Use of 3-demethylcolchicine in medicine

Abstract

The application discloses application of 3-demethylcolchicine in medicine, and experiment proves that the 3-demethylcolchicine can obviously reduce the body weight and liver weight of NAFLD mice, can obviously reduce the liver injury and liver lipid deposition of the NAFLD mice, and can improve the liver function and abnormal glucose metabolism of the NAFLD, and has a wide application prospect in NAFLD medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of 3-demethylcolchicine in the preparation of drugs for treating NAFLD, diabetes, and for lowering lipids and reducing weight. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. In recent years, with the improvement of people's living standards and changes in lifestyle and dietary structure, the incidence of obesity and diabetes has increased, and the incidence of NAFLD has also shown an upward trend, reaching 25%-30% in the general population. The liver morphology and function of NAFLD patients are affected by many adverse reactions, which are a series of continuous pathological processes, including simple fatty liver (FLD), steatohepatitis (NASH) and its related liver fibrosis and cirrhosis, and may even develop into liver cancer (HCC). Among them, 10% to 20% of NAFLD cases can develop into non-alcoholic steatohepatitis (NASH).

[0003] NAFLD has many causes, with diabetes and obesity being the main contributing factors. Furthermore, the liver plays a crucial role in glucose and lipid metabolism; therefore, NAFLD is a risk factor for many metabolic diseases, such as type 2 diabetes mellitus (T2DM), hyperlipidemia, hyperinsulinemia, and atherosclerosis. The presence of T2DM further increases the risk of liver diseases, including NAFLD. Obesity is also closely associated with T2DM. Obesity and insulin resistance (IR) are known to be two important characteristics of T2DM and also the most significant pathogenic factors for NAFLD. In fact, 90% of patients with T2DM will develop NAFLD. When T2DM and obesity coexist, 66% of NAFLD patients will develop advanced fibrosis.

[0004] Furthermore, the pathophysiological mechanisms of NAFLD are highly complex. Besides diabetes, obesity, and insulin resistance mentioned above, it is closely related to various pathological processes such as oxidative stress, inflammation, lipotoxicity, apoptosis, and fibrosis, although the specific mechanisms are not yet fully elucidated. Currently, there are no FDA-approved drugs specifically for the treatment of NAFLD in clinical practice. While some drugs, such as weight-loss and hypoglycemic agents, can be used clinically, they often have significant side effects and are prone to causing adverse reactions. Therefore, it is essential to find a drug that can effectively treat NAFLD while simultaneously lowering blood sugar, reducing fat / weight, protecting the liver, and having minimal toxic side effects.

[0005] Colchicine, also known as colchicine extract, is a plant alkaloid originally extracted from the seeds and bulbs of the autumn crocus. It is a white or pale yellow powder or needle-like crystals. Colchicine effectively inhibits the formation of the spindle apparatus during cell division, thereby inhibiting mitosis. However, colchicine has significant side effects, including kidney and intestinal failure and neutropenia. High doses of colchicine can also damage bone marrow and cause anemia. Given the severe side effects it causes, colchicine is not suitable for cancer treatment. Currently, it is mainly used clinically to treat gout.

[0006] 3-Demethylcolchicine (3DMC), a metabolite of colchicine, possesses free radical scavenging, anti-inflammatory, and anticancer properties. A review of existing literature and patents reveals that 3DMC has significantly lower toxicity and side effects compared to colchicine, and there are no reports of 3DMC being used to treat liver damage or NAFLD. Summary of the Invention

[0007] The problem to be solved by this invention is the application of 3-demethylcolchicine in the preparation of drugs for treating NAFLD, wherein the chemical structural formula of 3-demethylcolchicine is:

[0008]

[0009] The problem to be solved by the present invention is the application of 3-demethylcolchicine in weight loss and fat reduction drugs, wherein the chemical structural formula of 3-demethylcolchicine is (1).

[0010] The problem to be solved by the present invention is the application of 3-demethylcolchicine in the treatment of abnormal glucose metabolism, wherein the chemical structural formula of 3-demethylcolchicine is (1).

[0011] The technical solution adopted to achieve the purpose of this invention is:

[0012] The use of 3-demethylcolchicine or its pharmaceutically acceptable salt in the treatment of NAFLD, characterized in that: the chemical structural formula of the 3-demethylcolchicine is:

[0013]

[0014] The application of 3-demethylcolchicine or its pharmaceutically acceptable salt in weight loss and fat reduction drugs, characterized in that: the chemical structural formula of the 3-demethylcolchicine is (1).

[0015] The use of 3-demethylcolchicine or its pharmaceutically acceptable salt in drugs for treating abnormal glucose metabolism, characterized in that: the chemical structural formula of the 3-demethylcolchicine is (1).

