RU2448732C1 - Radiopaque preparation - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: radiopaque preparation (ROP) relates to novel medications, namely to preparations for contrasting internal organs and can be applied in radiodiagnostics in angiography, computer tomography and lymphography for diagnostics and treatment of tumours of parenchymatous organs. ROP represents oil contrast substance with viscosity 15-30 cps, obtained from oleic acid by its etherification and further iodination, and contains in its composition not less than 65% of ethyl esters of iodine- and diiodineoctadecanoic acids, from remaining 35% not more than 18% are ethyl esters of oleic, linoleic, lauric, myristic and palmitic acids and not more than 17% of unidentified admixtures.

EFFECT: ROP possesses high radiopaque ability compatible with such of used in clinical practice lipiodol and has at the same time lower viscosity, which makes it possible to carry out safe intravascular introduction.

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Description

The invention relates to new drugs, namely to means of contrasting the internal organs, and can find application in radiation diagnostics for angiography, computed tomography and lymphography for the diagnosis and treatment of tumors of parenchymal organs.

The possibilities of radiation diagnostics are increased with the use of radiopaque agents (RKS), but it is necessary that such drugs have the ability to absorb x-rays, be pharmacologically inert, chemically stable and organ-specific, and also have low viscosity and toxicity, minimal osmotic activity and quickly excreted from the body. All 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 RCC molecule does 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, research methods using oil radiopaque substances 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. It is necessary to create new iodine-containing radiopaque preparations with good visualization quality, low toxicity and viscosity to reduce the likelihood of potential complications that may arise during their use.

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 iodization of vegetable oils: refined sunflower oil (iodolipol), colic (campiodol), the aforementioned sesame seeds (iodipine), poppy seeds (lipiodol) and many others.

Since 1961, the widespread use of oil-based CSWs for direct clinical lymphography began, with lipiodol being predominantly used [C. Mayorovici et al., Pharmazie, 1956, v. 4, N2, p. 126-130]. It is obtained from poppy oil, which is a mixture of stearic, palmitic, oleic and linoleic acids. The oil is iodized and then thoroughly and thoroughly cleaned. In clinical practice, ultra-liquid lipiodol (lipiodol fluid), whose radiopacity (densitometric density on the Hounsfield scale) is 2984, has found widespread use. The viscosity of lipiodol is 34-70 cPs, specific weight. - 1.268-1.290 g / ml, iodine content - 37-39%. [Certificate of Conformity No. РОСС FR 02.А 36863 (certified in the State Standard of Russia Lipiodol Ultra-Fluid)]. The drug is widely used to contrast the internal organs, lymphatic channel and lymph nodes.

However, the use of lipiodol due to its high viscosity can lead to fat embolism, as well as to a decrease in the capillary volume of the pulmonary arteries - a dangerous side effect. In addition, a sufficiently high viscosity of the drug leads to a long injection time and requires electric injectors for its introduction. The drug also does not give clear images of some groups of lymph nodes. A serious drawback of lipiodol is the complex composition and high content of unidentifiable impurities in it.

Closest to the proposed one is a radiopaque agent [RF Patent No. 2336904 of 10.27.2008 for “X-ray contrast agent], containing ethyl diodlinoleic acid as an active ingredient, and linoleic acid ethyl ester as a stabilizer in the following ratio of components , wt.%:

diiodlinoleic acid ethyl ester 95% linoleic acid ethyl ester 5%

This drug has a lower viscosity (15-30 cPs) compared with lipiodol. This provides greater fluidity of the drug through the catheter and vessels of the parenchymal organs. The drug has a high radiopacity, a moderate allergenic effect, in diagnostic doses does not lead to an immunotoxic effect.

Linoleic acid, which is esterified with ethanol in the presence of concentrated sulfuric acid, served as the feedstock for this preparation. The obtained ethyl ether, located in the upper layer of the mixture, is separated and washed with distilled water until neutral, and then dried with calcined sodium sulfate. The resulting oil is subjected to vacuum distillation - the fraction with a boiling point of 160-170 ° C is a pure ethyl ester of linoleic acid. The ether is then iodinated in petroleum ether with an aqueous 57% solution of hydroiodic acid in the presence of phosphoric anhydride. The interaction of phosphoric anhydride with water produces a lot of heat, requiring a large amount of refrigerant, which complicates and lengthens the process. The resulting reaction mass is kept until separation: the upper layer contains diiodolinoleic acid ester, which, after distillation of the petroleum ether, is washed and dried. 5% of the starting ethyl ester of linoleic acid is added to the resulting product, and the desired PKC is obtained. The drug has a high radiopacity, a sufficiently low viscosity and is suitable for intravascular administration. However, the process of obtaining it is time-consuming, energy-intensive, but, most importantly, the drug contains a large amount (more than 50%) of unidentifiable impurities. At the time of the creation of this preparation, a chromatographic analysis of it was not carried out, and a high content of unidentifiable impurities in it was found by us after recording its chromatogram in order to compare it with those of the newly created RCC.

