WO2024012531A1 - 一种吡啶酮衍生物的应用 - Google Patents

一种吡啶酮衍生物的应用 Download PDF

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WO2024012531A1
WO2024012531A1 PCT/CN2023/107252 CN2023107252W WO2024012531A1 WO 2024012531 A1 WO2024012531 A1 WO 2024012531A1 CN 2023107252 W CN2023107252 W CN 2023107252W WO 2024012531 A1 WO2024012531 A1 WO 2024012531A1
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drug
substance
mass ratio
fibrosis
patient
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PCT/CN2023/107252
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English (en)
French (fr)
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李永国
陈小宁
宋浩亮
李洪蛟
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广州嘉越医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention relates to the technical field of biomedicine, and specifically relates to the application of pyridone derivatives in the preparation of drugs for treating fibrosis.
  • Fibrosis is a systemic disease that can affect any organ, more commonly seen in the heart, lungs, kidneys, liver and skin. It is a disorder of tissue repair response due to various types of tissue damage, especially during chronic inflammatory diseases. The outcome of fibrosis in different fibroproliferative diseases has its own unique mechanism. Fibrosis is reported to account for 45% of all deaths in the industrialized world.
  • intestinal fibrosis The main drivers of intestinal fibrosis are soluble molecules (such as cytokines and growth factors), G protein-coupled receptors, epithelial-to-mesenchymal transition, endothelial-to-mesenchymal transition, and intestinal microbiota.
  • immunosuppressants and anti-inflammatory biologics can relieve inflammation and improve related clinical symptoms, they have no direct anti-fibrotic effect and cannot reduce the intestinal fibrosis that has already occurred or prevent the subsequent fibrosis process. Therefore, there are currently no drugs approved for the treatment of intestinal fibrosis, and the treatment options for intestinal stenosis caused by intestinal fibrosis are still mainly surgery and endoscopic treatment (D'Alessio S et al, Nat Rev Gastroenterol Hepatol. 2022Mar; 19 (3):169-184.; Hu Fan et al., Chinese Journal of Medical Frontiers (Electronic Edition). 2021, Volume 13, Issue 7: Pages 14-21).
  • Non-alcoholic liver fibrosis refers to the abnormal proliferation of connective tissue in the liver caused by other pathogenic factors besides alcohol.
  • Fibrosis has long been thought to be irreversible, but preclinical models and clinical trials have shown that Obviously, it is a highly dynamic process. Pirfenidone was initially approved for use in idiopathic pulmonary fibrosis (IPF), and subsequent studies have shown that it also has a role in treating fibrotic diseases in other models of fibrosis. However, pirfenidone has poor efficacy and strong side effects, so it is urgent to find new anti-fibrotic drugs.
  • IPF idiopathic pulmonary fibrosis
  • the technical problem to be solved by the present invention is that the existing drugs for treating intestinal fibrosis or non-alcoholic liver fibrosis have large dosages.
  • the present invention provides an application of pyridone derivatives, which can treat and/or prevent intestinal fibrosis and non-alcoholic liver fibrosis with less use of such compounds.
  • the present invention provides the use of substance A in the preparation of medicines.
  • the substance A is a compound shown in formula I or a pharmaceutically acceptable salt thereof; the medicine is for the treatment and/or prevention of intestinal fibrosis or non-alcoholic Drugs for liver fibrosis;
  • the intestinal fibrosis is preferably intestinal fibrosis caused by inflammatory bowel disease.
  • the intestinal fibrosis is more preferably Crohn's disease intestinal fibrosis.
  • the administration frequency of the drug can be once/day, 2 times/day or 3 times/day; preferably, it is 2 times/day.
  • the unit dose of the drug can be determined according to the effective dose of the subject/patient; the unit dose of the substance A can be 25-900 mg, preferably 50-600 mg, more preferably 200-400 mg. .
  • the single dose of the drug can be determined according to the actual therapeutic effect of the subject/patient; preferably, the single dose of the drug is the substance A. and
  • the mass ratio of subject/patient the mass ratio may be 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the single dose of the drug can be the mass ratio of the substance A to the subject/patient, and the mass ratio can be It is 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the single dose of the drug may be the mass ratio of the substance A to the subject/patient, and the The mass ratio may be 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the daily dose of the drug can be determined according to the actual therapeutic effect of the subject/patient; preferably, the daily dose of the drug is the substance A.
  • the mass ratio of the subject/patient can be 3-80mg/kg, preferably 3mg/kg, 6mg/kg, 12mg/kg, 15mg/kg, 30mg/kg, 40mg/kg, 60mg/ kg or 80mg/kg.
  • the daily dosage of the drug can be the mass ratio of the substance A to the subject/patient, and the mass ratio can be It is 3-80 mg/kg, preferably 3 mg/kg, 6 mg/kg, 12 mg/kg, 15 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg or 80 mg/kg.
  • the daily dose of the drug may be the mass ratio of the substance A to the subject/patient, and the The mass ratio is 3-80 mg/kg, preferably 3 mg/kg, 6 mg/kg, 12 mg/kg, 15 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg or 80 mg/kg.
  • the concentration of the compound represented by Formula I in the drug may be 0.3-3 mg/mL; preferably 0.3 mg/mL, 0.6 mg/mL, 1 mg/mL, 1.5 mg/mL or 3 mg. /mL.
  • the medicament may comprise substance A and its pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a conventional carrier in the art.
  • the drug can be administered via the gastrointestinal tract, preferably orally.
  • the dosage form of the drug can be a conventional dosage form in this field, and the dosage form of the drug can be a solid preparation.
  • the mass fraction of the compound represented by Formula I in the drug is 3%-50%, preferably 10-20%.
  • the present invention also provides a pharmaceutical composition, including substance A and a pharmaceutically acceptable carrier thereof; the substance A is a compound shown in Formula I or a pharmaceutically acceptable salt thereof;
  • the mass fraction of the compound represented by formula I is 3%-50%.
  • the mass fraction of the compound represented by formula I may be 10%-20%.
  • the present invention also provides the use of a pharmaceutical composition in the preparation of drugs for treating and/or preventing intestinal fibrosis or non-alcoholic liver fibrosis.
  • the intestinal fibrosis is preferably intestinal fibrosis caused by inflammatory bowel disease; more preferably, it is Crohn's intestinal fibrosis.
  • the present invention also provides a method for treating and/or preventing intestinal fibrosis or non-alcoholic liver fibrosis, the method comprising: administering a therapeutically effective amount of substance A or a drug to a subject/patient, the drug comprising a substance A and its pharmaceutically acceptable carrier; the substance A is a compound shown in Formula I or a pharmaceutically acceptable salt thereof;
  • the pharmaceutically acceptable carrier is a conventional carrier in this field.
  • the intestinal fibrosis is preferably intestinal fibrosis caused by inflammatory bowel disease; more preferably, it is Crohn's intestinal fibrosis.
  • the administration frequency of the drug can be 1 time/day, 2 times/day or 3 times/day, preferably 2 times/day.
  • the unit dosage of the drug can be determined according to the effective dose of the subject/patient; preferably, the unit dosage of the substance A can be 25-900 mg, preferably 50-600 mg, more preferably 200-400mg.
  • the single dose of the drug can be determined according to the actual therapeutic effect of the subject/patient; preferably, the single dose of the drug can be the substance
  • the mass ratio of A to the subject/patient may be 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the single dose of the drug can be the mass ratio of the substance A to the subject/patient, and the mass ratio can be It is 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the single dose of the drug may be the mass ratio of the substance A to the subject/patient, and the The mass ratio may be 3-40 mg/kg, preferably 3 mg/kg, 6 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg or 40 mg/kg.
