Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
Title of the Invention
to
Method for Diagnosis of Alzheimer's Disease
Cross-Reference to Related Application
This application claims priority from United States
provisional applications Serial Nos. 60/133,161 filed May 7,
1999 and 60/179,975 filed February 3, 2000, the contents
both of which are hereby incorporated by reference.
Field of the Invention
The present invention provides a method for diagnosing
Alzheimer's disease (AD) in a subject. More particularly, a
2o method for diagnosing AD by completely or nearly-completely
releasing (3-amyloidl-92 or A~i3pE from plasma or serum
proteins and then analyzing the quantity of elevated levels
of (3-amyloidl-92 or A~3pE found in AD patients, or those who
are in the early stages of AD, or who will develop AD in the
future.
Background of the Invention
Neurodegenerative disorders such as Alzheimer's
3o disease (AD) and Parkinson' s disease (PD) afflict
humanity with great suffering and financial loss. AD is
characterized by neurofibrillary tangles, neuritic
plaques, and neuronal cell death. AD appears as either
the familial, early onset (<60 yrs) or late-onset (>60
yrs) forms, with the latter being more prevalent. AD is
the major cause of age-related dementia and cognitive
impairment (Wisniewski, T.; Ghiso, J.; Frangione, B.
Neurobiol. of Disease 1997, 4, 313-328). The amyloid
1
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s various recently identified mutations accounting for
certain types of inherited AD. Such mutations in the
presenilin (PS1 and PS2) genes are probably the cause of
the most frequent form of familial, early-onset AD
(Rogaev, E. I. Molecular Biology 1998, 32, 58). In
1o these cases, as with APP mutations, more A~31_~2 is
observed relative to A(3140. Extensive studies have
shown that A(31_~~ has a greater ability than A(31_qo to
aggregate into the amyloid fibrils that constitute the
plaques characteristic of AD (Lansbury, P. T., Jr.
15 Accts. Ch em. Res. 1996, 29, 317) . Even though A(31_qo is
generally present to a much larger degree in the
cerebrospinal fluid than A(31_42, it is A(31_Yz that is
present to a greater degree in AD plaques. Another
major amyloid related peptide found in plaques is an N-
2o terminally truncated variant in which the two N-terminal
amino acids are removed, and glutamic acid at position
three has formed a pyroglutamyl residue. (Saido, T. C.;
Iwatsubo, T.; Mann, D. M. A.; Shimada, H.; Ihara, Y.;
Kawashima, S. Neuron 1995, 14, 457. Iwatsubo, T.;
25 Saido, T. C.; Mann, D. M. A.; Lee, V. M.-Y.;
Trojanowski, J. Q. Am. J. Path. 1996, 149, 1823. Russo,
C.; Saido, T. C.; DeBusk, L. M.; Tabaton, M.; Gambetti,
P.; Teller, J. K. FEBS Lett. 1997, 409, 411.) This
peptide is termed A(33pE - pyroglutamyl at what was
3o previously residue 3 of either A(31_qo or A~31_4z. In fact,
some plaques may contain this particular pyroglutamyl
derivative to the extent of >500 (Kuo, Y.-M.; Emmerling,
M. R.; Woods, A. S.; Cotter, R. J.; Roher, A. E.
Biochem. Biophys. Res. Commun. 1997, 237, 188). The
35 A~33pE has an increased tendency to form ~3-pleated sheets
and aggregate than the parent amyloid peptides (He, W.;
z
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s Barrow, C. J. Biochemistry 1999, 38, 10871). Another
minor, related amyloid peptide is that in which the
aspartic acid at residue one is present as the
isoaspartic acid.
io The A(3 peptides can inhibit cholinergic
neurotransmitter function independent of neurotoxicity
(Auld, D. S.; Kar, S.; Quirion, R. Trends Neurosci.
1998, 21, 43). A(3 peptides bind to a number of natural
substances such as apoE3, apoE4, apoJ, transthyretin,
15 and albumin. In addition, A~3 has been reported to
interact with a membrane-bound receptor for advanced
glycation end products and to the class A scavenger
receptor (SR) associated with the production of reactive
oxygen species.
Recently, it has been described that circulating
levels of (3-amyloidl_42 in plasma are elevated ca. 6X in
patients with AD compared to age-matched controls (Kuo,
Y.-M.; Emmerling, M. R.; Lampert, H. C.; Hempelman, S.
R.; Kokjohn, T. A.; Woods, A. S.; Cotter, R. J.; Roher,
A. E. Biochem. Biophys. Res. Commun. 1999, 257, 787-791).
The pretreatment of plasma with formic acid was found to
be absolutely critical in dissociating the (3-amyloidl_42
from plasma proteins, prior to evaluation of the (3-
3o amyloidl_42 levels. Without the pretreatment, levels of (3-
amyloidl_~~ are significantly lower. The present invention
incorporates a step involving the complete or nearly-
complete dissociation of (3-amyloidl_42, or related amyloid
peptides such as A~33pE, from serum or plasma samples
obtained from humans, prior to analysis of ~3-amyloidl_42 or
3
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s A(33pE levels, as part of a diagnostic assay for the early
detection of AD, or the propensity to develop AD in the
future.
