CN118873667B

CN118873667B - Application of LAT1 inhibitors in the preparation of drugs for treating atherosclerosis

Info

Publication number: CN118873667B
Application number: CN202411289115.7A
Authority: CN
Inventors: 田进伟; 钟珊; 王思奇; 王帆; 王雪羽
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2024-09-14
Filing date: 2024-09-14
Publication date: 2025-08-05
Anticipated expiration: 2044-09-14
Also published as: CN118873667A

Abstract

The present invention relates to the use of LAT1 inhibitors in the preparation of medicaments for treating atherosclerosis, belonging to the field of medical technology. To address the clinical problem that advanced atherosclerosis still presents a significant residual risk under current treatment strategies, the present invention provides the use of LAT1 inhibitors in the preparation of medicaments for treating atherosclerosis. The present invention demonstrates that LAT1 inhibitors inhibit the entry of leucine into macrophages, reducing plaque area and necrotic core, thereby slowing the progression of atherosclerosis. LAT1 inhibitors can also enhance macrophage mitochondrial function, increase fatty acid oxidation, reduce lipid deposition within atherosclerotic plaques, increase plaque stability, and slow the progression of atherosclerosis induced by a high-fat diet. LAT1 inhibitors can also be used in patients with concurrent gastrointestinal bleeding, aortic aneurysms, or tumors, for whom the use of other anti-atherosclerotic drugs is limited.

Description

Application of LAT1 inhibitor in preparation of medicines for treating atherosclerosis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a LAT1 inhibitor in preparation of medicines for treating atherosclerosis.

Background

Atherosclerosis (atherosclerosis, AS) is a chronic vascular disease process in which macrophages gradually accumulate on the dilated arterial wall, partially in the lesion, phagocytizing lipids, generating various inflammatory mediators, accelerating the formation of advanced atherosclerosis. Advanced atherosclerosis has a relatively larger necrotic core and thin fibrous cap, and is more prone to rupture leading to the onset of acute cardiovascular events. Meanwhile, macrophages in the plaque are increased due to hypoxia in the plaque, so that lipid accumulation and chronic inflammation are increased, lipid metabolism capacity of the macrophages is weakened, apoptosis is increased, lipid accumulation in the plaque is further increased, and plaque vulnerability is increased.

Atherosclerotic heart disease (Atherosclerotic cardiovascular disease, ASCVD) is a special manifestation of atherosclerosis on the coronary arteries of the heart. When atherosclerosis occurs in coronary arteries, the coronary artery can cause lumen stenosis or occlusion, thereby causing myocardial ischemia, hypoxia or necrosis, and the coronary artery is a chronic cardiovascular disease with very high incidence rate at present.

The cause of atherosclerosis is complex, and hypertension, hyperlipidemia and diabetes are all important causative factors of atherosclerosis. The clinical application of the medicine for treating atherosclerosis at the present stage mainly comprises lipid-lowering, anti-thrombus and anticoagulation medicines. However, lipid-lowering drugs are prone to drug resistance, and anti-thrombus and anticoagulation drugs are prone to bleeding risk, so that the application of the lipid-lowering drugs in patients with combined gastrointestinal bleeding, aortic aneurysm or tumor is very limited, and the clinical treatment difficulty is greatly increased.

Disclosure of Invention

In order to solve the clinical problem that advanced atherosclerosis still has a large residual risk under the current treatment strategy, the invention provides the application of the LAT1 inhibitor in preparing medicines for treating atherosclerosis.

The technical scheme of the invention is as follows:

use of an inhibitor of LAT1 for the preparation of a medicament for the treatment of atherosclerosis.

Further, the medicament for treating atherosclerosis takes the LAT1 inhibitor as the only active ingredient or one of the active ingredients.

Further, the LAT1 inhibitor in the medicine for treating atherosclerosis accounts for 0.1-99 wt%.

Further, the LAT1 inhibitor in the medicine for treating atherosclerosis is one of JPH203, interfering RNA of LAT1 and leucine non-selective inhibitor 2-amino bicyclo- (2, 1) -heptane-2-carboxylic acid.