  • the daily dose of the drug can be determined according to the actual therapeutic effect of the subject/patient; preferably, the daily dose of the drug can be the substance
  • the mass ratio of A to the subject/patient, the mass ratio can be 3-80 mg/kg, preferably 3 mg/kg, 6 mg/kg, 12 mg/kg, 15 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg /kg or 80mg/kg.
  • the daily dose of the drug can be the mass ratio of the substance A to the subject/patient, and the mass ratio can be for 3- 80 mg/kg, preferably 3 mg/kg, 6 mg/kg, 12 mg/kg, 15 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg or 80 mg/kg.
  • the daily dose of the drug may be the mass ratio of the substance A to the subject/patient, and the The mass ratio may be 3-80 mg/kg, preferably 3 mg/kg, 6 mg/kg, 12 mg/kg, 15 mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg or 80 mg/kg.
  • the concentration of the compound represented by Formula I in the drug can be 0.3-3 mg/mL; preferably, it is 0.3 mg/mL, 0.6 mg/mL, 1 mg/mL, 1.5 mg/mL or 3 mg. /mL.
  • the drug can be administered via the gastrointestinal tract; preferably orally.
  • the dosage form of the drug can be a conventional dosage form in the art, and the dosage form of the drug can be a solid preparation.
  • the term "pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue. , without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient.
  • Representative carriers include water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These matrices include suspending agents, viscosifiers, transdermal penetration enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicine. Additional information regarding vectors may be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.
  • treatment refers to therapeutic therapy.
  • treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that causes or causes the condition or (b) ) one or more biological manifestations of a condition, (3) amelioration of one or more symptoms, effects, or side effects associated with the condition, or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slow the progression of a condition or one or more biological manifestations of a condition.
  • prevention refers to the reduction of the risk of acquiring or developing a disease or disorder.
  • terapéuticaally effective amount refers to an amount of a compound sufficient to effectively treat a disease or condition described herein when administered to a patient.
  • the “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as necessary by one skilled in the art.
  • patient refers to any animal, preferably mammals, most preferably humans, to which a compound is or has been administered in accordance with embodiments of the present invention.
  • mammal includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being the most preferred.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the compound represented by the formula I of the present invention can treat/or prevent intestinal fibrosis and non-alcoholic liver fibrosis and can achieve good medicinal effect at a smaller dose. Furthermore, the effective dose of the compound represented by Formula I of the present invention in treating/or preventing intestinal fibrosis and non-alcoholic liver fibrosis is 3.0 mg/kg.
  • Figure 1 is Example 1, the DAI score results of animals in each experimental group during the test period; all data are statistically calculated using the mean ⁇ standard deviation (SD);
  • Figure 2A is Example 1, the histopathological diagnosis scoring results of ulcers in the middle colon tissue of animals in each experimental group;
  • Figure 2B is Example 1, the histopathological scoring results of the inflammatory response of the mid-colon tissue of animals in each experimental group; all data in Figure 2 are statistically analyzed using the mean ⁇ standard deviation (SD), #P ⁇ 0.05,# #P ⁇ 0.01, ###P ⁇ 0.001, ####P ⁇ 0.0001 represents the statistical analysis results of animals in different treatment groups and the model control group;
  • SD standard deviation
  • Figure 3 is Example 1; representative pictures of ulcers in the colon tissue of animals in each experimental group; representative pictures of histopathology in the middle section of the colon of animals in each experimental group.
  • the histopathological diagnosis and treatment of the colon were performed under 100 ⁇ and 200 ⁇ optical microscopes respectively. For analysis, select 3 individual animals from each test to display the results.
  • the image scales are 1mm, 250 ⁇ m and 100 ⁇ m respectively;
  • Figure 4A shows the CVF% of colon tissue of animals in each experimental group in Example 1;
  • Figure 4B shows the HYP detection results in the mid-colon of animals in each experimental group in Example 1; all data in Figure 4 are statistically analyzed using the mean ⁇ standard deviation (SD), *P ⁇ 0.05, **P ⁇ 0.01, * **P ⁇ 0.001, ****P ⁇ 0.0001 represents the statistical analysis results of different experimental groups and normal control group, #P ⁇ 0.05, ##P ⁇ 0.01, ###P ⁇ 0.001, ####P ⁇ 0.0001 represents the statistical analysis results of animals in different treatment groups and model control groups;
  • FIG. 5 shows Example 2, Sirius red staining and diagnostic results of animal livers in each experimental group during the test period; Sirius red staining results of liver tissue of animals in each experimental group, the livers were organized under 10 ⁇ , 100 ⁇ and 200 ⁇ optical microscopes respectively. For pathological diagnosis and analysis, 3 individual animals from each test were selected to display the results.
  • the image scales are 1mm, 250 ⁇ m and 500 ⁇ m respectively;
  • Figure 6A shows the liver fibrosis scoring results of animals in each experimental group in Example 2.
  • Figure 6B shows the calculation results of liver CVF% of animals in each experimental group in Example 2;
  • Figure 6C shows the HYP detection results of animal livers in each experimental group in Example 2; all data are statistically analyzed using the mean ⁇ standard deviation (SD), *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 indicates the statistical analysis results of different experimental groups and the normal control group, #P ⁇ 0.05,##P ⁇ 0.01,###P ⁇ 0.001,####P ⁇ 0.0001 indicates different Statistical analysis results of animals in the treatment group and model control group.
  • Example 1 Experiment on the efficacy of TNBS-induced chronic colitis in rats with intestinal fibrosis model
  • test compound I To evaluate the therapeutic effect of the test compound I on the TNBS-induced chronic colitis model accompanied by intestinal fibrosis in SD rats.
  • Test drug Compound I, provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd.
  • TNBS two enema method Experimental animals undergo weight screening after passing the adaptation period test and observation, and are randomly divided into 6 experimental groups according to their weight, including normal control group, model control group, positive drug treatment group, and low-dose treatment of test article group, a medium-dose treatment group of the test article and a high-dose treatment group of the test article, with 10 mice in each group. On the day of grouping, except for the normal control group which was given 50% ethanol solution, all the other experimental animals were given TNBS ethanol solution via enema. The dosage of TNBS was every 30mg per animal, the frequency of modeling is once every two weeks, a total of 2 model inductions. The experimental period lasted for 4 weeks, and all animals drank and were fed normally.
  • the solvent of the test sample is 0.5% (w/v, the mass of methylcellulose accounts for the percentage of the volume of the solution) methylcellulose and 0.2% (v/v, the volume of Tween 80 accounts for the percentage of the volume of the solution) Tween 80 aqueous solution;
  • DAI disease activity index
  • the DAI score is based on the extent of weight loss, stool morphology and blood in the stool, and the three scoring indicators are used for comprehensive statistical analysis.
  • the colon was removed from the end of the cecum to the anus. After flushing the intestinal segment with physiological saline, the entire colon was divided into upper, middle and lower sections; the middle section of the colon was The tissue was cut into pieces, and a part of the tissue was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for testing the total hydroxyproline level of the mid-colon samples of animals in each group. The remaining mid-section tissue was fixed with 10% formaldehyde, embedded in paraffin, and cut into sections.
  • Image J was used to count the collagen-stained positive area and total sample area of the mid-colon samples (submucosa and muscular layer) of animals in each experimental group, and calculate the collagen volume fraction CVF% of each sample.
  • hydroxyproline detection kit to process the mid-colon samples according to the operating instructions, and detect the total hydroxyproline level in the mid-colon samples of animals in each group.
  • results The DAI score results after the first induction of TNBS (D0-D10) showed that the animals in the model control group showed obvious disease characteristics of colitis after TNBS intervention. The specific manifestations were that the weight growth was significantly inhibited, the animal activity level decreased, and the feces appeared It becomes mushy and thin, and the perianal area becomes inflamed and swollen.
  • the AUC (0-10d) of the model control group was calculated to be 45.75 ⁇ 4.72, which was significantly statistically different from the normal control group animals in the same period (P ⁇ 0.05).
