U.S. patent application number 14/345390 was filed with the patent office on 2014-11-27 for methods for the diagnosis and treatment of neurological and neurodegenerative diseases, disorders and associated processes.
The applicant listed for this patent is C2N Diagnostics. Invention is credited to Andrew C. Paoletti, Tim West.
Application Number | 20140349308 14/345390 |
Document ID | / |
Family ID | 47914834 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140349308 |
Kind Code |
A1 |
West; Tim ; et al. |
November 27, 2014 |
Methods for the Diagnosis and Treatment of Neurological and
Neurodegenerative Diseases, Disorders and Associated Processes
Abstract
Methods for the diagnosis, prognosis, treatment and determining
the efficacy of a therapeutic regimen for neurological and
neurodegenerative diseases, disorders, and associated processes
include using a detectable label to measure levels of
alpha-synuclein.
Inventors: |
West; Tim; (Saint Louis,
MO) ; Paoletti; Andrew C.; (Saint Louis, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
C2N Diagnostics |
Saint Louis |
MO |
US |
|
|
Family ID: |
47914834 |
Appl. No.: |
14/345390 |
Filed: |
September 19, 2012 |
PCT Filed: |
September 19, 2012 |
PCT NO: |
PCT/US12/56144 |
371 Date: |
March 17, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61536300 |
Sep 19, 2011 |
|
|
|
Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61P 21/02 20180101;
G01N 2800/28 20130101; G01N 2800/2835 20130101; A61P 25/28
20180101; A61P 9/00 20180101; A61P 25/16 20180101; G01N 33/6896
20130101; G01N 2800/2871 20130101; G01N 2800/52 20130101; G01N
2333/47 20130101; A61P 25/00 20180101; G01N 2800/2821 20130101;
G01N 2500/04 20130101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method to determine prognosis, diagnosis or efficacy of a
therapeutic regimen in a subject comprising contacting a biological
sample from a subject with a detectable isotope to detect the level
of alpha-synuclein in the sample wherein the level is compared with
a reference standard level [to determine the prognosis, diagnosis
or efficacy of the therapeutic regimen.
2. A method to diagnose an alpha-synuclein related disease or
disorder comprising: (a) administration of a labeled moiety to a
subject suspected of having an alpha-synuclein related disease or
disorder; (b) collection of a biological sample from the subject
and a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample; wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of positive diagnosis of an alpha-synuclein related disease or
disorder; thereby diagnosing an alpha-synuclein related disease or
disorder.
3. A method to determine the prognosis of an alpha-synuclein
related disease or disorder comprising: (a) administration of a
labeled moiety to a subject suspected of having an alpha-synuclein
related disease or disorder; (b) collection of a biological sample
from the subject and a corresponding normal sample; (c) measurement
of labeled alpha-synuclein and unlabeled alpha-synuclein from the
subject and corresponding normal sample; (d) determination of the
ratio of labeled alpha-synuclein to unlabeled alpha-synuclein from
the subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample; wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of positive prognosis of an alpha-synuclein related disease or
disorder; thereby prognosing an alpha-synuclein related disease or
disorder.
4. A method to determine the efficacy of a therapeutic regimen to
treat an alpha-synuclein related disease or disorder comprising:
(a) administration of a labeled moiety and a therapeutic agent to a
subject having an alpha-synuclein related disease or disorder; (b)
collection of a biological sample from the subject and a
corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample; wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of efficacy of a therapeutic regimen of a therapeutic agent to
treat an alpha-synuclein related disease or disorder; thereby
determining the efficacy of a therapeutic regimen to treat an
alpha-synuclein related disease or disorder.
5. An in vivo method to identify a therapeutic agent to treat an
alpha-synuclein related disease or disorder comprising: (a)
administration of a labeled moiety and a therapeutic agent to a
subject suspected of having an alpha-synuclein related disease or
disorder; (b) collection of a biological sample from the subject
and a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample; wherein a change in alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of the identification of a therapeutic agent to treat an
alpha-synuclein related disease or disorder; thereby identifying a
therapeutic agent to treat an alpha-synuclein related disease or
disorder an alpha-synuclein related disease or disorder.
6. An in vitro method to identify a therapeutic agent to treat an
alpha-synuclein related disease or disorder comprising: (a)
administration of a labeled moiety and a therapeutic agent to
cells; (b) collection of alpha-synuclein from the cells and a
corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the cells and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the cells
and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the cells to the
alpha-synuclein metabolism from the corresponding normal sample;
wherein a change in alpha-synuclein metabolism of the cells
compared to the corresponding normal sample is indicative of the
identification of a therapeutic agent to treat an alpha-synuclein
related disease or disorder; thereby identifying a therapeutic
agent to treat an alpha-synuclein related disease or disorder an
alpha-synuclein related disease or disorder.
7. A method to predict subject response to a therapeutic agent to
treat an alpha-synuclein related disease or disorder comprising:
(a) administration of a labeled moiety and a therapeutic agent to a
subject suspected of having an alpha-synuclein related disease or
disorder (b) collection of a biological sample from the subject and
a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample; wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of the identification of a therapeutic agent to treat an
alpha-synuclein related disease or disorder; thereby diagnosing an
alpha-synuclein related disease or disorder.
8. The method of claims 2-7, wherein the labeled moiety is a
labeled amino acid or labeled water.
9. The method of claim 8, wherein the amino acid is labeled with a
radioisotope or a non-radio-labeled isotope.
10. The method of claim 9 wherein the amino acid is labeled with a
non-radio labeled isotope.
11. The method of claim 10, wherein the non-radio labeled isotope
is selected from the group consisting of: .sup.2H, .sup.13C,
.sup.15N, .sup.17 or .sup.18O and .sup.33, 34 or 36S.
12. The method of claim 8, wherein the amino acid is selected from
the group consisting of leucine, isoleucine and phenylalanine.
13. The method of claim 12, wherein the labeled amino acid is
selected from the group consisting of one or more of:
.sup.15N.sub.x labeled leucine, wherein x=1-6; .sup.13C.sub.x
labeled phenylalanine, wherein x=1-9 and .sup.13C.sub.x labeled
isoleucine, wherein x=1-6.
14. The method of claims 2-7, wherein the labeled moiety is a
labeled water.
15. The method of claim 14, wherein the labeled water is selected
from the group consisting of deuterated water or oxygen 18
water.
16. The method of claims 2-7, wherein the biological sample is
selected from the group consisting of a bodily fluid or a tissue
sample.
17. The method of claim 16, wherein the bodily fluid is selected
from the group consisting of: blood, plasma, blood serum, cerebral
spinal fluid (CSF), urine, saliva, perspiration and tears.
18. The method of claim 17, wherein the bodily fluid is CSF.
19. The method of claim 16, wherein the tissue sample is a CNS
sample.
20. The method of claim 19, wherein the CNS sample is selected from
the group consisting of: tissue from the CNS system, brain tissue,
the forebrain tissue, the interbrain tissue, the midbrain tissue,
the hindbrain tissue and the spinal cord tissue.
