Use of bufalin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating reperfusion arrhythmia

Abstract

The application belongs to the field of heart disease treatment and natural medicine compound, and provides application of bufalin or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating reperfusion arrhythmia. Experiments in vitro and in vivo prove that bufalin inhibits L-type calcium channels in a concentration-dependent manner, reduces extracellular calcium ion inflow, and further reduces the incidence of ventricular arrhythmia, arrhythmia, reduces ventricular conduction time, increases left ventricular conduction velocity, and reduces ventricular conduction dispersion; and provides a new drug selection for the treatment of reperfusion arrhythmia.

Description

Technical Field

[0001] This application pertains to the fields of cardiac treatment and natural drug compounds. Specifically, this application provides the use of bufotoxin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating reperfusion arrhythmias. Background Technology

[0002] Currently, the treatment of reperfusion arrhythmias mainly focuses on symptomatic and supportive care. Commonly used drugs include amiodarone and lidocaine, which can control symptoms to some extent, but their efficacy is greatly affected by the timing of administration and are often accompanied by adverse reactions such as the risk of proarrhythmias, negative inotropic effects, and extracardiac organ toxicity, making it difficult to meet the urgent clinical need for safe and highly effective therapeutic drugs. In recent years, with the continuous deepening of the modernization of traditional Chinese medicine, scientists have begun to focus on targeted therapy using natural drugs or their extracts.

[0003] Bufotalin is a sterene compound isolated from toad venom, with the molecular formula C2. 24 H 32 O6, CAS Registry Number 464-74-4, has the structure shown in Equation I:

[0004]

[0005] Formula I

[0006] Current research on sand bull toxin mainly focuses on its anti-tumor effects, and there are no reports of its use in treating reperfusion arrhythmias. Summary of the Invention

[0007] On the one hand, this application provides the use of bufotoxin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating reperfusion arrhythmias.

[0008] Furthermore, the reperfusion arrhythmia is myocardial infarction reperfusion arrhythmia.

[0009] Furthermore, the myocardial infarction reperfusion arrhythmia refers to reperfusion arrhythmia following myocardial infarction thrombolysis, coronary intervention, or surgery.

[0010] Furthermore, the reperfusion arrhythmia is a reperfusion ventricular arrhythmia.

[0011] Furthermore, the drug is in oral or injectable form.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the drug has one or more of the following effects:

[0014] (1) Reduce the incidence and frequency of ventricular arrhythmias;

[0015] (2) Reduce ventricular conduction time and increase left ventricular conduction velocity;

[0016] (3) Reduce ventricular conduction dispersion.

[0017] Furthermore, the drug inhibits L-type calcium channels in a concentration-dependent manner, reducing the influx of extracellular calcium ions.

[0018] On the other hand, this application provides a medicament for treating reperfusion arrhythmias, said medicament comprising bufotoxin or a pharmaceutically acceptable salt thereof.

[0019] Furthermore, bufotoxin or its pharmaceutically acceptable salt is the sole active ingredient in the drug.

[0020] The term "pharmaceutically acceptable salt" as used in this application refers to non-toxic salts formed by the compounds of this application through conventional pharmaceutical salt-forming methods, which are physiologically suitable for medicinal use. These include, but are not limited to, salts formed with inorganic acids such as phosphoric acid, sulfuric acid, and hydrochloric acid, or organic acids such as acetic acid, tartaric acid, citric acid, and malic acid, or with acidic amino acids; they can also be sodium, potassium, calcium, or ammonium salts formed with inorganic bases after esterification or amide formation with the aforementioned acids.

[0021] The arrhythmias described in this application include, but are not limited to, ventricular arrhythmias, atrial arrhythmias, sinus arrhythmias, atrioventricular junctional arrhythmias, and conduction blocks.

[0022] It should be noted that the reperfusion arrhythmia animal model constructed in this application is only a classic experimental model for research on diseases related to myocardial ischemia-reperfusion injury. In principle, any type of disease involving the restoration of myocardial blood supply (i.e., reperfusion) and thereby causing arrhythmia falls within the intended application scope of the technical solution provided by this invention.

