U.S. patent application number 14/750860 was filed with the patent office on 2016-05-26 for ice-cleaved alpha-synuclein as a biomarker.
This patent application is currently assigned to Brandeis University. The applicant listed for this patent is Brandeis University. Invention is credited to Quyen Hoang, Gregory A. Petsko, Dagmar Ringe.
Application Number | 20160143985 14/750860 |
Document ID | / |
Family ID | 46024857 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160143985 |
Kind Code |
A1 |
Ringe; Dagmar ; et
al. |
May 26, 2016 |
ICE-CLEAVED ALPHA-SYNUCLEIN AS A BIOMARKER
Abstract
The present disclosure provides ICE-cleaved alpha-synuclein
fragments as biomarkers for alpha-synuclein-associated disease or
disorder and/or for ICE-regulator therapy.
Inventors: |
Ringe; Dagmar; (Cambridge,
MA) ; Petsko; Gregory A.; (New York, NY) ;
Hoang; Quyen; (Carmel, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brandeis University |
Waltham |
MA |
US |
|
|
Assignee: |
Brandeis University
Waltham
MA
|
Family ID: |
46024857 |
Appl. No.: |
14/750860 |
Filed: |
June 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13883554 |
Jul 22, 2013 |
9116157 |
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PCT/US11/59465 |
Nov 4, 2011 |
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14750860 |
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61410856 |
Nov 5, 2010 |
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61410852 |
Nov 5, 2010 |
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Current U.S.
Class: |
514/21.9 ;
514/44R |
Current CPC
Class: |
A61P 43/00 20180101;
G01N 2800/2814 20130101; A61K 38/005 20130101; A61P 25/28 20180101;
A61P 25/00 20180101; G01N 2800/52 20130101; A61P 25/16 20180101;
A61K 38/06 20130101; C12N 15/113 20130101; C12Q 1/37 20130101; G01N
33/6896 20130101; G01N 2800/2835 20130101 |
International
Class: |
A61K 38/06 20060101
A61K038/06; C12N 15/113 20060101 C12N015/113 |
Claims
1-16. (canceled)
17. A method of treating a patient suffering from or susceptible to
a synucleinopathy disease, disorder, or condition, comprising:
administering to the patient a composition comprising an amount of
an ICE inhibitor sufficient to inhibit cleavage of
.alpha.-synuclein by ICE.
18. The method of claim 17, wherein the patient has been determined
to display a, within a sample from the patient, a ratio of a
fragment to full length .alpha.-synuclein that is elevated as
compared to a reference ratio.
19. The method of claim 18, wherein the fragment is about 120 amino
acids in length.
20. The method of claim 18, wherein the fragment is 115 amino acids
in length.
21. The method of claim 18, wherein the fragment is 119 amino acids
in length.
22. The method of claim 18, wherein the fragment is 121 amino acids
in length.
23. The method of claim 18, wherein the fragment is about 20 amino
acids in length.
24. The method of claim 17, wherein the synucleinopathy disease,
disorder or condition is Parkinson's disease, dementia, or multiple
system atrophy.
25. The method of claim 24, wherein the Parkinson's disease is an
autosomal-dominant Parkinson's disease.
26. The method of claim 24, wherein the synucleinopathy disease,
disorder or condition is characterized by the presence of Lewy
bodies.
27. The method of claim of 18, wherein the fragment of alpha
synuclein is undetectable in the reference standard.
28. The method of claim 17, wherein the ICE inhibitor is a small
molecule, peptide, or nucleic acid.
29. The method of claim 28, wherein the ICE inhibitor is of one of
the following structures: ##STR00008##
30. The method of claim 17, wherein the administered composition
inhibits cleavage of a full length .alpha.-synuclein polypeptide
into at least two fragments having lengths of about 120 amino acids
and about 20 amino acids.
31. The method of claim 17, wherein the administered composition
inhibits proteolytic cleavage at or around a site corresponding to
residue 120 in full length .alpha.-synuclein.
32. The method of claim 17, wherein the administered composition
causes a higher ratio of full-length to cleaved fragments of
.alpha.-synuclein as compared to a control.
33. The method of claim 32, wherein the displayed ratio is one,
two, three, four, five, six, seven, eight, nine, or ten times
higher than the reference ratio.
34. The method of claim 32, wherein the displayed ratio is at least
5%, at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, or at least 95%
higher than the reference ratio.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/410,856, filed Nov. 5, 2010, and U.S.
provisional application Ser. No. 61/410,852, filed Nov. 5, 2010,
the entirety of each of which are hereby incorporated herein by
reference.
BACKGROUND
[0002] Parkinson's disease is a neurodegenerative disorder that is
pathologically characterized by the presence of intracytoplasmic
Lewy bodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed.,
Springer, Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath.
Exp. Neurol. 52:183-191, 1993), the major components of which are
filaments consisting of .alpha.-synuclein (Spillantini et al.,
Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai et al.,
Neurosci. Lett. 259:83-86, 1999), a 140-amino acid protein (Ueda et
al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Two dominant
mutations in .alpha.-synuclein causing familial early onset
Parkinson's disease have been described, suggesting that Lewy
bodies contribute mechanistically to the degeneration of neurons in
Parkinson's disease and related disorders (Polymeropoulos et al.,
Science 276:2045-2047, 1997; Kruger et al., Nature Genet.
18:106-108, 1998; Zarranz et al., Ann Neurol. 55:164-173, 2004).
Triplication and duplication mutations of the .alpha.-synuclein
gene have been linked to early-onset of Parkinson's disease
(Singleton et al., Science 302:841, 2003; Chartier-Harlin at al.
Lancet 364:1167-1169, 2004; Ibanez et al., Lancet 364:1169-1171,
2004). In vitro studies have demonstrated that recombinant
.alpha.-synuclein can indeed form Lewy body-like fibrils (Conway et
al., Nature Med. 4:1318-1320, 1998; Hashimoto et al., Brain Res.
799:301-306, 1998; Nahri et al., J. Biol. Chem. 274:9843-9846,
1999). Both Parkinson's disease-linked .alpha.-synuclein mutations
accelerate this aggregation process, demonstrating that such in
vitro studies may have relevance for Parkinson's disease
pathogenesis. .alpha.-Synuclein aggregation and fibril formation
fulfill the criteria of a nucleation-dependent polymerization
process (Wood et al., J. Biol. Chem. 274:19509-19512, 1999).
SUMMARY OF THE INVENTION
[0003] The present invention encompasses the finding that caspase-1
(ICE) cleaves .alpha.-synuclein in vivo. As described herein, such
cleavage generates .alpha.-synuclein fragments that are prone to
toxic aggregate formation. The present invention pertains, among
other things, to ICE-dependent cleavage of .alpha.-synuclein, as
well as to related methods which are useful for the diagnosis
and/or treatment of diseases and disorders associated with
ICE-cleaved .alpha.-synuclein.
[0004] The present invention provides, in one aspect, methods for
identifying a patient who will likely to respond to a therapy with
an ICE inhibitor. A provided method comprises steps of determining
in a sample of a patient suffering from or susceptible to a
synucleinopathy disease, disorder or condition a ratio of a
fragment to a full-length .alpha.-synuclein; and, if the ratio is
elevated as compared to a reference standard, designating the
patient as a good candidate for a therapy with an ICE
inhibitor.
[0005] In some embodiments, a fragment of .alpha.-synuclein is
about 120 amino acids in length. For example, a fragment of
.alpha.-synuclein is 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, or 126 amino acids in
length.
[0006] In some embodiments, a fragment of .alpha.-synuclein is
about 20 amino acids in length, e.g., 15, 16, 17, 18, 19, 20, 21,
22, 23, or 24 amino acids.
[0007] According to the invention, a provided method is useful for
a patient suffering from or susceptible to synucleinopathy disease,
disorder or condition is Parkinson's disease, dementia, or multiple
system atrophy. For example, Parkinson's disease may be an
autosomal-dominant Parkinson's disease. In certain embodiments, the
synucleinopathy disease, disorder or condition is characterized by
the presence of Lewy bodies.
[0008] In some embodiments of the methods described herein, a ratio
of a fragment to a full-length .alpha.-synuclein in a sample of a
patient is above 0, where the ratio may be about 0.01, 0.02, 0.03,
0.04, 0.05, 0.1 or greater.
[0009] In some embodiments, ICE fragments of .alpha.-synuclein is
undetectable in the reference standard.
[0010] In some embodiments, samples according to the invention may
be a blood sample.
[0011] In one aspect, the invention provides methods for
identifying and/or characterizing compounds that inhibit ICE. For
example, in some embodiments, the invention provides methods
comprising steps of: (1) providing a plurality of test compounds;
(2) contacting test compounds from the plurality with full-length
.alpha.-synuclein in the presence of ICE; and (3) determining
whether one or more of the test compounds inhibits ICE cleavage of
the full-length .alpha.-synuclein into .alpha.-synuclein fragments.
In some embodiments, .alpha.-synuclein cleavage is determined by
measuring relative levels of full-length .alpha.-synuclein and
cleaved .alpha.-synuclein; in some embodiments a higher ratio of
full-length .alpha.-synuclein to cleaved .alpha.-synuclein in the
presence of the test compound as compared to the control indicates
that the test compound is an ICE inhibitor that inhibits ICE
cleavage of .alpha.-synuclein.
[0012] In some embodiments of the invention, relevant
.alpha.-synuclein fragments include a fragment of about 120 amino
acids in length. In some embodiments of the invention, relevant
.alpha.-synuclein fragments include a fragment of about 20 amino
acids in length. In some embodiments of the invention, relevant
.alpha.-synuclein fragments include both a fragment of about 120
amino acids in length and one of about 20 amino acids in length. In
some embodiments, an .alpha.-synuclein fragment of interest
corresponds to a polypeptide resulting from cleavage of full-length
.alpha.-synuclein fragments at a site corresponding to residue 120
of SEQ ID NO: 1.
[0013] In some embodiments, the invention provides antibodies
specific to one or more particular .alpha.-synuclein fragments. In
some embodiments, the invention provides antibodies specific to one
or more .alpha.-synuclein fragments generated by ICE-dependent
proteolysis of .alpha.-synuclein. In some embodiments, the present
invention provides In some embodiments, the present invention
provides antibodies that bind to a full-length .alpha.-synuclein
polypeptide, but do not bind to a fragment of that
.alpha.-synuclein polypeptide that would be generated by cleavage
of the full-length .alpha.-synuclein polypeptide by ICE. In some
embodiments, the present invention provides .alpha.-synuclein
antibodies that specifically bind to a fragment generated by ICE
cleavage of a full-length .alpha.-synuclein polypeptide, but not to
the full-length .alpha.-synuclein polypeptide itself. In some
embodiments, antibodies provided herein specifically bind to one or
more conformational epitoptes.
[0014] The present invention provides systems, including methods,
for identifying and/or characterizing .alpha.-synuclein cleaving
enzymes. For example, in some embodiments, the present invention
provides methods comprising steps of: (1) providing a plurality of
candidate enzymes (e.g., caspase enzymes) that are candidate
.alpha.-synuclein cleaving enzymes; (2) contacting candidate
enzymes from the plurality with a full-length .alpha.-synuclein;
and (3) determining whether one or more of the candidate enzymes
cleaves the full-length .alpha.-synuclein into fragments. In some
embodiments, the step of determining comprises determining whether
one or more of the candidate enzymes cleaves the full-length
.alpha.-synuclein into fragments including one that is about 120
amino acids in length. This step may involve measuring or detecting
relative amounts of .alpha.-synuclein species (e.g., full length
and/or fragments). Alternatively or additionally, the present
invention provides methods comprising steps of (1) contacting a
full-length .alpha.-synuclein polypeptide with a caspase enzyme
under conditions and for a time sufficient to permit cleavage of
the full-length .alpha.-synuclein polypeptide into fragments. In
some embodiments, at least one fragment is detected.
[0015] The present invention also provides methods directed to an
ICE inhibitor therapy. In some embodiments, the invention provides
methods comprising administering to a patient suffering from or
susceptible to a developing synucleinopathy disease, disorder or
condition, a composition comprising an amount of an ICE inhibitor
sufficient to inhibit cleavage of .alpha.-synuclein by ICE.
[0016] In certain embodiments, the present invention relates to a
synucleinopathy disease, disorder or condition, including, but not
limited to, Parkinson's disease (such as an autosomal-dominant
Parkinson's disease), dementia, or multiple system atrophy. In
certain embodiments, present invention relates to a synucleinopathy
disease, disorder or condition that is characterized by presence of
Lewy bodies.
DEFINITIONS
[0017] Alpha-synuclein polypeptide/.alpha.-synuclein polypeptide:
The term ".alpha.-synuclein polypeptide" or "alpha-synuclein," as
used herein, refers to a polypeptide that shows a high degree of
sequence identity with a wild type .alpha.-synuclein polypeptide
such as, for example, wild type human .alpha.-synuclein. The
wild-type, full-length form of human .alpha.-synuclein is a 140
amino acid polypeptide comprising the following amino acid sequence
(see, for example, Accession Number: NP_000336.1):
TABLE-US-00001 MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV
GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL
GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
(SEQ ID NO: 1). In some embodiments, an .alpha.-synuclein
polypeptide shows at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% overall sequence identity with SEQ
ID NO: 1. The full-length .alpha.-synuclein primary structure is
typically divided into three distinct domains: Residues
corresponding to residues 1-60 of SEQ ID NO: 1 represent an
amphipathic N-terminal region dominated by four 11-residue repeats
including the consensus sequence KTKEGV (SEQ ID NO: 2). This
sequence has been reported to have a structural alpha helix
propensity similar to apolipoproteins-binding domains; residues
61-95 correspond to a central hydrophobic region which includes the
non-amyloid component (NAC) region, involved in protein
aggregation; and, residues 96-140 make up a highly acidic and
proline-rich region which has no distinct structural propensity. In
some embodiments, an .alpha.-synuclein polypeptide may include one
or more point mutations as compared with SEQ ID NO:1, which are
associated with a disease, disorder or condition. For example,
certain monogenic point mutations, including but not limited to
A30P, A53T, and E46K, have been identified as causal factors of
early onset familial Parkinson disease.
[0018] .alpha.-synuclein fragment: The term ".alpha.-synuclein
fragment," as used herein, refers to a polypeptide having an amino
acid sequence that is substantially identical to that of an
.alpha.-synuclein polypeptide except that the fragment includes
less than all of the amino acid residues found in a full-length
.alpha.-synuclein polypeptide. In some embodiments a fragment lacks
one or more terminal residues or sections found in a full-length
.alpha.-synuclein polypeptide. In some embodiments, an
.alpha.-synuclein fragment is fewer than 140, 139, 138, 137, 136,
135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123,
122, 121, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110,
109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96,
95, 94, 93, 92, 92, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79,
78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62,
61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,
44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
or 10 amino acids long. In some embodiments, an .alpha.-synuclein
fragment is about 120 amino acids long. In some embodiments, an
.alpha.-synuclein fragment corresponds to a cleavage product of a
full-length .alpha.-synuclein polypeptide. In some embodiments, an
.alpha.-synuclein fragment corresponds to a product of cleavage of
a full-length .alpha.-synuclein polypeptide at a site corresponding
to approximately residue 120 of SEQ ID NO: 1.
[0019] Biological sample: The term "biological sample," as used
herein, refers to any solid or fluid sample obtained from, excreted
by, or secreted by any living organism, including single-celled
micro-organisms (such as bacteria and yeasts) and multicellular
organisms (such as plants and animals, for instance a vertebrate or
a mammal, and in particular a healthy or apparently healthy human
subject or a human patient affected by a condition or disease to be
diagnosed or investigated). A biological sample can be in any form,
including a solid material such as a tissue, cells, a cell pellet,
a cell extract, cell homogenates, or cell fractions; or a biopsy,
or a biological fluid. The biological fluid may be obtained from
any site (e.g., blood, saliva (or a mouth wash containing buccal
cells), tears, plasma, serum, urine, bile, cerebrospinal fluid,
amniotic fluid, peritoneal fluid, and pleural fluid, or cells
therefrom, aqueous or vitreous humor, or any bodily secretion), a
transudate, an exudate (e.g. fluid obtained from an abscess or any
other site of infection or inflammation), or fluid obtained from a
joint (e.g. a normal joint or a joint affected by disease such as
rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A
biological sample can be obtained from any organ or tissue
(including a biopsy or autopsy specimen) or may comprise cells
(whether primary cells or cultured cells) or medium conditioned by
any cell, tissue or organ. Biological samples may also include
sections of tissues such as frozen sections taken for histological
purposes. Biological samples also include mixtures of biological
molecules including proteins, lipids, carbohydrates and nucleic
acids generated by partial or complete fractionation of cell or
tissue homogenates. In certain embodiments, a biological sample is
a blood sample containing erythrocytes. Although a sample is
preferably taken from a human subject, biological samples may be
from any animal, plant, bacteria, virus, yeast, etc. The term
"animal," as used herein, refers to humans as well as non-human
animals, at any stage of development, including, for example,
mammals, birds, reptiles, amphibians, fish, worms and single cells.
Cell cultures and live tissue samples are considered to be
pluralities of animals. In certain exemplary embodiments, the
non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a
rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a
pig). An animal may be a transgenic animal or a human clone. If
desired, a biological sample may be subjected to preliminary
processing, including preliminary separation techniques. In some
embodiments, a biological sample contains or is derived from one or
more cells or biopolymers. Cell-containing compositions include,
for example, mammalian blood, red cell concentrates, platelet
concentrates, leukocyte concentrates, blood cell proteins, blood
plasma, platelet-rich plasma, a plasma concentrate, a precipitate
from any fractionation of the plasma, a supernatant from any
fractionation of the plasma, blood plasma protein fractions,
purified or partially purified blood proteins or other components,
serum, semen, mammalian colostrum, milk, saliva, placental
extracts, a cryoprecipitate, a cryosupernatant, a cell lysate,
mammalian cell culture or culture medium, products of fermentation,
ascites fluid, proteins induced in blood cells, and products
produced in cell culture by normal or transformed cells (e.g., via
recombinant DNA or monoclonal antibody technology). Biological
compositions can be cell-free. In certain embodiments, a suitable
biological composition or biological sample is a red blood cell
suspension. In some embodiments, a blood cell suspension includes
mammalian blood cells. In certain embodiments, blood cells are
obtained from a human, a non-human primate, a dog, a cat, a horse,
a cow, a goat, a sheep or a pig. In certain embodiments, a blood
cell suspension includes red blood cells and/or platelets and/or
leukocytes and/or bone marrow cells.
[0020] Characteristic sequence element: As used herein, a
"characteristic sequence element" of a protein or polypeptide is
one that contains a continuous stretch of amino acids, or a
collection of continuous stretches of amino acids, that together
are characteristic of a protein or polypeptide. Each such
continuous stretch generally will contain at least two amino acids.
Furthermore, those of ordinary skill in the art will appreciate
that typically at least 5, at least 10, at least 15, at least 20 or
more amino acids are required to be characteristic of a protein. In
general, a characteristic sequence element is one that, in addition
to the sequence identity specified above, shares at least one
functional characteristic (e.g., biological activity, epitope, etc)
with the relevant intact protein. In many embodiments, a
characteristic sequence element is one that is present in all
members of a family of polypeptides, and can be used to define such
members.
[0021] Combination therapy: The term "combination therapy," as used
herein, refers to those situations in which two or more different
pharmaceutical agents are administered in overlapping regimens so
that the subject is simultaneously exposed to both agents.
[0022] Determine: Many methodologies described herein include a
step of "determining." Those of ordinary skill in the art, reading
the present specification, will appreciate that such "determining"
can utilize any of a variety of techniques available to those
skilled in the art, including, for example, specific techniques
explicitly referred to herein. In some embodiments, a determination
involves manipulation of a physical sample. In some embodiments, a
determination involves consideration and/or manipulation of data or
information, for example utilizing a computer or other processing
unit adapted to perform a relevant analysis. In some embodiments, a
determination involves receiving relevant information and/or
materials from a source.
