In vitro system for determining formation of abeta amyloid

Abstract

The invention relates to rapid methods for determining formation of Aβ amyloid and screening compounds which inhibit formation of Aβ amyloid in vitro, as well as kits for carrying out the present methods. Such an agent used in vivo may prevent, ameliorate or reverse the symptoms of Alzheimer's disease and Aβ amyloidotic disorders related to Alzheimer's disease, Down's syndrome, and Guamanian amyotrophic lateral sclerosis/Parkinson's dementia complex. The process described in this invention involves the rapid induction of Aβ amyloid by a heavy metal cation capable of binding to a polypeptide comprising at least amino acids 6 to 28 of Aβ, such as zinc to form amyloid and determination of formation of tinctorial Aβ amyloid. Moreover, a method of determining effectiveness of a candidate anti-amyloidotic agent for prevention or treatment of Aβ amyloidosis is described which uses cell cultures which express at least a human Aβ peptide.

What is claimed is:  
     
         1 . A rapid analytical method for detection of Aβ amyloid formation in a biological fluid which comprises: 
 (a) preparing a first set of reaction mixtures comprising neat biological fluid from a control human subject, and serial dilutions of the same made in aqueous buffer or physiological solution;  
 (b) preparing a second set of reaction mixtures comprising neat biological fluid from a human patient suspected of amyloidosis, and serial dilutions of the same made in aqueous buffer or physiological solution;  
 (c) adding an equal amount of Aβ peptide comprising at least amino acids 6 to 28 of Aβ to each serial dilution sample;  
 (d) contacting each of the first and the second set of reaction mixtures with an amount greater than 300 nM of a heavy metal cation capable of binding to an AD peptide comprising at least amino acids 6 to 28 of Aβ;  
 (e) centrifuging each of the first and the second sets of reaction mixtures to give a first and a second set of pellets, respectively; and  
 (f) comparing the amount of amyloid in the first and the second set of pellets and thereby detecting excessive Aβ amyloid formation in the biological fluid from the human patient suspected of amyloidosis.  
 
     
     
         2 . A rapid analytical method for detection of Aβ amyloid formation in a biological fluid as claimed in  claim 1 , wherein said biological fluid is CSF.  
     
     
         3 . A rapid analytical method for detection of Aβ amyloid formation in a biological fluid as claimed in  claim 2 , wherein in step (c), said heavy metal cation capable of binding to an AO peptide comprising at least amino acids 6 to 28 of Aβ is zinc.  
     
     
         4 . A method for determining whether a compound inhibits formation of Aβ amyloid which comprises: 
 (a) pre-filtering an aqueous buffer solution of Aβ peptide, which comprises at least the region in the Aβ peptide from amino acid number 6 to 28 to give a first filtrate;  
 (b) measuring the amount of Aβ peptide in the first filtrate obtained in step (a);  
 (c) contacting the first filtrate obtained in step (a) with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Aβ to give a reaction mixture;  
 (d) contacting the reaction mixture obtained in step (c) with a candidate anti-amyloidotic agent;  
 (e) filtering the reaction mixture obtained in step (d) to give a second filtrate; and  
 (f) comparing the amount of AD peptide in the second filtrate with the amount of Aβ peptide in the first filtrate, thereby determining whether the candidate compound inhibits formation of Aβ amyloid.  
 
     
     
         5 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 4 , wherein the heavy metal cation is selected from the group consisting of metalochloride salts of zinc, copper, and mercury.  
     
     
         6 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 4 , wherein the heavy metal cation is zinc chloride.  
     
     
         7 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 6 , wherein said Aβ peptide is selected from the group consisting of Aβ 1-39 , Aβ 1-40 , Aβ 1-41 , Aβ 1-42 , and Aβ 1-43 .  
     
     
         8 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 6 , wherein said Aβ peptide is Aβ 1-40 .  
     
     
         9 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 4 , wherein the pH of the reaction mixtures are between 6.8 to 7.8.  
     
     
         10 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 4 , wherein the pH of the reaction mixtures are about 7.4.  
     
     
         11 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 4 , wherein the concentration of the Aβ peptide is about 0.8 μM.  
     
     
         12 . A method for determining whether a compound inhibits formation of Aβ amyloid which comprises: 
 (a) assembling a first and a second reaction mixture, wherein each reaction mixture comprises an equal amount of a pre-filtered Aβ peptide solution, which comprises at least the region in the Aβ peptide from amino acid number 6 to 28, and an aqueous buffer or physiological solution;  
 (b) contacting each of the first and the second reaction mixtures with an equal amount of a candidate anti-amyloidotic agent;  
 (c) contacting the first reaction mixture with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Aβ;  
 (d) contacting the second reaction mixture with EDTA; and  
 (e) comparing the amount of amyloid formed in the first reaction mixture with that in the second reaction mixture, thereby determining whether the candidate compound inhibits the formation of Aβ amyloid.  
 
     
     
         13 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein the concentration of Aβ peptide in the reaction mixture is about 0.8 μM.  
     
     
         14 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein step (d) comprises the steps of: 
 (i) centrifuging the first and the second reaction mixtures, so that the soluble Aβ peptides are separated from the insoluble amyloid and a pellet is formed; and  
 (ii) comparing the amount of soluble Aβ peptide in the first reaction mixture with the soluble Aβ peptide in the second reaction mixture, thereby determining effectiveness of the candidate anti-amyloidotic agent.  
 
     
     
         15 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein the pH of the reaction mixtures are about 6.8 to 7.8.  
     
