WO2018053954A1 - Use of niclosamide ethanolamine salt in preparing medicine for type 2 diabetes - Google Patents

Abstract

Provided is a use of niclosamide ethanolamine salt in preparing medicine for preventing and treating type 2 diabetes. The niclosamide ethanolamine salt can reduce urinary albumin excretion rate of a type 2 diabetic db/db mice, improve pathological injury of glomeruli and renal tubular, and also has a protective effect on the liver.

Description

Application of niclosamide ethanolamine salt in prevention and treatment of type 2 diabetic nephropathy Technical field

The present invention relates to a novel application of Niclosamide ethanolamine salt (NEN) and, more particularly, to the use of NEN for the preparation of a medicament for the prevention and treatment of type 2 diabetic nephropathy.

Background technique

Diabetic nephropathy (DN) is one of the most important microvascular complications of diabetes. With the increase of diabetes incidence, DN has surpassed glomerular nephritis-related chronic kidney disease (CKD), becoming the primary of CKD. Cause. DN is insidious, and once it enters a large amount of proteinuria, it will progress to end stage renal disease (ESRD). The main clinical manifestations of early DN are glomerular hyperfiltration and microalbuminuria. Kidney hypertrophy is the main pathological feature of this stage, mainly characterized by glomerular hypertrophy, glomerular and tubular basement membrane thickening and mesangial area. Extracellular matrix deposition. The dysfunction of the mTOR/4E-BP1 signaling pathway plays an important role in the progression of DN.

Niclosamide is an anti-insecticide recommended by WHO. Because the drug is difficult to dissolve in water, it is made into the form of ethanolamine salt. In recent years, studies have shown that these drugs have obvious anti-tumor effects. However, NEN has not been reported on the efficacy of DN.

Summary of the invention

The present invention is based on the study of NEN for type 2 DN, and found that NEN has a strong preventive effect on type 2 DN. The object of the present invention is to provide a use of NEN in the preparation of a medicament for preventing and treating type 2 DN.

In order to achieve the above object, the present invention provides the following technical solutions:

1. To study the effect of NEN treatment on urinary albumin excretion rate and creatinine clearance rate in db/db mice;

2. To study the effects of NEN treatment on physiological metabolic indexes and pancreas in db/db mice;

3. To study the effect of NEN treatment on glomerular injury in db/db mice;

4. To study the effect of NEN treatment on renal tubular injury in db/db mice;

5. To study the effect of NEN treatment on mTOR/4E-BP1 signaling pathway in db/db mice;

6. To study the effect of NEN treatment on liver function in db/db mice.

The present invention has the following beneficial effects: the present invention aims to investigate the protective effect of NEN on type 2 DN and its mechanism of action. The results show that NEN can reduce the urinary albumin excretion rate of type 2 diabetes db/db mice and improve its Drink more, more urine symptoms, lower blood sugar, glycosylated hemoglobin, urine sugar levels, increase serum insulin levels, improve pancreatic pathological damage. NEN can alleviate the pathological damage of glomeruli and renal tubules, reduce the excretion of urine NAG, NGAL and TGF-β1. NEN can also inhibit the activation of mTOR/4E-BP1 signaling pathway and also play a protective role in the liver. According to the above studies, it can be concluded that NEN has a certain protective effect on type 2 DN, and its mechanism of action is related to inhibition of mTOR/4E-BP1 signaling pathway. NEN is prepared into an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form using an existing preparation process, or a tablet, a capsule, a granule, an oral solution, a patch or a coagulant. Glue medications can be used to prevent and treat type 2 diabetic nephropathy.

DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

Figure 1 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance in mice. Figure 1 (a) shows the effect of NEN on urinary albumin excretion rate in mice, and Figure 1 (b) shows the effect of NEN on small results Effect of rat creatinine clearance; wherein, compared with the wild type group, *** P <0.001; compared with db / db ^{group, # P <0.05, ### P} <0.001;

Figure 2 shows the effect of NEN on metabolic index and islet area in mice. Figure 2(a) shows the effect of NEN on fasting blood glucose in mice, and Figure 2(b) shows the effect of NEN on glycosylated hemoglobin in mice. (c) The effect of NEN on urine glucose in mice, Figure 2(d) shows the effect of NEN on serum insulin in mice, and Figure 2(e) shows the effect of NEN on serum glucagon in mice. 2 (f) Effect result NEN mouse islet area; wherein, compared with the wild type group, * P <0.05, ** P <0.01, *** P <0.001; compared with db / db group, ^# P <0.05, ^## P<0.01, ^### P<0.001;

