Method for preclinical study of cardiotropic antiarrhythmic drug

FIELD: medicine.

SUBSTANCE: invention represents a method for preclinical study of cardiotropic antiarrhythmic drugs, involving determining the bioelectric parameters in isolated multicellular perfused preparations and measuring an action potential duration, differing by the fact that the isolated multicellular perfused preparations are presented by rat's pulmonary vein myocardium; the parameters are measured in three operation modes of the multicellular preparations; a resting potential is additionally measured; varying APD 90%, related APD 50%/APD 90%, a spontaneous shear velocity of the resting potential, the most positive membrane potential in the resting preparation, a spontaneous activity train repetition rate, spontaneous action potential train repetition and variability frequency, post-depolarisation number and intensity, as well as a shear membrane potential corresponding to the beginning of train activity are used to evaluate the signs of antiarrhythmic and arrhythmogenic action.

EFFECT: more reliable prediction of the antiarrhythmic action of the potential pharmacological agents and reduction of experimental phase time.

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The present invention relates to experimental biology, in particular for experimental pharmacology and cardiology, concerns preclinical studies cardiotropic antiarrhythmic drugs and other cardiotropic funds, security definitions, i.e., the absence of arrhythmogenic activity of drugs.

At the present time to treat rhythm disorders of various types are widely used antiarrhythmic drugs. Continuously being developed to a considerable number of compounds, claiming new antiaritmikov. The effects of most anti-arrhythmic drugs is multifactorial, implemented at the molecular, cellular and tissue levels: it is due to the influence of molecular membrane and intracellular complexes, the bioelectric activity of individual cardiomyocytes, the electrical properties of cardiac tissue. Antiarrhythmic agent have an impact on all parts of the heart, including pacemaker, work infarction, vascular system, but unequally.

Antiarrhythmic agent, as well as other cardiotropic means cause side effects that may have undesirable properties, for example, under certain conditions, can lead to arrhythmias, i.e., to have arrhythmogenic effect.

In connection with slozhnosti.imenno steps and a plurality of target detection and predicting antiarrhythmic actions as well as side, arrhythmogenic properties of antiarrhythmic (and cardiotropic drugs is an extremely difficult task.

There are factors and scope, critical for the implementation of various types of anti-arrhythmic activity. The most common forms of arrhythmia is atrial flutter and flicker (atrial fibrillation) atrial. According to the most common at the present time the concept of ectopic activity in the field of myocardial plates ("arms") of the pulmonary veins, which interacts with the electrical activity in the Atria, is the cause of atrial fibrillation (AF). Myocardial tissue in the pulmonary veins differs from atrial, has a number of morphological features and characteristics of bioelectric activity. Myocardial sleeves of the pulmonary veins in the experimental conditions, and in vivo are foci triggering activity, abnormal automatie, circulation of excitation, i.e., the factors leading to the different types of arrhythmias, in particular atrial fibrillation.

Changes of bioelectric activity in the myocardium pulmonary veins under the action of pharmacological agents is critical, the Central element of the mechanism of initiation of atrial fibrillation, and prevention.

There are several in vivo and in vitro the ways of identifying, predicting antiarrhythmic activity. This model chloralkali arrhythmias, model gloriamarie arrhythmias, adrenaline, continua model arrhythmias. The essence of these methods is the introduction of high doses of calcium, barium, aconitine, oubain or strofantina anesthetized or awake animals.

(Gabriel R. U. Guidance on experimental (preclinical) study of new pharmacological substances, M.: Medicine, 2005.)

Infusion of calcium chloride to simulate ventricular and supraventricular arrhythmias proposed in the 1950's. Model chloralkali arrhythmias based on a generalized activation of the sympathetic division of the autonomic nervous system and subsequent release of norepinephrine from sympathetic terminala that, in turn, leads to the development of arrhythmia and abnormal automatie. Similar in nature is "adrenaline model arrhythmia", differing from the previous approach only the immediate injection of adrenaline.

