U.S. patent application number 12/150628 was filed with the patent office on 2010-03-18 for regulation of gsk-3alpha activity for the treatment or prevention of alzheimer's disease.
This patent application is currently assigned to Trustees of the University of Pennsylvania. Invention is credited to Peter S. Klein, Virginia M-Y. Lee, Christopher J. Phiel, Christina A. Wilson.
Application Number | 20100068308 12/150628 |
Document ID | / |
Family ID | 32474177 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068308 |
Kind Code |
A1 |
Phiel; Christopher J. ; et
al. |
March 18, 2010 |
Regulation of GSK-3alpha activity for the treatment or prevention
of Alzheimer's disease
Abstract
Provided is a novel use of therapeutic concentrations of an
inhibitor of glycogen synthase kinase-3 (GSK-3), including lithium
or any other GSK-3 inhibitor, to block, reduce or inhibit
processing of amyloid precursor proteins to beta-amyloid (A.beta.)
peptides, which are now believed to be the principal cause of
Alzheimer's disease, thereby providing methods useful for the
prevention, inhibition or reversal of the disease. Also provided
are methods of using agents that specifically target the .alpha.
isoform of GSK-3, which is responsible for APP processing, making
such selective GSK-3.alpha.-specific inhibitors especially useful
in the treatment, prevention, and possible reversal of Alzheimer's
disease. Further provided are kits and screening methods associated
with the present methods.
Inventors: |
Phiel; Christopher J.;
(Royersford, PA) ; Wilson; Christina A.;
(Lansdowne, PA) ; Lee; Virginia M-Y.;
(Philadelphia, PA) ; Klein; Peter S.; (Wynnewood,
PA) |
Correspondence
Address: |
MONTGOMERY, MCCRACKEN, WALKER & RHOADS, LLP
123 SOUTH BROAD STREET, AVENUE OF THE ARTS
PHILADELPHIA
PA
19109
US
|
Assignee: |
Trustees of the University of
Pennsylvania
|
Family ID: |
32474177 |
Appl. No.: |
12/150628 |
Filed: |
April 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10368769 |
Feb 19, 2003 |
7378111 |
|
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12150628 |
|
|
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60359290 |
Feb 20, 2002 |
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Current U.S.
Class: |
424/722 ; 435/15;
435/375 |
Current CPC
Class: |
Y10S 514/879 20130101;
A61K 33/00 20130101; A61P 25/28 20180101; A61K 31/00 20130101; A61K
31/19 20130101; A61K 31/19 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/722 ;
435/375; 435/15 |
International
Class: |
A61K 33/00 20060101
A61K033/00; C12N 5/00 20060101 C12N005/00; C12Q 1/48 20060101
C12Q001/48; A61P 25/28 20060101 A61P025/28 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was supported in part by Grant Nos. AG11542
and RO1 MH58324 from the National Institutes of Health.
Accordingly, the Government may have certain rights in this
invention.
Claims
1. A method of inhibiting or reducing symptoms of a condition,
disease or disorder in a patient mediated by GSK-3 or GSK-3.alpha.
activity, exclusive of Alzheimer's disease, the method comprising
the steps of: administering to the patient a therapeutically
effective composition, comprising a pharmaceutically acceptable
amount of a GSK-3 inhibitor or GSK-3.alpha.-specific inhibitor,
respectively; measuring the blocking, inhibiting or reducing
production of A.beta. amyloid peptides in the patient following
administering the pharmaceutically acceptable amount of the GSK-3
inhibitor or GSK-3.alpha.-specific inhibitor, respectively; and
determining that the administered inhibitor is sufficient to block
or inhibit GSK-3 or GSK-3.alpha. activity in the patient, such that
measured formation of A.beta. amyloid peptides in the patient is
reduced in the range of 30% to 60%.
2. The method of claim 1, wherein the patient is a mammal.
3. The method of claim 2, wherein the mammal is human.
4. The method of claim 1, wherein the GSK-3 inhibitor or
GSK-3.alpha.-specific inhibitor, respectively, comprises lithium or
a salt thereof or kenpaullone.
5. The method of claim 1, wherein the GSK-3 inhibitor comprises a
composition exclusive of lithium or kenpaullone.
6. The method of claim 1, wherein the patient's requirement for
other medication to treat the condition, disease or disorder and
the patient's symptoms related thereto is reduced.
7. The method of claim 1, further comprising: measuring the
blocking, inhibiting or reducing production of soluble
A.beta..sub.40 and A.beta..sub.42 peptide levels in the patient
following administering the pharmaceutically acceptable amount of
the GSK-3 inhibitor or GSK-3.alpha._inhibitor; and determining that
the administered inhibitor is effective for inhibiting or reducing
symptoms of the condition, disease or disorder in the patient, such
that measured soluble or insoluble A.beta..sub.40 and
A.beta..sub.42 peptide levels are reduced in the range of 30% to
60%.
8. The method of claim 1, wherein blocking, inhibiting or reducing
GSK-3.alpha. activity disrupts .gamma.-secretase cleavage of APP,
without inhibiting Notch processing.
9. The method comprising treating in vitro the condition, disease
or disorder of the patient of claim 1, wherein the condition,
disease or disorder is mediated by GSK-3 or GSK-3.alpha. activity,
respectively, in brain cells or tissues, the treatment comprising
exposing the brain cells or tissue to the therapeutically effective
composition, comprising a pharmaceutically acceptable amount of
GSK-3 inhibitor or GSK-3.alpha.-specific inhibitor, respectively,
that is sufficient to block or inhibit activity of GSK-3 or
GSK-3.alpha., respectively.
10. The method of claim 9, wherein the GSK-3 inhibitor or
GSK-3.alpha.-specific inhibitor, respectively, comprises lithium or
a salt thereof or kenpaullone.
11. The method of claim 9, wherein the brain cells or tissues are
from a non-Alzheimer's disease patient.
12. A method for stabilizing a patient susceptible to a condition,
disease or disorder in a patient mediated by GSK-3 or GSK-3.alpha.
activity, the method comprising the steps of: administering to the
patient a therapeutically effective composition, comprising a
pharmaceutically acceptable amount of a GSK-3 inhibitor or
GSK-3.alpha.-specific inhibitor, respectively, based upon
predetermined effective amounts of each composition; and
determining that the administered inhibitor is sufficient to block
or inhibit GSK-3 or GSK-3.alpha. activity in the patient.
13. The method of claim 12, wherein the patient is susceptible to
Alzheimer's disease.
14. The method of claim 12, wherein the patient is a mammal.
15. The method of claim 14, wherein the mammal is human.
16. The method of claim 1, wherein the GSK-3 inhibitor or
GSK-3.alpha.-specific inhibitor, respectively, comprises lithium or
a salt thereof or kenpaullone.
17. The method of claim 1, wherein the GSK-3 inhibitor comprises a
composition exclusive of lithium or kenpaullone.
18. The method of claim 12, wherein stabilizing the patient
comprises delaying onset of symptoms or preventing the condition,
disease or disorder mediated by GSK-3 or GSK-3.alpha.activity.
19. A kit for selectively inhibiting GSK-3 or GSK-3.alpha.
activity, respectively, comprising a therapeutically effective
composition, comprising a pharmaceutically acceptable amount of
GSK-3 inhibitor or GSK-3.alpha.-specific inhibitor, respectively,
that is sufficient to block or inhibit activity of GSK-3 or
GSK-3.alpha., respectively, and an instructional material regarding
the use thereof to treat a condition, disease or disorder mediated
by GSK-3 or GSK-3.alpha. activity in a patient, or in brain cells
or tissues of a patient.
20. A method of detecting a composition of matter that blocks,
inhibits or reduces GSK-3 or GSK-3.alpha. kinase activity,
comprising testing the composition in brain cells or brain tissue
in vitro from a subject for its capability of blocking, reducing or
inhibiting the processing of amyloid precursor proteins to
.beta.-amyloid peptides of the type characterizing Alzheimer's
disease and comparing the effectiveness of such composition for
disrupting the production of the A.beta. peptides with the
effectiveness of a known GSK-3 inhibitor, and selecting the
composition having such effective blocking, inhibiting or reducing
capability.
21. The method of claim 20, further comprising testing the
composition in brain cells or brain tissue from a subject for GSK-3
or GSK-3.alpha. specificity; and testing its capability of
blocking, reducing or inhibiting .gamma.-secretase mediated-APP
processing, then comparing effectiveness of such composition for
disrupting APP processing with effectiveness of the known
GSK-3.alpha.inhibitors; and selecting the composition having
effective blocking, inhibiting or reducing capability.
22. The method of claim 21, further comprising testing the
composition for its capability of blocking or inhibiting
GSK-3.beta. activity, and selecting the composition that blocks,
inhibits or reduces GSK-3.alpha. activity without affecting
GSK-3.beta. activity.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 60/359,290, filed Feb. 20, 2002 and application
Ser. No. 10/368,769, filed Feb. 19, 2003, the content of which is
herein incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods
useful in the treatment and prevention of Alzheimer's disease,
specifically relating to inhibiting the activity of glycogen
synthase kinase-3 and/or glycogen synthase kinase-3.alpha..
BACKGROUND OF THE INVENTION
[0004] Alzheimer's Disease (AD) is a degenerative brain disorder
associated with extensive loss of specific neuronal cellular
subpopulations, and characterized clinically by progressive loss of
memory, cognition, reasoning, judgment and emotional stability that
gradually leads to profound mental deterioration and ultimately
death. The disease currently affects as many as four million
individuals in the United States alone. To date, the disease has
proven to be incurable, and presently causes up to 100,000 deaths
annually.
[0005] The brains of individuals with AD exhibit neuronal
degeneration and characteristic lesions variously referred to as
amyloidogenic plaques, vascular amyloid angiopathy, and
neurofibrillary tangles. It is presently believed that progressive
cerebral deposition of particular amyloidogenic proteins,
beta-amyloid proteins, play a seminal role in the pathogenesis of
AD and can precede cognitive symptoms and onset of dementia by
years or possibly even decades.
[0006] Alzheimer's disease is associated with aberrant processing
of the amyloid precursor protein (APP), leading to increased
production and aggregation of amyloid-.beta. (A.beta.) peptides.
Amyloid plaques are composed primarily of 40 and 42 amino acid
peptides (A.beta..sub.40 and A.beta..sub.42, respectively) (Selkoe,
Proc. Nat'l. Acad. Sci. USA 98:11039-11041 (2001)) derived from APP
by sequential proteolysis catalyzed by the aspartyl protease, BACE
(Vassar et al., Science 286:735-741 (1999)), followed by
presenilin-dependent .gamma.-secretase cleavage (De Strooper et
al., Nature 391:387-390 (1998)). A.beta..sub.42 is less soluble
than A.beta..sub.40 and is the predominant A.beta. species in
amyloid plaques (Iwatsubo et al., Neuron 13:45-53 (1994)).
[0007] Presenilins 1 and 2 (PS1 and PS2) are integral membrane
proteins proposed to have inherent .gamma.-secretase activity
(Wolfe et al., Nature 398:513-517 (1999)) and interact in a
functional complex with nicastrin (Esler et al., Proc. Nat'l. Acad.
Sci. USA 99:2720-2725 (2002); Edbauer et al., Proc. Nat'l. Acad.
Sci. USA 99:8666-8671 (2002)), aph-1, and pen-2 (Francis et al.,
Dev. Cell 3:85-97 (2002)). Presenilins also interact with a number
of other proteins, including .alpha.-catenin and .beta.-catenin
(Soriano et al., J. Cell Biol. 152:785-794 (2001); Yu et al.,
Nature 407:48-54 (2000)). Presenilin 1, which is required for
.gamma.-secretase mediated processing of APP (De Strooper et al.,
1998), interacts with glycogen synthase kinase-3 (GSK-3)(Takashima
et al., Proc. Nat'l. Acad. Sci. USA 95:9637-9641 (1998); Kang et
al., J. Neurosci. 19:4229-4237 (1999); Kang et al., Cell
110:751-762 (2002)), although a functional role for this proteins
in .gamma.-secretase function has not been previously
established.
