Application of epoxidase type II inhibitor

Abstract

The invention discloses an application of an epoxidase type II inhibitor, the application is the application in preparation of drugs for treating liver injury, hepatitis, hepatic fibrosis, or liver function decline or complications thereof, and the application discovers that the existing COX-2 selective inhibitors such as celecoxib and the like and a novel efficient COX-2 selective inhibitor 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, 17, the 2, 8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid disclosed by the invention has the effects of remarkably relieving liver injury, reducing necrosis focus and inflammatory cell infiltration of liver cells and resisting cholestatic hepatic fibrosis; in addition, hepatocyte lipid accumulation and ballooning induced by high fat diet can be reduced, and the progress of lipid hepatitis is remarkably inhibited; it is found for the first time that the traditional Chinese medicine composition can significantly reduce the concentration of serum bilirubin and play a role in relieving hyperbilirubinemia caused by liver injury or liver insufficiency; the traditional Chinese medicine composition can be used for treating neonatal jaundice, congenital bile duct atresia, cholestatic cirrhosis, acute toxic hepatic failure, bilirubin increase caused by acute attack of chronic hepatitis, chronic hepatic failure or hepatic encephalopathy and the like; therefore, the method has obvious economic benefit and clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, the present invention relates to a COX-2 enzyme (cyclooxygenase type II) inhibitor, especially the use of deuterated benzopyran compounds in the preparation of drugs for treating liver damage, hepatitis, liver fibrosis, or liver function decline or its complications. Background Art

[0002] The chemical toxic pressure brought by environmental pollution, food mold and the widespread use of food additives, combined with factors such as alcoholic and non-alcoholic excessive diet, induces chronic liver damage such as liver inflammation, fatty degeneration and fibrosis. Among them, about eight million people develop major diseases such as cirrhosis, liver cancer and even liver failure each year. This number is still increasing year by year, causing huge social and medical pressure and family burden.

[0003] Imbalance in liver tissue damage and repair leads to liver fibrosis, which is the common pathological basis for different types of liver damage to develop into chronic liver disease. The activation of hepatic stellate cells (HSCs) is a key event in the development of liver fibrosis. The apoptosis or necrosis of liver parenchymal cells (including hepatocytes and bile duct cells) caused by damage produces a large number of active peroxide compounds and causes the release of inflammatory mediators, forming liver cell necrosis foci. Subsequently, immune cells are recruited to the site of injury to phagocytose and clean up dead cells, while releasing more proinflammatory cytokines to recruit more inflammatory cells, prompting further cascade amplification of the inflammatory response. On the other hand, proinflammatory factors can directly activate HSCs and initiate liver damage repair or fibrosis. In this process, three types of cells are mainly involved: dead hepatocytes, immune cells, and HSCs. Among them, the factors or chemical small molecules released by dead hepatocytes and immune cells form a positive feedback regulatory network. Recruiting activated HSCs in the liver tissue injury foci and promoting their proliferation are key events in liver tissue repair and / or fibrosis.

[0004] At present, the most prioritized and effective treatment strategy for liver fibrosis is the treatment of the primary disease, that is, the application of basic means in the early stage of the disease to delay or block the process of fibrosis, such as antiviral treatment, copper removal treatment, exercise therapy, and changes in diet and lifestyle habits. Recently, many anti-liver fibrosis drugs have entered the verification stage of clinical trials. As of May 2023, there are 395 clinical studies on liver fibrosis on ClinicalTrials.gov, including 216 interventional studies and 121 drug studies. The main links in the pathogenesis of liver fibrosis targeted include anti-inflammatory and antioxidant, specific cytokines and their intracellular signal transduction, inhibition of fibrosis formation and promotion of fibrosis reduction.

[0005] Cholestatic liver fibrosis mainly harms newborns with biliary atresia and the growing number of patients with primary biliary cholangitis. The former is often treated with laparoscopic surgery or anti-infective drugs such as ganciclovir; the latter still has chronic progression in about 40% of patients after standardized treatment. First- and second-line drugs for the treatment of cholestasis include ursodeoxycholic acid (inhibits cholesterol reabsorption), obeticholic acid (inhibits bile acid synthesis), S-adenosylmethionine (SAMe, regulates liver cell membrane fluidity and protects cells) and tripibuton tablets (promotes bile excretion).

[0006] Nonsteroidal anti-inflammatory drugs (NASIDs), especially COX-2 (cyclooxygenase type II) selective inhibitors, including celecoxib, meloxicam, NS-398, indomethcin and dideoxyfluridine (DFU), can partially relieve biliary liver fibrosis induced by bile duct obstruction. Among them, those with good effects can relieve the increase of ALT, AST and IL-6 caused by cholestatic liver injury, about 20% to 40%, but whether they can reduce the expression of COX-2 and the accumulation of bilirubin varies. A few opposing opinions believe that COX-2 inhibitors have no effect on liver fibrosis.

