The effect of the drug “Bendamine” on the clinical and morphological parameters of dogs in heart failure

1Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, Pekarska Str., 50, Lviv, 79010, Ukraine 2Rivne State University for the Humanities, Plastova Str., 29-a, Rivne, 33028, Ukraine 3Dnipro State Agrarian and Economic University, S. Efremov Str. 25, 49600, Dnipro, Ukraine 4National University of Life and Environmental Sciences of Ukraine, Heroyiv Oborony Str., 15, Kyiv, 03041, Ukraine 5Institute of Agriculture of Carpathian region of Ukraine, NAAS of Ukraine, Grushevskogo Str., 5, Obroshino, 81115, Ukraine


Introduction
The results of research and summarizing the literature indicate that more than 10 % of domestic diseases account for the share of cardiovascular disease (Borgarelli et al., 2004;2007;Borgarelli & Buchanan, 2012;Reynolds et al., 2012;Hollmer et al., 2017). It is worth noting that heart disease in pets is not always clinically manifested, so pet owners often learn about cardiovascular disease in their pets too late. When detecting cardiovascular pathology in animals, it is essential to establish the degree of hemodynamic disorders, as well as diseases of other organs, which may be crucial for the course and prognosis (Cornell et al., 2004;Chetboul & Tissier, 2012;Fox, 2012;Lopez-Alvarez et al., 2015;Larouche-Lebel et al., 2019).
Experimental studies obtained in recent years indicate the urgency of developing drugs of complex action to prevent the development of cardiovascular disease in dogs. This requires a much deeper study of the pathogenesis of heart failure, including cardiomyopathy (Tilley et al., 2008;Merveille et al., 2015;Ramírez et al., 2016;Menciotti et al., 2017;Yata et al., 2019;Morgan et al., 2020).
Given the analysis of data from domestic and foreign researchers, the development of an easy-to-use and safe complex cardiac drug, the use of which will increase the effectiveness of treatment of animals and a wide range of cardiovascular pathologies, is on time (Undhad et al., 2012;Trofimjak & Slivinska, 2016;Zhulikova, 2016). In this aspect, it should be noted that the introduction of new cardiac drugs in the practice of veterinary medicine is exceptionally relevant and promising. Among the cardiopharmaceuticals in veterinary medicine, drugs based on pimobendan are of interest, as their action is to increase the contractile capacity of the heart and thus improve trophism. Due to positive inotropic and vasodilatory effects on the heart, failure pimobendan increases the strength of heart rate and reduces both preload and post-load (Oldach et al., 2019;Tjostheim et al., 2019;Varcholyak & Gutyi, 2019).
Given the positive effects of pimobendan on the body of animals in developing heart failure for the treatment and prevention of this pathology, it is necessary to use drugs containing the above active substance (Trofimiak & Slivinska, 2021).
According to some scientists, the primary pathogenetic changes in the formation of the pathology of the cardiovascular system are closely related to the overstrain of the mechanisms of the utilization of reactive oxygen species and the degree of disturbances in the system POL -AOC and are primarily determined by the immune system (Keene et al., 2019). Their imbalance underlies metabolic disorders in general, which significantly affects the state of cell membranes. In addition, among the metabolism indicators in the development of pathology, the system "Lipid peroxidation -Antioxidant protection system" is characterized by early deviations Nazaruk et al., 2021;Martyshuk & Hutyi, 2021). They play a leading role in adopting a healthy body to extreme conditions and the development of much deeper disorders of pathology . In particular, it is considered proven that changes in the course of lipid peroxidation processes are one of the pathogenetic mechanisms of cardiovascular pathology .
Violation of the antioxidant defense system of dogs under the influence of toxic substances and its correction with the use of various combinations of pharmacological and natural antioxidants is relevant and timely (Lavryshyn et al., 2016).
In veterinary medicine, one of the most promising drugs in the group of antioxidants are derivatives of 3oxypyridine, in particular ethylmethylhydroxypyridine succinate -a direct-acting antioxidant. The mechanism of action of this drug is antioxidant and membrane-protective action . It inhibits the processes of lipid peroxidation and promotes the activity of superoxide dismutase, which neutralizes the starting radical of free radical processes. Under conditions of tissue ischemia, this drug enhances the compensatory activation of aerobic glycolysis, reduces the degree of inhibition of oxidative processes in the Krebs cycle, and promotes the activation of ATP synthesis. Under the influence of ethylmethylhydroxypyridine succinate, a decrease in cardiomyocytes of a specific protein was found, which accelerates apoptosis. Transmembrane ionic currents are also modulated: slow current through calcium channels is slowed down, fast sodium channel blockade is averted, and the fast-activated potassium current component of delayed rectification (JKR) is blocked (Varkholiak & Gutyj, 2018;Varkholiak et al., 2019;.
Thus, ethylmethylhydroxypyridine succinate has a complex effect on the metabolism of the myocardium of animals.
A combined approach to treating animals with heart failure, which is based on the use of phosphodiesterase-3 inhibitors and antioxidants, is relevant. Because antioxidant compounds can protect healthy cells and tissues of dogs from the development of unwanted side effects caused by the development of heart failure. In addition, it is known that the development of this pathology is characterized by oxidative stress, which causes a particular metabolic activity of antioxidants (Martyshuk, 2016;Varkholiak & Gutyj, 2019).
Thus, the use of antioxidants in the heart for heart failure in dogs can improve the redox adaptation of heart muscle cells and protect them from the harmful effects of free radicals.
The study aimed to investigate the effect of the drug "Bendamine" on dogs' clinical and morphological parameters in heart failure.

