CN110604735B

CN110604735B - Compound for treating hepatic fibrosis and scleroderma and application thereof

Info

Publication number: CN110604735B
Application number: CN201910881985.6A
Authority: CN
Inventors: 卢伟强; 章涵堃; 陈思; 姜兴武; 蒋蓓尔; 刘明耀
Original assignee: East China Normal University; Bioray Laboratories Inc
Current assignee: East China Normal University; Bioray Laboratories Inc
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2022-10-25
Anticipated expiration: 2039-09-18
Also published as: CN110604735A

Abstract

The invention belongs to the field of medicines, and particularly discloses a compound LQ-2-191 for treating fibrosis related diseases. The invention provides application of a target point with cannabinoid receptor CB2 as an action in the development of anti-fibrosis drugs. The LQ-2-191 can specifically activate CB2 in vivo and in vitro, inhibit inflammatory reaction at a fibrosis part by acting on the CB2 and effectively relieve the fibrosis symptom. Animal experiments prove that the LQ-2-191 can effectively relieve rat hepatic fibrosis induced by bile duct ligation and mouse scleroderma model fibrosis induced by bleomycin. The anti-fibrosis drug takes cannabinoid receptor CB2 as an action target, can obviously inhibit the occurrence and the development of hepatic fibrosis and scleroderma, and can be used for preparing the drugs for resisting hepatic fibrosis and preventing and treating liver cirrhosis and scleroderma.

Description

Compound for treating hepatic fibrosis and scleroderma and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a compound for treating hepatic fibrosis and scleroderma and application thereof.

Background

Fibrosis, a pathological condition caused by excessive tissue repair, generally occurs during wound healing after tissue injury. Examples of conditions associated with tissue fibrosis are numerous and generally include the formation of skin scarring, liver fibrosis, kidney fibrosis, pulmonary interstitial fibrosis, glomerulonephritis, heart failure (ischemic and non-ischemic), scleroderma, and the like.

Scleroderma, systemic sclerosis, is an autoimmune disease, the most prominent external features being skin thickening and arteriolar damage due to collagen accumulation. Two forms of sclerosis are currently common in the clinic: local sclerosis and systemic sclerosis. Topical sclerosis affects mainly the skin of the face, hands and feet. Systemic sclerosis may also develop in internal organs including the kidney, heart, lung and gastrointestinal tract, in addition to the skin. The current data indicate a 10-year survival rate of 75% for patients with localized systemic sclerosis; nearly 10% of patients develop pulmonary hypertension within 10 to 20 years. The 10-year survival rate for systemic sclerosis patients is only 55%, the most common cause of death being failure of the lungs, heart and kidneys. In addition, these patients are at a slightly increased risk of developing cancer.

Hepatic fibrosis is one of the key processes of the progression of various acute and chronic liver diseases such as liver cirrhosis and is also the most important risk factor of liver cancer. Liver cancer is the cancer with the highest mortality in the world, the pathogenesis process of the liver cancer is generally acute and chronic liver injury caused by alcohol, virus and other pathogenic factors, the liver is excessively repaired under the conditions of continuous injury and strong inflammation reaction, the balance of collagen fiber synthesis and degradation is broken, a large amount of extracellular matrix protein is excessively accumulated in the liver, liver fibrosis and liver cirrhosis are caused, and the liver cancer is possibly developed finally, and the pathogenesis mode is the main pathogenesis mode of liver cancer in China. Liver fibrosis is a necessary way for the development of various chronic liver diseases such as hepatitis, non-alcoholic fatty liver disease and the like to cirrhosis, so that early prevention of liver fibrosis can slow down the progress of various liver diseases. A number of animal experiments and clinical trials have demonstrated that liver fibrosis is a reversible process. Improving liver fibrosis can not only prevent inflammatory necrosis of liver cells and reduce abnormal liver metabolism, but also slow down the progress of cirrhosis and liver cancer, thereby effectively increasing survival time of patients and improving the life quality of patients.

