EA015508B1 - Pharmaceutical composition stimulating biosynthesis of s-adenosylmethionine and peroral drug - Google Patents

Abstract

An invention is referred to pharmacy and medicine and concerns a pharmaceutical composition stimulating biosynthesis of S-adenosylmethionine and also concerns a peroral drug in tablet dosage form which has a hepatoprotective effect and can be used for treatment of the diseases accompanied with liver injury. The objective of this invention is to develop a stable peroral drug in tablet dosage form based on a new non-toxic pharmaceutical composition and having a hepatoprotective effect due to stimulation of biosynthesis of S-adenosylmethionine in the hepatic cells. The set objective is solved by the development of a pharmaceutical composition stimulating biosynthesis of S-adenosylmethionine which is characterized by the fact that it contains the mixture of methionine, succinic acid, inosine and taurin in effective quantities and also by the development the peroral drug in tablet dosage form based on it, in peroral drug in tablet dosage form, which has a hepatoprotective effect. The new drug developed on the base of the pharmaceutical composition stimulating biosynthesis of S-adenosylmethionine is highly effective and safe, has stability of the pharmaceutical dosage form and may be used in long-term courses for therapy of different diseases.

Description

The invention relates to pharmaceuticals and medicine, relates to a pharmaceutical composition that stimulates the biosynthesis of 8-adenosylmethionine, and also relates to an oral drug that has a hepatoprotective effect and can be used in the treatment of diseases accompanied by liver damage.

State of the art

Currently, the creation of new highly effective and safe drugs of hepatoprotective action, which can be used with long courses of treatment of various diseases, is an urgent task.

It should be noted that the liver often suffers from the pathology of other organs - heart, kidney, digestive and hematopoietic diseases, infectious, skin, rheumatic and other diseases. On the other hand, the onset or exacerbation of various diseases, especially metabolic, skin, allergic, autoimmune, can occur precisely because of a violation of the normal functioning of the liver.

The hepatoprotective effect of a number of known drugs is due to their effect on the synthesis of 8-adenosylmethionine in liver cells. 8-adenosylmethionine (8-adenosyl-L-methionine, 8ΆΜ) is a biological substance that is part of all tissues and body fluids; it is synthesized from the essential amino acid methionine by cascading directed biochemical reactions.

Known preparations containing 8-adenosylmethionine in their composition make up for its deficiency and stimulate the production of endogenous 8-adenosylmethionine. However, with the exogenous administration of 8-adenosylmethionine, its high activity as a methyl group donor can lead to non-selective methylation of proteins, polysaccharides, amino acids with the formation of toxic metabolites and the occurrence of side effects (L1 £ e 8c1. 2008. Va1. 83 (25-26), p. 821-827).

On the other hand, the main problem of reducing the pharmacological activity of exogenous 8-adenosylmethionine in drugs is racemization - the transition from the active (8.8) - (+) 8-adenosyl-L-methionine form to inactive (B, 8) - (+) 8-adenosyl-b-methionine, catalyzed by temperature and humidity.

In addition, 8-adenosylmethionine is chemically unstable and is easily hydrolyzed to form adenine, adenosine, methylthioadenosine and a number of other sulfur-containing organic compounds.

In order to stabilize 8-adenosylmethionine in various dosage forms, its suspensions in anhydrous alcohols are used (1P 2008013509; 1P 2008011803 or various additives, for example, ascorbic acid or its salts (I8 2010168048) or a trialose polysaccharide (VyusNysh. Vyryuk. Ac1a, 2002. Yo1. 1573 (2), p. 105-108), as well as dextrins (1P 2010018596).

Quite often, various more stable derivatives of 8-adenosyl-b-methionine are used - organic and inorganic salts 8AM (Ιηΐ. 1oit. Ryagt. 2000. Yo1. 194 (1), pp. 61-68; VI 3490997; 8I 676169; I8 2003/0032796 ), including double salts (and 8 4057686; I8 3954726; I8 4465672).

As a medicine or nutritional supplement, 8AM can be used in parenteral and oral dosage forms.

With the parenteral administration of exogenous 8AM, it is found mainly in liver cells, but practically does not penetrate the blood-brain barrier (1 st. No. 1. Ieigokigd Rkusyuu. 1990. Wo. 53 (12): 1096-1098).

It should be noted that the treatment of chronic liver diseases with cytolysis syndrome (viral hepatitis, cirrhosis, fatty liver disease) requires long multi-month courses of 8AM, which does not allow the effective use of its parenteral dosage form for intravenous administration, and when administered orally, the bioavailability of 8AM is significantly lower compared with parenteral, which is due to poor transport of 8AM through the mucous membrane of the gastrointestinal tract and the membrane of the cells of the liver and mitochondria (Ο1ίη. Tyeg. 20 09. Yo1. 31 (2): 311320).

Thus, an increase in the synthesis of 8AM with oral administration can be realized either by increasing the frequency of its administration and dosage of the active substance, 8-adenosylmethionine (which is economically inexpedient and poses a risk of side effects), or by creating alternative drugs that stimulate the synthesis of endogenous 8AM.

