CN111329860A

CN111329860A - Application of Vanoxeine in medicine for treating persistent atrial fibrillation

Info

Publication number: CN111329860A
Application number: CN201910155431.8A
Authority: CN
Inventors: 张之颢; 付声姣
Original assignee: Hangzhou Jianshi Pharmaceutical Co ltd
Current assignee: Hangzhou Jianshi Pharmaceutical Co ltd
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2020-06-26

Abstract

The invention discloses an application of Vanoxeine in a medicine for treating persistent atrial fibrillation, and experiments show that a medicine VX can prolong the activation time (ACT) of acetylcholine (Ach) induced atrial fibrillation, prolong the Effective Refractory Period (ERP) time interval and improve the threshold value (AFT) of atrial fibrillation in a concentration-dependent manner; in addition, the medicine has stronger time-dependent inhibition effect on potassium ion channels of atrial fibrillation targets Kv1.5 (Ikur); in addition, VX had no significant effect on IK1 and IKs (both IC50 were greater than 300 μ M), but had inhibitory effects on hERG (IC 50=54.07 nM), Nav1.5 (IC 50=1.95 μ M), and Cav1.2 (IC 50=11.1 μ M), which were mutually offset by some single ion channel drug blocking effect, and it was found that VX at appropriate doses only slightly prolonged APD90 and had no or slight frequency dependence. It is shown that VX can effectively inhibit persistent atrial fibrillation at appropriate concentrations and does not induce ventricular arrhythmia due to prolonged ventricular APD90 and excessively prolonged QTc.

Description

Application of Vanoxeine in medicine for treating persistent atrial fibrillation

Technical Field

The invention belongs to the field of research and development of new drugs, and particularly provides a novel drug Vanoxeine (VX) capable of inhibiting persistent atrial fibrillation, and an action mechanism of the drug Vanoxeine (VX) is explained.

Background

Atrial Fibrillation (AF) and Atrial Flutter (AFL) are a global epidemic of arrhythmia disease that is growing as the world population ages, especially over the age of 40. According to the forecast about closing the door, the population with atrial fibrillation in the United states will reach 560 million people and China will reach 2000 million people by 2050. Therefore, huge market development space is brought, and the attention of a plurality of pharmaceutical factories is attracted.

There are three types of atrial fibrillation, one is paroxysmal atrial fibrillation, which is characterized by sudden increase of heartbeat, the duration time is within a week, usually not more than 24 hours, and the atrial fibrillation can be automatically stopped; secondly, the atrial fibrillation is sustained, the duration of abnormal heartbeat exceeds one week, the heartbeat rarely returns to normal automatically, and the normal state can be recovered only by treatment; and thirdly, permanent atrial fibrillation, which still cannot recover normal heartbeat after treatment, and can become permanent as paroxysmal atrial fibrillation and persistent atrial fibrillation along with the progress of diseases. Atrial fibrillation has two major hazards, causing cerebral stroke and heart failure. When the atrial fibrillation lasts for more than 48 hours, thrombus is formed in an atrium, and when the normal heartbeat is recovered, the thrombus is easy to fall off, so that stroke is caused.

Until now, there has been no satisfactory drug or surgical treatment to restore Normal Sinus Rhythm (NSR) for the time being, for the number of people who need acute treatment and relapse prevention, drugs for treating atrial fibrillation, which are generally administered from the group consisting of controlling ventricular rate, restoring and maintaining sinus rhythm, and anticoagulation therapy, wherein the common drugs for controlling ventricular rate include atenolol, metoprolol, nadolol, propranolol, sotalol, diltiazem, verapamil, and digitalis drugs such as digoxin, which are β receptor blockers, and the drugs for restoring and maintaining sinus rhythm include amiodarone, propafenone, flecainide, and the like, and the drugs for anticoagulation therapy include warfarin, dabigatran, rivaroxaban, apixaban, andxaban, and the like.

Kv1.5 (Ikur) overspeed delay essential potassium channel current is widely expressed in atria, and this channel plays an important role in atrial repolarization, involving phases I and II of atrial repolarization, affecting the normal rhythm and frequency of the atria by affecting the atrial myocyte Action Potential Duration (APD) and the Effective Refractory Period (ERP), which is largely open in AF patients, and thus Kv1.5 becomes an effective and hot ion channel target for the treatment of atrial fibrillation.

