Nrf2 activators and their use in the manufacture of a medicament

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to an Nrf2 activator and application thereof in preparation of medicine. The Nrf2 activator comprises a compound I and a traditional Chinese medicine extract; a mass ratio of the compound I to the traditional Chinese medicine extract is 1-10:0.5-5; the traditional Chinese medicine extract is one or more of an astragalus extract, a radix ginseng extract, a radix salviae miltiorrhizae extract and a radix smilacae extract. The Nrf2 activator is prepared into medicine for preventing or treating diabetic nephropathy together with a pharmaceutically acceptable carrier or excipient. The Nrf2 activator can target and activate an Nrf2 signal pathway, significantly inhibit accumulation of collagen and oxidative stress, and prevent and treat diabetic nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to an Nrf2 activator and its application in drug preparation. Background Technology

[0002] Diabetic nephropathy is one of the most important complications of diabetes, and its incidence is on the rise in my country. The prevalence of diabetic nephropathy in adults with type 2 diabetes is 10-40%, making it a leading cause of death worldwide for patients with type 1 or type 2 diabetes. Due to its complex metabolic disturbances, once it progresses to end-stage renal disease, it is often more difficult to treat than other kidney diseases. Therefore, timely prevention and treatment are crucial for delaying the progression of diabetic nephropathy. Increasing research indicates that diabetic nephropathy is characterized by thickening of the basement membrane, hypertrophy of renal tubules and glomeruli, and accumulation of extracellular matrix (ECM). These abnormal pathological changes are caused by the complex interaction of multiple factors. The accumulation of reactive oxygen species (ROS) can lead to vascular endothelial abnormalities and promote the expression of transforming growth factor (TGF)-β1, thereby promoting fibrosis in diabetic nephropathy. Inhibiting oxidative stress with antioxidants can effectively reduce high glucose (HG)-induced ECM deposition and fibrosis, thereby improving diabetic nephropathy. The transcription factor nuclear factor E2-associated factor 2 (Nrf2) is known as a major regulator of the cellular defense system, playing a crucial role in regulating it, particularly in the regulation of oxidative stress. ECH-associated protein 1 (Keap1), similar to Kelch, is considered the major repressor of Nrf2. Inhibition of Keap1 suppresses Nrf2 degradation, leading to nuclear translocation of Nrf2, which subsequently induces the transcription of various antioxidant genes and activates the cellular defense system. Improving Nrf2 activity can provide protection against various chronic diseases, including diabetic nephropathy. Currently, treatment for kidney disease primarily involves hemodialysis, kidney transplantation, and drug therapy. Once a patient's kidney function is impaired, long-term hemodialysis is necessary to maintain kidney function. However, this method only removes metabolites accumulated in the body due to kidney dysfunction from the blood and cannot restore severely impaired kidney function. Therefore, developing drugs that can effectively treat kidney disease, especially those that can prevent and treat kidney disease in its early stages, remains a pressing technical challenge. Currently, commonly used medications are mainly symptomatic treatments, such as those for diabetes, hypertension, and hyperlipidemia, or immunosuppressants and diuretics. These primarily restore kidney function by promoting diuresis and reducing proteinuria. However, long-term use of these medications can cause significant side effects such as liver damage, gastrointestinal dysfunction, and bone marrow suppression. Therefore, safe and effective drugs for treating kidney disease still need further development. Currently available diabetic nephropathy medications are mostly designed to control blood sugar, prevent coagulation, control blood pressure, and reduce proteinuria, but they lack specificity and effectiveness, and long-term use can lead to side effects. Traditional Chinese medicine treatment alone is slow to take effect and its efficacy is not significant.

[0003] Therefore, there is an urgent need for a precise, targeted, safe, and effective drug to prevent or treat diabetic nephropathy. Summary of the Invention

[0004] In view of this, the present invention provides an Nrf2 activator that can effectively activate the Nrf2 signaling pathway.