21. The method of claims 4-7, where in the therapeutic agent is
selected from the group consisting of small molecule inhibitors of
alpha-synuclein, antibodies against alpha-synuclein,
alpha-synuclein clearance activators, sirtuin 2 inhibitors,
proteomsome inhibitors, small molecule inhibitors of
alpha-synuclein polymerization, L-DOPA, cholesterylester transfer
protein (CEPT) inhibitors, metalloprotease inhibitors,
cholinesterase inhibitors, NMDA receptor antagonists, hormones,
neuroprotective agents and cell death inhibitors.
22. The method of claim 21, wherein the therapeutic agent is
L-DOPA.
23. A kit for determine prognosis, diagnosis or efficacy of a
therapeutic regimen in a subject having or suspected of having an
alpha-synuclein related disease or disorder.
24. The kit of claim 23, wherein the kit comprises one or more
labeled moieties and a means for administering the one or more
moieties to a subject.
25. The kit of claim 23, further comprising a means for obtaining a
biological sample and instructions for determining the ratio of
labeled to unlabeled alpha-synuclein.
Description
FIELD OF THE INVENTION
[0001] The invention generally relates to methods for the diagnosis
and treatment of neurological and neurodegenerative diseases,
disorders, and associated processes, and more specifically, to
levels of alpha synuclein and correlation with disease.
BACKGROUND INFORMATION
[0002] The protein alpha-synuclein has been implicated in
Parkinson's Disease (PD) through a variety of human and animal
studies. Recent studies have shown that the CSF concentration of
alpha-synuclein is significantly lower in patients with PD than in
control subjects, suggesting that the metabolism of alpha-synuclein
is altered in patients with PD. Like other protein misfolding
diseases, protein misfolding is concentration-dependent. Thus,
decreasing synthesis or increasing clearance of synuclein is a
potential way to develop treatments for PD and drug companies have
focused on the metabolism of this protein as a drug target.
[0003] The stable isotope labeling kinetic (SILK) assay relies on
the ability to detect metabolic incorporation of stable isotope
labeled amino acids into proteins and peptides. Stable isotopes add
a small amount of weight (2-100 Daltons) to peptides containing the
stable isotope and this additional weight can be measured by a mass
spectrometer. By measuring metabolic incorporation of stable
isotopes into proteins in the CSF at various times after
administration of a stable isotope, the SILK assay can be used to
measure production and clearance of proteins in the human central
nervous system.
[0004] The following describes the protocol for measuring the
metabolism of brain derived alpha-synuclein in a human subject. A
study participant is identified and enrolled in the study. On the
first day of the study the participant will have IV and lumbar
catheters placed and will be administered a stable isotope for a
pre-determined amount of time. Samples of plasma and CSF will be
drawn through the catheters at pre-determined times.
Alpha-synuclein will then be isolated from the biological samples
and the incorporation of the stable isotope into the protein will
be measured by a mass spectrometer. The change in labeled to
unlabeled alpha-synuclein over time will allow for calculation of
production and clearance rates for the protein.
[0005] Measuring alpha-synuclein metabolism can provide results to
inform about synthesis and clearance rates of alpha-synuclein in
normal as well as disease states, as well as provide a method to
directly determine the effects in humans of treatments which target
alpha-synuclein synthesis and clearance.
SUMMARY OF THE INVENTION
[0006] In one embodiment of the invention, we describe the methods
by which alpha-synuclein is isolated from biological samples by
immunoprecipitation using an antibody that recognizes
alpha-synuclein. In this embodiment, the isolated protein is eluted
from the antibody, for example by using formic acid and then
digested with trypsin or another protease. Incorporation of stable
isotopes into alpha-synuclein peptides is then analyzed on a mass
spectrometer and a ratio of labeled to unlabeled alpha-synuclein is
calculated.
[0007] In one embodiment, the present invention is a method to
determine prognosis, diagnosis or efficacy of a therapeutic regimen
in a subject comprising contacting a biological sample from a
subject with a detectable isotope to detect the level of
alpha-synuclein in the sample.
[0008] In an embodiment, the present invention is a method to
diagnose an alpha-synuclein related disease or disorder comprising:
(a) administration of a labeled moiety to a subject suspected of
having an alpha-synuclein related disease or disorder; (b)
collection of a biological sample from the subject and a
corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample, wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of positive diagnosis of an alpha-synuclein related disease or
disorder, thereby diagnosing an alpha-synuclein related disease or
disorder.
[0009] In another embodiment, the present invention is a method to
determine the prognosis an alpha-synuclein related disease or
disorder comprising: (a) administration of a labeled moiety to a
subject suspected of having an alpha-synuclein related disease or
disorder; (b) collection of a biological sample from the subject
and a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample, wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of positive prognosis of an alpha-synuclein related disease or
disorder, thereby prognosing an alpha-synuclein related disease or
disorder.
[0010] In an additional embodiment, the present invention is a
method to determine the efficacy of a therapeutic regimen to treat
an alpha-synuclein related disease or disorder comprising: (a)
administration of a labeled moiety and a therapeutic agent to a
subject suspected of having an alpha-synuclein related disease or
disorder; (b) collection of a biological sample from the subject
and a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; and (e) determination of
alpha-synuclein metabolism from the ratios of step (d); (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample, wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of efficacy of a therapeutic regimen of a therapeutic agent to
treat an alpha-synuclein related disease or disorder, thereby
determining the efficacy of a therapeutic regimen to treat an
alpha-synuclein related disease or disorder.
[0011] In an embodiment, the present invention is an in vivo method
to identify a therapeutic agent to treat an alpha-synuclein related
disease or disorder comprising: (a) administration of a labeled
moiety and a therapeutic agent to a subject suspected of having an
alpha-synuclein related disease or disorder; (b) collection of a
biological sample from the subject and a corresponding normal
sample; (c) measurement of labeled alpha-synuclein and unlabeled
alpha-synuclein from the subject and corresponding normal sample;
(d) determination of the ratio of labeled alpha-synuclein to
unlabeled alpha-synuclein from the subject and corresponding normal
sample; (e) determination of alpha-synuclein metabolism from the
ratios of step (d); and (f) comparison of the alpha-synuclein
metabolism from the subject to the alpha-synuclein metabolism from
the corresponding normal sample, wherein a change in
alpha-synuclein metabolism of the subject compared to the
corresponding normal sample is indicative of the identification of
a therapeutic agent to treat an alpha-synuclein related disease or
disorder, thereby identifying a therapeutic agent to treat an
alpha-synuclein related disease or disorder.
[0012] In one embodiment, the present invention is an in vitro
method to identify a therapeutic agent to treat an alpha-synuclein
related disease or disorder comprising: (a) administration of a
labeled moiety and a therapeutic agent to cells; (b) collection of
alpha-synuclein from the cells and a corresponding normal sample;
(c) measurement of labeled alpha-synuclein and unlabeled
alpha-synuclein from the cells and corresponding normal sample; (d)
determination of the ratio of labeled alpha-synuclein to unlabeled
alpha-synuclein from the cells and corresponding normal sample; (e)
determination of alpha-synuclein metabolism from the ratios of step
(d); and (f) comparison of the alpha-synuclein metabolism from the
cells to the alpha-synuclein metabolism from the corresponding
normal sample, wherein a change in alpha-synuclein metabolism of
the cells compared to the corresponding normal sample is indicative
of the identification of a therapeutic agent to treat an
alpha-synuclein related disease or disorder, thereby identifying a
therapeutic agent to treat an alpha-synuclein related disease or
disorder an alpha-synuclein related disease or disorder.