Figure 3 shows the effect of NEN on the physiological indexes of mice. Figure 3(a) shows the effect of NEN on the 24-hour water consumption of mice, and Figure 3(b) shows the effect of NEN on the 24-hour urine volume of mice. 3(c) is the effect of NEN on the body weight of mice; among them, ***P<0.001 compared with the wild type group; compared with the db/db group, ^## P<0.01;

Figure 4 shows the effect of NEN on glomerular injury in mice. Figure 4(a) shows the effect of NEN on kidney weight in mice, and Figure 4(b) shows the effect of NEN on glomerular vasospasm in mice. Figure 4(c) shows the effect of NEN on the volume of glomerular vasospasm in mice. Figure 4(d) shows the effect of NEN on the ratio of mesangial matrix in mice. Figure 4(e) shows the effect of NEN on The effect of glomerular basement membrane thickness in mice, Fig. 4(f) shows the effect of NEN on the width of the foot process in mice, and Fig. 4(g) shows the effect of NEN on the glomerulus of mice by PAS staining and electron microscopy. ; wherein, compared with the wild type group, ** P <0.01, *** P <0.001; compared with db / db ^{group, # P <0.05, ## P} <0.01;

Figure 5 shows the effect of NEN on renal tubular injury in mice. Figure 5(a) shows the effect of NEN on urine NAG in mice, and Figure 5(b) shows the effect of NEN on urine NGAL in mice. (c) The effect of NEN on the urine TGF-β1 in mice, Figure 5(d) shows the effect of NEN on the proximal tubular area of mice, and Figure 5(e) shows the proximal renal tubules of NEN in mice. The effect of lumen area, Figure 5 (f) is the effect of NEN on the proximal tubule wall area of mice, and Figure 5 (g) is the effect of NEN on the thickness of the renal tubule basement membrane in mice, Figure 5 ( h) shows the impact results for the mouse kidney tubules NEN on PAS staining and electron microscopy; wherein, compared with the wild type group, ** P <0.01, *** P <0.001; compared with db / db group, ^{# P} <0.05 , ^## P<0.01;

Figure 6 shows the effect of NEN on the mTOR/4E-BP1 signaling pathway in mouse renal cortex. Figure 6(a) shows the effect of NEN on the mTOR/4E-BP1 signaling pathway protein by immunoblotting. Figure 6(b) shows the effect of NEN on mTOR/4E-BP1 signaling pathway protein. The effect of NEN on mouse p-mTOR (Ser2448), Figure 6(c) shows the effect of NEN on mouse p-4E-BP1 (Thr37/46), and Figure 6(d) shows NEN vs. mouse p- 4E-BP1 (Thr70) effect; among them, compared with the wild type group, *P<0.05, ***P<0.001; compared with the db/db group, ^## P<0.01, ^### P<0.001;

Figure 7 shows the effect of NEN on liver function in mice. Figure 7(a) shows the effect of NEN on serum alanine aminotransferase (ALT), and Figure 7(b) shows the effect of NEN on serum aspartate aminotransferase (AST) in mice. As a result, Fig. 7(c) shows the effect of NEN on serum total protein (TP), and Fig. 7(d) shows the effect of NEN on serum albumin (ALB) in mice; among them, compared with the wild type group, ** P < 0.01, *** P <0.001; compared with the db/db group, ^## P < 0.01.

detailed description

The embodiments of the present invention will be specifically described below with reference to the accompanying drawings.

Early DN is mainly characterized by glomerular hyperfiltration and microalbuminuria. The pathological changes are mainly glomerular, tubular basement membrane thickening and mesangial matrix increase. The pathogenesis of DN is still unclear and there is a lack of treatment. The mTOR/4E-BP1 signaling pathway plays an important role in cell growth and proliferation, and its dysregulation is closely related to cancer, diabetes, cardiovascular and neurological diseases. Recent studies have shown that over-activation of this pathway is closely related to DN progression. NEN is an ethanolamine salt form of niclosamide, which is mainly used for anti-parasitic infections. Recent studies have shown that these drugs also have obvious anti-tumor effects. However, NEN has not been reported on the efficacy of DN.

The present invention aims to observe the protective effect of NEN on DN nephropathy and its effect on mTOR/4E-BP1 related signaling pathway.