Figure 1 shows the RCC chromatogram of the prototype and the table with the results of calculating the areas of the recorded peaks shows that the sum of unidentified impurities is more than 50% (see peaks 5.87, 6.11, 6.29, 6.48, 8.09, 8.31, 8.66, 10.65, 10.96 and 11.43 which correspond to areas 1.4, 1.39, 1.03, 0.58, 2.36, 1.29, 17.35, 2.80, 2.99 and 23.50, the sum of which is 54.69%) and only about 45% are identifiable, which are given under the chromatogram (see peaks 4.89, 5.43 , 7.21, 7.42, 9.15, 9.47, 10.07, which correspond to areas of 0.13, 0.48, 1.45, 2.75, 19.92, 13.73, 5.96. The sum of these areas is 44.42%).

The technical task of the present invention was to obtain RKS, which is an easily identifiable compound with high radiopacity, low viscosity and a minimum content of unidentifiable impurities due to the use of oleic acid as a feedstock.

The analysis of foreign literature indicates intensive research in the field of creating more advanced oil radiopaque preparations. In our country, the production of CSWs is absent and only imported products are used in clinical practice. In this regard, the creation of domestic CSW is extremely relevant and was the task of the present invention.

This problem was solved by obtaining an oil-based RKS, which is characterized by the fact that it is an oil contrast agent with a viscosity of 15-30 cPs obtained from oleic acid by its esterification and subsequent iodination with the isolation of the target product containing at least 65% ethyl esters of iodine and diiodoctadecanoic acids, of the remaining 35%, not more than 18% are ethyl esters of oleic, linoleic, lauric, myristic and palmitic acids and not more than 17% are unidentified impurities.

Figure 2 shows the chromatogram of one of the series of the obtained CSW. According to the results of calculating the peak areas, iodinated ethers are at least 65% (see peaks 9.44 and 10.73, which correspond to areas 27.92 and 37.57, i.e. 66%). Other identified products are ethyl esters of oleic, linoleic, lauric, myristic and palmitic acids and minor amounts of oleic and linoleic acids (see peaks 4.97, 6.62, 6.85, 15.17, 4.05, 5.33, 5.73, 10.08, 7.27, 10.33 shown in the chromatogram ) and make up no more than 18%. Non-identifiable impurities (peaks from 7.57 to 9.08) make up no more than 17% (see peaks 7.57, 7.70, 7.82, 7.97, 8.18, 8.37, 8.57, 8.85, 9.08 and their corresponding areas 3.7, 3.10, 2.37, 3.45, 0.79, 0.19 , 1.48, 0.92, 0.32, the sum of which is 16.32%).

The non-identifiable impurities contained in the proposed CSW, the amount of which is small (not more than 17%, while in the prototype there are more than 50%), do not significantly affect the viscosity of the drug as a whole, which is 15-30 cPs, i.e. comparable to the viscosity of the prototype and significantly less compared to lipiodol. This makes this PKC suitable for safe intravascular administration - in the arteries of parenchymal organs.

Get the claimed PKC as follows.

