Isosieverside and its compositions and uses

Abstract

The present application discloses isoschaftoside and its compositions and uses. Experiments have proven that isoschaftoside can significantly improve dextran sulfate sodium-induced colitis in mice, specifically manifested as significantly alleviating the weight loss and shortening of colon length caused by the model establishment; significantly reducing the disease activity index and spleen weight of mice; significantly alleviating the pathological changes in the colon tissues of mice, including reducing inflammatory cell infiltration, restoring the crypt structure of the tissue and the number of intestinal glands, and having significant anti-ulcerative colitis activity, showing good application prospects in the preparation of drugs for treating ulcerative colitis.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technologies, and particularly to isoschaftoside, its composition, and uses thereof. Background Art

[0002] Inflammatory bowel disease is a chronic and recurrent inflammatory disease caused by multiple factors such as genetics and environment, and mainly has two phenotypes: Crohn's disease (CD) and ulcerative colitis (UC). The main clinical manifestations are abdominal pain, diarrhea, mucus, bloody stools, tenesmus, etc. The lesion sites are mainly concentrated in the mucosa and submucosa of the colon, showing a continuous and diffuse distribution, mostly involving the rectum and sigmoid colon. The disease course is relatively long, often protracted and difficult to cure, resulting in repeated intestinal injuries, forming ulcers, polyps, and even malignant transformation into colorectal cancer. Epidemiological statistics at home and abroad show that the incidence and prevalence of UC have both shown an obvious increasing trend in recent years, and it has been listed as one of the modern intractable diseases by the World Health Organization.

[0003] Currently, the first-line drugs for treating inflammatory bowel disease include non-steroidal anti-inflammatory drugs, corticosteroids, and immunomodulators, etc. However, only 30 - 50% of patients benefit from the current FDA-approved treatment methods, and they are all associated with serious adverse reactions. Therefore, there is an urgent need to find new drugs with higher safety. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, this application provides the use of isoschaftoside in the preparation of a drug for treating and / or preventing inflammatory bowel disease.

[0005] Specifically, this application relates to the following aspects:

[0006] 1. The use of isoschaftoside in the preparation of a drug for treating and / or preventing inflammatory bowel disease.

[0007] 2. The use according to item 1, wherein the inflammatory bowel disease is ulcerative colitis.

[0008] 3. The use according to item 1 or 2, wherein the administration dose of isoschaftoside is 1 - 2 times a day, and 6 - 13 mg / kg of the subject each time.

[0009] 4. The use according to any one of items 1 - 3, wherein the single dose of the drug is 300 mg - 800 mg.

[0010] 5. The use according to item 3, wherein the subject is a human.

[0011] 6. The use according to any one of items 1 - 5, wherein the drug further comprises one or more other active substances.

[0012] 7. A composition for treating and / or preventing inflammatory bowel disease, wherein the composition contains isoschaftoside, and isoschaftoside is the only active ingredient in the composition.

[0013] 8. The composition according to item 7, wherein the inflammatory bowel disease is ulcerative colitis.

[0014] 9. The composition according to item 7 or 8, wherein the composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0015] 10. The composition according to any one of items 7-9, wherein the composition is a pill, capsule, tablet, powder, granule, injection or compound preparation.

[0016] This application provides a new use of isoschaftoside. Experiments have proved that isoschaftoside can significantly improve dextran sulfate sodium-induced colitis in mice, specifically manifested as significant weight loss and shortened colon length caused by the establishment of the model; significantly reduce the disease activity index and spleen weight of mice; significantly relieve the pathological changes of mouse colon tissue, including reducing inflammatory cell infiltration, restoring the crypt structure and the number of intestinal glands of the tissue, and has significant anti-ulcerative colitis activity, showing good application prospects in the preparation of drugs for treating ulcerative colitis. Brief Description of the Drawings

[0017] The drawings are used to better understand this application and do not constitute an improper limitation to this application. Among them:

[0018] Figure 1 It is a schematic diagram of the results of the colon length of the UC model mice induced by DSS with isoschaftoside of this application;

