Use of anethol blue light in the preparation of a medicament for the prevention and treatment of fibrotic diseases

Abstract

This invention provides the application of amphetamine hydrochloride in the preparation of drugs for the prevention and treatment of fibrotic diseases. The amphetamine hydrochloride provided by this invention can inhibit the synthesis of fibronectin and type I collagen in hepatic stellate cells, and exhibits significant anti-hepatic fibrosis therapeutic effects in a mouse model of liver fibrosis induced by bile duct ligation and carbon tetrachloride. This discovery provides a potential drug molecule with a new mechanism of action for the prevention and treatment of liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and pharmaceuticals, and relates to the application of amine acridine hydrochloride in the preparation of drugs for the prevention and treatment of fibrosis. Background Technology

[0002] Liver fibrosis is a key pathological feature of chronic liver disease, often triggered by medical procedures, drugs and chemicals, viral infections, alcohol abuse, metabolic disorders, cholestasis, non-alcoholic fatty liver disease (NASH), autoimmune diseases, or genetic disorders. These factors lead to abnormal extracellular matrix deposition, resulting in liver structural remodeling. The increasing global burden of liver disease, particularly in regions with high prevalence of hepatitis B and C, has led to a continuous rise in the incidence of liver fibrosis. The prevalence of non-alcoholic fatty liver disease (NASH) further exacerbates this trend. Current treatment strategies primarily focus on controlling the underlying causes, using anti-inflammatory drugs, antioxidants, and hepatoprotective agents to indirectly slow the progression of fibrosis. However, the clinical efficacy of these treatments is often limited and accompanied by varying degrees of adverse reactions. Therefore, the development of novel, highly effective anti-fibrotic drugs is of significant clinical importance.

[0003] Currently, drugs for treating liver fibrosis are still under development. Only Efruxifermin has entered phase 3 clinical trials, primarily for the treatment of non-alcoholic steatohepatitis (NAH). Efruxifermin is a fibroblast growth factor 21 (FGF21) analogue, which improves liver metabolism and reduces inflammation by mimicking the effects of endogenous FGF21 and has the potential to reverse liver fibrosis. However, its therapeutic effect on liver fibrosis caused by other factors remains unknown.

[0004] Activation of hepatic stellate cells (HSCs) plays a crucial role in the occurrence and development of liver fibrosis. HSCs are a non-parenchymal cell population, accounting for approximately 5-8% of all hepatocytes in healthy organs. Under normal circumstances, HSCs are in a quiescent state. However, when the liver suffers chronic injury, HSCs are stimulated by pro-fibrotic factors such as TGF-β, inflammatory mediators, and oxidative activation pathways, activating and transforming into fibroblast-like cells. These cells secrete large amounts of extracellular matrix proteins (such as collagen and fibronectin) and various pro-fibrotic factors, leading to the formation of fibrotic tissue. Since hepatic stellate cells are the core regulatory cells in the formation of liver fibrosis, targeting the activation of hepatic stellate cells holds promise as a therapeutic strategy for treating liver fibrosis. However, the development of anti-fibrotic drugs targeting this mechanism of action is currently still in the theoretical stage, and there is an urgent need for drug molecules with clear efficacy.

[0005] Amsacrine hydrochloride is a chemotherapy drug approved for cancer treatment in Canada, primarily for the treatment of acute lymphoblastic leukemia (ALL) and certain types of acute myeloid leukemia (AML). As a topoisomerase II inhibitor, amsacrine hydrochloride binds to DNA, inhibiting DNA replication and transcription, thereby inducing cell cycle arrest and cell death. There are no existing reports on the use of amsacrine hydrochloride in liver fibrosis.

[0006]

[0007] Tripterygium wilfordii is a natural compound extracted from the traditional Chinese medicinal herb Tripterygium wilfordii, and it is widely used in anti-inflammatory, immunomodulatory, and antitumor fields. Recent studies have shown that triptolide has a significant effect on organ fibrosis, and its pharmacological mechanisms include preventing myofibroblast activation, inhibiting epithelial-mesenchymal transition, resisting oxidative stress, inhibiting inflammatory responses, 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 the 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 action of triptolide against organ fibrosis [J]. Chinese Traditional and Herbal Drugs, 2024, 55(10):3569-3578.).

[0008]

[0009] PF-06873600 is an orally administered small-molecule cyclin-dependent kinase (CDK) inhibitor developed by Pfizer, primarily targeting CDK2, CDK4, and CDK6. PF06873600 is intended to treat various cancer types, particularly cancers associated with dysregulation of cell cycle regulation, such as breast 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 cancer cell proliferation and inducing apoptosis. This drug is currently undergoing clinical trials, and preliminary data show promising antitumor activity and a tolerable safety profile.