The feedstock is extra pure oleic acid (according to EC-No.204-007-1, Czech Republic) - an affordable, affordable product (the cost of 1 kg of linoleic acid is 10,221 rubles, and oleic acid is 2,200 rubles). It contains C18 (oleic acid) - not less than 60%, C18 (linoleic acid) - not more than 25%, C12 (lauric acid) - not more than 2%, C14 (myristic acid) - not more than 2-5% , C16 (palmitic acid) - not more than 9-15%. Commercial oleic acid is preliminarily purified from saturated acids with C12, C14, C16 by freezing at a temperature of 0-5 ° C, reducing their amount to about 10%. The oleic acid thus purified is esterified with ethanol in the presence of concentrated sulfuric acid at a temperature of 80 ° C. with stirring for 2-3 hours. The reaction mixture is kept until it is divided into 2 layers: the upper layer, a mixture of ethyl ethers and alcohol, is separated off, washed from sulfuric acid to a neutral reaction and subjected to vacuum (at a residual pressure of 2-3 mm Hg) distillation - fraction at 190-205 ° C is a mixture of ethyl esters of oleic and linoleic acids. The resulting ethyl esters are iodinated with gaseous hydrogen iodide - previously the entire technological system is purged with argon until air is displaced. Hydrogen iodide gas is obtained by the reaction of iodine with red phosphorus - for this, a suspension of red phosphorus in water is added dropwise to an aqueous suspension of powdered iodine. The resulting gaseous hydrogen iodide by argon flow is bubbled through a layer of oleic and linoleic acid ethyl esters. The reaction is carried out at a temperature of 4-10 ° C for 2 hours and is controlled by the weight gain of the sample, which should correspond to the calculated amount of attached hydrogen iodide over unsaturated bonds of ethyl esters of oleic and linoleic acids. The resulting iodinated ethyl esters are washed until neutral, treated with saturated aqueous thiosulfate and dried by centrifugation.

To obtain a dosage form from this preparation (substance), which constituted the subject of the present invention and received the trade name LINOYODOL, ethyl oleic acid ethyl ester was added to it at the rate of 5 parts by weight ether per 100 parts by weight linoiodol.

The composition of the RCC is controlled by chromatography. It has the following properties (in accordance with the requirements of the enterprise FSP project): the amount of bound iodine is 0.3-0.45 g / ml, the density is 1.2-1.3 g / cm ³ (at 20 ° C), the dynamic viscosity is 15-30 cPs (at 20 ° C), densitometric density - 2100-3100, unidentified impurities - not more than 17%, free iodine and iodides are absent.

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 the "Rules for the preclinical safety assessment of pharmacological agents (Good Laboratory Practice - GLP)" Doses of the drug in mg / kg were calculated on the basis of ethyl esters of iodine and diiodoctadecanoic acids. As a comparison drug, lipiodol fluid, widely used in clinical practice, was used.

Drug safety study

1. Acute toxicity

The acute toxicity of the dosage form of the drug was studied in sexually mature white mice and white Wistar rats.

In experiments on rodents, animals were randomized into groups using 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, 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 substance with intraperitoneal administration were 2000, 3500 and 5000 mg / kg, with intravenous administration - 1000, 2000 and 3000 mg / kg. The volume of drug administered did not exceed 0.2 ml for mice and 1.2 ml for rats.

After an acute experiment, rodents were observed for 14 days (1st day - continuous observation). Estimated integral indicators:

the appearance of the animals (condition of the hair and skin, injection site), behavior, symptoms of intoxication, weight gain (determination of the initial mass, on days 7 and 14), food and water consumption per day (determination of the initial consumption, on days 7 and 14 ) We evaluated the excretory system (frequency of urination, color of urine), the cardiovascular and respiratory system (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 quantity per day, on days 7 and 14), of the central and peripheral nervous system (behavioral reactions, the intensity and nature of motor activity, the presence and nature of seizures, motor coordination, and tactile reactions e, sound, pain and light 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.

1.1 Toxicometry

When administered intraperitoneally to white mice, doses of 2000, 3500, 5000 mg / kg were used. Mortality of white mice during 14 days of observation (females n = 5, males n = 5) was absent. With the intravenous administration of the drug to Wistar rats at doses of 1000, 2000, 3000 mg / kg (females n = 5, males n = 5) mortality was observed (14 days of observation) only at a dose of 3000 mg / kg. Throughout the observation period, the drug was administered to mice and rats daily. In the surviving animals, no abnormalities were noted in general condition and behavior.

Calculation of acute toxicity by probit analysis

probit-line zp = 4.681 * ln (d) + (- 4.890)

Doses (mg / kg)

Effective Effective Dose (ED): ED16 = 2296, ED50 = 2842, ED84 = 3519. LD50 = 76.7 ml / kg or 88.2 g / kg

Thus, the drug has an extremely low toxicity.

A lethal outcome in groups of animals with intravenous administration of the drug occurred during the first 15 minutes after drug administration - against the background of lethargy, severe shortness of breath, and the release of white foam from the nose, respiratory arrest occurred. The clinical picture testified to the development of pulmonary vascular thromboembolism.

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

Throughout the observation period (14 days), there were no deviations from the norm in general condition and behavior in the surviving animals of the experimental groups. With intravenous administration of drugs at a dose of 3000 mg / kg, all parameters were determined on the surviving animals (2 females, 2 males, the total number of animals - 4). In the remaining groups (with intraperitoneal administration, the doses were 2000, 3500 and 5000 mg / kg, with intravenous - 1000, 2000 and 3000 mg / kg), the parameters were determined for 10 animals per dose (5 males and 5 females).