(Role of the sympathetic-adrenal system in the mechanisms of genesis of cardiac fibrillation induced by calcium chloride. Kardiologiia. 1974 Jun; 14 (6): 78-82.)

"Hloridnaja model of arrhythmia based on the ability of barium disrupt the functioning of mitochondria, the conductivity of several groups of ion channels.

(Delfino G, Amerini S, Mugelli A. Barium cardiotoxicity: Relationship between ultrastructural damage and mechanica effects. Toxicol In Vitro. 1988; 2 (1): 49-55.)

"Continua model" and "Stroganina model" is based on the systemic administration of specific toxins: aconitine, suppressing the inactivation of sodium potentialcustomers channels of cardiomyocytes, oubain, suppressing the activity of Na/K-ATPase cardiomyocytes, respectively.

(Chan TY. Aconite poisoning. Clin Toxicol (Phila). 2009 Apr; 47 (4): 279-85.)

All of the above approaches practically have nothing to do with the physiological mechanisms leading to the occurrence of arrhythmias in vivo, and induced rhythm disturbances characteristic of the "agonistic" or acute intoxication. Using "aconitine" and "strofantino" models is justified only in the case of the development of products to treat rare genetic conditions. When using the above approaches it is impossible to establish the mechanisms and potential antiarrhythmic actions of pharmacological tools, safety. At the present time in the world practice the methods described above are practically not used.

Known methods based on surgical and/or mechanical disruption of the blood supply of the myocardium, aimed at identifying the antiarrhythmic activity. Small mammals, such as rats, verification of results using these approaches is extremely difficult, sleds is of a well-developed collateral circulation. When using large mammals experiments become extremely expensive, technical support experiments overly difficult.

Known methods of identifying pharmacological antiarrhythmic activity funds, which are based on electrical stimulation of the heart and enforce rhythm, alone or in combination with parasympathetic stimulation.

(Bhatt L. K., Nandakumar, K., S. L. Bodhankar Experimental animal models to induce cardiac arrhythmias. Ind. J. Pharm. 2005. 37. 348-357.)

These methods are effective to measure the ability to suppress persistent arrhythmia, but uninformative in terms of forecasting preventing ability and safety of antiarrhythmic drugs.

The closest way of the same purposes of the claimed invention is a method for predicting the efficacy and safety of antiarrhythmic drugs, which carry out the determination of the bioelectric parameters in isolated multicellular perfuziruemah preparations in vitro, representing sections of the working myocardium of the heart - the Atria, papillary muscles. This assess changes in the duration of action potentials (APS), refractoriness after application of the investigated compounds. Changing the duration of PD in the working myocardium is an element of regulation of cardiac activity in and of itself to them is no small predictive value: can talk about reducing, and increase the likelihood of arrhythmias.

(L. M. Hondeghem, L. Carlsson, G. Duker, Instability and triangulation of the action potential predict serious proarrhythmia, but action potential duration prolongation is antiarrhythmic. Circulation, 2001; 103, 2004-2013.)

The disadvantages of the above method are the low reliability and relevance with the actual pathophysiological processes that cause arrhythmias. You should also specify that this is a normal working myocardium of the heart and does not assess the effect of antiarrhythmic drugs on "natural arrhythmogenic substrate", which is the infarction pulmonary veins.

The present invention is to create an efficient method of pre-clinical studies cardiotropic antiarrhythmic drugs, increase reliability of predicting antiarrhythmic action, the security of potential pharmacological agents for this purpose, reducing the duration of the experimental phase and accelerates the transition to clinical research.

The technical result of the invention is to improve the reliability of predicting antiarrhythmic action potential pharmacological agents and the reduction in the duration of the experimental phase.

