the method comprising administrating the agent or composition; and a method for determining whether intrauterine bacterial flora has been improved or normalized.

Claims

1. An agent for improving bacterial flora in the uterus, comprising lactoferrin or a salt thereof as an active ingredient.

2. A therapeutic or prophylactic agent for a disease caused by a disturbance in the intrauterine bacterial flora, such as infertility, miscarriage, premature birth, recurrent pregnancy loss, endometriosis, endometrial cancer, cervicitis, endometritis, and/or chorioamnionitis, comprising lactoferrin or a salt thereof as an active ingredient.

3. A composition comprising the agent according to the above-described claim 1.

4. The composition according to the above-described claim 3, which is a pharmaceutical composition.

5. The composition according to the above-described claim 3, which is a food or drink composition.

6. A method for determining whether the intrauterine bacterial flora has been improved or normalized, comprising a step of measuring the Lactobacillus occupancy rate in the intrauterine bacterial flora by a bacterial flora analysis technique based on the amplification of the 16S ribosome RNA gene using a plurality of specimens collected from the inside of the uterus of a subject at two or more time points selected from before the administration or ingestion, during the administration or ingestion, and after the administration or ingestion, of the agent or composition according to claim 1; and a step of calculating the differences between the occupancy rates of the plurality of specimens.

7. The method according to the above-described claim 6, wherein the bacterial flora analysis technique is an amplicon sequencing method of using the 16S ribosome V4 region as an amplification target.

8. A method for treating or preventing a disease caused by a disturbance in the intrauterine bacterial flora, comprising administering an agent for improving the intrauterine bacterial flora comprising lactoferrin or a salt thereof as an active ingredient, or a composition comprising said agent to a subject.

9. The method according to the above-described claim 8, wherein the disease caused by a disturbance in the intrauterine bacterial flora is any one or more of infertility, miscarriage, premature birth, recurrent pregnancy loss, endometriosis, endometrial cancer, cervicitis, endometritis, and/or chorioamnionitis.

Description

MODE(S) FOR CARRYING OUT THE INVENTION

[0027] The agent of the present invention comprises lactoferrin or a salt thereof as an active ingredient. Furthermore, the composition of the present invention, for example, a pharmaceutical composition or a food or drink composition, is such a composition comprising the agent of the present invention comprising lactoferrin as an active ingredient. The agent or the composition of the present invention, for example, a pharmaceutical composition or a food or drink composition, is effective for an improvement of the intrauterine bacterial flora. The improvement of the intrauterine bacterial flora according to the present invention implies increasing of the Lactobacillus occupancy rate in the intrauterine bacterial flora. The method for determining improvement or normalization of the intrauterine bacterial flora will be described below.

[0028] Lactoferrin is a macromolecule having a molecular weight of about 80,000 and has a property of forming a chelate with two trivalent iron ions; however, the “lactoferrin” as used in. the present invention is not limited to biologically derived lactoferrin extracted from the milk and the like of mammals, and any lactoferrin may be used as long as it exhibits the biological activity of lactoferrin, particularly the action of improving the endometrial bacterial flora. Examples thereof include naturally occurring lactoferrins (for example, bovine lactoferrin included in bovine milk) obtainable from various mammals including human being (for example, cow, horse, pig, sheep, goat, and camel etc.), apolactoferrin (iron ion-free type) obtained by removing iron from lactoferrin by a routine method, metal-saturated or unsaturated lactoferrin obtained by chelating metal (iron, copper, zinc, manganese, or the like) ions with apolactoferrin, genetically recombined lactoferrins produced by genetic engineering technology, and products obtained by conjugating polyethylene glycol chains to these lactoferrins. in addition, genetically recombined lactoferrins include a recombinant lactoferrin produced based on modified lactoferrin gene, as well as lactoferrin secreted by transgenic animals, functional equivalents of active fragments of lactoferrin, and the like.

[0029] A salt of lactoferrin that can be used for the present invention is a physiologically acceptable salt of any arbitrary lactoferrin such. as described above, and examples include sodium salt, potassium salt, sulfuric acid salt, and phosphoric acid salt.

