Compositions of protectors against acute and chronic hepatic encephalopathies and method of treating acute and chronic hepatic encephalopathies. RU patent 2491062.

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and aims at treating and preventing hepatic encephalopathies. There are presented composite formulations containing various combinations of L-carnitine, acetyl-L-carnitine, succinate, L-glutamate, L-arginine, betaine and creatine phosphate, N-acetylcysteine, coenzyme Q₁₀ and dihydroquercetin; S-adenosylmethionine; coenzyme Q₁₀ and dihydroquercetin, dihydroquercetin and lipoamide.

EFFECT: group of inventions enables relieving behavioural, neurological and psychic disorders by activating the osmoprotective and energetic systems, detoxification reaction systems of ammonium ions and other toxins, as well as the antioxidant systems of cerebral and hepatic cells.

5 cl, 3 ex, 4 tbl, 3 dwg

The invention relates to medicine and can be used as in the food industry in the form of a biologically active additives to food, and pharmaceutical industries for the decision of problems of treatment of hepatic encephalopathy: providing reverse the development of behavioral, neurological and psychiatric disorders, prevention of development of a coma patients, and death. For treatment of acute and chronic hepatic encephalopathy offers integrated structures, whose components are effect on various metabolic and signaling system, including the activation: and energy systems, systems reactions detoxification ammonium ion and other toxins, as well as antioxidant systems of cells of the brain, the liver and other tissues and organs.

PE - reversible metabolic abnormalities in brain function (range of neuropsychiatric disorders), caused by the failure of the liver. In the basis of acute PE is the acute lack of the liver - the inability of the liver to provide detoxification ammonium ion (NH 4 + ) and other toxins, which enter the brain, bypassing the liver. The obligatory attribute of acute PE is (1, 2).

Chronic PE most likely to occur when cirrhosis of the liver, caused by alcoholic or non-alcoholic , viral hepatitis and other factors. Sign of chronic forms of Paix is also , often seen in combination with the increasing concentrations of proinflammatory cytokines in blood (1-4).

Severe forms of chronic PE (III and IV) and acute PE often can lead to coma and death of the organism (1-6).

To distinguish between three types of PE (1-5)caused by:

And - acute (acute) liver failure;

In the presence of Porto-systemic shunt (by pass) without liver disease;

With cirrhosis (chirrhosis).

In accordance with the criteria West , classify (1-4) the following stage of PI:

I - minimum (latent) PE, without visible signs of PE;

1 (I) - inversion sleep, asterisk, and others;

2 (II) - with lethargy apathy, disorientation in time, muddled speech and other;

3 (III) - drowse (stupor), complete disorientation, eccentric behavior, hyperreflexia;

4 (IV) coma.

Preclinical stage I, I and II is diagnosed using special psychological and neuropsychological tests, and stage III and IV - clinically (2). 60-80% of patients with cirrhosis have PE, half of them has expressed PE (I-IV), and the annual risk of developing PE patients with cirrhosis reaches 20% (3, 4).

Comatose state caused by acute PE is usually associated with the development of toxic or oedema in the brain, with dysfunction and swelling astrocytes and (or) with endothelial dysfunction and violation of the permeability of the blood brain barrier (4, 7, 8).

In the previous decade prevailed view of (7, 9-12, 130)that excessive activity the glutamate receptor (NMDA), occurs in the presence of excess NH 4 + causes: - accumulation of Ca 2 + and activation of neuronal NOS (nNOS), with the subsequent accumulation NO, cGMP and activation PKG; - activation of the Na-K ATPase and the depletion of ATP; a decrease in the concentration of glutamate and accumulation of glutamine, swelling astrocytes and, thus, development of toxic oedema (swelling).

As the main toxic considered glutamine (13, 14), which is in reaction:

Output of main astrocytes buffer organic - , taurine and betaine such conditions do not compensate for the accumulation of glutamine («Trojan horse»), leading to increased cells due to the ingress of water, to oxidative and stress and education mitochondrial pores of permeable by hydrolysis of glutamine and accumulation NH 4 + in mitochondria (14).

In the most popular is the idea that the swelling of astrocytes and swelling of the brain associated with the development of oxidative and stress, activation of NADPH-oxidase (15), NFβ (16), activation arachidonic acid metabolism (17) and the depletion of glutathione - GSH (18, 19). It is believed that stress leads to a decline in activity in the brain (A), one of the key enzymes cleaning NH 4 + in the body (20, 21). It is also known that the Peh, in the presence of inflammatory processes, there is also the activation of inducible iNOS(2, 4, 7, 8, 12, 23, 116).

Oxidation of NAD-dependent substrates in the mitochondria of the cells of the brain at the Peh suppressed (12, 28). There is a significant decrease of ATP (12, 29, 30, 124) and accumulation of lactate and glutamine in the brain(12, 29, 30, 48). The old hypothesis that NH 4 + can substitute K + some transport processes (24, 25) and cause depolarization cells in the brain, received experimental confirmation only recently (26, 27). Metabolic and signaling pathways involved in the mechanism of the toxicity of NH 4 + investigated insufficiently.

In acute liver failure, for example, when a portal hypertension and bypass hepatic blood flow, excess NH 4 + and other toxins produced by the microflora of the gastro-intestinal tract (gastro-intestinal bleeding),- lead to a breach of brain functions and the development of acute PE. To number of such secondary toxins may include fatty acids, mercaptans, manganese, false transmitters type of tyramine and (1, 33), ligands peripheral benzodiazepine (BDZ) receptor - and others (2-4, 7, 8, 33).

In the case of the development of severe forms of chronic PE (III and IV), swelling and dysfunction astrocytes not always observed, or Vice versa, a hypertrophy and hyperfunction of astrocytes. However, in any case oxidative and stress is seen as a root cause of the dysfunction of the brain at the Peh (1-4, 7, 8, 15).

