Mtor inhibitors with porous nanostructure and methods of making and pharmaceutical uses thereof

Abstract

The application discloses an mTOR inhibitor with a porous nano structure and a preparation method and pharmaceutical application thereof, and belongs to the technical field of biological medicines. 3+ The application obtains a nano catalyst targeting mTOR through aqueous phase synthesis, and a cerium phytate nanoparticle formed by coordination of phytic acid and cerium ions carries a large number of Ce The large number of holes in the material effectively promote adsorption of active oxygen free radicals, so that the cerium phytate has high efficient antioxidant performance and can be applied to treatment of various diseases related to active oxygen. In addition, phytic acid, as a ligand molecule constituting the cerium phytate, has six phosphate groups and can efficiently coordinate with cerium ions to form the cerium phytate material. Phytic acid also has anti-inflammatory, antioxidant and lipid metabolism regulating activities and can effectively enhance the antioxidant performance of the cerium-based material in the body. Therefore, the mTOR inhibitor prepared by the application has good biocompatibility and anti-inflammatory and antioxidant activities and is expected to be used for disease treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a nano catalyst targeting mTOR, a preparation method thereof and application of the nano catalyst as a drug for relieving NASH progression. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury disease closely related to insulin resistance and genetic susceptibility, and the disease spectrum includes non-alcoholic liver steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis and hepatocellular carcinoma. At present, NAFLD is a global epidemic trend, and is one of the causes of increasing cirrhosis and liver cancer, and has become the first chronic liver disease globally recognized. According to the literature, the global prevalence rate of NAFLD is 25.24%, and the prevalence rate is increasing year by year. Cardiovascular disease, malignant tumor and liver cirrhosis decompensation are common causes of death in patients with NAFLD. Despite the urgent medical needs, no drug has been approved for the treatment of NASH so far, and the development of NASH drugs is imminent.

[0003] The current widely accepted pathogenesis of NASH is the multiple-hit hypothesis. The first hit is due to a high-fat diet, which directly provides a large amount of free fatty acids (FFA) to the liver, and metabolic carbohydrates cause the synthesis of excess free fatty acids from acetyl-CoA. Meanwhile, excessive glucose consumption can also generate free fatty acids through the activation of carbohydrate reactions, promoting the generation of liver fat. Abnormal lipid metabolism in the liver, and a large accumulation of lipids, activates multiple links in hepatocyte peroxidation stress, endoplasmic reticulum stress, and the inflammatory cascade in hepatocytes. The increased free fatty acids in the plasma flow into the liver, and ectopic fat accumulation promotes the increase in hepatocyte cytokine secretion, increasing gluconeogenesis, thereby inhibiting insulin signaling and reducing glycogen production. Insulin resistance caused by increased fat forms the second hit in the mechanism of NASH in the liver. After experiencing the second hit, the liver appears to have a microcirculatory disorder, and the increasing plasma free fatty acids further cause tissue lipid metabolism disorder. As the disease progresses, hepatocyte damage promotes fatty hepatitis, hepatocyte oxidative necrosis, hepatocyte stress apoptosis, mitochondrial dysfunction, and endoplasmic reticulum stress, ultimately activating hepatic stellate cells (HSCs), collagen deposition, and causing liver fibrosis, hepatocyte ischemia and necrosis, and liver lobule reconstruction, leading to cirrhosis. These are referred to as the third hit of NASH. Currently, research and development enterprises basically start from the three aspects of metabolism, inflammation, and fibrosis when selecting targets, among which the metabolic targets can be further divided into three targets of lipids, glucose, and bile acids. The inflammation targets include oxidative stress, inflammation, and immune system targets. Although there are many target points for NASH drugs, new compounds for the treatment of NASH have entered the II / III phase of clinical trials. Among them, obeticholic acid (Obeticholic acid) has entered the III phase of clinical trials, but the high-dose group of the drug only has an improvement effect on liver fibrosis in 23% of patients, only 10% higher than the placebo group, and has obvious side effects, such as severe itching reactions, and some patients also have serious cardiovascular events. In addition, there is no particularly effective drug for reducing liver lipid damage. The existing auxiliary drugs, such as drugs that improve insulin resistance, drugs that regulate lipid metabolism and reduce liver lipid deposition, anti-oxidation and anti-inflammatory drugs, have weak and non-specific effects on liver lipid damage. mTOR is related to the occurrence and development of NASH. Studies have shown that lipid accumulation in the liver activates mTORCl, thereby promoting liver steatosis. mTORCl promotes metabolic reprogramming, which accelerates the occurrence and development of obesity-related liver cancer. In addition, by selectively inhibiting mTORCl signaling in mice, the activation of TFE3 in the liver is promoted without affecting other mTORCl targets, which can delay the occurrence and development of NAFLD and NASH. The activation of mTOR depends on the phosphorylation modification of the serine site at position 2446.

