WO2023274095A1 - Application of kaurane compound in preparation of drug for preventing and treating inflammatory bowel disease - Google Patents

Abstract

An application of a kaurane compound in the preparation of a drug for treating inflammatory bowel disease. The kaurane compound can mitigate the inflammatory reaction of dextran sodium sulfate (DSS)-induced inflammatory bowel disease model mice and improve the severity of the inflammatory bowel disease, and can be used for preparing a drug or health care product for treating the inflammatory bowel disease.

Description

Application of Kauritanes in the Preparation of Medicines for Preventing and Treating Inflammatory Bowel Diseases field of invention

The invention discloses a kauritanes compound for alleviating and treating inflammatory bowel disease. The invention discloses that the kauritanes compound can obviously reduce the body weight of enteritis model mice induced by dextran sodium sulfate (DSS), improve the shortening of the colon length of the colitis model mice, and reduce the infiltration of inflammatory cells in the colon tissue of the colitis model , reduce the increase of white blood cells, neutrophils, lymphocytes and monocytes in colitis model mice, and reduce the expression of inflammatory factors. The invention discloses the regulating effect of the kauritanes on macrophages and T cells when inflammatory bowel disease occurs.

Background technique

Inflammatory bowel disease is a chronic intestinal inflammatory disease with unclear pathogenesis, mainly including ulcerative colitis (Ulcerative Colitis, UC) and Crohn's disease (Crohn's disease, CD). UC is a chronic non-specific colonic inflammation. The lesions mainly involve the colonic mucosa, mostly starting from the distal colon, and can develop to the proximal end, and even involve the entire colon. The main clinical manifestations are diarrhea, abdominal pain, and mucus, pus and blood in the stool. CD is a kind of chronic granulomatous inflammation, and the lesions can involve all parts of the gastrointestinal tract, but it is more likely to occur in the terminal ileum and right colon; the main clinical manifestations are abdominal pain, diarrhea, and intestinal obstruction (Peixoto Armando, ACG Case Reports Journal, 2017, 4(1):e46). IBD is common in western countries, and the prevalence rate in European and North American countries is as high as 120-200/100,000. It is a common disease in the digestive field. According to statistics, the incidence of IBD has been increasing rapidly in Africa, Asia and South America since 1990. In Brazil, the annual growth rate of the incidence of UC and CD is as high as 15% and 11% respectively; in my country, the incidence of IBD is also showing an increasing trend year by year, and the annual growth rate of the incidence of UC and CD in Taiwan is 5% and 4% respectively (Kamm Michael A, The Lancet, 2017, 390(10114):2741-2742), IBD has become a common disease of the digestive system in my country and the main cause of chronic diarrhea and blood in the stool. Its etiology and pathogenesis are unknown, but it is generally believed to be related to heredity, abnormal intestinal mucosal immune regulation, persistent intestinal infection, and intestinal mucosal barrier defect. Most patients with inflammatory bowel disease are accompanied by extraintestinal manifestations of autoimmune diseases, such as erythema nodosum and arthritis. The course of inflammatory bowel disease is protracted, and some even last for decades. There is also the possibility of cancer. Current studies have found that in patients with inflammatory bowel disease, the local humoral or cellular immunity is activated, and a large number of inflammatory cells infiltrate in the lamina propria of the intestinal mucosa. Anti-colonic epithelial cell antibodies can be detected in the serum of some patients. Abnormal immune response in intestinal mucosal tissue is an important cause of intestinal mucosal tissue damage. The main method of IBD treatment is to restore the balance between pro-inflammatory factors and anti-inflammatory factors and regulate intestinal mucosal immune abnormalities. The immune response includes intestinal mucosal adaptation. Sexual immune response (such as T cells) and innate immune response (such as macrophages) (Khor B, Nature. 2011.474(7351): 307-17).

T cells, including Th1, Th2 and Th17 cells, are involved in the pathogenesis of DSS-induced enteritis. In the past, it was believed that the main effector cells leading to intestinal mucosal inflammation were CD4+ T cells, in which CD was considered to be dominated by Th1-type mucosal inflammation; UC was dominated by Th2-type. However, with the deepening of research, there is still a lot of controversy about the corresponding relationship between CD and UC and Th1 and Th2 cells. Th17 cell subsets are a type of CD4+T cell subsets discovered in recent years. In recent studies, it has been found that IBD patients have a large number of Th17 cell infiltration, which opens up a new field of research on inflammatory bowel disease. Studies have found that a variety of cytokines are related to the regulation of Th17 cell differentiation. The role of Th17 cells includes clearing specific extracellular pathogens, thereby playing a protective role; also including causing inflammation and autoimmune diseases. The main function of Th17 cells is to secrete cytokines, including IL-17A, IL-21, etc., which play an important role in the occurrence of various autoimmune diseases and induce inflammatory responses in IBD immune regulation. By controlling the secretion of Th7 cell-related cytokines, the clinical symptoms of IBD patients can be significantly alleviated (Yu-Fang Wang, World journal of gastroenterol, 2013, 19(11): 1827-33).

