Application of NBI-31772 in the preparation of drugs for improving diabetic muscular atrophy

Abstract

The application of NBI‑31772 in the preparation of drugs to improve diabetic muscular atrophy can effectively inhibit the occurrence of diabetic muscular atrophy and improve muscle strength and function by reducing the accumulation of IGFBP3 protein in cells. It can also improve insulin sensitivity, relieve hyperlipidemia, and inhibit fatty liver. This small molecule can be used as a clinical drug for diabetic muscular atrophy, and can also be used in combination with clinical diabetes drugs to achieve comprehensive improvement of diabetes and related complications.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and medicine, and particularly relates to the application of NBI-31772 in the preparation of a drug for improving diabetic amyotrophy. Background Art

[0002] Diabetes is a major chronic disease that is well known to everyone. Among its many complications, muscle disease, represented by muscle atrophy, is an important complication that is often overlooked in clinical practice. The accelerated decline of muscle function leads to a sharp increase in the risk of disability and death, making the treatment and rehabilitation of diabetic patients face more severe challenges. Unfortunately, the current understanding of the pathogenesis of diabetic muscle atrophy is relatively limited, and clinical treatment strategies are seriously lacking.

[0003] Early studies pointed out that the imbalance of protein synthesis and degradation in muscle cells is the key mechanism leading to muscle atrophy, and protein degradation-related E3 ubiquitin ligases such as MuRF1 and Atrogin-1 are considered to be key effector molecules, and their expression is significantly increased in patients with diabetes and obesity. Insulin resistance and increased inflammation in diabetic patients are considered to be key factors promoting muscle pathology, but this hypothesis has not been effectively supported by clinical studies. The effects of insulin sensitizers such as metformin on diabetic muscle atrophy are inconsistent in various studies. Some studies have shown that the grip strength of elderly patients improved after metformin treatment, but in another study, overweight patients with type 2 diabetes and chronic obstructive pulmonary disease had a significant decrease in grip strength after 24 weeks of metformin treatment. Glucagon-like peptide-1 (GLP-1) receptor agonist drugs such as liraglutide also have no consistent effect on the skeletal muscle mass index in patients with type 2 diabetes. Treatment with the PPARγ agonist pioglitazone did not significantly improve muscle loss in patients with type 2 diabetes. Even in individuals without type 2 diabetes, it was found that taking pioglitazone caused thigh muscle loss in men. The above studies fully indicate that the existing technology has a serious lack of understanding of the pathogenesis of diabetic muscle atrophy, and the treatment of diabetic muscle atrophy lacks a clear targeted strategy.

[0004] Sherryline Jogie-Brahim et al. pointed out in the article "Endocrine reviews, doi: 10.1210 / er.2008-0028" that IGFBP3 (insulin-like growth factor-1 binding protein 3) is a secretory protein whose main function is believed to be to bind to IGF-1 protein (insulin-like growth factor-1) in the blood circulation, protect IGF-1 from rapid degradation so that it can stably reach the target cells and activate downstream Akt and other signaling pathways by binding to IGF-1R (IGF-1 receptor) to regulate protein synthesis, cell proliferation and survival. The latest research suggests that IGFBP3 may have IGF-1-independent functions in addition to its IGF-1-dependent cell functions. In the process of diabetic muscle atrophy, a large amount of IGFBP3 protein accumulates in muscle cells, suggesting that muscle accumulation of IGFBP3 may be a key factor in promoting diabetic muscle atrophy.

[0005] NBI-31772

[0006] (1-[(3,4-dioxocyclohexa-1,5-dien-1-yl)-hydroxymethylidene]-6,7-dihydroxy-2H-isoquinoline-3-carboxylic acid) is a non-selective inhibitor of IGFBP family proteins, CAS No. 374620-70-9, but whether it can be used as a clinical drug for diabetic muscular atrophy has not been studied or reported. Summary of the invention

[0007] In order to overcome the defects of the above-mentioned prior art, the purpose of the present invention is to provide the use of NBI-31772 in the preparation of drugs for improving diabetic muscle atrophy, which can effectively inhibit the occurrence of diabetic muscle atrophy and improve muscle strength and function by reducing the accumulation of IGFBP3 protein in cells. This small molecule can be used as a clinical drug for diabetic muscle atrophy, and can also be used in combination with clinical diabetes drugs to achieve comprehensive improvement of diabetes and related complications.

