Use of CBL inhibitor NX1607 in the preparation of a medicament for treating atherosclerosis

Abstract

The present invention relates to the use of a CBL inhibitor in the preparation of a medicament for treating atherosclerosis. The CBL inhibitor medicament is preferably NX1607, and the medicament can inhibit the formation of plaque necrotic cores in a non-lipid-dependent manner, thereby stabilizing atherosclerotic plaques and delaying the progression of the disease. The present invention breaks through the limitations of traditional lipid-lowering drugs, directly intervenes in the key pathological link of plaque instability by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, and provides a new targeted strategy for the treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of the CBL inhibitor NX1607 in the preparation of drugs for treating atherosclerosis. Background Art

[0002] NX1607 is an oral CBL inhibitor with the following structure: , with an IC50 of <1 nM, and 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 glue, NX-1607 can bind to CBL-B (Casitas B-lineage lymphoma proto-oncogene) and promote its binding to different subunits, thus locking CBL-B in an inactive conformation and preventing its conversion to the active state. This mechanism effectively inhibits the function of CBL-B.

[0003] Atherosclerosis (hereinafter referred to as AS) is a vascular disease characterized by chronic inflammation and lipid metabolism imbalance. Its pathogenesis involves multiple links such as endothelial injury, deposition of oxidized low-density lipoprotein (ox-LDL), infiltration of monocytes / macrophages, formation of foam cells, and proliferation of smooth muscle cells. Although lipid abnormalities (such as elevated LDL and defective HDL function) and inflammatory responses are considered the core driving factors of AS, existing mainstream therapies (such as statins) mainly act by reducing circulating lipid levels and have limited effects on improving plaque stability. Clinical data show that even after receiving intensive lipid-lowering treatment, patients still have significant cardiovascular residual risks (residual risk rates reach 30%-40%), indicating that non-lipid-dependent mechanisms (such as dysregulation of inflammatory signals and defective efferocytosis) play an important role in the progression of AS.

[0004] Currently, only anti-lipid drugs or anti-inflammatory drugs combined with anti-thrombotic drugs are used clinically to delay the development of atherosclerosis, and cannot effectively reverse atherosclerosis caused by different reasons. There is no application of NX1607 in the preparation of drugs for treating AS disease in the prior art. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of a CBL inhibitor drug NX1607 in the preparation of a drug for treating atherosclerosis. The drug can inhibit the formation of plaque necrotic cores in a lipid-independent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. Its mechanism of action is independent of lipid metabolism regulation and mainly exerts its anti-AS effect through the following pathways. Compared with traditional lipid-lowering drugs, the innovation of NX1607 lies in: (1) not relying on LDL / HDL metabolism regulation, being applicable to patients with different lipid levels, especially high-risk patients with normal lipid levels but unstable plaques; (2) targeting the plaque microenvironment and inhibiting the formation of necrotic cores from the root; (3) avoiding the systemic side effects of broad-spectrum anti-inflammatory drugs by specifically inhibiting CBL.

[0006] The present invention provides an application 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] Formula I.

[0008] The CBL inhibitor is preferably NX1607.

[0009] The atherosclerosis is selected from one or more of coronary atherosclerosis, carotid atherosclerosis, cerebral atherosclerosis, aortic atherosclerosis, and lower extremity atherosclerosis, preferably coronary atherosclerosis and carotid atherosclerosis.

[0010] The atherosclerosis is selected from stable atherosclerotic plaques or unstable atherosclerotic plaques, preferably unstable atherosclerotic plaques.

[0011] The atherosclerosis is further preferably atherosclerosis with normal lipid levels but unstable plaques.

