Application of CBL inhibitor NX1607 in preparation of medicine for treating atherosclerosis

Abstract

The invention relates to application of a CBL inhibitor in preparation of a medicine for treating atherosclerosis, the CBL inhibitor medicine is preferably NX1607, and the medicine can inhibit plaque necrosis core formation in a non-lipid-dependent mode, so that atherosclerotic plaques are stabilized, and disease progression is delayed. The invention breaks through the limitation of the traditional lipid-lowering drug, directly intervenes the key pathological link of plaque instability by regulating and controlling the apoptotic cell removal mechanism in the inflammatory microenvironment, and provides a brand new targeting strategy for atherosclerosis treatment.

Description

Technical Field

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

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

[0003] Atherosclerosis (AS) is a vascular disease characterized by chronic inflammation and lipid metabolism imbalance. Its pathogenesis involves multiple links such as endothelial damage, oxidized low-density lipoprotein (ox-LDL) deposition, monocyte / macrophage infiltration, foam cell formation and smooth muscle cell proliferation. Although lipid abnormalities (such as elevated LDL and HDL functional defects) and inflammatory responses are considered to be the core driving factors of AS, existing mainstream therapies (such as statins) mainly work by reducing circulating lipid levels and have limited effects on improving plaque stability. Clinical data show that even with intensive lipid-lowering treatment, patients still have significant residual cardiovascular risk (residual risk rate of 30%-40%), suggesting that non-lipid-dependent mechanisms (such as dysregulated inflammatory signals and defective cell death function) play an important role in the progression of AS.

[0004] Currently, only anti-lipid drugs or anti-inflammatory drugs combined with anti-thrombotic drugs can delay the development of atherosclerosis, but they cannot effectively reverse atherosclerosis caused by different reasons. There is no application of NX1607 in the preparation of drugs for treating AS diseases in the prior art. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a CBL inhibitor NX1607 drug for use in the preparation of a drug for treating atherosclerosis, wherein the drug can inhibit the formation of plaque necrotic core in a lipid-independent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. Its mechanism of action is independent of lipid metabolism regulation, and it exerts anti-AS effects mainly through the following pathways. Compared with traditional lipid-lowering drugs, the innovation of NX1607 is reflected in: (1) it is independent of LDL / HDL metabolic regulation and is suitable for patients with different lipid levels, especially for high-risk patients with normal lipid levels but unstable plaques; (2) it targets the plaque microenvironment and inhibits the formation of necrotic core from the root; (3) it avoids 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. The CBL inhibitor is a compound having a chemical structure of Formula I or a pharmaceutically acceptable salt thereof or a precursor thereof or a metabolite thereof. Formula I.

[0007] The CBL inhibitor is preferably NX1607.

[0008] The atherosclerosis is selected from one or more of coronary atherosclerosis, carotid atherosclerosis, cerebral atherosclerosis, aortic atherosclerosis, and lower limb atherosclerosis, preferably coronary atherosclerosis and carotid atherosclerosis.

[0009] The atherosclerosis is selected from stable atherosclerotic plaques or unstable atherosclerotic plaques, preferably unstable atherosclerotic plaques.

[0010] The atherosclerosis is further preferably atherosclerosis with normal lipid levels but unstable plaques.

[0011] In addition, AS is usually divided into eight types clinically, among which types I-III are early lesions and can disappear. Types IV to VI are advanced lesions, among which type IV-V lesions are plaques containing large necrotic lipid cores and covered with fibrous caps, accompanied by a small amount of calcification. It can lead to luminal stenosis; type VI lesions have surface ulcers or intraplaque bleeding and thrombosis, and also show thin and uneven fibrous caps. In severe cases, the plaques rupture at the shoulder where the fibrous cap is the thinnest and the foam cells are the most infiltrated, and are considered to be vulnerable plaques of 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 be caused by the regression or change of lipids in stage VI lesions. Based on the fact that the present invention can directly intervene in the key pathological link of plaque instability by regulating the apoptotic cell clearance mechanism in the inflammatory microenvironment, target the plaque microenvironment, and inhibit the formation of necrotic cores from the root. Therefore, the drug of the present invention can be used to treat atherosclerosis types I to VI, preferably one or more of types I, II, III, IV, V or VI.

[0012] At the same time, the present invention also provides the use 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 lipid-free manner.

[0013] The present invention also provides the use of the above-mentioned drug in preparing a drug for inhibiting plaque lipid accumulation or reducing inflammatory infiltration.

[0014] To facilitate administration of the drug, the drug of the present invention further comprises pharmaceutically acceptable excipients.

[0015] The drug of the present invention is selected from oral preparations or parenteral preparations.

