RU2336904C1 - Radiopaque agent - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns new medicinal preparations, be more exact to agents of contrasting of internal organs, and can be used in radial diagnostics at an angiography, computer tomography and a lymphography. The radiopaque agent (ROA) represents an oil contrast agent consisting from diiodoethyl aether of linoleic acid with addition of 5% of aether aethylicus of initial linoleic acid. The iodine maintenance in it makes 48%, its density 1.18 (at 20°C), viscosity 15.2 cP (at 20°C), and providing good radiopaque properties.

EFFECT: creation of the radiopaque agent possessing moderate allergenic action.

Description

The invention relates to new drugs, namely to means of contrasting internal organs, and can find application in radiation diagnostics (LD) for angiography, computed tomography (CT) and lymphography.

It is known that the possibilities of LD significantly increase with the use of radiopaque agents (RKS). At the same time, it is necessary that RKS combine the maximum ability to absorb X-rays, pharmacological inertness, chemical stability, organ-specificity, low viscosity, minimal osmotic activity and toxicity, and rapid elimination from the body.

The most acceptable component of such agents is iodine due to its high density, relatively low toxicity, and strong covalent binding in an organic molecule.

These requirements are most met by iodized oil-based RKS, since they

- are not salts and do not dissociate into ions in the aquatic environment;

- have a long-lasting effect;

- easily assimilated by the body;

- due to the strong binding of iodine in the PKC molecule, they do not cause poisoning of the body with free iodine;

- characterized by low absorption by the walls of blood vessels, which reduces their toxic effect on the body.

In recent years, X-ray contrast research methods using oil contrast agents have been widely used in experimental and clinical medicine. The traditional areas of their application are lymphography, sialography, X-ray endovascular occlusion and chemoembolization of malignant neoplasms of various localizations, etc.

However, the number of oil-based CSWs suitable for these purposes is currently insufficient. The main task in the development of new iodine-containing radiopaque preparations is to reduce toxicity, viscosity, improve the quality of imaging and reduce the likelihood of complications that may arise when using them.

Iodized oils have been known since 1896 when an iodine sesame oil called iodipine was obtained. In 1901, iodized poppy seed oil, called lipiodol, was made. About 20 similar preparations are known that are obtained by the iodination of vegetable oils: refined sunflower oil (iodolipol), colic (campiodol), the aforementioned sesame (iodipine) and poppy (lipiodol), and many others. Since 1961, the widespread use of oil-based RKS for direct clinical lymphography began, with lipiodol being primarily used.

Lipiodol is obtained from poppy oil, which is a mixture of stearic, palmitic, oleic and linoleic acids. The oil is iodized and then thoroughly cleaned for a long time. The iodine content in lipiodol is 40%, beats. its weight is 1.350, viscosity is 4.3 P.

A serious drawback of lipiodol is, first of all, the high viscosity and the risk of fat embolism, as well as its complex composition, since it is a mixture of iodinated and non-iodized products, which makes it difficult to identify.

The use of lipiodol and other oil-based contrast agents causes a number of complications depending on the area of their application (high viscosity of oil-based PKCs, as mentioned above, can cause fat embolism, the most dangerous complication, allergic reactions are observed in some patients, oil-based PKCs undergo fragmentation, which causes intermittent image of the lymphatic vessels). In this regard, the efforts of researchers are aimed at creating CSFs with lower viscosity, suitable for lymphography.

An analysis of foreign literature indicates intensive research in the field of creating more advanced oil contrast preparations. In our country, the production of CSWs is absent and only imported drugs are used in clinical practice, and therefore the creation of domestic CSWs is extremely important and was the object of the present invention.

Closest to the proposed CSW is a super-liquid lipiodol (Lipiodol ultra fluid) (prototype) [C. Mayorovici et al., Pharmazie, 1956, v.4, N2, p.126-130], whose viscosity is 38 cP (at 25 ° C), beats. the weight. - 1.27 (at 25 ° C), the iodine content is 48%. X-ray contrast (densitometric density on the Hounsfield scale) - 2984. The drug is widely used for contrasting internal organs, lymphatic channel and lymph nodes.

