WO2022188444A1 - 一种新型抗代谢紊乱的fgf类似物及其应用 - Google Patents

一种新型抗代谢紊乱的fgf类似物及其应用 Download PDF

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WO2022188444A1
WO2022188444A1 PCT/CN2021/128690 CN2021128690W WO2022188444A1 WO 2022188444 A1 WO2022188444 A1 WO 2022188444A1 CN 2021128690 W CN2021128690 W CN 2021128690W WO 2022188444 A1 WO2022188444 A1 WO 2022188444A1
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fgf19
liver
injection
hepatitis
ngm282
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朱升龙
陈永泉
王振
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江南大学
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Priority to US17/819,766 priority patent/US12030921B2/en
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    • 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]
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    • 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
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
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    • A—HUMAN NECESSITIES
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a novel anti-metabolic disorder FGF analog and application thereof, and belongs to the technical field of medicine.
  • Fibroblast growth factor 19 is a newly discovered metabolic regulator that stimulates intestinal secretion and expression after being secreted into the intestine by bile acids. After being secreted from the intestine, FGF19 can enter the liver with the circulation and combine with FGFR4 in the liver. It has a hormone-like effect and plays an important metabolic regulatory role, such as regulating bile acid metabolism, regulating gallbladder filling, improving energy metabolism and reducing weight, improve blood sugar, etc. Since several previous studies have shown that FGF19 has a mitogenic effect, FGFR4 can promote the proliferation of FGF19 in the liver and has a cancer-promoting effect.
  • NGM282 is a non-tumorigenic engineered variant of human FGF19, a mutant belonging to the N-terminal modification of FGF19.
  • NGM282 has just completed a phase II clinical study in the United States, and the results show that 79% of patients achieved the primary treatment endpoint and 34% of patients achieved normal liver fat content at 12 weeks.
  • the mutant improved serum biomarkers of liver function, lipid metabolism, and fibrosis in patients, showing efficacy in the treatment of metabolic diseases.
  • FGF19 mutant NGM282 can significantly increase cholesterol levels after injection, and many studies have shown that elevated cholesterol levels are metabolic diseases It is one of the significant risk factors for metabolic diseases, which is a huge risk for the treatment of metabolic diseases.
  • FGF19 can also cause symptoms such as anorexia and decreased appetite, and there are certain hidden dangers in the future treatment process.
  • the present invention constructs four mutant proteins through prediction and experiment based on the original non-carcinogenic sequence, and prepares four biologically active FGF19 mutant proteins by optimizing the production and purification process.
  • the results show that the four mutants can play a role in the treatment of obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, as well as non-alcoholic steatohepatitis (NASH), atherosclerosis, liver damage, liver cirrhosis,
  • NASH non-alcoholic steatohepatitis
  • the efficacy of liver cancer, primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), and the treatment effect of 4 mutants was significantly better than that of NGM282 protein.
  • the present invention provides FGF19 protein analogs, and the amino acid sequence of the FGF19 protein analogs is shown in any of SEQ ID NO. 1-4.
  • the gene encoding the FGF19 protein analog is a gene encoding the FGF19 protein analog.
  • nucleotide sequences of the coding genes corresponding to the amino acids shown in SEQ ID NO. 1-4 are shown in SEQ ID NO. 5-8, respectively.
  • the present invention provides vectors and/or host cells carrying the genes.
  • the present invention provides a medicine or a pharmaceutical composition for treating diabetes or obesity, comprising the FGF19 protein analog.
  • the medicament or pharmaceutical composition further includes a pharmaceutically acceptable carrier or adjuvant.
  • the treatment of diabetes or obesity comprises inhibiting weight gain, lowering blood lipids and blood glucose, and increasing insulin sensitivity.
  • the present invention provides a medicine or a pharmaceutical composition for treating hepatitis or related diseases, comprising the FGF19 protein analog.
  • the medicament or pharmaceutical composition further includes a pharmaceutically acceptable carrier or adjuvant.
  • the treatment of hepatitis or related diseases includes reducing liver weight and liver triglyceride content, repairing liver damage, inhibiting the expression of inflammatory factors, improving non-alcoholic steatohepatitis, atherosclerosis, liver Injury, liver cirrhosis and liver cancer Primary biliary cholangitis and/or primary sclerosing cholangitis.
  • the present invention provides the application of the FGF19 protein analog in preparing a medicament for treating one or more diseases in diabetes, obesity, hepatitis or hepatitis-related diseases.
  • the medicament or pharmaceutical composition further includes a pharmaceutically acceptable carrier or adjuvant.
  • the dose of the FGF19 protein analog is 0.2-100 mg/kg.
  • the dose of the FGF19 protein analog is 0.2-3 mg/kg.
