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Polypeptides having bradykinin receptor binding activity and uses thereof

A technology of receptor binding and bradykinin, which is applied in the fields of pharmacy and biology, can solve the problems of poor stability of L-type polypeptides, achieve the effects of increasing accumulation, prolonging median survival, and reducing damage

Active Publication Date: 2019-10-08
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the poor stability of the L-type polypeptide in vivo, there are many defects in its research and development as a drug

Method used

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  • Polypeptides having bradykinin receptor binding activity and uses thereof
  • Polypeptides having bradykinin receptor binding activity and uses thereof
  • Polypeptides having bradykinin receptor binding activity and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1RI-B

[0029] Embodiment 1RI-BK, the preparation and characterization of RI-KD and RI-L-BK

[0030] Using the solid-phase synthesis method, deprotect the PAM-Boc resin with trifluoroacetic acid (TFA) for 1 minute, twice, and react with Boc-protected amino acids in turn. Wash the resin with / MeOH (1 / 1), dry it in vacuum, put the resin into a polypeptide cutting tube, add an appropriate amount of P-cresol, then pass through HF, stir and react in an ice bath for 1 hour, and remove the HF in the tube under reduced pressure after the reaction is completed. The precipitate was washed with glacial ether for 3 times, the residual precipitate was dissolved in 20% acetonitrile and then rotary evaporated, separated and purified with acetonitrile / water (containing 0.1% TFA) system, HPLC and ESI-MS were used to characterize RI-BK, RI-KD and RI-L- The purity and molecular weight of BK; Its HPLC collection of illustrative plates and mass spectrogram are as figure 1 As shown, the T of RI-BK RI-BK ...

Embodiment 2

[0032] Uptake of RI-BK-FITC by C6, HEK, U87, HUVEC and bEnd.3 cells

[0033] C6, U87, HUVEC, and bEnd.3 cells were inoculated in 12-well plates, and 24 hours after the cells adhered, the DMEM containing serum was discarded, and DMEM without serum and RI-BK-FITC fluorescein mother solution were added to each well. , the final concentration of fluorescein was controlled to be 5×10 -6 mol / L. Continue culturing for 2 hours, take it out, wash twice with PBS, fix with formaldehyde for 15 minutes, wash twice with PBS, stain with DAPI for 10 minutes, wash twice with PBS, add buffered glycerol at the end, and take photos with a fluorescent microscope. The test results show that C6, U87, HUVEC Both have significant uptake of RI-BK-FITC, and bEnd.3 has basically no uptake of RI-BK-FITC (such as figure 2 shown).

Embodiment 3

[0035] RI-BK, RI-KD and RI-L-BK inhibited the uptake of RI-BK-FITC in C6 cells by fluorescence photos and flow cytometry results

[0036] C6 cells were seeded in 12-well plates. After the cells adhered to the wall, the DMEM containing serum was discarded, and DMEM without serum and polypeptides RI-BK, RI-KD and RI-L-BK were added to each well to control the concentration of the polypeptides. The final concentration was 5×10 -3 mol / L and incubated for half an hour, then add RI-BK-FITC fluorescein mother solution to control the final concentration of fluorescein to be 5×10 -6 mol / L; take it out after continuing to culture for 2 hours, wash twice with PBS, fix with formaldehyde for 15 minutes, wash twice with PBS, stain with DAPI for 10 minutes, wash twice with PBS, and finally add buffered glycerol; take pictures under a fluorescent microscope; the test results are as follows image 3 At the same time, trypsinized C6 cells, centrifuged at 1000 rpm for 5 minutes, washed once wit...

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Abstract

The invention belongs to a field of pharmacy, relates to a field of protein polypeptide biological technology, and more specifically relates to a polypeptide which specifically binds to a bradykinin acceptor. The invention employs a polypeptide sequence overturn principle, and the bradykinin is used as a template for designing a polypeptide, whose amino acid sequences are as follows: RI-BK is RFPSFGPPR; RI-KD is PFPSFGPPRK; RRI-L-BK is RFPSFGPPRL. Experiments show that: the polypeptide has a high affinity for the bradykinin acceptor, can selectively open blood-brain tumor barriers, substantially improves accumulation of the medicament in the brain tumor and prolonging the lifetime of a glioma in-situ model nude mouse. The polypeptide of the present invention can be used for preparing adjuvant drugs of chemotherapeutics, and is used for preparing a combination drug for treating brain tumor together with a polymer micelle administration system which envelops the tumor medicament.

Description

technical field [0001] The invention belongs to the field of pharmacy and biotechnology, and relates to protein polypeptides, in particular to polypeptides that can specifically bind to bradykinin receptors; Use in combination medicine for tumors. Background technique [0002] The prior art discloses that the kinin family is a class of polypeptides with potent vasoactive and pro-inflammatory properties, including bradykinin (Bradykinin, BK), pancreatic kinin (Kallidin, KD) and their active metabolites. In the 1970s, Regoli et al. discovered that there are two kinds of kinin receptors B1 and B2; studies also showed that kinin activates the kinin receptors B1 and B2 coupled with G protein, affecting the kallikrein-kinin system, And then produce a variety of physiological effects. Kinins have been implicated in cardiovascular homeostasis, smooth muscle contraction and relaxation, inflammation, and nociceptive perception. In addition, the role of kinins in cell proliferation,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/18A61K38/08A61K47/42A61K9/107A61K9/127A61K9/14A61P35/00A61P25/00
Inventor 刘敏谢作旭陆伟跃谢操李雪
Owner FUDAN UNIV
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