U.S. patent application number 15/144323 was filed with the patent office on 2017-03-30 for identification of compounds that disperse tdp-43 inclusions.
The applicant listed for this patent is AQUINNAH PHARMACEUTICALS, INC., THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Justin Boyd, Gregory D. Cuny, Marcie Glicksman, Benjamin Wolozin.
Application Number | 20170088515 15/144323 |
Document ID | / |
Family ID | 47217664 |
Filed Date | 2017-03-30 |
United States Patent
Application |
20170088515 |
Kind Code |
A1 |
Wolozin; Benjamin ; et
al. |
March 30, 2017 |
IDENTIFICATION OF COMPOUNDS THAT DISPERSE TDP-43 INCLUSIONS
Abstract
Herein, compounds and compositions for use in treating diseases
relating to inclusion formation and stress granules are described.
Methods for screening for modulation of TDP-43 aggregation are also
described.
Inventors: |
Wolozin; Benjamin; (Newton,
MA) ; Glicksman; Marcie; (Winchester, MA) ;
Cuny; Gregory D.; (Houston, TX) ; Boyd; Justin;
(Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AQUINNAH PHARMACEUTICALS, INC.
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Newton
Boston |
MA
MA |
US
US |
|
|
Family ID: |
47217664 |
Appl. No.: |
15/144323 |
Filed: |
May 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14118628 |
Nov 19, 2013 |
9359363 |
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PCT/US2012/038861 |
May 21, 2012 |
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15144323 |
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61488468 |
May 20, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 211/34 20130101;
C07D 211/26 20130101; C07D 487/04 20130101; C07D 417/04 20130101;
A61P 25/28 20180101; A61K 31/365 20130101; A61K 31/505 20130101;
A61K 31/519 20130101; A61P 31/12 20180101; C07D 401/12 20130101;
A61K 31/4545 20130101; C07D 215/227 20130101; C07D 239/94 20130101;
A61K 31/47 20130101; G01N 33/5058 20130101; A61K 31/445 20130101;
C07D 215/36 20130101; A61K 31/517 20130101; A61K 31/454 20130101;
C07D 239/52 20130101; A61K 31/4025 20130101; C07D 401/04 20130101;
C07D 211/52 20130101; C07D 207/36 20130101; A61K 31/704 20130101;
A61P 35/00 20180101; A61K 31/551 20130101; C07D 403/06
20130101 |
International
Class: |
C07D 207/36 20060101
C07D207/36; C07D 401/12 20060101 C07D401/12; C07D 211/34 20060101
C07D211/34; C07D 487/04 20060101 C07D487/04; C07D 215/36 20060101
C07D215/36; G01N 33/50 20060101 G01N033/50; C07D 211/26 20060101
C07D211/26; C07D 211/52 20060101 C07D211/52; C07D 239/94 20060101
C07D239/94; C07D 215/227 20060101 C07D215/227; C07D 403/06 20060101
C07D403/06; C07D 417/04 20060101 C07D417/04; C07D 239/52 20060101
C07D239/52 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
Contract No. NS066108 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1-36. (canceled)
37. A method of treating a neurodegenerative disease or a viral
infection in a subject in need thereof, the method comprising
administering an effective amount of a compound selected from:
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## or
a pharmaceutically acceptable salt thereof.
38. The method of claim 37, wherein the compound is selected from:
##STR00031## ##STR00032## or a pharmaceutically acceptable salt
thereof.
39. The method of claim 37, wherein the compound is selected from:
##STR00033## ##STR00034## or a pharmaceutically acceptable salt
thereof.
40. The method of claim 37, wherein the neurodegenerative disease
is selected from Alzheimer's disease, frontotemporal dementia,
FTLD-U, amyotrophic lateral sclerosis (ALS), Huntington's chorea,
Creutzfeld-Jacob disease, trinucleotide repeat diseases, cerebral
degenerative diseases presenile dementia, senile dementia,
Parkinsonism linked to chromosome 17 (FTDP-17), progressive
supranuclear palsy (PSP), Huntington's disease (HD), Pick's
disease, primary progressive aphasia, corticobasal dementia,
Parkinson's disease, Parkinson's disease with dementia, dementia
with Lewy bodies, Down's syndrome, multiple system atrophy, spinal
muscular atrophy (SMA), spinocerebellar ataxia, spinal degenerative
disease/motor neuron degenerative diseases, Hallervorden-Spatz
syndrome, cerebral infarct, cerebral trauma, chronic traumatic
encephalopathy, and transient ischemic attack, or any combination
thereof.
41. The method of claim 40, wherein the neurodegenerative disease
is selected from Alzheimer's disease, frontotemporal dementia,
FTLD-U, amyotrophic lateral sclerosis (ALS), and Huntington's
chorea.
42. The method of claim 37, wherein viral infection is caused by a
virus selected from the group consisting of West Nile virus,
Respiratory Syncitial Virus (RSV), herpes simplex virus 1, herpes
simplex virus 2, Epstein-Barr virus (EBV), hepatitis virus A,
hepatitis virus B, hepatitis virus C, influenza viruses, chicken
pox, avian flu viruses, smallpox, polio viruses, HIV-1, and HIV-2,
or any combinations thereof.
43. The method of claim 37, wherein the subject is a mammal.
44. The method of claim 37, wherein the subject is a human.
45. The method of claim 37, further comprising the step of
diagnosing the subject for said neurodegenerative disease or said
viral infection prior to the onset of administration.
46. The method of claim 37, wherein the pathology of said
neurodegenerative disease or said viral infection comprises stress
granules.
47. The method of claim 37, wherein the pathology of said
neurodegenerative disease or said viral infection comprises stress
granules or TDP-43 inclusion formation.
48. A compound selected from: ##STR00035## ##STR00036##
##STR00037## ##STR00038## ##STR00039## or a pharmaceutically
acceptable salt thereof.
49. The compound of claim 48, wherein the compound is selected
from: ##STR00040## ##STR00041## or a pharmaceutically acceptable
salt thereof.
50. The compound of claim 48, wherein the compound is selected
from: ##STR00042## ##STR00043## or a pharmaceutically acceptable
salt thereof.
51. A pharmaceutical composition comprising a compound of claim 48
or a pharmaceutically acceptable salt thereof.
Description
[0001] This application claims priority to U.S. patent application
Ser. No. 14/118,628, filed Nov. 19, 2013, which is a national stage
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/US2012/038861, filed May 21, 2012, which claims priority to
U.S. Provisional Application No. 61/488,468, filed May 20, 2011.
The entire contents of each of the foregoing applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to methods and compositions modulating
inclusion formation and stress granules in cells, and for treatment
of neurodegenerative diseases, cancer and viral infections.
BACKGROUND OF THE INVENTION
[0004] TDP-43 was recently identified as one of the major proteins
that accumulate in inclusions in Amyotrophic Lateral Sclerosis
(ALS) and in Fronto-temporal lobar dementia with ubiquitin
inclusions (FTLD-U). Abnormalities in TDP-43 biology appear to be
sufficient to cause neurodegenerative disease because mutations in
TDP-43 occur in familial ALS. The prevalence of TDP-43 deposits in
diseases such as ALS and FTLD-U, combined with the ability of
abnormal TDP-43 to cause disease places TDP-43 in the class of
proteins that are major components of neurodegenerative disease.
This class includes tau, .alpha.-synuclein, huntingtin and
.beta.-amyloid. Analysis of the biology of the major proteins that
accumulate in other neurodegenerative diseases has lead to major
advances in our understanding of the pathophysiology of the disease
and also development of new drug discovery platforms. During the
course of studying TDP-43, we discovered that this protein is part
of the stress granule machinery. This work lead us to important
discoveries about how to model the pathophysiology of ALS and
FTLD-U in cell culture.
[0005] Currently, it is believed that aggregates that accumulate in
neurodegenerative diseases like ALS, FTLD-U, Parkinson's disease
and Huntington's disease accumulate slowly and are very difficult
to disaggregate or perhaps can't be disaggregated. Thus, there is a
need in the art for compostions and methods that can rapidly
disaggregate stress granules and/or inhbibt the formation of stress
granules.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention provides methods for treatment
of a neurodegenerative disease or disorder, a cancer, and/or a
viral infection in a subject, the method comprising administering a
stress granule modulator to a subject in need thereof.
[0007] In another aspect, the invention provides methods of
diagnosing a neurodegenerative disease in a subject, the method
comprising administering a stress granule marker to the subject.
For use in diagnosing a stress granule marker can be labeled with a
label.
[0008] In another aspect, the invention provides methods of
modulating stress granules comprising contacting a cell with a
TDP-43 inclusion inhibiting compound.
[0009] In another aspect, the invention provides methods of
modulating TDP-43 inclusion formation comprising contacting a cell
with a TDP-43 inclusion inhibitor.
[0010] In yet another aspect, the invention provides a method of
screening for modulators of TDP-43 aggregation comprising
contacting a compound with the cell that expresses TDB-43 and
develops spontaneous inclusions.
[0011] In still another aspect, the invention provides a cell that
expresses wild-type TDB-43 and develops spontaneous inclusions.
[0012] Still other objects and advantages of the invention will
become apparent to those of skill in the art from the disclosure
herein, which is simply illustrative and not restrictive. Thus,
other embodiments will be recognized by the skilled artisan without
departing from the spirit and scope of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A to 1B shows images of TDP-43 inclusions in PC12
cell lines after three days of induction. (FIG. 1A) Raw image of
TDP-43: GFP, nuclei are stained with DAPI. (FIG. 1B) Computer-based
image processing differentiates between cytoplasmic inclusions
(inset) and nuclear TDP-43 expression (inset).
[0014] FIGS. 2A to 12D are dose response curves showing inhibition
of TDP-43 inclusion formation with little toxicity by some of the
compounds described herein (FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A,
10A, 11A, 12A, and 12B). Structures of the compounds are also shown
(FIGS. 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12C, and 12D).
[0015] FIGS. 13A to 13C show structure activity analysis for
derivatives of compound 8. (FIG. 13A) Analogues of compound 8.
(FIG. 13B) Dose response curve for compounds 8 and 8c. (FIG. 13C)
Inhibition of inclusion formation in TDP-43::GFP PC12 cells by
analogs (3.5 uM).
[0016] FIGS. 14A to 14H shows inclusion formation in primary
neurons (DVI6) overexpressing TDP-43 and TIA. Map2 reactivity is
endogenous.
[0017] FIGS. 15A-1 to 15A-16, 15B-1 to 15B-16, and 15C show
induction of TDP-43 inclusions. FIGS. 15A-1 to 15A-16, stresses,
such as arsenite (0.5 mM, 1 hr) induce TDP-43 to translocate from
the nucleus to the cytoplasm where it forms granules that
co-localize with SG markers (e.g., TIA-1). Disease-linked mutations
in TDP-43 increase the amount of cytoplasmic translocation and
inclusion formation. Arrows point to cytoplasmic SG. FIGS. 15B-1 to
15B-16, show quantification of cells with SGs.
[0018] FIGS. 16A-1 to 16A-15, 16-B1 to 16-B15, 16C-1-16C-8, and
16D-1 to 16D-10 show co-localization of TDP-43 inclusions with
(FIGS. 16A-1 to 16A-15) TIA-1 in spinal cord of ALS patients and
(FIGS. 16B-1 to 16B-15) eIF3 in frontal cortex of patients with
FTLD-U.; (FIGS. 16C-1 to 16C-8) immunoadsorption with epitope
peptide removes reactivity, showing specificity.; (FIGS. 16D-1 to
16D-10) phospho-TDP-43 also co-localizes with SG markers. Bar=3
m.
[0019] FIGS. 17A-1 to 17A-8, 17B-1 to 17B-9, and 17C-1 to 17C-8
show derivation and characterization of human iPS (hiPS) cell
clones generated using a floxed single lentiviral stem cell
cassette (STEMCCA).; (FIGS. 17A-1 to 17A-8) Panel 1: Human
fibroblasts in culture and their reprogrammed iPSC progeny, derived
from individuals with alpha-1 antitrypsin deficiency, cystic
fibrosus, sickle cell disease, and scleroderma (SSc).; (FIGS. 17B-1
to 17B-9) Panel 2: Characterization of reprogrammed human clones by
RT-PCT (vs. hES and HDF controls); (FIGS. 17C-1 to 17C-8) Panel 3:
Characterization of reprogrammed human clones (intra vital
immunostaining on live cells).
[0020] FIG. 18 shows differentiated iPSCs stained markers: Neuronal
class III b-Tubulin (green) and homeobox protein DLX4/HB9 (red,
motor neuron specific).
[0021] FIGS. 19A-19D show motor function effects of LDN-0130436 in
C. elegans expressing human WT TDP-43 (FIG. 19A), non-transgenic N2
(FIG. 19B), A315T (FIG. 19C), and A315T (FIG. 19D).
[0022] FIGS. 20A-20D shows (FIG. 20A) C. elegans normally have 19
motor neurons, each of which shows strong connectivity; (FIG. 20B)
C. elegans line expressing human A315T TDP-43 shows a dramatic loss
of motor neurons by adult day 2; (FIG. 20C) when grown in the
presence of compound 8, motor neuron survival is strongly
increased. This is evident by counting the number of neuronal cell
bodies or counting the number of neurons lacking connections
(Dose=34.8 .mu.M); (FIG. 20D) quantification of neuronal loss (left
panel) and loss of connectivity (right panel) shows 50% and 70%
decreases with compound 8.
[0023] FIGS. 21A-21C shows results showing motor improvement in
lines of C. elegans that express human WT TDP-43 (CK405), A315T
TDP-43 (CK426) or non-transgenic (N2) (Dose=34.8 .mu.M).
[0024] FIGS. 22A-22B show treatment of PC12 cells expressing A315T
TDP-43::GFP show reduced levels of insoluble TDP-43 after treatment
with compound 8.
[0025] FIGS. 23A-23B shows rat cortical neuron (DIV7) transfected
with A315T TDP-43 and treated +/- compound 8 for 18 hr. Treatment
with compound 8 induces nuclear localization of TDP-43.
[0026] FIG. 24 shows compound 8 protects against induction of
caspase activity in hippocampal neurons expressing WT TDP-43.
[0027] FIG. 25 shows inhibition of HIV replication with
compounds.
[0028] FIG. 26 shows inhibition of HIV replication with
compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0029] ALS occurs with an incidence of approximately 1/100,000
(Lancet, 2007. 369: 2031-41; herein incorporated by reference in
its entirety). There is currently no therapy for ALS and it is
universally fatal. ALS presents with motor weakness in the distal
limbs that rapidly progresses proximally (Lancet, 2007. 369:
2031-41; Trends Mol Med, 2004. 10: 275-82; each herein incorporated
by reference in its entirety). TDP-43 is the major protein that
accumulates in affected motor neurons in sporadic ALS (Science,
2006. 314: 130-3; herein incorporated by reference in its
entirety). The causes of sporadic ALS are not known, but
identification of the major pathological species accumulating in
the spinal cord of ALS patients represents a seminal advance for
ALS research. TDP-43 is the only protein in ALS that is both
genetically and pathologically linked with sporadic ALS, which is
the predominant form of the disease. Multiple papers have
identified mutations in TDP-43 associated with sporadic and
familial ALS (Science, 2008. 319: 1668-72; Ann Neurol, 2008, 63(4),
535-538; each herein incorporated by reference in its entirety).
Inhibitors of cell death and inclusions linked to TDP-43 represents
a novel therapeutic approach to ALS, and could also elucidate
biochemical pathway linked to TDP-43 biology.
[0030] TDP-43 is one of the most promising targets for
pharmacotherapy of Amyotrophic Lateral Sclerosis (ALS). TDP-43 is
one of the major proteins that accumulate in inclusions in ALS, and
mutations in TDP-43 cause familial ALS, which indicates that
abnormalities in TDP-43 biology are sufficient to cause disease
(Science, 2006. 314: 130-3; herein incorporated by reference in its
entirety).
[0031] Formation of cytoplasmic TDP-43 inclusions appears to be
intimately linked to the RNA metabolism, and specifically the
biology of stress granules (SGs). SGs are protein-mRNA aggregates
that form in response to stress (Trends Biochem Sci, 2008. 33:
141-50; Biochem Soc Trans, 2002. 30: 963-9; Hum Mol Genet, 2010.
19: R46-64; each herein incorporated by reference in its entirety).
Studies from our laboratory, and other laboratories, show that
TDP-43 inclusions human brain (as well as in cell culture)
co-localize with SGs, and that agents that inhibit SG formation
also inhibit formation of TDP-43 inclusions (PLoS ONE, October 2010
5(10), e13250; herein incorporated by reference in its entirety).
Results presented herein demonstrate that neurodegeneration
mediated by TDP-43 is linked to the regulation of protein
translation and stress granule biology. The relationship between
TDP-43 and stress granule biology is important because it provides
a novel approach for dispersing TDP-43 inclusions using
physiological pathways that normally regulate this reversible
process rather than direct physical disruption of protein
aggregation by a small molecule pharmaceutical. Stress granule
biology also regulates autophagy and apoptosis, both of which are
linked to neurodegeneration. Hence, chemicals inhibiting TDP-43
aggregation can also inhibit neurodegeneration.
[0032] The inventors have completed a high throughput screen for
small molecules that inhibit TDP-43 aggregation using PC12 cells
that inducibly express TDP-43. They identified a number of
compounds (on different scaffolds) that inhibit TDP-43 inclusion
formation in a reproducible and dose-dependent manner, while
showing little to no cytotoxicity. These compounds can be further
tested to determine which of these lead compounds can inhibit
inclusion formation and neurodegeneration in secondary assays, and
then to do chemical optimization to increase the potency and
optimize the pharmacological properties of the two most promising
leads.
[0033] Accordingly, in one aspect the invention provides a method
of modulating stress granule formation, the method comprising
contacting a cell with a modulator of stress granule. As used
herein, the terms "modulator of stress granule" and "stress granule
modulator" refer to compounds and compositions that modulate the
formation and/or disaggregation of stress granules.
[0034] In one aspect, the invention provides methods for treatment
of a neurodegenerative disease or disorder, a cancer, and/or a
viral infection in a subject, the method comprising administering a
stress granule modulator to a subject in need thereof.
[0035] In another aspect, the invention provides methods of
diagnosing a neurodegenerative disease in a subject, the method
comprising administering a stress granule marker to the subject.
For use in diagnosing a stress granule marker can be labeled with a
label.
[0036] In another aspect, the invention provides methods of
modulating stress granules comprising contacting a cell with a
TDP-43 inclusion inhibiting compound.
[0037] In another aspect, the invention provides methods of
modulating TDP-43 inclusion formation comprising contacting a cell
with a TDP-43 inclusion inhibitor.
