Application of aminophenacridine hydrochloride in preparation of medicine for preventing and treating fibrosis diseases

Abstract

The invention provides application of aminophenacridine hydrochloride in preparation of a medicine for preventing and treating fibrosis diseases. The aminophenacridine hydrochloride provided by the invention can be used for inhibiting the synthesis of fibronectin (Fibronectin) and type I collagen (CollagenI) in hepatic stellate cells, and shows a remarkable anti-hepatic fibrosis treatment effect in a hepatic fibrosis mouse model induced by bile duct ligation and carbon tetrachloride; the discovery provides a potential drug molecule with a new action mechanism for prevention and treatment of hepatic fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and pharmacy, and relates to the application of amifenacil hydrochloride in the preparation of medicines for preventing and treating fibrotic diseases. Background Art

[0002] Liver fibrosis is an important pathological feature of chronic liver disease, which is often caused by medical surgery, drugs and chemical reagents, viral infection, alcohol abuse, metabolic disorders, cholestasis, non-alcoholic fatty liver disease, autoimmune diseases or genetic diseases, leading to abnormal deposition of extracellular matrix and then liver structural remodeling. The increasing burden of liver disease worldwide, especially in areas with high incidence of hepatitis B and C, has led to a continuous increase in the incidence of liver fibrosis. The prevalence of non-alcoholic fatty liver disease (NASH) has further promoted this trend. Existing treatment strategies mainly focus on controlling the cause and indirectly slowing down the progression of fibrosis by using anti-inflammatory, antioxidant drugs, liver protectants, etc. However, the clinical effects of these treatments are often limited and are accompanied by adverse reactions of varying degrees. Therefore, the development of new anti-fibrosis drugs with high efficacy is of great clinical significance.

[0003] Currently, drugs for the treatment of liver fibrosis are still under development, and only Efruxifermin has entered Phase 3 clinical trials, mainly for the treatment of non-alcoholic fatty liver disease. Efruxifermin is a fibroblast growth factor 21 (FGF21) analog that improves liver metabolism and reduces inflammation by simulating the effects of endogenous FGF21. It also has the potential to reverse liver fibrosis, but it is not yet known whether it has a therapeutic effect on liver fibrosis caused by other factors.

[0004] The activation of hepatic stellate cells (HSCs) plays a key role in the occurrence and development of liver fibrosis. HSCs are a non-parenchymal cell population that accounts for about 5-8% of all liver cells in healthy organs. Under normal circumstances, HSCs are in a quiescent state. However, when the liver is chronically damaged, HSCs are stimulated by pro-fibrotic factors such as TGF-β, inflammatory mediators, oxidative activation pathways, etc., and are activated and transformed into fibroblast-like cells, secreting large amounts of extracellular matrix proteins (such as collagen and fibronectin) and a variety of pro-fibrotic factors, leading to the formation of fibrotic tissue. Since hepatic stellate cells are the core regulatory cells for the formation of liver fibrosis, targeting the activation of hepatic stellate cells is expected to become a therapeutic strategy for the treatment of liver fibrosis, but the development of anti-fibrotic drugs targeting this mechanism of action is still in the theoretical stage, and drug molecules with clear efficacy are urgently needed.

[0005] Amsacrine hydrochloride is a chemotherapy drug used for cancer treatment. It has been approved for marketing in Canada and is mainly used to treat acute lymphoblastic leukemia (ALL) and certain types of acute myeloid leukemia (AML). As a topoisomerase II inhibitor, amsacrine hydrochloride inhibits DNA replication and transcription by binding to DNA, thereby inducing cell cycle arrest and cell death. The prior art has not reported the use of amsacrine hydrochloride in liver fibrosis.