[0023] Dosing regimen: A "dosing regimen" (or "therapeutic
regimen"), as that term is used herein, is a set of unit doses
(typically more than one) that are administered individually to a
subject, typically separated by periods of time. In some
embodiments, a given therapeutic agent has a recommended dosing
regiment, which may involve one or more doses. In some embodiments,
a dosing regimen comprises a plurality of doses each of which are
separated from one another by a time period of the same length; in
some embodiments, a dosing regime comprises a plurality of doses
and at least two different time periods separating individual
doses.
[0024] Isolated: The term "isolated," as used herein, refers to an
agent or entity that has either (i) been separated from at least
some of the components with which it was associated when initially
produced (whether in nature or in an experimental setting); or (ii)
produced by the hand of man. Isolated agents or entities may be
separated from at least about 10%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, or
more of the other components with which they were initially
associated. In some embodiments, isolated agents are more than 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.
[0025] Polypeptide: A "polypeptide," generally speaking, is a
string of at least two amino acids attached to one another by a
peptide bond. In some embodiments, a polypeptide may include at
least 3-5 amino acids, each of which is attached to others by way
of at least one peptide bond. Those of ordinary skill in the art
will appreciate that polypeptides sometimes include "non-natural"
amino acids or other entities that nonetheless are capable of
integrating into a polypeptide chain, optionally.
[0026] Prevention: The term "prevention," as used herein, refers to
a delay of onset, and/or reduction in frequency and/or severity of
one or more symptoms of a particular disease, disorder or condition
(e.g., infection for example with influenza virus). In some
embodiments, prevention is assessed on a population basis such that
an agent is considered to "prevent" a particular disease, disorder
or condition if a statistically significant decrease in the
development, frequency, and/or intensity of one or more symptoms of
the disease, disorder or condition is observed in a population
susceptible to the disease, disorder, or condition.
[0027] Substantial homology: The phrase "substantial homology" is
used herein to refer to a comparison between amino acid or nucleic
acid sequences. As will be appreciated by those of ordinary skill
in the art, two sequences are generally considered to be
"substantially homologous" if they contain homologous residues in
corresponding positions. Homologous residues may be identical
residues. Alternatively, homologous residues may be non-identical
residues that share one or more structural and/or functional
characteristics. For example, as is well known by those of ordinary
skill in the art, certain amino acids are typically classified as
"hydrophobic" or "hydrophilic" amino acids, and/or as having
"polar" or "non-polar" side chains In some embodiments,
substitution of one amino acid for another of the same type is
considered a "homologous" substitution. Typical amino acid
categorizations are summarized below:
TABLE-US-00002 Alanine Ala A nonpolar neutral 1.8 Arginine Arg R
polar positive -4.5 Asparagine Asn N polar neutral -3.5 Aspartic
Asp D polar negative -3.5 acid Cysteine Cys C nonpolar neutral 2.5
Glutamic Glu E polar negative -3.5 acid Glutamine Gln Q polar
neutral -3.5 Glycine Gly G nonpolar neutral -0.4 Histidine His H
polar positive -3.2 Isoleucine Ile I nonpolar neutral 4.5 Leucine
Leu L nonpolar neutral 3.8 Lysine Lys K polar positive -3.9
Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar
neutral 2.8 Proline Pro P nonpolar neutral -1.6 Serine Ser S polar
neutral -0.8 Threonine Thr T polar neutral -0.7 Tryptophan Trp W
nonpolar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val
V nonpolar neutral 4.2 Ambiguous Amino Acids 3-Letter 1-Letter
Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx Z
Leucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa
X
[0028] As is well known in this art, amino acid or nucleic acid
sequences may be compared using any of a variety of algorithms,
including those available in commercial computer programs such as
BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and
PSI-BLAST for amino acid sequences. Exemplary such programs are
described in Altschul, et al., Basic local alignment search tool,
J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in
Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical
Guide to the Analysis of Genes and Proteins, Wiley, 1998; and
Misener, et al., (eds.), Bioinformatics Methods and Protocols
(Methods in Molecular Biology, Vol. 132), Humana Press, 1999; all
of the foregoing of which are incorporated herein by reference. In
addition to identifying homologous sequences, the programs
mentioned above typically provide an indication of the degree of
homology. In some embodiments, two sequences are considered to be
substantially homologous if at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% or more of their corresponding residues are
homologous over a relevant stretch of residues. In some
embodiments, the relevant stretch is a complete sequence. In some
embodiments, the relevant stretch is at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, at least 50, at least 55, at least 60, at least 65, at
least 70, at least 75, at least 80, at least 85, at least 90, at
least 95, at least 100, at least 125, at least 150, at least 175,
at least 200, at least 225, at least 250, at least 275, at least
300, at least 325, at least 350, at least 375, at least 400, at
least 425, at least 450, at least 475, at least 500 or more
residues.
[0029] Substantial identity: The phrase "substantial identity" is
used herein to refer to a comparison between amino acid or nucleic
acid sequences. As will be appreciated by those of ordinary skill
in the art, two sequences are generally considered to be
"substantially identical" if they contain identical residues in
corresponding positions. As is well known in this art, amino acid
or nucleic acid sequences may be compared using any of a variety of
algorithms, including those available in commercial computer
programs such as BLASTN for nucleotide sequences and BLASTP, gapped
BLAST, and PSI-BLAST for amino acid sequences. Exemplary such
programs are described in Altschul, et al., Basic local alignment
search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et
al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs",
Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,
Bioinformatics: A Practical Guide to the Analysis of Genes and
Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics
Methods and Protocols (Methods in Molecular Biology, Vol. 132),
Humana Press, 1999; all of the foregoing of which are incorporated
herein by reference. In addition to identifying identical
sequences, the programs mentioned above typically provide an
indication of the degree of identity. In some embodiments, two
sequences are considered to be substantially identical if at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% or more of their
corresponding residues are identical over a relevant stretch of
residues. In some embodiments, the relevant stretch is a complete
sequence. In some embodiments, the relevant stretch is at least 10,
at least 15, at least 20, at least 25, at least 30, at least 35, at
least 40, at least 45, at least 50, at least 55, at least 60, at
least 65, at least 70, at least 75, at least 80, at least 85, at
least 90, at least 95, at least 100, at least 125, at least 150, at
least 175, at least 200, at least 225, at least 250, at least 275,
at least 300, at least 325, at least 350, at least 375, at least
400, at least 425, at least 450, at least 475, at least 500 or more
residues.
[0030] Therapeutic agent: As used herein, the phrase "therapeutic
agent" refers to any agent that elicits a desired biological or
pharmacological effect.
[0031] Treatment: As used herein, the term "treatment" refers to
any method used to alleviate, delay onset, reduce severity or
incidence, or yield prophylaxis of one or more symptoms or aspects
of a disease, disorder, or condition. For the purposes of the
present invention, treatment can be administered before, during,
and/or after the onset of symptoms.
[0032] Unit dose: The expression "unit dose" as used herein refers
to a physically discrete unit of a pharmaceutical composition,
formulated for administration to a subject. In many embodiments, a
unit dose contains a predetermined quantity of an active agent. In
some embodiments, a unit dose contains an entire single dose of the
agent. In some embodiments, more than one unit dose is administered
to achieve a total single dose. In some embodiments, administration
of multiple doses is required, or expected to be required, in order
to achieve an intended effect. The unit dose may be, for example, a
volume of liquid (e.g., an acceptable carrier) containing a
predetermined quantity of one or more therapeutic agents, a
predetermined amount of one or more therapeutic agents in solid
form, a sustained release formulation or drug delivery device
containing a predetermined amount of one or more therapeutic
agents, etc. It will be appreciated that a unit dose may contain a
variety of components in addition to the therapeutic agent(s). For
example, acceptable carriers (e.g., pharmaceutically acceptable
carriers), diluents, stabilizers, buffers, preservatives, etc., may
be included as described infra. It will be understood, however,
that the total daily usage of a formulation of the present
disclosure will often be decided by the attending physician within
the scope of sound medical judgment. In some embodiments, the
specific effective dose level for any particular subject or
organism may depend upon a variety of factors including the
disorder being treated and the severity of the disorder; activity
of specific active compound employed; specific composition
employed; age, body weight, general health, sex and diet of the
subject; time of administration, and rate of excretion of the
specific active compound employed; duration of the treatment; drugs
and/or additional therapies used in combination or coincidental
with specific compound(s) employed, and like factors well known in
the medical arts.
BRIEF DESCRIPTION OF THE DRAWING
[0033] FIG. 1 provides a set of SDS-PAGE images demonstrating that
purified, activated caspase-1 cleaves alpha-synuclein in vitro.
[0034] FIG. 2 provides a set of SDS-PAGE images demonstrating that
caspase-1 inhibitor blocks the fragmentation of
alpha-synuclein.
[0035] FIG. 3 provides a set of SDS-PAGE images demonstrating that
oxidative stress activates caspase-1 and generates the
alpha-synuclein fragment in neuroblastoma cells.
[0036] FIG. 4 provides graphs depicting results from assays
measuring caspase-1 cleavage of alpha-synuclein in vitro.
[0037] FIG. 5 provides an image of a Western blot of ICE digested
alpha-synuclein. Lane 1; molecular weight marker, lane 2;
.alpha.Syn alone, lane 3 to 6; .alpha.Syn plus increasing amounts
of ICE (2 to 12 .mu.g). Appearance of a smaller fragment of
synuclein below 17 kD with intensity increasing with amount of ICE
indicate that ICE was generating the fragment.
[0038] FIG. 6 provides an image of a Western blot of ICE digested
.alpha.-syn plus inhibitor. Lane 1; molecular weight marker, lane
2; .alpha.Syn alone, lane 3; .alpha.Syn plus ICE (10 ug), lane 4;
.alpha.Syn plus ICE (10 .mu.g) and 20 .mu.M of NCG inhibitor from
Graig Thomas at NIH. Appearance of a smaller fragment of synuclein
below 17 kD in lane 3 indicate that ICE was generating the
fragment. This fragment is demished in lane 4 containing ICE
specific inhibitor, indicating that ICE specifically was generating
the fragment.
[0039] FIG. 7 provides MALDI-TOF mass spec analysis of ICE digested
.alpha.-syn. The fragment generated by ICE was determined to be
13167 Da which corresponds to residues 1-121.
[0040] FIG. 8 provides graphs depicting M17 cell viability of M17
cells with pcDNA3 empty vector in the presence of menadione and M17
cells overexpressing alpha-synuclein in the presence of
menadione.
[0041] FIG. 9 provides an image of a Western blot of menadione
treated M17 cells. Lane 1; molecular weight marker, lane 2; M17
cells treated with 1% DMSO as control, lane 3; M17 cells treated
with 12 .mu.M menadione in 1% DMSO, lane 4; M17 cells treated with
14 .mu.M menadione in 1% DMSO. Appearance of a smaller fragment of
synuclein below 17 kD in lane 3 and 4 indicate that menadione
induced fragmentation of .alpha.-synuclein.
[0042] FIG. 10 provides a bar graph depicting the effects of
caspase-1 inhibition and knockdown on cell viability Inhibition of
ICE with VX765 or knockdown of ICE gene with shRNA rescued M17
cells overexpressing alpha-synuclein from alpha-synuclein-induced
toxicity.
[0043] FIG. 11 provides a graph of the aggregation of truncated
alpha-synuclein over the course of 70 hours.
DETAILED DESCRIPTION
[0044] Based on the novel finding that .alpha.-synuclein is an in
vivo substrate for the ICE protease, the present invention provides
methods and technologies directed to use of ICE-cleaved fragments
of .alpha.-synuclein as a marker for detecting certain
biological/clinical conditions and/or for identifying individuals
likely to respond (or not) to therapy with one or more ICE
inhibitors or other ICE regulating agents. Other methods, reagents,
systems and technologies based on presently described developments
are included within accompanying patent application entitled
"ICE-inhibiting compounds and uses thereof", filed on even date
herewith.
Alpha-Synuclein
[0045] Synucleins are a family of proteins composed of .alpha.-,
.beta.-, and .gamma.-synucleins. In neurons, the synuclein proteins
are localized predominantly at the presynaptic sites. Among the
synuclein proteins, .alpha.-Synuclein is a small lipid-binding
protein involved in vesicle trafficking whose function is poorly
characterized. It is of great interest from a clinical perspective
because .alpha.-synuclein dysfunction has been implicated in the
pathogenesis of several neurodegenerative disorders, including
Parkinson's disease (PD) (Ian et al., Clinical Neurosc. Res.
1:445-455, 2001; Trojanowski and Lee, Neurotoxicology 23:457-460,
2002).
[0046] .alpha.-Synuclein recombinant protein, and non-A.beta.
component (known as NAC), which is a 35-amino acid peptide fragment
of .alpha.-synuclein, both have the ability to form fibrils when
incubated at 37.degree. C., and are positive with amyloid stains
such as Congo red (demonstrating a red/green birefringence when
viewed under polarized light) and Thioflavin S (demonstrating
positive fluorescence) (Hashimoto et al., Brain Res. 799:301-306,
1998; Ueda et al., Proc. Natl. Acad. Sci. USA 90:11282-11286,
1993).
[0047] Diseases and disorders that are associated with
.alpha.-synuclein aggregates are collectively referred to as
synucleinopathies. Pathologically, .alpha.-synuclein has been
identified as a major component of Lewy bodies, the hallmark
inclusions of Parkinson's disease, and a fragment thereof was
isolated from amyloid plaques of a different neurological disease,
Alzheimer's disease. Biochemically, recombinant .alpha.-synuclein
has been shown to form amyloid-like fibrils that recapitulated the
ultrastructural features of .alpha.-synuclein isolated from
patients with dementia with Lewy bodies, Parkinson's disease and
multiple system atrophy. Additionally, the identification of
mutations within the .alpha.-synuclein gene, albeit in rare cases
of familial Parkinson's disease, have demonstrated a strong link
between synuclein pathology and neurodegenerative diseases. Thus,
dysfunction of .alpha.-synuclein appears to be a common link
amongst the pathogenesis underlying a spectrum of diseases such as
Parkinson's disease, dementia with Lewy bodies, multiple system
atrophy and the Lewy body variant of Alzheimer's disease.
[0048] Fibrillization and aggregation of .alpha.-synuclein is
thought to play major role in neuronal dysfunction and death of
dopaminergic neurons in Parkinson's disease. It has been suggested
that mutations in .alpha.-synuclein or genomic triplication of wild
type .alpha.-synuclein (leading to its overexpression) can cause
certain rare familial forms of Parkinson's disease. A number of
reports have indicated that over-expression of wild-type
.alpha.-synuclein induces neuronal cell death. See, e.g.,
Polymeropoulos, et al. (1997) Science 276(5321):2045-7, Kruger, et
al. (1998) Nat Genet. 18(2):106-8, Singleton, et al. (2003) Science
302(5646):841, Miller, et al. (2004) Neurology 62(10):1835-8,
Hashimoto, et al. (2003) Ann N Y Acad Sci. 991:171-88, Lo Bianco,
et al. (2002) Proc Natl Acad Sci USA. 99(16):10813-8, Lee, et al.
(2002) Proc Natl Acad Sci USA. 99(13):8968-73, Masliah, et al.
(2000) Science 287(5456): 1265-9, Auluck, et al. (2002) Science
295(5556):865-8, Oluwatosin-Chigbu et al. (2003) Biochem Biophys
Res Commun 309(3): 679-84, Klucken et al. (2004) J Biol Chem.
279(24):25497-502. While it has been suggested that protecting
neurons from the toxic effects of .alpha.-synuclein would be a
promising strategy for treating Parkinson's disease and other
synucleinopathies such as Lewy body dementia, the exact targets
relevant to the in vivo regulation of .alpha.-synuclein have been
largely unknown.
[0049] At least three isoforms of synuclein are produced through
alternative splicing (Beyer K (September 2006). "Alpha-synuclein
structure, posttranslational modification and alternative splicing
as aggregation enhancers". Acta Neuropathol. 112 (3): 237-51). The
majority form of the protein, and the one most investigated, is the
full 140 amino acid-long polypeptide, generally referred to as
full-length .alpha.-synuclein. Other isoforms are
.alpha.-synuclein-126, where exon 3 is lost and lacks residues
41-54; and .alpha.-synuclein-112 (Ueda K, Saitoh T, Mori H
(December 1994). "Tissue-dependent alternative splicing of mRNA for
NACP, the precursor of non-A beta component of Alzheimer's disease
amyloid.". Biochem. Biophys. Res. Commun. 205 (2): 1366-72), which
lacks residue 103-130 due to loss of exon 5 (Beyer K (September
2006). "Alpha-synuclein structure, posttranslational modification
and alternative splicing as aggregation enhancers". Acta
Neuropathol. 112 (3): 237-51).
[0050] .alpha.-Synuclein is also known as SNCA. In humans,
.alpha.-synuclein is encoded by the SNCA gene. (Ueda K, Fukushima
H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero D A, Kondo J, Ihara
Y, Saitoh T (December 1993). "Molecular cloning of cDNA encoding an
unrecognized component of amyloid in Alzheimer disease". Proc.
Natl. Acad. Sci. U.S.A. 90 (23): 11282-6; Xia Y, Saitoh T, Ueda K,
Tanaka S, Chen X, Hashimoto M, Hsu L, Conrad C, Sundsmo M,
Yoshimoto M, Thal L, Katzman R, Masliah E (October 2001).
"Characterization of the human alpha-synuclein gene: Genomic
structure, transcription start site, promoter region and
polymorphisms". J. Alzheimers Dis. 3 (5): 485-494; Xia Y, Saitoh T,
Ueda K, Tanaka S, Chen X, Hashimoto M, Hsu L, Conrad C, Sundsmo M,
Yoshimoto M, Thal L, Katzman R, Masliah E (2002). "Characterization
of the human alpha-synuclein gene: Genomic structure, transcription
start site, promoter region and polymorphisms: Erratum p 489 FIG.
3". J. Alzheimers Dis. 4 (4): 337).
[0051] An .alpha.-synuclein fragment has been shown to be present
in Alzheimer's disease amyloid. Originally identified as an unknown
non-Abeta (or "non-A.beta.") component (NAC) in an amyloid-enriched
fraction, this fragment was ultimately shown, through cloning of
the full-length cDNA that encodes it, to be a fragment of a
precursor protein, known as NACP (Ueda K, Fukushima H, Masliah E,
Xia Y, Iwai A, Yoshimoto M, Otero D A, Kondo J, Ihara Y, Saitoh T
(December 1993). "Molecular cloning of cDNA encoding an
unrecognized component of amyloid in Alzheimer disease". Proc.
Natl. Acad. Sci. U.S.A. 90 (23): 11282-6.
doi:10.1073/pnas.90.23.11282. PMID 8248242. PMC 47966.
http://www.pnas.org/content/90/23/11282). Subsequent to this
cloning, it was determined that NACP was the human homologue of
Torpedo synuclein. Therefore, NACP is now referred to as human
alpha-synuclein.
