WO2022018705A1 - A pharmaceutical composition for treating cardiovascular and cerebrovascular disorders,diseases in pregnant women and improving exercise tolerance - Google Patents

A pharmaceutical composition for treating cardiovascular and cerebrovascular disorders,diseases in pregnant women and improving exercise tolerance Download PDF

Info

Publication number
WO2022018705A1
WO2022018705A1 PCT/IB2021/056729 IB2021056729W WO2022018705A1 WO 2022018705 A1 WO2022018705 A1 WO 2022018705A1 IB 2021056729 W IB2021056729 W IB 2021056729W WO 2022018705 A1 WO2022018705 A1 WO 2022018705A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
groups
treatment
study
group
Prior art date
Application number
PCT/IB2021/056729
Other languages
French (fr)
Inventor
Mykola Ivanovych GUMENIUK
Original Assignee
M.T.K. Medical Center Limited Liability Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from UAA202004724A external-priority patent/UA127789C2/en
Application filed by M.T.K. Medical Center Limited Liability Company filed Critical M.T.K. Medical Center Limited Liability Company
Publication of WO2022018705A1 publication Critical patent/WO2022018705A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to the field of medicine, namely to the drugs for prevention and treatment of diseases of various etiologies, in particular, the diseases of the cardiovascular system (coronary heart disease, stable angina pectoris, peripheral artery disease), acute cerebrovascular disorders, chronic cerebrovascular disorders, the diseases in pregnant women and fetal development during pregnancy (preeclampsia in pregnant women, fetal distress, intrauterine growth restriction), as well as for improvement of exercise tolerance of human and status of human body in asthenia.
  • diseases of the cardiovascular system coronary heart disease, stable angina pectoris, peripheral artery disease
  • acute cerebrovascular disorders chronic cerebrovascular disorders
  • the diseases in pregnant women and fetal development during pregnancy preeclampsia in pregnant women, fetal distress, intrauterine growth restriction
  • the cardiovascular system diseases include coronary heart disease, stable angina pectoris, and peripheral artery disease.
  • CHD coronary heart disease
  • Myocardial demand in oxygen is primarily determined by heart rate, myocardial contractility, heart size and blood pressure. An increase in any of these parameters increases myocardial demand in oxygen.
  • coronary arteries have sufficient reserve of dilation, which provides, if necessary, a fivefold increase in coronary circulation. Restrictions on myocardial blood supply occur due to a decrease in the coronary artery lumen by more than 50%. Mismatch between coronary circulation and metabolic demands of the heart muscle is always accompanied by myocardial ischemia manifested clinically by angina attack, severe heart rhythm and conduction disorders, and, in some cases, myocardial infarction and sudden death. According to the Order N° 54 of the Ministry of Health of Ukraine on 2001 , the following forms of coronary heart disease are distinguished:
  • vasospastic angina angiospastic, spontaneous, variant, Princemetal
  • Cardiosclerosis focal cardiosclerosis: o postinfarction cardiosclerosis (with indication of the myocardial infarction history, its location and time of development); o chronic heart aneurysm; o focal cardiosclerosis (without indication of myocardial infarction).
  • the main etiological factor of CHD is coronary artery atherosclerosis.
  • factors that contribute to its development important are: hyperlipidemia, hypertension, high-calorie diet, obesity, diabetes, smoking, hypodynamics, genetic predisposition, age, and male gender.
  • Myocardial ischemia associated with impairment of the coronary arteries of another nature (ex., rheumatism, septic endocarditis, etc.), as well as hemodynamic disorders of non-coronary nature (aortic valve disease)
  • Coronary artery atherosclerosis is found in 95% of patients with CHD. Growing atherosclerotic plaque, hemorrhage into the plaque base with its disintegration, and the formed thrombus leads to a narrowing of the lumen or complete obstruction, resulting in organic obstruction of the coronary artery.
  • Atherosclerosis is a common cardiovascular system disease. This pathological process underlies the frequent causes of mortality and disability, such as coronary heart disease, ishemic stroke, chronic forms of cerebrovascular insufficiency, peripheral thrombosis etc. Atherosclerosis is a systemic disease and often leads to simultaneous impairment of the vessels of the brain, heart, kidneys, and limbs. Often, the first signs of venous insufficiency are manifested in old age but can be manifested in middle and even young age.
  • Atherosclerosis is a chronic disease of the vessels of elastic and muscular- elastic type, i.e., large arteries.
  • the main pathogenetic events of atherosclerosis are lipid, or rather cholesterol, intima infiltration and growth of connective tissue throughout the vascular wall. According to some authors, it is serum cholesterol that is an actual risk factor for both atherosclerosis in general and its main consequence - coronary heart disease.
  • lipid infiltration has the form of a so-called fat strip, which does not rise above the surface of the vascular wall and does not manifest itself clinically.
  • vascular endothelium produces vasodilators, such as prostaglandin, prostacyclin, and nitric oxide (NO) endothelium-derived relaxing factor (abbreviated EDRF), which also are antiplatelet agents.
  • vasodilators such as prostaglandin, prostacyclin, and nitric oxide (NO) endothelium-derived relaxing factor (abbreviated EDRF), which also are antiplatelet agents.
  • Arginine d-guanidine-a-aminovaleric acid
  • Arginine is a basic a-amino acid, the L- form of which is a semi-essential amino acid.
  • Arginine interacts with SH groups (nitrate receptors) in vascular smooth muscle cells, including coronary arteries, to form nitric oxide (NO), which is similar to EDRF in structure and effect. Due to its properties, arginine dilates arterioles and peripheral veins, reduces total peripheral vascular resistance, reduces venous outflow, and dilates pulmonary vessels, which reduces resistance in the small circulation and leads to regression of the symptoms in case of pulmonary edema, and reduces end-diastolic pressure and ventricular volume, thereby reducing myocardial oxygen demand. Additionally, arginine dilates the coronary arteries and prevents their spasm, reduces the diastolic tension of the ventricular wall, resulting in improved coronary circulation in the ischemic area.
  • SH groups nitrate receptors
  • NO nitric oxide
  • Tivortin drug comprising arginine in the form of a salt, such as arginine hydrochloride, and water for injections, is known in the prior art for a combination therapy of coronary heart disease.
  • the disease can be of varying severity (e.g, mild or severe), and depending on the disease severity, a patient may need treatment or inpatient care in specialized facilities or may be treated on an outpatient basis. In certain cases, for example, when a patient suffers from a relatively mild angina, treatment of a patient in inpatient facilities is pointless, otherwise treatment of a patient on an outpatient basis is advisable. It is known from open sources that substances of the so-called metabolic corrector class, such as inhibitors of oxidation of free fatty acids that affect the activity of enzymes involved in biochemical reactions, are widely used in cardiometabolic therapy.
  • levocarnitine also known as L- carnitine.
  • Levocarnitine facilitates the supply of long-chain fatty acids to the mitochondria of cells, thus providing a substrate for oxidation and energy production, which significantly improves the restoration of heart muscle cells in myocardial infarction.
  • Levocarnitine inhibits the formation of atherosclerotic plaques in the blood vessels and promotes the resorption of the plaques that have already formed.
  • levocarnitine reduces the contribution of the above factors, such as hyperlipidemia, high-calorie diet, obesity, diabetes, smoking, hypodynamics, and age, to the development of coronary heart disease.
  • compositions for the treatment of coronary heart disease containing both arginine and levocarnitine are unknown in the prior art. Publications mention the use of a drug containing arginine and a drug containing levocarnitine in the treatment of coronary heart disease. However, these drugs are not used simultaneously, rather there is a time interval between the administration of two different drugs.
  • Acute cerebrovascular disorders and chronic cerebrovascular disorders include, in particular, acute form or chronic form of cerebrovascular insufficiency.
  • Acute or chronic form of cerebrovascular insufficiency is the result of progressive insufficiency of blood supply to the brain, which leads to the development of multiple small focal necrosis in the brain tissue and the gradual dysfunction of the brain.
  • the main reasons that cause the onset and development of chronic cerebrovascular insufficiency are hypertension and atherosclerosis, depending on which hypertensive and atherosclerotic encephalopathy are distinguished.
  • Atherosclerosis is a chronic disease of the arteries, which develops due to impaired lipid and protein metabolism and is accompanied by the deposition of various fractions of cholesterol and proteins in the form of the plaques in the blood vessels, followed by the growth of the connective tissue (sclerosis), and calcification (calcium deposition), which results in deformation of the vessels and narrowing of the vessel lumen, in some cases, up to obturation (blockage of the vessel).
  • Atherosclerosis leads to organ and/or general circulatory disorders. Depending on the severity of atherosclerosis and its localization in the vascular system, certain clinical manifestations are formed, and some of them are distinguished into individual syndromes and even nosological forms.
  • Factors that lead to atherosclerosis are divided into endogenous (heredity, sex, and age) and exogenous (intoxication, hypertension, metabolic diseases, overeating, etc.). The following factors can lead to atherosclerosis: - age;
  • Atherosclerosis of the brain vessels is a pathological process characterized by the deposition of the plaques (referred to atherosclerotic plaques) on the walls of the large brain vessels, with their subsequent growth and replacement by connective tissue.
  • the brain vessel lumen is narrowing gradually, and circulation insufficiency is developing. Lesions of the internal and external carotid arteries are the most common. The cause of this condition lies in the impairment of lipid metabolism and protein metabolism.
  • Atherosclerotic plaques are detected in young people aged 20 years, but the highest prevalence of the disease is observed in adults aged 50 years or more, and more often in men than in women. The high prevalence of this disease among the population associates it even with one of the manifestations of aging.
  • Symptoms of cerebrovascular atherosclerosis include certain symptoms associated with patient complaints, which in severe cases significantly reduce the quality of life, namely sleep problems, such as insomnia, disturbing dreams, difficulty with waking up and problems with re-falling asleep; decreased body sensitivity; headaches; often repeated; gait change and incoordination; vision problems, tinnitus; emotional changes, such as irritability, depression, tearfulness, anxiety; hot flashes and facial sweating; rapid fatigue, constant weakness and inattention; trembling of the chin and limbs; memory problems or disorders, short-term memory problems.
  • sleep problems such as insomnia, disturbing dreams, difficulty with waking up and problems with re-falling asleep
  • decreased body sensitivity headaches; often repeated; gait change and incoordination
  • vision problems, tinnitus emotional changes, such as irritability, depression, tearfulness, anxiety; hot flashes and facial sweating; rapid fatigue, constant weakness and inattention; trembling of the chin and limbs; memory problems or disorders, short
  • Treatment for this disease is aimed at restoring metabolic processes, and prevention of bad cholesterol deposition on the blood vessel walls. Attention is also paid to restoration of the blood circulation and normalization of the brain tissue supply. Treatment is comprehensive and includes lifestyle changes and drug therapy.
  • the lifestyle changes include choosing a diet that restricts the intake of lipids, breaking bad habits, increasing physical activity, avoiding stress, and reducing the level of psycho-emotional stress.
  • the drug therapy focuses on the use of lipid-lowering drugs, antiplatelet, antihypertensive and antioxidant drugs, drugs for microcirculation improvement, and symptomatic therapy.
  • the drug therapy usually takes a long period of time and depends on the disease severity.
  • Ischemia is a temporary dysfunction or permanent impairment of the organ tissue or the whole organ due to a local decrease in blood supply caused by a vascular factor (narrowing or complete obstruction of the artery lumen).
  • ischemia depend on the extent and rate of circulation parameters reduction, duration of ischemia, tissue sensitivity to hypoxia, and general status of the body.
  • the most sensitive to ischemia are the central nervous system organs, myocardium, and kidney tissue.
  • Ischemia differs from hypoxia, which is an oxygen starvation of the organ tissue due to disorders of external and internal (tissue, cellular) respiration.
  • Ischemia is characterized by relative or absolute blood supply insufficiency, which is manifested by both local tissue hypoxia and metabolic disorders due to insufficient supply of the nutrients.
  • Ischemia is a dynamic, and usually potentially reversible, process.
  • the possibility of ischemic necrosis (heart attack) of the organ tissue directly depends on the duration and extent of the local circulation decrease.
  • Chronic cerebral ischemia is a slowly progressive brain dysfunction caused by diffuse or small-cell damage of the brain tissue during long-standing cerebral insufficiency.
  • the treatment for chronic cerebral ischemia involves a combination therapy including: antiplatelet drugs, such as the drugs that reduce the blood ability to thicken and improve the blood rheological properties by preventing the aggregation of erythrocytes and platelets; antioxidants and antihypoxants, such as the drugs that bind free radicals, delay oxidative processes, increase the body resistance to oxygen deficiency, affect intracellular redox processes indirectly, facilitating the transport of oxygen from the blood to the tissue, and improving the blood supply to the brain.
  • antiplatelet drugs such as the drugs that reduce the blood ability to thicken and improve the blood rheological properties by preventing the aggregation of erythrocytes and platelets
  • antioxidants and antihypoxants such as the drugs that bind free radicals, delay oxidative processes, increase the body resistance to oxygen deficiency, affect intracellular redox
  • This clinical protocol describes the use of magnesium sulfate and acetylsalicylic acid for the treatment of chronic cerebral ischemia of the different severity.
  • Preeclampsia in pregnant women is a manifestation of either hypertension after the 20th week of the pregnancy in a woman whose blood pressure was normal before, or an increase in hypertension that existed before the 20th week of the pregnancy, or development of proteinuria, or both, and may be accompanied with the signs of other organs/systems impairment in the pregnant woman body.
  • This disease affects both a mother and a fetus. The disease can result in the simultaneous manifestation of increased systemic vascular resistance, increased propensity of platelets to aggregate, increased activation of the coagulation system, as well as endothelial dysfunction.
  • the reason is a dysfunction of the placenta or impairment of the placenta implantation, which is confirmed by the rapid cessation of this condition after childbirth. Functional and morphological changes occur in the kidneys of pregnant women, glomerular filtration decreases, and symptoms of kidney impairment may appear.
  • the method of treatment of preeclampsia in pregnant women depends on the degree of threat to the woman and fetus, the duration of the pregnancy and the degree of the fetal development.
  • Antihypertensive drugs and magnesium sulfate are used the most commonly (see article titled DIAGNOSIS, EVALUATION AND MANAGEMENT OF HYPERTENSIVE DISORDERS IN PREGNANCY: BASIC CONCLUSIONS. Clinical Practical Guide of the Society of Obstetricians and Gynecologists of Canada, 2014//Reproductive Endocrinology.-2014.-N° 4(18).- P.74-85.).
  • Drugs containing nifedipine, hydralazine, and labetalol are used as antihypertensive drugs. In mild disease, at gestational age ⁇ 34 weeks, outpatient or inpatient treatment is possible. However, careful monitoring of the pregnant woman and fetus is required.
  • fetal distress is diagnosed according to the results of the fetal heart rate monitoring and fetal cardiac abnormalities recording, compilation and analysis of its biophysical profile, observation of umbilical cord circulation and record of umbilical cord circulation disorders.
  • fetal distress is characterized by impaired fetus functional status due to acute or repeated restriction of oxygen access to the fetus or impaired ability of the fetus to use oxygen in cellular metabolism (metabolic acidosis).
  • fetal distress progression it is divided into:
  • chronic fetal distress that develops due to the constant influence of a pathogenic factor (e.g., anemia in pregnant women, intrauterine infection (IUI), hypertension in pregnant women, etc.);
  • a pathogenic factor e.g., anemia in pregnant women, intrauterine infection (IUI), hypertension in pregnant women, etc.
  • - diabetic angiopathy occlusive vascular disorders in the placental area, microthrombosis
  • umbilical factors umbilical cord prolapse and compression, the true node of the umbilical cord, and umbilical cord entanglement
  • - congenital anomalies of cardiovascular system in the fetus and congenital disorders of nervous regulation in the fetus.
  • the utility model patent of Ukraine UA 44055 U (description of utility model patent published on 10.09.2009) describes a method of treating antenatal fetal distress including the use of anticoagulants with a drug containing polyunsaturated fatty acids, 2 times a day, with meals, for 29-30 days.
  • Intrauterine growth restriction syndrome is diagnosed in the fetus and neonates who have low birth weight or low body weight and height for their gestational age.
  • the variety of causes determines the heterogeneity of the pathogenesis of intrauterine growth restriction syndrome.
  • Intrauterine growth restriction can develop at different stages of the fetal development. Thus, low birth weight of a full-term neonate indicates that the factor that restricted the intrauterine fetal growth was acting during the last 2-3 months of pregnancy, but if at the same time there is a deficit in body length (below the 10th percentile for this period of the pregnancy), then unfavorable conditions for the fetus developed in the II trimester of the pregnancy.
  • the first type of intrauterine growth restriction is called hypotrophic, the second - hypoplastic.
  • the most common cause of the hypotrophic type of fetal intrauterine growth restriction is severe toxicosis at the second half of the pregnancy due to placental insufficiency, and, for hypoplastic type, it is multiple pregnancy, familial low birth weight, adolescent mother, minor nutritional deficiencies without deep hypovitaminosis.
  • Literature and publications note that the most common cause of intrauterine growth restriction is placental insufficiency. The correlation between the severity of placental insufficiency in pregnant women and the degree of intrauterine growth restriction was shown, and the treatment of placental insufficiency was shown to reduce fetal intrauterine growth restriction.
  • Asthenia also referred to as asthenic condition, asthenic syndrome, asthenic reaction, asthenodepressive syndrome, asthenoneurotic syndrome
  • Asthenia is a pathological condition manifested by increased fatigue and exhaustion with a very unstable mood, self-control reduction, impatience, restlessness, sleep disorder, inability to endure prolonged mental and physical stress, intolerance of loud sounds, bright light, and pungent odors.
  • Asthenic condition can be constitutionally conditioned (asthenic psychopathy), but can occur because of malnutrition, vitamin deficiency, excessive physical and mental stress, vascular, organic, and endocrine diseases, as well as during convalescence of infections, intoxications, and injuries.
  • Asthenic condition is characterized by affective lability, increased excitability, which is replaced by exhaustion (weakness with irritability), and hyperesthesia (acute sensitivity to sound, light, tactile stimuli). Often headaches and sleep disorders (increased drowsiness during the day and insomnia at night) develop, as well as a dramatic change in well-being depending on changes in weather. Asthenic condition that develops as a result of various organic diseases is characterized by mild memory impairment, generally, with impairment of memory of recent events. Asthenic disorders develop slowly, increasing in intensity. Sometimes the first manifestations of asthenic condition are increased fatigue and irritability, which are combined with impatience and constant desire to work, even in an environment that means rest.
  • asthenic disorders may be accompanied by passivity and adynamia.
  • the clinical picture of asthenic condition has specific features and depends on the reasons that caused it. Asthenic condition after somatic diseases acquires the form of emotional-hyperesthetic weakness, in which fatigue and affective lability are combined with intolerance of emotional stress. After a traumatic brain injury, asthenic condition is manifested by weakness with irritability, headache, mentism (flow of thoughts) and autonomic disorders. In endocrinopathy, asthenic condition is expressed by the increased fatigue, exhaustion, and inactivity. Asthenic condition in schizophrenia is manifested by the predominance of mental exhaustion and inconsistency of the latter with the degree of mental stress.
  • asthenic condition In atherosclerosis, the main signs of asthenic condition are strongly expressed fatigue, weakness with irritability, decrease in mood, and tearfulness. In arterial hypertension, asthenic condition acquires the form of "restless fatigue.” In progressive palsy, the combination of the increased fatigue with mild dimming (obnubilation) is observed. To correct asthenic conditions, treatment depending on the condition origin and aiming at the underlying disease, adequate nutrition, and sleep, is needed. According to the indications, the use of actoprotectors, nootropic drugs, psychostimulants, adaptogens, multivitamins is possible.
  • Pharmacology of sports medicine is a relatively new, but very actively advancing field of the clinical and experimental pharmacology in recent years.
  • the sports pharmacology aims at development, study, and practical implementation of drugs (DR) and dietary supplements (DS) to increase the adaptation of athletes to high-intensity exercise, and one of the main objectives of this field is to identify and correct factors that limit the physical fitness of athletes.
  • DR drug
  • DS dietary supplements
  • the sport training system especially in the high-achievement sports, means exceptionally high training and competition loads accompanied by high levels of emotional stress. It is natural that such high loads are the most powerful factor in mobilizing the body functional reserves, stimulating intensive adaptation processes, increasing endurance, strength, speed and, obviously, the increase in sports results. In this case, an important role in improving physical fitness, preventing fatigue, and accelerating the recovery process after exercise belongs to a balanced diet.
  • the first object of the invention is to develop a pharmaceutical composition for the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human, treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human, prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, treatment of asthenia in a human, and improvement of the exercise tolerance in a human; to expand a range of the drugs for the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human, treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human, prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, treatment of asthenia in a human, and improvement of the exercise tolerance in a human; and to increase the effectiveness of the treatment of chronic coronary heart disease, or stable angina pector
  • the second object of the invention is to improve the method of the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human; to expand a range of the drugs for the treatment of chronic ischemic heart disease, or stable angina pectoris or peripheral artery disease in a human; and to increase the effectiveness of the treatment of coronary heart disease, or stable angina pectoris or peripheral artery disease in a human.
  • the third object of the invention is to improve the method of the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human; to expand a range of the drugs for the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human; and to increase the effectiveness of the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human.
  • the fourth object of the invention is to improve the method of the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction; to expand a range of the drugs for the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction; and to increase the effectiveness of the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction.
  • the fifth object of the invention is to improve the method of the increasing exercise tolerance in a human being exposed to physical load, and to improve the human body status in asthenia syndrome; to expand a range of the drugs for the increasing exercise tolerance and improving the human body status in asthenia syndrome; and to increase the drug effect on a human to improve exercise tolerance in a human and human body status in asthenia syndrome.
  • the first object is achieved by a pharmaceutical composition having an oral solution dosage form and comprising active ingredients and excipients, the pharmaceutical composition comprising levocarnitine and arginine salt, such as arginine aspartate, as active ingredients, and pH adjusting agent that is an acidifier, sweetening agent and preservative as excipients, in the following ratio, mg/ml: arginine aspartate 180-320 levocarnitine 50-150 pH adjusting agent that is an acidifier 1.5-6.0 sweetening agent 0.4-1 .2 preservative 0.5-2.0 water balance to 1 ml
  • the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 240-300 levocarnitine 80-120 pH adjusting agent that is an acidifier 2.5-4.5 sweetening agent 0.6-1.0 preservative 1.0-1 .5 water balance to 1 ml
  • the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 264 levocarnitine 100 pH adjusting agent that is an acidifier 3 sweetening agent 0.8 preservative 1 water balance to 1 ml
  • the pharmaceutical composition may comprise malic acid as pH adjusting agent that is an acidifier.
  • the pharmaceutical composition may comprise sodium saccharin as the sweetening agent.
  • the pharmaceutical composition may comprise methyl parahydroxybenzoate and/or propyl parahydroxybenzoate as the preservative.
  • the pharmaceutical composition may comprise water for injections as water.
  • the pharmaceutical composition may have density of 1.1 g/ml, solution pH of 5-6.5, and dynamic viscosity of 2.5 sP at 20 °C.
  • the second object is achieved by a method of treating chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease in a human suffering from chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease, including administering to the said human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the treatment of chronic coronary heart disease, stable angina pectoris, and/or peripheral artery disease.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered as part of a combination therapy of chronic coronary heart disease, stable angina pectoris, and/or peripheral artery disease.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
  • the third object is achieved by a method of treating acute cerebrovascular disorders and/or chronic cerebrovascular disorders in a human suffering from acute cerebrovascular disorders and/or chronic cerebrovascular disorders, including administering to the said human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the treatment of acute cerebrovascular disorders and/or chronic cerebrovascular disorders.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered as part of a combination therapy of acute cerebrovascular disorders and/or chronic cerebrovascular disorders.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
  • the forth object is achieved by a method of prevention and treatment of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction, including administering to a pregnant woman a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the prevention and treatment of preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
  • the fifth object is achieved by a method of increasing exercise tolerance in a human being exposed to physical load, and/or improving the human body status in asthenia syndrome, including administering to a human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for increasing exercise tolerance, and/or improving the human body status in asthenia syndrome.
  • the human may be exposed to the physical load in sports.
  • the human body status in asthenia syndrome is a condition that occurs after the human previous disease.
  • the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
  • the sweetening agent corrects the taste properties of the pharmaceutical composition. Certain ratio of malic acid and the sweetening agent in the pharmaceutical composition provides a pleasant taste for most people.
  • sodium saccharin that is approximately 500 sweeter than sugar, is used as the sweetening agent.
  • the preservative provides stability to the solution of the pharmaceutical composition.
  • Methyl parahydroxybenzoate and/or propyl parahydroxybenzoate can be used as the preservative.
  • arginine is often used in the form of a salt, such as arginine aspartate.
  • arginine means arginine aspartate.
  • the pharmaceutical composition according to the invention is a clear solution, and the dosage form for the use is the oral solution.
  • a method of manufacturing of the pharmaceutical composition according to the invention is given below in Examples 1 -20.
  • the pharmaceutical composition according to the invention is prepared by mixing the components of the pharmaceutical composition in water.
  • the stainless steel reactor is charged with 180 L of water for injections heated at 80 °C. 0.08 kg of methyl parahydroxybenzoate and 0.02 kg of propyl parahydroxybenzoate are then charged to the reactor and stirred until complete dissolution.
  • the liquid in the reactor is then cooled at 40 °C. 36 kg of arginine aspartate is then added to the reactor and stirred vigorously for a period of time sufficient to obtain a clear solution.
  • the reactor is then charged with 10 kg of levocarnitine and stirred vigorously for period of time sufficient to obtain a clear solution.
  • 0.3 kg of malic acid is then charged to the reactor and stirred until complete dissolution.
  • 0.08 kg of sodium saccharin is added to the reactor and stirred until the components are dissolved and a clear solution is obtained.
  • Water for injections is then added to the reactor, diluting the volume of the solution to 200 L.
  • the resulting solution is cooled, saturated with nitrogen to residual oxygen content less than 300 ppm, following with the filtration of the solution through a membrane filter. After the filtration, the solution is poured into glass or polymer containers (bottles).
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 36 kg of arginine aspartate and 15 kg of levocarnitine, 0.6 kg of malic acid, 0.13 kg of sodium saccharin, 0.1 kg of methyl parahydroxybenzoate and 0.1 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 36 kg of arginine aspartate and 25 kg of levocarnitine, 0.9 kg of malic acid, 0.18 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 36 kg of arginine aspartate and 30 kg of levocarnitine, 1.2 kg of malic acid, 0.24 kg of sodium saccharin, 0.1 kg of methyl parahydroxybenzoate and 0.3 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 10 kg of levocarnitine, 0.4 kg of malic acid, 0.09 kg of sodium saccharin and 0.1 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 44 kg of arginine aspartate and 15 kg of levocarnitine, 0.7 kg of malic acid, 0.12 kg of sodium saccharin and 0.2 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 25 kg of levocarnitine, 0.9 kg of malic acid, 0.17 kg of sodium saccharin and 0.3 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 30 kg of levocarnitine, 1.2 kg of malic acid, 0.24 kg of sodium saccharin, 0.3 kg of methyl parahydroxybenzoate and 0.1 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 10 kg of levocarnitine, 0.5 kg of malic acid, 0.1 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 15 kg of levocarnitine, 0.7 kg of malic acid, 0.14 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate.
  • Example 11
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 53 kg of arginine aspartate and 25 kg of levocarnitine, 1 .0 kg of malic acid, 0.19 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 75 °C, 58 kg of arginine aspartate and 10 kg of levocarnitine, 0.6 kg of malic acid, 0.12 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 15 kg of levocarnitine, 0.8 kg of malic acid, 0.16 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 25 kg of levocarnitine, 1 .0 kg of malic acid, 0.20 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 10 kg of levocarnitine, 0.7 kg of malic acid, 0.14 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 15 kg of levocarnitine, 0.9 kg of malic acid, 0.18 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 25 kg of levocarnitine, 1.1 kg of malic acid, 0.22 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
  • the pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 .
  • the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
  • composition according to the invention can also be prepared by other methods.
  • the aim of the study was to investigate the acute toxicity parameters of the PC being the solution for oral administration when administered intragastrically to rats of both sexes (males, females).
  • the study techniques included toxicological, laboratory, pathomorphological and statistical techniques.
  • the PC being the solution for oral administration.
  • Composition levocarnitine and L-arginine aspartate as the active ingredients (1 ml of the solution comprises 100 mg of levocarnitine and 264 mg of arginine aspartate) and malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216) and water for injections as the excipients.
  • composition malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216) and water for injections.
  • the dry mixture of the PC (solution for oral administration) substances The composition of the mixture is given for 368.8 g, which corresponds to 1000 ml of the PC in the form of the solution for oral administration.
  • the active ingredients are levocarnitine - 100 g, and arginine aspartate - 264 g; and the excipients are malic acid - 3.0 g, sodium saccharin - 0.8 g, methyl parahydroxybenzoate (E218) - 0.8 g, and propyl parahydroxybenzoate (E216) - 0.2 g.
  • the duration of the animal acclimatization period was 14 days. During this period, each animal was examined on the daily basis (general condition, morbidity). Prior to the study, animals that met the inclusion criteria were randomized into groups according to the weight. Animals that did not meet the inclusion criteria were excluded from the experiment.
  • the animals were maintained in a separate room with controlled microclimate parameters: air temperature + 20-24 °C, humidity 45-65%, "12 hours day/night” light regime; in standard plastic cages, 6 animals per cage.
  • the animals were managed according to the scientific and methodological recommendations for maintaining laboratory animals and working with them / Yu.M. Kozhemyakin, O.S. Khromov, M.A. Filonenko, G.A. Saifetdinova. - K Avitsena, 2002. - 156 c.
  • each animal was subjected to a daily examination including an assessment of the animal general behavior and condition. Visually visible pathological formations were palpated.
  • Acute toxicity experiments were performed on the nonlinear outbred white rats of both sexes weighing 200 ⁇ 20 g, aged 13-15 weeks. A total of 210 rats were used in the experiment. All animals were divided into groups of 12 animals (6 males, 6 females). Each animal was assigned an individual number. Prior to the experiment, the animals were numbered continuously from 1 to 210. The groups were randomized according to the body weight as a leading feature (the spread in the initial weight between and within the groups did not exceed 10%). The study design is presented in Table 1.
  • the animals in the experimental groups were administered with 2 ml/100 g of the weight of the samples per one injection with 2-hour interval between the injections. Immediately after the administration, the animals were monitored for signs of intoxication (if any). The nature of respiration, motor activity, the presence/absence of seizures, vomiting, diarrhea, ophthalmic symptoms, cardiovascular symptoms, salivation, piloerection, and muscle tone were recorded. The animals were allowed to eat 2 hours after the last injection of the test sample (hereinafter abbreviated TS), whereas access to water was free.
  • TS test sample
  • the animals were being monitored for 14 days.
  • the animal weight was recorded before the experiment and on 3, 7 and 14 days of the experiment.
  • the average lethal dose LDso was calculated based on the percentage of the animal mortality in each group depending on the dose administered, using Kerber method (Preclinical studies of drugs (guidelines))/Edited by Stefanova O.V. - Publishing House “Avitsena", 2001. - 527 p.).
  • WF Weight factors
  • the histological structure of the liver, kidneys, lungs, myocardium, thymus, spleen, adrenal glands, pancreas, esophagus, stomach, small intestine and rectum, testicles, and ovaries of the rats was studied on 14th day after intragastric administration of 40 ml/kg of the PC solution and compared with the histological structure of the similar organs of the rats administered with 40 ml/kg of placebo (solvent) (negative control), and the intact animals.
  • the autopsed organ samples were fixed in 10% formalin solution, dehydrated in alcohols of the growing concentration, and embedded into paraffin.
  • the sections were dyed with hematoxylin and eosin.
  • the micropreparations were examined using Granum microscope.
  • the microscopic images were photographed using a Granum DSM 310 digital video camera.
  • the photographs were processed on a 2.4GHz Pentium computer with Toup View power.
  • the primary data obtained were processed using generally accepted methods of analysis of variance using parametric criteria for comparison of quantitative indicators (ANOVA analysis of variance, Newman-Keuls criterion) and nonparametric criteria (Kruskal-Wallis method, Mann-Whitney criterion). To compare qualitative variables, the criterion c2 was used. Before using parametric criteria, the hypothesis for the normality of the random variable distribution was tested using Leuven test.
  • the significance level p ⁇ 0.050 was adopted.
  • the Bonferroni correction was used, according to which the level of significance was p ⁇ 0.0250.
  • the standard package of statistical programs "Statistica 6.0" was used for mathematical calculations.
  • the PC being the solution for the oral administration and placebo (solvent) were administered intragastrically for one day at a dose of 2 ml/100 g of the animal, 2 times per day with 2-hour interval.
  • the volume of the TS administered was 40 ml/kg. 20-30 minutes after the first administration of the TS, the animals demonstrated a decrease in motor activity, which is, obviously, associated with a large volume of the solutions administered. These symptoms disappeared in 1 hour, and, after that, the animal behavior did not differ from the behavior of the animals in the IC group. Repeated administration of the solutions to the animals also caused similar symptoms that disappeared in 1 hour.
  • an important indicator is the animal body weight, the change of which characterizes the severity of the drug toxic effects. According to the data obtained, positive dynamics of the body weight was observed in all the experimental groups (Table. 3). Weight gain in both male and female rats did not differ from the weight gain in the 1C group.
  • n the number of the animals in each group.
  • n the number of the animals in each group.
  • n the number of the animals in each group.
  • Fecal masses were formed, the anus and vaginal opening were not contaminated, the testicles were located in the scrotum and were movable.
  • the location of the mediastinal organs in thoracic and abdominal cavities conformed to the norm for all the rats.
  • the thymus varied slightly in size and was gray-pink.
  • the heart had regular configuration and size, with a typical location of coronary arteries and veins.
  • the epicardium surface did not have any specific features, the myocardium section was dense.
  • the lungs occupied the entire pleural cavity, had pale pink color, were airy, and did not have adhesions between the leaves of the pleura.
  • Thoracic lymph nodes were not enlarged.
  • Peritoneum was transparent and smooth. No foreign content was found in the abdominal cavity.
  • the liver had uniform reddish-brown color, the capsule was not tense, and the edges of the lobes were not rounded.
  • the surface of the organ was smooth.
  • Pancreas did not have signs of hemorrhage, sclerosis, fatty necrosis; had pale pinkish-yellowish color and a form of a loose, weakly branched strand scattered along the gastrosplenic ligament. The spleen was full-blooded and had red-cherry color.
  • Renal capsule was easily removed. Dense layers with preserved patterns were clearly visible on the organ section. Adrenal glands without any specific features. Peritoneal lymph nodes were not enlarged. The mucous membrane of the glandular stomach had a typical relief of the gastric folds, normal color, no hemorrhages, edema, or erosive lesions. The mucous membrane of the small and large intestine had regular color, and the content conformed to the parts. Testicles, testicular appendages, prostate, seminal vesicles in the male rats, and uterine horns and ovaries in the female rats had no pathology. The bladder was small with a thin wall.
  • the border between the hepatic lobes was lubricated, zones of the portal tracts (triads) were narrow, and radial orientation of the hepatocyte strands was preserved.
  • Kidneys The condition of the glomeruli and the system of convoluted and straight tubules were comparable in the experimental and control rats.
  • the glomeruli varied moderately in size, and their density was regular.
  • the lumen of the capsule was free.
  • the nuclear saturation of the glomerular capillaries and the clarity of the capillary network pattern was moderate.
  • Epithelium of proximal and distal part of the nephron tubules had no changes.
  • the level of fluffiness of apical segments of the cells was comparable.
  • the tubules of the cerebral layer were regular.
  • the myocardium retained its normal histological structure. Cardiac muscle fibers were uniformly colored and arranged quite densely. In cardiomyocytes, the transverse striated myofibrils, occupying the whole sarcoplasm free from the nucleus, were moderately expressed. Nucleus had oblong-rounded shape and were normochromic. Inter-beam spaces were not large. Vessels of the venous type were often full-blooded. Stromal cellular reaction was not visible (Fig. 3).
  • the histological structure of the thymus in the experimental rats did not differ from the structure in the control animals.
  • the lobes were well formed, and the density of lymphocytes in the cortex and medulla was typical. Thymic corpuscles were small and not numerous. Many rats in both groups had a moderate pattern of "starry sky".
  • Spleen lymphoid nodules were regular in the size and number. Periarterial T- dependent and marginal B-dependent zones, as well as germinative centers, were clearly visible in the lymphoid nodules. Numerous erythrocytes and nuclear forms of cells were visible in the red pulp.
  • the adrenal glands of the experimental rats maintained their inherent histological structure. Signs of the changes in histological characteristics indicating a change in the production of mineral- and glucocorticoids were not observed when compared with the control micropreparations.
  • the functional status of neuroendocrinocytes varied moderately within physiologically normal limits, and the sinuses were full-blooded.
  • Excretory glandular cells of acinuses had typical two-zone coloring of cytoplasm, and cytoplasm ratio in the experimental and control rats coincided.
  • the islet apparatus was represented by pancreatic islets of different size, filled with insulinocytes evenly and densely enough.
  • the mucous membrane of the rectum was lined with a cubic single-layer epithelium with a clear cuticular border and significant infusion of the goblet cells.
  • the nuclei of epithelial cells were located at the same level, intestinal crypts were moderately deep, and the area of mitosis in the intestinal crypts was restricted by the bottom area. Lymphoid cell saturation of the stroma of the mucous membrane was moderate.
  • the PC belongs to the toxicity class VI, such as relatively safe substances, with LD50 more than 15 ml/kg. Since the exact value of LD50 for the finished dosage form of the PC could not be measured, the median lethal dose was assessed using the content of the active ingredients in the drug. To do this, a number of the doses of the dry mixture of the PC substances, which were administered to the animals in the same volume (100 ml/kg), were used. Due to the large volume, the obtained solutions were administered to the animals in divided doses with the maximal allowable volume for the rats being 2 ml/100 g of the animal per one administration, 5 times a day with 2- hour intervals between the administrations.
  • the toxicity class VI such as relatively safe substances
  • n the number of the animals in each group.
  • n the number of the animals in each group.
  • the study results showed that the administration of 40 ml/kg (maximal dose) of the PC and placebo (solvent) does not cause animal death and does not lead to statistically significant changes in the relative weight of the internal organs of the rats of both sexes. Histological examination confirms the absence of the toxic effects of the PC and placebo on the internal organs when administered at the toxic doses.
  • the administration of the maximal doses of both the dry mixture of the PC substances and individual PC substances caused the animal death, and this allowed calculating the median lethal doses.
  • the median lethal dose of the dry mixture of the PC substances for the males was 33.333 mg/kg, for the females - 38.750 mg/kg.
  • the median lethal dose of arginine aspartate substance for the male rats was 29.792 mg/kg, for the females - 30.833 mg/kg, and the median lethal dose of levocarnitine substance for the males and females was 17.833 mg/kg.
  • the obtained mortality results allowed calculating the median lethal doses LD50.
  • LD50 was 33.333 mg/kg
  • LD50 was 38.750 mg/kg.
  • the mixture of the PC substances when administered to the laboratory rats (males and females) intragastrically, does not cause death and does not affect the physiological processes in the animals.
  • the mixture of the PC substances belongs to the toxicity class VI, such as relatively safe substances, with LD50 more than 15 mg/kg.
  • the study techniques included general clinical, laboratory, physiological, biochemical, pathomorphological, and statistical techniques.
  • the object of the study was PC being the solution for the oral administration, whereas 1 ml of the solution comprises the active ingredients, such as 100 mg of levocarnitine and 264 mg of arginine aspartate, and the excipients, such as malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216), and water for injections.
  • active ingredients such as 100 mg of levocarnitine and 264 mg of arginine aspartate
  • excipients such as malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216), and water for injections.
  • the experiments involved intragastric administration.
  • the test sample was administered to the rats once a day.
  • the solvent placed in a separate group of the animals, such as group No. 2 (6 males and 6 females).
  • the study design is provided in Table 9.
  • Methods of the physiological research included daily monitoring of the animal behavior, general condition, food, and water intake, as well as record of the body weight in the dynamics.
  • the myocardium electrophysiological activity was assessed using electrocardiography, and the functional status of the central nervous system (CNS) was assessed using "Open Field” test.
  • the animal body weight was assessed in the dynamics, such as initial weight and the weight on 7, 14, 21 and 28 day.
  • the effect of the test object on the CNS status of the rats was assessed based on the motor activity (number of square intersections), orientation and research activity (number of mink sightings, number of balances), and emotional activity (a number of urinations, defecations, washings) using the "Open Field” test at the end of the administration period (28 days).
  • a whole impact on the CNS was assessed using "Sum of activities" integrated parameter.
  • the PC effect on the cardiovascular system (CVS) status of the animals was studied at the end of the administration period (28 days) using EK1T-03 M2 electrocardiograph.
  • ECG was recorded in the II standard lead.
  • the following parameters were considered: RR - duration of the complete cardiac cycle; the duration of the PQ interval, the propagation of excitation in the atrium, which characterizes the time; the duration of the QRS ventricular complex and electrical systole of the ventricles, such as Q - T interval; voltage of P, T and R waves.
  • heart rate 60/RR, beats/min
  • SP systolic index
  • the concentration of hemoglobin, the number of erythrocytes and leukocytes, percentage of different forms of leukocytes (band and segmented neutrophils, lymphocytes, eosinophils, monocytes) in the peripheral blood were assessed.
  • the blood was sampled from the rat tail vein at the end of the administration period (day 28).
  • the blood hemoglobin level was determined using cyanmethemoglobin method (the set available from "Phyllisit-diagnostics” company, Ukraine). Erythrocyte level was determined using colorimetric method. Leukocyte level was determined in Goryaev's chamber. White cell count was calculated using CJ1-01 blood cell counter according to the common method (Laboratory research in the clinic / ed. V.V. Menshikova. - M Meditsyna, 1987. - 365 c.).
  • ALT and AST alanine and aspartate aminotransferase
  • the glucose level was measured using the kits available from "Phyllisit-diagnostician" (Ukraine) and glucose oxidase assay; total protein was measured using biuret method; creatinine concentration was measured using reaction with picric acid (the method is based on Jaffy reaction); urea level was measured using urease method (Biosystems, Spain); and chlorides were measured using photometric method using the kit available from "Phyllisit-diagnostics” (Ukraine).
  • the blood ability to coagulate was determined using Althausen method. It is known that blood clotting time depends on the prothrombin time changes, calcium ion level, fibrinogen level, fluctuations in fibrinolytic activity, structural and functional features of a number of other factors of pro- and anticoagulant action, as well as blood cells.
  • prothrombin time PT
  • thrombin time TT
  • active partial thrombin time APTT
  • the tissue samples were fixed in 10% neutral formalin solution and embedded into celloidin-paraffin.
  • the histological preparations were dyed with hematoxylin and eosin and examined using Micros 400 microscope. Microphotographing of the microscopic images was performed using Nicon Col Pix 4500 digital camera. The photographs were processed on a Pentium 2.4 GHz computer using Nicon View 5.
  • Descriptive statistics were provided for the recorded variables: (i) for quantitative indicators, sample size (n), arithmetic mean, and standard error (m) were provided; (ii) for variables that are not a subject to the law of normal distribution, sample size (n), arithmetic mean, minimum (Min) and maximum value (Max) were provided; (iii) for qualitative indicators, frequency and percentage were provided.
  • quantitative indicators the hypothesis of normal distribution of the data in groups was tested using the Leven test. If the data in the groups for certain parameters were distributed normally, the experimental groups were compared with the control for these parameters using the Newman-Keuls test (p ⁇ 0.050) for independent samples. In the case of an abnormal distribution, the Kruskal-Wallis test (analog of analysis of variance for nonparametric data) and Mann-Whitney criterion with Bonferroni correction (p ⁇ 0.00167) were used.
  • the male group administered with different doses of the PC solution demonstrated 19% increase in the rat body weight relative to the baseline, the intact group - 23%, and the PC group - 24%. There were no statistically significant differences concerning the IC group.
  • the female groups administered with 2 and 20 ml/kg of the PC solution also demonstrated 14% and 12% increase in the body weight, respectively, relative to the baseline, the intact group - 15%, and the PC group - 12%.
  • p1 NK is a statistical significance level of the differences in comparison with the baselines (Newman-Keuls criterion)
  • n is a number of the animals in the group.
  • Table 12 presents the data on the effect of the test drug and its solvent on the CNS functional status of the rats.
  • p K_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
  • p2 M u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p ⁇ 0.00167);
  • n is a number of the animals in the group.
  • p K_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
  • p2 M u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p ⁇ 0.00167);
  • n is a number of the animals in the group.
  • the white cell count for the experimental animals did not differ from the one for the IC group. No pathological changes were recorded.
  • p1 M_u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p ⁇ 0.00167);
  • n is a number of the animals in the group.
  • n is a number of animals in the group.
  • the data in Table 15 show that erythrocyte, leukocyte, and hemoglobin level in the females of all the experimental groups did not changed statistically significantly when compared to the control parameters.
  • the white cell count for the experimental animals did not differ from the one for the IC group. No pathological changes were recorded.
  • n is a number of the animals in the group.
  • n is a number of the animals in the group.
  • hemostasis was studied using general (clotting time) and specific methods (APTT). Also, prothrombin time, thrombin time, and fibrinogen level were assessed, which give a differential picture of possible changes in the hemostasis system when the PC is administered on the long-term basis and allow assuming the tendency to hyper- or hypocoagulation in general.
  • pANOVA is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA);
  • n is a number of the animals in the group.
  • p2NK is a statistical significance level of the differences when comparing with the IC group (Newman-Keuls criterion);
  • n is a number of the animals in the group.
  • Tables 20 and 21 show the results of the analysis of carbohydrate and lipid metabolism, which also define the liver functional status. According to the glucose blood level, an approximate conclusion about the status of the processes involved in the carbohydrate metabolism was drawn, and the status of lipid metabolism was assessed according to the cholesterol, HDL and LDL levels.
  • n is a number of the animals in the group.
  • pANOVA is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA);
  • n is a number of the animals in the group.
  • n is a number of the animals in the group.
  • n is a number of the animals in the group.
  • Tables 22 and 23 indicate that the PC drug and placebo when administered on the long-term basis (28 days) did not cause nephrotoxic effects.
  • Data on the effect of the PC solution on the heart rate and ECG parameters are given in Tables 24 and 25. All the animals maintained the correct sinus rhythm. In the II standard lead, a positive P wave was always located before the characteristic ventricular QRS complex.
  • n is a number of the animals in the group.
  • n is a number of the animals in the group.
  • p K_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
  • p2 M_u is a statistical significance level of the differences when comparing with the IC group (Mann-Whitney criterion);
  • n is a number of the animals in the group.
  • the myocardium of the left and right ventricles of the heart had no signs of edema or redness.
  • Individual rats in both control and experimental groups had single small focal lymphomacrophagous infiltrates located between cardiomyocytes. Cardiomyocytes had regular sizes and color of cytoplasm and nuclei; the cross- outlined myofibrils were well traced.
  • the lungs of the control rats had moderate hyperemia of blood vessels and capillaries, focal dystelectasis (partial collapse of the alveoli walls at the opening of the thoracic cavity) and foci of emphysema-like stretching of the alveoli (agonal origin).
  • the activity of lymphoid tissue associated with the bronchi was moderate.
  • the lumens of the lung alveoli were free.
  • the administration of 2 ml/kg and 20 ml/kg of the PC had no significant effect on the organ structure, such as there were no signs of increased redness, dystrophic changes of pneumocytes of the 1 st and 2nd orders, necrobiotic changes in the cells, inflammatory stroma reaction, or increased activity of peribronchial lymphoid tissue.
  • the lumen of the tubules of some parts of the cerebral layer contained small microliths of calcium salts and small focal round cell infiltrates localized near, in the stroma, which may be due to the nature of the animal nutrition.
  • liver lobulation was pronounced, the cord structure was preserved, and the liver cords were located within a short distance from each other.
  • liver stroma had a form of thin layers of portal connective tissue with vessels penetrating them and bile ducts.
  • Hepatocytes had regular form, with evenly colored cytoplasm. The boundaries between the cells were well defined. The hepatocyte nuclei were clearly visible and located mainly in the center of the cells. Sinusoidal capillaries were narrow, poorly filled with blood both in the central areas and on the periphery of the lobules.
  • Some rats of both sexes in the different groups had small lymphoid-cell clusters periportally, and small foci of hepatocyte necrosis. According to the literature, a possible cause of such deviations may be local damage caused by toxic substances coming from the intestine.
  • periportal hepatocytes of the female rats treated with 2 ml/kg (60%) and 20 ml/kg (40%) of the PC had moderate (2 ml/kg) and weak (ml/kg) small-drop cytoplasmic vacuolation without compromising cell integrity.
  • pancreatic tissue of all the rats acinuses had a regular structure. Zymogenic zone of pancreatocyte cytoplasm was well colored; cell nuclei were big, well-defined, and located at a basal membrane. The exfoliation of the epithelium of the excretory ducts was not observed. Islets of Langerhans had various sizes; capillaries thereof were expanded moderately; and secretory cells had no signs of dystrophy.
  • the size and number of white pulp follicles in the spleen of all the rats were regular, and their structure after the administration of the solvent or the PC did not change when compared to the intact.
  • Red pulp was moderately full-blooded, contained macrophages, megakaryocytes and lymphocytes.
  • the size and ratio of cross-sections of the cortical areas of the adrenal glands in all the rats were regular. Histoarchitectonics of each zone of the cortex was maintained. Signs of the change in the adrenocorticocyte morphological features that indicates an impaired production of mineral and glucocorticoids were not observed. The levels of lipoidization of the glomerular and reticular layer cells were comparable for the control and experimental animals. Chromaffin cells of the brain had no specific features.
  • testes of all the male rats had no signs of edema, redness or infiltration, or atrophy. All spermatogenic epithelium layers were represented in the twisted seminal tubules. Epithelium exfoliation was not detected. The number and location of Leydig cells were regular.
