U.S. patent application number 09/956980 was filed with the patent office on 2002-06-27 for agents for use in the treatment of alzheimer's disease.
Invention is credited to Atwood, Craig S., Bush, Ashley I., Huang, Xudong, Tanzi, Rudolph E..
Application Number | 20020082273 09/956980 |
Document ID | / |
Family ID | 21898356 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082273 |
Kind Code |
A1 |
Bush, Ashley I. ; et
al. |
June 27, 2002 |
Agents for use in the treatment of alzheimer's disease
Abstract
The invention relates to the identification of pharmacological
agents to be used in the treatment of Alzheimer's disease and
related pathological conditions and compositions for treatment of
conditions caused by amyloidosis, A.beta.-mediated formation of
ROS, or both, such as Alzheimer's disease, are disclosed.
Inventors: |
Bush, Ashley I.;
(Somerville, MA) ; Huang, Xudong; (Cambridge,
MA) ; Atwood, Craig S.; (Somerville, MA) ;
Tanzi, Rudolph E.; (Canton, MA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Family ID: |
21898356 |
Appl. No.: |
09/956980 |
Filed: |
September 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09956980 |
Sep 21, 2001 |
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09038154 |
Mar 11, 1998 |
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Current U.S.
Class: |
514/291 ;
514/298; 514/420; 514/562; 514/566; 514/707 |
Current CPC
Class: |
A61K 31/555 20130101;
A61P 25/28 20180101; A61K 31/555 20130101; A61K 31/555 20130101;
A61K 31/47 20130101; A61K 31/47 20130101; A61K 31/47 20130101; A61K
31/4745 20130101; A61P 43/00 20180101; A61K 31/19 20130101; A61K
45/06 20130101; A61K 2300/00 20130101; A61K 31/47 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/291 ;
514/298; 514/562; 514/566; 514/420; 514/707 |
International
Class: |
A61K 031/4745; A61K
031/473; A61K 031/195; A61K 031/198; A61K 031/405; A61K
031/105 |
Goverment Interests
[0001] Part of the work performed during the development of this
invention utilized U.S. Government Funds under Grant No. R29AG12686
from the National Institutes of Health. The government may have
certain rights in this invention.
Claims
What is claimed is:
1. 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 group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol, for a time and under conditions to bring about
said treatment; wherein said combination reduces, inhibits or
otherwise interferes with A.beta.-mediated production of radical
oxygen species.
2. The method of claim 1 wherein the metal chelator is
bathocuproine.
3. The method of claim 1 further comprising administering a
supplement selected from the group consisting of: ammonium salt,
calcium salt, magnesium salt, and sodium salt.
4. The method of claim 3 wherein the supplement is magnesium
salt.
5. The method of claim 1 further comprising administering to the
subject an effective amount of a compound selected from the group
consisting of: rifampicin, disulfiram, and indomethacin, or a
pharmaceutically acceptable salt thereof.
6. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) a salt of a metal chelator, wherein said
chelator is selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof, and (b) clioquinol;
wherein said salt of the metal chelator is selected from the group
consisting of: ammonium, calcium, magnesium, and sodium; and
wherein said combination reduces, inhibits or otherwise interferes
with A.beta.-mediated production of radical oxygen species.
7. The method of claim 6 wherein the metal chelator is
bathocuproine.
8. The method of claim 6 wherein the salt of a metal chelator is a
magnesium salt.
9. The method of claim 6 further comprising administering to said
subject a compound selected from the group consisting of:
rifampicin, disulfiram, and indomethacin, or a pharmaceutically
acceptable salt thereof.
10. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) a chelator specific for copper, and (b)
clioquinol; wherein said combination reduces, inhibits or otherwise
interferes with A.beta.-mediated production of radical oxygen
species.
11. The method of claim 10 wherein the chelator specific for copper
is specific for the reduced form of copper.
12. The method of claim 11 wherein the chelator is bathocuproine or
a hydrophobic derivative thereof.
13. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) an alkalinizing agent and (b) clioquinol;
wherein said combination reduces, inhibits or otherwise interferes
with A.beta.-mediated production of radical oxygen species.
14. The method of claim 13 wherein the alkalinizing agent is
magnesium citrate.
15. The method of claim 13 wherein the alkalinizing agent is
calcium citrate.
16. 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 group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol, for a time and under conditions to bring about
said treatment; wherein said combination prevents formation of
A.beta. amyloid, promotes, induces or otherwise facilitates
resolubilization of A.beta. deposits, or both.
17. The method of claim 16 wherein the metal chelator is
bathocuproine.
18. The method of claim 16 further comprising administering a
supplement selected from the group consisting of: ammonium salt,
calcium salt, magnesium salt, and sodium salt.
19. The method of claim 18 wherein the supplement is magnesium
salt.
20. The method of claim 16 further comprising administering to the
subject an effective amount of a compound selected from the group
consisting of: rifampicin, disulfiram, and indomethacin, or a
pharmaceutically acceptable salt thereof.
21. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) a salt of a metal chelator, wherein said
chelator is selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof, and (b) clioquinol;
wherein said salt of the metal chelator is selected from the group
consisting of: ammonium, calcium, magnesium, and sodium; and
wherein said combination prevents formation of A.beta. amyloid,
promotes, induces or otherwise facilitates resolubilization of
A.beta. deposits, or both.
22. The method of claim 21 wherein the metal chelator is
bathocuproine.
23. The method of claim 21 wherein the salt of the metal chelator
is a magnesium salt.
24. The method of claim 21 further comprising administering to said
subject a compound selected from the group consisting of:
rifampicin, disulfiram, and indomethacin, or a pharmaceutically
acceptable salt thereof.
25. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) a chelator specific for copper, and (b)
clioquinol; wherein said combination prevents formation of A.beta.
amyloid, promotes, induces or otherwise facilitates
resolubilization of A.beta. deposits, or both.
26. The method of claim 25 wherein the chelator specific for copper
is specific for the reduced form of copper.
27. The method of claim 26 wherein the chelator is bathocuproine or
a hydrophobic derivative thereof.
28. A method of treating amyloidosis in a subject, said method
comprising administering to said subject an effective amount of a
combination of (a) an alkalinizing agent and (b) clioquinol;
wherein said combination prevents formation of A.beta. amyloid,
promotes, induces or otherwise facilitates resolubilization of
A.beta. deposits, or both.
29. The method of claim 28 wherein the alkalinizing agent is
magnesium citrate.
30. The method of claim 28 wherein the alkalinizing agent is
calcium citrate.
31. A pharmaceutical composition for treatment of conditions caused
by amyloidosis, A.beta.-mediated ROS formation, or both,
comprising: (a) a metal chelator selected from the group consisting
of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol, together with one or more pharmaceutically
acceptable carriers or diluents.
32. The pharmaceutical composition of claim 31 wherein the metal
chelator is bathocuproine.
33. The pharmaceutical composition of claim 31 further comprising a
supplement selected from the group consisting of: ammonium salt,
calcium salt, magnesium salt, and sodium salt.
34. The pharmaceutical composition of claim 33 wherein the
supplement is a magnesium salt.
35. The pharmaceutical composition of claim 31 further comprising a
compound selected from the group consisting of: rifampicin,
disulfiram, and indomethacin.
36. A pharmaceutical composition for treatment of conditions caused
by amyloidosis, A.beta.-mediated ROS formation, or both, comprising
a combination of (a) a salt of a metal chelator selected from the
group consisting of: bathocuproine, bathophenanthroline, DTPA,
EDTA, EGTA, penicillamine, TETA, and TPEN, or hydrophobic
derivatives thereof; and (b) clioquinol; wherein said salt of the
metal chelator is selected from the group consisting of: ammonium,
calcium, magnesium, and sodium, together with one or more
pharmaceutically acceptable carriers or diluents.
37. The pharmaceutical composition of claim 36 wherein the metal
chelator is bathocuproine.
38. The pharmaceutical composition of claim 36 wherein the salt of
the metal chelator is a magnesium salt.
39. The pharmaceutical composition of claim 36 further comprising a
compound selected from the group consisting of: rifampicin,
disulfiram, and indomethacin.
40. A pharmaceutical composition for treatment of conditions caused
by amyloidosis, A.beta.-mediated ROS formation, or both, comprising
a chelator specific for copper, with one or more pharmaceutically
acceptable carriers or diluents.
41. The pharmaceutical composition of claim 40 wherein the chelator
is specific for the reduced form of copper.
42. The pharmaceutical composition of claim 41 wherein the chelator
specific for the reduced form of copper is bathocuproine or a
hydrophobic derivative thereof.
43. A pharmaceutical composition for treatment of conditions caused
by amyloidosis, A.beta.-mediated ROS formation, or both, comprising
a combination of (a) an alkalinizing agent and (b) clioquinol;
together with one or more pharmaceutically acceptable carriers or
diluents.
44. The pharmaceutical composition of claim 43 wherein the
alkalinizing agent is magnesium citrate.
45. The pharmaceutical composition of claim 43 wherein the
alkalinizing agent is calcium citrate.
46. A composition of matter comprising: (a) a metal chelator
selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof; and (b) clioquinol.
47. The composition of claim 46 wherein the metal chelator is
bathocuproine.
48. The composition of claim 46 further comprising a supplement
selected from the group consisting of: ammonium salt, calcium salt,
magnesium salt, and sodium salt.
49. The composition of claim 48 wherein the supplement is a
magnesium salt.
50. The composition of claim 46 further comprising a compound
selected from the group consisting of: rifampicin, disulfiram, and
indomethacin.
51. A composition of matter comprising a combination of (a) a salt
of a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol; wherein said salt of the metal chelator is
selected from the group consisting of: ammonium, calcium,
magnesium, and sodium.
52. The composition of claim 51 wherein the metal chelator is
bathocuproine.
53. The composition of claim 51 wherein the salt of the chelator is
a magnesium salt.
54. The composition of claim 51 further comprising a compound
selected from the group consisting of: rifampicin, disulfiram, and
indomethacin.
55. A composition of matter comprising a combination of (a) an
alkalinizing agent and (b) clioquinol.
56. The composition of claim 55 wherein the alkalinizing agent is
magnesium citrate.
57. The composition of claim 55 wherein the alkalinizing agent is
calcium citrate.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is in the field of medicinal chemistry. In
particular, the invention is related to compositions for treatment
of Alzheimer's disease.
[0004] 2. Related Art
[0005] Polymers of Abeta (A.beta.), the 4.3 kD, 39-43 amino acid
peptide product of the transmembrane protein, amyloid protein
precursor (APP), are the main components extracted from the
neuritic and vascular amyloid of Alzheimer's disease (AD) brains.
A.beta. 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. 4:2757
(1985); Masters, C. L. et al., Proc. Natl. Acad. Sci. USA 82: 4245
(1985)). Growing evidence suggests that amyloid deposits are
intimately associated with the neuronal demise that leads to
dementia in the disorder.
[0006] The presence of an enrichment of the 42 residue species of
A.beta. in these deposits suggests that this species is more
pathogenic. The 42 residue form of A.beta. (A.beta..sub.1-42),
while a minor component of biological fluids, is highly enriched in
amyloid, and genetic studies strongly implicate this protein in the
etiopathogenesis of AD. Amyloid deposits are decorated with
inflammatory response proteins, but biochemical markers of severe
oxidative stress such as peroxidation adducts, advanced glycation
end-products, and protein cross-linking are seen in proximity to
the lesions. To date, the cause of A.beta. deposits is unknown,
although it is believed that preventing these deposits may be a
means of treating the disorder.
[0007] When polymers of A.beta. are placed into culture with rat
hippocampal a neurons, they are neurotoxic (Kuo, Y -M., et al., J.
Biol. Chem. 271:4077-81 (1996); Roher, A. E., et al., Journal of
Biological Chemistry 271:20631-20635 (1996)). The mechanism
underlying the formation of these neurotoxic polymeric A.beta.
species remains unresolved. The overexpression of A.beta. alone
cannot sufficiently explain amyloid formation, since the
concentration of A.beta. required for precipitation is not
physiologically plausible. That alterations in the neurochemical
environment are required for amyloid formation is indicated by its
solubility in neural phosphate buffer at concentrations of up to 16
mg/ml (Tomski, S. & Murphy, R. M., Archives of Biochemistry and
Biophysics 294:630 (1992)), biological fluids such as cerebrospinal
fluid (CSF) (Shoji, M., et al., Science 258:126 (1992); Golde, T.
E., et al. Science, 255(5045):728-730 (1992); Seubert, P., et al.,
Nature 359:325 (1992); Haass, C., et al., Nature 359:322 (1992))
and in the plaque-free brains of Down's syndrome patients (Teller,
J. K., et al., Nature Medicine 2:93-95 (1996)).
[0008] Studies into the neurochemical vulnerability of A.beta. to
form amyloid have suggested altered zinc and [H.sup.+] homeostasis
as the most likely explanations for amyloid deposition. A.beta. is
rapidly precipitated under mildly acidic conditions in vitro (pH
3.5-6.5) (Barrow, C. J. & Zagorski, M. G., Science 253:179-182
(1991); Fraser, P. E., et al., Biophys. J. 60:1190-1201 (1991);
Barrow, C. J., et al., J. Mol. Biol. 225:1075-1093 (1992); Burdick,
D., J. Biol. Chem. 267:546-554 (1992); Zagorski, M. G. &
Barrow, C. J., Biochemistry 31:5621-5631 (1992); Kirshenbaum, K.
& Daggett, V., Biochemistry 34:7629-7639 (1995); Wood, S. J.,
et al., J. Mol. Biol. 256:870-877 (1996)). Recently, it has been
shown that the presence of certain biometals, in particular redox
inactive Zn.sup.2+ and, to a lesser extent, redox active Cu.sup.2+
and Fe.sup.3+, markedly increases the precipitation of soluble
A.beta. (Bush, A. I., et al, J. Biol. Chem. 268:16109 (1993); Bush,
A. I., et al., J. Biol. Chem. 269:12152(1994); Bush, A. I., et al.,
Science 265:1464(1994); Bush, A. I., et al., Science 268:1921
(1995)). At physiological pH, A.beta..sub.1-40 specifically and
saturably binds Zn.sup.2+, manifesting high affinity binding
(KD=107 nM) with a 1:1 (Zn.sup.2+:A.beta.) stoichiometry, and low
affinity binding (KD=5.2 .mu.M) with a 2:1 stoichiometry.
[0009] The reduction by APP of copper (II) to copper (I) may lead
to irreversible A.beta. aggregation and SDS-resistant
polymerization. This reaction may promote an environment that would
enhance the production of hydroxyl radicals, which may contribute
to oxidative stress in AD (Multhaup, G., et al., Science 271:
1406-1409 (1996)). A precedence for abnormal Cu metabolism already
exists in the neurodegenerative disorders of Wilson's disease,
Menkes' syndrome and possibly familial amyotrophic lateral
sclerosis (Tanzi, R. E. et al., Nature Genetics 5:344 (1993); Bull,
P. C., et al., Nature Genetics 5:327 (1993); Vulpe, C., et al.,
Nature Genetics 3:7 (1993); Yamaguchi, Y., et al., Biochem.
Biophys. Res. Commun. 197:271 (1993); Chelly, J., et al., Nature
Genetics 3:14 (1993); Wang, D. & Munoz, D. G., J. Neuropathol.
Exp. Neurol. 54:548 (1995); Beckman, J. S., et al., Nature 364:584
(1993); Hartmann, H. A. & Evenson, M. A., Med. Hypotheses 38:75
(1992)).
[0010] Although much fundamental pathology, genetic susceptibility
and biology associated with AD is becoming clearer, a rational
chemical and structural basis for developing effective drugs to
prevent or cure the disease remains elusive. While the genetics of
the disorder indicates that the metabolism of A.beta. is intimately
associated with the etiopatholgenesis of the disease, drugs for the
treatment of AD have so far focused on "cognition enhancers" which
do not address the underlying disease processes.
SUMMARY OF THE INVENTION
[0011] An 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.
[0012] 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.beta..
[0013] In one aspect, the free radicals are reactive oxygen species
such as O.sub.2 or OH. In another aspect, the free radicals include
forms of A.beta..
[0014] The agent of this aspect of the present invention may
contain one or more than one compound such as a metal chelator or
metal complexing compound such as but not limited to DTPA,
bathocuproine, bathophenanthroline, clioquinol, penicillamine, or
derivatives, homologues or analogues thereof. Alternatively, or in
addition, the agent may comprise an antioxidant or other molecule
capable of interfering with A.beta. peptide-mediated radical
formation.
[0015] One aspect of the present invention comprises an agent for
use in treating AD in a subject comprising a metal chelator, metal
complexing compound and/or a compound capable of inhibiting free
radical formation by interaction of A.beta. peptides and biometals,
said agent optionally further comprising one or more
pharmaceutically acceptable carriers and/or diluents.
[0016] In one aspect, 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 group consisting of: bathocuproine, bathophenanthroline, DTPA,
EDTA, EGTA, penicillamine, TETA, and TPEN, or hydrophobic
derivatives thereof; and (b) clioquinol, for a time and under
conditions to bring about said treatment; wherein said combination
reduces, inhibits or otherwise interferes with A.beta.-mediated
production of radical oxygen species.
[0017] In a preferred embodiment, the metal chelator is
bathocuproine.
[0018] In another aspect, said method further comprises
administering a supplement selected from the group consisting of:
ammonium salt, calcium salt, magnesium salt, and sodium salt.
[0019] In a preferred embodiment, the supplement is magnesium
salt.
[0020] In another aspect, said method further comprises
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.
[0021] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) a salt of a metal chelator, wherein said chelator is
selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof, and (b) clioquinol;
wherein said salt of the metal chelator is selected from the group
consisting of: ammonium, calcium, magnesium, and sodium; and
wherein said combination reduces, inhibits or otherwise interferes
with A.beta.-mediated production of radical oxygen species.
[0022] In a preferred embodiment, the metal chelator is
bathocuproine.
[0023] In another preferred embodiment, the salt of the metal
chelator is a magnesium salt.
[0024] In another aspect, said method further comprises
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.
[0025] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) a chelator specific for copper, and (b) clioquinol; wherein
said combination reduces, inhibits or otherwise interferes with
A.beta.-mediated production of radical oxygen species.
[0026] In a preferred embodiment, the chelator specific for copper
is specific for the reduced form of copper. Most preferrably, the
chelator is bathocuproine or a hydrophobic derivative thereof.
[0027] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) an alkalinizing agent and (b) clioquinol; wherein said
combination reduces, inhibits or otherwise interferes with
A.beta.-mediated production of radical oxygen species.
[0028] In a preferred embodiment, the alkalinizing agent is
magnesium citrate. In another preferred embodiment, the
alkalinizing agent is calcium citrate.
[0029] Still another aspect of the present invention contemplates a
method of treating AD in a subject comprising administering to said
subject an agent capable of promoting, inducing or otherwise
facilitating resolubilization of A.beta. deposits in the brain for
a time and under conditions to effect said treatment.
[0030] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject a combination of (a) a metal chelator
selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof; and (b) clioquinol, for a
time and under conditions to bring about said treatment; wherein
said combination prevents formation of A.beta. amyloid, promotes,
induces or otherwise facilitates resolubilization of A.beta.
deposits, or both.
[0031] In a preferred embodiment, the metal chelator is
bathocuproine.
[0032] In another aspect, said method further comprises
administering a supplement selected from the group consisting of:
ammonium salt, calcium salt, magnesium salt, and sodium salt.
[0033] In a preferred embodiment, the supplement is magnesium
salt.
[0034] In another aspect, said method further comprises
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.
[0035] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) a salt of a metal chelator, wherein said chelator is
selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and
TPEN, or hydrophobic derivatives thereof, and (b) clioquinol;
wherein said salt of the metal chelator is selected from the group
consisting of: ammonium, calcium, magnesium, and sodium; and
wherein said combination prevents formation of A.beta. amyloid,
promotes, induces or otherwise facilitates resolubilization of
A.beta. deposits, or both.
[0036] In a preferred embodiment, the metal chelator is
bathocuproine. In another preferred embodiment, the salt of the
metal chelator is a magnesium salt.
[0037] In another aspect, said method further comprises
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.
[0038] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) a chelator specific for copper, and (b) clioquinol; wherein
said combination prevents formation of A.beta. amyloid, promotes,
induces or otherwise facilitates resolubilization of A.beta.
deposits, or both.
[0039] In a preferred embodiment, the chelator specific for copper
is specific for the reduced form of copper. Most preferrably, the
chelator is bathocuproine or a hydrophobic derivative thereof.
[0040] In yet another aspect, the invention relates to a method of
treating amyloidosis in a subject, said method comprising
administering to said subject an effective amount of a combination
of (a) an alkalinizing agent and (b) clioquinol; wherein said
combination prevents formation of A.beta. amyloid, promotes,
induces or otherwise facilitates resolubilization of A.beta.
deposits, or both.
[0041] In a preferred embodiment, the alkalinizing agent is
magnesium citrate. In another preferred embodiment, the
alkalinizing agent is calcium citrate.
[0042] Still another aspect of the invention relates to a
pharmaceutical composition for treatment of conditions caused by
amyloidosis, A.beta.-mediated ROS formation, or both, comprising:
(a) a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol, together with one or more pharmaceutically
acceptable carriers or diluents.
[0043] In a preferred embodiment, the metal chelator is
bathocuproine.
[0044] In another aspect, said method further comprises
administering a supplement selected from the group consisting of:
ammonium salt, calcium salt, magnesium salt, and sodium salt.
[0045] In a preferred embodiment, the supplement is magnesium
salt.
[0046] In another aspect, said composition further comprises a
compound selected from the group consisting of: rifampicin,
disulfiram, and indomethacin, or a pharmaceutically acceptable salt
thereof.
[0047] In yet another aspect, the invention relates to a
pharmaceutical composition for treatment of conditions caused by
amyloidosis, A.beta.-mediated ROS formation, or both, comprising a
combination of (a) a salt of a metal chelator selected from the
group consisting of: bathocuproine, bathophenanthroline, DTPA,
EDTA, EGTA, penicillamine, TETA, and TPEN, or hydrophobic
derivatives thereof; and (b) clioquinol; wherein said salt of the
metal chelator is selected from the group consisting of: ammonium,
calcium, magnesium, and sodium, together with one or more
pharmaceutically acceptable carriers or diluents.
[0048] In a preferred embodiment, the metal chelator is
bathocuproine. In another preferred embodiment, the salt of the
metal chelator is a magnesium salt.
[0049] In another aspect, said composition further comprises a
compound selected from the group consisting of: rifampicin,
disulfiram, and indomethacin, or a pharmaceutically acceptable salt
thereof.
[0050] In yet another aspect, the invention relates to a
pharmaceutical composition for treatment of conditions caused by
amyloidosis, A.beta.-mediated ROS formation, or both, comprising a
chelator specific for copper, with one or more pharmaceutically
acceptable carriers or diluents.
[0051] In a preferred embodiment, the chelator specific for copper
is specific for the reduced form of copper. Most preferrably, the
chelator is bathocuproine or a hydrophobic derivative thereof.
[0052] In yet another aspect, the invention relates to a
pharmaceutical composition for treatment of conditions caused by
amyloidosis, A.beta.-mediated ROS formation, or both, comprising a
combination of (a) an alkalinizing agent and (b) clioquinol;
together with one or more pharmaceutically acceptable carriers or
diluents.
[0053] In a preferred embodiment, the alkalinizing agent is
magnesium citrate. In another preferred embodiment, the
alkalinizing agent is calcium citrate.
[0054] Still another aspect of the invention relates to a
composition of matter comprising: (a) a metal chelator selected
from the group consisting of: bathocuproine, bathophenanthroline,
DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, or hydrophobic
derivatives thereof; and (b) clioquinol.
[0055] In a preferred embodiment, the metal chelator is
bathocuproine.
[0056] In another aspect, said method further comprises
administering a supplement selected from the group consisting of:
ammonium salt, calcium salt, magnesium salt, and sodium salt.
[0057] In a preferred embodiment, the supplement is magnesium
salt.
[0058] In another aspect, said composition further comprises an
effective amount of a compound selected from the group consisting
of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically
acceptable salt thereof.
[0059] In yet another aspect, the invention relates to a
composition of matter comprising a combination of (a) a salt of a
metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
and (b) clioquinol; wherein said salt of the metal chelator is
selected from the group consisting of: ammonium, calcium,
magnesium, and sodium.
[0060] In a preferred embodiment, the metal chelator is
bathocuproine. In another preferred embodiment, the salt of said
metal chelator is a magnesium salt.
[0061] In another aspect, said composition further comprises an
effective amount of a compound selected from the group consisting
of: rifampicin, disulfiram, and indomethacin, or a pharmaceutically
acceptable salt thereof.
[0062] In yet another aspect, the invention relates to a
composition of matter comprising a combination of (a) an
alkalinizing agent and (b) clioquinol.
BRIEF DESCRIPTION OF THE FIGURES
[0063] FIG. 1 is a graph showing the proportion of soluble
A.beta..sub.1-40 remaining following centrifugation of reaction
mixtures.
[0064] FIGS. 2A-2C:
[0065] FIG. 2A is a graph showing the proportion of soluble
A.beta..sub.1-40 remaining in the supernatant after incubation with
various metal ions.
[0066] FIG. 2B is a graph showing a turbidometric analysis of pH
effect on metal ion-induced A.beta..sub.1-40 aggregation.
[0067] FIG. 2C is a graph showing the proportion of soluble
A.beta..sub.1-40 remaining in the supernatant after incubation with
various metal ions, where high metal ion concentrations were
used.
[0068] FIG. 3 is a graph showing a competition analysis of
A.beta..sub.1-40 binding to Cu.sup.2+.
[0069] FIGS. 4A-4C:
[0070] FIG. 4A is a graph showing the proportion of soluble
A.beta..sub.1-40 remaining in the supernatant following incubation
at various pHs in PBS.+-.Zn.sup.2+ or Cu.sup.2+.
[0071] FIG. 4B is a graph showing the proportion of soluble
A.beta..sub.1-40 remaining in the supernatant following incubation
at various pHs with different Cu.sup.2+ concentrations.
[0072] FIG. 4C is a graph showing the relative aggregation of nM
concentrations of A.beta..sub.1-40 at pH 7.4 and 6.6 with different
Cu.sup.2+ concentrations.
[0073] FIGS. 5A and 5B:
[0074] FIG. 5A is a graph showing a turbidometric analysis of
Cu.sup.2+-induced A.beta..sub.1-40 aggregation at pH 7.4 reversed
by successive cycles of chelator.
[0075] FIG. 5B is a graph showing a turbidometric analysis of the
reversibility of Cu.sup.2+-induced A.beta..sub.1-40 aggregation as
the pH cycles between 7.4 and 6.6.
[0076] FIG. 6 shows the amino acid sequence of APP.sub.669-716 near
A.beta..sub.1-42. Rat A.beta. is mutated (R5G, Y10F, H13R; bold).
Possible metal-binding residues are underlined.
[0077] FIG. 7 is a graph showing the effects of pH, Zn.sup.2+ or
Cu.sup.2+ upon A.beta. deposit formation.
[0078] FIG. 8 is a western blot showing the extraction of A.beta.
from post-mortem brain tissue.
[0079] FIG. 9 is a western blot showing A.beta. SDS-resistant
polymerization by copper.
[0080] FIG. 10 is a graph showing Cu.sup.+ generation by
A.beta..
[0081] FIG. 11 is a graph showing H.sub.2O.sub.2 production by
A.beta..
[0082] FIG. 12 is a graphical representation showing a model for
the generation of reduced metal ions, O.sub.2.sup.-,
H.sub.2O.sub.2, and OH. by A.beta. peptides. Note that A.beta.
facilitates two consecutive steps in the pathway: the reduction of
metal ions, and the reaction of O.sub.2 with reduced metal ions.