Macrophages are a group of highly heterogeneous cells, which can be divided into two subpopulations: classically activated macrophages (M1 type) and alternatively activated macrophages (M2 type) according to their activation status (Besedovsky H, Clinical and experimental immunology, 1977; 27(1):1-12). The polarization of macrophages is influenced by various cytokines in the microenvironment and has considerable plasticity. M1 macrophages are mainly activated by IFN-γ, TNF-α, LPS and other stimuli, and can secrete a large number of inflammatory cytokines, such as IL-1β, IL-13, TNF-α, etc., as well as reactive oxygen products. These cellular mediators can promote the activation of Th1 and Th17 cells, promote local inflammatory responses, and accelerate the clearance of intracellular pathogens. If the activity of M1 macrophages is not controlled, it will lead to excessive inflammatory response and cause tissue damage (Knobloch HS, Frontiers in behavioral neuroscience, 2014; 8:31). Under the action of IL-4 and IL-13 produced by granulocytes or Th2 cells, macrophages are polarized into M2 macrophages, which are insensitive to LPS stimulation and secrete growth factors, IL-10, TGF-β, etc. Recruit Th2 and regulatory T cells (Treg), downregulate local inflammatory response, promote tissue repair and clearance of parasites. When the regulation of M2 macrophages is out of balance, it will enhance the allergic reaction, promote local fibrosis and tumor formation (KimYS, Cells, tissues, organs, 2012; 195(5):428-42). Therefore, regulating the balance between the two macrophage subpopulations of M1 and M2 plays a very important role in maintaining the immune homeostasis of the internal environment. The polarization of macrophages is regulated by various signaling pathways in the cell. More and more studies have found that IBD patients have abnormal polarization of intestinal macrophages, which may play a central role in the occurrence and development of IBD; restoring the balance between the two subtypes of intestinal macrophages may become a clinical An important means of treating IBD. Therefore, from the perspective of immune regulation, especially Th cell subsets and macrophage polarization, it is of great significance to further elucidate the immunogenesis mechanism of IBD by in-depth understanding of the multi-factor multi-target pathogenesis of IBD.

At present, there is no drug that can completely cure IBD. The main purpose of clinically used drugs for treatment is to relieve symptoms and improve the quality of life of patients. Therapeutic drugs include aminosalicylic acid preparations, glucocorticoids, immunosuppressants and various biological agents (such as TNF-α monoclonal antibody, etc.). There are deficiencies in each of the above drugs. For example, aminosalicylic acid drugs are likely to cause drug resistance in patients, and glucocorticoids will relapse after drug withdrawal. The cost of TNF antibody production and treatment is high, and it is prone to failure after long-term use, and it is easy to cause patients infection (Paolo Gionchetti, Dig Liver Dis, 2017, 49(6):604-17). Therefore, finding safer and more effective anti-colitis drugs is one of the hotspots in current IBD research, and it is an urgent clinical need that has not been met.

Compound A is a bayonene terpenoid isolated from stevioside. Stevia is a well-known traditional plant in South America and is a widely used sweetener worldwide. The effect of stevia on metabolism and cardiovascular system (Geuns JMC.Stevioside.Phytochemistry.2003; 64(5):913-21) has also been reported.

Previous studies have shown that the kaurane-like compounds represented by compound A have protective effects on heart and brain tissue, and can be used to treat myocardial ischemia and cerebral infarction (patent 1: CN100508962 C). In addition, compound A and related kaurane-type compounds can also inhibit tissue damage leading to inflammation, and inhibit fibrosis of myocardial and lung tissue (patent 2: CN108348481 A). Compound A may also be used for metabolic diseases and diabetic myocarditis. Studies have also proved that compound A also has inhibitory effect on some cytokines such as TNF-α, interleukin IL-6 and so on.

However, compound A and related kauritanes have not been reported in the treatment of inflammatory bowel disease. It is now known that immune dysfunction is the main cause of inflammatory bowel disease, and the imbalance of macrophages and T cells plays a key role in the initiation and development of the above-mentioned cytokine storm. However, compound A and related kaurane compounds have no reports on the immune regulation effects of the above-mentioned immune dysfunction and macrophages and T cells.

In this invention, we propose for the first time that compound A and related kauritanes can be used to treat inflammatory bowel disease; they can improve inflammatory bowel disease. Compound A and related kauritanes can also regulate the abnormal immune inflammatory response by inhibiting the polarization of macrophages and the differentiation of T cells caused by inflammatory bowel disease, and inhibiting the expression of various cytokines and chemical toxic substances. To achieve the effect of treating inflammatory bowel disease.