[0006] Triptolide is a natural compound extracted from the traditional Chinese medicinal material Tripterygium wilfordii, which is widely used in anti-inflammatory, immunomodulatory and anti-tumor fields. The latest studies have shown that triptolide has a significant effect on anti-organ fibrosis. Its pharmacological mechanism includes preventing myofibroblast activation, inhibiting the process of epithelial-mesenchymal transition, anti-oxidative stress, inhibiting inflammatory response, alleviating extracellular matrix deposition and regulating autophagy to prevent and treat organ fibrosis. In liver fibrosis, triptolide may improve fibrosis symptoms through multiple pathways such as allosteric activation of AMPK signaling pathway, inhibition of IL-1β and TNF-α and hepatic stellate cell activation, and anti-NF-κB activation (Liu Xin, Qiu Mingliang, Hu Xiaofeng, et al. Research progress on the mechanism of triptolide in anti-organ fibrosis [J]. Chinese Herbal Medicine, 2024, 55(10): 3569-3578.).

[0007] PF-06873600 is an oral small molecule cyclin-dependent kinase (CDK) inhibitor developed by Pfizer, which mainly targets CDK2, CDK4 and CDK6. PF06873600 is developed to treat a variety of tumor types, especially cancers associated with uncontrolled cell cycle regulation, such as breast cancer and prostate cancer. By inhibiting CDK2 / 4 / 6, PF06873600 can prevent tumor cells from entering the G1 / S phase of the cell cycle, thereby inhibiting the proliferation of cancer cells and inducing apoptosis. The drug is undergoing clinical trials, and preliminary data show certain anti-tumor activity and tolerable safety. The prior art has not reported the application of PF-06873600 in liver fibrosis. Summary of the invention

[0008] The purpose of the present invention is to provide an application of amifenesulfonyl hydrochloride in the preparation of a drug for preventing and treating fibrotic diseases. The CAS number of amifenesulfonyl hydrochloride is 54301-15-4, and the molecular structure is shown in Formula I:

[0009] The fibrotic disease is preferably liver fibrosis, and the pathogenic factors of liver fibrosis are selected from one or more of cholestasis, drugs and chemical reagents, viral infection, alcohol abuse, metabolic abnormalities, and autoimmune reactions.

[0010] The liver fibrosis is preferably liver fibrosis caused by overactivation of hepatic stellate cells; the liver fibrosis may also be preferably liver fibrosis caused by excessive synthesis of fibronectin and type I collagen; the liver fibrosis caused by cholestasis refers to liver fibrosis caused by abnormal bile excretion due to bile duct destruction, and the liver fibrosis caused by drugs and chemical reagents may further be preferably liver fibrosis caused by carbon tetrachloride.

[0011] The amifenecridine hydrochloride provided by the present invention can inhibit the synthesis of fibronectin and type I collagen in hepatic stellate cells, and exhibits significant anti-liver fibrosis therapeutic effects in bile duct ligation and carbon tetrachloride-induced liver fibrosis mouse models. This discovery provides a potential drug molecule with a new mechanism of action for the prevention and treatment of liver fibrosis.

[0012] The present invention also provides a pharmaceutical composition comprising amikacin hydrochloride and pharmaceutically acceptable excipients, and an excipient. The pharmaceutical composition and the excipient are in the form of one or more of capsules, tablets, oral suspensions, injections or freeze-dried powder injections.

[0013] The dosage form of the amifeneacrine hydrochloride of the present invention is preferably an injection or a lyophilized powder injection; the administration method is more preferably twice a week.

[0014] The present invention provides a new technical solution for preparing drugs for preventing or treating liver fibrosis. Compared with the prior art, the amifenecridine hydrochloride provided by the technical solution of the present invention is a new active ingredient, and its therapeutic effect is equivalent to or even better than that of the positive drug triptolide, and this therapeutic effect has a different mechanism of action compared with the existing anti-liver fibrosis drugs. The amifenecridine hydrochloride provided by the present invention inhibits the activation of hepatic stellate cells, the core regulatory cells of liver fibrosis, and reduces the synthesis of fibronectin and type I collagen, thereby providing a new strategy for the prevention and treatment of liver fibrosis. The present invention provides a new option for the prevention and treatment of liver fibrosis and fills the gaps in existing treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention induces a liver fibrosis model in mice by bile duct ligation, and finds that amphenacidine hydrochloride significantly alleviates the symptoms of the cholestatic liver fibrosis mouse model.