This is achieved by the fact that in the present method preclinical studies cardiotropic antiarrhythmic drugs, on the expectation definition bioelectric parameters in isolated multicellular perfuziruemah drugs and assessment of the change in the duration of action potentials according to the invention, as isolated multicellular perfuziruemah drugs used infarction pulmonary veins of the rats, and the changes of parameters are available in three modes multicellular preparations, additionally evaluate the resting potential and to increase DPD 90%, increase the ratio DRD 50%/DPD 90%, the decrease in the rate of spontaneous shift of the resting potential, reducing the most positive values of the membrane potential at rest the drug, to reduce repetition frequency packs spontaneous activity, reduce the frequency and variability of following the spontaneous PD in the stack, reducing the number and intensity reduction of postdeparture, the displacement of the membrane potential corresponding to the beginning of the burst of activity in the negative direction, assess signs of anti-arrhythmic action, and to reduce DPD 90% reduction relations DRD 50%/DPD 90%, increase in the rate of spontaneous shift of the resting potential, the increase in the most positive values of the membrane potential at rest the drug, increasing the repetition frequency packs spontaneous activity, increased frequency and variability of following the spontaneous PD in the stack, to increase the number and increase the intensity of postdeparture, the displacement of the membrane potential corresponding to the beginning of the burst activity in a positive way, appreciate the arrhythmogenic activity.

The technical result is achieved due to the use of patterns, with increased arrhythmogenic activity, namely infarction pulmonary veins of the rats.

Myocardial lining the pulmonary veins of the rats has a sufficient length, up to the bifurcations of the second order and intra-lungs plots, myocardial lining the pulmonary veins of the rats has properties characteristic of arrhythmogenic foci.

Myocardial lining the pulmonary veins Guinea pigs functionally indistinguishable from the atrial myocardium. Pulmonary vein of a rabbit do not have extensive myocardial plates, respectively, isolated preparations obtained from data mammal species, have low aritmogennosti and may not be used to evaluate the antiarrhythmic activity.

Use multicellular preparations, pulmonary veins, so as of great importance for the implementation of anti-arrhythmic action has a tissue level, factor interconnectedness of cardiomyocytes in tissue.

The implementation of the method

Multicellular preparations of pulmonary veins of the rats was obtained as follows: necrotizing animal, open chest, get the heart along with the lungs and placed in preparevalue bath (100 ml), filled with the perfusion solution at room temperature. Heart washed perfusion is astora (intraorale) containing heparin (or 0.2 units/ml). Ofpreparation the left atrium and pulmonary veins of the left lobe of the lung.

The pulmonary vein is separated from the proximal side in the area of the mouth, with the distal side in the area of the first bifurcation, cut along the longitudinal axis and deploy in a perfusion chamber, receiving the flat product (the area of drug ≈0.6-0.8 mm2). The drug deploy and fixed in the perfusion chamber endocardial side up. The procedure of preparation 5 minutes.

In Fig.1 shows a schematic representation of the preparation of the left atrium, pulmonary veins and the lobes of my lungs in rats, where SFM - the left atrial appendage, PL - free wall of the left atrium, LV - pulmonary vein, LDL - left lobe of the lung, DDL - incremental share of the lung. The dashed circled the area being examined.

After selection of a multicellular preparations are perfusion. Perfusion is performed at a temperature of 37°C and the velocity of the flow 15 ml/min (volume 3 cameras in 1 minute, which is necessary for adequate oxygenation of drugs). Perfusion solution continuously Oxygenium with Carbogen (95% O2and 5% CO2). Use of perfusion solution of standard composition: (mm): NaCl 133.47, KCl 4.69, NaH2PO4·2H2O 1.35, NaHCO316.31, MgSO4·7H2O 1.18, CaCl2·2H2O 2.5, glucose 7.77, pH of 7.2 to 7.4. Water mediterranei up to 1 litre.

Register potential generation is, action potentials. Action potentials away from "endocardial" side of the pulmonary veins. To register PD using glass microelectrodes filled with 3M KCl (resistance 1-30 Mω) connected to the amplifier. The amplified signal is fed to analog-to-digital Converter and a computer.