Cannabinoid receptors and their downstream pathways are intimately involved in fibrosis caused by a variety of inflammations. In the liver, after the CB1 receptor in the fibroblast is activated, the hepatic stellate cell can be activated, the division speed of the myofibroblast is accelerated, and the hepatic fibrosis is promoted. After the CB2 receptor expressed in the Kunpan cells is activated, the anti-inflammatory factor which promotes the apoptosis of hepatic stellate cells can be secreted, and myofibroblast apoptosis can be induced, so that the effect of inhibiting hepatic fibrosis is achieved. Thus, inhibition of the CB1 receptor or activation of the CB2 receptor may be effective in treating liver fibrosis. Also, for skin fibrosis caused by inflammation, inflammation can be effectively inhibited by activating the CB2 receptor, thereby effectively inhibiting the occurrence and development of skin fibrosis. However, there is a great deal of clinical evidence that antagonists that inhibit the CB1 receptor can cause serious side effects such as depression, suicidal ideation tendencies, and the like. Therefore, the development of CB2 selective agonists is a promising new strategy for hepatic fibrosis treatment.

A number of cannabinoid receptor agonists are currently entering clinical research for anti-fibrosis. The Anabasum is a natural artificial cannabinoid analogue which enters the clinic and is used for treating the fibrotic diseases, and achieves more remarkable effects. However, the general selectivity of Anabasum for different cannabinoid receptor subtypes has limited its clinical utility. Therefore, there is a great need to develop novel highly active, highly selective CB2 receptor agonists for use in the treatment of fibrotic diseases. Among them, CB2 receptor activation can inhibit or even reverse the occurrence of fibrosis.

Disclosure of Invention

Through large-scale drug screening, the invention discovers that LQ-2-191 can efficiently activate a CB2 downstream G protein channel, thereby effectively improving hepatic fibrosis and scleroderma. The LQ-2-191 inhibits inflammatory reaction of a fibrosis part by activating CB2 and effectively relieves the symptom of fibrosis.

The technical problem to be solved by the invention is to provide a compound for treating hepatic fibrosis and scleroderma.

The invention provides application of a compound LQ-2-191 in preparation of a medicament related to treatment of a fibrotic disease.

Wherein, the fibrosis-related disease drug takes cannabinoid receptor CB2 as an action target.

The drug is an anti-fibrosis drug named as LQ-2-191, and the molecular formula of the LQ-2-191 is as follows: c ₁₈ H ₂₃ F ₃ N ₄ O ₂ The structure is shown as formula (1):

in the invention, the LQ-2-191 can reduce inflammation of liver and skin by inhibiting the generation or infiltration of inflammatory mediators.

Wherein the inflammatory mediator is TNF-alpha, TGF-beta.

In the invention, the LQ-2-191 improves the fibrosis symptoms by reducing collagen deposition.

In the invention, the LQ-2-191 is administered in a dose of 1-20mg per kg of body weight of the subject.

The invention also provides a pharmaceutical composition, which comprises an effective amount of a compound shown as the formula (1) and a pharmaceutically acceptable carrier:

in the invention, the dosage form of the pharmaceutical composition is oral liquid.

The invention also provides application of the pharmaceutical composition in preparation of medicines for resisting hepatic fibrosis and scleroderma.

In the invention, the LQ-2-191 is injected into an animal individual (such as gavage), and the final concentration of the LQ-2-191 is 1-20mg/kg. Preferably, the final concentration of the LQ-2-191 is 20mg/kg.

The anti-fibrosis drug of the present invention can provide different effects when administered (administered) therapeutically. Generally, anti-fibrotic drugs can be formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carrier media to form pharmaceutical compositions. Wherein the aqueous carrier medium is normal saline, phosphate buffer, dextrose solution, distilled water, glycerol, ethanol, and combinations thereof.

Wherein the pharmaceutical composition typically has a pH of about 5 to about 8; preferably, the pH is about 6-8, which may vary depending on the nature of the substance being formulated and the condition being treated.

LQ-2-191 of the present invention can be prepared in the form of an oral preparation, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1-20mg/kg per day. The specific dosage should also take into account such factors as the route of administration, the health of the patient, etc. Furthermore, LQ-2-191 of the present invention may also be used with other therapeutic agents.