Among the preparations for oral administration, affecting the synthesis of 8-adenosylmethionine, methionine itself is known as the precursor 8AM (Me1aLo118sh. 1970. Yo1. 19 (5), p. 381-391).

Mathematical models are known that describe the synthesis of 8AM and its metabolites with methionine in the liver (VyuSyush. Vyryuk. Ac1a. 2010. Yo1. 1804 (1), p. 89-96). With the oral administration of methionine to rats in dosages from 0.6 to 4 g / kg, the concentration of methionine in the liver does not increase significantly and fluctuates at a level of 400 nmol / g, while the concentration of 8AM in the liver increases from 150 to 180 cM / g. A further increase in the dose of methionine over 4 g / kg leads to a metabolic jump - a sharp increase

- 1 015508 concentration δΑΜ is 4-10 times and reaches the level of 1000 μΜ / g (1. Thieog. ΒίοΙ. 2000, 204: 521-532). However, administration to animals of methionine at a dose of even 4 g / kg, when converted, corresponds to a single dose in humans of up to 40 g of methionine per day (Eagtako1. Tokyko1. 1981. Uo1. 44 (4), p. 389-395), which exceeds the recommended physiological and treatment dose of methionine several times and poses a danger of toxic effect (1ig. Liu. 2006. Yo1. 136, p. 1722-1725).

Thus, it is obvious that the achievement of acceptable concentrations of δ-adenosylmethionine in the liver using pure methionine is not possible due to the toxic effects of the latter.

There is evidence that a number of vitamins: B ₆ (pyridoxine), betaine and B ₁₂ increase the level of endogenous δ-adenosylmethionine (1ig. ΙιιΙγ. Δοί. Uyatsho 1. (Tokuo). 2001. Uo1. 47 (3), p. 188-194 )

It is also known that the biosynthesis of δ-adenosylmethionine can be increased by sequential administration of methionine and, after 4-6 hours, derivatives of adenine - 2-dioxopropyladenine (δυ 1552073). However, the need for separate administration of these active substances does not allow us to create a convenient oral dosage form.

As an active ingredient, δΑΜ is offered in various oral dosage forms. For example, pellet granules obtained by spray drying (I8 2003/0050274), sustained release tablets (υδ 2009/088404), enteric-coated gelatin capsules (υδ 6759395), granules (υδ 2008/0274997), effervescent tablets and granules (EP 2189154), lozenges (EP 2193787).

Also described is an oral dosage form of δ-adenosylmethionine resistant to hydrolysis and temperature (υδ 2009/0110729). This composition has significant drawbacks - the weight of one tablet with a content of δ 400 mg reaches 1300 mg, which is inconvenient for patients to take because of the large geometric dimensions of the tablets, in addition, the technology of tablets is difficult for industrial production.

As the closest analogue of the claimed invention can be selected therapeutic composition for oral use, which is described in patent EP 0136464. The composition can be made in the form of a tablet coated with an enteric-soluble membrane of the following composition:

MG % Active component BAME sulfate para-toluene sulfonate, 384 72.0 (equivalent to 200 mg of 8-adenosylmethionine) Pelevelo £> Avki 60 H, 4 Silica 77 14.6 Starch thirty 5.7 MCC anhydrous 10 1.9 Magnesium stearate core mass: 507 96.0 Enteric Coating: Polyacrylate fifteen 2.85 Polyethylene glycol one 0.19 Talc 2,5 0.5 Magic stearate 2,5 0.5 coating weight: 21 4.0 Tablet weight: 528 100.0

The specified drug is widely used in medical practice and can be used to treat liver diseases.

However, this tablet has a low bioavailability of δΑΜ (about 5%) due to the low level of transport of δΑΜ through the mucous membrane of the gastrointestinal tract (Cl. Thyeg. 2009. Wo. 31 (2), p. 311-320). In addition, after absorption from the intestine into the bloodstream, δΑΜ rapidly degrades, and a small amount of absorbed δ прон penetrates into the hepatocytes, which requires repeated doses of the drug every 6-8 hours to maintain the minimum therapeutic level of δΑΜ in blood plasma (Aegidkh. 1990. Vo1. 40. δirr1. 3, p. 124-128).

When storing such a preparation, a decrease in the content of the δ формы active form is observed due to its racemization process, in which the pharmacologically inactive isomer (S, δ) - (+) δ-adenosyl-L-methionine is formed.

These shortcomings reduce the therapeutic efficacy of drugs containing exogenous δΑΜ and confirm the need for the development of an alternative drug.

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SUMMARY OF THE INVENTION

An object of the present invention is to provide, on the basis of a new non-toxic pharmaceutical composition, a stable oral drug having a hepatoprotective effect by stimulating the biosynthesis of 8-adenosylmethionine in liver cells.

The problem is solved by creating a pharmaceutical composition that stimulates the biosynthesis of 8-adenosylmethionine, which according to the invention is characterized in that it contains a mixture of methionine, succinic acid, inosine and taurine in effective amounts.

The problem is also solved by creating an oral drug in the form of a tablet containing the active and auxiliary components, which according to the invention contains the above-described pharmaceutical composition as an active component.

