Application of all-transretinoic acid in preparation of medicine for treating ejection fraction retention type heart failure

Abstract

The invention relates to the technical field of medicine research and development, in particular to application of all-transretinoic acid in preparation of a medicine for treating ejection fraction retention type heart failure. The invention discloses that ATRA can significantly improve lipid metabolism, reduce blood pressure and effectively inhibit myocardial hypertrophy and fibrosis processes for the first time, and shows clear HFpEF treatment benefits under a safe administration dosage. A brand new treatment strategy based on new use of old medicines is provided for the disease, and good clinical transformation prospects and application values are achieved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of drug research and development, and in particular to application of all-trans retinoic acid in preparation of a drug for treating heart failure with preserved ejection fraction. BACKGROUND

[0002] Heart failure with preserved ejection fraction (HFpEF) is a subtype of heart failure with left ventricular ejection fraction (LVEF) not less than 50%, and the type of heart failure is increasing year by year and accounts for a high proportion in heart failure patients. Population aging and the widespread increase of risk factors such as obesity, metabolic syndrome and type 2 diabetes mellitus are the main reasons for the aggravation of the disease burden. The high incidence of HFpEF not only seriously affects the quality of life of patients, but also brings a heavy social burden. However, the existing drug treatment scheme, such as spironolactone and sacubitril valsartan, fails to significantly improve the main endpoints in key clinical trials; SGLT2 inhibitors and GLP-1 receptor agonists show certain curative effects, but have limitations in applicable population, and it is difficult to comprehensively cope with the complex pathological mechanism of HFpEF. Therefore, it is of important clinical significance to develop a drug with a new mechanism and capable of effectively improving the prognosis of HFpEF patients.

[0003] All-trans retinoic acid (ATRA) is a natural derivative of vitamin A. ATRA is currently mainly used for the treatment of acute promyelocytic leukemia in clinical application. The prior art does not find the application of ATRA in cardiovascular diseases, and ATRA is not found to be applied in heart failure with preserved ejection fraction. SUMMARY

[0004] The present application verifies the application potential of ATRA in the treatment of HFpEF for the first time in view of the deficiencies in the existing treatment of heart failure with preserved ejection fraction.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides application of all-trans retinoic acid in preparation of a drug for treating heart failure with preserved ejection fraction.

[0007] The present application also provides application of all-trans retinoic acid in preparation of a drug for improving diastolic function of the heart.

[0008] The present application also provides application of all-trans retinoic acid in preparation of a drug for improving myocardial hypertrophy and cardiac fibrosis.

[0009] Preferably, the dosage form of the drug includes tablets, pills, capsules, oral liquids or injection solutions.

[0010] The present application also provides a method for constructing an animal model of heart failure with preserved ejection fraction, comprising the following steps:

[0011] The animal is fed with high-fat feed and the water containing 0.4-0.6 g / L Nω-nitro-L-arginine methyl ester is used as the drinking water of the animal for 5-8 weeks.

[0012] Preferably, the animal is a male mouse.

[0013] The content of fat in the high-fat feed is ≥60%.

[0014] The application also provides application of the animal model of heart failure with preserved ejection fraction constructed by the construction method in screening of drugs for treating heart failure with preserved ejection fraction.

[0015] Compared with the prior art, the application has the following advantages:

[0016] ATRA can significantly improve lipid metabolism, reduce blood pressure, and effectively inhibit the progress of myocardial hypertrophy and fibrosis, and show clear treatment benefits for HFpEF at a safe administration dose.

