EP1007048A1 - Identification of agents for use in the treatment of alzheimer's disease - Google Patents
Identification of agents for use in the treatment of alzheimer's diseaseInfo
- Publication number
- EP1007048A1 EP1007048A1 EP98911551A EP98911551A EP1007048A1 EP 1007048 A1 EP1007048 A1 EP 1007048A1 EP 98911551 A EP98911551 A EP 98911551A EP 98911551 A EP98911551 A EP 98911551A EP 1007048 A1 EP1007048 A1 EP 1007048A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sample
- agent
- salt
- chelator
- amount
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Definitions
- This invention is in the field of medicinal chemistry.
- 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.
- a ⁇ Abeta
- APP amyloid protein precursor
- a ⁇ deposits are usually most concentrated in regions of high neuronal cell death, and may be present in various morphologies, including amorphous deposits, neurophil plaque amyloid, and amyloid congophilic angiopathy (Masters, C.L., et al, EMBO J. 4:2151 (1985); Masters, C.L. et al, Proc. Natl Acad. Sci. USA 82: 4245 (1985)).
- 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 A ⁇ in these deposits suggests that this species is more pathogenic.
- the 42 residue form of A ⁇ (A ⁇ ,. 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 gly cation end-products, and protein cross-linking are seen in proximity to the lesions. To date, the cause of A ⁇ deposits is unknown, although it is believed that preventing these deposits may be a means of treating the disorder.
- CSF cerebrospinal fluid
- 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 A ⁇ , the method comprising:
- the amount of Cu + present in said first and said second sample is determined by
- step (c) calculating the concentration of Cu + in said first and said second sample using the absorbancy obtained in step (b).
- the complexing agent is bathocuproinedisulfonic (BC) anion.
- the concentration of Cu + produced by A ⁇ 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.
- 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 A ⁇ .
- said A ⁇ samples of step 1(a) and step 1(c) are biological samples.
- said biological samples are CSF.
- 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 A ⁇ , said method comprising:
- the amount of Fe 2+ present is determined by using a spectrophotometric method analogous to that used for the determination of Cu + , above.
- the complexing agent is batho- phenanthrolinedisulfonic (BP) anion.
- the concentration of Fe 2+ -BP produced by A ⁇ 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.
- said method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader.
- two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of Fe 2+ by A ⁇ .
- said A ⁇ samples of step 1 (a) and step 1(c) are biological samples.
- the biological sample is CSF.
- 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 O 2 by A ⁇ , said method comprising: (a) adding Cu 2* or Fe 3+ to a first A ⁇ sample; (b) allowing said first sample to incubate for an amount of time sufficient to allow said first sample to generate H 2 O 2 ;
- step (a) adding catalase to a first aliquot of said first sample obtained in step (a) above in an amount sufficient to break down all of the H 2 O 2 generated by said sample;
- step (iii) said second sample obtained in step (b) above; (c) 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 ;
- step (d) adding DTNB to said samples obtained in step (c);
- step (e) incubating said samples obtained in step (d) for an amount of time sufficient to generate TMB; (f) measuring the absorbancy at about 407 to about 417 nm of said samples obtained in step (e);
- step (g) calculating the concentration of H 2 O 2 in said first and said second sample using the absorbancies obtained in step (f).
- the absorbancy of TMB is measured at about 412 nm.
- said method is performed in a microtiter plate, and the absorbancy measurement is performed by a plate reader.
- two or more different test candidate agents are simultaneously evaluated for an ability to alter the production of H 2 O 2 by A ⁇ .
- 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 O 2 by A ⁇ , said method comprising:
- the determination of the amount of O 2 present in said samples is accomplished by measuring the absorbancy of the sample at about 250 nm.
- 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 O 2 and
- a ⁇ to produce O 2 without interfering with the SOD-like activity of A ⁇ , said method comprising:
- 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 A ⁇ .
- AD Alzheimer's disease
- 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 A ⁇ , said method comprising:
- the neurotoxicity of A ⁇ is determined by using an MTT assay.
- the neurotoxicity of A ⁇ is determined by using an LDH release assay.
- the neurotoxicity of A ⁇ is determined by using a Live/Dead assay.
- said cells utilized in the assays are rat cancer cells. Even more preferrably said cells are rat primary frontal neuronal cells.
- kits for determining whether an agent is capable of altering the production of Cu + by A ⁇ which comprises a carrier means being compartmentalized to receive in close confinement therein one or more container means wherein (a) the first container means contains a peptide comprising A ⁇ peptide;
- a second container means contains a Cu 2+ salt
- a third container means contains BC anion.
- said A ⁇ peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 ⁇ M.
- the invention in another aspect relates to a kit for determining whether an agent is capable of altering the production of Fe 2+ by A ⁇ 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 A ⁇ peptide
- a second container means contains an Fe 3+ salt
- a third container means contains BP anion.
- said A ⁇ peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 ⁇ M..
- the invention in another aspect, relates to a kit for determining whether an agent is capable of altering the production of H 2 O 2 by A ⁇ 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 A ⁇ peptide
- a second container means contains a Cu 2+ salt
- a third container means contains TCEP; and (d) a fourth container means contains DTNB.
- said A ⁇ peptide is present as a solution in an aqueous buffer or a physiological solution, at a concentration above about 10 ⁇ M.
- 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 inhibiting redox-reactive metal-mediated polymerization of A ⁇ , said method comprising:
- step (f) 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 subj ect, said method comprising administering to said subj ect 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 A ⁇ .
- the free radicals are reactive oxygen species such as O " 2 or OH-.
- the free radicals include forms of A ⁇ .
- 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 A ⁇ deposits in the brain for a time and under conditions to effect said treatment.
- the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of (a) a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and (b) 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 A ⁇ - mediated 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.
- 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.
- 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, DTP A, 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 A ⁇ -mediated production of radical oxygen species.
- the metal chelator is EGTA.
- the metal chelator is TPEN.
- the salt of a metal chelator is a magnesium salt.
- 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.
- 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 A ⁇ -mediated production of radical oxygen species.
- the chelator specific for copper is specific for the reduced form of copper.
- the chelator is bathocuproine or a hydrophobic derivative thereof.
- 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 A ⁇ -mediated production of radical oxygen species.
- an alkalinizing agent is magnesium citrate.
- the alkalinizing agent is calcium citrate.
- Still another aspect of the present invention contemplates a method of treating AD in a subj ect comprising administering to said subj ect an agent capable of preventing formation of A ⁇ amyloid, promoting, inducing or otherwise facilitating resolubilization of A ⁇ deposits in the brain, or both, for a time and under conditions to effect said treatment.
- the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject an effective amount of (a) a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or hydrophobic derivatives thereof; and (b) 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 A ⁇ amyloid, promotes, induces or otherwise facilitates resolubilization of A ⁇ deposits, or both.
- 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.
- the invention relates to a method of treating amyloidosis in a subject, said method comprising administering to said subject a combination of (a) a metal chelator selected from the following group: bathocuproine, bathophenanthroline, DTP A, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and (b) 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 A ⁇ amyloid, promotes, induces or otherwise facilitates resolubilization of A ⁇ deposits, or both.
- a metal chelator selected from the following group: bathocuproine, bathophenanthroline, DTP A, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof
- a supplement selected from the group consisting of: ammonium salt, calcium salt,
- 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 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.
- the invention in another aspect, 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, DTP A, 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 prevents formation of A ⁇ amyloid, promotes, induces or otherwise facilitates resolubilization of A ⁇ deposits, or both.
- a salt of a metal chelator wherein said chelator is selected from the group consisting of: bathocuproine, bathophenanthroline, DTP A, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein said salt
- the metal chelator is EGTA. In another preferred embodiment, the metal chelator is TPEN. In yet 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.
- the invention in another aspect, 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 A ⁇ deposits, or both.
- the chelator specific for copper is specific for the reduced form of copper.
- the chelator is bathocuproine or a hydrophobic derivative thereof.
- 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 A ⁇ amyloid, promotes, induces or otherwise facilitates resolubilization of A ⁇ deposits, or both.
- an alkalinizing agent is magnesium citrate.
