WO2019144692A1 - 一种胰高血糖素保护剂及其应用 - Google Patents

一种胰高血糖素保护剂及其应用 Download PDF

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WO2019144692A1
WO2019144692A1 PCT/CN2018/117401 CN2018117401W WO2019144692A1 WO 2019144692 A1 WO2019144692 A1 WO 2019144692A1 CN 2018117401 W CN2018117401 W CN 2018117401W WO 2019144692 A1 WO2019144692 A1 WO 2019144692A1
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glucagon
sample
protectant
present application
blood
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PCT/CN2018/117401
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English (en)
French (fr)
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蔺涛
李民友
董超
张玲
罗宁
齐宗献
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广州市进德生物科技有限公司
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Priority to JP2020538994A priority Critical patent/JP7158063B2/ja
Publication of WO2019144692A1 publication Critical patent/WO2019144692A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

Definitions

  • the present application relates to the field of polypeptide preservation, and in particular to a glucagon protective agent and application thereof.
  • Glucagon also known as glycemic glycoside, is a hormone secreted by pancreatic islet alpha cells, consisting of 29 amino acids, a linear polypeptide with a molecular weight of 3,485.
  • Diabetes is a group of metabolic abnormalities characterized by elevated levels of chronic blood sugar. Insulin and glucagon are important for maintaining proper blood sugar levels. Insulin acts on the liver and peripheral tissues by increasing glucose peripheral intake and reducing glucose output from the liver to lower blood sugar levels. At the same time, glucagon binds to glucagon receptors on hepatocytes, causing hepatic glycogen Decomposes to release glucose stored in glycogen form, helping to maintain glucose levels in the blood. As these stores are depleted, glucagon stimulates the liver to synthesize additional glucose through gluconeogenesis, which is released into the bloodstream, preventing hypoglycemia.
  • Glucagon plays an important role in clinical practice. Studies have confirmed that glucagon can promote the biosynthesis of cyclic adenosine monophosphate (c-AMP) in hepatocytes, evaluate liver reserve function in patients with cirrhosis, and promote glycogenolysis and Glucose, increase blood sugar, treat hypoglycemia; inhibit gastrointestinal motility, use in gastrointestinal angiography and endoscopic cholangiopancreatography; activate adenylate cyclase, enhance myocardial contractility, treat refractory heart failure; To evaluate the clinical significance of islet ⁇ -cell function in diabetic patients.
  • c-AMP cyclic adenosine monophosphate
  • glucagon is extremely unstable in blood samples and has a great impact on testing. How to maintain the stability of glucagon in clinical samples has become an important factor in the quantitative detection of glucagon. It has been reported that aprotinin is added to the anticoagulant sample to protect the stability of glucagon. The single aprotinin solution itself is unstable and inconvenient to use. At the same time, the sample of aprotinin is added to the pancreatic hyperglycemia. It is not stable enough to meet the needs of clinical testing.
  • the purpose of the present application is to provide a glucagon protectant which is mainly used for the protection of glucagon in blood samples.
  • the protective agent has stable performance and has a significant effect on improving the stability of glucagon.
  • a glucagon protecting agent comprising the following components, the content of each component being:
  • the antioxidant is one or more of curcumin, glutathione, and L-methionine.
  • the serine protease inhibitor is one or more of aprotinin and trypsin.
  • the anticoagulant is one or more of ethylenediaminetetraacetate and citrate;
  • the surfactant is one or more of polysorbate fatty acid glyceride and fatty acid sorbitan
  • the DPP-IV inhibitor is one or more of sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, and tregliel;
  • the buffer is a glycine-sodium hydroxide buffer;
  • the biological preservative is one or more of ProClin300, KY-100.
  • the glucagon protective agent is used in an amount of 1.5 to 2.5% of the volume of the blood sample.
  • glucagon protectant for increasing glucagon stability in a blood sample.
  • the anticoagulant in the glucagon protective agent of the present application is one or more of ethylenediaminetetraacetate and citrate; the ethylenediaminetetraacetate may be disodium, dipotassium and tris Potassium salt.
  • Anticoagulants can effectively sequester calcium ions in blood samples and prevent blood coagulation; while EDTA salts can inhibit the activity of metalloproteinases and reduce the hydrolysis of glucagon by metalloproteinases.
  • the anticoagulant is contained in an amount of 10 to 100 g/L; preferably, 20 to 100 g/L.
  • the antioxidant is one or more of curcumin, glutathione, and L-methionine; preferably, it is a combination of curcumin, glutathione, and L-methionine.
  • Glucagon is a linear polypeptide formed by 29 amino acids, including amino acids such as methionine and arginine which are easily oxidized. These amino acids are sensitive to oxygen free radicals and are easily oxidized, leading to structural changes in glucagon and promoting amyloidization and fibrosis of glucagon, causing aggregation of glucagon.
  • Curcumin is an acidic polyphenolic substance extracted from the rhizome of the ginger family, such as turmeric, and has an antioxidant effect. Studies have shown that curcumin can directly remove free radicals from blood samples in vitro and in vivo. Curcumin can inhibit the production of free radicals by increasing the level of intracellular reduced glutathione. Moreover, curcumin can also scavenge free radicals involved in peroxidation, inhibit lipid peroxidation, and maintain various antioxidant enzymes. Activity. Curcumin also promotes the breakdown of amyloid and prevents the formation of amyloid.
