AU748768B2

AU748768B2 - Identification of agents for use in the treatment of Alzheimer's disease

Info

Publication number: AU748768B2
Application number: AU65484/98A
Authority: AU
Inventors: Craig S. Atwood; Ashley I Bush; Xudong Huang; Rudolph E Tanzi
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 1997-03-11
Filing date: 1998-03-11
Publication date: 2002-06-13
Anticipated expiration: 2018-03-11
Also published as: EP1007048A4; CA2284170A1; WO1998040071A1; EP1007048A1; JP2001514661A; AU6548498A; CA2284170C

Description

1 Identification of Agents for Use in the Treatment of Alzheimer's Disease Background of the Invention Field of the Invention This invention is in the field of medicinal chemistry. In particular, the invention is related to the detection of drugs useful in the treatment of Alzheimer's disease. The invention is also related to compositions for treatment of Alzheimer's disease.

Related Art Polymers of Abeta the 4.3 kD, 39-43 amino acid peptide product of the transmembrane protein, amyloid protein precursor (APP), are the main omponents extracted from the neuritic and vascular amyloid of Alzheimer's disease (AD) brains. Ap deposits are usually most concentrated in regions of high neuronal cell death, and may be present in 25 various morphologies, including amorphous deposits, neurophil plaque amyloid, and amyloid congophilic angiopathy (Masters, et al., EMBO J. 4:2757 (1985); Masters, C.L. et al., Proc. Natl. Acad. Sci.

;USA 82: 4245 (1985)). Growing evidence suggests S: 30 that amyloid deposits are intimately associated with the neuronal demise that leads to dementia in the disorder.

The presence of an enrichment of the 42 residue species of AP in these deposits suggests that this species is more pathogenic. The 42 residue form of A A3 (APi- 42 while a minor component of biological SS H:\lauraw\Keep\65484-98.doc 21/08/01 la fluids, is highly enriched in amyloid, and genetic studies strongly implicate this protein in the etiopathogenesis of AD. Amyloid deposits are decorated with inflammatory response proteins, but biochemical markers of severe oxidative stress such as peroxidation adducts, advanced glycation endproducts, and protein cross-linking are seen in proximity to the lesions. To date, the cause of AP deposits is unknown, although it is believed that preventing these deposits may be a means of treating the disorder.

H:\lauraw\Keep\65484-98.doc 21/08/01 WO 98/40071 PCT/US98/04683 -2- The presence of an enrichment of the 42 residue species of AP in these deposits suggests that this species is more pathogenic. The 42 residue form of Ap (Ap-42), while a minor component of biological fluids, is highly enriched in amyloid, and genetic studies strongly implicate this protein in the etiopathogenesis of AD. Amyloid deposits are decorated with inflammatory response proteins, but biochemical markers of severe oxidative stress such as peroxidation adducts, advanced glycation end-products, and protein cross-linking are seen in proximity to the lesions. To date, the cause of AP deposits is unknown, although it is believed that preventing these deposits may be a means of treating the disorder.

When polymers of Ap are placed into culture with rat hippocampal neurons, they are neurotoxic (Kuo, et al., J. Biol. Chem. 271:4077-81 (1996); Roher, et al., Journal of Biological Chemistry 271:20631-20635 (1996)). The mechanism underlying the formation of these neurotoxic polymeric AP species remains unresolved. The overexpression of AP alone cannot sufficiently explain amyloid formation, since the concentration of AP required for precipitation is not physiologically plausible. That alterations in the neurochemical environment are required for amyloid formation is indicated by its solubility in neural phosphate buffer at concentrations of up to 16 mg/ml (Tomski, S. Murphy, R.M. Archives ofBiochemistry and Biophysics 294:630 (1992)), biological fluids such as cerebrospinal fluid (CSF) (Shoji, et al., Science 258:126 (1992); Golde et al. Science, 255(5045):728-730 (1992); Seubert, et al., Nature 359:325 (1992); Haass, et al., Nature 359:322 (1992)) and in the plaque-free brains of Down's syndrome patients (Teller, J.K., et al., Nature Medicine 2:93-95 (1996)).

Studies into the neurochemical vulnerability of AP to form amyloid have suggested altered zinc and homeostasis as the most likely explanations for amyloid deposition. AP is rapidly precipitated under mildly acidic conditions in vitro (pH 3.5-6.5) (Barrow, C.J. Zagorski, Science 253:179-182 (1991); WO 98/40071 PCT/US98/04683 Fraser, et al., Biophys. J. 60:1190-1201 (1991); Barrow, el al., J. Mol.

Biol. 225:1075-1093 (1992); Burdick, J. Biol. Chem. 267:546-554 (1992); Zagorski, M.G. Barrow, Biochemistry 31:5621-5631 (1992); Kirshenbaum, K. Daggett, Biochemistry 34:7629-7639 (1995); Wood, S.J., /ct al.. .J Mol. Biol. 256:870-877 (1996)). Recently, it has been shown that the presence of certain biometals, in particular redox inactive Zn 2 and, to a lesser extent. redox active Cu 2 and Fe, markedly increases the precipitation of soluble AP (Bush. et J. Biol. Chem. 268:16109 (1993); Bush, et al., J. Biol.

Chem. 269:12152 (1994); Bush, et al., Science 265:1464 (1994); Bush, A.I., ci al.. Science 268:1921 (1995)). At physiological pH, AP.

4 0 specifically and saturably binds Zn 2 manifesting high affinity binding (KD 107 nM) with a 1:1 (Zn :Ap) stoichiometry, and low affinity binding (KD 5.2 p.M) with a 2:1 stoichiometry.

The reduction by APP of copper (II) to copper may lead to irreversible AP aggregation and crosslinking. This reaction may promote an environment that would enhance the production of hydroxyl radicals, which may contribute to oxidative stress in AD (Multhaup, et al., Science 271:1406-1409 (1996)). A precedence for abnormal Cu metabolism already exists in the neurodegenerative disorders of Wilson's disease, Menkes' syndrome and possibly familial amyotrophic lateral sclerosis (Tanzi, R.E. et al., Nature Genetics 5:344 (1993); Bull. et al., Nature Genetics 5:327 (1993); Vulpe, et al., Nature Genetics 3:7 (1993); Yamaguchi, et al., Biochem. Biophys. Res. Commun. 197:271 (1993); Chelly, et al., Nature Genetics 3:14 (1993); Wang, D. Munoz, D.G., J. Neuropathol. Exp. Neurol. 54:548 (1995); Beckman, et al., Nature 364:584 (1993); Hartmann, H.A. Evenson, Med. Hypotheses 38:75 (1992)).

Although much fundamental pathology, genetic susceptibility and biology associated with AD is becoming clearer, a rational chemical and structural basis for developing effective drugs to prevent or cure the disease remains elusive.

While the genetics of the disorder indicates that the metabolism of Ap is WO 98/40071 PCT/US98/04683 -4intimately associated with the etiopatholgenesis of the disease, drugs for the treatment of AD have so far focused on "cognition enhancers" which do not address the underlying disease processes.

Summary of the Invention In one aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein the agent is capable of altering the production of Cu' by AP, the method comprising: adding Cu 2 to a first A3 sample; allowing the first sample to incubate for an amount of time sufficient to allow said first sample to generate Cu'; adding Cu 2 to a second AP sample, the second sample additionally comprising a candidate pharmacological agent; allowing the second sample to incubate for the same amount of time as the first sample; determining the amount of Cu produced by the first sample and the second sample; and comparing the amount of Cu produced by the first sample to the amount of Cu' produced by the second sample; whereby a difference in the amount of Cu' produced by the first sample as compared to the second sample indicates that the candidate pharmacological agent has altered the production of Cu 4 by AP.

In a preferred embodiment, the amount of Cu' present in said first and said second sample is determined by adding a complexing agent to said first and said second sample, wherein said complexing agent is capable of combining with Cu' to form a complex compound, wherein said complex compound has an optimal visible absorption wavelength; WO 98/40071 PCT/US98/04683 measuring the absorbancy of said first and said second sample; and calculating the concentration of Cu' in said first and said second sample using the absorbancy obtained in step In a more preferred embodiment, the complexing agent is bathocuproinedisulfonic (BC) anion. The concentration of Cu' produced by AP may then be calculated on the basis of the absorbance of the sample at about 478 nm to about 488 nm, more preferable about 480 to about 486 nm, and most preferably about 483 nm. In another preferred embodiment, the method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader. Most preferrably, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Cu* by AP. In another preferred embodiment, said AP samples of step l(a) and step 1(c) are biological samples. Most preferrably, said biological samples are CSF.

In another aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of Fe 2 by Ap, said method comprising: adding Fe 3 to a first AP sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate Fe 2 adding Fe 3 to a second Ap sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of Fe 2 4 produced by said first sample and said second sample; and comparing the amount ofFe 2 present in said first sample to the amount of Fe 2 present in said second sample; WO 98/40071 PCT/US98/04683 -6whereby a difference in the amount of Fe 2 1 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of Fe 2 by Ap.

In a preferred embodiment, the amount of Fe 2 present is determined by using a spectrophotometric method analogous to that used for the determination of Cu above. In this method, the complexing agent is bathophenanthrolinedisulfonic (BP) anion. The concentration of Fe 21 -BP produced by AP may then be calculated on the basis of the absorbance of the sample at about 530 to about 540 nm, more preferably about 533 nm to about 538 nm, and most preferably about 535 nm. In another preferred embodiment, said method is performed in a microtiter plate. and the absorbancy measurement is performed by a plate reader. Most preferrably, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Fe 2 by AP.

In another preferred embodiment, said AP samples of step 1(a) and step 1(c) are biological samples. Most preferrably, the biological sample is CSF.

In vet another aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of H 2 0, by Ap, said method comprising: adding Cu 2 or Fe' to a first AP sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate H 2 0 2 adding Cu 2 or Fe" to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of H 2 0 2 produced by said first sample and said second sample; and comparing the amount ofH 2 0, present in said first sample to the amount of H 2 0 2 present in said second sample; WO 98/40071 PCT/US98/04683 -7whereby a difference in the amount of H,0 2 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of H,0 2 by Ap. In a preferred embodiment, the determination of the amount of HzO present in said first and said second sample is determined by adding catalase to a first aliquot of said first sample obtained in step above in an amount sufficient to break down all of the HO, generated by said sample; adding TCEP, in an amount sufficient to capture all of the H-02 present in said samples, to said first aliquot (ii) a second aliquot of said first sample obtained in step above; and (iii) said second sample obtained in step above; incubating the samples obtained in step for an amount of time sufficient to allow the TCEP to capture all of the H202; adding DTNB to said samples obtained in step incubating said samples obtained in step for an amount of time sufficient to generate TMB; measuring the absorbancy at about 407 to about 417 nm of said samples obtained in step and calculating the concentration ofHO, in said first and said second sample using the absorbancies obtained in step In a preferred embodiment, the absorbancy of TMB is measured at about 412 nm. In preferred embodiment, said method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader. Most preferrably, two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of H20 2 by AP.

WO 98/40071 PCTIUS98/04683 -8- In another aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of decreasing the production of O0 by AP, said method comprising: adding AP and to a first buffer sample having an 02 tension greater than 0; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate adding AP and a candidate pharmacological agent to a second buffer sample having an 02 tension greater than 0; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of O0 produced by said first sample and said second sample; and comparing the amount of 0O present in said first sample to the amount of O present in said second sample; whereby a difference in the amount of O; present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of 0O by A3. In a preferred embodiment, the AP used is A01-42.

In a preferred embodiment, the determination of the amount ofO present in said samples is accomplished by measuring the absorbancy of the sample at about 250 nm.

Because the ability of AP to generate H 2 0 2 from O; may in many instances be beneficial. Therefore, in a preferred embodiment, the invention also relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of interfering with the interaction of 0, and AP3 to produce O0, without interfering with the SOD-like activity of A3, said method comprising: identifying an agent capable of decreasing the production of O; by AP; and WO 98/40071 PCT/US98/04683 -9determining the ability of said agent to alter the SOD-like activity ofAP. In a preferred embodiment, the determination of the ability of said agent to alter the SOD-like activity of AP is made by determining whether AP is capable of catalytically producing Cu', Fe 2 or H 2 0,.

In another aspect the invention relates to a method for the identification of agents useful in the treatment of Alzheimer's disease (AD) because they are capable of reducing the toxicity of A3.

In one aspect the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of reducing the toxicity of A3, said method comprising: adding AP to a first cell culture; adding AP to a second cell culture, said second cell culture additionally containing a candidate pharmacological agent; determining the level of neurotoxicity of AP in said first and said second samples; and comparing the level ofneurotoxicity of AP in said first and said second samples, whereby a lower neurotoxicity level in said second sample as compared to said first sample indicates that said candidate pharmacological agent has reduced the neurotoxicity of AP, and is thereby capable of being used to treat AD. In a preferred embodiment, the neurotoxicity of AP is determined by using an MTT assay. In another preferred embodiment, the neurotoxicity of AP is determined by using an LDH release assay. In still another preferred embodiment, the neurotoxicity of AP is determined by using a Live/Dead assay. Preferrably said cells utilized in the assays are rat cancer cells. Even more preferrably said cells are rat primary frontal neuronal cells.

Yet another aspect of the invention relates to a kit for determining whether an agent is capable of altering the production of Cu' by Ap which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein WO 98/40071 PCT/US98/04683 the first container means contains a peptide comprising A3 peptide; a second container means contains a Cu 2 salt; and a third container means contains BC anion.

Preferrably, said AP peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 jM.

In another aspect the invention relates to a kit for determining whether an agent is capable of altering the production of Fe2' by AP which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein the first container means contains a peptide comprising A3 peptide; a second container means contains an Fe 3 salt; and a third container means contains BP anion.

Preferrably, said AP peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 pM..

In another aspect, the invention relates to a kit for determining whether an agent is capable of altering the production of H 2 0 2 by AP which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein the first container means contains a peptide comprising A3 peptide; a second container means contains a CU 2 salt; a third container means contains TCEP; and a fourth container means contains DTNB.

Preferably, said A3 peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 [M.

In yet another aspect the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent WO 98/40071 PCT/US98/04683 -Ilis capable of inhibiting redox-reactive metal-mediated polymerization of A3, said method comprising: adding a redox-reactive metal to a first AP sample; allowing said first sample to incubate for an amount of time sufficient to allow AP polymerization; adding said redox-reactive metal to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; removing an aliquot from each of said first and said second sample; and determining presence or absence of polymerization in said first and second samples, whereby an absence of AP polymerization in said second sample as compared to said first sample indicates that said candidate pharmacological agent has inhibited AP polymerization. Preferrably, at step a western blot analysis is performed to determine the presence or absence of polymerization in the first and the second sample.

Another aspect of the present invention contemplates a method for treating AD in a subject, said method comprising administering to said subject an effective amount of an agent which is capable of inhibiting or otherwise reducing metal-mediated production of free radicals.

The present invention provides a method for treating AD in a subject, said method comprising administering to said subject an effective amount of an agent comprising a metal chelator and/or a metal complexing compound for a time and under conditions sufficient to inhibit or otherwise reduce metal-mediated production of free radicals by AP. In one aspect, the free radicals are reactive oxygen species such as 0 or OH'. In another aspect, the free radicals include forms of AP.

WO 98/40071 PCT/US98/04683 -12- Still another aspect of the present invention relates to a method of treating AD in a subject comprising administering to said subject an agent capable of preventing, reducing or otherwise inhibiting ROS production by Ap deposits in the brain for a time and under conditions to effect said treatment.

In one aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and one or more pharmaceutically acceptable carriers or diluents; for a time and under conditions to bring about said treatment; and wherein said chelator reduces, inhibits or otherwise interferes with Apmediated production of radical oxygen species. The invention also relates to said method further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject a combination of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt, for a time and under conditions to bring about said treatment; and wherein said chelator reduces, inhibits or otherwise interferes with Ap-mediated production of radical oxygen species. In a preferred embodiment, the metal chelator is EGTA.

In another preferred embodiment, the metal chelator is TPEN. In yet another preferred embodiment, the supplement is magnesium salt.

In yet another aspect, the invention relates to said method further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

WO 98/40071 PCT/US98/04683 13- In yet another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a salt of a metal chelator, wherein said chelator is selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein said salt is selected from the group consisting of: ammonium, calcium, magnesium, and sodium; and wherein said salt of a metal chelator reduces, inhibits or otherwise interferes with Ap-mediated production of radical oxygen species. In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the the metal chelator is TPEN. In yet another preferred embodiment, the salt of a metal chelator is a magnesium salt. In yet another aspect, the invention relates to said method further comprising administering to said subject a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a chelator specific for copper; wherein said chelator reduces, inhibits or otherwise interferes with Ap-mediated production of radical oxygen species.

In a preferred embodiment, the chelator specific for copper is specific for the reduced form of copper. Most preferreably, the chelator is bathocuproine or a hydrophobic derivative thereof.

In yet another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of an alkalinizing agent, wherein said alkalinizing agent reduces, inhibits or otherwise interferes with Ap-mediated production of radical oxygen species. In a preferred embodiment, the alkalinizing agent is magnesium citrate. In another preferred embodiment, the alkalinizing agent is calcium citrate.

Still another aspect of the present invention contemplates a method of treating AD in a subject comprising administering to said subject an agent capable of preventing formation of AP amyloid, promoting, inducing or otherwise WO 98/40071 PCT/US98/04683 -14facilitating resolubilization of AP deposits in the brain, or both, for a time and under conditions to effect said treatment.

In one aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and one or more pharmaceutically acceptable carriers or diluents; for a time and under conditions to bring about said treatment; and wherein said chelator prevents formation of AP amyloid, promotes, induces or otherwise facilitates resolubilization of AP deposits, or both. In another aspect, the invention relates to said method further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject a combination of a metal chelator selected from the following group: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt, for a time and under conditions to bring about said treatment; and wherein said combination prevents formation of AP amyloid, promotes, induces or otherwise facilitates resolubilization of A deposits, or both.

In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the the metal chelator is TPEN. In yet another preferred embodiment, the supplement is a magnesium salt. In another aspect, the invention relates to said method further comprising administering to the subject an effective amount of a compound selected from the group consisting of: WO 98/40071 PCT/US98/04683 rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a salt of a metal chelator, wherein said chelator is selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, pcnacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein said salt is selected from the group consisting of: ammonium, calcium, magnesium. and sodium; and wherein said salt of a metal chelator prevents tormation of AP amyloid, promotes, induces or otherwise facilitates rcsolubilization of Ap deposits, or both. In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the the metal chelator is TPEN. In vet another preferred embodiment, the salt of a metal chelator is a magnesium salt. In another aspect, the invention relates to said method further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention relates to a method of treating amyloidosis in a subject. said method comprising administering to said subject an effective amount of a chelator specific for copper; wherein said chelator prevents formation of A amyloid, promotes, induces or otherwise facilitates resolubilization of AP deposits, or both. In a preferred embodiment, the chelator specific for copper is specific for the reduced form of copper. Most preferrably, the chelator is bathocuproine or a hydrophobic derivative thereof.

In yet another aspect, the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of an alkalinizing agent, wherein said alkalinizing agent prevents formation of AP amyloid, promotes, induces or otherwise facilitates resolubilization of AP deposits, or both. In a preferred embodiment, the WO 98/40071 PCT/US98/04683 16alkalinizing agent is magnesium citrate. In another preferred embodiment, the alkalinizing agent is calcium citrate.

Still another aspect contemplates pharmaceutical compositions for the prevention, reduction or inhibition of ROS production by AP deposits, or the prevention of formation of AP amyloid, promoting, inducing or otherwise facilitating the resolubilization of AP deposits, or both, in the brain.

In one aspect, the invention relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, AP-mediated ROS formation, or both, comprising: a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt, together with one or more pharmaceutically acceptable carriers or diluents.

In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the metal chelator is TPEN. In yet another preferred embodiment, the supplement is a magnesium salt.

In another aspect, the invention relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, Ap-mediated ROS formation, or both, comprising a salt of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and wherein said salt is selected from the group consisting of: ammonium, calcium, magnesium, and sodium, together with one or more pharmaceutically acceptable carriers or diluents. In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the the metal chelator is TPEN. In yet another preferred embodiment, the salt of a metal chelator is a magnesium salt.

In yet another aspect, the invention relates to pharmaceutical composition for treatment of conditions caused by amyloidosis, Ap-mediated ROS formation, or both, comprising a chelator specific for copper, with one or more WO 98/40071 PCT/US98/04683 -17pharmaceutically acceptable carriers or diluents. In a preferred embodiment, the chelator is specific for the reduced form of copper. Most preferrably, the chelator specific for the reduced form of copper is bathocuproine.

In another aspect, the invention relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, AP-mediated ROS formation, or both, comprising an alkalinizing agent, with one or more pharmaceutically acceptable carriers or diluents. In a preferred embodiment, the alkalinizing agent is magnesium citrate. In another preferred embodiment, the alkalinizing agent is calcium citrate.

In yet another aspect, the invention relates to a composition of matter comprising: a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin.

In still another aspect, the invention relates to a composition of matter comprising: a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt. In a preferred embodiment, the metal chelator is EGTA. In another preferred embodiment, the metal chelator is TPEN. In yet another preferred embodiment, the supplement is a magnesium salt.

Still another aspect, the invention relates to a method for determining which metal chelators used in the treatment of amyloidosis, should be supplemented with ammonium, calcium, magnesium, or sodium salts, comprising: contacting AP aggregates with solutions containing a range of concentrations of said metal chelators; preparing a dilution curve from data obtained in step WO 98/40071 PCT/US98/04683 18 selecting chelators which solubilize less AP aggregates at higher concentrations than at lower or intermiate concentrations; contacting AP aggregates with chelators selected in step(c), in the presence of an ammonium, calcium, magnesium or sodium salt; and determining if resolubilization is increased in the presence of said salt; thereby determining whether a metal chelator used in the treatment of amyloidosis should be supplemented with ammonium, calcium, magnesium, or sodium salts.

Brief Description of the Figures Figure 1 is a graph showing the proportion of soluble AP- 40 remaining following centrifugation of reaction mixtures.

Figures 2A-2C: Figure 2A is a graph showing the proportion of soluble Ap,.4, remaining in the supernatant after incubation with various metal ions.

Figure 2B is a graph showing a turbidometric analysis of pH effect on metal ioninduced A,- 1 4 aggregation. Figure 2C is a graph showing the proportion of soluble Ap- 40 remaining in the supernatant after incubation with various metal ions, where high metal ion concentrations were used.

Figure 3 is a graph showing a competition analysis of AP 40 binding to Cu 2 Figures 4A-4C: Figure 4A is a graph showing the proportion of soluble

AP-

4 0 remaining in the supernatant following incubation at various pHs in PBS Zn 2 or Cu 2 Figure 4B is a graph showing the proportion of soluble Ap i 40 remaining in the supernatant following incubation at various pHs with different Cu 2 concentrations. Figure 4C is a graph showing the relative aggregation ofnM concentrations of A 1.

40 at pH 7.4 and 6.6 with different Cu 2 concentrations.

Figures 5A and 5B: Figure 5A is a graph showing a turbidometric analysis of Cu 2 '-induced AP, 40 aggregation at pH 7.4 reversed by successive cycles of chelator. Figure 5B is a graph showing a turbidometric analysis of the WO 98/40071 PCT/US98/04683 -19reversibility of Cu 2 -induced AP|.

0 aggregation as the pH cycles between 7.4 and 6.6.

Figure 6 shows the amino acid sequence ofAPP 669 7 1 6 near A 1-42. Rat AP is mutated (R5G, Y10F, H13R; bold). Possible metal-binding residues are underlined.

Figure 7 is a graph showing the effects of pH, Zn 2 or Cu 2 upon AP deposit formation.

Figure 8 is a western blot showing the extraction of AP from post-mortem brain tissue.

Figure 9 is a western blot showing AP SDS-resistant polymerization by copper.

Figure 10 is a graph showing Cu' generation by Ap.

Figure 11 is a graph showing H202 production by Ap.

Figure 12 is a graphical representation showing a model for the generation of reduced metal ions, O0, H 2 0 2 and OH- by AP peptides. Note that AP facilitates two consecutive steps in the pathway: the reduction of metal ions, and the reaction of 02 with reduced metal ions. The peptide does not appear to be consumed or modified in a one hour time frame by participation in these reactions.

Figures 13A and 13B are graphical representations showing Fe" or Cu 2 reduction by Ap peptides. Figure 13A illustrates the reducing capacity of AP species (10 pM), compared to Vitamin C and insulin (Sigma) (all 10 gM) towards Fe 3 or Cu 2 (10 pM) in PBS, pH 7.4, after 1 hour co-incubation, 37 0 C. Data indicate concentration of reduced metal ions generated. Figure 13B shows the effect of oxygen tension and chelation upon AP,.4 metal reduction. A,42 was incubated as in Figure 13A under various buffer gas conditions. "Ambient" no efforts were made to adjust the gas tension in the bench preparations of the buffer vehicle, 100% 02 was continuously bubbled through the PBS vehicle for 2 hours (at 20 0 before the remainder of the incubation components were added, "Ar" 100% Ar was continuously bubbled through the PBS vehicle for WO 98/40071 PCT/US98/04683 2 hours (at 20 0 before the remainder of the incubation components were added. "+DFO or TETA" Desferrioxamine (DFO, Sigma, 200 pM) was added to the Ap, 4 incubation in the presence of Fe 3 10 riM, or triethylenetetramine dihydrochloride (TETA, Sigma, 200 pM) was added to the AP.

4 2 incubation in the presence ofCu 2 4 10 pM, under ambient oxygen conditions. All data points are means ±SD, n 3.

Figures 14A-14E are graphical representations showing production of

H

2 O, from the incubation of AP in the presence of substoichiometric amounts of Fe 3 or Cu 2 Figure 14A shows HO0 2 produced by AP, 4 2 (in PBS, pH 7.4, under ambient gas conditions, 1 hour, 37 0 C) following co-incubation with various concentrations of catalase in the presence of 1 pM Fe 3 Figure 14B shows a comparison of H 2 0 2 generation by variant AP species: A 1 4 2

AP.

40 rat AP- 40 Ap 40 and Ap,.28 (vehicle conditions as in Figure 14A). Figure 14C shows the effect of metal chelators (200 pM) on H,0 2 production from A,.42 when incubated in the presence of Fe 3 or Cu 2 (1 pM) (vehicle conditions as in Figure 14A). BC Bathocuproinedisulfonate, BP Bathophenanthrolinedisulfonate. The effects of DFO were assessed in the presence of Fe 3 and TETA was assessed in the presence of Cu 2 as indicated. Figure 14D shows H 2 0 2 produced by A 1- 42 A 1- 4 0 and Vitamin C in the presence of Fe 3 (1 pM) (in PBS, pH 7.4 buffer, 1 hr, 37°C) under various dissolved gas conditions (described in Figure 13B): ambient air, 0, enrichment, and anaerobic (Ar) conditions, as indicated. Figure 14E shows H 2 0 2 produced by APi 42 AP 40 and Vitamin C in the presence of Cu 2 (1 pM) (in PBS, pH 7.4 buffer, 1 hr, 37°C) under various dissolved gas conditions (as in Figure 14D). All data points are means ±SD, n =3.

Figure 15A and 15B are graphical representations showing superoxide anion detection. Figure 15A shows the spectrophotometric absorbance at 250 nm (after subtracting buffer blanks) for AP,42 (10 4M, in PBS, pH 7.4, with 1 pM Fe 3 incubated 1 hr, 37 C) under ambient air 100 U/mL superoxide dismutase, SOD), 0, enrichment, and anaerobic (Ar) buffer gas conditions (described in WO 98/40071 PCT/US98/04683 -21 Figure 13B). Figure 15B shows the spectrophotometric absorbance at 250 nm (after subtracting buffer blanks) for variant AP peptides: A, 42 Ap,- 40 rat AP -4 0 Ap 40 and Ap 1 2 (10 pM in PBS, pH 7.4, with 1 pM Fe 3 1, incubated 1 hr, 37°C, under ambient buffer gas conditions). All data points are means ±SD, n 3.

