RU2726927C1 - Method for simulating of combined drug-induced heart and kidneys injury in rats - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to experimental medicine, pathophysiology and pharmacology and can be used for studying mechanisms of formation, progression and therapy of combined cardiac and renal involvement in preclinical studies. Disclosed is a method for creating an experimental model of a toxic multiple organ pathology in the form of combined heart and kidney involvement in experimental animals. Cardiotoxicity is manifested by development of syndrome of elongated QT interval without morphological signs of heart tissue changes. Renal involvement was characterized by functional (reduced creatinine clearance, hyperuricemia) and coarse morphological changes of the tubular epithelium. Modeling of the toxic combined pathology of the heart and kidneys is based on a complex effect of a number of anti-tuberculosis preparations with cardiotoxic potential of a different mechanism of action and a combination with a direct nephrotoxic drug preparation, and factors (sex, age of animals) initiating a cascade of functional disorders leading to the syndrome of an elongated QT interval on a background of organic renal involvement.EFFECT: method provides absence of lethal outcome of experimental animals and short period of pathological condition development.5 cl, 2 dwg, 6 tbl, 1 ex

Description

The technical field to which the invention relates

The invention relates to experimental medicine, pathophysiology and pharmacology and can be used to study the mechanisms of formation, progression and therapy of combined lesions of the heart and kidneys in preclinical studies.

State of the art

Experimental models of toxic multiple organ damage to internal organs, in particular, kidneys and heart, are used to study the mechanisms of formation of pathological processes, to clarify the role of organs and systems, and especially their relationships under the influence of various factors on the body, to identify the leading role of electrolyte nephropathy, leading to combined disorders in the tissues of the kidneys and in the conducting system of the heart, and make it possible to find means for rational therapeutic intervention and prevention.

The risk of drug-induced damage to internal organs increases significantly in the presence of a number of factors: the need to use multicomponent treatment regimens for a number of diseases; a similar safety profile of the circuit components, the presence of comorbidities, the age of the patients. In conditions of polypharmacy, the risk of combined undesirable side effects, including those from the cardiovascular system and kidneys, increases.

For a long time, it was believed that only class IA and III antiarrhythmic drugs (for example, quinidine, sotalol, amiodarone) have a toxic effect on the cardiac conduction system (for example, quinidine, sotalol, amiodarone), however, over the past 20 years, there have been many evidences of an acquired change in the duration of the QT interval on an electrocardiogram (ECG) on the background of the use of various drugs of other pharmacological groups: fluoroquinolones, macrolides, antifungal and anti-tuberculosis drugs. The cardiotoxic effect of drugs, in particular fluoroquinolones, is more often observed in the elderly with renal failure, diarrhea or intensive diuretic therapy with loss of electrolytes [Stahlmann R., Lode H. Fluoroquinolones in the elderly: safety considerations // Drugs. Aging. - 2003. - Vol. 20, No 4 - P. 289-302]. Elderly patients are more vulnerable to drug-induced acute renal disease (Chen D, Luo C, Tang Z, Zhou Y, Chen H, Zeng C, Liu Z: Delayed renal function recovery from drug-induced acute interstitial nephritis. Am J Med Sci 2012 , 343 (1): 36-39).

In the research of a team of authors (A. Ore, E. T. Olayinka. Influence of Moxifloxacin on Hepatic Redox Status and Plasma Biomarkers of Hepatotoxicity and Nephrotoxicity in Rat // Biochemistry Research International-Volume 2015, Article ID 192724, 8 ps) and ET Olayinka , A. Ore, and OS Ola, "Influence of different doses of levofloxacin on antioxidant defense systems and markers of renal and hepatic dysfunctions in rats," Advances in Toxicology, vol. 2015, Article ID 385023, 7 pages, 2015) shows a combined drug damage to the kidneys and liver under the influence of fluoroquinolones in an experiment. Wistar rats were injected with moxifloxacin at doses of 4, 8, 16 mg / kg or levofloxacin at doses from 5 to 20 mg / kg for 7 days and a dose-dependent increase in the level of urea in the blood plasma and enzymes ALT, ACT, ALP was observed compared to intact rats ... At the same time, the levels of lipid profile indices (molonic dialdehyde) in the blood were increased and the levels of enzyme antioxidants (SOD, glutathione-S-transferase) and non-enzymatic antioxidants (ascorbic acid, reduced glutathione) in liver cells were significantly reduced. In this model, for a short time of using only one drug from the group of fluoroquinolones, a combined violation of the functional state of the kidneys and liver was obtained, but the functional state of the cardiovascular system was not investigated. A method for simulating cardiotoxicity in an experiment on rats using combinations of anti-tuberculosis drugs (PTP) is described (Mordyk A.V. Pathogenesis and substantiation of methods for correcting the cardiotoxic effect of anti-tuberculosis drugs (clinical and experimental study). - Abstract of the thesis ... Doctor of Medical Sciences ... - 2008). The essence of the method consists in the introduction of Wistar male rats weighing 180-250 g of 3 combinations of anti-TB drugs in therapeutic doses for 2 months with an assessment of the bioelectrical activity of the heart, heart rate variability and central hemodynamics. As a result of the study, it was found that the most pronounced manifestations of cardiotoxicity in the form of the appearance of single extrasystoles were observed when using a combination of the main anti-TB drugs (isoniazid + rifampicin + pyrazinamide + ethambutol). However, this method of modeling cardiotoxicity has disadvantages: a long modeling period (2 months). Also, this study did not investigate the nephrotoxicity of this regimen.