Contents of the invention

The object of the present invention is to provide the application of a kauritanes compound in the preparation of medicaments for the treatment and prevention of inflammatory bowel disease. The invention discloses a novel medicine for treating and/or preventing inflammatory bowel disease.

The invention discloses kaurane compounds, such as compound A (structural formula (I)), which are used for treating sepsis and multiple organ failure. Structural formula (I) represents a class of natural, synthetic or semi-synthetic compounds. Many of these compounds are already known to the public (Kinghorn AD, 2002, p86-137; Sinder BB et al., 1998; Chang FR et al., 1998; Hsu FL et al., 2002). Compounds of formula (I) may have one or more asymmetric centers and may exist as different stereoisomers.

in

ii. R1: hydrogen, hydroxyl or alkoxy.

iii. R2: carboxyl group, carboxylate, acid halide, aldehyde group, methylol group, and ester group, acrylamide group, acyl group or ether bond group that can generate carboxyl group.

iv. R3, R4, R5, R6, R8: Oxygen, hydroxyl, hydroxymethyl, and ester groups or alkoxymethyl groups that can be hydrolyzed to form hydroxymethyl.

v. R7: methyl group, hydroxyl group, and ester group or alkoxymethyl group that can be hydrolyzed to form hydroxymethyl group.

vi.R9: methylene or oxygen

The structure of a group of preferred compounds is shown in formula (I'). The compound has a kaurane structure, which is substituted at C13 and derivatized at C17 and C18. The compounds may possess multiple asymmetric centers and exist as different stereoisomers or diastereomers. The absolute configurations of positions 8 and 13 are (8R, 13S) or (8S, 13R).

in

vii. R2: Carboxyl, carboxylate, aldehyde, hydroxymethyl, methyl ester, acylmethyl, acid halide.

viii. R7: methyl, hydroxymethyl or methyl ether.

ix. R9: methylene or oxygen.

Compound A can be obtained after natural stevioside hydrolysis. Compound B is the aglycone of stevioside, and stevioside is the glycoside of compound B. Compounds A and B are isomers. Compound B can be obtained by hydrolysis and oxidation of stevioside, or by catalyzed reaction of intestinal bacteria in animals.

The molecular formula of compound A is C ₂₀ H ₃₀ O ₃ , and the chemical name is (4α, 8β, 13β)-13-methyl-16-oxo-17-norkauran-18-oic acid. Compound A is also known as ent-16-ketobeyran-18-oic acid. The compound is a tetracyclic diterpenoid compound containing a kaurane structure, wherein the absolute configuration of the asymmetric carbon atom is: (4R, 5S, 8R, 9R, 10s, 13s), and the carbon 13 position is substituted with a methyl group group, a carbonyl group at carbon 16, and a carboxyl group at carbon 18 (Rodrigues et al., 1988).

The molecular formula of compound B is C ₂₀ H ₃₀ O ₃ , and the chemical name is ent-13-hyrdoxykaur-16-en-18-oicacid, which is also known as steviol. This compound is also a tetracyclic diterpenoid containing a kaurane structure. Among them, the absolute configuration of the chiral carbon atom is (4R, 5S, 8R, 9R, 10S, 13S), the hydroxyl group is connected to the carbon 13, the methylene is connected to the double bond adjacent to the carbon 16, and the carboxyl group is connected to the carbon 18 (Rodrigues et al., 1993).

Compound A or B can also exist in the form of carboxylate at carbon 18, wherein the carboxylate is sodium and alkali metal or chloride and halogen. Both compounds A and B are kaurane compounds containing a kaurene structure. Compound A is a preferred compound of the invention. The present invention discloses that compound A or B has similar therapeutic effects in the treatment and prevention of cardiac hypertrophy and pulmonary hypertension. It can be concluded that all other compounds of formula (I) also have the same therapeutic effect as compound A. It is reported that Compound B is mutagenic under certain conditions in vitro. Therefore, compared with compound B, compound A is more suitable as a therapeutic drug. Compound A used in the present invention is the sodium salt of Compound A with better solubility.

The invention discloses the application of compound A structural formula (I) in the treatment and prevention of inflammatory bowel disease. After DSS induced inflammatory bowel disease in mice, the mice lost weight and had hematochezia. After administration of DSS, compound A was administered intraperitoneally, the body weight of the mice recovered, and the blood in the stool was significantly reduced. In an experiment of the present invention, DSS was added in free drinking water. For seven consecutive days, the mice lost weight significantly, had blood in the stool, diarrhea, and histopathological scores increased; /kg), the body weight of the mice recovered, and the hematochezia and diarrhea were relieved in a dose-dependent manner.

