TW202140007A - Novel modifying reagents for the presence ratio of intestinal microflora - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for improving intestinal flora, comprising a quinolone compound as an active ingredient.

Description

Novel modifying reagent for the existence ratio of intestinal microflora

The present invention relates to a medical product for treating and/or preventing diseases, which is expected to have therapeutic benefits by changing the intestinal flora. More specifically, the present invention relates to medicinal products for the treatment and/or prevention of obesity, diabetes, etc., in which quinolinone compounds are the active ingredients.

Recently, the relationship between intestinal bacteria and various diseases has been reported, and these reports include the relationship between Akkermansia muciniphila and obesity or diabetes. Akkermansia is a novel genus proposed in 2004 (Non-Patent Document 1). The strains belonging to this genus are about one genus, and its representative species is Akkermansia muciniphila. This bacterium is a gram-negative obligate anaerobe (gram-negative obligate anaerobe), which is a non-motile, non-spore-forming Eubacterium ellipsoidea. The main feature of this bacterium is that it is a mucin-metabolizing bacterium (hence its name), and it is believed that mucin is used as a carbon source necessary for its culture conditions.

It is known that the occupancy rate of Akkermansia muciniphila in the intestinal flora decreases in diabetic or obese patients. It has been reported that when the content of Akkermansia muciniphila in the intestinal flora of high-fat diet mice increases to the average content of the control group, the body weight decreases, the body fat rate decreases, and the intestinal mucus layer thickens (Non-Patent Documents 2 and 3). In addition, according to other previous documents, it is known that the increase in the content of Akkermansia muciniphila in the intestinal flora is suitable for increasing the mucin layer, improving the intestinal barrier function and treating inflammatory bowel disease (Non-Patent Documents 4 and 5) , Fatty liver, hepatitis, appendicitis (Non-Patent Document 6) and Diabetes (Non-Patent Document 7). In addition, the relationship between Akkermansia muciniphila and central nervous system diseases such as epilepsy and amyotrophic lateral sclerosis (ALS) has also been studied (Non-Patent Documents 8 and 9).

Patent Document 1 discloses a specific quinolinone antimicrobial agent, which exhibits antibacterial activity against Clostridioides difficile living in the intestine. [Citation List] [Patent Literature]

[PL 1] WO2013/029548 [Non-Patent Literature]

[NPL 1] M Derrien, M. International Journal of Systematic and Evolutionary Microbiology (2004). Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. [NPL 2] Karlsson CL, et al., Obes (Silver Spring) (2012). The microbiota of the gut in preschool children with normal and excessive body weight [NPL 3] Dao MC, et al., Gut (2016). Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology. [NPL 4] Png CW, et al., Am J Gastroenterol (2010). Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. [NPL 5] Lyra A, et al., World J Gastroenterol (2012). Comparison of bacterial quantities in left and right colon biopsies and faeces. [NPL 6] Swidsinski A, et al., Gut (2011). Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum [NPL 7] Zhang X, et al., PLoS One (2013). Human gut microbiota changes reveal the progression of glucose intolerance. [NPL 8] Christine A. Olson,; Helen E. Vuong,; Jessica M. Yano,; Qingxing Y. Liang,; David J. Nusbaum,; Elaine Y. Hsiao, et al., The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet, Cell, 2018, 173 [NPL 9] Eran Blacher,; Stavros Bashiardes,; Hagit Shapiro,; Daphna Rothschild,; Uria Mor,; Mally Dori-Bachash, et al., Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature, 2019, No. Volume 572

[technical challenge]

The main purpose of the present invention is to provide a novel medicament, which is expected to have therapeutic benefits for some diseases, such as obesity, diabetes, etc., by changing the intestinal flora, especially increasing the intestinal mucus layer. [Solution to the problem]

The present inventors have thoroughly studied, and subsequently discovered that the known quinolinone antimicrobial agent, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino- 3-cyano-5-pyridyl)-4-oxo-3-quinoline-carboxylic acid can improve the intestinal flora, especially significantly increase the occupancy rate of Akkermansia which is known to thicken the mucin layer , And it can be effectively used to treat diseases that are expected to be treatable by this effect, such as obesity and diabetes. Based on the new findings, the present invention has been completed.

The present invention includes the following embodiments. (Article 1) A pharmaceutical composition for improving intestinal flora, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5 -Pyridyl)-4-Pendoxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient.

(Article 2) The pharmaceutical composition of Clause 1, wherein the intestinal flora is improved by increasing the intestinal occupancy rate of Akkermansia.

(Article 3) The pharmaceutical composition according to Clause 1, wherein the intestinal mucus layer is thickened by improving the intestinal flora.

(Article 4) An agent for treating and/or preventing diseases expected to be improved by increasing the intestinal occupancy rate of Akkermansia, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro -8-Methyl-7-(2-amino-3-cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof is used as the active ingredient.

(Article 5) An agent for treating and/or preventing diseases that are expected to be improved by thickening the intestinal mucus layer, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7 -(2-Amino-3-cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient.

(Article 6) The agent of Clause 4 or 5, wherein the disease is obesity and/or diabetes.

(Article 7) A medicament for treating and/or preventing obesity and/or diabetes, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3 -Cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient.

(Article 8) Such as the medicament of any one of clauses 4 to 7, which is for oral administration.

(Article 9) The medicament according to any one of clauses 4 to 8, wherein the daily dose of the active ingredient is 0.1 mg to 30000 mg.

(Article 10) A method for treating and/or preventing obesity and/or diabetes, which comprises administering a therapeutically effective amount of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl to a patient in need Group-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof.

(Article 11) A kind of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3- The use of quinoline-formic acid or a pharmaceutically acceptable salt thereof for the manufacture of medicaments for the treatment and/or prevention of obesity and/or diabetes.