  • Glutathione is a naturally occurring tripeptide in human cells composed of glutamic acid, cysteine and glycine.
  • the reduced glutathione can reduce the oxidation of the oxidizing agent by combining with peroxides and free radicals, and can also inhibit the oxidation of free radicals by combining curcumin.
  • L-methionine is a sulfur-containing amino acid that is reductive. L-methionine protects methionine in glucagon and maintains glucagon structure stability.
  • the addition of 2.5-150 g/L of antioxidant to the protective agent can well maintain the stability of the glucagon structure in the blood sample; preferably, the antioxidant is used in an amount of 30-130 g/L. Still more preferably, the controlled amount is: curcumin 10 to 30 g/L; L-methionine 10 to 50 g/L; and glutathione 10 to 50 g/L.
  • Glucagon is a linear polypeptide structure, and amino acids are exposed, which is easily decomposed by proteases and causes structural changes. In order to maintain the stability of the glucagon structure, it is necessary to inhibit proteases that may cause structural changes and inactivate these proteases. Therefore, it is necessary to add a certain amount of serine protease inhibitor and DPP-IV inhibitor to the protective agent.
  • the serine protease inhibitor is one or more of aprotinin and trypsin; preferably, it is an aprotinin.
  • Aprotinin is a single-chain polypeptide extracted from bovine lung. It is a broad-spectrum protease inhibitor and has inhibitory effects on various kininogens, trypsin, chymotrypsin, plasmin and pepsin. Aprotinin forms a reversible inhibitor-enzyme complex with a inhibited enzyme at a stoichiometric ratio, inhibiting and reducing the activity of the enzyme.
  • DPP-IV is an important serine hydrolase distributed in mammals. It is involved in various biologically active peptides (such as glucagon, incretin, neuropeptide, gastrin releasing peptide, growth hormone releasing hormone, etc.). Hydrolysis, in turn, results in partial or complete inactivation. DPP-IV inhibitors can specifically inhibit the enzymatic activity of DPP-IV and reduce its decomposition of glucagon. In the present application, the DPP-IV inhibitor is one or more of sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, and telliptin.
  • the content of the serine protease inhibitor is 1 to 100 g/L, preferably 1 to 50 g/L, and still more preferably 1 to 20 g/L.
  • the DPP-IV inhibitor is added in an amount of 0.02 to 4 mmol/L, preferably 0.02 to 2 mmol/L. It was found through experiments that the addition of the above content of serine protease inhibitor and DPP-IV inhibitor can synergistically inhibit the degradation and aggregation of glucagon.
  • the surfactant is one or more of polysorbate (Tween) fatty acid glyceride and fatty acid sorbitan (Span); preferably, Tween-80 is added.
  • Tween polysorbate
  • Span fatty acid sorbitan
  • the addition of 0.1 to 5 g/L of Tween-80 can increase the solubility of glucagon and curcumin; preferably, 0.1 to 3 g/L is added.
  • the protective agent of the present application uses a glycine-sodium hydroxide buffer.
  • Glycine-sodium hydroxide buffer can maintain a stable alkaline environment, inactivate the aspartic acid amino acid protease in the blood, prevent glucagon degradation, and glycine has a certain inhibitory effect on the oxidation of glucagon.
  • the content of the buffer is preferably 20 to 200 mmol/L, preferably 60 to 200 mmol/L.
  • the biological preservative is one or more of ProClin300 and KY-100; the biological preservative has broad-spectrum antibacterial activity, can inhibit the growth of microorganisms such as bacteria and fungi in a relatively long period of time, and can maintain the activity of the enzyme in the system. .
  • the amount added is from 0.1 to 10 ml/L, preferably from 0.1 to 3 ml/L.
  • the glucagon protective agent of the present application is used for improving the stability of glucagon in a blood sample, and the dosage is 1.5 to 2.5% of the volume of the blood sample, which is much lower than the addition amount of 5 to 10% of the general liquid additive.
  • the advantage is to reduce the change of the sample volume and reduce the impact on the detection results; in addition, the cost can be reduced, and the cost of the protective agent added per mL of blood sample is only about 0.2 to 0.5 yuan.
  • This application has a significant effect on improving the stability of glucagon in blood samples.
  • the sample protection agent of the present application has stable performance and is convenient for storage and use.
  • composition of the application is simple, the material is cheap and easy to obtain; the preparation process is simple, and it is suitable for industrial production.
  • the application provides a glucagon protective agent, comprising the following components, the content of each component is: anticoagulant 10 ⁇ 100g / L; antioxidant 2.5 ⁇ 150g / L; serine protease inhibitor 1 ⁇ 100g / L Surfactant 0.1 ⁇ 5g / L; DPP-IV inhibitor 0.02 ⁇ 4mmol / L; buffer 20 ⁇ 200mmol / L; biological preservative 0.1 ⁇ 10ml / L.
  • the content of each component is: anticoagulant 20 ⁇ 100g / L; antioxidant 30 ⁇ 130g / L; serine protease inhibitor 1 ⁇ 50g / L; surfactant 0.1 ⁇ 3g / L; DPP-IV inhibition Agent 0.02 ⁇ 2mmol / L; buffer 60 ⁇ 200mmol / L; biological preservative 0.1 ⁇ 3ml / L.
