WO2004112700A2

WO2004112700A2 - Methods and compositions for modulating amyloid precursor protein translation

Info

Publication number: WO2004112700A2
Application number: PCT/US2004/018158
Authority: WO
Inventors: Jack Rogers; Sandra Payton; Steve Gullans; Jeff Randall; Satinder Sarang
Original assignee: The General Hospital Corporation; The Brigham And Women's Hospital, Inc.
Priority date: 2003-06-06
Filing date: 2004-06-07
Publication date: 2004-12-29
Also published as: WO2004112700A8; WO2004112700A3

Abstract

The invention relates to products and methods for modulating amyloid precursor protein (APP) translation and/or amyloid ß(Aß) secretion. The methods of the invention are useful to prevent and treat APP translation and/or Aß secretion-associated neurological disorders such as Alzheimer’s disease, Down’s syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

Description

METHODS AND COMPOSITIONS FOR MODULATING AMYLOID PRECURSOR PROTEIN TRANSLATION

Related Application

This application claims the benefit of US Provisional Patent Application serial number 60/476,720 filed June 6, 2003, the entire contents of which is incorporated by reference herein.

Field of the Invention

The invention relates to methods and products for modulating production of amyloid β in the nervous system. The invention is useful for preventing and treating amyloid /3 production associated diseases such as Alzheimer's disease. The invention also relates in part to assays that are useful for identifying and testing candidate compounds for modulation production of amyloid β.

Background of the Invention

Alzheimer's disease (AD) is a disorder that causes the gradual loss of brain cells. AD is named after Dr. Alois Alzheimer, who in 1906 noticed changes in the brain tissue of a woman who had died of an unusual mental illness. Upon examine, Dr. Alzheimer found abnormal clumps and tangled bundles of fibers, which are now known as amyloid plaques and neurofibrillary tangles, respectively. Today, these plaques and tangles in the brain are considered hallmarks of AD.

AD results in damage in brain regions associated with thought, memory, and language. Symptoms of AD are progressive and include dementia, which includes characteristics such as loss of memory, problems with reasoning or judgment, disorientation, difficulty in learning, loss of language skills, and decline in the ability to perform routine tasks. Additional AD symptoms may include personality changes, agitation, anxiety, delusions, and hallucinations. The risk of AD in the population increases with age. It is believed that up to 4 million

Americans have AD. The onset of AD is generally after age 60, but in rare instances younger individuals may be afflicted. It is generally believed that approximately 3 percent of men and women ages 65 to 74, and almost half of those age 85 and older have AD. The intracellular and extracellular plaque formation by amyloid β (Aβ) in the hippocampus is an important hallmark in Alzheimer's disease. Associated with the formation of plaque is the transcription of amyloid precursor protein (APP) and the secretion of Aβ. There is ongoing research into identifying compounds that are effective for preventing or reducing plaque formation and that are suitable for use in human patients. Problems have arisen regarding toxicity of new Alzheimer's drugs. Thus, there are very limited treatment options available for patients suspected of having and/or diagnosed as having AD. Several drugs have been approved in the US for treatment of early and mid-stage AD, but they have significant detrimental side effects and limited efficacy. The lack of effective treatments for AD means that the therapeutic options are quite limited. Thus, there is a significant need for effective compounds and methods for preventing and/or treating AD.

Summary of the Invention

We have identified FDA-approved drugs that have unexpected properties of being able to modulate the translation of the Alzheimer's amyloid precursor protein (APP) gene and/or the secretion of Aβ. The invention relates in part to the use of these newly identified compounds in methods and compositions to prevent and/or treat Alzheimer's disease. We have identified di-mercaptopropanol, docusate, quinoline gluconate, tamsulosin, and netilmicin as compounds that modulate the translation of the APP gene and/or the secretion of Aβ. Di-mercaptopropanol, docusate, quinoline gluconate, tamsulosin, and netilmicin have not been used in methods to modulate translation of the APP and/or Aβ secretion and the invention includes the use of these compounds to reduce the secretion of Aβ and in the treatment of AB secretion-associated disorders.

Thus, the invention includes methods for treating disorders resulting from or including aberrant translation of the APP gene and/or the secretion of A/3 and compositions for treating such disorders. In addition we have identified methods of assaying candidate compounds for the ability to modulate translation of the APP gene and/or the secretion of Aβ.

According to one aspect of the invention, methods for preventing or treating an Aβ- associated disease in a subject are provided. The methods include administering to a subject in need of such treatment an effective amount of a Group 1 A/3-modulating compound to treat the AjS-associated disease, wherein the subject is otherwise free of indications for treatment with the Group 1 Aj8-modulating compound that is administered. In some embodiments, the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In certain embodiments, the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In some embodiments, the Group 1 Aβ- modulating compound is quinoline gluconate. In some embodiments, the Group 1 A/3- modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In certain embodiments, the Group 1 Aj8-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A3 secretion. In some embodiments of the foregoing aspects of the invention, the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. In some embodiments of the foregoing aspects of the invention, the subject is human. In some embodiments of the foregoing aspects of the invention the A/3-modulating compound is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In some embodiments of the foregoing aspects of the invention, the A/3-modulating compound is a pro-drug. In some embodiments of the foregoing aspects of the invention the A/3-modulating compound is administered prophylactically to a subject at risk of having an A/3-associated disease. In certain embodiments of the foregoing aspects of the invention the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration. In some embodiments of the foregoing aspects of the invention the A 3-modulating compound is administered in combination with an additional drug for treating an A/3-associated disease. In some embodiments of the foregoing aspects of the invention also include administering a A/3-modulating compound selected from the group consisting of Group 2 A/3-modulating compounds and Group 3 A/3-modulating compounds. In some embodiments, the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In certain embodiments, the Group 3 Aβ- modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetyltransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP translation and/or A/3 secretion. According to another aspect of the invention, methods for treating a subject having a condition characterized by A/3 production are provided. The methods include administering to a subject in need of such treatment a Group 1 A/3-modulating compound, in an amount effective to decrease APP translation and/or Aβ secretion, wherein the subject is free of symptoms otherwise calling for treatment with the A/3-modulating compound that is administered. In some embodiments, the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In certain embodiments, the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is quinoline gluconate. In some embodiments, the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In some embodiments, the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion.

In some embodiments of the foregoing aspects of the invention, the condition characterized by A/3 production is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. h some embodiments of the foregoing aspects of the invention, the subject is human. In some embodiments, the A/3-modulating compound is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In some embodiments, the A/3-modulating compound is administered prophylactically to a subject at risk of having the condition characterized by Aβ production. In certain embodiments, the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration. In some embodiments, the A/3-modulating compound is administered in combination with an additional drug for treating an Aβ- associated disease. In some embodiments, the methods also include comprising administering an A/3-modulating compound selected from the group consisting of Group 2 A/3-modulating compounds and Group 3 A/3-modulating compounds. In some embodiments the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 3 A/3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetyltransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP translation and/or A/3 secretion.

According to another aspect of the invention, methods of evaluating the effect of candidate pharmacological agents on APP translation and/or Aβ secretion are provided. The methods include contacting a cell sample with a candidate pharmacological agent; determining the effect of the candidate pharmacological agent on the level of APP translation and/or A/3 secretion in the cell sample relative to the level of APP translation and/or A/3 secretion in a cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the level of APP translation and/or Aβ secretion in the test cell sample as compared with the control level indicates modulation of APP translation and/or Aβ secretion by the candidate pharmacological agent. In some embodiments, the cell samples comprise a cell that produces A/3. In some embodiments, the cell samples comprise a cell that is capable of producing A/3. In certain embodiments, the cell samples comprise a pGALA-transfected neuroblastoma cell with a reporter gene construct. In some embodiments, the reporter gene construct is a Green Fluorescent Protein construct. In certain embodiments, the amount of APP translation and/or AjS secretion is determined by measuring the level of Green Fluorescent Protein (GFP) expression in the cell samples. In some embodiments, a relative decrease in the level of APP translation and/or Aβ secretion in the cell sample contacted with the candidate pharmacological agent compared to the level of APP translation and/or A/3 secretion in the cell sample not contacted with the candidate pharmacological agent indicates the candidate pharmacological agent is an APP translation- inhibiting and/or A/3 secretion-inhibiting agent. In certain embodiments, a relative increase in the level of APP translation and/or Aβ secretion in the cell sample contacted with the candidate pharmacological agent compared to the level of APP translation and/or Aβ secretion in the cell sample not contacted with the candidate pharmacological agent indicates that the modulator is an APP translation-enhancing and/or A/3 secretion-enhancing agent.

According to another aspect of the invention, methods for preparing an animal model of a disease characterized by APP translation and/or A/3 secretion are provided. The methods include introducing an APP translation-enhancing and/or Aβ secretion-enhancing agent into a non-human animal. In some embodiments, the methods also include detecting in the non- human animal symptoms of a disorder characterized by APP translation and/or Aβ secretion. In some embodiments, the animal model is a model for a neurological disease. In some embodiments, the neurological disease selected from the group consisting of: Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. According to yet another aspect of the invention kits for treating a subject in accordance with the methods of any of the foregoing aspects of the invention are provided. The kits include a package housing a first container containing at least one dose of a Group 1 A/3-modulating compound, and instructions for using the A/3-modulating compound in the prevention and/or treatment of an A/3-associated disease. In some embodiments, the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In some embodiments, the Group 1 Aβ- modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is quinoline gluconate. In some embodiments, the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. According to some of the foregoing embodiments of the invention, the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with

Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. In some embodiments, the kits also include a container containing at least one dose of a Group 2 A/3-modulating compound selected from the group consisting of paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion and instructions for using the compound for prevention and/or treatment of an A/3-associated disease. In some embodiments, the kits also include a container containing at least one dose of a Group 3 A/3-modulating compound selected from the group consisting of tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP translation and/or A/3 secretion, and instructions for using the compound for prevention and/or treatment of an A/3-associated disease. In some embodiments, the A/3-modulating compound is formulated for delivery to neuronal cells. In some embodiments, the A/3- modulating compound is formulated for sustained release. According to another aspect of the invention, compositions are provided. The compositions include a Group 1 A/3-modulating compound and a Group 2 A/3-modulating compound and/or a Group 3 A/3-modulating compound. In some embodiments, the Group 1 A/3-modulating compound is dimercaptopropanol, docusate, quinoline gluconate, tamsulosin, or netilmicin, or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion. In certain embodiments, the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, or diphenylhydramine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 3 A/3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, or pilocarpine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. hi some embodiments, the compound is linked to a targeting molecule. In certain embodiments, the targeting molecule's target is a neuronal cell. In some embodiments, the compound is formulated for implantation, mucosal administration, injection, inhalation, or oral administration.

