Application of arginine diester in preparation of medicine for preventing and treating cardiovascular diseases
By preparing arginine diester derivatives with high cardiac targeting, the problem of L-arginine's poor effect in treating myocardial infarction was solved, and the cardiac function and ventricular remodeling after myocardial infarction was significantly improved, and a new treatment plan was provided.
Patent Information
- Application Number
- CN202510460431.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, L-arginine cannot significantly improve the outcome of cardiovascular disease after treatment of myocardial infarction, and may increase post-infarction mortality due to lack of cardiac targeting and insufficient bioavailability.
Arginine diester derivatives were developed to prepare cardiac-targeting and high bioavailability arginine diesters, including A2S, A3S, A4S and A6S, for the preparation of drugs to prevent and treat cardiovascular diseases.
Arginine diester significantly improves cardiac function after myocardial infarction, reduces ventricular remodeling, reduces myocardial fibrosis, enhances cardiac contraction function, and has good safety, providing a new therapeutic option for ventricular remodeling after myocardial infarction.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of arginine diester in the preparation of drugs for preventing and treating cardiovascular diseases. Background Art
[0002] With the changes in lifestyle and the development of population aging, the incidence and mortality of myocardial infarction have been on the rise year by year. Although reperfusion therapy and drugs for improving ventricular remodeling can significantly delay the progression of the disease and improve the quality of life of patients, the therapeutic interventions for promoting cardiac tissue healing are still limited.
[0003] L-arginine is the only substrate for synthesizing nitric oxide (NO) in the body, and its vascular effects are mainly mediated by NO. NO can protect the cardiovascular system by dilating blood vessels, inhibiting leukocyte adhesion, reducing platelet aggregation, and preventing plaque formation. In addition, L-arginine also has NO-independent vascular effects such as promoting insulin release, reducing blood sugar, reducing blood viscosity, inhibiting the activity of angiotensin-converting enzyme, reducing blood pressure, and regulating the functions of immune cells (macrophages and / or T cells). Clinical studies have shown that the reduced bioavailability of arginine and the increased catabolism of arginine lead to an increased risk of cardiovascular diseases. Compared with patients without coronary atherosclerotic heart disease (CAD), the plasma arginine level of CAD patients is significantly reduced. Therefore, improving the bioavailability of arginine has great clinical therapeutic potential in reducing the risk of cardiovascular diseases. In the early 19th century, people showed great interest in using L-arginine to prevent and treat cardiovascular diseases. Experimental animal models and preliminary clinical studies have both shown that intra-arterial or intravenous infusion of arginine can improve the function of coronary endothelial cells and increase the production of NO in arteries.
[0004] However, the results of studies on arginine supplementation therapy in terms of myocardial infarction outcomes have been unsatisfactory. A prospective study (VINTAGE MI) published in JAMA in 2009 showed (Schulman SP, Becker LC, Kass DA, Champion HC, Terrin ML, Forman S, Ernst KV, Kelemen MD, Townsend SN, Capriotti A, Hare JM, Gerstenblith G. L-arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial. Jama 2006;295:58-64.): Adding L-arginine to standard post-myocardial infarction treatment did not improve vascular stiffness or ejection fraction in patients and was potentially associated with a higher post-infarction mortality rate. Therefore, standardized treatment with L-arginine is not recommended after acute myocardial infarction. The reasons why arginine supplementation therapy cannot improve cardiovascular disease outcomes may be as follows: (1) Due to the lack of organ targeting, exogenous arginine supplementation increases plasma L-arginine levels but does not increase the intracellular arginine bioavailability in the heart to a level sufficient to elicit a biological effect. (2) The potential benefit of arginine supplementation in enhancing NO production may be inhibited by the increased levels of disease-related arginase and endogenous NOS inhibitors. Therefore, arginine needs to be modified to improve its bioavailability, enable it to more effectively exert biological effects, and have stronger cardiac targeting so that it can more effectively act on cardiac tissue. Summary of the Invention
[0005] An object of the present invention is to overcome the deficiencies of the prior art and provide an arginine derivative, arginine diester, and the application of arginine diester in the preparation of a drug for preventing and treating cardiovascular diseases.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] The present invention provides the application of arginine diester in the preparation of a drug for preventing and treating cardiovascular diseases, and the structural formula of the arginine diester is shown in Formula I:
[0008]
[0009] Among them, y = 2, 3, 4 or 6. When y = 2, the arginine diester is A2S; when y = 3, the arginine diester is A3S; when y = 4, the arginine diester is A4S; when y = 6, the arginine diester is A6S. The present invention has developed a new arginine diester derivative that can stably exist in the circulation for a long time, with stronger cardiac targeting and higher bioavailability of intracellular arginine in the heart. The arginine diester significantly reduces the degree of ventricular remodeling after myocardial infarction in mice, improves myocardial fibrosis, and enhances the cardiac contractile function. Compared with L-arginine, it has a more significant effect on ventricular remodeling after myocardial infarction and has good safety, which can provide a new treatment option for patients with ventricular remodeling after myocardial infarction and has important clinical application prospects.