[0023] The pharmaceutically acceptable excipients described in this application include, but are not limited to, solvents, solubilizers, suspending agents, diluents, antioxidants, preservatives, pH adjusters, osmotic pressure adjusters, coating agents, capsule shells, fillers, binders, disintegrants, lubricants, sustained-release agents, controlled-release agents, flavor masking agents, and taste agents. Those skilled in the art can use common pharmaceutical knowledge and the properties of bufotalin or its pharmaceutically acceptable salts to prepare various dosage forms, including but not limited to oral dosage forms such as tablets, capsules, and oral liquids, or injectable dosage forms such as water injections and powder injections. Attached Figure Description

[0024] Figure 1A This displays representative graphs of conduction time and conduction dispersion acquired by multichannel electrophysiological mapping in each group during ex vivo layer experiments.

[0025] Figure 1BThis displays representative electrocardiogram recordings and phase diagrams during reperfusion in each group during the ex vivo layer experiment.

[0026] Figure 1C This shows the incidence of spontaneous VT / VF within 30 minutes of reperfusion in an ex vivo layer experiment (n=6).

[0027] Figure 1D This shows the duration of spontaneous VT / VF within 30 minutes of reperfusion in an ex vivo layer experiment (n=6).

[0028] Figure 1E This shows the statistical results of left ventricular conduction time in the ex vivo layer experiment (n=6).

[0029] Figure 1F This shows the statistical results of conduction velocity in the ex vivo layer experiment (n=6).

[0030] Figure 1G This displays the statistical results of conduction dispersion in the ex vivo layer experiment (n=6).

[0031] Figure 2A The images show representative electrocardiogram recordings and phase diagrams under 50Hz stimulation in a body-level experiment.

[0032] Figure 2B Representative graphs showing left ventricular conduction time and conduction dispersion acquired by multi-channel electrophysiological mapping in each group during in vivo experiments.

[0033] Figure 2C The induction rate of ventricular arrhythmias under 50Hz stimulation in the in vivo experiment was shown (n=6).

[0034] Figure 2D The duration of ventricular arrhythmias under 50Hz stimulation in the in vivo experiment was shown (n=6).

[0035] Figure 2E The statistical results of left ventricular conduction time in the body-level experiment are shown (n=6).

[0036] Figure 2F The statistical results of conduction velocity in the bulk experiment are shown (n=6).

[0037] Figure 2G The statistical results of conduction dispersion in volumetric experiments are shown (n=6).

[0038] In the attached figure, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns indicates not significant. Detailed Implementation

[0039] Example 1: Preparation and treatment of animal models (in vitro)

[0040] Main instruments and materials:

[0041] SD rats: Male SD rats aged 6-8 weeks (220-240 g) were purchased from Beijing SPAF Biotechnology Co., Ltd.

[0042] Bufotoxin: Purchased from Chengdu Efa Biotechnology (CAS No.: 464-74-4), purity > 98%.

[0043] Amiodarone hydrochloride tablets: purchased from Sanofi (Hangzhou) Pharmaceutical Co., Ltd., product batch number: H31021872.

[0044] Construction of an isolated heart reperfusion arrhythmia model:

[0045] Male SD rats were injected intraperitoneally with heparin sodium (3125 U / kg) for 15 minutes, then anesthetized with isoflurane and euthanized by cervical dislocation. The heart was quickly removed via thoracotomy and immediately placed in pre-cooled oxygen-saturated modified Krebs-Henseleit (KH solution) buffer at 4°C for trimming. The aorta was then quickly connected to a perfusion needle, and retrograde perfusion was initiated using KH buffer saturated with an oxygen-mixed gas (95% O2 / 5% CO2) at 37°C. The rats were then transferred to a Langendorff perfusion system for retrograde perfusion at a rate of 10 ml / min. Once the heartbeat stabilized, a 6-0 suture was used to locate the left anterior descending branch below the left atrial appendage and ligated with a 2-0 suture slipknot. Successful ligation was indicated by weakened left ventricular contraction, cyanosis, and ST-segment elevation on lead II ECG. After 30 minutes of ischemia, the suture was released to restore perfusion.