[0030] The agent or composition of the present invention may comprise only one kind of lactoferrin such as described above or may comprise two or more kinds thereof. Since lactoferrin is a known substance, a commercially available product can be used. Furthermore, a lactoferrin purified from milk containing lactoferrin or the like by a known method, for example, a method for purifying lactoferrin using a sulfonated carrier (JP H3-109400 A), can be used. In addition, depending on the use application, a fraction from milk or the like, which contains lactoferrin at a high concentration (for example, a fraction obtained by removing saccharides from milk), can also be used.

[0031] The agent of the present invention comprises lactoferrin. as the only essential component; however, the agent or composition of the present invention. may comprise various components and additives that are known in the pharmaceutical or food industries, as desired. The form of the pharmaceutical composition and the food or drink composition of the present invention is not particularly limited. The food or drink composition of the present invention is to be orally administered, and the form of the composition may be various forms such as foods, food materials, beverages, and drinks. The composition of the present invention is preferably in the form of a dosage form convenient for oral administration, such as a powder preparation, a powdered drug, a granular preparation, a tablet, or a capsule.

[0032] Examples of additives that may be contained in a composition such as the pharmaceutical composition or the food or drink composition of the present invention include an excipient, a disintegrant, a lubricating agent, a binding agent, a surfactant, a fluidity promoter, a colorant, and a fragrance, which are conventionally used in pharmaceutical or food industries. These additives are appropriately selected according to the desired dosage form or the like.

[0033] For example, in a case in which the pharmaceutical composition or the food or drink composition of the present invention is in the form of a powdered preparation, a granular preparation, a tablet, a capsule, or the like, examples of the excipient used include monosaccharides and disaccharides such as lactose, sucrose, glucose, sorbitol, and lactitol; starches such as corn starch and potato starch; crystalline cellulose; and inorganic substances such as light silica gel, synthetic aluminum silicate, magnesium metasilicate aluminate, calcium hydrogen phosphate, and silicon dioxide. Furthermore, is addition to the excipient, a binding agent, a disintegrant, a surfactant, a lubricating agent, a fluidity promoter, a colorant, a fragrance, and the like can be appropriately used as necessary.

[0034] Examples of the disintegrant include starches, carboxymethyl cellulose (CMC), hydroxypropyl cellulose (EEC), carboxymethyl cellulose sodium salt, and polyvinyipyrrolidone. Regarding the lubricating agent, sucrose fatty acid esters, calcium stearate, magnesium stearate, and the like can be used.

[0035] Examples of the binding agent include starch, dextrin, powdered gum arabic, gelatin, hydroxypropyl starch, sodium carboxymethyl cellulose, methyl cellulose, crystalline cellulose, ethyl cellulose, and polyvinylpyrrolidone.

[0036] Examples of the surfactant include soybean lecithin and sucrose fatty acid esters; examples of the lubricating agent include talc, wax, sucrose fatty acid esters, hydrogenated vegetable oils, calcium stearate, and magnesium stearate; and examples of the fluidity promoter include anhydrous silicic acid, dry aluminum hydroxide, and magnesium silicate.

[0037] Since lactoferrin is unstable under a high-temperature and highly humid environment, it is preferable that the composition of the present invention is formulated in a dry state.

[0038] Since a lactoferrin powder usually has a very low specific gravity and cannot be directly tableted, in order to obtain the composition of the present invention as a preparation that is more stable and has a well-maintained a pharmacological effect, for example, the active ingredient is mixed with an excipient, a binding agent, and a disintegrant, the mixture is compression-molded with a slug machine to produce a large thin flat disc, the disc is crushed and sieved, and granules having a certain size are obtained. In the case of producing tablets, a lubricating agent is added to the granules, the mixture is tableted, and tablets may be covered with a coating film as desired to be manufactured as commercial products. Furthermore, in the case of producing capsules, a capsule may be filled. with a certain amount of the granules to be produced as capsules.