  • the route of administration of the drug includes intradermal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intravenous drip, intraarterial injection, intrabody injection and/or oral administration.
  • the present invention provides the application of the gene encoding the FGF19 protein analog in screening drugs for treating diabetes, obesity, hepatitis or hepatitis-related diseases.
  • nucleotide sequences of the genes are shown in SEQ ID NOs. 5-8, respectively.
  • the 4 novel FGF19 analogs of the present invention have longer effect, more stability and better treatment of obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, Efficacy in nonalcoholic steatohepatitis (NASH), atherosclerosis, liver injury, cirrhosis, liver cancer, primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).
  • NASH nonalcoholic steatohepatitis
  • PBC primary biliary cholangitis
  • PSC primary sclerosing cholangitis
  • Figure 1 is the SDS-PAGE electrophoresis analysis diagram of the purified protein expression in E. coli, which are FGF19-1, FGF19-2, FGF19-3, FGF19-4 and NGM282 proteins respectively;
  • Figure 2 is a comparison chart of the in vivo half-life of five proteins
  • Figure 3 is a graph showing the effects of five proteins on body weight and diet of db/db mice
  • Figure 4 is a graph showing the effects of five proteins on blood lipids in db/db mice
  • Figure 5 is a graph showing the effects of five proteins on diabetes-related indicators in db/db mice.
  • Figure 6 is a graph showing the effects of five proteins on related indicators such as steatohepatitis and liver fibrosis in NASH model mice;
  • Figure 7 is a graph showing the effects of five proteins on tumor proliferation in mice with liver cancer xenografts.
  • nude mice and db/db mice were purchased from Shanghai Slack Company. The animals were reared in the Animal Center of Jiangnan University Wuxi Medical College under alternating lighting every 12 hours at a temperature of 20 ⁇ 2°C.
  • Hepatoma cell line HepG2 was provided by Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; DMEM and 0.05% Trypsin were purchased from Boster Company; fetal bovine serum was purchased from Sijiqing Company.
  • the hepatocellular carcinoma cell line HepG2 was adherently grown in DMEM medium containing 10% fetal bovine serum, cultured at 37°C in a humidified incubator with 5% CO 2 , and passaged every other day.
  • mice were injected with 2 mg/kg of FGF21 protein, and the corresponding human dose was 0.2 mg/kg; rabbits were injected with 30 mg/kg of FGF21 protein, and the corresponding human dose was 3 mg/kg.
  • FGF19-1 nucleotide sequence shown in SEQ ID NO. 5
  • FGF19-2 nucleotide sequence shown in SEQ ID NO. ID NO. 6
  • FGF19-3 nucleotide sequence shown in SEQ ID NO. 7
  • FGF19-4 nucleotide sequence shown in SEQ ID NO.8.
  • the four genes were sent to Shanghai Jierui Biological Company for synthesis, and NdeI and BamHI restriction sites were designed at both ends of each gene.
  • the four synthetic vectors containing their respective target gene fragments and pET30a(+) were digested with NdeI and BamHI, respectively.
  • the four target fragments were ligated with the prokaryotic expression vector pET30a(+) using T4 DNA ligase.
  • the ligation reaction system was 10 ⁇ L, mixed well, ligated at 4°C overnight, and then transformed into E. coli DH5 ⁇ .
  • the positive clones were picked and identified by enzyme digestion, and four recombinant plasmids pET30a-FGF19-1, pET30a-FGF19-2, pET30a-FGF19-3 and pET30a-FGF19-4 were respectively constructed.
  • the correctly sequenced recombinant plasmids pET30a-FGF19-1, pET30a-FGF19-2, pET30a-FGF19-3 and pET30a-FGF19-4 were transformed into the expression strain Rosseta (DE3) competent cells.
  • the transformed single colonies were inoculated into 20 mL of LB medium containing Kan (50 ⁇ g/mL), and after culturing at 37°C for 8 hours, the bacterial solution was inoculated into another 20 mL of Kan (50 ⁇ g/mL) at a volume ratio of 1:100.
  • lysozyme (1 mg/mL) was added to the cells, placed on ice for 30 min, and the cells were disrupted by ultrasonic cells (working 1 s, interval 1 s, 4 min/time, 3 cycles in total). After the cells were completely broken, the cell broken liquid was treated with a Quix Stand pretreatment system (750kD ultrafiltration hollow fiber column), the inclusion bodies were enriched, and the liquid at the membrane permeable end was discarded. When the total volume is about 60mL, add 100mL washbuffer (20mmol/L Tris, 2mol/L Urea, 150mmol/L NaCl, pH8.0) to wash inclusion bodies.