[0046] Animal grouping and administration:

[0047] Rats were randomly divided into a control group, a model group, an amiodarone hydrochloride tablet (positive control) group, a high-dose group of bufotoxin, and a low-dose group, with 6 rats in each group. Control group (Sham): The left anterior descending artery was ligated but not ligated, and perfusion was continued for the same duration. Model group: The left anterior descending artery was ligated for 30 min, and then the ligation was released and perfusion was resumed for 30 min. High-dose bufotalin group (ARE-H): Bufotalin (final concentration 1.5 μg / mL) was added to the perfusion fluid before ligation of ischemia, and after 15 min of circulation perfusion, ischemia was performed for 30 min and reperfusion for 30 min. Low-dose bufotalin group (ARE-L): Bufotalin (final concentration 750 ng / mL) was added to the perfusion fluid before ligation of ischemia, and after 15 min of circulation perfusion, ischemia was performed for 30 min and reperfusion for 30 min. Positive drug group (AMI): Amiodarone (final concentration 10 μM) was added to the perfusion fluid before ligation of ischemia, and after 15 min of circulation perfusion, ischemia was performed for 30 min and reperfusion for 30 min.

[0048] Experimental indicators and results to be verified:

[0049] After stabilizing for 15 minutes, the heart returned to a normal rhythm for the experiment. Electrocardiogram (ECG) electrodes were carefully attached to the right atrium and left ventricle of the isolated heart. Two 64-channel pen electrodes were attached to the right and left ventricles for mapping, with the stimulation electrode placed at the apex. Spontaneous and frequency stimulation, S1S2 field potential signals, and ECG signals were recorded. Two cascaded EMS64-USB-1003 (MappingLab, UK) units were used to record the field potential signals of a normal heart. The recording software was EMapRecord 5.8.3 (MappingLab, UK), and the sampling frequency was 10 kHz.

[0050] Example 2: Preparation and treatment of animal models (in vivo)

[0051] Main instruments and materials:

[0052] SD rats: Male SD rats aged 6-8 weeks (220-240 g) were purchased from Beijing SPAF Biotechnology Co., Ltd.

[0053] Bufotoxin: Purchased from Chengdu Efa Biotechnology (CAS No.: 464-74-4), purity > 98%.

[0054] Amiodarone hydrochloride tablets: purchased from Sanofi (Hangzhou) Pharmaceutical Co., Ltd., product batch number: H31021872.

[0055] Construction of an in vivo cardiac reperfusion arrhythmia model:

[0056] Male SD rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (3.5 mL / kg), then intubated and connected to a small animal ventilator (respiratory rate 100 breaths / min, tidal volume 12.5 mL / kg, inspiratory-to-expiratory ratio 1:1). A thoracotomy was performed at the left third and fourth intercostal spaces to expose the heart. The pericardium was opened, and a 6-0 suture with a needle was passed through the left anterior descending branch below the root of the left atrial appendage, and ligated with a 2-0 suture. Successful ligation was indicated by ST segment elevation and left ventricular cyanosis on the electrocardiogram. After 30 minutes of ischemia, the ligation was carefully loosened to restore perfusion, and the chest was closed layer by layer. Postoperatively, penicillin sodium was administered intramuscularly to prevent infection, and the rats were kept warm until awake.

[0057] Animal grouping and administration:

[0058] Rats were randomly divided into a control group, a model group, an amiodarone hydrochloride tablet (positive control) group, a high-dose bufotalin group, a medium-dose group, and a low-dose group, with 6 rats in each group. Seven days prior to administration, rats were given different doses of the drug via gavage once daily. The sham-operated group (Sham) received only sutures without ligation; the model group (Model) received ligation of the left anterior descending artery (LAD) for 30 minutes, followed by reperfusion for 24 hours; the high-dose bufotalin group (ARE-H) received 8 mg / kg / day of bufotalin via gavage for 7 consecutive days; the medium-dose bufotalin group (ARE-M) received 4 mg / kg / day of bufotalin via gavage for 7 consecutive days; the low-dose bufotalin group (ARE-L) received 2 mg / kg / day of bufotalin via gavage for 7 consecutive days; and the positive control group (AMI) received 50 mg / kg / day of amiodarone via gavage for 7 consecutive days. After the last administration, the left anterior descending artery of the heart was ligated for 30 minutes, followed by ligation and reperfusion for 24 hours.

[0059] Experimental indicators and results to be verified:

[0060] Twenty-four hours after reperfusion, the rats were anesthetized again and connected to an in vivo ECG. Cardiac function was assessed using a small animal ultrasound imaging system, and the heart was exposed again by thoracotomy for multi-channel electrophysiological mapping, just as in the in vitro experiment. Conduction parameters at 6 Hz, S1-S2 refractory period, and arrhythmia induction under 50 Hz stimulation were recorded.