[0008] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0009] The use of NBI-31772 in the preparation of a drug for improving diabetic muscular atrophy, wherein the NBI-31772 is a small molecule inhibitor that specifically inhibits the activity and functional localization of IGFBP3 protein, reduces the accumulation of IGFBP3 protein in cells, and inhibits the occurrence of diabetic muscular atrophy.

[0010] The NBI-31772 can improve the loss of muscle strength in diabetic patients.

[0011] The NBI-31772 can improve muscle strength and function in diabetic patients.

[0012] As long as the structurally modified and modified compounds based on NBI-31772 can inhibit the activity and intracellular localization of IGFBP3 protein, they can be used to prepare drugs for improving diabetic muscular atrophy.

[0013] The NBI-31772 can improve insulin sensitivity and lower blood sugar.

[0014] The NBI-31772 can improve the hyperlipidemia symptoms in diabetic patients.

[0015] The NBI-31772 can reduce the symptoms of non-alcoholic fatty liver disease in diabetic patients.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects:

[0017] 1. NBI-31772 of the present invention can effectively inhibit the occurrence of diabetic muscle atrophy and improve muscle strength and function by reducing the accumulation of IGFBP3 protein in cells. This small molecule can be used as a clinical drug for diabetic muscle atrophy, and can also be used in combination with clinical diabetes drugs to achieve comprehensive improvement of diabetes and related complications. NBI-31772 and modified compounds based on it have good application prospects.

[0018] 2. The present invention confirms for the first time that NBI-31772 specifically inhibits the activity of IGFBP3 protein and reduces the accumulation of IGFBP3 in cells; the inhibitory target of NBI-31772 is more specific and highly unique. At the same time, the small molecule has a short metabolic cycle, is easily excreted through urine, has less accumulation in the body, and has no side effects.

[0019] In summary, NBI-31772 can reduce the cellular accumulation of IGFBP3 protein, improve insulin sensitivity, lower blood lipids, improve fatty liver symptoms, inhibit muscle atrophy, and improve muscle strength and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the structural formula of NBI-31772.

[0021] Figure 2 This is a schematic diagram of NBI-31772 reducing the accumulation of IGFBP3 protein in muscle cells of diabetic mice; immunofluorescence analysis of muscle sections showed that the IGFBP3 protein content was significantly reduced after NBI-31772 treatment, and the red fluorescence represents the IGFBP3 protein.

[0022] Figure 3 This is a schematic diagram of the improvement effect of NBI-31772 on muscle atrophy in diabetic mice; Figure 3 A in the figure shows the improvement of muscle fiber structure in diabetic mice after 8 weeks of NBI-31772 treatment; Figure 3 B in the figure shows that protein degradation in muscle cells of diabetic mice was significantly reduced after NBI-31772 was applied for 8 weeks; Figure 3 Figure C shows that the fibrin content in muscle cells of diabetic mice significantly increased after 8 weeks of NBI-31772 treatment. TA represents tibialis anterior muscle, GA represents gastrocnemius muscle, and QC represents quadriceps femoris muscle.

[0023] Figure 4 This is a schematic diagram of the effect of NBI-31772 on improving muscle function in diabetic mice; Figure 4 A in the figure shows that after NBI-31772 was applied to diabetic mice for 8 weeks, the mouse rolling wheel experiment confirmed that muscle strength was improved; Figure 4 B shows that after 8 weeks of NBI-31772 treatment of diabetic mice, the grip strength meter confirmed that the mice's forelimb strength increased.

