Application of neratinib in the preparation of drugs for preventing or treating atherosclerosis

Abstract

The present invention discloses the use of neratinib or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a medicament for preventing or treating atherosclerotic disease. Atherosclerotic disease is a chronic inflammatory disease of the vascular lining. Low-density lipoprotein and inflammatory cells accumulate in damaged areas of the vascular lining, gradually forming lipid streaks that further develop into lipid plaques, i.e., atherosclerosis. Neratinib has the effect of inhibiting inflammatory responses and is therefore useful in the preparation of a medicament for preventing or treating atherosclerotic disease.

Description

Technical Field

[0001] The present disclosure relates to the technical field of medicine, and in particular to a use of neratinib in preparing a drug for preventing or treating atherosclerosis. Background Art

[0002] Atherosclerotic disease refers to a disease in which lipid deposits form on the inner walls of medium or large arteries, causing narrowing of the lumen, reduced blood flow or blood blockage. It is an important cause of cardiovascular diseases including myocardial infarction and stroke, as well as other peripheral vascular diseases related to arterial blockage.

[0003] Atherosclerosis is considered a chronic inflammatory disease. Oxidized low-density lipoprotein (Ox-LDL) in the blood accumulates on the blood vessel walls, causing damage to the vascular endothelium and triggering a chronic inflammatory response in the blood vessels. Macrophages phagocytize excess lipids to form foam cells that participate in the formation of the lipid core and release inflammatory factors such as TNF-α, IL-1β, and IL-6. Low-density lipoprotein and inflammatory cells continue to accumulate at the damaged site of the endothelium, gradually forming lipid streaks, which further develop into lipid plaques, namely atherosclerosis. The microenvironment inside the plaque induces smooth muscle cells to proliferate and migrate into the lipid core, forming fibrous atherosclerotic plaques. In the late stage of plaque formation, apoptotic and necrotic cells in the plaque activate a local inflammatory response. Under the stimulation of factors such as blood flow, the plaque may rupture and fall off, forming a thrombus, causing diseases such as myocardial infarction and cerebral infarction, which seriously threaten health.

[0004] Inflammation plays a crucial role in the development and progression of atherosclerosis. According to the CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) trial, while hyperlipidemia and inflammation contribute to atherosclerotic disease, residual inflammatory risk plays a more significant role in determining the risk of recurrent atherosclerotic events, providing greater guidance for disease treatment. In statin-intolerant patients, inflammation assessed by high-sensitivity C-reactive protein (hs-CRP) is a better predictor of future cardiovascular events and mortality than hyperlipidemia assessed by low-density lipoprotein cholesterol (LDL-C). Therefore, targeting inflammation is becoming an important complementary strategy for the prevention and treatment of atherosclerotic cardiovascular disease. Currently, clinically used anti-inflammatory drugs include canakinumab and colchicine. The development or discovery of additional anti-inflammatory drugs holds significant clinical significance and application value for the treatment of atherosclerotic disease. Summary of the Invention

[0005] In view of this, the main purpose of the present invention is to propose a use of neratinib in the preparation of a drug for preventing or treating atherosclerosis, in order to at least partially solve the above technical problems.

[0006] In order to achieve the above-mentioned object, as one aspect of the present invention, a use of neratinib or a pharmaceutically acceptable salt or hydrate thereof in a drug for preventing or treating atherosclerotic diseases is provided.

[0007] Neratinib and its pharmaceutically acceptable salts or hydrates include maleate, nitrate, hydrochloride, phosphate, and carbonate.

[0008] Atherosclerotic disease is caused by chronic inflammatory response of the vascular lining, which leads to damage to the vascular endothelium.

[0009] The damage to the vascular endothelium is manifested as the continuous accumulation of low-density lipoprotein and inflammatory cells in the damaged areas of the vascular endothelium, gradually forming lipid streaks and further developing into lipid plaques, namely atherosclerosis.

[0010] Atherosclerotic diseases manifest as coronary heart disease, myocardial infarction, stroke, cerebral infarction, cerebral ischemia, refractory hypertension, lower limb artery stenosis, peripheral vascular disease and organ ischemic lesions.