It is obtained by iodizing refined poppy oil with gaseous hydroiodic acid. An iodinated oil with a viscosity of 4.3 P (lipiodol) is esterified with ethyl alcohol to give a less viscous product called super-liquid lipiodol (Guebert, France).

However, the use of this CSW can still lead to fat embolism, as well as to a decrease in the capillary volume of the pulmonary arteries - a dangerous side effect. A sufficiently high viscosity of the drug leads to a long injection time and requires electric injectors for its introduction. In addition, the drug does not give clear images of some groups of lymph nodes. In addition, it is a mixture of iodinated fatty acid esters and therefore difficult to identify.

The technical task of the present invention was to obtain domestic RKS, which is an easily identifiable individual compound with a lower viscosity, which makes it suitable for safe systemic use.

This problem is solved by obtaining an oil-based RKS, which is diol ethylenediamine of linoleic acid with the addition of 5% ethyl ester of linoleic acid.

Get the claimed PKC as follows.

The raw material for the production of linoleic acid diiodoethyl ester is individual linoleic acid (a commercial product), which, after distillation in vacuo, is treated with ethanol in benzene in the presence of a catalyst (sulfuric acid) with stirring for 3 hours and a temperature of 80 ° C. To increase the yield of ether during the reaction, azeotropic distillation of water is carried out to completely remove it and excess ethyl alcohol. Then the obtained ethyl ester of linoleic acid is separated (fatty layer), washed with distilled water, neutralized with saturated soda solution and washed with water again, after which it is dried with calcined sodium sulfate. The fraction with a boiling point of 160-170 ° C during vacuum distillation of the resulting reaction mass is ethyl ester of linoleic acid.

Linoleic acid ethyl ester in petroleum ether is treated with a 57% solution of hydroiodic acid in the presence of phosphoric anhydride P ₂ O ₅ at a temperature of 20 ° C, cooling the reaction mixture with ice and salt. After that, the lower layer of the reaction mixture is separated (phosphoric acid), and the upper (diiodinated linoleic acid ether in petroleum ether) is neutralized, dehalogenated, washed and dried, and then the petroleum ether is distilled off. In the obtained iodinated linoleic acid ester, 5% of the starting linoleic acid ethyl ester is added (to increase its stability). The iodine content in the finished product is 48%, its density is 1.18, the viscosity is 15.2 cP, the radiopacity is 2958 (for comparison, the viscosity of super-liquid lipiodol is 38 cP, the density is 1.27, the radiopacity is 2984, and the iodine content is 48%).

We present the results of a preclinical study of the claimed CSW.

The scope of the tests was determined by the “Guidelines for the experimental (preclinical) study of new pharmacological substances” (Ministry of Health of the Russian Federation, M., 2000, 398 pp.) And “Rules for the preclinical safety assessment of pharmacological agents Good Laboratory Practice (GLP).” Doses of the drug (in mg / kg) were calculated based on diiodoethyl ether of linoleic acid.

STUDY OF SAFETY OF THE APPLIED DRUGS

1. ACUTE TOXICITY

Acute toxicity of the drug was studied in sexually mature white mice and white Wistar rats.

In rodent experiments, animals were randomized into groups by randomization. The absence of signs of disease in animals and the homogeneity of groups (5 animals each) by sex and body weight (± 20%) were considered as a criterion for the acceptability of randomization. In white mice, the drug was administered intraperitoneally, and in rats, intravenously in increasing doses according to Litchfold-Wilcoxon (dosing accuracy was achieved by changing the volume of the drug administered). Doses of the drug (based on linoleic acid diiodoethyl ether) with intraperitoneal administration were 2000, 3500 and 5000 mg / kg, with intravenous administration - 1000, 2000 and 3000 mg / kg.

After an acute experiment, rodents were observed for 14 days (1st day - continuous observation). The integral indicators were evaluated: the appearance of the animals (condition of the hair and skin, injection site), behavior, symptoms of intoxication, weight gain (determination of initial weight, on days 7 and 14), food and water consumption per day (determination of initial consumption, on 7 and 14 days). We also evaluated the excretory system (frequency of urination, color of urine), the cardiovascular and respiratory systems (respiratory rate, heart rate - determine the initial frequency, on days 7 and 14), the digestive system (number and consistency of fecal masses - determine the initial amounts per day, on days 7 and 14), the central and peripheral nervous system (behavioral reactions, the intensity and nature of motor activity, the presence and nature of seizures, coordination of movements, reaction to yl, sound, pain and visual stimuli).

