Application of acemetacin in preparation of medicine for preventing and treating diabetes and hyperlipemia

Abstract

The invention belongs to the field of new application of known medicines, and relates to application of acemetacin in preparation of medicines for preventing and treating diabetes and hyperlipidemia. The acemetacin and a medicinal carrier can form a medicinal composition. The acemetacin or the pharmaceutical composition thereof can be used independently or combined with other medicines, and has the advantages of remarkable curative effect and safety in use in the aspect of treating diabetes and complications thereof as well as hyperlipidemia.

Description

Technical Field

[0001] The invention belongs to the field of new uses of known drugs and relates to the use of acemetacin in preparing drugs for preventing and treating diabetes and hyperlipidemia. Background Art

[0002] Diabetes is a common metabolic disorder of the endocrine system, mainly due to the disorder of sugar metabolism in the patient's body, which leads to the increase of blood sugar level and glycosylated hemoglobin in the blood. In recent years, the number of diabetes patients has increased significantly worldwide, and the number of patients with type 2 diabetes accounts for more than 90% of the total number of diabetic people. Once diabetes is diagnosed and not treated in time, it is very easy to cause various complications, such as diabetic nephropathy, diabetic cardiomyopathy, diabetic foot and hyperlipidemia, which seriously threaten the physical and mental health of patients and even cause death. Among them, diabetic cardiomyopathy and diabetic nephropathy are closely related to heart failure and high mortality. Diabetic cardiomyopathy is a cardiovascular complication of diabetes independent of other heart diseases; diabetic nephropathy is a renal microvascular lesion caused by poor blood sugar control. The deterioration of the course of both diseases is inseparable from the changes in the internal environment of the body induced by hyperlipidemia and hyperglycemia. Therefore, effective treatment of hyperlipidemia is an important measure to prevent and treat cardiovascular and cerebrovascular complications of diabetes.

[0003] Hyperlipidemia is a common indirect complication of diabetes, characterized by an increase in TG / TC (triglycerides / total cholesterol) and low-density lipoprotein cholesterol (LDL-C) in plasma, usually accompanied by a decrease in high-density lipoprotein cholesterol (HDL-C). With the changes in people's lifestyles and eating habits, the incidence of hyperlipidemia is increasing worldwide, and it can directly cause diseases that endanger human health, such as cerebral infarction, atherosclerosis, hypertension, coronary heart disease, myocardial infarction and other cardiovascular and cerebrovascular diseases.

[0004] Diabetic patients are often accompanied by lipid metabolism disorders, so patients need to take lipid-lowering drugs at the same time. Hypoglycemic and lipid-lowering drugs often have potential drug interactions, and long-term use alone or in combination can induce many adverse reactions. Therefore, it is urgent to develop drugs that have both hypoglycemic and hypoglycemic effects. Acemetacin (ACM) is a widely used non-steroidal anti-inflammatory analgesic that exerts anti-inflammatory and analgesic effects by inhibiting the release of hyaluronidase and inflammatory mediators. The absorption half-life of ACM is 0.86h, the elimination half-life is 2.62h, and the peak blood concentration is 1.49μg / ml. So far, there have been no reports on the research effects of ACM on the prevention and treatment of diabetes and its complications and hyperlipidemia. Summary of the invention

[0005] The technical problem to be solved by the present invention is to address the lack of drugs that can prevent and treat diabetes and hyperlipidemia. The present invention provides a new use field of known drugs and relates to the use of acemetacin in the preparation of drugs for preventing and treating diabetes and hyperlipidemia and complications of diabetes and hyperlipidemia.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the above technical problems is:

[0007] The invention discloses an application of acemetacin in preparing a medicine for preventing and treating diabetes and hyperlipidemia.

[0008] The application of acemetacin in the preparation of drugs for preventing and treating type I diabetes and / or non-insulin dependent type II diabetes.

[0009] The application of acemetacin in preparing medicine for preventing and treating diabetic complications.

[0010] The application of acemetacin in preparing medicine for preventing and treating hyperlipidemia.

