WO2016070798A1 - 一种抑制脂肪细胞分化和胰岛素耐受的药物 - Google Patents

一种抑制脂肪细胞分化和胰岛素耐受的药物 Download PDF

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WO2016070798A1
WO2016070798A1 PCT/CN2015/093726 CN2015093726W WO2016070798A1 WO 2016070798 A1 WO2016070798 A1 WO 2016070798A1 CN 2015093726 W CN2015093726 W CN 2015093726W WO 2016070798 A1 WO2016070798 A1 WO 2016070798A1
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group
mice
mes
fat diet
functional variant
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PCT/CN2015/093726
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French (fr)
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罗永章
王慧
李辉
鲁薪安
付彦
占顺利
周代福
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清华大学
北京普罗吉生物科技发展有限公司
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Priority to AU2015342324A priority Critical patent/AU2015342324B2/en
Priority to CN201580059881.7A priority patent/CN107148277B/zh
Priority to CA3003760A priority patent/CA3003760A1/en
Priority to JP2017524040A priority patent/JP2018501195A/ja
Priority to US15/524,094 priority patent/US20180015148A1/en
Priority to EP15857606.6A priority patent/EP3246043A4/en
Priority to CN202210191378.9A priority patent/CN114558111A/zh
Publication of WO2016070798A1 publication Critical patent/WO2016070798A1/zh
Priority to US17/869,348 priority patent/US20220409703A1/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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • 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
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • the present invention relates to the novel function of endostatin. Specifically, the present inventors have found that endostatin can significantly inhibit adipocyte differentiation and alleviate insulin resistance.
  • the invention also provides the use of endostatin in the treatment of diseases such as dietary obesity, nonalcoholic fatty liver disease, insulin resistance, glucose intolerance and the like.
  • the accumulation of fat causes the expansion of adipose tissue, which means an increase in the number and volume of fat cells, a process that is accompanied by angiogenesis (Cristancho AG et al. Nat Rev Mol Cell Biol 2011; 12:722-734; Daquinag AC Et al. Trends Pharmacol Sci 2011; 32: 300-307).
  • Endostatin Endostatin
  • YH-16 is an ES variant obtained by adding 9 additional amino acids (MGGSHHHHH) to the N-terminus of ES. It has been awarded the National Class I New Drug Certificate in 2005 for the treatment of non-small cell lung cancer (Fu Y et al. IUBMB Life 2009; 61: 613-626; Wang J et al. Zhongguo fei ai za zhi 2005; 8: 283-290; Han B et al. J Thorac Oncol 2011; 6(6): 1104-1109).
  • Polyethylene glycol (PEG) modified ES and YH-16 were named mES and mYH-16, respectively.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • the product obtained from the YH-16 molecule, the site of coupling is the activated mPEG-ALD aldehyde group and the N-terminal ⁇ -amino group of ES or YH-16.
  • angiogenesis inhibitors can inhibit obesity by inhibiting angiogenesis in adipose tissue (Rupnick MA et al. Proc Natl Acad Sci U S A 2002; 99: 10730-10735; Kim MY et al. Int J Obes (Lond). 2010; 34: 820-830).
  • Folkman Laboratories reported that several different vascular inhibitors can inhibit hereditary obesity in mice, including ES (Rupnick MA et al. Proc Natl Acad Sci U S A 2002; 99: 10730-10735).
  • adipocytes The increase in the number of adipocytes is directly dependent on the differentiation of adipocytes (Cristancho AG et al. Nat Rev Mol Cell Biol 2011; 12: 722-734), which is a very complex regulatory process.
  • PPAR ⁇ peroxisome proliferator-activated receptor gamma
  • the present invention relates to a novel activity of the known vasopressin protein Endostatin (ES), that is, an activity of inhibiting adipocyte differentiation, and provides ES based on the activity in the treatment of dietary obesity, fatty liver, insulin New uses in metabolic disorders such as resistance and glucose intolerance.
  • ES vasopressin protein Endostatin
  • ES can directly act on adipose precursor cells, and can inhibit adipocyte differentiation by inhibiting the expression of the adipocyte differentiation center regulators PPAR ⁇ 1 and/or PPAR ⁇ 2.
  • ES can inhibit the weight gain of mice caused by a high-fat diet, which is achieved by inhibiting the accumulation of fat in mice.
  • ES can inhibit the increase in liver weight and fat deposition in mice caused by a high-fat diet, thereby preventing and treating fatty liver.
  • ES can increase insulin response in mice by enhancing phosphorylation of Akt, and improve insulin resistance and glucose intolerance in mice.
  • ES mutants such as YH-16, 003, 007 and Z101 have activity comparable to ES in the above experiments, and polyethylene modified by polyethylene glycol (PEG) and its variant YH-16 003, 007, and Z101 (mES, mYH-16, m003, m007, and mZ101) have similar activities to unmodified proteins.
  • PEG polyethylene glycol
  • FIG. 2B Liver tissue sections of ES and its variant YH-16 treated mice.
  • ES and its variant YH-16 polyethylene glycol modified ES and its variant YH-16 (mES and mYH-16) are all capable of directly inhibiting adipocyte differentiation.
  • FIG. 5B polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007) significantly inhibited the increase in mouse adipose tissue weight caused by a high fat diet.
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001.
  • polyethylene glycol modified ES and its variants 003 and 007 had no effect on the weight of lung, heart and kidney in high fat diet mice.
  • FIG. 6A polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007) significantly inhibited liver weight gain in mice induced by a high fat diet.
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01.
  • FIG. 6B liver tissue sections of mice treated with polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007).
  • polyethylene glycol modified ES and its variants 003 and 007 are all capable of directly inhibiting the differentiation of adipocytes.
  • polyethylene glycol modified ES variant Z101 (mZ101) is capable of directly inhibiting the differentiation of adipocytes.
  • polyethylene glycol modified ES variants 009 and S03 can significantly inhibit high fat drink Diet-induced weight gain in mice. ** represents P ⁇ 0.01, *** represents P ⁇ 0.001
  • polyethylene glycol modified ES variants 36 and 249 had no effect on the weight of lung, heart and kidney in high fat diet mice.
  • Figure 20 The amino acid sequence of ES.
  • Figure 21 Amino acid sequence of ES variant YH-16.
  • Figure 22 Amino acid sequences of ES variants 381, 57, 114 and 124.
  • Figure 23 Amino acid sequences of ES variants 125, 160, 163 and 119.
  • the present invention provides the use of endostatin or a functional variant thereof for the preparation of a medicament for the treatment of dietary obesity, nonalcoholic fatty liver, insulin resistance or glucose intolerance.
  • the present invention provides a preparation for inhibiting differentiation of adipocytes by endostatin or a functional variant thereof Use in the body.
  • the functional variants can be YH-16, 003, 007, Z101, ES006, ES008, ES011, S02, S09, Z006, Z008, ZN1, 009, S03, 36, 249, 381, 57, 114, 124, 125, 160, 163, 119, mES, mYH-16, m003, m007, mZ101, mES006, mES008, mES011, mS02, mS09, mZ006, mZ008, mZN1, m009, mS03, m36, m249, M381, m57, m114, m124, m125, m160, m163 or m119.
  • the functional variants may be YH-16, 003, 007, Z101, 009, S03, 36, 249, mES, mYH-16, m003, m007, mZ101, m009, mS03 , m36 or m249.
  • the term "functional variant” as used herein includes one or more (eg, 1-5, 1-10, or 1 to 15 in the amino acid sequence, specifically, for example, may be 1, 2, 3, 4) , 5, 6, 7, 8, 9, 10, 12 or more) mutants of amino acid substitutions, deletions or additions of endostatin, and chemical modification of endostatin or its mutants, For example, a derivative obtained by modifying polyethylene glycol, and the mutant and derivative have substantially the same activity of inhibiting adipocyte differentiation as endostatin.
  • polyethylene glycol (PEG) modified ES and YH-16 are named mES and mYH-16, respectively, which are modified by monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) with a molecular weight of 20 kDa.
  • mPEG-ALD monomethoxypolyethylene glycol propionaldehyde
  • the product obtained from the ES or YH-16 molecule, the site of coupling is the activated mPEG-ALD aldehyde group and the N-terminal ⁇ -amino group of ES or YH-16 (other ES mutants and polyethylene II of the mutant) Alcohol-modified derivatives adopt similar modification schemes and nomenclature).
