RU2785582C2 - Use of pegylated recombinant chimeric mouse fibroblast-21 growth factor or its pharmaceutically acceptable salt in composition of drugs for treatment of non-alcoholic steatohepatitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to medicine, namely to the use of a pegylated recombinant chimeric mouse fibroblast-21 growth factor. The use of the pegylated recombinant chimeric mouse fibroblast-21 growth factor or its pharmaceutically acceptable salt in a composition of drugs for the treatment of non-alcoholic steatohepatitis is described, where the use is selected from reduction in lobular inflammation.

EFFECT: above-described use allows for effective reduction in lobular inflammation.

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Description

Field of invention

The present invention relates to the field of biomedicine and, in particular, relates to the use of pegylated recombinant chimeric mouse fibroblast growth factor-21 or its pharmaceutically acceptable salt in the composition of drugs for the treatment of non-alcoholic steatohepatitis.

Background of the invention

Non-alcoholic fatty liver disease (NAFLD), also known as fatty liver disease, has many causes. In this disease, lesions predominantly occur in the liver lobule, which is characterized by steatosis of liver parenchyma cells and accumulation of triglycerides (TG) (fat content in liver tissues is more than 5% of the wet weight of the liver, or more than 1/3 of liver cells show steatosis, confirmed histological examination). In terms of pathological changes, NAFLD is similar to alcoholic liver disease (ALD), except for the absence of a history of excessive alcohol consumption (equivalent to ethanol consumption for men <140 g/week, women <70 g/week) and other clinical pathological syndromes caused by specific factors that cause liver damage. NAFLD and NASH have become major causes of liver disease in Western countries over the past 20 years. Without timely control, NAFLD can easily progress to NASH as excess fat accumulation continues. In addition to excess fat accumulation, NASH differs from NAFLD in the presence of inflammatory cell infiltration into the liver, the degree of liver fibrosis, and the degree of damage to liver cells. In addition, if NASH continues to progress without effective control, it is highly likely to lead to fibrosis, cirrhosis, and even liver cancer.

Currently, there are no drugs for the treatment of NASH that can be safely and effectively used in clinical practice for a long time.

Fibroblast growth factor-21 (FGF-21) is another metabolic regulator recently discovered in vivo. It belongs to the FGF family and specifically affects liver cells, fat cells, and pancreatic islet cells. FGF-21 can effectively and safely regulate blood glucose and blood lipid levels independent of insulin, which has received much attention from researchers. It has also been reported that FGF-21 can effectively prevent and treat NAFLD induced in vitro (Liu Min et al., Effect of fibroblast growth factor-21 on lipid metabolism of non-alcoholic fatty liver cell model induced in vitro. Journal of Jilin University , May 2012, Vol. 38, No. 3, 477-481.)

CN2013101152100 (publication number CN103193878A; Applicant: Harbin Boao Biomedical Technology Development Co., Ltd.) discloses a novel long-acting mutant human FGF and its polyethylene glycol (PEG) conjugate. The protein structure of said FGF-PEG conjugate is shown in Sequence 3, and the method for preparing it is shown in Embodiment 4. The inventors later gave this conjugate the name FG (English name: PEGylated recombinant chimeric human-mouse fibroblast growth factor-21 or PEG- for short). hmFGF21).

The functions of FG are to regulate blood glucose, lower blood triglycerides, and regulate total cholesterol, etc. However, to date there have been no reports of the use of FG in the treatment of NASH.

Description of the invention

This document discloses the use of sustained release mouse FGF mutant in medicaments for the treatment of NASH.

As used herein, sustained-release mutant mouse FGF refers to pegylated recombinant chimeric mouse fibroblast growth factor-21, or a pharmaceutically acceptable salt thereof, having the sequence set forth in sequence 3 of patent application CN2013101152100 (publication number: CN103193878A).

In the context of this document, the use contemplates that PEGylated recombinant chimeric mouse fibroblast growth factor-21, or a pharmaceutically acceptable salt thereof, can reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, reduce steatosis and lobular inflammation, reduce hepatocellular ballooning dystrophy, and reduce liver damage.

In the context of this document, NASH includes, without limitation, hepatitis-induced NASH, obesity-induced NASH, diabetes-induced NASH, insulin resistance-induced NASH, hypertriglyceridemia-induced NASH, abetalipoproteinemia-induced NASH, glycogen storage disease-induced NASH, Wake's disease-induced NASH, Wolman's disease-induced NASH , and lipodystrophy-induced NASH, etc.