Further, the medicine for treating atherosclerosis also comprises pharmaceutically acceptable auxiliary materials and/or carriers.

Further, the medicine for treating atherosclerosis is a gastrointestinal tract administration dosage form, in particular an oral administration dosage form.

Further, the oral administration 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.

Further, the LAT1 inhibitor is administered in an amount of 25mg/kg body weight per 5 days of continuous administration at 9 days intervals in the medicament for treating atherosclerosis.

Further, the agent for treating atherosclerosis reduces plaque area and necrotic core by inhibiting leucine from entering macrophages, alleviating the progression of atherosclerosis.

Further, the medicament for treating atherosclerosis increases fatty acid oxidation, reduces lipid deposition in atherosclerotic plaques, increases stability of atherosclerotic plaques, and delays high-fat diet-induced atherosclerosis diseases by enhancing mitochondrial function.

The invention has the beneficial effects that:

Large neutral amino acid transporter small subunit 1 (Large neutral amino acids transporter small subunit, LAT1/SLC7A 5) is the predominant transporter for leucine in cells. Leucine transporter inhibitors, LAT1 inhibitors, have been shown to be effective in inhibiting leucine entry into cells and drugs using them as the main ingredient have been put into clinical trials for the treatment of biliary tract cancer and idiopathic pulmonary fibrosis, indicating that they have been clinically data-supported in terms of safety and efficacy.

The invention provides a new application of LAT1 inhibitor in treating atherosclerosis for the first time. Animal experiments prove that the LAT1 inhibitor can inhibit leucine from entering macrophages, reduce plaque areas and necrotic cores and delay the development of atherosclerosis, and meanwhile, the LAT1 inhibitor can also enhance the mitochondrial function of the macrophages, increase fatty acid oxidation, reduce lipid deposition in the atherosclerotic plaque, increase the stability of the atherosclerotic plaque and delay the atherosclerosis diseases induced by high-fat diet.

The invention uses macrophage specific Slc7a5 knockout mice to prove that targeting inhibition of macrophage leucine transport is a safer, effective and feasible strategy for treating atherosclerosis by intervening metabolism. The LAT1 inhibitor provides a treatment scheme capable of overcoming atherosclerosis caused by metabolic abnormality due to factors such as obesity, high protein diet and the like, so that the LAT1 inhibitor can be applied to patients with limited application of other types of anti-atherosclerosis drugs due to the combination of digestive tract hemorrhage or the combination of tumor diseases with bleeding tendency or the generation of drug resistance to lipid-lowering drugs.

Drawings

FIG. 1 is a photograph of pathological staining of aortic oil red O in two groups of mice in example 1;

FIG. 2 is a graph showing the results of a vascular tissue mRNA sequencing result pathway enrichment analysis of two groups of mice in example 1;

FIG. 3 is a photograph of pathological staining of aortic oil red O in three groups of mice in example 2;

FIG. 4 is an aortic root oil red O staining picture of three groups of mice in example 2;

FIG. 5 is a graph of HE staining of aortic root of three groups of mice in example 2, P being the area of aortic root plaque, NC being the necrotic core, L being the lumen;

FIG. 6 is a graph showing the comparison of mitochondrial membrane potential of macrophages after stimulation with JPH203 or PBS for ox-LDL in example 3;

FIG. 7 is a graph showing comparison of mRNA expression of key enzyme for macrophage fatty acid beta oxidation after stimulation with JPH203 or PBS for ox-LDL in example 3;

FIG. 8 is a photograph of macrophage oil red O staining after stimulation with JPH203 or PBS for ox-LDL in example 3;

FIG. 9 is a comparison of HE staining and O staining of atherosclerotic plaques at the aortic root of two mice in example 4.

Detailed Description

The following embodiments are used for further illustrating the technical scheme of the present invention, but not limited thereto, and all modifications and equivalents of the technical scheme of the present invention are included in the scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are all conventional equipment or apparatus in the art, and the raw materials and the like used in the examples of the present invention are commercially available unless otherwise specified, and the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

Example 1

This example provides the construction of a mouse model of advanced atherosclerosis and transcriptome detection results.