  • test product compound I and the positive drug sulfasalazine were administered to the mice of the respective corresponding groups according to the set treatment regimen.
  • AUC (0-10d) data of the respective groups compound I and sulfasalazine had chronic effects on the model animals.
  • the disease process of colitis was improved to some extent.
  • the DAI score AUC (0-10d) of animals in each treatment group was calculated to be 37.15 ⁇ 4.93, 36.05 ⁇ 4.87, 33.50 ⁇ 3.97 and 29.80 ⁇ 2.79, respectively, which decreased by 18.80% compared to the model control group in the same period. , 21.20%, 26.78% and 34.86%.
  • the effective dose of test compound I is smaller, 3.0mg/kg.
  • the DAI score results after the second induction of TNBS showed that the DAI score increased significantly, and the animals in the model control group showed obvious disease characteristics of chronic colitis, mainly characterized by mushy and thin feces and obvious perianal changes.
  • the degree of weight loss was reduced compared with the first round of induction, and there was a significant statistical difference compared with the normal control group during the same period (P ⁇ 0.05).
  • the score gradually decreased and returned to normal on the 24th day, which was the same as that of the normal control group during the same period. No significant difference was found compared with the normal control group. Its AUC (14-24d) was calculated to be 24.05 ⁇ 2.93.
  • the test product compound I and the positive drug sulfasalazine still improved the chronic colitis disease in model animals to a certain extent.
  • the DAI scores of the animals in each treatment group showed a certain degree of decrease.
  • the DAI score AUC (14-24d) during the scoring period was calculated to be 19.30 ⁇ 2.79, 17.25 ⁇ 3.16, 15.40 ⁇ 3.50 and 11.70 ⁇ 1.69 respectively, which were decreased by 19.75%, 28.27%, 35.97% and 51.35% respectively compared with the model control group in the same period. .
  • the histopathological diagnosis results showed that under this experimental system, the colon tissue structure of the normal control group animals was clear and layered, and no obvious histological abnormal changes were found.
  • the animals in the model control group showed obvious histological changes of colitis, and the pathological changes in the middle section of the colon were particularly obvious. Therefore, pathological diagnosis and analysis were performed on the middle section of the colon.
  • the pathological changes are mainly manifested as significant thickening of the colon wall, the formation of fissure-like ulcers on the mucosal surface of the intestinal wall, and typical granulomas in the submucosa and muscle layer, accompanied by infiltration of inflammatory cells mainly lymphocytes and plasma cells.
  • small vessel endoarteritis and fibroblast activation can be seen, small artery wall thickening and lumen stenosis, obvious massive connective tissue hyperplasia and severe fibrosis process appear in the submucosa and muscle layer, fibers are distributed in bundles, and ulcers are at the same time Characteristics such as destruction of crypt structure and bleeding can be seen in the surrounding area, and the average histopathological scores of tissue ulceration and inflammatory response are 2.00 ⁇ 0.00 points.
  • the positive control drug sulfasalazine has a significant inhibitory effect on the process of colon ulceration and inflammatory response in model animals under the set treatment plan.
  • the degree of ulceration and inflammation in the colon tissue of the animals in the corresponding treatment group was significantly slowed down.
  • the mean pathological scores were 0.50 ⁇ 0.53 points and 0.60 ⁇ 0.52 points respectively, and there was a significant statistical difference in the degree of improvement (P ⁇ 0.05).
  • the colon submucosa and muscularis connective tissue hyperplasia and fibrosis process of animals in the positive drug treatment group showed Significant improvement and healing performance.
  • the test compound I has a significant inhibitory effect on the synchronization of colon ulcer and inflammatory response in model animals under the set treatment plan.
  • the mean ulcer scores of the colon tissue of animals in each dose treatment group were 0.80 ⁇ 0.63 points, 0.70 ⁇ 0.67 points and 0.70 respectively. ⁇ 0.67 points, mean colon inflammatory response score respectively The scores were 1.00 ⁇ 0.67 points, 0.70 ⁇ 0.67 points and 0.80 ⁇ 0.79 points.
  • the degree of improvement was statistically significantly different (P ⁇ 0.05).
  • the animals in the treatment groups at each dose of the test compound I Tissue healing and fibrosis were slowed down in the colon.
  • the pathological scoring results of colon tissue ulceration and inflammatory response of animals in each experimental group are shown in Figures 2A and 2B. Representative pictures of ulcers in the colon tissue of animals in each experimental group are shown in Figure 3.
  • test compound I and the positive control drug sulfasalazine had a significant inhibitory effect on the colon fibrosis process of model animals under the set treatment plan, which was mainly manifested as inhibiting the proliferation of submucosal fibrous connective tissue and improving muscle layer ulcers. Fibrosis.
  • the analysis results of the collagen volume fraction (CVF%, fibrosis area/total area of colon submucosa and muscular layer) of the submucosa and muscular layer of the mid-colon showed that under the conditions of this experiment, the CVF% of the colon of normal control animals was 0.74 ⁇ 0.32 %, the CVF% of the colon of animals in the model control group was significantly increased under the same detection and analysis conditions, which was 6.39 ⁇ 3.16%, with significant statistical difference compared with the normal control group (P ⁇ 0.05).
  • the test product compound I and the positive control drug sulfasalazine have a significant inhibitory effect on the increase in CVF% of the colon of model animals under the set treatment plan. Under the same analysis conditions, their average CVF% values are 2.97 ⁇ 1.78% respectively. , 2.36 ⁇ 1.47%, 2.37 ⁇ 1.33% and 2.67 ⁇ 1.06%, all had significant statistical differences compared with the model control group (P ⁇ 0.05).
  • the HYP test results of the mid-colon tissue showed that compared with the normal control group (1141.78 ⁇ 106.42 ⁇ g/g), the colon HYP content of the model control group animals (2109.99 ⁇ 385.85 ⁇ g/g) was significantly increased, and There was a significant difference compared with the normal control group (P ⁇ 0.05).
  • the positive control drug sulfasalazine has an effect on colon fibrosis in model animals under the set treatment plan. The degree of dimensionality has a significant improvement effect. Under the same detection conditions, the colon HYP content (1299.79 ⁇ 161.27 ⁇ g/g) showed a significant decrease reaction, which was statistically different from the model control group (P ⁇ 0.05).
  • the test compound I also had a significant therapeutic effect on the colon fibrosis process of model animals under the set treatment plan.
  • the levels of HYP in the colon tissue of the animals in each dose treatment group showed a significant decrease, respectively. were 1333.43 ⁇ 155.33 ⁇ g/g, 1228.40 ⁇ 235.44 ⁇ g/g and 1270.56 ⁇ 287.01 ⁇ g/g.
  • the HYP content levels of the corresponding colon segments of animals in the model control group there were significant statistical differences (P ⁇ 0.05), and their improvement
  • the effect has a certain dose dependence, with the onset dose being 3.0mg/kg.
  • the fibrosis levels of colon tissue of animals in each experimental group are shown in Figure 4A and Figure 4B.
  • This experiment uses TNBS to perform enema intervention on male SD rats, once every 2 weeks, for 2 consecutive rounds, to induce lesions in their colon, and to establish a rat model of chronic colitis with intestinal fibrosis that is consistent with clinical onset characteristics.
  • the test compound I (3.0, 6.0 and 15.0 mg/kg, twice a day) and the positive control drug sulfasalazine (50.0 mg/kg, twice a day) were treated starting from the day of model induction.
  • test compound I has significant therapeutic effect.
  • test compound I To evaluate the therapeutic effect of the test compound I on the carbon tetrachloride-induced liver fibrosis model of SD rats.
  • Test drug Compound I, provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd.