[0019] Figure 2 It is a picture of the colon of the UC model mice induced by DSS with isoschaftoside of this application;

[0020] Figure 3 It is a schematic diagram of the results of the spleen weight of the UC model mice induced by DSS with isoschaftoside of this application;

[0021] Figure 4 It is a schematic diagram of the results of the spleen index of the UC model mice induced by DSS with isoschaftoside of this application;

[0022] Figure 5 It is a schematic diagram of the results of the weight change of the UC model mice induced by DSS with isoschaftoside of this application;

[0023] Figure 6 It is a schematic diagram of the results of the DAI score of the UC model mice induced by DSS with isoschaftoside of this application;

[0024] Figure 7This is the pathological section diagram of the colon of mice with UC model induced by DSS in this application with isopicroside. Detailed implementation manners

[0025] The following further illustrates this application with examples. It should be understood that the examples are only used to further illustrate and explain this application, and are not used to limit this application.

[0026] Unless otherwise defined, the technical and scientific terms in this specification have the same meanings as those commonly understood by those skilled in the art in this field. Although methods and materials similar or identical to those described herein can be applied in experiments or practical applications, the materials and methods are still described below. In case of conflict, this specification including its definitions shall prevail. Additionally, the materials, methods, and examples are for illustration only and are not restrictive. The following further illustrates this application with specific examples, but does not limit the scope of this application.

[0027] This application provides the use of isopicroside in drugs for treating and / or preventing inflammatory bowel disease.

[0028] This application also provides the use of isopicroside in the preparation of drugs for treating and / or preventing inflammatory bowel disease.

[0029] This application innovatively discovers that isopicroside has a therapeutic or preventive effect on inflammatory bowel disease.

[0030] Isopicroside is also known as isopicrotoxin, and it is a flavonoid compound that can be extracted from Desmodium styracifolium, Rabdosia serra, and Isodon lophanthoides var. gerardianus. The specific structure of isopicroside is shown as follows:

[0031]

[0032] The isopicroside of this application can be obtained through commercial purchase or can be prepared by known techniques in this field.

[0033] The inflammatory bowel disease in this application can cover Crohn's disease and ulcerative colitis.

[0034] In a specific implementation manner, the inflammatory bowel disease is ulcerative colitis.

[0035] As used herein, "treatment" refers to any improvement in any consequence of a disease, disorder or condition, such as extended survival, lower morbidity and / or alleviation of side effects caused by alternative treatment modalities. In some embodiments, treatment includes delaying or improving a disease, disorder or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In some embodiments, treatment includes delaying, alleviating or improving at least one physical parameter of a disease, disorder or condition, including physical parameters that may not be distinguishable to the patient. In some embodiments, treatment includes physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters) or both modulating a disease, disorder or condition.

[0036] As used herein, the term "prevention" refers to prophylactic treatment of a disease or condition; or delaying the onset or progression of the disease, disorder or condition.

[0037] The pharmaceutical composition can be used in a patient or other animal that receives the pharmaceutical composition of the present application for treating, preventing, alleviating and / or relieving the diseases or conditions described herein. In a specific embodiment, the pharmaceutical composition is used in mammals, including but not limited to humans, cows, horses, sheep, pigs, goats, rabbits, cats, dogs, mice, and any other mammal having a liver and capable of developing liver injury.

[0038] In a specific embodiment, the pharmaceutical composition is used in mice and humans.

[0039] The dosage of the pharmaceutical composition of the present application depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the route of administration and the frequency of administration, etc. The above dosage can be administered in a single dose form or divided into several, for example, two, three or four dose forms. The dosage level must be selected according to the specific route of administration, the severity of the condition to be treated and the condition and medical history of the patient to be treated.

