Use of CBL inhibitor NX1607 in preparation of drug for treating atherosclerosis

Abstract

Provided in the present invention is use of a CBL inhibitor in the preparation of a drug for treating atherosclerosis. The CBL inhibitor drug is preferably NX1607, and the drug can inhibit necrotic core formation in plaques in a lipid-independent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. By means of regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, the present invention directly intervenes in the key pathological link of plaque instability, thereby treating atherosclerosis.

Description

Application of CBL inhibitor NX1607 in the preparation of drugs for treating atherosclerosis Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CBL inhibitor NX1607 in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] NX1607 is an oral CBL inhibitor with the following structure: With an IC50 of < 1 nM, NX-1607 has been shown to enhance antigen recall, reduce T cell exhaustion, and increase cytokine production upon T cell receptor stimulation, overcoming inhibitory signals from the tumor microenvironment. As a unique molecular adhesive, NX-1607 binds to CBL-B (Casitas B-lineage lymphoma proto-oncogene) and promotes its binding to different subunits, thereby locking CBL-B in an inactive conformation and preventing its conversion to an active state. This mechanism effectively inhibits CBL-B function.

[0003] Atherosclerosis (AS) is a vascular disease characterized by chronic inflammation and lipid metabolism imbalance. Its pathogenesis involves multiple stages, including endothelial damage, oxidized low-density lipoprotein (ox-LDL) deposition, mononuclear / macrophage infiltration, foam cell formation, and smooth muscle cell proliferation. Although lipid abnormalities (such as elevated LDL and impaired HDL function) and inflammatory responses are considered core drivers of AS, current mainstream therapies (such as statins) primarily work by lowering circulating lipid levels, with limited effectiveness in improving plaque stability. Clinical data show that even with intensive lipid-lowering therapy, patients still have significant residual cardiovascular risk (residual risk rate of 30%-40%), suggesting that non-lipid-dependent mechanisms (such as dysregulation of inflammatory signaling and impaired cell burial function) play a crucial role in AS progression.

[0004] Currently, in clinical practice, only antilipidemia or anti-inflammatory drugs combined with antithrombotic drugs can delay the progression of atherosclerosis, but they cannot effectively reverse atherosclerosis caused by different reasons. There is currently no application of NX1607 in the preparation of drugs for treating AS. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of the CBL inhibitor NX1607 in the preparation of drugs for treating atherosclerosis. This drug inhibits plaque necrosis core formation in a non-lipid-dependent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. Its mechanism of action is independent of lipid metabolism regulation, primarily exerting its anti-AS effect through the following pathways. Compared with traditional lipid-lowering drugs, the innovation of NX1607 lies in: (1) it is independent of LDL / HDL metabolic regulation, making it suitable for patients with different lipid levels, especially high-risk patients with normal lipid levels but unstable plaques; (2) it targets the plaque microenvironment, inhibiting necrosis core formation at its source; and (3) by specifically inhibiting CBL, it avoids the systemic side effects of broad-spectrum anti-inflammatory drugs.

[0006] This invention provides the use of a CBL inhibitor drug in the preparation of a drug for treating atherosclerosis, wherein the CBL inhibitor is a compound having the chemical structure of Formula I or a pharmaceutically acceptable salt thereof, or a precursor thereof, or a metabolite thereof.

[0007]

[0008] Formula I.

[0009] The CBL inhibitor is preferably NX1607.

[0010] The atherosclerosis is selected from one or more of coronary atherosclerosis, carotid atherosclerosis, cerebral atherosclerosis, aortic atherosclerosis, and lower extremity atherosclerosis, with coronary atherosclerosis and carotid atherosclerosis being preferred.

[0011] The atherosclerosis is selected from stable atherosclerotic plaques or unstable atherosclerotic plaques, with unstable atherosclerotic plaques being preferred.

[0012] The atherosclerosis is further preferred to be atherosclerosis with normal lipid levels but unstable plaques.

