WO2018232810A1 - 长效降糖调脂多肽及其用途 - Google Patents

长效降糖调脂多肽及其用途 Download PDF

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WO2018232810A1
WO2018232810A1 PCT/CN2017/094009 CN2017094009W WO2018232810A1 WO 2018232810 A1 WO2018232810 A1 WO 2018232810A1 CN 2017094009 W CN2017094009 W CN 2017094009W WO 2018232810 A1 WO2018232810 A1 WO 2018232810A1
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blood glucose
peptide
polypeptide
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金亮
高华山
赵茜
张艳峰
卢世平
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China Pharmaceutical University
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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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

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  • the invention relates to a long-acting hypoglycemic and lipid-lowering polypeptide, which has a longer duration of drug efficacy and at the same time has immunomodulatory function, and belongs to the technical field of biochemical pharmacy.
  • type 1 diabetes is a disease in which insulin is absolutely deficient due to destruction of pancreatic beta cells.
  • the research on type 1 diabetes is mainly divided into the early diagnosis and prevention stage, as well as the middle and late stage of treatment.
  • diagnosis and prevention mainly study the corresponding diagnostic methods and preventive measures for the pathogenesis of diabetes.
  • the pathogenesis of type 1 diabetes involves T cell-mediated autoimmune attack and B lymphocyte production in beta cells in susceptible individuals, so patients have a long-term presence in the body before clinical symptoms appear.
  • the autoimmune response of islet ⁇ cells exists, which provides a basis for the diagnosis and prevention of diseases.
  • autoimmune antigens can be detected months or years before the onset of hyperglycemia. Aiming at these antigens, a variety of vaccines have been developed, the targets of which are mainly concentrated on autoimmune T cells; these vaccines can induce protective immune tolerance and improve pathological autoimmunity; immune tolerance through inhibition of effector T cells, And activate regulatory T cells (Treg cells) to improve the effect of pathological autoimmunity and delay the development of diabetes.
  • Treg cells activate regulatory T cells
  • Type 1 diabetes patients who have been diagnosed with hyperglycemia are mainly treated mainly.
  • Insulin is still the main drug for clinical treatment.
  • Intensive insulin therapy is also the main treatment for clinical control of type 1 diabetes, and islet transplantation, stem cell transplantation, etc.
  • the method has gradually emerged, but it is still in the research stage, and there is still a certain distance from the application. Therefore, the treatment of other defects of T1DM has become a good treatment, especially the common side effects of T1DM patients, such as weight gain, increased insulin dose, number of injections and hypoglycemia. Due to the lack of new treatments, T1DM patients are constantly trying to control their blood sugar.
  • the glucagon-like peptide-1 receptor agonist has been recognized as a concomitant therapy for insulin in patients with T1DM because they are able to maintain or reduce body weight, reduce the risk of hypoglycemia, and lower glucagon levels.
  • GLP-1RA protects beta cells, so GLP-1RA can increase the amount of beta cells in T1DM patients by stopping autoimmune destruction or increasing beta cell regeneration.
  • most patients with type 1 diabetes still have the ability to secrete insulin, which is equivalent to 20-30% of non-diabetic individuals. Therefore, there is an opportunity to maintain and increase the amount of beta cells, which can be achieved by the treatment of GLP-1RA. , This will also improve the risk of hypoglycemia, as the involvement of functional beta cells is more conducive to glucagon response.
  • GLP-1 analogue can effectively reduce fasting blood glucose and improve body weight in type 1 diabetic mice, and can be used as an adjuvant therapy for type 1 diabetes.
  • Heat shock protein 60 (Hsp60) is a major autoantigen, and high levels of anti-Hsp60 antibodies have been detected in patients with type 1 diabetes. Antibody titers are positively correlated with disease severity.
  • the P277 peptide a specific polypeptide of 437 to 460 on Hsp60, has a sequence of VLGGGCALLRCIPALDSLTPANED, which is an antigenic determinant and can specifically react with effector T cells.
  • P277 peptide has the same side effects as vascular endothelial injury caused by Hsp60. C57BL/6 mice immunized with P227 peptide can cause severe vascular endothelial damage.