The known "Method for modeling infarctoid cardiovasopathy in rats" (RU 21092048, IPC 7 G09B 23/28) ETC. Novgorodtseva (2000). The modeling of the infarct-like state in rats is based on electrolyte and steroid imbalance, accompanied by cardiac necrosis. The modeling of cardivasopathy included several pathogenic factors triggering disorders of various origins. The triggering mechanism for the development of this pathological state of the cardiovascular system was unilateral nephrectomy, leading to a gross violation of electrolyte metabolism, kidney function and aggravated by the used electrolyte imbalance with food due to excess sodium and calcium salts against a background of potassium and magnesium deficiency and the effect of cold stress was the resolving agent ... The disadvantage of modeling cardivasopathy is the death of animals in the postoperative period in 2% of cases due to renal failure, significant electrolyte imbalance, myocardial dystrophy and necrosis of the heart muscle.

Closest to the claimed one is a method for modeling cardiovasorenal hypertension in rats, based on the introduction of a cardiovaso-nephropathogenic semisynthetic diet for 2 weeks in experimental animals, enriched with cholesterol, salts of disubstituted aqueous sodium phosphate and replacing drinking water with 1% sodium chloride solution, and with a deficiency of potassium and magnesium [RU 2327228, 20.06.2008]. Also, a suspension of hydrocortisone acetate is injected daily intramuscularly at the rate of 1.5 mg per 100 g of animal weight. Subsequently, the rats were exposed to cold exposure for 4 h at 4 ° C. As a result, organic damage to the heart tissue and renal epithelium develops.

In this model, a combined damage to the cardiovascular system and kidneys was obtained when using a special semi-synthetic diet, which led to an electrolyte imbalance, and the subsequent effect of a physical factor, which became a triggering stress agent. This modeling method according to the main pathogenetic mechanism - electrolyte imbalance - is closest to the claimed modeling method. However, with this method of modeling, organic damage to the tissues of the kidneys and heart developed, caused by multifactorial effects. In addition, this model cannot be classified as toxic combined renal and cardiac lesions of drug etiology.

The technical problem to be solved by the claimed invention is the development of a method for modeling toxic damage to the kidneys and heart with predominantly functional disorders of the conducting system, toxic etiology and pathogenesis of the development of acquired syndrome of an extended QT interval, in a short time (2 weeks) and without lethality of animals.

The technical problem is solved by the claimed method by administering to experimental animals a complex of anti-tuberculosis drugs moxifloxacin, bedaquiline, linezolid, protionamide and capreomycin, while female rats aged 10-12 months were used for modeling as experimental animals. and weighing 330-350 g.

Disclosure of invention

The technical result of the claimed invention is to create a model that makes it possible to obtain in rats a violation of the functional state of the heart in the form of an extended QT interval syndrome, in combination with morphological and functional changes in the kidneys, caused by drug polypharmacy, close to the clinical course of these disorders in patients receiving complex therapy, for short term - 2 weeks.

The technical result is achieved by a method of modeling toxic damage to the kidneys and heart, which consists in the fact that toxic damage to the kidneys and heart in animals is caused by daily administration of a complex of anti-tuberculosis drugs moxifloxacin, bedaquiline, linezolid, protionamide and capreomycin to laboratory animals for at least 2 weeks, when In this case, female rats aged 10-12 months and weighing 330-350 g were used for modeling, drugs moxifloxacin, bedaquiline, linezolid, prothionamide are administered intragastrically in 0.1% starch gel, and capreomycin is administered intramuscularly. The drugs are administered in the following doses: moxifloxacin - 36 mg / kg, bedaquiline - 36 mg / kg, linezolid - 54 mg / kg, prothionamide - 90 mg / kg, capreomycin - 90 mg / kg and almost simultaneously.

