Use of proteasome inhibitor ixazomib in preparing medicine for treating diabetes

Abstract

The present invention belongs to the field of biomedicine technology, and relates to a new use of the proteasome inhibitor ixazomib in pharmaceutical manufacturing, and specifically relates to the use of the proteasome inhibitor ixazomib in the preparation of a drug for treating diabetes, wherein the drug category is a drug for improving insulin resistance in type 2 diabetes, reducing fasting blood sugar, reducing random blood sugar, reducing glycosylated hemoglobin, reducing blood lipids, or improving glucose tolerance. The present invention also provides a method for improving insulin resistance in cells in vitro, and a diabetes drug with ixazomib as an active ingredient provides a new tool and approach for the treatment of diabetes. The tissue chip treated with ixazomib in the present invention provides a series of gene targets related to diabetes, which provides a basis for subsequent research and drug screening.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the use of ixazomib in preparing a drug for treating diabetes, and specifically relates to the use of the proteasome inhibitor ixazomib in preparing a drug for metabolic inflammation and insulin resistance. Background Art

[0002] The prior art discloses that, according to the 2019 IDF Global Diabetes Map, the global prevalence of diabetes has risen to 9.3%, of which more than 90% are type 2 diabetes, and patients with type 2 diabetes are mainly concentrated in the Asia-Pacific region, including China. According to statistics, the total number of diabetes patients in China currently accounts for about a quarter of the world's total [1]. Diabetes and its complications have greatly increased the burden of death and disability worldwide. The 2013 Global Burden of Disease Study showed that diabetes has become the ninth leading cause of shortened life expectancy. A 2016 study showed that the 8-year reduction in life expectancy in the United States is closely related to type 2 diabetes [2].

[0003] Studies have shown that insulin resistance and insufficient insulin secretion are two key factors in the development of type 2 diabetes, but the specific mechanism has not yet been fully elucidated. There is increasing evidence that type 2 diabetes is a chronic low-grade inflammatory disease [3]. This type of inflammation is called metabolic inflammation, which is a series of chronic inflammatory states mediated by traditional inflammatory molecules and their related signaling pathways, triggered by excess nutrition and energy [4]. Cross-sectional studies have found that serum inflammatory markers such as TNF-α, CRP, IL-6, and SAA in patients with type 2 diabetes are significantly higher than those in healthy non-diabetic subjects [5, 6]. In addition, prospective studies have found that healthy non-diabetic subjects with high serum CRP and IL-6 have a risk of developing type 2 diabetes that is 4 times and 2 times that of the control group, respectively [7]. The risk of developing type 2 diabetes in patients with elevated serum IL-1β and IL-6 is 3 times that of the control group [8]. The above evidence fully demonstrates that inflammation is involved in the development of type 2 diabetes. In addition to the increased levels of inflammatory factors such as TNF-α, IL-6, and IL-1β in serum, the levels of pro-inflammatory factors such as TNF-α in the liver, fat[9], muscle

[10] , pancreatic islets

[11] , etc. of patients with type 2 diabetes also increased significantly, and macrophage infiltration increased. In summary, the industry consensus is that type 2 diabetes is essentially a chronic low-grade inflammatory disease.

[0004] Some studies have explored how metabolic inflammation is initiated. Although adipocytes and pancreatic β cells can synthesize and secrete inflammatory factors, the increase in inflammatory factors in patients with type 2 diabetes mainly comes from M1 macrophages infiltrating in fat, liver, muscle, and pancreatic islets [3], mainly in fat and liver. Excessive energy intake leads to fat accumulation in the body and an increase in free fatty acids. Free fatty acids bind to Toll-like receptors 2 or 4 (TLR2 / TLR4) on the surface of macrophages, causing macrophages to polarize to the pro-inflammatory M1 type

[12] , synthesizing and secreting pro-inflammatory factors such as TNF-α and IL-1β. Inflammatory factors further bind to corresponding antibodies on the surface of insulin-sensitive cells such as adipocytes and hepatocytes, causing the cells to produce insulin resistance. IL-1β can also bind to pancreatic β cells, inducing cell apoptosis, leading to insufficient insulin secretion. At the same time, inflammatory factors can further promote adipocytes, macrophages, etc. to secrete chemokines such as monocyte chemoattractant protein 1 (MCP-1), which attract monocytes, T lymphocytes, etc. to infiltrate fat, further expanding inflammation. The degree of macrophage infiltration in fat is positively correlated with the degree of obesity

[13] . At the same time, changes in dietary structure and lifestyle can also lead to intestinal flora disorders in the human body, a decrease in the number of Gram-positive bacteria, an increase in the number of Gram-negative bacteria, and an increase in the permeability between intestinal epithelial cells, thereby causing an increase in serum LPS. LPS binds to TLR4 on the surface of macrophages in fat, liver, etc., activating macrophages [14-16], and can also initiate metabolic inflammation.