First, the experimental method

1. Animal model: 8 week old male wild type mice (wild type) and db/db mice (BKS.Cg-Dock7 ^m +/+Lepr ^db /JNju) were purchased from the Model Animal Research Center of Nanjing University. Animal research experiments are carried out in strict accordance with the guidelines and regulations of animal ethics of Guangzhou University of Traditional Chinese Medicine. The experimental animals were controlled to freely ingest and drink at a constant room temperature of 20 ± 1 ° C, 12 hours light and 12 hours dark cycle. Experimental mice were randomly assigned to the following groups (8-10 per group): normal control mice were fed conventional food (wild type mice as normal group, ie, wild type group in the chart of the present invention), db/db mice were fed Conventional food (db/db group), db/db mice were fed with NEN-added food (db/db+NEN group). NEN was purchased from Hubei Shengtian Hengchuang Biotechnology Co., Ltd., and was added to mouse food at a standard rate of 10g/kg. The above experimental treatment lasted for 12 weeks.

2. Physiological and metabolic parameters: The body weight of the mice was weighed at 12 weeks, and the blood glucose of each group was measured using a blood glucose meter (Roche, Basel, Switzerland), and urine was collected using a metabolic cage (Tenibus, Italy). . After 12 weeks of treatment, the mice were sacrificed and blood samples, pancreas and kidney tissue samples were taken. The content of glycated hemoglobin (HbA _1C ) was measured using an Ultra2 glycated hemoglobin analyzer. Measurement of biochemical indicators of urine and blood using Roche's automatic biochemical analyzer, including urinary creatinine, glucose, NAG (urinary N-acetyl-β-glucosidase), serum creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) ), total protein (TP), albumin (ALB). Creatinine clearance (Ccr) was calculated by urine creatinine x urine volume x 1000 / serum creatinine / 1440 and expressed in microliters per minute.

3. Tissue preparation: Immediately after the mice were sacrificed, the pancreas was removed and fixed with 10% formalin. The kidneys were then removed, weighed, rinsed in phosphate buffer, and a certain amount of kidney tissue was cut along the longitudinal section and fixed with 10% formalin. One cubic millimeter of renal cortical tissue was fixed in 2.5% glutaraldehyde solution, then treated with 1% citric acid, and analyzed under an electron microscope (Electron Microscope, EM). Each group was randomly selected from 3 samples, each sample. Randomized 8-10 electron micrographs were used to measure the glomerular basement membrane, renal tubular basement membrane, and foot process width. The length of the scale in the figure was 200 nm. The remaining kidney tissue was immediately frozen in liquid nitrogen and stored at -80 °C for subsequent experimental studies.

4, light pathology: pancreatic paraffin section (3μm thick) and kidney paraffin section (2μm thick) HE staining and PAS staining were used to evaluate the pathological damage of islets and glomeruli. Areas of islet area and glomerular vasospasm were counted using the 4.10 version of NIS-Elements image processing software (Nikon, Japan) for image processing analysis. Each pancreatic tissue section measures 5-10 islet area, and each kidney tissue section is measured. 40-50 glomerular tuft area (GTA), 30 glomerular mesangial matrix area, 40-50 renal tubules and tubular lumen area (length to short axis ratio less than 1.5). The glomerular tuft volume (GTV) is measured by the method invented by Weibel. This measurement requires only the average of the random cross-sectional area of the glomerulus and is calculated according to the following formula: V _G =Area ^1.5 ×β/K, where _VG is the glomerular volume, β=1.38, and K (distribution coefficient) is set to 1.10. The tubular wall area is equal to the area of the renal tubule minus the area of the tubular lumen. The scale length in the illustration is 25 μm.

5, ELISA: ELISA test according to the manufacturer's instructions, serum insulin (Merck, Germany), glucagon (American R & Dsystem), urinary albumin (Bethyl, USA), urine TGF-β1 (Daktronic, Shenzhen, China) ) and the content of urine NGAL (American R&Dsystem).

6. Western blotting: The frozen renal cortex tissue was homogenized in lysis buffer, the total protein concentration was balanced, and then separated by SDS-PAGE gel electrophoresis, and the protein was transferred to the PVDF membrane to PVDF. The membrane was placed in TBS buffer containing 0.5 g/l skim milk powder, and reacted at room temperature for 1 hour to block a non-specific site on the membrane, then a primary antibody was added, and the mixture was incubated at 4 ° C overnight. After washing, the imaging system using the ChemiDoc ^TM MP (Bio-Rad, USA) protein bands were detected and analyzed, the results of using β-actin as an internal control for comparison. p-mTOR (Ser2448), p-4E-BP1 (Thr37/46), and p-4E-BP1 (Thr70) antibodies were purchased from CST Corporation of the United States; β-actin antibody was purchased from Sigma USA.

7, statistical analysis

Measurement data are expressed as mean ± standard deviation. Statistical differences between the two groups of samples were analyzed using independent sample t-test. Comparisons between groups of samples were performed using one-way ANOVA, and statistical analysis was performed using SPSS 16.0 statistical software. A statistically significant difference was considered when P < 0.05.

