RU2760685C1 - Composition for injection based on phosphotidylcholine, methionine, vitamin e and sodium selenite - "hepars", which has hepatoprotective properties, and a method for its preparation - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to the field of pharmaceuticals. The hepatoprotective injection pharmaceutical composition contains phosphatidylcholine, vitamin E, methionine and sodium selenite, as well as the surfactant TWIN-80, the organic pharmacopoeial solvent 2-pyrrolidone, benzyl alcohol and water, from which the micellar form is formed, using the following ratio of substances: phosphatidylcholine 0.24 g, vitamin E 0.02 g, methionine 0.04 g, sodium selenite 0.0011 g, benzyl alcohol 0.1 ml, 2-pyrrolidone 3.5 ml, TWIN-80 0.8 ml, water 5.3 ml.EFFECT: composition is characterized by low toxicity, the ability to target transport, controlled release in the target area, as well as chemical and physical stability during storage.1 cl, 38 tbl

Description

I. Scope

The invention relates to the field of pharmaceuticals and concerns the creation of a pharmaceutical composition based on phosphotidylcholine, methionine, vitamin E and sodium selenite - "Hepars", which has hepatoprotective activity and normalizes metabolic processes, and can be used in the manufacture of a drug in the form of solutions for injection and oral administration for the treatment and prevention of liver diseases.

According to statistical data of recent years, the largest share of all pathologies of domestic animals are diseases of non-infectious etiology. This is due to the negative effects of industrial and natural toxins and medicines. It should also be noted the role in the development of non-infectious pathology of unsatisfactory conditions of detention and imbalance of the diet in terms of nutrients, macro-, microelements and vitamins. Analyzing the work carried out in this direction, it can be noted that under the influence of the above factors, primarily the hepatobiliary system is affected.

The disadvantages of many new effective drugs are poor water solubility, short circulation time, low bioavailability, and overall high toxicity. One of the solutions to these problems is the use of micelles as carriers of active substances in preparations. At the same time, stringent requirements are imposed on such structures, such as low toxicity, good capacity in relation to the active substance, the ability to target transport, controlled release in the target area, chemical and physical stability during storage, as well as an acceptable price comparable to similar drugs.

II. Prior art

On the basis of phospholipids, and in particular, phosphotidylcholine, a number of hepatoprotective drugs have been created. So in the known drugs of the specified action: "Essentiale forte N" (Registration number: P N011496 / 01) and "Phosphogliv" (Registration number: P N002528 / 01) the main active principle is phosphatidylcholine.

□ - phosphatidylcholine

There are other compositions of hepatoprotective action based on: biomass of lactobacilli Lactobacillus acidophilus and products of their metabolism (RU 2290941); essential phospholipids and prebiotics (RU 2318538); 2,6-dimethyl-3-hydroxypyridine adamantyl carboxylate (RU 23325158); multicomponent systems from natural compounds (RU 2491062); hydrochloride of N-isopropylamide-2-benzylaminoethanesulfonic acid (RU 2614691); “Silymarin” (“Silibinin” instructions for medical use - RU No. LSR-002848/09 dated 2009-04-09) is a natural composition contained in the fruits of milk thistle and consisting of seven flavonoids and one flavolignan: silybin A, silybin B, isosilybin A, isosilybin B, silicristin, isosilicristin, silidianin, taxifolin.

According to the mechanism of action, hepatoprotectors can be divided into several main groups. We'll stop at three.

The first group is represented by antioxidants - plant polyphenols, α-tocopherol, retinol, cysteine and others.

The second group is represented by agents that repair hepatocyte membranes - phospholipid preparations - Essentiale, Phosphogliv.

The third group is represented by stimulants of liver parenchyma regeneration - methionine, uridine, cytidine, orotic acid.