[0005] By activating the Nrf2 signaling pathway, the expression of Nrf2 protein in the cell nucleus can be increased, while the expression levels of collagen I, TGF-β1, and Keap1 proteins in kidney tissue can be reduced. This inhibits collagen accumulation and oxidative stress, significantly alleviating STZ-induced diabetic nephropathy. Consequently, it reduces the levels of total cholesterol (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), β2-microglobulin (β2-MG), blood urea nitrogen (BUN), and creatinine (CR), as well as the urinary albumin to creatinine ratio (UACR), thereby preventing or treating diabetic nephropathy.

[0006] Traditional Chinese medicine (TCM) believes that the main pathological mechanism of diabetic nephropathy is deficiency of both Qi and Yin, kidney deficiency and blood stasis, with deficiency as the primary factor, accompanied by excess pathogenic factors such as dampness, turbidity, and heat. Commonly used TCM herbs for treating diabetic nephropathy include Astragalus membranaceus, Salvia miltiorrhiza, Paeonia lactiflora, and Rheum palmatum; however, using these herbs alone is relatively slow to take effect and the results are not obvious.

[0007] The Nrf2 activator comprises compound I and a traditional Chinese medicine extract; the mass ratio of compound I to the traditional Chinese medicine extract is 1-10:0.5-5; the traditional Chinese medicine extract is one or more of Astragalus membranaceus extract, Codonopsis pilosula extract, Salvia miltiorrhiza extract, and Dioscorea opposita extract; the structural formula of compound I is as follows: Formula I.

[0008]

[0009] Wherein, R is one of H, β-glucose, or 6-sulfoglucose.

[0010] Preferably, the mass ratio of compound I to the traditional Chinese medicine extract is 2:1.

[0011] Preferably, R is H.

[0012] Furthermore, the herbal extracts comprise, by weight, 10-60 parts of Astragalus membranaceus extract, 10-60 parts of Codonopsis pilosula extract, 10-60 parts of Salvia miltiorrhiza extract, and 10-60 parts of Dioscorea opposita extract.

[0013] Preferably, the herbal extract comprises, by weight, 40 parts of Astragalus membranaceus extract, 20 parts of Codonopsis pilosula extract, 20 parts of Salvia miltiorrhiza extract, and 20 parts of Dioscorea opposita extract.

[0014] Preferably, the herbal extract comprises, by weight, 50 parts of Astragalus membranaceus extract, 20 parts of Codonopsis pilosula extract, 10 parts of Salvia miltiorrhiza extract, and 20 parts of Dioscorea opposita extract.

[0015] Furthermore, the preparation process of the herbal extract is as follows: weigh each raw material separately, mix them, add 6-16 times the amount of water and decoct for 0.5-3 hours, filter to obtain filtrate, concentrate the filtrate under reduced pressure at a pressure of 0.01-0.1 MPa and a temperature of 50-70℃ for 1.5-2.5 hours, and then spray dry and pulverize to obtain dry powder of the herbal extract.

[0016] The present invention also aims to provide a medicament for the prevention or treatment of diabetic nephropathy, characterized in that it comprises the aforementioned Nrf2 activator and / or a pharmaceutically acceptable carrier or excipient.

[0017] Furthermore, the drug for preventing or treating diabetic nephropathy, preferably diabetic nephropathy, can be administered orally, sublingually, systemically, locally, or rectally.

[0018] Furthermore, the drugs for preventing or treating diabetic nephropathy are one or more of the following: tablets, oral liquids, injections, transdermal preparations, ointments, gels, creams, emulsions, microencapsulated drugs, granules, microspheres, targeted release drugs, and pills.

[0019] The present invention also provides a method for activating Nrf2, the method comprising activation using compound I or the aforementioned Nrf2 activator; the structural formula of compound I is as follows: Formula I.

[0020]

[0021] Wherein, R is one of H, β-glucose, or 6-sulfoglucose.