(Article 12) A kind of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3- Quinoline-formic acid or a pharmaceutically acceptable salt thereof is used for the treatment and/or prevention of obesity and/or diabetes. [Impact of Invention]

Oral administration of the compound of the present invention can quickly and greatly increase the occupancy rate of Akkermansia in the intestinal flora, and reorganize the intestinal flora, in which Akkermansia mucinophila is dominant. And restoring the intestinal flora can increase mucin production. Therefore, the present invention is expected to be an agent for treating and/or preventing obesity or diabetes by improving the above intestinal flora. In addition, the compound of the present invention is a poorly absorbable drug, and therefore it is distributed in the intestinal tract at a high concentration when it is orally administered, but its blood metastasis is low. Therefore, the compound of the present invention also has the advantage that the risk of systemic side effects (which is a problem with existing quinolinone antibacterial agents) is low.

The compound of the present invention 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo- 3-quinoline-carboxylic acid has a structure of formula (1), which is disclosed as compound numbers 2 to 18 in Patent Document 1 , which also discloses its manufacturing process and its antibacterial activity against Clostridium difficile. [Chemical formula 1]

The compounds of the present invention may be in the form of hydrates and/or solvates, and therefore, the compounds of the present invention also encompass their hydrates and/or solvates. In addition, the compounds of the present invention in which any one or more ¹ H atoms ^{are replaced by 2} H (D) atoms are also within the scope of the present invention. Polymorphism may exist in the crystals of the compound of the present invention or a pharmaceutically acceptable salt thereof, and therefore such crystal polymorphism is also within the scope of the present invention.

"Pharmaceutically acceptable salts" include: salts with inorganic acids as acid addition salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate and phosphate; and Salts of organic acids, such as oxalate, malonate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoro Acetate, acetate, methanesulfonate, p-toluenesulfonate and triflate; and salts with amino acids, such as glutamate and aspartate; and salts with bases, Alkali metal salts, such as sodium salt and potassium salt; alkaline earth metal salts, such as calcium salt; and ammonium salt.

"Intestinal flora" is a general term for the group of bacteria that live in the intestines of humans or animals. "Intestinal flora" in this context means a complex intestinal microbial ecosystem composed of about 100 trillion intestinal bacteria. The intestinal bacteria are composed of about 1,000 bacterial species. They mainly live in the large intestine and maintain their relationship with each other. Close relationship and balance. In the present invention, "improvement of intestinal flora" means to increase the intestinal occupancy rate of Akkermansia as the main improvement effect, and the representative species of Akkermansia is Akkermansia muciniphila . Akkermansia is a mucin metabolizing bacterium, and it is expected that the increase in the intestinal occupancy rate of Akkermansia can accelerate the increase of the mucin layer and the improvement of the intestinal barrier function, which will further lead to obesity, diabetes, and inflammation. Bowel disease, fatty liver, hepatitis, appendicitis, diabetes, epilepsy, amyotrophic lateral sclerosis (ALS), autism, atopic dermatitis, etc. and anti-cancer activity (such as immune checkpoint inhibitors such as PD1 antibody) Enhance the therapeutic effect of).

The administration route of the compound of the present invention can be selected from oral administration or direct rectal administration, such as enema preparations and suppositories, preferably oral administration. The daily dose depends on the compound, the route of administration, the patient's condition, the patient's age, and the like. For example, in the case of oral administration, it can generally be from about 0.02 mg to about 500 mg per kilogram of human or mammal body weight, preferably from about 0.01 mg to about 200 mg, more preferably from about 1 mg to about 1 mg. A dose of about 10 mg, and even more preferably about 2 mg to about 20 mg, is administered in one to several servings. For example, the daily dose for humans includes about 0.1 mg to about 30000 mg, preferably about 5 mg to about 12000 mg, more preferably about 50 mg to about 6000 mg, even more preferably about 100 mg to about 1200 mg.

The dosage forms of the present invention include tablets, capsules, granules, powders, liquids, syrups, suspensions, enemas and suppositories. These dosage forms can be prepared in a conventional manner. If the dosage form is in liquid form, it can be a formulation to prepare a solution or suspension by mixing it with water, a suitable aqueous solution, or other suitable solvent. Tablets and granules can be coated in a well-known manner. The dosage form can be prepared in a known manner together with pharmaceutically acceptable additives. According to the intended use, the additives used herein include excipients, disintegrants, binders, fluidizers, lubricants, coating agents, colorants, solubilizers, cosolvents, thickeners, dispersants, stabilizers, Sweeteners and flavoring agents. For example, it includes lactose, mannitol, dibasic calcium phosphate, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, calcium carboxymethyl cellulose, croscarmellose Sodium cellulose, crosprovidone, sodium starch glycolate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, calcium stearate, Sodium stearyl fumarate, polyethylene glycol, propylene glycol, titanium oxide, talc, ferric oxide and yellow iron oxide.

In the case where the compound of the present invention is formulated into a single dosage form, the dosage form may include 0.1 to 85% (w/w) of the compound of the present invention relative to the entire composition, but the present invention is not limited thereto. Preferably, it is 10 to 70% (w/w) relative to the entire composition.

In addition, the compound of the present invention can be used in combination with another drug or as a combination with another drug for the purpose of enhancing the effect and/or alleviating side effects. [Example]

The present invention is explained in more detail below with reference to examples, however, the present invention is not limited thereto. The compounds of the present invention (hereinafter referred to as "test substances") and reference drugs used herein are obtained as shown below. Test substance [1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo- 3-quinoline-formic acid] was obtained from Otsuka Pharmaceutical Co., Ltd. in Japan.

