The efficiency of the drug based on butaphosphane, B vitamins and L-carnitine in the treatment of horses

An important feature of all living organisms, acquired in the process of evolution, is the ability to adapt to various external influences and changes in the environment, to maintain homeostasis and regulatory processes in organs, systems and an organism as a whole. Modern methods of intensive animal husbandry on farms of different types with the use of highly productive breeds and the introduction of industrial technology of animal husbandry differ significantly from traditional methods of breeding. In this case, the physiological capabilities of an animal's organism often can’t change as quickly as required by the conditions of industrialized livestock, which leads to stresses. An animal under stress loses weight, becomes weaker, the resistance to diseases reduces, reproduction ability and metabolism changes, in particular, energy that might be used for growth and development of the organism is lost. It has been established that due to stress in animals, livestock enterprises suffer significant economic losses (Hrabovskyi, 2012; Suprun, 2012; Martyshuk et al., 2019; 2020; Slivinska et al., 2020; Grymak et al., 2020; Leskiv et al., 2021; Nazaruk et al., 2021).

The range of veterinary preparations and feed additives at the market of Ukraine is being actively expanded with new versions of domestic and imported products. Today most of domestic manufacturers of veterinary medicinal products produce generics (analogue medicinal products) of different pharmaceutical groups. To improve the resistance to stress situations, prevent malnutrition, improve resistance, preservation and productivity of agricultural animals, veterinary specialists widely use veterinary medicines and biologically active supplements (Zhyla et al., 2015; Chalmeh et al., 2020).

The adherence to the system of preclinical and clinical trials of veterinary medicines that meets international requirements is a key guarantee for the development of effective, safe and competitive drugs. It is important to assess the impact of the drug on metabolic processes in the animal organism estimating hematological, biochemical and immunological parameters, which objectively characterize safety of the drug, clinical picture and recovery, side effects and recurrence (VICH GL9, 2000; Kotsiumbas et al., 2006; VICH GL43, 2008; Zhyla, 2011; Kotsiumbas et al., 2013; Zhyla et al., 2016).

L-Cyn, a solution for injection, is a domestic drug (AB-09278-01-19), which effect is due to the combination of active substances, namely: butaphosphane, B vitamins (vitamin B12, vitamin B3) and L-carnitine, ATC vet classification code — QA12C. Other mineral additives, QA12CX91 Butaphosfan.

Butaphosphane is an organic compound of phosphorus, discovered by Bayer in 1926, affects a number of assimilation processes in animals, stimulates protein synthesis, accelerates the growth and development of animals, normalizes liver function, increases non-specific resistance of an organism, promotes the formation of bone tissue. In stressful situations, butaphosphane normalizes the level of stress hormone — hydrocortisone, thereby improving blood glucose utilization and helping to conserve the body's energy resources (Martyshuk & Gutyj, 2019).

Vitamin B12 (cyanocobalamin) plays an important role in the regulation of hematopoietic functions, participates in the purine synthesis, pyrimidine bases, nucleic acids necessary for the erythropoiesis, and normal protein; fat and carbohydrate metabolism of vitamin B are impossible without B12.

The effectiveness of the combination of butaphosphane and cyanocobalamin has been studied and described in the scientific literature (Kreipe et al., 2011; Nuber et al., 2015; Chalmeh et al., 2020). Veterinary drugs combining butaphosphane and cyanocobalamin, such as Catosal (Bayer HealthCare LLC, USA), Cyanophore (Vetsintez LLC, Ukraine), Vetozal (O.L.KAR, Ukraine) are heavily marketed. Combinations of these drugs with carnitine or nicotinamide are also known.

Nicotinamide (vitamin B3, (PP)) stimulates the production of nicotinadine dinucleotide phosphate (NADP) and nicotinadenine dinucleotide (NAD), which regulates the course of most redox reactions, providing normalization of many types of metabolism (including the energetic one). This vitamin takes part in the metabolism of fats, proteins, amino acids, purines, tissue respiration, glycogenolysis. Nicotinamide has detoxifying properties, is rapidly distributed in tissues and crosses the placental barrier (Lourens & Benyt, 1991; Nelson & Cox, 2017).

L-carnitine (vitamin B11) which is a product of the biosynthesis of lysine and methionine performs a number of important functions in an organism, including: detoxification, stimulation of metabolic processes, strengthening of blood vessels, stimulation of tissue regeneration. Its use helps to increase the endurance of an organism, improves heart function, increases muscle mass by accelerating metabolic processes in cells, promotes the penetration of vitamins and minerals and the excretion of breakdown products into cells, reducing the risk of fatty liver disease (Lourens & Benyt, 1991; Nelson & Cox, 2017; Tarasenko et al., 2007).