The invention carries out weight monitoring on an induced fibrosis animal model, and detects the content of collagen I (colla I), alpha smooth muscle actin (alpha-SMA) and hydroxyproline in tissues, and the experimental result shows that the LQ-2-191 can reduce infiltration of inflammatory cells at a fibrosis part and deposition of collagen, thereby improving fibrosis symptoms.

The invention also provides application of LQ-2-191 or the pharmaceutical composition in preparing a medicament for inhibiting and/or treating inflammatory mediators generated in the processes of hepatic fibrosis and scleroderma.

Wherein the inflammatory mediator is TNF-alpha, TGF-beta.

Wherein, the medicine is used for treating liver diseases, and the liver diseases refer to diseases related to liver fibrosis; the liver disease is chronic hepatitis, non-alcoholic fatty liver, liver cirrhosis or liver cancer and diseases caused by liver injury.

Wherein the medicament is for reducing the stiffness and thickness of the skin.

Wherein the medicament is for use in treating scleroderma; the scleroderma is an autoimmune disease, which is mainly characterized by skin thickening and arteriolar lesions caused by collagen accumulation and fibrotic degeneration of heart, liver and lung organs.

The invention also provides application of the LQ-2-191 or the pharmaceutical composition in preparing a medicament for reducing and/or treating collagen deposited in the liver fibrosis and scleroderma processes.

Wherein the LQ-2-191 or the pharmaceutical composition is used for reducing the expression level of ALT, AST and total bilirubin in serum.

The beneficial effects of the invention include: the invention provides application of LQ-2-191 or a pharmaceutical composition in preparation of medicines for resisting hepatic fibrosis and scleroderma. The LQ-2-191 or the pharmaceutical composition can obviously inhibit the development of hepatic fibrosis and scleroderma. The invention develops the LQ-2-191 as a new anti-fibrosis drug or an auxiliary component thereof, has obvious anti-fibrosis effect and stable property, and provides a new way and means for treating hepatic fibrosis and scleroderma.

Drawings

FIG. 1 shows the results of the calcium flux test of example 1LQ-2-191 at the CB2 receptor; wherein, fig. 1a shows the calcium flow activity of a yang drug CP55940, and fig. 1b shows the calcium flow activity of LQ-2-191.

FIG. 2 is a graph showing that the activation of CB2 receptor by LQ-2-191 of example 1 is inhibited by the CB2 receptor antagonist AM 630.

FIG. 3 is a schematic representation of example 1, LQ-2-191, being capable of causing desensitization of the CB2 receptor; wherein, FIG. 3a is calcium flux activity induced by DMSO, CP55940 and LQ-2-191 stimulating CB2 twice in sequence, and FIG. 3b is a statistical analysis chart of 3 a.

FIG. 4 shows the results of HTRF testing of example 1LQ-2-191 on CB2 receptors; wherein, FIG. 4a is HTRF test data of the positive drug CP55940, and FIG. 4b is HTRF test result of LQ-2-191.

FIG. 5 is a histopathological map of liver tissues of the control group stained with 2H and E and of the animals administered with LQ-2-191.

FIG. 6 is a pathological view of liver tissues of animals treated with the control group stained with sirius red and administered with LQ-2-191 of example 2.

FIG. 7 shows the liver function markers including alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and total bilirubin (T-BIL) in the serum of the rat in example 2.

FIG. 8 is a graph of example 2LQ-2-191 reducing the amount of rat liver fibrosis-associated markers, including TGF- β, col1a1, α -SMA and TNF- α.

FIG. 9 is a graph showing that example 2LQ-2-191 reduces liver fibrosis in rat serum, including type III Procollagen (PCIII), type IV Collagen (CIV), laminin (Laminin), and Hyaluronic Acid (HA).

FIG. 10 is a graph of the skin histopathology of the control group stained with 3H and E and animals given LQ-2-191.

FIG. 11 is the skin histopathology of the control group stained with sirius red and the animals given LQ-2-191 of example 3.

FIG. 12 is a photograph of the skin histopathology of the control group and animals given LQ-2-191 in immunohistochemical fluorescent staining of example 3.