Instance 1. Effect of test substance on intestinal flora in normal mice The test substance was administered to normal Balb/c mice over 21 days, and the feces excreted during this period were analyzed to evaluate the influence of the test substance on the intestinal flora. (Test Methods) Balb/c mice were orally administered with 5% gum arabic or test substance (10 mg/kg) as an administration vehicle once a day, and feces were collected on the 7th and 21st days. The collected feces were ground/homogenized with buffer in EZ-beads (Promega), and used Maxwell according to its instruction manual.^TM RSC automatic nucleic acid purification system (Promag) processes the supernatant to extract bacterial genomic DNA. Use DropSense96 (SCRUM) to measure the concentration of bacterial genomic DNA samples. Then the concentration of the sample was configured to 5 ng/μL, and it was used as a PCR template to prepare amplicons by PCR enrichment of the V4 region of the ribosomal RNA gene. Mix the prepared amplicon (5 μL), Nextera Index 1 adapter (N7xx) (5 μL), Nextera Index 2 adapter (S5xx) (5 μL), 2×KAPA HiFi HS ReadyMix (25 μL) And nuclease-free water (10 μL) to prepare a reaction solution, and the reaction solution was circulated 8 times (95°C×30 seconds, 55°C×30 seconds, 72°C× 30 seconds) and 72°C×5 minutes to prepare a library. The obtained library was purified with Agencourt AMPure XP, its concentration was measured with DropSense 96, and then its quality was evaluated with 2100 bioanalyzer electrophoresis system (Agilent). The concentration of each library was configured to 1 nM, and the same amount of each was mixed to prepare a sequencing library (NGS library) for analysis. The NGS library was denatured with 0.1 N NaOH to obtain a single-stranded product, the product was neutralized, and the concentration of the neutralized product was re-adjusted to about 1.5 pM, and analyzed with the next-generation sequencer MiniSeq (Illumina) according to its instruction manual. The sequencing step is performed by using the next generation sequencer (MiniSeq), and the nucleotide sequence file (.fastq) is automatically generated in the sequencer. Use the above sequence file (.fastq) of the V4 region on the 16S rRNA gene to classify the bacterial species in the intestinal flora by using the application software 16S Metagenomics (Illumina, Inc.) for metagenomics.

(Results) The analysis results of the intestinal flora on the 7th and 21st days are shown in Tables 1 and 2 below. For mice administered with vehicle, there was no change in the intestinal flora over time. On the other hand, compared with the vehicle control group, the intestinal flora of the mice administered the test substance changed significantly on the 7th day. The occupancy rate of Akkermansia in the fecal intestinal flora of the test substance administration group increased to more than 50%. On day 21, the occupancy rate decreased, but the rate was still higher than that of the vehicle control group. Table 1. Results on the 7th day (%) Belong to Vehicle Control 1 Vehicle Control 2 Vehicle Control 3 Test substance 1 Test substance 2 Test substance 3 Akkermansia 2.0 2.5 1.5 70.5 54.4 69.4 Lactobacillus 33.0 63.7 48.8 16.8 28.5 12.9 Blautia 16.7 5.7 11.4 0.3 4.8 2.8 Coprobacillus 0.2 0.1 0.5 0.0 0.0 0.0 Prosthecobacter 0.3 0.4 0.2 5.4 4.5 5.3 Bacteroides 7.5 6.5 4.4 0.5 0.5 1.5 Clostridium 2.0 0.8 3.2 0.3 0.5 0.2 Parabacteroides 4.5 3.9 2.8 0.3 0.3 0.9 Acetobacterium 0.2 0.2 0.1 0.0 0.0 0.0 Luteolibacter 0.0 0.0 0.0 1.1 0.9 1.1 Erysipelothrix 0.4 0.1 0.8 0.1 0.1 0.0 Alkaliphilus 4.4 1.2 4.0 0.1 0.0 0.1 Ruminococcus (Ruminococcus) 3.8 1.4 3.0 0.0 0.0 0.1 Rubritea (Rubritalea) 0.0 0.0 0.0 0.9 0.8 0.9 Mucispirillum 0.0 0.0 0.0 0.0 0.0 0.0 Pediococcus 0.8 1.5 0.9 0.1 0.1 0.1 Macrococcus (Macrococcus) 0.1 0.0 0.1 0.0 0.0 0.0 Oscillospira 1.8 1.0 1.2 0.0 0.1 0.1 other 13.4 5.8 10.0 1.0 1.6 1.8 uncategorized 8.8 5.4 7.1 2.6 2.8 2.8 Table 2. Results on Day 21 (%) Belong to Vehicle Control 1 Vehicle Control 2 Vehicle Control 3 Test substance 1 Test substance 2 Test substance 3 Akkermansia 1.3 1.4 1.7 23.4 17.5 18.6 Lactobacillus 50.0 60.3 34.9 13.6 20.7 18.4 Broutella 10.9 6.0 18.2 38.6 31.3 35.0 Faecium 0.1 0.0 0.3 6.6 12.4 10.5 Pseudomonas 0.1 0.3 0.3 2.0 1.4 1.6 Bacteroides 4.6 6.4 6.5 0.6 0.6 0.7 Clostridium spp 0.9 1.7 1.5 2.1 1.3 1.1 Parabacteroides 2.6 4.3 3.5 0.2 0.2 0.2 Acetobacter 0.1 0.1 0.2 1.3 0.9 1.3 Lutlibacter 0.0 0.0 0.0 0.4 0.3 0.3 Erysipelas 0.9 0.7 1.1 0.6 1.1 1.0 Alkalis 1.6 1.1 1.8 0.1 0.1 0.1 Rumenococcus 1.9 0.9 3.2 0.0 0.1 0.1 Lubliella 0.0 0.1 0.0 0.3 0.3 0.3 Pseudomonas 0.0 0.0 0.0 1.0 0.5 0.5 Pediococcus 0.9 1.5 1.1 0.2 0.3 0.3 Macrococcus 0.0 0.0 0.1 0.5 0.9 0.8 Oscillatoria 1.4 0.6 2.0 0.1 0.1 0.1 other 16.4 7.4 14.6 4.1 4.6 4.0 uncategorized 6.1 7.3 8.8 4.1 5.6 5.2

