阅读说明：本技术 一种高精度离子通道评估心脏安全风险的优化方法 (Optimization method for evaluating heart safety risk through high-precision ion channel ) 是由 张之颢 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种高精度离子通道评估心脏安全风险的优化方法,本发明的实验方法及流程接近人体心脏活动规律,简单及与临床相关系好,便于在实际应用中的推广和使用,既可以便于对临床使用前的药物进行TdP风险的确认,同时也可避免由于假阳性检测结果的出现而致使药品研发厂商的巨大损失,同时可以减轻国家和医疗机构的不必要医疗开支。(The invention discloses an optimization method for evaluating heart safety risk by a high-precision ion channel, the experimental method and the process of the invention are close to the heart activity rule of a human body, are simple and have good clinical correlation, are convenient to popularize and use in practical application, can not only facilitate the confirmation of TdP risk of the medicine before clinical use, but also avoid the huge loss of medicine research and development manufacturers caused by the occurrence of false positive detection results, and can also reduce the unnecessary medical expenses of the country and medical institutions.)

1. An optimization method for evaluating heart safety risk by using a high-precision ion channel is characterized by comprising the following steps: the method comprises the following steps:

cardiac potassium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, the hERG ion channel of the heart is activated by a voltage method, the duration of a +40mV depolarization voltage is about 500 ms, the depolarization voltage is repeated once every 5 s, the duration of a voltage repolarization stage is 100 ms and ranges from +40mV to-80 mV, so that Ramp is formed, the voltage is changed to-1.2V/s, a hyperpolarization small pulse of a previous-period voltage ranging from-80 mV to-90 mV is used for calculating input resistance according to ohm's law, and the maximum positive current recorded during Ramp is the current amplitude of the hERG channel to be recorded;

step 2: drug evaluation effect, continuous recording until stable pre-and post-dosing hERG current is obtained, and cells remain stable, as determined by current maintained at-80 mV and input resistance, each cell recommends perfusing two concentrations of drug, testing at least 4 concentrations for each drug, and the percent current inhibition associated with these concentrations ranges from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac calcium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, a Cav1.2 calcium ion channel of the heart is activated by a voltage method, the duration time of a +30 mV depolarization voltage is about 200 ms, the depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization phase is 100 ms, the voltage is from +30 mV to-80 mV, so that Ramp is formed, the voltage is changed to-1.1V/s, a hyperpolarization small pulse of a previous-period voltage of-80 to-90 mV is used for calculating input resistance according to ohm's law, and the maximum negative current recorded during Ramp is the current amplitude of the calcium channel to be recorded;

step 2: in drug evaluation, the effect was continuously recorded until a stable Cav1.2 current before and after dosing was obtained and the cells remained stable, as determined by the current held at-80 mV and the input resistance, two concentrations of drug were perfused per cell, at least 4 concentrations were tested for each drug, and the percent inhibition of current associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac late sodium channel:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, a voltage method is used for activating a late sodium ion channel of the heart, the duration time of a +40mV depolarization voltage is about 200 ms, the depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization stage is 100 ms, the voltage repolarization stage ranges from +40mV to-95 mV, Ramp is formed, the voltage is changed to-1.35V/s, a hyperpolarization small pulse with the early voltage ranging from-95 mV to-120 mV is used for calculating input resistance according to ohm's law, and the maximum negative current recorded during Ramp is the current amplitude of the late sodium ion channel to be recorded;

step 2: in drug evaluation, the effect was continuously recorded until a steady late sodium current before and after dosing was obtained and the cells remained stable, as determined by the current maintained at-80 mV and the input resistance, two concentrations of drug were perfused per cell, at least 4 concentrations were tested for each drug, and the percent current inhibition associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50.

Technical Field

The invention relates to the technical field of heart safety evaluation of medicines in new medicine research and development, in particular to an optimization method for evaluating heart safety risk by using a high-precision ion channel.

Background

The prolongation of QTc by drugs, and hence the resultant arrhythmia, is known as torsades de Point TdP. Clinical data show that TdP due to QT prolongation is the leading cause of drug market ban and restrictions. Due to the rarity and danger of the risk in clinic, European, American, Japanese and other government offices have issued corresponding preclinical guidelines. Because the probability of generation of TdP is very low and cannot be detected in most clinical experiments, the current guidelines for cardiac safety assessment S7B and CFDA in china predict the risk of occurrence of TdP clinically using indirect indicators before clinical practice. The indexes are mainly hERG, QTc and other main ion channels on the heart including 8-10 ion channels such as Na channels, Ca channels, K channels and the like, and action potential, in-vitro heart QTc and the like, wherein the hERG and the in-vivo Qtc of the dog are one of the data which must be provided by new drug clinical application at present.