[0013] In addition, clinically, AS is usually histologically classified into eight types. Types I-III are early lesions that can regress. Types IV-VI are progressive lesions. Types IV-V are plaques with large necrotic lipid cores and fibrous caps, accompanied by a small amount of calcification, which can lead to luminal stenosis. Type VI lesions have surface ulceration or intraplaque hemorrhage and thrombosis, and also show a thin and uneven fibrous cap. In severe cases, the plaque ruptures at the shoulder where the fibrous cap is thinnest and foam cell infiltration is most abundant, and is considered a vulnerable plaque in AS. Type VII lesions are simple calcified plaques. Type VIII lesions are fibrous plaques without lipid cores, which may be accompanied by a small amount of calcification. Type VIII lesions can develop from lipid regression or changes within stage VI lesions. Based on this invention, it can directly intervene in the key pathological link of plaque instability by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, targeting the plaque microenvironment, and inhibiting the formation of necrotic cores from the root. Therefore, the drug of the present invention can be used to treat atherosclerosis of types I to VI, preferably one or more of types I, II, III, IV, V or VI.

[0014] The present invention also provides the application of the above-mentioned drug in the preparation of a drug for inhibiting the formation of necrotic core of atherosclerotic plaques, preferably inhibiting the formation of necrotic core of plaques in a non-lipid-dependent manner.

[0015] The present invention also provides the use of the above-mentioned drug in the preparation of drugs that inhibit plaque lipid accumulation or reduce inflammatory infiltration.

[0016] To facilitate drug administration, the drug of the present invention also includes pharmaceutically acceptable excipients.

[0017] The drug of this invention is selected from oral preparations or parenteral preparations.

[0018] Alternatively, the drug of the present invention may be selected from solid dosage forms, preferably one of capsules, tablets, granules, or pills.

[0019] Alternatively, the drug of this invention may be selected from oral liquids or emulsions.

[0020] Alternatively, the drug of this invention may be selected from one of the following: injection solution, lyophilized powder injection, or inhalation solution.

[0021] The beneficial technical effects of this invention include:

[0022] 1. The drug of this invention inhibits the formation of plaque necrosis core in a non-lipid-dependent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. Breaking through the limitations of traditional lipid-lowering drugs, it directly intervenes in the key pathological link of plaque instability by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, providing a novel targeted strategy for the treatment of atherosclerosis.

[0023] 2. Compared with traditional lipid-lowering drugs, the innovation of the CBL inhibitor in this invention, especially NX1607, is reflected in: (1) It does not depend on LDL / HDL metabolic regulation and is suitable for high-risk patients with normal lipid levels but unstable plaques; (2) It targets the plaque microenvironment and inhibits the formation of necrotic cores from the root; (3) By specifically inhibiting CBL, it avoids the systemic side effects of broad-spectrum anti-inflammatory drugs. Attached Figure Description

[0024] Figure 1 shows the diet of Ldlr gene knockout mice. - / - The group represents Ldlr knockout mice. The solvent gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0025] Figure 2 shows Ldlr - / - Statistical charts of total cholesterol and plasma triglyceride levels in knockout mice. The left chart shows the total cholesterol level in plasma, and the right chart shows the triglyceride level in plasma. The solvent-gavaged control group used 0.5% sodium carboxymethyl cellulose as a control. NX1607 represents the NX1607 drug gavage treatment group.

[0026] Figure 3 shows Ldlr - / - The distribution of plasma lipoproteins in knockout mice is shown in the figure. The VLDL / CM peak represents the content of very low-density lipoprotein and chylomicrons, LDL represents the content of low-density lipoprotein, and HDL represents the content of high-density lipoprotein. The solvent gavage control group used 0.5% sodium carboxymethyl cellulose as a control. NX1607 represents the NX1607 drug gavage treatment group.

[0027] Figure 4 shows Ldlr - / - The levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in knockout mice were measured. The control group administered the solvent via gavage used 0.5% sodium carboxymethyl cellulose as a control. NX1607 represents the NX1607 drug gavage treatment group.

[0028] Figure 5 shows Ldlr - / - Inflammation-related results in the blood routine tests of knockout mice were measured, including white blood cell count, neutrophil count, lymphocyte count, and monocyte count. The solvent-based gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0029] Figure 6 shows Ldlr - / - The erythrocyte-related results in the complete blood count of knockout mice were as follows: erythrocyte count, hemoglobin content, hematocrit, mean erythrocyte volume, mean erythrocyte content, and mean erythrocyte concentration. The solvent-based gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0030] Figure 7 shows Ldlr - / - Platelet-related results in the complete blood count of knockout mice included platelet count, platelet distribution width, mean platelet volume, and plateletcrit. The solvent-based gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0031] Figure 8 shows Ldlr - / - Gross Oil Red O staining of blood vessels in knockout mice. The solvent-gavage control group used 0.5% sodium carboxymethyl cellulose as a control. NX1607 represents the NX1607 drug gavage treatment group.