[0010] Preferably, in the structural formula of the arginine diester, y = 2 or 3. A2S and A6S can significantly improve cardiac function and ventricular remodeling after myocardial infarction, reduce myocardial fibrosis after myocardial infarction in mice, increase the number of M2 macrophages after myocardial infarction, and have high biosafety.
[0011] Furthermore, the drug is a drug for preventing and treating myocardial infarction.
[0012] Even further, the drug is a drug for improving cardiac function and ventricular remodeling after myocardial infarction.
[0013] Even further, the drug is a drug for increasing left ventricular ejection fraction and / or shortening fraction of the minor axis.
[0014] Even further, the drug is a drug for reducing the left ventricular end-systolic diameter.
[0015] Even further, the drug is a drug for reducing myocardial fibrosis.
[0016] Even further, the drug is a drug for promoting macrophage polarization into M2 type.
[0017] Furthermore, the preparation method of the arginine diester is: reflux and condense L-arginine, diol monomer and toluenesulfonic acid monohydrate in toluene, and carry out esterification dehydration reaction to obtain arginine diester. The diol monomer includes ethylene glycol, 1,3-propanediol, 1,4-butanediol or 1,6-hexanediol.
[0018] When the diol monomer is ethylene glycol, y = 2 in formula I, and the arginine diester is A2S; when the diol monomer is 1,3-propanediol, y = 3 in formula I, and the arginine diester is A3S; when the diol monomer is 1,4-butanediol, y = 4 in formula I, and the arginine diester is A4S; when the diol monomer is 1,6-hexanediol, y = 6 in formula I, and the arginine diester is A6S.
[0019] Preferably, the diol monomer is ethylene glycol or 1,3-propanediol.
[0020] Furthermore, the reflux condensation temperature is 120 - 130°C, and the thermal esterification dehydration reaction lasts for 16 - 24 h.
[0021] In a specific embodiment of the present invention, the reflux condensation temperature is 130°C, and the thermal esterification dehydration reaction lasts for 24 h.
[0022] Furthermore, the molar ratio of L-arginine, diol monomer and monohydrate of toluenesulfonic acid is L-arginine: diol monomer: monohydrate of toluenesulfonic acid = 0.02:0.01:0.041.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0024] In terms of technology: Arginine diester significantly improves the bioavailability of arginine in local cardiac cells, exerts biological effects more effectively, has stronger cardiac targeting, and acts on cardiac tissue more effectively. The research results of animal experiments show that compared with L-arginine, arginine diester significantly improves cardiac function indexes such as ejection fraction and shortening fraction of minor axis after myocardial infarction, and ventricular remodeling, enhances cardiac contractile function, improves myocardial hypertrophy, significantly reduces the fibrotic area of the heart after myocardial infarction, increases the proportion of reparative macrophages in the heart after myocardial infarction, has high biological safety, and has no obvious influence on the structure and function of liver, lung, kidney and body weight, etc., providing new ideas and new solutions for the clinical treatment of ventricular remodeling after acute myocardial infarction and laying a reliable foundation for clinical application.