In addition, the task is solved in that the above-described oral drug according to the invention has a hepatoprotective effect.

The authors of the present invention for the first time found that a composition comprising the active components: methionine, succinic acid, inosine and taurine, specifically stimulates the biosynthesis of 8-adenosylmethionine in liver cells.

Known formulations of methionine with metabolites, for example with succinic acid, pectin, amino acids and glucose (KI 2105502) or methionine with succinic acid, amino acids and vitamins (KI 2222996), have a general normalizing effect on metabolic processes in the body and are used as food additives. In addition, the well-known combination of methionine with inosine, succinic acid, meglumine and salts of sodium, potassium and magnesium exhibits detoxifying activity and is used as an infusion solution (KI 2240116).

In addition, it was unexpected that the greatest influence on the stimulation of the biosynthesis of 8-adenosylmethionine is exerted not by the separate use of methionine, but by its combination with substances that are not directly involved in the biosynthesis of 8-adenosylmethionine.

The authors also showed that increased synthesis of 8-adenosylmethionine in the liver tissues is achieved by using active components in effective amounts due to the positive synergy of their interaction. Moreover, according to the invention, the generally accepted minimum therapeutic amounts of the active components are effective.

Due to the fact that the increase in the biosynthesis of 8-adenosylmethionine is observed with a significant decrease in the dosage of methionine in the composition compared to its individual use, it became possible to eliminate its toxic effect on the body and reduce the risk of side effects with prolonged use of this pharmaceutical composition.

Based on the claimed composition, an oral drug in the form of a tablet can be created that contains the desired additives and an enteric coating.

As target additives, lactose, sucrose, sorbitol, methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, pectin, starch, alginates, crospovidones, calcium or magnesium stearates, stearic acid, polyethylene glycol polyvinyl chloride, colloidal vinylidene oxide can be used. polysorbate 80 and other ingredients generally accepted in pharmaceutical technology.

Copolymers of methyl acrylic acid, acetylphthalyl cellulose, shellac and other polymers resistant to gastric juice in combination with talc, titanium dioxide, iron oxides and various plasticizers, such as propylene glycol, triethyl citrate and other ingredients commonly used in pharmaceutical technology, can be used as an enteric coating.

Targeted additives and enteric coating provide pharmaceutical stability of the dosage form, its complete disintegration and dissolution in the intestine, protection against gastric juice, due to which the active components are released in the duodenum, where they are most rapidly and completely absorbed.

The hepatoprotective effect of the indicated oral drug is due to the directed influence of its active component, which is a mixture of methionine, succinic acid, inosine and taurine in effective amounts, on the stimulation of the synthesis of 8-adenosylmethionine in the liver, which leads to an improvement in the rates of regeneration and proliferation of hepatocytes and allows its use in the treatment of various diseases accompanied by liver damage.

Thus, the claimed invention as a therapeutic agent is an effective alternative to exogenous administration of 8-adenosylmethionine and eliminates its inherent disadvantages such as low bioavailability of the active component, hydrolytic, thermal instability and a tendency to racemization 8AM.

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DETAILED DESCRIPTION OF THE INVENTION

The quantitative and qualitative composition of the claimed composition was empirically selected in the course of studying the effect on the synthesis of 8-adenosylmethionine of various doses of methionine and a number of substances taken in minimal, single, generally accepted therapeutic amounts.

As a result of a study using a human HepC2 hepatoma cell culture from the ATTS cell culture collection (USA), a number of active substances were selected that caused a significant increase in the synthesis of 8-adenosylmethionine in the following concentration ranges, mg / kg:

Methionine 5-50

Inosine 20-75

Succinic acid 75-125 Taurine 10-50

The data obtained were used in the following experiments.

Experience 1.

To select the composition of the composition and the preferred method of its administration, the content of 8-adenosylmethionine in the liver homogenates of experimental animals was determined after administration of various combinations of previously selected active components.

The experiments were performed on 400 white Wistar rats in accordance with the requirements of the ethics committee and the Laboratory Practice Rules in the Russian Federation using average dosages of methionine, inosine, succinic acid and taurine from the previously obtained concentration ranges of 20, 40, 100 and 20 mg / kg, respectively.

The content of 8-adenosylmethionine in the liver was determined by intragastric and intraduodenal administration of compositions in the form of suspensions prepared in a 10% starch paste using an atraumatic probe.

Experimental animals were divided into 20 groups of 20 animals each. Groups 1-10 were administered the studied compositions intragastrically, and groups 11-20 were administered intraduodenally. Studies have been conducted taking into account the coefficient of dose transfer from humans to rodents. After 2, 4, 6, and 8 hours after drug administration, 5 animals were killed in each group and the liver was removed for analysis on the content of 8AM.

Groups 1 and 11 (control) received a 10% starch suspension without drugs.

Groups 2 and 12 received methionine at a dose of 20 mg / kg.

Groups 2 and 13 received a mixture of methionine 20 mg / kg + inosine 40 mg / kg. Groups 4 and 14 received a mixture of methionine 20 mg / kg + succinic acid 100 mg / kg.