[0022] Specifically, the activation of the Nrf2 signaling pathway can be carried out in the constructed antioxidant molecular regulatory mechanism model.

[0023] The present invention also aims to provide a method for inhibiting the expression of collagen I and / or TGF-β1 and / or Keap1 proteins, the method comprising using compound I or the aforementioned Nrf2 activator as an inhibitor for inhibition; the structural formula of compound I is as follows: Formula I.

[0024]

[0025] Wherein, R is one of H, β-glucose, or 6-sulfoglucose.

[0026] The present invention also provides the use of compound I in the preparation of a drug for the prevention or treatment of diabetic nephropathy, as an Nrf2 activator or an inhibitor of collagen I and / or TGF-β1 and / or Keap1 protein expression. The structural formula of compound I is as follows: Formula I.

[0027]

[0028] Wherein, R is one of H, β-glucose, or 6-sulfoglucose.

[0029] Preferably, R is H.

[0030] In this invention, numerical values ​​such as "weight", "temperature" and "time" are involved, but numerical differences caused by instrument errors or operational errors are not included. That is to say, numerical differences caused by instrument errors or operational errors are also included in the technical solution of this invention.

[0031] In this invention, terms such as "filtration," "cooling," "reduced pressure concentration," and "spray drying" are all conventional operations used by those skilled in the art. That is to say, processes that are inconsistent with the technical solution of this invention but have the same operation are all included in the technical solution of this invention.

[0032] The beneficial effects of this invention are as follows:

[0033] Compound I in the Nrf2 activator provided by this invention can activate the Nrf2 signaling pathway, significantly alleviate metabolic disorders and kidney disease caused by renal insufficiency in mice, and the extracts of traditional Chinese medicine such as Astragalus membranaceus can replenish qi and blood, promote diuresis and reduce swelling, and can play a synergistic role in the prevention and treatment of diabetic nephropathy. Attached Figure Description

[0034] Figure 1 The effect of the drug composition on serum TC levels in db / db mice.

[0035] Figure 2 The effect of the drug composition on serum TG levels in db / db mice.

[0036] Figure 3 The effect of the drug composition on LDL-C levels in the serum of db / db mice.

[0037] Figure 4 The effect of the drug composition on HDL-C levels in the serum of db / db mice.

[0038] Figure 5The effect of the drug composition on VLDL-C levels in the serum of db / db mice.

[0039] Figure 6 The effect of the drug composition on serum BUN levels in db / db mice.

[0040] Figure 7 The effect of the drug composition on the serum β2-MG level in db / db mice.

[0041] Figure 8 The effect of the drug composition on UACR levels in db / db mice.

[0042] Figure 9 The effect of the drug composition on the pathological changes of kidney tissue in db / db mice.

[0043] Figure 10 The effect of the drug composition on the expression levels of nephrin and podocin in the kidney tissue of db / db mice.

[0044] Figure 11 The effect of the drug composition on STZ-induced abnormal pathological changes in mouse kidney tissue.

[0045] Figure 12 The effect of the drug composition on the expression of relevant proteins in STZ-induced diabetic nephropathy mice.

[0046] Figure 13 The drug composition activates the Nrf2 signaling pathway to alleviate ROS and fibrosis in podocytes incubated with high glucose. Detailed Implementation

[0047] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0048] In this embodiment of the invention, the type 1 diabetes animal model was established by raising male C57BL / 6 mice aged 6-8 weeks, weighing 20-25g, in a controlled environment (25℃±2℃, 50%±5% humidity) in a specific pathogen-free (SPF) barrier facility with a standard 12-hour light-dark cycle. Mice were allowed free access to standard food and water in their cages. Diabetes was induced in the mice by intraperitoneal injection of 50mg / kg streptozotocin (STZ) for 5 consecutive days. A high-fat diet (60kcal% fat) was then administered for 8 weeks to further develop diabetic nephropathy.

