Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOUNDS AND THEIR PREPARATION FOR THE TREATMENT OF
ALZHEIMER'S DISEASE BY INHIBITING BETA-AMYLOID PEPTIDE PRODUCTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Nonprovisional Application
No.
10/961,346 filed October 7, 2004; which claims the benefit of U.S. Provisional
Application
No. 60/588,433 filed July 16, 2004; which are incorporation herein by
reference thereto.
FIELD OF THE INVENTION
[0002] The present invention provides novel ginsenoside compounds,
compositions
(e.g. pharmaceutical compositions) comprising the ginsenoside compounds, and
methods for
the synthesis of these ginsenoside compounds. Additionally, the present
invention provides
methods for inhibiting beta-amyloid peptide production and methods for
treating or
preventing a pathological condition, particularly, neurodegeneration diseases
(e.g.
Alzheimer's disease), using these ginsenoside compounds.
STATEMENT OF GOVERNMENT INTEREST
[0003] This invention was made in part with government support under NIH Grant
No. ROI N543467. As such, the United States government may have certain rights
in this
invention.
BACKGROUND OF THE INVENTION
[0004] Alzheimer's disease (AD) is a neurodegenerative disease characterized
by a
progressive, inexorable loss of cognitive function (Francis, et al.,
Neuregulins and ErbB
receptors in cultured neonatal astrocytes. J. Neurosci. Res., 57:487-94, 1999)
that eventually
leads to an inability to maintain normal social and/or occupational
performance. Alzheimer's
disease is the most common form of age-related dementia, and one of the most
serious health
problems, in the United States. Approximately 4 million Americans suffer from
Alzheimer's
disease, at an annual cost of at least $100 billion - making Alzheimer's
disease one of the
costliest disorders of aging. Alzheimer's disease is about twice as common in
women as in
men, and accounts for more than 65% of the dementias in the elderly.
Alzheimer's disease is
the fourth leading cause of death in the United States. To date, a cure for
Alzheimer's disease
is not available, and cognitive decline is inevitable. Although the disease
can last for as many
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as 20 years, AD patients usually live from 8 to 10 years, on average, after
being diagnosed
with the disease.
[0005] The pathogenesis of Alzheimer's disease is associated with an excessive
amount of neurofibrillary tangles (composed of paired helical filaments and
tau proteins) and
neuritic or senile plaques (composed of neurites, astrocytes, and glial cells
around an amyloid
core) in the cerebral cortex. While senile plaques and neurofibrillary tangles
occur with
normal aging, they are much more prevalent in persons with Alzheimer's
disease. Specific
protein abnormalities also occur in Alzheimer's disease. In particular, AD is
characterized by
the deposition of the amyloid 0-peptide (AP) into amyloid plaques in the brain
(Selkoe, et al.
(2001) Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 81, 741-
66; Hardy and
Selkoe (2002). The amyloid hypothesis of Alzheimer's disease: progress and
problems on the
road to therapeutics. Science 297, 2209). A(3 is produced by sequential
proteolytic cleavages
of amyloid precursor protein (APP) by a set of membrane-bound proteases termed
0- and y-
secretases (Vassar and Citron (2000) Abeta-generating enzymes: recent advances
in beta- and
gamma-secretase research. Neuron 27, 419-422; John, et al. (2003) Human beta-
secretase
(BACE) and BACE inhibitors. .l. Med Chem. 46, 4625-4630; Selkoe and Kopan
(2003)
Notch and Presenilin: regulated intramembrane proteolysis links development
and
degeneration. Annu. Rev Neurosci. 26, 565-597; Medina and Dotti (2003) ripped
out by
presenilin-dependent gamma-secretase. Cell Signal 15, 829-841). Heterogeneous
J3-secretase
cleavage at the C-terminal end of A(3 produces two major isoforms of A(3, A040
and A042.
While A040 is the predominant cleavage product, the less abundant, highly
amyloidogenic
A042 is believed to be one of the key pathogenic agents in AD (Selkoe (2001)
Alzheimer's
disease: genes, proteins, and therapy. Pliysiol Rev. 81, 741-66) and increased
cerebrocorical
AR42 is closely related to synaptic/neuronal dysfunction associated with AD
(Selkoe,
Alzheimer's disease is a synaptic failure, Science 298:789-791, 2002).