[0038] In yet another aspect, the invention provides a method of
screening for modulators of TDP-43 aggregation comprising
contacting a compound with the cell that expresses TDB-43 and
develops spontaneous inclusions.
[0039] In still another aspect, the invention provides a cell that
expresses wild-type TDB-43 and develops spontaneous inclusions.
[0040] In some embodiments, the stress granule modulator inhibits
the formation of a stress granule. The stress granule modulator can
inhibit the formation of a stress granule by at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, or 100% (i.e.,
complete inhibition) relative to a control.
[0041] In some embodiments, the stress granule modulator
disaggregates a stress granule. The stress granule modulator can
disperses or disaggregate a stress granule by at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, or 100%
(i.e., complete dispersal) relative to a control.
[0042] In some embodiments, the stress granule comprises TDP-43,
i.e., is a TDP-43 inclusion. Accordingly, in some embodiments, a
modulator of stress granule is a modulator of TDP-43 inclusions. As
used herein, the terms "modulator of TDP-43 inclusion" and "TDP-43
inclusion modulator" refer to compounds and compositions that
modulate the formation and/or disaggregation of cytoplasmic TDP-43
inclusions. By TDP-43 inclusion is meant protein-mRNA aggregates
comprise a TDP-43 protein. The TDP-43 protein in a stress granule
can be wild-type or a mutant form of TDP-43
[0043] In some embodiments, the stress granule comprises a mutant
FUS protein.
[0044] In some embodiments, TDP-43 is inducibly expressed.
[0045] In some embodiments, the cell line is a neuronal cell
line.
[0046] In some embodiments, the cell is treated with a
physiochemical stressor. In some embodiments, the physicochemical
stressor is selected from arsenite, nutrient deprivation, heat
shock, osmotic shock, a virus, genotoxic stress, radiation,
oxidative stress, oxidative stress, a mitochondrial inhibitor, and
an endoplasmic reticular stressor. In some embodiments, the
physicochemical stressor is ultraviolet or x-ray radiation. In some
embodiments, the physicochemical stressor is oxidative stress
induced by FeCl.sub.2 or CuCl.sub.2 and a peroxide.
[0047] Method of Treatment
[0048] In another aspect, the invention provides a method for
treatment of a neurodegenerative disease or disorder, a caner,
and/or a viral infections, the method comprising administering an
effective amount of a modulator of stress granule formation to a
subject in need thereof.
[0049] In some embodiments, stress granule formation is inhibited.
In some embodiments, the stress granule is disaggregated. In some
embodiments, stress granule formation is stimulated.
[0050] In some embodiments, the stress granule comprises tar DNA
binding protein-43 (TDP-43), T-cell intracellular antigen 1
(TIA-1), TIA1 cytotoxic granule-associated RNA binding protein-like
1 (TIAR), GTPase activating protein binding protein 1 (G3BP-1),
GTPase activating protein binding protein 2 (G3BP-2), tris
tetraprolin (TTP), fused in sarcoma (FUS), or fragile X mental
retardation protein (FMRP).
[0051] In some embodiments, the stress granule comprises tar DNA
binding protein-43 (TDP-43), T-cell intracellular antigen 1
(TIA-1), TIA1 cytotoxic granule-associated RNA binding protein-like
1 (TIAR), GTPase activating protein binding protein 1 (G3BP-1),
GTPase activating protein binding protein 2 (G3BP-2), fused in
sarcoma (FUS), or fragile X mental retardation protein (FMRP).
[0052] In some embodiments, the stress granule comprises tar DNA
binding protein-43 (TDP-43), T-cell intracellular antigen 1
(TIA-1), TIA1 cytotoxic granule-associated RNA binding protein-like
1 (TIAR), GTPase activating protein binding protein 1 (G3BP-1),
GTPase activating protein binding protein 2 (G3BP-2), or fused in
sarcoma (FUS).
[0053] In some embodiments, the stress granule comprises tar DNA
binding protein-43 (TDP-43).
[0054] In some embodiments, the stress granule comprises T-cell
intracellular antigen 1 (TIA-1).
[0055] In some embodiments, the stress granule comprises TIA1
cytotoxic granule-associated RNA binding protein-like 1 (TIAR).
[0056] In some embodiments, the stress granule comprises GTPase
activating protein binding protein 1 (G3BP-1).
[0057] In some embodiments, the stress granule comprises GTPase
activating protein binding protein 2 (G3BP-2).
[0058] In some embodiments, the stress granule comprises tris
tetraprolin (TTP).
[0059] In some embodiments, the stress granule comprises fused in
sarcoma (FUS).
[0060] In some embodiments, the stress granule comprises fragile X
mental retardation protein (FMRP).
[0061] In some embodiments, the methods are performed in a subject
suffering from a neurodegenerative disease or disorder, a cancer,
and/or a viral infection. In some embodiments, the methods are
performed in a subject suffering from a neurodegenerative disease
or disorder. In some embodiments, the methods are performed in a
subject suffering from a cancer. In some embodiments, the methods
are performed in a subject suffering from a viral infection.
[0062] In some embodiments, the methods comprise administering a
stress granule modulator to a subject in need thereof. In some
embodiments, the subject is a mammal. In some embodiments, the
subject is a nematode. In some embodiments, the subject is
human.
[0063] In some embodiments, the methods further comprise the step
of diagnosing the subject for the neurodegenerative disease or
disorder prior to onset of administration of a stress granule
modulator.
[0064] In some embodiments, the neurodegenerative disease is
selected from the group consisting of Alzheimer's disease,
frontotemporal dementia, FTLD-U (a frontotemporal dementia caused
by mutations in progranulin protein), amyotrophic lateral sclerosis
(ALS), Huntington's chorea, Creutzfeld-Jacob disease, trinucleotide
repeat diseases, cerebral degenerative diseases presenile dementia,
senile dementia, Parkinsonism linked to chromosome 17 (FTDP-17),
progressive supranuclear palsy (PSP), Huntington's disease (HD),
Pick's disease, primary progressive aphasia, corticobasal dementia,
Parkinson's disease, Parkinson's disease with dementia, dementia
with Lewy bodies, Down's syndrome, multiple system atrophy, spinal
muscular atrophy (SMA), spinocerebellar ataxia, spinal degenerative
disease/motor neuron degenerative diseases, Hallervorden-Spatz
syndrome, cerebral infarct, cerebral trauma, chronic traumatic
encephalopathy, transient ischemic attack, and any combination
thereof.
[0065] In some embodiments, the neurodegenerative disease is
selected from the group consisting of Alzheimer's disease,
frontotemporal dementia, FTLD-U (a frontotemporal dementia caused
by mutations in progranulin protein), amyotrophic lateral sclerosis
(ALS), Huntington's chorea, Creutzfeld-Jacob disease, senile
dementia, Parkinsonism linked to chromosome 17 (FTDP-17),
progressive supranuclear palsy (PSP), Huntington's disease (HD),
Pick's disease, primary progressive aphasia, corticobasal dementia,
Parkinson's disease, Parkinson's disease with dementia, dementia
with Lewy bodies, Down's syndrome, multiple system atrophy, spinal
muscular atrophy (SMA), spinocerebellar ataxia, spinal degenerative
disease/motor neuron degenerative diseases, Hallervorden-Spatz
syndrome, cerebral infarct, cerebral trauma, chronic traumatic
encephalopathy, transient ischemic attack, and any combination
thereof.
[0066] In some embodiments, the neurodegenerative disease is
Alzheimer's disease or amyotrophic lateral sclerosis (ALS).
[0067] In some embodiments, the cancer is selected from the group
consisting of breast cancer, a melanoma, adrenal gland cancer,
biliary tract cancer, bladder cancer, brain or central nervous
system cancer, bronchus cancer, blastoma, carcinoma, a
chondrosarcoma, cancer of the oral cavity or pharynx, cervical
cancer, colon cancer, colorectal cancer, esophageal cancer,
gastrointestinal cancer, glioblastoma, hepatic carcinoma, hepatoma,
kidney cancer, leukemia, liver cancer, lung cancer, lymphoma,
non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreas
cancer, peripheral nervous system cancer, prostate cancer, sarcoma,
salivary gland cancer, small bowel or appendix cancer, small-cell
lung cancer, squamous cell cancer, stomach cancer, testis cancer,
thyroid cancer, urinary bladder cancer, uterine or endometrial
cancer, vulval cancer, and any combination thereof.
[0068] In some embodiments, the cancer is selected from the group
consisting of blastoma, carcinoma, a glioblastoma, hepatic
carcinoma, leukemia, and any combination thereof.
[0069] In some embodiments, the viral infection is caused by a
virus selected from the group consisting of West Nile virus,
Respiratory Syncitial Virus (RSV), herpes simplex virus 1, herpes
simplex virus 2, Epstein-Barr virus (EBV), hepatitis virus A,
hepatitis virus B, hepatitis virus C, influenza viruses, chicken
pox, avian flu viruses, smallpox, polio viruses, HIV-1, HIV-2, and
any combination thereof.
[0070] In some embodiments, the viral infection is caused by a
virus selected from the group consisting of herpes simplex virus 1,
herpes simplex virus 2, Epstein-Barr virus (EBV), hepatitis virus
A, hepatitis virus B, hepatitis virus C, HIV-1, HIV-2, and any
combination thereof.
[0071] In some embodiments, the viral infection is HIV-1 or
HIV-2.
[0072] In some embodiments, the pathology of the neurodegenerative
disease or disorder, cancer, and/or viral infection comprises
stress granules.
[0073] In some embodiments, pathology of the disease or disorder
comprises stress granules. By comprising stress granules is meant
that number of stress granules in a cell in the subject is changed
relative to a control and/or healthy subject or relative to before
onset of said disease or disorder. Exemplary diseases and disorders
pathology of which incorporate stress granules include, but are not
limited to, neurodegenerative diseases, cancers, and viral
infections.
[0074] Stress response follows a U shaped curve. Too much (such as
is induced in neurodegenerative diseases) is bad. Too little,
though, is also bad under other conditions (such as with an acute
stress such as a stroke). Thus some diseases benefit from
inhibiting stress granule formation, while other diseases benefit
from stimulating stress granule formation.
[0075] TDP-43 and other RNA-binding proteins appear to act in the
cytoplasm to process mRNA, such as by splicing mRNA, cleaving mRNA
introns, cleaving untranslated regions of mRNA or modifying protein
translation at the synapse, axon, dendrite or soma. For instance,
FMPR is a protein that causes mental retardation, and the signaling
systems that affect TDP-43 might also affect this protein and
improve cognitive function. This can be particularly important at
the synapse where neurons communicate. Without wishing to be bound
by a theory, the signaling systems that modulators of TDP-43
inclusions target can also modify these processes. These processes
could play a role in neurodegeneration or mental health illnesses
(e.g., schizophrenia). Thus, modulators of TDP-43 inclusions can
also act by modifying these RNA processing and protein translation
processes.
[0076] Neurodegenerative Diseases:
[0077] Without wishing to be bound by a theory, modulators of
TDP-43 inclusions, such as stress granules, can be used to delay
the progression of neurodegenerative illnesses where the pathology
incorporates stress granules. Such illnesses include ALS (where
mutations in TDP-43 cause ALS), and frontotemporal dementia (where
TDP-43 is the main protein that accumulates) where TDP-43 is the
main protein that accumulates to form the pathology. This group
also includes Alzheimer's disease and FTLD-U (a frontotemporal
dementia caused by mutations in tau protein), where TDP-43 and
other stress granule proteins co-localize with tau pathology.
Because modulators of TDP-43 inclusions can act to block the
enzymes that SIGNAL stress granule formation, such as the three
enzymes that phosphorylate eIF2a-PERK, GCN2 and HRI, modulators of
TDP-43 inclusions can also reverse stress granules that might not
include TDP-43. Accordingly, modulators of TDP-43 can be used for
treatment of neurodegenerative diseases and disorders pathology of
which incorporates stress granules. This can include Huntington's
chorea and Creutzfeld-Jacob disease--both quite rare.
[0078] The term "neurodegenerative disease" as used herein, refers
to a neurological disease characterized by loss or degeneration of
neurons. The term "neurodegenerative disease" includes diseases
caused by the involvement of genetic factors or the cell death
(apoptosis) of neurons attributed to abnormal protein accumulation
and so on. Additionally, neurodegenerative diseases include
neurodegenerative movement disorders and neurodegenerative
conditions relating to memory loss and/or dementia.
Neurodegenerative diseases include tauopathies and
a-synucleopathies. Exemplary neurodegenerative diseases include,
but are not limited to, Alzheimer's disease, frontotemporal
dementia, FTLD-U (a frontotemporal dementia caused by mutations in
tau protein), amyotrophic lateral sclerosis (ALS), Huntington's
chorea, Creutzfeld-Jacob disease, trinucleotide repeat diseases,
cerebral degenerative diseases presenile dementia, senile dementia,
Parkinsonism linked to chromosome 17 (FTDP-17), progressive
supranuclear palsy (PSP), Huntington's disease (HD), Pick's
disease, primary progressive aphasia, corticobasal dementia,
Parkinson's disease, Parkinson's disease with dementia, dementia
with Lewy bodies, Down's syndrome, multiple system atrophy, spinal
muscular atrophy (SMA), spinocerebellar ataxia, spinal degenerative
disease/motor neuron degenerative diseases, and Hallervorden-Spatz
syndrome.
[0079] As used herein, the term "a-synucleopathy" refers to a
neurodegenerative disorder or disease involving aggregation of
a-synuclein or abnormal a-synuclein in nerve cells in the brain.
a-Synucleopathies include, but are not limited to, Parkinson's
disease, Parkinson's disease with dementia, dementia with Lewy
bodies, Pick's disease, Down's syndrome, multiple system atrophy,
amylotrophic lateral sclerosis (ALS) and Hallervorden-Spatz
syndrome.
[0080] Cancers:
[0081] Cancer cells grow quickly and in low oxygen environments by
activating different elements of their stress response. Researchers
have shown that drugs targeting different elements of the stress
response can be anti-neoplastic. Rapamycin blocks mTOR, upregulates
autophagy and inhibits some types of tumors. Proteasomal
inhibitors, such as velcade (Millenium Pharma) are used to treat
some cancers ($1 billion/yr). HSP90 inhibitors, such as
17-allylaminogeldanamycin (17AAG), are in clinical trials for
cancer. Without wishing to be bound by a theory, modulators of
TDP-43 inclusions can also be used for treatment of cancer.
Additionally, TDP-43 modulators ca be combined with one or more
cancer therapies, such as chemotherapy and radiation therapy.
[0082] A "cancer" in subject refers to the presence of cells
possessing characteristics typical of cancer-causing cells, such as
uncontrolled proliferation, immortality, metastatic potential,
rapid growth and proliferation rate, and certain characteristic
morphological features. Often, cancer cells will be in the form of
a tumor, but such cells may exist alone within an animal, or may be
a non-tumorigenic cancer cell, such as a leukemia cell. In some
circumstances, cancer cells will be in the form of a tumor; such
cells may exist locally within an animal, or circulate in the blood
stream as independent cells, for example, leukemic cells. Examples
of cancer include but are not limited to breast cancer, a melanoma,
adrenal gland cancer, biliary tract cancer, bladder cancer, brain
or central nervous system cancer, bronchus cancer, blastoma,
carcinoma, a chondrosarcoma, cancer of the oral cavity or pharynx,
cervical cancer, colon cancer, colorectal cancer, esophageal
cancer, gastrointestinal cancer, glioblastoma, hepatic carcinoma,
hepatoma, kidney cancer, leukemia, liver cancer, lung cancer,
lymphoma, non-small cell lung cancer, osteosarcoma, ovarian cancer,
pancreas cancer, peripheral nervous system cancer, prostate cancer,
sarcoma, salivary gland cancer, small bowel or appendix cancer,
small-cell lung cancer, squamous cell cancer, stomach cancer,
testis cancer, thyroid cancer, urinary bladder cancer, uterine or
endometrial cancer, vulval cancer, and the like.
[0083] Other exemplary cancers include, but are not limited to,
ACTH-producing tumors, acute lymphocytic leukemia, acute
nonlymphocytic leukemia, cancer of the adrenal cortex, bladder
cancer, brain cancer, breast cancer, cervical cancer, chronic
lymphocytic leukemia, chronic myelocytic leukemia, colorectal
cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal
cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia,
head & neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,
kidney cancer, liver cancer, lung cancer (small and/or non-small
cell), malignant peritoneal effusion, malignant pleural effusion,
melanoma, mesothelioma, multiple myeloma, neuroblastoma,
non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovary (germ
cell) cancer, prostate cancer, pancreatic cancer, penile cancer,
retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell
carcinomas, stomach cancer, testicular cancer, thyroid cancer,
trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of
the vulva, Wilm's tumor, and the like
[0084] Viral Infections:
[0085] SGs often form during viral illnesses. Accordingly, some
viruses might mobilize SGs to subvert the cellular machinery
towards production of viral proteins. In this case, inhibitors of
stress granules can be useful for interfering with viral function.
Other viruses appear to inhibit SG formation to prevent the cell
from mobilizing a stress response. In such a case, inducer of
stress granules can interfere with viral activity and help combat
viral infections. One exemplary stress granule inducer is
Salubrinal, a PERK inhibitor. Two viruses for which SG biology has
been investigated include West Nile virus and Respiratory Syncitial
Virus (RSV). See for example. M. E Emara & M. A. Brinton, Proc.
Natl. Acad. Sci. USA, 104(21): 9041-9046 (2007); incorporated
herein by reference in its entirety.
[0086] Exemplary viruses include, but are not limited to, West Nile
virus, Respiratory Syncitial Virus (RSV), Epstein-Barr virus (EBV,
a member of the herpesvirus family), the hepatitis A, B, C, and D
viruses, influenza viruses, chicken pox, avian flu viruses,
smallpox, polio viruses, HIV, and the like.
[0087] Imaging
[0088] The compounds described herein are useful for detection
and/or diagnosis of stress granules. Accordingly, they can be used
as in vivo imaging agents of tissues and organs in various
biomedical applications. When used in imaging applications, the
compounds described herein typically comprise an imaging agent,
which can be covalently or noncovalently attached to the
compound.
[0089] As used herein, the term "imaging agent" refers to an
element or functional group in a molecule that allows for the
detection, imaging, and/or monitoring of the presence and/or
progression of a condition(s), pathological disorder(s), and/or
disease(s). The imaging agent may be an echogenic substance (either
liquid or gas), non-metallic isotope, an optical reporter, a boron
neutron absorber, a paramagnetic metal ion, a ferromagnetic metal,
a gamma-emitting radioisotope, a positron-emitting radioisotope, or
an x-ray absorber.
