Methods of identifying neuroprotective compounds for retinal ganglion cells

Patent metadata

Field	Value
Publication number	US-9771331-B2
Application number	US-91898706-A
Country	US
Kind code	B2
Filing date	Apr 24, 2006
Priority date	Apr 22, 2005
Publication date	Sep 26, 2017
Grant date	Sep 26, 2017

Abstract

Neuroprotective compounds for treating optic neuropathies and screening methods for identifying neuroprotective compounds.

First claim

The invention claimed is:

1. A high throughput method of identifying a neuroprotective compound, comprising:
(1) contacting dissociated retinal cells with a magnetic bead coupled to an anti-cd11 b/c antibody:
(2) separating cells bound to the anti-cd11 b/c antibody from other retinal cells to obtain a macrophage-depleted cell population;
(3) contacting the macrophage-depleted cell population with a magnetic bead coupled to an anti-Thy1 antibody;
(4) retaining cells bound to the anti-Thy1 antibody as a test enriched population of retinal ganglion cells;
(5) culturing the test enriched population of retinal ganglion cells in the absence of growth factors;
(6) contacting the test enriched population of retinal ganglion cells with a test compound previously unknown to have any neuroprotective pharmacological activity;
(7) staining the test enriched population of retinal ganglion cells with at least a dye capable of staining living cells and their neurites and a dye capable of staining the nuclei of dead cells:
(8) imaging retinal ganglion cells in the test enriched population to quantify the number of live cells and the extent of neurite outgrowth present in the test enriched population;
(9) comparing the number of live cells and the extent of neurite outgrowth present in the test enriched population of ganglion cells contacted with the test compound to a control number of live cells and extent of neurite outgrowth present in a control enriched population of retinal ganglion cells cultured in the presence of BDNF and forskolin and the absence of the test compound; and
(10) identifying the test compound as a neuroprotective compound if the number of live cells and/or the extent of neurite outgrowth in the compound treated population of retinal ganglion cells is statistically greater than the control number of live cells and/or extent of neurite outgrowth in the control enriched population of retinal ganglion cells grown in the presence of BDNF and forskolin and the absence of the test compound;

wherein steps (1) to (10) are performed as a high throughput screen in a multi-well plate.

2. The method of claim 1 wherein test compound is a small molecule.

3. The method of claim 1 wherein the test compound is a cDNA expression product.

4. The method of claim 1 wherein the multi-well plate comprises a multi-well microtiter plate.

Assignees

• Kerrison John Barnwell
• Zack Donald J
• Univ Johns Hopkins

Inventors

• Kerrison John Barnwell
• Zack Donald J

Classifications

• C07C239/16

  having nitrogen atoms of hydroxylamino groups further bound to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups · CPC title

• C07C233/25

  having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton · CPC title

• C07C2602/20

  the ring system containing seven carbon atoms · CPC title

• C07D333/22

  Radicals substituted by doubly bound hetero atoms, or by two hetero atoms other than halogen singly bound to the same carbon atom · CPC title

• G01N33/5058

  Neurological cells · CPC title