The peptide does not appear to be consumed or modified in a one
hour time frame by participation in these reactions.
[0083] FIGS. 13A and 13B are graphical representations showing
Fe.sup.3+ or Cu.sup.2+ reduction by A.beta. peptides.
[0084] FIG. 13A illustrates the reducing capacity of A.beta.
species (10 .mu.M), compared to Vitamin C and insulin (Sigma) (all
10 .mu.M) towards Fe.sup.3+ or Cu.sup.2+ (10 .mu.M) in PBS, pH 7.4,
after 1 hour co-incubation, 37.degree. C. Data indicate
concentration of reduced metal ions generated.
[0085] FIG. 13B shows the effect of oxygen tension and chelation
upon A.beta..sub.1-42 metal reduction. A.beta..sub.1-42 was
incubated as in FIG. 13A under various buffer gas conditions.
"Ambient"=no efforts were made to adjust the gas tension in the
bench preparations of the buffer vehicle, "O.sub.2"=100% O.sub.2
was continuously bubbled through the PBS vehicle for 2 hours (at
20.degree. C.), before the remainder of the incubation components
were added, "Ar"=100% Ar was continuously bubbled through the PBS
vehicle for 2 hours (at 20.degree. C.), before the remainder of the
incubation components were added. "+DFO or TETA"=Desferrioxamine
(DFO, Sigma, 200 .mu.M) was added to the A.beta..sub.1-42
incubation in the presence of Fe.sup.3+ 10 .mu.M, or
triethylenetetramine dihydrochloride (TETA, Sigma, 200 .mu.M) was
added to the A.beta..sub.1-42 incubation in the presence of
Cu.sup.2+ 10 .mu.M, under ambient oxygen conditions. All data
points are means .+-.SD, n=3.
[0086] FIGS. 14A-14E are graphical representations showing
production of H.sub.2O.sub.2 from the incubation of A.beta. in the
presence of substoichiometric amounts of Fe.sup.3+ or
Cu.sup.2+.
[0087] FIG. 14A shows H.sub.2O.sub.2 produced by A.beta..sub.1-42
(in PBS, pH 7.4, under ambient gas conditions, 1 hour, 37.degree.
C.) following co-incubation with various concentrations of catalase
in the presence of 1 .mu.M Fe.sup.3+.
[0088] FIG. 14B shows a comparison of H.sub.2O.sub.2 generation by
variant A.beta. species: A.beta..sub.1-42, A.beta..sub.1-40, rat
A.beta..sub.1-40, A.beta..sub.40-1, and A.beta..sub.1-28, (vehicle
conditions as in FIG. 14A).
[0089] FIG. 14C shows the effect of metal chelators (200 .mu.M) on
H.sub.2O.sub.2 production from A.beta..sub.1-42 when incubated in
the presence of Fe.sup.3+ or Cu.sup.2+ (1 .mu.M) (vehicle
conditions as in FIG. 14A). BC=Bathocuproinedisulfonate,
BP=Bathophenanthrolinedisulfonate- . The effects of DFO were
assessed in the presence of Fe.sup.3+, and TETA was assessed in the
presence of Cu.sup.2+, as indicated.
[0090] FIG. 14D shows H.sub.2O.sub.2 produced by A.beta..sub.1-42,
A.beta..sub.1-40, and Vitamin C in the presence of Fe.sup.3+ (1
.mu.M) (in PBS, pH 7.4 buffer, 1 hr, 37.degree. C.) under various
dissolved gas conditions (described in FIG. 13B): ambient air,
O.sub.2 enrichment, and anaerobic (Ar) conditions, as
indicated.
[0091] FIG. 14E shows H.sub.2O.sub.2 produced by A.beta..sub.1-2,
A.beta..sub.1-40, and Vitamin C in the presence of Cu.sup.2+ (1
.mu.M) (in PBS, pH 7.4 buffer, 1 hr, 37.degree. C.) under various
dissolved gas conditions (as in FIG. 14D). All data points are
means .+-.SD, n=3.
[0092] FIG. 15A and 15B are graphical representations showing
superoxide anion detection.
[0093] FIG. 15A shows the spectrophotometric absorbance at 250 nm
(after subtracting buffer blanks) for A.beta..sub.1-42 (10 .mu.M,
in PBS, pH 7.4, with 1 .mu.M Fe.sup.3+, incubated 1 hr, 37.degree.
C.) underambientair (+100 U/mL superoxide dismutase, SOD), O.sub.2
enrichment, and anaerobic (Ar). buffer gas conditions (described in
FIG. 13B).
[0094] FIG. 15B shows the spectrophotometric absorbance at 250 nm
(after subtracting buffer blanks) for variant A.beta. peptides:
A.beta..sub.1-42, A.beta..sub.1-40, rat A.beta..sub.1-40,
A.beta..sub.40-1, and A.beta..sub.1-28 (10 .mu.M in PBS, pH 7.4,
with 1 .mu.M Fe.sup.3+, incubated 1 hr, 37.degree. C., under
ambient buffer gas conditions). All data points are means .+-.SD,
n=3.
[0095] FIG. 16A and 16B are graphical representations showing
production of the hydroxyl radical (OH.) from the incubation of
A.beta. in the presence of substoichiometric amounts of Fe.sup.3+
or Cu.sup.2+.
[0096] FIG. 16A shows the signal from the TBARS assay of OHS
produced from Vitamin C (100 .mu.M) and variant A.beta. species (10
.mu.M): A.beta..sub.1-42, A.beta..sub.1-40, rat A.beta..sub.1-40,
A.beta..sub.40-1, and A.beta..sub.1-28 (in PBS, pH 7.4, with 1
.mu.M Fe.sup.3+ or Cu.sup.2+ as indicated, incubated 1 hr,
37.degree. C., under ambient buffer gas conditions).
[0097] FIG. 16B illustrates the effect of OH.-specific scavengers
upon OH. generation by Vitamin C and A.beta..sub.1-42. Mannitol (5
mM, Sigma) or dimethyl sulfoxide (DMSO, 5 mM, Sigma), was
co-incubated with Vitamin C (10 .mu.M+500 .mu.M H.sub.2O.sub.2) or
A.beta..sub.1-42 (10 .mu.M) (conditions as for FIG. 16A). All data
points are means .+-.SD, n=3.
[0098] FIG. 17 shows the reversibility of zinc-induced
A.beta..sub.1-40 aggregation with EDTA. Aggregation induced by pH
5.5 was not reversable in the same manner.
[0099] FIG. 18 shows the reversibility of zinc-induced aggregation
of A.beta..sub.1-40 mixed with 5% A.beta..sub.1-42.
[0100] FIGS. 19A-19C show dilution curves for TPEN, EGTA, and
bathocuproine, respectively, used in extracting a representative AD
brain sample. FIGS. 19A-19C show that metal chelators promote the
solubilization of A.beta. from human brain sample homogenates.
[0101] FIGS. 20A and 20B
[0102] FIG. 20A shows a western blot of chelation response in a
typical AD brain.
[0103] FIG. 20B shows a western blot comparing extracted A.beta.
from an AD brain (AD) to that of sedimentable deposits from healthy
brain tissue (young control--C). In the experiments of
[0104] FIG. 20B, TBS buffer was used rather than PBS.
[0105] FIG. 21 shows an indicative blot from AD brain extract. The
blot shows that chelation treatment results in disproportionate
solubilization of A.beta. dimers, while PBS alone does not.
[0106] FIG. 22 shows that recovery of total soluble protein is not
affected by the presence of chelators in the homogenization
step.
[0107] FIG. 23 is a graphical representation of resolubilization of
Zn, Cu, or pH induced aggregates in vitro. Values are expressed as
a percentage of A.beta. signal after washing with TBS alone.
[0108] FIG. 24 shows extraction of A.beta. from brain tissue with
clioquinol. Undiluted (100%) clioquinol is 1.6 .mu.M. S1 and S2
represent two sequential extractions from AD-affected tissue.
[0109] FIGS. 25A and 25B:
[0110] FIG. 24A shows a western blot of A.beta. extracted from
brain tissue by various concentrations of clioquinol.
[0111] FIG. 24B is a graphic representation of solubilization of
A.beta. by clioquinol.
[0112] FIG. 26 is a graph showing the proportion of total A.beta.
extracted utilizing PBS buffer alone, clioquinol (CQ),
bathocuproine (BC), or clioquinol together with bathocuproine
(CQ+BC).
[0113] FIG. 27 shows that extraction volume affects A.beta.
solubilisation.
[0114] FIGS. 28A and 28B
[0115] FIG. 28A shows the effect of metals upon the solubility of
brain-derived A.beta.: copper and zinc can inhibit the
solubilization of A.beta..
[0116] FIG. 28B shows that A.beta. solubility in metal-depleted
tissue is restored by supplementing with magnesium.
[0117] FIGS. 29A and 29B
[0118] FIG. 29A shows that patterns of chelator-promoted
solubilisation of A.beta. differ in AD and aged-matched, non-AD
tissue.
[0119] Upper panel: representative blot from AD specimen.
[0120] Lower panel: representative blot from aged non-AD tissue
bearing a similar total A.beta. load.
[0121] FIG. 29B shows soluble A.beta. resulting from chelation
treatment for AD and aged-matched, non-AD tissue, expressed as a
percentage of the PBS-only treatment group.
[0122] FIG. 30 shows that chelation promotes the solubilization of
A.beta..sub.1-40 and A.beta..sub.1-42 from AD and non-AD tissue.
Representative AD (left panels) and aged-matched control specimens
(right panels) were prepared as described in PBS or 5 mM BC.
Identical gels were run and Western blots were probed with mAbs WO2
(raised against residues 5-16, recognizes A.beta..sub.1-40 and
A.beta..sub.1-42) G210 (raised against residues 35-40, recognizes
A.beta..sub.1-40) or G211 (raised against residues 35-42,
recognizes A.beta..sub.1-42) (See Ida, N., et al., J. Biol. Chem.,
271:22908 (1996)).
[0123] FIG. 31A and 31B
[0124] FIG. 31A shows SDS-resistant polymerization of human
A.beta..sub.1-40 versus human A.beta..sub.1-42 with Zn.sup.2+ or
Cu.sup.2+.
[0125] FIG. 31B shows SDS-resistant polymerization of rat
A.beta..sub.1-40 with Cu.sup.2+ or Fe.sup.3+.
[0126] FIGS. 32A-32C
[0127] FIG. 32A shows H.sub.2O.sub.2/Cu induced SDS-resistant
polymerization of A.beta..sub.1-42 (2.5 .mu.M).
[0128] FIG. 32B shows H.sub.2O.sub.2/Fe induced SDS-resistant
polymerization of A.beta..sub.1-42 (2.5 .mu.M).
[0129] FIG. 32C shows that BC attenuates SDS-resistant
polymerization of A.beta..sub.1-42 (2.5 .mu.M).
[0130] FIGS. 33A and 33B show that H.sub.2O.sub.2 generation is
required for SDS-resistant polymerization of human
A.beta..sub.1-42. Solution concentrations of metal ion and
H.sub.2O.sub.2 were 30 .mu.M and 100 .mu.M, respectively.
[0131] FIG. 33A shows that TCEP (Tris(2-Carboxyethyl)-Phosphine
Hydrochloride) attenuates SDS-resistant A.beta..sub.1-42
polymerization. A.beta..sub.1-42 (2.5 .mu.M), H.sub.2O.sub.2 (100
.mu.M), ascorbic acid (100 .mu.M), TCEP (100 .mu.M).
[0132] FIG. 33B shows that anoxic conditions prevent SDS-resistant
A.beta. polymerization. A.beta..sub.1-42 (2.5 .mu.M) was incubated
with no metal or Cu.sup.2+ at either pH 7.4 or 6.6 and incubated
for 60 min. at 25.degree. C. under normal or argon purged
conditions. Argon was continuously bubbled through the buffer for 2
h (at 20.degree. C.) before the remainder of the incubation
components were added.
[0133] FIGS. 34A-34E show dissolution of SDS-resistant A.beta.
polymers.
[0134] FIG. 34A shows that chaotrophic agents are unable to disrupt
polymerization.
[0135] FIG. 34B shows that metal ion chelators disrupt
SDS-resistant A.beta..sub.1-40 polymers.
[0136] FIG. 34C shows that metal ion chelators disrupt
SDS-resistant A.beta..sub.1-42 polymers. The chelators, their log
stability constant, and their molecular weight, respectively, are
as follows: TETA (tetraethylenediamine), 20.4, 146; EDTA
(ethylenediaminetetra acetic acid), 18.1, 292; DTPA
(diethylenetriaminopenta acetic acid), 21.1, 393; CDTA
(trans-1,2-diaminocyclohexanetetra acetic acid), 22.0, 346; and NTA
(nitrilotriacetic acid), 13.1, 191.
[0137] FIG. 34D shows that .alpha.-helical promoting solvents and
low pH disrupt polymers. Aliquots of A.beta..sub.1-42 were
incubated at pH 1 or with DMSO/HFIP (75%:25%) for 2 h (30 min.,
37.degree. C.).
[0138] FIG. 34E shows that metal ion chelators disrupt
SDS-resistant A.beta. polymers extracted from AD brains. Aliquots
of SDS-resistant A.beta. polymers extracted from AD brains were
incubated with no chelator, TETA (1 mM or 5 mM) or BC (1 mM or 5
mM) for 2 h (30 min., 37.degree. C.) and aliquots collected for
analysis. Monomer A.beta..sub.40 is indicated.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0139] Definitions
[0140] In the description that follows, a number of terms are
utilized extensively. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided.
[0141] A.beta. peptide is also known in the art as A.beta., P
protein, .beta.-A4 and A4. In the present invention, the A.beta.
peptide may be comprised of peptides A.beta..sub.1-39
A.beta..sub.1-40 A.beta..sub.1-41, A.beta..sub.1-42, and
A.beta..sub.1-43. The most preferred embodiment of the invention
makes use of A.beta..sub.1-40. However, any of the A.beta. peptides
may be employed according to the present invention. A.beta.
peptides of the invention include A.beta..sub.X-39,
A.beta..sub.X-40, A.beta..sub.X-42, and A.beta..sub.X-43, where X
is less than or equal to 17; and A.beta..sub.Y-17, where Y is less
than or equal to 5. The sequence of A.beta. peptide is found in
Hilbich, C., et al., J. Mol. Biol. 228:460-473 (1992).
[0142] Amyloid as is commonly known in the art, and as is intended
in the present specification, is a form of aggregated protein.
[0143] Amyloidosis is any disease characterized by the
extracellular accumulation of amyloid in various organs and tissues
of the body.
[0144] A.beta. Amyloid is an aggregated A.beta. peptide. It is
found in the brains of patients afflicted with AD and DS and may
accumulate following head injuries.
[0145] Biological fluid means fluid obtained from a person or
animal which is produced by said person or animal. Examples of
biological fluids include but are not limited to cerebrospinal
fluid (CSF), blood, serum, and plasma. In the present invention,
biological fluid includes whole or any fraction of such fluids
derived by purification by any means, e.g., by ultrafiltration or
chromatography.
[0146] Copper(II), unless otherwise indicated, means salts of
Cu.sup.2+, i.e., Cu.sup.2+ in any form, soluble or insoluble.
[0147] Copper(I), unless otherwise indicated, means salts of
Cu.sup.+, i.e., Cu.sup.+ in any form, soluble or insoluble.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] Zinc, unless otherwise indicated, means salts of zinc, i.e.,
Zn.sup.2+ in any form, soluble or insoluble.
[0152] Methods for Identifying Agents Useful in the Treatment of
AD
[0153] The aim of the present invention is to clarify both the
factors which contribute to the neurotoxicity of A.beta. 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.beta., as well as the formation of
A.beta. polymers, and (ii) utilize such agents to develop methods
of preventing, treating or alleviating the symptoms of AD and
related disorders.
[0154] The present invention relates to the unexpected discovery
that A.beta. peptides directly produce oxidative stress through the
generation of abundant reactive oxygen species (ROS), which include
hydroxyl radical (OH.) and hydrogen peroxide (H.sub.2O.sub.2). The
production of ROS occurs by a metal (Cu, Fe) dependant, pH mediated
mechanism, wherein the reduction of Cu.sup.2+ to Cu.sup.+, or
Fe.sup.3+ to Fe.sup.2+, is catalyzed by A.beta.. A.beta. is highly
efficient at reducing Cu.sup.2+ and Fe.sup.3+.
[0155] All the redox properties of A.beta..sub.1-40 (the most
abundant form of soluble A.beta.) are exaggerated in
A.beta..sub.1-42. Additionally, A.beta..sub.1-42, but not
A.beta..sub.1-40, recruits O.sub.2 into spontaneous generation of
another ROS, O.sub.2.sup.-, which also occurs in a metal-dependent
manner. The exaggerated redox activity of A.beta..sub.1-42 and its
enhanced ability to generate ROS are likely to be the explanation
for its neurotoxic properties. Interestingly, the rat homologue of
A.beta., which has 3 substitutions that have been shown to
attenuate zinc binding and zinc-mediated precipitation, also
exhibits less redox activity than its human counterpart. This may
explain why the rat is exceptional in that it is the only mammal
that does not exhibit amyloid pathology with age. All other mammals
analyzed to date possess the human A.beta. sequence.
[0156] The sequence of ROS generation by A.beta. follows the
pathway of superoxide-dismutation, which leads to hydrogen peroxide
production in a Cu/Fe-dependent manner. After forming
H.sub.2O.sub.2, 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.beta. peptide, causing it to
polymerize. This is very likely to be the chemical mechanism that
causes the SDS-resistant polymerization that is seen in mature
plaque amyloid. Importantly, the redox activity of A.beta. is not
attenuated by precipitation of the peptide, suggesting that, in
vivo, amyloid deposits could be capable of generating ROS in situ
on an enduring basis. This suggests that the major source of the
oxidative stress in an AD-affected brain are amyloid deposits.
[0157] A model for free radical and amyloid formation in AD is
shown in FIG. 12. The proposed mechanism is explained as
follows.
[0158] (1) Soluble and precipitated A.beta. species possess
superoxide dismutase (SOD)-like activity. Superoxide (O.sub.2), the
substrate for the dismutation, is generated both by spillover from
mitochondrial respiratory metabolism, and by A.beta..sub.1-42
itself. A.beta.-mediated dismutation produces hydrogen peroxide
(H.sub.2O.sub.2)(see FIG. 11), requiring Cu.sup.2+ or Fe.sup.3+,
which are reduced during the reaction. Since H.sup.+ is required
for H.sub.2O.sub.2 production, an acidotic environment will
increase the reaction.
[0159] (2) H.sub.2O.sub.2 is relatively stable, and freely
permeable across cell membranes. Normally, it will be broken down
by intercellular catalase or glutathione peroxidase.
[0160] (3) In aging and AD, levels of H.sub.2O.sub.2 are high, and
catalase and peroxidase activities are low. If H.sub.2O.sub.2 is
not completely catalyzed, it will react with reduced Cu.sup.+ and
Fe.sup.2+ in the vicinity of A.beta. to generate the highly
reactive hydroxyl radical (OH.) by Fenton chemistry.
[0161] (4) OH. engenders a non-specific stress and inflammatory
response in local tissue. Among the neurochemicals that are
released from microglia and possibly neurons in the response are
Zn.sup.2+, Cu.sup.2+ and soluble A.beta.. Familial AD increases the
likelihood that A.beta..sub.1-42 will be released at this point.
Local acidosis is also part of the stress/inflammatory response.
These factors combine to make A.beta. precipitate and accumulate,
presumably so that it may function in situ as an SOD, since these
factors induce reversible aggregation. Hence, more soluble A.beta.
species decorate the perimeter of the accumulating plaque
deposits.
[0162] (5) If A.beta. encounters OH., it will cause covalently
cross-linking 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.
[0163] (6) If A.beta..sub.1-42 accumulates, it has the property of
recruiting O.sub.2 as a substrate for the abundant production of
O.sub.2.sup.- by a process that is still not understood. Since
O.sub.2 is abundant in the brain, A.beta..sub.1-42 is responsible
for setting off a vicious cycle in which the accumulation of
covalently linked A.beta. is a product of the unusual ability of
A.beta. to reduce O.sub.2, and feed an abundant substrate
(O.sub.2.sup.-) to itself for dismutation, leading to OH.
formation. The production of abundant free radicals by the
accumulating amyloid may further damage many systems including
metal regulatory proteins, thus compounding the problem. This
suggests that the major source of the oxidative stress in an
AD-affected brain are amyloid deposits.
[0164] The metal-dependent chemistry of A.beta.-mediated superoxide
dismutation is reminiscent of the activity of superoxide dismutase
(SOD). Interestingly, mutations of SOD cause amyotrophic lateral
sclerosis, another neurodegenerative disorder. SOD is predominantly
intracellular, whereas A.beta. is constitutively found in the
extracellular spaces where it accumulates. Investigation of A.beta.
by laser flash photolysis confirmed the peptide's SOD-like
activity, suggesting that A.beta. may be an anti-oxidant under
physiological circumstances. Since H.sub.2O.sub.2 has been shown to
induce the production of A.beta., the accumulation of A.beta. in AD
may reflect a response to an oxidant stress paradoxically caused by
A.beta. excess. This may cause and, in turn, be compounded by,
damage to the biometal homeostatic mechanisms in the brain
environment.
[0165] Thus, it has recently been discovered (i) that much of the
A.beta. aggregate in AD-affected brain is held together by zinc and
copper, (ii) that A.beta. peptides exhibit Fe/Cu-dependent redox
activity similar to that of SOD, (iii) that A.beta..sub.1-42 is
especially redox reactive and has the unusual property of reducing
O.sub.2 to O.sub.2.sup.-, and (iv) that deregulation of A.beta.
redox reactivity causes the peptide to conveniently polymerize.
Since these reactions must be strongly implicated in the
pathogenetic events of AD, they offer promising targets for
therapeutic drug design.
[0166] The discovery that A.beta. can generate H.sub.2O.sub.2 and
Cui, both of which are associated with neurotoxic effects, offers
an explanation for the neurotoxicity of A.beta. polymers. These
findings suggest that it may be possible to lessen the
neurotoxicity of A.beta. by controlling factors which alter the
concentrations of Cu.sup.+ and ROS, including hydrogen peroxide,
being generated by accumulated and soluble A.beta.. It has been
discovered that manipulation of factors such as zinc, copper, and
pH can result in altered Cu.sup.+ and H.sub.2O.sub.2 production by
A.beta.. Therefore, agents identified as being useful for the
adjustment of the pH and levels of zinc and copper of the brain
interstitium can be used to adjust the concentration of Cu.sup.+
and H.sub.2O.sub.2, and can therefore be used to reduce the
neurotoxic burden. Such agents will thus be a means of treating
Alzheimer's disease.
[0167] Agents Useful in the Treatment of AD
[0168] 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.sub.2.sup.- and
biometal-dependent pathway of free radical generation by A.beta.
peptides. The radicals of this aspect of the present invention may
comprise reactive oxygen species (ROS) such as but not limited to
O.sub.2.sup.- and OH as well as radicalized A.beta. peptides. It is
proposed, according to the present invention, that by interfering
in the radical generating pathway, the neurotoxicity of the A.beta.
peptides is reduced.
[0169] Accordingly, one aspect of the present invention
contemplates a method for treating Alzheimer's disease (AD) in a
subject, said method comprising administering to said subject an
effective amount of an agent which is capable of inhibiting or
otherwise reducing metal-mediated production of free radicals.
[0170] The preferred agents according to this aspect are metal
chelators, metal complexing compounds, antioxidants and compounds
capable of reducing radical formation of A.beta. peptides or
mediated by A.beta. peptides. Particularly preferred metal
chelators and metal complexors are capable of interacting with
metals (M) having either a reduced charge state M.sup.n+ or an
oxidized state of M.sup.(n+1)+. Even more particularly, M is Fe
and/or Cu.
[0171] It is proposed that interactions of A.beta. with Fe and Cu
are of significance to the genesis of the oxidation insults that
are observed in the AD-affected brain. This is due to redox-active
metal ions being concentrated in brain neurons and participating in
the generation of ROS or other radicals by transferring electrons
in their reduced state and described in the following
reactions:
[0172] Reduced Fe/Cu reacts with molecular oxygen to generate the
superoxide anion.
M.sup.n++O.sub.2.fwdarw.M.sup.(n+1)++O.sub.2.sup.- Reaction (1)
[0173] The O.sub.2.sup.- generated undergoes dismutation to
H.sub.2O.sub.2 either catalyzed by SOD or spontaneously.
O.sub.2.sup.-+O.sub.2.sup.-+2H.sup.+.fwdarw.H.sub.2O.sub.2+O.sub.2
Reaction (2)
[0174] The reaction of reduced metals with H.sub.2O.sub.2 generates
the highly reactive hydroxyl radical by the Fenton reaction.
M.sup.n++H.sub.2O.sub.2.fwdarw.M.sup.(n+1)++OH+OH.sup.- Reaction
(3)
[0175] Additionally, the Haber-Weiss reaction can form OH in a
reaction catalyzed by M.sup.(n+1)+/M.sup.n+ (Miller et al.,
1990).
O.sub.2.sup.-+H.sub.2O.sub.2.fwdarw.OH+OH.sup.-+O.sub.2 Reaction
(4)
[0176] Still more preferably, the agent comprises one or more of
bathocuproine and/or bathophenanthroline or compounds related
thereto at the structural and/or functional levels. Reference to
compounds such as bathocuproine and bathophenanthroline include
functional derivatives, homologues and analogues thereof.
[0177] Accordingly, another aspect of the present invention
provides a method for treating AD in a subject said method
comprising administering to said subject Ian 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 I or otherwise reduce metal-mediated production of free
radicals.
[0178] In one aspect, the free radicals are reactive oxygen species
such as O.sub.2.sup.- or OH. In another aspect, the free radicals
include forms of A.beta.. However, in a broader sense, it has been
found that the metal-mediated A.beta. reactions in the brain of AD
patients results in the generation of reduced metals and hydrogen
peroxide, as well as superoxide and hydroxyl radicals. Furthermore,
formation of any other radical or reactive oxygen species by
interaction of any of these products with any other metabolic
substrate (e.g., superoxide+nitric acid=peroxynitrite) contributes
to the pathology observed in AD and Down's syndrome patients.
Cu.sup.2+ reaction with A.beta. generates Cu.sup.+, A.beta..,
O.sub.2.sup.-, H.sub.2O.sub.2, and OH., all of which not only
directly damage the cells, but also react with biochemical
substrates like nitric oxide.
[0179] 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.beta. peptides for a time and under
conditions sufficient to inhibit or otherwise reduce production of
radicals.
[0180] 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.sup.+) and
iron (Fe.sup.2+).
[0181] Another mechanism elucidated in accordance with the present
invention concerns the formation of aggregates of A.beta., as in
conditions involving amyloidosis. In a preferred embodiment, the
aggregates are those of amyloid plaques occurring in the brains of
AD-affected subjects.
[0182] 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.beta.
aggregates.
[0183] The data indicate that Zn-induced A.beta.1-40 aggregation is
completely reversible in the presence of divalent metal ion
chelating agents. This suggests that zinc binding may be a
reversible, normal function of A.beta. and implicates other
neurochemical mechanisms in the formation of amyloid. A process
involving irreversible A.beta. aggregation, such as the
polymerization of A.beta. monomers, in the formation of polymeric
species of A.beta. that are present in amyloid plaques is thus a
more plausible explanation for the formation of neurotoxic
polymeric A.beta. species.
[0184] According to this aspect of the present invention, there is
provided a method of treating AD in a subject comprising
administering to said subject an agent capable of promoting,
inducing or otherwise facilitating resolubilization of amyloid
deposits for a time and under conditions to effect said
treatment.