This may be violations of various neural networks in the brain related to the following: by Hyper-activation (in case of acute PE) and with subsequent suppression (chronic PE) alarm in the chain of glutamate o NMDA o Ca 2+ o cGMP o PKG (9-11); violation in the system of exchange of monoamines (34); abuses of the system of transmission of signals with the participation of acetylcholine (Ach) (35, 36); with the dominance of the alarm with the participation of brake transmitter gamma-aminobutyric acid (GABA) by excessive activation of the peripheral BDZ receptors (4, 7, 33) and the excess production of mitochondria (7), and other

In acute liver failure caused by drug poisoning, primary toxins become activated derivatives of these compounds (in particular in the form of derivatives coenzyme A)that cause a rapid obesity, reduced GSH, oxidative stress and necrosis of the liver cells and inhibition of energy for different cell types as a result of inhibition of the Krebs Cycle,-oxidation of fatty acids (LCD) and Urea Cycle (in the liver). The growth of the LCD in these conditions leads to the development of steatosis of the liver (37, 38). Accumulating COA derivative long-chain FA in these conditions themselves become toxins, causing further collapse of various energy systems and the death of the organism (39, 130). Such scenario is when the poisoning of acetylsalicylic acid (Reye's syndrome and disease (37-39)). Blocking reactions Urea Cycle in the liver in these conditions leads to the development of acute secondary .

Similar mechanisms may be implemented in a number of congenital diseases associated with the violation systems oxidation of branched chain amino acids and with the accumulation of toxic COA derivative branched ketokislot (propionic, and common valeric and others) (40,41, 130). It should be noted that short-chain LCD with an odd number of carbon atoms (propionic and lactate by intestinal bacteria, can also be important secondary toxins, reinforcing the toxic action of surplus NH 4 + (2,33). The role of such secondary toxins is not taken into account.

Among misunderstood secondary toxins, classical forms of PE are also long-chain LCD (myristic, palmitic, oleic, linolenic and arachidonic), which can be formed in conditions of excess NH 4 + from triglycerides or at the hydrolysis of the phospholipids membranes, as is the case when a heart attack or stroke (ischemia/reperfusion), when the local increase of concentrations of toxic LCD can reach tens of (42, 43). Shown (44, 45), that oxidise Palmitoyl carnitine in concentration of 10-20 micrometers, may to cause the death of cells of different types, by the infringement of Ca2+ homeostasis, activation lipo- oxidation pathways acids and cell death, due to nonspecific cation conductivity . In the presence of NH 4 + toxic action long-LCD increases (46).

Be aware that a significant minority of patients with PE in history has steatohepatitis caused by alcoholic or non-alcoholic fatty degeneration of the liver.

Under such conditions it becomes clear that the lack of a strict correlation between the levels of NH 4 + in the blood and the severity of the development of a PE can be connected, as with heterogeneous sample of patients and presence of secondary toxins in the liver and the brain (fatty acids, and others). Thus, in one a unique study conducted in India, when a homogeneous sample of patients with acute PE (in the absence of their medical support in the first three days after hospitalization!), it is shown that the correlation between the levels of NH 4 + in the blood, the severity of the Peh, and the risk of death of patients, due to the development of acute PE (47).

Thus, it is now recognized that the ammonium ion, inflammation and infection underlie the pathogenesis of PE cirrhosis of the liver, and act synergistically (4, 7, 8).

Existing approaches in the treatment of patients with PE are well known (1-6). Effective treatment of acute PE and severe forms of chronic PE no. A final way in treatment of acute and severe forms of the Peh is considered for liver transplantation (1-4).

2) the activation of the systems of cleaning NH 4 + in the body, primarily due to the activation of Urea Cycle in the liver and in various tissues and organs.

Less common are: application antagonist GABA receptor, transfusion therapy or radical method of transplantation of the liver (1-4).

There are two patented the drug, with the first and foremost goal activation cleaning NH 4 + in the body and declared as: hepatoprotectors, means to fight and protectors of hepatic encephalopathy:

1. Product company «Merz» (Germany) - «» (52).

«», more than 15 years used in world clinical practice. In the «» - L-ornithine and L-aspartate, owing to what its name in the research community LOLA. Daily intake of acute PE: up to 20-40 g sometimes up to 80,

2. «» (53) - Ukrainian drug, developed by the SSC medicinal products of Ukraine in 2001 « 2005 is used as a drug. In the «»: L-glutamate and L-arginine. The recommended daily intake of up to 6-10,

The action of both drugs is similar and is primarily directed at the activation of the main system cleaning NH 4 + liver - Urea Cycle, as well as .

Both compositions, LOLA and «», contain connection.

L-arginine and L-ornithine are directly substrates (intermediates) Urea Cycle, and L-aspartate, ATF and - . The General equation of balance in Cycle of Urea has the form:

L-glutamate in a quasi-equilibrium reactions quickly in the L-aspartate:

and is source L-ornithine in other reaction:

L-glutamate reactions in the brain, muscle, kidney and hepatocytes involved in cleaning NH 4 + education glutamate-Mina (reaction). L-arginine, also a substrate of NO-synthase (NOS) and a source of NO in the body.

The combined use of probiotics, L-glutamate and L-arginine in the treatment of PE, studied in 50-60s (55). In 60-ies in the number of patents, to accelerate the cleaning NH 4 , instead of a pair of L-glutamate and L-arginine, suggests the use of the L-ornithine and L-aspartate (51).

The drug « was not widely used in the world medical practice, although actively advertised in Ukraine as a hepatoprotector, antioxidant and drug (53).

The drug «» (LOLA) actively investigated in different medical centers, including the Russian Federation(1-6, 56-62), however, data on the efficacy of highly contradictory. Sometimes there is a decrease in the level of NH 4 + and improvement of patients (5, 56-58), sometimes reversion only mild forms of PE (5, 59). In other studies, the effectiveness of LOLA is not confirmed (60-62). In Russia registered a patent for a method of treatment of patients with PE with lactulose, probiotics and LOLA (63). In domestic practice synonym LOLA - .

Recent attempts to use as the protector of the Peh, the combination of L-ornithine and etc (64). Several clinical studies are used L-carnitine or acetyl-L-carnitine (at doses of 2-4 g), which also have a protector properties in milder forms of PE (minimum PE; I, II). Sometimes there is a decrease in NH 4 + in the blood and improving the condition of patients (65-67).