[0007] Regarding the mechanism of COX-2 involved in regulating liver fibrosis, one study found that conjugated bile acid up-regulated COX-2 through EGFR and promoted HSCs proliferation; another study found that Celecoxib, NS-398 and DFU (50-100μM) could inhibit HSCs proliferation, and all three could reduce the production of prostaglandin E2 and prostaglandin I2 to block their promotion of HSCs proliferation and ERK activation. Celecoxib relied on inhibiting Akt activity to induce HSCs apoptosis; Meloxicam could inhibit the expression of TGFβ1 and MMP. However, a different opinion proposed that PGE2 induced HSCs apoptosis, and 5μM PGE2 treatment of HSCs reduced α-SMA and COL1 (type I collagenase), and it was speculated that PGE2 derived from hepatocytes played an anti-liver fibrosis role.

[0008] The novel highly effective COX-2 selective inhibitors designed by the applicant's drug research and development center, deuterated benzopyran compounds, have the structural characteristics of formula (I): It has been confirmed in animal arthritis, pain, swelling and tumors, and has been authorized by the following patent: Deuterated benzopyran compounds and their applications (CN102757417A).

[0009] It would be of great significance if this type of new COX-2 enzyme inhibitor deuterated benzopyran compound could be developed as a drug for preventing liver damage, hepatitis, liver fibrosis, or liver function decline or its complications. Summary of the invention

[0010] Based on this, the object of the present invention is to provide a use of a cyclooxygenase type II inhibitor in the preparation of a medicament for treating liver injury, hepatitis, liver fibrosis, or decreased liver function or its complications.

[0011] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0012] The first aspect of the present invention provides the use of a cyclooxygenase type II inhibitor in the preparation of a medicament for treating liver damage, hepatitis, liver fibrosis, or decreased liver function or its complications.

[0013] The second aspect of the present invention provides a drug for treating liver damage, hepatitis, liver fibrosis, or decreased liver function or its complications, wherein the drug comprises a cyclooxygenase type II inhibitor.

[0014] The present invention finds that existing COX-2 selective inhibitors such as celecoxib and the new high-efficiency COX-2 selective inhibitor 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid (GIBH-1014) can effectively inhibit hepatocyte necrosis and inflammatory cell infiltration in the liver tissue of bile duct ligation model mice, reduce the staining signal of deposited collagen fibers in the tissue, and reduce the number of activated HSCs cells, and have a significant anti-cholestatic liver fibrosis effect, which accumulates basic data for the time window and drugs for future clinical intervention; at the same time, GIBH -1014 can also reduce ballooning caused by lipid accumulation, reduce the area percentage of ballooning lipid-degenerated cells, and inhibit the progression of steatohepatitis; and for the first time, it was found that GIBH-1014 can significantly reduce serum bilirubin concentration and can be used to treat neonatal jaundice, congenital biliary atresia, cholestatic cirrhosis, acute toxic liver failure, increased bilirubin caused by acute attack of chronic hepatitis, chronic liver failure or hepatic encephalopathy; therefore, COX-2 selective inhibitors, especially deuterated benzopyran compounds, have obvious economic benefits and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The changes in the RNA expression level and protein of COX-2 in the bile duct ligation model mice 6 hours to 18 days after surgery in Example 1 of the present invention.

[0016] Figure 2 The figure shows the changes of PGs in serum and liver of bile duct ligation model mice in Example 1 of the present invention.

[0017] Figure 3 This is the change in relative mRNA expression of PGE2 receptor Ep1-4 in Example 1 of the present invention.

[0018] Figure 4 This is a graph showing the expression of COX-2 protein in the liver of mice after two weeks of treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0019] Figure 5 This is a graph showing changes in PGE2 in the liver and serum after treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0020] Figure 6 The changes in the degree of liver fibrosis after treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0021] Figure 7 The fibrosis grade, inflammation grade and ImageJ statistical results of liver fibrosis after treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0022] Figure 8 These are the results of changes in liver physiological function of BDL model mice after treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0023] Fig. 9 The jaundice symptoms, liver color, and white fibrosis spots of mice treated with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention are shown.

[0024] Fig.10 The figure shows the changes in the number of lymphocytes, M1 and M2 macrophages in the liver of mice after treatment with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 1 of the present invention.