[0011] The diabetes mellitus is a common disease of the endocrine system, which is mainly caused by the disorder of sugar metabolism in the patient's body, resulting in the increase of blood sugar and glycosylated hemoglobin in the blood.

[0012] The diabetic complications include diabetic complications mainly including hyperlipidemia, diabetic nephropathy, diabetic cardiomyopathy and diabetic foot, which may threaten the patient's physical and mental health and even cause the patient's death in severe cases.

[0013] The hyperlipidemia is characterized by elevated blood lipids, and long-term development may lead to atherosclerosis, which in turn causes ischemia of organs such as the heart, brain, and kidneys.

[0014] The system containing acemetacin is used as an active ingredient and mixed with a pharmaceutical carrier to prepare a corresponding pharmaceutical dosage form.

[0015] The dosage form contains 1-99% (mass) active ingredient and 1-99% (mass) pharmaceutical carrier, preferably 10-90% (mass) acemetacin and 10-90% (mass) pharmaceutical carrier.

[0016] The dosage form is an aqueous solution, a non-aqueous solution, a suspension, a tablet, a capsule, a granule or an infusion.

[0017] The dosage of acemetacin used in treatment is determined according to the age and condition of the patient. The commonly used daily dosage is 10-500 mg, the optimal daily dosage is 50-200 mg, and the administration frequency is once or several times a day.

[0018] The advantages of the present invention are:

[0019] The present invention constructs in vitro and in vivo hyperglycemia and hyperlipidemia models to investigate the use of the test substance acemetacin in the prevention and treatment of diabetes and its complications and hyperlipidemia and its complications. The experimental results show that acemetacin has both hypoglycemic and lipid-lowering effects, is highly safe, has a broad spectrum of biological activities, and can prevent or treat the following diseases: diabetes and hyperlipidemia and complications of diabetes and hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a study on the effect of ACM in inhibiting insulin resistance in vitro, where Figure A is the determination of glucose consumption in HepG2 cells after intervention with different concentrations of ACM; Figure B is the determination of glucose uptake capacity in HepG2 cells after intervention with different concentrations of ACM; Figure C is a quantitative graph of glucose uptake capacity in HepG2 cells after intervention with different concentrations of ACM; Figure D is the determination of glycogen content in HepG2 cells after intervention with different concentrations of ACM.

[0021] Figure 2 These are the results of pathological examination of ACM on type 2 diabetic mice with diabetic cardiomyopathy and diabetic nephropathy, among which, A is the result of H&E and Masson staining of cross-section of heart tissue of type 2 diabetic mice; B is the result of H&E and Masson staining of longitudinal section of heart tissue of type 2 diabetic mice; C is the result of H&E and Masson staining of cross-section of kidney tissue of type 2 diabetic mice. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0023] In the following examples, the ACM used was purchased from Bidex Pharmaceuticals, with a CAS number of 53164-05-9. Example 1: Study on the effect of ACM on inhibiting insulin resistance in vitro

[0024] 1. Model preparation and drug administration:

[0025] HepG2 cells were cultured in high-glucose (4500 mg / L) DMEM medium containing 10% serum + 1% double antibody, placed in an incubator at 37°C, 5% CO2, and 95% relative saturated humidity for routine culture and passage, and cells in the logarithmic growth phase were taken for subsequent experiments.

[0026] The insulin resistance model of HepG2 cells was established. The glucose concentration was determined by the glucose oxidase method, and the success of the model was determined by calculating the glucose consumption rate. The specific operation was as follows: After digesting the cells in the logarithmic growth phase, about 1×10 5The cells were inoculated in a 96-well culture plate. After the cells were cultured to 70%-80% fusion, they were intervened with 0.2μmol / L, 0.25μmol / L and 0.3μmol / L palmitic acid, respectively. There were 6 sub-wells for each concentration of palmitic acid. After 24 hours of intervention, the glucose concentration was determined by the glucose oxidase method. By calculating the glucose consumption rate, the condition for successful modeling, i.e., palmitic acid with a concentration of 0.3μmol / L, was selected for subsequent experiments.