[0039] The oral (pharmaceutical or food and drink) composition of the present invention may also be produced as an enteric-coated preparation. It is known that lactoferrin is decomposed by pepsin, which is a proteolytic enzyme in the stomach, in an acidic state with gastric acid, and it is considered that the absorption action site of lactoferrin exists in the intestinal tract centered on the small intestine. The method for processing lactoferrin into an enteric-coated preparation is not particularly limited. For example, in order to prepare an enteric-coated preparation, an enteric-coated capsule may be prepared with a coating film containing a base that is resistant to gastric fluid and dissolves in the small intestine, for example, a base selected from the group consisting of shellac, hydroxypropyl methylcellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, a methacrylic acid copolymer, water-insoluble ethyl cellulose, and an aminoalkyl methacrylate copolymer, as a main component, and the capsule may be filled with granules comprising an active ingredient, or a lubricating agent may be added to granules comprising an active ingredient, and the mixture may be tableted, and the tablet thus obtained may be coated. with the above-described coating film.

[0040] Whether the prepared composition is enteric-coated can be checked by testing the disintegrability using the liquid No.1 (pH 1.2, Japanese Pharmacopoeia, General Testing Methods 41) prepared by adding hydrochloric acid and water to 2.0 g of sodium chloride, dissolving the salt, and making the solution up to 1000 ml, and the liquid No.2 (pH 6.8) prepared by adding 118 ml of 0.2 N sodium hydroxide test solution and water to 250 mi of a 0.2 N potassium dihydrogen phosphate test solution up to 1000 ml. Tablets or granules that do not disintegrate when immersed in the liquid No.1 for 120 minutes and disintegrate when immersed in the liquid No.2 for 60 minutes, do not dissolve in the stomach but start to disintegrate only after flowing into the duodenum, so that the active ingredient is eluted, and the tablets or granules can be considered to be enteric-coated.

[0041] Regarding a method capable of delivering lactoferrin to the intestinal tract other than the use of an enteric-coated preparation, the use of a liposome preparation composed of a lipid bilayer is mentioned. A liposome preparation also does not disintegrate in the stomach but is emulsified by bile juice and disintegrates in the small intestine, and therefore, a liposome preparation can deliver lactoferrin to the absorption site in the intestinal tract. Alternatively, a method of simply increasing the pH in the stomach, that is using an alkali agent in combination, to prevent pepsin from acting on lactoferrin, may also be used.

[0042] Examples of the route of administration of the agent or composition of the present invention include oral, transdermal, injection, transintestinal, and intrarectal administration. A preferred route of administration for the pharmaceutical composition and the food or drink composition of the present invention is the oral route.

[0043] The agent or composition of the present invention may be administered alone or may be used in combination with other agent. Furthermore, in a case in which the agent or composition of the present invention is used in combination with other agent, the two may be used simultaneously or may be used one after the other.

[0044] The amount of administration or ingestion per day of the agent or composition of the present invention effective for improving the intrauterine bacterial flora varies depending on the preparation form, the method of administration, the age and body weight of the subject, and the like; however, in the human being, generally, about 0.1 mg to about 5,000 mg, preferably about 0.5 mg to about 2,000 mg, and most preferably about 10 mg to about 1,000 mg, as lactoferrin per day as an active ingredient can be administered all at once or in divisions, before a meal, after a meal, between meals, and/or before bedtime, or the like.

[0045] The agent or composition of the present invention can be administered to human beings and non-human animals, and preferably mammals. Examples of the non-human animals include domestic animals such as cow, horse, pig, and sheep, and companion animals such as dog and cat. The amount of administration or ingestion in a mammal such as a pet (excluding human being) is preferably 0.2 mg to 300 mg/kg of body weight/day as lactoferrin.

[0046] Whether the intrauterine bacterial flora has been improved or normalized. by the agent or composition of the present invention can be determined by measuring the Lactobacillus occupancy rate in the intrauterine bacterial flora by a bacterial flora analysis technique based on the amplification of the 16S rRNA gene using a specimen collected from the uterus of a test subject at two or more time points selected from before administration or ingestion, during administration or ingestion, or after administration or ingestion of the agent or composition of the present invention, comparing the occupancy rates of a plurality of specimens collected at different time points of collection, and calculating the differences.

[0047] The specimen is a sample collected from the inside of the uterus and may be a sample including the intrauterine bacteria, and the method of collection is also not limited; however, for example, the uterine cavity fluid collected using a suction pipette, or the like is used.