[0025] In terms of economy: The present invention provides a way to reduce the long-term treatment cost of cardiovascular diseases through arginine diester and reduce the medical expenses caused by complications after myocardial infarction. With the increasing incidence of cardiovascular diseases, the demand for related drugs is also increasing. The introduction of innovative therapies may occupy market share and bring economic benefits. A technical breakthrough for the major clinical problem of poor therapeutic effect of L-arginine can attract more investors' attention and enhance the commercial value of enterprises and research institutions.
[0026] In terms of society: The present invention can not only provide new treatment options for patients with ventricular remodeling after myocardial infarction, but also may improve the overall health status and quality of life of patients. By improving cardiac function and reducing the risk of complications, it may indirectly reduce the mortality related to cardiovascular diseases. The success of the new therapy may enhance the public's attention and prevention awareness of cardiovascular diseases and promote the implementation of relevant health education and policies. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is the preparation process and structural formula of arginine diester.
[0028] Figure 2 Arginine diester improved cardiac function and ventricular remodeling in mice after myocardial infarction. Among them, A is the representative image of M-mode echocardiogram of each group; B is the left ventricular ejection fraction (EF) of each group; C is the fractional shortening (FS) of each group; D is the left ventricular end-systolic diameter (LVID-s) of each group; E is the left ventricular end-diastolic diameter (LVID-d) of each group. The results are shown as mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, compared with PBS, n = 6 in each group.
[0029] Figure 3 Oral administration of arginine diester significantly improved cardiac function and ventricular remodeling in mice after myocardial infarction. Among them, A is the experimental flow chart; B is the representative image of M-mode echocardiogram of each group; C is the left ventricular ejection fraction (EF) on the 7th, 14th, 21st, and 28th days after myocardial infarction after oral administration of each drug; D is the fractional shortening (FS) on the 7th, 14th, 21st, and 28th days after myocardial infarction after oral administration of each drug; E is the left ventricular ejection fraction (EF) 28 days after administration by three different routes of administration: intravenous injection (iv), intraperitoneal injection (ip), and oral administration (op). **P ≤ 0.01, ****P ≤ 0.0001, compared with PBS; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.001, compared with L-Arg.
[0030] Figure 4 Arginine diester alleviated myocardial fibrosis in mice after myocardial infarction. Among them, A is the schematic diagram of Masson staining of the heart in each group; B is the statistical result of quantitative analysis of the fibrotic area in each group. The results are shown as mean ± SEM, ***P ≤ 0.001, ****P ≤ 0.0001, compared with PBS, n = 6 in each group.
[0031] Figure 5 Arginine diester increased the number of M2 macrophages after myocardial infarction and promoted the polarization of M2 macrophages after myocardial infarction. Among them, A is the gating strategy for immune cells of CD45-positive cells; B is the gating strategy for macrophages positive for F4 / 80 CD11; C is the representative diagram of flow cytometry of CD206-positive macrophages in the hearts of each group 5 days after myocardial infarction; D is the statistical chart of the proportion of CD206-positive macrophages in the hearts of each group 5 days after myocardial infarction. ****P ≤ 0.0001, compared with PBS, n = 5 in each group.
[0032] Figure 6Arginine diester has high biosafety in vivo. Among them, A is a representative picture of the HE staining results of the lungs, livers, and kidneys in each group; B is the expression level of serum ALT (alanine aminotransferase) in each group of mice; C is the expression level of serum AST (aspartate aminotransferase) in each group of mice; D is the expression level of serum BUN (blood urea nitrogen) in each group of mice; E is the expression level of serum CREA (creatinine) in each group of mice. Detailed implementation manners
[0033] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.