A macroscopic study of the internal organs of dead animals (brain, heart, liver, kidneys, lungs, spleen, adrenal glands, stomach, intestines, bladder, pancreas, thymus) was performed. With delayed death, a microscopic examination of the organs was carried out.

Acute toxicity study results

1.1. Toxicometry

Mortality of white mice with intraperitoneal administration of the drug within 14 days of observation was absent.

Mortality of Wistar rats with intravenous administration was noted only at a dose of 3000 mg / kg and amounted to 6 rats (3 males and 3 females) by day 14.

Calculation of acute toxicity by probit analysis

Doses (mg / kg)

ED16 = 2296 ED50 = 2842 ED84 = 3519

According to the data obtained, the drug has an extremely low toxicity.

1.2. The effect of a single injection of the drug on the integral and functional indicators.

Throughout the observation period (administered doses of the drug daily for 14 days, white mice intraperitoneally at 2000, 3500 and 5000 mg / kg, for rats - intravenously at 1000, 2000 and 3000 mg / kg) deviations from the norm in general condition and behavior in survivors animals of the experimental groups were not noted. A uniform increase in the mass of animals was observed during the entire observation period, which proved the absence of a delayed toxic effect of the drug.

To register the consumption of water and food by animals in the same time periods, 2 hours after administration of the drug, animals were planted in metabolic cells for a day.

Studies have shown that with the introduction of the drug in all doses, there was a slight increase in feed intake by 14 days after administration. In groups of animals with intravenous administration of the drug at doses of 2000 and 3000 mg / kg, an increase in water consumption was also observed.

Evaluation of renal excretory function was carried out by the number of daily urine of rats. Urine frequency, color, and urine transparency were also evaluated.

Urine in all groups at all periods of observation was transparent, the color ranged from light yellow to dark yellow. The dynamics of the daily amount of urine in rats led to the conclusion that there is no effect of the drug on its daily amount. There was no statistically significant difference between baseline and daily urine after administration of the drug.

The parameter for evaluating the cardiovascular system (CVS) was selected heart rate (HR). The study showed that in animals (rats) with a single intravenous administration of the drug at doses of 1000, 2000 and 3000 mg / kg, only with a dose of 3000 mg / kg, there is a tendency to tachycardia.

The parameter for evaluating the respiratory system (DS) of rats was the respiratory rate (NPV) with a single intravenous administration of the drug at doses of 1000, 2000 and 3000 mg / kg. The results showed that at all doses, there were no statistically significant differences with the initial values for the NPV parameter.

The function of the digestive system in white mice and rats was evaluated by the number and consistency of fecal matter. With intraperitoneal and intravenous administration of the drug in all doses, there were no changes in the number and consistency of fecal matter in white mice or rats.

1.3. Pathological studies

Animals of all experimental groups after the observation period (14 days) were withdrawn from the experiment. Doses of the drug with intraperitoneal administration were 2000, 3500 and 5000 mg / kg, with intravenous administration - 1000, 2000, 3000 mg / kg (10 animals for each group - 5 females, 5 males). External differences between the animals of the experimental groups were not identified.

The coat of mice and rats had a neat appearance, was shiny, without foci of baldness. Animal nutrition is satisfactory. The skin and subcutaneous tissue at the injection site did not represent any changes. Infiltrates, irritation, or tissue necrosis at the injection site were not observed.

A macroscopic examination of the distinct effect of the drug on the condition of internal organs was not established. When examining the thoracic and abdominal cavities, there were no violations in the location of the internal organs.

The thyroid gland closely adjoined the larynx, had the usual sizes and density, pinkish-red color. The thymus was triangular in shape, whitish in color and moderately dense.

The size and shape of the heart had no changes. The heart muscle was brown, dense.

The surface of the lungs was pale pink; lungs collapsed when opening the chest. The sectional tissue also had a uniform pale pink color. The mucous membrane outside the pulmonary bronchi was smooth, shiny, pale pink.

The stomach had the usual shape and size, the lumen was filled with dense food contents. The mucous membrane of the stomach was pale pink, shiny, folded. The mucosa of the small and large intestines was shiny, smooth.