[0048] The combination of the time point of collecting specimens is not particularly limited and is acceptable so long as there is an interval of one day or longer between the time points, and the time points may be any time points before administration or ingestion, during administration or ingestion, or after administration or ingestion of the agent or composition of the present invention. It is preferable that the specimens obtained at two or more time points include at least one specimen obtained. during administration or ingestion, or after administration or ingestion of the agent or composition of the present invention. For example, in the case of collecting specimens at two time points, it is preferable to collect at any combination of before administration or ingestion and during or after administration. or ingestion. of the agent or composition of the present invention.; two different time points during administration or ingestion; during administration or ingestion and after administration or ingestion; and two different time points after administration. or ingestion.

[0049] The bacterial flora analysis technique used for the determination method of the present invention generally comprises the following steps: (1) the genomic DNA in a specimen collected from a test subject is extracted, and partial or full lengths of the DNA sequences of bacteria-derived 16S rRNA gene is amplified; (2) a library in which an adapter sequence is added to the DNA amplification products is produced, and sequencing is performed with a next-generation sequencer; and (3) the sequence reads thus obtained. are combined. with a data analysis software, and the origin of bacteria and the occupancy rates in the specimen are estimated.

[0050] Regarding the bacterial flora analysis technique based. on the amplification. of the 16S rRNA gene, for example, a 16S amplicon sequencing method, a shotgun sequencing method, a full-length 16S amplicon sequencing method, or any bacterial flora analysis methods equivalent to those techniques is mentioned. Regarding the amplification target region of the 16S rRNA gene, it is preferable to use the variable regions of V1-V2, V1-V5, and/or V4, which are generally used for 16S rRNA sequencing of human specimens. Particularly, it is preferable to use a primer set intended for the V4 region, with which the bacteria existing in the genital organs can be detected with high sensitivity, and amplicon sequencing methods intended for the V4 region are most preferred because a wide range of microorganisms including Pseudomonas, Escherichia, Salmonella, Lactobacillus, Enterococcus, Listeria, Bacillus, Gardnerella, Bifidobacterium, and two kinds of yeast can be detected.

[0051] According to the present invention, the state in which the intrauterine bacterial flora has been normalized refers to a state in which when a specimen collected from. the inside of the uterus is measured by a bacterial flora analysis technique based on amplification of the 16S rRNA gene as described above, the Lactobacillus occupancy rate is 50% or higher, and this Lactobacillus occupancy rate is preferably 70% or higher, and more preferably 90% or higher. In a case in which the difference between measured values obtained by various methods for the same specimen exceeds the general range of error, the value measured by a bacterial flora analysis method. according to the 16S amplicon sequencing method of using a next-generation sequencer (for example, trade name “MiSeq” (Illumine K.K.)) is used as a reference.

[0052] According to the present invention, the state in which the intrauterine bacterial flora has been improved implies that in a case in which specimens collected from the inside of the uterus at two time points are measured by the bacterial flora analysis method as described above, the Lactobacillus occupancy rate in the specimen collected later has increased as compared to the specimen collected earlier.

EXAMPLES

[0053] Hereinafter, the present invention will be described more specifically by way of Examples.

Example 1

[0054] The inventors of the present invention conducted an intrauterine bacterial flora analysis on in vitro fertilization patients of age 45 or less who consulted an infertility treatment clinic, and as a result, patients (test subjects) whose intrauterine bacterial balance was disturbed were subjected to oral administration of lactoferrin to examine the effect.

[0055] <Intrauterine Bacterial Flora Analysis>

[0056] The intrauterine bacterial flora analysis was carried out as follows, basically according to the method described in Non-Patent Document 6. The mucus of the cervical canal and the uterine cervix of a test subject was wiped off, a catheter for embryo transplantation (trade name “Kitazato (registered trademark) IUI catheter” (Kitazato Corporation, Japan)) was carefully inserted through the vagina into the uterus, and the uterine cavity fluid was collected as a specimen. The collected uterine cavity fluid was transferred into 1 mL of a preservative solution for bacterial inactivation and stabilization (trade name “MMB collection tube” (DNA Genotek Inc., Canada)).

[0057] In order to extract the bacterial genomic DNA, Proteinase K and lysozyme were added to the preservative solution to dissolve bacteria. The genomic DNA was extracted using a DNA extraction kit (trade name “Agencourt Genfind v2 Blood & Serum DNA Isolation Kit” (Beckman Coulter, Inc., USA)). The concentration of the extracted DNA was measured using trade name “(Dubit dsDNA HS Assay Kit” (Thermo Fisher Scientific K.K.).