A uniform increase in the mass of animals was observed during the entire observation period, which indicates the absence of delayed toxic effects of the drug. To register the consumption of water and food by animals during the same time periods, 2 hours after administration of the drug, rats were landed in metabolic cells for 24 hours. With the introduction of the drug in all doses, there was a slight increase in feed intake by 14 days after administration of the drug. In groups of animals with intravenous administration of the drug at doses of 2000 and 3000 mg / kg, an increase in water consumption was observed.

The excretory function of the kidneys in rats was assessed by the amount of daily urine. Urinary frequency, color, and urine transparency were also evaluated. The urine of animals in all groups at all periods of observation was transparent, the color ranged from light yellow to dark yellow. There was no significant difference between baseline and daily urine after administration of the drug.

The parameter for assessing the cardiovascular system of rats was selected heart rate (HR). In animals with intravenous administration of the drug at a dose of 3000 mg / kg, a tendency to tachycardia was noted.

The respiration rate (RR) was chosen as the parameter for evaluating the respiratory system of rats; there were no significant differences with the initial values for the RR parameter.

The function of the digestive system was evaluated by the amount 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 masses

1.3. Pathological studies

Animals of all experimental groups after the observation period (14 days) were withdrawn from the experiment. Doses of the substance with intraperitoneal administration were 2000, 3500 and 5000 mg / kg, with intravenous administration - 1,000, 2,000, 3,000 (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 were not changed. 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 intravenous administration of the drug at a dose of 3000 mg / kg (with a fatal outcome), the following changes were found during the autopsy of animals. A foamy liquid of pale pink color appears in the lungs on the surface of the incision. The vessels of the meninges are full-blooded. The liver is moderately enlarged, the tissue is pale in color, granular consistency.

1.4. Acute Toxicity Research Summary

A randomized study of acute toxicity in rodents — white nonlinear mice and white Wistar rats — showed that the toxic doses (mg / kg) of the test drug are comparable to those of the approved radiopaque oil iodine-containing agents and, with a single intravenous administration of the drug, are ED16 = 2296; ED50 = 2842; ED84 = 3519. The tests conducted indicate the absence of contraindications in terms of acute toxicity for medical use of the drug.

2. Subacute toxicity

Experiments to study subacute toxicity of the drug were performed on 40 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 (in the tail veins) in the following doses: 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 performed 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 administration of the drug. Since during the first 14 days from the moment of the introduction of mortality among animals, even with doses of intraperitoneal and intravenous administration of 1800 and 1400 mg / kg, no drug administration dates were extended to 28 days. Animals died on the background of respiratory failure with intravenous administration and against the background of general intoxication with intraperitoneal administration.

Calculation of subacute toxicity during intraperitoneal administration was carried out by Probit-Analys metod Litchfild-Wilcoxon, dose - 1800 mg / kg Doses (mg / kg) in males were ED16 = 28046 ED50 = 35622 ED84 = 45246, in females - ED16 = 21570 ED50 = 32588 ED84 = 49236.

The results of the studies showed that the studied compound has low toxicity with intraperitoneal administration. The total lethal dose of the substance with intraperitoneal administration was 32588 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.

Calculation of subacute toxicity with intravenous administration was carried out by Probit-Analys metod Litchfild-Wilcoxon, dose - 1400 mg / kg. Doses (mg / kg) in males were ED16 = 23966 ED50 = 28007 ED84-32729, in females - ED16-21245 ED50-26928 ED84 = 32132.

The results of the studies showed that the studied compound has low toxicity when administered intravenously. The data obtained made it possible to calculate the cumulative activity coefficient LD _{50 (N)} / LD _{50 (1)} . which amounted to about 10, which corresponds to a very weak cumulative activity (almost no). Analysis of sexual sensitivity showed the absence of statistically significant differences in the compared groups.

Thus, the drug belongs to hazard class 4 according to the degree of exposure to the body.

2.2. Effect on rat body mass dynamics and feed intake

The constant and positive dynamics of rat body weight is an integral indicator of animal health. The decrease in body weight in animals with intraperitoneal administration up to 14 days can be explained by the stressful effect of daily administration of the drug. However, by day 28 from the time of administration, an increase in body weight of experimental animals was noted. With intravenous administration, a statistically significant increase in rat body weight was observed in all groups.

