Use of LAT1 inhibitor in the preparation of a medicament for treating atherosclerosis

Abstract

The present invention relates to the use of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis, belonging to the field of pharmaceutical technology. To solve the clinical problem that there is still a relatively high residual risk in advanced atherosclerosis under the current treatment strategies, the present invention provides the use of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis. The present invention has confirmed that LAT1 inhibitors delay the progression of atherosclerosis by inhibiting the entry of leucine into macrophages, reducing plaque area and necrotic core; at the same time, LAT1 inhibitors can also enhance the mitochondrial function of macrophages, increase fatty acid oxidation, reduce lipid deposition in atherosclerotic plaques, increase the stability of atherosclerotic plaques, delay the development of atherosclerosis induced by a high-fat diet, and can be applied to patients in whom the use of other types of anti-atherosclerotic drugs is limited due to diseases such as combined gastrointestinal bleeding, aortic aneurysm or tumor.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a chronic vascular lesion process. During the chronic inflammatory reaction process, macrophages gradually accumulate on the dilated arterial wall, some in the lesions, phagocytize lipids, produce various inflammatory mediators, and accelerate the formation of advanced atherosclerosis. Advanced atherosclerosis has a relatively larger necrotic core and a thin fibrous cap, and is more likely to rupture, leading to the occurrence of acute cardiovascular events. At the same time, macrophages in the plaque have weakened lipid metabolism ability and increased apoptosis due to increased hypoxia, lipid accumulation, and chronic inflammation in the plaque, further leading to increased lipid accumulation in the plaque and increased plaque vulnerability.

[0003] Atherosclerotic cardiovascular disease (ASCVD) is a special manifestation of atherosclerosis in the coronary arteries of the heart. When atherosclerosis occurs in the coronary arteries, it will lead to lumen stenosis or occlusion, thereby causing myocardial ischemia, hypoxia, or necrosis, and it is a type of chronic cardiovascular disease with a very high incidence rate at present.

[0004] The etiology of atherosclerosis is complex. Hypertension, hyperlipidemia, and diabetes are all important pathogenic factors of atherosclerosis. At present, the drugs clinically used to treat atherosclerosis mainly include lipid-lowering, antithrombotic, and anticoagulant drugs. However, lipid-lowering drugs are prone to drug resistance, and the application of antithrombotic and anticoagulant drugs is prone to bleeding risks, and their application in patients with combined gastrointestinal bleeding, aortic aneurysm, or tumors is very limited, greatly increasing the clinical treatment difficulty. Summary of the Invention

[0005] To solve the clinical problem that there is still a relatively large residual risk in advanced atherosclerosis under the current treatment strategy, the present invention provides the application of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis.

[0006] Technical Solution of the Present Invention:

[0007] The application of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis.

[0008] Further, the drug for treating atherosclerosis takes the LAT1 inhibitor as the sole active ingredient or one of the active ingredients.

[0009] Further, the content of the LAT1 inhibitor in the drug for treating atherosclerosis is 0.1wt% - 99wt%.

[0010] Further, the LAT1 inhibitor in the drug for treating atherosclerosis is one of JPH203, interfering RNA of LAT1, and leucine non-selective inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid.

[0011] Further, the drug for treating atherosclerosis further comprises pharmaceutically acceptable excipients and / or carriers.

[0012] Further, the drug for treating atherosclerosis is a dosage form for gastrointestinal administration, specifically an oral dosage form.

[0013] Further, the oral dosage form is a granule dosage form, a tablet dosage form, a capsule dosage form, a pill dosage form or an oral liquid dosage form.

[0014] Further, the dosage of the LAT1 inhibitor in the drug for treating atherosclerosis is 25 mg / kg body weight, and it is administered continuously for 5 days every time, with an interval of 9 days.

[0015] Further, the drug for treating atherosclerosis reduces the plaque area and necrotic core by inhibiting the entry of leucine into macrophages, and alleviates the progression of atherosclerosis.

[0016] Further, the drug for treating atherosclerosis enhances mitochondrial function, increases fatty acid oxidation, reduces lipid deposition in atherosclerotic plaques, increases the stability of atherosclerotic plaques, and delays atherosclerotic diseases induced by a high-fat diet.