[0052] Alpha-synuclein is primarily found in neural tissue, making
up to 1% of all proteins in the cytosol (Iwai A, Masliah E,
Yoshimoto M, Ge N, Flanagan L, de Silva H A, Kittel A, Saitoh T
(February 1995). "The precursor protein of non-A beta component of
Alzheimer's disease amyloid is a presynaptic protein of the central
nervous system". Neuron 14 (2): 467-75). It is predominantly
expressed in the neocortex, hippocampus, substantia nigra,
thalamus, and cerebellum. It is predominantly a neuronal protein
but can also be found in glial cells. In melanocytic cells, SNCA
protein expression may be regulated by MITF (Hoek K S, Schlegel N
C, Eichhoff O M, et al. (2008). "Novel MITF targets identified
using a two-step DNA microarray strategy". Pigment Cell Melanoma
Res. 21 (6): 665-76). It has been established that alpha-synuclein
is extensively localized in the nucleus of mammalian brain neurons,
suggesting a role of alpha-synuclein in the nucleus (Yu S, Li X,
Liu G, Han J, Zhang C, Li Y, Xu S, Liu C, Gao Y, Yang H, Ueda K,
Chan P (March 2007). "Extensive nuclear localization of
alpha-synuclein in normal rat brain neurons revealed by a novel
monoclonal antibody". Neuroscience 145 (2): 539-55). Synuclein is,
however, found predominantly in the presynaptic termini, in both
free or membrane-bound forms (McLean P J, Kawamata H, Ribich S,
Hyman B T (March 2000). "Membrane association and protein
conformation of alpha-synuclein in intact neurons. Effect of
Parkinson's disease-linked mutations". J. Biol. Chem. 275 (12):
8812-6) with roughly 15% of synuclein being membrane-bound in any
moment in neurons (Lee H J, Choi C, Lee S J (January 2002).
"Membrane-bound alpha-synuclein has a high aggregation propensity
and the ability to seed the aggregation of the cytosolic form". J.
Biol. Chem. 277 (1): 671-8).
[0053] It has also been shown that alpha-synuclein localizes in
neuronal mitochondria (Zhang L, Zhang C, Zhu Y, Cai Q, Chan P, Ueda
K, Yu S, Yang H (December 2008) "Semi-quantitative analysis of
alpha-synuclein in subcellular pools of rat brain neurons: an
immunogold electron microscopic study using a C-terminal specific
monoclonal antibody". Brain Res 1244: 40-52; Liu G, Zhang C, Yin J,
Li X, Cheng F, Li Y, Yang H, Ueda K, Chan P, Yu S (May 2009).
"Alpha-Synuclein is differentially expressed in mitochondria from
different rat brain regions and dose-dependently down-regulates
complex I activity". Neurosci. Lett. 454 (3): 187-92).
Alpha-synuclein is highly expressed in the mitochondria in
olfactory bulb, hippocampus, striatum, and thalamus, where the
cytosolic alpha-synuclein is also rich; the cerebral cortex and
cerebellum are two exceptions, by contrast contain rich cytosolic
alpha-synuclein but very low levels of mitochondrial
alpha-synuclein. Within the mitochondria, it has been shown that
alpha-synuclein is localized in the inner membrane of mitochondria,
and that the inhibitory effect of alpha-synuclein on complex I
activity of mitochondrial respiratory chain is dose-dependent.
Thus, it is suggested that alpha-synuclein in mitochondria is
differentially expressed in different brain regions and the
background levels of mitochondrial alpha-synuclein may be a
potential factor affecting mitochondrial function and predisposing
some neurons to degeneration.
[0054] It has been shown that alpha-synuclein significantly
interacts with tubulin (Alim M A, Hossain M S, Arima K, Takeda K,
Izumiyama Y, Nakamura M, Kaji H, Shinoda T, Hisanaga S, Ueda K.
(January 2002). "Tubulin seeds alpha-synuclein fibril formation.".
J. Biol. Chem. 277 (3): 2112-7), and that alpha-synuclein may have
an activity as potential microtubule-associated protein like tau
(Alim M A, Ma Q L, Takeda K, Aizawa T, Matsubara M, Nakamura M,
Asada A, Saito T, Kaji H, Yoshii M, Hisanaga S, Ueda K (August
2004). "Demonstration of a role for alpha-synuclein as a functional
microtubule-associated protein". J. Alzheimers Dis. 6 (4): 435-42;
discussion 443-9).
[0055] Recent evidence suggests that alpha-synuclein functions as a
molecular chaperone in the formation of SNARE complexes (Bonini N
M, Giasson B I (November 2005). "Snaring the function of
alpha-synuclein". Cell 123 (3): 359-61; Chandra S, Gallardo G,
Fernandez-Chacon R, Schluter O M, Sudhof T C (November 2005).
"Alpha-synuclein cooperates with CSPalpha in preventing
neurodegeneration". Cell 123 (3): 383-96). Indeed, there is growing
evidence that alpha-synuclein is involved in the functioning of the
neuronal Golgi apparatus and vesicle trafficking (A. A. Cooper, A.
D. Gitler, A. Cashikar, C. M. Haynes, K. J. Hill, B. Bhullar, K.
Liu, K. Xu, K. E. Strathearn, F. Liu, S. Cao, K. A. Caldwell, G. A.
Caldwell, G. Marsischky, R. D. Kolodner, J. Labaer, J. C. Rochet,
N. M. Bonini, and S. Lindquist. (2006). "Alpha-synuclein blocks
ER-golgi traffic and Rab1 rescues neuron loss in Parkinson's
models". Science 313 (5785): 324-328).
[0056] Experimental evidence has been collected on the interaction
of alpha-synuclein with membrane and its involvement with membrane
composition and turnover. Yeast genome screening has found that
several genes that deal with lipid metabolism play a role in
alpha-synuclein toxicity (Willingham S, Outeiro T F, DeVit M J,
Lindquist S L, Muchowski P J (December 2003). "Yeast genes that
enhance the toxicity of a mutant huntingtin fragment or
alpha-synuclein". Science 302 (5651): 1769-72). Conversely,
alpha-synuclein expression levels can affect the viscosity and the
relative amount of fatty acids in the lipid bilayer (Uversky V N
(October 2007). "Neuropathology, biochemistry, and biophysics of
alpha-synuclein aggregation". J. Neurochem. 103 (1): 17-37).
Alpha-synuclein is known to directly bind to lipid membranes,
associating with the negatively charged surfaces of phospholipids
(Uversky V N (October 2007). "Neuropathology, biochemistry, and
biophysics of alpha-synuclein aggregation". J. Neurochem. 103 (1):
17-37). A preferential binding to small vesicles has been found
(Zhu M, Li J, Fink A L (October 2003). "The association of
alpha-synuclein with membranes affects bilayer structure,
stability, and fibril formation". J. Biol. Chem. 278 (41):
40186-97). The binding of alpha-synuclein to lipid membranes has
complex effects on the latter, altering the bilayer structure and
leading to the formation of small vesicles (Madine J, Doig A J,
Middleton D A (May 2006). "A study of the regional effects of
alpha-synuclein on the organization and stability of phospholipid
bilayers". Biochemistry45 (18): 5783-92). Alpha-synuclein has been
shown to bend membranes of negatively charged phospholipid vesicles
and form tubules from large lipid vesicles (Varkey J, Isas J M,
Mizuno N, Jensen M B, Bhatia V K, Jao C C, Petrlova J, Voss J,
Stamou D, Steven A C, Langen R (August 2010). "Membrane curvature
induction and tubulation is a common feature of synucleins and
apolipoproteins". J Biol Chem). Studies have also suggested a
possible antioxidant activity of alpha-synuclein in the membrane
(Zhu M, Qin Z J, Hu D, Munishkina L A, Fink A L (July 2006).
"Alpha-synuclein can function as an antioxidant preventing
oxidation of unsaturated lipid in vesicles". Biochemistry 45 (26):
8135-42).
[0057] As described in more detail herein, the inventors of the
instant disclosure have discovered that .alpha.-synuclein is an in
vivo target of the protease, caspase-1. Caspase-1 is a member of
the cysteine protease family of enzymes and is also commonly
referred to as ICE. Caspase-1/ICE has been widely studied for its
involvement in the regulation of apoptosis and inflammatory
responses (e.g., cytokine production). Evidence presented herein
shows that ICE cleaves the full-length .alpha.-synuclein
polypeptide into at least two fragments, which are about 120 amino
acids and about 20 amino acids, respectively, and that the
ICE-mediated proteolytic site is localized toward the C-terminus of
the .alpha.-synuclein polypeptide.
.alpha.-Synuclein antibodies
[0058] The invention also includes antibodies that are specific to
.alpha.-synuclein fragments, including for example fragments which
are the ICE-dependent cleavage products of the full-length
.alpha.-synuclein. In some embodiments, the invention provides
.alpha.-synuclein antibodies that specifically recognize
.alpha.-synuclein that is not or has not been cleaved by ICE. It is
known that cysteine proteases including ICE catalyze the cleavage
of their substrates following an aspartic acid residue (Asp or D)
present on the target. The .alpha.-synuclein primary sequence
reveals that there are three aspartic acid residues which are
potential targets for ICE-dependent proteolysis, which are at
residues 115, 119 and 121 (shown in bold below).
[0059] Antibodies may be generated against a peptide based on the
amino acid sequence of .alpha.-synuclein around residues 114-122
(114E, 115D, 116M, 117P, 118V, 119D, 120P, 121D and 122N; shown
with dotted underline above). Such antibodies can be generated by
immunizing a laboratory animal with alpha-synuclein or a fragment
thereof to induce antibodies, and screening the resulting
antibodies to identify those having the desired binding
specificity. The antibody technology is highly developed and is
well known to one of ordinary skill in the art. Typically, an
antigenic peptide should contain at least 5-6 amino acid residues
to confer specificity. For example, in some embodiments, the
antigenic peptide used to generate .alpha.-synuclein antibodies
includes residues 114-116. In some embodiments, the antigenic
peptide includes residues 118-120. In some embodiments, the
antigenic peptide includes residues 120-122.
[0060] It is also possible to generate an antibody specific to the
C-terminal fragment of .alpha.-synuclein corresponding generally to
the last 20 amino acid residues, such that the antibody will
recognize and bind to both full-length .alpha.-synuclein and the
ICE-cleaved .alpha.-synuclein of approximately 20 amino acids.
[0061] Alternatively or additionally, .alpha.-synuclein antibodies
may be generated in accordance with the present invention against
the N-terminus/central portions of .alpha.-synuclein such that both
the full-length and the larger (e.g., .about.120 amino acids)
fragment of .alpha.-synuclein generated by ICE proteolysis can be
detected.
[0062] .alpha.-Synuclein antibodies of the invention shall embrace,
in addition to full length immunoglobulins, various antigen-binding
fragments thereof, which recognize full-length .alpha.-synuclein,
and/or specific fragments of .alpha.-synuclein generated by
proteolysis.
[0063] The term "antibody" is used herein in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g. bispecific antibodies)
formed from at least two intact antibodies, antibody fragments, so
long as they exhibit the desired biological activity, and antibody
like molecules such as scFv. A native antibody usually refers to
heterotetrameric glycoproteins composed of two identical light (L)
chains and two identical heavy (H) chains. Each heavy and light
chain has regularly spaced intrachain disulfide bridges. Each heavy
chain has at one end a variable domain (VH) followed by a number of
constant domains. Each light chain has a variable domain at one end
(VL) and a constant domain at its other end; the constant domain of
the light chain is aligned with the first constant domain of the
heavy chain, and the light-chain variable domain is aligned with
the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light- and
heavy-chain variable domains.
[0064] Certain portions of the variable domains differ extensively
in sequence among antibodies and are used in the binding and
specificity of each particular antibody for its particular antigen.
However, the variability is not evenly distributed throughout the
variable domains of antibodies. It is concentrated in three or four
segments called "complementarity-determining regions" (CDRs) or
"hypervariable regions" in both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four or five
FR regions, largely adopting a .beta.-sheet configuration,
connected by the CDRs, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The CDRs in each
chain are held together in close proximity by the FR regions and,
with the CDRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., NIH Publ.
No. 91-3242, Vol. I, pages 647-669 (1991)). The constant domains
are not necessarily involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0065] A hypervariable region or CDR as used herein defines a
subregion within the variable region of extreme sequence
variability of the antibody, which form the antigen-binding site
and are the main determinants of antigen specificity. According to
one definition, they can be residues (Kabat nomenclature) 24-34
(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region
and residues (Kabat nomenclature 31-35 (H1), 50-65 (H2), 95-102
(H3) in the heavy chain variable region. Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institute of Health, Bethesda, Md. [1991]). An "intact"
antibody is one which comprises an antigen-binding variable region
as well as a light chain constant domain (CL) and heavy chain
constant domains, CHI, CH2 and CH.sub.3. The constant domains may
be native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector functions.
Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from antibody
phage libraries. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
[0066] According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. "Antibody
fragments" comprise a portion of an intact antibody, preferably the
antigen binding or variable region of the intact antibody. Examples
of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv
fragments; diabodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. Papain
digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments, each with a single
antigen-binding site, and a residual "Fc" fragment, whose name
reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen. "Fv" is the
minimum antibody fragment which contains a complete
antigen-recognition and -binding site. This region consists of a
dimer of one heavy- and one light-chain variable domain in tight,
non-covalent association. It is in this configuration that the
three CDRs of each variable domain interact to define an
antigen-binding site on the surface of the VH-VL dimer.
Collectively, the six CDRs confer antigen-binding specificity to
the antibody. However, even a single variable domain (or half of an
Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0067] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0068] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at about position Cys226, or from about
position Pro230, to the carboxyl-terminus of the Fc region. The Fc
region of an immunoglobulin generally comprises two constant
domains, a CH2 domain and a CH3 domain, and optionally comprises a
CH4 domain. By "Fc region chain" herein is meant one of the two
polypeptide chains of an Fc region.
[0069] The "hinge region," and variations thereof, as used herein,
includes the meaning known in the art, which is illustrated in, for
example, Janeway et al., Immuno Biology: the immune system in
health and disease (Elsevier Science Ltd., NY) (4th ed., 1999).
Depending on the amino acid sequence of the constant domain of
their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known. The "light chains" of antibodies
(immunoglobulins) from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa (K) and lambda
(.lamda.), based on the amino acid sequences of their constant
domains.
Target Populations
[0070] Subjects who are candidates for the ICE inhibitor therapy
described herein may be at present symptomatic or asymptomatic of
one for more forms of synucleinopathies. In some embodiments, the
candidate subject (e.g., patient) has been diagnosed as suffering
from or susceptible to at least one form of synucleinopathy, such
as Parkinson's disease.
[0071] In some embodiments, the candidate subject (e.g., patient)
has not been diagnosed with a synucleinopathy but is considered at
risk of developing at least one form of synucleinopathy. For
example, the subject may carry a genetic allele that renders him or
her susceptible to such a disease. In some cases, the subject's
family history may indicate the risk.
[0072] It has been known for several years that Lewy Bodies, the
aggregates found in the dying neurons of Parkinson's Disease (PD)
patients, contain, in addition to ubiqutin and full-length
.alpha.-synuclein, a fragment of .alpha.-synuclein that appears to
have been produced by specific proteolytic cleavage at around
residue 120. Notably, several in vitro studies have shown that this
fragment aggregates more readily than the full-length protein,
leading a number of investigators to speculate that the fragment
may nucleate aggregation in vivo (1) Inhibition of the proteolytic
cleavage that produces the more toxic fragment would represent an
attractive new strategy for preventing or arresting the disease;
however, until now, the identity of the in vivo enzyme(s)
responsible for .alpha.-synuclein cleavage remained unknown. As
described in Exemplification below, the inventors of the instant
application have for the first time identified ICE to be at least
one of the target enzymes, which can be inhibited to reduce the
proteolytic cleavage of .alpha.-synuclein.
[0073] According to the present invention, individuals for whom
therapy with one or more ICE regulators, and particularly with one
or more ICE inhibitors, is indicted include individuals in which
.alpha.-synuclein is cleaved by ICE and/or .alpha.-synuclein
aggregates are generated as a result of ICE activity. Such
individuals can be identified using any of a number of
methodologies including, for example, those that permit detection
and/or quantification of levels and/or activity of ICE and/or of
products of ICE cleavage.
[0074] As described herein, levels of .alpha.-synuclein cleavage
products, and particularly of .alpha.-synuclein fragments produced
by cleavage of .alpha.-synuclein by ICE, can act as biomarkers that
indicate presence of and/or susceptibility to a disease, disorder
or condition, and/or as biomarkers that identify patients likely
(or unlikely) to respond to therapy with ICE regulators (e.g., ICE
inhibitors).
[0075] Among other things, the present invention provide kits
comprising reagents suitable for detection and/or quantification of
relevant biomarkers as described herein. In some embodiments, such
reagents include one or more antibodies, including, for example,
one or more antibodies that detects an .alpha.-synuclein fragment
and/or full-length .alpha.-synuclein as described herein.
ICE Regulators
[0076] The present invention methods for screening for,
identifying, and or characterizing agents (e.g., a compound or
compounds) that inhibit (or stimulate) ICE cleavage of
.alpha.-synuclein. Such compounds can be used to treat a variety of
diseases or conditions associated with abnormal .alpha.-synuclein
processing and/or aggregation.
[0077] According to the invention, a provided method for
identifying and/or characterizing an ICE inhibitor comprises the
following steps: (1) a plurality of test compounds are provided,
where the test compounds contain candidate ICE inhibitor(s); (2)
the test compounds are contacted with full-length .alpha.-synuclein
in the presence of ICE (e.g., in a proteolysis reaction); and (3)
it is determined whether one or more of the test compounds inhibit
ICE-dependent cleavage of the full-length .alpha.-synuclein.
[0078] Typically, relative degree of proteolysis of
.alpha.-synuclein is determined by measuring relative levels of
full-length .alpha.-synuclein and cleaved .alpha.-synuclein in a
reaction. A test compound which is an ICE inhibitor can reduce the
amount of ICE-induced cleavage of .alpha.-synuclein under otherwise
identical conditions. Therefore, a higher ratio of full-length
.alpha.-synuclein to cleaved (fragment) .alpha.-synuclein in the
presence of the test compound as compared to a suitable control
indicates that the test compound is an ICE inhibitor that inhibits
ICE cleavage of .alpha.-synuclein. "A suitable control" may be a
compound known to be inert or otherwise inactive as to modulating
ICE activity, or may simply comprise the vehicle (e.g., reaction
buffer) alone.
[0079] Relative levels of full-length and cleaved .alpha.-synuclein
may be measured by any suitable methods, such as protein
immuno-blotting (e.g., Western blot) and mass spectrometry. A
number of suitable techniques are known to those skilled in the
art.
[0080] A plurality of test compounds to be screened, identified,
and/or characterized may comprise any variety of molecules. The
term "compound" or "chemical compound" as used herein can include
organometallic compounds, organic compounds, metals, transitional
metal complexes, and small molecules. In certain embodiments,
polynucleotides are excluded from the definition of compounds. In
certain embodiments, polynucleotides and peptides are excluded from
the definition of compounds. In a certain embodiment, the term
compounds refers to small molecules (e.g., preferably, non-peptidic
and non-oligomeric) and excludes peptides, polynucleotides,
transition metal complexes, metals, and organometallic
compounds.
[0081] Thus, candidate molecules may be one or more of a small
molecule, a peptide, or a nucleic acid. The nucleic acids may be,
for example, an RNA or DNA molecule, e.g., mRNA, RNAi, siRNA or an
oligonucleotide.
[0082] "Small Molecule": As used herein, the term "small molecule"
refers to a non-peptidic, non-oligomeric organic compound either
synthesized in the laboratory or found in nature. Small molecules,
as used herein, can refer to compounds that are "natural
product-like", however, the term "small molecule" is not limited to
"natural product-like" compounds. Rather, a small molecule is
typically characterized in that it contains several carbon-carbon
bonds, and has a molecular weight of less than 1500, although this
characterization is not intended to be limiting for the purposes of
the present invention. Examples of "small molecules" that occur in
nature include, but are not limited to, taxol, dynemicin, and
rapamycin. Examples of "small molecules" that are synthesized in
the laboratory include, but are not limited to, compounds described
in Tan et al., ("Stereoselective Synthesis of over Two Million
Compounds Having Structural Features Both Reminiscent of Natural
Products and Compatible with Miniaturized Cell-Based Assays" J. Am.
Chem. Soc. 120:8565, 1998; incorporated herein by reference). In
certain embodiments, natural-product-like small molecules are
utilized.