Instance 2. Effect of test substance on intestinal flora in mice with colitis (Test Methods) Use untreated CD4+ T cells to isolate the set of mice (Miltenyi Biotec), remove untreated T cells (CD4 + CD62L + CD44− cells) from the spleen of Balb/c mice, and use 500 μL/body (5×105 Individual cells/body) were transplanted into the abdominal cavity of immunocompromised mice (SCID mice) to prepare colitis model mice. 14 days after cell transplantation, mice were divided into groups based on their body weight. To each group of mice, 5% gum arabic or test substance (10 mg/kg) was orally administered as an administration vehicle once a day for 21 days. Untreated mice means SCID mice that have not received transplantation. The next generation sequencer was used to analyze the intestinal flora in the collected feces. The detailed analysis and analysis method are the same as in Example 1.

(Results) The analysis results of the 21-day intestinal flora are shown in Tables 3 to 5 below. For the vehicle control group and the untreated group, there was no significant change in the intestinal flora over time. On the other hand, the intestinal flora of the mice administered with the test substance changed significantly. On day 7 or later, the occupancy rate of Akkermansia in the intestinal flora of feces in the test substance administration group increased significantly. Table 3. Vehicle control group (%) Belong to 0 days 1 day 3 days 7 days 14 days 21 days Akkermansia 0.0 0.0 0.0 0.0 0.0 0.0 Bacteroides 26.6 28.5 31.5 24.2 19.7 11.7 Lactobacillus 7.6 18.8 18.5 32.0 23.8 48.5 Broutella 29.4 19.9 14.0 14.9 11.8 4.7 Parabacteroides 7.3 7.0 10.5 7.8 17.3 9.0 Rumenococcus 4.6 2.9 3.2 2.8 2.6 1.4 Adlercreutzia 2.9 3.5 3.4 2.8 1.5 1.1 Oscillatoria 2.8 1.8 2.2 1.6 1.8 0.6 Alkalis 0.5 1.9 1.7 1.5 2.0 0.4 Johnsonella 1.0 0.6 0.8 0.3 0.4 0.2 Rhodothermus 1.1 0.6 0.5 0.4 0.3 0.0 Clostridium spp 0.8 0.6 0.8 0.5 0.7 0.7 Pseudobutyrivibrio 1.7 1.0 1.1 0.4 0.3 0.0 Sphingobacterium 0.7 0.8 0.9 0.7 0.7 0.4 Slackia 0.8 0.7 0.6 0.5 0.3 0.2 Pediococcus 0.4 0.6 0.5 0.6 0.5 0.7 Escherichia 0.1 0.2 0.2 0.1 0.8 3.9 Emeticia (Emticicia) 0.6 0.3 0.2 0.3 0.6 0.3 other 5.0 4.8 4.0 3.9 5.6 7.7 uncategorized 6.2 5.6 5.6 4.7 9.3 8.3 Table 4. Test substance administration group (10 mg/kg) (%) Belong to 0 days 1 day 3 days 7 days 14 days 21 days Akkermansia 0.0 0.0 0.0 13.7 26.2 21.1 Bacteroides 15.1 71.9 80.8 70.0 49.2 37.7 Lactobacillus 5.0 1.3 0.1 0.1 4.4 4.2 Broutella 39.4 6.0 3.4 0.9 0.5 0.9 Parabacteroides 3.2 8.1 7.1 6.6 9.6 25.3 Rumenococcus 6.9 1.3 0.9 0.1 0.0 0.0 Anderella 3.2 0.0 0.0 0.0 0.0 0.0 Oscillatoria 3.7 1.8 0.2 0.1 0.1 0.4 Alkalis 0.8 0.1 0.0 0.0 0.0 0.0 Johansenia 1.6 0.4 0.1 0.1 0.1 0.1 Rhodothermophilus 1.4 0.2 0.1 0.0 0.0 0.0 Clostridium spp 0.8 1.2 0.5 0.4 0.6 0.9 Pseudobutyl Vibrio 1.9 0.1 0.0 0.0 0.0 0.0 Sphingomyces 0.3 0.7 0.6 0.7 0.6 0.4 Schreckia 1.0 0.1 0.0 0.0 0.0 0.0 Pediococcus 0.4 0.1 0.0 0.0 0.2 0.1 Escherichia 0.1 0.9 0.0 0.0 0.0 0.0 Ermitia 0.0 0.0 0.0 0.0 0.0 0.0 other 8.0 2.1 2.6 3.7 4.5 3.9 uncategorized 7.2 3.7 3.5 3.7 4.0 4.9 Table 5. Untreated group (%) Belong to 0 days 1 day 3 days 7 days 14 days 21 days Akkermansia 0.0 0.0 0.0 0.0 0.1 0.0 Bacteroides 21.7 21.2 27.0 10.1 12.8 6.9 Lactobacillus 38.1 41.4 9.2 33.5 45.4 22.4 Broutella 14.5 14.8 29.3 27.4 17.7 23.4 Parabacteroides 3.5 2.8 4.6 1.6 2.2 13.5 Rumenococcus 3.3 3.3 6.0 4.7 3.0 5.3 Anderella 3.3 2.0 2.0 2.0 2.7 1.5 Oscillatoria 1.5 1.2 3.3 2.7 1.4 2.5 Alkalis 0.3 0.3 0.6 0.5 0.8 4.8 Johansenia 0.9 0.7 1.7 1.9 1.1 0.6 Rhodothermophilus 0.9 0.8 1.3 1.4 1.0 1.4 Clostridium spp 0.5 0.4 1.1 0.8 0.4 0.7 Pseudobutyl Vibrio 0.2 0.3 0.6 0.6 0.2 0.1 Sphingomyces 0.4 0.3 0.4 0.2 0.2 0.3 Schreckia 0.7 0.6 0.7 0.8 0.7 0.7 Pediococcus 0.8 0.7 0.3 0.6 0.8 0.4 Escherichia 0.0 0.0 0.0 0.0 0.0 0.0 Ermitia 0.0 0.0 0.0 0.0 0.4 0.6 other 4.0 4.4 5.6 5.4 4.3 6.4 uncategorized 5.3 4.8 6.3 5.9 5.1 8.4

