KR102569081B1 - Oral pharmaceutical form for lactitol and non-alcoholic fatty liver disease - Google Patents

Abstract

The present invention relates to the pharmaceutical field, in particular to the use of lactitol in a therapeutically effective amount. Preferably, it relates to the use of lactitol in a dose of 1-20 g/day for the prevention and treatment of non-alcoholic fatty liver disease in a mammal in need thereof. The present invention provides an extension of the range of effective means for the treatment and prevention of non-alcoholic fatty liver disease.

Description

Oral pharmaceutical form for lactitol and non-alcoholic fatty liver disease

The present invention relates to the field of pharmaceuticals, and in particular to drugs for the treatment and prevention of non-alcoholic fatty liver disease.

Fatty hepatosis or non-alcoholic fatty liver disease-NAFLD (liver steatosis, fatty infiltration, fatty degeneration of the liver) is a condition in which more than 5% of the liver mass is fat, mainly neutral It is a fat state. When the fat content exceeds 10% of the organ weight, more than 50% of hepatocytes contain fat, and fat accumulation is distributed throughout the liver tissue.

The causes of fatty liver dysfunction are metabolic syndrome-metabolic disorders and hormonal changes. In this case, there is a risk of developing diabetes mellitus and cardiovascular complications due to increased levels of lipids in the blood.

Fatty degeneration of hepatocytes can be caused by:

- alcohol abuse;

- obesity;

- certain viral infections (hepatitis B and C viruses);

- nutritional disorder;

- metabolic disorders in diabetes mellitus;

- increased liver enzymes (ALT, AST, GGT);

- genetic defects of the urea cycle and oxidation of fatty acids;

- genetic factors;

- Certain medications, eg non-steroidal anti-inflammatory drugs

NAFLD is based on insulin resistance (cellular immunity to insulin) and metabolic disorders, primarily lipid and carbohydrate disorders. Fatty degeneration of the liver occurs because of increased uptake of fatty acids in the liver with food or under increased lipolysis (breakdown of fat in adipose tissue).

NAFLD is a multifactorial disease subject to a combination of risk factors:

- abdominal obesity (waist size greater than 94 cm for men and greater than 80 cm for women);

- Abuse of alcohol and harmful foods;

- atherosclerosis due to high blood lipid levels, increased blood triglycerides to levels above 1.7 mmol/L, increased cholesterol levels and decreased levels of high-density lipoproteins;

- an increase in blood pressure to values greater than 130/85 mmHg;

-impaired glucose tolerance, long-term hyperglycemia (diabetes mellitus type 2);

- insulin resistance;

- Working with toxic substances.

- Taking multiple medications;

- after surgery on the gastrointestinal tract;

- Suffering from hereditary chronic diseases

NAFLD includes the following developmental stages:

1) hepatic steatosis - accumulation of fat inclusions in hepatocytes;

2) non-alcoholic steatohepatitis - the number of fat inclusions increases;

3) Fibrosis - Excessive fat content destroys liver cells and forms fatty cysts that promote replacement of liver tissue with fibrous tissue.

4) develop liver cirrhosis as a result of NAFLD, with an increased risk of developing hepatocellular carcinoma.

NAFLD manifests itself in the following stages:

- hepatocyte steatosis - hepatocytes are not destroyed and organ function is practically intact;

- hepatocyte necrobiosis - cell death due to accumulation of fat, cyst formation, mesenchymal cell response occurs;

- pre-cirrhotic stage - connective tissue of the organ is replaced by mesenchymal tissue;