[0004] However, the existing inhibitors of mTOR have side effects such as cytotoxicity and systemic metabolic disorders. Therefore, mTOR inhibitors with less side effects and high targeting are in urgent need. Nanomaterials play a huge role in medical imaging, detection of intracellular substances to be tested, and targeted delivery of drugs due to their unique physical and chemical properties. The physiological characteristics of the liver that intercept most nanoparticles are the natural advantages of nanobiomaterials targeting the liver. Cerium-based nanoszymes have natural phosphodiesterase activity and can dephosphorylate a variety of proteins. However, there is currently a lack of reports on cerium-based nanoszymes and mTOR and NASH. SUMMARY

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide an mTOR inhibitor with a porous nanostructure and a preparation method and pharmaceutical applications thereof.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application discloses a preparation method of an mTOR inhibitor with a porous nanostructure, comprising: under stirring conditions, adding an aqueous cerium ammonium nitrate solution to an aqueous phytic acid solution, stirring uniformly, filtering, washing, and separating to obtain a CePA material, and preparing the mTOR inhibitor with a porous nanostructure by preparing the CePA material with a buffer solution at a concentration of 40-60 mg / mL.

[0008] Preferably, the aqueous cerium ammonium nitrate solution is prepared by dissolving 10-15 g of cerium ammonium nitrate hexahydrate in 200 mL of pure water.

[0009] Further preferably, the mass ratio of cerium ammonium nitrate hexahydrate to phytic acid is 10-15:8.

[0010] Preferably, the flow rate of the aqueous cerium ammonium nitrate solution is controlled at 5-8 mL / min.

[0011] Preferably, the CePA material is dispersed in PBS buffer to a concentration of 40-60 mg / mL.

[0012] The present application also discloses an mTOR inhibitor with a porous nanostructure prepared by the above-mentioned preparation method, which is a nano-catalyst targeting mTOR with a particle size of 30-50 nm.

[0013] The present application also discloses the use of the above-mentioned mTOR inhibitor with a porous nanostructure in the preparation of a drug for treating / preventing non-alcoholic steatohepatitis.

[0014] Preferably, the drug is a drug that exerts anti-inflammatory and lipid metabolism regulating effects by inhibiting the phosphorylation level of mTOR molecules.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The present application obtains a nano-catalyst targeting mTOR, i.e., a porous cerium phytate nanoparticle, by aqueous phase synthesis. The cerium phytate nanoparticle formed by the coordination of phytic acid and cerium ions has a large number of Ce 3+ , which is the basis for the anti-inflammatory and antioxidant activities of cerium phytate. At the same time, a large number of pores in the material effectively promote the adsorption of active oxygen free radicals, so that it has high antioxidant performance and can be applied to the treatment of various diseases related to active oxygen. In addition, phytic acid, as a ligand molecule constituting CePA, has six phosphate groups and can efficiently coordinate with cerium ions to form CePA material. Phytic acid also has anti-inflammatory, antioxidant and lipid metabolism regulating activities and can effectively enhance the antioxidant performance of cerium-based materials in vivo. Therefore, the nano-catalyst targeting mTOR prepared by the present application has good biocompatibility and anti-inflammatory and antioxidant activities and is expected to be used for disease treatment.

[0017] The nano-catalyst targeting mTOR, i.e., CePA, prepared by the present application can target liver tissues accompanied by lipid metabolism disorders and inflammation and enter liver cells, exert anti-inflammatory and lipid metabolism regulating effects by inhibiting the phosphorylation level of mTOR molecules, reduce the inflammation level and lipid deposition in liver tissues, and relieve the degree of fibrosis of liver tissues. Therefore, CePA can simultaneously exert the effects of metabolic reprogramming and immune microenvironment remodeling, so as to inhibit the progression of NASH. It can be inferred that the inhibitory effect of the product on mTOR signals can also be applied to the treatment of other pathological changes and abnormalities caused by excessive activation of mTOR. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 TEM image of CePA prepared by the present application;

[0019] Figure 2 X-ray photoelectron spectrogram of CePA;

[0020] Figure 3 Specific surface area analysis graph;

[0021] Figure 4 Electron microscope image of CePA in a digestive fluid;

