U.S. patent application number 13/721254 was filed with the patent office on 2013-06-27 for method of diagnosing, preventing and/or treating dementia & related disorders.
This patent application is currently assigned to Meso Scale Technologies, LLC. The applicant listed for this patent is Pankaj Oberoi, Robert M. Umek. Invention is credited to Pankaj Oberoi, Robert M. Umek.
Application Number | 20130164217 13/721254 |
Document ID | / |
Family ID | 48654768 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164217 |
Kind Code |
A1 |
Umek; Robert M. ; et
al. |
June 27, 2013 |
METHOD OF DIAGNOSING, PREVENTING AND/OR TREATING DEMENTIA &
RELATED DISORDERS
Abstract
Described are assay methods, modules and kits useful in the
detection, treatment and/or prevention of dementia and related
conditions, including but not limited to Alzheimer's disease and
mild cognitive disorders.
Inventors: |
Umek; Robert M.; (Silver
Spring, MD) ; Oberoi; Pankaj; (Rockville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Umek; Robert M.
Oberoi; Pankaj |
Silver Spring
Rockville |
MD
MD |
US
US |
|
|
Assignee: |
Meso Scale Technologies,
LLC
Gaithersburg
MD
|
Family ID: |
48654768 |
Appl. No.: |
13/721254 |
Filed: |
December 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61578344 |
Dec 21, 2011 |
|
|
|
Current U.S.
Class: |
424/1.89 ;
424/1.11; 424/1.81; 435/6.16; 435/7.9; 436/501; 506/9 |
Current CPC
Class: |
G01N 2800/2821 20130101;
G01N 2800/52 20130101; G01N 33/6896 20130101; G01N 2333/4709
20130101 |
Class at
Publication: |
424/1.89 ; 506/9;
436/501; 435/6.16; 435/7.9; 424/1.11; 424/1.81 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for diagnosing Alzheimer's-related dementia in a
patient comprising (a) obtaining a test sample from a patient; (b)
measuring a level of a first biomarker in said test sample, wherein
said first biomarker comprises A.beta. 16, A.beta. 17, or
combinations thereof; and (c) diagnosing from said measuring step
the presence, absence, and/or progression of Alzheimer's-related
dementia in said patient.
2. The method of claim 1 wherein said method further comprises
measuring a level of at least one additional biomarker in said
sample and determining from said level of said first biomarker and
said level of said at least one additional biomarker the presence,
absence, and/or progression of Alzheimer's-related dementia in said
patient.
3. The method of claim 2 wherein said method further comprises
comparing said level of said first biomarker and said at least one
additional biomarker(s) in said sample to a level of said first
biomarker and said at least one additional biomarker(s) in a normal
control sample and diagnosing the presence, absence and/or
progression of Alzheimer's-related dementia in said patient based
on said comparison.
4. The method of claim 2 wherein said at least one additional
biomarker comprises A.beta. 42, A.beta. 40, A.beta. 38, A.beta. 39,
A.beta. 37, A.beta. 34, A.beta. 43, total tau, P-tau, C-tau, or
combinations thereof.
5. The method of claim 1 further comprising conducting a PET scan
of said patient's brain using a radioactive diagnostic agent to
estimate plaque density in said patient, and said diagnosing step
further comprises diagnosing the presence, absence, and/or
progression of Alzheimer's-related dementia in said patient based
on said PET scan and said biomarker levels.
6. The method of claim 5 wherein said radioactive diagnostic agent
is Amyvid, Pittsburgh Compound B, or combinations thereof.
7. A method for monitoring the disease progression of and/or
efficacy of a treatment regimen for Alzheimer's-related dementia in
a patient, said method comprising (a) obtaining two or more test
samples from a patient undergoing said treatment regimen for
Alzheimer's-related dementia, wherein said two or more test samples
include (i) a first test sample obtained from said patient at time
t and (ii) at least one additional test sample obtained at time
(t+n), wherein n is an integer greater than 1; (b) measuring the
level(s) of a first biomarker in said two or more test samples,
wherein said first biomarker comprises A.beta. 16, A.beta. 17, or
combinations thereof; and (c) determining from said level(s) of
said first biomarker the progression of and/or efficacy of said
treatment regimen in said patient.
8. The method of claim 7 wherein said method further comprises
measuring a level of at least one additional biomarker in said two
or more test samples and determining from said level(s) of said
first biomarker and said at least one additional biomarker the
disease progression and/or efficacy of said treatment regimen for
Alzheimer's-related dementia in said patient.
9. The method of claim 8 wherein said method further comprises
comparing said level(s) of said first biomarker and said at least
one additional biomarker(s) in said two or more test samples to a
level(s) of said first biomarker and said at least one additional
biomarker(s) in a normal control sample and determining said
disease progression and/or treatment efficacy based on said
comparison.
10. The method of claim 8 wherein said at least one additional
biomarker comprises A.beta. 42, A.beta. 40, A.beta. 38, A.beta. 39,
A.beta. 37, A.beta. 34, A.beta. 43, total tau, P-tau, C-tau, or
combinations thereof.
11. The method of claim 8 further comprising conducting a PET scan
of said patient's brain using a radioactive diagnostic agent to
estimate plaque density in said patient, and said diagnosing step
further comprises diagnosing disease progression and/or treatment
efficacy based on said PET scan and said biomarker levels.
12. The method of claim 11 wherein said radioactive diagnostic
agent is Amyvid, Pittsburgh Compound B, or combinations
thereof.