[0008] Glycogen synthase kinase-3 (GSK-3) is a serine/threonine
protein kinase having a monomeric structure and a size of
approximately 47 kilodaltons. It is one of several protein kinases
which phosphorylate glycogen synthase (Embi et al., Eur. J.
Biochem. 107:519-527 (1980); Hemmings et al., Eur. J. Biochem.
119:443-451 (1982)). GSK-3 is also referred to in the literature as
factor A (F.sub.A) in the context of its ability to regulate
F.sub.C, a protein phosphatase (Vandenheede et al., J. Biol. Chem.
255:11768-11774 (1980)). Other names for GSK-3 and homologs thereof
include: zeste-white3/shaggy (zw3/sgg; the Drosophila melanogaster
homolog), ATP-citrate lyase kinase (ACLK or MFPK; Ramakrishna et
al., Biochem. 28:856-860 (1989); Ramakrishna et al., J. Biol. Chem.
260:12280-12286 (1985), GSKA (the Dictyostelum homolog; Harwood et
al., Cell 80:139-48 (1995), and MDSI, MCK1, and others (yeast
homologs; Hunter et al., TIBS 22:18-22 (1997)), tau protein kinase
(mammalian) and GSKA (Dictyostelium).
[0009] The gene encoding GSK-3 is highly conserved across diverse
phyla. In vertebrates, GSK-3 exists in two isoforms, designated
GSK-3.alpha. (51 kDa) and GSK-3.beta. (47 kDa). These two isoforms
are the products of distinct genes. The amino acid identity among
vertebrate homologs of GSK-3 is in excess of 98% within the
catalytic domain (Plyte et al., Biochim. Biophys. Acta 1114:147-162
(1992)), although GSK-3.alpha. is known to be slightly larger than
GSK-3.beta.. Sun et al., J. Biol. Chem. 277(14):11933-11940 (April
2002) have reported that in brain extracts and in MAP fractions,
the amounts of GSK-3.alpha. and GSK-3.beta. are almost equal, but
that there are profound differences between the amounts of each
kinase complexed with tau, further distinguishing the functions of
the two. The authors determined that 6-fold more tau is complexed
with GSK-3.beta. than with GSK-3.alpha. in the brain, and that
GSK-3.beta. is bound to tau within an approximately 400-kDa
micro-tubule-associated complex. Thus, GSK-3.beta. associates with
the microtubules via tau.
[0010] GSK-3 phosphorylates numerous proteins in vitro, including
beta-catenin, glycogen synthase, phosphatase inhibitor I-2, the
type-II subunit of cAMP-dependent protein kinase, the G-subunit of
phosphatase-1, ATP-citrate lyase, acetyl coenzyme A carboxylase,
myelin basic protein, a microtubule-associated protein, a
neurofilament protein, an N-CAM cell adhesion molecule, nerve
growth factor receptor, c-Jun transcription factor, JunD
transcription factor, c-Myb transcription factor, c-Myc
transcription factor, L-myc transcription factor, adenomatous
polyposis coli tumor suppressor protein, and tau protein (Plyte et
al., 1992; Korinek et al., Science 275:1784-1787 (1997); Miller et
al., Genes & Dev. 10:2527-2539 (1996)). The phosphorylation
site recognized by GSK-3 has been determined in several of these
proteins (Plyte et al., 1992). The diversity of these proteins
suggests a wide role for GSK-3 in the control of cellular
metabolism, regulation, growth, and development. GSK-3 tends to
phosphorylate serine and threonine residues in a proline-rich
environment, but does not display the absolute dependence upon
these amino acids which is displayed by protein kinases which are
members of the mitogen-activated protein (MAP) kinase or cdc2
families of kinases.
[0011] U.S. Pat. No. 6,441,053 (Klein et al.) teaches a method of
identifying inhibitors of GSK-3 and for treating a GSK-3-related
disorders--other than Alzheimer's disease in an animal. The method
comprises providing a mixture comprising GSK-3, a phosphate source,
and a GSK-3 substrate, incubating the mixture in the presence or
absence of a test compound, and assessing the activity of GSK-3 in
the mixture. A reduction of GSK-3 activity following incubation of
the mixture in the presence of the test compound is an indication
that the test compound is an inhibitor of GSK-3. In the '053
patent, however, the GSK-3 inhibitor is expressly not lithium.
[0012] U.S. Pat. No. 6,057,117 (Harrison et al.) teaches a
pharmaceutical composition comprising a selective GSK-3 inhibitor
identified by: (a) contacting a first radiolabeled peptide
substrate comprising an isolated nucleotide sequence, in which the
N-terminal serine is the target of phosphorylation by GSK-3 and the
C-terminal serine is prephosphorylated, coupled to an anchor ligand
with GSK-3 in the presence of radiolabeled phosphate-.gamma.ATP, a
substrate anchor, and a candidate inhibitor, then (b) contacting a
second radiolabeled peptide substrate coupled to an anchor ligand
with GSK-3 in the presence of radiolabeled phosphate-.gamma.ATP,
and a substrate anchor, and (c) identifying an inhibitor of GSK-3
kinase activity by a reduction of radiolabel incorporation in step
(a) compared to step (b). The identified composition is also used
to treat a subject having a condition mediated by GSK-3 activity or
susceptible to such a condition. In an alternative embodiment of
the '117 patent a second therapeutic compound may be added, wherein
the compound may be lithium. However, no lithium therapy is
suggested with regard to blocking or inhibiting the activity of the
GSK-3.alpha. isoform. See also Stambolic et al., Current Biology
6(12):1664-1668 (1996).
[0013] The activity of both GSK-3.alpha. and -3.beta. has been
reported to be inhibited by lithium (e.g., Klein et al., Proc.
Natl. Acad. Sci. USA 93:8455-8459 (1996); Hedgepeth et al., Dev.
Biol. 185:82-91 (1997); Phiel et al., Annu. Rev. Pharmacol.
Toxicol. 41:789-813 (2001); US Publ. Patent Appl. 20010052137
(Klein et al.)), yet specific inhibitors of the activity of the
GSK-3.alpha.isoform alone (without affecting GSK-3.beta.) remain
unknown. Inhibition of GSK-3.beta. is a physiological mechanism by
which lithium exerts its therapeutic effects in animals (e.g.,
humans) afflicted with a variety of disorders. For example, lithium
is an effective drug for treatment of bipolar (manic-depressive)
disorder (Price et al., New Eng. J. Med. 331:591-598 (1994);
Goodwin et al., (1990) In: Manic-Depressive Illness, New York:
Oxford University Press), and can be used to treat profound
depression in some cases, although it is not known whether lithium
works through GSK-3 in the treatment of bipolar disorder. Despite
the remarkable efficacy of lithium observed during decades of its
use, the molecular mechanism(s) underlying its therapeutic actions
have not been fully elucidated (Bunney et al., (1987) In:
Psychopharmacology: The Third Generation of Progress, (Hy, ed.) New
York, Raven Press, 553-565; Jope et al., Biochem. Pharmacol.
47:429-441 (1994); Risby et al., Arch. Gen. Psychiatry 48:513-524
(1991); Wood et al., Psychol. Med. 17:570-600 (1987)).
[0014] Lithium is a fixed monovalent cation and the lightest of the
alkali metals (group la of the Periodic Table of the elements).
Li.sup.+ has the highest energy of hydration of the alkali metals
and, as such, can substitute for Na.sup.+ (and to a lesser extent
K.sup.+) for ion transport by biological systems. Lithium is both
electroactive and hydrophilic, and trace amounts of Li.sup.+ are
found in human tissues; typical human blood plasma concentrations
of Li.sup.+ are about 17 .mu.g/L.
[0015] Unlike other psychotropic drugs, Li.sup.+ has no discernible
psychotropic effects in normal man, although the therapeutic
efficacy of lithium in the treatment of acute mania and the
prophylactic management of bipolar (manic/depressive) disorder has
been consistently demonstrated. The oral and parenteral
administration of lithium salts, such as lithium carbonate and
lithium citrate, has also found widespread use in the current
treatment of, for example, alcoholism, aggression, schizophrenia,
unipolar depression, skin disorders, immunological disorders,
asthma, multiple sclerosis, rheumatoid arthritis, Crohn's disease,
ulcerative colitis, and irritable bowel syndrome, as well as for
use in many other diseases and conditions.
[0016] Unfortunately, no drug treatments for Alzheimer's disease
have, to date, proven to be very satisfactory, and demonstrating
the effectiveness of such drugs in the treatment of dementias can
be quite difficult, see, e.g., Handbook of Dementing Illnesses,
(John Morris, Ed.), Marcel Dekker 1994, p. 591. Part of this
difficulty arises from the fact that it can often be difficult to
clearly diagnose the type of dementia with which the patient is
afflicted. Thus, there exists a pressing need to identify
compositions that have a blocking or inhibiting effect in humans on
the control of GSK-3.alpha. specifically required for APP
processing to (A.beta.) peptides, and/or to reduce formation of
both amyloid plaques and neurofibrillary tangles, recognized as two
pathological hallmarks of Alzheimer's disease. By finding a
GSK-3.alpha.-specific inhibitor, preferably that does not also
affect GSK-3.beta., it will be possible to treat Alzheimer's
disease in a patient without inhibiting GSK-3.beta., which serves
many critical functions in cells.
SUMMARY OF THE INVENTION
[0017] The present invention provides a novel approach using
therapeutic concentrations of an inhibitor of GSK-3 specifically to
reduce processing of amyloid precursor proteins to beta-amyloid
(A.beta.) peptides, and thus to prevent, inhibit or reverse
Alzheimer's disease. In one aspect of the invention, lithium
treatment is shown to inhibit production of (A.beta.) peptides in
cultured cells, and in whole animals carrying familial Alzheimer's
disease mutations. Moreover, this effect of lithium is mediated
through the inhibition of GSK-3.alpha..
[0018] GSK-3 has previously been shown to phosphorylate tau
protein, a component of paired helical filaments once thought to be
a cause of Alzheimer's disease, and therefore GSK-3 inhibitors were
proposed as potential therapy for Alzheimer's disease. However, no
one has previously reported or suggested that GSK-3 inhibitors,
such as lithium or any other GSK-3 inhibitor, act to block or
inhibit the production of A.beta. peptides, which are now believed
to be the principal cause of Alzheimer's.
[0019] Therapeutic concentrations of lithium block or inhibit
production of A.beta..sub.40 and A.beta..sub.42 peptides by
interfering with .gamma.-secretase cleavage of APP, but they do not
inhibit Notch processing. Notch is a distinct signaling molecule
that is likely required for multiple functions. The fact that APP
processing is blocked without affecting Notch processing means that
one need not worry about potential side effects arising from
inhibition of Notch processing. Importantly, the lithium
compositions block accumulation of A.beta. peptides in the brains
of mice that otherwise overproduce A.beta. peptides. Thus, in
accordance with one aspect of the invention there are provided
GSK-3 inhibitor compositions, e.g., a pharmaceutically acceptable
lithium salt and a physiologically acceptable carrier, at
therapeutically effective concentrations that are sufficient to
inhibit, block, or even reverse, A.beta. peptide accumulation.
[0020] A second important aspect of this invention was the
discovery that the .alpha. isoform of GSK-3 (GSK-3.alpha.) is
specifically responsible for APP processing. Therefore, agents that
specifically target GSK-3.alpha. will be especially useful in the
treatment, prevention, and possible reversal of Alzheimer's
disease. Further provided are methods for treating a condition
mediated by GSK-3.alpha. activity by administering a selective
inhibitor of GSK-3.alpha., which is preferably a therapeutically
effective amount of a composition that specifically inhibits
GSK-3.alpha., but not GSK-3.beta. or other lithium sensitive
enzymes, or a pharmaceutically acceptable salt thereof, and a
physiologically acceptable carrier.