[0034] Example 1 Preparation method and structural formula of arginine diester (Arg-y-S)
[0035] 1. Synthesize arginine diester A2S: Using L-arginine (0.04 mol), ethylene glycol (0.02 mol), and toluenesulfonic acid monohydrate (TsOH·H2O, 0.082 mol) as raw materials, add them to toluene and reflux and condense to 130 °C, fully heat for the esterification dehydration reaction for 24 h, and perform recrystallization purification with isopropanol as the solvent, and vacuum dry to obtain arginine diester A2S.
[0036] 2. Synthesize arginine diester A3S: Replace the ethylene glycol in step 1 with 1,3-propanediol, and keep other methods unchanged to obtain arginine diester A3S.
[0037] 3. Synthesize arginine diester A4S: Replace the ethylene glycol in step 1 with 1,4-butanediol, and keep other methods unchanged to obtain arginine diester A4S.
[0038] 4. Synthesize arginine diester A6S: Replace the ethylene glycol in step 1 with 1,6-hexanediol, and keep other methods unchanged to obtain arginine diester A6S.
[0039] The chemical reaction equation for synthesizing arginine diester (Arg-y-S) is as Figure 1 shown, where "y" represents the number of methylene groups on the diol.
[0040] Example 2 Arginine diester significantly improves cardiac function and ventricular remodeling after myocardial infarction in mice
[0041] I. Experimental method
[0042] To investigate whether arginine diester can improve ventricular remodeling after acute myocardial infarction in mice and which type of arginine diester has a more significant effect, in this invention, permanent ligation of the left anterior descending coronary artery was performed on mice. After the operation, the mice were randomly divided into 9 groups, with 6 mice in each group. Since the day after the operation, equal volumes of PBS or different types of arginine diester (PBS solution of arginine diester) were given for treatment. After continuous intraperitoneal injection for 7 days, the cardiac function was detected on the 28th day after the operation. The groups were the sham operation sham group, the blank control PBS group, the L-Arg group (300 mg / kg), the D-Arg group (300 mg / kg), the A2S group (300 mg / kg), the A3S group (300 mg / kg), the A4S group (300 mg / kg), the A6S group (300 mg / kg), and the positive control captopril group (20 mg / kg, captopril).
[0043] II. Experimental Results
[0044] Compared with the PBS group, the cardiac function and the structure of the heart in the A2S group, A3S group, A4S group, and A6S group were improved to varying degrees (Table 1, Figure 2 A~ Figure 2 E), and the drug effects in the A2S group and A3S group were the most significant.
[0045] Compared with the PBS group, the left ventricular ejection fraction ( Figure 2 B, A2S group: 63.73±0.12%, PBS group: 41.75±0.15%, P<0.0001) and the fractional shortening ( Figure 2 C, A2S group: 34.40±0.075%, PBS group: 20.29±0.10%, P<0.0001) in the A2S group were significantly improved 7 days after drug administration, and the left ventricular end-systolic diameter ( Figure 2 D, A2S group: 2.72±0.01 mm, PBS group: 3.54±0.02 mm, P<0.05,) were significantly reduced.
[0046] Compared with the PBS group, the left ventricular ejection fraction ( Figure 2 B, A3S group: 64.04±0.15%, PBS group: 41.75±0.15%, P<0.001) and the fractional shortening ( Figure 2 C, A3S group: 33.61±0.15%, PBS group: 20.29±0.10%, P<0.01) in the A3S group were significantly improved 7 days after drug administration, and the left ventricular end-systolic diameter ( Figure 2 D, A3S group: 2.57±0.01 mm, PBS group: 3.54±0.02 mm, P<0.01) were significantly reduced.
[0047] The L-Arg group and the D-Arg group had no significant therapeutic effect. These results indicate that arginine diester significantly improves important indicators of cardiac function and cardiac structure in mice, thereby improving ventricular remodeling after acute myocardial infarction.