  • the anticoagulant is one or more of ethylenediaminetetraacetate and citrate; and the antioxidant is one or more of curcumin, glutathione, and L-methionine;
  • the serine protease inhibitor is one or more of aprotinin and trypsin;
  • the surfactant is one or more of polysorbate fatty acid glyceride, fatty acid sorbitan;
  • DPP-IV inhibitor is sigrid One or more of statins, vildagliptin, saxagliptin, alogliptin, linagliptin, and treglieline;
  • the biological preservative is one or more of ProClin300, KY-100.
  • each component is: ethylenediaminetetraacetate 20-100 g/L; curcumin 10-30 g/L; L-methionine 10-50 g/L; glutathione 10-50 g /L; aprotinin 1 ⁇ 20g / L; surfactant 0.1 ⁇ 3g / L; DPP-IV inhibitor 0.02 ⁇ 2mmol / L; glycine - sodium hydroxide buffer 60 ⁇ 200mmol / L; ProClin300 0.1 ⁇ 3ml / L.
  • aprotinin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
  • curcumin was purchased from Sigma
  • DPP-IV inhibitor Telilide
  • Table 1 shows the specific formulation compositions of some of the examples. It should be noted that the material formulation of the protective agent of the present application is not limited to the data in Table 1.
  • the main instrument enzyme standard analyzer, model RT-6000, produced by Shenzhen Leidu Life Science Co., Ltd.
  • Glucagon test kit (Cat. No. 10-1271-01), produced by Mercodia, Sweden.
  • the disposable human venous blood sample collection container (EDTA2K) is produced by Liuyang Sanli Medical Technology Development Co., Ltd.
  • Glucagon protectant Example 1-7 glucagon protectant.
  • the blood sample was obtained from a cynomolgus monkey and was provided by the South China Primate Research Center.
  • the cynomolgus monkeys were fasted for more than 12 hours, and blood was collected using a disposable human venous blood sample collection container. The same cynomolgus monkey took 7 tubes of blood, 2 mL per tube. Immediately after blood collection, 40 ⁇ L of the glucagon protective agent of Examples 1-7 was added, mixed, centrifuged, and the supernatant plasma was taken and dispensed to measure the stability of glucagon at 2-8 °C.
  • Example 3 Example 6 and Example 7 have the strongest protective effect.
  • the main instrument enzyme standard analyzer, model RT-6000, produced by Shenzhen Leidu Life Science Co., Ltd.
  • Glucagon test kit (Cat. No. 10-1271-01), produced by Mercodia, Sweden.
  • the disposable human venous blood sample collection container (EDTA2K) is produced by Liuyang Sanli Medical Technology Development Co., Ltd.
  • Glucagon protective agent provided by Guangzhou Jinde Biotechnology Co., Ltd.
  • the blood sample was obtained from a cynomolgus monkey and was provided by the South China Primate Research Center.
  • the cynomolgus monkeys were fasted for more than 12 hours, and blood was collected using a disposable human venous blood sample collection container.
  • the cynomolgus monkeys were numbered 1-6, and each cynomolgus monkey took 5 tubes of blood, 2 mL per tube.
  • 40 ⁇ L of 0.9% sodium chloride solution control group 1 was added, and immediately after the second tube was collected, 40 ⁇ L of aprotinin solution (control group 2) was added, and the third to fifth tubes were respectively added with pancreatic hyperglycemia after blood collection.
  • 40 ⁇ L of the protective agent (experimental group, respectively, Example 3, Example 6 and Example 7), after mixing, centrifuged, and the supernatant plasma was taken and dispensed for testing.
  • the sample is tested according to the following storage conditions:
  • Example 6 The sample of Example 6 was tested after being placed at 2 to 8 ° C for 24, 48, 72, 96 and 120 hours.
  • Table 3 shows the preservation of the control group 1, the control group 2, and the experimental group immediately after centrifugation, and the unit is pmol/L.
  • control group 2 and the experimental group were relatively close, indicating that the addition of aprotinin and the glucagon protecting agent of the present application were equivalent to the protective effect of glucagon in the blood sample.
  • Table 4 shows the preservation of the control group 1, the control group 2, and the experimental group samples at 37 ° C for 4 hours, and the unit was pmol/L.
  • Table 5 shows the storage conditions of the control group 1, the control group 2, and the experimental group samples after standing at 2 to 8 ° C for 24 hours, and the unit was pmol/L.
  • the protective agent of Example 6 was used as an experimental group to observe the protection after standing at 2 to 8 ° C for 24, 48, 72, 96 and 120 hours, and the unit was pmol/L.
  • the main instrument enzyme standard analyzer, model RT-6000, produced by Shenzhen Leidu Life Science Co., Ltd.
  • Glucagon test kit (Cat. No. 10-1271-01), produced by Mercodia, Sweden.
  • the disposable human venous blood sample collection container (EDTA2K) is produced by Liuyang Sanli Medical Technology Development Co., Ltd.
  • Glucagon protective agent provided by Guangzhou Jinde Biotechnology Co., Ltd.
  • Sample source Healthy volunteers.
  • the sample is tested according to the following storage conditions:
  • Example 6 The sample of Example 6 was tested after being placed at 2 to 8 ° C for 24, 48, 72, 96 and 120 hours.