[0024] Figure 5 This is a schematic diagram showing that NBI-31772 improves insulin sensitivity in diabetic mice; Figure 5 A in the figure is the body weight of diabetic mice (db / db) after 4 weeks of NBI-31772 treatment; Figure 5 B in the figure is the body weight of diabetic mice (db / db) after 8 weeks of NBI-31772 treatment; Figure 5 C in the figure is the insulin tolerance test of diabetic mice (db / db) after 4 weeks of NBI-31772 treatment; Figure 5 D in the figure is the insulin tolerance test of diabetic mice (db / db) after 8 weeks of NBI-31772 treatment.

[0025] Figure 6 This is a schematic diagram of the improvement of blood lipids by NBI-31772 in diabetic mice; Figure 6 A in the figure is the change of total cholesterol content in diabetic mice after 8 weeks of NBI-31772 treatment; Figure 6 B in the figure is the change in triglyceride content after 8 weeks of NBI-31772 treatment.

[0026] Figure 7 This is a schematic diagram of the improvement effect of NBI-31772 on fatty liver in diabetic mice; Figure 7 A in the figure is the change in liver weight of diabetic mice after 8 weeks of NBI-31772 treatment; Figure 7B in the figure shows the changes in HE staining of liver sections of diabetic mice after 8 weeks of NBI-31772 treatment; the white vacuoles in the sections represent large lipid droplets, and the white dots represent small lipid droplets in liver cells. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0028] 1. Experimental Materials

[0029] NBI-31772 Figure 1 The structural formula shown was purchased from Taoshu Company; cholesterol and triglyceride determination kits were purchased from Nanjing Jiancheng Bioengineering Institute; TRIzol reagent was purchased from Invitrogen; RNA reverse transcription kit and SYBR fluorescent dye were purchased from Dalian Bao Biotechnology Company. RNA primer sequences were ordered and synthesized from Beijing Aoke Dingsheng Biotechnology Co., Ltd.

[0030] 2. Experimental Animal Breeding and Model Establishment

[0031] db / db male mice were purchased from Nanjing Jicui Pharmaceutical Animal Co., Ltd. The mice were housed in a room with controlled temperature (22 to 28 degrees) and humidity (60%). The lighting in the room was maintained in a 12-hour day and 12-hour night cycle. The mice were able to freely eat and drink water during the experiment. The experimental mice were divided into two groups, with 8 mice in each group. The two groups were: (1) the control group, normal diet, intraperitoneal injection of normal saline (6 mg / kg / day) every 3 days (2) the drug intervention group, normal diet, intraperitoneal injection of NBI-31772 (6 mg / kg / day) every 3 days. The experimental period was 2 months, and an insulin tolerance test was performed at 4 weeks and 8 weeks of drug intervention, respectively. At the same time, muscle function tests such as rolling wheel test, grip strength test, and gait analysis were performed at the 8th week.

[0032] 3. Experimental Methods

[0033] (1) Insulin tolerance test

[0034] Insulin tolerance test (ITT) was performed according to published standard protocols. Mice were fasted for 6 h and then injected with insulin (0.7 U / kg in normal diet) intraperitoneally, and blood glucose levels were measured at 0, 15, 30, 45, 60, 90, and 120 min.

[0035] (2) Roller test

[0036] The motor coordination and balance ability of the mouse model were evaluated by the rotarod test. The mice were trained daily for 3 consecutive days on a rotating rod at a constant speed of 4 revolutions per minute. On the fourth day, they were tested for accelerated rotarods. The test started at 4 revolutions per minute and gradually increased to 40 revolutions per minute within 5 minutes. During the test, if the experimental animal fell off the rotating rod or grabbed the rod twice in a row instead of trying to run on it, the test was terminated and its time on the rod was recorded. The data are presented as the maximum value of the mouse's residence time on the rotating rod (from three trials).

[0037] (3) Grip force detection

[0038] The maximum grip strength of mice was measured using a Bioseb grip tester (BIO-GS3, Bioseb, Florida, USA). The mice were placed on a test grid so that they could grasp the grid with their forelimbs or both forelimbs and hindlimbs. The tail of the mouse was then gently pulled in the direction of the force detected by the device. When the mouse released the grid, the detector recorded its peak grip strength, which was expressed in grams (g). Each mouse was tested three times, and the maximum value was recorded as the mouse's grip strength.

