LV14677B - Agent for restoration of membranes of hepatocytes in case of toxic liver damage - Google Patents

Abstract

Invention relates to the field of pharmacology and particularly compositions for restoration of membranes of hepatocytes in case of toxic liver damage. The invention provides a composition comprising metoprolol and essential phospholipids.

Description

DESCRIPTION OF THE INVENTION

The invention relates to the pharmaceutical industry and the medical industry, and relates to the issue of regeneration of hepatocyte membranes in the event of liver toxicity, in particular to the development of compositions for repairing hepatocyte membranes in the event of liver toxicity.

TECHNICAL LEVEL

The liver plays a leading role in the biotransformation and excretion of many chemicals, and is sensitive to the toxic effects of drugs and other substances. Hepatic toxicity causes both subclinical and severe forms of hepatitis. Various terms are used to denote hepatic toxicity: toxic hepatitis, hepatosis, toxic hepatopathy. Hepatic toxicity is manifested as damage and / or collapse of liver cells - cytolysis syndrome. The term "cytolysis" refers to the entire spectrum of changes in membrane permeability of hepatocytes, from minor disruptions to collapse of plasma and intracellular membranes, thus the destruction of the hepatocyte itself [1].

The problem of liver toxic damage is a current medical problem, as mortality in severe forms reaches 70-90% and there is a high risk of hepatocyte pathological processes being synchronized. The rapid increase in the number of medicinal products, the changes in the immunological status of the patients, the increase in the number of elderly patients, contribute to the increase in the number of side-effects of the medicinal products, especially concerning toxic liver damage. In most cases, progression of the causal factor (such as alcohol intoxication) is likely to progress the disease and develop cirrhosis of the liver.

Currently, the problem of hepatic toxic damage is becoming more and more acute due to the increase in alcohol and its surrogate consumption [1-6].

Among the etiological risks of developing hepatic toxic injury, pharmaceuticals are of particular importance. More than 900 cures and about 6 million chemicals with proven hepatotoxicity are known. True hepatotoxins include direct-acting preparations (paracetamol) and intermediate-acting antimetabolites and similar compounds that cause hepatocyte damage. Paracetamol, aspirin, diclofenac, and the like are the drugs most commonly used to cause hepatocyte toxicity. [7-9].

Paracetamol is a component of many preparations. Patients who have been forced to take paracetamol for a long time (for years) are predominantly people with chronic inflammatory and degenerative connective tissue disorders: rheumatologic patients, elderly patients with osteoarthritis, arthritis, and the like. diseases [10-13]. Studies in volunteers have shown that administration of a therapeutic dose of paracetamol for more than 14 days contributes to an increase in liver injury rates [14-15].

Until the 20th century In the early 1980s, works describing liver damage in patients treated with aspirin (acetylsalicylic acid) [15] were rife. Use of aspirin is also associated with the so-called Ray's syndrome, in which acute liver damage is observed in children and adolescents. Subsequent studies confirmed that patients on long-term treatment with non-steroidal anti-inflammatory drugs have severe hepatocyte dysfunction with more than 8-fold higher transaminases. Monitoring of liver transaminases every 4-8 weeks is recommended in these patients. French researchers in the late 1980s published a report on 83 cases of acute hepatitis (6 fatalities) associated with the use of non-steroidal anti-inflammatory drugs (NSAIDs). The authors recommended that liver tests be performed in the case of long-term use of NSAIDs, especially during the first 6 months [16],

Drug toxicity is the cause of liver injury and associated jaundice in 2-5% of hospitalized patients, hepatitis in 50% of patients> 40 years and severe liver failure in 25% of patients.

According to the recommendations of the American and Canadian Gastroenterological Society, the damage to toxic hepatocyte membranes should be determined by the total of the following criteria [16-17]:

1. Triple and multiple elevations of transaminases (alanine aminotransferase, ALT (EC 2.6.1.2) and aspartate aminotransferase, ASAT (EC 2.6.1.1)). It confirms toxic damage to hepatocyte membranes. Physiologically, serum alanine aminotransferase and aspartate aminotransferase are less than 35 U / l. A double increase in alanine aminotransferase and aspartate aminotransferase upon repeated administration is a toxic sign of hepatocyte membrane damage due to the hepatotoxicity of the product used. Alanine aminotransferase and serum aspartate aminotransferase are determined by spectrophotometry using the Warburg optical test. The principle of the assay is based on the difference in absorbance of the reduced form of nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD) at 340 nm [18-20].