[0083] In certain embodiments, the combinatorial libraries are
small organic molecule libraries including, but not limited to,
benzodiazepines, isoprenoids, thiazolidinones, metathiazanones,
pyrrolidines, morpholino compounds, and benzodiazepines. In another
embodiment, the combinatorial libraries comprise peptides; random
bio-oligomers; benzodiazepines; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates; peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
or carbohydrate libraries. Combinatorial libraries are themselves
commercially available (see, e.g., ComGenex, Princeton, N.J.;
Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd,
Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences,
Columbia, Md.; etc.).
Compounds
[0084] Suitable compounds described herein for use according to the
present invention include compounds incorporated herein by
reference, and pharmaceutically acceptable derivatives thereof,
that are particularly effective in the treatment and/or prevention
of diseases, disorders, and/or conditions of the present invention.
For instance, in some embodiments described compounds are useful in
the treatment and/or prevention of Parkinson's disease (including
idiopathic Parkinson's disease (PD)), Diffuse Lewy Body Disease
(DLBD) also known as Dementia with Lewy Bodies (DLB), combined
Alzheimer's and Parkinson disease and/or multiple system atrophy
(MSA).
[0085] In some embodiments, described compounds for use in
accordance with the present invention include any compound that
inhibits ICE.
[0086] In some embodiments, described compounds are those that
inhibit ICE selectively. In some embodiments, described compounds
are those that inhibit one or more enzymes in the caspase or
ICE/CED-3 family in addition or as an alternative to ICE.
[0087] In some embodiments, described compounds for use in
accordance with the present invention include, but are not limited
to, "WO 2005/117846" compounds. The phrase "WO 2005/117846
compounds" as used herein, refers to compounds as described and
depicted in any one of the following documents: WO 03/068242, WO
03/042169, WO 98/16505, WO 93/09135, WO 03/106460, WO 03/103677, WO
03/104231, WO 02/085899, WO 00/55114, WO 00/55127, WO 00/61542, WO
01/05772, WO 01/10383, WO 01/16093, WO 01/42216, WO 01/72707, WO
01/90070, WO 01/94351, WO 02/094263, WO 02/42278, U.S. Pat. No.
6,184,210, U.S. Pat. No. 6,184,244, U.S. Pat. No. 6,187,771, U.S.
Pat. No. 6,197,750, U.S. Pat. No. 6,242,422, April 2001 American
Chemical Society (ACS) Meeting in San Diego, Calif., USA, WO
02/22611, US 2002/0058630, WO 02/12638, WO 95/35308, U.S. Pat. No.
5,716,929, WO 97/22619, U.S. Pat. No. 6,204,261, WO 99/47545, WO
01/90063, US Patent Publication 2004/0014753, US Patent Publication
2004/0009966, US Patent Publication 2003/0236296, US Patent
Publication 2003/0096737, US Patent Publication 2003/0092703, US
Patent Publication 2002/0169177, U.S. Pat. No. 6,693,096, U.S. Pat.
No. 6,610,683, U.S. Pat. No. 6,531,467, U.S. Pat. No. 6,528,506,
U.S. Pat. No. 6,200,969, WO 2003/072528, WO 2003/032918, WO
01/00658, WO 98/10778, U.S. Pat. No. 6,716,818, U.S. Pat. No.
6,620,782, U.S. Pat. No. 6,566,338, U.S. Pat. No. 6,495,522, U.S.
Pat. Nos. 6,355,618, 6,153,591, WO 2005/003100, WO 2004/002401, WO
00/61542, WO 00/55114, WO 99/47154, U.S. Pat. No. 6,083,981, U.S.
Pat. No. 5,932,549, U.S. Pat. No. 5,919,790, U.S. Pat. No.
5,744,451, WO 2002/089749, WO 99/36426, WO 98/16505, WO 98/16504,
WO 98/16502, U.S. Pat. No. 6,316,415, U.S. Pat. No. 5,932,549, U.S.
Pat. No. 5,919,790, U.S. Pat. No. 5,744,451, EP 1082127, EP
1049703, EP 0932600, EP 0932598, WO 99/56765, WO 93/05071, EP
0600800 and EP 1378573 (which, as set forth herein, are all
incorporated by reference herein). In one embodiment, compounds for
use in this invention include those of WO 00/55114, WO 00/55127, WO
00/61542, WO 00/61542, WO 01/05772, WO 01/10383, WO 01/16093, WO
01/42216, WO 01/72707, WO 01/90070, WO 01/94351, US Publication
2003/0092703, WO 02/094263, US Publication 2002/0169177, U.S. Pat.
No. 6,184,210, U.S. Pat. No. 6,184,244, U.S. Pat. No. 6,187,771,
U.S. Pat. No. 6,197,750, U.S. Pat. No. 6,242,422, April 2001
American Chemical Society (ACS), meeting in San Diego, Calif.,
USA<WO 02/22611, US Publication 2002/0058630, US Publication
2003/0096737, WO 95/35308, WO 97/22619, WO 99/47545, and WO
01/90063. In another embodiment, compounds for use in this
invention include those of WO 04/058718, WO 04/002961, WO 95/35308,
WO 97/22619, WO 99/47545, and WO 01/90063. Alternately, compounds
for use in this invention include those of WO 95/35308, WO
97/22619, WO 99/47545, and WO 01/90063. Preferred compounds are
those recited in the claims of the above-referenced documents.
These compounds may be obtained by methods known to skilled
practitioners in the methods disclosed in documents cited
herein.
[0088] In some embodiments, described compounds for use in
accordance with the present invention include, but are not limited
to, "Wannamaker" compounds. The phrase "Wannamaker compounds" as
used herein, refers to compounds as described and depicted in any
one of the following documents: U.S. Ser. No. 12/165,838, WO
91/15577, WO 93/05071, WO 93/09135, WO 93/12076, WO 93/14777, WO
93/16710, WO 95/35308, WO 96/30395, WO 9633209 and WO 98/01133;
European patent applications 503, 561, 547, 699, 618, 223, 623,
592, and 623 606, and U.S. Pat. Nos. 5,434,248, 5,710,153,
5,716,929, 5,744,451, WO 95/26958; U.S. Pat. No. 5,552,400; and
Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996) (which, as
set forth herein, are all incorporated by reference herein).
[0089] In some embodiments, described "Wannamaker" compounds for
use in accordance with the present invention are peptide and/or
peptidyl inhibitors of ICE.
[0090] In some embodiments, described "Wannamaker" compounds for
use in accordance with the present invention are non-peptidyl
inhibitors of ICE.
[0091] In some embodiments, described "Wannamaker" compounds for
use in accordance with the present invention are inhibitors of ICE
that are reported to have a favorable in vivo profile. Exemplary
such compounds include, but are not limited to, compounds of the
following formula:
##STR00001##
wherein the various substituents are as defined and described in
U.S. Ser. No. 12/165,838.
[0092] In some embodiments, described compounds for use in
accordance with the present invention are pro-drugs of inhibitors
of ICE including, but not limited to, compounds as described and
defined in U.S. Ser. No. 12/165,838 (now U.S. Pat. No. 8,022,041,
the entirety of which is incorporated herein by reference).
[0093] In some embodiments, described compounds for use in
accordance with the present invention are of either of the
following formulae:
##STR00002##
[0094] In certain embodiments, described compounds for use in
accordance with the present invention are of either of the
following formulae:
##STR00003##
[0095] In certain embodiments, the compound is VX-765:
##STR00004##
[0096] In some embodiments, the compound is NCGC00185682:
##STR00005##
[0097] In some embodiments, described compounds for use in
accordance with the present invention include, but are not limited
to, "Zhang" compounds. The phrase "Zhang compounds" as used herein,
refers to compounds as described and depicted in Zhang et al.,
World J. Gastroenterol. 2007, 13(4): 623-627 (the entirety of which
is incorporated herein by reference). In some embodiments, a
described "Zhang" compound for use in accordance with the present
invention is
Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone.
[0098] In some embodiments, described compounds for use in
accordance with the present invention include, but are not limited
to, "Corasaniti" compounds. The phrase "Corasaniti compounds" as
used herein, refers to compounds as described and depicted in
Corasaniti et al., Toxicol. Lett. 2003, 4:139(2-3):213-9 (the
entirety of which is incorporated herein by reference). In some
embodiments, a described "Corasaniti" compound for use in
accordance with the present invention is
Ac-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) or
t-butoxycarbonyl-L-aspartic acid benzyl ester chloromethylketone
(Boc-Asp-(OBzl)-CMK).
[0099] In some embodiments, described compounds for use in
accordance with the present invention include aspartic acid analogs
as described and defined in WO 96/03982 (the entirety of which is
incorporated herein by reference).
[0100] In some embodiments, described compounds are characterized
in that they cause a detectable decrease (e.g., of at least an
amount such as at least 5%, at least 6%, at least 7%, at least 9%,
at least 10%, at least 11%, at least 12%, at least 13%, at least
14%, at least 15%, at least 16%, at least 17%, at least 18%, at
least 19%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or more) in the severity or frequency
of one or more symptoms of the disease, disorder, or condition of
the present invention, and/or delay of onset of one or more
symptoms of a disease, disorder, or condition of the present
invention.
[0101] In some embodiments, described compounds are characterized
in that they cause a detectable change the levels of biomarkers
associated with ICE inhibition.
[0102] In some embodiments, described compounds are characterized
in that they can inhibit or block pathophysiological effects of
certain diseases as set forth herein.
[0103] In some embodiments, described compounds, by inhibiting ICE,
directly facilitate the arrest or resolution of certain diseases
described herein, and/or facilitate the restoration of normal
functioning.
[0104] In some embodiments, described compounds are characterized
in that they inhibit cleavage of a full-length .alpha.-synuclein
polypeptide into two or more fragments. In certain embodiments,
described compounds are characterized in that the inhibit cleavage
of a full length .alpha.-synuclein polypeptide into at least two
fragments having lengths of about 120 amino acids and about 20
amino acids, or lengths of specifically 120 amino acids and 20
amino acids.
[0105] In some embodiments, described compounds are characterized
in that they inhibit proteolytic .alpha.-synuclein cleavage. In
certain embodiments, described compounds are characterized in that
they inhibit proteolytic cleavage at or around a site corresponding
to residue 120 in full length .alpha.-synuclein. In some
embodiments, described compounds are characterized in that they
lessen the degree of proteolytic .alpha.-synuclein cleavage.
[0106] In some embodiments, described compounds are characterized
in that they cause a higher ratio of full-length to cleaved
fragments of .alpha.-synuclein in the cell as compared to control.
In some embodiments, described compounds are characterized in that
they cause a higher ratio of full-length to cleaved fragments of
.alpha.-synuclein in the cell as compared to control. In certain
embodiments, a "higher ratio" is when the ratio of full-length to
cleaved fragments of .alpha.-synuclein in a treated cell is one,
two, three, four, five, six, seven, eight, nine, or ten times
higher than as compared to the control. In certain embodiments, a
"higher ratio" is when the ratio of full-length to cleaved
fragments of .alpha.-synuclein in a treated cell is at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% higher than
as compared to the control.
[0107] In some embodiments, described compounds are characterized
in that they are capable of penetrating the blood-brain barrier
(BBB) in a therapeutically effective amount. In some embodiments,
described compounds are formulated as prodrugs, wherein the prodrug
is capable of penetrating the blood-brain barrier (BBB), for
example, in an amount greater than that of the described compound
when not in prodrug form. In some embodiments, the prodrug passes
readily through the blood brain barrier. In certain embodiments,
the prodrug has a brain penetration index of at least one, two,
three, four, five, six seven, eight, nine, or ten times the brain
penetration index of the drug alone. In some embodiments, the
prodrug is stable in the environment of both the stomach and the
bloodstream and may be delivered by ingestion.
[0108] In some embodiments, a prodrug comprises a hydrolyzable
carrier. Once in the central nervous system, the prodrug, which
preferably is inactive, is hydrolyzed into the carrier and a
provided compound or analog thereof (and optionally another drug).
In some embodiments, the carrier is a normal component of the
central nervous system and is inactive and harmless. The compound
and/or drug, referred to herein as the "payload," once released
from the carrier, is active. In some embodiments, the carrier is a
fatty acid and comprises a partially-saturated straight chain
molecule having between about 16 and 26 carbon atoms, and more
preferably 20 and 24 carbon atoms. Examples of fatty acid carriers
are provided in U.S. Pat. Nos. 4,939,174; 4,933,324; 5,994,932;
6,107,499; 6,258,836; and 6,407,137, the disclosures of which are
incorporated herein by reference in their entirety.
[0109] In some embodiments, a described compound is a targeted
compound. As used herein, the phrase "targeted compound" refers to
any compound comprising a targeting moiety and a payload. In some
embodiments, a targeting moiety and payload are the same moiety
and/or compound. In some embodiments, a targeting moiety and
payload are different moieties and/or compounds. In some
embodiments, a targeting moiety and payload are encapsulated. In
some embodiments, a targeting moiety and a payload are covalently
bound. In some embodiments, a targeting moiety and a payload are
non-covalently bound. In some embodiments, a targeting moiety and a
payload are reversibly bound. In some embodiments, a targeting
moiety and a payload are irreversibly bound. A "targeting" moiety,
as used herein, is any moiety that facilitates delivery of a
payload to a desired site with greater selectivity and/or
specificity for that site than would be achieved in the absence of
the targeting moiety. In some embodiments, a targeting moiety
facilitates penetration of the blood-brain barrier.
[0110] A "payload," as used herein, is any one or more compounds
used in the treatment and/or prevention of diseases, disorders,
and/or conditions of the present invention. In some embodiments, a
payload is a WO 2005/117846 compound. In some embodiments, a
payload is a Wannamaker compound. In some embodiments, the
Wannamaker compound is selected from the compounds disclosed in
U.S. Ser. No. 12/165,838 (now U.S. Pat. No. 8,022,041). In some
embodiments, a payload is the Vertex prodrug VX-765, depicted
below:
##STR00006##
In some embodiments, a payload is a Zhang compound. In some
embodiments, a payload is a Corasaniti compound. In some
embodiments, a payload is an aspartic acid analog as described and
defined in WO 96/03982.
[0111] In some embodiments, a payload is the NIH compound
NCGC00185682, depicted below:
##STR00007##
In some embodiments, a payload is any one of the ICE inhibitors
described and defined herein and/or incorporated by reference
herein. In some embodiments, a payload is an antioxidant. In some
embodiments, a payload is an antioxidant that is capable of
reducing oxidative stress such that activation of caspase-1 is
inhibited. Compounds to be Screened, Identified, and/or
Characterized
[0112] Compounds to be screened, identified, and/or characterized
using one or more methods described herein can be of any of a
variety of chemical classes. In some embodiments, such compounds
are small organic molecules having a molecular weight in the range
of 50 to 2,500 daltons. Such compounds can comprise functional
groups involved in structural interaction with proteins (e.g.,
hydrogen bonding), and typically include at least an amine,
carbonyl, hydroxyl, or carboxyl group, and preferably at least two
such functional chemical groups. Such compounds often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures (e.g., purine core) substituted with one or
more of the above functional groups.
[0113] In some embodiments, compounds are biomolecules such as, for
example, polypeptides, peptidomimetics (e.g., peptoids), amino
acids, amino acid analogs, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives or structural analogues thereof,
polynucleotides, nucleic acid aptamers, polynucleotide analogs,
carbohydrates, lipids, etc., or combinations thereof. In some
embodiments, compounds are antioxidants. In some embodiments,
compounds are antioxidants and are screened for the ability to
reduce oxidative stress such that activation of caspase-1 is
inhibited.
[0114] Compounds can be obtained or provided from any of a number
of potential sources, including: chemical libraries, natural
product libraries, and combinatorial libraries comprised of random
peptides, oligonucleotides, or organic molecules. Chemical
libraries consist of diverse chemical structures, some of which are
analogs of known compounds or analogs or compounds that have been
identified as "hits" or "leads" in other drug discovery screens,
while others are derived from natural products, and still others
arise from non-directed synthetic organic chemistry. Natural
product libraries re collections of microorganisms, animals,
plants, or marine organisms which are used to create mixtures for
screening by: (1) fermentation and extraction of broths from soil,
plant or marine microorganisms, or (2) extraction of plants or
marine organisms. Natural product libraries include polypeptides,
non-ribosomal peptides, and variants (non-naturally occurring)
thereof. For a review, see Science 282:63-68 (1998). Combinatorial
libraries are composed or large numbers of peptides,
oligonucleotides, or organic compounds as a mixture. These
libraries are relatively easy to prepare by traditional automated
synthesis methods, PCR, cloning, or proprietary synthetic methods.
Still other libraries of interest include peptide, protein,
peptidomimetic, multiparallel synthetic collection,
recombinatorial, and polypeptide libraries. In some embodiments, a
chemical "library" contains only compounds that are structurally
related to one another (e.g., share at least one common structural
moiety; in many embodiments, a common core). In some embodiments, a
chemical "library" contains a plurality, and in some embodiments, a
majority of compounds that are structurally related. In some
embodiments, a chemical "library" contains a least one compound
that is not structurally related (or not structurally significantly
related) to other compounds in the library.
[0115] For a review of combinatorial chemistry and libraries
created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707
(1997). Identification of test compounds through the use of the
various libraries herein permits subsequent modification of the
test compound "hit" or "lead" to optimize the capacity of the "hit"
or "lead" to inhibit ICE in a mammalian cell.
[0116] Compounds for use in accordance with the present invention
can be synthesized by any chemical or biological method. The
compounds identified above can also be pure, or may be in a
heterologous composition (e.g., a pharmaceutical composition), and
can be prepared in an assay-, physiologic-, or
pharmaceutically-acceptable diluent or carrier as described in
further detail herein (see Pharmaceutical Compositions and Methods
of Treatment below).
[0117] Pharmaceutical compositions and methods provided herein are
useful for treating various conditions associated with
ICE-dependent .alpha.-synuclein proteolysis.
[0118] Pharmaceutical compositions and methods provided herein are
useful for treating or preventing the various diseases, disorders,
and conditions as set forth herein.
[0119] The invention provides several screening methods to identify
agents having a pharmacological activity useful in treating a
synucleinopathy. The methods include screens that can be performed
in vitro, in cells or transgenic animals, and which test a variety
of parameters as an indication of activity. Agents determined to
have an activity in these screens can be retested in secondary
screens of animal models of synucleinopathy or in clinical trials
to determine activity against behavioral or other symptoms of these
diseases.
[0120] As outlined below, the screening, identifying, and/or
characterizing methods contemplated herein include in vitro as well
as in vivo (e.g., cell and animal) assay systems. In some
embodiments, a compound is considered to be an inhibitor if
reduction of cleaved .alpha.-synuclein of at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or more is observed in one or
more assays as described herein. In some embodiments, a compound is
considered to be an inhibitor if reduction of cleaved
.alpha.-synuclein of at least 2, 3, 4, 5, 6, 7, 8, 9 or more times
is observed in one or more assays as described herein.
In Vitro Assays
[0121] Thus, in certain embodiments, enzymatic (e.g., proteolysis)
assays are carried out in vitro in which ICE cleavage of
.alpha.-synuclein is measured in the presence or absence of various
test compounds. The assay is performed in the presence of a
specific concentration of the test agent or appropriate control
under specific conditions. Specific conditions include types of
buffers, concentrations of agent, solvent agent is dissolved or
suspended in, pH, temperature, time of incubation, etc. These
particular parameters may be determined by the operator or
scientist conducting the assay as would be appreciated by one of
skill in this art. Modulatory effects of any test compound on
.alpha.-synuclein cleavage by ICE can be determined by measuring
relative levels of full-length verses cleaved products of
.alpha.-synuclein in the sample. For example, if in the presence of
a test compound, there is less degree of .alpha.-synuclein
cleavage, then the test compound is a candidate inhibitor of ICE.
In some embodiments, a candidate inhibitor of ICE is an
antioxidant. In some embodiments, a candidate inhibitor of ICE is
an antioxidant that inhibits activation of ICE.