[0021] Also included in the invention are methods of treating a
GSK-3.alpha.-related disorder in an animal, comprising
administering to the animal (or to the brain, brains cells or brain
tissue of an animal) a GSK-3.alpha. inhibitor suspended in a
pharmaceutically acceptable carrier. Preferably, the animal is a
mammal, and more preferably, the mammal is a human. The
GSK-3.alpha. related disorder, which is treated according to the
methods of the invention, is preferably Alzheimer's disease.
Nevertheless, selective inhibition of GSK-3.alpha. may be useful to
treat or inhibit other disorders mediated by GSK-3.alpha. activity.
Thus, the invention provides methods for treating a biological
condition mediated by GSK-3.alpha. activity by administering an
effective amount of a pharmaceutical composition comprising a
selective GSK-3.alpha. inhibitor to a subject having a condition
mediated by GSK-3.alpha. activity or susceptible to such a
condition, e.g., Alzheimer's disease, wherein the production of
A.beta. peptides is blocked or inhibited.
[0022] The invention also provides an in vitro method of
identifying an inhibitor of GSK-3.alpha. kinase activity that will
block or inhibit the production of A.beta. peptides, without
blocking or inhibiting GSK-3.beta., and includes a pharmaceutical
composition comprising an inhibitor identified by this in vitro
method.
[0023] One aspect of the present invention provides methods for the
treatment of a subject having Alzheimer's disease, or the
conditions related thereto, wherein the methods comprise
administering to the subject (or to a subject's brain cells or
brain tissue) an inhibitor of GSK-3 kinase activity, such as
lithium, in an amount sufficient to block or inhibit the production
of A.beta. peptides. Moreover, methods using those agents that
specifically target GSK-3.alpha. to disrupt the .gamma.-secretase
mediated processing of APP will be especially useful in the
treatment, prevention and possible reversal of Alzheimer's
disease.
[0024] In accordance with still another aspect of the present
invention, there are further provided methods for stabilizing a
subject susceptible to Alzheimer's disease or the formation of
amyloid plaques and neurofibrillary tangles or accumulated A.beta.
peptides (particularly A.beta..sub.40 and A.beta..sub.42 peptides)
in the brain, thereby also stopping or inhibiting the processing of
APP in the brain, or in brain cells or tissue, wherein the methods
comprise administering to the subject a stabilizing amount of
inhibitor of GSK-3 kinase activity, such as lithium, sufficient to
block or inhibit the production of A.beta. peptides. Further
provided are methods using those agents that specifically target
GSK-3.alpha. to disrupt the .gamma.-secretase mediated processing
of APP to stabilize the subject susceptible to Alzheimer's
disease.
[0025] In accordance with yet another aspect of the present
invention, there are provided methods for preventing Alzheimer's
disease in a susceptible subject or preventing the formation of
amyloid plaques and neurofibrillary tangles or accumulated A.beta.
peptides (particularly A.beta..sub.40 and A.beta..sub.42 peptides)
in the brain, by administering an inhibitor of GSK-3 kinase
activity, such as lithium, sufficient to block or inhibit the
production of A.beta. peptides. Also provided are methods using
those agents that specifically target GSK-3.alpha. to disrupt the
.gamma.-secretase mediated processing of APP to prevent Alzheimer's
disease in a subject susceptible to the disease.
[0026] In a further aspect of the present invention, there are
provided methods for reversing the recognized hallmark effects in a
subject having Alzheimer's disease, by preventing or inhibiting the
continued formation of amyloid plaques and neurofibrillary tangles
or accumulated A.beta.peptides (particularly A.beta..sub.40 and
A.beta..sub.42 peptides) in the brain, by administering an
inhibitor of GSK-3 kinase activity, such as lithium, sufficient to
block or inhibit the production of A.beta. peptides causing a
reversal of the Alzheimer's disease state, and/or using those
agents that specifically target GSK-3.alpha. to disrupt the
.gamma.-secretase mediated processing of APP to further reverse the
Alzheimer's disease state in the subject.
[0027] All combinations, sources and amounts of the active
ingredients discussed herein in conjunction with the compositions
of the present invention are contemplated as being administered in
accordance with the method taught herein. Preferably the foregoing
methods further comprise monitoring such subject's A.beta. peptide
levels, particularly A.beta..sub.40 and A.beta..sub.42 peptides, or
GSK-3.alpha. levels, or the production or processing of APP.
Included are in vivo and in vitro methods.
[0028] Also provided are kits for administering an inhibitor of
GSK-3 kinase activity, such as lithium, in an amount sufficient to
block, reduce or inhibit the production of A.beta. peptides in a
subject of Alzheimer's disease, or one that is susceptible thereto,
and/or for administering those agents that specifically target
GSK-3.alpha. to disrupt the .gamma.-secretase mediated processing
of APP in the brain, brain cells or brain tissue of a subject.
[0029] Additional objects, advantages and novel features of the
invention will be set forth in part in the description, examples
and figures which follow, all of which are intended to be for
illustrative purposes only, and not intended in any way to limit
the invention, and in part will become apparent to those skilled in
the art on examination of the following, or may be learned by
practice of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0030] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended figures.
[0031] FIGS. 1a-1d show that lithium reduces secreted
A.beta..sub.40 and A.beta..sub.42 levels in a dose-dependent manner
and causes accumulation of APP C-terminal fragments. FIG. 1a is a
histogram depicting the effect of treating CHO-APP.sub.695 cells
with sodium chloride (NaCl) or lithium chloride (LiCl), then
measuring A.beta. levels by an A.beta. sandwich ELISA. The
histogram represents normalized levels (fm A.beta./intracellular
full length APP) of A.beta..sub.40 (light bars) and A.beta..sub.42
(dark bars). Error bars represent standard deviation. FIG. 1b
depicts a PhosphorImager visualization of an SDS-PAGE separation of
.sup.35S-methionine labeled, immunoprecipitated A.beta. secreted
from CHO-APP.sub.695 cells in the presence of LiCl (lane 2) or
DFK-167 (lane 3). FIG. 1c depicts a PhosphorImager visualization of
SDS-PAGE separated, immunoprecipitated intracellular APP
holoprotein and C-terminal fragments (C99, C89, and C83) from
CHO-APP.sub.695 cells after treatment with LiCl or DFK-167. FIG. 1d
depicts a western blot as assessed by myc (9E10), showing
expression of Notch constructs (each having a C-terminal myc tag)
after CHO-APP.sub.695 cells were transfected with .DELTA.E-Notch
and treated with either LiCl or DFK-167 for 24 hours. Notch
intracellular domain (ICD) beginning at residue 1744 is a positive
control for cleavage (lane 2). .DELTA.E-Notch V1744K with a point
mutation in the .gamma.-secretase cleavage site of .DELTA.E-Notch
is a negative control for cleavage (lane 3).
[0032] FIGS. 2a-2d show that GSK-3 inhibitors reduce A.beta.,
independent of .beta.-catenin stabilization. FIG. 2a graphically
shows the effect of treating CHO-APP.sub.695 cells with kenpaullone
(a GSK-3 inhibitor), or with roscovitine (a cdk inhibitor that does
not inhibit GSK-3). Secreted A.beta. levels (A.beta..sub.40=light
bars; A.beta..sub.42=dark bars). FIG. 2b depicts a western blot
showing that in CHO-APP.sub.695 cells treated with either
kenpaullone or lithium, GSK-3 was inhibited, causing an
accumulation of .beta.-catenin protein. Roscovitine was added as a
control. Western blot for actin is shown as a loading control.
Con=untreated cells; D=DMSO control; Ken=kenpaullone;
Rosco=roscovitine. FIG. 2c graphically shows the effect of
transfecting CHO-APP.sub.695 cells with a .beta.-catenin-responsive
luciferase reporter construct, OT-Luc (light bars), or with a
mutated luciferase reporter, OF-Luc (dark bars), followed by
treatment with LiCl, kenpaullone, or roscovitine. FIG. 2d
graphically shows effect of overexpression of .beta.-catenin on
A.beta. production. CHO-APP.sub.695 cells were transfected with
either GFP (control) or .beta.-catenin in pCS2. Secreted
A.beta..sub.40=light bars and A.beta..sub.42=dark bars. Inset in
FIG. 2d shows western blot of endogenous GFP and overexpressed
(.beta.-cat) .beta.-catenin in duplicate lanes.
[0033] FIGS. 3a-3b show that GSK-3.alpha. is required for A.beta.
production. FIG. 3a depicts a western blot for GSK-3.alpha. and
GSK-3.beta. of lysates from CHO-APP.sub.695 cells transfected with
siRNAs. siRNA against pGL3-luciferase (Pp-Luc) is control
transfection. GSK-3.alpha. siRNA selectively reduces GSK-3.alpha.
protein (closed arrow) and GSK-3.beta. directed siRNA selectively
reduces GSK-3.beta. (open arrow). FIG. 3b graphically shows A.beta.
levels (A.beta..sub.40=light bars; A.beta..sub.42=dark bars)
secreted from siRNA transfected cells. Error bars represent
standard deviation. Asterisks indicate a significant difference
from control, as determined by one-way ANOVA (p<0.05).
[0034] FIGS. 4a-4c graphically show that lithium blocks A.beta.
accumulation in cultured neurons and in the brains of mice
overproducing A.beta. peptides. FIG. 4a shows the effect of lithium
treatment on embryonic mouse cortical neurons infected with a
Semliki Forest Virus containing either wild-type APP (APP-WT) or
APP with the pathogenic Swedish mutation (KM670/671NL).
A.beta..sub.40=light bars; and A.beta..sub.42=dark bars. FIGS. 4b
and 4c show cortical A.beta..sub.40 and A.beta..sub.42 accumulation
in both RIPA-extracted (soluble) and formic acid (FA)-extracted
(insoluble) fractions of cortical tissue from lithium-treated or
NaCl-treated subject animals that were heterozygous for both the
APP-Swedish transgene (Tg2576) and the PS1 P264L knock-in. Error
bars represent SEM. Asterisks in panel b and c denote significant
difference from NaCl treated animals when assessed by a one-way
ANOVA with p<0.05.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0035] The present invention provides a novel approach using an
inhibitor of GSK-3 kinase activity, such as lithium, administered
to a subject (or to the brain cells or brain tissues of a subject)
sufficient to block or inhibit, or even reverse, the production of
A.beta. peptides in the brain, or brain cells or tissues, of the
subject, thereby reducing the formation of both amyloid plaques and
neurofibrillary tangles in the brains and brain tissue of
Alzheimer's disease patients Thus, methods of the invention
inhibit, prevent or reverse Alzheimer's disease in the subject as
shown in the Examples that follow, wherein the production of
(A.beta.) peptides was inhibited in cultured cells by the
administration of lithium, and in whole animals carrying familial
Alzheimer's disease mutations. This effect of using GSK-3
inhibitors, such as lithium or any other GSK-3 inhibitor, to block,
inhibit or reverse the production of A.beta. peptides (now
recognized as the principal cause of the Alzheimer's disease
state), has never before been reported.