[0005] Other studies have shown that inflammation causes insulin resistance in patients with type 2 diabetes. Insulin works by activating the insulin signaling pathway. Insulin recognizes and binds to the insulin receptor IR on the cell membrane, activating the tyrosine kinase activity of IR and promoting phosphorylation of the tyrosine site of IR itself. Phosphorylated IR can recruit the insulin receptor substrate IRS and promote its tyrosine phosphorylation, further activating its downstream AKT and other signaling pathways, inhibiting gluconeogenesis, promoting glycolysis and glycogen synthesis

[17] , and lowering blood sugar. The specific molecular mechanism by which various inflammatory factors cause insulin resistance is mainly through the activation of the IKK-β / NFκB

[18] and JNK (Jun N-terminal kinases) inflammatory signaling pathways. These two inflammatory pathways interfere with the insulin signaling pathway in the following two different ways. A large number of clinical trials and animal experiments have confirmed that inhibiting IKK-β can reduce the phosphorylation level of nuclear factor κB (NFκB) and improve insulin resistance [18-20]. NFκB is one of the most important nuclear transcription factors. Under normal circumstances, NF-κB binds to its inhibitory factor κBα (IκBα) protein and is free in the cytoplasm. After stimulation by inflammatory factors, free fatty acids or LPS, IκBα protein kinase (IKK) is activated, thereby phosphorylating the serine site of IκBα, which is further ubiquitinated and degraded by the proteasome. The nuclear localization sequence of NFκB is released and transferred to the nucleus as a transcription factor to participate in the transcription of proinflammatory factors such as TNF-α and IL-6

[21] . Inflammatory factors further activate the IKK-β / NFκB and JNK signaling pathways, leading to insulin resistance. Unlike IKK-β / NFκB, JNK inhibits IRS1 tyrosine phosphorylation by directly phosphorylating the serine site of IRS1. Serine-phosphorylated IRS1 is further ubiquitinated and degraded by the proteasome, blocking the insulin signaling pathway and leading to insulin resistance

[22] .

[0006] Studies have confirmed that type 2 diabetes, atherosclerosis, obesity, and non-alcoholic fatty liver disease are metabolic inflammatory diseases closely related to chronic low-grade inflammation. Some researchers have proposed a new concept of metabolic inflammatory syndrome: patients with two of the above four metabolic diseases can be diagnosed with metabolic inflammatory syndrome (MIS). [23-24] This concept is conducive to the same treatment and prevention of different diseases. Clinical studies have used anti-inflammatory therapy to treat type 2 diabetes and obtained preliminary results. For example, non-steroidal anti-inflammatory drugs can lower blood sugar in patients with type 2 diabetes and obesity, but the dose required for lowering blood sugar is far more than the commonly used clinical dose by about 10 times. At the same time, high doses can also cause many serious adverse reactions, such as salicylic acid causing liver damage and deafness, and aspirin causing liver damage and coagulation dysfunction.

[25] Therefore, these commonly used anti-inflammatory drugs cannot be used to treat type 2 diabetes in clinical practice. In addition to nonsteroidal anti-inflammatory drugs, studies have also proposed specific inhibitors of inflammatory factors, such as anti-TNFα monoclonal antibodies, anti-IL1β monoclonal antibodies and IL-1 receptor antagonists. However, clinical trials have shown that the hypoglycemic effect is weak

[26] . The reason may be that inhibiting a single inflammatory factor is not enough to completely improve metabolic inflammation. Therefore, it is necessary to find new anti-inflammatory targets for the treatment of type 2 diabetes and related metabolic diseases (MIS).

[0007] It is not difficult to see from the mechanism of inflammation leading to insulin resistance that the proteasome plays a vital role in it. It can not only degrade ubiquitinated IκBα to activate NFκB, promote the transcription of inflammatory factors, and lead to insulin resistance, but also degrade ubiquitinated IRS1, block the insulin signaling pathway, and lead to insulin resistance. Whether the use of proteasome inhibitors can reverse the metabolic inflammation and insulin resistance of type 2 diabetes has become a research focus in the industry.

