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WO2018171557A1 - 一种长效化突变的人源成纤维生长因子的新用途 - Google Patents

一种长效化突变的人源成纤维生长因子的新用途 Download PDF

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Publication number
WO2018171557A1
WO2018171557A1 PCT/CN2018/079482 CN2018079482W WO2018171557A1 WO 2018171557 A1 WO2018171557 A1 WO 2018171557A1 CN 2018079482 W CN2018079482 W CN 2018079482W WO 2018171557 A1 WO2018171557 A1 WO 2018171557A1
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group
caused
use according
steatohepatitis
nonalcoholic
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French (fr)
Inventor
李剑
韩君
马晓慧
太平
王根辈
曹小丹
黄瑞晶
金永杰
李静
陈晨
贾国勇
王媛媛
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Tasly Pharmaceutical Group Co Ltd
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Tasly Pharmaceutical Group Co Ltd
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Priority to EP18771156.9A priority Critical patent/EP3603660B1/en
Priority to CN201880013016.2A priority patent/CN110520146A/zh
Priority to JP2020500949A priority patent/JP7491839B2/ja
Priority to CA3049703A priority patent/CA3049703A1/en
Priority to RU2019125343A priority patent/RU2785582C2/ru
Publication of WO2018171557A1 publication Critical patent/WO2018171557A1/zh
Priority to US16/520,053 priority patent/US12152059B2/en
Anticipated expiration legal-status Critical
Priority to US18/920,806 priority patent/US20250042964A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a long-acting mutant human fibroblast growth factor-21 for treating non-alcoholic steatohepatitis drugs.
  • Non-alcoholic fatty liver disease also known as non-alcoholic fatty liver
  • NAFLD non-alcoholic fatty liver
  • TG Degeneration and triglyceride
  • pathological changes and alcoholic liver disease is similar, but no history of excessive drinking (equivalent to ethanol, male ⁇ 140g / week, female ⁇ 70g / week) and other clear pathological factors of liver damage.
  • nonalcoholic fatty liver disease includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and nonalcoholic fatty liver cirrhosis ( Non-alcoholic cirrhosis (NAC) and hepatocellular carcinoma.
  • Non-alcoholic fatty liver disease NAFLD
  • NASH nonalcoholic steatohepatitis
  • Non-alcoholic fatty liver disease can not be controlled in time, and the fat continues to accumulate and degenerate easily to non-alcoholic steatohepatitis.
  • Non-alcoholic steatohepatitis differs from nonalcoholic fatty liver disease in that in addition to excessive accumulation of fat, inflammatory cell infiltration, varying degrees of fibrosis, and damage to liver cells occur in the liver.
  • non-alcoholic steatohepatitis continues to progress without effective control, it is highly likely to cause liver fibrosis, cirrhosis and even liver cancer.
  • Fibroblast growth factor-21 is another recently discovered metabolic regulator in vivo, belonging to the family of fibroblast growth factors, which specifically act on liver, fat, and islet cells and not researchers are reliant on the ability of insulin to effectively and safely regulate blood sugar and blood lipids. It has also been reported that fibroblast growth factor-21 (FGF-21) can effectively prevent and control NAFLD induced in vitro (Liu Min et al., fibroblast growth factor- Effect of 21 on lipid metabolism in a non-alcoholic fatty liver cell model induced in vitro, Journal of Jilin University, May 2012, Vol. 38, No. 3, 477-481)
  • CN2013101152100 (publication number is CN103193878A) affiliated to Harbin Boao Biomedical Technology Development Co., Ltd., discloses a new long-acting mutant human fibroblast growth factor and cross-linking of polyethylene glycol, polyethylene glycol
  • the protein structure of the crosslinked product is Sequence 3, and its preparation method is shown in Example 4 in the literature, and the inventors of the present invention later named it as a liposaccharide (English name: PEGylation Recombinant Human-mouse Chimeric Fibroblast Growth Factor 21). , referred to as PEG-hmFGF21).