13. A method for detecting plaques in the brain of a patient, said
method comprising (a) obtaining two or more test samples from a
patient undergoing a treatment regimen for dementia, wherein said
two or more test samples include (i) a first test sample obtained
from said patient at time t and (ii) at least one additional test
sample obtained at time (t+n), wherein n is an integer greater than
1; (b) measuring the level(s) of a first biomarker in said two or
more test samples, wherein said first biomarker comprises A.beta.
16, A.beta. 17, A.beta. 42, A.beta. 40, A.beta. 38, A.beta. 39,
A.beta. 37, A.beta. 34, A.beta. 43, total tau, P-tau, C-tau, or
combinations thereof; and (c) determining from said level(s) of
said first biomarker the presence and/or absence of plaques in the
brain of said patient.
14. The method of claim 13 wherein said method further comprises
measuring a level of at least one additional biomarker in said two
or more test samples, wherein said additional biomarker comprises
A.beta. 16, A.beta. 17, A.beta. 42, A.beta. 40, A.beta. 38, A.beta.
39, A.beta. 37, A.beta. 34, A.beta. 43, total tau, P-tau, C-tau, or
combinations thereof, and determining from said level(s) of said
first biomarker and said level(s) of said at least one additional
biomarker the presence and/or absence of plaques in the brain of
said patient.
15. The method of claim 14 wherein said method further comprises
comparing said level(s) of said first biomarker and said at least
one additional biomarker(s) in said two or more test samples to a
level of said first biomarker and said at least one additional
biomarker(s) in a normal control sample and determining the
presence and/or absence of plaques in the brain of said patient
based on said comparison.
16. A method for diagnosing Alzheimer's-related dementia and/or
plaque formation in a patient comprising (a) obtaining a test
sample from a patient; (b) measuring a level of an autoantibody to
a first biomarker in said test sample, wherein said first biomarker
is an A-beta peptide, tau, a fragment or isoform of an A-beta
peptide or tau, or combinations thereof; (c) diagnosing from said
measuring step the presence, absence, and/or progression of
Alzheimer's-related dementia and/or plaque formation in said
patient.
17. The method of claim 16 wherein said autoantibody binds to an
A-beta peptide or a fragment or isoform thereof.
18. The method of claim 17 wherein said autoantibody binds to an
A-beta peptide selected from the group consisting of A.beta. 42,
A.beta. 40, A.beta. 38, A.beta. 39, A.beta. 37, A.beta. 34, A.beta.
43, A.beta. 16, A.beta. 17 or combinations thereof.
19. The method of claim 16 wherein said autoantibody binds to tau
or a fragment or isoform thereof.
20. The method of claim 19 wherein said autoantibody binds to a tau
protein selected from total tau, P-tau, C-tau, or combinations
thereof.
21. The method of claim 16 wherein said method further comprises
measuring a level of an additional autoantibody to at least one
supplemental biomarker in said sample, wherein said supplemental
biomarker comprises one or more of the following: an A.beta.
peptide, tau, a fragment or isoform of an A.beta. peptide or tau,
or combinations thereof, and said first biomarker and said
supplemental biomarker are different, and determining from said
levels of autoantibody and additional autoantibody the presence,
absence, and/or progression of Alzheimer's-related dementia and/or
plaque formation in said patient.
22. The method of claim 21 wherein said method further comprises
comparing said levels of autoantibody and additional autoantibody
in said sample to levels of autoantibody and additional
autoantibody in a normal control sample and diagnosing the
presence, absence and/or progression of Alzheimer's-related
dementia and/or plaque formation in said patient based on said
comparison.
23. The method of claim 21 wherein said at least one additional
biomarker comprises A-beta peptide or a fragment or isoform
thereof.
24. The method of claim 23 wherein said A-beta peptide is selected
from the group consisting of A.beta. 42, A.beta. 40, A.beta. 38,
A.beta. 39, A.beta. 37, A.beta. 34, A.beta. 43, A.beta. 16, A.beta.
17 or combinations thereof.
25. The method of claim 21, wherein said at least one additional
biomarker comprises tau or a fragment or isoform thereof.
26. The method of claim 25 wherein said at least one additional
biomarker is selected from the group consisting of total tau,
P-tau, C-tau, or combinations thereof.
27. The method of claim 16 further comprising conducting a PET scan
of said patient's brain using a radioactive diagnostic agent to
estimate plaque density in said patient, and said diagnosing step
further comprises diagnosing the presence, absence, and/or
progression of Alzheimer's-related dementia and/or plaque formation
in said patient based on said PET scan and said autoantibody
levels.
28. The method of claim 27 wherein said radioactive diagnostic
agent is Amyvid, Pittsburgh Compound B, or combinations thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims benefit of U.S. Provisional
Application No. 61/578,344 filed on Dec. 21, 2011, the entire
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to assay methods, modules and kits for
diagnostic assays useful in the detection, treatment and/or
prevention of dementia and related conditions, including but not
limited to Alzheimer's disease and mild cognitive disorders. In
particular, the assays of the invention are useful in the
diagnosis, treatment and/or prevention of a disease or condition
associated with an abnormal level of one or more isoforms of
amyloid beta peptides ("A.beta.") and/or with a changed ratio of
levels of A.beta. isoforms and/or with the formation of plaques
containing one or more A.beta. isoforms in a mammal.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease is characterized by a gradual loss of
memory, decline in the ability to perform routine tasks,
disorientation, difficulty in learning, loss of language skills,
impaired judgment and ability to plan, and personality changes.
Over time, these changes become so severe that they interfere with
an individual's daily functioning, resulting eventually in death.