Figure 16A and 16B are graphical representations showing production of the hydroxyl radical from the incubation of AP in the presence of substoichiometric amounts of Fe 3 or Cu 2 Figure 16A shows the signal from the TBARS assay of OH* produced from Vitamin C (100 p.M) and variant AP species pM): AP 1 42 Api-o, rat Ap,-4o, Ap 40 and Ap .28 (in PBS, pH 7.4, with 1 pM Fe" or Cu 2 as indicated, incubated 1 hr, 37°C, under ambient buffer gas conditions). Figure 16B illustrates the effect of OH*-specific scavengers upon OH- generation by Vitamin C and API- 4 2 Mannitol (5 mM, Sigma) or dimethyl sulfoxide (DMSO, 5 mM, Sigma), was co-incubated with Vitamin C (10 p.M 500 uLM H 2 0 2 or A 1 42 (10 UiM) (conditions as for Figure 16A). All data points are means ±SD, n 3.

Figure 17 shows the reversibility of zinc-induced A 40 aggregation with EDTA. Aggregation induced by pH 5.5 was not reversable in the same manner (data not shown).

Figure 18 shows the reversibility of zinc-induced aggregation of Api- 40 mixed with 5% AP-42.

Figures 19A-19C shows dilution curves for TPEN, EGTA, and bathocuproine, respectively, used in extracting a representative AD brain sample.

Figures 19A-19C show that metal chelators promote the solubilization of AP from human brain sample homogenates.

Figures 20A and 20B Figure 20A shows a western blot of chelation response in a typical AD brain. Figure 20B shows a western blot comparing extracted AP from an AD brain (AD) to that of sedimentable deposits from healthy brain tissue (young control In the experiments of Figure 20B, TBS buffer was used rather than PBS.

WO 98/40071 PCT/US98/04683 -22- Figure 21 shows an indicative blot from AD brain extract. The blot shows that chelation treatment results in disproportionate solubilization of AP dimers, while PBS alone does not.

Figure 22 shows that recovery of total soluble protein is not affected by the presence of chelators in the homogenization step.

Figure 23 shows that extraction volume affects AP solubilization.

Figures 24A and 24B Figure 24A shows the effect of metals upon the solubility of brain-derived Ap: copper and zinc can inhibit the solubilization of AP. Figure 24B shows that AP solubility in metal-depleted tissue is restored by the addition of magnesium.

Figures 25A and 25B Figure 25A shows that patterns of chelatorpromoted solubilization of AP differ in AD and aged-matched, non-AD tissue.

Upper panel: representative blot from AD specimen.

Lower panel: representative blot from aged non-AD tissue bearing a similar total A3 load.

Figure 25B shows soluble AP resulting from chelation treatment for AD and aged-matched, non-AD tissue, expressed as a percentage of the PBS-only treatment group.

Figure 26 shows that chelation promotes the solubilization of Ap, 40 and AP,.4 from AD and non-AD tissue. Representative AD (left panels) and agedmatched control specimens (right panels) were prepared as described in PBS or mM BC. Identical gels were run and Western blots were probed with mAbs W02 (raised against residues 5-16, recognizes A 1-40 and A ,1-42) G210 (raised against residues 35-40, recognizes AP 40 or G211 (raised against residues 35-42, recognizes Ap 1 42) (See Ida, N. et al., J. Biol. Chem. 271:22908 1996).

Figure 27A and 27B Figure 27A shows SDS-resistant polymerization of human API_ 40 versus human Ap1, 42 with Cu 2 'or Fe3'. Figure 27B shows SDSresistant polymerization of rat Ap,_ 40 with Cu 2 or Fe 3 Figures 28A 28C Figure 28A shows H,0 2 /Cu induced SDS-resistant polymerization of AP_ 4 2 (2.5 pM). Figure 28B shows H 2 02/Fe induced SDS- WO 98/40071 PCT/US98/04683 -23 resistant polymerization of AP 1 42 (2.5 Figure 28C shows that BC attenuates SDS-resistant polymerization of AP_ 42 (2.5 AuM).

Figures 29A and 29B show that H0 2 O generation is required for SDSresistant polymerization of human AP,_ 42 Solution concentrations of metal ion and 11,02 were 30 /M and 100 respectively. Figure 29A shows that TCEP (Tris(2-Carboxyethyl)-Phosphine Hydrochloride) attenuates SDS-resistant

A

1 -42 polymerization. A1, _42 (2.5 yM), H-202 (100 gM), ascorbic acid (100 4M), TCEP (100 pM). Figure 29B shows that anoxic conditions prevent SDS-resistant AP polymerization. AP, 1 .4 (2.5 was incubated with no metal or Cu 2 at either pH 7.4 or 6.6 and incubated for 60 min. at 25 C under normal or argon purged conditions. Argon was continuously bubbled through the buffer for 2 h (at 20 C) before the remainder of the incubation components were added.

Figures 30A-30E show dissolution of SDS-resistant Ap polymers. Figure shows that chaotrophic agents are unable to disrupt polymerization. Figure 30B shows that metal ion chelators disrupt SDS-resistant AP, 40 polymers. Figure shows that metal ion chelators disrupt SDS-resistant AP,-42 polymers. The chelators, their log stability constant, and their molecular weight, respectively, are as follows: TETA (tetraethylenediamine), 20.4, 146; EDTA (ethylenediaminetetra acetic acid), 18.1, 292; DTPA (diethylenetriaminopenta acetic acid), 21.1, 393; CDTA (trans-1, 2 -diaminocyclohexanetetra acetic acid), 22.0, 346; and NTA (nitrilotriacetic acid), 13.1, 191. Figure 30D shows that a-helical promoting solvents and low pH disrupt polymers. Aliquots of A 1_ 42 were incubated at pH 1 or with DMSO/HFIP for 2 h (30 min., 37 0 Figure 30E shows that metal ion chelators disrupt SDS-resistant AP polymers extracted from AD brains. Aliquots of SDS-resistant AP polymers extracted from AD brains were incubated with no chelator, TETA (1 mM or 5 mM) or BC (1 mM or 5 mM) for 2 h (30 min., 37°C) and aliquots collected for analysis. Monomer Ap,_ 4 0 is indicated.

WO 98/40071 PCT/US98/04683 -24- Detailed Description of the Preferred Embodiments Definitions In the description that follows, a number of terms are utilized extensively.

In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

AP peptide is also known in the art as AP, p protein, P-A4 and A4. In the present invention, the A3 peptide may be comprised of peptides Ap3, 39 A1,.

4 0 AP A .42, and A 143. The most preferred embodiment of the invention makes use of Aip,, 4 However, any of the AP peptides may be employed according to the present invention. The sequence of AP peptide is found in Hilbich, el a., J. Mol. Biol. 228:460-473 (1992).

Amyloid as is commonly known in the art, and as is intended in the present specification, is a form of aggregated protein.

Amyloidosis is any disease characterized by the extracellular accumulation of amyloid in various organs and tissues of the body.

AP Amyloid is an aggregated AP peptide. It is found in the brains of patients afflicted with AD and DS and may accumulate following head injuries.

Biological fluid means fluid obtained from a person or animal which is produced by said person or animal. Examples of biological fluids include but are not limited to cerebrospinal fluid (CSF), blood, serum, and plasma. In the present invention, biological fluid includes whole or any fraction of such fluids derived by purification by any means, by ultrafiltration or chromatography.

Copper(II), unless otherwise indicated, means salts of Cu 2 Cu 2 in any form, soluble or insoluble.

Copper(I), unless otherwise indicated, means salts ofCu', Cu'in any form, soluble or insoluble.

Metal chelators include metal-binding molecules characterized by two or more polar groups which participate in forming a complex with a metal ion, 9 9* 9999 o oo 25 9.

9 9 9 *9 .9 9 9 9 9** eg and are generally well-known in the art for their ability to bind metals competitively.

Physiological solution as used in the present specification means a solution which comprises compounds at physiological pH, about 7.4, which closely represents a bodily or biological fluid, such as CSF, blood, plasma, et cetera.

Treatment: delay or prevention of onset, slowing down or stopping the progression, aggravation, or deterioration of the symptoms and signs of Alzheimer's disease, as well as amelioration of the symptoms and signs, or curing the disease by reversing the physiological and anatomical damage.

Zinc, unless otherwise indicated, means salts of zinc, Zn 2 in any form, soluble or insoluble.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word "comprising" H:\lauraw\Keep\65484-98.doc 21/08/01 **9999 o o*99 9 9 9999 9.* o• 99 99 9 99 9 9 25a means "including but not limited to", and that the word "comprises" has a corresponding meaning.

Methods for Identifying Agents Useful in the Treatment of AD The aim of the present invention is to clarify both the factors which contribute to the neurotoxicity of AP polymers and the mechanism which underlies their formation. These findings can then be used to identify agents that can be used to decrease the neurotoxicity of AP, as well as the formation of AP polymers, and (ii) utilize such agents to develop methods of preventing, treating or alleviating the symptoms of AD and related disorders.

The present invention relates to the unexpected discovery that AP peptides directly produce oxidative stress through the generation of abundant reactive oxygen species (ROS), which include hydroxyl radical and hydrogen peroxide (H 2 0 2 The production of ROS occurs by a metal (Cu, Fe) dependant, pH mediated mechanism, wherein the reduction of Cu 2 to Cu or Fe 3 to Fe 2 is catalyzed by AP. AP is highly efficient at reducing Cu 2 and Fe 3 *e F All the redox properties of AP 1 4 0 (the most abundant form of soluble AP) are exaggerated in AP 1 42. Additionally, AP1- 42 but not API140, recruits 02 30 into spontaneous generation of another ROS, 02, which also occurs in a metalo9 9*o H:\1auraw\Keep\65484-98.doc 21/08/01 WO 98/40071 PCT/US98/04683 -26dependent manner. The exaggerated redox activity of A, 1 42 and its enhanced ability to generate ROS are likely to be the explanation for its neurotoxic properties. Interestingly, the rat homologue of A, which has 3 substitutions that have been shown to attenuate zinc binding and zinc-mediated precipitation, also exhibits less redox activity than its human counterpart. This may explain why the rat is exceptional in that it is the only mammal that does not exhibit amyloid pathology with age. All other mammals analyzed to date possess the human AP sequence.

The sequence of ROS generation by AP follows the pathway of superoxide-dismutation, which leads to hydrogen peroxide production in a Cu/Fedependent manner. After forming H 2 0 2 the hydroxyl radical is rapidly formed by a Fenton reaction with the Fe or Cu that is present, even when these metals are only at trace concentrations. The OH- radical is very reactive and rapidly attacks the'AP peptide, causing it to cross-link and polymerize. This is very likely to be the chemical mechanism that causes the covalent cross-linking that is seen in mature plaque amyloid. Importantly, the redox activity of Ap is not attenuated by precipitation of the peptide, suggesting that, in vivo, amyloid deposits could be capable of generating ROS in situ on an enduring basis. This suggests that the major source of the oxidative stress in an AD-affected brain are amyloid deposits.

A model for free radical and amyloid formation in AD is shown in Figure 12. The proposed mechanism is explained as follows.

Soluble and precipitated AP species possess superoxide dismutase (SOD)-like activity. Superoxide the substrate for the dismutation, is generated both by spillover from mitochondrial respiratory metabolism, and by

AP,

42 itself. Ap-mediated dismutation produces hydrogen peroxide (H0 2 ,)(see Figure 11), requiring Cu 2 or Fe 3 which are reduced during the reaction. Since H' is required for H 2 0 2 production, an acidotic environment will increase the reaction.

WO 98/40071 PCT/US98/04683 -27-

H

2 0 2 is relatively stable, and freely permeable across cell membranes. Normally, it will be broken down by intercellular catalase or glutathione peroxidase.

In aging and AD, levels of H 2 02 are high, and catalase and peroxidase activities are low. If H-0 2 is not completely catalyzed, it will react with reduced Cu' and Fe 2 in the vicinity of AP to generate the highly reactive hydroxyl radical by Fenton chemistry.

OH* engenders a non-specific stress and inflammatory response in local tissue. Among the neurochemicals that are released from microglia and possibly neurons in the response are Zn 2 Cu 2 and soluble Ap. Familial AD increases the likelihood that will be released at this point. Local acidosis is also part of the stress/inflammatory response. These factors combine to make Ap precipitate and accumulate, presumably so that it may function in situ as an SOD. since these factors induce reversible aggregation. Hence, more soluble AP species decorate the perimeter of the accumulating plaque deposits.

If AP encounters OH*, it will covalently cross-link during the oligomerization process, making it a more difficult accumulation to resolubilize, and leading to the formation of SDS-resistant oligomers characteristic of plaque amvloid.

If A~,42 accumulates, it has the property of recruiting 0, as a substrate for the abundant production of O; by a process that is still not understood. Since 02 is abundant in the brain, A,.

42 is responsible for setting off a vicious cycle in which the accumulation of covalently linked AD is a product of the unusual ability of AP to reduce 02, and feed an abundant substrate to itself for dismutation, leading to OH. formation. The production of abundant free radicals by the accumulating amyloid may further damage many systems including metal regulatory proteins, thus compounding the problem. This suggests that the major source of the oxidative stress in an AD-affected brain are amyloid deposits.

WO 98/40071 PCT/US98/04683 -28- The metal-dependent chemistry of Ap-mediated superoxide dismutation is reminiscent of the activity of superoxide dismutase (SOD). Interestingly, mutations of SOD cause amyotrophic lateral sclerosis, another neurodegenerative disorder. SOD is predominantly intracellular, whereas AP is constitutively found in the extracellular spaces where it accumulates. Investigation of AP by laser flash photolysis confirmed the peptide's SOD-like activity, suggesting that AP may be an anti-oxidant under physiological circumstances. Since H 2 has been shown to induce the production of Ap, the accumulation of AP in AD may reflect a response to an oxidant stress paradoxically caused by AP excess. This may cause and, in turn, be compounded by, damage to the biometal homeostatic mechanisms in the brain environment.

Thus, it has recently been discovered that much of the AP aggregate in AD-affected brain is held together by zinc and copper, (ii) that A3 peptides exhibit Fe/Cu-dependent redox activity similar to that of SOD, (iii) that AP,.4 is especially redox reactive and has the unusual property of reducing 0, to O, and (iv) that deregulation of AP redox reactivity causes the peptide to conveniently polymerize. Since these reactions must be strongly implicated in the pathogenetic events of AD, they offer promising targets for therapeutic drug design.

The discovery that AP can generate H 2 0 2 and Cu', both of which are associated with neurotoxic effects, offers an explanation for the neurotoxicity of AP polymers. These findings suggest that it may be possible to lessen the neurotoxicity of AP by controlling factors which alter the concentrations of Cu' and ROS, including hydrogen peroxide, being generated by accumulated and soluble Ap. It has been discovered that manipulation of factors such as zinc, copper, and pH can result in altered Cu' and H 2 0, production by Ap. Therefore, agents identified as being useful for the adjustment of the pH and levels of zinc and copper of the brain interstitium can be used to adjust the concentration of Cu' and H 2 0 2 and can therefore be used to reduce the neurotoxic burden. Such agents will thus be a means of treating Alzheimer's disease.

WO 98/40071 PCT/US98/04683 -29- Thus, one object of the present invention is to provide a method for the identification of agents to be used in the treatment of AD. As may be understood by reference to the Examples below, agents to be used in the treatment of AD include: agents that reduce the amount of Cu or Fe 2 produced by A3; agents that promote or inhibit the production of hydrogen peroxide by AP; agents that inhibit the production of O0 by A3; agents that inhibit the production of OH,.

Of course, as aggregation and especially crosslinking of A3 contributes to the neurotoxic burden, agents which have been identified to have the activities listed above may then also be subjected to tests which determine if an agent is capable of inhibiting AP plaque deposition or facilitating plaque resolubilization (see Example 1).

Agents identified as having the above-listed activities may then be tested for their ability to reduce the neurotoxicity of both soluble and crosslinked Ap.

Thus, in one aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein the agent is capable of altering, and preferably decreasing, the production of Cu' by AP, the method comprising: adding Cu 2 to a first AP sample; allowing the first sample to incubate for an amount of time sufficient to allow said first sample to generate Cu'; adding Cu 2 4 to a second AP sample, the second sample additionally comprising a candidate pharmacological agent; allowing the second sample to incubate for the same amount of time as the first sample; determining the amount of Cu' produced by the first sample and the second sample; and WO 98/40071 PCT/US98/04683 comparing the amount ofCu' produced by the first sample to the amount of Cu' produced by the second sample; whereby a difference in the amount of Cu' produced by the first sample as compared to the second sample indicates that the candidate pharmacological agent has altered the production of Cu 4 by AP. Of course, where the amount of Cu' is lower in the second sample then in the first sample, this will indicate that the agent has decreased Cu' production.

In a preferred embodiment, the amount of Cu* present in said first and said second sample is determined by adding a complexing agent to said first and said second sample. wherein said complexing agent is capable of combining with Cu' to form a complex compound, wherein said complex compound has an optimal visible absorption wavelength; measuring the absorbancy of said first and said second sample: and calculating the concentration of Cu' in said first and said second sample using the absorbancy obtained in step In a more preferred embodiment, the complexing agent is bathocuproinedisulfonic (BC) anion. The concentration of Cu' produced by AP may then be calculated on the basis of the absorbance of the sample at about 478 nm to about 488 nm, more preferable about 480 to about 486 nm, and most preferably about 483 nm.

In an even more preferred embodiment, the above-described method may be performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader, thus allowing large numbers of candidate pharmacological compounds to be tested simultaneously.

In another aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering, and preferably decreasing, the production of Fe2' by AP, said method comprising: WO 98/40071 PCT/US98/04683 -31 adding Fe 3 to a first A3 sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate Cu'; adding Fe 3 1 to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of Fe 2 produced by said first sample and said second sample; and comparing the amount of Fe 2 present in said first sample to the amount of Fe 2 present in said second sample; whereby a difference in the amount of Fe 2 1 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of Fe2' by AP. Of course, where the amount of Fe 2 1 is lower in the second sample than in the first sample, this will indicate that the agent has decreased Fe 2 4 production.

In a preferred embodiment, the amount of Fe 2 present is determined by using a spectrophotometric method analogous to that used for the determination of Cu', above. In this method, the complexing agent is bathophenanthrolinedisulfonic (BP) anion. The concentration of Fe 2 4 -BP produced by AP may then be calculated on the basis of the absorbance of the sample at about 530 to about 540 nm, more preferably about 533 nm to about 538 nm, and most preferably about 535 nm.

In yet another aspect, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of H 2 0 2 by Ap, said method comprising: WO 98/40071 PCT/US98/04683 -32adding Cu 2 1 or Fe 3 1 to a first AP sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate H20 2 adding Cu 2 or Fe 3 to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of H 2 02 produced by said first sample and said second sample; and comparing the amount ofH 2 0 2 present in said first sample to the amount of 1-1H 2 0 2 present in said second sample; whereby a difference in the amount of H 2 0 2 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of H 2 0 2 by AP. As will be understood by one of ordinary skill in the art, this method may be used to detect agents which decrease the amount of H 2 0 2 produced (in which case the amount of H2,0 2 will be lower in the second sample than in the first sample), or to increase the amount of H 2 0, produced (in which case the amount ofH,O, will be lower in the first sample than in the second sample).

In a preferred embodiment, the determination of the amount of H 2 0,, present in said first and said second sample is determined by adding catalase to a first aliquot of said first sample obtained in step of claim 1 in an amount sufficient to break down all of the

H

2 0 2 generated by said sample; adding TCEP, in an amount sufficient to capture all of the H-02 generated by said samples, to said first aliquot (ii) a second aliquot of said first sample obtained in step of claim I; and (iii) said second sample obtained in step of claim 1; WO 98/40071 PCT/US98/04683 -33incubating the samples obtained in step for an amount of time sufficient to allow the TCEP to capture all of the H 2 02,; adding DTNB to said samples obtained in step incubating said samples obtained in step for an amount of time sufficient to generate TMB; measuring the absorbancy at about 407 to about 417 nm of said samples obtained in step and calculating the concentration ofH 2 0, in said first and said second sample using the absorbancies obtained in step In a preferred embodiment, the absorbancy ofTMB is measured at about 412 nm.

In a preferred embodiment, the above-described method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader, thus making it possible to screen large numbers of candidate pharmacological agent simultaneously.

In another embodiment, the invention provides a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of decreasing the production of O; by AP, said method comprising: adding AP and to a first buffer sample having an 02 tension greater than 0; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate O0; adding AP and a candidate pharmacological agent to a second buffer sample having an 02 tension greater than 0; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of O0 produced by said first sample and said second sample; and comparing the amount of 0O present in said first sample to the amount of 02 present in said second sample; WO 98/40071 PCT/US98/04683 -34whereby a difference in the amount of O; present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of 0O by Ap. In a preferred embodiment, the AP used is AP|-42- Of course, the amount of 02 produced by AP may be measured by any method known to those of ordinary skill in the art. In a preferred embodiment, the determination of the amount of O present in said samples is accomplished by measuring the absorbancy of the sample at about 250 nm.

Because the ability of AP to generate H,0 2 from 0O may in many instances be beneficial, in a preferred embodiment, the invention also relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of interfering with the interaction of 02 and Ap to produce without interfering with the SOD-like activity of AP, said method comprising: identifying an agent capable of decreasing the production of 02 by Ap; and determining the ability of said agent to alter the SOD-like activity of Ap. In a preferred embodiment, the determination of the ability of said agent to alter the SOD-like activity of AP is made by determining whether AP is capable of catalytically producing Cu*, Fe 2 or H,0 2 Methods, besides those which are disclosed elsewhere in this application, for determining if AP is capable of catalytically producing Cu', Fe 2 or H 2 0 2 are well known to those of ordinary skill in the art. In particular, the catalytic production of H 2 0, may be determined by using laser flash photolysis or pulse radiolysis (Peters, G. Rodgers, M.A. J., Biochim. Biophys. Acta 637:43-52 (1981).

In another aspect, candidate pharmacological agents which have been identified by one or more of the above screening assays can undergo further screening to determine if the agents are capable of altering, and preferably reducing or eliminating, Ap-mediated toxicity in cell culture. Such assays include the MTT assay, which measures the reduction of 3-(4,5-dimethylthiazol- WO 98/40071 PCT/US98/04683 2-yl)-2,5, diphenyl tetrazolium bromide (MTT) to a colored formazon (Hansen et al., JImmunol Methods, 119:203-210 (1989)). Although alternatives have not been ruled out (see Burdon et al., Free Radic Res Commun.,18(6):369-380 (1993)), the major site of MTT reduction is thought to be at two stages of electron transport, the cytochrome oxidase and ubiquinone of mitochondria (Slater et al., 1963). A second cytotoxic assay is the release of lactic dehydrogenase (LDH) from cells, a measurement routinely used to quantitate cytotoxicity in cultured CNS cells (Koh, J.Y. and D.W. Choi, J. Neurosci. Meth. 20:83-90 (1987). While MTT measures primarily early redox changes within the cell reflecting the integrity of the electron transport chain, the release of LDH is thought to be through cell lysis. A third assay is visual counting in conjunction with trypan blue exclusion. Other commercially available assays for neurotoxicity, including the Live-Dead assay, may also be used to determine if a candidate compound which alters Cu', Fe 2

H

2 0 2 OH-, and 02 production, or alters copper-induced, pH dependent aggregation and crosslinking of AP, is also capable of reducing the neurotoxicity of A3.

Thus, in another preferred embodiment, the invention relates to a method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of reducing the toxicity of AP, said method comprising: adding AP to a first cell culture; adding Ap to a second cell culture, said second cell culture additionally containing a candidate pharmacological agent; determining the level of neurotoxicity of AP in said first and said second samples; and comparing the level ofneurotoxicity of A in said first and said second samples, whereby a lower neurotoxicity level in said second sample as compared to said first sample indicates that said candidate pharmacological agent has reduced the neurotoxicity of Ap, and is thereby capable of being used to treat AD.

WO 98/40071 PCT/US98/04683 -36- Assays which can be used to determine the neurotoxicity of a candidate agent include, but are not limited to, the MTT assay and the LDH release assay, as described in Behl et al., Cell 77:817-827 (1994), and the Live/Dead EukoLight Viability/Cytotoxicity Assay, commercially available from Molecular Probes, Inc.

(Eugene, OR).

Cells types which may be used for these neurotoxicity assays include both cancer cells and primary cells, such as rat primary frontal neuronal cells.

Candidate pharmacological agents to be tested in any of the abovedescribed methods will be broad-ranging but can be classified as follows: Candidate pharmacological agents for the alteration of the SOD-like activity of AP will be broad-ranging but can be classified as follows: Agents Which Modify the Availability of Zn or Cu for Interaction with Af They include chelating agents such as desferrioxamine, but also include amino acids histidine and cysteine which bind free zinc, and are thought to be involved in bringing zinc from the plasma across the blood-brain barrier (BBB).

These agents include all classes of specific zinc chelating agents, and combinations of non-specific chelating agents capable of chelating zinc such as EDTA (Edetic acid, N,N'-1,2-Ethane diylbis[N-(carboxymethyl)glycine] or (ethylenedinitrilo)tetraacetic acid, entry 3490 in Merck Index 10th edition) and all salts of EDTA, and/or phytic acid [myo-lnositol hexakis(dihydrogen phosphate), entry 7269 in the Merck Index 10th edition] and phytate salts.

Preferred candidate agents within this class include bathocuproine and bathophenanthroline.

Miscellaneous Because there is no precedent for an effective anti-amyloidotic pharmaceutical, it is reasonable to serendipitously try out compounds which may WO 98/40071 PCT/US98/04683 -37have access to the brain compartment for their ability to inhibit either Cu' or H 2 0 2 production by Ap. These compounds include dye compounds, heparin, heparin sulfate, and anti-oxidants, ascorbate, trolox and tocopherols.

In the present invention, the Ap used may be any form of A3. In a preferred embodiment, the AP used is selected from the group consisting of Ap 39 Api- 40

AP,

4 1

A,.

42 and A,- 4 3 Even more preferably, the AP used is

AP.

4 0 or API42. The most preferred embodiment of the invention makes use of Ap- 40 The sequence of AP peptide is found in Hilbich, et al., J. Mol. Biol.

228:460-473 (1992).

The pH of the various reaction mixtures are preferably close to neutral (about The pH, therefore, may range from about 6.6 to about 8, preferably from about 6.6 to about 7.8, and most preferably about 7.4.

Buffers which can be used in the methods of the present invention include, but are not limited to, PBS, Tris-chloride and Tris-base, MOPS, HEPES, bicarbonate, Krebs, and Tyrode's. The concentration of the buffers may be between about 10 mM and about 500 mM. Because of the nature of the assays which are included in the methods of the claimed invention, when choosing a buffer, it must be borne in mind that spontaneous free radical production within a given buffer might interfere with the reactions. For this reason, PBS is the preferred buffer for use in the methods of the invention, although other buffers may be used provided that proper controls are used to correct for the abovementioned free radical formation of a given buffer.

Cu 2 must be present in the reaction mixture for A3 to produce Cu'. Any salt of Cu 2 may be used to satisfy this requirement, including, but not limited to, CuCl 2 Cu(N0 3 2 etc. Concentrations of copper from at least about 1 PM may be used; most preferable, a copper concentration of about 10 pM is to be included in the reaction mixture.

Similarly, a redox active metal such as Cu 2 or Fe 3 must be present in the reaction mixture for Ap to catalytically produce H 2 0 2 Any salt of Cu 2 may be used to satisfy this requirement, including, but not limited to, CuCd, Cu(NO 3 2 WO 98/40071 PCT/US98/04683 -38etc. Similarly, and salt of Fe 3 1 may be used in accordance with the invention, such as FeCl3. Concentrations of copper or iron from at least about 1 pM may be used; most preferably, a copper or iron concentration of about 10 pM is to be included in the reaction mixture.

