Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation
AssigneeThe General Hospital Corporation
InventorsAshley I. Bush, Xudong Huang, Craig S. Atwood, Rudolph E. Tanzi
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Abstract
The invention relates to methods for identifying candidate pharmacological agents to be used in the treatment and/or prevention of Alzheimer's disease and/or related pathological conditions.
Claims9
A method for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity, but does not inhibit the superoxide-dependent hydrogen peroxide formation, the method comprising:
- (a) adding a candidate agent to an amyloid β (Aβ)-containing sample;
- (b) determining whether said candidate agent inhibits dissolved oxygen-dependent hydrogen peroxide formation;
- and
- (c) determining whether said candidate agent does not inhibit the superoxide-dependent hydrogen peroxide formation.
A method for identifying an agent that alters the production of H2O2 by Aβ, said method comprising:
- (a) adding Cu(II) or Fe(III) to a first Aβ sample;
- (b) allowing said first sample to incubate for an amount of time sufficient to generate H2O2;
- (c) adding Cu(II) or Fe(III) to a second Aβ sample, said second sample additionally comprising a candidate pharmacological agent;
- (d) allowing said second sample to incubate for the same amount of time as said first sample;
- (e) determining the amount of H2O2 produced by said first sample and second sample;
- and
- (f) comparing the amount of H2O2 present in said first sample to the amount of H2O2 present in said second sample;
- whereby a difference in the amount of H2O2 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of H2O2 by Aβ.
The method of claimed 5, wherein said biological fluid is cerebrospinal fluid (CSF).
The method of claimed 4, wherein the determination of the amount of H2O2 present in said first and second samples is determined by
- (a) adding catalase to a first aliquot of said first sample in an amount sufficient to break down all of the H2O2 generated by said sample;
- (b) adding tris(2-carboxyethyl)-phosphine hydrochloride (TCEP), in an amount sufficient to capture all of the H2O2 generated by said samples, to
- (i) a first aliquot of said first sample;
- (ii) a second aliquot of said first sample;
- and
- (iii) said second sample;
- (c) incubating the samples obtained in (b) for an amount of time sufficient to allow the TCEP to capture all of the H2O2;
- (d) adding 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to said samples obtained in (c);
- (e) incubating said samples obtained in (d) for an amount of time sufficient to generate 2-nitro-5-thiobenzoic acid (TMB);
- (f) measuring the absorbencies at 412 nm of said samples obtained in (e);
- and
- (g) calculating the concentration of H2O2 in said first and second samples using the absorbencies obtained in (f).
Dependencies are inferred from the claim text.