The present invention also discloses that in mice with inflammatory bowel disease induced by DSS, the results of routine blood test show that the values of white blood cells, neutrophils and monocytes in the mice are significantly higher than those in the normal group, showing that the DSS model group The mice had an inflammatory response; after administration of compound A, the white blood cells, neutrophils and monocytes of the mice were significantly lower than those of the DSS model group, and at the same time approached the normal level. The effect is better than or equivalent to the commercially available drugs 5-aminosalicylic acid (5-ASA), dexamethasone (Dex) and infest interest (IFX). It shows that compound A has obvious regulating effect on immune dysfunction caused by inflammatory bowel disease, and makes it return to normal. In another implementation disclosed by the present invention, compound B in structural formula (I) also has similar effects to compound A. The above disclosed content has not been reported in the past, nor can it be predicted and deduced by industry insiders, and should be regarded as novel and creative.

On the other hand, this study also disclosed a surprising finding: in the DSS-induced inflammatory bowel disease mice, after administration of Dex, the neutrophils increased further compared with the inflammatory bowel disease mice. It shows that immune dysfunction not only did not return to normal, but aggravated the disorder of immune function. In addition, the spleen is an important organ for regulating immune antibodies, and the various immunoglobulins it produces are crucial for the body to fight against pathogens. The spleen/body weight ratio of mice with inflammatory bowel disease increased significantly. However, with hormone therapy, this proportion decreased significantly. and lower than normal control levels. The body's ability to fight pathogens is reduced. The present invention discloses for the first time that the use of compound A to treat inflammatory bowel disease can avoid the toxic and side effects of clinical use of corticosteroids to treat inflammatory bowel disease. In another implementation disclosed by the present invention, compound B in structural formula (I) also has similar effects to compound A. The above disclosed content has not been reported in the past, nor can it be predicted and deduced by industry insiders, and should be regarded as novel and creative.

The systemic massive production of cytokines in the body (cytokine storm) is an important reason for the occurrence and development of inflammatory bowel disease. In the experiment disclosed in the present invention, after the seventh day of DSS-induced inflammatory bowel disease mice, the levels of TNF-α, IL-1β and IFN-γ in the serum of the detected mice were significantly increased compared with the normal group, and different doses of compound A were given The TNF-α, IL-1β and IFN-γ in the post-treatment serum decreased significantly. Existing literature reports that compound A can inhibit the increase of the above-mentioned inflammatory factors in ischemic injury. The present invention discloses for the first time that compound A can inhibit the increase of cytokines caused by inflammatory bowel disease. The etiology and pathology of ischemia and inflammatory bowel disease are completely different. In another experiment disclosed by the present invention, compound B in the structural formula (I) also has similar effects to compound A. The above-mentioned content disclosed by the present invention is not predictable and deducible by those in the industry, and should be regarded as having novelty and creativity.

The present invention also discloses that the spleen macrophages M1 and M2 increase significantly during the inflammatory bowel disease. The above-mentioned biomarkers of M1 and M2 macrophages and the number of macrophages were significantly decreased after compound A was administered. Compound A can regulate the inflammatory response caused by macrophages by inhibiting M1 and M2 macrophages.

The invention also discloses the influence of the inflammatory bowel disease on spleen T cells. In the DSS model group, Th17 cells increased significantly, while Treg cells decreased. After compound A was administered, the above-mentioned Th17 cells decreased and Treg cells increased. Compound A can slow down the inflammatory response by regulating T cells.

Description of drawings

Figure 1 is the effect of Compound A in Example 1 of the present invention on blood in the stool of mice with inflammatory bowel disease.

Figure 2 is the effect of intraperitoneal injection of Compound A of different doses on the body weight of DSS-induced inflammatory bowel disease mice in Example 1 of the present invention

Fig. 3 is the effect of Compound A in Example 1 of the present invention on the colon length of mice with inflammatory bowel disease.

Fig. 4 is the effect of Compound A in Example 1 of the present invention on the spleen weight of mice with inflammatory bowel disease.

Fig. 5 is HE staining of compound A in Example 1 of the present invention on mouse tissue with inflammatory bowel disease.

Fig. 6 is the effect of Compound A in Example 1 of the present invention on the histopathological score of mice with inflammatory bowel disease.

Fig. 7 is the effect of Compound A in Example 2 of the present invention on the intestinal permeability of mice with inflammatory bowel disease.

Fig. 8 is the effect of Compound A in Example 3 of the present invention on the blood routine of mice with inflammatory bowel disease.

Figure 9 is the effect of Compound A in Example 4 of the present invention on inflammatory factors in mice with inflammatory bowel disease

Figure 10 is the effect of Compound A in Example 5 of the present invention on Th7 cells in mice with inflammatory bowel disease

Figure 11 is the effect of Compound A in Example 5 of the present invention on FoxP3 cells in mice with inflammatory bowel disease

Figure 12 is the effect of Compound A in Example 5 of the present invention on macrophage M1 of mice with inflammatory bowel disease

Fig. 13 is the effect of Compound A in Example 5 of the present invention on macrophage M2 in mice with inflammatory bowel disease.