Alzheimer's disease is a type of dementia and it is only confirmed
upon autopsy when the characteristic plaques and tangles are
observed.
[0004] Biomarkers that can be used to diagnose Alzheimer's disease
and/or other forms of dementia are in demand. Particularly useful
are biomarkers that can diagnose Alzheimer's disease with greater
than 95% precision and accuracy before autopsy, biomarkers that can
distinguish among various forms of dementia, biomarkers that
reflect modulation of the disease by therapeutics, and biomarkers
that predict the progression of Alzheimer's disease well in advance
of neurological symptoms. For example, studies suggest that by the
time A.beta. 42 or tau are altered in abundance in cerebral spinal
fluid, there is already significant neuronal damage in the brain.
The hope is that early stage biomarkers can be found to justify
prophylactic intervention early in a patient's life.
SUMMARY OF THE INVENTION
[0005] The invention provides a method for diagnosing dementia in a
patient comprising (a) measuring a level of a first biomarker in a
test sample obtained from a patient, wherein said first biomarker
is selected from the group consisting of A.beta. 16, A.beta. 17,
and combinations thereof; (b) and diagnosing from said measuring
step the presence, absence, and/or progression of dementia in said
patient.
[0006] Also provided is a method for monitoring the progression of
and/or efficacy of treatment for dementia in a patient, said method
comprising (a) measuring the level(s) of a first biomarker in
samples obtained at different times from said patient, wherein said
first biomarker is selected from the group consisting of A.beta.
16, A.beta. 17, and combinations thereof; and (b) determining from
said level(s) of said first biomarker the progression of and/or
efficacy of treatment for dementia in said patient.
[0007] Still further, the invention provides a method for
diagnosing, treating and/or preventing a disease or condition
associated with an abnormal level of one or more isoforms of
amyloid beta peptides in a patient comprising (a) measuring a level
of a first biomarker in a test sample obtained from a patient,
wherein said first biomarker is selected from the group consisting
of A.beta. 16, A.beta. 17, and combinations thereof; and (b)
diagnosing from said measuring step the presence, absence, and/or
progression of said condition in said patient.
[0008] The invention contemplates a method for diagnosing, treating
and/or preventing a disease or condition associated with an
abnormal level of one or more isoforms of amyloid beta peptides,
said method comprising (a) measuring the level(s) of a first
biomarker in samples obtained at different times from said patient,
wherein said first biomarker is selected from the group consisting
of A.beta. 16, A.beta. 17, and combinations thereof; and (b)
determining from said level(s) of said first biomarker the
progression of and/or efficacy of treatment for said condition in
said patient.
[0009] Also provided is a method for diagnosing Alzheimer's disease
in a patient comprising (a) measuring a level of a first biomarker
in a test sample obtained from a patient, wherein said first
biomarker is selected from the group consisting of A.beta. 16,
A.beta. 17, and combinations thereof; (b) and diagnosing from said
measuring step the presence, absence, and/or progression of
Alzheimer's disease in said patient.
[0010] And further provided is a method for monitoring the
progression of and/or efficacy of treatment for Alzheimer's disease
in a patient, said method comprising (a) measuring the level(s) of
a first biomarker in samples obtained at different times from said
patient, wherein said first biomarker is selected from the group
consisting of A.beta. 16, A.beta. 17, and combinations thereof; and
(b) determining from said level(s) of said first biomarker the
progression of and/or efficacy of treatment for Alzheimer's disease
in said patient.
[0011] The methods of the present invention may further include
evaluating one or more additional biomarker selected from the group
consisting of A.beta. 42, A.beta. 40, A.beta. 38, A.beta. 39,
A.beta. 37, A.beta. 34, A.beta. 43, tau, fragments and isoforms of
A.beta. peptides and tau, and combinations thereof.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0012] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. The articles "a" and "an" are used herein to refer to one
or to more than one (i.e., to at least one) of the grammatical
object of the article. By way of example, "an element" means one
element or more than one element.
[0013] "Dementia" refers to a group of similar age-related
disorders that result in diminished cognitive functioning (there
are also injury- and AIDS-associated dementia that are not age
related). Dementia is usually chronic or progressive in nature and
characterized by disturbance of multiple higher cortical functions,
including memory, thinking, orientation, comprehension,
calculation, learning capacity, language, and judgment. Impairments
of cognitive function are commonly accompanied, and occasionally
preceded, by deterioration in emotional control, social behavior,
or motivation. This syndrome occurs in Alzheimer's disease, in
cerebrovascular disease, AIDS, and in other conditions primarily or
secondarily affecting the brain (World Health Organization ICD-10,
1992).
[0014] Alzheimer's disease is one form of dementia. Like other
forms of dementia, diagnosis of Alzheimer's disease is based on
clinical and neuropsychological evaluation with the exclusion of
secondary causes of memory loss, with final confirmation by
autopsy. Like other forms of dementia, Alzheimer's disease
progresses through three main stages: mild, moderate, and severe.
Neuropsychological symptoms of mild or early stage Alzheimer's
disease may include but are not limited to difficulty learning and
remembering new information; difficulty managing finances, planning
meals, taking medication on schedule; depression symptoms (sadness,
decreased interest in usual activities, loss of energy); and
disorientation in otherwise familiar places. In moderate
Alzheimer's disease, the damaging processes occurring in the brain
worsen and spread to other areas that control language, reasoning,
sensory processing, and thought. In this stage, neuropsychological
symptoms of Alzheimer's disease become more pronounced and
behavioral problems may become more obvious; these symptoms include
but are not limited to forgetfulness, continually repeating stories
and/or asking the same questions repeatedly, difficulty performing
routine daily tasks, following written instructions, agitation,
restlessness, repetitive movements, wandering, paranoia, delusions,
hallucinations, deficits in intellect and reasoning, lack of
concern for appearance, hygiene, and sleep become more noticeable.