  • YH-16, 003, 007, Z101, 009, S03, 36 and 249 are particularly preferred mutants of endostatin; mES, mYH-16, m003, m007, mZ101, m009, mS03, m36 and m249 are preferred derivatives of ES, YH-16, 003, 007, Z101, 009, S03, 36 and 249, respectively.
  • Various variants of endostatin, such as ES006, ES008, ES011, S02, S09, Z006, are also provided in PCT International Application No. PCT/CN2012/081210, which is incorporated herein in its entirety by reference. Z008, ZN1, etc.
  • the term "functional variant” or "variant” as used herein encompasses mutants and derivatives of endostatin.
  • the invention also provides a method of treating dietary obesity, nonalcoholic fatty liver disease, insulin resistance or glucose intolerance comprising administering to a subject a therapeutically effective amount of endostatin or a functional variant thereof.
  • terapéuticaally effective amount refers to an amount of active compound that is sufficient to cause a biological or medical response desired by a clinician in a subject.
  • the "therapeutically effective amount” of endostatin or a functional variant thereof can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, condition, and the like. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg of body weight.
  • the medicament provided by the present invention can be formulated into a clinically acceptable dosage form such as a powder or an injection, and administered by a conventional means such as injection.
  • the invention also provides a method of inhibiting adipocyte differentiation comprising administering to a subject a therapeutically effective amount of endostatin or a functional variant thereof.
  • the invention also provides a method for treating dietary obesity, nonalcoholic fatty liver, insulin resistance or glucose A poorly tolerated drug comprising an endostatin as a active ingredient or a functional variant thereof.
  • Dietary obesity refers to obesity caused by excess calories in the form of fat stored in the body due to the calories in the diet exceeding the body's energy consumption.
  • Nonalcoholic fatty liver disease is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. Its pathological changes are similar to those of alcoholic liver disease (ALD), but the patient has no history of excessive drinking. Syndrome.
  • Insulin resistance also known as insulin resistance, refers to the body's insensitivity to insulin response, so that the biological effect of insulin-promoting glucose uptake and utilization is lower than normal, that is, the body needs higher Insulin concentration can respond to insulin. Hyperinsulin and high glucose levels in plasma caused by insulin resistance often lead to metabolic syndrome, gout and type 2 diabetes.
  • Glucose intolerance refers to a decrease in the ability to regulate blood glucose concentration caused by a decrease in glucose metabolism in the body. It is manifested that the blood glucose level cannot be adjusted to normal in a timely manner after a large intake of glucose, and may progress to diabetes if not treated in time.
  • Mouse liver weight inhibition rate ⁇ 1 - liver weight of the drug-administered group / liver weight of the high-fat diet group ⁇ 100%.
  • liver fat deposition in mice ⁇ 1 - liver cytoplasmic vacuole rate in the drug-administered group / hepatic cytoplasmic vacuole rate in the high-fat diet group ⁇ 100%.
  • the ES and its variant proteins used in the examples of the present invention are all supplied by Beijing Proji Company.
  • Example 1 ES and YH-16 significantly inhibited weight gain in mice induced by high fat diet
  • mice Twenty-four healthy C57BL/6 mice (7 weeks old, male, purchased from Beijing Weitong Lihua Company) were divided into 4 groups, 8 in each group, which were treated as follows:
  • the first group the normal diet group
  • the second group high fat diet group
  • the third group high fat diet + ES group (administration group);
  • the fourth group high fat diet + YH-16 group (administration group).
  • mice In the normal diet group, 10% of the calorie-intake calorie-derived feed (D12450J, Research Diets, USA) was used to feed the mice; the high-fat diet to consume 60% of the calorie-derived calorie-derived feed (D12492, Research) Diets, USA) were fed mice.
  • the administration method was as follows: the third group and the fourth group were intraperitoneally injected with ES or YH-16 (Protgen) once a day at a dose of 12 mg/kg/day, the second group was injected with an equal volume of normal saline, and the first group was not injected, and the cycle was 60 days.
  • the time of the first injection was set to day 0, the last administration was completed on the 59th day, the body weight of the mice was weighed once every three days, and the last weight measurement was performed on the 60th day (ie, one day after the last administration).
  • Draw a weight curve (Figure 1A).
  • the results showed that both ES and YH-16 significantly inhibited the weight gain caused by the high-fat diet, with inhibition rates of 37.5% and 30.6%, respectively. (Table 1).
  • mice After completion of the glucose tolerance test on day 61, the mice were sacrificed, whole body adipose tissue was isolated, and weighed (Fig. 1B, Table 1). The results showed that the weight of adipose tissue in ES and YH-16 mice was significantly lower than that in the non-administered high-fat diet group. The inhibition rates of ES and YH-16 on fat accumulation in mice induced by high-fat diet were 47.7% and 42.2%, respectively. Table 1).
  • mice Mouse lungs, heart and kidneys were isolated and weighed (Fig. 1C, Table 1). The results showed that there was no significant difference in lung, heart and kidney weight between the four groups of mice, indicating that ES and YH-16 had no effect on the lung, heart and kidney of the mice.
  • Example 2 ES and YH-16 significantly inhibited liver weight gain and fat deposition in mice induced by high fat diet
  • mice in Example 1 were subjected to the glucose tolerance test on the 61st day, and the liver tissues were taken out and weighed (Fig. 2A, Table 1).
  • ES and YH-16 inhibited the increase in liver weight of mice induced by high-fat diet, and the inhibition rates were 23.8% and 20.5%, respectively.
  • the liver tissue was fixed and embedded in paraffin, and a section of 8 ⁇ m thick was prepared for tissue HE staining.
  • the main steps include: sectioning dewaxing and rehydration, followed by staining with hematoxylin and eosin, followed by conventional dehydration, sealing, and observation using a conventional optical microscope (Olympus IX71 microscope) and photographing (Fig. 2B).
  • HE staining showed that hepatic cytoplasmic fat vacuoles appeared in the liver sections of the high-fat diet group, indicating that the high-fat diet can cause fat deposition in the liver of mice, while the ES and YH-16-administered mice in the treatment group
  • the fat deposition was significantly less than that of the unadministered high-fat diet group, and the inhibition rates were 78.9% and 75.2%, respectively, and the inhibition rate (Fig. 2C). This indicates that ES and YH-16 have significant inhibitory effects on nonalcoholic fatty liver disease.
  • Example 3 ES and YH-16 significantly improve insulin resistance and glucose intolerance in mice
  • mice in Example 1 were subjected to an insulin resistance test 6 hours after the completion of administration on the 59th day.
  • the specific steps include: cutting the tail of the mouse to take blood, detecting the basic blood sugar level (Roche handheld blood glucose meter), and setting the monitoring point time to 0 minutes.
  • mice in Example 1 were subjected to starvation overnight treatment on the 60th day, and the glucose tolerance test was performed on the 61st day.
  • the specific steps include: cutting the tail of the mouse to take blood, detecting the basic blood sugar level (Roche handheld blood glucose meter), and setting the monitoring point time to 0 minutes.
  • the mice were intragastrically administered with glucose solution (20 mg/ml), and each mouse was perfused with 1 mg of glucose per gram of body weight. Blood was taken at 20 minutes, 40 minutes, 60 minutes, and 80 minutes after gavage, and blood glucose was measured. Curve ( Figure 3B). After the glucose was administered, the blood glucose level of the high-fat diet increased rapidly and the recovery rate was slower than that of the normal diet group. This indicated that the high-fat diet caused glucose tolerance in mice, while ES and Glucose intolerance was significantly improved in the YH-16-administered group.
  • mice After completing the glucose tolerance test on day 61, the mice were sacrificed, systemic adipose tissue was isolated, and Akt phosphorylation levels in adipose tissue were detected by Western blot (Fig. 3C).
  • the results showed that the Akt phosphorylation level was lower in the high-fat diet group compared with the normal diet group, while the Akt phosphorylation level was higher in the ES-administered group than in the high-fat diet group.
  • the Akt pathway is an important glycemic regulatory pathway downstream of insulin. Insulin resistance and decreased levels of Akt phosphorylation are often accompanied. This is consistent with the results of ES being able to effectively improve insulin resistance and glucose intolerance.