[0185] With respect to this aspect of the present invention, it is
proposed that a metal chelator or metal complexing agent be
administered. A.beta. 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 70%.
[0186] In addition, the agent of this aspect of the present
invention may comprise a metal chelator or metal complexing agent
alone or in combination with another active ingredient such as but
not limited to rifampicin, disulfiram, indomethacin or related
compounds. Preferred metal chelators are DTPA, bathocuproine,
bathophenanthroline, and penicillamine or related compounds.
[0187] 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.
[0188] Accordingly, the present invention contemplates compositions
such as pharmaceutical compositions comprising an active agent and
one or more pharmaceutically, acceptable carriers and/or diluents.
The active agent may be a single compound such as a metal chelator
or metal complexing agent or may be a combination of compounds such
as a metal chelating or complexing compound and another compound.
Preferred active agents include, for reducing radical formation,
bathocuproine and/or bathophenanthroline; and for promoting
resolubilization, DTPA, bathocuproine, bathophenanthroline, and
penicillamine or derivatives, homologues or analogues thereof, or
any combination thereof.
[0189] Further, it has been found that the agents of the present
invention may be administered along with the compound clioquinol.
Clioquinol has been shown to be particularly effective in
resolubilizing A.beta. aggregates in combination with other
chelators. Most preferrably clioquinol is administered in
combination with bathocuproine.
[0190] It has also been found that there is a clioquinol
concentration "window" within which the A.beta. aggregates are
dissolved. Increasing the concentration of clioquinol above the
optimal window concentration not only is toxic to the patient but
also sharply drops the dissolution effect of clioquinol on the
A.beta. amyloid. Similarly, amounts below that of the window are
too small to result in any dissolution.
[0191] Therefore, for each given patient, the attending physician
need be mindful of the window effect and attend to varying the
dosages of clioquinol so that during the course of administration,
clioquinol concentrations would be varied frequently to randomly
allow achieving the most effective concentration for dissolving
A.beta. amyloid deposits in the given patient.
[0192] It is, therefore, desired that the plasma levels of
clioquinol not be steady state, but be kept fluctuating between
0.01 .mu.M, but not greater than 2 .mu.M. Since the drug is
absorbed to reach peak plasma levels within 30 minutes of oral
ingestion, and since the excretion half life is about 1-3 hours,
the best way to dose the patient is with oral doses 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.
[0193] An oral dose of 200 mg/kg achieves 5 .mu.M plasma levels in
rats, and 10-30 .mu.M in dogs. An oral dose of 500 mg/kg achieves
20-70 .mu.M in monkeys. The drug is freely permeable into the brain
and is rapidly excreted.
[0194] Therefore, in humans, it is expected that a plasma level of
0.5 .mu.M would be achieved within 30 minutes of ingesting 10 mg/kg
body weight. In a 70 kg person this is 700 mg of clioquinol.
Therefore, a dose of 700 mg four times a day (2800 mg/day) would be
therapeutic.
[0195] However, sustained treatment with doses of clioquinol at a
dose as low as 10 mg/kg/day causes the neurological side effect,
subacute myclo-optic neuritis. Therefore, dosage that high is
undesirable. This is equivalent to 700 mg/day. The side effect is
believed to be due to loss of vitamin B12. Therefore, co-therapy
with vitamin B12 100 .mu.M/day orally or, preferably, 1000
.mu.M/month intramuscularly, is to be administered with clioquinol
treatment to abolish this side effect.
[0196] To minimize the chances of this side effect, a lower dose of
clioquinol can also be used -100 mg, three or four times a day
would achieve peak plasma levels of about 0.1 .mu.M, and is likely
to be therapeutic without putting the patient at risk for
neurological side effects. Nevertheless, co-administration of
Vitamin B12 should be mandatory.
[0197] For the treatment of moderately affected or severely
affected patients, where risking the neurological side effects is
less of a concern since the quality of their life is very poor, the
patient may be put on a program of treatment (after informed
consent) consisting of high dose clioquinol 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 clioquinol therapy, the treatment should be
discontinued. All regimens would be accompanied by Vitamin B12
co-therapy.
[0198] Another typical case would be the treatment of a mildly
affected individual. Such a patient would be treated with low dose
clioquinol for up to 12 months. If after 6 months no clinical gains
have been made, the patient could then be placed on the high/low
alternation regimen for up to another 12 months.
[0199] Particular concentrations and modes of therapy will vary
depending on the particular clioquinol-containing combination
administered.
[0200] Accordingly, the present invention contemplates compositions
such as pharmaceutical compositions comprising an active agent and
one or more pharmaceutically, acceptable carriers and/or diluents.
The active agent may be clioquinol or a combination of clioquinol
and another metal chelating compound.
[0201] The pharmaceutical forms containing the active agents may be
administered in any convenient manner either orally or parenteraly
such as by intravenous, intraperitoneal, subcutaneous, rectal,
implant, transdermal, slow release, intrabuccal, intracerebral or
intranasal administration. Generally, the active agents need to
pass the blood brain barrier and may have to be chemically
modified, e.g. made hydrophobic, to facilitate this or be
administered directly to the brain or via other suitable routes.
For injectable use, sterile aqueous solutions (where water soluble)
are generally used or alternatively sterile powders for the
extemporaneous preparation of sterile injectable solutions may be
used. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol, and the like), suitable mixtures thereof, and
vegetable oils. The preventions of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thirmerosal and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0202] 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. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and the freeze-drying technique which yield a
powder of the active ingredient plus any additional desired
ingredient from previously sterile-filtered solution thereof.
Preferred compositions or preparations according to the present
invention are prepared so that an injectable dosage unit contains
between about 0.25 .mu.g and 500 mg of active compound.
[0203] When the active agents are suitably protected they may be
orally administered, for example, with an inert diluent or with an
assimilable edible carrier, or it may be enclosed in hard or soft
shell gelatin capsule, or it may be compressed into tablets, or it
may be incorporated directly with the food of the diet. For oral
therapeutic administration, the active compound may be incorporated
with excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least 1% by weight of active compound. The percentage of the
compositions and preparations may, of course, be varied and may
conveniently be between about 5 to about 80% of the weight of the
unit. The amount of active compound in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
Preferred compositions or preparations according to the present
invention are prepared so that an oral dosage unit form contains
between about 1.0 .mu.g and 2000 mg of active compound.
[0204] The tablets, troches, pills, capsules and the like may also
contain other components such as listed hereafter: A binder such as
gum, acacia, corn starch or gelatin; excipients such as dicalcium
phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid and the like; a lubricant such as magnesium
stearate; and a sweetening agent such a sucrose, lactose or
saccharin may be added or a flavoring agent such as peppermint, oil
of wintergreen, or cherry flavoring. When the dosage unit form is a
capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills, or capsules may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propylparabens
as preservatives, a dye and flavoring such as cherry or orange
flavor. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compound(s) may be
incorporated into sustained-release preparations and
formulations.
[0205] 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.
[0206] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit
forms of the invention are dictated by and directly dependent on
(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.
[0207] 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
fig 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.
[0208] 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.
[0209] The pharmaceutical compositions of the invention may be
administered to any animal which may experience the beneficial
effects of the compounds of the invention. Foremost among such
animals are mammals, e.g., humans, although the invention is not
intended to be so limited.
[0210] The following examples are provided by way of illustration
to further describe certain preferred embodiments of the invention,
and are not intended to be limiting of the present invention,
unless specified.
EXAMPLES
Example 1
Copper-induced, pH Dependent Aggregation of A.beta.
[0211] Materials and Methods
[0212] a) Preparation of A.beta. Stock
[0213] Human A.beta. 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, Conn.). Synthetic A.beta.
peptide solutions were dissolved in trifluoroethanol (30% in
Milli-Q water (Millipore Corporation, Milford, Mass.)) or 20 mM
HEPES (pH 8.5) at a concentration of 0.5-1.0 g/ml, centrifuged for
20 min. at 10,000 g and the supernatant (stock A.beta.) used for
subsequent aggregation assays on the day of the experiment. The
concentration of stock A.beta. was determined by UV spectroscopy at
214 nm or by Micro BCA protein assay (Pierce, Rockford, Ill.). The
Micro BCA assay was performed by adding 10 .mu.l of stock A.beta.
(or bovine serum albumin standard) to 140 .mu.l of distilled water,
and then adding an equal volume of supernatant (150 .mu.l) to a
96-well plate and measuring the absorbance at 562 nm. The
concentration of A.beta. was determined from the BSA standard
curve. Prior to use all buffers and stock solutions of metal ions
were filtered though a 0.22 .mu.m filter (Gelan Sciences, Ann
Arbor, Mich.) to remove any particulate matter. All metal ions were
the chloride salt, except lead nitrate.
[0214] b) Aggregation Assays
[0215] A.beta. stock was diluted to 2.5 .mu.M in 150 mM NaCl and 20
mM glycine (pH 3-4.5), MES (pH 5-6.2) or HEPES (pH 6.4-8.8), with
or without metal ions, incubated (30 min., 37.degree. C.),
centrifuged (20 min., 10000 g). The amount of protein in the
supernatant was determined by the Micro BCA protein assay as
described above.
[0216] c) Turbidometric Assays
[0217] Turbidity measurements were performed as described by Huang,
X., et al., J. Biol. Chem. 272:26464-26470 (1997), except A.beta.
stock was brought to 10 .mu.M (300 .mu.l) in 20 mM HEPES buffer,
150 mM NaCl (pH 6.6, 6.8 or 7.4) with or without metal ions prior
to incubation (30 min., 37.degree. C.). To investigate the pH
reversibility of Cu.sup.2+-induced A.beta. aggregation, 25 .mu.M
A.mu..sub.1-40 and 25 .mu.M Cu.sup.2+ were mixed in 67 mM phosphate
buffer, 150 mM NaCl (pH 7.4) and turbidity measurements were taken
at four 1 min. intervals. Subsequently, 20 .mu.l aliquots of 10 mM
EDTA or 10 mM Cu.sup.2+ were added into the wells alternatively,
and, following a 2 min. delay, a further four readings were taken
at 1 min. intervals. After the final EDTA addition and turbidity
reading, the mixtures were incubated for an additional 30 min.
before taking final readings. To investigate the reversibility of
pH mediated Cu.sup.2+-induced A.beta..sub.1-40 aggregation, 10
.mu.M A.beta. and 30 .mu.M Cu.sup.2+ were mixed in 67 mM phosphate
buffer, 150 mM NaCl (pH 7.4) and an initial turbidity measurement
taken. Subsequently, the pH of the solution was successively
decreased to 6.6 and then increased back to 7.5. The pH of the
reaction was monitored with a microprobe (Lazar Research
Laboratories Inc., Los Angeles, Calif.) and the turbidity read at 5
min. intervals for up to 30 min. This cycle was repeated three
times.
[0218] d) Immunofittration Detection of Low Concentrations of
A.beta..sub.1-40 Aggregate
[0219] Physiological concentrations of A.beta. (8 nM) were brought
to 150 mM NaCl, 20 mM HEPES (pH 6.6 or 7.4), 100 nM BSA with
CuCl.sub.2 (0, 0.1, 0.2, 0.5 and 2 .mu.M) and incubated (30 min.,
37.degree. C.). The reaction mixtures (200 .mu.l) were then placed
into the 96-well Easy-Titer ELIFA system (Pierce, Rockford, Ill.)
and filtered through a 0.22 .mu.m cellulose acetate filter (MSI,
Westboro, Mass.). Aggregated particles were fixed to the membrane
(0.1% glutaraldehyde, 15 min.), washed thoroughly and then probed
with the anti-A.beta. mAB 6E10 (Senetek, Maryland Heights, Mich.).
Blots were washed and exposed to film in the presence of ECL
chemiluminescence reagents (Amersham, Buckinghamshire, England).
Immunoreactivity was quantified by transmitance analysis of ECL
film from the immunoblots.
[0220] e) A.beta. Metal-capture ELISA
[0221] A.beta. (1.5 ng/well) was incubated (37.degree. C., 2 hr) in
the wells of Cut coated microtiter plates (Xenopore, Hawthorne,
N.J.) with increasing concentrations of Cu.sup.2+ (1-100 nM).
Remaining ligand binding sites on well surfaces were blocked with
2% gelatin in tris-buffered saline (TBS) (3 hr at 37.degree. C.)
prior to overnight incubation at room temperature with the
anti-A.beta. mAb 6E10 (Senetek, Maryland Heights, Mich.).
Anti-mouse IgG coupled to horseradishperoxidase was then added to
each well and incubated for 3 hr at 37.degree. C. Bound antibodies
were detected by a 30 minute incubation with stable peroxidase
substrate buffer/3,3',5,5'-Tetramethyl benzidine (SPSB/TMB) buffer,
followed by the addition of 2 M sulfuric acid and measurement of
the increase in absorbance at 450 nm.
[0222] f) Extraction of A.beta. from Post-mortem Brain Tissue
[0223] Identical regions of frontal cortex (0.5 g) 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.beta. content by western blotting using monoclonal antibody (mAb)
WO2. The data shows a typical (of n=12 comparisons) result
comparing the amount of A.beta. extracted into the supernatant
phase in AD compared to control (young adult) samples.
N,N,N',N'-tetrakis [2-pyridyl-methyl] ethylenediamine (TPEN) (5
.mu.M) allows the visualization of a population of pelletable
A.beta. that had not previously been recognized in unaffected brain
samples (FIG. 8).
[0224] g) A.beta. Polymerization by Copper
[0225] Cu.sup.2+-induced SDS-resistant oligomerization of A.beta.:
A.beta. (2.5 .mu.M), 150 mM NaCl, 20 mM hepes (pH 6.6, 7.4, 9) with
or without ZnCl.sub.2 or CuCl.sub.2. Following incubation
(37.degree. C.), aliquots of each reaction (2 ng peptide) were
collected at 0 d, 1 d, 3 d and 5 d and western blotted using
anti-A.beta. monoclonal antibody 8E10 (FIG. 9). Migration of the
molecular size markers are indicated (kDa). The dimer formed under
these conditions has been found to be SDS-resistant. Cu.sup.2+
(2-30 .mu.M) induced SDS-resistant polymerization of peptide.
Co-incubation with similar concentrations of Zn.sup.2+ accelerates
the polymerization, but zinc alone has no effect. The antioxidant
sodium metabisulfite moderately attenuates the reaction, while
ascorbic acid dramatically accelerates A.beta. polymerization. This
suggests reduction of Cu.sup.2+ to Cu.sup.+ with the latter
mediating SDS-resistant polymerization of A.beta.. Mannitol also
abolishes the polymerization, suggesting that the polymerization is
mediated by the generation of the hydroxyl radical by a Fenton
reaction that recruits Cu.sup.+. It should be noted that other
means of visualizing and/or determining the presence or absence of
polymerization other than western blot analysis may be used. Such
other means include but are not limited to density sedimentation by
centrifugation of the samples.
[0226] Results
[0227] It has previously been reported that Zn.sup.2+ induces rapid
precipitation of A.beta.in vitro (Bush, A. I., et al., Science
265:1464 (1994)). This metal has an abnormal metabolism in AD and
is highly concentrated in brain regions where A.beta. precipitates.
The present data indicate that under very slightly acidic
conditions, such as in the lactic acidotic AD brain, Cu.sup.2+
strikingly induces the precipitation of A.beta. through an unknown
conformational shift. pH alone dramatically affects A.beta.
solubility, inducing precipitation when the pH of the incubation
approaches the pl of the peptide (pH 5-6). Zinc induces 40-50% of
the peptide to precipitate at pH>6.2, below pH 6.2 the
precipitating effects of Zn.sup.2+ and acid are not summative. At
pH<5, Zn.sup.2+ has little effect upon A.beta. solubility.
Cu.sup.2+ is more effective than Zn.sup.2+ in precipitating A.beta.
and even induces precipitation at the physiologically relevant pH
6-7. Copper-induced precipitation of A.beta. occurs as the pH falls
below 7.0, comparable with conditions of acidosis (Yates, C. M., et
al., J. Neurochem. 55:1624 (1990)) in the AD brain. Investigation
of the precipitating effects of a host or other metal ions in this
system indicated that metal ion precipitation of A.beta. was
limited to copper and zinc, as illustrated, although Fe.sup.2+
possesses a partial capacity to induce precipitation (Bush, A. I.,
et al., Science 268:1921 (1995)).
[0228] On the basis these in vitro findings, the possibility that
A.beta. deposits in the AD-affected brain may be held in assembly
by zinc and copper ions was investigated. Roher and colleagues have
recently shown that much of the A.beta. that deposits in
AD-affected cortex can be solubilized in water (Roher, A. E, et
al., J. Biol. Chem. 271:20631 (1996)). Supporting the clinical
relevance of in vitro findings, it has recently been demonstrated
that metal chelators increase the amount of A.beta. extracted by
Roher's technique (in neutral saline buffer), and that the
extraction of A.beta. is increased as the chelator employed has a
higher affinity for zinc or copper. Hence TPEN is highly efficient
in extracting A.beta., 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.mu.M). Zinc and
copper ions (5-50 .mu.M) added back to the extracting solution
abolish the recovery of A.beta. (which is subsequently extracted by
the SDS sample buffer in the pellet fraction of the centrifuged
brain homogenate suspension), but Ca.sup.2+ and Mg.sup.2+ added
back to the chelator-mediated extracts of A.beta. cannot abolish
A.beta. resolubilization from AD-affected tissue even when these
metal ions are present in millimolar concentrations.
[0229] Importantly, atomic absorption spectrophotometry assays of
the metal content of the chelator-mediated extracts confirms that
Cu and Zn are co-released with A.beta. by the chelators, along with
lower concentrations of Fe. These data strongly indicate that
A.beta. deposits (probably of the amorphous type) are held together
by Cu and Zn and may also contain Fe. Interestingly, A.beta. is not
extractable from control brain without the use of chelators. This
suggests that metal-assembled A.beta. deposits may be the earliest
step in the evolution of A.beta. plaque pathology.
[0230] These findings propelled further inquiries into chemistry of
metal ion-A.beta. interaction. The precipitating effects upon
A.beta. of Zn.sup.2+ and Cu.sup.2+ were found to be qualitatively
different. Zn-mediated aggregation is reversible with chelation and
is not associated with neurotoxicity in primary neuronal cell
cultures, whereas Cu-mediated aggregation is accompanied by the
slow formation of covalently-bonded SDS-resistant dimers and
induction of neurotoxicity. These neurotoxic SDS-resistant dimers
are similar to those described by Roher (Roher, A. E, et al., J.
Biol. Chem. 271:20631(1996)).
[0231] To accurately quantitate the effects of different metals and
pH on A.beta. solubility, synthetic human A.beta..sub.1-40 (2.5
.mu.M) was incubated (37.degree. C.) in the presence of metal ions
at various pH for 30 min. The resultant aggregated particles were
sedimented by centrifugation to permit determination of soluble
A.beta..sub.1-40 in the supernatant. To determine the
centrifugation time required to completely sediment the aggregated
particles generated under these conditions, A.beta..sub.1-40 was
incubated for 30 min at 37.degree. C. with no metal, Zn.sup.2+ (100
.mu.M), Cu.sup.2+ (100 .mu.M) and pH (5.5). Reaction mixtures were
centrifuged at 10000 g for different times, or ultracentrifuged at
100000 g for 1 h. (FIG. 1). FIG. 1 shows the proportion of soluble
A.beta..sub.1-40 remaining following centrifugation of reaction
mixtures. A.beta..sub.1-40 was incubated (30 min., 37.degree. C.)
with no metal, under acidic conditions (pH 5.5), Zn.sup.2+ (100
.mu.M) or Cu.sup.2+ (100 .mu.M), and centrifuged at 10000 g for
different time intervals, or at 100,000 g (ultracentrifuged) for 1
h for comparison. All data points are means .+-.SD, n=3.
[0232] Given that conformational changes within the N-terminal
domain of A.beta. are induced by modulating [H.sup.+] (Soto, C., et
al., J. Neurochem. 63:1191-1198 (1994)), and that there is a metal
(Zn.sup.2+) binding domain in the same region, experiments were
designed to determine whether there was a synergistic effect of pH
on metal ion-induced A.beta. aggregation. A.beta..sub.1-40 was
incubated with different bioessential metal ions at pH 6.6, 6.8 and
7.4. The results are show in FIG. 2A, where "all metals" indicates
incubation with a combination containing each metal ion at the
nominated concentrations, concurrently. FIG. 2A shows the
proportion of soluble A.beta..sub.1-40 remaining in the supernatant
after incubation (30 min., 37.degree. C.) with various metals ions
at pH 6.6, 6.8 or 7.4 after centrifugation (10,000 g, 20 min.).
[0233] The [H.sup.+] chosen represented the most extreme, yet
physiologically plausible [H.sup.+] that A.beta..sub.1-40 would be
likely to encounter in vivo. The ability of different bioessential
metal ions to aggregate A.beta..sub.1-40 at increasing H.sup.+
concentrations fell into two groups; Mg.sup.2+, Ca.sup.2+,
Al.sup.3+, Co.sup.2+, Hg.sup.2+, Fe.sup.3+, Pb.sup.2+ and Cu.sup.2+
showed increasing sensitivity to induce
A.beta..sub.1-40,aggregation, while Fe.sup.2+, Mn.sup.2+,
Ni.sup.2+, and Zn.sup.2+ were insensitive to alterations in
[H.sup.+] in their ability to aggregate A.beta..sub.1-40. Cu.sup.2+
and Hg.sup.2+ induced most aggregation as the [H.sup.+] increased,
although the [H.sup.+] insensitive Zn.sup.2+-induced aggregation
produced a similar amount of aggregation. Fe.sup.2+, but not
Fe.sup.3+, also induced considerable aggregation as the [H.sup.+]
increased, possibly reflecting increased aggregation as a result of
increased crosslinking of the peptide.
[0234] Similar results were obtained when these experiments were
repeated using turbidometry as an index of aggregation (FIG. 2B).
The data indicate the absorbance changes between reaction mixtures
with and without metal ions at pH 6.6, 6.8 or 7.4. Thus,
A.beta..sub.1-40 has both a pH insensitive and a pH sensitive metal
binding site. At higher concentrations of metal ions this pattern
was repeated, except Co.sup.2+ and Al.sup.3+-induced A.beta.
aggregation became pH insensitive, and Mn became sensitive (FIG.
2C).
[0235] Since .sup.64Cu is impractically short-lived (t1/2=13 h), a
novel metal-capture ELISA assay was used to perform competition
analysis of A.beta..sub.1-40 binding to a microtiter plate
impregnated with Cu.sup.2+, as described in Materials and Methods.
Results are shown in FIG. 3. All assays were performed in
triplicate and are means .+-.SD, n=3. Competition analysis revealed
that A.beta..sub.1-40 has at least one high affinity, saturable
Cu.sup.2+ binding site with a Kd=900 pM at pH 7.4 (FIG. 3). The
affinity of A.beta. for Cu.sup.2+ is higher than that for Zn.sup.2+
(Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994)). Since
Cu.sup.2+ does not decrease Zn.sup.2+-induced aggregation (Bush, A.
I., et al., J. Biol. Chem. 269:12152 (1994)), indicating Cu.sup.2+
does not displace bound Zn.sup.2+, there are likely to be two
separate metal binding sites. This is supported by the fact that
there is both a pH sensitive and insensitive interaction with
different metal ions.
[0236] Since the conformational state and solubility of A.beta. is
altered at different pH (Soto, C., et al., J. Neurochem.
63:1191-1198 (1994)), the effects of [H.sup.+] on Zn.sup.2+- and
Cu.sup.+-induced A.beta..sub.1-40 aggregation were studied. Results
are shown in FIGS. 4A, 4B and 4C. FIG. 4A shows the proportion of
soluble A.beta..sub.1-40 remaining in the supernatant following
incubation (30 min., 37.degree. C.) at pH 3.0-8.8 in buffered
saline .+-.Zn.sup.2+ (30 .mu.M) or Cu.sup.2+ (30 .mu.M) and
centrifugation (10000 g, 20 min.), expressed as a percentage of
starting peptide. All data points are means .+-.SD, n=3. [H.sup.+]
alone precipitates A.beta..sub.1-40 (2.5 .mu.M) as the solution is
lowered below pH 7.4, and dramatically once the pH falls below 6.3
(FIG. 4A). At pH 5.0, 80% of the peptide is precipitated, but the
peptide is not aggregated by acidic environments below pH 5,
confirming and extending earlier reports on the effect of pH on
A.beta. solubility (Burdick, D., J. Biol. Chem. 267:546-554
(1992)). Zn.sup.2+ (30 .mu.M) induced a constant level (.about.50%)
of aggregation between pH 6.2-8.5, while below pH 6.0, aggregation
could be explained solely by the effect of [H.sup.+].
[0237] In the presence of Cu.sup.2+ (30 .mu.M), a decrease in pH
from 8.8 to 7.4 induced a marked drop in A.beta..sub.1-40
solubility, while a slight decrease below pH 7.4 strikingly
potentiated the effect of Cu.sup.2+ on the peptide's aggregation.
Surprisingly, Cu.sup.2+ caused >85% of the available peptide to
aggregate by pH 6.8, a pH which plausibly represents a mildly
acidotic environment. Thus, conformational changes in A.beta.
brought about by small increases in [H.sup.+] result in the
unmasking of a second metal binding site that leads to its rapid
self-aggregation. Below pH 5.0, the ability of both Zn.sup.2+ and
Cu.sup.2+ to aggregate A.beta. was diminished, consistent with the
fact that Zn binding to A.beta. is abolished below pH 6.0 (Bush, A.
I., et al., J. Biol. Chem. 269:12152 (1994)), probably due to
protonation of histidine residues.
[0238] The relationship between pH and Cu.sup.2+ on
A.beta..sub.1-40 solubility was then further defined by the
following experiments (FIG. 4B). The proportion of soluble
A.beta..sub.1-40 remaining in the supernatant after incubation (30
min., 37.degree. C.) at pH 5.4-7.8 with different Cu.sup.2+
concentrations (0, 5, 10, 20, 30 .mu.M), and centrifugation (10,000
g, 20 min.), was measured and expressed as a percentage of starting
peptide. All data points are means .+-.SD, n=3. At pH 7.4,
Cu.sup.2+-induced A.beta.aggregation was 50% less than that induced
by Zn.sup.2+ over the same concentration range, consistent with
earlier reports (Bush, A. I., et al., J. Biol. Chem. 269:12152
(1994)). There was a potentiating relationship between [H.sup.+]
and [Cu.sup.2+] in producing A.beta. aggregation; as the pH fell,
less Cu.sup.2+ was required to induce the same level of
aggregation, suggesting that [H.sup.+] is controlling
Cu.sup.2+induced A.beta..sub.1-40 aggregation.
[0239] To confirm that this reaction occurs at physiological
concentrations of A.beta..sub.1-40 and Cu.sup.2+, a novel
filtration immunodetection system was employed. This technique
enabled the determination of the relative amount of
A.beta..sub.1-40 aggregation in the presence of different
concentrations of H.sup.+ and Cu.sup.2+ (FIG. 4C). The relative
aggregation of nM concentrations of A.beta..sub.1-40 at pH 7.4 and
pH 6.6 in the presence of different Cu.sup.2+ concentrations (0,
0.1, 0.2, 0.5 .mu.M) were determined by this method. Data represent
mean reflectance values of immunoblot densitometry expressed as a
ratio of the signal obtained when the peptide is treated in the
absence of Cu.sup.2+. All data points are means .+-.SD, n=2.
[0240] This sensitive technique confirmed that physiological
concentrations of A.beta..sub.1-40 are aggregated under mildly
acidic conditions and that aggregation was greatly enhanced by the
presence of Cu.sup.2+ at concentrations as low as 200 .mu.M.