Summary of the Invention
The present invention is directed to a method of
diagnosing Alzheimer's disease involving analysis of a
plasma or serum sample ir~ such a way that ~3-amyloidl42 or
A~33pE is completely or nearly completely (ie.,
thoroughly) dissociated from binding proteins prior to
the analysis of the levels of (3-amyloidl_~2 or A(33pE.
We propose to diagnose patients with AD, or those
who are gradually developing AD, or those who might
2o develop AD in the future, by a procedure which involves
analysis of plasma by completely or nearly-completely
dissociating ~3-amyloidl_42 or A(33pE from plasma proteins,
such as albumin, followed by evaluation of the levels of
(3-amyloidl_42 or A(33pE. Those patients whose (3-amyloidl_YZ
z5 or A(33pE levels are determined to be above a certain
threshold level would be considered to either have AD or
have a high probability of developing AD in the future.
Further, the procedure could be done at different times,
and the relative increase in the levels of (3-amyloidl_42 or
3o A(33pE could be taken as a diagnostic component as to the
presence of AD or the liability for developing AD in the
future. Also, the relative increase in the levels of (3-
amyloidl_42 or A~33pE could be taken as a prognostic marker
and a marker to monitor the progression of the disease.
35 Levels of (3-amyloid,_42 or A~33pE could be determined along
4
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
with standard cognitive testing for additional assessment
for prognosis and progression of the disease.
Detailed Description of the Invention
io The present invention provides a method of diagnosing
Alzheimer's disease in a subject in need thereof
comprising:
(a) obtaining a plasma or serum sample from a subject
wherein the plasma or serum sample contains an A(3 peptide
and a binding protein;
(b) contacting the plasma or serum sample with a
dissociation reagent thereby thoroughly dissociating the A(3
peptide from the binding protein; and
(c) measuring the quantity of A~3 peptide in the plasma
or serum sample.
Alternatively, the method may be further defined by
adding a step to neutralize the dissociation reagent prior
to analysis. This method is useful where the assay used to
measure the quantity of A(3 peptide may be affected by the
presence of an active dissociation reagent. For example a
highly acidic or basic dissociation reagent may alter the
pH of the plasma or serum sample such that an immunoassay
technique is not possible. The present invention also
3o provides a method of diagnosing Alzheimer's disease in a
subject in need thereof comprising:
(a) obtaining a plasma or serum sample from a subject
wherein the plasma or serum sample contains an A(3 peptide
and a binding protein;
5
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
(b) contacting the plasma or serum sample with a
dissociation reagent thereby thoroughly dissociating the A~3
peptide from the binding protein;
(c) contacting the plasma or serum sample with a
neutralizing reagent; and
to (d) measuring the quantity of A(3 peptide in the plasma
or serum sample.
The term "subject" as used herein, refers to an
animal, preferably a mammal, most preferably a human, who
has been the object of treatment, observation, biochemical
screening, or experiment.
As used herein the term "A(3 peptide" is a ~3-amyloid
peptidel_4z or enzymatically modified (3-amyloid peptide, such
as where aspartic acid at position one is modified to
2o isoaspartic acid. Particularly preferred A(3 peptides are
selected from the group consisting of (3-amyloid,_s2 and
A(33pE .
As used herein the term "binding protein" is any
protein to which ~3-amyloid peptides naturally bind, such as
apoE3, apoE4, apoJ, transthyretin, and albumin. The
inventors contemplate that there are cell surface receptors
that also bind A(3 peptides.
The term "dissociation reagent" is a material,
preferably a solution, that causes A(3 peptide to dissociate
3o from the binding protein, and optionally causes
denaturation of the binding protein. Preferred
dissociation reagents include acids, including formic acid,
hydrochloric acid, acetic acid, sulfuric acid, bases,
including sodium hydroxide, miscible organic solvents,
including ethanol, methanol, DMSO, and DMF. The
6
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
dissociation reagent may also be a detergent or surfactant
including Triton X-100, Tween 20, Tween 80, and sodium
dodecyl sulfate ("SDS") or a chaotropic agent icluding
urea, and guanidinium chloride. Yet another type of
dissociation reagent is a compound that inhibits (3-amyloid
to aggregation, such as raloxifene and those compounds
described in Curr. Med. Chem. 1997, 4, 159; and Exp. Opin.
Ther. Pat. 1997, 7, 1115.
