According to literature statistics , the cardiotoxicity of anti-tumor drug treatment mainly includes 9 aspects: heart insufficiency and heart failure, coronary heart disease, heart valve disease, arrhythmia (especially prolonged QT interval), hypertension, thromboembolic Disease, peripheral vascular disease and stroke, pulmonary hypertension and pericardial complications. Among them, the side effects of prolonged QT interval have become an important issue that threatens the safety of the public’s medication, and it is also an issue of drug heart safety that is currently widely concerned by drug regulatory authorities and new drug research and development companies.
Part 1: Cardiac Safety Data of Drugs
First of all, let’s take a look at a picture. Readers who have read our last issue will definitely find it familiar… Yes, that’s the picture. This is “Can the pharmaceutical industry reduce attrition rates?” published by Kola in 2004. . This article counts AstraZeneca, BMS, Novartis, Pfizer and other top 10 pharmaceutical companies in Europe and the United States that entered the clinical stage of pipeline development from 1991 to 2000. Among them, the proportion of drug development failures due to safety is Toxicology (21%) + Clinical safety (12%) = 33% of all failures.
Look at another picture, this is “Drug Safety Sciences and the Bottleneck in Drug Development” published by PB Watkins in 2011. This article points out that the failure of clinical trials and the withdrawal of approved drugs are mainly related to the cardiovascular system and liver. Among them, the cardiovascular adverse events that are most likely to lead to withdrawal are the potentially fatal arrhythmia, which is known as torsion de pointes. Tachycardia (TdP) can now be identified by monitoring the QT interval of the ECG in a phase 1 clinical trial.
Among the 79 drugs that failed clinical trials in Phase I-III, the cardiovascular system accounted for 22%, the liver toxicity accounted for 22%, and the central nervous system accounted for 22%;
Among the 47 drugs withdrawn from the market after FDA approval, TdP accounted for 33%, other cardiovascular systems accounted for 12%, and liver toxicity accounted for 32%.
Examples of withdrawal drugs-terfenadine (SELDANE):
Terfenadine, a long-acting antihistamine, was first marketed in the UK, France, and West Germany in 1982, and was approved for listing in the United States in 1985. It has been quickly promoted and is widely used worldwide for the treatment of allergies. Rhinitis. This drug is the first antihistamine used to relieve allergy symptoms without causing drowsiness. After reporting the first death case caused by a therapeutic dose in 1990 and a large number of patients with arrhythmia side effects, the FDA had to give terfenadine a warning about arrhythmia side effects; in 1992, a total of 15 deaths were collected. Cases and 83 cases of torsade de pointes (TdP) cases, the FDA once again upgraded to a serious “black box warning”; in 1997, it has caused about 350 deaths, and the FDA finally withdrew terfenadine from the market, ending the original The life of medicine.
Why is this happening? This is because pharmacologists had little knowledge of hERG inhibition at that time, and did not consider the need to test terfenadine for cardiotoxicity. It is impossible to find that terfenadine molecules themselves have strong inhibitory effects on potassium ion channels. Effect (ie hERG inhibition).
Later, in order to avoid the occurrence of the above-mentioned serious adverse events, the European and American drug regulatory authorities required pharmaceutical companies to comprehensively evaluate the potential cardiotoxicity of drugs during the development of new drugs.
At the same time, ICH issued the “S7A: Safety Pharmacology Studies for Human Pharmaceuticals” guide in November 2000, which clearly stated that the safety evaluation of drugs should include the effects on cardiac repolarization and QT interval;
In 2005, the guideline “S7B: The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) By Human Pharmaceuticals” was released. This is a supplement to S7A. It proposes a pre-clinical evaluation method including 4 levels of models. The first one is to evaluate the effect of drugs on the rapid activation of delayed rectifier potassium current (IKr) on isolated animal or human cardiomyocytes or transfected HERG expression channels;
In 2006, the “E14: The Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs” was proposed, requiring a comprehensive QT study of all new drugs that act on the whole body, and quantitative evaluation of the impact of candidate drugs on the QT interval .
In 2014, my country issued the “Technical Guidelines for Non-clinical Research on Potential Effects of Drug QT Interval Extension”, which also stipulated non-clinical research on drugs. So far, my country has not yet issued technical guidelines for QT interval clinical research.
Clinically, torsade de pointes ventricular tachycardia caused by QT prolongation caused by drugs has also attracted more and more attention. In 2010, the AHA/ ACC published guidelines for the prevention of torsades de pointes in hospitals, which clearly stated that torsades de pointes induced by prolonged QT interval is a serious malignant arrhythmia, and positive measures must be taken to prevent it. Control. It can be seen that a consensus has been reached in preclinical and clinical evaluation of the effects of drugs on myocardial repolarization. Drug management agencies and clinicians at home and abroad have paid great attention to the side effects of drugs that cause QT interval prolongation.
The fundamental purpose of the guidelines on cardiac QT interval prolongation promulgated above is to evaluate whether compounds have cardiac risks in the early stages of new drug development, so as to reduce the risk of later development. Since the ICH E14/S7B guidelines were issued in 2005, no marketed drug has been withdrawn from the market due to QT-related adverse reactions. At the same time, the reports of non-antiarrhythmic drugs leading to TdP have also been significantly reduced after the market. However, the guidelines still have limitations. On the one hand, hERG channel blockade is not the only factor in QT interval prolongation. On the other hand, QT interval prolongation does not necessarily lead to TdP.