Instance 3. Analyze the intestinal flora and measure the test substance administered twice a day (100 mg/kg) The amount of mucin in the feces of the rat The vehicle (5% acacia) or the test substance (100 mg/kg) was administered to normal SD rats twice a day, and the stool was collected daily, and the intestinal flora in the stool was analyzed and the amount in the stool was measured. The amount of mucin. (Test Methods) SD rats were orally administered 5% gum arabic or test substance (100 mg/kg) as the administration vehicle twice a day, and feces were collected every day. The collected feces are ground/homogenized in EZ-beads (Promag) with buffer solution, and use Maxwell according to its instruction manual.^TM RSC automatic nucleic acid purification machine (Promag) processes the obtained supernatant to extract bacterial genomic DNA. The concentration of the bacterial genomic DNA sample obtained was analyzed with DropSense96 (SCRUM), the concentration of the sample was configured to be 5 ng/μL, and then the V4 region of the ribosomal RNA gene was concentrated by PCR to prepare amplicons. Mix the prepared amplicon (5 μL), Nextera Index 1 adapter (N7xx) (5 μL), Nextera Index 2 adapter (S5xx) (5 μL), 2×KAPA HiFi HS ReadyMix (25 μL) And nuclease-free water (10 μL) to prepare a reaction solution, and the reaction solution was circulated 8 times (95°C×30 seconds, 55°C×30 seconds, 72°C× 30 seconds) and 72°C×5 minutes to prepare a library. The obtained library was purified with Agencourt AMPure XP, its concentration was analyzed with DropSense 96, and then its quality was evaluated with 2100 bioanalyzer electrophoresis system (Agilent). The concentration of each library was configured to 1 nM, and the same amount of each was mixed to prepare an NGS library. The NGS library was denatured with 0.1 N NaOH to obtain a single-stranded product, the product was neutralized, and the concentration of the neutralized product was re-adjusted to about 1.5 pM, and analyzed with the next-generation sequencer MiniSeq (Illumina) according to its instruction manual. Use the nucleotide sequence file of the V4 region to classify the bacterial species in the intestinal flora by using the application software 16S Metagenomics (Illumina, Inc.) for metagenomics. To analyze the amount of mucin in feces, feces were collected daily, lyophilized, and the lyophilized samples were measured with FecalMucin analysis kit (Cosmo Bio Co., Ltd.).

(Results) The results of the vehicle control group and the test substance administration group are shown in Tables 6 and 7 below. The results showed that the stool of the vehicle control group had a variety of intestinal flora, but there was no significant change from day 0 to day 7. The results also show that the occupancy rate of Akkermansia is extremely low. On the other hand, the test substance administration group (100 mg/kg) showed the diversity of intestinal flora/temporary reduction of bacterial species. On the first day, the occupancy rate of Akkermansia increased significantly, Lactobacillus increased, and Parabacteroides increased slightly. These three species account for 90%, and the diversity of the intestinal flora is reduced. On the 3rd day, the occupancy rate of Akkermansia was further increased to about 40%, Lactobacillus was significantly reduced, and Parabacteroides was slightly increased. Subsequently, on the 7th day, the occupancy rate of Akkermansia spp. slightly decreased, Parabacteroides spp. increased slightly, and the occupancy rate of other species also increased. Diversity has a tendency to recover. Table 6. Vehicle control group (%) Belong to 0 days 1 day 3 days 5 days 7 days Parabacteroides 19.7 22.8 20.6 27.2 24.2 Lactobacillus 16.7 15.8 14.5 11.8 11.4 Broutella 12.9 12.0 15.1 11.3 12.4 Akkermansia 0.8 0.9 0.6 0.8 0.4 Clostridium spp 6.6 6.1 5.4 3.8 5.0 Rumenococcus 3.7 3.8 4.8 4.8 5.2 Bacteroides 2.8 2.9 2.4 3.6 3.0 Prevotella 2.9 4.1 3.0 4.4 3.7 Turicibacter 2.9 3.2 4.0 2.9 5.1 Dysgonomonas 2.0 2.1 1.9 2.5 1.7 Oscillatoria 2.0 2.8 2.9 3.2 3.3 Lachnospira 2.1 2.0 2.2 1.6 1.6 Sarcina 3.3 2.2 1.6 0.7 1.0 Alkalis 1.7 1.8 1.9 2.1 2.3 Pseudomonas 0.1 0.1 0.2 0.1 0.1 Phascolarctobacterium 0.0 0.0 0.1 0.1 0.0 Providencia 0.0 0.0 0.0 0.0 0.0 Johansenia 1.5 1.5 1.8 1.5 1.7 Table 7. Test substance (100 mg/kg) administration group (%) Belong to 0 days 1 day 3 days 5 days 7 days Parabacteroides 20.8 31.4 35.3 41.0 44.2 Lactobacillus 14.5 28.6 5.2 3.6 3.1 Broutella 15.2 0.8 0.1 0.9 0.9 Akkermansia 1.3 23.3 41.2 20.0 16.0 Clostridium spp 5.0 1.3 0.0 0.2 0.1 Rumenococcus 4.2 0.3 0.0 0.0 0.0 Bacteroides 3.1 0.6 2.2 6.1 5.6 Prevotella 3.3 0.1 0.0 0.1 0.1 Tribacter 1.5 0.4 0.0 0.0 0.0 Abnormal development bacteria 2.2 1.9 2.0 4.2 2.5 Oscillatoria 2.6 0.4 0.0 0.1 0.5 Trichospira 2.7 0.1 0.0 0.0 0.0 Sarcina 2.0 0.6 0.0 0.0 0.0 Alkalis 1.3 0.1 0.0 0.0 0.0 Pseudomonas 0.3 3.0 5.6 4.3 2.1 Koala 0.0 0.0 0.3 5.0 9.8 Providencia 0.0 0.0 1.7 5.5 6.6 Johansenia 1.8 0.1 0.0 0.0 0.0

