RU2790828C1 - Hepatoprotective effect of low molecular weight mimetics ngf - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and pharmacology and concerns a means for the treatment of non-alcoholic fatty liver disease (NAFLD). The use of the low molecular weight mimetic NGF hexamethylenediamide (bis-L-monosuccinyl-L-glutamyl-L-lysine) as a treatment for NAFLD has been proposed. In an experimental model of NAFLD caused by a high-fat diet in Wistar rats, hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) at a dose of 5 mg/kg orally eliminates morphological disorders in the liver characteristic of NAFLD. Hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) reduces abdominal obesity, reduces cholesterol in the blood by 30%, triglycerides by 28% in comparison with animals on a fat diet, but not treated.

EFFECT: expansion of the arsenal of medicines for the treatment of NAFLD, obtaining a more effective, safe, non-toxic drug, suitable for oral use.

1 cl, 4 tbl, 1 dwg, 4 ex

Description

Field of invention

SUBSTANCE: invention relates to medicine and pharmacology, and concerns an agent for the treatment of non-alcoholic fatty liver disease (NAFLD).

State of the art

In recent decades, a consistently high incidence of NAFLD has been recorded in the world. This pathology is more common among people with obesity, dyslipidemia and metabolic syndrome [Hamaguchi M., Koima T., Takeda N., et al. The metabolic syndromes a predictor of nonalcoholic fatty liver disease / Ann. Intern. Med. 2005; 143:772-8]. The natural course of processes in the altered liver parenchyma can lead to the development of terminal stages of the disease - cirrhosis and liver cancer [Pavlov Ch.S., Glushenkov D.V., Bulichenko M.A. et al. Non-alcoholic fatty liver disease in the clinic of internal diseases / BC. 2011; 6:11-17].

In the progression of NAFLD, regardless of its etiology, the leading factor is the accumulation of free fatty acids, which are a highly active substrate of lipid peroxidation (LPO). Against the background of a decrease in the activity of antioxidant defense enzymes, LPO leads to membrane damage and cell necrosis. LPO products such as 4-hydroxynonenal and malondialdehyde are able to activate hepatic stellate cells, which are the main producers of collagen, and also cause cross-linking of cytokeratins with the formation of Mallory bodies and stimulate neutrophil chemotaxis. Superoxide anions induce an increase in the production of cytokines - TNF-α, TGF-β and interleukin-8. TNF-α, depleting the antioxidant defense system. They interact with mitochondrial reactive oxygen radicals, which leads to the death of hepatocytes [Donnelly K.L., Smith C.I., Schwarzenberg S.J., et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with non-alcoholic fatty liver disease / J. Clin. Invest. 2005; 115(5): 1343-51].

Е., Madrigal-Bujaidar Е.,

et al. Review of natural products with hepatoprotective effects / World J. Gastroenterol. 2014; 20(40): 14787-14804]. Несмотря на широкий арсенал используемых гепатопротекторных препаратов, большинство из них не лишены недостатков. Ряд гепатопротекторов имеют крайне низкую биодоступность: у S-аденозилметионина она составляет 2-3% [Goren J.L., Stoll A.L., Damico K.E., et al. Bioavailability and lack of toxicity of S-adenosyl-L-methionine (SAMe) in humans / Pharmacotherapy. 2004; 24(11): 1501-1507], силимарина - 13% (при пероральном введении у крыс) [Morazzoni P., Montalbetti A., Malandrino S., Pifferi G. Comparative pharmacokinetics of silipide and silymarin in rats / Eur. J. Drug. Metab. Pharmacokinet. 1993; 18(3):289-97]. Препараты флавонолигнанов (силимарин) и эссенциальных фосфолипидов усиливают холестаз и обладают высоким аллергогенным потенциалом [Морозов С.В. Гепатопротекторы в клинической практике: рациональные аспекты использования: пособие для врачей / С.В. Морозов, Ю.А. Кучерявый. М.: 4ТЕ Арт, 2011. 28 с].The existing drug therapy for NAFLD is based on the use of antioxidants, mainly of plant origin, amino acid derivatives and drugs with membrane stabilizing properties [