The present invention may be practiced at temperatures ranging from about 25 °C to about 40°C. The preferred temperature range is from about to about 40°C. The most preferred temperature for the practice of the present invention is about 37°C, human body temperature.

The production of Cu' and H,O, by AP peptide occurs at nearinstantaneous rate. Hence, the measurement of the concentration of Cu or H 2 0, produced may be performed by the present methods substantially immediately after the addition of Cu 2 to the AP peptide. However, if desired, the reaction may be allowed to proceed longer. In a preferred embodiment of the invention, the reaction is carried out for about 30 minutes.

The invention may also be carried out in the presence of biological fluids, such as the preferred biological fluid, CSF, to closely simulate actual physiological conditions. Of course, such fluids will already contain Ap, so that where the methods of the invention are to be carried out utilizing a biological fluid such as CSF, no further AP peptide will be added to the sample. The biological fluid may be used directly or diluted from about 1:1,000 to about fold.

The amount ofH 2 2 O, Cu' or Fe 2 produced by a sample may be measured by any standard assay for H 2 02, Cu' or Fe 2 For example, the PeroXOquant Quantitative Peroxide Assay (Pierce, Rockford, IL) may be used to determine the amount of H 2 0 2 produced. Fe 2 1 may be determined using the spectrophotometric method of Linert et al., Biochim. Biophys. Acta 1316:160-168 (1996). Other such methods will be readily apparent to those of ordinary skill in the art.

In a preferred embodiment, the H,0 2 or Cu' produced by the sample is complexed with a complexing agent having an optimal visible absorption WO 98/40071 PCT/US98/04683 -39wavelength. The amount of H,,0 or Cu produced by a sample is then detected using optical spectrophotometry (see Example 2).

In a preferred embodiment, the complexing agent to be used for the determination of the amount of Cu' produced is bathocuproinedisulfonic anion (see Example the complex Cu'-BC has an optimal visible absorption wavelength of about 483 nm. As is mentioned above, AP will produce H,O, and Cu' almost immediately following the addition of Cu 2 and Zn 2 to the reaction mixture. Thus, BC may be added to the reaction immediately following the addition of Cu 2 and Zn 2 to the Ap samples. The concentration of BC to be achieved in a sample is between about 10 p.M to about 400 pM, more preferably about 75 upM to about 300 piM, and still more preferably about 150 PM to about 275 upM. In the most preferred embodiment, the concentration of BC to be achieved in a sample is about 200 pM. Of course, one of ordinary skill in the art can easily optimize the concentration of BC to be added with no more than routine experimentation.

Where the amount of Fe 2 produced is to be determined, the complexing agent to be used for the determination of the amount of Fe 2 z produced is bathophenanthrolinedisulfonic (BP) anion, (see Example the complex Fe 2

-BP

has an optimal visible absorption wavelength of about 535 nm. As is mentioned above, AP will produce H 2 ,O and Fe2' almost immediately following the addition of Fe 3 and Zn 2 1 to the reaction mixture. Thus, BP may be added to the reaction immediately following the addition of Fe 3 and Zn 2 1 to the AP samples. The concentration of BP to be achieved in a sample is between about 10 pM to about 400 uiM, more preferably about 75 pM to about 300 pM, and still more preferably about 150 pM to about 275 pM. In the most preferred embodiment, the concentration of BP to be achieved in a sample is about 200 uM. Of course, one of ordinary skill in the art can easily optimize the concentration of BP to be added with no more than routine experimentation.

The above-described spectrophotometric assays may be used to determine the concentration of Cu' or Fe 2 as is described in Example 2.

WO 98/40071 PCT/US98/04683 Each of the assays of the present invention is ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement therein one or more container means, such as vials, tubes, and the like, each of said container means comprising one of the separate elements of the assay to be used in the method. For example, there may be provided a container means containing standard solutions of the AP peptide or lyophilized AP peptide and a container means containing a standard solution or varying amounts of a salt of redox active metal, such as Cu 2 or Fe 3 1, in any form, in solution or dried, soluble or insoluble, in addition to further carrier means containing varying amounts and/or concentrations of reagents used in the present methods. For example, solutions to be used for the determination of Cu' or Fe 2 as described in Example 2 will include BC anion and BP anion, respectively. Similarly, solutions to be used for the determination of H 2 0 2 as described in Example 2 include TCEP and DTNB, as well as catalase Standard solutions of AP peptide preferably have concentrations above about tM, more preferably from about 10 to about 25 .M or if the peptide is provided in its lyophilized form, it is provided in an amount which can be solubilized to said concentrations by adding an aqueous buffer or physiological solution. The standard solutions of analytes may be used to prepare control and test reaction mixtures for comparison, according to the methods of the present invention.

Agents Useful in the Treatment of AD A further aspect of the present invention is predicted in part on the elucidation of mechanisms of neurotoxicity in the brain in AD subjects. One mechanism involves a novel 0; and biometal-dependent pathway of free radical generation by AP peptides. The radicals of this aspect of the present invention may comprise reactive oxygen species (ROS) such as but not limited to 0_ and OH as well as radicalized AP peptides. It is proposed, according to the present WO 98/40071 PCTIUS98/04683 -41 invention, that by interfering in the radical generating pathway, the neurotoxicity of the AP peptides is reduced.

Accordingly, one aspect of the present invention contemplates a method for treating Alzheimer's disease (AD) in a subject, said method comprising administering to said subject an effective amount of an agent which is capable of inhibiting or otherwise reducing metal-mediated production of free radicals.

The preferred agents according to this aspect are metal chelators, metal complcxing compounds, antioxidants and compounds capable of reducing radical formation of AP peptides or mediated by AP peptides. Particularly preferred metal chelators and metal complexors are capable of interacting with metals (M) having either a reduced charge state (M or an oxidized state Even more particularly, M is Fe and/or Cu.

It is proposed that interactions of AP with Fe and Cu are of significance to the genesis of the oxidation insults that are observed in the AD-affected brain.

This is due to redox-active metal ions being concentrated in brain neurons and participating in the generation of ROS or other radicals by transferring electrons in their reduced state and described in the following reactions: Reduced Fe/Cu reacts with molecular oxygen to generate the superoxide anion.

M' O, M n) 02 Reaction (1) The 0, generated undergoes dismutation to H 2 0, either catalyzed by SOD or spontaneously.

0O 0O 2H' H,20 0 2 Reaction (2) The reaction of reduced metals with H 2 0 2 generates the highly reactive hydroxyl radical by the Fenton reaction.

WO 98/40071 PCTUS98/04683 -42- M" H,0 2 M OH OH- Reaction (3) Additionally, the Haber-Weiss reaction can form OH in a reaction catalyzed by (Miller el al., 1990).

O; H 2 O OH OH- 02 Reaction (4) Still more preferably, the agent comprises one or more of bathocuproine and/or bathophenanthroline or compounds related thereto at the structural and/or functional levels. Reference to compounds such as bathocuproine and bathophenanthroline include functional derivatives, homologues and analogues thereof.

Accordingly, another aspect of the present invention provides a method for treating AD in a subject said method comprising administering to said subject an effective amount of an agent comprising at least one metal chelator and/or metal complexing compound for a time and under conditions sufficient to inhibit or otherwise reduce metal-mediated production of free radicals.

In one aspect, the free radicals are reactive oxygen species such as O, or OH.. In another aspect, the free radicals include forms of Ap. In another aspect, the free radicals include forms of Ap. However, in a broader sense, it has been found that the metal-mediated AD reactions in the brain of AD patients results in the generation of reduced metals and hydrogen peroxide, as well as superoxide and hydroxyl radicals. Furthermore, formation of any other radical or reactive oxygen species by interaction of any of these products with any other metabolic substrate superoxide nitric acid peroxynitrite) contributes to the pathology observed in AD and Down's syndrome patients. Cu 2 reaction with AP generates Cu', AP., H 2 0 2 and OH., all of which not only directly damage the cells, but also react with biochemical substrates like nitric oxide.

Yet a further aspect of the present invention is directed to a method for treating AD in a subject, said method comprising administering to said subject an WO 98/40071 PCT/US98/04683 -43 effective amount of an agent, said agent comprising a metal chelator, metal complexing compound or a compound capable of interfering with metal mediated free radical formation mediated by Ap peptides for a time and under conditions sufficient to inhibit or otherwise reduce production of radicals.

The preferred metals according to these aspects of the present invention include Cu and Fe and their various oxidation states. Most preferred are reduced forms of copper and iron (Fe 2 Another mechanism elucidated in accordance with the present invention concerns the formation of aggregates of Ap, as in conditions involving amyloidosis. In a preferred embodiment, the aggregates are those of amyloid plaques occurring in the brains of AD-affected subjects.

The aggregates according to this aspect of the present invention are nonfibrillary and fibrillary aggregates and are held together by the presence of a metal such as zinc and copper. A method of treatment involves resolubilizing these AP aggregates.

The data indicate that Zn-induced Api 40 aggregation is completely reversible in the presence of divalent metal ion chelating agents. This suggests that zinc binding may be a reversible, normal function of AP and implicates other neurochemical mechanisms in the formation of amyloid. A process involving irreversible AP aggregation, such as the polymerization of AP monomers, in the formation of polymeric species of AP that are present in amyloid plaques is thus a more plausible explanation for the formation of neurotoxic polymeric AP species.

According to this aspect of the present invention, there is provided a method of treating AD in a subject comprising administering to said subject an agent capable of promoting, inducing or otherwise facilitating resolubilization of amyloid deposits for a time and under conditions to effect said treatment.

With respect to this aspect of the present invention, it is proposed that a metal chelator or metal complexing agent be administered. AP deposits which are composed of fibrillary and non-fibrillary aggregates may be resolubilized by WO 98/40071 PCT/US98/04683 -44the metal chelating or metal complexing agents, according to this aspect. While fibrile aggregations per se, may not be fully disassociated by administration of such agents, overall deposit resolubilization approaches In addition, the agent of this aspect of the present invention may comprise a metal chelator or metal complexing agent alone or in combination with another active ingredient such as but not limited to rifampicin, disulfiram, indomethacin or related compounds. Preferred metal chelators are bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof.

A "related" compound according to these and other aspects of the present invention are compounds related to the levels of structure or function and include derivatives, homologues and analogues thereof.

Accordingly, the present invention contemplates compositions such as pharmaceutical compositions comprising an active agent and one or more pharmaceutically, acceptable carriers and/or diluents. The active agent may be a single compound such as a metal chelator or metal complexing agent or may be a combination of compounds such as a metal chelating or complexing compound and another compound. Preferred active agents include, for reducing radical formation and for promoting resolubilization, bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof, or any combination thereof.

It has been found that for some chelators there is an optimal concentration "window" within which the AP aggregates are dissolved (see Example 5 below).

Increasing the concentration of chelators above the concentration window may not only be toxic to the patient, but also can sharply decrease the dissolution effect of chelators on the AP amyloid. Similarly, amounts below the optimal concentration window are too small to result in significant dissolution.

Although the data indicate that higher concentrations of chelators may be effective in dissolution of AP aggregates when supplemented by certain substances which favor dissolution, e.g. magnesium, it is expected that there will WO 98/40071 PCT/US98/04683 still be an optimally effective window of chelator concentration. Within the optimal dissolution window, it will be important to balance optimal dissolution against possible side effects or toxicity inherent in the use of chelators as pharmaceutical compositions.

Therefore, for each given patient, the attending physician need be mindful of the window effect and attend to varying the dosages of chelator compsositions so that during the course of administration, chelator concentrations will be varied frequently to randomly allow achieving the most effective concentration for dissolving AP amyloid deposits in the given patient.

It is, therefore, desired that the plasma levels of chelators not be steady state, but be kept fluctuating, so that transiently optimal concentrations occur in the patient. The best way to dose the patient is no more often than every three hours, preferably every six hours or eight hours, but as infrequently as once every day or once every two days are expected to be therapeutic.

For the treatment of moderately affected or severely affected patients, where risking the neurological side effects is less of a concern since the quality of their life is very poor, the patient may be put on a program of treatment consisting of high dose chelator compositions for 1 to 21 days, but preferably no more than 14 days, followed by a period of low dose therapy for seven days to three months. A convenient schedule would be two weeks of high dose therapy followed by two weeks of low dose therapy, oscillating between high and low dose periods for up to 12 months. If after 12 months the patient has made no clinical gains on high/low chelator therapy, the treatment should be discontinued.

Another typical case would be the treatment of a mildly affected individual. Such a patient would be treated with low dose chelators for up to 12 months. If after 6 months no clinical gains have been made, the patient could then be placed on the high/low alternation regimen for up to another 12 months.

Accordingly, the present invention contemplates compositions such as pharmaceutical compositions comprising an active agent and one or more pharmaceutically, acceptable carriers and/or diluents. The active agent may be a WO 98/40071 PCT/US98/04683 -46metal chelator or a combination of a metal chelator and another active agent, e.g.

an antioxidant or an alkalinizing agent Most preferrably, the invention involves the co-administration hydrophobic and hydrophillic derivatives of chelators. Also most preferrably, the invention involves the co-administration of chelators of oxidized metals and chelators of reduced metals. Various permutations of both classes of chelators may be administered to achieve optimal results.

The pharmaceutical forms containing the active agents may be administered in any convenient manner either orally or parenteraly, such as by intravenous, intraperitoneal, subcutaneous, rectal, implant, transdermal, slow release, intrabuccal, intracerebral or intranasal administration. Generally, the active agents need to pass the blood brain barrier and may have to be chemically modified, e.g. made hydrophobic, to facilitate this or be administered directly to the brain or via other suitable routes. For injectable use, sterile aqueous solutions (where water soluble) are generally used or alternatively sterile powders for the extemporaneous preparation of sterile injectable solutions may be used. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization by, for WO 98/40071 PCT/US98/04683 -47example, filtration or irradiation. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof. Preferred compositions or preparations according to the present invention are prepared so that an injectable dosage unit contains between about 0.25 jg and 500 mg of active compound.

When the active agents are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 1 tg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain other components such as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings WO 98/40071 PCT/US98/04683 -48or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active material and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 jig WO 98/40071 PCT/US98/04683 -49to about 2000 mg. Alternatively, amounts ranging from 200 ng/kg/body weight to above 10 mg/kg/body weight may be administered. The amounts may be for individual active agents or for the combined total of active agents.

Compositions of the present invention include all compositions wherein the compounds of the present invention are contained in an amount which is effective to achieve their intended purpose. They may be administered by any means that achieve their intended purpose. The dosage administered will depend on the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of the treatment, and the nature of the effect desired. The dosage of the various compositions can be modified by comparing the relative in vivo potencies of the drugs and the bioavailability using no more than routine experimentation.

The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are mammals, humans, although the invention is not intended to be so limited.

The following examples are provided by way of illustration to further describe certain preferred embodiments of the invention, and are not intended to be limiting of the present invention, unless specified.

WO 98/40071 PCT/US98/04683 Examples Example 1 Copper-Induced, pH Dependent Aggregation of AP Materials and Methods a) Preparation of Af Stock Human AP 40 peptide was synthesized, purified and characterized by HPLC analysis, amino acid analysis and mass spectroscopy by W.M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT). Synthetic AP peptide solutions were dissolved in trifluoroethanol (30 in Milli-Q water (Millipore Corporation, Milford, MA)) or 20 mM HEPES (pH at a concentration of 0.5-1.0 g/ml, centrifuged for 20 min. at 10,000g and the supernatant (stock AP 1-40) used for subsequent aggregation assays on the day of the experiment. The concentration of stock AP I 40 was determined by UV spectroscopy at 214 nm or by Micro BCA protein assay (Pierce, Rockford, IL).

The Micro BCA assay was performed by adding 10 l of stock Ap i 40 (or bovine serum albumin standard) to 140 tl of distilled water, and then adding an equal volume of supernatant (150utl) to a 96-well plate and measuring the absorbance at 562 nm. The concentration of Ap- 4 0 was determined from the BSA standard curve. Prior to use all buffers and stock solutions of metal ions were filtered though a 0.22 tm filter (Gelan Sciences, Ann Arbor, MI) to remove any particulate matter. All metal ions were the chloride salt, except lead nitrate.

b) Aggregation Assays Ap- 40 stock was diluted to 2.5 pM in 150 mM NaCl and 20 mM glycine (pH MES (pH 5-6.2) or HEPES (pH with or without metal ions, incubated (30 min., 37 centrifuged (20 min., 10,000g). The amount of protein WO 98/40071 PCT/US98/04683 -51 in the supernatant was determined by the Micro BCA protein assay as described above.

c) Turbidometric Assays Turbidity measurements were performed as described by Huang, et al., J. Biol. Chem. 272:26464-26470 (1997), except AP1-40 stock was brought to 10 pM (300 pl) in 20 mM HEPES buffer, 150 mM NaCI (pH 6.6, 6.8 or 7.4) with or without metal ions prior to incubation (30 min., 37°C). To investigate the pH reversibility ofCu 2 -induced AP aggregation, 25 ktM Ap, 40 and 25 M Cu 2 were mixed in 67 mM phosphate buffer, 150 mM NaCI (pH 7.4) and turbidity measurements were taken at four 1 min. intervals. Subsequently, 20 pl aliquots of 10 mM EDTA or 10 mM Cu 2 were added into the wells alternatively, and, following a 2 min. delay, a further four readings were taken at 1 min. intervals.

After the final EDTA addition and turbidity reading, the mixtures were incubated for an additional 30 min. before taking final readings. To investigate the reversibility of pH mediated Cu2'-induced A 1 40 aggregation, 10 pM AP1- 40 and upM Cu 2 were mixed in 67 mM phosphate buffer, 150 mM NaCI (pH 7.4) and an initial turbidity measurement taken. Subsequently, the pH of the solution was successively decreased to 6.6 and then increased back to 7.5. The pH of the reaction was monitored with a microprobe (Lazar Research Laboratories Inc., Los Angeles, CA) and the turbidity read at 5 min. intervals for up to 30 min. This cycle was repeated three times.

d) Immunofiltration Detection of Low Concentrations ofA Aggregate Physiological concentrations of API-40 (8 nM) were brought to 150 mM NaCI, 20 mM HEPES (pH 6.6 or 100 nM BSA with CuCl 2 0.1, 0.2, and 2 uM) and incubated (30 min., 37°C). The reaction mixtures (200 pl) were then placed into the 96-well Easy-Titer ELIFA system (Pierce, Rockford, IL) and WO 98/40071 PCT/US98/04683 -52filtered through a 0.22 [m cellulose acetate filter (MSI, Westboro, MA).

Aggregated particles were fixed to the membrane glutaraldehyde, 15 min.), washed thoroughly and then probed with the anti-AP mAB 6E10 (Senetek, Maryland Heights, MI). Blots were washed and exposed to film in the presence of ECL chemiluminescence reagents (Amersham, Buckinghamshire, England).

Immunoreactivity was quantified by transmitance analysis of ECL film from the immunoblots.

e) A Metal-capture ELISA

AP,

40 (1.5 ng/well) was incubated (37°C, 2 hr) in the wells of Cu 2 coated microtiter plates (Xenopore, Hawthorne, NJ) with increasing concentrations of Cu 2 (1-100 nM) as described by Moir et al., .Journal of Biological Chemistry (submitted). Remaining ligand binding sites on well surfaces were blocked with 2% gelatin in tris-buffered saline' (TBS) (3 hr at 37 0 C) prior to overnight incubation at room temperature with the anti-AP mAb 6E1 0 (Senetek, Maryland Heights, MI). Anti-mouse IgG coupled to horseradish peroxidase was then added to each well and incubated for 3 hr at 37 0 C. Bound antibodies were detected by a 30 minute incubation with stable peroxidase substrate buffer/3,3',5,5'- Tetramethyl benzidine (SPSB/TMB) buffer, followed by the addition of 2 M sulfuric acid and measurement of the increase in absorbance at 450 nm.

f) Extraction of Af from Post-mortem Brain Tissue Identical regions of frontal cortex (0.5g) from post-mortem brains of individuals with AD, as well as non-AD conditions, were homogenized in TBS, pH 4.7 ±metal chelators. The homogenate was centrifuged and samples of the soluble supernatant as well as the pellet were extracted into SDS sample buffer and assayed for AP content by western blotting using monoclonal antibody (mAb) W02. The data shows a typical (of n=12 comparisons) result comparing the WO 98/40071 PCT/US98/04683 -53 amount of AP extracted into the supernatant phase in AD compared to control (young adult) samples. N,N,N',N'-tetrakis [2-pyridyl-methyl] ethylenediamine (TPEN) (5 M) allows the visualization of a population of pelletable AP that had not previously been recognized in unaffected brain samples Figure 8).

g) A P Cross-linking by Copper CuL -induced SDS-resistant oligomerization of AP: Ap,,40 (2.5 utM), 150 mMN NaCI. 20 mM hepes (pl- 6.6. 7.4, 9) with or without ZnC1, or CuC,.

Following incubation (37 0 aliquots of each reaction (2 ng peptide) were collected at 0 d. 1 d. 3 d and 5 d and western blotted using anti-AP monoclonal antibody SL 10 Figure Migration of the molecular size markers are indicated The dimer formed under these conditions has been found to be SDSresistant. Cu' (2-30 pM) induced SDS-resistant polymerization of peptide. Coincubation with similar concentrations ofZn accelerates the polymerization, but zinc alone has no effect. The antioxidant sodium metabisulfite moderately attenuates the reaction, while ascorbic acid dramatically accelerates Ap polymerization. This suggests reduction of Cu 2 to Cu' with the latter mediating SDS-resistant polymerization of Ap. Mannitol also abolishes the polymerization, suggesting that the bridging is mediated by the generation of the hydroxyl radical by a Fenton reaction that recruits Cu'. It should be noted that other means of visualizing and/or determining the presence or absence of polymerization other than western blot analysis may be used. Such other means include but are not limited to density sedimentation by centrifugation of the samples.

Results It has previously been reported that Zn 2 induces rapid precipitation of AP in vitro Bush, el al., J. Biol. Chem. 269:12152 (1994)). This metal has an abnormal metabolism in AD and is highly concentrated in brain regions where AP WO 98/40071 PCT/US98/04683 54precipitates. The present data indicate that under very slightly acidic conditions, such as in the lactic acidotic AD brain, Cu 24 strikingly induces the precipitation of AP through an unknown conformational shift. pH alone dramatically affects AP solubility, inducing precipitation when the pH of the incubation approaches the pi of the peptide (pH Zinc induces 40-50% of the peptide to precipitate at pH 6.2, below pH 6.2 the precipitating effects of Zn 2 and acid are not summative.

At pH 5, Zn 2 has little effect upon AP solubility. Cu 2 is more effective than Zn 2 in precipitating A3 and even induces precipitation at the physiologically relevant pH 6-7. Copper-induced precipitation of AP occurs as the pH falls below 7.0, comparable with conditions of acidosis (Yates, el al., J. Neurochem.

55:1624 (1990)) in the AD brain. Investigation of the precipitating effects of a host or other metal ions in this system indicated that metal ion precipitation of Ap was limited to copper and zinc, as illustrated, although Fe 2 possesses a partial capacity to induce precipitation (Bush, et al., Science 268:1921 (1995)).

On the basis these in vitro findings, the possibility that AP deposits in the AD-affected brain may be held in assembly by zinc and copper ions was investigated. Roher and colleagues have recently shown that much of the AP that deposits in AD-affected cortex can be solubilized in water (Roher, A.E, et al., J Biol. Chem. 271:20631 (1996)). Supporting the clinical relevance of in vitro findings, it has recently been demonstrated that metal chelators increase the amount of AP extracted by Roher's technique (in neutral saline buffer), and that the extraction of AP is increased as the chelator employed has a higher affinity for zinc or copper. Hence TPEN is highly efficient in extracting AP, as are TETA, and bathocuproine, EGTA and EDTA are less efficient, requiring higher concentrations 91 mm) to achieve the same level of recovery as say, TPEN Zinc and copper ions (5-50 M) added back to the extracting solution abolish the recovery of AP (which is subsequently extracted by the SDS sample buffer in the pellet fraction of the centrifuged brain homogenate suspension), but Ca 2 and Mg 2 added back to the chelator-mediated extracts of AP cannot abolish AP WO 98/40071 PCT/US98/04683 resolubilization from AD-affected tissue even when these metal ions are present in millimolar concentrations.

Importantly, atomic absorption spectrophotometry assays of the metal content of the chelator-mediated extracts confirms that Cu and Zn are co-released with AP by the chelators, along with lower concentrations of Fe. These data strongly indicate that Ap deposits (probably of the amorphous type) are held together by Cu and Zn and may also contain Fe. Interestingly, AP is not extractable from control brain without the use of chelators. This suggests that metal-assembled AP deposits may be the earliest step in the evolution of A3 plaque pathology.

These findings propelled further inquiries into chemistry of metal ion- Ap interaction. The precipitating effects upon AP ofZn 2 and Cu 2 were found to be qualitatively different. Zn-mediated aggregation is reversible with chelation and is not associated with neurotoxicity in primary neuronal cell cultures, whereas Cumediated aggregation is accompanied by the slow formation of covalently-bonded SDS-resistant dimers and induction of neurotoxicity. These neurotoxic SDSresistant dimers are similar to those described by Roher (Roher, A.E, et al., J. Biol.

Chem. 271:20631 (1996)).

To accurately quantitate the effects of different metals and pH on AP solubility, synthetic human Ap 40 (2.5 UM) was incubated (3 7C) in the presence of metal ions at various pH for 30 min. The resultant aggregated particles were sedimented by centrifugation to permit determination of soluble A 1 40 in the supernatant. To determine the centrifugation time required to completely sediment the aggregated particles generated under these conditions, AP 1-40 was incubated for 30 min at 37 0 C with no metal, Zn 2 (100 1 Cu 2 (100 U.M) and pH Reaction mixtures were centrifuged at 10 000g for different times, or ultracentrifuged at 100 000g for 1 h. (Figure Figure 1 shows the proportion of soluble AP,- 4 0 remaining following centrifugation of reaction mixtures. AP- 40 was incubated (30 min., 37°C) with no metal, under acidic conditions (pH Zn 2 (100 pM) or Cu 2 (100 and centrifuged at 10 000g for different time WO 98/40071 PCT/US98/04683 -56intervals, or at 100,000g (ultracentrifuged) for I h for comparison. All data points are means SD, n 3.

Given that conformational changes within the N-terminal domain of AP are induced by modulating (Soto, et al., J. Neurochem. 63:1191-1198 (1994)). and that there is a metal (Zn 2 binding domain in the same region, experiments were designed to determine whether there was a synergistic effect of pl on metal ion-induced AP aggregation. ApI- 40 was incubated with different bioessential metal ions at pH 6.6, 6.8 and 7.4. The results are show in Figure 2A, where "all metals" indicates incubation with a combination containing each metal ion at the nominated concentrations, concurrently. Figure 2A shows the proportion of soluble remaining in the supernatant after incubation (30 min., 37°C) with various metals ions at pH- 6.6, 6.8 or 7.4 after centrifugation (10,000g, min.).

The [H chosen represented the most extreme, yet physiologically plausible that AP, 4 0 would be likely to encounter in vivo. The ability of different bioessential metal ions to aggregate A,.

4 0 at increasing H' concentrations fell into two groups; Mg 2 Ca, Al 3, Co 2 Hg Fe 3 Pb and Cu 2 showed increasing sensitivity to induce A,- 40 aggregation, while Fe 2 Mn, 2 N i 2 and Zn 2 were insensitive to alterations in [H4] in their ability to aggregate Ap,- 40 Cu 2 and -lHg induced most aggregation as the increased, although the insensitive Zn -induced aggregation produced a similar amount of aggregation. Fe 2 but not Fe". also induced considerable aggregation as the increased, possibly reflecting increased aggregation as a result of increased crosslinking of the peptide.