[0010] There are no existing reports on the application of PF-06873600 in liver fibrosis.

[0011] Summary of the Invention

[0012] The purpose of this invention is to provide the application of amine benzoyl hydrochloride in the preparation of a drug for the prevention and treatment of fibrosis, wherein the CAS number of amine benzoyl hydrochloride is 54301-15-4, and its molecular structure is shown in Formula I:

[0013]

[0014] 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.

[0015] The liver fibrosis is preferably caused by excessive activation of hepatic stellate cells; the liver fibrosis can also be preferably 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 damage; the liver fibrosis caused by drugs and chemical reagents can be further preferably caused by carbon tetrachloride.

[0016] The acridine hydrochloride provided by this invention can inhibit the synthesis of fibronectin and type I collagen in hepatic stellate cells, and shows significant anti-hepatic fibrosis therapeutic effects in a mouse model of bile duct ligation and carbon tetrachloride-induced liver fibrosis. This discovery provides a potential drug molecule with a new mechanism of action for the prevention and treatment of liver fibrosis.

[0017] The present invention also provides a pharmaceutical composition comprising amine acridine hydrochloride and pharmaceutically acceptable excipients, and comprising excipients. The pharmaceutical dosage form comprising the pharmaceutical composition and excipients is one or more of the following: capsules, tablets, oral suspensions, injections, or lyophilized powder for injection.

[0018] The preferred dosage form of the amine acridine hydrochloride of the present invention is an injection solution or a lyophilized powder for injection; the preferred administration method is twice a week.

[0019] This invention provides a new technical solution for preparing drugs to prevent or treat liver fibrosis. Compared with existing technologies, the acetaminophen hydrochloride provided by this invention is a novel pharmacologically active ingredient, with therapeutic effects comparable to or even superior to those of the positive control drug triptolide. Furthermore, this therapeutic effect has a different mechanism of action compared to existing anti-liver fibrosis drugs. The acetaminophen hydrochloride provided by this invention works by inhibiting the activation of hepatic stellate cells—the core regulatory cells of liver fibrosis—and reducing the synthesis of fibronectin and type I collagen, thereby providing a new strategy for the prevention and treatment of liver fibrosis. This invention provides a new option for the prevention and treatment of liver fibrosis and fills the gaps in existing treatment methods. Attached Figure Description

[0020] Figure 1 This invention uses a mouse model of liver fibrosis induced by bile duct ligation to find that acetaminophen hydrochloride significantly alleviates the symptoms of cholestatic liver fibrosis in mouse models.

[0021] Figure 2 This invention demonstrates that by inducing a mouse model of liver fibrosis using carbon tetrachloride, amine benzoyl hydrochloride significantly alleviated the symptoms of liver fibrosis in the mouse model.

[0022] Figure 3 Amphetamine hydrochloride inhibits the transcription of genes related to fibronectin and type I collagen in the hepatic stellate cell line LX-2.

[0023] Figure 4 Amphetamine hydrochloride inhibits the synthesis of fibronectin and type I collagen in the hepatic stellate cell line LX-2. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following embodiments are commercially available, generally under conventional conditions or as recommended by the manufacturer. Statistical analysis was performed using Graph Pad Prism 8.0.1 software. Data are expressed as X±SD. t-tests were used to analyze differences between two groups, and one-way ANOVA was used for analysis of variance of multiple groups. *, P<0.05; **, P<0.01; ***, P<0.001; ns, P>0.05.

[0025] Example 1: The therapeutic effect of acetaminophen hydrochloride on a mouse model of liver fibrosis induced by bile duct ligation.

[0026] Experimental materials: C57BL / 6 mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., all of which were male and aged 8-10 weeks; Amphetamine hydrochloride, Tripterygium wilfordii, and PF-06873600 drug powder were purchased from Taoshu Biotechnology Co., Ltd.; Sodium pentobarbital powder was purchased from Sigma-Aldrich.

[0027] Experimental methods:

[0028] (1) Drug preparation: Amphetamine hydrochloride was dissolved in 2% DMSO, 40% PEG300, 5% Tween-80 and 53% physiological saline in sequence to a final concentration of 0.1 mg / mL, vortexed thoroughly and administered within 30 minutes of preparation; Tripterygium wilfordii was dissolved in DMSO to a stock solution of 0.5 mg / mL, and diluted 100 times with physiological saline to a final concentration of 5 μg / mL before administration; PF-06873600 was dissolved in 40% PEG300 and 60% physiological saline to a final concentration of 5 mg / mL.