  • Liposide regulates blood sugar, lowers blood triglycerides and regulates overall cholesterol, but there are no reports of lipolysin in the treatment of nonalcoholic steatohepatitis.
  • the present invention provides the use of a long-acting mutant human fibroblast growth factor for the treatment of non-alcoholic steatohepatitis.
  • the long-acting mutant human fibroblast growth factor is a lipoflavin or a salt thereof, the sequence of which is shown in Sequence 3 of CN2013101152100 (publication number CN103193878A):
  • lipoflavin or a salt thereof can lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, improve the degree of steatosis, improve the degree of hepatic lobular inflammation, and reduce the degree of hepatocyte ballooning and Improve the degree of liver damage.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • the nonalcoholic steatohepatitis according to the present invention includes, but is not limited to, nonalcoholic steatohepatitis caused by hepatitis, nonalcoholic steatohepatitis caused by obesity, nonalcoholic steatohepatitis caused by diabetes, nonalcohol caused by insulin resistance
  • Non-alcoholic steatohepatitis caused by steatohepatitis hypertriglyceridemia, nonalcoholic steatohepatitis caused by no beta lipoproteinemia, nonalcoholic steatohepatitis caused by glycogen storage disease, Wake disease
  • Nonalcoholic steatohepatitis, nonalcoholic steatohepatitis caused by Wolman's disease, and nonalcoholic steatohepatitis caused by lipodystrophy Nonalcoholic steatohepatitis caused by hepatitis, nonalcoholic steatohepatitis caused by obesity, nonalcoholic steatohepatitis caused by diabetes, nonalcohol caused by insulin resistance
  • the drug is a pharmaceutical composition comprising the lipoflavin of the present invention or a pharmaceutically acceptable salt thereof as a pharmaceutically active ingredient
  • the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form, including: a tablet, Capsules, granules, pills, powders, ointments, dandruffs, powders, solutions, injections, suppositories, sprays, drops, patches
  • drugs of the invention are preferably injectable drugs, such as powder or liquid needles
  • the liquid needle is a water needle, an organic solvent needle, a suspension needle or the like.
  • the pharmaceutical composition of the present invention may contain conventional excipients such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent, and a moisturizing agent.
  • Suitable fillers include starch, sucrose, cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Solid oral compositions can be prepared by conventional methods such as mixing, filling, tableting, and the like.
  • common excipient ingredients include: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride , thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, 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, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, poly Vinyl pyrrolidone, glycerin, Tween 80, agar, calcium carbon
  • the pharmaceutical composition of the present invention determines the dosage according to the condition of the disease at the time of use, and can be administered 1-6 times a day for 1-10 dosage units each, and each dosage unit may be 0.1 mg to 1000 mg.
  • LPS can significantly reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels) to improve the degree of steatosis, improve the degree of hepatic lobular inflammation, reduce the degree of hepatocyte ballooning, and improve the degree of liver damage.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • composition liposin protein (concentration: 10 mg/mL), histidine (pharmaceutical grade, concentration: 10 mg/mL), citric acid-sodium citrate buffer (20 mM sodium citrate-citric acid, 100 mM NaCl) , pH 5.5 ⁇ 0.1).
  • the concentration is 12.5 mg / mL lipoprotein protein stock solution, the buffer system is citric acid - sodium citrate, the pH is adjusted to 5.5 ⁇ 0.1, standby;
  • the concentration is 50mg/mL histidine mother liquor, the buffer system is citric acid-sodium citrate buffer, the pH is adjusted to pH 5.5 ⁇ 0.1, and the standby is used;
  • step 1) The lipoprotein protein solution of step 1) and the histidine mother liquor of step 2) were mixed at a volume ratio of 4:1 to a 2 mL vial at a rate of 500 ⁇ L/piece.
  • the methionine choline-deficient feed was used to induce the mouse NASH model, mainly because the application of this kind of feed has been more than 40 years old, and its production process has become more mature and the characterization has been verified by many parties. Its rapidity (approximately 7 weeks can cause NASH-related symptoms) and effectiveness (induced symptoms are very similar to human NASH).