[0036] Moreover, this effect of lithium is mediated through the
inhibition of GSK-3.alpha.. Therapeutic concentrations of lithium
block or inhibit production of A.beta..sub.40 and A.beta..sub.42
peptides by interfering with .gamma.-secretase cleavage of APP, as
shown in the Examples by the effect on CTF accumulation. Moreover,
by using those agents that specifically target GSK-3.alpha. to
disrupt the .gamma.-secretase mediated processing of APP, the
Alzheimer's disease state in the subject (or in the brain cells or
tissue of the subject) is treated, prevented, or even in some
cases, reversed. It further blocks or inhibits generation of
A.beta. peptides through inhibition of GSK-3.alpha., or reduces
those A.beta. peptides that have already begun to form. In support
of this conclusion: 1) lithium was shown to reduce A.beta.
production in cultured cells and in the brains of mice that
overproduce A.beta.peptides; 2) kenpaullone, an alternative
GSK-3.alpha. inhibitor that acts through a distinct mechanism, also
inhibits A.beta. production; 3) RNAi mediated depletion of
GSK-3.alpha. reduces A.beta. production; and 4) moderate
overexpression of GSK-3.alpha. increases A.beta. production.
Furthermore, when lithium is administered as a GSK-3.alpha.
inhibitor, it inhibits APP processing at the .gamma.-secretase
step.
[0037] In contrast, in the examples that follow, it is shown that
reduction of GSK-3.beta. does not attenuate, and may enhance,
A.beta. production. These observations are surprising, as the
sequences of GSK-3.alpha. and 13 are 97% identical within the
kinase domains and appear to be redundant in the Wnt pathway,
although not in the regulation of NF-.kappa.B (Hoeflich et al.,
Nature 406:86-90 (2000)). However, the amino- and carboxy-terminal
sequences of GSK-3.alpha. and GSK-3.beta. are quite dissimilar, and
this divergence may account for functional differences in the
regulation of APP processing, perhaps reflecting differences in
protein-protein interactions.
[0038] A number of molecules have been identified recently that are
required in addition to presenilin for .gamma.-secretase activity,
including nicastrin (Esler et al., 2002; Edbauer et al., 2002),
aph-1, and pen-2 (Francis et al., 2002). Although their respective
roles in the regulation of .gamma.-secretase activity have not yet
been defined, loss of any of these components affects both APP and
Notch processing. In contrast, as shown in the examples that
follow, lithium does not inhibit Notch processing, indicating that
lithium is not a general inhibitor of .gamma.-secretase. While
consensus sites for GSK-3 phosphorylation have been identified in
PS1 that are important for PS1 stability, mutation of these sites
does not affect .gamma.-secretase activity (Kirschenbaum et al., J.
Biol. Chem. 276:7366-7375 (2001)). Thus, it is unlikely that
GSK-3.alpha. plays a role in the biogenesis, stability, or overall
activity of the .gamma.-secretase complex. Rather, GSK-3.alpha.
appears to regulate .gamma.-secretase activity toward specific
substrates or access of these substrates to the .gamma.-secretase
complex.
[0039] Recently, a subset of non-steroidal anti-inflammatory drugs
(NSAIDs) has also been shown to reduce A.beta..sub.42 levels
without affecting Notch cleavage (Weggen et al., Nature 414:212-216
(2001), see also U.S. Pat. No. 6,160,018 suggesting lithium as a
possible counterions in the generation of an enantiomeric form of a
NSAID for the treatment of Alzheimer's disease). While the target
of these NSAIDs has not been determined in this context, the
mechanisms of lithium and NSAIDs appear to differ, as NSAIDs shift
.gamma.-secretase cleavage of APP to increase A.beta..sub.38 at the
expense of A.beta..sub.42, while lithium inhibits .gamma.-secretase
cleavage, reducing A.beta..sub.40 and A.beta..sub.42. This apparent
difference in mechanism suggests that combination therapy of the
lithium as used in the present invention with an NSAID could have
an enhanced effect in reducing A.beta.peptide accumulation, and
also in the production of GSK-3.alpha..
[0040] Both GSK-3.alpha. and GSK-3.beta. phosphorylate tau protein,
which is a microtubule-binding protein that, in its
hyperphosphorylated state, is the major component of
neurofibrillary tangles (Alvarez et al., Bipolar Disord. 4:153-165
(2002)). Lithium inhibits GSK-3 mediated tau phosphorylation (Phiel
et al., 2001). However, that function is not the focus of the
present treatment of Alzheimer's disease. Rather by focussing on
GSK-3.alpha. as a target, the present use of a GSK-3.alpha.
inhibitor, e.g., lithium, provides an unforeseen method for
reducing the formation of amyloid plaques and neurofibrillary
tangles, the two primary pathological features of Alzheimer's
disease. Lithium has also been demonstrated to protect neurons from
proapoptotic stimuli, and thus, may also reduce neuronal cell death
associated with Alzheimer's (reviewed in Alvarez et al., 2002).
[0041] Lithium has been used for more than fifty years to treat
bipolar disorder, but has a narrow therapeutic window and a higher
frequency of side effects in older patients. Thus, while lithium
might be considered to prevent progression of AD symptoms,
especially in younger patients carrying FAD mutations or Down's
syndrome patients, new agents that specifically target GSK-3.alpha.
may prove valuable in the treatment of AD.
[0042] The brains of individuals with AD exhibit neuronal
degeneration and characteristic lesions variously referred to as
amyloidogenic plaques, vascular amyloid angiopathy, and
neurofibrillary tangles. Large numbers of these lesions,
particularly amyloidogenic plaques and neurofibrillary tangles, are
generally found in several areas of the human brain important for
memory and cognitive function in patients with AD. Smaller numbers
of these lesions in a more restricted anatomical distribution are
found in the brains of most aged humans who do not have clinical
AD, as well as patients suffering from Down's Syndrome and
Hereditary Cerebral Hemorrhage with Amyloidosis of the
Dutch-Type.
[0043] The term "subject" is used interchangeably herein with
"patient" and is intended to include living organisms in which
Alzheimer's disease, or any recognized condition that may be
related thereto, may develop, and in which lithium provides a
treatment therefor in accordance with the present invention, e.g.,
preferably mammals, most preferably humans. Examples of subjects
include humans, dogs, cats, mice, rats, and transgenic species
thereof. For example, animals within the scope of the invention
include animals of agricultural interest, such as livestock and
fowl.
[0044] For practicing the methods of the invention, particularly in
vivo, the lithium or lithium salt compositions and carriers
therefor, are administered to the subjects in a biologically
compatible form suitable for pharmaceutical administration in vivo.
By "biologically compatible form suitable for administration in
vivo" is meant, e.g., lithium or lithium salts prepared as
described herein, to be administered in any circumstance in which
any toxic effects are outweighed by the therapeutic effects of the
treatment. Also included are compositions, such as lithium, that
act intracellularly to inhibit or reduce levels of a subject's
formation of amyloid plaques and neurofibrillary tangles or
accumulation of A.beta. peptide levels, particularly A.beta..sub.40
and A.beta..sub.42 peptides, or GSK-3.alpha. levels, or the
production or processing of APP. Moreover, as will be understood by
those skilled in the art, "bioavailable" or "biocompatible," as
used herein, means that a particular element or compound such as
lithium is, for example by its particular oxidation state or the
components with which it is complexed, in a form which allows for
the element or compound to be absorbed, incorporated or be
otherwise physiologically available to the individual to whom it is
administered. Any bioavailable sources are contemplated for use in
the practice of the present invention. When lithium is administered
to a subject, lithium salts are preferred.
[0045] The methods are also useful for research purposes, wherein
GSK-3 inhibitors, such as lithium or any other GSK-3 inhibitor, and
carriers therefor are administered to brain tissue or cells of any
species in vitro. Such treated cells or tissues may also be
returned to the subject or another subject of any species, in which
case the applications are acceptably used ex vivo.
[0046] Also embodied in the invention are methods of treating a
GSK-3.alpha.-related disorder in an animal, wherein the methods
comprise administering to the animal (or to the brain, brains cells
or brain tissue of an animal) a GSK-3.alpha. inhibitor suspended in
a pharmaceutically acceptable carrier. The GSK-3.alpha. related
disorder, which is treated according to the method of the
invention, may be any disorder mediated by GSK-3.alpha. activity,
but is preferably Alzheimer's disease. Thus, the invention is a
method for treating a biological condition mediated by GSK-3.alpha.
activity by administering an effective amount of a pharmaceutical
composition comprising a selective GSK-3.alpha. inhibitor to a
subject having a condition mediated by GSK-3.alpha. activity or
susceptible to such a condition, e.g., Alzheimer's disease. The
selective inhibitor of GSK-3.alpha.activity, includes lithium, or a
pharmaceutically acceptable salt thereof, and a physiologically
acceptable carrier.
[0047] The invention is also embodied by an in vitro method of
identifying an inhibitor of GSK-3.alpha. kinase activity, and
includes a pharmaceutical composition comprising an inhibitor
identified by this in vitro method.
[0048] In yet another embodiment, the invention provides methods
for the treatment of a subject having Alzheimer's disease, wherein
the methods comprise administering to the subject (or to the brains
cells or tissue thereof) an effective amount of an inhibitor of
GSK-3, such as lithium, specifically to reduce processing of
amyloid precursor proteins to beta-amyloid (A.beta.) peptides, and
thus to prevent, inhibit or reverse Alzheimer's disease. Moreover,
this effect of lithium is mediated through the inhibition of
GSK-3.alpha.. Because the .alpha. isoform of GSK-3 (GSK-3.alpha.)
has been found herein to be specifically responsible for APP
processing, in another embodiment of this invention, agents that
specifically target GSK-3.alpha. are especially useful in the
treatment, prevention, and possible reversal of Alzheimer's
disease. Further provided are methods for treating a condition
mediated by GSK-3.alpha. activity by administering a selective
inhibitor of GSK-3.alpha., which is preferably a therapeutically
effective amount of a composition that specifically inhibits
GSK-3.alpha., but does not inhibit or block GSK-3.beta. or other
lithium sensitive enzymes. In an embodiment of the invention there
are provided one or more bioavailable sources of the GSK-3.alpha.)
inhibitor, such as lithium, or a pharmaceutically acceptable salt
thereof, and a physiologically acceptable carrier.
[0049] Also embodied are methods for treating in a subject
Alzheimer's disease, or the conditions relating thereto, wherein
the methods comprise administering to the subject (or to the brains
cells or tissue thereof) an Alzheimer's disease-reducing or
disease-inhibiting amount of an inhibitor of GSK-3, such as
lithium, and a physiologically acceptable carrier. Also provided
are methods for treating, inhibiting or reducing Alzheimer's
disease related conditions, such as the formation of amyloid
plaques and neurofibrillary tangles or accumulation of A.beta.
peptides (particularly A.beta..sub.40 and A.beta..sub.42 peptides)
in the brain or in brain cells or tissue, as well as methods for
preventing or inhibiting the .gamma.-secretase cleavage of APP in
the brain.
[0050] In yet another embodiment of the invention, there are
further provided methods for stabilizing a subject susceptible to
Alzheimer's disease or the formation of amyloid plaques and
neurofibrillary tangles or accumulated A.beta. peptides
(particularly A.beta..sub.40 and A.beta..sub.42 peptides) in the
brain, thereby also stopping or inhibiting the .gamma.-secretase
cleavage of APP, wherein the methods comprise administering to the
subject (or to the brains cells or tissue thereof) a stabilizing
amount of an inhibitor of GSK-3, such as lithium and a
physiologically acceptable carrier.
[0051] In yet another embodiment, there are provided methods for
treating a subject susceptible to Alzheimer's disease or the
formation of amyloid plaques and neurofibrillary tangles or
accumulated A.beta. peptides (particularly A.beta..sub.40 and
A.beta..sub.42 peptides) in the brain, thereby also preventing the
.gamma.-secretase cleavage of APP in the brain, wherein the methods
comprise administering to a subject (or to the brains cells or
tissue thereof) a preventing amount of an inhibitor of GSK-3, such
as lithium and a physiologically acceptable carrier.
[0052] Also embodied in the invention is a composition that
inhibits GSK-3.alpha. activity in vivo, such as a pharmaceutically
acceptable lithium composition. All combinations, sources and
amounts of the active ingredients discussed herein in conjunction
with the compositions of the present invention are also
contemplated as being administered in accordance with the foregoing
methods.