[0006] Presenilins are required for intramembrane proteolysis of selected type-
I
nlembrane proteins, including amyloid-beta precursor protein (APP), to yield
amyloid-beta
protein (De Strooper et al., Deficiency of presenilin-1 inhibits the normal
cleavage of
amyloid precursor protein. Nature 391:387-90, 1998; Steiner and Haass,
Intramembrane
proteolysis by presenilins. Nat. Rev. Mol. Cell. Biol. 1:217-24, 2000; Ebinu
and Yankner, A
rip tide in neuronal signal transduction. Neuron 34:499-502, 2002; De Strooper
and Annaert,
Presenilins and the intramembrane proteolysis of proteins: facts and fiction.
Nat. Cell Biol.
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3:E221-25, 2001; Sisodia and George-Hyslop, y-Secretase, Notch, a-beta and
Alzheimer's
disease: where do the presenilins fit in? Nat. Rev. Neurosci. 3:281-90, 2002).
Such
proteolysis may be mediated by presenilin-dependent (3-secretase machinery,
which is known
to be highly conserved across species, including nematodes, flies, and mammals
(L'Hernault
and Arduengo, Mutation of a putative sperm membrane protein in Caenorhabditis
elegans
prevents sperm differentiation but not its associated meiotic divisions. J.
Cell. Biol. 119:55-
58, 1992; Levitan and Greenwald, Facilitation of lin-12-mediated signaling by
sel-12, a
Caenorhabditis elegans S 182 Alzheimer's disease gene. Nature 377:351-54,
1999; Li and
Greenwald, HOP-l, a Caenorhabditis elegans presenilin, appears to be
functionally redundant
with SEL-12 presenilin and to facilitate LIN-12 and GLP-l signaling. Proc.
Natl. Acad. Sci.
USA 94:12204-209, 1997; Steiner and Haass, Intramembrane proteolysis by
presenilins. Nat.
Rev. Mol. Cell. Biol. 1:217-24, 2000; Sisodia and George-Hyslop, y-Secretase,
Notch, a-beta
and Alzheimer's disease: where do the presenilins fit in? Nat. Rev. Neurosci.
3:281-90,
2002).
[0007] y-Secretase, a high-molecular-weight, multi-protein complex harboring
presenilin heterodimers and nicastrin, mediates the final step in A(3
production in Alzheimer's
disease (Li, et al., Presenilin 1 is linked with 0-secretase activity in the
detergent solubilized
state. Proc. Natl. Acad. Sci. USA 97:6138-43, 2000; Esler, et al., Activity-
dependent
isolation of the presenilin-y-secretase complex reveals nicastrin and a gamma
substrate.
Proc. Natl. Acad .Sci. USA 99:2720-25, 2002). The stabilization of presenilin
heterodimers
(converted from a short-lived pool to a long-lived pool) and other undefined
core components
appears to be critical for y-secretase activity (Thinakaran, et al., Evidence
that levels of
presenilins (PS 1 and PS2) are coordinately regulated by competition for
limiting cellular
factors. J. Biol. Chem. 272:28415-422, 1997; Tomita, et al., The first proline
of PALP motif
at the C terminus of presenilins is obligatory for stabilization, complex
formation, and
gamma-secretase activities of presenilins. J. Biol. Chem. 276:33273-281,
2001). y-Secretase
activity displays very loose sequence specificity near the target
transmembrane cleavage site
and has been shown to mediate the intramembrane cleavage of other non-APP type-
I
membrane substrates, including Notch (Schroeter, E.H., et al. (1998) Notch-1
signaling
requires ligand-induced proteolytic release of intracellular domain. Nature
393, 382-386; De
Strooper, et al. (1999) Presenilin-l-dependent gamma-secretase-like protease
mediates
release of Notch intracellular domain. Nature 398:518-522), ErbB4 (Lee, et al.
(2002)
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Presenilin-dependent gamma-secretase-like intramembrane cleavage of ErbB4. J.