  • Example 4 ES and YH-16 significantly inhibited the differentiation of adipose precursor cells into adipocytes
  • 3T3-L1 fat precursor cells in good condition were selected, resuspended in DMEM medium containing 10% calf serum, seeded into a six-well plate, and placed in a cell culture incubator with 5% carbon dioxide at 37 °C. to cultivate.
  • the differentiation is induced: the first step, MDI induction medium induction (this time is defined as the first day of cell culture differentiation); the second and second days, after changing the insulin induction medium, continue to culture two
  • the third step was to continue the culture with DMEM medium containing 10% FBS until the 8th day, 3T3-L1 differentiated into adipocytes.
  • the experiment is divided into 5 groups:
  • the first group the control group
  • the second group ES treatment group
  • the third group YH-16 treatment group
  • the fourth group mES processing group
  • Group 5 Myh-16 treatment group.
  • the drug-treated group was additionally added with 50 ⁇ g/ml of ES, YH-16, mES or mYH-16 during the induction of differentiation (ie, from day 1 to day 8), and the control group was added with an equal volume of protein buffer.
  • the liquid exchange was re-added to the above drugs and the control treatment.
  • the MDI induction solution was added with 1 ⁇ M dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 10 ⁇ g/mL bovine insulin in DMEM containing 10% FBS; the insulin induction medium was 10%. 10 ⁇ g/mL bovine insulin was added to the DMEM medium of FBS.
  • the medium in the six-well plate was discarded, and after fixation, it was stained with oil red for 10 minutes. Decolorize and rinse three times with PBS to remove excess dye. Oil red can identify oils in fat cells, and dye them red.
  • the digital camera took a picture of the six-well plate and observed it with an inverted microscope (Olympus IX71 microscope) and photographed it (Fig. 4A). The results showed that ES, YH-16, mES and mYH-16 directly inhibited the differentiation of adipose precursor cells into adipocytes (Fig. 4B).
  • the cell differentiation center controls the expression levels of PPAR ⁇ 1 and PPAR ⁇ 2 (Fig. 4C). It was found that ES, YH-16, mES and mYH-16 inhibited the expression levels of the transcription factors PPAR ⁇ 1 and PPAR ⁇ 2 in the adipocyte differentiation center during adipocyte differentiation.
  • RNA expression levels of PPAR ⁇ 1 and PPAR ⁇ 2 were examined: before the induction of 3T3-L1 (day 0) and on the sixth day of induction, total cellular RNA was extracted according to the standard procedure of the TRIZOL reagent (purchased from Invitrogen). Reverse transcription was performed using Fermentas reverse transcription kit (RevertAid TM First Strand cDNA Synthesis Kits ), reaction was carried out according to standard instructions.
  • Fluorescence quantitative Real-Time PCR was used to detect PPAR ⁇ 1/2, a regulator of the adipocyte differentiation center. Fluorometric Real-Time PCR using the Stratagene kit (Brilliant II) Green QRT-PCR Master Mix), the fluorescent quantitative PCR instrument is MX3000P (purchased from Stratagene), the fluorescent dye is SYBR Green, the reaction system is 20 ⁇ L, and the number of reaction cycles is 40.
  • reaction primers are as follows:
  • PPAR ⁇ 1 forward primer (5'-3'): ACAAGATTTGAAAGAAGCGGTGA
  • PPAR ⁇ 1 reverse primer (5'-3'): GCTTGATGTCAAAGGAATGCGAAGGA
  • PPAR2 forward primer (5'-3'): CGCTGATGCACTGCCTATGAG
  • PPAR2 reverse primer (5'-3'): TGGGTCAGCTCTTGTGAATGGAA
  • the ⁇ Ct value was obtained from the fluorescence map given by the instrument, and the relative ⁇ ( ⁇ Ct) value was calculated, and the relative changes in the mRNA levels of PPAR ⁇ 1 and PPAR ⁇ 2 were calculated (Fig. 4D). It was found that during the differentiation of adipocytes, ES inhibited the expression of transcription factors PPAR ⁇ 1 and PPAR ⁇ 2 mRNA in the adipocyte differentiation center.
  • Example 5 polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007) significantly inhibited weight gain in mice induced by a high-fat diet
  • the first group the normal diet group
  • the second group high fat diet group
  • the third group high fat diet + mES group (administration group);
  • the fourth group high fat diet + m003 group (administration group);
  • the fifth group high fat diet + m007 group (administration group).
  • Each diet group feed was the same as in Example 1.
  • the administration method was as follows: the third group, the fourth group and the fifth group were injected with mES, m003 or m007 (Protgen) once a week at a dose of 50 mg/kg/week, and the second group was injected with an equal volume of physiological saline, first. The group was not injected and the cycle was 8 weeks. The time of the first injection was set to week 0, the last dose was completed in the 7th week, the body weight of the mice was weighed once a week, and the body weight of the mice was weighed the last time in the 8th week, and the body weight curve was drawn (Fig. 5A). . The results showed that mES, m003 and m007 were able to significantly inhibit the weight gain caused by the high-fat diet, with inhibition rates of 33.7%, 22.9% and 42.9%, respectively (Table 2).
  • mice After weighing the mice for the last time in the 8th week, the mice were sacrificed, and the adipose tissue was isolated and weighed (Fig. 5B, Table 2). The results showed that the weight of adipose tissue in the mES, m003 and m007 groups was significantly lower than that in the non-administered high-fat diet group.
  • the inhibition rates of mES, m003 and m007 on the fat accumulation in mice induced by high-fat diet were 41.4%, 31.9% and 40.5% (Table 2).
  • mice Mouse lungs, heart and kidneys were isolated and weighed (Fig. 5C, Table 2). The results showed that there was no significant difference in lung, heart and kidney weight between the five groups of mice, indicating that mES, m003 and m007 had no effect on the lung, heart and kidney of the mice.
  • Example 6 polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007) significantly inhibited liver weight gain and fat deposition in mice induced by a high-fat diet
  • Liver tissues were fixed according to the experimental method of Example 2 and embedded in paraffin sections for tissue HE staining. Liver tissue morphology was observed and photographed using a conventional optical microscope (Olympus IX71 microscope) (Fig. 6B). HE staining showed that the fat deposition in the liver of the mice treated with the mES, m003, and m007 treatment groups was significantly less than that of the unadministered high-fat diet group, and the inhibition rates were 70.6%, 56.1%, and 73.1%, respectively (Fig. 6C). This indicates that mES, m003 and m007 have significant inhibitory effects on nonalcoholic fatty liver.
  • Example 7 polyethylene glycol modified ES and its variants 003 and 007 (mES, m003 and m007) significantly inhibited the differentiation of adipose precursor cells into adipocytes
  • 3T3-L1 adipose precursor cells were cultured and induced to differentiate as in Example 4. The experiment is divided into 4 groups:
  • the first group the control group
  • the second group mES processing group
  • the third group m003 treatment group
  • the fourth group m007 processing group.
  • the drug-treated group was additionally added with 50 ⁇ g/ml of mES, m003 or m007 during the induction of differentiation (ie, from day 1 to day 8), and the control group was added with an equal volume of protein buffer, and each time the solution was re-added. Drug and control treatment.
  • Example 7 After the end of the induction, the cells were subjected to oil red staining according to the experimental method in Example 4.
  • the digital camera took a picture of the six-well plate and observed it with an inverted microscope (Olympus IX71 microscope) and photographed it (Fig. 7A).
  • the results showed that mES, m003 and m007 could directly inhibit the differentiation of adipose precursor cells into adipocytes, and mES and m007 inhibited better than m003 (Fig. 7B). This is consistent with the results of the animal experiments in Example 6, which also explains why mES and m007 have a better inhibitory effect on weight gain in high fat diet animals than m003.
  • Example 8 polyethylene glycol modified ES variant Z101 (mZ101) significantly inhibited weight gain in mice induced by high fat diet
  • mice per group mice per group
  • diet feed
  • mode of administration mice per group
  • dosing cycle mice per group
  • mouse body weight weighing mice per group
  • the first group the normal diet group
  • the second group high fat diet group
  • the third group high fat diet + mZ101 group (administration group).
  • the dose administered was 12 mg/kg/week.