Second, the results

1. NEN can reduce the excretion rate of urinary albuminuria in db/db mice, and has no significant effect on the creatinine clearance rate of mice.

Figure 1 shows the effect of NEN on urinary albumin excretion rate and creatinine clearance in mice. Figure 1 (a) shows the effect of NEN on urinary albumin excretion rate in mice (n=6-8 per group). Figure 1(b) shows the effect of NEN on creatinine clearance in mice (n=6-8 per group); compared with the wild type group, urinary albumin in the db/db group increased significantly and gradually increased. At 12 weeks, the creatinine clearance of db/db mice was significantly increased. After 8 weeks of NEN intervention, the urinary albumin excretion rate of the db/db+NEN group was significantly reduced, and the effect was maintained until 12 weeks. However, after 12 weeks of intervention, there was no significant change in creatinine clearance.

2, NEN can reduce fasting blood glucose, glycosylated hemoglobin, urine sugar in db/db mice, increase serum insulin levels, and improve pancreatic pathological damage.

Figure 2 shows the effect of NEN on the metabolic index and islet area of mice at 12 weeks. Figure 2 (a) shows the effect of NEN on fasting blood glucose in mice (n=6-8 per group), and Figure 2(b) shows The effect of NEN on glycosylated hemoglobin in mice (n=6-8 per group), Figure 2(c) shows the effect of NEN on urine glucose in mice (n=6-8 per group), Figure 2(d) is The effect of NEN on serum insulin in mice (n=6-8 per group), Figure 2(e) shows the effect of NEN on serum glucagon in mice (n=5 per group), Figure 2(f) The effect of NEN on the islet area of mice (n=4 per group), at 12 weeks, with wild type Compared with the mice in the group, the blood glucose, glycated hemoglobin and urine glucose levels of the db/db group increased significantly (Fig. 2a, b, c), serum insulin and glucagon levels were significantly increased (Fig. 2d, e), islet area Significantly larger (Fig. 2f); compared with the db/db group, blood glucose, glycated hemoglobin, and urinary glucose levels were significantly lower in the db/db+NEN group (Fig. 2a, b, c), and serum insulin was significantly elevated (Fig. 2d). Glucagon was slightly elevated, but it was not statistically significant (Fig. 2e), and the islet area was significantly smaller (Fig. 2f).

3, NEN can improve the symptoms of polydipsia and polyuria in db/db mice.

Figure 3 shows the effect of NEN on the physiological indexes of mice at 12 weeks, Figure 3(a) shows the effect of NEN on the 24-hour water consumption of mice, and Figure 3(b) shows the effect of NEN on the 24-hour urine volume of mice. As a result, Fig. 3(c) shows the effect of NEN on the body weight of mice; n=6-8 per group, at 12 weeks, compared with the wild type group, the db/db group showed polydipsia, polyuria and body weight. Symptoms were increased (Fig. 3a, b, c); compared with the db/db group, the db/db+NEN group showed significant improvement in polydipsia and polyuria (Fig. 3a, b).

4, NEN can improve glomerular injury in db/db mice

Figure 4 shows the effect of NEN on glomerular injury in db/db mice. Figure 4(a) shows the effect of NEN on kidney weight in mice (n=6-8 per group), and Figure 4(b) shows NEN. Results of glomerular vasospasm in mice (n=6 per group), Figure 4(c) shows the effect of NEN on glomerular vasospasm volume in mice (n=6 per group), Figure 4 ( d) the effect of NEN on the ratio of glomerular mesangial matrix in mice (n=6 per group), and Fig. 4(e) shows the effect of NEN on the thickness of glomerular basement membrane in mice (n=3 per group) Fig. 4(f) shows the effect of NEN on the width of the foot process in mice (n=3 per group), and Fig. 4(g) shows the PAS staining picture (the scale length is 25 μm in the figure) and the SEM image (illustration) The length of the middle scale is 200 nm), which shows the effect of NEN on mouse glomeruli; at 12 weeks, the kidney weight of db/db mice increases, the area and volume of glomerular vasospasm increase, and the proportion of mesangial matrix increases. Large, glomerular basement membrane thickening, wide foot mutation. These changes were improved after 12 weeks of NEN intervention. The PAS staining image (the scale length of the scale is 25 μm) and the SEM image (the scale length of the scale is 200 nm) show the characteristics of each group.