E., Madrigal-Bujaidar E.,

et al. Review of natural products with hepatoprotective effects / World J. Gastroenterol. 2014; 20(40): 14787-14804]. Despite the wide arsenal of hepatoprotective drugs used, most of them are not without drawbacks. A number of hepatoprotectors have extremely low bioavailability: in S-adenosylmethionine it is 2-3% [Goren JL, Stoll AL, Damico KE, et al. Bioavailability and lack of toxicity of S-adenosyl-L-methionine (SAMe) in humans / Pharmacotherapy. 2004; 24(11): 1501-1507], silymarin - 13% (when administered orally in rats) [Morazzoni P., Montalbetti A., Malandrino S., Pifferi G. Comparative pharmacokinetics of silipide and silymarin in rats / Eur. J. Drug. Metab. Pharmacokinet. 1993; 18(3):289-97]. Preparations of flavonolignans (silymarin) and essential phospholipids increase cholestasis and have a high allergenic potential [Morozov S.V. Hepatoprotectors in clinical practice: rational aspects of use: a guide for doctors / S.V. Morozov, Yu.A. Curly. M.: 4TE Art, 2011. 28 p.].

F.T. Alpha-lipoic acid intoxication in an adolescent girl: Case report and review of the literature / Case Reports Turk. Pediatri. Ars. 2020; 55(3): 328-330]. Терапевтическая доза α-липоевой кислоты достигает 600 мг/сут; уже при 1200 мг/сут наблюдаются токсические эффекты, что свидетельствует о низком терапевтическом индексе соединения [Han Т., Bai J., Liu W., Hu Y. A systematic review and meta-analysis of α-lipoic acid in the treatment of diabetic peripheral neuropathy / Eur. J. Endocrinol. 2012, 167: 465-471]. Указанные факторы определяют целесообразность поиска альтернативных эффективных и безопасных средств для лечения НАЖБП.Data on the effectiveness of antioxidants for the prevention and treatment of NAFLD are ambiguous [Okovity S.V., Sukhanov D.S., Romantsov M.G. Hepatotropic drugs: the current state of the problem / Ter. archive. 2012; 2: 62-68]. Despite the relatively large range of agents for suppressing free radical oxidation reactions, their use is hampered by such problems as the toxicity of some phenolic antioxidants and the displacement of endogenous antioxidants when using synthetic compounds [Aleksandrova A.E. Antihypoxic activity and mechanisms of action of some synthetic and natural compounds / Exp. wedge, farm. 2005; 68(5): 72-78]. The widespread use of one of the known antioxidant drugs, α-lipoic acid, is limited by the occurrence of such adverse events as gastro- and nephrotoxicity, headache when ingested, skin irritation and pruritus when administered parenterally [Spain RI, Andeen NK, Gibson R.C. Lipoic acid supplementation associated with neural epidermal growth factor-like 1 (NELLl)-associated membranous nephropathy / Kidney Int. 2021; 100(6):1208-1213. Polat S.,

FT Alpha-lipoic acid intoxication in an adolescent girl: Case report and review of the literature / Case Reports Turk. Pediatri. Ars. 2020; 55(3): 328-330]. The therapeutic dose of α-lipoic acid reaches 600 mg / day; already at 1200 mg/day, toxic effects are observed, which indicates a low therapeutic index of the compound [Han T., Bai J., Liu W., Hu Y. A systematic review and meta-analysis of α-lipoic acid in the treatment of diabetic peripheral neuropathy / Eur. J. Endocrinol. 2012, 167: 465-471]. These factors determine the feasibility of finding alternative effective and safe drugs for the treatment of NAFLD.