  • P277 peptide immunity induces severe atherosclerosis. It hardens and a large amount of P277 antibody is found to accumulate around the atherosclerotic plaque, indicating that the P277 peptide not only has a T epitope that regulates the immune response, but also has an A atherogenic B epitope.
  • the inventor applied for a Chinese invention patent application with the application number CN201610949156.3, the application publication number CN106518987A, and the design and use of the novel diabetes immunomodulatory peptide-VP peptide, which was scanned by an epitope at 2016.10.28.
  • Technique accurate scanning of the B epitope of P277 peptide, under the premise of ensuring the integrity of T epitope and the spatial structure of B epitope, the B epitope can be modified by amino acid mutation to obtain VP peptide, which can maintain immunomodulatory effect. At the same time, eliminate the side effects of vascular endothelial damage.
  • the inventors found in further practice that although the duration of the drug effect of Progly peptide is not short, up to 2 hours, if the duration of the drug effect can be greatly extended by further improvement, The formation of long-acting drugs is more conducive to the marketing of the drug.
  • the technical problem to be solved by the present invention is to overcome the problems existing in the prior art and provide a long-acting hypoglycemic and lipid-lowering polypeptide, which can greatly prolong the duration of the drug effect of the Progly peptide.
  • the main research process of the present invention is as follows: In the practical research, the inventors want to extend the duration of the drug effect of the Progly peptide by means of the fusion peptide, but it is quite difficult to select which peptide to fuse. Press In the past research experience, the selection of peptides with longer half-life (or duration of drug effect) for fusion should prolong the duration of Progly peptides. However, after trying to fuse multiple peptides, they were found to be counterproductive. Progly peptides work synergistically.
  • the inventors expanded the range of choices, tried some peptides with half-life (or duration of drug effect) that were not too long, and finally screened out VP peptides (the duration of which lasted only 3-4 hours), VP peptides and The fusion peptide of Progly peptide can not only exert the pharmacological effect of Progly peptide, but also has a long-lasting effect of up to 10 hours. It is a long-acting hypoglycemic and lipid-lowering polypeptide that meets the inventors' expectations and has immunomodulatory functions.
  • a long-acting hypoglycemic lipid-lowering polypeptide characterized in that the amino acid sequence of the polypeptide is:
  • the pharmaceutical composition comprises a pharmaceutical carrier and/or a pharmaceutically active substance.
  • the long-acting hypoglycemic lipid-lowering polypeptide of the present invention can achieve a drug efficacy duration of up to 10 hours.
  • the polypeptide is a bifunctional polypeptide: it can reduce the blood sugar and reduce glycated hemoglobin in patients with type 1 diabetes, reduce blood sugar in patients with type 2 diabetes, inhibit eating and lower glycated hemoglobin, and be the same as VP peptide. Regulate immunity and inhibit pathological immune attacks.
  • FIG. 1 is a view showing the results of an experiment of PGLP-1 peptide of Example 1 of the present invention.
  • * indicates P ⁇ 0.05, *** indicates P ⁇ 0.001;
  • GLP-1 group was compared with the control group, ⁇ indicates P ⁇ 0.001;
  • PGLP-1 When compared with the GLP-1 group, $ indicates P ⁇ 0.05, $$ indicates P ⁇ 0.01, and $$$ indicates P ⁇ 0.001;
  • GTT refers to the glucose tolerance test;
  • AUC refers to the area under the curve.
  • Example 2 is a schematic diagram showing the results of an experiment of VP peptide according to Example 1 of the present invention.
  • FIG. 3 is a schematic diagram showing the results of the P15 peptide experiment of Example 1 of the present invention.
  • PGLP-1-VP refers to P15 peptide; when compared with the control group, *** indicates P ⁇ 0.001; GLP-1 group compared with the control group When ⁇ indicates P ⁇ 0.001; GTT refers to the glucose tolerance test; AUC refers to the area under the curve.
  • Example 4 is a schematic diagram showing the cumulative eating effect of Example 2 of the present invention.
  • Fig. 5 is a view showing the results of blood glucose effect in Example 2 of the present invention.
  • Fig. 6 is a view showing the results of the influence of triglyceride and free fatty acid of Example 2 of the present invention.
  • Fig. 7 is a view showing the results of the effects of morbidity and mortality of NOD mice according to Example 2 of the present invention.