[0061] Statistical analysis:

[0062] All data were statistically analyzed using SPSS 27.0 software. Quantitative data are expressed as mean ± standard deviation (x̄ ± s). Mean ± standard deviation (S) is used. One-way ANOVA was used for comparisons among multiple groups. If variances were homogeneous, the LSD method was used for post-hoc multiple comparisons; if variances were unequal, the Dunnett T3 method was used. Independent samples t-tests were used for comparisons between two groups. Fisher's exact test was used to compare the incidence of cardiac arrhythmias. A p-value < 0.05 was considered statistically significant. Statistical graphs were created using GraphPad Prism 10.1.2 software.

[0063] Example 3: Experimental Results and Analysis

[0064] Figure 1A This diagram shows representative images of conduction time and conduction dispersion acquired by multi-channel electrophysiological mapping in each group during the ex vivo pleural model experiment. The pseudo-color image shows the propagation sequence of the excitation wave on the surface of the left ventricle; red represents the excitation initiation region, and blue represents the excitation delay region. Compared with the control group, the model group showed prolonged conduction time and increased dispersion; the high-dose bufotoxin group (ARE-H) and the positive control group (AMI) significantly shortened conduction time and reduced dispersion, indicating that the drugs improved electrical conduction synchronicity after ischemia-reperfusion.

[0065] Figure 1B The diagram shows representative electrocardiogram (ECG) recordings and phase diagrams during reperfusion in each group during the ex vivo layer experiment. Lead II ECG recordings showed typical ventricular tachycardia (VT) and ventricular fibrillation (VF) during reperfusion in the model group, with wide and abnormal QRS complexes and rhythm disturbances. Phase diagrams further show the propagation path of the electrical excitation wave on the ventricular surface; abnormal re-entrant excitation was observed in the model group. The high-dose bufotoxin group (ARE-H) and the positive control group (AMI) only experienced occasional premature ventricular contractions (PVCs) without persistent VT / VF; phase diagrams showed orderly excitation propagation.

[0066] Figure 1C The study showed the incidence of spontaneous VT / VF within 30 minutes of reperfusion in the ex vivo layer experiment. The incidence of VT / VF was 100% (6 / 6) in the model group, decreased to 16.7% (1 / 6) in the high-dose bufotalin group (ARE-H), 33.3% (2 / 6) in the low-dose group (ARE-L), and 50% (3 / 6) in the positive control group (AMI). Compared with the model group, P<0.05, **P<0.001, indicating that bufotalin dose-dependently reduces the risk of reperfusion arrhythmias.

[0067] Figure 1DThis study shows the duration of spontaneous VT / VF within 30 minutes of reperfusion in the ex vivo layer experiment. Compared with the control group, the VT / VF duration was significantly prolonged (>15 min) in the model group, while the VT / VF duration was significantly shortened in the high-dose bufotoxin group (ARE-H) and the positive control group (AMI). Compared with the control group, P<0.001; compared with the model group, P<0.05, *P<0.001.

[0068] Figure 1E The left ventricular field potential conduction time was recorded under 6 Hz stimulation. The conduction time in the model group was significantly prolonged compared to the control group (P<0.001), suggesting that myocardial ischemia-reperfusion injury leads to slowed electrical conduction. The high-dose bufotoxin group (ARE-H) significantly shortened the conduction time (*P<0.05 compared to the model group), while the low-dose group (ARE-L) and the positive control group (AMI) showed a shortening trend, but the difference was not statistically significant. ANOVA: P<0.01.

[0069] Figure 1F The results of conduction velocity statistics in the ex vivo layer experiment are shown. The conduction velocity in the model group was significantly slower than that in the control group, while the high-dose group of bufotoxin (ARE-H) significantly increased the conduction velocity (*P<0.05 compared with the model group), indicating that the drug improved the electrical conduction function of the myocardium after ischemia-reperfusion.

[0070] Figure 1G The statistical results of conduction dispersion in the ex vivo layer experiment are shown. The dispersion in the model group was significantly greater than that in the control group (P<0.001), suggesting that ischemia-reperfusion leads to increased spatial heterogeneity of electrical activity, providing a matrix for reentry formation. The dispersion was significantly reduced in the high-dose group (ARE-H), low-dose group (ARE-L), and positive control group (AMI) compared with the model group (**P<0.01), indicating that the drug can improve the spatial homogeneity of myocardial electrical activity.