[0039] (4) mRNA content detection

[0040] The reverse transcription RNA-real-time fluorescence quantitative PCR method was used for detection. The specific method is as follows: RNA extraction: 50 mg of liver tissue, add 800 μL of TRIzol reagent, and manually grind the liver tissue. Shake at room temperature for 5 minutes, add 200 μl of chloroform (1 / 5 of the total volume) to extract protein, mix vigorously for 15 seconds, leave at room temperature for 15 minutes, and centrifuge at 13,000g, 4℃ for 20 minutes. Transfer the upper aqueous phase to another RNase-Free EP tube, add an equal volume of isopropanol, mix and leave at room temperature for 15 minutes, centrifuge at 13,000g, 4℃ for 20 minutes, and discard the supernatant. Add 500 μL of pre-cooled 75% ethanol, mix by inversion, and centrifuge at 13,000g, 4℃ for 10 minutes. Discard the supernatant, let the ethanol evaporate completely, and dissolve in 10-20 μl DEPC water. Use a UV spectrophotometer to measure its concentration for reverse transcription. RNA reverse transcription: The transcription volume is 20 μl, take out 500 ng RNA, add 4 μl 5X Master Mix, add DEPC water to 20 μl volume, incubate at 37℃ for 60 min, and then at 80℃ for 15 s. Store at -20℃ for later use.

[0041] Real-time PCR: SYBR Green method was used, and the reaction system included 1 μl cDNA, 5 μl Premix Ex Taq TMII, 0.5 μl upstream and downstream primer mixture (10 μM), add sterile water to 10 μl. The reaction conditions are as per the instructions, 95°C for 10 min, 40 cycles of PCR (each cycle includes 95°C for 30 s, 55°C for 30 s, 72°C for 20 s), and finally observe the melting curve (95°C for 15 s, 60°C for 15 s, 95°C for 15 s).

[0042] (5) Statistical analysis

[0043] Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad). Pairwise comparisons were performed using a two-tailed Student's T test. Statistically significant values ​​were *p<0.05, **p<0.01.

[0044] Embodiment 1

[0045] NBI-31772 reduces the accumulation of IGFBP3 protein in cells

[0046] IGFBP3 protein is mainly present in the blood circulation, and the content of IGFBP3 protein in cells is low under normal conditions. Figure 2 As shown, immunofluorescence analysis of muscle sections showed that there was significant accumulation of IGFBP3 protein in the tibialis anterior and gastrocnemius muscle cells of diabetic mice (red fluorescent signal represents IGFBP3 protein). The db / db mice used in the experiment are a mouse model commonly used in basic scientific research on diabetes and clinical drug development, and are a classic model for studying diabetes in the scientific community. After 8 weeks of NBI-31772 treatment, the protein content of IGFBP3 in muscle was significantly reduced. These data confirm that NBI-31772 can inhibit the functional activity of IGFBP3 entering cells.

[0047] Embodiment 2

[0048] NBI-31772 can improve the symptoms of diabetic amyotrophy

[0049] Muscle atrophy is a typical complication of diabetes, with increased protein degradation in muscle cells and decreased muscle mass. Figure 3 As shown in Figure A, muscle sections of db / db mice showed that there were a large number of atrophic muscle fibers in various muscles. After 8 weeks of NBI-31772 treatment, the atrophic muscle fibers disappeared significantly and the muscle structure was normal. Figure 3 As shown in Figure B, after 8 weeks of NBI-31772 treatment, the expression of key genes regulating muscular atrophy in muscle cells decreased significantly. Figure 3 As shown in Figure C, the content of the key fibroblast protein MYHC in muscle cells increased significantly after 8 weeks of NBI-31772 treatment, indicating an increase in muscle mass. These data confirm that NBI-31772 can significantly improve the symptoms of muscle atrophy.

[0050] Embodiment 3

[0051] NBI-31772 improves muscle strength in diabetic patients

[0052] Diabetic muscle atrophy is mainly accompanied by a decrease in muscle strength, which is a key factor leading to various mechanical injuries and accidents. Figure 4 As shown in Figure A, the wheel test showed that after 8 weeks of NBI-31772 treatment, the body strength and balance ability of diabetic mice were significantly improved. Figure 4 As shown in Figure B, the grip strength test showed that after 8 weeks of NBI-31772 treatment, the muscle strength of the mouse forelimbs increased significantly. These data all confirm that NBI-31772 can significantly improve muscle strength and body balance.