Groups 5 and 15 received a mixture of methionine 20 mg / kg + taurine 20 mg / kg Groups 6 and 16 received a mixture of methionine 20 mg / kg + inosine 40 mg / kg + taurine 20 mg / kg.

Groups 7 and 17 received a mixture of methionine 20 mg / kg + inosine 40 mg / kg + succinic acid 100 mg / kg.

Groups 8 and 18 received a mixture of methionine 20 mg / kg + taurine 20 mg / kg + succinic acid 100 mg / kg.

Groups 9 and 19 received a mixture of methionine 20 mg / kg + inosine 40 mg / kg + succinic acid 100 mg / kg + taurine 20 mg / kg.

Groups 10 and 20 received the substance 8AM at a dose of 160 mg / kg.

The quantitative content of 8AM in the liver was determined using an 81ι хроматιη liquid chromatograph; · ιάζι.ι ЬС-20 Ргошшепсе. For each sample, 3 reproducible chromatograms were obtained. Statistical processing was performed using the software packages Exce1, Ogary Rab Prt. MYSHA! on PC RS Repyish II. Differences between the samples were evaluated using Student's test (I).

The composition was considered effective with a significant increase in the synthesis of 8-adenosylmethionine in rat liver homogenate compared with animal groups that received only methionine or 8AM.

Presented in the table. 1 and 2, the data indicate a significant increase in the synthesis of endogenous 8-adenosylmethionine with the introduction of various combinations with methionine compared with its separate use in non-toxic therapeutic doses.

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Table 1

ZAM content in rat liver homogenates with intragastric administration of components

Group number Experimental groups of animals (n = 20) The content of ZAM in the liver cg / g (M + t) 2 h 4 hours 6 h 8 41 one Control group 8.2 + 0.2 8.4 + 0.2 8.3 + 0.2 8.4 + 0.2 2 Methionine 10.4 + 0.2 12.4 + 0.3 11.1 + 0.2 9.3 + 0.3 3 Methionine + inosine 12.5 + 0.3 19.9 + 0.2 20.1 + 0.2 4 Methionine + UC 13.1 + 0.2 18.1 ± 0.3 20.2 + 0.3 14.5 + 0.3 5 Methionine + Taurine 12.2 + 0.2 17.8 + 0.4 16.5 + 0.3 11.2 + 0.2 6 Methionine + Inosine + Taurine 15.4 + 0.3 21.6 + 0.5 17.8 + 0.2 11.9 + 0.2 7 Methionine + Inosine + UC 16.2 + 0.3 24.4 + 0.4 22.0 + 0.3 16.4 + 0.3 8 Methionine + Taurine + UC 14.9 + 0.2 19.2 + 0.3 17.0 + 0.2 12.2 + 0.2 nine Methionine + Inosine + UC + Taurine 15.7 + 0.2 28.1 + 0.4 27.0 + 0.4 18.0 + 0.3 10 ZAM dose 32 mg / kg 8.6 + 0.2 11.4 + 0.3 12.1 + 0.2 11.2 + 0.3

table 2

ZAM content in rat liver homogenates with intraduodenal administration of components

Group number Experimental groups of animals (n = 20) The content of RAM in the liver rg / g (M ± t) 2h 4 hours 6h 8h eleven Control group 8.4 + 0.2 8.5 + 0.2 8.1 + 0.3 8.3 + 0.2 12 Methionine 12.5 + 0.2 16.1 + 0.2 13.5 + 0.2 8.9 + 0.3 thirteen Methionine + inosine 15.2 + 0.2 21,4 +, 03 22.9 + 0.4 14.6 + 0.3 14 Methionine + UC 12.8 + 0.3 21.9 + 0.4 16.3 + 0.3 9.2 + 0.3 fifteen Methionine + Taurine 16.3 + 0.2 17.8 + 03 16.4 + 0.3 11.7 + 0.2 sixteen Methionine + Inosine + Taurine 17.5 + 0.4 24.6 + 0.2 26.2 + 0.3 13.1 + 0.2 17 Methionine + Inosine + UC 17.9 + 0.3 27.9 + 0.3 24.0 + 0.3 15.2 + 0.3 eighteen Methionine + Taurine + UC 16.5 + 0.2 25.4 + 0.4 17.3 + 0.4 12.0 + 0.2 nineteen Methionine + Inosine + UC + Taurine 20.1 + 0.4 35.1 + 0.5 32.6 + 0.5 16.4 + 0.3 twenty ZAM dose 32 mg / kg 12.3 + 0.2 15.7 + 0.4 14.4 + 0.2 11.2 + 0.2

Thus, it was found that the composition, which is a mixture of methionine, inosine, succinic acid and taurine, with intraduodenal administration provides the highest level of ZAM in the liver in the longest time interval and determines the use of an enteric coating for the oral form. In this case, the stimulation of the biosynthesis of ZAM in the liver with the introduction of the claimed composition is provided due to the manifestation of the super-total effect of the interaction of active components.