  • Fig. 8 is a view showing the results of effect of fasting blood glucose in NOD mice according to Example 2 of the present invention.
  • Fig. 9 is a view showing the results of the effects of pancreas weight and pancreatitis in NOD mice according to Example 2 of the present invention.
  • Fig. 10 is a view showing the results of the effect of the immunomodulatory factor Th1/Th2 of NOD mice in Example 2 of the present invention.
  • mice Eight-week-old male C57BL/6 mice were randomly divided into 5 groups, 10 in each group, which were solvent control group (negative control group), PGLP-1 group (1 nmol/kg), and PGLP-1 group (30 nmol/ Glucose tolerance test was performed in kg), PGLP-1 group (100 nmol/kg), and GLP-1 group (30 nmol/kg).
  • solvent control group negative control group
  • PGLP-1 group 1 nmol/kg
  • PGLP-1 group (30 nmol/ Glucose tolerance test was performed in kg
  • PGLP-1 group 100 nmol/kg
  • GLP-1 group (30 nmol/kg).
  • PGLP-1 is a Progly peptide
  • GLP-1 is a glucagon-like peptide-1.
  • GLP-1 (30nmol/kg) and PGLP-1 (30nmol/kg) were administered subcutaneously, respectively, and then glucose was administered at 0.5h, 1h, 2h, 4h after administration. (1.5 g/kg), blood glucose was detected 15 minutes after the administration of the sugar, and as shown in Fig. 1B, PGLP-1 was still significantly different from the control group after 2 hours of administration (p ⁇ 0.05), indicating that PGLP -1 can continue to reduce sugar for 2h.
  • the GLP-1 hypoglycemic effect lasted only 0.5h.
  • mice Eight male mice were injected subcutaneously with VP peptide at a dose of 50 nmol/kg. At different times after administration At the inter-point (0h, 0.5h, 1h, 2h, 3h, 4h), blood was taken from the eyelids, and the blood samples of the mice were collected. After standing at room temperature for 10 min, 3000 rpm ⁇ 15 min. The upper serum was carefully aspirated and stored frozen at -20 °C.
  • the content of VP peptide in mouse serum was determined by ELISA kit, and the detection sensitivity was 0.02 nmol/mL.
  • the blood concentration-time curve of the VP peptide in mice is shown in Figure 2.
  • the detectable concentration of the subcutaneous injection can reach 4 hours, indicating that the duration of the subcutaneous injection of the VP peptide is 3-4 h.
  • GLP-1 (30nmol/kg) and P15 (30nmol/kg) were administered subcutaneously, respectively, and then 0.5h, 1h, 2h, 4h, 6h, 8h, 10h after administration, respectively.
  • Glucose (1.5 g/kg) was administered for 12 hours, and blood glucose was measured 15 minutes after the administration of the sugar.
  • P15 was still significantly different from the control group after administration for 10 hours (p ⁇ 0.001). The efficacy of P15 lasted for 10 hours.
  • mice were randomly divided into 4 groups, 12 in each group, which were solvent control group (negative control group), PGLP-1 group (positive control group), VP group (positive control group), and P15 group (experimental group).
  • the dose was 30 nmol/kg, 2 times a day, the solvent was physiological saline, 0.1 ml was injected subcutaneously, and the drug was administered at a fixed time point, AM9:00-10:00, PM8:00-9:00.
  • the negative control group was injected subcutaneously with 0.1 ml of physiological saline.
  • mice were fasted for 8 hours before administration, fasted at AM8:00, fasting blood glucose was detected at the tail vein of PM4:00, and a blood glucose tester (OHA-231 type of Omron blood glucose meter) was used for blood glucose administration for 0 weeks. Then, at 1, 2, 3, and 4 weeks after administration, fasting blood glucose was measured, and the fasting time and the detection time were the same as 0 weeks.
  • a blood glucose tester OAA-231 type of Omron blood glucose meter
  • TG triglyceride
  • FFA free fatty acid
  • fasting blood glucose (fasting 6h) was measured from the 5th week and administered (subgroups and experimental groups PGLP-1, VP, P15). Blood glucose was measured at intervals of 2 weeks. Two consecutive ⁇ 11mmol/L were considered to be morbid.