[0013] In one embodiment, the present invention is a method to
predict subject response to a therapeutic agent to treat an
alpha-synuclein related disease or disorder comprising: (a)
administration of a labeled moiety and a therapeutic agent to a
subject suspected of having an alpha-synuclein related disease or
disorder; (b) collection of a biological sample from the subject
and a corresponding normal sample; (c) measurement of labeled
alpha-synuclein and unlabeled alpha-synuclein from the subject and
corresponding normal sample; (d) determination of the ratio of
labeled alpha-synuclein to unlabeled alpha-synuclein from the
subject and corresponding normal sample; (e) determination of
alpha-synuclein metabolism from the ratios of step (d); and (f)
comparison of the alpha-synuclein metabolism from the subject to
the alpha-synuclein metabolism from the corresponding normal
sample, wherein a change in the alpha-synuclein metabolism of the
subject compared to the corresponding normal sample is indicative
of the identification of a therapeutic agent to treat an
alpha-synuclein related disease or disorder, thereby diagnosing an
alpha-synuclein related disease or disorder.
[0014] In one aspect, the labeled moiety is a labeled amino acid.
In a further aspect, the amino acid is labeled with a radioisotope
or a non-radio labeled isotope. In one aspect, the amino acid is
labeled with a non-radio labeled isotope. In an additional aspect,
the non-radio labeled isotope can be .sup.2H, .sup.13C, .sup.15N,
.sup.17 or .sup.18O and .sup.33, 34 or 36S. In a further aspect,
the amino acid can be leucine, isoleucine and phenylalanine.
Further, the labeled amino acid maybe one or more of .sup.15N.sub.x
labeled leucine, wherein x=1-6; .sup.13C.sub.x labeled
phenylalanine, wherein x=1-9 and .sup.13C.sub.x labeled isoleucine,
wherein x=1-6. In one aspect, the labeled moiety is labeled water.
In a further aspect, the labeled water is deuterated water
(.sup.2H.sub.2O), oxygen 18 water (H.sub.2.sup.18O) or other
similar molecules. In one aspect, the biological sample is a bodily
fluid or a tissue sample. In a further aspect, the bodily fluid can
be blood, plasma, blood serum, cerebral spinal fluid (CSF), urine,
saliva, perspiration and tears. In one aspect, the bodily fluid is
CSF. In an additional aspect, the tissue sample is a CNS sample. In
a further aspect, the CNS sample can be tissue from the CNS system,
brain tissue, the forebrain tissue, the interbrain tissue, the
midbrain tissue, the hindbrain tissue and the spinal cord
tissue.
[0015] In one aspect the therapeutic agent can be small molecule
inhibitors of alpha-synuclein, antibodies against alpha-synuclein,
alpha-synuclein clearance activators, sirtuin 2 inhibitors,
proteomsome inhibitors, small molecule inhibitors of
alpha-synuclein polymerization, L-DOPA, cholesterylester transfer
protein (CEPT) inhibitors, metalloprotease inhibitors,
cholinesterase inhibitors, NMDA receptor antagonists, hormones,
neuroprotective agents and cell death inhibitors. In one aspect,
the therapeutic agent is L-DOPA.
[0016] In one embodiment, the present invention is a kit for
determine prognosis, diagnosis or efficacy of a therapeutic regimen
in a subject having or suspected of having an alpha-synuclein
related disease or disorder. In one aspect, the kit comprises one
or more labeled moieties and a means for administering the one or
more moieties to a subject. In an additional aspect, the kit
further comprises a means for obtaining a biological sample and
instructions for determining the ratio of labeled to unlabeled
alpha-synuclein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the amino acid sequence of alpha-synuclein (SEQ
ID NO:1) showing the tryptic cleavage sites.
[0018] FIG. 2 shows a multiple sequence comparison (SEQ ID NOs:1-3)
showing differences in the amino acid sequence of alpha-, beta- and
gamma-synuclein.
[0019] FIG. 3 shows tryptic peptides originating from
alpha-synuclein (SEQ ID NOs:4-13) observed on the mass
spectrometer. We have analyzed alpha-synuclein isolated from
biological sources such as cerebrospinal fluid, conditioned cell
culture media, and cell lysates as well as recombinant
alpha-synuclein.
[0020] FIG. 4 shows the mass spectrum of alpha-synuclein 81-96
peptide (SEQ ID NO:13). Contains essential amino acid phenylalanine
(F). The presence of a phenylalanine in the 81-96 tryptic peptide
makes this peptide a peptide that can be used to monitor metabolism
of alpha-synuclein by administration of stable isotope labeled
phenylalanine.
[0021] FIG. 5 shows the mass spectrum of alpha-synuclein 33-43
peptide (SEQ ID NO:6). Contains essential amino acid leucine (L).
The presence of a leucine in the 33-43 tryptic peptide makes this
peptide a peptide that can be used to monitor metabolism of
alpha-synuclein by administration of stable isotope labeled
leucine.
[0022] FIG. 6 shows the mass spectrum of alpha-synuclein 35-43
peptide (SEQ ID NO:7). Contains essential amino acid leucine (L).
The presence of a leucine in the 35-43 tryptic peptide makes this
peptide a peptide that can be used to monitor metabolism of
alpha-synuclein by administration of stable isotope labeled
leucine.
[0023] FIG. 7 shows alpha-synuclein standard curves derived from
samples taken 3, 5 and 7 days after administration of the labeled
moiety.
[0024] FIG. 8 shows the relative intensity of alpha-synuclein ions
derived from samples taken 3, 5 and 7 days after administration of
the labeled moiety.
[0025] FIG. 9 shows the relative ion intensity of alpha-synuclein
measured in labeled and unlabeled samples.
[0026] FIG. 10 shows the levels of amyloid beta protein and
alpha-synuclein detected by the SILK assay over 36 hours post
administration of the labeled moiety.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is based, in part, on the discovery
that stable isotope labeling of biomolecules leads to small
differences in molecular weight of the biomolecules, but does not
alter the physical or chemical properties of the biomolecules.
Using the techniques provided herein, analysis of biomolecules can
be used to diagnose and/or treat a subject having or at risk of
developing a neurological or neurodegenerative disorder.
Accordingly, the present invention provides methods and kits useful
for measuring the metabolism of alpha-synuclein in a subject.
[0028] The invention also provides a method to assess whether a
therapeutic agent affects the production or clearance rate of
alpha-synuclein in the subject. Accordingly, the method may be used
to determine the optimal doses and/or optimal dosing regimens of
the therapeutic agent. Additionally, the method may be used to
determine which subjects respond better to a particular therapeutic
agent. For example, subjects with increased production of
alpha-synuclein may respond better to one therapeutic agent,
whereas subjects with decreased clearance of alpha-synuclein may
respond better to another therapeutic agent. Alternatively,
subjects with one particular genotype may respond better to a
particular therapeutic agent than those with a different genotype.