A separate group are drugs with a hepatoprotective properties, action which is most often not stated. The effect of these drugs is aimed at restoring the functions of the liver cirrhosis and hepatitis of different etiology.

Presented recently in the domestic market «», includes phospholipid phosphatidylcholine and acid ( of milk Thistle) (6, 68). In the world medical practice and physiology of sport have long been used drugs (BAD) similar compositions (such as «Liver-Pro» company «Santegra, USA). It is believed that the phospholipids and significantly enhance antioxidant and protective mechanisms of reparative liver cells.

Recently patented products, in which in addition to the above compounds are bile acids and other compounds (69). However, the question about the methods of reversion of fibrosis and cirrhosis of the liver is still open (Nobel prize). Currently promising in treatment of such diseases is considered approach aimed at activation of metabolism and cells of the liver, with the participation of cannabinoid receptors.

Thus, in the world of medical practice, the most popular protector Peh is the drug LOLA, which is taken as a prototype.

The main disadvantage of the existing approaches in the search for methods of correction of PE are attempts to find the minimal set of tools capable of providing effective protection of the organism from an excess of NH4, both in acute and chronic Peh.

This refers to the attempts of application of LOLA (1-6, 51-63), «» (53), L-carnitine (65 to 67, 70, 71), inhibitors of NMDA (10) or GABA receptors (33, 50), a combination of NMDA receptor inhibitors and L-carnitine (128), NOS inhibitors (129), inhibitors of (13, 14), the introduction of encapsulated (125, 126), creatine (127), and other

There is not a one-two compounds able to be a panacea in the conditions of the Peh.

Acute and chronic PE represent «the metabolic Syndrome, including complex chemical (metabolic) and mental (signal) violations, the correction of which requires comprehensive protection.

Leaving aside the existing effective ways to reduce production NH 4 + bacteria in the gastrointestinal tract (1), in our opinion, effective protectors of metabolic disorders in acute and chronic PE should be comprehensive and have the following properties:

1) activate system cells in the brain and liver, participating in regulation of the volume of cells and promote the excretion of toxic : glutamine, lactate and framework, i.e. preventing the development of brain edema;

2) activate the energy of the mitochondria of different cells, because the active functioning: (A), Urea Cycle (In), as well as a number of transport (involved in the regulation of Ca 2+ and Na + /K + /Cl - homeostasis in the brain, kidney, liver and muscles) is processes;

3) activate cleaning NH 4 + in the brain, kidney, muscle and liver ( hepatocytes) with the participation of , as well as in the liver with the participation of the Urea Cycle ( hepatocytes);

4) to reduce activity of the functioning of the various neural networks in the brain involving alarm excitatory neurotransmitter glutamate (NMDA AMP A) and activate the alarm with the participation of brake transmitter GABA and glycine (in cases of acute PE). Restore activity alarm arginine o NO o cGMP o PKG and alarm systems with the participation of acetylcholine (in conditions of chronic PE);

5) activate the antioxidant and anti-inflammatory system cells in the brain and liver;

6) to reduce the accumulation of toxic COA derivative and other substances;

7) to induce metabolic and signal systems, aimed at the activation of the transcription and synthesis of enzymes de novo a number of metabolic pathways, including: the reaction of oxidative phosphorylation mitochondrial Krebs Cycle, the Cycle of Urea, exchange of glutamate, exchange of GSH, bypass, and other (chronic PE).

Long-term search protectors PE using thousands of animals allowed us to detect unexpected result is that a combination of certain metabolites have effects of their action on various metabolic and signaling systems. The combination of a relatively small concentrations of these compounds increases the effect of the actions of each of them.

This applies to the combined effect of:

- succinate, glutamate and acetyl-E-carnitine on the energy in the cells;

- (L-arginine and creatine phosphate) and (L-carnitine, acetyl-L-carnitine and betaine) on different signaling and transport systems with the participation of Central A2 m2 and receptors;

- succinate, coenzyme Q10 and , as well as L-arginine and L-glutamate on the activation of transcription factors for various metabolic and signaling pathways.

- L-glutamate, N-acetylcysteine (or S-adenosyl-methionine) and betaine activation of antioxidant systems of cells;

- L-glutamate and acetyl-L-carnitine on the activation of synthesis of N- (a key activator Urea Cycle) and the activation of synthesis of N- (NAAG) - brake dipeptide, ligand mGluRII (2) - metabotropic glutamate receptors and other

Characteristic of components of the proposed compositions protectors PE

All components are natural compounds are well studied and are standard components of food and pharmaceutical products.

L-carnitine, acetyl-L-carnitine and betaine

L-carnitine coenzyme binding transfer acyl groups in the cells of animals and plants with carbon chain length of C 2 (acetate) and C 3 (propionate) to C 24-26 . L-carnitine is an essential buffer acyl groups. Because of this exogenous L-carnitine is able to reduce the concentrations of toxic or COA derived medicines and bile acids and increase the concentration of free coenzyme A.

L-carnitine is devoted to a huge number of publications. It is also indicated for use in cardiovascular disease, obesity and diabetes, at the Peh pain syndromes, in the treatment of neurological diseases in children, when a heart attack/stroke. Widely used in sport and is advertised as the burner weight.

Mechanisms and pathways involved in the regulation of cell volume, is poorly studied. L-carnitine was one of the first compounds, which increased the survival of animals at the model of acute intoxication NH 4 + (70), although this is not always confirmed (71). It is shown that all Quaternary amines, including L-carnitine, acetyl-L-carnitine and betaine are (71, 72) and holinomimetikami (71-75).

However, by itself, L-carnitine even at high concentrations (15-30 g) is not an effective protector of the Peh.

In our opinion, are important key 3 the properties of L-carnitine:

- (Central m 2-holinoretseptora) and (together with the α 2-agonists - L-arginine and creatine phosphate);

- buffer acyl groups substrates, oxidized in the Cycle Krebsa in the form of acetyl or , i.e. participants of the energy metabolism;

acceptor - acyl and other groups, toxic derivatives long-LCD and other compounds that in the form of COA derivative (including bile acids).

Claimed by the compositions of the present invention, the number of L-carnitine 1 dose of 4-6 g or 2.5-3 g (acute PE), or 1.5-2 g (chronic PE).