[0040] It should be recognized, however, that the total daily dosage of the pharmaceutical composition of the present application must be decided by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion; the duration of the treatment; drugs used in combination with or concurrently with the composition; and like factors well known in the medical arts. For example, it is the practice in the art to start the dosage at a level below that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0041] In a specific embodiment, the administration dosage of isoscoparin is 1 - 2 times per day, 6 - 13 mg / kg of the subject each time. For example, it can be 6 mg / kg of the subject, 7 mg / kg of the subject, 8 mg / kg of the subject, 9 mg / kg of the subject, 10 mg / kg of the subject, 11 mg / kg of the subject, 12 mg / kg of the subject, 13 mg / kg of the subject each time.

[0042] In a specific embodiment, the subject is a human.

[0043] Those skilled in the art can understand that the administration frequency and dosage can be adjusted according to the specific conditions of the subject or the severity of inflammatory bowel disease.

[0044] In a specific embodiment, continuous administration is carried out for 3 to 10 days, such as 3 days, 5 days, 7 days, 10 days, etc., and any value between these values. In a specific embodiment, the administration dosage of isoscoparin is 1 time per day, 6 - 13 mg / kg of the subject each time, and continuous administration is carried out for 3 to 10 days.

[0045] In a specific embodiment, the drug is administered in a single-dose form, that is, a single-dose form. In a specific embodiment, the single dose for administration is 300 mg - 800 mg. For example, it can be 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, and any value between these values. Those skilled in the art should understand that usually, the body weight of a human is calculated as 60 kg. If the body weight of the human is not 60 kg, it can be converted according to this standard.

[0046] The drugs of the present application can be administered in unit-dose forms, and the administration routes can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, buccal mucosa, skin, peritoneal or rectal, etc. The dosage forms for administration are, for example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation and various particulate drug delivery systems.

[0047] In a specific embodiment, the drug is a pill, a capsule, a tablet, a powder, a granule, an injection or a compound preparation.

[0048] In a specific embodiment, the drug may further optionally contain a pharmaceutically acceptable carrier or excipient, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gel, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc., but not limited thereto. In addition to the above components, the drug of the present application may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives, etc.

[0049] In a specific embodiment, the drug of the present application may further contain one or more other active substances. The other active substances refer to substances other than isoschaftoside that have activity and produce certain functions. The active substances may be active substances for treating or preventing inflammatory bowel disease, or may be active substances for assisting in treating or preventing inflammatory bowel disease, or may be active substances that do not treat inflammatory bowel disease, but it can play a positive effect or function in other aspects.

[0050] The present application also provides a composition for treating and / or preventing inflammatory bowel disease, wherein the composition contains isoschaftoside, and isoschaftoside is the only active ingredient in the composition. That is, isoschaftoside is the only active ingredient for the composition to treat and / or prevent inflammatory bowel disease, and the composition does not contain other active ingredients that can treat and / or prevent inflammatory bowel disease.

[0051] The term "composition" means a product comprising a product containing specified amounts of each specified component, and any product directly or indirectly resulting from the combination of the specified amounts of each specified component.

[0052] In a specific embodiment, the composition further contains one or more pharmaceutically acceptable carriers or excipients. The one or more pharmaceutically acceptable carriers or excipients are the carriers or excipients as described above.

[0053] In a specific embodiment, the inflammatory bowel disease is ulcerative colitis.

[0054] The composition of the present application can be in the form of pills, capsules, tablets, powders, granules, injections or compound preparations.

[0055] The compositions of the present application can be administered in single-dose forms, and the administration routes can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal, or rectal, etc. The dosage forms for administration are, for example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. They can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle drug delivery systems.

[0056] The administration dose of the composition of the present application can be converted with reference to the administration dose of isoschaftoside as described above.

[0057] The isoschaftoside involved in the present application has the efficacy of treating and / or preventing inflammatory bowel disease. The present application verified through mouse model experiments that isoschaftoside can significantly improve dextran sulfate sodium-induced colitis in mice, specifically manifested as significant weight loss and shortened colon length caused by modeling; significantly reducing the disease activity index and spleen weight of mice; significantly alleviating the pathological changes of mouse colon tissues, including reducing inflammatory cell infiltration, restoring tissue crypt structure and the number of intestinal glands, and having significant anti-ulcerative colitis activity.