[0090] Suitable optical reporters include, but are not limited to,
fluorescent reporters and chemiluminescent groups. A wide variety
of fluorescent reporter dyes are known in the art. Typically, the
fluorophore is an aromatic or heteroaromatic compound and can be a
pyrene, anthracene, naphthalene, acridine, stilbene, indole,
benzindole, oxazole, thiazole, benzothiazole, cyanine,
carbocyanine, salicylate, anthranilate, coumarin, fluorescein,
rhodamine or other like compound. Suitable fluorescent reporters
include xanthene dyes, such as fluorescein or rhodamine dyes,
including, but not limited to, Alexa Fluor.RTM. dyes
(InvitrogenCorp.; Carlsbad, Calif.), fluorescein, fluorescein
isothiocyanate (FITC), Oregon Green.TM., rhodamine, Texas red,
tetrarhodamine isothiocynate (TRITC), 5-carboxyfluorescein (FAM),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),
N,N,N,N'-tetramefhyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX). Suitable fluorescent reporters also
include the naphthylamine dyes that have an amino group in the
alpha or beta position. For example, naphthylamino compounds
include 1-dimethylamino-naphthyl-5-sulfonate,
1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene
sulfonate, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS). Other fluorescent reporter dyes include coumarins, such as
3-phenyl-7-isocyanatocoumarin; acridines, such as
9-isothiocyanatoacridine and acridine orange;
N-(p(2-benzoxazolyl)phenyl)maleimide; cyanines, such as Cy2,
indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),
indodicarbocyanine 5.5 (Cy5.5),
3-(-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CyA);
1H,5H,11H, 15H-Xantheno[2,3,4-ij:5,6,7-i'j']diquinolizin-18-ium,
9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]
amino]sulfonyl]-4(or
2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-inner salt (TR or
Texas Red); BODIPY.TM. dyes; benzoxadiazoles; stilbenes; pyrenes;
and the like. Many suitable forms of these fluorescent compounds
are available and can be used.
[0091] Examples of fluorescent proteins suitable for use as imaging
agents include, but are not limited to, green fluorescent protein,
red fluorescent protein (e.g., DsRed), yellow fluorescent protein,
cyan fluorescent protein, blue fluorescent protein, and variants
thereof (see, e.g., U.S. Pat. Nos. 6,403,374, 6,800,733, and
7,157,566; each herein incorporated by reference in its entirety).
Specific examples of GFP variants include, but are not limited to,
enhanced GFP (EGFP), destabilized EGFP, the GFP variants described
in Doan et al, Mol. Microbiol, 55:1767-1781 (2005), the GFP variant
described in Crameri et al, Nat. Biotechnol., 14:315319 (1996), the
cerulean fluorescent proteins described in Rizzo et al, Nat.
Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509
(1998), and the yellow fluorescent protein described in Nagal et
al, Nat. Biotechnol., 20:87-90 (2002) (each herein incorporated by
reference in its entirety). DsRed variants are described in, e.g.,
Shaner et al, Nat. Biotechnol., 22:1567-1572 (2004) (herein
incorporated by reference in its entirety), and include
mStrawberry, mCherry, morange, mBanana, mHoneydew, and mTangerine.
Additional DsRed variants are described in, e.g., Wang et al, Proc.
Natl. Acad. Sci. U.S.A., 101:16745-16749 (2004) (herein
incorporated by reference in its entirety), and include mRaspberry
and mPlum. Further examples of DsRed variants include mRFPmars
described in Fischer et al, FEBS Lett., 577:227-232 (2004) and
mRFPruby described in Fischer et al, FEBS Lett, 580:2495-2502
(2006) (each herein incorporated by reference in its entirety).
[0092] Suitable echogenic gases include, but are not limited to, a
sulfur hexafluoride or perfluorocarbon gas, such as
perfluoromethane, perfluoroethane, perfluoropropane,
perfluorobutane, perfluorocyclobutane, perfluropentane, or
perfluorohexane.
[0093] Suitable non-metallic isotopes include, but are not limited
to, .sup.11C, .sup.14C, .sup.13N, .sup.18F, .sup.123I, .sup.124I,
and .sup.125I.
[0094] Suitable radioisotopes include, but are not limited to,
.sup.99mTc, .sup.95Tc, .sup.111In, .sup.62Cu, .sup.64Cu, Ga,
.sup.68Ga, and .sup.153Gd.
[0095] Suitable paramagnetic metal ions include, but are not
limited to, Gd(III), Dy(III), Fe(III), and Mn(II).
[0096] Suitable X-ray absorbers include, but are not limited to,
Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag,
and Ir.
[0097] In some embodiments, the radionuclide is bound to a
chelating agent or chelating agent-linker attached to the
aggregate. Suitable radionuclides for direct conjugation include,
without limitation, .sup.18F, .sup.124I, .sup.125I, .sup.131I, and
mixtures thereof. Suitable radionuclides for use with a chelating
agent include, without limitation, .sup.47Sc, .sup.64Cu, .sup.67Cu,
.sup.89Sr, .sup.86Y, .sup.87Y, .sup.90Y, .sup.105Rh, .sup.111Ag,
.sup.111In, .sup.117mSn, .sup.149Pm, .sup.153Sm, .sup.166Ho,
.sup.177Lu, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Bi, and
mixtures thereof. Suitable chelating agents include, but are not
limited to, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their
phosphonate analogs, and mixtures thereof. One of skill in the art
will be familiar with methods for attaching radionuclides,
chelating agents, and chelating agent-linkers to the
nanoparticles.
[0098] A detectable response generally refers to a change in, or
occurrence of, a signal that is detectable either by observation or
instrumentally. In certain instances, the detectable response is
fluorescence or a change in fluorescence, e.g., a change in
fluorescence intensity, fluorescence excitation or emission
wavelength distribution, fluorescence lifetime, and/or fluorescence
polarization. One of skill in the art will appreciate that the
degree and/or location of labeling in a subject or sample can be
compared to a standard or control (e.g., healthy tissue or organ).
In certain other instances, the detectable response the detectable
response is radioactivity (i.e., radiation), including alpha
particles, beta particles, nucleons, electrons, positrons,
neutrinos, and gamma rays emitted by a radioactive substance such
as a radionuclide.
[0099] Specific devices or methods known in the art for the in vivo
detection of fluorescence, e.g., from fluorophores or fluorescent
proteins, include, but are not limited to, in vivo near-infrared
fluorescence (see, e.g., Frangioni, Curr. Opin. Chem. Biol,
7:626-634 (2003)), the Maestro.TM. in vivo fluorescence imaging
system (Cambridge Research & Instrumentation, Inc.; Woburn,
Mass.; herein incorporated by reference in its entirety), in vivo
fluorescence imaging using a flying-spot scanner (see, e.g.,
Ramanujam et al, IEEE Transactions on Biomedical Engineering,
48:1034-1041 (2001); herein incorporated by reference in its
entirety, and the like. Other methods or devices for detecting an
optical response include, without limitation, visual inspection,
CCD cameras, video cameras, photographic film, laser-scanning
devices, fluorometers, photodiodes, quantum counters,
epifluorescence microscopes, scanning microscopes, flow cytometers,
fluorescence microplate readers, or signal amplification using
photomultiplier tubes.
[0100] Any device or method known in the art for detecting the
radioactive emissions of radionuclides in a subject is suitable for
use in the present invention. For example, methods such as Single
Photon Emission Computerized Tomography (SPECT), which detects the
radiation from a single photon gamma-emitting radionuclide using a
rotating gamma camera, and radionuclide scintigraphy, which obtains
an image or series of sequential images of the distribution of a
radionuclide in tissues, organs, or body systems using a
scintillation gamma camera, may be used for detecting the radiation
emitted from a radiolabeled aggregate. Positron emission tomography
(PET) is another suitable technique for detecting radiation in a
subject.
[0101] Magnetic resonance imaging (MRI), nuclear magnetic resonance
imaging (NMRI), or magnetic resonance tomography (MRT) is a medical
imaging technique used in radiology to visualize detailed internal
structures. MRI makes use of the property of Nuclear magnetic
resonance (NMR) to image nuclei of atoms inside the body. Thus,
labels having magnetic properties can be detected using MRI and/or
related technologies.
[0102] SG proteins, such as TDP-43, undergo translocation to the
cytoplasm and aggregate. Translocation likely requires a
post-translational modification as well as binding to a transport
protein. Aggregation is associated with a change in protein
conformation. Modulators of TDP-43 can bind to SG proteins
specifically under states of cytoplasmic translocation (for
instance, because they recognize a binding site enabled by a
post-translational modification) or SG proteins that are in an
aggregated state associated with SGs. Thus, modulators of TDP-43
inclusions can be used image areas in a subject's body that have
increased levels of SGs--either physiological or pathological. For
instance, in ALS and Alzheimer's disease, inventors have
demonstrated that TDP-43 associates with the pathology that
accumulates. Thus, compounds that recognize aggregated TDP-43 can
be used to image pathology, much like the imaging agent PiB, which
is currently used in Alzheimer's research. However, PiB has a
problem because it recognizes amyloid, whose accumulation occurs
both in patients with Alzheimer's disease and in many non-demented
people. However, an agent that recognizes SGs would specifically
identify patients that have intracellular pathology (such as
neurofibrillary tangles, which the inventor has shown are
associated with SGs). Such agents can be used to diagnose patients
with or at risk for neurodegenerative illnesses.
[0103] Additionally, imaging of SGs in a subject can be used to
localize pain. For example, a modulator can be administered to a
subject having a pain and the pain is difficult to localize, and
subsequent imaging can localize the area of the body that is
diseased or injured. This can greatly speed diagnosis and can be
generally applicable throughout the medical arts.
[0104] Further, the compounds described herein can be used to image
organs for transplants. Organs are harvested for transplants--such
as kidneys and hearts. A problem in the field is that surgeons
don't know how well the organ survived the harvesting and transport
to the receiving hospital. Sometimes, organs are transplanted only
to have them fail because they were injured in transport. A quick
cytologic stain with a stress granule marker can be a large advance
for the field. Accordingly, modulators of TDP-43 inclusions can be
used as stress granule markers.
[0105] Modulators
[0106] In some embodiments, the compound is of formula (I):
##STR00001##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0107] In one embodiment, the compound is of formula (I). In
another embodiment, the compound is an isomer of formula (I). In
still another embodiment, the compound is an analog of formula (I).
In yet another embodiment, the compound is a prodrug of a compound
of formula (I). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula (I).
[0108] In compounds of formula (I), R.sup.11 and R.sup.12 are
independently H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, C(O)R.sup.17, or C(O)OR.sup.17, each of which can
be optionally substituted. In some embodiments, R.sup.11 is an
optionally substituted aryl or heteroaryl.
[0109] In one embodiment, R.sup.11 is an optionally substituted
pyridinyl. The pyridinyl group can be substituted at the 2, 3, 4,
5, 6, or any combinations of those positions. When the pyridinyl
group is substituted by two or more substituents, said two or more
substituents can be same, all different, or a combination of same
and different substituents. Additionally, the pyridinyl group can
be attached to the rest of formula (I) by 2, 3, 4, 5, or 6
positions. In one embodiment of this, R.sup.11 is
##STR00002##
[0110] In another embodiment, R.sup.11 is an optionally substituted
benzothiazolyl. The benzothiazolyl group can be substituted at the
2, 4, 5, 6, 7, or any combinations of those positions. When the
benzothiazolyl group is substituted by two or more substituents,
said two or more substituents can be same, all different, or a
combination of same and different substituents. Additionally, the
benzothiazolyl group can be attached to the rest of formula (I) by
2, 4, 5, 6, or 7 position. In one embodiment of this R.sup.11
is
##STR00003##
[0111] In some embodiments, R.sup.12 is H or an optionally
substituted C.sub.1-C.sub.6 alkyl.
[0112] In compounds of formula (I), R.sup.13 and R.sup.14 are
independently H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, OR.sup.17, C(O)R.sup.17, or C(O)OR.sup.17,
N(R.sup.17).sub.2, each of which can be optionally substituted.
[0113] In one embodiment, at least one of R.sup.13 and R.sup.14 is
H. When R.sup.13 and R.sup.14 are different, the carbon atom to
which they are attached is chiral. Thus, when R.sup.13 and R.sup.14
are different from each other, the carbon to which they are
attached can have the R- or the S-configuration.
[0114] In another embodiment, both of R.sup.13 and R.sup.14 are
H.
[0115] Variable b, in the compounds of formula (I), can be 0, 1, 2,
3, or 4. In one embodiment b is 0.
[0116] When present each R.sup.15 is independently, halo, alkyl,
alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl, NO.sub.2,
OR.sup.17, OC(O)R.sup.17, OC(O)OR.sup.17, N(R.sup.17).sub.2,
NHC(O)R.sup.17, NHC(O)OR.sup.17, C(O)R.sup.17, C(O)OR.sup.17,
SR.sup.17, or SO.sub.2R.sup.17, each of which can be optionally
substituted.
[0117] The variable c, in the compounds of formula (I), can be 0,
1, 2, 3, 4, or 5. It is to be understood that when present a
R.sup.16 substituent can be located at the 2, 3, 4, 5, or 6
position of the phenyl group. When more than one R.sup.16 is
present, they can be located at any combination of 2, 3, 4, 5, and
6 positions of the phenyl group. In one embodiment of this, when b
is 1, substituent R.sup.16 is located at position 2 of the phenyl
group. Accordingly, a compound of formula (I) is of formula
(Ia):
##STR00004##
[0118] When present, each R.sup.16 is independently, halo, alkyl,
alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl, NO.sub.2,
OR.sup.17, OC(O)R.sup.17, OC(O)OR.sup.17, N(R.sup.17).sub.2,
NHC(O)R.sup.17, NHC(O)OR.sup.17, C(O)R.sup.17, C(O)OR.sup.17,
SR.sup.17, or SO.sub.2R.sup.17, each of which can be optionally
substituted. In one embodiment, R.sup.16 is OR.sup.17. In one
embodiment, R.sup.16 is methoxy.
[0119] In compounds of formula (I), substituent R.sup.17 is
independently for each occurrence, H, alkyl, alkenyl, alkynyl,
cyclyl, heterocyclyl, aryl, or heteroaryl, each of which can be
optionally substituted. In one embodiment, R.sup.17 is an
optionally substituted C.sub.1-C.sub.6 alkyl. In one further
embodiment of this, R.sup.17 is a methyl, ethyl, propyl, or
butyl.
[0120] Compounds of formula (I) can be synthesized by following the
outlined shown in Scheme I.
##STR00005##
[0121] In one embodiment, a compound of formula (I) is LDN-0118790
or LDN-0121669. Structure of LDN-0118790 is shown in FIG. 2B and
structure of LDN-0121669 is shown in FIG. 3B.
[0122] In one embodiment, a compound of formula (I) is an analog of
LDN-0118790 or LDN-0121669.
[0123] In one embodiment, a compound of formula (I) is an isomer of
LDN-0118790 or LDN-0121669.
[0124] In one embodiment, a compound of formula (I) is a prodrug of
LDN-0118790 or LDN-0121669.
[0125] In one embodiment, a compound of formula (I) is a
pharmaceutically acceptable salt of LDN-0118790 or LDN-0121669.
[0126] In some embodiments, the compound is of formula (II):
##STR00006##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0127] In one embodiment, the compound is of formula (II). In
another embodiment, the compound is an isomer of formula (II). In
still another embodiment, the compound is an analog of formula
(II). In yet another embodiment, the compound is a prodrug of a
compound of formula (II). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula (II).
[0128] In compounds of formula (II), R.sup.21 and R.sup.23 are
independently H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, C(O)R.sup.27, or C(O)OR.sup.27, each of which can
be optionally substituted. In some embodiments, R.sup.21 and
R.sup.23 are both H.
[0129] Substituent R.sup.22 can be H or alkyl. In some embodiments,
R.sup.22 is an optionally substituted C.sub.1-C.sub.6 alkyl. In one
further embodiment of this, R.sup.22 is methyl or ethyl.
[0130] When present, R.sup.24, R.sup.25, and R.sup.26 are
independently halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl,
heteroaryl, NO.sub.2, OR.sup.27, OC(O)R.sup.27, OC(O)OR.sup.27,
N(R.sup.27).sub.2, NHC(O)R.sup.27, NHC(O)OR.sup.27, C(O)R.sup.27,
C(O)OR.sup.27, SR.sup.27, or SO.sub.2R.sup.27, each of which can be
optionally substituted. In one embodiment, both of R.sup.25 and
R.sup.26 are OR.sup.27. In one embodiment, both of R.sup.25 and
R.sup.26 are methoxy.
[0131] It is to be understood that when present, R.sup.24,
R.sup.25, and R.sup.26 can be attached any position on the ring
they are linked to. For example, R.sup.25 and/or R.sup.26
substituents can be attached to position 2, 3, 4, 5, or 6 position
of the phenyl group. When more than one of R.sup.25 or R.sup.26 is
present, they can be located at any combination of 2, 3, 4, 5, and
6 positions of the respective phenyl group. Additionally, when two
or more of R.sup.24 are present all can be same, all different, or
a combination of same and different. Similarly, when two or more of
R.sup.25 are present all can be same, all different, or a
combination of same and different. Likewise, when two or more of
R.sup.26 are present all can be same, all different, or a
combination of same and different.
[0132] Variable d can be 0, 1, 2, 3, or 4. In one embodiment, d is
0.
[0133] Variables e and f are independently 0, 1, 2, 3, 4, or 5. In
one embodiment both of e and f are 1. In one embodiment, when e is
1, R.sup.25 is attached to position 4 of the phenyl group. In one
embodiment, when f is 1, R.sup.26 is attached to position 2 of the
phenyl group.
[0134] In some embodiments, a compound of formula (II) is of
formula (IIa):
##STR00007##
[0135] Substituent R.sup.27 in compounds of formula (II) is
independently for each occurrence H, alkyl, alkenyl, alkynyl,
cyclyl, heterocyclyl, aryl, or heteroaryl, each of which can be
optionally substituted. In one embodiment, R.sup.27 is an
optionally substituted C.sub.1-C.sub.6 alkyl. In one further
embodiment of this, R.sup.27 is a methyl, ethyl, propyl, or
butyl.
[0136] In one embodiment, a compound of formula (II) is LDN-0124614
as shown in FIG. 4B.
[0137] In one embodiment, a compound of formula (I) is an analog of
LDN-0124614.
[0138] In one embodiment, a compound of formula (I) is an isomer of
LDN-0124614.
[0139] In one embodiment, a compound of formula (I) is a prodrug of
LDN-0124614.