[0025] Fig.11 These are the results of the treatment of liver fibrosis induced by steatosis with COX-2 inhibitors (GIBH-1014 and Celecoxib) in Example 2 of the present invention.

[0026] Fig.12 This is the effect of PGE2 on the activation and proliferation of HSCs in Example 3 of the present invention.

[0027] Fig.13 Schematic diagram of the scratch experiment on primary HSCs in Example 3 of the present invention.

[0028] Fig.14 This is the effect of PGE2 on HSCs migration in Example 3 of the present invention.

[0029] Fig.15 This is the effect of PGE2 on the expression of Cd163 and Arg-1 in M2 macrophages in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0032] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0033] The inventors of the present invention found that bile duct ligation model mice induce COX-2 elevation due to cholestasis, and PGE2 in the liver and serum increases, and excessive production of PGE2 leads to the occurrence and development of liver fibrosis, and interferes with the clearance of hemoglobin (upstream of bilirubin) by M2 macrophages. After treatment with the existing COX-2 selective inhibitor Celecoxib and the new high-efficiency COX-2 selective inhibitor deuterated benzopyran compounds designed by the applicant's drug research and development center, its anti-liver fibrosis effect is significantly better than the existing NSAIDs, and it can effectively inhibit the progression of fatty hepatitis, accumulating basic data for the time window and drugs for future clinical intervention, and for the first time found that it can significantly reduce serum bilirubin concentration.

[0034] In the following examples, the deuterated benzopyran compound used is specifically 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid (hereinafter labeled as GIBH-1014), which is prepared according to patent number CN102757417A. GIBH-1014 is different from Celecoxib (Celebrex, Pfizer) and Vioxx (Vioxx, Merck), two COX-2 inhibitors. The latter two interfere with the efficiency of COX-2 catalytic synthesis of PGs by binding to the side pocket of the COX-2 active domain, while GIBH-1014 is directly inserted into the key active domain of COX-2. This combination significantly improves the inhibition efficiency of COX-2 and the pharmacokinetic properties in vivo. In terms of efficacy, GIBH-1014 has a strong inhibitory efficiency on the enzymatic activity of hCOX-2 (IC50=39nM), and this concentration has almost no interference with the enzymatic activity of hCOX-1 (IC50>100μM). Rats were rapidly absorbed after oral gavage of GIBH-1014, with a peak time Tmax of only 15min, a bioavailability of nearly 100%, and a long retention time in the body, with a T1 / 2 of up to 5hr. In the future, long-term clinical use with low doses can be achieved, reducing side effects and ensuring safety. In animal models of inflammation, swelling and pain, the excellent IC50 and EC50 values ​​and effectiveness of GIBH-1014 have been confirmed.

[0035] The main scientific research achievements of the present invention include the following:

[0036] 1. Reduced the level of PGE2 in mouse liver tissue and serum;

[0037] 2. It increased the number of M2 macrophages in the liver that were damaged and attacked. It is speculated that GIBH-1014 may alleviate liver fibrosis by increasing the number and proportion of anti-inflammatory and repairing M2 macrophages in the liver;

[0038] 3. Inhibits the progression of liver fibrosis, specifically:

[0039] (a) The hepatocyte necrosis foci and inflammatory cell infiltration in liver tissue were significantly reduced, the staining signal of deposited collagen fibers in the tissue was significantly reduced, the number of activated HSCs cells was significantly reduced, and the fibrosis grade and inflammation grade were reduced;

[0040] (b) It reduces the increase in bilirubin caused by bile duct damage (it can be used to treat neonatal jaundice, congenital biliary atresia, cholestatic cirrhosis, acute toxic liver failure, increased bilirubin caused by acute attack of chronic hepatitis, chronic liver failure or hepatic encephalopathy); ALT, AST, TBIL, DBIL and IBIL all return to normal levels, maintaining normal liver physiological function;

[0041] (c) The jaundice symptoms (ears, palms, soles, and tail) caused by cholestasis are relieved, and the liver is normal in color, without obvious enlargement and white fibrosis spots;

[0042] (d) Significantly improved the survival rate of mice.

[0043] 4. Inhibits the progression of fatty liver hepatitis, which is specifically reflected in:

[0044] (a) Inhibited liver damage and inflammatory cell infiltration, and reduced fibrosis and inflammation grades;

[0045] (b) Ballooning caused by lipid accumulation was reduced, and the area percentage of ballooning lipid degeneration cells was reduced;

[0046] (c) ALT, AST, TC and HDL all returned to normal levels, and LDL was significantly reduced, effectively reducing liver damage and cholesterol accumulation, maintaining normal liver physiological function, and avoiding abnormal liver function.