The changes in the amount of each mucin in the rats in the vehicle control group and the test substance administration group are shown in FIG. 1. The amount of mucin in feces of the vehicle control group did not change significantly from day 0 to day 7. On the other hand, the amount of mucin in feces of the test substance administration group (100 mg/kg) increased from the first day, the amount reached approximately the maximum on the third day, and reached the peak on the seventh day. The results indicate that there is a significant and strong relationship between the increase in the occupancy rate of Akkermansia in the test substance administration group and the amount of mucin in the intestine (r=0.52, P<0.05).

Instance 4. Dosing the test substance twice a day (1 , 3 , 10 mg/kg) , SASP (300 mg/kg) , CPFX (500 mg/kg) Or once a day DEX (1 mg/kg) The classics DSS Analysis of intestinal flora in feces of induced colitis model rats A vehicle (5% gum arabic) or test substance (1, 3, 10 mg/kg) was administered to colitis model rats prepared by oral administration of dextran sulfate sodium (DSS) twice a day , And collect the feces of each administration group every day to analyze the intestinal flora in the feces. In addition, SASP (sulfasalazine), CPFX (ciprofloxacin) and DEX (dexamethasone) were also evaluated as comparative agents for the test substance of the present invention. (Test Methods) The rats were allowed to adapt to an environment accessible to water bottles for three days, and then they were divided into groups. After grouping, the rats were allowed to freely drink the 3% DSS solution placed in the water bottle to induce the symptoms of colitis (the starting day of drinking water was set to 0 days). For the rats in the untreated group, freely drink the water for injection in the water bottle during the test. From the next day of grouping to the last day of the test, use the test substance (1, 3, 10 mg/kg), SASP (300 mg/kg) or CPFX (500 mg/kg) as the contrast reagent or the vehicle (5% Gum Arabic) was administered to rats orally with a feeding needle twice a day. The control agent DEX (1 mg/kg) was orally administered to rats with a gastric tube once a day. The feces of each rat were collected daily, and the intestinal flora in the collected feces was analyzed by the next generation sequencer. The detailed analysis and analysis method are the same as in Example 3.