[0048] Table 1 Analysis of cardiac function parameters after arginine diester treatment in myocardial infarction
[0049]
[0050] n = 6 / group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs PBS group
[0051] HR: Heart rate; EF: Left ventricular ejection fraction; FS: Fractional shortening; LVIDs: Left ventricular internal diameter at end-systole; LVIDd: Left ventricular internal diameter at end-diastole; LV vol s: Left ventricular volume at end-systole; LV vol d: Left ventricular volume at end-diastole; LVAWs: Left ventricular anterior wall thickness at end-systole; LVAWd: Left ventricular anterior wall thickness at end-diastole; LVPWs: Left ventricular posterior wall thickness at end-systole; LVPWd: Left ventricular posterior wall thickness at end-diastole.
[0052] Example 3 Oral administration of arginine diester significantly improves cardiac function and ventricular remodeling in mice after myocardial infarction
[0053] I. Experimental method
[0054] Clinically, arginine drugs are usually administered orally. Next, the effects of orally administered A2S and A3S on cardiac function in mice were detected. Starting from the first day after myocardial infarction in mice, A2S and A3S were orally administered at a dose of 900 mg / kg. The same dose of PBS was injected as a blank control group, and the same dose of L-Arg was used as a negative control. Echocardiography was performed on the cardiac function of mice on the 0th, 7th, 14th, 21st, and 28th days after administration, and tissue and biochemical tests were performed on the 28th day ([ Figure 3 A).
[0055] II. Experimental results
[0056] The results showed that the cardiac function and cardiac structure of A2S and A3S were improved to varying degrees after 7 days of oral administration ([ Figure 3 B), and significant improvement was observed in the ejection fraction and shortening fraction of the heart in mice after myocardial infarction ([ Figure 3 C~ Figure 3 E), and the effect was significantly better than that of L-Arg, and this effect became more and more significant over time.
[0057] Example 4 Arginine diester reduces myocardial fibrosis in mice after myocardial infarction
[0058] I. Experimental methods
[0059] When myocardial tissue is damaged or necrotic after myocardial infarction, the necrotic cells will be replaced by fibrous tissue. The damaged myocardial tissue is similar to a wound and needs to heal. Myocardial fibrosis occurs at this time, producing scars that have a certain protective effect on myocardial tissue to a certain extent. However, the healing of this scar is mainly controlled by the human body itself, and the damaged part will be replaced by many fibroblasts, resulting in excessive fibrotic repair, leading to myocardial hypertrophy and ventricular remodeling; ultimately, the heart will lose its compensatory ability.
[0060] To study whether A2S and A3S affect the progression of fibrosis after myocardial infarction, a mouse myocardial infarction model was established. According to the method of Example 3, the mice were randomly divided into 4 groups and treated with PBS or different types of arginine diesters daily for 28 consecutive days by oral administration. On the 28th day after myocardial infarction, the mouse heart tissue was taken for Masson staining. The groups were the blank control PBS group, the L-Arg group (900 mg / kg), the A2S group (900 mg / kg), and the A3S group (900 mg / kg).
[0061] II. Experimental results
[0062] Masson staining showed that there was a large accumulation of collagen in the left ventricular tissue of the PBS group ( Figure 4 A), while A2S ( Figure 4 B, A2S group: 6.11 ± 0.02%, PBS group: 17.82 ± 0.12%, P < 0.0001) and A3S ( Figure 4 B, A3S group: 7.94 ± 0.05%, PBS group: 17.82 ± 0.12%, P < 0.001) treatment reduced the collagen accumulation. The L-Arg group had no significant therapeutic effect on reducing myocardial fibrosis after myocardial infarction. These results indicate that A2S and A3S alleviate cardiac function damage caused by ventricular remodeling by reducing myocardial fibrosis.