[0071] Figure 2A The images show representative electrocardiogram (ECG) recordings and phase diagrams under 50 Hz stimulation in the in vivo experiments. Twenty-four hours after reperfusion, arrhythmias were induced using 50 Hz high-frequency stimulation. The model group immediately exhibited persistent VT / VF after stimulation, with ECGs showing wide and abnormal QRS complexes, and phase diagrams showing reentry excitation. The high-dose bufotoxin group (ARE-H) and the positive control group (AMI) showed only short runs of premature ventricular contractions or no significant arrhythmias, and phase diagrams showed orderly electrical activity.

[0072] Figure 2BRepresentative graphs of left ventricular conduction time and conduction dispersion acquired by multi-channel electrophysiological mapping in each group during in vivo experiments are shown. Consistent with the results of in vitro experiments, the model group showed prolonged left ventricular conduction time and increased dispersion in the in vivo heart, with pseudo-color images indicating uneven propagation of the excitation wave. All dose groups of sand frog toxin and the positive control group improved conduction parameters to varying degrees, with the high-dose group showing the most significant effect.

[0073] Figure 2C This study showed the induction rate of ventricular arrhythmias under 50Hz stimulation in a somatic experiment. The induction rate was 100% (6 / 6) in the model group, decreased to 16.7% (1 / 6) in the high-dose bufotoxin group (ARE-H), 33.3% (2 / 6) in the medium-dose group (ARE-M), 66.7% (4 / 6) in the low-dose group (ARE-L), and 33.3% (2 / 6) in the positive control group (AMI). Compared with the model group, P<0.05, **P<0.001, ****P<0.0001, indicating that bufotoxin dose-dependently reduces cardiac susceptibility to high-frequency stimulation.

[0074] Figure 2D The duration of ventricular arrhythmias under 50 Hz stimulation was shown in the in vivo experiments. The model group had the longest VT / VF duration (>20 s), while the high-dose group (ARE-H), medium-dose group (ARE-M), and positive control group (AMI) all significantly shortened the VT / VF duration. Compared with the model group, ***P<0.001.

[0075] Figure 2E Left ventricular conduction time was recorded under 6 Hz stimulation of the heart in vivo. The model group showed a significantly longer conduction time than the control group (P<0.01). The high, medium, and low dose groups of bufotalin and the positive control group all significantly shortened the conduction time (**P<0.01 compared with the model group), indicating that bufotalin can also improve electrical conduction function after ischemia-reperfusion in vivo.

[0076] Figure 2F The statistical results of conduction velocity in the in vivo experiment are shown. The conduction velocity in the model group was significantly slower than that in the control group. The conduction velocity was increased to varying degrees in all dose groups of sand bufotoxin and the positive control group, and the differences were statistically significant compared with the model group (*P<0.05, **P<0.01).

[0077] Figure 2G The statistical results of conduction dispersion in the in vivo experiment are shown. It can be seen that the dispersion of the model group is significantly increased compared with the control group (P<0.05). The high-dose group of bufotoxin (ARE-H) and the positive control group (AMI) can significantly reduce the dispersion (compared with the model group, *P<0.05). The medium and low dose groups show a decreasing trend, but the difference is not statistically significant.

Claims

1. The use of bufotalin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating reperfusion ventricular arrhythmias; wherein the reperfusion ventricular arrhythmias are myocardial infarction reperfusion ventricular arrhythmias; wherein the myocardial infarction reperfusion ventricular arrhythmias are reperfusion ventricular arrhythmias following myocardial infarction thrombolysis, coronary intervention or surgery.

2. The application according to claim 1, wherein the drug is an oral or injectable dosage form.

3. The application according to claim 2, wherein the medicament further comprises pharmaceutically acceptable excipients.

4. According to claim 1, the drug has one or more of the following effects: (1) Reduce the incidence and frequency of ventricular arrhythmias; (2) Reduce ventricular conduction time and increase left ventricular conduction velocity; (3) Reduce ventricular conduction dispersion.

5. In the application according to claim 1, the drug inhibits L-type calcium channels in a concentration-dependent manner, reducing the influx of extracellular calcium ions.

Images