CN112957449B - Pharmaceutical composition for preventing and/or treating cardiomyopathy and nephropathy and preparation method and application thereof - Google Patents

Abstract

The invention provides a pharmaceutical composition for preventing and/or treating cardiomyopathy and nephropathy and a preparation method and application thereof, belonging to the technical field of biological medicines. The research of the invention finds that Ang IV can be applied to the treatment of cardiomyopathy and nephropathy and further prepared into a nano-particle medicinal preparation, and experiments prove that the Ang IV can obviously improve the contraction and relaxation functions of the left ventricle of a diabetic mouse, relieve myocardial fibrosis of the diabetic mouse, reduce urine protein and improve the clearance rate of renal creatinine, and meanwhile, the preparation process is simple and has good practical application value.

Description

A kind of pharmaceutical composition for preventing and/or treating cardiomyopathy and nephropathy and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a pharmaceutical composition for preventing and/or treating cardiomyopathy and nephropathy, and a preparation method and application thereof.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

In recent years, due to problems such as obesity and unbalanced diet, the incidence of diabetes, hypertension, and coronary heart disease has continued to rise, and the trend of younger onset is obvious. There are many complications of these diseases and great harm, mainly including cardiomyopathy, cerebral infarction and other cardiovascular and cerebrovascular diseases, as well as nephropathy, retinopathy and peripheral neuropathy. Cardiomyopathy and nephropathy are important complications of diabetes, hypertension and coronary heart disease, which can reduce the quality of life of patients, increase the economic burden, and eventually lead to death. Therefore, delaying the remodeling and dysfunction of the heart and kidneys, improving their quality of life and reducing their economic burden is a major livelihood and social problem in our country, with great social significance and economic benefits. It is known that the pathological mechanism of cardio-renal remodeling caused by diabetes involves microvascular damage, interstitial fibrosis, apoptosis and other links, but the key regulatory targets in the pathogenesis and progression of the disease are not yet clear. Therefore, further elucidating the mechanism of cardio-renal remodeling and finding effective intervention measures is a major issue to be solved urgently in the field of cardiovascular disease. Previous studies have confirmed that abnormal renin-angiotensin system (RAS) plays an important role in cardiorenal remodeling. Finding new members of RAS is a difficult scientific research and an urgent need for myocardial remodeling and targeted therapy for heart failure.

Traditional RAS includes renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin I (AngI), angiotensin II (Ang II), Ang-II type 1 receptor (AT ₁ R) and aldosterone, Ang II and aldosterone play key roles in the pathogenesis of diabetic cardiomyopathy. In recent years, scholars have begun to explore the role and mechanism of new members of RAS in myocardial remodeling and heart failure. These new members include: angiotensin-converting enzyme 2 (ACE2), angiotensin (1–7) [Ang-( 1–7)], Mas receptor (MasR), AT ₂ receptor (AT ₂ R), angiotensin (1–9) [Ang-(1–9)], angiotensin (1–5) [ Ang-(1–5)], angiotensin III (Ang III), angiotensin IV (Ang IV), etc.

In recent years, domestic and foreign studies have reported that Ang II, Ang-(1–7), AT ₁ R, AT ₂ R and MasR are involved in myocardial cell injury and protection. Although Ang IV is thought to have the opposite biological effect of Ang II, the research fields of Ang IV pharmacology, formulation and translational medicine are still rarely reported.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the present invention provides a pharmaceutical composition for preventing and/or treating cardiomyopathy and nephropathy, and a preparation method and application thereof. According to the research of the present invention, Ang IV can be applied to the treatment of cardiomyopathy and nephropathy, and can be further prepared into nanoparticle pharmaceutical preparations. It has been proved by experiments that it can significantly improve the left ventricular systolic and diastolic functions of diabetic mice and reduce myocardial fibers in diabetic mice. and reduce urinary protein and improve renal creatinine clearance rate, thereby completing the present invention.