Nerve growth factor (NGF) is the most important member of the neurotrophin group. It is known that NGF is expressed not only in neurons, but also in cells of various peripheral organs, including the liver. There is evidence of the ability of NGF to protect hepatocytes from oxidative damage caused by xenobiotics. This effect is eliminated by antibodies to NGF, which enhance the damaging effect of oxidative stress on liver cells [Valdovinos-Flores C., Gonsebatt M.E. Nerve growth factor exhibits an antioxidant and an autocrine activity in mouse liver that is modulated by buthionine sulfoximine, arsenic, and acetaminophen / Free Radic. Res. 2013; 47(5): 404-412]. However, due to the low enzymatic stability of native neurotrophins and the inability to penetrate biological barriers under conditions of systemic administration, their practical use is impossible. The effect of NGF on the liver was studied only under conditions of its local application to primary cultures of hepatocytes or with intraperitoneal administration of the recombinant molecule.

At the Research Institute of Pharmacology named after V.V. Zakusova has been developing an original strategy for a number of years, which consists in constructing systemically active low-molecular-weight mimetics of individual neurotrophin loops [Gudasheva T.A., Antipova T.A., Seredenin SB Novel low-molecular-weight mimetics of the nerve growth factor / Dokl. Biochem. Biophys. 2010; 434:262-265. Gudasheva TA, Ostrovskaya RU, Seredenin SB Novel technologies for dipeptide drugs design and their implantation / Curr. Pharm. Des. 2018; 24(26): 3020-3027]. Based on the sequence Asp ⁹³ -Glu ⁹⁴ -Lys ⁹⁵ -Gln ⁹⁶ β-bend of the 4th loop of NGF, a dipeptide mimetic - hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) was constructed [RF Patent No. 2410392, 2010; Patent US 9.683.014 B2, 2017; Patent CN 102365294 B, 2016]. The neuroprotective activity of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) was studied in vitro both in immortalized and primary cell cultures. It has been shown that hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine in micro- and nanomolar concentrations increases the survival of neuronal cells under toxic effects of hydrogen peroxide, glutamic acid, 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine [Gudasheva T.A., Antipova T.A., Seredenin S.B. New low molecular weight nerve growth factor mimetics / DAN. 2010; 434: 4: 262-265]. -N-monosuccinyl-L-glutamyl-L-lysine) were also obtained in experiments in vivo.The effectiveness of this compound in various models of Alzheimer's disease, as well as focal and global cerebral ischemia and hemorrhagic stroke [Seredenin S.B., Gudasheva T. A. Creation of a pharmacologically active small molecule with the properties of nerve growth factor / S.S. Korsakov Journal of Neurology and Psychology 2015; 115(6): 63-70].

et al. Biochemical and nutritional overview of diet-induced metabolic syndrome models in rats: what is the best choice? / Nutr. and Diab. 2020; 10: 24]. Исходя из этого, представляется важным оценить эффективность гексаметилендиамид (бис-N-моносукцинил-L-глутамил-L-лизина) на модели НАЖБП, развивающейся при использовании ВЖД.One of the most commonly used and effective methods for modeling NAFLD in laboratory animals is the use of a high-fat diet (HFD), which mimics the intake of excess free fatty acids and cholesterol [Zhang M., Lv XY, Li J., et al. The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model / Exp. Diab. Res. 2008; ID 704045.

et al. Biochemical and nutritional overview of diet-induced metabolic syndrome models in rats: what is the best choice? /nutr. and Diab. 2020; 10:24]. Based on this, it seems important to evaluate the effectiveness of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) in the model of NAFLD that develops with the use of HD.

The essence of the invention

The essence of the invention is the use of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) as an agent for the treatment of NAFLD, which is active at a dose of 5 mg/kg orally in relation to the functional and morphological state of the liver.

The technical result of the invention is the expansion of the arsenal of drugs for the treatment of NAFLD, obtaining a more effective, safe, non-toxic, suitable for oral use.