[0017] The application first discloses and verifies the treatment potential of ATRA in an animal model of HFpEF, provides a new treatment strategy for the disease based on old drugs, and has good clinical conversion prospects and application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Heart ultrasound data for modeling heart failure with preserved ejection fraction (A represents representative ultrasound images of M type, PW, and Tissue mode of mice in the control group and the model group; B represents changes in left ventricular ejection fraction of mice in the control group and the model group; C represents left ventricular short-axis shortening rate of mice in the control group and the model group; D represents the average value E' of the E peak velocity of mitral valve blood flow divided by the early diastolic velocity of the lateral wall and septum at the mitral annulus; ** represents P<0.01, and ns represents no statistical difference);

[0019] Figure 2 Heart ultrasound detection results (A is representative ultrasound images of M type, PW, and Tissue mode of mice in the control group, the model group, and the treatment group; B is changes in left ventricular ejection fraction of mice in the control group, the model group, and the treatment group; C is left ventricular short-axis shortening rate of mice in the control group, the model group, and the treatment group; D is the average value E' of the E peak velocity of mitral valve blood flow divided by the early diastolic velocity of the lateral wall and septum at the mitral annulus; * represents P<0.05, ** represents P<0.01, and ns represents no statistical difference);

[0020] Figure 3For the results of cardiac gross and histological detection (A represents the representative pictures of cardiac gross, HE, WGA and Sirius red staining of control group, model group and treatment group mice; B represents the ratio of heart weight (HW) / tibial length (TL) statistical chart; C represents the myocardial cell size statistical chart of HE staining; D represents the myocardial cell size statistical chart of WGA staining; E represents the perivascular fibrosis statistical chart of Sirius red staining; ** represents P<0.01, and ns represents no statistical difference);

[0021] Figure 4 For the results of plasma TC, TG, blood pressure and heart failure index detection (A represents the plasma TC detection result statistical chart; B represents the plasma TG result detection statistical chart; C represents the blood pressure (SBP) detection result statistical chart; D represents the transcription level results statistical chart of heart failure indexes Nppa, Nppb and Myh7; * represents P<0.05, ** represents P<0.01, and ns represents no statistical difference);

[0022] Figure 5 For the results of liver function treatment detection (A represents the ALT detection result statistical chart; B represents the plasma ST detection result statistical chart; ns represents no statistical difference). DETAILED DESCRIPTION

[0023] In the present application, the dosage of ATRA used in the treatment of HFpEF is 5 mg / kg / d, and ATRA is dissolved in physiological saline containing 10% dimethyl sulfoxide (DMSO) + 90% sulfobutyl ether-β-cyclodextrin (SBE-β-CD).

[0024] The schemes provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0025] Example 1

[0026] 1. Experimental process

[0027] 1.1 Construction of animal model

[0028] The experiment used 8-week-old male C57BL / 6N mice provided by the Pesticide Toxicology Research Center of Tongji Medical College, Huazhong University of Science and Technology, with experimental unit license number SYXK (E) 2019-0046. The feeding environment temperature was controlled at 23±3℃, and the light-dark cycle was 12 hours / 12 hours, with free feeding and drinking water. All animal experiments were approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

[0029] The HFpEF mouse model was constructed by adding 0.5 g / L of Nω-nitro-L-arginine methyl ester (L-NAME) in drinking water and feeding with high-fat feed (D12492, 60% fat energy supply) for 7 weeks.

[0030] 1.2 Animal grouping and administration method

[0031] The animal experiment was divided into the following three groups: (1) control group (CON): fed with ordinary feed (AIN93M, 10% fat function) for 7 weeks; (2) model group (HFpEF): L-NAME plus high-fat diet for 7 weeks, starting from the fifth week, the same amount of solvent (10% DMSO + 90% SBE-β-CD normal saline) was given by gavage, lasting for three weeks; (3) ATRA treatment group (HFpEF+ATRA): L-NAME plus high-fat diet for 7 weeks, starting from the fifth week, ATRA (5 mg / kg / d, dissolved in 10% DMSO + 90% SBE-β-CD normal saline) was given by gavage, lasting for three weeks.