  • Carbon tetrachloride intraperitoneal injection method Experimental animals undergo weight screening after passing the adaptation period test and observation, and are randomly divided into normal control group and model induction group according to their weight. The animals in the model induction group are given 20% carbon tetrachloride intraperitoneally on the day of the first grouping. Chlorinated carbon corn oil solution, the modeling frequency is 2 times/week for 6 consecutive weeks. Induced with carbon tetrachloride for 2 weeks, the animals in the model induction group were randomly divided into five experimental groups according to their alanine aminotransferase (ALT) levels (body weight index was used as a calibration factor), including the model control group, the positive drug treatment group, and the test group.
  • ALT alanine aminotransferase
  • the low-dose treatment group of the test product The low-dose treatment group of the test product, the middle-dose treatment group of the test product and the high-dose treatment group of the test product. Animals in the normal control group were given corresponding solvents. The experimental period lasted for 6 weeks, and all animals drank and were fed normally.
  • the solvent of the test sample is 0.5% (w/v) methylcellulose and 0.2% (v/v) Tween 80 aqueous solution;
  • liver was removed, washed with physiological saline, and divided into three segments: the large lobe, the left lobe, and the right middle lobe; the large lobe tissue was quickly frozen in liquid nitrogen and stored in -80°C refrigerator was used to detect the total hydroxyproline level of liver samples from each group of animals; the left lobe and right middle lobe of the liver were fixed with 10% formaldehyde, embedded in paraffin, cut into 3 ⁇ m thick sections, and then in xylene Dewaxing, hydration with graded ethanol, Sirius red staining, dehydration, clearing, mounting, observing and photographing histopathological changes under a microscope; routine tissue cryopreservation.
  • liver lobe samples were processed using a hydroxyproline detection kit according to the operating instructions, and the total hydroxyproline levels of the liver samples of animals in each group were detected.
  • the model animal livers showed obvious liver fibrosis lesions induced by carbon tetrachloride, including disordered lobular structure, obvious collagen hyperplasia at the interface of the liver plate, and thickening of connective tissue in the portal area and central vein area. Formation of pseudolobular structure.
  • the test compound I can be seen under 200 ⁇ and 400 ⁇ light microscopes.
  • the pseudolobular structure of the liver of some animals in the medium-dose and high-dose treatment groups showed a trend of disintegration, which was mainly manifested in the fiber bundles at the hepatic fiber bridges. There are two aspects: narrowing and reduction in the number of pseudolobules.
  • the degree of hepatic fibrosis of the animals in the model control group was obvious. Their fibrosis stages were all S3, and the average fibrosis score was 4.90 ⁇ 0.88 points. Most of the animals' liver portal ducts and portal ducts and/or portal ducts and central vein areas had severe fibrosis. Pseudolobular structures formed by fibrous bridges were visible, and a few animals showed obvious signs of liver cirrhosis. The test compound I has a certain inhibitory effect on the liver fibrosis process of model animals under the set treatment plan. The mean liver fibrosis scores of the animals in the low, medium and high dose groups were 4.40 ⁇ 0.70 points, 4.00 ⁇ 0.94 points and 4.20 respectively.
  • liver collagen volume fraction (CVF%) of animals in each experimental group showed that compared with the normal control group (1.24 ⁇ 0.23%), the liver CVF% of animals in the model control group was significantly increased, which was 8.75 ⁇ 2.39%.
  • the test compound I had a significant inhibitory effect on the liver fibrosis process of model animals under the set treatment plan. Its CVF% were 5.02 ⁇ 1.42%, 4.87 ⁇ 1.68% and 5.60 ⁇ 1.92% respectively, which were compared with the model control group. There is a significant difference (P ⁇ 0.05).
  • the positive control drug Pirfenidone had a significant inhibitory effect on the degree of liver fibrosis in model animals. Its liver CVF% was 6.58 ⁇ 2.20%, which was significantly different from the model control group (P ⁇ 0.05).
  • the detection results of hydroxyproline in the liver tissues of animals in each experimental group showed that the HYP content level in the livers of animals in the model control group was 702.84 ⁇ 173.44 ⁇ g/g, which was significantly higher than that of animals in the normal control group (307.30 ⁇ 78.13 ⁇ g/g). Significant difference (P ⁇ 0.05).
  • the test compound I medium and high dose groups and the positive control drug pirfenidone had a certain inhibitory effect on the increase in liver hydroxyproline levels in model animals under the set treatment plan, and their liver HYP levels showed a downward trend. They were 560.60 ⁇ 114.04 ⁇ g/g, 607.79 ⁇ 112.74 ⁇ g/g and 611.19 ⁇ 183.66 ⁇ g/g respectively.
  • the improvement effect of the test compound I at the medium dose was the most obvious, and there was a significant difference compared with the model control group (P ⁇ 0.05).
  • the liver tissue fibrosis levels of animals in each experimental group are shown in Figure 6.
  • This experiment induces liver lesions in male SD rats through intraperitoneal injection of 20% carbon tetrachloride corn oil solution (twice a week for 6 consecutive weeks), and establishes a rat liver fibrosis model consistent with clinical onset characteristics.
  • treatment with test compound I (3.0, 10.0 and 30.0 mg/kg, twice a day) and positive control drug pirfenidone (150.0 mg/kg, twice a day) was started. Medication for 4 weeks.
  • the liver pathological fibrosis score results showed that the test compound I could inhibit the liver fibrosis process of model animals after 4 weeks of treatment.
  • the mean liver fibrosis scores of the animals in the low, medium and high dose groups were 4.40 ⁇ 0.70, 4.00 ⁇ 0.94 and 0.94 respectively. 4.20 ⁇ 0.92 points, both significantly lower than the model control group (4.90 ⁇ 0.88 points).
  • Quantitative analysis of collagen volume fraction (CVF%) of liver slices showed that the CVF% levels of the test compound I low, medium and high dose groups and the positive control drug pirfenidone group were 5.02 ⁇ 1.42% and 4.87 ⁇ 1.68 respectively. %, 5.60 ⁇ 1.92% and 6.58 ⁇ 2.20%, all significantly lower than the model Type control group (8.75 ⁇ 2.39%, P ⁇ 0.05), indicating that each treatment group has a significant inhibitory effect on liver fibrosis.
  • the liver tissue hydroxyproline level of the test compound I medium-dose group was also significantly lower than that of the model control group (560.60 ⁇ 114.04 ⁇ g/g vs 702.84 ⁇ 173.44 ⁇ g/g, P ⁇ 0.05 ).
  • the results of this efficacy evaluation show that the test compound I has significant therapeutic effect.