Similar results were obtained when these experiments were repeated using turbidometry as an index of aggregation (Figure 2B). The data indicate the absorbance changes between reaction mixtures with and without metal ions at pH 6.6, 6.8 or 7.4. Thus, AP.

4 0 has both a pH insensitive and a pH sensitive metal binding site. At higher concentrations of metal ions this pattern was repeated, except Co 2 and Al 3 -induced AP aggregation became pH insensitive, and Mn became sensitive (Figure 2C).

WO 98/40071 PCT/US98/04683 -57- Since 6 4 Cu is impractically short-lived (tl/2 13 a novel metal-capture ELISA assay was used to perform competition analysis of Ap.4 0 binding to a microtiter plate impregnated with Cu 2 as described in Materials and Methods.

Results are shown in Figure 3. All assays were performed in triplicate and are means SD, n=3. Competition analysis revealed that Ap-.40 has at least one high affinity, saturable Cu 2 binding site with a Kd 900 pM at pH 7.4 (Figure The affinity of AP for Cu 2 is higher than that for Zn 2 (Bush, et al., J. Biol. Chem.

269:12152 (1994)). Since Cu 2 does not decrease Zn 2 *-induced aggregation (Bush, et al., J. Biol. Chem. 269:12152 (1994)), indicating Cu 2 does not displace bound Zn 2 there are likely to be two separate metal binding sites. This is supported by the fact that there is both a pH sensitive and insensitive interaction with different metal ions.

Since the conformational state and solubility of AP is altered at different pH (Soto, et al., J. Neurochem. 63:1191-1198 (1994)), the effects of[H'] on Zn and Cu'-induced AP1-40 aggregation were studied. Results are shown in Figures 4A, 4B and 4C. Figure 4A shows the proportion of soluble AP 40 remaining in the supernatant following incubation (30 min., 37°C) at pH 3.0-8.8 in buffered saline Zn 2 (30 M) or Cu 2 (30 uM) and centrifugation (10 000g, min.), expressed as a percentage of starting peptide. All data points are means SD, n=3. alone precipitates AP,.

4 0 (2.5 uM) as the solution is lowered below pH 7.4, and dramatically once the pH falls below 6.3 Figure 4A). At pH 80% of the peptide is precipitated, but the peptide is not aggregated by acidic environments below pH 5, confirming and extending earlier reports on the effect ofpH on AP solubility (Burdick, J. Biol. Chem. 267:546-554 (1992)). Zn 2 pM) induced a constant level of aggregation between pH 6.2-8.5, while below pH 6.0, aggregation could be explained solely by the effect of In the presence of Cu 2 (30 pM), a decrease in pH from 8.8 to 7.4 induced a marked drop in A 1-4 0 solubility, while a slight decrease below pH 7.4 strikingly potentiated the effect of Cu 2 on the peptide's aggregation. Surprisingly, Cu2'caused >85 of the available peptide to aggregate by pH 6.8, a pH which WO 98/40071 PCT/US98/04683 -58plausibly represents a mildly acidotic environment. Thus, conformational changes in Ap brought about by small increases in result in the unmasking of a second metal binding site that leads to its rapid self-aggregation. Below pH 5.0, the ability of both Zn 2 and Cu' to aggregate AP was diminished, consistent with the fact that Zn binding to AP is abolished below pH 6.0 (Bush, et al., J. Biol.

Chem. 269:12152 (1994)), probably due to protonation of histidine residues.

The relationship between pH and Cu 2 on AP 1 40 solubility was then further defined by the following experiments (Figure 4B). The proportion of soluble AP,.

,0 remaining in the supernatant after incubation (30 min., 37 0 C) at pH 5.4-7.8 with different Cu 2 concentrations 5, 10, 20, 30 pM), and centrifugation (10,000g, min.), was measured and expressed as a percentage of starting peptide. All data points are means SD, n=3. At pH 7.4, Cu2'-induced AP aggregation was less than that induced by Zn 2 over the same concentration range, consistent with earlier reports (Bush, et al., J. Biol. Chem. 269:12152 (1994)). There was a potentiating relationship between and [Cu 2 in producing AP aggregation; as the pH fell, less Cu 2 +was required to induce the same level of aggregation, suggesting that [Hf] is controlling Cu 2 induced A, 40 aggregation.

To confirm that this reaction occurs at physiological concentrations of AP 1 and Cu 2 4 a novel filtration immunodetection system was employed. This technique enabled the determination of the relative amount of AP, 1 40 aggregation in the presence of different concentrations of H' and Cu 2 (Figure 4C). The relative aggregation of nM concentrations of Ap, 1 40 at pH 7.4 and pH 6.6 in the presence of different Cu 2 concentrations 0.1, 0.2, 0.5 uM) were determined by this method. Data represent mean reflectance values ofimmunoblot densitometry expressed as a ratio of the signal obtained when the peptide is treated in the absence of Cu 2 All data points are means SD, n 2.

This sensitive technique confirmed that physiological concentrations of AP1-40 are aggregated under mildly acidic conditions and that aggregation was greatly enhanced by the presence of Cu 2 at concentrations as low as 200 nM.

Furthermore, as previously observed at higher A 40 concentrations, a decrease in WO 98/40071 PCT/US98/04683 -59pH from 7.4 to 6.6 potentiated the effect of Cu 2 on aggregation of physiological concentrations of AP-40. Thus, A .40 aggregation is concentration independent down to 8 nM where Cu 2 is available.

It has recently been shown that Zn 2 mediated A 40 aggregation is reversible whereas A 1 4 0 aggregation induced by pH 5.5 was irreversible.

Therefore, experiments were performed to determine whether Cu '/pH-mediated AP 40 aggregation was reversible. Cu2-induced A,- 40 aggregation at pH 7.4 was reversible following EDTA chelation, although for each new aggregation cycle, complete resolubilization of the aggregates required a longer incubation. This result suggested that a more complex aggregate is formed during each subsequent aggregation cycle, preventing the chelator access to remove Cu 2 from the peptide.

This is supported by the fact that complete resolubilization occurs with time, and indicates that the peptide is not adopting a structural conformation that is insensitive to Cu' 2 -induced aggregation/EDTA-resolubilization.

The reversibility of pH potentiated Cu 2 -induced A3 140 aggregation was studied by turbidometry between pH 7.5 to 6.6, representing H concentration extremes that might be found in vivo (Figures 5A and 5B). Unlike the irreversible aggregation of AP 4 0 observed at pH 5.5. Cu'-induced Ap,3 40 aggregation was fully reversible as the pH oscillated between pH 7.4 and 6.6. Figure 5A shows the turbidometric analysis of Cu 2 -induced AP 40 aggregation at pH 7.4 reversed by successive cycles of chelator (EDTA), as indicated. Figure 5B shows turbidometric analysis of the reversibility of Cu2-induced Ap340 as the pH cycles between 7.4 and 6.6. Thus, subtle conformational changes within the peptide induced by changing within a narrow pH window, that corresponds to physiologically plausible allows the aggregation or resolubilization of the peptide in the presence of Cu 2 WO 98/40071 PCT/US98/04683 Discussion These results suggest that subtle conformational changes in AP induced by promote the interaction of AP,3 4 with metal ions, in particular Cu> and Hg 2 l allowing self-aggregate or resolubilize depending on the (Figures 2A-2C, 4A- 4C). A decrease in pH below 7.0 increases the P-sheet conformation (Soto, el al., J. Neurochem. 63:1191-1198 (1994)), and this may allow the binding of Cu 2 to soluble A3 that could further alter the conformation of the peptide allowing for self aggregation, or simply help coordinate adjacent AP molecules in the assembly of the peptides into aggregates. Conversely, increasing pH above 7.0 promotes the a-helical conformation (Soto, et al. J. Neurochem. 63:1191-1198 (1994)), which may alter the conformational state of the dimeric aggregated peptide, releasing Cu and thereby destabilizing the aggregate with the resultant release of AP into solution. Thus, in the presence of Cu 2 A 40 oscillates between an aggregated and soluble state dependent upon the

AP

40 aggregation by Co 2 1, like Zn 2 was pH insensitive and per mole induced a similar level of aggregation. Unlike Zn 2

A,-

40 binding of Co 2 may be employed for the structural determination of the pH insensitive binding site given its nuclear magnetic capabilities (See Figure 2C).

The biphasic relationship of AP solubility with pH mirrors the conformational changes previously observed by CD spectra within the N-terminal fragment (residues 1-28) of AP (reviewed in (Soto, et al., J. Neurochem.

63:1191-1198 (1994)); a-helical between pH 1-4 and but P-sheet between pH 4-7. The irreversible aggregates of A3 formed at pH 5.5 supports the hypothesis that the P-sheet conformation is a pathway for A3 aggregation into amyloid fibrils.

Since aggregates produced by Zn 2 and Cu 2 under mildly acidic conditions (Figures 5A and 5B) are chelator/pH reversible, their conformation may be the higher energy a-helical conformation.

These results now indicate that there are three physiologically plausible conditions which could aggregate Ap: pH (Figures 1, 4A-4C; Fraser, et al., WO 98/40071 PCT/US98/04683 -61 Biophys. J. 60:1190-1201 (1991); Barrow, C.J. and Zagorski, Science 253:179-182 (1991); Burdick, J. Biol. Chem. 267:546-554 (1992); Barrow, et al., J. Mol. Biol. 225:1075-1093 (1992); Zagorski, M.G. and Barrow, C.J., Biochemistry 31:5621-5631 (1992); Kirshenbaum, K. and Daggett, V., Biochemistry 34:7629-7639 (1995); Wood, et al., J. Mol. Biol. 256:870-877 (1996), [Zn 2 Figures 1, 2A and 2B, 4A-4C; Bush, et al., J. Biol. Chem.

269:12152 (1994); Bush, et al., Science 265:1464 (1994); Bush, et al., Science 268:1921 (1995); Wood, et al., J. Mol. Biol. 256:870-877 (1996))and under mildly acidic conditions, [Cu 2 (Figures 2A, 4A-4C, 5B). Interestingly, changes in metal ion concentrations and pH are common features of the inflammatory response to injury. Therefore, the binding of Cu 2 and Zn 2 to AP may be of particular importance during inflammatory processes, since local sites of inflammation can become acidic (Trehauf, P.S. McCarty, Arthr. Rheum.

14:475-484 (1971); Menkin, Am. J. Pathol. 10:193-210 (1934)) and both Zn 2 and Cu 2 are rapidly mobilized in response to inflammation (Lindeman, et al., J. Lab. Clin. Med 81:194-204 (1973); Terhune, M.W. Sandstead, H.H., Science 177:68-69 (1972); Hsu, et al., J. Nutrition 99:425-432 (1969); Haley, J. Surg. Res. 27:168-174 (1979); Milaninio, et al., Advances in Inflammation Research 1:281-291 (1979); Frieden, in Inflammatory Diseases and Copper, Sorenson, ed, Humana Press, New Jersey (1980), pp. 159- 169).

Serum copper levels increase during inflammation, associated with increases in ceruloplasmin, a Cu 2 transporting protein that may donate Cu 2 4 to enzymes active in processes of basic metabolism and wound healing such as cytochrome oxidase and lysyl oxidase (Giampaolo, et al., in Inflammatory Diseases and Copper, Sorenson, ed, Humana Press, New Jersey (1980), pp.

329-345; Peacock, E.E. and vanWinkle, in WoundRepair, W.B. Saunders Co., Philadelphia, pp. 145-155) (1976)). Since the release of Cu 2 from ceruloplasmin is greatly facilitated by acidic environments where the cupric ion is reduced to its cuprous form (Owen, Jr., Proc. Soc. Exp. Biol. Med. 149:681-682 (1975)), WO 98/40071 PCT/US98/04683 -62periods of mild acidosis may promote an environment of increased free Cu 2 Similarily, aggregation of another amyloid protein, the acute phase reactant serum amyloid P component (SAP) to the cell wall polysaccharide, zymosan, has been observed with Cu 2 at acidic pH (Potempa, et al., Journal of Biological Chemistry 260:12142-12147 (1985)). Thus, exchange of Cu 2 to A,.

4 0 during times of decreased pH may provide a mechanism for altering the biochemical reactivity of the protein required by the cell under mildly acidic conditions. Such a function may involve alterations in the aggregation/adhesive properties (Figures or oxidative functions of AP at local sites of inflammation.

While the pathogenic nature of AP- 4 2 in AD is well described (Maury, Lab. Investig. 72:4-16 (1995); Multhaup, et al.. Nature 325:733-736 (1987)), the function of the smaller A, 4 0 remains unclear. The present data suggest that Cu2+-binding and aggregation of AP will occur when the pH of the microenvironment rises. This conclusion can be based on the finding that the reaction is and [Cu 2 dependent and reversible within a narrow, physiologically plausible, pH window. This is further supported by the specificity and high affinity ofCu 2 binding under mildly acidic conditions compared to the constant Zn -induced aggregation (and binding) of A p 40 over a wide pH range The brain contains high levels of both Zn 2 (-150 uM; Frederickson, C.J. International Review of Neurobiology 31:145-237 (1989)) and Cu 2 (-100 gM; Warren, et al., Brain 83:709-717 (1960); Owen, Physiological Aspects of Copper, Noyes Publications, Park Ridge, New Jersey (1982), pp160- 191). Intracellular concentrations are approximately 1000 and 100 fold higher than extracellular concentrations. This large gradient between intracellular and extracellular compartments suggests a highly energy dependent mechanism is required in order to sequester these metals within neurons. Therefore, any alterations in energy metabolism, or injury, may affect the reuptake of these metal ions and promote their release into the extracellular space, and together with the synergistic affects of decreased pH (see above) induce membrane bound Ap.

40 to aggregate. Since increased concentrations of Zn 2 and Cu 2 and decreased pH, are WO 98/40071 PCT/US98/04683 -63 common features of all forms of cellular insult, the initiation of AD,-40 function likely occurs in a coordinated fashion to alter adhesive and/or oxidative properties of this membrane protein essential for maintaining cell integrity and viability.

That A 1.40 has such a high affinity for these metal ions, indicates a protein that has evolved to respond to slight changes in the concentration of extracellular metal ions. This is supported by the fact that aggregation in the presence of Cu is approx. 30% at pH 7.1, the pH of the brain (Yates, et al., J. Neurochem.

55:1624-1630 (1990)), but 85% at pH 6.8. Taken together, our present results indicate that Ap3- 4 0 may have evolved to respond to biochemical changes associated with neuronal damage as part of the locally mediated response to inflammation or cell injury. Thus, it is possible that Cu 2 mediated Ap 4 0 binding and aggregation might be a purposive cellular response to an environment of mild acidosis.

The deposition of amyloid systemically is usually associated with an inflammatory response (Pepys, M.B. Baltz, Adv. Immunol. 34:141-212 (1983); Cohen, in Arthritis and Allied Conditions, D.J. McCarty, ed., Lea and Febiger, Philadelphia, pp. 1273-1293 (1989); Kisilevsky, Lab. Investig.

49:381-390 (1983)). For example, serum amyloid A, one of the major acute phase reactant proteins that is elevated during inflammation, is the precursor of amyloid A protein that is deposited in various tissues during chronic inflammation, leading to secondary amyloidosis (Gorevic, et al., Ann. NY Acad Sci. 380.393 (1982)). An involvement of inflammatory mechanisms has been suggested as contributing to plaque formation in AD (Kisilevsky, Mol. Neurobiol. 49:65-66 (1994)). Acute-phase proteins such as alpha 1-antichymotrypsin and c-reactive protein, elements of the complement system and activated microglial and astroglial cells are consistently found in AD brains.

The rapid appearance, within days of AP deposits and APP immunoreactivity following head injury (Roberts, et al., Lancet. 338:1422- 1423 (1991); Pierce, et al., JournalofNeuroscience 16:1083-1090 (1996)), rather than the more gradual accumulation of A3 into more dense core amyloid WO 98/40071 PCT/US98/04683 -64plaques over months or years in AD may be compatible with the release of Zn', Cu 2 and mild acidosis in this time frame. Thus, pH/metal ion mediated aggregation may form the basis for the amorphous AP deposits observed in the aging brain and following head injury, allowing the maintenance of endothelial and neuronal integrity while limiting the oxidative stress associated with injury that may lead to a diminishment of structural function.

Since decreased cerebral pH is a complication of aging (Yates, et al., J. Neurochem. 55:1624-1630 (1990)), these data indicate that Cu and Zn mediated AP aggregation may be a normal cellular response to an environment of mild acidosis. However, prolonged exposure of Ap to an environment of lowered cerebral pH may promote increased concentrations of free metal ions and reactive oxygen species, and the inappropriate interaction of Ap42 over time promoting the formation of irreversible AP oligomers and it's subsequent deposition as amyloid in AD. The reversibility of this pH mediated Cu 2 aggregation does however present the potential for therapeutic intervention. Thus, cerebral alkalinization may be explored as a therapeutic modality for the reversibility of amyloid deposition in vivo.

Example 2 Free Radical Formation and SOD-like activity ofAlzheimer's A/ Peptides Materials and Methods a) Determination of Cu' and Fe 2 This method is modified from a protocol assaying serum copper and iron (Landers, et al., Amer. J. Clin. Path. 29:590 (1958)). It is based on the fact that there are optimal visible absorption wavelengths of 483 nm and 535 nm for Cu' complexed with bathocuproinedisulfonic (BC) anion and Fe 2 coordinated by bathophenanthrolinedisulfonic (BP) anion, respectively.

WO 98/40071 PCT/US98/04683 Determination of molar absorption of these two complexes was accomplished essentially as follows. An aliquot of 500 pl of each complex (500 pM. in PBS pH 7.4, with ligands in excess) was pipetted into 1 cm-pathlength quartz cuvette, and their absorbances were measured. Their molar absorbancy was determined based on Beer-Lambert's Law. Cu'-BC has a molar absorbancy of 2762 M cm while Fe-BP has a molar absorbancy of 7124 M' cm Determination of the equivalent vertical pathlength for Cu'-BC and Fe" -BP in a 96-well plate was carried out essentially as follows. Absorbances of the two complexes with a 500 pM, 100 pM, 50 [pM, and 10 pM concentration of relevant metal ions Fc 2 were determined both by 96-well plate readers (300 1Fl.) and I 1V-vis spectrometer (500 pL), with PBS, pH 7.4, as the control blank.

lhe resulting absorbancies from the plate reader regress against absorbancies by a LUV-vis spectrometer. The slope k from the linear regression line is equivalent to the vertical pathlength if the measurement is carried out on a plate. The results are: k(cm) r Cu'-BC 1.049 0.998 Fe>-BP 0.856 0.999 With molar absorbancy and equivalent vertical pathlength in hand, the concentrations (pM) of Cu' or Fe' can be deduced based on Beer-Lambert's Law, using proper buffers as controls.

for Fe 2 [Fe2-] (535nm) x 10 6 (7124 x 0.856) for Cu': [Cu (WM) AA(483nm) x 10 6 (2762 x 1.049) where AA is absorbancy difference between sample and control blank.

WO 98/40071 PCT/US98/04683 -66b) Determination of This method is modified from a H 2 0, assay reported recently (Han, J.C., et al., Anal. Biochem. 234:107 (1996)). The advantages of this modified HO, assay on 96-well plate include high throughput, excellent sensitivity IM), and the elimination of the need for a standard curve of HO,, which is problematic due to the labile chemical property of HO,.

AP peptides were co-incubated with a H 2 02-trapping reagent (Tris(2carboxyethyl)-phosphine hydrochloride, TCEP, 100 iM) in PBS (pH 7.4 or at 37 C for 30 mins. Then 5,5'-dithio-bis(2-nitrobenzoic acid) (DBTNB, 100 M) was added to react with remaining TCEP. The product of this reaction has a characteristic absorbance maximum of 412 nm The assay was adapted to a 96-well format using a standard absorbance range (see Figure 11).

The chemical scheme for this novel method is: Scheme I:

CH

2

CH

2 COO-

CH

2

CH

2

COO

I

"OOCCH

2

CH

2 -P

H

2 0 2

OOCCH

2

CH

2 -P=O

H

2 0

CH

2

CH

2 COO"'

CH

2

CHCOO

(TCEP) [Tris (2-carboxethyl) phosphine] WO 98/40071 WO 9840071PCT/US98/04683 67 Scheme 11:

CH

2

CH

2

COO.

CH

2

CH

2

COO

remaining TCEP

CH

2

CH

2

COO-

OOCCH

2 L CH 2 -0

CM

2

CH

2

COO

H

2 0~

COO-

COO'

2S- O NO 2 2H+ has characteristic optimal absorption peak at 412 nm with 14,150 M-1 cm- 1 molar extinction coefficient TCEP*HCl was synthesized by hydrolyzing tris (2-cyno-ethyl) phosphine (purchased from Johnson-Mathey (Waydhill, in refluxing aqueous HC1 (Burns, et al., J Org. Chem. 56:2648 (1991)) as shown below.

aq HCI

(NCCH

2

CH

2 3 P -elu a (HO 2

CCH

2 CH9) 3 PH C[ refluxTCEP. HCl In order to carry out the above-described assay in a 96-well plate, it was necessary to calculate the equivalent vertical pathlength of acid (TMB) in a 96-well plate. This determination was carried out essentially as described for Cu'-BC and Fe 2 '-BP in Example 2. The resulting absorbancies from the plate reader regress against absorbancies by a UV-vis spectrometer. The WO 98/40071 PCT/US98/04683 -68slope k from the linear regression line is equivalent to the vertical pathlength if the measurement is carried out on a plate. The results are: k r2 0.875 1.00 The concentration of H202 can then be deduced from the difference in absorbance between the sample and the control (sample plus 1000 U/tl catalase)

[H

2 02, (0/h

A

AA (412nm) (2 x 0.875 x 14150) c) Determination of OH.

Determination of OH* was performed as described in Gutteridge et al.

Biochim. Biophys. Acta 759: 38-41 (1983).

d) Ci' Generation by Aft: Influence of Zn" and pH AP (10 pM in PBS, pH 7.4 or 6.8, as shown) was incubated for minutes (37°C) in the presence of Cu 2 10 piM Zn 2 10 gM). Cu' levels (n=3, ±SD) were assayed against a standard curve. These data indicate that the presence of Zn 2 can mediate the reduction of Cu 2 in a mildly acidic environment. The effects of zinc upon these reactions are strongly in evidence but complex. Since the presence of 10 uM zinc will precipitate the peptide, it is clear that the peptide possesses redox activity even when it is not in the soluble phase, suggesting that cortical Ap deposits will not be inert in terms of generating these highly reactive products. Cerebral zinc metabolism is deregulated in AD, and therefore levels of interstitial zinc may play an important role in adjusting the WO 98/40071 PCT/US98/04683 -69- Cu' and H02, production generated by Ap. The rat homologue of AP,.

40 does not manifest the redox reactivity of the human equivalent. Insulin, a histidinecontaining peptide that can bind copper and zinc, exhibits no Cu 2 reduction.

e) Hydrogen Peroxide Production by A/ species

AP,

1 42 (10 was incubated for 1 hr at 37°C, pH 7.4 in ambient air (first bar), continuous argon purging continuous oxygen enrichment at pH or in the presence of the iron chelator desferioxamine (220 PM; DFO).

Variant A3 species (10 ptM) were tested: A. -40 4 0 rat ApI- 40 (rAP3-40), and scrambled Ap, 4 0 (sAPl40) were incubated for 1 hr at 37 0 C, pH 7.4 in ambient air.

Values (mean ±SD, n=3) represent triplicate samples minus values derived from control samples run under identical conditions in the presence of catalase U/ml). The details of the experiment are as follows: AP peptides were coincubated with a H 2 0 2 -trapping reagent (Tris(2-carboxyethyl)-phosphine hydrochloride, TCEP, 100 pM) in PBS (pH 7.4 or 7.0) at 37 0 C for 30 mins.

Then 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 100 UtM) was added to react with remaining TCEP, the product has a characteristic absorbance maximum of 412 nm. The assay was adapted to a 96-well format using a standard absorbance range.

Results and Discussion A/t exhibits metal-dependent and independent redox activity Because Ap was observed to be covalently linked by Cu, the ability of the peptide to reduce metals and generate hydroxyl radicals was studied. The bathocuproine and bathophenanthroline reduced metal assay technique employed by Multhaup et al. was used in order to determine that APP itself possesses a Cu 2 reducing site on its ectodomain (Multhaup, et al., Science 271:1406 (1996)).

It has been discovered that A3 possesses a striking ability to reduce both Fe" to WO 98/40071 PCT/US98/04683 Fe 2 and Cu 2 to Cu modulated by Zn 2 and pH (See Figure 10). It is of great interest that the relative redox activity of the peptides studied correlates so well with their relative pathogenicity viz AP 1 -4>AP- 40 >ratAp in all redox assays studied. Since one of the caveats in using the reduced metals assay is that the detection agents can exaggerate the oxidation potential of Cu' or Fe (III), other redox products were explored by assays where no metal ion indicators were necessary. It was discovered that hydrogen peroxide was rapidly formed by AP species (Figure 11). Thus, AP produces both H 2 0 2 and reduced metals whilst also binding zinc. Structurally, this is difficult to envisage for a small peptide, but we have recently shown that AP is dimeric in physiological buffers. Since HO, and reduced metal species are produced in the same vicinity, these reaction products are liable to produce the highly toxic hydroxyl radical by Fenton chemistry, and the formation of hydroxyl radicals from these peptides has now been shown with the thiobarbituric acid assay. The formation of hydroxyl radicals correlates with the covalent polymerization of the peptide (Figure 9) and can be blocked by hydroxyl scavengers. Thus the concentrations of Fe, Cu, Zn H' in the brain interstitial milieu could be important in facilitating precipitation and neurotoxicity for AP by direct (dimer formation) and indirect (Fe 2 /Cu' and HO, formation) mechanisms.

H

2 ,O production by AP explains the mechanism by which H 2 O, has been described to mediate neurotoxicity (Behl, C. et al., Cell 77:827 (1994)), previously thought to be the product of cellular overproduction alone.

Interestingly, the scrambled AP peptide (same size and residue content as Figure 6) produces appreciable H,0 2 but no hydroxyl radicals. This is because the scrambled AP peptide is unable to reduce metal ions. This leads to the conclusion that what makes Ap such a potent neurotoxin is its capacity to produce both reduced metals and HO 2 at the same time. This "double whammy" can then produce hydroxyl radicals by the Fenton reaction, especially if the H20, is not rapidly removed from the vicinity of the peptide. Catalase and glutathione peroxidase are the principal means of catabolizing HO 2 and their levels are low WO 98/40071 PCT/US98/04683 -71 in the brain, especially in AD, perhaps explaining the propensity of AP to accumulate in brain tissue.

Figure 11 shows that the production of H202 is oxygen dependent, and further investigation has indicated that AP can spontaneously produce the superoxide radical in the absence of metal ions. This property of Ap is particularly exaggerated in the case of AP, 42 probably explaining why this peptide is more neurotoxic and more enriched than A 40 in amyloid. 02 generation will be subject to spontaneous dismutation to generate H 2 0 2 however, this is a relatively slow reaction, although it may account for the majority of the

HO

2 detected in our AP assays. 0; is reactive, and the function of superoxide dismutase (SOD) is to accelerate the dismutation to produce HO, which is then catabolized by catalase and peroxidases into oxygen and water. The most abundant form of SOD is Cu/Zn SOD, mutations of which cause another neurodegenerative disease, amyotrophic lateral sclerosis (Rosen, et al., Nature 364:362 (1993)). Since Ap, like Cu/Zn SOD, is a dimeric protein that binds Cu and Zn and reduces Cu 2 and Fe 3 we studied the 0; dismutation behavior of AP in the j sec time-scale using laser pulse photolysis. These experiments have shown that AP exhibits Fe/Cu-dependent SOD-like activity with rate constants of dismutation at 108 M- sec', which are strikingly similar to SOD. Hence, AP appears to be a good candidate to possess the same function as SOD, and therefore may function as an antioxidant. This may explain why oxidative stresses cause it to be released by cells (Frederikse, et al., Journal of Biological Chemistry 271: 10169 (1996)). However, if AP 4 2 is involved in the reaction to oxidative stress, or if the Hz0 2 clearance is compromised at the cellular level, Ap will accumulate, recruiting more 0, and producing more O0 leading to a vicious cycle and localizing tissue peroxidation damage and protein cross-linking.