Vanoxerine (VX, 1- [2- [ bis (4-fluorophenyl) methoxy ] ethyl ] -4- (3-phenylpropyl) piperazine dihydrochloride) is an inhibitor of dopamine receptor, has a molecular formula of C14H11N4O4Cl, and has a structural formula shown in the following figure. Drugs have been used to treat parkinson's disease and depression, but the clinical effects are unclear. The compounds all have inhibition effects on hERG, hCav1.2 and hNav1.5, but can mutually offset the blocking effect of a single ion channel drug under the condition of proper administration dosage. VX has a similar stimulus potential for currents that block the outward fast repolarization IKr produced by the hERG channel and low threshold inward currents produced by the hcav1.2 ion channel; and much lower for INa, so that VX does not cause arrhythmia in normal patients at a given dose. Appropriate doses of VX have been reported in the literature to be safe and effective in converting AF/AFL patients to Normal Sinus Rhythm (NSR).

Vanoxeine structural formula

Disclosure of Invention

In order to solve the problems, the invention discloses application of Vanoxerine in the medicine of the persistent atrial fibrillation.

To better understand the essence of the present invention, the pharmacological experiments and results of Vanoxeine (VX) are presented below to demonstrate a novel effect in the treatment of the inhibition of persistent atrial fibrillation.

The invention aims to provide application of Vanoxeine (VX) in treatment and inhibition of persistent atrial fibrillation.

The adopted technical scheme is that the change of each parameter of the hybrid dog with atrial fibrillation induced by processing acetylcholine by VX is observed; (ii) inhibition of Kv1.5 ion channel by VX; effect of VX on hERG, nav1.5, IKs, IK1 and cav1.2, and on rabbit cattail fiber action potential.

Experiments show that the drug VX can prolong the activation time (ACT) of acetylcholine (Ach) induced atrial fibrillation, prolong the Effective Refractory Period (ERP) time and improve the threshold value (AFT) of atrial fibrillation in a concentration-dependent manner; in addition, the medicine has stronger time-dependent inhibition effect on potassium ion channels of atrial fibrillation targets Kv1.5 (Ikur); in addition, VX had no significant effect on IK1 and IKs (both IC50 were greater than 300 μ M), but had inhibitory effects on hERG (IC 50=54.07 nM), Nav1.5 (IC 50=1.95 μ M), and Cav1.2 (IC 50=11.1 μ M), which were mutually offset by some single ion channel drug blocking effect, and it was found that VX at appropriate doses only slightly prolonged APD90 and had no or slight frequency dependence. It is shown that VX can effectively inhibit persistent atrial fibrillation at appropriate concentrations and does not induce ventricular arrhythmia due to prolonged ventricular APD90 and excessively prolonged QTc.

At appropriate concentrations, VX is effective in treating persistent atrial fibrillation without inducing arrhythmia due to prolongation of APD90 and excessive prolongation of QTc, and has an effective effect in inhibiting persistent atrial fibrillation.

Drawings

FIG. 1 is a waveform of Atrial Fibrillation (AF) induction;

FIG. 2 is a graph of 3 μ MVX acting on acetylcholine (Ach) -induced AF;

FIG. 3 is a waveform of the effect of VX on ERP in atrial fibrillation;

FIG. 4 is a graph of IC50 versus current for VX blocking Kv1.5;

FIG. 5 is a graph of IC50 versus current for VX blocking hERG;

FIG. 6 is a graph of IC50 versus current for VX blocking Cav1.2;

FIG. 7 is a graph of IC50 versus current for VX blocking hNav1.5;

FIG. 8 is a graph of IC50 versus current for VX blocking IK 1;

FIG. 9 is a graph of IC50 versus current for VX blocking IKs;

FIG. 10 is a graph of the effect of VX blockade on the action potential of calamus fibers;

FIG. 11 is a graph of the effect of VX on ACT, ERP, AFT values;

FIG. 12 is a graph of the effect of VX on rabbit cattail fibers APD60 and APD 90.

Detailed Description

For the purpose of enhancing the understanding of the present invention, the following detailed description of the present invention will be given in conjunction with examples and drawings, which are provided for the purpose of illustration only and are not intended to limit the scope of the present invention.

The experiment included the following steps:

example 1: hybrid dogs with acetylcholine-induced atrial fibrillation were perfused with VX.