Biol.
Chern. 277, 6318-6323; Ni, et al. (2001) Gamma -Secretase cleavage and nuclear
localization
of ErbB-4 receptor tyrosine kinase. Science 294, 2179-2181), and p75
neurotrophin receptor
(p75NTR) (Jung, et al. (2003) Regulated intramembrane proteolysis of the p75
neurotrophin
receptor modulates its association with the TrkA receptor. J Biol Chem. 278,
42161-42169).
It is predicted that general blockage of [i-secretase activity not only
abolishes A(3 generation
but also inhibits normal processing of other cellular r3-secretase substrates,
required for the
relevant cellular function of these substrates. Thus, complete inhibition of y-
secretase
activity could potentially lead to severe side-effects (Doerfler, et al.,
Links Free in PMC
Presenilin-dependent gamma-secretase activity modulates thymocyte development.
(2001)
Proc Natl. Acad. Sci USA 98, 9312-9317; Hadland, et al. Gamma -secretase
inhibitors repress
thyinocyte development. Proc Natl. Acad. Sci USA 98, 7487-7491). A safer
approach would
ideally be to use reagents which can selectively reduce A[i42 generation
without affecting the
intramembrane proteolysis of other y-secretase substrates. As an example, a
subset of
nonsteroidal anti-inflammatory drugs (NSAIDs) was shown to decrease the
production of
AP42 (Weggen, et al. (2001). A subset of NSAIDs lower amyloidogenic Abeta42
independently of cyclooxygenase activity. Nature 414, 212-216), without
significantly
affecting y-secretase-mediated cleavage of ErbB4 (Weggen, et al. (2003).
Abeta42-lowering
nonsteroidal anti-inflammatory drugs preserve intraniembrane cleavage of the
amyloid
precursor protein (APP) and ErbB-4 receptor and signaling through the APP
intracellular
domain. J. Biol. Chem. 278, 30748-30754). Accordingly, small molecules which
are able to
selectively reduce A042 production (without affecting the cleavage of other y-
secretase
substrates) are attractive and promising as therapeutic reagents for treating
AD.
[0008] Most cases of early-onset familial Alzheimer's disease (FAD) are caused
by
mutations in two related genes encoding presenilin proteins: PS 1 and PS2
(Tanzi, et al., The
gene defects responsible for familial Alzheimer's disease. Neurobiol. Dis.
3:159-68, 1996;
Hardy, J., Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci.
20:154-59,
1997; Selkoe, D.J., Alzlieimer's disease: genes, proteins, and therapy.
Physiol. Rev. 81:741-
66, 2001). FAD-associated mutations in the presenilins give rise to an
increased production
of a longer (42 amino acid residues), more amyloidogenic form of amyloid-beta
(A[i42).
Deciphering the pathobiology associated with the presenilins provides a unique
opportunity
to elucidate a molecular basis for Alzheimer's disease. It is suspected that
excess beta-
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amyloid production causes the neuronal degeneration underlying dementia
characteristic of
AD.
[0009] Ginseng is the common name given to the dried roots of plants of the
genus
Panax which has been used extensively in Asia for thousands of years as a
general health
5 tonic and medicine for treating an array of diseases (Cho, et al. (1995)
Phamiacological
action of Korean ginseng. In the Society for Korean Ginseng (eds.):
Understanding Korean
Ginseng, Seoul: Hanlim Publishers, pp 35-54; Shibata S. (2001) Chemistry and
cancer
preventing activities of ginseng saponins and some related triterpenoid
compounds. J Korean
Med Sci. 16 Suppl:S28-37; Attele, et al. (1999); Ginseng pharmacology:
multiple constituents
and multiple actions. Biochem Pharmacol. 58:1685-1693; Coleman, et al. (2003).
The
effects of Panax ginseng on quality of life. J. Clin. Pharm. Ther. 28, 5-15;
Coon and Ernst
(2002). Panax ginseng: a systematic review of adverse effects and drug
interactions. Drug
Saf. 25:323-44). The Panax genus contains about six species native to eastern
Asia and two
species native to eastern North America. Panax ginseng (Asian ginseng) and
Panax
quinquefolius L. (North American ginseng) are the two species most commonly
used in
nutraceutical and pharmaceutical compositions. The roots and their extracts
contain a variety
of substances including saponins.