Of scientific interest is the study of the effectiveness of a new combination of a balanced complex of active substances, such as butaphosphane, vitamin B12, nicotinamide (vitamin B3) and L-carnitine (vitamin B11). The aim of our work was to study the therapeutic efficacy of L-Cyn as a tonic in horses, with an original combination of four active substances under the influence of various stress factors: physical exertion, and as a part of a comprehensive treatment of multifunctional diseases.

Material and methods of research

Clinical trials of the drug were performed at the Komarnivsky Research Center (Peremozhne village, Horodok district, Lviv region) on working horses aged 5–15 of different sexes. Laboratory studies were performed in the laboratory of clinical and biological studies of State Scientific and Research Control Institute of Veterinary Medicinal Products and Feed Additives.

To solve this problem, two experiments were conducted: the first one studied the effectiveness of L-Cyn in horses by physical exertion; the second one studied the effectiveness of L-Cyn in a stallion as a part of complex therapy in the treatment of intoxication with symptoms of acute renal failure.

Experiment 1. During the clinical and diagnostic examination of livestock on the farm, 5 working horses of different sex and age were selected. The general clinical condition and fatness of the selected animals were satisfactory. Blood samples were taken from the experimental horses before the beginning of L-Cyn administration and 10 days after the last administration of the drug.

The drug was administered intramuscularly at a dose of 20 ml once a day for 5 consecutive days. The clinical condition of the experimental animals was observed for 21 days from the start of the administration of the drug.

Experiment 2. Treatment of acute intoxication in a stallion. After a clinical examination of the sick stallion, the blood was taken from the jugular vein for laboratory examination. Based on clinical data and the results of blood tests, the doctor diagnosed intoxication with symptoms of acute renal failure and prescribed the treatment, which included analgetics, antispastics, anti-inflammatory agents, antihistamines and rehydration therapy. From the second day of treatment, the complex therapy also included the studied drug at a dose of 25 ml with intravenous administration for 5 consecutive days.

The morpho-functional state of the animals before and after the drug administration was assessed by the clinical condition, morphological and biochemical parameters of the blood, which were determined according to generally accepted methods (Lapovets et al., 2011; Vlizlo et al., 2012; Kotsiumbas et al., 2014). To assess the general condition and characterize changes in the main parameters of the animal body, a number of laboratory tests that met the requirements for clinical trials of drugs of this pharmacotherapeutic group were selected (European Treaty Series/123; VICH GL9, 2000; Levchenko et al., 2002; Horbatiuk, 2004; VICH GL43, 2008; Lapovets et al., 2011; Kotsiumbas et al., 2013). EDTA-stabilized horse blood samples were used for morphological studies, while serum was used for biochemical studies. Hematological examinations (number of erythrocytes, leukocytes, hemoglobin content, hematocrit, erythrocyte indices (МСНС, МСV, МСН) were performed using Mythic 18 Vet automatic analyzer using the principle of impedance. The leukogram was examined by microscopy of blood smears stained with Romanovsky-Gimza dye.

The content of total protein, glucose, urea, creatinine, activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (LF), lactate dehydrogenase (LDH), creatine kinase (analysis of blood) in the serum of horses was determined using a semi-automatic biochemical analyzer HumaLyzer 3000, using standardized kits Human Diagnostics Worldwide (Germany).

The obtained results were processed statistically, estimating the probability of the difference of parameters (P < 0.05) according to Student's criterion. The statistically significant result was the difference between the values at which the level of probability of difference (P) didn’t exceed 0.05, which is a common approach in laboratory studies.

The presence and the degree of significant differences in the studied parameters before and after the administration of the drug indicate the intensity of the specific pharmacological effect of the drug (CVMP/EWP/81976/2010).

The Results and their interpretation

The first experiment examined the effectiveness of L-Cyn in horses during physical exertion. The laboratory studies before the use of the drug demonstrated that most hematological and biochemical parameters of the blood corresponded to the physiological norm for such animals (Tables 1, 2).

