Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-12-21
2003-07-08
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S375000, C435S368000, C435S354000
Reexamination Certificate
active
06589747
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention provides, among other things, assays and methods of diagnosis and treatment that are based on the surprising observation of an interaction between amyloid &bgr; or its aggregates with sodium channels. In particular, the present invention provides methods to identify compounds that modulate this interaction, and methods of diagnosis and treatment that are based on this interaction.
BACKGROUND OF THE INVENTION
Certain neurodegenerative diseases such as Alzheimer's disease and Down's syndrome are characterized by the presence of insoluble aggregates of amyloid &bgr; (A&bgr;). This 39-43 amino acid peptide is derived by abnormal proteolysis from the Amyloid Precursor Protein (APP). It is well known that the amyloid &bgr; aggregates/plaques widely found in the brain and intraneuronal neurofibrillary tangles of Alzheimer's disease patients can modulate neurite outgrowth, synaptogenesis, synaptic plasticity or cause neuronal death (Mattson et al,
Trends Neurosci.,
16, 406-415 (1993)). The mechanism(s) by which these effects are accomplished remain elusive, however (Frazer et al,
Trends Neurosci.,
20, 67-72 (1997)).
Presently, one theory maintains that these deleterious effects may originate from amyloid &bgr;'s ability to promote chronic calcium influx into neurons through modulation of neuronal calcium ion channels (Daidson et al.,
Brain Res.,
643, 324-327 (1994)), or to form transmembrane cation-permeable channels (Kawahara et al.,
Biophysiol. J.,
73, 67-75 (1997)) that indiscriminately allow calcium, sodium, and other cations to flood a cell's interior and destroy its calcium homeostasis. Calcium channel blockers such as nimodipine that can cross the blood-brain barrier have been shown to slow the progression of Alzheimer's disease in some patients (Tollefson,
Biol. Psychiatry,
27, 1133-1142 (1990)). Similarly, zinc cation, which is known to bind at specific amyloid &bgr; 1-40 sites (Bush et al,
Science,
265, 1464-1467 (1994)), can block the calcium influx through amyloid &bgr; 1-40 channels (Arispe et al.,
Proc. Natl. Acad. Sci.,
93, 1710-1715 (1996)).
In addition, amyloid &bgr; aggregates have been shown to induce abnormal potassium ion channel activity. In cultured hippocampal neurons, amyloid &bgr; opens a calcium-sensitive, large conductance potassium channel (i.e., iberitoxin-sensitive BK) (Furukawa et al.,
Nature,
379, 74-78 (1996)), which could lead to chronic loss of cytoplasmic potassium and destroy the ability of the neurons to generate and propagate action potentials critical for brain signaling functions. Such aberrant potassium channel activity has in fact been noted in Alzheimer's disease patients (Etchebemgaray et al.,
Proc. Natl. Acad. Sci.,
90, 8209-8213 (1993)). Likewise, in the cultured cholinergic septal cell line SN56, amyloid &bgr; (e.g., amyloid &bgr; 1-40) causes cell death by impacting a TEA-sensitive potassium channel (Colom et al.,
J. Neurochemistry,
70, 1925-1934 (1998)).
These studies suggest that amyloid &bgr; aggregates exert their effects through multiple targets. None of the targets that have been described, however, would result in the rapid and complete inhibition of neuronal electrical impulses critical for brain function that is observed with advanced stage Alzheimer's disease. Furthermore, the ion channel studies described above were performed on cultured neurons, whose properties may have been changed significantly through dedifferentiation in culture conditions. Therefore, the data currently available likely does not represent the true response of native neurons.
Recent genetic studies have shown that mutations in the amyloid protein precursor and presenilin genes affect the processing and production of amyloid &bgr; and thus are related to age of onset and susceptibility to Alzheimer's disease. Additional studies have shown that variants in ApoE genes also affect susceptibility and age of onset for Alzheimer's disease. Spontaneous Alzheimer's disease is hypothesized to be under the control of other genes and environmental factors that have yet to be identified.
Additionally, inhibition of amyloid &bgr; aggregation, and hence toxicity, is believed to be beneficial therapeutically in the treatment of Alzheimer's disease. Several reports have appeared providing evidence that small, diffusable aggregates (Lambert et al.,
Proc. Natl. Acad. Sci.,
95, 6448-6453 (1998); PCT International Application WO 98/33815), and protofibrils (Harper et al.,
Chem. Biol.,
4, 119-125 (1997); Harper et al.,
Chem. Biol.,
4, 951-959 (1997); Walsh et al.,
J. Biol. Chem.,
272, 22364-22374 (1997)), and not the completely fibrillar peptide, might be the A&bgr; species toxic to living cells. The physical differences in the alleged toxic species identified by these different research groups “supports the concept that different A&bgr; assemblies have distinct neurobiological activities, which may be manifested differently using an electrophysiological readout” (Hartley et al.,
J. Neuroscience,
19, 8876-8884 (1999)). Thus, the etiology of Alzheimer's disease may be quite complex, and may warrant a variety of different avenues of diagnosis and treatment.
Along these lines, U.S. Pat. No. 5,876,948 describes screening methods to identify inhibitors of the neurotoxic effect of amyloid &bgr;. In particular, the '948 patent purportedly provides a method of screening candidate compounds, wherein the method comprises obtaining a cell such as a primary neuron, a neuronal cell, or a cell developmentally derived from neuronal tissue. The cell is contacted with a candidate compound in the presence of a neurotoxin selected from the group consisting of amyloid &bgr;1-38, &bgr;1-40, &bgr;1-43, and &bgr;29-35, and it is determined whether the compound reduces the effect of the neurotoxin (e.g., reduces cell death), or reduces accumulation of amyloid &bgr; on the cell surface. Accordingly, this patent provides no information on how to screen for compounds that target cell events that precede cell death, apart from accumulation of the peptide on the cell surface. A need thus remains for useful assays to screen for compounds that exert protective effects at a stage prior to cell death mediated by amyloid &bgr;, and for compounds that exert their effect apart from interference with amyloid &bgr; deposition on the cell surface. Furthermore, the '948 patent does not appear to consider any effect of the aggregation state of amyloid &bgr; on neurotoxicity. A related patent, U.S. Pat. No. 5,137,873, claims a method for treating a disease that is characterized by accumulation of amyloid &bgr;, which comprises administration of a therapeutically effective amount of a tachykinin agonist such as substance P.
U.S. Pat. No. 5,892,018 pertains to DNA sequences encoding a novel subfamily of amiloride-sensitive sodium channel proteins from the human central nervous system. PCT International Application WO 98/54316 describes a new class of sodium channel protein that may function as a receptor for endogenous transmitters. Both the '018 patent and the '316 application suggest that ion channels may play some role in the pathogenesis or treatment of Alzheimer's disease (see, '316 application, pages 1 and 15; see, '018 patent, column 1, lines 50-54). However, neither document presents any assay for diagnosis of Alzheimer's disease, much less any methods for its treatment.
Thus, there remains a need for further understanding of the functioning of amyloid &bgr; or its aggregates, and means to diagnose, predict, prevent and treat diseases, disorders, and conditions that result from amyloid &bgr; or its aggregates. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to a surprising observation of an interaction between amyloid &bgr; or its agg
Buhl Allen E.
Epps Dennis E.
Lee Kai S.
Sun Xiao-Dong
Taylor Bruce M.
Darnley , Jr. James D.
Kunz Gary
Pharmacia & Upjohn
Pharmacia & Upjohn Company
Turner Sharon L
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