[0010] Ginseng has been well known to have specific pharmacological effects
including improvement of liver function and immune enhancement, as well as
anti-
arteriosclerotic, anti-thrombotic, anti-stress, anti-diabetic, anti-
hypertensive and antitumor
effects. Among several classes of compounds isolated from the ginseng root,
ginseng
saponins are known to be the chemical constituents that contribute to its
pharmacological
effects. These compounds are triterpene glycosides named ginsenosides Rx (x is
index "a" to
"k" depending on its polarity). The polarity is determined by their mobility
on thin-layer
chromatography plates and is a function of the number of monosaccharide
residues in the
molecule's sugar chain.
[0011] To date, at least 31 ginsenosides have been isolated from white and red
ginseng. All of the ginsenosides can be divided into three groups depending on
their
aglycons: protopanaxadiol-type ginsenosides (e.g., Rbl, Rb2, Rc, Rd, (20R)Rg3,
(20S)Rg3,
Rh2), protopanaxatriol-type ginsenosides (e.g., Re, Rf, Rgl, Rg2, Rhl), and
oleanolic acid-
type ginsenosides (e.g., Ro). Both protopanaxadiol-type and protopanaxatriol-
type
ginsenosides have a triterpene backbone structure, known as dammarane (Attele,
et al. (1999)
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Ginseng pharmacology: multiple constituents and multiple actions. Biochem.
Pharmacol.
58:1685-1693). Rkl, Rg5 (20R)Rg3 and (20S)Rg3 are ginsenosides that are almost
uniquely
present in heat-processed ginseng, but not found to exist as trace elements in
unprocessed
ginseng (Kwon, et al. (2001) Liquid chromatographic determination of less
polar
ginsenosides in processed ginseng. J. Chromatogr. A. 921;335-339; Park, et al.
(2002);
Cytotoxic dammarane glycosides from processed ginseng. Chem. Pharm. Bul. 50,
538-540
Park, et al. (2002); Three new dammarane glycosides from heat-processed
ginseng. Arch.
Pharm. Res. 25, 428-432; Kim, et al. (2000); Steaming of ginseng at high
temperature
enhances biological activity. J. Nat. Prod. 63:1702-1702). Carbohydrates
including
glucopyranosyl, arabinopyranosyl, arabinofuranosyl and rhamnopyranosyl may
also be
chemically associated with a particular ginsenoside.
[0012] Processing of ginseng with steam at high temperature further enhances
the
content of these unique ginsenosides Rkl, Rg5, (20R)Rg3 and (20S)Rg3, which
appear to
possess novel pharmacological activities. At least some of the beneficial
qualities of ginseng
can be attributed to its triterpene saponin content, a mixture of glucosides
referred to
collectively as ginsenosides.
[0013] U.S. Patent 5,776,460 ("the '460 patent") discloses a processed ginseng
product having enhanced pharmacological effects. This ginseng product,
commercially
known as "sun ginseng," contains increased levels of effective pharmacological
components
due to heat-treating of the ginseng at a high temperature for a particular
period of time. As
specifically disclosed in the '460 patent, heat treatment of ginseng may be
perfornled at a
temperature of 120 to 180 C for 0.5 to 20 hours, and is preferably performed
at a
temperature of 120 to 140 C for 2 to 5 hours. The heating time varies
depending on the
heating temperature such that lower heating temperatures require longer
heating times while
higher heating temperatures require comparatively shorter heating times. The
'460 patent
also discloses that the processed ginseng product has pharmacological
properties specifically
including anti-oxidant activity and vasodilation activity.
[0014] Recently, Tae-Wan Kim et al. demonstrated that the unique components of
the
heat-processed ginseng product disclosed in the '460 patent significantly
lower the production
A042 in cells (patent application pending). Specifically, the inventors
discovered that at least
three ginsenosides Rkl, (20S)Rg3, and Rg5, unique components of the heat-
processed
ginseng known as "Sun Ginseng," as well as Rgk351, which is a mixture of
(20R)Rg3,
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(20S)Rg3, Rg5, and Rkl, lower the production of A(342 in mammalian cells.