Finally, in the advanced stages of Alzheimer's disease, damage to
the brain's nerve cells is widespread. People with severe
Alzheimer's may exhibit neuropsychological symptoms may include
difficulty walking and communicating coherently, refusal to eat or
drink, an inability to recognize family or faces, and difficulty
with all essential activities of daily living.
[0015] Alzheimer's disease is characterized by two major pathologic
observations in the brain: neurofibrillary tangles (NFT) and beta
amyloid (or neuritic) plaques, comprised predominantly of an
aggregate of amyloid beta (A.beta.). Individuals with Alzheimer's
disease exhibit characteristic beta-amyloid deposits in the brain
(beta amyloid plaques) and in cerebral blood vessels (beta amyloid
angiopathy) as well as neurofibrillary tangles. Neurofibrillary
tangles occur not only in Alzheimer's disease but also in other
dementia-inducing disorders. On autopsy, large numbers of these
lesions are generally found in areas of the human brain important
for memory and cognition.
[0016] Beta-amyloid is a neurotoxic peptide that exists in several
isoforms, now believed to be a causative precursor or factor in the
development of disease. Deposition of A.beta. in areas of the brain
responsible for cognitive activities is a major factor in the
development of Alzheimer's disease. Beta-amyloid plaques are
predominantly composed of amyloid beta peptide. The various
fragments of A.beta. peptide are derived by sequential proteolysis
of the amyloid precursor protein (APP). Several proteases called
secretases are involved in the processing of APP. The processing of
APP leads to various fragments of A.beta., including but not
limited to A.beta. 40, A.beta. 42, A.beta. 39, A.beta. 38, A.beta.
37, A.beta. 43, A.beta. 34, A.beta. 17, and A.beta. 16.
[0017] The sequence for human APP is found at GenBank Accession No.
XM047793. The sequences of the various preferred isoforms of human
Ab are provided in Table 1 below:
TABLE-US-00001 TABLE 1 A.beta. 43
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G-G-V-V-I-A-T (SEQ ID NO: 1) A.beta. 42
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G-G-V-V-I-A (SEQ ID NO: 2) A.beta. 40
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G-G-V-V (SEQ ID NO: 3) A.beta. 39
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G-G-V (SEQ ID NO: 4) A.beta. 38
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G-G (SEQ ID NO: 5) A.beta. 37
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L-M-V-G (SEQ ID NO: 6) A.beta. 34
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-
G-A-I-I-G-L (SEQ ID NO: 7) A.beta. 17
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L (SEQ ID NO: 8) A.beta. 16
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K (SEQ ID NO: 9)
[0018] It will be understood that other isoforms of A.beta. are
within the scope of the invention, e.g., those isoforms having a
C-terminus at position 16, 17, 34, 37, 38, 39, and 40 relative to
the numbering for A.beta. 1-42 and truncated n-termini.
[0019] Cleavage of APP at the N-terminus of the A.beta. peptide by
beta-secretase (BACE) and at the C-terminus by one or more
gamma-secretases constitutes the beta-amyloidogenic pathway, i.e.,
the pathway by which A.beta. is formed. Cleavage of APP by
alpha-secretase produces alpha-sAPP, a secreted form of APP that
does not result in beta-amyloid plaque formation. This alternate
pathway precludes the formation of A.beta. peptide.
[0020] In addition to the numerous isoforms of A.beta., various
proteins have been associated with the diagnosis of dementia or
Alzheimer's disease. Table 2 summarizes these markers. (See Flirski
et al., Current Alzheimer Research (2005) 2: 47-64, the disclosure
of which is incorporated herein by reference in its entirety.)
TABLE-US-00002 TABLE 2 Up- or down- regulated in Marker Sample
Alzheimer's Patients A.beta. 42 CSF Down Total tau CSF Up P-tau CSF
Up ACT CSF, plasma Up F2-iPs CSF Up Non-enzymatic Plasma Down
antioxidants SOD RBC Up Cu Plasma Up 24-OHC CSF Up NTP CSF, urine
Up F4-iPs CSF Up DNA 8-OHG/ CSF Up free 8-OHG RNA 8-OHG CSF Up
3-nitrotyrosine CSF Up tTG CSF Up CD95 Plasma Up Glyc-AChE CSF Up
Glyc-BuChE ST/PI CSF Down hK10 CSF Down A.beta.3-44/ A.beta.3-47
CSF Down C-tau CSF Up A.beta.42 Plasma Up IL-6, CRP, Plasma Up
TNF-alpha Haptoglobin, CRP Plasma Up TNF-alpha Plasma Up
antioxidants Plasma Up Homocystein Plasma Up Folate, vitamin B6,
Plasma Down vitamin B12 Cholesterol Up Lanosterol, Up
lathosterol
[0021] Therefore, in one embodiment, the invention provides a
method for diagnosing Alzheimer's-related dementia and/or plaque
formation in the brain of a patient comprising (a) measuring a
level of a first biomarker in a test sample obtained from a
patient, wherein said first biomarker is selected from the group
consisting of A.beta. 16 and/or A.beta. 17, and combinations
thereof; (b) diagnosing from said measuring step the presence,
absence, and/or progression of disease (i.e., Alzheimer's-related
dementia and/or plaque formation) in said patient. This method may
further comprise measuring one or more additional biomarkers, e.g.,
one or more isoforms of A.beta., total tau, P-tau, C-tau, ACT,
F2-iPs, non-enzymatic antioxidants, SOD, Cu, 24-OHC, NTP, F4-iPs,
DNA 8-OHG/free 8-OHG, RNA 8-OHG, 3-nitrotyrosine, tTG, CD95,
Glyc-AChE, Glyc-BuChE, ST/PI, hK10, IL-6, CRP, TNF-alpha, and
combinations thereof. In addition, the present invention also
contemplates measuring the level of autoantibodies in a patient
sample to a biomarker associated with Alzheimer's-related dementia
and/or plaque formation, including but not limited to an A.beta.