  • Table 7 shows the preservation of the control group 1, the control group 2, and the experimental group immediately after centrifugation, and the unit was pmol/L.
  • control group 1 was significantly lower than that of the control group 2 and the experimental group, indicating that no aprotinin was added during the treatment of the clinical blood sample, and the glucagon in the sample was largely degraded.
  • control group 2 and the experimental group were relatively close, indicating that the addition of aprotinin and the glucagon protective agent of the present application have comparable protective effects on glucagon in the blood sample.
  • Table 8 shows the preservation of the control group 1, the control group 2, and the experimental group samples at 37 ° C for 4 hours, and the unit was pmol/L.
  • Table 9 shows the storage conditions of the control group 1, the control group 2, and the experimental group samples after standing at 2 to 8 ° C for 24 hours, and the unit was pmol/L.
  • the glucagon protective agent of the present application has a greater protective effect on glucagon in the sample than in the control group 2 with aprotinin at 2-8 °C.
  • the sample of the experimental group (Example 6) was taken, and the glucagon stable type was observed after being placed at 2 to 8 ° C for 24, 48, 72, 96 and 120 hours, and the unit was pmol/L.
  • the glucagon protective agent provided by the present application has stable performance, is convenient for storage and use, and has obvious effects on improving the stability of glucagon; and the preparation process of the protective agent is simple, the composition is simple, and the material is easy to obtain. ,cheap price.
  • the glucagon protective agent of the present application has a significant effect on improving the stability of glucagon in a blood sample; and the sample protective agent has stable performance and is convenient for storage and use; the protective agent has simple composition, and the material is cheap and easy to obtain; Simple, suitable for industrial production.

Abstract

本文公开了一种胰高血糖素保护剂，包括抗凝剂10～100g/L；抗氧化剂2.5～150g/L；丝氨酸蛋白酶抑制剂1～100g/L；表面活性剂0.1～5g/L；DPP-IV抑制剂0.02～4mmol/L；缓冲液20～200mmol/L；生物防腐剂0.1～10ml/L。该保护剂主要用于血液样本中胰高血糖素的保护，自身性能稳定，同时对提高胰高血糖素的稳定性有明显作用。

Description

一种胰高血糖素保护剂及其应用