[0058] Examples

[0059] Experimental materials

[0060] Animals: SPF-grade male C57 BL / 6 mice, weighing 22 (±2) g, 7 weeks old. Purchased from Zhuhai Bestone Biotechnology Co., Ltd., animal license number: [SCXK (Guangdong) 2022-0051].

[0061] Isoschaftoside, from Chengdu Kloma Co., Ltd.;

[0062] Dextran sulfate sodium (DSS), from MP Biomedical, USA, product number: CHB201109;

[0063] Sodium carboxymethyl cellulose, from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0064] Fecal occult blood test kit, from Beijing Regen Biotechnology Co., Ltd.

[0065] Example 1 Effect of isoschaftoside on treating ulcerative colitis induced by dextran sulfate sodium

[0066] 1. Preparation of main reagents

[0067] Preparation of 3.5% DSS solution

[0068] Weigh 3.5 g of DSS, dissolve it in 100 mL of distilled water, mix well to make it fully dissolve, prepare it freshly for each use, and replace it once a day.

[0069] Preparation of Isoschaftoside Suspension

[0070] Isoschaftoside low-dose group solution: Take an appropriate amount of pure isoschaftoside, and prepare a 50 mg / kg solution with 0.5% sodium carboxymethylcellulose, and administer it by gavage in equal volume.

[0071] Isoschaftoside high-dose group solution: Take an appropriate amount of pure isoschaftoside, and prepare a 100 mg / kg solution with 0.5% sodium carboxymethylcellulose, and administer it by gavage in equal volume.

[0072] 2. Experimental methods

[0073] 2.1 Animal grouping, modeling and drug administration

[0074] Animals were randomly divided into a normal group (i.e., blank group NC), a model group (DSS), an isoschaftoside low-dose group (DSS+L-ISS) (50 mg / kg), and an isoschaftoside high-dose group (DSS+H-ISS) (100 mg / kg), with 6 animals in each group. The first day of the experiment was recorded as day 0. Both the normal group and the model group were given 0.5% sodium carboxymethylcellulose solution by gavage in equal volume, and the other groups were given the corresponding drugs by gavage in equal volume. Except for the normal group, the remaining groups were allowed to freely drink 3.5% DSS solution from day 0 of the experiment. 12 hours before sample collection, the animals were fasted but allowed to drink water.

[0075] 2.2 Scoring of mouse disease activity index (DAI)

[0076] During the DSS feeding period, the animals were weighed once a day, the water intake of each cage of animals was calculated every day, and the fecal occult blood test was performed every day. Evaluation and scoring were carried out from three aspects: body weight, fecal viscosity, fecal occult blood and other indicators, and the DAI score was the comprehensive score of the three indicators. The scoring rules are shown in Table 1:

[0077] Table 1

[0078]

[0079] 2.3 Sample collection

[0080] On the 7th day of the experiment, the mice were fasted but allowed to drink water for 12 hours. On the 8th day, blood was collected by eye socket puncture, left to stand at room temperature for half an hour, centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant was collected and stored at -80°C for later use. After blood collection, the mice were sacrificed by cervical dislocation. The spleen and the entire colon were dissected from the mice. After cleaning the mesentery, they were neatly placed on A4 paper, photographed, and the colon length was recorded. 1 cm of colon tissue was cut near the anal end and fixed in 4% paraformaldehyde solution, and the remaining colon tissue was rinsed with normal saline and stored at -80°C for later use.

[0081] 2.4 Colonic pathological examination

[0082] Colon tissues 1 cm from the anal end of different mice were fixed in 4% paraformaldehyde solution; paraffin sections were prepared through steps such as washing, dehydration, clearing, infiltration with paraffin wax, embedding, sectioning and adhering sections, dewaxing, H&E staining, dehydration, clearing, and mounting, and the degree of colon tissue inflammation, crypt damage, inflammation depth, inflammation infiltration area, and lesion depth were observed under an optical microscope.