Preferred effective amounts of the active components in the composition are, wt.%:

Methionine 4.5-16.7

Inosine 18.2-25.0

Succinic acid 41.7-68.2

Taurine 9.1-16.7

The inventive composition containing a mixture of methionine, inosine, succinic acid and taurine in effective amounts can be implemented by preparing an oral drug, for example in the form of a tablet.

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Experience 2.

For the manufacture of tablets receive the core, which is coated with an enteric coating.

To obtain tablet cores, they mix: 50 g of methionine, 100 g of inosine, 250 g of succinic acid, 50 g of taurine - the active ingredients and target additives - starch, lactose, microcrystalline cellulose, colloidal silicon oxide and / or other pharmacologically inactive auxiliary substances used in the preparation of tablet dosage forms. The resulting mixture is granulated in a granulator dryer with a solution of at least one granulating agent from the series: methyl cellulose, povidone, hydroxypropyl methyl cellulose, starch, etc. The granulate is sieved, dusted with calcium or magnesium stearate, stearic acid or another lubricant. About 600 g of tablets with an average weight of 634 mg are obtained from the obtained granulate.

The obtained tablet cores in a fluidized bed or drum type apparatus are coated with an enteric-film coating up to an average weight of one tablet of about 694 mg, which provides protection against low pH of gastric juice and consisting of a copolymer of methacrylic acid and ethyl acrylate, or acetylphthalyl cellulose, or shellac, or other gastric juice-resistant polymer, as well as talc, titanium dioxide, iron oxides, propylene glycol, triethyl citrate or other fillers and plasticizers commonly used in pharmaceutical technology Hur, necessary for the formation of high-quality film coating.

At the same time, one coated tablet contains:

mg mao. % succinic acid 250 35.97 Inosine one hundred 14.39 Taurine fifty 7.19 Methionine fifty 7.19 Targeted: Supplements 184 26.48 Enteric coating 61 8.78 Total: 695 100.00

Experience 3.

A study of the toxicity of the claimed composition was carried out on a combination containing the active components in the maximum effective amounts.

The acute toxicity study conducted according to the standard method for 100 Wistar rats by the oral administration of ground tablets having the composition in accordance with experiment 2 showed that intragastric administration of drugs in doses up to 10,000 mg / kg in the finished dosage form to rats does not cause behavioral changes and the general condition of animals, mass coefficients of organs relative to control animals, macroscopic changes in the brain, internal and endocrine organs of experimental animals, and is not accompanied by dissection zheniem or necrosis at the site of the main injection - the mucosa of the stomach and intestines.

When studying chronic toxicity, it was shown that oral administration of the drug at doses of 152 and 1520 mg / kg to rats and at doses of 25.7 and 257 mg / kg to rabbits of both sexes (according to the composition of active principles) daily for 120 days does not lead to the development of pathological changes from the general condition and behavior of animals, it does not have a toxic effect on cardiovascular activity, morphological composition of peripheral blood and bone marrow, on the functional state of the liver and kidneys, protein, carbohydrate, fatty and electrolyte species BMENA substances causes no dystrophic, destructive, focal sclerotic changes in parenchymal cells and stroma of the internal organs, and is not accompanied by locally irritating action.

Thus, the claimed composition has a low level of general toxicity and in accordance with the generally accepted conversion factor (Guskova T.A. Safety assessment of drugs at the preclinical stage. // Chemical-Pharmaceutical Journal. - 1990. - No. 7. P. 10- 15) can be used for humans in a dose of up to 1800 mg of a mixture of active ingredients per day.

Experience 4.

The study of the pharmaceutical stability of the inventive oral drug in the form of tablets in comparison with the prototype.

The stability study of the inventive oral medicinal product in the form of tablets obtained in accordance with experiment 2 and the prototype tablets described in Example E with a film coating проводили was carried out under stressful conditions (1CH O1L. Europe 2008).

The tablets were packaged in A1i / A1i blisters. The drug was subjected to stressful influence in the Vshbeg KVE-240 climatic chamber at T = 40 ° C and a relative humidity of 75% for 6 months. The results of the study are presented in table. 3.

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Table 3

Characterization of tablets of the claimed composition and prototype under stress

The drug, quality indicators Characterization of tablets during storage under stressful conditions 0 month 1 month 3 months 6 months Prototype Moisture contents, % 0.35 + 0.05 0.37 + 0.06 0.40 + 0.06 0.42 + 0.05 Quantitative content ZAM ion, mg 407.2 + 4.2 401.4 + 5.1 401.5 + 4.2 396.0 + 4.7 (8.3) - (+) 8-adenosyl-b-methionine,% 82.6 + 3.9 79.8 + 3.4 74.2 + 3.5 72.1 + 2.1 Impurities: Adenosine,% 0.25 + 0.01 0.36 + 0.02 0.46 + 0.02 0.55 + 0.03 Methylthioadenosine,% 0.33 + 0.06 0.45 + 0.03 0.62 + 0.06 0.77 + 0.02 Declared Moisture contents, % 0.77 + 0.03 0.82 + 0.05 0.82 + 0.06 0.80 + 0.05 Methionine 51.5 + 0.3 52.0 + 0.3 52.20 + 0.3 51.3 + 0.3 succinic acid 249.0 + 4.6 252.4 + 4.8 248.4 + 4.2 252.9 + 5.3 Inosine 102.7 + 0.5 101.9 + 0.4 102.1 + 0.4 102.3 + 0.5 Taurine 50.3 + 0.8 49.8 + 0.7 49.8 + 0.7 50.0 + 0.8 Impurities: Hypoxanthin,% 0.15 + 0.01 0.14 + 0.02 0, 16 + 0,01 0.15 + 0.01 Guanosine,% 0.10 + 0.02 0.13 + 0.03 0.12 + 0.02 0.13 + 0.04 Unidentified impurities 0.45 + 0.05 0.43 + 0.02 0.55 + 0.04 0.50 + 0.02