[0017] Advantages of the present invention:

[0018] Large neutral amino acid transporter small subunit 1 (LAT1 / SLC7A5) is the main transporter of leucine in cells. Leucine transporter inhibitor - LAT1 inhibitor has been proven to effectively inhibit the entry of leucine into cells. Drugs using it as the main component have been put into clinical trials for the treatment of cholangiocarcinoma and idiopathic pulmonary fibrosis, indicating that clinical data support its safety and effectiveness.

[0019] The present invention for the first time provides a new use of LAT1 inhibitors for treating atherosclerosis. Through animal experiments, the present invention has confirmed that LAT1 inhibitors delay the progression of atherosclerosis by inhibiting the entry of leucine into macrophages, reducing plaque area and necrotic core; meanwhile, LAT1 inhibitors can also enhance the mitochondrial function of macrophages, increase fatty acid oxidation, reduce lipid deposition in atherosclerotic plaques, increase the stability of atherosclerotic plaques, and delay atherosclerotic diseases induced by high-fat diet.

[0020] The present invention has confirmed, by using macrophage-specific Slc7a5 knockout mice, that targeted inhibition of leucine transport in macrophages is an effective and feasible strategy for treating atherosclerosis by intervening in metabolism with higher safety. The LAT1 inhibitor provides a treatment regimen that can overcome atherosclerosis caused by metabolic abnormalities due to factors such as obesity and high-protein diet, making it applicable to patients in whom the application of other types of anti-atherosclerotic drugs is limited due to complications such as gastrointestinal bleeding, or tumors with a bleeding tendency, or drug resistance to lipid-lowering drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Pathological staining pictures of Oil Red O of the aorta of two groups of mice in Example 1;

[0022] Figure 2 Result graph of pathway enrichment analysis of mRNA sequencing of vascular tissues of two groups of mice in Example 1;

[0023] Figure 3 Pathological staining pictures of Oil Red O of the aorta of three groups of mice in Example 2;

[0024] Figure 4 Oil Red O staining pictures of the aortic root of three groups of mice in Example 2;

[0025] Figure 5 HE staining pictures of the aortic root of three groups of mice in Example 2, where P is the plaque area of the aortic root, NC is the necrotic core, and L is the lumen;

[0026] Figure 6 Comparison graph of mitochondrial membrane potential of macrophages after treatment with JPH203 or PBS for ox-LDL stimulation in Example 3;

[0027] Figure 7 Comparison graph of mRNA expression of key enzymes for fatty acid β-oxidation of macrophages after treatment with JPH203 or PBS for ox-LDL stimulation in Example 3;

[0028] Figure 8 Oil Red O staining pictures of macrophages after treatment with JPH203 or PBS for ox-LDL stimulation in Example 3;

[0029] Figure 9 It is a comparison diagram of HE staining and Oil Red O staining of atherosclerotic plaques at the aortic root of two kinds of mice in Example 4. Detailed implementation manners

[0030] The technical solutions of the present invention will be further described below in conjunction with embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, shall be covered by the protection scope of the present invention. For the process equipment or devices not specifically noted in the following embodiments, conventional equipment or devices in the field are used. If not specifically indicated, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically indicated, the technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0031] Example 1

[0032] This example provides the construction of a late atherosclerotic mouse model and the results of transcriptome detection.

[0033] 1. Materials:

[0034] 1.1 Animals

[0035] The Apoe - / - mice in this example were purchased from Cyagen Biosciences (Suzhou). The experimental animals were randomly grouped and fed a normal diet (NOR group) and a high-fat diet (AS group) respectively from 8 weeks of age. The aortas of the two groups of mice were sampled at 24 weeks of age. The use of experimental animals was approved by the Ethics Committee of Harbin Medical University.

[0036] 1.2 Reagents

[0037] The high-fat feed (D12079B, 40% fat for energy, 0.15% cholesterol) was purchased from Keao Xieli Feed Co., Ltd., and the modified Oil Red O staining solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0038] 2. Methods

[0039] 2.1 Oil Red O staining of the aortic root of mice:

[0040] Select 10-μm frozen sections, equilibrate at room temperature for 5 minutes, and rinse repeatedly with dd water 5 times; dry the slides, add 60% isopropanol for 30 - 40 seconds, discard the liquid; add Oil Red O and place it in the incubation membrane for 35 minutes, observe, and place it in water after obvious coloring; add hematoxylin staining for 3 minutes, terminate the staining with running water, observe under the microscope, and blue back.