The invention is illustrated by the drawing, described in detail in example 3, which shows the effect of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) on the morphological structure of the liver: fig. 1 - Micrographs of histological preparations of rat liver tissue. A - control group (standard food); B - VZhD + FR group; C - group VZhD + A (hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine)). Hematoxylin-eosin staining.

The following are specific examples that illustrate, but do not limit the present invention.

Example 1

As follows from the materials shown in table 1, hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) has a number of advantages in comparison with existing hepatoprotective agents.

Thus, among the existing hepatoprotective agents, hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) has the lowest therapeutic dose (5 mg) when administered orally and the highest toxic dose LD ₅₀ (714 mg/kg), which testifies to the wide breadth of its therapeutic action. It has been experimentally established that the bioavailability of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) in rats when administered intraperitoneally is 84.6%.

Example 2 Effect of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) on cholesterol and triglyceride levels.

The experiments were performed on adult male Wistar rats with an initial body weight of 250–270 g obtained from the Stolbovaya nursery. The animals were kept in accordance with SP 2.2.1.3218-14 and GOST 33215-2014 subject to the light regime (12 hours of light and 12 hours of darkness), an average temperature of 22±2°C and 60% humidity. The experiments were carried out in accordance with the requirements of GOST 33044-2014 and Directive of the Council of the European Community 86/609/EEC on the use of animals for experimental research.

Animals (n=24) were randomly divided into three equal groups, one of which remained on a standard diet of 290 kcal/100 g (n=8, control), and the other two groups were kept on a high fat diet for 12 weeks. . The VZD was prepared ex tempore according to the described method [Shaykhutdinova E.R., Palikov V.A., Palikova Y.A., et al. Effect of standard and high-fat diets during modeling of streptozotocin-induced diabetes in Rats on the development of complications / Bull. Exp. Biol. Med. 2021; 170(6): 737-740] by adding to 450 g of standard feed 150 g of melted lard, 5 g of common salt and 15 g of monosodium glutamate. This diet is characterized by the content of 45% fat, 35% carbohydrates, 20% protein and a total calorie content of 516 kcal/100 g. 0.9% NaCl solution (HVD + FR), another group (n=8) in the same regimen orally hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) at a dose of 5 mg/kg (HVD + A ). Animals had free access to food (with the exception of 12 hours prior to the measurement of cholesterol and triglycerides in the blood) and to drinking water ad libitum. Determination of the level of cholesterol and triglycerides in the blood was carried out using the express analyzer Accutrend Plus (Roche, Germany) on the first and last day of therapy.

Statistical processing of the results was carried out using the Biostat program. The nature of the data distribution was determined by the Shapiro-Wilk test. The statistical significance of differences between groups was assessed by ANOVA followed by Bonferroni's test. Data are presented as mean ± standard error of the mean (M ± SEM). The difference in indicators was considered significant at p<0.05.

In animals of the HD+FR group, the level of cholesterol was 80%, and triglycerides - almost 95% higher than in animals that received standard food. The introduction of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) helps to reduce cholesterol levels by 30% compared with the HD+FR group. The level of triglycerides in the blood of rats with IVD treated with the compound of the invention was lower by 28% lower than in rats of the IVD+FR group (Table 1).

Thus, the data obtained indicate a violation of lipid metabolism in the conditions of the use of HRP and the normalizing effect of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) on its parameters.

Example 3 Effect of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) on the morphological structure of the liver

The experiments were performed on adult male Wistar rats with an initial body weight of 250–270 g obtained from the Stolbovaya nursery. The animals were kept in accordance with SP 2.2.1.3218-14 and GOST 33215-2014 subject to the light regime (12 hours of light and 12 hours of darkness), an average temperature of 22±2°C and 60% humidity. The experiments were carried out in accordance with the requirements of GOST 33044-2014 and Directive of the Council of the European Community 86/609/EEC on the use of animals for experimental research.