The anti-tuberculosis drugs that were used to create the model have a pronounced potential for cardiotoxicity with a different mechanism of toxic action, in combination with the intramuscular administration of an anti-tuberculosis drug that causes a direct nephrotoxic effect and electrolyte imbalance. The use of sexually mature female rats aged 10-12 months and weighing 330-350 g increases the development of cardiotoxicity and nephrotoxicity. For the experiments, healthy rats were selected that had passed the quarantine observation period for 2 weeks, with a small, up to 20 grams, spread in body weight. Before the administration of the complex of drugs, the rats underwent functional studies.

Pathological changes were manifested by a decrease in the heart rate from 354.7 ± 8.4 to 320.6 ± 15.3, the appearance of single extrasystoles, a violation of the heart rhythm associated with electrical conduction - an increase in QT intervals from 0.043 ± 0.002 ms to 0.056 ± 0.002 ( P = 0.03) and QRS from 0.022 ± 0.001 to 0.028 ± 0.003 ms against the background of a decrease in the content of electrolytes - potassium to 2.83 + 0.11 at a rate of 4.37 + 0.26 and calcium to 1.19 + 0, 01 at a rate of 1.27 + 0.02 mmol / l and changes in urine and blood renal profile. An increase in the level of uric acid was observed in the blood serum (9.78 ± 1.28 versus 6.8 ± 0.41 in the control), a 1.5-fold increase in creatinine. The creatinine clearance decreased to 0.62 + 0.07 versus 0.77 + 0.02. In all rats, kidney changes were of varying severity. In the cortical substance of the kidneys, there was a strong expansion of the lumen of the tubules, disaggregation and desquamation, death and atrophy of the epithelium of the tubules. In some areas of the intact epithelium of the convoluted tubules - signs of granular dystrophy. In the lumen of some tubules - accumulations of dead epithelium (granular casts) or accumulations of homogeneous eosinophilic fluid (primary urine). Separate damaged vascular glomeruli were found, which looked like structureless and dense lumps. In the medulla, granular casts and accumulations of homogeneous eosinophilic fluid (urine) were found.

EFFECT: method allows to obtain a new experimental model of combined toxic lesion of kidneys and heart of drug origin with predominant disorders of the cardiac conduction system, which most accurately reflects the mechanisms of development of acquired syndrome of prolonged QT interval, ventricular arrhythmias and electrolyte imbalance. The proposed model is simple to execute, has good reproducibility, no lethal outcome in experimental animals and the complexity of the impact on the animal's body.

Brief Description of Drawings

The invention is illustrated by the following drawings.

In FIG. 1 is a photograph of a fragment of the abdominal cavity of a rat showing discoloration (whitish) and an increase in the size of the kidneys.

In FIG. 2 shows an ECG of a rat, which shows changes, namely, a decrease in heart rate, an increase in QT and QRS intervals compared to the control, extrasystoles.

Implementation of the invention

All reagents used are commercially available, all procedures, unless otherwise stated, were carried out at room temperature or ambient temperature, that is, in the range from 18 to 25 ° C.

The possibility of implementing the claimed invention is shown, but not limited, in the examples of specific implementation.

Before the administration of the complex of drugs, the rats underwent functional studies and biochemical studies. The ECG was used to assess the heart rate (HR), indicators of time intervals of heart contractions (RR, PQ, QRS, QT). In blood and urine tests, the content of electrolytes, creatinine and creatinine clearance were determined.

We used non-linear female rats aged 11 months weighing 330-350 g, which daily for 2 weeks were injected intragastrically in 0.1% starch gel PTP complex in the following doses: moxifloxacin (36 mg / kg) + bedaquiline (36 mg / kg) + linezolid (54 mg / kg) + prothionamide (90 mg / kg). Capreomycin was administered intramuscularly at a dose of 90 mg / kg. Doses of drugs corresponded to the maximum therapeutic doses for humans.

The complex of preparations was prepared ex tempore as follows: the tablet of each preparation was thoroughly ground in porcelain mortars and the required volume of 0.1% starch gel was added so that the required amount of the preparation was contained in 0.5 ml of starch suspension. Immediately before administration, all preparations were drawn into one syringe and simultaneously injected in a total volume of 2 ml into the stomach of a rat. Capreomycin was injected into the muscle in a volume of 0.5 ml almost simultaneously with the intragastric administration of PTP tablets.