  • mice At the end of the 39-week observation period, the mouse death dates were recorded and plotted. The results showed that the control mice started to die at 21 weeks, and at 39 weeks, 7 died, with a mortality rate of 58.44%, while the experimental group PGLP- The mortality rates of group 1, VP group and P15 group were 33.33%, 30%, and 15.39%, respectively. Compared with the control group, the mortality rate of the experimental group P15 group was significantly lower, p ⁇ 0.05, the experimental group PGLP-1 group, VP group. Although the group reduced the incidence, there was no significant difference (Fig. 7B).
  • polypeptide P15 of the present invention can significantly reduce the morbidity and mortality of NOD mice.
  • fasting blood glucose (fasting 6 h) was measured from the 5th week and administered (subgroups and experimental groups PGLP-1, VP, and P15), and blood glucose was measured at intervals of 2 weeks. Blood glucose was measured periodically for each group of mice for 24 weeks and continued for 10 weeks.
  • Diabetes did not occur in the P15 group, however, diabetes was present in the blank control group, PGLP-1 or VP group (Fig. 8A). These data suggest that P15 can prevent the recurrence of diabetes.
  • the blood glucose level of the P15 group was maintained at a low level from the 29th week (stopping the administration), and at the 39th week, the blood glucose level of the P15 group was significantly lower than that of the control group and the VP group (excluding diabetic mice) (p ⁇ 0.05). 8B).
  • mice were sacrificed, the mouse pancreas was taken, the pancreas weight was weighed, and the difference between the groups was analyzed.
  • mice were sacrificed, the spleen was taken, and antibody staining was performed to analyze the ratio of Th1 and Th2 and the ratio of Th2/Th1 by flow cytometry.