Animals (n=24) were randomly divided into three equal groups, one of which remained on a standard diet of 290 kcal/100 g (n=8, control), and the other two groups were kept on a high fat diet for 12 weeks. . The VZD was prepared ex tempore according to the described method [Shaykhutdinova E.R., Palikov V.A., Palikova Y.A., et al. Effect of standard and high-fat diets during modeling of streptozotocin-induced diabetes in Rats on the development of complications / Bull. Exp. Biol. Med. 2021; 170(6): 737-740] by adding to 450 g of standard feed 150 g of melted lard, 5 g of common salt and 15 g of monosodium glutamate. This diet is characterized by the content of 45% fat, 35% carbohydrates, 20% protein and a total calorie content of 516 kcal/100 g. 0.9% NaCl solution (HVD+FR), another group (n=8) was orally administered hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) at a dose of 5 mg/kg (HVD+ A). The animals had free access to food and drinking water ad libitum. Animals were sacrificed by decapitation under ether anesthesia. Fragments from the central parts of the liver up to 1 cm in size were fixed in 10% neutral formalin (pH=7.4) (Sigma, USA) for 24 h, dehydrated, and embedded in paraffin blocks. Sections 5 μm thick were made using a microtome (Micritome Jung RM2035, Germany). The resulting sections were stained with hematoxylin-eosin according to the manufacturer's method (Blik MedicalProduction, Russia), after which the preparations were dehydrated and placed under coverslips.

Microscopic analysis was carried out in 10 fields of view of each preparation using an Olympus CX 31 microscope with an Olympus CX 30 digital camera (Olympus, Japan), a personal computer and Arstek Image View software (Arstek, Russia) with an eyepiece x10 magnification and objectives x10, x40. The architectonics of the organ, the condition of the vessels, signs of the inflammatory process, and the presence of binuclear hepatocytes were assessed. To assess the prevalence of inflammatory-dystrophic reactions in the parenchyma of the organ, an evaluation scale proposed by J. Zhao et al. [Zhao J., Zhang Y., Wan Y., et al. Pien Tze Huang Gan Bao attenuates carbon tetrachloride-induced hepatocyte apoptosis in rats, associated with suppression of p53 activation and oxidative stress / Mol. Med. Rep. 2017; 16(3): 2611-2619]:

0 - there are no signs of an inflammatory reaction and degenerative phenomena of hepatocytes;

+ - inflammation and dystrophy cover no more than 33% of the liver parenchyma;

++ - inflammatory process and dystrophic phenomena in hepatocytes spread to an area equal to 33-66% of the parenchyma of the organ;

+++ - more than 66% of the area of the liver parenchyma are involved in the inflammatory-dystrophic process.

Statistical processing of the results was carried out using the Biostat program. Differences in the severity of morphological changes in the structure of the liver were analyzed using the chi-square test.

Morphological analysis of the liver, carried out at the end of the experiment, showed that in rats fed with standard food, the morphological structure of the liver corresponded to the norm. The portal tracts are moderately expressed, the walls of the vessels are clear and thin. Aggregation of erythrocytes and sludge was not detected. The lobule structure and beam structure of the parenchyma were established. Polygonal hepatocytes with clear oval or spherical nuclei located in the center of the cell, eosinophilic cytoplasm. The hepatic plates start from the central vein, the liver lobules are fan-shaped from the center to the periphery (Fig. 1A).

In micropreparations of the liver of rats of the VZhD+FR group, tissue plethora with vasodilation and sinusoids, aggregation and partial intravascular hemolysis of erythrocytes were noted. The lobular structure of the liver tissue is blurred, the structure of the hepatic beams is broken. Binuclear hepatocytes were noted. Balloon dystrophy of hepatocytes was revealed, characterized by swelling and a noticeable increase in cells with a rarefied cytoplasm, acidophilic granules and clumps of intermediate filaments. These changes, which are characteristic of NAFLD, affected more than 2/3 of the liver parenchyma. On the periphery of the lobule there are unstructured hepatocytes, between which the phenomena of leuko- and lymphocytic infiltration are visible (Fig. 1B).