Furthermore, as previously observed at higher A.beta..sub.1-40
concentrations, a decrease in pH from 7.4 to 6.6 potentiated the
effect of Cu.sup.2+ on aggregation of physiological concentrations
of A.beta..sub.1-40. Thus, A.beta..sub.1-40 aggregation is
concentration independent down to 8 nM where Cu.sup.2+ is
available.
[0241] It has recently been shown that Zn.sup.2+ mediated
A.beta..sub.1-40 aggregation is reversible whereas A.beta..sub.1-40
aggregation induced by pH 5.5 was irreversible. Therefore,
experiments were performed to determine whether
Cu.sup.2+/pH-mediated A.beta..sub.1-40 aggregation was reversible.
Cu.sup.2+-induced A.beta..sub.1-40 aggregation at pH 7.4 was
reversible following EDTA chelation, although for each new
aggregation cycle, complete resolubilization of the aggregates
required a longer incubation. This result suggested that a more
complex aggregate is formed during each subsequent aggregation
cycle, preventing the chelator access to remove Cu.sup.2+ from the
peptide. This is supported by the fact that complete
resolubilization occurs with time, and indicates that the peptide
is not adopting a structural conformation that is insensitive to
Cu.sup.2+-induced aggregation/EDTA-resolubilization.
[0242] The reversibility of pH potentiated Cu.sup.2+-induced
A.beta..sub.1-40 aggregation was studied by turbidometry between pH
7.5 to 6.6, representing H.sup.+ concentration extremes that might
be found in vivo (FIGS. 5A and 5B). Unlike the irreversible
aggregation of A.beta..sub.1-40 observed at pH 5.5.
Cu.sup.2+-induced A.beta.1-40 aggregation was fully reversible as
the pH oscillated between pH 7.4 and 6.6. FIG. 5A shows the
turbidometric analysis of Cu.sup.2+-induced A.beta..sub.1-40
aggregation at pH 7.4 reversed by successive cycles of chelator
(EDTA), as indicated. FIG. 5B shows turbidometric analysis of the
reversibility of Cu.sup.2+-induced A.beta..sub.1-40 as the pH
cycles between 7.4 and 6.6. Thus, subtle conformational changes
within the peptide induced by changing [H.sup.+] within a narrow pH
window, that corresponds to physiologically plausible [H.sup.+],
allows the aggregation or resolubilization of the peptide in the
presence of Cu.sup.2+.
[0243] Discussion
[0244] These results suggest that subtle conformational changes in
A.beta. induced by [H.sup.+] promote the interaction of
A.beta..sub.1-40 with metal ions, in particular Cu.sup.2+and
Hg.sup.2+ allowing self-aggregate or resolubilize depending on the
[H.sup.+] (FIGS. 2A-2C, 4A-4C). A decrease in pH below 7.0
increases the P-sheet conformation (Soto, C., et al., J. Neurochem.
63:1191-1198 (1994)), and this may allow the binding of Cu.sup.2+
to soluble A.beta. that could further alter the conformation of the
peptide allowing for self aggregation, or simply help coordinate
adjacent A.beta. molecules in the assembly of the peptides into
aggregates. Conversely, increasing pH above 7.0 promotes the
.alpha.-helical conformation (Soto, C., et al., J. Neurochem.
63:1191-1198 (1994)), which may alter the conformational state of
the dimeric aggregated peptide, releasing Cu and thereby
destabilizing the aggregate with the resultant release of A.beta.
into solution. Thus, in the presence of Cu.sup.2+, A.beta..sub.1-40
oscillates between an aggregated and soluble state dependent upon
the [H.sup.+].
[0245] A.beta..sub.1-40 aggregation by Co.sup.2+, like Zn.sup.2+,
was pH insensitive and per mole induced a similar level of
aggregation. Unlike Zn.sup.2+, A.beta..sub.1-40 binding of
Co.sup.2+ may be employed for the structural determination of the
pH insensitive binding site given its nuclear magnetic capabilities
(See FIG. 2C).
[0246] The biphasic relationship of A.beta. solubility with pH
mirrors the conformational changes previously observed by CD
spectra within the N-terminal fragment (residues 1-28) of AD
(reviewed in (Soto, C., et al., J. Neurochem. 63:1191-1198 (1994));
.alpha.-helical between pH 1-4 and >7, but .beta.-sheet between
pH 4-7. The irreversible aggregates of A.beta. formed at pH 5.5
supports the hypothesis that the 1-sheet conformation is a pathway
for A.beta. aggregation into amyloid fibrils. Since aggregates
produced by Zn.sup.2+ and Cu.sup.2+ under mildly acidic conditions
(FIGS. 5A and 5B) are chelator/pH reversible, their conformation
may be the higher energy .alpha.-helical conformation.
[0247] These results now indicate that there are three
physiologically plausible conditions which could aggregate A.beta.:
pH (FIGS. 1, 4A-4C; Fraser, P. E., et al., Biophys. J. 60:1190-1201
(1991); Barrow, C. J. and Zagorski, M. G., Science 253:179-182
(1991); Burdick, D., J. Biol. Chem. 267:546-554 (1992); Barrow, C.
J., et al., J. Mol. Biol. 225:1075-1093 (1992); Zagorski, M. G. and
Barrow, C. J., Biochemistry 31:5621-5631 (1992); Kirshenbaum, K.
and Daggett, V., Biochemistry 34:7629-7639 (1995); Wood, S. J., et
al., J. Mol. Biol. 256:870-877 (1996), [Zn.sup.2+] (FIGS. 1, 2A and
2B, 4A-4C; Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994);
Bush, A. I., et al., Science 265:1464 (1994); Bush, A. I., et al.,
Science 268:1921 (1995); Wood, S. J., et al., J. Mol. Biol.
256:870-877 (1996)) and under mildly acidic conditions, [Cu.sup.2+]
(FIGS. 2A, 4A-4C, 5B). Interestingly, changes in metal ion
concentrations and pH are common features of the inflammatory
response to injury. Therefore, the binding of Cu.sup.2+ and
Zn.sup.2+ to A.beta. may be of particular importance during
inflammatory processes, since local sites of inflammation can
become acidic (Trehauf, P. S. & McCarty, D. J., Arthr. Rheum.
14:475-484 (1971); Menkin, V., Am. J. Pathol. 10:193-210 (1934))
and both Zn.sup.2+ and Cu.sup.2+ are rapidly mobilized in response
to inflammation (Lindeman, R. D., et al., J. Lab. Clin. Med.
81:194-204 (1973); Terhune, M. W. & Sandstead, H. H., Science
177:68-69 (1972); Hsu, J. M., et al., J. Nutrition 99:425-432
(1969); Haley, J. V., J. Surg. Res. 27:168-174 (1979); Milaninio,
R., et al., Advances in Inflammation Research 1:281-291 (1979);
Frieden, E., in Inflammatory Diseases and Copper, Sorenson, J. R.
J., ed, Humana Press, New Jersey (1980), pp. 159-169).
[0248] Serum copper levels increase during inflammation, associated
with increases in ceruloplasmin, a Cu.sup.2+ transporting protein
that may donate Cu.sup.2+ to enzymes active in processes of basic
metabolism and wound healing such as cytochrome oxidase and lysyl
oxidase (Giampaolo, V., et al., in Inflammatory Diseases and
Copper, Sorenson, J. R. J., ed, Humana Press, New Jersey (1980),
pp.329-345; Peacock, E. E. and vanWinkle, W., in Wound Repair, W.
B. Saunders Co., Philadelphia, pp. 145-155) (1976)). Since the
release of Cu.sup.2+ from ceruloplasmin is greatly facilitated by
acidic environments where the cupric ion is reduced to its cuprous
form (Owen, C. A., Jr., Proc. Soc. Exp. Biol. Med. 149:681-682
(1975)), periods of mild acidosis may promote an environment of
increased free Cu.sup.2+. Similarily, aggregation of another
amyloid protein, the acute phase reactant serum amyloid P component
(SAP) to the cell wall polysaccharide, zymosan, has been observed
with Cu.sup.2+ at acidic pH (Potempa, L. A., et al., Journal of
Biological Chemistry 260:12142-12147 (1985)). Thus, exchange of
Cu.sup.2+ to A.beta..sub.1-40 during times of decreased pH may
provide a mechanism for altering the biochemical reactivity of the
protein required by the cell under mildly acidic conditions. Such a
function may involve alterations in the aggregation/adhesive
properties (FIGS. 1-5B) or oxidative functions of A.beta. at local
sites of inflammation.
[0249] While the pathogenic nature of A.beta..sub.42 in AD is well
described (Maury, C.P.J., Lab. Investig. 72:4-16 (1995); Multhaup,
G., et al., Nature 325:733-736 (1987)), the function of the smaller
A.beta..sub.1-40 remains unclear. The present data suggest that
Cu.sup.2+-binding and aggregation of A.beta. will occur when the pH
of the microenvironment rises. This conclusion can be based on the
finding that the reaction is [H.sup.+] and [Cu.sup.2+] dependent
and reversible within a narrow, physiologically plausible, pH
window. This is further supported by the specificity and high
affinity of Cu.sup.2+ binding under mildly acidic conditions
compared to the constant Zn.sup.2+-induced aggregation (and
binding) of A.beta..sub.1-40 over a wide pH range (6.2-8.5). The
brain contains high levels of both Zn.sup.2+ (.about.150 .mu.M;
Frederickson, C. J. International Review of Neurobiology 31:145-237
(1989)) and Cu.sup.2+ (.about.100 .mu.M; Warren, P. J., et al.,
Brain 83:709-717 (1960); Owen, C. A., Physiological Aspects of
Copper, Noyes Publications, Park Ridge, N.J. (1982), pp160-191).
Intracellular concentrations are approximately 1000 and 100 fold
higher than extracellular concentrations. This large gradient
between intracellular and extracellular compartments suggests a
highly energy dependent mechanism is required in order to sequester
these metals within neurons. Therefore, any alterations in energy
metabolism, or injury, may affect the reuptake of these metal ions
and promote their release into the extracellular space, and
together with the synergistic affects of decreased pH (see above)
induce membrane bound A.beta..sub.1-40 to aggregate. Since
increased concentrations of Zn.sup.2+ and Cu.sup.2+, and decreased
pH, are common features of all forms of cellular insult, the
initiation of A.beta..sub.1-40 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.beta..sub.1-40 has such a high affinity for
these metal ions, indicates a protein that has evolved to respond
to slight changes in the concentration of extracellular metal ions.
This is supported by the fact that aggregation in the presence of
Cu is approx. 30% at pH 7.1, the pH of the brain (Yates, C. M., et
al., J. Neurochem. 55:1624-1630 (1990)), but 85% at pH 6.8. Taken
together, our present results indicate that A.beta..sub.1-40 may
have evolved to respond to biochemical changes associated with
neuronal damage as part of the locally mediated response to
inflammation or cell injury. Thus, it is possible that Cu.sup.2+
mediated A.beta..sub.1-40 binding and aggregation might be a
purposive cellular response to an environment of mild acidosis.
[0250] The deposition of amyloid systemically is usually associated
with an inflammatory response (Pepys, M. B. & Baltz, M. L.,
Adv. Immunol. 34:141-212 (1983); Cohen, A. S., in Arthritis and
Allied Conditions, D. J. McCarty, ed., Lea and Febiger,
Philadelphia, pp. 1273-1293 (1989); Kisilevsky, R., Lab. Investig.
49:381-390 (1983)). For example, serum amyloid A, one of the major
acute phase reactant proteins that is elevated during inflammation,
is the precursor of amyloid A protein that is deposited in various
tissues during chronic inflammation, leading to secondary
amyloidosis (Gorevic, P. D., et al., Ann. NY Acad. Sci. 380:393
(1982)). An involvement of inflammatory mechanisms has been
suggested as contributing to plaque formation in AD (Kisilevsky,
R., Mol. Neurobiol 49:65-66 (1994)). Acute-phase proteins such as
alpha 1-antichymotrypsin and c-reactive protein, elements of the
complement system and activated microglial and astroglial cells are
consistently found in AD brains.
[0251] The rapid appearance, within days of A.beta. deposits and
APP immunoreactivity following head injury (Roberts, G. W., et al.,
Lancet. 338:1422-1423 (1991); Pierce, J. E. S., et al., Journal of
Neuroscience 16:1083-1090 (1996)), rather than the more gradual
accumulation of A.beta. into more dense core amyloid plaques over
months or years in AD may be compatible with the release of
Zn.sup.2+, Cu.sup.2+ and mild acidosis in this time frame. Thus,
pH/metal ion mediated aggregation may form the basis for the
amorphous A.beta. 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.
[0252] Since decreased cerebral pH is a complication of aging
(Yates, C. M., et al., J. Neurochem. 55:1624-1630 (1990)), these
data indicate that Cu and Zn mediated A.beta. aggregation may be a
normal cellular response to an environment of mild acidosis.
However, prolonged exposure of A.beta. to an environment of lowered
cerebral pH may promote increased concentrations of free metal ions
and reactive oxygen species, and the inappropriate interaction of
A.beta..sub.1-42 over time promoting the formation of irreversible
A.beta. oligomers and it's subsequent deposition as amyloid in AD.
The reversibility of this pH mediated Cu.sup.2+ aggregation does
however present the potential for therapeutic intervention. Thus,
cerebral alkalinization may be explored as a therapeutic modality
for the reversibility of amyloid deposition in vivo.
Example 2
[0253] Free Radical Formation and SOD-like Activity of Alzheimer's
A.beta. Peptides
[0254] Materials and Methods
[0255] a) Determination of Cu.sup.+ and Fe.sup.2+
[0256] This method is modified from a protocol assaying serum
copper and iron (Landers, J. W., et al., Amer. Clin. Path. 29:590
(1958)). It is based on the fact that there are optimal visible
absorption wavelengths of 483 nm and 535 nm for Cu.sup.+ complexed
with bathocuproinedisulfonic (BC) anion and Fe.sup.2+ coordinated
by bathophenanthrolinedisulfonic (BP) anion, respectively.
[0257] Determination of molar absorption of these two complexes was
accomplished essentially as follows. An aliquot of 500 .mu.l of
each complex (500 .mu.M, in PBS pH 7.4, with ligands in excess) was
pipetted into 1 cm-pathlength quartz cuvette, and their absorbances
were measured. Their molar absorbancy was determined based on
Beer-Lambert's Law. Cu.sup.+-BC has a molar absorbancy of 2762
M.sup.-1 cm.sup.-1, while Fe.sup.2+-BP has a molar absorbancy of
7124 M.sup.-1 cm.sup.-1.
[0258] Determination of the equivalent vertical pathlength for
Cu.sup.+-BC and Fe.sup.2+-BP in a 96-well plate was carried out
essentially as follows. Absorbances of the two complexes with a 500
.mu.M, 100 .mu.M, 50 .mu.M, and 10 .mu.M concentration of relevant
metal ions (Cu.sup.+, Fe.sup.2+) were determined both by 96-well
plate readers (300 .mu.L) and UV-vis spectrometer (500 .mu.L), with
PBS, pH 7.4, as the control blank. The resulting absorbancies from
the plate reader regress against absorbancies by a UV-vis
spectrometer. The slope k from the linear regression line is
equivalent to the vertical pathlength if the measurement is carried
out on a plate. The results are:
1 k(cm) r.sup.2 Cu.sup.+--BC 1.049 0.998 Fe.sup.2+--BP 0.856
0.999
[0259] With molar absorbancy and equivalent vertical pathlength in
hand, the concentrations (.mu.M) of Cu.sup.+ or Fe.sup.2+ can be
deduced based on Beer-Lambert's Law, using proper buffers as
controls. 1 for Fe 2 + : [ Fe 2 + ] ( M ) = A ( 535 nm ) ( 7124
.times. 0.856 ) .times. 10 6 for Cu + : [ Cu + ] ( M ) = A ( 483 nm
) ( 2762 .times. 1.049 ) .times. 10 6
[0260] where .DELTA.A is absorbancy difference between sample and
control blank.
[0261] b) Determination of H.sub.2O.sub.2
[0262] This method is modified from a H.sub.2O.sub.2 assay reported
recently (Han, J. C., et al., Anal. Biochem. 234:107 (1996)). The
advantages of this modified H.sub.2O.sub.2 assay on 96-well plate
include high throughput, excellent sensitivity (.about.1 .mu.M),
and the elimination of the need for a standard curve of
H.sub.2O.sub.2, which is problematic due to the labile chemical
property of H.sub.2O.sub.2.
[0263] A.beta. peptides were co-incubated with a
H.sub.2O.sub.2-trapping reagent (Tris(2-carboxyethyl)-phosphine
hydrochloride, TCEP, 100 .mu.M) in PBS (pH 7.4 or 7.0) at
37.degree. C. for 30 mins. Then 5,5'-dithio-bis(2-nitrobenzoic
acid) (DBTNB, 100 .mu.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 FIG. 11).
[0264] The chemical scheme for this novel method is: 1
[0265] TCEP.HCl was synthesized by hydrolyzing tris (2-cyno-ethyl)
phosphine (purchased from Johnson-Mathey (Waydhill, Mass.)), in
refluxing aqueous HCl (Burns, J. A. et al., J. Org. Chem. 56:2648
(1991)) as shown below. 2
[0266] In order to carry out the above-described assay in a 96-well
plate, it was necessary to calculate the equivalent vertical
pathlength of 2-nitro-5-thiobenzoic acid (TMB) in a 96-well plate.
This determination was carried out essentially as described for
Cu.sup.+-BC and Fe.sup.2+-BP in Example 5. The resulting
absorbancies from the plate reader regress against absorbancies by
a UV-vis spectrometer. The slope k from the linear regression line
is equivalent to the vertical pathlength if the measurement is
carried out on a plate. The results are:
2 k r.sup.2 0.875 1.00
[0267] The concentration of H.sub.2O.sub.2 can then be deduced from
the difference in absorbance between the sample and the control
(sample plus 1000 U/.mu.l catalase) 2 [ H 2 O 2 ] ( M ) = A ( 412
nm ) ( 2 .times. 0.875 .times. 14150 )
[0268] c) Determination of OH.
[0269] Determination of OH. was performed as described in
Gufferidge et al. Biochim. Biophys. Acta 759: 38-41(1983).
[0270] d) Cu.sup.+ Generation by A.beta.: Influence of Zn.sup.2+
and pH
[0271] A.beta. (10 .mu.M in PBS, pH 7.4 or 6.8, as shown) was
incubated for 30 minutes (37.degree. C.) in the presence of
Cu.sup.2+10 .mu.M .+-.Zn.sup.2+ 10 .mu.M). Cu.sup.30 levels (n=3,
.+-.SD) were assayed against a standard curve. These data indicate
that the presence of Zn.sup.2+ can mediate the reduction of
Cu.sup.2+ in a mildly acidic environment. The effects of zinc upon
these reactions are strongly in evidence but complex. Since the
presence of 10 .mu.M zinc will precipitate the peptide, it is clear
that the peptide possesses redox activity even when it is not in
the soluble phase, suggesting that cortical A.beta. deposits will
not be inert in terms of generating these highly reactive products.
Cerebral zinc metabolism is deregulated in AD, and therefore levels
of interstitial zinc may play an important role in adjusting the
Cu.sup.+ and H.sub.2O.sub.2 production generated by A.beta.. The
rat homologue of A.beta..sub.1-40 does not manifest the redox
reactivity of the human equivalent. Insulin, a histidine-containing
peptide that can bind copper and zinc, exhibits no Cu.sup.2+
reduction.
[0272] e) Hydrogen Peroxide Production by A.beta. Species
[0273] A.beta..sub.1-42 (10 .mu.M) was incubated for 1 hr at
37.degree. C., pH 7.4 in ambient air (first bar), continuous argon
purging (Ar), continuous oxygen enrichment (O.sub.2) at pH 7.0
(7.0), or in the presence of the iron chelator desferioxamine (220
.mu.M; DFO). Variant A.beta. species (10 .mu.M) were tested:
A.beta..sub.1-40 (A.beta..sub.1-40), rat A.beta..sub.1-40
(rA.beta..sub.1-40), and scrambled A.beta..sub.1-40
(sA.beta..sub.1-40) were incubated for 1 hr at 37.degree. C., pH
7.4 in ambient air. Values (mean .+-.SD, n=3) represent triplicate
samples minus values derived from control samples run under
identical conditions in the presence of catalase (10 U/ml). The
details of the experiment are as follows: A.beta. peptides were
co-incubated with a H.sub.2O.sub.2-trapping reagent
(Tris(2-carboxyethyl)-phosphine hydrochloride, TCEP, 100 .mu.M) in
PBS (pH 7.4 or 7.0) at 37.degree. C. for 30 mins. Then
5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 100 .mu.M) was added to
react with remaining TCEP, the product has a characteristic
absorbance maximum of 412 nm. The assay was adapted to a 96-well
format using a standard absorbance range.
[0274] Results and Discussion
[0275] A.beta. exhibits metal-dependent and independent redox
activity
[0276] Because A.beta. was observed to be covalently linked by Cu,
the ability of the peptide to reduce metals and generate hydroxyl
radicals was studied. The bathocuproine and bathophenanthroline
reduced metal assay technique employed by Multhaup et al. was used
in order to determine that APP itself possesses a Cu.sup.2+
reducing site on its ectodomain (Multhaup, G., et al., Science
271:1406 (1996)). It has been discovered that A.beta. possesses a
striking ability to reduce both Fe.sup.3+ to Fe.sup.2+, and
Cu.sup.2+ to Cu.sup.+, modulated by Zn.sup.2+ and pH (6.6-7.4) (See
FIG. 10). It is of great interest that the relative redox activity
of the peptides studied correlates so well with their relative
pathogenicity viz A.beta..sub.1-42>A.beta..sub.1-40>ratA.b-
eta. in all redox assays studied. Since one of the caveats in using
the reduced metals assay is that the detection agents can
exaggerate the oxidation potential of Cu.sup.2+ or Fe (III), other
redox products were explored by assays where no metal ion
indicators were necessary. It was discovered that hydrogen peroxide
was rapidly formed by A.beta.species (FIG. 11). Thus, A.beta.
produces both H.sub.2O.sub.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.beta. is dimeric in
physiological buffers. Since H.sub.2O.sub.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 (FIG. 9) and can be blocked by
hydroxyl scavengers. Thus the concentrations of Fe, Cu, Zn &
H.sup.+ in the brain interstitial milieu could be important in
facilitating precipitation and neurotoxicity for A.beta. by direct
(dimer formation) and indirect (Fe.sup.2+/Cu.sup.+ and
H.sub.2O.sub.2 formation) mechanisms.
[0277] H.sub.2O.sub.2 production by A.beta. explains the mechanism
by which H.sub.2O.sub.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. Interestingly, the
scrambled A.beta. peptide (same size and residue content as FIG. 6)
produces appreciable H.sub.2O.sub.2 but no hydroxyl radicals. This
is because the scrambled A.beta. peptide is unable to reduce metal
ions. This leads to the conclusion that what makes A.beta. such a
potent neurotoxin is its capacity to produce both reduced metals
and H.sub.2O.sub.2 at the same time. This "double whammy" can then
produce hydroxyl radicals by the Fenton reaction, especially if the
H.sub.2O.sub.2 is not rapidly removed from the vicinity of the
peptide. Catalase and glutathione peroxidase are the principal
means of catabolizing H.sub.2O.sub.2, and their levels are low in
the brain, especially in AD, perhaps explaining the propensity of
A.beta. to accumulate in brain tissue.
[0278] FIG. 11 shows that the production of H.sub.2O.sub.2 is
oxygen dependent, and further investigation has indicated that
A.beta. can spontaneously produce the superoxide radical (O.sub.2)
in the absence of metal ions. This property of A.beta. is
particularly exaggerated in the case of A.beta..sub.1-42, probably
explaining why this peptide is more neurotoxic and more enriched
than A.beta..sub.1-40 in amyloid. O.sub.2 generation will be
subject to spontaneous dismutation to generate H.sub.2O.sub.2,
however, this is a relatively slow reaction, although it may
account for the majority of the H.sub.2O.sub.2 detected in our
A.beta. assays. O.sub.2 is reactive, and the function of superoxide
dismutase (SOD) is to accelerate the dismutation to produce
H.sub.2O.sub.2 which is then catabolized by catalase and
peroxidases into oxygen and water. The most abundant form of SOD is
Cu/Zn SOD, mutations of which cause another neurodegenerative
disease, amyotrophic lateral sclerosis (Rosen, D., et al., Nature
364:362 (1993)). Since A.beta., like Cu/Zn SOD, is a dimeric
protein that binds Cu and Zn and reduces Cu.sup.2+ and Fe.sup.3+,
we studied the O.sub.2 dismutation behavior of A.beta. in the sec
time-scale using laser pulse photolysis. These experiments have
shown that A.beta. exhibits Fe/Cu-dependent SOD-like activity with
rate constants of dismutation at .apprxeq.10.sup.8 M.sup.-1
sec.sup.-1, which are strikingly similar to SOD. Hence, A.beta.
appears to be a good candidate to possess the same function as SOD,
and therefore may function as an antioxidant. This may explain why
oxidative stresses cause it to be released by cells (Frederikse, P.
H., et al., Journal of Biological Chemistry 271: 10169 (1996)).
However, if A.beta..sub.1-42 is involved in the reaction to
oxidative stress, or if the H.sub.2O.sub.2 clearance is compromised
at the cellular level, A.beta. will accumulate, recruiting more
O.sub.2 and producing more O.sub.2 leading to a vicious cycle and
localizing tissue peroxidation damage and protein
cross-linking.
Example 3
Therapeutic Agents for Inhibition of Metal-mediated Production of
Reactive Oxygen Species
[0279] Materials and Methods
[0280] a) Synthesis of Peptides
[0281] Synthetic A.beta. peptides A.beta..sub.1-40 and
A.beta..sub.1-42 were synthesized by the W. Keck Laboratory, Yale,
Conn. In order to verify the reproducibility of the data obtained
with these peptides, confirmatory data were obtained by reproducing
experiments with these A.beta. peptides synthesized and obtained
from other sources: Glabe laboratory, University of California,
Irvine, Calif., Multhaup Laboratory, University of Heidelberg, U.S.
Peptides, Bachem, and Sigma. Rat A.beta. was synthesized and
characterized by the Multhaup Laboratory, University of Heidelberg.
A.beta..sub.1-28 was purchased from U.S. Peptides, Bachem, and
Sigma. A.beta. peptide stock solutions were prepared in chelex-100
resin (BioRad) treated water and quantified.
[0282] b) Metal Reduction Assay
[0283] The metal reduction assay was performed using a 96-well
microtiter plate (Costar) based upon a modification of established
protocols (Landers, J. W., et al., Amer. Clin. Path. 29:590 (1958);
Landers, J. W., et al., Clinica Chimica Acta 3:329 (1958)).
Polypeptides (10 .mu.M) or Vitamin C (100 .mu.M), metal ions (10
.mu.M, Fe(NO.sub.3).sub.3 or Cu(NO.sub.3).sub.2), and reduced metal
ion indicators, bathophenanthrolinedisulfonic acid (BP, for
Fe.sup.2+, Sigma, 200 .mu.M) or bathocuproinedisulfonic acid (BC,
for Cu.sup.+, Sigma, 200 .mu.M), were coincubated in phosphate
buffered saline (PBS), pH 7.4, for 1 hr at 37.degree. C. The metal
ion solutions were prepared by direct dilution in the buffer from
their aqueous stocks purchased from National Institute of Standards
and Technology (NIST). Absorbances were then measured at 536 mn
(Fe.sup.2+-BP complex) and 483 nm (Cu.sup.+-BC complex),
respectively, using a 96-well plate reader (SPECTRAmax 250,
Molecular Devices, CA). In control samples, both metal ion and
indicator were present to determine the background buffer signal.