WO 98/40071 PCT/US98/04683 72- Example 3 Cell Culture and Cytotoxic Assays Several different assays may be utilized to determine whether a candidate pharmacological agent identified by any of the above-summarized assays is capable of altering the neurotoxicity of Ap. The first is the MTT assay, which measures the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide (MTT) to a colored formazon (Hansen et al., J Immunol Methods, 119:203-210 (1989)). A second cytotoxic assay is the release of lactic dehydrogenase (LDH) from cells, a measurement routinely used to quantitate cytotoxicity in cultured CNS cells (Koh, J.Y. and D.W. Choi, J. Neurosci. Meth.

20:83-90 (1987). While MTT measures primarily early redox changes within the cell reflecting the integrity of the electron transport chain, the release of LDH is thought to be through cell lysis. A third assay is visual counting in conjunction with trypan blue exclusion. Yet another assay is the Live/Dead EukoLight Viability/Cytotoxicity Assay (Molecular Probes, Inc., Eugene, OR).

Example 4 Therapeutic Agents for Inhibition of Metal-Mediated Production of Reactive Oxygen Species Materials and Methods a) Synthesis of Peptides Synthetic A3 peptides A 1 40 and AP 4 2 were synthesized by the W. Keck Laboratory, Yale, CT. In order to verify the reproducibility of the data obtained with these peptides, confirmatory data were obtained by reproducing experiments with these A3 peptides synthesized and obtained from other sources: Glabe WO 98/40071 PCT/US98/04683 -73 laboratory, University of California, Irvine, CA, Multhaup Laboratory, University of Heidelberg, U.S. Peptides, Bachem, and Sigma. Rat AP was synthesized and characterized by the Multhaup Laboratory, University of Heidelberg. A 1 2 was purchased from U.S. Peptides, Bachem, and Sigma. AP peptide stock solutions were prepared in chelex-100 resin (BioRad) treated water and quantified.

b) Metal Reduction Assay The metal reduction assay was performed using a 96-well microtiter plate (Costar) based upon a modification of established protocols (Landers, et al., Amer. Clin. Path. 29:590 (1958); Landers, et al., Clinica Chimica Acta 3:329 (1958)). Polypeptides (10 uM) or Vitamin C (100 pM), metal ions pM, Fe(N0 3 3 or Cu(N0 3 2 and reduced metal ion indicators, bathophenanthrolinedisulfonic acid (BP, for Fe 2 Sigma, 200pM) or bathocuproinedisulfonic acid (BC, for Cu', Sigma, 200 RM), were coincubated in phosphate buffered saline (PBS), pH 7.4, for 1 hr at 37°C. The metal ion solutions were prepared by direct dilution in the buffer from their aqueous stocks purchased from National Institute of Standards and Technology (NIST).

Absorbances were then measured at 536 nm (Fe 2 *-BP complex) and 483 nm (Cu'-BC complex), respectively, using a 96-well plate reader (SPECTRAmax 250, Molecular Devices, CA). In control samples, both metal ion and indicator were present to determine the background buffer signal. As a further control, both metal ion and peptide were present in the absence of indicator to estimate the contribution of light scattering due to turbidity to the absorbance reading at these wavelengths. The net absorbances (AA) at 536 nm or 483 nm were obtained by deducting the absorbances from these controls from the absorbances generated by the peptide and metal in the presence of the respective indicator.

The concentrations of reduced metal ions (Fe 2 1 or Cu') were quantified based on the formula: Fe 2 or Cu' A* 10 6 where L is the measured equivalent vertical pathlength for a well of 300 L volume as described in the WO 98/40071 PCT/US98/04683 -74instrument's specifications manual (0.856 cm for Fe 2; 1.049 cm for M is the known molecular absorbance which is 7124 (for Fe 2 -BP complex) or 2762 (for Cu'-BC complex).

c) H 2 0, Assay The HO2, assay was performed in a UV-transparent 96-well microtiter plate (Molecular Devices, CA), according to a modification of an existing protocol (Han, et al., Anal. Biochem. 234:107 (1996); Han et al., Anal.

Biochem. 220: 5-10 (1994)). Polypeptides (10 pM) or Vitamin C (10 tM), Fe 3 or Cu 2 (1 M) and a H 2 0, trapping agent- Tris(2-Carboxyethyl)Phosphine Hydrochloride (TCEP, Pierce, 50 uiLM)- were co-incubated in PBS buffer (300 uL), pH 7.4, for 1 hour at 37 C. Under identical conditions, catalase (Sigma, 100 U/mL) was substituted for the polypeptides, to serve as a control signal representing 0 LM H 2 0 2 Following incubation, the unreacted TCEP was detected by 5,5-Dithio-bis(2-Nitrobenzoic acid) (DTNB, Sigma, 50 KLM) which generates 2 moles of the coloured product. The reactions are: TCEP H 2 0 2 -TCEP=O H,0, then the remaining TCEP is reacted with DTNB: TCEP DTNB H 2 0 TCEP=O 2NTB The amount of H 2 0 produced was quantified based on the formula: HO, hA* 106/(2*L*M), where hA is the absolute absorbance difference between a sample and catalase-only control at 412 nm wavelength; L 0.875 cm, the equivalent vertical pathlength obtained from the platereader manufacturer's specifications; M is the molecular absorbance for NTB (14150 cm-' at 412 nm).

WO 98/40071 PCT/US98/04683 TCEP is a strong reducing agent, and, hence, will artifactually react with polypeptides that contain disulfide bonds. This was determined not to be a source of artifact for the measurement of H,O, generation from Ap, which does not possess a disulfide bond.

d) Estimation of 02 The spectrophotometric absorption peak for O; is 250 nm where its extinction coefficient is much greater than that of HO, (Bielski et al., Philos Trans R Soc Lond B Biol Sci. 311: 473-482 (1985)). The production of 0; was estimated by measuring the spectrophotometric absorption of polypeptides gM, 300 pL) after incubation for one hour in PBS, pH 7.4, at 37°C, using a 96-well plate reader. The corresponding blank was the signal from PBS alone.

An absolute baseline for the signal generated by the peptide was not achievable in this assay since the absorption peak for tyrosine (residue 10 of AP) is close (254 nm) to the absorption peak for However, attenuation of the absorbance by co-incubation with superoxide dismutase (100 U/mL) indicated that the majority of the absorbance signal was due to the presence of O.

e) Thiobarbituric Acid Reaction Substance (TBARS) Assay OH' The Thiobarbituric Acid-Reactive Substance (TBARS) assay for incubation mixtures with Fe 3 or Cu 2 was performed in a 96-well microtiter format modified from established protocols (Gutteridge et al. Biochim. Biophys.

Acta 759: 38-41 (1983)). Ap peptide species (10 iM) or Vitamin C (100 PM), were incubated with Fe 3 or Cu 2 (1 IM) and deoxyribose (7.5 mM, Sigma) in PBS, pH 7.4. Following incubation (37 C, 1 hour), glacial (17 M) acetic acid and 2-thiobarburic acid w/v in 0.05 M NaOH, Sigma) were added and heated (100°C, 10 min). The final mixtures were placed on ice for 1-3 minutes before absorbances at 532 nm were measured. The net absorbance change for each WO 98/40071 PCT/US98/04683 -76sample were obtained by deducting the absorbance from a control sample consisting of identical chemical components except for the Vitamin C or AP peptides.

Results and Discussion Oxygen radical involvement in human aging, the predominant risk factor for Alzheimer's disease was first proposed by Harman in 1956 (Harman, J. Gerontol. 11:298 (1956)) and increasing evidence has implicated oxidative stress in the pathogenesis of AD. Apart from metabolic signs of oxidative stress in AD-affected neocortex such as increased glucose-6-phosphate dehydrogenase activity (Martins, et al., J. Neurochem. 46:1042-1045 (1986)) and increased heme oxygenase-1 levels (Smith, et al., Am. J. Pathol. 145:42 (1994)), there are also numerous signs of oxygen radical-mediated chemical attack such as increased protein and free carbonyls (Smith, etal., Proc. Nail. Acad Sci.

USA 88:10540 (1991); Hensley, et al., J. Neurochem. 65:2146 (1995); Smith, et al., Nature 382:120 (1996)), lipid peroxidation adducts (Palmer, A.M.

Burns, Brain Res. 645:338 (1994); Sayre, L.M. et al., J. Neurochem.

68:2092 (1997)), peroxynitrite-mediated protein nitration (Good, et al., Am.

J Pathol. 149:21 (1996)); Smith, et al., Proc. Natl. Acad. Sci. USA 94:9866 (1997)), and mitochondrial and nuclear DNA oxidation adducts (Mecocci, et al., Ann. Neurol., 34:609-616 (1993); Mecocci, et al., Ann.

Neurol., 36:747-751 (1994)). Recently, treatment of individuals with the antioxidant vitamin E has been reported to delay the progression of clinical AD (Sano, M. et al., N.Engl. J. Med. 336:1216 (1997)).

A relationship seems likely to exist between the signs of oxidative stress and the characteristic AP collections (Glenner, G.G. Wong, Biochem.

Biophys. Res. Commun. 120:885 (1984)) found in the cortical interstitium and cerebrovascular intima media in AD. The brain regional variation of oxidation biomarkers corresponds with amyloid plaque density (Hensley, et al., WO 98/40071 PCT/US98/04683 -77- .J Neurochem. 65:2146 (1995)). Indeed, neurons cultured from subjects with Down's syndrome, a condition complicated by the invariable premature deposition of cerebral AP (Rumble, et al., N. Engl. J. Med. 320:1446 (1989)) and the overexpression of soluble AP 1-42 in early life (Teller, et al., Nature MI'edicine 2:93 (1996)), exhibit lipid peroxidation and apoptotic cell death caused by increased generation of hydrogen peroxide (Busciglio, J. Yankner, B.A., Naturc 378:776 (1995)). Synthetic AP peptides have been shown to induce lipid peroxidation of synaptosomes (Butterfield, et al., Biochem. Biophys. Res.

(omnmnn. 200:710 (1994)), and to exert neurotoxicity (Behl, et al., Cell "7:817 (1994); Mattson, et al., J. Neurochem. 65:1740 (1995)) or vascular endothclial toxicity through a mechanism that involves the generation of cellular superoxide/hydrogen peroxide and is abolished by the presence of SOD (Thomas. et al.. Nature 380:168 (1996) or catalytic synthetic O0,HO, scavengers (Bruce. et al., Proc. Natl. Acad Sci. USA 93:2312 (1996)).

Antioxidant vitamin E and the spin-trap compound PBN have been shown to protect against Ap-mediated neurotoxicity in vitro (Goodman, Mattson, LExp. Neurol. 128:1 (1994); Harris, et al., Exp. Neurol. 131:193 (1995)).

Ap. a 39-43 amino acid peptide, is produced (Haass, et al., Nature 359:322 (1992); Seubert, et al., Nature 359:325 (1992); Shoji, et al., Science 258:126 (1992)) by constitutive cleavage of the amyloid protein precursor (APP) (Kang, et al., Nature 325:733 (1987); Tanzi, et al., Nature Genet (1993)) as a mixture of polypeptides manifesting carboxyl-terminal heterogeneity.

is the major soluble AP species in biological fluids (Vigo-Pelfrey, C., et al., J Neurochem. 61:1965 (1993)) and AP, 4 2 is a minor soluble species, but is heavily enriched in interstitial plaque amyloid (Masters, et al., Proc. Natl.

Acad. Sci. USA 82:4245 (1985); Kang, J. et al., Nature 325:733 (1987); Prelli, F., et al., J. Neurochem. 51:648 (1988); Roher et al., J. Cell Biol. 107:2703-2716 WO 98/40071 PCT/US98/04683 -78- (1988); Roher et al., J. Neurochem. 61:1916-1926 (1993); Miller, et al., Arch. Biochem. Biophys. 301:41 (1993)). The discovery of pathogenic mutations of APP close to or within the AP domain (van Broeckhoven, et al., Science 248:1120 (1990); Levy, et al., Science 248:1124 (1990); Goate, et al., Nature 349:704 (1991); Murrell, et al., Science 254:94 (1991); Mullan, el al., Nature Genet 1:345 (1992)) indicates that the metabolism of AP is involved with the pathophysiology of this predominantly sporadic disease. Familial ADlinked mutations of APP, presenilin-1 and presenilin-2 correlate with increased cortical amyloid burden and appear to induce an increase in the ratio of as part of their common pathogenic mechanism (Suzuki, et al., Science 264:1336 (1994); Scheuner et al., Nat Med., 2(8):864-870 (1996); Citron, et al., Nature Medicine 3:67 (1997)). However, the mechanism by which AP, 4 2 exerts more neurotoxicity than AP 40 and other AP peptides (Dor6, et al., Proc. Natl. Acad.

Sci. USA 94:4772 (1997)) remains unclear.

One of the models proposed for AP neurotoxicity is based on a series of observations of Ap-generated oxyradicals generated by a putative AP peptide fragmentation mechanism which is O0-dependent, metal-independent and involves the sulfoxation of the methionine at AP residue 35 (Butterfield,

D.A.,

el al., Biochem. Biophys. Res. Commun. 200:710 (1994); Hensley, et al., Proc. Natl. Acad. Sci. USA 91:3270 (1994); Hensley, et al., Ann N YAcad Sci., 786: 120-134 (1996). AP 25 35 peptide has been reported to exhibit H,O,-like reactivity towards aqueous Fe 2 nitroxide spin probes, and synaptosomal membrane proteins (Butterfield, et al., Life Sci. 58:217 (1996)), and A 1 4 0 has also been reported to generate the hydroxyl radical by mechanisms that are unclear (Tomiyama, et J. Biol. Chem. 271:6839 (1996)). However, there has been no quantitative appraisal of the ROS-generating capacity of AP ,42 versus that of Ai .40 and other AP variants, to date.

AP is a metal binding protein which saturably binds zinc via a histidinemediated specific high affinity site (KD 107 nM) as well as by low affinity WO 98/40071 PCT/US98/04683 -79binding (K D 5.2 pM). The high-affinity zinc binding site was mapped to a stretch of contiguous residues between positions 6-28 of the AP sequence (Bush, et al., J. Biol. Chem. 269:12152 (1994)). Concentrations of zinc 2 1 uM rapidly induce aggregation of human APl 4 0 solutions (Bush, et al., Science 265:1464 (1994)), in reversible manner which is dependent upon the dimerization of peptide in solution, its alpha-helical content, and the concentration of NaCI (Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)). Rat/mouse ("rat Ap", with substitutions o Rs-G, Yof-F, and H,3-R, as compared to human AP) binds zinc less avidly (a single binding site, KA=3.8 M) and, unlike the human peptide, is not precipitated by zinc at concentrations 25 uM. Since zinc is concentrated in the neocortex, we hypothesized that the differential solubility of the rat/mouse A3 peptide in the presence of zinc may explain the scarcity with which these animals form cerebral AP deposits (Johnstone, et al., Mol.

Brain Res. 10:299 (1991); Shivers, et al., EMBO J. 7:1365 (1988)).

We have also observed interactions of AP with Cu 2 which stabilizes dimerization of on gel chromatography (Bush, et al., J. Biol. Chem.

269:12152 (1994)), and which binds to the peptide with an affinity estimated to be in the low picomolar range. Fe 2 has been observed to induce partial aggregation of AP (Bush, et al., Science 268:1921 (1995)), and to induce SDS-resistant polymerization of the peptide (Dyrks, el al., J. Biol. Chem.

267:18210-18217 (1992)). We hypothesized that the interactions of Ap with Fe and Cu may contribute to the genesis of the oxidation insults that are observed in the AD-affected brain. This is because Fe 3 and Cu 2 are redox-active metal ions that are concentrated in brain neurons, and may participate in the generation of ROS by transferring electrons in their reduced state (reviewed in Markesbery, 1997).

The levels of Cu and Fe, and their binding proteins, are dysregulated in AD (Diebel, et al., J. Neurol. Sci. 143:137 (1996); Good, et al., Ann.

Neurol. 31:286 (1992); Robinson, et al., Alzheimer's Research 1:191 (1995); Thompson, et al., Neurotoxicology 9:1 (1988); Kennard, et WO 98/40071 PCT/US98/04683 al.. Nature Medicine 2:1230 (1996); Connor, et al., Neurosci. Lett. 159:88 (1993)) and may therefore lead to conditions that could promote ROS production.

While a direct role for AP in metal-dependent ROS generation has not been described, the peptide's physiochemical interation with transition metals, the presence of ferritin (Grudke-lqbal, et al., Acta Neuropathol. 81:105 (1990)) and rcdox reactive iron (Smith, et al., Proc. Natl. Acad Sci. USA 94:9866 (1997)) in amyloid lesions, and the facilitation of Ap- 40 neurotoxicity in cell culture by nanomolar concentrations of iron (Schubert, D. Chevion, M., Biocihwm. Biophys. Res. Commun. 216:702 (1995)), collectively support such a possibility.

We report the simultaneous production of H 2 0, and reduced metal ions by Ah. with the consequent generation of the hydroxyl radical. The amounts of reduced metal and ROS were both greatest when generated by AP 1 2 >A3 40 >>rat AP3 4 0, and AI- 28, a chemical relationship that correlates with the relative neurotoxicity of these peptides. These data describe a novel, 0 and biometaldependent pathway of ROS generation by Alzheimer A3 peptides which may explain the occurrence of oxidative stress in AD brain.

a) Metal Ion Reduction by A/f Peptides T'o determine whether AP peptides possess metal-reducing properties, the ability of AP peptides (Example 1) to reduce Fe 3 and Cu 2 compared to Vitamin C and other polypeptides (Example 2) was measured. Vitamin C, serving as a positive control, reduced Cu 2 efficiently (Figure 13A). However, the reduction of Cu 2 by AP,1 42 was as efficient, reducing all of the available Cu 2 during the incubation period. A,- 4 0 reduced 60% of the available Cu 2 whereas rat A,- 40 and AP3 12 8 reduced no Cu 2 The reduction of Cu 2 by BSA and insulin was less efficient than that by the human AP peptides, and was not unexpected since these polypeptides possess cysteine residues and reduce Cu 2 in the process of forming disulfide bonds.

WO 98/40071 PCT/US98/04683 -81 Fe 3 '/Fe 2 has lower standard reduction potential (0.11 V) than Cu 2 */Cu' (0.15 V) does under our experimental conditions (Miller, et al., Free Radical Biology Medicine 8:95 (1990)), and, in general, Fe 3 was reduced with less efficiency by Vitamin C and the polypeptides that reduced Cu Vitamin C reduced 15% of the available Fe 3 however Ap 42 was the most efficient of the agents tested for Fe> 3 reduction, reducing more Fe 3 in the incubation period than Vitamin C A1-40 and BSA Rat AP.

4 0 Ap, 2 8 and insulin did not significantly facilitate the reduction ofFe 3 4 Analysis of A, and

AP.

40 after incubation with Cu> 2 and Fe 3 under these conditions revealed that there was no apparent mass modification of the peptides on mass spectrometry, and no change in its migration pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), nor evidence for increased aggregation of the peptides by turbidometry or sedimentation analysis, suggesting that the peptides were not consumed or modified during the reduction reaction. Under these conditions, the complete kinetics of the peptide-mediated reactions cannot be appreciated (the presence of AP -42 induced the total consumption of the Cu 2 substrate within the incubation period), but a striking relationship exists between the relative efficiencies of the various AP peptides to reduce Cu 2 '/Fe 3 in this system and their respective participation in amyloid neuropathology.

Since the dissolved 02 in the buffer vehicle may be expected to react with the reduced metals being generated [Reaction the effect of modulating the 0, tension in the buffer upon the generation of reduced metals by the AP peptides (Figure 13B) was examined. Prior to the addition of Vitamin C or polypeptide, the buffer vehicle was continuously bubbled for 2 hours at 20 0

C

with 100% 02 to create conditions of increased 0 2 tension, or Argon to create anaerobic conditions. Increasing the 0, tension slightly reduced the levels of reduced metals being detected, probably due to the diversion of a fraction of the Fe 2 /Cu' being generated to Reaction and, if HO0 2 is being produced as a product of Reaction the recruitment of Fe 2 '/Cu into the Fenton reaction [Reaction However, performing the reaction under anaerobic (Argon WO 98/40071 PCT/US98/04683 -82purged) conditions also slightly reduced the levels of reduced metals being detected. This may be because some of the reduction of Fe3/Cu" is due to reaction with 0;: M O- M" O2 Reaction To determine whether the reduction of metal ions in the presence of Ap was due to the action of the peptide or the generation of O; by the peptide, the effects of metal ion chelators on the generation of reduced metal ions (Figure 13B) was studied. It was found that coincubation of AP, 1 42 with the relatively Fe 3 -specific chelator desferrioxamine (DFO) under ambient oxygenation conditions nearly halved the production ofFe Coincubation of AP,- 42 with the high-affinity Cu 2 chelator TETA abolished 95% of the Cu 4 generated by the peptide under ambient oxygenation conditions. These data indicate that the majority of the Cu* and a significant amount of the Fe 2 produced by A,.

4 2 are due to the direct action of the peptide and not indirectly due to the production of Oi.

The inhibitory effects of chelation upon Ap-mediated reduction of metal ions indicates that AP probably directly coordinates Fe 3 and Cu 2 and also that these chelating agents are not potentiating the redox potential of the metals ions, suggested to be an artifactual mechanism for the generation of reduced metal species (Sayre, L.M. et al., Science 274:1933 (1996)). The reasons for DFO being less effective than TETA in attenuating metal reduction may relate to the respective (unknown) binding affinities for Fe" and Cu 2 to the AP peptide, the stereochemistry of the coordination of the metal ions by the peptide, and the abilities of the chelating agents to affect electron transfer after coordinating the metal ion.

The reduction of metal ions by AP must leave the peptide, at least transiently, radicalized, in agreement with the electron paramagnetic resonance WO 98/40071 PCT/US98/04683 -83 (EPR) findings ofHensley et al., Proc. Natl. Acad. Sci. 91:3270 (1994). In their report, DFO, EDTA or Chelex 100 could not abolish the EPR signal generated by Ap 2 5 35 in PBS, leading these investigators to conclude that the radicalization of AP was metal-independent. However, the inventors have found that after treatment with Chelex 100 the concentrations of Fe and Cu in PBS are still as high as =0.5 pM which could be sufficient to induce the radicalization of the peptide after metal reduction. Since DFO does not abolish the reduction of Fe 3 by AP,3 42 (Figure 13B), and since EDTA has been observed to potentiate Femediated Fenton chemistry (Samuni et al., Eur. J. Biochem. 137: 19-124(1983)), it is suspected that Hensley and colleagues may have inadvertently overlooked the contribution of metal reduction to Ap-mediated radical formation.

Rat Ap- 4 0 did not reduce metal ions, and has been shown to have attenuated binding of Zn 2 (Bush et al., Science, 265:1464 (1994)). A similar attenuation of Cu 2 and Fe3' binding by rat Ap i 40 compared to human AP 40 is anticipated. These data also indicate that the rat AP substitutions in human Ap's zinc binding domain towards the peptide's amino terminus (Bush et al., J. Biol.

Chem., 269:12152 (1994)) involve residues that mediate the metal-reducing properties of the peptide. However, the hydrophobic carboxyl-terminal residues were also critical to the reduction properties of Ap. That Api.2, did not reduce metal ions indicates that an intact Zn '-binding site (Bush el al., J. Biol. Chem.

269:12152 (1994)) is insufficient to facilitate the metal reduction reaction. The mechanism by which the two additional hydrophobic residues (Ile and Ala) on API.4 2 so substantially enhance the peptide's redox activity compared to Ap.4 0 is still unclear.

It has been observed that sulfoxation of the methionine residue at Ap position 35 accompanies the EPR changes seen during the incubation of Ap 2 5 3 for 3 hours in PBS at 37 0 C (Hensley, et al., Ann N YAcadSci., 786: 120-134 (1996)), however, no evidence was found for a modification of Ap, 40 and A1-42 after mass spectrophotometric examination of the peptides incubated under conditions as described. Therefore, AP-mediated metal reduction, and the WO 98/40071 PCT/US98/04683 -84subsequent Ap-mediated redox reactions described below, appear to be achieved by a mechanism that differs from that previously reported.

b) Production of H 2 02 by A/t Peptides The reduced metal ions produced by Ap were expected to generate O0 and

H

2 0 2 by Reactions and To study this, a novel assay was developed (Example 2) which detected the generation of 10 |iM HO, by AP,.4 in the presence of 1 M Fe" under ambient 02 conditions (Figure 14A). To validate the assay, coincubation with catalase was observed to abolish the H,O, signal in a dose dependent manner. The amount of H 2 0, produced by the various AP peptides was studied, and observed that the order of the production of H,O, by the AP variants was Ap, 42 Ap,40 rat A 4 0 AP,.2 (Figure 14B), paralleling the amounts of metal reduction by the same peptides (Figure 13A).

H

2 02 formation is likely to be mediated first by O,-dependent O0 formation [Reaction followed by dismutation [Reaction To appraise the contribution of Reaction to H 2 0 2 formation, HO, formation by AP~i42 in the presence of chelators was measured (Figure 14C). The amount of H,O, formed in the presence of 1 1M Cu 2 was 25% greater than the amount formed in the presence of 1 uM Fe 3 Coincubation with DFO had no effect on H,O, formation in the presence of 1 pM Fe 3 However, TETA, and the Cu'-specific indicator BC, both substantially inhibited the formation of H,,0 in the presence of 1 pM Cu 2 The reasons why DFO partially inhibited Fe 3 reduction, but was unable to inhibit H 2 0, formation are unclear. These data indicate that the formation of HO, by AP is dependent upon the presence of substoichiometric amounts of The possibility that formation of H 2 0, in the presence of Fe 3 was due to the presence of trace quantities of Cu 2 cannot be excluded.

BC and BP, agents that specifically complex reduced metal ions, were far more effective than DFO and TETA at inhibiting H 2 0 2 formation by Ap (Figure 14C) but the reasons for this are not clear. The relatively Fe2+-specific WO 98/40071 PCT/US98/04683 complexing agent, BP, inhibited H 2 0, formation in the presence of Cu 2 and the relatively Cu'-specific complexing agent, BC, inhibited HI 2 0 formation in the present ofFe 3 suggesting that these agents are not totally specific in their metal ion affinities. The formation ofH 2 O, by Ap in the absence ofBC or BP confirms that the reduction of metals is not contingent upon the artifactual enhancement of the metal ions' redox potentials (Sayre, Science 274:1933 (1996)).

To determine whether the formation of O/HO, by AP is merely due to the reduction of metal ions, or whether AP also facilitates the recruitment of the substrates in Reaction the generation of H 2 02 by A 1 -42, API- 4 0 and Vitamin C under different 02 tensions in the presence of 1 lM Fe 3 (Figure 14D) or 1 uM Cu 2 (Figure 14E) was studied. The presence of Vitamin C was used as a control measure to determine the amount H 2 0 2 that is generated by the presence of reduced metals alone. In the presence of either metal ion, there was a significant increase in the amount ofH 2 0, produced under higher 0, tensions. The presence of either AP1-42 and A 1.