The term "neutralizing reagent" is a material,
preferably a solution, that counteracts the dissociation
reagents so that the final plasma or serum sample
composition is suitable for an assay to measure the
dissociated A(3 peptide. Where the dissociation reagent is
an acid, a suitable amount of a base, as the neutralizing
reagent, may be added to the plasma or serum sample to
2o prepare it for an assay. Alternatively where the
dissociation reagent is a base, a suitable amount of acid,
as the neutralizing reagent, may be added to the plasma or
serum sample. Not all dissociation reagents will require a
neutralizing reagent, including miscible organic solvents
or compounds that inhibit ~3-amyloid aggregation.
The terms "thoroughly dissociated" and/or "thoroughly
dissociating" as used herein when referring to dissociation
of A(3 peptide from binding protein means that the A(3
peptide is completely or nearly completely dissociated from
3o the binding protein; that is, greater than 80% (preferably,
>90%, more preferably, >95%).
The levels of A~i peptide are measured by methods known
in the art including, but not limited to, immunoassay
techniques, HPLC analysis, HPLC/MS (mass spectral)
analysis, MALDI/TOF (matrix-assisted laser desorption /
time-of-flight) mass spectral analysis, size exclusion
7
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s chromatography, thioflavin-T or Congo Red staining, or
ES/MS (electrospray ionization mass spectral) analysis. By
comparing the levels of (3-amyloidl_4Z or A~33pE with normal
(control) patients, one of ordinary skill in the art can
readily determine whether the patient is suffering from AD,
to is at risk for developing AD, or one can monitor the
progression of AD.
A particularly preferred assay format is the
immunoassay format, wherein the A(3 peptide is isolated
from the plasma or serum sample using one affinity capture
15 reagent, and is detected with a affinity label reagent.
The affinity capture or affinity label reagents comprise
compounds capable of specific interaction with the A(3
peptide to the exclusion of similar compounds. These
compounds include, for example, synthetic or natural amino
2o acid polypeptides, proteins (including antibodies), small
synthetic organic molecules, or deoxy- or ribo- nucleic
acid sequences with about 20-fold or greater affinity for
the A(3 peptide compared to other proteins or peptides.
The labeled compounds useful in the present invention
2s may be labeled compounds, with means of direct detection,
or may be detected by an indirect means, for example by a
second labeled compound.
The phrase "labeled compound" refers to moieties
capable of measurement comprising radioactive atoms,
3o enzymes, fluorescent molecules, or alternative tags, for
example biotin. Particular radioisotopes useful as a
label in the present invention are 3H, 125I , 13~I , 3sS 32p or
33p, particular examples of enzymes suitable for use in
the present invention are horseradish peroxidase, alkaline
35 phosphatase, or luciferase. A preferred example of a
detectable label is an enzyme that cleaves a substrate to
8
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s yield a chromogenic or luminescent product. Particular
examples of fluorescent molecules are fluorescein (FITC),
rhodamine, R-phycoerythrin (PE)
or AlexaT~ dyes (Molecular Probes). Direct measurement is
conducted by observing the presence of the radioactive atom
to or flourogenic molecule, or by observation of enzymatic
activity of a colorimetric or luminescent substrate.
Indirect measurement is conducted by adding an additional
compound including a label to the plasma or serum sample so
that it can interact with the compound bound to the plasma
15 or serum sample. A well-known example is when the labeled
compound comprises biotin, and a second compound comprises
avidin or streptavidin and a detectable label. A second
well-known example is when a first antibody is used to bind
to the A(3 peptide and is detected with an anti-antibody
2o comprising a detectable label. In this case the first
antibody comprises a label in that there are specific
regions capable of detection within the structure of the
first antibody.
z5 Also included in the invention is a diagnostic kit in
which all of the components required for a viable
diagnostic determination are packaged together sufficient
for complete or nearly-complete dissociation of A(3 peptide
from a plasma or serum sample and subsequent analysis of
3o the levels of the A(3 peptide such as by an ELISA (enzyme
linked immunosorbant assay). The components of an
immunoassay diagnostic kit include a dissociation reagent,
optionally a neutralizing reagent, and an affinity capture
reagent, for instance, an affinity coated resin, an
35 affinity label reagent, and optionally immunoassay control
reagents. Immunoassay control reagents are those that
9
CA 02373150 2001-11-06
WO 00/68694 PCT/US00/12167
s confirm proper function of the assay system and serve to
validate interpretation of the sample. A negative control
is one that yields no signal from the affinity label
reagent, and is often used to determine the background
noise of an assay system or used to calculate the signal to
io noise ratio for a positive sample using calculations well
known in the art. A positive control is one that yields a
signal from the affinity label reagent, and is often used
to validate the assay system or to compare to a sample to
interpret the status of the sample. This can be done
15 empirically using a "yes/no" system or can be made
quantitative by comparing the signal generated by control
samples containing known quantities of AD peptide with a
test sample.
While the foregoing specification teaches the
principles of the present invention, with examples provided
for the purpose of illustration, it will be understood that
the practice of the invention encompasses all of the usual
variations, adaptations and/or modifications as come within
the scope of the following claims and their equivalents.
to