With intraperitoneal and intravenous administration of the drug in animals, there was a loss of appetite for up to 14 days from the time of administration. By 28 days, the indicator returned to normal.

To register the consumption of water and food by animals during the same time periods, 2 hours after administration of the drug, rats were landed in metabolic cells for 24 hours. Change in water consumption with intraperitoneal and intravenous administration of the drug was not noted.

2.3. Impact on the functional state of the kidneys

Evaluation of renal excretory function was carried out by the amount of daily urine. Urinary frequency, color, and urine transparency were also evaluated. The urine of animals in all groups at all periods of observation was transparent, the color ranged from light yellow to dark yellow. The effect of intraperitoneal and intravenous administration of the drug on the daily amount of urine in rats was not detected.

2.4. Effect on the state of the cardiovascular system

The heart rate was selected as an evaluation parameter. In animals with intraperitoneal and intravenous administration of the drug, there was a tendency to tachycardia.

2.5. Impact on the state of the respiratory system

The evaluation parameter was selected BH. In animals with intraperitoneal administration of the drug, a decrease in BH was observed, while in groups of animals with intravenous administration, a significant increase in the frequency of respiratory movements was observed.

2.6. The effect of the drug on the functions of the digestive system

The function of the digestive system was evaluated by the amount 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 masses.

2.7. Influence on laboratory parameters of 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 guillotination of animals.

It should be noted leukocytosis and an increase in the relative content of neutrophils in the peripheral blood of rats on day 28 after the start of administration, more pronounced with the intraperitoneal administration of the drug, which should be regarded as a sign of a reactive inflammatory reaction of the peritoneum (peritonitis) as a result of prolonged administration of the drug.

Daily intravenous and intraperitoneal administration of the drug does not lead to significant deviations of biochemical parameters of peripheral blood.

2.8. The results of pathomorphological studies

Pathomorphological studies were carried out with intraperitoneal administration of the drug at doses of 900 mg / kg and 1800 mg / kg and intravenous administration (into the tail veins) in the following doses: 140 mg / kg and 1400 mg / kg (10 animals per group, 5 females, 5 males).

Rats in the groups of intraperitoneal administration at a dose of 900 mg / kg and intravenous administration of the drug at a dose of 140 mg / kg, respectively, of proper physique, satisfactory nutrition. The coat is shiny, neat-looking, foci of baldness is not determined. With intravenous administration of the drug at a dose of 1400 mg / kg and intraperitoneal administration at a dose of 1800 mg / kg in animals, the coat is dull and unkempt.

The following are generalized pathomorphological data on rats of all groups. Cases of data discrepancies between comparison groups are discussed additionally.

Teeth saved. Visible mucous membranes are pale in color, shiny. The mammary glands of females without seals to the touch, there were no discharge from the nipples. The genitals of males are developed correctly. There is no deformation or swelling of the limbs. When administered intraperitoneally in the abdominal cavity contains a significant amount of the test substance, evenly distributed throughout all intraperitoneal organs. With intravenous administration, the thoracic and abdominal cavities do not contain effusion. The position of the internal organs of the thoracic and abdominal cavities is not a violation. The parietal and visceral pleura and peritoneum are thin, shiny, smooth. The submandibular lymph nodes and salivary glands are oval, pale yellow or pinkish in color, with a smooth surface, a thin capsule, not fused together and to the underlying tissues. The cut surface is uniform in color. The thyroid gland is reddish in color, of normal size and shape, moderately dense consistency. The thymus is triangular in shape, whitish in color, moderately dense, of normal size. Intima of the aorta is smooth, shiny, whitish in color. The diameter of the aorta is not changed. The leaves of the pericardium are thin, transparent, smooth. The size and shape of the heart do not represent changes. The left ventricle is reduced, the right contains a small amount of dark liquid blood. Heart valves are thin, shiny, smooth. The muscle of the heart in the context of a uniform brown color, moderately dense. The lumen of the trachea and large bronchi is not changed, the mucous membrane is shiny, smooth, pale in color. Light airy, without seals to the touch, pale pink color. In the case of intravenous administration of the drug at a dose of 1400 mg / kg, lungs of reduced airiness, a small amount of pinkish liquid is secreted during the incision.