[0011] The above drugs also include lipid-lowering drugs, which are used in combination with neratinib. Lipid-lowering drugs include statins, ezetimibe, bepedic acid and PCSK9 inhibitors.

[0012] The above-mentioned medicine further includes a pharmaceutically acceptable carrier.

[0013] Neratinib can reduce the area of ​​atherosclerotic plaques.

[0014] The above-mentioned drugs are in the form of preparations, including tablets, capsules, granules, powders, pills, lozenges, powders, solutions, syrups, suspensions, emulsions and elixirs.

[0015] As another aspect of the present invention, a pharmaceutical composition for preventing or treating atherosclerotic diseases is provided, comprising nartinib or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0016] The neratinib or its pharmaceutically acceptable salt or hydrate provided by the present invention can significantly reduce the LDLR of high cholesterol-fed patients by using neratinib. - / -The atherosclerotic plaque area and the levels of interleukin-1β and high-sensitivity C-reactive protein in the serum of the mouse model indicate that neratinib or a pharmaceutically acceptable salt or hydrate thereof of the present invention can reduce the atherosclerotic plaque area and inhibit the inflammatory response, thereby achieving the effect of preventing or treating atherosclerotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the anti-inflammatory drug screening process used in the present invention;

[0018] Figure 2 This is a flow chart of the mouse experiment in Example 1 of the present invention;

[0019] Figure 3 This is a micrograph of the aortic arch of the vehicle control group and the drug-treated group in Example 1 of the present invention;

[0020] Figure 4 This is a comparative diagram of aortic arch plaque area analysis between the vehicle control group and the drug-treated group in Example 1 of the present invention;

[0021] Figure 5 This is a micrograph of the aortic sinus of the vehicle control group and the drug-treated group in Example 2 of the present invention;

[0022] Figure 6 This is a comparative diagram of aortic sinus plaque area analysis between the vehicle control group and the drug-treated group in Example 2 of the present invention;

[0023] Figure 7 This is a comparative analysis of serum interleukin-1β, interleukin-6, and high-sensitivity C-reactive protein levels in the vehicle control group and the drug-treated group in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0025] Drug repositioning, or repurposing, is a strategy to identify new uses for approved or investigational drugs, with the advantages of low risk of failure, improved safety, and shortened development time.

[0026] The present invention screens drug molecules for anti-endothelial inflammatory response through a drug-disease association map (Connectivity Map, CMap), the process is shown as follows Figure 1 As shown, it is divided into three steps: gene identification, CMap screening and verification, which specifically include:

[0027] Gene identification: Human umbilical vein endothelial cells (HUVEC) were treated with inflammatory factors TNF-α and IL-1β, and differentially expressed genes were analyzed by transcriptome sequencing. The top 50 differentially expressed genes were screened to establish an inflammatory model.

[0028] CMap screening: The differentially expressed genes obtained by transcriptome sequencing are input into the CMap database to match the corresponding compound data set; compounds with negative correlation coefficients between two inflammatory factors in the data set are selected as potential anti-inflammatory drugs; drugs common in the database with negative correlation coefficients between the two inflammatory factors are screened to obtain candidate drugs.

[0029] Verification: The drug's effectiveness in preventing or treating atherosclerosis is evaluated and determined through in vitro cell experiments and in vivo animal experiments.

[0030] Through the above process, it was discovered that the small molecule inhibitor neratinib has the effect of significantly inhibiting endothelial inflammatory response.

[0031] Among them, neratinib, as an oral small molecule tyrosine kinase inhibitor, is known to achieve therapeutic effects by inhibiting the proliferation of pancreatic cancer cells. In 2020, the FDA approved it for the treatment of HER2-positive breast cancer.

[0032] According to an embodiment of the present invention, there is provided a use of neratinib or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a medicament for preventing or treating atherosclerotic diseases.