  • washbuffer 20mmol/L Tris, 2mol/L Urea, 150mmol/L NaCl, pH8.0
  • the denatured mFGF21 was concentrated with a 5KD hollow fiber column, renatured to a volume of 80mL, and the container containing the renaturation solution (20mmol/L Tris, 50mmol/L NaCl, pH8.0) was filled with a rubber tube and a hollow fiber. Column reservoir connection. After the liquid reservoir is sealed, after the liquid flows out from the permeable end, due to the negative pressure generated in the reservoir, the renaturing liquid is added dropwise to the denaturing liquid at a certain speed, and the renaturing liquid is slowly and uniformly renatured.
  • IEX buffer A (20 mmol/L Tris, 10 mmol/L NaCl, pH 8.0) on a Capto Q column (packed in XK16/20 empty column, column height 10 cm) by AKTApurifier100 system.
  • Twenty-five rabbits weighing about 2 kg were selected and randomly divided into 5 groups. Each group was subcutaneously injected with 5 proteins NGM282, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, at a dose of 30 mg/kg, at 0h, 1h, 3h, 5h, 7h, and 24h after administration. About 800 ⁇ L of blood was collected from the marginal vein. Centrifuge at 12,000 r/m for 10 min, and store the supernatant at -20°C for later use.
  • the in vivo half-life of five proteins was determined by ELISA indirect method: NGM282, FGF19-1, FGF19-2, FGF19-3 and FGF19-4 proteins were diluted with different concentrations (20 ⁇ g/mL, 2 ⁇ g/mL, 200ng/mL, 20ng/mL). mL and 2ng/mL) to establish a standard curve of protein concentration, respectively, coat the diluted standard protein and serum on an enzyme-labeled plate, and use the ELISA indirect method to determine the content of the target protein in each serum. Statistical analysis and calculation of 6 kinds of In vivo half-life of proteins.
  • Example 3 Effects of recombinant protein on body weight, diet, blood lipids and diabetes-related indicators
  • FGF19-1, FGF19-2, FGF19-3 and FGF19-4 were prepared according to the method of Example 1.
  • mice Fifty SPF 8-week-old male db/db mice were pre-bred for 1 week and weighed. The next day, they were fasted for 6 hours. Blood was collected from the tail vein to measure the fasting blood glucose of the mice, and the mice with abnormal body weight were excluded. 42 model mice whose blood glucose and body weight values were relatively close to the mean were screened and randomly divided into saline injection group (Saline), NGM282 group, FGF19-1 group, FGF19-2 group, FGF19-3 group and FGF19-4 group Groups, 6 in each group.
  • Saline saline injection group
  • NGM282 group FGF19-1 group
  • FGF19-2 group FGF19-3 group
  • FGF19-4 group Groups 6 in each group.
  • the experimental group was given the corresponding test substance once every day at about 8:30 am, intraperitoneally, at a dose of 2 mg/kg, and the normal saline group was injected with the same volume of normal saline for 8 consecutive weeks.
  • food and water were free to drink.
  • Mice diet and body weight status were monitored during the period.
  • the mice in each experimental group were sacrificed (fasted the night before), and blood was collected from the eyeball to measure the blood glucose, triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) of the experimental mice. ) and high-density lipoprotein (HDL-C) levels.
  • Statistical analysis was performed on the obtained experimental data.
  • the experimental detection data are shown in Figure 3 to Figure 5.
  • the results in Figure 3 show that, compared with the normal saline control group, the NGM282 protein and the mutated four new proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 All of them can significantly reduce the body weight of mice, but NGM282 protein injection can significantly reduce the amount of mice's diet and suppress their appetite. After the drug, it not only inhibited body weight more strongly and significantly, but also did not affect the mice's eating, indicating that this mutation successfully improved the side effects of the original FGF19 diet.
  • Example 4 Influence of recombinant protein on non-alcoholic steatohepatitis (NASH) related indicators
  • FGF19-1, FGF19-2, FGF19-3 and FGF19-4 were prepared according to the method of Example 1.
  • mice 60 SPF grade 8-week-old male C57BL/6 mice were pre-bred for 1 week and then fed with methionine choline deficient MCD diet. Forty-two animals were randomly divided into normal saline injection group (Saline), NGM282 group, FGF19-1 group, FGF19-2 group, FGF19-3 group and FGF19-4 group, with 6 animals in each group.
  • the experimental group was given the corresponding test substance once every day at about 8:30 am, intraperitoneally, at a dose of 2 mg/kg, and the normal saline group was injected with the same volume of normal saline for 8 consecutive weeks. During the experiment, food and water were free to drink.
  • mice in each experimental group were sacrificed (fasted the night before), and the levels of triglyceride (TG), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) in the liver of the experimental mice were measured and histological sections were stained. and inflammatory markers. The obtained experimental data were subjected to statistical analysis.