  • the results of the histological studies indicate that 2 ml/kg and 20 ml/kg of the PC, and placebo when administered intragastrically on the long-term basis, did not cause any morphological manifestations of the toxic effects in the heart muscle, liver, kidneys, lungs, adrenal glands, pancreas, spleen, and reproductive system of the rats.
  • the thymus (being an organ that, among all the organs of the lymphoid system, has excessive lability of its morphological structure, and easily and rapidly occurring reactive morphological changes in response to various factors) was marked with the complex of the adaptive reactions that conformed to 1 -2 stages of accidental transformation. According to the literature, these stages are reversible and disappear after the cessation of the stress. Additionally, moderate/weak vacuolation of hepatocytes of some female rats treated with the solvent or the PC was not dose-dependent and, apparently, was associated with the nature of the food consumed by the animals, and their greater sensitivity when compared to the males.
  • the following studies relate to determination of the effective dose of the PC, in the form of the solution for the oral administration, on a rat model of acute asphyxia.
  • Hypoxia is a universal pathological process that accompanies and defines the development of various pathologies, such as coronary heart disease, myocardial infarction, chronic heart failure, myocardiopathy, cerebrovascular disorders, chronic obstructive pulmonary disease, and asthenic condition, etc.
  • hypoxia can be defined as a cellular imbalance between energy demand and energy production in the mitochondrial oxidative phosphorylation system.
  • the reasons for the energy production impairment in the cell under hypoxic conditions are various: disorders of external respiration, blood circulation in the lungs, transport function of blood oxygen, impaired systemic, regional blood circulation, and microcirculation, decreased oxygen supply to the mitochondria in most pathological conditions.
  • inhibition of mitochondrial oxidation develops.
  • the activity of NAD-dependent oxidases (dehydrogenases) of the Krebs cycle is inhibited during the initial maintanance of the activity of FAD- dependent succinatoxidase, which is inhibited in more severe hypoxia.
  • Mitochondrial oxidation impairment leads to inhibition of associated phosphorylation and causes a progressive deficiency of ATP, a universal source of energy in the cell.
  • Energy deficiency is the essence of any form of hypoxia and causes qualitatively similar metabolic and structural disorders in various organs and tissues. Reducing the concentration of ATP in the cell leads to a weakening of its inhibitory effect on one of the key enzymes of glycolysis, such as phosphofructokinase. Glycolysis, which is activated in hypoxia, partially compensates for ATP deficiency, but causes quick accumulation of lactate and the development of acidosis, followed by autoinhibition of glycolysis.
  • hypoxia leads to a complex modification of the functions of the biological membranes, which affects both the lipid bilayer and membrane enzymes.
  • the main functions of the membranes (barrier, receptor, and catalytic) are impaired or modified.
  • the aim of this preclinical study was to determine the effect of the PC on the bioelectrical activity of the rat heart (BEAH) on a model of acute asphyxia. This is important to assess the severity of organotropic antihypoxic effects on the heart when administered inside, and to determine effective doses.
  • the animals were being acclimatized for 14 days in the test room.
  • the groups were randomized according to the body weight as the main feature.
  • the animals were maintained in the plastic cages in a room with controlled microclimate parameters, such as air temperature 20-24 °C, humidity 55 ⁇ 10%, "12 hours day/night" light regime.
  • the room was air-conditioned and sterilized on the daily basis using a quartz lamp.
  • the animals had free access to water.
  • Settled tap water was used for drinking.
  • Granulated balanced feed was used for the animal feeding (TY.Y 15.7-2123600159-001 : 2007).
  • the animals were managed according to standard operating procedures.
  • Group 1 (positive control) included 6 animals.
  • Group 2 (first test sample) included 6 animals, wherein the animals were administered with the solution containing 100 mg/ml of levocarnitine and 264 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate was 100/264 mg/kg of body weight, respectively.
  • Group 3 (second test sample) included 6 animals, wherein the animals were administered with the solution containing 200 mg/ml of levocarnitine and 528 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 200/528 mg/kg of body weight, respectively.
  • Group 4 (third test sample) included 6 animals, wherein the animals were administered with the solution containing 300 mg/ml of levocarnitine and 792 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 300/792 mg/kg of body weight, respectively.
  • Group 5 (fourth test sample) included 6 animals, wherein the animals were administered with the solution containing 400 mg/ml of levocarnitine and 1056 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 400/1056 mg/kg of body weight, respectively.
  • the solution was administered intragastrically at doses of 1 , 2, 3 and 4 ml/kg for 3 days.
  • the doses for the animals were recalculated from the daily dose for humans (30 ml/day according to the instructions for use in the clinical practice) using the coefficients of dose recalculation and I.P. Ulanova method taking into account the body surface (I.P. Ulanova, K.K. Sidorov, A.l. Khalepo On the question of taking into account the body surface of experimental animals in toxicological research. Edited by A. A. Letaveta and I.V. Sanotsky. - L. Ed. "Meditsyna", 1968, issue 10. - pp. 18-25.). Since the PC is intended for the oral administration in the clinical practice, the drug was administered to the laboratory animals intragastrically through a metal probe using a syringe.
  • Acute asphyxia was modeled using the method described in N.G. Stepanyuk. Comparative evaluation of antihypoxic properties of cordarone, benzofurocaine, vinborone and emoxipin in the experiment / N.G. Stepanyuk, V.V. Yushkova, V.B. Mudrytsky [etc.] // Bulletin of Vinnytsia National Medical University. - 2007. - N° 11 (2/1 ) - P. 576-579., and G.l. Stepanyuk. Search for effective antihypoxants among derivatives of succinic acid / G.l. Stepanyuk, O.P. Drachuk, S.A. Oliynyk, O.G. Yushkovskaya // Sports Medicine.
  • Quantitative values were processed using methods of variation statistics (mean value, its standard error, median, upper and lower quartiles) using parametric (one-way analysis of variance ANOVA, Newman-Keuls criterion) and nonparametric analysis methods (Kruskal-Wallis and Mann-Whitney criteria). The accepted significance level was p ⁇ 0.05. A standard Statistica software package (version 6) was used to obtain statistical conclusions.
  • p is the level of statistical significance when compared with the group of positive control, Mann-Whitney criterion; n is the number of the animals in the group.
  • the study aimed at assessing the effect of the combined use of levocarnitine and arginine aspartate in the form of the solutions for the oral administration in comparison with the use of arginine aspartate in the form of the solution for the oral administration, on physical fitness and psychological status of the qualified athletes, such as representatives of power and cyclic sports, during the standard training process.
  • Objectives of the study were following: to assess the effect of the combined use of levocarnitine and arginine aspartate in the form of the solutions for the oral administration in comparison with the use of arginine aspartate in the form of the solution for the oral administration, on the physical fitness and psychological status of the qualified athletes; to evaluate the tolerability and possible side effects of the study solutions; to compare the results of the study solutions obtained in the two main and control groups of the healthy athletes, according to the parameters of the impact on the physical fitness and psychological status of the athletes.
  • the study was performed according to the requirements of the State Expert Center of the Ministry of Health of Ukraine for clinical trials of drugs.
  • the study was an open, randomized, comparative, parallel study in three groups.
  • the study drugs were following: arginine aspartate in the form of the solution for the oral administration.
  • Composition 5 ml of the solution contained 1 g of arginine aspartate (arginine - 0.57 g, aspartic acid - 0.43 g), and excipients: sorbitol (E 420), sodium saccharin (E 954), methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), "Caramel” food flavor, and purified water. levocarnitine in the form of the solution for the oral administration.
  • Composition 5 ml of the solution contained 1 g of levocarnitine, and excipients: methylparaben (E 218), propylparaben (E 216), sucrose, sorbitol (E 420), "Banana” flavor, and purified water.
  • Randomization of the athletes of both groups (1 group included 36 track and field athletes and 2 group included 33 weightlifters) into subgroups was done according to the randomization scheme presented in the form of a table based on random numbers generated by the procedure of generating evenly distributed random numbers of the built-in Application Analysis Package Microsoft Excel.
  • the researcher received a notification of a subgroup for monitoring/research for this athlete.
  • the researcher put the randomized number and identifying information for the athlete included in the study in the Journal of Randomization.
  • the athletes (almost healthy individuals, all men) included in the study were divided into the main (1 A, 1 B, 2A, 2B) and control (1 C and 2C) subgroups in a ratio of 1 :1 :1 to study the effectiveness of the pharmacological drugs used.
  • the athletes in the control subgroups 1 C and 2C did not receive any drugs during the training process.
  • the athletes in the main subgroups 1 A and 2A were prescribed with the study arginine aspartate drug, being the solution for the oral administration, in a daily dose of 40 ml, divided into 2 doses of 20 ml, immediately after meals, for 21 days during the training process.
  • the athletes in the main subgroups 1 B and 2B were prescribed with the study arginine aspartate drug, being the solution for the oral administration, and the study levocarnitine drug, being the solution for the oral administration, in a daily dose of 15 ml, divided into 2 doses of 7.5 ml, 2 times a day, immediately after meals, for 21 days of the study during the training process.
  • Neurometabolic, nootropic, adaptogenic, anabolic and cardioprotective drugs were not allowed during the study.
  • the athletes continued to train normally, keep a regular diet and a healthy lifestyle (complete abstinence from alcohol and smoking).
  • a clinical examination was performed including: measurement of heart rate, arterial blood pressure; palpation and percussion of the abdominal cavity through the anterior abdominal wall; auscultation of the heart and lungs; examination of the skin and visible mucous membranes; assessment of the musculoskeletal system and urinary system status.
  • the athletes were subjected to an objective examination including measurement of the heart rate, arterial blood pressure, auscultation of the heart and lungs, examination of the skin and visible mucous membranes, assessment of the musculoskeletal system and urinary system, at 1 and 2 appointments.
  • the following methods were used to examine athletes: record of the demographic data; collection of sports history (type of sports, discipline, stage (period) of training); physical examination; laboratory diagnostics (standard hematological and biochemical parameters); assessment of prooxidant-antioxidant balance in erythrocyte membranes; psychodiagnostics; determination of the specific physical fitness parameters according to the type of sports.
  • Severity of the symptoms is assessed in points on a scale:
  • erythrocyte hematological analysis
  • erythrocyte parameters including the average absolute content and average concentration of hemoglobin in erythrocytes, average erythrocyte volume, erythrocyte anisocytosis
  • biochemical blood analysis total protein, bilirubin, urea, creatinine, glucose, potassium, sodium, and ionized calcium level
  • activity of ALT, AST, GGT and alkaline phosphatase marker enzymes APTT; assessment of prooxidant- antioxidant balance at the membrane level ("shadow of erythrocytes") with the assessment of malonic dialdehyde and reduced glutathione levels).
  • Psychodiagnostics included assessment of the psychological status of the athletes (type of temperament and severity of psychophysiological stress in points).
  • the main criterion for the effect of the drugs used was an increase in the specific physical fitness parameters and improve in the psychophysiological status (severity of psychophysiological stress) of the athlete.
  • Drug tolerability was assessed based on the subjective symptoms and feelings reported by the athlete, and objective data obtained by the researcher during the use of the study drugs in the athlete training process. The dynamics of the laboratory parameters, as well as the frequency and nature of adverse reactions were considered.
  • erythrocyte hematological analysis (leukocyte, platelet, erythrocyte and hemoglobin level, hematocrit, erythrocyte parameters, including the average absolute level and average concentration of hemoglobin in erythrocytes, average erythrocyte volume, and erythrocyte anisocytosis);
  • biochemical blood analysis total protein, bilirubin, urea, creatinine, glucose, potassium, sodium, and ionized calcium level; activity of ALT, AST, GGT and alkaline phosphatase marker enzymes; APTT; assessment of prooxidant- antioxidant balance (PAB) at the membrane level ("shadow of erythrocytes") with the assessment of malonic dialdehyde and reduced glutathione levels, and calculation of prooxidant-antioxidant coefficient (Cpa).
  • PAB prooxidant- antioxidant balance
  • any clinically significant negative deviation of the laboratory parameters was considered as a side effect, if it is not caused by the nature of the disease progression or the predicted effect of the use of concomitant therapy.
  • a bilateral critical area was used to evaluate the results. Compliance with the normal distribution was assessed using Shapiro-Wilk tests. The samples were compared using Student test for parametric data and Wilcoxon test for nonparametric data. ANOVA analysis of variance and the Kruskal-Wallis test were used for multiple comparisons, and the Marasquilo-Lyakh-Gurianov test was used for qualitative parameters. Rank correlation and Spearman correlation index were used for correlation analysis.
  • the athlete aerobic fitness was studied using PWC170 physical working capacity test, at 170 bpm using "Kettler E-3" bicycle ergometer ("KETTLER", Germany). Two loads of different moderate capacities (W1 and W2) were used to determine PWC170 value. At the last minute of these loads, heart rate was determined (f1 and f2, respectively). Physical working capacity in kgm/min was calculated as follows:
  • PWC1 70 W1 + (W2 - W1) (170 - f1) / (f2 - f1 ), wherein PWC170 is exercise capacity, at which the heart rate is equal to 170 bpm; W1 and W2 is a capacity of the first and second loads, respectively, kgm/min; and f1 and f2 is heart rate at the end of the first and second loads.
  • the athlete sitting on a bicycle ergometer, rested for 3 minutes, then he was offered to do the second, more intense, load session.
  • the duration of the first and second loads was 5 minutes
  • the whole study procedure took about 13 minutes.
  • the time for the athletes to pass the simulated competition distances of 800 and 1500 m was recorded using an electronic chronometer.
  • the specific physical fitness parameters for the weightlifters were studied using a modified Abalakov device, which is a barbell connected to a movable vertical ruler, measuring the height (cm) and time (s) for the athlete to do control exercises (height of the jump with the barbell, and height of the barbell lift in a jerk). Measurements were performed before training without doing warm-up under the standard conditions.
  • Fps wherein Fps is a psychophysiological stress factor; ⁇ ps is the sum of the points for the psychological stress severity; and ⁇ ph is the sum of the points for the physiological stress severity.
  • 1 B and 1C groups differ the most significantly.
  • 1 A group is the closest to 1 B group, but not so much regarding changes in the studied parameters.
  • 1C group is characterized by the opposite dynamics of the fitness parameter changes (or lack thereof) relative to 1 A and 1 B groups.
  • Tables 31 and 32 obviously show that arginine aspartate leads to an increase in the specific fitness parameters in both sports after the course of the drug, which is almost not observed in the control groups under the training conditions. To even greater extent, this direction of the fitness changes is expressed for the combined use of arginine aspartate and levocarnitine. It should be noted that a fairly short duration of the selected monitoring time (21 days), due to the period of the drug administration recommended in the manufacturer's instructions, allows rejecting the hypothesis of the targeted influence of the training process on the severity of the physical fitness changes.
  • one of the most important variables that determine the degree of the impact on the athlete fitness such as the severity of oxidative stress, which correlate with the degree of impairment/improvement of PAB, directly indicates the high effectiveness of the drugs for the groups of different sports (track and field and weightlifting), since the resulting parameter of the severity of PAB impairment in the body decreases, primarily at the membrane level (Fps).
  • the decrease in this prooxidant-antioxidant factor value clearly indicates the normalization of the balance between oxidative factors associated with the accumulation of active oxygen radicals, and antioxidant, primarily non-enzymatic, factors for the athlete when using arginine aspartate, and especially combination of aspartate and levocarnitine, during the intense and regular exercises.
  • the determining parameter of the effect on PAB is a decrease in MDA, as the study drugs function as antioxidants, i.e. substances that primarily decrease the severity of prooxidant reactions.
  • Table 37 shows that, generally, a significant decrease in the severity of psychophysiological stress and both its components is observed in 2A and 2B subgroups, although not substantial.
  • the decrease in the psychophysiological stress severity is much more noticeable for the main subgroups of the athletes (both track and field athletes and weightlifters), who received a combination of arginine aspartate and levocarnitine. While arginine aspartate is a powerful angioprotective and antioxidative agent, levocarnitine adds its neuroprotective and energetic effect, which causes the predominant effect of two drugs combination on the decrease in the psychophysiological stress severity.
  • the study drug effectiveness was assessed based on the increase in the physical fitness parameters (for the future, accordingly, based on the results of competitions).
  • the results of the statistical analysis show that, for the representatives of both sports (track and field athletes and weightlifters), the regimen administration of both study drugs, namely individual arginine aspartate and the combination of arginine aspartate and levocarnitine, had no significant negative impact on the main studied parameters of homeostasis. This indicates a high safety profile of the drugs when used in the dynamics of the real intense training loads.
  • the body response to any pharmacological action is usually reflected in the results of the studies of the blood biochemical parameters indicating the status of protein, lipid, and carbohydrate metabolism, functional status of the liver and pancreas, kidneys and biliary tract.
  • the results of the dynamic studies of the serum biochemical parameters for the track and field athletes and weightlifters under the influence of the study drugs are presented in Tables 40 and 41.
  • Arginine aspartate and especially its combined use with levocarnitine, is very important for the manifestation of the ergogenic action. Arginine aspartate has a pronounced antioxidant effect based on the reduction in the oxidative stress manifestations caused by activation of the antioxidant membrane-associated enzyme function, as shown in the recent studies.
  • arginine-containing pharmacological agents reduce sarcolemma postload impairment by more than 2 times, accompanied by a 25% reduction in muscle desmin loss and in mRNA m- calpain regulation, which eventually leads to acceleration of the recovery process after the training.
  • the data obtained using the methods of the descriptive statistics indicate the absence of significant differences in the most important indicators of the cell blood count for the main and control subgroups for both the track and field athletes and weightlifters.
  • the same absence of any negative dynamics is noted for hematocrit values, average volume of erythrocytes, their relative and absolute saturation with hemoglobin and degree of anisocytosis, and thrombocytes.
  • heart rate values are reference for the athletes for whom bradycardia refers to the adaptive responses to the training process and indicates the economization of the heart.
  • Tables 42 and 43 show that heart rate, systolic and diastolic arterial blood pressure of the athletes did not differ significantly throughout the study in the main and control subgroups. Thus, no negative changes in the main parameters of the functional status of the cardiovascular system were recorded for the representatives of both sports, such as the track and field athletes and weightlifters, during the regimen administration of arginine aspartate, and combination of arginine aspartate and levocarnitine, which confirms the high safety profile of the study drugs.
  • the study drugs have a convenient form for use by the athletes in training camp conditions and at the training stages before competitions, as they do not require dilution or other dosage forms than specified by the manufacturer.
  • the study drug use does not lead to negative changes in the general status of the athletes according to the results of the physical examination and does not cause impairment of the functional status of the cardiovascular system and main laboratory parameters.
  • the study drug use results in the stimulation of the physical fitness of the representatives of various sports, regardless of the specifics of the predominant mechanism of the energy supply (aerobic or anaerobic glycolytic), which is expressed in the increase of the parameters of the general and specific physical fitness.
  • the study drug use leads to a significant decrease in the oxidative stress manifestations, primarily at the cell membrane level, which aids in avoiding the development of further pathobiochemical changes inherent in the final stage of the qualified athlete training.
  • the studied drugs are well tolerated by the athletes, cause essentially no side effects and can be recommended as allowed ergogenic agents for the training of the athletes, such as the representatives of the cyclic and power sports in amateur and high-achievement sports.
  • the aim of the study was to investigate the effect of the PC on the exercise tolerance and quality of life in patients with stable angina of > functional class II.
  • the patients were randomized into treatment groups using block randomization with a 1 :1 ratio for the use of the PC in combination with basic therapy or only basic therapy for 21 days. The final appointment was held on the 22nd day.
  • the primary criterion of the therapy effectiveness was the change in the duration of the physical exercise performed during the exercise test (TDT) according to the R. Bruce protocol at the end of the 21 -day treatment regimen in comparison with the baseline, which is an indication of the drug pharmacodynamic effect.
  • the standard basic therapy which, according to the unified clinical protocol of the Ministry of Health (2015), is the optimal drug therapy, was chosen as a control.
  • the patients in the main group received 20 ml of the study pharmaceutical composition according to the invention 2 times a day for 10 days.
  • the number of the patients in the study was following: the number of patients randomized was 110. ITT population was 110. PP population was 91 , and safety population was 110.
  • CHD coronary heart disease
  • the study therapy was the therapy with the PC being the solution for the oral administration.
  • 1 ml of the solution contains: 100 mg of levocarnitine and 264 mg of arginine aspartate; and excipients: malic acid, sodium saccharin, methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), and water for injections.
  • Method of the administration included oral administration, before meals, 2 nebulas 2 times a day, in the morning and in the evening, in combination with the standard therapy.
  • the therapy duration was 21 days.
  • the primary effectiveness criterion is the change in the duration of the exercise performed during the exercise test (TDT) according to the protocol of R. Bruce at the end of a 21 -day treatment regimen compared with the baseline.
  • ITT Intent-to-treat population
  • PP Per protocol
  • the main population for the assessment of the primary effectiveness criterion was the PP population. Additionally, the primary effectiveness criterion was assessed for the ITT population. The main population for the assessment of secondary effectiveness criteria was the ITT population. Additionally, secondary effectiveness criteria were assessed for the PP population. The main population for the safety assessment was the safety population. Demographic data, effectiveness and safety parameters are given for the treatment groups using the descriptive statistics:
  • SD Standard deviation
  • the homogeneity of the groups was assessed according to the main initial parameters (demographic and anthropometric characteristics of the patients) to evaluate the success of the randomization (assessment of the parameter balance in the groups).
  • the groups were compared using Student t-test for independent samples or using Mann-Whitney test depending on the results of the assessment of the data distribution normality in the groups using Shapiro-Wilk test.
  • group comparisons were performed using the Pearson xi-square test (c2).
  • MFI (2015) is the optimal drug therapy, and includes at least one drug that affects the symptoms of angina, and drugs that affect the prevention of the disease complications.
  • glyceryl trinitrate short acting nitroglycerin
  • beta blockers for long-term symptom control and prevention of the attacks for patients with stable angina pectoris included 1 st line drugs: beta blockers, calcium channel blockers, adequate doses of which reduce heart rate considering the side effects and contraindications. In case of insufficient effectiveness of the therapy, it was recommended to replace the beta blocker with a calcium channel blocker or to prescribe a combination of the beta blocker and dihydropyridine calcium channel blockers.
  • Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers are prescribed to the patients with stable coronary heart disease and diabetes mellitus, hypertension, chronic heart failure or asymptomatic left ventricular dysfunction.
  • the drug was the most effective at doses of 1 and 2 ml/kg of body weight when administered intragastrically on the rat model of acute asphyxia.
  • the optimal dose for oral arginine is 6 g per day, 40 ml of the PC was decided to be used in a clinical study as the optimal daily dose.
  • Assessment of the primary effectiveness criterion for the claimed pharmaceutical composition was made as follows.
  • null hypothesis (HO) was formulated as follows:
  • the null hypothesis (HO) states that the effectiveness of the therapy including the claimed pharmaceutical composition will be inferior to the standard therapy effectiveness or will be equal to it;
  • H H : e ⁇ d H a : e> d.
  • d > 0 is the value of the clinically significant differences, that allows assuming that the therapy including the study drug is more effective than the basic therapy;
  • the dynamics in the groups was assessed using the paired Student t-test or Wilcoxon sign rank criterion in each group, depending on the results of the test on the difference distribution normality [Tapp5 - TappO] using the Shapiro-Wilk test.
  • the relative increase / decrease of the primary effectiveness criterion was calculated, and evaluated the paired Student t-test or Wilcoxon sign rank criterion in each group, depending on the results of the test on the difference distribution normality [Tapp5 - TappO] using the Shapiro-Wilk test.
  • the dichotomous variable such as an increase in duration of the load before the end of the treatment regimen for 1 minute (increase presence / increase absence), was created.
  • parameters of the descriptive statistics for each group were provided.
  • the comparisons between the study groups were performed using the Pearson xi- square test (>$2) (with Yates correction for dichotomous (binary) variables) or a two- way version of Fisher exact test (at expected frequencies less than 5).
  • the dichotomous variable such as an increase in duration of the load before the end of the treatment regimen for 2 minutes (increase presence / increase absence), was created.
  • parameters of the descriptive statistics for each group were provided.
  • the comparisons between the study groups were performed using the Pearson xi- square test (>$2) (with Yates correction for dichotomous (binary) variables) or a two- way version of Fisher exact test (at expected frequencies less than 5).
  • the dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
  • the dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test. For the “number of angina attacks per week and their dynamics” criterion:
  • a categorical variable which can be used to assess the presence / absence of a 50% decrease in the parameter value at the end of the treatment (Tapp5) compared to TappO (categories: 50% decrease or more / no 50% decrease), was created.
  • parameters of the descriptive statistics were provided for each group (frequency and part in %).
  • Pearson xi-square test >$2 (with Yates correction for dichotomous) or a two-way version of Fisher exact test (at expected frequencies less than 5) were used.
  • a categorical variable which can be used to assess the presence / absence of a 50% decrease in the parameter value at the end of the treatment (Tapp5) compared to TappO (categories: 50% decrease or more / no 50% decrease), was created.
  • parameters of the descriptive statistics were provided for each group (frequency and part in %).
  • Pearson xi-square test >$2 (with Yates correction for dichotomous) or a two-way version of Fisher exact test (at expected frequencies less than 5) were used.
  • the dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
  • the dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
  • the results of the laboratory tests (complete blood count, urinalysis, biochemical blood test parameters), measuring the heart rate (FIR) and arterial blood pressure (BP) are presented for the treatment groups using the descriptive statistics.
  • the descriptive statistics parameters were calculated for each safety endpoint for each group. Comparison of the groups by the number of the patients with SE was made using Pearson xi-square test (>$2) (with Yates correction for the dichotomous (binary) variables) or the two-way version of Fisher exact test (at expected frequencies less than 5).
  • the treadmill test was performed during appointment 0 and appointment 5 on the VALIANT treadmill (Lode BV, the Netherlands) or its analogue.
  • the study was performed according to R. Bruce modified protocol.
  • the following parameters were calculated: exercise tolerance and functional class of angina. High tolerance is for I functional class with the energy intensity of the physical activity performed more than 7.0 MET. Average tolerance to PA is for II - III functional class (energy intensity of II class - 4.0-6.9 MET, III - 2.0-3.9 MET). Low tolerance is for IV functional class (metabolic cost of the load less than 2.0 MET).
  • the following parameters were calculated: total load time and peak oxygen consumption. TDT was interpreted by a cardiologist.
  • the recording was carried out at appointment 0 and appointment 5 using diaries recording angina pectoris attack. The results were interpreted by the cardiologist.
  • the flow-dependent vasodilation test was performed at appointment 0 and appointment 5 using Philips HD11XE ultrasound machine or its analogue with a 3- 12 MHz linear sensor.
  • the right brachial artery was scanned 2-10 cm above the elbow, and the tonometer cuff was positioned on the forearm below the location of the artery.
  • the diameter of the brachial artery and circulation velocity were measured.
  • the brachial artery diameter was measured as the distance between the anterior and posterior walls of the artery at the border of the intimate vessel in the normal-diastole phase of the circulation, which was determined at the time of the R wave on the ECG synchronized with the ultrasound image.
  • PZVD was measured at the 60th second after 5 minutes of brachial artery compression with a pressure 50 mm Hg higher than the level of systolic arterial blood pressure of the patient by calculating the percentage change in the diameter of the artery compared to the baseline Daily ECG monitoring
  • the daily ECG was monitored on the Diacard DC 03250 v2.1 system or its analogue at appointment 0 and appointment 5. During this test, the following parameters were recorded: average daily heart rate, maximum and minimum heart rate, number and characteristics of ventricular and supraventricular arrhythmias, QTc interval variability, heart rate variables: SDNN, SDANN, RMSSD. The results were interpreted by the cardiologist.
  • the safety and tolerability of the 21 -day PC (oral solution) administration was assessed based on subjective symptoms and feelings reported by the patient to the researcher, and objective data obtained during the treatment.
  • Heart rate was measured by heart auscultation simultaneously with the measurement of pulse rate in the radial artery (or carotid artery if pulsation in the radial artery was weak) for one minute in a sitting position. In case of pulse deficit both parameters (heart rate and frequency) were recorded.
  • Respiratory rate was measured at rest in a supine position for one minute, recording the respiratory movements of the chest or abdominal wall, without attracting the patient attention.
  • Arterial blood pressure was measured on the brachial artery in the supine position using Korotkov method with a certified sphygmomanometer or tonometer and a cuff length and width adjusted to the length and circumference of the patient shoulder according to the recommendations for measuring arterial blood pressure RMOAG/BHOK, 2010.
  • the cuff size has to conform to the arm size.
  • the rubber part of the inflatable cuff must cover at least 80% of the shoulder circumference.
  • a cuff 12-13 cm wide and 30-35 cm long (average size) was used; but it was necessary to have a larger and a smaller cuff for full and thin arms, respectively.
  • the mercury column or the tonometer needle should have been at the zero mark before the start of the measurement.
  • phase 5 of Korotkov tones corresponds to the DBP; in children, adolescents and young people immediately after exercise; in pregnant women and adults with some pathological conditions, when it is impossible to determine the 5th phase, 4th phase of Korotkov tones, which is characterized by a significant weakening of tones, should be identified;
  • the patient should raise the hand and perform a few compressive movements with the wrist, then repeat the measurement, while not squeezing the artery with a membrane of the phonendoscope;
  • the blood for the analysis was sampled in the morning on an empty stomach (10-12 hours after the last meal) from the ulnar vein with a disposable sterile syringe under aseptic / antiseptic conditions.
  • the tests required a collection of approximately 12 ml of the blood (1 tablespoon) at appointment 0 and appointment 5.
  • the morning average portion was collected after adequate perineal hygiene.
  • the urine was to be delivered to the laboratory within two hours after the collection.
  • Complete blood count (hemoglobin, erythrocytes, leukocytes levels, white blood cell count, ESR), biochemical blood test (glucose, lactate and pyruvateserum levels, urea and creatinine levels, bilirubin, ALT, AHF, AST, and total albumin level), blood glucose from the ulnar vein and urinalysis (color, transparency, relative density, pH, glucose, protein, ketone bodies) were performed in the laboratory of the research center.
  • the study schedule (Table 45) shows the stages of the study and the procedures that were performed for each study participant.
  • the absolute average difference ( ⁇ SD) in the duration of the exercise performed during the treadmill test after the treatment compared to the baseline was 0.87 ⁇ 1.21 minutes (95% Cl 0.51 ; 1.23) in the main group, and 0.28 ⁇ 1.30 minutes (95% Cl - 0.10; 0.67) in the control group (differences between the groups are statistically significant, p ⁇ 0.001 ).
  • the average difference between the groups was 0.589 minutes (95% Cl 0.067; 1.111) (differences between the groups were statistically significant).
  • the absolute average difference ( ⁇ SD) of the duration of the exercise during the treadmill test after the treatment compared to the baseline was 0.93 ⁇ 1.15 minutes (95% Cl 0.62; 1.25) in the main group, and 0.21 ⁇ 1.22 minutes (95% Cl 0.12; 0.54) in control group (differences between the groups were statistically significant, p ⁇ 0.001 ).
  • the average difference between the groups was 0.724 minutes (95% Cl 0.272; 1.176) (differences between the groups were statistically significant).
  • the relative average difference ( ⁇ SD) of the duration of the exercise during the treadmill test after the treatment compared to the baseline was 24.32 ⁇ 36.64 % (95% Cl 14.31 ; 34.32) in the main group, and 6.51 ⁇ 24.04 % (95% Cl 0.01 ; 13.01) in the control group (differences between the groups were statistically significant, p ⁇ 0.001).
  • ITT population allows concluding that the duration of the exercise increased statistically significantly in the main group compared with the control group.
  • the absolute mean difference ( ⁇ SD) of the peak oxygen consumption after the treatment compared with the baseline was 2.16 ⁇ 4.40 ml/min/kg (95% Cl 0.96; 3.36) in the main group, and 0.49 ⁇ 3.38 ml/min/kg (95% Cl -0.42; 1 .41 ) in the control group (differences between the groups were statistically significant, p ⁇ 0.001 ).
  • the relative average difference ( ⁇ SD) of the peak oxygen consumption after the treatment compared with the baseline was 18.93 ⁇ 47.58 % (95% Cl 5.95; 31 .92) in the main group, and 7.25 ⁇ 34.03 % (95% Cl - 1.95; 16.45) in the control group (differences between the groups were statistically significant, p ⁇ 0.001 ).
  • the average value ( ⁇ SD) of the threshold load capacity during the treadmill test initially was 6.34 ⁇ 2.18 MET in the main group, and 6.51 ⁇ 2.09 MET in the control group (no statistically significant differences between the groups, p 0.936).
  • the absolute average difference ( ⁇ SD) in the threshold load capacity during the treadmill test after the treatment compared with the baseline was 1 .15 ⁇ 1 .85 MET (95% Cl 0.64; 1 .65) in the main group, and 0.06 ⁇ 1 .20 MET (95% Cl - 0.26; 0.38) in the control group (differences between the groups were statistically significant, p ⁇ 0.001).
  • the relative average difference ( ⁇ SD) of the threshold load capacity during the treadmill test after the treatment compared with the baseline was 20.53 ⁇ 32.80 % (95% Cl 11 .58; 29.48) in the main group, and 2.02 ⁇ 14.76 % (95% Cl - 1 .97; 6.01 ) in the control group (differences between the groups were statistically significant, p ⁇ 0.001).
  • the average value ( ⁇ SD) of the threshold load capacity during the treadmill test initially was 6.28 ⁇ 2.37 MET in the main group, and 6.45 ⁇ 2.26 MET in the control group (no statistically significant differences between the groups, p 0.948).
  • the average value ( ⁇ SD) of the flow-dependent vasodilation (FDVD) initially was 19.03 ⁇ 25.85 % in the main group, and 17.88 ⁇ 23.99 % in the control group (no statistically significant differences between the groups, p 0.552).
  • the average value ( ⁇ SD) of the flow-dependent vasodilation (PZVD) initially was 21 65 ⁇ 27.95 % in the main group, and 20.22 ⁇ 25.61 % in the control group (no statistically significant differences between the groups, p 0.732).
  • the average difference ( ⁇ SD) in the number of ventricular arrhythmias ( ⁇ SD) between the appointments, such as after the treatment and at the baseline, was 57.31 ( ⁇ 519.79) in the main group (95% Cl - 199.19; 84.56), and 145.60 ( ⁇ 1037.12) in the control group (95% Cl - 134.77; 425.97) (no statistically significant differences between the groups, p 0.339).
  • the mean difference ( ⁇ SD) in the number of supraventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 16.11 ( ⁇ 393.90) (95% Cl - 91.40; 123.63) in the main group, and 56.05 ( ⁇ 454.68) (95% Cl - 178.97; 66.86) in the control group (no statistically significant differences between the groups, p 0.652).
  • the average difference ( ⁇ SD) in the number of mixed arrhythmias between the appointments, such as after the treatment and at the baseline, was 0.04 ( ⁇ 2.92) (95% Cl - 0.76; 0.83) in the main group, and 12.82 ( ⁇ 94.33) (95% Cl - 38.32; 12.68) in the control group (no statistically significant differences between the groups, p 1.00).
  • the average difference ( ⁇ SD) of QT variability values between the appointments, such as after the treatment and at the baseline, was 5.77 ( ⁇ 101 .93) (95% Cl - 36.39; 24.86) sec in the main group, and 0.74 ( ⁇ 90.55) (95% Cl - 26.15; 27.63) sec in the control group (no statistically significant differences between the groups, p 0.306).
  • the average difference ( ⁇ SD) in the number of ventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 53.62 ( ⁇ 565.42) (95% Cl - 223.49; 116.25) in the main group, and 181 .96 ( ⁇ 1131 .43) (95% Cl - 154.04; 517.95) in the control group (no statistically significant differences between the groups, p 0.450).
  • the average difference ( ⁇ SD) in the number of supraventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 49.58 ( ⁇ 379.66) (95% Cl -64.49; 163.64) in the main group, and 71.22 ( ⁇ 494.59) (95% Cl -218.09; 75.66) in the control group (no statistically significant differences between the groups, p 0.849).
  • the average difference ( ⁇ SD) in the number of mixed arrhythmias between the appointments, such as after the treatment and at the baseline, was 0.04 ( ⁇ 3.20) (95% Cl - 0.92; 1 .01 ) in the main group, and 15.33 ( ⁇ 103.14) (95% Cl - 45.96; 15.30) in the control group (no statistically significant differences between the groups, p 1.00).
  • hypoglycemic drugs were prescribed the most often as concomitant drugs. Except insulin, the second most frequently prescribed drug in the main group was acetylsalicylic acid.
  • the absolute change was 0.87 ⁇ 1 .21 min (95% Cl 0.51 ; 1 .23) in the main group, and 0.28 ⁇ 1 .30 min (95% Cl - 0.10; 0. 67) in the control group (differences between the groups were statistically significant, p ⁇ 0.001 ).
  • the average difference between the groups was 0.589 minutes (95% Cl 0.067; 1.111) (differences between the groups were statistically significant).
  • the conclusion on the exceeding effectiveness of the combination therapy (PC and the standard therapy) in the main group compared to the standard therapy alone can be drawn based on the positive statistically significant differences between the groups in the primary effectiveness variable in favor of the main group. This allows concluding that the therapy with the study PC (oral solution) on the background of the standard therapy exceeds the effectiveness of the standard therapy alone concerning the duration of the exercise performed.
  • the average increase in FDVD in the main group was also significantly more pronounced than in the control group.
  • the average difference in FDVD was 1.13 ⁇ 2.51 % (95% Cl 0.37; 1 .88) in the main group of the PP population, and 0.58 ⁇ 2.74 % (95% Cl - 0.23; 1 .40) in the control group of the PP population; and 1.51 ⁇ 2.52 % (95% Cl 0.82; 2.20) in the main group of the ITT population, and 0.62 ⁇ 2.52 % (95% Cl - 0.06; 1 .30) in the control group of the ITT population.
  • the pharmaceutical composition according to the invention in the oral solution form containing two active substances, such as arginine and levocarnitine, increases the effectiveness of the combination therapy of coronary heart disease and improves the quality of life by reducing the frequency of the angina attacks.
  • the aim of the clinical study was to study the effectiveness of the PC containing 100 mg of levocarnitine and 264 mg of arginine aspartate in 1 ml of the solution as a part of the combination therapy of such a disease as chronic cerebrovascular disorder (CCD).
  • CCD chronic cerebrovascular disorder
  • the study design was as follows.
  • the patients were divided into three groups, wherein the first group was the main group, the second group was the first control group, the third group was the second control group.
  • the groups were statistically homogeneous by sex and age, as well as by comorbidities. In particular, all the examined patients had a history of hypertension, wherein the groups did not differ in the duration of the disease, baseline systolic and diastolic blood pressure, and heart rate. In all three groups, approximately equal numbers of the patients received antihypertensive therapy. All the patients were diagnosed with atherosclerosis (cardiosclerosis, carotid artery disease). Frequencies of obesity and disorders of the blood lipid spectrum were similar.
  • the patients in the main group were prescribed with:
  • statins in the presence of concomitant hypertension
  • the PC comprising 100 mg of levocarnitine and 264 mg of arginine aspartate in 1 ml of the solution, orally, 10 ml three times a day, before meals, for 21 days.
  • 1 ml of the PC oral solution comprised 264 mg of arginine aspartate and 100 mg of levocarnitine, and excipients, such as malic acid, sodium saccharin, methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), and water for injections.
  • the daily dose of the PC was 30 ml
  • the daily dose of arginine aspartate was 7.92 g
  • the daily dose of levocarnitine was 3 g.
  • the patients in the first control group were prescribed with:
  • statins in the presence of concomitant hypertension
  • Tivortin aspartate drug orally, 10 ml 4 times a day, for 21 days.
  • 1 ml of Tivortin oral solution comprised 200 mg of arginine aspartate and excipients, such as sorbitol (E 420), sodium saccharin (E954), methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), "Caramel” food flavor, and water for injections.
  • the daily dose of arginine aspartate was 8 g.
  • the patients in the second control group were prescribed with:
  • statins in the presence of concomitant hypertension
  • levocarnitine solution in the form of the solution for oral administration, 5 ml 3 times a day, for 21 days.
  • 1 ml of levocarnitine solution for the oral administration comprised 200 mg of levocarnitine, and excipients, such as methylparaben (E 218), propylparaben (E 216), sucrose, sorbitol (E 420), "Banana" flavor, and purified water.
  • the daily dose of levocarnitine was 3 g
  • the patients of all three groups demonstrated positive clinical dynamics during the period of the inpatient treatment.
  • the patients complained of clinical manifestations of CCD Stages l-ll.
  • all the patient groups showed a positive trend being a regression of most of the complaints made before the treatment.
  • Table 47 shows the results of the changes in the patient complaints. Positive dynamics of the complaints was observed in all the groups.
  • the frequency of the complaints of weakness, fatigue, memory impairment, and headache decreased significantly (p ⁇ 0.05). There was no significant dynamics in the "noise in the head", “change of mood”, “gait instability” and “sleep disturbance” parameters (p> 0.05).
  • Table 49 Data from neuropsychological examination of the patients based on MMSE scale before and after the treatment are shown in Table 49.
  • Table 49 shows that statistically significant improvement after the treatment in the main group was confirmed by the overall score according to MMSE scale, and according to "attention and calculation", “word reproduction” and “ speech functions” subscales (p ⁇ 0.05).
  • significant improvement after the treatment was confirmed by the overall score, and the "speech functions” test (p ⁇ 0.05).
  • the second control group significant improvement after the treatment was confirmed by the overall score and the "attention and calculation” and “speech functions” tests (p ⁇ 0.05).
  • the overall improvement according to the " speech functions” test was 25% in the main group, 10% in the first control group, and 11% in the second control group.
  • the overall improvement according to the overall score was 12% in the main group, 5% in the first control group, 5% in the second control group.
  • the level of cognitive impairment was assessed according to the Montreal scale of assessment of cognitive impairment (MoCA) before and after the treatment (Table 50).
  • the scale allows evaluating a number of cognitive functions, such as short-term memory, recollection, attention, working memory, and abstract thinking.
  • the patients in all the groups improved the average task performance score compared with the baseline (p ⁇ 0.05).
  • the patients in the main group demonstrated more pronounced dynamics in the task performing (p ⁇ 0.05).
  • the parameter decreased the least.
  • the overall improvement was 38% in the main group, 12% in the first control group, and 23% in the second control group.
  • HADS Hospital Anxiety and Depression Scale
  • Chronic cerebrovascular disorder is a daunting manifestation that develops in the middle and old age. This disorder has no pronounced symptoms and develops gradually, therefore, it often manifests late, when the degradation of personality has already begun.
  • the pharmacotherapeutic effect should be as comprehensive as possible and aimed at restoring regular circulation in the affected area and activating energy processes in the human brain, improving mental performance and memory, normalizing the blood supply and resistance of the brain cells to oxygen starvation.
  • CCD complex of the clinical symptom of CCD included general cerebral symptoms, vestibulo-cochlear disorders, pyramidal and cortico-nuclear insufficiency, autonomic and coordination disorders specifically attributed to this disease.
  • Cognitive impairment was typical for all the patients: some patients demonstrated psycho-emotional disorders in the form of anxiety, and several patients demonstrated depression. Asthenic symptoms were observed in all the studied patients.
  • the main neurological symptoms of Stages l-ll of CCD are essentially expressed in the asthenic syndrome and anxiety. Headache, dizziness, general weakness, increased fatigue, emotional lability, sleep disturbances, reduced efficiency - all this set of the complaints is typical for the initial stages of CCD.
  • the treatment with the PC maximized the efficiency and daily activity of the patients, enhanced memory, reduced headache and anxiety compared with the first and second control groups of arginine or levocarnitine.
  • Analysis of the data in Table 47-51 shows that the claimed pharmaceutical composition according to the invention had a more pronounced protective effect and had an unexpected technical result in comparison with comparative drugs containing arginine aspartate or levocarnitine alone. Analysis of the data obtained during the studies allows claiming that the claimed pharmaceutical composition had an unexpected synergistic effect.
  • potentiation In pharmacology, a separate case of synergism, in which the effect of simultaneous use of two or more active substances exceeds the total effect of each of these substances alone, is called potentiation.
  • the use of the combination of the components, such as arginine aspartate and levocarnitine, in the claimed pharmaceutical composition gives the effect of potentiation, therefore, the pharmaceutical composition according to the invention has an unpredictable synergistic effect.
  • the enhancement in the overall score was 12% in the main group, 5% in the first control, and 5% in the second control group.
  • the cognitive impairment dynamics based on the Montreal scale of assessment of cognitive impairment were more pronounced for the patients treated with the pharmaceutical composition compared with the monotherapy.
  • the overall improvement was 38% in the main group, 12% in the first control group, and 23% in the second control group.
  • NADS Hospital Anxiety and Depression Scale
  • Levocarnitine increases the rate of fat oxidation in mitochondria and plays a key role in the body metabolism, such as it provides transport of long-chain fatty acids into the mitochondrial matrix, controls and modulates the intracellular pool of coenzyme in the cell, participates in detoxification of organic acids and xenobiotics.
  • levocarnitine transports acetyl residues from mitochondria to the cytosol, thus participating in the synthesis of acetylcholine and acetylcarnitine.
  • acetylcarnitine The neurobiological effects of acetylcarnitine include direct effects on energy metabolism and phospholipid metabolism, synoptic morphology, and transmission of numerous neurotransmitters.
  • arginine aspartate being an active donor of NO, improves endothelium-dependent vasodilation, reduces platelet aggregation and reduces endothelium-dependent adhesion of monocytes. This helps to restore cerebral circulation, normalize hemodynamics, reduce oxidative stress in the lesion and, accordingly, reduce neurological deficit.
  • the pharmaceutical composition comprising two active substances, such as arginine aspartate and levocarnitine, increases the effectiveness of the combination therapy of CCD.
  • the PC being the part of the combination treatment of CCD, showed significantly greater effectiveness than the use of arginine or aspartate alone.
  • the aim of the preclinical trial was to study the effectiveness of the PC being the solution for the oral administration and comprising 264 mg of arginine hydrochloride and 100 mg of levocarnitine in 1 ml, for the treatment and prevention of preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction.
  • the trial design was as follows.
  • preeclampsia According to a directive of the International Society for the Study of Hypertension in Pregnancy, clinical features of preeclampsia include severe hypertension after 20th week of gestation, proteinuria with or without edema and changes in laboratory parameters, as well as kidney, liver and brain impairment.
  • NO nitric oxide
  • Powerful NO vasodilator is synthesized from L-arginine by nitric oxide synthase (NOS).
  • L-NAME N-nitro-L-arginine methyl ester
  • the drug being the PC according to the invention and comprising 264 mg of arginine hydrochloride and 100 mg of levocarnitine in 1 ml;
  • the first comparative drug comprising 264 mg ⁇ ml of arginine aspartate
  • the second comparative drug comprising 200 mg/ml of levocarnitine
  • the trial was conducted on 8 groups of animals.
  • the average body weight of non-pregnant rats was 200 ⁇ 10 g.
  • the volume and quantity of the study drugs are indicated based on 200g weight of the rats.
  • Group 1 intact control
  • included non-pregnant rats treated with saline (n
  • Pregnancy modeling was performed before the trial using monitoring the animal estrous cycle and subsequent fertilization with the identification of the first day of fertilization based on the presence of sperm in the animal vaginal swab.
  • Pregnant and virgin nonlinear animals were housed individually in standard metabolic cells, which allowed urine to be collected, and food and water intake to be recorded throughout the trial.
  • laboratory animals in the groups No. 4-7 started to be administered with the study / control drug intragastrically, once a day, using a syringe through a metal probe. The administration was continued until delivery.
  • One arterial and one venous catheter were implanted on the fourteenth day of the gestation (delivery from day 21 to day 22).
  • Abnormalities of fetal developmental were assessed by weighing neonates and assessing neonatal mortality. Measurements of the average blood pressure of the non-anesthetized animals that was not restricted in their movements were performed daily, at a fixed time, using an electronic manometer and a pressure transducer.
  • Sample collection and analysis were performed as follows. The animal body weight, water and food intake, and urine volume were monitored daily. The urine samples collected over twenty-four hours were centrifuged at 3000 G for 15 minutes and stored in a freezer at -20 °C before albumin chemical analysis (usually ⁇ 2 weeks). Shortly before infusion, blood samples were taken into syringes from a catheter implanted in the aorta (for baseline values), and, 4 days later, to assess the effect of L-NAME on nitrate and nitrite levels, and platelet count.
  • Histological analysis of the kidneys was performed as follows. 4 days after the start of the treatment, the animals were anesthetized, and both kidneys were removed and weighed, the left kidney being treated for microscopic examination. The coronary sections of the kidneys were fixed in 10% formalin and imbedded into paraffin blocks. The sections (thickness 3 pm) were dyed. The sections were examined on a blinded basis for lesions of the glomerular zone.
  • Preeclampsia is characterized by hypertension, proteinuria, as well as impairment of the kidneys and liver of the mother, and, consequently, possible abnormalities of the fetal development.
  • body weight of the females receiving L-NAME (Group 3) was less than body weight of the pregnant animals in the control group (croup No. 2) that received saline, which may reflect significant intrauterine growth restriction.
  • the animals treated with the pharmaceutical composition according to the invention (Groups No. 6 and 7) showed a significant increase in body weight, indicating normalization of the metabolic processes and, most probably, impact on the fetal weight restoration (see Table 52).
  • the animals receiving the pharmaceutical composition according to the invention demonstrated a statistically significant restoration of the average urine volume compared with the Group 3 and the comparative groups (Group 4 and 5) receiving L-NAME, which may indicate normalization of the renal function and lowering of the arterial blood pressure caused by restoration of the blood nitric oxide level.
  • mean arterial blood pressure indeed indicate the antihypertensive effect of arginine, levocarnitine and three doses of the claimed pharmaceutical composition.
  • the effect of the claimed pharmaceutical composition according to the invention was more pronounced, compared with the similar effect of the comparative drugs (Table 52).
  • the most pronounced pharmaceutical effect according to the urine volume and mean arterial blood pressure was seen when the amount of arginine / levocarnitine was increased to 520/200 mg, respectively.
  • Kidney impairment is one of the main prognostic markers in preeclampsia progression.
  • the kidney status was analyzed by histological analysis, and by the level of proteinuria (urinary albumin level). Inhibition of nitric oxide synthesis can cause glomerular capillary hypertension, which leads to sclerotic lesions of the kidney glomerular zone.
  • the administration of L-NAME was accompanied by severe morphological changes in the kidney glomerular zone of the pregnant animals. Glomerular capillary lumens were segmentally occluded by intraluminal masses of eosinophilic composition. Extraglomerular lumens were filled with protein. In addition, mild diffuse interstitial edema and sparse interstitial lymphocyte infiltration were observed. For the groups receiving the pharmaceutical composition, such renal changes were not observed, in contrast to the individual administration of arginine and levocarnitine, for which pathological changes were recorded on the preparations.
  • Urinary glomerular proteins of intermediate size such as albumin
  • the average daily urinary albumin excretion increased sharply in the positive control group (Group 3) administered with L-NAME alone (mg/24 h) from 8.3 ⁇ 1.5 to 56.3 ⁇ 14.3 mg/24 h (p ⁇ 0.005).
  • arginine, levocarnitine, and two doses of the claimed pharmaceutical composition according to the invention in addition to L-NAME was shown to decrease the urinary albumin level, and, for the groups receiving the pharmaceutical composition according to the invention, proteinuria was least pronounced, which confirms the synergistic nephroprotective effect of the composition in comparison with the individual administration of arginine and levocarnitine (see Table 53).