[0049] In this embodiment of the invention, the type 2 diabetes animal model was an 8-week-old male C57BL / KsJ (db / db) mouse, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The db / db mouse is a genetic model of early type 2 diabetic nephropathy, characterized by hyperglycemia and enhanced urinary albumin excretion. Before animal experiments, all mice were placed in a specific pathogen-free (SPF) isolation facility with controlled temperature (25±2℃) and humidity (50±5%) during a standard 12-hour light-dark cycle, and were given free access to standard food and water in their cages.

[0050] In this embodiment of the invention, the immortalized mouse podocyte cell line was obtained from AcceGen Biotechnology Corporation (USA). The podocytes were cultured in a solution containing 10% fetal bovine serum at a concentration of 1×10⁻⁶ mcg / mL. 5 The cells were cultured in RPMI 1640 medium containing U / L streptomycin sulfate and recombinant interferon-c. They were then incubated at 33°C and 5% CO2. Next, the fusion cultures were digested with trypsin and maintained in interferon-free 6-well, 12-well, or 96-well plates, and cultured for another 2 weeks at a constant temperature of 37°C and 5% CO2. For glucose treatment, podocytes were cultured in RPMI 1640 medium containing 5 mM normal glucose (referred to as the LG group) or 30 mM high glucose medium (referred to as the HG group). Synthesized Nrf2 siRNA (si-Nrf2) and negative control siRNA (NC) were purchased from Shanghai Jierui Biotechnology Co., Ltd., and then... 3000 transfected si-Nrf2 to a final concentration of 50 nM into podocytes. TGF-β1 was used to induce cell fibrosis.

[0051] In this embodiment of the invention, the preparation process of the traditional Chinese medicine extract is as follows: each raw material is weighed separately, mixed, and then decocted with 6-16 times the amount of water for 0.5-3 hours. The mixture is filtered to obtain a filtrate. The filtrate is concentrated under reduced pressure at a pressure of 0.01-0.1 MPa and a temperature of 50-70°C for 1.5-2.5 hours. Then, it is spray-dried and pulverized to obtain a dry powder of the traditional Chinese medicine extract.

[0052] Example 1: Preparation of Traditional Chinese Medicine Extracts

[0053] Chinese herbal extract 1

[0054] Weigh out 50g of Astragalus membranaceus, 20g of Codonopsis pilosula, 10g of Salvia miltiorrhiza, and 20g of Dioscorea opposita, totaling 100g. Mix them together, add 1000g of water, soak for 30 minutes, then decoct for 2 hours and filter to obtain the filtrate. Add 800g of water to the residue, decoct for 2 hours, and filter to obtain the filtrate again. Combine the two filtrates and concentrate under reduced pressure at 0.05 MPa and 50℃ for 1.5 hours. Spray dry the concentrate to obtain the dry powder of Chinese herbal extract 1.

[0055] Chinese herbal extract 2

[0056] Weigh out 40g of Astragalus membranaceus, 20g of Codonopsis pilosula, 20g of Salvia miltiorrhiza, and 20g of Dioscorea opposita, totaling 100g. Mix them, add 1500g of water, soak for 30 minutes, then decoct for 2 hours and filter to obtain the filtrate. Add another 1000g of water to the residue, decoct for 1 hour, and filter to obtain the filtrate again. Combine the two filtrates and concentrate under reduced pressure at 0.1 MPa and 60℃ for 2.5 hours. Spray dry the concentrate to obtain the dry powder of herbal extract 2.

[0057] Example 2 Preparation of the composition

[0058] Composition 1

[0059] Weigh out 15g of compound I (betalain) with R as H and 15g of dry powder of traditional Chinese medicine extract 1, mix them evenly to obtain composition 1.

[0060] Composition 2

[0061] Weigh out 20g of compound I (betalain) with R as H and 10g of dry powder of traditional Chinese medicine extract 2, mix them evenly to obtain composition 2.