  • TG triglyceride
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • the experimental data are shown in Figure 6.
  • the results in Figure 6A show that, compared with the normal saline control group (Saline), the NGM282 protein and the four novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 after the mutation All of them can significantly reduce liver weight and liver triglyceride (TG) content in mice, but the therapeutic effect of four novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 is significantly better than that of NGM282.
  • Figure 6B The transaminase results further showed that the protective function of four novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 against liver injury was significantly better than that of NGM282.
  • FIG. 6C is the result of Sirius red staining, which is used to observe the deposition of collagen fibers in the liver and reflect liver fibrosis.
  • Example 5 Effect of recombinant protein on liver cancer
  • FGF19-1, FGF19-2, FGF19-3 and FGF19-4 were prepared according to the method of Example 1.
  • HepG2 cells were inoculated subcutaneously into 6-week-old male nude mice at 1 ⁇ 10 6 cells/a, and when the tumors grew to 200 mm 3 , they were randomly divided into saline injection group (Saline), NGM282 group, and FGF19-1 group. , FGF19-2 group, FGF19-3 group and FGF19-4 group, 6 in each group.
  • the experimental group was given the corresponding test substance once, intraperitoneally, at a dose of 2 mg/kg, and the normal saline group was injected with the same volume of normal saline for 21 consecutive days. Tumor volume was monitored daily, mice were sacrificed three weeks later, and tumors were weighed. The results showed that all five proteins could inhibit the volume of the transplanted tumor and the final tumor weight, but the inhibitory effect of NGM282 was significantly lower than that of the mutant recombinant protein (as shown in Figure 7).

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Abstract

本发明公开了一种新型抗代谢紊乱的FGF类似物及其应用，属于医药技术领域。本发明在FGF19突变体NGM282基础上进行改造，得到的新型FGF19类似物的与NGM282相比具有更长效、更稳定的效果，能更好的改善肝脏损伤及纠正代谢紊乱、胖症、超重、代谢综合征、糖尿病、血脂异常等病症，且治疗过程中均未出现原始FGF19突变体NGM282治疗过程引起胆固醇升高及饮食下降的副作用。

Description