4 0 generated more H20, (AP 1- 4 2 A3 1 -40) than Vitamin C under any 0, tension studied, and generated H 2 0, under conditions where Vitamin C produced none, even though reduced metal ions must be present due to the activity of Vitamin C. Therefore, under these ambient and argon-purged conditions, the reduction of metal ions is insufficient to produce H 2 0 2 These data indicate that AP indeed facilitates the recruitment of 02 into Reaction more than would be expected by the interaction of the metals reduced by AP with the passively dissolved 02. Under relatively anaerobic conditions, the Ap peptides were observed to still produce H 2 0 in the presence of Cu 2 (Figure 14E). This is probably due to the ability of A3 to recruit 02 into Reaction under conditions of very low 02 tension. Since 02 is preferentially dissolved in hydrophobic environments (Halliwell and Gutteridge, Biochem. 219:1-14 (1984)), it seems that the hydrophobic carboxyl-terminus of Ap could attract Oz, serving as a reservoir for the substrate.

WO 98/40071 PCT/US98/04683 86c) Evidence of the Superoxide Anion Formed by the Af6 -metal Complex.

To confirm the production of O; by Ap, the absorbance of the peptide in solution at 250 nm, the absorbance peak of O (Figure 15A) was measured. The absorbance generated by AP, 1 4 2 in the presence of 1 LM Fe" was 60% reduced when co-incubated with SOD, increased in the presence of high 02 tension and abolished under anaerobic conditions. These data support the likelihood that AP generates H,0 2 by first generating O,.

The absorbance changes at 250 nm for the various A3 peptides in PBS (Figure 15B) paralleled the production of HO 2 from the same peptides (Figure 14B), but the reason for the A 25 0 being much greater for Ap, 42 compared to AP,- 40 is unclear. It is likely that a fraction of the total H202 generated by AP is decomposed by the Fenton reaction [Reaction Therefore, the amount of HO, detected may be an attenuated reflection of the amount of 0O detected.

d) Detection ofHydroxyl Radicals Generatedfrom theAfP-metal Complex Having demonstrated that human AP peptides simultaneously produce

H

2 0, and reduced metals, we proceeded to determine whether the hydroxyl radical was formed by the Fenton or Haber-Weiss reactions [Reactions and A modified TBARS assay was employed to detect OH* released from co-incubation mixtures of AP peptides and 1 ~M Fe 3 or Cu 2 As expected,

AP,.

4 2 produced more OH* than A 4 0 and rat AP did not generate OH* (Figure 16A). In contrast to the amount of Fe 2 and Cu' produced (Figure 13A), AP generated more OH* in the presence of Fe 3 than in the presence of Cu 2 This may be because Fe 2 is more stable than Cu', which may be more rapidly oxidized by Reaction Therefore, the Fe 2 generated by AP may have a greater opportunity than the Cu' generated to react with H 2 0 2 It is also possible that the contribution of the Haber-Weiss reaction to the production of OH* [Reaction is greater in the presence of Fe 3 than in the presence of Cu 2 4 WO 98/40071 PCT/US98/04683 -87- The effects of the OH* scavengers, dimethyl sulfoxide (DMSO) and mannitol, upon Ap, 42 -mediated OH- generation were studied. Whereas these agents suppressed the generation of OH* by Vitamin C in the presence of Fe 3 and DMSO suppressed the generation of OH* by Vitamin C in the presence of Cu 2 4 neither were able to quench the generation of OH* by A 142, whether in the presence of Fe" or Cu 2 (Figure 16B). This suggests that these scavengers cannot encounter the OH* generated by AP before the TBARS reagent does.

e) Similarity Between Bleomycin-Fe and A -Fe/Cu Complexes The present Examples provide evidence for a model by which Fe/Cu and 02 are mediators and substrates for the production of OH* by AP (Figures 16A and 16B) in a manner that depends upon the presence and length of the peptide's carboxyl terminus. The brain neocortex is an environment that is rich in both 02 and Fe/Cu, which may explain why this organ is predisposed to Ap-mediated neurotoxicity, if this mechanism is confirmed in vivo. The transport of Fe, Cu and Zn in the brain is largely energy-dependent. For example, the copper-transporting gene for Wilson's disease is an ATPase (Tanzi, R.E. et al., Nature Genetics 5:344 (1993)), and the re-uptake of zinc following neurotransmission is highly energy-dependent (Assaf, S.Y. S.H. Chung, Nature, 308:734-736 (1984); Howell et al., Nature, 308:736-738 (1984)).

There is increasing evidence for lesions of brain energy metabolism in aging and AD (Parker et al., Neurology, 40:1302-1303 (1990); (Mecocci et al., Ann. Neurol. 34:609-616 (1993); Beal, M.F. Neurobiol. Aging (Suppl 2):S171-S174(1994)). Therefore, damage to energy-dependent brain metal homeostasis may be an upstream lesion for the genesis of A deposition in AD.

Most brain biometals are bound to proteins or other ligands, however, according to our findings, only AP small fraction of the available metals needs to be derailed to the Ap-containing compartment to precipitate the peptide and to activate its ROS-generating activities. The generation of ROS described herein WO 98/40071 PCT/US98/04683 -88depends upon the sub-stoichiometric amounts of Fe 3 /Cu 2 (1:10, metal:AP), and it was estimated that 1% of the zinc that is released during neurotransmission would be sufficient to precipitate soluble AP in the synaptic vicinity (Huang, X.

et al., J. Biol. Chem. 272:26464-26470 (1997)).

A polypeptide which generates both substrates of the Fenton reaction in sufficient quantities to form significant amounts of the OH* radical is unusual.

Therefore, AP collections in the AD-affected brain are likely to be a major source of the oxidation stress seen in the effected tissue. One recent report describes that AP is released by the treatment of the mammalian lens in culture with H,O, (Frederikse, et al., J. Biol. Chem. 271:10169 (1996)). If a similar response mechanism to HO, stress exists in neocortex, then the increasing H,O, concentration generated by the accumulating AP mass in the AD-affected brain may induce the production of even more AP leading to a vicious cycle of AP accumulation and ROS stress.

The simultaneous production of Fenton substrates by AP is a chemical property that is brought into therapeutic application in the oxidation mechanism of the bleomycin-iron complex. Bleomycin is a glycopeptide antibiotic produced by Streptomyces verticillus and is a potent antitumor agent. It acts by complexing Fe 3 and then binding to tumor nuclear DNA which is degraded in situ by the generation of OH- (Sugiura, et al., Biochem. Biophys. Res. Commun.

105:1511 (1997)). Similar to Ap-Fe*/Cu 2 complexes, incubation of bleomycin in aqueous solution also engenders the production of H 2 0 2 and OH* in an Fe 3 -dependent manner. DFO could not inhibit H,0 2 production from the Ap-Fe 3 /Cu 2 complex, and similarly, DFO does not inhibit the OH*-mediated DNA damage caused by the bleomycin-Fe 3 complex. Also, low-molecular-mass OH* scavengers mannitol and DMSO were unable to inhibit the generation of OH- by Ap-Fe 3 '/Cu 2 and are similarly unable to inhibit OH* production from bleomycin-Fe It is proposed herein that inhibition of Ap-mediated OH* provides means of treatment, e.g. therapy, by compounds that are Fe or Cu chelators. The clinical WO 98/40071 PCT/US98/04683 -89administration of DFO was reported as being effective in preventing the progression of AD (Crapper-McLachlan, D.R. et al., Lancet 337:1304 (1991)); however, since DFO chelates Zn 2 as well as Fe 3 and AI(III), the effect, if verifiable, may not have been due to the abolition of the redox activity of AP, but may have been due to the disaggregation ofZn 2 -mediated AP deposits (Chemy, R.A. et al., Soc. Neurosci. Abstr. 23:(abstract)(1997)) which may have reduced cortical AP burden and, consequently, oxidation stress.

j) Oxidative Stress and Alzheimer's Disease Pathology Autopsy tissue from AD subjects has been reported to exhibit higher basal TBARS formation than control material (Subbarao, K.V. et al., J. Neurochem.

55:342(1990); Balazs, L. and M. Leon, Neurochem. Res. 19:1131 (1994); Lovell et al., Neurology 45:1594 (1995)). These observations could be explained, on the basis of the present findings, as being due to the reactivity of the AP content within the tissue. A 1- 4 0 recently has been shown to generate TBARS in a dosedependent manner when incubated in cell culture, however TBARS reactivity was reduced by pre-treating the cells with trypsin which also abolished the binding of the peptide to the RAGE receptor (Yan et al., Nature 382:685 (1996)). One possibility for this result is that the RAGE receptor tethers an AP microaggregate sufficiently close to the cell to permit increased penetration of the cell by H,0 2 which may then combine with reduced metals within the cell to generate the Fenton reaction. Alternatively, AP may generate the Fenton chemistry at the RAGE receptor. The resulting attack of the cell surface by the highly reactive OH radical, which reacts within nanometers of its generation, may have been the source of the positive TBARS assay.

APP also reduces Cu 2 but not Fe 3 at a site in its amino terminus (Multhaup, et al., Science 271:1406-1409 (1996)), adjacent to a functional and specific Zn 2 -binding site that modulates heparin binding and protease inhibition WO 98/40071 PCT/US98/04683 (Bush el al., 1993; Van Nostrand, 1995). Therefore, the amino terminus of APP reiterates an association with transition metal ions that is found in the A3 domain.

This intriguing theme of tandem Cu/Zn interaction and associated redox activity found in two soluble fragments of the parent protein may indicate that the function and metabolism of APP could be related to biometal homeostasis and associated redox environments.

The present findings indicate that the manipulation of the brain biometal environment with specific agents acting directly chelators and antioxidants) or indirectly by improving cerebral energy metabolism) holds promise as a means for therapeutic intervention in the prevention and treatment ofAlzheimer's disease.

Example Resolubilization of A/6 Considerable evidence now indicates that the accumulation of AP in the brain cortex is very closely related to the cause of Alzheimer's disease. AP is a normal component of biological fluids whose function is unknown. AP accumulates in a number of morphologies varying from highly insoluble amyloid to deposits that can be extracted from post-mortem tissue in aqueous buffer. The factors behind the accumulation are unknown, but the inventors have systematically appraised the solubility of synthetic A3 peptide in order to get some clues as to what kind of pathological environment could induce the peptide to precipitate.

It was found that A3 has three principal vulnerabilities: zinc, copper and low pH. The precipitation of AP by copper is dramatically exaggerated under mildly acidic conditions pH suggesting that the cerebral lactic acidosis that complicates Alzheimer's disease could contribute to the precipitation of AP were this event to be mediated by copper. A consideration of the involvement of WO 98/40071 PCT/US98/04683 -91 zinc and copper in plaque pathology is contemplatable since the regulation of these metals in the brain has been shown to be abnormal in AD.

Recently direct evidence has been obtained indicating that these metals are integral components of the Ap deposits in the brain in AD. It was found that zinc- and copper-specific chelators dramatically redissolve a significant proportion (up to 70%) of AP extracted from post-mortem AD affected brain tissue, compared to the amount extracted from the tissue by buffer in the absence of chelators.

These data support a strategy of redissolving AP deposits in vivo by chelation. Interestingly, a reported success in attempting to slow down the progression of Alzheimer's disease used a chelation strategy with desferrioxamine. The authors (Crapper-McLachlan, et al., 337:1304 (1991), thought that they were chelating aluminum, but desferrioxamine is also a chelator of copper and zinc. Treatment with desferrioxamine is impractical because the therapy requires twice daily deep intramuscular injections which are very painful, and also causes side effects such as anaemia due to iron chelation.

A/t Extraction from Human Brain Post-Mortem Samples The inventors have recently characterized zinc-mediated AP deposits in human brain (Cherny, et al., Soc. Neurosci Abstr. 23:(Abstract) (1997)).

It was recently reported that there is a population of water-extractable AP deposit in the AD-affected brain (Kuo, et al., J. Biol. Chem. 271:4077-81 (1996)).

The inventors hypothesized that homogenization of brain tissue in water may dilute the metal content in the tissue, so lowering the putative zinc concentration in AP collections, and liberating soluble A3 subunits by freeing Ap complexed with zinc To test this hypothesis, the brain tissue preparation protocol of Kuo and colleagues was replicated, but phosphate-buffered saline pH 7.4 (PBS) was substituted as the extraction buffer, achieving similar results. Highly sensitive WO 98/40071 PCT/US98/04683 -92and specific anti-Ap monoclonal antibodies (Ida, N. et al., J. Biol. Chem.

271:22908 1996) were used to assay AP extraction by western blot. Next, the extraction of the same material was repeated with PBS in the presence of chelators of varying specificities (Table and it was determined that the presence of a chelator increased the amount of AP in the soluble extract severalfold (Figures 19A-19C, 20A and 20B, 25A; Table 2).

The amount of AP detected in the pellet fraction of each sample is correspondingly lower (data not shown), indicating that the effect of the chelator is upon the disassembly of the A3 aggregate, and not by inhibition of an Apcleaving metalloprotease (such as insulin degrading enzyme cleavage of AP reported recently by Dennis Selkoe at the 2 7 th Annual Meeting for the Society for Neuroscience. New Orleans). The extraction ofsedimentable AP into the soluble phase correlated only with the extraction of zinc from the pellet, and not with any other metal assayed (Table Examination of the total amount of protein released by the treatments revealed that chelation was not merely liberating more proteins in a non-specific manner.

WO 98/40071 PCT/US98/04683 -93- Table 1. Dissociation Constants for Metal Ions of Various Chelators Used to Extract Human Brain AP.

CHELATOR Ca Cu Mg Fe Zn Al Co EGTA 10.9 17.6 5.3 11.8 12.6 13.9 12.4 EDTA 10.7 18.8 8.9 14.3 16.5 16.5 16.5 Pcnicillamine 0 18.2 0 0 10.2 0 0 TPEN 3.0 20.2 0 14.4 15.4 0 0 Bathophenanthrolin 0 8.8 0 5.6 6.9 0 0 e Bathocuproine (BC) 0 19.1 0 0 4.1 0 4.2 (C u') LogK is illustrated for the chelators, where K= Different chelators have greatly differing affinities for metal ions, as shown. TPEN is relatively specific for Zn and Cu, and has no affinity for Ca and Mg (which are far more abundant metal ions in tissues). Bathocuproine (BC) has high affinity for zinc and for cuprous ions. Whereas all the chelators examined have a significant affinity for zinc, EGTA and EDTA have significant affinities for Ca and Mg.

The ability of chelators to extract AP from post-mortem brain tissue was studied in over 40 cases (25 AD, 15 age-matched and young adult controls, all confirmed by histopathology). While there is a lot of variation between samples as to what is the best concentration of given chelator for the optimum extraction of Ap, there are no cases where a chelator does not, at some concentration, extract far more AP than PBS alone.

Figure 19 shows that metal chelators promote the solubilization of AP from human brain sample homogenates. Representative curves for three chelators (TPEN, EGTA, Bathocuproine) used in extracting the same representative AD brain sample are shown. 0.5 g of prefrontal cortex was dissected and homogenized in PBS chelator as indicated. The homogenate was then centrifuged (100,000 g) and the supernatant removed, and a sample taken for WO 98/40071 PCT/US98/04683 -94western blot assay using anti-Ap specific antibodies after Tricine PAGE.

Densitometry was performed against synthetic peptide standards. The blots shown here represent typical results. Similar results were achieved whether or not protease inhibitors were included in the PBS (extraction was at 4 0

C).

Furthermore, similar results were achieved when the brain sample was homogenized in PBS and then pelleted before treated with PBS chelator.

There is also a complex relationship between the dose of the chelator and the resultant resolubilization of AP (Figures 19A-C). For the same given sample, neither TPEN nor EGTA could increase the extraction of AP in a does-dependent manner. Rather, although concentrations of chelators could be very effective in the low micromolar range TPEN 4 utM, Figure 19A), higher concentrations induced a paradoxical loss of recovery. This kind of response was found in every case examined. The extraction of AP is abolished by adding exogenous zinc, but is enhanced by adding magnesium. Preliminary in vitro data indicate that whereas Mg has no effect on the precipitation of AP, its presence enhances the peptide's resolubilization following zinc-induced precipitation. Therefore, the "polyphasic" profile of chelator extraction of AP, with higher concentrations of TPEN and EGTA inducing a loss of recovery, may be explained by the chelation of Mg that is only expected to occur after the chelation of zinc when the relative abundance of Mg in the sample, and the relative dissociation constants of TPEN and EGTA are considered.

In contrast, bathocuproine (BC) exhibits a clear dose-dependent increase in AP extraction from human brain, probably due to its relatively high specificity for zinc, although an interaction with trace amounts of Cu or other metals not yet assayed, cannot be excluded.

Western blot analysis of extracts using A,.

4 2-specific monoclonals revealed the presence of abundant A -42 species. It was observed that =20% of AD cases exhibit clear SDS-resistant Ap dimers in the soluble extract after treatment with chelators. These dimers are reminiscent of the neurotoxic A 4 2 dimers that were extracted by Roher and colleagues from AD-affected brain WO 98/40071 PCT/US98/04683 (Roher, et al., Journal ofBiological Chemistry 271:20631-20635 (1996)).

An estimation of the proportion of total precipitated AP in the sample was achieved by extracting the homogenate pellet following centrifugation, into formic acid, and then performing a western blot on the extract following neutralization. The proportion of pelletable AP that is released by chelation treatment varies considerably from case to case, from as little as 30% to as much as 80%. In the absence of a chelator, no more than 10% of the total pelletable AP is extracted by PBS alone.

One preliminary emerging trend is that samples with a greater proportion of diffuse or vascular AP deposit are more likely to have their pelletable AP resolubilized by chelation treatment. Also, extraction of the tissue homogenate overnight with agitation greatly increases the amount of AP extracted in the presence of chelators (compared to PBS alone), when compared to briefer periods of extraction indicating that the disassembly of AP deposits by chelation treatment is a time-dependent reaction and is unlikely to be due to inhibition of a protease. A study of brain cortical tissue from one amyloid-bearing

APP

transgenic mouse indicates that, like human brain, homogenization in the presence of a chelator enhances the extraction of pelletable Ap.

Effects of various chelators on the extraction of AP into the supernatant as a percentage change from control extractions is summarized below in Table 2.

Table 2. Effects of Various Chelators Upon Extraction of Ap.

Effect of Chelators change from control) TPEN EGTA

BATHOCUP

0.1mM 2.0mM 0.1mM 2.0mM 0.1mM Mean 182 241 207 46 301 400 +-SD 79 81 115 48 190 181 WO 98/40071 PCT/US98/04683 -96- Densitometry of AP western blots (Figures 19A- 19C) was performed for a series of 6 AD brain samples homogenized in the presence of chelators as indicated. The mean increases in signal, above the signal generated by PBS extraction alone, are indicated in Table 2. A significantly increased amount of chelator-induced AP resolubilization was achieved by a 16 hour extraction with agitation in subsequent studies.

Table 3 shows a comparison between pellets of post-centrifugation homogenates in the presence and absence of a chelator (TPEN).

Table 3. Residual Metals in Pellets of Post-Centrifugation Homogenatesin the Presence and Absence of Chelator.

METAL Zn Cu Fe Ca Mg Al PBS 50.7 11.9 227 202 197 44 alone (12.0) (69) (69) (94) (111) mg/kg

(SD)

+TPEN 33.2* 9.8 239 (210) 230 mg/kg (76) (89) (94) (108) (SD) Frontal cortex from AD and healthy controls was homogenized in the presence and absence of PBS TPEN (0.1 mM). After ultracentrifugation of the homogenate, the pellets were extracted into concentrated HCI and measured for metal content by ion coupled plasma atomic emission spectroscopy (ICP-AES).

Using the same technique, zinc-mediated assembly of AP in normal brains was shown. Figures 20A and 20B show sedimentable AP deposits in healthy brain tissue. The effects of chelators in enhancing AP extraction from brain homogenates is also observed in normal tissue. Figure 20A illustrates a western blot with anti-Ap antibody of material extracted from a 27-year-old individual with no history of neurological disorder. T= TPEN, E= EGTA, B= bathocuproine. Bathocuproine is much less effective in extracting AP from control tissue than from AD tissue. These data are typical of 15 cases.

WO 98/40071 PCT/US98/04683 -97- As expected, far less total AP is present in normal brain samples compared to AD brain samples, although the content of AP increases with age.

It is possible that these findings in young adult brains represent the zinc-mediated initiation of amyloid formation in deposits that, in youth, are too diffuse to be detected by immunohistochemistry.

Roher and others have suggested that dimers of AP are the toxic component of amyloid. As shown in Figure 21, dimers appear in response to chelation in disproportion to the monomeric signal (treatment with PBS alone does not generate soluble dimers). This su97ggests that Ap deposits are being dismantled by the chelators into SDS-resistant dimeric structural units.

Figure 22 shows that the recovery of total soluble protein is not affected by the presence of chelators in the homogenization step. The proportionality of extracted subfractions, calculated based on total protein as determined by formic acid extraction, should not be prone to artifact based on chelator-specific affects.

Example 6 Differential Effects of Chelation of Cerebral Ap Deposits in AD-Affected Subjects Versus Age-Matched Controls and the Effect of Magnesium Experiments involving extraction of cerebral tissue from AD-affected subjects and non-AD, age-matched controls by chelation indicate different resolubilization responses of amyloid deposits between the two sample groups with regard to extraction by specific chelators.

Higher concentrations of chelators with relatively broad specificity (e.g.

EGTA) result in less resolubilization of AP deposits. Experiments show that chelation of magnesium negatively affects resolubilzation of Ap deposits.

WO 98/40071 PCTIUS98/04683 -98- Materials and Methods Cortical tissue was dissected from the frontal poles of frozen AD and agematched normal brains for which histopathological and clinical documentation were provided. AD tissue was selected according to CERAD criteria (Mirra et al., Neurology 41:479-486 (1991)) with particular attention paid to the presence of neuritic plaques and neurofibrillary tangles. Histological examination of AP levels in normal specimens ranged from immunohistochemically undetectable to substantially present in the form of diffuse plaques.

Suitable quantities of gray matter from each subject were minced to serve as pools of homogenous tissue. Equal portions (0.5 g unless otherwise specified) were homogenized (Ika Ultaturax T-25, Janke and Kunkel, Staufen, Germany) for 3 x 30 second periods at full speed with a 30 second rest between runs in 3 ml of ice-cold phosphate-buffered saline (PBS pH 7.4) containing a cocktail ofprotease inhibitors (Biorad, Hercules, CA. Note: EDTA was not included in the protease inhibitor mixture) or in the presence of chelators or metal ions prepared in PBS.

To obtain the soluble fraction, the homogenates were centrifuged at 100,000 x g for 30 min (Beckman J180, Beckman instruments, Fullerton, CA) and the supernatant collected in 1 ml aliquots and stored on ice or immediately frozen at C. In each experiment, all protein was precipitated from 1 ml of supernatant from each treatment group using 1:5 ice cold 10% trichloracetic acid and pelleted in a bench top microfuge (Heraeus, Osteroder, Germany) at 10,000 x g. The remaining pellet was frozen at -70 C.

The efficiency of the precipitation was validated by applying the technique to a sample of whole human serum, diluted 1:10, to which had been added 2 ag of synthetic A,- 4 0 or AP 1 4 2 Keck Laboratory, Yale University New Haven, CT). Protein in the TCA pellet was estimated using the Pierce BCA kit (Pierce, Rockford, IL). The total AP load ofunextracted cortex was obtained by dissolving g of grey matter in 2 ml of 90% formic acid, followed by vacuum drying and neutralization with 30% ammonia.

WO 98/40071 PCT/US98/04683 -99- Precipitated protein was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) on Novex pre-cast 10-20% Tris-Tricine gels followed by Western transfer onto 0.2 im nitrocellulose membrane (Biorad, Hercules, CA). AP was detected using the W02, G210 or G211 monoclonal antibodies (Ida, N. et al., J. Biol. Chem. 271:22908 (1996)) in combination with HRP-conjugated rabbit anti-mouse IgG (Dako, Denmark), and visualized using chemiluminescence (ECL, Amersham Life Science, Little Chalfont, Buckinghamshire, UK). Each gel included two or more lanes containing known quantities of synthetic Ap which served as internal reference standards. Blot images were captured by a Relisys scanner with transparency adapter (Teco Information Systems, Taiwan, ROC) and densitometry conducted using the NIH Image 1.6 program (National Institutes for Health, USA., Modified for PC by Scion Corporation, Frederick, MD), calibrated using a step diffusion chart. For quantitation of AP in brain extracts, the internal reference standards of synthetic AP were utilized to produce standard curves from which values were interpolated.

In the experiments corresponding to the results shown in Figure 23, duplicate 0.2 g samples of AD cortical tissue were homogenized and subjected to ultracentrifugation as described, but using either 1 ml or 2 ml of extraction buffer (PBS). Protein was precipitated from the entire supernatant and redissolved in 100 pl of sample buffer. Equal volumes of TCA-precipitated protein were subjected to Tris-Tricine SDS-PAGE and A3 was visualized as described above.

In the experiments corresponding to the results shown in Figure 24A, 0.2 g specimens of frontal cortex from AD brain were homogenized in the presence of 2 ml of PBS or varying concentrations of Cu 2 (Cu(S04) 2 or Zn 2 (Zn(SO4) 2 AP in the high speed supernatant was visualized as described above.

In the experiments corresponding to the results shown in Figure 24B, 0.2 g specimens of frontal cortex from AD brain were homogenized in the presence of 2 ml or PBS or 2 mM EGTA. The homogenates were then spun at 100,000 x g for 30 min and the supernatant discarded. The remaining (metal depleted) pellets were rehomogenized in a further 2 ml of either PBS alone, EGTA alone, 2 mM WO 98/40071 PCT/US98/04683 -100- Mg 24 (Mg(Cl) 2 *6H,O) in PBS or 2 mM Ca 2 (CaCI 2 .2H 2 0) in PBS and the homogenate subjected to ultracentrifugation. Ap in the soluble fraction was visualized as described above.

In the experiments corresponding to the results shown in Figures 25A and 25B, frontal cortex from AD and age-matched, amyloid-positive subjects were treated with PBS, TPEN, EGTA or BC (0.1 mM and 2 mM) and soluble AP assessed as described above.

In the experiments corresponding to the results shown in Figure 26, representative AD (left panels) and aged-matched control specimens (right panels) were prepared as described in PBS or 5mM BC. Identical gels were run and Western blots were probed with mAbs W02 (raised against residues 5-16, recognizes A 140 and Ap G210 (raised against residues 35-40, recognizes Ap, or G211 (raised against residues 35-42, recognizes AP.

4 2 (Ida, N. et al., J.

Biol. Chem. 271:22908 (1996).

Results and Discussion To further explore the involvement of metal ions in the deposition and architecture of amyloid deposits, the inventors extracted brain tissue from histologically-confirmed AD-affected subjects and from subjects that were agematched to AD-affected subjects but were not clinically demented (age-matched controls, in the presence of a variety of chelating agents and metals.

Chelators were selected which displayed high respective affinities for zinc and/or copper relative to more abundant metal ions such as calcium and magnesium. See Table 4 below.

Table 4. Stability constants of metal chelators WO 98/40071 PCT/US98/04683 101 BC n/a Cu 2 n/a n/a 4.1 n/a 4.2 6.1 Cu' 19.1 logKl0 where K=[Metal.Ligand]/[Metal][Ligand]. From: NIST database of critically selected stability constants for metal complexes Version 2.0 1995.

A series of titration curves were prepared to determine the chelator concentration at which maximal response was obtained. In these experiments, selected chelators were limited to EGTA, TPEN and BC. Figures 19A-C show interesting dose-dependent patterns of chelator solubilization of Ap.

It was found that EGTA and TPEN elicited a significant enhancement in solubilization of Ap in a pattern of response typified by peak values at or near 0.004 mM and 0.1 mM, and lower values at concentrations in between. Both chelators were increasingly ineffective at concentrations over 1 mM, and at 2 mM, EGTA virtually abolished the signal for AP. In contrast, BC elicited a typical concentration-dependent response with no decline in effectiveness in the low millmolar range even when extended to 20 mM. Total TCA-precipitated protein in the supernatant was assayed and found to be unaffected by either chelator kind or concentration.