Abstract

The invention relates to the field of medicine, namely to the drugs for the treatment of diseases of various etiologies. The claimed pharmaceutical composition has an oral solution dosage form and contains active ingredients, excipients, and water, and contains levocarnitine and arginine aspartate as active ingredients, pH adjusting agent being an acidifier, sweetening agent and preservative excipients. Additionally, a method of treating chronic coronary heart disease, stable angina pectoris, peripheral artery disease, acute cerebrovascular disorders and chronic cerebrovascular disorders in a human is provided, including administering to the said human the above pharmaceutical composition. The pharmaceutical composition is highly effective and promising for implementation in medical practice as a mean for the outpatient treatment of the diseases of various etiologies and their consequences. Additionally, the pharmaceutical composition expands the range of the drugs for the treatment of the diseases of various etiologies and improves the quality of the patient life.

Description

A PHARMACEUTICAL COMPOSITION FOR TREATING CARDIOVASCULAR AND CEREBROVASCULAR DISORDERS, DISEASES IN PREGNANT WOMEN AND IMPROVING EXERCISE TOLERANCE
DESCRIPTION
FIELD OF THE INVENTION
The invention relates to the field of medicine, namely to the drugs for prevention and treatment of diseases of various etiologies, in particular, the diseases of the cardiovascular system (coronary heart disease, stable angina pectoris, peripheral artery disease), acute cerebrovascular disorders, chronic cerebrovascular disorders, the diseases in pregnant women and fetal development during pregnancy (preeclampsia in pregnant women, fetal distress, intrauterine growth restriction), as well as for improvement of exercise tolerance of human and status of human body in asthenia.
BACKGROUND OF THE INVENTION
The cardiovascular system diseases include coronary heart disease, stable angina pectoris, and peripheral artery disease.
The term “coronary heart disease” (abbreviated as CHD) refers to a group of cardiovascular diseases caused by myocardial impairment as a result of coronary heart disorders that occur due to an imbalance between the delivery of oxygen and metabolic demand of the heart muscle in oxygen.
Myocardial demand in oxygen is primarily determined by heart rate, myocardial contractility, heart size and blood pressure. An increase in any of these parameters increases myocardial demand in oxygen. Under normal conditions, coronary arteries have sufficient reserve of dilation, which provides, if necessary, a fivefold increase in coronary circulation. Restrictions on myocardial blood supply occur due to a decrease in the coronary artery lumen by more than 50%. Mismatch between coronary circulation and metabolic demands of the heart muscle is always accompanied by myocardial ischemia manifested clinically by angina attack, severe heart rhythm and conduction disorders, and, in some cases, myocardial infarction and sudden death. According to the Order N° 54 of the Ministry of Health of Ukraine on 2001 , the following forms of coronary heart disease are distinguished:
1. Sudden coronary death:
- sudden clinical coronary death with successful resuscitation;
- sudden coronary death (fatal).
2. Angina:
- stable angina pectoris with assessment of functional class;
- stable angina pectoris, angiographically intact vessels (coronary syndrome x);
- vasospastic angina (angiospastic, spontaneous, variant, Princemetal).
3. Unstable angina:
- primary angina;
- progressive angina;
- early postinfarction angina (3 to 28 days of myocardial infarction).
4. Acute myocardial infarction:
- acute myocardial infarction with pathological Q wave;
- acute myocardial infarction without pathological Q wave;
- acute myocardial infarction (undetermined);
- recurrent myocardial infarction (3 to 28 days);
- recurrent myocardial infarction (after 28 days);
- acute coronary insufficiency.
5. Complications of myocardial infarction (with indication of the onset time):
- acute heart failure (classes l-IV according to T. Killip);
- heart rhythm and conduction disorders;
- external heart rupture (with hemopericardium, without hemopericardium) and internal heart rupture (ventricular septal defect, atrial septal defect, chordae tendineae rupture, papillary muscle rupture);
- thromboembolism with different localization;
- acute heart aneurysm;
- Dressier syndrome;
- postinfarction angina (3 to 28 days).
6. Cardiosclerosis. focal cardiosclerosis: o postinfarction cardiosclerosis (with indication of the myocardial infarction history, its location and time of development); o chronic heart aneurysm; o focal cardiosclerosis (without indication of myocardial infarction).
- diffuse cardiosclerosis.
7. Painless form of CHD.
The main etiological factor of CHD is coronary artery atherosclerosis. Among the factors that contribute to its development, important are: hyperlipidemia, hypertension, high-calorie diet, obesity, diabetes, smoking, hypodynamics, genetic predisposition, age, and male gender. Myocardial ischemia associated with impairment of the coronary arteries of another nature (ex., rheumatism, septic endocarditis, etc.), as well as hemodynamic disorders of non-coronary nature (aortic valve disease), do not relate to CHD and is considered as a secondary syndrome according to nosological forms.
Coronary artery atherosclerosis is found in 95% of patients with CHD. Growing atherosclerotic plaque, hemorrhage into the plaque base with its disintegration, and the formed thrombus leads to a narrowing of the lumen or complete obstruction, resulting in organic obstruction of the coronary artery.
Atherosclerosis is a common cardiovascular system disease. This pathological process underlies the frequent causes of mortality and disability, such as coronary heart disease, ishemic stroke, chronic forms of cerebrovascular insufficiency, peripheral thrombosis etc. Atherosclerosis is a systemic disease and often leads to simultaneous impairment of the vessels of the brain, heart, kidneys, and limbs. Often, the first signs of venous insufficiency are manifested in old age but can be manifested in middle and even young age.
Atherosclerosis is a chronic disease of the vessels of elastic and muscular- elastic type, i.e., large arteries. The main pathogenetic events of atherosclerosis are lipid, or rather cholesterol, intima infiltration and growth of connective tissue throughout the vascular wall. According to some authors, it is serum cholesterol that is an actual risk factor for both atherosclerosis in general and its main consequence - coronary heart disease. At the early stages of the pathological process, lipid infiltration has the form of a so-called fat strip, which does not rise above the surface of the vascular wall and does not manifest itself clinically. However, further on atheroma and connective tissue start growing, which leads to the formation of atherosclerotic plaque that reduces the vessel lumen and may rupture and become covered with ulcers causing thromboembolic complications. The vessel lumen narrowing by 70% or more is considered hemodynamically significant stenosis, in which the risk of ischemic complications is very high. In the presence of a large atherosclerotic plaque there is a significant risk of impairment of the vascular wall integrity with the subsequent development of thrombosis. Additionally, vascular endothelium produces vasodilators, such as prostaglandin, prostacyclin, and nitric oxide (NO) endothelium-derived relaxing factor (abbreviated EDRF), which also are antiplatelet agents. In patients with coronary heart disease, the dynamic balance between endothelial vasodilator factors and antiplatelet factors, on the one hand, and vasoconstrictive factors and proaggregating factors, on the other, is impaired. The latter begins to predominate leading to the development of coronary artery spasm and increased platelet aggregation. Impairment of the hemostasis system, such as changes in platelet function, increased blood viscosity, and inhibition of fibrinolysis, which can lead to the development of intravascular thrombosis, is essential in the progression of coronary heart disease. Additionally, insufficiently developed network of collateral coronary blood supply is important. It has been proven that hyperproduction of catecholamines that occurs in stressful situations can cause myocardial damage. Additionally, attention should be paid to the consequences of functional physical overload of the heart.
One of the drugs for the treatment of patients with coronary heart disease is arginine. Arginine (d-guanidine-a-aminovaleric acid) is a basic a-amino acid, the L- form of which is a semi-essential amino acid.
Arginine interacts with SH groups (nitrate receptors) in vascular smooth muscle cells, including coronary arteries, to form nitric oxide (NO), which is similar to EDRF in structure and effect. Due to its properties, arginine dilates arterioles and peripheral veins, reduces total peripheral vascular resistance, reduces venous outflow, and dilates pulmonary vessels, which reduces resistance in the small circulation and leads to regression of the symptoms in case of pulmonary edema, and reduces end-diastolic pressure and ventricular volume, thereby reducing myocardial oxygen demand. Additionally, arginine dilates the coronary arteries and prevents their spasm, reduces the diastolic tension of the ventricular wall, resulting in improved coronary circulation in the ischemic area.
Tivortin drug comprising arginine in the form of a salt, such as arginine hydrochloride, and water for injections, is known in the prior art for a combination therapy of coronary heart disease.
The disadvantage of this drug is that it has the form of the solution for infusion. The use of such the drug is possible only for the inpatient treatment, and administration of the drug requires the presence of qualified experienced staff in the inpatient facility.
The disease can be of varying severity (e.g, mild or severe), and depending on the disease severity, a patient may need treatment or inpatient care in specialized facilities or may be treated on an outpatient basis. In certain cases, for example, when a patient suffers from a relatively mild angina, treatment of a patient in inpatient facilities is pointless, otherwise treatment of a patient on an outpatient basis is advisable. It is known from open sources that substances of the so-called metabolic corrector class, such as inhibitors of oxidation of free fatty acids that affect the activity of enzymes involved in biochemical reactions, are widely used in cardiometabolic therapy.
A known representative of these inhibitors is levocarnitine (also known as L- carnitine). Levocarnitine facilitates the supply of long-chain fatty acids to the mitochondria of cells, thus providing a substrate for oxidation and energy production, which significantly improves the restoration of heart muscle cells in myocardial infarction. Levocarnitine inhibits the formation of atherosclerotic plaques in the blood vessels and promotes the resorption of the plaques that have already formed.
Thus, due to its properties, levocarnitine reduces the contribution of the above factors, such as hyperlipidemia, high-calorie diet, obesity, diabetes, smoking, hypodynamics, and age, to the development of coronary heart disease.
Pharmaceutical compositions for the treatment of coronary heart disease containing both arginine and levocarnitine are unknown in the prior art. Publications mention the use of a drug containing arginine and a drug containing levocarnitine in the treatment of coronary heart disease. However, these drugs are not used simultaneously, rather there is a time interval between the administration of two different drugs.
Acute cerebrovascular disorders and chronic cerebrovascular disorders include, in particular, acute form or chronic form of cerebrovascular insufficiency.
Acute or chronic form of cerebrovascular insufficiency is the result of progressive insufficiency of blood supply to the brain, which leads to the development of multiple small focal necrosis in the brain tissue and the gradual dysfunction of the brain. The main reasons that cause the onset and development of chronic cerebrovascular insufficiency are hypertension and atherosclerosis, depending on which hypertensive and atherosclerotic encephalopathy are distinguished.
Atherosclerosis is a chronic disease of the arteries, which develops due to impaired lipid and protein metabolism and is accompanied by the deposition of various fractions of cholesterol and proteins in the form of the plaques in the blood vessels, followed by the growth of the connective tissue (sclerosis), and calcification (calcium deposition), which results in deformation of the vessels and narrowing of the vessel lumen, in some cases, up to obturation (blockage of the vessel). Atherosclerosis leads to organ and/or general circulatory disorders. Depending on the severity of atherosclerosis and its localization in the vascular system, certain clinical manifestations are formed, and some of them are distinguished into individual syndromes and even nosological forms. Factors that lead to atherosclerosis are divided into endogenous (heredity, sex, and age) and exogenous (intoxication, hypertension, metabolic diseases, overeating, etc.). The following factors can lead to atherosclerosis: - age;
- genetic predisposition;
- food with high cholesterol;
- sedentary lifestyle;
- overweight;
- chronic diseases, such as hypertension and diabetes;
- frequent psycho-emotional loads.
In the pathogenesis of atherosclerosis, the leading role belongs to the impairment of lipid metabolism and protein metabolism. An important factor is the quality of fat consumed and the composition of food. Esterification of cholesterol with unsaturated fatty acids (linoleic, linolenic, and arachidonic) contained in oils or fish oil, causes the formation of insoluble cholesterol esters, which easily precipitate from the blood. Recently, in the development of atherosclerosis, changes of the ratio of various components of the so-called lipoprotein system (fat-protein complexes, which consist mainly of cholesterol-protein masses, i.e. fatty substances linked to proteins) in the blood have been considered to be important. It is the fat-protein complexes, which formed and deposited in the blood vessel intima, at the early stages of the disease that can be observed with light microscopy. Deposits in the intima of the aorta and large arteries are visible in the form of spots and stripes.
Atherosclerosis of the brain vessels (also referred to cerebrovascular atherosclerosis) is a pathological process characterized by the deposition of the plaques (referred to atherosclerotic plaques) on the walls of the large brain vessels, with their subsequent growth and replacement by connective tissue. The brain vessel lumen is narrowing gradually, and circulation insufficiency is developing. Lesions of the internal and external carotid arteries are the most common. The cause of this condition lies in the impairment of lipid metabolism and protein metabolism. Atherosclerotic plaques are detected in young people aged 20 years, but the highest prevalence of the disease is observed in adults aged 50 years or more, and more often in men than in women. The high prevalence of this disease among the population associates it even with one of the manifestations of aging.
Clinical signs of the disease do not appear immediately. This happens long after cholesterol started to deposit. Symptoms appear after narrowing of the lumen of the brain arteries and capillaries to such extent that the organs receive less blood by 15% or more.
Symptoms of cerebrovascular atherosclerosis include certain symptoms associated with patient complaints, which in severe cases significantly reduce the quality of life, namely sleep problems, such as insomnia, disturbing dreams, difficulty with waking up and problems with re-falling asleep; decreased body sensitivity; headaches; often repeated; gait change and incoordination; vision problems, tinnitus; emotional changes, such as irritability, depression, tearfulness, anxiety; hot flashes and facial sweating; rapid fatigue, constant weakness and inattention; trembling of the chin and limbs; memory problems or disorders, short-term memory problems.
Treatment for this disease is aimed at restoring metabolic processes, and prevention of bad cholesterol deposition on the blood vessel walls. Attention is also paid to restoration of the blood circulation and normalization of the brain tissue supply. Treatment is comprehensive and includes lifestyle changes and drug therapy. The lifestyle changes include choosing a diet that restricts the intake of lipids, breaking bad habits, increasing physical activity, avoiding stress, and reducing the level of psycho-emotional stress. The drug therapy focuses on the use of lipid-lowering drugs, antiplatelet, antihypertensive and antioxidant drugs, drugs for microcirculation improvement, and symptomatic therapy. The drug therapy usually takes a long period of time and depends on the disease severity.
The publication entitled "Cerebral atherosclerosis" on the website https://diseases.medelement.com/disease/cerebralny-ateroscleroz/12935 describes the treatment of cerebrovascular atherosclerosis using combination therapy that include the use of statin drugs, such as lovastatin (daily dose in the range of 20 to 80 mg), pravastatin (daily dose in the range of 20 to 80 mg), and the use of antiplatelet agents, such as acetylsalicylic acid (daily dose in the range of 50 to 100 mg).
Ischemia is a temporary dysfunction or permanent impairment of the organ tissue or the whole organ due to a local decrease in blood supply caused by a vascular factor (narrowing or complete obstruction of the artery lumen). The consequences of ischemia depend on the extent and rate of circulation parameters reduction, duration of ischemia, tissue sensitivity to hypoxia, and general status of the body. The most sensitive to ischemia are the central nervous system organs, myocardium, and kidney tissue. Ischemia differs from hypoxia, which is an oxygen starvation of the organ tissue due to disorders of external and internal (tissue, cellular) respiration. Ischemia is characterized by relative or absolute blood supply insufficiency, which is manifested by both local tissue hypoxia and metabolic disorders due to insufficient supply of the nutrients. Ischemia is a dynamic, and usually potentially reversible, process. The possibility of ischemic necrosis (heart attack) of the organ tissue directly depends on the duration and extent of the local circulation decrease.
Chronic cerebral ischemia is a slowly progressive brain dysfunction caused by diffuse or small-cell damage of the brain tissue during long-standing cerebral insufficiency. In the clinical protocol titled "DIAGNOSIS AND TREATMENT CLINICAL PROTOCOL FOR CHRONIC CEREBRAL ISCHEMIA", recommended by the Expert Council of the RSE on the REU "Republican Center for Health Development", Ministry of Health and Social Development of the Republic of Kazakhstan, the treatment for chronic cerebral ischemia involves a combination therapy including: antiplatelet drugs, such as the drugs that reduce the blood ability to thicken and improve the blood rheological properties by preventing the aggregation of erythrocytes and platelets; antioxidants and antihypoxants, such as the drugs that bind free radicals, delay oxidative processes, increase the body resistance to oxygen deficiency, affect intracellular redox processes indirectly, facilitating the transport of oxygen from the blood to the tissue, and improving the blood supply to the brain.
This clinical protocol describes the use of magnesium sulfate and acetylsalicylic acid for the treatment of chronic cerebral ischemia of the different severity.
Preeclampsia in pregnant women (formerly called toxemia in pregnant women) is a manifestation of either hypertension after the 20th week of the pregnancy in a woman whose blood pressure was normal before, or an increase in hypertension that existed before the 20th week of the pregnancy, or development of proteinuria, or both, and may be accompanied with the signs of other organs/systems impairment in the pregnant woman body. This disease affects both a mother and a fetus. The disease can result in the simultaneous manifestation of increased systemic vascular resistance, increased propensity of platelets to aggregate, increased activation of the coagulation system, as well as endothelial dysfunction. The reason is a dysfunction of the placenta or impairment of the placenta implantation, which is confirmed by the rapid cessation of this condition after childbirth. Functional and morphological changes occur in the kidneys of pregnant women, glomerular filtration decreases, and symptoms of kidney impairment may appear.
The method of treatment of preeclampsia in pregnant women depends on the degree of threat to the woman and fetus, the duration of the pregnancy and the degree of the fetal development. Antihypertensive drugs and magnesium sulfate are used the most commonly (see article titled DIAGNOSIS, EVALUATION AND MANAGEMENT OF HYPERTENSIVE DISORDERS IN PREGNANCY: BASIC CONCLUSIONS. Clinical Practical Guide of the Society of Obstetricians and Gynecologists of Canada, 2014//Reproductive Endocrinology.-2014.-N° 4(18).- P.74-85.). Drugs containing nifedipine, hydralazine, and labetalol are used as antihypertensive drugs. In mild disease, at gestational age <34 weeks, outpatient or inpatient treatment is possible. However, careful monitoring of the pregnant woman and fetus is required.
According to the Clinical Protocol on Obstetrics "Fetal Distress during Pregnancy and Childbirth", approved by the order of the Ministry of Health of Ukraine N° 900 on 27.12.2006, all disorders of the fetus functional status are referred to as "fetal distress". This term has come to be used as a generic term for all the fetus functional disorders, formerly referred to as "chronic fetal hypoxia" and "acute fetal hypoxia." Fetal distress is diagnosed according to the results of the fetal heart rate monitoring and fetal cardiac abnormalities recording, compilation and analysis of its biophysical profile, observation of umbilical cord circulation and record of umbilical cord circulation disorders. Detecting the reasons for the fetal cardiac abnormalities, its biophysical profile and umbilical cord circulation may be very problematic when using modern noninvasive methods of research. Publications and literature indicate that fetal distress is characterized by impaired fetus functional status due to acute or repeated restriction of oxygen access to the fetus or impaired ability of the fetus to use oxygen in cellular metabolism (metabolic acidosis). Depending on the rate of the fetal distress progression, it is divided into:
• chronic fetal distress that develops due to the constant influence of a pathogenic factor (e.g., anemia in pregnant women, intrauterine infection (IUI), hypertension in pregnant women, etc.);
• acute fetal distress that develops due to acute disorders of uterine-placental and placental-fetal circulation (e.g., placental abruption, umbilical factors, acute maternal hypotension (anaphylactic shock), uterine rupture, uterine tetany).
Etiological factors that may lead to fetal distress are divided into:
1 ) Preplacental factors that include conditions leading to:
- impairment of oxygen transport to the uterus and placenta;
- cardiovascular and pulmonary maternal pathology;
- anemia in pregnant women Hb <100 g/L;
- hypertension in pregnant women, hypotension in pregnant women, preeclampsia with a predominance of the hypertensive component,
- previous inflammatory diseases of the endometrium and abortion resulting in pathological changes in the spiral arteries and in the placental area;
- diabetic angiopathy (occlusive vascular disorders in the placental area, microthrombosis);
- overdue pregnancy and preeclampsia (microthrombosis, trophoblastic embolism and peripheral vasospasm in the area of the spiral arteries).
2) Placental factors:
- primary placental insufficiency (small placenta, placental hemangiomas, etc.);
- premature abruption of the normally located placenta;
- hyperplasia of the placenta due to an infectious toxic agent at the late stages.
3) Postplacental factors:
- umbilical factors (umbilical cord prolapse and compression, the true node of the umbilical cord, and umbilical cord entanglement); - congenital anomalies of cardiovascular system in the fetus and congenital disorders of nervous regulation in the fetus.
The observations in the publications suggest that, in most cases, the factors of fetal distress are placental factors that are associated with the circulation.
The utility model patent of Ukraine UA 44055 U (description of utility model patent published on 10.09.2009) describes a method of treating antenatal fetal distress including the use of anticoagulants with a drug containing polyunsaturated fatty acids, 2 times a day, with meals, for 29-30 days.
Intrauterine growth restriction syndrome is diagnosed in the fetus and neonates who have low birth weight or low body weight and height for their gestational age. The variety of causes determines the heterogeneity of the pathogenesis of intrauterine growth restriction syndrome. Intrauterine growth restriction can develop at different stages of the fetal development. Thus, low birth weight of a full-term neonate indicates that the factor that restricted the intrauterine fetal growth was acting during the last 2-3 months of pregnancy, but if at the same time there is a deficit in body length (below the 10th percentile for this period of the pregnancy), then unfavorable conditions for the fetus developed in the II trimester of the pregnancy. The first type of intrauterine growth restriction is called hypotrophic, the second - hypoplastic. The most common cause of the hypotrophic type of fetal intrauterine growth restriction is severe toxicosis at the second half of the pregnancy due to placental insufficiency, and, for hypoplastic type, it is multiple pregnancy, familial low birth weight, adolescent mother, minor nutritional deficiencies without deep hypovitaminosis.
Literature and publications note that the most common cause of intrauterine growth restriction is placental insufficiency. The correlation between the severity of placental insufficiency in pregnant women and the degree of intrauterine growth restriction was shown, and the treatment of placental insufficiency was shown to reduce fetal intrauterine growth restriction.
For the prevention of intrauterine growth restriction in pregnant women, conventional treatment and prevention measures are used, including hormonal correction, vitamin therapy, use of antioxidants, antispasmodics and antiplatelet agents, sedatives, tocolytic drugs, antibacterial drugs, and vasoactive drugs. The patent US 8389483 B2 (description of the invention was published on 05.03.2013) describes a method of preventing intrauterine growth restriction due to placental vascular insufficiency, including intravenous bolus of 50-100 cubic meters of at least 10-20% hypertensive glucose to a pregnant woman two or three times a day.
Asthenia (also referred to as asthenic condition, asthenic syndrome, asthenic reaction, asthenodepressive syndrome, asthenoneurotic syndrome) is a pathological condition manifested by increased fatigue and exhaustion with a very unstable mood, self-control reduction, impatience, restlessness, sleep disorder, inability to endure prolonged mental and physical stress, intolerance of loud sounds, bright light, and pungent odors. Asthenic condition can be constitutionally conditioned (asthenic psychopathy), but can occur because of malnutrition, vitamin deficiency, excessive physical and mental stress, vascular, organic, and endocrine diseases, as well as during convalescence of infections, intoxications, and injuries. Asthenic condition is characterized by affective lability, increased excitability, which is replaced by exhaustion (weakness with irritability), and hyperesthesia (acute sensitivity to sound, light, tactile stimuli). Often headaches and sleep disorders (increased drowsiness during the day and insomnia at night) develop, as well as a dramatic change in well-being depending on changes in weather. Asthenic condition that develops as a result of various organic diseases is characterized by mild memory impairment, generally, with impairment of memory of recent events. Asthenic disorders develop slowly, increasing in intensity. Sometimes the first manifestations of asthenic condition are increased fatigue and irritability, which are combined with impatience and constant desire to work, even in an environment that means rest. In severe cases, asthenic disorders may be accompanied by passivity and adynamia. The clinical picture of asthenic condition has specific features and depends on the reasons that caused it. Asthenic condition after somatic diseases acquires the form of emotional-hyperesthetic weakness, in which fatigue and affective lability are combined with intolerance of emotional stress. After a traumatic brain injury, asthenic condition is manifested by weakness with irritability, headache, mentism (flow of thoughts) and autonomic disorders. In endocrinopathy, asthenic condition is expressed by the increased fatigue, exhaustion, and inactivity. Asthenic condition in schizophrenia is manifested by the predominance of mental exhaustion and inconsistency of the latter with the degree of mental stress. In atherosclerosis, the main signs of asthenic condition are strongly expressed fatigue, weakness with irritability, decrease in mood, and tearfulness. In arterial hypertension, asthenic condition acquires the form of "restless fatigue." In progressive palsy, the combination of the increased fatigue with mild dimming (obnubilation) is observed. To correct asthenic conditions, treatment depending on the condition origin and aiming at the underlying disease, adequate nutrition, and sleep, is needed. According to the indications, the use of actoprotectors, nootropic drugs, psychostimulants, adaptogens, multivitamins is possible.
In general, patients develop weakness with irritability, expressed by increased excitability rapidly followed by exhaustion, affective lability with a predominance of low mood with features of moodiness and dissatisfaction, as well as tearfulness. This syndrome is a manifestation of various disorders.
Asthenia onset as a result of exhaustion, diseases of internal organs, infectious diseases, immune system disorders, anemia, endocrine (hormonal) disorders, intoxications, side effects of the treatment with certain drugs, emotional, mental and physical overload, improperly organized work, rest, nutrition, and also in nervous and mental illnesses.
There is a known method of treating asthenic syndrome (see the description of the patent of Russian Federation RU 2463045 C1 , publ. 10.10.2012) including prescribing to a patient a drug being a composition containing 2.0 ml of cytoflavin and 5 ml of novocaine and is administered suboccipitally by interstitial injection into the posterior region of the neck. The disadvantage of the known method of the asthenic syndrome treatment is the drug dosage form, namely interstitial injection. Thus, to perform the interstitial injection of the drug, qualified medical personnel is required.
Modern sports science requires the development and use of adequate pharmacological support to accelerate adaptation to high-intensity exercise, stimulation of physical fitness, especially in the high-achievement sports, prevention of overtraining and sports injuries. With a huge variety of existing means of pharmacological support for the physical fitness, their systematization and knowledge of the mechanisms of influence and the main points of application are necessary.
Pharmacology of sports medicine (or sports pharmacology) is a relatively new, but very actively advancing field of the clinical and experimental pharmacology in recent years. The sports pharmacology aims at development, study, and practical implementation of drugs (DR) and dietary supplements (DS) to increase the adaptation of athletes to high-intensity exercise, and one of the main objectives of this field is to identify and correct factors that limit the physical fitness of athletes.
Currently, the sport training system, especially in the high-achievement sports, means exceptionally high training and competition loads accompanied by high levels of emotional stress. It is natural that such high loads are the most powerful factor in mobilizing the body functional reserves, stimulating intensive adaptation processes, increasing endurance, strength, speed and, obviously, the increase in sports results. In this case, an important role in improving physical fitness, preventing fatigue, and accelerating the recovery process after exercise belongs to a balanced diet.
There is a known method of improving the physical fitness and physical endurance of a human (see the description of the patent of Russian Federation RU 2642673 C1 , publ. on 25.01.2018) that include administering to a human a composition containing vitamin A, vitamin E, succinic acid, dry guarana extract and chocolate mass in a certain ratio. The disadvantage of this method of increasing physical endurance is the low effectiveness of this composition in the case of significant physical load, for example, when the human is exposed to physical load in sports.
SUMMARY OF THE INVENTION
The first object of the invention is to develop a pharmaceutical composition for the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human, treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human, prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, treatment of asthenia in a human, and improvement of the exercise tolerance in a human; to expand a range of the drugs for the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human, treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human, prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, treatment of asthenia in a human, and improvement of the exercise tolerance in a human; and to increase the effectiveness of the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human, treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human, prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, treatment of asthenia in a human, and improvement of the exercise tolerance in a human.
The second object of the invention is to improve the method of the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral artery disease in a human; to expand a range of the drugs for the treatment of chronic ischemic heart disease, or stable angina pectoris or peripheral artery disease in a human; and to increase the effectiveness of the treatment of coronary heart disease, or stable angina pectoris or peripheral artery disease in a human.
The third object of the invention is to improve the method of the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human; to expand a range of the drugs for the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human; and to increase the effectiveness of the treatment of acute cerebrovascular disorders and chronic cerebrovascular disorders in a human.
The fourth object of the invention is to improve the method of the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction; to expand a range of the drugs for the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction; and to increase the effectiveness of the prevention of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction.
The fifth object of the invention is to improve the method of the increasing exercise tolerance in a human being exposed to physical load, and to improve the human body status in asthenia syndrome; to expand a range of the drugs for the increasing exercise tolerance and improving the human body status in asthenia syndrome; and to increase the drug effect on a human to improve exercise tolerance in a human and human body status in asthenia syndrome.
The first object is achieved by a pharmaceutical composition having an oral solution dosage form and comprising active ingredients and excipients, the pharmaceutical composition comprising levocarnitine and arginine salt, such as arginine aspartate, as active ingredients, and pH adjusting agent that is an acidifier, sweetening agent and preservative as excipients, in the following ratio, mg/ml: arginine aspartate 180-320 levocarnitine 50-150 pH adjusting agent that is an acidifier 1.5-6.0 sweetening agent 0.4-1 .2 preservative 0.5-2.0 water balance to 1 ml
In addition, according to one embodiment of the first object of the invention, the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 240-300 levocarnitine 80-120 pH adjusting agent that is an acidifier 2.5-4.5 sweetening agent 0.6-1.0 preservative 1.0-1 .5 water balance to 1 ml
In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 264 levocarnitine 100 pH adjusting agent that is an acidifier 3 sweetening agent 0.8 preservative 1 water balance to 1 ml In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition may comprise malic acid as pH adjusting agent that is an acidifier.
In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition may comprise sodium saccharin as the sweetening agent.
In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition may comprise methyl parahydroxybenzoate and/or propyl parahydroxybenzoate as the preservative.
In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition may comprise water for injections as water.
In addition, according to one of the embodiments of the first object of the invention, the pharmaceutical composition may have density of 1.1 g/ml, solution pH of 5-6.5, and dynamic viscosity of 2.5 sP at 20 °C.
The second object is achieved by a method of treating chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease in a human suffering from chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease, including administering to the said human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the treatment of chronic coronary heart disease, stable angina pectoris, and/or peripheral artery disease.
In addition, according to one of the embodiments of the second object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered as part of a combination therapy of chronic coronary heart disease, stable angina pectoris, and/or peripheral artery disease.
In addition, according to one of the embodiments of the second object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
The third object is achieved by a method of treating acute cerebrovascular disorders and/or chronic cerebrovascular disorders in a human suffering from acute cerebrovascular disorders and/or chronic cerebrovascular disorders, including administering to the said human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the treatment of acute cerebrovascular disorders and/or chronic cerebrovascular disorders.
In addition, according to one of the embodiments of the third object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered as part of a combination therapy of acute cerebrovascular disorders and/or chronic cerebrovascular disorders. In addition, according to one of the embodiments of the third object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
The forth object is achieved by a method of prevention and treatment of the diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction, including administering to a pregnant woman a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for the prevention and treatment of preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction.
In addition, according to one of the embodiments of the forth object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
The fifth object is achieved by a method of increasing exercise tolerance in a human being exposed to physical load, and/or improving the human body status in asthenia syndrome, including administering to a human a drug being the pharmaceutical composition according to one of the embodiments of the first object of the invention, in an amount effective for increasing exercise tolerance, and/or improving the human body status in asthenia syndrome.
In addition, according to one of the embodiments of the fifth object of the invention, the human may be exposed to the physical load in sports.
In addition, according to one of the embodiments of the fifth object of the invention, the human body status in asthenia syndrome is a condition that occurs after the human previous disease.
In addition, according to one of the embodiments of the fifth object of the invention, the pharmaceutical composition according to one of the embodiments of the first object of the invention may be administered in a daily dose of 20-40 ml.
DETAILED DESCRIPTION OF THE INVENTION
The sweetening agent corrects the taste properties of the pharmaceutical composition. Certain ratio of malic acid and the sweetening agent in the pharmaceutical composition provides a pleasant taste for most people. In the preferred embodiment, sodium saccharin, that is approximately 500 sweeter than sugar, is used as the sweetening agent.
The preservative provides stability to the solution of the pharmaceutical composition. Methyl parahydroxybenzoate and/or propyl parahydroxybenzoate can be used as the preservative.
The content of the auxiliary components is adjusted based on the object of the achieving a high concentration of the active ingredients and achieving the solution stability. In pharmacy, arginine is often used in the form of a salt, such as arginine aspartate. Hereinafter in the text, the term "arginine" means arginine aspartate.
The pharmaceutical composition according to the invention is a clear solution, and the dosage form for the use is the oral solution. A method of manufacturing of the pharmaceutical composition according to the invention is given below in Examples 1 -20.
Example 1
The pharmaceutical composition according to the invention is prepared by mixing the components of the pharmaceutical composition in water.
The stainless steel reactor is charged with 180 L of water for injections heated at 80 °C. 0.08 kg of methyl parahydroxybenzoate and 0.02 kg of propyl parahydroxybenzoate are then charged to the reactor and stirred until complete dissolution. The liquid in the reactor is then cooled at 40 °C. 36 kg of arginine aspartate is then added to the reactor and stirred vigorously for a period of time sufficient to obtain a clear solution. The reactor is then charged with 10 kg of levocarnitine and stirred vigorously for period of time sufficient to obtain a clear solution. 0.3 kg of malic acid is then charged to the reactor and stirred until complete dissolution. Then 0.08 kg of sodium saccharin is added to the reactor and stirred until the components are dissolved and a clear solution is obtained. Water for injections is then added to the reactor, diluting the volume of the solution to 200 L. The resulting solution is cooled, saturated with nitrogen to residual oxygen content less than 300 ppm, following with the filtration of the solution through a membrane filter. After the filtration, the solution is poured into glass or polymer containers (bottles).
Example 2
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 36 kg of arginine aspartate and 15 kg of levocarnitine, 0.6 kg of malic acid, 0.13 kg of sodium saccharin, 0.1 kg of methyl parahydroxybenzoate and 0.1 kg of propyl parahydroxybenzoate.
Example 3
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 36 kg of arginine aspartate and 25 kg of levocarnitine, 0.9 kg of malic acid, 0.18 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
Example 4
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 36 kg of arginine aspartate and 30 kg of levocarnitine, 1.2 kg of malic acid, 0.24 kg of sodium saccharin, 0.1 kg of methyl parahydroxybenzoate and 0.3 kg of propyl parahydroxybenzoate.
Example 5
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 10 kg of levocarnitine, 0.4 kg of malic acid, 0.09 kg of sodium saccharin and 0.1 kg of methyl parahydroxybenzoate.
Example 6
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 44 kg of arginine aspartate and 15 kg of levocarnitine, 0.7 kg of malic acid, 0.12 kg of sodium saccharin and 0.2 kg of propyl parahydroxybenzoate.
Example 7
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 25 kg of levocarnitine, 0.9 kg of malic acid, 0.17 kg of sodium saccharin and 0.3 kg of methyl parahydroxybenzoate.
Example 8
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 44 kg of arginine aspartate and 30 kg of levocarnitine, 1.2 kg of malic acid, 0.24 kg of sodium saccharin, 0.3 kg of methyl parahydroxybenzoate and 0.1 kg of propyl parahydroxybenzoate.
Example 9
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 10 kg of levocarnitine, 0.5 kg of malic acid, 0.1 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
Example 10
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 15 kg of levocarnitine, 0.7 kg of malic acid, 0.14 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate. Example 11
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 53 kg of arginine aspartate and 25 kg of levocarnitine, 1 .0 kg of malic acid, 0.19 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
Example 12
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 53 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
Example 13
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 75 °C, 58 kg of arginine aspartate and 10 kg of levocarnitine, 0.6 kg of malic acid, 0.12 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
Example 14
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 15 kg of levocarnitine, 0.8 kg of malic acid, 0.16 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate.
Example 15
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 25 kg of levocarnitine, 1 .0 kg of malic acid, 0.20 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
Example 16
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 58 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
Example 17
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 10 kg of levocarnitine, 0.7 kg of malic acid, 0.14 kg of sodium saccharin and 0.1 kg of propyl parahydroxybenzoate.
Example 18
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 15 kg of levocarnitine, 0.9 kg of malic acid, 0.18 kg of sodium saccharin and 0.2 kg of methyl parahydroxybenzoate.
Example 19
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 25 kg of levocarnitine, 1.1 kg of malic acid, 0.22 kg of sodium saccharin and 0.3 kg of propyl parahydroxybenzoate.
Example 20
The pharmaceutical composition according to the invention is prepared similarly to the method described in Example 1 . However, the reactor is charged with water for injections heated at 80 °C, 64 kg of arginine aspartate and 30 kg of levocarnitine, 1 .2 kg of malic acid, 0.24 kg of sodium saccharin and 0.4 kg of methyl parahydroxybenzoate.
The pharmaceutical composition according to the invention can also be prepared by other methods.
To study the pharmaceutical effect of the claimed pharmaceutical composition according to the invention, a number of studies were conducted.
To study toxicity and therapeutic effect concerning the treatment of various diseases, a number of preclinical and clinical trials were conducted. The design and results of the conducted trials are given hereinafter. The pharmaceutical composition being the first object of the invention is abbreviated as PC hereinafter.
To study the toxicity of the PC, the following study was performed.
The aim of the study was to investigate the acute toxicity parameters of the PC being the solution for oral administration when administered intragastrically to rats of both sexes (males, females).
The aim of the study included:
- assessment of the acute toxicity class for the PC in the rats of both sexes, when the PC is administered intragastrically;
- assessment of the acute toxicity class for the dry mixture of the PC substances in the rats of both sexes, when the PC is administered intragastrically. When lethality is observed, LD50 for the dry mixture of the PC substances was calculated.
The study techniques included toxicological, laboratory, pathomorphological and statistical techniques.
The objects of the study included the PC being the solution for the oral administration, placebo (solvent) and the dry mixture of the substances.
The study was conducted according to the Guidelines for experimental (preclinical) study of new drugs (2001 ), Order N° 944 of State Expert Centers of the Ministry of Health of Ukraine and meets the requirements of Good Laboratory Practice (GLP).
Tested objects were:
1 ) The PC being the solution for oral administration. Composition: levocarnitine and L-arginine aspartate as the active ingredients (1 ml of the solution comprises 100 mg of levocarnitine and 264 mg of arginine aspartate) and malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216) and water for injections as the excipients.
2) Placebo (solvent). Composition: malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216) and water for injections.
3) The dry mixture of the PC (solution for oral administration) substances. The composition of the mixture is given for 368.8 g, which corresponds to 1000 ml of the PC in the form of the solution for oral administration. The active ingredients are levocarnitine - 100 g, and arginine aspartate - 264 g; and the excipients are malic acid - 3.0 g, sodium saccharin - 0.8 g, methyl parahydroxybenzoate (E218) - 0.8 g, and propyl parahydroxybenzoate (E216) - 0.2 g.
Animal acclimatization and maintenance.
The duration of the animal acclimatization period was 14 days. During this period, each animal was examined on the daily basis (general condition, morbidity). Prior to the study, animals that met the inclusion criteria were randomized into groups according to the weight. Animals that did not meet the inclusion criteria were excluded from the experiment.
The animals were maintained in a separate room with controlled microclimate parameters: air temperature + 20-24 °C, humidity 45-65%, "12 hours day/night" light regime; in standard plastic cages, 6 animals per cage. The animals were managed according to the scientific and methodological recommendations for maintaining laboratory animals and working with them / Yu.M. Kozhemyakin, O.S. Khromov, M.A. Filonenko, G.A. Saifetdinova. - K Avitsena, 2002. - 156 c.
The animals had free access to water. Settled tap water from glass drinkers was used for drinking. Granular complete feed TM "MOUNTAIN" (corresponds to TU.U15.7-2123600159-001 : 2007) was used as feed. Animals were managed according to the rules of the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes.
Monitoring of the animals
During the study, each animal was subjected to a daily examination including an assessment of the animal general behavior and condition. Visually visible pathological formations were palpated.
Acute toxicity experiments were performed on the nonlinear outbred white rats of both sexes weighing 200±20 g, aged 13-15 weeks. A total of 210 rats were used in the experiment. All animals were divided into groups of 12 animals (6 males, 6 females). Each animal was assigned an individual number. Prior to the experiment, the animals were numbered continuously from 1 to 210. The groups were randomized according to the body weight as a leading feature (the spread in the initial weight between and within the groups did not exceed 10%). The study design is presented in Table 1.
Table 1
The study design
Figure imgf000023_0001
Figure imgf000024_0001
The animals in the experimental groups were administered with 2 ml/100 g of the weight of the samples per one injection with 2-hour interval between the injections. Immediately after the administration, the animals were monitored for signs of intoxication (if any). The nature of respiration, motor activity, the presence/absence of seizures, vomiting, diarrhea, ophthalmic symptoms, cardiovascular symptoms, salivation, piloerection, and muscle tone were recorded. The animals were allowed to eat 2 hours after the last injection of the test sample (hereinafter abbreviated TS), whereas access to water was free.
The animals were being monitored for 14 days. The animal weight was recorded before the experiment and on 3, 7 and 14 days of the experiment. The average lethal dose LDso was calculated based on the percentage of the animal mortality in each group depending on the dose administered, using Kerber method (Preclinical studies of drugs (guidelines))/Edited by Stefanova O.V. - Publishing House "Avitsena", 2001. - 527 p.).
On day 14, all the survived animals were sacrificed under inhalation anesthesia. At autopsy, the animal internal organs status was assessed macroscopically. The absolute mass of the heart, liver, spleen, lungs, kidneys, adrenal glands, testes, and thymus was measured. Weight factors (WF), such as relative weight of the organs, was calculated according to the formula:
Figure imgf000025_0001
The histological structure of the liver, kidneys, lungs, myocardium, thymus, spleen, adrenal glands, pancreas, esophagus, stomach, small intestine and rectum, testicles, and ovaries of the rats was studied on 14th day after intragastric administration of 40 ml/kg of the PC solution and compared with the histological structure of the similar organs of the rats administered with 40 ml/kg of placebo (solvent) (negative control), and the intact animals.
The autopsed organ samples were fixed in 10% formalin solution, dehydrated in alcohols of the growing concentration, and embedded into paraffin. The sections were dyed with hematoxylin and eosin. The micropreparations were examined using Granum microscope. The microscopic images were photographed using a Granum DSM 310 digital video camera. The photographs were processed on a 2.4GHz Pentium computer with Toup View power.