2. Double and multiple increases in gamma glutamyltranspeptidase, GGT (EC 2.3.22). Gammagglutamyltranspeptidase activity is determined during the treatment process for hepatocyte toxic injury. Physiologically, serum gamma glutamyltranspeptidase activity ranges from 11 to 49 U / l [18-20]. The principle of the method for determining the level of gamma-glutamyltransferase is based on the transfer of the residual glutamyl from gamma-L - (+) glutamyl-4-nitroaniline to the dipeptide acceptor, glycylglycine, which is also a buffer. GGT concentration is determined photometrically by tint intensity [20-21].

Phospholipids and plant flavonoids are known for the treatment of toxic damage to hepatocyte membranes:

• Preparations containing essential phospholipids: Esenciale and others [21, 22, 23, 24] • Preparations containing flavonoids of thistle plants: silymarin, cariesil [25,26] • Preparations containing other plant flavonoids: Hofitol (artichoke extract) • Animal organ preparations: cinepars (liver extract).

Of herbal preparations, silymarin is used, which is a thistle extract (Silybum marianum). Silymarin is used in acute and chronic liver disease.

Esenciale Forte is a combination product containing essential phospholipids as its main active ingredients as well as a balanced vitamin complex. In clinical practice, preparations of essential phospholipids are used in cases of hepatic toxicity (especially alcohol), and in the case of hepatotoxic medicinal products. Since 1989, five clinical trials have been conducted to demonstrate the efficacy of phospholipids in alcoholic liver disease and other causes of hepatic steatosis and in the use of hepatotoxic agents. However, no positive effect of these products on liver function has been observed.

The treatment of hepatic toxic injury is complicated by the fact that in most cases it is difficult to reverse or reduce the dose of the hepatotoxic medicinal product (eg in the treatment of tuberculosis with isoniazid). In many cases, paracetamol withdrawal is accompanied by chronic pain syndrome. Problems of finding hepatocyte function restorative agents associated with hepatic toxicity are well known to those skilled in the art of restoring hepatocyte function in the event of hepatic toxicity.

Metoprolol (Metoproloy), a beta blocker without intrinsic sympathomimetic activity, is known. Metoprolol has hypotensive, antianginal and antiarrhythmic effects. Metoprolol is used in arterial hypertension, angina, arrhythmia [27,28],

SUMMARY OF THE INVENTION

Preventing toxic damage to hepatocyte membranes is a complex problem. The first signs of hepatocyte damage are manifested by hepatocyte nuclear dystrophy and necrosis. One of the first and objective causes of hepatocyte injury is a double and greater elevation of enzyme-indicator levels (ALT, ASAT, GGT) in the blood: ALT and ASAT from 70 U / l and higher, GGT - above 100 U / l. Hitherto known preparations for the treatment of liver injury do not allow to reduce abnormally high levels of ALT, ASAT and GGT. Known preparations do not have the specificity of restoring hepatocyte membranes in the event of hepatic toxicity.

The object and technical result of the invention is the development of a composition for the regeneration of hepatocyte membranes in the event of liver toxicity.

The objective is achieved by the development of a novel agent containing metoprolol, essential phospholipids and emulsifier in the following amounts (mg):

metoprolol 100.0 - 110.0 essential phospholipids 100.0-200.0 emulsifier 2.0-10.0.

Metoprolol (Metoproloy) is a Zeta-blocker without intrinsic sympathomimetic activity. Metoprolol is a white, crystalline powder, freely soluble in water.

Essential Phospholipids - (EPL). The product obtained from linseed oil (Oleum Lini) is an ethyl ether containing diglyceride residues, phosphoric acid and choline residues in combination with a mixture of unsaturated fatty acid ethyl ethers: oleic acid (approximately 15%), linoleic acid (approximately 15%) and linoleic acid (approximately 57%). ). The content of saturated fatty acids reaches 9-11%. Yellowish, mobile, oily liquid with a slightly bitter taste. Do not mix with water, mix with alcohol and ether in any proportion.