- the alkalinizing agent is calcium citrate.
- Still another aspect contemplates pharmaceutical compositions for the prevention, reduction or inhibition of ROS production by A ⁇ deposits, or the prevention of formation of A ⁇ amyloid, promoting, inducing or otherwise facilitating the resolubilization of A ⁇ deposits, or both, in the brain.
- the invention relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, A ⁇ -mediated ROS formation, or both, comprising: (a) a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTP A, EDTA, EGTA, penacillamine,
- TETA, and TPEN or hydrophobic derivatives thereof; and (b) 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.
- the metal chelator is EGTA.
- the metal chelator is TPEN.
- the supplement is a magnesium salt.
- the invention relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, A ⁇ -mediated ROS formation, or both, comprising a salt of a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTP A, 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.
- the metal chelator is EGTA.
- the metal chelator is TPEN.
- the salt of a metal chelator is a magnesium salt.
- the invention relates to pharmaceutical composition for treatment of conditions caused by amyloidosis, A ⁇ -mediated ROS formation, or both, comprising a chelator specific for copper, with one or more pharmaceutically acceptable carriers or diluents.
- the chelator is specific for the reduced form of copper.
- the chelator specific for the reduced form of copper is bathocuproine.
- the invention in another aspect, relates to a pharmaceutical composition for treatment of conditions caused by amyloidosis, A ⁇ -mediated ROS formation, or both, comprising an alkalinizing agent, with one or more pharmaceutically acceptable carriers or diluents.
- the alkalinizing agent is magnesium citrate.
- the alkalinizing agent is calcium citrate.
- the invention relates to a composition of matter comprising: (a) a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and (b) a compound selected from the group consisting of: rifampicin, disulfiram, and indomethacin.
- the invention relates to a composition of matter comprising: (a) a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and (b) a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt.
- a metal chelator selected from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof
- a supplement selected from the group consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt.
- the metal chelator is EGTA.
- the metal chelator is TPEN.
- the supplement is a magnesium salt.
- 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:
- step (b) preparing a dilution curve from data obtained in step (a); (c) selecting chelators which solubilize less A ⁇ aggregates at higher concentrations than at lower or intermiate concentrations;
- step(d) contacting A ⁇ aggregates with chelators selected in step(c), in the presence of an ammonium, calcium, magnesium or sodium salt; and (e) 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.
- Figure 1 is a graph showing the proportion of soluble A ⁇ M0 remaining following centrifugation of reaction mixtures.
- Figures 2A-2C Figure 2A is a graph showing the proportion of soluble A ⁇ ,. 40 remaining in the supernatant after incubation with various metal ions.
- Figure 2B is a graph showing a turbidometric analysis of pH effect on metal ion- induced A ⁇ M0 aggregation.
- Figure 2C is a graph showing the proportion of soluble A ⁇ ,. 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 A ⁇ ,_ 40 binding to Cu + .
- Figures 4A-4C Figure 4 A is a graph showing the proportion of soluble
- Figure 4B is a graph showing the proportion of soluble A ⁇ j.40 remaining in the supernatant following incubation at various pHs with different Cu 2+ concentrations.
- Figure 4C is a graph showing the relative aggregation of nM concentrations of A ⁇ M0 at pH 7.4 and 6.6 with different Cu 2* concentrations.
- Figures 5A and 5B Figure 5 A is a graph showing a turbidometric analysis of Cu 2 -induced A ⁇ ,. 40 aggregation at pH 7.4 reversed by successive cycles of chelator.
- Figure 5B is a graph showing a turbidometric analysis of the reversibility of Cu + -induced A ⁇ 0 aggregation as the pH cycles between 7.4 and 6.6.
- Figure 6 shows the amino acid sequence of APP 669 . 7]6 near A ⁇ M2 .
- Rat A ⁇ 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 A ⁇ deposit formation.
- Figure 8 is a western blot showing the extraction of A ⁇ from post-mortem brain tissue.
- Figure 9 is a western blot showing A ⁇ SDS-resistant polymerization by copper.
- Figure 10 is a graph showing Cu + generation by A ⁇ .
- Figure 11 is a graph showing H 2 O 2 production by A ⁇ .
- Figure 12 is a graphical representation showing a model for the generation of reduced metal ions, O 2 , H 2 O 2 , and OH* by A ⁇ peptides. Note that
- FIGS 13A and 13B are graphical representations showing Fe 3+ or Cu 2+ reduction by A ⁇ peptides.
- Figure 13A illustrates the reducing capacity of A ⁇ species (10 ⁇ M), compared to Vitamin C and insulin (Sigma) (all 10 ⁇ M) towards
- FIG. 13B shows the effect of oxygen tension and chelation upon A ⁇ M2 metal reduction.
- a ⁇ ,_ 42 was incubated as in Figure 13 A under various buffer gas conditions.
- “Ambient” no efforts were made to adjust the gas tension in the bench preparations of the buffer vehicle,
- "O 2 " 100% O 2 was continuously bubbled through the PBS vehicle for
- Figures 14A-14E are graphical representations showing production of H 2 O 2 from the incubation of A ⁇ in the presence of substoichiometric amounts of Fe 3+ or Cu 2* .
- Figure 14A shows H 2 O 2 produced by A ⁇ M2 (in PBS, pH 7.4, under ambient gas conditions, 1 hour, 37 °C) following co-incubation with various concentrations of catalase in the presence of 1 ⁇ M Fe 3+ .
- Figure 14B shows a comparison of H 2 O 2 generation by variant A ⁇ species: A ⁇ ,_ 42 , A ⁇ M0 , rat A ⁇ , .40 , A ⁇ 40 ., and A ⁇ , .28 (vehicle conditions as in Figure 14A).
- Figure 14C shows the effect of metal chelators (200 ⁇ M) on H 2 O 2 production from A ⁇ ,_ 42 when incubated in the presence of Fe 3* or Cu 2+ (1 ⁇ M) (vehicle conditions as in
- FIG. 14A shows H 2 O 2 produced by A ⁇ , _ 42 , A ⁇ 0 , and Vitamin C in the presence of Fe 3+ (1 ⁇ M) (in PBS, pH 7.4 buffer, 1 fir, 37 °C) under various dissolved gas conditions (described in
- Figure 13B ambient air, O 2 enrichment, and anaerobic (Ar) conditions, as indicated.
- Figure 15A and 15B are graphical representations showing superoxide anion detection.
- Figure 15 A shows the spectrophotometric absorbance at 250 nm (after subtracting buffer blanks) for A ⁇ , _ 42 (10 ⁇ M, in PBS, pH 7.4, with 1 ⁇ M Fe 3+ , incubated 1 hr, 37 °C) under ambient air (+ 100 U/mL superoxide dismutase, SOD), O 2 enrichment, and anaerobic (Ar) buffer gas conditions (described in Figure 13B).
- Figure 15B shows the spectrophotometric absorbance at 250 nm (after subtracting buffer blanks) for variant A ⁇ peptides: A ⁇ ,_ 42 , A ⁇ ,. 40 , rat A ⁇ ,.
- Figure 16A and 16B are graphical representations showing production of the hydroxyl radical (OH») from the incubation of A ⁇ 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 ⁇ M) and variant A ⁇ species (10 ⁇ M): A ⁇ ,. 42 , A ⁇ ,. 40 , rat A ⁇ , .40 , A ⁇ 40.
- Figure 16B illustrates the effect of OH'-specific scavengers upon OH* generation by Vitamin C and A ⁇ , .42 .
- 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 A ⁇ 0 mixed with 5% A ⁇ , .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 A ⁇ 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 A ⁇ from an AD brain (AD) to that of sedimentable deposits from healthy brain tissue (young control - C).
- TBS buffer was used rather than PBS.
- Figure 21 shows an indicative blot from AD brain extract. The blot shows that chelation treatment results in disproportionate solubilization of A ⁇ 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 A ⁇ solubilization.
- Figures 24A and 24B - Figure 24A shows the effect of metals upon the solubility of brain-derived A ⁇ : copper and zinc can inhibit the solubilization of A ⁇ .