[0027] The experiment was divided into 5 groups, namely, control group (addition of equal amount of solvent), model group (addition of 0.3 μmol / L palmitic acid), low concentration ACM group (addition of 50 μmol / L ACM + 0.3 μmol / L palmitic acid), high concentration ACM group (addition of 100 μmol / L ACM + 0.3 μmol / L palmitic acid) and positive drug group (addition of 5 μmol / L RSG + 0.3 μmol / L palmitic acid), with 6 replicate wells in each group.

[0028] 2. Determination of glucose consumption in HepG2 cells:

[0029] 2×10 6 HepG2 cells were grown and grouped according to step 1 above after the cells adhered to the wall. The culture medium was replaced with serum-free high-glucose DMEM containing palmitic acid and different drugs (except the control group) and cultured for 12 hours. After 12 hours, the supernatant of each group of culture medium was collected into different 1mL EP tubes and labeled according to the group. The culture medium was centrifuged at 1000r / min for 10 minutes at room temperature, and the glucose content in the culture medium was calculated according to the operation of the kit. Glucose content (mmol / L) = (A determination-A blank) / (A control-A blank) × C standard × sample dilution multiple, the results are shown in Figure 1 A.

[0030] 3. Determination of glucose uptake capacity of HepG2 cells:

[0031] 2×10 6HepG2 cells were added to each well. After the cells adhered to the wall, they were grouped according to the above step 1. The culture medium was replaced with serum-free high-glucose DMEM containing palmitic acid and different drugs (except the control group) and continued to be cultured for 23 hours. After 23 hours, each group was added with a final concentration of 150 μmol / L glucose fluorescent probe 2-NBDG and continued to be cultured for 1 hour. After 1 hour, the culture medium was discarded and washed twice with PBS. 80 μl of lysis solution was added to each well. After low-temperature lysis for 5 minutes, the cells were scraped and collected in a 1mL EP tube and centrifuged at 4℃12000r / min for 10 minutes. The supernatant was transferred to a black ELISA plate and the fluorescence intensity was measured with an ELISA instrument at Ex / EM=475nm / 550nm (excitation wavelength of 475nm and emission wavelength of 550nm). At the same time, a blank group was set before measuring the absorbance value. The treatment method was to add an equal amount of PBS to a blank well of the black ELISA plate. Relative glucose uptake (mmol / L) = (A assay - A blank) / (A control - A blank) × C standard × sample dilution factor. For results, see Figure 1 B. Figure 1 C.

[0032] 4. Determination of glycogen content in HepG2 cells:

[0033] 2×10 6 HepG2 cells were grown, and after the cells adhered, they were grouped according to step 1 above, and the culture medium was replaced with serum-free high-glucose DMEM containing palmitic acid and different drugs (except the control group) and cultured for 12 hours. After 12 hours, each group of cells was washed three times with pre-cooled PBS, and 80 μl of lysis solution was added to each well. After low-temperature lysis for 5 minutes, the cells were scraped and collected in a 1 mL EP tube, one part of which was used for protein quantification and one part for glycogen determination. The cell lysate used for glycogen determination was first placed in an 80°C water bath for 1 hour, and after cooling to room temperature, 2.5 times the volume of anhydrous ethanol was added. The mixture was centrifuged at 12000 r / min for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The glycogen content in the sample was determined according to the method in the kit instructions. Relative glycogen content (%) = (glycogen content in the experimental group / protein concentration in the experimental group) / (glycogen content in the control group / protein concentration in the control group) × 100%, see for results. Figure 1 D.

[0034] 5. Evaluation results of ACM's inhibitory effect on insulin resistance in vitro:

[0035] Figure 1 AThe results showed that compared with the control group, the glucose content in the culture medium of the model group was increased; compared with the model group, the glucose content in the culture medium was decreased after low and high concentration ACM intervention.

[0036] Figure 1 B. Figure 1C The results showed that compared with the control group, the intracellular glucose uptake capacity of the model group was reduced; compared with the model group, the intracellular glucose uptake capacity increased after low and high concentration ACM intervention.

[0037] Figure 1 D The results showed that compared with the control group, the intracellular glycogen synthesis capacity of the model group decreased; compared with the model group, the intracellular glycogen synthesis capacity was enhanced after low and high concentration ACM intervention.