Example

Methods and embodiments of the invention are provided in detail in the following examples.

detailed description

To further illustrate the techniques used to achieve the objects of the present invention, detailed methods, techniques, procedures and features for determining and identifying the pharmaceutical and therapeutic uses of the compounds of the present invention are described below. The case provides the experimental methods and results used to support and validate the animal models used in the present invention. Appropriate control group experiments and statistical analysis methods were used for the cases involved. The following cases are used to describe rather than limit the application of the present invention. The methods and techniques involved in these cases can be used to screen and determine the therapeutic effect of such compound preparations. The same method can be used to evaluate the therapeutic effect of other such compound preparations.

The cases cited in the present invention are used to support the experimental methods and results of the present invention, and to verify the animal models used in the present invention. Appropriate controls and statistical tests were used in all experiments of the present invention. The following examples are offered to illustrate, but not to limit, the invention. These examples illustrate the methods and techniques used to screen and identify certain kaurane compounds of formula (I) with specific pharmacological activity. The therapeutic use of other compounds of formula (I) can also be determined in the same way.

Experimental Materials

Experimental animals: adult male Balb/c mice, weighing 20g±5g, aged 6-8 weeks. The rearing environment included constant temperature, humidity, and strict dark-to-light cycles, with ad libitum feeding.

Chemical reagent: Compound A (ent-17-norkaurane-16-oxo-18-oic acid, molecular formula, C ₂₀ H ₄₀ O ₃ , molecular weight: 318.5) is obtained from stevioside through acid hydrolysis and crystallization purification. The sodium salt of compound A can be obtained by adding NaOH or other sodium-containing bases; the purity of the sodium salt of compound A measured by high performance liquid chromatography is greater than 99%. The administration mode of the test compound: intravenous injection or intraperitoneal injection or oral administration. Dosage: compound A (or its sodium salt), 10 mg/kg to 15 mg/kg.

Statistical Analysis

The differences among multiple groups were compared by analysis of variance (one-way analysis of variance) followed by Fisher's test. The P values of all tests were two-tailed, and P<0.05 was considered to be statistically different.

Example 1

This case mainly observes the effect of intraperitoneal injection of different doses of compound A on dextran sodium sulfate (DSS)-induced ulcerative colitis in mice.

Clean grade Balb/c mice, 6-8 weeks old, male. Divide into eight groups at random, establish normal group (give normal saline), model group (free drinking 3.5%DSS), compound A (5mg/kg) blank group, compound A (10mg/kg)+DSS group, compound A (15mg /kg)+DSS group, positive control group 5-ASA(50mg/kg)+DSS group, positive control group Dex(10mg/kg)+DSS group, positive control group IFX(1mg/kg)+DSS group, each group 15 only. After grouping, the mice in the experimental group were intraperitoneally injected with compound A, Dex and IFX, and 5-ASA, and the mice in the control group were injected with normal saline intraperitoneally. The experimental group was administered for 2 consecutive days. From the third day, both the experimental group and the control group began to drink 3.5% DSS aqueous solution freely, and the daily drinking volume of each mouse was calculated as 7 mL, and fresh DSS solution was changed every two days. DSS was given for 7 days in total, and the mice were observed every day. Weight changes, blood in the stool and stool consistency. On the 10th day, the mice were blood-collected and killed, and the colorectal tissues of the mice were taken and measured for length. Part of the tissues were formalin-fixed and the remaining tissues were frozen at -80°C for subsequent histology. Analytical and molecular biology experiments.

It can be seen from Figure 1 that the mice in the model group of the DSS group had severe hematochezia, and after compound A was administered, the hematochezia of the mice was alleviated.

It can be seen from Fig. 2 that on day 0, the average body weights of animals in all 7 groups were basically the same. In the following 7 days, the body weight of the normal group and the 15mg/kg control group increased steadily. In the first three days, the body weight of all groups increased slowly, but from the fourth day after free drinking of DSS, the body weight of all other groups except the normal group and the 15mg/kg control group showed a trend of steep decline. Compared with other groups, the body weight of model group and Dex group lost the most. The body weight of the compound A treatment group also decreased. The two positive control drugs, 5-ASA and Infliximab groups, lost less body weight than the model group, and the Infliximab group lost more weight than the 5-ASA group. It can be seen that Compound A has a good effect on maintaining body weight.