[0016] Alternatively, the drug of the present invention is selected from solid preparations, preferably one of capsules, tablets, granules and pills.

[0017] Or the medicine of the present invention is selected from one of oral liquid and emulsion.

[0018] Or the drug of the present invention is selected from one of injection, lyophilized powder injection and inhalation.

[0019] The beneficial technical effects of the present invention include:

[0020] 1. The drug of the present invention inhibits the formation of plaque necrotic core in a lipid-independent manner, thereby stabilizing atherosclerotic plaques and delaying disease progression. 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.

[0021] 2. Compared with traditional lipid-lowering drugs, the innovative features of the CBL inhibitors in the present invention, especially NX1607, are as follows: (1) they are independent of LDL / HDL metabolic regulation and are suitable for high-risk patients with normal lipid levels but unstable plaques; (2) they target the plaque microenvironment and inhibit the formation of necrotic cores from the root; and (3) they avoid the systemic side effects of broad-spectrum anti-inflammatory drugs by specifically inhibiting CBL. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the dietary pattern of Ldlr gene knockout mice. - / - represents Ldlr knockout mice, the vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0023] Figure 2 For Ldlr - / - Statistical graphs of total cholesterol and plasma triglyceride levels in knockout mice. The left graph shows the plasma total cholesterol content, and the right graph shows the plasma triglyceride content. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0024] Figure 3 For Ldlr - / - Distribution of plasma lipoproteins in knockout mice, where VLDL / CM peak represents very low density lipoprotein and chylomicron content, LDL represents low density lipoprotein content, HDL represents high density lipoprotein content, the vehicle gavage control group uses 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0025] Figure 4 For Ldlr - / - Plasma alanine aminotransferase and aspartate aminotransferase levels of knockout mice, the vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represented the NX1607 drug gavage treatment group.

[0026] Figure 5 For Ldlr - / - The inflammation-related results in the routine blood test of knockout mice were the number of white blood cells, neutrophils, lymphocytes, and monocytes. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represented the NX1607 drug gavage treatment group.

[0027] Figure 6 For Ldlr - / -The results of red blood cells in the routine blood test of knockout mice were red blood cell count, hemoglobin content, hematocrit, mean corpuscular volume, mean hemoglobin content, and mean hemoglobin concentration. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represented the NX1607 drug gavage treatment group.

[0028] Figure 7 For Ldlr - / - The platelet-related results in the routine blood test of knockout mice were platelet count, platelet distribution width, mean platelet volume, and platelet volume. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represented the NX1607 drug gavage treatment group.

[0029] Figure 8 For Ldlr - / - Gross Oil Red O staining of blood vessels in knockout mice. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group.

[0030] Fig. 9 For Ldlr - / - HE, Oil Red O, CD68, and TUNEL staining of the outflow tract of knockout mice. The scale bar is 100 μm. The vehicle gavage control group used 0.5% sodium carboxymethyl cellulose as a control, and NX1607 represents the NX1607 drug gavage treatment group. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention will be further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention.

[0032] In addition, 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.

[0033] 1. Experimental Animals and Their Husbandry Experimental animal species, sex, age and source: Ldlr gene (gene number 16835, gene function: involved in lipid transport; regulation of inflammatory response; and regulation of lipid metabolism), male, 8 weeks old, weight 19-25g. Ldlr gene knockout mice (Ldlr - / - ) were purchased from GemPharmatech (Nanjing, China).

[0034] 2. Experimental Animal Feed Formula Western Diet (WD) was purchased from Research Diet, catalog number: D12108C.

[0035] 3. Animal feeding and grouping All experiments were conducted in accordance with the principles of laboratory animal care (NIH publication No. 85Y23, revised in 1996) and approved by the Peking University Laboratory Animal Ethics Committee (LA2023460). All animals were bred and raised in the SPF animal room of Peking University Health Science Center (license number: SYXK (Beijing) 2022-0037), with a daily 12-h light cycle, 25±2℃ and 40±5 humidity. All mice were free to eat and drink unless otherwise specified. Figure 1 The figure shows the dosing and diet diagram of Ldlr knockout mice. Eight-week-old mice with normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given NX1607 by gavage for 16 weeks (NX1607 dosage: 5 mg / kg body weight / day). They were divided into two groups: the vehicle control group and the drug treatment group, and the vehicle used was 0.5% sodium carboxymethyl cellulose.

[0036] 4. Determination of plasma total cholesterol and triglyceride levels Eight-week-old mice fed a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given vehicle control or NX1607 gavage treatment for 16 weeks. The mice were fasted for 4 h, and anticoagulant or non-anticoagulant blood samples were collected from the orbital vein. Plasma was separated by centrifugation at 4 °C for 10 min. Plasma total cholesterol (TC) and triglyceride (TG) concentrations were measured using commercially available kits from Biopharmaceuticals (Beijing, China).