According to another aspect of the invention, compositions are provided. The compositions include a Group 2 A/3-modulating compound and a Group 3 A/3-modulating compound. In some embodiments, the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, or diphenylhydramine, or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion. In some embodiments, the Group 3 A/3- modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, or pilocarpine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. In some embodiments, the compound is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In certain embodiments, the compound is formulated for implantation, mucosal administration, injection, inhalation, or oral administration.

According to yet another aspect of the invention, therapeutic methods are provided. The methods include administering a Group 1 A/3-modulating compound to a subject based on a diagnosis of an A/3-associated disease in the subject, in an amount effective to treat the disease. In some embodiments, the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In certain embodiments, the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is quinoline gluconate. In some embodiments, the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion, h certain embodiments, the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

In some embodiments of the foregoing aspects of the invention, the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. In some embodiments of the foregoing aspects of the invention, the subject is human. In some embodiments of the foregoing aspects of the invention the A/3-modulating compound is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In some embodiments of the foregoing aspects of the invention, the A/3-modulating compound is a pro-drug, hi some embodiments of the foregoing aspects of the invention the A/3-modulating compound is administered prophylactically to a subject at risk of having an A -associated disease. In certain embodiments of the foregoing aspects of the invention the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration. In some embodiments of the foregoing aspects of the invention the A/3-modulating compound is administered in combination with an additional drug for treating an A/3-associated disease. In some embodiments of the foregoing aspects of the invention also include administering a A/3-modulating compound selected from the group consisting of Group 2 A/3-modulating compounds and Group 3 A/3-modulating compounds. In some embodiments, the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion. In certain embodiments, the Group 3 A/3- modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP translation and/or A/3 secretion.

According to yet another aspect of the invention, therapeutic methods are provided. The methods include administering a Group 1 A/3-modulating compound to a subject based on a diagnosis of a condition characterized by A/3 production, in an amount effective to decrease APP translation and/or A3 secretion. In some embodiments, the Group 1 Aβ- modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In certain embodiments, the Group 1 A/3- modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is quinoline gluconate. In some embodiments, the Group 1 A -modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

In some embodiments of the foregoing aspects of the invention, the condition characterized by A/3 production is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. In some embodiments of the foregoing aspects of the invention, the subject is human. In some embodiments, the A 3-modulating compound is linked to a targeting molecule. In some embodiments, the targeting molecule's target is a neuronal cell. In some embodiments, the A/3-modulating compound is administered prophylactically to a subject at risk of having the condition characterized by A/3 production. In certain embodiments, the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration. In some embodiments, the A -modulating compound is administered in combination with an additional drug for treating an Aβ- associated disease. In some embodiments, the methods also include comprising administering an A/3-modulating compound selected from the group consisting of Group 2 A/3-modulating compounds and Group 3 A/3-modulating compounds. In some embodiments the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion. In some embodiments, the Group 3 A/3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetyltransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP translation and/or AjS secretion.

The use of the foregoing compositions in the preparation of a medicament, particularly a medicament for prevention and/or treatment of an A/3-associated disorder. These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Drawings

Fig. 1. shows a diagram of the APP untranslated region, which is a target for small molecules Fig. IB and Fig. 1C are drawings of the chemical structures of the small molecules desferrioxamine and phenserine, respectively. These molecules exemplify small compounds that interfere with the activation of APP5'UTR by its cognate binding protein, iron-regulatory protein (IRP) to efficiently translate APP. Fig. 1 A illustrates the predicted secondary structure folded from the 5' untranslated region of APP mRNA (SEQ ID NO:l) (Zuker et al., Science 244:48-52, 1989).

Fig. 2 provides drawings of the APP 5 'UTR stemloop and screening constructions. Fig. 2A shows the APP 5 'UTR stemloop (SEQ ID NO:2) with the homology to the ferritin Iron-responsive Element shown in bold lettering. Fig. 2B shows drawings of the luciferase constructs used to screen for FDA-preapproved medicinal compounds. The luciferase expression constructs pGAL (APP 5 'UTR) and pGALA (APP 5' & 3' UTR), are derived from pGL3 (Promega, Madison, WT). The luciferase gene is translationally driven by APP 5 'UTR and 3 'UTR regulatory sequences. These constructs were used to screen for FDA- drugs that suppress APP 5 'UTR directed translation.

Fig. 3 shows results indicating the effect of paroxetine on the intracellular level of

APP. Fig. 3 A shows digitized images of Western blots demonstrating that paroxetine significantly lowered intracellular APP levels (30% reduced at 5μM after 72 hour treatment of SY5Y neuroblastoma cells). Fig. 3B shows a diagram of the structure of paroxetine. Fig. 3C shows a graph illustrating the difference between the paroxetine affect on APP levels versus that seen in untreated cells.

Fig. 4 shows results indicating the effect of dimercaptopropanol on the intracellular level of APP. Fig. 4 A shows digitized images of Western blots demonstrating that dimercaptopropanol significantly lowered intracellular APP levels. Fig. 4B shows a diagram of the structure of dimercaptopropanol. Fig. 4C is a graph illustrating that at a dose of 0.1 μM dimercaptopropanol suppressed neuroblastoma APP levels by >50% compared to untreated neuroblastoma counterparts. At a lower dose of 0.01 μM dimercaptopropanol the APP levels were diminished by 50% relative to control levels.

Fig. 5 is a graph illustrating results of ELISA tests to determine the effect of 0.0/ M, lOμM, 15/xM, and 20μM concentrations of paroxetine on the secretion of A/3(l-40) in lens B3 cells. Secretion was reduced by up to 30% following a 4 day treatment with 15μM paroxetine.

Fig. 6 is a graph illustrating results of ELISA tests to determine the effect of O.OμM,

O.lμM, O.OlμM, and 0.001/ M concentrations of dimercaptopropanol on the secretion of A/3(l-40) in lens B3 cells. Cells were treated for 72 hours. Secretion was reduced by from 20% to 50% relative to control treated cells.

Fig. 7 is a graph illustrating results of ELISA tests to determine the effect of O.OμM,

O.lμM, 0.01/ M, and 0.00 lμM concentrations of dimercaptopropanol on the secretion of A/3(l-42) in lens B3 cells. Cells were treated for 72 hours. Secretion was reduced by from 20% to 50% relative to control-treated cells. Fig. 8 is a diagram of the dicistronic construct (pJR-1) used to screen for APP 5 'UTR directed drugs. Neuroblastoma cells were stably transfected with pJRl and cells were exposed to increasing concentrations of lead drugs candidates (lμM, lOμM, 100 μM). The percent inhibition values and IC-50 values of 6 lead drugs to suppress APP 5'UTR directed translation was calculated and each value was standardized by dicistronic GFP co-expression (standardizing ratio). A luciferase gene was used in place of the RFP gene shown. The sequence: gcgguggcggcgcgggcagagcaaggacgcggcggaucccacucgc is from the 5' UTR of APP and is SEQ ID NO:2.

Detailed Description of the Invention

The methods of the invention involve the administration of compounds that modulate the translation of amyloid precursor protein (APP) and/or the secretion of amyloid /3 (A/3) in neuronal tissues. The term "AjS" is used herein interchangeably with the term "Abeta" and each means amyloid β. As used herein the term "modulate" means enhance or inhibit. Compositions of the invention include compounds that modulate the level of translation of amyloid precursor protein (APP) and/or the secretion of amyloid β (A/3) in cells, tissues, and subjects. As used herein, the term "A/3-modulating compounds" means compounds that modulate the level of translation of amyloid precursor protein (APP) and/or the secretion of amyloid β (A/3) in cells, tissues, and subjects. The methods of the invention involve the administration of A/3-modulating compounds and therefore are useful to reduce or prevent the accumulation of A/3 in Alzheimer's disease and other disorders. As used herein, the term "A/3-associated disorder" includes, but is not limited to: Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis. We have discovered that the deleterious effects seen in these disorders that are triggered by the accumulation of A/3 can be ameliorated by the administration of the compositions of the invention. The compositions of the invention include compounds that modulate translation of amyloid precursor protein (APP) and/or the secretion of amyloid /3 (AjS) in cells and/or tissues, thereby reducing the cell and tissue damage and clinical manifestations of A/3-associated disorders.

As used herein, the term "subject" means any mammal that may be in need of treatment with an AjS-modulating compound of the invention. Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, rats, etc. Table 1 provides A/3-modulating compounds of the invention and includes: dimercaptopropanol, docusate, quinoline-gluconate, tamsulosin and netilmicin.

Table 1. A/3-modulating compounds.