Acetyl-L-carnitine

Also, L-carnitine, widely used in medicine and sport. It is known that in the conditions of acute PE (PE model) is a strong decrease of speeds oxidation of NAD-dependent substrates in the Krebs Cycle, including pyruvate, malate, oxidise Palmitoyl carnitine and others in the mitochondria, in comparison with oxidation succinate and glutamate (12, 28).

In the conditions of reduced speeds oxidation of pyruvate ketone bodies and the LCD when the Peh, education of acetyl-L-carnitine - is of great importance to ensure a high speed of work of the Krebs Cycle and the achievements of ATP in the mitochondria (if succinate and glutamate).

Use in the composition acetyl-L-carnitine has several goals:

- the action as (Central m 2-holinoretseptora) and (together with the α 2-agonists - L-arginine and creatine phosphate);

donor acetyl groups in the synthesis of Acetylcholine in the presynaptic terminal ;

donor acetyl groups in the Krebs Cycle (education and activation of the Krebs Cycle, together with suktinatom and glutamate);

donor acetyl groups in the synthesis of N- {jointly with glutamate), the predecessor of the brake of a dipeptide NAAG;

donor acetyl groups in the synthesis of N- (together glutamate) - a key activator Urea Cycle.

In the claimed the compositions of the present invention, the number of acetyl-L-carnitine 1 dose is 4 to 5 g or 1-2 g (with acute PE) and 0.5-1.5 g (chronic PE).

Betaine (Trimethylglycine) is widely used: in the food industry in the amount of 0.3-3 g (as gainer); as a hepatoprotector; for injuries; diseases of the bowel and other Betaine is a product of the disintegration of choline, a predecessor of glycine and serine. Bipolar is a molecule capable of keeping the water. Along with the m & e- and taurine is an endogenous , because in the conditions of accumulation of toxic glutamine, lactate and alanine - output betaine and other prevents increasing cells.

Betaine,a good donor music outfits groups, which is of great importance in the different reactions of methylation, including the induction of synthesis of enzymes of different metabolic pathways de novo. Along with L-carnitine and acetyl-L-carnitine is (75, 78).

Thus betaine joined as:

- and ;

donor music outfits groups.

Claimed by the compositions of the present invention, the number of betaine () 1 dose is 0.5-2 g (acute and chronic PE).

L-arginine, L-glutamate

L-arginine is one of the intermediates of the Urea Cycle, substrate in the reactions of NO synthesis and polyamines. The predecessor of creatine. Arginine - the many faces of Janus, which is written not less than . Widely used in medicine in sport (as gainer).

Known « paradox» (89), which is caused by the fact that increasing concentrations of arginine more than 1 mm causes activation of NOS and NO growth, despite the high affinity (n, e, and i) NOS to arginine is (1-5 microns). Explain such property is the presence of a competitive inhibitor NOS - asymmetric (ADMA) in the cells (89). Found that the concentration of ADMA in the conditions of different types of pathologies can reach 100-300 microns (90, 91). However, there are other mechanisms of action of arginine. It is known that arginine has an affinity to α 2-, since the introduction of arginine in the blood causes the hypotensive effect (92). It is also shown (31, 32)that L-arginine, acting through α 2 adrenergic receptors, causes activation of eNOS in , enhancing the alarm system: G VH o PIP3K O the ACT o eNOS. Similarly Supplement L-arginine is also manifested in its antagonism with norepinephrine (NE) on adipocytes (78).

Thus, the introduction of L-arginine in the composition provides:

- activation of substrate and activator N--synthase) Cycle

Urea in the liver;

- activation of the Central α 2 adrenoreceptor who (together with m 2 cholinergic receptors, may be important:

- in astrocytes and neurons;

- in the presynaptic inhibition in different neural networks;

- in suppressing the alarm with the participation of the camp Ca 2+ ;

- the activation and recovery alarm in the chain NO o cGMP o PKG conditions of chronic PE;

- restoration of a pool of creatine;

- activation of the alarm with the participation of PIP3K, an ACT, which results in the synthesis of various proteins, reparative processes and .

Claimed by the compositions of the present invention, the number of L-arginine 1 dose of 1.5-2.5 g (for acute and chronic PE).

L-glutamate - a key amino acid, in different amino acids (i.e., source and ornithine aspartate in Cycle of Urea), excitatory neurotransmitter in the brain, the predecessor of the brake transmitter GABA and braking peptide NAAG. Well oxidized in the Cycle Krebsa in pair with suktinatom (3 has a maximum speed of breath). Source α-Ketoglutarate for the Krebs Cycle in and reactions (b, C) and the substrate in the synthesis of glutamine in reaction (A). In the conditions of acute and chronic Peh there is reduction in the concentration of glutamate, accumulation of glutamine and development of oedema (4, 7, 12, 93-95).

- activation (reaction);

- activation of the Urea Cycle through education an important regulator Cycle - N-acetyl-glutamate (together with acetyl-L-carnitine);

- activation of the Urea Cycle through education Cycle L-aspartate (reaction);

- activation Cycle Krebs by recycling (reaction) substrate in the Krebs Cycle and inhibitor maximum rate of oxidation of succinate in the Krebs Cycle (together with suktinatom and acetyl-L-carnitine);

- activation of the Krebs Cycle, by preventing the depletion αKetoglutarate (reaction);

- synthesis of brake transmitter GABA and braking dipeptide NAAG (together with acetyl-L-carnitine)participating in the presynaptic inhibition through glutamate receptors type II - mGluR II (2) (87, 88);

- activation of the alarm with the participation of mTOR and synthesis proteins;

- participation in the synthesis of GSH (jointly with N-acetylcysteine and betaine).