[0037] The results are as follows Figure 2 As shown in , NX1607 treatment had no effect on the plasma total cholesterol and triglyceride levels of mice.

[0038] 5. Rapid Liquid Chromatography Determination of Plasma Lipoprotein Distribution Eight-week-old mice fed a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given NX1607 by gavage for 16 weeks. After 4 h of fasting, blood samples with or without anticoagulants were collected from the orbital vein. Plasma was separated by centrifugation at 4 °C for 10 min. Lipoprotein profiles were determined by rapid liquid chromatography, and the contents of samples (TC) and triglycerides (TG) were measured using commercially available kits from Biopharm (Beijing, China).

[0039] The results are as follows Figure 3 As shown in the results, NX1607 treatment had no significant effect on the distribution content of various lipoproteins in mouse plasma.

[0040] 6. Determination of plasma alanine aminotransferase and aspartate aminotransferase levels Eight-week-old mice with normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given 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. Plasma was separated by centrifugation at 4°C for 10 minutes. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the samples were measured using commercially available kits from Nanjing Jiancheng.

[0041] The results are as follows Figure 4 As shown in the results, NX1607 treatment had no significant effect on the level of alanine aminotransferase (ALT) in mouse plasma, and no significant effect on the level of aspartate aminotransferase (AST), which proves that the drug does not cause liver toxicity.

[0042] VII. Determination of mouse whole blood routine Eight-week-old mice with normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given NX1607 by gavage for 16 weeks. Anticoagulant blood samples were collected from the orbital vein. The blood must be fully mixed with the anticoagulant and diluent at the time of blood collection or immediately after blood collection for measurement. Mouse blood samples were measured and analyzed using the animal blood routine analyzer provided by Furui Runze (Beijing).

[0043] The results are as follows Figure 5-7 As shown in , NX1607 treatment had no significant effect on the inflammation-related indicators (leukocytes, neutrophils, lymphocytes, and 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 mouse blood ( Figure 6 ); had no significant effect on platelet-related indicators in mouse blood (platelet count, platelet distribution width, mean platelet volume, platelet volume) ( Figure 7 ). Figure 5-Figure 7 The results showed that NX1607 had no side effects on whole blood biochemical indicators.

[0044] 8. Oil red O staining of blood vessels Eight-week-old mice fed a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given NX1607 by oral gavage for 16 weeks. The whole-length aorta of the mice was obtained, fixed with paraformaldehyde, dehydrated with 20% sucrose, and stained with Oil Red O.

[0045] The results are as follows Figure 8 As shown, the results of the macroscopic Oil Red O staining of the aorta showed that the lipid accumulation (red part) of the mice in the NX1607 treatment group was reduced, indicating that the aortic plaques were significantly reduced. The above pathological results show that the atherosclerotic plaques in the aorta were significantly reduced after the treatment of NX1607, and the drug has a significant therapeutic effect on atherosclerosis.

[0046] IX. HE, Oil Red O, Bodipy, and CD68 staining of aortic outflow tract Eight-week-old mice fed a normal diet (CD) were given the above-mentioned Western diet (WD) for 4 weeks and then given NX1607 by oral gavage for 16 weeks. The frozen samples of the aortic outflow tract (aortic root) were selected and sectioned at a thickness of 7 μm for HE and Oil Red O staining as well as CD68 and TUNEL immunofluorescence staining.

[0047] The results are as follows Fig. 9 As shown, the Oil Red O staining results of the aortic outflow tract plaques in NX1607-treated mice compared with the control group mice showed that lipid accumulation in the outflow tract was reduced (ORO staining red part), indicating that the aortic root plaques were significantly reduced, the HE staining results and the TUNEL staining positive signal area (green part) showed that the necrotic core area of ​​the aortic root was significantly reduced, and the CD68 positive signal area indicated that the inflammatory infiltration in the aortic root plaque area was reduced.

[0048] The above pathological results collectively indicate that after treatment with NX1607, atherosclerotic plaques were significantly reduced, necrotic areas were significantly reduced, and inflammation was significantly alleviated. This drug has a significant therapeutic effect on atherosclerosis.

[0049] In summary, CBL inhibitors represented by NX1607 have the effect of treating atherosclerosis and can be used in the preparation of drugs for treating 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 or a precursor thereof or a metabolite thereof, Formula I.

2. The use according to claim 1, wherein the CBL inhibitor is NX1607.

3. 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.

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

5. 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.

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

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

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

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

10. 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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