Finally, by allowing isoform specific quantitation, the method may
be used to determine whether a therapeutic agent can modulate the
production of an alpha-synuclein by switching production of one
isoform to another isoform.
[0029] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described.
[0031] The term "subject" as used herein refers to any individual
or patient to which the subject methods are performed. Generally
the subject is human, although as will be appreciated by those in
the art, the subject may be an animal. Thus other animals,
including mammals such as rodents (including mice, rats, hamsters
and guinea pigs), cats, dogs, rabbits, farm animals including cows,
horses, goats, sheep, pigs, etc., and primates (including monkeys,
chimpanzees, orangutans and gorillas) are included within the
definition of subject. In addition, the term "subject" may refer to
a culture of cells, where the methods of the invention are
performed in vitro to assess, for example, efficacy of a
therapeutic agent.
[0032] As used herein, the terms "sample" and "biological sample"
refer to any sample suitable for the methods provided by the
present invention. A sample of cells used in the present method can
be obtained from tissue samples or bodily fluid from a subject, or
tissue obtained by a biopsy procedure (e.g., a needle biopsy) or a
surgical procedure. In certain embodiments, the biological sample
of the present invention is a sample of bodily fluid, e.g.,
cerebral spinal fluid (CSF), blood, plasma, urine, saliva, and
tears.
[0033] The term "antibody" as used in this invention is meant to
include intact molecules of polyclonal or monoclonal antibodies, as
well as fragments thereof, such as Fab and F(ab').sub.2, Fv and SCA
fragments which are capable of binding an epitopic determinant. The
term "specifically binds" or "specifically interacts," when used in
reference to an antibody means that an interaction of the antibody
and a particular epitope has a dissociation constant of at least
about 1.times.10.sup.-6, generally at least about
1.times.10.sup.-7, usually at least about 1.times.10.sup.-8, and
particularly at least about 1.times.10.sup.-9 or 1.times.10-10 or
less.
[0034] As used herein the term "alpha-synuclein related disease or
disorder" refers to any disease or disorder in which circulating
levels or production of alpha-synuclein or alpha-synuclein
metabolism is changed from normal. This change can be an increase
or decrease in alpha-synuclein levels or metabolism compared to
normal.
[0035] As disclosed herein, stable isotope labeling of
alpha-synuclein leads to small differences in molecular weight of
alpha-synuclein, but does not alter the general physical or
chemical properties of alpha-synuclein. Thus, alpha-synuclein will
bind to antibodies and elute off a liquid chromatography column in
an identical fashion. Only sensitive instruments, such as mass
spectrometers, provide the ability to measure the small differences
in weight between labeled and unlabeled alpha-synuclein.
[0036] Several different moieties may be used to label
alpha-synuclein. Generally speaking, the two types of labeling
moieties utilized in the method of the invention are radioactive
isotopes and non-radioactive (stable) isotopes. In one embodiment,
non-radioactive isotopes may be used and measured by mass
spectrometry. Preferred stable isotopes include deuterium
(.sup.2H), .sup.13C, .sup.15N, .sup.17 or 18O and .sup.33, 34, or
36S, but it is recognized that a number of other stable isotopes
that change the mass of an atom by more or less neutrons than is
seen in the prevalent native form would also be effective. A
suitable label generally will change the mass of alpha-synuclein
such that it can be detected in a mass spectrometer. Alternatively,
a radioactive isotope may be used, and the labeled alpha-synuclein
may be measured with a scintillation counter (or via nuclear
scintigraphy) as well as by a mass spectrometer. One or more
labeled moieties may be used simultaneously or in sequence.
[0037] Thus, in one embodiment, when the method is employed to
measure the metabolism of alpha-synuclein, the labeled moiety
typically will be an amino acid. Those of skill in the art will
appreciate that several amino acids may be used to provide the
label of alpha-synuclein. Generally, the choice of amino acid is
based on a variety of factors such as: (1) The amino acid generally
is present in at least one residue of alpha-synuclein. (2) The
amino acid is generally able to quickly reach the site of protein
production and rapidly equilibrate across the blood-brain barrier
or other tissue or cellular barriers. (3) The amino acid label
generally does not influence the metabolism of the protein of
interest (e.g., very large doses of leucine may affect muscle
metabolism). And (4) availability of the desired amino acid (i.e.,
some amino acids are much more expensive or harder to manufacture
than others).
[0038] In one embodiment, the amino acid is an essential amino acid
(not produced by the body), so that a higher percent of labeling
may be achieved. In another embodiment, the amino acid is a
non-essential amino acid. Exemplary amino acids include, but are
not limited to, leucine, isoleucine, and phenylalanine. As such, in
one embodiment, the labeled amino acid is one or more of a
.sup.15N-labeled amino acid, a .sup.13C.sub.x-labeled
phenylalanine, where x=1 to 9, a .sup.13C.sub.x-labeled isoleucine,
where x=1 to 6. For example, .sup.13C.sub.6-phenylalanine, which
contains six .sup.13C atoms, may be used to label alpha-synuclein.
In another embodiment, .sup.13C.sub.6-leucine may be used to label
alpha-synuclein.
[0039] There are numerous commercial sources of labeled amino
acids, both non-radioactive isotopes and radioactive isotopes.
Generally, the labeled amino acids may be produced either
biologically or synthetically. Biologically produced amino acids
may be obtained from an organism (e.g., kelp/seaweed) grown in an
enriched mixture of .sup.13C, .sup.15N, or another isotope that is
incorporated into amino acids as the organism produces proteins.
The amino acids are then separated and purified. Alternatively,
amino acids may be made with known synthetic chemical
processes.
[0040] In one embodiment, when the method is employed to measure
the metabolism of alpha-synuclein, the labeled moiety typically
will be labeled water. In one aspect the labeled water is
deuterated water (.sup.2H.sub.2O). In another aspect, the labeled
water is oxygen 18 water (H.sub.2.sup.18O). In a further aspect,
the labeled water is a molecule similar to deuterated water or
oxygen 18 water.
[0041] The labeled moiety (e.g., labeled amino acid) may be
administered to a subject by several methods. Suitable routes of
administration include intravenously, intra-arterially,
subcutaneously, intraperitoneally, intramuscularly, or orally. In
one embodiment, the labeled moiety may be administered by
intravenous infusion. In another embodiment, the labeled moiety may
be orally ingested.
