Application of dihydrocapsaicin in treatment of vascular calcification

Abstract

The invention discloses an application of dihydrocapsaicin in treatment of vascular calcification. Specifically, the invention discloses application of dihydrocapsaicin or pharmaceutically acceptable salt thereof, the dihydrocapsaicin or the pharmaceutically acceptable salt thereof is used for preparing a preparation or a composition, and the preparation or the composition is used for treating vascular calcification. Particularly, calcification of vascular smooth muscle cells can be effectively treated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and in particular relates to the application of dihydrocapsaicin in treating vascular calcification. BACKGROUND

[0002] Vascular calcification (VC) involves the accumulation and deposition of hydroxyapatite crystals in the cells of the arterial wall, leading to increased stiffness, decreased compliance, thrombosis and plaque rupture of the vascular wall. VC is a common adverse phenotype of various vascular diseases, and is a phenotypic feature of atherosclerosis, hypertension, aortic valve stenosis, coronary heart disease, diabetes and chronic kidney disease, etc. It is also an independent risk factor for disease occurrence, progression and death.

[0003] In addition, VC is also an independent risk factor and marker of disease aggravation and aging. Therefore, it is a fatal factor threatening human health.

[0004] However, there is currently no recognized effective treatment strategy for reversing or curing VC in clinical practice, especially the lack of specific drugs.

[0005] Therefore, it is of great significance and clinical value to develop new drugs for inhibiting or treating VC. SUMMARY

[0006] The present application provides a drug for inhibiting or treating vascular calcification.

[0007] In a first aspect of the present application, the use of dihydrocapsaicin or a pharmaceutically acceptable salt thereof for the preparation of a preparation or composition for treating vascular calcification is provided.

[0008] In another preferred embodiment, the treatment comprises reducing the degree of vascular calcification and inhibiting the continued calcification.

[0009] In another preferred embodiment, the vascular calcification comprises intimal calcification and medial calcification of blood vessels or calcification of vascular smooth muscle cells.

[0010] In another preferred embodiment, the treatment comprises inhibiting or reducing the calcium concentration in vascular cells, including vascular smooth muscle cells.

[0011] In another preferred embodiment, the dihydrocapsaicin has the following formula (1):

[0012]

[0013] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0014] In another preferred embodiment, the preparation comprises a laboratory preparation.

[0015] In another preferred embodiment, the dosage form of the pharmaceutical composition comprises: oral preparations, injections, lyophilized preparations.

[0016] In another preferred embodiment, the pharmaceutical composition is a solid preparation, a liquid preparation, or a semi-solid preparation.

[0017] In another preferred embodiment, the pharmaceutical is administered by oral, intravenous, intramuscular, subcutaneous, and inhalation administration.

[0018] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, preferably a tablet, a capsule, and a granule.

[0019] In a second aspect of the present application, there is provided a method for inhibiting the calcification of vascular smooth muscle cells in vitro, comprising the step of: adding dihydrocapsaicin to a calcification model established by simulating the calcium-phosphorus metabolic disorder caused by insufficient blood supply, i.e., a serum-free and calcium-added vascular smooth muscle cell calcification model, thereby directly inhibiting the calcification of vascular smooth muscle cells.

[0020] In another preferred embodiment, the calcium used in the model is CaCl2.

[0021] In another preferred embodiment, the extent of calcification is detected using alizarin red.

[0022] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0023] In a third aspect of the present application, there is provided a pharmaceutical composition comprising:

[0024] (a1) a first active ingredient, wherein the first active ingredient is dihydrocapsaicin or a pharmaceutically acceptable salt thereof;

[0025] (a2) a second active ingredient, wherein the second active ingredient is an additional drug for treating vascular calcification; and

[0026] (b) a pharmaceutically acceptable carrier.

[0027] In a fourth aspect of the present application, there is provided a kit comprising:

[0028] (Z1) a first pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a1) a first active ingredient, wherein the first active ingredient is dihydrocapsaicin or a pharmaceutically acceptable salt thereof;

[0029] (Z2) a second pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a2) a second active ingredient, wherein the second active ingredient is an additional drug for treating vascular calcification.

