Application of asparagine endopeptidase inhibitor in preparation of medicine for improving insulin resistance

Abstract

The invention relates to application of an asparagine endopeptidase inhibitor in preparation of a medicine for improving insulin resistance. According to the application, starting from inhibiting the aging driving factor AEP, the AEP inhibitor is tried to be applied to the research of insulin resistance for the first time, and the AEP inhibitor is found to be capable of remarkably improving glucose tolerance and insulin sensitivity. Therefore, the invention provides the application of the asparagine endopeptidase inhibitor in preparation of the medicine for improving insulin resistance. Experiments prove that the AEP inhibitor not only can significantly enhance insulin sensitivity of old mice and improve glucose tolerance, but also can reduce serum low-density lipoprotein cholesterol and lactic dehydrogenase levels, shows multiple improvement effects on glucose metabolism and lipid metabolism, and also can be used for preparing the AEP inhibitor. After long-term administration, obvious toxic and side effects are not found in pathological examinations of blood routine examination, urine routine examination, heart, liver, spleen, lung, kidney and other important organs, so that the traditional Chinese medicine composition is proved to have good safety and is suitable for long-term intervention of chronic diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of asparagine endopeptidase inhibitors in the preparation of drugs that improve insulin resistance. Background Technology

[0002] Insulin resistance is a pathological condition in which target tissues (mainly skeletal muscle, liver, and white adipose tissue) are less sensitive to insulin. Normal levels of insulin are insufficient to promote glucose uptake by peripheral tissues, inhibit hepatic gluconeogenesis, and regulate lipid metabolism. The body then needs to compensate by secreting supraphysiological doses of insulin to maintain blood glucose homeostasis.

[0003] However, most existing drug intervention strategies for insulin resistance target specific downstream effector molecules: metformin activates AMP-activated protein kinase (AMPK), and thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor γ (PPARγ). While these drugs improve insulin sensitivity to some extent, they fail to address the root cause upstream and have significant side effects: thiazolidinediones can lead to weight gain and increased fracture risk; although metformin has a relatively good safety profile, gastrointestinal side effects still limit long-term use for some patients. Therefore, there is an urgent clinical need for novel intervention strategies that can target upstream driving factors and systematically improve insulin resistance at its root. Summary of the Invention

[0004] Studies have found that aging itself is an independent risk factor for insulin resistance. Mitochondrial dysfunction, chronic low-grade inflammation, and NAD+ are also contributing factors. + The synergistic effect of multiple aging markers, such as decreased insulin levels and impaired autophagy, jointly drives age-related decline in insulin sensitivity. Asparagine endopeptidase (AEP), a lysosomal cysteine ​​protease, has been proven to be a key molecule driving pathological aging. As a protease that cleaves multiple substrates, it has potential applications in neurodegenerative diseases. This application, starting with the inhibition of the aging driver AEP, is the first attempt to apply AEP inhibitors to research on insulin resistance, finding that AEP inhibitors can significantly improve glucose tolerance and insulin sensitivity. Based on this, this application provides the application of asparagine endopeptidase inhibitors in the preparation of drugs to improve insulin resistance.

[0005] In a first aspect, this application provides the use of asparagine endopeptidase inhibitors in the preparation of drugs that improve insulin resistance.

[0006] The experimental verification of this application shows that AEP inhibitors not only significantly enhance insulin sensitivity and improve glucose tolerance in aged mice, but also reduce serum low-density lipoprotein cholesterol and lactate dehydrogenase levels, demonstrating multiple effects on improving glucose and lipid metabolism. Furthermore, no obvious toxic side effects were observed in routine blood tests, routine urine tests, and pathological examinations of important organs such as the heart, liver, spleen, lungs, and kidneys after long-term administration, confirming that it has good safety and is suitable for long-term intervention in chronic diseases.