Information on the difficulty of formulating a hepatocyte membrane reconstitution composition for hepatic toxicity is well known to those of skill in the art, pharmacists, therapists, and hepatologists involved in the repair of hepatocytes and their membranes in the event of hepatic toxicity.

Suggested Examples for Executing the Invention

The complexity of this invention is due to the lack of knowledge of the components of the novel agent and the effective interrelationship between these components to ensure the ability of the novel agent to restore hepatocytes in the event of hepatic toxicity of the membrane. The desired pharmacological form of the product was also unknown. The complexity of the invention was also exacerbated by the fact that the components of the novel agent are both hydrophilic (metoprolol) and hydrophobic (essential phospholipids).

In order to develop and obtain the optimal ratios of the components of the new agent, the composition with different ratios of metoprolol, essential phospholipids and emulsifier was studied.

The pharmacological forms of the new agent were investigated experimentally. Specifically, the capsule form was used.

Decreasing the amount of metoprolol by less than 100 mg resulted in a decrease in the efficacy of the agent in restoring hepatocyte membranes in hepatic toxic injury: high enzyme-indicator activity: ASAT and ALT above 150 U / L, GGT above 200 U / L. Increasing the amount of metoprolol by more than 100 mg caused a decrease in blood pressure in some patients;

Reduction of the amount of essential phospholipids by less than 100 mg resulted in a decrease in the efficacy of the agent in the restoration of hepatocyte membranes in hepatic toxicity: the activity of enzyme-inducers in the blood decreased slightly but remained high: ASAT and ALT above 150 U / L, GGT - above 200 U / L. Increasing the amount of essential phospholipids by more than 200 mg caused difficulties in the production of the capsule formulation of the new agent, which became unstable.

Each component of a proposed product has only its specific properties. The combined effect of the components makes it possible to obtain an effect that can lead to the development of a new agent that restores hepatocytes in the event of toxic liver damage.

The emulsifier reduces the surface tension at the phase boundary of the dispersion system. Complex glycerol essential oils and high molecular fatty acids, glycerol monooleate, sorbitan ethers and polyoxyethylene sorbitan and higher fatty acids, distilled monoglycerides, natural phospholipids and combinations thereof may be used in place of the emulsifier. Reducing the amount of emulsifier by less than 2 gr or increasing the amount of emulsifier by more than 10 gr causes difficulties in technological processes - it is difficult to obtain a homogeneous mass.

The mutual composition of the experimentally obtained novel agent gave a stable formulation suitable for production in capsule form, maintaining the technological properties and stability for 24 months.

EXAMPLES I-II

Example I

The new product includes:

metoprolol 100.0 mg essential phospholipids 100.0 mg emulsifier 2.0 mg.

Method of preparation of the composition:

To prepare the product, weigh 100 mg of metoprolol succinate, place it in a laboratory glass and heat in a water bath to 45-50 ° C. Weigh separately and add 2 mg of the emulsifier (eg glycerol monoolacetate), add 100 mg of essential phospholipid, heat the mixture in a water bath to 45-50 ° C, stirring constantly until a homogeneous mass is obtained. The production of capsules requires special equipment for pressing the capsules by rotary-matrix method.

Example II

The new product includes:

metoprolol 110.0 mg essential phospholipids 200.0 mg emulsifier 10.0 mg.

Formulation method - analogous to Example I.

The agent containing metoprolol, essential phospholipids and emulsifier obtained according to the described technology was successfully used to prepare a dosage form in the form of soft gelatine capsules containing 202.0-320.0 mg of the new agent.

To confirm the study on the restoration of hepatocyte membrane function in the event of hepatic toxic injury, an experimental study was conducted with mice (25-30 g) kept under standard vivarium conditions with free access to food and water.

The effect of hepatocyte membrane repair was investigated histologically: by the number of stored hepatocytes in liver histology. A series of experiments were performed on 24 animals divided into 3 groups:

- control group (A) - 8 mice with induced toxic liver injury (mice poisoned with acetaminophen).

- experimental group (B) - 8 mice with induced toxic liver damage (mice poisoned with acetaminophen), a new agent was given to the mice.