[0016] Figure 2 The present invention uses carbon tetrachloride to induce a liver fibrosis model in mice, and finds that amphenacidine hydrochloride can significantly alleviate the symptoms of the liver fibrosis mouse model.

[0017] Figure 3 Afenac hydrochloride inhibits the transcription of fibronectin and type I collagen related genes in the hepatic stellate cell line LX-2 cells.

[0018] Figure 4 Afenac hydrochloride inhibits the synthesis of fibronectin and type I collagen in the hepatic stellate cell line LX-2. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents, instruments and methods used in the following examples are generally obtained through commercial channels under conventional conditions or according to the conditions recommended by the manufacturer without special instructions. Graph Pad Prism 8.0.1 software was used for statistical analysis, and the data were expressed as X±SD. The t-tests test was used to analyze the differences between the two groups, and One-way ANOVA was used for variance analysis of multiple groups of data. *, P<0.05; **, P<0.01; ***, P<0.001; ns, P>0.05.

[0020] Example 1: The therapeutic effect of amphenacidine hydrochloride on the bile duct ligation-induced liver fibrosis model in mice.

[0021] Experimental materials: C57BL / 6 mice were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., all of which were male and 8-10 weeks old; amphenacidine hydrochloride, triptolide, and PF-06873600 drug powders were purchased from Taoshu Biotechnology Co., Ltd.; pentobarbital sodium powder was purchased from Sigma.

[0022] Experimental methods:

[0023] (1) Drug preparation: Amitraz hydrochloride was dissolved in 2% DMSO, 40% PEG300, 5% Tween-80, and 53% saline in sequence to a final concentration of 0.1 mg / mL, vortexed thoroughly, and administered within 30 minutes of preparation; triptolide was dissolved in DMSO to a 0.5 mg / mL stock solution, and diluted 100 times with saline to a final concentration of 5 μg / mL when administered; PF-06873600 was dissolved in 40% PEG300 and 60% saline to a final concentration of 5 mg / mL.

[0024] (2) Construction of bile duct ligation model and subsequent treatment: C57BL / 6 mice were randomly divided into 5 experimental groups, namely blank control group, vehicle control group and amphenicol hydrochloride, triptolide, and PF-06873600 administration groups, with 5 mice in each group. The blank control group was not treated. On the day of modeling, the skin and muscle tissues of the other groups were cut open layer by layer with surgical scissors to expose the liver tissue of the mice. The liver lobe was gently opened, and it was found that the bile duct was often attached to a small section of the intestine. A small section of the bile duct was bluntly separated with surgical forceps, and two surgical sutures were buried and tied with surgical knots respectively. The bile duct was cut off from the middle of the two ligatures. After confirming that there was no organ damage or bleeding in the abdominal cavity, the liver lobe and intestine were gently repositioned with cotton swabs, and the abdomen was sutured layer by layer with surgical sutures. After the skin was sutured, it was disinfected with iodine and alcohol cotton balls. The vehicle control group underwent the same surgery but the bile duct was not ligated. From the first day after modeling, the mice were given the following regimen: amikacin hydrochloride (1 mg / kg, intravenous injection, twice a week), triptolide (50 μg / kg, intravenous injection, twice a week), PF-06873600 (50 mg / kg, oral gavage, twice a day), and the vehicle control group was given the same volume of vehicle. After 12 days, liver tissue was collected, paraffin sections were sliced, and H&E staining and Masson staining were performed to evaluate the degree of liver fibrosis in mice. Mouse liver tissue samples were lysed and RNA was extracted, and rt-qPCR was used to detect the gene transcription of type I collagen and α-smooth muscle actin in cells.