[0083] 2.5 Data statistical analysis

[0084] The experimental data were analyzed and processed using Graphpad prism 9.5 software. When the measurement data met the normal distribution, one-way ANOVA was used; when it did not, Tamhanes T2(M) test was used. During data analysis, the number of each group was n = 6. Compared with the blank group, #, P < 0.05; ##, P < 0.01; , P < 0.001; compared with the model group, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0085] 3. Experimental results

[0086] 3.1 Effect of isoscoparin on the colon length of DSS-induced UC mouse model

[0087] The measurement results of the colon length of each group of mice are as Figure 1 and Figure 2 shown. The results showed that compared with the blank group, the colon length of the model group mice was significantly reduced; compared with the model group, the colon lengths of the high-dose isoscoparin group (H-ISS) and low-dose isoscoparin group (L-ISS) mice were significantly increased.

[0088] 3.2 Effect of isoscoparin on the spleen weight of DSS-induced UC mouse model

[0089] The measurement results of the spleen weight of each group of mice are as Figure 3 shown. The results showed that compared with the blank group, the spleen weight of the model group mice was significantly increased; compared with the model group, the spleen weight of the high-dose isoscoparin group mice was significantly reduced.

[0090] 3.3 Effect of isoscoparin on the spleen index of DSS-induced UC mouse model

[0091] The measurement results of the spleen index of each group of mice are as Figure 4 shown. The results showed that compared with the blank group, the spleen index of the model group mice was significantly increased; compared with the model group, the spleen index of the high-dose isoscoparin group mice was significantly reduced.

[0092] 3.4 Effect of isoscoparin on the body weight change of DSS-induced UC mouse model

[0093] The line graphs of the body weights of each group of mice are as Figure 5 shown. The results show that the body weights of the mice in the blank group did not change significantly during the test period. Compared with the blank group, the body weights of the mice in the model group decreased significantly during the experiment. Compared with the model group, the downward trends of the body weights of the mice in the high- and low-dose isoschaftoside groups were effectively controlled.

[0094] 3.5 Effect of isoschaftoside on the disease activity index (DAI) of UC mouse models induced by DSS

[0095] Since the 0th day of the experiment, except for the normal group, each group of mice was allowed to drink 3.5% DSS aqueous solution freely. The mice gradually showed signs of decreased activity, weight loss, and reduced activity. Starting from the 4th day of the experiment, some mice showed diarrhea and bloody stools, and most mice showed the above conditions on the 6th day of the experiment. 12 h before sampling, all 3.5% DSS aqueous solutions were replaced with drinking water.

[0096] The DAI results of each group of mice are as Figure 6 shown. The results show that the high- and low-dose isoschaftoside groups of this application can reduce the DAI scores of UC model mice.

[0097] 3.6 Results of colon pathological sections of isoschaftoside

[0098] The results of colon pathological sections of each group of mice are as Figure 7 shown. The results show that compared with the blank group, the colons of the mice in the model group showed obvious lesions and enhanced inflammatory infiltration. Compared with the model group, the colon lesions of the mice in the high- and low-dose isoschaftoside groups were significantly alleviated and the inflammatory infiltration was improved.

[0099] Although the embodiments of the present application have been described above in combination, the present application is not limited to the above specific embodiments and application fields. The above specific embodiments are merely illustrative and guiding, rather than restrictive. Those of ordinary skill in the art can also make many forms under the inspiration of this specification and without departing from the scope protected by the claims of the present application, and these all belong to the scope of protection of the present application.

Claims

1. Use of isoschaftoside in the preparation of a drug for treating and / or preventing ulcerative colitis, wherein isoschaftoside is used as the sole active ingredient.

2. The use according to claim 1, wherein the administration dose of isoschaftoside is 1-2 times a day, 6-13 mg / kg of the subject each time.

3. The use according to claim 1, wherein the single dose of the drug is 300 mg - 800 mg.

4. The use according to claim 2, wherein the subject is a human.

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