[0033] According to the embodiments of the present invention, drug screening and mouse model experiments have verified that neratinib can reduce the area of ​​atherosclerotic plaques and inhibit inflammatory responses, thereby achieving the effect of preventing or treating atherosclerotic diseases.

[0034] According to an embodiment of the present invention, the chemical formula of neratinib is shown as formula (I).

[0035]

[0036] According to an embodiment of the present invention, neratinib can be used to prepare drugs for preventing or treating atherosclerotic diseases using its pharmaceutically acceptable salts and hydrates, including soluble salts and insoluble salts.

[0037] According to an embodiment of the present invention, neratinib or a pharmaceutically acceptable salt thereof includes maleate, nitrate, hydrochloride, phosphate, and carbonate.

[0038] According to an embodiment of the present invention, atherosclerotic disease is atherosclerosis caused by damage to the vascular intima due to chronic inflammatory response of the vascular lining.

[0039] According to an embodiment of the present invention, the damage to the vascular endothelium is manifested as the continuous accumulation of low-density lipoprotein and inflammatory cells in the damaged part of the vascular endothelium, which gradually forms lipid streaks and further develops into lipid plaques, namely atherosclerosis.

[0040] According to an embodiment of the present invention, endothelial cells are a single layer of cells lining the blood vessels. Endothelial cells act as a physical barrier to blood vessels and have antioxidant, anti-inflammatory, and anti-thrombotic functions. Endothelial cells produce nitric oxide (NO) to regulate blood pressure and blood flow, resist vascular inflammatory responses, and endothelial cell dysfunction promotes atherosclerosis. Ox-LDL in the blood causes an inflammatory response in endothelial cells. Endothelial cells upregulate the expression of intercellular adhesion molecule (ICAM1) and vascular cell adhesion molecule (VCAM1), which recruits monocytes to adhere to endothelial cells and promotes the formation of atherosclerotic plaques. Therefore, resisting endothelial inflammation can alleviate atherosclerosis.

[0041] According to an embodiment of the present invention, atherosclerotic diseases are manifested as coronary heart disease, myocardial infarction, stroke, cerebral infarction, cerebral ischemia, refractory hypertension, lower limb artery stenosis, peripheral vascular disease and organ ischemic lesions.

[0042] According to an embodiment of the present invention, atherosclerotic diseases are formed on the inner wall of arterial blood vessels, and arteries of various organs may develop atherosclerosis, including the aorta, carotid arteries, coronary arteries, intracranial arteries, renal arteries, lower limb arteries, etc., thereby manifesting related diseases as organ ischemic lesions.

[0043] According to an embodiment of the present invention, the drug further includes a lipid-lowering drug used in combination with neratinib, and the lipid-lowering drug includes statins, ezetimibe, bepedic acid and PCSK9 inhibitors.

[0044] According to an embodiment of the present invention, the prevention or treatment direction of atherosclerotic disease drugs is achieved through lipid-lowering and anti-inflammatory pathways. Lowering lipids while reducing inflammation can achieve better effects. Statins are commonly used lipid-lowering drugs, and when used in combination with neratinib, better prevention or treatment effects can be achieved.

[0045] According to an embodiment of the present invention, the preparation of a drug for preventing or treating atherosclerotic diseases further includes a pharmaceutically acceptable carrier.

[0046] According to an embodiment of the present invention, the carriers used in the pharmaceutical composition of the present invention are common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically).

[0047] According to an embodiment of the present invention, neratinib can significantly reduce the LDLR of mice fed with a high cholesterol diet. - / - The atherosclerotic plaque area of ​​the mouse model was measured using the following experimental procedures: Figure 2 shown.

[0048] According to an embodiment of the present invention, the drug for preventing or treating atherosclerotic diseases is prepared in the form of a preparation including tablets, capsules, granules, powders, pills, lozenges, powders, solutions, syrups, suspensions, emulsions and elixirs.

[0049] According to an embodiment of the present invention, a pharmaceutical composition for preventing or treating atherosclerotic diseases includes neratinib or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0050] Furthermore, drugs that lower LDLR - / - Levels of interleukin-1β and high-sensitivity C-reactive protein in mouse serum.