[0007] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0008] In a second aspect, this application provides the use of asparagine endopeptidase inhibitors in the preparation of drugs that enhance insulin sensitivity and / or regulate glucose tolerance.

[0009] In some embodiments, the drug can also reduce serum levels of at least one of the following substances: low-density lipoprotein, cholesterol, and lactate dehydrogenase.

[0010] A third aspect of this application provides the use of asparagine endopeptidase inhibitors in the preparation of medicaments for the prevention or treatment of metabolic diseases.

[0011] In some embodiments, the metabolic disease is an age-related metabolic disease; Furthermore, the metabolic diseases include insulin resistance caused by natural aging.

[0012] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0013] In some embodiments, the drug is suitable for oral administration; And / or, the effective dose of the asparagine endopeptidase inhibitor is 7.5 mg / kg.

[0014] In a fourth aspect, this application provides a medicament for improving insulin resistance, comprising an asparagine endopeptidase inhibitor.

[0015] In some embodiments, it also contains pharmaceutically acceptable excipients; And / or, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ) below: Equation (Ⅰ). Attached Figure Description

[0016] Figure 1The results of glucose tolerance test and insulin tolerance test to assess metabolic function, as well as the results of serum alanine aminotransferase, aspartate aminotransferase, low-density lipoprotein cholesterol and lactate dehydrogenase concentrations, are shown in the examples.

[0017] Figure 2 The images show the detection results of the concentration of drug #11a in serum and representative H&E staining images of the heart, liver, spleen, lungs and kidneys in the examples. Detailed Implementation

[0018] The technical solution of this application will be further described in detail below with reference to specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0020] As used herein, the terms “and / or,” “or / and,” and “and / or” may include any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0021] In this article, "one or at least one" means any one, any two, or any two or more of the listed items.

[0022] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0023] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0024] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0025] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0026] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0027] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0028] Currently, drugs for treating insulin resistance have a single mechanism of action, targeting only specific pathways. For example, metformin activates AMP-activated protein kinase (AMPK), and thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor γ (PPARγ). These drugs improve insulin sensitivity to some extent, but their targets are not the core drivers of the aging process. Insulin resistance is essentially a manifestation of the aging process in peripheral metabolic organs, and its occurrence involves mitochondrial dysfunction, chronic inflammation, and NAD+. +Synergistic effects of multiple aging markers such as exhaustion and autophagy insufficiency. Simply intervening in downstream effector molecules like AMPK or PPARγ is insufficient to reverse systemic metabolic disorders caused by upstream aging drivers, resulting in limited efficacy and difficulty in achieving fundamental improvement. Furthermore, these drugs have significant side effects; long-term use carries risks of liver and kidney toxicity, weight gain, and fractures, making them unsuitable for long-term use. Asparagine endopeptidase (AEP) is a lysosomal cysteine ​​protease that has recently been identified as a key molecule driving pathological aging. As a protease that cleaves multiple substrates, it can simultaneously act on multiple aging-related substrates. While existing technologies have disclosed the role of AEP in neurodegenerative diseases, they completely fail to address its function in regulating insulin sensitivity in peripheral tissues (such as muscle, fat, and liver), and there is no disclosure that AEP inhibitors can improve insulin resistance.

[0029] This application, starting with the inhibition of the aging driver factor AEP, is the first attempt to apply AEP inhibitors to research on insulin resistance, finding that AEP inhibitors can significantly improve glucose tolerance and insulin sensitivity. Based on this, this application provides the use of asparagine endopeptidase inhibitors in the preparation of drugs that improve insulin resistance.

[0030] The technical solution of this application is described in detail below: In a first aspect, this application provides the use of asparagine endopeptidase inhibitors in the preparation of drugs that improve insulin resistance.