  • MCD feeds Two weeks after adaptive feeding of eight-week-old C57BL male mice, methionine and choline deficiency (MCD) feeds were started. MCD feeds were randomly divided into four groups according to animal body weight after two weeks of feeding.
  • the solvent group FGF-21
  • the group the low-dose group of lipopolysaccharide, the high-dose group of liposin, 10 animals in each group, was administered subcutaneously once a day for administration, and the administration period was 2 weeks.
  • mice were sacrificed and relevant tissues were removed for subsequent analysis.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • liver morphology changes of mice were observed by H&E staining method. The specific steps were as follows: freshly removed small pieces of liver were fixed in formalin solution overnight, dehydrated by gradient dehydration, and embedded in sections to 5 ⁇ m, using hematoxylin - The red staining solution was used to stain the liver sections, and finally the morphology of the liver tissues of each mouse was observed under a microscope.
  • the NASH Clinical Research Network scores are clinically defined, including three aspects: steatosis, lobular inflammation, and hepatocyte ballooning. (ballooning).
  • mice were randomly selected from each group, and five fields of view were randomly selected from the liver tissue H&E stained sections of the corresponding mice to score from the above three aspects, and the degree of hepatitis lesions in each group of mice was objectively evaluated.
  • mice with FGF-21 in non-alcoholic steatohepatitis decreased to 100.2 ⁇ 20.6 U/L, and was significantly higher than that of the solvent group of 199.8 ⁇ 30.8 U/L.
  • the difference (P ⁇ 0.05) ALT levels in low-dose and high-dose groups of lipopolysaccharide decreased to 51.1 ⁇ 7.7U/L and 39.9 ⁇ 4.1U/L, respectively, and were significantly different from the solvent group (P ⁇ 0.001).
  • P ⁇ 0.05 compared with the FGF-21 group, indicating that the lipopolysaccharide not only has the effect of lowering the ALT level but also has better efficacy than the FGF-21 group.
  • Table 4 shows the improvement of NASH clinical relevance score
  • the low-dose leptin group and/or the high-dose leptin group were significantly superior to the FGF-21-treated group in steatosis, hepatic lobular inflammation, hepatocyte ballooning, and NASH total score.
  • the above results indicate that liposaccharide not only has the effects of improving hepatic lobular inflammation, hepatocyte balloon-like degeneration and NASH total score, but also has a significantly better effect than the FGF-21 group.
  • low-dose leptin or / and high-dose leptin group can significantly reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase in serum compared with solvent group and FGF-21 group.
  • AST serum alanine aminotransferase
  • AST aspartate aminotransferase
  • liposaccharide has a role in the treatment of nonalcoholic steatohepatitis.
  • Lactosin has clinical application value in the treatment of nonalcoholic steatohepatitis and is superior to the prior art.