     
         16 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein the pH of the reaction mixtures are about 7.4.  
     
     
         17 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 14 , wherein in step (ii), said pellets are stained with an amyloid-staining dye.  
     
     
         18 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 14 , wherein said heavy metal cation is selected from the group consisting of salts of zinc, copper, and mercury.  
     
     
         19 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 14 , wherein said heavy metal cation is a zinc salt.  
     
     
         20 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein step (a), the Aβ peptide solution is prefiltered before assembling said first and second reaction mixtures; and wherein step (d) comprises the steps of: 
 (i) filtering the first and the second reaction mixtures, separately, and  
 (ii) comparing the amount of Aβ peptide in the filtrate, thereby determining effectiveness of the candidate anti-amyloidotic agent.  
 
     
     
         21 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 20 , wherein step (d)(ii) comprises the step of measuring the fraction of Aβ peptide in the filtrate by calculating ratio of the filtrate OD 214  relative to the OD 214  of the prefiltered Aβ peptide solution at step (a), thereby determining whether the compound inhibits formation of Aβ amyloid.  
     
     
         22 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 2   1 , wherein said Aβ peptide is selected from the group consisting of Aβ 1-39 , Aβ 1-40 , Aβ 1-41 , Aβ 1-2 , and Aβ 1-43 .  
     
     
         23 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 21 , wherein said Aβ peptide is Aβ 1-40 .  
     
     
         24 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 21 , wherein all filtering is done using filters with a pore size that allows passage of the soluble Aβ peptide used in the reaction mixtures but does not allow passage of amyloid.  
     
     
         25 . A method for determining whether a compound inhibits formation of Aβ amyloid which comprises: 
 (a) assembling a first and a second reaction mixture, wherein each reaction mixture comprises an equal amount of a prefiltered Aβ peptide solution, which contains at least the region in the Aβ peptide from amino acid number 6 to 28, and an aqueous buffer or physiological solution;  
 (b) contacting each of the first and the second reaction mixtures with an equal amount of a candidate anti-amyloidotic agent;  
 (c) contacting only the first reaction mixture with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of Aβ; and  
 (d) comparing the amount of amyloid formed in the first reaction mixture with that in the second reaction mixture, thereby determining whether the compound inhibits formation of Aβ amyloid.  
 
     
     
         26 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 25 , wherein the concentration of Aβ peptide in the reaction mixture is about 0.8 μM.  
     
     
         27 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 25 , wherein step (d) comprises the steps of: 
 (i) filtering the first and the second reaction mixtures, separately, through filters with a pore size that allows passage of the soluble AP peptide used in the reaction mixtures but does not allow passage of amyloid; and  
 (ii) comparing the amount of amyloid accumulated at step (i) on the filters, thereby determining effectiveness of the candidate anti-amyloidotic agent.  
 
     
     
         28 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 12 , wherein said physiological solution is CSF.  
     
     
         29 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 25 , wherein said physiological solution is CSF.  
     
     
         30 . A method for determining whether a compound inhibits formation of Aβ amyloid which comprises: 
 (a) establishing a first and a second cell culture comprising a cell line which expresses at least a human AD peptide comprising at least the region of the AD peptide from amino acid number 6 to 28;  
 (b) contacting equal concentrations of zinc to each cell culture;  
 (c) contacting the first cell culture with the candidate agent, and contacting the second cell culture with a heavy metal chelating agent; and  
 (d) comparing the amount of amyloid and zinc-induced AP aggregates in each cell culture, thereby determining effectiveness of the candidate anti-amyloidotic agent.  
 
     
     
         31 . A method for determining whether a compound inhibits formation of Aβ amyloid as claimed in  claim 30 , wherein said heavy metal chelating agent is EDTA or Desferrioxamine.  
     
     
         32 . A method for determining whether a compound inhibits formation of Aβ amyloid which comprises: 
 (a) establishing a first and a second cell culture comprising a cell line which expresses at least a human Aβ peptide comprising at least the region of the Aβ peptide from amino acid number 6 to 28;  
 (b) contacting the first cell culture with zinc to give a first reaction mixture;  
 (c) contacting the first reaction mixture and the second cell culture with the candidate agent; and  
 (d) comparing the amount of amyloid and zinc-induced AO aggregates in each cell culture, thereby determining effectiveness of the candidate anti-amyloidotic agent.  
 
     
     
         33 . A kit for determining whether a compound inhibits formation of Aβ amyloid which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein 
 (a) the first container means contains a peptide comprising at least the region of the Aβ peptide from amino acid number 6 to 28; and  
 (b) a second container means contains a heavy metal cation.  
 
     
     
         34 . The kit of  claim 33 , wherein said Aβ peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 μM.  
     
     
         35 . The kit of  claim 34 , wherein said concentration is about 10 to about 25 μM.  
     
     
         36 . The kit of  claim 33 , wherein said Aβ peptide is present in lyophilized form.  
     
     
         37 . The kit of  claim 33 , wherein said heavy metal cation is present as a metallochloride solution, at a concentration above about 300 nM.  
     
     
         38 . The kit of  claim 37 , wherein said concentration is about 25 μM.  
     
     
         39 . The kit of  claim 37 , wherein said heavy metal cation is zinc.  
     
     
         40 . The kit of  claim 38 , wherein said heavy metal cation is zinc.  
     
     
         41 . The kit of  claim 33 , further comprising 
 (c) one or more container means containing standard solutions of chelators of heavy metal cations.    
     
     
         42 . The kit of claim  41 , further comprising 
 (d) one or more container means containing standard solutions of amyloid-staining dyes.