[0030] In a fifth aspect of the present application, there is provided a method for preventing and / or treating vascular calcification, comprising administering dihydrocapsaicin or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0031] In another preferred embodiment, the vascular calcification comprises intimal calcification and medial calcification (i.e. vascular smooth muscle cell calcification) of blood vessels.

[0032] In another preferred embodiment, the vascular calcification is vascular smooth muscle cell calcification.

[0033] In another preferred embodiment, the subject comprises a human and a non-human mammal.

[0034] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A schematic diagram showing the effect of dihydrocapsaicin on vascular smooth muscle cells.

[0036] Figure 2 DHC inhibits cell calcification. (A) DHC molecular structure (from BindingDB); (B) DHC target genes; (C) DHC chemical-phenotype analysis; (D) DHC target genes gene ontology (GO) cellular component analysis; (E) DHC target genes disease analysis; (F) DHC inhibits cell calcification. f0: hVSMC normal culture, DHC-f1: 0 μL; f2: 0.5 μL; f3: 2 μL; f4: 8 μL. DHC: dihydrocapsaicin; hVSMC: human vascular smooth muscle cells; BM: basal medium; detection condition: Ca1.2 UL + DHC, 4.5 h, 4 mM DHC. The picture is drawn by SANGERBOX and HIPLOT.

[0037] Figure 3 Functional and pathway analysis of target genes. (A) DHC target genes gene ontology (GO) molecular function (MF) analysis; (B) DHC target genes gene ontology (GO) biological process (BP) analysis; (C) DHC target genes KEGG pathway analysis; (D) DHC target genes REACTOME analysis. MF: molecular function; BP: biological process; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; DHC: dihydrocapsaicin. DETAILED DESCRIPTION

[0038] The present inventors have made extensive and intensive studies, and have found for the first time that dihydrocapsaicin is useful for effectively treating vascular calcification through a large number of screenings. The experiments of the present invention show that dihydrocapsaicin can significantly inhibit vascular calcification, particularly vascular media calcification or vascular smooth muscle cell calcification Figure 1

[0039] Definitions

[0040] To enable a better understanding of the present disclosure, certain terms are defined first. As used in this application, and unless specifically identified otherwise, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

[0041] The term "about" can refer to a value or a composition that is within an acceptable error range for the specific value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101, and all values in between.

[0042] As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0043] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the entire range of values as well as the values within that range (e.g., one tenth and one hundredth of an integer).

[0044] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the associated listed items.

[0045] Dihydrocapsaicin

[0046] Dihydrocapsaicin (DHC, C 18 H 29 NO3) is the main pungent component of natural capsaicinoids in chili peppers. It has a molecular formula as shown in A of Figure 2 DHC has diverse pharmacological and physiological properties, such as analgesic, anticancer, anti-inflammatory, antioxidant, and anti-obesity effects. These properties suggest its potential clinical application value in pain relief, cancer prevention, and weight loss, and benefits to the cardiovascular and gastrointestinal systems. Therefore, the study of the pharmacological and physiological effects of DHC and its mechanisms has become a research hotspot in the fields of food and pharmacological sciences.

[0047] Vascular calcification

[0048] ​Vascular calcification (VC) is a common adverse phenotype of vascular diseases and an independent risk factor for disease progression. VC involves the accumulation and deposition of hydroxyapatite crystals within the cells of the arterial wall, leading to increased stiffness, decreased compliance, thrombosis, and plaque rupture. VC is a common adverse phenotype of multiple vascular diseases, a phenotypic characteristic of atherosclerosis, hypertension, aortic valve stenosis, coronary heart disease, diabetes, and chronic kidney disease, and an independent risk factor for disease occurrence, progression, and death. VC includes intimal calcification and medial calcification (i.e., vascular smooth muscle cell calcification).

[0049] Vascular smooth muscle cells are mainly present in the medial layer of the vascular wall, including arterial, venous, and lymphatic vessel structures. The main function is to regulate the caliber of blood vessels by contraction and relaxation, thereby controlling blood flow resistance and blood pressure. In addition, smooth muscle cells are also involved in the development, remodeling, and repair processes of blood vessels. In pathological processes such as atherosclerosis, smooth muscle cells can migrate to the intima, proliferate, and synthesize extracellular matrix to form a fibrous cap.