Since the assessment of the safety of the heart of a new drug preclinical is of great importance in relation to the safety of patients, G7 developed countries have started to execute the S7B regulation for the assessment of heart safety, which is jointly established by drug regulatory agencies in the european union, japan and the usa and large pharmaceutical companies, in 2005, with enormous profits. The preclinical heart safety assessment of the drug greatly reduces the side effect of the new drug on the heart, and enables European and American pharmaceutical companies to save more than 150 billion dollars of research and development cost each year. Over time, the S7B regulation will become increasingly accepted by the drug administration and pharmaceutical companies of various countries.

China is a growing developing country, the biopharmaceutical industry has achieved a rapid achievement in the last decade and gradually shortens the gap with the western countries, and the execution of the S7B regulation from now on opens a convenient door for the medicines in China to move to the world, and especially plays a very key role in the utilization, development and clinical application of traditional Chinese medicines in China. The taking and injection of traditional Chinese medicine are the biggest characteristics of the medicine in China, but the traditional Chinese medicine injection is often reported to have serious adverse reactions, and many of the adverse reactions are complications caused by arrhythmia.

However, over 10 years of extensive data and experience have shown that defects in early preclinical cardiac evaluation designs or direct neglect of cardiac evaluation can have very serious consequences, which directly result in a ten-fold or one-hundred-fold economic loss, which is irretrievable in many cases. The compound has the potential risk of heart, and the lead compound can greatly increase the research and development resource requirements, including a large amount of time, cost and the like. Meanwhile, the risk of the heart evaluation directly leads to the blurring of the benefit of the medicine so that the risk is greater than the benefit. The risk can directly lead to use warnings of new drugs. These all correspondingly reduce the competitive power of new drugs after marketing. The research and development of new drugs for small and medium enterprises can directly lead to the reduction of compound value, increase the difficulty of financing and searching for partners. The most common result is that cardiac risk of the drug directly leads to the termination of development, a result that is costly for the company. Conversely, if the experimental design is not rigorous, false positives will increase and a compound with good potential will be discarded, and the loss to the company is immeasurable.

Disclosure of Invention

The invention aims to provide an optimization method for evaluating the heart safety risk by using a high-precision ion channel.

The technical scheme adopted by the invention is as follows:

an optimization method for evaluating heart safety risk by using a high-precision ion channel is characterized by comprising the following steps: the method comprises the following steps:

cardiac potassium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, the hERG ion channel of the heart is activated by a voltage method, the duration of a +40mV depolarization voltage is about 500 ms, the depolarization voltage is repeated once every 5 s, the duration of a voltage repolarization stage is 100 ms and ranges from +40mV to-80 mV, so that Ramp is formed, the voltage is changed to-1.2V/s, a hyperpolarization small pulse of a previous-period voltage ranging from-80 mV to-90 mV is used for calculating input resistance according to ohm's law, and the maximum positive current recorded during Ramp is the current amplitude of the hERG channel to be recorded;

step 2: drug evaluation effect, continuous recording until stable pre-and post-dosing hERG current is obtained, and cells remain stable, as determined by current maintained at-80 mV and input resistance, each cell recommends perfusing two concentrations of drug, testing at least 4 concentrations for each drug, and the percent current inhibition associated with these concentrations ranges from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac calcium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, a Cav1.2 calcium ion channel of the heart is activated by a voltage method, the duration time of a +30 mV depolarization voltage is about 200 ms, the depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization phase is 100 ms, the voltage is from +30 mV to-80 mV, so that Ramp is formed, the voltage is changed to-1.1V/s, a hyperpolarization small pulse of a previous-period voltage of-80 to-90 mV is used for calculating input resistance according to ohm's law, and the maximum negative current recorded during Ramp is the current amplitude of the calcium channel to be recorded;