[0140] In one embodiment, a compound of formula (I) is a
pharmaceutically acceptable salt of LDN-0124614.
[0141] In some embodiments, the compound is of formula (III):
##STR00008##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0142] In one embodiment, the compound is of formula (III). In
another embodiment, the compound is an isomer of formula (III). In
still another embodiment, the compound is an analog of formula
(III). In yet another embodiment, the compound is a prodrug of a
compound of formula (III). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula
(III).
[0143] In compounds of formula (III), R.sup.31 and R.sup.32 are
independently, H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, C(O)R.sup.36, or C(O)OR.sup.36, each of which can
be optionally substituted. In some embodiments, R.sup.31 and
R.sup.32 are both H.
[0144] When present, R.sup.33, R.sup.34, and R.sup.35 are
independently halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl,
heteroaryl, NO.sub.2, OR.sup.36, OC(O)R.sup.36, OC(O)OR.sup.36,
N(R.sup.36).sub.2, NHC(O)R.sup.36, NHC(O)OR.sup.36, C(O)R.sup.36,
C(O)OR.sup.36, SR.sup.36, or SO.sub.2R.sup.36, each of which can be
optionally substituted. In one embodiment, R.sup.35 is alkyl or
OR.sup.36. In one embodiment, R.sup.35 is ethyl or methoxy.
[0145] It is to be understood that when present, R.sup.33,
R.sup.34, and R.sup.36 can be attached to any available position on
the ring they are linked to. For example, R.sup.34 and/or R.sup.35
substituents can be attached to position 2, 3, 4, 5, or 6 position
of the phenyl group. When more than one of R.sup.34 or R.sup.35 is
present, they can be located at any combination of 2, 3, 4, 5, and
6 positions of the respective phenyl group. Additionally, when two
or more of R.sup.33 are present all can be same, all different, or
a combination of same and different. Similarly, when two or more of
R.sup.34 are present all can be same, all different, or a
combination of same and different. Likewise, when two or more of
R.sup.35 are present all can be same, all different, or a
combination of same and different.
[0146] Variable g in compounds of formula (III) can be 0, 1, 2, 3,
4, 5, 6, or 7. In one embodiment, g is 0.
[0147] Variables h and i in compounds of formula (III) are
independently 0, 1, 2, 3, 4, or 5. In some embodiments, h is 0. In
some other embodiments, i is 1. In one embodiment, h is 0 and i is
1.
[0148] When i is 1, R.sup.35 can be attached to position 2, 3, 4,
5, or 6 of the phenyl group. In one embodiment, R.sup.35 is
attached at position 4 of the phenyl group.
[0149] Substituent R.sup.36 is independently for each occurrence H,
alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or heteroaryl,
each of which can be optionally substituted. In one embodiment,
R.sup.36 is an optionally substituted C.sub.1-C.sub.6 alkyl. In one
further embodiment of this, R.sup.36 is a methyl, ethyl, propyl, or
butyl.
[0150] In some embodiments, a compound of formula (III) is of
formula (Ma):
##STR00009##
[0151] In one embodiment, a compound of formula (III) is
LDN-0125734, shown in FIG. 5B, or LDN-01215735, shown in FIG.
6B.
[0152] In one embodiment, a compound of formula (III) is an analog
of LDN-0125734 or LDN-01215735.
[0153] In one embodiment, a compound of formula (III) is an isomer
of LDN-0125734 or LDN-01215735.
[0154] In one embodiment, a compound of formula (III) is a prodrug
of LDN-0125734 or LDN-01215735.
[0155] In one embodiment, a compound of formula (III) is a
pharmaceutically acceptable salt of LDN-0125734 or
LDN-01215735.
[0156] In some embodiments, the compound is of formula (IV):
##STR00010##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0157] In one embodiment, the compound is of formula (IV). In
another embodiment, the compound is an isomer of formula (IV). In
still another embodiment, the compound is an analog of formula
(IV). In yet another embodiment, the compound is a prodrug of a
compound of formula (IV). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula (IV).
[0158] R.sup.41 is H, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, heteroaryl, C(O)R.sup.47, C(O)OR.sup.47,
SO.sub.2R.sup.47, each of which can be optionally substituted. In
some embodiments, R.sup.41 is H or C.sub.1-C.sub.6 alkyl. In one
embodiment, R.sup.41 is methyl.
[0159] R.sup.42 and R.sup.43 are independently H, halo, alkyl,
alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl, NO.sub.2,
OR.sup.47, OC(O)R.sup.47, OC(O)OR.sup.47, N(R.sup.47).sub.2,
NHC(O)R.sup.47, NHC(O)OR.sup.47, C(O)R.sup.47, C(O)OR.sup.47,
SR.sup.47, or SO.sub.2R.sup.47, each of which can be optionally
substituted. In one embodiment, both of R.sup.42 and R.sup.43 are
C.sub.1-C.sub.6 alkyl. In one further embodiment of this, both of
R.sup.42 and R.sup.43 are methyl.
[0160] In compounds of formula (IV), R.sup.44 can be H, alkyl,
alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl,
C(O)R.sup.47, C(O)OR.sup.47, SO.sub.2R.sup.47, each of which can be
optionally substituted. In some embodiments, R.sup.44 is H or
C.sub.1-C.sub.6 alkyl.
[0161] When present, each R.sup.45 is independently halo, alkyl,
alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl, NO.sub.2,
OR.sup.47, OC(O)R.sup.47, OC(O)OR.sup.47, N(R.sup.47).sub.2,
NHC(O)R.sup.47, NHC(O)OR.sup.17, C(O)R.sup.47, C(O)OR.sup.47,
SR.sup.47, or SO.sub.2R.sup.47, each of which can be optionally
substituted.
[0162] Similarly, when present, each R.sup.46 is independently
halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl,
NO.sub.2, OR.sup.47, OC(O)R.sup.47, OC(O)OR.sup.47,
N(R.sup.47).sub.2, NHC(O)R.sup.47, NHC(O)OR.sup.47, C(O)R.sup.47,
C(O)OR.sup.47, SR.sup.47, or SO.sub.2R.sup.47, each of which can be
optionally substituted. In some embodiments, R.sup.46 is alkyl or
halo. In one embodiment R.sup.46 is C.sub.1-C.sub.6 alkyl. In one
embodiment, R.sup.46 is methyl. In one embodiment R.sup.46 is
chloro (Cl).
[0163] It is to be understood that when present, R.sup.45 and
R.sup.46 can be attached to any available position on the ring they
are linked to. For example, R.sup.45 substituent can be attached to
position 2, 3, 4 or 5 of the pyrrolidine ring. When more than one
of R.sup.45 is present, they can be located at any combination of
2, 3, 4, and 5 positions of the pyrrolidine ring. Similarly,
R.sup.46 substituent can be attached to position 2, 3, 4, 5, or 6
of the phenyl ring. Again, when more than one of R.sup.46 is
present, they can be located at any combination of 2, 3, 4, 5, and
6 positions of the phenyl group. Additionally, when two or more of
R.sup.45 are present all can be same, all different, or a
combination of same and different. Similarly, when two or more of
R.sup.46 are present all can be same, all different, or a
combination of same and different.
[0164] Variable j can be 0, 1, 2, 3, or 4. In one embodiment, j is
0.
[0165] Variable k can be 0, 1, 2, 3, 4, or 5. In some embodiments,
k is 2. When k is 2, one R.sup.46 can be attached to position 2 and
the other can be at position 3, 4, 5, or 6 of the phenyl ring; one
R.sup.46 can be at position 3 and the other R.sup.46 can be at
position 4 or 5. In one embodiment, when k is two, one R.sup.46 is
at position 2 and the other is at position 5 of the phenyl
ring.
[0166] When k is 2 both R.sup.46 groups can be the same or
different. In some embodiment of this, one R.sup.46 is
C.sub.1-C.sub.6 alkyl and the other is halo. In one embodiment of
this, one R.sup.46 is methyl and the other is Cl.
[0167] Substituent R.sup.47 is independently for each occurrence H,
alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or heteroaryl,
each of which can be optionally substituted.
[0168] In some embodiments, a compound of formula (IV) is of
formula (IVa):
##STR00011##
[0169] In some further embodiments of this, a compound of formula
(Iva) is of formula (IVb):
##STR00012##
[0170] In one embodiment, a compound of formula (IV) is LDN-0130436
as shown in FIG. 7B.
[0171] In one embodiment, a compound of formula (IV) is an analog
of LDN-0130436.
[0172] In one embodiment, a compound of formula (IV) is
LDN-0130436, LDN-0130436B, LDN-0130436C, LDN-0130436D,
LDN-0130436E, LDN-0130436F, LDN-0130436G, or LDN-0130436H.
[0173] In one embodiment, a compound of formula (IV) is
LDN-0130436B.
[0174] In one embodiment, a compound of formula (IV) is
LDN-0130436C.
[0175] In one embodiment, a compound of formula (IV) is
LDN-0130436D.
[0176] In one embodiment, a compound of formula (IV) is
LDN-0130436E.
[0177] In one embodiment, a compound of formula (IV) is
LDN-0130436F.
[0178] In one embodiment, a compound of formula (IV) is
LDN-0130436G.
[0179] In one embodiment, a compound of formula (IV) is
LDN-0130436H.
[0180] In one embodiment, a compound of formula (IV) is an isomer
of LDN-0130436.
[0181] In one embodiment, a compound of formula (IV) is a prodrug
of LDN-0130436.
[0182] In one embodiment, a compound of formula (IV) is a
pharmaceutically acceptable salt of LDN-0130436.
[0183] In some embodiments, the compound is of formula (V):
##STR00013##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0184] In one embodiment, the compound is of formula (V). In
another embodiment, the compound is an isomer of formula (V). In
still another embodiment, the compound is an analog of formula (V).
In yet another embodiment, the compound is a prodrug of a compound
of formula (V). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula (V).
[0185] R.sup.51 can be H, alkyl, alkenyl, alkynyl, cyclyl,
heterocyclyl, aryl, heteroaryl, C(O)R.sup.55, C(O)OR.sup.55,
SO.sub.2R.sup.55, each of which can be optionally substituted. In
some embodiments, R.sup.51 is C.sub.1-C.sub.6 alkyl or H.
[0186] When present, each of R.sup.52, R.sup.53, and R.sup.54 is
independently halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl,
heteroaryl, NO.sub.2, OR.sup.55, OC(O)R.sup.55, OC(O)OR.sup.55,
N(R.sup.55).sub.2, NHC(O)R.sup.55, NHC(O)OR.sup.55, C(O)R.sup.55,
C(O)OR.sup.55, SR.sup.55, or SO.sub.2R.sup.5, each of which can be
optionally substituted.
[0187] In some embodiments, R.sup.52 is a C.sub.1-C.sub.6 alkyl. In
one embodiment, R.sup.52 is t-butyl.
[0188] In some embodiments, R.sup.54 is OR.sup.55. In one
embodiment, R.sup.54 is OCH.sub.2CF.sub.3.
[0189] It is to be understood that when present, R.sup.52,
R.sup.53, and R.sup.55 can be attached to any available position on
the ring they are linked to. When two or more of R.sup.52 are
present all can be same, all different, or a combination of same
and different. Similarly, when two or more of R.sup.53 are present
all can be same, all different, or a combination of same and
different. Likewise, when two or more of R.sup.54 are present all
can be same, all different, or a combination of same and
different.
[0190] Variable 1 can be 0, 1, 2, 3, or 4. In one embodiment, 1 is
1. In one embodiment, when 1 is 1, R.sup.52 is attached at position
4 of the phenyl ring. In one embodiment, 1 is 1 and R.sup.52 is
t-butyl.
[0191] Variable m can be 0, 1, 2, 3, or 4. In one embodiment, m is
0.
[0192] Variable n can be 0, 1, 2, or 3. In one embodiment, n is 2.
When m is 2, the two R.sup.54 can be attached to neighboring
carbons of the ring. Additionally, when n is 2, both R.sup.54
groups can be the same or different. In some embodiment, both
R.sup.54 are OR.sup.55. In one embodiment of R.sup.55 is an
optionally substituted C.sub.1-C.sub.6 alkyl. In one embodiment,
both R.sup.54 are OCH.sub.2CF.sub.3.
[0193] Substituent R.sup.55 is independently for each occurrence H,
alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or heteroaryl,
each of which can be optionally substituted.
[0194] In one embodiment, a compound of formula (V) is LDN-0015257
as shown in FIG. 8B.
[0195] In one embodiment, a compound of formula (V) is an analog of
LDN-0015257.
[0196] In one embodiment, a compound of formula (V) is an isomer of
LDN-0015257.
[0197] In one embodiment, a compound of formula (V) is a prodrug of
LDN-0015257.
[0198] In one embodiment, a compound of formula (V) is a
pharmaceutically acceptable salt of LDN-0015257.
[0199] In some embodiments, the compound is of formula (VI):
##STR00014##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0200] In one embodiment, the compound is of formula (VI). In
another embodiment, the compound is an isomer of formula (VI). In
still another embodiment, the compound is an analog of formula
(VI). In yet another embodiment, the compound is a prodrug of a
compound of formula (VI). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula (VI).
[0201] In compounds of formula (VI), p can be 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10. In one embodiment, p is 4.
[0202] R.sup.61 and R.sup.62 are independently H, alkyl, alkenyl,
alkynyl, cyclyl, heterocyclyl, aryl, heteroaryl, C(O)R.sup.65,
C(O)OR.sup.65, or SO.sub.2R.sup.65 each of which can be optionally
substituted, or R.sup.61 and R.sup.62 together with the nitrogen
they are attached to form a 5-8 membered optionally substituted
heterocyclyl.
[0203] In one embodiment, one of R.sup.61 and R.sup.62 is H and the
other is an optionally substituted C.sub.1-C.sub.6 alkyl.
[0204] In one embodiment, one of one of R.sup.61 and R.sup.62 is H
and the other is --(CH.sub.2).sub.tN(R.sup.66).sub.2. Each R.sup.66
is independently H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, C(O)R.sup.65, C(O)OR.sup.65, or SO.sub.2R.sup.65
each of which can be optionally substituted. In some embodiments,
one R.sup.66 is H and the other is C(O)R.sup.65. In some further
embodiments of this, one of R.sup.66 is C(O)R.sup.65 and R.sup.65
is an optionally substituted aryl. In one embodiment, the
optionally substituted aryl is a substituted phenyl, e.g.,
4-fluoro-phenyl, i.e.,
##STR00015##
[0205] Variable t can 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one
embodiment, t is 2.
[0206] When present, each of R.sup.63 and R.sup.64 is independently
halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl,
NO.sub.2, OR.sup.65, OC(O)R.sup.65, OC(O)OR.sup.65,
N(R.sup.65).sub.2, NHC(O)R.sup.65, NHC(O)OR.sup.65, C(O)R.sup.65,
C(O)OR.sup.65, SR.sup.65, or SO.sub.2R.sup.65, each of which can be
optionally substituted. In some embodiments, both of R.sup.63 and
R.sup.64 are halo. In one embodiment, both of R.sup.63 and R.sup.64
are F.
[0207] When present, R.sup.63 and R.sup.64 can be attached to any
available position on the ring they are linked to. For example,
R.sup.63 and/or R.sup.64 can be attached to position 2, 3, 4, 5 or
6 of the respective phenyl group they are linked to. In one
embodiment, R.sup.63 and R.sup.64 are attached to position 4 of the
respective phenyl ring.
[0208] When two or more of R.sup.63 are present all can be same,
all different, or a combination of same and different. Similarly,
when two or more of R.sup.64 are present all can be same, all
different, or a combination of same and different.
[0209] When present, R.sup.65 is independently for each occurrence
H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or
heteroaryl, each of which can be optionally substituted.
[0210] Variables q and r are independently 0, 1, 2, 3, 4, or 5. In
some embodiments, q is 1. In some embodiments, r is 1. In one
embodiment, q and r are both 1. When q and r are both 1, R.sup.63
and R.sup.64 can be same or different.
[0211] In one embodiment, R.sup.61 and R.sup.62 together with the
nitrogen they are attached to form an optionally substituted
six-membered ring. Accordingly, in some embodiments, a compound of
formula (VI) is of formula (VIa):
##STR00016##
wherein R.sup.66 is an optionally substituted aryl or heteroaryl,
and R.sup.67 is a H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, or OR.sup.65, each of which can be optionally
substituted.
[0212] In some embodiments, R.sup.66 is an optionally substituted
phenyl.
[0213] In some embodiments, R.sup.67 is OH or
CH.sub.2NHCH.sub.2C.ident.CH.
[0214] In one embodiment, R.sup.66 is an optionally substituted
phenyl and R.sup.67 is OH.
[0215] In another embodiment, R.sup.66 is an optionally substituted
phenyl and R.sup.67 is CH.sub.2NHCH.sub.2C.ident.CH.
[0216] In one embodiment, a compound of formula (VI) is LDN-0052997
(shown in FIG. 12), LDN-0052998 (shown in FIG. 12), LDN-0057218
(shown in FIG. 9), or LDN-0057325 (shown in FIG. 10).
[0217] In one embodiment, a compound of formula (VI) is an analog
of LDN-0052997, LDN-0052998, LDN-0057218, or LDN-0057325.
[0218] In one embodiment, a compound of formula (VI) is an isomer
of LDN-0052997, LDN-0052998, LDN-0057218, or LDN-0057325.
[0219] In one embodiment, a compound of formula (VI) is a prodrug
of LDN-0052997, LDN-0052998, LDN-0057218, or LDN-0057325.
[0220] In one embodiment, a compound of formula (VI) is a
pharmaceutically acceptable salt of LDN-0052997, LDN-0052998,
LDN-0057218, or LDN-0057325.
[0221] In some embodiments, the compound is of formula (VII):
##STR00017##
and analogs, derivatives, isomers, prodrugs, and pharmaceutically
acceptable salts thereof.
[0222] In one embodiment, the compound is of formula (VII). In
another embodiment, the compound is an isomer of formula (VII). In
still another embodiment, the compound is an analog of formula
(VII). In yet another embodiment, the compound is a prodrug of a
compound of formula (VII). In another embodiment, the compound is a
pharmaceutically acceptable salt of a compound of formula
(VII).
[0223] Variable u in compounds of formula (VII) can be 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10. In one embodiment, u is 2.
[0224] In compounds of formula (VII), R.sup.71 and R.sup.72 are
independently H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl,
aryl, heteroaryl, C(O)R.sup.75, C(O)OR.sup.75, or SO.sub.2R.sup.75
each of which can be optionally substituted. In one embodiment,
both of R.sup.71 and R.sup.72 are H.