  • the P15 peptide of the present invention can effectively exert a sugar-dependent insulin secretion (IPTGG); reduce blood glucose in STZ diabetic mice, inhibit eating in STZ diabetic mice; reduce morbidity and mortality in NOD mice Rate, increase pancreatic weight, inhibit pancreatitis; regulate immune balance in mice.
  • IPTGG sugar-dependent insulin secretion

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Abstract

提供了一种长效降糖调脂多肽及其用途，该多肽的氨基酸序列为HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGPGVLGGGCRCIPALDSLTPANED，该多肽可用于制备治疗或预防1型糖尿病、2型糖尿病药物或药物组合物，也可用于制备免疫调节药物或药物组合物。

Description

长效降糖调脂多肽及其用途 技术领域
本发明涉及一种长效降糖调脂多肽，具有更长的药效持续时间，同时兼具免疫调节功能，属于生化药学技术领域。
背景技术
据发明人所知，1型糖尿病(T1DM)是一种由于胰腺β细胞被破坏而导致胰岛素绝对缺乏的疾病。目前，1型糖尿病的研究主要分为前期诊断和预防阶段，以及中后期的治疗阶段。其中，诊断和预防主要针对糖尿病的发病机理而研究相应的诊断方法和预防措施。1型糖尿病的发病机制涉及T细胞介导的自身免疫攻击以及在易感个体中B淋巴细胞产生针对β细胞的抗体反应，因此患者在出现临床症状前的较长一段时间内，体内就有针对胰岛β细胞的自身免疫反应存在，这就为疾病的诊断和预防提供了基础。目前研究者已经发现，在出现高血糖临床症状之前，自身免疫抗原可在数月或数年之前就可以被检测到。针对这些抗原，目前已经研制了多种疫苗，其靶点主要集中在自身免疫性T细胞；这些疫苗能够诱导保护性免疫耐受，改善病理性的自身免疫；免疫耐受通过抑制效应T细胞，并激活调节性T细胞(Treg细胞)，起到改善病理性自身免疫的效果，延缓糖尿病发展。
已经确诊为高血糖的1型糖尿病患者主要以治疗为主，胰岛素仍然是临床治疗的主要药物，加强的胰岛素治疗也是目前临床上控制1型糖尿病的主要的治疗手段，而胰岛移植、干细胞移植等方法也逐渐出现，但是都处于研究阶段，离应用还存在一定的距离。因此靶向T1DM其他缺陷的治疗也成为一种良好的治疗方式，特别是T1DM患者常见的治疗副作用，如体重增加，胰岛素剂量的增加，注射次数的和低血糖等。由于缺乏新的治疗方式，导致T1DM患者要不断地努力控制血糖。胰高血糖素样肽-1受体激动剂(GLP-1RA)已被认为是T1DM患者胰岛素的伴随疗法，因为它们能够保持或减轻体重、降低低血糖风险以及降低胰高血糖素水平等。GLP-1RA能够保护β细胞，因此GLP-1RA可以通过停止自身免疫破坏或增加β细胞再生来增加T1DM患者的β细胞量。在诊断时，大多数1型糖尿病患者仍然具有分泌胰岛素的能力，相当于非糖尿病个体的20-30％，因此，存在保持和增加β细胞量的机会，这可以通过GLP-1RA的治疗来实现， 这也将改善低血糖的风险，因为功能性β细胞的参与更有利于胰高血糖素的反应。
发明人于2016.12.20申请了申请号为CN201611222521.7、申请公布号为CN106519016A、名称为《降糖调脂肽——Progly肽的设计及其用途》的中国发明专利申请，其中涉及的Progly肽为GLP-1类似物，能够有效降低1型糖尿病小鼠的空腹血糖，改善体重等，可以作为1型糖尿病的辅助治疗药物。
热休克蛋白60(Hsp60)是一种主要的自身抗原，在1型糖尿病患者体内已经检测到高水平的抗Hsp60抗体，抗体的滴度与疾病严重程度正相关。其中Hsp60上437位～460位的一段特异性多肽P277肽，其序列为VLGGGCALLRCIPALDSLTPANED，是一种抗原决定簇，能特异地与效应T细胞反应。P277肽与Hsp60一样具有引起血管内皮损伤的副作用，P227肽免疫的C57BL/6小鼠，能够引起严重的血管内皮损伤，在高脂饲养新西兰大白兔模型上，P277肽免疫诱发了严重动脉粥样硬化，并且发现大量P277抗体聚集在动脉硬化斑块周围，说明P277肽不仅存在调节免疫反应的T表位，还存在致动脉粥样硬化的B表位。