To create the model, the animals were randomly assigned to 3 groups. The first group of animals (8 animals) was injected with the PTP complex for 14 days, the second group of animals (8 animals) was injected with the PTP complex for 28 days, then they were removed from the experiment, after all the necessary tests had been carried out. The control group (10 individuals) was injected daily orally with a 0.1% solution of starch paste in a volume of 2.0 ml (corresponding to the volume of the administered PTP complex) and intramuscularly with 0.9% sodium chloride solution in a volume of 0.5 ml (corresponding the volume of capreomycin injection), then removed from the experiment, having previously carried out all the necessary tests.

The introduction of drugs for a longer period (28 days) is impractical, because death of animals appears.

The criteria for assessing toxicity were: the number of dead animals and the timing of their death, the dynamics of body weight, the clinical picture of intoxication, ECG data, indicators of daily urine output, studies of blood electrolytes and pathological changes in tissues and internal organs, detected during autopsy of dead and surviving animals experiment at the end of the experiment.

Research results.

As a result of the studies, it was found that the animals tolerated the administration of the complex of drugs satisfactorily during the first 14 days of administration. There were no deaths of animals during this period. In rats, a decrease in appetite, a change in the color and consistency of feces, and piloerection were noted. With further continuation of the introduction, the condition of the animals worsened. Death of 1 rat was noted on the 20th day of administration of the complex of drugs.

To determine the dynamics of body weight, the animals were weighed twice - at the beginning of the experiment and before withdrawal from the experiment. The body weight of the rats was measured using an AND EK-600i laboratory balance (maximum weighing limit 600 g, readout resolution 0.1 g), manufactured by AND Company Limited, Japan. It was found that the applied anti-drug therapy complex caused a progressive decrease in the body weight of the animals, in contrast to the rats of the control group, in which a physiological increase in body weight was observed (table 1).

To determine the indicators of the electrical activity of the heart in rats, ECG indicators were recorded on a SHILLER AT-1 electrocardiograph (Switzerland) at a tape pulling speed of 50 mm / sec in standard leads I, II and III and in enhanced unipolar leads from the extremities in aVL aVR and aVF.

The heart rate (HR), indicators of time intervals of heart contractions (RR, PQ, QRS, QT) were assessed.

Changes in the indicators of time intervals of heart contractions were revealed in rats of the experimental groups in comparison with the control group of animals. The heart rate decreased in the rats of the experimental groups after 14 and 28 injections of the combination of drugs in comparison with the control group of animals (table 2).

The PQ interval did not change in the rats of the experimental groups throughout the entire experiment as compared with the control group of animals (Table 3).

The QT interval increased to 0.056 ± 0.002 and 0.067 ± 0.003 sec in the experimental group of rats on days 14 and 28 of the experiment (respectively) compared with the control group of animals (0.043 ± 0.002 sec).

The QRS interval increased after the 14th injection to 0.028 ± 0.003 sec compared to the control group of animals (0.022 ± 0.001 sec).

Qualitative changes were revealed: after the 14th administration of a complex of anti-tuberculosis drugs in one of eight rats, a heart rhythm disorder associated with electrical conduction was noted: the loss of the third or fifth R wave, in the presence of a P wave.

After 28 administration of a complex of anti-tuberculosis drugs, one of seven rats showed a high domed T wave in leads II, III and aVF.

The studied combination of drugs causes a decrease in the heart rate, the appearance of a high domed T wave, a violation of the heart rhythm associated with electrical conduction, an increase in the QT and QRS intervals.

To assess the functional state of the kidneys under conditions of spontaneous diuresis, rats were placed in special exchange cells, where urine was collected during the day. The daily water balance (the ratio between the amount of fluid introduced into the body and the amount of fluid excreted from the body during the day) was assessed, for which a container with a fixed amount of fluid was placed in the exchange cell, the remainder of which can be measured at the end of the experiment.

As a result of the study, the effect of the anti-TB drugs complex on the functional state of the kidneys was established. In the experimental group of animals, progressive polyuria was noted, both on the 14th and 28th days of observation, which indicates renal pathology (table 3).

Biochemical studies of blood and urine revealed hyperuricemia after 14 and, especially, after 28 days of PTP administration and a significant decrease in creatinine clearance compared to intact rats (Table 4).

Macroscopic examination of the animals withdrawn from the experiment after 14 and 28 days showed a change in the size and color of the kidneys (Fig. 1). Measurement of the mass of individual organs at autopsy for the purpose of morphometry and determination of their mass coefficients was carried out using an electronic laboratory balance E 2000D (maximum weighing limit 2000 g, readout resolution 0.001 g), manufacturer: Firm "Sartorius" AG, Germany. A progressive increase in the weight of the kidneys and liver of animals was established relative to the data of the control group. The results of the morphometric analysis of the relative mass of the internal organs of rats are presented in Table 4.