[0225] When present, each R.sup.73 and R.sup.74 is independently
halo, alkyl, alkenyl, cyclyl, heterocyclyl, aryl, heteroaryl,
NO.sub.2, OR.sup.75, OC(O)R.sup.75, OC(O)OR.sup.75,
N(R.sup.75).sub.2, NHC(O)R.sup.75, NHC(O)OR.sup.75, C(O)R.sup.75,
C(O)OR.sup.75, SR.sup.75, or SO.sub.2R.sup.75, each of which can be
optionally substituted. In some embodiments, R.sup.73 is a
C.sub.1-C.sub.6 alkyl. In one embodiment, R.sup.73 is methyl. In
some embodiments, R.sup.74 is a halogen. In one embodiment,
R.sup.74 is Cl.
[0226] When present, R.sup.73 and R.sup.74 can be attached to any
available position on the ring they are linked to. For example,
R.sup.73 can be linked to position 4, 5, 6, 7, or 8 of quinolinone
to which it is attached. Similarly, R.sup.74 can be attached to
position 2, 3, 4, 5 or 6 of the phenyl to which it is attached.
When two or more of R.sup.73 are present all can be same, all
different, or a combination of same and different. Similarly, when
two or more of R.sup.74 are present all can be same, all different,
or a combination of same and different.
[0227] When present, R.sup.75 is independently for each occurrence
H, alkyl, alkenyl, alkynyl, cyclyl, heterocyclyl, aryl, or
heteroaryl, each of which can be optionally substituted.
[0228] Variables v and w are independently 0, 1, 2, 3, 4, or 5. In
some embodiments, v is 2. In some embodiments, w is 1. In one
embodiment, v is 2 and w is 1.
[0229] In some embodiments, a compound of formula (VII) is of
formula (VIIa):
##STR00018##
[0230] In one embodiment, a compound of formula (VII) is
LDN-0076437 shown in FIG. 11.
[0231] In one embodiment, a compound of formula (VI) is an analog
of LDN-0076437.
[0232] In one embodiment, a compound of formula (VI) is an isomer
of LDN-0076437.
[0233] In one embodiment, a compound of formula (VI) is a prodrug
of LDN-0076437.
[0234] In one embodiment, a compound of formula (VI) is a
pharmaceutically acceptable salt of LDN-0076437.
[0235] Exemplary embodiments of compounds of formulas (I)-(VI) may
be obtained commercially from ChemDiv (San Diego, Calif.;
http://us.chemdiv.com), ChemBridge (San Diego, Calif.;
http://www.chembridge.com), and/or Peakdale (Chapel-en-le-Frith,
Derbyshire, UK; www.peakdale.co.uk). For example, LDN-0125735 (pdt
no. C737-1968), LDN-0130436 (pdt no. G243-0036), LDN-0130436B (pdt
no. G243-0026), LDN-0130436C (pdt no. G243-0049), LDN-0130436D (pdt
no. G243-0053), LDN-0130436E (pdt no. G243-0093), LDN-0130436F (pdt
no. G243-0212), LDN-0130436G (pdt no. G243-0223), LDN-0130436H (pdt
no. G243-0064), LDN-0124614 (pdt no. C202-0879), LDN-0125734 (pdt
no. C737-1949), and LDN-0076437 (pdt. no. C279-0807) are available
from ChemDiv. LDN-0118790 (pdt. no. 51516001), LDN-0118870 (pdt.
no. 52164389), LDN-0119629 (pdt. no. 59052230), LDN-0121669 (pdt.
no. 81328507), LDN-0196125 (pdt. no. 10570989), LDN-0066337 (pdt.
no. 6140545), LDN-0202779 (pdt. no. 40784194), and LDN-0209285
(pdt. no. 94103550) are available from ChemBridge. LDN-0015257
(pdt. no. 3001137) is available from Peakdale.
[0236] Other exemplary embodiments of compounds of formulas
(I)-(VI) such as LDN-0057218 and LDN-0057325 can be synthesized as
discussed in, for example, Choi, et al., Bioorganic & Medicinal
Chemistry 10 (2002) 4091-4102; herein incorporated by reference in
its entirety.
[0237] Other exemplary inclusion modulators or inhibitors include
mithramycin A, parthenolide and mycophenolic acid.
[0238] In some embodiments, the inclusion inhibitor is selected
from mithramycin A, parthenolide and mycophenolic acid.
[0239] In some embodiments, the inclusion inhibitor is selected
from the group consisting of
##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023##
or a pharmaceutically acceptable salt thereof.
[0240] In some embodiments, the inclusion inhibitor is selected
from the group consisting of
##STR00024## ##STR00025##
or a pharmaceutically acceptable salt thereof.
[0241] Pharmaceutical Compositions
[0242] For administration to a subject, the compounds of the
invention can be provided in pharmaceutically acceptable
compositions. These pharmaceutically acceptable compositions
comprise a therapeutically-effective amount of one or more of the
compounds described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention can be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), lozenges,
dragees, capsules, pills, tablets (e.g., those targeted for buccal,
sublingual, and systemic absorption), boluses, powders, granules,
pastes for application to the tongue; (2) parenteral
administration, for example, by subcutaneous, intramuscular,
intravenous or epidural injection as, for example, a sterile
solution or suspension, or sustained-release formulation; (3)
topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8)
transmucosally; or (9) nasally. Additionally, compounds can be
implanted into a patient or injected using a drug delivery system.
See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol.
24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides
and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Pat. No.
3,773,919; and U.S. Pat. No. 35 3,270,960, each of which are herein
incorporated by reference in its entirety.
[0243] As used here, the term "pharmaceutically acceptable" refers
to those compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0244] As used here, the term "pharmaceutically-acceptable carrier"
means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents,
such as magnesium stearate, sodium lauryl sulfate and talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl
oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; (22) bulking agents, such as
polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; (22) C.sub.2-C.sub.12 alchols, such as
ethanol; and (23) other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, coloring agents,
release agents, coating agents, sweetening agents, flavoring
agents, perfuming agents, preservative and antioxidants can also be
present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable carrier" or the like are
used interchangeably herein.
[0245] The phrase "therapeutically-effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect in at least a
sub-population of cells in an animal at a reasonable benefit/risk
ratio applicable to any medical treatment. For example, an amount
of a compound administered to a subject that is sufficient to
produce a statistically significant, measurable change in at least
one symptom of inflammation.
[0246] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art. Generally, a
therapeutically effective amount can vary with the subject's
history, age, condition, sex, as well as the severity and type of
the medical condition in the subject, and administration of other
pharmaceutically active agents.
[0247] As used herein, the term "administer" refers to the
placement of a composition into a subject by a method or route
which results in at least partial localization of the composition
at a desired site such that desired effect is produced. A compound
or composition described herein can be administered by any
appropriate route known in the art including, but not limited to,
oral or parenteral routes, including intravenous, intramuscular,
subcutaneous, transdermal, airway (aerosol), pulmonary, nasal,
rectal, and topical (including buccal and sublingual)
administration.
[0248] Exemplary modes of administration include, but are not
limited to, injection, infusion, instillation, inhalation, or
ingestion. "Injection" includes, without limitation, intravenous,
intramuscular, intraarterial, intrathecal, intraventricular,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, sub capsular, subarachnoid, intraspinal,
intracerebro spinal, and intrasternal injection and infusion. In
some embodiments, the compositions are administered by intravenous
infusion or injection.
[0249] By "treatment", "prevention" or "amelioration" of a disease
or disorder is meant delaying or preventing the onset of such a
disease or disorder, reversing, alleviating, ameliorating,
inhibiting, slowing down or stopping the progression, aggravation
or deterioration the progression or severity of a condition
associated with such a disease or disorder. In one embodiment, at
least one symptom of a disease or disorder is alleviated by at
least 5%, at least 10%, at least 20%, at least 30%, at least 40%,
or at least 50%.
[0250] As used herein, the terms "effective" and "effectiveness"
includes both pharmacological effectiveness and physiological
safety. Pharmacological effectiveness refers to the ability of the
treatment to result in a desired biological effect in the patient.
Physiological safety refers to the level of toxicity, or other
adverse physiological effects at the cellular, organ and/or
organism level (often referred to as side-effects) resulting from
administration of the treatment. "Less effective" means that the
treatment results in a therapeutically significant lower level of
pharmacological effectiveness and/or a therapeutically greater
level of adverse physiological effects.
[0251] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, canine species, e.g.,
dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and
fish, e.g., trout, catfish and salmon. Patient or subject includes
any subset of the foregoing, e.g., all of the above, but excluding
one or more groups or species such as humans, primates or rodents.
In certain embodiments, the subject is a mammal, e.g., a primate,
e.g., a human. The terms, "patient" and "subject" are used
interchangeably herein. The terms, "patient" and "subject" are used
interchangeably herein.
[0252] Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
are not limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
disorders associated with neurodegenerative disease or disorder,
cancer, or viral infections.
[0253] In addition, the methods described herein can be used to
treat domesticated animals and/or pets. A subject can be male or
female. A subject can be one who has been previously diagnosed with
or identified as suffering from or having a neurodegenerative
disease or disorder, a disease or disorder associated with cancer,
a disease or disorder associated with viral infection, or one or
more complications related to such diseases or disorders but need
not have already undergone treatment.
[0254] The compound can be administrated to a subject in
combination with a pharmaceutically active agent. Exemplary
pharmaceutically active compound include, but are not limited to,
those found in Harrison's Principles of Internal Medicine,
13.sup.th Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY;
Physicians Desk Reference, 50.sup.th Edition, 1997, Oradell N.J.,
Medical Economics Co.; Pharmacological Basis of Therapeutics,
8.sup.th Edition, Goodman and Gilman, 1990; United States
Pharmacopeia, The National Formulary, USP XII NF XVII, 1990;
current edition of Goodman and Oilman's The Pharmacological Basis
of Therapeutics; and current edition of The Merck Index, each of
which are herein incorporated by reference in its entirety. In some
embodiments, pharmaceutically active agent include those agents
known in the art for treatment of cancer, inflammation or
inflammation associated disorders, or infections.
[0255] The compound and the pharmaceutically active agent can be
administrated to the subject in the same pharmaceutical composition
or in different pharmaceutical compositions (at the same time or at
different times). When administrated at different times, the
compound and the pharmaceutically active agent can be administered
within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3
hours, 4, hours, 8 hours, 12 hours, 24 hours of administration of
the other When the inhibitor and the pharmaceutically active agent
are administered in different pharmaceutical compositions, routes
of administration can be different.
[0256] The amount of compound that can be combined with a carrier
material to produce a single dosage form will generally be that
amount of the inhibitor that produces a therapeutic effect.
Generally out of one hundred percent, this amount will range from
about 0.1% to 99% of inhibitor, preferably from about 5% to about
70%, most preferably from 10% to about 30%.
[0257] Toxicity and therapeutic efficacy can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of
the population) and the ED50 (the dose therapeutically effective in
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD50/ED50. Compositions that exhibit large
therapeutic indices, are preferred.
[0258] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration
utilized.
[0259] The therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC50 (i.e., the concentration of the therapeutic
which achieves a half-maximal inhibition of symptoms) as determined
in cell culture. Levels in plasma may be measured, for example, by
high performance liquid chromatography. The effects of any
particular dosage can be monitored by a suitable bioassay.
[0260] The dosage may be determined by a physician and adjusted, as
necessary, to suit observed effects of the treatment. Generally,
the compositions are administered so that inflammasome inhibitor is
given at a dose from 1 .mu.g/kg to 150 mg/kg, 1 .mu.g/kg to 100
mg/kg, 1 .mu.g/kg to 50 mg/kg, 1 .mu.g/kg to 20 mg/kg, 1 .mu.g/kg
to 10 mg/kg, 1 .mu.g/kg to 1 mg/kg, 100 .mu.g/kg to 100 mg/kg, 100
.mu.g/kg to 50 mg/kg, 100 .mu.g/kg to 20 mg/kg, 100 .mu.g/kg to 10
mg/kg, 100 .mu.g/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50
mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100
mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be
understood that ranges given here include all intermediate ranges,
for example, the range 1 tmg/kg to 10 mg/kg includes 1 mg/kg to 2
mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg,
1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg
to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10
mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7 mg/kg to 10
mg/kg,8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and the like. It is
to be further undertood that the ranges intermediate to the given
above are also within the scope of this invention, for example, in
the range 1 mg/kg to 10 mg/kg, dose ranges such as 2 mg/kg to 8
mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, and the like.
[0261] With respect to duration and frequency of treatment, it is
typical for skilled clinicians to monitor subjects in order to
determine when the treatment is providing therapeutic benefit, and
to determine whether to increase or decrease dosage, increase or
decrease administration frequency, discontinue treatment, resume
treatment or make other alteration to treatment regimen. The dosing
schedule can vary from once a week to daily depending on a number
of clinical factors, such as the subject's sensitivity to the
polypeptides. The desired dose can be administered at one time or
divided into subdoses, e.g., 2-4 subdoses and administered over a
period of time, e.g., at appropriate intervals through the day or
other appropriate schedule. Such sub-doses can be administered as
unit dosage forms. In some embodiments, administration is chronic,
e.g., one or more doses daily over a period of weeks or months.
Examples of dosing schedules are administration daily, twice daily,
three times daily or four or more times daily over a period of 1
week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4
months, 5 months, or 6 months or more.
DEFINITIONS
[0262] Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
Unless explicitly stated otherwise, or apparent from context, the
terms and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the
singular.
[0263] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0264] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the invention.
[0265] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0266] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%.
[0267] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise.
[0268] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
this disclosure, suitable methods and materials are described
below. The term "comprises" means "includes." The abbreviation,
"e.g." is derived from the Latin exempli gratia, and is used herein
to indicate a non-limiting example. Thus, the abbreviation "e.g."
is synonymous with the term "for example."
[0269] The terms "decrease", "reduced", "reduction", "decrease" or
"inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
""reduced", "reduction" or "decrease" or "inhibit" means a decrease
by at least 10% as compared to a reference level, for example a
decrease by at least about 20%, or at least about 30%, or at least
about 40%, or at least about 50%, or at least about 60%, or at
least about 70%, or at least about 80%, or at least about 90% or up
to and including a 100% decrease (e.g. absent level as compared to
a reference sample), or any decrease between 10-100% as compared to
a reference level.
[0270] The terms "increased", "increase" or "enhance" or "activate"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of any doubt, the terms
"increased", "increase" or "enhance" or "activate" means an
increase of at least 10% as compared to a reference level, for
example an increase of at least about 20%, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about
a 2-fold, or at least about a 3-fold, or at least about a 4-fold,
or at least about a 5-fold or at least about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a
reference level.
[0271] The term "statistically significant" or "significantly"
refers to statistical significance and generally means a two
standard deviation (2SD) below normal, or lower, concentration of
the marker. The term refers to statistical evidence that there is a
difference. It is defined as the probability of making a decision
to reject the null hypothesis when the null hypothesis is actually
true. The decision is often made using the p-value.
[0272] For simplicity, chemical moieties are defined and referred
to throughout can be univalent chemical moieties (e.g., alkyl,
aryl, etc.) or multivalent moieties under the appropriate
structural circumstances clear to those skilled in the art. For
example, an "alkyl" moiety can be referred to a monovalent radical
(e.g. CH.sub.3--CH.sub.2--), or in other instances, a bivalent
linking moiety can be "alkyl," in which case those skilled in the
art will understand the alkyl to be a divalent radical (e.g.,
--CH.sub.2--CH.sub.2--), which is equivalent to the term
"alkylene." Similarly, in circumstances in which divalent moieties
are required and are stated as being "alkoxy", "alkylamino",
"aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic",
"alkyl" "alkenyl", "alkynyl", "aliphatic", or "cycloalkyl", those
skilled in the art will understand that the terms "alkoxy",
"alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl",
"heterocyclic", "alkyl", "alkenyl", "alkynyl", "aliphatic", or
"cycloalkyl" refer to the corresponding divalent moiety.
[0273] The term "halogen" refers to any radical of fluorine,
chlorine, bromine or iodine.
[0274] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents. Exemplary acyl groups include, but are
not limited to, (C.sub.1-C.sub.6)alkanoyl (e.g., formyl, acetyl,
propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),
(C.sub.3-C.sub.6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,
cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),
heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,
pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl,
piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g.,
benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl,
thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl,
1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,
benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl,
cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl
group may be any one of the groups described in the respective
definitions.
[0275] The term "alkyl" refers to saturated non-aromatic
hydrocarbon chains that may be a straight chain or branched chain,
containing the indicated number of carbon atoms (these include
without limitation propyl, allyl, or propargyl), which may be
optionally inserted with N, O, or S. For example, C.sub.1-C.sub.6
indicates that the group may have from 1 to 6 (inclusive) carbon
atoms in it.
[0276] The term "alkenyl" refers to an alkyl that comprises at
least one double bond. Exemplary alkenyl groups include, but are
not limited to, for example, ethenyl, propenyl, butenyl,
l-methyl-2-buten-1-yl and the like.
[0277] The term "alkynyl" refers to an alkyl that comprises at
least one triple bond.
[0278] The term "alkoxy" refers to an --O-alkyl radical.
[0279] The term "aminoalkyl" refers to an alkyl substituted with an
amino.
[0280] The term "mercapto" refers to an --SH radical.
[0281] The term "thioalkoxy" refers to an --S-alkyl radical.
[0282] The term "aryl" refers to monocyclic, bicyclic, or tricyclic
aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring
may be substituted by a substituent. Examplary aryl groups include,
but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl,
fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl,
and the like.
[0283] The term "arylalkyl" refers to alkyl substituted with an
aryl.
[0284] The term "cyclyl" or "cycloalkyl" refers to saturated and
partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, for example, 3 to 8 carbons, and, for example, 3 to 6
carbons, wherein the cycloalkyl group additionally may be
optionally substituted. Exemplary cycloalkyl groups include, but
are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl,
and the like.
[0285] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by a substituent. Examplary heteroaryl groups include,
but are not limited to, pyridyl, furyl or furanyl, imidazolyl,
benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl,
pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the
like.
[0286] The term "heteroarylalkyl" refers to an alkyl substituted
with a heteroaryl.
[0287] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring may be
substituted by a substituent. Examplary heterocyclyl groups
include, but are not limited to piperazinyl, pyrrolidinyl,
dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
[0288] The term "haloalkyl" refers to an alkyl group having one,
two, three or more halogen atoms attached thereto. Exemplary
haloalkyl groups incude, but are not limited to chloromethyl,
bromoethyl, trifluoromethyl, and the like.