- Cirrhotic stage

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases in the world. Therefore, according to epidemiological studies, 10-40% of the world population suffers from NAFLD ( Buyeverov AO: Chronic liver diseases: a brief guide for practitioners. 2nd ed. ― M.: Medical News Agency, 2014, 137 p. ;Fatty liver degeneration and ischemic heart disease. , modern concept of pathogenesis, clinical features, principles of diagnosis and treatment) / Textbook. - SPb., 2011, 53 p.; Trukhan DI: Non-alcoholic fatty liver disease in the practice of a "first contact" physician / Clinical prospects of gastroenterology, hepatology, 2012, N.1, pp.3-9) . Sometimes, NAFLD is the first organ damage of metabolic syndrome and is an important predictor of the development and progression of diabetes mellitus or cardiovascular disease (Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP, M .: Concept Design LLC, 2014, 346 p.; Azizov VA et al.: Non-alcoholic fatty liver disease and cardiovascular complications: what is the relationship? / Eurasian Journal of Cardiology, 2013, N.1, pp. 63-69; Schiff, Yu.R. et al. Alcoholic, medicinal, genetic and metabolic diseases / trans. from English; ed. by Mukhin NA et al., M., GEOTAR-Media, 2011, 480 p.). Fatty disease usually develops between the ages of 40 and 60, and women suffer more often. Fatty diseases also affect children. In the case of overweight with an index of 30 (weight (kg) divided by height (m) squared), the probability of fatty liver dysfunction is 40%.

NAFLD combines a variety of liver damage including hepatic steatosis, nonalcoholic steatohepatitis (NASH), steatofibrosis and steatocirrhosis (Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP , M., Concept Design LLC, 2014, 346 p.; Trukhan DI Non-alcoholic fatty liver disease in the practice of a "first contact" physician/Clinical prospects of gastroenterology, hepatology, 2012, N.1, pp.3- 9; Schiff Yu.R. et al. Alcoholic, medicinal, genetic and metabolic diseases / trans. from English; ed. by Mukhin NA et al., M., GEOTAR-Media, 2011, 480 p.; Korneeva EV et al. Pathophysiology of metabolic syndrome / Monograph, M., "Higher Education and Science" Publishing House, 2012, 136 p. ). NASH is known to be a comorbid of the metabolic syndrome and is actually a hepatic manifestation of the metabolic syndrome ( Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP, M., Concept Design LLC, 2014, 346 p.; Korneeva EV et al. Pathophysiology of metabolic syndrome / Monograph. - M., "Higher Education and Science" Publishing House, 2012, 136 p.; Stelmakh VV et al. Energotropic pathogenetically oriented therapy with succinate-containing drugs in non-alcoholic fatty liver disease: Methodological recommendations, SPb., Tactic Studio, 2014, 40 p. ). The onset of NASH with metabolic syndrome was associated with 37.5% of cases ( Arapkina OM: Non-alcoholic fatty liver disease - metabolic syndrome / Medical Almanac, 2010, N.1, pp.162-163 ). Due to obesity, NASH occurs in 20-47% of cases, 15% of cases with type 2 diabetes mellitus, and 20-80% of cases with dyslipidemia. If NASH is left untreated, NASH is known to progress to cirrhosis with the development of portal hypertension, hepatic cell failure, and hepatocellular carcinoma in 5-25% of cases ( Fatty liver degeneration and ischemic heart disease. English; ed. by Mukhin NA et al., M., GEOTAR-Media, 2011, 480 p.; Shaposhnikov AV Hepatooncoprevention. Concept and principles of implementation / Manual for physicians, M., Forte print, 2013, 80 p.) .

NASH is actively involved in the symptoms and exacerbation of atherogenic dyslipidemia (Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP, M., Concept Design LLC, 2014, 346 p. Schiff Yu.R. et al. Alcoholic, medicinal, genetic and metabolic diseases / trans. from English; ed. by Mukhin NA et al., M., GEOTAR-Media, 2011, 480 p.; Drapkina OM et al. : Atherogenic dyslipidemia and the liver/Medical alphabet, 2012, V.4, N.24, pp.30-35 ). Atherosclerotic dyslipidemia is associated with the possibility of ischemic heart disease (IHD) and acute myocardial infarction (AMI) and acute cerebrovascular accident (ACVA) (stroke). Increases complications ( Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP, M., Concept Design LLC, 2014, 346 p.; Azizov VA et al.: Non-alcoholic fatty liver disease and cardiovascular complications: what is the relationship? / Eurasian Journal of Cardiology, 2013, N.1, pp. 63-69 ). NASH is also very important in the progression of carbohydrate metabolism disorders ( Fatty liver degeneration and ischemic heart disease. Geriatric aspects. Monograph / ed. Khoroshinina LP, M., Concept Design LLC, 2014, 346 p.; Schiff Yu.R. et al Alcoholic, medicinal, genetic and metabolic diseases / trans. from English; ed. by Mukhin NA et al., M., GEOTAR-Media, 2011, 480 p. ). Chronic hyperglycemia likewise causes a high risk of cardiovascular pathology.