- Figure 24B shows that A ⁇ solubility in metal-depleted tissue is restored by the addition of magnesium.
- Figures 25A and 25B - Figure 25A shows that patterns of chelator- promoted solubilization of A ⁇ differ in AD and aged-matched, non-AD tissue.
- Figure 25B shows soluble A ⁇ 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 A ⁇ ,. 40 and A ⁇ , .42 from AD and non-AD tissue.
- Representative AD left panels
- aged- matched control specimens right panels
- Identical gels were run and Western blots were probed with mAbs WO2 (raised against residues 5-16, recognizes A ⁇ ,. 40 and A ⁇ ,_ 42 )
- G210 raised against residues 35-40, recognizes A ⁇ , .40
- G211 raised against residues 35-42, recognizes A ⁇ ,. 42
- Figure 27A and 27B - Figure 27A shows SDS-resistant polymerization of human A ⁇ ,_ 40 versus human A ⁇ ,_ 42 with Cu 2+ or Fe 3+ .
- Figure 27B shows SDS- resistant polymerization of rat A ⁇ ,_ 40 with Cu 2+ or Fe 3+ .
- Figures 28A - 28C - Figure 28A shows H 2 O 2 /Cu induced SDS-resistant polymerization of A ⁇ ,_ 42 (2.5 ⁇ M).
- Figure 28B shows H 2 O 2 .
- Figure 28C shows that BC attenuates SDS-resistant polymerization of A ⁇ ,_ 42 (2.5 ⁇ M).
- Figures 29A and 29B show that H 2 O 2 generation is required for SDS- resistant polymerization of human A ⁇ ,_ 42 .
- Solution concentrations of metal ion and H 2 O 2 were 30 ⁇ M and 100 ⁇ M, respectively.
- Figure 29A shows that TCEP
- Figures 30A-30E show dissolution of SDS-resistant A ⁇ polymers.
- Figure 30A shows that chaotrophic agents are unable to disrupt polymerization.
- Figure 30B shows that metal ion chelators disrupt SDS-resistant A ⁇ , _ 40 polymers.
- TETA tetraethylenediamine
- EDTA ethylenediaminetetra acetic acid
- DTPA diethylenetriaminopenta acetic acid
- CDTA tn .-l,2-diaminocyclohexanetetra acetic acid
- FIG. 30E shows that metal ion chelators disrupt SDS-resistant A ⁇ polymers extracted from AD brains. Aliquots of SDS-resistant A ⁇ polymers extracted from AD brains were incubated with no chelator, TETA (1 mM or 5 mM) or BC (1 mM or 5 mM) for
- a ⁇ peptide is also known in the art as A ⁇ , ⁇ protein, ⁇ -A4 and A4.
- the A ⁇ peptide may be comprised of peptides A ⁇ , .39 A ⁇ M0 A ⁇ i . 4 , , A ⁇ , _ 42 , and A ⁇ , _ 43 .
- the most preferred embodiment of the invention makes use of A ⁇ 0 .
- any of the A ⁇ peptides may be employed according to the present invention.
- the sequence of A ⁇ peptide is found in Hilbich, C, et al, J. Mol. Biol. 228:460-413 (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.
- a ⁇ Amyloid is an aggregated A ⁇ 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.
- biological fluid includes whole or any fraction of such fluids derived by purification by any means, e.g., by ultrafiltration or chromatography.
- Copper(II), unless otherwise indicated, means salts of Cu 2+ , i.e., Cu 2* in any form, soluble or insoluble.
- Copper(I) unless otherwise indicated, means salts of Cu + , i.e., 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, 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 means salts of zinc, i.e., Zn 2* in any form, soluble or insoluble.
- the aim of the present invention is to clarify both the factors which contribute to the neurotoxicity of A ⁇ polymers and the mechanism which underlies their formation. These findings can then be used to (i) identify agents that can be used to decrease the neurotoxicity of A ⁇ , as well as the formation of
- a ⁇ 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 A ⁇ peptides directly produce oxidative stress through the generation of abundant reactive oxygen species (ROS), which include hydroxyl radical (OH») and hydrogen peroxide (H 2 O 2 ).
- ROS reactive oxygen species
- 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 A ⁇ .
- a ⁇ is highly efficient at reducing Cu 2+ and Fe 3 ".
- the sequence of ROS generation by A ⁇ follows the pathway of superoxide-dismutation, which leads to hydrogen peroxide production in a Cu/Fe- dependent manner.
- the hydroxyl radical (OH « ) 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 A ⁇ 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.
- amyloid deposits are amyloid deposits.
- H 2 O 2 hydrogen peroxide
- Cu 2+ or Fe 3+ copper
- H + is required for H 2 O 2 production, an acidotic environment will increase the reaction.
- H 2 O 2 is relatively stable, and freely permeable across cell membranes. Normally, it will be broken down by intercellular catalase or glutathione peroxidase.
- OH* engenders a non-specific stress and inflammatory response in local tissue.
- the neurochemicals that are released from microglia and possibly neurons in the response are Zn 2+ , Cu 2+ and soluble A ⁇ . Familial AD increases the likelihood that A ⁇ ,. 42 will be released at this point. Local acidosis is also part of the stress/inflammatory response. These factors combine to make A ⁇ precipitate and accumulate, presumably so that it may function in situ as an SOD, since these factors induce reversible aggregation. Hence, more soluble A ⁇ species decorate the perimeter of the accumulating plaque deposits.
- a ⁇ ,. 42 If A ⁇ 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 amyloid.
- a ⁇ ,. 42 If A ⁇ ,. 42 accumulates, it has the property of recruiting O 2 as a substrate for the abundant production of O 2 by a process that is still not understood. Since O 2 is abundant in the brain, A ⁇ ,_ 42 is responsible for setting off a vicious cycle in which the accumulation of covalently linked A ⁇ is a product of the unusual ability of A ⁇ to reduce O 2 , and feed an abundant substrate (O 2 ) to itself for dismutation, leading to OH* formation.
- one object of the present invention is to provide a method for the identification of agents to be used in the treatment of AD.
- agents to be used in the treatment of AD include: (a) agents that reduce the amount of Cu * or Fe 2+ produced by A ⁇ ;
- 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 A ⁇ 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 A ⁇ .
- 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 A ⁇ , the method comprising:
- Cu + is lower in the second sample than in the first sample, this will indicate that the agent has decreased Cu * production.
- the amount of Cu + present in said first and said second sample is determined by (a) 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;
- step (c) calculating the concentration of CA in said first and said second sample using the absorbancy obtained in step (b).
- the complexing agent is bathocuproinedisulfonic (BC) anion.
- concentration of Cu * produced by A ⁇ may then be calculated on the basis of the absorbance of the sample at about
- 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.
- 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 Fe 2+ by A ⁇ , said method comprising: (a) adding Fe 3+ to a first A ⁇ sample;
- the amount of Fe 2+ present is determined by using a spectrophotometric method analogous to that used for the determination of Cu + , above.
- the complexing agent is batho- phenanthrolinedisulfonic (BP) anion.
- the concentration of Fe 2+ -BP produced by A ⁇ 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.
- 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.
- 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 O 2 by A ⁇ , said method comprising: (a) adding Cu 2+ or Fe 3+ to a first A ⁇ sample;
- this method may be used to detect agents which decrease the amount of H 2 O 2 produced (in which case the amount of H 2 O 2 will be lower in the second sample than in the first sample), or to increase the amount of H 2 O 2 produced (in which case the amount of H 2 O 2 will be lower in the first sample than in the second sample).
- the determination of the amount of H 2 O 2 present in said first and said second sample is determined by
- step (a) adding catalase to a first aliquot of said first sample obtained in step (a) of claim 1 in an amount sufficient to break down all of the
- step (ii) a second aliquot of said first sample obtained in step (a) of claim 1 ; and (iii) said second sample obtained in step (b) of claim 1 ; (c) 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 ;
- step (d) adding DTNB to said samples obtained in step (c);
- step (e) incubating said samples obtained in step (d) for an amount of time sufficient to generate TMB;
- step (f) measuring the absorbancy at about 407 to about 417 nm of said samples obtained in step (e);
- step (g) calculating the concentration of H 2 O 2 in said first and said second sample using the absorbancies obtained in step (f).