FIG. 13 is a graph of example 3LQ-2-191 reducing the amount of mouse skin fibrosis-associated markers, including TGF- β, col1a1, α -SMA, and TNF- α.

Detailed Description

The present invention will be further described in detail with reference to the following specific examples, but the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1LQ-2-191 is a selective agonist of cannabinoid receptor CB2

All compounds were tested at a single concentration, selected at 10 μ M, using the method for testing calcium flux. 252 compounds are tested in batches, and the compounds which can activate the CB2 receptor at 10 mu M and reach the efficiency of over 30 percent of the positive drug CP55940 are subjected to 8-concentration gradient rescreening, so that the LQ-2-191 has better activity (EC) on the CB2 receptor ₅₀ =15.73nM, as shown in fig. 1), superior to the positive drug CP55940 (EC) ₅₀ =21.69 nM), and EC at the CB1 receptor ₅₀ Greater than 10. Mu.M, indicating that LQ-2-191 is a selective agonist of cannabinoid receptor CB 2.

To confirm that the compound is intracellular calcium flux induced by the CB2 receptor, the present invention was immediately validated using the CB2 receptor specific antagonist AM630, the results of which are shown in fig. 2. After the cells are treated by adding the AM630, the cells added with the positive drugs CP55940 and LQ-2-191 can not cause intracellular calcium flow signals, but the cells added with only the CP55940 and the LQ-2-191 can detect the signals without the AM630 treatment, which indicates that the LQ-2-191 causes the intracellular calcium flow by activating CB2 receptors, namely the LQ-2-191 is an agonist of the CB2 receptors.

Receptor desensitization is a feature shared by many membrane receptors. After GPCRs are activated by ligands, in order to avoid sustained activation of receptors, the body generally has its own negative regulatory mechanisms, receptor desensitization being one of the most common modes of regulation. After the receptor is activated to complete signal transduction, endocytosis can occur to desensitize the receptor, once the receptor is endocytosed, the receptor can not receive the stimulation of extracellular signals any more, and therefore the signal path is prevented from being continuously activated. In the desensitization experiment, the invention discovers that LQ-2-191 can cause the desensitization of the CB2 receptor, when the CB2 receptor is activated by using LQ-2-191 for the first time and is incubated for ten minutes, the cells are stimulated by using the CP55940 positive drug of the CB2 receptor again, and the intracellular calcium flow signal response cannot be caused, so that the desensitization of the receptor is caused after the LQ-2-191 is activated, the CB2 receptor cannot be activated by using the positive drug again (the result is shown in a figure 3), and the LQ-2-191 is proved to be the agonist of the CB2 receptor again.

Since CB2 receptors naturally transmit downstream signals via coupling G α i, the present invention also detects changes in intracellular cAMP upon activation of CB2 receptors by LQ-2-191. Activation of G.alpha.i inhibits adenylate cyclase activity and thus reduces intracellular cAMP levels, so the present invention is first stimulated with 2.5. Mu.M forskolin for testing. The present invention verifies the compound using HTRF (homogeneous time-resolved fluorescence) method, and the results are shown in fig. 4, and the present invention found that the HTRF results are substantially consistent with the calcium flux results. One indicates that the test used to screen compounds is robust and the other indicates that LQ-2-191 does act via the CB2 receptor and is a potent agonist of the CB2 receptor.

Example 2LQ-2-191 alleviate biliary ligation-induced liver fibrosis symptoms in rats via CB2 receptors

Approximately 200g CB2 knockout rats, 4-6 weeks old, were selected and starved for 6 hours prior to surgery. African Ding Mazui rats were used, each rat was injected intraperitoneally with 2mL of avertin. After the rat is anesthetized, finding a bile duct at an abdominal opening, and tying the bile duct by using a No. 5 thread for the model building group, wherein tying is performed twice to avoid dropping of the tying thread; the control group was opened only to the abdominal cavity and the bile duct was isolated and not ligated. After the ligation was completed, the internal organs of the rats were recovered, and then the internal organs were rinsed with physiological saline containing double antibody. Finally, the wound is closed. And the rats were placed on a 37 ℃ thermostatic table until their anaesthesia was recovered. The pad is changed every day within three days after operation, and the iodine tincture is used for disinfecting the wound to avoid wound infection. LQ-2-191 (oral, 20 mg/kg) treatment was given starting on the third day after surgery, daily for two weeks, after which the rat was sacrificed. Detecting blood index and hepatic fibrosis index of rat, and evaluating the effect of the medicine.