[0041] 2.2 mRNA high-throughput sequencing technology for mouse aortic tissue:

[0042] Six NOR group samples and six AS group samples were entrusted to Novogene Bioinformatics Technology Co., Ltd. in Beijing to complete the high-throughput sequencing and subsequent analysis related work.

[0043] 2.3 Differentially expressed mRNAs between the two groups of tissues were obtained according to the sequencing, and KEGG enrichment was performed.

[0044] 3. Results

[0045] 3.1 Observation and photography were performed under a pathological microscope. The results are as Figure 1 shown, indicating that the model was successfully established.

[0046] 3.2 The results are as Figure 2 shown. In this embodiment, analysis of the mRNA sequencing results showed that the downregulated genes in the AS group were significantly enriched in the branched-chain amino acid degradation pathway, suggesting impaired branched-chain amino acid metabolism during the progression of atherosclerosis.

[0047] Example 2

[0048] This example investigated the therapeutic effect of JPH203 on atherosclerosis in a mouse model of atherosclerosis fed a high-fat diet.

[0049] 1. Materials

[0050] Reagents: L-leucine was purchased from Sigma, and the LAT1 selective inhibitor JPH203 (Nanvuranlat) was purchased from TargetMol.

[0051] 2. Methods

[0052] The experimental animals used in this example were randomly divided into 3 groups. All were fed a high-fat diet starting from 8 weeks of age. Starting from 20 weeks of age, they were respectively gavaged with JPH203 (at a dose of 25 mg / kg, once every other day), fed with leucine (prepared at a concentration of 8 mg / ml, and the drinking water was changed every 2 days), or fed with normal water. At 24 weeks of age, the aortas and hearts of the two groups of mice were harvested. Gross oil red O staining was performed on the aorta, and sections of the aortic sinus of the heart were stained with HE and oil red O to detect the degree of atherosclerotic lesions.

[0053] 3. Results

[0054] 3.1 This example confirmed that under the treatment of JPH203, the atherosclerotic plaque area and the lipid content within the plaque (indicated by the proportion of oil red O) formed in mice fed the same high-fat diet were smaller than those in the control group; while under the feeding of excessive leucine, the lesion area and lipid content were increased compared with the control group, as Figure 3 and Figure 4 shown.

[0055] 3.2 This embodiment confirmed that the atherosclerotic plaque area and necrotic core in the aortic root decreased under JPH203 treatment compared with the control group; while under excessive leucine feeding, the plaque area and necrotic core increased compared with the control group, as Figure 5 shown.

[0056] Example 3

[0057] This embodiment investigated the effect of JPH203 on the mouse macrophage cell line RAW264.7.

[0058] 1. Materials

[0059] 1.1. Cells:

[0060] The mouse macrophage cell line RAW264.7 cells used in this embodiment were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0061] The cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum (Science Cell, USA), penicillin (100 U / mL), and streptomycin (100 μg / mL) at 37 °C and 5% CO2.

[0062] 1.2. Reagents:

[0063] Oxidized low-density lipoprotein (ox-LDL) was purchased from Guangzhou Yiyuan Biotechnology Co., Ltd.;

[0064] The mitochondrial membrane potential detection kit (JC-1) was purchased from Beyotime Biotechnology Co., Ltd.;

[0065] TRIzol Reagent was purchased from Invitrogen, USA;

[0066] The reverse transcription kit (04897030001) was purchased from Roche, Germany;

[0067] The SYBR Green Master (ROX) kit used for fluorescence real-time (Real-time) quantitative PCR (polymerase chain reaction) was purchased from Roche, Germany;

[0068] The Real-Time PCR specific primers were designed and synthesized by Ribobio Co., Ltd.

[0069] 2. Methods

[0070] 2.1 JC-1 staining for detecting mitochondrial membrane potential:

[0071] RAW264.7 cells were divided into two groups, both stimulated with ox-LDL for 24 hours, and simultaneously given PBS (control group) or JPH203 treatment (treatment group). Cells were collected after 24 hours.