本申请要求在2018年01月24日提交中国专利局、申请号为201810066819.6、发明名称为“一种胰高血糖素保护剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及多肽保存领域，具体涉及一种胰高血糖素保护剂及其应用。
背景技术
胰高血糖素，也称升糖素，是一种由胰脏胰岛α细胞分泌的激素，由29个氨基酸组成直链多肽，分子量为3485。
糖尿病是一组以慢性血糖水平增高为特征的代谢异常综合征，胰岛素和胰高血糖素对于维持适当的血糖水平很重要。胰岛素通过增加葡萄糖外周摄取和减少自肝脏输出的葡萄糖以降低血糖水平而作用肝脏和外周组织，与此同时，胰高血糖素通过与肝细胞上的胰高血糖素受体结合，引起肝糖原分解而释放以糖原形式贮藏的葡萄糖，从而帮助维持血液中的葡萄糖水平。随着这些贮藏被耗尽，胰高血糖素刺激肝脏通过糖异生合成额外的葡萄糖，该葡萄糖释放到血流中，阻止低血糖的发生。
胰高血糖素在临床上具有重要作用，研究已证实胰高血糖素能够促进肝细胞内环磷腺苷(c-AMP)的生物合成，评价肝硬化患者的肝脏储备功能；促进糖原分解和糖异生，升高血糖，治疗低血糖；抑制胃肠蠕动，用于胃肠造影及内镜下胰胆管造影；激活腺苷酸环化酶，增强心肌收缩力，治疗难治性心衰；评价糖尿病患者胰岛β细胞功能等临床意义。
近年来，多个制药公司致力于胰高血糖素类似物的研制，有的在进行临床试验，有的已经完成了临床试验进入市场，表明胰高血糖素在不久的将来必定成为糖尿病、心血管等疾病的常用治疗药物之一。
准确地监测胰高血糖素含量变化对于评价胰岛β细胞、血糖控制及相关代谢疾病的治疗是必不可少的前提。但是，胰高血糖素在血液样本中极不稳定，给检测带来了很大影响。如何保持胰高血糖素在临床样本中的稳定性，成为胰高血糖素定量检测过程中的重要因素。有报道在抗凝血液样本中添加抑肽酶来保护胰高血糖素的稳定性，单一的抑肽酶溶液本身不稳定，使用不方便；同时经添加抑肽酶的样本，其中的胰高血糖素不太稳定，难以满足临床检测的需求。
发明内容
本申请的目的是提供一种胰高血糖素保护剂，主要应用于血液样本中胰高血糖素的保护。该保护剂性能稳定，同时对提高胰高血糖素的稳定性有明显作用。
根据本申请的一个方面，提供了一种胰高血糖素保护剂，包括以下组分，所述各组分的含量为：
Figure PCTCN2018117401-appb-000001
其中，包括以下组分，所述各组分的含量为：
Figure PCTCN2018117401-appb-000002
其中，所述抗氧化剂为姜黄素、谷胱甘肽、L-甲硫氨酸中的一种或多种。
其中，所述丝氨酸蛋白酶抑制剂为抑肽酶、胰蛋白酶中的一种或多种。
其中，所述抗凝剂为乙二胺四乙酸盐、柠檬酸盐中的一种或多种；所述表面活性剂为聚山梨酯脂肪酸甘油酯，脂肪酸山梨坦中的一种或多种；所述DPP-IV抑制剂为西格列汀、维格列汀、沙格列汀、阿格列汀、利格列汀、特力利汀中的一种或多种；所述缓冲液为甘氨酸-氢氧化钠缓冲液；所述生物防腐剂为ProClin300、KY-100中的一种或多种。
其中，包括以下组分，所述各组分的含量为：
Figure PCTCN2018117401-appb-000003
Figure PCTCN2018117401-appb-000004
其中，包括以下组分，所述各组分的含量为：
Figure PCTCN2018117401-appb-000005
其中，包括以下组分，所述各组分的含量为：
Figure PCTCN2018117401-appb-000006
其中，所述胰高血糖素保护剂的用量为血液样本体积的1.5～2.5％。
根据本申请的另一方面提供的胰高血糖素保护剂在提高血液样本胰高血糖素稳定性上的应用。
本申请的胰高血糖素保护剂中的抗凝剂为乙二胺四乙酸盐、柠檬酸盐中的一种或多种；乙二胺四乙酸盐可以为二钠、二钾和三钾盐。抗凝剂可以有效地螯合血液标本中钙离子，阻 止血液凝固；而EDTA盐可以抑制金属蛋白酶的活性，降低金属蛋白酶对胰高血糖素的水解作用。在本申请保护剂中，抗凝剂的含量为10-100g/L；优选的，为20～100g/L。
抗氧化剂为姜黄素、谷胱甘肽、L-甲硫氨酸中的一种或多种；优选的，为姜黄素、谷胱甘肽、L-甲硫氨酸的组合物。胰高血糖素是由29个氨基酸形成的直链多肽，其中包括甲硫氨酸、精氨酸等容易被氧化的氨基酸。这些氨基酸对氧自由基比较敏感，容易被氧化，会导致胰高血糖素的结构改变，并促进胰高血糖素的淀粉样化和纤维化，引起胰高血糖素的聚集。
姜黄素是从姜科植物姜黄等的根茎中提取的一种酸性多酚类物质，具有抗氧化作用。研究证明，姜黄素在体内外，可以直接清除血液样本中的自由基。姜黄素能够通过增加细胞内还原型谷胱甘肽水平，来抑制自由基的产生，而且，姜黄素还可以清除参与过氧化反应的自由基，抑制脂质过氧化反应，维持各种抗氧化酶的活性。姜黄素还可以促进淀粉样蛋白的分解，并防止淀粉样蛋白的形成。
谷胱甘肽是人类细胞中自然合成的一种三肽，由谷氨酸、半胱氨酸和甘氨酸组成。其中的还原型谷胱甘肽，可以通过与过氧化物及自由基结合，减少氧化剂的氧化作用，同时可以配合姜黄素，抑制自由基的氧化作用。
L-甲硫氨酸是一种含硫氨基酸，有还原性。L-甲硫氨酸可以对胰高血糖素中的甲硫氨酸起到保护作用，维持胰高血糖素结构的稳定。
通过实验验证，保护剂中添加2.5-150g/L的抗氧化剂，可以很好地保持血液样本中胰高血糖素结构的稳定；优选的，抗氧化剂用量为30～130g/L。更进一步优选的，控制用量：姜黄素10～30g/L；L-甲硫氨酸10～50g/L；谷胱甘肽10～50g/L。