[0047] In the present invention, the deuterated benzopyran compounds, the term "deuterium" means a single deuterium atom, the deuterium free radical is attached to a carbon atom or an oxygen atom to form a deuterated compound, the deuterated benzopyran compounds themselves have a carboxylic acid group but do not interact with the hydrophobic assembly band of the COX-2 active site. The deuterated benzopyran compounds are directly inserted into the key active domain of COX-2, and this combination significantly improves the inhibition efficiency of COX-2 and the pharmacokinetic properties in vivo. Therefore, all deuterated benzopyran compounds have similar effects to GIBH-1014 and are within the protection scope of the present invention.

[0048] In some of the embodiments of the present invention, the use of cyclooxygenase type II inhibitors in the preparation of drugs for treating liver damage, hepatitis, liver fibrosis, or decreased liver function or its complications is disclosed.

[0049] In some embodiments, the cyclooxygenase type II inhibitors include but are not limited to deuterated benzopyran compounds, and marketed drugs such as celecoxib, meloxicam, indomethacin and dideoxyfluridine.

[0050] In some embodiments, the deuterated benzopyran compound is 6-bromo-5,7-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 8-chloro-6-deuterated methyl-2-(trifluoromethyl)-2H-2H-benzopyran-3-carboxylic acid or 6-bromo-8-deuterated methyl-2-trifluoromethyl-2H-benzopyran-3-carboxylic acid; the deuterated benzopyran compound is preferably 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid.

[0051] In some embodiments, the liver damage is caused by physical, chemical or biological factors; the hepatitis is an inflammatory response of liver tissue caused by physical, chemical or biological factors.

[0052] In some embodiments, the hepatitis includes but is not limited to steatohepatitis and cholestatic hepatitis.

[0053] In some embodiments, the liver fibrosis includes but is not limited to cholestatic liver fibrosis and liver fibrosis caused by severe fatty liver.

[0054] In some embodiments, the decreased liver function is an increase in AST, ALT, ALP, GGT, TBA, TBIL, DBIL or IBIL; the complication of decreased liver function is hyperbilirubinemia or hepatic encephalopathy caused by hyperbilirubinemia.

[0055] In some embodiments, the hyperbilirubinemia includes but is not limited to neonatal jaundice, congenital biliary atresia, cholestatic cirrhosis, acute toxic liver failure, increased bilirubin caused by acute attack of chronic hepatitis, chronic liver failure and hepatic encephalopathy.

[0056] In other embodiments of the present invention, a drug for treating liver damage, hepatitis, liver fibrosis, or decreased liver function or its complications is disclosed, wherein the drug comprises a cyclooxygenase type II inhibitor.

[0057] In some embodiments, the cyclooxygenase inhibitors include but are not limited to deuterated benzopyran compounds or pharmaceutically acceptable salts or stereoisomers thereof, and marketed drugs such as celecoxib, meloxicam, indomethacin, dideoxyfluridine, etc.

[0058] In some embodiments, the deuterated benzopyran compound is 6-bromo-5,7-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 8-chloro-6-deuterated methyl-2-(trifluoromethyl)-2H-2H-benzopyran-3-carboxylic acid or 6-bromo-8-deuterated methyl-2-trifluoromethyl-2H-benzopyran-3-carboxylic acid, more preferably 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid.

[0059] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0060] In some embodiments, the drug is in the form of tablets, pills, capsules or injections.

[0061] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0062] Example 1 COX-2 inhibitors alleviate liver fibrosis in the BDL model

[0063] 1. Modeling of bile duct ligation

[0064] After the common bile duct ligation surgery in mice, jaundice symptoms can be observed within 7 days, with occasional ascites and death. The model can usually be successfully established within two weeks. Dissection of mice with cholestatic liver injury model showed abnormal enlargement of the common bile duct and gallbladder, and a large amount of bile accumulated, resulting in a yellow-green or even dark green color. The knot tied during the surgery did not loosen, and the liver was yellowish overall, possibly with small white spots of necrotic fibrosis. Bile overflow caused severe adhesion of the tissues around the liver, a significant increase in serum bilirubin, and deterioration of other liver biochemical indicators.

[0065] This liver fibrosis model has the advantages of short modeling time, clear damage source and process, stable fibrosis area, and obvious pseudolobule characteristics. The morphological observation and dynamic changes of COX-2 in the whole course of the bile duct ligation model mice were analyzed to determine the intervention time window and length of COX-2 inhibitors (GIBH-1014 and Celecoxib).

