Abeta42 LOWERING AGENTS

Abstract

The invention provides a method of preventing, delaying, or reversing the progression of Alzheimer's disease by administering an Aβ 42 lowering agent to a mammal under conditions in which levels of Aβ 42 are selectively reduced, levels of Aβ 38 are increased, and levels of Aβ 40 are unchanged. The invention provides methods and materials for developing and identifying Aβ 42 lowering agents. In addition, the invention provides methods for identifying agents that increase the risk of developing, or hasten progression of, Alzheimer's disease. The invention also provides compositions of Aβ 42 lowering agents and antioxidants, Aβ 42 lowering agents and non-selective secretase inhibitors, as well as Aβ 42 lowering agents and acetylcholinesterase inhibitors. The invention also provides kits containing Aβ 42 lowering agents, antioxidants, non-selective secretase inhibitors, and/or acetylcholinesterase inhibitors as well as instructions related to dose regimens for Aβ 42 lowering agents, antioxidants, non-selective secretase inhibitors, and acetylcholinesterase inhibitors.

1 . A method of identifying an Aβ 42  lowering agent comprising: (a) contacting a compound with a biological composition that produces Aβ 42  and Aβ 40 ; (b) comparing the ratio of Aβ 42  to Aβ 40  in said biological composition contacted with said candidate Aβ 42  lowering agent to the ratio of Aβ 42  to Aβ 40  in said biological composition not contacted with said compound; (c) identifying said compound as an Aβ 42  lowering agent if a reduction in the ratio of Aβ 42  to Aβ 40  in said biological composition contacted with said compound is observed when compared with the ratio of Aβ 42  to Aβ 40  in said biological composition not contacted with said compound.  
     
     
         2 . The method of  claim 1 , wherein said biological composition that produces Aβ 42  comprises a cell-free composition, an in vitro cell-based composition, or an in vivo animal-based composition.  
     
     
         3 . The method of  claim 2  wherein said biological composition is a cell-free composition.  
     
     
         4 . The method of  claim 3  wherein said cell-free biological composition comprises a purified enzyme preparation, a partially purified enzyme preparation, or a cell lysate.  
     
     
         5 . The method of  claim 2 , wherein said biological composition is an in vitro cell-based composition.  
     
     
         6 . The method of  claim 2 , wherein said biological composition is an in vitro cell-based composition or an in vivo animal-based composition, and wherein said comparing the ratio of Aβ 42  to Aβ 40  in said biological composition contacted with said candidate Aβ 42  lowering agent to the ratio of Aβ 42  to Aβ 40  in said biological composition not contacted with said compound comprises measuring the level of secreted Aβ 42  and Aβ 40 .  
     
     
         7 . The method of  claim 5 , wherein said in vitro cell-based biological composition is a cell culture.  
     
     
         8 . The method of  claim 5 , wherein said in vitro cell-based biological composition comprises cells that express one or more familial Alzheimer's disease genes selected from the group consisting of a mutant amyloid precursor protein (APP) gene, a mutant presenilin-1 gene, and a mutant presenilin-2 gene.  
     
     
         9 . The method of  claim 5 , wherein said in vitro cell-based biological composition comprises cells that express a mutant APP having the Swedish mutation (APP695NL).  
     
     
         10 . The method of  claim 7 , wherein the cells of said cell culture secrete one or more Aβ peptides selected from the group consisting of Aβ 34 , Aβ 36 , Aβ 37 , Aβ 38 , Aβ 39 , Aβ 40  and Aβ 42 .  
     
     
         11 . The method of  claim 7 , wherein the cells of said cell culture secrete Aβ 42 .  
     
     
         12 . The method of  claim 1 , wherein the step of comparing the ratio of Aβ 42  to Aβ 40  produced by said biological composition comprises determining the level of Aβ 42  and Aβ 40  with one or more techniques selected from the group consisting of an immunoprecipitation, western hybridization, sandwich enzyme-linked immunosorbent assays (ELISA), and mass-spectrometry.  
     
     
         13 . The method of  claim 1 , wherein said Aβ 42  to Aβ 40  ratio is determined by measuring Aβ 42  and Aβ 40  levels with an antibody based assay.  
     
     
         14 . The method of  claim 2 , wherein said biological composition is an in vivo animal-based composition.  
     
     
         15 . The method of  claim 1 , wherein said biological composition is a transgenic animal.  
     