[0031] As a cysteine ​​protease, the abnormal activation of AEP is both a direct consequence of the aging microenvironment (such as lysosomal stress, oxidative damage, and chronic inflammation) and a direct trigger for promoting the decline of multiple tissue functions by producing toxic protein fragments, amplifying SASP, and inducing the accumulation of DNA damage. Whether AEP acts as a bridge connecting the basic aging process with the degenerative diseases of multiple organs and affects insulin metabolism in the context of natural aging still lacks direct in vivo experimental evidence.

[0032] The choice of long-term administration method requires comprehensive consideration of factors such as the physicochemical properties of the drug, administration frequency, animal stress response, and experimental objectives. Dietary supplementation (feed mixing) offers advantages such as ease of operation, no operational stress on animals, suitability for long-term chronic administration, and the ability to simulate the once-daily or multiple-dose oral administration pattern of clinical patients. Furthermore, it avoids the stress interference caused by daily capture and gavage, making it more suitable for aging studies and long-term survival experiments. For long-term chronic intervention studies aiming to delay aging and improve insulin resistance, dietary supplementation has irreplaceable advantages due to its low stress level, close resemblance to clinical reality, and suitability for survival analysis.

[0033] The experimental verification of this application shows that AEP inhibitors not only significantly enhance insulin sensitivity and improve glucose tolerance in aged mice, but also reduce serum low-density lipoprotein cholesterol and lactate dehydrogenase levels, demonstrating multiple effects on improving glucose and lipid metabolism. Furthermore, no obvious toxic side effects were observed in routine blood tests, routine urine tests, and pathological examinations of important organs such as the heart, liver, spleen, lungs, and kidneys after long-term administration, confirming that it has good safety and is suitable for long-term intervention in chronic diseases.

[0034] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0035] The asparagine endopeptidase inhibitor with the above-described structure is an AEP small molecule inhibitor (compound #11a), which can significantly improve glucose tolerance and insulin sensitivity in naturally aging mice and has the potential to improve insulin resistance. It should be noted that the asparagine endopeptidase inhibitor is not limited to the AEP small molecule inhibitor (compound #11a), but can also be other asparagine endopeptidase inhibitors.

[0036] In some embodiments, the drug is suitable for oral administration. Oral administration is convenient, safe, and economical. This method eliminates the need for injections, is painless and non-invasive, greatly improving patient adherence to long-term medication; at the same time, the administration method is gentle, allowing for timely intervention in case of overdose, thus enhancing safety. Furthermore, oral formulations have lower production costs, and patients can take them independently without the assistance of professional medical personnel, saving medical resources. With the help of diverse dosage form designs such as sustained-release and enteric coatings, it can also achieve stable drug release or provide local treatment for gastrointestinal diseases.

[0037] In some embodiments, the effective dose of asparagine endopeptidase inhibitor is 7.5 mg / kg. At this dose, the asparagine endopeptidase inhibitor significantly improves glucose tolerance and insulin sensitivity in naturally aging mice.

[0038] In a second aspect, this application provides the use of asparagine endopeptidase inhibitors in the preparation of drugs that enhance insulin sensitivity and / or regulate glucose tolerance.

[0039] This application study found that asparagine endopeptidase inhibitors can enhance insulin sensitivity and / or regulate glucose tolerance, and can be used to prepare drugs that enhance insulin sensitivity and / or regulate glucose tolerance.

[0040] In some embodiments, the drug can also reduce serum levels of at least one of the following substances: low-density lipoprotein, cholesterol, and lactate dehydrogenase.

[0041] This application study found that asparagine endopeptidase inhibitors can also reduce the level of at least one of the following substances in serum: low-density lipoprotein, cholesterol and lactate dehydrogenase, for use in the preparation of drugs that reduce the levels of the above three substances in serum.

[0042] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0043] The asparagine endopeptidase inhibitor with the above-described structure is an AEP small molecule inhibitor (compound #11a), which can significantly improve glucose tolerance and insulin sensitivity in naturally aging mice and has the potential to improve insulin resistance. It should be noted that the asparagine endopeptidase inhibitor is not limited to the AEP small molecule inhibitor (compound #11a), but can also be other asparagine endopeptidase inhibitors.