Determination of rat blood serum electrolytes was performed on a GemPremier 3500 device (USA). The results of the studies revealed a significant decrease in the content of potassium and calcium in the blood serum of experimental rats after 14 days of administration of PTP compared with control animals (Table 6). Differences in potassium content after 28 days with control were insignificant.

In the study of histological preparations of the heart tissues of rats that received the PTP complex for 14 days, no significant changes were found in comparison with the control. In the cortical substance of the kidneys of these rats, a rather pronounced expansion of the lumen of the convoluted and rectangular tubules, focal disaggregation, desquamation and death of the tubular epithelium, accumulation of homogeneous eosinophilic fluid (primary urine), the presence of granular casts in a small amount and signs of granular dystrophy in the epithelium of individual tubules were noted. and wrinkling of some glomeruli. In the medulla of individual rats in the rectal tubules of the accumulation of homogeneous eosinophilic fluid (urine).

In the study of histological preparations of the heart tissues of rats that received the PTP complex for 28 days, no significant changes were revealed in comparison with the control in 7 out of 8 rats. In 1 rat, the heart wall was thinned, stretched, cardiomyocytes with signs of granular dystrophy were found in the myocardium of the left ventricle. In the histological preparations of the kidneys in most of these rats, disaggregation and desquamation and atrophy of the tubular epithelium are expressed. There are groups of cells of the tubular epithelium wall with signs of vacuolar and granular degeneration. In the lumen of part of the tubules, granular casts or accumulation of homogeneous eosinophilic fluid. Some damaged shriveled and compacted glomeruli are found. In all rats, areas of a loose structure with dilated tubules alternate with areas of dense arrangement of tubules. Granular and homogeneous casts are found in the medulla.

Conclusions: Used complex of anti-TB drugs (moxifloxacin (36 mg / kg) + bedaquiline (36 mg / kg) + linezolid (54 mg / kg) + prothionamide (90 mg / kg) intragastrically and capreomycin (90 mg / kg) intramuscularly) in rats , old females, in 14 days caused kidney damage and a cardiotoxic effect, which manifested itself in a decrease in heart rate, the appearance of single extrasystoles and a heart rhythm disorder associated with electrical conduction, an increase in QT and QRS intervals against a background of a decrease in the content of electrolytes - potassium and calcium compared with control and severe kidney damage.

Example 1. Rat No. 3, 14 days of administration of the PTP complex.

On the ECG: an increase in the intervals PQ (0.08 sec) and QT (0.06 sec).

In the analyzes: a decrease in the content of potassium in the blood to 2.7 μg / ml; decrease in creatinine clearance to 0.4.

On opening the abdominal cavity: discoloration (whitish), size and mass of the kidneys. Histological examination showed signs of tubulointerstitial nephritis. Morphological examination of the heart - compliance with the norm.

Thus, the studies carried out made it possible to obtain a new experimental model of toxic multiple organ pathology in the form of combined damage to the heart and kidneys in experimental animals. Cardiotoxicity was manifested by the development of prolonged QT interval syndrome without morphological signs of changes in heart tissues. Kidney damage was characterized by functional (decreased creatinine clearance, hyperuricemia) and gross morphological changes in the tubular epithelium. The modeling of toxic combined pathology of the heart and kidneys is based on the complex effect of a number of anti-tuberculosis drugs with cardiotoxic potential of various mechanisms of action and in combination with a drug of direct nephrotoxic action, and factors (gender, age of animals) that initiate a cascade of functional disorders leading to the development of prolonged QT interval syndrome against the background of organic kidney damage. The proposed model is simple to execute, has good reproducibility, no lethal outcome of experimental animals, and a short period of development of the pathological condition.

Claims (5)

1. A method of modeling toxic damage to the kidneys and heart, which consists in causing toxic damage to the kidneys and heart in animals by daily administration of a complex of anti-tuberculosis drugs moxifloxacin, bedaquiline, linezolid, prothionamide and capreomycin for at least 2 weeks, while for modeling used female rats aged 10-12 months. and weighing 330-350 g. 2. The method according to claim 1, characterized in that moxifloxacin, bedaquiline, linezolid, protionamide are administered intragastrically in 0.1% starch gel. 3. The method according to claim 1, characterized in that capreomycin is administered intramuscularly. 4. The method according to claim 1, characterized in that the drugs are administered in the following doses: moxifloxacin - 36 mg / kg, bedaquiline - 36 mg / kg, linezolid - 54 mg / kg, prothionamide - 90 mg / kg, capreomycin - 90 mg / kg. 5. The method according to claim 1, characterized in that the drugs are administered almost simultaneously.

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