Abstract

一种吡啶酮衍生物的应用。一种物质A在制备药物中的应用，所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；所述药物为治疗和/或预防肠纤维化或非酒精性肝纤维化的药物。所述物质A或包含物质A的药物对肠纤维化或非酒精性肝纤维化在较小剂量下即可达到较好的药效。

Description

一种吡啶酮衍生物的应用
本申请要求申请日为2022/7/14的中国专利申请2022108347679的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药技术领域，具体涉及吡啶酮衍生物在制备治疗纤维化的药物中的应用。
背景技术
纤维化(Fibrosis)是一种***性疾病，可以影响任何器官，更常见于心脏、肺、肾、肝和皮肤，是多种类型组织损伤尤其是在慢性炎症性疾病过程中，组织修复反应失调的结局，不同的纤维增生性疾病中的纤维化有各自的独特机制。据报道，在工业化世界中，因纤维化引起的死亡人数占总死亡人数比例的45％。
肠纤维化的主要驱动因素是可溶性分子(例如细胞因子和生长因子)、G蛋白偶联受体、上皮间质转化、内皮间质转化以及肠道微生物群等。免疫抑制剂和抗炎生物制剂虽然可以缓解炎症并改善相关临床症状，但其均无直接的抗纤维化效果，并不能减轻已发生的肠道纤维化或阻止后续的纤维化进程。因此，目前尚无针对肠纤维化治疗药物获批，肠纤维化引起的肠道狭窄治疗方案仍以手术和内镜下治疗为主(D'Alessio S et al,Nat Rev Gastroenterol Hepatol.2022Mar；19(3):169-184.；胡帆等，中国医学前沿杂志(电子版).2021年第13卷第7期：14-21页)。
非酒精性肝纤维化是指除酒精外的其他致病因素引起的肝脏内***异常增生。
长期以来，人们一直认为纤维化不可逆，但临床前模型和临床试验均表 明，它是一个高度动态变化的过程。吡非尼酮最初被批准用于特发性肺纤维化(IPF)，后续研究表明其在其他纤维化模型中也显示有治疗纤维化疾病的作用。但吡非尼酮药效较差且毒副作用较强，寻找新的抗纤维化药物迫在眉睫。
发明内容
本发明要解决的技术问题是现有治疗肠纤维化或非酒精性肝纤维化的药物用量大。本发明提供了一种吡啶酮衍生物的应用，使用较少的该类化合物就能够治疗和/或预防肠纤维化和非酒精性肝纤维化。
本发明提供了一种物质A在制备药物中的应用，所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；所述药物为治疗和/或预防肠纤维化或非酒精性肝纤维化的药物；
所述应用中，所述肠纤维化优选为炎症性肠病引起的肠纤维化。
所述应用中，所述肠纤维化更优选为克罗恩病肠纤维化。
所述应用中，所述药物的施用频率可为1次/日、2次/日或3次/日；优选为2次/日。
所述应用中，所述药物的单位剂量可根据受试者/患者的起效量来确定；所述物质A的单位剂量可为25-900mg，优选为50-600mg，更优选为200-400mg。
所述应用中，每次施用所述药物时，所述药物的单次剂量可根据受试者/患者的实际治疗效果来确定；较佳地，所述药物的单次剂量为所述物质A与 受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述应用中，当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的单次剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述应用中，当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的单次剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述应用中，每次施用所述药物时，所述药物的日用剂量可根据受试者/患者的实际治疗效果来确定；较佳地，所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比可为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述应用中，当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的日用剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述应用中，当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的日用剂量可为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述应用中，所述药物中如式Ⅰ所示化合物在药物中的浓度可为0.3-3mg/mL；优选为0.3mg/mL、0.6mg/mL、1mg/mL、1.5mg/mL或3mg/mL。
所述应用中，所述药物可包含物质A和其药学上可接受的载体。
所述应用中，所述药学上可接受的载体可为本领域常规载体。
所述应用中，所述药物可经胃肠道给药，优选口服。
所述应用中，所述药物的剂型可为本领域常规剂型，所述药物的剂型可为固体制剂。
所述应用中，所述如式Ⅰ所示化合物占所述药物的质量分数为3％-50％，优选10-20％。
本发明还提供一种药物组合物，包括物质A和其药学上可接受的载体；所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；
所述药物组合物中，所述如式Ⅰ所示化合物的质量分数为3％-50％。
所述药物组合物中，所述如式Ⅰ所示化合物的质量分数可为10％-20％。
本发明还提供一种药物组合物在制备治疗和/或预防肠纤维化或非酒精性肝纤维化药物中的应用。
所述应用中，所述肠纤维化优选为炎症性肠病引起的肠纤维化；更优选为克罗恩肠纤维化。
本发明还提供了一种治疗和/或预防肠纤维化或非酒精性肝纤维化的方法，所述方法包括：向受试者/患者施用治疗有效量物质A或药物，所述药物包含物质A和其药学上可接受的载体；所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；
所述方法中，所述药学上可接受的载体为本领域常规载体。
所述方法中，所述肠纤维化优选为炎症性肠病引起的肠纤维化；更优选为克罗恩肠纤维化。
所述方法中，所述药物的施用频率可为1次/日、2次/日或3次/日，优选为2次/日。
所述方法中，所述药物的单位剂量可根据受试者/患者的起效量来确定；优选地，所述物质A的单位剂量可为25-900mg，优选为50-600mg，更优选为200-400mg。
所述方法中，每次施用所述药物时，所述药物的单次剂量可根据受试者/患者的实际治疗效果来确定；较佳地，所述药物的单次剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述方法中，当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的单次剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述方法中，当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的单次剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg。
所述方法中，每次施用所述药物时，所述药物的日用剂量可根据受试者/患者的实际治疗效果来确定；较佳地，所述药物的日用剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述方法中，当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的日用剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3- 80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述方法中，当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的日用剂量可为所述物质A与受试者/患者的质量比，所述质量比可为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
所述方法中，所述药物中如式Ⅰ所示化合物在药物中的浓度可为0.3-3mg/mL；优选为0.3mg/mL、0.6mg/mL、1mg/mL、1.5mg/mL或3mg/mL。