step 2: in drug evaluation, the effect was continuously recorded until a stable Cav1.2 current before and after dosing was obtained and the cells remained stable, as determined by the current held at-80 mV and the input resistance, two concentrations of drug were perfused per cell, at least 4 concentrations were tested for each drug, and the percent inhibition of current associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac late sodium channel:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, a voltage method is used for activating a late sodium ion channel of the heart, the duration time of a +40mV depolarization voltage is about 200 ms, the depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization stage is 100 ms, the voltage repolarization stage ranges from +40mV to-95 mV, Ramp is formed, the voltage is changed to-1.35V/s, a hyperpolarization small pulse with the early voltage ranging from-95 mV to-120 mV is used for calculating input resistance according to ohm's law, and the maximum negative current recorded during Ramp is the current amplitude of the late sodium ion channel to be recorded;

step 2: in drug evaluation, the effect was continuously recorded until a steady late sodium current before and after dosing was obtained and the cells remained stable, as determined by the current maintained at-80 mV and the input resistance, two concentrations of drug were perfused per cell, at least 4 concentrations were tested for each drug, and the percent current inhibition associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50.

The invention has the advantages that: the experimental method and the flow are close to the heart activity rule of the human body, are simple and have good clinical relation, are convenient to popularize and use in practical application, can not only facilitate the confirmation of TdP risk of the medicine before clinical use, but also avoid the huge loss of medicine research and development manufacturers due to the occurrence of false positive detection results, and can also reduce the unnecessary medical expenses of the country and medical institutions.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic diagram of the voltage method in step 1 of the cardiac potassium channel of the present invention;

FIG. 2 is a graph of the drug test protocol recorded during step 2 of the cardiac potassium channel of the present invention;

FIG. 3 is a graph of the results of the drug assay analysis in step 2 of the cardiac potassium channel of the present invention;

FIG. 4 is a schematic diagram of the voltage method in step 1 of the cardiac calcium channel of the present invention;

FIG. 5 is a graph of the drug testing protocol during step 2 of the cardiac calcium channel of the present invention;

FIG. 6 is a graph showing the results of the drug assay in step 2 of the cardiac calcium channel of the present invention;

FIG. 7 is a schematic diagram of the voltage method in step 1 of the cardiac late sodium channel of the present invention;

FIG. 8 is a graph of the drug test protocol during step 2 of the cardiac late sodium channel of the present invention;

FIG. 9 is a graph of the results of the drug assay analysis in step 2 of the cardiac calcium channel of the present invention.

Detailed Description

The test method for evaluating arrhythmia caused by the action of a drug on a cardiac ion channel is a more comprehensive and more accurate innovative scheme of the current internationally commonly used method, and comprises the steps of stably transferring a human body hERG channel, Cav1.2 and late sodium Nav1.5 to HEK293 cells, recording the kinetic influence of a compound on the ion channel, analyzing and evaluating big data, and carrying out the following steps:

cell culture: the cell line is derived from HEK-293 cells and 5% CO at 37 ℃₂Culturing in an incubator. When the cell density reaches 80%, the cells are pre-washed by phosphate buffer, then digested by trypsin/EDTA, subcultured, and the cell density is 2X 10⁶One cell/dish. The culture medium comprises the following components: DMEM, 15% fetal bovine serum and 1% penicillin.

The experimental quality is guaranteed: the channel current was recorded using a whole-cell recording mode, with experimental temperatures of 36 ± 1 ℃. Firstly, perfusing extracellular fluid, and perfusing the extracellular fluid containing the drug to be detected when the current is stable, namely the current attenuation (Run-Down) is less than 5% within 5 min and the tail current is not less than 300pA, wherein the concentration is from low to high, and each concentration is perfused for 4 min.

Experimental data: collected with PATCHMASTER (HEKA Instruments IncD-67466 Lambrright/Pfalz Germany) and analyzed using Origin (Origin Lab Corporn, Northampton, Mass.) software, and data are expressed as mean + -SE.