     
         16 . The method of  claim 15 , wherein said transgenic animal expresses one or more familial Alzheimer's disease genes.  
     
     
         17 . The method of  claim 16 , wherein the one or more familial Alzheimer's disease genes are selected from a mutant APP, a mutant presenilin-1, and a mutant presenilin-2.  
     
     
         18 . The method of  claim 17 , wherein the familial Alzheimer's disease gene is APP having the Swedish mutation (APP695NL).  
     
     
         19 . The method of  claim 1 , wherein said biological composition comprises mammalian cells expressing APP are cultured under conditions that allow for APP expression, APP processing, and Aβ 42  secretion from said mammalian cells.  
     
     
         20 . The method of  claim 1 , wherein said biological composition comprises a cell line that expresses an APP selected from the group consisting of APP695 and APP751.  
     
     
         21 . The method of  claim 14 , wherein said cells are selected from the group consisting of CHO cells, human neuroglioma cells, fibroblast cells, HEK293 cells, and HS683 cells.  
     
     
         22 . The method of  claim 1 , wherein said biological composition comprises an enzyme.  
     
     
         23 . The method of  claim 1 , wherein said Aβ 42  lowering agent does not substantially change the level of Aβ 40 .  
     
     
         24 . The method of  claim 1 , wherein said Aβ 42  lowering agent increases the level of one or more of Aβ 34 , Aβ 36 , Aβ 37 , Aβ 38 , and Aβ 39 .  
     
     
         25 . A method for identifying an Aβ 42  lowering agent comprising: (a) providing a candidate Aβ 42  lowering agent which is a NSAID, NSAID derivative, or NSAID analogue; and (b) determining the effects of said candidate Aβ 42  lowering agent on levels of Aβ 42  in a biological composition following contact of said candidate Aβ 42  lowering agent with said biological composition, wherein a decrease in the level of Aβ 42  indicates that said candidate Aβ 42  lowering agent is an Aβ 42  lowering agent.  
     
     
         26 . The method of  claim 1 , wherein said compound is an NSAID is selected from the group consisting of amino aryl carboxylic acid derivatives, aryl acetic acid derivatives, and aryl propionic acid derivatives.  
     
     
         27 . The method of  claim 1 , wherein said compound is an NSAID derivative is selected from the group consisting of a derivative of an amino aryl carboxylic acid derivative, a derivative of an aryl acetic acid derivative, and a derivative of an aryl propionic acid derivative.  
     
     
         28 . The method of  claim 1 , wherein said compound is an NSAID analogue is selected from the group consisting of an analogue of an amino aryl carboxylic acid derivative, an analogue of an aryl acetic acid derivative, and an analogue of an aryl propionic acid derivative.  
     
     
         29 . The method of  claim 1 , wherein said compound is an NSAID, NSAID derivative, or NSAID analogue selected from the group consisting of NPPB, mefenamic acid, APHS, reservatrol, SC560, NS398, guaiazulene, ketorolac, benzylamine, ketoprofen, fenbufen, isoixicam, tenoxicam, tolfenamic acid, acemetacin, niflumic acid, dapsone, sulindac sulfone, nimesulide, suxibuzone, acetylsalicyclic acid, salicylic acid, carprofen, celecoxib, rofecoxib, fenoprofen, flurbiprofen, ibuprofen, naproxen, sulindac, sulindac sulfide, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, meloxicam, flufenamic acid, meclofenamic acid, and indomethacin.  
     
     
         30 . The method of  claim 1 , wherein said compound is a derivative or analogue of a NSAID selected from meclofenamic acid and flufenamic acid, wherein said NSAID derivative or analogue is modified from said NSAID by altering the position of the carboxylic acid group on the phenyl ring of said NSAID, altering the position or type of substituents on the phenyl ring opposite the carboxylic acid group of said NSAID, altering the bond connecting the two phenyl rings of said NSAID, altering the carboxylic acid group of said NSAID to propionic acid or another substituent, or performing any combination of these alterations, to generate a candidate Aβ 42  lowering agent.  
     
     
         31 . The method of  claim 1 , wherein said compound is a derivative or analogue of a NSAID selected from fenoprofen, flurbiprofen, and carprofen, wherein said NSAID derivative or analogue is modified from said NSAID by altering the position of the propionic acid group on the phenyl ring of said NSAID, altering the position or type of substituents on the phenyl ring opposite the propionic acid group of said NSAID, altering the bond connecting the two phenyl rings of said NSAID, altering the acetic acid group of said NSAID to carboxylic acid or another substituent, or performing any combination of these alterations, to generate a candidate Aβ 42  lowering agent.  
     