The mucous membrane of the esophagus is shiny, smooth, pale in color. The stomach is of normal size and shape, filled with food contents. The mucous membrane of the glandular part of the stomach is folded, pink, shiny. The mucous membrane of the body of the stomach is folded, pink, shiny. The mucous membrane of the small intestine is pale pink, shiny, smooth. The mucous membrane of the colon is grayish, shiny, smooth. The liver is moderately enlarged in rat groups with intraperitoneal and intravenous administration of the drug at doses of 1800 and 1400 mg / kg. The surface of the liver is smooth, uniform dark red in color, the capsule is thin transparent. The liver tissue in the section is full-blooded, moderately dense. The pancreas is flat in shape, pale pink in color, lobed, moderately dense consistency. The spleen is of usual shape, dark cherry in color, moderately dense consistency. The surface of the organ is smooth, the capsule is thin. On a section on a dark red background of the spleen, small grayish-colored follicles are visible. In the group of animals with intraperitoneal administration of the drug at a dose of 1800 mg / kg, an increase in the spleen was noted. The size and shape of the kidneys are not changed. The surface of the kidneys is brown, smooth, the capsule is thin, transparent, easily removable. On the section of the organ, the cortical and medulla are clearly distinguishable. The adrenal glands are round in shape, pale yellow in color, with a smooth surface, moderately dense. A dark-colored medulla is clearly visible in the section. The bladder is filled with clear urine. The mucous membrane of the bladder is smooth, shiny, pale in color. The body of the uterus of females of normal density, size and shape. The uterine horns are thin, the mucous membrane is shiny, pale. The ovaries are dark red in color, with an uneven surface, moderately dense. The testicles of males are whitish in color, of normal size and density. The membranes of the brain are thin, transparent. The substance of the brain is of normal density, the surface of the brain is smooth. On the frontal sections of the brain, gray and white matter are distinctly distinguished. The ventricles of the brain are of normal size, there is no expansion.

2.9. 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 with hematoxylin and eosin stains according to Van Gieson and Sudan III were presented. Sudan III staining is a selective method for determining fats in histological media. Thus, the detection of Sudan positive inclusions indicates the accumulation of the oil preparation in the tissues.

The study of the organs of animals included in the experiment (14th and 28th day) was carried out according to an algorithm that included the assessment of each of the signs according to a five-point system: 0 - no sign; 1 - minimum severity; 2 - weak severity; 3 - moderate severity; 4 - sign is expressed; 5 - sign is pronounced.

A 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. When examining the kidneys, heart, and brain of animals on the 14th and 28th day 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. 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.

2.10. Summary of Subacute Toxicity

The data on subacute toxicity allow us to recommend this drug for one-time systemic use (for lymphography, for intravascular administration - vascular embolization in the treatment of tumors). Intravenous administration of the drug is contraindicated because of the danger of embolization of the vessels of the lungs and brain, which is confirmed by the data of histological studies. Repeated administration of the drug can cause an inflammatory reaction.

3. The study of allergenic properties

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 Wistar rats with a body weight of 200-225 g), outbred white mice with a body weight of 18-20 g, albino guinea pigs (body weight of 250-300 g). The animals came from the nursery RAMS "Rappolovo", St. Petersburg.

All experimental animals were quarantined (acclimatization period) 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 experiment, animals meeting the criteria for inclusion in the study were divided into groups using the randomization method. Cages with experimental animals were in special rooms: light mode: 12 hours light, 12 hours - darkness; air temperature: 18-20 ° C; relative humidity: 50-70%. Daily air sterilization was performed using a quartz lamp.

3.1. General anaphylaxis reaction (anaphylactic shock)

The drug was administered to guinea pigs in an effective therapeutic 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, hair tousled);

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

The results obtained indicate that the drug in a therapeutic 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. 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, which were administered at 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 in a volume of 0.2 ml.

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 (in points according to S.V.Suvorov), where:

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.

When sensitizing animals with a therapeutic dose, no signs of reaction were found. In the case of its tenfold excess at the injection site, 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 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 into the base of the tail 60 μl of the emulsion of the drug in PAF. The control group (mice, 5 males, 5 females) was injected with PAF emulsion with Hanks solution. To detect sensitization after 5 days, mice were injected with 40 μl of HUMM preparation in the Hanks solution in the paw pad. The magnitude of the edema was recorded using an MK-0-25 engineering micrometer after 6, 22 and 24 hours. Animals of the control group were sensitized with PAF with Hanks solution according to the same scheme. The difference in the thickness of both legs was characterized by the magnitude of the edema, which can be used to judge the intensity of the "delayed type" hypersensitivity reaction. The “delayed-type” hypersensitivity 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 carried out on Balb mice weighing 18-20 g. The total number of animals was 60.

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 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.

