UA58565C2 - Cytoflavin injectional drug possessing cytoprotective properties - Google Patents

Abstract

The invention relates to the medicine, in particular to the injectional drug possessing cytoprotective properties. The composition represents the mixture of natural metabolites and co-enzymes dissolved in the solvent, the natural metabolites comprising succinic acid and riboxin (inosine) and co-enzymes comprising nicotine amide and sodium riboflavin-5-mononucleotide. The ingredients are dissolved in the water for injections. In addition the composition contains sodium hydroxide and N-methylglucamine.

Description

The study of the claimed medicinal product was carried out in comparison with the prototype.

Experiment 1. Study of cardioprotective effect on the model of antiarrhythmic activity

Considering that arrhythmias are one of the first indicators of the development of myocardial cell pathology in various cardiovascular diseases, which are accompanied by damage to myocardial cells, such as acute myocardial infarction and coronary heart disease, in order to evaluate the cytoprotective effect of "Ditoflavin" on myocardial cells, it seemed necessary to study and evaluate primarily the antiarrhythmic activity of the proposed drug.

The experiment was conducted on linear white SBA mice, which were divided into five groups of 25 animals each.

Experimental heart arrhythmia was modeled by the generally accepted method - subcutaneous administration of alkaloid aconitine in a single dose of 200 μg/kg of animal weight and propranol subcutaneously in a single dose of 4 Ω/kg.

The introduction of the aconitine alkaloid into the body causes a steady increase in the sodium permeability of cardiomyocyte membranes (Orlov B.N. Poisonous animals and plants of the USSR. -- M. -- Higher School, 1990, p. 192 - 194).

Administration of propranol causes bradycardia of the heart due to the blockade of p-adrenoceptors in the myocardium and changes in the intracellular ratio of potassium and sodium ions (UUoikipd Stor op AgpiNtiavz-Sigsshinyop. - 1991, M.84, Me 4, r.1831 - 1851). "Cytoflavin", prototype and control - 0.995 isotonic solution of sodium chloride was injected intravenously into the tail vein in a single dose of 1.2 ml/kg of animal weight, once five minutes before the administration of aconitine or propranol. Recording of the electrocardiogram in mice was carried out at the beginning of the experiment, and then - 10, 30 and 60 minutes after the administration of the drugs.

To determine the time of occurrence and duration of extrasystole during the experiment, the electrocardiogram was monitored for 60 minutes.

The obtained experimental data were processed statistically using the Student's test at Р « 0.05.

Data on testing the antiarrhythmic activity of the drugs are shown in Table 1.

Table 1

Effect of "Cytoflavin" solution and prototype on aconitine and propranolol arrhythmia in mice

The drug, which Number of mice with Latent period Duration Number ' -. | extrasystole, | to extrasystole, and - used in the experiment on / KhVyl. extrasystoles, waves extrasystoles itoflavin itoflavin

Note: 7 - the probability of differences at Р « 0.05.

When simulating aconitine arrhythmia, administration of aconitine caused polygonal extrasystole in 100905 animals (see Table 1).

With the introduction of "Cytoflavin" in a dose of 1.2 ml/kg in 5 minutes. before the introduction of aconitine, extrasystole developed only in 25956 mice, while the period of extrasystole decreased by 3595, namely, the number of ecstasystole decreased by 2.4 times.

Thus, "Cytoflavin" showed a pronounced cytoprotective antiarrhythmic effect on myocardial cells in the model of antinitin arrhythmia. Taking into account the mechanism of action of the alkaloid aconitine, the mechanism of the protective action of "Cytoflavin" is due to the stabilization of the membranes of cardiomyocytes cells and a decrease in their permeability to sodium ions.

On the contrary, when the prototype solution is introduced in 5 min. before the introduction of aconitine, a completely opposite picture developed: extrasystoles were observed in 7,595 mice, and the latent period, duration, and total number of extrasystoles did not change at all compared to aconitine.