- the absorbancy of TMB is measured at about
- 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.
- 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 2 by A ⁇ , said method comprising:
- the A ⁇ used is A ⁇ ,. 42 .
- the amount of O 2 produced by A ⁇ may be measured by any method known to those of ordinary skill in the art.
- the determination of the amount of O 2 present in said samples is accomplished by measuring the absorbancy of the sample at about 250 nm.
- 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 O 2 and A ⁇ to produce O 2 , without interfering with the SOD-like activity of A ⁇ , said method comprising: (a) identifying an agent capable of decreasing the production of O 2 by A ⁇ ; and
- the determination of the ability of said agent to alter the SOD-like activity of A ⁇ is made by determining whether A ⁇ is capable of catalytically producing Cu + , Fe 2+ or H 2 O 2 .
- Methods, besides those which are disclosed elsewhere in this application, for determining if A ⁇ is capable of catalytically producing Cu + , Fe 2+ or H 2 O 2 are well known to those of ordinary skill in the art.
- the catalytic production of H 2 O 2 may be determined by using laser flash photolysis or pulse radiolysis (Peters, G. & Rodgers, M.A. J, Biochim. Biophys. Ada 637:43-52 (1981).
- 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, A ⁇ -mediated toxicity in cell culture.
- assays include the MTT assay, which measures the reduction of 3-(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. 2(9:83-90 (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.
- Live-Dead assay may also be used to determine if a candidate compound which alters Cu + , Fe 2+ , H 2 O 2 , OH*, and O 2 production, or alters copper-induced, pH dependent aggregation and crosslinking of A ⁇ , is also capable of reducing the neurotoxicity of A ⁇ .
- 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 A ⁇ , said method comprising: (a) adding A ⁇ to a first cell culture;
- 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 etal. , Cell 77: ⁇ 7-827 (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 above- described methods will be broad-ranging but can be classified as follows:
- a ⁇ will be broad-ranging but can be classified as follows:
- BBB blood-brain barrier
- 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,NX1,2-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-Inositol hexakis(dihydrogen phosphate), entry 7269 in the Merck Index 10th edition] and phytate salts.
- Preferred candidate agents within this class include bathocuproine and bathophenanthroline .
- the A ⁇ used may be any form of A ⁇ .
- the A ⁇ used is selected from the group consisting of
- a ⁇ ,_ 39 A ⁇ , .40 A ⁇ M] , A ⁇ M2 , and A ⁇ ,_ 43 .
- the A ⁇ used is A ⁇ M0 or A ⁇ , legal 42 .
- the most preferred embodiment of the invention makes use of A ⁇ ,. 40 .
- the sequence of A ⁇ peptide is found in Hilbich, C, et al, J. Mol. Biol. 228:460-413 (1992).
- the pH of the various reaction mixtures are preferably close to neutral
- the pH 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.
- 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 above- mentioned free radical formation of a given buffer.
- Cu 2+ must be present in the reaction mixture for A ⁇ to produce Cu + .
- Any salt of Cu 2+ may be used to satisfy this requirement, including, but not limited to, CuCl 2 Cu(NO 3 ) 2 , etc.
- Concentrations of copper from at least about 1 ⁇ M may be used; most preferable, a copper concentration of about 10 ⁇ M is to be included in the reaction mixture.
- a redox active metal such as Cu + or Fe 3+ must be present in the reaction mixture for A ⁇ to catalytically produce H 2 O 2 .
- Any salt of Cu 2+ may be used to satisfy this requirement, including, but not limited to, ' CuCl 2 Cu(NO 3 ) 2 , etc.
- salt of Fe 3+ may be used in accordance with the invention, such as FeCl 3 .
- Concentrations of copper or iron from at least about 1 ⁇ M may be used; most preferably, a copper or iron concentration of about 10 ⁇ M 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 30 °C to about 40 °C.
- the most preferred temperature for the practice of the present invention is about 37 °C, i.e., human body temperature.
- the production of Cu + and H 2 O 2 by A ⁇ peptide occurs at near- instantaneous rate.
- the measurement of the concentration of Cu + or H 2 O 2 produced may be performed by the present methods substantially immediately after the addition of Cu 2+ to the A ⁇ peptide.
- 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 A ⁇ , so that where the methods of the invention are to be carried out utilizing a biological fluid such as CSF, no further A ⁇ peptide will be added to the sample.
- the biological fluid may be used directly or diluted from about 1 : 1 ,000 to about 1 :5 fold.
- the amount of H 2 O 2 , Cu + or Fe 2+ produced by a sample may be measured by any standard assay for H 2 O 2 , Cu + or Fe 2+ .
- the PeroXOquant Quantitative Peroxide Assay (Pierce, Rockford, IL) may be used to determine the amount of H 2 O 2 produced.
- Fe 2+ may be determined using the spectrophotometric method of Linert et al , Biochim. Biophys. Ada 1316: 160- 168 ( 1996). Other such methods will be readily apparent to those of ordinary skill in the art.
- the H 2 O 2 or Cu + produced by the sample is complexed with a complexing agent having an optimal visible absorption wavelength.
- the amount of H 2 O 2 or Cu + produced by a sample is then detected using optical spectrophotometry (see Example 2).
- the complexing agent to be used for the determination of the amount of Cu + produced is bathocuproinedisulfonic anion (BC), (see Example 2); the complex Cu + -BC has an optimal visible absorption wavelength of about 483 nm.
- BC may be added to the reaction immediately following the addition of Cu 2+ and Zn 2+ to the A ⁇ samples.
- the concentration of BC to be achieved in a sample is between about 10 ⁇ M to about 400 ⁇ M, more preferably about 75 ⁇ M to about 300 ⁇ M, and still more preferably about 150 ⁇ M to about 275 ⁇ M. In the most preferred embodiment, the concentration of BC to be achieved in a sample is about 200 ⁇ M.
- one of ordinary skill in the art can easily optimize the concentration of BC to be added with no more than routine experimentation.
- the complexing agent to be used for the determination of the amount of Fe 2+ produced is bathophenanthrolinedisulfonic (BP) anion, (see Example 2); the complex Fe 2+ -BP has an optimal visible absorption wavelength of about 535 nm.
- BP bathophenanthrolinedisulfonic
- the concentration of BP to be achieved in a sample is between about 10 ⁇ M to about 400 ⁇ M, more preferably about 75 ⁇ M to about 300 ⁇ M, and still more preferably about 150 ⁇ M to about 275 ⁇ M. In the most preferred embodiment, the concentration of BP to be achieved in a sample is about 200 ⁇ M.
- concentration of BP to be achieved in a sample is about 200 ⁇ M.
- one of ordinary skill in the art can easily optimize the concentration of BP to be added with no more than routine experimentation.
- kits 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.
- 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.
- solutions to be used for the determination of H 2 O 2 as described in Example 2 include TCEP and DTNB, as well as catalase (lOU/ml).
- Standard solutions of A ⁇ peptide preferably have concentrations above about 10 ⁇ M, 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.
- 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 O 2 and biometal-dependent pathway of free radical generation by A ⁇ peptides.
- the radicals of this aspect of the present invention may comprise reactive oxygen species (ROS) such as but not limited to O 2 and OH as well as radicalized A ⁇ peptides. It is proposed, according to the present invention, that by interfering in the radical generating pathway, the neurotoxicity of the A ⁇ peptides is reduced.
- ROS reactive oxygen species
- 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.
- AD Alzheimer's disease
- the preferred agents according to this aspect are metal chelators, metal complexing compounds, antioxidants and compounds capable of reducing radical formation of A ⁇ peptides or mediated by A ⁇ peptides.
- Particularly preferred metal chelators and metal complexors are capable of interacting with metals (M) having either a reduced charge state (M n+1 ) or an oxidized state of (M n* ')+.
- M is Fe and/or Cu.
- Reduced Fe/Cu reacts with molecular oxygen to generate the superoxide anion.
- the O 2 generated undergoes dismutation to H 2 O 2 either catalyzed by SOD or spontaneously.