Cell-Based Assays
[0122] In certain embodiments, ICE cleavage of .alpha.-synuclein
may be assayed using cellular systems. Thus, methods for
identifying and/or characterizing candidate compounds which may
inhibit ICE cleavage of .alpha.-synuclein in a cell comprise
contacting a cell expressing .alpha.-synuclein and ICE with a test
compound, and determining the modulatory effect of the test
compound on a phenotype of the cell with respect to the
presence/levels of full-length and cleaved products of
.alpha.-synuclein, and/or aggregate formation of .alpha.-synuclein
in the cell. In such methods, modulation of the phenotype is
indicative of the efficacy of the compound. In particular, if the
test compound causes a higher ratio of full-length to cleaved
fragments of .alpha.-synuclein in the cell as compared to control,
then the test compound is a candidate inhibitor of ICE. In some
embodiments, the phenotype of cells to be assayed includes
measuring .alpha.-synuclein aggregate levels.
[0123] The cell types suitable for use in the cellular screening,
identification, and/or characterization assays may be any cells
that express both .alpha.-synuclein and ICE. In some embodiments,
either one or both of .alpha.-synuclein and ICE are endogenously
expressed. In other embodiments, either one or both of
.alpha.-synuclein and ICE are introduced into the cells by
transfection or infection of exogenous genes. In some embodiments,
introduction of one or more exogenous genes or fragments thereof
involves a transgenic animal model (discussed in further detail
below). For example, cells suitable for described methods may be
obtained from a transgenic animal source.
[0124] The contacting may be by adding the candidate compound to
the media, directly to the cells, or as a fluid flowing over the
cell, e.g., in a lateral flow or a planar flow patch clamp device.
One of skill in the art would be able to identify other appropriate
methods having the benefit of this disclosure.
Animal Models
[0125] In certain embodiments, the screening methods of the
invention may employ one or more animal models. For example,
transgenic mice expressing various alleles of .alpha.-synuclein may
be used to screen for compounds that may inhibit ICE cleavage of
.alpha.-synuclein in vivo. A number of transgenic mouse lines that
exhibit Parkinson's disease-like phenotype are commercially
available. These include, without limitation, the following
strains:
[0126] B6.129P2-Sncg.sup.tm1Vlb/J;
[0127] B6.Cg-Tg(THY1-SNCA*A53T)F53Sud/J;
[0128] B6.Cg-Tg(THY1-SNCA*A53T)M53Sud/J;
[0129] B6;129-Gt(ROSA)26Sor.sup.tm1(SNCA*A53T)Djmo/TmdJ;
[0130] B6;129-Gt(ROSA)26Sor.sup.tm2(SNCA*119)Djmo/TmdJ;
[0131] B6;129-Gt(ROSA)26Sor.sup.tm3(SNCA*E46K)Djmo/TmdJ;
[0132] B6;129X1-Snca.sup.tm1Rosl/J;
[0133] B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J;
[0134] STOCK Tg(THY1-SNCA*A53T)F53Sud/J;
[0135] B6.129-Sncb.sup.tm1Sud/J;
[0136] B6;129-Snca.sup.tm1SudSncb.sup.tm1.1Sud/J;
[0137] B6;SJL-Tg(THY1-SNCA*A30P)M30Sud/J;
[0138] C57BL/6-Tg(THY1-SNCA)1Sud/J; and,
[0139] STOCK Tg(THY1-Snca)M1mSud/J (available from Jackson
Lab).
[0140] In certain embodiments, the method involves genetic
screening to identify a gene involved in .alpha.-synuclein
processing. For example, the gene knock-down or knockout technique
can be employed to rescue a cellular or systematic phenotype, such
as cellular abnormalities or pathogenic features that can be
detected. A number of model systems may be suitable, including but
are not limited to yeast and rodents, such as mice and rats.
Methods for Identifying Other Enzymes
[0141] Processing of full-length .alpha.-synuclein to truncated
fragments is catalyzed by one or more proteases. Therefore, the
present invention in a further aspect provides screening methods
for identifying enzymes (e.g., proteases) that cleave
.alpha.-synuclein in vitro and/or in vivo. The method used to
identify ICE as an .alpha.-synuclein protease is described in the
Exemplification section below. The invention contemplates
identifying additional proteases that cleave .alpha.-synuclein.
Such additional proteases are candidates for therapeutic targets
for the treatment of Parkinson's disease and other
.alpha.-synuclein-associated diseases and conditions.
[0142] In certain embodiments, the protease may be purified in
vitro using a substrate peptide (e.g., peptide inhibitor)
identified by the screening methods discussed above. A preferred
inhibitor is a peptide of alpha-synuclein of e.g., at least about 5
but up to 20 contiguous amino acids of full-length
.alpha.-synuclein. In some embodiments, the peptide includes
residues 113, 114, 115 116, 117, 118, 119, 120, 121, 122, 123, 124,
135, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139 and/or 140. In some embodiments, the peptide includes
residues 114-117, 111-126, 113-126, 113-119, 117-121 or 120-125, or
130-136, 132-138, 131-135, 133-134, 133-137, or 135-136, in which a
residue N-terminal to the cleavage site (e.g., between residues
115-116, 119-120, 122-123, 133-134 and 135-136) has been replaced
by a transition state analog. Such an inhibitor is used as an
affinity purification reagent to purify the protease from extracts
of brain cells. Such cells can be obtained from cadaver of a normal
individual or one who has suffered from a LBD disease. Levels of
protease may be elevated in the latter. The enzymatic activity of a
protease can be assayed by presenting it with an alpha-synuclein
substrate and monitoring formation of cleavage products.
End-specific antibodies described below are useful for detecting
cleavage products. A substrate can be, for example, the natural
human form of alpha-synuclein described above, a fragment thereof,
containing residues flanking both sides of the cleavage site, or a
mutant form thereof in which the mutation is associated with a
hereditary form of LBD. Optionally, the C-terminus of the substrate
can be immobilized to a solid phase, and the N-terminus to a label.
Cleavage of a substrate releases the label to a liquid phase. The
liquid phase can readily be separated from the solid phase, and the
amount of label quantified as a measure of proteolytic
activity.
Therapy
[0143] Based on the finding that ICE cleaves .alpha.-synuclein to
generate fragments that are more prone to aggregate to form Lewy
Bodies (LB), the invention provides methods for treating a subject
suffering from or at risk of developing at least one form of
synucleinopathies. Provided methods comprise administering to the
subject an effective amount of ICE inhibitor to inhibit
.alpha.-synuclein cleavage so as to reduce the formation of toxic
.alpha.-synuclein fragments in cells.
[0144] The term "synucleinopathy," as used herein, refers to a
disease, disorder or condition associated with abnormal expression,
stability, activities and/or cellular processing of
.alpha.-synuclein. Thus the term embraces so-called Lewy Body
Disease (LBD) which is characterized by degeneration of the
dopaminergic system, motor alterations, cognitive impairment, and
formation of Lewy bodies (LBs). (McKeith et al., Clinical and
pathological diagnosis of dementia with Lewy bodies (DLB): Report
of the CDLB International Workshop, Neurology (1996) 47:1113-24).
Lewy Bodies are spherical protein deposits found in affected nerve
cells. Their presence in the brain disrupts the brain's normal
function interrupting the action of neurotransmitters including
acetylcholine and dopamine Synucleinopathies include Parkinson's
disease (including idiopathic Parkinson's disease (PD)), Diffuse
Lewy Body Disease (DLBD) also known as Dementia with Lewy Bodies
(DLB), Combined Alzheimer's and Parkinson disease and multiple
system atrophy (MSA). DLBD shares symptoms of both Alzheimer's and
Parkinson's disease (including Parkinson's disease chemically
induced by exposure to environmental agents such as pesticides,
insecticides, or herbicides and/or metals such as manganese,
aluminum, cadmium, copper, or zinc, SNCA gene-linked Parkinson's
disease, sporadic or idiopathic Parkinson's disease, or Parkin- or
LRRK2-linked Parkinson's disease). DLBD differs from Parkinson's
disease mainly in the location of Lewy Bodies. In DLBD Lewy Bodies
form mainly in the cortex. In Parkinson's disease, they form mainly
in the substantia nigra. Other synucleinopathies include Pure
Autonomic Failure, Lewy body dysphagia, Incidental LBD, Inherited
LBD (e.g., mutations of the alpha-synuclein gene, PARK3 and PARK4),
and Multiple System Atrophy (e.g., Olivopontocerebellar Atrophy,
Striatonigral Degeneration and Shy-Drager Syndrome).
[0145] Thus, the compositions and methods described herein are
useful for treating various conditions associated with
ICE-dependent .alpha.-synuclein proteolysis.
Testing Biological Samples
[0146] As described herein, among other things the present
invention provides methods and systems relating to characterization
of biological samples, e.g., from subjects thought or known to be
suffering from or susceptible to one or more forms of
syucleinopathy and/or thought or known to be candidates for the ICE
inhibitor therapy. For example, in certain embodiments, a candidate
subject is tested for the presence of .alpha.-synuclein cleavage
products. The present inventors of the invention have found that it
is possible to detect .alpha.-synuclein in biological samples, such
as a blood sample.
[0147] For example, the present inventors have shown that
erythrocytes contain .alpha.-synuclein, which can be detected by
routine methods known to the art. Therefore, biological samples,
such as a blood sample can be collected from a patient, and the
presence and/or relative levels of full-length and cleaved
.alpha.-synuclein can be determined. In a biological sample
collected from a typical healthy human subject, the cleaved form of
.alpha.-synuclein is virtually undetectable. Therefore, the mere
presence (detectable amount) of .alpha.-synuclein cleavage products
in a sample collected from a patient should indicate the
pathogenesis of synucleinopathies. Amongst subjects who are
positive for .alpha.-synuclein cleavage products, elevated levels
of the cleaved fragments relative to full-length counterpart may
indicate corresponding severity of the disease.
[0148] Generally, a sample to be tested for the presence or amount
of .alpha.-synuclein (full-length or cleaved fragments) may be
collected from an animal (e g, mammal) or extracts thereof; and
blood, saliva, urine, feces, semen, tears, or other body fluids or
extracts thereof. For example, the term "biological sample" refers
to any solid or fluid sample obtained from, excreted by or secreted
by any living organism, including single-celled micro-organisms
(such as bacteria and yeasts) and multicellular organisms (such as
plants and animals, for instance a vertebrate or a mammal, and in
particular a healthy or apparently healthy human subject or a human
patient affected by a condition or disease to be diagnosed or
investigated). A biological sample can be in any form, including a
solid material such as a tissue, cells, a cell pellet, a cell
extract, cell homogenates, or cell fractions; or a biopsy, or a
biological fluid. A biological fluid may be obtained from any site
(e.g., blood, saliva (or a mouth wash containing buccal cells),
tears, plasma, serum, urine, bile, cerebrospinal fluid, amniotic
fluid, peritoneal fluid, and pleural fluid, or cells therefrom,
aqueous or vitreous humor, or any bodily secretion), a transudate,
an exudate (e.g. fluid obtained from an abscess or any other site
of infection or inflammation), or fluid obtained from a joint (e.g.
a normal joint or a joint affected by disease such as rheumatoid
arthritis, osteoarthritis, gout or septic arthritis). A biological
sample can be obtained from any organ or tissue (including a biopsy
or autopsy specimen) or may comprise cells (whether primary cells
or cultured cells) or medium conditioned by any cell, tissue or
organ. Biological samples may also include sections of tissues such
as frozen sections taken for histological purposes. Biological
samples also include mixtures of biological molecules including
proteins, lipids, carbohydrates and nucleic acids generated by
partial or complete fractionation of cell or tissue homogenates. In
certain embodiments, the biological sample is a blood sample
containing erythrocytes. Although the sample is preferably taken
from a human subject, biological samples may be from any animal,
plant, bacteria, virus, yeast, etc. The term animal, as used
herein, refers to humans as well as non-human animals, at any stage
of development, including, for example, mammals, birds, reptiles,
amphibians, fish, worms and single cells. Cell cultures and live
tissue samples are considered to be pluralities of animals. In
certain exemplary embodiments, the non-human animal is a mammal
(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat,
a sheep, cattle, a primate, or a pig). An animal may be a
transgenic animal or a human clone. If desired, the biological
sample may be subjected to preliminary processing, including
preliminary separation techniques.
[0149] The present invention also provides methods of in vivo
detection of synucleinopathy in a patient. Such methods are useful
to diagnose or confirm diagnosis of a synucleinopathy of PD or
susceptibility thereto. For example, such methods can be used on a
patient presenting with symptoms of dementia or motor impairment.
If the patient has LBs, then the patient is likely suffering from a
synucleinopathy. Such methods can also be used on asymptomatic
patients. Presence of abnormal deposits of amyloid indicates
susceptibility to future symptomatic disease. Such methods are also
useful for monitoring disease progression and/or response to
treatment in patients who have been previously diagnosed with a
synucleinopathy.
[0150] As stated above, patients amenable to treatment include
individuals at risk of disease of a synucleinopathy but not showing
symptoms, as well as patients presently showing symptoms.
Therefore, the present methods can be administered prophylactically
to individuals who have a known genetic risk of a synucleinopathy.
Such individuals include those having relatives who have
experienced this disease, and those whose risk is determined by
analysis of genetic or biochemical markers, as well as
environmental risk factors. Genetic markers of risk toward PD
include mutations in the alpha-synuclein or Parkin, UCHL1, and
CYP2D6 genes; particularly mutations at positions 30 and 53 of the
alpha-synuclein gene. Individuals presently suffering from
Parkinson's disease can be recognized from its clinical
manifestations including resting tremor, muscular rigidity,
bradykinesia and postural instability.
[0151] In some embodiments, a patient is free of clinical symptoms
or risk factors any amyloidogenic disease other than one
characterized by Lewy bodies. In some embodiments, a patient is
free of clinical symptoms or risk factors of any disease
characterized by extracellular amyloid deposits. In some
embodiments, a patient is free of diseases characterized by amyloid
deposits of A.beta. peptide. In some embodiments, a patient is free
of clinical symptoms and risk factors of Alzheimer's disease. In
some methods, a patient has concurrent Alzheimer's disease and a
disease characterized by Lewy bodies. In some embodiments, a
patient has concurrent Alzheimer's and Parkinson's disease.
[0152] In some embodiments, a candidate subject for receiving an
ICE inhibitor therapy described herein for the treatment of
synucleinopathy is not being treated for a known inflammatory
condition, where the ICE inhibitor is administered for purposes of
inhibiting pro-inflammatory cytokine production or signaling.
Common inflammatory conditions for which an ICE inhibitor is
administered for purposes of inhibiting pro-inflammatory cytokines
include arthritis, asthma and other allergic conditions. The most
common cellular ICE targets (substrates) for these conditions
include cytokines, such as precursors of IL-1.beta. and IL-18,
which promote the Th2 immunity upon cleavage by ICE and therefore
are pro-inflammatory.
[0153] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, 30, etc.). Usually, however, it is not necessary to
begin treatment until a patient reaches 40, 50, 60 or 70. Treatment
typically entails multiple dosages over a period of time.
Effectiveness of a treatment can be evaluated by determining a
subject's responsiveness to the treatment. In some embodiments, it
may be monitored by assaying relative amounts of full-length and
cleaved .alpha.-synuclein proteins in a biological sample collected
from the subject (e.g., patient). In certain embodiments, detecting
the mere presence of certain .alpha.-synuclein fragments (cleavage
products) in a biological sample may be indicative of a
pathological condition. In some embodiments, levels of antibody, or
activated T-cell or B-cell responses to a therapeutic agent (e.g.,
a truncated form of alpha-synuclein peptide) may be monitored over
time. In some embodiments, two or more parameters are combined to
confirm diagnosis or responsiveness to a therapy over time.
[0154] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a synucleinopathy in a regime comprising an
amount and frequency of administration of the composition or
medicament sufficient to eliminate or reduce the risk, lessen the
severity, or delay the outset of the disease, including
physiological, biochemical, histologic and/or behavioral symptoms
of the disease, its complications and intermediate pathological
phenotypes presenting during development of the disease. In
therapeutic applications, compositions or medicates are
administered to a patient suspected of, or already suffering from
such a disease in a regime comprising an amount and frequency of
administration of the composition sufficient to cure, or at least
partially arrest, the symptoms of the disease (physiological,
biochemical, histologic and/or behavioral), including its
complications and intermediate pathological phenotypes in
development of the disease.
[0155] An amount adequate to accomplish therapeutic or prophylactic
treatment is defined as a therapeutically- or
prophylactically-effective dose. A combination of amount and dosage
frequency adequate to accomplish therapeutic or prophylactic
treatment is defined as a therapeutically or
prophylactically-effective regime. In both prophylactic and
therapeutic regimes, agents are usually administered in several
dosages until a sufficient immune response has been achieved.
Typically, the immune response is monitored and repeated dosages
are given if the immune response starts to wane. For a subject with
a detectable level of ICE-cleaved .alpha.-synuclein in a biological
sample, e.g., blood sample, it is useful to monitor changes in the
levels of ICE-cleaved .alpha.-synuclein in samples collected over
time to determine the effectiveness of a therapy. For example, a
sample may be obtained from a subject having or at risk of
developing an .alpha.-synuclein-associated condition, and the
presence (e.g., levels) of ICE-cleaved .alpha.-synucleinis
measured. This is repeated after an interval, such as 2 weeks, 4
weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 24
months, etc. In some cases, levels of ICE-cleaved
.alpha.-synucleinis are measured before, during and/or after a
therapy. If a subject is responsive to a therapy, such as an ICE
inhibitor therapy, the level of ICE-cleaved .alpha.-synucleinis
detected in a biological sample following the therapy is expected
to fall.
[0156] In some embodiments, administration of an agent results in
reduction of intracellular levels of aggregated alpha-synuclein. In
some methods, administration of the agent results in a reduction in
levels of C-terminal truncated forms of alpha-synculein. In some
methods, administration of an agent results in improvement in a
clinical symptom of a synucleinopathy, such as motor or cognitive
function in the case of Parkinson's disease. In some methods,
reduction in intracellular levels of aggregated alpha-synuclein or
improvement in a clinical symptom of disease is monitored at
intervals after administration of an agent.
[0157] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the patient is a human but nonhuman mammals including
transgenic mammals can also be treated. Treatment dosages need to
be titrated to optimize safety and efficacy.
[0158] As provided further below, compounds described herein can
optionally be administered in combination with other agents that
are at least partly effective in treatment of synucleinopathy.
Compounds of the invention can also be administered in conjunction
with other agents that increase passage of the agents of the
invention across the blood-brain barrier.
Administration
[0159] The term "administration" or "administering" includes routes
of introducing the compound of the invention(s) to a subject to
perform their intended function. Examples of routes of
administration that may be used include injection (subcutaneous,
intravenous, parenterally, intraperitoneally, intrathecal), oral,
inhalation, rectal and transdermal. The pharmaceutical preparations
may be given by forms suitable for each administration route. For
example, these preparations are administered in tablets or capsule
form, by injection, inhalation, eye lotion, ointment, suppository,
etc., administration by injection, infusion or inhalation; topical
by lotion or ointment; and rectal by suppositories. Oral
administration is preferred. The injection can be bolus or can be
continuous infusion. Depending on the route of administration, the
compound of the invention can be coated with or disposed in a
selected material to protect it from natural conditions which may
detrimentally effect its ability to perform its intended function.
The compound of the invention can be administered alone, or in
conjunction with either another agent as described above or with a
pharmaceutically-acceptable carrier, or both. The compounds of the
invention can be administered prior to the administration of the
other agent, simultaneously with the agent, or after the
administration of the agent. Furthermore, the compound of the
invention can also be administered in a pro-form which is converted
into its active metabolite, or more active metabolite in vivo.
[0160] The language "biological activities" of a compound of the
invention includes all activities elicited by compound of the
inventions in a responsive cell. It includes genomic and
non-genomic activities elicited by these compounds.