[0066] 2. Experimental Grouping

[0067] According to the experimental needs, the mice were divided into 4 groups, namely, the empty operation group (open abdomen to expose the liver hilum, without ligation and subsequent suture), the bile duct ligation model group, the GIBH-1014 administration group and the Celecoxib administration group.

[0068] The empty operation group and the bile duct ligation model group were gavaged with the drug solvent methylcellulose (cmc) as a control, while the latter two were administered simultaneously in the bile duct ligation model. According to the preliminary experimental results, 10 mg / kg of GIBH-1014 or 15 mg / kg of Celecoxib were given by gavage every day from the fourth day after the ligation surgery. Mouse samples were collected after two weeks to identify the improvement effect of liver fibrosis.

[0069] 3. Experimental Results

[0070] 1. Cholestasis induces COX-2 elevation

[0071] At 6 hours, 24 hours, 48 ​​hours, 3 days, 5 days, 7 days, 10 days, 14 days, 16 days and 18 days after the common bile duct ligation surgery, the mice were treated and samples were collected (n≥3), and their livers were homogenized, and RNA and protein were extracted to analyze the expression of COX-2. Figure 1 shown.

[0072] from Figure 1It can be seen that the RNA expression level of COX-2 in bile duct ligation model (BDL) mice increased hundreds of times at least 6 hours after surgery and reached a peak on the 7th to 10th day, then gradually fell back to a lower level but was still higher than that of normal control mice ( Figure 1 The corresponding COX-2 protein expression level responded slowly and maintained for a long time, with the highest peak occurring on the 5th day. The BDL model group increased 2 to 3 times compared with the normal control mice ( Figure 1 B and C in ).

[0073] 2. Cholestatic injury leads to increased PGE2 in the liver and serum

[0074] The BDL model mouse samples that have been modeled were collected and analyzed. The results of liquid chromatography-mass spectrometry are as follows: Figure 2 shown.

[0075] Figure 2 The results showed that compared with the normal control group, the levels of PGD2 and PGE2 in serum of the model group increased by 8.5 times and 5.5 times, reaching 544.8 pg / mL and 25.55 pg / mL, respectively, but in the liver they only increased by about 1.3 to 1.5 times ( Figure 2 A and B in Figure 1); PGJ2 decreased by about 1.8 times in serum and about 2.3 times in liver ( Figure 2 C in the figure); the differences in the changes of the remaining PGs were insufficient ( Figure 2 DF in the figure). Based on the previous experience of BDL modeling and the expression changes of COX-2, we selected the model mouse samples on the fifth day, which is the key transformation stage of bile duct injury, to identify their PGs composition spectrum. The results showed that in the early stage of BDL-induced injury, PGE2 in liver tissue increased by about two times, while the level of PGE2 in serum did not change much ( Figure 2 G); PGD2 did not change significantly.

[0076] Binding to PGE2 receptors (Ep1-4) not only increases expression in the liver of cholestatic injury ( Figure 3 A), and gradually increased during the natural activation process of primary HSCs cultured in vitro ( Figure 3 B), suggesting that PGE2 is a key focus in the process of liver fibrosis, and it may regulate the proliferation, activation, migration and extracellular matrix production of HSCs through receptor binding.

[0077] 3. COX-2 inhibitors reduce PGE2 levels in BDL liver tissue and serum

[0078] Western blot was used to detect liver proteins in mice with cholestatic liver fibrosis treated with COX-2 inhibitors for two weeks. Figure 4 As shown, from Figure 4 The results showed that the use of COX-2 inhibitors could not reduce the increase in COX-2 caused by damaging stimulation.

[0079] Identification of the PGs composition spectrum after COX-2 inhibitor treatment Figure 5 As shown, from Figure 5 The results showed that the treatment group could reduce the accumulation of PGE2 in the liver to a near normal level in the early stage of damage. Figure 5 A in the figure can also relieve the increase of systemic PGE2 (in serum) in the later stage of disease progression. Figure 5 B).

[0080] 4. COX-2 inhibitors alleviate liver fibrosis induced by bile duct ligation in mice

[0081] Starting from the fourth day after the BDL operation, without removing the bile duct ligation of the mice, the mice in the experimental group were given 10 mg / kg of GIBH-1014 or 15 mg / kg of Celecoxib (the dosage was based on the bioavailability of the drug measured previously) by oral gavage every day to observe the changes in the degree of liver fibrosis after COX-2 inhibition (see the process for details). Figure 6 A in the figure). After two weeks of oral administration of two COX-2 inhibitors, the survival rate of mice in the drug-treated experimental groups was significantly increased (GIBH-1014 group, 91.7%; Celecoxib group, 75%; control group, 69.2%) ( Figure 6 B). Compared with the liver tissue of the control mice in the drug-treated group, the hepatocyte necrosis and inflammatory cell infiltration in the liver tissue of the drug-treated group were significantly reduced (HE staining), the staining signal of deposited collagen in the tissue was significantly reduced (Masson's staining and scarlet staining), and the number of activated HSCs cells was significantly reduced (α-SMA immunostaining) ( Figure 6 C).

