Methods and compositions for enhancing the uptake of therapeutic agents by target cells

a technology of target cells and therapeutic agents, applied in the field of medicaments, can solve the problems of preventing the formation of new blood vessels and lymphatic vessels, unclear specific mechanism, membrane perforation, etc., and achieve the effect of increasing the overall endocytosis of es

Active Publication Date: 2014-11-13
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]The invention is based on the following discovery. ES can be internalized by endothelial cells through both lipid raft / caveolae-dependent endocytic pathway and clathrin-coated pit endocytic pathway. The inventors surprisingly found that inhibiting lipid raft / caveolae-dependent endocytic pathway with NT leads to an unexpected result that the uptake of ES by angiogenic and lymphangiogenic endothelial cells is increased, resulting in an emhanced inhibitory effect of ES on endothelial cells, tumor angiogenesis, and tumor growth. Without being bound by theory, it is speculated that the inhibition of lipid raft / caveolae-dependent endocytic pathway leads to the compensatory activation of the highly efficient clathrin-coated pit endocytic pathway, resulting in an increased overall endocytosis of ES.
[0012]Similarly, ligands of EGFR have been shown to have a similar endocytic machinery to ES. For example, monoclonal antibody against EGFR is a potent antitumor agent that inhibits the signal activation and tumor proliferation by specifically binding to EGFR on tumor cell surface. The inventors found that, by affecting lipid raft / caveolae-dependent endocytosis, NT significantly enhances the uptake of EGFR monoclonal antibody by tumor cells, leading to enhanced therapeutic efficacy of this antibody. These findings indicate the potential use of NT in tumor therapy and imaging. Furthermore, there are many protein drugs sharing a similar endocytic pattern like ES, including endothelin receptor type A, cholera toxin, transforming growth factor-β receptors, B cell antigen receptor, bone morphogenetic protein receptor, HM 1.24, and integrins. These proteins all have the potential to be used in combination with NT to achieve an enhanced therapeutic effect.

Problems solved by technology

ES can inhibit the proliferation and migration of angiogenic and lymphangiogenic endothelial cells, and thus can impede the formation of new blood vessels and lymphatic vessels.
However, the specific mechanism remains unclear.
The mechanism of nystatin and its analogs (e.g., amphotericin B) is that they can bind with ergosterol on the fungus cell membrane, resulting in membrane perforation, potassium leakage, and subsequently the death of fungus.

Method used

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  • Methods and compositions for enhancing the uptake of therapeutic agents by target cells
  • Methods and compositions for enhancing the uptake of therapeutic agents by target cells
  • Methods and compositions for enhancing the uptake of therapeutic agents by target cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

NT Enhances the Uptake and Internalization of ES in Blood Vessel Endothelial Cells and Lymphatic Vessel Endothelial Cells

[0060]Human microvascular endothelial cells (HMECs) were from ATCC (accession number CRL 10636). Mouse lymphatic endothelial cells (mLEC) were isolated via digesting peritoneal lymphoma induced by incomplete Freund's adjuvant injection (Zhuo W. et al, Journal of Pathology; 222:249-260). Endu was obtained from Simcere-Medgenn Bio-pharmaceutical Co., Ltd. Endu is a recombinant ES containing an additional amino acid sequence (M)GGSHHHHH at N-terminal, the sequence of which is SEQ ID NO.3 or SEQ ID NO.4 because the first amino acid residue M will be randomly deleted when expressed in E. Coli. ES was obtained from Protgen Ltd., the amino acid sequence of which is SEQ ID NO.1 or SEQ ID NO.2 because the first amino acid residue M will be randomly deleted when expressed in E. Coli. Methoxy PEG propionaldehyde (mPEG-ALD) with an average molecular weight of 20 kDa (Jenkem T...

example 2

NT Increases the Inhibitory Effect of ES on Endothelial Cell Signal Pathways by Enhancing ES Internalization

[0066]Kim et al (2002) found that ES can inhibit the signal transduction pathways in endothelial cell mediated by extracellular regulated protein kinases (ERK), p38 mitogen-activated protein kinases (MAPK), and p125 focal adhesion kinase (p125 FAK), resulting in the inhibition of endothelial cell activity (Kim et al, J. Biol. Chem. 2002, 277, 27872-27879). In this example, the ERK and p38 MAPK pathways were taken as the markers of endothelial cell activation and it is found that the combination of NT and ES further elevated the inhibitory effect of ES on these pathways.