[0022] Figure 5 HE staining result graph of main organs in vivo of mice after different treatments;

[0023] Figure 6 Spatial conformation graph of the combination of CePA and mTOR protein;

[0024] Figure 7 Western blotting results of CePA significantly reducing the phosphorylation level of mTOR in hepatocytes;

[0025] Figure 8 CoIP results of CePA treatment significantly reducing the interaction of mTOR with its upstream signal Akt and mTOR-related regulatory proteins;

[0026] Figure 9 Oil red staining results of CePA inhibiting the accumulation of fatty acids in hepatocytes;

[0027] Figure 10 CePA inhibits the expression of genes related to fatty acid synthesis; where a is srebp1; b is acc; c is fasn;

[0028] Figure 11 CePA inhibits the production of pro-inflammatory factors and promotes the production of anti-inflammatory factors; where a is IL-1; b is IL-6; c is IL-10; d is TGF-β;

[0029] Figure 12 To observe the effect of CePA on the differentiation and phagocytic ability of macrophages treated with inflammatory stimulators;

[0030] Figure 13 Gene expression chart for further verification that CePA affects hepatocyte fatty acid synthesis and macrophage inflammatory factor release through the mTOR pathway after using mTOR inhibitors; where a is srebp1; b is acc; c is fasn; d is acly; e is iNOS; f is IL-6; g is IL-10; h is TGF-β;

[0031] Figure 14 Proteomic and transcriptomic detection PCA chart for mice fed with CePA-containing high-fat feed and simple high-fat mice; where a is the PCA result chart of proteomic detection; b is the PCA result chart of transcriptomic detection;

[0032] Figure 15 Omics data result chart; where a is CePA inhibiting lipid synthesis and inflammatory response levels; b is GSEA analysis of CePA reducing mTOR signal levels; c is a heat map of CePA reducing mTOR signal levels;

[0033] Figure 16 Mice fed with CePA-containing high-fat feed have improved liver function compared to high-fat diet mice; where a is ALT level; b is AST level;

[0034] Figure 17The lipid level and blood glucose level of the mouse fed with the high-fat feed containing CePA are improved compared with the high-fat diet mouse; wherein a is the CHO level; b is the blood glucose level;

[0035] Figure 18 The HE staining graph and the statistical graph of the mouse fed with the high-fat feed containing CePA, which is significantly improved in liver steatosis and inflammation; wherein a is the HE staining result graph; b is the NAS score statistical graph; c is the liver tissue balloon-like change statistical graph; d is the inflammation lesion statistical graph; e is the steatosis statistical graph;

[0036] Figure 19 The liver tissue oil red staining graph of the mouse fed with the high-fat feed containing CePA, which is significantly improved in lipid accumulation; wherein a is the oil red staining graph; b is the oil red staining statistical result graph;

[0037] Figure 20 The liver tissue fatty acid synthesis related molecule expression result graph, the fatty acid synthesis related molecule expression of the mouse fed with the high-fat feed containing CePA is reduced; wherein a is srebp1; b is acc; c is fasn; DETAILED DESCRIPTION

[0038] In order to make the person skilled in the art better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the scope of protection of the present application.

[0039] It should be noted that the terms "first", "second" and the like in the specification and claims of the present application and the above-described drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or a chronological sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not have to be limited to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0040] The present application will be described in further detail below in conjunction with the drawings:

[0041] The present application has prepared a kind of drug phytic acid cerium (CePA) with porous nanostructure composed of cerium ion and phytic acid molecule.

[0042] CePA was synthesized by dissolving 12.4 g of cerium ammonium nitrate hexahydrate in 200 mL of pure water, and then adding the cerium ammonium nitrate aqueous solution to the phytic acid aqueous solution (containing 8 g of phytic acid) under stirring, controlling the flow rate of the cerium ammonium nitrate aqueous solution to be 5 mL / min. After the end of the addition of the cerium ammonium nitrate aqueous solution, the stirring was continued for 12 h, and then the obtained CePA material was separated and washed by a suction filtration device, and finally dispersed in PBS buffer to a CePA concentration of 50 mg / mL, and stored at 4°C for use.

[0043] Abnormal fat accumulation or deposition in the liver (hepatic steatosis), liver inflammation and liver damage or liver tissue damage (liver fibrosis) are characteristics of NASH. In the development of NASH, a large amount of inflammatory factors and other substances are released into the microenvironment by liver tissue, domesticating the immune microenvironment and metabolic microenvironment, and accelerating the progression of NASH. CePA has the characteristics of inflammation driving, and can reach the liver tissue with inflammation caused by abnormal accumulation of lipids through peripheral blood, and through the dual effects of anti-inflammation and regulation of lipid metabolism, reduce the level of liver inflammation and lipid deposition, relieve the degree of liver tissue fibrosis, and ultimately slow down the progression of NASH. Therefore, CePA can inhibit the occurrence and development of NASH by simultaneously targeting metabolic reprogramming and immune microenvironment remodeling.