- experimental group (C) - 8 mice with induced toxic liver damage (mice poisoned with acetaminophen), the mice were given Essenciale.

Use of the new agent in experimental group (B), with drug-induced liver injury, improved the histology of liver parenchyma: 1) disappearance of hepatocyte lesion (collapse), 2) 30% improvement in hepatocyte regeneration, and 3) reduction of connective tissue formation in liver parenchyma .

Use of the new agent in the experimental group (C), with drug-induced liver injury, improved the histology of hepatic parenchymal histology by 10%, while the number of damaged hepatocytes was more than 30% than in group (B).

A prospective clinical study was conducted between 2009 and 2011 to investigate the effects of the new agent on restoring hepatocyte membrane function in hepatic toxicity due to long-term hepatotoxicity and / or long-term alcohol use. . Patients' blood serum had elevated enzymes ALAT, ASAT, GGT (ASAT and ALAT above 70 U / L, GGT above 100 U / L). The study was conducted using the Pauls Stradins Clinical University Hospital base.

In the first series, 40 patients were observed. Patients in the first series used paracetamol 1 g daily for chronic pain (2-3 months).

In the second series, 40 patients with chronic alcoholism were observed.

Patients in the first series who received long-term treatment with paracetamol (40 people in total) were divided into two subgroups who received 1) a new agent once a day (20 patients) and 2) Essenciale 400 mg 2 times a day (20 patients) ). During follow-up (2 months.), Serum levels of enzymes (ALT, ASAT, GGT) were tested.

Second-line patients (chronic alcoholism, 40 people) were also divided into two subgroups, with 1) a new agent once a day (20 patients) and Essenciale 400 mg twice a day (20 patients). During the follow-up (2 months), serum levels of enzymes (ALT, ASAT, GGT) were tested.

Enzyme activity was found to be normal in the AST - 18 U / L, ALT - 25 U / L (normal to 35 U / L), GGT - 38 U / L (normal 11-49 U / L) subgroups, additionally, the new agent was used, both in the subacute hepatic toxicity group with paracetamol and in the alcoholic liver subgroup. The results of the study showed that the new agent restores hepatocyte membrane function in the 40 (20 + 20) patients who initially had hepatic toxicity due to prolonged use of paracetamol (20 patients) or chronic alcoholism (20 patients).

In the subgroup of patients receiving Essentiale, enzyme-indicator activity declined slightly but was still high: AST and ALT greater than 150 U / L (normal to 35 U / L), GGT greater than 200 U / L (normal 11 -49 U / L). The results of the study confirmed the assumption that hepatocyte membranes do not regenerate with Essentiale.

Statistical analysis of the study results was performed using SPSS 16.0 software. Variance statistical methods were used, using paired and independent Student's t-criteria for correlation analysis. A p <0.05 level was considered as a confidence criterion. Nonparametric criteria were also used - Wilcoxon Signed Ranks Test. Reliable differences (p <0.01 and p <0.05) were considered when comparing the series of variations.

Research was done with permission of Riga Stradins University Ethics Commission. The study protocol, consent protocol, and membership form are in accordance with the European Directive on Good Clinical Practice and the Helsinki Declaration. There is no conflict of interest.

EXTREME USE

A new formulation containing metoprolol, essential phospholipids and an emulsifier was used as a remedy for hepatocyte membrane repair in the event of liver toxicity. The timing of the new product does not depend on the timing of the food. The new agent, which restores hepatocytes in the case of hepatic toxicity of the membrane, takes 4-6 months. It can be extended to 9 months and longer if needed, depending on the adverse effects.

Industrial form of agent that restores hepatocytes in the event of hepatic toxicity of the membrane:

• The new agent can be used in the form of capsules with a uniform oily suspension filling.

• tablets containing 100-110 mg metoprolol and capsules containing Esenciale (100-200 mg) can be taken at the same time.

Sources of information

1. Friedman, Scott E.; Grendell, James H.; McQuaid, Kenneth R. (2003). Current diagnosis & treatment in gastroenterology. New York: Lang Medical Books / McGraw-Hill. pp. 664-679.

2. McNally, Peter F. (2006). GELiver Secrets: with STUDENT CONSULT Access. Saint Louis: C.V. Mosby], [Lee W.M., Senior J.R. // Toxicol. Pathol. - 2005. V.33 (1) .— P.155-164.2, 8.21.