[0053] The invention also is embodied by a kit for inhibiting
glycogen synthase kinase 3.alpha. activity, preferably in vivo. The
kit comprises the GSK-3.alpha. activity-inhibiting composition
described above, such as lithium, and an instructional material.
The instructional material can, for example, be one selected from
the group consisting of an instructional material that describes
administration of the composition to an animal in order to inhibit
GSK-3.alpha. activity, or an instructional material that describes
administration of the composition to an animal in order to
alleviate a disorder known to be alleviated by administration of
the GSK-3.alpha. inhibitor, such as lithium.
[0054] Preferably the foregoing methods further comprise monitoring
such subject's A.beta. peptide levels, particularly A.beta..sub.40
and A.beta..sub.42 peptides, or GSK-3.alpha. levels, or the
.gamma.-secretase cleavage of APP.
[0055] Administration of an "effective amount" or a
"therapeutically effective amount" of a GSK-3 inhibitor or
GSK-3.alpha. inhibitor, respectively, of the present invention is
defined as an amount that is useful, at dosages and for periods of
time necessary to achieve the desired result. For example, a
therapeutically effective amount of a GSK-3 inhibitor or
GSK-3.alpha. inhibitor (also referred to as a "therapeutically
effective inhibitor composition") in accordance with the present
invention may vary according to factors, such as the disease state,
age, sex, and weight of the subject, and the ability of the agent
to elicit a desired response particularly to Alzheimer's disease in
the subject. Dosage regimens of a GSK-3 inhibitor or GSK-3.alpha.
inhibitor, such as lithium, in the patient may be adjusted to
provide the optimum therapeutic response. For example, several
divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0056] "Pharmaceutically acceptable GSK-3 inhibitor compositions,"
or "pharmaceutically acceptable GSK-3.alpha. inhibitor
compositions," "pharmaceutical inhibitor compositions" or simply
"pharmaceutical compositions" contemplated for use in the practice
of the present invention (such as those comprising a GSK-3
inhibitor or GSK-3.alpha. inhibitor, respectively) refer to those
compositions that are not harmful to a subject when administered in
vivo, and which when administered in therapeutically effective
amounts or concentrations are sufficient to inhibit, block, or even
reverse, A.beta. peptide accumulation or undesirable APP
processing, respectively. "Pharmaceutically acceptable lithium
salt(s)," refer to lithium salts prepared from pharmaceutically
acceptable, non-toxic acids or bases. The active compounds
contemplated for use herein, include pharmaceutical compositions or
other compounds in an amount sufficient to produce the desired
preventive, inhibitory or reversing effect upon Alzheimer's
disease, or processes or conditions related thereto, or to inhibit
or block A.beta. peptide accumulation or GSK-3.alpha. activity
relating to .gamma.-secretase cleavage of APP. The administration
to the patient of such compositions can be in the form of a solid,
a solution, an emulsion, a dispersion, a micelle, a liposome, and
the like, wherein the resulting GSK-3 inhibitor composition
contains one or more of the active compounds contemplated for use
herein, as active ingredients thereof, in admixture with an organic
or inorganic carrier or excipient suitable for nasal, enteral,
oral, inhalation, or transdermal applications (see, e.g., U.S. Pat.
Nos. 6,375,990 or 6,335,034), or parenteral applications, or
osmotic pump, or vaginal, rectal or ophthalmic administration, for
example, as such methods may already be administered in the
treatment of depression.
[0057] The term "pharmaceutically acceptable carrier" means a
chemical composition with which a pharmaceutically active agent can
be combined and which, following the combination, can be used to
administer the agent to a subject (e.g., a mammal, such as a
human). The term "physiologically acceptable" ester or salt means
an ester or salt form of a pharmaceutically active agent which is
compatible with any other ingredients of the pharmaceutical
composition and which is not deleterious to the subject to which
the composition is to be administered. In the active GSK-3
inhibitor, such as lithium, ingredients may be compounded, for
example, with the usual non-toxic, pharmaceutically and
physiologically acceptable carriers for tablets, pellets, capsules,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, suppositories, solutions, emulsions, suspensions, hard
or soft capsules, caplets or syrups or elixirs and any other form
suitable for use.
[0058] Oral administration is a preferred route of administration
of the GSK-3 inhibitor, such as lithium composition, or
GSK-3.alpha. activity-inhibiting or -blocking composition of the
present invention. A formulation of a pharmaceutical composition of
the invention suitable for oral administration may be prepared,
packaged, or sold in the form of a discrete solid dose unit
including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche, or a lozenge, each containing a predetermined
amount of the active ingredient. Other formulations suitable for
oral administration include, but are not limited to, a powdered or
granular formulation, an aqueous or oily suspension, an aqueous or
oily solution, or an emulsion.
[0059] As used herein, an "oily" liquid is one which comprises a
carbon-containing liquid molecule and which exhibits a less polar
character than water.
[0060] A tablet comprising the active ingredient may, for example,
be made by compressing or molding the active ingredient, optionally
with one or more additional ingredients. Compressed tablets may be
prepared by compressing, in a suitable device, the active
ingredient in a free-flowing form such as a powder or granular
preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing
agent. Molded tablets may be made by molding, in a suitable device,
a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at least sufficient liquid to moisten the mixture.
[0061] In addition, such compositions may contain one or more
agents selected from flavoring agents (such as peppermint, oil of
wintergreen or cherry) to create an acceptable or a pleasant taste
for optimal patient compliance, coloring agents, preserving agents,
and the like, in order to provide pharmaceutically elegant and
palatable preparations.
[0062] The carriers that can be used include, e.g., glucose,
lactose, gum acacia, gelatin, mannitol, starch paste, magnesium
trisilicate, talc, corn starch, keratin, colloidal silica, potato
starch, urea, medium chain length triglycerides, dextrans, and
other carriers suitable for use in manufacturing preparations, in
solid, semisolid, or liquid form. In addition auxiliary,
stabilizing, thickening and coloring agents may be used.
[0063] Tablets containing the active ingredients in admixture with
non-toxic pharmaceutically acceptable excipients may also be
manufactured by known methods. Pharmaceutically acceptable
excipients used in the manufacture of tablets include, but are not
limited to, inert diluents, granulating and disintegrating agents,
binding agents, and lubricating agents. The excipients used may be,
for example, (1) inert diluents, such as calcium carbonate,
lactose, calcium phosphate, sodium phosphate, and the like; (2)
granulating and disintegrating agents, such as corn starch, potato
starch, alginic acid, and the like; (3) binding agents, such as gum
tragacanth, corn starch, gelatin, acacia, and the like; and (4)
lubricating agents, such as magnesium stearate, stearic acid, talc,
and the like.
[0064] Known dispersing agents include, but are not limited to,
potato starch and sodium starch glycolate. Known surface active
agents include, but are not limited to, sodium lauryl sulfate.
Known diluents include, but are not limited to, calcium carbonate,
sodium carbonate, lactose, microcrystalline cellulose, calcium
phosphate, calcium hydrogen phosphate, and sodium phosphate. Known
granulating and disintegrating agents include, but are not limited
to, corn starch and alginic acid. Known binding agents include, but
are not limited to, gelatin, acacia, pre-gelatinized maize starch,
polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known
lubricating agents include, but are not limited to, magnesium
stearate, stearic acid, silica, and talc.
[0065] The tablets may be uncoated, or they may be preferably
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract, thereby providing sustained action
over a longer period. For example, a time delay material, such as
glyceryl monostearate or glyceryl distearate may be employed. The
tablets may also be coated by the techniques described in the U.S.
Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form osmotic
therapeutic tablets for controlled release.
[0066] Oral compositions may be made, using known technology, which
specifically release orally-administered agents in the small or
large intestines of a human patient. For example, formulations for
delivery to the gastrointestinal system, including the colon,
include enteric coated systems, based, e.g., on methacrylate
copolymers such as poly(methacrylic acid, methyl methacrylate),
which are only soluble at pH 6 and above, so that the polymer only
begins to dissolve on entry into the small intestine. The site
where such polymer formulations disintegrate is dependent on the
rate of intestinal transit and the amount of polymer present. For
example, a relatively thick polymer coating is used for delivery to
the proximal colon (Hardy et al., Aliment. Pharmacol. Therap.
1:273-280 (1987)). Polymers capable of providing site-specific
colonic delivery can also be used, wherein the polymer relies on
the bacterial flora of the large bowel to provide enzymatic
degradation of the polymer coat, and hence, release of the
drug.
[0067] When formulations for oral use are in the form of hard
gelatin capsules, the active ingredients may be mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate,
kaolin, or the like (see, e.g., U.S. Pat. No. 6,517,859). They may
also be in the form of soft gelatin capsules, wherein the active
ingredients are mixed with water or an oil medium, for example,
peanut oil, liquid paraffin, olive oil and the like.
[0068] Pulsed release technology, such as that described in U.S.
Pat. No. 4,777,049, may also be used to administer the active agent
to a specific location within the gastrointestinal tract. Such
systems permit drug delivery at a predetermined time and can be
used to deliver the active agent, optionally together with other
additives that my alter the local microenvironment to promote agent
stability and uptake, directly to the colon, without relying on
external conditions other than the presence of water to provide in
vivo release.
[0069] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be
prepared, packaged, and sold either in liquid form or in the form
of a dry product intended for reconstitution with water or another
suitable vehicle prior to use.
[0070] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, subcutaneous, intraperitoneal, intravenous,
intraarterial, intramuscular, or intrasternal injection and
intravenous, intraarterial, or kidney dialytic infusion
techniques.
[0071] The pharmaceutical compositions may also be in the form of a
sterile injectable suspension. Such a suspension may be formulated
according to known methods, using sterile aqueous solutions (where
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersion. Suitable dispersing or wetting agents and suspending
agents may be used. The composition must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The sterile injectable
preparation may also be a sterile injectable solution or suspension
in a non-toxic parenterally-acceptable diluent or solvent, for
example, as a solution in 1,4-butanediol. Sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil may be employed including synthetic
mono- or diglycerides, fatty acids (including oleic acid),
polyunsaturated fatty acids (such as dihomo-gamma-linolenic acid,
gamma-linolenic acid and linoleic acid), naturally occurring
vegetable oils or synthetic fatty vehicles like ethyl oleate or the
like (see, e.g., U.S. Pat. No. 5,252,333 teaching pharmaceutical
compositions containing lithium salts of C.sub.18-22
polyunsaturated fatty acids adapted for the treatment of
Alzheimer's disease). Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent that delays absorption, for example, aluminum monostearate
or gelatin. Buffers, preservatives, antioxidants, and the like can
be incorporated as required.
[0072] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations, such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient may be as high as the
solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0073] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles, wherein at least 98% of the particles
by weight have a diameter greater than 0.5 nanometers and at least
95% of the particles by number have a diameter less than 7
nanometers. More preferably, at least 95% of the particles by
weight have a diameter greater than 1 nanometer and at least 90% of
the particles by number have a diameter less than 6 nanometers. Dry
powder compositions preferably include a solid fine powder diluent
such as sugar and are conveniently provided in a unit dose
form.
[0074] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0075] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient, and may conveniently be administered using any
nebulizing or atomizing device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers.
[0076] The formulations described herein as being useful for
pulmonary delivery are also useful for intranasal delivery of a
pharmaceutical composition of the invention. Another formulation
suitable for intranasal administration is a coarse powder
comprising the active ingredient and having an average particle
from about 0.2 to 500 micrometers. Such a formulation is
administered in the manner in which snuff is taken, i.e., by rapid
inhalation through the nasal passage from a container of the powder
held close to the nares.
[0077] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of the active ingredient, and may further comprise one
or more of the additional ingredients described herein.