As a further control, both metal ion and peptide were present in
the absence of indicator to estimate the contribution of light
scattering due to turbidity to the absorbance reading at these
wavelengths. The net absorbances (.DELTA.A) at 536 nm or 483 nm
were obtained by deducting the absorbances from these controls from
the absorbances generated by the peptide and metal in the presence
of the respective indicator.
[0284] The concentrations of reduced metal ions (Fe.sup.2+ or
Cu.sup.+) were quantified based on the formula: Fe.sup.2+ or
Cu.sup.+ (.about.M)=A*10.sup.6/(L*M), where L is the measured
equivalent vertical pathlength for a well of 300 .mu.L volume as
described in the instrument's specifications manual (0.856 cm for
Fe.sup.2+; 1.049 cm for Cu.sup.+); M is the known molecular
absorbance (M.sup.-1 cm.sup.-1) which is 7124 (for Fe.sup.2+-BP
complex) or 2762 (for Cu.sup.+-BC complex).
[0285] c) H.sub.2O.sub.2 Assay
[0286] The H.sub.2O.sub.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)). Polypeptides (10 .mu.M) or Vitamin C (10 .mu.M), Fe.sup.3+
or Cu.sup.2+ (1 .mu.M) and a H.sub.2O.sub.2 trapping
agent-Tris(2-Carboxyethyl)Phosphine Hydrochloride (TCEP, Pierce, 50
.mu.M)-were co-incubated in PBS buffer (300 .mu.L), pH 7.4, for 1
hour at 37.degree. C. Under identical conditions, catalase (Sigma,
100 U/mL) was substituted for the polypeptides, to serve as a
control signal representing 0 .mu.M H.sub.2O.sub.2. Following
incubation, the unreacted TCEP was detected by
5,5-Dithio-bis(2-Nitrobenzoic acid) (DTNB, Sigma, 50 KLM) which
generates 2 moles of the coloured product. The reactions are:
TCEP+H.sub.2O.sub.2.fwdarw.TCEP=O+H.sub.2O,
[0287] then the remaining TCEP is reacted with DTNB:
TCEP+DTNB+H.sub.2O.fwdarw.TCEP=O+2NTB (2-nitro-5-thiobenzoate).
[0288] The amount of H.sub.2O.sub.2 produced was quantified based
on the formula: H.sub.2O.sub.2 (.mu.M)=hA*106/(2*L*M), where hA is
the absolute absorbance difference between a sample and
catalase-only control at 412 nm wavelength; L=0.875 cm, the
equivalent vertical pathlength obtained from the platereader
manufacturer's specifications; M is the molecular absorbance for
NTB (14150 M.sup.-1 cm.sup.-1 at 412 nm).
[0289] 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.sub.2O.sub.2 generation from A.beta., which does
not possess a disulfide bond.
[0290] d) Estimation of O.sub.2
[0291] The spectrophotometric absorption peak for O.sub.2 is 250 nm
where its extinction coefficient is much greater than that of
H.sub.2O.sub.2 (Bielski et al., Philos Trans R Soc Lond B Biol Sci.
311: 473-482 (1985)). The production of O.sub.2.sup.- was estimated
by measuring the spectrophotometric absorption of polypeptides (10
.mu.M, 300 .mu.L) after incubation for one hour in PBS, pH 7.4, at
37.degree. C., using a 96-well plate reader. The corresponding
blank was the signal from PBS alone. An absolute baseline for the
signal generated by the peptide was not achievable in this assay
since the absorption peak for tyrosine (residue 10 of A.beta.) is
close (254 nm) to the absorption peak for O.sub.2.sup.-. However,
attenuation of the absorbance by co-incubation with superoxide
dismutase (100 U/mL) indicated that the majority of the absorbance
signal was due to the presence of O.sub.2.sup.-.
[0292] e) Thiobarbituric Acid Reaction Substance (TBARS)
Assay--OH.
[0293] The Thiobarbituric Acid-Reactive Substance (TBARS) assay for
incubation mixtures with Fe.sup.3+ or Cu.sup.2+ was performed in a
96-well microtiter format modified from established protocols
(Gutteridge et al. Biochim. Biophys. Acta 759: 38-41 (1983)).
A.beta. peptide species (10 .mu.M) or Vitamin C (100 .mu.M), were
incubated with Fe.sup.3+ or Cu.sup.2+ (1 .mu.M) and deoxyribose
(7.5 mM, Sigma) in PBS, pH 7.4. Following incubation (37.degree.
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.degree.
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.beta. peptides.
[0294] Results and Discussion
[0295] Oxygen radical involvement in human aging, the predominant
risk factor for Alzheimer's disease (AD), was first proposed by
Harman in 1956 (Harman, D., J. Gerontol. 11:298 (1956)) and
increasing evidence has implicated oxidative stress in the
pathogenesis of AD. Apart from metabolic signs of oxidative stress
in AD-affected neocortex such as increased glucose-6-phosphate
dehydrogenase activity (Martins, R. N., et al., J. Neurochem.
46:1042-1045 (1986)) and increased heme oxygenase-1 levels (Smith,
M. A., et al., Am. J. Pathol. 145:42 (1994)), there are also
numerous signs of oxygen radical-mediated chemical attack such as
increased protein and free carbonyls (Smith, C. D., et al., Proc.
Natl. Acad. Sci. USA 88:10540 (1991); Hensley, K., et al., J.
Neurochem. 65:2146 (1995); Smith, M. A., et al., Nature 382:120
(1996)), lipid peroxidation adducts (Palmer, A. M. & Burns, M.
A., Brain Res. 645:338 (1994); Sayre, L. M. et al., J. Neurochem.
68:2092 (1997)), peroxynitrite-mediated protein nitration (Good, P.
F., et al., Am. J. Pathol. 149:21 (1996)); Smith, M. A., et al.,
Proc. Natl. Acad. Sci. USA 94:9866 (1997)), and mitochondrial and
nuclear DNA oxidation adducts (Mecocci, P., et al., Ann. Neurol.,
34:609-616 (1993); Mecocci, P., et al., Ann. Neurol., 36:747-751
(1994)). Recently, treatment of individuals with the antioxidant
vitamin E has been reported to delay the progression of clinical AD
(Sano, M. et al., N. Engl. J. Med. 336:1216 (1997)).
[0296] A relationship seems likely to exist between the signs of
oxidative stress and the characteristic A.beta. collections
(Glenner, G. G. & Wong, C., Biochem. Biophys. Res. Commun.
120:885 (1984)) found in the cortical interstitium and
cerebrovascular intima media in AD. The brain regional variation of
oxidation biomarkers corresponds with amyloid plaque density
(Hensley, K., et al., J. Neurochem. 65:2146 (1995)). Indeed,
neurons cultured from subjects with Down's syndrome, a condition
complicated by the invariable premature deposition of cerebral
A.beta. (Rumble, B., et al., N. Engl. J. Med. 320:1446 (1989)) and
the overexpression of soluble A.beta.1-42 in early life (Teller, J.
K., et al., Nature Medicine 2:93 (1996)), exhibit lipid
peroxidation and apoptotic cell death caused by increased
generation of hydrogen peroxide (Busciglio, J. & Yankner, B.
A., Nature 378:776 (1995)). Synthetic AD peptides have been shown
to induce lipid peroxidation of synaptosomes (Butterfield, D. A.,
et al., Biochem. Biophys. Res. Commun. 200:710 (1994)), and to
exert neurotoxicity (Behl, C., et al., Cell 77:817 (1994); Mattson,
M. P., et al., J. Neurochem. 65:1740 (1995)) or vascular
endothelial toxicity through a mechanism that involves the
generation of cellular superoxide/hydrogen peroxide
(O.sub.2.sup.-/H.sub.2O.sub.2) and is abolished by the presence of
SOD (Thomas, T., et al., Nature 380:168 (1996) or catalytic
synthetic O.sub.2.sup.-/H.sub.2O.sub.2 scavengers (Bruce, A. J., et
al., Proc. Natl. Acad. Sci. USA 93:2312 (1996)). Antioxidant
vitamin E and the spin-trap compound PBN have been shown to protect
against A.beta.-mediated neurotoxicity in vitro (Goodman, Y., &
Mattson, M. P., Exp. Neurol. 128:1 (1994); Harris, M. E., et al.,
Exp. Neurol. 131:193 (1995)).
[0297] A.beta., 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., Science 258:126 (1992)) by constitutive
cleavage of the amyloid protein precursor (APP) (Kang, J., et al.,
Nature 325:733 (1987); Tanzi, R. E., et al., Nature Genet (1993))
as a mixture of polypeptides manifesting carboxyl-terminal
heterogeneity. A.alpha.-40 is the major soluble A.beta. species in
biological fluids (Vigo-Pelfrey, C., et al., J. Neurochem. 61:1965
(1993)) and A.beta..sub.1-42 is a minor soluble species, but is
heavily enriched in interstitial plaque amyloid (Masters, C. L., et
al., Proc. Natl. Acad. Sci. USA 82:4245 (1985); Kang, J. et al.,
Nature 325:733 (1987); Prelli, F., et al., J. Neurochem. 51:648
(1988); Roher et al., J. Cell Biol. 107:2703-2716 (1988); Roher et
al., J. Neurochem. 61:1916-1926 (1993); Miller, D. L., et al.,
Arch. Biochem. Biophys. 301:41 (1993)). The discovery of pathogenic
mutations of APP close to or within the A.beta. domain (van
Broeckhoven, C., et al., Science 248:1120 (1990); Levy, E., et al.,
Science 248:1124 (1990); Goate, A., et al., Nature 349:704 (1991);
Murrell, J., et al., Science 254:94 (1991); Mullan, M., et al.,
Nature Genet 1:345 (1992)) indicates that the metabolism of A.beta.
is involved with the pathophysiology of this predominantly sporadic
disease. Familial AD-linked mutations of APP, presenilin-1 and
presenilin-2 correlate with increased cortical amyloid burden and
appear to induce an increase in the ratio of A.beta..sub.1-42 as
part of their common pathogenic mechanism (Suzuki, N., et al.,
Science 264:1336 (1994); Scheuner et al., Nat Med., 2(8):864-870
(1996); Citron, M., et al., Nature Medicine 3:67 (1997)). However,
the mechanism by which A.beta..sub.1-42 exerts more neurotoxicity
than A.beta..sub.1-40 and other A.beta. peptides (Dore, S., et al.,
Proc. Natl. Acad. Sci. USA 94:4772 (1997)) remains unclear.
[0298] One of the models proposed for A.beta. neurotoxicity is
based on a series of observations of A.beta.-generated oxyradicals
generated by a putative A.beta. peptide fragmentation mechanism
which is O.sub.2-dependent, metal-independent and involves the
sulfoxation of the methionine at A.beta. residue 35 (Butterfield,
D. A., et al., Biochem. Biophys. Res. Commun. 200:710 (1994);
Hensley, K., et al., Proc. Natl Acad. Sci. USA 91:3270 (1994);
Hensley, K., et al., Ann N Y Acad Sci., 786: 120-134 (1996).
A.beta..sub.25-35 peptide has been reported to exhibit
H.sub.2O.sub.2-like reactivity towards aqueous Fe.sup.2+, nitroxide
spin probes, and synaptosomal membrane proteins (Butterfield, D.
A., et al., Life Sci. 58:217 (1996)), and A.beta..sub.1-40 has also
been reported to generate the hydroxyl radical by mechanisms that
are unclear (Tomiyama, T., et al., J. Biol. Chem. 271:6839 (1996)).
However, there has been no quantitative appraisal of the
ROS-generating capacity of A.beta..sub.1-42 versus that of
A.beta..sub.1-40 and other A.beta. variants, to date.
[0299] A.beta. is a metal binding protein which saturably binds
zinc via a histidine-mediated specific high affinity site
(K.sub.D=107 nM) as well as by low affinity binding (K.sub.D=5.2
.mu.M). The high-affinity zinc binding site was mapped to a stretch
of contiguous residues between positions 6-28 of the A.beta.
sequence (Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994)).
Concentrations of zinc .gtoreq.1 .mu.M rapidly induce aggregation
of human A.beta..sub.1-40 solutions (Bush, A. I., et al., Science
265:1464 (1994)), in reversible manner which is dependent upon the
dimerization of peptide in solution, its alpha-helical content, and
the concentration of NaCl (Huang, X., et al., J. Biol. Chem.
272:26464-26470 (1997)). Rat/mouse A.beta..sub.1-40 ("rat A.beta.",
with substitutions o R.sub.5.fwdarw.G, Y.sub.10.fwdarw.F, and
H.sub.13.fwdarw.R, as compared to human A.beta.) binds zinc less
avidly (a single binding site, K.sub.A=3.8 .mu.M) and, unlike the
human peptide, is not precipitated by zinc at concentrations
.ltoreq.25 .mu.M. Since zinc is concentrated in the neocortex, we
hypothesized that the differential solubility of the rat/mouse
A.beta. peptide in the presence of zinc may explain the scarcity
with which these animals form cerebral A.beta. deposits (Johnstone,
E. M., et al., Mol. Brain Res. 10:299 (1991); Shivers, B. D., et
al., EMBO J. 7:1365 (1988)).
[0300] We have also observed interactions of A.beta. with
Cu.sup.2+, which stabilizes dimerization of A.beta..sub.1-40 on gel
chromatography (Bush, A. I., et al., J. Biol. Chem. 269:12152
(1994)), and which binds to the peptide with an affinity estimated
to be in the low picomolar range. Fe.sup.2+ has been observed to
induce partial aggregation of A.beta. (Bush, A. I., et al., Science
268:1921 (1995)), and to induce SDS-resistant polymerization of the
peptide (Dyrks, T., et al., J. Biol. Chem. 267:18210-18217 (1992)).
We hypothesized that the interactions of A.beta. with Fe and Cu may
contribute to the genesis of the oxidation insults that are
observed in the AD-affected brain. This is because Fe.sup.3+ and
Cu.sup.2+ are redox-active metal ions that are concentrated in
brain neurons, and may participate in the generation of ROS by
transferring electrons in their reduced state (reviewed in
Markesbery, 1997).
[0301] The levels of Cu and Fe, and their binding proteins, are
dysregulated in AD (Diebel, M. A., et al., J. Neurol. Sci. 143:137
(1996); Good, P. F., et al., Ann. Neurol. 31:286 (1992); Robinson,
S. R., et al., Alzheimer's Research ;1:191 (1995); Thompson, C. M.,
et al., Neurotoxicology 9:1 (1988); Kennard, M. L., et al., Nature
Medicine 2:1230 (1996); Connor, J. R., et al., Neurosci. Lett.
159:88 (1993)) and may therefore lead to conditions that could
promote ROS production. While a direct role for A.beta. in
metal-dependent ROS generation has not been described, the
peptide's physiochemical interation with transition metals, the
presence of ferritin (Grudke-Iqbal, I., et al., Acta Neuropathol.
81:105 (1990)) and redox reactive iron (Smith, M. A., et al., Proc.
Natl. Acad. Sci. USA 94:9866 (1997)) in amyloid lesions, and the
facilitation of A.beta..sub.1-40 neurotoxicity in cell culture by
nanomolar concentrations of iron (Schubert, D. & Chevion, M.,
Biochem. Biophys. Res. Commun. 216:702 (1995)), collectively
support such a possibility.
[0302] We report the simultaneous production of H.sub.2O.sub.2 and
reduced metal ions by A.beta., with the consequent generation of
the hydroxyl radical. The amounts of reduced metal and ROS were
both greatest when generated by
A.beta..sub.1-42>A.beta..sub.1-40>>rat A.beta..sub.1-40,
A.beta..sub.40-1 and A.beta..sub.1-28, a chemical relationship that
correlates with the relative neurotoxicity of these peptides. These
data describe a novel, O.sub.2.sup.- and biometal-dependent pathway
of ROS generation by Alzheimer A.beta. peptides which may explain
the occurrence of oxidative stress in AD brain.
[0303] a) Metal Ion Reduction by A.beta. Peptides
[0304] To determine whether A.beta. peptides possess metal-reducing
properties, the ability of A.beta. peptides (Example 1) to reduce
Fe.sup.3+ and Cu.sup.2+, compared to Vitamin C and other
polypeptides (Example 2) was measured. Vitamin C, serving as a
positive control, reduced Cu.sup.2+efficiently (FIG. 13A). However,
the reduction of Cu.sup.2+ by A.beta..sub.1-42 was as efficient,
reducing all of the available Cu.sup.2+ during the incubation
period. A.beta..sub.1-40 reduced 60% of the available Cu.sup.2+,
whereas rat A.beta..sub.1-40 and A.beta..sub.1-28 reduced no
Cu.sup.2+. The reduction of Cu.sup.2+ by BSA (25%) and insulin
(10%) was less efficient than that by the human A.beta. peptides,
and was not unexpected since these polypeptides possess cysteine
residues and reduce Cu.sup.2+ in the process of forming disulfide
bonds.
[0305] Fe.sup.3+/Fe.sup.2+ has lower standard reduction potential
(0.11 V) than Cu.sup.2+/Cu.sup.+ (0.15 V) does under our
experimental conditions (Miller, D. M., et al., Free Radical
Biology & Medicine 8:95 (1990)), and, in general, Fe.sup.3+ was
reduced with less efficiency by Vitamin C and the polypeptides that
reduced Cu.sup.2+. Vitamin C reduced 15% of the available
Fe.sup.3+, however A.beta..sub.1-42 was the most efficient (50%) of
the agents tested for Fe.sup.3+ reduction, reducing more Fe.sup.3+
in the incubation period than Vitamin C (15%), A.beta..sub.1-40
(12%) and BSA (8%). Rat A.beta..sub.1-40, A.beta..sub.1-28 and
insulin did not significantly facilitate the reduction of
Fe.sup.3+. Analysis of A.beta..sub.1-42 and A.beta..sub.1-40 after
incubation with Cu.sup.2+ and Fe.sup.3+ under these conditions
revealed that there was no apparent mass modification of the
peptides on mass spectrometry, and no change in its migration
pattern on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS PAGE), nor evidence for increased aggregation
of the peptides by turbidometry or sedimentation analysis,
suggesting that the peptides were not consumed or modified during
the reduction reaction. Under these conditions, the complete
kinetics of the peptide-mediated reactions cannot be appreciated
(the presence of A.beta..sub.1-42 induced the total consumption of
the Cu.sup.2+ substrate within the incubation period), but a
striking relationship exists between the relative efficiencies of
the various A.beta. peptides to reduce Cu.sup.2+/Fe.sup.3+ in this
system and their respective participation in amyloid
neuropathology.
[0306] Since the dissolved O.sub.2 in the buffer vehicle may be
expected to react with the reduced metals being generated [Reaction
(1)], the effect of modulating the O.sub.2 tension in the buffer
upon the generation of reduced metals by the A.beta. peptides (FIG.
13R) was examined. Prior to the addition of Vitamin C or
polypeptide, the buffer vehicle was continuously bubbled for 2
hours at 20.degree. C. with 100% O.sub.2 to create conditions of
increased O2 tension, or Argon to create anaerobic conditions.
Increasing the O.sub.2 tension slightly reduced the levels of
reduced metals being detected, probably due to the diversion of a
fraction of the Fe.sup.2+/Cu.sup.+ being generated to Reaction (1),
and, if H.sub.2O.sub.2 is being produced as a product of Reaction
(2), the recruitment of Fe.sup.2+/Cu.sup.+ into the Fenton reaction
[Reaction (3)]. However, 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.sup.3+/Cu.sup.2+ is due to reaction with
O.sub.2.sup.-:
M.sup.(n+1)+O.sub.2.sup.-.fwdarw.M.sup.n++O.sub.2 Reaction (5)
[0307] To determine whether the reduction of metal ions in the
presence of A.beta. was due to the action of the peptide or the
generation of O.sub.2- by the peptide, the effects of metal ion
chelators on the generation of reduced metal ions (FIG. 13B) was
studied. It was found that coincubation of A.beta..sub.1-42 with
the relatively Fe.sup.3+-specific chelator desferrioxamine (DFO)
under ambient oxygenation conditions nearly halved the production
of Fe.sup.2+. Coincubation of A.beta..sub.1-42 with the
high-affinity Cu.sup.2+ chelator TETA abolished 95% of the Cu.sup.+
generated by the peptide under ambient oxygenation conditions.
These data indicate that the majority of the Cu.sup.+ and a
significant amount of the Fe.sup.2+ produced by A.beta..sub.1-42
are due to the direct action of the peptide and not indirectly due
to the production of O.sub.2.sup.-.
[0308] The inhibitory effects of chelation upon A.beta.-mediated
reduction of metal ions indicates that A.beta. probably directly
coordinates Fe.sup.3+ and Cu.sup.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.sup.3+ and Cu.sup.2+ to the A.beta.
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.
[0309] The reduction of metal ions by A.beta. must leave the
peptide, at least transiently, radicalized, in agreement with the
electron paramagnetic resonance (EPR) findings of Hensley et al.,
Proc. Natl. Acad. Sci. 91:3270 (1994). In their report, DFO, EDTA
or Chelex 100 could not abolish the EPR signal generated by
A.beta..sub.25-35 in PBS, leading these investigators to conclude
that the radicalization of A.beta. was metal-independent. However,
the inventors have found that after treatment with Chelex 100 the
concentrations of Fe and Cu in PBS are still as high as
.apprxeq.0.5 .mu.M (8), which could be sufficient to induce the
radicalization of the peptide after metal reduction. Since DFO does
not abolish the reduction of Fe.sup.3+ by A.beta..sub.1-42 (FIG.
13B), and since EDTA has been observed to potentiate Fe-mediated
Fenton chemistry (Samuni et al., Eur. J. Biochem.
137:119-124(1983)), it is suspected that Hensley and colleagues may
have inadvertently overlooked the contribution of metal reduction
to A.beta.-mediated radical formation.
[0310] Rat A.beta..sub.1-40 did not reduce metal ions, and has been
shown to have attenuated binding of Zn.sup.2+ (Bush et al.,
Science, 265:1464 (1994)). A similar attenuation of Cu.sup.2+ and
Fe.sup.3+ binding by rat A.beta..sub.1-40 compared to human
A.beta..sub.1-41 is anticipated. These data also indicate that the
rat A.beta. substitutions in human A.beta.'s zinc binding domain
towards the peptide's amnino terminus (Bush et al., J. Biol. Chem.,
269:12152 (1994)) involve residues that mediate the metal-reducing
properties of the peptide. However, the hydrophobic
carboxyl-terminal residues were also critical to the reduction
properties of A.beta.. That A.beta..sub.1-28 did not reduce metal
ions indicates that an intact Zn.sup.2+-binding site (Bush et al.,
J. BioL. Chem. 269:12152 (1994)) is insufficient to facilitate the
metal reduction reaction. The mechanism by which the two additional
hydrophobic residues (Ile and Ala) on A.beta..sub.1-42 so
substantially enhance the peptide's redox activity compared to
A.beta..sub.1-40 is still unclear.
[0311] It has been observed that sulfoxation of the methionine
residue at AD position 35 accompanies the EPR changes seen during
the incubation of A.beta..sub.25-35 for 3 hours in PBS at
37.degree. C. (Hensley, K., et al., Ann N Y Acad Sci., 786: 120-134
(1996)), however, no evidence was found for a modification of
A.beta..sub.1-40 and A.beta..sub.1-42 afier mass spectrophotometric
examination of the peptides incubated under conditions as
described. Therefore, A.beta.-mediated metal reduction, and the
subsequent A.beta.-mediated redox reactions described below, appear
to be achieved by a mechanism that differs from that previously
reported.
[0312] b) Production of H.sub.2O.sub.2 by A.beta. Peptides
[0313] The reduced metal ions produced by A.beta. were expected to
generate O.sub.2 and H.sub.2O.sub.2 by Reactions (1) and (2). To
study this, a novel assay was developed (Example 3) which detected
the generation of 10 .mu.M H.sub.2O.sub.2 by A.beta..sub.1-42 in
the presence of 1 .mu.M Fe.sup.3+ under ambient O.sub.2 conditions
(FIG. 14A). To validate the assay, coineubation with eatalase was
observed to abolish the H.sub.2O.sub.2 signal in a dose dependent
manner. The amount of H.sub.2O.sub.2 produced by the various
A.beta. peptides was studied, and observed that the order of the
production of H.sub.2O.sub.2 by the A.beta. variants was
A.beta..sub.1-42>A.beta..sub.1-40,>rat A.beta..sub.1-40-
A.beta..sub.1-28 (FIG. 14B), paralleling the amounts of metal
reduction by the same peptides (FIG. 13A).
[0314] H.sub.2O.sub.2 formation is likely to be mediated first by
O.sub.2-dependent O.sub.2.sup.- formation [Reaction (1)], followed
by dismutation [Reaction (2)]. To appraise the contribution of
Reaction (1) to H.sub.2O.sub.2 formation, H.sub.2O.sub.2 formation
by A.beta..sub.1-42 in the presence of chelators was measured (FIG.
14C). The amount of H.sub.2O.sub.2 formed in the presence of 1
.mu.M Cu.sup.2+ was 25% greater than the amount formed in the
presence of 1 .mu.M Fe.sup.3+. Coincubation with DFO had no effect
on H.sub.2O.sub.2 formation in the presence of 1 .mu.M Fe.sup.3+.
However, TETA, and the Cu.sup.+-specific indicator BC, both
substantially inhibited the formation of H.sub.2O.sub.2 in the
presence of 1 .mu.M Cu.sup.2+. The reasons why DFO partially
inhibited Fe.sup.3+ reduction, but was unable to inhibit
H.sub.2O.sub.2 formation are unclear. These data indicate that the
formation of H.sub.2O.sub.2 by A.beta. is dependent upon the
presence of substoichiometric amounts of Cu.sup.+/(II). The
possibility that formation of H.sub.2O.sub.2 in the presence of
Fe.sup.3+ was due to the presence of trace quantities of Cu.sup.2+
cannot be excluded.
[0315] BC and BP, agents that specifically complex reduced metal
ions, were far more effective than DFO and TETA at inhibiting
H.sub.2O.sub.2 formation by A.beta. (FIG. 14C) but the reasons for
this are not clear. The relatively Fe.sup.2+-specific complexing
agent, BP, inhibited H.sub.2O.sub.2 formation in the presence of
Cu.sup.2+, and the relatively Cu.sup.+-specific complexing agent,
BC, inhibited H.sub.2O.sub.2 formation in the present of Fe.sup.3+,
suggesting that these agents are not totally specific in their
metal ion affinities. The formation of H.sub.2O.sub.2 by A.beta. in
the absence of BC or BP confirms that the reduction of metals is
not contingent upon the artifactual enhancement of the metal ions'
redox potentials (Sayre, L. M., Science 274:1933 (1996)).
[0316] To determine whether the formation of
O.sub.2.sup.-/H.sub.2O.sub.2 by A.beta. is merely due to the
reduction of metal ions, or whether A.beta. also facilitates the
recruitment of the substrates in Reaction (1), the generation of
H.sub.2O.sub.2 by A.beta..sub.1-42, A.beta..sub.1-40 and Vitamin C
under different O.sub.2 tensions in the presence of 1 .mu.M
Fe.sup.3+ (FIG. 14D) or 1 .mu.M Cu.sup.2+ (FIG. 14E) was studied.
The presence of Vitamin C was used as a control measure to
determine the amount H.sub.2O.sub.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.sub.2O.sub.2 produced under higher O.sub.2 tensions. The presence
of either A.beta..sub.1-42 and A.beta..sub.1-40 generated more
H.sub.2O.sub.2 (A.beta..sub.1-42 >A.sym..sub.1-40) than Vitamin
C under any O.sub.2 tension studied, and generated H.sub.2O.sub.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.sub.2O.sub.2.