It should be noted that the activity of vitamin E, a representative of the first group of hepatoprotectors, is significantly increased in the presence of selenium preparations, which are also indirectly antioxidants due to the activation of glutathione peroxidase and a number of other enzymes (Augustyniak Agnieszka, Bartosz Grzegorz, Cipak Ana, Duburs Gunars, Horakov L'Ubica, Luczaj Wojciech, Majekova Magdalena, Odysseos Andreani D., Rackova Lucia, Skrzydlewska Elzbieta, Stefek Milan, Strosov Miriam, Tirzitis Gunars, Venskutonis Petras Rimantas, Viskupicova Jana. An updated overview. // Free Radical Research. 2010, Vol. 44, Issue 10, P. 1216-1262.). The effectiveness of the combined use of vitamin E and selenium-containing preparations has been established to increase resistance to oxidative stress (Erkekoglu Pinar, Giray Belma Kocer, Kizilgtin Murat, Rachidi Walid, Hininger-Favier Isabelle, Roussel Anne-Marie, Favier Alain, and Hincal Filiz. Di (2 -ethylhexyl) phthalate-induced renal oxidative stress in rats and protective ect of selenium. // Toxicology Mechanisms and Methods, 2012; vol. 22, Issue 6: P. 415-423.), higher production of thyroid hormones (Zhao Na ; Sang Zhong-na; Tan Long; Zhang Jin-xiang; Yao Zhai-xiao; Zhang Wan-qi. Effects of selenium on thyroid function and deiodinase activities in EAT rats. // Huanjing Yu Jiankang Zazhi, vol. 27, Issue 2 , P.108-110, C3 (Chinese) 2010 Huanjing Yu Jiankang Zazhi Bianjibu.), Diabetes treatment (Sokmen BB, Basaraner H. and Yanardag R. Combined effects of treatment with vitamin C, vitamin E and selenium on the skin of diabetic rats. // Human and Experimental Toxicology vol ... 32, Issue 4, P. 379-384.), Etc.

Known inventions in which selenium compounds are patented as a hepatoprotective agent, for example: "Antioxidant agent with a hepatoprotective effect based on nanostructured selenium and methods for its production and use" (RU 2557992).

Known patent, which combined substances of a flavonoid nature ("Silymarin") and selenium (RU 2645092).

The closest in technical essence is a pharmaceutical composition for the treatment and / or prevention of liver diseases (RF patent No. 2725622, MGZh A61K31 / 685, A61K31 / 704, A61K31 / 355, A61R1 / 16, publ. 03.06.2020, bull. No. 19, containing phospholipids, glycyrrhizic acid or sodium glycyrrhizinate, vitamin E and water in the following ratio of components, mg per 1 ml:

phospholipids 5 ... 5 00 glycyrrhizic acid or its sodium salt 2 ... 200 vitamin E ODZ ... W water the rest is up to 1 ml

In addition, the composition may contain phosphatidylcholine as phospholipids, and alpha-tocopherol or alpha-tocopherol acetate as vitamin E.

However, no drugs are known that combine components from all the groups described.

To obtain a water-soluble combination of biologically active substances, it was decided to use the process of micelle formation - spontaneous association of surfactant molecules in a solution. As a result, micelles - associates of a characteristic structure, consisting of dozens of amphiphilic molecules with long-chain hydrophobic radicals and polar hydrophilic groups - appear in the surfactant - solvent system. The micelles formed during micelle formation are polydisperse and are characterized by a size distribution.

Therefore, the aim of the present invention is a water-soluble pharmaceutical combination, consisting of: phosphatidylcholine, vitamin E, methionine and sodium selenite, which is characterized by low toxicity, good capacity in relation to the active substance, the ability to targeted transport, controlled release in the target area, chemical and physical stability during storage, at an acceptable price comparable to similar preparations.

III. The essence of the invention

Micelle formation is typical for various types of surfactants. In our case, the surfactant is TWIN-80. Micelle formation occurs within a certain temperature range for each surfactant, the most important characteristics of which are the Kraft point and the cloud point. The Krafft point is the lower temperature limit of surfactant micelle formation (it is usually equal to 283-293 K; at temperatures below the Craft point, the surfactant solubility is insufficient for the formation of micelles). The cloud point is the upper temperature limit of surfactant micelle formation, its usual values are 323-333 K (at higher temperatures the surfactant-solvent system loses its stability and stratifies into two macrophases. Micelles disintegrate at high temperatures (388-503 K)).

To successfully create the composition, we chose a narrow temperature range suitable for the main components. It ranged from 325-329 K (52-65 ° C). This interval allows maintaining the stability of the micellar form and ensuring further profitability of the formulation.

The preparation of micellar solutions was carried out in 4 stages. At the first stage, phosphotidylcholine and vitamin E were dissolved in an organic solvent (the pharmacopoeial solvent 2-pyrrolidone was used) at a temperature of 60 degrees, then the corresponding surfactant was added to this solution, and then a solution of water with methionine, sodium selenite and benzyl was added dropwise at a constant temperature alcohol.

Table 1 shows the compositions of the compositions for the selection of optimal active substances, organic with stability.

After receiving the compositions, we tested various storage conditions.