[0042] The labeled moiety may be administered slowly over a period
of time, as a large single dose depending upon the type of analysis
chosen (e.g., steady state or bolus/chase), or slowly over a period
of time after an initial bolus dose. To achieve steady-state levels
of the labeled alpha-synuclein, the labeling time generally should
be of sufficient duration so that the labeled alpha-synuclein may
be reliably quantified. In one embodiment, the labeled moiety is
administered as a single oral dose. In another embodiment, the
labeled moiety is administered for a period of time ranging from
about one hour to about 36 hours. In another embodiment, the
labeled moiety is administered for a period of time ranging from
about 6 hours to about 12 hours. In yet another embodiment, the
labeled moiety is administered for a period of time ranging from
about 9 hours to about 12 hours. In yet another embodiment, the
labeled moiety is administered for a period of time ranging from
about 9 hours to about 24 hours. The rate of administration of the
labeled moiety may range from about 0.5 mg/kg/hr to about 5
mg/kg/hr. In one embodiment, the rate of administration of labeled
leucine is from about 1 mg/kg/hr to about 3 mg/kg/hr. In another
embodiment, the rate of administration of labeled leucine is from
1.8 mg/kg/hr to about 2.5 mg/kg/hr. In another embodiment, the
labeled leucine may be administered as a bolus of between about 50
and about 500 mg/kg body weight of the subject, between about 50
and about 300 mg/kg body weight of the subject, or between about
100 and about 300 mg/kg body weight of the subject. In yet another
embodiment, the labeled leucine may be administered as a bolus of
about 200 mg/kg body weight of the subject. In an alternate
embodiment, the labeled leucine may be administered intravenously
as detailed above after an initial bolus of between about 0.5 to
about 10 mg/kg, between about 1 to about 4 mg/kg, or about 2 mg/kg
body weight of the subject. In another embodiment the water will be
administered daily over 1-7 days.
[0043] Those of skill in the art will appreciate that the amount
(or dose) of the labeled moiety can and will vary. Generally, the
amount is dependent on (and estimated by) the following factors:
(1) The type of analysis desired. For example, to achieve a steady
state of about 15% labeled leucine in plasma requires about 2
mg/kg/hr over about 9 hr after an initial bolus of 3 mg/kg over 10
min. In contrast, if no steady state is required, a large bolus of
labeled moiety (e.g., 1 or 5 grams of labeled leucine) may be given
initially. (2) The rate of metabolism of alpha-synuclein. For
example, if alpha-synuclein is being produced rapidly, then less
labeling time may be needed and less label may be needed--perhaps
as little as 0.5 mg/kg over 1 hour. However, most proteins have
half-lives of hours to days and, so more likely, a continuous
infusion for 9, 12 or 24 hours may be used at 0.5 mg/kg to 4 mg/kg.
And (3) the sensitivity of detection of the label. For example, as
the sensitivity of label detection increases, the amount of label
that is needed may decrease.
[0044] It should be understood that more than one labeled moiety
may be used in a single subject. This would allow multiple labeling
of alpha-synuclein and may provide information on the production or
clearance of alpha-synuclein at different times. For example, a
first label may be given to subject over an initial time period,
followed by a pharmacologic agent (drug), and then a second label
may be administered. In general, analysis of the samples obtained
from the subject would provide a measurement of metabolism of
alpha-synuclein before AND after drug administration, directly
measuring the pharmacodynamic effect of the drug in the same
subject. Alternatively, multiple labels may be used at the same
time to increase labeling of alpha-synuclein.
[0045] The method of the invention provides that a sample be
obtained from the subject such that the metabolism of
alpha-synuclein can be determined. In one embodiment, the sample is
a body fluid. Suitable body fluids include, but are not limited to,
cerebral spinal fluid (CSF), blood plasma, blood serum, urine,
saliva, perspiration, and tears. In another embodiment, the sample
is a tissue sample, such as a sample of tissue from the central
nervous system (CNS). The sample generally will be collected using
standard procedures well known to those of skill in the art.
[0046] In one embodiment, the sample is a CNS sample, which
includes, but is not limited to, tissue from the central nervous
system, which comprises brain tissue and spinal cord tissue. In one
embodiment of the invention, the CNS sample may be taken from brain
tissue, including, but not limited to, tissue from the forebrain
(e.g., cerebral cortex, basal ganglia, hippocampus), the interbrain
(e.g., thalamus, hypothalamus, subthalamus), the midbrain (e.g.,
tectum, tegmentum), or the hindbrain (e.g., pons, cerebellum,
medulla oblongata). In another embodiment, the CNS sample may be
collected from spinal cord tissue. In still other embodiments, CNS
samples from more than one CNS region may be taken. Accordingly,
the metabolism of alpha-synuclein may be measured in different CNS
samples, e.g., in the cortex and the hippocampus,
simultaneously.
[0047] CNS samples may be obtained by known techniques. For
instance, brain tissue or spinal cord tissue may be obtained via
dissection or resection. Alternatively, CNS samples may be obtained
using laser microdissection. The subject may or may not have to be
sacrificed to obtain the sample, depending on the CNS sample
desired and the subject utilized.
[0048] In one embodiment, the sample is obtained from the subject
at a single predetermined time point, for example, within an hour
of labeling. In general, for proteins with fast metabolism, samples
obtained during the first 12-18 hours after the start of
administration of the labeled moiety may be used to determine the
rate of production of alpha-synuclein, and samples taken during
24-36 hrs after the start of administration of the labeled moiety
may be used to determine the clearance rate of alpha-synuclein. In
general, for proteins with slow metabolism, samples obtained during
the first 1-4 days after the start of administration of the labeled
moiety may be used to determine the rate of production of
alpha-synuclein, and samples taken during 4-14 days after the start
of administration of the labeled moiety may be used to determine
the clearance rate of alpha-synuclein. In another embodiment, the
sample is obtained from the subject hourly from 0 to 12 hours, 0 to
24 hours, or 0 to 36 hours. In yet another embodiment, samples may
be taken from an hour to days or even weeks apart depending upon
the production and clearance rates of alpha-synuclein.
[0049] Those of skill in the art will appreciate that the labeled
moiety should be administered in a timely fashion which will allow
observation of incorporation of the labeled moiety into
alpha-synuclein. The labeling of alpha-synuclein will take place as
the protein is synthesized inside the cell. But the incorporation
of the label into alpha-synuclein is only measured once the protein
has exited the cell and entered the cerebrospinal fluid or the
blood stream. If the protein undergoes complex processing in order
to exit the cell, the time from synthesis until appearance in the
bodily fluid could be significant. Thus if it takes 24-48 hours for
alpha-synuclein to show up in CSF, administration of label may have
to occur 24-48 hours before the start of CSF sampling.
Alternatively, if it takes 48-72 hours for alpha-synuclein to show
up in CSF, administration of label may have to occur 48-72 hours
before the start of CSF sampling. Further, if it takes 3 days to
one week for alpha-synuclein to show up in CSF, administration of
label may have to occur 3 days to one week before the start of CSF
sampling.
[0050] It should be understood that if samples at different
time-points are desired, more than one subject may be used. For
instance, one subject may be used for a baseline sample, another
subject for a time-point of one hour post administration of the
labeled moiety, another subject for a time-point six hours post
administration of the labeled moiety.