发明人于2016.10.28申请了申请号为CN201610949156.3、申请公布号为CN106518987A、名称为《新型糖尿病免疫调节肽-VP肽的设计及其用途》的中国发明专利申请，其中，通过表位扫描技术，精确扫描到P277肽的B表位，在最大程度保证T表位完整性和B表位空间结构的前提下，通过氨基酸突变来改造B表位，得到VP肽，能够保持免疫调节作用的同时，消除血管内皮损伤的副作用。
在上述已有成果的基础上，发明人在进一步地实践研究中发现，虽然Progly肽的药效持续时间不短，可达2小时，但是如果能通过进一步改进使其药效持续时间大幅延长，形成长效药物，则更加有利于该药物的市场推广。
发明内容
本发明所要解决的技术问题是：克服现有技术存在的问题，提供一种长效降糖调脂多肽，能大幅延长Progly肽的药效持续时间。
本发明的主要研究过程如下：发明人在实践研究中想要通过融合肽的方式来延长Progly肽的药效持续时间，然而在选择融合哪种肽时则颇费一番周折。按 以往研究经验来看，选择半衰期(或药效持续时间)较长的肽进行融合，应当能延长Progly肽的药效持续时间，然而在尝试多个这样的肽进行融合后，发现它们反而不利于Progly肽发挥药效。于是，发明人扩大了选择范围，尝试一些半衰期(或药效持续时间)不太长的肽进行融合，终于从中筛选出VP肽(其药效持续时间仅达3-4小时)，VP肽与Progly肽的融合肽不但能正常发挥Progly肽的药效，而且药效持续时间长达10小时，是符合发明人预期的长效降糖调脂多肽，同时兼具免疫调节功能。
本发明的技术方案如下：
一种长效降糖调脂多肽，其特征是，所述多肽的氨基酸序列为：
Figure PCTCN2017094009-appb-000001
上述长效降糖调脂多肽用于制备治疗或预防1型糖尿病药物或药物组合物的用途。
上述长效降糖调脂多肽用于制备治疗或预防2型糖尿病药物或药物组合物的用途。
上述长效降糖调脂多肽用于制备免疫调节药物或药物组合物的用途。
上述用途中，所述药物组合物包括药物载体和/或药物活性物质。
本发明的长效降糖调脂多肽可实现长达10小时的药效持续时间。该多肽为双功能多肽：既能与GLP-1类似物一样，降低1型糖尿病病人的血糖并降低糖化血红蛋白，降低2型糖尿病病人的血糖、抑制进食并降低糖化血红蛋白，又能与VP肽一样，调节免疫，抑制病理性的免疫攻击。
附图说明
图1为本发明实施例1的PGLP-1肽实验结果示意图。图中，PGLP-1组与对照组相比时，*表示P<0.05，***表示P<0.001；GLP-1组与对照组相比时，^^^表示P<0.001；PGLP-1组与GLP-1组相比时，$表示P<0.05，$$表示P<0.01，$$$表示P<0.001；GTT指糖耐量实验；AUC指曲线下面积。
图2为本发明实施例1的VP肽实验结果示意图。
图3为本发明实施例1的P15肽实验结果示意图。图中，PGLP-1-VP即指P15肽；PGLP-1组与对照组相比时，***表示P<0.001；GLP-1组与对照组相比 时，^^^表示P<0.001；GTT指糖耐量实验；AUC指曲线下面积。
图4为本发明实施例2的累计进食影响结果示意图。
图5为本发明实施例2的血糖影响结果示意图。
图6为本发明实施例2的甘油三酯和游离脂肪酸影响结果示意图。
图7为本发明实施例2的NOD小鼠发病率和死亡率影响结果示意图。
图8为本发明实施例2的NOD小鼠空腹血糖影响结果示意图。
图9为本发明实施例2的NOD小鼠胰腺重量和胰腺炎影响结果示意图。
图10为本发明实施例2的NOD小鼠免疫调节因子Th1/Th2影响结果示意图。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1 本发明多肽与Progly肽、VP肽的药效持续时间比较
(1)Progly肽对正常小鼠的急性和持续降糖效果
8周龄的雄性C57BL/6小鼠，随机分为5组，每组10只，分别为溶剂对照组(阴性对照组)、PGLP-1组(1nmol/kg)、PGLP-1组(30nmol/kg)、PGLP-1组(100nmol/kg)、GLP-1组(30nmol/kg)进行糖耐量实验。其中，PGLP-1即Progly肽，GLP-1即胰高血糖素样肽-1。
结果如图1A所示，与对照组相比，GLP-1、PGLP-1均能够显著降低血糖值，曲线下面积与对照组相比，明显降低，说明PGLP-1能够与GLP-1一样具有糖依赖性的促胰岛素的分泌作用。同时PGLP-1(1、30、100nmol/kg)能够剂量依赖性地降低血糖，最小起效浓度为1nmol/kg。