Rgk351 and
Rkl are most effective in reducing A(342 levels. Furthermore, Rkl was also
shown to inhibit
the A(342 production in a cell-free assay using a partially purified y-
secretase complex,
suggesting that Rkl modulates either specificity and/or activity of the y-
secretase enzyme. In
addition, Tae-Wan Kim et al. found that certain ginsenosides which harbor no
A[i42-reducing
activity in vitro, are effective in reducing A(342 in vivo. For example, some
of the 20(S)-
protopanaxatriol (PPT) group ginsenosides, such as Rgl, can be converted into
PPT after oral
ingestion. Thus, while Rgl generally has no amyloid-reducing activity in
vitro, Rgl may be
converted into an active amyloid-reducing compound PPT in vivo.
SUMMARY OF THE INVENTION
[0015] The present invention provides compositions and methods for preventing
and
treating neurodegenerative diseases, such as Alzheimer's disease.
[0016] In one aspect, the present invention provides a compound having the
general
formula:
R3 R
4
R5
Rl
R2
wherein Rr is selected from the group consisting of a-OH, (3-OH, a-O-X, P-O-X,
a-R6COO-,
(3-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate containing one
or more
sugars or acylated derivatives thereof, and R6 is an alkenyl, aryl, or alkyl
I; R2 is selected
from the group consisting of H, OH, OAc, and O-X, wherein X is a carbohydrate
containing
one or more sugars or acylated derivatives thereof; R3 is selected from the
group consisting of
H, OH, and OAc; R4 is an alkenyl, aryl, or alkyl II; and R5 is H or OH. The
alkyl I group
may further contains oxygen, nitrogen, or phosphorus and the alkyl II group
may further
contain a functional group selected from the group consisting of hydroxyl,
ether, ketone,
oxime, hydrazone, imine, and Schiff base. In one embodiment, the sugar group
is selected
from the group consisting of Glc, Ara(pyr), Ara(fur), Rha, and Xyl. In another
embodiment,
R4 is selected from the group consisting of:
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O
O O O )t,~~X
O
N" N" N" X'
I I ~ _,~I
OR'
OR' 47~/ ' OR' ~ X
+ O
HO
OR' O'
O
OH 5 wherein the configuration of any stereo-center is R or S; X is OR or NR,
wherein R is alkyl
or aryl; X' is alkyl, OR, or NR, wherein R is alkyl or aryl; and R' is H,
alkyl, or acyl. In
another embodiment, the present invention provides a composition,
particularly, a
pharinaceutical composition, comprising a compound having the general formula:
R3 R4
R5
R, 4; R2
wherein RI is selected from the group consisting of a-OH, (3-OH, a-O-X, (3-O-
X, a-R6COO-,
[I-R6COO-, a-R6P03-, and (3-R6PO3-, wherein X is a carbohydrate containing one
or more
sugars or acylated derivatives thereof, and R6 is alkenyl, aryl, or alkyl I;
R2 is selected from
the group consisting of H, OH, OAc, and O-X, wherein X is a cairbohydrate
containing one or
more sugars or acylated derivatives thereof; R3 is selected from the group
consisting of H,
OH, and OAc; R4 is alkenyl, aryl, or alkyl II; and R5 is H or OH.
[0017] The present invention also provides a method for the synthesis of a
compound
having formula:
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R3 R
R,
4i 4
HO
R2
which comprises the steps of:
a) treating a compound having formula:
R3 R4
R,
HO
4-
R2
b) with an oxidizing agent, to form a compound having formula:
R3 R
4
Rl
R2
c) treating the compound formed in step (a) with a reducing agent, to form a
compound having formula:
R3 R4
RI
HO
R2
wherein Rl is H or OH; R2 is selected from the group consisting of H, OH, OAc,
and O-X,
wherein X is a carbohydrate containing one or more sugars or acylated
derivatives thereof; R3
is selected from the group consisting of H, OH, and OAc; and R4 is alkenyl,
aryl, or alkyl. In
one embodiment, the oxidizing agent is chromic anhydride and the reducing
agent is NaBH4.