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Abstract

本发明公开了一种长效化突变的人源成纤维生长因子的新用途，其中所述长效化突变的人源成纤维生长因子为脂糖素，其中所述新用途是在制备治疗非酒精性脂肪肝炎的药物中的应用。

Description

一种长效化突变的人源成纤维生长因子的新用途 技术领域
本发明属于生物医药领域，具体涉及一种长效化突变的人源成纤维细胞生长因子-21在治疗非酒精性脂肪肝炎药物中的应用。
背景技术
非酒精性脂肪性肝病(non-alcoholic fatty liver disease，NAFLD)，又称非酒精性脂肪肝(non-alcoholic fatty liver)，是由多种原因引起，病变主体在肝小叶，以肝实质细胞脂肪变性和甘油三酯(triglyceride，TG)蓄积(肝组织脂含量超过肝湿量的5％以上，或在组织学上有1/3以上肝细胞脂肪化)为特征，病理学改变与酒精性肝病(alcoholic liver disease，ALD)相似，但无过量饮酒史(折合乙醇量男性<140g/周、女性<70g/周)和其他明确的损肝因素的临床病理综合征。
非酒精性脂肪性肝病的疾病谱包括非酒精性单纯性脂肪肝(non-alcoholic fatty liver，NAFL)、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis，NASH)及非酒精性脂肪性肝硬化(non-alcoholic cirrhosis，NAC)和肝细胞癌。
近20年来非酒精性脂肪肝病(NAFLD)以及非酒精性脂肪肝炎(NASH)已成为西方国家肝病的首要病因。非酒精性脂肪肝病如此时未能得以及时控制，脂肪继续过度累积变性易进展为非酒精性脂肪肝炎。非酒精性脂肪肝炎与非酒精性脂肪肝病的区别在于除脂肪的过度积累以外，肝部出现炎症细胞浸润、不同程度的纤维化以及肝细胞的损害。另外，如果非酒精性脂肪肝炎未得到有效控制而继续进展，则极易导致肝纤维化、肝硬化甚至肝癌的发生。
目前对于NASH疾病临床尚无可长期应用，安全有效的药物。
成纤维细胞生长因子-21(fibroblast growth factor-21，FGF-21)是近期发现的体内又一个代谢调节因子，属于成纤维细胞生长因子家族，其特异性作用于肝脏、脂肪、胰岛细胞且不依赖于胰岛素有效安全地调节血糖血脂的能力深得研究人员青睐，也有报道成纤维细胞生长因子-21(FGF-21)可以有效的防治体外诱导的NAFLD(刘敏等，成纤维细胞生长因子-21对体外诱导的非酒精性脂肪肝细胞模型脂代谢的影响，吉林大学学报，2012年5月，第38卷第3期，477-481)
CN2013101152100(公开号为CN103193878A)隶属于哈尔滨博翱生物医药技术开发有限公司，公开了一种新的长效化突变的人源成纤维生长因子及聚乙二醇的交联物，聚乙二醇交 联物的蛋白质结构为序列3，其制备方法见该文献中的实施例4，后来该发明的发明人将其命名为脂糖素(英文名为：PEGylation Recombinant Human-mouse Chimeric Fibroblast Growth Factor 21，简称PEG-hmFGF21)。
脂糖素有调节血糖，降低血液中甘油三酯和调节总体胆固醇等功效，但是目前没有使用脂糖素治疗非酒精性脂肪肝炎方面的报道。
发明内容
本发明提供了长效化突变的人源成纤维生长因子在治疗非酒精性脂肪肝炎的药物中的应用。
所述长效化突变的人源成纤维生长因子为脂糖素或其盐，其序列见CN2013101152100(公开号为CN103193878A)的序列3：
本发明所述应用，包括：脂糖素或其盐可以降低血清中谷丙转氨酶(ALT)及谷草转氨酶(AST)水平、改善脂肪变性程度、改善肝小叶炎症程度、降低肝细胞气球样变性程度和改善肝脏损伤程度。