[0082] The above results were graded according to the International Association for Hepatology and the modified Ishak score. The results showed that the fibrosis grade and inflammation grade of the COX-2 inhibitor treatment group were lower than those of the control group. ImageJ statistical results also showed that the COX-2 inhibitor treatment group had a smaller area of ​​necrosis, especially the GIBH-1014 treatment group ( Figure 7 ).

[0083] At the same time, COX-2 inhibitor treatment significantly improved the liver physiological function of BDL model mice, and the ALT, AST, TBIL, DBIL and IBIL of the treatment group returned to normal levels ( Figure 8 ), indicating that COX-2 inhibitors can effectively reduce the increase in bilirubin caused by liver damage and bile duct damage and maintain normal liver physiological function.

[0084] On the other hand, COX-2 inhibitors relieved the jaundice symptoms caused by cholestasis (ears, palms, soles, tail), and the liver was normal in color, without obvious enlargement and white fibrosis spots ( Fig. 9 ).

[0085] In summary, the therapeutic use of COX-2 inhibitors not only inhibits liver damage and inflammatory cell infiltration in the BDL model, but also reduces the number of activated HSCs cells and the deposition of extracellular collagen fibers, thereby directly inhibiting the progression of liver fibrosis.

[0086] 5. GIBH-1014 increases the number of M2 macrophages in the liver

[0087] All non-parenchymal cells in the liver of normal mice, BDL one-week model mice, and mice treated with GIBH-1014 were isolated and extracted, and flow cytometry was used after immunostaining. Immunofluorescence staining of non-parenchymal cells was performed using flow cytometry antibodies CD45, CD11b, F4 / 80, CD86, and CD206.

[0088] The results are as follows Fig.10 The results showed that compared with normal mice, CD45 + The number of cells increased by 1.67 times ( Fig.10 A and B in the figure), CD86 + CD11b + The number of cells doubled ( Fig.10 C and D), but CD206 + The number of cells decreased to 39% of the original Fig.10 E and F); One week after GIBH-1014 was given to treat BDL, CD45 + Cells further increase, CD86 + CD11b + The number of cells also increased, which was 1.23 and 1.48 times of the BDL group, respectively, while CD206 + The number of cells recovered to nearly 71% of that in normal mice.

[0089] CD86 and CD206 are considered to be specific markers of M1 and M2 mononuclear macrophages, respectively. That is, when the liver is damaged by progressive cholestasis, the number of lymphocytes and M1 macrophages increases, while the number of M2 macrophages decreases. Compared with the BDL injury group, the number of lymphocytes, M1 and M2 macrophages in the liver increased after one week of GIBH-1014 treatment. The population of M2 macrophages that were damaged and attacked recovered after GIBH-1014 treatment. It can be speculated that GIBH-1014 alleviates liver fibrosis by increasing the proportion of anti-inflammatory and repairing M2 macrophages in the liver.

[0090] Example 2 COX-2 inhibitor significantly improves steatohepatitis in MASH mice

[0091] Similar to the cholestasis model, GIBH-1014 and Celecoxib were used to characterize their ability to interfere with steatosis-induced liver fibrosis.

[0092] From the twelfth week of MASH (metabolic steatohepatitis) mouse modeling, the high-fat diet combined with CCl4 injury ( Fig.11 In the case of A), the experimental group mice were given 10 mg / kg of GIBH-1014 or 15 mg / kg of Celecoxib by oral gavage every day to observe the changes in the degree of liver fibrosis after COX-2 inhibition (process as shown in Fig.11 B).

[0093] After seven weeks of oral administration of two COX-2 inhibitors, the body weight of the drug-treated group was significantly reduced ( Fig.11 C), balloon-like lipid degeneration and inflammatory cell infiltration in the liver tissue were significantly reduced compared with those in the control mice without drug administration (HE staining, Fig.11 D).

[0094] The above results were graded according to the International Association for the Study of Hepatology and the modified Ishak score. The results showed that the fibrosis grade and inflammation grade of the COX-2 inhibitor treatment group were significantly lower than those of the control group. Image J statistical results also showed that the area percentage of balloon-like lipid-degenerated cells was smaller, especially in the GIBH-1014 treatment group ( Fig.11 E).