[0063] Example 5 Arginine diester increased the number of M2 macrophages after myocardial infarction and stimulated the polarization of M2 macrophages after myocardial infarction
[0064] I. Experimental methods
[0065] Macrophages are one of the most active cell types at all stages after myocardial infarction, including stages such as cardiac inflammation and tissue repair. Macrophages mediate cardiac inflammation by secreting cytokines and chemokines, and these cytokines and chemokines play different roles at different stages of myocardial infarction. In the early stage of myocardial infarction, macrophages are mainly of the M1 type, with strong pro-inflammatory characteristics, which exacerbate cardiac injury and promote adverse ventricular wall remodeling after myocardial infarction. Three to seven days after myocardial infarction, macrophages begin to polarize into M2, repairing the cardiac tissue after myocardial infarction. Therefore, the proportion and number of M2 macrophages in the myocardial infarction repair stage are very important for preventing pathological ventricular wall remodeling and the dysfunction of viable myocardium. Quantify the proportion of macrophages in myocardial infarction mice treated with the blank control PBS group, L-Arg group, A2S group, and A3S group in Example 3 on the 5th day after myocardial infarction by flow cytometry.
[0066] II. Experimental Results
[0067] As Figure 5 shown in A, CD45-positive cells were defined as immune cells, and macrophages positive for CD11b and F4 / 80 were isolated ( Figure 5 B), and finally, the proportion of CD206-positive M2 macrophages / total macrophages was analyzed.
[0068] As Figure 5 shown in C and Figure 5 D, after treatment with A2S (A2S group: 22.32 ± 0.03%, PBS group: 9.26 ± 0.03%, P < 0.0001) and A3S (A3S group: 21.24 ± 0.07%, PBS group: 9.26 ± 0.03%, P < 0.0001), the proportion of CD206-positive M2 macrophages in the heart increased.
[0069] The above results indicate that after myocardial infarction treated with A2S and A3S, macrophages in the heart are polarized into the M2 repair type, thus reducing cardiac injury.
[0070] Example 6 Arginine Diester Has High Biosafety in Vivo
[0071] I. Experimental Methods
[0072] Take the sera, livers, lungs, and kidneys of mice 28 days after myocardial infarction treated with the blank control PBS group, L-Arg group, A2S group, and A3S group in Example 3. Detect the liver and kidney functions of the sera, make paraffin sections of the livers, lungs, and kidneys and perform HE staining to observe whether there are changes in the morphological structures of the organs to determine the biosafety of A2S and A3S.
[0073] II. Experimental Results
[0074] The results of HE staining showed that there were no significant differences in the morphological structures of the kidneys, livers, and lungs among each group and the PBS group ( Figure 6 A).
[0075] The results of liver and kidney function tests showed that there were no significant differences in the levels of serum ALT, AST, creatinine, and urea nitrogen between the A2S group and the A3S group and the PBS group ( Figure 6 B~ Figure 6 E).
[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. Use of arginine diester in the preparation of a drug for preventing and treating cardiovascular diseases, wherein the structural formula of the arginine diester is shown as Formula I: Among them, y = 2, 3, 4 or 6.
2. The application according to claim 1, characterized in that In the structural formula of the arginine diester, y = 2 or 3.
3. The application according to claim 1, wherein The drug is a drug for preventing and treating myocardial infarction.
4. The application according to claim 3, wherein The drug is a drug for improving cardiac function and ventricular remodeling after myocardial infarction.
5. The application according to claim 3, wherein The drug is a drug for increasing left ventricular ejection fraction and / or shortening fraction of minor axis.
6. The application according to claim 3, characterized in that, The drug is a drug for reducing left ventricular end-systolic diameter.
7. The application according to claim 3, wherein The drug is a drug for alleviating myocardial fibrosis.
8. The application according to claim 3, characterized in that, The drug is a drug for promoting macrophage polarization into M2 type.
9. The application according to claim 1, characterized in that, The preparation method of the arginine diester is as follows: reflux and condense L-arginine, diol monomer and toluenesulfonic acid monohydrate in toluene, and carry out esterification and dehydration reaction to obtain arginine diester, wherein the diol monomer includes ethylene glycol, 1,3-propanediol, 1,4-butanediol or 1,6-hexanediol.
10. The application according to claim 9, wherein The reflux and condensation temperature is 120-130 °C, and the thermal esterification and dehydration reaction is carried out for 16-24 h.