     
         32 . The method of  claim 1 , wherein said candidate Aβ 42  lowering agent is a derivative or analogue of indomethacin, wherein said indomethacin derivative or analogue is modified from said indomethacin by altering the carboxylic acid group of indomethacin to another substituent, altering the indole nitrogen to another substituent, or performing any combination of these alterations to generate a candidate Aβ 42  lowering agent.  
     
     
         33 . A method of identifying an Aβ 42  lowering agent comprising: (a) contacting a compound with a biological composition that secretes Aβ 42  and Aβ 40 ; (b) comparing the ratio of secreted Aβ 42  to Aβ 40  in said biological composition contacted with said candidate Aβ 42  lowering agent to the ratio of secreted Aβ 42  to Aβ 40  in said biological composition not contacted with said compound; (c) identifying said compound as an Aβ 42  lowering agent if a reduction in the ratio of secreted Aβ 42  to Aβ 40  in said biological composition contacted with said compound is observed when compared with the ratio of secreted Aβ 42  to Aβ 40  in said biological composition not contacted with said compound.  
     
     
         34 . The method of  claim 33 , wherein said biological composition that secretes Aβ 42  comprises in vitro cell-based composition or an in vivo animal-based composition.  
     
     
         35 . The method of  claim 34 , wherein said biological composition is an in vitro cell-based composition.  
     
     
         36 . The method of  claim 35 , wherein said in vitro cell-based biological composition is a cell culture.  
     
     
         37 . The method of  claim 35 , wherein said in vitro cell-based biological composition comprises cells that express one or more familial Alzheimer's disease genes selected from the group consisting of a mutant amyloid precursor protein (APP) gene, a mutant presenilin-1 gene, and a mutant presenilin-2 gene.  
     
     
         38 . The method of  claim 37 , wherein said in vitro cell-based biological composition comprises cells that express a mutant APP having the Swedish mutation (APP695NL).  
     
     
         39 . The method of  claim 38 , wherein the cells of said cell culture secrete one or more Aβ peptides selected from the group consisting of Aβ 34 , Aβ 36 , Aβ 37 , Aβ 38 , Aβ 39 , Aβ 40  and Aβ 42 .  
     
     
         40 . The method of  claim 39 , wherein the cells of said cell culture secrete Aβ 42 .  
     
     
         41 . The method of  claim 33 , wherein the step of comparing the ratio of Aβ 42  to Aβ 40  produced by said biological composition comprises determining the level of Aβ 42  and Aβ 40  with one or more techniques selected from the group consisting of an immunoprecipitation, western hybridization, sandwich enzyme-linked immunosorbent assays (ELISA), and mass-spectrometry.  
     
     
         42 . The method of  claim 33 , wherein said Aβ 42  to Aβ 40  ratio is determined by measuring Aβ 42  and Aβ 40  levels with an antibody based assay.  
     
     
         43 . The method of  claim 34 , wherein said biological composition is an in vivo animal-based composition.  
     
     
         44 . The method of  claim 33  wherein said biological composition is a transgenic animal.  
     
     
         45 . The method of  claim 44 , wherein said transgenic animal expresses one or more familial Alzheimer's disease genes.  
     
     
         46 . The method of  claim 45 , wherein the one or more familial Alzheimer's disease genes are selected from a mutant APP, a mutant presenilin-1, and a mutant presenilin-2.  
     
     
         47 . The method of  claim 46 , wherein the familial Alzheimer's disease gene is APP having the Swedish mutation (APP695NL).  
     
     
         48 . The method of  claim 33 , wherein said biological composition comprises mammalian cells expressing APP are cultured under conditions that allow for APP expression, APP processing, and Aβ 42  secretion from said mammalian cells.  
     
     
         49 . The method of  claim 33 , wherein said biological composition comprises a cell line that expresses an APP selected from the group consisting of APP695 and APP751.  
     
     
         50 . The method of  claim 33 , wherein said cells are selected from the group consisting of CHO cells, human neuroglioma cells, fibroblast cells, HEK293 cells, and HS683 cells.  
     
     
         51 . The method of  claim 33 , wherein said biological composition comprises an enzyme.  
     