The applicant of the present invention uses the above method to perform test research on 3 standard compounds on potassium, calcium and sodium ion channels of heart respectively by using an experimental scheme conforming to the fluctuation of human heart, the test dose is 5 concentrations, and the routine test screening program is as follows:

as shown in fig. 1 to 9, an optimization method for evaluating the cardiac safety risk by using a high-precision ion channel includes the following steps:

cardiac potassium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, as shown in figure 1, the hERG ion channel of the heart is activated by a voltage method simulating the fluctuation of the human heart, the duration time of a depolarization voltage of +40mV is about 500 ms, the depolarization voltage is repeated every 5 s, the duration time of a voltage repolarization phase is 100 ms, the voltage repolarization phase is from +40mV to-80 mV, Ramp is formed, the voltage is changed to-1.2V/s, a hyperpolarization small pulse of a previous-period voltage of-80 to-90 mV is used for calculating input resistance according to ohm's law, and the maximum positive current recorded during Ramp is the current amplitude of the hERG channel to be recorded;

step 2: in drug evaluation, recording is continued until stable control (control) and after dofetilide is obtained, the maximum positive current recorded during Ramp is determined to be the magnitude of the hERG channel current to be recorded, while the cells remain stable, as determined by the current maintained at-80 mV and input resistance, two concentrations of drug are perfused per cell, as shown in fig. 2, 3, at least 5 concentrations are tested for each drug, and the percent inhibition of current associated with these concentrations ranges from 20% to 80% inhibition, to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac calcium ion channels:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, as shown in figure 4, a Cav1.2 calcium ion channel of the heart is activated by a novel combined voltage method which accords with the heart fluctuation of a human body, the duration time of a depolarization voltage of +30 mV is about 200 ms, the depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization stage is 100 ms, Ramp is formed from +30 mV to-80 mV, the voltage is changed into-1.1V/s, a small hyperpolarization pulse of a previous voltage of-80 to-90 mV is used for calculating input resistance according to ohm law, and the maximum negative current recorded during Ramp is the current amplitude of the calcium channel to be recorded;

step 2: in drug evaluation, the cav1.2 current before and after steady dosing was recorded and the cells remained steady, as determined by the current maintained at-80 mV and input resistance, two concentrations of drug were perfused per cell, as shown in fig. 5, 6, at least 4 concentrations were tested for each drug, and the percent inhibition of current associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50;

cardiac late sodium channel:

step 1: the sealing resistance of the glass electrode and the cell membrane is enabled to be more than or equal to 1G omega, as shown in figure 7, a novel voltage method conforming to the combined voltage method of human heart fluctuation is used for activating the late sodium ion channel of the heart, the duration time of a +40mV depolarization voltage is about 200 ms, the voltage depolarization voltage is repeated once every 5 s, the duration time of a voltage repolarization stage is 100 ms, Ramp is formed from +40mV to-95 mV, the voltage is changed to-1.35V/s, a hyperpolarization small pulse with an early voltage of-95 to-120 mV is used for calculating input resistance according to ohm's law, and the maximum negative current recorded during Ramp is the current amplitude of the late sodium ion channel to be recorded;

step 2: in drug evaluation, the effect was continuously recorded until a steady late sodium current before and after dosing was obtained and the cells remained stable, as determined by the current maintained at-80 mV and the input resistance, two concentrations of drug were perfused per cell, as shown in fig. 8, 9, at least 4 concentrations were tested for each drug, and the percent inhibition of current associated with these concentrations ranged from 20% to 80% inhibition, in order to better estimate the concentration associated with 50% current inhibition, IC 50.

In the 3 known structure of hERG, Cav and late Nav1.5 ion channel blocker validation: the hERG blocker Dofetilide (Dofetilide) showed an IC150 of 0.15 uM with a slope k of 1.5. Dorphyrinthide had no effect on the time at which the hERG current peaked in the ramp test. The Cav1.2 calcium channel blocker verapamil showed an IC150 of 9.05 uM with a slope k of 0.83. Verapamil had no effect on the time to peak inward calcium current in the ramp test. Late sodium current Nav1.5 channel blocker Ranolazine showed an IC150 of 0.04 uM with a slope k of 1. Ranolazine had no effect on the time at which the inward calcium current peaked in the ramp test.

The applicant tests 3 medicines used in clinic by using 3 in-vitro cell test potential stimulation methods conforming to the cardiac fluctuation of human bodies, and the results are consistent with the clinical discovery results, so that the reliability of the compound cardiac safety assessment judged by the experimental method and the clinical relation are more excellent than those of the traditional method.

The experimental method and the process of the invention are close to the heart activity rule of the human body, are simple and have good clinical relevance, are convenient for popularization and use in practical application, can not only facilitate the confirmation of TdP risk of the medicine before clinical use, but also avoid the huge loss of medicine research and development manufacturers caused by the occurrence of false positive detection results, and can also reduce the unnecessary medical expenses of the nation and medical institutions.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should be included in the protection scope defined by the claims of the present invention.