The compounds dimercaptopropanol, docusate, quinoline gluconate, tamsulosin and netilmicin are known to act as agents in treatment of non-A/3-associated disorders. The foregoing compounds have never before been given to patients to modulate translation of amyloid precursor protein (APP) and/or the secretion of amyloid β (Aβ) in cells and/or tissues, and/or to treat or prevent A/3-associated disorders in subjects who are otherwise free of indications for their administration. Preferably, the dimercaptopropanol, docusate, quinoline-gluconate, tamsulosin and netilmicin compounds of the invention are administered to subjects that are free of indications for their previously determined use. By "free of indications for their previously determined use", it is meant that the subject does not have symptoms that call for treatment with one or more of the compounds of the invention for a previously determined use of that compound (see Table 1), other than the indication that exists as a result of this invention. As used herein the term "previously determined use" of a compound means the use of the compound that was previously identified. Thus, the previously determined use is not the use of modulating translation of amyloid precursor protein (APP) and/or the secretion of amyloid β (Aβ) in cells and/or tissues.

The methods of the invention include administration of an A/3-modulating compound that preferentially targets neuronal cells and/or tissues. In addition, the compounds can be specifically targeted to neuronal tissue (e.g. glial cells and/or neuronal cells) using various delivery methods, including, but not limited to: administration to neuronal tissue, the addition of targeting molecules to direct the compounds of the invention to neuronal tissues (e.g. glial cells and or neuronal cells), etc. Additional methods to specifically target molecules and compositions of the invention to brain tissue and/or neuronal tissues are known to those of ordinary skill in the art.

The invention involves, in part, the administration of a compound that modulates the level of translation of APP and/or the secretion of A/3 in cells, tissues, and/or subjects. As used herein, the term "translation of APP" means the translation of the APP gene and production of A/3 protein. As used herein, the term "secretion of A/3" means the release of AjS from cells. It is understood that the accumulation of intracellular AjS and extracellular plaque formation by A/3 is associated with AjS diseases such as Alzheimer's disease, and that the transcription of the APP gene is associated with the production of Aβ, hence plaque. As used herein the term "modulate" means either to inhibit or enhance. As used herein, the term "inhibit" means to decrease the level of franslation of APP and/or to decrease the level of secretion of A/3 to a level or amount that is statistically significantly less than a control level of APP translation and/or AjS secretion. In some cases, the decrease in the level of APP translation and/or A/3 secretion means the level of APP translation and/or A/3 secretion is reduced from an initial level to a level significantly lower than the initial level level. In some cases this reduced level may be zero. As used herein, the term "enhance" means to increase the level of translation of APP and/or the level of secretion of A/3 to a level or amount that is statistically significantly more than a control level of APP translation and/or A/3 secretion. In some cases, the increase in the level of APP translation and/or A/3 secretion means the level of APP translation and/or A/3 secretion is raised from zero to a level above zero, in other cases an increase in APP translation and/or A/3 secretion means an increase from a level that is above zero to a level significantly higher than that original or baseline level of activity.

A control level of APP franslation and/or A/3 secretion is the level that represents the normal level of APP translation and or A/3 secretion in a cell, tissue, and/or subject, hi some instances, a control level will be the level in a disorder-free cell, tissue, or subject, and may be useful, for example, to monitor an increase in the level of APP translation or A/3 secretion in a cell. In other instances a confrol level will be the level in a cell, tissue, or subject with a neurological disorder, e.g. an Alzheimer's disease, and may be useful, for example, to monitor a decrease in the level of APP translation or A/3 secretion in a cell. These types of control levels are useful in assays to assess the efficacy of an APP translation-modulating compound and/or A/3 secretion-modulating compound of the invention.

It will be understood by one of ordinary skill in the art that a control level of APP translation and/or A/3 secretion may be a predetermined value, which can take a variety of forms. It can be a single value, such as a median or mean. It can be established based upon comparative groups, such as in disease-free groups that have normal levels of APP translation and/or A/3 secretion. Other comparative groups may be groups of subjects with specific neurological disorders, e.g. Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, and inclusion body myositis. It will be understood that disease-free cells and/or tissues may be used as comparative groups for cells or tissues that have an A/3-associated disorder.

In some embodiments, a compound that inhibits and thereby reduces the level of APP translation and/or AjS secretion is an agent that reduces an A/3-associated disease or disorder. The level of APP translation and/or A/3 secretion may be one that is below the level seen in subjects with a neurological disorder, e.g. may be a level that is clinically asymptomatic. In other embodiments, a compound that enhances and thereby increases level of APP translation and/or A/3 secretion is an agent that increases or triggers the onset of an A/3-associated disease or disorder. Such an enhancing compound may be useful for the production of a cell or animal model of an APP translation and/or A/3 secretion-associated disorder. Such models are useful in research and for assays to assess potential diagnostic and/or treatment methods. The invention relates in part to the administration of a level of APP translation and/or A/3 secretion inhibiting compound of the invention in an amount effective to treat or prevent the production of A/3 plaques in cells, tissues, and/or subjects with an AjS-associated disease or disorder.

In some aspects of the invention, the APP translation-modulating and/or A/3 secretion- modulating compounds include functional analogs, derivatives, and variants of the APP translation-modulating and/or A/3 secretion-modulating compounds. For example, functional analogs, derivatives, and variants of the APP franslation-modulating and/or AjS secretion- modulating compounds of Table 1 can be made, for example, to enhance a property of a compound, such as stability. Functional analogs, derivatives, and variants of the compounds of Table 1 may also be made to provide a novel activity or property to a compound of Table 1, for example, to enhance detection, h some embodiments of the invention, modifications to an APP translation-modulating and/or A/3 secretion-modulating molecule of the invention, can be made to the structure or side groups of the compound and can include deletions, substitutions, and additions of atoms, or side groups. Alternatively, modifications can be made by addition of a linker molecule, addition of a detectable moiety, such as biotin or a fluorophore, chromophore, enzymatic, and/or radioactive label, and the like. Analogs of the dimercaptopropanol, docusate, quinoline gluconate, tamsulosin, and/or netilmicin molecules that retain some or all of the APP translation-modulating and/or A/3 secretion-modulating activity of the dimercaptopropanol, docusate, quinoline gluconate, tamsulosin, and/or netilmicin molecules, respectively, also can be used in accordance with the invention. In some embodiments, an analog of a molecule may have a higher level of APP translation-modulating and/or A/3 secretion-modulating activity than that molecule.

Chemical groups that can be added to or substituted in the molecules include: hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, acyl, amino, acyloxy, acylamino, carboalkoxy, carboxyamido, carboxyamido, halo and thio groups. Substitutions can replace one or more chemical groups or atoms on the molecules. Molecular terms, when used in this application, have their common meaning unless otherwise specified. The term "hydrido" denotes a single hydrogen atom (H). The term "acyl" is defined as a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group, examples of such radicals being acetyl and benzoyl. The term "amino" denotes a nitrogen radical containing two substituents independently selected from the group consisting of hydrido, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. The term "acyloxy" denotes an oxygen radical adjacent to an acyl group. The term "acylamino" denotes a nitrogen radical adjacent to an acyl group. The term "carboalkoxy" is defined as a carbonyl radical adjacent to an alkoxy or aryloxy group. The term "carboxyamido" denotes a carbonyl radical adjacent to an amino group. The term "carboxy" embraces a carbonyl radical adjacent to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group. The term "halo" is defined as a bromo, chloro, fluoro or iodo radical. The term "thio" denotes a radical containing a substituent group independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to a divalent sulfur atom, such as, methylthio and phenylthio.

The term "alkyl" is defined as a linear or branched, saturated radical having one to about ten carbon atoms unless otherwise specified. Preferred alkyl radicals are "lower alkyl" radicals having one to about five carbon atoms. One or more hydrogen atoms can also be replaced by a substitutent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkyl groups include methyl, tert-butyl, isopropyl, and mefhoxymethyl.

The term "alkenyl" embraces linear or branched radicals having two to about twenty carbon atoms, preferably three to about ten carbon atoms, and containing at least one carbon- carbon double bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkenyl groups include ethylenyl or phenyl ethylenyl.

The term "alkynyl" denotes linear or branched radicals having from two to about ten carbon atoms, and containing at least one carbon-carbon triple bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of alkynyl groups include propynyl.

The term "aryl" denotes aromatic radicals in a single or fused carbocyclic ring system, having from five to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of aryl groups include phenyl, naphthyl, biphenyl, and terphenyl. "Heteroaryl" embraces aromatic radicals which contain one to four hetero atoms selected from oxygen, nitrogen and sulfur in a single or fused heterocyclic ring system, having from five to fifteen ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of heteroaryl groups include, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups.

The term "cycloalkyl" is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of a cycloalkyl group include cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