The data presented show that during storage the quantitative content of 8AM in the prototype decreases, and the amount of impurities increases. In addition, racemization of 8AM is observed with the formation of the inactive optical isomer (I, 8) - (+) 8-adenosyl-L-methionine and a drop in the active (8.8) - (+) 8-adenosyl-L-methionine.

An oral drug based on the claimed composition exhibits higher stability - the number of active components does not decrease, and there is no significant increase in impurities. This indicates the advantage of the proposed drug in terms of maintaining the dosage of active components and reducing the risk of side effects as a result of the formation of impurities.

Experience 5.

The study of the bioavailability of the active components and the effectiveness of the oral dosage form based on the claimed composition in comparison with the prototype by studying 8AM-stimulating activity was carried out on 36 chinchilla rabbits.

All animals received standard granular food and were kept in a vivarium in accordance with internationally accepted standards for the handling of laboratory animals. Animals were divided into 3 groups of 12 individuals.

The animals of the first experimental group were administered one tablet obtained in experiment 2.

The animals of the second experimental group were injected with 2 tablets with a total content of 8AM 400 mg according to the prototype (example E with a film coating And).

The animals of the third experimental (control) group were given placebo tablets made from pharmacologically inactive ingredients: 400 mg of lactose, 400 mg of microcrystalline cellulose, hydroxypropyl methylcellulose, polysorbate 80, calcium stearate and coated with a copolymer of methacrylic acid and ethyl acrylate.

In each group of animals, 2, 4, 6, and 8 hours after oral administration of the drugs, 3 individuals were killed and the liver was removed to quantify 8AM. The concentration of 8AM in liver homogenates was determined by HPLC.

The data obtained are presented in table. 4, showed that the inventive composition in the form of tablets coated with an enteric-soluble membrane has a high bioavailability of the active components, since it provides a 1.7-2.5 times higher level of concentration of 8AM in the liver of rabbits compared to the prototype.

Table 4

Concentration of 8AM in rabbit liver homogenates by oral administration

A drug The concentration of BAM in liver homogenates cg / g (M ± W) 2 h 4 h 6 h 8h The claimed composition 19.1 + 0.3 41.0 + 0.5 54.4 + 0.5 31.6 + 0.5 Prototype 10.3 + 0.3 18.7 + 0.4 21.3 + 0.4 18.4 + 0.2 Control (placebo) 7.0 + 0.2 7.4 + 0.2 6.5 + 0.2 7.2 + 0.2

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Experience 6.

A study of the hepatoprotective effect of the claimed oral drug in a multi-organ alcoholic lesion model.

The experiment was based on a standard model of alcoholic multiple organ failure. The experiment used 80 individuals of male rats of the Wistar breed of standard weight (160-170 g). To simulate toxic hepatosis in 60 rats, ethanol was used, which was administered 3 times a day intragastrically through a metal atraumatic probe at a dose of 4 g / kg (half of LI5 ₀ ) for 5 days (ethanol was diluted in distilled water to 40% concentration). After 6 days, the presence of toxic fatty hepatosis was confirmed in some experimental animals, according to the morphological picture of the liver.

Ethanol intoxication disrupted the functional activity of the liver: protein-synthetic, detoxifying, liposynthetic. This was accompanied by biochemical signs of cytolysis. The activity of transaminases and phosphatases increased in the blood, the content of total protein, lipids, the reserve of sulfhydryl groups in the blood serum decreased, the levels of bilirubin and ceruloplasmin increased. In the liver, the stores of glycogen, reduced glutathione, and levels of detoxifying cytochromes decreased. Damage to the liver was accompanied by a decrease in its functional activity: the duration of hexenal sleep increased significantly, the excretion of bromosulfalein slowed down.

In total, 4 experimental groups of 20 animals were formed, which, after the formation of toxic alcoholic hepatosis for 10 days, were injected with a non-invasive probe once a day using the following drugs:

1) a group of intact animals without alcohol intoxication (control of the model of alcohol intoxication);

2) a group of animals with alcohol intoxication without treatment - they introduced a 10% starch suspension;

3) a group of animals with alcohol intoxication and treatment of the claimed composition, which was administered in the form of ground tablets, obtained in accordance with experiment 2, at a dose of 180 mg / kg by the sum of the active components in the composition of a 10% starch suspension;

4) a group of animals with alcohol intoxication and treatment with the prototype (example E with a film coating And) in the form of crushed tablets in a 10% starch suspension in a dose equivalent to 8AM 160 mg / kg

The hepatoprotective activity of the claimed drug in comparison with the prototype was evaluated by several groups of indicators: biochemical, morphometric, functional. Hepatoprotective efficacy was judged by the clinical picture, body weight dynamics, relative liver mass, bilirubin, transaminases, phosphatases, ceruloplasmin, total protein, serum lipids, glycogen, glutathione, 8H-groups, glutamyl transferase, cytochromes in the liver, exercise tests ( hexenal test, bromosulfoalein test) and histological picture of the liver. Statistical processing of the experimental results was carried out according to Student-Fisher.

