Preparation method of novel GLP-1 receptor stimulant and applications of novel GLP-1 receptor stimulant in treatment field of neurodegenerative diseases

A receptor agonist, neurodegenerative technology, applied in the field of biomedicine, to achieve stable blood drug concentration, low production cost, and reduce toxic effects

Inactive Publication Date: 2016-11-16
上海朗安生物技术有限公司
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  • Summary
  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Studies have shown that albiglutide cannot or can only pass through the blood-brain barrier to a limited extent [5], so it cannot exert therapeutic effects in the central nervous system

Method used

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  • Preparation method of novel GLP-1 receptor stimulant and applications of novel GLP-1 receptor stimulant in treatment field of neurodegenerative diseases
  • Preparation method of novel GLP-1 receptor stimulant and applications of novel GLP-1 receptor stimulant in treatment field of neurodegenerative diseases
  • Preparation method of novel GLP-1 receptor stimulant and applications of novel GLP-1 receptor stimulant in treatment field of neurodegenerative diseases

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] The preparation of embodiment 1 novel GLP-1 receptor agonist EHL

[0085] 1.1 Construction of EHL expression vector

[0086] The gene sequence of the gene synthesis EHL recombinant protein, the 3' end of the exenatide gene sequence is connected to the human fibronectin domain mutant (SEQ ID: 5 ) at the 5' end to obtain the EHL gene fragment, which was then constructed into an expression vector with a His tag and transformed into Escherichia coli.

[0087] 1.2 Induced expression of EHL

[0088] First, the Escherichia coli BL21 (DE3) expression strain transformed with the EHL recombinant plasmid was used for the first expansion culture with 2YT-Km medium) as the seed solution. Then, at a ratio of 0.1-0.5%, the seed solution was inoculated into a shake flask containing 2L 2YT medium for the second fermentation culture. When growing to the logarithmic growth phase, IPTG with a final concentration of 1mM was added and then induced overnight at 25 degrees. For expression, ...

Embodiment 2

[0103] Example 2 Detection of novel GLP-1 receptor agonist EHL half-life

[0104] 2.1 Grouping of experimental animals and preparation of specimens

[0105] (1) 5-month-old male SPF grade C57BL / 6J mice were selected and randomly divided into two groups: a novel GLP-1 receptor agonist group (EHL group) and an exenatide group (EXT group), with 5- Six rats were given a single intraperitoneal injection of EHL / EXT (125nmol / kg). At 7 time points (0.5, 1, 3, 6, 12, 24, 48 hours) after the administration, 100 μl of blood was collected from the orbit and placed in a 1.5ml centrifuge tube, overnight at 4°C.

[0106] (2) Centrifuge at 1000g for 20 minutes, label the supernatant, aliquot and store at -80°C to avoid repeated freezing and thawing.

[0107] 2.2 ELISA detection of Exendin-4 concentration in serum

[0108] (1) 25 minutes to 45 minutes before using the kit (PHOENIX, USA), make all the items in the kit reach room temperature.

[0109] (2) Dilute 50ml of 20× buffer, and dilut...

Embodiment 3

[0125] The therapeutic effect of embodiment 3 novel GLP-1 receptor agonists on AD model mice

[0126] 3.1 Test animals and their genotype identification

[0127] 5XFAD transgenic mice were bred and bred under standard conditions in an SPF grade animal room. The temperature was controlled at 25°C±2°C, the humidity was 45%-65%, free to eat and drink and water, and the light was cycled 12:12 hours a day and night. Newborn mice were tail-cut at 7-14 days after birth for genotype identification, and were divided into cages according to gender at 21-24 days after birth, with no more than 5 mice per cage. 5XFAD (heterozygous, HET) female mice aged 2-3 months were mated with C57BL / 6J male mice. 5XFAD heterozygous (HET) mice and wild-type (WT) mice were bred. 5XFAD heterozygous male mice were used for the experiment.

[0128] Genomic DNA extraction:

[0129] (1) Cut the tail of the mouse 7-14 days after birth, and place it in a 1.5mL EP tube;

[0130] (2) Prepare DNA lysate: ddH2...

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Abstract

The invention provides a novel GLP-1 receptor agonist, which is specifically a fusion protein formed by recombining GLP-1 receptor agonist molecules and non-antibody protein scaffold genes. The fusion protein can pass through the blood-brain barrier and enter the central nervous system Play a role. A linker molecule is also included between the GLP-1 receptor agonist molecule and the non-antibody protein scaffold. The non-antibody protein scaffold is derived from engineered human fibronectin domains with serum albumin targeting and reversible binding to human serum albumin. The novel GLP-1 receptor agonist has therapeutic effect in the field of neurodegenerative diseases. Using the method provided by the present invention to optimize exenatide, the half-life can reach 10 times that before the modification. When treating AD transgenic mice, intraperitoneal injection once a day greatly facilitates the treatment and stabilizes the blood drug concentration. The new GLP-1 receptor agonist is a prokaryotic expressed protein, which has low production cost, simple process and is more suitable for large-scale production.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a novel GLP-1 receptor agonist and its application in the field of neurodegenerative disease treatment. Background technique [0002] Neurodegenerative diseases are a group of progressive, disabling, severe and fatal complex diseases, which can be divided into acute neurodegenerative diseases and chronic neurodegenerative diseases. The former mainly includes stroke and brain injury; the latter Mainly including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), etc., which deteriorate over time and cause functional impairment. The brain pathological changes of such diseases mainly have two manifestations: one is the loss of a large number of neurons caused by apoptosis; the other is that there is no obvious decrease in the number of cell bodies in the nervous system, but the structure and function of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/22A61K47/48A61P25/28
CPCA61K45/00A61K38/22
Inventor 卢水秀滕灵艳史孟君黄金秦文远
Owner 上海朗安生物技术有限公司
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