  • mice After weighed the mice for the last time in the 8th week, the mice were sacrificed, and the adipose tissue and liver were isolated and weighed (Fig. 8B and C, Table 3). The results showed that the weight of adipose tissue in the mZ101 group was significantly lower than that in the non-administered high-fat diet group, and the inhibition rate of fat accumulation was 77.2% (Table 3). mZ101 also inhibited the increase in liver weight in mice caused by a high-fat diet with an inhibition rate of 21.5% (Table 3).
  • Example 9 polyethylene glycol modified ES variant Z101 (mZ101) significantly inhibited the differentiation of adipose precursor cells into adipocytes
  • 3T3-L1 adipose precursor cells were cultured and induced to differentiate as in Example 4. The experiment is divided into 2 groups:
  • the first group the control group
  • the second group mZ101 treatment group.
  • the drug-treated group was additionally added with 50 ⁇ g/ml of mZ101 (Protgen) during the induction of differentiation (ie, from day 1 to day 8), and the control group was added with an equal volume of protein buffer, and the above drugs were re-added each time. Control treatment.
  • Example 4 After the end of the induction, the cells were subjected to oil red staining according to the experimental method in Example 4.
  • the digital camera took a picture of the six-well plate and observed it with an inverted microscope (Olympus IX71 microscope) and photographed it (Fig. 9A).
  • the results showed that mZ101 directly inhibited the differentiation of adipose precursor cells into adipocytes (Fig. 9B).
  • Example 10 polyethylene glycol modified ES variants 009 and S03 (m009 and mS03) significantly inhibited the weight gain of mice induced by high fat diet, and the inhibitory effect of mS03 was better than m009
  • mice per group mice per group
  • diet feed
  • mode of administration mice per group
  • dosing cycle mice per group
  • mouse body weight weighing mice per group
  • the first group the normal diet group
  • the second group high fat diet group
  • the third group high fat diet + m009 group (administration group);
  • the fourth group high fat diet + mS03 group (administration group).
  • the dose administered was 12 mg/kg/week.
  • mice After weighed the mice for the last time in the 8th week, the mice were sacrificed, and the adipose tissue and liver were isolated and weighed (Fig. 10B and C, Table 4).
  • the results showed that the adipose tissue weight of the mice in the m009 and mS03 groups was significantly lower than that in the non-administered high-fat diet group, and the inhibition rates of m009 and mS03 on the fat accumulation in mice induced by the high-fat diet were 45.7% and 59.5%, respectively (Table 4).
  • M009 and mS03 were also able to inhibit the increase in liver weight in mice induced by a high-fat diet with inhibition rates of 16.7% and 25.7%, respectively (Table 4).
  • mice Mouse lungs, heart and kidneys were isolated and weighed (Fig. 10D, Table 4). The results showed that there was no significant difference in lung, heart and kidney weight between the 4 groups of mice, indicating that m009 and mS03 had no effect on the lung, heart and kidney of the mice.
  • Example 11 polyethylene glycol modified ES variants 009 and S03 (m009 and mS03) significantly inhibited the differentiation of adipose precursor cells into adipocytes
  • 3T3-L1 adipose precursor cells were cultured and induced to differentiate as in Example 4. The experiment is divided into 3 groups:
  • the first group the control group
  • the second group m009 treatment group
  • the third group mS03 treatment group.
  • the drug-treated group was additionally added with 50 ⁇ g/ml of m009 or mS03 during the induction of differentiation (ie, from day 1 to day 8), and the control group was added with an equal volume of protein buffer, and the above drugs were re-added each time. Control treatment.
  • Example 12 polyethylene glycol modified ES variants 36 and 249 (m36 and m249) significantly inhibited weight gain in mice induced by a high-fat diet
  • mice per group mice per group
  • diet feed
  • mode of administration mice per group
  • dosing cycle mice per group
  • mouse body weight weighing mice per group
  • the first group the normal diet group