From a biological point of view, NASH is explained by the so-called “multiple parallel hits” hypothesis, according to which the pathogenesis of NAFLD is a model of the interaction of factors. Examples of factors include insulin resistance, activation of lipid peroxidation processes, sustained production of pro-inflammatory cytokines such as TNF-α, IL-6, IL-8, etc., and adipose tissue production of the so - called adipocytokines: leptin, resistin, and adiponectin by Medical News Agency, 2014, 137 p. ).

The main causes of non-alcoholic fatty liver disease (NAFLD) are insulin resistance and oxidative stress. Since insulin resistance and consequent inability to use glucose as an energy source occur in the body, this is compensated by activation of lipolysis and extraction of fatty acids from peripheral tissues. When large amounts of free fatty acids (FFAs) enter the liver, it leads to fatty burden and steatosis formation in the hepatic parenchyma.

As a rule, NAFLD is dangerous because it progresses gradually and can progress to cirrhosis. It is estimated that within the next 20-30 years, fatty liver disease will become the most common cause of cirrhosis requiring transplantation ( "Transplantation of the liver", National Clinical Recommendations, 2013, 42 pp. ).

Hepatocytes are the main functional cells of the liver, and each individual hepatocyte is 15-30 microns in diameter. There are about 250 billion hepatocytes in the human liver. Severe damage to hepatocytes results in cell necrosis. In case of necrosis, hepatocytes can regenerate by dividing intact hepatocytes. Nevertheless, the alternation of necrosis and regeneration triggers the process of liver fibrosis, leading to cirrhosis, portal hypertension, hepatic encephalopathy, hemorrhage and other complications.

These diseases occur after the liver fibrosis stage. Liver fibrosis is a proliferation of connective tissue that occurs due to excessive accumulation of extracellular matrix proteins, including collagen. Hepatocytes are known to be composed of hepatocytes, sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells. Hepatic stellate cells play the most important role in liver fibrosis ( Francis JE et al. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex / American Journal of Physiology-Gastrointestinal and Liver Physiology, 2000, V.279, Nl, pp. G7-G11).

Hepatic stellate cells, which normally perform the function of storing retinoid, a precursor of vitamin A, account for 15% of all hepatocytes. When hepatocytes are damaged or die, Kupffer cells begin to engulf the damaged hepatocytes and secrete cytokines (TGF-beta, PDGF, FGF, HGF, PAF, and ET-1) for the proliferation of hepatic stellate cells. begin to secrete Hepatic stellate cells differentiate into myofibroblasts. Myofibroblasts induce liver fibrosis by synthesizing collagen that accumulates in the intercellular matrix. Thus, activation of hepatic stellate cells plays a dominant role in the progression of hepatic fibrosis.

Activation of hepatic stellate cells proceeds in three stages: a pre-inflammatory stage, an inflammatory stage, and a post-inflammatory stage.

In the pre-inflammatory stage, hepatocyte damage induces the synthesis of hormones that stimulate the proliferation of hepatic stellate cells or reduces the activity of arginase, a cell growth inhibitor, to enhance the proliferation of hepatic stellate cells. cause. The formation of acetaldehyde or lipid peroxide stimulates the expression of matrix genes.

In the inflammatory phase, under the influence of cytokines (TGF-beta, PDGF, FGF, HGF, PAF, and ET-1) secreted by excited Kupffer cells and platelets, hepatic stellate cells proliferate and additionally fibroblasts ( differentiate into myofibroblasts that can transform into fibrocytes ( Marie-Reine Losser et al. Mechanisms of Liver Damage / Seminars in Liver Disease, 1996, V.16, N.4, pp.357-367; Anke MBC Tiggelman et al. Transforming growth factor-β-induced collagen synthesis by human liver myofibroblasts is inhibited by α 2 -macroglobulin / Journal of Hepatology, 1997, V.26, N.6, pp. 1220-1228 ).