[0025] Experimental results: Figure 1 The results in Table 1 show that in the bile duct ligation-induced mouse liver fibrosis model, the degree of liver fibrosis damage in the mice in the amifenacil hydrochloride administration group was significantly lower than that in the modeling group, and the therapeutic effect of amifenacil hydrochloride was better than that in the triptolide administration group. The rt-qPCR results showed that the transcription of type I collagen and α-smooth muscle actin genes in the modeling mice was significantly increased, and the transcription of type I collagen and α-smooth muscle actin genes was reduced after amifenacil hydrochloride administration, which was equivalent to the effect of the triptolide group. These results indicate that amifenacil hydrochloride can significantly alleviate the symptoms of mice with liver fibrosis caused by cholestasis. The PF-06873600 administration group had no significant effect on liver fibrosis.

[0026] Table 1: Masson staining scores Group Liver fibrosis ratio (%) Blank control group 0.00±0.00 Vehicle control group 12.11±3.48 Amphenacrine hydrochloride group 4.68±2.18* Triptolide group 8.29±5.04 PF-06873600 Group 11.80±1.59

[0027] Example 2: The therapeutic effect of amphenacidine hydrochloride on the carbon tetrachloride-induced liver fibrosis model in mice.

[0028] Experimental materials: C57BL / 6 mice were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., all of which were male and 6-8 weeks old; the sources of amphenacidine hydrochloride, triptolide, and sodium pentobarbital powder were the same as those in Example 1; and carbon tetrachloride was purchased from Sigma.

[0029] Experimental method: The preparation method of amifenecridine hydrochloride and triptolide drug powder is the same as in Example 1. Carbon tetrachloride was diluted in olive oil at a ratio of 1:3. C57BL / 6 mice were randomly divided into 4 experimental groups, namely, a blank control group, a solvent control group, and an amifenecridine hydrochloride and triptolide administration group, with 5 mice in each group. The solvent control group and the administration group were orally administered CCL4 (1mL / kg) twice a week, and the blank control group was given an equal volume of olive oil. The administration group was divided into two groups at the same time and administered according to the following scheme: amifenecridine hydrochloride (1mg / kg, intravenous injection, twice a week), triptolide (50μg / kg, intravenous injection, twice a week), and liver tissue was collected after 6 weeks. The liver tissue samples were paraffin sectioned and then H&E stained and Sirius red stained to evaluate the degree of liver fibrosis in mice. The mouse liver tissue samples were lysed and RNA was extracted, and rt-qPCR was used to detect the gene transcription of type I collagen and α-smooth muscle actin in the cells.

[0030] Experimental results: Figure 2 The results in Table 2 show that in the carbon tetrachloride-induced mouse liver fibrosis model, the degree of liver fibrosis in the mice in the amifenecridine hydrochloride administration group was significantly lower than that in the modeling group, and was better than that in the triptolide administration group. The rt-qPCR results showed that the administration of amifenecridine hydrochloride significantly reversed the transcription levels of type I collagen and α-smooth muscle actin genes in mice with liver fibrosis, and the effect was comparable to that of triptolide. These results indicate that amifenecridine hydrochloride can significantly alleviate the symptoms of the carbon tetrachloride-induced liver fibrosis mouse model.

[0031] Table 2: Sirius red staining scores Group Liver fibrosis ratio (%) Blank control group 0.00±0.00 Vehicle control group 8.91±1.81 Amphenacrine hydrochloride group 3.51±0.40**** Triptolide group 5.92±0.86**

[0032] Example 3: Effect of amikacin hydrochloride on the transcription of fibronectin and type I collagen related genes in LX-2 cells.

[0033] Experimental materials: LX-2 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd.; TGF-β factor was purchased from Jinan Protein Technology Co., Ltd.; the sources of amifenesin hydrochloride and triptolide drug powders were the same as those in Example 1.

[0034] Experimental instruments: Fluorescence quantitative PCR instrument was purchased from Bio-Rad.