The term "heterocyclyl" embraces a saturated or partially unsaturated ring containing zero to four hetero atoms selected from oxygen, nifrogen and sulfur in a single or fused heterocyclic ring system having from three to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfoxy, and formyl. Examples of a heterocyclyl group include morpholinyl, piperidinyl, and pyrrolidinyl. The term "alkoxy" denotes oxy-containing radicals substituted with an alkyl, cycloalkyl or heterocyclyl group. Examples include methoxy, tert-butoxy, benzyloxy and cyclohexyloxy. The term "aryloxy" denotes oxy-containing radicals substituted with an aryl or heteroaryl group. Examples include phenoxy. The term "sulfoxy" is defined as a hexavalent sulfur radical bound to two or three substituents selected from the group consisting of oxo, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein at least one of said substituents is oxo. The APP translation-modulating and/or A/3 secretion-modulating compounds of the invention also include, but are not limited to any pharmaceutically acceptable salts, esters, or salts of an ester of the compound. Examples of salts that may be used, which is not intended to be limiting include: chloride, acetate, hydrochloride, methansulfonate or other salt of a compound of Table 1 or a functional analog, derivative, variant, or fragment of the compound. Derivatives of the compounds of Table 1 include compounds which, upon administration to a subject in need of such administration, deliver (directly or indirectly) a pharmaceutically active APP translation-modulating and/or A/3 secretion-modulating compound as described herein. An example of pharmaceutically active derivatives of the invention includes, but is not limited to, pro-drugs. A pro-drug is a derivative of a compound that contains an additional moiety that is susceptible to removal in vivo yielding the parent molecule as a pharmacologically active agent. An example of a pro-drug is an ester that is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known to those of ordinary skill in the art and may be adapted to the present invention. Analogs, variants, and derivatives of the compounds in Table 1 of the invention may be identified using standard methods known to those of ordinary skill in the art. Useful methods involve identification of compounds having similar chemical structure, similar active groups, chemical family relatedness, and other standard characteristics. For the purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics 75^th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito. 1999, the contents of which are incorporated herein by reference in their entirety. Using the structures of the compounds disclosed herein, one of ordinary skill in the art is enabled to make predictions of structural and chemical motifs for analogs, variants, and/or derivatives that possess similar functions of the compounds disclosed in Table 1. Using structural motifs as search, evaluation, or design criteria, one of ordinary skill in the art is enabled to identify classes of compounds (functional variants of the APP translation- modulating and/or A/3 secretion-modulating molecules or agents) that possess the modulatory function of the compounds disclosed herein. These compounds may be synthesized using standard synthetic methods and tested for activity as described herein. The invention also involves methods for determining the functional activity of APP translation-modulating and/or A/3 secretion-modulating compounds described herein.- The function or status of a compound as an APP translation-modulating and/or A/3 secretion- modulating compound can be determined according to assays known in the art or described herein. For example, cells can be contacted with a candidate APP translation-modulating and/or AjS secretion-modulating compound under conditions that produce APP franslation and/or A/3 secretion, and standard procedures can be used to determine whether APP translation and/or A/3 secretion activity is modulated by the compound and/or whether the A/3 levels are modulated by the compound. Such methods may also be utilized to determine the status of analogs, variants, and derivatives as inhibitors of APP translation and/or AjS secretion. Although not intended to be limiting, an example of a method with which the ability of an APP franslation-modulating and/or A/3 secretion-modulating compound to modulate APP translation and/or A/3 secretion activity can be tested, is an in vitro assay system provided herein in the Examples section. Using such assays the level of APP translation and/or AjS secretion activity can be measured in the system both before and after contacting the system with a candidate APP translation-modulating and/or A/3 secretion-modulating compound as an indication of the effect of the compound on the level of APP translation and/or A/3 secretion. Secondary screens may further be used to verify the compounds identified as enhancers or inhibitors of APP translation and/or AjS secretion.

In addition, analogs of APP translation-modulating and/or A/3 secretion-modulating compounds can be tested for their APP translation and/or AjS secretion-modulating activity by using an activity assay (see examples). An example of an assay method, although not intended to be limiting, is contacting a tissue or cell sample with an APP translation- modulating and/or AjS secretion-modulating compound and determining the compound's modulatory activity as described herein. Contacting a similar cell or tissue sample with an analog of the APP translation-modulating and/or A/3 secretion-modulating compound, determining its activity, and then comparing the two activity results as a measure of the efficacy of the analog's APP translation-modulating and/or A/3 secretion-modulating activity. In addition to the in vitro assays described above, an in vivo assay may be used to determine the functional activity of APP franslation-modulating and/or A/3 secretion- modulating compounds described herein. In such assays, animal models of AjS-associated disease can be treated with an APP franslation-modulating and/or AjS secretion-modulating compound of the invention. APP translation and/or A/3 secretion activity and/or accumulation of AjS may be assayed using methods described herein, which may include labeling or imaging methods. In addition, the deposition of A/3 may be assayed more directly by histopathologic examination of brains. Additionally, animals with and without APP translation-modulating and/or A/3 secretion-modulating compound treatment can be examined for behavior and/or survival as an indication of the effectiveness and/or efficacy of the compounds. Behavior may be assessed by examination of symptoms of aberrant A/3 deposition as described herein. These measurements can then be compared to corresponding measurements in control animals. For example, test and control animals may be examined following adminisfration of an APP translation-modulating and/or AjS secretion-modulating compound (enhancer or inhibitor) of the invention. In some embodiments, test animals are administered an APP franslation-modulating and/or A/3 secretion-modulating compound of the invention and control animals are not. Any resulting change in AjS production and/or deposition can then be determined for each type of animal using known methods in the art as described herein. Such assays may be used to compare levels of APP translation and/or A/3 secretion in animals administered the candidate APP translation-modulating and/or AjS secretion-modulating compound to control levels of A/3 production in animals not administered the APP translation-modulating and/or AjS secretion-modulating compound as an indication that the putative APP franslation-modulating and/or AjS secretion-modulating compound is effective to modulate APP translation and/or A/3 secretion activity.

Once one or more APP franslation-modulating and/or AjS secretion-modulating compounds are verified as modulating APP translation and/or A/3 secretion using assays as described herein (e.g., in Examples), further biochemical and molecular techniques may be used to identify the targets of these compounds and to elucidate the specific roles that these target molecules play in the process of APP translation and or A/3 secretion and in AjS- associated disease. An example, though not intended to be limiting, is that the compound(s) may be labeled and contacted with a cell to identify the host cell proteins with which these compounds interact. Such proteins may be purified, e.g., by labeling the compound with an immunoaffinity tag and applying the protein-bound compound to an immunoaffinity column. An APP franslation-modulating and/or A/3 secretion-modulating compound of the invention maybe delivered to the cell using standard methods known to those of ordinary skill in the art. Various techniques may be employed for introducing APP translation-modulating and/or AjS secretion-modulating compounds of the invention to cells, depending on whether the compounds are introduced in vitro or in vivo in a host.

When administered, the APP translation-modulating and/or A/3 secretion-modulating compounds (also referred to herein as therapeutic compounds and/or pharmaceutical compounds) of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.

The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The characteristics of the carrier will depend on the route of adminisfration.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The adminisfration may, for example, be oral, intravenous, intraperitoneal, intrathecal, intramuscular, infranasal, intracavity, subcutaneous, intradermal, or transdermal.

The therapeutic compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for parenteral adminisfration conveniently comprise a sterile aqueous preparation of the therapeutic agent, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

Compositions suitable for oral adminisfration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the therapeutic agent. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.

In some embodiments of the invention, an APP translation-modulating and/or A/3 secretion-modulating compound of the invention may be delivered in the form of a delivery complex. The delivery complex may deliver the APP franslation-modulating and/or AjS secretion-modulating compound into any cell type, or may be associated with a molecule for targeting a specific cell type. Examples of delivery complexes include an APP franslation- modulating and/or AjS secretion-modulating compound of the invention associated with: a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., an antibody, including but not limited to monoclonal antibodies, or a ligand recognized by target cell specific receptor). Some complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex can be cleavable under appropriate conditions within the cell so that the APP franslation-modulating and/or Aβ secretion-modulating compound is released in a functional form. An example of a targeting method, although not intended to be limiting, is the use of liposomes to deliver an APP franslation-modulating and/or A/3 secretion-modulating compound of the invention into a cell. Liposomes may be targeted to a particular tissue, such neuronal cells, (e.g. hippocampal cells, etc) by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein. Such proteins include proteins or fragments thereof specific for a particular cell type, antibodies for proteins that undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.

For certain uses, it may be desirable to target the compound to particular cells, for example specific neuronal cells, including specific tissue cell types, e.g. hippocampal cells or other tissue-specific nervous system cells, hi such instances, a vehicle (e.g. a liposome) used for delivering an APP franslation-modulating and/or AjS secretion-modulating compound of the invention to a cell type (e.g. a neuronal cell) may have a targeting molecule attached thereto that is an antibody specific for a surface membrane polypeptide of the cell type or may have attached thereto a ligand for a receptor on the cell type. Such a targeting molecule can be bound to or incorporated within the APP translation-modulating and/or AjS secretion- modulating compound delivery vehicle. Where liposomes are employed to deliver the APP translation-modulating and/or A/3 secretion-modulating compounds of the invention, proteins that bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.

Liposomes are commercially available from Invifrogen, for example, as LΓPOFECTL ™ and LIP OFECTACE™, which are formed of cationic lipids such as N-[l- (2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammomum bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications.

The invention provides a composition of the above-described agents for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo. Delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the therapeutic agent of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as polylactic and polyglycolic acid, poly(lactide-glycolide), copolyoxalates, polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polycaprolactone. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; phospholipids; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

In one particular embodiment, the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. WO 95/24929, entitled "Polymeric Gene Delivery System", describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient. In accordance with the instant invention, the compound(s) of the invention is encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in WO 95/24929. The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compound is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the compound is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the compounds of the invention include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is selected according to the method of delivery which is to be used. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents and compounds of the invention of the invention to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.

In general, the agents and/or compounds of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and mefhacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl mefhacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Use of a long-term sustained release implant may be particularly suitable for treatment of subjects with an established neurological disorder conditions as well as subjects at risk of developing a neurological disorder. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active • ingredient for at least 7 days, and preferably 30-60 days, and most preferably months or years. The implant may be positioned at or near the site of the neurological damage or the area of the brain or nervous system affected by or involved in the neurological disorder. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dexfrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. In the case of preventing or treating a disorder or condition that results in aberrant production and/or deposition of AjS, the desired response is reducing the onset, stage or progression of the aberrant production and/or deposition. This may involve only slowing the progression of the damage temporarily, although more preferably, it involves halting the progression of the damage permanently. An effective amount for preventing and/or treating the aberrant production and/or deposition of AjS is that amount that reduces the amount or level of APP franslation and/or AjS secretion activity, when the cell or subject is a cell or subject with an A/3-associated disease, with respect to that amount that would occur in the absence of the active compound.

