Patent Description:
Non-alcoholic fatty liver disease (NAFLD), also known as non-alcoholic fatty liver, is caused by a variety of reasons. Lesions of this disease mainly occur in the hepatic lobule characterized by steatosis of hepatic parenchymal cells and triglyceride (TG) accumulation (fat content in liver tissues accounting for over <NUM>% of liver wet weight or more than <NUM>/<NUM> hepatic cells presenting steatosis confirmed by histology). NAFLD is similar to alcoholic liver disease (ALD) in terms of pathological changes except absence of history of excessive drinking (equivalent to ethanol consumption, male <<NUM> / week, female <<NUM> / week) and other clinical pathological syndromes caused by specific liver-damaging factors. NAFLD and NASH have become the leading causes of liver disease in Western countries in the past <NUM> years. Without timely control, NAFLD can easily progress to NASH as fat continues to accumulate excessively. In addition to excessive accumulation of fat, NASH differs from NAFLD in the presence of inflammatory cell infiltration in liver, the degree of hepatic fibrosis, and the degree of liver cell damage. Furthermore, if NASH continues to progress without effective control, it is highly likely to cause hepatic fibrosis, cirrhosis and even liver cancer.

At present, there are no such drugs for NASH that can be clinically used for long term, safe and effective.

Fibroblast growth factor-<NUM> (FGF-<NUM>) is another metabolic regulator recently discovered in vivo. It belongs to FGF family and specifically acts on liver, fat, and islet cells. FGF-<NUM> can effectively and safely regulate blood glucose and blood fat independently of insulin, which has got researchers' a lot of attention. It has also been reported that FGF-<NUM> can effectively prevent and treat NAFLD induced in vitro (<NPL>. ) Further examples may be found in <CIT>; <CIT>; <CIT> and <NPL>).

<CIT> (Publication Number: <CIT>; Applicant: Harbin Boao Biomedical Technology Development Co. ) discloses a new long-acting mutant human FGF and the polyethylene glycol (PEG) conjugate thereof. The protein structure of the said FGF-PEG conjugate is shown in Sequence <NUM> and its preparation method is shown in Example <NUM>. This conjugate was later named by its inventors as PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> (or PEG-hmFGF21 for short).

PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> has the functions of regulating blood glucose, lowering blood triglycerides, and regulating total cholesterol, et al. However, there have been no reports of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> in the treatment of NASH so far.

Disclosed herein the use of a PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> in the preparation of drugs for treating NASH.

As used herein, the PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor 21has the amino acid sequence set forth in Sequence <NUM> disclosed in the patent application <CIT> (Publication Number: <CIT>) and is obtainable according to the preparation method disclosed in Example <NUM> of <CIT> (Publication Number: <CIT>).

As used herein, the use includes that PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> or its salt can lower the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, improve steatosis and lobular inflammation, reduce the degree of hepatocellular ballooning degeneration, and improve liver damage.

As used herein, the NASH includes, not limited to, hepatitis-induced NASH, obesity-induced NASH, diabetes-induced NASH, insulin resistance-induced NASH, hypertriglyceridemia-induced NASH, abetalipoproteinemia-induced NASH, NASH induced by glycogen storage disease, NASH induced by Wake's Disease, NASH induced by Wolman disease, and lipodystrophia-induced NASH, et al.

As used herein, the drugs mean pharmaceutical compositions containing PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> or a pharmaceutically acceptable salt thereof as the active pharmaceutical ingredient. The pharmaceutical compositions can be prepared into any pharmaceutically acceptable dosage form, including tablets, capsules, granules, pills, powders, paste, sublimed preparation, dustpowders, solutions, injections, suppositories, sprays, drops, patches, and drop pills. Drugs of the present disclosure are preferably prepared into injectable drugs, such as powder injections or liquid injections. The liquid injections include water injections, organic solvent injections, and suspension injections, et al.

The preparations of the pharmaceutical compositions for oral administration may contain conventional excipients such as the bonding agent, stuffing bulking agent, diluting agent, tablet compressing agent, lubricating agent, disintegrating agent, coloring agent, flavoring agent, and wetting agent. The suitable stuffing bulking agents include starch, sucrose, cellulose, mannitol, lactose, and other similar ones. The suitable disintegrating agents include starch, polyvinyl pyrrolidone and starch derivatives such as sodium starch glycolate. The suitable lubricating agents include, for example, magnesium stearate. The solid form of the compositions for oral administration can be prepared through conventional methods including mixing, filling, tablet compressing, et al. Repeated mixing allows the active pharmaceutical substance to be distributed throughout the compositions containing a large amount of stuffing bulking agent. The excipients commonly-used include mannitol, sorbitol, sodium pyrosulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium disodium edetate, carbonates of alkali metal and aqueous solutions thereof, acetates of alkali metal and aqueous solutions thereof, phosphates of alkali metal and aqueous solutions thereof, 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, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween <NUM>, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipids, kaolin, pulvistalci, calcium stearate , magnesium stearate, et al.

The usage and dosage of the pharmaceutical composition of the present disclosure are determined according to conditions of diseases while being used. For example, the pharmaceutical composition can be administered <NUM>-<NUM> times a day for <NUM>-<NUM> doses each, and each dose can be <NUM>-<NUM>.

The new use of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> provided by the present invention has the following advantages:
PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> can significantly lower the levels of ALT and AST in serum, improve steatosis and lobular inflammation, reduce the degree of hepatocellular ballooning degeneration, and improve the pathological score of liver damage. The final results demonstrate that the present invention can be used to treat NASH with superior effectiveness to prior art.

The present disclosure will now be further demonstrated using the following embodiments.