1. Materials:

1.1 animals

Apoe-/-mice of this example were purchased from Sai industry Co (Suzhou), experimental animals were randomly grouped and fed a normal diet (NOR group) and a high fat diet (AS group) from 8 weeks of age, respectively, and two groups of mice were obtained from the aorta at 24 weeks of age. The use of experimental animals was approved by the ethical committee of the university of halbine medical science.

1.2 Reagents

High fat diet (D12079B, 40% fat powered, 0.15% cholesterol) was purchased from co-feed limited, australia, family and modified oil red O staining fluid was purchased from beijing solibao.

2. Method of

2.1 Staining of aortic root oil red O:

selecting frozen slices of 10 microns, balancing at room temperature for 5 minutes, repeatedly washing with dd water for 5 times, wiping the slices, dripping 60% isopropanol for 30-40 seconds, discarding liquid, dripping oil red O into a hatching membrane for 35 minutes, observing, placing into water after obvious coloring, dripping sappan wood for 3 minutes, stopping the dyeing with flowing water, observing under a mirror, and turning blue.

2.2 High throughput sequencing technique of mRNA from rat aortic tissue:

6 NOR group samples and 6 AS group samples, and entrust Beijing Nobela source biological information technology Co., ltd.

2.3 Two sets of tissue differential mRNAs were obtained according to sequencing and KEGG enrichment was performed.

3. Results

3.1 Pathological microscope observation and photographing. The results are shown in FIG. 1, which illustrates the success of model building.

3.2 Results are shown in fig. 2, and analysis of the mRNA sequencing results in this example shows that the AS group downregulated genes are significantly enriched in branched-chain amino acid degradation pathways, suggesting that branched-chain amino acid metabolism is impaired during the progression of atherosclerosis.

Example 2

This example examines the therapeutic effect of JPH203 on atherosclerosis in a high fat diet fed atherosclerosis mouse model.

1. Material

Reagent L-leucine was purchased from Sigma and LAT1 selective inhibitor JPH203 (Nanvuranlat) was purchased from Tao Shu organism.

2. Method of

The experimental animals applied to this example were randomly divided into 3 groups, each fed a high fat diet from 8 weeks of age, and were fed with JPH203 (at 25mg/kg dose, once every other day), leucine (at 8mg/ml concentration, with water replaced every 2 days), or normal water, respectively, from 20 weeks of age, and two groups of mice were harvested at 24 weeks of age for aortic and heart. The aortic valve was stained with substantially oil red O, and the heart was sectioned at the aortic sinus region and HE, oil red O stained to detect the extent of atherosclerotic lesions.

3. Results

3.1 This example demonstrates that the same high fat diet fed mice had reduced atherosclerotic plaque area and intra-plaque lipid content (shown as oil red O ratio) under treatment with JPH203 compared to the control group, and increased lesion area and lipid content under excessive leucine feeding compared to the control group, as shown in fig. 3 and 4.

3.2 This example demonstrates that the atherosclerotic plaque area and necrotic core of the aortic root under treatment with JPH203 is reduced compared to the control group, while the plaque area and necrotic core is increased compared to the control group with excessive leucine feeding, as shown in fig. 5.

Example 3

This example examined the effect of JPH203 on RAW264.7 in the mouse macrophage cell line.

1. Material

1.1. And (3) cells:

the mouse macrophage cell line RAW264.7 cells used in this example were purchased from Shanghai cell bank of the national academy of sciences.

Cell culture in DMEM medium containing 10% inactivated fetal bovine serum (company SCIENCE CELL, USA), penicillin (100U/mL), streptomycin (100. Mu.g/mL) at 37℃under 5% CO ₂.

