In vitro assays for inhibition of microglial activation

Patent metadata

Field	Value
Publication number	US-2017003280-A1
Application number	US-201515109163-A
Country	US
Kind code	A1
Filing date	Jan 9, 2015
Priority date	Jan 11, 2014
Publication date	Jan 5, 2017
Grant date	—

Abstract

The present invention provides cell-based assays, including high throughput cell-based assays, for identification of candidate therapeutic agent with no known agents with the ability to inhibit microglial activation in vivo in response to different ligands.

First claim

What is claimed is:

1 . A cell-based assay for identification of candidate therapeutic agents that inhibit microglia activation in vivo, comprising:
providing in vitro activated microglial cells; contacting said activated microglial cells with one or more test agents; identifying one or more test agents that exhibit inhibition of the in vitro microglia activation; and determining toxicity of the identified test agents that exhibit inhibition of the in vitro microglia activation by determination of cell death upon exposure of the microglial cells to the test agents; wherein the test agents that exhibit both inhibition of the in vitro microglia activation and lack of toxicity are candidate therapeutic agents for the inhibition of microglial activation in vivo.

2 . The assay of claim 1 , further comprising the step of activating microglial cells in vitro using an agent associated with microglial activation.

3 . The assay of claims 1 and 2 , wherein the microglial cells are activated by in vitro exposure to an active fibrin composition.

4 . The assay of claims 1 and 2 , wherein the microglial cells are activated by in vitro exposure to LPS.

5 . The assay of claim 1 through 4 , wherein the candidate therapeutic agents exhibit at least a 50% inhibition of the in vitro microglia activation.

6 . The assay of claims 1 through 5 , wherein the toxicity is measured through identification of cell death of the activated microglial cells.

7 . The assay of claim 6 , wherein the cell death is identified through shrinkage of the cell surface area of the microglia.

8 . The assay of claims 6 and 7 , wherein the cell death is identified through shrinkage of the cell surface area of the microglia to 150 μm 2 or less.

9 . The assay of claims 1 through 8 , wherein the one or more test agents comprise agents with demonstrated affinity to targets involved in microglia activation.

10 . The assay of claims 1 through 9 , wherein the one or more test agents comprise known drugs with clinical evaluation data.

11 . The assay of claims 1 through 10 , wherein the one or more test agents comprise known anti-inflammatory and microglia-macrophage response modulators.

12 . The assay of claims 1 through 8 , wherein the one or more test agents comprise agents with no known activity or identified binding target.

13 . A high-throughput cell-based assay for identifying a candidate therapeutically active agent for clinical inhibition of microglia activation, comprising the steps of:
providing a panel of in vitro activated microglial cells; contacting said panel of microglial cells with one or more individual test agents; determining whether the test agents cause inhibition of the activation of the microglial cells treated with the test agents; and determining the level of cell death of the microglial cells treated with the test agents; wherein the one or more test agents that exhibit inhibition of the activation of the microglial cells and no increased cell death are candidate therapeutically active agents for clinical inhibition of microglia activation in vivo.

14 . The assay of claim 13 , further comprising the step of activating microglial cells in vitro using an agent associated with microglial activation.

15 . The assay of claim 14 , wherein the microglial cells are activated by in vitro exposure to an active fibrin composition.

16 . The assay of claim 14 , wherein the microglial cells are activated by in vitro exposure to LPS.

17 . The assay of claims 13 through 16 , wherein the toxicity is measured through identification of cell death of the activated microglial cells

18 . The assay of claim 17 , wherein the cell death is identified through shrinkage of the cell surface area of the microglia.

19 . The assay of claims 17 and 18 , wherein the cell death is identified through shrinkage of the cell surface area of the microglia to 150 μm 2 or less.

20 . The assay of claims 13 through 19 , wherein the one or more test agents comprise agents with demonstrated affinity to targets involved in microglia activation.

21 . The assay of claims 13 through 20 , wherein the one or more test agents comprise known drugs with clinical evaluation data.

22 . The assay of claims 13 through 21 , wherein the one or more test agents comprise known anti-inflammatory and microglia-macrophage response modulators.

23 . The assay of claims 13 through 19 , wherein the one or more test agents comprise agents with no known activity or identified binding target.

Assignees

• The J David Gladstone Inst
• Univ California

Inventors

• Akassoglou Katerina
• Arkin Michelle
• Ang Kean-Hooi
• Meyer-Franke Anke
• Wilson Christopher

Classifications

• G01N33/5047

  Cells of the immune system · CPC title

• G01N2800/28

  Neurological disorders · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• G01N2500/00

  Screening for compounds of potential therapeutic value · CPC title

• G01N33/5058 Primary

  Neurological cells · CPC title