[0044] I. Experimental methods

[0045] 1. Synthesis of CePA

[0046] CePA was synthesized by dissolving 12.4 g of cerium ammonium nitrate hexahydrate in 200 mL of pure water, and then adding the cerium ammonium nitrate aqueous solution to the phytic acid aqueous solution (containing 8 g of phytic acid) under stirring, controlling the flow rate of the cerium ammonium nitrate aqueous solution to be 5 mL / min. After the end of the addition of the cerium ammonium nitrate aqueous solution, the stirring was continued for 12 h, and then the obtained CePA material was separated and washed by a suction filtration device, and finally dispersed in PBS buffer to a CePA concentration of 50 mg / mL, and stored at 4°C for use.

[0047] 2. Characterization and biological safety verification

[0048] The morphology of cerium phytate nanoparticles obtained by TEM was 30-50 nm in size (see Figure 1 ). XRD of the material verified the formation of cerium phytate particles (see Figure 2 ). Through specific surface area analysis of the cerium phytate material, it was found that the obtained cerium phytate material had a large number of pores, which was beneficial to subsequent biological applications (see Figure 3 ).

[0049] 3. CePA stability and biological safety detection

[0050] CePA stability test results show that CePA materials are stable in the form of complexes in simulated gastric juice and small intestinal juice (see Figure 4 ). In vivo experiments were performed to evaluate the role of CePA in delaying the development of NASH. Side effect evaluation showed that CePA did not change the morphology of different tissues (see Figure 5 ).

[0051] II. CePA inhibits the phosphorylation and activation of mTOR through interaction

[0052] There is increasing evidence that mTOR is not only related to inflammation, but also involved in regulating lipid metabolism. In order to study the pathogenesis and potential therapeutic drugs of NASH, the present application synthesized CePA. According to the molecular lattice structure of CePA and the molecular docking calculation of interaction with mTOR, direct interaction between CePA and mTOR can occur, and the steric hindrance of mTOR 2446 serine is affected (see Figure 6 ). Further experiments confirmed that the phosphorylation level of mTOR 2446 serine in liver cells was significantly reduced after CePA treatment (see Figure 7 ). Phosphorylation modification of 2446 serine is crucial for mTOR signal activation, and this site modification depends on its direct binding and catalysis with Akt kinase. CoIP results show that CePA treatment significantly reduces the interaction of mTOR with Akt, thereby inhibiting its phosphorylation modification (see Figure 8 ).

[0053] III. CePA inhibits lipid synthesis and inflammatory response through mTOR-dependent pathways

[0054] Further in vitro experiments evaluated the effect of CePA on lipid metabolism and inflammatory response. Hepatocytes were cultured with PBS, phytic acid cerium (CePA), fatty acid (FFA), and phytic acid cerium + fatty acid (CePA + FFA), respectively. Then, the improvement effect of phytic acid cerium on lipid accumulation in hepatocytes was detected. The results of oil red staining of hepatocytes showed that the lipid in hepatocytes treated with phytic acid cerium was significantly reduced (see Figure 9 ), and the expression of molecules related to fatty acid synthesis was also reduced, and the lipid metabolism was significantly improved (see Figure 10). Primary macrophages were cultured with PBS, inflammatory stimuli (LPS + IFN-g), CePA, and CePA + inflammatory stimuli, respectively, to detect the expression of inflammation-related molecules and the differentiation and phagocytic capacity of macrophages. The results showed that, after treatment with CePA, the pro-inflammatory factors were significantly reduced compared with the positive control group, and the expression of anti-inflammatory factors was significantly increased (see Figure 11 ); and macrophages tended to differentiate into M2 anti-inflammatory macrophages (see Figure 12 ). To further confirm that CePA exerts regulatory effects by affecting the phosphorylation of mTOR, leucine was added to hepatocytes to effectively activate the phosphorylation of mTOR. The results showed that leucine can significantly reverse the decrease in the expression of lipid metabolism-related genes induced by CePA; at the same time, leucine can increase the expression of pro-inflammatory factors reduced by CePA and reduce the increase in the expression of anti-inflammatory factors caused by CePA (see Figure 13 ).