[0044] In some embodiments, the drug is suitable for oral administration. Oral administration is convenient, safe, and economical. This method eliminates the need for injections, is painless and non-invasive, greatly improving patient adherence to long-term medication; at the same time, the administration method is gentle, allowing for timely intervention in case of overdose, thus enhancing safety. Furthermore, oral formulations have lower production costs, and patients can take them independently without the assistance of professional medical personnel, saving medical resources. With the help of diverse dosage form designs such as sustained-release and enteric coatings, it can also achieve stable drug release or provide local treatment for gastrointestinal diseases.

[0045] In some embodiments, the effective dose of asparagine endopeptidase inhibitor is 7.5 mg / kg. At this dose, the asparagine endopeptidase inhibitor significantly improves glucose tolerance and insulin sensitivity in naturally aging mice.

[0046] A third aspect of this application provides the use of asparagine endopeptidase inhibitors in the preparation of medicaments for the prevention or treatment of metabolic diseases.

[0047] This application marks the first time that the application scope of AEP inhibitors has been expanded to the field of metabolic diseases.

[0048] In some embodiments, the metabolic disease is an age-related metabolic disease.

[0049] Furthermore, the metabolic diseases include insulin resistance caused by natural aging. It should be noted that the metabolic diseases are not limited to insulin resistance caused by natural aging, but may also include other age-related metabolic diseases.

[0050] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0051] The asparagine endopeptidase inhibitor with the above-described structure is an AEP small molecule inhibitor (compound #11a), which can significantly improve glucose tolerance and insulin sensitivity in naturally aging mice and has the potential to improve insulin resistance. It should be noted that the asparagine endopeptidase inhibitor is not limited to the AEP small molecule inhibitor (compound #11a), but can also be other asparagine endopeptidase inhibitors.

[0052] In some embodiments, the drug is suitable for oral administration. Oral administration is convenient, safe, and economical. This method eliminates the need for injections, is painless and non-invasive, greatly improving patient adherence to long-term medication; at the same time, the administration method is gentle, allowing for timely intervention in case of overdose, thus enhancing safety. Furthermore, oral formulations have lower production costs, and patients can take them independently without the assistance of professional medical personnel, saving medical resources. With the help of diverse dosage form designs such as sustained-release and enteric coatings, it can also achieve stable drug release or provide local treatment for gastrointestinal diseases.

[0053] In some embodiments, the effective dose of asparagine endopeptidase inhibitor is 7.5 mg / kg. At this dose, the asparagine endopeptidase inhibitor significantly improves glucose tolerance and insulin sensitivity in naturally aging mice.

[0054] In a fourth aspect, this application provides a medicament for improving insulin resistance, said medicament containing an asparagine endopeptidase inhibitor.

[0055] In some embodiments, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ): Equation (Ⅰ).

[0056] The asparagine endopeptidase inhibitor with the above-described structure is an AEP small molecule inhibitor (compound #11a), which can significantly improve glucose tolerance and insulin sensitivity in naturally aging mice and has the potential to improve insulin resistance. It should be noted that the asparagine endopeptidase inhibitor is not limited to the AEP small molecule inhibitor (compound #11a), but can also be other asparagine endopeptidase inhibitors.

[0057] In some embodiments, the aforementioned medicament further contains pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may be, for example, carriers, adjuvants, excipients, or diluents, and are well known to those skilled in the art and readily available to the public. Optionally, the pharmaceutically acceptable carrier is an excipient that is chemically inert to asparagine endopeptidase inhibitors and does not have harmful side effects or toxicity under the conditions of use.