It can be seen from Figure 3 that after administration of DSS to the animals, the colon of the experimental animals will lose weight and shorten due to the thinning of the intestinal wall. After the animal was sacrificed on the 9th day, the length of the colonic tissue was measured, and we found that after drug treatment, the length of the colonic tissue of the compound A group was significantly increased compared with the model group, while other administration groups also increased. But no significant difference.

Increased spleen weights in animals may correlate with the degree of inflammation. It can be seen from Figure 4 that after administration of DSS, the spleen weight of the mice will increase, showing splenomegaly. After being sacrificed on the 9th day, the weight of the spleen was weighed and recorded, and continued to be corrected by the weight of each animal before sacrifice. It can be found that the spleen size and weight of the mice in the model group are significantly different from those in the normal control group. Different degrees of splenomegaly were relieved in each administration group. Compared with the model group, whether the compound A, 5-ASA, Dex and infliximab groups decreased or not, there were significant differences compared with the model group.

Colonic histopathological changes are one of the strongest evidences for evaluating the efficacy of drugs. It can be seen from Figure 5 and Figure 6 that on the 9th day of DSS, the intestinal tissue structure of the mice in the model group was severely abnormal. The main manifestations were that the normal structure of the mucosa layer basically disappeared, the mucosal epithelial cells eroded and fell off, the lamina propria was exposed, and the mucosal layer was heavily inflamed. Cells, new blood vessels and fibrous tissue hyperplasia, hemorrhage, submucosa edema, and a large number of inflammatory cell infiltration can be seen. The pathological lesions in the compound A, 5-ASA and infliximab groups at each dose were partially similar to those in the model group, but to a lesser extent, while the Dex group had no significant difference compared with the model group.

Example 2

This case mainly observes the permeability of the colon of experimental mice in each group.

1) Principle: The permeability of animal intestine can be semi-quantitatively detected by using fluorescent tracer to detect the fluorescence intensity in serum.

2) Preparation of standard curve:

Take several normal mice, collect hemolysis-free serum, weigh 200 μg of FITC-dextran powder, dissolve it in 5ml serum, and dilute it in multiples, and then use a microplate reader to detect the fluorescence intensity to obtain the standard curve.

3) On the day of sacrificing the animals, fast 4 hours in advance.

4) Oral administration of the prepared FITC-dextran tracer at a dose of 60 mg/kg body weight.

5) Blood was collected from animals before sacrifice, and serum without hemolysis was collected.

6) Serum was added to a 96-well plate, and 100 μl per well was used to detect the fluorescence intensity with a microplate reader (excitation light 488 nm, emission light 520 nm).

7) The content of FITC-dextran in animal serum can be calculated by the standard curve formula.

FITC-dextran (fluorescein-labeled dextran) is a fluorescent dye. After exogenous administration of FITC-dextran to mice, the level of intestinal permeability can be reflected by detecting the fluorescence intensity of FITC-dextran in serum. A novel index for evaluating intestinal inflammation. Therefore, on the day of sacrificing the animals, the mice were fasted for more than 6 hours, and then given FITC-dextran by intragastric administration, and after 6 hours, the fluorescence content of FITC-dextran in the serum was detected. As shown in Figure 7, the FITC content in the serum of the mice in the normal control group was very low, indicating that the intestinal permeability was normal. In the model group, the FITC content increased significantly, indicating that the intestinal permeability increased and the intestinal wall was damaged. However, the content of FITCF in the compound A, 5-ASA, Dex and Infliximab groups was significantly reduced, indicating that compound A has a certain effect on protecting the intestinal tract.

Example 3

This case mainly illustrates the effect of compound A on the blood routine of inflammatory bowel disease.

After the 9th day, blood was collected from the mice, and gently dropped into the prepared EP tubes prepared with EDTA salt anticoagulant, and the 80-100 μL blood samples were mixed, and the blood routine was tested using an automatic blood analyzer, including red blood cell count ( RBC), monocytes (MONO), neutrophils (LYMPH), platelets (PLT), hemoglobin (HGB), hematocrit (HCT) and white blood cell count (WBC), etc.

As shown in Figure 8, compared with the normal group, WBC, NEUT, LYMPH and MONO in the model group were significantly increased. After the intervention of compound A, 5-ASA and Infliximab, WBC, NEUT, LYMPH and MONO were all decreased, but Dex There was no significant difference between the model group and the model group.

Example 4

This case mainly illustrates the effect of Compound A on inflammatory factors.

Serum TNF-α, IFN-γ and IL-1β levels were determined according to the instructions provided by the ELISA kit.

As shown in Figure 9, the contents of serum TNF-α, IFN-γ and IL-1β in the serum of mice in the normal control group were low, while the levels of serum TNF-α, IFN-γ and IL-1β in the serum of mice in the model group were content was significantly increased, with significant differences. The levels of TNF-α, IFN-γ and IL-1β in the serum of mice administered with compound A, 5-ASA group, Dex group and infliximab group all decreased, and compound A had significant differences compared with the model group , the content of IFN-γ was significantly different between 5-ASA and model group; the content of TNF-α was significantly different between Infliximab and model group. The results showed that compound A improved the high level expression of serum anti-inflammatory factors caused by inflammatory response.