[0072] Take 100,000 - 600,000 cells and resuspend them in 0.5 ml of cell culture medium, which can contain serum and phenol red. Add 0.5 ml of JC-1 staining working solution and mix by inverting several times.

[0073] Incubate in a cell culture incubator at 37ºC for 20 minutes. During the incubation, prepare an appropriate amount of JC-1 staining buffer (1X) by mixing 1 ml of JC-1 staining buffer (5X) with 4 ml of distilled water according to the ratio, and place it in an ice bath.

[0074] After the incubation at 37ºC, centrifuge at 600 g for 3 - 4 minutes at 4ºC to precipitate the cells. Discard the supernatant, taking care not to aspirate the cells as much as possible.

[0075] Wash twice with JC-1 staining buffer (1X):

[0076] Add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 600 g for 3 - 4 minutes at 4ºC to precipitate the cells, and discard the supernatant. Then add 1 ml of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 600 g for 3 - 4 minutes at 4ºC to precipitate the cells, and discard the supernatant. After resuspending with an appropriate amount of JC-1 staining buffer (1X), use a flow cytometer to collect data and analyze.

[0077] 2.2 Detection of the expression levels of key enzymes in fatty acid β-oxidation by extracting total RNA from macrophages after ox-LDL stimulation:

[0078] RAW264.7 cells were divided into two groups, both stimulated with ox-LDL for 24 hours, and simultaneously given PBS (control group) or JPH203 treatment (treatment group). Cells were collected after 24 hours.

[0079] Add 1 ml of TRIzol Reagent to each group of cell samples. Then grind thoroughly with a grinder and place at room temperature (15 - 30℃) to completely separate the nucleoprotein complex. After 3 minutes, add 0.2 ml of chloroform to each, tightly cap the lid, shake vigorously for 15 seconds, place at room temperature for 3 minutes, centrifuge at 12000 r / min for 15 minutes at 4℃, and then transfer the upper colorless aqueous phase (about 0.5 ml) to another 1.5 ml centrifuge tube (take care not to aspirate the middle layer). Add 0.5 ml of isopropanol to each tube, let stand at room temperature for 5 minutes, centrifuge at 12000 r / min for 10 minutes at 4℃, and discard the supernatant.

[0080] Wash the RNA precipitate with 1 ml of 75% ethanol per tube. Aspirate thoroughly to ensure that the RNA is fully contacted with the ethanol. Centrifuge at 12,000 r / min for 6 min at 4°C, and discard the supernatant. Wash the RNA precipitate with 1 ml of 100% ethanol per tube. Aspirate thoroughly, centrifuge at 12,000 r / min for 5 min at 4°C, and discard the supernatant.

[0081] After air drying for 10 minutes, dissolve the RNA with RNase-free DEPC water. Aspirate repeatedly to fully dissolve the RNA. Use an ultraviolet spectrophotometer to measure the RNA concentration. After reverse transcription to obtain cDNA, perform fluorescence quantitative PCR to detect the mRNA expression levels of the key fatty acid oxidation enzymes ACOX1 and HADHA to evaluate the fatty acid oxidation function of macrophages.

[0082] 2.3 Oil Red O staining of cells:

[0083] First, prepare the Oil Red O working solution. Dilute the stock solution of 5% Oil Red O staining solution and double-distilled water at a ratio of 3:2 to obtain a mixture of 60 ml of Oil Red O staining solution and 40 ml of double-distilled water. Filter the mixture and let it stand at room temperature until there is no precipitate.

[0084] Add the cells to the well plate. Add a small amount of 60% isopropanol to the well plate, cover the cells for 15 - 20 seconds, then aspirate the isopropanol and let the cells dry slightly. Add the Oil Red O staining working solution to the well plate to cover the cells. Stain for 30 minutes at room temperature in the dark. Remove the staining solution. Place the cells under the microscope for observation and photography.

[0085] 3. Results

[0086] 3.1 This example confirms that after treatment with JPH203, the decrease in mitochondrial membrane potential is reduced, indicating that mitochondrial function is restored to a certain extent. As Figure 6 shown, the area within the box represents the decrease in mitochondrial membrane potential with green fluorescence positive.