[0050] Diseases caused by vascular smooth muscle cell calcification include atherosclerosis, chronic kidney disease-related vascular calcification, diabetic vasculopathy, hypercalcemia-related vascular calcification, and senile vascular calcification.

[0051] The vascular calcification of the present application is significantly different from heart valve calcification.

[0052] Heart valve cells are located on the heart valve, including the mitral valve, tricuspid valve, aortic valve, and pulmonary valve, and are the main cell type that constitutes the heart valve. In normal physiological conditions, they are responsible for maintaining the structural integrity and function of the valve, and by synthesizing and remodeling the extracellular matrix, they ensure the normal opening and closing of the valve. In pathological conditions, heart valve interstitial cells (VICs) are activated and participate in the repair and remodeling of the valve, such as regulating the synthesis and degradation of extracellular matrix in response to inflammation, injury, etc.

[0053] Diseases related to heart valve calcification include aortic valve stenosis, mitral valve stenosis, aortic valve insufficiency, valve calcification after infective endocarditis, and congenital heart valve disease.

[0054] Compositions and uses thereof

[0055] Based on the unexpected discovery of the present application, the present inventors provide various different uses of the active ingredients of the present application.

[0056] Typically, the active ingredients of the present application can be used to prepare a preparation or composition for treating vascular calcification, including reducing the degree of vascular calcification and inhibiting continued calcification.

[0057] The present application also provides a variety of different compositions containing the active ingredients of the present application, including but not limited to: pharmaceutical compositions, nutraceutical compositions.

[0058] In the case of pharmaceutical compositions, the term "active ingredient" as used herein refers to memantine or a pharmaceutically acceptable salt thereof useful in the present application.

[0059] The term "effective amount" or "effective dose" as used herein refers to an amount that is functional or active and acceptable to a human and / or animal.

[0060] The term "pharmaceutically acceptable" as used herein refers to those substances that are suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio, i.e., substances that are acceptable to the U.S. Food and Drug Administration as well as the European Medicines Agency. The term "pharmaceutically acceptable carrier" refers to a carrier for therapeutic administration including various excipients and diluents.

[0061] The pharmaceutical compositions of the present application contain a safe and effective amount of the active ingredients of the present application and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulations are generally matched to the mode of administration, and the dosage forms of the pharmaceutical compositions of the present application are injectable, oral (tablets, capsules, oral solutions), transdermal, and sustained release. For example, they are prepared by conventional methods using, for example, physiological saline or aqueous solutions containing dextrose and other adjunct agents. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0062] The effective amount of the active ingredients of the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) based on various factors. Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc. of the active ingredients; the severity of the disease to be treated; the body weight of the patient; the immune status of the patient; the route of administration; etc. Generally, satisfactory results are indicated to be obtained when the active ingredients of the present application are administered at a dosage of about 0.00001 mg to 50 mg / kg, preferably 0.0001 mg to 10 mg / kg, of animal body weight per day. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0063] The pharmaceutically acceptable carriers of the present application include, but are not limited to: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The selection of the carrier should be matched to the mode of administration, which is well known to one of ordinary skill in the art.

[0064] The main advantages of the present application include:

[0065] (a) The present application has found that the natural compound dihydrocapsaicin from chili pepper has excellent effect on treating vascular calcification, especially the calcification of medial vascular smooth muscle cells.

[0066] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0067] Example 1 Experimental study

[0068] 1.1 Experimental content:

[0069] In this example, the drug screening experiment was carried out using the previously established vascular calcification (VC) cell model of the present application. The experiment included treating the vascular calcification cells with different drugs, and finally found that dihydrocapsaicin (DHC) had an inhibitory effect on vascular calcification. Other compounds including atorvastatin, vitamin D and sodium chloride (NaCl) had no obvious effect on treating vascular calcification.

[0070] 1.2 Construction method of vascular calcification cell model

[0071] The present inventors have previously studied the effects of various factors on the calcification of human vascular smooth muscle cells (HSMC). It was found that three key factors were needed to establish a rapid and simple vascular calcification cell model: serum, Ca 2+ and PO43-. Serum deprivation is essential for the ultrafast calcification of HSMC. PO43- is essential for the formation of calcium nodules of HSMC, and the efficient calcification of HSMC depends on the concentration of phosphorus. Exogenous Ca 2+ supplementation is also essential for the formation of HSMC calcium nodules, and the effect is concentration-dependent. Of course, the efficient calcification of HSMC also depends on the induction time, which only takes a few hours.