所述方法中，所述药物可经胃肠道给药；优选口服。
所述方法中，所述药物的剂型可为本领域常规剂型，所述药物的剂型可为固体制剂。
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
术语“治疗”指治疗性疗法。涉及具体病症时，治疗指：(1)缓解疾病或者病症的一种或多种生物学表现，(2)干扰(a)导致或引起病症的生物级联中的一个或多个点或(b)病症的一种或多种生物学表现，(3)改善与病症相关的一种或多种症状、影响或副作用，或者与病症或其治疗相关的一种或多种症状、影响或副作用，或(4)减缓病症或者病症的一种或多种生物学表现发展。
术语“预防”是指获得或发生疾病或障碍的风险降低。
术语“治疗有效量”是指在给予患者时足以有效治疗本文所述疾病或病症的化合物的量。“治疗有效量”将根据化合物、病症及其严重度、以及欲治疗患者的年龄而变化，但可由本领域技术人员根据需要进行调整。
术语“患者”是指根据本发明的实施例，即将或已经接受了该化合物给药的任何动物，哺乳动物为优，人类最优。术语“哺乳动物”包括任何哺乳动物。哺乳动物的实例包括但不限于牛、马、羊、猪、猫、狗、小鼠、大鼠、家兔、豚鼠、猴、人等，以人类为最优。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明如式Ⅰ所示化合物治疗/或预防肠纤维化和非酒精性肝纤维化较小剂量即可达到很好的药效。进一步，本发明如式Ⅰ所示化合物在治疗/或预防肠纤维化和非酒精性肝纤维化起改善效应的起效剂量为3.0mg/kg。
附图说明
图1为实施例1，试验周期内各实验组动物DAI评分结果；所有数据均采用平均值(Mean)±标准差(SD)进行统计；
图2A为实施例1，各实验组动物结肠中段组织溃疡的组织病理学诊断评分结果；
图2B为实施例1，各实验组动物结肠中段组织炎症反应的组织病理学评分结果；图2所有数据均采用平均值(Mean)±标准差(SD)进行统计分析，#P<0.05,##P<0.01,###P<0.001,####P<0.0001表示不同治疗组动物与模型对照组的统计分析结果；
图3为实施例1；各实验组动物结肠组织溃疡灶代表性图片；各实验组动物结肠中段组织病理学代表性图片，分别于100×及200×光学显微镜下对结肠进行组织病理学诊断及分析，选取各试验3只动物个体进行结果展示，图像标尺分别为1mm、250μm和100μm；
图4A为实施例1，各实验组动物结肠组织CVF％；
图4B为实施例1，各实验组动物结肠中段HYP检测结果；图4所有数据均采用平均值(Mean)±标准差(SD)进行统计分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001表示不同实验组与正常对照组的统计分析结果，#P<0.05，##P<0.01，###P<0.001，####P<0.0001表示不同治疗组动物与模型对照组的统计分析结果；
图5为实施例2，试验周期内各实验组动物肝脏天狼星红染色及诊断结果；各实验组动物肝脏组织天狼星红染色结果，分别于10×、100×及200×光学显微镜下对肝脏进行组织病理学诊断及分析，选取各试验3只动物个体进行结果展示，图像标尺分别为1mm、250μm和500μm；
图6A为实施例2，各实验组动物肝脏纤维化评分结果；
图6B为实施例2，各实验组动物肝脏CVF％计算结果；
图6C为实施例2，各实验组动物肝脏HYP检测结果；所有数据均采用平均值(Mean)±标准差(SD)进行统计分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001表示不同实验组与正常对照组的统计分析结果，#P<0.05,##P<0.01,###P<0.001,####P<0.0001表示不同治疗组动物与模型对照组的统计分析结果。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 TNBS诱导的大鼠慢性结肠炎伴肠纤维化模型药效实验
1.实验目的
评价供试品化合物Ⅰ对TNBS诱导SD大鼠慢性结肠炎伴随肠纤维化模型的治疗作用。
2.实验方法
1)动物：SD大鼠，无特定病原体(SPF)级，雄性，动物分组时7～8周龄，每组10只，由北京华阜康生物科技股份有限公司提供。
2)试剂、阳性药物及受试药物
A.试剂
表1试剂来源
B.阳性药物：柳氮磺吡啶结肠溶胶囊，提供单位为广东强基药业有限公司。
C.受试药物：化合物Ⅰ，提供单位为广州嘉越医药科技有限公司。
3)造模方法
TNBS两次灌肠法：实验动物经适应期检验及观察合格后进行体重筛选，根据体重随机分为6个实验组，包括正常对照组、模型对照组、阳性药物治疗组、供试品低剂量治疗组、供试品中剂量治疗组和供试品高剂量治疗组，每组10只小鼠。分组当天，除正常对照组给予50％乙醇溶液外，其余各实验动物均经灌肠方式给予TNBS乙醇溶液，TNBS给药剂量为每 只动物30mg，造模频率为两周一次，共2次模型诱导。实验周期共4周，所有动物均采用正常饮水及喂食。
4)分组及给药
表2分组及给药情况
备注：
a:供试品溶媒为0.5％(w/v，甲基纤维素质量占溶液体积的百分数)甲基纤维素和0.2％(v/v，吐温80体积占溶液体积的百分数)吐温80水溶液；
b:采用0.5％CMC Na(羧甲基纤维素钠)生理盐水溶液作为溶媒，配制至所需浓度；
c:采用0.5％甲基纤维素(w/v)和0.2％(v/v)吐温80水溶液作为溶媒，配制至所需浓度。
5)临床指征测定
基于动物体重、粪便形态及性状、隐血或便血情况等指标，对各组实验动物进行疾病活动指数(DAI)评分。在模型诱导后进行，每天一次，连续10天，共2轮。DAI评分标准见下表3；
表3 DAI评分标准
备注：基于体重下降幅度、粪便形态及便血进程进行DAI评分，采用三个评分指标综合进行统计分析。
6)终点样品收集、组织病理学分析和羟脯氨酸含量分析
A.动物经戊巴比妥钠麻醉安乐死后，取出结肠，即盲肠末端至***段，使用生理盐水进行肠段冲洗后，将整段结肠等分为上、中、下三段；对结肠中段进行切分，一部分组织使用液氮速冻后储存于-80℃冰箱，用于检测各组动物结肠中段样本的总羟脯氨酸水平，其余中段组织使用10％甲醛固定，石蜡包埋，切成3μm厚的切片，然后在二甲苯中脱蜡，梯度乙醇水化，H&E染色和Masson染色，脱水、透明化，封固，显微镜下观察并拍摄组织病理学改变；常规组织冻存。
B.组织病理学分析
表4结肠组织学损伤评分标准
备注：
a：基于Masson Stain结果，采用Image J对各实验组动物结肠中段样本(黏膜下层及肌层)胶原染色阳性区域面积和样本总面积进行统计，计算各样本胶原容积分数CVF％。
C.结肠羟脯氨酸水平检测
用羟脯氨酸检测试剂盒，根据操作说明书对结肠中段样本进行处理，检测各组动物结肠中段样本的总羟脯氨酸水平。
4.实验结果
1)临床评分
表5试验周期内各实验组动物DAI评分的AUC(曲线下面积)

结果：TNBS第一次诱导后(D0-D10)的DAI评分结果显示，模型对照组动物于TNBS干预后出现明显结肠炎疾病特征，具体表现为体重增长明显受到抑制，动物活动水平减退，粪便呈现糊状及稀薄样变，肛周发炎肿胀。模型对照组AUC(0-10d)经计算为45.75±4.72，与同期正常对照组动物相比均具有明显统计学差异(P<0.05)。
供试品化合物Ⅰ及阳性药物柳氮磺吡啶按照设定的治疗方案施用于各自对应组的小鼠，根据各自组的AUC(0-10d)数据，化合物Ⅰ和柳氮磺吡啶对模型动物慢性结肠炎疾病进程均具有一定的改善。与阳性药物对照组相比，各治疗组动物DAI评分AUC(0-10d)经计算分别为37.15±4.93、36.05±4.87、33.50±3.97和29.80±2.79，相对于同期模型对照组分别下降18.80％、21.20％、26.78％和34.86％，供试品化合物Ⅰ的起效剂量较小，为3.0mg/kg。
TNBS第二次诱导后(D14-D24)的DAI评分结果显示，DAI评分明显升高，模型对照组动物出现明显慢性结肠炎疾病特征，主要表现为粪便呈现糊状及稀薄样变和肛周明显发炎肿胀两个方面，体重下降程度较首轮诱导有所减轻，与同期正常对照组相比具有明显统计学差异(P<0.05)，随后评分逐渐降低，于第24天恢复至正常状态，与正常对照组相比未见明显差异。其AUC(14-24d)经计算为24.05±2.93。