[0053] The above experimental results prove that NBI-31772 can alleviate and improve diabetic muscle atrophy by inhibiting the cellular accumulation of IGFBP3 protein, reducing the activity of IGFBP3 protein, improving insulin sensitivity, lowering blood lipids, improving fatty liver, inhibiting the progression of diabetic muscle atrophy, and enhancing muscle strength and function.

[0054] Embodiment 4

[0055] Effect of NBI-31772 on improving insulin sensitivity in diabetic mice

[0056] The db / db mouse is a mouse model commonly used in basic scientific research on diabetes and clinical drug development, and is a classic model for studying diabetes in the scientific community. Figure 5 As shown, db / db mice were divided into two groups, one as a control group and the other as a drug intervention group. The drug intervention lasted for 8 weeks in total, and the changes in the weight difference and insulin sensitivity of the mice were detected in the fourth week. Figure 5 As shown, compound intervention was effective at 4 weeks ( Figure 5 A) and 8 weeks ( Figure 5 B) There was no effect on the weight of mice at both time points. However, the insulin sensitivity of mice was significantly improved after 4 weeks of drug intervention ( Figure 5 C), also showed an improvement in insulin sensitivity after 8 weeks of drug intervention ( Figure 5 D in the figure). This indicates that NBI-31772 can rapidly and continuously improve insulin sensitivity and has the effect of lowering blood sugar.

[0057] Embodiment 5

[0058] Effect of NBI-31772 on improving hyperlipidemia in diabetic mice

[0059] The most obvious sign of diabetes is the marked increase in cholesterol and triglyceride levels in the blood. Figure 6 As shown in Figure A, after 8 weeks of NBI-31772 treatment, the total cholesterol level in the blood was significantly reduced. Figure 6 Figure B shows that after 8 weeks of NBI-31772 treatment, triglycerides in the blood also decreased significantly. These all suggest that NBI-31772 can significantly improve the hyperlipidemia symptoms associated with diabetes.

[0060] Embodiment 6

[0061] NBI-31772 improves symptoms of nonalcoholic fatty liver disease

[0062] The development of non-alcoholic fatty liver disease is also accompanied by the onset of obesity and diabetes. Figure 7 As shown in Figure A, after 8 weeks of NBI-31772 treatment, the liver weight was significantly reduced. Figure 7 As shown in B, HE staining of liver sections showed that there was excessive accumulation of lipid droplets in the liver of db / db mice, and after 8 weeks of NBI-31772 treatment, the lipid droplets in the liver were significantly reduced. These data confirm that NBI-31772 can significantly improve the symptoms of fatty liver.

[0063] In summary, NBI-31772 can be used for the following applications:

[0064] Application of NBI-31772 in the preparation of diabetic amyotrophy drugs.

[0065] Application of NBI-31772 in the preparation of therapeutic drugs for lowering blood sugar and improving insulin sensitivity.

[0066] Application of NBI-31772 in the preparation of lipid-lowering drugs.

[0067] Application of NBI-31772 in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

[0068] The present invention is not limited to the above embodiments. Any non-essential additions and substitutions made by those skilled in the art based on the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. The use of NBI-31772 in the preparation of a drug for improving diabetic amyotrophy, characterized in that: The NBI-31772 is a small molecule inhibitor that specifically inhibits the activity and functional localization of the IGFBP3 protein, reduces the accumulation of the IGFBP3 protein in cells, and inhibits the occurrence of diabetic muscular atrophy; The NBI-31772 can improve the loss of muscle strength in diabetic patients; The NBI-31772 can improve muscle strength and function in diabetic patients.

2. The use of NBI-31772 according to claim 1 in the preparation of a drug for improving diabetic amyotrophy, characterized in that: The NBI-31772 can improve insulin sensitivity and lower blood sugar.

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