Part 2: Evaluation method of cardiac safety
2.1 Normal ECG and QT interval
The impulse emitted by the sinus node produces a continuous process of depolarization and repolarization of myocardial autonomic cells and contractile cells through the myocardial conduction system, causing a series of potential changes, which are recorded by body surface electrodes and become an electrocardiogram.
The so-called QT interval of the heart refers to the period from the beginning of the QRS complex to the end of the T wave, including the process of ventricular depolarization and repolarization. The extension of the QT interval is receiving more and more attention and is considered It is one of the key indicators for the safety evaluation of new drugs. The prolonged QT interval easily leads to an increased risk of malignant arrhythmias, and most often triggers torsades de pointes (TdP), which can easily evolve into ventricular fibrillation and cause sudden death.
2.2 Electrophysiological changes of myocardial cell membrane
The formation of an electrocardiogram is based on the changes in potential during the depolarization and repolarization of cardiomyocytes. In the resting state, the inner membrane potential is negative than the outer membrane, about -90mV, that is, it is in a polarized state. When myocardial cells are stimulated and excited, the membrane potential changes to form an action potential.
2.3 hERG gene and coding protein channel
The hERG gene was originally isolated and identified from the human hippocampal cDNA library in 1994 by Warmke et al. , which has homology with the Drosophila EAG gene. The hERG gene is located on human chromosome 7 (7q35～q36), about 55kb, has 16 exons, and encodes 1159 amino acid residues. It can be expressed in human body tissues such as myocardium, brain, liver, spleen, etc., with the highest expression in myocardial tissue. The protein product encoded by the hERG gene is the key membrane-bound potassium ion channel forming part in the myocardial tissue. It is controlled by the membrane potential and regulates the potassium outflow of the cell in a gated manner. When the compound binds to the hERG potassium channel, the outflow of potassium ions is blocked, which can prolong the repolarization time of cardiomyocytes, which is reflected in the electrocardiogram, that is, the QT interval is prolonged, which may induce the fatal risk of TdP.
hERG inhibition is divided into two types: hereditary (primary) and non-hereditary (secondary). Hereditary prolonged QT interval mainly includes gene mutations that can produce ion channel dysfunction and congenital prolonged QT syndrome; non-hereditary prolonged QT interval can be caused by metabolic abnormalities, diseases and drugs.
In 1995, Sanguinetti et al. for the first time transfected the hERG gene to express the hERG protein channel on the cell, and also confirmed that the hERG channel of pure hERG gene expression, its biological characteristics are completely consistent with the normal IKr channel . This is the theoretical basis for evaluating the effect of drugs on IKr in vitro using cells transfected with hERG gene.
2.4 How to evaluate the effect of drugs on hERG channel current?
Theoretically, the most ideal method for hERG channel current is to directly measure IKr on cardiomyocytes, but this has many problems:
(1) The application of enzymatic hydrolysis for the acute separation of cardiomyocytes requires higher technology and more time. At the same time as digestion, potassium channel protein may be partially digested ;
(2) In addition to IKr, cardiomyocytes also have many sodium and calcium currents, which can confuse IKr;
(3) Myocardium is contractile and sensitive to calcium. Therefore, it is necessary to form a very good high-resistance seal during patch clamp recording to perform current measurement .
On the contrary, how to use hERG gene to express hERG current on suitable cells, and then determine the effect of drugs on the rapid extension of rectifier potassium current, seems to be a better choice. Therefore, according to the ICH S7B guidelines to evaluate whether drugs block IKr, IKr can be measured directly on isolated cardiomyocytes or hERG current can be measured on cells transfected with hERG gene.
At the earliest, Xenopus oocytes and Chinese hamster ovary cells (CHO) were used to transfect hERG genes to express potassium channel currents, and it was found that the former expressed higher potassium currents than the latter [6-7]; at the same time, these two cells They are all heterogeneous cells, and there are racial differences between human cells. Therefore, in 1996, Snyders et al. first proposed that HEK293 cells can stably express the hERG gene . Now it has become a reliable in vitro evaluation method and is widely used.
2.5 hERG inhibition in vitro evaluation technology and influencing factors
The patch clamp technique is known as the “gold standard” for studying ion channels and is the most important technique for studying ion channels. With the continuous deepening of research, the current patch clamp technology has developed from conventional patch clamp technology to automatic patch clamp technology and FluxORTM Thallium Assay, etc., which are used to study the mechanism of action of compounds and ion channels, and are also used for new drug declarations. During the process, the toxicity evaluation of the candidate drug and the structure optimization of the lead compound.
When evaluating the inhibitory effect of drugs on current intensity, the most important indicator is the half inhibitory concentration IC50. This indicator generally requires the determination of multiple concentrations of drugs (5-8 cells/concentration), and then the Hill equation fitting get.
Therefore, there are inevitably many influencing factors in the measurement process of this indicator, including but not limited to the following aspects: (1) The temperature of the experiment can affect the IC50; (2) Different voltage stimulation schemes will also affect the IC50 result; ( 3) The cell carrier of the experiment will also affect IC59; (4) The influence of the electrode inner and extracellular fluid used for measurement. Therefore, this reminds us that when evaluating the effect of drugs on hERG current, we must first ensure the consistency of experimental conditions in order to obtain a more objective and correct drug inhibitory effect.
For example, in the previous projects we investigated, the hERG IC50 measurement results differed by more than three times between the two measurements, because it was measured by two different laboratories.