Example 5

This example illustrates the effect of Compound A on the polarization of T cells and macrophages.

1) Grouping of experimental animals: 70 6-8 week-old Balb/c male mice were adaptively fed for one week, and randomly divided into 8 groups with 10 mice in each group, which were normal group, compound A normal control group, DSS model group, Compound A group, 5-ASA group, Dex group and infliximab group. Drink DSS freely for seven consecutive days.

2) Extraction of spleen-peritoneal macrophages and T cells: mice were killed by necking on the eighth day, and spleen T cells and macrophages were extracted.

3) Detection of macrophages by flow cytometry: block the macrophages with MACS for 20 minutes on ice, centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, and add 0.2 μl of PE-anti-mouse F4/ 80 antibody and BV421-anti-CD11c antibody, incubated on ice in the dark for 30min, washed once with PBS, fixed on ice with 50μl of fixative for 10min, 50μl of 1x permeabilization solution was used to permeate the membrane, centrifuged at 1000rpm, 4°C for 5min, and then added 100μl of 1x Permeabilization solution to rupture the membrane, centrifuge, add 0.2 μl of FITC-anti-mouse CD206 antibody, incubate on ice in the dark for 30 minutes, add 100 μl of permeation solution and centrifuge, finally add 1ml PBS to wash once, centrifuge, collect the precipitate, and re-incubate in 200 μl PBS suspension, FACSCelesta flow cytometry detection, FlowJo 7.6.1 software analysis M1 type (F4/80 ⁺ CD11c ⁺ CD206 ^- ) and M2 type (F4/80 ⁺ CD11c ^- CD206 ⁺ ).

4) Flow cytometric detection of T cells: After blocking the macrophages with MACS for 20 minutes on ice, centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, add 0.2 μl of CD4, CD3 and CD25 antibodies to the cell suspension, and place on ice After incubating in the dark for 30 minutes, wash once with PBS, fix with 50 μl of fixative solution on ice for 10 minutes, rupture the membrane with 50 μl of 1x permeabilization solution, centrifuge at 1000 rpm, 4°C for 5 minutes, then add 100 μl of 1x permeation solution to rupture the membrane, centrifuge, and add 0.2 μl The IL-17 and FoxP3 antibodies were incubated on ice in the dark for 30 minutes, added 100 μl permeabilization solution and centrifuged, and finally washed once with 1ml PBS, centrifuged, collected the precipitate, resuspended in 200 μl PBS, and detected by FACSCelesta flow cytometry, FlowJo 7.6. 1 software to analyze Th17 and Treg cells.

As shown in Figure 10 and Figure 11, compared with the DSS model group, Th17 cells were significantly reduced after compound A, 5-ASA group, Dex group and infliximab intervention, and Treg cells were significantly increased, indicating that compound A plays an important role in the regulation of T cells important.

As shown in Figure 12 and Figure 13, compared with the DSS model group, after the intervention of compound A, 5-ASA group, Dex group and infliximab, both M1 and M2 peritoneal macrophages were significantly reduced, indicating that the effect of compound A on M1 and M2 macrophages was significantly reduced. The imbalance of phagocytes plays a regulatory role to maintain the balance of macrophages in the body.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can also be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (26)