[0067]In this example, endothelial cells were separated into four groups: (1) negative control group, neither NT treatment, nor ES treatment; (2) NT group, treatment with NT at 50 μg / mL for 20 min, without ES treatment; (3) ES group, treatment with ES at 5 μg / mL for 30 min, without NT treatment; (4) NT plus ES g...

example 3

NT Enhances the Inhibitory Effect of ES on Endothelial Cell Migration

[0068]Cell migration assay: HMEC (2×104 cells per well) were seeded into DMEM (Hyclone) containing 0.5% FBS (Hyclone) and 10 ng / mL VEGF (PeproTech) on the upper layer of Transwell chambers (8 μm pores, Costar). ES (40 μg / mL) or NT (50 μg / mL) was added into both the upper and lower layers, and then the chambers were incubated in a 5% CO2 atmosphere at 37° C. for 6 hours to allow cell migration. After glutaraldehyde fixation and crystal violet staining, 5 randomly selected fields in each well were observed with a microscopy (400× magnification, Olympus IX71). The numbers of the cells migrated into the lower layer were counted and mean values were calculated. Experiments were performed in triplicate and each experiment was repeated twice.

[0069]In this example, endothelial cells were separated into four groups: (1) negative control group, neither NT treatment, nor ES treatment; (2) NT group, treatment with NT at 50 μg / ...

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Abstract

The present invention relates to a new use of a known medicament. Specifically, the invention relates to methods and compositions for enhancing the therapeutic efficacy of a therapeutic agent by increasing the uptake of the therapeutic agent by target cells, and in particular relates to a pharmaceutical composition comprising a regulating agent of lipid raft / caveolae-dependent endocytic pathway and some therapeutic agents, such as anti-tumor agents. The invention also relates to a method for screening a regulating agent of lipid raft / caveolae-dependent endocytic pathway capable of enhancing the therapeutic efficacy of anti-tumor agents.

Description

CROSS-REFERENCE OF THE RELATED APPLICATION[0001]The present application is a 35 U.S.C. §371 National Phase Entry Application of PCT / CN2012 / 073202, filed 28 Mar. 2012, designating the United States, which in turn claims priority to Chinese Patent Application No. 201110085338.8, filed on 28 Mar. 2011, both of which are incorporated herein by reference.FIELD OF INVENTION[0002]The present invention relates to a new use of a known medicament. Specifically, the invention relates to methods and compositions for enhancing the therapeutic efficacy of a therapeutic agent by increasing the uptake of the therapeutic agent by target cells, and in particular relates to a pharmaceutical composition comprising a regulating agent of lipid raft / caveolae-dependent endocytic pathway and some therapeutic agents, such as anti-tumor agents. The invention also relates to a method for screening a regulating agent of lipid raft / caveolae-dependent endocytic pathway capable of enhancing the therapeutic efficac...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/365A61K47/48A61K31/724A61K31/7048A61K38/17
CPCA61K31/7048A61K38/1709A61K31/724A61K31/365A61K47/48215A61K9/0019A61K31/065A61K9/127A61K38/39A61K39/395A61K45/06A61K31/718A61K47/60A61P35/00A61P43/00A61P9/00A61K2300/00
Inventor LUO, YONGZHANGCHEN, YANGFU, YANJIA, LINCHANG, GUODONG
Owner TSINGHUA UNIV
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