[0032] 1.3 Mouse heart ultrasound detection

[0033] After the gavage experiment, the mouse heart ultrasound detection was performed. Before the experiment, the mouse hair in the precordial region was carefully removed with a cotton swab dipped in depilatory cream. Then, the mouse was firmly fixed on the test bench in a supine position with adhesive tape. A small amount of ultrasonic coupling agent was applied to the limbs of the mouse to ensure good contact with the electrodes of the test bench, providing a stable electrical signal transmission environment for subsequent detection. After the mouse was fixed, isoflurane was used for continuous gas anesthesia assisted by a respirator. During the anesthesia process, the heart rate of the mouse was closely monitored, and the dose of anesthetic was finely adjusted to stabilize the heart rate of the mouse at 500-600 bpm. After the heart rate of the mouse was stabilized, an appropriate amount of ultrasonic coupling agent was applied to the precordial region, and two-dimensional echocardiogram and M-mode echocardiogram of long axis and short axis were recorded with the aid of ultrasonic equipment. These images will be used to accurately evaluate the cardiac systolic function of the mouse. After completing the data collection related to cardiac systolic function, the ultrasonic probe was moved to the apex of the mouse. During this process, the dose of anesthetic was adjusted again to control the heart rate of the mouse at 500 bpm. Then the ratio of left ventricular early diastolic blood flow velocity peak to mitral annulus early diastolic motion velocity peak (E / E' ratio) was obtained. By analyzing the E / E' ratio, the diastolic function of the left ventricle of the mouse was effectively evaluated.

[0034] 1.4 Mouse blood pressure measurement

[0035] After the gavage experiment, the blood pressure of the mouse was detected. The tail blood pressure was measured using a non-invasive blood pressure meter. The mouse blood pressure measurement was performed using the multi-channel real-time blood pressure monitoring system. First, open the CODA controller, turn on the heating platform and set the appropriate heating level according to the room temperature, open the CODA software, select the CODA device and perform the controller diagnostic test, and close the device test window after the test is completed. In the "Specimen" tab, name the animal and select the animal type as mouse; in the "Basic Session Information" set the number of cycles, set the adaptation cycle to 5 cycles, and set the measurement cycle to 10 cycles. Lift the mouse tail and gently place it in the back of the holder, with the animal facing the nose cone opening. Carefully secure the back door of the holder and slide the nose cone towards the back door to restrict the animal's movement, but be careful not to pinch the tail or other body parts. Thread the tail through the "occlusion cuff" as close to the tail base as possible without forcing it, and secure the "occlusion cuff tube" in the notch at the back of the holder top. Thread the tail through the "VPR cuff" so that it is located 2 mm inside the "occlusion cuff", and secure the "VPR cuff tube" in the notch at the back of the holder top. Finally, connect the cuffs to the CODA controller. The holder should be placed in the designated position on the heating platform. If multiple tests are performed on the same group of animals over a period of time, rotate the holder on the heating platform to improve the consistency and accuracy of blood pressure measurements.

[0036] 1.5 Histological examination

[0037] After the end of the gavage experiment, the mice were sacrificed, and the heart tissue was taken for gross observation, then fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned for WGA, Sirius red and HE staining, respectively, to evaluate the myocardial cell structure and fibrosis degree. The mRNA expression levels of Nppa, Nppb and Myh7 in myocardial tissue were detected by qPCR.

[0038] 2. Results

[0039] 2.1 HFpEF model was successfully constructed

[0040] Before the mice were sacrificed, echocardiography was performed. Echocardiography showed that the left ventricular ejection fraction (LVEF) and short-axis shortening fraction (LVFS) of the model group mice did not change significantly, while the E / E' ratio increased significantly, indicating impaired diastolic function, and the HFpEF model was successfully constructed. Figure 1

[0041] 2.2 ATRA improved the diastolic function of the mouse HFpEF model

[0042] By comparing the echocardiography results of the three groups of mice, it was found that the LVEF and LVFS of the three groups were within the normal range, and ATRA treatment significantly improved the E / E' index of HFpEF mice. Figure 2 ​). This suggests that ATRA can significantly improve diastolic dysfunction in the HFpEF mouse model.