[0161] The term "effective amount" as used herein includes an
amount effective, at dosages and for periods of time necessary, to
achieve the desired result, e.g., sufficient to treat a disorder.
An effective amount of compound of the invention may vary according
to factors such as the disease state, age, and weight of the
subject, and the ability of the compound of the invention to elicit
a desired response in the subject. Dosage regimens may be adjusted
to provide the optimum therapeutic response. An effective amount is
also one in which any toxic or detrimental effects (e.g., side
effects) of the compound of the invention are outweighed by the
therapeutically beneficial effects.
[0162] A therapeutically effective amount of compound of the
invention (e.g., an effective dosage) may range from about 0.001 to
30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body
weight, more preferably about 0.1 to 20 mg/kg body weight, and even
more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4
to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will
appreciate that certain factors may influence the dosage required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of a compound of the invention can include a
single treatment or, preferably, can include a series of
treatments. In one example, a subject is treated with a compound of
the invention in the range of between about 0.1 to 20 mg/kg body
weight, one time per week for between about 1 to 10 weeks,
preferably between 2 to 8 weeks, more preferably between about 3 to
7 weeks, and even more preferably for about 4, 5, or 6 weeks. It
will also be appreciated that the effective dosage of a compound of
the invention used for treatment may increase or decrease over the
course of a particular treatment.
Combination Therapy
[0163] It is further contemplated that the treatment method
comprising an ICE inhibitor described herein may be used in
combination with one or more additional therapeutics for the
treatment of synucleinopathy, such that the ICE inhibitor is
administered to a subject in conjunction with a synucleinopathy
therapy other than an ICE inhibitor. Additional therapeutic agents
that are normally administered to treat a particular disease or
condition may be referred to as "agents appropriate for the
disease, or condition, being treated."
[0164] "In conjunction with" means that the ICE inhibitor and
additional therapy or therapies are administered to a subject in
combination. The administrations may be simultaneous administration
or separate administrations.
[0165] Thus, in some embodiments of the present invention,
compounds described herein may be administered in combination with
one or more additional therapeutic agents. Such additional
therapeutic agents may be administered separately from a described
compound-containing composition, as part of a multiple dosage
regimen. Alternatively or additionally, such agents may be part of
a single dosage form, mixed together with a described compound in a
single composition. If administered as part of a multiple dosage
regime, the two active agents may be submitted simultaneously,
sequentially or within a period of time from one another normally
within five hours from one another.
[0166] As used herein, the terms "combination," "combined," and
related terms refer to the simultaneous or sequential
administration of therapeutic agents in accordance with this
invention. For example, a described compound may be administered
with another therapeutic agent simultaneously or sequentially in
separate unit dosage forms or together in a single unit dosage
form. Accordingly, the present invention provides a single unit
dosage form comprising a described compound, an additional
therapeutic agent, and a pharmaceutically acceptable carrier,
adjuvant, or vehicle. Two or more agents are typically considered
to be administered "in combination" when a patient or individual is
simultaneously exposed to both agents. In many embodiments, two or
more agents are considered to be administered "in combination" when
a patient or individual simultaneously shows therapeutically
relevant levels of the agents in a particular target tissue or
sample (e.g., in brain, in serum, etc.).
[0167] The amount of both a described compound and additional
therapeutic agent (in those compositions which comprise an
additional therapeutic agent as described above) that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. Preferably, compositions in accordance with the
invention should be formulated so that a dosage of between 0.01-100
mg/kg body weight/day of a described compound can be
administered.
[0168] In some embodiments of the invention, agents that are
utilized in combination may act synergistically. Therefore, the
amount of either agent utilized in such situations may be less than
that typically utilized or required in a monotherapy involving only
that therapeutic agent. Commonly, a dosage of between 0.01-1,000
rig/kg body weight/day of the additional therapeutic agent can be
administered.
[0169] The amount of additional therapeutic agent present utilized
in combination therapy according to the present invention typically
will be no more than the amount that would normally be administered
in a composition comprising that therapeutic agent as the only
active agent. Preferably the amount of additional therapeutic agent
utilized will range from about 50% to 100% of the amount normally
utilized in therapies involving that agent as the only
therapeutically active agent. Established dosing regimens for known
therapeutic agents are known in the art and incorporated herein by
reference.
[0170] For example, compounds described herein, or pharmaceutically
acceptable compositions thereof, can be administered in combination
with one or more treatments for Parkinson's Disease such as
L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,
bromocriptine, pergolide, trihexephendyl, and amantadine; For
example, methods of the present invention can be used in
combination with medications for treating PD. Such therapeutic
agents include levodopa, carbodopa, levodopa (Sinemet and Sinemet
CR), Stalevo (carbodopa, levodopa, and entacapone),
anticholinergics (trihexyphenidyl, benztropine mesylate,
procyclidine, artane, cogentin), bromocriptidine (Parlodel),
pergolide (Permax), ropinirol (Requip), pramipexole (Mirapex),
cabergoline (Dostinex), apomorphine (Apokyn), rotigotine (Neupro),
Ergolide, Mirapex or Requip.
[0171] In some embodiments, described compositions and formulations
may be administered in combination with one or more treatments for
Parkinson's Disease such as ACR-343, rotigotine(Schwarz),
rotigotine patch (UCB), apomorphine (Amarin), apomorphine
(Archimedes), AZD-3241 (Astra Zeneca), creatine (Avicena), AV-201
(Avigen), lisuride (Axxonis/Biovail), nebicapone (BIAL Group),
apomorphine (Mylan), CERE-120 (Ceregene), melevodopa+carbidopa
(Cita Neuropharmaceuticals), piclozotan (Daiichi), GM1 Ganglioside
(Fidia Farmaceutici), Altropane (Harvard University), Fluoratec
(Harvard University), fipamezole (Juvantia Pharma), istradefylline
(Kyowa Hakko Kogyo), GPI-1485 (MGI GP), Neu-120 (Neurim
Pharmaceuticals), NGN-9076 (NeuroGeneration Inc), NLX-P101
(Neurologix), AFQ-056 (Novartis), arundic acid (Ono/Merck &
Co), COMT inhibitor (Orion), ProSavin (Oxford Biomedica),
safinamide (Pharmacia & Upjohn), PYM-50028 (Phytopharm),
PTX-200 (Phytix), 123I-iometopane (Research Triangle Institute),
SYN-115 (Roche Holding), preladenant (Schering Plough), ST-1535
(Sigma-Tau Ind. Farm), ropinirole (SmithKline Beecham), pardoprunox
(Solvay), SPN-803 (Supernus Pharmaceuticals), nitisinone
(Syngenta), TAK-065 (Takeda), cell therapy (Titan Pharmaceuticals),
PD gene therapy (University of Auckland/Weill Medical College),
18F-AV-133 (University of Michigan), mitoquinone/mitoquinol redox
mixture (Antipodean Pharmaceuticals), 99m-Tc-tropantiol (University
of Pennsylvania), apomorphine (Vectura), BIIB-014 (Vernalis Group),
aplindore (Wyeth), and XP-21279 (XenoPort Inc).
[0172] Alternatively or additionally, in some embodiments,
described compositions and formulations may be administered in
combination with one or more treatments for Alzheimer's disease
such as Aricept.RTM. and Excelon.RTM.. In some embodiments,
described compositions and formulations may be administered in
combination with one or more treatments for Parkinson's Disease
such as ABT-126 (Abbott Laboratories), pozanicline (Abbott
Laboratories), MABT-5102A (AC Immune), Affitope AD-01 (AFFiRiS
GmbH), Affitope AD-02 (AFFiRiS GmbH), davunetide (Allon
Therapeutics Inc), nilvadipine derivative (Archer Pharmaceuticals),
Anapsos (ASAC Pharmaceutical International AIE), ASP-2535 (Astellas
Pharma Inc), ASP-2905 (Astellas Pharma Inc), 11C-AZD-2184
(AstraZeneca plc), 11C-AZD-2995 (AstraZeneca plc), 18F-AZD-4694
(AstraZeneca plc), AV-965 (Avera Pharmaceuticals Inc), AVN-101
(Avineuro Pharmaceuticals Inc), immune globulin intravenous (Baxter
International Inc), EVP-6124 (Bayer AG), nimodipine (Bayer AG),
BMS-708163 (Bristol-Myers Squibb Co), CERE-110 (Ceregene Inc),
CLL-502 (CLL Pharma), CAD-106 (Cytos Biotechnology AG), mimopezil
((Debiopharm SA), DCB-AD1 (Development Centre for Biotechnology),
EGb-761 ((Dr Willmar Schwabe GmbH & Co), E-2012 (Eisai Co Ltd),
ACC-001 (Elan Corp plc), bapineuzumab (Elan Corp plc), ELND-006
(Elan Pharmaceuticals Inc), atomoxetine (Eli Lilly & Co),
LY-2811376 (Eli Lilly & Co), LY-451395 (Eli Lilly & Co),
m266 (Eli Lilly & Co), semagacestat (Eli Lilly & Co),
solanezumab (Eli Lilly & Co), AZD-103 (Ellipsis
Neurotherapeutics Inc), FGLL (ENKAM Pharmaceuticals A/S), EHT-0202
(ExonHit Therapeutics SA), celecoxib (GD Searle & Co),
GSK-933776A (GlaxoSmithKline plc), rosiglitazone XR
(GlaxoSmithKline plc), SB-742457 (GlaxoSmithKline plc), R-1578
(Hoffmann-La Roche AG), HF-0220 (Hunter-Fleming Ltd), oxiracetam
(ISF Societa Per Azioni), KD-501 (Kwang Dong Pharmaceutical Co
Ltd), NGX-267 (Life Science Research Israel), huperzine A (Mayo
Foundation), Dimebon (Medivation Inc), MEM-1414 (Memory
Pharmaceuticals Corp), MEM-3454 (Memory Pharmaceuticals Corp),
MEM-63908 (Memory Pharmaceuticals Corp), MK-0249 (Merck & Co
Inc), MK-0752 (Merck & Co Inc), simvastatin (Merck & Co
Inc), V-950 (Merck & Co Inc), memantine (Merz & Co GmbH),
neramexane (Merz & Co GmbH), Epadel (Mochida Pharmaceutical Co
Ltd), 123I-MNI-330 (Molecular Neuroimaging Llc), gantenerumab
(MorphoSys AG), NIC5-15 (Mount Sinai School of Medicine), huperzine
A (Neuro-Hitech Inc), OXIGON (New York University), NP-12 (Noscira
SA), NP-61 (Noscira SA), rivastigmine (Novartis AG), ECT-AD (NsGene
A/S), arundic acid (Ono Pharmaceutical Co Ltd), PF-3084014 (Pfizer
Inc), PF-3654746 (Pfizer Inc), RQ-00000009 (Pfizer Inc), PYM-50028
(Phytopharm plc), Gero-46 (PN Gerolymatos SA), PBT-2 (Prana
Biotechnology Ltd), PRX-03140 (Predix Pharmaceuticals Inc),
Exebryl-1 (ProteoTech Inc), PF-4360365 (Rinat Neuroscience Corp),
HuCAL anti-beta amyloid monoclonal antibodies (Roche AG), EVT-302
(Roche Holding AG), nilvadipine (Roskamp Institute), galantamine
(Sanochemia Pharmazeutika AG), SAR-110894 (sanofi-aventis), INM-176
(Scigenic & Scigen Harvest), mimopezil (Shanghai Institute of
Materia Medica of the Chinese Academy of Sciences), NEBO-178
(Stegram Pharmaceuticals), SUVN-502 (Suven Life Sciences), TAK-065
(Takeda Pharmaceutical), ispronicline (Targacept Inc), rasagiline
(Teva Pharmaceutical Industries), T-817MA (Toyama Chemical),
PF-4494700 (TransTech Pharma Inc), CX-717 (University of
California), 18F-FDDNP (University of California Los Angeles),
GTS-21 (University of Florida), 18F-AV-133 (University of
Michigan), 18F-AV-45 (University of Michigan), tetrathiomolybdate
(University of Michigan), 123I-IMPY (University of Pennsylvania),
18F-AV-1/ZK (University of Pennsylvania), 11C-6-Me-BTA-1
(University of Pittsburgh), 18F-6-OH-BTA-1 (University of
Pittsburgh), MCD-386 (University of Toledo), leuprolide acetate
implant (Voyager Pharmaceutical Corp), aleplasinin (Wyeth),
begacestat (Wyeth), GSI-136 (Wyeth), NSA-789 (Wyeth), SAM-531
(Wyeth), CTS-21166 (Zapaq), and ZSET-1446 (Zenyaku Kogyo).
[0173] Alternatively or additionally, in some embodiments,
described compositions and formulations may be administered in
combination with one or more treatments for motor neuronal
disorders, such as AEOL-10150 (Aeolus Pharmaceuticals Inc),
riluzole (Aventis Pharma AG), ALS-08 (Avicena Group Inc), creatine
(Avicena Group Inc), arimoclomol (Biorex Research and Development
Co), mecobalamin (Eisai Co Ltd), talampanel (Eli Lilly & Co),
R-7010 (F Hoffmann-La Roche Ltd), edaravone (Mitsubishi-Tokyo
Pharmaceuticals Inc), arundic acid (Ono Pharmaceutical Co Ltd),
PYM-50018 (Phytopharm plc), RPI-MN (ReceptoPharm Inc), SB-509
(Sangamo BioSciences Inc), olesoxime (Trophos SA), sodium
phenylbutyrate (Ucyclyd Pharma Inc), and R-pramipexole (University
of Virginia).
[0174] Alternatively or additionally, in some embodiments,
described compositions and formulations may be administered in
combination with one or more antioxidants. In some embodiments,
described compositions and formulations may be administered in
combination with one or more antioxidants capable of reducing
oxidative stress such that activation of caspase-1 is inhibited.
Exemplary antioxidants are known in the chemical and medicinal arts
and are identified using methods described above and herein.
Pharmaceutical Compositions
[0175] Agents of the invention are often administered as
pharmaceutical compositions comprising an active therapeutic agent,
i.e., and a variety of other pharmaceutically acceptable
components. See Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa., 1980). The preferred form depends
on the intended mode of administration and therapeutic application.
The compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0176] In some embodiments, the present invention provides
pharmaceutically acceptable compositions comprising a
therapeutically effective amount of one or more of a described
compound, formulated together with one or more pharmaceutically
acceptable carriers (additives) and/or diluents for use in treating
Parkinson's disease (including idiopathic Parkinson's disease
(PD)), Diffuse Lewy Body Disease (DLBD) also known as Dementia with
Lewy Bodies (DLB), Combined Alzheimer's and Parkinson disease,
multiple system atrophy (MSA), or any other diseases, disorders, or
conditions associated with .alpha.-synuclein. As described in
detail, pharmaceutical compositions of the present invention may be
specially formulated for administration in solid or liquid form,
including those adapted for the following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection
as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as
a cream, ointment, or a controlled-release patch or spray applied
to the skin, lungs, or oral cavity; intravaginally or
intrarectally, for example, as a pessary, cream or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary and to
other mucosal surfaces.
[0177] Pharmaceutically acceptable salts of compounds described
herein include conventional nontoxic salts or quaternary ammonium
salts of a compound, e.g., from non-toxic organic or inorganic
acids. For example, such conventional nontoxic salts include those
derived from inorganic acids such as hydrochloride, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts
prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,
palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,
salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isothionic, and the
like.
[0178] In other cases, described compounds may contain one or more
acidic functional groups and, thus, are capable of forming
pharmaceutically-acceptable salts with pharmaceutically-acceptable
bases. These salts can likewise be prepared in situ in the
administration vehicle or the dosage form manufacturing process, or
by separately reacting the purified compound in its free acid form
with a suitable base, such as the hydroxide, carbonate or
bicarbonate of a pharmaceutically-acceptable metal cation, with
ammonia, or with a pharmaceutically-acceptable organic primary,
secondary or tertiary amine Representative alkali or alkaline earth
salts include the lithium, sodium, potassium, calcium, magnesium,
and aluminum salts and the like. Representative organic amines
useful for the formation of base addition salts include ethylamine,
diethylamine, ethylenediamine, ethanolamine, diethanolamine,
piperazine and the like. See, for example, Berge et al., supra.
[0179] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0180] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0181] Formulations for use in accordance with the present
invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or
parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the host being treated, and
the particular mode of administration. The amount of active
ingredient that can be combined with a carrier material to produce
a single dosage form will generally be that amount of the compound
which produces a therapeutic effect. Generally, this amount will
range from about 1% to about 99% of active ingredient, preferably
from about 5% to about 70%, most preferably from about 10% to about
30%.
[0182] In certain embodiments, a formulation as described herein
comprises an excipient selected from the group consisting of
cyclodextrins, liposomes, micelle forming agents, e.g., bile acids,
and polymeric carriers, e.g., polyesters and polyanhydrides; and a
compound of the present invention. In certain embodiments, an
aforementioned formulation renders orally bioavailable a described
compound of the present invention.
[0183] Methods of preparing formulations or compositions comprising
described compounds include a step of bringing into association a
compound of the present invention with the carrier and, optionally,
one or more accessory ingredients. In general, formulations may be
prepared by uniformly and intimately bringing into association a
compound of the present invention with liquid carriers, or finely
divided solid carriers, or both, and then, if necessary, shaping
the product.
[0184] Formulations described herein suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. Compounds described
herein may also be administered as a bolus, electuary or paste.
[0185] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), an active
ingredient is mixed with one or more pharmaceutically-acceptable
carriers, such as sodium citrate or dicalcium phosphate, and/or any
of the following: fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; binders, such as,
for example, carboxymethylcellulose, alginates, gelatin, polyvinyl
pyrrolidone, sucrose and/or acacia; humectants, such as glycerol;
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol, glycerol monostearate,
and non-ionic surfactants; absorbents, such as kaolin and bentonite
clay; lubricants, such as talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, and
mixtures thereof; and coloring agents. In the case of capsules,
tablets and pills, the pharmaceutical compositions may also
comprise buffering agents. Solid compositions of a similar type may
also be employed as fillers in soft and hard-shelled gelatin
capsules using such excipients as lactose or milk sugars, as well
as high molecular weight polyethylene glycols and the like.
[0186] Tablets may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made in a suitable machine in which a mixture
of the powdered compound is moistened with an inert liquid
diluent.
[0187] Tablets and other solid dosage forms, such as dragees,
capsules, pills and granules, may optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well known in the pharmaceutical-formulating art. They may
alternatively or additionally be formulated so as to provide slow
or controlled release of the active ingredient therein using, for
example, hydroxypropylmethyl cellulose in varying proportions to
provide the desired release profile, other polymer matrices,
liposomes and/or microspheres. They may be formulated for rapid
release, e.g., freeze-dried. They may be sterilized by, for
example, filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions that can be dissolved in sterile water, or some other
sterile injectable medium immediately before use. These
compositions may also optionally contain opacifying agents and may
be of a composition that they release the active ingredient(s)
only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner Examples of
embedding compositions that can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0188] Liquid dosage forms for oral administration of compounds of
the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0189] Besides inert diluents, oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, coloring, perfuming and preservative
agents.
[0190] Suspensions, in addition to active compounds, may contain
suspending agents as, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and mixtures thereof.
[0191] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active compound.
[0192] Dosage forms for topical or transdermal administration of a
compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0193] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0194] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0195] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Dissolving or dispersing the compound in the proper medium
can make such dosage forms. Absorption enhancers can also be used
to increase the flux of the compound across the skin. Either
providing a rate controlling membrane or dispersing the compound in
a polymer matrix or gel can control the rate of such flux.
[0196] Examples of suitable aqueous and nonaqueous carriers, which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0197] Such compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Inclusion of one or more antibacterial and/or and
antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like, may be desirable in certain embodiments.