一种新型抗代谢紊乱的FGF类似物及其应用 技术领域
本发明涉及一种新型抗代谢紊乱的FGF类似物及其应用，属于医药技术领域。
背景技术
成纤维细胞生长因子19(FGF19)是新近发现的一种代谢调节因子，由胆汁酸分泌进入肠道后刺激肠道分泌和表达。FGF19经肠道分泌后可随循环进入肝脏并与肝脏中的FGFR4结合起作用，它具有激素样作用，发挥着重要的代谢调节作用，如调节胆汁酸代谢、调节胆囊的充盈、提高能量代谢降低体质量、改善血糖等。由于前期多项研究表明FGF19具有促有丝分裂作用，FGFR4能促进FGF19在肝脏中的增殖并具有促癌作用。2014年有研究发现FGF19N末端结构域是与FGFR相互作用至关重要的区域，因此，选择性地敲除识别FGFR4受体的区域，能消除FGF19促有丝分裂的活性，因此，多篇文章集中在FGF19的N端进行突变。
NGM282是人FGF19的非致瘤性工程化变体，是属于FGF19N端改造的一种突变体。NGM282刚刚在美国完成II期临床研究，结果显示，79％的患者达到主要治疗终点，34％的患者在12周时达到正常的肝脏脂肪含量。该突变体改善了患者肝功能、脂质代谢和纤维化的血清生物标志物，显示出治疗代谢类疾病的疗效。但是在上述临床研究中除了观察到一些常见的消化道症状、恶心和注射部位红斑外，还发现FGF19突变体NGM282注射后可以显著提高胆固醇含量，而多项研究表明胆固醇含量升高是代谢类疾病的显著高危因素之一，这对代谢类疾病治疗来说是个巨大的风险。此外，FGF19还会引起厌食，食欲降低等症状，对未来治疗过程存在一定的隐患。
发明内容
鉴此，本发明通过预测及试验，在原始非致癌序列基础上进行改造，构建出4种突变蛋白，通过优化生产及纯化工艺制备了4种具有生物学活性的FGF19突变蛋白。结果显示4种突变体均可发挥治疗肥胖症、超重、代谢综合征、糖尿病、高血糖症、血脂异常，以及非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肝损伤、肝硬化、肝癌、原发性胆汁性胆管炎(PBC)及原发性硬化性胆管炎(PSC)的功效，且4种突变体治疗效果显著优于NGM282蛋白。
本发明提供了FGF19蛋白类似物，所述FGF19蛋白类似物氨基酸序列如SEQ ID NO.1～4任一所示。
在一种实施方式中，编码所述FGF19蛋白类似物的基因。
在一种实施方式中，SEQ ID NO.1～4所示氨基酸对应的编码基因核苷酸序列分别如SEQ ID NO.5～8所示。
本发明提供了携带所述基因的载体和/或宿主细胞。
本发明提供了治疗糖尿病或肥胖的药物或药物组合物，含有所述FGF19蛋白类似物。
在一种实施方式中，所述药物或药物组合物还包括药学上可接受的载剂或辅料。
在一种实施方式中，所述治疗糖尿病或肥胖包括抑制体重增加、降低血脂和血糖、提高胰岛素敏感性。
本发明提供了治疗肝炎或相关疾病的药物或药物组合物，含有所述FGF19蛋白类似物。
在一种实施方式中，所述药物或药物组合物还包括药学上可接受的载剂或辅料。
在一种实施方式中，所述治疗肝炎或相关疾病包括降低肝脏重量和肝脏甘油三酯的含量、修复肝脏损伤、抑制炎症因子的表达，改善非酒精性脂肪性肝炎、动脉粥样硬化、肝损伤、肝硬化及肝癌原发性胆汁性胆管炎和/或原发性硬化性胆管炎。
本发明提供了所述FGF19蛋白类似物在制备治疗糖尿病、肥胖、肝炎或肝炎相关疾病中的一种或多种疾病的药物中的应用。
在一种实施方式中，所述药物或药物组合物还包括药学上可接受的载剂或辅料。
在一种实施方式中，所述FGF19蛋白类似物的剂量为0.2～100mg/kg。
在一种实施方式中，所述FGF19蛋白类似物的剂量为0.2～3mg/kg。
在一种实施方式中，所述药物的给药途径包括皮内注射、皮下注射、静脉注射、肌肉注射、腹腔注射、静脉滴注、动脉注射、体腔内注射和/或口服。
本发明提供了所述编码所述FGF19蛋白类似物的基因在筛选具有治疗糖尿病、肥胖、肝炎或肝炎相关疾病的药物中的应用。
在一种实施方式中，所述基因的核苷酸序列分别如SEQ ID NO.5～8所示。
本发明的有益效果：
(1)本发明的4种新型FGF19类似物与原始FGF19突变体NGM282相比具有更长效、更稳定、更好的治疗胖症，超重，代谢综合征，糖尿病，高血糖症，血脂异常，非酒精性脂肪性肝炎(NASH)、动脉粥样硬化、肝损伤、肝硬化、肝癌、原发性胆汁性胆管炎(PBC)及原发性硬化性胆管炎(PSC)的功效。
(2)本发明的4种新型FGF19类似物治疗过程中均未出现原始FGF19突变体NGM282治疗过程引起胆固醇升高及饮食下降的副作用，对机体正常生命活动影响较小。
附图说明
图1是纯化后的蛋白在大肠杆菌中表达量的SDS-PAGE电泳分析图，分别为FGF19-1、FGF19-2、FGF19-3、FGF19-4和NGM282蛋白；
图2是5种蛋白的体内半衰期比较图；
图3是5种蛋白对db/db小鼠体重和饮食的影响图；
图4是5种蛋白对db/db小鼠血脂的影响图；
图5是5种蛋白对db/db小鼠糖尿病相关指标的影响图；
图6是5种蛋白对NASH模型小鼠脂肪性肝炎及肝纤维化等相关指标的影响图；
图7是5种蛋白对肝癌移植瘤小鼠肿瘤增殖的影响图。
具体实施方式
