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Method for quantitative determination of in vivo activity of recombination human endothelial chalone

A technology for quantitative determination of endostatin, applied in anti-angiogenesis therapy, in the field of quantitative determination of in vivo activity, can solve the problems of difficult quantification, not obvious dose-effect relationship, and only qualitative, etc., to achieve stable results, Good repeatability and stable experimental results

Inactive Publication Date: 2007-10-03
江苏省基因药物工程技术研究中心
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Problems solved by technology

[0003] At present, the method used for the determination of endostatin activity in vivo is chicken embryo allantoic membrane angiogenesis test, but the dose-effect relationship of this method is not very obvious, and generally it can only be qualitative, and it is difficult to quantify

Method used

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  • Method for quantitative determination of in vivo activity of recombination human endothelial chalone
  • Method for quantitative determination of in vivo activity of recombination human endothelial chalone
  • Method for quantitative determination of in vivo activity of recombination human endothelial chalone

Examples

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Embodiment 1

[0021] Example 1: Establishment of an animal model for the in vivo activity determination of recombinant endostatin

[0022] The main function of recombinant endostatin is to inhibit the growth of tumor vascular endothelial cells and other abnormally proliferating vascular endothelial cells. When measuring its activity in vivo, it is considered that the abnormal proliferation of vascular endothelial cells in tumor tissues is the fastest and the tissue with the largest blood vessel density is the first choice. Liver cancer The density of blood vessels is large, the proliferation of vascular endothelial cells is fast, and there is obvious migration phenomenon, so it is more appropriate to choose mouse liver cancer as an animal model for in vivo testing. However, it does not exclude other solid tumors as animal models for quantitative determination of activity in vivo as the scope of protection of this patent.

[0023] (1) Mouse liver cancer H 22 (Ascites type) tumor type passag...

Embodiment 2

[0040] Embodiment 2: the ED of recombinant human endostatin activity quantitative determination working reference substance 50 and determination of potency

[0041] (1) Determination of tumor inhibition rate

[0042] According to the above-mentioned method for measuring the activity of recombinant human endostatin, the reference substance was used to carry out 6 independent activity measurements, and the results of 6 tumor inhibition rate measurements were obtained (see Table 1). Figure 6.

[0043] (2) ED of 6 experiments of the reference substance 50 and potency calculation

[0044] The regression curve was made with the tumor inhibition rate against the dose, and x was ED 50 When the actual dose, in mg / kg, when calculating the ED 50 When the value of y is 50, the formula is y=a+bx, where a is the intercept and b is the slope. Through the regression curve, the correlation coefficient r can be obtained. Taking the results of the control experiment 1 as an example, use a ...

Embodiment 3

[0052] Example 3: In vivo activity determination and potency calculation of recombinant human endostatin to be tested

[0053] Measure the randomized block design method by volume response parallel line to carry out recombinant human endostatin titer determination (tumor inhibition test method), obtain the tumor inhibition test result in recombinant human endostatin titer determination (tumor weight, see Table 3), Activity assay titers were calculated by the formula. Among them, S is the recombinant human endostatin active reference substance, the titer is 86.2U, and the diluent ds 1 : 0.2mg / ml, ds 2 : 0.4mg / ml, ds 3 : 0.8mg / ml; T is the finished product of recombinant human endostatin, the marked potency is 80U, the diluent dt 1 : 0.2mg / ml, dt 2 : 0.4mg / ml, dt 3 : 0.8mg / ml, r=0.8:0.4=2, I=lgr=lg2=0.3010. The calculated results are shown in the table below (tumor inhibition weight is the average tumor weight of the negative control group minus each tumor weight):

[005...

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Abstract

A method for quantitatively testing the in vivo activity of recombinant human endostatin includes such steps as using mouse' s liver cancer as animal model, quantitatively converting the tumor suppressing rate to activity units, measuring the valence of reference, testing the reliability , calculating the confidence, tumor suppressing experiment of the specimen to be tested and reference specimen, and calculating the confidence of the valence of the specimen to be tested to quantitatively measure said in vivo activity and error.

Description

technical field [0001] The invention belongs to the field of genetic engineering biological products, and relates to a method for quantitatively measuring the in vivo activity of highly active soluble recombinant human endostatin (Endostatin) produced by genetic engineering methods, and the method is used in the anti-angiogenesis-dependent diseases. Applications in Angiogenesis Therapy. The term of the present invention relates to: recombinant human endostatin protein, recombinant human endostatin protein active fragment and PEGylated long-acting recombinant human endostatin and active fragment. Background technique [0002] Human endostatin recombinant protein can be produced and purified from Escherichia coli expression system, yeast expression system, insect expression system, eukaryotic cell expression system and virus expression system, and the recombinant human endostatin activity produced by different ways or different preparation methods Often not the same. The rec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00C12N15/09
Inventor 徐根兴朱浩文徐家明荣志刚赵延发钟慎政
Owner 江苏省基因药物工程技术研究中心
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