胰高血糖素为直链多肽结构，氨基酸暴露在外，非常容易被蛋白酶分解而导致结构的改变。为了维持胰高血糖素结构的稳定，就需要对可能造成结构改变的蛋白酶进行抑制，使这些蛋白酶失活。所以需要在保护剂中添加一定含量的丝氨酸蛋白酶抑制剂和DPP-IV抑制剂。
丝氨酸蛋白酶抑制剂为抑肽酶、胰蛋白酶中的一种或多种；优选的，为抑肽酶。抑肽酶是由牛肺提取的单链多肽，为广谱蛋白酶抑制剂，对各种激肽酶原、胰蛋白酶、糜蛋白酶、纤溶酶、胃蛋白酶等均有抑制作用。抑肽酶按一定化学比例与被抑制的酶形成可逆的抑制物-酶复合物，抑制和降低酶的活性。
DPP-IV是哺乳动物体内分布的一种重要的丝氨酸水解酶，其参与体内多种生物活性多肽(如胰高血糖素、肠促胰岛素、神经肽、胃泌素释放肽、生长激素释放激素等)的水解进而导致其部分或完全失活。DPP-IV抑制剂可以特异性的抑制DPP-IV的酶活性，降低其对胰高血糖素的分解作用。本申请中，DPP-IV抑制剂为西格列汀、维格列汀、沙格列汀、阿格列汀、利格列汀、特力利汀的一种或多种。
丝氨酸蛋白酶抑制剂的含量为1～100g/L，优选的1～50g/L，更进一步优选的1～20g/L。DPP-IV抑制剂的添加量为0.02～4mmol/L，优选的0.02～2mmol/L。通过实验发现，添加以上含量的丝氨酸蛋白酶抑制剂和DPP-IV抑制剂，两者协同作用，可以很好地抑制胰高血糖素的降解和聚集。
表面活性剂为聚山梨酯(吐温)脂肪酸甘油酯，脂肪酸山梨坦(司盘)中的一种或多种；优选的，添加吐温-80。添加0.1～5g/L的吐温-80可以增加胰高血糖素和姜黄素的溶解性；优选的，添加0.1～3g/L。
本申请的保护剂使用的是甘氨酸-氢氧化钠缓冲液。甘氨酸-氢氧化钠缓冲液可以维持稳定的碱性环境，使血液中的天门冬氨基酸蛋白酶失活，防止胰高血糖素降解，甘氨酸对胰高血糖素的氧化有一定的抑制作用。缓冲液的含量以20～200mmol/L为宜，优选的，60～200mmol/L。
生物防腐剂为ProClin300、KY-100中的一种或多种；生物防腐剂具有广谱抗菌活性，能在比较长的时间内抑制细菌、真菌等微生物的生长，同时可以保持体系中酶的活性。添加量在0.1～10ml/L，优选0.1～3ml/L。
本申请的胰高血糖素保护剂应用在提高血液样本胰高血糖素稳定性上，用量为血液样本体积的1.5～2.5％，远低于一般液体添加剂5～10％的添加量。其优势是减少对样本体积的改变，降低对检测结果的影响；另外可以降低成本，每mL血液样本添加的保护剂费用仅在0.2～0.5元左右。
与已有技术相比，本申请的胰高血糖素保护剂的有益效果主要体现在：
1、本申请对提高血液样本中胰高血糖素的稳定性有明显作用。
2、本申请的样本保护剂性能稳定，便于贮存和使用。
3、本申请成份简单，物料便宜、易得；制备工艺简单，适合工业化生产。
具体实施方式
下面将结合实施例，对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互任意组合。
本申请提供一种胰高血糖素保护剂，包括以下组分，各组分的含量为：抗凝剂10～100g/L；抗氧化剂2.5～150g/L；丝氨酸蛋白酶抑制剂1～100g/L；表面活性剂0.1～5g/L；DPP-IV抑制剂0.02～4mmol/L；缓冲液20～200mmol/L；生物防腐剂0.1～10ml/L。
优选的，各组分的含量为：抗凝剂20～100g/L；抗氧化剂30～130g/L；丝氨酸蛋白酶抑制剂1～50g/L；表面活性剂0.1～3g/L；DPP-IV抑制剂0.02～2mmol/L；缓冲液60～200mmol/L；生物防腐剂0.1～3ml/L。
其中，抗凝剂为乙二胺四乙酸盐、柠檬酸盐中的一种或多种；抗氧化剂为姜黄素、谷胱甘肽、L-甲硫氨酸中的一种或多种；丝氨酸蛋白酶抑制剂为抑肽酶、胰蛋白酶中的一种或多种；表面活性剂为聚山梨酯脂肪酸甘油酯，脂肪酸山梨坦中的一种或多种；DPP-IV抑制剂为西格列汀、维格列汀、沙格列汀、阿格列汀、利格列汀、特力利汀中的一种或多种；生物防腐剂为ProClin300、KY-100中的一种或多种。
具体的，各组分的含量为：乙二胺四乙酸盐20～100g/L；姜黄素10～30g/L；L-甲硫氨酸10～50g/L；谷胱甘肽10～50g/L；抑肽酶1～20g/L；表面活性剂0.1～3g/L；DPP-IV抑制剂0.02～2mmol/L；甘氨酸-氢氧化钠缓冲液60～200mmol/L；ProClin300 0.1～3ml/L。
实施例
保护剂制备过程：
1、配制100mL浓度为20～200mmol/L的甘氨酸-氢氧化钠缓冲液；
2、分别称取各自预定含量的姜黄素、乙二胺四乙酸二钾、L-甲硫氨酸、谷胱甘肽、吐温-80，并依次溶于甘氨酸-氢氧化钠缓冲液；量取0.01～1ml ProClin300，溶于上述溶液；混匀、过滤，放置2～8℃备用。
3、取10ml上述溶液，精确称取预定含量的抑肽酶，混匀溶解；量取2～400μl，浓度为0.1mmol/mL的DPP-IV抑制剂(特力利汀)，加入上述溶液，混匀，2～8℃保存。
上述物料均为市售品；其中，抑肽酶购于上海阿拉丁生化科技股份有限公司，姜黄素购于Sigma公司，DPP-IV抑制剂(特力利汀)购于MedChemexpress公司。
表1示出了一些实施例的具体配方组成。需要指出的是，本申请的保护剂的物料配方并不局限于表1中数据。
表1胰高血糖素保护剂实施例
Figure PCTCN2018117401-appb-000007