The results show that: after the bile duct ligation operation, the liver of both the CB2 gene knockout rat and the wild rat is obviously enlarged, and the weight of the liver is obviously increased, but after the wild rat is treated by the LQ-2-191, the weight of the liver of the LQ-2-191 treated group is obviously reduced compared with that of a bile duct ligation model induced group. Meanwhile, the liver weight index is also obviously reduced, which shows that LQ-2-191 has certain effect on inhibiting liver injury, and the liver weight index is hardly changed before and after treatment on rats with CB2 receptor deletion. Meanwhile, the invention discovers that ALT, AST and T-BIL in serum of both wild rats and CB2 receptor-deficient rats are remarkably increased after the biliary ligation operation is carried out (as shown in figure 7). However, after the wild rat is treated by the LQ-2-191, the ALT and AST content is remarkably reduced, which indicates that the liver injury induced by ligation of the bile duct of the rat is relieved by the LQ-2-191, besides, the T-BIL in serum is also remarkably reduced, but the rat with the CB2 receptor deletion is not protected, and the CB2 receptor plays an important role in the treatment process of the LQ-2-191, namely the LQ-2-191 plays a role in protecting the liver by activating the CB2 receptor.

In addition to testing for basal liver function impairment in serum, the present invention also measures four basic indicators for assessing liver fibrosis, including type III Procollagen (PCIII), type IV Collagen (CIV), laminin (lamin), and Hyaluronic Acid (HA). PCIII procollagen is an intermediate product in the process of fibrosis, and the content of the PCIII procollagen in serum is in positive correlation with the liver fibrosis degree, so the liver fibrosis degree can be judged by detecting the amount of PCIII in the serum. CIV, laminin and HA are all components constituting basement membrane, and the content in serum can reflect the degree of liver fibrosis. From the results (as shown in fig. 9), the four indices of liver fibrosis in the serum of the wild-type rat and the CB2 receptor-deficient rat were all significantly increased after bile duct ligation, but only the wild-type rat was relieved after LQ-2-191 treatment, and the four indices of fibrosis in the serum of the CB2 receptor-deficient rat were not relieved after compound treatment.

Hepatic fibrosis is caused by excessive liver self-repair after the liver is damaged. During the process of fibrosis, inflammatory lymphocytes play an important role, and it is due to the excessive inflammation in the liver that the excessive repair of the liver is caused. Therefore, lymphocyte infiltration is positively correlated with the severity of liver inflammation and the degree of fibrosis. From the results of H & E staining of rat liver sections of this batch (as shown in fig. 5), the infiltration of inflammatory cells in wild-type rats was significantly reduced after LQ-2-191 treatment, while the infiltration of inflammatory cells in CB2 receptor-deficient rats was not reduced. In addition, the number of hydropsy liver cell deaths after bile duct ligation was significantly increased in liver cell morphology (as shown in fig. 5), while wild-type rats were relieved after drug treatment, but the compound-treated group of rats with CB2 receptor deletion was not significantly different from the control group.

Collagen deposition is the most important phenotype in the hepatic fibrosis process, and then the invention uses sirius red staining to observe the collagen deposition condition in rat liver, the result shows (as shown in figure 6) that the collagen deposition amount of wild rat and CB2 receptor-deficient rat is obviously increased after bile duct ligation, but the collagen deposition amount in liver of wild rat is obviously reduced after LQ-2-191 treatment, while the collagen deposition amount of CB2 receptor-deficient rat treatment group is not obviously changed compared with the control group, meanwhile, the invention also finds that the hepatic fibrosis degree is obviously increased after CB2 knockout, which shows that the self hepatic fibrosis resistance of rat is reduced after CB2 knockout.