[0029] (2) Construction and subsequent treatment of bile duct ligation model: C57BL / 6 mice were randomly divided into 5 experimental groups: blank control group, solvent control group, and groups treated with amphetamine hydrochloride, triptolide, and PF-06873600, 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 layer by layer with surgical scissors to expose the liver tissue of the mice. The liver lobe was gently separated, and the bile duct was often attached to a small section of the intestine. The bile duct was bluntly separated with surgical forceps, two surgical sutures were buried and tied, and the bile duct was cut from the middle of the two ligation points. 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 closed layer by layer with surgical sutures. After the skin was sutured, it was disinfected with iodine and alcohol cotton balls. The solvent control group underwent the same operation but without bile duct ligation. From day one post-modeling, mice were administered the following medications: amphetamine hydrochloride (1 mg / kg, intravenous injection, twice a week), triptolide (50 μg / kg, intravenous injection, twice a week), and PF-06873600 (50 mg / kg, gavage, twice a day). The solvent control group received the same volume of solvent. Liver tissue was harvested after 12 days. Paraffin sections were prepared for H&E staining and Masson's staining to assess the degree of liver fibrosis. RNA was extracted from mouse liver tissue samples by lysis, and rt-qPCR was used to detect the transcription of type I collagen and α-smooth muscle actin genes in the cells.

[0030] Experimental results: such as Figure 1 The results in Table 1 show that in the bile duct ligation-induced liver fibrosis model in mice, the degree of liver fibrosis damage in the aminopyrine hydrochloride group was significantly lower than that in the model group, indicating that the efficacy of aminopyrine hydrochloride was superior to that of the triptolide group. rt-qPCR results showed that the transcription of type I collagen and α-smooth muscle actin genes was significantly increased in the model mice, while the transcription of type I collagen and α-smooth muscle actin genes decreased after aminopyrine hydrochloride administration, with effects comparable to those of the triptolide group. These results indicate that aminopyrine hydrochloride can significantly alleviate the symptoms of liver fibrosis in mice caused by cholestasis. The PF-06873600 group had no significant effect on liver fibrosis.

[0031] Table 1: Masson staining scores

[0032] Group Percentage of liver fibrosis (%) Blank control group 0.00±0.00 Solvent control group 12.11±3.48 Amine acridine hydrochloride group 4.68±2.18* Tripterygium wilfordii methyl group 8.29±5.04 PF-06873600 group 11.80±1.59

[0033] Example 2: The therapeutic effect of amine acridine hydrochloride on a carbon tetrachloride-induced mouse model of liver fibrosis.

[0034] Experimental materials: C57BL / 6 mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., all of which were male and 6-8 weeks old; the sources of acridine hydrochloride, triptolide, and sodium pentobarbital powder were the same as in Example 1; carbon tetrachloride was purchased from Sigma.

[0035] Experimental Methods: The preparation methods for the acridine hydrochloride and triptolide powders were 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 four experimental groups: a blank control group, a solvent control group, and a acridine hydrochloride and triptolide treatment group, with five mice in each group. The solvent control group and the treatment group were orally administered CCL4 (1 mL / kg) twice a week, while the blank control group was given an equal volume of olive oil. The treatment group was simultaneously divided into two subgroups and administered acridine hydrochloride (1 mg / kg, intravenous injection, twice a week) and triptolide (50 μg / kg, intravenous injection, twice a week) according to the following regimen. After 6 weeks, liver tissue was collected, and the liver tissue samples were paraffin-embedded and stained with H&E and Sirius red to assess the degree of liver fibrosis in mice. The liver tissue samples were lysed and RNA was extracted. The gene transcription of type I collagen and α-smooth muscle actin in cells was detected by rt-qPCR.

[0036] Experimental results: such as Figure 2 The results in Table 2 show that in the carbon tetrachloride-induced mouse model of liver fibrosis, the degree of liver fibrosis in the aminopyrine hydrochloride-treated group was significantly lower than that in the model group, and superior to that in the triptolide-treated group. rt-qPCR results showed that aminopyrine hydrochloride administration significantly reversed the transcription levels of type I collagen and α-smooth muscle actin genes in mice with liver fibrosis, with effects comparable to triptolide. These results indicate that aminopyrine hydrochloride can significantly alleviate the symptoms of the carbon tetrachloride-induced liver fibrosis mouse model.