[0051] Accordingly, the present invention provides that detection
of the amount of labeled alpha-synuclein and the amount of
unlabeled alpha-synuclein in the sample may be used to determine
the ratio of labeled alpha-synuclein to unlabeled alpha-synuclein,
which in turn, may be used to estimate the production and clearance
rates of alpha-synuclein in the subject. Exemplary means for
detecting differences in mass between the labeled and unlabeled
alpha-synuclein include, but are not limited to, liquid
chromatography mass spectrometry, gas chromatography mass
spectrometry, MALDI-TOF mass spectrometry, and tandem mass
spectrometry.
[0052] However, prior to detecting the ratio of labeled
alpha-synuclein to unlabeled alpha-synuclein, it may be desirable
to isolate and/or separate alpha-synuclein from other biomolecules
in the sample. Thus, in one embodiment, immunoprecipitation may be
used to isolate and purify alpha-synuclein before it is analyzed.
In another embodiment, alpha-synuclein may be isolated or purified
by affinity chromatography or immunoaffinity chromatography.
Alternatively, mass spectrometers having chromatography setups may
be used to separate biomolecules without immunoprecipitation, and
then alpha-synuclein may be measured directly. In an exemplary
embodiment, alpha-synuclein may be immunoprecipitated and then
analyzed by a liquid chromatography system interfaced with a tandem
MS unit equipped with an electrospray ionization source
(LC-ESI-tandem MS).
[0053] In another aspect, the invention provides that the
metabolism of multiple biomolecules in the same sample may be
measured simultaneously. That is, both the amount of unlabeled and
labeled biomolecule may be detected and measured separately or at
the same time for multiple biomolecules. As such, the invention
provides a useful method for screening changes in production and
clearance of one or more biomolecules on a large scale (i.e.,
proteomics/metabolomics) and provides a sensitive means to detect
and measure biomolecules involved in the underlying
pathophysiology. In one aspect, the invention also provides a means
to measure multiple types of biomolecules. In this context, for
example, a protein and a lipid may be measured simultaneously or
sequentially. For example, both alpha-synuclein and A.beta. could
be isolated from a CSF sample and the production and clearance of
the two individual proteins be determined in the same subject.
[0054] Once the amount of labeled and unlabeled alpha-synuclein has
been detected in a sample, the ratio or percent of labeled
alpha-synuclein to unlabeled alpha-synuclein may be determined by
dividing the amount of labeled alpha-synuclein with the amount of
unlabeled alpha-synuclein. If a mass spectrometer is used for
detection of alpha-synuclein, the ratio would be calculated by
dividing the ion intensity of labeled alpha-synuclein with the ion
intensity of unlabeled alpha-synuclein.
[0055] The invention allows measurement of the labeled and
unlabeled protein at the same time, so that the ratio of labeled to
unlabeled protein, as well as other calculations, may be made. As
measurements of labeling ratios are combined over different
sampling times after infusion of the stable isotope, the data can
be combined to form a metabolic profile. Those of skill in the art
will be familiar with the first order kinetic models of labeling
that may be used with the method of the invention. For example, the
fractional synthesis rate (FSR) may be calculated. The FSR equals
the initial rate of increase of labeled to unlabeled protein
divided by the precursor enrichment. Likewise, the fractional
clearance rate (FCR) may be calculated. In addition, other
parameters, such as fractional turnover rate (FTR), lag time, and
isotopic tracer steady state, may be determined and used as
measurements of the protein's metabolism and physiology. Also,
modeling may be performed on the data to fit multiple compartment
models to estimate transfer between compartments. Of course, the
type of mathematical modeling chosen will depend on the individual
synthesis and clearance parameters (e.g., one-pool, multiple pools,
steady state, non-steady-state, compartmental modeling, etc.). As
used herein, "steady state" refers to a state during which there is
insignificant change in the measured parameter over a specified
period of time.
[0056] Stable isotope kinetic labeling (SILK) methodology has been
shown to detect metabolic incorporation of stable (non-radioactive)
isotopes into newly synthesized proteins in the cerebrospinal fluid
of living subject. For detailed information regarding SILK, see
U.S. Pub. Nos. 2008/0145941 and 2009/0142766, and International PCT
Pub. No. WO 2006/107814, the entire content of each of which is
incorporated herein by reference). SILK makes it possible to
measure the production and clearance rates of proteins in the
central nervous system. Thus far, this methodology has been applied
to measuring the production and clearance of the amyloid beta
protein (A.beta.) implicated in Alzheimer's disease (AD).
[0057] However, until now, the current version of the SILK assay
measures only the metabolism of A.beta.. In the data demonstrated
here we show that the SILK method can also be applied to
alpha-synuclein. This assay is distinct in the use of an antibody
that specifically binds to alpha-synuclein for isolation of
alpha-synuclein from the biological fluid and in the selection of
alpha-synuclein specific peptides for monitoring of stable isotope
incorporation into alpha-synuclein.
[0058] Accordingly, the production of protein is typically based
upon the rate of increase of the labeled/unlabeled protein ratio
over time (i.e., the slope, the exponential fit curve, or a
compartmental model fit defines the rate of protein production).
For these calculations, a minimum of one sample is typically
required (one could estimate the baseline label), two are
preferred, and multiple samples are more preferred to calculate an
accurate curve of the uptake of the label into the protein (i.e.,
the production rate). If multiple samples are used or preferred,
the samples need not be taken from the same subject. For instance,
proteins may be labeled in five different subjects at time point
zero, and then a single sample taken from each subject at a
different time point post-labeling.
[0059] Conversely, after the administration of labeled amino acid
is terminated, the rate of decrease of the ratio of labeled to
unlabeled protein typically reflects the clearance rate of that
protein. For these calculations, a minimum of one sample is
typically required (one could estimate the baseline label), two are
preferred, and multiple samples are more preferred to calculate an
accurate curve of the decrease of the label from the protein over
time (i.e., the clearance rate). If multiple samples are used or
preferred, the samples need not be taken from the same subject. For
instance, proteins may be labeled in five different subjects at
time point zero, and then a single sample taken from each subject
at a different time point post-labeling. The amount of labeled
protein in a CNS sample at a given time reflects the production
rate or the clearance rate (i.e., removal or destruction) and is
usually expressed as percent per hour or the mass/time (e.g.,
mg/hr) of the protein in the subject.
[0060] The method of the invention may be used to diagnose or
monitor the progression of a neurological or neurodegenerative
disease by measuring the in vivo metabolism of alpha-synuclein in a
subject. Additionally, the methods of the invention may be used to
monitor the treatment of a neurological or neurodegenerative
disease by measuring the in vivo metabolism of alpha-synuclein in a
subject. The metabolism of alpha-synuclein may be linked to a
neurological or neurodegenerative disease such that any increase or
decrease may be indicative of the presence or progression of the
disease. Thus, the metabolism of alpha-synuclein may be compared to
the metabolism of alpha-synuclein in a corresponding normal sample,
to the metabolism of alpha-synuclein in a subject of known
neurological or neurodegenerative disease state, to the metabolism
of alpha-synuclein from the same subject determined at an earlier
time, or any combination thereof.
[0061] In addition, such methods may help identify an individual as
having a predisposition for the development of the disease, or may
provide a means for detecting the disease prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this
type may allow health professionals to employ preventative measures
or aggressive treatment earlier thereby preventing the development
or further progression of the disease.