[0051] The effective dosage of the drug compound used in the present invention can be adjusted accordingly according to the mode of administration and the severity of the disease to be treated. Based on the body surface area conversion of the 20 mg / kg / d dose in the mouse model, the daily dosage for adults is estimated to be 2.2 mg / kg. For example, the daily dosage for a 70 kg adult is 154 mg, which is lower than the current clinical dosage of neratinib (240 mg / d).

[0052] Preferably, the effective dose can be determined by a person skilled in the art based on various factors, including but not limited to the pharmacokinetic parameters of neratinib, the health condition, body weight, and route of administration of the patient being treated.

[0053] The present invention provides the use of neratinib in preventing atherosclerotic diseases, offers a new solution for the preparation of drugs for preventing or treating atherosclerotic diseases, and has good promotion and application value.

[0054] The technical solutions of the present invention are further described below by means of specific examples in conjunction with the accompanying drawings. It should be noted that the following specific examples are merely illustrative and the scope of protection of the present invention is not limited thereto. The drugs or reagents used in the following examples are all commercially available or homemade by known preparation methods. The methods used in the following examples, such as Image J, are all well-known methods in the art and can be carried out by referring to the descriptions in textbooks or relevant literature, and will not be described in detail.

[0055] All data were processed and analyzed using Graphpad Prism 9.0 software. Means of all samples were compared using the independent sample t-test. P < 0.05 indicated statistical significance.

[0056] Example 1

[0057] Neratinib for LDLR - / - Effects of aortic arch lesions in mice

[0058] Preparation of experimental mice: Commercially purchased SPF grade LDLR - / - Mice (7 weeks old, female) were housed on sterile bedding changed once a week and had free access to food and water.

[0059] Animal dosing and treatment: LDLR - / - After one week of acclimation in the animal house, mice were fed a high-cholesterol (1.25% cholesterol) diet and randomly divided into two groups: a vehicle control group and a neratinib-treated group (20 mg / kg / day). Neratinib was administered orally for 14 weeks. Neratinib was purchased commercially and stored as a powder at -80°C. Before administration, the drug was prepared in 0.5% sodium carboxymethylcellulose to a concentration of 6 mg / ml.

[0060] During the administration period, the mice were weighed once a week, and samples were collected after the 14th week for subsequent molecular and pathological experiments.

[0061] Experimental results and analysis:

[0062] After 14 weeks of treatment, the two groups of mice were treated accordingly, and the aorta (including the aortic arch and thoracic aorta) was removed. After the fresh aortic specimen was removed, the peripheral excess fat and tissue were stripped under a stereomicroscope, and the specimen was fixed in 4% paraformaldehyde overnight. The specimen was removed the next day, washed with PBS, opened longitudinally, and placed in PBS. Before staining with Oil Red O, the specimen was taken out and placed in 60% isopropanol for 10 seconds, then stained in 0.3% Oil Red O for 5 minutes, rinsed in 60% isopropanol until the blood vessel wall turned white, rinsed with PBS, and photographed with a camera. The photographs are shown in the figure below. Figure 3 shown

[0063] Image J software was used to analyze the plaque deposition in the aorta. The plaque area ratio was calculated as the ratio of the plaque deposition area to the total aortic area. The results were as follows: Figure 4 shown.

[0064] according to Figure 3 and Figure 4 It can be seen that compared with the vehicle control group, the aortic arch plaque area in the drug-treated group was significantly reduced.

[0065] Example 2

[0066] Neratinib for LDLR - / - Effects of aortic sinus lesions in mice

[0067] The preparation, feeding and administration of mice were the same as in Example 1.

[0068] After 14 weeks of treatment, the hearts of the two groups of mice were removed and placed in 4% paraformaldehyde overnight. The next day, they were washed with PBS and dehydrated in 30% sucrose. On the third day, the hearts were embedded in cryoembedding medium and serially sectioned using a freezing microtome to obtain sections of the aortic sinus. The sections were stored at -80°C and allowed to warm to room temperature for 20 minutes before use.

