Use of Jun inhibitors in the preparation of medications for treating heart failure with preserved ejection fraction

Abstract

The present invention relates to the use of Jun inhibitors in the manufacture of a medicament for treating heart failure with preserved ejection fraction (HFpEF).

Description

Technical Field

[0001] The present invention relates to the use of Jun inhibitors in the manufacture of agents for treating heart failure with preserved ejection fraction (HFpEF).

Background Art

[0002] Cardiovascular diseases are one of the medical problems. Heart failure (HF) is the main cause of death in patients with cardiovascular diseases and is a major clinical problem. Heart failure includes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF).

[0003] Currently, HFpEF accounts for approximately 50% of all heart failure patients, and its morbidity is increasing at a surprising rate and is also the main cause of the increase in mortality due to cardiovascular diseases. HFpEF is a syndrome accompanied by multiple organ disorders and is a disorder or result jointly caused by the heart, lungs, kidneys, bones, immunity, inflammation, metabolism, and other factors, and often accompanies symptoms such as obesity, hypertension, myocardial hypertrophy, diabetes, and atrial fibrillation. HFpEF is a syndrome with high morbidity and mortality. According to clinical statistics, the mortality rate due to HF is 35%, and the proportion of deaths due to HFpEF among them accounts for 57%. However, so far, there are few drug therapies and medical devices that have been proven to change the disease progression and prognosis of HFpEF patients. Currently, there is an urgent need in the art to develop agents and / or therapies that can effectively treat HFpEF.

[0004] Jun is a transcription factor and a member of the AP1 family having chromatin-binding activity and transcriptional cis-regulatory region-binding activity, and is involved in the regulation of processes including animal organ development, protein phosphorylation, and cell proliferation. A Jun inhibitor refers to an inhibitor that can inhibit the expression of the Jun gene, reduce the DNA-binding activity of Jun, reduce the level of the Jun gene expression product, and prevent or block Jun signal transduction. Currently, studies have shown that Jun inhibitors have therapeutic effects in animal models such as endometriosis, breast cancer, and sepsis.

Summary of the Invention

[0005] (Disclosure of the invention) The inventors of this invention were the first to discover that suppressing the high expression of Jun using a Jun inhibitor can provide preventive and therapeutic effects on HFpEF, and thus completed the present invention. [Means for solving the problem]

[0006] Therefore, the technical solution of the present invention includes the use of Jun inhibitors in the manufacture of agents for treating or preventing heart failure with preserved ejection fraction (HFpEF).

[0007] In a preferred embodiment, the Jun inhibitor is selected from T-5224, MLN44, SR11302, veratramine, KCR motif peptide-1-{N-[2-succinamidylethyl]amino}anthraquinone, NY2267, cFos LZ, anti-Jun, anti-Fos SZ, FosW, FosWCANDI, CPW, FosUisCan, A-Fos, JNK inhibitor VIII, IQ3, tandicertib (CC-930), or pharmaceutically acceptable salts of the above substances. The above substances have been reported to have Jun inhibitory activity, and their structures are shown below.

[0008] T-5224: [ka]

[0009] MLN944: [ka]

[0010] SR11302: [ka]

[0011] Veratramine:

Chem.

[0012] KCR Motif Peptide-1-{N-[2-Succinimidylethyl]amino}anthraquinone:

Chem.

[0013] NY2267:

Chem.

[0014] cFos LZ:

Chem.

[0015] Anti-Jun SZ:

Chem.

[0020] FosUisCan: [ka]

[0021] A-Fos: [ka]

[0022] JNK inhibitor VIII: [ka]

[0023] IQ 3: [ka]

[0024] Tandicertive (CC-930): [ka]

[0025] In a more preferred embodiment, the Jun inhibitor is T-5224 or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 shows the structure of the HFpEF model, with A: schematic diagram of the experimental procedure, B: results of detecting contractile function in mice after five weeks of feeding, and C: results of detecting expansion function in mice after five weeks of feeding. [Figure 2]Figure 2 shows the relative expression of Jun in cardiomyocytes 15 weeks after HFD+L-NAME ingestion, indicating that Jun is highly expressed in HFpEF model mice. [Figure 3] Figure 3 shows that T-5224 can effectively mitigate the onset and progression of HFpEF, with A: detection results of contractile function in mice at various time points, B: detection results of diastolic function in mice at various time points, C: changes in body weight in mice treated with various therapies, and D: changes in Jun expression in cardiomyocytes of mice treated with various therapies.