Specifically, the present invention relates to the following technical solutions:

The first aspect of the present invention provides the use of Ang IV polypeptide in the preparation of medicaments for preventing and/or treating cardiomyopathy and nephropathy.

According to the present invention, the concept of "prophylaxis and/or treatment" refers to any measure suitable for the treatment of cardiomyopathy and renal disease and related diseases, or the preventive treatment of such diseases or symptoms of such manifestations, or the avoidance of such diseases. Relapse of a disease, such as after the end of a treatment period or treatment of symptoms of a disease that has already occurred, or pre-interventional prevention or inhibition or reduction of the occurrence of such disease or symptoms.

Wherein, the cardiomyopathy is diabetic cardiomyopathy, and the nephropathy is diabetic nephropathy.

According to the present invention, not only the application of Ang IV polypeptide in the preparation of medicaments for preventing and/or treating cardiomyopathy and nephropathy is disclosed, but also the application of Ang IV polypeptide in combination with at least one other active pharmaceutical ingredient can enhance the kind of effect. As an alternative to or in addition to other pharmaceutically active ingredients, Ang IV polypeptides can also be used in combination with other non-pharmaceutically active ingredients.

In view of this, the second aspect of the present invention provides a pharmaceutical composition whose active ingredient is composed of Ang IV polypeptide and at least one other active pharmaceutical ingredient and/or at least one other non-pharmaceutical active ingredient constitute.

The third aspect of the present invention provides a nanoparticle comprising the above-mentioned Ang IV polypeptide or the above-mentioned pharmaceutical composition.

The above-mentioned nanoparticles are chitosan nanoparticles, and the chitosan encapsulates the above-mentioned Ang IV polypeptide or the above-mentioned pharmaceutical composition. The particle size of the nanoparticles is controlled to be 150-450 nm.

A fourth aspect of the present invention provides a preparation method of the above-mentioned nanoparticles, the preparation method comprising:

S1, chitosan is dissolved in glacial acetic acid solution, adjust pH with saturated sodium hydroxide solution, obtain solution I;

S2, adding the Ang IV polypeptide into the sodium tripolyphosphate (TPP) aqueous solution to dissolve, adding the Ang IV polypeptide and the sodium tripolyphosphate mixed solution dropwise to the above solution I, and forming nanoparticles spontaneously by the electrostatic action of anions and cations; Wash and freeze-dry.

The fifth aspect of the present invention provides the application of the above-mentioned Ang IV polypeptide, pharmaceutical composition and/or nanoparticle in the preparation of a medicine with any one or more of the following functions:

1) Improve left ventricular systolic and diastolic function in patients with diabetes;

2) Reduce cardiac interstitial deposition in diabetic patients;

3) Reduce perivascular collagen deposition in diabetic patients;

4) Reduce myocardial fibrosis in diabetic patients;

5) Reduce the urinary protein of nephropathy in diabetic patients;

6) Improve renal creatinine clearance.

Beneficial technical effects of one or more of the above technical solutions:

The above technical scheme reports for the first time that Ang IV polypeptide has the effect of treating diabetic cardiomyopathy and nephropathy, and further prepares nanoparticles with chitosan. By optimizing the preparation process, the final encapsulation rate can reach 88.2%; The particle size is concentrated between 150 and 450 nm.

It has been verified by experiments that the nanoparticle preparation prepared by the above technical scheme can significantly improve the left ventricular systolic and diastolic function of diabetic mice, reduce myocardial fibrosis in diabetic mice, reduce urinary protein, and improve renal creatinine clearance rate, so it has good practical application. value.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

Fig. 1 is the particle size distribution diagram of Ang IV chitosan nanoparticles in the embodiment of the present invention;