实验动物及饲养：裸鼠及db/db小鼠购于上海斯莱克公司。饲养于江南大无锡医学院动物中心，每12小时交替照明，温度20±2℃。
细胞培养：肝癌细胞系HepG2由中国科学院生物化学与细胞生物学研究所提供；DMEM、0.05％Trypsin，购于博士德公司；胎牛血清购于四季青公司。
其他药品为国产分析纯。
肝癌细胞系HepG2贴壁生长于含10％胎牛血清的DMEM培养液中，于37℃，5％CO 2湿化培养箱中培养，隔天传代一次。
下述实施例中对小鼠注射2mg/kg的FGF21蛋白，对应的人体剂量为0.2mg/kg；对家兔注射30mg/kg的FGF21蛋白，对应的人体剂量为3mg/kg。
实施例1：重组蛋白的构建、表达及纯化
(1)FGF19-1、FGF19-2、FGF19-3和FGF19-4表达载体的构建
根据计算机模拟替换及大肠杆菌密码子偏好性，设计出4种新型FGF19基因，分别为FGF19-1(核苷酸序列如SEQ ID NO.5所示)、FGF19-2(核苷酸序列如SEQ ID NO.6所示)、FGF19-3(核苷酸序列如SEQ ID NO.7所示)、FGF19-4(核苷酸序列如SEQ ID NO.8所示)。将这4种基因送至上海捷瑞生物公司合成，同时在各基因两端分别设计NdeI与BamHI酶切位点。将4种合成的含有各自目的基因片段的载体和pET30a(+)分别用NdeI与BamHI双酶切，酶切完毕后，胶回收各自需要的目标片段。使用T4DNA连接酶将4种目的片段分别与原核表达载体pET30a(+)连接，连接反应体系为10μL，混匀，4℃连接过夜，然后各自转化至大肠杆菌DH5α中。挑取阳性克隆，经过酶切鉴定后，即分别构建得到4种重组质粒pET30a-FGF19-1、pET30a-FGF19-2、pET30a-FGF19-3和pET30a-FGF19-4。
(2)蛋白的表达及纯化
将测序正确的重组质粒pET30a-FGF19-1、pET30a-FGF19-2、pET30a-FGF19-3和pET30a-FGF19-4转化至表达菌株Rosseta(DE3)感受态细胞中。转化后的单菌落分别接种至20mL含Kan(50μg/mL)的LB培养基中，37℃培养8h后，将菌液以体积比为1:100接种于 另一20mL含Kan(50μg/mL)的新鲜LB培养基中，在37℃培养，当A600在0.35左右时，加入IPTG至终浓度为0.25mmol/L进行诱导，诱导温度为30℃，诱导5h后取菌体，用Lysis buffer(20mmol/L Tris，150mmol/L NaCl，pH8.0)重悬菌体，破碎菌体后离心，分别取上清和沉淀进行12wt％SDS-PAGE电泳分析。结果显示FGF19-1、FGF19-2、FGF19-3和FGF19-4蛋白在大肠杆菌中表达量显著增加，目标蛋白大部分以包涵体形式存在。
收集大量诱导后的菌体，向菌体中加入溶菌酶(1mg/mL)，冰上放置30min，超声波细胞破碎菌体细胞(工作1s，间隔1s，4min/次，共3次循环)。菌体破碎彻底后，利用Quix Stand预处理系统(750kD超滤中空纤维柱)处理细胞破碎液，富集包涵体，弃去膜透过端液体。当总体积约为60mL时，加入100mLwashbuffer(20mmol/L Tris，2mol/L Urea，150mmol/L NaCl，pH8.0)洗涤包涵体。当溶液体积为50mL，再向其中加入洗涤液100mL，重复上述实验4次。洗涤完毕后，当溶液体积为50mL，关闭透过端，向洗涤后的包涵体中加入150mL的变性液(20mmol/L Tris，10mol/L Urea，150mmol/L NaCl，pH8.0)，循环变性2小时。打开透过端，膜透过端收集液即为mFGF21变性液。用5KD中空纤维柱对变性后的mFGF21进行浓缩，至体积80mL后进行复性，将装有复性液(20mmol/L Tris，50mmol/L NaCl，pH8.0)的容器用胶皮管与中空纤维柱的储液器连接。储液器密封后，透过端流出液体后，由于储存器中产生负压，使复性液以一定的速度滴加至变性液中，缓慢匀速复性。当加入复性液体积为变性液6倍时，即复性完毕，8000rpm/min，4℃离心20min，收集上清。复性上清液经AKTApurifier100系统，与5倍柱体积IEX buffer A(20mmol/L Tris、10mmol/L NaCl，pH8.0)平衡好的Capto Q柱(装于XK16/20空柱，柱高10cm，流速300cm/h)完全结合后，用3-4倍柱体积IEX buffer A冲洗；当紫外曲线达到稳定的基线时，利用IEX buffer A和IEX buffer B(20mmol/L Tris，1mol/L NaCl，pH8.0)混合液洗脱，15wt％和100wt％IEX buffer B液冲洗杂蛋白，18.5wt％-19wt％IEX buffer B液洗脱目标蛋白，收集各洗脱峰，并进行15wt％SDS PAGE电泳分析。结果显示纯化后蛋白纯度在95％以上，如图1所示，泳道1为蛋白标准分子量Marker；泳道2-6分别为纯化后的FGF19-1、FGF19-2、FGF19-3和FGF19-4。
实施例2：重组蛋白体内半衰期的检测