These compositions (for acute and chronic PE) according to the present invention, the number of L-glutamate (glutamic acid) 1 dose is 0.75 to 1.5,

Succinic acid (succinate) - one of intermediates of the Krebs Cycle, the substrate reaction (SDG). SDG is one of the most fast reactions of the Krebs Cycle and, in contrast to the key regulatory reactions Cycle - a- and reactions - SDG speed weakly depends on the relationship NAD/NADH and ATP/ADP in the mitochondria. Inhibition of SDG different also poorly expressed. Therefore, in the conditions of hypoxia and other pathological conditions, oxidation succinate is of particular importance. Paired with glutamate (providing cleaning inhibitor SDG - and education α-Ketoglutarate) reach the maximum speed of the mitochondrial respiration in condition 3 () (12). In conditions of acute PE, respiratory depression mitochondria for this pair substrates expressed much weaker than for all NAD-dependent substrates (pyruvate, malate, ketone bodies, oxidise Palmitoyl carnitine) (12, 28).

The study of the role of succinic acid in various conditions, owing primarily to the efforts of Professor M.N. Kondrashova, many papers, symposia and conferences (96,97).

Succinic acid is widely used in medicine and sport. Even low pharmacological number of succinic acid and its derivatives (e.g Mexidol or succinate ammonium) have an important regulatory effect on the metabolism of man and animals (97). This may be due to its action through cell receptors GPR91 with participation of G i /G o G q proteins (98). expression GPR91 suktinatom takes place during ischemia. The important role of succinic acid has stabilize education hypoxic protein factor HIF-1 (hypoxia-induced factor), i.e. in the regulation of de novo activity of metabolic pathways, to ensure efficient production of energy by the mitochondria in the conditions of hypoxia and at various stress conditions, including oxidative stress (99, 100).

Thus, in the composition of the protectors of the Peh, amber acid provides:

- high speed of the Krebs Cycle and uptime of ATP in MiTo- (in combination with glutamate and acetyl-L-carnitine);

- under oxidative and stress;

- induction of metabolic pathways de novo (through the HIF and the receptor GPR91)providing for the formulation of the FADH and NADH.

However, by itself, amber acid, even at high concentrations (up to 5-10 mg/kg), is not effective protector of acute ammonium intoxication (101).

Claimed by the compositions of the present invention, the number of succinate (succinic acid) 1 dose is 0.75 to 1.5,

Creatine phosphate is an important cofactor transfer of energy-rich phosphate groups, buffer ATP in the cell:

KrF is important when you run marginal anaerobic activity athletes, because the pool of creatine in the muscle cells and the brain is 4-5 times higher than the total content of adenylates. Widely used in sports biochemistry. In the conditions of the Peh is the place to the collapse of the creatine to creatinine, creatinine level in the blood increases (4, 93).

In their tread PE is used for:

- recovery pool creatine+phosphocreatine in the cells;

- as that through α 2 adrenergic can participate in cells and in the presynaptic inhibition (together with L-arginine and m 2 holinomimetikami).

With the introduction of large doses of creatine phosphate may, as , in combination with L-carnitine and ethanol, replace the L-arginine (see below).

Claimed by the compositions of the present invention, the number of creatine phosphate 1 dose of 4-6 g (acute PE), or 0.5-1.5 g (chronic PE)

Ethanol, one of the the most popular connections in chronic reception is addictive and fatty liver (alcoholic steatohepatitis), due to the recovery of NADH and sustainable suppression oxidation of NAD-dependent substrates, including with the participation of transcription factors. In acute introduction of large doses of ethanol poisoning occurs . However, in some cases with acute PE caused by acetaminophen, shows the positive effect of the introduction of ethanol N-acetylcysteine (102).

In their tread acute PE, ethanol can be used as a regulator of NMDA receptors, GABA and glycine, due to the fact that even small doses of ethanol activate the brake And GABA-receptors, and large doses (50 mm and above) leads to inhibition of the activity of the NMDA receptor (80, 123) and activation receptors (123).

Claimed by the compositions of the present invention, the amount of ethanol on 1 dose is 20-50 ml

N-acetylcysteine (NAC) - the amino acid cysteine. Plays an important role in the reactions of synthesis of glutathione GSH de novo along with glutamate and glycine, as well as in various reactions .

In conditions of acute PE caused by excess ammonium ion in the presence of oxidative and stress(2, 4, 8, 12, 15), the activity of antioxidant systems of cells of the liver and brain falls. This decreases the activity of glutathione peroxidase and glutathione reductase (12). On the experimental model of the Peh with the introduction of acetaminophen and other toxins, is shown, that is a drop in the restored GSH, due to the development of oxidative stress (103, 104). In experiments carried out on the cultures of neurons and astrocytes also shown to cause cell death due to oxidative stress, is associated with the depletion of GSH (18, 19).

The level of GSH may decrease due to its transformation into GSSG in antioxidant reactions, and through education conjugates with different toxins or in the reactions of synthesis of leukotrienes, when activated ways with the participation of arachidonic acid.

NAC is used as an antidote for poisoning acetaminophen, ensuring active recovery pool glutathione level GSH (103, 104). Combination of NAC, glutamate and glycine also capable of providing effective resynthesis of GSH de novo. Use GSH is impractical because first, it breaks down in the kidneys, and then the original compounds used by the body for resynthesis GSH (105).

Therefore, as part of hepatoprotectors PE NAC:

- provides, in combination with L-glutamate, resynthesis of GSH;

- participates in reactions ;

- regulates anti-inflammatory processes.

However, the NAC, even in large concentrations (15-20 g) is not an effective protector of acute PE caused by primary .

Claimed by the compositions of the present invention quantity Q 10 1 dose is 20-30 mg (chronic PE).

DGK - flavonoids, extractable from larch - well-known-tion and widely used antioxidant (108) - is used as part of treading on its essential purpose,as an antioxidant. DHA can be replaced obtained from grapes of Resveratrol (109)) or from green tea.

Claimed by the compositions of the present invention, the number of DHA per 1 dose of 15-20 mg (chronic PE).

Formulations and doses declared songs protectors PE

The following examples serve to illustrate the claimed invention, and not to limit the scope of the claims. All components are well studied, are natural compounds are widely used in food and pharmaceutical products.

The proposed formulations protectors PE can be used as a dietary Supplement and is executed in the form of powders or pellets, suitable for oral administration. Compositions protectors PE receive direct mixing, divide into portions or granulated and divide into portions and Packed in foil bags with the use of traditional auxiliary components and techniques under the standard for this area of technology.