In other embodiments of the invention, (e.g. for making animal models), an effective amount of the pharmaceutical compound is that amount effective to enhance APP translation and/or A/3 secretion activity. Such enhancements can be determined using standard assays as described above herein. Measurements of APP translation and/or Aβ secretion activity and/or measurements of APP translation and/or A/3 secretion-related damage, are provided herein and are known to those of ordinary skill in the art and may vary depending on the specific AjS-associated disease. The pharmaceutical compound dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease or disorder. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. The pharmaceutical compounds of the invention may be administered alone, in combination with each other, and/or in combination with other drug therapies that are administered to subjects with neurological disorders or trauma. Additional drug therapies (for treatment and/or prophylaxis) that may be administered with pharmaceutical compounds of the invention include, but are not limited to: trophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurofrophin 3 (NT3), glial cell line- derived neurotrophic factor (GDNF), or ciliary neurotrophic factor (CNTF). Other growth factors that may be delivered to the brain and spinal cord include: neurofrophin 4/5 (NT4/5), leukemia inhibitory factor (LIF), cardiotrophin (CT-1), insulin-like growth factors 1 and 2 (IGF-1, IGF-2), transforming growth factor alpha (TGF-alpha), transforming growth factor beta 1-3 (TGF-betal, TGF beta2, TGF-beta3), neurturin (NTN), artemin (ART), persephin (PSP), acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), fibroblast growth factor-5 (FGF-5), platelet-derived growth factor (PDGF) and stem cell factor (SCF). Other drug therapies (for treatment and/or prophylaxis) that may be administered with pharmaceutical compounds of the invention include amyloid degrading enzymes for Alzheimer's Disease (e.g., the neprilysin (NEP) family of zinc metalloproteinases, such as NEP and endothelin-converting enzyme, insulysin, angiotensin- converting enzyme, matrix metalloproteinases, plasmin and thimet oligopeptidase (endopeptidase-24.15)); glutamate degrading enzymes; anti-oxidants including SODl, SOD2, glutathione peroxidase and catalase; anti-apoptotics including Bcl-2, CrmA, baculoviral IAPs and mammalian IAPs (inhibitor of apoptosis proteins including naip, xiap/hilp/miha, c- iapl/hiap-2/mihb, c-iap2/hiap-l/mihc); proteasome enhancers; kinase inhibitors; glutamate transport enhancers (e.g., EAAT2/ GLT1); glutamate metabolizers (e.g., glutamate decarboxylase); beta-amyloid protein antibodies; neurofransmitter synthesizing enzymes including GAD, choline acetyl-fransferase and tyrosine hydroxylase; compounds that inhibit caspase activity including caspase inhibitors (e.g., Z-Val-Ala-Asp-fluoromethylketone (Z- VAD-frnk); Z-VDVAD-fmk, Z-DEVD-fmk, and Z-Asp-cmk (Z-Asp-2,6-dichlorobenzoyl- oxymethylketone)), minocycline and dominant negative caspase mutants; haloperidol; phenothiazines; benzodiazepines; acetylcholine esterase inhibitors (including donepezil, rivastigmine and galantamine); tetrahydroacridinamine (Tacrine); beta- and gamma-secretase inhibitors; Abeta vaccines; Cu-Zn chelators; cholesterol-lowering drugs; non-steroidal anti- inflammatory drugs; carbidopa and/or levodopa with or without a catechol-O-methyl fransferase (COMT) inhibitors such as Comtan or Tasmar; dopamine agonists including pramipexole, pergolide, and ropinerol; amantadine; selegiline; gabapentin; lamotrigine; topiramate; vigabatrin; Rilutek® (riluzole); Neurontin® (gabapentin); cholinergic agents including pyridostigmine; beta blockers including timolol, levobunolol and betaxolol; parasympathomimetics including pilocarpine, carbachol and phospholine iodide; alpha agonists including apraclonidine, brimonidine and epinephrine; carbonic anhydrase inhibitors including dorzolamide and latanoprost.

The above-described drug therapies are known to those of ordinary skill in the art and are administered by modes known to those of skill in the art. The drug therapies are administered in amounts that are effective to achieve the physiological goals (to reduce symptoms and damage from A/3-associated disease in a subject, e.g. cell death), in combination with the pharmaceutical compounds of the invention. Thus, it is contemplated that the drug therapies may be administered in amounts which are not capable of preventing or reducing the physiological consequences of the A/3-associated disorder when the drug therapies are administered alone, but which are capable of preventing or reducing the physiological consequences of A/3-associated disease when administered in combination with the APP translation-modulating and/or AjS secretion-modulating compounds of the invention. Diagnostic tests known to those of ordinary skill in the art maybe used to assess the level of APP translation and/or A/3 secretion activity and or the level of A/3 in a subject and to evaluate a therapeutically effective amount of a pharmaceutical compound administered. Examples of diagnostic tests are set forth below. A first determination of APP translation and/or A/3 secretion activity and/or the level of A/3 in a cell and/or tissue may be obtained using one of the methods described herein (or other methods known in the art), and a second, subsequent determination of the level of APP translation and/or AjS secretion and/or A/3 level may be done. A comparison of the APP translation and/or AjS secretion activity and/or the level of A/3 may be used to assess the effectiveness of adminisfration of a pharmaceutical compound of the invention as a prophylactic or a treatment of the A/3-associated disease. Family history or prior occurrence of an AjS-associated neurological disease, even if the AjS- associated neurological disease is absent in a subject at present, may be an indication for prophylactic intervention by administering a pharmaceutical compound described herein to reduce or prevent aberrant AjS production and/or deposition.

An example of a method of diagnosis of aberrant Aβ production and/or deposition which can be performed using standard methods such as, but not limited to: imaging methods, electrophysiological methods, and histological methods. Additional methods of diagnosis and assessment of AjS-associated disease and the resulting cell death or damage are known to those of skill in the art.

In addition to the diagnostic tests described above, clinical features of AjS-associated disorders can be monitored for assessment of APP translation and/or A/3 secretion levels following onset of an AjS-associated disease. These features include, but are not limited to: assessment of the presence of plaque accumulation, neuronal cell lesions, spinal cord lesions, brain lesions, and behavioral abnormalities. Such assessment can be done with methods known to one of ordinary skill in the art, such as behavioral testing and imaging studies, such as radiologic studies, CT scans, PET scans, etc.

The invention also provides a pharmaceutical kit comprising one or more containers comprising one or more of the APP translation-modulating and/or A/3 secretion-modulating compounds of the invention and/or formulations of the invention. The kit may also include instructions for the use of the one or more APP franslation-modulating and/or A/3 secretion- modulating compounds or formulations of the invention for the treatment of an AjS-associated disease. The kits of the invention may also comprise additional drugs for preventing and/or treating an A/3-associated disease. hi another aspect of the invention, cell models and/or non-human animal models of AjS production enhancement and/or AjS-associated disease may be produced by administering an enhancer of APP translation and/or A/3 secretion to an animal or contacting a cell with the enhancer of APP translation and/or A/3 secretion. Such models may be useful for testing treatment strategies, monitoring clinical features of disease, or as tools to assess strategies for the prevention of A/3-production and/or deposition damage in AjS-associated disease. In some embodiments, enhancing APP translation and/or AjS secretion is increasing A/3 production and/or deposition and increases the manifestation of an AjS-associated disease. This increase in AjS production and/or deposition may be an increase above a confrol level of A/3 production and/or deposition, but may be one that does not result in cell death or other clinical characteristics indicative of an AjS-associated disease in a cell, tissue, or subject.

Cells and animal models made using enhancing compounds of the invention may also be useful for assessing the ability of lead compounds to inhibit APP franslation and/or A/3 secretion. For example, a cell contacted with an enhancer of APP translation and/or A/3 secretion may be further contacted with putative agents that are candidate or lead compounds for treating or preventing an AjS-associated disorder. The ability of the lead or candidate compound to prevent or treat the A/3-associated disease may be evaluated in the model cell or animal. In addition the enhancers may serve as lead compounds in that if their targets (by definition functionally important) can be identified and characterized, it may subsequently be possible to rationally design new compounds that act as inhibitors of these targets.

The invention also relates in some aspects to the identification and testing of candidate APP translation-modulating and/or A/3 secretion-modulating compounds The APP translation-modulating and/or Aβ secretion-modulating compounds of the invention can be screened for modulating (enhancing or inhibiting) APP franslation and/or AjS secretion using the same type of assays as described herein (e.g., in the Example section). Using such assays, the APP translation-modulating and/or A/3 secretion-modulating compounds that have the best inhibitory activity can be identified. It is understood that any mechanism of action described herein for the APP franslation-modulating and/or AjS secretion-modulating compounds is not intended to be limiting, and the scope of the invention is not bound by any such mechanistic descriptions provided herein.

The invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents and compounds that modulate APP translation and/or AjS secretion. Generally, the screening methods involve assaying for compounds which modulate (enhance or inhibit) the level of APP translation and or AjS secretion. As will be understood by one of ordinary skill in the art, the screening methods may measure the level of APP franslation and/or A/3 secretion directly, e.g., screening methods described herein. In addition, screening methods maybe utilized that measure a secondary effect of APP translation and/or A/3 secretion, for example the level of plaque deposition and/or neuronal cell death in a cell or tissue sample or by measuring behavioral characteristics of A/3- associated disease. A wide variety of assays for pharmacological agents can be used in accordance with this aspect of the invention, including, APP translation assays, AjS secretion assays, AjS production assays, cell viability assays, cell-based assays, etc. As used herein, the term "pharmacological agent" means APP translation-modulating compounds and/or AjS secretion- modulating compounds. An example of such an assay that is useful to test candidate APP translation-modulating and/or AjS secretion-modulating compounds is provided in the

Examples section. In such assays, the assay mixture comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.

Candidate compounds useful in accordance with the invention encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate compounds comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid molecule, the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules are also contemplated.

It is contemplated that cell-based assays as described herein can be performed using cell samples and/or cultured cells. Biopsy cells and tissues as well as cell lines grown in culture are useful in the methods of the invention.

Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological compounds may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the compounds. Candidate compounds also include analogs, derivatives, and/or variants of the APP translation and/or A/3 secretion modulating compounds described herein.