Thus, the prototype showed weak protective antiarrhythmic activity on myocardial cells in the model of antinitin arrhythmia and did not have a stabilizing effect on cardiomyocyte cells in this model.

In arrhythmia caused by the administration of propranol, polytonic extrasystole was also induced in 10,095 mice.

With the preliminary introduction of "Ditoflavin" in a dose of 1.2 ml/kg into the tail vein in 5 minutes. before the introduction of propanol, extrasystole developed in 4095 animals, while the latent period of extrasystole decreased by 2995, and the total number of extrasystole decreased by 3.2 times. The obtained data indicate a high cytoprotective effect of "Cytoflavin" on myocardial cells in the model of arrhythmia caused by the administration of propranol. The mechanism of the protective action is due to the normalization of the biochemistry of sodium/potassium exchange through the cell membrane.

When the prototype solution was administered, polytomous extrasystole was detected in 5095 mice, the latent period of extrasystole practically did not change, and the average number of extrasystole decreased only 1.7 times (Table 1).

This indicates a moderate protective effect of the prototype on myocardial cells.

Thus, on the propranolol arrhythmia model, "Ditoflavin" also exhibits a pronounced cytoprotective antiarrhythmic effect, while the prototype solution has only a weak protective antiarrhythmic effect.

This difference is explained by the creation of an optimal combination of natural metabolites - succinic acid (an energy component), riboxin (a substrate for ATP synthesis) and coenzymes-vitamins to obtain a cytoprotective effect. As a result of the acceleration of the assimilation of metabolites by the cells of the body, there is an acceleration of biochemical processes inside the cell and a protective effect is exerted on the cells of the myocardium - cardiomyocytes.

Experiment 2. Study of the cardioprotective effect on a model of myocardial ischemia

Ischemia (lack of oxygen) in the cells of the myocardium leads to a violation of biochemical processes inside cardiomyocytes, which are associated with energy consumption, followed by necrosis (destruction) of the cell membrane.

The study of the anti-ischemic activity of "CDitoflavin" in comparison with the prototype solution was carried out on white linear rats of the Vistar breed. All animals were divided into three groups of 25 animals.

For research, we used a commonly accepted model of ischemia caused by occlusion (surgical reduction of the vessel diameter - ligation) of the left coronary artery (M/ipviom/ E. MeiShiovz yug Pe adeyesiiop apa azzevztepi oi apiapnuytis asiimyu. / Rpappasoiodu apa (Shegaru. -- 1984. M.84, p.401 - 433).

This model best illustrates the development of acute myocardial infarction in humans.

Rats were anesthetized by intraperitoneal administration of ethaminal sodium in a dose of 5 Ω/kg, and the electrocardiogram was fixed and registered.

Rats with a rhythm disorder were removed from the experiment. A total of 36 rats were selected, divided into three groups of 12 animals each.

In the remaining animals, femoral vein and femoral artery were catheterized. Then, during artificial ventilation, the lungs of the rats were opened and the descending branch of the left coronary artery was ligated at the lower edge of the left atrial appendage. In all animals, mean arterial pressure in the femoral artery was recorded using a mercury manometer, and electrocardiogram monitoring was performed for 30 minutes to detect early gastric arrhythmias that develop on the background of myocardial ischemia.

The first group of animals was injected with "Cytoflavin" in the tail vein at a dose of 1.2 ml/kg five minutes after ligation.

The second group was injected with the prototype solution at a dose of 1.2 ml/kg.

The third control group was injected into the tail vein with 0.995 physiological sodium chloride solution at a dose of 1.2 ml/kg.

Artificial ventilation of the lungs was continued throughout the experiment in order to prevent respiratory arrest caused by postoperative pneumothorax. According to electrocardiogram data, heart rate, mean arterial pressure, frequency of occurrence and total duration of periods of fibrillation and gastric tachycardia were evaluated.

The effectiveness of the drugs on general protective cytoprotective effect on body cells was evaluated by lethality in each group.

Throughout the experiment, blood pressure in the femoral vein was recorded in all animals, and an electrocardiogram was recorded 1, 5, 15, 30, 45, and 60 minutes after the start of the introduction of solutions into the tail vein.