[0072] Human smooth muscle cells (HSMC) were purchased from ATCC (Manassas, VA) and cultured in 24-well plates using α-MEM complete medium (α-MEM with 10% fetal bovine serum). When the cells reached 80-90% confluence, they were induced to calcify by adding 0.38 ml of α-MEM base medium + 20 μl of CaCl2solution (100 mM) per well in a 24-well plate.

[0073] After 3-6 hours of induction, the medium was aspirated and the cells were washed twice with 1 x TBS.

[0074] Subsequently, the cells were fixed with 4% neutral formaldehyde solution for 30 minutes. After washing twice with 1 x TBS, the cells were stained with alizarin red for 5 minutes.

[0075] Finally, the cells were washed three times with 1 x TBS and the staining was observed under a microscope (Nikon TS100, Tokyo, Japan) and digital images were taken using a CCD camera.

[0076] Calcium nodules stained with alizarin red reflect the calcification phenomenon.

[0077] 1.3 Experimental method

[0078] When the cells reached 80-90% confluence, 1.2 μl of 100 mM calcium ions (CaCl2) was added to the total cell volume (about 0.5 x 10 5 cells) in each well of a 24-well plate as a calcification inducer for 3-5 hours, in combination with different concentrations (0 μl, 0.5 μl, 2 μl, 8 μl) of 4 mM dihydrocapsaicin (DHC) as a calcification modulator. Finally, the calcium nodules were stained with alizarin red to assess the level of calcification.

[0079] wherein the total number of cells in each of fl to f4 is the same (about 0.5 x 10 5 Each of fl to f4 was repeated in 4 wells.

[0080] 1.4 Experimental results

[0081] The results are shown in F of Table 1. Figure 2 The results show that, in fl to f4, the degree of calcium nodules stained with alizarin red is lighter and the degree of vascular calcification (or vascular smooth muscle cell calcification) gradually decreases as the concentration or amount of dihydrocapsaicin increases. This indicates the significant inhibitory effect of DHC on VC.

[0082] Example 2 Mechanism study

[0083] In this example, to investigate the role and pathways of DHC in VC, the inventors extracted experimentally identified (not computer-predicted) target genes of DHC using Comparative Toxicogenomics Database (CTD) and its online tools. Subsequently, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to analyze the cellular functions and signaling pathways of these genes.

[0084] The results showed that DHC( Figure 2 has 20 target genes. Figure 2 B) Chemico-phenotype analysis of DHC identified cell death phenotypes such as apoptosis process and autophagy Figure 2 C) in VC. Cellular component analysis showed that these target genes are mainly located in the cellular membrane system. The highest GO level includes CHOP-ATF4 complex, endoplasmic reticulum membrane, and intracellular membrane-bounded organelle Figure 2 D) in VC.

[0085] Disease analysis found three types of common diseases: necrosis, tumor, and cardiomyopathy Figure 2 E) in VC. Molecular function analysis highlighted energy and substance metabolism, stress response, antioxidant mechanism, enzyme activity, and signal transduction Figure 3 A) in VC. Biological process analysis identified processes related to metabolism, apoptosis, and response to various stimuli Figure 3 B) in VC. KEGG analysis showed that the pathways are mainly concentrated in metabolism, cell death, and key signaling pathways such as MAPK, ErbB, HIF-1, FoxO, and sphingolipid signaling pathways Figure 3 C) in VC.

[0086] REACTOME analysis confirmed the results of GO and KEGG analyses Figure 3 D) in VC.

[0087] These findings suggest that the role and signaling pathways of DHC are highly relevant to VC, revealing the mechanism of the experimental findings of the present application, i.e., the principle of the inhibitory effect of DHC on VC.