[0018] The present invention further provides a method for the synthesis of a
compound having formula:
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R3 R
4
R5
R11
$-
R2
which comprises the steps of:
(a) treating a compound having formula:
R3 R4
R5
HO 4 $-
R2
5 with an oxidizing agent, to form a compound having formula:
R3 R
4
R5
R2
(b) treating the compound formed in step (a) with a reducing agent, to form a
compound having formula:
R3 R
4
R5
HO
R2
10 (c) optionally, treating the compound formed in step (b) with protected Rl
derivative, to form a compound having formula:
R3 R4
R5
R, Protected
R2
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(d) treating the compound formed in step (c) with deprotection agent, to form
a
compound having formula:
R3 R4
R5
R11
$-
R2
wherein Rl is selected from the group consisting of a-OH, (3-OH, a-O-X, (3-O-
X, a-R6COO-
,(3-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate containing
one or more
sugars or acylated derivatives thereof, and R6 is alkenyl, aryl, or alkyl I;
R2 is selected from
the group consisting of H, OH, OAc, and O-X, wherein X is a carbohydrate
containing one or
more sugars or acylated derivatives thereof; R3 is selected from the group
consisting of H,
OH, and OAc; R4 is alkenyl, aryl, or alkyl II; and R5 is H or OH.
[0019] Additionally, the invention provides a method for the synthesis of a
compound
having formula:
OH ---
O 0
GIcGIc H
wherein the method comprises the steps of:
(a) treating a compound having formula:
HO
HO OH
H
with an oxidizing agent, to form a compound having formula:
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HO
OH
O
H
(b) treating the compound formed in step (a) with a protecting agent, to form
a
compound having formula:
HO
O OAc
H
(c) treating the compound formed in step (b) with a reducing agent, to form a
compound having formula:
HO
HO OAc
H
(d) treating the compound formed in step (c) with Ac8-Glc-Glc-Br, to form a
compound having formula:
HO
OAc 0
Ac$'GICGIcO H
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(e) treating the compound formed in step (d) with deprotection agent, to form
a
compound having formula:
HO
OH
O 0
GIcGIc H
(f) further modifying the compound formed in step (e) to form a compound
having formula:
,
I 4 OH ', ---
O
GIcGIc H
HO /
HO OH
H
In one embodiment, the starting material, betulafolienetriol, is obtained from
a plant, such as,
for example, common birch.
[0020] In one aspect, the present invention provides a method for the
synthesis of a
compound having formula:
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HO ~
HO
H
wherein the method comprises the step of treating a compound having formula:
HO
0
H
with a reducing agent, such as NaBH4.
[0021] In another aspect, the present invention provides a method for the
synthesis of
a compound having formula:
HO
0
GIcGIc H
wherein the method comprises the steps of:
(a) treating a compound having formula:
HO
O
H
with a reducing agent, to form a compound having formula:
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HO
HO
H
(b) treating the compound formed in step (a) with Ac8-Glc-Glc-Br, to form a
compound having formula:
5
HO
Ac$,GIcGIc O H
(c) treating the compound formed in step (d) with deprotection agent, to form
a
compound having formula:
HO
O
GIcGIc H
10 [0022] Additionally, the present invention provides a method for treating
or
preventing a pathological condition in a subject, comprising administering a
compound
having the general formula:
R3 R
4
R5
Rl R2
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to the subject, wherein Rl is selected from the group consisting of a-OH, (3-
OH, a-O-X, (3-
O-X, a-R6COO-, (3-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate
containing one or more sugars or acylated derivatives thereof, and R6 is
alkenyl, aryl, or alkyl
I; R2 is selected from the group consisting of H, OH, OAc, and O-X, wherein X
is a
carbohydrate containing one or more sugars or acylated derivatives thereof; R3
is selected
from the group consisting of H, OH, and OAc; R4 is alkenyl, aryl, or alkyl II;
and R5 is H or
OH. In one embodiment, the pathological condition is neurodegeneration,
preferably,
Alzheimer's disease and A(342-related disorder.