Recent findings have demonstrated the presence of neurotoxic dimers in the soluble (Kuo, et al., J. Biol. Chem. 271:4077-81 (1996)) and insoluble (Roher, et al., Journal of Biological Chemistry 271:20631-20635 (1996); Giulian, D. et al., J Neurosci., 16:6021-6037 (1996)) fractions of AP extracts of the brains of AD individuals. Figure 21 shows that chelator-promoted solubilization of AP elicits SDS-resistant dimers. Under the preparation conditions used, SDS-resistant dimers were not generally observed in the extracts with PBS alone. Dimers were found to appear, however, in response to chelatorpromoted solubilization of Ap.

The signal for dimeric AP was frequently disproportionate to that of monomeric AP and the ratio varied with both the type and concentration of chelator used (Figure 21). In contrast, when synthetic AP_, 4 was run under WO 98/40071 PCT/US98/04683 102identical conditions, the monomer:dimer ratio reflected a predictable and reproducible concentration-dependent relationship. These data suggest that the dimers observed in extracts of human brain are predominantly an intermediate structural unit generated by the dissolution of amyloid, resulting in turn from the sequestration of metals by chelating agents.

Figure 24A shows the effect of metals upon the solubility of brain-derived Ap. Precipitation ofAP was induced by adding either copper or zinc to unchelated extracts. The resulting signal for soluble AP was attenuated, the threshold concentration being between 20 and 50 piM for copper and between 5 and 20 tM for zinc. At concentrations greater than 100 pM solubility was abolished.

Interestingly, at lower concentrations of copper there appears to be a transitional stage where AP is present in the dimeric form prior to complete aggregation, mirroring the intermediate stage dimers elicited by chelator-mediated solubilization.

In order to confirm that the chelators were effective at sequestering metals at the concentrations employed in these experiments, ICP-AES was used to determine the residual levels of several metals in the post-centrifugation pellets retained from the experiment described in Figures 19A-19C. Of the six metals tested, zinc levels were reduced by TPEN in a dose dependent manner, whereas EGTA affected calcium and magnesium, particularly at higher concentrations. See Table 5 below.

Table 5. Residual Metal Levels in Post-centrifugation (Extracted) Pellets Mg Al Ca Fe Zn Cu (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) PBS 202 36 573 411 60 13 0.004 147 22 322 317 28 TPEN 0.001 192 34 490 512 42 12

TPEN

(mM) 0.04 201 22 956 322 22 0.1 200 60 708 389 21 12 200 148 419 376 19 11 205 16 377 307 17 WO 98/40071 PCT/US98/04683 103- Mg Al Ca Fe Zn Cu (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) PBS 223 52 1186 266 45 11 0.004 228 73 795 247 53 11 0.001 237 43 862 281 49 12

EGTA

S0.04 247 104 1402 438 71 13 0.01 213 61 675 272 54 13 191 62 519 238 27 13 168 27 455 230 18 12 0.004 234 33 489 231 47 12 0.001 225 88 1306 275 47 13 BC 0.04 226 38 753 248 56 (mM) 0.01 223 73 762 256 49 13 254 42 1602 271 49 14 238 38 912 249 53 Metal levels were measured in 10 AD specimens treated with 0.1 mM TPEN. See Table 6 below. The observed increase in extractable AP correlated with significant depletion in zinc in every case and to a lesser extent, copper, when compared with PBS-treated tissue. No other metal tested was significantly influenced by treatment at this concentration.

Table 6. Residual Metal Levels (Based on 10 AD Specimens) Zn Cu Fe Ca Mg Al PBS 50.7 11.9 227 202 197 44 SEM) (28.8) (28.3) (39.1) (46.2) TPEN 33.2 9.8 239 210 230 (31.7) (37.0) (39.2) (45.0) Given the precipitous decline in extractable AP observed when employing high concentrations of TPEN or EGTA (see Figure 19A and 19B), it was hypothesized that magnesium or calcium might also have a significant role in the AP solubility equilibrium. Magnesium or calcium added to the homogenization buffer produced no appreciable alteration in soluble AP. However, using an extract previously depleted of metals by high levels of EGTA, the addition of WO 98/40071 PCT/US98/04683 104magnesium, and to a lesser extent calcium, led to resolubilization of the precipitated Ap. Figure 24B shows that AP solubility in metal-depleted tissue samples is restored by supplementing with magnesium.

Mindful of the high variability observed between individual subjects, 6 AD and 5 aged-matched control brains were chosen at random to determine if the observed phenomena were broadly applicable. These specimens were subjected to chelation treatment at selected concentrations of 0.1 or 2.0 mM or with PBS alone. Figure 25A shows that patterns of chelator-promoted solubilization of AP differ in AD and aged, non-AD tissue. The chelator-promoted solubilization of AP from AD brains represented an increase of up to 7-fold over that seen with PBS alone; the mean increase for BC being around 4 fold, and that for TPEN around 2 fold. Treatment with EGTA at 2 mM always produced a diminution in AP signal below that observed for the PBS control (See Figure The effects observed with non-demented, aged-matched controls were similar with respect to EGTA and TPEN. However, it is noteworthy that the effect of BC was much reduced. In some cases (Figure 25A, lower panel), BC treatment caused an attenuation in soluble AP suggesting that the amyloid deposits in ADaffected brain respond to this chelator in a different fashion than the deposits predominating in non-demented elderly brain.

For each subject in the experiments of Figures 25A and 25B, the extractable AP was derived and calculated as a proportion of the total preextraction AP load See Table 7 and 8 below.

Table 7. AD-affected Tissue AD 1 2 3 4 5 6 X +/-SEM X C/PBS Total.AP (pg/g) 10.8 77.0 80.3 6.0 14.4 16.8 43.0 14.1 PBS pg/g 0.74 1.39 1.04 0.07 3.0 0.06 1.05 0.44 of total) (0.3) TPEN 2mM pg/g 0.21 3.40 1.80 5.50 5.00 0.28 2.73 0.85 2.60 of total) (2.25) (1.75) (0.9) WO 98/40071 PCT/US98/04683 105- BC2mM pg/g 0.31 5.54 3.62 6.05 6.03 0.54 4.10 0.86 3.90 of total) (10.0) (1.2) Table 8. Age-Matched Control Tissue AC 1 2 3 4 5 X +/-SEM X C/PBS Total AP (gg/g) 0.7 4.2 2.7 3.2 3.6 2.8 0.60 PBS pg/g 0.17 0.13 0.18 0.10 0.66 0.25 0.10 of total) (25.0) (18.3) (11.3) (4.4) TPEN 2mM Vg/g 0.22 0.38 0.26 0.09 1.06 0.40 0.17 1.6 of total) (32.0) (29.5) (16.7) (5.1) BC 2mM pg/g 0.03 0.24 0.29 0.08 0.98 0.32 0.16 1.28 of total) (11.0) (27.2) (10.3) (4.6) Total AP for AD brains ranged from 6 80ug/g wet weight tissue. The percentage of AP extractable (one extraction/centrifugation sequence) ranged from 0.33 The corresponding values for aged-matched control brains were 0.68 4.2gg/g total AP and 2.6 29.5% extractable.

In order to further investigate these different responses to chelators, triplicate blots of AD tissue and control tissue which displayed cerebrovascular and diffuse amyloid deposits were compared using antibodies specific for Ap, 4 0 and Api-42. Figure 26 shows that chelation promotes the solubilization of Ap.

40 and API- 42 from AD and non-AD tissue. Using 3 different monoclonal antibodies, attempts to detect whether any particular species of AP were selectively affected by chelation were performed. Both A,1-40 and A3 1 were liberated by chelation, however the dimeric form of AP 1 40 in both AD and control tissue predominated.

As reported by Roher et al., Proc. Natl. Acad. Sci., 90:10836-10840 (1993) the predominant form of cerebrovascular amyloid is AP 1 -42. Somewhat surprisingly, the dimeric form of this highly aggregating species is absent in the (control) tissue in which it is most favored.

WO 98/40071 PCT/US98/04683 106- It has recently been reported that the zinc-dependent Insulin Degrading Enzyme (IDE) has significant A3 cleavage activity (Perez et al., Proc Soc. for Neuroscience 20: Abstract 321.13 (1997)). In the experiments presented here, the disassembly of amyloid is reflected in the intermediate dimeric species which result from conversion between soluble and insoluble forms. Thus, simple inhibition of catalytic enzyme activity cannot account for the observed increase in soluble Ap. However, in the event that a proportion of the chelator-mediated augmcntation of AP solubilization was due to inhibition of this enzyme, homogenisations were conducted both in the presence of 1 mM n-ethyl amimide (NEM a potent inhibitor of IDE. and at 37 C. No enhancement of Ap signal was observed above that of PBS alone for NEM nor was there any diminution of signal after incubation at 37 0

C.

Discussion Metal chelators offer a powerful tool for investigating the role of metals in the complex environment of the brain, however the strengths of these compounds may also define their limitations. The broad metal affinities of most chelators make them rather a blunt instrument. Attempts were made to sharpen the focus of the use of chelators by selecting chelators with a range of affinities for the metals of interest. These differences may be exploited by appropriate dilution, thereby favoring the binding of the relatively high affinity ligand (metal for which the chelator has the highest affinity).

The dilution profiles exhibited by EGTA and TPEN (Figure 19A and 19B) possibly reflect a series of equilibria between different metal ligands and the chelators, whereby the influence of low affinity, but abundant, metals is observed at high chelator concentrations and that of the high affinity, but more scarce, metals predominates at low concentrations of chelator. In the case of AP itself, this explanation is further complicated by the presence of low and high affinity binding WO 98/40071 PCT/US98/04683 107sites for zinc (and copper) (Bush, A.I. et al., J. Biol. Chem., 269:12152-12158 (1994)).

The results shown in Figure 19A and 19B coupled with the hypothesis that lower affinity metals are removed at higher concentrations of chelators implies a role for lower affinity metals in meditation of AP solubility. Metals such as Mg and Ca may be increasingly removed at higher chelator concentrations. Figure 24B shows that Mg 2 and to a lesser extent, Ca 2 restore sobulitity to metal depleted AP aggregate pellets. This indicates that these metals may function to mediate an A3 solubility equilibrium in vivo.

Bathocuproine with its low affinity for metals other than Cu- is effective at solubilizing AP through a dilution range over 3 orders of magnitude, and interestingly, does not diminish in effectiveness at the highest levels tested. The particular affinity of BC for Cu" has been exploited to demonstrate that in the process of binding to APP, Cu 2 is reduced to Cu' resulting in the liberation of potentially destructive free radicals (Multhaup, et al., Science 271:1406-1409 (1996)). It has also been shown that AP has a similar propensity for reducing copper with consequent free radical generation (Huang, el al., J. Biol. Chem.

272:26464-26470 (1997)).

Although the predicted reduction in copper in extraction pellets treated with BC has not been demonstrated, it is possible that the ratio ofCu> 2 to Cu has been affected. At this stage, however, the means to evaluate' the relative contributions of divalent and reduced forms to the total copper content of such extraction pellets are not available.

In addition to their primary metal binding characteristics, chelators are a class of compounds which vary in hydrophobicity and solubility. Their capacity to infiltrate the highly hydrophobic amyloid deposits may therefore be an important factor in the disassembly of aggregated Ap. It is also possible that the chelators are also acting to liberate intracellular stores of A3 in vesicular compartments as metal-bound aggregates. Preliminary data indicates that this may be the case with platelets.

WO 98/40071 PCT/US98/04683 108- The variability between subjects is consistent, reflecting the heterogeneity of the disease in its clinical and histopathological expression. Despite this, a consistent pattern of response to the actions of chelators by tissue from both AD and non-AD subjects is observed. This universality of the phenomenon of chelator-mediated solubilization is strongly suggestive that metals are also involved in the assembly of amyloid deposits in normal individuals, although the dissimilar patterns of response suggest that different mechanisms are operating in the disease and non-pathological states.

On the basis of the evidence presented here and the in vitro data, it is proposed that zinc functions in the healthy individual to promote the reversible aggregation of Ap, counteracted by magnesium acting to maintain A3 solubility.

Further, the disease state is characterized by an unregulated interaction with copper resulting in the generation of free radicals.

A functional homoeostatic mechanism implies equilibrium between intracellular copper and zinc (and perhaps other metals) normally present in trace amounts, for which AP has strong affinity, and more abundant metals which bind less strongly to Ap. Zinc is of particular interest because the anatomical distribution of zinc correlates with the cortical regions most susceptible to amyloid plaque formation (Assaf, S.Y. Chung, Nature, 308:734-736 (1984)).

It has recently been demonstrated (Huang, et al., J. Biol. Chem.

272:26464-26470 (1997)) that zinc-promoted aggregation of synthetic AP is reversible by the application of EDTA. The tightly-regulated neurocortical zinc transport system might provide a physiological parallel for this chelator-mediated disaggregation by moving zinc quickly in and out of the intraneuronal spaces.

Copper, while binding less avidly to AP than zinc (Bush, et al.. J.

Biol. Chem. 269:12152-12158 (1994)) has greater potential to inflict damage via free radical generation, resulting polymers are SDS-resistant (see Example 7, below). Slight alterations in the transportation and/or metabolism of metals resulting from age-related deterioration of cellular processes may provide the environment for a rapid escalation of metal-mediated AP accretion which WO 98/40071 PCT/US98/04683 109eventually overwhelms regulatory and clearance mechanisms. In describing a mechanism for AP homeostasis this model for amyloid deposition implies a possible physiological role for AP whereby aggregation and disaggregation may be effected through regulation or cortical metal levels and that the predominantly sporadic character of AD reflects individual differences in the brain milieu. Such a mechanism by no means rules out other genetic, environmental, inflammatory or other processes influencing the progression of the disease. Furthermore, in demonstrating the effectiveness ofchelators in solubilising amyloid, it is suggested herein that suitable agents of this type are useful for therapeutic or prophylactic use in AD.

Example 7 Formation of SDS-Resistant Aft Polymers The cause for the permanent deposition of A in states such as Alzheimer's Disease (AD) and Down's Syndrome (DS) are unknown, but the extraction of Ap from the brains of AD and DS patients indicates that there are forms of AP that can be resolubilized in water and run as a monomer on SDS-PAGE (Kuo, Y-M., et al., J. Biol. Chem. 271:4077-4081 (1996)), and forms that manifest SDS-, ureaand formic acid-resistant polymers on PAGE (Masters, C.L. et al., Proc. Natl.

Acad. Sci. USA 82:4245-4249 (1985); Dyrks, et al., J. Biol. Chem. 267:18210- 18217 (1992); Roher, et al., Journal of Biological Chemistry 271:20631- 20635 (1996). Thus, the extraction of SDS-resistant AP polymers from plaques implicates polymerization as a pathogenic mechanism that promotes the formation of AD amyloid.

The exact mechanism underlying the formation of SDS-resistant polymeric AP species remains unresolved. Recently, Huang, et al. have shown that AP reduces both Cu 2 and Fe 3 (Huang, et al., J. Biol. Chem. 272:26464-26470 (1997)), providing a mechanism whereby a highly reactive species could promote WO 98/40071 PCT/US98/04683 110the modification of proteins via an oxidative mechanism. Here, the inventors tested the ability of Cu 2 and Fe 3 to promote SDS-resistant AP polymerization.

Materials and Methods Human AP,3 40 peptide was synthesized, purified and characterized as described above. Rat AP, -40 was obtained from Quality Control Biochemicals, Inc.

(Hopkinton, MA). Peptides were analyzed and stock solutions prepared as described above.

As above, electronic images captured using the Fluoro-S Image Analysis System (Bio-Rad, Hercules, CA) were analyzed using Multi-Analyst Software (Bio-Rad, Hercules, CA). This chemiluminescent image analysis system is linear over 2 orders of magnitude and has comparable sensitivity to film.

Human AD derived SDS-resistant polymers were solublized in formic acid, and then dialyzed with 5 changes of 100 mM ammonium bicarbonate, pH 7.5. The solublized peptide was then used for subsequent chelation experiments.

Results and Discussion The generation of SDS-resistant AP polymers by metal ions was tested by incubating Cu" 2 (30 pM) or Zn 2 (30 at pH 6.6, 7.4 and 9.0 with AP,_ 40 As shown in Figure 9, Western blot analysis of samples incubated with Cu and run under SDS denaturing and P-mercaptoethanol reducing conditions revealed an increase in dimeric, trimeric and higher oligomeric AP species over time. The dimer and trimer had molecular weights of approximately 8.5 kD and 13.0 kD, respectively. Image analysis indicated 42% and 9% conversion of the monomer to dimer and trimer, respectively, in samples incubated at pH 7.4 after 5 d. The conversion of monomer to the dimer and trimer was 29% and respectively, at pH 6.6 after 5 d.

WO 98/40071 PCT/US98/04683 -111 In contrast, changes in alone did not induce SDS-resistant

AI

1 4 0 polymerization. Less than 4% of the peptide was converted to the SDS-resistant dimer after 5 d in samples incubated at pH 6.6, 7.4 or 9.0, most likely as a result of contaminating Cu 2 in the buffer and AD solutions. Cu 2 contamination of chelex-treated PBS was up to 0.5 /M as determined by ion coupled plasma-atomic emission spectroscopy (ICP-AES). Although Zn' 2 induces rapid aggregation of

AP,_

40 (Bush, A et al., J. Biol. Chem. 268:16109 (1993); Bush, el al., J.

Biol. Chem. 269:12152 (1994); Bush, et al., Science 265:1464-1467 (1994); Bush, el al., Science 268:1921-1922 (1995); Atwood et al., submitted; Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)), it did not induce SDSresistant Ap polymerization (Figure 9) as previously reported (Bush, et al., Science 268:1921-1922 (1995)).

AP I -42 is the predominant species found in amyloid plaques (Masters, C.L.

el al., Proc. Natl. Acad. Sci. USA 82: 4245 (1985); Murphy, et al., Am. J.

Pathol. 144:1082-1088 (1994); Mak, et al., Brain Res. 667:138-142 (1994); lwatsubo, et al., Ann. Neurol. 37:294-299 (1995); Mann et al., Ann. Neurol.

40:149-156 (1996)). Therefore, the ability of 40 and AP,3_4 to form SDSresistant polymers was compared.

In contrast to Cu 2 -induced SDS-resistant A, 4 0 polymerization over days, SDS-resisitant AP,- 42 polymerization occurred within minutes in the presence of Cu 2 (Figure 27A). Unlike Ap 4 0 where Cu 2 induces the formation of a SDSresistant dimeric species first, AP,342 initially forms an apparent trimer species in the presence of Cu 2 Over time, dimeric and higher polymeric species also appear in AP 42 incubations with Cu 2 at both pH 7.4 and 6.6. The greater Cu 2 induced Ap 4 2 polymerization observed at pH 6.6 compared with pH 7.4 in samples incubated for 30 min. was reversed after 5 d. At pH 6.6, both Ap,_ 4 0 and AP,.

42 exist in an aggregated form within minutes. Therefore, the formation of these polymeric species occurs within AP aggregates and the formation of SDS-resistant A[ polymers is independent of aggregation state (see below). Similar results were obtained using the monoclonal antibody 4G8.

WO 98/40071 PCT/US98/04683 112- Since redox active Fe (Smith, et al., Proc. Natl. Acad. Sci. USA 94:9866 (1997)) and ferritin (Grudke-Iqbal, et al., Acta Neuropathol. 81:105 (1990)) are found in amyloid lesions, experiments were performed to determine if Fe 3 could induce SDS-resistant polymerization of A,_ 1 4 0 and AP,3 4 2 (Figure 27A). Fe3' did not induce A,_40 polymerization above background levels with either peptide. The small increase in polymeric 4 0 and -40 in samples with no metal ions reflects a small contaminating concentration of Cu 2 The formation of amyloid plaques is not a feature of aged rats (Johnstone, et al., Mol. Brain Res. 10:229 (1991); Shivers et al. (1988)). To test whether rat AP-4, would form SDS-resistant AP polymers, rat API, 40 was incubated with Cu 2 and Fe 3 at pH 7.4 and 6.6 (Figure 27B). Neither metal ion induced SDSresistant Ap polymers Huang, X. el al., J. Biol. Chem. 272:26464-26470 (1997)).

The binding and reduction of Cu 2 by rat Ap 40 is markedly decreased compared to that of human A, 1 40 (Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)). This result suggests that the generation of SDS-resistant AP polymers is dependent upon the binding and reduction of Cu 2 by Ap.

Tests were performed to determine the concentration of Cu 2 required to induce the formation of SDS-resistant A 40 and Ap,_ 42 polymers. Ap,_ 40 and Ap,_42 were incubated with different [Cu 2 (0-30 pM) at pH 7.4 and 6.6 and the samples analyzed by Western blot and the signal quantitated using the Fluoro-S Image Analysis System (Bio-Rad, Hercules, CA) as previously described.

At pH 7.4, the increase in polymerization of AP-.

40 was barely detectable as [Cu 2 was increased from 0.5 to 1 M but under mildly acidic conditions (pH SDS-resistant polymerization could be detected (over 3-fold increase in dimerization)(Table 9A).

Table 9A. Cu 2 Induced SDS-Resistant Polymers of AP 4 0 WO 98/40071 WO 98/007 1PCTIUS98/04683 113- 94.8 4.9 0.3 0 0 1 93.6 5.9 0.6 0 0 84.3 14.2 1.5 0 0 85.2 13.2 1.6 0 0 76.2 19.1 4.7 0 0 pH 6.6 [Cu21i Monomer Dimer Trimer Tetramer Pentamer 0 97.9 2.1 1 0 0 97.6 2.2 0.2 0 0 1 92.6 7. 3 0.1 0 0 90.1 9.8 0.1 0 0 79.4 16.1 4.5 0 0 74.5 13.2 12.2 0 0 A similar CU 2 1 concentration and pH dependent increase in SDS-resistant AP, 1 2 polymers also was observed (Table 913), but SDS-resistant polymerization occurred at much lower [Cu 2 Table 9B. Cu 24 Induced SDS-rcsistant Polymers of Ap,.

4 2 7.4 [Cu 2 1] Monomer Dimer Trimer Tetramer Pentamer 0 76.61 0 16.0 5.5 1.9 70.7 0 20.5 6.2 1 64.9 0 23.6 7.4 56.1 0 31.8 8.7 4.1 55.1 0 30.3 10.3 4. 3 57.1 0 31.1 8.3 4.2 pH 6.6 [Cu 2 Monomer JDimer Trimer Tetramner Pentamer 0 61.0 j 0 27.3 8.6 j 3.8 52.1 j 0 33.8 12.0 J 3.

WO 98/40071 PCT/US98/04683 114pH 7.4 Monomer Dimer Trimer Tetramer Pentamer 59.6 0 30.0 7.1 3.2 52.3 0 31.7 13.6 2.2 polymerization was not detected with increasing Fe" concentrations at any pH. Therefore, of the metal ions known to interact with AP, only Cu whose ability to aggregate and bind Cu> under mildly acidic conditions is enhanced, is capable of inducing SDS-resistant Ap polymerization.

Oxygen radical mediated chemical attack has been correlated with an increase in protein and free carbonyls (Smith, et al., Proc. Natl. Acad Sci.

USA 88:10540 (1991); Hensley, et al., J. Neurochem. 65:2146 (1995); Smith, et al., Nature 382:120 (1996)) and peroxynitrite-mediated protein nitration (Good, et al., Am. J. Pathol. 149:21 (1996); Smith, et al., Proc. Natl.

Acad. Sci. USA 94:9866 (1997)).

AP is capable of reducing Cu> and H20, is produced in solutions containing AP and Cu> or Fe 3 (Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)). As shown above, the generation of SDS-resistant AP polymers in the order Ap, 1 _4 AP 1 40 rat AP,_ 4 0 in the presence of Cu> correlates well with the generation of Cu' and reactive oxygen species (ROS; OH-, H,O, and 0,: Huang, et al., J Biol. Chem. 272:26464-26470 (1997)) by each peptide.

The increased generation of SDS-resistant AP polymers in the presence of Cu> compared to Fe 3 also was correlated with the generation of the reduced metal ions, respectively (Huang, X. et al., J. Biol. Chem. 2 72:26464-26470 (1997)). The increase in SDS-resistant AP polymerization seen under mildly acidic conditions may be a result of the higher driving the production of HO, dismutated from O; with the subsequent generation of OH' via Fenton-like chemistry inducing a modification of AP that results in SDS-resistant AP polymers (see Figure 12 showing a schematic of the proposed mechanism of AP-mediated reduced metal/ROS production).

WO 98/40071 PCT/US98/04683 -115- To confirm whether ROS were involved in the generation ofSDS-resistant polymers, experiments were performed to determine whether Cu in the presence or absence of H20, could promote AP polymerization (Figure 28A). A similar level of Ap, _42 polymerization was observed in the presence of Cu 2 or Cu indicating that the reduced metal ion alone was not capable of increasing Ap polymerization. Likewise, polymerization of AP_ 42 in the presence of HO was low and equivalent to control levels. However, the addition of Cu 2 or Cu' to AP in the presence of H,O, induced a similar, marked increase in dimers, trimers and tetramers within 1 hour. After 1 day, higher molecular weight polymers 18 kD) were generated (from the oligomers), with a subsequent reduction in the levels of monomer. dimer. trimer and tetramer only with the coincubation of HO, and Cu 2 Both the reduced and oxidized forms of Cu produced similar levels of polymerization in the presence of H,0 2 In contrast, neither Fe> nor Fe 2 induced as much polymerization as Cu 2 in the presence of H20, after 1 day incubation (Figures 28A and 28B). Since Fe" is not reduced as efficiently as Cu2 by AP (Huang, el al., Biol. Chem. 272:26464-26470 (1997)), and Cu' is rapidly converted to Cu 2 in solution, these results suggest that the reduction reaction is required for the polymerization reaction to proceed.

It was confirmed that the reduction of Cu 2 was required for generating SDS-resistant AP polymerization by incubating Ap, 42 and Cu 2 with and without bathocupoinedisulfonic acid a Cu* specific chelator (Figure 28C). There was a marked decrease in polymerization, indicating that Cu generation was crucial for the polymerization of Ap. It is possible that the decreased polymerization may be due to chelation of Cu2 by BC, however given the low binding affinity of BC for Cu 2 compared with Ap, it seems likely that the chelation of Cu' by BC prevents it from inducing SDS-resistant AP polymerization. Therefore, AP may undergo a hydroxyl radical modification that promotes its assembly into SDS-resistant polymers.

If H,0 2 is required for the polymerization reaction under physiological conditions, the removal of H20, and it's precursors 02 and O0 (Huang, et al., WO 98/40071 PCT/US98/04683 116- J. Biol. Chem. 272:26464-26470 (1997)) should decrease SDS-resistant polymerization. To confirm that H202 generated in the presence of A and Cu" 2 was required for the polymerization reaction, A, 42 was incubated with or without Cu 2 4 in the presence of TCEP (Figure 29A). TCEP significantly reduced the level of polymerization in samples with and without Cu 2 over 3 days. This indicates that the generation of HO, is required for the polymerization of Ap.

To confirm that the generation of 02 was required for SDS-resistant AP polymerization, AP,_ 42 was incubated with and without Cu 2 at pH 7.4 and 6.6 under argon in order to decrease the reduction of molecular 0, (Figure 29B).

Argon-purging of the solution markedly decreased AP,, 42 polymerization under each condition, indicating that the generation of ROS is required for the polymerization of Ap.