Fig. 2 shows that Ang IV chitosan nanoparticles by intragastric administration improves the left ventricular systolic and diastolic function of diabetic mice in the embodiment of the present invention. A: Schematic diagram of echocardiography results; B-E: Quantitative analysis of left ventricular ejection fraction (LVEF), fractional shortening (FS), mitral valve early-to-late diastolic flow velocity ratio (E/A) and mitral valve Ratio of early to late peak velocities in ring diastole (E'/A'). *P<0.05; **P<0.01; ***P<0.001;

Figure 3 shows that Ang IV chitosan nanoparticles by gavage improves left ventricular myocardial fibrosis in diabetic mice in the embodiment of the present invention. A: Schematic diagram of MASSON staining results; B: Quantitative analysis of collagen volume fraction (CVF) and the ratio of perivascular collagen area/luminal area. *P<0.05; **P<0.01; ***P<0.001;

Figure 4 shows the effect of intragastric administration of Ang IV chitosan nanoparticles on the quantification of urinary protein and creatinine clearance in diabetic mice in the example of the present invention.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

At present, the application of pharmaceutical preparations developed based on Ang IV in diabetic cardiomyopathy and nephropathy has not been reported.

In view of this, in a specific embodiment of the present invention, there is provided the use of Ang IV polypeptide in the preparation of medicaments for preventing and/or treating cardiomyopathy and nephropathy.

According to the present invention, the concept of "prophylaxis and/or treatment" refers to any measure suitable for the treatment of cardiomyopathy and nephropathy-related diseases, or the preventive treatment of such diseases or symptoms of such manifestations, or the avoidance of such diseases. Relapse of a disease, eg, after a treatment period has ended or treatment of symptoms of an already onset disease, or pre-interventional prevention or suppression or reduction of the occurrence of such disease or symptoms.

In another specific embodiment of the present invention, the cardiomyopathy is diabetic cardiomyopathy, and the nephropathy is diabetic nephropathy.

According to the present invention, not only the application of Ang IV polypeptide in the preparation of medicaments for preventing and/or treating cardiomyopathy and nephropathy is disclosed, but also the application of Ang IV polypeptide in combination with at least one other active pharmaceutical ingredient can enhance the kind of effect. As an alternative to or in addition to other pharmaceutically active ingredients, Ang IV polypeptides can also be used in combination with other non-pharmaceutically active ingredients.

In view of this, in another specific embodiment of the present invention, there is provided a pharmaceutical composition, the active ingredient of which is composed of Ang IV polypeptide and at least one other active pharmaceutical ingredient and/or at least one other non-drug Active ingredient composition.

Other non-pharmaceutically active ingredients include, but are not limited to, pharmaceutically acceptable carriers, excipients and/or diluents.

In yet another specific embodiment of the present invention, a nanoparticle is provided, which comprises the above-mentioned Ang IV polypeptide or the above-mentioned pharmaceutical composition.

The above-mentioned nanoparticles are chitosan nanoparticles, and the chitosan encapsulates the above-mentioned Ang IV polypeptide or the above-mentioned pharmaceutical composition. The particle size of the nanoparticles is controlled to be 150-450 nm.

In another specific embodiment of the present invention, the molecular weight of chitosan is 200-2000kDa, more preferably 250-1200kDa, most preferably 600kDa, and the degree of deacetylation is greater than 90%.

As a small molecule polypeptide, Ang IV has low bioavailability when administered orally, and has short half-life and inconvenient administration when administered intravenously, subcutaneously or intramuscularly. Chitosan can attach to the surface of mucosa and can open tight junctions of epithelial cells, therefore, chitosan can be used as a promoter of macromolecular drugs to improve their bioavailability. In order to improve the disadvantage of low oral bioavailability of Ang IV and achieve the purpose of slow release and long-term effect, the present invention adopts ion crosslinking method to prepare it into chitosan nanoparticles. During the test, it was found that different molecular weights of chitosan and pH of chitosan solution would affect the encapsulation effect of Ang IV. Among them, the molecular weight of chitosan was 250-1200kDa, the degree of deacetylation was greater than 90%, and the chitosan molecular weight range was 250-1200kDa. When the pH value of the solution is in the range of 4.5-6.0, the encapsulation efficiency of Ang IV is as high as 88.2%; when the molecular weight of chitosan is less than 250kDa or more than 1200kDa, the encapsulation efficiency will gradually decrease.