[0032] Figure 9 shows Ldlr - / - HE, Oil Red O, CD68, and TUNEL staining images of the outflow tract of knockout mice, with a scale bar of 100 μm. The solvent-gavage control group used 0.5% sodium carboxymethyl cellulose as a control. NX1607 represents the NX1607 drug gavage treatment group. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0034] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] I. Laboratory Animals and Their Care

[0036] Laboratory animal species, sex, age, and origin: Ldlr gene (gene number 16835, gene function: involved in lipid transport; regulation of inflammatory responses; and regulation of lipid metabolism processes), male, 8 weeks old, weighing 19-25g. Ldlr gene knockout mice (Ldlr... - / - Purchased from GemPharmatech (Nanjing, China).

[0037] II. Laboratory animal feed formulation

[0038] The Western Diet (WD) was purchased from Research Diet, product number: D12108C.

[0039] III. Animal husbandry and grouping

[0040] All experiments were performed in accordance with the principles for laboratory animal care (revised 1996, NIH Publication No. 85Y23) and approved by the Experimental Animal Ethics Committee of Peking University (approval number LA2023460). All animals were bred and housed in the SPF-level animal facility at Peking University Health Science Center (license number: SYXK (Jing) 2022-0037), with a 12-hour daily light-dark cycle, a temperature of 25±2°C and a humidity of 40±5%. Unless otherwise specified, all mice had ad libitum access to food and water. As shown in Figure 1, it is a schematic diagram of the administration and diet of Ldlr knockout mice. Eight-week-old mice fed with a chow diet (CD) were fed the aforementioned Western diet (WD) for 4 weeks, and then received intragastric administration of NX1607 for 16 weeks (dosage of NX1607: 5 mg / kg body weight per day). The mice were divided into two groups: the vehicle control group and the administration group, and 0.5% sodium carboxymethyl cellulose was used as the vehicle.

[0041] IV. Determination of plasma total cholesterol and triglyceride contents

[0042] After eight-week-old mice on a chow diet (CD) were fed the aforementioned Western diet (WD) for 4 weeks and then received 16 weeks of intragastric administration of vehicle control or NX1607, the mice were fasted for 4 hours, and blood samples with or without anticoagulant were collected from the orbital vein. Blood was centrifuged at 4°C for 10 min to separate plasma. Plasma concentrations of total cholesterol (TC) and triglyceride (TG) were measured using commercial kits purchased from Zhong Sheng Bei Kong (Beijing, China).

[0043] The results are shown in Figure 2, and NX1607 treatment has no effect on the contents of plasma total cholesterol and triglyceride in mice.

[0044] V. Determination of plasma lipoprotein distribution by fast liquid chromatography

[0045] After eight-week-old mice on a chow diet (CD) were fed the aforementioned Western diet (WD) for 4 weeks and then received 16 weeks of intragastric administration of NX1607, the mice were fasted for 4 hours, and blood samples with or without anticoagulant were collected from the orbital vein. Blood was centrifuged at 4°C for 10 min to separate plasma. Lipoprotein distribution was determined using a fast liquid chromatography system, and the contents of sample TC and triglyceride (TG) were measured using commercial kits from Zhong Sheng Bei Kong (Beijing, China).

[0046] The results are shown in Figure 3, NX1607 treatment has no significant effect on the distribution and content of various lipoproteins in mouse plasma.

[0047] VI. Determination of plasma alanine aminotransferase and aspartate aminotransferase contents

[0048] Eight-week-old mice on a normal diet (CD) were given the Western diet (WD) for 4 weeks, followed by 16 weeks of gavage treatment with NX1607. After fasting for 4 hours, blood samples were collected from the orbital vein using either anticoagulant or non-anticoagulant methods. Plasma was separated by centrifugation at 4°C for 10 minutes. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the samples were measured using a commercially available kit developed in Nanjing.

[0049] As shown in Figure 4, NX1607 treatment had no significant effect on the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse plasma. This demonstrates that the drug does not cause hepatotoxicity.