- the Haber- Weiss reaction can form OH in a reaction catalyzed by
- 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.
- 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.
- the free radicals are reactive oxygen species such as O " 2 or
- the free radicals include forms of A ⁇ .
- the free radicals include forms of A ⁇ .
- the free radicals include forms of A ⁇ .
- Cu 2+ reaction with A ⁇ generates Cu + , A ⁇ *, O 2 , H 2 O 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 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 A ⁇ 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 (Cu + ) and iron (Fe 2+ ).
- Another mechanism elucidated in accordance with the present invention concerns the formation of aggregates of A ⁇ , as in conditions involving amyloidosis.
- 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 non- fibrillary 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 A ⁇ aggregates.
- 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.
- a ⁇ deposits which are composed of fibrillary and non-fibrillary aggregates may be resolubilized by the 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 10%.
- 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.
- 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.
- 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.
- 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.
- 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 metal chelator or a combination of a metal chelator and another active agent, e.g. an antioxidant or an alkalinizing agent
- 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.
- 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.
- sterile aqueous solutions where water soluble
- sterile powders for the extemporaneous preparation of sterile injectable solutions may be used.
- 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 example, filtration or irradiation.
- 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 ⁇ g and 500 mg of active compound.
- the active agents 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.
- the active compound 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 ⁇ g and 2000 mg of
- 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.
- 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
- a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen
- 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.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- 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.
- Dosage unit form 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 (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) 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 ⁇ g to 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.
- compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention.
- animals are mammals, e.g., humans, although the invention is not intended to be so limited.
- Human A ⁇ 0 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 A ⁇ peptide solutions were dissolved in trifluoroethanol (30 % in Milli-Q water (Millipore Corporation, Milford, MA)) or 20 mM HEPES (pH 8.5) at a concentration of 0.5-1.0 g/ml, centrifuged for 20 min. at 10,000g and the supernatant (stock A ⁇ M0 ) used for subsequent aggregation assays on the day of the experiment.
- trifluoroethanol 30 % in Milli-Q water (Millipore Corporation, Milford, MA)
- 20 mM HEPES pH 8.5
- the concentration of stock A ⁇ M0 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 lO ⁇ l of stock A ⁇ 0 (or bovine serum albumin standard) to 140 ⁇ l of distilled water, and then adding an equal volume of supernatant (150 ⁇ l) to a 96-well plate and measuring the absorbance at 562 nm.
- the concentration of A ⁇ ,. 40 was determined from the BSA standard curve. Prior to use all buffers and stock solutions of metal ions were filtered though a 0.22 ⁇ m filter (Gelan Sciences, Ann Arbor, MI) to remove any paniculate matter. All metal ions were the chloride salt, except lead nitrate.
- a ⁇ M0 stock was diluted to 2.5 ⁇ M in 150 mM NaCl and 20 mM glycine
- Turbidity measurements were performed as described by Huang, X., et al, J Biol. Chem. 272:26464-26470 (1997), except A ⁇ M0 stock was brought to 10 ⁇ M
- Immunoreactivity was quantified by transmitance analysis of ECL film from the immunoblots.
- a ⁇ M0 (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
- 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.
- SPSB/TMB stable peroxidase substrate buffer/3, 3', 5,5'- Tetramethyl benzidine
- 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 A ⁇ content by western blotting using monoclonal antibody (mAb)
- N,N,N',N'-tetrakis [2-pyridyl-methyl] ethylenediamine (TPEN) (5 ⁇ M) allows the visualization of a population of pelletable A ⁇ that had not previously been recognized in unaffected brain samples ( Figure 8).
- 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 A ⁇ that deposits in AD-affected cortex can be solubilized in water (Roher, A.E, et a , J. Biol. Chem. 277:20631 (1996)).
- metal chelators increase the amount of A ⁇ extracted by Roher's technique (in neutral saline buffer), and that the extraction of A ⁇ is increased as the chelator employed has a higher affinity for zinc or copper.
- TPEN is highly efficient in extracting A ⁇ , 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 (5 ⁇ M).
- FIG. 1 shows the proportion of soluble A ⁇ M0 remaining following centrifugation of reaction mixtures.
- Figure 5B shows turbidometric analysis of the reversibility of Cu 2+ -induced A ⁇ ,. 40 as the pH cycles between 7.4 and 6.6.
- a ⁇ ,. 40 oscillates between an aggregated and soluble state dependent upon the [H + ].
- a ⁇ M0 aggregation by Co 2+ like Zn 2+ , was pH insensitive and per mole induced a similar level of aggregation.
- 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).
- Figures 5A and 5B are chelator/pH reversible, their conformation may be the higher energy a -helical conformation.
- the binding of Cu 2 f and Zn 2+ to A ⁇ may be of particular importance during inflammatory processes, since local sites of inflammation can become acidic (Trehauf, P.S. & McCarty, D.J. ,Arthr. Rheum. J4-A15-4S4 (1971); Menkin, V.,Am. J. Pathol 70:193-210 (1934)) and both Zn 2+ and Cu 2+ are rapidly mobilized in response to inflammation (Lindeman, R.D., et al, J. Lab. Clin. Med. 57:194-204 (1973); Terhune, M.W.
- 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.
- 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 A ⁇ , .40 to aggregate.
- increased concentrations of Zn 2+ and Cu 2+ , and decreased pH are common features of all forms of cellular insult, the initiation of A ⁇ ,. 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 ⁇ ,. 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.
- amyloid systemically is usually associated with an inflammatory response (Pepys, M.B. & Baltz, M.L., Adv. Immunol. 34:141-212
- 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.
- pH/metal ion mediated aggregation may form the basis for the amorphous A ⁇ 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.
- This method is modified from a protocol assaying serum copper and iron
- the concentrations ( ⁇ M) of Cu + or Fe 2+ can be deduced based on Beer-Lambert's Law, using proper buffers as controls.
- This method is modified from a H 2 O 2 assay reported recently (Han, J.C, et al, Anal. Biochem. 234:101 (1996)).
- the advantages of this modified H 2 O 2 assay on 96-well plate include high throughput, excellent sensitivity (-1 ⁇ M), and the elimination of the need for a standard curve of H 2 O 2 , which is problematic due to the labile chemical property of H 2 O 2 .
- a ⁇ peptides were co-incubated with a H 2 O 2 -trapping reagent (Tris(2- carboxyethyl)-phosphine hydrochloride, TCEP, 100 ⁇ M) in PBS (pH 7.4 or 7.0) at37°C for30mins. 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 [18].
- the assay was adapted to a 96-well format using a standard absorbance range (see Figure 11).
- OOCCH 2 CH 2 — P + H 2 0 2 ⁇ OOCCH 2 CH 2 — P 0 + H,0
- TCEP'HCl was synthesized by hydrolyzing tris (2-cyno-ethyl) phosphine (purchased from Johnson-Mathey (Waydhill, MA)), in refluxing aqueous HCl (Burns, J.A.. et al, J. Org. Chem. 56:2648 (1991)) as shown below.
- TMB 2-nitro-5-thiobenzoic acid
- the concentration of H 2 O 2 can then be deduced from the difference in absorbance between the sample and the control (sample plus 1000 U/ ⁇ l catalase)
- Cu + levels (n 3, ⁇ SD) were assayed against a standard curve.
- Cerebral zinc metabolism is deregulated in AD, and therefore levels of interstitial zinc may play an important role in adj usting the Cu + and H 2 O 2 production generated by A ⁇ .
- the rat homologue of A ⁇ ,. 40 does not manifest the redox reactivity of the human equivalent. Insulin, a histidine- containing peptide that can bind copper and zinc, exhibits no Cu 2+ reduction.
- a ⁇ ,. 42 (10 ⁇ M) was incubated for 1 hr at 37 °C, pH 7.4 in ambient air (first bar), continuous argon purging (Ar), continuous oxygen enrichment (O 2 ) at pH 7.0 (7.0), or in the presence of the iron chelator desferioxamine (220 ⁇ M; DFO).