To predict the effectiveness and safety of potential antiarrhythmic drugs used perfusion of drugs pulmonary veins of the rats in three modes:

1. "Rhythmically excited medication"

The drug continuously stimulate rectangular pulses of 2 MS interval S=300 MS and an amplitude equal to two thresholds (1-3 B), which leads to occurrence of action potentials in the drugs. If necessary use other intervals of stimulation. Stimulating silver electrodes (D=0.5 mm) is placed in the proximal region of the drug.

Calculate the duration of PD at 50% and 90% repolarization (DRD 50%, DPD 90%) when the stimulation interval S=300 MS. Determine the increase in DPD 90% in the experimental groups compared with the DPD 90% in the control group.

2. "Resting drug"

After a 10-minute period of continuous stimulation and rhythmic activity cease stimulation. Termination of stimulation in the infarction pulmonary veins rats leads to spontaneous shift p of the building of peace in the region more positive values (Fig.2). Assess the degree and rate of spontaneous shift of the resting potential in the control groups, as well as after the action of the investigational pharmacological compounds.

In Fig.2 shows examples of action potentials and spontaneous shift of the resting potential in the myocardium of the pulmonary veins of the rats when the drug is in a resting state. 1, 2, 3 - ways to amend paragraphs at the termination of stimulation in the pulmonary veins (LV), 4 - atrial part of the drug (PL). The arrow indicates the moment of cessation of the stimulation.

3. "Spontaneously active drug"

The drug during the experiment perfusion 1 µm phenylephrine, resulting in infarction pulmonary vein of the rat develops a periodic train of spontaneous activity; spontaneous action potentials in packs related to early postdeposition.

In Fig.3 shows the cyclical nature of spontaneous burst activity and potential changes in the myocardium of the pulmonary veins of the rats under the action of phenylephrine: periods of spontaneous activity are accompanied by hyperpolarization, the rest period is accompanied by depolarization, where the bottom depicts a single PD preceding burst activity (0), the first DD in the stack, followed by early postdeposition (1 and 2), a typical train-PD (3), last DD tutu (67).

Assess the impact of the investigational pharmacological compounds on cha is Tautou repetition packs spontaneous activity, the most negative potential in the rest period, frequency, and variability of following the spontaneous PD in the stack, the intensity of postdeparture, the value of the membrane potential corresponding to the beginning of the burst activity.

Criteria for predicting the efficacy and safety of potential anti-arrhythmic and other pharmacological tools evaluation can serve the following bioelectric parameters that is possible only in the pulmonary veins of rats.

Namely, as signs of antiarrhythmic efficacy and safety are increasing DPD 90%, the increase of the ratio DRD 50%/DPD 90%, the decrease in the rate of spontaneous shift of the resting potential, the reduction in the most positive values of the membrane potential at rest the drug, reducing repetition frequency packs spontaneous activity, reduce the frequency and variability of following the spontaneous PD in the stack, reducing the number and a decrease in the intensity of postdeparture, the displacement of the membrane potential corresponding to the beginning of the burst of activity in the negative.

As features of arrhythmogenic activity are reduced DPD 90%, the decrease of the ratio of DPD 50%/DPD 90% of the increase in the rate of spontaneous shift of the resting potential, the increase in the most positive values of the membrane potential in oedema the drug, the increase in the repetition frequency packs spontaneous activity, an increase in the frequency and variability of following the spontaneous PD in the stack, increasing the number and the intensity of postdeparture, the displacement of the membrane potential corresponding to the beginning of the burst activity in a positive way.

Thus, the inventive method is effective, increases the reliability of predicting antiarrhythmic action, the security of potential pharmacological agents for this purpose reduces the duration of the experimental phase and accelerates the transition to clinical research.