Animals were sacrificed by means of cervical vertebral dislocation, 4 lymph nodes (2 inguinal and 2 axillary), thymus and spleen were excised from each animal. The organs were weighed on a VT type torsion balance. Cell suspensions were prepared from the spleen, thymus and lymph nodes. Counting the number of cells in suspension was carried out after lysis of red blood cells on a conductometric particle counter CMK-2.

Colloidal carcass particles were used to evaluate phagocytosis. 0.05% carcass suspension in a volume of 2 ml was administered intraperitoneally. After 10 minutes, 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.

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. The mass of lymphoid organs of animals of the experimental groups did not differ significantly from the corresponding indicator of animals in the control group. The cellularity of lymphoid organs after administration of the drug remained within the control values. 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. 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 amount - 28 pcs.). 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 presence of a mutagenic effect of the drug in doses ten times the effective therapeutic dose was revealed. With single and multiple use of the drug in a dosage of 140 mg / kg, a mutagenic effect was not detected.

6. Effect on reproductive function

In the process of drug research, a justification of reproductive safety was carried out on the basis of information on the effect on reproductive organs, fertility and early embryogenesis (males and females), embryonic and postnatal development. The study was conducted in the experimental laboratory of the St. Petersburg Institute of Pharmacy on outbred rats (RAMS nursery laboratory animals "Rappolovo").

The aim of the study was to determine the negative effect of the drug on generative function during perinodular administration in two experimental doses to male and female laboratory outbred rats. The drug was administered to animals periodically weekly: for females for 15 days, for males - 49 days in doses of 0.25 ml / kg and 0.50 ml / kg. Total number of animals: 50 male rats and 100 female rats. Animals were divided into two groups: experimental and intact.

Since the drug is supposed to be used mainly as a radiopaque agent and as material for embolization, daily administration of the drug was considered inappropriate. Dose calculation:

the estimated maximum dose administered intraarterially through a catheter for a person weighing 80 kg is 20 ml, i.e. 0.25 ml / kg, calculated without taking into account the metabolic coefficient, taking into account the fact that the drug is delayed in the vessels of the tumor long after administration. Doses selected for reproductive toxicity studies (0.25 and 0.50 ml / kg) are adequate for the maximum therapeutic dose and the most safely tolerated dose, i.e. minimal toxic.

At the end of the drug administration period, experimental animals were mated with intact animals, having previously checked the estrous cycle in the ratio of males: females = 1: 2, for a period of two estrous cycles. On the 16-19th day of pregnancy, female necropsy was performed. Males were not euthanized. The number of corpus luteum in the ovaries, implantation sites in the uterus, live fetuses and dead fetuses was counted in females euthanized during 16-19 pregnancy. Preimplantation mortality (embryotoxic effect of the drug) was evaluated - the difference between the number of corpus luteum in the ovaries and the number of implantation sites in the uterus and post-implantation mortality (fetotoxic effect of the drug) - the difference between the number of implantation sites in the uterus and the number of live fetuses. As a result of studies of reproductive toxicity in females, it was shown that the drug does not have a toxic effect.

The males did not undergo euthanasia, assessed their ability to fertilize and conceive. The ratio of the number of pregnant females to the number of mated females was calculated, in percent. As a result of the study of the reproductive toxicity of the drug with its daily intravenous administration for 49 days to males at doses of 0.25 and 0.5 ml / kg, it was shown that the test drug does not have a toxic effect on the generative function of experimental male rats.

The next goal of the study was to determine the embryo- and fetotoxic effects of the drug observed in the antenatal period with perinodular administration in two doses to pregnant female outbred rats. The study was conducted on 90 pregnant females, which were divided into two groups: the control group, who received an intravenous solvent, placebo, and two experimental women who received the drug at doses of 0.25 and 0.50 ml / kg. The studies evaluated pre- and postnatal embryonic mortality, recorded anatomical malformations, and the overall development of the fetus. The offspring evaluated physical development, the maturation rate of sensory-motor reflexes, motor and emotional behavior, and the ability to coordinate movements. The results showed that the drug, when administered 3 times intravenously at doses of 0.25 and 0.50 ml / kg, does not exert a teratogenic effect to pregnant females recorded in the antenatal period. A fetotoxic effect of the drug was found when it was used at a dose of 0.50 ml / kg, which manifested itself in an increase in post-implantation mortality, which made it possible to give advice on the undesirability of using the drug during pregnancy.