In the post-inflammatory phase, cytokines and growth factors are secreted by already differentiated myofibroblasts to activate undifferentiated hepatic stellate cells and secrete extracellular matrices. Myofibroblasts activated and differentiated under the influence of hepatic stellate cells form collagen and accumulate in the intercellular matrix. Collagen monomers are very unstable and are easily degraded at body temperature, and the degraded monomers polymerize to cause hepatic fibrosis ( Brenner DA et al. Type I collagen gene regulation and the molecular pathogenesis of cirrhosis / American Journal of Physiology-Gastrointestinal and Liver Physiology, 1993, V.264, N.4, pp.G589-G595 ).

Cirrhosis of the liver as a result of liver fibrosis occurs due to the polymerization of accumulated collagen. The resulting collagen is delivered as insoluble fibers. Cirrhosis is caused by permanent inflammation in the liver, such as when hepatocyte destruction occurs, when hepatocyte regeneration is replaced by connective tissue, when alcohol is consumed, in hepatitis, when there are toxic effects, etc. triggered and/or supported. Liver cirrhosis is a serious pathology and causes fatal complications such as portal hypertension, esophageal and gastric bleeding, hepatoma, hepatic encephalopathy, and coma ( Gines Ð. et al. Management of cirrhosis). and ascites / The New England Journal of Medicine, 2004, V.350, N. 16, pp. 1646-1654).

In connection with the foregoing, prevention and treatment of NAFLD, including all stages of development, is a significant and socially important factor.

The object of the proposed invention is to find a new agent that is effective in the prevention and treatment of non-alcoholic fatty liver disease.

The present invention proposes to use lactitol as this agent.

Lactitol (systematic name: 4-O-alpha-D-galactopyranosyl-D-glucitol) is a sugar alcohol synthesized from milk sugar (lactose), a disaccharide ( disaccharides), has a long experience of use, and has unique properties.

In the EU, USA and Japan, lactitol is used as a sweetener in the food industry and has an energy value as low as 2 kcal/g. Therefore, it is considered an ideal sweetener for next-generation functional nutritional products. A major advantage of lactitol compared to other prebiotics is also its resistance to high temperatures and low pH values. These properties allow the use of lactitol in foods exposed to high temperature processing, especially in the bakery and confectionery industries ( Artyukhova SI et al.: Use of lactitol in biotechnology of functional food products / Modern high technologies, 2013, N .3, pp.87-88 ).

According to pharmacological indicators, lactitol belongs to the "Laxatives group". According to the ATC, lactitol belongs to the "osmotic laxatives" group and has the code A06AD12. Lactitol differs from other osmotic laxatives (except for lactulose-based drugs) in that its osmotic properties are not produced by the active substance but by its metabolites in the colon. Since the human intestines do not have an enzyme system capable of breaking down lactitol, lactitol is not absorbed in the small intestine and enters the intestine in an almost unchanged form. Already in the ascending colon, lactitol is actively assimilated by the saccharolytic microflora. Low-molecular-weight short-chain aliphatic carboxylic acids (SCFAs) (lactic, acetic, butyric, and propionic) formed during microbial metabolism lead to: 1) decrease in intestinal pH promoting activation of propulsive peristalsis; 2) Increased volume and dilution of intestinal contents due to increased osmotic pressure and fluid retention, facilitating movement through the intestine.

In addition to the dose-dependent laxative effect, lactitol has a prebiotic effect (prebiotics are not digested and not absorbed from the upper gastrointestinal tract, but the microflora of the human large intestine ) is a food component that is fermented by stimulating the growth and vitality of colonic microorganisms). Lactitol's prebiotic effect is that when it enters the colon unchanged, lactitol is used as an energy source by probiotic intestinal microbes, and bacteria of the Escherichia coli group. This is due to the fact that it inhibits the growth of proteolytic bacteria by inhibiting their attachment to the epithelial cell walls. Lactitol enhances protein synthesis by bacteria and slows the formation of toxins. Depending on the type of metabolism, lactitol is similar to dietary fibers, is hydrolyzed and is not absorbed in the stomach and small intestine, and is fermented in the large intestine by glycolytic microorganisms and, as already known, produces low fatty acids, carbon dioxide, and hydrogen. and converted to biomass. The products of lactitol metabolism are SCFAs, mainly acetic acid and butyric acid in a 2:1 ratio. A fundamental difference between lactitol and other disaccharides is the different profile of SCFAs synthesized by microorganisms. Thus, compared to lactulose, lactitol metabolism produces 10 times more butyric acid. As a result, organic acids are absorbed by the body and provide an energy value of 2 kcal/g. The total energy value of lactitol is 2.4-2.6 kcal/g (as opposed to carbohydrate - 4 kcal/g). Acetic acid molecules cross the intestinal wall and enter the liver, where they are distributed as an energy substrate to muscle tissue and internal organs (heart, kidneys, brain, etc.). SCFAs are major energy donors and modulators of colonocyte metabolism.