[0050] VII. Determination of complete blood count in mice

[0051] Eight-week-old mice on a normal diet (CD) were given the Western diet (WD) for 4 weeks, followed by 16 weeks of gavage treatment with NX1607. Anticoagulant blood samples were collected from the orbital vein. The blood samples were thoroughly mixed with the anticoagulant and diluent at the time of collection or immediately after collection before analysis. Mice blood samples were analyzed using an animal blood routine analyzer provided by Furui Runze (Beijing).

[0052] The results are shown in Figures 5-7. NX1607 treatment had no significant effect on inflammation-related indicators (white blood cells, neutrophils, lymphocytes, and monocytes) in mouse blood (Figure 5); no significant effect on erythrocyte-related indicators (erythrocyte count, hemoglobin content, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin content), and mean corpuscular hemoglobin concentration (Figure 6); and no significant effect on platelet-related indicators (platelet count, platelet distribution width, mean platelet volume, and plateletcrit) in mouse blood (Figure 7). The results in Figures 5-7 show that NX1607 has no side effects on whole blood biochemical indicators.

[0053] 8. Gross Oil Red O staining of blood vessels

[0054] Eight-week-old mice on a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks, and then treated with NX1607 by gavage for 16 weeks. The entire length of the mouse aorta was taken, fixed with paraformaldehyde, dehydrated with 20% sucrose, and stained with Oil Red O.

[0055] The results are shown in Figure 8. Gross Oil Red O staining of the aorta revealed reduced lipid accumulation (red portion) in the NX1607-treated mice, indicating a significant reduction in aortic plaque. These pathological results demonstrate that NX1607 treatment significantly reduced atherosclerotic plaques in the aorta, indicating that the drug has a significant therapeutic effect on atherosclerosis.

[0056] IX. Aortic outflow tract HE, Oil Red O, Bodipy, and CD68 staining

[0057] Eight-week-old mice on a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks, and then treated with NX1607 by gavage for 16 weeks. Frozen samples of the aortic outflow tract (aortic root) were collected with a thickness of 7 μm and stained with HE, Oil Red O, CD68, and TUNEL immunofluorescence.

[0058] The results are shown in Figure 9. Compared with the control group, the Oil Red O staining results of the aortic outflow tract plaque in NX1607 treated mice showed a reduction in lipid accumulation in the outflow tract (red part of ORO staining), indicating a significant reduction in plaque in the aortic root. HE staining results and TUNEL staining positive signal area (green part) showed a significant reduction in the necrotic core area of ​​the aortic root. CD68 positive signal area indicated a reduction in inflammatory infiltration in the aortic root plaque area.

[0059] The above pathological results collectively demonstrate that after treatment with NX1607, atherosclerotic plaques were significantly reduced, necrotic areas were significantly reduced, and inflammation was significantly alleviated, indicating that the drug has a significant therapeutic effect on atherosclerosis.

[0060] In summary, CBL inhibitors, represented by NX1607, have therapeutic effects on atherosclerosis and can be used in the preparation of drugs for treating atherosclerosis.

Claims

1. The application of a CBL inhibitor drug in the preparation of drugs for treating atherosclerosis, characterized in that, The CBL inhibitor is a compound having the chemical structure of Formula I or a pharmaceutically acceptable salt thereof. 。 2. The application according to claim 1, characterized in that, The atherosclerosis is selected from one or more of the following: coronary atherosclerosis, carotid atherosclerosis, cerebral atherosclerosis, aortic atherosclerosis, and lower extremity atherosclerosis.

3. Use according to claim 2, characterized in that, The atherosclerosis is selected from stable atherosclerotic plaques or unstable atherosclerotic plaques.

4. The application according to claim 1, characterized in that, The atherosclerosis is selected from one of the following types: atherosclerosis I, II, III, IV, V, or VI.

5. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The drug is selected from oral and / or parenteral preparations.

7. The application according to claim 1, characterized in that, The drug is selected from solid dosage forms, including capsules, tablets, granules, and pills.

8. The application according to claim 1, characterized in that, The drug is selected from oral liquids and emulsions.

9. The application according to claim 1, characterized in that, The drug is selected from one of the following: injection solution, lyophilized powder injection, and inhalation solution.

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