5, NEN can improve renal tubular damage in db / db mice

Figure 5 shows the effect of NEN on renal tubular injury in db/db mice. Figure 5(a) shows the effect of NEN on urine NAG in mice (n=6-8 per group), and Figure 5(b) shows NEN. Results of urine NGAL in mice (n=6-8 per group), Figure 5(c) shows the effect of NEN on urine TGF-β1 in mice (n=6-8 per group), Figure 5 ( d) the effect of NEN on the proximal tubule area of mice (n=6 per Group 5, Figure 5(e) shows the effect of NEN on the lumen area of the proximal renal tubules in mice (n=6 per group), and Figure 5(f) shows the effect of NEN on the proximal tubule wall area in mice. Results (n=6 per group), Fig. 5(g) shows the effect of NEN on the thickness of the renal tubular basement membrane in mice (n=3 per group), and Fig. 5(h) is the PAS staining picture (the scale length in the illustration) The results of NEN on mouse renal tubules were shown by 25 μm and electron microscopy images (200 nm in the figure). At 12 weeks, NAG, NGAL, and TGF-β1 excretion were significantly higher in the db/db group than in the wild type group. Increased, proximal tubules, lumen and wall area increased, renal tubular basement membrane thickened significantly. NEN intervention can significantly reduce NAG, NGAL, TGF-β1 excretion after 12 weeks of intervention, reduce renal tubular, lumen and wall area, and reduce basement membrane thickness. The PAS stained image (the scale length of the scale is 25 μm) and the SEM image (the scale length of the scale is 200 nm) show the characteristics of each group.

6. NEN inhibits the activation of mTOR/4E-BP1 signaling pathway in renal cortex of db/db mice

Figure 6 shows the effect of NEN on the mTOR/4E-BP1 signaling pathway in mouse renal cortex. Figure 6(a) shows the effect of NEN on the mTOR/4E-BP1 signaling pathway protein by immunoblotting. Figure 6(b) shows the effect of NEN on mTOR/4E-BP1 signaling pathway protein. The effect of NEN on p-mTOR (Ser2448) (n=4 per group), Figure 6(c) shows the effect of NEN on p-4E-BP1 (Thr37/46) (n=4 per group), Figure 6 (d) The effect of NEN on p-4E-BP1 (Thr70) (n=4 per group); at 12 weeks, db/db mouse renal cortex p-mTOR (Ser2448), p-4E-BP1 (Thr37) /46), p-4E-BP1 (Thr70) content increased significantly, NEN intervention can significantly reduce the expression levels of these proteins.

7. NEN intervention in db/db mice showed no hepatotoxicity after 12 weeks

Figure 7 shows the effect of NEN on liver function in mice. Figure 7(a) shows the effect of NEN on serum ALT (n=6-8 per group), and Figure 7(b) shows the effect of NEN on serum AST in mice. Results (n=6-8 per group); Figure 7(c) shows the effect of NEN on serum TP (n=6-8 per group), and Figure 7(d) shows the effect of NEN on serum ALB in mice ( n=6-8 per group); at 12 weeks, compared with the wild type group, blood biochemistry of db/db mice showed a significant increase in ALT, TP, and ALB, and AST changes were not obvious, compared with the db/db group, db/ After 12 weeks of NEN intervention in the db+NEN group, serum ALT and AST decreased significantly, and serum TP and ALB did not change significantly.

The above experimental results show that NEN can reduce urinary albumin excretion rate in type 2 diabetes db/db mice, improve polydipsia, polyuria symptoms, and reduce fasting blood glucose, glycated hemoglobin and urine sugar in db/db mice. Level, increase serum insulin levels, reduce pathological damage to the pancreas. NEN can also improve the pathological damage of glomeruli and renal tubules, reduce the excretion of urine NAG, NGAL and TGF-β1, inhibit the activation of mTOR/4E-BP1 signaling pathway, and also have a certain protective effect on the liver. In summary, NEN has a certain protective effect on type 2 DN, and its mechanism of action is related to the inhibition of mTOR/4E-BP1 signaling pathway.

The embodiments of the present invention have been described above with reference to the drawings, but the present invention is not limited to the specific embodiments described above, and the specific embodiments described above are merely illustrative and not restrictive, and those skilled in the art In the light of the present invention, many forms may be made without departing from the spirit and scope of the invention as claimed.

Claims (3)

The application of niclosamide ethanolamine salt in the preparation of a medicament for preventing and treating type 2 diabetic nephropathy. The use according to claim 1, wherein the drug is an oral administration form, an injection administration form, a mucosal administration form or a transdermal administration form. The use according to claim 1, wherein the drug is a tablet, a capsule, a granule, an oral solution, a patch or a gel.

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