The primary data obtained were processed using generally accepted methods of analysis of variance using parametric criteria for comparison of quantitative indicators (ANOVA analysis of variance, Newman-Keuls criterion) and nonparametric criteria (Kruskal-Wallis method, Mann-Whitney criterion). To compare qualitative variables, the criterion c2 was used. Before using parametric criteria, the hypothesis for the normality of the random variable distribution was tested using Leuven test.
For multiple comparisons, the significance level p <0.050 was adopted. When using nonparametric methods for the comparison, the Bonferroni correction was used, according to which the level of significance was p <0.0250. The standard package of statistical programs "Statistica 6.0" was used for mathematical calculations.
The effect of the PC on the survival of the white rats
The PC being the solution for the oral administration and placebo (solvent) were administered intragastrically for one day at a dose of 2 ml/100 g of the animal, 2 times per day with 2-hour interval. In total, the volume of the TS administered was 40 ml/kg. 20-30 minutes after the first administration of the TS, the animals demonstrated a decrease in motor activity, which is, obviously, associated with a large volume of the solutions administered. These symptoms disappeared in 1 hour, and, after that, the animal behavior did not differ from the behavior of the animals in the IC group. Repeated administration of the solutions to the animals also caused similar symptoms that disappeared in 1 hour.
Subsequent monitoring for 14 days showed that all animals were active, had a satisfactory appetite, responded normally to sound and light stimuli. Urination and defecation were adequate. There were no signs of respiratory distress. No seizures were observed.
For the entire monitoring period, death of the animals was not recorded in any of the experimental groups (Table. 2).
Table 2
The results of the mortality study after the intragastric administration of the TS to the rats of both sexes
Figure imgf000026_0001
According to the methodological recommendations, an important indicator is the animal body weight, the change of which characterizes the severity of the drug toxic effects. According to the data obtained, positive dynamics of the body weight was observed in all the experimental groups (Table. 3). Weight gain in both male and female rats did not differ from the weight gain in the 1C group.
Table 3
The results of the TS effect on the dynamics of the rat body (g), n = 6
(M±m)
Figure imgf000026_0002
Figure imgf000027_0001
Note: 1. pANOVA - statistical significance level between the experimental groups or within each experimental group (one-way analysis of variance ANOVA)
2 pi N-K _ statistical significance level regarding the result (Newman-Keuls criterion)
3. n - the number of the animals in each group.
Analysis of the internal organ weight factors for the animals indicates the absence of the toxic effects of the PC solution and placebo in acute overdose. The internal organ weight factors are within the physiological norm and do not differ statistically significantly from the factors for the animals in the IC group (Table. 4, 5).
Table 4
The results of the TS effect on the internal organ weight factors (%) for male rats, n = 6, M (Mmin ÷ Mmax)
Figure imgf000027_0002
Figure imgf000028_0001
Note: 1. pK u - statistical significance level between the experimental groups (Kruskal-Wallis criterion);
2. n - the number of the animals in each group.
Table 5
The results of the TS effect on the internal organ weight factors (%) for female rats, n = 6, M (Mm in ÷ Mmax)
Figure imgf000028_0002
Figure imgf000029_0001
Note: 1. pK_u - statistical significance level between the experimental groups (Kruskal-Wallis criterion);
2 P2m U - statistical significance level regarding the IC group (Kruskal-Wallis criterion);
3. n - the number of the animals in each group.
The results of the macroscopic examination.
Appearance of the hair body coat and mucous membranes of natural openings for the animals in the experimental groups (No. 1 -3) did not differ from those for the rats in the intact group and the group of the animals treated with placebo.
Fecal masses were formed, the anus and vaginal opening were not contaminated, the testicles were located in the scrotum and were movable. At autopsy, the location of the mediastinal organs in thoracic and abdominal cavities conformed to the norm for all the rats. The thymus varied slightly in size and was gray-pink.
The heart had regular configuration and size, with a typical location of coronary arteries and veins. The epicardium surface did not have any specific features, the myocardium section was dense. The lungs occupied the entire pleural cavity, had pale pink color, were airy, and did not have adhesions between the leaves of the pleura. Thoracic lymph nodes were not enlarged. Peritoneum was transparent and smooth. No foreign content was found in the abdominal cavity. The liver had uniform reddish-brown color, the capsule was not tense, and the edges of the lobes were not rounded. The surface of the organ was smooth. Pancreas did not have signs of hemorrhage, sclerosis, fatty necrosis; had pale pinkish-yellowish color and a form of a loose, weakly branched strand scattered along the gastrosplenic ligament. The spleen was full-blooded and had red-cherry color.
Renal capsule was easily removed. Dense layers with preserved patterns were clearly visible on the organ section. Adrenal glands without any specific features. Peritoneal lymph nodes were not enlarged. The mucous membrane of the glandular stomach had a typical relief of the gastric folds, normal color, no hemorrhages, edema, or erosive lesions. The mucous membrane of the small and large intestine had regular color, and the content conformed to the parts. Testicles, testicular appendages, prostate, seminal vesicles in the male rats, and uterine horns and ovaries in the female rats had no pathology. The bladder was small with a thin wall.
Pathomorphological examination of the rat internal organs Histological examination revealed that, 14 days after the administration of 40 ml/kg of the PC solution and placebo (solvent), the liver of the rats did not differ structurally from the liver of the intact animals.
The border between the hepatic lobes was lubricated, zones of the portal tracts (triads) were narrow, and radial orientation of the hepatocyte strands was preserved.
Signs typical for the functional stress, activation, or suppression of hepatocytes, such as changes in the size and location of the nuclei, the number of nucleoli in the nucleus, and the condition of the chromatin substance, were not detected. The population of dinuclear hepatocytes and the condition of the microcirculatory tract were not visually altered. Apoptosis, mitotic activity, and activation of Kupffer cells were not increased.
Kidneys. The condition of the glomeruli and the system of convoluted and straight tubules were comparable in the experimental and control rats. The glomeruli varied moderately in size, and their density was regular. The lumen of the capsule was free. The nuclear saturation of the glomerular capillaries and the clarity of the capillary network pattern was moderate. Epithelium of proximal and distal part of the nephron tubules had no changes. The level of fluffiness of apical segments of the cells was comparable. The tubules of the cerebral layer were regular.
The myocardium retained its normal histological structure. Cardiac muscle fibers were uniformly colored and arranged quite densely. In cardiomyocytes, the transverse striated myofibrils, occupying the whole sarcoplasm free from the nucleus, were moderately expressed. Nucleus had oblong-rounded shape and were normochromic. Inter-beam spaces were not large. Vessels of the venous type were often full-blooded. Stromal cellular reaction was not visible (Fig. 3).
In the respiratory segment of the lungs of the experimental and control rats, the alveolar pattern of the parenchyma was clear. Signs of alveolar edema and increase in cell saturation of the interalveolar septa were not observed. Lymphocytic reaction in the stroma of the alveolar tree conformed to the norm in all the rats. The bronchial epithelium and bronchioles were intact.
The histological structure of the thymus in the experimental rats did not differ from the structure in the control animals. The lobes were well formed, and the density of lymphocytes in the cortex and medulla was typical. Thymic corpuscles were small and not numerous. Many rats in both groups had a moderate pattern of "starry sky".
Spleen lymphoid nodules were regular in the size and number. Periarterial T- dependent and marginal B-dependent zones, as well as germinative centers, were clearly visible in the lymphoid nodules. Numerous erythrocytes and nuclear forms of cells were visible in the red pulp.
The adrenal glands of the experimental rats maintained their inherent histological structure. Signs of the changes in histological characteristics indicating a change in the production of mineral- and glucocorticoids were not observed when compared with the control micropreparations. In the cerebral layer, the functional status of neuroendocrinocytes (chromaffin cells) varied moderately within physiologically normal limits, and the sinuses were full-blooded.
Exo- and endocrine segments were clearly visible in the pancreas.
Excretory glandular cells of acinuses had typical two-zone coloring of cytoplasm, and cytoplasm ratio in the experimental and control rats coincided. The islet apparatus was represented by pancreatic islets of different size, filled with insulinocytes evenly and densely enough.
In the esophagus of all the studied rats, the structure of the multilayered keratinizing epithelium was not impaired. No signs of irritation in the stroma of the mucous membrane and in the submucosal layer were recorded.
After the administration of 40 ml/kg of the PC solution, desquamative processes in the lining epithelium of the mucous membrane of the stomach studied area were absent, and the epithelium in the pits was regular. Stomach glands were straight, long, with a typical arrangement of the main, lining, and mucous cells.
In the large intestine, the appearance of the villi of the mucous membrane was regular. Epithelial cells lining the villi and intestinal crypts were not altered, goblet cells were sufficient in number and were at different stages of secretion.
In all the experimental rats, the mucous membrane of the rectum was lined with a cubic single-layer epithelium with a clear cuticular border and significant infusion of the goblet cells. The nuclei of epithelial cells were located at the same level, intestinal crypts were moderately deep, and the area of mitosis in the intestinal crypts was restricted by the bottom area. Lymphoid cell saturation of the stroma of the mucous membrane was moderate.
Testicles in all the males at the time of the study had no pathological changes. The strip of spermatogenic epithelium was quite wide and contained 3 to 5 rows of germ cells arranged in regular concentric layers, according to the stages of the sexual cycle. All stages of spermiogenesis and spermatogenesis were traced. Sertoli cells and Leydig cells were not visually altered.
In the ovaries of the female rats in the experimental and control groups egg follicles of different stages of development and corpora lutea were clearly visible. After the administration of 40 ml/kg of the PC and placebo (solvent), optically visible changes in the quality and number of the egg follicles and corpora lutea were not observed.
The macro- and microscopic data obtained allow concluding that 14 days after the intragastric administration of 40 ml/kg of the PC and placebo (solvent), they did not lead to significant changes in the histological structure of the studied internal organs of the rats in comparison with the intact control. Thus, the results obtained indicate that the PC and placebo (solvent) when administered intragastrically to the laboratory rats (males and females) at the dose of 40 ml/kg does not cause death and does not affect the physiological processes in the animals.
According to the classification of toxic substances, the PC belongs to the toxicity class VI, such as relatively safe substances, with LD50 more than 15 ml/kg. Since the exact value of LD50 for the finished dosage form of the PC could not be measured, the median lethal dose was assessed using the content of the active ingredients in the drug. To do this, a number of the doses of the dry mixture of the PC substances, which were administered to the animals in the same volume (100 ml/kg), were used. Due to the large volume, the obtained solutions were administered to the animals in divided doses with the maximal allowable volume for the rats being 2 ml/100 g of the animal per one administration, 5 times a day with 2- hour intervals between the administrations. 10-20 minutes after the first administration of the studied doses, the animals demonstrated a decrease in motor activity. The second and third administrations caused gastric overflow in the animals, whereas fecal masses were soft and not formed, followed by diarrhea. The fourth and fifth administrations had a strengthening effect on the general condition of the animals resulting in an open form of diarrhea, mucus secretion, wet and dirty tail. With increasing the dose, the animals weakened faster. An increase in severe toxic effects, which resulted in the animal death, were observed. For the males, mortality occurred for the range of 30.000 - 60.000 mg/kg, for the females - 40.000 - 60.000 mg/kg. Animal mortality was observed on the 1 st day after the PC administration. Animal mortality is presented in Table 6.
Table 6
Study of the lethal effects of the dry mixture of the PC substances administered intragastrically to the rats
Figure imgf000032_0001
Figure imgf000033_0001
Further monitoring of the survived animals revealed a gradual recovery of all the physiological processes in the animals. On 3-14 days, the animals were active, had a satisfactory appetite, responded normally to sound and light stimuli. Urination and defecation were also normal. Respiratory disorders and seizures were not observed.
The effect of the dry mixture of the PC substances on the animal weight are provided in Tables 7 and 8.
Table 7
The effect of the dry mixture of the PC substances on the body weight (g) dynamics for the male rats, n = 6, M±m
Figure imgf000033_0002
Note: 1. pANOVA - statistical significance level in each experimental group (one way analysis of variance ANOVA)
2. pi N K — statistical significance level regarding the result (Newman-Keuls criterion)
3. n - the number of the animals in each group.
Table 8
The effect of the dry mixture of the PC substances on the body weight (g) dynamics for the female rats, n = 6, M±m
Figure imgf000034_0001
Note: 1. pANOVA - statistical significance level in each experimental group (one way analysis of variance ANOVA)
2 pi N-K _ statistical significance level regarding the result (Newman-Keuls criterion) 3. n - the number of the animals in each group.
The study results showed that the administration of 40 ml/kg (maximal dose) of the PC and placebo (solvent) does not cause animal death and does not lead to statistically significant changes in the relative weight of the internal organs of the rats of both sexes. Histological examination confirms the absence of the toxic effects of the PC and placebo on the internal organs when administered at the toxic doses. The administration of the maximal doses of both the dry mixture of the PC substances and individual PC substances caused the animal death, and this allowed calculating the median lethal doses. The median lethal dose of the dry mixture of the PC substances for the males was 33.333 mg/kg, for the females - 38.750 mg/kg. The median lethal dose of arginine aspartate substance for the male rats was 29.792 mg/kg, for the females - 30.833 mg/kg, and the median lethal dose of levocarnitine substance for the males and females was 17.833 mg/kg.
The administration of the high doses of the dry mixture of the PC substances caused gastrointestinal dysfunction in the animals, which in turn led to dehydration and weight loss. On the third day, the animal weight decreased in all groups, both for the males and females, compared with the initial value. On the 7th day the animal weight was restored, and, by the end of the monitoring period, the weight exceeded the initial values.
The obtained mortality results allowed calculating the median lethal doses LD50. For the males, LD50 was 33.333 mg/kg, and for the females LD50 was 38.750 mg/kg. Thus, the mixture of the PC substances, when administered to the laboratory rats (males and females) intragastrically, does not cause death and does not affect the physiological processes in the animals. According to the classification of toxic substances, the mixture of the PC substances belongs to the toxicity class VI, such as relatively safe substances, with LD50 more than 15 mg/kg.
The subsequent studies aimed at investigating the possible toxic effects of the drug when administered repeatedly for 28 days.
The study techniques included general clinical, laboratory, physiological, biochemical, pathomorphological, and statistical techniques.
The object of the study was PC being the solution for the oral administration, whereas 1 ml of the solution comprises the active ingredients, such as 100 mg of levocarnitine and 264 mg of arginine aspartate, and the excipients, such as malic acid, sodium saccharin, methyl parahydroxybenzoate (E218), propyl parahydroxybenzoate (E216), and water for injections.
This study aimed at identifying physiological and structural changes caused by prolonged intragastrical administration of the PC being the solution for the oral administration, with studying the effects on the gastrointestinal tract, in the experiments on the rats of both sexes, and determining the dose dependence of these changes.
The objects of the study included:
1 ) Studying of the toxic effects of 2 and 20 ml/kg of body weight of the PC being the solution for the oral administration on the rats (males, females).
2) Studying of the effect of the PC on the gastrointestinal tract status, when administered on the long-term basis.
The studies were conducted according to the Guidelines for experimental (preclinical) study of new drugs (2001 ), Order N° 944 of State Expert Centers of the Ministry of Health of Ukraine and met the requirements of Good Laboratory Practice (GLP).
Experiments to study subacute toxicity were performed on 48 nonlinear outbred white rats of both sexes weighing 170-210 g, aged 3.5-4 months. All the animals were divided into 4 groups, 12 individuals per group (6 males, 6 females).
An experimental study on a rat model of acute asphyxia found that 1 and 2 ml/kg of body weight of the PC show the greatest and almost identical activity. To study the PC toxic effect, the dose of 2 ml/kg was determined as a conditionally therapeutic dose in the experimental study, and it was close to the dose calculated from the dose for humans, as well as the dose of 20 ml/kg was selected, which exceeds the conditionally therapeutic dose 10 times. According to the guidelines for the study of toxicity with repeated administration, the period of the PC administration was 28 days (Experimental study of the toxic effects of potential drugs / V.M. Kovalenko, O.V. Stefanov, Y.M. Maksimov, I.M. Trakhtenberg // Methodical recommendations - Kyiv, 2000. - 74-97 p.).
For a more complete assessment of the toxic effect of the PC solution, the experiments involved intragastric administration. The test sample was administered to the rats once a day. To assess the toxic effect of the active ingredients and excipients of the PC, the solvent (placebo) was studied on a separate group of the animals, such as group No. 2 (6 males and 6 females). The study design is provided in Table 9.
Table 9
Distribution of the animals into groups in the toxicological study of the PC
Figure imgf000036_0001
In this experiment, physiological parameters, morphological and biochemical parameters of the blood and urine of the animals were studied.
Methods of the physiological research included daily monitoring of the animal behavior, general condition, food, and water intake, as well as record of the body weight in the dynamics. The myocardium electrophysiological activity was assessed using electrocardiography, and the functional status of the central nervous system (CNS) was assessed using "Open Field" test.
The animal body weight was assessed in the dynamics, such as initial weight and the weight on 7, 14, 21 and 28 day. The effect of the test object on the CNS status of the rats was assessed based on the motor activity (number of square intersections), orientation and research activity (number of mink sightings, number of balances), and emotional activity (a number of urinations, defecations, washings) using the "Open Field" test at the end of the administration period (28 days). A whole impact on the CNS was assessed using "Sum of activities" integrated parameter.
The PC effect on the cardiovascular system (CVS) status of the animals was studied at the end of the administration period (28 days) using EK1T-03 M2 electrocardiograph.
ECG was recorded in the II standard lead. When deciphering the electrocardiograms, the following parameters were considered: RR - duration of the complete cardiac cycle; the duration of the PQ interval, the propagation of excitation in the atrium, which characterizes the time; the duration of the QRS ventricular complex and electrical systole of the ventricles, such as Q - T interval; voltage of P, T and R waves. Additionally, heart rate (60/RR, beats/min) and systolic index (SP, QT/RR%), which reflects the contractile function of the myocardium, were calculated.
The concentration of hemoglobin, the number of erythrocytes and leukocytes, percentage of different forms of leukocytes (band and segmented neutrophils, lymphocytes, eosinophils, monocytes) in the peripheral blood were assessed. The blood was sampled from the rat tail vein at the end of the administration period (day 28).
The blood hemoglobin level was determined using cyanmethemoglobin method (the set available from "Phyllisit-diagnostics" company, Ukraine). Erythrocyte level was determined using colorimetric method. Leukocyte level was determined in Goryaev's chamber. White cell count was calculated using CJ1-01 blood cell counter according to the common method (Laboratory research in the clinic / ed. V.V. Menshikova. - M Meditsyna, 1987. - 365 c.).
The effect of the study drug on the liver functional status was assessed using a number of the blood biochemical parameters. The activity of alanine and aspartate aminotransferase (ALT and AST) was measured using enzymatic-photometric method with 2,4-dinitrophenylhydrazine using the kits available from "PLIVA - Lachema Diagnostica sro" (Czech Republic). Cholesterol, HDL and LDL were measured using enzymatic method (Biosystems, Spain). The glucose level was measured using the kits available from "Phyllisit-diagnostician" (Ukraine) and glucose oxidase assay; total protein was measured using biuret method; creatinine concentration was measured using reaction with picric acid (the method is based on Jaffy reaction); urea level was measured using urease method (Biosystems, Spain); and chlorides were measured using photometric method using the kit available from "Phyllisit-diagnostics" (Ukraine).
The blood ability to coagulate was determined using Althausen method. It is known that blood clotting time depends on the prothrombin time changes, calcium ion level, fibrinogen level, fluctuations in fibrinolytic activity, structural and functional features of a number of other factors of pro- and anticoagulant action, as well as blood cells.
Therefore, prothrombin time (PT), thrombin time (TT), active partial thrombin time (APTT) was determined in the blood plasma using a coagulograph and the kits available from RENAM (Russian Federation). The fibrinogen level was determined using Rutberg method.
2.5% of the water load was used to assess the secretory function of the renal tubules. After intragastric administration of the water (2.5 ml/100 g of weight), the rats were placed in individual cells for 3 hours to collect urine. The amount of urine (ml) excreted was calculated per 100 g of the animal weight per 1 hour. Urea, creatinine, and chlorides levels were determined based on the volume of the urine collected (pmol/3 hours). Urine pH was determined using diagnostic strips ("PLIVA - Lachema Diagnostica sro", Czech Republic), and urine density was determined using weight method.
Animals were sacrificed using subpulmonary anesthesia with chloroform and dissected. The internal organs and systems (heart, lungs, liver, kidneys, spleen, adrenal glands, testicles, thymus, testes/ovaries, esophagus, stomach, colon and rectum) were evaluated on the microscopic level. Absolute masses of the internal organs (heart, lungs, liver, kidneys, spleen, adrenal glands, testicles, thymus, testes) were measured to calculate the weight factors (WF) according to formula (1 ):
Figure imgf000038_0001
Samples of the heart, thymus, lungs, liver, spleen, kidneys, adrenal glands, pancreas, testes, and ovaries tissues were subjected to the histological examination. The effect of the TS on the gastrointestinal mucosa (esophagus, stomach, colon and rectum) was also studied. Similar organs of the animals from the intact control group were considered as the norm.
The tissue samples were fixed in 10% neutral formalin solution and embedded into celloidin-paraffin. The histological preparations were dyed with hematoxylin and eosin and examined using Micros 400 microscope. Microphotographing of the microscopic images was performed using Nicon Col Pix 4500 digital camera. The photographs were processed on a Pentium 2.4 GHz computer using Nicon View 5.
Descriptive statistics were provided for the recorded variables: (i) for quantitative indicators, sample size (n), arithmetic mean, and standard error (m) were provided; (ii) for variables that are not a subject to the law of normal distribution, sample size (n), arithmetic mean, minimum (Min) and maximum value (Max) were provided; (iii) for qualitative indicators, frequency and percentage were provided. For quantitative indicators, the hypothesis of normal distribution of the data in groups was tested using the Leven test. If the data in the groups for certain parameters were distributed normally, the experimental groups were compared with the control for these parameters using the Newman-Keuls test (p <0.050) for independent samples. In the case of an abnormal distribution, the Kruskal-Wallis test (analog of analysis of variance for nonparametric data) and Mann-Whitney criterion with Bonferroni correction (p <0.00167) were used.
After administration of the study drug, the animals were constantly supervised by the experimenter. No signs of intoxication in the animals were observed. They were active, had no signs of aggression. Character and behavior were individual for each animal. The defecation and urination processes were adequate. The animals approached the food and took it willingly. Palpation of all the experimental animal did not reveal any neoplasms. Regional lymph nodes were not enlarged, the testicles were movable and located in the scrotum, the size of the scrotum is limited by the size of the testicles. No animal deaths were observed (Table 10).
Table 10
Survival of the laboratory rats after intragastric administration of the PC
Figure imgf000039_0001
An important indicator of the toxic effects of the drugs is the animal body weight. The dynamics of the white rat body weight influenced by the PC are presented in Table 11.
After 28 days of the monitoring, the male group administered with different doses of the PC solution demonstrated 19% increase in the rat body weight relative to the baseline, the intact group - 23%, and the PC group - 24%. There were no statistically significant differences concerning the IC group.
The female groups administered with 2 and 20 ml/kg of the PC solution also demonstrated 14% and 12% increase in the body weight, respectively, relative to the baseline, the intact group - 15%, and the PC group - 12%.
Statistical analysis also found no significant differences concerning the IC group.
Table 11
The effect of the PC intragastric administration on the rat body weight (g)
(M±m)
Figure imgf000040_0001
Figure imgf000041_0001
Note: 1. pANOVA is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA)
2. pdynamics is a statistical significance level of the differences within the experimental group in the dynamics (analysis of variance ANOVA)
3. p1 NK is a statistical significance level of the differences in comparison with the baselines (Newman-Keuls criterion)
4. n is a number of the animals in the group.
Thus, the animals of all the experimental groups demonstrated positive weight gain dynamics. Statistically significant deviations from the corresponding values for the intact and negative control were absent in both males and females.
Table 12 presents the data on the effect of the test drug and its solvent on the CNS functional status of the rats.
When comparing the behavioral responses of the studied male groups (Table 12) with the parameters for the respective IC groups after 28 days of the TS administration at different doses, the differences were not significant.
Table 12
Influence of the PC on the CNS functional status of the male rats M(Mmin ÷
Mmax), n = 6
Figure imgf000041_0002
Figure imgf000042_0001
Note: 1. pK_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
2. p2M u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p <0.00167);
3. n is a number of the animals in the group.
The results of the PC effect on the CNS functional status of the female rats, when PC is administered on the long-term basis, are shown in Table 13. According to the data obtained, the females influenced by 2 and 20 ml/kg of the TS for 28 days demonstrated a statistically significant decrease in the number of upright stands and inspected holes. The data obtained indicate a slight decrease in the research activity. The most pronounced change was recorded for the group of the animals administered with 20 ml/kg of body weight of the PC (Table.13). However, the sum of the activities was not affected by the recorded changes, which allows concluding that the PC has no toxic effect on the female rats. Table 13
Influence of the PC on the CNS functional status of the female rats M (Mmin
÷ Mmax), n = 6
Figure imgf000043_0001
Note: 1. pK_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
2. p2M u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p <0.00167);
3. n is a number of the animals in the group.
Thus, intragastric administration of the PC and its solvent for 28 days did not have a neurotoxic effect on the male and female rats. The results of the study of the PC solution effect on hematological parameters are presented in Tables 14 and 15.
The data in Table 14 show that, for all the experimental groups of the male rats, administration of two doses of the PC solution and its solvent caused a moderate long-term increase in erythrocyte level and hemoglobin level compared with the IC group, but the values did not exceed physiological norms. This may indicate an erythropoietic effect of the PC.
The white cell count for the experimental animals did not differ from the one for the IC group. No pathological changes were recorded.
Table 14
Influence of the PC on hematological parameters for the male rats, (M±m),
M(Mmin÷Mmax), n = 6
Figure imgf000044_0001
Note: 1 . pANOVA / pK_u is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion)
2. p1 M_u is a statistical significance level of the differences concerning the IC group (Mann-Whitney criterion p <0.00167);
3. n is a number of the animals in the group.
Table 15
Influence of the PC on hematological parameters for the female rats, (M±m), M(Mmin÷Mmax), n = 6
Figure imgf000045_0001
Note: 1 . pANOVA / pK_u is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion)
2. n is a number of animals in the group. The data in Table 15 show that erythrocyte, leukocyte, and hemoglobin level in the females of all the experimental groups did not changed statistically significantly when compared to the control parameters. The white cell count for the experimental animals did not differ from the one for the IC group. No pathological changes were recorded.
Assessment of the potential hepatotoxicity of 2 and 20 mg/kg of the studied objects during the long-term administration was carried out according to the generally accepted range of parameters that are widely used in the laboratory practice and give an idea of the status of protein, lipid, and carbohydrate metabolism, as well as hemostasis, liver synthetic function, and allow assessing the specific enzymological spectrum.
The results of the studies shown in Tables 16 and 17 indicate the absence of a negative effect of the PC and placebo on the liver functional status.
The total protein level in the serum did not change during the experiment for both males and females, which indicates the absence of a negative effect of the TS on protein synthesis processes in the liver. Adequate levels of aminotransferases indicate the maintaining of hepatocyte integrity at the physiological level.
Table 16
The effect of the PC on the biochemical parameters of the male rat serum,
(M±m), n = 6
Figure imgf000046_0001
Note: 1 . pANOVA / pK_u is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion)
2. n is a number of the animals in the group. Table 17
The effect of the PC on the biochemical parameters of the female rat serum, (M±m), n = 6
Figure imgf000047_0001
Note: 1 . pANOVA / pK_u is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group.
For an advanced description of the general trophic process status, hemostasis was studied using general (clotting time) and specific methods (APTT). Also, prothrombin time, thrombin time, and fibrinogen level were assessed, which give a differential picture of possible changes in the hemostasis system when the PC is administered on the long-term basis and allow assuming the tendency to hyper- or hypocoagulation in general.
The results are shown in Tables 18 and 19.
It was found that the long-term administration of different doses of the PC solution does not affect the clotting time and APTT in both males and females. Prothrombin and thrombin times were studied to detect impairment of coagulation factors and to assess liver function. They remained within the physiological norm for both males and females, indicating no effect on the thrombin activity. Plasma fibrinogen level fluctuated within physiological values for both males and females. Table 18
The effect of the PC drug on hemostasis in the male rats, (M±m), n = 6
Figure imgf000048_0001
Note: 1. pANOVA is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA);
2. n is a number of the animals in the group.
Table 19
The effect of the PC on hemostasis in the female rats, (M±m), n = 6
Figure imgf000048_0002
Figure imgf000049_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. p2NK is a statistical significance level of the differences when comparing with the IC group (Newman-Keuls criterion);
3. n is a number of the animals in the group.
According to the results (Tables 18 and 19), the animals treated intragastrically with 2 and 20 ml/kg of the PC solution and placebo for 28 days demonstrated no changes in hemostasis. All the studied parameters (clotting time, APTT, prothrombin and thrombin time, and fibrinogen level) for males and females were in the range analogues to the values for the intact control groups.
Thus, it can be concluded that the daily intragastric administration of the PC and placebo does not cause impairment of the coagulation system.
Tables 20 and 21 show the results of the analysis of carbohydrate and lipid metabolism, which also define the liver functional status. According to the glucose blood level, an approximate conclusion about the status of the processes involved in the carbohydrate metabolism was drawn, and the status of lipid metabolism was assessed according to the cholesterol, HDL and LDL levels.
Table 20
Influence of the PC on the blood serum biochemical parameters of the lipid and carbohydrate metabolism for the male rats, n = 6 (M±m)
Figure imgf000049_0002
Figure imgf000050_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group.
Analysis of the data (Tables 20 and 21 ) shows that, for both male and female rats, prolonged administration of both doses of the PC solution and placebo did not cause changes in the said lipid and carbohydrate metabolism parameters. The values were within the range of the parameters for the IC group.
Table 21
Influence of the PC on the blood serum biochemical parameters of lipid and carbohydrate metabolism for the female rats (M±m), n = 6
Figure imgf000050_0002
Note: 1. pANOVA is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA);
2. n is a number of the animals in the group.
Thus, the studied PC and placebo when administered intragastrically did not have a toxic effect on the rat metabolism.
The results of the study of the kidney functional activity affected by the administration of the studied objects are presented in Tables 22 and 23. According to the results obtained, intragastric administration of the PC solution and placebo to the rats (males and females) for 28 days did not have a significant effect on the renal excretory function. All the studied parameters did not differ significantly from the parameters for the control groups.
Table 22
The effect of PC on the kidney functional status of the male rats (M±m), n =
6
Figure imgf000051_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group. Table 23
The effect of the PC on the kidney functional status of the female rats
(M±m), n = 6
Figure imgf000052_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group.
Thus, the data in Tables 22 and 23 indicate that the PC drug and placebo when administered on the long-term basis (28 days) did not cause nephrotoxic effects. Data on the effect of the PC solution on the heart rate and ECG parameters are given in Tables 24 and 25. All the animals maintained the correct sinus rhythm. In the II standard lead, a positive P wave was always located before the characteristic ventricular QRS complex.
The administration of the PC and placebo to the male rats for 28 days did not lead to significant changes in the parameters that define the myocardium electrophysiological activity (Table 24).
Table 24
Influence of the PC on the ECG parameters for the male rats, (M±m), n = 6
Figure imgf000053_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group.
Similarly, intragastric administration of the PC solution and placebo to the female rats for 28 days (Table 25) did not lead to the changes in the parameters that define the myocardium electrophysiological activity. Table 25
Influence of the PC on the ECG parameters for the female rats, (M±m), n =
6
Figure imgf000054_0001
Note: 1 . pANOVA / pK U is a statistical significance level of the differences between the experimental groups (analysis of variance ANOVA or Kruskal-Wallis criterion);
2. n is a number of the animals in the group.
Thus, the data obtained allow concluding that the PC solution when administered intragastrically for 28 days does not cause significant changes in the ECG in neither males nor females.
Autopsy showed that the position of the organs in the abdominal and thoracic cavities is anatomically correct. The appearance of the body coat and mucous membrane of the natural openings of the rats in all the experimental groups (intact control, negative control (placebo) 20 ml/kg, PC 2 ml/kg and 20 ml/kg) were the same. Regional lymph nodes were not enlarged. When examining the body cavities, pathological content and adhesions were not revealed. Color, consistency of the organs and tissues, and appearance of serous membranes conformed to the norm without any signs of hypo- or hypertrophy of the organs, circulatory disorders, inflammation, or tumor growth.
To calculate the weight factors, the absolute masses of the internal organs were measured. The results are given in Table 26.
Table 26
The effect of the PC solution on the weight factors of the internal organs of the male and female rats M (Mmin÷Mmax), n = 6
Figure imgf000055_0001
Figure imgf000056_0001
Note: 1. pK_u is a statistical significance level of the differences between the experimental groups (Kruskal-Wallis criterion);
2. p2M_u is a statistical significance level of the differences when comparing with the IC group (Mann-Whitney criterion);
3. n is a number of the animals in the group.
It was found that the intragastric administration of the studied objects to the male rats for 28 days did not cause statistically significant changes in WF in comparison with the male rats in the 1C group.
However, the females in the 1C groups and the animals administered with 20 mg/kg of the PC demonstrated an increase in WF for the liver and kidneys.
Microscopic examination of the internal organs of all the rats treated with different doses of the PC showed regular structure and did not differ from those in the animals in the negative and intact control groups.
The myocardium of the left and right ventricles of the heart had no signs of edema or redness. Individual rats in both control and experimental groups had single small focal lymphomacrophagous infiltrates located between cardiomyocytes. Cardiomyocytes had regular sizes and color of cytoplasm and nuclei; the cross- outlined myofibrils were well traced. The lungs of the control rats had moderate hyperemia of blood vessels and capillaries, focal dystelectasis (partial collapse of the alveoli walls at the opening of the thoracic cavity) and foci of emphysema-like stretching of the alveoli (agonal origin). The activity of lymphoid tissue associated with the bronchi was moderate. The lumens of the lung alveoli were free. The administration of 2 ml/kg and 20 ml/kg of the PC had no significant effect on the organ structure, such as there were no signs of increased redness, dystrophic changes of pneumocytes of the 1 st and 2nd orders, necrobiotic changes in the cells, inflammatory stroma reaction, or increased activity of peribronchial lymphoid tissue.
The kidneys of the rats in the negative control group and groups receiving different doses of the PC, as well as the rats in the IC group, had no signs of circulatory disorders, dystrophic and necrobiotic changes of the nephrotheli of the tubular nephron, or intermediate inflammation. The nephron glomeruli had normal structure and cellularity, and the capillaries were moderately full-blooded. In some rats in all the groups, the lumen of the tubules of some parts of the cerebral layer contained small microliths of calcium salts and small focal round cell infiltrates localized near, in the stroma, which may be due to the nature of the animal nutrition.
The liver lobulation was pronounced, the cord structure was preserved, and the liver cords were located within a short distance from each other.
In all the groups, liver stroma had a form of thin layers of portal connective tissue with vessels penetrating them and bile ducts. Hepatocytes had regular form, with evenly colored cytoplasm. The boundaries between the cells were well defined. The hepatocyte nuclei were clearly visible and located mainly in the center of the cells. Sinusoidal capillaries were narrow, poorly filled with blood both in the central areas and on the periphery of the lobules. Some rats of both sexes in the different groups had small lymphoid-cell clusters periportally, and small foci of hepatocyte necrosis. According to the literature, a possible cause of such deviations may be local damage caused by toxic substances coming from the intestine. In addition, periportal hepatocytes of the female rats treated with 2 ml/kg (60%) and 20 ml/kg (40%) of the PC had moderate (2 ml/kg) and weak (ml/kg) small-drop cytoplasmic vacuolation without compromising cell integrity.
In pancreatic tissue of all the rats, acinuses had a regular structure. Zymogenic zone of pancreatocyte cytoplasm was well colored; cell nuclei were big, well-defined, and located at a basal membrane. The exfoliation of the epithelium of the excretory ducts was not observed. Islets of Langerhans had various sizes; capillaries thereof were expanded moderately; and secretory cells had no signs of dystrophy.
The thymus lobes of the rats in the control groups, and after the administration of different doses of the PC, were moderately large; the layers of the connective tissue between the lobes were relatively thin; the lobes were well divided into cortical and cerebral zones, whereas cellularity of the zones was regular. Quite often, the animals receiving placebo and the PC drug had a picture of "starry sky", enlargement of the cerebral layer and cystic enlargement with keratinization of thymic cells. These changes resemble the 1 -2 stages of accidental involution of the thymus, which develops due to several factors, including stress. In this case, repeated administration of a sufficiently large volume of the solvent or the PC was a stress factor for the animals, in response to which they formed a complex of adaptive reactions.
The size and number of white pulp follicles in the spleen of all the rats were regular, and their structure after the administration of the solvent or the PC did not change when compared to the intact. Red pulp was moderately full-blooded, contained macrophages, megakaryocytes and lymphocytes.
The size and ratio of cross-sections of the cortical areas of the adrenal glands in all the rats were regular. Histoarchitectonics of each zone of the cortex was maintained. Signs of the change in the adrenocorticocyte morphological features that indicates an impaired production of mineral and glucocorticoids were not observed. The levels of lipoidization of the glomerular and reticular layer cells were comparable for the control and experimental animals. Chromaffin cells of the brain had no specific features.
The testes of all the male rats had no signs of edema, redness or infiltration, or atrophy. All spermatogenic epithelium layers were represented in the twisted seminal tubules. Epithelium exfoliation was not detected. The number and location of Leydig cells were regular.
Similarly, hemodynamic abnormalities were not recorded in the ovaries of the rats. All the rats had follicles and corpora lutea at all stages of development, as well as atretic follicles.
Thus, the results of the histological studies indicate that 2 ml/kg and 20 ml/kg of the PC, and placebo when administered intragastrically on the long-term basis, did not cause any morphological manifestations of the toxic effects in the heart muscle, liver, kidneys, lungs, adrenal glands, pancreas, spleen, and reproductive system of the rats.
The thymus (being an organ that, among all the organs of the lymphoid system, has excessive lability of its morphological structure, and easily and rapidly occurring reactive morphological changes in response to various factors) was marked with the complex of the adaptive reactions that conformed to 1 -2 stages of accidental transformation. According to the literature, these stages are reversible and disappear after the cessation of the stress. Additionally, moderate/weak vacuolation of hepatocytes of some female rats treated with the solvent or the PC was not dose-dependent and, apparently, was associated with the nature of the food consumed by the animals, and their greater sensitivity when compared to the males.
The study results showed that 2 and 20 ml/kg of the studied PC when administered on the long-term basis did not trigger signs of intoxication in the animals, did not affect the general trophic processes, hematological parameters, and biochemical parameters of the blood and urine, and did not affect the central nervous system and cardiovascular system of the laboratory animals. The histological examinations confirmed the absence of the PC toxic effects on the internal organs and systems of the laboratory animals. Specific cases in the thymus and liver did not have a dose-dependent effect and indicate reversible processes.
Long-term administration of the PC did not trigger a local irritation effect on the gastrointestinal mucosa of the laboratory animals.
The results of the study of the PC toxicity allow concluding that the long-term administration of 2 and 20 ml/kg of PC did not cause signs of intoxication in the animals, did not affect general trophic processes, hematological parameters, biochemical parameters of the blood and urine, and physiological status of CNS and CVS. Individual fluctuations observed throughout the study were within the physiological norm and were adaptive.
The histological study of the internal organs in subacute toxicity (28 days) indicated the absence of signs of cardiotoxic, nephrotoxic and hepatotoxic effects on the rats. Morphofunctional status of the lung tissue and peripheral organ of the immune system (the spleen) were not impaired.
Intragastric administration of the PC and placebo to the laboratory rats did not cause irritation to the esophagus and stomach of the animals, as confirmed by the histological examination.
The following studies relate to determination of the effective dose of the PC, in the form of the solution for the oral administration, on a rat model of acute asphyxia.
Hypoxia is a universal pathological process that accompanies and defines the development of various pathologies, such as coronary heart disease, myocardial infarction, chronic heart failure, myocardiopathy, cerebrovascular disorders, chronic obstructive pulmonary disease, and asthenic condition, etc.
In general, hypoxia can be defined as a cellular imbalance between energy demand and energy production in the mitochondrial oxidative phosphorylation system. The reasons for the energy production impairment in the cell under hypoxic conditions are various: disorders of external respiration, blood circulation in the lungs, transport function of blood oxygen, impaired systemic, regional blood circulation, and microcirculation, decreased oxygen supply to the mitochondria in most pathological conditions. As a result, inhibition of mitochondrial oxidation develops. First, the activity of NAD-dependent oxidases (dehydrogenases) of the Krebs cycle is inhibited during the initial maintanance of the activity of FAD- dependent succinatoxidase, which is inhibited in more severe hypoxia. Mitochondrial oxidation impairment leads to inhibition of associated phosphorylation and causes a progressive deficiency of ATP, a universal source of energy in the cell.
Energy deficiency is the essence of any form of hypoxia and causes qualitatively similar metabolic and structural disorders in various organs and tissues. Reducing the concentration of ATP in the cell leads to a weakening of its inhibitory effect on one of the key enzymes of glycolysis, such as phosphofructokinase. Glycolysis, which is activated in hypoxia, partially compensates for ATP deficiency, but causes quick accumulation of lactate and the development of acidosis, followed by autoinhibition of glycolysis.
Hypoxia leads to a complex modification of the functions of the biological membranes, which affects both the lipid bilayer and membrane enzymes. The main functions of the membranes (barrier, receptor, and catalytic) are impaired or modified.
The aim of this preclinical study was to determine the effect of the PC on the bioelectrical activity of the rat heart (BEAH) on a model of acute asphyxia. This is important to assess the severity of organotropic antihypoxic effects on the heart when administered inside, and to determine effective doses.
The preclinical study of the PC in the form of the solution for the oral administration was conducted in compliance with the requirements of Good Laboratory Practice (GLP), and according to the guidelines (Stefanov, 2001) and Orders of the Ministry of Health of Ukraine N° 944 om 14.12.2009 and N° 95 on 16.02 .2009.
The study design was following.
Before the experiment, the animals were being acclimatized for 14 days in the test room. The groups were randomized according to the body weight as the main feature. The animals were maintained in the plastic cages in a room with controlled microclimate parameters, such as air temperature 20-24 °C, humidity 55±10%, "12 hours day/night" light regime. The room was air-conditioned and sterilized on the daily basis using a quartz lamp. The animals had free access to water. Settled tap water was used for drinking. Granulated balanced feed was used for the animal feeding (TY.Y 15.7-2123600159-001 : 2007). The animals were managed according to standard operating procedures.
The experiments were performed on adult male rats weighing 180-220 g.
The study was conducted on 5 groups of the animals:
Group 1 (positive control) included 6 animals.
Group 2 (first test sample) included 6 animals, wherein the animals were administered with the solution containing 100 mg/ml of levocarnitine and 264 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate was 100/264 mg/kg of body weight, respectively.
Group 3 (second test sample) included 6 animals, wherein the animals were administered with the solution containing 200 mg/ml of levocarnitine and 528 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 200/528 mg/kg of body weight, respectively. Group 4 (third test sample) included 6 animals, wherein the animals were administered with the solution containing 300 mg/ml of levocarnitine and 792 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 300/792 mg/kg of body weight, respectively.