In the context of this document, medicinal products means pharmaceutical compositions containing pegylated recombinant chimeric mouse fibroblast growth factor-21 or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient. Pharmaceutical compositions may be in any pharmaceutically acceptable dosage form, including tablets, capsules, granules, pills, powders, ointments, freeze-dried preparations, pulverized powders, solutions, injectables, suppositories, sprays, drops, patches. and dragee. The medicaments of the present invention are preferably prepared as injectable medicaments such as injectable powders or liquid injectable preparations. Liquid injection preparations include aqueous injection preparations, injection preparations containing an organic solvent, suspension injection preparations, etc.

Formulations of pharmaceutical compositions for oral use may contain conventional excipients such as a binder, a bulking agent, a diluting agent, a tablet compression aid, a lubricant, a tablet disintegrating agent, a coloring agent, a flavoring agent, and a wetting agent. Suitable bulking agents include starch, sucrose, cellulose, mannitol, lactose, and the like. Suitable tablet disintegrating agents include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Solid form compositions for oral use can be obtained using conventional methods, including mixing, filling, compressing tablets, etc.

Repeated mixing allows the active pharmaceutical substance to be distributed throughout the volume of compositions containing a large amount of filling bulking agent. Common excipients include mannitol, sorbitol, sodium pyrosulfite, sodium hydrosulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, EDTA disodium, calcium disodium edetate, alkali metal carbonates and their aqueous solutions, alkali metal acetates and their aqueous solutions. , alkali metal phosphates and their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, D-mannitol, organosilicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipids, kaolin, talc , calcium stearate, magnesium stearate, etc.

The use and dosage of the pharmaceutical composition of the present invention is determined based on the stages of the disease at the time of use. For example, the pharmaceutical composition may be administered 1-6 times per day in 1-10 doses each time, each dose being 0.1 mg-1000 mg.

The use of PEGylated recombinant chimeric mouse fibroblast growth factor-21 of the present invention has the following advantages.

Pegylated recombinant chimeric mouse fibroblast growth factor-21 can significantly reduce serum levels of ALT and AST, reduce steatosis and lobular inflammation, reduce the degree of hepatocellular ballooning dystrophy, and reduce the pathological index of liver damage. The end results show that the present invention can be used to treat NASH with an efficacy that is superior to that of the prior art.

Detailed description of the invention

The present invention will now be further described with the following embodiments.

The method for producing FG used in the present invention is described in Embodiment 4 of CN2013101152100 (publication number: CN103193878A).

Embodiment 1. Obtaining aqueous injectable preparations

1. Composition: FG protein (concentration: 10mg/ml), histidine (pharmaceutical grade, concentration: 10mg/ml), citric acid-sodium citrate buffer (20mM sodium citrate-citric acid, 100mM NaCl, pH5.5± 0.1).

2. How to receive

1) a 12.5 mg/ml solution of the FG protein is obtained for further use in a buffer system based on citric acid-sodium citrate and the pH is adjusted to 5.5±0.1;

2) receive for further use 50 mg/ml stock solution of histidine in a buffer system based on citric acid-sodium citrate, pH adjusted to 5.5±0.1;

3) mix the FG protein solution from step 1) with the histidine stock solution from step 2) at a 4:1 volume ratio and transfer 500 μl of the mixed solution into 2 ml penicillin vials.

Experimental Example 1 Therapeutic effect of FG over a range of effective doses in a mouse model of diet-induced NASH with MCD (Methionine Choline Deficiency).

A mouse model of diet-induced NASH with MCD was used in this experiment, primarily because this model has been in use for over 40 years and the process for producing it is fairly well established. In addition, there is a lot of evidence for the condition induced by this diet in terms of rate of onset (i.e. NASH-related symptoms may appear after about 4 weeks) and efficacy (i.e., the symptoms induced are very similar to NASH symptoms in person).

1. Method of conducting the experiment

After two weeks of adaptive feeding, eight-week-old male C57BL mice were fed an MCD diet. After feeding on the MCD diet for two weeks, mice were randomly divided into 4 groups according to body weight: solvent treatment group, FGF-21 group, low dose FG group, high dose FG group. Each group included 10 mice and were injected subcutaneously daily for two weeks.