In another specific embodiment of the present invention, there is provided a preparation method of the above-mentioned nanoparticles, the preparation method comprising:

S1, chitosan is dissolved in glacial acetic acid solution, adjust pH with saturated sodium hydroxide solution, obtain solution I;

S2, adding the Ang IV polypeptide into the sodium tripolyphosphate (TPP) aqueous solution to dissolve, adding the Ang IV polypeptide and the sodium tripolyphosphate mixed solution dropwise to the above solution I, and forming nanoparticles spontaneously by the electrostatic action of anions and cations; Wash and freeze-dry.

In another specific embodiment of the present invention, the molecular weight of chitosan is 200-2000kDa, more preferably 250-1200kDa, most preferably 600kDa, and the degree of deacetylation is greater than 90%.

In another specific embodiment of the present invention, in step S1, the pH value is adjusted to 4.5-6.0, preferably, the pH value is adjusted to 5.5.

In yet another specific embodiment of the present invention, in step S2, the mass ratio of sodium tripolyphosphate to chitosan is 1:10-30, preferably 1:30; by optimizing the quality of sodium tripolyphosphate and chitosan ratio, and at the same time with other parameters, so as to prepare nanoparticles with good particle size and shape.

In yet another specific embodiment of the present invention, the particle size of the nanoparticles is 150-450 nm.

In another specific embodiment of the present invention, the application of the above-mentioned Ang IV polypeptide, pharmaceutical composition and/or nanoparticle in the preparation of a medicine with any one or more of the following functions is provided:

1) Improve left ventricular systolic and diastolic function in patients with diabetes;

2) Reduce cardiac interstitial deposition in diabetic patients;

3) Reduce perivascular collagen deposition in diabetic patients;

4) Reduce myocardial fibrosis in diabetic patients;

5) Reduce the urinary protein of nephropathy in diabetic patients;

6) Improve renal creatinine clearance.

In another specific embodiment of the present invention, the patients include human and non-human mammals, such as rats, mice, rabbits, monkeys, orangutans and the like.

The present invention is further explained and illustrated by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention.

Embodiment 1 A kind of pharmaceutical composition for treating and/or preventing cardiomyopathy and nephropathy

The pharmaceutical composition is chitosan nanoparticles encapsulating Ang IV polypeptide.

The preparation method of the chitosan nanoparticles encapsulating the Ang IV polypeptide, comprising the following steps:

(1) 30 mg of chitosan with a molecular weight of 600 kDa was dissolved in 8 mL of 10% glacial acetic acid solution, and the pH was adjusted to 5.5 with saturated sodium hydroxide solution to obtain solution I;

(2) Take 2 mL of Ang IV polypeptide solution with a concentration of 1.5 μmol/dL and 2 mL of 0.5 mg/mL sodium tripolyphosphate (TPP) aqueous solution and mix and stir (1000 rpm) evenly. Mix 1 drop of Ang IV polypeptide and TPP solution. It is added dropwise into the above solution I at a speed of /3 to 5 seconds, and nanoparticles are formed spontaneously through the electrostatic action of anions and cations; and they are obtained after centrifugation, washing, and freeze drying. The suspension was centrifuged at high speed at 4°C for 20 minutes (15,000 rpm), the precipitate was collected, washed three times with triple distilled water for 3 minutes each time, and freeze-dried.

Embodiment 2 A kind of pharmaceutical composition for treating and/or preventing cardiomyopathy and nephropathy

The pharmaceutical composition is chitosan nanoparticles encapsulating Ang IV polypeptide.