Since lactitol is a food source for sugar-degrading colonic bacteria ( Lactobacillus spp., Lactobacillus bifidus, Lactobacillus acidophilus, Bifidobacteria ), it selectively increases the biomass of glycolytic microorganisms and is commonly used in constipation. Inhibits the growth of Enterobacteria and Enterococi proteolytic bacteria with increasing numbers. In a comparative study of the effects of lactitol and lactulose on probiotic and conditionally pathogenic and pathogenic bacteria, the effect of lactitol was found to be more selective. In particular, unlike lactulose, lactitol is not fermented by Escherichia coli ( E. coli ) and is degraded by much smaller numbers of Staphylococci ( St. aureus ) and Clostridia ( Cl. perfringens ). A randomized clinical trial comparing the efficacy and safety of lactitol and lactulose in the treatment of constipation (Amit Maydeo. Lactitol or lactulose in the treatment of chronic constipation: Result of a systematic / Journal of the Indian Medical Association, 2010, V.108 , N.11, pp.789-792) showed that lactitol was better tolerated by patients, with almost the same laxative effect. Compared to lactulose, fewer side effects were observed with lactitol (31.20 ± 0.80% for lactitol, 62.10 ± 1.10% for lactulose, p = 0.0019). In addition, the effective dose of lactitol used in pediatrics was almost twice less than that of lactulose (effective dose of lactitol 250-400 mg/kg/day, effective dose of lactulose 500-750 mg/kg/day). kg/day).

The prior art (international patent application WO 0239832 A1, published on May 23, 2002) is a food (or food supplement) for improving intestinal microflora, which is used to treat intestinal infections, colon cancer, and diarrhea. ) Suggests the use of lactitol for prevention or immunity enhancement. Preferably, the use of lactitol in a dose of 5-15 g per day is suggested.

Lactitol is also approved for use in hepatic encephalopathy and hyperammonemia as an agent to help reduce the formation and absorption of ammonia in the intestine.

Accordingly, a patent document (patent GB 2113998 A, publ. 08/17/1983) discloses the use of lactitol for the treatment of port-systemic encephalopathy, a central nervous disorder caused by complications of progressive cirrhosis. The use is suggested, preferably the use of lactitol in a dose of 20-200 g per day. In patients suffering from this disease, nitrogenous toxic substances produced in the intestine, preferably ammonia, are not neutralized in the liver and enter the blood circulation and brain, damaging nerve cells. In this document, the indicated therapeutic effect of lactitol is also related to reduced formation and absorption of ammonia.

However, the use of lactitol for the treatment of non-alcoholic fatty liver disease (NAFLD) is not disclosed in the prior art. To date, no direct study has been conducted on the effects of lactitol in the case of fatty liver dysfunction.

The technical effect of the present invention is to expand the range of means used for the prevention and treatment of non-alcoholic fatty liver disease.

These technical effects are obtained by using a therapeutically effective amount of 4-O-alpha-D-galactopyranosyl-D-glusyl for the prevention and treatment of non-alcoholic fatty liver disease in a mammal in need thereof. It is achieved by using tol (lactitol).

As a preferred embodiment, the mammal is a human.

A therapeutically effective amount (or therapeutic dose) is an amount (or dose) of an active agent used for therapeutic or prophylactic purposes. For example, a therapeutically effective amount of lactitol can be 1-20 g/day.

Lactitol can be administered to mammals, preferably to humans, at any stage of the development of non-alcoholic fatty liver disease.

Also, in one embodiment of the present invention, lactitol at a dose of 1-20 g/day may be for preventing and treating liver cirrhosis resulting from non-alcoholic fatty liver disease.

In one embodiment, the use of lactitol may be in an oral dosage form.