peptide, tau, a fragment or isoform of an A.beta. peptide or tau,
or combinations thereof. The method can also include measuring
autoantibodies to one or more biomarker including but not limited
to: A.beta. 16, A.beta. 17, A.beta. 40, A.beta. 42, A.beta. 39,
A.beta. 38, A.beta. 37, A.beta. 34, A.beta. 43, total tau, P-tau,
C-tau, ACT, F2-iPs, non-enzymatic antioxidants, SOD, Cu, 24-OHC,
NTP, F4-iPs, DNA 8-OHG/free 8-OHG, RNA 8-OHG, 3-nitrotyrosine, tTG,
CD95, Glyc-AChE, Glyc-BuChE, ST/PI, hK10, IL-6, CRP, TNF-alpha, and
combinations thereof. The method can include measuring
autoantibodies level(s) as well as protein biomarker levels of one
or more of the Alzheimer's-related/plaque-related biomarkers
identified herein. In addition to measuring protein biomarker and
autoantibody levels in a patient sample, the method of the present
invention can also include measuring BACE and gamma-secretase
activity in a patient sample. In this regard, the patient's profile
of autoantibody and biomarker levels, as well as BACE and/or
gamma-secretase activities is considered in making a diagnosis of
Alzheimer's-related dementia and/or plaque formation, alone or in
combination with the clinical evaluation of one or more
neuropsychological symptoms listed hereinabove.
[0022] In one embodiment, the method comprises measuring A.beta. 16
and/or A.beta. 17, and one or more of A.beta. 40, A.beta. 42,
A.beta. 39, A.beta. 38, A.beta. 37, A.beta. 34, A.beta. 43, tau,
and combinations thereof. In another embodiment, the method
comprises measuring A.beta. 16 and/or A.beta. 17, and one or more
of A.beta. 40, A.beta. 42, A.beta. 38, tau, and combinations
thereof. In these embodiments, tau may be total tau, P-tau, C-tau
or combinations thereof. Measurements are taken on samples selected
from the group consisting of urine, blood and/or cerebrospinal
fluid (CSF) and combinations thereof, depending on the biomarker(s)
selected.
[0023] In addition, the invention provides a method for diagnosing
Alzheimer's-related dementia, and/or the development of plaques in
the brain of a patient comprising (a) measuring a level of a first
biomarker in a test sample obtained from a patient, wherein said
first biomarker is selected from the group consisting of A.beta. 16
and/or A.beta. 17, and combinations thereof; (b) assessing one or
more neuropsychological symptoms associated with
Alzheimer's-related dementia and/or plaque formation; and (c)
diagnosing from said measuring and assessing steps the presence,
absence, and/or progression of Alzheimer's-related dementia, and/or
plaque formation in said patient. This method may further comprise
measuring one or more additional biomarkers, e.g., one or more
fragments and/or isoforms of A.beta. (A.beta. 40, A.beta. 42,
A.beta. 39, A.beta. 38, A.beta. 37, A.beta. 34, A.beta.43), forms
of tau (e.g., total tau, P-tau, C-tau, etc.), ACT, F2-iPs,
non-enzymatic antioxidants, SOD, Cu, 24-OHC, NTP, F4-iPs, DNA
8-OHG/free 8-OHG, RNA 8-OHG, 3-nitrotyrosine, tTG, CD95, Glyc-AChE,
Glyc-BuChE, ST/PI, hK10, IL-6, CRP, TNF-alpha, and combinations
thereof, as well as BACE and/or gamma-secretase activity.
[0024] In one embodiment, biomarkers of dementia, e.g., Alzheimer's
disease, and/or plaque formation reflect a central pathogenic
process of the disorder, e.g., degeneration of neurons and synapses
or the development of typical lesions as neuritic plaques and
neurofibrillary tangles. The biomarkers used in the method of the
present invention have a sensitivity of at least 75%, preferably at
least 80%, and more preferably at least 85% for detecting
Alzheimer's-related dementia, and/or plaque formation, and a
specificity of at least 75%, preferably at least 80%, and more
preferably at least 85% for distinguishing other dementias. In a
preferred embodiment, the biomarkers identified herein can be used
to differentiate various stages of Alzheimer's-related dementia and
in so doing, can be used to assess a patient's relative progression
from one stage of Alzheimer's-related dementia to the next. For
example, while CSF Ab42 levels alone can be used to identify the
presence of amyloid plaques in asymptomatic patients, evaluation of
(i) A.beta.42 levels in relation to other isoforms of A.beta.,
e.g., A.beta. 40, A.beta. 39, A.beta. 38, A.beta. 37, A.beta. 34,
A.beta. 43, A.beta.16, A.beta.17, (ii) A.beta. peptide patterns,
i.e., the relative levels of each of each individual A.beta.
peptide and/or the relative ratio of A.beta. peptides, preferably
measured over time, and/or (iii) A.beta. peptide levels in relation
to other diagnostic biomarkers, e.g., tau (e.g., p-tau and c-tau),
preferably measured over time, can be informative of the disease
progression. In a preferred embodiment, disease progression is
monitored over time by monitoring biomarker levels at a first time
point, t, and repeating that measurement at a second time point,
t+n, wherein n is one or more, in order to assess the relative
change in the biomarker(s) levels in the patient over a given time
interval. The time interval can be measured over the span of hours,
days, months, and/or years.