[0087] 3.2 This example confirms that JPH203 increases the expression levels of the key fatty acid oxidation enzymes in macrophages under ox-LDL stimulation, as Figure 7 shown.

[0088] 3.3 This example confirms that JPH203 reduces the lipid content in macrophage-derived foam cells induced by ox-LDL, as Figure 8 shown.

[0089] Example 4

[0090] This example confirms that targeted inhibition of macrophage leucine transport can treat atherosclerosis through experiments on macrophage-specific Slc7a5 knockout mouse models.

[0091] 1. Materials

[0092] Animal:

[0093] Macrophage-specific Slc7a5 Mouse atherosclerosis model: Slc7a5 flox / flox The mice were constructed by Cyagen Biosciences (Suzhou) based on CRISPR / Cas9 technology.

[0094] To drive Slc7a5 macrophage-specific knockout, Slc7a5 flox / flox the mice were crossed with Lyz2 -cre mice (Cyagen Biosciences). The Slc7a5 flox / flox Lyz2 -cre mice were verified by PCR genotyping. To construct Apoe - / - background atherosclerosis model mice, Slc7a5 flox / flox Lyz2 the Apoe - / - -cre mice were crossed with Slc7a5 flox / flox Lyz2 -cre; Apoe - / - the mice were crossed with Slc7a5 flox / flox ; Apoe - / - mice.

[0095] 2. Methods

[0096] 8-week-old Slc7a5 flox / flox Lyz2 -cre; Apoe - / - the mice were fed a high-fat diet for 16 weeks together with Slc7a5 flox / flox ; Apoe - / - mice (control group) to induce atherosclerosis.

[0097] 3. Results

[0098] This example demonstrates that in Slc7a5 flox / flox Lyz2 -cre; Apoe - / - mice, the atherosclerotic plaque area, necrotic core, and lipid content within the plaque (shown by the proportion of Oil Red O) in the aortic root were all reduced compared with the control group, as Figure 9 shown.

Claims

1. Use of a LAT1 inhibitor in the preparation of a drug for treating atherosclerosis, characterized in that: The LAT1 inhibitor in the drug for treating atherosclerosis is JPH203. The drug for treating atherosclerosis reduces the plaque area and necrotic core by inhibiting the entry of leucine into macrophages, thereby alleviating the progression of atherosclerosis.

2. Use of the LAT1 inhibitor according to claim 1 in the preparation of a medicament for treating atherosclerosis, characterized in that: The drug for treating atherosclerosis uses the LAT1 inhibitor as the only active ingredient or one of the active ingredients.

3. Use of the LAT1 inhibitor according to claim 1 or 2 in the preparation of a medicament for treating atherosclerosis, characterized in that: The content of the LAT1 inhibitor in the drug for treating atherosclerosis is 0.1 wt% to 99 wt%.

4. Use of the LAT1 inhibitor according to claim 3 in the preparation of a medicament for treating atherosclerosis, characterized in that: The drug for treating atherosclerosis further comprises pharmaceutically acceptable excipients and / or carriers.

5. Use of the LAT1 inhibitor according to claim 4 in the preparation of a medicament for treating atherosclerosis, characterized in that: The drug for treating atherosclerosis is in the form of a gastrointestinal tract administration dosage form, specifically an oral administration dosage form.

6. Use of the LAT1 inhibitor according to claim 5 in the preparation of a medicament for treating atherosclerosis, characterized in that: The oral dosage form is a granule dosage form, a tablet dosage form, a capsule dosage form, a pill dosage form or an oral liquid dosage form.

7. Use of the LAT1 inhibitor according to claim 6 in the preparation of a medicament for treating atherosclerosis, characterized in that: The dosage of the LAT1 inhibitor in the drug for treating atherosclerosis is 25 mg / kg body weight, and the drug is administered for 5 consecutive days with an interval of 9 days.

8. Use of the LAT1 inhibitor according to claim 7 in the preparation of a medicament for treating atherosclerosis, characterized in that: The drug for treating atherosclerosis increases fatty acid oxidation by enhancing mitochondrial function, reduces lipid deposition in atherosclerotic plaques, increases the stability of atherosclerotic plaques, and delays atherosclerotic diseases induced by a high-fat diet.

Images