Statistical significance was assessed using the Student's test. The summarized data on the experiment are shown in Table 2.

Table 2

Anti-ischemic effect of "Cytoflavin"

TVKVR psapist o Frequency of occurrence P duration. Ge) 1,

Prototype | 221157 | 100 | 90 6077 | oz (Control | 3595247 | 100 | 127237 | 86 2 4 4B2zh | 2854

Note: 7 - the probability of differences at Р « 0.05.

The results of the study of the anti-ischemic activity of "CDitoflavin" showed that in case of heart ischemia, the drug probably reduces the number of ventricular extrasystoles by 2.62 times compared to the control and by 1.6 times compared to the prototype.

Tachycardia duration was reduced 3-fold compared to control and 2.1-fold compared to prototype, although the frequency of tachycardia was not reduced in all cases. "Cytoflavin" also reliably reduced the frequency of ventricular fibrillation by 2.2 times compared to the control and 1.5 times compared to the prototype, although it practically did not affect the duration of the fibrillation time. The obtained data indicate a high cytoprotective activity of "Ditoflavin" on myocardial cells under conditions of ischemia.

The data on the survival of animals in the experimental conditions also testify to the high cytoprotective activity of "Cytoflavin", with the introduction of which 10095 (12 rats) survived after the ligation operation, with the introduction of the prototype - 5095 (6 rats), and with the introduction of the control physiological solution - only 2595 (3 rats) (see Table 3).

Table C

The influence of "Cytoflavin" on the survival of animals in the conditions of the occurrence of early postischemic arrhythmias in reparit rats

Prototype ІІ 777777771711112 І7777771717171717611111111117111111111150111

Monitoring of mean arterial pressure and heart rate (see Table 4) showed that "Cytoflavin" restored mean arterial pressure faster than the prototype in the early period after occlusion, with virtually no effect on heart rate. This effect indicates a more pronounced compensatory effect of "Cytoflavin" in comparison with the control and with the prototype in the restoration of biochemical processes occurring in cells during ischemia of cardiomyocytes, and, therefore, about the general cytoprotective effect on the myocardium.

Table 4

The effect of "Cytoflavin" on the mean arterial pressure (SBP) and heart rate (HR) under the conditions of the occurrence of early postischemic arrhythmias and retria Go 71 5 Y 15 | from | 45 | 60 "I

SAT At 5 before primary school

CheS in 95 to

Note: 7 - probability of differences at Р« 0.05

Experiment 3. Study of the hepatoprotective and repoprotective activity of "Ditoflavin" in ethylene glycol poisoning

The well-known model of toxic hepatitis and nephroso-nephritis - 1,2-ethylene glycol poisoning - was used to simulate the affected cells of the liver and kidneys. (Reproduction of diseases in animals for experimental and therapeutic research. Edited by N.V. Lazareva. Moscow: "Medgiz", 1964, 3926.).

In the experiment, 6 experimental groups of animals of 12 female rats each were formed: A - intact animals, which were injected with physiological solution; B - intact animals injected with "Cytoflavin"; B - intact animals that were injected with the prototype; G - animals with ethylene glycol intoxication without treatment; D - animals with intoxication and treatment with "Cytoflavin"; E - animals with ethylene glycol intoxication and prototype treatment.

Ethylene glycol was administered once intragastrically through an atraumatic probe in a dose of 1.5 g/kg. "Cytoflavin", prototype and physiological solution in the control were injected into the tail vein of rats once a day for five days at a dose of 1.2 ml/kg. Biological studies were carried out according to standard methods (Stalnaya

I.D., Harishvili T.G. Modern methods in biochemistry. M.: Medicine, 1978. 95 p., Clinical evaluation of laboratory tests / Ed. WELL. Tytsa, M.: Medicine, 1986, 480 p.). The obtained experimental data were processed by Student-Fisher statistical methods.