A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which maybe used to facilitate optimal binding, or to reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used. An exemplary APP translation and/or A/3 secretion assay is described herein, which may be used to identify candidate compounds that modulate APP franslation and/or A/3 secretion. In general, the mixture of the foregoing assay materials is incubated under conditions whereby, but for the presence of the candidate pharmacological compound, there is an above-normal level of APP franslation and/or A/3 secretion, although in some embodiments the candidate compound may be one that increases the level of APP translation and/or A/3 secretion. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.

After incubation, the level of APP translation and/or A/3 secretion may be detected by any convenient method available to the user. Detection may be effected in any convenient way for cell-based assays. For cell-based assays, one of the components usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, etc.).

A variety of methods may be used to detect the label, depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, sfrepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments of the invention and are not to be construed to limit the scope of the invention.

Examples

Example 1

Introduction

Our goal was to use newly discovered compounds directed to the 5' untranslated region of APP mRNA (5 'UTR) (Fig. 1) to limit amyloid-/3 (A/3)-peptide output in cell-culture systems, and subsequently to test therapeutically these compounds in transgenic mouse models for APP and A/3-peptide overexpression. We developed and utilized a screen to identify APP 5'UTR-binding compounds from a unique library of 1,200 FDA pre-approved drugs, arranged in a format that is convenient for transferring drugs to cells growing in 96- well plates. Using a new transfection-based assay, in which APP - 5 'UTR and 3 'UTR sequences drive the translation of a luciferase reporter gene, we have screened for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well characterized. New and powerful combinations of these APP-directed, FDA- approved drug hits can then be tested at low combinatorial doses to test efficacy using an APP-transgenic mouse model. These drugs can be examined in clinical trials to test their therapeutic benefit to AD patients and subject with, or at risk of, AjS-associated disorders. Mefhods

Construct Preparation

We made two constructs, designated as pGAL and pGALA (Fig. 2). The original pGAL construct encodes the complete 146 nucleotide 5 'UTR of the APP gene. This PCR generated cassette was inserted in between the H άTII and Ncol sites in front of the luciferase gene in the pGL-3 vector (Promega, Madison, WI). pGAL transcribes a hybrid luciferase reporter that encodes the previously described IL-1 responsive 90 nucleotide element (Rogers et al., J. Biol. Chem. 274 , 6421-31, 1999), but also an additional upstream 55 nucleotides immediately downstream from the 5' cap site of APP mRΝA. The pGALA construct was prepared by cloning the complete 1.2 Kb APP 3 'UTR into a convenient Xbal site immediately downsfream of the luciferase gene in the pGAL construct. Hybrid APP- luciferase mRΝAs expressed from pGALA transfectants transcribe the 146 nt APP 5 'UTR sequence element inserted in front of the reporter gene start codon, and an additional 1.2 kB of APP 3 'UTR sequences downstream from the luciferase stop codon (Mbella et al., Mol Cell Biol, 20(13):4572-9, 2000). Therefore, the hybrid APP-luciferase mRΝA expressed in pGALA transfectants exhibits the natural arrangement of APP gene 5' and 3' untranslated regions to provide an authentic and novel target for the purpose of drug screening.

Transfections

Neuroblastoma cells were co-transfected with the pGALA construct (encoding APP 5 'UTR and 3 'UTR sequences) and with a Green Fluorescent Protein (GFP) construct. Reporter genes were expressed fro m an SV40 promoter. In four separate screening assays transfections were performed in the presence of lipofectamine-2000 according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Typically neuroblastoma cells

(SY5Y) were grown in 3 separate flasks (100 mm²), and each flask was then transfected (12 h) with pGALA (6 mg DNA) and pGFP(3 mg DNA). After transfection, cells were subsequently passaged equally into 96-well flasks to be exposed to our library of FDA pre- approved compounds. All 1,200 compounds were arranged on 96-well templates in 12 plates, and were stored at -20°C. Compounds were present in a 10 mL volume at a uniform 10 mM concentration within each well. Prior to screening, drugs were reconstituted in Dulbecco's modified Eagle's medium (DMEM) (without FCS) for 1 h at 37°C to maximize solubility at the final 1 mM concentration. Resuspended drugs were multipipetted from the 96-well templates and transferred to the pGALA transfectants (80% confluent cells growing in each well). In these screens, cells were exposed to 100 mM doses of each drug for 48 h. After the screen was completed cell viability was established by a microscopic examination of each well. Relative GFP gene expression was established for cells in each well of the 96- well plates by reading at 500 nM wavelength (GFP) using an automated Wallac 1420 multilabel counter. After obtaining a GFP readout, the cells in each 96-well plate were lysed in 50 mL Reporter Lysis Buffer (Promega ). Luciferase assays were performed over a 15-s readout timeframe using the Walacl420 counter. As a positive experimental control for our screens, the cells in the last two selected rows of each 96-well plate were exposed either to desferrioxamine (concentrations of 1 mM to 100 mM in row 11), or the divalent cation chelator, EDTA (1 mM to 100 mM in the adjacent row 12). Desferrioxamine was employed as a positive confrol compound that is known to suppress APP 5 'UTR driven franslation of the downstream luciferase reporter transcript. Matching concentrations of EDTA were employed as a negative confrol because this divalent cation chelator (e.g., Mg²⁺, Ca²⁺) does not inhibit translation driven by APP 5 'UTR sequences. Drug "hits" were identified as those compounds that suppressed luciferase expression similar to the effect of desferrioxamine.

Results

We have used pGALA transfected neuroblastoma cells to screen the complete library of 1,200 FDA pre-approved compounds. These transfectants express a luciferase coding- block fused between APP 5 'UTR and 3 'UTR sequences. A list of top APP-5'UTR directed compounds (Table 1) can now be tested for their capacity to lower APP and A/3-peptide levels in neuronal derived cells. It was our goal to identify new compounds from this list that are more potent than phenserine (Yu et al., J. Med. Chem. 42,1855-1861, 1999; Haroutunian et al., Brain Res. Mol. Brain Res. 46, 161-168,1997), and have greater clinical efficacy than the use of the iron chelator desferrioxamine (Part et al, J. Pediatr. Surg. 25, 224-227, 1990; Crapper McLachlan et al., Lancet 337,1304-1308, 1991). Using drug-treatment protocols on cell cultures similar to conditions described for the use of desferrioxamine, we exposed pGALA-pGFP co-fransfected neuroblastoma cells (SY5Y) to 1,200 FDA pre-approved compounds. Medicinal compounds that caused >95% reduction in luciferase gene expression were identified, and a preliminary frequency distribution of the drug classes is shown in Table 2. Table 2. Drug classes

a Screened to cause > 95% reduction of APP-luciferase expression in pGALA-rransfected neuroblastoma cells.

We used a microscopic examination of cells to ensure that we only scored as positives any wells that displayed 90-100% cell viability (n = 3). Cofransfected GFP expression was also 90%-100% of GFP gene expression present in untreated samples (n = 7). Thus far, these APP 5 'UTR directed compounds are the best candidate compounds from the library of 1200 FDA-approved drugs. This list represents the most active one or two compounds from each of the twelve 96-well plates that were screened. The compounds that exhibited the highest capacity to suppress APP 5 'UTR driven translation of the luciferase reporter gene were categorized into groups (Table 2). These groups of compounds represent the panel of drugs that most significantly suppressed translation of a transfected luciferase reporter gene ligated in between APP 5 'UTR and 3 'UTR sequences. Members of the first group exert their clinically relevant pharmacological action by inhibiting receptor function in the brain (Table 2), and most of these drugs move rapidly across the blood-brain barrier (BBB). Members of the second group are antibiotics that are known to be bactericidal, based on their capacity to bind to bacterial ribosomal RNA and suppress bacterial franslation. The third group includes compounds that bind and detoxify metals, and act as interchelating agents. Actinomycin D (AcfD) was found to suppress APP-driven translation in our assay. ActD represents a useful positive confrol that validates our screen because Ac D is an RNA Polymerase II inhibitor that interchelates between the bases stacked into the helices formed by DNA and ribosomal RNAs. The fourth group of compounds was drugs associated with lipid metabolism. The identification of these compounds as lead drugs from "the translational-suppression screen" was particularly unexpected. Other compounds tested from the FDA library exhibited a less stringent 90% threshold for lowering APP 5 'UTR driven luciferase gene expression. Example 2 Introduction

Our laboratory identified 18 lead FDA-pre-approved drugs as potentially useful agents for AD therapeutics. These agents may suppress APP translation and thus limit pathogenic A/3 peptide secretion. In a preliminary characterization paroxetine and dimercaptopropanol were found to lower intracellular APP while maintaining amyloid- precursor-like-protein 1 (APLP-1) levels. This finding provides validation for our fransfection-based reporter assay to screen drugs that suppress APP 5 'UTR conferred franslation of a luciferase reporter transcript. Interestingly azithromycin represents subset of APP 5'UTR-directed drugs that do alter the cleavage of APP resulting in the increased production of the cytoplasmic tail fragment of APP. This finding may be best explained as an unexpected link between 7-secretase (or perhaps α-secretase) activation and APP an 5'UTR effector protein (Rogers et al, JBiol Chem 274:6421-6431, 1999; Buxbaum, et al. PNAS 89:10075-10078, 1992; Cao, et al., Science 293:115-120, 2001).

Methods Cell culture

Neuroblastoma cells (SY5Y) were exposed to paroxetine for 72 hr at 1 μM, 5 μM, andlO μM concentrations. Then dimercaptopropanol was administered for 72 hr at a 0.001- 0.1 μM (concentration range).

Construct Preparation

The pGAL and pGALA screening constructs shown in Fig. 2 were prepared as previously described (Rogers et al., JMol Neurosci 19:77-82, 2002). To generate the dicistronic construct, pJR-1, a PCR-generated DNA cassette encoding the 146 bp APP was cloned into the multiple cloning site (MCS) of pIRES2 between unique XhoI/EcoRI sites (Clonetech Laboratories, Inc., Palo Alto, CA). The downsfream EcoRI/BamHI sites were then used to ligate in the luciferase reporter gene (Luc)(Promega, Madison, WI). Stable transfectants were prepared by co-transfecting pJR-1 with a rSV2Neomycin plasmid and selecting G418 resistant colonies.