  • the second group high fat diet group
  • the third group high fat diet + m36 group, the dose was 6mg / kg / week (administration group);
  • the fourth group high-fat diet + m36 group, the dose was 12mg / kg / week (administration group);
  • the fifth group high fat diet + m249 group, the dose was 6mg / kg / week (administration group);
  • the sixth group high fat diet + m249 group, the dose was 12 mg / kg / week (administration group).
  • mice After weighing the mice for the last time in the 8th week, the mice were sacrificed, and the adipose tissue and liver were isolated and weighed (Fig. 12B and C, Table 5).
  • the results showed that the weight of adipose tissue in the low-dose m36 (6 mg/kg/week) group and the high-dose m249 (12 mg/kg/week) group was significantly lower than that in the non-administered high-fat diet group, and the low-dose m36 (6 mg/kg/week).
  • the inhibition rates of high fat diet m249 (12 mg/kg/week) on fat accumulation in mice induced by high fat diet were 30% and 38.4%, respectively (Table 5).

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Abstract

本发明提供了血管内皮抑制素或其功能性变体在制备用于治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的药物中的用途。在本发明的实施方案中，所述功能性变体可以是YH-16、mES、mYH-16、m003、m007、mZ101等。

Description

一种抑制脂肪细胞分化和胰岛素耐受的药物 技术领域
本发明涉及血管内皮抑制素的新功能。具体而言，本发明发现血管内皮抑制素可以显著抑制脂肪细胞分化并缓解胰岛素耐受。本发明还提供了血管内皮抑制素在治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗、葡萄糖耐受不良等疾病中的用途。
背景技术
脂肪的积累会引起脂肪组织的扩张，这意味着脂肪细胞的数量增多和体积增大，这个过程伴随着血管新生(Cristancho AG et al.Nat Rev Mol Cell Biol 2011；12:722-734；Daquinag AC et al.Trends Pharmacol Sci 2011；32:300-307)。
1997年，Folkman实验室发现了内源性血管抑制剂Endostatin(血管内皮抑制素，简称ES)，可直接靶向血管内皮细胞，具有抑制新生血管生成和治疗肿瘤的活性(O'Reilly MS et al.Cell 1997；88:277-285；Boehm T.et al.Nature 1997；390:404-407)。
YH-16是在ES的N-末端添加了9个额外氨基酸(MGGSHHHHH)而得到的ES变体，已经于2005年获得国家一类新药证书，用于治疗非小细胞肺癌(Fu Y et al.IUBMB Life 2009；61:613-626；Wang J et al.Zhongguo fei ai za zhi 2005；8:283-290；Han B et al.J Thorac Oncol 2011；6(6):1104-1109)。聚乙二醇(PEG)修饰的ES和YH-16分别命名为mES和mYH-16，它们分别是一个分子量为20kDa的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰一个ES或YH-16分子得到的产物，偶联的位点是活化的mPEG-ALD醛基与ES或YH-16的N-末端α-氨基。
已有报道表明，血管生成抑制因子可以通过抑制脂肪组织中的血管新生来抑制肥胖(Rupnick MA et al.Proc Natl Acad Sci U S A 2002；99:10730-10735；Kim MY et al.Int J Obes(Lond).2010；34:820-830)。2002年，Folkman实验室报道了几个不同的血管抑制剂可以抑制小鼠遗传性肥胖，其中包括ES(Rupnick MA et al.Proc Natl Acad Sci U S A 2002；99:10730-10735)。
脂肪细胞数量的增多直接依赖于脂肪细胞的分化(Cristancho AG et al.Nat Rev Mol Cell Biol 2011；12:722-734)，这是一个非常复杂的调控过程。研究表明，过氧化物酶体增殖剂激活受体γ(Peroxisome proliferator-activated receptor gamma，PPARγ)是脂肪细胞分化的中心控制因子(Tang QQ et al.Annu Rev Biochem2012；81:715-736)，它能够通过调控下游脂肪细胞表型控制基因(包括CD36,ap2,Glut4,LPL and LXR等)的表达来调控脂肪细胞的分化(Cristancho AG et al.Nat Rev Mol Cell Biol 2011；12:722-734；Lee J.et al.J Cell Biochem 2012；113:2488-2499)。
大量流行病学研究表明，肥胖能够引起代谢紊乱，是代谢综合征的一个重要临床表现，也是引起非酒精性脂肪肝、胰岛素抵抗、葡萄糖耐受不良、和II型糖尿病的重要危 险因素(Malik VS et al.Nat Rev Endocrinol 2013；9:13-27)。
发明内容
本发明涉及已知血管抑制剂蛋白血管内皮抑制素(Endostatin，ES)的新活性，即抑制脂肪细胞分化的活性，并提供了以该活性为基础的ES在治疗饮食性肥胖、脂肪肝、胰岛素抵抗和葡萄糖耐受不良等代谢紊乱疾病中的新用途。
发明人发现，ES可直接作用于脂肪前体细胞，并且可以通过抑制脂肪细胞分化中心调控因子PPARγ1和/或PPARγ2的表达抑制脂肪细胞分化。
发明人发现，ES可以抑制由于高脂饮食引起的小鼠体重增加，这种抑制作用是通过抑制小鼠体内脂肪的积累而实现的。
发明人发现，ES可以抑制高脂饮食引起的小鼠肝脏重量增加和脂肪沉积，从而预防和治疗脂肪肝。
发明人还发现，ES可以通过增强Akt的磷酸化提高小鼠对胰岛素的响应，改善小鼠的胰岛素抵抗和葡萄糖耐受不良。
发明人还发现，ES的突变体例如YH-16、003、007和Z101等在上述实验中具有与ES相当的活性，而经过聚乙二醇(PEG)修饰的ES及其变体YH-16、003、007和Z101(mES、mYH-16、m003、m007和mZ101)等与未修饰的蛋白具有相类似的活性。
附图说明
图1A，ES及其变体YH-16可显著抑制高脂饮食引起的小鼠体重增长。***代表P<0.001。
图1B，ES及其变体YH-16可显著抑制高脂饮食引起的小鼠脂肪组织重量增加。*代表P<0.05，***代表P<0.001。
图1C，ES及其变体YH-16对高脂饮食小鼠的肺脏、心脏和肾脏的重量没有影响。
图2A，ES及其变体YH-16可显著抑制高脂饮食引起的小鼠肝脏重量增加。*代表P<0.05，**代表P<0.01。
图2B，ES及其变体YH-16处理组小鼠肝脏组织切片。
图2C，ES及其变体YH-16可显著抑制高脂饮食引起的小鼠肝脏脂肪沉积。***代表P<0.001。
图3A，ES及其变体YH-16可显著改善小鼠的胰岛素抵抗。***代表P<0.001。
图3B，ES及其变体YH-16可显著提高小鼠对葡萄糖的耐受能力。***代表P<0.001。
图3C，ES可显著提高胰岛素信号通路的下游因子Akt的磷酸化水平。
图4A，ES及其变体YH-16、聚乙二醇修饰的ES及其变体YH-16(mES和mYH-16)都能够直接抑制脂肪细胞的分化。
图4B，ES及其变体YH-16、聚乙二醇修饰的ES及其变体YH-16(mES和mYH-16)抑制脂肪细胞分化的定量统计结果。***代表P<0.001。
图4C，ES及其变体YH-16，聚乙二醇修饰的ES及其变体YH-16(mES和mYH-16)可显著抑制脂肪细胞分化中心调控因子PPARγ1/2的蛋白表达水平。
图4D，ES抑制脂肪细胞分化中心调控因子PPARγ1/2的mRNA表达水平。*代表P<0.05；***代表P<0.001。
图5A，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)可显著抑制高脂饮食引起的小鼠体重增长。***代表P<0.001。
图5B，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)可显著抑制高脂饮食引起的小鼠脂肪组织重量增加。*代表P<0.05，**代表P<0.01，***代表P<0.001。
图5C，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)对高脂饮食小鼠的肺脏、心脏和肾脏的重量没有影响。