The liver is moderately enlarged. The capsule of the liver was thin, transparent. The liver tissue was brownish in color and moderately dense.

The pancreas was pale pink, lobed.

The size and shape of the kidneys did not differ from the control, the capsule was easily removed. The surface of the organ was smooth, uniform brownish-gray in color. In the section of the kidneys, the cortical and medulla were clearly distinguished.

The shape, size and density of the adrenal glands, ovaries or testicles did not differ from the usual ones.

The spleen had a dark cherry color, smooth surface and dense texture.

The membranes of the brain were thin, transparent. The substance of the brain had a moderate density. No ventricular dilatation was observed.

In rats with a fatal outcome with an intravenous administration of a drug at a dose of 3000 mg / kg, the following changes were detected at autopsy:

- in the lungs, a foamy liquid of pale pink color appears on the surface of the incision;

- vessels of the meninges are full-blooded;

- the liver is moderately enlarged, the tissue is pale in color, granular consistency.

The tests carried out indicate the absence of contraindications for the medical use of the studied drug in terms of acute toxicity.

2. SUBSTRATE TOXICITY

Experiments to study subacute toxicity of the drug were performed on 48 sexually mature rats of both sexes of the Wistar line, combined into randomized groups for the study.

The studied drug was administered daily for 28 days intraperitoneally at doses of 900 mg / kg and 1800 mg / kg and intravenously (into the tail veins) at doses of 140 mg / kg and 1400 mg / kg. Each group of animals consisted of 5 males and 5 females.

The total duration of observation of animals was 28 days. The condition of animals (condition of hair and skin, injection site, behavior, intensity and nature of motor activity, the presence and nature of seizures, coordination of movements, symptoms of intoxication) was recorded daily. Weighing of animals, consumption of food and water per day was performed once a week.

Physiological and biochemical studies were carried out before the start of the experiment (background values), on days 7 and 14.

After the introduction of the drug, animals of all experimental groups were euthanized and sent for autopsy and pathomorphological examination.

2.1 Toxicometry

When the drug was administered in doses of 900 mg / kg intraperitoneally and 140 mg / kg intravenously, no mortality of animals was observed during the 28-day daily administration of the drug. Since during the first 14 days from the moment of daily administration of lethality among animals, even with doses of intraperitoneal and intravenous administration of 1800 and 1400 mg / kg, respectively, no time periods for administration of the drug were extended to 28 days. After 16 days, the animals died (4 females and 4 males) on the background of respiratory failure with intravenous administration and against the background of general intoxication with intraperitoneal administration.

Calculation of subacute toxicity with intraperitoneal administration

DOSE - 1800 mg / kg

Males: Doses (mg / kg)

ED16 = 28046 ED50 = 35622 ED84 = 45246

Females: Doses (mg / kg)

ED16 = 21570 ED50 = 32588 ED84 = 49236

Conclusion: the results of the studies showed that the studied compound has low toxicity with intraperitoneal administration. The total lethal dose of the drug with intraperitoneal administration was 32588 mg / kg, which corresponds to class VI toxicity (relatively harmless). Analysis of sexual sensitivity showed the absence of statistically significant differences in the compared groups.

Calculation of subacute toxicity with intravenous administration of the drug

DOSE - 1400 mg / kg

Males: Doses (mg / kg)

ED16 = 23966 ED50 = 28007 ED84 = 32729

Females: Doses (mg / kg)

ED16 = 21245 ED50 = 26928 ED84 = 34132

Conclusion: the results of the studies showed that the studied compound has low toxicity when administered intravenously. The data obtained allowed us to calculate the coefficient of cumulative activity of LD _{50 (N)} / LD _{50 (1)} , which was about 10, which corresponds to a very weak cumulative activity (almost none). Analysis of sexual sensitivity showed the absence of statistically significant differences in the compared groups.

General conclusion: the substance belongs to the 4th hazard class according to the degree of exposure to the body.

2.2. The effect of the drug on laboratory parameters of rat peripheral blood.

Biochemical and hematological studies were performed on days 14 and 28 from the start of drug administration. Blood was obtained by puncture of the tail vein or (at the end of the study) after simultaneous guillotine of animals.