The clinical picture in the group of rats that received ethylene glycol without treatment was characterized by hypodynamism, inhibition of reactions, disheveled fur, and animal unkemptness. On the 2nd day, they developed macrohematuria, and from the 4th day - oliguria. Biochemical and histological analysis of the liver and kidneys confirmed the development of kidney and liver damage - nephroso-nephritis and toxic hepatitis.

Ethylene glycol intoxication disturbed all functions of the liver: protein-synthesizing and pigmentary, which was accompanied by biochemical signs of cytolysis.

All animals, without exception, developed pronounced symptoms of kidney damage: erythrocytes, protein, cylinders, calcium oxalate crystals, renal epithelial cells were determined in the urine analysis. All functions of the kidneys - excretory, excretory and secretory - suffered, and the products of nitrogenous metabolism and cytolysis indicators increased in the blood.

Morphological examination revealed degenerative changes and small hemorrhages in the parenchyma of the kidneys, fatty dystrophy of the neuroepithelium was observed, fat droplets were visible in the cytoplasm of convoluted tubule cells.

The hepatoprotective effectiveness of "Ditoflavin" in comparison with the prototype was determined by the clinical picture, the content of total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, total protein, serum lipids, glycogen, glutathione in the liver and a loading sample - hexanal test.

Experimental data on the hepatoprotective effect of "Ditoflavin" are shown in Table 5.

Table 5

Hepatoprotective effect of "Cytoflavin" in comparison with the prototype

A B in D d E physical. itoflavin solution Prototype Without treatment itoflavin Prototype

Total 64 2 65 25 64 16 40 w 4" 58 xx 46 w 57 protein, g/l

AST, mkat/l 0.60 - 0.05 0.60 -0.06 | 0.60 - 0.06 0.990.127 | 0.73 0.067 | 0.88 0.127 0.69 and 0.11 0692012 | 0692011 | 2.65 20.247 | 0.73 50.04 | 0.96 0.147 0.74 50.12 0.750011 | 0.732012 | 1,990,207 | 0.72 0.047 | 0.96 011 1.46 of 0.04 148-003 | 1.43 0.12 5.77 50267 1.56 50.127 | 2.88 5 0.137

ALT, μcat/l 0.20 0.03 0.20 0.02 | 0.25 50.04 1.68 5 0.117 0.32 0.067 | 0.52 - 0.087 160 of 5 155 55 170 of 12 70-10" 130 of 107 100 of 107 liver, mg 9o

Glycogen same same and o 2500 of 100 2400 of 90 2460 of 110 850 of 80 2000 of 150 1800 of 200 liver, mg 9o 25.0 1.5 26.0 w 1.8 24.0 with 1.6 46.5 62.57 27.0 51.57 35.2 1.07 dream, min.

General 3003 ЗО жо 32102 6.8 ж 04 General | dovo bilirubin 77777771 71717111 ntactntvaryny.i////// | Research animals.//7/7/

Note: 7 - the probability of differences at Р « 0.05

AST - aspartate aminotransferase

Alkaline phosphatase

KF - creatine phosphate

ALT - alanine aminotransferase

Experiments showed that the administration of "Cytoflavin" and the prototype to healthy animals (groups A, B, B) did not cause violations of liver functions and behavioral reactions of rats.

The introduction of ethylene glycol (group G) was accompanied by serious impairment of all liver functions.

A decrease in total protein and lipids and glycogen reserves in the liver indicates a violation of the protein-synthesizing function of the liver.

An increase in the level of aminotransferases, alkaline phosphatase, bilirubin and thymon test indicates a significant destruction of liver cells - hepatocytes and a violation of pigment function.

An increase in the time of hexane sleep, a decrease in the level of renewable glutathione and creatine phosphate shows a decrease in the detoxifying function of the liver.

The use of "Cytoflavin" for the treatment of liver damage caused by ethylene glycol (group D) showed a probable normalization of all functions of the protein-synthesizing liver (total protein, lipids, glycogen) to 80 - 9195 of normal, pigment (bilirubin, aminotransferases, alkaline phosphate, thymol test) to 62 - 9795 from the norm, detoxifying (hexenal sleep, glutathione, creatine phosphate) to the level of 82 - 9795 from the norm (group A).