[0062] As used herein a "corresponding normal sample" refers to a
sample from the same organ and/or of the same type as the sample
being examined. In one aspect, the corresponding normal sample
comprises a sample of cells obtained from a healthy individual.
Such a corresponding normal sample can, but need not be, from an
individual that is age-matched and/or of the same sex as the
individual providing the sample being examined. In another aspect,
the corresponding normal sample comprises a sample of cells
obtained from an otherwise healthy portion of tissue of the subject
from which the sample being tested is obtained.
[0063] Reference to the metabolism of alpha-synuclein in a subject
of known neurological or neurodegenerative disease state includes a
predetermined metabolism of alpha-synuclein linked to a
neurological or neurodegenerative disease. Thus, the metabolism may
be compared to a known metabolism of alpha-synuclein obtained from
a sample of a single individual or may be from an established cell
line of the same type as that of the subject. In one aspect, the
established cell line can be one of a panel of such cell lines,
wherein the panel can include different cell lines of the same type
of disease and/or different cell lines of different diseases
associated with alpha-synuclein. Such a panel of cell lines can be
useful, for example, to practice the present method when only a
small number of cells can be obtained from the subject to be
treated, thus providing a surrogate sample of the subject's cells,
and also can be useful to include as control samples in practicing
the present methods.
[0064] Exemplary neurological or neurodegenerative diseases that
may be linked to the metabolism of alpha-synuclein include, but are
not limited to, Alzheimer's Disease, Parkinson's Disease, stroke,
frontal temporal dementias (FTDs), aging-related disorders and
dementias, Lewy Body Disease, Traumatic Brain Injury (TBI), and
Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease). It is
also envisioned that the method of the invention may be used to
study the normal physiology, metabolism, and function of the
CNS.
[0065] In another aspect, the present invention provides a method
for assessing whether a therapeutic agent used to treat a
neurological or neurodegenerative disease affects the metabolism of
alpha-synuclein in the subject. For example, the metabolism of
alpha-synuclein may be measured to determine if a given therapeutic
agent results in an increase, or a decrease in the production or
clearance of alpha-synuclein. In one embodiment, the method is
performed in vivo, as herein described. In another embodiment, the
method is performed in vitro utilizing a culture of cells, where
the culture of cells is the "subject" in the methods described
herein. Accordingly, use of the methods provided herein will allow
those of skill in the art to accurately determine the degree of
change in the metabolism of alpha-synuclein, and correlate these
measurements with the clinical outcome of the disease modifying
treatment. Results from this aspect of the invention, therefore,
may help determine the optimal doses and frequency of doses of a
therapeutic agent, may assist in the decision-making regarding the
design of clinical trials, and may ultimately accelerate validation
of effective therapeutic agents for the treatment of neurological
or neurodegenerative diseases.
[0066] Thus, the method of the invention may be used to predict
which subjects will respond to a particular therapeutic agent. For
example, subjects with increased metabolism of alpha-synuclein may
respond to a particular therapeutic agent differently than subjects
with decreased metabolism of alpha-synuclein. In particular,
results from the method may be used to select the appropriate
treatment (e.g., an agent that blocks the production of
alpha-synuclein or an agent that increases the clearance
alpha-synuclein) for a particular subject. Similarly, results from
the method may be used to select the appropriate treatment for a
subject having a particular genotype.
[0067] The method for predicting which subjects will respond to a
particular therapeutic agent include administering a therapeutic
agent and a labeled moiety to the subject, wherein the labeled
moiety is incorporated into alpha-synuclein as it is produced in
the subject. In one embodiment, the therapeutic agent may be
administered to the subject prior to the administration of the
labeled moiety. In another embodiment, the labeled moiety may be
administered to the subject prior to the administration of the
therapeutic agent. The period of time between the administration of
each may be several minutes, an hour, several hours, or many hours.
In still another embodiment, the therapeutic agent and the labeled
moiety may be administered simultaneously. The method further
includes collecting at least one biological sample, which includes
labeled and unlabeled alpha-synuclein, determining a ratio of the
labeled alpha-synuclein and unlabeled alpha-synuclein in the
sample, and calculating the metabolism of alpha-synuclein in the
subject. Thereafter, a comparison of the calculated metabolism to a
control value will determine whether the therapeutic agent alters
the metabolism (e.g., by altering the rate of production or the
rate of clearance) of alpha-synuclein in the subject.
[0068] Those of skill in the art will appreciate that the
therapeutic agent can and will vary depending upon the neurological
or neurodegenerative disease or disorder to be treated.
Non-limiting examples of suitable therapeutic agents include small
molecule inhibitors of alpha-synuclein production, humanized
antibodies against alpha-synuclein, alpha-synuclein CNS clearance
activators, sirtuin 2 inhibitors, proteosome inhibitors, small
molecule inhibitors of alpha-synuclein polymerization.
[0069] Other suitable AD therapeutic agents include
cholesterylester transfer protein (CETP) inhibitors,
metalloprotease inhibitors, cholinesterase inhibitors, NMDA
receptor antagonists, hormones, neuroprotective agents, A.beta.
production inhibitors such as inhibitors and modulators of gamma
and beta secretases, anti-A.beta. antibodies, anti-Tau antibodies,
and cell death inhibitors. Many of the above mentioned therapeutic
agents may also affect the in vivo metabolism of other proteins
implicated in neurodegenerative disorders.
[0070] The therapeutic agent may be administered to the subject in
accord with known methods. Typically, the therapeutic agent will be
administered orally, but other routes of administration such as
parenteral or topical may also be used. The amount of therapeutic
agent that is administered to the subject can and will vary
depending upon the type of agent, the subject, and the particular
mode of administration. Those skilled in the art will appreciate
that dosages may be determined with guidance from Goodman &
Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition
(2001), Appendix II, pp. 475-493, and the Physicians' Desk
Reference.
[0071] In another aspect, the invention provides a kit for
performing the methods of the invention. In one embodiment, a kit
is provided for diagnosing and/or monitoring the progression or
treatment of a neurological or neurodegenerative disease in a
subject. The kit includes one or more labeled moieties (e.g.,
labeled amino acids) and a means for administering the one or more
amino acids to the subject. The kit may further include a means for
obtaining a biological sample at regular time intervals from the
subject. In certain embodiments, the kit will also include
instructions for detecting and determining the ratio of labeled to
unlabeled alpha-synuclein over time and for calculating the
metabolism of alpha-synuclein. In one embodiment, the instructions
will disclose methods for comparing the calculated concentration to
certain standards and/or controls as disclosed herein.
[0072] In another embodiment, the kit of the invention provides a
compartmentalized carrier including one or more containers
containing the labeled moiety and the various means for performing
the methods of the invention.