[0078] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, 0.1 to 20% (w/w) active ingredient, the balance
comprising an orally dissolvable or degradable composition and,
optionally, one or more of the additional ingredients described
herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0079] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1-1.0% (w/w)
solution or suspension of the active ingredient in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other ophthalmically-administrable formulations
include those comprising the active ingredient in microcrystalline
form or in a liposomal preparation.
[0080] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for vaginal
administration. Such a composition may be in the form of, for
example, a suppository, an impregnated or coated
vaginally-insertable material, such as a tampon, a douche
preparation, or a solution for vaginal irrigation. Methods for
impregnating or coating a material with a chemical composition are
known in the art, and include, but are not limited to methods of
depositing or binding a chemical composition onto a surface,
methods of incorporating a chemical composition into the structure
of a material during the synthesis of the material (i.e., such as
with a physiologically degradable material), and methods of
absorbing an aqueous or oily solution or suspension into an
absorbent material, with or without subsequent drying. Douche
preparations or solutions for vaginal irrigation may be made by
combining the active ingredient with a pharmaceutically acceptable
liquid carrier. As is well known in the art, such preparations may
be administered using, and may be packaged within, a delivery
device adapted to the vaginal anatomy of the subject. Such
preparations may further comprise various additional ingredients
including, but not limited to, antioxidants, antibiotics,
antifungal agents, and preservatives.
[0081] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for rectal
administration. Such a composition may be in the form of, for
example, a suppository, a retention enema preparation, and a
solution for rectal or colonic irrigation. These compositions may
be prepared by mixing the active ingredients with a suitable
non-irritating excipient, such as cocoa butter, synthetic glyceride
esters of polyethylene glycols (which are solid at ordinary
temperatures, but which liquefy and/or dissolve in the rectal
cavity to release the active ingredients), and the like.
[0082] In addition, sustained release systems, including
semi-permeable polymer matrices in the form of shaped articles
(e.g., films or microcapsules) can also be used for the
administration of the active compound employed herein.
[0083] As will be appreciated by those of skill in the art,
Alzheimer's disease presents a complicated array of conditions and
symptoms. Because of the inter-relatedness of these conditions and
symptoms, invention compositions are useful in treating many of
them. In addition, there are a number of precursor conditions which
portend the development of Alzheimer's disease and which can be
treated by administration of compositions as described herein.
Therefore, in accordance with another aspect of the present
invention, there are provided methods of using GSK-3 inhibitors,
such as lithium or other such inhibitors, for reducing or
minimizing in the brain or brain tissue the formation of amyloid
plaques and neurofibrillary tangles or accumulated A.beta.
peptides, for blocking or inhibiting production of A.beta..sub.40
and A.beta..sub.42 peptides by interfering with .gamma.-secretase
cleavage of APP, and for specifically blocking or inhibiting
GSK-3.alpha. activity, which is specifically required for maximal
processing of APP, thereby reducing the dosage of other
anti-Alzheimer's disease agents that the subject may be taking.
Thus, the general well-being of the Alzheimer's disease patient is
in general improved, wherein the methods comprise administration of
compositions as described herein.
[0084] Since individual subjects may present a wide variation in
severity of symptoms and each active ingredient has its unique
therapeutic characteristics, it is up to the practitioner to
determine a subject's response to treatment and vary the dosages of
the active ingredients accordingly. It is especially advantageous
to formulate compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on the unique characteristics of the composition or salt prepared
in accordance with the present invention and the particular
therapeutic effect to be achieved.
[0085] A pharmaceutical composition of the invention may be
administered to deliver a dose of between 500 picograms per
kilogram body weight per day and 1 milligrams per kilogram body
weight per day to a subject. However, lithium should generally not
be administered to patients having significant renal or
cardiovascular disease, severe debilitation or dehydration, sodium
depletion, or to patients receiving diuretics, since the risk of
lithium toxicity can be high in such patients (Physicians Desk
Reference, (1997) pp. 2352), as are numerous other side effects
(detailed in the Physicians Desk Reference (1997) pp. 2352, 2658),
although the mechanism(s) by which lithium exerts these diverse
effects are unclear.
[0086] It is understood that the ordinarily skilled physician or
veterinarian will readily determine and prescribe an effective
amount of the compound to alleviate a disorder associated with
aberrant GSK-3.alpha. activity in the subject. In so proceeding,
the physician or veterinarian may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. It is further
understood, however, that the specific dose level for any
particular subject will depend upon a variety of factors including
the activity of the specific compound employed, the age, body
weight, general health, gender, and diet of the subject, the time
of administration, the route of administration, the rate of
excretion, any drug combination, and the severity of the disorder
being treated. Since lithium compounds have be well-tested in the
treatment of human patients suffering from depression, dosage
amounts and safety concerns are already known, or can be readily
determined, without undue experimentation by those skilled in the
art of treating patients (see also Phiel et al., 2001; U.S. Pat.
No. 4,556,068).
[0087] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed., 1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference.
[0088] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0089] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient. A unit dose of a pharmaceutical composition of the
invention generally comprises from about 1 nanogram to about 1 gram
of the active ingredient, and preferably comprises from about 50
nanograms to about 10 milligrams of the active ingredient.
[0090] In addition to the active GSK-3 inhibitor or
GSK-3.alpha.-specific inhibitor component, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents. Particularly
contemplated additional agents include virus particles comprising
one or more polypeptides or polynucleotide(s) encoding such a
polypeptide. The polypeptides can also be administered as fusion
proteins, such as proteins that would facilitate entry into
cells.
[0091] Another embodiment of the invention relates to a kit
comprising a pharmaceutical composition of the invention and an
instructional material. As used herein, an "instructional material"
includes a publication, a recording, a diagram, or any other medium
of expression used to communicate the usefulness of the
pharmaceutical composition of the invention for inhibiting GSK-3 or
GSK-3.alpha.-specific activity in a subject. The instructional
material may also, for example, describe an appropriate dose of the
pharmaceutical composition of the invention. The instructional
material of the kit of the invention may, for example, be affixed
to a container containing a pharmaceutical composition of the
invention or be shipped together with a container containing the
pharmaceutical composition. Alternatively, the instructional
material may be shipped separately from the container with the
intention that the instructional material and the pharmaceutical
composition be used cooperatively by the recipient.
[0092] The invention is further embodied by a kit comprising a
pharmaceutical composition of the invention and a delivery device
for delivering the GSK-3.alpha. inhibiting composition to a
subject. By way of example, the delivery device may be a squeezable
spray bottle, a metered-dose spray bottle, an aerosol spray device,
an atomizer, a dry powder delivery device, a self-propelling
solvent/powder-dispensing device, a syringe, a needle, a tampon, or
a dosage-measuring container. The kit may further comprise an
instructional material as described herein.
[0093] The invention includes transgenic (preferably non-human)
animals, which comprise a transgene encoding a polypeptide
GSK-3.alpha. inhibiting composition described in this disclosure.
The polypeptide is able to interact with GSK-3.alpha., and inhibit
GSK-3.alpha. activity, thereby preventing or inhibiting normal tau
phosphorylation associated with GSK-3.alpha.. Thus, expression of
the transgene can mimic the effect of GSK-3 inhibitor
administration in the animal. The transgene preferably comprises a
promoter from which initiation of transcription can be controlled.
Numerous examples of controllable promoters are known in the art,
and include inducible promoters, repressible promoters,
temperature-sensitive promoters, and tissue-specific promoters. A
preferred promoter is the calcium-calmodulin dependent protein
kinase II alpha (CaMKIIalpha) promoter. Expression of polypeptide s
operably linked with this promoter sequence is generally limited to
adult neurons of the forebrain, including neurons of the neocortex,
the hypothalamus, the amygdala, and the basal ganglia. The
transgenic animal can be of any species for which transgenic
generation methods are known (i.e., including at least mammals such
as cows, goats, pigs, sheep, and rodents such as rats and
mice).
[0094] The present invention is further described by example. These
examples are provided for purposes of illustration only, and are
not intended to be limiting unless otherwise specified. The various
scenarios are relevant for many practical situations, and are
intended to be merely exemplary to those skilled in the art. These
examples are not to be construed as limiting the scope of the
appended claims, rather such claims should be construed to
encompass any and all variations that become evident as a result of
the teachings provided herein.
EXAMPLES
Example 1
Role of GSK-3 and Effect of Lithium on Production of A.beta..sub.40
and A.beta..sub.42 Peptides
[0095] To investigate the role of GSK-3 in the production of
peptides A.beta..sub.40 and A.beta..sub.42, Chinese hamster ovary
(CHO) cells stably expressing APP (CHO-APP.sub.695) were treated
with lithium chloride (LiCl), which is a direct inhibitor of
GSK-3.alpha. and 13 (Phiel et al., 2001). CHO.sub.695 cells
(available commercially, e.g., American Type Culture Collection
(ATCC), Manassas, Va.) were maintained in culture in MEM.alpha.+5%
fetal bovine serum (FBS) (e.g., BioWhittaker, Walkersville, Md.)
with added penicillin/streptomycin and glutamine. Stocks of lithium
chloride were prepared in sterile water. To quantify the
A.beta..sub.40 and A.beta..sub.42 secretion following treatment
with LiCl, the CHO-APP.sub.695 cells were plated in 6-well dishes
at a density of 5.times.10.sup.5 cells per well. LiCl was added to
the cells in fresh medium, and media and cells were collected 24
hours later. A.beta. determinations from the media were made by
sandwich ELISA in femtomoles using the method of Suzuki et al.,
Science 264:1336-1340 (1994).
[0096] Notably, although CHO cells were used, one is not limited to
such cells. CHO cells are acceptable models for human cellular
responses (Sahasrabudhe et al., J. Biol. Cell 267:25602-25608
(1992)). For example, human cells overexpressing APP (293Ts for
example) could be used. In fact, it is shown herein that lithium
reduces A.beta. levels in neurons derived from human embryonic
carcinoma cells (NTera2/D1 or NT2 cell line from which mouse and
human primary neurons were derived). CHO.sub.695 cells were chosen
because they were readily available and generate A.beta. peptides
at levels that are sufficient for the detection methods used.
[0097] To visualize the APP fragments, CHO.sub.695 cells plated on
6-well dishes were methionine-deprived for 30 minutes by incubation
in methionine-free DMEM (Life Technologies, Inc., Grand Island,
N.Y.) before adding 500 .mu.Ci .sup.35S-methionine (Perkin Elmer
Life Sciences, Inc., Boston Mass.) per ml of DMEM with 5% dialyzed
FBS (Invitrogen) and mg/ml L-proline for 2.5 hours. Medium was
collected and cells (2 wells/sample) were rinsed twice with PBS
(phosphate buffered saline), then scraped into RIPA buffer (a
standard immunoprecipitation buffer, see, Harlow and Lane (eds.),
In Antibodies: a laboratory manual, Cold Spring Harbor Laboratory
Press, NY (1988)). Lysates were sonicated and centrifuged at
100,000.times.g for 20 minutes. Using protein A/G agarose beads
(Santa Cruz Biotechnologies, Santa Cruz, Calif.), media was
immunoprecipitated with the antibody BAN-50 (Suzuki et al., 1994)
that recognizes A.beta. amino acids 1-10, and cell lysates were
immunoprecipitated with antibody 2493 (Lee et al., J. Biol. Chem.
278:4458-2466 (2003)), which is a rabbit polyclonal antibody that
recognizes the C-terminus of APP. Proteins were resolved by
electrophoresis on 10/16% step gradient Tris-tricine polyacrylamide
gels. Gels were fixed in 50% methanol+5% glycerol, dried, and
exposed to a PhosphorImager screen.
[0098] For comparison purposes, the results were normalized to
levels of intracellular full-length APP. APP levels were quantified
by SDS-PAGE followed by western immunoblotting with the
amino-terminal APP antibody Karen (Turner et al., J. Biol. Chem.