[0069] Step 1: Soak the slides in PBS for 5 minutes, stand the slides upright and dry them with filter paper, then draw circles with a histochemical pen;

[0070] Step 2: Wash with 60% isopropanol for 10 seconds, discard the supernatant, and stand the slice upright to dry with filter paper;

[0071] Step 3: Immerse tissue sections in 0.3% Oil Red O for 5 minutes;

[0072] Step 4: Wash with 60% isopropanol for 10 seconds and discard the supernatant.

[0073] Step 5: Wash slowly with PBS, discard the floating liquid, and dry with paper;

[0074] Step 6: Seal the slide with glycerol gelatin (preheated at 75°C), drop a drop on the slide, and cover with a coverslip.

[0075] After completion, take a photo with a microscope, as shown in the following figure Figure 5 As shown; Image J software was used to analyze the plaque deposition in the aortic sinus, and the plaque area ratio was calculated as the ratio of the aortic sinus atherosclerotic plaque area to the total lumen area, as shown Figure 6 shown.

[0076] according to Figure 5 and Figure 6 It can be seen that compared with the vehicle control group, the aortic sinus plaque area in the drug-treated group was significantly reduced.

[0077] Example 3

[0078] Neratinib for LDLR - / - Effects of serum inflammatory factor levels in mice

[0079] The preparation, feeding and administration of mice were the same as in Example 1.

[0080] After 14 weeks of treatment, the two groups of mice were treated with the corresponding treatment. One eyeball was removed and blood was collected into a 1.5 ml centrifuge tube and placed at 4 °C for 4 h to coagulate.

[0081] The coagulated blood was placed in a centrifuge and centrifuged at 8000 g for 10 min at 4°C. The supernatant was separated to obtain serum samples, which were stored at −80°C.

[0082] ELISA (enzyme-linked immunosorbent assay) kits were used to measure the levels of serum IL-1β, IL-6, and high-sensitivity C-reactive protein.

[0083] The values ​​were read using a microplate reader. After the measurement was completed, Graphpad Prism 9.0 software was used for processing and statistical analysis. The means of all samples were compared using independent sample t-test analysis. p < 0.05 indicated statistically significant differences. The results are shown in Figure 2. Figure 7 shown.

[0084] according to Figure 7 It can be seen that compared with the vehicle control group, the levels of IL-1β and high-sensitivity C-reactive protein in the serum of the drug-treated group were significantly decreased.

[0085] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of neratinib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating atherosclerotic diseases.

2. The use according to claim 1, wherein the salt comprises maleate, nitrate, hydrochloride, phosphate, or carbonate.

3. The use according to claim 1, wherein the atherosclerotic disease is atherosclerosis caused by vascular intima damage due to chronic inflammatory response of the vascular endothelium.

4. The use according to claim 3, wherein the vascular endothelial damage is manifested as the continuous accumulation of low-density lipoprotein and inflammatory cells in the damaged part of the vascular endothelium, gradually forming lipid streaks, and further developing into lipid plaques, i.e., atherosclerosis.

5. The use according to claim 3, wherein the atherosclerotic disease is manifested as coronary heart disease, myocardial infarction, stroke, cerebral infarction, cerebral ischemia, refractory hypertension, lower limb artery stenosis, peripheral vascular disease and organ ischemic lesions.

6. The use according to claim 1, wherein the drug further comprises a lipid-lowering drug used in combination with neratinib, wherein the lipid-lowering drug comprises a statin, ezetimibe, bepedic acid and a PCSK9 inhibitor.

7. The use according to claim 1 or 6, wherein the medicine further comprises a pharmaceutically acceptable carrier.

8. The use according to claim 1, wherein neratinib can reduce the area of ​​atherosclerotic plaques.

9. The use according to claim 1 or 6, wherein the drug is in the form of a preparation, including tablets, capsules, granules, powders, pills, lozenges, powders, solutions, syrups, suspensions, emulsions and elixirs.

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