[0073] In this embodiment of the invention, we demonstrate the
feasibility of a stable isotope labeling kinetics (SILK)
alpha-synuclein assay. FIG. 1 shows the tryptic cleavage sites in
the alpha-synuclein sequence and thus a list of possible tryptic
peptides originating from alpha-synuclein. Other proteases can be
used instead of trypsin and will yield different cleavage patterns
and different peptides. When digesting recombinant alpha-synuclein
or alpha-synuclein isolated from biological sources we observed
several tryptic peptides originating from alpha-synuclein. FIG. 3
shows a list of peptides that we have observed either from
recombinant alpha-synuclein or from alpha-synuclein that has been
isolated from CSF or other biological sources.
[0074] The following examples are provided to further illustrate
the advantages and features of the present invention, but are not
intended to limit the scope of the invention. While they are
typical of those that might be used, other procedures,
methodologies, or techniques known to those skilled in the art may
alternatively be used.
EXAMPLES
[0075] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
Measurement of Incorporation of .sup.13C.sub.6 Leucine into
Alpha-Synuclein Produced by Cells
[0076] Tissue culture cells (SH-SY5Y) were stably transfected with
a construct that leads to over-expression of alpha-synuclein. The
cells were grown in media containing known ratios of .sup.13C.sub.6
to .sup.12C.sub.6 labeled leucine (tracer to tracee ratio; TTR)
(TTR=0.00, 0.0127, 0.0256, 0.0526, 0.111, 0.25). Media was
collected after 3, 5, and 7 days of growing in the labeled media.
Alpha-synuclein was isolated from media samples by
immunoprecipitation using C2N-ASMAB3. Isolated proteins were
digested with trypsin and analyzed on a TSQ-Vantage triple
quadruple mass spectrometer setup to monitor the labeled and
unlabeled alpha-synuclein 35-43 peptide. The ratio of labeled to
unlabeled alpha-synuclein was calculated for each sample. The
approximate concentration of alpha-synuclein as measured by the
intensity of the labeled and unlabeled alpha-synuclein increased
from the initial collection to the subsequent collections as the
cells grew more confluent. In addition, the ratio of labeled to
unlabeled alpha-synuclein more closely matched the expected
concentrations as the cells turned over the originally unlabeled
alpha-synuclein and produced new alpha-synuclein using the mixture
of labeled and unlabeled amino acids.
Example 2
Measurement of Incorporation of .sup.13C.sub.6 Leucine into
Alpha-Synuclein Produced by Humans
[0077] A human volunteer was administered .sup.13C.sub.6 leucine
for 9 hours and had CSF samples taken every hour for 36 hours
starting at the time of leucine infusion. Alpha-synuclein was
isolated from the CSF samples as well as samples from one
.sup.13C.sub.6 leucine alpha-synuclein standard curve by
immunoprecipitation and digested with trypsin. Incorporation of
.sup.13C.sub.6 leucine into the 35-43 peptide was analyzed for each
sample by mass spectrometer.
[0078] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
131140PRTHomo sapiens 1Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala
Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr Lys Gln Gly
Val Ala Glu Ala Ala Gly Lys 20 25 30 Thr Lys Glu Gly Val Leu Tyr
Val Gly Ser Lys Thr Lys Glu Gly Val 35 40 45 Val His Gly Val Ala
Thr Val Ala Glu Lys Thr Lys Glu Gln Val Thr 50 55 60 Asn Val Gly
Gly Ala Val Val Thr Gly Val Thr Ala Val Ala Gln Lys 65 70 75 80 Thr
Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 85 90
95 Lys Asp Gln Leu Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu Gly Ile
100 105 110 Leu Glu Asp Met Pro Val Asp Pro Asp Asn Glu Ala Tyr Glu
Met Pro 115 120 125 Ser Glu Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala
130 135 140 2134PRTHomo sapiens 2Met Asp Val Phe Met Lys Gly Leu
Ser Met Ala Lys Glu Gly Val Val 1 5 10 15 Ala Ala Ala Glu Lys Thr
Lys Gln Gly Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly
Val Leu Tyr Val Gly Ser Lys Thr Arg Glu Gly Val 35 40 45 Val Gln
Gly Val Ala Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Ser 50 55 60
His Leu Gly Gly Ala Val Phe Ser Gly Ala Gly Asn Ile Ala Ala Ala 65
70 75 80 Thr Gly Leu Val Lys Arg Glu Glu Phe Pro Thr Asp Leu Lys
Pro Glu 85 90 95 Glu Val Ala Gln Glu Ala Ala Glu Glu Pro Leu Ile
Glu Pro Leu Met 100 105 110 Glu Pro Glu Gly Glu Ser Tyr Glu Asp Pro
Pro Gln Glu Glu Tyr Gln 115 120 125 Glu Tyr Glu Pro Glu Ala 130
3127PRTHomo sapiens 3Met Asp Val Phe Lys Lys Gly Phe Ser Ile Ala
Lys Glu Gly Val Val 1 5 10 15 Gly Ala Val Glu Lys Thr Lys Gln Gly
Val Thr Glu Ala Ala Glu Lys 20 25 30 Thr Lys Glu Gly Val Met Tyr
Val Gly Ala Lys Thr Lys Glu Asn Val 35 40 45 Val Gln Ser Val Thr
Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Asn 50 55 60 Ala Val Ser
Glu Ala Val Val Ser Ser Val Asn Thr Val Ala Thr Lys 65 70 75 80 Thr
Val Glu Glu Ala Glu Asn Ile Ala Val Thr Ser Gly Val Val Arg 85 90
95 Lys Glu Asp Leu Arg Pro Ser Ala Pro Gln Gln Glu Gly Val Ala Ser
100 105 110 Lys Glu Lys Glu Glu Val Ala Glu Glu Ala Gln Ser Gly Gly
Asp 115 120 125 413PRTHomo sapiens 4Ala Lys Glu Gly Val Val Ala Ala
Ala Glu Lys Thr Lys 1 5 10 511PRTHomo sapiens 5Ala Lys Glu Gly Val
Val Ala Ala Ala Glu Lys 1 5 10 611PRTHomo sapiens 6Thr Lys Glu Gly
Val Leu Tyr Val Gly Ser Lys 1 5 10 79PRTHomo sapiens 7Glu Gly Val
Leu Tyr Val Gly Ser Lys 1 5 815PRTHomo sapiens 8Thr Lys Glu Gly Val
Val His Gly Val Ala Thr Val Ala Glu Lys 1 5 10 15 913PRTHomo
sapiens 9Glu Gly Val Val His Gly Val Ala Thr Val Ala Glu Lys 1 5 10
1022PRTHomo sapiens 10Thr Lys Glu Gln Val Thr Asn Val Gly Gly Ala
Val Val Thr Gly Val 1 5 10 15 Thr Ala Val Ala Gln Lys 20
1120PRTHomo sapiens 11Glu Gln Val Thr Asn Val Gly Gly Ala Val Val
Thr Gly Val Thr Ala 1 5 10 15 Val Ala Gln Lys 20 1217PRTHomo
sapiens 12Thr Val Glu Gly Ala Gly Ser Ile Ala Ala Ala Thr Gly Phe
Val Lys 1 5 10 15 Lys 1316PRTHomo sapiens 13Thr Val Glu Gly Ala Gly
Ser Ile Ala Ala Ala Thr Gly Phe Val Lys 1 5 10 15
* * * * *