[0043] 2.3 ATRA significantly improved myocardial hypertrophy and cardiac fibrosis in the mouse HFpEF model

[0044] After the mice were sacrificed, the heart tissue was photographed and then immersed in a 4% paraformaldehyde solution for paraffin-embedded sectioning. By comparing the heart body and calculating the ratio of heart weight / tibia length, it was found that the heart of the model group was significantly hypertrophic, and ATRA improved this pathological phenotype. The resulting sections were stained with HE and WGA to observe the changes in myocardial cell morphology. By comparing the morphology of the two groups, it can be seen that the myocardial cells of the HFpEF group of mice were significantly hypertrophic, and ATRA treatment reversed this pathological process. In addition, the mouse heart tissue was stained with Sirius red to detect the degree of cardiac fibrosis, suggesting that the model group of mice had significant fibrosis around the heart blood vessels, and ATRA treatment improved the cardiac fibrosis Figure 3 ).

[0045] 2.4 ATRA improved TC, blood pressure, and heart failure indicators in HFpEF mice

[0046] After the mice were anesthetized, blood was taken from the eye and the plasma was separated. The total cholesterol (TC) and triglyceride (TG) kits were used to detect the TC and TG content in the mouse plasma. The results showed that in the model group, TC and TG were significantly increased, and ATRA could reduce the TC content, but had no significant effect on TG. The Coda blood pressure meter was used to measure the blood pressure of the mice, and the results showed that the blood pressure of the model group increased, and ATRA could improve the blood pressure. After extracting the RNA from the mouse heart tissue, qPCR was performed to detect the transcription level of the heart failure indicators Nppa, Nppb and Myh7. The results showed that in the model group, the expression levels of the heart failure indicators were significantly increased, and ATRA treatment improved this situation Figure 4 ).

[0047] 2.5 Effect on liver function

[0048] After the mice were anesthetized, blood was taken from the eye and the plasma was separated. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) kits were used to detect the AST and ALT content in the mouse plasma. There was no statistical difference in the AST and ALT levels among the three groups, indicating that ATRA did not cause liver toxicity at this dose Figure 5 ).

[0049] From the above examples, ATRA can significantly improve lipid metabolism, reduce blood pressure, and effectively inhibit the progression of myocardial hypertrophy and fibrosis, and show clear therapeutic benefits for HFpEF at a safe dose. ATRA, as an endogenous vitamin A metabolite, has been widely used in the treatment of acute promyelocytic leukemia (APL) in clinical practice, and its safety has been fully proven. The present application first discloses and verifies the therapeutic potential of ATRA in an animal model of HFpEF, providing a new treatment strategy for the disease based on the use of old drugs, and has good clinical conversion prospects and application value.

[0050] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Application of all-trans retinoic acid in the preparation of drugs for treating heart failure with preserved ejection fraction.

2. Application of all-trans retinoic acid in the preparation of drugs that improve cardiac diastolic function.

3. Application of all-trans retinoic acid in the preparation of drugs to improve myocardial hypertrophy and cardiac fibrosis.

4. The application according to any one of claims 1 to 3, characterized in that, The dosage forms of the drug include tablets, pills, capsules, oral liquids, or injections.

5. A method for constructing an animal model of heart failure with preserved ejection fraction, characterized in that, Includes the following steps: An aqueous solution containing 0.4–0.6 g / L of Nω-nitro-L-arginine methyl ester was used as drinking water for the animals, while they were fed a high-fat diet for 5–8 weeks.

6. The construction method according to claim 5, characterized in that, The animal in question was a male mouse; The high-fat feed contains ≥60% fat.

7. The application of the animal model of heart failure with preserved ejection fraction constructed by the method of claim 5 or 6 in screening drugs for the treatment of heart failure with preserved ejection fraction.

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