[0016] In the above technical solution, the pharmaceutically usable salt is one of bromate, hydrochloride and fluoroate.

[0017] In the above technical solution, the dosage form of the drug can be any medically recognized dosage form, such as powder, injection, capsule, tablet, or oral liquid.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention provides the application of 3-demethylcolchicine in pharmaceuticals. Experiments have confirmed that 3-demethylcolchicine can significantly reduce body weight and liver weight in NAFLD mice, alleviate liver pathological damage and liver lipid deposition, improve liver function, and improve abnormal glucose metabolism in NAFLD. It has broad application prospects in NAFLD drugs. Attached Figure Description

[0020] Figure 1 The data shown is the weight measurement data of the mice;

[0021] Where, a is a graph showing the trend of mouse weight change during drug administration; b is the fasting weight of the mouse before it was killed;

[0022] Figure 2 The data shown is the fat weight data of the mice;

[0023] Where a is the weight of subcutaneous fat in mice; b is the weight of epididymal fat in mice;

[0024] Figure 3 The figure shows the liver weight of mice in each group;

[0025] Figure 4 The image shown is an HE-stained image of the right lobe of a mouse liver.

[0026] Figure 5 The image shown is an Oil Red O staining image of the right lobe of a mouse liver.

[0027] Figure 6 The figures show the levels of AST(a) and ALT(b) in mouse serum;

[0028] Figure 7 The figure shows the insulin content in mouse serum;

[0029] Figure 8 The results of the blood glucose level test in the mice are shown.

[0030] Where a is the fasting blood glucose (Fast) value of mice; b is the random postprandial blood glucose (Feed) value of mice; c is the glucose tolerance test (GTT) of mice; d is the area under the GTT curve; e is the insulin tolerance test (ITT) of mice; and f is the area under the ITT curve. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] The instrument models and manufacturers involved in the following embodiments are as follows:

[0033] Instrument Name model factory Fluorescence upright microscope Model BX53 Olympus GmbH Fully Automated Biochemical Analyzer Fully automated biochemical analyzer Model SMT100 Beijing Pulang New Technology Co., Ltd.

[0034] The sources of the drugs involved in the following examples are as follows:

[0035]

[0036]

[0037] Example 1 (3DMC administration group)

[0038] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. The model was established by inducing a 60% high-fat diet (HFD) for 8 weeks.

[0039] Over the next 8 weeks, the purchased 3DMC powder was dissolved in autoclaved physiological saline to prepare a specific concentration. Mice were then injected intraperitoneally twice daily, morning and evening, at a dose of 0.0375 mg / kg, with 0.1 mL of the 3DMC solution per 10 g of body weight. This treatment continued for 8 weeks, during which time mice were fed a fixed amount of 60% high-fat (HFD) diet.

[0040] Example 2 (3DMC administration group)

[0041] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. The model was established by inducing a 60% high-fat diet (HFD) for 8 weeks.

[0042] Over the next 8 weeks, the purchased 3DMC powder was dissolved in autoclaved physiological saline to prepare a specific concentration. Mice were then injected intraperitoneally twice daily, morning and evening, at a dose of 0.075 mg / kg, with 0.1 mL of the 3DMC solution per 10 g of body weight. This treatment continued for 8 weeks, during which time mice were fed a fixed amount of 60% high-fat (HFD) diet.

[0043] Example 3 (3DMC administration group)

[0044] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. The model was established by inducing a 60% high-fat diet (HFD) for 8 weeks.

[0045] Over the next 8 weeks, the purchased 3DMC powder was dissolved in autoclaved physiological saline to prepare a specific concentration. Mice were then injected intraperitoneally twice daily, morning and evening, at a dose of 0.15 mg / kg, with 0.1 mL of the 3DMC solution per 10 g of body weight. This treatment continued for 8 weeks, during which time mice were fed a fixed amount of 60% high-fat (HFD) diet.

[0046] Comparative Example 1 (Model Group)

[0047] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. The model was established by inducing a 60% high-fat diet (HFD) for 8 weeks.

[0048] Over the next 8 weeks, mice were intraperitoneally injected with physiological saline at a rate of 0.1 mL per 10 g body weight, twice daily, morning and evening. The administration was continued for 8 weeks, during which time mice were fed a diet equal to 60% of the high-fat (HFD) diet found in Examples 1-3.

[0049] Comparative Example 2 (Normal Group CNTR)

[0050] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. They were then fed a normal diet for eight weeks.

[0051] Over the next 8 weeks, mice were injected intraperitoneally with physiological saline at a rate of 0.1 mL per 10 g body weight, twice daily, morning and evening. During the 8-week administration period, mice were given the same amount of normal diet as in Examples 1-3.

[0052] Comparative Example 3 (Positive Group)

[0053] Eight healthy male SPF-grade C57BL / 6J mice weighing 20±2g were selected and acclimatized for one week. The model was established by inducing a 60% high-fat diet (HFD) for 8 weeks.