[0062] Example 3

[0063] The db / db mice were randomly divided into 6 groups according to Table 1:

[0064] Table 1 Grouping of db / db mouse models

[0065] Model group Experimental methods Diabetic nephropathy model group (Ctrl) Standard feed feeding Extract group 1 (TQW1) Extract 1 was mixed with feed at a ratio of 5g / kg and then fed to the animal. Extract group 2 (TQW2) Extract 2 was mixed with feed at a ratio of 5g / kg and then fed to the animal. Beetroot red group (BCN) Mix betalains with feed at a ratio of 1g / kg and feed. Composition Group 1 (ZHW1) Mix composition 1 with feed at a ratio of 1.5 g / kg and feed. Composition 2 (ZHW2) Mix composition 2 with feed at a ratio of 1.5 g / kg and feed.

[0066] Feeding was performed according to the grouping and methods outlined in Table 1 for 8 weeks. Blood samples were collected to measure the levels of total cholesterol (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), β2-microglobulin (β2-MG), blood urea nitrogen (BUN), and creatinine (CR). 24-hour urine samples were collected to measure urinary albumin, and the urinary albumin-to-creatinine ratio (UACR) was calculated. The results are shown below. Figure 1-8 As shown.

[0067] Example 4

[0068] Mice fed according to the grouping and methods in Table 1 were examined by HE and PAS staining and transmission electron microscopy (TEM). The results showed that, compared with the diabetic nephropathy group, the combined group mice exhibited significantly reduced glomerular mesangial expansion and glomerular sclerosis index, and significantly decreased glomerular basement membrane thickness (GB MT). The results are as follows: Figure 9As shown; IHC staining results indicated that, compared with the kidneys of diabetic nephropathy mice, composition 2 significantly increased the expression of nephrin and podocin, as shown in the figure. Figure 10 As shown. Therefore, composition 2 can effectively protect the kidney structure of diabetic mice and prevent pathological changes.

[0069] Example 5

[0070] Normal mice and type 1 diabetic animal model mice were divided into 3 groups according to Table 2 below:

[0071] Table 2 Grouping of normal mice and type 1 diabetes animal models

[0072] Model group Element Normal control group (Ctrl) Standard feed feeding STZ-induced group (STZ) Standard feed feeding STZ+ZHW2 group (STZ+ZHW2) Mix composition 2 with feed at a ratio of 1.5 g / kg and feed.

[0073] Kidney tissues from mice in the normal control group, STZ group, and STZ+ZHW2 group were stained with HE, PAS, Sirius red, Masson's trichrome staining, and examined by TEM. The results showed that STZ induction induced significant pathological changes in the kidney tissue, with a significant increase in glomerular mesangial matrix and glomerular sclerosis index. Figure 11 AC). Furthermore, STZ mice showed significantly increased GBMT (glomerular matrix membrane thickness) and foot process width. Figure 11 (A, D, E), significant collagen accumulation was observed in the kidney sections. Figure 11 A, F, G). Intervention with composition 2 (ZHW2) can reverse these abnormal pathological changes and significantly reduce the glomerular mesangial matrix and glomerular sclerosis index. Figure 11 AC), reducing GBMT and foot process width ( Figure 11 A, D, E) inhibited collagen accumulation. Figure 11 (A, F, G), SOD activity was significantly rescued. Figure 11 H), STZ-induced renal ROS production was significantly reduced ( Figure 11 Therefore, ZHW2 significantly alleviated the symptoms of diabetic nephropathy.