These data indicate that A.beta. indeed facilitates the recruitment
of O.sub.2 into Reaction (1) more than would be expected by the
interaction of the metals reduced by A.beta. with the passively
dissolved O.sub.2. Under relatively anaerobic conditions, the
A.beta. peptides were observed to still produce H.sub.2O.sub.2 in
the presence of Cu.sup.2+ (FIG. 14E). This is probably due to the
ability of A.beta. to recruit O.sub.2 into Reaction (1) under
conditions of very low O.sub.2 tension. Since O.sub.2 is
preferentially dissolved in hydrophobic environments (Halliwell and
Gutteridge, Biochem. J., 219:1-14 (1984)), it seems that the
hydrophobic carboxyl-terminus of A.beta. could attract O.sub.2,
serving as a reservoir for the substrate.
[0317] c) Evidence of the Superoxide Anion Formed by tile
A.beta.-metal Complex
[0318] To confirm the production of O.sub.2.sup.- by A.beta., the
absorbance of the peptide in solution at 250 nm, the absorbance
peak of O.sub.2.sup.- (FIG. 15A) was measured. The absorbance
generated by A.beta..sub.1-42 in the presence of 1 .mu.M Fe.sup.3+
was 60% reduced when co-incubated with SOD, increased in the
presence of high O.sub.2 tension and abolished under anaerobic
conditions. These data support the likelihood that A.beta.
generates H.sub.2O.sub.2 by first generating O.sub.2.sup.-.
[0319] The absorbance changes at 250 nm for the various AD peptides
in PBS (FIG. 15B) paralleled the production of H.sub.2O.sub.2 from
the same peptides (FIG. 14B), but the reason for the A.sub.250
being much greater for A.beta..sub.1-42 compared to
A.beta..sub.1-40 is unclear. It is likely that a fraction of the
total H.sub.2O.sub.2 generated by A.beta. is decomposed by the
Fenton reaction [Reaction (3)]. Therefore, the amount of
H.sub.2O.sub.2 detected may be an attenuated reflection of the
amount of O.sub.2.sup.- detected.
[0320] d) Detection of Hydroxyl Radicals Generatedfrom the
A.beta.-metal Complex
[0321] Having demonstrated that human A.beta. peptides
simultaneously produce H.sub.2O.sub.2 and reduced metals, it was
determined whether the hydroxyl radical was formed by the Fenton or
Haber-Weiss reactions [Reactions (3) and (4)]. A modified TBARS
assay was employed to detect OH released from co-incubation
mixtures of A.beta. peptides and 1 .mu.M Fe.sup.3+ or Cu.sup.2+. As
expected, A.beta..sub.1-42 produced more OH than A.beta..sub.1-40,
and rat A.beta. did not generate OH (FIG. 16A). In contrast to the
amount of Fe.sup.2+ and Cu.sup.+ produced (FIG. 13A), A.beta.
generated more OH. in the presence of Fe.sup.3+ than in the
presence of Cu.sup.2+. This may be because Fe.sup.2+ is more stable
than Cu.sup.+, which may be more rapidly oxidized by Reaction (1).
Therefore, the Fe.sup.2+ generated by A.beta. may have a greater
opportunity than the Cu.sup.+ generated to react with
H.sub.2O.sub.2. It is also possible that the contribution of the
Haber-Weiss reaction to the production of OH. [Reaction (5)] is
greater in the presence of Fe.sup.3+ than in the presence of
Cu.sup.2+.
[0322] The effects of the OHS scavengers, dimethyl sulfoxide (DMSO)
and mannitol, upon A.beta..sub.1-42-mediated OHS generation were
studied. Whereas these agents suppressed the generation of OH. by
Vitamin C in the presence of Fe and DMSO suppressed the generation
of OH by Vitamin C in the presence of Cu.sup.2+, neither were able
to quench the generation of OH. by A.beta..sub.1-42, whether in the
presence of Fe.sup.3+ or Cu.sup.2+ (FIG. 16B). This suggests that
these scavengers cannot encounter the OH. generated by A.beta.
before the TBARS reagent does.
[0323] e) Similarity Between Bleomycin-Fe and A.beta.-Fe/Cu
Complexes
[0324] The present Examples provide evidence for a model by which
Fe/Cu and O.sub.2 are mediators and substrates for the production
of OH. by A.beta. (FIGS. 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.sub.2 and
Fe/Cu, which may explain why this organ is predisposed to
A.beta.-mediated neurotoxicity, if this mechanism is confirmed in
vivo. The transport of Fe, Cu and Zn in the brain is largely
energy-dependent. For example, the copper-transporting gene for
Wilson's disease is an ATPase (Tanzi, R. E. et al., Nature Genetics
5:344 (1993)), and the re-uptake of zinc following
neurotransmission is highly energy-dependent (Assaf, S. Y. & S.
H. Chung, Nature, 308:734-736 (1984); Howell et al., Nature,
308:736-738 (1984)).
[0325] There is increasing evidence for lesions of brain energy
metabolism in aging and AD (Parker et al., Neurology, 40:1302-1303
(1990); (Mecocci et al., Ann. Neurol. 34:609-616 (1993); Beal, M.
F. Neurobiol. Aging 15 (Suppl 2):S171-S174(1994)). Therefore,
damage to energy-dependent brain metal homeostasis may be an
upstream lesion for the genesis of A.beta. deposition in AD. Most
brain biometals are bound to proteins or other ligands, however,
according to our findings, only A.beta. small fraction of the
available metals needs to be derailed to the A.beta.-containing
compartment to precipitate the peptide and to activate its
ROS-generating activities. The generation of ROS described herein
depends upon the sub-stoichiometric amounts of Fe.sup.+/Cu2+ (1:10,
metal:A.beta.), and it was estimated that 1% of the zinc that is
released during neurotransmission would be sufficient to
precipitate soluble A.beta. in the synaptic vicinity (Huang, X. et
al., J. Biol. Chem. 272:26464-26470 (1997)).
[0326] A polypeptide which generates both substrates of the Fenton
reaction in sufficient quantities to form significant amounts of
the OH. radical is unusual. Therefore, A.beta. collections in the
AD-affected brain are likely to be a major source of the oxidation
stress seen in the effected tissue. One recent report describes
that A.beta. is released by the treatment of the mammalian lens in
culture with H.sub.2O.sub.2 (Frederikse, P. H., et al., J. Biol.
Chem. 271:10169 (1996)). If a similar response mechanism to
H.sub.2O.sub.2 stress exists in neocortex, then the increasing
H.sub.2O.sub.2 concentration generated by the accumulating A.beta.
mass in the AD-affected brain may induce the production of even
more A.beta. leading to a vicious cycle of A.beta. accumulation and
ROS stress.
[0327] The simultaneous production of Fenton substrates by A.beta.
is a chemical property that is brought into therapeutic application
in the oxidation mechanism of the bleomycin-iron complex. Bleomycin
is a glycopeptide antibiotic produced by Streptomyces verticillus
and is a potent antitumor agent. It acts by complexing Fe.sup.3+
and then binding to tumor nuclear DNA which is degraded in situ by
the generation of OHS (Sugiura, Y., et al., Biochem. Biophys. Res.
Commun. 105:1511(1997)). Similar to A.beta.-Fe.sup.3+/Cu.sup.2+
complexes, incubation of bleomycin in aqueous solution also
engenders the production of O.sub.2.sup.-, H.sub.2O.sub.2 and OH.
in an Fe.sup.+-dependent manner. DFO could not inhibit
H.sub.2O.sub.2 production from the A.beta.-Fe.sup.+/Cu.sup.2+
complex, and similarly, DFO does not inhibit the OH.-mediated DNA
damage caused by the bleomycin-Fe.sup.3+ complex. Also,
low-molecular-mass OH. scavengers mannitol and DMSO were unable to
inhibit the generation of OH. by A.beta.-Fe.sup.3+/Cu.sup.2+, and
are similarly unable to inhibit OH. production from
bleomycin-Fe.sup.3+.
[0328] It is proposed herein that inhibition of A.beta.-mediated
OHS 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.sup.2+ as well as Fe.sup.3+ and Al(III), the
effect, if verifiable, may not have been due to the abolition of
the redox activity of A.beta., but may have been due to the
disaggregation of Zn.sup.2+-mediated A.beta. deposits (Chemy, R. A.
et al., Soc. Neurosci. Abstr. 23:(abstract)(1997)) which may have
reduced cortical A.beta. burden and, consequently, oxidation
stress.
[0329] f) Oxidative Stress and Alzheimer's Disease Pathology
[0330] Autopsy tissue from AD subjects has been reported to exhibit
higher basal TBARS formation than control material (Subbarao, K. V.
et al., J. Neurochem. 55:342 (1990); Balazs, L. and M. Leon,
Neurochem. Res. 19:1131 (1994); Lovell et al., Neurology 45:1594
(1995)). These observations could be explained, on the basis of the
present findings, as being due to the reactivity of the A.beta.
content within the tissue. A.beta..sub.1-40 recently has been shown
to generate TBARS in a dose-dependent manner when incubated in cell
culture, however TBARS reactivity was reduced by pre-treating the
cells with trypsin which also abolished the binding of the peptide
to the RAGE receptor (Yan et al., Nature 382:685 (1996)). One
possibility for this result is that the RAGE receptor tethers an
A.beta. microaggregate sufficiently close to the cell to permit
increased penetration of the cell by H.sub.2O.sub.2 which may then
combine with reduced metals within the cell to generate the Fenton
reaction. Alternatively, A.beta. 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.
[0331] APP also reduces Cu.sup.2+, but not Fe.sup.3+, at a site in
its amino terminus (Multhaup, G., et al., Science 271:1406-1409
(1996)), adjacent to a functional and specific Zn.sup.2+-binding
site that modulates heparin binding and protease inhibition (Bush
et al., 1993; Van Nostrand, 1995). Therefore, the amino terminus of
APP reiterates an association with transition metal ions that is
found in the A.beta. domain. This intriguing theme of tandem Cu/Zn
interaction and associated redox activity found in two soluble
fragments of the parent protein may indicate that the function and
metabolism of APP could be related to biometal homeostasis and
associated redox environments.
[0332] 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.
Example 4
Resolubilization of A.beta.
[0333] Considerable evidence now indicates that the accumulation of
A.beta. in the brain cortex is very closely related to the cause of
Alzheimer's disease. A.beta. is a normal component of biological
fluids whose function is unknown. A.beta. accumulates in a number
of morphologies varying from highly insoluble amyloid to deposits
that can be extracted from post-mortem tissue in aqueous buffer.
The factors behind the accumulation are unknown, but the inventors
have systematically appraised the solubility of synthetic A.beta.
peptide in order to get some clues as to what kind of pathological
environment could induce the peptide to precipitate.
[0334] It was found that A.beta. has three principal
vulnerabilities--zinc, copper and low pH. The precipitation of
A.beta. 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 AD 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.
[0335] Recently direct evidence has been obtained indicating that
these metals are integral components of the A.beta. deposits in the
brain in AD. It was found that zinc- and copper-specific chelators
(including clioquinol) dramatically redissolve a significant
proportion (up to 70%) of A.beta. extracted from post-mortem AD
affected brain tissue, compared to the amount extracted from the
tissue by buffer in the absence of chelators.
[0336] These data support a strategy of redissolving A.beta.
deposits in vivo by chelation. Therefore, clioquinol is an
excellent candidate for further development since it chelates both
copper and zinc, and since it is hydrophobic, is enriched in the
brain. Interestingly, a reported success in attempting to slow down
the progression of Alzheimer's disease used a chelation strategy
with desferrioxamine. The authors (Crapper-McLachlan, D. R., et
al., 337:1304 (1991), thought that they were chelating aluminum,
but desferrioxamine is also a chelator of copper and zinc.
Treatment with desferrioxamine is impractical because the therapy
requires twice daily deep intramuscular injections which are very
painful, and also causes side effects such as anaemia due to iron
chelation.
[0337] Resolubilization of Metal-induced A.beta. Aggregates by
Chelators
[0338] A.beta. (10 ng/well in TBS) aggregation was induced by
addition of ZnCl.sub.2 (25 .mu.M), CuCl.sub.2 (5 .mu.M) or acidic
conditions (pH 5.5). Aggregates were transferred to a 0.2 .mu.
nylon membrane by filtration. The aggregates were then washed (200
.mu.l/well) with TBS alone, TBS containing 2 .mu.M EDTA, or TBS
containing 2 .mu.M clioquinol. The membrane was fixed, probed with
the anti-A.beta. monoclonal antibody 6E10, and developed for
exposure to ECL-film. FIG. 17 shows relative signal strength as
determined by transmittance analysis of the ECL-film, calibrated
against known amounts of the peptide. Values are expressed as a
percentage of A.beta. signal after washing with TBS alone.
[0339] Both EDTA and clioquinol treatments were more effective than
TBS alone at resolubilizing the retained (aggregated) A.beta. when
the peptide was precipitated by Zn or Cu (see FIG. 17). When
A.beta. was precipitated by pH 5.5 however, it was not
resolubilized more readily by either chelator compared to TBS
washing alone. The pH 5.5 precipitate contains a much greater
proportion of beta-sheet amyloid than the A.beta. precipitates
formed by Zn or Cu.
Example 5
A.beta. Extractionfrom Human Brain Post-mortem Samples
[0340] The inventors have recently characterized zinc-mediated
A.beta. deposits in human brain (Cherny, R. A., et al., Soc.
Neurosci 4bstr. 23:(Abstract) (1997)). It was recently reported
that there is a population of water-extractable A.beta. deposit in
the AD-affected brain (Kuo, Y -M., et al., J. Biol. Chem.
271:4077-81 (1996)). The inventors hypothesized that homogenization
of brain tissue in water may dilute the metal content in the
tissue, so lowering the putative zinc concentration in A.beta.
collections, and liberating soluble A.beta. subunits by freeing
A.beta. complexed with zinc [Zn.sup.2+].
[0341] To test this hypothesis, the brain tissue preparation
protocol of Kuo and colleagues was replicated, but
phosphate-buffered saline pH 7.4 (PBS) was substituted as the
extraction buffer, achieving similar results. Highly sensitive and
specific anti-A.beta. monoclonal antibodies (Ida, N., et al., J.
Biol. Chem., 271:22908 (1996)) were used to assay A.beta.
extraction by western blot. Next, the extraction of the same
material was repeated with PBS in the presence of chelators of
varying specificities (Table 1), and it was determined that the
presence of a chelator increased the amount of A.beta. in the
soluble extract several-fold (FIGS. 19A-19C, 20A and 20B, 25A;
Table 2).
[0342] The amount of A.beta. detected in the pellet fraction of
each sample is correspondingly lower, indicating that the effect of
the chelator is upon the disassembly of the A.beta. aggregate, and
not by inhibition of an A.beta.-cleaving metalloprotease (such as
insulin degrading enzyme cleavage of A.beta. reported recently by
Dennis Selkoe at the 27.sup.th Annual Meeting for the Society for
Neuroscience, New Orleans). The extraction of sedimentable A.beta.
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.
3TABLE 1 Dissociation Constants for Metal Ions of Various Chelators
Used to Extract Human Brain A.beta.. CHELATOR Ca Cu Mg Fe Zn Al Co
EGTA 10.9 17.6 5.3 11.8 12.6 13.9 12.4 EDTA 10.7 18.8 8.9 14.3 16.5
16.5 16.5 Penicillamine 0 18.2 0 0 10.2 0 0 TPEN 3.0 20.2 0 14.4
15.4 0 0 Bathophenanthroline 0 8.8 0 5.6 6.9 0 0 Bathocuproine (BC)
0 19.1 0 0 4.1 0 4.2 (Cu.sup.+)
[0343] LogK is illustrated for the chelators, where K=[ML]/[M][L].
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.
[0344] The ability of chelators to extract A.beta. from post-mortem
brain tissue was studied in over 40 cases (25 AD, 15 age-matched
and young adult controls, all confirmed by histopathology). While
there is a lot of variation between samples as to what is the best
concentration of given chelator for the optimum extraction of
A.beta., there are no cases where a chelator does not, at some
concentration, extract far more A.beta. than PBS alone.
[0345] FIG. 19 shows that metal chelators promote the
solubilization of A.beta. 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.beta. 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.degree.
C.). Furthermore, similar results were achieved when the brain
sample was homogenized in PBS and then pelleted before treated with
PBS .+-.chelator.
[0346] There is also a complex relationship between the dose of the
chelator and the resultant resolubilization of A.beta. (FIGS.
19A-C). For the same given sample, neither TPEN nor EGTA could
increase the extraction of A.beta. in a does-dependent manner.
Rather, although concentrations of chelators could be very
effective in the low micromolar range (e.g., TPEN 4 .mu.M, FIG.
19A), higher concentrations induced a paradoxical loss of recovery.
This kind of response was found in every case examined. The
extraction of A.beta. is abolished by adding exogenous zinc, but is
enhanced by adding magnesium. Preliminary in vitro data indicate
that whereas Mg has no effect on the precipitation of A.beta., its
presence enhances the peptide's resolubilization following
zinc-induced precipitation. Therefore, the "polyphasic" profile of
chelator extraction of A.beta., with higher concentrations of TPEN
and EGTA inducing a loss of recovery, may be explained by the
chelation of Mg that is only expected to occur after the chelation
of zinc when the relative abundance of Mg in the sample, and the
relative dissociation constants of TPEN and EGTA are
considered.
[0347] In contrast, bathocuproine (BC) exhibits a clear
dose-dependent increase in A.beta. extraction from human brain,
probably due to its relatively high specificity for zinc, although
an interaction with trace amounts of Cu.sup.+ or other metals not
yet assayed, cannot be excluded.
[0348] Western blot analysis of extracts using
A.beta..sub.1-42-specific monoclonals revealed the presence of
abundant A.beta..sub.1-42 species. It was observed that
.apprxeq.20% of AD cases exhibit clear SDS-resistant A.beta. dimers
in the soluble extract after treatment with chelators. These dimers
are reminiscent of the neurotoxic A.beta..sub.1-42 dimers that were
extracted by Roher and colleagues from AD-affected brain (Roher, A.
E., et al., Journal of Biological Chemistry 271:20631-20635
(1996)). An estimation of the proportion of total precipitated
A.beta. in the sample was achieved by extracting the homogenate
pellet following centrifligation, into formic acid, and then
performing a western blot on the extract following neutralization.
The proportion of pelletable A.beta. that is released by chelation
treatment varies considerably from case to case, from as little as
30% to as much as 80%. In the absence of a chelator, no more than
10% of the total pelletable A.beta. is extracted by PBS alone.
[0349] One preliminary emerging trend is that samples with a
greater proportion of diffuse or vascular A.beta. deposit are more
likely to have their pelletable A.beta. resolubilized by chelation
treatment. Also, extraction of the tissue homogenate overnight with
agitation greatly increases the amount of A.beta. extracted in the
presence of chelators (compared to PBS alone), when compared to
briefer periods of extraction indicating that the disassembly of
A.beta. deposits by chelation treatment is a time-dependent
reaction and is unlikely to be due to inhibition of a protease. A
study of brain cortical tissue from one amyloid-bearing APP
transgenic mouse indicates that, like human brain, homogenization
in the presence of a chelator enhances the extraction of pelletable
A.beta..
[0350] Effects of various chelators on the extraction of A.beta.
into the supernatant as a percentage change from control
extractions is summarized below in Table 2.
4TABLE 2 Effects of Various Chelators Upon Extraction of A.beta..
Effect of Chelators (% change from control) TPEN EGTA BATHOCUP 0.1
mM 2.0 mM 0.1 mM 2.0 mM 0.1 mM 2.0 mM Mean (n = 6) 182 241 207 46
301 400 +/-SD 79 81 115 48 190 181
[0351] Densitometry of A.beta. western blots (FIGS. 19A-19C) was
performed for a series of 6 AD brain samples homogenized in the
presence of chelators as indicated. The mean (.+-.SD) increases in
signal, above the signal generated by PBS extraction alone, are
indicated in Table 2. A significantly increased amount of
chelator-induced A.beta. resolubilization was achieved by a 16 hour
extraction with agitation in subsequent studies.
[0352] Table 3 shows a comparison between pellets of
post-centrifugation homogenates in the presence and absence of a
chelator (TPEN).
5TABLE 3 Residual Metals in Pellets of Post-Centrifugation
Homogenatesin the Presence and Absence of Chelator. METAL Zn Cu Fe
Ca Mg Al PBS 50.7 11.9 227 202 197 44 alone (12.0) (3.5) (69) (69)
(94) (111) mg/kg (SD) +TPEN 33.2* 9.8 239 (210) 230 65 mg/kg (9.8)
(3.1) (76) (89) (94) (108) (SD)
[0353] Frontal cortex from AD (n=6) and healthy controls (n=4) was
homogenized in the presence and absence of PBS i TPEN (0.1 mM).
After ultracentrifugation of the homogenate, the pellets were
extracted into concentrated HCl and measured for metal content by
ion coupled plasma--atomic emission spectroscopy (ICP-AES).
[0354] Using the same technique, zinc-mediated assembly of A.beta.
in normal brains was shown. FIGS. 20A and 20B show sedimentable
A.beta. deposits in healthy brain tissue. The effects of chelators
in enhancing A.beta. extraction from brain homogenates is also
observed in normal tissue. FIG. 20A illustrates a western blot with
anti-A.beta. 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.beta. from control tissue than from AD tissue. These
data are typical of 15 cases.
[0355] As expected, far less total A.beta. is present in normal
brain samples compared to AD brain samples, although the content of
A.beta. increases with age. It is possible that these findings in
young adult brains represent the zinc-mediated initiation of
amyloid formation in deposits that, in youth, are too diffuse to be
detected by immunohistochemistry.
[0356] Roher and others have suggested that dimers of A.beta. are
the toxic component of amyloid. As shown in FIG. 21, dimers appear
in response to chelation in disproportion to the monomeric signal
(treatment with PBS alone does not generate soluble dimers). This
suggests that A.beta. deposits are being dismantled by the
chelators into SDS-resistant dimeric structural units.
[0357] FIG. 22 shows that the recovery of total soluble protein is
not affected by the presence of chelators in the homogenization
step. The proportionality of extracted subfractions, calculated
based on total protein as determined by formic acid extraction,
should not be prone to artifact based on chelator-specific
affects.
Example 6
Resolubilization of A.beta. by Clioquinol
[0358] In one previous attempt to use metal chelation as a
therapeutic for AD, Crapper-McLachlan and colleagues
(Crapper-McLachlan, D. R., et al., 337:1304 (1991)) administered
intramuscular desferrioxamine (DFO) daily to a small cohort of AD
patients, and reported that their treatment attenuated the
progression of the disease. Replication of this study has not been
attempted.
[0359] The inventors attributed the beneficial effect to the
removal of aluminum; however, they have conceded in presentations
at meetings (e.g. International Conference on Alzheimer's Disease,
1992, Padua) that post-mortem metal analysis on brain tissue from
subjects in the study indicated that although aluminum levels were
lowered than placebo controls, zinc and iron levels were also lower
in the brains of subjects treated with DFO. This is because, like
all chelators, DFO has only a relative specificity for aluminum,
but with also complex with zinc and iron. There appears to be no
report on a histopathological analysis of post-mortem brain A.beta.
content in the subjects who took DFO compared to the controls.
[0360] The administration of DFO, a painful intramuscular
injection, is fraught with complications including the non-specific
problems of chelation therapies (e.g. anemia). Although the results
of Crapper-McLachlan and colleagues remain contentious and have not
yet been reproduced, the possibility that the beneficial effects
they reported were due to the partial removal of zinc from brain
A.beta. collections cannot be excluded. DFO is a charged molecule
that does not easily penetrate the blood-brain barrier, and, as
such, is not an ideal candidate for the removal of zinc from
A.beta. deposits, especially as its affinity for zinc is relatively
low. Therefore, a more suitable candidate compound to attempt a
trial of A.beta. Dissolution in APP Tgs was sought.
[0361] Clioquinol (iodochlorhydroxyquin,
5-chloro-7-iodo-8-hydroxyquinolin- e, MW 305.5) is a USP drug that
chelates zinc [K(Zn)=12.5, K(Cu)=15.8, K(Ca)=8.1, K(Mg)=8.6], is
hydrophobic, has a low general toxicity profile, and crosses the
blood brain barrier (Padmanabhan et al., 1989). It therefore
possesses some of the ideal prototypic properties for a candidate
agent that could solubilize zinc-assembled A.beta. deposits in
vivo. It has been used as an oral antiamebic antibiotic, and as a
topical antibiotic.
[0362] It has been demonstrated that clioquinol is rapidly absorbed
from the gut of rats and mice where blood levels reached 1-10 .mu.M
within one hour of ingestion (Kotaki et al., J Pharmacobiodyn,
6(11):881-887 (1983)). Since the drug is hydrophobic, it passes
rapidly into the brain, and then is rapidly excreted, so that a
bolus dose of clioquinol is almost completely removed from the
brain within three hours. It appears to be safe in many mammalian
species, including rat and mouse (Tateishi, J., et al., Lancet,
2(7786):1096 (1972); Tateishi, J., et al., Acta Neuropathol.,
(Berl), 24(4):304-320 (1973)), and is still used as a veterinary
antibiotic (Entero Vioform).
[0363] Clioquinol was withdrawn from use as an oral antibiotic for
humans in the early 1970's when its ingestion in Japan was linked
to a mysterious condition called subacute myelo-optic neuritis
(SMON), a condition that resembles subacute combined degeneration
of the cord caused by vitamin B12 deficiency. The mechanism of SMON
has never been elucidated, but in the 1970's a considerable
literature developed exploring the pathophysiology of clioquinol
ingestion (Tateishi, J., et al., Lancet, 2(7786):1096 (1972);
Tateishi, J., et al., Acta Neuropathol., (Berl), 24(4):304-320
(1973)). Several reports have demonstrated that clioquinol
complexes with zinc in the brain, especially in areas enriched in
synaptic vesicular zinc such as the temporal lobe (Shiraki, H.
Handbook of Clinical Neurology, Vol. 37 (1979)). Indeed, over
ingestion of clioquinol has been reported to induce amnesia in
humans (Shiraki, H. Handbook of Clinical Neurology, Vol. 37
(1979)).
[0364] Because of its relatively safe profile in mice, and because
there is a large literature on its pharmacology in this animal,
clioquinol was chosen for study as a means to specifically chelate
zinc from A.beta. deposits in vitro (induced aggregates and brain
samples). It is possible that the low concentrations of clioquinol
shown to be effective in resolubilizing A.beta. in the present
invention may avoid the adverse SMON effect noted above. Thus,
given its other pharmacological properties, clioquinol may hold
promise as a effective agent in the treatment of AD in humans.
[0365] Dissolving Clioquinol
[0366] In order to obtain a solution of clioquinol in PBS, the
following protocol was followed: 5.3 grams of clioquinol was
suspended with agitation in 200 milliliter of n-decane. The
undissolved material was settled, air dried, and weighed, based on
which it was determined that only 2% of the clioquinol had
dissolved in the n-decane. 100 milliliter of the supernatant (light
yellow) was agitated in 100 milliliter of PBS, pH 7.4. Next, the
phases were allowed to separate. The lower phase (PBS) was
collected and filtered to remove the residue which had formed at
the phase interface upon extraction with the organic solvent. The
concentration of clioquinol in the PBS was determined to be 800
nanomolar. This number was arrived at based on two assumptions: (1)
2% of the clioquinol was dissolved in the n-decane; and (2) the
partitioning coefficient is 1/1750 with PBS at 1:1 mixture of
n-decane to clioquinol.
[0367] Resolubilization of in vitro Metal-induced A.beta.
Aggregates
[0368] First, in order to appraise the efficacy of clioquinol in
resolubilizing A.beta. aggregates, its ability to resolubilize
A.beta. aggregates formed in vitro by the action of Cu.sup.2+ or
Zn.sup.2+ upon A.beta..sub.1-40 was examined FIG. 23 shows
resolubilization of metal-induced A.beta. aggregate by chelators.