1. A pharmaceutical preparation for the prevention and treatment of inflammatory bowel disease and extraintestinal manifestations using a kaurane compound and a pharmaceutically acceptable salt thereof or in combination with an immunosuppressive drug, an antibody, an antibiotic or an S1P receptor modulator in the application.
2. The inflammatory bowel disease according to the method of claim 1 includes ulcerative colitis and Crohn's disease.
3. The perianal diseases included in the Crohn's disease of claim 2 include: perianal erythema, abscess, ulcer and perianal fissure or fistula.
4. The perianal symptoms according to claim 3 include inflammation symptoms of hemorrhoids.
5. The Crohn's disease as claimed in claim 2 includes fibrosis and intestinal lumen stricture.
6. According to claim 1, the parenteral manifestations are oral Crohn's disease, amyloidosis, episcleritis, scleromalacia, corneal ulcer, primary sclerosing cholangitis, lupus and pulmonary inflammatory disease.
7. The parenteral manifestation according to claim 1 is arthritis, osteoarthritis and ankylosing spondylitis.
8. According to claim 1, the parenteral manifestations are inflammatory skin diseases, including atypical dermatitis, psoriasis, rosacea, prickly heat, acne, pyoderma gangrene, sweet syndrome, intestinal-related dermatosis-arthritis syndrome ( BADAS), proliferative purulent dermatitis-purulent stomatitis (PPV), hypersensitivity vasculitis.
9. According to claim 1, the parenteral manifestations are autoimmune skin diseases, including urticaria, vitiligo and alopecia areata.
10. Prevention and treatment according to claim 1, characterized in that the mechanism of action of the prevention and treatment involves inhibition of cytokine production or inhibition of cytokine storm. Cytokines include TNF-α, IL-1β and IFN-γ, etc.
11. According to the described anti-inflammatory drug of claim 1, it is characterized in that comprising sulfasalazine, mesalazine, 5-aminosalicylate of balsalazide olsalazine and comprising prednisone, budesonide, fluticasone, fluni Corticosteroids such as acetaminophen, ciclesonide, mometasone, beclomethasone, and dexamethasone.
12. According to the described anti-inflammatory medicine of claim 1, it is characterized in that comprising the 5-amino salicylate of sulfasalazine, mesalazine, balsalazide and olsalazine.
13. The antibiotic according to claim 1 is aminoglycoside antibiotics, ansamycin, carbacephem, carbapenem, cephalosporin, glycopeptide, lincosamide, lipopeptide, macrolide, monobactamycin , nitrofurans, oxazolones, penicillins, peptides, quinolones, sulfonamides, tetracyclines, chloramphenicol, phosphonic acid antibiotics, and a mycobacterial antibiotic.
14. The antibody according to claim 1, which is infliximab, adalimumab, golimumab, vedolizumab, certolizumab, natalizumab, ustekinumab and Bm-ca.
15. Prevention and treatment according to claim 1, characterized in that its mechanism of action involves inhibition and regulation of macrophage activation and proliferation.
16. Prevention and treatment according to claim 1, characterized in that its mechanism of action involves inhibition of activation and proliferation of inflammatory cells including leukocytes, neutrophils, monocytes and lymphocytes.
17. The treatment of claim 1 includes inhibiting the activation of intestinal innate lymphocytes and CD4, CD8 T lymphocytes.
18. The treatment according to claim 1 includes metabolic reprogramming of macrophages, innate lymphocytes and CD4, CD8 T lymphocytes.
19. The compound according to the method of claim 1 is a compound represented by structural formula (I).

    Compounds of formula (I) may have one or more asymmetric centers and may exist as different stereoisomers.

    

    in

    ii. R1: hydrogen, hydroxyl or alkoxy.

    iii. R2: carboxyl group, carboxylate, acid halide, aldehyde group, methylol group, and ester group, acrylamide group, acyl group or ether bond group that can generate carboxyl group.

    iv. R3, R4, R5, R6, R8: Oxygen, hydroxyl, hydroxymethyl, and ester groups or alkoxymethyl groups that can be hydrolyzed to form hydroxymethyl.

    v. R7: methyl group, hydroxyl group, and ester group or alkoxymethyl group that can be hydrolyzed to form hydroxymethyl group.

    vi. R9: methylene or oxygen.
20. According to the compound described in the method in claim 12, it is characterized in that wherein said compound of structural formula (I) is the compound shown in structural formula (II). The compounds may possess multiple asymmetric centers and exist as different stereoisomers or diastereomers. The absolute configurations of positions 8 and 13 are (8R, 13S) or (8S, 13R).

    

    in

    vii. R2: Carboxyl, carboxylate, aldehyde, hydroxymethyl, methyl ester, acylmethyl, acid halide.

    viii. R7: methyl, hydroxymethyl or methyl ether.

    ix. R9: methylene or oxygen.
21. The compound according to claim 12, characterized in that the compound of the structural formula (I) is the compound shown in the structural formula A.

    
22. According to the compound described in claim 12, it is characterized in that wherein said compound of structural formula (I) is the compound shown in structural formula B.

    
23. The pharmaceutical preparation according to claim 1 includes: tablets, capsules, granules, suppositories, ointments, patches, aqueous injections, and sustained and controlled release preparations through oral, parenteral or implantation.
24. According to the pharmaceutical preparation described in claim 1, it is characterized in that it is aerosol inhalation, metered-dose aerosol or dry powder inhalation through lung or nose.
25. According to the method described in claim 1, the pharmaceutical preparation is characterized in that the pharmaceutical standard liquid injection or infusion or other suitable dosage forms are used to deliver to the patients in need through methods such as muscle, vein, abdominal cavity, interventional catheter and ventilator.
26. The pharmaceutical combination according to claim 1, characterized in that it is administered orally, inhaled, nasal spray, injection, external, eye drops, rectal or vaginal, and in solid polar, solution dosage form, spray aerosol dosage form, injection Type, ointment dosage form, skin patch dosage form, thin film dosage form, soft capsule dosage form, suppository dosage form give the patients who need it.

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