[0012] In addition, AS is usually histologically divided into eight types clinically. Among them, types I-III belong to early lesions and can regress. Types IV-VI belong to advanced lesions. Among them, types IV-V lesions are plaques containing a large necrotic lipid core covered with a fibrous cap, accompanied by a small amount of calcification, which can lead to lumen stenosis. Type VI lesions have surface ulcers 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 the foam cell infiltration is the most, which is considered an AS vulnerable plaque. Type VII lesions are simple calcified plaques. Type VIII lesions are fibrous plaques without a lipid core, which may be accompanied by a small amount of calcification. Type VIII lesions can be derived from the regression or change of lipids in type VI lesions. Based on the present invention, by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, the key pathological link of plaque instability can be directly intervened, targeting the plaque microenvironment, and the formation of the necrotic core can be inhibited from the root. Therefore, the drug of the present invention can be used to treat atherosclerosis types I-VI, preferably one or more of types I, II, III, IV, V or VI.

[0013] Meanwhile, the present invention also provides the application of the above drug in the preparation of a drug for inhibiting the formation of the necrotic core of atherosclerotic plaques, preferably inhibiting the formation of the necrotic core of plaques in a non-lipid-dependent manner.

[0014] The present invention also provides the application of the above drug in the preparation of a drug for inhibiting plaque lipid accumulation or reducing inflammatory infiltration.

[0015] For the convenience of drug administration, the drug of the present invention also includes pharmaceutically acceptable excipients.

[0016] The drug of the present invention is selected from oral preparations or parenteral preparations.

[0017] Or the drug of the present invention is selected from solid preparations, preferably one of capsules, tablets, granules, pills.

[0018] Or the drug of the present invention is selected from one of oral liquids, emulsions.

[0019] Or the drug of the present invention is selected from one of injections, freeze-dried powder injections, inhalants.

[0020] The beneficial technical effects of the present invention include:

[0021] 1. The drug of the present invention inhibits the formation of the necrotic core of plaques in a non-lipid-dependent manner, thereby stabilizing atherosclerotic plaques and delaying the progression of the disease. It breaks through the limitations of traditional lipid-lowering drugs, directly intervenes in the key pathological link of plaque instability by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, and provides a new targeted strategy for the treatment of atherosclerosis.

[0022] 2. Compared with traditional lipid-lowering drugs, the innovation of CBL inhibitors, especially NX1607, in the present invention is reflected in: (1) It does not rely on LDL / HDL metabolism regulation and is applicable to 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram of the diet pattern of Ldlr gene knockout mice. Ldlr - / - represents Ldlr knockout mice. The vehicle gavage control group uses 0.5% sodium carboxymethylcellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0024] Figure 2 for Ldlr - / - It is a statistical chart of the total cholesterol and plasma triglyceride contents of Ldlr

[0025] Figure 3 for Ldlr - / - knockout mice. The left chart is the plasma total cholesterol content, and the right chart is the plasma triglyceride content. The vehicle gavage control group uses 0.5% sodium carboxymethylcellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0026] Figure 4 for Ldlr - / - It is a chart of the alanine aminotransferase and aspartate aminotransferase contents in the plasma of Ldlr

[0027] Figure 5 for Ldlr - / - It is the results related to inflammation in the blood routine of Ldlr knockout mice, including the number of white blood cells, neutrophils, lymphocytes, and monocytes. The vehicle gavage control group uses 0.5% sodium carboxymethylcellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0028] Figure 6 for Ldlr - / -Results related to red blood cells in the blood routine of knockout mice include the number of red blood cells, hemoglobin content, hematocrit, mean corpuscular volume, mean hemoglobin content, and mean hemoglobin concentration. The solvent gavage control group used 0.5% sodium carboxymethylcellulose as the control, and NX1607 represents the NX1607 drug gavage treatment group.

[0029] Figure 7 is Ldlr - / - Results related to platelets in the blood routine of knockout mice include the number of platelets, platelet distribution width, mean platelet volume, and platelet hematocrit. The solvent gavage control group used 0.5% sodium carboxymethylcellulose as the control, and NX1607 represents the NX1607 drug gavage treatment group.

[0030] Figure 8 is Ldlr - / - Gross oil red O staining map of the blood vessels of knockout mice. The solvent gavage control group used 0.5% sodium carboxymethylcellulose as the control, and NX1607 represents the NX1607 drug gavage treatment group.