[0058] In some embodiments, the drug is suitable for oral administration. Oral administration is convenient, safe, and economical. This method eliminates the need for injections, is painless and non-invasive, greatly improving patient adherence to long-term medication; at the same time, the administration method is gentle, allowing for timely intervention in case of overdose, thus enhancing safety. Furthermore, oral formulations have lower production costs, and patients can take them independently without the assistance of professional medical personnel, saving medical resources. With the help of diverse dosage form designs such as sustained-release and enteric coatings, it can also achieve stable drug release or provide local treatment for gastrointestinal diseases.

[0059] In some embodiments, the effective dose of asparagine endopeptidase inhibitor is 7.5 mg / kg. At this dose, the asparagine endopeptidase inhibitor significantly improves glucose tolerance and insulin sensitivity in naturally aging mice.

[0060] In some embodiments, the drug is in the form of a liquid or a solid dosage form. The dosage form of the drug can be adjusted using the pharmaceutically acceptable excipients described above.

[0061] The technical solution of this application has at least the following beneficial effects: This application is the first to discover AEP as a novel target for regulating peripheral insulin sensitivity; it provides a novel anti-aging intervention strategy that differs from traditional hypoglycemic drugs. Unlike existing symptomatic strategies that simply lower blood sugar, this application addresses the root cause by inhibiting AEP, a newly confirmed driver of aging, thereby fundamentally improving age-related systemic metabolic decline. AEP not only significantly enhances insulin sensitivity and improves glucose tolerance in aged mice, but also reduces serum low-density lipoprotein cholesterol and lactate dehydrogenase levels, demonstrating multiple ameliorative effects on glucose and lipid metabolism. More importantly, no significant toxic side effects were observed in blood routine tests, urine routine tests, and pathological examinations of important organs such as the heart, liver, spleen, lungs, and kidneys after long-term administration, confirming its good safety profile and suitability for long-term intervention in chronic diseases.

[0062] This application provides for the first time a technical solution and experimental evidence for AEP inhibitors to improve insulin resistance, and expands the clinical application of AEP inhibitors to the field of metabolic diseases for the first time.

[0063] The following is a specific embodiment.

[0064] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0065] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0066] Unless otherwise specified, the structural formula of the AEP small molecule inhibitor (compound #11a, hereinafter referred to as: AEP inhibitor #11a) involved in the following examples is shown in formula (Ⅰ), and it was synthesized by Sandia Pharmaceutical Technology (Shanghai) Co., Ltd.: Equation (Ⅰ).

[0067] Unless otherwise specified, the 18-month-old C57BL / 6 mice used in the following examples were obtained from Jiangsu Airingfei Biotechnology Co., Ltd.

[0068] Example 1 The experimental design of this embodiment is as follows: An 18-month-old C57BL / 6 mouse model of natural aging was constructed to simulate the age-related insulin resistance process. The experimental group mice were fed a diet containing 7.5 mg / kg of the AEP inhibitor #11a for 5 consecutive months, while the control group was given a normal diet. After the administration, the glucose homeostasis and insulin sensitivity of the mice were assessed using the intraperitoneal glucose tolerance test (GTT) and the intraperitoneal insulin tolerance test (ITT), followed by serum biochemical analysis of physiological changes. The specific experimental procedure is as follows: 1. Eighteen-month-old C57BL / 6 mice were housed under a 12-hour light-dark cycle, with the ambient temperature maintained at 20-22℃. The mice were randomly divided into two groups: (1) Control group: fed standard feed (standard feed from Nantong Trofi Feed Technology Co., Ltd., product code LAD3001M). (2) Experimental group (i.e. #11a group): AEP inhibitor #11a (7.5 mg / kg body weight) was added to the same standard feed. Nantong Trofi Feed Technology Co., Ltd. was commissioned to add AEP inhibitor #11a to the standard feed at a dose of 7.5 mg / kg.