Such oral dosage forms may be powders free of further adjuvants and/or active substances (in addition to the active substance, lactitol). The powder may be placed in a sachet. Optionally, oral dosage forms (in addition to lactitol) may additionally contain pharmaceutically acceptable adjuvants, such as powders, tablets (including modified release tablets), capsules, granules ), syrup, drink, gel, plate, soft gelatin candy, and the like. As pharmaceutically acceptable adjuvants, various additives may be added which are used in the preparation of prepared oral dosage forms, are compatible with the active agent and other adjuvants, and do not adversely affect the patient being prevented or treated.

The following tests were performed to confirm the possibility of using lactitol as a drug for the treatment and prevention of non-alcoholic fatty liver disease, including the prevention and treatment of cirrhosis resulting from NAFLD.

Example 1.

For the study, mice with a defect in the leptin gene, a hyperphage protein in obesity, were selected. Hepatic steatosis can form very rapidly in these mice.

Obese male mice of approximately 8 weeks of age with the genetic defect were randomly assigned to groups of 7 mice each. Seven plain white laboratory mice served as controls. All animals were maintained at a constant temperature of 23 ± 0.5 °C, humidity of 50 ± 5% and light corresponding to daylight hours (7:00-19:00). Animals had free access to water and food. All procedures and experiments on mice were performed in accordance with International Rules for the Care of Animals.

The mice were divided into study groups, and L-ornithine-L-aspartate was administered to the mice at a dose similar to the daily dose of lactitol (7.5, 15.0 or 30.0 mg/kg). did. L-ornithine-L-aspartate is known to be used for the treatment of steatosis (of various origins) and steatohepatitis ( Zhuravleva LV et al.: L-ornithine-L-aspartate in the treatment of non-alcoholic fatty liver disease in obese patients with signs of metabolic syndrome / Medical Practicer, 2015, pp.25-31 ).

Mice with a leptin gene defect develop chronic inflammatory hepatic steatosis and represent an animal model of non-alcoholic steatohepatitis (NASH). In the case of NASH, an increase in fatty inclusions occurs and the potential for inflammatory damage increases. An important criterion for NASH, in the absence of viral infection or alcoholism, is the serum transferases released by damaged hepatocytes: alanine aminotransferase (ALT), aspartate aminotransferase It is an elevated level of indicators of aminotransferase (AST) and sorbit dehydrogenase (SDH). The content of these enzymes is increased in genetically defective mice as a result of hepatic steatosis and concomitant secondary inflammation.

In [Table 1], ALT, AST and SDH indices and normal low weight of serum obtained from mice administered with lactitol and L-ornithine-G-aspartate (LoLa) The levels of these enzymes in serum from mice (control group) and diabetic mice (diabetic group) administered with physiological saline were summarized. The levels of ALT, AST and SDH were markedly increased in genetically defective obese diabetic mice compared to normal mice.

Data obtained as summarized in Table 1 indicate that ALT, AST and SDH decreased after administration of L-ornithine-L-aspartate at doses of 7.5-30.0 mg/kg/day. Concomitantly, treatment with lactitol at doses of 7.5-30.0 mg/kg/day resulted in a dose-dependent decrease of these liver enzymes in serum.

The obtained profiles of liver enzymes correlate with liver histology. Therefore, in the genetically defective obese diabetic mice of the diabetic group administered with saline, intracellular fat accumulation in the liver in the form of fat droplets was observed. Similar to the group of mice given L-ornithine-L-aspartate (LoLa), daily administration of lactitol at therapeutic and subtherapeutic doses for 4 weeks resulted in fat accumulation in the liver. this markedly decreased.

Example 2.

In addition, body weight gain was evaluated in mice of all groups participating in the study of Example 1. The obtained data is shown in [Table 2].

Mice in the diabetic group showed weight gain during the entire 4-week study period. Moreover, untreated diabetic mice (neither lactitol nor LoLa) gained weight compared to mice treated with either the study drug (lactitol) or the reference drug (LoLa). The data obtained show that administration of lactitol to diabetic mice induces a dose dependent weight loss.

Example 3.