- Variant A ⁇ species (10 ⁇ M) were tested: A ⁇ , .40 (A ⁇ M0 ), rat A ⁇ M0 (rA ⁇ M0 ), and scrambled A ⁇ ,. 40 (sA ⁇ M0 ) were incubated for 1 hr at 37°C, pH 7.4 in ambient air.
- a ⁇ exhibits metal-dependent and independent redox activity
- a ⁇ produces both H 2 O 2 and reduced metals whilst also binding zinc. Structurally, this is difficult to envisage for a small peptide, but we have recently shown that A ⁇ is dimeric in physiological buffers. Since H 2 O 2 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.
- H 2 O 2 production by A ⁇ explains the mechanism by which H 2 O 2 has been described to mediate neurotoxicity (Behl, C. et al, Cell 77:827 (1994)), previously thought to be the product of cellular overproduction alone.
- the scrambled A ⁇ peptide (same size and residue content as Figure 6) produces appreciable H 2 O 2 but no hydroxyl radicals. This is because the scrambled A ⁇ peptide is unable to reduce metal ions.
- FIG 11 shows that the production of H 2 O 2 is oxygen dependent, and further investigation has indicated that A ⁇ can spontaneously produce the superoxide radical (O 2 ) in the absence of metal ions.
- This property of A ⁇ is particularly exaggerated in the case of A ⁇ M2 , probably explaining why this peptide is more neurotoxic and more enriched than A ⁇ 0 in amyloid.
- O 2 generation will be subject to spontaneous dismutation to generate H 2 O 2 , however, this is a relatively slow reaction, although it may account for the majority of the H 2 O 2 detected in our A ⁇ assays.
- SOD superoxide dismutase
- MTT 3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide (MTT) to a colored formazon
- 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.
- MTT 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).
- Synthetic A ⁇ peptides A ⁇ , .40 and A ⁇ M2 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 A ⁇ peptides synthesized and obtained from other sources: Glabe laboratory, University of California, Irvine, CA, Multhaup Laboratory, University of Heidelberg, U.S. Peptides, Bachem, and Sigma. Rat A ⁇ was synthesized and characterized by the Multhaup Laboratory, University of Heidelberg. A ⁇ ,. 28 was purchased from U.S. Peptides, Bachem, and Sigma. A ⁇ peptide stock solutions were prepared in chelex-100 resin (BioRad) treated water and quantified.
- the metal reduction assay was performed using a 96-well microtiter plate (Costar) based upon a modification of established protocols (Landers, J.W., etal, Amer. Clin. Path. 29:590 (1958); Landers, J.W., et al, Clinica Chimica Ada 3:329 (1958)).
- PBS phosphate buffered saline
- 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).
- the H 2 O 2 assay was performed in a UV-transparent 96-well microtiter plate (Molecular Devices, CA), according to a modification of an existing protocol (Han, J.C, et al, Anal. Biochem. 234:107 (1996); Han et al, Anal Biochem. 220: 5-10 (1994)).
- 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 2 O 2 generation from A ⁇ , which does not possess a disulfide bond.
- the spectrophotometric absorption peak for O 2 is 250 nm where its extinction coefficient is much greater than that of H 2 O 2 (Bielski et al, Philos Trans R Soc Lond B Biol Sci. 311: 473-482 (1985)).
- the production of O 2 was estimated by measuring the spectrophotometric absorption of polypeptides (10 ⁇ M, 300 ⁇ L) after incubation for one hour in PBS, pH 7.4, at 37°C, using a
- TBARS Thiobarbituric Acid-Reactive Substance
- a ⁇ peptide species (10 ⁇ M) or Vitamin C (100 ⁇ M) were incubated with Fe 3+ or Cu 2+ (1 ⁇ M) 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 (1%, 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 sample were obtained by deducting the absorbance from a control sample consisting of identical chemical components except for the Vitamin C or A ⁇ peptides.
- Antioxidant vitamin E and the spin-trap compound PB ⁇ have been shown to protect against A ⁇ -mediated neurotoxicity in vitro (Goodman, Y., & Mattson, M.P., Ex ?. Neurol. 128: ⁇ (1994); Harris, M. ⁇ ., et al, Exp. Neurol 737:193 (1995)).
- a ⁇ a 39-43 amino acid peptide, is produced (Haass, C, et al, Nature 359:322 (1992); Seubert, P., et al, Nature 359:325 (1992); Shoji, M., et al,
- a ⁇ -40 is the major soluble A ⁇ species in biological fluids (Vigo-Pelfrey, C, et al, J. Neurochem. 67:1965 (1993)) and A ⁇ ,_ 42 is a minor soluble species, but is heavily enriched in interstitial plaque amyloid (Masters, C.L., etal, Proc. Natl. Acad. Sci.
- K D 107 nM
- Vitamin C serving as a positive control, reduced Cu 2+ efficiently (Figure 13 A).
- the reduction of Cu 2+ by A ⁇ ,. 42 was as efficient, reducing all of the available Cu 2+ during the incubation period.
- a ⁇ ,. 40 reduced 60% of the available Cu 2+
- rat A ⁇ , .40 and A ⁇ ,. 2g reduced no Cu 2+ .
- Fe 3+ /Fe 2+ has lower standard reduction potential (0.11 V) than Cu 2 7Cu + (0.15 V) does under our experimental conditions (Miller, D.M., et al, Free Radical Biology & Medicine 5:95 (1990)), and, in general, Fe + was reduced with less efficiency by Vitamin C and the polypeptides that reduced Cu 2+ . Vitamin C reduced 15% of the available Fe 3+ , however A ⁇ ,.
- Rat A ⁇ ,. 40 , A ⁇ ,. 28 and insulin did not significantly facilitate the reduction of Fe 3+ .
- the buffer vehicle Prior to the addition of Vitamin C or polypeptide, the buffer vehicle was continuously bubbled for 2 hours at 20 °C with 100%) O 2 to create conditions of increased O 2 tension, or Argon to create anaerobic conditions.
- O 2 tension slightly reduced the levels of reduced metals being detected, probably due to the diversion of a fraction of the Fe 2 7Cu + being generated to Reaction (1), and, if H 2 O 2 is being produced as a product of Reaction (2), the recruitment of Fe 2 7Cu + into the Fenton reaction [Reaction (3)].
- performing the reaction under anaerobic (Argon purged) conditions also slightly reduced the levels of reduced metals being detected. This may be because some of the reduction of Fe 3 7Cu 2+ is due to reaction with O " 2 :
- the inhibitory effects of chelation upon A ⁇ -mediated reduction of metal ions indicates that A ⁇ 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 3+ and Cu 2+ to the A ⁇ 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.
- Rat A ⁇ ,. 40 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 Fe 3+ binding by rat A ⁇ ,. 40 compared to human A ⁇ ,. 40 is anticipated. These data also indicate that the rat A ⁇ substitutions in human A ⁇ '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 carboxy 1-terminal residues were also critical to the reduction properties of A ⁇ . That A ⁇ ,_ 28 did not reduce metal ions indicates that an intact Zn 2+ -binding site (Bush et al, J. Biol Chem.
- Example 2 which detected the generation of 10 ⁇ M H,O 2 by A ⁇ , .42 in the presence of 1 ⁇ M Fe 3+ under ambient O 2 conditions ( Figure 14A).
- coincubation with catalase was observed to abolish the H 2 O 2 signal in a dose dependent manner.
- the amount of H 2 O 2 produced by the various A ⁇ peptides was studied, and observed that the order of the production of H 2 O 2 by the
- a ⁇ variants was A ⁇ , relief 42 > A ⁇ , .40 » rat A ⁇ , .40 - A ⁇ ,_ 28 ( Figure 14B), paralleling the amounts of metal reduction by the same peptides ( Figure 13 A).
- H 2 O 2 formation is likely to be mediated first by O 2 -dependent O " 2 formation [Reaction (1)], followed by dismutation [Reaction (2)].
- Reaction (1) O 2 -dependent O " 2 formation [Reaction (1)]
- dismutation (2) dismutation [Reaction (2)]
- H 2 O 2 formation by A ⁇ ,_ 42 in the presence of chelators was measured ( Figure 14C).
