Enhancement of drug therapy by mirna

A therapeutic and biological technology that can be applied to DNA/RNA fragments, drug combinations, recombinant DNA technology, etc., and can solve problems such as interference with mitosis

Inactive Publication Date: 2011-05-25
ABRAXIS BIOSCI LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, platinum reagents have been postulated to also react with cellular proteins, particularly HMG domain proteins, thereby further interfering with mitosis

Method used

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  • Enhancement of drug therapy by mirna
  • Enhancement of drug therapy by mirna
  • Enhancement of drug therapy by mirna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0215] This example demonstrates the apoptotic activity of the HSP90 inhibitor 17-AAG.

[0216] The homogeneous caspase-3 / 7 assay (Promega, Madison, WI) uses a proprietary lysis / activation buffer together with (Z-DEVD) 2-rhodamine 110 substrate, allowing for use in adherent, A simple "add-mix-read" format for the detection of caspase-3 and -7 in suspension and primary culture cells or in purified caspase preparations. The assay uses a rhodamine 110-based substrate that allows for extreme sensitivity previously unobtainable with conventional colorimetric or fluorometric assays.

[0217] In particular, 100 μl Caspase-3 / 7 reagent was added to each well of a white or black 96-well plate containing 100 μl of blank, control or cultured cells. Cover the plate with a plate sealer for extended periods of incubation (>4 hours). To perform the assay in a 384-well plate, the Caspase-3 / 7 reagent:sample 1:1 volume ratio. The contents of the wells were mixed using a plate shaker at ...

Embodiment 2

[0220] This example demonstrates that the HSP90 inhibitor 17-AAG inhibits Her2 expression.

[0221] 10,000 BT474 cells / well were seeded into microtiter plates and grown for 48 hours. After this pre-incubation, BT474 cells were treated with various concentrations of 17-AAG and its analogs for 24 hours. At the end of this incubation, medium was removed from each well, each well was washed twice with ice-cold Tris-buffered saline (containing 0.1% Tween 20), and cells were fixed with methanol (ice-cold) at 40°C 10 minutes. Fixed BT474 cells were immunostained with anti-Her2 antibody. The presence of Her2 protein was determined by measuring the absorbance at 405 nm in a plate reader.

[0222] like figure 2 As shown in , the IC50 for 17-AAG is close to 32 nM for the Her2 inhibition assay. This result suggests that 17-AAG strongly inhibits Her2 protein expression.

Embodiment 3

[0224] This example demonstrates the internalization and degradation of Her2 following 17-AAG inhibition of HSP90.

[0225] 17-AAG-treated BT474 cells were examined by confocal imaging system. BT474 cells were seeded on glass slides and treated at IC50 concentrations for 24 hours. 17-AAG-treated and control BT474 cells were fixed with methanol, stained with Her2 antibody, and analyzed by confocal imaging. like image 3 As shown in , after 17-AAG treatment, Her2 protein expression was abolished from its cell surface sublocalization and sublocalized to the cytoplasm.

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PUM

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Abstract

This invention provides methods and compositions for screening of microRNA capable of modulating gene expression in the apoptotic pathway in the presence of HSP90 inhibitor. The use of miRNA for enhancing the activity of therapeutic agents not limited to HSP90 inhibitor is also disclosed. The diagnostic use of miRNA for predicting response to therapy not limited to therapeutic agents is also disclosed. A method for the identification and therapeutic application of small molecules which are modulators of these nucleic acids are also included in this application.

Description

Background of the invention [0001] Although tremendous progress has been made in elucidating the genomic abnormalities that give rise to malignant cancer cells, currently available chemotherapy is still unsatisfactory and the prognosis for most patients diagnosed with cancer remains dismal. Thus, there is a continuing need to develop new therapies, especially new therapies that work well together, if not synergistically, with other agents and treatments. [0002] Heat shock proteins (HSPs) are a class of chaperone proteins that are upregulated in response to elevated temperature and other environmental stresses such as ultraviolet light, nutrient deprivation and hypoxia. HSPs act as molecular chaperones for other cellular proteins (called "client" proteins) and contribute to their proper folding and repair of client proteins. There are several known families of HSPs, each with its own set of client proteins. The HSP90 family is one of the most abundant HSP families, accounti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K31/713A61K31/00A61P35/00
CPCC12N2320/31C12N15/111C12N2330/10C12N2310/141A61P25/00A61P35/00
Inventor V·特里鲁L·黄N·德塞
Owner ABRAXIS BIOSCI LLC
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