本发明所述的非酒精性脂肪肝炎，其包括但不限于肝炎引起的非酒精性脂肪肝炎、肥胖症引起的非酒精性脂肪肝炎、糖尿病引起的非酒精性脂肪肝炎、胰岛素抵抗引起的非酒精性脂肪肝炎、高甘油三酯血症引起的非酒精性脂肪肝炎、无β脂蛋白血症引起的非酒精性脂肪肝炎、糖原贮积病引起的非酒精性脂肪肝炎、韦克病引起的非酒精性脂肪肝炎、沃尔曼病引起的非酒精性脂肪肝炎和脂肪营养不良所引起的非酒精性脂肪肝炎等。
其中所述药物是含有本发明脂糖素或其药用盐作为药物活性成分的药物组合物，所述药物组合物可以被制备成任何一种可药用的剂型，这些剂型包括：片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、丹剂、粉剂、溶液剂、注射剂、栓剂、喷雾剂、滴剂、贴剂，本发明的药物优选注射用药物，如制备成粉针或液体针，所述液体针如水针，有机溶剂针，混悬液针等。
本发明的药物组合物，其口服给药的制剂可含有常用的赋形剂，诸如粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂。适用的填充剂包括淀粉、蔗糖、纤维素、甘露糖醇、乳糖和其它类似的填充剂。适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠。适宜的润滑剂包括，例如硬脂酸镁。可通过混合，填充，压片等常用的方法制备固体口服组合物。进行反复混合可使活性物质分布在整个使用大量填充剂的那些组合物中；常用的辅料成分包括：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠， 一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、吐温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
本发明的药物组合物在使用时根据疾病的状况确定用法用量，可以每日服1-6次，每次1-10个剂量单位，每个剂量单位可以是0.1mg-1000mg。
本发明提供的脂糖素的新用途具有以下优点：
脂糖素可以显著降低血清中谷丙转氨酶(ALT)及谷草转氨酶(AST)水平)改善脂肪变性程度、改善肝小叶炎症程度、降低肝细胞气球样变性程度、改善肝脏损伤程度的病理评分。最终结果证明本发明可以用于治疗非酒精性脂肪肝炎，且效果优于现有技术。
具体实施方式
以下通过实施例进一步说明本发明。
本发明使用的脂糖素，其制备方法可以参见CN2013101152100(公开号为CN103193878A)的实施例4。
实施例1：水针的制备
1、组成：脂糖素蛋白(浓度为10mg/mL)、组氨酸(药用级，浓度为10mg/mL)、柠檬酸-柠檬酸钠缓冲液(20mM柠檬酸钠-柠檬酸，100mM NaCl，pH5.5±0.1)。
2、制备方法：
1)浓度为12.5mg/mL脂糖素蛋白原液，缓冲体系为柠檬酸-柠檬酸钠，调节pH为5.5±0.1，备用；
2)浓度为50mg/mL组氨酸母液，缓冲体系为柠檬酸-柠檬酸钠缓冲液，调节pH为pH5.5±0.1，备用；
3)将步骤1)的脂糖素蛋白原液与步骤2)的组氨酸母液按照体积比为4：1配比，以500μL/支的规格灌装至2mL西林瓶。
实验例1：脂糖素在有效剂量范围内对蛋氨酸及胆碱缺乏饮食(MCD diet)诱导的非酒精性脂肪性肝炎(non-alcoholic steatohepatitis，NASH)小鼠模型的治疗作用。
本实验选用蛋氨酸胆碱缺乏饲料(MCD)诱导小鼠NASH模型，主要是由于该种饲料的应用已经有超过四十年的历史，其生产工艺越发成熟且引发表征也已经得到了多方验证，因其快速性(约4周即可引发NASH相关症状)和有效性(诱发症状与人类NASH有很大相似性)。
1、实验方法：
将八周龄C57BL雄性小鼠适应性饲养两周后开始蛋氨酸及胆碱缺乏(MCD)饲料饲喂，MCD饲料饲喂两周后根据动物体重随机分为四组，为溶剂组，FGF-21组，脂糖素低剂量组，脂糖素高剂量组，每组动物10只，给药方式为皮下注射每日1次，给药，给药周期为2周。