- the amount of H 2 O 2 formed in the presence of 1 ⁇ M Cu 2+ was 25% greater than the amount formed in the presence of 1 ⁇ M FeA
- Coincubation with DFO had no effect on H 2 O 2 formation in the presence of 1 ⁇ M FeA
- TETA, and the Cu + -specific indicator BC both substantially inhibited the formation of H 2 O 2 in the presence of 1 ⁇ M Cu 2* .
- Vitamin C was used as a control measure to determine the amount H 2 O 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 of H 2 O 2 produced under higher O 2 tensions.
- the presence of either A ⁇ M2 and A ⁇ ,. 40 generated more H 2 O 2 (A ⁇ ,. 42 > A ⁇ ,. 40 ) than Vitamin C under any O 2 tension studied, and generated H 2 O 2 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 .
- the present Examples provide evidence for a model by which Fe/Cu and O 2 are mediators and substrates for the production of OH* by A ⁇ ( 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 O 2 and Fe/Cu, which may explain why this organ is predisposed to A ⁇ -mediated neurotoxicity, if this mechanism is confirmed in vivo.
- the transport of Fe, Cu and Zn in the brain is largely energy-dependent.
- the copper-transporting gene for Wilson's disease is an ATPase (Tanzi, R.E.
- OH* scavengers mannitol and DMSO were unable to inhibit the generation of OH* by A ⁇ -Fe 3 7Cu 2+ , and are similarly unable to inhibit OH* production from bleomycin-Fe 3+ .
- a ⁇ -mediated OH* provides means of treatment, e.g. therapy, by compounds that are Fe or Cu chelators.
- the clinical administration 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 Al(III), the effect, if verifiable, may not have been due to the abolition of the redox activity of A ⁇ , but may have been due to the disaggregation of Zn 2+ -mediated A ⁇ deposits (Cherny,
- a ⁇ 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
- the present findings indicate that the manipulation of the brain biometal environment with specific agents acting directly (e.g. chelators and antioxidants) or indirectly (e.g. by improving cerebral energy metabolism) holds promise as a means for therapeutic intervention in the prevention and treatment of Alzheimer ' s disease.
- specific agents acting directly e.g. chelators and antioxidants
- indirectly e.g. by improving cerebral energy metabolism
- a ⁇ is a normal component of biological fluids whose function is unknown. A ⁇ accumulates in a number of mo ⁇ hologies 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 A ⁇ peptide in order to get some clues as to what kind of pathological environment could induce the peptide to precipitate.
- a ⁇ has three principal vulnerabilities: zinc, copper and low pH.
- the precipitation of A ⁇ by copper is dramatically exaggerated under mildly acidic conditions (e.g., pH 6.9), suggesting that the cerebral lactic acidosis that complicates Alzheimer's disease could contribute to the precipitation of A ⁇ were this event to be mediated by copper.
- a consideration of the involvement of 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.
- the inventors have recently characterized zinc-mediated A ⁇ deposits in human brain (Cherny, R.A., et al, Soc. Neurosci Abstr. 23:(Abstract) (1997)). It was recently reported that there is a population of water-extractable A ⁇ deposit in the AD-affected brain (Kuo, Y-M., et ⁇ /., J. Biol. Chem. 277:4077-81 (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 A ⁇ collections, and liberating soluble A ⁇ subunits by freeing A ⁇ complexed with zinc [Zn 2+ ].
- the amount of A ⁇ 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 A ⁇ aggregate, and not by inhibition of an A ⁇ - cleaving metalloprotease (such as insulin degrading enzyme cleavage of A ⁇ reported recently by Dennis Selkoe at the 27 th Annual Meeting for the Society for Neuroscience, New Jersey).
- a ⁇ - cleaving metalloprotease such as insulin degrading enzyme cleavage of A ⁇ reported recently by Dennis Selkoe at the 27 th Annual Meeting for the Society for Neuroscience, New La.
- the extraction of sedimentable A ⁇ into the soluble phase correlated only with the extraction of zinc from the pellet, and not with any other metal assayed (Table 3). 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.
- 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.
- Figure 19 shows that metal chelators promote the solubilization of A ⁇ 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 western blot assay using anti-A ⁇ 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°C). Furthermore, similar results were achieved when the brain sample was homogenized in PBS and then pelleted before treated with PBS ⁇ chelator.
- bathocuproine exhibits a clear dose-dependent increase in A ⁇ 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.
- Table 3 shows a comparison between pellets of post-centrifugation homogenates in the presence and absence of a chelator (TPEN).
- FIG. 20A illustrates a western blot with anti-A ⁇ 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 A ⁇ from control tissue than from AD tissue. These data are typical of 15 cases. As expected, far less total A ⁇ is present in normal brain samples compared to AD brain samples, although the content of A ⁇ increases with age.
- dimers of A ⁇ 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 A ⁇ 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.
- AD tissue was dissected from the frontal poles of frozen AD and age- matched normal brains for which histopathological and clinical documentation were provided.
- AD tissue was selected according to CERAD criteria (Mirra et al., Neurology 47:479-486 (1991)) with particular attention paid to the presence of neuritic plaques and neurofibrillary tangles. Histological examination of A ⁇ 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 of protease 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.
- 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 -70 °C
- 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
- PB S Protein was precipitated from the entire supernatant and redissolved in 100 ⁇ l of sample buffer. Equal volumes of TCA-precipitated protein were subjected to Tris-Tricine SDS-PAGE and A ⁇ was visualized as described above.
- the inventors extracted brain tissue from histologically-confirmed AD-affected subjects and from subjects that were age- matched to AD-affected subjects but were not clinically demented (age-matched controls, "AC") 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.
- Figure 24A shows the effect of metals upon the solubility of brain-derived A ⁇ .
- Precipitation of A ⁇ was induced by adding either copper or zinc to unchelated extracts.
- the resulting signal for soluble A ⁇ was attenuated, the threshold concentration being between 20 and 50 ⁇ M for copper and between 5 and 20 ⁇ M for zinc.
- concentrations greater than 100 ⁇ M solubility was abolished.
- concentrations greater than 100 ⁇ M solubility was abolished.
- at lower concentrations of copper there appears to be a transitional stage where A ⁇ is present in the dimeric form prior to complete aggregation, mirroring the intermediate stage dimers elicited by chelator-mediated solubilization.
- 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.
- 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.
- FIG. 25A shows that patterns of chelator-promoted solubilization of A ⁇ differ in AD and aged, non-AD tissue.
- the chelator-promoted solubilization of A ⁇ 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 A ⁇ signal below that observed for the PBS control (See Figure 25B).
- Total A ⁇ for AD brains ranged from 6 - 80 ⁇ g/g wet weight tissue.
- the percentage of A ⁇ extractable (one extraction centrifugation sequence) ranged from 0.33 - 10%.
- the corresponding values for aged-matched control brains were 0.68 - 4.2 ⁇ g/g total A ⁇ and 2.6 - 29.5% extractable.
- 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 sha ⁇ en 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).
- 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 A ⁇ . It is also possible that the chelators are also acting to liberate intracellular stores of A ⁇ in vesicular compartments as metal -bound aggregates. Preliminary data indicates that this may be the case with platelets. 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.
- a functional homoeostatic mechanism implies equilibrium between intracellular copper and zinc (and perhaps other metals) normally present in trace amounts, for which A ⁇ has strong affinity, and more abundant metals which bind less strongly to A ⁇ .
- 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, S.H., Nature, 308:134-136 (1984)). It has recently been demonstrated (Huang, X., et al, J. Biol Chem.
- AD Alzheimer' s Disease
- DS Down's Syndrome
- Rat A ⁇ ,_ 40 peptide was synthesized, purified and characterized as described above. Rat A ⁇ , _ 40 was obtained from Quality Control Biochemicals, Inc.
- 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.
- a ⁇ ,_ 42 is the predominant species found in amyloid plaques (Masters, C.L. et al, Proc. Natl. Acad. Sci. USA 82: 4245 (1985); Mu ⁇ hy, G.M., et al, Am. J. Pathol. 744:1082-1088 (1994); Mak, K., et al, Brain Res. 667:138-142 (1994);
- Tests were performed to determine the concentration of Cu 2* required to induce the formation of SDS-resistant A ⁇ ,_ 40 and A ⁇ ,_ 42 polymers.