In micropreparations of the liver of rats on HVD and hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) therapy, moderate vascular plethora with less pronounced erythrocyte aggregation in their lumens compared to healthy animals was noted. The lobular structure and beam structure of the hepatic lobule was preserved. Portal tracts are slightly dilated, with weak focal lymphoid infiltration. The number of binuclear hepatocytes is reduced (Fig. 1C). Against the background of therapy, the number of leukocyte infiltrates and the severity of balloon dystrophy were also reduced (Table 3).

Thus, if rats on a standard diet have no signs of an inflammatory reaction and dystrophic phenomena of hepatocytes, then under conditions of keeping on the HDR, significant dystrophic changes are noted affecting from 1/3 to 2/3 of the liver parenchyma. During therapy with hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine), a significant decrease in the number of animals with significant (more than 2/3 of the area of the organ) liver damage was noted, which indicates a hepatoprotective effect of the compound.

Example 4 Effect of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) on rates of abdominal obesity

The experiments were performed on adult male Wistar rats with an initial body weight of 250–270 g obtained from the Stolbovaya nursery. The animals were kept in accordance with SP 2.2.1.3218-14 and GOST 33215-2014 subject to the light regime (12 hours of light and 12 hours of darkness), an average temperature of 22±2°C and 60% humidity. The experiments were carried out in accordance with the requirements of GOST 33044-2014 and Directive of the Council of the European Community 86/609/EEC on the use of animals for experimental research.

Animals (n=24) were randomly divided into three equal groups, one of which remained on a standard diet of 290 kcal/100 g (n=8, control), and the other two groups were kept on a high fat diet for 12 weeks. . The VZD was prepared ex tempore according to the described method [Shaykhutdinova E.R., Palikov V.A., Palikova Y.A., et al. Effect of standard and high-fat diets during modeling of streptozotocin-induced diabetes in Rats on the development of complications / Bull. Exp. Biol. Med. 2021; 170(6): 737-740] by adding to 450 g of standard feed 150 g of melted lard, 5 g of common salt and 15 g of monosodium glutamate. This diet is characterized by the content of 45% fats, 35% carbohydrates, 20% proteins and a total calorie content of 516 kcal/100 g. 0.9% NaCl solution (HVD+FR), the other group (n=8) was orally administered hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) at a dose of 5 mg/kg (HVD+ A). The animals had free access to food and drinking water ad libitum. At the end of the experiment, the body weight and length from the tip of the nose to the beginning of the tail were assessed. These indicators were used to calculate the body mass index (BMI), as well as the circumference of the chest (CO) and abdomen (CO) to calculate the ratio of CO/OG [Novelli E.L., Diniz Y.S., Galhardi C.M., et al. Anthropometrical parameters and markers of obesity in rats / Lab. Anim. 2007; 41(1): 111-119].

Statistical processing of the results was carried out using the Biostat program. The nature of the data distribution was determined by the Shapiro-Wilk test. The statistical significance of differences between groups was assessed by ANOVA followed by Bonferroni's test. Data are presented as mean ± standard error of the mean (M ± SEM). The difference in indicators was considered significant at p<0.05.

HD promoted the growth of adipose tissue mass and abdominal obesity, which was reflected in an increase in the ratio of OB/OE by 11% in animals on IV, compared with the control. The BMI of the animals on the VZhD was 14% higher than the BMI of the animals fed the standard feed. BMI and OB/OE of rats treated with hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) did not differ significantly from BMI of healthy animals (Table 3).

Thus, VAD caused the development of abdominal obesity, characterized by an increase in BMI and the ratio of OB / OG, and the use of hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) contributed to the normalization of these indicators.

Claims (1)

The use of low molecular weight NGF mimetic hexamethylenediamide (bis-N-monosuccinyl-L-glutamyl-L-lysine) as a treatment for non-alcoholic fatty liver disease.

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