Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-04-28
2003-10-28
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C436S080000, C436S084000, C436S127000, C530S350000
Reexamination Certificate
active
06638711
ABSTRACT:
BACKGROUND OF THE INVENTION
The neuropathology of Alzheimer's disease (AD) is characterized by marked neocortical Abeta deposition and signs of oxidative stress. Metabolic signs of oxidative stress in the neocortex of AD patients, widespread oxygen radical-mediated brain damage, systemic signs of oxidative stress and the response of antioxidant systems have all been observed in AD (Martins, R. N., et al.,
J. Neurochem
. 46:1042-1045 (1986); Smith, M. A., et al.,
Nature
382:120-121 (1996); Ceballos-Picot, I., et al.,
Free Radic. Biol. Med
. 20(4):579-87 (1996); Nunomura, A., et al.,
J. Neurosci
. 19(6):1959-64 (1999)). In fact, amelioration of oxidation injury maybe the basis for the clinical benefit of vitamin E treatment in AD subjects (Sano, M., et al.,
N. Engl. J. Med
. 336:1216-1222 (1997)).
A&bgr; is a dimer that simultaneously binds Cu and Zn. (Huang, X., et al.,
J. Biol. Chem
. 272:26464-26470 (1997); Atwood, C. S., et al.,
Journal of Biological Chemistry
273:12817-12826 (1998); Lovell, M. A., et al.,
J. Neurol. Sci
. 158(1):47-52 (1998); Huang, X., et al.,
Biochemistry
38:7609-7616 (1999); Garzon-Rodriguez, W., et al.,
J. Biol. Chem
. 272:21037-21044 (1997)). It is released from cells by oxidative stress, but its normal function and role in AD are unclear. Polymers of Abeta (A&bgr;), 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 deposits found in the brains of those afflicted with AD. A&bgr; deposits are usually most concentrated in regions of high neuronal cell death, and may be present in various morphologies, including amorphous deposits, 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)). A&bgr; deposits are decorated with inflammatory response proteins. In addition, biochemical markers of severe oxidative stress, such as peroxidation adducts, advanced glycation end-products, and protein crosslinking, are located in close proximity to the deposits.
To date, the cause of A&bgr; deposits is unknown. However, it is believed that preventing the deposit formation may be a means of treating AD since growing evidence suggests that A&bgr; deposits are intimately associated with the neuronal demise that leads to dementia in AD. More specifically, genetic studies have strongly implicated the 42 residue form of A&bgr; (A&bgr;
1-42
) in the pathogenesis of AD (Maury, C. P. J.,
Lab. Investig
. 72:4-16 (1995); Multhaup, G., et al.,
Nature
325:733-736 (1987)). A&bgr;
1-42
, while a minor component of biological fluids, is highly concentrated in A&bgr; deposits. This suggests that A&bgr;
1-42
is more pathogenic than other neurotoxic A&bgr; species. See, e.g., Kuo, Y-M., et al.,
J. Biol. Chem
. 271:4077-81 (1996); Roher, A. E., et al.,
J. Biol. Chem
. 271:20631-20635 (1996).
The systemic deposition of amyloid is usually associated with an inflammatory response (Pepys, M. B. and 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 (1989), pp. 1273-1293; 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 I -antichymotrypsin and c-reactive protein, elements of the complement system and activated microglial and astroglial cells are consistently found in AD brains.
The mechanism underlying the formation of neurotoxic A&bgr; amyloid remains unresolved. The overexpression of A&bgr; alone cannot sufficiently explain amyloid formation, since the concentration of A&bgr; required for aggregation is not physiologically plausible. Moreover, alterations in the neurochemical environment are required for amyloid formation since the presence of A&bgr;
1-42
is normal in biological fluids such as cerebrospinal fluid (CSF) (Shoji, M.,
Science
258: 126 (1992); Golde et al.,
Science
255(5045): 728-730 (1992); Seubert, P. et al.,
Nature
359: 325 (1992); Haass et al.,
Nature
359: 322 (1992)).
Studies into the neurochemical vulnerability of A&bgr; to form amyloid suggest altered zinc and [H
+
] homeostasis as the most likely explanations for amyloid formation since A&bgr; 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. 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)), and since the presence of redox inactive Zn(II) and, to a lesser extent, redox active Cu(II) and Fe(III), markedly increases the precipitation of soluble A&bgr; (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)). Zinc has an abnormal metabolism in AD and is highly concentrated in brain regions where A&bgr; aggregates.
However, the complete reversibility of Zn(II)-induced A&bgr;
1-40
aggregation in the presence of divalent metal ion chelating agents suggests that zinc binding is a reversible, normal function of A&bgr; and implicates other neurochemical mechanisms in the formation of A&bgr; deposits. A process involving irreversible A&bgr; aggregation, such as the crosslinking of A&bgr; monomers in the formation of A&bgr; polymeric species present in amyloid plaques, is thus a more plausible explanation for the formation of neurotoxic A&bgr; deposits.
The reduction by APP of copper (II) to copper (I) may lead to irreversible A&bgr; aggregation and crosslinking. More specifically, this reaction may promote an environment that enhances the production of hydroxyl radicals, which may contribute to oxidative stress in AD (Multhaup, G., et al.,
Science
271:1406-1409 (1996)). A precedent for abnormal Cu metabolism already exists in the neurodegenerative disorders of Wilson's disease and Menkes' syndrome and possibly in familial amyotrophic lateral sclerosis (Tanzi, R. E. et al.,
Nature Genetics
5:344 (1993)).
Although the fundamental pathology, genetic susceptibility and biology associated with AD are becoming clearer, a rational chemical and structural basis for developing effective drugs to prevent or cure the disease remains elusive. While the genetics of AD indicate that the metabolism of A&bgr; is intimately associated with the pathogenesis of the disease as indicated above, 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
The present invention is directed to the identification of agents that can be used to decrease the neurotoxicity of A&bgr; and the formation of A&bgr; polymers, and to the use of such agents to develop methods of preventing, treating or alleviating AD and/or the symptoms of AD. More specifically, the present invention is directed to the identification of agents that could be used to treat AD.
Because the ability of A&bgr; to function as an antioxidant, i.e., to generate H
2
O
2
from O
2
−
may, in many instances, be beneficial, the invention also relates to a method for identifying an agent to be used in the treatment and/or prevention of AD and symptoms th
Atwood Craig S.
Bush Ashley I.
Huang Xudong
Tanzi Rudolph E.
Bunner Bridget E.
Kemmerer Elizabeth
Sterne Kessler Goldstein & Fox P.L.L.C.
The General Hospital Corporation
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