- a ⁇ ,_ 40 and A ⁇ ,_ 42 were incubated with different [Cu 2+ ] (0-30 ⁇ M) 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.
- N ⁇ ⁇ - 4 o polymerization was not detected with increasing Fe 3+ concentrations at any pH. Therefore, of the metal ions known to interact with A ⁇ , only Cu 2+ , whose ability to aggregate and bind Cu 2 " under mildly acidic conditions is enhanced, is capable of inducing SDS-resistant A ⁇ polymerization.
- Oxygen radical mediated chemical attack has been correlated with an increase in protein and free carbonyls (Smith, CD., et al, Proc. Natl. Acad. Sci. USA 55:10540 (1991); Hensley, K., et al, J. Neurochem. 65:2146 (1995); Smith, M.A., et al, Nature 352: 120 (1996)) and peroxynitrite -mediated protein nitration (Good, P.F., et al, Am. J. Pathol 749:21 (1996); Smith, M.A., et al, Proc. Natl Acad. Sci. USA 94:9866 (1997)).
- a ⁇ is capable of reducing Cu 2+ and H 2 O 2 is produced in solutions containing A ⁇ and Cu 2+ or Fe 3 " (Huang, X. et al, J. Biol Chem. 272:26464-26470 (1997)).
- the generation of SDS-resistant A ⁇ polymers in the order A ⁇ ,_ 42 » A ⁇ ,_ 40 » rat A ⁇ ,_ 40 in the presence of Cu 2* correlates well with the generation of Cu * and reactive oxygen species (ROS; OH “ , H 2 O 2 and O " 2 : Huang, X.. et al, J. Biol Chem. 272:26464-26410 (1997)) by each peptide.
- ROS reactive oxygen species
- H 2 O 2 is required for the polymerization reaction under physiological conditions
- the removal of H 2 O 2 and it's precursors O 2 and O " should decrease SDS-resistant polymerization.
- a ⁇ , _ 42 was incubated with or without Cu 2+ 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 H 2 O 2 is required for the polymerization of A ⁇ .
- a ⁇ ,_ 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 O 2 (Figure 29B).
- Argon-purging of the solution markedly decreased A ⁇ , remind 42 polymerization under each condition, indicating that the generation of ROS is required for the polymerization of A ⁇ .
- SDS-resistant polymers of A ⁇ by this metal-catalyzed oxidative mechanism strongly suggested that a chemical modification to the peptide backbone allows the formation of the polymer species.
- SDS-resistant polymers generated by incubating A ⁇ ,_ 42 with Cu 2+ at pH 74 and 6.6, or A ⁇ ,_ 42 with Cu 2 " plus H 2 O 2 were subjected to treatment with urea ( Figure 30 A) and guanidine HCl, chao trophic agents known to disrupt H-bonding.
- the SDS-resistant polymers generated with Cu 2* are similar to those extracted from post-mortem AD brains (Roher, A.E., et al, Journal of Biological
- TETA and BC were incubated with A ⁇ oligomers extracted from human brain.
- Figure 30E shows that both TETA and BC significantly increased the amount of monomer A ⁇ in samples treated with these chelators. Although the increase in the amount of monomer was small, these results suggest that human oligomeric A ⁇ species are partially held together with metal ions. Importantly, this result indicates the potential of chelation therapy as a means of reducing amyloidosis.
- SDS-resistant A ⁇ 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 A ⁇ in amyloid plaques.
- Incubation of SDS-resistant A ⁇ polymers with proteinase K resulted in complete degradation of both monomer and oligomeric A ⁇ 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 A ⁇ oligomers compared to the Cu-mediated SDS-resistant polymers generated in vitro.
- Soluble A ⁇ ,_ 40 and A ⁇ ,_ 42 both exist in phosphate buffered saline as noncovalent dimers (Huang, X., et al, J. Biol. Chem. 272:26464-26470 (1997); and unpublished observations).
- Disruption of ionic and hydrogen bonding of A ⁇ in the soluble and aggregated forms (pH or Zn 2+ ) by the ionic detergent SDS results in the complete dissociation of A ⁇ into the monomer species as detected on SDS- PAGE ( Figures 9, 32-34).
- the formation of SDS-resistant polymers of A ⁇ over time in the presence of Cu 2+ ( Figures 9, 27A-27B, 28A-28C) suggests that conformational or structural alterations allow for the formation of a thermodynamically more stable complex.
- a chemical modification to the peptide may increase the affinity of the polymer for Cu 2+ and the formation of a stable complex.
- 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 CuA
- the binding of Cu 2+ to A ⁇ 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, X., etal, J. Biol. Chem. 272:26464- 26470 (1997)) and correlated with the generation of SDS-resistant polymers for each species.
- Fe 2* is found in much higher concentrations in the brains of AD patients compared with age-matched controls (Ehmann, W.D., et al, Neurotoxicol. 7: 197- 206 (1986); Dedman, D.J., et ⁇ /., Biochem. J. 257:509-514 (1992); Joshi, J.G., et al, Environ. Health Per sped. 702:207-213 (1994)). This is partly attributable to the increased ferritin rich microglia and oligodendrocytes that localize to amyloid plaques (Grudke-Iqbal, I., et al, Ada Neuropathol. 57:105 (1990); Conner, J.R., et al, J. Neurosci. Res. 37:75-83 (1992); Sadowki, M., et al, Alzheimer's Res. 7:71-76 (1995)).
- Structural differences between A ⁇ ,_ 40 and A ⁇ ,_ 42 may allow for the formation of a thermodynamically stable dimer in the case of A ⁇ ,_ 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 A ⁇ ,_ 42 compared to A ⁇ ,_ 40 is most likely explained by the increased ability of A ⁇ ,_ 42 to reduce Cu and generate ROS.
- the present invention indicates that the manipulation of the brain biometal environment with specific agents acting directly (e.g., chelators and antioxidants) or indirectly (e.g. , by improving cerebral energy metabolism) provides a means for therapeutic intervention in the prevention and treatment of Alzheimer's disease.
- specific agents acting directly e.g., chelators and antioxidants
- indirectly e.g. , by improving cerebral energy metabolism
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CA2301706C (en) | 1997-08-21 | 2005-10-04 | P.N. Gerolymatos S.A. | Use of phanquinone for the treatment of alzheimer's disease |
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US6323218B1 (en) * | 1998-03-11 | 2001-11-27 | The General Hospital Corporation | Agents for use in the treatment of Alzheimer's disease |
US6638711B1 (en) | 1999-04-29 | 2003-10-28 | The General Hospital Corporation | Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation |
WO2000018392A1 (en) | 1998-09-25 | 2000-04-06 | Glycox Corporation Limited | Fructosamine oxidase: antagonists and inhibitors |
BR0008241A (en) | 1999-01-07 | 2001-10-30 | Gerolymatos P N Sa | Use of phanquinone for the preparation of a pharmaceutical composition for the treatment of memory impairment and treatment method |
DE19909357A1 (en) * | 1999-03-03 | 2000-09-07 | Gerd Multhaup | Copper agonist that binds to the APP copper binding site and / or has an inhibitory effect on the release of the amyloid-Aß peptide |
JP4796725B2 (en) * | 1999-08-04 | 2011-10-19 | ユニバーシティ オブ サザン カリフォルニア | Amyloid β protein (spherical assembly and use thereof) |
EP1266884A4 (en) * | 2000-03-22 | 2003-04-23 | Bf Res Inst Inc | Image diagnosis probe based on substituted azobenzene or analogue thereof for disease attributable to amyloid accumulation and composition for image diagnosis containing the same |
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US20060100278A1 (en) | 2002-08-20 | 2006-05-11 | Cooper Garth J S | Dosage forms and related therapies |
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US8338089B2 (en) | 2006-11-20 | 2012-12-25 | The Johns Hopkins University | Method of inhibiting lentiviral infectivity utilizing zinc chelation to inhibit Vif activity |
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WO1998040071A1 (en) | 1998-09-17 |
AU6548498A (en) | 1998-09-29 |
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