[0058] The bacterial flora analysis was carried out according to the 16S amplicon sequencing method of using a next-generation sequencer. Based on. the protocol of “Earth Microbiome Project” (Non-Patent Document 12), amplification of bacterial DNA was carried out using a primer in which a sequence that amplifies the V4 region of the 16S rRNA gene and an Illumina Nextera KT adapter sequence were linked (Non-Patent Document 6). A mixed liquid of 25 ng/μL of DNA for PCR, 200 μmol/L of deoxyribonucleotide triphosphate, 400 nmol/L of each primer, 2.5 U of trade name “FastStart HiFi polymerase”, 20 mg/mL of BSA (Sigma), 0.5 mol/L of betaine (Sigma), and a buffer including MgCl.sub.2 (Roche) was produced. PCR was performed with a thermal cycler (trade name “SimpliAmp Thermal Cycler” (Thermo Fisher)), and the DNA was denatured at 94° C. for 2 minutes, subsequently subjected to 30 cycles, each cycle including maintaining for 20 seconds at 94° C., 30 seconds at 50° C., and 1 minute at 72° C., and finally the DNA was reacted for 5 minutes at 72° C. The PCR product was purified with trade name “Agencourt AMPure XL)” (Beckman Coulter, Inc., USA). based on “Illumina 16S Metagenomic Sequencing Library Preparation protocol” (https://support.illumina.com/documents/documentation/chemistrydocumentation/163/16s-metagenomic-library-prep-quide-15044223-b.pdf), a library was created using trade name “Nextera XT Index kit” (Illumina, Inc., USA). For the library thus created, sequencing was performed by pair-end sequencing of 2×200-bp using trade name “MiSeq Reagent Kit v3” (Illumina K.K.). Regarding the data analysis, a quality check of the entire sequence was performed using “fastqc2C” (https://www.bonformatics.babraham.ac.uk/projects/fastqc/), and identification of bacteria to the genus level was performed using “USEARCH” (https://www.drive5.com/usearch/) and “QIIME” (http://qiime.org/).

[0059] <Case 1>

[0060] In this test subject, a non-Lactobacillus Dominant Microbiota composed of 40% of Lactobacillus bacteria and 60% of Streptococcus bacteria was recognized. This test subject was administered with. 750 mg/day of amoxcillin, a penicillin antibiotic, for 7 days. Subsequently, a commercially available Lactoferrin preparation (trade name “LACTOFERRIN GX” (manufactured by NRL Pharma, Inc.)) was orally administered at a dose of 300 mg/day as the amount of lactoferrin. After 50 days of continuous lactoferrin administration, a second intrauterine bacterial flora analysis was performed, and as a result, Lactobacillus bacteria increased to 100%, and an improvement of the intrauterine bacterial flora was recognized.

[0061] Amoxicillin has a weak effect on anaerobic bacteria, amoxicillin alone has a low effect of increasing Lactobacillus bacteria, and rather, there is a possibility that Lactobacillus bacteria may be a target of amoxicillin. Therefore, it is considered that by the administration of lactoferrin, Lactobacillus bacteria proliferated in the uterus, and the intrauterine bacterial flora was improved.

[0062] <Case 2>

[0063] In this test subject, the intrauterine bacterial flora was extremely unbalanced, and the results of the initial intrauterine bacterial flora analysis were 0.1% of Lactobacillus bacteria, 94.7% of Streptococcus bacteria, and 5.2% of others. This test subject was administered with 750 mg/day of amoxicillin for 14 days. From the 5th day after the initiation of administration of amoxicillin (32.sup.nd day after the initial examination), the same lactoferrin preparation as that of Case 1 was continuously orally administered at a dose of 300 mg/ day as the amount of lactoferrin. In the second intrauterine bacterial flora analysis carried out after completion of the administration of amoxicillin (57.sup.th day after the initial examination/25.sup.th day after the initiation of administration of lactoferrin), Streptococci were reduced to 17%, and Lactobacilli were increased to 16.2%; however, a noticeable increase in other bacteria such as Escherichia bacteria was recognized. Since the effect of amoxicillin alone was not recognized, 300 mg/day of cefdinir, a cephem antibiotic, was administered for 7 days from the 53.sup.rd day after the initiation of administration. of lactoferrin (85.sup.th day after the initial examination), and also, a probiotic tampon (trade name “Florgynal Tampon Probiotigue” (Laboratoires IPRAD, Paris, France)) was used in combination. After completion. of these treatments, only the administration of the lactoferrin preparation was continued, and as a result, the proportion of Lactobacillus bacteria was improved to 98.1% in the third examination carried out on the 119.sup.th day from the initial examination. The administration of the lactoferrin preparation was continued for 142 days at a dose of 300 mg/day as the amount of lactoferrin. This test subject subsequently naturally conceived.