Compositions are used in the form of beverages with addition of the required quantities of sodium bicarbonate (equimolar content of acids). 1 dose proposed compounds dissolved in 150-250 ml of water for preparation of Fizz.

Compositions of protectors of the Peh of the administered oral dose of 1-2 to 2-3 times a day, depending on the degree of development of PE and condition of the patient.

Coenzyme Q 10 and (DHA) is dissolved in vegetable oil (preferably olive oil) and Packed in soft gelatin capsules standard for this area of technology and used in combination with the basic composition of the tread chronic PE, according to the same scheme.

and Q 10 , Packed in beta- or other water-soluble forms may be entered in the main part in appropriate quantities, without dissolution in oil.

Examples of the protectors PE

Made in the form of powders or granules in the bag-bags

Example 1

Example 2

L-carnitine

2 g

L-carnitine

1.5 g

Acetyl-L-carnitine

1 g

Acetyl-L-carnitine

1.5 g

Succinate

1 g

Succinate

1 g

L-glutamate

1 g

L-glutamate

1,25 g

L-arginine

2 g

L-arginine

1.5 g

Betaine

0.5 g

Betaine

1 g

Phosphocreatine

1 g

N-acetyl-L-cysteine

1 g

The composition of the bag dissolves in 150-200 ml of water. Reception per os 1-6 doses per day.

Example 3

Sasha-bags (granules or powder).

L-carnitine

1.5 g

Acetyl-L-carnitine

1.5 g

Succinate

0.8 g

L-glutamate

1 g

L-arginine

1,25 g

Betaine

0.6 g

Additionally

in the form of soft capsules

Q 10

30 mg

DGK

20 mg

Composition paketika dissolved in 150-200 ml of water. Used 1-6 doses per day per os, and also 2-6 capsules a day.

5. Test protectors PE in vivo

Model of PE on animals

Used classic model (9-12, 71) with the introduction of ip lethal dose NH 4 Cl (NH 4 Ac), with simultaneous introduction of NaCl. For NH 4 Cl defined LD100 is 16.5 mmol/kg After the occurrence of clonic and then tonic convulsions and coma animals died within 10-15 minutes.

Solution NH 4 Cl with a pH of 7.4 (NaOH) and solutions protectors PE prepared on the basis of introduction of 25-30 ml/kg solutions I.P. Pavlova. Given the steep dependence percent survival of animals of the administered dose NH 4 Cl at values close to the LD 100, in acute experiments entered dose NH 4 C1 amounted to 110-115% of LD100, and amounted to 18.0-18,5 mmol/kg (LD100+). The death of animals occurs in 6-7 minutes This allows you to avoid the influence of non-specific factors on the survival of animals at introduction of protectors of the Peh. Protectors most acute PE injected I.P. Pavlova. after 20-30 seconds after NH 4 Cl. The sequence of the introduction of protectors and toxins specified in the tables 1-3.

On the basis of the dynamics of changes concentrations of ammonium ion NH 4 + , glucose and urea in the blood of mice with the introduction of toxin NH4C1 and the protectors of LoLa and Σ 1 clearly shows that the protector Σ 1 causes a decrease in concentrations of toxin ammonium ion NH 4 + more effectively than existing pharmaceutical market the drug LoLa («»). While there has been less increase of concentration of glucose and urea in the blood associated with both drugs Urea Cycle and Gluconeogenesis in the liver.

1. Models with liver failure

Because the liver healthy animal does not reflect the state with hepatic insufficiency, arising from different types of PE used: several common models (114, 115) acute or chronic liver failure, leading to the development of the Peh, as well as, the recommended classification of stages of PE for laboratory animals (116).

2.1 Acute liver failure, leading to necrosis and fatty degeneration of the liver cells, oxidative stress - caused by the introduction of CCL 4 (2-4 ml/kg subcutaneously or I.P. Pavlova.), acetaminophen - AAF (300-500 mg/kg I.P. Pavlova.) or thioacetamide (TAA, 300-500 mg/kg I.P. Pavlova.), in accordance with the known data (103, 104, 116-118). Also used the model of acute multiple organ failure, with I.P. Pavlova. the introduction of the propionic acid (119, 120).

2.2 Chronic failure of the liver, caused by the introduction of CCl 4 (TAA), followed by the introduction of 24 hours NH4C1, or used a model of type 2 diabetes (115),followed by the introduction of CCL (or TAA).

2.2.1 models obese animals within 1-2 months along with standard food, fed lard (with the control of lipid composition) at the rate of up to 300-500 mg / 1 pet per day. Controlled levels of glucose, insulin, ALT, bilirubin, glycerol, triglycerides and LCD in the blood and liver, conducted histological examination of the liver.

2.2.2 In the model of type 2 Diabetes, in accordance with the data of work (115), in 1 - 2 months feeding fat, the animals were injected I.P. Pavlova. 50mg/kg (to inhibit the activity of beta-cells) and then in 15 days, after the control of the main indicators of blood used in the experiments.

Examples of the protectors of PE in vivo

Compositions protectors PE mg per 1 kg of animal weight:

Σ 1 (example 1)

Σ 2 (example 2)

L-carnitine

300

L-carnitine

300

Acetyl-L-carnitine

300

Acetyl-L-carnitine

300

Succinate

150

Succinate

150

L-glutamate

200

L-glutamate

200

L-arginine

250

L-arginine

300

Betaine

125

Betaine

125

Phosphocreatine

200

N-acetyl-L-cysteine

100

Σ 3 (example 3)

L-carnitine

300

Acetyl-L-carnitine

200

Succinate

150

L-glutamate

200

L-arginine

300

Betaine

100

Additionally

in the form of soft capsules

Q 10

50

DGK

60

LOLA2: L-ornithine = 750 L-aspartate = 500.

Acute PE

Summary trials protectors of acute PE given in Table 1. It is seen that the simultaneous introduction of toxin NH 4 Cl and different protectors causes the survival of animal parts. The effect varies greatly, not depending on the sex of animals. Introduction LOLA, which aims to reduce the concentration NH 4 Cl (in the body), ensures the survival of 60-80% of the animals. The best results has the composition of the Σ 3 (70,100%). The introduction of half doses of both protectors for 15 minutes prior to the introduction of NH 4 Cl provides higher levels of efficiency Σ 2 and LOLA (Table 1, line 6, 7). However, the net model of acute ammonium intoxication with animals from the intact liver does not reflect the status of the protection mechanisms in animals with liver failure.