[0054] Over the following 8 weeks, 15 mg of pioglitazone hydrochloride tablets were dissolved in 76.923 mL of double-distilled water to prepare a pioglitazone hydrochloride aqueous solution with a concentration of 0.195 mg / mL. Mice were administered 0.1 mL of the pioglitazone hydrochloride aqueous solution per 10 g of body weight by gavage once daily. The administration was continued for 8 weeks, during which time mice were given a fixed amount of 60% high-fat diet (HFD).

[0055] Pioglitazone hydrochloride, as an insulin sensitizer, can regulate the transcription of many insulin-related genes that control glucose and lipid metabolism. It increases the post-receptor level, thereby increasing tissue sensitivity to insulin and reducing insulin resistance, thus achieving the goal of lowering blood sugar.

[0056] In the above examples and comparative examples, the weight of all mice was recorded weekly, and the dosage varied with changes in body weight.

[0057] Mice were monitored for fasting blood glucose and postprandial blood glucose levels every two weeks. At week eight, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. Eight weeks after drug administration, blood, liver tissue, subcutaneous fat, and epididymal fat were collected from each group of mice. Serum was obtained after centrifugation of the blood, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and insulin were measured to evaluate liver function in each group. Liver tissue was weighed, the right lobe was embedded, sectioned, and then stained with hematoxylin and eosin (HE) and Oil Red O to observe the degree of liver damage and steatosis in each group of mice.

[0058] The above indicators from the model group show that after 8 weeks of induction with a 60% high-fat diet (HFD), the HE staining results of the mouse liver showed severe damage to hepatocyte morphology, with obvious swelling and a large area of ​​lipid droplets; Oil Red O staining results showed significant hepatic steatosis, lipid deposition, and tissue lesions; and liver function indicators AST and ALT showed a significant increase. These changes indicate that after 8 weeks of induction with a 60% high-fat diet (HFD), the mouse liver was severely damaged, exhibiting obvious NAFLD disease manifestations.

[0059] The testing process and results are as follows:

[0060] (1) 3DMC can significantly reduce the weight of NAFLD mice.

[0061] Figure 1 Figure a shows the trend of body weight change in mice during the drug administration period. The body weight of mice in the model group (Model) was significantly higher than that in the normal group (CNTR). After 8 weeks of drug administration, the body weight of mice in each example group and the positive group decreased significantly compared with the model group, and the decrease was greater in each example group than in the positive group. Figure 1b represents the fasting body weight of the mice before sacrifice. After drug administration, the fasting body weight of mice in each example group and the positive control group before sacrifice decreased significantly compared to the model group, and the decrease was greater in each example group than in the positive control group. This indicates that both 3DMC and pioglitazone hydrochloride can reduce the body weight of mice, but 3DMC has a better weight-loss effect than pioglitazone hydrochloride.

[0062] Figure 2 Figure a shows the subcutaneous fat weight of mice. Compared with the normal group, the subcutaneous fat of the model group mice was significantly increased. After administration, the subcutaneous fat weight of mice in each example group and the positive group decreased to varying degrees. Figure 2 Figure b shows the epididymal fat weight of mice. The epididymal fat weight of the model group mice was significantly increased compared with the normal group. After the administration, the epididymal fat weight of mice in each example group decreased to varying degrees. The results indicate that 3DMC can play a significant role in reducing lipids, and its weight-reducing effect may be related to the inhibition of fat accumulation in NAFLD mice.

[0063] (2) 3DMC can significantly reduce liver weight, alleviate liver pathological damage and lipid deposition, and improve liver function in NAFLD mice.

[0064] Figure 3 The figures show the liver weight of mice in each group. The liver weight of the model group mice was significantly increased compared with the normal group. After the drug administration was completed, the liver weight of mice in each drug administration group was significantly reduced. The results demonstrate that 3DMC can significantly reduce the liver weight of NAFLD mice.

[0065] Figure 4 The images shown are HE staining images of the right lobe of the liver tissue from mice. Figure A shows the HE staining image of the right lobe of the liver tissue from the normal group; Figure B shows the HE staining image of the right lobe of the liver tissue from the model group; Figure C shows the HE staining image of the right lobe of the liver tissue from the positive group; Figure D shows the HE staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 1) for NAFLD; Figure E shows the HE staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 2) for NAFLD; and Figure F shows the HE staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 3) for NAFLD. HE staining results show that the liver tissue structure of the normal group mice was intact, with tightly packed hepatocytes and clearly stained nuclei (Figure A), while the cell morphology of the model group was severely damaged, with some cells showing significant swelling and a larger area of ​​lipid droplets (Figure B). After administration, the number of lipid droplets in the liver of the positive group was significantly reduced, and the morphology of the liver cells was significantly improved (Figure C); after administration, the number of lipid droplets in the liver of each of the example groups was significantly reduced, and the morphology of the liver cells was significantly improved, with intact cell structure (Figures D, E, and F).