评估PGLP-1的在体内的代谢水平，分别在皮下给予GLP-1(30nmol/kg)、PGLP-1(30nmol/kg)，然后分别在给药后的0.5h、1h、2h、4h给予葡萄糖(1.5g/kg)，在给糖之后的15min检测血糖，结果如图1B所示，PGLP-1在给药2h后，与对照组相比仍然具有显著性差异(p<0.05)，说明PGLP-1能够持续降糖达到2h之久。而GLP-1降糖效果仅仅持续0.5h。
(2)VP肽的药效持续时间
将8只雄性小鼠皮下注射VP肽，给药剂量50nmol/kg。给药后在不同的时 间点(0h，0.5h，1h，2h，3h，4h)眼眶取血，采集小鼠血样，室温静置10min后，3000rpm×15min。小心吸取上层血清，-20℃冻存。
采用ELISA试剂盒测定小鼠血清中VP肽的含量，检测灵敏度为0.02nmol/mL。VP肽在小鼠体内的血药浓度-时间曲线如图2所示，皮下注射的可检测浓度可以达到4小时，说明皮下注射VP肽的药效持续时间为3-4h。
(3)本发明多肽的药效持续时间
8周龄的雄性C57BL/6小鼠，随机分为6组，每组10只，分别为溶剂对照组(阴性对照组)、P15组(0.1nmol/kg)、P15组(1nmol/kg)、P15组(30nmol/kg)、P15组(100nmol/kg)、GLP-1组(30nmol/kg)进行糖耐量实验。P15即本发明长效降糖调脂多肽。
结果如图3A所示，与对照组相比，GLP-1、P15均能够显著降低血糖值，曲线下面积与对照组相比，明显降低，说明P15与GLP-1一样具有糖依赖性的促胰岛素分泌作用。同时P15(0.1、1、30、100nmol/kg)能够剂量依赖性的降低血糖，最小起效浓度为0.1nmol/kg。
评估P15在体内的代谢水平，分别在皮下给予GLP-1(30nmol/kg)、P15(30nmol/kg)，然后分别在给药后的0.5h、1h、2h、4h、6h、8h、10h、12h给予葡萄糖(1.5g/kg)，在给糖之后的15min检测血糖，结果如图3B所示，P15在给药10h后，与对照组相比仍然具有显著性差异(p<0.001)，说明P15的药效持续时间达到10h之久。
实施例2 本发明多肽的药效学评价
(1)STZ糖尿病模型小鼠造模，及分组
80只6周龄的雄性c57小鼠(购自扬州大学实比较医学中心，许可证号：SCXK(苏)2012-0004)，适应性饲养1周。实验前空腹12小时，按50mg/kg的剂量腹腔注射STZ溶液(pH＝5.2的柠檬酸缓冲液)，连续注射5天，注射后1周、2周分别检测空腹血糖，血糖值均超过11.0mmol/L，为造模成功。
将成模小鼠随机分成4组，每组12只，分别为溶剂对照组(阴性对照组)、PGLP-1组(阳性对照组)、VP组(阳性对照组)、P15组(实验组)。给药剂量为30nmol/kg，每天2次，溶剂为生理盐水，皮下注射0.1ml，固定时间点给药，AM9：00—10:00，PM8:00—9:00。阴性对照组，皮下注射0.1ml生理盐水。
(2)对STZ糖尿病模型小鼠累计进食的影响
从给药开始，每组分别记录初始添食量，隔2～3天检测剩食量，并继续添加新的鼠粮，并记录，依次类推，到4周给药结束，记录小鼠的总进食量，并绘制累计进食曲线，结果如图4所示，P15组同PGLP-1阳性对照组具有相同的抑制进食效果，明显少于溶剂组和VP组。
(3)对STZ糖尿病模型小鼠血糖的影响
给药前小鼠空腹8小时，AM8:00开始禁食，PM4:00尾静脉检测空腹血糖，检测用血糖检测仪(欧姆龙血糖仪HEA-231型)，作为给药0周血糖。之后分别在给药1、2、3、4周，检测空腹血糖，禁食时间和检测时间同0周方法。
结果如图5所示，连续给药2周后，P15组与阴性对照组相比，血糖明显降低(p<0.01，与对照组比较)，到第4周，平均血糖值只有9.3mmol/L(p<0.01，与对照组比较)，几乎接近正常值，与阳性对照组PGLP-1相似。说明P15处理能够发挥GLP-1RA的作用，很好地降低STZ糖尿病模型小鼠的血糖。
(4)对STZ糖尿病模型小鼠甘油三酯和游离脂肪酸的影响
给药4周后，眼眶取血，静置收集血清，试剂盒分别检测甘油三酯(TG)和游离脂肪酸(FFA)水平，结果如图6所示。
结果显示，在血清甘油三酯水平方面，PGLP-1和P15组明显降低血中的甘油三酯含量，与模型组比较，p≤0.001，存在极明显差异；在血清游离脂肪酸水平方面，PGLP-1和P15组明显降低血中的甘油三酯含量，与模型组比较，p≤0.01，存在显著性差异。