The drug was administered to pregnant females on the 4th (pre-implantation period), on the 12th (organogenesis) and 17th (fetogenesis) days of pregnancy at doses of 0.25 and 0.50 ml / kg. The total number of animals is 45 female rats. On the 20-21th day of pregnancy, females necropsy was performed. In females who underwent euthanasia on days 20-21 of pregnancy, the number was counted: yellow bodies in the ovaries, implantation sites in the uterus, live and dead fetuses. Preimplantation and postimplantation mortality were evaluated. The morphological (anatomical) malformations, as well as the overall retardation of fetal development, were evaluated.

When assessing the teratogenic effect of the drug, the number of fetuses with abnormalities that were noticeable during external examination was calculated, then the fruits were divided into 2 groups and the condition of the internal organs was examined in some fruits, and the condition of the skeleton in others. The fruits were weighed and their craniocaudal size was determined. A clinical examination of each animal was carried out daily, manifestations and severity of signs of intoxication were noted. Body weight was recorded before the administration of the drug, on the 10-11th day of pregnancy and before necropsy.

Next, the antenatal damaging effect of the drug was detected, recorded in the postnatal period of development with perinodular administration in two doses to female outbred rats on the 12th (organogenesis) and 17th (fetogenesis) days of pregnancy and during lactation weekly for 3 weeks in doses 0.25 and 0.5 ml / kg (number of animals - 45 female rats).

In each litter the number of baby rats was counted, then 6-8 newborns were left (approximately the same number of females and males). On 25-30 days after birth, the rat pups were separated from their mothers. Evaluated: 1) the physical development of the offspring, 2) the maturation rate of sensory-motor reflexes during feeding by the female, 3) motor and emotional behavior and the ability to coordinate movements of the offspring after feeding, 4) learning and development of memory: the development of conditioned reflexes as with positive and negative reinforcement and preservation of acquired skills (2 rats were taken from each litter for this study).

Conclusions: As a result of studies of the reproductive activity of the drug, it was shown that the studied drug does not have a toxic effect on the generative function of experimental male rats and the reproductive function of females. There was no negative effect on pre- and postnatal development of offspring. The study of the antenatal damaging effect of the drug, registered only in the postnatal period, showed that the drug does not affect the physical, sensory-motor, emotional and zoosocial development of the offspring.

7. Local and resorptive action with intravascular administration

The goal was to study the local and resorptive effect of the drug with the intravascular method of administration. The following routes of administration were used: in the portal vein of the liver, in the abdominal aorta and intravenously (in the tail vein of rats) in the following doses - 140 mg / kg and 1400 mg / kg. Each group of animals consisted of 5 males and 5 females of mature Wistar rats. The objects of observation were the aorta at the injection site and the portal vein immediately after administration and one hour after administration.

Methodology of the morphological study: for the study, a part of the abdominal aorta was taken at the injection site, as well as a segment of the aorta distal to the injection site of the drug; portal vein along with the adjacent part of the liver.

Results: the wall of the abdominal aorta at the injection site, the portal vein is not changed; no signs of mechanical damage and inflammation were found; unchanged red blood cells were found in the lumen of blood vessels; the intima, muscle membrane and adventitia of the studied vessels in experimental animals are not changed.

8. The study of radiopaque properties of the drug

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

When administered subcutaneously, a series of radiographs shows a radiopaque formation at the injection site (soft tissue drug imbibition), 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 injection of the drug lipiodol-fluid, with a visual examination, there were no significant differences with the drug.

Summary

According to the results of a preclinical study of the proposed CSW in comparison with lipiodol fluid, we can conclude that the drug has low toxicity (Grade 4), high radiopaque, comparable with the reference drug and the prototype. Other advantages of the claimed PKC are presented above.

According to the results of preclinical studies, it became possible to determine the scope of the claimed RCC: hepatography, hysterosalpingography, vesiculography, lymphography, sialography, fistulography and palliative treatment of primary and metastatic tumors of the liver, kidneys by oil chemoembolization.

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

All the necessary documents have been submitted to the claimed CSW, including the draft Federal Drug Administration for the substance and dosage form, in M3 - reg. number 2720.

Claims (1)

X-ray contrast agent, characterized in that it is an oil contrast agent with a viscosity of 15-30 cPs obtained from oleic acid by its esterification and subsequent iodination with the isolation of the target product containing at least 65% ethyl esters of iodine and diiodoctadecanoic acids Of the remaining 35%, not more than 18% are ethyl esters of oleic, linoleic, lauric, myristic and palmitic acids and not more than 17% are unidentified impurities.

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