[0035] Experimental method: LX-2 cells were selected and inoculated into 6-well plates (3.0×10 6The cells were cultured in a 37°C, 5% CO2 incubator overnight. TGF-β (5 ng / mL) was given the next day, and drugs (concentrations were amikacin hydrochloride: 1.25, 2.5, 5.0 μM; triptolide: 1.6, 8, 40 nM) were given at the same time. The cells were collected and RNA was extracted. RT-qPCR was used to detect the transcription of fibronectin and type I collagen related genes in the cells.

[0036] Experimental results: The results are as follows Figure 3 As shown in the data, compared with the control group, the transcription of fibronectin and type I collagen in LX-2 cells was significantly increased after TGF-β stimulation, and the transcription of fibronectin and type I collagen in LX-2 cells was significantly decreased after administration of amifenecridine hydrochloride, which was equivalent to the effect of the triptolide group, indicating that amifenecridine hydrochloride inhibits the transcription of fibronectin and type I collagen related genes in hepatic stellate cells.

[0037] Example 4: Effect of amikacin hydrochloride on the synthesis of fibronectin and type I collagen in LX-2 cells.

[0038] Experimental materials: LX-2 cells were sourced from the same source as in Example 3; TGF-β factor was sourced from the same source as in Example 3; and amifenecridine hydrochloride and triptolide drug powders were sourced from the same source as in Example 1.

[0039] Experimental method: LX-2 cells were seeded in 12-well plates (1.5×10 6 The cells were cultured in a 37°C, 5% CO2 incubator overnight. TGF-β (5 ng / mL) was given the next day, and drugs (concentrations were amikacin hydrochloride: 1.25, 2.5, 5.0 μM; triptolide: 1.6, 8, 40 nM) were given at the same time. The cells were collected and lysed, and the proteins were collected. The expression of fibronectin and type I collagen in the cells was detected by Western blot.

[0040] Experimental results: Figure 4 As shown in the data, compared with the control group, the expression of fibronectin and type I collagen in LX-2 cells was significantly increased after TGF-β stimulation. After administration of amphenacrine hydrochloride, the expression of fibronectin and type I collagen decreased in a concentration-dependent manner, and the effect was better than that of the triptolide group, indicating that amphenacrine hydrochloride inhibits the expression of fibronectin and type I collagen in hepatic stellate cells.

Claims

1. A use of amifenesulfonyl hydrochloride in the preparation of a drug for preventing and treating fibrotic diseases, wherein the molecular structure of amifenesulfonyl hydrochloride is as shown in Formula I:

2. The use according to claim 1, characterized in that: The organ fibrosis refers to liver fibrosis, which is caused by one or more pathogenic factors including medical surgery, drugs and chemical reagents, viral infection, alcohol abuse, metabolic disorder, cholestasis, non-alcoholic fatty liver disease, autoimmune disease or genetic disease.

3. The use according to claim 2, characterized in that: The liver fibrosis refers to liver fibrosis caused by excessive activation of hepatic stellate cells.

4. The use according to claim 2, characterized in that: The liver fibrosis refers to liver fibrosis caused by excessive synthesis of fibronectin and type I collagen.

5. The use according to claim 2, wherein the liver fibrosis caused by cholestasis refers to liver fibrosis caused by abnormal bile excretion due to bile duct destruction.

6. The use according to claim 2, wherein the liver fibrosis caused by drugs and chemical reagents refers to liver fibrosis caused by carbon tetrachloride.

7. The use according to claim 1, characterized in that: The amifenacil hydrochloride inhibits the activation of hepatic stellate cells, the core regulatory cells of liver fibrosis, and reduces the synthesis of fibronectin and type I collagen, thereby preventing liver fibrosis.

8. The use according to claim 1, characterized in that: The medicine is prepared from amikacin hydrochloride and pharmaceutically acceptable excipients.

9. The use according to claim 1, characterized in that: The dosage form of the drug is one or more of injection solution and lyophilized powder injection.

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