[0055] IV. CePA alleviates the progression of NASH by inhibiting mTOR signaling

[0056] C57BL / 6 mice were fed with high-fat diet (HFD) for 16 weeks to establish a NASH model, and HFD supplemented with 1% CePA was fed to evaluate the effect of CePA. The livers of mice fed with CePA-containing high-fat feed and simple high-fat mice were taken for proteome and transcriptome detection. The PCA plots of the proteome and transcriptome showed that the intra-group difference of the two sample groups was small, and the inter-group difference was large, indicating that the samples had good uniformity (see Figure 14 ). In addition, CePA inhibited the levels of lipid synthesis and inflammatory response and reduced the level of mTOR signaling (see Figure 15 ); by measuring the serum ALT and AST levels, the results showed that the degree of liver damage induced by high-fat diet containing CePA was significantly lower than that of the HFD group (see Figure 16 ). The results of serum CHO level determination and blood glucose detection showed that the lipid content and blood glucose content induced by high-fat diet containing CePA were significantly lower than those of the HFD group (see Figure 17 ). These data indicate that CePA plays an important regulatory role in diet-induced NASH mouse models.

[0057] The characteristics of NASH are hepatic steatosis, hepatocyte injury (balloon degeneration), inflammation, and fibrosis. We mainly observed the protective effect of CePA on NASH through histological staining. H&E staining was used to observe the damage to the liver tissue of mice. The results showed that the NAS score of HFD mice increased, and there was different degree of balloon degeneration, interlobular inflammatory infiltration, and fatty degeneration, indicating that HFD can significantly aggravate liver damage, and HFD feed containing CePA can significantly reverse the above phenomena (see Figure 17Oil red O staining results showed that CePA could effectively inhibit the significant accumulation of lipid droplets in HFD-induced mouse liver tissues (see Figure 18 ) Then, the expression of genes related to lipid synthesis (srebpl, acc and fasn) in liver tissues was detected, and the results showed that CePA could effectively reduce the increase in the expression of HFD-induced lipid synthesis-related genes (see Figure 19 ) These results showed that CePA could regulate liver steatosis, reduce liver fat deposition, and protect mice from HFD-induced liver damage. The prognosis of NASH and liver-specific diseases is closely related to the degree of liver fibrosis. About 40% of NASH patients will develop fibrosis progression. Although liver fibrosis is not a clinical diagnostic criterion for NASH, the degree of liver fibrosis can predict the prognosis of NASH, including the mortality rate of NASH patients. Therefore, by detecting the degree of HFD-induced liver tissue fibrosis through Massan and Sirius red staining, it was found that CePA could effectively improve the HFD-induced liver tissue fibrosis (see Figure 20 ) In summary, the research results of the present application showed that CePA could protect HFD-induced NASH by regulating lipid droplet aggregation and fat deposition in liver tissues.

[0058] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. A method for preparing an mTOR inhibitor having a porous nanostructure, characterized by, Comprise: Under stirring condition, the cerium ammonium nitrate aqueous solution is added into the phytic acid aqueous solution, and stirred uniformly, then filtered, washed, and separated to obtain the CePA material, which is prepared into a concentration of 40-60 mg / mL with a buffer solution, and the CePA can be combined with mTOR, so as to prepare the mTOR inhibitor with porous nanostructure; The cerium ammonium nitrate aqueous solution is prepared by dissolving 10-15 g of cerium nitrate hexahydrate in 200 mL of pure water; the mass ratio of cerium nitrate hexahydrate to phytic acid is 10-15:

8.

2. The method of claim 1, wherein the mTOR inhibitor having a porous nanostructure is prepared by, The flow rate of the cerium ammonium nitrate aqueous solution is controlled to be 5-8 mL / min.

3. The method of claim 1, wherein the mTOR inhibitor having a porous nanostructure is prepared by, The CePA material is dispersed into a concentration of 40-60 mg / mL with a PBS buffer solution.

4. The mTOR inhibitor having a porous nanostructure, prepared by the preparation method according to any one of claims 1 to 3, characterized in that, The mTOR inhibitor with porous nanostructure is a nano catalyst targeting mTOR, and the particle size is 30-50 nm.

5. The use of the mTOR inhibitor with porous nanostructure in the preparation of a drug for treating / preventing non-alcoholic steatohepatitis according to claim 4.

6. The use according to claim 5, wherein the compound is ###0002### The drug is a drug for playing an anti-inflammatory and lipid metabolism regulating role by inhibiting the phosphorylation level of mTOR molecules.

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