Figure PCTCN2018117401-appb-000008
测试例
下面给出实施例1-7胰高血糖素保护剂的保存效果检测结果。
1、仪器、试剂和样本
主要仪器：酶标分析仪，型号RT-6000，深圳雷杜生命科学股份有限公司生产。
主要试剂：胰高血糖素检测试剂盒(货号10-1271-01)，由瑞典Mercodia公司生产。
一次性人体静脉血样采集容器(EDTA2K)，由浏阳市三力医用科技发展有限公司生产。
胰高血糖素保护剂：实施例1-7胰高血糖素保护剂。
样本：血液样本来源于食蟹猴，由华南灵长类研究中心提供。
2、实验方案：
食蟹猴禁食12小时以上，使用一次性人体静脉血样采集容器采血。同一食蟹猴采血7管，每管2mL。采血后立即加入40μL实施例1-7的胰高血糖素保护剂，混匀，离心，取上清血浆，分装，检测2～8℃下胰高血糖素稳定性。
表2实施例1-7检测结果(单位：pmol/L)
Figure PCTCN2018117401-appb-000009
从表2可以看出，样本在2～8℃下，随着时间的增长，样本中的胰高血糖素检测结果没有明显变化，说明本申请的胰高血糖素保护剂对样本中胰高血糖素的保护作用非常强。其中，
实施例3、实施例6以及实施例7保护作用最强。
对比例
对比例1
1、仪器、试剂和样本
主要仪器：酶标分析仪，型号RT-6000，深圳雷杜生命科学股份有限公司生产。
主要试剂：胰高血糖素检测试剂盒(货号10-1271-01)，由瑞典Mercodia公司生产。
一次性人体静脉血样采集容器(EDTA2K)，由浏阳市三力医用科技发展有限公司生产。
胰高血糖素保护剂，由广州市进德生物科技有限公司提供。
样本：血液样本来源于食蟹猴，由华南灵长类研究中心提供。
2、实验方案：
食蟹猴禁食12小时以上，使用一次性人体静脉血样采集容器采血。将食蟹猴编号1-6，每只食蟹猴采血5管，每管2mL。其中第1管采血后加入0.9％氯化钠溶液40μL(对照组1)，第2管采血后立即加入抑肽酶溶液40μL(对照组2)，第3至5管采血后分别加入胰高血糖素保护剂40μL(实验组，分别为实施例3、实施例6和实施例7)，混匀后，离心，取上清血浆，分装，供检测使用。
样本按下面保存条件检测：
(1)离心后立即检测，
(2)37℃放置4小时后检测，
(3)2～8℃放置24小时后检测
(4)实施例6样本在2～8℃放置24、48、72、96和120小时后检测。
样本检测，按照胰高血糖素检测试剂盒(货号10-1271-01)说明书执行。
3、实验结果：
表3为对照组1、对照组2以及实验组样本离心后立即检测的保存情况，单位：pmol/L。
表3样本检测结果
Figure PCTCN2018117401-appb-000010
Figure PCTCN2018117401-appb-000011
从表3可以看出，对照组1的检测值明显低于对照组2和实验组；表明血液样本处理过程中，不添加抑肽酶，样本中胰高血糖素会有较大程度降解。
对照组2和实验组的检测值比较接近，表明添加抑肽酶和本申请的胰高血糖素保护剂对血液样本中胰高血糖素保护作用相当。
表4为对照组1、对照组2以及实验组样本37℃放置4小时的保存情况，单位：pmol/L。
表4样本检测结果
Figure PCTCN2018117401-appb-000012
从表4可以看出，样本在37℃放置4小时后，对照组1的检测结果降低非常明显，接近检测低限。对照组2的检测结果也有大幅度的下降，实验组检测结果下降程度较小，说明本申请的胰高血糖素保护剂在37℃条件下，对样本中胰高血糖素的保护作用要明显大于添加抑肽酶组。
表5为对照组1、对照组2以及实验组样本2～8℃放置24小时后的保存情况，单位：pmol/L。
表5样本检测结果
Figure PCTCN2018117401-appb-000013
从表5可以看出，样本在2～8℃放置24小时后，对照组1的检测结果降低非常明显，对照组2的检测结果也有大幅度的下降，实验组检测结果没有明显变化，说明本申请的胰高血糖素保护剂在2～8℃条件下，对样本中胰高血糖素的保护作用要明显大于添加抑肽酶组。
取实施例6保护剂作为实验组观察2～8℃放置24、48、72、96和120小时后的保护情况，单位：pmol/L。
表6样本检测结果
Figure PCTCN2018117401-appb-000014
从表6可以看出，样本在2～8℃放置120小时，样本中的胰高血糖素检测结果没有明显变化，说明本申请的胰高血糖素保护剂在2～8℃条件下，对样本中胰高血糖素的保护作用非常强，更加满足临床样本检测的需求。
对比例2
1、仪器、试剂和样本
主要仪器：酶标分析仪，型号RT-6000，深圳雷杜生命科学股份有限公司生产。
主要试剂：胰高血糖素检测试剂盒(货号10-1271-01)，由瑞典Mercodia公司生产。
一次性人体静脉血样采集容器(EDTA2K)，由浏阳市三力医用科技发展有限公司生产。
胰高血糖素保护剂：由广州市进德生物科技有限公司提供。
样本来源：健康志愿者。
2、实验方案：
健康志愿者，男性，10人。空腹12小时以上，使用一次性人体静脉血样采集容器采血。编号1-10，每人采血3管，每管2mL。其中第1管采血后加入0.9％氯化钠溶液40μL(对照组1)，第2管采血后立即加入抑肽酶溶液40μL(对照组2)，第3采血后分别加入胰高血糖素保护剂40μL(实验组，实施例6)，混匀后，离心，取上清血浆，分装，供检测使用。
样本按下面保存条件检测：