After daily intraperitoneal administration of the drug at doses of 900 mg / kg and 1800 mg / kg and intravenous administration at doses of 140 mg / kg and 1400 mg / kg in the peripheral blood of rats, the following laboratory parameters were determined: red blood cells, hemoglobin, hematocrit, color index, average volume erythrocytes, the average concentration of hemoglobin in the erythrocyte, platelets, white blood cells, neutrophils, basophils, eosinophils, monocytes, lymphocytes, coagulation time, the beginning and end of blood coagulation.

The data obtained showed that daily intraperitoneal administration of the drug does not lead to significant deviations, in laboratory blood counts on day 14, on day 28, leukocytosis and an increase in the relative content of neutrophils were noted, which should be regarded as a sign of a reactive inflammatory reaction of the peritoneum (peritonitis) as a result prolonged administration of the drug. With the intravenous administration of the drug at a dose of 140 mg / kg, deviations from the norm in laboratory parameters were not observed. With the introduction of the drug in a dose of 1400 mg / kg on day 14, deviations were also not observed, and by day 28 there was a 100% mortality of animals.

Blood biochemical parameters were determined after daily intraperitoneal administration of the drug in doses of 900 and 1800 mg / kg and intravenous doses of 140 and 1400 mg / kg, which included total protein, creatinine, urea, cholesterol, glucose, total bilirubin, alanine aminotransferase, alkaline phosphatase, sodium and potassium also after 14 and 28 days from the start of drug administration.

The data obtained showed that daily intravenous and intraperitoneal administration of the drug does not lead to significant deviations of the biochemical parameters of peripheral blood in surviving animals.

2.3. Histological findings

The objects of study were fixed in a 10% formalin solution and embedded in paraffin. For histological examination, preparations of the brain, liver, heart, kidneys and lungs were used in hematoxylin and eosin stains, according to Van Gieson and Sudan Sh.

A study of the preparations of animals that died during the experiment with the intravenous administration of the drug at a dose of 1400 mg / kg showed the following:

In the liver, pronounced plethora, especially in the central sections of the lobules. The beam and lobular structure of the liver is preserved. The hepatocyte cytoplasm is flaky or finely vacuolated. Kupffer cells are not numerous.

In the brain, pronounced plethora of the vessels of the brain and membranes is pronounced. In the part of the vessels of the brain, focal extravasates. Reversible dystrophic changes in neurons. Around the vessels there are individual cells in a state of apoptosis (apoptotic bodies and fragments of nuclear chromatin).

In the spleen, plethora is noted. Lymphoid follicles are quite large. There are no bright centers. The periarterial zones are wide. In the red pulp, diffusely located macrophages in moderation.

Thus, the study of the organs of animals that died during the experiment revealed pronounced circulatory disorders in the studied organs, which was accompanied by dystrophic changes in parenchymal elements in the brain and liver.

In the study of the kidneys, heart, brain of animals on days 14 and 28 from the administration of the drug, no significant difference between the studied parameters was obtained. No dependence of the structure of these organs on the dose and route of administration, as well as on the timing of the experiment.

In the liver, the state of hepatocytes was characterized by a greater severity of dystrophic changes on the 14th day (variants of protein dystrophy) compared with 28 days. There were no convincing signs of fatty degeneration or accumulation of the drug in the liver in any animal.

In the lungs, in the later stages of the experiment, regardless of the route of administration of the drug, accumulation of fat-containing substances (preparation) in small-caliber vessels and / or macrophages was constantly detected (in 6 out of 7 animals against 2 out of 8 in the early stages). This was not accompanied by significant changes in the structure of the bronchi or respiratory departments. No enrichment with free cells was noted.

Summary of Subacute Toxicity

The above results of experiments on sexually mature Wistar rats showed that the studied compound has low toxicity in the intraperitoneal and intravenous routes of administration. The total lethal dose with its intraperitoneal administration was 32588 mg / kg, with an intravenous dose of 26928 mg / kg, which corresponds to toxicity class VI (relatively harmless). Analysis of sexual sensitivity showed the absence of statistically significant differences in the compared groups.

The inventive RCC refers to the 4th hazard class according to the degree of impact on the body. Repeated administration of the drug can cause an inflammatory reaction.

