Novel glucagon analogue and application thereof

A technology of glucagon and analogues, which is applied in the field of polypeptide drugs, can solve the problems of no clinical use value, etc., and achieve the effect of enhancing biological stability, good stability and long-term activity in vivo

Active Publication Date: 2019-05-17
清紫生物科技(深圳)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biologically active GLP-1 in the human body is mainly GLP-1(7-36) amide and GLP-1(7-37), but both are rapidly hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV) (half-life Less than 5min), no clinical value

Method used

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  • Novel glucagon analogue and application thereof
  • Novel glucagon analogue and application thereof
  • Novel glucagon analogue and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1. Newly discovered peptide SEQ.ID NO.1 (His-Ser-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ser-Tyr-Leu-Glu-Gly-Lys-Ala -Thr-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Leu-Glu) and SEQ.ID NO.2 (His-Ser-Glu-Gly-Thr-Phe-Thr-Ser -Asp-Phe-Ser-Ser-Tyr-Leu-Asp-Gly-Lys-Ala-Ala-Lys-Glu-Phe-Val-Ala-Trp-Leu-Val-Lys-Ser-Leu-Glu) can effectively stimulate GLP-1 receptor and can resist the hydrolysis of DPP4 in humans

[0147] In order to verify whether the sequences SEQ.ID NO.1 and SEQ.ID NO.2 can act as GLP-1 receptor agonists, we chemically synthesized polypeptides with a purity greater than 95%. The polypeptide was diluted to a concentration of 100 nM in Hank's Balanced Salt Solution (HBSS, pH 7.4) containing 20 mM HEPES and 2.5 mM Sulfamide. In order to detect whether the synthesized GLP-1 polypeptide can activate the GLP-1 receptor, Millipore purchased ready-to-use cells for GLP-1 detection (product number HTS163RTA) and corresponding complete medium. The cells are derived fr...

Embodiment 2

[0153] Example 2, SEQ.ID NO.1 and SEQ.ID NO.2 can regulate insulin secretion

[0154] The INS-1 832 / 13 cell line is derived from rat β cells transformed with the human proinsulin gene. Expression of GLP1 receptors, after being stimulated by active GLP1 or its analogues, and under high concentration glucose conditions, insulin secretion is increased. We have demonstrated that the GLP1 analogs to be tested can activate GLP-1R through calcium influx experiments. In this experiment, the INS-1 832 / 13 cell line was used to detect whether the GLP1 analogue to be tested could promote insulin release by activating GLP-1R.

[0155] The complete medium of the INS-1 832 / 13 cell line is RPMI 1640 supplemented with 10% fetal bovine serum, 50IU / ml penicillin, 50mg / L streptomycin, 10mM HEPES, 2mM L-glutamine, 1mM sodium pyruvate , and 50uM β-mercaptoethanol. INS-1 832 / 13 cells were passaged twice a week and cultured in a 5% CO2, 37°C incubator.

[0156] 1x105 INS-1 832 / 13 cells were seede...

Embodiment 3

[0160] Example 3, SEQ.ID NO.1 and SEQ.ID NO.2 can regulate the insulin level of obese mice (BKS db) and reduce blood sugar.

[0161] In order to verify whether the peptides SEQ.ID NO.1 and SEQ.ID NO.2 can promote insulin secretion and reduce blood sugar concentration in type 2 diabetes model animals, we choose BKS.Cg-Dock7 + / +Lepr / J(db / db) male obese mice were used as experimental subjects (The Jackson Laboratory). Firstly, the experimental animals were starved for 18 hours, and then intraperitoneally injected with 25nmol / kg polypeptide concentration (SEQ.ID NO.1, SEQ.ID NO.2, Exendin-4) and 18mmol / kg glucose concentration. The control group was injected with normal saline. Blood glucose concentrations were measured before injection and then at 15-minute intervals (0 min, 15 min, 30 min, 45 min, 60 min). Rat tail blood was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the concentration of insulin, and a blood glucose tester was used to me...

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Abstract

The invention relates to a novel glucagon analogue and application thereof. Compared with wild-type human GLP1, the novel glucagon analogue has a longer half-life period and a better insulin secretionpromotion activity, and a strong long-acting blood glucose reducing effect.

Description

technical field [0001] The invention belongs to the technical field of polypeptide medicine. It specifically involves the application of new GLP-1 receptor agonist drugs in promoting insulin secretion and lowering blood sugar, as well as the application in the treatment of type II diabetes and obesity. Background technique [0002] Type II diabetes is a long-term metabolic disorder characterized by hyperglycemia, insulin resistance and relative lack of insulin. Long-term complications caused by high blood sugar in patients include heart disease, stroke, diabetic retinopathy, and even blindness, kidney failure, poor blood flow in extremities, and even amputation. Type II diabetes accounts for approximately 90% of diabetes cases. Since 1960, the incidence of type 2 diabetes has increased significantly. As of 2017, approximately 450 million people were diagnosed with type 2 diabetes, compared with approximately 30 million in 1985. The World Health Organization predicts that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605A61K38/26A61P3/10
Inventor 张相民马丹军
Owner 清紫生物科技(深圳)有限公司
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