图6A，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)可显著抑制高脂饮食引起的小鼠肝脏重量增加。*代表P<0.05，**代表P<0.01。
图6B，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)处理组小鼠肝脏组织切片。
图6C，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)可显著抑制高脂饮食引起的小鼠肝脏脂肪沉积。***代表P<0.001。
图7A，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)都能够直接抑制脂肪细胞的分化。
图7B，聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)抑制脂肪细胞分化的定量结果。***代表P<0.001。
图8A，聚乙二醇修饰的ES变体Z101(mZ101)可显著抑制高脂饮食引起的小鼠体重增长。**代表P<0.01，***代表P<0.001。
图8B，聚乙二醇修饰的ES变体Z101(mZ101)可显著抑制高脂饮食引起的小鼠脂肪组织重量增加。***代表P<0.001。
图8C，聚乙二醇修饰的ES变体Z101(mZ101)可显著抑制高脂饮食引起的小鼠肝脏重量增加。*代表P<0.05。
图8D，聚乙二醇修饰的ES变体Z101(mZ101)对高脂饮食小鼠的肺脏、心脏和肾脏的重量没有影响。
图9A，聚乙二醇修饰的ES变体Z101(mZ101)能够直接抑制脂肪细胞的分化。
图9B，聚乙二醇修饰的ES变体Z101(mZ101)抑制脂肪细胞分化的定量结果。***代表P<0.001。
图10A，聚乙二醇修饰的ES变体009和S03(m009和mS03)可显著抑制高脂饮 食引起的小鼠体重增长。**代表P<0.01，***代表P<0.001
图10B，聚乙二醇修饰的ES变体009和S03(m009和mS03)可显著抑制高脂饮食引起的小鼠脂肪组织重量增加。***代表P<0.001
图10C，聚乙二醇修饰的ES变体009和S03(m009和mS03)可显著抑制高脂饮食引起的小鼠肝脏重量增加。*代表P<0.05
图10D，聚乙二醇修饰的ES变体009和S03(m009和mS03)对高脂饮食小鼠的肺脏、心脏和肾脏的重量没有影响。
图11A，聚乙二醇修饰的ES变体009和S03(m009和mS03)都能够直接抑制脂肪细胞的分化。
图11B，聚乙二醇修饰的ES变体009和S03(m009和mS03)抑制脂肪细胞分化的定量结果。***代表P<0.001
图12A，聚乙二醇修饰的ES变体36和249(m36和m249)可显著抑制高脂饮食引起的小鼠体重增长。**代表P<0.01，***代表P<0.001
图12B，聚乙二醇修饰的ES变体36和249(m36和m249)可显著抑制高脂饮食引起的小鼠脂肪组织重量增加。*代表P<0.05，**代表P<0.01，***代表P<0.05
图12C，聚乙二醇修饰的ES变体36和249(m36和m249)可显著抑制高脂饮食引起的小鼠肝脏重量增加。*代表P<0.05，**代表P<0.01
图12D，聚乙二醇修饰的ES变体36和249(m36和m249)对高脂饮食小鼠的肺脏、心脏和肾脏的重量没有影响。
图13，ES变体003的氨基酸序列。
图14，ES变体007的氨基酸序列。
图15，ES变体Z101的氨基酸序列。
图16，ES变体009的氨基酸序列。
图17，ES变体S03的氨基酸序列。
图18，ES变体36的氨基酸序列。
图19，ES变体249的氨基酸序列。
图20，ES的氨基酸序列。
图21，ES变体YH-16的氨基酸序列。
图22，ES变体381、57、114和124的氨基酸序列。
图23，ES变体125、160、163和119的氨基酸序列。
具体实施方式
本发明提供了血管内皮抑制素或其功能性变体在制备用于治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的药物中的用途。
本发明提供了血管内皮抑制素或其功能性变体在制备用于抑制脂肪细胞分化的药 物中的用途。
在一些实施方案中，所述功能性变体可以是YH-16、003、007、Z101、ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163或m119。在本发明的优选实施方案中，所述功能性变体可以是YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36或m249。
本文所用的术语“功能性变体”包括在氨基酸序列中含有一个或多个(例如1-5个、1-10个或者1到15个，具体地，例如可以是1、2、3、4、5、6、7、8、9、10、12个甚至更多个)氨基酸的取代、缺失或添加的血管内皮抑制素的突变体，以及对血管内皮抑制素或其突变体进行化学修饰，例如聚乙二醇修饰，而获得的衍生物，并且所述突变体和衍生物具有与血管内皮抑制素基本相同的抑制脂肪细胞分化的活性。例如，聚乙二醇(PEG)修饰的ES和YH-16分别命名为mES和mYH-16，它们分别是一个分子量为20kDa的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰一个ES或YH-16分子得到的产物，偶联的位点是活化的mPEG-ALD醛基与ES或YH-16的N-末端α-氨基(其他的ES突变体以及该突变体的聚乙二醇修饰后的衍生物采用相似的修饰方案及命名方式)。例如，在本发明的具体实施方案中，YH-16、003、007、Z101、009、S03、36和249是血管内皮抑制素的特别优选的突变体；mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249分别是ES、YH-16、003、007、Z101、009、S03、36和249的优选的衍生物。PCT国际申请PCT/CN2012/081210(在此以引用的方式将其全部内容并入本文)中还提供了血管内皮抑制素的多种突变体，例如ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1等。本文所用的术语“功能性变体”或“变体”涵盖血管内皮抑制素的突变体及衍生物。
本发明还提供了一种治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的方法，包括给予受试者治疗有效量的血管内皮抑制素或其功能性变体。
本文所用的术语“治疗有效量”指的是足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。血管内皮抑制素或其功能性变体的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
本发明所提供的药物可以制成粉末、注射剂等临床可接受的剂型，并通过注射等常规方式给药。
本发明还提供了一种抑制脂肪细胞分化的方法，包括给予受试者治疗有效量的血管内皮抑制素或其功能性变体。
本发明还提供了一种用于治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖 耐受不良的药物，包含作为活性成分的血管内皮抑制素或其功能性变体。
饮食性肥胖是指由于饮食中的热量超过机体能量消耗，导致过剩的热量以脂肪的形式储存在体内而引起的肥胖。
非酒精性脂肪肝(NAFLD)是一种与胰岛素抵抗和遗传易感密切相关的代谢应激性肝脏损伤，其病理学改变与酒精性肝病(ALD)相似，但患者无过量饮酒史的临床病理综合征。
胰岛素抵抗(insulin resistance)，也称胰岛素耐受，是指机体对胰岛素的反应不敏感，从而使得胰岛素促进葡萄糖的摄取和利用的生物学效应低于正常水平的现象，亦即机体需要更高的胰岛素浓度才能对胰岛素产生反应。胰岛素抵抗引起的血浆中高胰岛素和高糖含量经常导致代谢综合征、痛风和2型糖尿病。
葡萄糖耐受不良是指机体葡萄糖代谢能力下降导致的对血糖浓度调节能力的下降，表现为在大量摄取葡萄糖后不能及时将血糖水平调节至正常，如不及时治疗干预可发展为糖尿病。
小鼠体重抑制率＝{1-给药组体重增加/高脂饮食组体重增加}100％。
小鼠脂肪积累抑制率＝{1-给药组脂肪组织重量/高脂饮食组脂肪组织重量}100％。
小鼠肝脏重量抑制率＝{1-给药组肝脏重量/高脂饮食组肝脏重量}100％。
小鼠肝脏脂肪沉积抑制率＝{1-给药组肝胞质空泡率/高脂饮食组肝胞质空泡率}100％。
本发明的实施例中使用的ES及其变体蛋白均由北京普罗吉公司提供。
实施例1、ES和YH-16显著抑制高脂饮食引起的小鼠体重增加
取24只健康的C57BL/6小鼠(7周龄，雄性，购自北京维通利华公司)，分为4组，每组8只，分别作如下处理：
第一组：正常饮食组；
第二组：高脂饮食组；
第三组：高脂饮食+ES组(给药组)；
第四组：高脂饮食+YH-16组(给药组)。
正常饮食组以摄入热卡的10％来自于脂肪成分的饲料(D12450J，Research Diets，USA)饲养小鼠；高脂饮食以摄入热卡的60％来自于脂肪成分的饲料(D12492，Research Diets，USA)饲养小鼠。
给药方式为：第三组和第四组每天腹腔注射一次ES或YH-16(Protgen)，剂量为12mg/kg/天，第二组注射等体积生理盐水，第一组不注射，周期为60天。将第一次注射的时间设为第0天，第59天完成最后一次给药，每三天称量小鼠体重一次，第60天(即最后一次给药后一天)进行最后一次体重测量，绘制体重曲线(图1A)。结果显示，ES和YH-16均能够显著抑制高脂饮食引起的体重增加，抑制率分别为37.5％和30.6％ (表1)。
第61天完成葡萄糖耐受测试后，处死小鼠，分离全身脂肪组织，并称重(图1B，表1)。结果显示，ES和YH-16组小鼠脂肪组织重量明显小于未给药高脂饮食组，ES和YH-16对于高脂饮食引起的小鼠脂肪积累的抑制率分别为47.7％和42.2％(表1)。