(Results) The results of each group are shown in Tables 8 to 15 below. The results showed that the changes of intestinal flora in feces were different from group to group. The intestinal flora in the feces of the untreated group had a variety of intestinal flora, but there was no significant change from day 0 to day 7. In the vehicle control group, the occupancy rate of Bacteroides increased over time. On the other hand, in the test substance administration group (1, 3, 10 mg/kg), the occupancy rate of Akkermansia was significantly increased from the first day. A dose-dependent occupancy of Akkermansia was observed. In the comparative reagent administration group (SASP-based and DEX groups), there was a time course change in the intestinal flora, but the occupancy rate of Akkermansia did not change significantly. In the CPFX group, the occupancy rate of Akkermansia did not change significantly until day 7, despite the extremely high dose (ie, 500 mg/kg). Table 8. Untreated group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 16.9 17.8 12.3 9.1 Parabacteroides 17.1 18.8 12.0 14.3 Broutella 9.3 6.3 8.1 6.0 Akkermansia 3.6 3.5 0.9 1.7 Bacteroides 3.0 3.5 6.9 10.3 Clostridium spp 4.9 4.6 4.6 3.5 Allobaculum 2.5 2.7 1.2 0.7 Prevotella 2.2 3.3 2.6 5.5 Abnormal development bacteria 2.8 3.2 4.8 5.7 Schreckia 1.7 1.4 1.8 1.0 Rumenococcus 2.3 2.0 0.5 1.0 Tribacter 2.1 3.4 2.8 2.3 Pediococcus 1.3 1.5 2.4 1.8 Oscillatoria 1.2 1.0 1.0 1.9 Bifidobacterium 0.6 0.7 1.5 1.1 Table 9. Solvent control group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 14.4 14.7 16.5 12.6 Parabacteroides 19.9 19.8 13.7 4.6 Broutella 11.6 10.4 9.5 11.7 Akkermansia 1.3 1.3 1.8 0.3 Bacteroides 3.1 2.6 11.2 20.2 Clostridium spp 5.1 5.6 4.2 3.5 Isobacterium 1.7 4.4 5.0 5.1 Prevotella 2.8 2.7 3.7 0.1 Abnormal development bacteria 2.8 2.5 1.5 1.5 Schreckia 1.5 0.6 0.5 1.7 Rumenococcus 2.1 2.9 2.2 0.3 Tribacter 1.2 1.9 2.5 2.1 Pediococcus 1.4 1.5 1.5 2.0 Oscillatoria 2.2 3.8 3.3 2.1 Bifidobacterium 0.6 0.7 0.6 1.6 Table 10. SASP group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 20.9 30.6 18.6 16.0 Parabacteroides 18.3 15.5 1.1 0.1 Broutella 9.3 3.5 11.3 13.9 Akkermansia 2.0 0.6 0.0 0.0 Bacteroides 3.3 2.6 1.3 1.7 Clostridium spp 5.1 2.7 2.6 1.5 Isobacterium 1.7 7.9 8.1 8.2 Prevotella 2.8 0.3 0.0 0.1 Abnormal development bacteria 3.0 3.1 0.8 0.1 Schreckia 1.6 2.6 1.6 0.3 Rumenococcus 2.2 0.8 0.2 0.2 Tribacter 1.1 0.8 1.5 4.2 Pediococcus 1.4 3.1 3.0 2.4 Oscillatoria 1.0 1.0 0.5 0.5 Bifidobacterium 0.5 3.5 16.7 13.3 Table 11. DEX group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 20.3 21.8 16.9 11.2 Parabacteroides 20.4 13.0 5.5 7.3 Broutella 10.6 6.5 6.4 4.8 Akkermansia 1.5 3.0 1.6 1.4 Bacteroides 2.7 2.7 11.2 19.6 Clostridium spp 4.1 4.7 4.3 4.8 Isobacterium 2.0 5.0 3.3 5.7 Prevotella 2.3 2.3 2.6 0.3 Abnormal development bacteria 2.7 3.6 1.8 2.1 Schreckia 1.5 1.0 0.7 1.0 Rumenococcus 2.0 0.9 0.2 0.3 Tribacter 1.9 2.1 2.6 2.8 Pediococcus 1.6 3.5 3.6 2.5 Oscillatoria 1.3 1.7 2.1 1.1 Bifidobacterium 0.6 1.4 2.4 2.5 Table 12. CPFX group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 15.5 53.3 66.6 51.7 Parabacteroides 19.7 3.1 0.0 0.2 Broutella 10.1 6.0 1.1 2.0 Akkermansia 1.2 0.8 0.0 1.0 Bacteroides 3.3 0.4 0.9 8.6 Clostridium spp 4.0 3.7 1.8 0.7 Isobacterium 3.8 1.9 0.4 0.1 Prevotella 2.5 0.3 0.1 0.0 Abnormal development bacteria 2.6 0.5 0.1 0.1 Schreckia 1.5 0.6 0.1 0.1 Rumenococcus 2.2 2.4 0.0 0.0 Tribacter 3.8 2.3 0.4 0.1 Pediococcus 1.0 3.8 8.2 6.9 Oscillatoria 1.1 2.2 0.8 0.3 Bifidobacterium 1.4 0.7 1.3 0.4 Table 13. Test substance (1 mg/kg) group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 18.2 24.1 15.2 6.0 Parabacteroides 18.7 23.1 16.8 11.1 Broutella 9.5 4.3 0.5 3.0 Akkermansia 2.0 4.9 12.4 7.1 Bacteroides 3.0 4.7 11.2 8.5 Clostridium spp 5.2 3.3 1.2 6.2 Isobacterium 2.4 0.8 0.1 0.0 Prevotella 2.7 5.8 1.3 0.1 Abnormal development bacteria 2.9 3.4 1.5 3.2 Schreckia 1.6 0.7 7.3 1.0 Rumenococcus 1.9 1.2 0.5 1.1 Tribacter 1.1 0.5 0.3 0.1 Pediococcus 1.5 1.6 0.8 1.0 Oscillatoria 1.3 1.6 0.6 0.5 Bifidobacterium 0.6 0.4 5.6 5.4 Table 14. Test substance (3 mg/kg) group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 17.3 27.3 14.4 5.5 Parabacteroides 16.6 27.7 20.9 12.5 Broutella 9.7 1.4 0.6 1.8 Akkermansia 1.5 6.1 16.3 5.2 Bacteroides 3.4 3.1 5.3 11.4 Clostridium spp 5.1 1.4 0.5 5.9 Isobacterium 2.9 0.4 0.1 0.0 Prevotella 2.2 6.2 0.3 0.2 Abnormal development bacteria 2.5 3.8 1.4 3.1 Schreckia 1.6 1.7 11.1 0.6 Rumenococcus 1.5 1.1 0.6 1.1 Tribacter 1.7 0.3 0.0 0.0 Pediococcus 1.6 1.6 0.7 0.7 Oscillatoria 1.8 0.8 0.1 0.3 Bifidobacterium 0.9 0.2 5.5 5.2 Table 15. Test substance (10 mg/kg) group (%) Belong to 0 days 1 day 3 days 7 days Lactobacillus 18.9 19.1 12.6 5.7 Parabacteroides 16.7 28.1 20.9 14.2 Broutella 7.8 0.6 0.5 1.3 Akkermansia 3.7 20.2 20.0 6.4 Bacteroides 3.3 2.4 0.8 7.5 Clostridium spp 4.8 0.7 0.2 4.1 Isobacterium 2.0 0.1 0.0 0.0 Prevotella 1.8 1.8 0.0 0.2 Abnormal development bacteria 2.8 2.9 1.4 3.9 Schreckia 1.6 1.5 14.6 1.3 Rumenococcus 2.0 0.3 0.0 0.2 Tribacter 3.3 0.4 0.0 0.0 Pediococcus 1.7 1.1 0.6 0.6 Oscillatoria 0.9 0.4 0.0 0.2 Bifidobacterium 0.5 0.1 4.9 5.5

Instance 5. Effect of test substance on obesity model mice induced by high-fat diet In order to evaluate the efficacy of the test substance on obesity or diabetes, the test substance was orally administered to obesity model mice induced by a high-fat diet, and the blood sugar content and hemoglobin A1c (HbA1c) were analyzed. (Test Methods) C57BL/6J mice were divided into groups based on their body weight, blood glucose content and HbA1c content. Shortly after grouping, a high-fat diet was started, and vehicle (control) or test substance (10 mg/kg) was administered orally once a day. Twice a week, weigh your body weight and food consumption. On the first day of administration, the blood glucose level was analyzed by blood obtained from the tail vein and then measured every two weeks. The HbA1c content was analyzed by measuring the blood obtained from the tail vein every 4 weeks with DCA Vantage (Siemens Healthcare Diagnostics).