[0069] 2. Mice were fed according to the above feeding method for 5 months. At the 5th month, glucose homeostasis and insulin sensitivity were assessed using an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin tolerance test (IPITT). Blood glucose levels were measured at specified time points after injection using a blood glucose meter. For the IPGTT, mice were fasted for 12 hours before receiving an intraperitoneal injection of sterile glucose solution (2 g / kg). For the IPITT, mice were fasted for 6 hours before receiving an intraperitoneal injection of human insulin (0.5 U / kg). Blood glucose levels were measured using a Roche blood glucose meter via tail vein sampling before injection (0 minutes) and at specific time points after injection (0, 15, 30, 60, 90, 120 minutes).

[0070] 3. After 5 months of dietary feeding, mice were deeply anesthetized with isoflurane at an induction concentration of 5% (v / v) and a maintenance concentration of 2% (v / v). They were then euthanized via cardiac puncture. Whole blood was collected in EDTA anticoagulant tubes and centrifuged at 4 °C, 2000 × g for 10 min to separate plasma. The serum concentration of drug #11a was determined by LC-MS / MS. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), low-density lipoprotein cholesterol (LDL-C), and lactate dehydrogenase (LDH) concentrations were determined by Wuhan Saiwei Biotechnology Co., Ltd. using standard automated biochemical analysis methods. The mice were then perfused with a peristaltic pump system, receiving 20-30 mL of chilled PBS (4 °C, pH 7.4 phosphate buffer) at a flow rate of approximately 5 mL / min until the right atrial effluent became clear and the liver turned white, indicating complete blood removal. Major organs, including the brain, liver, lungs, kidneys, and quadriceps femoris muscle, were rapidly dissected. Samples used for histological examination were fixed by immersion in 4% paraformaldehyde at 4 °C for 48 h, and then transferred to 75% (v / v) ethanol for long-term storage.

[0071] The chromatographic conditions for determining the serum concentration of drug #11a using LC-MS / MS are as follows (all percentages are volume percentages (v / v)): Chromatographic column: ACOUITY BEH C18 column, 1.7 μm, 2.1 mm × 100 mm. Mobile phase: 0.1% formic acid aqueous solution (solvent A) and 0.1% formic acid acetonitrile solution (solvent B). Gradient elution program: 60% solvent B (0–0.5 min), 60–98% solvent B (0.5–1 min), 98% solvent B (1–3 min). 60% solvent B (3-5 min). Flow rate: 0.2 mL / min, temperature: 40 ℃, injection: 1 μL. The mass spectrometer #11a uses electrospray ionization for ionization and scans in negative ion multiple reaction monitoring (MRM) mode. The precursor ion molecular weight is 362.15, and the product ion molecular weights are 274.15, 188.10, and 119.05.

[0072] The above test results are as follows Figure 1 and Figure 2 As shown.

[0073] Figure 1 The results of the AEP inhibitor #11a promoting metabolic homeostasis (AB) were obtained from the glucose tolerance test (GTT) and insulin tolerance test (ITT) to assess metabolic function, as well as the results of serum alanine aminotransferase, aspartate aminotransferase, low-density lipoprotein cholesterol and lactate dehydrogenase concentrations. Figure 1 In Figure (A), the left plot shows the blood glucose level (mM) change curve after intraperitoneal injection of glucose at a dose of 2 g / kg; the right plot shows the area under the GTT curve (AUC) quantitative results. The left plot of Figure (B) shows the blood glucose level (mM) change curve after intraperitoneal injection of insulin at a dose of 0.5 U / kg; the right plot shows the area under the ITT curve (AUC) quantitative results. In Figures (A) and (B), the horizontal axis represents the time (in minutes) of insulin injection. Figure (CF) shows the serum alanine aminotransferase (ALT) (Figure C), aspartate aminotransferase (AST) (Figure D), low-density lipoprotein cholesterol (LDL-C) (Figure E), and lactate dehydrogenase (LDH) (Figure F) concentrations measured using standard automated biochemical analysis methods. Significance levels are marked as: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. All data are expressed as mean ± standard deviation.