[0289] The term "optionally substituted" means that the specified
group or moiety, such as an alkyl group, alkenyl group, alkynyl
group, cyclyl group, heterocyclyl group, aryl group, heteroaryl
group and the like, is unsubstituted or is substituted with one or
more (typically 1-4 substituents) independently selected from the
group of substituents listed below in the definition for
"substituents" or otherwise specified.
[0290] The term "substituents" refers to a group "substituted" on
an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, or
heteroaryl group at any atom of that group. Suitable substituents
include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl,
alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,
amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl,
alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl,
arenesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In
some cases, two substituents, together with the carbons to which
they are attached to can form a ring.
[0291] In many cases, protecting groups are used during preparation
of the compounds of the invention. As used herein, the term
"protected" means that the indicated moiety has a protecting group
appended thereon. In some preferred embodiments of the invention,
compounds contain one or more protecting groups. A wide variety of
protecting groups can be employed in the methods of the invention.
In general, protecting groups render chemical functionalities inert
to specific reaction conditions, and can be appended to and removed
from such functionalities in a molecule without substantially
damaging the remainder of the molecule.
[0292] Representative protecting groups, are disclosed in Greene
and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d
ed., John Wiley & Sons, New York, 199; herein incorporated by
reference in its entirety. Examples of hydroxyl protecting groups
include, but are not limited to, t-butyl, t-butoxymethyl,
methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p'-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,
chloroacetate, trichloroacetate, trifluoroacetate, pivaloate,
benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate
and tosylate. Exemplary amino-protecting groups include, but are
not limited to, carbamate protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and
imine and cyclic imide protecting groups, such as phthalimido and
dithiasuccinoyl.
[0293] As used here in the term "isomer" refers to compounds having
the same molecular formula but differing in structure. Isomers
which differ only in configuration and/or conformation are referred
to as "stereoisomers." The term "isomer" is also used to refer to
an enantiomer.
[0294] The term "enantiomer" is used to describe one of a pair of
molecular isomers which are mirror images of each other and
non-superimposable. Other terms used to designate or refer to
enantiomers include "stereoisomers" (because of the different
arrangement or stereochemistry around the chiral center; although
all enantiomers are stereoisomers, not all stereoisomers are
enantiomers) or "optical isomers" (because of the optical activity
of pure enantiomers, which is the ability of different pure
enantiomers to rotate planepolarized light in different
directions). Enantiomers generally have identical physical
properties, such as melting points and boiling points, and also
have identical spectroscopic properties. Enantiomers can differ
from each other with respect to their interaction with
plane-polarized light and with respect to biological activity.
[0295] The designations "R and S" are used to denote the absolute
configuration of the molecule about its chiral center(s). The
designations may appear as a prefix or as a suffix; they may or may
not be separated from the isomer by a hyphen; they may or may not
be hyphenated; and they may or may not be surrounded by
parentheses.
[0296] The designations or prefixes "(+) and (-)" are employed to
designate the sign of rotation of plane-polarized light by the
compound, with (-) meaning that the compound is levorotatory
(rotates to the left). A compound prefixed with (+) is
dextrorotatory (rotates to the right).
[0297] The term "racemic mixture," "racemic compound" or "racemate"
refers to a mixture of the two enantiomers of one compound. An
ideal racemic mixture is one wherein there is a 50:50 mixture of
both enantiomers of a compound such that the optical rotation of
the (+) enantiomer cancels out the optical rotation of the (-)
enantiomer.
[0298] The term "resolving" or "resolution" when used in reference
to a racemic mixture refers to the separation of a racemate into
its two enantiomorphic forms (i.e., (+) and (-); 65 (R) and (S)
forms). The terms can also refer to enantioselective conversion of
one isomer of a racemate to a product.
[0299] The term "enantiomeric excess" or "ee" refers to a reaction
product wherein one enantiomer is produced in excess of the other,
and is defined for a mixture of (+)- and (-)-enantiomers, with
composition given as the mole or weight or volume fraction
F.sub.(+) and F.sub.(-) (where the sum of F.sub.(+) and
F.sub.(-)=1). The enantiomeric excess is defined as
*F.sub.(+)-F.sub.(-)* and the percent enantiomeric excess by
100.times.*F.sub.(+)-F.sub.(-)*. The "purity" of an enantiomer is
described by its ee or percent ee value (% ee).
[0300] Whether expressed as a "purified enantiomer" or a "pure
enantiomer" or a "resolved enantiomer" or "a compound in
enantiomeric excess", the terms are meant to indicate that the
amount of one enantiomer exceeds the amount of the other. Thus,
when referring to an enantiomer preparation, both (or either) of
the percent of the major enantiomer (e.g. by mole or by weight or
by volume) and (or) the percent enantiomeric excess of the major
enantiomer may be used to determine whether the preparation
represents a purified enantiomer preparation.
[0301] The term "enantiomeric purity" or "enantiomer purity" of an
isomer refers to a qualitative or quantitative measure of the
purified enantiomer; typically, the measurement is expressed on the
basis of ee or enantiomeric excess.
[0302] The terms "substantially purified enantiomer,"
"substantially resolved enantiomer" "substantially purified
enantiomer preparation" are meant to indicate a preparation (e.g.
derived from non optically active starting material, substrate, or
intermediate) wherein one enantiomer has been enriched over the
other, and more preferably, wherein the other enantiomer represents
less than 20%, more preferably less than 10%, and more preferably
less than 5%, and still more preferably, less than 2% of the
enantiomer or enantiomer preparation.
[0303] The terms "purified enantiomer," "resolved enantiomer" and
"purified enantiomer preparation" are meant to indicate a
preparation (e.g. derived from non optically active starting
material, substrates or intermediates) wherein one enantiomer (for
example, the R-enantiomer) is enriched over the other, and more
preferably, wherein the other enantiomer (for example the
S-enantiomer) represents less than 30%, preferably less than 20%,
more preferably less than 10% (e.g. in this particular instance,
the R-enantiomer is substantially free of the S-enantiomer), and
more preferably less than 5% and still more preferably, less than
2% of the preparation. A purified enantiomer may be synthesized
substantially free of the other enantiomer, or a purified
enantiomer may be synthesized in a stereopreferred procedure,
followed by separation steps, or a purified enantiomer may be
derived from a racemic mixture.
[0304] The term "enantioselectivity," also called the enantiomeric
ratio indicated by the symbol "E," refers to the selective capacity
of an enzyme to generate from a racemic substrate one enantiomer
relative to the other in a product racemic mixture; in other words,
it is a measure of the ability of the enzyme to distinguish between
enantiomers. A nonselective reaction has an E of 1, while
resolutions with E's above 20 are generally considered useful for
synthesis or resolution. The enantioselectivity resides in a
difference in conversion rates between the enantiomers in question.
Reaction products are obtained that are enriched in one of the
enantiomers; conversely, remaining substrates are enriched in the
other enantiomer. For practical purposes it is generally desirable
for one of the enantiomers to be obtained in large excess. This is
achieved by terminating the conversion process at a certain degree
of conversion.
[0305] The term "analog" as used herein refers to a compound that
results from substitution, replacement or deletion of various
organic groups or hydrogen atoms from a parent compound. As such,
some monoterpenoids can be considered to be analogs of
monoterpenes, or in some cases, analogs of other monoterpenoids,
including derivatives of monoterpenes. An analog is structurally
similar to the parent compound, but can differ by even a single
element of the same valence and group of the periodic table as the
element it replaces.
[0306] The term "derivative" as used herein refers to a chemical
substance related structurally to another, i.e., an "original"
substance, which can be referred to as a "parent" compound. A
"derivative" can be made from the structurally-related parent
compound in one or more steps. The phrase "closely related
derivative" means a derivative whose molecular weight does not
exceed the weight of the parent compound by more than 50%. The
general physical and chemical properties of a closely related
derivative are also similar to the parent compound.
[0307] As used herein, a "prodrug" refers to compounds that can be
converted via some chemical or physiological process (e.g.,
enzymatic processes and metabolic hydrolysis) to a therapeutic
agent. Thus, the term "prodrug" also refers to a precursor of a
biologically active compound that is pharmaceutically acceptable. A
prodrug may be inactive when administered to a subject, i.e. an
ester, but is converted in vivo to an active compound, for example,
by hydrolysis to the free carboxylic acid or free hydroxyl. The
prodrug compound often offers advantages of solubility, tissue
compatibility or delayed release in an organism. The term "prodrug"
is also meant to include any covalently bonded carriers, which
release the active compound in vivo when such prodrug is
administered to a subject. Prodrugs of an active compound may be
prepared by modifying functional groups present in the active
compound in such a way that the modifications are cleaved, either
in routine manipulation or in vivo, to the parent active compound.
Prodrugs include compounds wherein a hydroxy, amino or mercapto
group is bonded to any group that, when the prodrug of the active
compound is administered to a subject, cleaves to form a free
hydroxy, free amino or free mercapto group, respectively. Examples
of prodrugs include, but are not limited to, acetate, formate and
benzoate derivatives of an alcohol or acetamide, formamide and
benzamide derivatives of an amine functional group in the active
compound and the like. See Harper, "Drug Latentiation" in Jucker,
ed. Progress in Drug Research 4:221-294 (1962); Morozowich et al,
"Application of Physical Organic Principles to Prodrug Design" in
E. B. Roche ed. Design of Biopharmaceutical Properties through
Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977);
Bioreversible Carriers in Drug in Drug Design, Theory and
Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987);
Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al.
"Prodrug approaches to the improved delivery of peptide drug" in
Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti et al. (1997)
Improvement in peptide bioavailability: Peptidomimetics and Prodrug
Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al.
(1998) "The Use of Esters as Prodrugs for Oral Delivery of
(3-Lactam antibiotics," Pharm. Biotech. ll:345-365; Gaignault et
al. (1996) "Designing Prodrugs and Bioprecursors I. Carrier
Prodrugs," Pract. Med. Chem. 671-696; Asgharnejad, "Improving Oral
Drug Transport", in Transport Processes in Pharmaceutical Systems,
G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p.
185-218 (2000); Balant et al., "Prodrugs for the improvement of
drug absorption via different routes of administration", Eur. J.
Drug Metab. Pharmacokinet., 15(2): 143-53 (1990); Balimane and
Sinko, "Involvement of multiple transporters in the oral absorption
of nucleoside analogues", Adv. Drug Delivery Rev., 39(1-3): 183-209
(1999); Browne, "Fosphenytoin (Cerebyx)", Clin. Neuropharmacol.
20(1): 1-12 (1997); Bundgaard, "Bioreversible derivatization of
drugs--principle and applicability to improve the therapeutic
effects of drugs", Arch. Pharm. Chemi 86(1): 1-39 (1979); Bundgaard
H. "Improved drug delivery by the prodrug approach", Controlled
Drug Delivery 17: 179-96 (1987); Bundgaard H. "Prodrugs as a means
to improve the delivery of peptide drugs", Arfv. Drug Delivery Rev.
8(1): 1-38 (1992); Fleisher et al. "Improved oral drug delivery:
solubility limitations overcome by the use of prodrugs", Arfv. Drug
Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. "Design of
prodrugs for improved gastrointestinal absorption by intestinal
enzyme targeting", Methods Enzymol. 112 (Drug Enzyme Targeting, Pt.
A): 360-81, (1985); Farquhar D, et al., "Biologically Reversible
Phosphate-Protective Groups", Pharm. Sci., 72(3): 324-325 (1983);
Freeman S, et al., "Bioreversible Protection for the Phospho Group:
Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)
Methylphosphonate with Carboxyesterase," Chem. Soc., Chem. Commun.,
875-877 (1991); Friis and Bundgaard, "Prodrugs of phosphates and
phosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives
of phosphate- or phosphonate containing drugs masking the negative
charges of these groups", Eur. J. Pharm. Sci. 4: 49-59 (1996);
Gangwar et al., "Pro-drug, molecular structure and percutaneous
delivery", Des. Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting
Date 1976, 409-21. (1977); Nathwani and Wood, "Penicillins: a
current review of their clinical pharmacology and therapeutic use",
Drugs 45(6): 866-94 (1993); Sinhababu and Thakker, "Prodrugs of
anticancer agents", Adv. Drug Delivery Rev. 19(2): 241-273 (1996);
Stella et al., "Prodrugs. Do they have advantages in clinical
practice?", Drugs 29(5): 455-73 (1985); Tan et al. "Development and
optimization of anti-HIV nucleoside analogs and prodrugs: A review
of their cellular pharmacology, structure-activity relationships
and pharmacokinetics", Adv. Drug Delivery Rev. 39(1-3): 117-151
(1999); Taylor, "Improved passive oral drug delivery via prodrugs",
Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino and
Borchardt, "Prodrug strategies to enhance the intestinal absorption
of peptides", Drug Discovery Today 2(4): 148-155 (1997); Wiebe and
Knaus, "Concepts for the design of anti-HIV nucleoside prodrugs for
treating cephalic HIV infection", Adv. Drug Delivery Rev.:
39(1-3):63-80 (1999); Waller et al., "Prodrugs", Br. J. Clin.
Pharmac. 28: 497-507 (1989), each herein incorporated by reference
in its entirety.
[0308] As used herein, the term "pharmaceutically-acceptable salts"
refers to the conventional nontoxic salts or quaternary ammonium
salts of therapeutic agents, e.g., from non-toxic organic or
inorganic acids. These salts can be prepared in situ in the
administration vehicle or the dosage form manufacturing process, or
by separately reacting a therapeutic agent in its free base or acid
form with a suitable organic or inorganic acid or base, and
isolating the salt thus formed during subsequent purification.
Conventional nontoxic salts include those derived from inorganic
acids such as 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. See, for example, Berge et al.,
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19 (1977), herein
incorporated by reference in its entirety.
[0309] In some embodiments of the aspects described herein,
representative salts include the hydrobromide, hydrochloride,
sulfate, bisulfate, phosphate, nitrate, acetate, succinate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like.
[0310] TDP-43 is the principle component of inclusions in
amyotrophic lateral sclerosis (ALS) and in some frontotemporal
dementia (FTLD-U). TDP-43 is a nuclear RNA binding protein, which
translocates to the cytoplasm during stress where it forms
cytoplasmic granules. Our results indicate that these cytoplasmic
TDP-43 inclusions co-localize with RNA granules termed "stress
granules" (SGs), both in cells and in human ALS spinal cord, and
TDP-43 inclusions can be reversed by chemicals that reverse SGs
(PLoS ONE, October 2010 5(10), e13250; herein incorporated by
reference in its entirety). Under many conditions (e.g., arsenite
treatment, nutrient deprivation) co-localization with SGs
approaches 100%. Disease-linked mutations in TDP-43 increase
cytoplasmic inclusion formation. This linkage to SGs appears to
generalize to other ALS-linked genes because FUS, ataxin-2 and SMN
all are associated with ALS or motor neuron diseases, also
translocate to the cytoplasm, also form inclusions co-localized
with SGs, and (for FUS & ataxin-2) also form complexes
associated with TDP-43 (Nature, 2010, 466, 1069-75; Proc Natl Acad
Sci USA, 2010, 107, 13318-23; each herein incorporated by reference
in its entirety). These data point to a strong biological
connection between SGs and TDP-43. Discovery of the association
between TDP-43 and SGs paves the way for novel insights into TDP-43
biology, and also suggests mechanisms by which mutations in TDP-43
cause disease. Accordingly, SG biology stimulates formation of
TDP-43 inclusions, and that pathogenic factors linked to ALS
increase TDP-43 inclusion formation through a process mediated by
SG pathways.
[0311] ALS is a devastating, rapidly fatal neurodegenerative
disease that strikes people, and currently has no disease modifying
treatments. Discovery of the putative association between TDP-43
and SG biology links TDP-43 to a biological pathway whose biology
is profoundly important to neuronal function, and that offers many
potential targets for pharmacological intervention. A striking
number of proteins linked to ALS are RNA binding proteins, and most
of these participate in SG biology. Thus, understanding the role of
SG biology in the pathophysiology of TDP-43 will likely provide
insights into the pathophysiology of other proteins linked to ALS,
including FUS, ataxin-2 and VCP. SG biology is also fundamentally
interesting because it is one of the rare examples of a normal
physiological process that is based on reversible aggregation of
proteins; one aspect of this story that is fascinating is the large
number of SG proteins that share homology to yeast prion proteins,
which raises the possibility that understanding SG biology will
also help to illuminate the biology of diseases resulting from
prion proteins. The reversible nature of SG-based aggregation
offers a biological pathway that can be applied to reverse the
pathology and toxicity associated with TDP-43 inclusion formation.
Preliminary results by the authors already demonstrate that
chemicals that reverse SG formation also reverse formation of
TDP-43 inclusions. These chemicals though are quite toxic (they
inhibit protein translation), but the SG pathway offers many other
targets able to reverse SG biology that are not toxic to the cell.
Investigating the particular elements of the SG pathway that
regulate TDP-43 inclusion formation can identify selective
approaches for therapeutic intervention to delay or halt the
progression of ALS.
[0312] Results presented herein demonstrate, for the first time,
that TDP-43 pathology in the human CNS is associated with SG
markers. Regulation of protein translation is clearly critical for
healthy brain functioning, and SG biology plays a fundamental role
in this regulatory axis.
[0313] TDP-43:
[0314] TDP-43 is also known as Tar DNA binding protein (TARDBP).
TDP-43 is a major protein component of inclusions in ALS and FTLD-U
(Science, 2006, 314, 130-3; herein incorporated by reference in its
entirety). TDP-43 is a 414 amino acid nuclear protein encoded by
the TARDBP gene on chromosome 1. It is ubiquitously expressed in
all tissues (J Biol Chem, 2001, 276, 36337-43; herein incorporated
by reference in its entirety). It contains two RNA recognition
motifs and a glycine rich domain at the C-terminus. Nuclear
functions associated with TDP-43 include acting as a
transcriptional repressor (such as for the SP-10 gene),
contributing to exon skipping (as shown for the cystic fibrosis
transmembrane conductance regulator gene), and acting as a scaffold
protein for nuclear bodies in concert with survival motor neuron
protein (SMN) (J Biol Chem, 2005, 280, 37572-84; J Biol Chem, 2007,
282, 36143-54; Proc Natl Acad Sci USA, 2002, 99, 13583-8; each
herein incorporated by reference in its entirety). Interestingly,
many other proteins associated with ALS or motor neuron diseases
are also RNA binding proteins, including FUS, SMN, ataxin-2, VCP.
This suggests a systematic connection between RNA binding proteins
and motor neuron diseases.