The preliminary introduction of animals propionic acid suppresses energy cells of various organs and tissues and detoxification reactions NH 4 in the body due to accumulation of toxic derivatives and (119, 121) and a model of propionic and and multiorgan failure. With the introduction of animal 20-25 mmol/kg N, is a drop of ATP in the cells and accumulation NN 4 in the blood by inhibiting Urea Cycle (92). Table 1 (line 8) shows that the introduction of non-lethal doses NH 4 Cl in such conditions leads to a quick death of the animals. Even with the introduction of large doses NH 4 Cl, introduction of protectors ensures the survival of animals (Table 2, line 10, 11). Best figures of protection has protector Σ 2

In Table 1 (lines 12-14), see also the summary data obtained on models Peh, in which non-fatal dose CCl 4 , cause steatohepatitis and necrosis of the liver cells that simulates the presence of hepatic insufficiency. Introduction of various doses of NH 4 (12-14 mmol/kg) 24 hours after the introduction of these toxins can cause the development of acute PE and death of animals. However, this effect depends on the dose used toxins CCl 4 . It is seen that when small doses of CCl 4 (2 ml/kg) for the introduction of lethal for the intact animals dose NH 4 , almost all of the animals survive (80%), i.e. a peculiar effect of preconditioning.

It is known that with the introduction of CCl 4 , AAF, or TAA, reparative processes begin in few hours. Therefore, the choice of such models and conditions for demonstration of the effectiveness of actions protectors should be carefully monitored, since at low doses of toxins variety protectors will have a positive impact, as can be seen from a number of published experiments (56, 117, 118).

It is seen that in our situation the introduction of protectors in varying degrees ensures the survival of animals. The most effective is Σ 2 .

Chronic PE

Animal studies

With the introduction of large doses of CCL 4 (TAA), the subsequent introduction of non-lethal doses NH 4 Cl 24 hours (Table 2, line 1) led to the death of all the animals in 3-4 days, with the development of signs of chronic PE for animals (loss activity, lethargy, lack of reactions to painful stimuli and coma).

Introduction of protectors Σ 3 and LOLA 24 hours after the CCl 4 (1 per day) resulted in a decrease in mortality and prevent the development of symptoms of chronic Peh. Indicators for Σ 3 were higher than for the protector LOLA (Tab 2 rows 2, 3).

And, finally, in the model of type 2 Diabetes, non-fatal dose CCl 4 (for healthy animals) cause death of animals within 3-4 days, due to the development of and coma, with all registrable signs of chronic Peh. Daily administration of the protectors also causes the survival of animal parts. Composition of Ultrasonic again proves to be more effective than LOLA (Table 2, line 5, 6).

Table 3 shows the data on parameters of the blood of animals with type 2 Diabetes, received from control animals (before the introduction of the CCL 4 ), and in 72 hours after introduction of the CCL 4 and the subsequent introduction of protectors. The comparison shows that the composition of ultrasonic has a high efficiency of hepatoprotector and tread chronic Peh.

The chronic toxicity of compounds Σ 2 and Σ 3 was determined in experiments with the introduction of these protectors within 1 month per os. Blood parameters are normal, histological changes in the liver (Fig.1) and brain is not detected. These data show that the investigated compounds are not toxic.

The chronic toxicity of compounds Σ 2 and Σ 3 was determined in experiments with the introduction of these protectors within 1 month per os. Blood parameters are normal, histological changes in the liver (Fig.1) and brain is not detected. These data show that the investigated compounds are not toxic.

Clinical studies on patients

Research of these compounds have also been conducted in patients with chronic hepatic encephalopathy due to hepatic cirrhosis (HepB). Tested the ability of hepatoprotectors reduce and reduce the degree of development of hepatic encephalopathy, estimated to record the frequency of critical flicker in seconds (KCHM test). On Fig. 2, 3 presents data a month of receiving the drug, made by the Example 1 the rate of 8 g 1 reception 2 times a day for 30 minutes before eating. It is seen that use of the drug leads to a decrease in the concentration of the toxin NH 4 + plasma arterial blood. 11 patients took place 2-3-fold lower concentration NH 4 + , in 10 patients, a decline of 30 to 60%. Transaminaz activity in the blood remained within the normal range in 18 patients, 3 patients is decreased. Evaluation stages of the Peh, recorded using the test KCHM, 16 patients was an increase in the indicator KCHM and improvement stages of PE (transition from the stage of the Peh of the II stage of the Peh, I, 0), and 5 patients stage PE I persisted. The General condition and assessment of the health of patients significantly improved.

In such a way investigational drugs are effective with liver protecting drugs and protectors of the Peh, ensuring the effective reduction of the concentration of endotoxin NH 4 + as in the conditions of acute and chronic forms of PE; the survival of the animal model in acute experiments; improvement of the patients and produces reversionary stages of chronic PE in patients with liver failure caused by cirrhosis.

The proposed formulations songs

There were identified the following set of features to ensure the achievement of the result:

1. The composition of the protectors of acute and chronic forms of hepatic encephalopathy, containing per dose of L-carnitine is 1.5-3 g, acetyl-L-carnitine - 1-3 g succinate (amber acid) - 0,75-1,5 g L-glutamate (glutamic acid) - 0,75-1,5 g L-arginine - 1.5-2.5 g, betaine - 0,25-2,

3. The composition of the protectors of acute and chronic forms of hepatic encephalopathy, containing per dose of L-carnitine is 1.5-3 g, acetyl-L-carnitine - 1-3 g succinate - 0,75-1,5 g L-glutamate - 0,75-1,5 g L-arginine - 1.5-2.5 g, betaine - 0,25-2 g, coenzyme Q 10 - 20-30 mg and - 15-20 mg

4. Composition according to claim 1, characterized in that it is executed in the form of powders or granules with the addition of Na 3 in equimolar acids doses, as well as other daughter components, reducing adhesion of components of powders or granules, powders or granules implemented with the possibility of dissolution in water for use as a Fizz and packaged in sachets-packages.