Treatment with the prototype (group E) also had a positive effect after ethylene glycol poisoning, however, the prototype restored the protein-synthesizing function of the liver by 60 - 9095, pigment - by 68 - 7195, and detoxification - only by 50 - 7095.

Thus, "Cytoflavin" has a pronounced cytoprotective effect on liver cells - hepatocytes in case of ethylene glycol poisoning (see Table 5).

The effectiveness of "Cytoflavin" as a renoprotector (protection of kidney cells) was evaluated by a set of biochemical indicators in comparison with the norm, a control group and a prototype: relative kidney mass, daily diuresis, urine protein, urine chlorides, urine sugar, urea, creatinine, lactate dehydrogenase.

Experimental data on the renoprotective effect of "Ditoflavin" are shown in Table 6.

Table 6

Renoprotective effect of "Cytoflavin" in comparison with the prototype, M z m

Experimental groups of physics. solution | cytoflavin Prototype treatment Cytoflavin Prototype 184 6 183 57 188 x 11 154 5117 181 w 11 160 w 11 7.9 250.9 7.812 7.7 1.0 11.2 51.57 8.4 0.7 10.8 1, 1

Daily diuresis, ml 3.520.3 3.5 50.4 1.9 0.37 2.804" 1.9 20.5 440.5 4.504 4.504 12.4 1.37 6.0 0.67 8.7 0.87 4.0 20.3 3804 4.0 20.3 1.2 0.37 3.3 0.3 2.5 z 0.Ah 1.5 20.5 14203 16201 4.2 0 ...

oh 0.91 x w 1.00 w mmol/h/l 77717111 ntactntvariniid///// | research animals.//

Note: 7 - probable differences from intact animals at P "0.05

Experimental data showed that the introduction of both "Cytoflavin" and the prototype to healthy animals (groups A, B, B) did not have a negative effect on the functional activity of the kidneys.

Under conditions of pathology - poisoning with ethylene glycol (group G), the weight of the kidneys increased in rats, which indicates edema. A probable increase in the content of protein, chlorides, sugar, urea in the urine and an increase in the density of urine indicate a violation of the excretory and secretory function of the kidneys. A sharp decrease in the daily volume of urine against the background of an increase in the level of creatine and the enzyme lactate dehydrogenase indicates a violation of the cells of the renal tubules, the development of nephroso-nephritis and, as a result, anuria.

The use of "Cytoflavin" (group D) for the treatment of kidney damage caused by ethylene glycol showed the restoration of the functional activity of the kidneys according to all indicators: swelling decreased sharply (kidney mass became 9495 of the norm), excretory function (diuresis, urine density, creatinine) recovered by 62 - 10095, secretory function (content of urine protein, sugar, chlorides, urea, lactate dehydrogenase level) improved to 62 - 8295 from normal.

The use of the prototype (group E) for treatment also had a positive therapeutic effect in nephronephritis, but it was less effective than "Ditoflavin" according to various indicators: the kidney mass was 73905 from normal, the excretory function was restored by 51 - 9395, and the secretory function by 45 - 6095 from the norm (see

Table 6).

The analysis of histological data in animals of groups D and E also showed a decrease in dystrophic changes in kidney and liver tissue, which was more pronounced in group D when treated with "TsDitoflavin".

Thus, "Cytoflavin" exhibits a pronounced cytoprotective effect on nephroepithelial cells and hepatocytes, which exceeds the effect of the prototype in experimental hepatitis and nephroso-nephritis caused by the use of ethylene glycol.

Thus, the study of the biological activity of the drug showed that it has a cytoprotective effect, that is, it has a protective effect on the cells of the heart and liver, normalizing the work of these organs in various pathologies.

To create a stable dosage form, the necessary amounts of M-methylglucamine and sodium hydroxide were experimentally selected. These components are not active components of the dosage form, but provide the necessary physicochemical parameters.