[0027] In Figures 1-3, "CHOW" represents a normal mouse, "HFD+L-NAME" represents a model control group mouse, and "HFD+L-NAME+T-5524" represents a model treatment group mouse. In Figures 3A-3C, "CHOW" represents a normal mouse and "HFD+L-NAME" represents a model control group mouse, and the bar graphs at each time point are arranged from left to right in the order of "CHOW," "HFD+L-NAME," and "HFD+L-NAME+T-5524." [Modes for carrying out the invention]

[0028] The following examples illustrate specific embodiments of the present invention and verify the effects achieved by the present invention. It should be understood that the following examples are merely illustrative, and the technical solutions of the present invention are not limited to these examples.

[0029] (Examples)

[0030] 1. Materials and Reagents In this example, C57BL / 6N wild-type mice were purchased from Beijing Vital River. The suppliers of the reagents are shown in the table below.

[0031] [Table 1]

[0032] In addition to the above, the other materials and reagents used in this embodiment were also commercially available.

[0033] 2. Guidelines for Animal Experiments In this example, all animal experiments were conducted under the guidance of the Laboratory Animal Center of the Institutional Animal Care and Use Committee of Fuwai Hospital, National Center for Cardiovascular Diseases, China. All mice were bred and reared in the same environment, and during the experiments, the mice were randomly divided into groups. Echocardiographic analysis was performed by independent researchers who were unaware of the purpose of this study.

[0034] 3. Induction of a heart failure model with preserved ejection fraction Eight-to-ten-week-old male C57BL / 6N wild-type mice were divided into three groups: a normal group (normal diet and drinking water), a model control group (high-fat diet and administration of N-nitro-L-arginine methyl ester), and a model treatment group (high-fat diet and administration of N-nitro-L-arginine methyl ester, plus treatment with T-5224). Of these, the model control group and the model treatment group were modeled according to the following literature: Gabriele G. Schiattarella et al., Nitrosative stress drives heart failure with preserved ejection fraction, https: / / doi.org / 10.1038 / s41586-019-1100-z. Specifically, we induced heart failure with preserved ejection fraction using a high-fat diet (HFD) (60% kcal, derived from fat (lard)) and N-nitro-L-arginine methyl ester (abbreviated as L-NAME, 0.5 g / L in drinking water), thereby obtaining an animal model of HFpEF.

[0035] At five weeks of model induction, the systolic function parameter LVEF in mice did not change, but the diastolic parameter (E / E') significantly increased at five weeks of model induction, indicating that a heart failure model with preserved ejection fraction, as recorded in the aforementioned literature, was successfully obtained. At the same time, there was no significant difference in the diastolic parameter (E / E') between the model control group and the model treatment group at five weeks. Subsequent drug administration was performed at the same baseline, as shown in Figure 1.

[0036] 4. Jun expression was correlated with HFpEF. At 15 weeks of model induction, cardiomyocytes from the normal group and the model control group were separated by perfusion, and quantitative RCR detection was performed. The specific procedure was as follows:

[0037] 4.1. Isolation of adult mouse cardiomyocytes: To isolate cardiomyocytes from the hearts of adult mice, the classical perfusion method was used. Specifically, to prevent the heart from coagulating during surgery, which would increase the difficulty of digestion, 100 μL of heparin sodium (1000 units in 50 mL) was injected into the mice 20 minutes before euthanasia. The mice were then anesthetized and euthanized, the hearts were removed and washed in a calcium-free solution. Next, digestion was performed using the Langendorff method. Using a Langendorff apparatus, the hearts were perfused with a calcium-free solution for 5 minutes, and then digested with a digestive enzyme solution (calcium-free solution containing 0.7 mg / mL type II collagenase and 0.7 mg / mL bovine serum albumin) for approximately 30 minutes. The hearts were constantly touched for approximately 20 minutes. When the heart became soft and slippery, it indicated that digestion was basically complete, and then tissue was collected from the ventricles, finely dissected, and gently pipetted to separate into single cells. The cells were precipitated, the supernatant was collected to remove undigested and adherent tissue, and the cell was centrifuged at 100G at 4°C for 2 minutes to obtain a cardiomyocyte pellet. The supernatant consisted almost entirely of non-cardiomyocytes. The cardiomyocytes were resuspended in a calcium-free solution containing 10% FBS and used in subsequent experiments. The non-cardiomyocytes could be resuspended with culture medium or PBS and used in subsequent experiments. If purer cardiomyocytes and non-cardiomyocytes were required, the cell suspension could be separated by centrifuging three times (100G, 2 minutes at room temperature). Cardiomyocytes were collected for further experiments.