[0315] Mutations in TDP-43 are increasingly associated with
disease. Two papers initially identified different point mutations
in TDP-43 (A315T, M337V) that are associated with ALS, and multiple
papers have expanded upon these findings to identify other
mutations associated with sporadic and familial ALS (Science, 2008,
319, 1668-72; Ann Neurol. 2008, 63(4), 535-538; each herein
incorporated by reference in its entirety). Association of
mutations with ALS indicates that abnormalities in TDP-43 are
sufficient to cause disease. Increasingly, studies suggest a link
between TDP-43 and cell death. Acute expression of TDP-43 in chick
spinal cord elicits apoptosis in neurons (Science, 2008, 319,
1668-72; herein incorporated by reference in its entirety).
Transgenic models of TDP-43 (WT or mutant) in mouse, Drosophila and
C. elegans all show evidence of neurodegeneration (J Neurosci,
2010, 30, 10851-9; Proc Natl Acad Sci USA, 2010, 107, 3858-63; J
Exp Med, 2010, 207, 1661-73; Neurobiol Dis. 2010, 40(2), 404-414;
Neuroscience, 2010, 167, 774-85; Proc Natl Acad Sci USA, 2009, 106,
18809-14; J Biol Chem, 2010, 285, 11068-72; Proc Natl Acad Sci USA.
2010, 107, 7, 3169-74; and Hum Mol Genet, 2010, 19 (16): 3206-3218;
each herein incorporated by reference in its entirety). The
mechanisms of toxicity are unknown but cleavage is associated with
TDP-43 pathology. Brains from subjects with ALS and FTD show
smaller bands at 25 KD and 35 KD that appear to be cleavage
products containing the carboxy domain of TDP-43 (Science, 314,
130-3; herein incorporated by reference in its entirety). TDP-43
can be cleaved by caspases in vitro, and forms cytoplasmic
inclusions in response to apoptotic stimuli (J Neurosci, 2007, 27,
10530-4; herein incorporated by reference in its entirety).
[0316] A recent publication by co-authored by the inventors
describes the relationship between TDP-43 and SGs (PLoS ONE,
October 2010 5(10), e13250; herein incorporated by reference in its
entirety); George Murphy has published on a novel system for the
efficient production of clinically relevant, transgene-free human
iPSCs (Stem Cells. 2010, 28 (10), 1728-1740; herein incorporated by
reference in its entirety). Leonard Petrucelli has published on
transgenic mice expressing WT TDP-43 (J Neurosci, 2010, 30,
10851-9; herein incorporated by reference in its entirety).
[0317] RNA Binding Proteins:
[0318] mRNA binding proteins facilitate mRNA trafficking from the
nucleus to the cytoplasm as part of the biological machinery that
regulates mRNA metabolism, such as RNA decay and protein
translation. RNA decay is a constitutive process that occurs in
cytoplasmic compartments termed processing bodies (P-bodies).
However, under stressful conditions mRNA binding proteins
consolidate mRNA in cytoplasmic compartments, termed the stress
granules (SGs); this recruitment is mediated by multiple proteins,
including T-cell intracellular antigen 1 (TIA-1), RasGAP-associated
endoribnuclease (G3BP), elongation initiation factor 3 (eIF3) and
poly-A binding protein (PABP) (Trends Biochem Sci, 2008, 33,
141-50; herein incorporated by reference in its entirety). SGs
function in part to triage RNA and sequester transcripts not needed
for coping with the stress (Trends Biochem Sci, 2008, 33, 141-50;
herein incorporated by reference in its entirety). The mechanism of
SG formation is striking because it results from the regulated,
reversible aggregation process of mRNA binding proteins with
prion-like domains, such as TIA-1, TIAR and G3BP (Mol Biol Cell,
2004, 15, 5383-98; herein incorporated by reference in its
entirety).
[0319] It will recognized that one or more features of any
embodiments disclosed herein may be combined and/or rearranged
within the scope of the invention to produce further embodiments
that are also within the scope of the invention.
[0320] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be within the scope of the
present invention.
[0321] The invention is further described by the following
non-limiting Examples.
Examples
[0322] Examples are provided below to facilitate a more complete
understanding of the invention. The following examples illustrate
the exemplary modes of making and practicing the invention.
However, the scope of the invention is not limited to specific
embodiments disclosed in these Examples, which are for purposes of
illustration only, since alternative methods can be utilized to
obtain similar results.
[0323] Establishing the PC12-TDP-43 Model. Generation of
TDP-43::GFP Inducible PC12 Cell Lines:
[0324] We generated a Tet-Off inducible PC12 cell line (Clontech;
the parent line stably expresses high levels of the Tetracycline
binding protein) that is stably transfected with a WT TDP-43::GFP
(where the GFP is at the C-terminus). Newly induced TDP-43::GFP (24
hrs) is initially observable with a diffuse nuclear localization;
after 72 hrs of expression, cytoplasmic and nuclear aggregates of
TDP-43 become readily apparent (FIGS. 1A to 1B, arrows). The
expression of TDP-43 in this line appears to occur in a fraction of
the cells but we believe this is due to auto-regulation because
expression of TDP-43::GFP can be induced in all of the cells by
some of the compounds we have identified. Treatment with arsenite
(50 .mu.M, 18 hrs) increased the rate (>80 of cells) and
consistency of inclusion formation. The high-throughput screen used
the arsenite (50 .mu.M, 18 hrs) treatment protocol, where arsenite
was added 1 hr after the test compounds.
[0325] Preliminary Screens to Identify Inhibitors of TDP-43
Inclusion Formation:
[0326] We carried out the high-throughput screen in collaboration
with the Laboratory for Drug Discovery in Neurodegeneration (LDDN),
which is an integral part of the Harvard NeuroDiscovery Center
(HNDC) (www.neuridiscovery.harvard.edu). LDDN has a permanent staff
of industry-seasoned scientists with specialties in assay
development, laboratory automation, informatics, and medicinal
chemistry. The LDDN has a compound library of 75,000 compounds
selected with a series of filters for "drug-like" properties
including the physical properties that predict their likelihood to
cross the blood brain barrier. LDDN has completed over fifty high
throughput screens. We used the automated GE IN Cell Analyzer 1000
microscope system for high throughput analyses. Test chemicals were
added to the cells 48 hrs after induction of TDP-43 (by removal of
doxycycline). After another 24 hrs, the cells were fixed and
double-stained with DAPI (to detect nuclei). Inclusions present in
a collar around the nucleus but not fully co-localized with the
DAPI stain (FIGS. 1A to 1B) were identified by the IN-Cell analyzer
as inclusions. The computer counted the number of cells (based on
DAPI-positive nuclei), TDP-43 levels and inclusions per field. The
counts from 6 different fields within a well were averaged to
develop a measure of the mean number of inclusions per field. This
was repeated for every well in a 384 well plate and every plate in
the library. We screened a 1600 compound library of FDA approved
chemicals, known bioactives, and purified natural products, and an
additional 75,000 chemicals from the general compound library (the
libraries are described in the Resources section). Sixteen out of
the 75,000 compounds were also selected as leads because they
reduced TDP-43 inclusions by more than three standard deviations
beyond the mean, showed <20% toxicity (based on counting total
cell numbers), repeated on subsequent evaluations using fresh
powder compound stocks and showed a dose dependent concentration
curve for inhibition of TDP-43 aggregation using both 5 point and
12 point dose response curves (FIGS. 2-12). We also identified
additional compounds from the library of FDA/bioactive chemicals.
Representative EC50 values are shown in Table 1. Additional
representative compounds are shown in FIGS. 13A to 13C. The
compounds identified represented several different structural
classes, are generally Lipinski rule compliant, and have no known
toxicophores or reactive groups.
TABLE-US-00001 TABLE 1 Inhibition of TDP-43 Inclusion Formation.
Cmpd No. Code No. EC50 (nM) 1 LDN-0118790 38 2 LDN-0118870 74 3
LDN-0119629 39 4 LDN-0121669 98 5 LDN-0124614 306 6 LDN-0125734 15
7 LDN-0125735 139 8 LDN-0130436 174 9 LDN-0196125 <1 2-1
LDN-0015257 152 2-2 LDN-0057218 290 2-3 LDN-0057325 1920 2-4
LDN-0066337 10 2-5 LDN-0076437 1290 2-7 LDN-0202779 11 2-8
LDN-0209285 180 Mithramycin A LDN-0052881 2000 Parthenolide
LDN-0014143 3000 Mycophenolic acid LDN-0014149 1500
[0327] Expressing TDP-43 in Primary Neurons Leads to Inclusion
Formation:
[0328] An important element of a drug screening is to examine
toxicity and inclusion formation in the primary neuronal cells
grown in culture, which is based on the results of multiple groups
studying primary neurons grown in culture (PLoS One, 2010. 5:
e15878; herein incorporated by reference in its entirety). Initial
results of examining expression of TDP-43 in primary neuronal
cultures show that TDP-43 readily forms inclusions (FIGS. 14A-14H),
and that the process of inclusion formation and toxicity is
enhanced by arsenite (50 uM, 18 hrs), much like in our
high-throughput screen, and by others (PLoS ONE, October 2010
5(10), e13250; J Neurosci, 2010. 30: 639-49; each herein
incorporated by reference in its entirety). The field of stress
granules (SG) classically used an acute arsenite treatment of 0.5
mM for 30-60 min. While this procedure works in PC12 cells (and in
induced pluripotent stem cells), the inventors have also taken care
to modify the treatment to enable the screening and analysis of the
drugs. Thus, a treatment of 15-50 uM for a much longer time (18-24
hrs) to induce the SGs and TDP-43 inclusions turns out to be
important when looking in hippocampal neurons grown in culture (the
inventors have used down to 7 uM) and induced pluripotent stem
cells. TDP-43 inclusion induction has also been analyzed in iPSPs,
and they can be induced well by puromycin. This shows improvements
over H.sub.2O.sub.2 and wortmannin.
[0329] Accordingly, one can readily quantify toxicity using
analyses of neurite length, arborization and cell size, using
approaches similar to Przedborsky and colleagues (Nat Neurosci,
2007,10: 615-22; herein incorporated by reference in its
entirety).
[0330] Accordingly, the invention provides a novel neuronal cell
line that inducibly expresses WT TDP-43 and develops spontaneous
inclusions, which provides a novel approach for high throughput
screening of inhibitors of TDP-43 cytoplasmic inclusions. The
inducible nature of the screening obviates potential toxicity that
is commonly observed with stable overexpression of TDP-43.
[0331] Additionally, the data presented herein indicates that that
TDP-43 inclusions form in conjunction with the SG pathway, and that
inhibitors of SG formation can also inhibit TDP-43 inclusion
formation (PLoS ONE, October 2010 5(10), e13250; herein
incorporated by reference in its entirety). Accordingly, a compound
identified by the screening method described herein can be used to
interrogate the role of TDP-43 in SG formation and the role of SG
formation in the pathophysiology of ALS and frontotemporal dementia
(FTD).
[0332] Determination of Effect of Compounds on Formation of TDP-43
Inclusions:
[0333] Primary cultures of cortical and motor neurons, transduced
the neurons with TDP-43 (WT, A315T or A343T) are generated and the
viability and inclusion formation during exposure to each of the
lead compounds is followed. In one example, cortical and motor
neurons are examined because TDP-43 forms inclusions in cortical
neurons (frontotemporal dementia) and in motor neurons (ALS).
Cortical neurons: Rat embryos are harvested at E18 and placed in
cell culture. At DIV 3, the cells were transduced with lentivirus
TDP-43 (WT, A315T or A343T) using a multiplicity of infection of 5.
Motor neurons: We use the mouse ChAT::GFP line that selectively
expresses GFP in cholinergic neurons; a colony of these mice is
currently available at Boston University in the laboratory of
Krzystof Blusztajn (Nat. Protoc, 2008. 3: 34-40; herein
incorporated by reference in its entirety). Mouse fetuses are
harvested at E12.5 as described previously, and sorted by FACS as
described by Dr. Blusztajn's group (Nat. Protoc, 2008. 3: 34-40;
Nat Neurosci, 2007. 10: 615-22; each herein incorporated by
reference in its entirety). CNS tissue are dissociated and GFP
positive neurons are isolated by FACS. The GFP-positive neurons are
placed in culture. Neuronal identity will be ascertained by
complementing the GFP fluorescence with staining for MAP2, as
described by Nagai, et al (Nat Neurosci, 2007. 10: 615-22; herein
incorporated by reference in its entirety). At day 3 in vitro
(DIV3), the neurons are transduced with lentivirus TDP-43 (WT,
A315T or A343T) using a multiplicity of infection of 5.
[0334] Treatment:
[0335] The test compounds are added to the cultures 24 hrs after
viral transduction (DIV4), and maintained throughout the treatment
period; fresh compound in new medium is added every 2 days. The
neurons are imaged at DIV6 to measure outcomes under basal
conditions. On DIV7, arsenite (50 .mu.M, 18 hrs) is added, and then
the neurons are imaged after 18 hrs and the outcomes quantified.
For each lead compound generate an 8 point dose response curve is
generated, using a range corresponding to 2 log units above and
below the IC.sub.50 for each compound (determined based on the
studies in PC12 cells); generally this corresponds to a range of 10
nM to 10 .mu.M. Fresh compound is be added every third day until
termination of the assay by replacing 50% of the medium with medium
containing fresh compounds. Toxicity and inclusion formation is
followed as described below.
[0336] Toxicity:
[0337] Neurotoxicity/neurodegeneration is followed using protocols
similar to those described by Przedborsky and colleagues (Nat
Neurosci, 2007. 10: 615-22; herein incorporated by reference in its
entirety). At days 1, 3 and 7 after infection toxicity is
quantified. The number of neurons, size of the cell bodies, process
length (determined by counting the number of processes >700
.mu.m), and analysis by the neurite tracer plugin for the image J
application (J Neurosci Methods, 2008. 168: 134-9; herein
incorporated by reference in its entirety). For cortical neurons,
toxicity of each condition is analyzed by LDH assay normalized to
protein content at the end of the assay.
[0338] Inclusion Formation Assay:
[0339] Cortical and motor neurons transduced with TDP-43
spontaneously develop inclusions after treatment with 50 .mu.M
arsenite, 18 hr. At T=24 hrs test compounds are added (dose=Ki)
.+-.0.5 mM arsenite (1 hr), where Ki refers to 50% inhibition of
inclusion formation from the PC12 primary screen assay. At days 1,
3 and 7 the cells are fixed and inclusion formation quantified
using the IN Cell analyzer.
[0340] Analysis of Endogenous TDP-43:
[0341] The effect of compounds on inclusion formation in neurons
that do not over-express TDP-43 to mimic the environment of the
brain normally can be examined as follows. Cortical and motor
neurons are grown in culture. At DIV 7, the neurons are treated
with the test compounds (dose=1 & 10.times.IC.sub.50) .+-.0.5
mM arsenite (1 hr) as described in the "Inclusion Formation Assay"
above. Following fixation the cells are probed with anti-TDP-43
antibody (Santa Cruz Labs). Total TDP-43 levels and cytoplasmic
inclusion formation are analyzed by confocal microscopy. The
effects of compounds that appear to reduce endogenous TDP-43 levels
can be further examined by treating cortical and motor neurons with
the compound (0, 1 hr and 24 hrs, dose=1 and 3.times.Ki) and
immunoblotting the TDP-43 to quantify the levels of TDP-43 and
compare to levels of other proteins, such as TIA-1 (a stress
granule protein) and actin (a housekeeping protein).
[0342] Determine how the Lead Compounds Affect Stress Granule
Formation:
[0343] TDP-43 is transduced into primary cultures of spinal cord
neurons with lentiviruses .+-.test compounds (dose: 1 &
5.times.IC.sub.50). After 24 hrs treatment with test compounds,
neurons are treated .+-.0.5 mM arsenite, 1 hr, and fixed. Following
fixation, colocalzation of TDP-43 with SG markers (TIA-1 or eIF3)
is determined by immunocytochemistry as described by us previously
(PLoS ONE, October 2010 5(10), e13250; herein incorporated by
reference in its entirety).
[0344] Whether the compounds inhibit aggregation of recombinant
TDP-43 in vitro can be tested as follow. Fresh recombinant TDP-43
(3 .mu.M) is incubated in solution .+-.test compounds
(dose=Ki*(0.5, 1, 2, 4 or 10)), and aggregation is followed
spectrophotometrically by the increase in turbidity at 395 nm over
1 hr (J Biol Chem, 2009. 284: 20329-39; herein incorporated by
reference in its entirety).
[0345] The strong correlation between neurodegeneration and
inclusion formation translates into identifying compounds that
inhibit neurodegeneration in addition to inhibiting inclusion
formation. Without wishing to be bound by a theory, the excellent
potency arises because the assay is modulating the signaling
systems that regulate stress granule formation because enzymatic
reactions such as kinase reactions are commonly very sensitive to
small molecule therapeutics. There can be multiple pathways for
inhibiting TDP-43 inclusion formation, including: 1) inhibition of
stress granule formation, 2) inhibition of nuclear TDP-43 export,
3) homomeric inhibition TDP-43 binding (one TDP-43 molecule to
another), 4) heteromeric inhibition TDP-43 binding (binding of
TDP-43 to other aggregating stress granule proteins, such as TIA-1
or eIF3). Inhibiting nuclear export is unlikely because the
compounds also inhibit intra-nuclear inclusions.
[0346] Cell Culture:
[0347] Primary motor neuron cultures are generated as described
previously (Neuroscience, 2009. 159: 647-56; herein incorporated by
reference in its entirety). Mouse spinal cord neurons are isolated
from embryonic day 12.5 pups. Cholinergic neurons are isolated by
FACS and cultured at 5000 cells per cm.sup.2. Motoneurons were
plated in the presence of a cocktail of neurotrophic factors
(referred as "NTFs": 1 ng/ml BDNF, 100 pg/ml GDNF, 10 ng/ml CNTF)
in neural basal medium added at the time of cell seeding (Neuron,
2002. 35: 1067-83; herein incorporated by reference in its
entirety). Cortical neurons are isolated from E17.5 mouse brains.
Neurons were plated in neurobasal medium supplemented with B27, 0.5
mM glutamine and penicillin/streptomycin onto poly-D-lysine/laminin
coated dishes (J Biol Chem, 2004. 279: 46915-20; herein
incorporated by reference in its entirety). PC12 cells are grown in
DMEM, 10% NBS/Pen-Strep, 100 .mu.g/ml hygromycin, 50 .mu.M
puromycin and 50 .mu.g/ml doxycycline (the latter is removed for
TDP-43 induction).
[0348] Immunocytochemistry:
[0349] can be performed as described previously (J Neurochem, 2010,
112, 6, 1593-1604; herein incorporated by reference in its
entirety).
[0350] LDH Assay:
[0351] can be performed as described previously (J. Neuroscience,
2000. 20: 6048-54; herein incorporated by reference in its
entirety).