[0038] 6. Statistical methods:

[0039] SPSS software was used for statistical analysis, and the data were expressed as mean ± standard deviation. The comparison among multiple groups was performed by one-way ANOVA followed by Scheffe test. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0040] Example 2: Study on the hypoglycemic and hypolipidemic effects of ACM in vivo

[0041] 1. Preparation and administration of hyperglycemia and hyperlipidemia mouse model:

[0042] After 7 days of adaptive feeding of 60 SD male mice, 10 mice continued to be fed with ordinary feed and served as the control group, and the other mice were fed with high-fat feed (high-fat feed is to adjust the ingredients of ordinary feed so that the system contains 20wt% protein, 20wt% carbohydrates and 60wt% fat). After 4 weeks, the weight of mice increased significantly. In the 5th week, the control group mice were intraperitoneally injected with the following equal dose of citric acid buffer. At the same time, the other mice except the control group were intraperitoneally injected with 40mg / kg body weight of streptozotocin (STZ) for three consecutive days to induce hyperglycemia and hyperlipidemia model. After 1 week, the fasting blood glucose of all mice was measured. First, the mice were fasted for 12h, then the tails were cut and blood was collected, and the fasting blood glucose level was detected using a blood glucose meter. Mice with fasting blood glucose greater than 11.1mmol / L were selected from the high-fat feeding group, which were hyperglycemia and hyperlipidemia mice. The screened hyperglycemic and hyperlipidemic mice were divided into a model group (corn oil gavage, n = 10), a low-dose ACM group (corn oil + 2.5 mg / kg ACM gavage, n = 10), a high-dose ACM group (corn oil + 5 mg / kg ACM gavage, n = 10) and a positive drug group (corn oil + 10 mg / kg RSG gavage, n = 10) according to their blood sugar levels. Among them, the control group mice were gavaged with corn oil every day and fed with ordinary feed, and the model group and the drug group were fed with the above-mentioned high-fat feed. After 4 weeks of drug administration, the mice were fasted for 12 hours, and then the tails were cut to collect blood, and the fasting blood sugar level was tested using a blood glucose meter; then the orbital blood was collected, and the blood samples were collected and centrifuged for analysis of the corresponding indicators in the serum (see Table 1-3).

[0043] 2. Related blood index detection:

[0044] After oral administration of ACM and high-fat diet for 4 weeks, fasting but not water was performed for 12 hours. After 12 hours, blood glucose (GLU) was measured by blood sampling from the tail vein using a blood glucose meter. Blood was collected by orbital blood sampling and centrifuged for 10 minutes (3500r / min) to obtain serum, one part of which was stored at -80°C for future use, and the other part was tested for total cholesterol (TC), triglycerides (TG), free fatty acids (FFA), creatinine (CRE), and blood urea nitrogen (BUN). All indicators were operated according to the instructions of the kit.

[0045] 3. Evaluation results of ACM's hypoglycemic and hypolipidemic effects in vivo:

[0046] Table 1

[0047]

[0048] Table 2

[0049]

[0050] Table 3

[0051]

[0052] After being fed a high-fat diet and injected with STZ, the fasting blood glucose of mice was greater than 11.1mmol / L, and the blood TC, TG, and FFA indicators increased significantly, verifying that the hyperglycemic and hyperlipidemia mouse model was successfully established. The experimental results are shown in Table 1-3:

[0053] Table 1 shows the detection of total cholesterol (TC) and triglyceride (TG) levels after oral administration of ACM. Compared with the model group, the TC and TG levels in the serum of mice in the ACM and positive drug groups were significantly reduced (P < 0.05).

[0054] Table 2 shows the detection of glucose (Glu) and free fatty acid (FFA) levels after oral administration of ACM. Compared with the model group, the Glu and FFA levels in the serum of mice in the ACM and positive drug groups were significantly reduced (P < 0.05).

[0055] Table 3 shows the detection of serum creatinine (CRE) and urea nitrogen (BUN) levels after oral administration of ACM. Compared with the model group, the CRE and BUN levels in the serum of mice in the ACM and positive drug groups were significantly reduced (P < 0.05).