[0023] The present invention further provides a method for inhibiting (3-
amyloid
production in subject, including inhibiting [3-amyloid production in an in
vitro context,
comprising administering a compound having the general formula:
R3 R
4
R5
R,
4-
R2
to the subject, wherein Rl is selected from the group consisting of a-OH, (3-
OH, a-O-X, (3-
O-X, a-R6COO-, (3-R6COO-, a-R6PO3-, and (3-R6PO3-, wherein X is a carbohydrate
containing one or more sugars or acylated derivatives thereof, and R6 is
alkenyl, aryl, or alkyl
I; R2 is selected from the group consisting of H, OH, OAc, and O-X, wherein X
is a
carbohydrate containing one or more sugars or acylated derivatives thereof; R3
is selected
from the group consisting of H, OH, and OAc; R4 is alkenyl, aryl, or alkyl II;
and R5 is H or
OH.
[0024] Additional aspects of the present invention will be apparent in view of
the
description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 depicts sequential proteolytic processing of (3-amyloid
precursor
protein (APP), mediated by (3- and y-secretases.
[0026] FIG. 2 shows the HPLC profile of (a)White Ginseng; (b) Red Ginseng; and
(c)
Sun Ginseng (heat processed ginseng).
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[0027] FIG. 3 illustrates the general chemical formula of: (a) Rg3, (b) Rkl
and (c)
Rg5.
[0028] FIG. 4 shows that Rgk351, (20R)Rg3, Rkl and Rg5 reduce the generation
of
A042 in CHO cells stably transfected with human APP695. The CHO cells were
treated with
the indicated compounds (at 50 g/ml) for 8 hrs. A042 levels in the medium
were measured
by ELISA and normalized to intracellular full-length APP.
[0029] FIG. 5 shows that treatment with Rgk351, Rkl and Rg5 reduced AJ342 in
the
medium of CHO cells expressing human APP in a dose-dependent manner.
[0030] FIG. 6 demonstrates that treatment of Rgk351, Rkl and Rg5
preferentially
reduced A[i42 (vs. A(340) in the medium of CHO cells expressing human APP in a
dose-
dependent manner. The relative levels of A(3 and A042 were normalized to
values obtained
from non-treated and vehicle-treated cells. Similar data were obtained using
Neuro2a-sw
(mouse Neuro2a cells expressing Swedish familial Alzheimer's disease mutant
form of APP)
and 293 cells expressing human APP.
[0031] FIG. 7 depicts an analysis of cell lysates and shows that Rgk351, Rkl
and Rg5
caused the increased accumulation of APP C-terminal fragments (y-secretase
substrates),
while the full-length holoAPP levels were not affected.
[0032] FIG. 8 demonstrates that treatment of Rgk351 and Rkl reduced the A[342
levels in CHO cells co-expressing human APP together with either wild-type
presenilin 1 or
familial Alzheimer-linked mutant forms of presenilin 1 (delta E9 ad L286V).
The effects of
Rg5 on the AP42 generation were much smaller as compared to Rgk351 and Rk1.
[0033] FIG. 9 shows effects of Rkl(R1) and Rg5(R5) on A042-specific y-
secretase
activity. Naproxen (NP) and sulindac sulfide (SS) were tested in parallel.
[0034] FIG. 10 depicts the effects of native ginsenosides on A(342 production.
The
structures of seven standard ginsenosides studied (Rbl, Rb2, Rc, Rd, Re, Rgl,
and Rg2) are
shown in Table 1. CHO cells stably transfected with human APP695 together with
either
wild-type (A, CHO-APP/PS1 cells) or DE9 FAD mutant (B, CHO-APP/AE9PS1 cells)
forms
of PS 1 were used. Cells were treated with the indicated compounds (at 50 M)
for 8 hrs.
Levels of secreted A040 and A042 in the medium were determined by ELISA and
normalized to intracellular full-length APP. In CHO-APP/PS1 cells, average A(3
amounts in
control samples were 320 pM for A[i40 and 79 pM for A(342. The relative levels
of A[i and
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A(342 were normalized to values obtained from non-treated and vehicle-treated
cells and are
shown as % to control + s.d.). One of three representative experiments are
shown.