However, the number of leukocytes corresponded to the maximum physiological norm, and the activity of LDH exceeded the norm more than twice. This high activity of the enzyme is apparently the result of chronic physical exertion, because LDH is an enzyme of carbohydrate metabolism, which is the most active in skeletal and cardiac muscles, kidneys, liver and erythrocytes. LDH catalyzes one of the most important glycolysis reactions — the interconversion of pyruvic and lactic acids and is an important biochemical diagnostic test for assessing muscle function under conditions of anaerobic glycolysis by physical exertion (Tarasenko et al., 2007; Lapovets et al., 2011; Nelson & Cox, 2017).

On the 10th day after the administration of the drug the analysis of hematological parameters in horses demonstrated a probable increase, within the norm, of hemoglobin by 12% (P ≤ 0.05), tendency to increase in the number of erythrocytes and hematocrit and probable decrease in the number of leukocytes and eosinophils by 19.4 and 41.9%, respectively compared with the parameters before the drug administration. (Table 1).

The analysis of biochemical parameters during this period, showed a probable 4.1 times (P ≤ 0.05) increase in the activity of creatinephosphoric acid (catalyzes the formation of high-energy compound creatine phosphate from ATP and creatine, which is consumed by the body during physical exertion), 1,6 times (P ≤ 0.05) increase in the activity of AST (aminotransferase, which demonstrated the greatest enzymatic activity in the heart muscle, is contained in significant quantities in the muscles of the musculoskeletal system and hepatocytes) and a 1.6 times decrease (P ≤ 0.05) in LDH activity. The enzymes CK, AST and LDH are markers of cardiomyocytes and the myocardium in general (Tarasenko et al., 2007; Lapovets et al., 2011; Nelson & Cox, 2017).

At the same time, a tendency to increase the content of creatinine and total bilirubin was observed in serum. Glucose and urea levels did not change significantly. It should be noted that the fluctuations of all these parameters did not exceed their physiological norm for horses (Table 2).

Table 1

Hematological parameters of horses after administration L-tsyn, (М ± m, n = 5)

Note: hereinafter* – Р ≤ 0,05 compared to the period of use of the drug

Table 2

Biochemical parameters of horse serum when using L-tsyn (M ± m, n = 5)

On the 8th-10th day after the last administration of the drug, the clinical condition of the animals improved, the horses became more active, and their appetite increased. No newly diagnosed cases have been reported in animals during the clinical trial period. No adverse reactions or negative events were observed in horses during the administration of the drug and after treatment.

In the second experiment, a veterinary of the farm during a clinical examination of a sick animal diagnosed behavioral disorders, anxiety, loss of appetite, muscle tremor, abdominal wall tension, painful on palpation, thin stool, decreased urination. The process was acute. The animal's body temperature ranged from 36.0 to 36.5 °C, and the respiratory rate was 18 breaths movements / min, pulse - 40 beats per minute.

The clinical condition of the animals was constantly monitored. The improvement of the clinical condition was observed on the 3rd day of treatment. Clinical symptoms of the disease disappeared on the 7th-8th day.

Analyzing the results of morphological and biochemical parameters an increase in the number of leukocytes was observed in the blood of the seek horse, the leukogram demonstrated an increase in the percentage of stab neutrophils and a decrease in the number of erythrocytes (Table 3).

Table 3

Results of morphological and biochemical studies of a sick horse

In examining biochemical parameters in serum, the increase of total protein, urea, creatinine, total cholesterol and phosphorus was observed, simultaneously with a decrease of calcium, as well as a significant increase in the activity of aminotransaminases and AP. Normalization of blood parameters of the sick animal was observed on the 10th day after the beginning of treatment (Table 3).

Thus, based on the results of the treatment, it can be concluded that as part of the therapy in treatment of intoxication in horses, L-Cyn helped to improve the clinical condition, normalizing morphofunctional parameters of the body.

Conclusions

By physical exertion in working horses, L-Cyn, a solution for injection, demonstrated therapeutic efficacy, activating the processes of hematopoiesis, improving the physiological state of the body and helping to improve the overall tone. The drug did not demonstrated unexpected negative local and general effects.

The use of L-Cyn in horses as a part of complex therapy in the treatment of intoxication contributed to the rapid improvement of the clinical condition and normalization of morpho-functional parameters of the body. The drug demonstrated hepato- and nephroprotective properties, reduced the symptoms of intoxication, helped to restore the normal physiological state of the animal.

The use of L-Cyn in clinical veterinary practice will expand the list of drugs in this group and allow veterinary practitioners to use it both for monotherapy and for the comprehensive treatment of multifunctional diseases in horses.

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