Materials and methods
Two groups of dogs were formed for research: control (healthy) and experimental (sick). The 30-day experiment included ten dogs of the control group and ten dogs of the experimental (sick) breed of spaniel and dogs 10-12 years old.
The treatment regimen for sick dogs: furosemide was administered orally on an empty stomach at 2 mg/kg every 12 hours; enalapril was administered orally at 0.5 mg/kg every 12 hours; bendamine was administered orally on an empty stomach at 20 mg/kg every 24 hours. It is recommended to divide the dose into two doses.
The following parameters were determined in heparinstabilized blood: the number of erythrocytes was photoelectrocolometrically according to the method of E. S. Gavrilets et al. (1966); the number of leukocytes -with the help of Goryaev's grid in the counting chamber; hemoglobin content -by the method of L. M. Pimenova et al. (1975). The hematocrit value was determined by the micro method in the modification of Todorov J. According to the formulas, blood indices were mathematically calculated according to the number of erythrocytes, blood hemoglobin content, and hematocrit content. The leukogram was determined by morphological parameters of the number of white blood cells with differentiated counting of different forms of leukocytes (Vlizlo et al., 2012).
All animal manipulations were carried out following the European Convention for the Protection of Vertebrate Ani-mals Used for Experimental and Scientific Purposes (Strasbourg, 1986).
The analysis of research results was performed using the software package Statistica 6.0. Student's t-test assessed the probability of differences. The results were considered plausible at P ≤ 0.05.

Results and discussion
When determining the dynamics of the incidence of dogs with cardiovascular pathology based on the analysis of journal reports in veterinary clinics of Lviv, it was found that in the period 2016-2019, this pathology was manifested in 7.8-12.7 % of surveyed animals, with less than half of the cases, were diagnosed with chronic heart failure (5.3-8.1 % of the examined animals). The disease had no seasonal dynamics. The development of heart failure in dogs was accompanied by various clinical symptoms depending on the stage of the process. Initially, most owners observed an increase in the frequency of respiratory movements during sleep, then -a decrease in endurance during walks, shortness of breath, and cough. With the development of the pathological process, there was pulmonary edema (recorded in one dog), then -ascites. Decreased myocardial contractility, blood stasis, and hemodynamic disturbances lead to the development of hypoxia, so in sick animals, there was a decrease in the rate of filling the capillaries of mucous membranes, pallor, cyanosis. Body temperature in sick animals was within physiological parameters, while heart rate and respiration were increased compared to healthy dogs due to the activation of compensatory mechanisms.
Monitoring the frequency of respiratory movements in dogs during sleep is a relatively new indicator used in clinical practice. Raising the CDR with high specificity and sensitivity can predict the likelihood of pulmonary edema, assess the effectiveness of diuretic therapy in congestive heart failure, select the minimum effective dose of diuretic, predict the onset of decompensation, and timely adjust the treatment regimen.

Table 1
Indicators of the frequency of respiratory movements of dogs with the development of heart failure and the action of the drug "Bendamine" (M ± m; n = 10)

Control
Research Before treatment After treatment Frequency of respiratory movements for 1 min in a state of sleep 12-20 30-58 20-35 The therapy application in the experimental group significantly reduced the frequency and severity of shortness of breath and increased endurance during exercise. There was also a decrease in the frequency and duration of cough attacks.
After treatment, signs, and intensity of ascites decreased in dogs of the experimental group. It has been suggested that this is due to an increase in myocardial contractile function due to the positive ionotropic action of bendamine.
The owners also recorded an improvement in appetite in animals after 30 days of therapy. Even in the dogs of the experimental group, after treatment, physical activity and emotional reactions improved. They were less depressed and more inclined to communicate with the owners.
A decrease in cardiac coefficients was found during radiography in the experimental group after the therapy application (Table 2). An electrocardiographic study showed a reduction in heart rate in 10 dogs, which may be due to the negative chronotropic effect of the drug, which is characteristic of the group of phosphoesterases. The decrease in heart rate, in turn, is due to peripheral dilatation, which is achieved by inhibiting phosphoesterase III. In six dogs out of ten, ventricular ectopic contractions were detected, the diversity of which was 18-40 for 20 min and was reduced to 10-27, respectively, on day 30 of the recommended therapy (Fig. 1).