Transfection-based drug screens We employed a transient transfection-based assay to screen a complete library of 1,200 FDA pre-approved compounds for APP 5'UTR-directed drugs in neuroblastoma cells. For monitoring luciferase gene expression, transfected cells were passaged equally into 96- wells. Six lead drugs were added to the stably transfected neuroblastoma cells for 24 hr and 48 hr (at concentrations of 1 μM, 10 μM, and 100 μM of each drug). Experimentally, compounds (10 μM stocks) were diluted 1/100 to 1/10,000 into 2ml DMEM (without FCS) for 1 hour to maximize solubility. Each individual drug (100 μl volume) was then added to the 96 wells, and their effects on reporter expression were tested in cells grown in triplicate or quadruplet wells. After drug treatment, cell viability was established by a microscopic examination of each well. Maintenance of GFP expression was used to monitor cell viability. To monitor GFP expression we read each 96-well plate at 480nm/509nm wavelength (GFP) ' using an automated Wallac-1420 multilabel counter. After obtaining a GFP readout, the cells in each 96 well plate were lysed in 50 μl Reporter Lysis buffer (Promega, Madison, WI) followed by luciferase assay (also using the Wallac-1420 counter). Eighteen hits were identified (Rogers et al, JMol Neurosci 19:77-82, 2002). To validate the initial screen we counter-screened the 18 leads in SY5Y neuroblastoma cells stably transfected with the pJRl dicistronic luciferase/GFP construct (Fig. 8). For monitoring luciferase gene expression, transfected cells were passaged equally into 96-wells. Six lead drugs were added to the stably transfected neuroblastoma cells for 24 hr and 48 hr (at concentrations of 1 μM, 10 μM and 100 μM of each drug).

Western blotting

Protein blotting was performed on a BioRad apparatus (BioRad Laboratories, Hercules, CA) to fractionate the proteins (10 μg uniform loading), run at 100 volts, and transferred at 200 volts according to manufacturer's instructions. Prior to loading, protein content was determined by protein BCA assays (Pierce Biotechnology, Rockford, IL) and standardized to allow equal loading. The primary antibody was A8717 (APP) C-terminal rabbit polyclonal antibody (Sigma- Aldrich, St. Louis, MO). Blots were incubated at 1 :2000 dilution overnight in a cold room, on a shaker. For detection, anti-rabbit antibody at 1 :25,000 was added to the blots for 45 minutes. The blots were then washed for 2 to 3 hours in 30 minute intervals. After the wash, the blots were developed using chemiluminescence. To standardize for specificity of drug action on APP expression, we monitored APLP-1 levels in response to drug treatment. We loaded 10 μg of the same lysates and blotted with the primary antibody to APLP-1 (Calbiochem-Novabiochem, San Diego,CA). The same anti- rabbit secondary antibody monitored APLP-1 protein levels as used for APP-specific detection (A8717). Blots were quantitated using NTH-Image 1.62 software.

Aβ Assays

The degree to which paroxetine and dimercaptopropanol reduced A/3 was assayed using an ELISA assays (Dr. Dennis Selkoe, Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA). Conditioned medium was collected from untreated and drug-treated human lens epithelia cells (B3 cells). A/3 (1-40) and A/3 (1-42) levels were measured using a standard sandwich ELISA assay where 2G3 and 21F12 capture antibodies were used for A/3(l-40) and A/3(l-42) assays, respectively; and 266B antibody was used for detection.

Results Eighteen lead drugs hits suppress APP 5 'UTR conferred translation of a luciferase reporter transcript

Using a transient fransfection-based assay (constructs in figure 1) we identified 18 drug "hits" that >95% suppressed franslation of the hybrid APP 5'UTR-Luciferase transcript in neuroblastoma (SY5Y) cells (n=5) (Rogers et al. J Mol. Neurosci. 19:77-82, 2002). The positive hits included dimercaptopropanol, docusate, quinoline-gluconate, tamsulosin, netilmicin, paroxetine, prochloroperazine, diphenylhydramine, tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mytomycin C, carnetine acetylfransferase, and pilocarpine. Six of the hits were validated to selectively reduce luciferase expression translationally driven from the APP 5 'UTR but maintain co- expression of GFP with an internally controlled viral Internal Ribosome Entry Site (Figure 2, Table 3). For this purpose SY5Y neuroblastoma cells were stably transfected with the dicistronic construct (pJRl). Cells were passaged equally into 96-wells and drug treatments were performed (4 wells /assay). Table 3 shows the extent to which azithromycin, quinoline- gluconate, dimercaptopropanol (dimercaprol), paroxetine (Paxel®), tamsulosin, and atorvastatin suppressed luciferase reporter mRNA translation via the 146 nt APP 5 'UTR sequence. Luciferase activity (APP 5 'UTR specific) was expressed as a ratio of GFP expression (viral IRES was an internal experimental confrol) (1, 10, 100 μM doses were added to stable dicistronic transfectants for 24 and 48 hours). The most active drug was found to be dimercaptopropanol (5% residual luciferase activity after 48 hour dimercaptopropanol treatment (100 μM). Paroxetine treated transfectants exhibited 65% of the APP 5 'UTR conferred enhancement present in untreated counterparts (Table 3). Treatment of the same dicistronic transfectants with azithromycin (antibiotic), atovastatin (HMGCoA reductase inhibitor), quinoline-gluconate (analogue of the anti-malarial drug, cloroquine), tamsulosin (alpha-adenergic receptor blocker), each inhibited translational enhancement by the APP 5 'UTR element (Table 3). Another drug, pilocarpine (cholinergic agonist), lowered APP 5 'UTR directed franslation. Pilocarpine caused a 40-45% translational inhibition relative to untreated cells. None of the drug leads suppressed dicistronic GFP gene expression (internal specificity confrol).

Table 3: Suppression of APP 5 'UTR conferred translation of a dicistronic luciferase reporter gene expressed in pJRl stably transfected SY5Y cells (Figure 2). Data is the luciferase expression remaining after drug treatment (percentage relative to untreated cells (Luc/GFP)).

Paroxetine and dimercaptopropanol reduce intracellular APP levels.

We focused on the capacity of paroxetine and dimercaptopropanol to suppress intracellular APP levels. Western blot analysis showed that paroxetine significantly lowered intracellular APP levels (30% reduced at 5 μM after 72 h treatment of SY5Y neuroblastoma cells (n=3) Fig. 3). Dimercaptopropanol (0.1 μM Hg chelator) suppressed neuroblastoma APP levels by >50% compared to untreated neuroblastoma counterparts. At the lower dose of dimercaptopropanol (0.01 μM) APP levels were diminished by 50 % (n=3) (Fig. 4). Higher concenfrations (lμM) of dimercaptopropanol were found to be toxic to cells whereas equivalent doses of paroxetine did not effect cell viability. Paroxetine and dimercaptopanol treated SY5Y cells expressed equivalent levels of APLP-1 as their untreated counterparts. Table 4 summarizes our finding that paroxetine and dimercaptopropanol selectively target the translation of APP (but not APLP-1) in neuroblastoma cells. This result was critical to account for the specificity and selectivity we need to identify new of APP 5'UTR-directed drugs. Table 4: The effect of paroxetene and dimercaptopropanol to decrease APP levels. Azithromycin alters the APP cleavage pattern in neuroblastoma cells leading to the enhanced appearance of the cytoplasmic tail of APP (n=4).

Azithromycin alters APP cleavage pathways.

Azithromycin treatment of neuroblastoma cells induced a dose dependent increase of the amount of the cytoplasmic tail fragment of APP (6-12 kDa) in both neuroblastoma cells and in human lens B3-epithelial cells (Cao and Sudhof, Science 293;115-120, 2001). In neuroblastoma cells, 1-5 μM doses of azithromycin caused this cleavage of APP although the drug no longer exerted any pharmacological effect at concentrations greater than 10 μM. In B3 lens epithelial cells, by contrast, azithromycin signaled cleavage APP to generate the enhanced presence of the cytoplasmic tail fragment of APP at all dose ranges. Therefore azithromycin stimulates either α-secretase and/or γ-secretase cleavage of nascent intracellular APP. The action of azithromycin to inhibit APP 5 'UTR conferred franslation, and at the same time to alter the cleavage of APP, supports a model whereby common proteins are involved both in the translation of APP and the production of secreted APP (APP(s)) or the cytoplasmic intracellular domain (AICD) of APP. Evidently other APP mRNA sequences (3 'UTR and coding sequences) mediate azithromycin dependent translation of APP (i.e., total APP expression was unaffected by azithromycin).

Aβ peptide output in response to Paroxetine and Dimercaptopropanol.

Paroxetine (paxil) and dimercaptopropanol reduced APP translation, whereas azithromycin was observed to alter APP processing. Therefore we tested the capacity of these agents to lower secreted A/3 peptide levels. Lens epithelial cells (B3) were an excellent endogenous cell line for this purpose since B3 cells generate greater levels of basal A/3 relative to neuroblastoma cells (i.e., SY5Y and SKNSH cells) (Dr Lee Goldstein, Mass. General Hospital, Boston, MA, Personal communications). Table 5 summarizes the results of ELISAs that suggest paroxetine lowered A/3(l-40) secretion into the conditioned medium of lens B3 cells by up to 30% after a 4 day treatment (15 μM dose paroxetine) (see Fig.5). Human lens epithelial cells were used as a model, and were freated with dimercaptopropanol. The dimercaptopropanol freatment reduced A/3(l-40) and A/3(l-42) levels by 20% to 50% relative to control freated cells (72 hour freatment of the cells) (see Figs. 6 and 7). To confrol for selectivity drugs in the same class as paroxetine (i.e., prozac, another serotonin re-uptake blocker) are tested to determine whether they also reduce AjS secretion. In sum, our current results showed that drugs that lower APP franslation (i.e., paroxetine and dimercaptopropanol) also reduce the secretion of A/3(l-40) and A0(l-42).

