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Modified recombinant human endostatin and use thereof

A technology of endostatin and weight-average molecular weight, which is applied to medical preparations containing non-active ingredients, medical preparations containing active ingredients, drug combinations, etc., can solve the problems of difficult purification of biologically active mixtures, and achieve anti-endothelial cell proliferation Effects of increased activity, reduced economic burden, and reduced dosing frequency

Inactive Publication Date: 2009-03-11
JIANGSU SIMCERE PHARMACEUTICAL R & D CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is also the problem of isomers in single-molecule modification. The isomers may have large differences in biological activity and the mixture of isomers is usually difficult to purify.

Method used

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  • Modified recombinant human endostatin and use thereof
  • Modified recombinant human endostatin and use thereof
  • Modified recombinant human endostatin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, CH with a weight average molecular weight of 20kd 3 O-(CH 2 CH 2 O) 20kD -CH 2 CH 2 CH(OCH 2 CH 3 ) 2 Conjugates with Endostar (abbreviated as PEG 20 -ENDO) preparation:

[0040] (1)CH 3 O-(CH 2 CH 2 O) 20kD -CH 2 CH 2 CH(OCH 2 CH 3 ) 2 Conjugation to Endostar

[0041] 20ml Endostar stock solution (PH5.0~5.5, HAC / NaAC Buffer, protein content 5mg / ml), add reducing agent sodium cyanoborohydride NaCNBH 3 To a final concentration of 20mM, add 100mg of monomethoxypolyethylene glycol-3,3-diethoxypropane to make the molar ratio of Endostar to 1:1, and stir overnight at 4°C to form PEG 20 -ENDO modification, the product was identified by SDS-PAGE, see the identification results figure 1 .

[0042] (2)PEG 20 -Purification of ENDO

[0043] The above reaction products were separated and purified with CM-Sepharose column and AKTA chromatography. The sample was diluted with 20mM, pH 7.0 PB buffer and loaded, equilibrated to the baseline with 20m...

Embodiment 2

[0044] Embodiment 2, weight average molecular weight is the CH of 40kd 3 O-(CH 2 CH 2 O) 40kD -CH 2 CH 2 Conjugates of CHO and Endostar (abbreviated as PEG 40 -ENDO) preparation:

[0045] (1)CH 3 O-(CH 2 CH 2 O) 40kD -CH 2 CH 2 Coupling of CHO and Endostar 20ml Endostar stock solution (PH5.0~5.5, HAC / NaAC Buffer, protein content 5mg / ml), add reducing agent sodium cyanoborohydride NaCNBH 3 To a final concentration of 20mM, add 400mg of monomethoxypolyethylene glycol-3,3-diethoxypropane to make the molar ratio of Endostar to 1:2, and stir overnight at 4°C to form PEG 40 -ENDO modification, the product was identified by SDS-PAGE. ( Figure 4 )

[0046] (2)PEG 40 -Purification of ENDO

[0047] The above reaction products were separated and purified with CM-Sepharose column and AKTA chromatography. The sample was diluted with 20mM, pH 7.0 PB buffer and then loaded, equilibrated to the baseline with 20mM, pH 7.0 PB buffer, and then gradient with 20mM, pH 7.0 PB buf...

Embodiment 3

[0048] Example 3, Pharmacokinetic study of polyethylene glycol-modified recombinant human endostatin

[0049]Fifteen male SD rats were randomly divided into 3 groups and administered at 4.5 mg / kg body weight. Group A is unmodified Endostar, group B is PEG 20 -ENDO, Group C is PEG 40 -ENDO. Blood was collected at 5min, 10min, 15min, 30min, 1h, 4h, 6h, 12h, 24h, 48h, and 96h after administration, anticoagulated with heparin, and immediately centrifuged at 6000r / min for 10min to obtain plasma samples. Using Human Endostatin Protein The EIA kit was used to determine the content of samples in plasma. The effective blood drug concentration of endostatin after the determination is modified is increased by more than 20 times ( Figure 7 ), the half-life of Endostar is 8 hours, PEG 20 -ENDO has an in vivo half-life of 18h, PEG 40 -ENDO has an in vivo half-life of 97h. Compared with Endostar, PEG 20 -ENDO's AUC increased by at least 10 times, PEG 40 -ENDO's AUC increased by ...

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Abstract

The invention discloses modified recombinant human endostatin and an application thereof. The modified recombinant human endostatin has the following structure: CH3O-(CH2CH2O)m-CH2CH2CH2-N<alpha>H-Endosta, wherein the weight average molecular weight of CH3O-(CH2CH2O)m-CH2CH2CH2- is between 20 and 40Kd. The modified recombinant human endostatin strengthens in vivo stability, improves plasma concentration, prolongs half decay period, and remarkably increases the proliferative activity of anti-endothelial cells, thereby reducing administration dosage, reducing administration frequency, and relieving the body pain and the economical burden of patients. The modified recombinant human endostatin can be used to prepare antineoplastic medicine.

Description

technical field [0001] The invention belongs to the technical field of biological product pharmacy, and relates to a modified recombinant human endostatin and its application. Background technique [0002] In 1971, Professor Folkman put forward the view that "tumor growth depends on angiogenesis". By blocking angiogenesis, it is possible to control tumor growth and achieve the purpose of curbing tumor invasion, recurrence, and metastasis. of new fields. [0003] In 1997, Harvard University O'Reilly et al. found that the culture medium of mouse hemangioendothelioma cells had an inhibitory effect on endothelial cells. Through purification, it was found that the active substance was specific to endothelial cells within a certain concentration range (100ng / ml~600ng / ml). Exhibited dose-dependent growth inhibition. N-terminal amino acid sequencing showed that the substance was the C-terminal fragment of collagen XVIII, with a molecular weight of about 20 kD, and this protein was...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/08A61K38/17A61K47/48A61P35/00
CPCA61K47/48215C07K14/78A61K38/00A61K47/60A61P35/00
Inventor 姚文兵田浤许向阳李海瑞童玥高向东
Owner JIANGSU SIMCERE PHARMACEUTICAL R & D CO LTD
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