[0066] Figure 5The images show Oil Red O staining images of the right lobe of the liver tissue from mice. Figure G shows the Oil Red O staining image of the right lobe of the liver tissue from the normal group of mice; Figure H shows the Oil Red O staining image of the right lobe of the liver tissue from the model group of mice; Figure I shows the Oil Red O staining image of the right lobe of the liver tissue from the positive group of mice; Figure J shows the Oil Red O staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 1) for NAFLD; Figure K shows the Oil Red O staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 2) for NAFLD; and Figure L shows the Oil Red O staining image of the right lobe of the liver tissue from the group treated with 3DMC (as described in Example 3) for NAFLD. The Oil Red O staining sections show that the hepatocytes in the normal group have normal structure, pale blue cytoplasm, and no red lipid droplets (Figure G). However, the hepatocytes in the model group show a large number of red lipid droplets of varying sizes, indicating hepatic steatosis and lipid deposition (Figure H). After treatment, fatty degeneration was significantly reduced in all example groups and the positive group, and the number of red lipid droplets in the cytoplasm was also significantly reduced (Figures I, J, K, L), with the reduction being more significant in the example group than in the positive group.

[0067] Figure 4 Figure 5 The results showed that 3DMC could reduce lipid deposition in the liver of NAFLD mice.

[0068] also, Figure 6 The figures show the serum AST(a) and ALT(b) levels in mice. Compared with the normal group, the serum AST and ALT levels in the model group were significantly increased, while the serum AST and ALT levels in each of the example groups were significantly decreased compared with the model group, indicating that 3DMC can effectively improve liver function in NAFLD mice.

[0069] (3) 3DMC can improve abnormal glucose metabolism in NAFLD.

[0070] Figure 7 The figure shows the insulin content in mouse serum. Compared with the normal group, NAFLD mice showed a significant decrease in serum insulin content, exhibiting obvious insulin resistance. After administration, the serum insulin content in the positive control group was higher than that in the model group, indicating that pioglitazone hydrochloride can reduce insulin resistance. Furthermore, after administration, the serum insulin content in all groups of the examples was significantly higher than that in the model group, especially in Examples 2 and 3, where the increase was more pronounced than in the positive control group. These results demonstrate that 3DMC can effectively reduce insulin resistance in mice, and at a certain dose, its effect on insulin resistance is more significant than that of pioglitazone hydrochloride.

[0071] Figure 8 The results of the blood glucose level test in mice are shown. Figure 8 'a' represents the fasting blood glucose (Fast) value in mice. Compared with the model group, the fasting blood glucose levels in each embodiment group were significantly lower, and there was no dose-dependent relationship. Figure 8 b represents the random postprandial blood glucose (Feed) value of mice. Compared with the model group, the random postprandial blood glucose in each embodiment group was also significantly reduced. Figure 8 c represents the mouse glucose tolerance test (GTT). Figure 8 d represents the area under the GTT curve; Figures c and d show that, compared with the model group, each embodiment group can better improve the glucose tolerance of mice. Figure 8 e represents the mouse insulin resistance (ITT) experiment. Figure 8 f represents the area under the ITT curve. Figures e and f show that, compared with the model group, each embodiment group can better improve glucose tolerance in mice. In summary, this demonstrates that 3DMC has a significant effect in reducing insulin resistance and lowering blood glucose, and can improve abnormal glucose metabolism in NAFLD mice.

[0072] In conclusion, 3DMC can improve the weight and liver weight gain and abnormal glucose and lipid metabolism associated with NAFLD mice, and has a significant effect on reducing weight and lowering blood sugar.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of 3-demethylcolchicine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of NAFLD, characterized in that: The chemical structural formula of the 3-demethylcolchicine is: 。 2. Use of 3-demethylcolchicine or a pharmaceutically acceptable salt thereof for the manufacture of a drug for weight loss and fat reduction, characterized in that: The chemical structural formula of the 3-demethylcolchicine is: 。 3. Use of 3-demethylcolchicine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of abnormal glucose metabolism, characterized in that: The chemical structural formula of the 3-demethylcolchicine is: 。 4. Use according to any one of claims 1 to 3, characterized in that: The pharmaceutically acceptable salt is bromate, hydrochloride or fluoric acid salt.

5. The application as described in any one of claims 1-3, characterized in that, The dosage form of the medicine is any one of the dosage forms recognized by medicine.

6. Use according to any one of claim 5, wherein The dosage form of the medicine is one of powder, injection, capsule, tablet and oral liquid.

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