[0031] Figure 9 is Ldlr - / - HE, oil red O, CD68, and TUNEL staining maps of the outflow tract of knockout mice. The scale bar is 100 μm. The solvent gavage control group used 0.5% sodium carboxymethylcellulose as the control, and NX1607 represents the NX1607 drug gavage treatment group. Detailed implementation manners

[0032] The following further illustrates the detailed implementation manners of the present invention in conjunction with the accompanying drawings. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention.

[0033] In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] I. Experimental animals and feeding

[0035] Species, sex, age in weeks, and source of experimental animals: Ldlr gene (gene number is 16835, gene function: involved in lipid transport; regulation of inflammatory response; and regulation of lipid metabolism process), male, 8 weeks old, body weight 19 - 25 g. Ldlr gene knockout mice (Ldlr - / - ) were purchased from GemPharmatech (Nanjing, China).

[0036] II. Feed formula for experimental animals

[0037] The Western Diet (WD) was purchased from Research Diets, Inc., catalog number: D12108C.

[0038] III. Animal Rearing and Grouping

[0039] All experiments were conducted in accordance with the principles of laboratory animal care (NIH publication No. 85-23, revised in 1996) and approved by the Laboratory Animal Ethics Committee of Peking University (LA2023460). All animals were bred and raised in the SPF-class animal facility of the Peking University Health Science Center (license number: SYXK (Beijing) 2022-0037), ensuring a 12-hour light-dark cycle per day, maintaining a temperature of 25 ± 2 °C and a humidity of 40 ± 5%. Without special instructions, all mice had free access to food and water. As Figure 1 shown in the diagram of drug administration and diet for Ldlr gene knockout mice, eight-week-old mice on a normal diet (CD) were given the above Western Diet (WD) for 4 weeks and then treated with NX1607 by gavage for 16 weeks (dosage of NX1607: 5 mg / kg body weight / day). They were divided into two groups: a vehicle control group and a drug administration group, with the vehicle being 0.5% sodium carboxymethylcellulose.

[0040] IV. Determination of Plasma Total Cholesterol and Triglyceride Levels

[0041] After eight-week-old mice on a normal diet (CD) were given the above Western Diet (WD) for 4 weeks and then treated with vehicle control or NX1607 by gavage for 16 weeks, the mice were fasted for 4 hours, and anticoagulant or non-anticoagulant blood samples were collected from the orbital vein. The samples were centrifuged at 4 °C for 10 minutes to separate the plasma. The plasma total cholesterol (TC) and triglyceride (TG) concentrations were measured using commercially available kits from ZhongSheng Beikong (Beijing, China).

[0042] The results are as Figure 2 shown, and NX1607 treatment had no effect on the plasma total cholesterol and triglyceride levels in mice.

[0043] V. Determination of Plasma Lipoprotein Distribution by Fast Liquid Chromatography

[0044] After eight-week-old mice on a normal diet (CD) were given the above Western Diet (WD) for 4 weeks and then treated with NX1607 by gavage for 16 weeks, the mice were fasted for 4 hours, and anticoagulant or non-anticoagulant blood samples were collected from the orbital vein. The samples were centrifuged at 4 °C for 10 minutes to separate the plasma. The lipoprotein distribution was determined using a fast liquid chromatograph, and the sample (TC) and triglyceride (TG) contents were measured using commercially available kits from ZhongSheng Beikong (Beijing, China).

[0045] The results are as Figure 3 shown, and NX1607 treatment had no significant effect on the distribution of various lipoproteins in the plasma of mice.

[0046] VI. Determination of the contents of alanine aminotransferase and aspartate aminotransferase in plasma

[0047] After 4 weeks of feeding the above-mentioned Western diet (WD) to 8-week-old mice on a normal diet (CD) and then administering intragastric treatment with NX1607 for 16 weeks, the mice were fasted for 4 h, and anticoagulant or non-anticoagulant blood samples were collected from the orbital vein. The samples were centrifuged at 4 °C for 10 min to separate the plasma. The contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the samples were measured using commercially available kits from Nanjing Jiancheng.