(1)离心后立即检测，
(2)37℃放置4小时后检测，
(3)2～8℃放置24小时后检测
(4)实施例6样本在2～8℃放置24、48、72、96和120小时后检测。
样本检测，按照胰高血糖素检测试剂盒(货号10-1271-01)说明书执行。
3、实验结果：
表7为对照组1、对照组2以及实验组样本离心后立即检测的保存情况，单位：pmol/L。
表7样本检测结果
Figure PCTCN2018117401-appb-000015
从表7可以看出，对照组1的检测值明显低于对照组2和实验组，表明临床血液样本处理过程中，不添加抑肽酶，样本中胰高血糖素会有较大程度降解。
对照组2和实验组的检测值比较接近，表明添加抑肽酶和本申请的胰高血糖素保护剂对血液样本中胰高血糖素的保护作用相当。
表8为对照组1、对照组2以及实验组样本37℃放置4小时的保存情况，单位：pmol/L。
表8样本检测结果
Figure PCTCN2018117401-appb-000016
Figure PCTCN2018117401-appb-000017
从表8可以看出，样本在37℃放置4小时后，对照组1的检测结果降低非常明显，接近检测低限。对照组2的检测结果也有大幅度的下降，实验组检测结果下降程度较小，说明本申请的胰高血糖素保护剂在37℃条件下，对样本中胰高血糖素的保护作用要明显大于添加抑肽酶组。
表9为对照组1、对照组2以及实验组样本2～8℃放置24小时后的保存情况，单位：pmol/L。
表9样本检测结果
Figure PCTCN2018117401-appb-000018
从表9可以看出，样本在2～8℃放置24小时后，对照组1的检测结果降低非常明显，对照组2的检测结果也有一定程度的下降，实验组检测结果则没有明显变化，说明本申请的胰高血糖素保护剂在2～8℃条件下，对样本中胰高血糖素的保护作用要明显大于添加抑肽酶的对照组2。
取实验组(实施例6)样本，观察2～8℃放置24、48、72、96和120小时后的胰高血糖素稳定型，单位：pmol/L。
表10样本检测结果
Figure PCTCN2018117401-appb-000019
从表10可以看出，样本在2～8℃放置120小时，临床样本中的胰高血糖素检测结果没有明显变化，说明本申请的胰高血糖素保护剂在2～8℃条件下，对临床样本中胰高血糖素的保护作用非常强，更加满足临床样本检测的需求。
综上所述，本申请提供的胰高血糖素保护剂性能稳定，便于贮存和使用；对提高胰高血糖素的稳定性有明显作用；并且该保护剂制备工艺简单，成份简单，物料易得，价格便宜。
上面描述的内容可以单独地或者以各种方式组合起来实施，而这些变型方式都在本申请的保护范围之内。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制。尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
工业实用性
本申请的胰高血糖素保护剂对提高血液样本中胰高血糖素的稳定性有明显作用；且样本保护剂性能稳定，便于贮存和使用；保护剂成份简单，物料便宜、易得；制备工艺简单，适合工业化生产。

Claims (10)

  1. 一种胰高血糖素保护剂，其特征在于，包括以下组分，所述各组分的含量为：
    Figure PCTCN2018117401-appb-100001
  2. 如权利要求1所述的胰高血糖素保护剂，其特征在于，包括以下组分，所述各组分的含量为：
    Figure PCTCN2018117401-appb-100002
  3. 如权利要求1或2所述的胰高血糖素保护剂，其特征在于，所述抗氧化剂为姜黄素、谷胱甘肽、L-甲硫氨酸中的一种或多种。
  4. 如权利要求3所述的胰高血糖素保护剂，其特征在于，所述丝氨酸蛋白酶抑制剂为抑肽酶、胰蛋白酶中的一种或多种。
  5. 如权利要求4所述的胰高血糖素保护剂，其特征在于，
    所述抗凝剂为乙二胺四乙酸盐、柠檬酸盐中的一种或多种；
    所述表面活性剂为聚山梨酯脂肪酸甘油酯，脂肪酸山梨坦中的一种或多种；
    所述DPP-IV抑制剂为西格列汀、维格列汀、沙格列汀、阿格列汀、利格列汀、特力利汀中的一种或多种；
    所述缓冲液为甘氨酸-氢氧化钠缓冲液；
    所述生物防腐剂为ProClin300、KY-100中的一种或多种。
  6. 如权利要求5所述的胰高血糖素保护剂，其特征在于，包括以下组分，所述各组分的 含量为：
    Figure PCTCN2018117401-appb-100003
  7. 如权利要求6所述的胰高血糖素保护剂，其特征在于，包括以下组分，所述各组分的含量为：
    Figure PCTCN2018117401-appb-100004
  8. 如权利要求6所述的胰高血糖素保护剂，其特征在于，包括以下组分，所述各组分的含量为：
    Figure PCTCN2018117401-appb-100005
    Figure PCTCN2018117401-appb-100006
  9. 如权利要求1-8任一项所述的胰高血糖素保护剂，其特征在于，所述胰高血糖素保护剂的用量为血液样本体积的1.5～2.5％。
  10. 一种如权利要求1-8任一项所述的胰高血糖素保护剂在提高血液样本胰高血糖素稳定性上的应用。
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