Details of the distribution of animals into groups and the introduction are given in table 1.

Table 1. Distribution of animals into groups and administration

Group n Dose Administration period (days) Solvent Treatment Group ten Solvent in the same volume 14 days Group FGF-21 ten 0.4 mg/kg/day 14 days Low dose FG group ten 0.125 mg/kg/day 14 days High dose FG group ten 2 mg/kg/day 14 days

After the end of the administration, the mice were sacrificed and their respective tissues were removed for further analyses.

2. Definition of indexes

2.1. Serum was separated and various biochemical indices were determined: Serum ALT and AST levels were determined using a clinical assay kit (provided by Shanghai Shensuo-UNF Medical. Diagnostic Articles Co., Ltd).

2.2. Histomorphological study of mouse liver tissue

Morphological changes in mouse liver were observed by staining with hematoxylin-eosin. The specific steps were as follows. A freshly extracted small fragment of the liver was fixed in a formalin solution overnight and, after gradual dehydration, embedded in paraffin, and then sliced 5 µm thick. Liver sections were stained with hematoxylin-eosin and, finally, the liver tissue morphology of each mouse was examined under a microscope.

2.3. Detection of collagen fibers in mouse liver tissue

Collagen deposition in mouse liver was examined by the Sirius Red (SR) staining method. The specific steps were as follows. Liver sections from the paraffin blocks were stained with the Sirius Red staining kit and finally, the fibrosis of the liver tissue of each mouse was examined under a microscope.

In clinical practice, there is a system of quantitative indicators for determining the severity of NASH, i.e. indicators of the Network of NASH Research Centers, mainly covering three parameters: steatosis, lobular inflammation and ballooning dystrophy. In this experiment, five mice were randomly selected from each group, and within the hematoxylin-eosin-stained sections of liver tissue of the corresponding mouse, five fields of view of the microscope were randomly selected, based on which the indicators were evaluated for the three above parameters in order to objectively assess the degree of hepatitis in mice in each group.

3. SPSS16.0 statistical software was used to determine statistics and analyze data. A t-test was used to compare the difference between the two data groups. One-way analysis of variance and multiple linear regression analysis were used to compare differences between multiple data groups. The difference was statistically significant with P<0.05.

4. Experimental results

4.1. The effect of FG on serum ALT levels is shown in Table 2.

Table 2. Effect of FG on serum ALT levels

Solvent Treatment Group Group FGF-21 Low dose FG group High dose FG group Serum ALT (U/l) 199.8±30.8 100.2±20.6* 51.1±7.7***Δ 39.9±4.1***Δ

The data shown in the table is the mean ± standard error; (2) *vs solvent treatment group P<0.05; *** vs. solvent treatment group P<0.001; Δ vs FGF 21 group P<0.05.

As shown in Table 2, after two weeks of treatment, the ALT level of NASH mice in the FGF 21 group decreased to 100.2 ± 20.6 U/L, and there was a significant difference compared to the ALT level in the vehicle treatment group of 199 .8±30.8 U/L (P<0.05). ALT levels in the low dose FG group and the high dose FG group decreased to 51.1 ± 7.7 U/L and 39.9 ± 4.1 U/L, respectively, and showed a significant difference compared to the vehicle treatment group ( P<0.001), as well as the FGF 21 group (P<0.05).

These results mean that FG is not only effective in lowering ALT levels, but, in this case, is also more effective than FGF 21.

4.2. The effect of FG on serum AST levels is shown in Table 3.

Table 3. Effect of FG on serum AST levels

Solvent Treatment Group Group FGF-21 Low dose FG group High dose FG group Serum ALT (U/L) 196.9±29.3 115.7±15.9* 74.4±7.9**Δ 73.7±7.8**Δ

(1) The data given in the table is the mean ± standard error; (2) *vs solvent treatment group P<0.05; ** in comparison with the solvent treatment group P<0.01; Δ vs FGF-21 group P<0.05.

As shown in Table 3, after two weeks of treatment, the AST level in NASH mice in the FGF-21 group decreased to 115.7 ± 15.9 U/L, and there was a significant difference compared to the AST level in the vehicle treatment group of 196 .9±29.3 U/L (P<0.05). AST levels in the low dose FG group and the high dose FG group decreased to 74.4 ± 7.9 U/L and 73.7 ± 7.8 U/L, respectively, and showed a significant difference compared to the vehicle treatment group ( P<0.01), as well as the FGF-21 group (P<0.05). These results mean that FG is not only effective in lowering ALT levels, but, in this case, is also more effective than FGF-21.