动物分组及给药情况具体情况见表1所示：
表1：动物分组及给药情况
组别 动物数目(只) 剂量 给药周期(天)
溶剂组 10 同体积溶剂 14天
FGF-21组 10 0.4mg/kg/d 14天
脂糖素低剂量组 10 0.125mg/kg/d 14天
脂糖素高剂量组 10 2mg/kg/d 14天
给药结束后处死小鼠并摘取相关组织进行后续分析。
2、检测指标
2.1分离血清，检测血清中各种生化指标：血清谷丙转氨酶(ALT)，谷草转氨酶(AST)含量使用临床通用测定试剂盒测定(申索佑福)；
2.2小鼠肝脏组织形态学检测。通过H&E染色方法观察小鼠肝脏形态变化，其具体步骤为：将新鲜取下的小块肝脏固定在福尔马林溶液中过夜，经过梯度脱水后再浸蜡，包埋切片至5μm，采用苏木素-伊红染液将肝脏切片进行染色，最后在显微镜下观察各个小鼠肝脏组织的形态。
2.3小鼠肝脏组织胶原纤维检测。通过天狼星红(Sirius Red，SR)染色方法观察小鼠肝脏的胶原沉积，其具体步骤为：将石蜡块切出的小鼠肝脏切片采用Sirius Red染色试剂盒进行染色，最后在显微镜下观察各个小鼠肝脏组织的纤维化情况。
临床上设定了对非酒精性脂肪性肝炎的严重程度的评分标准(NASH Clinical Research Network scores)，主要包括三方面：脂肪变性(steatosis)，肝小叶炎症(lobular inflammation)和肝细胞气球样变性(ballooning)。本试验将从每组随机选五只小鼠，在对应小鼠的肝组织H&E染色切片上随机选五个视野从上述三方面进行评分，客观评价各组小鼠的肝炎病变程 度。
3、数据统计和分析应用SPSS16.0统计学软件分析，两组数据之间采用t检验、多组数据之间采用One-wayANOVA检验、多元线性回归分析，以P<0.05为差异有统计学意义。
4、实验结果：
4.1脂糖素对血清中谷丙转氨酶(ALT)水平的影响：见表2
表2脂糖素对血清中谷丙转氨酶(ALT)水平的影响
  溶剂组 FGF-21组 脂糖素低剂量组 脂糖素高剂量组
血清ALT(U/L) 199.8±30.8 100.2±20.6* 51.1±7.7***Δ 39.9±4.1***Δ
注：(1)表中所示数据均为均值±标准误；(2)*，与溶剂组相比P<0.05；***，与溶剂组相比P<0.001；Δ，与FGF-21组相比P<0.05。
表2结果显示：经2周治疗后发现，FGF-21组非酒精性脂肪性肝炎小鼠ALT水平降低至100.2±20.6U/L，且与溶剂组199.8±30.8U/L相比有显著性差异(P<0.05)，脂糖素低剂量组及高剂量组ALT水平分别降低至51.1±7.7U/L及39.9±4.1U/L，且与溶剂组有极显著差异(P<0.001)，与FGF-21组相比也有显著差异(P<0.05)，均说明脂糖素不但具有降低ALT水平的药效而且其药效优于FGF-21组。
4.2脂糖素对血清中血清中谷草转氨酶(AST)水平的影响：见表3
表3脂糖素对血清中血清中谷草转氨酶(AST)水平的影响
Figure PCTCN2018079482-appb-000001
注：(1)表中所示数据均为均值±标准误；(2)*，与溶剂组相比P<0.05；**，与溶剂组相比P<0.01； Δ，与FGF-21组相比P<0.05。
表3结果显示：经2周治疗后发现，FGF-21组非酒精性脂肪性肝炎小鼠AST水平降低至115.7±15.9U/L，且与溶剂组196.9±29.3U/L相比有显著性差异(P<0.05)，脂糖素低剂量组及高剂量组ALT水平分别降低至74.4±7.9U/L及73.7±7.8U/L，且与溶剂组有极显著差异(P<0.01)，与FGF-21组相比也有显著差异(P<0.05)，说明脂糖素不但具有降低ALT水平的药效而且其药效优于FGF-21组。
4.3对NASH临床相关评分的改善作用：见表4
表4对NASH临床相关评分的改善作用
  脂肪变性 肝小叶炎症 肝细胞气球样变性 总评分