Anaesthetizing hybrid dogs with sodium pentobarbital, performing tracheal intubation, opening the chest cavity, fixing four electrodes on a high left/right atrium and a low left/right atrium respectively, determining a pacing threshold, stimulating by 2 times of the pacing threshold, recording a basic state and a pacing atrial map by a multi-conductive physiological instrument, continuously stimulating by 8 basic stimulators with a stimulation period of 200ms (8S 1 pulse) by a program stimulator, stimulating by 50 Hz high frequency for 1 sec (50 Hz × 1 sec), adding 0.1 mu M of acetylcholine (Ach) into coronary arteries, stimulating and inducing Atrial Fibrillation (AF) of the hybrid dogs by an S1S2 program, then perfusing VX with different concentrations, and recording normal and non-stimulated atrial waveforms (normal), induced and successful atrial fibrillation waveforms, ACT, ERP duration and AFT threshold voltage respectively;

experimental results as shown in fig. 1-3 and fig. 11, when VX was added at various concentrations, it was found that the drug extended the duration of ACT and ERP, and increased the AFT value for induction of atrial fibrillation, in a concentration-dependent increase. Particularly, when the concentration of the medicine is more than 0.3 mu M, ACT and ERP can be obviously prolonged, the AFT value is improved, and VX can effectively inhibit atrial fibrillation.

Example 2: different concentrations of VX-treated hkv1.5 (IKur) potassium channels were recorded using the whole-cell patch clamp technique.

The method comprises the following specific steps: culturing HEK-293 cells of over-expression human Kv1.5 potassium ion channels by adopting 83% DMEM, 15% fetal calf serum, 1% non-essential amino acid and 1% 100x penicillin-streptomycin, depolarizing the cells to 0mv/500ms by using a standard whole-cell patch clamp technical recording method to activate the Kv1.5 (IKur) ion channels, then clamping the cells back to-80 mv/s, wherein the stimulation frequency is 10s once, and the maximum current recorded at 0mv is the current amplitude of the Kv1.5 channels to be recorded;

the experimental results are shown in fig. 4, which contains IC50 fitting curve and current curve graph of VX inhibiting kv1.5, from which VX is found to be able to inhibit kv1.5 channel at low dose and the inhibition is time dependent, the effect is stronger with increasing action time. Because Kv1.5 is one of the targets for treating atrial fibrillation, the time-dependent inhibition effect of VX on Kv1.5 further indicates that VX has the effect of treating persistent atrial fibrillation.

Example 3: different concentrations of VX-treated hERG, IK1 (Kir2.1), hNav1.5, IKs (KvLQT 1/minK), Cav1.2 ion channels were recorded using whole-cell patch clamp techniques.

The method comprises the following specific steps: over-expressing human hERG potassium channel HEK-293 cells were cultured with 84% DMEM, 15% fetal bovine serum and 1% 100x penicillin-streptomycin, 89% DMEM/F12, 10% fetal bovine serum, 1% 100x penicillin-streptomycin and G418, over-expressing human hNav1.5 potassium channel HEK-293 cells were cultured, and cell membrane currents were recorded using standard whole-cell patch clamp techniques. The hERG recording method is to depolarize the cell to 40mv/5ms to activate hERG ion channel, the cells were then clamped back to-50 mv/s to record the inactivation of the channel, with a stimulation frequency of 15s once, the maximum current recorded at-50 mv was the hERG channel current amplitude to be recorded, the hNav1.5 recording program was to depolarize the cell to-10 mv/100ms to activate the hNav1.5 ion channel, then clamping the cells back to-130 mv/10ms, clamping for 8s at-80 mv, then clamp back to-130 mv/5ms, depolarize the cell to-10 mv/100ms again to activate hNav1.5 ion channel, finally clamp back to-130 mv, stimulation frequency is once for 30s, the maximum inward current recorded at-10 mv of two clamping is the Nav1.5 channel current amplitude to be recorded.