The preparation method of the chitosan nanoparticles encapsulating the Ang IV polypeptide, comprising the following steps:

(1) 10 mg of chitosan with a molecular weight of 250 kDa was dissolved in 8 mL of 10% glacial acetic acid solution, and the pH was adjusted to 5.5 with saturated sodium hydroxide solution to obtain solution I;

(2) Take 2 mL of Ang IV polypeptide solution with a concentration of 1.5 μmol/dL and 2 mL of 0.5 mg/mL TPP aqueous solution and mix and stir (1000 rpm) evenly. Speed dropwise added to the above solution I, through the electrostatic action of anions and cations to spontaneously form nanoparticles; after centrifugation, washing, freeze-drying. The suspension was centrifuged at high speed at 4°C for 20 minutes (15,000 rpm), the precipitate was collected, washed three times with triple distilled water for 3 minutes each time, and freeze-dried.

Embodiment 3 A kind of pharmaceutical composition for treating and/or preventing cardiomyopathy and nephropathy

The pharmaceutical composition is chitosan nanoparticles encapsulating Ang IV polypeptide.

The preparation method of the chitosan nanoparticles encapsulating the Ang IV polypeptide, comprising the following steps:

(1) 20 mg of chitosan with a molecular weight of 1200 kDa was dissolved in 8 mL of 10% glacial acetic acid solution, and the pH was adjusted to 5.5 with saturated sodium hydroxide solution to obtain solution I;

(2) Take 2 mL of Ang IV polypeptide solution with a concentration of 1.5 μmol/dL and 2 mL of 0.5 mg/mL TPP aqueous solution and mix and stir (1000 rpm) evenly. Speed dropwise added to the above solution I, through the electrostatic action of anions and cations to spontaneously form nanoparticles; after centrifugation, washing, freeze-drying. The suspension was centrifuged at high speed at 4°C for 20 minutes (15,000 rpm), the precipitate was collected, washed three times with triple distilled water for 3 minutes each time, and freeze-dried.

Test Example 1 Optimization of the preparation process of Ang IV chitosan nanoparticles

The Ang IV chitosan nanoparticle suspension was dropped onto a copper grid and stained with 2% sodium phosphotungstate. The particle size range of the nanoparticle preparation was measured by a light scattering analyzer. The results showed that the particle size of Ang IV chitosan nanoparticles was less than 600 nm, and concentrated between 150 and 450 nm. As shown in Figure 1.

Table 1 shows the effects of different factors on the physical properties and drug-loading properties of chitosan nanoparticles encapsulated with Ang IV polypeptide.

Test Example 2 Study on the effect of Ang IV chitosan nanoparticles on the heart and kidney of diabetic mice

(1) Experimental method

(1) Animal group intervention: 8-week-old male C57BL/6 mice were selected and fed with normal diet. The C57 mice were divided into the following groups: normal control group, diabetes model group, low-dose Ang IV group, medium-dose Ang IV group and high-dose Ang IV group. An animal model of diabetes was established by intraperitoneal injection of streptozotocin (STZ). STZ was dissolved in 0.1mol/L sterile citric acid-sodium citrate buffer solution (pH 4.2-4.5) at a concentration of 1%, and the mice in the diabetic group were intraperitoneally injected with STZ solution (55 mg/kg·day) for 5 consecutive days to induce diabetes. Mice in the normal control group were injected with the same dose of blank vehicle in parallel. 2 months after STZ injection, Ang IV was started for 4 months, and the subcutaneous osmotic pump was used for continuous infusion. ·day, the dose of high-dose Ang IV group was 2.88 mg/kg·day. Mice were euthanized at the end of the 6th month after injection of STZ, and the heart and kidney were removed, and histopathological changes such as fibrosis were detected. 24h urine protein quantification and creatinine clearance were measured.