It may alternatively or additionally be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into
the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0198] In some cases, in order to prolong the effect of a drug, it
may be desirable to slow the absorption of the drug from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material having poor water solubility. The rate of absorption of
the drug then depends upon its rate of dissolution, which in turn,
may depend upon crystal size and crystalline form. Alternatively,
delayed absorption of a parenterally-administered drug form is
accomplished by dissolving or suspending the drug in an oil
vehicle.
[0199] Injectable depot forms are made by forming microencapsule
matrices of the described compounds in biodegradable polymers such
as polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions, which are
compatible with body tissue.
[0200] In certain embodiments, a described compound or
pharmaceutical preparation is administered orally. In other
embodiments, a described compound or pharmaceutical preparation is
administered intravenously. Alternative routs of administration
include sublingual, intramuscular, and transdermal
administrations.
[0201] When compounds described herein are administered as
pharmaceuticals, to humans and animals, they can be given per se or
as a pharmaceutical composition containing, for example, 0.1% to
99.5% (more preferably, 0.5% to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0202] Preparations described herein may be given orally,
parenterally, topically, or rectally. They are of course given in
forms suitable for the relevant administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administrations are
preferred.
[0203] Such compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0204] Regardless of the route of administration selected,
compounds described herein which may be used in a suitable hydrated
form, and/or the pharmaceutical compositions of the present
invention, are formulated into pharmaceutically-acceptable dosage
forms by conventional methods known to those of skill in the
art.
[0205] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the invention may be varied so as to
obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0206] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular compound being
employed, the duration of the treatment, other drugs, compounds
and/or materials used in combination with the particular compound
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0207] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of described compounds employed in
the pharmaceutical composition at levels lower than that required
to achieve the desired therapeutic effect and then gradually
increasing the dosage until the desired effect is achieved.
[0208] In some embodiments, one or more described compounds, or
pharmaceutical compositions thereof, is provided to a
synucleinopathic subject chronically. Chronic treatments include
any form of repeated administration for an extended period of time,
such as repeated administrations for one or more months, between a
month and a year, one or more years, or longer. In many
embodiments, chronic treatment involves administering one or more
described compounds, or pharmaceutical compositions thereof,
repeatedly over the life of the subject. Preferred chronic
treatments involve regular administrations, for example one or more
times a day, one or more times a week, or one or more times a
month. In general, a suitable dose such as a daily dose of one or
more described compounds, or pharmaceutical compositions thereof,
will be that amount of the one or more described compound that is
the lowest dose effective to produce a therapeutic effect. Such an
effective dose will generally depend upon the factors described
above. Generally doses of the compounds of this invention for a
patient, when used for the indicated effects, will range from about
0.0001 to about 100 mg per kg of body weight per day. Preferably,
the daily dosage will range from 0.001 to 50 mg of compound per kg
of body weight, and even more preferably from 0.01 to 10 mg of
compound per kg of body weight. However, lower or higher doses can
be used. In some embodiments, the dose administered to a subject
may be modified as the physiology of the subject changes due to
age, disease progression, weight, or other factors.
[0209] If desired, the effective daily dose of one or more
described compounds may be administered as two, three, four, five,
six, or more sub-doses administered separately at appropriate
intervals throughout the day, optionally, in unit dosage forms.
[0210] While it is possible for a described compound to be
administered alone, it is preferable to administer a described
compound as a pharmaceutical formulation (composition) as described
above.
[0211] Described compounds may be formulated for administration in
any convenient way for use in human or veterinary medicine, by
analogy with other pharmaceuticals.
[0212] According to the invention, described compounds for treating
neurological conditions or diseases can be formulated or
administered using methods that help the compounds cross the
blood-brain barrier (BBB). The vertebrate brain (and CNS) has a
unique capillary system unlike that in any other organ in the body.
The unique capillary system has morphologic characteristics which
make up the blood-brain barrier (BBB). The blood-brain barrier acts
as a system-wide cellular membrane that separates the brain
interstitial space from the blood.
[0213] The unique morphologic characteristics of the brain
capillaries that make up the BBB are: (a) epithelial-like high
resistance tight junctions which literally cement all endothelia of
brain capillaries together, and (b) scanty pinocytosis or
transendothelial channels, which are abundant in endothelia of
peripheral organs. Due to the unique characteristics of the
blood-brain barrier, hydrophilic drugs and peptides that readily
gain access to other tissues in the body are barred from entry into
the brain or their rates of entry and/or accumulation in the brain
are very low.
[0214] In one aspect of the invention, described compounds that
cross the BBB are particularly useful for treating
synucleinopathies. In one embodiment, described compounds that
cross the BBB are particularly useful for treating Parkinson's
Disease (PD). Therefore it will be appreciated by a person of
ordinary skill in the art that some of the compounds of the
invention might readily cross the BBB. Alternatively, the compounds
of the invention can be modified, for example, by the addition of
various substitutuents that would make them less hydrophilic and
allow them to more readily cross the BBB.
[0215] Various strategies have been developed for introducing those
drugs into the brain which otherwise would not cross the
blood-brain barrier. Widely used strategies involve invasive
procedures where the drug is delivered directly into the brain. One
such procedure is the implantation of a catheter into the
ventricular system to bypass the blood-brain barrier and deliver
the drug directly to the brain. These procedures have been used in
the treatment of brain diseases which have a predilection for the
meninges, e.g., leukemic involvement of the brain (U.S. Pat. No.
4,902,505, incorporated herein in its entirety by reference).
[0216] Although invasive procedures for the direct delivery of
drugs to the brain ventricles have experienced some success, they
are limited in that they may only distribute the drug to
superficial areas of the brain tissues, and not to the structures
deep within the brain. Further, the invasive procedures are
potentially harmful to the patient.
[0217] Other approaches to circumventing the blood-brain barrier
utilize pharmacologic-based procedures involving drug latentiation
or the conversion of hydrophilic drugs into lipid-soluble drugs.
The majority of the latentiation approaches involve blocking the
hydroxyl, carboxyl and primary amine groups on the drug to make it
more lipid-soluble and therefore more easily able to cross the
blood-brain barrier.
[0218] Another approach to increasing the permeability of the BBB
to drugs involves the intra-arterial infusion of hypertonic
substances which transiently open the blood-brain barrier to allow
passage of hydrophilic drugs. However, hypertonic substances are
potentially toxic and may damage the blood-brain barrier.
[0219] Antibodies are another method for delivery of compositions
of the invention. For example, an antibody that is reactive with a
transferrin receptor present on a brain capillary endothelial cell,
can be conjugated to a neuropharmaceutical agent to produce an
antibody-neuropharmaceutical agent conjugate (U.S. Pat. No.
5,004,697, incorporated herein in its entirety by reference). Such
methods are conducted under conditions whereby the antibody binds
to the transferrin receptor on the brain capillary endothelial cell
and the neuropharmaceutical agent is transferred across the blood
brain barrier in a pharmaceutically active form. The uptake or
transport of antibodies into the brain can also be greatly
increased by cationizing the antibodies to form cationized
antibodies having an isoelectric point of between about 8.0 to 11.0
(U.S. Pat. No. 5,527,527, incorporated herein in its entirety by
reference).
[0220] A ligand-neuropharmaceutical agent fusion protein is another
method useful for delivery of compositions to a host (U.S. Pat. No.
5,977,307, incorporated herein in its entirety by reference). The
ligand is reactive with a brain capillary endothelial cell
receptor. The method is conducted under conditions whereby the
ligand binds to the receptor on a brain capillary endothelial cell
and the neuropharmaceutical agent is transferred across the blood
brain barrier in a pharmaceutically active form. In some
embodiments, a ligand-neuropharmaceutical agent fusion protein,
which has both ligand binding and neuropharmaceutical
characteristics, can be produced as a contiguous protein by using
genetic engineering techniques. Gene constructs can be prepared
comprising DNA encoding the ligand fused to DNA encoding the
protein, polypeptide or peptide to be delivered across the blood
brain barrier. The ligand coding sequence and the agent coding
sequence are inserted in the expression vectors in a suitable
manner for proper expression of the desired fusion protein. The
gene fusion is expressed as a contiguous protein molecule
containing both a ligand portion and a neuropharmaceutical agent
portion.
[0221] The permeability of the blood brain barrier can be increased
by administering a blood brain barrier agonist, for example
bradykinin (U.S. Pat. No. 5,112,596, incorporated herein in its
entirety by reference), or polypeptides called receptor mediated
permeabilizers (RMP) (U.S. Pat. No. 5,268,164, incorporated herein
in its entirety by reference). Exogenous molecules can be
administered to the host's bloodstream parenterally by
subcutaneous, intravenous or intramuscular injection or by
absorption through a bodily tissue, such as the digestive tract,
the respiratory system or the skin. The form in which the molecule
is administered (e.g., capsule, tablet, solution, emulsion)
depends, at least in part, on the route by which it is
administered. The administration of the exogenous molecule to the
host's bloodstream and the intravenous injection of the agonist of
blood-brain barrier permeability can occur simultaneously or
sequentially in time. For example, a therapeutic drug can be
administered orally in tablet form while the intravenous
administration of an agonist of blood-brain barrier permeability is
given later (e.g., between 30 minutes later and several hours
later). This allows time for the drug to be absorbed in the
gastrointestinal tract and taken up by the bloodstream before the
agonist is given to increase the permeability of the blood-brain
barrier to the drug. On the other hand, an agonist of blood-brain
barrier permeability (e.g., bradykinin) can be administered before
or at the same time as an intravenous injection of a drug. Thus,
the term "co-administration" is used herein to mean that the
agonist of blood-brain barrier and the exogenous molecule will be
administered at times that will achieve significant concentrations
in the blood for producing the simultaneous effects of increasing
the permeability of the blood-brain barrier and allowing the
maximum passage of the exogenous molecule from the blood to the
cells of the central nervous system.
[0222] In other embodiments, a described compound can be formulated
as a prodrug with a fatty acid carrier (and optionally with another
neuroactive drug). The prodrug is stable in the environment of both
the stomach and the bloodstream and may be delivered by ingestion.
The prodrug passes readily through the blood brain barrier. The
prodrug preferably has a brain penetration index of at least two
times the brain penetration index of the drug alone. Once in the
central nervous system, the prodrug, which preferably is inactive,
is hydrolyzed into the fatty acid carrier and a described compound
or analog thereof (and optionally another drug). The carrier
preferably is a normal component of the central nervous system and
is inactive and harmless. The compound and/or drug, once released
from the fatty acid carrier, is active. Preferably, the fatty acid
carrier is a partially-saturated straight chain molecule having
between about 16 and 26 carbon atoms, and more preferably 20 and 24
carbon atoms. Examples of fatty acid carriers are provided in U.S.
Pat. Nos. 4,939,174; 4,933,324; 5,994,932; 6,107,499; 6,258,836;
and 6,407,137, the disclosures of which are incorporated herein by
reference in their entirety.
[0223] Administration of agents of the present invention may be for
either prophylactic or therapeutic purposes. When provided
prophylactically, the agent is provided in advance of disease
symptoms. The prophylactic administration of the agent serves to
prevent or reduce the rate of onset of symptoms of Parkinson's
disease (including idiopathic Parkinson's disease (PD)), Diffuse
Lewy Body Disease (DLBD) also known as Dementia with Lewy Bodies
(DLB), Combined Alzheimer's and Parkinson disease and multiple
system atrophy (MSA). When provided therapeutically, the agent is
provided at (or shortly after) the onset of the appearance of
symptoms of actual disease. In some embodiments, the therapeutic
administration of the agent serves to reduce the severity and
duration of the disease.
[0224] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (e.g.,
adjuvants).
[0225] For parenteral administration, agents of the invention can
be administered as injectable dosages of a solution or suspension
of the substance in a physiologically acceptable diluent with a
pharmaceutical carrier that can be a sterile liquid such as water
oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl. Compositions for parenteral
administration are typically substantially sterile, substantially
isotonic and manufactured under GMP conditions of the FDA or
similar body.
[0226] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above (see Langer, Science 249, 1527 (1990) and Hanes,
Advanced Drug Delivery Reviews 28, 97-119 (1997). The agents of
this invention can be administered in the form of a depot injection
or implant preparation which can be formulated in such a manner as
to permit a sustained or pulsatile release of the active
ingredient.
[0227] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications. For
suppositories, binders and carriers include, for example,
polyalkylene glycols or triglycerides; such suppositories can be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0228] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins
(See Glenn et al., Nature 391, 851 (1998)). Co-administration can
be achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein. Alternatively, transdermal delivery can be achieved
using a skin path or using transferosomes (Paul et al., Eur. J.
Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta
1368, 201-15 (1998)).
EXEMPLIFICATIONS
[0229] Provided below is an exemplary embodiment of the present
invention, demonstrating inhibition of caspase-1/ICE as a promising
therapeutic approach to treat Parkinson's disease. It should not be
construed to be limiting in any way.
Background
[0230] The protein .alpha.-synuclein is associated with multiple
neurological disorders, including the two most prevalent
neurodegenerative diseases, Parkinson disease and Alzheimer
disease. Collectively, these .alpha.-synuclein associated disorders
are referred to as synucleinophathies, and most are characterized
by the presence of insoluble .alpha.-synuclein-rich aggregates
called Lewy bodies (1-3). The presence of Lewy bodies in neurons of
the substantia nigra is the histopathological hallmark of Parkinson
disease, and is currently used to differentiate Parkinson disease
from other neurological disorders with overlapping clinical
symptoms (4). In addition to .alpha.-synuclein being the major
component of Lewy bodies found in the sporadic form of Parkinson
disease (4), monogenic point mutations (A30P, A53T, and E46K) as
well as gene duplication and triplication of the .alpha.-synuclein
locus have been identified as causal factors of early onset
familial Parkinson disease (5-7). As such, .alpha.-synuclein is
likely involved in a pathogenic pathway common to both sporadic and
familial forms of synucleinopathies. The role of .alpha.-synuclein
in normal brain function is still poorly understood. There is
evidence that it plays a role in synaptic vesicle transport and
possibly in mitochondrial fusion and fission; it is also important
for memory and learning in mice and song birds, respectively (8,
9). Overexpression of human .alpha.-synuclein in yeast and C.
elegans (neither of which expresses .alpha.-synuclein naturally)
results in defective ER-Golgi vesicular transport, a result of
deregulation of the Rab1 GTPase (3, 10). .alpha.-synuclein is small
(140 residues) and highly conserved in vertebrates. Its sequence
contains multiple KTKE (SEQ ID NO: 3) or EKTK (SEQ ID NO: 4)
imperfect amino acid repeats spanning the first 2/3 of the protein
(residues 1 to 83), while the C-terminal region (residues 100-140)
is highly acidic. The repeat segments have high .alpha.-helical
propensity and helical structure is detected by circular dichroism
(CD) and nuclear magnetic resonance (NMR) when .alpha.-synuclein is
incubated with some detergents and lipid vesicles (11, 12).
[0231] It has been known for several years that Lewy Bodies, the
aggregates found in the dying neurons of Parkinson's Disease (PD)
patients, contain, in addition to ubiquitin and full-length
.alpha.-synuclein, a fragment of .alpha.-synuclein that appears to
have been produced by specific proteolytic cleavage at around
residue 120. Several in vitro studies have shown that this fragment
aggregates more readily than the full-length protein, leading a
number of investigators to speculate that the fragment may nucleate
aggregation in vivo (1) Inhibition of the proteolytic cleavage that
produces the fragment would represent an attractive new strategy
for preventing or arresting the disease. However, there are
hundreds of proteases in the human genome and many are essential
genes; as such, it has not been possible to identify the target
enzyme.
[0232] To identify the target enzyme(s) responsible for cleaving
.alpha.-synuclein in vivo, we turned to yeast. In contrast to the
situation in mammalian cells, yeast has fewer than 60 proteases and
none is an essential gene. Yeast also has no brain, which one would
think might make it a poor model organism for PD research, but
Lindquist's lab showed that overexpression of human
.alpha.-synuclein in yeast resulted in aggregation and
cytotoxicity, and went on to show that genes that suppressed this
toxicity when overexpressed along with synuclein could suppress
synuclein toxicity in mouse and cell culture models of Parkinson's
Disease (2). These and other observations suggested to us that
yeast might represent a model organism capable of simplifying the
protease hunt, so we set out first to find the protease in yeast
and then to validate that enzyme in human cells as a PD target.
Example 1
Toxic .alpha.-Synuclein Fragments
[0233] The present invention demonstrated that the aggregates
formed in yeast when human .alpha.-synuclein is overexpressed
contain the same fragment of the protein that is found in Lewy
Bodies.
Example 2
Demonstration of the Involvement of Certain Cysteine Proteases
[0234] Next, each of the more than 50 yeast proteases in the
synuclein-overexpression strain were systematically deleted in
order to see if any of the deletions rescued yeast from synuclein
toxicity. Two deletions did: deletion of RIM13, the sole yeast
homolgoue of the human cysteine protease calpain, and YCA1, the
sole yeast homologue of the human caspase family of cysteine
proteases. Since in yeast RIM13 activates YCA1, it seemed possible
that only a single enzyme was cleaving synuclein in yeast. Loss of
either protease not only abolished synuclein toxicity, it also
eliminated the production of synuclein fragments and aggregates.
The involvement of cysteine proteases was confirmed by screening a
battery of protease inhibitors to see if any of them would prevent
synuclein toxicity in yeast. Only the non-specific cysteine
protease inhibitors were effective.
[0235] Humans have 28 calpain and caspase isozymes--a large but not
impossible number to test. RNAi was used to knock down each of
these in turn in a neuronal cell culture model of PD. It is a
neuroblastoma cell line (BE(2)-M17) carrying a wild-type
.alpha.-synuclein overexpressing vector. BE(2)-M17 is a clone of
the SK-N-BE(2) cell line that was established in November of 1972
from a bone marrow biopsy taken from a 2 year old boy with
neuroblastoma. Synuclein on its own is not toxic to these cells,
but it is toxic when combined with an oxidative stress agent such
as rotenone or menadione. Greater than 90% reduction of mRNA levels
for each of the 29 protease genes was routinely achieved, and
greater than 50% reduction in protein levels (typically, about 70%
reduction) was routinely achieved.
Example 3
Identification of ICE as a Target Protease
[0236] The present example demonstrates that ICE is a target
protease. It was determined that in this cell culture model not
only did synuclein form Lewy Body-like aggregates, but it also gave
rise to the same fragment found in yeast and PD brains. Each of the
knockdown cell lines were examined for the loss of this fragment,
and it was found that reduction in the amount of only one of the
human calpains and caspases abolished fragment production:
caspase-1.
[0237] Caspase-1 is not an essential gene. Moreover, there is a
crystal structure already known for this protein, which is
sometimes referred to as interleukin-1-beta converting enzyme
(ICE). It is expressed in brain, including the neurons of the nigra
pars compacta. Due to its importance in inflammation, caspase-1 was
investigated thoroughly by several drug companies as a possible
target for the treatment of chronic inflammatory diseases. A number
of very potent, highly specific inhibitors of the enzyme were
developed (3). Although none of those drugs reached the market, at
least two, from Vertex Corporation, passed Phase 1 clinical trials
and were determined to be safe for use in humans. None of these
drugs have ever been tested as a possible treatment for Parkinson's
Disease.
Example 4
Confirmation that ICE Cleaves Alpha-Synuclein In Vitro
[0238] The present invention further demonstrates that purified
caspase-1/ICE can be used to verify that caspase-1/ICE cleaves
.alpha.-synuclein in vitro, and produces the expected
fragmentation. Purified, activated caspase-1 does indeed cleave
alpha-synuclein (FIG. 5). In this set of in vitro assays, a 60 ul
reaction mixture consisting of 21 ug of synuclein, various amount
of ICE in 100 mM HEPES, pH 7.4, 0.1% OG, 10% glycerol, 150 mM NaCl.
Incubated at 37.degree. C. for 2 hours. 20 ul were withdrawn from
the reaction for SDS-PAGE followed by western blotting with
anti-synuclein antibody (FIG. 5).