Table 2
Radiographic parameters in dogs with the development of heart failure and the action of the drug "Bendamine" (M ± m; n = 10) Echocardiographic examination of all dogs showed an increase in the ratio of LP/Ao, which indicates the presence of a trio dilation, which is characteristic of patients with this stage of heart failure. After treatment, there was a slight decrease in the ratio of LP/Ao. The positive effect of the drug is associated with increased myocardial contractile function and decreased postload (positive ionotropic and vasodilating effect of bendamine). After all, a decrease in pressure in the left ventricle leads to a decrease in pressure in the left atrium and pulmonary veins.
The index of left ventricular contractility increased in the experimental group after treatment, which correlates with data on the pharmacodynamic properties of the drug (Fig. 2  and 3).
Thus, echocardiographic changes in treated dogs were manifested by an increase in left ventricular systolic function and a decrease in left atriodilation.
In treating dogs with heart failure, we used the cardiac drug "Bendamine", which was fed to animals for 30 days. It was found that before treatment, the number of erythrocytes in the blood of dogs with clinical signs of heart failure was higher by 18.2 %, while the hemoglobin level was lower by 1.8 % compared to the control group of dogs.
After the course of treatment, it was found that in the blood of dogs of the experimental group, the number of erythrocytes and hemoglobin levels ranged between 5.8 ± 0.06 T/L and 143.5 ± 4.7 g/L, respectively (Table 3).
It was also found that the hematocrit value in the control group of dogs ranged from 0.43 ± 0.04 L/L, while in the experimental group of dogs with clinical signs of heart failure, it decreased by 11.6 %. On the 30th day of the experiment, after a course of treatment and use of the drug "Bendamine" in the blood of the experimental group of dogs found, an increase in hematocrit by 26.3 % compared with the blood taken before treatment of animals.  Hematological parameters of the blood of dogs with heart failure and the action of the drug "Bendamine" (M ± m; n = 10) When determining red blood cell indices in dogs of the control group, it was found that the average hemoglobin content in one erythrocyte was 23.7 ± 0.65 pg, the average concentration of hemoglobin in erythrocytes ranged from 30.3 ± 0.58 g/100 ml. In contrast, the average erythrocyte volume was 78.2 ± 3.4 μm 3 . In patients of dogs of the experimental group, red blood cell index values probably changed, namely: the average erythrocyte volume and the average hemoglobin content in one erythrocyte probably decreased by 25.3 and 16.9 %, while the average hemoglobin concentration in erythrocytes probably increased by 11.2 % relative to the control group of dogs.
After a course of treatment for dogs with heart failure, it was found that on the 30th day of the experiment, the indicators of red blood cell indices of the experimental group of dogs fluctuated within physiological limits.
When determining the number of leukocytes in the blood of dogs with heart failure, we found a slight increase throughout the experiment, were at the beginning of the investigation, they increased by 5.5 %, and on the 30th day of treatment -by 7.7 % compared with the control group (Table 4).

Table 4
Blood leukogram of dogs with heart failure and the action of the drug "Bendamine" (M ± m; n = 10)
The analysis of the leukogram of sick animals of the experimental group showed an increase in the percentage of monocytes to 6.1 ± 0.6 %, while after treatment, this figure was 5.1 ± 0.8 %.
During the treatment period, a relatively good prognostic indicator is an increase in the percentage of lymphocytes in the blood to 15.3 ± 2.9 % and eosinophils to 3.0 ± 0.4 %.
After treatment in the blood of the experimental group of dogs on the 30th day of the experiment, the percentage of both segmental and rod-shaped neutrophils, compared to the period before treatment, decreased by 6.8 and 1.2 %, respectively.

Conclusions
Studies confirm the feasibility of using the drug "Bendamine" in dogs with the development of heart failure. The use of the cardiac drug "Bendamine" in dogs with heart failure positively affected the restoration of hematopoiesis and the normalization of morphological parameters of the blood.