5种蛋白NGM282、FGF19-1、FGF19-2、FGF19-3和FGF19-4的体内半衰期检测。
选取体重约2kg的家兔25只，随机分为5组。每组分别皮下注射5种蛋白NGM282、FGF19-1、FGF19-2、FGF19-3和FGF19-4，剂量30mg/kg，在给药后的0h、1h、3h、5h、7h、24h，于耳缘静脉采血800μL左右。12000r/m离心10min，取上清保存于-20℃备用。ELISA间接法测定5种蛋白的体内半衰期：用稀释好不同浓度的NGM282、FGF19-1、FGF19-2、 FGF19-3和FGF19-4蛋白(20μg/mL、2μg/mL、200ng/mL、20ng/mL和2ng/mL)分别建立蛋白浓度含量的标准曲线，将稀释后的标准蛋白和血清包被酶标板，应用ELISA间接法测定各血清中目标蛋白的含量，统计学分析并计算出6种蛋白的体内半衰期。
体内半衰期t 1/2＝0.301*(t2-t1)/log(OD1/OD2)，其中OD1和OD2分别表示t1和t2时取出血清所对应酶标板上的平均光吸收值。
结果如图2所示，经公式计算出NGM282蛋白和突变改造后的蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4的体内半衰期分别约为36min、79min、66min、67min、69min，说明了4种新型FGF19-1、FGF19-2、FGF19-3和FGF19-4体内半衰期显著增加。
实施例3：重组蛋白对体重、饮食、血脂及糖尿病相关指标的影响
按照实施例1的方法制备FGF19-1、FGF19-2、FGF19-3和FGF19-4这4种蛋白。
取SPF级8周龄雄性db/db小鼠50只，预饲养1周后称重，次日禁食不禁水6h，从尾部的静脉中取血测定小鼠的空腹血糖，剔除体重异常的小鼠，筛选血糖及体重值相对接近均值的成模小鼠42只，随机分为生理盐水注射组(Saline)、NGM282组、FGF19-1组、FGF19-2组、FGF19-3组和FGF19-4组，每组6只。于每天早上8点半左右给予实验组相应的受试物一次，腹腔注射，剂量2mg/kg，生理盐水组注射相同体积的生理盐水，连续给药8周。实验过程中自由饮食、饮水。期间监测小鼠饮食和体重状况。给药8周后，各实验组小鼠处死(前夜禁食)，从眼球部取血测定实验小鼠血糖、甘油三脂(TG)、总胆固醇(TC)、低密度脂蛋白(LDL-C)和高密度脂蛋白(HDL-C)水平。将所得实验数据进行统计学分析。
实验检测数据如图3至图5所示，图3结果表明，相对于生理盐水对照组，NGM282蛋白和突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4均可显著降低小鼠体重，但NGM282蛋白注射后可显著降低小鼠饮食量，抑制其食欲，而相对于NGM282，4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4给药后不仅能更为强劲和显著的抑制体重外，且并不影响小鼠进食，表明此种突变改造成功的改善了原始FGF19饮食下降的副作用。
给药8周后，各实验组小鼠血清血脂水平结果如图4所示，相对于生理盐水组，NGM282组中的小鼠血清中TG、TC及LDL-c的含量显著升高，而各种HDL-c的含量无明显差异，这与之前的诸多临床报道一致，多项研究表明胆固醇及血脂含量升高是代谢类疾病的显著高危因素之一，这对代谢类疾病治疗来说是个巨大的风险。而经过改造后，4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4注射后不仅没有原始FGF19蛋白升高TG、TC及LDL-c的副作用，还可显著降低血清中TG的含量，这些结果进一步表明这些突变改造成功的改善了原 始FGF19血脂升高的副作用，极大的增加了FGF19临床应用的安全性和有效性。
给药期间，分别在0周，2周，4周及8周测量空腹血糖，各实验组小鼠空腹血糖水平结果如图5A所示，治疗2周后，NGM282并无明显改善血糖效果，而FGF19-1，FGF19-3已显著降低小鼠空腹血糖；治疗4周后，NGM282才开始发挥降低血糖的作用，但治疗效果显著低于FGF19-3和FGF19-4，8周后几组之间无明显差异，结果表明突变后的重组FGF19蛋白降糖作用起效快且优于原始NGM282。给药8周后，进行了葡萄糖耐量试验和胰岛素耐量试验，结果如图5B和5C所示，相对于NGM282蛋白，突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4可更为显著的改善糖尿病小鼠葡萄糖敏感性和胰岛素敏感性。
实施例4：重组蛋白对非酒精性脂肪性肝炎(NASH)相关指标的影响
按照实施例1的方法制备FGF19-1、FGF19-2、FGF19-3和FGF19-4这4种蛋白。