结果说明，PGLP-1和P15处理后能够明显降低STZ糖尿病模型小鼠的甘油三酯和游离脂肪酸水平。
(5)对NOD小鼠发病率和死亡率的影响
采用NOD小鼠，从第5周开始，检测空腹血糖(空腹6h)，并给药(分对照组和实验组PGLP-1组、VP组、P15组)，期间间隔2周检测血糖，血糖值连续两次≥11mmol/L认为发病。
结果显示，对照组小鼠从15周开始发病，到39周时，发病率为66.67％，而实验组PGLP-1组、VP组、P15组分别在17周、21周、17周发病，说明VP组能够延缓发病时间。等观察期结束，实验组PGLP-1组、VP组、P15组的发病 率分别为33.33％、40％、15.39％，说明在后期，VP组抑制发病的作用开始减少，与对照组比较，实验组P15组的发病率显著降低，p<0.05，实验组PGLP-1组、VP组虽然降低发病率，但是无显著性差异(图7A)。
39周观察期结束，分别记录小鼠死亡日期，并作图，结果显示，对照组小鼠，21周开始死亡，到39周时，死亡7只，死亡率为58.44％，而实验组PGLP-1组、VP组、P15组的死亡率分别为33.33％、30％、15.39％，与对照组比较，实验组P15组的死亡率率显著降低，p<0.05，实验组PGLP-1组、VP组虽然降低发病率，但是无显著性差异(图7B)。
结果说明本发明多肽P15能够显著降低NOD小鼠的发病率与死亡率。
(6)对NOD小鼠空腹血糖的影响
采用NOD小鼠，从第5周开始，检测空腹血糖(空腹6h)，并给药(分对照组和实验组PGLP-1组、VP组、P15组)，期间间隔2周检测血糖。定期测定每组每只小鼠的血糖经过24周的治疗，继续观察10周。
P15组未发生糖尿病，然而，空白对照组、PGLP-1或VP组都相继有糖尿病的发生(图8A)。这些数据表明，P15能够预防糖尿病的再次发生。另外，P15组血糖值从29周开始维持在较低水平(停止给药)，第39周，P15组血糖值明显低于对照组和VP组(排除糖尿病小鼠)(p<0.05)(图8B)。
(7)对NOD小鼠胰腺重量和胰腺炎的影响
作为(6)的延续，39周观察期结束后，处死小鼠，取小鼠胰腺，称胰腺重量，并分析各组之间的差异。
结果显示，P15能够显著增加胰腺重量，与对照组相比，p<0.05，其他组均有升高，但是差异性不显著(图9A)。将胰腺进行HE染色，分析小鼠胰腺炎的程度，结果如图所示，对照组全部伴随胰腺炎的发生，而实验组PGLP-1、VP、P15组分别有1只、1只、3只无胰腺炎(图9B)。结果说明，P15肽能够抑制胰腺炎的发生。
(8)对NOD小鼠免疫调节因子Th1/Th2的影响
作为(6)的延续，39周观察期结束后，处死小鼠，取脾脏，经过抗体染色，流式分析Th1和Th2的比例，以及Th2/Th1的比值。
结果显示，实验组VP组、P15组能够显著增加Th2的比例，与对照组比 较，p<0.05，PGLP-1组有轻微升高，但是差异不显著(图10A和C)；实验组PGLP-1组、P15组、VP组对Th1无影响，各组之间无显著性差异(图10A和B)；实验组VP组、P15组的Treg/Th17的比值显著升高，p<0.05，PGLP-1组有轻微升高，但是差异不显著(图10A和D)。结果说明，P15肽能通过提高Th2的比例，以及Th2/Th1的比值来调节小鼠体内免疫平衡。
综合来看，本发明P15肽能够有效地发挥糖依赖性促胰岛素分泌作用(IPTGG)；降低STZ糖尿病模型小鼠的血糖，抑制STZ糖尿病模型小鼠的进食；降低NOD小鼠的发病率和死亡率，增加胰腺重量，抑制胰腺炎；调节小鼠体内免疫平衡。
Figure PCTCN2017094009-appb-000002

Claims (5)

  1. 一种长效降糖调脂多肽，其特征是，所述多肽的氨基酸序列为：HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGPGVLGGGCRCIPALDSLTPANED。
  2. 权利要求1述及长效降糖调脂多肽用于制备治疗或预防1型糖尿病药物或药物组合物的用途。
  3. 权利要求1述及长效降糖调脂多肽用于制备治疗或预防2型糖尿病药物或药物组合物的用途。
  4. 权利要求1述及长效降糖调脂多肽用于制备免疫调节药物或药物组合物的用途。
  5. 根据权利要求2或3或4述及的用途，其特征是，所述药物组合物包括药物载体和/或药物活性物质。
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