For the preparation method of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> used in the present invention, see Embodiment <NUM> of <CIT> (Publication Number: <CIT>).

A mouse model of MCD diet-induced NASH is used in this experiment, mainly because this diet has been used for more than <NUM> years and its production process has become more mature. In addition, the characterization this diet induced has obtained multiple verification in terms of its rapidity (i.e. NASH related symptoms can be triggered in about <NUM> weeks) and effectiveness (i.e. symptoms induced have great similarity to those of human NASH).

After adaptive feeding for two weeks, C57BL male mice of eight-week-old were began to be fed MCD diet. After MCD diet fed for two weeks, the mice were randomly divided into <NUM> groups according to the body weight: a solvent group, a FGF-<NUM> group, a low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group, and a high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group. There were <NUM> mice in each group and they were administered once a day via hypodermic injection for two weeks.

Details of animal grouping and administration are shown in Table <NUM>:.

After the administration, the mice were killed and their relevant tissues were removed for subsequent analyses.

<NUM> The blood serum was separated and various biochemical indices therein were detected: levels of ALT and AST in serum were determined using a clinically general assay kit (provided by Shanghai Shensuo-UNF Medical. Diagnostic Articles Co.

Morphological changes in mouse liver were observed through H&E staining method. The specific steps were as follows: The freshly removed small piece of liver was fixed in formalin solution overnight, and embedded in paraffin after gradient dehydration, then sectioned into <NUM> slices. The liver sections were stained with hematoxylin-eosin, and finally the morphology of liver tissue of each mouse was observed under a microscope.

The collagen deposition in mouse liver was observed through Sirius Red (SR) staining method. The specific steps were as follows: The liver sections from paraffin blocks were stained using the Sirius Red staining kit, and finally the fibrosis of liver tissue of each mouse was observed under a microscope.

There is a scoring system for assessing the severity of NASH in clinical practice, i.e., NASH Clinical Research Network Scores, mainly including three aspects: steatosis, lobular inflammation, and ballooning degeneration. In this experiment, five mice were randomly selected from each group, and five microscopic fields of view within the H&E stained liver tissue sections of the corresponding mouse were randomly selected based on which a score is given from the above three aspects, objectively evaluating the degree of hepatitis of mice in each group.

<NUM> statistical software was used for statistics and data analysis. A t-test was used to compare the difference between two groups of data. One-way ANOVA test and multiple linear regression analysis were used to compare differences among multiple groups of data. The difference was statistically significant with P<<NUM>.

<NUM> Effect of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> on serum ALT level as shown in Table <NUM>:.

As shown in Table <NUM>, after two weeks of treatment, the ALT level of mice with NASH in the FGF-21group decreased to <NUM>±<NUM> U/L, and there was a significant difference compared with the solvent group with the ALT level <NUM>±<NUM> U/L (P<<NUM>). The levels of ALT in the low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group and high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group decreased to <NUM> ±<NUM> U/L and <NUM>±<NUM> U/L, respectively, and a significant difference was found compared with the solvent group (P<<NUM>) as well as the FGF-<NUM> group (P<<NUM>). These results indicate that PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> not only has the efficacy of lowering ALT levels but also its efficacy herein is better than FGF-<NUM>.

<NUM> Effect of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> on serum AST level as shown in Table <NUM>:.

As shown in Table <NUM>, after two weeks of treatment, the AST level of mice with NASH in the FGF-21group decreased to <NUM>±<NUM>. 9U/L, and there was a significant difference compared with the solvent group with the AST level <NUM>±<NUM> U/L (P<<NUM>). The levels of AST in the low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group and high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group decreased to <NUM>±<NUM>. 9U/L and <NUM>±<NUM>. 8U/L, respectively, and a significant difference was found compared with the solvent group (P<<NUM>) as well as the FGF-<NUM> group (P<<NUM>). These results indicate that PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> not only has the efficacy of lowering ALT levels but also its efficacy herein is better than FGF-<NUM>.

<NUM> Effect of improvement on NASH clinical scores as shown in Table <NUM>:.

As shown in Table <NUM>, after two weeks of treatment, both the scores for ballooning and NASH total scores in FGF-<NUM> group had improved significantly (P<<NUM>) compared with the solvent group, whereas no significant improvement in scores for lobular inflammation had been observed (P><NUM>). Compared with the solvent group, there was a significant improvement in both low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group and high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group in terms of scores for lobular inflammation, scores for ballooning and NASH total scores. Scores for steatosis in high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group had been significantly improved compared with the solvent group. In addition, in comparison with FGF-<NUM> group, all scores for steatosis, lobular inflammation, ballooning and the total thereof had been significantly improved in low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group and/or high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group. These results indicate that PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> not only has the efficacy of improving lobular inflammation, ballooning, and NASH total scores but also its efficacy herein is better than FGF-<NUM>.

Detections after injection of PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> show that compared with the solvent group and FGF-<NUM> group, low-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group and/or high-dose PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> group can significantly lower the levels of ALT and AST in serum, improve steatosis and lobular inflammation, reduce the degree of hepatocellular ballooning degeneration, and improve the pathological score of liver damage. These results indicate that PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> has the effect of treating NASH.

Claim 1:
A PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> or a pharmaceutically acceptable salt thereof for use in treating non-alcoholic steatohepatitis, wherein the PEGylated Recombinant Human-mouse Chimeric Fibroblast Growth Factor <NUM> is obtainable by providing a peptide consisting of the amino acid sequence according to:
<IMG>
; and PEGylating said peptide at pH <NUM> using mPEG-ALD having a molecular weight of 20kD and sodium cyanoborohydride.