A.beta. (10 ng/well in buffered saline) aggregation was induced by
addition of metals (5 .mu.M) or acidic conditions (pH 5.5).
Aggregates were transferred to a 0.2 .mu. nylon membrane by
filtration. The aggregates were then washed (200 .mu.l/well) with
TBS alone, TBS containing 2 .mu.M EDTA or TBS with 2 .mu.M
clioquinol. The membrane was then fixed, probed with anti-A.beta.
monoclonal antibody 6E10 and developed for exposure to ECL-film.
FIG. 23 shows the relative signal as determined by densitometric
analysis of the ECL-film, calibrated against known amounts of the
peptide. Values are expressed as a % of A.beta. signal remaining on
the filter after washing with TBS alone. Clioquinol is hydrophobic,
so that the reagent must first be solubilized in an organic
solvent, and then partitioned into the aqueous buffer according to
established protocols.
[0369] Like EDTA (FIG. 17), clioquinol significantly resolubilized
precipitated A.beta.. Cu.sup.2+ partially precipitates
A.beta..sub.1-40 (Bush, A. I., et al., Science 268:1921 (1995)) at
pH 7.4. EDTA (2 .mu.M) resolubilized 35% of a Zn.sup.2+-induced
A.beta. precipitate, 60% of a Cu.sup.2+-induced precipitate, and
15% of a pH 5.5-induced precipitate. In contrast, clioquinol (2
.mu.M) was more effective at resolubilizing the Zn.sup.2+- and
Cu.sup.2+-induced A.beta. precipitates (50%, and 85%,
respectively), but was also ineffective at resolubilizing the pH
5.5 precipitate (10%). Since the aggregate at pH 5.5 is
predominantly .beta.-shect (Wood, S. J. et al., J. Mol Bio.,
256:870-877 (1996)), these data indicate that the resolubilization
of A.beta. by clioquinol/EDTA is likely to be due to specific
chelation effects.
[0370] Extraction of Agfrom Samples of AD-affected Brains
[0371] Next the ability of clioquinol to extract A.beta. deposits
from human brain was examined. It was found that, like other zinc
chelators, clioquinol efficiently increases the resolubilization of
AD, compared to the amount of A.beta. resolubilized from the pellet
fraction of brain homogenate by PBS alone. FIG. 24 shows the effect
of clioquinol upon the extraction of A.beta. from AD-affected
brain. Fragments of prefrontal cortex from individual post-mortem
samples with the histopathological diagnosis of AD were homogenized
in PBS, pH 7.4, and then pelleted after centrifugation. The pellets
were then washed with agitation twice for 30 minutes, 4.degree. C.,
with PBS or PBS containing clioquinol (100% =0.8 .mu.M clioquinol).
The suspension was then pelleted (10,000 g for 30 minutes) and the
supernatant removed (S1) for western blot analysis using
A.beta.-specific antibodies (illustrated). The pellet was treated a
second time in this experiment with agitation and centrifugation,
and the second supernatant (S2) analysed. The data show typical
results by western blot.
[0372] In agreement with earlier findings which showed that the
optimal concentration of chelator for the extraction of A.beta. is
idiosyncractic from case to case, and that there is a paradoxical
diminution of A.beta. extraction when the chelator concentration
rises above the optimum, it was found that optimal clioquinol
concentrations for A.beta. resolubilization vary in a similar
manner (e.g., Specimen #1=0.08 .mu.M, #2=0.8 .mu.M). It was also
observed that apparently dimeric A.beta. was more frequently
observed on SDS-PAGE (illustrated), and that in these cases (e.g.,
Specimen #2) the first wash did not resolubilize much A.beta., but
the second wash was very efficient at resolubilizing the peptide.
It was surmised that the pellet mass may be coated with
adventitial, non-A.beta., proteins that are removed by the first
wash, allowing the second treatment access to the A.beta.
collection. Indeed, further studies have shown that both sustained
(for 16 hours) and repeated exposure to the chelator increases the
resolubilization of A.beta. significantly.
[0373] FIG. 25A and 25B show the western blot and accompanying
densitometric analysis of reolubilization of A.beta. from
AD-affected brain. FIG. 25A is a western blot showing the effect of
clioquinol upon the resolubilization of A.beta. from AD-affected
brain. In this study, the brain specimen (from a different case
than that of FIG. 24) was homogenized according to the protocols in
FIG. 19. In this case a dose-dependent response to clioquinol was
observed. Synthetic peptide standards that were used to calibrate
densitometric quantification are shown in the two right-most
lanes.
[0374] FIG. 25B is a chart showing densitometry performed upon the
results in FIG. 25A, above. Proportional change in the amount of
A.beta. recovered in the extraction of A.beta. by clioquinol from
human brain is shown. As little as a 1% dilution of clioquinol in
PBS (100% =0.8 .mu.M) or 8 nM clioquinol is capable of doubling the
recovery of A.beta. in the soluble phase.
[0375] In sequential extraction experiments, as described above,
clioquinol (1.12 .mu.M) has been shown to result in a 2.5 fold
increase in solubilization of A.beta. relative to PBS alone (see
FIGS. 25A and 25B). Significantly, the findings the present
invention show that very low (8 nM) concentrations of clioquinol
may resolubilize more than twice the amount of A.beta. compared to
PBS buffer alone (see FIGS. 25A and 25B). This suggests that such
low concentrations may be therapeutically effective in treating
amyloidosis, preferrably that occurring in AD-affected human
subjects.
Example 7
Potentiation of Resolubilization of Amyloidfrom AD-affected Brain
Tissue
[0376] A.beta. was extracted from cortical tissue obtained from
three subjects with clinically and histopathologically confirmed
Alzheimer's disease in the presence of 1.6 .mu.M clioquinol (CQ), 2
mM bathocuproine (BC), CQ+BC or PBS. Soluble A.beta. (ng/g tissue)
was determined as described. Total A.beta. was determined following
formic acid extraction of otherwise untreated tissue.
[0377] FIG. 26 illustrates the potentiation of A.beta.
resolubilization using clioquinol in combination with bathocuproine
by graphically showing the proportion of total A.beta. extracted.
Table 4 below shows the data depicted in FIG. 26 and, in addition,
shows each chelator or chelator combination in PBS buffer.
6TABLE 4 Potentiation of Chelator-Promoted A.beta. Solubilization
BC + CQ + Subject PBS CQ BC CQ + BC CQ + PBS PBS BC + PBS 1 0.74
1.85 3.1 5 0.11 2.36 4.26 2 1.8 4.5 7.2 11.2 2.7 5.4 9.4 3 2.3 3.4
6 12.7 1.1 3.7 10.4 (% of total A.beta. extracted)
[0378] The effect of clioquinol and bathocuproine combined is seen
to be much more than additive. In subject 3, for example, the
potentiated effect was over twice that of a simple additive effect
(10.4 compared to 1.1+3.7 or 4.8). These data suggest that
combinations of clioquinol and bathocuproine may be particularly
effective therapeutic combinations for the treatment of
amyloidosis, in particular, the pathological A.beta.-aggregation
manifest in brains of those afflicted with Alzheimer's disease.
Example 8
Differential Effects of Chelation of Cerebral A.beta. Deposits in
AD-affected Subjects Versus Age-matched Controls and the Effect of
Magnesium
[0379] Experiments involving extraction of cerebral tissue from
AD-affected subjects and non-AD, age-matched controls by chelation
indicate different resolubilization responses of amyloid deposits
between the two sample groups with regard to extraction by specific
chelators.
[0380] Higher concentrations of chelators with relatively broad
specificity (e.g. EGTA) result in less resolubilization of A.beta.
deposits. Experiments show that chelation of magnesium negatively
affects resolubilzation of A.beta. deposits.
[0381] Materials and Methods
[0382] Cortical 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 41:479-486
(1991)) with particular attention paid to the presence of neuritic
plaques and neurofibrillary tangles. Histological examination of
A.beta. levels in normal specimens ranged from
immunohistochemically undetectable to substantially present in the
form of diffuse plaques.
[0383] 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.times.30s 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, California.--Note: EDTA was
not included in the protease inhibitor mixture) or in the presence
of chelators or metal ions prepared in PBS. To obtain the soluble
fraction, the homogenates were centrifuged at 100,000.times. g for
30 min (Beckman J180, Beckman instruments, Fullerton, California)
and the supernatant collected in 1 ml aliquots and stored on ice or
immediately frozen at -70.degree. C. In each experiment, all
protein was precipitated from 1 ml of supernatant from each
treatment group using 1:5 ice cold 10% trichloracetic acid and
pelleted in a bench top microfuge (Heraeus, Osteroder, Germany) at
10,000.times. g. The remaining pellet was frozen at -70.degree.
C.
[0384] The efficiency of the precipitation was validated by
applying the technique to a sample of whole human serum, diluted
1:10, to which had been added 2 .mu.g of synthetic A.beta..sub.1-40
or A.beta..sub.1-42 (W. Keck Laboratory, Yale University New Haven,
Conn.). Protein in the TCA pellet was estimated using the Pierce
BCA kit (Pierce, Rockford, Ill.). The total A.beta. load of
unextracted cortex was obtained by dissolving 0.5 g of grey matter
in 2 ml of 90% formic acid, followed by vacuum drying and
neutralization with 30% ammonia.
[0385] Precipitated protein was subjected to SDS polyacrylamide gel
electrophoresis (SDS-PAGE) on Novex pre-cast 10-20% Tris-Tricine
gels followed by Western transfer onto 0.2 .mu.m nitrocellulose
membrane (Biorad, Hercules, Calif.). A.beta. was detected using the
WO2, G210 or G211 monoclonal antibodies (Ida, N., et al., J. Biol.
Chem., 271:22908 (1996)) in combination with HRP-conjugated rabbit
anti-mouse IgG (Dako, Denmark), and visualized using
chemiluminescence (ECL, Amersham Life Science, Little Chalfont,
Buckinghamshire, UK). Each gel included two or more lanes
containing known quantities of synthetic A.beta. which served as
internal reference standards. Blot images were captured by a
Relisys scanner with transparency adapter (Teco Information
Systems, Taiwan, ROC) and densitometry conducted using the NIH
Image 1.6 program (National Institutes for Health, USA. Modified
for PC by Scion Corporation, Frederick, Md.), calibrated using a
step diffusion chart. For quantitation of A.beta. in brain
extracts, the internal reference standards of synthetic A.beta.
were utilized to produce standard curves from which values were
interpolated.
[0386] In the experiments corresponding to the results shown in
FIG. 27, duplicate 0.2 g samples of AD cortical tissue were
homogenized and subjected to ultracentrifugation as described, but
using either 1 ml or 2 ml of extraction buffer (PBS). Protein was
precipitated from the entire supernatant and redissolved in 100
.mu.l of sample buffer. Equal volumes of TCA-precipitated protein
were subjected to Tris-Tricine SDS-PAGE and A.beta. was visualized
as described above.
[0387] In the experiments corresponding to the results shown in
FIG. 28A, 0.2 g specimens of frontal cortex from AD brain were
homogenized in the presence of 2 ml of PBS or varying
concentrations of Cu.sup.2+ (Cu(SO.sub.4).sub.2) or Zn.sup.2+
(Zn(SO.sub.4).sub.2). A.beta. in the high speed supernatant was
visualized as described above.
[0388] In the experiments corresponding to the results shown in
FIG. 28B, 0.2 g specimens of frontal cortex from AD brain were
homogenized in the presence of 2 ml or PBS or 2 mM EGTA. The
homogenates were spun at 100,000.times. g for 30 min and the
supernatant discarded. The remaining (metal depleted) pellets were
rehomogenized in a further 2 ml of either PBS alone EGTA alone, 2
mM Mg.sup.2+ (Ng(Cl).sub.2.6H.sub.2O) in PBS or 2 mM Ca.sup.2+
(CaCl.sub.2.2H.sub.2O) in PBS and the homogenate subjected to
ultracentrifugation. A.beta. in the soluble fraction was visualized
as described above.
[0389] In the experiments corresponding to the results shown in
FIGS. 29A and 29B, frontal cortex from AD (n=6) and age-matched,
amyloid-positive (n=5) subjects were treated with PBS, TPEN, EGTA
or BC (0.1 mM and 2 mM) and soluble A.beta. assessed as described
above.
[0390] In the experiments corresponding to the results shown in
FIG. 30, representative AD (left panels) and aged-matched control
specimens (right panels) were prepared as described in PBS or 5 mM
BC. Identical gels were run and Western blots were probed with mAbs
WO2 (raised against residues 5-16, recognizes A.beta..sub.1-40 and
A.beta..sub.1-42), G210 (raised against residues 35-40, recognizes
A.beta..sub.1-40), or G211 (raised against residues 35-42,
recognizes A.beta..sub.1-42) (See Ida, N., et al., J. Biol. Chem.,
271:22908 (1996)).
[0391] Results and Discussion
[0392] To further explore the involvement of metal ions in the
deposition and architecture of amyloid deposits, the inventors
extracted brain tissue from histologically-confirmed AD-affected
subjects and from subjects that were 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 5 below.
7TABLE 5 Stability constants of metal chelators Ca Cu Mg Fe Zn Al
Co EGTA 10.86 17.57 5.28 11.8 12.6 13.9 12.35 TPEN 3 20.2 n/a 14.4
15.4 n/a n/a BC n/a Cu.sup.2+ n/a n/a 4.1 n/a 4.2 6.1 Cu.sup.+
19.1
[0393] logK10 where K=[Metal.Ligand]/[Metal][Ligand]. From: NIST
database of critically selected stability constants for metal
complexes Version 2.0 1995.
[0394] A series of titration curves were prepared to determine the
chelator concentration at which maximal response was obtained. In
these experiments, selected chelators were limited to EGTA, TPEN
and BC. FIGS. 19A-C show that chelators affect the solubilization
of A.beta. in a dose-dependent manner.
[0395] It was found that EGTA and TPEN elicited a significant
enhancement in solubilization of A.beta. in a pattern of response
typified by peak values at or near 0.004 mM and 0.1 mM, and lower
values at concentrations in between. Both chelators were
increasingly ineffective at concentrations over 1 mM, and at 2 mM,
EGTA virtually abolished the signal for A.beta.. In contrast, BC
elicited a typical concentration-dependent response with no decline
in effectiveness in the low millmolar range even when extended to
20 mM. Total TCA-precipitated protein in the supernatant was
assayed and found to be unaffected by either chelator kind or
concentration.
[0396] Recent findings have demonstrated the presence of neurotoxic
dimers in the soluble (Kuo, Y -M., et al., J. Biol. Chem.
271:4077-81 (1996)) and insoluble (Roher, A. E., et al., Journal of
Biological Chemistry 271:20631-20635 (1996); Giulian, D. et al., J.
Neurosci., 16:6021-6037 (1996)) fractions of A.beta. extracts of
the brains of AD individuals. FIG. 21 shows that chelator-promoted
solubilization of A.beta. elicits SDS-resistant dimers. Under the
preparation conditions used, SDS-resistant dimers were not
generally observed in the extracts with PBS alone. Dimers were
found to appear in response to chelator-promoted solubilization of
A.beta. however.
[0397] The signal for dimeric A.beta. was frequently
disproportionate to that of monomeric A.beta. and the ratio varied
with both the type and concentration of chelator used (FIG. 21). In
contrast, when synthetic A.beta..sub.1-40 was run under identical
conditions, the monomer:dimer ratio reflected a predictable and
reproducible concentration-dependent relationship. These data
suggest that the dimers observed in extracts of human brain are
predominantly an intermediate structural unit generated by the
dissolution of amyloid, resulting in turn from the sequestration of
metals by chelating agents.
[0398] FIG. 28A shows the effect of metals upon the solubility of
brain-derived A.beta.. Precipitation of A.beta. was induced by
adding either copper or zinc to unchelated extracts. The resulting
signal for soluble A.beta. was attenuated, the threshold
concentration being between 20 and 50 .mu.M for copper and between
5 and 20 .mu.M for zinc. At concentrations greater than 100 .mu.M
solubility was abolished. Interestingly, at lower concentrations of
copper there appears to be a transitional stage where A.beta. is
present in the dimeric form prior to complete aggregation,
mirroring the intermediate stage dimers elicited by
chelator-mediated solubilization.
[0399] In order to confirm that the chelators were effective at
sequestering metals at the concentrations employed in these
experiments, ICP was used to determine the residual levels of
several metals in the post-centrifugation pellets retained from the
experiment described in FIGS. 19A-19C. Of thesix metals tested,
zinc levels were reduced by TPEN in a dose dependent manner,
whereas EGTA affected calcium and magnesium, particularly at higher
concentrations.
8TABLE 6 Residual Metal Levels in Post-Centrifugation (Extracted)
Pellets Mg Al Ca Fe Zn Cu (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)
(mg/kg) TPEN (mM) PBS 202 36 573 411 60 13 0.004 147 22 322 317 28
10 0.001 192 34 490 512 42 12 0.04 201 22 956 322 22 10 0.1 200 60
708 389 21 12 2.0 200 148 419 376 19 11 5.0 205 16 377 307 17 10
EGTA (mM) PBS 223 52 1186 266 45 11 0.004 228 73 795 247 53 11
0.001 237 43 862 281 49 12 0.04 247 104 1402 438 71 13 0.01 213 61
675 272 54 13 2.0 191 62 519 238 27 13 5.0 168 27 455 230 18 12 BC
(mM) 0.004 234 33 489 231 47 12 0.001 225 88 1306 275 47 13 0.04
226 38 753 248 56 15 0.01 223 73 762 256 49 13 2.0 254 42 1602 271
49 14 5.0 238 38 912 249 53 15
[0400] Metal levels were measured in 10 AD specimens treated with
0.1 mM TPEN. See Table 7 below. The observed increase in
extractable A.beta. correlated with significant depletion in zinc
in every case and to a lesser extent, copper, when compared with
PBS-treated tissue. No other metal tested was significantly
influenced by treatment at this concentration.
9TABLE 7 Residual Metal Levels (Based on 10 AD Specimens) Zn Cu Fe
Ca Mg Al PBS 50.7 11.9 227 202 197 44 (+/-SEM) (4.9) (1.5) (28.8)
(28.3) (39.1) (46.2) TPEN 33.2 9.8 239 210 230 65 (+/-SEM) (4.1)
(1.7) (31.7) (37.0) (39.2) (45.0)
[0401] Given the precipitous decline in extractable A.beta.
observed when employing high concentrations of TPEN or EGTA (see
FIG. 9), it was hypothesized that magnesium or calcium might also
have a significant role in the A.beta. solubility equilibrium.
Magnesium or calcium added to the homogenization buffer produced no
appreciable alteration in soluble A.beta.. However, using an
extract previously depleted of metals by high levels of EGTA, the
addition of magnesium, and to a much lesser extent calcium, led to
resolubilization of the precipitated A.beta.. FIG. 28B shows that
A.beta. solubility in metal-depleted tissue samples is restored by
supplementing with magnesium.
[0402] Mindful of the high variability observed between individual
subjects, 6 AD and 5 aged-matched control brains were chosen at
random to determine if the observed phenomena were broadly
applicable. These specimens were subjected to chelation treatment
at selected concentrations of 0.1 or 2.0 mM or with PBS alone. FIG.
29A shows that patterns of chelator-promoted solubilization of
A.beta. differ in AD and aged, non-AD tissue. The chelator-promoted
solubilization of A.beta. 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.beta. signal
below that observed for the PBS control (See FIG. 29B).
[0403] The effects observed with non-demented, aged-matched
controls were similar with respect to EGTA and TPEN. However, it is
noteworthy that the effect of BC was much reduced. In some cases
(FIG. 29A, lower panel), BC treatment caused an attenuation in
soluble A.beta. suggesting that the amyloid deposits in AD-affected
brain respond to this chelator in a different fashion than the
deposits predominating in non-demented elderly brain.
[0404] For each subject in the experiments of FIGS. 29A and 29B,
the extractable A.beta. was derived and calculated as a proportion
of the total pre-extraction A.beta. load See Table 8 and 9
below.
10TABLE 8 AD-Affected Tissue AD 1 2 3 4 5 6 X +/-SEM X C/PBS Total
A.beta.(.mu.g/g) 10.8 77.0 80.3 6.0 14.4 16.8 43.0 14.1 PBS .mu.g/g
0.74 1.39 1.04 0.07 3.0 0.06 1.05 0.44 (% of total) (0.1) (1.8)
(1.3) (1.1) (2.1) (0.4) (1.2) (0.3) TPEN 2 mM .mu.g/g 0.21 3.40
1.80 5.50 5.00 0.28 2.73 0.85 2.60 (% of total) (0.2) (4.4) (2.25)
(9.2) (3.5) (1.75) (4.6) (0.9) BC 2 mM .mu.g/g 0.31 5.54 3.62 6.05
6.03 0.54 4.10 0.86 3.90 (% of total) (0.3) (7.2) (4.5) (10.0)
(4.2) (3.4) (5.4) (1.2)
[0405]
11TABLE 9 Age-Matched Control Tissue AC 1 2 3 4 5 X +/-SEM X C/PBS
Total A.beta.(.mu.g/g) 0.7 4.2 2.7 3.2 3.6 2.8 0.60 PBS .mu.g/g
0.17 0.13 0.18 0.10 0.66 0.25 0.10 (% of total) (25.0) (3.1) (6.7)
(3.3) (18.3) (11.3) (4.4) TPEN 2 mM .mu.g/g 0.22 0.38 0.26 0.09
1.06 0.40 0.17 1.6 (% of total) (32.0) (9.0) (9.7) (3.0) (29.5)
(16.7) (5.1) BC 2 mM .mu.g/g 0.03 0.24 0.29 0.08 0.98 0.32 0.16
1.28 (% of total) (5) (5.7) (11.0) (2.6) (27.2) (10.3) (4.6)
[0406] Total A.beta. for AD brains ranged from 6-80.mu.g/g wet
weight tissue. The percentage of A.beta. extractable (one
extraction/centrifugation sequence) ranged from 0.33-10%. The
corresponding values for aged-matched control brains were 0.68-4.2
.mu.g/g total A.beta. and 2.6-29.5% extractable.
[0407] In order to further investigate these different responses to
chelators, triplicate blots of AD tissue and control tissue which
displayed cerebrovascular and diffuse amyloid deposits were
compared. FIG. 30 shows that chelation promotes the solubilization
of A.beta..sub.1-40 and A.beta..sub.1-42 from AD and non-AD tissue.
Using 3 different monoclonal antibodies, attempts to detect whether
any particular species of A.beta. were selectively affected by
chelation were performed. Both A.beta..sub.1-40 and
A.beta..sub.1-42 were liberated by chelation, however the dimeric
form of A.beta..sub.1-40 in both AD and control tissue
predominated. As reported by Roher, A. E., et al., PNAS 90:
10,836-10,840 (1993), the predominant form of cerebrovascular
amyloid is A.beta..sub.1-42. Somewhat surprisingly, the dimeric
form of this highly aggregating species is absent in the (control)
tissue in which it is most favored.
[0408] It has recently been reported that the zinc-dependent
Insulin Degrading Enzyme (IDE) has significant A.beta. cleavage
activity (Perez et al., Proc Soc. for Neuroscience 20: Abstract
321.13 (1997))23. In the experiments presented here, the
disassembly of amyloid is reflected in the intermediate dimeric
species which result from conversion between soluble and insoluble
forms. Thus, simple inhibition of catalytic enzyme activity cannot
account for the observed increase in soluble A.beta.. However, in
the event that a proportion of the chelator-mediated augmentation
of A.beta. solubilization was due to inhibition of this enzyme,
homogenisations were conducted both in the presence of 1 mM n-ethyl
amimide (NEM), a potent inhibitor of IDE, and at 37.degree. C. No
enhancement of A.beta. signal was observed above that of PBS alone
for NEM, nor was there any diminution of signal after incubation at
37.degree. C.
[0409] Discussion
[0410] Metal chelators offer a powerful tool for investigating the
role of metals in the complex environment of the brain, however the
strengths of these compounds may also define their limitations. The
broad metal affinities of most chelators make them rather a blunt
instrument. Attempts were made to sharpen the focus of the use of
chelators by selecting chelators with a range of affinities for the
metals of interest. These differences may be exploited by
appropriate dilution, thereby favoring the binding of the
relatively high affinity ligand (metal for which the chelator has
the highest affinity).
[0411] The dilution profiles exhibited by EGTA and TPEN possibly
reflect a series of equilibria between different metal ligands and
the chelator, whereby the influence of abundant but low affinity
metals is observed at high chelator concentrations and that of the
high affinity, but more scarce, metals is predominates at low
concentrations of chelator. In the case of A.beta. itself, this
explanation is further complicated by the presence of low and high
affinity binding sites for zinc (and copper) (Bush, A. I. et al.,
J. Biol. Chem., 269:12152-12158 (1994)).
[0412] Bathocuproine with its low affinity for metals other than
Cu.sup.+ is effective at solubilizing A.beta. through a dilution
range over 3 orders of magnitude, and interestingly, does not
diminish in effectiveness at the highest levels tested. The
particular affinity of BC for Cu.sup.+ has been exploited to
demonstrate that in the process of binding to APP, Cu.sup.2+ is
reduced to Cu.sup.+ resulting in the liberation of potentially
destructive free radicals (Multhaup, G., et al., Science
271:1406-1409 (1996)). It has been shown that A.beta. has a similar
propensity for reducing copper with consequent free radical
generation (Huang, X., et al., J. Biol. Chem. 272:26464-26470
(1997)).
[0413] Although the predicted reduction in copper in extraction
pellets treated with BC has not been demonstrated, it is possible
that the ratio of Cu.sup.2+ to Cu.sup.+ has been affected. At this
stage, however, the means to evaluate the relative contributions of
divalent and reduced forms to the total copper content of such
extraction pellets are not available.
[0414] In addition to their primary metal binding characteristics,
chelators are a class of compounds which vary in hydrophobicity and
solubility. Their capacity to infiltrate the highly hydrophobic
amyloid deposits may therefore be an important factor in the
disassembly of aggregated A.beta.. It is also possible that the
chelators are also acting to liberate intracellular stores of
A.beta. in vesicular compartments as metal-bound aggregates.
Preliminary data from our laboratory indicates that this may be the
case with platelets.
[0415] 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.
[0416] On the basis of the evidence presented here and the in vitro
data, it is proposed that zinc functions in the healthy individual
to promote the reversible aggregation of A.beta., counteracted by
magnesium acting to maintain A.beta. solubility. Further, the
disease state is characterized by an unregulated interaction with
copper resulting in the generation of free radicals.
[0417] A functional homoeostatic mechanism implies equilibrium
between intracellular copper and zinc (and perhaps other metals)
normally present in trace amounts, for which A.beta. has strong
affinity, and more abundant metals which bind less strongly to
A.beta.. 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, 308:734-736 (1984)).
[0418] It has recently been demonstrated (Huang, X., et a., J.
Biol. Chem. 272:26464-26470 (1997)) that zinc-promoted aggregation
of synthetic A.beta. is reversible by the application of EDTA. The
tightly-regulated neurocortical zinc transport system might provide
a physiological parallel for this chelator-mediated disaggregation
by moving zinc quickly in and out of the intraneuronal spaces.
[0419] Copper, wvhile binding less avidly to A.beta. than zinc
(Bush, A. I., et al., J. Biol. Chem. 269:12152-12158 (1994)) has
greater potential to inflict damage via free radical generation,
resulting polymers are not reversible (see Example 10, below).