Group 5 (fourth test sample) included 6 animals, wherein the animals were administered with the solution containing 400 mg/ml of levocarnitine and 1056 mg/ml of arginine aspartate, the dose of levocarnitine/arginine aspartate, was 400/1056 mg/kg of body weight, respectively.
The solution was administered intragastrically at doses of 1 , 2, 3 and 4 ml/kg for 3 days. The doses for the animals were recalculated from the daily dose for humans (30 ml/day according to the instructions for use in the clinical practice) using the coefficients of dose recalculation and I.P. Ulanova method taking into account the body surface (I.P. Ulanova, K.K. Sidorov, A.l. Khalepo On the question of taking into account the body surface of experimental animals in toxicological research. Edited by A. A. Letaveta and I.V. Sanotsky. - L. Ed. "Meditsyna", 1968, issue 10. - pp. 18-25.). Since the PC is intended for the oral administration in the clinical practice, the drug was administered to the laboratory animals intragastrically through a metal probe using a syringe.
Acute asphyxia was modeled using the method described in N.G. Stepanyuk. Comparative evaluation of antihypoxic properties of cordarone, benzofurocaine, vinborone and emoxipin in the experiment / N.G. Stepanyuk, V.V. Yushkova, V.B. Mudrytsky [etc.] // Bulletin of Vinnytsia National Medical University. - 2007. - N° 11 (2/1 ) - P. 576-579., and G.l. Stepanyuk. Search for effective antihypoxants among derivatives of succinic acid / G.l. Stepanyuk, O.P. Drachuk, S.A. Oliynyk, O.G. Yushkovskaya // Sports Medicine. - 2010. - N°4 (17). - P.56-59.). On the 4th day of the drug administration, the anesthetized animals were immobilized on a dissecting table. The skin, fascia, and neck muscles were dissected with scissors and tweezers. Electrocardiograph electrodes were positioned, the trachea was clamped with a surgical clamp, and the duration of BEAH was recorded to the last systole using ECG.
Quantitative values were processed using methods of variation statistics (mean value, its standard error, median, upper and lower quartiles) using parametric (one-way analysis of variance ANOVA, Newman-Keuls criterion) and nonparametric analysis methods (Kruskal-Wallis and Mann-Whitney criteria). The accepted significance level was p <0.05. A standard Statistica software package (version 6) was used to obtain statistical conclusions.
The results of assessment of the PC (solution for oral administration) effect on the rat BEAH in acute asphyxia are shown in Table 27. Table 27
The effect of the PC (solution for oral administration) on the duration of bioelectrical activity of the rat heart (BEAH) in acute asphyxia
Figure imgf000062_0001
Notes: p is the level of statistical significance when compared with the group of positive control, Mann-Whitney criterion; n is the number of the animals in the group.
According to the data obtained, the administration of 1 and 2 ml/kg of body weight of the study drug statistically significantly increased the life expectancy of the animals by 31% and 24%, respectively (Table 27). Increase of the test sample dose did not increase the effectiveness of the drug: the duration of BEAH of the rats that was administered with 3 and 4 mg/kg of body weight of the PC remained at the positive control level. Therefore, the data obtained indicate that the preventive administration of the PC being the solution for the oral administration had a positive effect on the rat BEAH.
Thus, in acute asphyxia caused by compression of the rat trachea, the PC was found to support the rat BEAH, which is reflected in increased life expectancy of the animals in asphyxia. Presumably, this cardioprotective effect is due to the antihypoxic effect of the PC on the myocardium and the conduction system of the heart. Even though, quantitatively, the effectiveness of 2 ml/kg of body weight of the drug was slightly lower than 1 ml/kg of body weight, there were no statistically significant differences between the groups. Based on the data obtained, it can be stated that the greatest, almost identical activity of the PC is found for the doses of
1 and 2 ml/kg of body weight, and they are worth using for the further studies.
The study results allow concluding that, in the model of acute asphyxia, the PC had a positive effect on the bioelectrical activity of the rat heart, prolonging it by 24-31%. The greatest effectiveness of the PC was recorded for the doses of 1 and
2 ml/kg of body weight. To study the effect of the PC on increase of the exercise tolerance in a human being exposed to physical load, the following study was conducted.
Study of the combined use of arginine aspartate and levocarnitine in the form of the solution for the oral administration vs. the use of arginine aspartate in the form of the solution for the oral administration to assess the stimulation of the physical fitness of the healthy athletes in high-achievement sports.
The study aimed at assessing the effect of the combined use of levocarnitine and arginine aspartate in the form of the solutions for the oral administration in comparison with the use of arginine aspartate in the form of the solution for the oral administration, on physical fitness and psychological status of the qualified athletes, such as representatives of power and cyclic sports, during the standard training process.
Objectives of the study were following: to assess the effect of the combined use of levocarnitine and arginine aspartate in the form of the solutions for the oral administration in comparison with the use of arginine aspartate in the form of the solution for the oral administration, on the physical fitness and psychological status of the qualified athletes; to evaluate the tolerability and possible side effects of the study solutions; to compare the results of the study solutions obtained in the two main and control groups of the healthy athletes, according to the parameters of the impact on the physical fitness and psychological status of the athletes.
The study design was following:
The study was performed according to the requirements of the State Expert Center of the Ministry of Health of Ukraine for clinical trials of drugs. The study was an open, randomized, comparative, parallel study in three groups.
69 athletes were included in the study. During the inclusion of the athletes, clinical and laboratory, psychophysiological and pedagogical examination of each subject was conducted. After the examination, the athletes, who gave written informed consent to participate in the study and who met the inclusion/exclusion criteria, were included in the study.
The study drugs were following: arginine aspartate in the form of the solution for the oral administration. Composition: 5 ml of the solution contained 1 g of arginine aspartate (arginine - 0.57 g, aspartic acid - 0.43 g), and excipients: sorbitol (E 420), sodium saccharin (E 954), methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), "Caramel" food flavor, and purified water. levocarnitine in the form of the solution for the oral administration. Composition: 5 ml of the solution contained 1 g of levocarnitine, and excipients: methylparaben (E 218), propylparaben (E 216), sucrose, sorbitol (E 420), "Banana" flavor, and purified water.
Randomization of the athletes of both groups (1 group included 36 track and field athletes and 2 group included 33 weightlifters) into subgroups was done according to the randomization scheme presented in the form of a table based on random numbers generated by the procedure of generating evenly distributed random numbers of the built-in Application Analysis Package Microsoft Excel.
The study included 69 healthy athletes, who were in a general preparatory phase of the preparatory period of the annual macrocycle with a standard training regime and meet the inclusion/exclusion criteria described in the protocol. All the athletes, who met the inclusion criteria, were simply randomized into main (46 athletes) and control (23 athletes) subgroups.
The athletes were randomized after they had provided the informed consent. To randomize the athlete into any subgroup for monitoring/ research, a responsible researcher reported the identifying information about the athlete (name and age / year of birth) to the study monitor by phone.
In response, the researcher received a notification of a subgroup for monitoring/research for this athlete. The researcher put the randomized number and identifying information for the athlete included in the study in the Journal of Randomization.
The athletes (almost healthy individuals, all men) included in the study were divided into the main (1 A, 1 B, 2A, 2B) and control (1 C and 2C) subgroups in a ratio of 1 :1 :1 to study the effectiveness of the pharmacological drugs used.
The athletes in the main subgroups 1A and 1 B received the study arginine aspartate drug, being the solution for the oral administration, for 21 days during the training process.
The athletes in the main subgroups 2A and 2B received simultaneously the study arginine aspartate drug, being the solution for the oral administration, and study levocarnitine drug for 21 days during the training process.
The athletes in the control subgroups 1 C and 2C did not receive any drugs during the training process.
The athletes in the main subgroups 1 A and 2A were prescribed with the study arginine aspartate drug, being the solution for the oral administration, in a daily dose of 40 ml, divided into 2 doses of 20 ml, immediately after meals, for 21 days during the training process.
The athletes in the main subgroups 1 B and 2B were prescribed with the study arginine aspartate drug, being the solution for the oral administration, and the study levocarnitine drug, being the solution for the oral administration, in a daily dose of 15 ml, divided into 2 doses of 7.5 ml, 2 times a day, immediately after meals, for 21 days of the study during the training process. Neurometabolic, nootropic, adaptogenic, anabolic and cardioprotective drugs were not allowed during the study. During the study, the athletes continued to train normally, keep a regular diet and a healthy lifestyle (complete abstinence from alcohol and smoking).
During the screening period, a clinical examination was performed including: measurement of heart rate, arterial blood pressure; palpation and percussion of the abdominal cavity through the anterior abdominal wall; auscultation of the heart and lungs; examination of the skin and visible mucous membranes; assessment of the musculoskeletal system and urinary system status.
Parameters of hematological homeostasis and biochemical homeostasis were determined based on the laboratory parameters.
To assess the baseline parameters of the athlete fitness, psychophysiological and pedagogical studies of the specific physical fitness parameters were studied.
Throughout the study, the athletes were subjected to an objective examination including measurement of the heart rate, arterial blood pressure, auscultation of the heart and lungs, examination of the skin and visible mucous membranes, assessment of the musculoskeletal system and urinary system, at 1 and 2 appointments.
At the end of the studied drugs administration (3, final, appointment), in addition to an objective examination, the athletes were re-examined for hematological homeostasis and biochemical homeostasis parameters. The psychophysiological and pedagogical studies of the specific physical fitness parameters were re-investigated.
All the data related to the prescription of the drugs, surveys and examinations of the athletes were put in the Individual registration form of the athlete.
The study included the following steps: screening and period of the study drugs administration (21 days). The monitoring data were recorded according to the following scheme (Table 28).
Table 28
Scheme of the recording of the study monitoring data
Figure imgf000066_0001
Inclusion criteria:
- qualified male athletes, age 18 to 26 years, representatives of different groups of sports, such as cyclic sports (36 track and field athletes, such as middle distance runners) and power sports (33 weightlifters) in the process of the general preparatory stage of the preparatory period of the annual macrocycle with the standard training process, who did not have the manifestations of any acute respiratory viral infections at the time of the study, as well as did not have a history of diseases of the cardiorespiratory, digestive, and excretory systems with clinical manifestations, except for functional changes due to professional activities;
- athlete’s informed written consent to participate in the study;
- the ability of the participant to cooperate in the research process adequately.
Exclusion criteria:
- SARS at the onset of the study;
- hypersensitivity to the components of the study drugs;
- allergic reactions or acute conditions associated with a history of medication;
- concomitant participation in any another clinical study (or similar in ergogenic action mechanism) of the substance or any study drug during the previous 30 days prior to inclusion in this study (any drug capable of influencing the assessment of therapeutic effect; taking any drug, the use of which is unacceptable for 2 months before participating in the study);
- participation in competitions at the time of the examination;
- trauma;
- chronic diseases, including type II diabetes mellitus;
- significant changes in complete blood count and biochemical blood analysis.
Conditions for withdrawal of each individual athlete from the study or discontinuation of the drug were the following:
- individual intolerance of the study drug;
- development of severe and/or unexpected side effects during the study;
- significant deterioration of the general condition during the study period;
- non-compliance with the study regimen of the drug (s) use;
- refusal of the athlete to participate in the study.
In case of early withdrawal of the athlete from the study, the researcher did not replace the athlete. The reasons for early withdrawal from the study are indicated in the individual registration form.
Study methods used
The following methods were used to examine athletes: record of the demographic data; collection of sports history (type of sports, discipline, stage (period) of training); physical examination; laboratory diagnostics (standard hematological and biochemical parameters); assessment of prooxidant-antioxidant balance in erythrocyte membranes; psychodiagnostics; determination of the specific physical fitness parameters according to the type of sports.
Demographic data and sports history:
- age, body weight, and height;
- specialization and sports qualification;
- stage (period) of the preparation;
- the presence (absence) of the training the day before.
Clinical examination:
- measurement of t-body;
- measurement of arterial blood pressure and heart rate;
- physical examination (percussion of the anterior chest wall and auscultation of the heart and aorta; palpation of the abdominal organs; examination of the skin and visible mucous membranes; visual assessment of the musculoskeletal system).
Record of subjective complaints:
- cough, runny nose;
- headache;
- general weakness.
Severity of the symptoms is assessed in points on a scale:
- 0 - absent;
- 1 - insignificant severity;
- 2 - moderate severity;
- 3 - significant severity.
Laboratory blood tests:
- hematological analysis (leukocyte, platelet and erythrocyte levels, hemoglobin level, hematocrit, erythrocyte parameters, including the average absolute content and average concentration of hemoglobin in erythrocytes, average erythrocyte volume, erythrocyte anisocytosis); - biochemical blood analysis (total protein, bilirubin, urea, creatinine, glucose, potassium, sodium, and ionized calcium level; activity of ALT, AST, GGT and alkaline phosphatase marker enzymes; APTT; assessment of prooxidant- antioxidant balance at the membrane level ("shadow of erythrocytes") with the assessment of malonic dialdehyde and reduced glutathione levels).
Psychodiagnostics included assessment of the psychological status of the athletes (type of temperament and severity of psychophysiological stress in points).
Common specific physical fitness parameters based on the specifics of muscular activity:
- for the track and field athletes (middle distance runners): aerobic fitness assessed using PWC170 test; time of passing the simulated competition distances of 800 and 1500 m;
- for the weightlifters: the height of the standing jump and the height of the barbell lifting in a jerk, as well as the time of doing the exercise.
The main criterion for the effect of the drugs used was an increase in the specific physical fitness parameters and improve in the psychophysiological status (severity of psychophysiological stress) of the athlete.
The evaluation of the study drug effectiveness was made by the researcher based on the above parameter on the following scale (Table 29).
Table 29
Description of the categories of variable overall effectiveness of the drugs
Figure imgf000069_0001
The drug effectiveness was evaluated based on the positive dynamics of the studied parameters in comparison with similar parameters for the control subgroups of the athletes (without the use of drugs).
Drug tolerability was assessed based on the subjective symptoms and feelings reported by the athlete, and objective data obtained by the researcher during the use of the study drugs in the athlete training process. The dynamics of the laboratory parameters, as well as the frequency and nature of adverse reactions were considered.
Assessment of the tolerability was based on:
1. Objective data obtained by the researcher during the study. For this purpose, an objective examination of the athletes was conducted during each visit. In this case, any clinically significant negative manifestation should be considered as a side effect, if it is not caused by the nature of the training process (or athlete acute disease during the study) or the predicted effect of the drug.
2. Data of the laboratory examination before and after the administration of the study drugs:
- hematological analysis (leukocyte, platelet, erythrocyte and hemoglobin level, hematocrit, erythrocyte parameters, including the average absolute level and average concentration of hemoglobin in erythrocytes, average erythrocyte volume, and erythrocyte anisocytosis);
- biochemical blood analysis (total protein, bilirubin, urea, creatinine, glucose, potassium, sodium, and ionized calcium level; activity of ALT, AST, GGT and alkaline phosphatase marker enzymes; APTT; assessment of prooxidant- antioxidant balance (PAB) at the membrane level ("shadow of erythrocytes") with the assessment of malonic dialdehyde and reduced glutathione levels, and calculation of prooxidant-antioxidant coefficient (Cpa).
In this case, any clinically significant negative deviation of the laboratory parameters was considered as a side effect, if it is not caused by the nature of the disease progression or the predicted effect of the use of concomitant therapy.
3. Subjective feelings of the athlete. During each visit, the researcher examined the athlete considering the presence and severity of both predicted and unpredicted adverse reactions.
Drug tolerability was assessed by the researcher on the following scale (Table 30). Table 30
Drug tolerability scale
Figure imgf000071_0001
Statistical analysis was performed by a statistician appointed by the sponsor and independently by the researcher. The analysis included:
- description of the athletes included in the study;
- the number of subjects who withdraw from the study;
- number of undesirable/side effects;
- analysis of the initial homogeneity of the main and control subgroups;
- analysis of the effectiveness in each group;
- comparison of the effectiveness between the main and control subgroups;
- assessment of the tolerability in each group;
- assessment of over- effectiveness. - statistical conclusions.
Data were processed using software packages Statistica 8.0 and MedStat using parametric and nonparametric methods of the statistical analysis.
The difference was considered statistically significant at p <0.05. A bilateral critical area was used to evaluate the results. Compliance with the normal distribution was assessed using Shapiro-Wilk tests. The samples were compared using Student test for parametric data and Wilcoxon test for nonparametric data. ANOVA analysis of variance and the Kruskal-Wallis test were used for multiple comparisons, and the Marasquilo-Lyakh-Gurianov test was used for qualitative parameters. Rank correlation and Spearman correlation index were used for correlation analysis.
At the beginning of the study, the values of the specific physical fitness parameters and the severity of manifestations of psychophysiological stress were studied. A similar research algorithm was implemented at the end of the study drug administration, and in the control subgroups algorithm was implemented at the end of the study period without any drug administration including ergogenic.
Methods of the specific fitness were applied based on specificity of the muscular activity. For the weightlifters, it was an estimation of the height of the standing jump and height of the barbell lift in a jerk, as well as time of doing the exercises. For the track and field athletes (middle distance runners) it included aerobic fitness assessed using PWC170 test, and the time for passing the simulated competition distances of 800 and 1500 m.
The athlete aerobic fitness was studied using PWC170 physical working capacity test, at 170 bpm using "Kettler E-3" bicycle ergometer ("KETTLER", Germany). Two loads of different moderate capacities (W1 and W2) were used to determine PWC170 value. At the last minute of these loads, heart rate was determined (f1 and f2, respectively). Physical working capacity in kgm/min was calculated as follows:
PWC1 70 = W1 + (W2 - W1) (170 - f1) / (f2 - f1 ), wherein PWC170 is exercise capacity, at which the heart rate is equal to 170 bpm; W1 and W2 is a capacity of the first and second loads, respectively, kgm/min; and f1 and f2 is heart rate at the end of the first and second loads.
After the first load of low capacity, the athlete, sitting on a bicycle ergometer, rested for 3 minutes, then he was offered to do the second, more intense, load session. The duration of the first and second loads was 5 minutes The whole study procedure took about 13 minutes. The time for the athletes to pass the simulated competition distances of 800 and 1500 m was recorded using an electronic chronometer.
The specific physical fitness parameters for the weightlifters were studied using a modified Abalakov device, which is a barbell connected to a movable vertical ruler, measuring the height (cm) and time (s) for the athlete to do control exercises (height of the jump with the barbell, and height of the barbell lift in a jerk). Measurements were performed before training without doing warm-up under the standard conditions.
Psychological research included the assessment of the type of temperament using the Eisenko personal questionnaire and assessment of the severity of psychophysiological stress using Ivanchenko standard test modified by the authors. In addition, the psychophysiological stress factor (Fps) was calculated. The calculation of this factor is used when the athlete is undergoing current research, and the degree of his fitness for the competition is determined. Fps is defined by the following formula as the ratio of the sum of the points for the psychological stress severity to the sum of the points for the physiological stress severity.
Fps
Figure imgf000073_0001
wherein Fps is a psychophysiological stress factor; åps is the sum of the points for the psychological stress severity; and åph is the sum of the points for the physiological stress severity.
Analysis of the dynamics of the specific physical fitness parameters for the athletes specializing in cyclic (track and field) and power (weightlifting) sports in the monitored subgroups are given in Tables 31 and 32 using the methods of the descriptive statistics.
Table 31
Analysis of the dynamics of the specific physical fitness in the subgroups of the track and field athletes using the methods of the descriptive statistics
Figure imgf000073_0002
Figure imgf000074_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance. Table 32
Analysis of the dynamics of the specific physical fitness in the subgroups of the weightlifters using the methods of the descriptive statistics
Figure imgf000075_0001
Figure imgf000076_0001
The results obtained allow concluding that 1 B and 1C groups differ the most significantly. 1 A group is the closest to 1 B group, but not so much regarding changes in the studied parameters. 1C group is characterized by the opposite dynamics of the fitness parameter changes (or lack thereof) relative to 1 A and 1 B groups.
Tables 31 and 32 obviously show that arginine aspartate leads to an increase in the specific fitness parameters in both sports after the course of the drug, which is almost not observed in the control groups under the training conditions. To even greater extent, this direction of the fitness changes is expressed for the combined use of arginine aspartate and levocarnitine. It should be noted that a fairly short duration of the selected monitoring time (21 days), due to the period of the drug administration recommended in the manufacturer's instructions, allows rejecting the hypothesis of the targeted influence of the training process on the severity of the physical fitness changes.
To assess the degree of the impact not only on the changes of the specific physical fitness parameters, but also the homeostasis parameters, that mediate the fitness, the analysis of the changes in prooxidant-antioxidant balance at the membrane level was conducted using "shadows" of erythrocytes. The results of the analysis in the groups and within the subgroups in the monitoring dynamics are given in Tables 33 and 34.
Table 33
Analysis of the dynamics of prooxidant-antioxidant balance (PAB) in the subgroups of the track and field athletes using the methods of the descriptive statistics
Figure imgf000077_0001
Figure imgf000078_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance.
Table 34
Analysis of the dynamics of prooxidant-antioxidant balance (PAB) in the subgroups of the weightlifters using the methods of the descriptive statistics
Figure imgf000079_0001
2C subgroup, control
Figure imgf000080_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance.
Thus, one of the most important variables that determine the degree of the impact on the athlete fitness, such as the severity of oxidative stress, which correlate with the degree of impairment/improvement of PAB, directly indicates the high effectiveness of the drugs for the groups of different sports (track and field and weightlifting), since the resulting parameter of the severity of PAB impairment in the body decreases, primarily at the membrane level (Fps). The decrease in this prooxidant-antioxidant factor value clearly indicates the normalization of the balance between oxidative factors associated with the accumulation of active oxygen radicals, and antioxidant, primarily non-enzymatic, factors for the athlete when using arginine aspartate, and especially combination of aspartate and levocarnitine, during the intense and regular exercises.
In addition to the specific physical fitness parameters, additional criteria of the effectiveness of the study drugs were data of the changes in the severity of oxidative stress. Since the differences were distributed mostly normally, the comparison of the studied parameters values in each group and within the subgroups at Appointment 3 and Appointment 1 was performed using the paired Student t-test (Table 35).
The statistical analysis of the obtained data shows significant positive changes in the PAB parameters for the main 1A, 1 B, 2A and 2B subgroups in contrast to the control values in the respective subgroups of the track and field athletes and weightlifters. Table 35
The results of the comparison of the PAB parameters for each subgroup of the track and field athletes and weightlifters using paired Student t-test
Figure imgf000081_0001
Figure imgf000082_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance.
Regarding the lack of a significant increase in the natural GSH antioxidant level in the main 1 A subgroup of the track and field athletes, it should be noted that the determining parameter of the effect on PAB is a decrease in MDA, as the study drugs function as antioxidants, i.e. substances that primarily decrease the severity of prooxidant reactions. These data confirm the effective impact of arginine aspartate (individually) and, especially in combination with levocarnitine, on the oxidative stress parameters, which limits the development of the ergogenic properties of the body, and, consequently, on special and general physical fitness.
Analysis of the degree of the study drugs impact on psychophysiological stress in general, and its components on the representatives of different sports (track and field athletes and weightlifters) are provided in Tables 36 and 37.
Table 36
Analysis of the dynamics of the psychophysiological stress values and its components in the subgroups of the track and field athletes using the methods of the descriptive statistics
Figure imgf000082_0002
Figure imgf000083_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance. Data in Table 36 shows that the track and field athletes in 1A and 1B main subgroups demonstrate significant changes in the severity of psychophysiological stress and its components, such as reduction. On the contrary, in 1C control subgroup, the athletes, who did not receive any pharmacological support during the training process, demonstrated an increase in the manifestation of psychophysiological stress and its components, which is quite clear in terms of increasing psychophysiological tension to the end of the studied mesocycle of the preparation.
Table 37 shows that, generally, a significant decrease in the severity of psychophysiological stress and both its components is observed in 2A and 2B subgroups, although not substantial.
Table 37
Analysis of the dynamics of the psychophysiological stress values and its components in the subgroups of the weightlifters using the methods of the descriptive statistics
Figure imgf000084_0001
Figure imgf000085_0001
* The conclusion on the significance of the differences is drawn at p <0.05 level of significance.
It should be noted that the decrease in the psychophysiological stress severity is much more noticeable for the main subgroups of the athletes (both track and field athletes and weightlifters), who received a combination of arginine aspartate and levocarnitine. While arginine aspartate is a powerful angioprotective and antioxidative agent, levocarnitine adds its neuroprotective and energetic effect, which causes the predominant effect of two drugs combination on the decrease in the psychophysiological stress severity.
For the athletes in 2C subgroup, who did not receive the drugs, the severity of the psychophysiological stress during the studied 21 -day mesocycle of the preparation increased, which may further cause the decrease in not only physical fitness, but also the competition results. For both track and field athletes and weightlifters, not only the reduction in the psychophysiological stress is important, but also the decrease in the stress severity from "high" to "medium", which increases the level of psychological stability of the athlete, who prepares for the competition. Thus, reduction in the psychophysiological stress severity affected by both individual arginine aspartate and combination with levocarnitine, has a pronounced neuroprotective effect on the skilled athletes during a real training process.
The obtained reduction in the psychophysiological stress severity creates the preconditions for the reasonable co-use of arginine aspartate and levocarnitine as allowed means for stimulation of the athlete fitness.
Concerning the temperament type, as well as motivational components, they had virtually no effect on the physical fitness and the psychophysiological stress severity, did not change significantly throughout the study, and, therefore, were not assesses as a determining ergogenic factor.
The study drug effectiveness was assessed based on the increase in the physical fitness parameters (for the future, accordingly, based on the results of competitions).
Since the change was not dichotomous, the superior efficacy of the co-use of arginine aspartate and levocarnitine was assessed in comparison with the results obtained for the athletes, who received only arginine aspartate during the 21 -day mesocycle of the monitoring, and for the athletes in the control group, who did not receive any pharmacological means throughout the training process.
The results of the analysis of the data obtained using the study drugs for the track and field athletes, allow concluding that the PWC170 overall aerobic fitness parameter was essentially the same throughout the study for individual arginine aspartate and combination with levocarnitine. However, in both cases, PWC170 parameters exceeded the increase in the parameters for the control group of the athletes. The time for passing the simulated competition distance of 800 m was significantly reduced for the athletes, who received arginine aspartate, in comparison to the control data.
An even more pronounced decrease in the time for passing this distance was observed when the combination of arginine aspartate and levocarnitine was used in a regimen fashion.
Similar dynamics in the reduction of the time for passing the competition distance is observed for 1500 m. The data obtained allow concluding that the regimen use of the combined arginine aspartate and levocarnitine is more effective than the individual use of arginine aspartate. In general, it should be noted that, according to the canons of sports science, reduction in the time for passing the distances of 800 and 1500 m by 1.5-2.0 seconds, when recording time with an electronic chronometer allows, the runner to significantly increase the whole result of the competition. Concerning the results of evaluating the study drug effectiveness for the weightlifters, it can be noted that the absolute significant change in the studied fitness parameters for the weightlifters when using a combination of arginine aspartate and levocarnitine compared with the athletes, who receive arginine aspartate, is recorded only for the increase in the height of the jump with a barbell in a jerk. Other changes in physical fitness values in 2A and 2B groups of the athletes were equivalent, which indicates the absence of excessive effectiveness of the two-drug concomitant regimen administration when compared to individual arginine aspartate. Regarding the physical fitness parameters for the weightlifters in the control group, a clear significant increase in the parameters of the height of the standing jump with a barbell and the height of the barbell lift in a jerk was shown for the athletes in the main subgroups while reducing exercise time, which indicates improvement of the specific physical fitness of the representatives of the power sports, who used pharmacological support.
The results obtained allow concluding that it is more reasonable to use the regimen administration of individual arginine aspartate for the track and field athletes and combined regimen administration of arginine aspartate and levocarnitine for the representatives of power sports.
Assessment of the safety and tolerability of the study drugs based on dynamic clinical monitoring of subjective and objective data on the condition of the athletes, regardless their relation to the study drug (s), was conducted throughout the study. Assessment of the drug tolerability was made based on the athlete subjective feelings and the results of the physical examination by a sports doctor with record of heart rate, blood pressure and laboratory data.
The athletes tolerated oral administration of the drugs well. This form is the most convenient for use in training camp and competition conditions. The use of the study drugs in the regimen mode throughout the standard training process did not have a negative impact on the quantitative level of the main constants of homeostasis, including data from complete blood count, and parameters of liver, carbohydrate, lipid, and nitrogen metabolism. Data from laboratory studies are presented in Tables 38 and 39.
Table 38
The results of the dynamic evaluation of the complete blood count parameters in the subgroups of the track and field athletes using the methods of the descriptive statistics
Figure imgf000088_0001
1C control group
Figure imgf000089_0001
* The conclusion on the significance of the changes can be made at p<0.05.
Table 39
The results of the dynamic evaluation of the complete blood count parameters in the subgroups of the weightlifters using the methods of the descriptive statistics
Figure imgf000089_0002
Figure imgf000090_0001
* The conclusion on the significance of the changes can be made at p<0.05.
Thus, the results of the statistical analysis show that, for the representatives of both sports (track and field athletes and weightlifters), the regimen administration of both study drugs, namely individual arginine aspartate and the combination of arginine aspartate and levocarnitine, had no significant negative impact on the main studied parameters of homeostasis. This indicates a high safety profile of the drugs when used in the dynamics of the real intense training loads.
The body response to any pharmacological action is usually reflected in the results of the studies of the blood biochemical parameters indicating the status of protein, lipid, and carbohydrate metabolism, functional status of the liver and pancreas, kidneys and biliary tract. The results of the dynamic studies of the serum biochemical parameters for the track and field athletes and weightlifters under the influence of the study drugs are presented in Tables 40 and 41.
Table 40
The results of the dynamic assessment of the main parameters of the biochemical blood test in the subgroups of the athletes using the methods of the descriptive statistics
Figure imgf000091_0001
Figure imgf000092_0001
1 B main subgroup
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
* The conclusion on the significance of the changes can be made at p<0.05.
Assessing the impact on all studied parameters of biochemical homeostasis of the track and field athletes, it should be noted that the negative changes due to the regimen administration of the study drugs were not observed. On the contrary, it should be noted that there was a decrease in the activity of AST and ALT marker liver enzymes in the main groups, which indicates an improvement in the liver functional status. The influence on the creatinine and urea level is essentially absent, which indicates a favorable safety profile of the said drugs and is important when selecting the drugs for the athlete pharmacological support. The important result of this clinical study is the confirmed reduction in APTT, which reflects the beneficial effect on the blood physical status and indirect effect on the rate of oxygen transfer.
Arginine aspartate, and especially its combined use with levocarnitine, is very important for the manifestation of the ergogenic action. Arginine aspartate has a pronounced antioxidant effect based on the reduction in the oxidative stress manifestations caused by activation of the antioxidant membrane-associated enzyme function, as shown in the recent studies.
In addition, it has recently been found that arginine-containing pharmacological agents reduce sarcolemma postload impairment by more than 2 times, accompanied by a 25% reduction in muscle desmin loss and in mRNA m- calpain regulation, which eventually leads to acceleration of the recovery process after the training.
No significant changes were found in the level of not only the main potassium and sodium electrolytes, but also magnesium, inorganic phosphorus, etc., except for cholesterol, lipid metabolism (triglycerides) and iron metabolism parameters (saturation of transferrin with iron, total serum iron binding capacity). Table 41
The results of the dynamic assessment of the main parameters of the biochemical blood test in the subgroups of the weightlifters using the methods of the descriptive statistics
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
* The conclusion on the significance of the changes can be made at p<0.05.
The data obtained on the safety of the study drugs for the weightlifters indicate that there were no negative changes in biochemical homeostasis in intense muscular activity.
Extremely small increases in the activity of marker enzymes of the pancreas and liver, such as a-amylase and ALT, were within the error of the assessment and were not significant.
Thus, the data obtained using the methods of the descriptive statistics indicate the absence of significant differences in the most important indicators of the cell blood count for the main and control subgroups for both the track and field athletes and weightlifters. The same absence of any negative dynamics is noted for hematocrit values, average volume of erythrocytes, their relative and absolute saturation with hemoglobin and degree of anisocytosis, and thrombocytes. This clearly indicates the absence of a negative reaction of the athlete blood system on the regimen administration of individual arginine aspartate, and the combination of arginine aspartate and levocarnitine.
An objective study of the athletes was conducted during the ergogenic examination of the athletes with the measurement of the physiological parameters. At medical control, the heart rate, arterial pressure, condition of the skin and visible mucous membranes, etc. were examined. During the study, the most important parameters of hemodynamics in the main and control subgroups of the representatives of both sports remained stable (Table 42 and 43).
Table 42
Dynamic assessment of the main parameters of the hemodynamics in the subgroups of the track and field athletes using the methods of the descriptive statistics
Figure imgf000103_0001
1C control subgroup
Figure imgf000104_0001
* The conclusion on the significance of the changes can be made at p<0.05.
Table 43
Dynamic assessment of the main parameters of the hemodynamics in the subgroups of weightlifters using the methods of descriptive statistics
Figure imgf000104_0002
2B main subgroup
Figure imgf000105_0001
* The conclusion on the significance of the changes can be made at p<0.05.
It should be noted that heart rate values are reference for the athletes for whom bradycardia refers to the adaptive responses to the training process and indicates the economization of the heart. Tables 42 and 43 show that heart rate, systolic and diastolic arterial blood pressure of the athletes did not differ significantly throughout the study in the main and control subgroups. Thus, no negative changes in the main parameters of the functional status of the cardiovascular system were recorded for the representatives of both sports, such as the track and field athletes and weightlifters, during the regimen administration of arginine aspartate, and combination of arginine aspartate and levocarnitine, which confirms the high safety profile of the study drugs.
The results of the study allow drawing the following conclusions:
1 . The study drugs have a convenient form for use by the athletes in training camp conditions and at the training stages before competitions, as they do not require dilution or other dosage forms than specified by the manufacturer.
2. The study drug use does not lead to negative changes in the general status of the athletes according to the results of the physical examination and does not cause impairment of the functional status of the cardiovascular system and main laboratory parameters.
3. The study drug use results in the stimulation of the physical fitness of the representatives of various sports, regardless of the specifics of the predominant mechanism of the energy supply (aerobic or anaerobic glycolytic), which is expressed in the increase of the parameters of the general and specific physical fitness.
4. The study drug use is accompanied with a significant tendency to the increase in the psychophysiological stress severity, which is one of the most important factors in the deterioration of the results of the training and competion activities.
5. The study drug use leads to a significant decrease in the oxidative stress manifestations, primarily at the cell membrane level, which aids in avoiding the development of further pathobiochemical changes inherent in the final stage of the qualified athlete training.
6. The studied drugs are well tolerated by the athletes, cause essentially no side effects and can be recommended as allowed ergogenic agents for the training of the athletes, such as the representatives of the cyclic and power sports in amateur and high-achievement sports.
To study the use of the PC for the treatment of chronic coronary heart disease, or stable angina pectoris or peripheral arterial disease, the following study was conducted.
The aim of the study was to investigate the effect of the PC on the exercise tolerance and quality of life in patients with stable angina of > functional class II.
The study objects:
1 . To study the effect of the regimen administration of the PC on the patients with chronic coronary heart disease, such as:
- tolerance to physical activity.
- frequency of angina attacks;
- the number of nitroglycerin pills needed to eliminate angina attacks; - quality of life.
General design of the study was the following.
This study was an open-label, randomized, comparative phase III study in parallel groups.
After signing the informed consent, screening procedures were performed (2 days before randomization).
After the inclusion criteria had been confirmed, the patients were randomized into treatment groups using block randomization with a 1 :1 ratio for the use of the PC in combination with basic therapy or only basic therapy for 21 days. The final appointment was held on the 22nd day.
The scheme of the study design is given in Table 44.
Table 44
The scheme of the study design
Figure imgf000107_0001
To assess the effectiveness and safety of the PC, a study design was selected to meet the designated objects, namely randomized, comparative, parallel, and multicenter.
To ensure minimization of systematic inclusion error, a randomized parallel study design was chosen.
The primary criterion of the therapy effectiveness was the change in the duration of the physical exercise performed during the exercise test (TDT) according to the R. Bruce protocol at the end of the 21 -day treatment regimen in comparison with the baseline, which is an indication of the drug pharmacodynamic effect.
The standard basic therapy, which, according to the unified clinical protocol of the Ministry of Health (2015), is the optimal drug therapy, was chosen as a control.
In addition to the basic therapy, the patients in the main group received 20 ml of the study pharmaceutical composition according to the invention 2 times a day for 10 days.
The number of the patients in the study was following: the number of patients randomized was 110. ITT population was 110. PP population was 91 , and safety population was 110.
Diagnosis and basic inclusion criteria were following.
The study included men and women aged 45 to 75 years with diagnosed coronary heart disease (CHD), who met all inclusion/exclusion criteria and signed the informed consent. Most of the patients received the outpatient treatment and monitoring, a smaller number of the patients were in the hospital.
The study included the patients who met all the following criteria:
1. Men and women aged 45-75 years with diagnosed CHD, wherein stable angina pectoris of > II FS was verified by cardiac ventriculography, stress tests or the presence of postinfarction cardiosclerosis.
2. Positive results of the treadmill test: ST segment elevation (> 1.0 mm); horizontal or oblique ST segment depression > 1 mm after 0.06 s after j point in at least two adjacent leads in three consecutive complexes.
3. Readiness and ability of the patient to fulfill the requirements of the study protocol.
4. The patients whose treatment regimen or condition is expected to remain stable throughout the study period.
5. Negative pregnancy test result and consent to use adequate contraception throughout the study (used for women of childbearing potential).
6. The patient-signed informed consent to participate in the study.
The study therapy was the therapy with the PC being the solution for the oral administration. 1 ml of the solution contains: 100 mg of levocarnitine and 264 mg of arginine aspartate; and excipients: malic acid, sodium saccharin, methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), and water for injections. Method of the administration included oral administration, before meals, 2 nebulas 2 times a day, in the morning and in the evening, in combination with the standard therapy.
The therapy duration was 21 days.
For the control group, the standard therapy was prescribed. Criteria for evaluating the effectiveness and safety of the claimed pharmaceutical composition when used in the standard therapy were the following:
The primary effectiveness criterion is the change in the duration of the exercise performed during the exercise test (TDT) according to the protocol of R. Bruce at the end of a 21 -day treatment regimen compared with the baseline.
Secondary effectiveness criteria:
1 . Increase in the duration of exercise performed during the exercise test (TDT) according to the protocol of R. Bruce at the end of the 21 -day treatment regimen for 1 minute;
2. Increase in the duration of exercise performed during the exercise test (TDT) according to the protocol of R. Bruce at the end of the 21 -day treatment regimen for 2 minutes;
3. Dynamics of peak oxygen consumption according to the TDT results at the end of the 21 -day treatment regimen compared with the baseline;
4. Change in the power of the threshold load during the TDT exercise test at the end of the 21 -day treatment regimen compared with the baseline;
5. The number of angina attacks per week and their dynamics;
6. The number of doses (pills) of nitroglycerin per week and their dynamics; and reduction in the number of nitroglycerin pills taken per week to eliminate angina attacks at the end of the 21 -day treatment regimen;
7. Reduction in the number of angina attacks before the end of the treatment by 50% compared to the baseline;
8. Reduction in the number of nitroglycerin pills (doses) consumed to eliminate angina attacks before the end of the treatment by 50% compared to the baseline;
9. Change in the level of flow-dependent vasodilation at the end of the 21 - day treatment regimen compared with the baseline;
10. Change in QTc dispersion, the number of arrhythmic events according to the results of daily ECG monitoring at the end of the 21 -day treatment regimen;
11 . Assessment of the quality of life according to the HeartQol questionnaire and its dynamics at the end of the 21 -day treatment regimen compared to the baseline.
The safety criteria for the claimed pharmaceutical composition were as follows:
1 . Overall frequency of the side effects;
2. Frequency of the serious side effects;
3. Frequency of the side effects associated with the use of the study drug; 4. Frequency of the side effects that led to the patient withdrawal from the study;
5. Frequency of the side effects not previously described in the instructions for use of the study drug.
Statistical methods used during the study were following:
Data analysis was performed in the following populations:
- Intent-to-treat population (ITT) included all randomized patients who received at least one dose of the study drug / standard therapy.
- Per protocol (PP) population included all randomized patients who underwent complete regimen of the treatment with the study drugs and did not have significant deviations from the protocol.
- Safety population included all randomized patients who have received at least one dose of the study drug / standard therapy.
The main population for the assessment of the primary effectiveness criterion was the PP population. Additionally, the primary effectiveness criterion was assessed for the ITT population. The main population for the assessment of secondary effectiveness criteria was the ITT population. Additionally, secondary effectiveness criteria were assessed for the PP population. The main population for the safety assessment was the safety population. Demographic data, effectiveness and safety parameters are given for the treatment groups using the descriptive statistics:
Continuous (quantitative) data are provided using the following parameters:
- Number of observations (n);
- Average (M);
- Standard deviation (SD);
- 95% confidence interval for the average (95% Cl);
- Minimum value (min);
- Maximum value (max);
- Median (Me);
- Interquartile range (IQR).
Absolute (n) and relative (%) frequencies for each category, as well as 95% Cl, were calculated for the variables presented in the form of qualitative and ordinal parameters. At the beginning of the statistical analysis, the homogeneity of the groups was assessed according to the main initial parameters (demographic and anthropometric characteristics of the patients) to evaluate the success of the randomization (assessment of the parameter balance in the groups). For quantitative indicators, the groups were compared using Student t-test for independent samples or using Mann-Whitney test depending on the results of the assessment of the data distribution normality in the groups using Shapiro-Wilk test. For the categorical parameters, group comparisons were performed using the Pearson xi-square test (c2). If the expected frequencies in any of the elements of the correlation table were less than 5, Fisher exact criterion was used for the comparison. When applying Pearson xi-square criterion for dichotomous (binary) variables, Yates correction (continuity correction) was used.