[0095] On the other hand, COX-2 inhibitor treatment significantly improved the liver physiological function indicators of MASH model mice. ALT, AST, TC and HDL in the treatment experimental group returned to normal levels, and LDL was significantly reduced ( Fig.11 F), indicating that COX-2 inhibitor treatment effectively reduces liver damage and cholesterol accumulation, and maintains normal liver physiological function.

[0096] In summary, treatment with COX-2 inhibitors inhibits liver injury and inflammatory cell infiltration in the MASH model, reduces ballooning caused by lipid accumulation, and avoids abnormal liver function.

[0097] Example 3 Principle of the effect of PGE2 level on fibrosis progression

[0098] In order to understand how elevated PGE2 levels after injury affect the process of fibrosis, primary HSCs naturally activated in vitro for 7 days were selected for experiments.

[0099] 1. PGE2 promotes the activation, proliferation and migration of HSCs

[0100] Immunofluorescence staining results Fig.12 The results showed that after 24 hours of LPS stimulation, HSCs proliferation was activated and the number increased by about 22% ( Fig.12 At the same time, the statistical results of the average cell fluorescence intensity proved that the production and accumulation of PGE2 in HSCs increased, and the expression of α-SMA protein also increased ( Fig.12 A, C and D in the figure). When GIBH-1014 was given at the same time as LPS stimulation, it was found that its proliferation effect was inhibited, and the production and accumulation of PGE2 in HSCs cells were reduced, and the expression of α-SMA protein was reduced accordingly ( Fig.12 A, C and D in the figure.

[0101] The above results show that the PGE2 and α-SMA levels in HSCs increased after LPS stimulation, and decreased after COX-2 inhibitor treatment. The PGE2 level was positively correlated with the HSCs activation marker (α-SMA).

[0102] After verifying that the increase of endogenous PGE2 is related to the promotion phenotype of HSCs' own proliferation and activation, we tried to add different concentrations of exogenous PGE2 to clarify the order in which it affects the behavior of surrounding HSCs through the paracrine pathway. Similarly, in vitro activated HSCs were added with 9 concentrations of PGE2 ranging from 1pM to 100mM for 24 hours. CCK-8 detection found that when the exogenous PGE2 reached more than 1μM, the number of HSCs increased by 14-26% ( Fig.12 E), that is, the cell proliferation of HSCs showed a positive dose effect with PGE2.

[0103] Interestingly, cell migration experiments showed that 10 nM and higher concentrations of PGE2 can promote the migration ability of primary HSCs. Primary HSCs under the same conditions were selected for scratch experiments, and 4 concentrations of PGE2 were added for 8 hours each ( Fig.13), calculate and compare the areas of their migration towards each other, and make statistics such as Fig.14 As shown, the results showed that compared with the control group with no obvious effect, the area of ​​migration in the 10nM group after 8 hours was 28%, and the 100nM and 1000nM groups were also 20%, indicating that low concentrations of PGE2 can significantly promote the migration of HSCs, and this concentration (10nM) is consistent with the concentration of HSCs proliferation caused by damage in the previous article. It is speculated that damage leads to an increase in endogenous PGE2 in HSCs, thereby promoting the cell's own proliferation and activation-related phenotypes. At the same time, the level of PGE2 excreted by HSCs is sufficient to trigger the migration of surrounding HSCs, thereby aggravating fibrosis.

[0104] In summary, elevated PGE2 levels, including endogenous increases after LPS stimulation and direct exogenous addition, can promote the activation, proliferation and migration of HSCs.

[0105] 2. PGE2 inhibits the expression of Cd163 and Arg-1 in M2 macrophages

[0106] To explore the relationship between excessive PGE2 production and the reduction in the number or functional loss of M2 macrophages in progressive cholestatic injury, we exogenously added different concentrations of PGE2 to observe whether it could change the polarization and related phenotypes of monocytes.

[0107] The results showed that treatment with PGE2 at 100 nM for 24 h could inhibit the expression of CD163 in mononuclear phagocytes, whether in M0 / M1 / M2 type before or after polarization ( Fig.15 The most significant effect was the inhibition of PGE2 on M2 monocyte CD163.

[0108] PGE2 receptor antagonists were used to observe whether the above effects could be compensated. ONO-8130, PF04418948, L-798106 and ONO-AE3-208 are EP1 / 2 / 3 / 4 receptor antagonists, respectively. The results showed that EP1 / 2 / 4 antagonists can increase the expression of CD163 in M2 monocytes and relieve the inhibitory effect of PGE2 ( Fig.15 C). Based on the above results, it is speculated that the large amount of PGE2 produced by HSCs induced by injury pathway EP1 / 2 / 4 inhibits the expression of CD163 in monocytes, especially M2 type.