[0025] In one embodiment, the biological marker is present in body
fluids that are easily accessible, including but not limited to
urine, blood or cerebrospinal fluid (CSF). The assays of the
present invention may be conducted by any suitable method. In one
embodiment, the measuring step is conducted on a single sample, and
it may also be conducted in a single assay chamber, including but
not limited to a single well of an assay plate. The assay chamber
may also be an assay chamber of a cartridge. As used herein, the
term "sample" is intended to mean any biological fluid, cell,
tissue, organ or combinations or portions thereof, which includes
or potentially includes a biomarker of a disease of interest. For
example, a sample can be a histologic section of a specimen
obtained by biopsy, or cells that are placed in or adapted to
tissue culture. A sample further can be a subcellular fraction or
extract, or a crude or substantially pure nucleic acid molecule or
protein preparation. In one embodiment, the samples that may be
analyzed in the assays of the present invention include but are not
limited to blood or blood fractions such as, cerebral spinal fluid,
serum and plasma. In one embodiment, the level is measured using an
immunoassay.
[0026] As used herein, a "biomarker" is a substance that is
associated with a particular disease. A change in the expression
levels of a biomarker may correlate with the risk or progression of
a disease or with the susceptibility of the disease to a given
treatment. A biomarker may be useful in the diagnosis of disease
risk or the presence of disease in an individual, or to tailor
treatments for the disease in an individual (choices of drug
treatment or administration regimes). In evaluating potential drug
therapies, a biomarker may be used as a surrogate for a natural
endpoint such as survival or irreversible morbidity. If a treatment
alters the biomarker, which has a direct connection to improved
health, the biomarker serves as a "surrogate endpoint" for
evaluating clinical benefit. A sample that is assayed in the
diagnostic methods of the present invention may be obtained from
any suitable patient, including but not limited to a patient
suspected of having a disorder associated with abnormal A.beta.
levels, as described herein or a patient having a predisposition to
a condition associated with abnormal A.beta. levels. The patient
may or may not exhibit clinical symptoms associated with a
condition associated with abnormal A.beta. levels.
[0027] As used herein, the term "level" refers to mean the amount,
concentration, accumulation or rate of a biomarker molecule. A
level can be represented, for example, by the amount or synthesis
rate of messenger RNA (mRNA) encoded by a gene, the amount or
synthesis rate of polypeptide corresponding to a given amino acid
sequence encoded by a gene, or the amount or synthesis rate of a
biochemical form of a molecule accumulated in a cell, including,
for example, the amount of particular post-synthetic modifications
of a molecule such as a polypeptide, nucleic acid or small
molecule. The term can be used to refer to an absolute amount of a
molecule in a sample or to a relative amount of the molecule,
including amounts or concentration determined under steady-state or
non-steady-state conditions. Level may also refer to an assay
signal that correlates with the amount, concentration, accumulation
or rate of a biomarker molecule. The expression level of a molecule
can be determined relative to a control molecule in a sample.
According to one aspect of the invention, the levels or levels of
biomarker(s) are measured in the samples collected from individuals
clinically diagnosed with or suspected of or at risk of developing
a condition associated with abnormal A.beta. levels using
conventional methods, e.g., biopsy or other conventional diagnostic
methods, as well as from healthy individuals. It may also be used
to screen for disease in a broad population of asymptomatic
individuals. For example, specific biomarkers valuable in
distinguishing between normal and diseased patients could be
identified by visual inspection of the data, for example, data
plotted on a one-dimensional or multidimensional graph, or using
methods of statistical analysis, such as a statistically weighted
difference between control individuals and diseased patients and/or
Receiver Operating Characteristic (ROC) curve analysis.
[0028] For example and without limitation, diagnostically valuable
biomarkers may be first identified using a statistically weighted
difference between control individuals and diseased patients,
calculated as
D - N .sigma. D * .sigma. N ##EQU00001##
[0029] wherein D is the median level of a biomarker in patients
diagnosed as having, for example, breast cancer or ovarian cancer,
N is the median of the control individuals, (TD is the standard
deviation of D and (TN is the standard deviation of N. The larger
the magnitude, the greater the statistical difference between the
diseased and normal populations.
[0030] According to one embodiment of the invention, biomarkers
resulting in a statistically weighted difference between control
individuals and diseased patients of greater than, e.g., 1, 15.2,
2.5 or 3 could be identified as diagnostically valuable
markers.
[0031] Another method of statistical analysis for identifying
biomarkers is the use of z scores, e.g., as described in Skates et
al. (2007) Cancer Epidemiol. Biomarkers Prey. 16(2):334-341.