[0038] 4.2. Quantitative PCR detection: Total RNA was extracted from cells using the GeneJet RNA purification kit (Thermo Scientific, K0732), and 0.1 μg of total RNA was reverse transcribed using the iScript™ cDNA synthesis kit (Bio-Rad, 1708890) to generate cDNA. qPCR was performed using the iTaqUniversal SYBR Green supermix (1725121, Bio-Rad) on the ABI Vii7 real-time system (Life Technologies, Q6), and β-Actin was used for standardized quantitative analysis. As shown in Figure 2, significantly higher expression of Jun was observed in the mouse HFpEF animal model compared to normal mice. This indicates that Jun expression in mice correlates with HFpEF, suggesting that Jun is highly expressed in HFpEF.

[0039] 5. Method of administering T-5224 After obtaining animal models, i.e., from the fifth week after inducing the HFpEF model, the model treatment group was treated with T-5224, while the model control group was treated with a drug-free solvent. Mice that received a normal diet and drinking water throughout the entire induction process were used as negative controls. Mice were administered the drug when they were five weeks old. The treatment group received 250 mg / kg of T-5224 depending on the mouse's body weight. Specifically, administration was performed every other day, with 0.8 mg of T-5224 dissolved in 200 μL of 1% PVP solution each time. Administration began at the fifth week and ended at the thirteenth week (a total of 15 administrations), with a total dose of 250 mg / kg. The control group received the same volume of 1% PVP solution, and all other treatments were the same.

[0040] 6. Conventional echocardiography and Doppler imaging All mice were housed under different conditions for five weeks, after which conventional echocardiography and Doppler imaging were initiated every two weeks until the end of week fifteen. Specifically, transthoracic echocardiography was performed using a VisualSonics Vevo 2100 system with an MS400 transducer (Visual Sonics). Left ventricular ejection fraction (LVEF) and other systolic function indicators were obtained from short-axis M-mode scans at the mid-ventricular level, indicated by the presence of papillary muscle, in conscious, gently restrained mice. Apical four-chamber images of the heart were obtained in anesthetized mice and used for pulsed wave diastolic measurement and tissue Doppler imaging at the mitral valve level. Anesthesia was induced with 2.5% isoflurane and confirmed by no response to strong compression of one of the hind legs. During echocardiography acquisition (under temperature control), isoflurane was reduced to 1.0%–1.5% and adjusted to maintain a heart rate in the range of 500 beats / min. The parameters collected were heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, end-diastolic interventricular septal thickness, left ventricular end-diastolic posterior wall diameter, left ventricular shortening ratio, LVEF, peak Doppler blood flow velocity across the mitral valve in prediastole, peak Doppler blood flow velocity across the mitral valve in late diastole, isovolumetric relaxation time, tissue Doppler peak of myocardial relaxation velocity at the mitral annulus in prediastole, and early filling deceleration time. At the end of the procedure, all mice recovered from anesthesia without abnormalities. All parameters were measured at least three times, and mean values ​​were provided. Echocardiography and Doppler imaging were used to detect systolic and diastolic function.

[0041] 7. Experimental Results and Conclusions Firstly, when the contractile and diastolic functions of the mice were detected at five weeks, no significant changes were observed in contractile function, but the diastolic parameter E / E' increased significantly, demonstrating impaired diastolic function and showing that the model described in the aforementioned literature was successfully obtained. At the same time, with five weeks as the starting point for drug administration, there was no significant difference in cardiac diastolic function between the model control group and the model treatment group before drug administration (Figure 1A-C), and drug administration was carried out based on this.

[0042] Secondly, Jun expression was upregulated in the model control group compared to the normal group, indicating a correlation between Jun expression and HFpEF. In the HFpEF mouse model, Jun was highly expressed (Figure 2). Based on this correlation, we were able to conclude that Jun inhibitors can be used for the prevention and treatment of HFpEF.

[0043] Furthermore, the efficacy of the Jun inhibitor T-5224 in the prevention and treatment of HFpEF was investigated. After successful model construction and confirmation that the baselines of the model control group and the model treatment group were identical, the model treatment group was treated with T-5224. Cardiac function detection results showed that the onset and progression of HFpEF were sufficiently suppressed in the model treatment group (after T-5224 administration). Specifically, diastolic function in mice treated with T-5224 in combination with a high-fat diet and L-NAME (HFD + 0.5g / LL-NAME) was significantly improved and maintained until week 15. However, continuous deterioration of diastolic function was observed in model control mice that were not treated with T-5224 (Figure 3A-B). At the same time, Jun expression was downregulated in the model treatment group compared to the model control group (Figure 3D), and mouse obesity was improved (Figure 3C). This suggests that the Jun inhibitor T-5224 may have preventive and therapeutic effects on HFpEF in a mouse HFpEF model.

Claims

[Claim 1] A pharmaceutical composition for treating or preventing heart failure with preserved ejection fraction, comprising a Jun inhibitor, wherein the Jun inhibitor is selected from T-5224 or a pharmaceutically acceptable salt thereof.

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