271:8966-8970 (1996)), and visualized using .sup.125I-labelled
secondary antibodies by PhosphorImager analysis and ImageQuant
software (Amersham Biosciences Corp, Piscataway, N.J.).
[0099] LiCl robustly inhibited production of both A.beta..sub.40
and A.beta..sub.42, with an IC.sub.50 between 1-2 mM, well within
the therapeutic range of lithium for bipolar disorder. Meanwhile,
sodium chloride (NaCl) was found to have no effect on APP
processing (FIG. 1a), which is consistent with a recent report
using transient overexpression of the APP carboxyl terminus C100 in
COST cells (Sun et al., Neurosci. Lett. 321:61-64 (2002)).
Example 2
Lithium Inhibits A.beta..sub.40 and A.beta..sub.42 Production at
the Level of .gamma.-secretase
[0100] To confirm the effect of lithium on the level of APP
peptides, the accumulation of APP processing intermediates was
measured in the presence of LiCl. The cleavage of APP by .alpha. or
.beta.-secretase generates APP C terminal fragments (CTFs), which
are then further cleaved by .gamma.-secretase. If .gamma.-secretase
is inhibited, for example with a known inhibitor of
.gamma.-secretase activity, DFK-167, then APP CTFs accumulate (see,
Wolfe et al., 1999).
[0101] The reduction of A.beta. was confirmed by the metabolic
.sup.35S-methionine labeling of CHO-APP.sub.695 cells in either the
presence or absence of LiCl. CHO-APP.sub.695 cells were maintained,
cultured and visualized as described in Example 1, as were the LiCl
treatments. Stocks of the .gamma.-secretase inhibitor, DFK-167
(Enzyme Systems Products, Livermore, Calif.) were prepared in DMSO
(see, Wolfe et al., J. Med. Chem. 41:6-9 (1998)).
[0102] CHO-APP.sub.695 cells were treated with 5 mM LiCl or with
the, 100 .mu.M DFK-167 for 24 hours. At the end of the 24 hour
exposure, the APP fragments were .sup.35S-methionine labeled and
treated as in Example 1. The labeled A.beta. secreted from
CHO-APP.sub.695 cells was immunoprecipitated from the medium, then
SDS-PAGE separated and PhosphorImager visualized. Control cells
were similarly handled, but without treatment with LiCl or DFK-167.
Both LiCl and DFK-167 were found to dramatically reduce the
steady-state levels of secreted A.beta. peptides (see, FIG. 1b,
lanes 2 and 3 respectively).
[0103] Accordingly, lithium did not affect steady-state levels of
APP (FIG. 1c) or the levels of the N- and C-terminal fragments of
PS1 (data not shown), nor did it interfere with the detection of
A.beta. peptides (not shown). Lithium, therefore, functionally
reduced the level of A.beta. peptides, apparently at a
post-translational step, such as APP processing or A.beta.
stability.
[0104] To confirm that lithium inhibits A.beta..sub.40 and
A.beta..sub.42 production at the level of .gamma.-secretase,
CHO-APP.sub.695 cells were treated, as above, with 5 mM LiCl or 100
.mu.M DFK-167 for 24 hours. The intracellular APP from the
CHO-APP.sub.695 cells was immunoprecipitated from the medium, then
SDS-PAGE separated. APP holoprotein and APP C-terminal fragments
(C99, C89, and C83) were PhosphorImager visualized (see, FIG.
1c).
[0105] As shown in FIG. 1c, exposure of CHO-APP.sub.695 cells to
lithium for 24 hours caused an accumulation of APP CTFs similar to
the effect shown as a result of treatment with DFK-167. Thus, the
accumulation of the APP CTFs demonstrates that lithium inhibits
A.beta..sub.40 and A.beta..sub.42 production at the level of
.gamma.-secretase.
Example 3
Lithium is Neither an Inhibitor of Notch Processing or
.gamma.-Secretase
[0106] The .gamma.-secretase activity is also required for the
release of the Notch intracellular domain (NICD) (De Strooper et
al., Nature 398:518-522 (1999)). Since lithium inhibits APP
processing at the level of .gamma.-secretase, the effects of
lithium were examined on Notch processing. .DELTA.E-Notch has been
constructed to lack most of its extracellular domain, but it
retains the transmembrane domain containing the .gamma.-secretase
cleavage site. Thus, it is constitutively cleaved (Schroeter et
al., Nature 393:382-386 (1998)).
[0107] CHO-APP.sub.695 cells were plated at a density of
1.times.10.sup.5 cells per well of 6-well dishes. Cells were
transfected with 2 .mu.g of either Notch-ICV or Notch-.DELTA.E as
indicated in FIG. 1d, see lanes 4-11. Notch .DELTA.E in pCS2MT and
Notch ICV in pCS2MT were kindly provided by R. Kopan (published
construct). Notch .DELTA.E V 1744K in pCS2MT was created by
site-directed mutagenesis using a QuikChange.TM. mutagenesis kit
(Stratagene, La Jolla, Calif.) and confirmed by sequencing APP-WT
and APP-Swedish (KM670/671NL) in pSFV as described previously
(Forman et al., J. Biol. Chem. 272:32247-32253 (1997)).
[0108] Twenty four (24) hours later, media was changed and drugs
were added--either lithium (ranging 0.5 mM to 5 mM LiCl) or DFK-167
(ranging 10 .mu.M to 50 .mu.M) and allowed o remain for 24 hours.
After exposure to the drugs, the cells were harvested as described
above, and immunoblotted with either increasing concentrations of
myc (9E10) antibody or cleaved Notch antibody that recognizes the
cleavage product (Cell Signaling Technologies, Beverly, Mass.) (see
concentrations shown in FIG. 1d). The myc 9E10 antibody only
recognizes Notch 1 processed at the .gamma.-secretase cleavage site
(between residues 1743-1744 in full-length Notch). Notch
intracellular domain (NICD), beginning at residue 1744 of
full-length Notch, was used as a positive control for cleavage
(FIG. 1d, lane 2). .DELTA.E-Notch V1744K (see, Schroeter et al.,
1998) has a point mutation in the .gamma.-secretase cleavage site
of .DELTA.E-Notch, and accordingly was used as a negative control
for cleavage (FIG. 1d, lane 3). All Notch constructs had a
C-terminal myc tag. Neither the overexpression of GSK-3.alpha. or
GSK-3.beta. had any effect on Notch cleavage (data not shown).
[0109] Lithium at the studied concentrations did not inhibit Notch
processing as assessed by western blotting. .DELTA.E-V1744K was not
processed because of the point mutation at the .gamma.-secretase
cleavage site, and therefore, is not detected with the antibody
specific for cleaved Notch (FIG. 1d, lane 3). Thus, lithium was
shown not to be a general inhibitor of .gamma.-secretase under
these conditions.
Example 4
Lithium Reduces A.beta. Production because it Inhibits GSK-3
[0110] While lithium is a direct and highly selective inhibitor of
GSK-3 (Phiel et al., Annu. Rev. Pharmacol. Toxicol. 41:789-813
(2001)), it also inhibits inositol monophosphatase (IMPase), as
well as structurally related phosphomonoesterases, and
phosphoglucomutase (Phiel et al., 2001). To confirm that lithium
reduces A.beta. production through inhibition of GSK-3, rather than
a reduction resulting from inhibition of IMPase, or the
structurally related phosphomonoesterases or phosphoglucomutase,
the CHO-APP.sub.695 cells were treated with the structurally
unrelated GSK-3 inhibitor, kenpaullone (Leost et al., Eur. J.
Biochem. 267:5983-5994 (2001)).
[0111] Stocks of kenpaullone (Calbiochem, La Jolla, Calif.) were
prepared in DMSO. Secreted labeled A.beta. secreted from
CHO-APP.sub.695 cells was obtained as described in the preceding
examples. Drugs were added to fresh medium, and medium and cells
were collected, as described above, 24 hours later.
Immunoprecipitates from the medium were SDS-PAGE separated and
PhosphorImager visualized. As shown in FIG. 2a, kenpaullone (tested
at concentrations of 2.0 .mu.M and 5.0 .mu.M, respectively)
dramatically reduced both A.beta..sub.40 and A.beta..sub.42
secretion, resulting in a 50% reduction at 2.0 .mu.M kenpaullone.
At 5.0 .mu.M, kenpaullone caused a reduction of greater than
90%.
[0112] However, kenpaullone is also known to inhibit
cyclin-dependent kinases (cdks), although such inhibition requires
at least 20-fold higher concentrations in vitro than the
concentration needed for inhibition of GSK-3 (Leost et al., 2001).
Nevertheless, to confirm that the effect seen was actually caused
by the lithium and not by a reaction with another composition,
CHO-APP.sub.695 cells were treated with roscovitine, a cdk
inhibitor that does not inhibit GSK-3 (Leclerc et al., J. Biol.
Chem. 276:251-260 (2001)). Stocks of roscovitine (Calbiochem) were
prepared in DMSO. As expected, the roscovitine (tested at
concentrations of 2.0 .mu.M and 5.0 .mu.M, respectively) had no
effect on A.beta..sub.40 and A.beta..sub.42, as shown in FIG. 2a.
Thus, neither kenpaullone nor roscovitine inhibited A.beta.
production through inhibition of cdks.
[0113] Inhibition of GSK-3 also causes accumulation of
.beta.-catenin protein. This was confirmed by western blot, in
CHO-APP.sub.695 cells treated with kenpaullone (tested at
concentrations of 0.5 .mu.M, 2.0 .mu.M and 5.0 .mu.M, respectively)
or lithium (0.5 mM, 2.0 mM and 5.0 mM LiCl) as shown in FIG. 2b.
Roscovitine was added as a control (tested at concentrations of 0.5
.mu.M, 2.0 .mu.M and 5.0 .mu.M, respectively) in FIG. 2b, as had
been in the study summarized in FIG. 2a. A western blot for actin
is was used as a loading control (see FIG. 2b).
Example 5
Effect of Increasing .beta.-catenin Protein on PS1 and
A.beta..sub.40 and A.beta..sub.42 Production
[0114] PS1 also interacts with .beta.-catenin and has been shown to
regulate .beta.-catenin protein levels (Zhang et al., Nature
395:698-702 (1998)) and subcellular localization (Kang et al.,
2002). Because GSK-3 inhibitors such as lithium and kenpaullone
cause accumulation of .beta.-catenin protein (Phiel et al., 2001),
it is technically possible that .beta.-catenin could play a role as
either a downstream effector of PS1 or a direct modulator of PS1
function. Therefore, tests were conducted to determine whether
increasing .beta.-catenin protein could mimic the effect of lithium
and kenpaullone on A.beta..sub.40 and A.beta..sub.42
production.
[0115] CHO-APP.sub.695 cells were transfected with a
.beta.-catenin-responsive reporter construct, OT-luciferase
(OT-Luc) (shown in FIG. 2c as light bars), or a mutated reporter,
OF-luciferase (OF-Luc) (shown in FIG. 2c as dark bars).
OT-luciferase was provided by K. Kinzler and B Vogelstein.
[0116] After transfection, cells were treated with lithium (at
concentrations of 0.5 mM, 2.0 mM and 5 mM LiCl, respectively),
kenpaullone (at concentrations of 0.5 mM, 2.0 mM and 5 mM,
respectively) or roscovitine (at concentrations of 0.5 mM, 2.0 mM
and 5 mM) for 24 hours. Cells were harvested and luciferase assays
performed.
[0117] From the assays, it is clear that both kenpaullone and
lithium inhibited GSK-3 in the CHO-APP.sub.695 cells, as indicated
by an accumulation of .beta.-catenin protein (FIG. 2b; see Example
4), and by the activation of a .beta.-catenin/Tcf-dependent
reporter, Lef-OT (FIG. 2c).
[0118] Xenopus .beta.-catenin (Fagatto et al., J. Cell. Biol.