[0074] Figure 2 The results show the detection results of serum drug concentration of #11a determined by LC-MS / MS and representative H&E staining images of heart, liver, spleen, lung and kidney tissues. Figure 2In the figure, (A) shows the serum concentration of AEP inhibitor #11a after 5 months of continuous administration without toxic side effects, determined by LC-MS / MS; (B) shows representative H&E staining images of heart, liver, spleen, lung, and kidney tissues. Scale bar: 50 µm. Significance levels are marked as: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. All data are expressed as mean ± standard deviation.

[0075] from Figure 1 Eighteen-month-old C57BL / 6 mice were randomly divided into a control group and a #11a group (n = 12 / group). The #11a group received the drug (7.5 mg / kg) mixed into their feed for 5 consecutive months. IPGTT and IPITT were performed at 23 months of age. The #11a group showed significantly lower blood glucose levels at all time points compared to the control group, with a significantly reduced area under the glucose-time curve (AUC). The #11a group showed a more significant decrease in blood glucose after insulin injection, with a significantly lower AUC than the control group. Serum LDL-C and LDH levels were significantly reduced in the #11a group. In summary, #11a improves insulin sensitivity, resulting in a better blood glucose response, without affecting liver function, blood lipids, or cardiac function.

[0076] from Figure 2 As shown in Figure A, after 5 months of feeding, a certain amount of #11a remained in the blood (as shown in Figure A). Figure 2 As shown in section B, compared with the control group, no pathological abnormalities were found in the stained sections of the heart, liver, spleen, lungs, and kidneys of the experimental group mice. In summary, after long-term administration of #11a, no significant toxic side effects were observed in blood routine tests, urine routine tests, and pathological examinations of important organs such as the heart, liver, spleen, lungs, and kidneys, confirming that the AEP inhibitor #11a has good safety and is suitable for long-term intervention in chronic diseases.

[0077] In summary, the AEP inhibitor of this application can significantly enhance the body's insulin sensitivity and improve glucose tolerance, showing the potential to improve insulin resistance. At the same time, the AEP inhibitor can also reduce serum low-density lipoprotein cholesterol and lactate dehydrogenase levels, demonstrating multiple improving effects on glucose and lipid metabolism. Furthermore, no obvious toxic side effects were found in blood routine, urine routine, and pathological examinations of important organs such as the heart, liver, spleen, lungs, and kidneys after long-term administration of the AEP inhibitor, confirming its good safety and suitability for long-term intervention in chronic diseases.

[0078] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of asparagine endopeptidase inhibitors in the preparation of drugs to improve insulin resistance.

2. The application according to claim 1, characterized in that, The structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ) below: Equation (Ⅰ).

3. Application of asparagine endopeptidase inhibitors in the preparation of drugs that enhance insulin sensitivity and / or regulate glucose tolerance.

4. The application according to claim 3, characterized in that, The drug can also reduce the level of at least one of the following substances in serum: low-density lipoprotein, cholesterol, and lactate dehydrogenase.

5. Application of asparagine endopeptidase inhibitors in the preparation of drugs for the prevention or treatment of metabolic diseases.

6. The application according to claim 5, wherein the metabolic disease is an age-related metabolic disease; Furthermore, the metabolic diseases include insulin resistance caused by natural aging.

7. The application according to any one of claims 3-6, characterized in that, The structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ) below: Equation (Ⅰ).

8. The application according to any one of claims 1-6, characterized in that, The drug is suitable for oral administration; And / or, the effective dose of the asparagine endopeptidase inhibitor is 7.5 mg / kg.

9. A drug for improving insulin resistance, characterized in that, It contains an asparagine endopeptidase inhibitor.

10. The medicament according to claim 9, characterized in that, It also contains pharmaceutically acceptable excipients; And / or, the structural formula of the asparagine endopeptidase inhibitor is shown in formula (Ⅰ) below: Equation (Ⅰ).

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