[0008] The proteasome is an important component of the ubiquitin proteasome pathway, a classic protein degradation pathway in nucleated cells. It is a multimer composed of multiple subunits, including a barrel-shaped 20s core particle and two upper and lower 19s regulatory subunits. The core particle is composed of four axially stacked isoheptane rings (two outer α rings and two inner β rings), each of which is connected by seven subunits. The β1, β2 and β5 subunits of the β ring contain peptidyl glutamyl hydrolase, trypsin and chymotrypsin activities, respectively. Proteins are degraded into small molecular peptides here. The regulatory subunit is also composed of multiple subunits and has the function of recognizing and binding ubiquitin on the substrate and transporting the substrate to the core particle

[27] . The proteasome is involved in regulating processes such as cell apoptosis and proliferation in the body. Proteasome inhibitors have been used clinically as an effective anti-multiple myeloma drug. However, in the past 10 years, more and more studies have found that the proteasome is also involved in antigen presentation, inflammatory response and metabolic regulation

[28] . So, as type 2 diabetes is a metabolic inflammatory disease, can proteasome inhibitors be used as new anti-inflammatory drugs to treat type 2 diabetes? Most of the known related studies focus on the use of proteasome inhibitors to treat chronic inflammatory diseases, including arthritis, encephalomyelitis, and lupus erythematosus

[30] . The results of several studies related to diabetes are inconsistent. It has been reported that the proteasome inhibitor bortezomib can prevent NOD mice from developing type 1 diabetes, but one week of medication failed to reduce the blood sugar of NOD mice that have already developed the disease

[30] . Curcumin, an extract of the traditional Chinese medicine turmeric, can reduce the blood sugar of db / db mice by inhibiting proteasome activity

[31] . The hypoglycemic drug rosiglitazone can inhibit proteasome activity

[33] . Aghdam et al. also found that proteasome inhibitors can improve diabetic nephropathy

[33] . However, there are also reports that proteasome inhibitors increase endoplasmic reticulum stress in hepatocytes and reduce AKT phosphorylation levels

[34] . However, there is no direct evidence that proteasome inhibitors can improve metabolic inflammation and insulin resistance in type 2 diabetes, and the indirect evidence is inconsistent.

[0009] Based on the foundation and current status of the prior art, the inventors of the present application intend to provide a new drug for treating diabetes, involving the use of ixazomib in the preparation of drugs for treating diabetes, especially the use of the proteasome inhibitor ixazomib in the preparation of drugs for metabolic inflammation and insulin resistance.

[0010] References related to the present invention include:

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[0031] 21.Hadi Khodabandehloo,Gorgani-Firuzjaee Sattar,Panahi Ghodratollah,et al.Molecular and cellular mechanisms linking inflammation to insulinresistance and beta-cell dysfunction[J].TRANSLATIONAL RESEARCH,2016,167(1):228-256.

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[0039] 30.G Mondanelli,Albini E,Pallotta M-T,et al.The Proteasome InhibitorBortezomib Controls Indoleamine 2,3-Dioxygenase 1Breakdown and RestoresImmune Regulation in Autoimmune Diabetes[J].Front Immunol,2017,8428.

[0040] 31. S Weisberg, Leibel R, Tortoriello D-V. Proteasome inhibitors, including curcumin, improve pancreatic beta-cell function and insulin sensitivity in diabetic mice[J]. Nutr Diabetes, 2016, 6e205.

[0041] 32. X Wang, Hu Z, Hu J, et al. Insulin resistance accelerates muscle protein degradation: Activation of the ubiquitin-proteasome pathway by defects in muscle cell signaling[J]. Endocrinology, 2006, 147(9): 4160-4168.

[0042] 33. S-Y Aghdam, Gurel Z, Ghaffarieh A, et al. High glucose and diabetes modulate cellular proteasome function: Implications in the pathogenesis of diabetes complications[J]. Biochem Biophys Res Commun, 2013, 432(2): 339-344.

[0043] 34. T Otoda, Takamura T, Misu H, et al. Proteasome dysfunction mediates obesity-induced endoplasmic reticulum stress and insulin resistance in the liver[J]. Diabetes, 2013, 62(3): 811-824. Summary of the Invention

[0044] The purpose of the present invention is to provide a new drug for treating diabetes based on the existing technology and the current status, which relates to the use of ixazomib in the preparation of drugs for treating diabetes, especially the use of the proteasome inhibitor ixazomib in the preparation of drugs for metabolic inflammation and insulin resistance.

[0045] The present invention provides a new drug for treating or alleviating diabetes, and specifically provides use of the proteasome inhibitor ixazomib in preparing drugs for metabolic inflammation and insulin resistance.