溶剂组 2.2±0.2 2.6±0.1 1.3±0.1 6.0±0.21
FGF-21组 2.0±0.3 2.1±0.2 0.9±0.1 ** 4.7±0.5 *
脂糖素低剂量组 1.8±0.3 0.7±0.1 ***ΔΔΔ 0.7±0.1 ** 3.2±0.4 ***Δ
脂糖素高剂量组 0.9±0.3 **Δ 0.6±0.2 ***ΔΔΔ 0.4±0.1 ***ΔΔ 1.9±0.5 ***ΔΔ
注：(1)表中所示数据均为均值±标准误；(2)*，与溶剂组相比P<0.05；**，与溶剂组相比P<0.01；***，与溶剂组相比P<0.001； Δ，与FGF-21组相比P<0.05； ΔΔ，与FGF-21组相比P<0.01； ΔΔΔ，与FGF21组相比P<0.001。
表4结果显示：经2周治疗后，FGF-21组与溶剂组相比，尽管肝细胞气球样变性以及NASH总评分均有显著改善(P<0.05)，但对肝细胞炎症无显著改善(P>0.05)。脂糖素低剂量组及高剂量组与溶剂组相比在肝小叶炎症、肝细胞气球样变性以及NASH总评分上均有极显著改善。另外，脂糖素高剂量组在脂肪变性水平上也显著优于溶剂组。另外，脂糖素低剂量组或/和脂糖素高剂量组在脂肪变性、肝小叶炎症、肝细胞气球样变性以及NASH总评分上均显著优于FGF-21处理组。以上结果说明脂糖素不但具有改善肝小叶炎症、肝细胞气球样变性以及NASH总评分的药效而且其药效显著优于FGF-21组。
5、实验结果：
通过在注射脂糖素检测发现，脂糖素低剂量组或/和脂糖素高剂量组与溶剂组及FGF-21组相比可以显著降低血清中血清中谷丙转氨酶(ALT)及谷草转氨酶(AST)水平，改善脂肪变性程度，肝小叶炎症程度，降低肝细胞气球样变性程度，改善肝脏损伤程度的病理评分。以上结果说明脂糖素具有治疗非酒精性脂肪肝炎的作用。
6、结论：脂糖素具有治疗非酒精性脂肪肝炎的临床应用价值，且优于现有技术。

Claims (9)

  1. 一种长效化突变的人源成纤维生长因子在制备治疗非酒精性脂肪肝炎的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述长效化突变的人源成纤维生长因子为脂糖素或其药用盐。
  3. 根据权利要求2所述的应用，其特征在于，所述应用选自：脂糖素在降低血清中谷丙转氨酶及谷草转氨酶水平、改善脂肪变性程度、改善肝小叶炎症程度、降低肝细胞气球样变性程度和改善肝脏损伤程度中的应用。
  4. 根据权利要求1所述的应用，其特征在于，所述的非酒精性脂肪肝炎，选自肝炎引起的非酒精性脂肪肝炎、肥胖症引起的非酒精性脂肪肝炎、糖尿病引起的非酒精性脂肪肝炎、胰岛素抵抗引起的非酒精性脂肪肝炎、高甘油三酯血症引起的非酒精性脂肪肝炎、无β脂蛋白血症引起的非酒精性脂肪肝炎、糖原贮积病引起的非酒精性脂肪肝炎、韦克病引起的非酒精性脂肪肝炎、沃尔曼病引起的非酒精性脂肪肝炎或脂肪营养不良所引起的非酒精性脂肪肝炎。
  5. 根据权利要求2所述的应用，其特征在于，所述药物为含有脂糖素或其药用盐作为药物活性成分的药物组合物。
  6. 根据权利要求5所述的应用，其特征在于，所述药物组合物可以被制备成任何一种可药用的剂型。
  7. 根据权利要求6所述的应用，其特征在于，所述剂型选自：片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、丹剂、注射剂、栓剂、喷雾剂、滴剂、贴剂、滴丸剂。
  8. 根据权利要求7所述的应用，其特征在于，所述剂型选自粉针或液体针。
  9. 根据权利要求8所述的应用，其特征在于，所述液体针选自水针，有机溶剂针，混悬液针。
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CN108619490A (zh) 2018-10-09
US20190367574A1 (en) 2019-12-05
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