4.3. The effect of reducing NASH clinical scores is shown in Table 4.

Table 4. Effect of reducing NASH clinical scores

Steatosis Lobular inflammation Ballooning dystrophy Total indicator Solvent Treatment Group 2.2±0.2 2.6±0.1 1.3±0.1 6.0±0.21 Group FGF-21 2.0±0.3 2.1±0.2 0.9±0.1** 4.7±0.5* Low dose FG group 1.8±0.3 0.7±0.1***ΔΔΔ 0.7±0.1** 3.2±0.4***Δ High dose FG group 0.9±0.3**Δ 0.6±0.2***ΔΔΔ 0.4±0.1***ΔΔ 1.9±0.5***ΔΔ

(1) The data given in the table is the mean ± standard error; (2) *vs solvent treatment group P<0.05; ** in comparison with the solvent treatment group P<0.01; *** vs. solvent treatment group P<0.001; Δ in comparison with the FGF-21 group P<0.05; ΔΔ vs FGF-21 group P<0.01; ΔΔΔ versus FGF-21 group P<0.001.

As shown in Table 4, after two weeks of treatment, both ballooning dystrophy score and total NASH score in the FGF-21 group were significantly reduced (P<0.05) compared to the vehicle treatment group, while there was a significant reduction in lobular inflammation scores. not observed (P>0.05). Compared to the solvent treatment group, a significant decrease was observed in both the low dose FG group and the high dose FG group in terms of lobular inflammation scores, ballooning dystrophy scores and total NASH scores. Steatosis scores in the high dose FG group were significantly reduced compared to the vehicle treatment group. In addition, compared to the FGF-21 group, all scores of steatosis, lobular inflammation, ballooning dystrophy and their total score significantly decreased in the low FG dose group and/or the high FG dose group. These results mean that FG is not only effective in reducing lobular inflammation, ballooning dystrophy and total NASH, but, in this case, is also more effective than FGF-21.

5. Experimental results

Findings after FG injection show that compared to the vehicle treatment group and the FGF-21 group, the low dose FG group and/or the high dose FG group can significantly decrease serum ALT and AST levels, decrease steatosis and lobular inflammation, decrease the degree of hepatocellular ballooning dystrophy and decrease the pathological indicator of liver damage. These results indicate that FG is effective in the treatment of NASH.

6. Conclusion. FG has potential for clinical use in the treatment of NASH that is superior to that of the prior art.

Claims (8)

1. Use of pegylated recombinant chimeric mouse fibroblast growth factor-21 or a pharmaceutically acceptable salt thereof in medicaments for the treatment of non-alcoholic steatohepatitis, wherein the use is selected from the reduction of lobular inflammation. 2. Use according to claim 1, wherein the use is further selected from reducing serum levels of alanine aminotransferase and aspartate aminotransferase, reducing steatosis, reducing the degree of hepatocellular ballooning dystrophy, and reducing liver damage. 3. Use according to claim 1, wherein non-alcoholic steatohepatitis (NASH) is selected from hepatitis induced NASH, obesity induced NASH, diabetes induced NASH, insulin resistance induced NASH, hypertriglyceridemia induced NASH, abetalipoproteinemia induced NASH, NASH glycogen storage disease induced, NASH induced Wake's disease, Wolman's disease-induced NASH, or lipodystrophy-induced NASH. 4. Use according to claim 1, wherein the medicaments are pharmaceutical compositions containing pegylated recombinant chimeric mouse fibroblast growth factor-21 or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient. 5. Use according to claim 4, wherein the pharmaceutical compositions may be in any pharmaceutically acceptable dosage form. 6. Use according to claim 5, wherein the dosage forms are selected from tablets, capsules, granules, pills, powders, ointments, freeze-dried preparation, pulverized powders, solutions, injectables, suppositories, spray solutions, drops, patches and dragee. 7. Use according to claim 6, wherein the dosage forms are selected from injectable powders or liquid injectable preparations. 8. Use according to claim 7, wherein the liquid injectables are selected from aqueous injectables, organic solvent-containing injectables, and suspension injectables.