[0352] Animals:
[0353] Timed pregnant female C57/B6 mice can be used for isolating
primary neurons. Approximately 78 pregnant mice to are needed to
generate primary cultures of cortical neurons. This number is based
on the use of 26 pregnant mice in the first year (1/wk for 6
months) and 52 pregnant mice in the second year (1/wk). Pregnant
mice can be purchased (for non-transgenic mice) or generated from
the colony of ChAT::GFP mice maintained at Boston University School
of Medicine by Krzysztof Blusztajn. In one embodiment, C57/B6 WT
and ChAT::GFP are used because of published studies utilizing mouse
primary cultured neurons to investigate the pathogenesis of
ALS/FTLD-U and TDP-43 expression and function, and the utility of
having mice with labeled cholinergic neurons. The mice can be
killed by inhalation of carbon dioxide, which is an approved,
humane method of sacrifice. These methods are consistent with the
recommendation of the Panel on Euthanasia of the American
Veterinary Medical Association.
[0354] Determining whether compounds delay deterioration of motor
function in C. elegans expressing TDP-43 in C. elegans: C. elegans
is generally used as a simple in vivo model of disease bridging the
gap between in vitro studies and in vivo studies in rodents (J Biol
Chem, 2005. 280: 42655-68; J Neurosci, 2009. 29: 9210-8;
Neurodegener Dis, 2010. 7: 68-75; each herein incorporated by
reference in its entirety). Accordingly, C. elegans lines
expressing TDP-43 (WT, G294A and A315T), obtained from Brian
Kraemer, can be used to determine whether the indentified compounds
ameliorate motor dysfunction associated with TDP-43 expression (J
Neurosci, 2010. 30: 16208-19; herein incorporated by reference in
its entirety). For the experiments, lines of C. elegans (30 per
plate, 3 plates per dose) are synchronized and plated onto NGM
plates containing the test compound at L3, using dose ranges of 0,
1, 10 and 100.times.IC.sub.50; higher doses are used because C.
elegans are typically less sensitive to compounds than cultured
cells. The nematodes are aged on the plates, and transferred to
fresh plates every other day. Movement and survival are calculated
at adult days 1, 3 and 5, using methods described in the art (see,
e.g., J Neurosci. 2010 Dec. 1, 30(48): 16208-19; IEEE Trans Biomed
Eng. 2004 Oct 51(10):1811-20, each herein incorporated by reference
in its entirety).
[0355] In one example, eggs (C. elegans expressing A315T TDP-43,
line CK426) were plated on agar containing the test compound. On
day 4, movement of C. elegans was quantified and moved to plates
with fresh compounds. On day 5, movement of C. elegans was again
quantified.
[0356] Expressed or Endogenous TDP-43 Forms Cytoplasmic Inclusions
that Co-Localize with SG:
[0357] The inventors transfected human BE-M17 neuroblastoma cells
with WT TDP-43, TDP-43.sub.86-414 or TDP-43.sub.216-414 constructs
N-terminally tagged with GFP. Full length WT TDP-43 localized to
the nucleus under basal conditions (FIGS. 15A-1 to 15A-16 and 15B-1
to 15B-16, WT TDP-43 shown). To investigate TDP-43 aggregation
under the stressful conditions, cells were exposed to arsenite, an
agent classically used to induce SGs (Biochem Soc Trans, 2002, 30,
963-9; J Cell Biol, 2000, 151, 1257-68; J Cell Biol, 1999, 147,
1431-42; each herein incorporated by reference in its entirety).
Arsenite causes stress through multiple mechanisms (Toxicol Appl
Pharmacol, 2001, 177, 132-48; herein incorporaed by reference in
its entirety). Arsenite directly induces oxidative stress by
reacting with oxygen in a reaction similar to the Fenton reaction,
and arsenite also uses up glutathione, which causes further
oxidative stress (Toxicol Appl Pharmacol, 2001, 177, 132-48; herein
incorporaed by reference in its entirety). Upon exposure to
arsenite (1 hr) WT TDP-43 remained largely nuclear, but a small
amount translocated to the cytoplasm where it formed inclusions
(FIGS. 15B-1 to 15B-16, right panels, arrows). To determine whether
the inclusions co-localized with SGs, we co-labeled the cells with
antibodies to SG markers, including TIA-1, eIF3 and poly-A binding
protein (PABP) (FIGS. 15A-1 to 15A-16 and 15B-1 to 15B-16). Double
labeling experiments indicated that inclusions composed of WT
TDP-43 co-localized with SG markers under arsenite-induced
conditions (FIGS. 15B-2, 15B-6, 15B-10, 15B-14, TIA-1 shown as SG
marker, arrows); TDP-43 inclusions also co-localized with SG
markers under basal conditions, but the fraction of cells (<10%)
exhibiting TDP-43 inclusions under basal conditions.
Co-localization with other SG markers is also observed. Similar
results were obtained when experiments were performed using HEK 293
cells (not shown).
[0358] Disease-Linked Mutations Enhance Cytoplasmic Translocation
and SG Formation:
[0359] The strong link between TDP-43 and SG biology prompted us to
examine whether disease-linked mutations in TDP-43 also enhance
formation of inclusions through processes linked to SGs. GFP-tagged
TDP-43 (WT, G294A, A315T, Q331K, Q343R) were transfected into
BE-M17 cells, and inclusion formation was examined after treatment
with arsenite (0.5 mM, 1 hr) in the presence or absence of
cycloheximide (50 .mu.g/ml, 1 hr, FIGS. 15A-1 to 15A-16). The
mutations moderately increased TDP-43 inclusion formation under
basal conditions (FIG. 15B). Arsenite treatment was associated with
more inclusion formation for mutant TDP-43 constructs than for WT
TDP-43 (FIGS. 15A-1 to 15A-16 & 15B-1 to 15B-16). The
inclusions that formed in response to arsenite fully co-localized
with TIA-1, suggesting that inclusion formed by mutant TDP-43 were
also SGs (FIGS. 15A-1 to 15A-16). In each case, formation of
inclusions composed of mutant TDP-43 constructs was reversible by
cylcoheximide (10 .mu.g/ml, 1 hr, FIGS. 15A-1 to 15A-16 & B-1
to 15B-16). Importantly, each of the mutations also showed a
striking decrease in nuclear localization in response to arsenite
treatment, suggesting that the mutations increased the degree of
nuclear export (FIGS. 15A-1 to 15A-16 & 15B-1 to 15B-16). The
enhanced stress-induced cytoplasmic localization associated with
these mutants might contribute to their strong tendency to form
inclusions. These data demonstrate that enhancement of inclusions
with properties resembling SGs is a common feature of TDP-43
mutations associated with ALS. In addition, toxicity studies
examining the vulnerability of neurons expressing mutant TDP-43
results provide evidence that disease-linked TDP-43 mutations
increase cell death processes and SG formation.
[0360] TDP-43 Inclusions in Brain Tissue from ALS and FTLD-U Donors
Co-Localize with SG Markers:
[0361] Finally we examined whether TDP-43 pathology present in ALS
and FTLD-U cases were associated with SG markers.
Immunocytochemistry was performed on cases of ALS and FTLD-U using
antibodies to TDP-43 and SG markers, including eIF3 and TIA-1.
Sudan black was used to remove endogenous autofluorescence due to
lipofuscin (data not shown); this method greatly increased the
ability to distinguish between fluorescence related to the antibody
signal and fluorescence caused by lipofuscin. Using sudan black to
remove autofluorescence, we were able to readily visualize TDP-43
positive inclusions that showed co-labeling with these SG markers
in ALS spinal cord tissue and FTLD-U brain (FIGS. 16A-1 to 16A-15
& 16B-1 to 16B-15). We also observed co-localization between
phospho-TDP-43 inclusions and eIF3 or TIA-1 (FIGS. 16D-1 to
16D-10). The specificity of eIF3 staining was tested by
immuno-adsorption; pre-absorption of TDP-43 antibodies with the
antigenic peptide eliminated all reactivity, indicating the
specificity of the antibody (FIGS. 16C-1 to 16C-8). The absence of
reactivity following pre-adsorption also demonstrated that labeling
of SG markers was not due to the artifact of "bleed-through" from
the green channel. No co-labeling was observed with antibody to a
different class of RNA-binding protein, the P-body marker anti-Dcp1
(data not shown). Thus inclusions containing TDP-43 in the FTLD-U
brain and ALS spinal cord also contain other SG proteins, which is
consistent with a hypothesis that SG biology is intimately linked
to the mechanisms underlying TDP-43 inclusion formation.
[0362] Induced Pluripotent Stem Cells (iPSCs) Form Motor
Neurons:
[0363] To establish a simplified method for the derivation of
iPSCs, we sought to develop a vector that would result in efficient
reprogramming with a single reagent, without the need for
concurrent additional vectors, transgenes, or chemical exposures.
Importantly, the use of a single polycistronic vector, expressing
Oct4, Klf4, Sox2, and c-Myc, allowed us to reprogram post-natal
somatic cells with an efficiency 50 fold greater than previously
published methods, and with a single viral integration (Stem Cells.
2010, 28 (10), 1728-1740; Stem Cells, 2009, 27, 543-9; each herein
incorporated by reference in its entirety). We have now adapted
this vector (STEMCCA-loxP) by replacing all four reprogramming
factors with their corresponding human counterparts to create a
humanized STEMCCA-loxP vector. This vector allows for the efficient
derivation of human iPSCs (FIGS. 17A-1 to 17A-8, 17B-1 to 17B-9,
and 17C-1 to 17C-8).
[0364] Directed Differentiation of Human iPSCs into Motor
Neurons.
[0365] To develop a novel source of motor neurons for
neurodegenerative research, we established conditions for the
efficient directed differentiation of human iPSCs into motor
neurons based on our protocol initially used for human Embryonic
Stem cells.sup.12 (Nat Biotechnol., 2009 27(3): 275-80, herein
incorporated by reference in its entirety). For this adaptation,
human iPSCs were cultured on OP9 feeder cells in differentiation
media (IMDM, 20% FBS, 100 ng/ml ROCK-1) for 5 days, followed by
passaging and further differentiation in NIM media supplemented
with retinoic acid (0.1 uM), ascorbic acid (0.4 ug/ml), dbcAMP (1
uM), and 0.1 uM Human hedgehog Agonist (hAg) until day 24, transfer
to medium with B-27 (1.times.), BDNF, GDNF, IGF-1 and CTNF (10
ng/mL) for 3 days, and plating on laminin for before experimental
analysis. Using this co-culture protocol, motor neurons emerged,
were collected and characterized as shown in FIG. 18 to confirm
expression of two accepted motor neuron markers.
[0366] Modification of motor function in nematodes over-exspressing
TDP-43 with the compounds.
[0367] Some of the exemplary compounds described herein were tested
in a in vivo system using C. elegans expressing WT or A315T TDP-43.
The nematode lines were hatched on agar plates containing varying
doses of test compound; we used doses that were 10-200.times. the
IC50 observed with cell lines because C. elegans tend to be much
less sensitive to exogenous compounds than cells grown in culture.
Worms typically need doses of compound that are 10-100 fold greater
than in mammals due to the environment (dirt) and have strong
protective mechanisms--such as a thick cuticle that is resistant to
chemicals. Interestingly, many of the compounds tested modify motor
function, but the effects varied depending on the compound. For
instance, compound 8 (LDN-0130436) improved motor function to
similar degrees in C. elegans expressing human WT or A315T TDP-43
(FIG. 19A, WT shown), but exhibited little effect on the
non-transgenic N2 line (FIG. 19B). In contrast, compound 8 strongly
inhibited motor function in C. elegans expressing WT TDP-43 (FIG.
19C) or in the N2line, but had only a modest effect on the A315T
line (FIG. 19D). Another compound increased motor function by about
6-fold in all the lines. Each of these compounds dispersed TDP-43
inclusions in PC12 cells and in other cell lines, yet exhibited
disparate actions in C. elegans. Multiple different pathways are
known to modulate formation of SGs, including the pathways mediated
by kinases that phosphorylate eIF2A, PERK, HRI and GCNA, as well as
pathways that proceed independently of eIF2A. Thus, without wishing
to be bound by a theory, the differential motor phenotypes observed
for the different compounds in C. elegans can reflect different
mechanisms of action for each compound.
[0368] Using these GFP-labeled C. elegans lines, we observe that
compound 8 increases survival of motor neurons (FIGS. 20A to 20D).
We used the nematode line expressing A315T TDP-43 for the study
because this line shows the most age-dependent visible loss of
motor neurons. The nematodes were hatched and grown on plates
containing compound 8 (dose=34.8 .mu.M, which corresponds to
200.times.IC50). Pictures were taken at adult day 2 (FIGS. 20A-C)
and neuronal loss was quantified (FIG. 20D). Two different measures
were used for quantifying neuronal loss. One method was to count
the number of visible neuronal cell bodies (FIG. 20D). Using this
method we observed that compound 8 elicited an 50% decrease in
neuronal loss, which was highly significant (FIG. 20D). The second
method quantified the number of neurons that did not have visible
connections to other neurons. This measure is readily observable in
FIG. 20D. Note that the vehicle treated nematodes have some neurons
that exist as isolated cell bodies without visible processes
connecting them to other neurons; such neurons were counted as
"lacking connections". In contrast, nematodes treated with compound
8 show very few (if any) neurons lacking connections. These results
are quantified in FIG. 20D. Using this method we observed that
compound 8 elicited reduced the number of neurons lacking
connections by almost 70% (69.3%, FIG. 20D).
[0369] Compound 8 also improves motor function by a similar
percentage in C. elegans expressing human WT or A315T TDP-43 (FIGS.
21A to 21C), but exhibits little effect on the non-transgenic N2
line. Nematodes expressing human WT TDP-43 show about a 65% loss of
function, and this functional loss is restored by treatment with
compound 8 (FIGS. 21A to 21C, middle panel). Nematodes expressing
human A315T TDP-43 show a more severe loss of motor function
(.about.93%). Compound 8 improves motor function by a percentage
similar to that of WT TDP-43, but this is not sufficient to restore
motor function up to the normal level of functioning (FIG. 21C,
middle panel). One of the aspects of compound 8 action that
captures our attention is that it has no affect on motor function
in nematodes that do not express TDP-43, suggesting that it is
selective for TDP-43. In contrast, compound 7 improves motor
function in non-transgenic AND transgenic TDP-43.
[0370] Reduction of Levels of Insoluble TDP-43:
[0371] The previous study demonstrates that the amount of insoluble
TDP-43 increases in response to treatment with arsenic, which
corresponds to with induction of stress granules. We used the
tetracycline inducible PC12 cells expressing human WT TDP-43::GFP.
Using these cells, we induced TDP-43 expression, and treated with
arsenic (0.5 M, 1 hr) .+-.compound 8 (3.5 .mu.M). The cells were
then lysed, fractionated into soluble/insoluble and then
immunoblotted. The results in FIGS. 22A and 22B show that arsenite
increases amount of aggregated TDP-43::GFP, which is consistent
with our prior results. Cells treated with compound 8 show an
absolutely striking reduction in levels of high molecular weight
aggregated TDP-43 under basal conditions or after treatment with
arsenite (FIGS. 22A and 22B). One can also see that expressing
TDP-43 leads to formation of a lower molecular weight TDP-43 band,
which might be a TDP-43 cleavage fragment (arrow, FIGS. 22A and
22B). Treatment with compound 8 eliminates this cleavage fragment
(FIGS. 22A and 22B). We also fractionated the cell lysates and
demonstrated that compound 8 causes an equally impressive
translocation of TDP-43::GFP from the insoluble to the soluble
fraction.
[0372] Translocation of TDP-43::GFP from the Cytoplasm to the
Nucleus:
[0373] Increasing data suggest that the process of ALS leads to
loss of TDP-43 expression in the nucleus and increased expression
in the cytoplasm. It is hypothesized that this loss of TDP-43
nuclear expression leads to the neurodegeneration associated with
ALS. We examined the effects of compound 8 on the localization of
TDP-43 in rat hippocampal neurons transfected with human A315T
TDP-43 and treated with arsenite (FIGS. 23A and 23B). Hippocampal
neurons show increased cytoplasmic translocation of TDP-43 under
conditions of arsenite treatment compared to basal conditions, and
of A315T TDP-43 compared to WT TDP-43. FIG. 23 demonstrates the
striking effects of compound 8, which causes a dramatic shift in
localization of TDP-43 from the cytoplasm to the nucleus. This
contrasts with the theory that loss of nuclear TDP-43 is actually
what causes the disease. Thus, compound 8 might have the ability to
increase levels of nuclear TDP-43.
[0374] Compound 8 protects against neurotoxicity: An important
question is whether the compounds will work on protect against
toxicity induced by TDP-43. To test this, we transfected primary
cultures of hippocampal neurons with EGFP or WT-TDP-43;
transfection efficiency was 30%. The following day we measured
caspase activity. There was a moderate level of baseline activity
evident in the EGFP transfected cells. However, cells transfected
with TDP-43 showed more caspase activity, and about half of this
increase was reversed by pretreatment with compound 8 (FIG. 24).
Without being bound by theory, this strongly suggests that compound
8 might be protect against toxicity related to TDP-43, and protect
neurons. Inclusions are one thing, but the bottom line is neuron
death. The nematode assay provides strong evidence of
neuroprotection. FIG. 24 shows neuroprotection in primary cultures
of hippocampal neurons. This was done using a fluorescent assay
using a substrate for caspase 3 that fluoresces after cleavage
(sold by Promega Corp. and Biotum). This assay has also been
performed using an antibody that only detects cleaved caspase 3
(Cell Signaling Inc).
[0375] Compounds for TDP-43 aggregation inhibition also inhibit
replication of HIV. Several compounds were assayed to explore
inhibition of HIV replication. The assay used was a p24 ELISA assay
(Proc Natl Acad Sci USA. 2008 May 6; 105(18):6684-9; herein
incorporated by reference in its entirety). Inhibition of HIV
replication for several compounds (listed on axis) is shown in
FIGS. 25 and 26.
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[0441] Although the invention has been described and illustrated in
the foregoing illustrative embodiments, it is understood that the
present disclosure has been made only by way of example, and that
numerous changes in the details of implementation of the invention
can be made without departing from the spirit and scope of the
invention, which is limited only by the claims that follow.
Features of the disclosed embodiments can be combined and/or
rearranged in various ways within the scope and spirit of the
invention to produce further embodiments that are also within the
scope of the invention. Those skilled in the art will recognize, or
be able to ascertain, using no more than routine experimentation,
numerous equivalents to the specific embodiments described
specifically in this disclosure. Such equivalents are intended to
be encompassed in the scope of the following claims.
* * * * *
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