5. The composition according to claim 2, characterized in that it is executed in the form of powders or granules with the addition of NaHCO 3 in equimolar acids doses, and other components, reducing adhesion of components of powders or granules, powders or granules are made with the ability of dissolution in water for use as a Fizz and packaged in sachets-packages.

6. The composition according to claim 3, wherein it is made in the form of powders or granules with the addition of N 3 in equimolar acids doses, as well as other daughter components, reducing adhesion of components of powders or granules, powders or granules are made with the ability of dissolution in water for use as a Fizz and packaged in sachets-packages.

7. The method of treatment of acute and chronic forms of hepatic encephalopathy, and liver disease, selected from acute and chronic hepatitis and steatohepatitis and cirrhosis of different etiology, including oral administration to patients from 1 to 6 doses per day, in any combination, according to claims 1 to 6, depending on patient's weight and individual tolerance components of compositions.

Table 1

Modeling of acute PE and protectors action in the experiment on mice in vivo

No.

Background condition of animals

Toxin Dose

Protector

Number of animals in the experiment 1

The time before the destruction of all or animal parts (min)

% survival

1

control

LD100 NH 4 Cl 16.5 mmol/kg

NaCl

10

10-15

0

2

-||-

LD100+NH 4 Cl 18.5 mmol/kg

NaCl

10

8-12

0

3

-||-

LD100+NH 4 Cl

Σ 1

10, 10

20-40

40, 70

4

-||-

LD100+NH 4 Cl

Σ 2

10, 10

15-20

70, 100

5

-||-

LD100+NH 4 Cl

LOLA

10, 10

15-20

60, 80

6

50% Σ 2 15 min

LD100+NH 4 Cl

NaCL

10

12-20

100

7

50% LOLA 15 min

LD100+NH 4 Cl

NaCL

10

12-20

70

8

Propionate Na 25 mmol/kg 15 minutes before the NH 4 Cl

LD30 NH 4 Cl 14 mmol/kg

NaCl

10, 10

15-20

0

9

-||-

LD100 NH 4 Cl

NaCl

10

8-12

0

10

-||-

LD100 NH 4 Cl

Σ 2

10,10

20-40

60, 80

11

-||-

LD100 NH 4 Cl

LOLA

10, 10

20-30

30, 40

12

Acute hepatitis CCl 4 2 ml/kg in 24 hours

LD100+NH 4 Cl

NaCl

10

10-12

80

13

CCl 4 3 ml/kg in 24 hours

LD100+NH 4 Cl

NaCl

10

15-30

50

14

CCl 4 4 ml/kg in 24 hours

LD100+NH 4 Cl

NaCl

10

7-12

0

Table comparison 2

Modeling of chronic PE and effectiveness of the action of the protectors of the experiment on mice in vivo

No.

Background condition of animals

Toxin dose

Protector*

Number of animals in the experiment 1

The time before the destruction of all or parts of animals (hours)

% survival

1

CCl 4 1 time I.P. Pavlova. 4 mg/kg

NH 4 Cl 10 mmol/kg

NaCl

10

72-96

0

2

-||-

-||-

Σ 3 1 time per day

10

72-132

70

3

-||-

-||-

LOLA 1 time per day

10

72-120

30

4

Type 2 diabetes

CCl 4 , 2 ml/kg

NaCl

10 10

72-96 72

0

5

-||-

-||-

Σ 3

10 10

84-120 72

60

6

-||-

-||-

LOLA

10 10

84-108 72

20

Introduction 24 hours after the toxin within 3 days.

Table 3 comparison

Comparison of the effectiveness of the drugs Σ 3 and LOLA in surviving animals with type 2 diabetes, after the introduction of the CCl 4 and subsequent daily introduction of protectors (in accordance with the Protocol on claims 4-6, PL. 2).

AAT plasma U/liter

Plasma glucose mmol

SJK plasma mmol

Control

85 of + 20

6,6 approximately 0.5

0,41±0,15

Diabetes mellitus type 2 (D2)

16,8 about 1.1

1,8±0,35

D2, CCl 4 , the introduction of NaCl 2 days

420±50

18,3 ą1.5

2,1±0,56

D2, CCl 4 , introduction Σ 3 3 days

150±35

11,1±0,8

1,5±0,32

D2, CCl 4 , introduction LOLA 3 days

280±40

13,8 about 1.1

1,7±0,41

1. The composition of the protectors of acute and chronic forms of hepatic encephalopathy, containing per dose: L-carnitine is 1.5-3 g, acetyl-L-carnitine - 1-3 g succinate (amber acid) - 0,75-1,5 g L-glutamate (glutamic acid) - 0.75-1.5 g, L-arginine is 1.5-2.5 g, betaine -0,25-2,

2. The composition of the protectors of acute and chronic forms of hepatic encephalopathy, containing per dose: L-carnitine is 1.5-3 g, acetyl-L-carnitine - 1-3 g succinate (amber acid) - 0,75-1,5 g L-glutamate (glutamic acid) - 0.75-1.5 g, L-arginine is 1.5-2.5 g, betaine - 0,25-2 g, phosphocreatine, 2-3 g, N-acetylcysteine - 0,25-1,5,

3. The composition of the protectors of acute and chronic forms of hepatic encephalopathy, containing per dose: L-carnitine is 1.5-3 g, acetyl-L-carnitine - 1-3 g succinate - 0,75-1,5 g L-glutamate - 0,75-1,5 g L-arginine - 1.5-2.5 g, betaine - 0,25-2 g, coenzyme Q 10 - 20-30 mg and - 15-20 mg

4. Composition according to claim 1-3, wherein it is made in the form of powders or granules with the addition of NaHCO 3 in equimolar acids doses, as well as other daughter components, reducing adhesion of components of powders or granules, powders or granules are made with the ability of dissolution in water for use as a Fizz and packaged in sachets-packages.

5. The method of treatment of acute and chronic forms of hepatic encephalopathy, including the introduction of patients composition selected from the music by : 1-4.