US 9,771,331 B2Granted

Methods of identifying neuroprotective compounds for retinal ganglion cells

AssigneeThe Johns Hopkins University

InventorsJohn Barnwell Kerrison, Donald J. Zack

Life of this patent

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Abstract

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

Claims4

Dependent claims

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.

9 more elements

Dependencies are inferred from the claim text.

Facts

Application: 11/918,987Filed 24 Apr 2006 · us provisional application
Priority: 22 Apr 2005
Published: US 2009/0215765 A127 Aug 2009
Granted: 26 Sep 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification16 codes

C07D 239/50
Two or more oxygen, sulphur or nitrogen atoms: three nitrogen atoms
C07C 233/25
With the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring:…
C07C 239/16
Hydroxylamino compounds or their ethers or esters: having nitrogen atoms of hydroxylamino groups further bound to carbon atoms of hydrocarbon…
C07C 251/86
Hydrazones: having doubly-bound carbon atoms of hydrazone groups bound to carbon atoms of six-membered aromatic rings

Family

Application60/674,202Us provisional application · filed 22 Apr 2005
US 2009/0215765 A1Pre-grant publication · published 27 Aug 2009
US 9,771,331 B2This patent

From the US filing history. Foreign family members aren’t listed.

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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.

9 more elements

Facts

Application

11/918,987Filed 24 Apr 2006 · us provisional application

Priority

22 Apr 2005

Published

US 2009/0215765 A127 Aug 2009

Granted

26 Sep 2017

Term ends

Not estimatedThe source records don’t support an estimate.

Classification16 codes

C07D 239/50
Two or more oxygen, sulphur or nitrogen atoms: three nitrogen atoms
C07C 233/25
With the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring:…
C07C 239/16
Hydroxylamino compounds or their ethers or esters: having nitrogen atoms of hydroxylamino groups further bound to carbon atoms of hydrocarbon…
C07C 251/86
Hydrazones: having doubly-bound carbon atoms of hydrazone groups bound to carbon atoms of six-membered aromatic rings