The way preclinical studies cardiotropic antiarrhythmic drugs, including the definition of bioelectric parameters in isolated multicellular perfuziruemah drugs and assessment of the change in the duration of action potentials, characterized in that the isolated multicellular perfuziruemah drugs used infarction pulmonary veins rats, additionally evaluate the resting potential, and the changes of parameters are available in three modes multicellular preparations:
1) the drug is continuously stimulated by rectangular pulses of 2 MS interval S=300 MS and an amplitude equal to two thresholds (1-3), calculate the duration sweat the capacity of action at the level of 50% and 90% repolarization (DRD 50%, DPD 90%) when the stimulation interval S=300 MS, determine the increase in DPD 90% in the experimental groups compared with the DPD 90% in the control group;
2) after a 10-minute period of continuous stimulation and rhythmic activity cease stimulation, assess the degree and rate of spontaneous shift of the resting potential in the control groups, as well as after the action of the investigational pharmacological compounds;
3) the drug perfusion 1 µm phenylephrine for development infarction pulmonary veins rats periodic train of spontaneous activity, and assess the impact of the investigational pharmacological compounds on the repetition rate of the packets of spontaneous activity, the most negative potential in the rest period, frequency, and sequence variability of spontaneous action potentials (APS) in the stack, the intensity of postdeparture, the value of the membrane potential corresponding to the beginning of the burst activity;
to increase DPD%, increase the ratio DPD%/DPD%, the decrease in the rate of spontaneous shift of the resting potential, reducing the most positive values of the membrane potential at rest the drug, to reduce repetition frequency packs spontaneous activity, reduce the frequency and variability of following the spontaneous PD in the stack, reducing the number and intensity reduction of postdeparture, offset memb the data capacity, corresponding to the beginning of the burst activity in a negative way, appreciate the signs of anti-arrhythmic action, and to reduce DPD% reduction relations DPD%/DPD%, increase in the rate of spontaneous shift of the resting potential, the increase in the most positive values of the membrane potential at rest the drug, increasing the repetition frequency packs spontaneous activity, increased frequency and variability of following the spontaneous PD in the stack, to increase the number and increase the intensity of postdeparture, the displacement of the membrane potential corresponding to the beginning of the burst activity in a positive way, appreciate the arrhythmogenic activity.



 

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10 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to medicinal drug quality control and provides a method of authenticating and determining the quantitative content of benzethonium chloride in medicinal drugs, which includes separating components of the drug via high-performance liquid chromatography, where the eluent used is a mixture of acetonitrile, tetrabutylammonium hydrophosphate, disubstituted potassium phosphate and water, with content of tetrabutylammonium hydrophosphate of 2.5 mM, content of disubstituted potassium phosphate of 5 mM, wherein separation of the components of the drug is carried out with column length of 125 mm.

EFFECT: high accuracy and faster analysis since there is no need for special sample preparation.

9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of screening a non-teratogenic substance comprising bringing a test substance into contact with cereblon or a fragment of cereblon, evaluating the capacity of the test substance to bind to cereblon or the fragment of cereblon, and selecting a test substance that does not bind to cereblon or the fragment of cereblon or a test substance exhibiting lower capacity to bind to cereblon or the fragment of cereblon compared with thalidomide.

EFFECT: improved method.

8 cl, 19 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method involves taking blood at 1 ml per a sample into the Vakutainer evacuated test tube with sprayed ethylene diaminetetraacetic acid 2.2 mg; a reference sample 1.0 ml and a sample 1.0 ml with weighted xenobiotics are taken from the total volume into the Eppendorf tubes; 30-minute incubation is followed by using a uniform procedure to prepare blood smears by applying on a dry slide to prepare a smear to be stained according to Leishman; blood corpuscles are analysed by the Zeiss light microscope at a magnification 1500. A microscope field showing echinocytes with styloid processes of the membrane not found in the reference sample is a sign of the xenobiotic membrane toxicity.

EFFECT: invention enables an instant assessment of the membrane toxicity of the substance.

FIELD: medicine.

SUBSTANCE: polysomnography is conducted. Slow sleep phases (SSP) and fast sleep phases (FSP) are determined. A maturity index of integrative sleeping apparatuses (MIS) is calculated by formula MIS = SSP/FSP. If the MIS is less than 1.5, a physiologically optimum structure of the nocturnal sleep is stated in a healthy child.

EFFECT: method enables assessing the quality of the nocturnal sleep in the children.

1 tbl, 2 ex

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