[0064] Therefore, it was revealed that lactoferrin can improve an imbalance of the intrauterine bacterial flora and can exhibit an effect of preventing or improving various problems caused by a disturbance in the intrauterine bacterial flora. Furthermore, during the implementation of this test, there were no reports of side effects. Therefore, it can be said that this method and the lactoferrin preparation used therefor are highly safe.

[Example 2] Production of Lactoferrin Tablets

[0065] To 20 kg of raw lactoferrin powder extracted from milk (purity of 95% or higher as a protein; lactoferrin in proteins was 90% or more), 18.5 kg of lactose, 19.7 kG of crystalline cellulose (trade name “Avicel”), 1.2 kg of carboxymethyl cellulose calcium salt, and 0.6 kg of sucrose fatty acid esters were added, a mixture thus obtained was pulverized with a mixer, and a powder that passed through 100-mesh sieve was obtained. This mixed powder was tableted using a tableting machine to obtain tablets each having a major axis of 9 mm and a weight of 300 mg. One tablet contained 100 mg of the raw lactoferrin powder.

[Example 3] Production of Enteric-Coated Lactoferrin Tablets (Part 1)

[0066] The tablets produced in Example 1 were introduced into a coating machine (manufactured by Freund Corporation, HICOATER HCT-48N). An enteric coating liquid composed of 9.6 wt % of shellac, 1.5 wt % of L-arginine, 1.9 wt % of sorbitol, 2.4 wt % of sucrose fatty acid esters, 4.8 wt % of ethanol, and 79.8% of purified water was sprayed on those tablets, and an enteric coating was provided at a ratio of 8% to 9% by mass with respect to the tablets to obtain a manufactured product.

[Example 4] Production of Enteric-Coated Lactoferrin Tablets (Part 2)

[0067] The tablets produced in Example 1 were introduced into a coating machine (manufactured by Freund Corporation, HICOATER HCT-48N). An enteric coating liquid composed of 9 wt% of carboxymethyl cellulose, 1 wt % of glycerin fatty acid esters, 45 wt % of ethanol, and 45 wt % of methylene chloride was sprayed. on those tablets, and an. enteric coating was provided at a ratio of 12% by mass with respect to the tablets to obtain a manufactured product.

[Example 5] Production of Enteric-Coated Lactoferrin Tablets (Part 3)

[0068] The tablets produced in Example 1 were introduced into a coating machine (manufactured by Freund Corporation, HICOATER HCT-48N). An enteric coating liquid obtained by dissolving 8 parts by mass of zein, which is a protein obtainable from corn kernel, and 2 parts by mass of glycerin in 150 parts by mass of a 70 wt % aqueous solution of ethanol was sprayed on those tablets, and tablets with a coating at a ratio of 10% by mass with respect to the tablets were obtained.

[Example 6] Production of Enteric-Coated Lactoferrin Tablets (Part 4)

[0069] The tablets produced in Example 1 were introduced into a coating machine (manufactured by Freund Corporation, HICOATER HCT-48N). An enteric coating liquid obtained by dissolving 30 parts by mass of shellac and 7 parts by mass of castor oil in 63 parts by mass of isopropanol was sprayed on those tablets, and tablets with a coating at a ratio of 10% by mass with respect to the tablets were obtained.

[0070] This patent application is based on Japanese Patent Application No. 2018-176473, filed on Sep. 20, 2018, and the subject matters described in the specification and the scope of the claims of Japanese Patent Application No. 2018-176473 are all incorporated in this specification.