The quality of the finished product depends to a large extent on the quality and purity of the starting pharmaceutical substances, therefore, it is extremely important to properly describe the characteristics and specifications of the starting material. High temperature processing or even steam sterilization of the finished product (if possible) can affect the composition of the prototype. It is necessary to control the various conditions of detention and storage. Starting materials must meet pharmacopoeial requirements if they have monographs, and, often, more detailed specifications of individual parameters may be required to confirm compliance with the original medicinal product. When using components from different sources, additional characterization and comparability studies will be required. The prototypes were tested for 6 months at 37 ° C. In this case, no precipitation and separation of the solution were observed. Also, in the course of the research, there was no deviation beyond the statistical error of the concentrations of phosphotidylcholine, vitamin E, sodium selenite and methionine from the initial values.

The most important indicator of any drug is the concentration of active ingredients. However, for its successful use in everyday practice, it must have a relatively low viscosity and stability over a wide temperature range. Therefore, we carried out studies of the relative viscosity of the most promising formulations. And found that OSB 7; 11 and 12 have a relative viscosity of 1.9; 2.4 and 1.3, respectively, which makes them easy to use in daily veterinary practice. In addition, we conducted studies of viscosity at 10 and 40 ° C and found a change of less than 35% of this indicator, while it was established that at 0 and 50 ° C these solutions do not freeze and do not delaminate. Work was carried out to determine the pH of both the formulations themselves and their solutions diluted in a ratio of 1 to 100 with distilled water, and it was found that the pH is in the range from 5 to 6.6, which will allow them to be used successfully.

Thus, the obtained micellar forms of the composition, consisting of: vitamin E 0.2%, methionine 0.4%, sodium selenite 0.011%; and phosphotidylcholine in the range from 1.2 to 4.75% in the pharmacopoeial solvent 2-pyrroldidone in the range from 21 to 79% using the surfactant TWIN-80 in the range from 4 to 8%, it is possible to create stable micellar forms. It has been established that the viscosity and pH value make it possible to use these formulations when testing on laboratory animals, and, in the long term, on target animals.

To conduct studies on model hepatitis, we have chosen the OSP 7 formulation - hereinafter “Hepars”.

To determine the hepatoprotective properties of the drug "Gepars" in case of experimental hepatitis induced by carbon tetrachloride, 4 groups of white non-linear mice were formed. The scheme of the experiment on the study of the hepatoprotective properties of the drug "Gepars" with tetrachloromethane-induced experimental hepatitis in mice is presented in table 2.

The first group of 20 animals was injected intraperitoneally with 50% solution of carbon tetrachloride in olive oil in a volume of 1.22 ml / kg of live weight. The animals of this group were not treated. To confirm the hepatotoxic effect of carbon tetrachloride, 10 animals were euthanized one day after injection.

The second group of 10 animals after the introduction of carbon tetrachloride was treated with the drug "Gepars" daily from the second to the sixth day.

After the introduction of carbon tetrachloride, the third out of 10 animals underwent hepatitis therapy with the reference drug Essentiale, every day from the second to the sixth day.

The volume of carbon tetrachloride for the provocation of hepatitis was administered at a 50% lethal dose.

The fourth group (control) - no injections were performed.

Euthanasia of all surviving animals was carried out upon reaching 50% death of animals in the first experimental group, by the method of cervical vertebrae translocation under gas anesthesia (isoflurane), followed by taking biological materials.

During the study of hepatoprotective properties on the model of acute toxic hepatitis, the mortality of laboratory animals for 7 days of the experiment was (table 3) in the 16th group, 50%, in the second and third - 10%, in the 46th group (background) no signs of intoxication and death of animals were noted. ...

When analyzing the hematological parameters of the peripheral blood of mice 24 hours after the start of the experiment, a significant increase in the total number of leukocytes was found in mice with carbon tetrachloride-induced hepatitis compared with control animals. The increase in the total number of leukocytes was due to granulocytic cells. This fact indicates the development of inflammatory processes in the body of animals, in particular in the liver, affected by the action of carbon tetrachloride. In addition, there is a significant decrease in erythrocytes and hemoglobin in the peripheral blood of mice of the first experimental group (Tables 4, 5).

When analyzing the biochemical parameters of the blood serum of mice (Table 5), a significant increase in the indicator liver enzymes of alanine and aspartic transaminases in the blood serum of mice was found 24 hours after the injection of a 50% solution of carbon tetrachloride in olive oil

relative mice of the control group. These changes indicate the ability of carbon tetrachloride to trigger the processes of lipid peroxidation and reduce the integral antiradical activity in the liver, which in turn leads to damage to the cell membranes of the centrilobular lobules of the liver. The consequence of this is the release of cytoplasmic enzymes, which include transaminases, into the intercellular space, from where they enter the bloodstream, where their activity increases sharply. Along with this, there is a significant increase in the activity of alkaline phosphatase, which indicates the processes of cholestasis due to compression of the bile ducts by exudate. In addition, there is a sharp decrease in the concentration of glucose in the blood serum of animals, which is a consequence of the increased energy consumption for compensation of mesenchymal-dystrophic processes in the body of mice.