取SPF级8周龄雄性C57BL/6小鼠60只，预饲养1周后喂食蛋氨酸胆碱缺失MCD饲料，喂食8周后，剔除体重异常，筛选血糖及体重值相对接近均值的成模小鼠42只，随机分为生理盐水注射组(Saline)、NGM282组、FGF19-1组、FGF19-2组、FGF19-3组和FGF19-4组，每组6只。于每天早上8点半左右给予实验组相应的受试物一次，腹腔注射，剂量2mg/kg，生理盐水组注射相同体积的生理盐水，连续给药8周。实验过程中自由饮食、饮水。给药8周后，各实验组小鼠处死(前夜禁食)，测定实验小鼠肝脏甘油三脂(TG)、碱性磷酸酶(ALP)、谷丙转氨酶(ALT)水平并进行组织切片染色和炎症指标检测。所得实验数据进行统计学分析。
实验检测数据如图6所示，图6A结果表明，相对于生理盐水对照组(Saline)，NGM282蛋白和突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4均可显著降低小鼠肝脏重量和肝脏甘油三酯(TG)含量，但4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4治疗效果要明显优于NGM282。图6B转氨酶结果进一步显示4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4对肝脏损伤的保护功能显著优于NGM282。此外，HE染色结果直接显示出4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4注射后可显著降低肝脏脂肪空泡，显微镜下几乎观察不到空泡，而NGM282治疗后还存在部分脂肪空泡(图6C)。图6D是天狼星红染色结果，用以观察肝脏胶原纤维沉积情况，反应肝脏纤维化，结果显示突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4均可逆转肝脏纤维化，而NGM282治疗会还存在部分纤维化状态，表明改造后的重组蛋白对肝脏纤维化的逆转效果显著优于NGM282。NASH的主要病理状态是肝脏存在炎症，通过qPCR检测标志性炎症因子表达情况，结果显示突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3 和FGF19-4均可显著抑制炎症因子表达，且抑制效果显著优于NGM282(图6E)。通过上述多项指标检测发现突变改造后的4种新型蛋白FGF19-1、FGF19-2、FGF19-3和FGF19-4针对NASH和肝脏损伤的治疗效果显著优于原始的NGM282。
实施例5：重组蛋白对肝癌的影响
按照实施例1的方法制备FGF19-1、FGF19-2、FGF19-3和FGF19-4这4种蛋白。
将人肝癌细胞HepG2细胞按照1×10 6个细胞/只接种6周龄的雄性裸鼠的皮下，待肿瘤长至200mm 3随机分为生理盐水注射组(Saline)、NGM282组、FGF19-1组、FGF19-2组、FGF19-3组和FGF19-4组，每组6只。于每天早上8点半左右给予实验组相应的受试物一次，腹腔注射，剂量2mg/kg，生理盐水组注射相同体积的生理盐水，连续给药21天。每天监测肿瘤体积，三周后处死小鼠，称量肿瘤重量。结果显示：5种蛋白均能抑制移植瘤体积和最终肿瘤重量，但NGM282抑制效果显著低于突变后的重组蛋白(如图7所示)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (11)

  1. FGF19蛋白类似物，其特征在于，所述FGF19蛋白类似物氨基酸序列如SEQ ID NO.1～4任一所示。
  2. 编码权利要求1所述FGF19蛋白类似物的基因。
  3. 携带权利要求2所述基因的载体和/或微生物细胞。
  4. 治疗糖尿病或肥胖的药物或药物组合物，其特征在于，含有权利要求1所述FGF19蛋白类似物。
  5. 根据权利要求4所述的药物或药物组合物，其特征在于，所述治疗糖尿病或肥胖包括但不限于抑制体重增加、降低血脂和血糖、提高胰岛素敏感性。
  6. 根据权利要求4或5所述的药物或药物组合物，其特征在于，所述药物中还含有人体可接受的修饰、药用载体和/或辅料。
  7. 治疗肝炎或相关疾病的药物或药物组合物，其他在于，含有权利要求1所述FGF19蛋白类似物；所述治疗肝炎或相关疾病包括降低肝脏重量和肝脏甘油三酯的含量、修复肝脏损伤、抑制炎症因子的表达，改善非酒精性脂肪性肝炎、动脉粥样硬化、肝损伤、肝硬化及肝癌、原发性胆汁性胆管炎和/或原发性硬化性胆管炎。
  8. 权利要求1所述FGF19蛋白类似物、或权利要求3所述载体或微生物细胞在制备治疗糖尿病、肥胖、肝炎或肝炎相关疾病中的一种或多种疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述FGF19蛋白类似物的剂量为0.2～100mg/kg。
  10. 根据权利要求8或9所述的应用，其特征在于，所述药物的给药途径包括皮内注射、皮下注射、静脉注射、肌肉注射、腹腔注射、静脉滴注、动脉注射、体腔内注射和/或口服。
  11. 权利要求2所述基因在筛选具有治疗糖尿病、肥胖、肝炎或肝炎相关疾病的药物中的应用。
PCT/CN2021/128690 2021-03-12 2021-11-04 一种新型抗代谢紊乱的fgf类似物及其应用 WO2022188444A1 (zh)

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