供试品化合物Ⅰ及阳性药物柳氮磺吡啶对模型动物慢性结肠炎疾病仍具有一定程度的改善，与同期模型对照动物相比，各治疗组动物DAI评分均呈现一定程度下降。评分周期内DAI评分AUC(14-24d)经计算分别为19.30±2.79、17.25±3.16、15.40±3.50和11.70±1.69，相对于同期模型对照组分别下降19.75％、28.27％、35.97％和51.35％。
试验周期内各实验组动物DAI评分结果如图1所示。
2)结肠组织病理
表6各实验组动物结肠组织病理学评分结果
结果：组织病理学诊断结果表明，在本实验体系下，正常对照组动物结肠组织结构清晰，分层明显，未见明显组织学异常改变。而模型对照组动物则出现明显的结肠炎组织学改变，以结肠中段的病理学改变特征尤为明显，因此，针对结肠中段进行病理学诊断及分析。病理学改变主要表现为结肠肠壁显著增厚，肠壁黏膜表面可见裂隙状溃疡灶形成，黏膜下层及肌层出现典型肉芽肿，内伴有以淋巴细胞及浆细胞为主的炎性细胞浸润，可见小血管动脉内膜炎和成纤维细胞激活，小动脉管壁增厚及官腔狭窄，黏膜下层及肌层出现明显大量***增生及重度纤维化进程，纤维呈束状分布，同时溃疡灶周围可见隐窝结构破坏和出血等特征，其组织溃疡和炎症反应组织病理学评分均值均为2.00±0.00分。阳性对照药物柳氮磺吡啶在设定治疗方案下对模型动物结肠溃疡和炎症反应进程具有明显的抑制作用，与模型对照组动物相比，其对应治疗组动物结肠组织溃疡及炎症程度均显著减缓，病理评分均值分别为0.50±0.53分和0.60±0.52分，改善程度具有明显统计学差异(P<0.05)，同时，阳性药物治疗组动物结肠黏膜下层及肌层***增生及纤维化进程呈现明显改善及愈合表现。
供试品化合物Ⅰ在设定治疗方案下对模型动物结肠溃疡和炎症反应进程同步具有明显的抑制作用，各剂量治疗组动物结肠组织溃疡评分均值分别为0.80±0.63分、0.70±0.67分和0.70±0.67分，结肠炎症反应评分均值分别 为1.00±0.67分、0.70±0.67分和0.80±0.79分，其改善程度与同期模型对照组相比均具备显著统计学差异(P<0.05)，同时，供试品化合物Ⅰ各剂量治疗组动物结肠均可见组织愈合及纤维化程度减缓。各实验组动物结肠组织溃疡及炎症反应病理评分结果如图2A和2B所示。各实验组动物结肠组织溃疡灶代表性图片如图3所示。
3)结肠组织纤维化水平
表7各实验组动物结肠组织CVF％及HYP含量水平
结果：供试品化合物Ⅰ及阳性对照药物柳氮磺吡啶在设定治疗方案下对模型动物结肠纤维化进程具有明显的抑制效应，主要表现为抑制黏膜下层纤维***增生和改善肌层溃疡灶纤维化。对结肠中段粘膜下层及肌层的胶原容积分数(CVF％，纤维化面积/结肠粘膜下层及肌层总面积)分析结果显示，在本实验条件下，正常对照组动物结肠CVF％为0.74±0.32％，模型对照组动物结肠CVF％在同等检测分析条件下显著增高，为6.39±3.16％，与正常对照组相比具有明显统计学差异(P<0.05)。供试品化合物Ⅰ及阳性对照药物柳氮磺吡啶在设定治疗方案下对模型动物结肠CVF％升高反应具有明显的抑制作用，于同等分析条件下，其CVF％均值分别为2.97±1.78％、2.36±1.47％、2.37±1.33％和2.67±1.06％，与模型对照组相比均具有明显统计学差异(P<0.05)。
与结肠CVF％结果一致，结肠中段组织HYP检测结果显示，与正常对照组(1141.78±106.42μg/g)相比，模型对照组动物结肠HYP含量(2109.99±385.85μg/g)明显升高，且与正常对照组相比呈显著性差异(P<0.05)。阳性对照药物柳氮磺吡啶在设定治疗方案下对模型动物结肠纤 维化程度具有明显的改善作用，于同等检测条件下，其结肠HYP含量(1299.79±161.27μg/g)呈现明显下降反应，与模型对照组相比具备明显统计学差异(P<0.05)。
供试品化合物Ⅰ在设定治疗方案下对模型动物结肠纤维化进程也同步具有明显的治疗效应，与模型对照组动物相比，各剂量治疗组动物结肠组织HYP水平均呈现明显下降反应，分别为1333.43±155.33μg/g、1228.40±235.44μg/g和1270.56±287.01μg/g，与模型对照组动物对应结肠段HYP含量水平相比均具备明显统计学差异(P<0.05)，并且其改善效应具有一定的剂量依赖关系，起效剂量为3.0mg/kg。各实验组动物结肠组织纤维化水平如图4A和图4B所示。
结论：本试验采用TNBS对实验动物雄性SD大鼠进行灌肠干预，1次/2周，连续2轮，诱导其结肠发生病变，建立符合临床发病特征的大鼠慢性结肠炎伴肠纤维化模型。模型诱导当天开始进行供试品化合物Ⅰ(3.0、6.0和15.0mg/kg，每天2次)和阳性对照药物柳氮磺吡啶(50.0mg/kg，每天2次)治疗，于治疗周期(连续4周)内，通过监测实验动物体重变化、结肠炎疾病活动度DAI评分、结肠组织病理学检测及结肠组织羟脯氨酸检测等指标，评价供试品化合物Ⅰ对TNBS诱导SD大鼠慢性结肠炎伴肠纤维化的治疗作用。本试验建立符合临床发病特征的大鼠慢性结肠炎伴肠纤维化模型，药效评价结果表明供试品化合物Ⅰ具备显著的治疗效果。
实施例2化合物对四氯化碳诱导的大鼠肝纤维化模型的体内药效学研究
1.实验目的
评价供试品化合物Ⅰ对四氯化碳诱导SD大鼠肝纤维化模型的治疗作用。
2.实验方法
1)动物：SD大鼠，无特定病原体(SPF)级，雄性，动物分组时7～8周龄，每组10只小鼠，北京华阜康生物科技股份有限公司提供。
2)试剂、阳性药物及受试药物
A.试剂
表9试剂来源来源
B.阳性药物：吡非尼酮(Pirfenidone)，提供单位为瀚香生物科技。
C.受试药物：化合物Ⅰ，提供单位为广州嘉越医药科技有限公司。
3)造模方法
四氯化碳腹腔注射法：实验动物经适应期检验及观察合格后进行体重筛选，根据体重随机分为正常对照组和模型诱导组，模型诱导组动物于首次分组当天经腹腔方式给予20％四氯化碳玉米油溶液，造模频率为2次/周，连续6周。用四氯化碳诱导2周，根据模型诱导组动物谷丙转氨酶(ALT)水平二次随机分组(体重指标作为校准因素)为5个实验组，包括模型对照组、阳性药物治疗组、供试品低剂量治疗组、供试品中剂量治疗组和供试品高剂量治疗组。正常对照组动物给予相应溶媒。实验周期共6周，所有动物均采用正常饮水及喂食。
4)分组及给药情况
表10分组及给药
备注：
a:供试品溶媒为0.5％(w/v)甲基纤维素和0.2％(v/v)吐温80水溶液；
b:采用0.5％(w/v)甲基纤维素和0.2％(v/v)吐温80水溶液作为溶媒，配制至所需浓度；
5)终点样品收集、组织病理学分析和羟脯氨酸含量分析。
A.动物经戊巴比妥钠麻醉安乐死后，取出肝脏，使用生理盐水进行冲洗后，将肝脏分为肝大叶、左叶和右中叶三段；肝大叶组织使用液氮速冻后储存于-80℃冰箱，用于检测各组动物肝脏样本的总羟脯氨酸水平；肝左叶和右中叶组织使用10％甲醛固定，石蜡包埋，切成3μm厚的切片，然后在二甲苯中脱蜡，梯度乙醇水化，天狼星红染色，脱水、透明化，封固，显微镜下观察并拍摄组织病理学改变；常规组织冻存。
B.肝组织切片纤维化水平分析；
表11肝脏纤维化的评分标准
基于肝脏切片天狼星红染色结果，1)进行纤维化分期评分，评分标准见表3；2)采用Image J对各实验组动物肝脏纤维化面积和样本总面积进行统计，计算各样本胶原容积分数CVF％。
C.肝脏羟脯氨酸水平检测
采用羟脯氨酸检测试剂盒根据操作说明书对肝脏大叶样本进行处理，检测各组动物肝脏样本的总羟脯氨酸水平。
4.实验结果
1)肝脏组织病理诊断结果；试验周期内各实验组动物肝脏天狼星红染色及诊断结果如图5所示。
结果：在本实验体系下，模型动物肝脏在四氯化碳诱导下出现明显的肝纤维化病变，包括小叶结构紊乱，肝板界面出现明显胶原增生，汇管区和中央静脉区***变厚，形成假小叶结构。供试品化合物Ⅰ在设定治疗方案下，于200×及400×光镜下可见，中剂量及高剂量治疗组部分动物肝脏假小叶结构呈现瓦解趋势，主要表现在肝纤维桥连处纤维束出现缩窄以及假小叶数量减少两个方面。同时，供试品各治疗组动物肝脏中央静脉区域肝细胞脂肪变性及肿胀变性程度均较模型对照组出现一定程度降低，以中剂量和高剂量最为明显，而阳性对照药物吡非尼酮治疗组则未见明显改善。
2)肝脏组织纤维化水平
表12各实验组动物肝纤维化进程评分
结果：模型对照组动物肝脏纤维化程度明显，其纤维化分期均为S3期，纤维化评分均值为4.90±0.88分，大部分动物肝脏门管与门管和/或门管与中央静脉区域均可见纤维桥接形成的假小叶结构，少数动物出现明显肝硬化表征。供试品化合物Ⅰ在设定治疗方案下对模型动物肝纤维化进程具有一定的抑制作用，低、中、高剂量组动物肝纤维化评分均值分别为4.40±0.70分、4.00±0.94分和4.20±0.92分，其中以中剂量对纤维化程度的改善效应最为明显，与模型对照组相比呈显著性差异(P<0.05)，而阳性对照药物吡非尼酮则对模型动物肝脏纤维化进程未见明显影响，其纤维化评分均值为5.40±0.52分，与模型对照组无显著性差异(P>0.05)。