According to the Unified Clinical Protocol, MFI (2015) is the optimal drug therapy, and includes at least one drug that affects the symptoms of angina, and drugs that affect the prevention of the disease complications.
- Drugs for short-term symptom control included glyceryl trinitrate (short acting nitroglycerin) in the form of pills or spray to stop or prevent angina.
- Therapy for long-term symptom control and prevention of the attacks for patients with stable angina pectoris included 1st line drugs: beta blockers, calcium channel blockers, adequate doses of which reduce heart rate considering the side effects and contraindications. In case of insufficient effectiveness of the therapy, it was recommended to replace the beta blocker with a calcium channel blocker or to prescribe a combination of the beta blocker and dihydropyridine calcium channel blockers.
In case of insufficient effectiveness of the 1 st line therapy for control of stenocardia symptoms, it was recommended to add one or a combination of the 2nd line drugs, such as adequate doses of nitrates of prolonged action, and ivabradine, to the therapy.
The drugs for the prevention of complications (according to the clinical protocol of the Ministry of Health (2015)):
- 75-150 mg/day of acetylsalicylic acid medicines. In case of intolerance to acetylsalicylic acid, 75 mg of clopidogrel medicines per day are prescribed in the absence of contraindications.
- Statins are prescribed to all patients diagnosed with CFID, regardless of lipid profile.
- Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers are prescribed to the patients with stable coronary heart disease and diabetes mellitus, hypertension, chronic heart failure or asymptomatic left ventricular dysfunction.
According to the preclinical study on the assessment of the PC (solution for oral administration) effective dose, the drug was the most effective at doses of 1 and 2 ml/kg of body weight when administered intragastrically on the rat model of acute asphyxia. Based on the results, as well as literature data, that indicate that, in terms of pharmacokinetics, pharmacodynamics, and safety, the optimal dose for oral arginine is 6 g per day, 40 ml of the PC was decided to be used in a clinical study as the optimal daily dose. Assessment of the primary effectiveness criterion for the claimed pharmaceutical composition was made as follows.
In accordance with the purpose and objects of the study, the null hypothesis (HO) was formulated as follows:
- The null hypothesis (HO) states that the effectiveness of the therapy including the claimed pharmaceutical composition will be inferior to the standard therapy effectiveness or will be equal to it;
- An alternative hypothesis (Ha) states that the effectiveness of the therapy including the claimed pharmaceutical composition will exceed the standard therapy effectiveness.
H : e < d Ha: e> d. wherein d > 0 is the value of the clinically significant differences, that allows assuming that the therapy including the study drug is more effective than the basic therapy; e is a difference of means [Tapp5 - TappO] = m (combination therapy) - m (standard therapy), wherein m is the arithmetic mean of the main variable for the corresponding group.
Student t-test or Mann-Whitney U test was used to assess the differences in the primary effectiveness criterion between the groups, depending on the results of the data distribution normality assessment in the groups using the Shapiro-Wilk test. The difference in the therapy effectiveness was assessed using bilateral confidence intervals (Cl). The calculation of 95% Cl was performed based on t-distribution. According to the protocol, the conclusion about the superior effectiveness of the combination therapy (including the claimed pharmaceutical composition) in the main group compared to the standard therapy in the control group was drawn based on positive statistically significant differences for the primary criterion between the groups in favor of the main group. The positive results were the greater increase in the duration of the load on the treadmill test in the main group than an increase in the duration of the load on the treadmill test in the control group.
The dynamics in the groups was assessed using the paired Student t-test or Wilcoxon sign rank criterion in each group, depending on the results of the test on the difference distribution normality [Tapp5 - TappO] using the Shapiro-Wilk test. The relative increase / decrease of the primary effectiveness criterion was calculated, and evaluated the paired Student t-test or Wilcoxon sign rank criterion in each group, depending on the results of the test on the difference distribution normality [Tapp5 - TappO] using the Shapiro-Wilk test.
Assessment of the secondary effectiveness criteria for the claimed pharmaceutical composition was performed as follows.
Ill For the “increase in the duration of exercise performed during the exercise test (treadmill test, TDT) according to the protocol of R. Bruce at the end of the 21 - day treatment regimen for 1 minute” criterion:
To compare the groups, the dichotomous variable, such as an increase in duration of the load before the end of the treatment regimen for 1 minute (increase presence / increase absence), was created. For this variable, parameters of the descriptive statistics for each group (frequency and share in %) were provided. The comparisons between the study groups were performed using the Pearson xi- square test (>$2) (with Yates correction for dichotomous (binary) variables) or a two- way version of Fisher exact test (at expected frequencies less than 5).
For the “increase in the duration of exercise performed during the exercise test (TDT) according to the protocol of R. Bruce at the end of the 21 -day treatment regimen for 2 minutes” criterion:
To compare the groups, the dichotomous variable, such as an increase in duration of the load before the end of the treatment regimen for 2 minutes (increase presence / increase absence), was created. For this variable, parameters of the descriptive statistics for each group (frequency and share in %) were provided. The comparisons between the study groups were performed using the Pearson xi- square test (>$2) (with Yates correction for dichotomous (binary) variables) or a two- way version of Fisher exact test (at expected frequencies less than 5).
For the “dynamics of peak oxygen consumption according to the results of TDT at the end of the 21 -day treatment regimen compared with the baseline” criterion:
The dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
For the “change in the capacity of the threshold load during the TDT loading at the end of the 21 -day treatment regimen compared with the baseline” criterion:
The dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test. For the “number of angina attacks per week and their dynamics” criterion:
Dynamics in the groups is presented graphically and using the descriptive statistics. To assess the differences in [Tapp5 - TappO] between the groups, Student t-test or Mann-Whitney U-test was used depending on the Shapiro-Wilk test results.
For the “number of nitroglycerin doses (pills) per week and their dynamics” criterion:
Group dynamics is presented graphically and using the descriptive statistics. To assess the differences in [Tapp5 - TappO] between the groups, Student t-test or Mann-Whitney U-test was used, depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
For the “reduction in the number of angina attacks before the end of treatment by 50% compared to the baseline” criterion:
To compare the groups, a categorical variable, which can be used to assess the presence / absence of a 50% decrease in the parameter value at the end of the treatment (Tapp5) compared to TappO (categories: 50% decrease or more / no 50% decrease), was created. For the variable, parameters of the descriptive statistics were provided for each group (frequency and part in %). To compare the study groups, Pearson xi-square test (>$2) (with Yates correction for dichotomous) or a two-way version of Fisher exact test (at expected frequencies less than 5) were used.
For the “50% reduction in the number of nitroglycerin pills (doses) consumed to eliminate angina attacks by the end of the treatment compared to the baseline” criterion:
To compare the groups, a categorical variable, which can be used to assess the presence / absence of a 50% decrease in the parameter value at the end of the treatment (Tapp5) compared to TappO (categories: 50% decrease or more / no 50% decrease), was created. For the variable, parameters of the descriptive statistics were provided for each group (frequency and part in %). To compare the study groups, Pearson xi-square test (>$2) (with Yates correction for dichotomous) or a two-way version of Fisher exact test (at expected frequencies less than 5) were used.
For the “change in the level of flow-dependent vasodilation at the end of the 21 -day treatment regimen compared with the baseline” criterion:
The dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test. For the “change in QTc variability and the number of arrhythmic events according to the results of the daily ECG monitoring at the end of the 21 -day treatment regimen” criterion:
The dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
For the “assessment of the quality of life according to the HeartQol questionnaire and its dynamics at the end of the 21 -day treatment regimen compared with the baseline” criterion:
The dynamics in the groups are presented graphically, and using the descriptive statistics, and evaluated using the paired Student t-test or Wilcoxon sign rank criterion for each group depending on the results of the Shapiro-Wilk test on the difference distribution normality [Tapp5 - TappO]. The relative increase / decrease in the parameters were calculated. To assess the differences between the groups, Student t-test or Mann-Whitney U-test was used depending on the results of the data distribution normality assessment for the groups using the Shapiro-Wilk test.
For all the criteria, a two-way version of the corresponding criterion with a significance level (a - probability of type I error) of 0.05 was used. A significance level of 0.01 was used for the Shapiro - Wilk test.
The safety analysis of the claimed pharmaceutical composition was performed as follows.
The results of the laboratory tests (complete blood count, urinalysis, biochemical blood test parameters), measuring the heart rate (FIR) and arterial blood pressure (BP) are presented for the treatment groups using the descriptive statistics.
The change in the above parameters for each group at the appointments and for the difference [Tapp5 - TappO] were assessed. The significance of the changes in the analyzed parameter dynamics for each group was assessed using Student t- test for paired data or Wilcoxon sign rank criterion depending on the results of the difference distribution normality assessment [Tapp5 - TappO] using Shapiro-Wilk test. Each parameter was converted into a categorical variable (norm / clinically insignificant deviation / clinically significant deviation) for which the frequency and part in % were calculated in each group and for each appointment according to the patient survey schedule. Previous comorbidities and SE were coded using the MedDRA classifier. Previous and concomitant therapy were encoded using the ATC classifier. The descriptive statistics parameters (frequency and part in % for each group) were calculated for each safety endpoint for each group. Comparison of the groups by the number of the patients with SE was made using Pearson xi-square test (>$2) (with Yates correction for the dichotomous (binary) variables) or the two-way version of Fisher exact test (at expected frequencies less than 5).
A total of 110 patients were included in the study. 110 patients were randomized and 109 completed the study. The ITT population consisted of 110 patients, the PP population consisted of 91 patients, and the safety population consisted of 110 patients.
The treadmill test (TDT) was performed during appointment 0 and appointment 5 on the VALIANT treadmill (Lode BV, the Netherlands) or its analogue. The study was performed according to R. Bruce modified protocol. The following parameters were calculated: exercise tolerance and functional class of angina. High tolerance is for I functional class with the energy intensity of the physical activity performed more than 7.0 MET. Average tolerance to PA is for II - III functional class (energy intensity of II class - 4.0-6.9 MET, III - 2.0-3.9 MET). Low tolerance is for IV functional class (metabolic cost of the load less than 2.0 MET). In addition, the following parameters were calculated: total load time and peak oxygen consumption. TDT was interpreted by a cardiologist.
Recording of the number of angina attacks and the amount of nitroglycerin taken
The recording was carried out at appointment 0 and appointment 5 using diaries recording angina pectoris attack. The results were interpreted by the cardiologist.
Flow-dependent vasodilation.
The flow-dependent vasodilation test was performed at appointment 0 and appointment 5 using Philips HD11XE ultrasound machine or its analogue with a 3- 12 MHz linear sensor. The right brachial artery was scanned 2-10 cm above the elbow, and the tonometer cuff was positioned on the forearm below the location of the artery. For the baseline, the diameter of the brachial artery and circulation velocity were measured. The brachial artery diameter was measured as the distance between the anterior and posterior walls of the artery at the border of the intimate vessel in the normal-diastole phase of the circulation, which was determined at the time of the R wave on the ECG synchronized with the ultrasound image. PZVD was measured at the 60th second after 5 minutes of brachial artery compression with a pressure 50 mm Hg higher than the level of systolic arterial blood pressure of the patient by calculating the percentage change in the diameter of the artery compared to the baseline Daily ECG monitoring
The daily ECG was monitored on the Diacard DC 03250 v2.1 system or its analogue at appointment 0 and appointment 5. During this test, the following parameters were recorded: average daily heart rate, maximum and minimum heart rate, number and characteristics of ventricular and supraventricular arrhythmias, QTc interval variability, heart rate variables: SDNN, SDANN, RMSSD. The results were interpreted by the cardiologist.
Quality of Life (HeartQol) Questionnaire.
The patient received HeartQoL quality of life questionnaire at appointment 0 and appointment 5. The results were interpreted by the cardiologist.
The following safety parameters were evaluated:
- total frequency of the side effects;
- frequency of the serious side effects;
- frequency of the side effects associated with the use of the study drug;
- frequency of the side effects that led to the patient withdrawal from the study;
- frequency of the side effects not previously described in the instructions for use of the study drug.
The safety and tolerability of the 21 -day PC (oral solution) administration was assessed based on subjective symptoms and feelings reported by the patient to the researcher, and objective data obtained during the treatment.
The safety analysis of the drug involved the data of physical, laboratory and instrumental examinations. Safety assessment was performed throughout the study.
Assessment of vital signs.
The main vital signs (heart rate, RR, arterial blood pressure, body temperature) were measured at rest before the physical examination (after 15 minutes of the rest, not earlier than an hour after smoking cigarettes and 2 hours after eating). Heart rate (HR) was measured by heart auscultation simultaneously with the measurement of pulse rate in the radial artery (or carotid artery if pulsation in the radial artery was weak) for one minute in a sitting position. In case of pulse deficit both parameters (heart rate and frequency) were recorded. Respiratory rate (RR) was measured at rest in a supine position for one minute, recording the respiratory movements of the chest or abdominal wall, without attracting the patient attention.
Arterial blood pressure was measured on the brachial artery in the supine position using Korotkov method with a certified sphygmomanometer or tonometer and a cuff length and width adjusted to the length and circumference of the patient shoulder according to the recommendations for measuring arterial blood pressure RMOAG/BHOK, 2010. The cuff size has to conform to the arm size. The rubber part of the inflatable cuff must cover at least 80% of the shoulder circumference. For the adults, a cuff 12-13 cm wide and 30-35 cm long (average size) was used; but it was necessary to have a larger and a smaller cuff for full and thin arms, respectively. The mercury column or the tonometer needle should have been at the zero mark before the start of the measurement. To assess the arterial blood pressure level on each hand it was necessary to perform at least two measurements with an interval of at least 1 minute; with a blood pressure difference > 5 mm Hg., one additional measurement was made; the final (recorded) value was taken as the minimum of three measurements.
Measurement technique:
- pump the air quickly into the cuff to a pressure level of 20 mm Hg higher than the SBP (after pulse ceasing);
- measure the blood pressure with an accuracy of 2 mm Hg;
- reduce the pressure in the cuff at a rate of approximately 2 mm Hg in 1 second;
- the pressure level which the 1 st tone appears, corresponds to SBP (1 phase of Korotkov's tones);
- the pressure level at which tones disappear (phase 5 of Korotkov tones) corresponds to the DBP; in children, adolescents and young people immediately after exercise; in pregnant women and adults with some pathological conditions, when it is impossible to determine the 5th phase, 4th phase of Korotkov tones, which is characterized by a significant weakening of tones, should be identified;
- if the tones are very weak, the patient should raise the hand and perform a few compressive movements with the wrist, then repeat the measurement, while not squeezing the artery with a membrane of the phonendoscope;
- at the initial examination of the patient the pressure on both hands should be measured; further measurements are performed on the hand on which the blood pressure is higher.
Laboratory tests
The blood for the analysis was sampled in the morning on an empty stomach (10-12 hours after the last meal) from the ulnar vein with a disposable sterile syringe under aseptic / antiseptic conditions. The tests required a collection of approximately 12 ml of the blood (1 tablespoon) at appointment 0 and appointment 5. For the urinalysis, the morning average portion was collected after adequate perineal hygiene. The urine was to be delivered to the laboratory within two hours after the collection.
Complete blood count (hemoglobin, erythrocytes, leukocytes levels, white blood cell count, ESR), biochemical blood test (glucose, lactate and pyruvateserum levels, urea and creatinine levels, bilirubin, ALT, AHF, AST, and total albumin level), blood glucose from the ulnar vein and urinalysis (color, transparency, relative density, pH, glucose, protein, ketone bodies) were performed in the laboratory of the research center.
The study schedule (Table 45) shows the stages of the study and the procedures that were performed for each study participant.
If the patient visited the research center outside of the scheduled appointments (unscheduled appointment), it did not affect the order of the scheduled appointments. All examinations conducted during the unscheduled appointment were documented in the primary documentation and the IRC.
Table 45
Schedule of appointments and procedures
Figure imgf000119_0001
Figure imgf000120_0001
The research results
Change in the duration of the exercise performed during the exercise test (TDT) according to R. Bruce protocol at the end of the 21 -day treatment regimen compared with the baseline:
PP population
The average (±SD) duration in the exercise performed during the treadmill test initially was 4.95±2.05 minutes in the main group, 5.11 ±1.82 minutes in the control group (there were no statistically significant differences between the groups, p = 0.695).
After the treatment, the average (±SD) duration in the exercise performed during the treadmill test was 5.82±2.15 minutes in the main group (differences compared to the baseline were statistically significant, p <0.001 ), and 5.39±2.02 minutes in the control group, (no statistically significant differences compared to the baseline, p = 0.160) (no statistically significant differences between the groups at appointment 5, p = 0.161 ).
The absolute average difference (±SD) in the duration of the exercise performed during the treadmill test after the treatment compared to the baseline was 0.87±1.21 minutes (95% Cl 0.51 ; 1.23) in the main group, and 0.28±1.30 minutes (95% Cl - 0.10; 0.67) in the control group (differences between the groups are statistically significant, p <0.001 ). The average difference between the groups was 0.589 minutes (95% Cl 0.067; 1.111) (differences between the groups were statistically significant).
The relative average difference (±SD) of the duration in the exercise performed during the treadmill test after the treatment compared with the baseline was 23.89±39.27 % (95% Cl 12.10; 35.69) in the main group, and 8.29±25.49 % (95% Cl 0.72; 15.85) in the control group (differences between the groups were statistically significant, p = 0.001).
Thus, assessment of the PP population allows concluding that the duration of the exercise increased statistically significantly in the main group compared with the control group. Thus, the hypothesis stating that the use of the PC in combination with the basic standard therapy is more beneficial than the basic therapy alone can be considered proven according to the primary effectiveness criterion.
ITT population
The average (±SD) duration of the exercise during the treadmill test initially was 4.98±1.95 minutes in the main group, and 5.20±1.74 minutes in the control group (no statistically significant differences between the groups, p = 0.506).
The average (±SD) duration of the exercise during the treadmill test after the treatment was 5.95±2.08 minutes in the main group (differences compared to baseline were statistically significant, p <0.001 ), and 5.41 ±1.92 minutes in the control group (no statistically significant differences compared to baseline, p = 0.267) (no statistically significant differences between the groups at appointment 5, p = 0.053).
The absolute average difference (±SD) of the duration of the exercise during the treadmill test after the treatment compared to the baseline was 0.93±1.15 minutes (95% Cl 0.62; 1.25) in the main group, and 0.21 ±1.22 minutes (95% Cl 0.12; 0.54) in control group (differences between the groups were statistically significant, p <0.001 ). The average difference between the groups was 0.724 minutes (95% Cl 0.272; 1.176) (differences between the groups were statistically significant).
The relative average difference (±SD) of the duration of the exercise during the treadmill test after the treatment compared to the baseline was 24.32±36.64 % (95% Cl 14.31 ; 34.32) in the main group, and 6.51 ±24.04 % (95% Cl 0.01 ; 13.01) in the control group (differences between the groups were statistically significant, p <0.001).
Thus, assessment of the ITT population allows concluding that the duration of the exercise increased statistically significantly in the main group compared with the control group. Increase in the exercise duration during the test (TDT) according to R. Bruce protocol at the end of the 21 -day treatment regimen for 1 minute in comparison with the baseline. ITT population
An increase in the duration of the exercise performed during the treadmill test after the treatment for 1 minute was recorded for 21/54 (38.89%) patients, and for in 8/55 (14.55%) patients in the control group (differences between the groups were statistically significant, p = 0.008). The average difference was 24.343 % (95% Cl 7.752; 39.387) (differences between the groups were statistically significant).
Thus, it can be concluded that the proportion of the patients in the main group for whom the increase in the duration of the exercise performed after the treatment compared with the initial level was 1 minute, statistically does not differ from the control group.
Increase in the exercise duration during the exercise test (TDT) according to R. Bruce protocol at the end of the 21 -day treatment regimen for 2 minutes compared to the initial status:
ITT population
An increase in the duration of the exercise performed during the treadmill test after the treatment for 2 minutes was recorded for 8/54 (14.81%) patients, and for 3/55 (5.45%) patients in the control group (no statistically significant differences between the groups, p = 0.191 ). The average difference was 9.360 (95% Cl - 2.426; 21 .678) (differences between the groups were not statistically significant).
Thus, it can be concluded that the proportions of the patients for whom the increase in the duration of the exercise performed after the treatment compared with the baseline was 2 minutes, did not differ statistically in the main and control groups.
PP population
An increase in the duration of the exercise performed during the treadmill test after the treatment for 2 minutes was recorded for 6/45 (13.33%) patients, and for 3/46 (6.52%) patients in the control group (no statistically significant differences between the groups, p = 0.315). The average difference was 6.812 (95% Cl - 6.252; 20.350) (differences between the groups were not statistically significant).
Thus, it can be concluded that the proportions of the patients for whom the increase in the exercise duration after the treatment compared with the baseline was 2 minutes, did not differ statistically in the main and control groups.
Dynamics of the peak oxygen consumption according to the TDT results at the end of the 21 -day treatment regimen compared with the baseline:
ITT population
The average value (±SD) of the peak oxygen consumption during the treadmill test initially was 15.05±7.98 ml/min/kg in the main group, and 14.69±7.36 ml/min/kg in the control group (no statistically significant differences between the groups, p = 0.995). The average value (±SD) of the peak oxygen consumption during the treadmill test after the treatment was 17.22±9.54 ml/min/kg in the main group (differences compared to the baseline statistically significant, p <0.001 ), and 15.18±7.48 ml/min/kg in the control group (no statistically significant differences compared to the baseline, p = 0.154) (no statistically significant differences between the groups at appointment 5, p = 0.228).
The absolute mean difference (±SD) of the peak oxygen consumption after the treatment compared with the baseline was 2.16±4.40 ml/min/kg (95% Cl 0.96; 3.36) in the main group, and 0.49±3.38 ml/min/kg (95% Cl -0.42; 1 .41 ) in the control group (differences between the groups were statistically significant, p <0.001 ).
The relative average difference (±SD) of the peak oxygen consumption after the treatment compared with the baseline was 18.93±47.58 % (95% Cl 5.95; 31 .92) in the main group, and 7.25±34.03 % (95% Cl - 1.95; 16.45) in the control group (differences between the groups were statistically significant, p <0.001 ).
Thus, there was a statistically significant difference in the increase in the peak oxygen consumption between the main and control groups after the treatment and before the treatment, both in absolute and relative values.
PP population
The average value (±SD) of the peak oxygen consumption during the treadmill test initially was 14.32±8.60 ml/min/kg in the main group, and 13.76±7.67 ml/min/kg in the control group (no statistically significant differences between the groups, p = 0.934).
The average value (±SD) of the peak oxygen consumption during the treadmill test after the treatment was 16.05±9.87 ml/min/kg in the main group (differences compared with the baseline were statistically significant, p <0.001 ), and 14.44±7.93 ml/min/kg in the control group (no statistically significant differences compared with the baseline, p = 0.066) (no statistically significant differences between the groups at appointment 5, p = 0.549).
The absolute average difference (±SD) in the peak oxygen consumption after the treatment compared with the baseline was 1.72±4.48 ml/min/kg (95% Cl 0.38; 3.07) in the main group, and 0.69±3.63 ml/min/kg (95% Cl - 0.39; 1 .77) in the control group (differences between the groups were statistically significant, p = 0.002).
The relative average difference (±SD) in the peak oxygen consumption after the treatment compared with the baseline was 18.19±51 .66 % in the main group (95% Cl 2.67; 33.72), and 9.16±36.87 % in the control group (95% Cl - 1 .79; 20.11 ) (differences between the groups were statistically significant, p = 0.001 ).
Thus, there was a statistically significant difference in the increase in the peak oxygen consumption between the main and control groups after the treatment and before the treatment, both in absolute and relative values. Change in the capacity of the threshold load during the TDT test at the end of the 21 -day treatment regimen compared with the baseline:
ITT population
The average value (±SD) of the threshold load capacity during the treadmill test initially was 6.34±2.18 MET in the main group, and 6.51 ±2.09 MET in the control group (no statistically significant differences between the groups, p = 0.936).
The average value (±SD) of the threshold load capacity during the treadmill test after the treatment was 7.52±2.71 MET in the main group (differences compared with the baseline were statistically significant, p <0.001 ), and 6.57±2.24 MET in the control group (no statistically significant differences compared with the baseline, p = 0.440) (no statistically significant differences between the groups at appointment 5, p = 0.073).
The absolute average difference (±SD) in the threshold load capacity during the treadmill test after the treatment compared with the baseline was 1 .15±1 .85 MET (95% Cl 0.64; 1 .65) in the main group, and 0.06±1 .20 MET (95% Cl - 0.26; 0.38) in the control group (differences between the groups were statistically significant, p <0.001).
The relative average difference (±SD) of the threshold load capacity during the treadmill test after the treatment compared with the baseline was 20.53±32.80 % (95% Cl 11 .58; 29.48) in the main group, and 2.02±14.76 % (95% Cl - 1 .97; 6.01 ) in the control group (differences between the groups were statistically significant, p <0.001).
Thus, a statistically significant difference was found in the increase in the threshold load capacity between the main and control groups after the treatment and before the treatment, in both absolute and relative values.
PP population
The average value (±SD) of the threshold load capacity during the treadmill test initially was 6.28±2.37 MET in the main group, and 6.45±2.26 MET in the control group (no statistically significant differences between the groups, p = 0.948).
The average value (±SD) of the threshold load capacity during the treadmill test after the treatment was 7.19±2.78 MET in the main group (differences compared with the baseline were statistically significant, p = 0.006), and 6.52±2.43 MET in the control group (no statistically significant differences compared with the baseline, p = 0.440) (no statistically significant differences between the groups at appointment 5, p = 0.311 ).
The absolute average difference (±SD) in the threshold load capacity during the treadmill test after the treatment compared with the baseline was 0.91 ±1 .85 MET (95% Cl 0.35; 1 .47) in the main group, 0.07±1 .31 MET (95% Cl - 0.32; 0.46) in the control group (differences between the groups were statistically significant, p = 0.040). The relative average difference (±SD) in the threshold load capacity during the treadmill test after the treatment compared with the baseline was 17.60±34.19 % (95% Cl 7.33; 27.88) in the main group, and 2.41 ±16.14 % (95% Cl - 2.38; 7.20) in the control group (differences between the groups were statistically significant, p = 0.044).
Thus, a statistically significant difference was found in the increase in the threshold load capacity between the main and control groups after the treatment and before the treatment, in both absolute and relative values.
The number of the angina attacks per week and their dynamics
ITT population
The average number (±SD) of the angina attacks in the last 7 days initially was 1 49±2.54 (median 0.00) in the main group, and 1 24±1 .54 (median 0.00) in the control group (no statistically significant differences between the groups, p = 0.969).
The average number (±SD) of the angina attacks in the last 7 days after the treatment was 1 .02±1 .95 (median 0.00) in the main group (differences compared with the baseline were statistically significant, p = 0.022), and 1 80±2.45 (median 1 .00) in the control group (differences compared with the baseline were statistically significant, p = 0.047) (no statistically significant differences between the groups at appointment 5, p = 0.072).
The average difference (±SD) in the number of the angina attacks in the last 7 days after the treatment compared with the baseline was 0.50±2.37 (95% Cl -1.15; 0.15) in the main group, and 0.56±2.03 (95% Cl 0.02-1.11 ) in the control group (differences between the groups were statistically significant, p = 0.003).
Thus, it can be concluded that there was a statistically significant difference in the dynamics of the angina attack number the week after the treatment. While the average number of the angina attacks in the main group decreased statistically, the number of the attacks in the control group increased statistically significantly.
PP population
The average number (±SD) of the angina attacks in the last 7 days initially was 1 82±2.71 (median 1 .00) in the main group, and 1 48±1 .57 (median 1 .00) in the control group (no statistically significant differences between the groups, p = 0.842).
The average number (±SD) of the angina attacks in the last 7 days after the treatment was 1.22±2.08 (median 0.00) in the main group (differences compared with the baseline were statistically significant, p = 0.022), and 2.15±2.54 (median 2.00) in the control group (differences compared with the baseline were statistically significant, p = 0.047) (no statistically significant differences between the groups at appointment 5, p = 0.049).
The average difference (±SD) in the number of the angina attacks in the last 7 days after the treatment compared with the baseline was 0.60±2.59 (95% Cl - 1 .38; 0.18) in the main group, and 0.67±2.20 (95% Cl 0.02-1 .33) in the control group (differences between the groups were statistically significant, p = 0.003).
Thus, it can be concluded that there was a statistically significant difference in the dynamics of the angina attack number the week after the treatment. While the average number of the angina attacks in the main group decreased statistically significantly, the attack number in the control group increased statistically significantly.
The number of nitroglycerin doses (pills) per week and their dynamics
ITT population
The average number (±SD) of nitroglycerin pills consumed in the last 7 days initially was 0.71 ±1.15 (median 0.00) in the main group, and 0.58±0.94 (median 0. 00) in the control group (no statistically significant differences between the groups, p = 0.733).
The average number (±SD) of nitroglycerin pills consumed in the last 7 days after the treatment was 0.39±0.74 (median 0.00) in the main group (differences compared with the baseline were statistically significant, p = 0.028), and 0.93±1 .97 (median 0.00) in the control group (no statistically significant differences compared with the baseline, p = 0.127) (no statistically significant differences between the groups at appointment 5, p = 0.234).
The average difference (±SD) in the number of nitroglycerin pills consumed in the last 7 days after the treatment compared with the baseline was 0.33±1.06 (95% Cl - 0.62; - 0.04) in the main group, and 0.35±1 .61 (95% Cl - 0.09; 0.78) in the control group (no statistically significant differences between the groups, p = 0.064).
Thus, it can be concluded that no statistically significant differences in the number of nitroglycerin doses (pills) in the dynamics after the treatment between the main and control group were found, although the average number of the pills decreased statistically significantly in the main group and slightly increased in the control group.
PP population
The average number (±SD) of nitroglycerin pills consumed in the last 7 days initially was 0.87±1.22 (median 0.00) in the main group, and 0.70±0.99 (median 0.00) in the control group (no statistically significant differences between the groups, p = 0.635).
The average number (±SD) of nitroglycerin pills consumed in the last 7 days after the treatment was 0.47±0.79 (median 0.00) in the main group (differences compared with the baseline were statistically significant, p = 0.028), and 1 .11 ±2.11 (median 0.00) in the control group (no statistically significant differences compared with the baseline, p = 0.127) (no statistically significant differences between the groups at appointment 5, p = 0.206). The average difference (±SD) in the number of nitroglycerin pills consumed in the last 7 days after the treatment compared with the baseline was 0.40±1.16 (95% Cl - 0.75; - 0.05) in the main group, 0.41 ±1.76 (95% Cl - 0.11 ; 0.94) in the control group (no statistically significant differences between the groups, p = 0.054).
Thus, it can be concluded that no statistically significant differences in the number of nitroglycerin doses (pills) in the dynamics after the treatment between the main and control group were found, although the average number of the pills decreased statistically significantly in the main group and slightly increased in the control group.
Reduction in the number of the angina attacks by 50% before the end of the treatment compared with the baseline
ITT population
The number of the patients who demonstrated a reduction in the number of the angina attacks by 50% before the end of the treatment compared with the baseline was 13/55 (23.64%) in the main group, and 7/55 (12.73%) in the control group (no statistically significant differences between the groups, p = 0.216).
Thus, statistically significant differences between the main and control groups in the number of the patients who achieved a reduction in the number of the angina attacks by 50% or more before the end of the treatment were not found.
PP population
The number of the patients who demonstrated the reduction in the number of the angina attacks by 50% before the end of the treatment compared with the baseline was 13/45 (28.89%) in the main group, and 7/46 (15.22%) in the control group (no statistically significant differences between the groups, p = 0.186).
Thus, statistically significant differences between the main and control groups in the number of the patients who achieved the reduction in the number of the angina attacks by 50% or more before the end of the treatment were not found.
Reduction in the number of nitroglycerin pills (doses) consumed to eliminate the angina attacks by 50% before the end of the treatment compared with the baseline
ITT population
The number of the patients who demonstrated a decrease in the number of nitroglycerin pills (doses) consumed to eliminate the angina attacks by 50% before the end of the treatment compared with the baseline was 11/55 (20%) in the main group, and 9/55 (16.36%) in the control group (no statistically significant differences between the groups, p = 0.805).
Thus, statistically significant differences between the main and control groups in the number of the patients who demonstrated the decrease in the number of nitroglycerin pills (doses) consumed to eliminate the angina attacks by 50% or more before the end of the treatment were not found. PP population
The number of the patients who demonstrated a decrease in the number of nitroglycerin pills (doses) consumed to eliminate the angina attacks by 50% by the end of the treatment compared with the baseline was 11/45 (24.44%) in the main group, and 9/46 (19.57%) in the control group (no statistically significant differences between the groups, p = 0.757).
Thus, statistically significant differences between the main and control groups in the number of the patients who demonstrated the decrease in the number of nitroglycerin pills (doses) consumed to eliminate the angina attacks by 50% or more before the end of the treatment were not found.
Change in the level of the flow-dependent vasodilation at the end of the 21 - day treatment regimen compared with the baseline
ITT population
The average value (±SD) of the flow-dependent vasodilation (FDVD) initially was 19.03±25.85 % in the main group, and 17.88±23.99 % in the control group (no statistically significant differences between the groups, p = 0.552).
At the end of the treatment (Appointment 5), the average FDVD value was 20.73±25.73 % in the main group (differences compared with the baseline were statistically significant, p <0.001 ), and 18.50±24.07 % in the control group (differences compared with the baseline were statistically significant, p = 0.016) (differences between the groups at Appointment 5 were statistically significant, p = 0.018).
The average difference (±SD) of PZVD values between the appointments after the end of the treatment and initially was 1 .51 ±2.52 % (95% Cl 0.82; 2.20) in the main group, and 0.62±2.52 % (95% Cl - 0.06; 1.30) in the control group (differences between the groups were statistically significant, p = 0.002).
Thus, there was a significant increase in FDVD in the main group after the end of the treatment, wherein the average increase in FDVD in the main group was more pronounced than in the control group.
PP population
The average value (±SD) of the flow-dependent vasodilation (PZVD) initially was 21 65±27.95 % in the main group, and 20.22±25.61 % in the control group (no statistically significant differences between the groups, p = 0.732).
At the end of the treatment (Appointment 5), the average PZVD value was 22.78±27.76 % in the main group (differences compared with the baseline were statistically significant, p <0.001 ), and 20.80±25.73 % in the control group (no statistically significant differences compared with the baseline, p = 0.098) (no statistically significant differences between the groups at Appointment 5, p = 0.133).
The average difference (±SD) of PZVD values between appointments after the treatment and initially was 1 .13±2.51 % (95% Cl 0.37; 1 .88) in the main group, and 0.58±2.74 % (95% Cl - 0.23; 1 .40) in the control group (differences between the groups were statistically significant, p = 0.039).
Thus, there was a significant increase in FDVD in the main group after the end of the treatment compared with the baseline. The average increase in FDVD in the main group was also more pronounced than in the control group.
Change in QTc variability, and the number of arrhythmic events according to the results of the daily ECG monitoring at the end of the 21 -day treatment regimen
ITT population
At the baseline (Appointment 0), the mean value of the QT variability (±SD) was 22.98±86.35 sec in the main group, and 11.81 ±59.21 sec in the control group (no statistically significant differences between the groups, p = 0.848).
At the end of the treatment (Appointment 5), the average value of the QT variability (±SD) was 18.59±82.49 sec in the main group (no statistically significant differences compared with the baseline, p = 0.346), and 12.43±57.79 sec in the control group (no statistically significant differences compared with the baseline, p = 0.360) (no statistically significant differences between the groups, p = 0.267).
The average difference (±SD) of the QT variability between the appointments after the treatment and at the baseline was 4.81 (±92.90) sec (95% Cl - 30.16; 20.55) in the main group, and 0.62 (±82.66) sec (95% Cl - 21.72; 22.97) in the control group (no statistically significant differences between the groups, p = 0.191 ).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the QT variability value after the treatment compared with the baseline.
At the baseline (Appointment 0), the average number of ventricular arrhythmias was 490.85±1120.16 in the main group, and 493.47±1100.80 in the control group (no statistically significant differences between the groups, p = 0.976).
At the end of the treatment (Appointment 5), the average number of ventricular arrhythmias was 442.63±1216.51 in the main group (differences compared with the baseline were statistically significant, p = 0.010), and 639.07±1355.16 in the control group (no statistically significant differences compared with the baseline, p = 0.539) (no statistically significant differences between the groups, p = 0.143).
The average difference (±SD) in the number of ventricular arrhythmias (±SD) between the appointments, such as after the treatment and at the baseline, was 57.31 (±519.79) in the main group (95% Cl - 199.19; 84.56), and 145.60 (±1037.12) in the control group (95% Cl - 134.77; 425.97) (no statistically significant differences between the groups, p = 0.339).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the number of ventricular arrhythmias after the treatment compared with the baseline. At the baseline (Appointment 0), the average number of supraventricular arrhythmias was 94.24±215.73 in the main group, and 226.47±497.55 in the control group (no statistically significant differences between the groups, p = 0.983).
At the end of the treatment (Appointment 5), the average number of supraventricular arrhythmias was 112.00±340.95 in the main group (no statistically significant differences compared with the baseline, p = 0.053), and 170.42±370.20 in the control group (no statistically significant differences compared with the baseline, p = 0.362) (no statistically significant differences between the groups, p = 0.709).
The mean difference (±SD) in the number of supraventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 16.11 (±393.90) (95% Cl - 91.40; 123.63) in the main group, and 56.05 (±454.68) (95% Cl - 178.97; 66.86) in the control group (no statistically significant differences between the groups, p = 0.652).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the number of supraventricular arrhythmias after the treatment compared with the baseline.
At the baseline (Appointment 0), the average number of mixed arrhythmias was 0.25±1.89 in the main group, and 14.24±94.36 in the control group (no statistically significant differences between the groups, p = 0.174).
At the end of the treatment (Appointment 5), the average number of mixed arrhythmias was 0.30±2.18 in the main group (no statistically significant differences compared with the baseline, p = 1.00), and 1.42±5.89 in the control group (no statistically significant differences compared with the baseline, p = 0.675) (no statistically significant differences between the groups, p = 0.176).
The average difference (±SD) in the number of mixed arrhythmias between the appointments, such as after the treatment and at the baseline, was 0.04 (±2.92) (95% Cl - 0.76; 0.83) in the main group, and 12.82 (±94.33) (95% Cl - 38.32; 12.68) in the control group (no statistically significant differences between the groups, p = 1.00).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the mixed arrhythmias number after the treatment compared with the baseline.
PP population
At the baseline (Appointment 0), the mean value of QT variability (±SD) was 27.99±94.92 sec in the main group, and 14.04±64.62 sec in the control group (no statistically significant differences between the groups, p = 0.790). At the end of the treatment (Appointment 5), the average value of the QT variability (±SD) was 22.23±90.09 sec in the main group (no statistically significant differences compared with the baseline, p = 0.728), and 14.78±63.03 sec in the control group (no statistically significant differences compared with the baseline, p = 0.267) (no statistically significant differences between the groups, p = 0.417).
The average difference (±SD) of QT variability values between the appointments, such as after the treatment and at the baseline, was 5.77 (±101 .93) (95% Cl - 36.39; 24.86) sec in the main group, and 0.74 (±90.55) (95% Cl - 26.15; 27.63) sec in the control group (no statistically significant differences between the groups, p = 0.306).
Thus, no statistically significant differences were found concerning the changes in the QT variability values between the treatment groups after the treatment compared with the baseline.
At the baseline (Appointment 0), the average number of ventricular arrhythmias was 534.91 ±1192.95 in the main group, and 567.00±1188.23 in the control group (no statistically significant differences between the groups, p = 0.971 ).
At the end of the treatment (Appointment 5), the average number of ventricular arrhythmias was 481 .29±1308.45 in the main group (no statistically significant differences compared with the baseline, p = 0.067), and 748.96±1457.15 in the control group, (no statistically significant differences compared with the baseline, p = 0.788) (differences between the groups at Appointment 5 were statistically significant, p = 0.046).
The average difference (±SD) in the number of ventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 53.62 (±565.42) (95% Cl - 223.49; 116.25) in the main group, and 181 .96 (±1131 .43) (95% Cl - 154.04; 517.95) in the control group (no statistically significant differences between the groups, p = 0.450).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the number of ventricular arrhythmias after the treatment compared with the baseline, although there were statistically significant differences in the number of ventricular arrhythmias after the treatment (at Appointment 5).
At the baseline (Appointment 0), the average number of supraventricular arrhythmias was 80.18±127.58 in the main group, and 244.59±521 .56 in the control group (no statistically significant differences between the groups, p = 0.787).
At the end of the treatment (Appointment 5), the average number of supraventricular arrhythmias was 129.76±371 .42 in the main group (no statistically significant differences compared with the baseline, p = 0.097), and 173.37±387.24 in the control group (no statistically significant differences compared with the baseline, p = 0.098) (no statistically significant differences between the groups, p = 0.915). The average difference (±SD) in the number of supraventricular arrhythmias between the appointments, such as after the treatment and at the baseline, was 49.58 (±379.66) (95% Cl -64.49; 163.64) in the main group, and 71.22 (±494.59) (95% Cl -218.09; 75.66) in the control group (no statistically significant differences between the groups, p = 0.849).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the number of supraventricular arrhythmias after the treatment compared with the baseline.
At the baseline (Appointment 0), the average number of mixed arrhythmias was 0.31 ±2.09 in the main group, and 17.02±103.13 in the control group (no statistically significant differences between the groups, p = 0.181 ).
At the end of the treatment (Appointment 5), the average number of mixed arrhythmias was 0.36±2.39 in the main group (no statistically significant differences compared with the baseline, p = 1.00), and 1.70±6.41 in the control group (no statistically significant differences compared with the baseline, p = 0.675) (no statistically significant differences between the groups, p = 0.175).
The average difference (±SD) in the number of mixed arrhythmias between the appointments, such as after the treatment and at the baseline, was 0.04 (±3.20) (95% Cl - 0.92; 1 .01 ) in the main group, and 15.33 (±103.14) (95% Cl - 45.96; 15.30) in the control group (no statistically significant differences between the groups, p = 1.00).
Thus, no statistically significant differences between the treatment groups were found concerning the changes in the number of mixed arrhythmias after the treatment compared with the baseline.
Assessment of the quality of life according to the HeartQoL questionnaire and its dynamics after the end of the 21 -day treatment regimen compared with the baseline
ITT population
At the baseline, the average value (±SD) for the quality of life according to the HeartQoL questionnaire was 1 .62±1 .53 points in the main group, and 1 .62±0.55 points in the control group (no statistically significant differences between the groups, p = 0.212).
After the treatment, the average value (±SD) for the quality of life according to the HeartQoL questionnaire was 1 84±0.84 points in the main group (differences compared with the baseline were statistically significant, p <0.001 ), and 1.62±0.59 points in the control group (no statistically significant differences compared with the baseline, p = 0.683) (no statistically significant differences between the groups at Appointment 5, p = 0.143).
The average difference (±SD) in the value for the quality of life according to the HeartQoL questionnaire compared with the baseline was 0.22±1 .72 points (95% Cl - 0.26; 0.69) in the main group, and 0.01 ±0.22 points (95% Cl - 0.05; 0.07) in the control group (differences between the groups were statistically significant, p = 0.002).