Slight alterations in the transportation and/or metabolism of
metals resulting from age-related deterioration of cellular
processes may provide the environment for a rapid escalation of
metal-mediated A.beta. accretion which eventually overwhelms
regulatory and clearance mechanisms. In describing a mechanism for
A.beta. homeostasis this model for amyloid deposition implies a
possible physiological role for A.beta. whereby aggregation and
disaggregation may be effected through regulation or cortical metal
levels and that the predominantly sporadic character of AD reflects
individual differences in the brain milieu. Such a mechanism by no
means rules out other genetic, environmental, inflammatory or other
processes influencing the progression of the disease. Furthermore,
in demonstrating the effectiveness of chelators in solubilising
amyloid, it is suggested herein that agents of this type are useful
for therapeutic or prophylactic use in AD.
Example 10
Formation of SDS-resistant A.beta. Polymers
[0420] The cause for the permanent deposition of A.beta. in states
such as Alzheimer's Disease (AD) and Down's Syndrome (DS) are
unknown, but the extraction of A.beta. from the brains of AD and DS
patients indicates that there are forms of A.beta. that can be
resolubilized in water and run as a monomer on SDS-PAGE (Kuo, Y
-M., et al., J. Biol. Chem. 271:4077-4081 (1996); see also Example
9 above), and forms that manifest SDS-, urea- and formic
acid-resistant polymers on PAGE (Masters, C. L. et al., Proc. Natl.
4cad. Sci. USA 82:4245-4249 (1985); Dyrks, T., et al., J. Biol.
Chem. 267:18210-18217 (1992); Roher, A. E., et al., Journal of
Biological Chemistry 271:20631-20635 (1996). Thus, the extraction
of SDS-resistant A.beta. polymers from plaques implicates
polymerization as a pathogenic mechanism that promotes the
formation of AD amyloid.
[0421] The exact mechanism underlying the formation of
SDS-resistant polymeric A.beta. species remains unresolved.
Recently, we found that A.beta. reduces both Cu.sup.2+ and
Fe.sup.3+ (Huang, X., et al., J. Biol. Chem. 272:26464-26470
(1997)), providing a mechanism whereby a highly reactive species
could promote the modification of proteins via an oxidative
mechanism. In this study we test the ability of Cu.sup.2+ and
Fe.sup.3+ to promote SDS-resistant A.beta. polymerization.
[0422] Materials and Methods
[0423] Human A.beta..sub.1-40 peptide was synthesized, purified and
characterized as described above. Rat A.beta..sub.1-40 was obtained
from Quality Control Biochemicals, Inc. (Hopkinton, Mass.).
Peptides were analyzed and stock solutions prepared as described
above.
[0424] As above, electronic images captured using the Fluoro-S
Image Analysis System (Bio-Rad, Hercules, Calif.) were analyzed
using Multi-Analyst Software (Bio-Rad, Hercules, Calif.). This
chemiluminescent image analysis system is linear over 2 orders of
magnitude and has comparable sensitivity to film.
[0425] 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.
[0426] Results and Discussion
[0427] The generation of SDS-resistant A.beta. polymers by metal
ions was tested by incubating Cu.sup.2+ (30 .mu.M) or Zn.sup.2+ (30
.mu.M) at pH 6.6, 7.4 and 9.0 with A.beta..sub.1-40. As shown in
FIG. 9, Western blot analysis of samples incubated with Cu.sup.2+
and run under SDS denaturing and P-mercaptoethanol reducing
conditions revealed an increase in dimeric, trimeric and higher
oligomeric A.beta. species over time. The dimer and trimer had
molecular weights of approximately 8.5 kD and 13.0 kD,
respectively. Image analysis indicated 42% and 9% conversion of the
monomer to dimer and trimer, respectively, in samples incubated at
pH 7.4 after 5 d. The conversion of monomer to the dimer and trimer
was 29% and 2%, respectively, at pH 6.6 after 5 d.
[0428] In contrast, changes in [H+] alone did not induce
SDS-resistant A.beta..sub.1-40 polymerization. Less than 4% of the
peptide was converted to the SDS-resistant dimer after 5 d in
samples incubated at pH 6.6, 7.4 or 9.0, most likely as a result of
contaminating Cu.sup.2+ in the buffer and A.beta. solutions.
Cu.sup.2+ contamination of chelex-treated PBS was up to 0.5,.mu.M
as determined by ion coupled plasma-atomic emission spectroscopy
(ICP-AES). Although Zn.sup.2+ induces rapid aggregation of
A.beta..sub.1-40 (Bush, A. I., et al., J. Biol. Chem. 268:16109
(1993); Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994); Bush,
A. I., et al., Science 265:1464-1467 (1994); Bush, A. I., et al.,
Science 268:1921-1922 (1995); Atwood et al., submitted; Huang, X.
et al., J. Biol. Chem. 272:26464-26470 (1997)), it did not induce
SDS-resistant A.beta. polymerization (FIG. 9) as previously
reported (Bush, A. I., et al., Science 268:1921-1922 (1995)).
[0429] A.beta..sub.1-42 is the predominant species found in amyloid
plaques (Masters, C. L. et al., Proc. Natl. Acad. Sci. USA 82: 4245
(1985); Murphy, G. M., et al., Am. J. Pathol. 144:1082-1088 (1994);
Mak, K., et al., Brain Res. 667:138-142 (1994); Iwatsubo, T., et
al., Ann. Neurol. 37:294-299 (1995); Mann et al., Ann. Neurol.
40:149-156 (1996)). Therefore, the ability of A.beta..sub.1-40 and
A.beta..sub.1-42 to form SDS-resistant polymers was compared.
[0430] In contrast to Cu.sup.2+-induced SDS-resistant
A.beta..sub.1-40 polymerization over days, SDS-resisitant
A.beta..sub.1-42 polymerization occurred within minutes in the
presence of Cu.sup.2+ (FIG. 31A). Unlike A.beta..sub.1-40 where
Cu.sup.2+ induces the formation of a SDS-resistant dimeric species
first, A.beta..sub.1-42 initially forms an apparent trimer species
in the presence of Cu.sup.2+. Over time, dimeric and higher
polymeric species also appear in A.beta..sub.1-42 incubations with
Cu.sup.2+ at both pH 7.4 and 6.6. The greater Cu.sup.2+ induced Add
.sub.42 polymerization observed at pH 6.6 compared with pH 7.4 in
samples incubated for 30 min. was reversed after 5 d. At pH 6.6,
both A.beta..sub.1-40 and A.beta..sub.1-42 exist in an aggregated
form within minutes. Therefore, the formation of these polymeric
species occurs within A.beta. aggregates and the formation of
SDS-resistant A.beta. polymers is independent of aggregation state
(see below). Similar results were obtained using the monoclonal
antibody 4G8.
[0431] Since redox active Fe (Smith, M. A., et al., Proc. Natl.
Acad. Sci. USA 94:9866 (1997)) and ferritin (Grudke-Iqbal, I., et
al., Acta Neuropathol. 81:105 (1990)) are found in amyloid lesions,
experiments were performed to determine if Fe could induce
SDS-resistant polymerization of A.beta..sub.1-40 and
A.beta..sub.1-42 (FIG. 31A). Fe.sup.3+ did not induce
A.beta..sub.1-40 polymerization above background levels with either
peptide. The small increase in polymeric A.beta..sub.1-40 and
A.beta..sub.1-40 in samples with no metal ions reflects a small
contaminating concentration of Cu.sup.2+.
[0432] The formation of amyloid plaques is not a feature of aged
rats (Johnstone, E. M., et al., Mol. Brain Res. 10:229 (1991);
Shivers et al., EMBO J., 7:1365-1370 (1988)). To test whether rat
A.beta..sub.1-40 would form SDS-resistant A.beta. polymers, rat
A.beta..sub.1-40 was incubated with Cu.sup.2+ and Fe.sup.3+ at pH
7.4 and 6.6 (FIG. 31B). Neither metal ion induced SDS-resistant
A.beta. polymers (Huang, X. et al., J. Biol. Chem. 272:26464-26470
(1997)). The binding and reduction of Cu.sup.2+ by rat
A.beta..sub.1-40 is markedly decreased compared to that of human
A.beta..sub.1-40 (Huang, X. et al., J. Biol. Chem. 272:26464-26470
(1997)). This result suggests that the generation of SDS-resistant
A.beta. polymers is dependent upon the binding and reduction of
Cu.sup.2+ by A.beta..
[0433] Tests were performed to determine the concentration of
Cu.sup.2+ required to induce the formation of SDS-resistant
A.beta..sub.1-40 and A.beta..sub.1-42 polymers. A.beta..sub.1-40
and A.beta..sub.1-42 were incubated with different [Cu.sup.2+]
(0-30 .mu.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, Calif.) as previously described.
[0434] At pH 7.4, the increase in polymerization was barely
detectable as [Cu.sup.2+] was increased from 0.5 to 1 .mu.M, but
under mildly acidic conditions (pH 6.6), SOS-resistant
polymerization could be detected (over 3-fold increase in
dimerization (Table 10A).
12TABLE 10A Cu.sup.2+ - Induced SDS-Resistant Polymers of
A.beta..sub.1-40 [Cu.sup.2+] Monomer Dimer Trimer Tetramer Pentamer
pH 7.4 0 96.8 3.2 <0.1 0 0 0.5 94.8 4.9 0.3 0 0 1 93.6 5.9 0.6 0
0 5 84.3 14.2 1.5 0 0 10 85.2 13.2 1.6 0 0 30 76.2 19.1 4.7 0 0 pH
6.6 0 97.9 2.1 <0.1 0 0 0.5 97.6 2.2 0.2 0 0 1 92.6 7.3 0.1 0 0
5 90.1 9.8 0.1 0 0 10 79.4 16.1 4.5 0 0 30 74.5 13.2 12.2 0 0
[0435] A similar Cu.sup.2+ concentration and pH dependent increase
in SDS-resistant A.beta..sub.1-42 polymers also was observed (Table
10B), but SDS-resistant polymerization occurred at much lower
[Cu.sup.+].
13TABLE 10B Cu.sup.2+--Induced SDS-Resistant Polymers of
A.beta..sub.1-42 [Cu.sup.2+] Monomer Dimer Trimer Tetramer Pentamer
pH 7.4 0 76.61 0 16.0 5.5 1.9 0.5 70.7 0 20.5 6.2 2.5 1 64.9 0 23.6
7.4 4.0 5 56.1 0 31.8 8.7 4.1 10 55.1 0 30.3 10.3 4.3 30 57.1 0
31.1 8.3 4.2 pH 6.6 0 61.0 0 27.3 8.6 3.8 0.5 52.1 0 33.8 12.0 3.0
5 59.6 0 30.0 7.1 3.2 10 52.3 0 31.7 13.6 2.2
[0436] A.beta..sub.1-40 polymerization was not detected with
increasing Fe.sup.3+ concentrations at any pH. Therefore, of the
metal ions known to interact with A.beta., only Cu.sup.2+, whose
ability to aggregate and bind Cu.sup.2+ under mildly acidic
conditions is enhanced, is capable of inducing SDS-resistant
A.beta. polymerization.
[0437] Oxygen radical mediated chemical attack has been correlated
with an increase in protein and free carbonyls (Smith, C. D., et
a., Proc. Natl. Acad. Sci. USA 88:10540 (1991); Hensley, K., et
al., J. Neurochem. 65:2146 (1995); Smith, M. A., et al., Nature
382:120 (1996)) and peroxynitrite-mediated protein nitration (Good,
P. F., et al., Am. J. Pathol. 149:21 (1996); Smith, M. A., et al.,
Proc. Natl. Acad. Sci. USA 94:9866 (1997)).
[0438] A.beta. is capable of reducing Cu.sup.2+ and H.sub.2O.sub.2
is produced in solutions containing A.beta. and Cu.sup.2+ or
Fe.sup.3+ (Huang, X. et al., i J. Biol. Chem. 272:26464-26470
(1997)). As shown above, the generation of SDS-resistant A.beta.
polymers in the order
A.beta..sub.1-42>>A.beta..sub.1-40>>rat
A.beta..sub.1-40 in the presence of Cu.sup.2+ correlates well with
the generation of Cu.sup.+ and reactive oxygen species (ROS;
OH.sup.-, H.sub.2O.sub.2 and O.sub.2.sup.-: Huang, X. et al., J.
Biol. Chem. 272:26464-26470 (1997)) by each peptide.
[0439] The increased generation of SDS-resistant A.beta. polymers
in the presence of Cu.sup.2+ compared to Fe.sup.3+ also was
correlated with the generation of the reduced metal ions,
respectively (Huang, X. et al., J. Biol. Chem. 272:26464-26470
(1997)). The increase in SDS-resistant A.beta. polymerization seen
under mildly acidic conditions may be a result of the higher
[H.sup.+] driving the production of H.sub.2O.sub.2 dismutated from
O.sub.2.sup.- with the subsequent generation of OH. via Fenton-like
chemistry inducing a modification of A.beta. that results in
SDS-resistant A.beta. polymers (see FIG. 12, showing a schematic of
the proposed mechanism of A.beta.-mediated reduced metal/ROS
production).
[0440] To confirm whether ROS were involved in the generation of
SDS-resistant polymers, experiments were performed to determine
whether Cu in the presence or absence of H.sub.2O.sub.2 could
promote A.beta. polymerization (FIG. 32A). A similar level of
A.beta..sub.1-42 polymerization was observed in the presence of
Cu.sup.2+ or Cu.sup.+, indicating that the reduced metal ion alone
was not capable of increasing A.beta. polymerization. Likewise,
polymerization of A.beta..sub.1-42 in the presence of
H.sub.2O.sub.2 was low and equivalent to control levels. However,
the addition of Cu.sup.2+ or Cu.sup.+ to A.beta. in the presence of
H.sub.2O.sub.2 induced a similar, marked increase in dimers,
trimers and tetramers within 1 hour. After 1 day, higher molecular
weight polymers (>18 kD) were generated (from the oligomers),
with a subsequent reduction in the levels of monomer, dimer, trimer
and tetramer only with the coincubation of H.sub.2O.sub.2 and
Cu.sup.2+.
[0441] Both the reduced and oxidized forms of Cu produced similar
levels of polymerization in the presence of H.sub.2O.sub.2. In
contrast, Fe.sup.3+ of Fe.sup.2+ did not induce as much
polymerization as Cu.sup.2+ in the presence of H.sub.2O.sub.2 after
1 day incubation (FIGS. 32A and 32B). Since Fe.sup.3+ is not
reduced as efficiently as Cu.sup.2+ by A.beta. (Huang, X., et al.,
J. Biol. Chem., 272:26464-26470 (1997)), and Cu.sup.+ is rapidly
converted to Cu.sup.2+ in solution, these results suggest that the
reduction reaction is required for the polymerization reaction to
proceed.
[0442] It was confirmed that the reduction of Cu.sup.2+ was
required for generating SDS-resistant A.beta. polymerization by
incubating A.beta..sub.1-42 and Cu.sup.2+ with and without
bathocupoinedisulfonic acid (BC), a Cu.sup.+ specific chelator
(FIG. 32C). There was a marked decrease in polymerization,
indicating that Cu.sup.+ generation was crucial for the
polymerization of A.beta.. It is possible that the decreased
polymerization may be due to chelation of Cu.sup.2+ by BC, however
given the low binding affinity of BC for Cu.sup.2+ compared with
A.beta., it seems likely that the chelation of Cu.sup.+ by BC
prevents it from inducing SDS-resistant Ad polymerization.
Therefore, A.beta. may undergo a hydroxyl radical modification that
promotes its assembly into SDS-resistant polymers.
[0443] If H.sub.2O.sub.2 is required for the polymerization
reaction under physiological conditions, the removal of
H.sub.2O.sub.2 and it's precursors O.sub.2 and O.sub.2.sup.-
(Huang, X., et al., J. Biol. Chem., 272:26464-26470 (1997)) should
decrease SDS-resistant polymerization. To confirm that
H.sub.2O.sub.2 generated in the presence of A.beta. and Cu.sup.2+
was required for the polymerization reaction, A.beta..sub.1-42 was
incubated with or without Cu.sup.2+ in the presence of TCEP (FIG.
33A). TCEP significantly reduced the level of polymerization in
samples with and without Cu.sup.2+ over 3 days. This indicates that
the generation of H.sub.2O.sub.2 is required for the polymerization
of A.beta..
[0444] To confirm that the generation of O.sub.2.sup.- was required
for SDS-resistant A.beta. polymerization, A.beta..sub.1-42 was
incubated with and without Cu.sup.2+ at pH 7.4 and 6.6 under argon
in order to decrease the reduction of molecular O.sub.2 (FIG. 33B).
Argon-purging of the solution markedly decreased A.beta..sub.1-42
polymerization under each condition, indicating that the generation
of ROS is required for the polymerization of A.beta..
[0445] Taken together, these results indicate that polymerization
occurs as a result of Haber-Weiss chemistry where the continual
reduction of Cu.sup.2+ by A.beta. provides a species for the
reduction of molecular O.sub.2 and the subsequent generation of
O.sub.2.sup.-, H.sub.2O.sub.2 and OH. The binding and reduction of
Cu.sup.2+ by A.beta. is supported by the finding that the
incubation of Fe.sup.3+, H.sub.2O.sub.2 and ascorbic acid with
A.beta..sub.1-40 (FIG. 33A) and A.beta..sub.1-42 does not induce
SDS-resistant polymerization equivalent to Cu.sup.2+ with
H.sub.2O.sub.2 alone. Since ascorbic acid effectively reduces
Fe.sup.3+, the reduction of a metal ion that is not bound to
A.beta. is insufficient to induce significant SDS-resistant
polymerization.
[0446] The formation of SDS-resistant polymers of A.beta. by this
metal-catalyzed oxidative mechanism strongly suggested that a
chemical modification to the peptide backbone allows the formation
of the polymer species. To test if the SDS-resistant polymers were
covalently linked, SDS-resistant polymers generated by incubating
A.beta..sub.1-42 with Cu.sup.2+ at pH 7 4 and 6.6, or
A.beta..sub.1-42 with Cu.sup.2+ plus H.sub.2O.sub.2 were subjected
to treatment with urea (FIG. 34A) and guanidine HCl, chaotrophic
agents known to disrupt H-bonding. Urea and guanidine HCl did not
disrupt the SDS-resistant polymers at 4.5 M, and only slightly at
9M, suggesting that the SDS-resistant polymers were held together
by high-affinity bonds, but not hydrogen bonding alone. HPLC-MS
analyses confirmed no covalent modification of the peptide and no
evidence of intermolecular covalent crosslinking.
[0447] Since covalent and/or hydrogen bonding were not involved in
polymer formation, experiments were performed to detemine whether
Cu.sup.2+ coordination of the complex by ionic interactions was
allowing for the formation of the SDS-resistant polymer species. To
disrupt these ionic interactions, different chelating agents were
added to a solution containing Cu.sup.2+-induced A.beta..sub.1-40
or A.beta..sub.1-42 SDS-resistant polymers generated at pH 7.4
(FIGS. 34B and 34C).
[0448] All chelators significantly reduced the amount of
A.beta..sub.1-40 or A.beta..sub.1-42 SDS-resistant polymers. EDTA
was less effective in destabilizing the polymers, possibly due to
its larger molecular mass, and lower affinity for Cu.sup.2+. EDTA
reduced the amount of A.beta..sub.1-40 polymers, but increased the
amount of A.beta..sub.1-40 polymers at pH 7.4. This may be due to
the fact that EDTA can enhance the redox potential of Cu under
certain conditions.
[0449] Cu.sup.2+-induced SDS-resistant polymers generated at pH 6.6
were also disrupted with chelation treatment to a similar extent.
These results suggest that the chelation of Cu.sup.2+ away from
A.beta. results in the disruption of the polymer complex and the
release of monomer species. Thus, there is an absolute requirement
for metal ions in the stabilization of the SDS-resistant polymer
complex.
[0450] The SDS-resistant polymers generated with Cu.sup.2+ are
similar to those extracted from post-mortem AD brains (Roher, A.
E., et al., Journal of Biological Chemistry 271:20631-20635
(1996)). To determine if these human oligomeric A.beta. species
could be disrupted by metal chelators, TETA and BC were incubated
with A.beta. oligomers extracted from human brain. FIG. 30E shows
that both TETA and BC significantly increased the amount of monomer
A.beta. in samples treated with these chelators. Although the
increase in the amount of monomer was small, these results suggest
that human oligomeric A.beta. species are partially held together
with metal ions. Importantly, this result indicates the potential
of chelation therapy as a means of reducing amyloidosis.
[0451] To examine whether conformational changes could disrupt the
SDS-resistant polymers, solutions of SDS-resistant A.beta..sub.1-42
polymers in the presence or absence of Cu.sup.2+ were incubated
with the .alpha.-helical promoting solvent system DMSO/HFIP, or
under acidic conditions (pH 1) (FIG. 34D). These conditions reduced
the amount of polymer compared to untreated controls at both pH 7.4
and 6.6, indicating that an alteration in the conformation of
A.beta..sub.1-42 to the .alpha.-helical conformation could disrupt
the strong A.beta.-Cu.sup.2+ ionic interactions. This provides
indirect evidence that the polymer structures are likely to be in
the more thermodynamically favorable .beta.-sheet conformation,
such as those found in neuritic plaques.
[0452] SDS-resistant A.beta. 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.beta. in
amyloid plaques. Incubation of SDS-resistant A.beta. polymers with
proteinase K resulted in complete degradation of both monomer and
oligomeric A.beta. species. Since protease treatment is incapable
of digesting hard core amyloid, some form of 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.beta. oligomers compared to the
Cu-mediated SDS-resistant polymers generated in vitro.
[0453] Soluble A.beta..sub.1-40 and A.beta..sub.1-42 both exist in
phosphate buffered saline as non-covalent dimers (Huang, X., et
al., J. Biol. Chem. 272:26464-26470 (1997); and unpublished
observations). Disruption of ionic and hydrogen bonding of A.beta.
in the soluble and aggregated forms (pH or Zn.sup.2+) by the ionic
detergent SDS results in the complete dissociation of A.beta. into
the monomer species as detected on SDS-PAGE (FIGS. 9, 28-30). The
formation of SDS-resistant polymers of A.beta. over time in the
presence of Cu.sup.2+ (FIGS. 9, 31A-31B, 32C) suggests that
conformational or structural alterations allow for the formation of
a thermodynamically more stable complex.
[0454] Although no covalent crosslinking between peptides was
detected, it is possible that a covalent modification(s) takes
place within the peptide backbone that allows for a high affinity
association to form between the peptide and Cu.sup.2+. Thus, a
chemical modification to the peptide may increase the affinity of
the polymer for Cu.sup.2+ and the formation of a stable complex.
Alternatively, the requirement for molecular oxygen suggests that
Cu may be coordinated by oxygen or ROS in the formation of
SDS-resistant polymers.
[0455] The formation of SDS-resistant polymers was dependent upon
the binding and reduction of Cu.sup.2+. The binding of Cu.sup.2+ to
A.beta. was confirmed by the detection of Cu.sup.2+ in both the
monomer and dimer following SDS-PAGE. The [Cu.sup.2+] of PVDF
membrane containing the immobilized peptide species was measured by
ICP-AES (unpublished observations; Huang, X., et al., J. Biol.
Chem. 272:26464-26470 (1997)) and correlated with the generation of
SDS-resistant polymers for each species.
[0456] Cu.sup.2+ coordination between A.beta. molecules was
required in order to maintain the structure since chelation
treatment disrupted the in vitro generated SDS-resistant polymer
(FIGS. 34B-34E). Human SDS-resistant A.beta. polymers also were
disrupted with the Cu.sup.+ specific chelator BC indicating Cu
coordination in the stabilization of these structures (FIG. 34E).
Together with the fact that Cu specific chelators can extract more
SDS-resistant A.beta. polymers from AD brains in aqueous buffer
(see Example 9), these results implicate Cu.sup.2+ in the
generation of SDS-resistant polymers in vivo.
[0457] Fe.sup.3+ did not induce the formation of SDS-resistant
polymers in vitro (FIGS. 31A) as previously reported except in the
presence of excess H.sub.2O.sub.2 or ascorbic acid as previously
reported (Dyrks, T., et al., J. Biol. Chem. 267:18210-18217 (1992);
data not shown). Dyrks, T., et al. did, however observe significant
increases in SDS-resistant polymerization with metal-catalyzed
oxidation systems (Fe-hemin, Fe-hemoglobin or Fe-EDTA) in the
presence of H.sub.2O.sub.2. The A.beta..sub.1-42 used in their
experiments was likely to be Cu-bound as it was extracted from a
wheat germ expression system and already was present as
SDS-resistant oligomers. Thus, it is possible that Cu-bound A.beta.
used in these experiments contributed to the increased
SDS-resistant polymerization observed in the Fe-catalyzed oxidation
systems. Although Fe.sup.3+ is reduced by A.beta. (Huang, X., et
al., J. Biol. Chem. 272:26464-26470 (1997)), it is unable to
effectively coordinate the complex like Cu (FIG. 32B).
[0458] Fe.sup.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
al., Biochem. J. 287:509-514 (1992); Joshi, J. G., et al., Environ.
Health Perspect. 102:207-213 (1994)). This is partly attributable
to the increased ferritin rich microglia and oligodendrocytes that
localize to amyloid plaques (Grudke-Iqbal, I., et al., Acta
Neuropathol. 81:105 (1990); Conner, J. R., et al., J. Neurosci.
Res. 31:75-83 (1992); Sadowki, M., et al., Alzheimer's Res. 1:71-76
(1995)).
[0459] Recently, redox active Fe was localized to amyloid lesions
(Smith, M. A., et al., Proc. Natl. Acad. Sc. USA 94:9866 (1997).
While Fe is normally sequestered by metalloproteins, this
localization of ferritin-rich cells around amyloid deposits, and
the very high concentrations of iron in amyloid plaques (Conner, J.
R., et al., J. Neurosci Res. 31:75-83 (1992); Markesbery, W. R. and
EBhmann, W. D., "Brain trace elements in Alzheimer's disease," in
Terry, R. D., et al., eds., Alzheimer Disease, Raven Press, New
York (1994), pp. 353-368) suggests that reduced Fe released from
ferritin and transferrin under mildly acidic conditions could be
available for Fenton chemistry and the formation of SDS-resistant
polymers. However, even in the presence of a Fe-ROS generating
system (ascorbic acid, H.sub.2O.sub.2 and Fe) the generation of
SDS-resistant Ad polymers in vitro was low (FIG. 33A) and may be
explained by Cu.sup.2+ contamination of the buffers.
[0460] Interestingly, diffuse plaques, which may represent the
earliest stages of plaque formation, are relatively free of
ferritin-rich cells (Ohgami, T., et al., Acta Neuropathol
81:242-247 (1991)). Therefore, the accretion of iron in amyloid
plaques may be a secondary response to the neurodegeneration caused
by the reduction of Cu.sup.2+ and the generation of ROS by A.beta.
and the formation of neurotoxic SDS-resistant A.beta. polymers.
[0461] Structural differences between A.beta..sub.1-40 and
A.beta..sub.1-42 may allow for the formation of a thermodynamically
stable dimer in the case of A.beta..sub.1-40 and trimer in the case
of A.beta..sub.1-42 (FIGS. 31A, 34B and 34C). Irrespective of this,
the increased generation of SDS-resistant polymers by
A.beta..sub.1-42 compared to A.beta..sub.1-40 is most likely
explained by the increased ability of A.beta..sub.1-42 to reduce Cu
and generate ROS. Since the addition of exogenous H.sub.2O.sub.2 to
A.beta..sub.1-42 increases the generation of dimeric SDS-resistant
polymers of A.beta..sub.1-42 (FIGS. 32A and 32B), this dimeric
species may be an integral intermediate in the formation of the
SDS-resistant A.beta. trimers, and may explain why A.beta..sub.1-40
polymerization occurs more slowly.
[0462] AD Pathology
[0463] 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.
[0464] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
[0465] All patents and publications cited in the present
specification are incorporated by reference herein in their
entirety.
* * * * *