各实验组动物肝脏胶原容积分数(CVF％)计算结果显示，与正常对照组(1.24±0.23％)相比，模型对照组动物肝脏CVF％显著升高，为8.75±2.39％。供试品化合物Ⅰ在设定治疗方案下对模型动物肝纤维化进程具有明显抑制效应，其CVF％分别为5.02±1.42％、4.87±1.68％和5.60±1.92％，与模型对照组相比均具显著性差异(P<0.05)。阳性对照药物Pirfenidone对模型动物肝纤维化程度具有显著的抑制效应，其肝脏CVF％为6.58±2.20％，与模型对照组相比具有显著性差异(P<0.05)。
各实验组动物肝脏组织羟脯氨酸检测结果表明，模型对照组动物肝脏HYP含量水平为702.84±173.44μg/g，与正常对照组动物(307.30±78.13μg/g)相比明显升高，呈显著性差异(P<0.05)。供试品化合物Ⅰ中、高剂量组及阳性对照药物吡非尼酮在设定治疗方案下对模型动物肝脏羟脯氨酸水平升高具有一定程度的抑制作用，其肝脏HYP水平呈现下降趋势，分别为560.60±114.04μg/g、607.79±112.74μg/g和611.19±183.66μg/g，其中以供试品化合物Ⅰ中剂量的改善效应最为明显，与模型对照组相比具有显著性差异(P<0.05)。各实验组动物肝脏组织纤维化水平如图6所示。
结论：本试验通过腹腔注射20％四氯化碳玉米油溶液(每周2次，连续6周)诱导实验动物雄性SD大鼠肝脏发生病变，建立符合临床发病特征的大鼠肝纤维化模型。于模型连续诱导2周后开始进行供试品化合物Ⅰ(3.0、10.0和30.0mg/kg，每天2次)和阳性对照药物吡非尼酮(150.0mg/kg，每天2次)治疗，连续给药4周。肝脏病理纤维化评分结果显示，供试品化合物Ⅰ治疗给药4周可抑制模型动物肝脏纤维化进程，低、中、高剂量组动物肝纤维化评分均值分别为4.40±0.70、4.00±0.94和4.20±0.92分，均明显低于模型对照组(4.90±0.88分)。肝脏切片的胶原容积分数(CVF％)定量分析结果显示，供试品化合物Ⅰ低、中、高剂量组及阳性对照药物吡非尼酮组的CVF％水平分别为5.02±1.42％、4.87±1.68％、5.60±1.92％和6.58±2.20％，均显著低于模 型对照组(8.75±2.39％，P<0.05),表明各治疗组对肝纤维化具有显著抑制作用。与肝脏病理纤维化分析结果一致，供试品化合物Ⅰ中剂量组的肝组织羟脯氨酸水平较模型对照组亦显著降低(560.60±114.04μg/g vs 702.84±173.44μg/g，P<0.05)。本药效评价结果表明供试品化合物Ⅰ具备显著的治疗效果。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不违背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种物质A在制备药物中的应用，其特征在于，所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；所述药物为治疗和/或预防肠纤维化或非酒精性肝纤维化的药物；
  2. 如权利要求1所述应用，其特征在于，所述应用满足如下条件中的一种或多种：
    ①所述肠纤维化为炎症性肠病引起的肠纤维化，优选为克罗恩肠纤维化；
    ②所述药物包含物质A和其药学上可接受的载体；
    ③所述药物经胃肠道给药，例如口服；
    ④所述药物的施用频率为1次/日、2次/日或3次/日，优选为2次/日；
    ⑤所述药物的剂型为固体制剂；
    ⑥所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg；
    ⑦所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg；
    和⑧所述物质A的单位剂量为25-900mg，优选为50-600mg，更优选为200-400mg。
  3. 如权利要求1所述应用，其特征在于，所述应用满足如下条件中的一种或多种：
    ①所述如式Ⅰ所示化合物在药物中的浓度为0.3-3mg/mL；优选为0.3mg/mL、0.6mg/mL、1mg/mL、1.5mg/mL或3mg/mL；
    ②当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg；
    ③当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg；
    ④当所述药物为制备治疗和/或预防肠纤维化的药物时，所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg；
    和⑤当所述药物为制备治疗和/或预防非酒精性肝纤维化的药物时，所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
  4. 如权利要求1所述应用，其特征在于，所述如式Ⅰ所示化合物占所述药物的质量分数为3％-50％，优选10％-20％。
  5. 一种药物组合物，其特征在于，包括物质A和其药学上可接受的载体；所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；
    所述药物组合物中，所述如式Ⅰ所示化合物的质量分数为3％-50％。
  6. 如权利要求5所述药物组合物，其特征在于，所述药物组合物中，所述如式Ⅰ所示化合物的质量分数为10％-20％。
  7. 一种如权利要求5或6所述药物组合物在制备治疗和/或预防肠纤维化或非酒精性肝纤维化药物中的应用；
    较佳地，
    所述肠纤维化为炎症性肠病引起的肠纤维化，优选为克罗恩肠纤维化。
  8. 一种治疗和/或预防肠纤维化或非酒精性肝纤维化的方法，其特征在于，所述方法包括：通过向受试者/患者施用治疗有效量的物质A或药物；
    所述药物包含物质A和其药学上可接受的载体；所述物质A为如式Ⅰ所示化合物或其药学上可接受的盐；
  9. 如权利要求8所述方法，其特征在于，所述方法满足如下条件中的一种或多种：
    ①所述肠纤维化为炎症性肠病引起的肠纤维化，优选为克罗恩肠纤维化；
    ②所述药物包含物质A和其药学上可接受的载体；
    ③所述药物经胃肠道给药，例如口服；
    ④所述药物的施用频率为1次/日、2次/日或3次/日，优选为2次/日；
    ⑤所述药物的剂型为固体制剂；
    ⑥所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或30mg/kg；
    ⑦所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg；
    和⑧所述物质A的单位剂量为25-900mg，优选为50-600mg，更优选为200-400mg。
  10. 如权利要求8所述方法，其特征在于，所述方法满足如下条件中的一种或多种：
    ①所述如式Ⅰ所示化合物在药物中的浓度为0.3-3mg/mL；优选为0.3mg/mL、0.6mg/mL、1mg/mL、1.5mg/mL或3mg/mL；
    ②当所述药物为治疗和/或预防肠纤维化的药物时，所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg；
    ③当所述药物为治疗和/或预防非酒精性肝纤维化的药物时，所述药物的单次剂量为所述物质A与受试者/患者的质量比，所述质量比为3-40mg/kg，优选为3mg/kg、6mg/kg、10mg/kg、15mg/kg、30mg/kg或40mg/kg；
    ④当所述药物为制备治疗和/或预防肠纤维化的药物时，所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3-80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg；
    和⑤当所述药物为制备治疗和/或预防非酒精性肝纤维化的药物时，所述药物的日用剂量为所述物质A与受试者/患者的质量比，所述质量比为3- 80mg/kg，优选为3mg/kg、6mg/kg、12mg/kg、15mg/kg、30mg/kg、40mg/kg、60mg/kg或80mg/kg。
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