Thus, it can be concluded that there was the increase in the quality of life in the main group after the end of the treatment, which was more pronounced compared with the control group.
PP population
At the baseline, the average value (±SD) for the quality of life according to the HeartQoL questionnaire was 1 .60±1 .68 points in the main group, and 1 54±0.52 points in the control group (no statistically significant differences between the groups, p = 0.212).
After the treatment, the average value (±SD) for the quality of life according to the HeartQoL questionnaire was 1.71 ±0.86 points in the main group (differences compared with the baseline were statistically significant, p = 0.014), and 1 .53±0.58 points in the control group (no statistically significant differences compared with the baseline, p = 0.966) (no statistically significant differences between the groups at Appointment 5, p = 0.470).
After the treatment, the average difference (±SD) in the value of the quality of life according to the HeartQoL questionnaire was 0.12±1 .87 points (95% Cl - 0.45; 0.68) in the main group, and 0.00±0.19 points (95% Cl - 0.06; 0.06) in the control group (no statistically significant differences between the groups, p = 0.061 ).
Thus, it can be concluded that there was the increase in the quality of life after the treatment in the main group, but the changes were not statistically significant compared with the control group.
Statistical / analytical results
Previous and concomitant diseases. Both in the main and the control group disorders of the cardiovascular system were most often as concomitant pathology: in 34/55 (61.82%) and 32/55 (58.18%) patients, respectively (no statistically significant differences between the groups, p = 0.846)
Preliminary and concomitant therapy
In the main and control groups, hypoglycemic drugs were prescribed the most often as concomitant drugs. Except insulin, the second most frequently prescribed drug in the main group was acetylsalicylic acid.
Conclusion on the effectiveness
According to the results of the primary effectiveness criterion assessment, such as the change in the duration of the exercise performed during the exercise test according to the R. Bruce protocol at the end of the 21 -day treatment regimen compared with the baseline, in the population according to the protocol (the main population for the primary effectiveness criterion assessment), the absolute change was 0.87±1 .21 min (95% Cl 0.51 ; 1 .23) in the main group, and 0.28±1 .30 min (95% Cl - 0.10; 0. 67) in the control group (differences between the groups were statistically significant, p <0.001 ). The average difference between the groups was 0.589 minutes (95% Cl 0.067; 1.111) (differences between the groups were statistically significant).
Similar results with a statistically significant difference between the groups in favor of the combination therapy were obtained for the ITT population.
According to the protocol, the conclusion on the exceeding effectiveness of the combination therapy (PC and the standard therapy) in the main group compared to the standard therapy alone can be drawn based on the positive statistically significant differences between the groups in the primary effectiveness variable in favor of the main group. This allows concluding that the therapy with the study PC (oral solution) on the background of the standard therapy exceeds the effectiveness of the standard therapy alone concerning the duration of the exercise performed.
Additionally, statistically significant differences were obtained in the analysis of the relative increase in the exercise duration, which was 23.89±39.27 % (95% Cl 12.10; 35.69) in the main group of the PP population, and 8.29±25.49 % (95% Cl 0.72; 15.85) in the control group of the PP population; and 24.32±36.64 % (95% Cl 14.31 ; 34.32) in the main group of the ITT population, and 6.51 ±24.04 % (0.01 ; 13.01 ) in the control group of the ITT population.
The assessment of additional effectiveness criteria revealed a statistically significantly greater number of the patients in the combination therapy group of the ITT population compared with the standard therapy who demonstrated the increase in the exercise duration for 1 minute after the 21 -day treatment regimen, (38.89% and 14.55%, respectively). In the population according to the protocol, the differences between the groups did not reach statistical significance (37.78% and 17.39%, respectively).
No statistically significant differences between the number of the patients in the main and control groups, who demonstrated the increase in the exercise duration for 2 minutes after the 21 -day treatment regimen, were found either in the PP population (13.33% and 6.52%, respectively), or in the ITT population (14.81% and 5.45%, respectively).
Statistically significant difference between the treatment groups was found concerning the increase in the peak oxygen consumption after the treatment compared with the baseline. In the PP population, the change was 1.72±4.48 ml/min/kg (95% Cl 0.38; 3.07) in the main group, and 0.69±3.63 (95% Cl - 0.39; 1.77) ml/min/kg in the control group; in the ITT population, the change was 2.16±4.40 ml/min/kg (95% Cl 0.96; 3.36) in the main group, and 0.49±3.38 ml/min/kg (95% Cl - 0.42; 1 .41 ) in the control group. Similarly, statistically significant differences between the groups were found for the relative change in the peak oxygen consumption: 18.19±51 .66 % (95% Cl 2.67; 33.72) in the main group of the PP population, and 9.16±36.87 % (95% Cl - 1 .79; 20.11 ) in the control group of the PP population; and 18.93±47.58 % (95% Cl 5.95; 31.92) in the main group of the ITT population, and 7.25±34.03 % (95% Cl - 1 .95; 16.45) in the control group of the ITT population.
Statistically significant difference between the treatment groups was found concerning the increase in the threshold load capacity after the 21 -day treatment regimen. The value was 0.91 ±1.85 MET (95% Cl 0.35; 1.47) in the main group of the PP population, and 0.07±1 .31 (95% Cl - 0.32; 0.46) MET in the control group of the PP population; 1 .15±1 .85 MET (95% Cl 0.64; 1 .65) in the main group of the ITT population, and 0.06±1 .20 MET (95% Cl - 0.26; 0.38) in the control group of the ITT population. Similarly, statistically significant differences between the groups were found for the relative increase in the threshold load capacity: 17.60±34.19 % (95% Cl 7.33; 27.88) in the main group of the PP population, and 2.41 ±16.14 % (95% Cl - 2.38; 7.20) in the control group of the PP population; 20.53±32.80 % (95% Cl 11 .58; 29.48) in the main group of the ITT population, and 2.02±14.76 % (95% Cl - 1 .97; 6.01 ) in the control group of the ITT population.
Statistically significant difference in the dynamics of the angina attack number per week after the treatment was found for both analyzed populations. While the average number of the angina attacks in the main group decreased statistically significantly (average change was -0.60±2.59 (95% Cl - 1.38; 0.18) in the PP population and -0.50±2.37 (95% Cl -1.15; 0.15) in the ITT population), in the control group, the number of the attacks increased statistically significantly (average change was 0.67±2.20 (95% Cl 0.02-1 .33) in the PP population and 0.56±2.03 (95% Cl 0.02-1 .11 ) in the ITT population).
No statistically significant differences between the treatment groups were found concerning the number of the patients who demonstrated the decrease in the number of the angina attacks by 50% per week after the treatment compared with the baseline. This result may be due to the fact that the average number of the angina attacks per week was relatively small at the baseline.
No statistically significant differences between the treatment groups were found concerning the number of nitroglycerin pills consumed per week in any of the analyzed populations.
Additionally, no statistically significant differences between the treatment groups were found concerning the number of the patients who achieved the decrease in the number of nitroglycerin pills consumed per week by 50% after the end of the treatment compared with the baseline.
In the main group, statistically significant increase in the mean value of FDVD was observed after the treatment compared with the baseline. The average increase in FDVD in the main group was also significantly more pronounced than in the control group. The average difference in FDVD was 1.13±2.51 % (95% Cl 0.37; 1 .88) in the main group of the PP population, and 0.58±2.74 % (95% Cl - 0.23; 1 .40) in the control group of the PP population; and 1.51 ±2.52 % (95% Cl 0.82; 2.20) in the main group of the ITT population, and 0.62±2.52 % (95% Cl - 0.06; 1 .30) in the control group of the ITT population.
No statistically significant differences between the treatment groups were found regarding the changes in QT variability, ventricular arrhythmias, supraventricular arrhythmias, and mixed arrhythmias after the treatment compared with the baseline in any of the analyzed populations.
In the main group of the ITT population, the increase in the quality of life according the HeartQoL questionnaire after the treatment, which was more pronounced compared to the control group, was observed. The average difference in the quality of life compared with the baseline was 0.22±1 .72 points (95% Cl - 0.26; 0.69) in the main group, and 0.01 ±0.22 points (95% Cl - 0.05; 0.07) in the control group (differences between the groups were statistically significant, p = 0.002). The population according to the protocol also demonstrated the increase in the quality of life after the treatment, but the changes were not statistically significant compared with the control group.
The study results allow concluding that the combination therapy with the claimed pharmaceutical composition on the background of the standard therapy exceeds the effectiveness of the standard therapy alone for the patients with coronary heart disease, and additionally has an acceptable safety profile comparable to the individual standard therapy.
Summarizing the clinical study data, it can be concluded that the pharmaceutical composition according to the invention in the oral solution form containing two active substances, such as arginine and levocarnitine, increases the effectiveness of the combination therapy of coronary heart disease and improves the quality of life by reducing the frequency of the angina attacks.
A clinical study was conducted to determine the possibility of the PC use for the treatment of diseases associated with cerebrovascular disorders, in particular, chronic cerebrovascular disorders, and the practicability of such use.
The aim of the clinical study was to study the effectiveness of the PC containing 100 mg of levocarnitine and 264 mg of arginine aspartate in 1 ml of the solution as a part of the combination therapy of such a disease as chronic cerebrovascular disorder (CCD).
The study design was as follows.
The study involved 75 patients of both sexes aged 55 to 75 years, who were divided into three groups, 25 patients per group. All the patients were diagnosed with chronic cerebrovascular disorders of Stages l-ll.
The patients were divided into three groups, wherein the first group was the main group, the second group was the first control group, the third group was the second control group. The groups were statistically homogeneous by sex and age, as well as by comorbidities. In particular, all the examined patients had a history of hypertension, wherein the groups did not differ in the duration of the disease, baseline systolic and diastolic blood pressure, and heart rate. In all three groups, approximately equal numbers of the patients received antihypertensive therapy. All the patients were diagnosed with atherosclerosis (cardiosclerosis, carotid artery disease). Frequencies of obesity and disorders of the blood lipid spectrum were similar.
The patients in the main group were prescribed with:
- a basic therapy including statins, antiplatelets, and antihypertensive drugs (in the presence of concomitant hypertension);
- the PC comprising 100 mg of levocarnitine and 264 mg of arginine aspartate in 1 ml of the solution, orally, 10 ml three times a day, before meals, for 21 days. 1 ml of the PC oral solution comprised 264 mg of arginine aspartate and 100 mg of levocarnitine, and excipients, such as malic acid, sodium saccharin, methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), and water for injections. The daily dose of the PC was 30 ml, the daily dose of arginine aspartate was 7.92 g, and the daily dose of levocarnitine was 3 g.
The patients in the first control group were prescribed with:
- a basic therapy including statins, antiplatelets, and antihypertensive drugs (in the presence of concomitant hypertension);
- arginine aspartate solution (Tivortin aspartate drug) orally, 10 ml 4 times a day, for 21 days. 1 ml of Tivortin oral solution comprised 200 mg of arginine aspartate and excipients, such as sorbitol (E 420), sodium saccharin (E954), methyl parahydroxybenzoate (E 218), propyl parahydroxybenzoate (E 216), "Caramel" food flavor, and water for injections. The daily dose of arginine aspartate was 8 g.
The patients in the second control group were prescribed with:
- a basic therapy including statins, antiplatelets, and antihypertensive drugs (in the presence of concomitant hypertension);
- levocarnitine solution in the form of the solution for oral administration, 5 ml 3 times a day, for 21 days. 1 ml of levocarnitine solution for the oral administration comprised 200 mg of levocarnitine, and excipients, such as methylparaben (E 218), propylparaben (E 216), sucrose, sorbitol (E 420), "Banana" flavor, and purified water. The daily dose of levocarnitine was 3 g
The design scheme of the study is presented in Table 46.
All the patients underwent somatic and neurological examination, which was supplemented by clinical and biochemical blood analysis, Doppler of cerebral vessels, electroencephalogram (EEG), and electrocardiogram (ECG). The full examination was performed 2 times - before the treatment and at the end of the 21 - day treatment regimen. Additionally, a number of scales were used, such as adapted quantitative neurological scale according to A. I. Fedin, the Mini Mental State Examination (MMSE), the Montreal Cognitive Assessment Scale (MoCA), and the Hospital Anxiety and Depression Scale (NADS).
Table 46
Research design scheme
Figure imgf000138_0001
According to the results, the patients of all three groups demonstrated positive clinical dynamics during the period of the inpatient treatment. The patients complained of clinical manifestations of CCD Stages l-ll. After the treatment regimen, all the patient groups showed a positive trend being a regression of most of the complaints made before the treatment. Table 47 shows the results of the changes in the patient complaints. Positive dynamics of the complaints was observed in all the groups. At the end of the treatment, the frequency of the complaints of weakness, fatigue, memory impairment, and headache decreased significantly (p <0.05). There was no significant dynamics in the "noise in the head", "change of mood", "gait instability" and "sleep disturbance" parameters (p> 0.05). In the main and control groups of the patients, a significant difference in the dynamics was observed for the complaints, such as general weakness, decreased ability to work, memory impairment, headache, dizziness, gait instability (p <0.05). The dynamics in other complaints in the main and control groups of the patients did not differ significantly (P> 0.05). Table 57 - The frequency of the patient complaints in three groups before and after the treatment, in %
Figure imgf000139_0001
* Significant difference before and after the treatment (p <0.05).
During the study, a number of positive general clinical changes was also recorded. Specifically, the neurological symptoms reversed faster in the main monitored group than in the control group. Analysis of the neurological disorder dynamics after the treatment regimen based on the adapted quantitative neurological scale according to A.l. Fedin showed positive changes in individual neurological symptoms and syndromes in all three groups (Table 48). Table 48 shows that significant changes in the parameters were observed in all the groups concerning "cerebral symptoms", "autonomic disorders", "movement disorders" and "overall score" (p <0.05). Compared with the first control group and the second control group, the main group demonstrated more pronounced dynamics in the cerebral and autonomic symptoms, as well as the overall score (P <0.05). The overall decrease in the score of neurological disorders was 34% in the main group, 15% in the first control group, and 17% in the second control group.
Table 48
Neurological disorder severity for the patients in three groups based on the adapted quantitative neurological scale according to A. I. Fedin before and after the treatment
Figure imgf000140_0001
* Significant difference before and after the treatment (p <0.05).
Data from neuropsychological examination of the patients based on MMSE scale before and after the treatment are shown in Table 49. Table 49 shows that statistically significant improvement after the treatment in the main group was confirmed by the overall score according to MMSE scale, and according to "attention and calculation", "word reproduction" and " speech functions" subscales (p <0.05). In the first control group, significant improvement after the treatment was confirmed by the overall score, and the "speech functions" test (p <0.05). In the second control group, significant improvement after the treatment was confirmed by the overall score and the "attention and calculation" and "speech functions" tests (p <0.05). Thus, the overall improvement according to the " speech functions" test was 25% in the main group, 10% in the first control group, and 11% in the second control group. The overall improvement according to the overall score was 12% in the main group, 5% in the first control group, 5% in the second control group.
Table 49
Comparison of the patients in three groups according to MMSE scale before and after the treatment
Figure imgf000141_0001
* Significant difference before and after the treatment (p <0.05).
For three groups of the patients, the level of cognitive impairment was assessed according to the Montreal scale of assessment of cognitive impairment (MoCA) before and after the treatment (Table 50). The scale allows evaluating a number of cognitive functions, such as short-term memory, recollection, attention, working memory, and abstract thinking. When being treated, the patients in all the groups improved the average task performance score compared with the baseline (p <0.05). At the same time, the patients in the main group demonstrated more pronounced dynamics in the task performing (p <0.05). In the first control group of the patients, the parameter decreased the least. Thus, according to Table 50. the overall improvement was 38% in the main group, 12% in the first control group, and 23% in the second control group.
Table 50
Comparison of the patients in three groups based on Montreal scale of assessment of cognitive impairment before and after the treatment
Figure imgf000142_0001
* Significant difference before and after the treatment (p <0.05).
Comparisons of anxiety and depression before and after the treatment recorded for the three groups of the patients on the Hospital Anxiety and Depression Scale (NADS) are shown in Table 51. The patients in all the groups showed a decrease in anxiety and depression (p <0.05). The average assessment of anxiety in all the groups conforms to clinically pronounced manifestations. When being treated, the overall level of anxiety decreased to subclinical levels. Decrease in the score of anxiety according to NADS Scale was 42% in the main group, 19% in the first control group, and 21% in the second control group. The average level of depression was significantly lower in all the groups and conformed to subclinical manifestations. When being treated, the average score of general depression normalized only in the main group. In the control groups, it decreased within the subclinical level. Decrease in score of depression was 35% in the main group, 13% in the first control group, and 20% in the second control group.
Table 51
Hospital Anxiety and Depression Scale (HADS) scores before and after the treatment for the patients in three groups
Figure imgf000143_0001
* Significant difference before and after the treatment (p <0.05).
The study PC showed a good level of safety and tolerability. No adverse reactions were recorded when treating with the PC. The treatment was well tolerated. Adherence to the therapy was high. None of the patients left the study.
Chronic cerebrovascular disorder is a formidable manifestation that develops in the middle and old age. This disorder has no pronounced symptoms and develops gradually, therefore, it often manifests late, when the degradation of personality has already begun. At any form of chronic cerebrovascular disorder, the pharmacotherapeutic effect should be as comprehensive as possible and aimed at restoring regular circulation in the affected area and activating energy processes in the human brain, improving mental performance and memory, normalizing the blood supply and resistance of the brain cells to oxygen starvation.
In the study, the complex of the clinical symptom of CCD included general cerebral symptoms, vestibulo-cochlear disorders, pyramidal and cortico-nuclear insufficiency, autonomic and coordination disorders specifically attributed to this disease. Cognitive impairment was typical for all the patients: some patients demonstrated psycho-emotional disorders in the form of anxiety, and several patients demonstrated depression. Asthenic symptoms were observed in all the studied patients.
The study revealed that all the study drugs had an effect on CCD treatment. However, the PC according to the invention showed the greatest effectiveness.
The main neurological symptoms of Stages l-ll of CCD are essentially expressed in the asthenic syndrome and anxiety. Headache, dizziness, general weakness, increased fatigue, emotional lability, sleep disturbances, reduced efficiency - all this set of the complaints is typical for the initial stages of CCD. The treatment with the PC maximized the efficiency and daily activity of the patients, enhanced memory, reduced headache and anxiety compared with the first and second control groups of arginine or levocarnitine. Analysis of the data in Table 47-51 shows that the claimed pharmaceutical composition according to the invention had a more pronounced protective effect and had an unexpected technical result in comparison with comparative drugs containing arginine aspartate or levocarnitine alone. Analysis of the data obtained during the studies allows claiming that the claimed pharmaceutical composition had an unexpected synergistic effect.
In pharmacology, a separate case of synergism, in which the effect of simultaneous use of two or more active substances exceeds the total effect of each of these substances alone, is called potentiation. As will be shown below by calculations, the use of the combination of the components, such as arginine aspartate and levocarnitine, in the claimed pharmaceutical composition gives the effect of potentiation, therefore, the pharmaceutical composition according to the invention has an unpredictable synergistic effect.
Analysis of the dynamics of the neurological status based on A. I. Fedin scale showed that the administration of the pharmaceutical composition led to a greater improvement in general cerebral and autonomic symptoms, as well as the overall score. The overall decrease in the severity of neurological disorders was 34% in the main group, 15% in the first control group, and 17% in the second control group. The expected total pharmaceutical effect of the two comparative drugs was 15% + 17% = 32%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention, was 34%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the reduction in the overall score of neurological status based on A. I. Fedin by 34% - 32% = 2%.
Greater effect of the pharmaceutical composition compared to the therapy in the first and second control groups was clearly observed in a significant improvement in cognitive function (based on MMSE scale results). Additionally, there were statistically significant differences in the overall score based on MMSE scale and the "speech functions" subscale after the treatment. The overall improvement in neuropsychological status of the patients based on the "speech functions" test and MMSE scale was 25% in the main group, 10% in the first control group, and 11% in the second control group. The expected total pharmaceutical effect of two comparative drugs was 10% + 11% = 21%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention was 25%, which exceeded the expected total pharmaceutical effect of two comparative drugs based on the "speech functions" test and MMSE scale by 25% - 21% = 4%. The enhancement in the overall score was 12% in the main group, 5% in the first control, and 5% in the second control group. The expected total pharmaceutical effect of two comparative drugs was 5% + 5% = 10%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention was 12%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the total score on MMSE scale by 12% - 10% = 2%.
The cognitive impairment dynamics based on the Montreal scale of assessment of cognitive impairment (MoCA) were more pronounced for the patients treated with the pharmaceutical composition compared with the monotherapy. The overall improvement was 38% in the main group, 12% in the first control group, and 23% in the second control group. The expected total pharmaceutical effect of two comparative drugs was 12% + 23% = 35%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention was 38%, which exceeded the expected total pharmaceutical effect of two comparative drugs on improvement of the cognitive impairment dynamics based on the Montreal scale by 38% - 35% = 3%.
The anxiety and depression dynamics according to the Hospital Anxiety and Depression Scale (NADS) were more pronounced for the patients in the main group, who were treated with the pharmaceutical composition, compared to the monotherapy. Decrease in anxiety score according to NADS Scale was 42% in the main group, 19% in the first control group, and 21% in the second control group. The expected total pharmaceutical effect of two comparative drugs was 19% + 21 % = 40%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention was 42%, which exceeded the expected total pharmaceutical effect of two comparative drugs on reduction in the anxiety score based on NADS Scale by 42% - 40% = 2%. Decrease in the depression score according to NADS Scale was 35% in the main group, 13% in the first control group, and 20% in the second control group. The expected total pharmaceutical effect of two comparative drugs was 13% + 20% = 33%. The pharmaceutical effect of the drug being the pharmaceutical composition according to the invention was 35%, which exceeded the expected total pharmaceutical effect of two comparative drugs on reduction in the depression score based on NADS Scale by 35% - 33% = 2%.
Such effects are associated with the synergism of arginine aspartate and levocarnitine in the PC. Levocarnitine increases the rate of fat oxidation in mitochondria and plays a key role in the body metabolism, such as it provides transport of long-chain fatty acids into the mitochondrial matrix, controls and modulates the intracellular pool of coenzyme in the cell, participates in detoxification of organic acids and xenobiotics. In brain tissues, levocarnitine transports acetyl residues from mitochondria to the cytosol, thus participating in the synthesis of acetylcholine and acetylcarnitine. The neurobiological effects of acetylcarnitine include direct effects on energy metabolism and phospholipid metabolism, synoptic morphology, and transmission of numerous neurotransmitters. The use of arginine aspartate, being an active donor of NO, improves endothelium-dependent vasodilation, reduces platelet aggregation and reduces endothelium-dependent adhesion of monocytes. This helps to restore cerebral circulation, normalize hemodynamics, reduce oxidative stress in the lesion and, accordingly, reduce neurological deficit.
Summarizing the data of the clinical studies, it can be concluded that the pharmaceutical composition comprising two active substances, such as arginine aspartate and levocarnitine, increases the effectiveness of the combination therapy of CCD. The PC, being the part of the combination treatment of CCD, showed significantly greater effectiveness than the use of arginine or aspartate alone.
To determine the possibility of the PC use for the treatment and prevention of the diseases in pregnant women and fetal development during pregnancy, in particular preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction, and the feasibility of such use, a preclinical trial was conducted.
The aim of the preclinical trial was to study the effectiveness of the PC being the solution for the oral administration and comprising 264 mg of arginine hydrochloride and 100 mg of levocarnitine in 1 ml, for the treatment and prevention of preeclampsia in pregnant women, fetal distress, and intrauterine growth restriction.
The trial design was as follows.
According to a directive of the International Society for the Study of Hypertension in Pregnancy, clinical features of preeclampsia include severe hypertension after 20th week of gestation, proteinuria with or without edema and changes in laboratory parameters, as well as kidney, liver and brain impairment. One of the key factors of this pathology is confirmed to be the inhibition of the synthesis of nitric oxide (NO) vasodilator. Powerful NO vasodilator is synthesized from L-arginine by nitric oxide synthase (NOS). Chronic inhibition of NOS by N-nitro- L-arginine methyl ester (L-NAME) in pregnant rats leads to the dose-dependent development of hypertension in combination with proteinuria, renal vasoconstriction, thrombocytopenia and maternal and fetal mortality. This emphasizes the importance of this molecule in pregnancy and is a useful preclinical model for assessing the role of NO in pregnancy and developing new drugs for the prevention and treatment of preeclampsia. In view of this, the trial on the PC, the pharmacodynamic effect of which is related to the presence of levocarnitine and arginine hydrochloride, and its effect on the clinical characteristics of preeclampsia, increase of nitric oxide synthesis in endothelial cells and the fetus condition, was conducted.
To compare the pharmaceutical effects of individual arginine, individual levocarnitine, and combined arginine and levocarnitine (PC), the preclinical trial was conducted on pregnant rats with chronic NOS inhibition by N-nitro-L-arginine methyl ester (abbreviated as L-NAME).
The following solutions were used for the trial:
- the drug being the PC according to the invention and comprising 264 mg of arginine hydrochloride and 100 mg of levocarnitine in 1 ml;
- the first comparative drug comprising 264 mg\ml of arginine aspartate;
- the second comparative drug comprising 200 mg/ml of levocarnitine;
The trial was conducted on 8 groups of animals. The average body weight of non-pregnant rats was 200 ± 10 g. The volume and quantity of the study drugs are indicated based on 200g weight of the rats. Group 1 (intact control) included non-pregnant rats treated with saline (n =
10).
Group 2 (negative control) included pregnant rats treated with saline (n = 10).
Group 3 (positive control) included pregnant rats administered with the solution of L-NAME nitric oxide synthase blocker (n = 10).
Group 4 (comparative group) included pregnant rats administered intragastrically with 2 ml (528 mg) of the reference sample N°1 (arginine aspartate), and then L-NAME nitric oxide synthase blocker (n = 10).
Group 5 (comparative group) included pregnant rats administered intragastrically with 2 ml (200 mg) of the reference sample N°1 (levocarnitine), and then L-NAME nitric oxide synthase blocker (n = 10).
Group 6 (experimental group / PC) included pregnant rats administered intragastrically with 2 ml of the PC test sample (equal to 528 mg of arginine aspartate and 200 mg of levocarnitine), and then L-NAME nitric oxide synthase blocker (n = 10).
Group 7 (experimental group / PC) included pregnant rats administered intragastrically with 1 ml of the PC test sample (equal to 264 mg of arginine aspartate and 100 mg of levocarnitine), and then L-NAME nitric oxide synthase blocker (n = 10).
Pregnancy modeling was performed before the trial using monitoring the animal estrous cycle and subsequent fertilization with the identification of the first day of fertilization based on the presence of sperm in the animal vaginal swab. Pregnant and virgin nonlinear animals were housed individually in standard metabolic cells, which allowed urine to be collected, and food and water intake to be recorded throughout the trial. Starting from the fifth day of the pregnancy, laboratory animals in the groups No. 4-7 started to be administered with the study / control drug intragastrically, once a day, using a syringe through a metal probe. The administration was continued until delivery. One arterial and one venous catheter were implanted on the fourteenth day of the gestation (delivery from day 21 to day 22). One day later, the groups No. 3-7 started to be administered with L-NAME dissolved in sterile saline through a catheter implanted into a vena cava at a rate of 0.5 mg in 0.05 ml per 100 g of body weight per hour. The groups No. 1 -2 were administered with 2 ml of saline only at the similar rate. The infusions were continued for 5 days.
Abnormalities of fetal developmental were assessed by weighing neonates and assessing neonatal mortality. Measurements of the average blood pressure of the non-anesthetized animals that was not restricted in their movements were performed daily, at a fixed time, using an electronic manometer and a pressure transducer.
Sample collection and analysis were performed as follows. The animal body weight, water and food intake, and urine volume were monitored daily. The urine samples collected over twenty-four hours were centrifuged at 3000 G for 15 minutes and stored in a freezer at -20 °C before albumin chemical analysis (usually <2 weeks). Shortly before infusion, blood samples were taken into syringes from a catheter implanted in the aorta (for baseline values), and, 4 days later, to assess the effect of L-NAME on nitrate and nitrite levels, and platelet count.
Histological analysis of the kidneys was performed as follows. 4 days after the start of the treatment, the animals were anesthetized, and both kidneys were removed and weighed, the left kidney being treated for microscopic examination. The coronary sections of the kidneys were fixed in 10% formalin and imbedded into paraffin blocks. The sections (thickness 3 pm) were dyed. The sections were examined on a blinded basis for lesions of the glomerular zone.
Statistical analysis. The results are presented as the mean±SEM. Comparison of the respective values for the pregnant and virgin rats was performed using the Student test. Probability <0.05 was considered statistically significant.
The results.
Preeclampsia is characterized by hypertension, proteinuria, as well as impairment of the kidneys and liver of the mother, and, consequently, possible abnormalities of the fetal development. There was no significant difference in the food intake between the pregnant animals in the control group and the animals in the groups No. 3-7. Despite almost identical food intake, body weight of the females receiving L-NAME (Group 3) was less than body weight of the pregnant animals in the control group (croup No. 2) that received saline, which may reflect significant intrauterine growth restriction. The animals treated with the pharmaceutical composition according to the invention (Groups No. 6 and 7) showed a significant increase in body weight, indicating normalization of the metabolic processes and, most probably, impact on the fetal weight restoration (see Table 52).
Table 52 Effect of chronic inhibition of NO synthase with L-NAME on the body weight, food and water intake, urine volume, and arterial blood pressure in pregnant and virgin rats
Figure imgf000149_0001
* The changes are statistically significant for the animals in the negative control group (* p <0.05, ** p <0.01 ).
# The changes are statistically significant for the animals in the positive control group (# p <0.05, ## p <0.01 ).
The administration of L-NAME (Group 3) did not cause significant changes in the water intake for the pregnant rats compared with the animals in the negative control group (Group 2). Additionally, there was no significant difference between the animals in the control groups (Groups No. 2-3), the comparative groups (Groups No. 4-5) and the study animal groups (Groups No. 6-7). Interestingly, with no significant difference in the water intake for the pregnant animals, infusion of L- NAME decreased daily urination of the pregnant animals significantly (Group 3), compared with the pregnant animals receiving saline only (Group 2) (Table 52). This may indicate a decrease in renal blood supply, which is caused by a vasoconstrictive effect, with a decrease in the blood nitric oxide level when administering L-NAME nitric oxide synthase blocker. The animals receiving the pharmaceutical composition according to the invention demonstrated a statistically significant restoration of the average urine volume compared with the Group 3 and the comparative groups (Group 4 and 5) receiving L-NAME, which may indicate normalization of the renal function and lowering of the arterial blood pressure caused by restoration of the blood nitric oxide level.
The obtained data of mean arterial blood pressure (MABP) indeed indicate the antihypertensive effect of arginine, levocarnitine and three doses of the claimed pharmaceutical composition. Moreover, the effect of the claimed pharmaceutical composition according to the invention was more pronounced, compared with the similar effect of the comparative drugs (Table 52). The most pronounced pharmaceutical effect according to the urine volume and mean arterial blood pressure was seen when the amount of arginine / levocarnitine was increased to 520/200 mg, respectively.
Kidney impairment is one of the main prognostic markers in preeclampsia progression. The kidney status was analyzed by histological analysis, and by the level of proteinuria (urinary albumin level). Inhibition of nitric oxide synthesis can cause glomerular capillary hypertension, which leads to sclerotic lesions of the kidney glomerular zone. The administration of L-NAME was accompanied by severe morphological changes in the kidney glomerular zone of the pregnant animals. Glomerular capillary lumens were segmentally occluded by intraluminal masses of eosinophilic composition. Extraglomerular lumens were filled with protein. In addition, mild diffuse interstitial edema and sparse interstitial lymphocyte infiltration were observed. For the groups receiving the pharmaceutical composition, such renal changes were not observed, in contrast to the individual administration of arginine and levocarnitine, for which pathological changes were recorded on the preparations.
Urinary glomerular proteins of intermediate size, such as albumin, indicate the renal tubules impairment, which can develop in severe preeclampsia. Thus, it was found that the average daily urinary albumin excretion increased sharply in the positive control group (Group 3) administered with L-NAME alone (mg/24 h) from 8.3±1.5 to 56.3±14.3 mg/24 h (p <0.005). The administration of arginine, levocarnitine, and two doses of the claimed pharmaceutical composition according to the invention in addition to L-NAME was shown to decrease the urinary albumin level, and, for the groups receiving the pharmaceutical composition according to the invention, proteinuria was least pronounced, which confirms the synergistic nephroprotective effect of the composition in comparison with the individual administration of arginine and levocarnitine (see Table 53).
Table 53 Effect of the study drugs on urinary albumin level upon chronic inhibition of
NO synthase with L-NAME
Figure imgf000151_0001
* The changes are statistically significant for the animals in the negative control group (* p <0.05, ** p <0.01 , *** p <0.005).
# The changes are statistically significant for the animals in the positive control group (* p <0.05, ** p <0.01 ).
Typically, thrombocytopenia is associated with a preeclamptic status due to increased aggregation and adhesion of platelets to the damaged endothelium. In the positive control group (Group 2), a statistically significant decrease in platelets in the blood as a result of the infusion of L-NAME was observed. Decreased platelets indicate the onset of the so-called HELLP syndrome, which is caused by vascular endothelium impairment in preeclampsia. Stable platelet level was recorded, when administering arginine (Group 4) and all the doses of PC (Groups No. 6-7), which indicates a protective effect on vascular endothelium (Table 53). Additionally, higher protective effect was recorded for Groups No. 6-7 (Table 53).
To investigate the abnormality of the fetal developmental, the effect of the test and reference samples on neonatal weight and offspring mortality was studied. The development of preeclampsia is associated with the risks of decreased fetal perfusion and decreased fetal growth. Significant embryoprotective effect of the PC was confirmed by the data on neonatal weight and the percentage of the offspring mortality. The infusion of L-NAME starting from the 15th day of the pregnancy caused significant intrauterine growth restriction without affecting the duration of the gestation compared to the animals in the control groups. Preventive administration of the claimed PC to the animals was found to lead to a statistically significant increase in fetal growth rates compared to the control and reference groups (Table 54). The offspring mortality was studied immediately after the birth. For Group 3 animals receiving L-NAME inhibitor only, a significant number of neonates were stillborn. Approximately 10.6% of all the neonates in Group 3 died (Table 54). Statistically significant retention of live fetuses was found for all the doses of the PC (Groups No. 6-7), which indicates a protective effect of the PC on pregnancy (Table 54).
Table 54
Effect of the study drugs on neonatal weight upon chronic inhibition of NO synthase with L-NAME
Figure imgf000152_0001
* The changes are reliable for the animals in the negative control group (* p <0.05, ** p <0.01 , *** p <0.005).
# The changes are statistically significant for the animals in the positive control group (# p <0.05, ## p <0.01 ).
Table 54 shows that the pharmaceutical composition according to the invention had the greatest embryoprotective effect, because, in addition to the restoration of the fetal body weight, it protected the offspring from death in preeclampsia. This effect can be associated with lowering of mean arterial blood pressure, restoration of the kidney function and reduction of the pro-inflammatory processes on the endothelial walls by restoring nitric oxide level.
Above mentioned allow concluding that the claimed pharmaceutical composition according to the invention comprising arginine hydrochloride and levocarnitine has a pronounced embryoprotective effect on the fetus and mother body by activating the nitric oxide system and optimizing the energy balance of the cells. Pronounced protective effect of the PC according to the invention on prevention of preeclamptic disorders is manifested in the restoration of nitric oxide blood level, subsequent restoration of mean arterial blood pressure, normalization of the renal function, and embryoprotective effect on the fetus development.
Analysis of the data provided in Tables 52-54 shows that, in comparison with the drugs containing either arginine or levocarnitine, the claimed pharmaceutical composition according to the invention has a more pronounced protective effect and has an unexpected technical result. The data show that the claimed pharmaceutical composition creates unexpected synergistic effect.
In pharmacology, a specific case of synergism, in which the effect of the simultaneous use of two or more active substances exceeds the estimated total effect of the use of each of these substances individually, is called potentiation. As will be shown below by the calculations, the use of the combination of arginine and levocarnitine in the claimed PC, provides the potentiation effect, therefore, the pharmaceutical composition according to the invention shows an unpredictable synergistic effect.
The calculation of the pharmaceutical effect for each of the reference and test samples and conclusion whether they have the potentiation effect, was performed according to the following technique. Initially, the maximum possible pharmaceutical effect that is the difference between the values of a body status parameter for Group 2 animals (negative control) and Group 3 animals (positive control), was calculated. The pharmaceutical effect for the first comparative drug was calculated as the difference between the value of a body status parameter for Group 3 animals (positive control, L-NAME) and the corresponding parameter for Group 4 animals (L-NAME + arginine), and was expressed as a percentage of the maximal possible pharmaceutical effect. The pharmaceutical effect for the second comparative drug (levocarnitine) was calculated as the difference between the value of a body status parameter for Group 3 animals (positive control, L-NAME) and the corresponding parameter for Group 5 animals (L-NAME + levocarnitine), and was expressed as a percentage of the maximal possible pharmaceutical effect. The pharmaceutical effect for the test sample of the PC No. 1 according to the invention (210 mg on arginine basis) was calculated as the difference between the value of a body status parameter for Group 3 animals (positive control, L-NAME) and the corresponding parameter for Group 6 animals (L-NAME+ PC 528 mg), and was expressed as a percentage of the maximal possible pharmaceutical effect. The pharmaceutical effect for the test sample of the PC No. 2 according to the invention (264 mg on arginine basis) was calculated as the difference between the value of a body status parameter for Group 3 animals (positive control, L-NAME) and the corresponding parameter for Group 7 animals (L-NAME+ PC 264 mg), and was expressed as a percentage of the maximal possible pharmaceutical effect. The expected total pharmaceutical effect of co-administration of the first comparative drug and the second comparative drug was then calculated by summing the pharmaceutical effects of the first comparative drug and the second comparative drug. The difference between the pharmaceutical effects of the pharmaceutical compositions No. 1 and No. 2 according to the invention, and the estimated expected total pharmaceutical effect of the co-administration of the first comparative drug and the second comparative drug was then calculated. If the pharmaceutical effect of the pharmaceutical composition according to the invention exceeded the estimated expected total pharmaceutical effect of the co-administration of the first comparative drug and the second comparative drug, it can be concluded that there is the potentiation of arginine effect and levocarnitine effect.
For example, according to the studies, the use of two reference samples and three test samples led to an increase in mean body weight. The maximal possible pharmaceutical effect was 30. The pharmaceutical effect of arginine drug was 7, which was 23% of 30. and the pharmaceutical effect of levocarnitine drug was 5, which was 16% of 30. Accordingly, the expected total pharmaceutical effect of the two comparative drugs was 23 % + 16% = 39%.
The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (420 mg on arginine basis) was 12 g, which was 40% of 30. The pharmaceutical effect of the PC test sample No. 2 being the pharmaceutical composition according to the invention exceeded the expected total pharmaceutical effect of of two comparative drugs by 40% - 39% = 1 %.
The pharmaceutical effects were calculated for each analyzed animal body status parameter.
The increase in animal urine volume was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 27%, and the percentage of the pharmaceutical effect for levocarnitine drug was 9%. The estimated expected total pharmaceutical effect of two comparative drugs was 27% + 9% = 36%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 59%, which exceeded the estimated expected total pharmaceutical effect of the use of two comparative drugs on the increase of urination by 59% - 36% = 13%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 45%, which exceeds the estimated expected total pharmaceutical effect of two comparative drugs on the increase of urination by 45% - 36% = 9%.
The decrease in animal mean arterial blood pressure was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 32%, and the percentage of pharmaceutical effect for levocarnitine drug was 18.7%. The expected total pharmaceutical effect of two comparative drugs was 32% + 18.7% = 50.7%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 78%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the decrease in mean arterial blood pressure by 78% - 50.7% = 27.3%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 61.5%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the decrease in mean arterial blood pressure by 61.5% - 50.7% = 10.8%.
The decrease in the animal albumin urinary level was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 30.6%, and the percentage of pharmaceutical effect for levocarnitine drug was 6.6%. The expected total pharmaceutical effect of two comparative drugs was 30.6% + 6.6% = 37.2%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 58%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the increase in the albumin urinary level by 58% - 37.2% = 20.8%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 54%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the increase in the albumin urinary level by 54% - 37.2% = 16.8%.
The increase in the animal platelet blood level was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 30%, and the percentage of pharmaceutical effect for levocarnitine drug was 8%. The expected total pharmaceutical effect of two comparative drugs was 30% + 8% = 38%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 55%, which exceeded the expected total pharmaceutical effect of the use of two comparative drugs on the increase in the platelet level by 55% - 38% = 17%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 48%, which exceeded the estimated expected total pharmaceutical effect of two comparative drugs on the increase in the platelet level by 48% - 38% = 10%.
The increase in the mean fetal weight was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 28%, and the percentage of pharmaceutical effect for levocarnitine drug was 19%. The expected total pharmaceutical effect of two comparative drugs was 28% + 19% = 47%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 68%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the increase in the mean fetal weight by 68% - 47% = 21%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 50%, which exceeded the expected total pharmaceutical effect of the use of two comparative drugs on the increase in the mean fetal weight by 50% - 47% = 3%.
The increase in the offspring survival was observed when using each test sample and reference sample. The percentage of the pharmaceutical effect for arginine drug was 44%, and the percentage of pharmaceutical effect for levocarnitine drug was 30%. The expected total pharmaceutical effect of two comparative drugs was 44%+ 30% = 74%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 1 according to the invention (528 mg on arginine basis) was 82.5%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the increase in the offspring survival by 82.5% - 74% = 8.5%. The pharmaceutical effect of the PC test sample being the pharmaceutical composition No. 2 according to the invention (264 mg on arginine basis) was 76%, which exceeded the expected total pharmaceutical effect of two comparative drugs on the increase in the offspring survival by 76% - 74% = 2%.
Thus, the calculations for each animal body status parameter during the trial clearly show the potentiation of the simultaneous arginine effect and levocarnitine effect in the pharmaceutical composition according to the invention. Most of the analyzed parameters indicate that the most pronounced pharmaceutical effect was observed for 2 ml of the PC (528 mg on arginine basis and 200 mg of levocarnitine).
The above shows that the pharmaceutical composition according to the claimed invention is highly effective and promising for implementation into medical practice as a mean for outpatient treatment of coronary heart disease and its consequences, for the treatment and prevention of the diseases in pregnant women and fetal development during pregnancy. Additionally, it expands the range of the drugs for the treatment of coronary heart disease and for the treatment and prevention of the diseases in pregnant women and fetal development during pregnancy; improves the quality of life of the patients with coronary heart disease by reducing the frequency of the angina attacks; reduces the number of neonates with respiratory distress syndrome and syndrome of intrauterine growth restriction.
These examples only illustrate the invention, but do not limit it.