Obese male mice, approximately 8 weeks of age, were randomly assigned to groups of 5-7 animals each, such that body weight (50-55 g) and serum glucose levels (after feeding >300 mg/dl (deciliter)) were similar between groups. Lean male mice served as controls. After arrival, mice were left alone for at least 7 days to acclimate. All animals were maintained at controlled temperature (23 ± 0.5 °C), relative humidity (50 ± 5%) and daylight hours (7:00-19:00). Animals had free access to food and water.

No drug was administered to mice in one of the groups of mice prone to obesity (obesity group). The remaining group of obese mice constituted a treatment group that received oral administration of lactitol or LoLa daily for 2 weeks. At the end of the treatment period, from mice in all study groups, 100 μL of venous blood was heparinized from the retrobulbar sinus of the mice to perform chemical analysis of the serum for triglycerides and free fatty acids ( Heparinized) capillaries were collected.

As summarized in [Table 3], based on the above results, it can be seen that the administration of lactitol to mice prone to obesity resulted in a dose-dependent decrease in triglyceride and free fatty acid contents.

Thus, the tests and studies conducted clearly indicate that lactitol affects the fatty degeneration process of the liver and has a positive therapeutic and preventive effect on fatty liver disease of non-alcoholic origin.

Example 4.

A test was conducted within the scope of the present invention to confirm the assumption that administration of lactitol would have a therapeutic effect in inhibiting the development of cirrhosis.

Experiments were performed in outbred white laboratory mice. Cirrhosis of the liver was simulated by intragastric administration of a 50% solution of CC1 ₄ in olive oil (a hepatotropic poison) at a dose of 2 mL/kg, twice a week, 6 times a week for 3 weeks. To enhance the pathological effect on the liver, animals were given a 10% ethyl alcohol solution for the entire duration of the experiment.

Lactitol (15 mg) was administered orally to groups of animals daily for 4 weeks prior to modeling liver pathology. Administration of lactitol was continued even after the onset of cirrhosis induction. A control group of animals was administered the same regimen and equivalent volume of distilled water instead of lactitol.

Liver status was assessed after the mice were killed. On day 41 of the experiment, biochemical studies were performed on aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in serum. In addition, the morphological status of the liver was evaluated on the 41st day of the experiment. Serum enzyme activity was determined conventionally using a biochemical assay and standard kits. Blood for the study was taken from the femoral artery via catheter. In liver histological preparations stained with hematoxylin and eosin, the number of infiltrating cells was determined using an Avtandilov eyepiece graticule containing 25 test points. The number of cells reaching the test points of the graticule was counted in 20 fields of view. The relative area of infiltration was calculated as the ratio of graticule points per infiltrating cell to all graticule points in 20 fields of view. The connective tissue area was determined by means of computer processing of the graphic data. For this purpose, the area of picrofuxin-stained structures was measured in a standard area of liver section (sequential micrographs of 10 fields taken with a micro video camera with a program to transfer the images to a computer). Percentages were calculated for selected standard areas. Results were performed by means of variation statistics using Student's t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

The experiment showed a significant mouse mortality of 48.9% in the control group, whereas the mortality rate was very low at 4.2% in the group of mice injected with lactitol against the background of liver cirrhosis modeling.

In the biochemical study of serum, the activities of ALT, AST and ALP were found to increase on the 40th day after the introduction of CC1 ₄ into the control group (Table 4). At the same time, stabilization of serum enzyme activity was observed in the group administered with lactitol.

Histological examination of the liver of the untreated (not receiving lactitol) mouse group showed clear violation of the lobular structure of the organ. Structural changes in hepatic venules, arterioles and bile ducts occurred. Granulation tissue replaced the dead liver cells. Neoplasms of blood vessels and hepatic ducts developed intensively. Fibrous cords and micro-nodes, so-called pseudolobes, were formed. The latter was a group of hepatocytes surrounded by fibrotic patches. Massive large droplet fatty degeneration was noted in the remaining hepatocytes. Administration of lactitol prevented the development of morphological signs of liver cirrhosis. Liver lobular structure was preserved. It should be noted that signs of droplet fatty degeneration and portal infiltration were recorded by the end of the observation period.

A significant decrease in the content of infiltrating cells and the area of connective tissue was demonstrated in the group of mice treated with lactitol (Table 5).

Example 5.