分离小鼠肺脏、心脏和肾脏，并称重(图1C,表1)。结果显示，四组小鼠之间肺脏、心脏和肾脏的重量没有显著性差异，说明ES和YH-16对小鼠肺脏、心脏和肾脏没有影响。
实施例2、ES和YH-16显著抑制高脂饮食引起的小鼠肝脏重量增加和脂肪沉积
实施例1中小鼠，第61天完成葡萄糖耐受测试后，取出肝脏组织，称重(图2A，表1)。ES和YH-16抑制高脂饮食引起的小鼠肝脏重量增加，抑制率分别为23.8％和20.5％。
将肝脏组织固定并用石蜡包埋，制成8μm厚的切片，进行组织HE染色。主要步骤包括：切片脱蜡复水后，依次使用入苏木素和伊红染色，后进行常规脱水，封片，使用常规光学显微镜(Olympus IX71显微镜)观察并拍照记录(图2B)。HE染色显示，高脂饮食组小鼠肝脏切片中出现肝细胞胞质脂肪空泡，表明高脂饮食能够引起小鼠肝脏中的脂肪沉积，而ES和YH-16给药处理组的小鼠肝脏中的脂肪沉积明显少于未给药高脂饮食组，抑制率分别是78.9％和75.2％，抑制率(图2C)。这说明ES和YH-16对非酒精性脂肪肝有明显抑制作用。
实施例3、ES和YH-16显著改善小鼠胰岛素抵抗和葡萄糖耐受不良
实施例1中的小鼠，第59天完成给药6小时后，进行胰岛素耐受测试。具体步骤包括：剪鼠尾取血，检测基础血糖值(罗氏手持式血糖仪)，将该监测点时间设为0分钟。腹腔注射人胰岛素(诺和灵R，诺和诺德)0.5U/kg，分别在注射胰岛素后20分钟，40分钟，60分钟，80分钟时取血，测小鼠血糖，绘制曲线(图3A)。发现注射胰岛素后，随着时间的推移正常饮食组小鼠血糖水平快速降低，高脂饮食组小鼠血糖水平降低速度慢，说明高脂饮食会引起小鼠的胰岛素抵抗，而ES和YH-16可显著缓解高脂饮食引起的胰岛素抵抗。
实施例1中的小鼠，第60天测量体重后，对小鼠进行饥饿过夜处理，第61天进行葡萄糖耐受测试。具体步骤包括：剪鼠尾取血，检测基础血糖值(罗氏手持式血糖仪)，将该监测点时间设为0分钟。对小鼠进行葡萄糖溶液(20mg/ml)灌胃，每只鼠每克体重灌1mg葡萄糖，分别在灌胃后20分钟，40分钟，60分钟，80分钟时取血，测小鼠血糖，绘制曲线(图3B)。发现葡萄糖灌胃后，随着时间的推移，与正常饮食组小鼠相比高脂饮食最小鼠血糖值快速上升且回复速度慢，说明高脂饮食会导致小鼠葡萄糖耐受不良，而ES和YH-16给药组小鼠葡萄糖耐受不良得到显著改善。
第61天完成葡萄糖耐受测试后，处死小鼠，分离全身脂肪组织，并用Western blot检测脂肪组织中Akt的磷酸化水平(图3C)。结果显示，与正常饮食组相比，高脂饮食组Akt磷酸化水平降低，而ES给药组Akt磷酸化水平高于高脂饮食组。Akt通路是胰岛素下游重要的血糖调控通路。胰岛素抵抗和Akt磷酸化水平降低往往伴随发生。这与ES能够有效改善胰岛素抵抗和葡萄糖耐受不良的结果相一致。
实施例4、ES和YH-16显著抑制脂肪前体细胞向脂肪细胞分化
选取状态良好的3T3-L1脂肪前体细胞，以含10％小牛血清的DMEM培养基重悬，种入六孔板中，将六孔板放入细胞培养箱，5％二氧化碳，37℃正常培养。细胞长满两天后，开始诱导分化：第一步、加MDI诱导培养基诱导(将此时定义为细胞培养分化的第1天)；第二步、两天后换胰岛素诱导培养基，继续培养两天；第三步、换含10％FBS的DMEM培养液继续培养，直到第8天，3T3-L1分化为脂肪细胞。实验分为5组：
第一组：对照组；
第二组：ES处理组；
第三组：YH-16处理组；
第四组：mES处理组；
第五组：Myh-16处理组。
其中，给药处理组在诱导分化期间(即第1天到第8天)分别额外加入50μg/ml的ES、YH-16、mES或mYH-16，对照组加等体积蛋白缓冲液，每次换液均重新加入以上药物及对照处理。
MDI诱导液是在含10％FBS的DMEM培养液中加入1μM地塞米松、0.5mM 3-异丁基-1-甲基黄嘌呤和10μg/mL牛胰岛素；胰岛素诱导培养基是在含10％FBS的DMEM培养液中加入10μg/mL牛胰岛素。
诱导结束后，弃六孔板内培养基，固定后加油红染色10分钟。脱色，用PBS漂洗三次，除去多余的染料。油红能够识别脂肪细胞中的油脂，比将其染成红色。数码相机对六孔板进行拍照，倒置显微镜(Olympus IX71显微镜)观察并拍照记录(图4A)。结果显示，ES、YH-16、mES和mYH-16均能直接抑制脂肪前体细胞分化为脂肪细胞(图4B)。
诱导过程中的第6天，弃培养基，收取细胞，加入100ul 2X SDS电泳上样缓冲液，100℃煮样15分钟，经电泳、转膜，用免疫印迹检测各组全细胞裂解液中脂肪细胞分化中心控制因子PPARγ1和PPARγ2的表达水平(图4C)。发现在脂肪细胞分化过程中，ES、YH-16、mES和mYH-16均能抑制脂肪细胞分化中心控制转录因子PPARγ1和PPARγ2的蛋白表达水平。
检测PPARγ1和PPARγ2的mRNA表达水平：3T3-L1诱导前(0天)和诱导过程中的第6天，按照TRIZOL试剂(购自Invitrogen)说明书的标准操作提取细胞总RNA。 用Fermentas反转录试剂盒(RevertAidTM First Strand cDNA Synthesis Kits)进行反转录，反应按照说明书标准进行。
使用荧光定量Real-Time PCR检测脂肪细胞分化中心调控因子PPARγ1/2。荧光定量Real-Time PCR使用Stratagene试剂盒(Brilliant II 
Figure PCTCN2015093726-appb-000001
Green QRT-PCR Master Mix)，荧光定量PCR仪器为MX3000P(购自Stratagene)，荧光染料为SYBR Green，反应体系为20μL，反应循环数为40个。
PCR运行程序：95℃变性，10秒；60℃退火延伸，30秒，反应体系为20μL，反应循环数40个循环，最后是72℃保持5分钟。内参为GAPDH。反应引物如下：
PPARγ1正向引物(5’-3’)：ACAAGATTTGAAAGAAGCGGTGA
PPARγ1反向引物(5’-3’)：GCTTGATGTCAAAGGAATGCGAAGGA
PPAR2正向引物(5’-3’)：CGCTGATGCACTGCCTATGAG
PPAR2反向引物(5’-3’)：TGGGTCAGCTCTTGTGAATGGAA
GAPDH正向引物(5’-3’)：CCAGCCTCGTCCCGTAGACA
GAPDH反向引物(5’-3’)：TGAATTTGCCGTGAGTGGAGTC
以内参为GAPDH，根据仪器给出的荧光图得到ΔCt值，计算出相对Δ(ΔCt)值，进而计算出PPARγ1和PPARγ2的mRNA水平的相对变化(图4D)。发现在脂肪细胞分化过程中，ES能抑制脂肪细胞分化中心控制转录因子PPARγ1和PPARγ2的mRNA表达水平。
实施例5、聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)显著抑制高脂饮食引起的小鼠体重增加
取40只健康的C57BL/6小鼠(7周龄，雄性，购自北京维通利华公司)，分为5组，每组8只，分别作如下处理：
第一组：正常饮食组；
第二组：高脂饮食组；
第三组：高脂饮食+mES组(给药组)；
第四组：高脂饮食+m003组(给药组)；
第五组：高脂饮食+m007组(给药组)。
各饮食组饲料同实施例1。
给药方式为：第三组、第四组和第五组每周尾静脉注射一次mES、m003或m007(Protgen)，剂量为50mg/kg/周，第二组注射等体积生理盐水，第一组不注射，周期为8周。将第一次注射的时间设为第0周，第7周完成最后一次给药，每周称量小鼠体重一次，第8周最后一次称量小鼠体重后，绘制体重曲线(图5A)。结果显示，mES、m003和m007均能够显著抑制高脂饮食引起的体重增加，抑制率分别为33.7％、22.9％和42.9％(表2)。
第8周最后一次称量小鼠体重后，处死小鼠，分离全身脂肪组织，并称重(图5B，表2)。结果显示，mES、m003和m007组小鼠脂肪组织重量明显小于未给药高脂饮食组，mES、m003和m007对于高脂饮食引起的小鼠脂肪积累的抑制率分别为41.4％、31.9％和40.5％(表2)。
分离小鼠肺脏、心脏和肾脏，并称重(图5C，表2)。结果显示，五组小鼠之间肺脏、心脏和肾脏的重量没有显著性差异，说明mES、m003和m007对小鼠肺脏、心脏和肾脏没有影响。
实施例6、聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)显著抑制高脂饮食引起的小鼠肝脏重量增加和脂肪沉积
实施例5中小鼠，第8周最后一次称量小鼠体重后，取出肝脏组织，称重(图6A，表2)。结果显示，mES、m003和m007抑制高脂饮食引起的小鼠肝脏重量增加，抑制率分别为21.3％、21.3％和25.2％(表2)。
按照实施例2中实验方法将肝脏组织固定并用石蜡包埋切片，进行组织HE染色。使用常规光学显微镜(Olympus IX71显微镜)观察并拍照记录肝脏组织形态(图6B)。HE染色显示，mES、m003和m007给药处理组的小鼠肝脏中的脂肪沉积明显少于未给药高脂饮食组，抑制率分别是70.6％、56.1％和73.1％(图6C)。这说明mES、m003和m007对非酒精性脂肪肝有明显抑制作用。
实施例7、聚乙二醇修饰的ES及其变体003和007(mES、m003和m007)显著抑制脂肪前体细胞向脂肪细胞分化
3T3-L1脂肪前体细胞培养、诱导分化同实施例4。实验分为4组：
第一组：对照组；
第二组：mES处理组；
第三组：m003处理组；
第四组：m007处理组。
其中，给药处理组在诱导分化期间(即第1天到第8天)分别额外加入50μg/ml的mES、m003或m007，对照组加等体积蛋白缓冲液，每次换液均重新加入以上药物及对照处理。
诱导结束后，按照实施例4中实验方法对细胞进行油红染色。数码相机对六孔板进行拍照，倒置显微镜(Olympus IX71显微镜)观察并拍照记录(图7A)。结果显示，mES、m003和m007均能直接抑制脂肪前体细胞分化为脂肪细胞，其中mES和m007抑制效果优于m003(图7B)。这与实施例6中的动物实验结果相一致，这也解释了mES和m007对于高脂饮食动物体重增长抑制效果好于m003的原因。