Table 5: The effect of paroxetine and dimercaptopropanol to decrease A/3 secretion from human B3 lens epithelial cells.

DRUG FDA ACTIVITY AjS INHIBITION %x+/-SEM

Paroxetene Blocks Serotonin uptake 30% +/- 15, (n=3)

Dimercaprol Detoxifies Pb, Cu Hg 50% +/- 10%, (n =3)

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

We claim:

Claims

1. A method for preventing or treating an A/3-associated disease in a subject, comprising: administering to a subject in need of such treatment an effective amount of a Group 1 AjS-modulating compound to freat the A/3-associated disease, wherein the subject is otherwise free of indications for freatment with the Group 1 A/3-modulating compound that is administered.

2. The method of claim 1, wherein the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

3. The method of claim 1, wherein the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

4. The method of claim 1, wherein the Group 1 AjS-modulating compound is quinoline gluconate.

5. The method of claim 1, wherein the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP franslation and/or A/3 secretion.

6. The method of claim 1, wherein the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

7. The method of claim 1, wherein the AjS-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

8. The method of claim 1 , wherein the subject is human.

9. The method of claim 1, wherein the A/3-modulating compound is linked to a targeting molecule.

10. The method of claim 9, wherein the targeting molecule's target is a neuronal cell.

11. The method of claim 1 , wherein the A/3-modulating compound is a pro-drug.

12. The method of claim 1, wherein the AjS-modulating compound is administered prophylactically to a subject at risk of having an A/3-associated disease.

13. The method of claim 1 , wherein the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral adminisfration.

14. The method of claim 1 , wherein the AjS-modulating compound is administered in combination with an additional drug for treating an AjS-associated disease.

15. The method of claim 1 further comprising administering a AjS-modulating compound selected from the group consisting of Group 2 A/3-modulating compounds and Group 3 Aj3- modulating compounds.

16. The method of claim 15 wherein the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

17. The method of claim 15 wherein the Group 3 Aj3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP franslation and/or AjS secretion.

18. A method for treating a subj ect having a condition characterized by A/3 production comprising administering to a subject in need of such treatment a Group 1 AjS-modulating compound, in an amount effective to decrease APP translation and/or A/3 secretion, wherein the subject is free of symptoms otherwise calling for freatment with the A/3-modulating compound that is administered.

19. The method of claim 18 wherein the Group 1 A/3-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

20. The method of claim 18, wherein the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

21. The method of claim 18 wherein the Group 1 AjS-modulating compound is quinoline gluconate.

22. The method of claim 18 wherein the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP translation and/or Aβ secretion.

23. The method of claim 18 wherein the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

24. The method of claim 18 wherein the condition characterized by A/3 production is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

25. The method of claim 18 wherein the subject is human.

26. The method of claim 18 wherein the AjS-modulating compound is linked to a targeting molecule.

27. The method of claim 18 wherein the targeting molecule's target is a neuronal cell.

28. The method of claim 18 wherein the A/3-modulating compound is administered prophylactically to a subject at risk of having the condition characterized by A/3 production.

29. The method of claim 18 wherein the mode of administration is selected from the group consisting of: implantation, mucosal administration, injection, inhalation, and oral administration.

30. The method of claim 18 wherein the A/3-modulating compound is administered in combination with an additional drug for treating an AjS-associated disease.

31. The method of claim 18 further comprising administering a AjS-modulating compound selected from the group consisting of Group 2 AjS-modulating compounds and Group 3 AjS- modulating compounds.

32. The method of claim 31 wherein the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

33. The method of claim 31 wherein the Group 3 A/3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP franslation and/or A/3 secretion.

34. A method of evaluating the effect of candidate pharmacological agents on APP franslation and or A/3 secretion, comprising: contacting a cell sample with a candidate pharmacological agent; determining the effect of the candidate pharmacological agent on the level of APP franslation and/or A/3 secretion in the cell sample relative to the level of APP franslation and/or A/3 secretion in a cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the level of APP translation and/or A/3 secretion in the test cell sample as compared with the control level indicates modulation of APP franslation and/or A/3 secretion by the candidate pharmacological agent.

35. The method of claim 34, wherein the cell samples comprise a cell that produces A/3.

36. The method of claim 34, wherein the cell samples comprise a cell that is capable of producing A/3.

37. The method of claim 34, wherein the cell samples comprise a pGALA-transfected neuroblastoma cell with a reporter gene construct.

38. The method of claim 37, wherein the reporter gene construct is a Green Fluorescent Protein construct.

39. The method of claim 37, wherein the amount of APP translation and/or A/3 secretion is determined by measuring the level of Green Fluorescent Protein (GFP) expression in the cell samples.

40. The method of claim 34, wherein a relative decrease in the level of APP translation and/or A/3 secretion in the cell sample contacted with the candidate pharmacological agent compared to the level of APP translation and/or AjS secretion in the cell sample not contacted with the candidate pharmacological agent indicates the candidate pharmacological agent is an APP translation-inhibiting and/or A/3 secretion-inhibiting agent.

41. The method of claim 34, wherein a relative increase in the level of APP franslation and/or AjS secretion in the cell sample contacted with the candidate pharmacological agent compared to the level of APP franslation and/or AjS secretion in the cell sample not contacted with the candidate pharmacological agent indicates that the modulator is an APP translation- enhancing and/or A/3 secretion-enhancing agent.

42. A method for preparing an animal model of a disease characterized by APP franslation and/or A/3 secretion, comprising introducing an APP translation-enhancing and/or AjS secretion-enhancing agent into a non-human animal.

43. The method of claim 42, further comprising detecting in the non-human animal symptoms of a disorder characterized by APP translation and/or A/3 secretion.

44. The method of claim 42, wherein the animal model is a model for a neurological disease.

45. The method of claim 44, wherein the neurological disease selected from the group consisting of: Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

46. A kit for treating a subject in accordance with the method of claim 1 , comprising a package housing a first container containing at least one dose of a Group 1 AjS- modulating compound, and instructions for using the A/3-modulating compound in the prevention and/or treatment of an A/3-associated disease.

47. The kit of claim 46, wherein the Group 1 AjS-modulating compound is dimercaptopropanol, or an analog, derivative, or variant thereof that decreases APP franslation and/or A/3 secretion.

48. The kit of claim 46, wherein the Group 1 A/3-modulating compound is docusate or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

49. The kit of claim 46, wherein the Group 1 AjS-modulating compound is quinoline gluconate.

50. The kit of claim 46, wherein the Group 1 A/3-modulating compound is tamsulosin or an analog, derivative, or variant thereof that decreases APP franslation and/or AjS secretion.

51. The kit of claim 46, wherein the Group 1 A/3-modulating compound is netilmicin or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

52. The kit of claim 46, wherein the AjS-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

53. The kit of claim 46, further comprising a container containing at least one dose of a Group 2 AjS-modulating compound selected from the group consisting of paroxetine, prochloroperazine, diphenylhydramine or an analog, derivative, or variant thereof that decreases APP franslation and/or A/3 secretion and instructions for using the compound for prevention and/or treatment of an A/3-associated disease.

54. The kit of claim 46, further comprising a container containing at least one dose of a Group 3 AjS-modulating compound selected from the group consisting of tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, pilocarpine or an analog, derivative, or variant thereof, that decreases APP franslation and/or Aβ secretion, and instructions for using the compound fro prevention and/or treatment of an AjS-associated disease.

55. The kit of claim 46, wherein the A/3-modulating compound is formulated for delivery to neuronal cells.

56. The kit of claim 46, wherein the AjS-modulating compound is formulated for sustained release.

57. A composition comprising a Group 1 AjS-modulating compound and a Group 2 AjS-modulating compound and/or a Group 3 A/3-modulating compound.

58. The composition of claim 57, wherein the Group 1 AjS-modulating compound is dimercaptopropanol, docusate, quinoline gluconate, tamsulosin, or netilmicin, or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

59. The composition of claim 57, wherein the Group 2 AjS-modulating compound is paroxetine, prochloroperazine, or diphenylhydramine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

60. The composition of claim 57, wherein the Group 3 A/3-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, or pilocarpine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

61. The composition of claim 57, wherein the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis,

Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis.

62. The composition of claim 57, wherein the compound is linked to a targeting molecule.

63. The composition of claim 62, wherein the targeting molecule's target is a neuronal cell.

64. The composition of claim 57, wherein the compound is formulated for implantation, mucosal administration, injection, inhalation, or oral administration.

65. A composition comprising a Group 2 A/3-modulating compound and a Group 3 AjS-modulating compound.

66. The composition of claim 65, wherein the Group 2 A/3-modulating compound is paroxetine, prochloroperazine, or diphenylhydramine, or an analog, derivative, or variant thereof that decreases APP translation and/or AjS secretion.

67. The composition of claim 65, wherein the Group 3 AjS-modulating compound is tetracycline, actinomycin D, benzyl alcohol, chloroquine, atorvastatin, azithromycin, diphtheria toxin, mitomycin C, carnetine acetylfransferase, or pilocarpine, or an analog, derivative, or variant thereof that decreases APP translation and/or A/3 secretion.

68. The composition of claim 65, wherein the A/3-associated disease is selected from the group consisting of Alzheimer's disease, Down's syndrome, cerebrovascular amyloidosis, Hereditary Amyloidosis with Cerebral Hemorrhage of the Dutch Type, vascular dementia, and inclusion body myositis .

69. The composition of claim 65, wherein the compound is linked to a targeting molecule.

70. The composition of claim 69, wherein the targeting molecule's target is a neuronal cell.

71. The composition of claim 65, wherein the compound is formulated for implantation, mucosal administration, injection, inhalation, or oral administration.