[0046] In order to solve the above technical problems, the present invention experimentally studied the potential target of ixazomib for the treatment of diabetes and metabolic inflammatory syndrome. The experimental results confirmed that ixazomib can reduce fasting blood sugar, random blood sugar, glycosylated hemoglobin and blood lipids in db / db mice, an animal model of type 2 diabetes, and improve the glucose tolerance of mice. The results showed that it mainly reduces blood sugar by improving the insulin sensitivity of db / db mice. Oral administration, twice a week, after 6 weeks of intervention, significantly reduced the HOMA-IR index and fasting insulin level of db / db mice. After intraperitoneal injection of insulin, the rate of blood sugar decline in the drug intervention group was significantly accelerated. In addition, the results showed that ixazomib significantly increased the phosphorylation level of Akt in the insulin signaling pathway of db / db mice liver, as well as the IRS1 protein content. The experimental results of the present invention confirmed that ixazomib increased the insulin sensitivity of type 2 diabetic mice.

[0047] Furthermore, the present invention prepared a protein chip for the liver of db / db mice intervened by ixazomib, and screened out 14 differentially expressed proteins, of which 11 were upregulated and 3 were downregulated. The two most obviously upregulated proteins, TRAIL and Axl, both had an inhibitory effect on inflammation, and the two most obviously downregulated proteins both had the effect of promoting inflammatory response, indicating that the proteasome inhibitor inhibits metabolic inflammation in type 2 diabetic mice through TRAIL or Axl, thereby improving insulin resistance and lowering blood sugar.

[0048] The present invention provides use of a proteasome inhibitor ixazomib in preparing a drug for treating diabetes, in particular, use of the proteasome inhibitor ixazomib in preparing a drug for treating metabolic inflammation and insulin resistance.

[0049] Preferably, the drug is a drug for improving insulin resistance in type 2 diabetes, lowering fasting blood sugar, lowering random blood sugar, lowering glycosylated hemoglobin, lowering blood lipids or improving glucose tolerance.

[0050] Preferably, the drug is a drug that increases the phosphorylation level of Akt and the content of IRS1 protein in the liver.

[0051] Preferably, the drug is a drug for improving metabolic inflammation.

[0052] Preferably, the drug can:

[0053] Upregulation of TRAIL, Axl, CD36, PF4, CD40, Fas, IL-17F, IL-2Ra, OPN, IGFBP-6, I-TAC gene expression; or

[0054] Downregulate NOV, CTLA4 or CD6 gene expression.

[0055] The present invention also provides a group of screening targets for diabetes drugs, wherein the screening targets are selected from one or more of the following genes:

[0056] TRAIL, Axl, CD36, PF4, CD40, Fas, IL-17F, IL-2Ra, OPN, IGFBP-6, I-TAC, NOV, CTLA4, or CD6.

[0057] In a preferred embodiment of the present invention, tissue samples before and after ixazomib treatment were compared, and the results showed that the expression of TRAIL, Axl, CD36, PF4, CD40, Fas, IL-17F, IL-2Ra, OPN, IGFBP-6, and I-TAC genes was upregulated; meanwhile, the expression of NOV, CTLA4 or CD6 genes was downregulated.

[0058] The present invention also provides a method for alleviating insulin resistance of cells cultured in vitro, the method comprising the following steps:

[0059] (1) collecting pancreatic islet cells and culturing them in an in vitro cell culture medium; and / or

[0060] (2) Add ixazomib to the cell culture medium and incubate.

[0061] On the other hand, the ixazomib of the present invention can be used alone or in combination with other solvents and diabetes drugs to prepare a drug for treating or alleviating diabetes.

[0062] Furthermore, the present invention provides a pharmaceutical composition for treating diabetes, wherein the pharmaceutical composition contains ixazomib and a commonly used injection solvent in medicine.

[0063] Preferably, the pharmaceutical composition further contains bortezomib.

[0064] The present invention studies have confirmed that the proteasome inhibitor ixazomib can improve insulin resistance and lower blood sugar in type 2 diabetic db / db mice, and at the same time confirmed that it is possible to inhibit metabolic inflammation in type 2 diabetic mice through TRAIL or Axl, thereby improving insulin resistance and lowering blood sugar. The ixazomib of the present invention can significantly improve clinical indicators of diabetes, improve insulin resistance in type 2 diabetes, reduce fasting blood sugar, reduce random blood sugar, reduce glycosylated hemoglobin, reduce blood lipids or improve glucose tolerance, and provide new intervention tools and approaches for the treatment of diabetes. The tissue chip treated with ixazomib in the present invention provides a series of gene targets related to diabetes, which provides a basis for subsequent research and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 :Proteasome inhibitors ixazomib and bortezomib (ps-341) affect the changes of db / db mouse parameters.