3. The study of the allergenic properties of the drug

The study of the allergenic properties of the drug was carried out in accordance with the "Methodological guidelines for assessing the allergenic properties of pharmacological substances" MH RF, 2000.

The experiments were carried out on white rats of the Wistar line with a body weight of 200-225 g), white outbred mice with a body weight of 18-20 g, albino guinea pigs (body weight of 250-300 g).

All experimental animals were quarantined for 14 days. During this period, animals were examined daily, their behavior and general condition were assessed, and patients with different or different behavior were rejected. Before the start of the experiment, animals that meet the criteria for inclusion in the study are distributed into groups using the randomization method.

When studying the allergenic properties of the drug, the reaction of general anaphylaxis (guinea pigs) was used, the reactions of the immune complexes (rats), the delayed-type hypersensitivity reaction (mice) were studied, and the cutaneous application method (guinea pigs) was used.

3.1. General anaphylaxis reaction (anaphylactic shock).

The drug was administered to guinea pigs in an effective diagnostic dose (140 mg / kg) and in a dose 10 times higher (1400 mg / kg): the first injection was subcutaneous, the next two intramuscularly every other day into the thigh area. The control group of animals was injected with saline in a volume corresponding to the volume of the study drug.

On the 21st day after the sensitizing injection, the animals were injected intravenously with a resolving dose of the drug equal to the total sensitizing dose. A similar dose was administered to guinea pigs of the control groups. Anaphylactic shock intensity was taken into account in Wiegle indices:

++++ - fatal shock;

+++ - severe shock (general cramps, asphyxiation, the animal loses its ability to stay on its paws, falls on its side, does not die);

++ - moderate shock (small convulsions, severe bronchospasm);

+ - weak shock (some anxiety, rapid breathing, scratching of the muzzle, involuntary urination, defecation, wool breathing, scratching of the muzzle, involuntary urination, defecation, hair tousled);

0 - shock did not develop, there are no signs of it.

The results obtained showed that the drug in a diagnostic dose has minor allergenic properties. Half of the animals showed mild shock (+ on the Wiegle scale). With the introduction of the drug at a dose of 1400 mg / kg, in some cases shock was fatal (++++ on the Wiegle scale). It should be noted that in the group of control animals that received the same permissive dose, shock was also observed with a fatal outcome.

3.2. The reaction of immune complexes

For the reaction, white Wistar rats were used, to which the drug was administered at doses of 140 and 1400 mg / kg. In the control groups used saline in the same volume. Sensitization was performed subcutaneously, five times with an interval of 6 days.

10 days after the last administration of the drug, a permissive dose was administered intradermally.

The resolving dose of the drug was determined on intact animals. The highest dilution of antigen, which did not cause visible changes in the skin one hour after administration, was taken as the permissive dose. In the control groups, the solvent was added in the same volume.

The reaction was recorded visually using a five-point system:

0 - no visible reaction;

1 - pale pink erythema throughout the site or its periphery;

2 - bright pink erythema throughout the site or its periphery;

3 - red erythema throughout the site;

4 - skin infiltration and swelling (thickening of the skin fold) in the presence or absence of erythema;

5 - erythema, severe infiltration, focal ulceration (necrosis), hemorrhages, crust formation are possible.

In the positive case, after 30 minutes, edema and hyperemia may develop at the injection site. After 2-3 hours, the inflammatory focus may thicken and the skin becomes brown-red. During a histological examination, in the case of a positive reaction, acute hemorrhagic inflammation can be detected, in the infiltrated area polymorphic-nuclear leukocytes can be seen.

From the data obtained as a result of the studies it follows that during sensitization of animals with a diagnostic dose of the drug, no signs of a reaction of immune complexes were found. In the case of its ten-fold excess at the injection site, only slight edema was observed without hyperemia.

According to a histological study, with a sensitization dose of 140 mg / kg, there are no signs of edema, plethora, or a cellular reaction. When stained according to Van Gieson, connective tissue is fuchsinophilic. Sudan-positive inclusions are located at the level of the appendages, sometimes reaching the papillary layer.

With sensitization of rats with a dose of 1400 mg / ml, histological preparations of the skin show edema and plethora of the dermis, focal hemorrhages. When stained according to Van Gieson, pronounced picrinophilia. Sudan-positive teardrop-shaped inclusions are located mainly at the level of the appendages of the skin, in places up to the papillary dermis. There is no cellular reaction.