The present inventors then examined the effect of compound LQ-2-191 on anti-fibrosis at the mRNA level (as shown in FIG. 8). The results are consistent with the previous results, the compound LQ-2-191 can also inhibit the hepatic fibrosis of wild rats induced by bile duct ligation at the mRNA level, and can reduce the occurrence of inflammation and the expression of collagen, but has no relieving effect on rats with CB2 receptor deletion, and further shows that the compound LQ-2-191 can inhibit the hepatic fibrosis induced by bile duct ligation by activating the CB2 receptor.

Example 3LQ-2-191 alleviate bleomycin-induced fibrosis of mouse skin

Specifically, LQ-2-191 can reduce bleomycin-induced skin thickness increase and collagen deposition.

50 Balb/c mice of 6-8 weeks old are taken and randomly divided into 5 groups, namely a control group, a model building group + LQ-2-1911mg/kg, a model building group + LQ-2-1915mg/kg and a model building group + Win55212-21 mg/kg (a positive control group).

Mice in the experimental group were subcutaneously injected with 100. Mu.L of 1mg/mL bleomycin (15 mg bleomycin dissolved in 15mLPBS buffer) per day as a building block, and mice in the control group were subcutaneously injected with physiological PBS buffer alone.

Drug treatment was initiated two weeks after induction and a daily bleomycin solution was injected simultaneously for four weeks.

The mice are sacrificed after four weeks of drug treatment, skin tissues are taken for fixation and embedding, the tissues are subjected to morphological staining, collagen staining and immunohistochemical staining after slicing, and the degree of skin fibrosis of the mice and the curative effect of drug treatment are evaluated.

Bleomycin (100 μ L, 1mg/ml in PBS buffer) treated with bleomycin induced a significant increase in skin thickness and collagen deposition in mice of the fibrosis group compared to the normal control group (PBS + Vehicle). However, the skin thickness of the LQ-2-191 group of mice treated with LQ-2-191 was significantly improved.

The H & E staining results showed (as shown in fig. 10) that after bleomycin induction, the skin of the mice was significantly thickened and the number of cells associated with inflammation in the dermis, adipose and dermato-muscular layers was significantly increased. However, in mice treated with LQ-2-191, the number of inflammatory-related lymphocyte infiltrates in the skin tissue was significantly reduced, and the skin thickness of the mice was significantly decreased relative to the bleomycin-induced group.

Meanwhile, after the skin tissue sections of mice of different treatment groups are respectively dyed by sirius red dye (as shown in figure 11), the invention discovers that the LQ-2-191 can obviously reduce the deposition of collagen in the skin under different dosages of 1mg/kg and 5 mg/kg. The invention also uses an immunohistochemical method to detect alpha-SMA which is another main index of fibrosis, and the immunohistochemical result is shown in figure 12, after the bleomycin is induced, the quantity of the alpha-SMA at the hair follicle of the mouse is obviously increased. And after the LQ-2-191 is used for treating the mice, the expression of the alpha-SMA in the hair follicle can be obviously inhibited. These results indicate that LQ-2-191 has a role in the treatment of bleomycin-induced skin fibrosis.

In order to further confirm the change of other fibrosis markers in the skin, the invention takes the affected skin of a mouse, extracts the total mRNA of the skin, and further detects three indexes closely related to fibrosis by a QPCR method, including TGF-beta, col1a1 and alpha-SMA, and an index TNF-alpha related to inflammation. The results show (as shown in figure 13) that the expression level of markers related to fibrosis and inflammation in the skin of the mouse is remarkably reduced after the LQ-2-191 is used for treatment, and further prove that the LQ-2-191 can be used for treating bleomycin-induced skin fibrosis of the mouse.

The present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

Claims

The application of LQ-2-191 or a composition in preparing a medicament for inhibiting and/or treating scleroderma is characterized in that the composition comprises an active ingredient LQ-2-191 and a pharmaceutically acceptable carrier, wherein the structure of LQ-2-191 is shown as a formula (1):

the LQ-2-191 is used for reducing the hardness and thickness of the skin.
2. The use of claim 1, wherein the medicament is for the treatment of scleroderma.