Production of oral dosage forms of lactitol in powder form packaged in sachets

Lactitol monohydrate in powder, sieved without auxiliary substances, was packaged in bags in an amount of 10 g and placed in cardboard boxes.

Example 6.

Production of oral dosage forms of lactitol in capsule form

Powdered lactitol monohydrate is sieved and mixed thoroughly with lactose powder in a ratio of 2:1. The resulting mixture in an amount of 500 mg is placed in an appropriately sized hard gelatin capsule.

Example 7.

Production of oral dosage forms of lactitol in the form of tablets

1,600 mg of starch, 1,600 mg of ground lactose, 400 mg of talc and 1,000 mg of lactitol are mixed and pressed into a bar. The resulting bar is ground into granules and sieved through a sieve to collect granules of 14-16 mesh size. The obtained granules are tableted in the form of suitable tablets each weighing 560 mg.

Thus, it has been conclusively confirmed that the administration of lactitol in the scope of the present application contributes, in particular, to the prevention of cirrhosis changes resulting from NAFLD.

[Table 1]

Effects of lactitol and L-ornithine-L-aspartate on serum markers of liver damage

[Table 2]

Effect of test compound (lactitol) and control drug (L-ornithine-L-aspartate) on body weight of diabetic mice

[Table 3]

Effects of administration of lactitol and L-ornithine-L-aspartate on triglyceride and free fatty acid content of blood serum

[Table 4]

Effect of lactitol on morphological parameters of the liver of male mice with liver cirrhosis on day 41 of the experiment.

* Significance of the difference between an indicator and its background value at p<0.05 is indicated.

** The significance of the difference from the indicator and its control value at p < 0.05 is indicated.

[Table 5]

Effect of lactitol on morphological parameters of the liver of male mice with liver cirrhosis on day 41 of the experiment.

* Significance of the difference between an indicator and its background value at p<0.05 is indicated.

** The significance of the difference from the indicator and its control value at p < 0.05 is indicated.

Claims (13)

A pharmaceutical composition for the prevention and treatment of non-alcoholic fatty liver disease in a mammal in need thereof, wherein the pharmaceutical composition comprises a therapeutically effective amount of 4-O-alpha-D-galactopira A pharmaceutical composition comprising nosyl-D-glucitol (lactitol).
According to claim 1,
The pharmaceutical composition, characterized in that the mammal is a human.
According to claim 1 or 2,
A pharmaceutical composition, characterized in that the therapeutically effective amount of lactitol is 1-20 g / day.
According to claim 1 or 2,
A pharmaceutical composition characterized in that lactitol is intended for administration in a dosage of 1-20 g/day to a mammal in need thereof at any stage of development of non-alcoholic fatty liver disease.
According to claim 1 or 2,
Lactitol at a dose of 1-20 g/day is a pharmaceutical composition, characterized in that for the prevention and treatment of liver cirrhosis caused by non-alcoholic fatty liver disease.
According to claim 1 or 2,
A pharmaceutical composition, characterized in that lactitol is used in an oral dosage form at a dosage of 1-20 g/day.
According to claim 6,
The pharmaceutical composition according to claim 1 , wherein the oral dosage form does not contain additional active substances.
According to claim 6,
The pharmaceutical composition according to claim 1 , wherein the oral dosage form is a powder without additional adjuvants.
According to claim 6,
The oral dosage forms include powders, tablets, capsules, granules, syrups, drinks, gels, plates, soft gelatin candies. gelatin candies), and a pharmaceutical composition characterized in that it further comprises a pharmaceutically acceptable adjuvant (adjuvants).
An oral dosage form for the prevention and treatment of non-alcoholic fatty liver disease in a mammal in need thereof, wherein the oral dosage form comprises a therapeutically effective amount of 4-O-alpha-D-galactopira An oral dosage form comprising nosyl-D-glucitol (lactitol) and optionally a pharmaceutically acceptable adjuvant.
According to claim 10,
An oral dosage form characterized in that it does not contain additional adjuvants.
According to claim 10,
Oral dosage form, characterized in that it does not contain further active substances.
According to claim 10,
The oral dosage forms include powders, tablets, capsules, granules, syrups, drinks, gels, plates, soft gelatin candies. gelatin candies), characterized in that it further comprises a pharmaceutically acceptable adjuvant.