Experiment 4. Study of the influence of the content of M-methylglucamine and sodium hydroxide on the stability of "Ditoflavin"

Taking into account the complex multicomponent composition of "Cytoflavin" and the presence in it of M-nucleoside - riboxin (inosine) and riboflavin mononucleotide, which are easily hydrolyzed in aqueous solutions by the M-glycosidic bond in acidic and alkaline environments (Biochemist's Handbook: translation from English/ Dawson R., Zllyot D.,

Jones K. -- M. MIR, 1991, p. 71 - 88, 115), a study of the effect of the content of the stabilizer and complexing agent M-methyleneglucamine on the pH of the solution and the stability (absence of decomposition of the components) during two-year storage at room temperature of the aqueous solution. "Cytoflavin" is a drug intended for intravenous administration. The solutions introduced into the bloodstream must be physiologically compatible with the physical and chemical parameters of the blood, in particular with the pH.

The introduction of acidic and/or alkaline solutions into the bloodstream is inadmissible, because it can lead to the formation of blood clots, damage to the walls of veins, excretion of medicinal substances into the sediment, and blockage of large and small blood vessels (I.L. Muravyov. -- Technology of drugs, vol. 2, M. -- Medicine, -- 1980, -- p. 622 - 689, State Pharmacology of the USSR, M. -- Medicine, 1990. -- p. 140).

The influence of the amount of sodium hydroxide on the pH value of the drug solution and the properties of the dosage form was investigated (Table 7).

Table 7

The influence of the sodium hydroxide content on the pH of the "Cytoflavin" solution

Variant and o Note hydroxide, 9o solution of the resulting solution | I'm a fan of Tsfevsheshnnian

And succinic acid for use 517 | with fe recoerennn of succinic acid for the use of e| ke Sh| 5 days of intravenous administration " " value of intravenous administration

SHE IN: DESTROY en " " the value of intravenous administration

71111111 | intravenous administration

The conducted studies showed that the optimum for this dosage form is the content of sodium hydroxide in the amount of 3.3 - 3.795. The experimental data of the experiment are given in Table 8.

Table 8

Effect of M-methylglucamine content on pH and stability of "Cytoflavin"

Variant Contents M- Stability characteristics Note p methylglucamine, 90 dosage form p

А Precipitates during storage - Acid hydrolysis does not occur, suitable for use 50 Precipitates during storage - Acid hydrolysis does not occur, suitable for use 100 The solution is stable during storage, Not suitable for " but has an acidic pH value of intravenous administration

The solution is stable during storage. Suitable for use

And ДоОЙ000017,93000000000 | The solution is stable during storage. Suitable for use

Е 20.0 The solution is stable during storage, Not suitable for " but has an alkaline pH value for intravenous administration, which is 250. The solution turns black during storage - For use, alkaline hydrolysis does not occur. Suitable

The conducted studies showed that the content is optimal for creating a stable medicinal product

M-methylglucamine in "Citaflavin" from 15.7 to 17.3905 - variants G, D (Table 8) and sodium hydroxide content from 3.3 to 3.790 - variants G, D (Table 7).

Experiment 5. Determination of acute toxicity of "Cytoflavin"

Determination of the acute toxicity of "CDitoflavin" was carried out on linear white male mice of SBA in groups of animals.

The drug was injected into the tail vein in increasing doses according to Wilkonson-Litchfield and the LdBvo indicator was determined - the dose of the drug that causes the death of 5095 animals.

The results of the study are shown in Table 9.

Table 9

Acute toxicity of "Ditoflavin"

Dose of "Cytoflavin", mg/kg 1000 1260 1580 2000 oVizhilomyshes.//-/-:/ | 7710 | 77767174 1110710

0 mice died 4 | 6 | yuyu | at

The results of the experiment showed that the drug "Cytoflavin" belongs to the low-toxic compounds: LdDvo is 1146 out of 124mg/kg.

Application No. 98708522 for the registration of a trademark in the Russian Federation was submitted for the word "Cytoflavin", date of submission 05/19/98.