[0109] Subsequently, the same in vitro experiment was performed on primary mouse liver Kupffer cells (KCs). KCs are macrophages that colonize the liver. The results showed that after KCs were induced to polarize to M2, the marker gene Cd163 was specifically expressed, and PGE2 could also inhibit Cd163 expression; similarly, the level of another M2 marker gene Arg-1 was also inhibited ( Fig.15 D and E in ).

[0110] CD163 and ARG-1 are not only specific evidence for the classification of M2 populations, but also important protein carriers for this type of cells to exert their anti-inflammatory function. PGE2 can significantly inhibit the expression of their mRNA, suggesting that PGE2 not only interferes with the differentiation of M2 polarity, but also destroys the maintenance of the M2 phenotype.

[0111] M2 monocytes play a major role in reducing inflammatory mediators, immunosuppressing inflammatory foci and tissue repair in the inflammatory process. COX-2 inhibitors may promote CD163 by reducing PGE2 content. + The increase in the number of M2 monocytes can exert anti-inflammatory effects, thereby alleviating liver fibrosis.

[0112] Another article found that the clearance of hemoglobin (upstream of bilirubin synthesis) depends on CD163 on the cell membrane of M2 monocytes. Both M2 cells with CD163 and CHO overexpressing cell lines with CD163 knocked in showed high affinity for hemoglobin and hemoglobin-haptoglobin complex, and it can be observed that both are engulfed into cells through CD163. + The increase in the number of M2 monocytes will also be associated with an increase in the efficiency of hemoglobin clearance and a decrease in the content of its downstream product bilirubin.

[0113] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0114] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of cyclooxygenase type II preparations in the preparation of drugs for treating liver damage, hepatitis, liver fibrosis, or decreased liver function or its complications.

2. The use according to claim 1, characterized in that: The cyclooxygenase type II inhibitor is a deuterated benzopyran compound, celecoxib, meloxicam, indomethacin or dideoxyfluridine.

3. The use according to claim 1, characterized in that: The deuterated benzopyran compound is 6-bromo-5,7-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 8-chloro-6-deuterated methyl-2-(trifluoromethyl)-2H-2H-benzopyran-3-carboxylic acid or 6-bromo-8-deuterated methyl-2-trifluoromethyl-2H-benzopyran-3-carboxylic acid; the deuterated benzopyran compound is preferably 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid.

4. The use according to any one of claims 1 to 3, characterized in that: The liver damage is liver damage caused by physical, chemical or biological factors; the hepatitis is an inflammatory reaction of liver tissue caused by physical, chemical or biological factors.

5. The use according to claim 4, characterized in that: The hepatitis is steatohepatitis or cholestatic hepatitis.

6. The use according to any one of claims 1 to 3, characterized in that: The liver fibrosis is cholestatic liver fibrosis or liver fibrosis caused by severe fatty liver.

7. The use according to any one of claims 1 to 3, characterized in that: The decreased liver function is an increase in AST, ALT, ALP, GGT, TBA, TBIL, DBIL or IBIL; the complication of decreased liver function is hyperbilirubinemia or hepatic encephalopathy caused by hyperbilirubinemia.

8. The use according to claim 7, characterized in that: The hyperbilirubinemia is neonatal jaundice, congenital biliary atresia, cholestatic cirrhosis, acute toxic liver failure, increased bilirubin caused by acute attack of chronic hepatitis, chronic liver failure or hepatic encephalopathy.

9. A drug for treating liver damage, hepatitis, liver fibrosis, or liver function decline or its complications, characterized in that: The drug comprises a cyclooxygenase type II inhibitor or a pharmaceutically acceptable salt or stereoisomer thereof; preferably, the cyclooxygenase type II inhibitor is a deuterated benzopyran compound, celecoxib, meloxicam, indomethacin or dideoxyfluridine; more preferably, the cyclooxygenase type II inhibitor is 6-bromo-5,7-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 6,8-deuterated dimethyl-2-(trifluoromethyl)-2H-benzopyran-3-carboxylic acid, 8-chloro-6-deuterated methyl-2-(trifluoromethyl)-2H-2H-benzopyran-3-carboxylic acid or 6-bromo-8-deuterated methyl-2-trifluoromethyl-2H-benzopyran-3-carboxylic acid.

10. The drug according to claim 9, characterized in that The medicine further comprises a pharmaceutically acceptable carrier; and / or the medicine is a tablet, a pill, a capsule or an injection.

Images