132:1105-1114 (1996)) was subcloned from plasmid pCS2+ into plasmid
pSFV. Secreted A.beta..sub.40 (FIG. 2d, light bars) and
A.beta..sub.42 (FIG. 2d, dark bars) levels were assessed as
described in the preceding Examples, and as shown in FIG. 1. The
FIG. 2d inset shows a western blot of endogenous green fluorescent
protein (GFP) and overexpressed (.beta.-cat) .beta.-catenin in
duplicate lanes. Overexpression of .beta.-catenin did not affect
A.beta. production (FIG. 2d) under conditions that activated the
.beta.-catenin dependent reporter Lef-OT (OT-luciferase)(data not
shown).
[0119] Semliki Forest Virus (SFV) vectors were also utilized to
overexpress .beta.-catenin and APP. SFV was prepared and titered in
BHK cells (as previously described by Cook et al., Nat. Med.
3:1021-1023 (1997)). Cells were infected with SFV in serum-free
DMEM at a multiplicity of infection of 10. One (1) hour after
infection, the medium was replaced with MEM.alpha.+5% FBS (for
CHO.sub.695 cells) or DMEM+B27 supplement (for murine primary
neurons). As with the previous findings relating to the
overexpression of .beta.-catenin and APP, infection with SFV
encoding .beta.-catenin had no effect on A.beta. production (data
not shown). These observations, particularly when combined, show
that an elevated level of .beta.-catenin is not sufficient to cause
the decrease in A.beta. seen with GSK-3 inhibitors.
Example 6
GSK-3 Regulates APP Processing and is Required for A.beta.
Production
[0120] The foregoing data show that two structurally unrelated
inhibitors of GSK-3 reduce production of secreted A.beta. peptides.
While the logical explanation is that these inhibitors act through
inhibition of GSK-3, and therefore that GSK-3 regulates A.beta.
production, the possibility remains that these two agents
fortuitously inhibit distinct, unknown targets involved in APP
processing. Thus, to confirm that GSK-3 regulates APP processing,
expression of endogenous GSK-3 was reduced using RNA interference
(RNAi) (see, Elbashir et al., Nature 411:494-498 (2001)).
[0121] CHO-APP.sub.695 cells were transfected with short
interfering RNAs (siRNAs) directed against GSK-3.alpha. and
GSK-3.beta. as follows. RNA oligonucleotides were synthesized by
Dharmacon, Inc. (Lafayette, Colo.) Sequences used were:
TABLE-US-00001 Pp-Luc sense- (SEQ ID NO: 1) 5' CUU ACG CUG AGU ACU
UCG AdTdT 3'; Pp-Luc antisense- (SEQ ID NO: 2) 5' UCG AAG UAC UCA
GCG UAA GdTdT; GSK-3.beta. sense- (SEQ ID NO: 3) 5' AUC UUU GGA GCC
ACU GAU UdTdT 3'; GSK-3.beta. antisense- (SEQ ID NO: 4) 5' AAU CAG
UGG CUC CAA AGA UdTdT 3'; GSK-3.alpha. sense- (SEQ ID NO: 5) 5' UUC
UAC UCC AGU GGU GAG AdTdT 3'; and GSK-3.alpha. antisense- (SEQ ID
NO: 6) 5' UCU CAC CAC UGG AGU AGA AdTdT 3'.
[0122] The dsRNA was produced using conditions described in
Elbashir et al., 2001, supra. The siRNA was transfected into the
CHO-APP.sub.695 cells plated in 6-well dishes using GenePORTER
transfection reagent. The siRNA (25 nM) was co-transfected with 2
.mu.g plasmid DNA. The siRNA against pGL3-luciferase (Pp-Luc)
represents control transfection. GSK-3.alpha.siRNA selectively
reduces GSK-3.alpha. protein (see FIG. 3a, closed arrow).
GSK-3.beta. directed siRNA selectively reduces GSK-3.beta. (see
FIG. 3a, open arrow). Following transfection, the cells were
cultured and harvested 48 hours later. GSK-3.alpha. and GSK-3.beta.
protein levels were examined using an antibody that recognized both
GSK-3 isoforms (Calbiochem), and western blotted as above.
[0123] While a control siRNA had no effect on GSK-3 expression,
GSK-3-directed siRNAs reduced GSK-3 protein in an isoform-specific
manner as shown in the western blot presented in FIG. 3a.
Accordingly, GSK-3.alpha. is required for A.beta. production.
[0124] As graphically presented in FIG. 3b, A.beta. levels
(A.beta..sub.40 shown in light bars; A.beta..sub.42 shown in dark
bars) secreted from siRNA and transfected cells in a representative
experiment, were assessed as in FIG. 1. Error bars represent
standard deviation. The experiment was repeated six times with
similar results. As a result, the selective reduction of GSK-3
.alpha. protein decreased A.beta..sub.40 and A.beta..sub.42 levels
by 45% and 43%, respectively (FIG. 3b). Asterisks indicate a
significant difference from control, as determined by one-way ANOVA
(p<0.05).
[0125] Surprisingly, reduction of GSK-3.beta. protein did not lead
to decreased A.beta..sub.40 and A.beta..sub.42 levels. To the
contrary, GSK-3.beta. reduction resulted in a modest increase in
levels of secreted A.beta..sub.40 and A.beta..sub.42. Thus,
although these data further confirm that GSK-3.alpha. is required
for maximal production of A.beta..sub.40 and A.beta..sub.42, they
also suggest that GSK-3.beta. may in certain settings antagonize
APP processing.
[0126] Since either lithium treatment or GSK-3.alpha. depletion was
shown to reduce A.beta..sub.40 and A.beta..sub.42 levels, it was
important to confirm whether raising GSK-3.alpha. levels would
similarly enhance A.beta..sub.40 and A.beta..sub.42 production as
expected. To do so, CHO-APP695 cells were transfected with green
fluorescent protein (GFP) or increasing amounts of GSK-3.alpha.
(0.5 .mu.g to 2.0 .mu.g), then secreted A.beta. levels were
assessed as in Example 1. Overexpression of GSK-3.alpha. in
CHO-APP.sub.695 cells was found to increase A.beta..sub.40 and
A.beta..sub.42 levels in a dose dependent manner (data not shown).
Thus, levels of A.beta. production were directly correlated with
GSK-3.alpha. expression in both loss of function and overexpression
approaches. The isoform specific depletion of GSK-3 by RNAi
together with the overexpression data confirmed that GSK-3.alpha.
is required for maximal APP processing.
Example 7
Effect of Lithium Treatments on A.beta. Production in Cultured
Neurons and in Vivo in Brain Tissue of Animal Models
[0127] In light of the foregoing examples, it was important to
demonstrate that lithium blocked or reduced A.beta. production in
cultured neurons and in the brains of recognized animal models
associated with Alzheimer's disease.
[0128] To generate NT2N neurons, a human embryonic carcinoma cell
line (NTera2/D1 or NT2) was grown and maintained, essentially as
described by Pleasure et al., J. Neurosci. 12:1802-1815 (1992). For
murine primary embryonic neurons, cortices from E15 mouse brains
were isolated and incubated in 0.1% trypsin/HBSS/0.5 mM EDTA,
without Ca.sup.2+ or Mg.sup.2+ (Invitrogen, Carlsbad, Calif.). HBSS
(Hank's Balanced Salt Solution; containing potassium chloride,
monobasic potassium phosphate, sodium chloride, dibasic sodium
phosphate, and D-glucose). Cells were mechanically dissociated
using a fire-polished pipette. Cells were plated in DMEM+10% FBS
(fetal bovine serum) in poly-D-lysine coated 6-well plates at a
density of 10.sup.6 cells/well. Twenty-four (24) hours after
plating, the medium was replaced with DMEM plus B27 (Invitrogen) to
promote neuronal survival and inhibit growth of non-neuronal cells.
Neurons were used for experiments after three (3) days in
culture.
[0129] Primary cultures of the embryonic mouse cortical neurons
were then infected with a Semliki Forest Virus (SFV) containing
either wild-type APP (APP-WT) or APP with the pathogenic Swedish
mutation (KM670/671NL), then treated with oral lithium (2.0 or 5.0
mM LiCl, as shown in FIG. 4a) for 24 hours. Media was collected and
levels of secreted A.beta..sub.40 (FIG. 4a; light bars) and
A.beta..sub.42 (FIG. 4a; dark bars) were measured as described
above.
[0130] Production of A.beta..sub.40 and A.beta..sub.42 was reduced
by 60% and 78%, respectively, for wild-type APP and to a similar
extent for the APP-Swedish mutation (FIG. 4a). A lower, clinically
relevant concentration of lithium (1.0 mM) also reduced
accumulation of endogenous A.beta. by as much as 60% after 3-days
of culture in the neuronal NT2N cell line (FIG. 4a).
[0131] To determine the effect of oral lithium treatments on brain
tissue in vivo, transgenic mice expressing pathogenic,
FAD-associated forms of APP (APP-Swedish/Tg2576) were crossed to
mice carrying a "knock-in" of the PS1.sup.P264L mutation
(PS1.sup.P264L/wt) (provided by Flood; see, Siman et al., J.
Neurosci. 20:8717-8726 (2000)). Three-month-old heterozygous female
Tg2576/PS1.sup.P264L/wt mice were administered LiCl (n=7) or NaCl
(n=7) (300 mg/kg in 0.4 ml water, by gastric gavage once daily for
3 weeks). Animals were allowed free access to food and water, and
lithium-treated mice were given free access to 450 mM NaCl
solution. Animals were sacrificed three (3) hours after the final
dose and tissue accumulated A.beta. levels were measured after
stepwise extraction of A.beta. from brain cortical tissues by both
RIPA-extraction (soluble fractions) and formic acid-extraction
(insoluble fractions).
[0132] Briefly, the mouse cerebral cortices were lysed in RIPA
buffer. Following centrifugation, insoluble material was extracted
in formic acid (FA) (essentially as described by Wilson et al.,
Nat. Neurosci. 5:849-855 (2002)). RIPA- and FA-extracted samples
were diluted and A.beta. levels were measured by sandwich ELISA as
above (A.beta. sandwich ELISA obtainable from Takeda
Pharmaceuticals, Osaka, Japan).
[0133] Under these conditions, serum lithium levels were 0.8-1.2 mM
(FIG. 4b), which was safely within the therapeutic range for
bipolar patients treated with lithium. In the lithium treated
group, soluble RIPA-extracted A.beta..sub.40 and A.beta..sub.42
levels were each reduced by 40% (FIG. 4b). Furthermore, levels of
insoluble A.beta..sub.40 and A.beta..sub.42 extractable with formic
acid were reduced by 62% and 51%, respectively (FIG. 4c).
Accordingly, this demonstrated that a clinically tolerated dose of
lithium markedly reduces the tissue level of A.beta. peptides
(A.beta. production) in both the neurons and in the brains of
subjects treated with oral lithium.
[0134] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0135] While the foregoing specification has been described with
regard to certain preferred embodiments, and many details have been
set forth for the purpose of illustration, it will be apparent to
those skilled in the art without departing from the spirit and
scope of the invention, that the invention may be subject to
various modifications and additional embodiments, and that certain
of the details described herein can be varied considerably without
departing from the basic principles of the invention. Such
modifications and additional embodiments are also intended to fall
within the scope of the appended claims.
Sequence CWU 1
1
6119RNAArtificialSynthetic formulation 1cuuacgcuga guacuucga
19219RNAArtificialSynthetic formulation 2ucgaaguacu cagcguaag
19319RNAArtificialSynthetic formulation 3aucuuuggag ccacugauu
19419RNAArtificialSynthetic formulation 4aaucaguggc uccaaagau
19519RNAArtificialSynthetic formulation 5uucuacucca guggugaga
19619RNAArtificialSynthetic formulation 6ucucaccacu ggaguagaa
19
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