[0037] Table 2: Sirius Red Staining Scores

[0038] Group Percentage of liver fibrosis (%) Blank control group 0.00±0.00 Solvent control group 8.91±1.81 Amine acridine hydrochloride group 3.51±0.40**** Tripterygium wilfordii methyl group 5.92±0.86**

[0039] Example 3: Effects of amine acridine hydrochloride on the transcription of fibronectin and type I collagen-related genes in LX-2 cells.

[0040] Experimental materials: LX-2 cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd.; TGF-β factor was purchased from Nearshore Protein Technology Co., Ltd.; the sources of the acridine hydrochloride and triptolide powder were the same as in Example 1.

[0041] Experimental instruments: Real-time PCR instrument purchased from Bio-Rad.

[0042] Experimental method: LX-2 cells were seeded in 6-well plates (3.0 × 10⁻⁶ cells per well). 6 Cells / well were incubated overnight at 37°C in a 5% CO2 incubator. The next day, TGF-β (5 ng / mL) was administered, along with drugs (concentrations of acridine hydrochloride: 1.25, 2.5, 5.0 μM; triptolide: 1.6, 8, 40 nM) for 24 h. Cells were then collected and RNA was extracted. RT-qPCR was used to detect the transcription of fibronectin and type I collagen-related genes in the cells.

[0043] Experimental results: The results are as follows Figure 3 As shown, compared with the control group, TGF-β stimulation significantly increased the transcription of fibronectin and type I collagen in LX-2 cells, while the administration of acetaminophen hydrochloride significantly decreased the transcription of fibronectin and type I collagen genes in LX-2 cells, with effects comparable to those of triptolide. This suggests that acetaminophen hydrochloride inhibits the transcription of fibronectin and type I collagen-related genes in hepatic stellate cells.

[0044] Example 4: Effects of amine acridine hydrochloride on the synthesis of fibronectin and type I collagen in LX-2 cells.

[0045] Experimental materials: LX-2 cells were sourced in the same way as in Example 3; TGF-β factor was sourced in the same way as in Example 3; and the powders of acridine hydrochloride and triptolide were sourced in the same way as in Example 1.

[0046] Experimental method: LX-2 cells were seeded in 12-well plates (1.5 × 10⁻⁶ cells per well). 6 Cells (cells / well) were incubated overnight at 37°C in a 5% CO2 incubator. The next day, TGF-β (5 ng / mL) was administered, along with drugs (concentrations of acridine hydrochloride: 1.25, 2.5, 5.0 μM; triptolide: 1.6, 8, 40 nM) for 24 h. Cells were then collected and lysed, and proteins were collected. Western blot was used to detect the expression of fibronectin and type I collagen in the cells.

[0047] Experimental results: such as Figure 4 As shown, compared with the control group, TGF-β stimulation significantly increased the expression of fibronectin and type I collagen in LX-2 cells. The expression of fibronectin and type I collagen decreased in a concentration-dependent manner after administration of amphetamine hydrochloride, which was more effective than the triptolide group, suggesting that amphetamine hydrochloride inhibits the expression of fibronectin and type I collagen in hepatic stellate cells.

Claims

1. The use of amphetamine hydrochloride in the preparation of a drug for the prevention and treatment of liver fibrosis, wherein the molecular structure of amphetamine hydrochloride is shown in Formula I: ; Its features are, The liver fibrosis mentioned refers to liver fibrosis caused by drugs and chemical reagents and / or cholestasis. The dosage form of the drug is one or more of the following: injection solution, lyophilized powder injection.

2. The application according to claim 1, characterized in that, The liver fibrosis mentioned refers to liver fibrosis caused by excessive activation of hepatic stellate cells.

3. The application according to claim 1, characterized in that, The liver fibrosis mentioned refers to liver fibrosis caused by excessive synthesis of fibronectin and type I collagen.

4. In the application according to claim 1, the liver fibrosis caused by cholestasis refers to liver fibrosis caused by abnormal bile excretion due to bile duct damage.

5. In the application according to claim 1, the liver fibrosis caused by the drug and chemical reagent refers to liver fibrosis caused by carbon tetrachloride.

6. The application according to claim 1, characterized in that, The aforementioned amphetamine hydrochloride prevents liver fibrosis by inhibiting the activation of hepatic stellate cells, the core regulatory cells of liver fibrosis, and reducing the synthesis of fibronectin and type I collagen.

7. The application according to claim 1, characterized in that, The drug is made from acetaminophen hydrochloride and pharmaceutically acceptable excipients.

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