Claims

1 . A pharmaceutical composition comprising an arginine salt as active ingredient and water, wherein the pharmaceutical composition having an oral solution dosage form and comprising active ingredients and excipients, the pharmaceutical composition comprising levocarnitine and arginine salt, such as arginine aspartate, as active ingredients, and pH adjusting agent that is an acidifier, sweetening agent and preservative as excipients, in the following ratio, mg/ml: arginine aspartate 180-320 levocarnitine 50-150 pH adjusting agent that is an acidifier 1.5-6.0 sweetening agent 0.4-1 .2 preservative 0.5-2.0 water balance to 1 ml
2. The pharmaceutical composition according to claim 1 , wherein the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 240-300 levocarnitine 80-120 pH adjusting agent that is an acidifier 2.5-4.5 sweetening agent 0.6-1.0 preservative 1.0-1 .5 water balance to 1 ml
3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition comprises arginine aspartate, levocarnitine, pH adjusting agent that is an acidifier, sweetening agent, preservative, and water, in the following ratio, mg/ml: arginine aspartate 264 levocarnitine 100 pH adjusting agent that is an acidifier 3 sweetening agent 0.8 preservative 1 water balance to 1 ml
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein pH adjusting agent that is an acidifier is malic acid.
5. The pharmaceutical composition according to any one of claims 1 to 4, wherein sweetening agent is sodium saccharin.
6. The pharmaceutical composition according to any one of claims 1 to 5, wherein preservative is methyl parahydroxybenzoate and/or propyl parahydroxybenzoate.
7. The pharmaceutical composition according to any one of claims 1 to 6, wherein water is water for injections.
8. The pharmaceutical composition according to any one of claims 1 to 7, wherein density of the pharmaceutical composition is 1.1 g/ml, pH of the solution is 5-6.5, and dynamic viscosity is 2.5 sP at 20 °C.
9. A method of treating chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease in human suffering from chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease, including administering to said human a drug, wherein the drug is the pharmaceutical composition according to any one of claims 1 to 8 in an amount that is effective for the treatment of chronic coronary heart disease, and/or stable angina pectoris, and/or peripheral artery disease.
10. The method according to claim 9, wherein the pharmaceutical composition according to any one of claims 1 -8 is administered as a part of a combination therapy of chronic coronary heart disease, stable angina pectoris, and/or peripheral artery disease.
11. The method according to any one of claims 9 to 10, wherein the pharmaceutical composition according to any one of claims 1 to 8 is administered in a daily dose of 20- 40 ml.
12. A method of treating acute cerebrovascular disorders and/or chronic cerebrovascular disorders in human suffering from acute cerebrovascular disorders and/or chronic cerebrovascular disorders, including administering to said human a drug being the pharmaceutical composition according to any one of claims 1 to 8 in an amount effective for the treatment of acute cerebrovascular disorders and/or chronic cerebrovascular disorders.
13. The method according to claim 12, wherein the pharmaceutical composition according to any one of claims 1 to 8 is administered as a part of a combination therapy of acute cerebrovascular disorders and/or chronic cerebrovascular disorders.
14. The method according to any one of claims 12-13, wherein the pharmaceutical composition according to any one of items 1 -8 is administered in a daily dose of 20-40 ml.
15. A method of prevention and treatment of diseases in pregnant women and fetal development during pregnancy, such as preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction, including administering to a pregnant woman a drug being the pharmaceutical composition according to any one of claims 1 to 8 in an amount effective for the prevention and treatment of preeclampsia in pregnant women, fetal distress, and/or intrauterine growth restriction.
16. The method according to claim 15, wherein the pharmaceutical composition according to any one of claims 1 to 8 is administered in a daily dose of 20-40 ml.
17. A method of increasing exercise tolerance in human being exposed to physical load, and/or improving the human body status in asthenia syndrome, including administering to the human a drug being the pharmaceutical composition according to any one of claims 1 to 8 in an amount effective for increasing exercise tolerance, and/or improving the human body status in asthenia syndrome.
18. The method according to claim 17, wherein the human is exposed to the physical load in sports.
19. The method according to claim 17, wherein the human body status in asthenia syndrome is a condition that occurs after the human previous disease.
20. The method according to any one of claims 17 to 19, wherein the pharmaceutical composition according to any one of claims 1 to 8 is administered in a daily dose of 20- 40 ml.
PCT/IB2021/056729 2020-07-24 2021-07-26 A pharmaceutical composition for treating cardiovascular and cerebrovascular disorders,diseases in pregnant women and improving exercise tolerance WO2022018705A1 (en)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
UA?202004724 2020-07-24
UAA202004724A UA127789C2 (en) 2020-07-24 2020-07-24 PHARMACEUTICAL COMPOSITION, METHOD OF INCREASING TOLERANCE TO PHYSICAL EXERCISES IN HUMANS AND IMPROVING HUMAN CONDITION WITH ASTHENY SYNDROME
UAU202004795 2020-07-27
UAU202004792 2020-07-27
UAU202004797 2020-07-27
UAU202004800 2020-07-27
UAU202004797 2020-07-27
UAU202004794 2020-07-27
UAU202004794 2020-07-27
UAU202004795 2020-07-27
UAU202004792 2020-07-27
UAU202004800 2020-07-27

Publications (1)

Publication Number Publication Date
WO2022018705A1 true WO2022018705A1 (en) 2022-01-27

Family

ID=79730026

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2021/056729 WO2022018705A1 (en) 2020-07-24 2021-07-26 A pharmaceutical composition for treating cardiovascular and cerebrovascular disorders,diseases in pregnant women and improving exercise tolerance

Country Status (1)

Country Link
WO (1) WO2022018705A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0059775B1 (en) * 1980-06-09 1984-09-19 Leopold &amp; Co. Chem. pharm. Fabrik Gesellschaft m.b.H. Infusion solution for the protection of the liver and the improvement of its functioning, and process for its production
DE10128934A1 (en) * 2001-06-18 2003-02-06 Bernhard Sibbe Well tolerated, natural product-based composition for prophylaxis of angina pectoris, containing specific aminoacids, vitamins and/or peptides, preferably L-arginine, L-carnitine, L-lysine and vitamin C
UA44055U (en) 2009-06-05 2009-09-10 Одесский Государственный Медицинский Университет Method for treating antenatal distress of fetus
RU2463045C1 (en) 2011-05-12 2012-10-10 Общество с ограниченной ответственностью "МЕЖДУНАРОДНЫЙ ЦЕНТР КЛИНИЧЕСКОЙ ЛИМФОЛОГИИ" Method of treating cerebrovascular diseases and asthenic syndrome
US8389483B2 (en) 2003-06-13 2013-03-05 Sumathi Paturu Intrauterine fetal growth restriction—the treatment modalities for clinical research, and the biochemical rationale
WO2017085642A1 (en) * 2015-11-17 2017-05-26 Товарыство З Обмэжэною Видповидальнистю Мэдычный Центр "Мтк" Pharmaceutical composition
RU2642673C1 (en) 2017-06-27 2018-01-25 Ольга Александровна Логинова Composition for increasing operability and physical durability

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0059775B1 (en) * 1980-06-09 1984-09-19 Leopold &amp; Co. Chem. pharm. Fabrik Gesellschaft m.b.H. Infusion solution for the protection of the liver and the improvement of its functioning, and process for its production
DE10128934A1 (en) * 2001-06-18 2003-02-06 Bernhard Sibbe Well tolerated, natural product-based composition for prophylaxis of angina pectoris, containing specific aminoacids, vitamins and/or peptides, preferably L-arginine, L-carnitine, L-lysine and vitamin C
US8389483B2 (en) 2003-06-13 2013-03-05 Sumathi Paturu Intrauterine fetal growth restriction—the treatment modalities for clinical research, and the biochemical rationale
UA44055U (en) 2009-06-05 2009-09-10 Одесский Государственный Медицинский Университет Method for treating antenatal distress of fetus
RU2463045C1 (en) 2011-05-12 2012-10-10 Общество с ограниченной ответственностью "МЕЖДУНАРОДНЫЙ ЦЕНТР КЛИНИЧЕСКОЙ ЛИМФОЛОГИИ" Method of treating cerebrovascular diseases and asthenic syndrome
WO2017085642A1 (en) * 2015-11-17 2017-05-26 Товарыство З Обмэжэною Видповидальнистю Мэдычный Центр "Мтк" Pharmaceutical composition
RU2642673C1 (en) 2017-06-27 2018-01-25 Ольга Александровна Логинова Composition for increasing operability and physical durability

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
" DIAGNOSIS, EVALUATION AND MANAGEMENT OF HYPERTENSIVE DISORDERS IN PREGNANCY: BASIC CONCLUSIONS", CLINICAL PRACTICAL GUIDE OF THE SOCIETY OF OBSTETRICIANS AND GYNECOLOGISTS OF CANADA, 2014
"Fetal Distress during Pregnancy and Childbirth", CLINICAL PROTOCOL ON OBSTETRICS, no. 900, 27 December 2006 (2006-12-27)
"Preclinical studies of drugs (guidelines", 2001, PUBLISHING HOUSE ''AVITSENA, pages: 527
BURTSCHER MARTIN ET AL: "The prolonged intake of L-arginine-L-aspartate reduces blood lactate accumulation and oxygen consumption during submaximal exercise", JOURNAL OF SPORTS SCIENCE & MEDICINE, 1 September 2005 (2005-09-01), Turkey, pages 314 - 322, XP055858629, Retrieved from the Internet <URL:https://www.jssm.org/volume04/iss3/cap/jssm-04-314.pdf> [retrieved on 20211108] *
DIAGNOSIS AND TREATMENT CLINICAL PROTOCOL FOR CHRONIC CEREBRAL ISCHEMIA
G.I. STEPANYUKO.P. DRACHUKS.A. OLIYNYKO.G. YUSHKOVSKAYA: "Search for effective antihypoxants among derivatives of succinic acid", SPORTS MEDICINE, vol. 4, no. 17, 2010, pages 56 - 59
I.P. ULANOVAK.K. SIDOROVA.I. KHALEPO: "Meditsyna", 1968, article "On the question of taking into account the body surface of experimental animals in toxicological research", pages: 365 c - 25
KARLIC HEIDRUN ET AL: "Supplementation of l-carnitine in athletes: does it make sense?", NUTRITION, vol. 20, no. 7-8, 1 July 2004 (2004-07-01), AMSTERDAM, NL, pages 709 - 715, XP055858632, ISSN: 0899-9007, DOI: 10.1016/j.nut.2004.04.003 *
KELLER U ET AL: "Carnitine status of pregnant women: effect of carnitine supplementation and correlation between iron status and plasma carnitine concentration", EUROPEAN JOURNAL OF CLINICAL NUTRITION, vol. 63, no. 9, 1 September 2009 (2009-09-01), GB, pages 1098 - 1105, XP055858636, ISSN: 0954-3007, Retrieved from the Internet <URL:https://www.nature.com/articles/ejcn200936.pdf> DOI: 10.1038/ejcn.2009.36 *
KURYATA OLEXANDR ET AL: "Effect of Combination of L-Arginine and L-Carnitine on Serum AGEs Level, Kidney and Endothelial Function in Patients with Chronic Heart Fa", JOURNAL OF NUTRITIONAL THERAPEUTICS, vol. 7, no. 3, 16 August 2018 (2018-08-16), pages 77 - 83, XP055857274, Retrieved from the Internet <URL:http://dx.doi.org/10.6000/1929-5634.2018.07.03.2> DOI: 10.6000/1929-5634.2018.07.03.2 *
LASSALA ARANTZATZU ET AL: "Parenteral Administration of L-Arginine Enhances Fetal Survival and Growth in Sheep Carrying Multiple Fetuses", THE JOURNAL OF NUTRITION, vol. 141, no. 5, 1 May 2011 (2011-05-01), US, pages 849 - 855, XP055858638, ISSN: 0022-3166, Retrieved from the Internet <URL:https://academic.oup.com/jn/article-pdf/141/5/849/29812622/849.pdf> DOI: 10.3945/jn.111.138172 *
N.G. STEPANYUKV.V. YUSHKOVAV.B. MUDRYTSKY: "Comparative evaluation of antihypoxic properties of cordarone, benzofurocaine, vinborone and emoxipin in the experiment", BULLETIN OF VINNYTSIA NATIONAL MEDICAL UNIVERSITY, vol. 11, no. 2/1, 2007, pages 576 - 579
OYAMA MUNENORI ET AL: "Neuroimaging pattern and pathophysiology of cerebellar stroke-like lesions in MELAS with m.3243A>G mutation: a case report", BMC NEUROLOGY, vol. 20, no. 1, 1 May 2020 (2020-05-01), pages 1 - 6, XP055850257, Retrieved from the Internet <URL:https://bmcneurol.biomedcentral.com/track/pdf/10.1186/s12883-020-01748-7.pdf> DOI: 10.1186/s12883-020-01748-7 *
REPRODUCTIVE ENDOCRINOLOGY, vol. 4, no. 18, 2014, pages 74 - 85
V.M.KOVALENKO, O.V. STEFANOV, Y.M. MAKSIMOV, I.M. TRAKHTENBERG: "Experimental study of the toxic effects of potential drugs", METHODICAL RECOMMENDATIONS - KYIV, 2000, pages 74 - 97

Similar Documents

Publication Publication Date Title
Stathatos et al. The controversy of the treatment of critically ill patients with thyroid hormone
Christodoulou et al. Early treatment of Menkes disease with parenteral Cooper‐Histidine: Long‐term follow‐up of four treated patients
Ramirez et al. Leukocyte ascorbic acid and its relationship to coronary artery disease in man
Ewer et al. The role of platelet activating factor in a neonatal piglet model of necrotising enterocolitis
US10925890B2 (en) Use of dextran sulfate
JPH06503359A (en) Use of AICA riboside compounds for the treatment and prevention of tissue damage resulting from decreased blood flow
WO2022018705A1 (en) A pharmaceutical composition for treating cardiovascular and cerebrovascular disorders,diseases in pregnant women and improving exercise tolerance
CA2507429C (en) Use of carnitines for the prevention and/or treatment of disorders caused by the andropause
Fernandez-Segoviano et al. Pulmonary vascular lesions in the toxic oil syndrome in Spain.
UA127789C2 (en) PHARMACEUTICAL COMPOSITION, METHOD OF INCREASING TOLERANCE TO PHYSICAL EXERCISES IN HUMANS AND IMPROVING HUMAN CONDITION WITH ASTHENY SYNDROME
UA146440U (en) PHARMACEUTICAL COMPOSITION
SCOTT et al. The Jarisch-Herxheimer phenomenon in late syphilis: Probable fatal reactions to penicillin
RATE Fetal heart rate monitoring
CN113143899A (en) Application of capsaicin in preparing medicine for treating ulcerative colitis
UA147644U (en) METHOD OF TREATMENT OF CHRONIC ISCHEMIC HEART DISEASE OR STABLE VOLTAGE ANGIA OR ATHEROSCLEROSIS OF PERIPHERAL VESSELS IN HUMAN
US7776913B2 (en) Carnitines for treating or preventing disorders caused by andropause
RU2763761C1 (en) Hepatoprotective humic agent
Porter et al. Primary pulmonary hypertension occurring in twins
Brebner Dissecting aneurysm of the aorta with renal complications
UA146441U (en) METHOD OF TREATMENT OF ACUTE DISORDERS OF CEREBRAL CIRCULATION AND CHRONIC DISORDERS OF CEREBRAL CIRCULATION IN HUMAN
Roh Subcapsular hematoma in fatty liver of pregnancy
CN109481446B (en) Composition for improving body functions of pregnant woman, lying-in woman and lactating mother and fetus and preparation process
UA146444U (en) METHOD OF INCREASING TOLERANCE TO EXERCISE IN HUMAN BEINGS AND IMPROVING THE CONDITION OF HUMANS WITH ASTHENIA SYNDROME
UA146442U (en) METHOD OF PREVENTION AND TREATMENT OF SUCH DISEASES OF PREGNANT WOMEN AND FETAL DEVELOPMENT DURING PREGNANCY, AS PREECLAMPSIA OF PREGNANT WOMEN
Singh et al. An Ayurvedic Approach to Stree Vandhyatva (Anovulation) Utilizing Uttar Basti

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21758756

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21758756

Country of ref document: EP

Kind code of ref document: A1