1.2. Reagent:

oxidized low density lipoprotein (ox-LDL) was purchased from guangzhou yiyuan biotechnology limited;

Mitochondrial membrane potential detection kit (JC-1) was purchased from peganum bio-limited;

TRIzol Reagent was purchased from Invitrogen, usa;

Reverse transcription kit (04897030001) was purchased from Roche company, germany;

SYBR GREEN MASTER (ROX) kit for fluorescent Real-time (Real-time) quantification PCR (polymerase chain reaction) was purchased from Roche, germany;

Real-Time PCR specific primers were designed and synthesized by Rui Boxing Biotechnology Inc.

2. Method of

2.1 JC-1 staining detects mitochondrial membrane potential:

RAW264.7 cells were divided into 2 groups, each stimulated with ox-LDL for 24 hours, and PBS (control group) or JPH203 treatment (treatment group) was given simultaneously, and cells were collected after 24 hours.

10-60 Ten thousand cells are taken and resuspended in 0.5ml of cell culture liquid, which can contain serum and phenol red. Adding 0.5mlJC-1 dyeing working solution, reversing for several times, and mixing.

Incubate for 20 min at 37℃in the cell incubator. During incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared in a ratio of 4ml distilled water per 1ml JC-1 staining buffer (5X) and placed in an ice bath.

After 37 ℃ incubation, 600g of 4 ℃ are centrifuged for 3-4 minutes, and cells are precipitated. The supernatant was discarded, taking care not to aspirate cells as much as possible.

Wash 2 times with JC-1 staining buffer (1X):

1ml JC-1 staining buffer (1X) was added to resuspend the cells, 600g 4C was centrifuged for 3-4 min, the cells were pelleted, and the supernatant was discarded. Then adding 1mlJC-1 staining buffer (1X) to re-suspend the cells, centrifuging 600g of 4 ℃ for 3-4 minutes, precipitating the cells, and discarding the supernatant. After re-suspending with an appropriate amount of JC-1 staining buffer (1X), data were collected and analyzed using a flow cytometer.

2.2 Extracting total RNA of macrophages after ox-LDL stimulation to detect the expression level of fatty acid beta oxidation key enzyme:

1 Ml TRIzol Reagent was added to each cell sample. Then the mixture is fully ground by a grinder and is subjected to complete separation of the nucleoprotein complex at room temperature (15-30 ℃). After 3 minutes, 0.2 ml of chloroform was added to each, the lid was closed, vigorously shaken for 15 seconds, left at room temperature for 3 minutes, centrifuged at 12000r/min for 15 minutes at 4℃and the upper colorless aqueous phase was transferred (about 0.5 ml) to another 1.5ml centrifuge tube (note not to be drawn into the middle layer). 0.5ml of isopropanol was added to each tube, allowed to stand at room temperature for 5 minutes, centrifuged at 12000r/min at 4℃for 10 minutes, and the supernatant was discarded.

RNA pellet was washed with 1ml of 75% ethanol per tube, and sufficient aspiration was performed to bring RNA into sufficient contact with ethanol, and centrifuged at 12000r/min for 6min at 4℃and the supernatant was discarded. RNA pellet was washed with 1ml of 100% ethanol per tube, well aspirated, centrifuged at 12000r/min for 5min at 4℃and the supernatant discarded.

After air-drying for 10 minutes, RNA was dissolved in DEPC water without RNase, and repeated aspiration was performed to dissolve RNA sufficiently. RNA concentration was determined using an ultraviolet spectrophotometer, reverse transcription was performed to obtain cDNA, and fluorescent quantitative PCR was performed to detect mRNA expression levels of fatty acid oxidation key enzymes ACOX1 and HADHA to assess macrophage fatty acid oxidation function.

2.3 Cell oil red O staining:

First, an oil red O working fluid was prepared. Diluting the stock solution 5% oil red O dye liquor and double distilled water according to the proportion of 3:2 to obtain a mixed liquor of 60ml oil red O dye liquor and 40ml double distilled water. The mixture was filtered and allowed to stand at room temperature until the liquid had no precipitate.

Cells were added to the well plate. And adding a small amount of 60% isopropanol into the pore plate, covering the cells for 15-20 seconds, then sucking off the isopropanol, and slightly airing the water. Oil red O staining working solution is added into the pore plate to cover cells. Dyeing for 30 minutes at room temperature in dark. And removing the staining solution. The cells were photographed under a microscope.