[0239] Moreover, the caspase-1 inhibitor from the NIH lab
completely blocks this cleavage in a dose-dependent manner (FIG.
6), demonstrating its specificity. Here, a 60 ul reaction mixture
consisting of 21 .mu.g of synuclein, 10 .mu.g ICE in 100 mM HEPES,
pH 7.4, 0.1% OG, 10% glycerol, 150 mM NaCl, with or without 20
.mu.M inhibitor was incubated at 37.degree. C. for 2 hours. 20
.mu.l were withdrawn from the reaction for SDS-PAGE followed by
western blotting with anti-synuclein antibody (FIG. 6). As shown in
FIG. 4, activity against a model substrate (Ac-YVAD-AMC, from Enzo,
Inc.) (SEQ ID NO: 5) was determined to be 110 .mu.M/min/mg.
Moreover, fragmentation of alpha-synuclein in vitro was shown to be
ICE specific (FIG. 6).
[0240] Mass spectrometer was used to determine the site of
cleavage. There was only one cut, at residue 121, exactly as is
observed in the fragment found in Lewy Bodies. The fragment
generated by ICE was determined to be 13167 Da which corresponds to
residues from 1-121 (FIG. 7). For mass spectrometry experiments, a
60 ul reaction mixture consisting of 21 .mu.g of synuclein, 10
.mu.g ICE in 100 mM HEPES, pH 7.4, 0.1% OG, 10% glycerol, 150 mM
NaCl. Incubated at 37.degree. C. for 2 hours. Mixed with matrix and
analyzed with MALDI-TOF mass spec. A representative datum is shown
in FIG. 7.
Example 5
Confirmation that ICE Cleaves Alpha-Synuclein In Vivo
[0241] The present example confirms that ICE cleaves
alpha-synuclein in vivo. While the in vivo assays have been more
challenging due to persistent caspase-1 activation at low levels in
unstressed cells (possibly because the cells are not really
unstressed), it is definitive that caspase-1 cleaves
alpha-synuclein both in vitro and in vivo and that
caspase-1-cleaved alpha-synuclein aggregates much faster than
full-length, wild type alpha-synuclein (e.g., SEQ ID NO: 1).
Example 6
Demonstration that Oxidative Stress Activates ICE to Cleave
Alpha-Synuclein
[0242] The present example demonstrates, for the first time, that
oxidative stress at the mitochondria can activate caspase-1 and
induce cleavage of alpha-synuclein, thereby providing a possible
explanation for the known PD-inducing agents such as rotenone and
MPTP (FIG. 3). In this set of experiments, M17 overexpressing
.alpha.-synuclein was grown in Optimem in presence or absence of
menadione to confluence. Cells were then scraped and lysed by
sonnication for SDS-PAGE followed by western blotting with
anti-synuclein antibody.
[0243] Menadione was shown to promote alpha-synuclein induced
toxicity in a neuronal cell line (M17). It was found that low
concentrations of menadione are not toxic to M17 cells carrying
empty pcDNA3 vector, but are toxic to M17 cells overexpressing
.alpha.-synuclein (FIG. 8 and FIG. 9).
[0244] To further support the biological significance of ICE on
.alpha.-synuclein processing, known inhibitors of ICE may be
utilized. For example, Vertex provides ICE inhibitors, which are
tested for their inhibitory activity that can block fragment
formation in vitro.
[0245] It may be further tested that the ICE inhibitor, e.g., the
Vertex compounds, can block synuclein fragment formation in the
Cookson neuroblastoma model of PD, or any other suitable PD model
systems, such as transgenic animal models. Additionally, it may be
examined that this same compound rescues yeast from
.alpha.-synuclein toxicity.
Example 7
Demonstration of Ability of RNAi and Chemical Inhibition of ICE to
Reduce Alpha-Synuclein Fragmentation in Nerve Cells
[0246] A cell viability assay was performed using LIVE/DEAD
(molecular probes) assay according to the manufacture instructions.
As summarized in FIG. 10, both RNAi knockdown of caspase-1/ICE and
treatment with the caspase-1/ICE inhibitor block synuclein fragment
formation in nerve cells and should inhibit synuclein aggregation
in neuronal cells derived from one or more mouse models of PD.
[0247] It is contemplated that once it has been determined that
inhibition of caspase-1 is protective in neurons derived from PD
mice, it is then tested that same inhibitor on the mice themselves.
The ability of an ICE inhibitor (e.g., the Vertex compound) to
penetrate the blood-brain barrier in animals may be also examined
using well-known techniques such as mass spectrometry, a technique
routinely used to measure drug penetration into the brain.
[0248] It would be necessary to determine the suitable dosing
protocol, and there would need to be several end-points, including
survival and reduction or abolition of fragment production. A
crucial question is just what effect one should be looking for. If
the fragment nucleates the production of toxic synuclein oligomers,
but does not effect the kinetics of aggregate propagation, it is
likely that inhibition of caspase-1/ICE would delay the onset of
disease but may not completely prevent it. In certain situations,
inhibiting the protease may affect disease progression once PD
symptoms have already appeared. Thus, caspase-1/ICE inhibition is a
potential treatment for diagnosed PD/as well as a possible way to
prevent or delay disease initiation. In sum, the above examples
confirm that (1) caspase-1 cleaves alpha-synuclein in vitro and in
vivo at the site found in the fragments of alpha-synuclein
(.alpha.-Syn) that are observed in Lewy Bodies; and (2) RNAi
knockdown or chemical inhibition of caspase-1 reduces fragment
formation in nerve cells in culture. These results demonstrate that
caspase-1 (ICE) inhibition appears to be a valid and promising
target for Parkinson's (PD) therapy.
Example 8
Compounds Screening
[0249] Yet further contemplated is to screen additional (novel) ICE
inhibitor compounds with preferred pharmacokinetics with respect to
brain penetration and half-life. A number of diversity libraries of
small molecules with known or likely ability to cross the
blood-brain barrier are available and may be used for these
screening
[0250] In some cases, in silico screening using the already
determined crystal structure may be employed.
[0251] In addition, screening may be done by direct
genetic/functional screening in our yeast model for synuclein
toxicity.
Example 9
Pretreatment with Caspase-1 Inhibitors
[0252] The present Example describes an approach for assessing the
ability of pretreatment with caspase-1 inhibitors to prevent or
delay the accumulation and aggregation of .alpha.Syn expression and
improve neurological functional recovery in rotenone-treated rats.
In particular, the present Example determines whether caspase-1
inhibition will prevent or delay .alpha.Syn fragment formation and
aggregation and prevent motor disability in the rotenone mouse PD
model. Of particular interest is the Vertex prodrug VX-765 and the
NIH compound NCGC00185682.
Research Design
[0253] Subjects: A total of 50 C57/BL mice comprise this
experiment.
[0254] Experimental procedure: Seven month old male Lewis rats
(300-350 g) are employed. For the pretreatments experiments (Groups
1-2), rats receive caspase-1 inhibitors (50 mg/kg based on
similarities to the Vertex compound) or vehicle for one week. Then,
Groups 1-2 (above) receive 3 mg/kg rotenone once per day for 10
days. This is a dosing paradigm that produces motor impairments,
loss of nigrostriatal dopamine, and alpha synuclein aggregation
(13) (Groups 5 and 6 receive the caspase-1 inhibitor or vehicle
followed by vehicle in lieu of the rotenone. This allows for
assessment of the effects of caspase-1 inhibitors upon the normal
animal and determination of whether caspase-1 treated animals
exhibit structural and functional neuroprotection to the level of
normal animals (vehicle-vehicle). Daily caspase-1 inhibitor or
vehicle treatment continues for 14 days following the rotenone (or
vehicle) treatment. A this point, the model becomes stable. From
this time point forward, motor function using the rotorod, rearing
(apomorphine stimulated and not) and postural instability tests are
evaluated. Animals are sacrificed two months after the last
rotenone injection for histological and biochemical analysis.
[0255] Outcome measures are: 1) behavioral data from the rotarod
test, rearing and postural instability tests; 2) stereological
counts of nigral DAT- and TH-ir neurons and .alpha.Syn-ir cells in
both aggregated and non-aggregated forms within the SN; 3) HPLC
measurements of dopamine and its metabolites, 4) measurement of the
optical density of TH-ir within striatum; 5) Western Blot Analysis
for .alpha.Syn protein expression and fragment formation; and 6)
Quantitative RT-PCR analysis for .alpha.Syn mRNA expression within
SN.
TABLE-US-00003 Group Number Group Description Total Numbers of
Animals 1 caspase-1 inhibitor 10 (histological pretreatment +
rotenone (left side of the brain) and biochemical (right side of
the brain) analysis) 2 vehicle pretreatment + 10 (histological
rotenone (left side of the brain) and biochemical (right side of
the brain) analysis) 3 caspase-1 inhibitor 10 (histological
posttreatment + rotenone (left side of the brain) and biochemical
(right side of the brain) analysis) 4 vehicle posttreatment + 10
(histological rotenone (left side of the brain) and biochemical
(right side of the brain) analysis) 5 caspase-1 5 (histological
inhibitor + vehicle (left side of the brain) and biochemical (right
side of the brain) analysis) 6 vehicle + vehicle 5 (histological
(left side of the brain) and biochemical (right side of the brain)
analysis)
Example 10
Treatment with Caspase-1 Inhibitors
[0256] The present Example describes an approach for determining
the ability of caspase-1 inhibitor treatment delivered after
rotenone treatment to reverse or retard the fragmentation and
aggregation of alpha-synuclein and thus improve neurological
functional recovery in the rotenone-treated rat.
[0257] Experiment 1b: All aspects of this experiment are identical
to Experiment 1a above, except that the caspase-1 inhibitor is
administered only at days 7-14 post-rotenone (or vehicle) to
determine whether this treatment can reverse an already established
synucleinopathy.
Example 11
Prevention with Caspase-1 Inhibitors
[0258] The present Example describes an approach for determining
the ability of caspase-1 inhibitor treatment to prevent or delay
the fragmentation and aggregation of .alpha.Syn and improve
neurological functional recovery in rats receiving intranigral
viral over-expression of alpha synuclein.
[0259] Experiment 2: Experiment 2 tests the hypothesis that a
caspase-1 inhibitor (at .about.50 mg/kg) reduces the .alpha.Syn
fragmentation and aggregation within the SN and improves functional
recovery in the AAV6-.alpha.Syn treated rat PD model. The AAV
vector empty plasmid is commercially available (pAAV-MCS) from
Stratagene. Full human wild type .alpha.Syn gene (including coding
region+3'UTR) are cloned into the AAV6 plasmid.
Research Design
[0260] Subjects: 100 young adult Sprague Dawley male rats comprise
this experiment.
[0261] Experimental procedure: For one week, rats receive daily
injections of caspase-1 inhibitors or vehicle. Then rats in groups
1-4 above receive vector injections comprised of 2 .mu.l of equally
titered AAV6-alpha syn or AAV6-GFP. Caspase-1 treatment is also
tested in rats receiving intranigral injection of vehicle due to
the potential general toxicity of these vectors (especially GFP)
for which caspase-1 inhibitor treatment may have effects. All rats
continue to receive caspase-1 or vehicle treatment for 6 weeks,
after which time they are sacrificed for histological and
biochemical analysis. Statistical analysis is similar to Experiment
1 above.
[0262] Outcome measures are: Same as Experiment 1 except analysis
of behavioral data of motor function will be performed on the
cylinder test.
[0263] Statistical Analysis: Behavioral, neuroanatomical,
neurochemical, and molecular measures comparisons between different
groups are assessed using a factorial ANOVA. Post-hoc tests
controlling for multiple comparisons are employed to test
individual group differences.
TABLE-US-00004 Group Number Group Description Total Numbers of
Animals 1 caspase-1 inhibitor + AAV6 20 (10 for histological
alpha-synuclein analysis and 10 for biochemical analysis) 2 vehicle
+ AAV6 alpha- 20 (10 for histological synuclein analysis and 10 for
biochemical analysis) 3 caspase-1 inhibitor + AAV6 20 (10 for
histological alpha-synuclein analysis and 10 for biochemical
analysis) 4 vehicle + AAV6-GFP 20 (10 for histological analysis and
10 for biochemical analysis) 5 caspase-1 inhibitor + vehicle 10 (5
for histological analysis and 5 for biochemical analysis) 6 vehicle
+ vehicle 10 (5 for histological analysis and 5 for biochemical
analysis)
[0264] Outcomes: It is contemplated that caspase-1 inhibition will
1) reduce the fragmentation and aggregation of .alpha.Syn protein
within the SN; 2) delay or block nigrostriatal dopaminergic
degeneration, and 3) prevent or delay the onset of motor disability
in one or both rat models of PD.
[0265] One aspect of this experiment design is that it relies on
the administration of a toxin to generate the PD-like model. A
supplemental protocol is therefore contemplated to include testing
in an alpha-synuclein transgenic model of PD. Also considered is
the use of an alpha-synuclein knockout mouse as a negative control
to address the role of alpha-synuclein in these studies. In some
embodiments, experimental designs that incorporate alpha-synuclein
transgenic (and knockout mice) include protocol wherein doses of
Caspase-1 inhibitor are varied.
Example 12
Blood-Brain Barrier (BBB) Penetration
[0266] The present example describes an approach for assessing is
the ability of, inter alia, the two compounds referenced above
(i.e., VX-765 and NCGC00185682) to cross the blood brain-barrier
(BBB). Preliminary tests suggest that BBB penetration in animals is
low for both compounds. In view of the fact that studies of VX-765
as a potential epilepsy therapeutic are currently underway at
Vertex, it is hypothesized that either BBB penetration in humans is
better or that the amount of compound in the CNS that is needed to
achieve the desired effect being measured in the studies is
small.
[0267] The following two-stage process may be used to determine BBB
permeability.
(1) Cell-Free Permeability
[0268] Collagen-coated, microporous, polycarbonate membranes in
12-well Costar Transwell.RTM. plates without MDR1-MDCK cells were
used for this study. The permeability assay buffer was Hanks
Balanced Salt Solution containing 10 mM HEPES with 15 mM glucose at
a pH of 7.4. The dosing solution concentration of the test
compounds was 1 .mu.M in the assay buffer. In duplicate, cell-free
inserts were dosed on the top (apical) chamber and incubated at
37.degree. C. with 5% CO.sub.2 in a humidified incubator. After 120
minutes, aliquots were taken from the receiver chambers. Samples
were taken from the donor chamber at 5 and 120 minutes. All samples
were assayed by LC-MS/MS. The apparent permeability, P.sub.app, and
percent recovery were calculated as follows:
P.sub.app=(dCr/dt).times.Vr/(A.times.C.sub.0) (1)
Percent
Recovery=100.times.((Vr.times.C.sub.rfinal)(Vd.times.C.sub.dfina-
l))/(Vd.times.C.sub.N) (2)
[0269] Where, dCr/dt is the slope of the cumulative concentration
in the receiver compartment versus time in .mu.M/s; Vr is the
volume of the receiver compartment in cm.sup.3; Vd is the volume of
the donor compartment in cm.sup.3; A is the area of the insert
(1.13 cm.sup.2 for 12-well Transwell.RTM.); C.sub.0 is the measured
0 minute donor concentration in 1 .mu.M; C.sub.N is the nominal
concentration of the dosing solution in 1 .mu.M; C.sub.rfinal is
the cumulative receiver concentration in 1 .mu.M at the end of the
incubation period; C.sub.dfinal is the concentration of the donor
in 1 .mu.M at the end of the incubation period.
(2) Permeability, MDR1-MDCK
[0270] MDR1-MDCK monolayers were grown to confluence on
collagen-coated, microporous, polycarbonate membranes in 12-well
Costar Transwell.RTM. plates. Details of the plates and their
certification are shown below. The permeability assay buffer was
Hanks Balanced Salt Solution containing 10 mM HEPES and 15 mM
glucose at a pH of 7.4 The dosing solution concentration was 5
.mu.M in the assay buffer. Cell monolayers were dosed on the apical
side (A-to-B) or basolateral side (B-to-A) and incubated at
37.degree. C. with 5% CO.sub.2 in a humidified incubator. At 60 and
120 minutes, aliquots were taken from the receiver chambers and
replaced with fresh assay buffer. Samples were taken from the donor
chamber at 120 minutes. Each determination was performed in
duplicate. The lucifer yellow flux was measured for each monolayer
after being subjected to the test compounds to ensure no damage was
inflicted to the cell monolayers during the flux period. All
samples were assayed by LC-MS/MS using electrospray ionization. The
apparent permeability, P.sub.app, and percent recovery were
calculated as follows:
P.sub.app=(dCr/dt).times.Vr/(A.times.C.sub.N) (1)
Percent
Recovery=100.times.((Vr.times.C.sub.rfinal)(Vd.times.C.sub.dfina-
l))/(Vd.times.C.sub.N) (2)
[0271] Where dCr/dt is the slope of the cumulative concentration in
the receiver compartment versus time in .mu.M/s; Vr is the volume
of the receiver compartment in cm.sup.3; Vd is the volume of the
donor compartment in cm.sup.3; A is the area of the cell monolayer
(1.13 cm.sup.2 for 12-well Transwell.RTM.); C.sub.N is the nominal
concentration of the dosing solution in 5 .mu.M; C.sub.rfinal is
the cumulative receiver concentration in 5 .mu.M at the end of the
incubation period; C.sub.dfinal is the concentration of the donor
in 5 .mu.M at the end of the incubation period.
REFERENCES
[0272] 1. Qin Z, Hu D, Han S, Hong D P, Fink A L. Role of different
regions of .alpha.-synuclein in the assembly of fibrils.
Biochemistry. 2007 Nov. 20; 46(46):13322-30. [0273] 2. Cooper A A,
Gitler A D, Cashikar A, Haynes C M, Hill K J, Bhullar B, Liu K, Xu
K, Strathearn K E, Liu F, Cao S, Caldwell K A, Caldwell G A,
Marsischky G, Kolodner R D, Labaer J, Rochet J C, Bonini N M,
Lindquist S. A-synuclein blocks ER-Golgi traffic and Rab1 rescues
neuron loss in Parkinson's models. Science. 2006 Jul. 21;
313(5785):324-8. [0274] 3. Siegmund B, Zeitz M. Pralnacasan (Vertex
Pharmaceuticals). IDrugs. 2003 February; 6(2):154-8.
EQUIVALENTS
[0275] The foregoing disclosure is considered to be sufficient to
enable one ordinary skilled in the art to practice the invention.
The present invention is not to be limited in scope by the examples
provided, since the examples are intended as mere illustrations of
one or more aspects of the invention. Other functionally equivalent
embodiments are considered within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description. Each of the limitations of the
invention can encompass various embodiments of the invention. It is
therefore anticipated that each of the limitations of the invention
involving any one element or combinations of elements can be
included in each aspect of the invention. This invention is not
limited in its application to the details of construction and the
arrangement of components set forth or illustrated in the drawing.
The invention is capable of other embodiments and of being
practiced or of being carried out in various ways.
[0276] Also, the phraseology and terminology used herein is for the
purpose of description and should not be regarded as limiting. The
use of "including" "comprising" or "having" "containing"
"involving" and variations thereof herein is meant to encompass the
items listed thereafter and equivalents thereof as well as
additional items.
[0277] All references, patents and patent applications that are
recited in this application are incorporated by reference in their
entirety.
Sequence CWU 1
1
51140PRTHomo 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 26PRTArtificial sequenceSynthetic polypeptide 2Lys Thr
Lys Glu Gly Val 1 5 34PRTArtificial sequenceSynthetic polypeptide
3Lys Thr Lys Glu 1 44PRTArtificial sequenceSynthetic polypeptide
4Glu Lys Thr Lys 1 54PRTArtificial sequenceSynthetic polypeptide
5Tyr Val Ala Asp 1
* * * * *
References