[0056] In summary, ACM can improve the blood glucose and blood lipid levels of hyperglycemic and hyperlipidemic mice.

[0057] 4. Pathological testing:

[0058] After 28 days of continuous administration, the mice were killed by cervical dislocation, and the heart and kidney tissues of the mice were stained with HE and MASSON (see Figure 2 ).

[0059] Figure 2 A (cross section) and 2B (longitudinal section) are the results of HE and MASSON staining of the hearts of mice with hyperglycemia and hyperlipidemia. The results of the HE staining experiment showed that the structure of the heart tissue of the animals in the blank control group was clear, and no fibrosis was observed. The heart tissue sections of the mice in the model group were severely fibrotic, as shown by the filling of myocardial fibrous connective tissue, accompanied by inflammatory cell infiltration, and a large number of fibroblasts and collagen tissue deposition. The heart tissue structure of the mice in the ACM-treated group was relatively complete compared with the model group, with no obvious inflammatory cells, a small number of fibrous foci, and a significantly reduced degree of fibrosis. The results of Masson staining showed that there was a small amount of collagen distributed in the heart tissue of the animals in the blank control group, and a significant increase in blue collagen fibers was observed in the model group. Compared with the model group, the collagen fiber deposition in the mice in the ACM-treated group was significantly reduced. The above pathological tissue section results show that ACM has a significant anti-diabetic cardiomyopathy effect in vivo.

[0060] Figure 2C shows the results of HE and MASSON staining of the kidneys of mice with hyperglycemia and hyperlipidemia. The results of HE staining experiments showed that the renal tissue structure of the animals in the blank control group was clear and no fibrosis was observed. The renal tissue sections of the mice in the model group were severely fibrotic, as shown by the destruction or disappearance of the tubular structure, the filling of the tubular interstitial fibrous connective tissue, and the infiltration of inflammatory cells; the tubular interstitium was significantly edematous and widened, and a large number of fibroblasts and collagen tissue were deposited. The renal tissue structure of the mice in the ACM administration group was relatively complete compared with the model group, no obvious inflammatory cells were found in the tubular interstitium, there were a small number of fibrous foci, and the degree of fibrosis was significantly reduced. The results of Masson staining showed that a small amount of collagen was distributed in the renal tissue of the animals in the blank control group. A significant increase in blue collagen fibers was observed in the tubular interstitium of the model group. Compared with the model group, the collagen fiber deposition in the mice in the ACM administration group was significantly reduced. The above pathological tissue section results show that ACM has a significant anti-diabetic nephropathy effect in vivo.

[0061] Based on the results of pathological tissue sections, ACM can alleviate kidney and heart damage induced by hyperglycemia and hyperlipidemia, and delay the progression of complications of diabetes and hyperlipidemia.

Claims

1. A use of acemetacin in preparing a drug for preventing and treating diabetes and hyperlipidemia.

2. The use according to claim 1, characterized in that: The application of acemetacin in the preparation of drugs for preventing and treating type I diabetes and / or non-insulin dependent type II diabetes.

3. The use according to claim 1, characterized in that: The acemetacin is used in the preparation of drugs for preventing and treating complications induced by diabetes such as diabetic nephropathy, diabetic cardiomyopathy, diabetic foot and hyperlipidemia.

4. The use according to claim 1, characterized in that: The acemetacin is used in the preparation of drugs for preventing and treating hyperlipidemia and complications such as coronary atherosclerosis, cerebral infarction, hypertension, myocardial infarction, etc. induced by hyperlipidemia.

5. The use according to any one of claims 1 to 4, characterized in that: The system containing acemetacin is used as an active ingredient and mixed with a pharmaceutical carrier to prepare a corresponding pharmaceutical dosage form.

6. The use according to claim 5, characterized in that: The dosage form is an aqueous solution, a non-aqueous solution, a suspension, a tablet, a capsule, a granule or an infusion.

7. The use according to any one of claims 1 to 6, characterized in that: The daily dose of acemetacin is 10-500 mg, and the optimal daily dose is 50-200 mg.

Images