Another method of statistical analysis that can be useful in the
inventive methods of the invention for determining the efficacy of
particular candidate analytes, such as particular biomarkers, for
acting as diagnostic marker(s) is ROC curve analysis. An ROC curve
is a graphical approach to looking at the effect of a cut-off
criterion, e.g., a cut-off value for a diagnostic indicator such as
an assay signal or the level or level of an analyte in a sample, on
the ability of a diagnostic to correctly identify positive or
negative samples or subjects. One axis of the ROC curve is the true
positive rate (TPR, the probability that a true positive
sample/subject will be correctly identified as positive, or
alternatively, the false negative rate (FNR=1-TPR, the probability
that a true positive sample/subject will be incorrectly identified
as a negative). The other axis is the true negative rate, i.e.,
TNR, the probability that a true negative sample will be correctly
identified as a negative, or alternatively, the false positive rate
(FPR=1-TNR, the probability that a true negative sample will be
incorrectly identified as positive). The ROC curve is generated
using assay results for a population of samples/subjects by varying
the diagnostic cut-off value used to identify samples/subjects as
positive or negative and plotting calculated values of TPR or FNR
and TNR or FPR for each cut-off value. The area under the curve
(referred to herein as the ROC area) is one indication of the
ability of the diagnostic to separate positive and negative
samples/subjects.
[0032] Diagnostic indicators analyzed by ROC curve analysis may be
a level or levels of an analyte, e.g., a biomarker, or an assay
signal. Alternatively, the diagnostic indicator may be a function
of multiple measured values, for example, a function of the
level/assay signal of a plurality of analytes, e.g, a plurality of
biomarkers, or a function that combines the level or level or assay
signal of one or more analytes with a patients scoring value that
is determined based on visual, radiological and/or histological
evaluation of a patient. The multi-parameter analysis may provide
more accurate diagnosis relative to analysis of a single
marker.
[0033] Candidates for a multi-analyte panel could be selected by
using criteria such as individual analyte ROC areas, median
difference between groups normalized by geometric interquartile
range (10R) etc. The objective is to partition the analyte space so
as to improve separation between groups (for example, normal and
disease populations) or to minimize the misclassification rate.
[0034] One approach is to define a panel response as a weighted
combination of individual analytes and then compute an objective
function like ROC area, product of sensitivity and specificity,
etc. See e.g., WO 2004/058055, as well as US 2006/0205012, the
disclosures of which are incorporated herein by reference in their
entireties.
[0035] Biomarker levels may be measured using any of a number of
techniques available to the person of ordinary skill in the art,
e.g., direct physical measurements (e.g., mass spectrometry) or
binding assays (e.g., immunoassays, agglutination assays and
immunochromatographic assays). The method may also comprise
measuring a signal that results from a chemical reactions, e.g., a
change in optical absorbance, a change in fluorescence, the
generation of chemiluminescence or electrochemiluminescence, a
change in reflectivity, refractive index or light scattering, the
accumulation or release of detectable labels from the surface, the
oxidation or reduction or redox species, an electrical current or
potential, changes in magnetic fields, etc. Suitable detection
techniques may detect binding events by measuring the participation
of labeled binding reagents through the measurement of the labels
via their photoluminescence (e.g., via measurement of fluorescence,
time-resolved fluorescence, evanescent wave fluorescence,
up-converting phosphors, multi-photon fluorescence, etc.),
chemiluminescence, electrochemiluminescence, light scattering,
optical absorbance, radioactivity, magnetic fields, enzymatic
activity (e.g., by measuring enzyme activity through enzymatic
reactions that cause changes in optical absorbance or fluorescence
or cause the emission of chemiluminescence). Alternatively,
detection techniques may be used that do not require the use of
labels, e.g., techniques based on measuring mass (e.g., surface
acoustic wave measurements), refractive index (e.g., surface
plasmon resonance measurements), or the inherent luminescence of an
analyte.
[0036] Binding assays for measuring biomarker levels may use solid
phase or homogenous formats. Suitable assay methods include
sandwich or competitive binding assays. Examples of sandwich
immunoassays are described in U.S. Pat. No. 4,168,146 and U.S. Pat.
No. 4,366,241, both of which are incorporated herein by reference
in their entireties. Examples of competitive immunoassays include
those disclosed in U.S. Pat. No. 4,235,601, U.S. Pat. No. 4,442,204
and U.S. Pat. No. 5,208,535, each of which are incorporated herein
by reference in their entireties.
[0037] Multiple biomarkers may be measured using a multiplexed
assay format, e.g., multiplexing through the use of binding reagent
arrays, multiplexing using spectral discrimination of labels,
multiplexing of flow cytometric analysis of binding assays carried
out on particles, e.g., using the Luminex.RTM. system. Suitable
multiplexing methods include array based binding assays using
patterned arrays of immobilized antibodies directed against the
biomarkers of interest. Various approaches for conducting
multiplexed assays have been described (See e.g., US 20040022677;
US 20050052646; US 20030207290; US 20030113713; US 20050142033; and
US 20040189311, each of which is incorporated herein by reference
in their entireties. One approach to multiplexing binding assays
involves the use of patterned arrays of binding reagents, e.g.,
U.S. Pat. Nos. 5,807,522 and 6,110,426; Delehanty J-B., Printing
functional protein microarrays using piezoelectric capillaries,
Methods Mol. Biol. (2004) 264: 135-43; Lue R Y et al.,
Site-specific immobilization of biotinylated proteins for protein
microarray analysis, Methods Mol. Biol. (2004) 264: 85-100; Lovett,
Toxicogenomics: Toxicologists Brace for Genomics Revolution,
Science (2000) 289: 536-537; Berns A, Cancer: Gene expression in
diagnosis, nature (2000) 403: 491-92; Walt, Molecular Biology:
Bead-based Fiber-Optic Arrays, Science (2000) 287: 451-52 for more
details). Another approach involves the use of binding reagents
coated on beads that can be individually identified and
interrogated. See e.g., WO 9926067, which describes the use of
magnetic particles that vary in size to assay multiple analytes;
particles belonging to different distinct size ranges are used to
assay different analytes. The particles are designed to be
distinguished and individually interrogated by flow cytometry.