The clinical picture in the second group of rats treated without ethanol was characterized by physical inactivity, lethargy, ruffled hair and untidiness of animals. They gained weight worse than the rest of the rats. In this group, by the 17th day of the study, 60% of the animals died.

In the third group of animals that received the claimed drug, the death rate was 20%, the manifestation of intoxication in rats caused by the introduction of ethanol decreased sharply - the animals were mobile, neat, had good appetite.

In the fourth group receiving the prototype, death was observed in 40% of the animals, an improvement in behavioral reactions was observed, however, they were less pronounced compared with animals of the third group.

Ethanol intoxication is accompanied by a decrease in body weight and an increase in the relative mass of the liver. Treatment with the claimed agent helps to normalize these indicators to a greater extent compared to the prototype (Table 5): the liver mass of rats of the third group was 25.7% less than the liver mass of rats treated with the prototype and practically did not differ from the relative mass of intact animals.

The mortality of animals in the prototype group in the model of alcoholic liver damage was 1.5 times higher, while the biochemical parameters of the surviving animals in the groups did not differ significantly; a significant difference in the compared groups was obtained according to the dynamics of AlAT, alkaline phosphatase and thymol samples. The obtained data correlated with the results of biochemical analyzes (Tables 5, 6).

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Table 5

Rat body weight and relative liver mass

The studied indicators (M ± t) Experimental groups Intact animals Without treatment The claimed composition Prototype treatment Body weight g 181.5 ± 4.8 161.5 ± 8.9 180.2 ± 8.4 172.0 ± 6.1 Relative liver mass, mg / 100 g 46.2 ± 2.3 71.1 ± 4.4 50.2 ± 3.4 63.1 ± 3.8

Table 6

Biochemical and functional indicators of the liver

The studied indicators (M ± t) Experimental groups Intact Without treatment Declared COMPOSITION Prototype Total protein, g / l 64 * 1 31 ± 2 * 63 ± 4 58 ± 5 Total lipid, g / l 3.4 ± 0.3 2.4 ± 0.2 * 3.6 ± 0.1 s, s * oe AlAT shge-ka, mmol / s.l 0.30 ± 0.01 2.49 ± 0.09 * 0.32 ± 0.08 ^th 0.57 ± 0.09 * AsAT serum, mcmol / (s.l) 0.19 ± 0.02 0.38 ± 0.02 * 0.22 ± 0.04 0.34 ± 0.11 CF, mkkat / l 0.73 ± 0.10 2.28 ± 0.18 * 0.71 ± 0.11 1.03 ± 0.11 * Alkaline phosphatase, kcat / l 0.68 ± 0.08 2.53 ± 0.22 * 0.69 ± 0.10 ^th 0.9b ± 0.14 LDH, mmol / h / l 4.82 ± 0.30 8.39 ± 0.35 * 4.68 ± 0.30 5.45 ± 0.36 Thymol test, turbidity 1.40 ± 0.01 5.88 ± 0.35 * 1.39 ± 0.1СР 2.91 ± 0.48 * 8H-groups, serum, mg% 1532 ± 64 258 ± 39 * 1568 ± 39 1619 ± 73 * Reconstituted glutathione, liver, mg% 167 ± 5 59 ± 10 * 172 ± 12 165 ± 8 P ₄ m, liver, mmol / mg protein xU ⁴ 1.15 ± 0.03 0.55 ± 0.08 * 1.19 ± 0.03 0.95 ± 0.03 ₅ , liver, mmol / mg protein xu ^ * 0.85 ± 0.04 0.57 ± 0.05 * 0.81 ± 0.01 0.71 ± 0.03 Glycogen, liver, mg% 2466 ± 100 722 ± 77 * 2413 ± 105 2180 ± 144 Hexenal sleep, sec 24.8 ± 5.9 99.2 ± 9.4 24.1 ± 5.6 29.9 ± 4.7 The concentration of bromosulfalein in the 10th minute after administration, serum, extinction 13.5 ± 0.8 40.3 ± 4.2 * 13.7 ± 0.5 13.3 ± 0.7 Ceruloplasmin, serum, mg / l 408 ± 20 980 ± 79 * 441 ± 21 484 ± 25 Cholesterol, serum, mmol / l 1.83 ± 0.22 1.29 ± 0.20 1.78 ± 0.34 1.64 ± 0.20 Bilirubin total, serum, mmol / l 3.1 ± 0.3 7.2 ± 0.2 * 3.2 ± 0.1 3.7 ± 0.4

Notes: * -P <0.05 compared with intact animals; ^Δ -P <0.05 compared with the prototype

Thus, on the model of multiple organ alcoholic lesions, it was found that the claimed drug has a hepatoprotective effect and exceeds the prototype in terms of animal mortality, the dynamics of normalization of morphological and biochemical parameters of liver damage.