Also, 24 hours after the start of the experiment, mice of group 1a showed a significant increase in urea and creatinine relative to the control group of animals. These changes indicate functional kidney failure in response to the action of xenobiotics. Thus, a decrease in the antitoxic function of the liver with the introduction of carbon tetrachloride led to the accumulation of toxic products in the body of animals, which have a negative effect on the kidneys, which leads to their damage.

Analysis of biochemical parameters of blood serum of animals for 6 days of the experiment (table 6) showed that the activity of indicator liver enzymes, alanine and aspartic transaminases in the second and third experimental groups is significantly higher than the values of the control group of animals. However, the activity of these enzymes is significantly lower than in the group of mice 1b, which were injected with the xenobiotic carbon tetrachloride without any therapeutic measures. This fact indicates the restoration of the structural and functional properties of hepatocytes, a decrease in inflammatory and destructive processes in the liver of the second and third experimental groups of mice under the influence of hepatoprotective drugs.

In the second experimental group of animals, which were used with the therapeutic purpose of the drug Gepars, a significant increase in the total protein and its fractions was noted relative to the animals of the third experimental group, which may be a consequence of the synergism of methionine and selenium, which contribute to an increase in the compensatory factors of the organism on the action of the xenobiotic. In addition, methionine, which is part of the drug, helps to remove excess fat from the liver, plays an important role in the synthesis of adrenaline, creatine, and a number of other biologically important compounds, takes part in the exchange of sulfur-containing amino acids, deamination, transmethylation, decarboxylation reactions; modulates the effect of vitamins (B9, B12, C) and hormones, activates the action of enzymes and proteins. And it is also a powerful antioxidant, thereby contributing to the neutralization of xenobiotics (in this case, carbon tetrachloride). Suppresses the release of histamine, thus, contributes to the restoration of liver tissue, by stimulating the recirculation of glutathione, regulates nitrogen balance.

At the same time, in the animals of the 16th experimental group, significantly lower albumin indices were noted, with overestimated concentrations of the globulin fractions of the protein relative to the control animals. These changes indicate a decrease in the albumin-synthesizing function of the liver in the body of animals against the background of the use of xenobiotics, and an increase in globulin fractions is a consequence of the release of proteins from the acute phase of inflammation into the blood, which indicates the presence of inflammatory and destructive processes in the liver parenchyma.

A decrease in total protein and albumin is also noted in the third experimental group of mice, which were prescribed a reference drug for therapeutic purposes.

This may be due to the increased energy consumption for the restoration of the functional activity of hepatocytes damaged by the action of the xenobiotic. Serum biochemical parameters are shown in Table 6.

When analyzing the dynamics of the increase in body weight of animals (table 7), a significant decrease in the average daily weight gain for 6 days of the experiment was found in animals of group 16. In this group, the weights of the animals remained at the initial level. At the same time, in the animals of the second and third experimental groups, the increase in body weight was significantly higher than in the 16th group, but significantly lower than in the control animals. This fact indicates the negative effect of the xenobiotic, leading to disruption of metabolic processes in the body of mice.

When analyzing the mass of the internal organs of mice, an increase in the coefficients of the mass of the liver and kidneys in 1a and 1 groups of animals relative to the control ones was reliably established. Whereas, in the second and third experimental groups of mice, which, after the introduction of the xenobiotic tetrachloromethane, were injected with hepatoprotective drugs, the organ weight changed within the statistical error, the organ coefficients showed a similar result. These indicators were within the physiological values for this type of animals and did not differ from the control group (table 8).

The use of the drug "Gepars" in animals with pathology of the hepatobiliary system caused by the action of xenobiotics (carbon tetrachloride), helps to reduce inflammatory destructive processes in the liver and kidneys and, as a consequence, to restore their functional activity.

More detailed experimental data are given in the appendix.

Claims (1)

Hepatoprotective injection pharmaceutical composition containing phosphatidylcholine and vitamin E, characterized in that it contains methionine and sodium selenite as biologically active substances, as well as a surfactant TWIN-80, an organic pharmacopoeial solvent 2-pyrrolidone, benzyl alcohol and water, of which a micellar form is formed, while the following ratio of substances is used: phosphatidylcholine - 0.24 g, vitamin E - 0.02 g, methionine - 0.04 g, sodium selenite - 0.0011 g, benzyl alcohol - 0.1 ml, 2 -pyrrolidone - 3.5 ml, TWIN-80 - 0.8 ml, water 5.3 ml.

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