Taken together, these results indicate that polymerization occurs as a result of Haber-Weiss chemistry where the continual reduction of Cu 2 by Ap provides a species for the reduction of molecular 0, and the subsequent generation of O;, HO, and OH-. The binding and reduction of Cu 2 by AP is supported by the finding that the incubation of Fe 3 H,0 2 and ascorbic acid with Ap 40 (Figure 29A) and AP 1 -42 does not induce SDS-resistant polymerization equivalent to Cu 2 with H 2 0, alone. Since ascorbic acid effectively reduces Fe", the reduction of a metal ion that is not bound to Ap is insufficient to induce significant SDS-resistant polymerization.

The formation of SDS-resistant polymers of AP by this metal-catalyzed oxidative mechanism strongly suggested that a chemical modification to the peptide backbone allows the formation of the polymer species. To test if the SDSresistant polymers were covalently linked, SDS-resistant polymers generated by incubating AP3, 42 with Cu 2 at pH 7 4 and 6.6, or AP 4 2 with Cu 2 plus H2,O were subjected to treatment with urea (Figure 30A) and guanidine HCI, chaotrophic agents known to disrupt H-bonding. Urea and guanidine HCI did not disrupt the SDS-resistant polymers at 4.5 M, and only slightly at 9M, suggesting that the SDSresistant polymers were held together by high-affinity bonds, but not hydrogen WO 98/40071 PCT/US98/04683 -117bonding alone. HPLC-MS analyses, however, confirmed no covalent modification of the peptide and no evidence of covalent crosslinking.

Since covalent and/or hydrogen bonding were not involved in polymer formation, experiments were performed to detemine whether Cu 2 coordination of the complex by ionic interactions was allowing for the formation of the SDSresistant polymer species. To disrupt these ionic interactions, different chelating agents were added to a solution containing Cu -induced Ap,_ 40 or AP, 42

SDS-

resistant polymers generated at pH 7.4 (Figures 30B and 30C; TETA, tetraethylenediamine; EDTA, ethylenediaminetetra acetic acid; DTPA, diethylenetriaminopenta acetic acid; CDTA, trans-1,2-diaminocyclohexanetetra acetic acid; NTA, nitrilotriacetic acid).

All chelators significantly reduced the amount of Ap_ 40 or AP 1 4

SDS-

resistant polymers. EDTA was less effective in destabilizing the polymers, possibly due to its larger molecular mass, and lower affinity for Cu 2

EDTA

reduced the amount of AP I 40 polymers, but increased the amount of AP,_ 40 polymers at pH 7.4. This may be due to the fact that EDTA can enhance the redox potential of Cu under certain conditions.

Cu 2 -induced SDS-resistant polymers generated at pH 6.6 were also disrupted with chelation treatment to a similar extent. These results suggest that the chelation of Cu 2 away from AP results in the disruption of the polymer complex and the release of monomer species. Thus, there is an absolute requirement for metal ions in the stabilization of the SDS-resistant polymer complex.

The SDS-resistant polymers generated with Cu 2 are similar to those extracted from post-mortem AD brains (Roher, et al., Journal ofBiological Chemistry 2 71:20631-20635 (1996)). To determine if these human oligomeric AP species could be disrupted by metal chelators, TETA and BC were incubated with AP oligomers extracted from human brain. Figure 30E shows that both TETA and BC significantly increased the amount of monomer AP in samples treated with these chelators. Although the increase in the amount of monomer was small, these WO 98/40071 PCT/US98/04683 118results suggest that human oligomeric AP species are partially held together with metal ions. Importantly, this result indicates the potential of chelation therapy as a means of reducing amyloidosis.

To examine whether conformational changes could disrupt the SDSresistant polymers, solutions of SDS-resistant polymers in the presence or absence of Cu 2 were incubated with the a-helical promoting solvent system DMSO/HFIP, or under acidic conditions (pH 1) (Figure 30D). These conditions reduced the amount of polymer compared to untreated controls at both pH 7.4 and 6.6. indicating that an alteration in the conformation of Ap 4 to the a-helical conlormation could disrupt the strong Ap-Cu 24 ionic interactions. This provides indirect evidence that the polymer structures are likely to be in the more thermodynamically favorable P-sheet conformation, such as those found in neuritic plaques.

SDS-resistant AP polymers, such as that found in the AD-affected brain, are likely to be more resilient to proteolytic degradation and may explain the permanent deposition of AP in amyloid plaques. Incubation of SDS-resistant AP polymers with proteinase K resulted in complete degradation of both monomer and oligomeric AP species. Since protease treatment is incapable of digesting hard core amyloid, covalent crosslinking of the peptide following its deposition may occur over time that prevents proteolytic digestion. This may explain the limited disruption of human SDS-resistant A3 oligomers compared to the Cu-mediated SDS-resistant polymers generated in vitro.

Soluble Ap,_ 40 and A,_4 2 both exist in phosphate buffered saline as noncovalent dimers (Huang, et al., J. Biol. Chem. 272:26464-26470 (1997); and unpublished observations). Disruption of ionic and hydrogen bonding of AP in the soluble and aggregated forms (pH or Zn 2 by the ionic detergent SDS results in the complete dissociation of AP into the monomer species as detected on SDS- PAGE (Figures 9, 32-34). The formation of SDS-resistant polymers of Ap over time in the presence of Cu 2 (Figures 9, 27A-27B, 28A-28C) suggests that WO 98/40071 PCT/US98/04683 119conformational or structural alterations allow for the formation of a thermodynamically more stable complex.

Although no covalent crosslinking between peptides was detected, it is possible that a covalent modification(s) takes place within the peptide backbone that allows for a high affinity association to form between the peptide and Cu 2 Thus, a chemical modification to the peptide may increase the affinity of the polymer for Cu 2 and the formation of a stable complex. Alternatively, the requirement for molecular oxygen suggests that Cu may be coordinated by oxygen or ROS in the formation of SDS-resistant polymers.

The formation of SDS-resistant polymers was dependent upon the binding and reduction of Cu 2 The binding ofCu 2 to AP was confirmed by the detection of Cu 2 in both the monomer and dimer following SDS-PAGE. The [Cu 2 of PVDF membrane containing the immobilized peptide species was measured by ICP-AES (unpublished observations; Huang, et al., J. Biol. Chem. 272:26464- 26470 (1997)) and correlated with the generation of SDS-resistant polymers for each species.

Cu 2 coordination between AP molecules was required in order to maintain the structure since chelation treatment disrupted the in vitro generated SDSresistant polymer (Figures 30B-30E). Human SDS-resistant AP polymers also were disrupted with the Cu'-specific chelator BC indicating Cu coordination in the stabilization of these structures (Figure 30E). Together with the fact that Cuspecific chelators can extract more SDS-resistant Ap polymers from AD brains in aqueous buffer (see Example these results implicate Cu 2 in the generation of SDS-resistant polymers in vivo. Fe 3 did not induce the formation of SDS-resistant polymers in vitro (Figures 27A) as previously reported except in the presence of excess H 2 0 2 or ascorbic acid as previously reported (Dyrks, et al., J. Biol. Chem. 267:18210- 18217 (1992); and data not shown). Dyrks, et al. did, however observe significant increases in SDS-resistant polymerization with metal-catalyzed oxidation systems (Fe-hemin, Fe-hemoglobin or Fe-EDTA) in the presence of WO 98/40071 PCT/US98/04683 120-

H

2 0 2 The A,_ 42 used in their experiments was likely to be Cu-bound as it was extracted from a wheat germ expression system and already was present as SDSresistant oligomers. Thus, it is possible that Cu-bound AP used in these experiments contributed to the increased SDS-resistant polymerization observed in the Fe-catalyzed oxidation systems. Although Fe 3 is reduced by AP (Huang, et al., J. Biol. Chem. 272:26464-26470 (1997)), it is unable to effectively coordinate the complex like Cu (Figure 28B).

Fe 2 is found in much higher concentrations in the brains of AD patients compared with age-matched controls (Ehmann, el al., Neurotoxicol. 7:197- 206 (1986); Dedman, et al., Biochem. J. 287:509-514 (1992); Joshi, el al., Environ. Health Perspect. 102:207-213 (1994)). This is partly attributable to the increased ferritin rich microglia and oligodendrocytes that localize to amyloid plaques (Grudke-Iqbal, et al., Acta Neuropathol. 81:105 (1990); Conner, J.R., et al., J. Neurosci. Res. 31:75-83 (1992); Sadowki, et al., Alzheimer's Res.

1:71-76 (1995)).

Recently, redox active Fe was localized to amyloid lesions (Smith, M.A., el al., Proc. Natl. Acad. Sci. USA 94:9866 (1997)). While Fe is normally sequestered by metalloproteins, this localization of ferritin-rich cells around amyloid deposits, and the very high concentrations of iron in amyloid plaques (Conner, et al., J. Neurosci. Res. 31:75-83 (1992); Markesbery, W.R. and Ehmann, "Brain trace elements in Alzheimer's disease," in Terry, et al., eds., Alzheimer Disease, Raven Press, New York (1994), pp. 353-368) suggests that reduced Fe released from ferritin and transferrin under mildly acidic conditions could be available for Fenton chemistry and the formation of SDSresistant polymers. However, even in the presence of a Fe-ROS generating system (ascorbic acid, H 2 0, and Fe) the generation of SDS-resistant Ap polymers in vitro was low (Figure 29A) and may be explained by Cu 2 contamination of the buffers.

Interestingly, diffuse plaques, which may represent the earliest stages of plaque formation, are relatively free of ferritin-rich cells (Ohgami, et al., Acta Neuropathol 81:242-247 (1991)). Therefore, the accretion of iron in amyloid WO 98/40071 PCT/US98/04683 121 plaques may be a secondary response to the neurodegeneration caused by the reduction of Cu 2 and the generation of ROS by AP and the formation of neurotoxic SDS-resistant AP polymers.

Structural differences between AP 40 and AP,_ 42 may allow for the formation of a thermodynamically stable dimer in the case of A,3_ 40 and trimer in the case of A, 42 (Figures 27A, 30B and 30C). Irrespective of this, the increased generation of SDS-resistant polymers by AP, 4 2 compared to AP, 40 is most likely explained by the increased ability of A,_ 42 to reduce Cu and generate ROS. Since the addition of exogenous H 2 0, to AP, 42 increases the generation ofdimeric SDSresistant polymers ofAp,3_4, (Figures 28A and 28B), this dimeric species may be an integral intermediate in the formation of the SDS-resistant AP trimers, and may explain why Ap 40 polymerization occurs more slowly.

AD Pathology The present invention indicates that the manipulation of the brain biometal environment with specific agents acting directly chelators and antioxidants) or indirectly by improving cerebral energy metabolism) provides a means for therapeutic intervention in the prevention and treatment of Alzheimer's disease.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

All patents and publications cited in the present specification are incorporated by reference herein in their entirety.

Claims

1. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and one or more pharmaceutically acceptable carriers or diluents; for a time and under conditions to bring about said treatment; and wherein said chelator reduces, inhibits or otherwise interferes with AS-mediated production of radical oxygen species.

2. A method of claim 1, further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a 20 pharmaceutically acceptable salt thereof.

3. A method of treating amyloidosis in a subject, said method comprising administering to said subject a combination of a metal chelator selected from the 25 group consisting of: bathocuproine, bathophenanthroline, g DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt, for a time and under conditions to bring about said treatment; and wherein said chelator reduces, inhibits or otherwise interferes with Ai-mediated production of radical oxygen species.

4. A method of claim 3, wherein the metal chelator is EGTA. \Pabral\Keep\speci\65484-98.doc 9/02/02 H=\Pcabra\Keep\speci\6S484-98.doc 19/02/02 123 A method of claim 3 wherein the metal chelator is TPEN.

6. A method of any one of claims 3 to 5, wherein the supplement is magnesium salt.

7. A method of any one of claims 3 to 6, further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

8. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a salt of a metal chelator, wherein said chelator is selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein said salt of a metal chelator is selected 20 from the group consisting of: ammonium, calcium, magnesium, and sodium; and wherein said salt of a metal chelator reduces, inhibits or otherwise interferes with A#-mediated production of radical oxygen species. 25 9. A method according to claim 8, wherein the metal chelator is EGTA. e

10. A method according to claim 8, wherein the metal chelator is TPEN.

11. A method according to any of claims 8 to wherein the salt of a metal chelator is a magnesium salt.

12. A method according to any one claims 8 to 11, further comprising administering to said subject a compound selected from the group consisting of: N H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 I 124 rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

13. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a chelator specific for copper; wherein said chelator reduces, inhibits or otherwise interferes with Aj-mediated production of radical oxygen species.

14. A method of claim 13, wherein the chelator specific for copper is specific for the reduced form of copper.

15. A method of claim 13 or claim 14, wherein the chelator is bathocuproine or a hydrophobic derivative thereof.

16. A method of treating amyloidosis in a subject, 20 said method comprising administering to said subject an effective amount of an alkalinizing agent, wherein said alkalinizing agent reduces, inhibits or otherwise interferes with Aj-mediated production of radical oxygen species.

17. A method of claim 16, wherein the alkalinizing agent is magnesium citrate.

18. A method of claim 16, wherein the alkalinizing agent is calcium citrate.

19. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and one or more pharmaceutically acceptable H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 125 carriers or diluents; for a time and under conditions to bring about said treatment; and wherein said chelator prevents formation of A# amyloid, promotes, induces or otherwise facilitates resolubilization of A deposits, or both. A method of claim 19, further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

21. A method of treating amyloidosis in a subject, said method comprising administering to said subject a combination of a metal chelator selected from the following group: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, S 20 magnesium salt, and sodium salt, for a time and under o conditions to bring about said treatment; and wherein said combination prevents formation of A/ amyloid, promotes, induces or otherwise facilitates resolubilization of A# deposits, or both.

22. A method of claim 21, wherein the metal chelator is EGTA.

23. A method of claim 21, wherein the metal chelator is TPEN.

24. A method of any one of claims 21 to 23, wherein the supplement is magnesium salt. i\ H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 ,T 126 A method of any one of claims 21 to 24, further comprising administering to the subject an effective amount of a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

26. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a salt of a metal chelator, wherein said chelator is selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein said salt of a metal chelator is selected from the group consisting of: ammonium, calcium, 15 magnesium, and sodium; and wherein said salt of a metal chelator prevents formation of Aj amyloid, promotes, induces or otherwise facilitates resolubilization of AP deposits, or both.

27. A method of claim 26, wherein the metal chelator :is EGTA.

28. A method of claim 26, wherein the metal chelator :is TPEN.

29. A method of any one of claims 26 to 28, wherein the salt of a metal chelator is a magnesium salt. A method of any one of claims 26 to 29, further comprising administering to said subject a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof. S'H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 -1 k 127

31. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of a chelator specific for copper; wherein said chelator prevents formation of A3 amyloid, promotes, induces or otherwise facilitates resolubilization of A3 deposits, or both.

32. A method of claim 31, wherein the chelator specific for copper is specific for the reduced form of copper.

33. A method of claim 31 or claim 32, wherein the chelator is bathocuproine or a hydrophobic derivative 15 thereof.

34. A method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of an alkalinizing agent, wherein said alkalinizing agent prevents formation of A3 amyloid, promotes, induces or otherwise facilitates resolubilization of A3 deposits, or both. A method of claim 34, wherein the alkalinizing 25 agent is magnesium citrate.

36. A method of claim 34, wherein the alkalinizing agent is calcium citrate.

37. A pharmaceutical composition comprising: a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and one H:\KarraR\Keep\speci\Biotech\65484-98.doc 11/04/02 ll 128 or more pharmaceutically acceptable carriers or diluents; wherein the metal chelator is present in an amount effective to treat conditions caused by amyloidosis, A#- mediated ROS formation, or both when used in a method of claim 1.

38. A pharmaceutical composition for treatment of conditions caused by amyloidosis, A#-mediated ROS formation, or both, comprising: a metal chelator selected from the group consisting of:bathocuproine, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; one or more pharmaceutically acceptable carriers or diluents; and a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.

39. A pharmaceutical composition for treatment of conditions caused by amyloidosis, A#-mediated ROS S 20 formation, or both, when used in a method of claim 3, wherein said composition comprises: a metal chelator selected from the group consisting of: bathocuproine, :bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and 25 a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt, together with one or more pharmaceutically acceptable carriers or diluents.

40. A pharmaceutical composition of claim 39, wherein the metal chelator is EGTA. SH:\Pcabral\Keep\speci\65484-98.doc 19/02/02 129

41. A pharmaceutical composition of claim 39, wherein the metal chelator is TPEN.

42. A pharmaceutical composition of any one of claims 39 to 41, wherein the supplement is a magnesium salt.

43. A pharmaceutical composition comprising a salt of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and wherein said salt of a metal chelator is selected from the group consisting of: ammonium, calcium, magnesium, and sodium, together with one or more pharmaceutically acceptable carriers or diluents; wherein said salt of a metal chelator is present in an amount effective to treat conditions caused by amyloidosis, A#- mediated ROS formation, or both, when used in a method of claim 8.

44. A pharmaceutical composition of claim 43, wherein the metal chelator is EGTA.

45. A pharmaceutical composition of claim 43, 25 wherein the metal chelator is TPEN.

46. A pharmaceutical composition of any one of claims 43 to 45, wherein the salt of the chelator is a magnesium salt.

47. A pharmaceutical composition comprising a chelator specific for copper, with one or more \pharmaceutically acceptable carriers or diluents; wherein H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 130 the copper chelator is present in an amount effective to treat conditions caused by amyloidosis, A/-mediated ROS formation, or both, when used in a method of claim 13.

48. A pharmaceutical composition of claim 47, wherein the chelator is specific for the reduced form of copper.

49. A pharmaceutical composition of claim 48, wherein the chelator specific for the reduced form of copper is bathocuproine. A pharmaceutical composition comprising an alkalinizing agent, with one or more pharmaceutically 15 acceptable carriers or diluents; wherein said alkalinizing agent is present in an amount effective to treat conditions caused by amyloidosis, A3-mediated ROS formation, or both, when used in a method of claim 16.

51. A pharmaceutical composition of claim wherein the alkalinizing agent is magnesium citrate.

52. A composition of matter comprising: a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin.

53. A composition of matter for treatment of conditions caused by amyloidosis, Ap-mediated ROS formation, or both, when used in a method of claim 3, O"/'wherein said composition comprises: a metal chelator H:\KarraR\Keep\speci\Biotech\65484-98.doc 11/04/02 41' r 131 selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt.

54. A composition of claim 53, wherein the metal chelator is EGTA. A composition of claim 53, wherein the metal chelator is TPEN.

56. A composition of any one of claims 53 to wherein the supplement is a magnesium salt. contacting A# aggregates with solutions containing a range of concentrations of said metal ochelators;

57. A method for detering a dilution curve from data 25 obtained in step selecting chelators which solubilize less A§ aggregates at higher concentrations than at lower or intermediate concentrations; contacting A# aggregates with chelators 30 selected in t he treatment of amyloidosis, should be supplemented with ammonium, calcium, magnesium or sodium salt; and H:\Pral\Keep\speci\654498.doc20 salts, comprising:19/02/02 contacting Ap aggregates with solutions containing a range of concentrations of said metal e.oeo ;chelators; preparing a dilution curve from data 25 obtained in step selecting chelators which solubilize less A<8 aggregates at higher concentrations than at lower or intermediate concentrations; contacting A aggregates with chelators selected in step(c), in the presence of an ammonium, calcium, magnesium or sodium salt; and J H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 132 determining if resolubilization is increased in the presence of said salt; thereby determining whether a metal chelator used in the treatment of amyloidosis should be supplemented with ammonium, calcium, magnesium, or sodium salts.

58. A method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of Cu(I) by said method comprising: adding Cu(II) to a first AP sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate Cu(I); adding Cu(II) to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for 0*0, the same amount of time as said first sample; determining the amount of Cu(I) produced by said first sample and said second sample; and comparing the amount of Cu(I) produced by 'i said first sample to the amount of Cu(I) produced by said second sample; whereby a difference in the amount of Cu(I) 25 produced by said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of Cu(I) by AP.

59. The method of claim 59, wherein the amount of Cu(I) present in said first and said second sample is determined by adding a complexing agent to said first and said second sample, wherein said complexing agent is H:\Pcabral\Keep\speci\65484-9 8 .doc 19/02/02 133 capable of combining with Cu(I) to form a complex compound, wherein said complex compound has an optimal visible absorption wavelength; measuring the absorbancy of said first and said second sample; and calculating the concentration of Cu(I) in said first and said second sample using the absorbancy obtained in step

60. A method of claim 59, wherein said complexing agent is bathocuproinedisulfonic anion.

61. A method of claim 59 or claim 60, wherein said method is performed in a microtiter plate, and the 15 absorbancy measurement is performed by a plate reader. *9

62. A method of any one of claims 59 to 61, wherein two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Cu(I) by A#. 9

63. A method of claim 58, wherein said first A sample of step and said second A# sample of step (c) is a biological sample.

64. A method of claim 63, wherein said biological sample is CSF. A method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of Fe(II) by A, said method comprising: adding Fe(III) to a first A# sample; i H\Pcabral\Keep\speci\65484-98.doc 19/02/02 r 134 allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate Fe(II); adding Fe(III) to a second A# sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; determining the amount of Fe(II) produced by said first sample and said second sample; and comparing the amount of Fe(II) present in said first sample to the amount of Fe(II) present in said second sample; whereby a difference in the amount of Fe(II) present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of Fe(II) by A#.

66. A method of claim 65, wherein the amount of Fe(II) present in said first and said second sample is determined by adding a complexing agent to said first and .t said second sample, wherein said complexing agent is t o* capable of combining with Fe(II) to form a complex 25 compound, wherein said complex compound has an optimal visible absorption wavelength; measuring the absorbancy of said first and said second sample; and calculating the concentration of Fe(II) in said first and said second sample using the absorbancy obtained in step -LU, Hi\Pcabral\Keep\speci\65484-98.doc 19/02/02 L: L i rC 135

67. A method of claim 66, wherein said complexing agent is bathophenanthrolinedisulfonic (BP) anion.

68. A method of claim 66 or claim 67, wherein said method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader.

69. A method of claim 68, wherein two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Fe(II) by AP. A method of claim 65, wherein said first AP sample of step and said second AP sample of step (c) 15 is a biological sample. 4, 0 o 71. A method of claim 70, wherein said biological sample is CSF. 0

72. A method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of altering the production of H 2 0 2 by AP, said 0 method comprising: adding Cu(II) or Fe(III) to a first AP 25 sample; allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate H 2 0 2 adding Cu(II) or Fe(III) to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 136 determining the amount of H 2 0 2 produced by said first sample and said second sample; and comparing the amount of H 2 0 2 present in said first sample to the amount of H 2 0 2 present in said second sample; whereby a difference in the amount of H 2 0 2 present in said first sample as compared to said second sample indicates that said candidate pharmacological agent has altered the production of H 2 0 2 by Aj.

73. A method of claim 72, wherein the A samples of steps and step are a biological fluid.

74. A method of claim 73, wherein said biological fluid is CSF.

75. A method of any one of claims 72 to 74, wherein the determination of step of the amount of H 2 0 2 present in said first and said second sample is determined by adding catalase to a first aliquot of said "first sample obtained in step of claim 1 in an amount sufficient to break down all of the H 2 0 2 generated by said sample; adding TCEP, in an amount sufficient to capture all of the H 2 0 2 generated by said samples, to said first aliquot (ii) a second aliquot of said first sample obtained in step of claim 1; and (iii) said second sample obtained in step of claim 1; H\Pcabral\Keep\speci\65484-98.doc 19/02/02 )v 137 incubating the samples obtained in step (b) for an amount of time sufficient to allow the TCEP to capture all of the H 2 0 2 adding DTNB to said samples obtained in step incubating said samples obtained in step for an amount of time sufficient to generate TMB; measuring the absorbancy at 412 nm of said samples obtained in step and calculating the concentration of H 2 0 2 in said first and said second sample using the absorbancies obtained in step

76. A method of claim 75, wherein said method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader.

77. A method of claim 76, wherein two or more different test candidate agents are simultaneously 20 evaluated for an ability to alter the production of H 2 0 2 by Aj3.

78. A method for the identification of an agent to be used in the treatment of AD, wherein said agent is 25 capable of interfering with the interaction of 02 and Aj to produce 02, without interfering with the SOD-like activity of Af, said method comprising: identifying an agent capable of decreasing the production of 02 by Af; and determining the ability of said agent to alter the SOD-like activity of A#. H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 138

79. A method of claim 78, wherein the determination of the ability of said agent to alter the SOD-like activity of AP is made by determining whether A is capable of catalytically producing Cu(I), Fe(II) or H 2 0 2 A method for the identification of an agent to be used in the treatment of AD, wherein said agent is capable of reducing the toxicity of AP, said method comprising: adding AP to a first cell culture; adding AP to a second cell culture, said second cell culture additionally containing a candidate pharmacological agent; determining the level of neurotoxicity of AP in said first and said second samples using LDH release assay or a Live/Dead assay; and comparing the level of neurotoxicity of A in said first and said second samples, whereby a lower neurotoxicity level in said second sample as compared to said first sample indicates that said candidate pharmacological agent has reduced the neurotoxicity of AP, and is thereby capable of being used to treat AD. o 25 81. A method of claim 80, wherein said cells are rat cancer cells.

82. A method of claim 80, wherein said cells are rat primary frontal neuronal cells.

83. A kit for determining whether an agent is capable of altering the production of Cu(I) by AP when used in a method of claim 58, wherein said kit comprises a H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 f I 139 carrier means being compartmentalized to receive in close confinement therein one or more container means wherein the first container means contains a peptide comprising A# peptide; a second container means contains a Cu(II) salt; and a third container means contains bathocuproinedisolfonic anion.

84. A kit of claim 83, wherein said A# peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about IM.

85. A kit for determining whether an agent is capable of altering the production of Fe (II) by AP when used in a method of claim 65, wherein said kit comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein 20 the first container means contains a peptide comprising A# peptide; •i a second container means contains an Fe(III) salt; and a third container means contains 25 bathophenanthrolinedisulfonic anion.

86. A kit of claim 85, wherein said A# peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about IM.

87. A kit for determining whether an agent is capable of altering the production of H 2 0 2 by AP when used H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 r 140 in a method of claim 73, wherein said kit comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein the first container means contains a peptide comprising A peptide; a second container means contains a Cu(II) salt; a third container means contains TCEP; and a fourth container means contains DTNB; wherein the kit is used to determine whether an agent is capable of altering the production of H 2 0 2 by Aj.

88. A kit of claim 87, wherein said A3 peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about pM. *e

89. A method for the identification of an agent to be used in the treatment of AD, wherein said agent is 20 capable of inhibiting redox-reactive metal -mediated crosslinking of Aj, said method comprising: adding a redox-reactive metal to a first A# sample; allowing said first sample to incubate for 25 an amount of time sufficient to allow A crosslinking; adding said redox-reactive metal to a second AP sample, said second sample additionally comprising a candidate pharmacological agent; allowing said second sample to incubate for the same amount of time as said first sample; removing an aliquot from each of said first and said second sample; and H:\Pcabral\Keep\speci\65484-98.doc 19/02/02 141 determining presence or absence of crosslinking in said first and second samples, whereby an absence of AS crosslinking in said second sample as compared to said first sample indicates that said candidate pharmacological agent has inhibited A/ crosslinking. A method of claim 89, wherein at step a western blot analysis is performed to determine the presence or absence of crosslinking in the first and the second sample.

91. A method according to any one of claims 1, 3, 8, 13, 16, 19, 21, 26, 31, 34, 57, 58, 59, 65, 72, 78, 15 87, 89, substantially as herein described with reference to any one of the examples or figures.

92. A composition according to claim 38 or claim 52, substantially as herein described with reference to any one of the examples or figures. Dated this 1 1 t h day of April 2002 THE GENERAL HOSPITAL CORPORATION 25 By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H\KarraR\Keep\speci\Biotech\65484-98.doc 11/04/02 H' H:\KarraR\Keep\speci\Biotech\65484-98.doc 11/04/02 I