3. Results

3.1 This example demonstrates a decrease in mitochondrial membrane potential following treatment with JPH203, representing a degree of restoration of mitochondrial function, as shown in fig. 6, the decrease in mitochondrial membrane potential, which represents a green fluorescence positive in the box.

3.2 This example demonstrates that JPH203 increases the expression level of key enzymes for macrophage fatty acid oxidation under ox-LDL stimulation, as shown in FIG. 7.

3.3 This example demonstrates that JPH203 reduces lipid content in macrophage-derived foam cells under ox-LDL induction, as shown in figure 8.

Example 4

In the embodiment, the macrophage specific Slc7a5 knockout mouse model experiment proves that the targeted inhibition of macrophage transport leucine can treat atherosclerosis.

1. Material

Animals:

Macrophage specific Slc7a5 mouse atherosclerosis model Slc7a5 ^flox/flox mice were constructed by Sai corporation (Suzhou) based on CRISPR/Cas9 technology.

To drive macrophage specific knockdown of Slc7a5, the Slc7a5 ^flox/flox mice were hybridized with Lyz2-cre mice (sei). Slc7a5 ^flox/flox Lyz2-cre mice were verified by PCR genotyping. To construct an atherosclerosis model mouse against Apoe ^-/- background, the Slc7a5 ^flox/flox Lyz2-cre mice were hybridized with Apoe ^-/- mice, ultimately resulting in Slc7a5 ^flox/floxLyz2-cre;Apoe^-/- mice and Slc7a5 ^flox/flox;Apoe^-/- mice.

2. Method of

The 8 week old Slc7a5 ^flox/floxLyz2-cre;Apoe^-/- mice were fed a 16 week high fat diet with the Slc7a5 ^flox/flox;Apoe^-/- mice (control group) to induce atherosclerosis.

3. Results

This example demonstrates that the area of atherosclerotic plaque, necrotic core and intra-plaque lipid content (shown as oil red O ratio) were all reduced in the aortic root of the Slc7a5 ^flox/floxLyz2-cre;Apoe^-/- mice compared to the control group, as shown in fig. 9.

Claims

Use of an inhibitor of LAT1 for the preparation of a medicament for the treatment of atherosclerosis, wherein the inhibitor of LAT1 in the medicament for the treatment of atherosclerosis is JPH203, which reduces plaque area and necrotic core by inhibiting leucine entry into macrophages, and reduces the progression of atherosclerosis.
2. Use of a LAT1 inhibitor according to claim 1 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the medicament for the treatment of atherosclerosis comprises the LAT1 inhibitor as the sole active ingredient or one of the active ingredients.
3. Use of a LAT1 inhibitor according to claim 1 or 2 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the content of LAT1 inhibitor in the medicament for the treatment of atherosclerosis is 0.1 wt. -% to 99 wt. -%.
4. Use of a LAT1 inhibitor according to claim 3 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the medicament for the treatment of atherosclerosis further comprises pharmaceutically acceptable excipients and/or carriers.
5. Use of LAT1 inhibitors according to claim 4 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the medicament for the treatment of atherosclerosis is in a form for administration via the gastrointestinal tract, in particular in a form for oral administration.
6. The use of LAT1 inhibitors according to claim 5 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the oral administration form is a granule form, a tablet form, a capsule form, a pill form or an oral liquid form.
7. Use of the LAT1 inhibitor according to claim 6 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that the amount of LAT1 inhibitor administered in the medicament for the treatment of atherosclerosis is 25mg/kg body weight, every 5 days of continuous administration, at intervals of 9 days.
8. The use of LAT1 inhibitors according to claim 7 for the preparation of a medicament for the treatment of atherosclerosis by increasing mitochondrial function, increasing fatty acid oxidation, decreasing lipid deposition in atherosclerotic plaques, increasing atherosclerotic plaque stability, delaying high fat diet induced atherosclerotic disease.