[0048] The results are as Figure 4 shown. NX1607 treatment had no significant effect on the content of alanine aminotransferase (ALT) in the plasma of mice, and had no significant effect on the content of aspartate aminotransferase (AST). It was proved that the use of this drug did not produce hepatotoxicity.

[0049] VII. Determination of the complete blood count of mice

[0050] After 4 weeks of feeding the above-mentioned Western diet (WD) to 8-week-old mice on a normal diet (CD) and then administering intragastric treatment with NX1607 for 16 weeks, anticoagulant blood samples were collected from the orbital vein. The blood must be fully mixed with anticoagulant and diluent immediately during or after blood collection and then analyzed on a machine. The blood samples of mice were measured and analyzed using an animal complete blood count analyzer provided by Furui Runze (Beijing).

[0051] The results are as Figures 5-7 shown. NX1607 treatment had no significant effect on the inflammation-related indicators (white blood cells, neutrophils, lymphocytes, monocytes) in the blood of mice ( Figure 5 ); had no significant effect on the red blood cell-related indicators (red blood cells, hemoglobin content, hematocrit, mean corpuscular volume, mean hemoglobin content), and mean hemoglobin concentration in the blood of mice ( Figure 6 ); had no significant effect on the platelet-related indicators (platelet count, platelet distribution width, mean platelet volume, platelet hematocrit) in the blood of mice ( Figure 7 ). Figures 5-7 The results

[0052] VIII. Gross oil red O staining of blood vessels

[0053] After 4 weeks of feeding the above-mentioned Western diet (WD) to 8-week-old mice on a normal diet (CD) and then administering intragastric treatment with NX1607 for 16 weeks, the mice were sacrificed. The entire length of the aorta of the mice was taken, fixed with paraformaldehyde, dehydrated with 20% sucrose, and stained with oil red O.

[0054] The results are as Figure 8As shown, the gross oil red O staining results of the aorta showed a reduction in lipid accumulation (red part) in the mice of the NX1607 treatment group, suggesting a significant reduction in aortic plaques. The above pathological results indicated that after treatment with NX1607, the atherosclerotic plaques in the aorta were significantly reduced, and the drug had a significant therapeutic effect on atherosclerosis.

[0055] IX. HE, Oil Red O, Bodipy, and CD68 Staining of the Aortic Outflow Tract

[0056] Eight-week-old mice on a normal diet (CD) were given the above Western diet (WD) for 4 weeks, then treated with intragastric administration of NX1607 for 16 weeks, and then samples were taken. Frozen sections of the aortic outflow tract (aortic root) with a thickness of 7 μm were used for HE, oil red O staining, and CD68 and TUNEL immunofluorescence staining.

[0057] The results were as Figure 9 shown. The oil red O staining results of the aortic outflow tract plaques in the NX1607-treated mice compared with those in the control group showed a reduction in lipid accumulation in the outflow tract (red part of the ORO staining), suggesting a significant reduction in the plaques at the aortic root. The HE staining results and the area of positive TUNEL staining signals (green part) showed a significant reduction in the area of the necrotic core at the aortic root. The area of positive CD68 signals indicated a reduction in inflammatory infiltration in the plaque area at the aortic root.

[0058] The above pathological results together indicated that after treatment with NX1607, atherosclerotic plaques were significantly reduced, the necrotic area was significantly reduced, and inflammation was significantly alleviated. The drug had a significant therapeutic effect on atherosclerosis.

[0059] In summary, CBL inhibitors represented by NX1607 have the effect of treating atherosclerosis and can be used in the preparation of drugs for the treatment of atherosclerosis.

Claims

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

3. The use according to claim 2, characterized in that: The atherosclerosis is selected from atherosclerotic stable plaque or atherosclerotic unstable plaque.

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

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

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

7. The use according to claim 1, characterized in that: The medicine is selected from solid preparations, including capsules, tablets, granules and pills.

8. The use according to claim 1, characterized in that: The medicine is selected from one of oral liquid and emulsion.

9. The use according to claim 1, characterized in that: The medicine is selected from one of injection, lyophilized powder injection and inhalant.

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