Experience 7.

The therapeutic efficacy of an oral drug based on the claimed composition was studied in the case of cytolytic syndrome in patients with pulmonary tuberculosis on the background of chemotherapy.

The effectiveness of the drug was evaluated in 65 patients with pulmonary tuberculosis in combination with hepatitis B and C in comparison with the prototype - tablets containing the active component of BAM 400 mg against active anti-tuberculosis therapy. The characteristics of patients when included in the study are presented in table. 7.

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Table 7

Clinical characteristics of patients before treatment

Patient Characterization The claimed composition Prototype Total number of patients 33 32 Number of men 21 17 Number of women eleven fifteen The average duration of chemotherapy for tuberculosis, months 1.4 1,5 The number of patients with chronic hepatitis B 6 8 The number of patients with chronic hepatitis C 25 twenty The number of patients with chronic combined hepatitis B and C 2 3

Patients receiving active anti-TB treatment in the intensive phase in accordance with WHO recommendations with a treatment period of more than 1 month were included in the study groups by randomization by the envelope method. Patients were included in the study at a level of AlAT exceeding the norm by 1.5 times, which, according to WHO recommendations, requires canceling chemotherapy or switching to a non-hepatotoxic regimen (with the appointment of three drugs: streptomycin, ethambutol and fluoroquinolones), which contributes to an increase in the drug resistance of MBT.

The course of treatment in the study group of patients (n = 33) was 2 tablets 2 times a day for 25 days. In the comparison group (n = 32), similar patients received a prototype of 2 tablets 2 times a day. The volume of etiotropic therapy was 4–6 anti-tuberculosis chemotherapy drugs, while the number of patients with MDR-MBT who received IV therapy was not significantly different in the study groups (36.4 and 34.4%, respectively). The effectiveness of treatment was evaluated at 26-28 days from the start of treatment. Tuberculosis therapy was maintained in full. The criteria for the effectiveness of the drug based on the claimed composition in comparison with the prototype were the following parameters.

1. The number of patients in whom the level of cytolysis decreased to 1-1.5 norms after the end of the course (complete biochemical remission) without the need for transfer to intravenous hepatoprotective therapy.

2. The number of patients in whom the level of cytolysis remained above 1.5 norms after the end of the course (partial biochemical remission) who require a transfer to intravenous hepatoprotective therapy.

3. The number of patients who did not respond to therapy, as well as patients with an increase in the level of cytolysis (lack of biochemical remission).

4. The number of side effects of the claimed drug in comparison with the prototype during the course of treatment.

In both groups, patients experienced similar side effects - pain in the epigastric region and headaches that did not require drug withdrawal.

The data obtained (table. 8, 9) indicate that the drug based on the claimed composition in the form of tablets coated with an enteric-soluble membrane (experiment 2) has a higher therapeutic efficacy against cytolytic and cholestatic syndromes compared to the prototype.

Table 8

The dynamics of biochemical parameters of cytolysis and cholestasis in patients with pulmonary tuberculosis on the background of chemotherapy in comparison with the prototype

Parameter The claimed composition (n = 33) Prototype (n = 32) Before treatment After treatment Before treatment After treatment ALT activity, ME / L 192.8 ± 18.3 62.4 ± 4.4 187.6 ± 15.3 121.9 ± 10.2 * Gammaglugamyltranspeptidase 120.6 ± 10.7 62D ± 5.9 122.7 ± 11.2 92.4 ± 8.6 *

* - differences in the compared indices are significant in different groups, p <0.05

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Table 9

The effectiveness of the claimed composition in cytolytic syndrome in patients with pulmonary tuberculosis on the background of chemotherapy in comparison with the prototype

Parameter The claimed composition (n = 33) Prototype (n = 32) The number of patients with complete biochemical remission, people (%) 26 (79) 8 (25) The number of patients with partial biochemical remission, people (%) 5 (15) 15 (47) The number of patients who did not respond to therapy (lack of biochemical remission), people (%) 2 (6) 9 (28) Purely patients with side effects, people (%) 2 (6) 2 (6)

Thus, an oral drug created on the basis of the claimed pharmaceutical composition that stimulates the biosynthesis of 8-adenosylmethionine has high hepatoprotective effect and stability of the dosage form in the absence of side effects, and can be used in the treatment of diseases accompanied by liver damage.

Claims (3)

CLAIM 1. Pharmaceutical composition that stimulates the biosynthesis of 8-adenosylmethionine, characterized in that it contains a mixture of methionine, succinic acid, inosine and taurine in effective amounts. 2. Oral drug in the form of a tablet containing the active and auxiliary components, characterized in that as an active component, the tablet contains a pharmaceutical composition according to claim 1. 3. Oral drug according to claim 2, having a hepatoprotective effect.