For cells recording IK1 (Kir2.1), IKs (KvLQT 1/minK), Cav1.2 ion channel currents from acutely isolated guinea pig cells, the cells were prepared as follows: rats were anesthetized intraperitoneally (250-. The aorta is reversely perfused at 36-37 ℃ and the normal Taiwanese's fluid saturated by 100% oxygen is perfused for 3-5 minutes, so that the hematocele in the cardiac muscle and the ventricular cavity is discharged as completely as possible. Then, the heart is perfused with the calcium-free Taiwanese liquid until the heart is completely relaxed, and the beating is stopped. Then changing the perfusion of the calcium-free Taiwanese liquid containing 0.2 mg/ml protease and 1.0 mg/ml collagenase for 4-6 minutes until the myocardium is soft. Finally, the perfusion was changed to modified KB solution for 5 minutes. The ventricular muscle was cut, minced in KB fluid and blown repeatedly to separate the cells. After filtration and precipitation, the cells were sequentially re-calcified with 0.3, 0.6, 1 mM CaCl2 in Taiwan's solution. Isolated cardiomyocytes were stored in calcium-containing taiwanese fluid at room temperature for subsequent electrophysiological experiments. Depolarizing the cell to 15mv/200ms to activate Cav1.2 ion channel, clamping the cell back to-40 mv/s, stimulating once at 5s frequency, and recording the maximum inward current at 15mv as the current amplitude of Cav1.2 channel to be recorded; polarizing the cell to-120 mv/500ms to activate the IK1 (Kir2.1) ion channel, then clamping the cell back to-80 mv/s, stimulating once at 2s, and recording the maximum inward current at-120 mv, which is the current amplitude of the IK1 channel to be recorded; the cell is depolarized to 60mv/5000ms to activate the IKs (KvLQT 1/minK) ion channel, then the cell is clamped back to-40 mv/s, the stimulation frequency is once for 10s, and the maximum inward current recorded at 60mv is the current amplitude of the IKs channel to be recorded.

The experimental results are shown in FIGS. 5-9, VX has inhibition effects on hERG (IC 50=54.07 nM), Nav1.5 (IC 50=1.95 μ M) and Cav1.2 (IC 50=11.1 μ M), especially on hERG, and can effectively inhibit the channel on nanomolar scale. The inhibition effect on the three ion channel is also positive, but the inhibition effect on a single ion channel drug can be mutually counteracted at the proper dosage, and in addition, VX has no obvious effect on IKs and IK1, and the IC50 is larger than 300 mu M.

Example 4: the action potential of VX-treated rabbit cattail fiber was recorded.

The method comprises the following specific steps: separating adult rabbit cattail fibers, sequentially and continuously perfusing VX liquid medicines with different concentrations on the cattail fibers for 15 minutes from low to high concentration, fixing the prepared tissue of the cattail fibers in a recording bath, controlling the perfusion speed to be 5-6 ml/min, keeping the temperature in the bath at 36 +/-1 ℃ through circulating water in a water bath kettle, and recording the action potential of the rabbit cattail fibers by adopting stimulation frequencies of 0.5, 1 and 2 HZ.

The experimental results are shown in fig. 10 and fig. 12, and when VX acts on the action potential of rabbit cattail fibers, the compound has no effect on APD60 and APD90 at the concentration of less than 0.3 mu M and has no obvious frequency dependence when the compound acts on 0.5HZ and 1HZ in normal physiological or pathological transition states of human beings. APD60 and APD90 with rabbit-pump fibers at 0.3-1 μ M had a mild effect and a slight frequency dependence.

In conclusion, it is found through experiments that drug VX can prolong the activation time (ACT) in acetylcholine (Ach) induced atrial fibrillation, prolong the Effective Refractory Period (ERP) time course and improve the Atrial Fibrillation Threshold (AFT) in a concentration-dependent manner; in addition, the medicine has stronger time-dependent inhibition effect on potassium ion channels of atrial fibrillation targets Kv1.5 (Ikur); in addition, VX had no significant effect on IK1 and IKs (both IC50 were greater than 300 μ M), but had inhibitory effects on hERG (IC 50=54.07 nM), Nav1.5 (IC 50=1.95 μ M), and Cav1.2 (IC 50=11.1 μ M), which were mutually offset by some single ion channel drug blocking effect, and it was found that VX at appropriate doses only slightly prolonged APD90 and had no or slight frequency dependence. It is shown that VX can effectively inhibit persistent atrial fibrillation at appropriate concentrations and does not induce ventricular arrhythmia due to prolonged ventricular APD90 and excessively prolonged QTc.

Claims

Use of vanoxerin in the treatment of persistent atrial fibrillation.