3. Pat. RU 2430682, Cl, 2011, A61B 8/06.

4. Pat. RU 2442981, Cl, 2012, G01N 33/48.

5. Pat. EP 2135557, Al, 2009, A61B 8/06.

6. Pat. RU 2267998, Cl, 2006, A61B 10/00.

7. Gunnell D, Murray V, Hawton K (2000). «Use of paracetamol (acetaminophen) for suicide and nonfatal poisoning: worldwide pattems of use and misuse». Suicide & life-threatening behavior 30 (4): 313-26.

8. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002). Mechanisms of hepatotoxicity. Toxicol. Sci. 65 (2): 166-76.

9. L.F. Perscott Salicylate hepatitis. In Davis M., Tredger J.M., Williams R., eds. Drug Reactions and the Liver. London: Pluman Medical, 1981,267-269.

10. Pat. WO 03/032973, A2, 2003, A61K 31/167.

11. Pat. WO 2006/016126, Al, A61K 9/16.

12. Pat. WO 2007/128413, Al, A61K 31/167.

13. Pat. RU 2442576, Cl, 2012, A61K 31/137.

14. Lee W.M. Senior J.R. // Toxicol. Pathol. - 2005. -V.33 (1). - P. 155-164.2, 8, 21.

15. Bell L. N., Chalasani N. Epidemiology of Idiosyncratic Drug-Induced Liver Injury // Semin Liver Dis. 2009; 29 (4): 337-347 Benichou C (1990). Criteria of drug-induced liver disorders. Report of an intemational consensus meeting.

J. Hepatol. 11 (2): 272-6.

16. Andrade RJ, Lucena Ml, Kaplowitz N, et al. (2006). Outcome of acute idiosyncratic drug-induced liver injury: Long-term follow-up in a hepatotoxicity registry. Hepatology44 (6); 1581-8.

17. R. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 8th Edition, Biomedical Publications, Foster City, CA, 2008, p. 1431-3.

18. Navarro V. J., Senior J. R. // New Engl. J. Med. - 2006. - V. 354. - P.731-739

19. Andrade RJ, Robles M, Femandez-Castaner A, Lopez-Ortega S, Lopez-Vega MC, Lucena Ml (2007). Evaluation of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists. World J. Gastroenterol. 13 (3): 329-40.

20. http: // www.eurolab.ua/hepatit/2134/.08.10.2012.

21. Keeffe, Emmet B; Friedman, Lawrence M. (2004). Handbook of liver diseases. Edinburgh: Churchill Livingstone. pp. 104-123.

22 Ejnorep Α.Φ., Mafiope Α.Ή, HccjieņoBaHne ochobhhx naToreHeTHHecKnx'jiHHHH nopaaceHHfl κηετοκ without taking b ycjioBnax KJIHHHHeCKOH Η ΟΚΟΠερΗΜβΗΤβΠΒΗΟΗ naTOJIOrHH Η ΠΟΑΧΟΛΗ κ peryJWUHH Η KynnpoBaHHio οτηχ npopeccoB // Υεπεχη renaTOJio-ran. 1982. - Βηπ. 10. - C. 12-34.

23. Pat. RU 2282457, Cl, 2006, A61K 36/74.

24. http://ru.wikipedia.org/wiki./ 10/7/2012.

25. Bergymeyer H.U., et al. // Clin. chem. 1987. V. 24. P. 58-73.

26. Bedi S., Goel A., Chand N., Rai J. A prospective study of evaluating the effect of Silymarin on hepatic function in tubercular patients receiving DOTS Chest 2009 136: 4.

27. Corrigan 0. The biopharmaceutical drug classification and drugs administered in extended release (ER) formulation. Adv Exp Med Biol 1997: 423: 111-28.

28. Pat. WO 02/072074, Al, 2002, A61K 9/16.

Claims (1)

A remedy for hepatocyte membrane repair in the event of liver toxicity is characterized by the presence of metoprolol, essential phospholipids and emulsifier in the following amounts (mg): metoprolol 100.0 -110.0 essential phospholipids 100.0 - 200.0 emulsifier 2.0-10.0.