实施例8、聚乙二醇修饰的ES变体Z101(mZ101)显著抑制高脂饮食引起的小鼠体重增加
实验小鼠准备(每组8只小鼠)、饮食(饲料)、给药方式、给药周期及小鼠体重称量均同实施例5。实验分组如下：
第一组：正常饮食组；
第二组：高脂饮食组；
第三组：高脂饮食+mZ101组(给药组)。
其中给药剂量为12mg/kg/周。
第8周最后一次称量小鼠体重后，绘制体重曲线(图8A)。结果显示，mZ101能够显著抑制高脂饮食引起的体重增加，抑制率为31％(表3)。
第8周最后一次称量小鼠体重后，处死小鼠，分离全身脂肪组织和肝脏，并称重(图8B和C，表3)。结果显示，mZ101组小鼠脂肪组织重量明显小于未给药高脂饮食组，脂肪积累的抑制率为77.2％(表3)。mZ101也能够抑制高脂饮食引起的小鼠肝脏重量增加，抑制率为21.5％(表3)。
分离小鼠肺脏、心脏和肾脏，并称重(图8D，表3)。结果显示，五组小鼠之间肺脏、心脏和肾脏的重量没有显著性差异，说明mZ101对小鼠肺脏、心脏和肾脏没有影响。
实施例9、聚乙二醇修饰的ES变体Z101(mZ101)显著抑制脂肪前体细胞向脂肪细胞分化
3T3-L1脂肪前体细胞培养、诱导分化同实施例4。实验分为2组：
第一组：对照组；
第二组：mZ101处理组。
其中，给药处理组在诱导分化期间(即第1天到第8天)额外加入50μg/ml的mZ101(Protgen)，对照组加等体积蛋白缓冲液，每次换液均重新加入以上药物及对照处理。
诱导结束后，按照实施例4中实验方法对细胞进行油红染色。数码相机对六孔板进行拍照，倒置显微镜(Olympus IX71显微镜)观察并拍照记录(图9A)。结果显示，mZ101能直接抑制脂肪前体细胞分化为脂肪细胞(图9B)。
实施例10、聚乙二醇修饰的ES变体009和S03(m009和mS03)显著抑制高脂饮食引起的小鼠体重增加，且mS03的抑制效果优于m009
实验小鼠准备(每组8只小鼠)、饮食(饲料)、给药方式、给药周期及小鼠体重称量均同实施例5。实验分组如下：
第一组：正常饮食组；
第二组：高脂饮食组；
第三组：高脂饮食+m009组(给药组)；
第四组：高脂饮食+mS03组(给药组)。
其中给药剂量为12mg/kg/周。
第8周最后一次称量小鼠体重后，绘制体重曲线(图10A)。结果显示，m009和mS03均能够显著抑制高脂饮食引起的体重增加，抑制率分别为10.6％和19.0％(表4)。
第8周最后一次称量小鼠体重后，处死小鼠，分离全身脂肪组织和肝脏，并称重(图10B和C，表4)。结果显示，m009和mS03组小鼠脂肪组织重量明显小于未给药高脂饮食组，m009和mS03对于高脂饮食引起的小鼠脂肪积累的抑制率分别为45.7％和59.5％(表4)。m009和mS03也能够抑制高脂饮食引起的小鼠肝脏重量增加，抑制率分别为16.7％和25.7％(表4)。
分离小鼠肺脏、心脏和肾脏，并称重(图10D，表4)。结果显示，4组小鼠之间肺脏、心脏和肾脏的重量没有显著性差异，说明m009和mS03对小鼠肺脏、心脏和肾脏没有影响。
实施例11、聚乙二醇修饰的ES变体009和S03(m009和mS03)显著抑制脂肪前体细胞向脂肪细胞分化
3T3-L1脂肪前体细胞培养、诱导分化同实施例4。实验分为3组：
第一组：对照组；
第二组：m009处理组；
第三组：mS03处理组。
其中，给药处理组在诱导分化期间(即第1天到第8天)分别额外加入50μg/ml的m009或mS03，对照组加等体积蛋白缓冲液，每次换液均重新加入以上药物及对照处理。
诱导结束后，按照实施例4中实验方法对细胞进行油红染色。数码相机对六孔板进行拍照，倒置显微镜(Olympus IX71显微镜)观察并拍照记录(图11A)。结果显示，m009和mS03均能直接抑制脂肪前体细胞分化为脂肪细胞，其中mS03的抑制效果优于m009(图11B)。这与实施例9中的动物实验结果相一致，这也解释了mS03对于高脂饮食动物体重增长抑制效果好于m009的原因。
实施例12、聚乙二醇修饰的ES变体36和249(m36和m249)显著抑制高脂饮食引起的小鼠体重增加
实验小鼠准备(每组8只小鼠)、饮食(饲料)、给药方式、给药周期及小鼠体重称量均同实施例5。实验分组如下：
第一组：正常饮食组；
第二组：高脂饮食组；
第三组：高脂饮食+m36组，给药剂量6mg/kg/周(给药组)；
第四组：高脂饮食+m36组，给药剂量12mg/kg/周(给药组)；
第五组：高脂饮食+m249组，给药剂量6mg/kg/周(给药组)；
第六组：高脂饮食+m249组，给药剂量12mg/kg/周(给药组)。
第8周最后一次称量小鼠体重后，绘制体重曲线(图12A)。结果显示，低剂量m36(6mg/kg/周)和高剂量m249(12mg/kg/周)均能够显著抑制高脂饮食引起的体重增加，抑制率分别为30.3％和50.3％(表5)。
第8周最后一次称量小鼠体重后，处死小鼠，分离全身脂肪组织和肝脏，并称重(图12B和C，表5)。结果显示，低剂量m36(6mg/kg/周)组和高剂量m249(12mg/kg/周)组小鼠脂肪组织重量明显小于未给药高脂饮食组，低剂量m36(6mg/kg/周)和高剂量m249(12mg/kg/周)对于高脂饮食引起的小鼠脂肪积累的抑制率分别为30％和38.4％(表5)。低剂量m36(6mg/kg/周)和高剂量m249(12mg/kg/周)也能够抑制高脂饮食引起的小鼠肝脏重量增加，抑制率分别为18.4％和22.9％(表5)。
分离小鼠肺脏、心脏和肾脏，并称重(图12D)。结果显示，六组小鼠之间肺脏、心脏和肾脏的重量没有显著性差异，说明m36和m249对小鼠肺脏、心脏和肾脏没有影响。
表1
Figure PCTCN2015093726-appb-000002
表2
Figure PCTCN2015093726-appb-000003
表3
Figure PCTCN2015093726-appb-000004
表4(图11)
Figure PCTCN2015093726-appb-000005
Figure PCTCN2015093726-appb-000006
Figure PCTCN2015093726-appb-000007
Figure PCTCN2015093726-appb-000008
Figure PCTCN2015093726-appb-000009

Claims (15)

  1. 血管内皮抑制素或其功能性变体在制备用于治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的药物中的用途。
  2. 权利要求1的用途，其中所述功能性变体选自由YH-16、003、007、Z101、ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163和m119构成的组。
  3. 权利要求1的用途，其中所述功能性变体选自由YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249构成的组。
  4. 血管内皮抑制素或其功能性变体在制备用于抑制脂肪细胞分化的药物中的用途。
  5. 权利要求4的用途，其中所述功能性变体选自由YH-16、003、007、Z101、ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163和m119构成的组。
  6. 权利要求4的用途，其中所述功能性变体选自由YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249构成的组。
  7. 治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的方法，包括给予受试者治疗有效量的血管内皮抑制素或其功能性变体。
  8. 权利要求7的方法，其中所述功能性变体选自由YH-16、003、007、Z101、ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163和m119构成的组。
  9. 权利要求7的方法，其中所述功能性变体选自由YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249构成的组。
  10. 抑制脂肪细胞分化的方法，包括给予受试者治疗有效量的血管内皮抑制素或其功能性变体。
  11. 权利要求10的方法，其中所述功能性变体选自由YH-16、003、007、Z101、 ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163和m119构成的组。
  12. 权利要求10的方法，其中所述功能性变体选自由YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249构成的组。
  13. 用于治疗饮食性肥胖、非酒精性脂肪肝、胰岛素抵抗或葡萄糖耐受不良的药物，包含作为活性成分的血管内皮抑制素或其功能性变体。
  14. 权利要求13的药物，其中所述功能性变体选自由YH-16、003、007、Z101、ES006、ES008、ES011、S02、S09、Z006、Z008、ZN1、009、S03、36、249、381、57、114、124、125、160、163、119、mES、mYH-16、m003、m007、mZ101、mES006、mES008、mES011、mS02、mS09、mZ006、mZ008、mZN1、m009、mS03、m36、m249、m381、m57、m114、m124、m125、m160、m163和m119构成的组。
  15. 权利要求13的药物，其中所述功能性变体选自由YH-16、003、007、Z101、009、S03、36、249、mES、mYH-16、m003、m007、mZ101、m009、mS03、m36和m249构成的组。
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