3.3. The method of cutaneous applications.

The study of the sensitizing effect of the drug was carried out by 10 repeated cutaneous applications on a portion of the lateral surface of the body of albino guinea pigs. Application of the drug to the skin in doses of 140 mg / kg and 1400 mg / kg was carried out 5 times a week for two weeks. The skin reaction was taken into account daily on the S.V.Suvorov skin test rating scale.

The occurrence of red erythema throughout the skin was observed in animals with the application of 1400 mg / kg after 7 applications. In animals with 140 mg / kg, only the appearance of transient pale pink erythema was observed throughout the site. In a number of animals of this group, it was not possible to visually detect changes in the skin.

Thus, the development of contact dermatitis with cutaneous application of the drug is possible only with a ten-fold increase in the daily dosage.

3.4. "Delayed type" hypersensitivity reaction

The experiment was conducted on 10 mice (5 males, 5 females). Mice were sensitized once by intradermal injection of the drug into the base of the tail in Freund's complete adjuvant (PAF). The control group (mice, 5 males, 5 females) was injected with PAF solution with Hanks solution. To detect sensitization after 5 days, mice were injected with 40 μl of a 10 mM Hanks solution in the paw pad. The magnitude of the edema was recorded using an engineering micrometer after 6, 22 and 24 hours. Animals of the control group were sensibilized with PAF and Hanks' solution according to the same scheme. The difference in the thickness of both legs characterizes the magnitude of the edema, which can be used to judge the intensity of the “delayed type” hypersensitivity reaction (HRT).

The data obtained indicated that the HRT reaction in the experimental group did not develop.

3.5. Allergy Assessment Summary

The drug has a moderate allergenic effect, however, if the dosage is exceeded, anaphylactic shock, contact dermatitis and hypersensitivity due to the formation of immune complexes may develop. In order to avoid the development of anaphylatic shock, the use of the drug in case of allergies to iodine-containing drugs should be excluded.

4. Immunotoxic effect

The study was conducted on Balb mice weighing 18-20 g. The total number of animals is 60 individuals.

Animals were divided into three groups:

1. Control group - animals (10 males and 10 females), which were injected subcutaneously with saline for two days.

2. Experimental animals (10 males and 10 females), which were injected subcutaneously with the test drug at a dose of 140 mg / kg of body weight.

3. Experimental animals (10 males and 10 females), which were injected subcutaneously with a dose of 1400 mg / kg of body weight.

On the 8th day after the first injection of the drug, the state of the immune system of the experimental and control animals was assessed. In some mice (15 males and 15 females), the mass and cellularity of lymphoid organs, as well as the phagocytic activity of peritoneal macrophages, were determined.

Another part of the animals (15 males and 15 females) was used to assess the immune response to sheep erythrocytes.

Assessment of the mass and cellularity of the organs of the immune system

1. Animals were sacrificed by means of cervical vertebrae dislocation, 4 lymph nodes (2 inguinal and 2 axillary), thymus and spleen were excised from each animal.

The organs were weighed on a torsion balance.

2. From the spleen, thymus and lymph nodes, cell suspensions were prepared. Counting the number of cells in suspension was carried out after lysis of red blood cells on a conductometric particle counter.

Determination of phagocytic activity of peritoneal macrophages.

For phagocytosis, colloidal carcass particles were used. 0.05% A carcass suspension in a volume of 2 ml was administered intraperitoneally. After 10 min, the cavity was washed with 5 ml of isotonic sodium chloride solution. The peritoneal exudate (KPI) cells thus obtained were washed three times, resuspended in 1 ml of physiological saline, and the content of nucleated cells and the percentage of phagocytic cells were calculated in the Goryaev chamber.

Determination of antibody titer in the serum of mice.

The mice of the control and two experimental groups were injected intraperitoneally with a suspension of sheep erythrocytes (EB) in physiological saline in a suboptimal dose of 5 × 10 ⁷ EB / mouse. On the 7th day after the introduction of red blood cells from mice, blood was taken and the titer of antibodies against EB was determined by hemagglutination.

RESULTS.