(2) Cardiac ultrasound detection

Cardiac ultrasonography was performed by a Vevo2100 ultra-high-resolution small animal ultrasound system by technicians unaware of the study content. Mice were given 2% isoflurane inhalation anesthesia, and were fixed in a supine position on a constant temperature heating plate at 37°C. Ultrasound was performed after depilation of the left chest, and the following parameters were recorded: left ventricular diameter, left ventricular end-diastolic volume (LVEDV), left ventricular end-diastolic volume (LVEDV), and left ventricular End-systolic volume (LVESV), left ventricular ejection fraction (LVEF) and fractional shortening (FS). The pulse wave Doppler measures the mitral valve annulus diastolic early (E) and late (A) blood flow velocities and their ratios; tissue Doppler measures the mitral valve annulus early (E') and late (A') peak velocities and its ratio.

(3) Statistical analysis

SPSS v21.0 software was used for data analysis. Continuous variables are expressed as mean ± standard error or median [interquartile range]. If it follows a normal distribution, the analysis of variance is used for comparison between groups. If the difference between groups is statistically significant, the Bonferroni method is further used. Pairwise comparison. If it did not obey the normal distribution, the Kruskal-Wallis rank sum test was used for comparison between groups. If the difference between groups was statistically significant, the Dwass-Steel-Critchlow-Fligner method was used for multiple comparisons. Categorical variables were tested using the chi-square test or the Kruskal-Wallis rank-sum test, depending on their unordered or ordered nature. A two-sided P<0.05 was considered statistically significant.

(2) Experimental results

The results of echocardiography in mice showed that intragastric administration of Ang IV chitosan nanoparticles could improve left ventricular systolic and diastolic function in diabetic mice. At the end of 2 months after STZ injection, LVEF, FS, E/A and E'/A' were similar between normal and diabetic mice. At the end of 4 and 6 months, LVEF, FS, E/A and E'/A' were significantly reduced in diabetic mice compared to normal mice. Ang IV chitosan nanoparticle intervention for 2 and 4 months can dose-dependently reverse left ventricular function. as shown in picture 2.

Compared with normal hearts, diabetic hearts show significant myocardial disturbances. However, these myocardial morphological abnormalities were dose-dependently alleviated by Ang IV chitosan nanoparticle intervention. Ang IV dose-dependently reduces interstitial and perivascular collagen deposition in diabetic hearts. These results suggest that Ang IV can dose-dependently attenuate myocardial fibrosis in diabetic mice. As shown in Figure 3.

As shown in Figure 4, gavage of Ang IV chitosan nanoparticles can dose-dependently reduce the 24-h urine protein level and increase the renal creatinine clearance in a dose-dependent manner in diabetic nephropathy mice.

Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, the The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (3)

1. The application of angiotensin IV polypeptide in the preparation of medicaments for treating diabetic cardiomyopathy and diabetic nephropathy. 2. The application of angiotensin IV polypeptide, pharmaceutical composition and/or nanoparticle in the preparation of a medicine with any one or more of the following functions: 1) Improve left ventricular systolic and diastolic function in diabetic patients; 2) Reduce cardiac interstitial deposition in diabetic patients; 3) Reduce perivascular collagen deposition in diabetic patients; 4) Reduce myocardial fibrosis in diabetic patients; 5) Reduce the urinary protein of nephropathy in diabetic patients; 6) Improve renal creatinine clearance in diabetic patients; The active ingredient of the pharmaceutical composition is composed of angiotensin IV polypeptide and at least one other pharmaceutical active ingredient and/or at least one other non-pharmaceutical active ingredient; other non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients agent and/or diluent; The nanoparticle comprises angiotensin IV polypeptide or the pharmaceutical composition; the nanoparticle is a chitosan nanoparticle, and the chitosan-encapsulated angiotensin IV polypeptide or the pharmaceutical composition; the nanoparticle particle The diameter of chitosan is controlled to be 150~450nm, and the molecular weight of chitosan is 200~2000kDa. 3. The application according to claim 2, wherein the molecular weight of the chitosan is 600 kDa, and the degree of deacetylation is greater than 90%.

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