[0066] Among them, a, random blood glucose; b, fasting blood glucose; e, glycosylated hemoglobin and f, serum total cholesterol. c, body weight and d, food intake of mice were not changed.

[0067] Figure 2 :IPGTT showed that proteasome inhibitor intervention improved the glucose tolerance of db / db mice.

[0068] Figure 3 :The changes of blood glucose and insulin levels in mice by insulin and ixazomib,

[0069] Among them, a, b, c, proteasome inhibitors increased the rate of blood glucose decline in db / db mice after intraperitoneal injection of insulin; d, ixazomib reduced the HOMA-IR index of db / db mice; e, ixazomib reduced the fasting insulin level of db / db mice.

[0070] Figure 4 :The effect of ixazomib on the changes of blood glucose related protein levels,

[0071] Among them, a, ixazomib increased the p-Akt level and b, IRS1 protein content in the liver of db / db mice; c, ixazomib did not change the expression of IRS1 in the liver of db / db mice.

[0072] Figure 5 :Differential protein result diagram,

[0073] Among them, the screening conditions are: p value is less than 0.05, and foldchange is greater than 1.2 or less than 0.83; i13, i14, i7, and i4 are ixazomib-intervention db / db mice; con2.14, con2.11, con2.13, and con2.12 are control db / db mice.

[0074] Figure 6 :GO analysis suggested that ixazomib improved liver inflammation in db / db mice. DETAILED DESCRIPTION

[0075] The technical solutions in the embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0076] Example 1

[0077] The type 2 diabetes animal model db / db mice were injected with ixazomib for intervention. The results showed that the proteasome inhibitor ixazomib reduced the fasting blood glucose, random blood glucose, glycated hemoglobin and blood lipids of db / db mice and improved glucose tolerance.

[0078] Figure 1 Figure 2 shows that the proteasome inhibitors ixazomib and bortezomib (PS-341) reduced a, random blood glucose; b, fasting blood glucose; e, glycosylated hemoglobin and f, serum total cholesterol in db / db mice, but did not change c, body weight and d, food intake of the mice.

[0079] Figure 2 As shown in the figure, IPGTT showed that proteasome inhibitor intervention improved glucose tolerance in db / db mice.

[0080] Example 2

[0081] Oral administration twice a week for 6 weeks significantly reduced the HOMA-IR index and fasting insulin levels of db / db mice. After intraperitoneal injection of insulin, the rate of blood sugar decline in the drug intervention group was also significantly accelerated. The proteasome inhibitor ixazomib improved the insulin sensitivity of db / db mice.

[0082] Figure 3a, b, and c show that proteasome inhibitors increased the rate of blood glucose decline in db / db mice after intraperitoneal injection of insulin; d shows that ixazomib reduced the HOMA-IR index of db / db mice; e shows that ixazomib reduced the fasting insulin level of db / db mice.

[0083] Figure 4 a, ixazomib increased the p-Akt level and b, IRS1 protein content in the liver of db / db mice; c, ixazomib did not change the expression of IRS1 in the liver of db / db mice.

[0084] Example 3

[0085] Raybiotech Mouse Cytokine Array GS4000 protein chip showed that the proteasome inhibitor ixazomib can improve liver inflammation in db / db mice.

[0086] in, Figure 5 Screening conditions for differentially expressed proteins: p value less than 0.05, and foldchange greater than 1.2 or less than 0.83; i13, i14, i7, and i4 are ixazomib-treated db / db mice; con2.14, con2.11, con2.13, and con2.12 are control db / db mice.

[0087] Table 1: 14 differentially expressed proteins screened by protein microarray

[0088]

[0089] Among them, most pro-inflammatory proteins were down-regulated, while anti-inflammatory proteins were up-regulated. +: promotes inflammation; -: inhibits inflammation; + / -: promotes and inhibits inflammation; UK: unknown.

Claims

1. Use of the proteasome inhibitor ixazomib in the preparation of a drug for treating an insulin resistance disease, wherein the insulin resistance disease is type 2 diabetes.

2. The use according to claim 1, characterized in that The medicine is a medicine for improving insulin resistance of type 2 diabetes, lowering fasting blood sugar, lowering random blood sugar, lowering glycosylated hemoglobin, lowering blood lipids or improving glucose tolerance.

3. The use according to claim 1, characterized in that The drug is a drug that increases the phosphorylation level of Akt and the content of IRS1 protein in the liver.

4. The use according to claim 1, characterized in that: The drug can: up-regulate the gene expression of TRAIL, Axl, CD36, PF4, CD40, Fas, IL-17F, IL-2 Ra, OPN, IGFBP-6, I-TAC; or, down-regulate NOV, CTLA4, or CD6 gene expression.

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