1. The mass of lymphoid organs of animals in experimental groups did not differ significantly from the corresponding indicator of animals in the control group.

2. Cellularity of lymphoid organs after administration of the drug remained within the control values.

3. A study of the phagocytic activity of peritoneal macrophages showed that the administration of the drug at a dose of 140 mg / kg did not affect either the number of cells in the abdominal cavity or their phagocytic activity. At the same time, administration of the drug at a dose of 1400 mg / kg reduced the number of cells in the cavity. In this case, the percentage of phagocytic cells increased by 2-3 times.

4. The determination of the titer of antibodies against sheep erythrocytes showed that in animals that received the drug at a dose of 140 mg / kg, antibody titers exactly correspond to the control values. In the group of animals that received the drug in a tenfold increased dose, in some individuals the antibody titer was 2-4 times higher than in the control. However, these deviations are within the error of the titration method and therefore they cannot be considered reliable.

5. Mutagenic properties

The study was conducted on mice weighing 18-20 g (total number - 28 individuals). In the first series of experiments, accounting for chromosome aberrations was carried out only on males, the drug was administered once subcutaneously at doses of 140 and 1400 mg / kg (4 mice per dose), physiological saline was administered to the control group (4 mice). Fixation of cellular material (red bone marrow and testes) was carried out 24 hours after administration.

In the second series of experiments, the drug at a dose of 140 mg / kg was administered subcutaneously daily for 5 days on males and females (8 mice, of which 4 males and 4 females, the control group - 8 mice, of which 4 males and 4 females). Fixation of cellular material (red bone marrow in males and females, testes in males) was carried out 24 hours after the last injection. Then a microscopic cytogenetic analysis of the prepared preparations was carried out.

The data obtained as a result of the study indicated the presence of a mutagenic effect of the drug in a dose ten times the effective diagnostic dose. With single and multiple use of the drug in a dosage of 140 mg / kg, a mutagenic effect was not detected.

6. The study of x-ray contrast properties

The study was conducted on sexually mature rats of the Wistar strain (20 individuals in total). An intravascular injection of the drug was carried out at a dose of 140 mg / kg (into the tail vein) with X-ray photographs 5 and 10 minutes after the injection (5 males were the experimental group, 5 males the control group with the introduction of ultra-fluid lipiodol). The second group of animals underwent subcutaneous administration of the test drug with x-rays 10 minutes after the injection (5 males were the experimental group, 5 males - the control group with the introduction of ultra-fluid lipiodol). The dose of the drug is 900 mg / kg. X-ray study was carried out using an X-ray apparatus "DIAGNOMAX M-25."

With subcutaneous administration of the drug, a radiopaque formation at the injection site (soft tissue imbibition) is visualized in a series of radiographs obtained, the size corresponding to the amount of drug administered.

With intravenous administration, the highest contrast was determined in the tail vein along the administration over 30 mm. A lower degree of contrast was determined in the vascular system of the liver, lungs, and brain. Such a picture persisted at all time periods of the study.

When comparing the results of an X-ray study obtained with a similar administration of ultra-fluid lipiodol, there were no significant differences with the study drug during visual examination.

Thus, we can conclude that the radiopaque properties of the studied drug are comparable to those of the comparison drug (Lipiodol Ultra-fluid).

Based on the studies, it became possible to determine the scope of the claimed drug: diagnosis by hepatography, hysterosalpingography, vesiculography, lymphography, sialography, fistulography and palliative treatment of primary and metastatic tumors of the liver, kidney by oil chemoembolization.

Recommended routes of administration: in the arteries of the liver, kidney, pancreas, portal vein, lymphatic vessel, salivary gland duct, arch of the foot or hand, limbography of the limb, sialography.

A draft FSP has been prepared for the claimed RCS, which has been submitted to the Federal State Unitary Enterprise “Scientific Center for Expertise of Medical Application Tools”.

Documents have been prepared for submission to the Pharmacological and Pharmacopoeial Committees for the registration of the drug as a CSW.

Claims (1)

X-ray contrast agent, characterized in that it is an oily contrast agent containing diodoethyl ether of linoleic acid and ethyl ester of linoleic acid in the following ratio of components, wt.%: linoleic acid diiodoethyl ester 95.0 linoleic acid ethyl ester 5,0