[0074] Example 6

[0075] According to Western blotting analysis ( Figure 12The results showed that the expression levels of collagen I, TGF-β1, and Keap1 proteins were significantly increased in the kidneys of diabetic mice, while the expression of Nrf2 protein in the cell nucleus was significantly decreased, indicating that STZ-induced collagen accumulation and oxidative stress lead to diabetic nephropathy. In mice treated with ZHW2, the expression levels of collagen I, TGF-β1, and Keap1 proteins were significantly decreased, while the expression of Nrf2 protein in the cell nucleus was significantly increased, approaching the normal levels of the control group. Compared with the STZ group, after intervention with composition 2, the expression of Gp91phox, p22phox, p47phox, and p40phox in mice was reduced, while the expression of GCLM, SOD1, and SOD2 was restored, showing that the intervention with composition 2 significantly reduced the oxidative stress induced by STZ. Figure 11 Histopathological results showed that ZHW2 can activate the Nrf2 signaling pathway, inhibit collagen accumulation and oxidative stress, thereby significantly alleviating STZ-induced diabetic nephropathy.

[0076] Example 7

[0077] Western blot and immunofluorescence (IF) staining analysis showed that podocin expression, which was reduced by high glucose (HG) induction, was restored after Nrf2 inhibition. In HG-incubated podocytes, Nrf2 silencing followed by increased ZHW2 addition led to a decrease in podocin expression. Figure 13 A, Figure 13 B). In podocytes incubated with HG, ROS is produced in large quantities, which is significantly reduced after ZHW2 intervention. However, silencing Nrf2 almost eliminates the antioxidant effect of ZHW2 in scavenging ROS. Figure 13 C). The results of quantitative real-time PCR further demonstrated that the mRNA expression levels of Gp91phox, p22phox, p47phox, and p40phox were significantly increased in podocytes incubated with HG, while the expression of GCLM, SOD1, and SOD2 was significantly decreased. ZHW2 intervention could restore these levels, exhibiting excellent inhibitory effects against oxidative stress. Figure 13 D). Silencing Nrf2 further promotes the mRNA expression of Gp91phox, p22phox, p47phox, and p40phox in HG-incubated podocytes, while further inhibiting the expression of GCLM, SOD1, and SOD2. However, after Nrf2 silencing in HG-incubated podocytes, the original ability of ZHW2 to inhibit oxidative stress is also greatly reduced. Figure 13D). We found that in HG-incubated podocytes, silencing Nrf2 also led to increased HO-1 expression and significantly weakened Keap1 and NOX1 effects due to ZHW2. Furthermore, in HG-incubated podocytes transfected with siNrf2, ZHW2 no longer exhibited anti-fibrotic activity, a finding further supported by the restoration of p-SMAD2, p-SMAD3, Collagen I, and TGF-β1 expression. Figure 13 E). These experimental results demonstrate that the ameliorative effect of ZHW2 on fibrosis and oxidative stress in HG-incubated podocytes is highly dependent on the activation of Nrf2.

[0078] In summary, the composition 2 (ZHW2) of the present invention significantly alleviates diabetic nephropathy by targeting and activating the Nrf2 signaling pathway to inhibit collagen accumulation and oxidative stress.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of Nrf2 activator in the preparation of drugs for the prevention or treatment of diabetic nephropathy, characterized in that, The Nrf2 activator comprises compound I and a traditional Chinese medicine extract; the mass ratio of compound I to the traditional Chinese medicine extract is (1-2):1; the traditional Chinese medicine extract comprises, by mass parts, 40 parts of Astragalus membranaceus extract, 20 parts of Codonopsis pilosula extract, 20 parts of Salvia miltiorrhiza extract, and 20 parts of Dioscorea opposita extract; the structural formula of compound I is as follows: Formula I. Wherein, R is H; The preparation process of the herbal extract is as follows: weigh each raw material separately, mix them, add 6-16 times the amount of water and decoct for 0.5-3 hours, filter to obtain filtrate, concentrate the filtrate under reduced pressure at a pressure of 0.01-0.1 MPa and a temperature of 50-70℃ for 1.5-2.5 hours, and then spray dry and pulverize to obtain dry powder of the herbal extract.

2. The application according to claim 1, characterized in that, The drug includes the Nrf2 activator and / or a pharmaceutically acceptable carrier or excipient.

3. The application according to claim 2, characterized in that, The drug is one or more of the following: tablets, oral liquids, and pills.

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