[0088] Discussion

[0089] Vascular calcification (VC) is not a passive irreversible complication, but an active process regulated by multiple factors. This provides a rare opportunity for active prevention or treatment of VC. Programmed cell death (such as apoptosis) is a key promoting factor of calcification. CHOP-ATF4 complex is a key regulator of osteoblast differentiation, and endoplasmic reticulum serves as a Ca 2+ pool, both of which are closely related to calcium signaling. Various stress factors, such as ischemia, exogenous toxins, and calcium and phosphorus metabolism disorders, can lead to cell calcification.

[0090] The analysis of the present invention shows that the role and signaling pathways of DHC-targeted genes are highly concentrated in metabolism, cell death, and responses to various stimuli, indicating a strong link between DHC and cell calcification. These are consistent with the conclusions of a review on the role of inflammatory responses, endoplasmic reticulum stress, mitochondrial dysfunction, iron homeostasis, metabolic imbalance, and some related signaling pathways in the progression of VC. The key point is that DHC receptors, members of the TRPV family (TRPV1-6), constitute a time-Ca 2+ dependent non-selective cation channel related to the TRP ion channel family.

[0091] This means that DHC can inhibit the influx of extracellular Ca 2+ . The vascular calcification cell model of the present invention is established under high calcium conditions without serum, effectively simulating ischemia, exogenous toxins, and calcium metabolism disorders, and can rapidly induce cell calcification within a few hours. This model is superior to traditional cell calcification models, which are complex and unstable and take up to a month. Therefore, it is very suitable for screening drugs that inhibit calcification.

[0092] The research of the present invention shows that DHC is the first natural product from the edible plant Capsicum that has been confirmed in this model as a potential calcification-inhibiting drug. Future animal and clinical experiments should be conducted to confirm the cellular results.

[0093] In summary, the research of the present invention elucidates the role and signaling pathways of DHC in cell calcification. By using various analytical tools and methods, it was found that the role and signaling pathways of DHC are mainly concentrated in cell death, metabolism, and responses to various stresses, which are highly relevant to the calcification process. Cell experiments confirmed its inhibitory effect on VC. The research of the present invention not only provides the first natural food-grade potential drug from Capsicum that has been confirmed by the model established by the present inventor to treat calcification-related diseases, but also provides a rapid strategy for screening drugs for various diseases related to calcium metabolism disorders.

[0094] All the documents mentioned in the present invention are cited in this application as references, as if each document is individually cited as a reference. In addition, it should be understood that those skilled in the art can make various modifications or modifications to the present invention after reading the above teachings of the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. The use of dihydrocapsaicin or a pharmaceutically acceptable salt thereof, characterized in that, Used to prepare formulations or compositions for the treatment of vascular calcification.

2. The use as described in claim 1, characterized in that, The treatment includes reducing the degree of vascular calcification and inhibiting further calcification.

3. The use as described in claim 1, characterized in that, The vascular calcification includes intimal calcification and medial calcification or vascular smooth muscle cell calcification.

4. The use as described in claim 1, characterized in that, The molecular formula of the dihydrocapsaicin is shown in formula (1) below:

5. The use as described in claim 1, characterized in that, The composition includes pharmaceutical compositions.

6. The use as described in claim 5, characterized in that, The dosage forms of the pharmaceutical composition include: oral preparations, injections, and lyophilized preparations.

7. A method for inhibiting calcification of vascular smooth muscle cells in vitro, characterized in that, The steps include: adding dihydrocapsaicin to a calcification model established to simulate insufficient blood supply and disordered calcium and phosphorus metabolism, namely a serum-free and calcium-added vascular smooth muscle cell calcification model, thereby directly inhibiting the calcification of vascular smooth muscle cells.

8. The method as described in claim 7, characterized in that, The method is non-diagnostic and non-therapeutic.

9. A pharmaceutical composition, characterized in that, include: (a1) The first active ingredient, wherein the first active ingredient is dihydrocapsaicin or a pharmaceutically acceptable salt thereof; (a2) A second active ingredient, wherein the second active ingredient is an additional drug for treating vascular calcification; and (b) Pharmaceutically acceptable carriers.

10. A medicine box, characterized in that, include: (Z1) A first pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a1) a first active ingredient, wherein the first active ingredient is dihydrocapsaicin or a pharmaceutically acceptable salt thereof; (Z2) A second pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a2) a second active ingredient, wherein the second active ingredient is an additional drug for treating vascular calcification.

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