(result) The weight of untreated mice (who took a common diet) increased slightly until day 63, while the weight gain of the vehicle control group taking a high-fat diet was significantly higher than that of the untreated group (on day 63, p<0.01) The weight increases. Compared with the weight gain of the vehicle control group (p<0.05 on day 63), the weight gain of the test substance administration group taking the high-fat diet was significantly suppressed. Regarding blood glucose levels, compared to the increase in blood glucose levels in the untreated group (p<0.01 on day 63), the vehicle control group taking a high-fat diet showed a significant increase in blood glucose levels. Compared with the increase in blood glucose level in the vehicle control group (at 63rd day, p<0.01), which was the same level as the untreated group, the increase in blood glucose level in the test substance administration group taking a high-fat diet was significantly suppressed . Also for HbA1c content, compared to the increase in HbA1c content of the untreated group (on day 63, p<0.01), the vehicle control group taking a high-fat diet showed a significant increase in HbA1c content. Compared with the increase in HbA1c content in the vehicle control group (on day 63, p<0.05), the increase in HbA1c content in the test substance administration group taking a high-fat diet was significantly suppressed. Compared with the solvent control group that took a high-fat diet, the administration of the test substance significantly suppressed weight gain, significantly suppressed the increase in blood sugar content and significantly suppressed the increase in HbA1c content, which indicates that the test substance is suitable for the treatment of obesity and diabetes.

Instance 6. Test substance on diabetic model mice (db/db Mouse ) Influence In order to evaluate the efficacy of the test substance on diabetes, the test substance was orally administered to diabetic model mice (db/db mice), and the blood glucose content and hemoglobin A1c (HbA1c) as indicators of diabetes were analyzed. (Test Methods) The db/db mice were grouped based on their body weight, blood glucose content and HbA1c content. Shortly after grouping, vehicle (control) or test substance (10 mg/kg) was administered orally once a day. After starting the administration, the blood glucose level was measured every two weeks and the HbA1c level was measured every four weeks. However, in the 8th and 12th weeks, the test mice were fasted from the previous day, and the measured blood glucose level was the fasting blood glucose level.

(result) The results are shown in Figures 5 to 7. Compared with the vehicle control group (on the 70th day of random blood glucose content, p<0.01; and on the 84th day of fasting blood glucose content, p<0.01), the random blood glucose content and fasting blood glucose content in the test substance group increased significantly Be suppressed. Regarding HbA1c, compared to the increase in the content of the vehicle control group (on day 84, p<0.01), the increase in the content of the test substance administration group was significantly suppressed. As compared with the vehicle control group, the administration of the test substance significantly suppressed the increase in blood glucose content and the increase in HbA1c content, which indicates that the test substance is suitable for the treatment of diabetes.

[Figure 1] Figure 1 shows the results of the change in the amount of mucin in Example 3. [Figure 2] Figure 2 shows the results of body weight changes in Example 5 ( ^** p<0.01, ^* p<0.05). [Figure 3] Figure 3 shows the results of changes in blood glucose levels in Example 5 ( ^** p<0.01). [Figure 4] Figure 4 shows the result of the change of HbA1c in Example 5 ( ^** p<0.01, ^* p<0.05). [Figure 5] Figure 5 shows the results of random blood glucose changes in Example 6 ( ^** p<0.01). [Figure 6] Figure 6 shows the results of changes in fasting blood glucose levels in Example 6 ( ^** p<0.01). [Figure 7] Figure 7 shows the result of the change of HbA1c in Example 6 ( ^** p<0.01).

Claims (12)

A pharmaceutical composition for improving intestinal flora, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5 -Pyridyl)-4-Pendoxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient. The pharmaceutical composition of claim 1, wherein the intestinal flora is improved by increasing the intestinal occupancy rate of Akkermansia. The pharmaceutical composition of claim 1, wherein the intestinal mucus layer is thickened by improving the intestinal flora. An agent for treating and/or preventing diseases expected to be improved by increasing the intestinal occupancy rate of Akkermansia, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro -8-Methyl-7-(2-amino-3-cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof is used as the active ingredient. An agent for treating and/or preventing diseases that are expected to be improved by thickening the intestinal mucus layer, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7 -(2-Amino-3-cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient. The agent of claim 4 or 5, wherein the disease is obesity and/or diabetes. A medicament for treating and/or preventing obesity and/or diabetes, which comprises 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3 -Cyano-5-pyridyl)-4- pendant oxy-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof as the active ingredient. Such as the medicine of any one of claims 4 to 7, which is for oral administration. The medicament according to any one of claims 4 to 8, wherein the daily dose of the active ingredient is 0.1 mg to 30000 mg. A method for treating and/or preventing obesity and/or diabetes, which comprises administering a therapeutically effective amount of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl to a patient in need Group-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3-quinoline-carboxylic acid or a pharmaceutically acceptable salt thereof. A kind of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3- The use of quinoline-formic acid or a pharmaceutically acceptable salt thereof for the manufacture of medicaments for the treatment and/or prevention of obesity and/or diabetes. A kind of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methyl-7-(2-amino-3-cyano-5-pyridyl)-4-oxo-3- Quinoline-formic acid or a pharmaceutically acceptable salt thereof is used for the treatment and/or prevention of obesity and/or diabetes.

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