     
         52 . The method of  claim 33 , wherein said Aβ 42  lowering agent does not substantially change the level of Aβ 40 .  
     
     
         53 . The method of  claim 33 , wherein said Aβ 42  lowering agent increases the level of one or more of Aβ 34 , Aβ 36 , Aβ 37 , Aβ 38 , and Aβ 39 .  
     
     
         54 . A method for identifying an Aβ 42  lowering agent comprising: (a) providing a candidate Aβ 42  lowering agent which is a NSAID, NSAID derivative, or NSAID analogue; and (b) determining the effects of said candidate Aβ 42  lowering agent on levels of secreted Aβ 42  on a biological composition following contact of said candidate Aβ 42  lowering agent with said biological composition, wherein a decrease in the level of secreted Aβ 42  indicates that said candidate Aβ 42  lowering agent is an Aβ 42  lowering agent.  
     
     
         55 . The method of  claim 33 , wherein said compound is an NSAID is selected from the group consisting of amino aryl carboxylic acid derivatives, aryl acetic acid derivatives, and aryl propionic acid derivatives.  
     
     
         56 . The method of  claim 33 , wherein said compound is an NSAID derivative is selected from the group consisting of a derivative of an amino aryl carboxylic acid derivative, a derivative of an aryl acetic acid derivative, and a derivative of an aryl propionic acid derivative.  
     
     
         57 . The method of  claim 33 , wherein said compound is an NSAID analogue is selected from the group consisting of an analogue of an amino aryl carboxylic acid derivative, an analogue of an aryl acetic acid derivative, and an analogue of an aryl propionic acid derivative.  
     
     
         58 . The method of  claim 33 , wherein said compound is an NSAID, NSAID derivative or NSAID analogue of an NSAID selected from the group consisting of NPPB, mefenamic acid, APHS, reservatrol, SC560, NS398, guaiazulene, ketorolac, benzylamine, ketoprofen, fenbufen, isoixicam, tenoxicam, tolfenamic acid, acemetacin, niflumic acid, dapsone, sulindac sulfone, nimesulide, suxibuzone, acetylsalicyclic acid, salicylic acid, carprofen, celecoxib, rofecoxib, fenoprofen, flurbiprofen, ibuprofen, naproxen, sulindac, sulindac sulfide, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, meloxicam, flufenamic acid, meclofenamic acid, and indomethacin.  
     
     
         59 . The method of  claim 33 , wherein said compound is a derivative or analogue of a NSAID selected from meclofenamic acid and flufenamic acid, wherein said NSAID derivative or analogue is modified from said NSAID by altering the position of the carboxylic acid group on the phenyl ring of said NSAID, altering the position or type of substituents on the phenyl ring opposite the carboxylic acid group of said NSAID, altering the bond connecting the two phenyl rings of said NSAID, altering the carboxylic acid group of said NSAID to propionic acid or another substituent, or performing any combination of these alterations, to generate a candidate Aβ 42  lowering agent.  
     
     
         60 . The method of  claim 33 , wherein said compound is a derivative or analogue of a NSAID selected from fenoprofen, flurbiprofen, and carprofen, wherein said NSAID derivative or analogue is modified from said NSAID by altering the position of the propionic acid group on the phenyl ring of said NSAID, altering the position or type of substituents on the phenyl ring opposite the propionic acid group of said NSAID, altering the bond connecting the two phenyl rings of said NSAID, altering the acetic acid group of said NSAID to carboxylic acid or another substituent, or performing any combination of these alterations, to generate a candidate Aβ 42  lowering agent.  
     
     
         61 . The method of  claim 33 , wherein said compound is a derivative or analogue of indomethacin, wherein said indomethacin derivative or analogue is modified from said indomethacin by altering the carboxylic acid group of indomethacin to another substituent, altering the indole nitrogen to another substituent, or performing any combination of these alterations to generate a candidate Aβ 42  lowering agent.  
     
     
         62 . The method of  claim 33 , wherein said compound is a derivative or analogue of sulindac sulfide, wherein said sulindac sulfide derivative or analogue is modified from said sulindac sulfide by altering the methylthiol group of sulindac sulfide to another substituent, altering the propionic acid group of sulindac sulfide to another substituent, altering the fluoride moiety of sulindac sulfide to another substituent, or performing any combination of these alterations, to generate a candidate Aβ 42  lowering agent.