Vignali has described a multiplex binding assay in which 64
different bead sets of microparticles are employed, each having a
uniform and distinct proportion of two dyes (Vignali, D. A A,
"Multiplexed Particle-Based Flow Cytometric Assays" J ImmunoL Meth.
(2000) 243: 243-55). A similar approach involving a set of 15
different beads of differing size and fluorescence has been
disclosed as useful for simultaneous typing of multiple
pneumococcal serotypes (Park, M. K et aI., "A Latex Bead-Based Flow
Cytometric Immunoassay Capable of Simultaneous Typing of Multiple
Pneumococcal Serotypes (Multibead Assay)" Clin. Diagn. Lab Immunol.
(2000) 7: 486-89. Bishop, J E et al. have described a multiplex
sandwich assay for simultaneous quantification of six human
cytokines (Bishop, L E. et al., "Simultaneous Quantification of Six
Human Cytokines in a Single Sample Using Microparticle-based Flow
Cytometric Technology," Clin. Chem (1999) 45:1693-1694. A
diagnostic test may be conducted in a single assay chamber, such as
a single well of an assay plate or an assay chamber that is an
assay chamber of a cartridge.
[0038] The assay modules, e.g., assay plates or cartridges or
multi-well assay plates), methods and apparatuses for conducting
assay measurements suitable for the present invention are described
for example, in US 20040022677; US 20050052646; US 20050142033; US
20040189311, each of which is incorporated herein by reference in
their entireties. Assay plates and plate readers are now
commercially available (MULTI-SPOT.RTM. and MULTI-ARRAY.RTM. plates
and SECTOR.RTM. instruments, Meso Scale Discovery, a division of
Meso Scale Diagnostics, LLC, Gaithersburg, Md.). In addition, the
various isoforms of A.beta. can be purchased from AnaSpec, Inc.
(www.anaspec.com, Fremont, Calif.) or rPeptide (www.rpeptide.com,
Bogart, Ga.). Antibodies specific for the various isoforms of
A-beta may be produced by methods known in the art, e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual. (Third Ed.)
(2000).
[0039] Briefly, mice are immunized with short peptides (7-9 amino
acids) corresponding to the c-terminal sequences of A-beta 16, 17,
34, 37, 38, 39, 40, 42, or 43 coupled to KLH. After fusion of
spleens, hybridomas are selected after analysis of binding
activities against a multiplex array of amyloid peptide fragments.
Specifically, A-beta 1-16, 1-17, 1-34, 1-37, 1-38, 1-39, 1-40,
1-42, and 1-43 are immobilized on independent electrodes in a
Multi-Spot plate. Supernatants from individual hybridoma clones are
introduced into wells containing the peptides arrayed at the
bottom. Antibody binding is detected through the use of a labeled
goat-anti-mouse antibody. Hybridomas are selected that exhibit a
high signal on only one of the peptides consistent with the
production of antibodies that bind well but with specificity for
each unique end. For each isoform, hybridomas are identified that
bind each of the peptides and not the other forms. In one
embodiment, the immunogens that may be used to produce antibodies
specific for A-beta 38, -40, and -42 are: 38: CIIGLMVGG (SEQ ID NO:
10); 40: CGLMVGGVV (SEQ ID NO: 11); and 42: LMVGGVVIA (SEQ ID NO:
12), respectively.
[0040] Patents, patent applications, publications, and test methods
cited in this disclosure are incorporated herein by reference in
their entirety.
[0041] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such modifications
are intended to fall within the scope of the claims.
[0042] A claim which recites "comprising" allows the inclusion of
other elements to be within the scope of the claim; the invention
is also described by such claims reciting the transitional phrases
"consisting essentially of" (i.e., allowing the inclusion of other
elements to be within the scope of the claim if they do not
materially affect operation of the invention) or "consisting of"
(i.e., allowing only the elements listed in the claim other than
impurities or inconsequential activities which are ordinarily
associated with the invention) instead of the "comprising" term.
Any of these three transitions can be used to claim the
invention.
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Sequence CWU 1
1
12143PRTArtificial Sequenceisoforms of human A beta 43 1Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 40
242PRTArtificial Sequenceisoforms of human A beta 42 2Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 340PRTArtificial
Sequenceisoforms of human A beta 40 3Asp Ala Glu Phe Arg His Asp
Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala
Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met
Val Gly Gly Val Val 35 40 439PRTArtificial Sequenceisoforms of
human A beta 39 4Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val 35
538PRTArtificial Sequenceisoforms of human A beta 38 5Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu Met Val Gly Gly 35 637PRTArtificial Sequenceisoforms of
human A beta 37 6Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val
His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser
Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly 35
734PRTArtificial Sequenceisoforms of human A beta 34 7Asp Ala Glu
Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu
Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu 817PRTArtificial Sequenceisoforms of human A beta 17
8Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1
5 10 15 Leu 916PRTArtificial Sequenceisoforms of human A beta 16
9Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1
5 10 15 109PRTArtificial Sequenceisoforms of human A beta 38 10Cys
Ile Ile Gly Leu Met Val Gly Gly 1 5 119PRTArtificial
Sequenceisoforms of human A beta 40 11Cys Gly Leu Met Val Gly Gly
Val Val 1 5 129PRTArtificial Sequenceisoforms of human A beta 42
12Leu Met Val Gly Gly Val Val Ile Ala 1 5
* * * * *
References