[0035] FIG. 11 shows A(342-lowering activity of several ginsenosides derived
from
heat- or steam-processed ginseng. CHO-APP/PS 1(A) and CHO-APP/AE9PS 1(B) cells
were
treated with the indicated compounds at 50 M for 8 hrs and the levels of
secreted A040 and
A(342 were determined as described in Figure 1. Note that the potency of A042-
reducing
activity was in order of Rkl >/= (20S)Rg3 > Rg5 > (20R)Rg3, and the effects of
Rhl and
Rg6 were not significant. Rh2 also exhibited AJ342-lowering effects although
the cell
viability was partially affected at 50 M treatment (data not shown). The PSi-
DE9 FAD
mutation diminished the A042 response to Rkl treatment (B).
[0036] FIG. 12 shows treatment with Rgk351, Rkl and Rg5 reduced A[i42 in the
medium of CHO-APP cells in a dose-dependent manner. (A) Dose-response of A(342
lowering activity of Rkl and Rg5. IC50 of Rkl was about 20 M. (B) Rkl
preferentially
lowers A042 (vs. A(340) in cultured CHO-APP cells and the A042-inhibition
pattern of Rkl
is similar to that of sulindac sulfide (SS). The relative levels of A(340 and
AP42 were
normalized to values obtained from non-treated and vehicle-treated cells.
Similar data were
obtained using Neuro2a-sw (mouse Neuro2a cells expressing Swedish familial
Alzheimer's
disease mutant form of APP) and 293 cells expressing human APP (data not
shown). The
effects of Rg5 on the A[342 generation were much smaller as compared to Rgk351
and Rkl.
[0037] FIG. 13. depicts an analysis of APP processing after Rkl treatment.
Steady-
state levels of full-length APP and APP C-terminal fragments (APP-CTFs) were
examined by
Western blot analysis using anti-Rl antibody. Rgk351(mixture of Rg3, Rg5 and
Rkl), Rkl
and Rg5 treatment resulted in increased accumulation of APP C-terminal
fragments (y-
secretase substrates) in CHO-APP cells and mouse neuroblastoma neuro2a cells
stably
expressing Swedish FAD mutant form (KM670/671NL) of APP (APPsw). Correlated
A(342
levels for each sample are shown in the bottom panel.
[0038] FIG. 14 shows that A[342-lowering ginsenoside Rkl does not
significantly
affect the production of intracellular domains (ICDs) from APP (A, AICD),
Notchl (B,
NICD) or p75 neurotrophin receptor (p75NTR, p75-ICD). Membrane fractions
isolated from
293 cells overexpressing either APP (A), Notch-AE (B) or p75-AE (C) and
incubated in the
presence of indicated compounds: Compound E(CpdE, general y-secretase
inhibitor),
Rgk351, Rkl and sulindac sulfide (SS). Very low amounts of AICD, NICD and p75-
ICD
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were detected in control samples (- Incubate) or in samples treated with
Cpd.E, but AICD,
NICD and p75-ICD were abundantly produced in samples incubated with Rgk351,
Rkl and
ss.
[0039] FIG. 15 shows that A[342-lowering ginsenoside Rkl and (20S)Rg3 inhibits
A(3
generation in a cell-free y-secretase assay. (A) CHAPSO-solubilized membrane
fractions
were incubated with recombinant y-secretase substrates together with the
indicated
compounds (at 100 M) and the levels of A(342 and A(340 were determined by
ELISA as
described (27-29). (B) Dose-response of A[340 and A[342-lowering activity of
Rkl and
(20S)Rg3 in a cell-free y-secretase assay. IC50 of Rkl was 27 + 3 M for AJ340
and 32 + 5
for A(342. ICso of (20S')Rg3 was 27 + 4 for A(340 and 26 + 7 for A(342.
[0040] FIG. 16 depicts the effects of two major metabolites of ginsenosides,
including
20(S)-protopanaxatriol (PPT) and 20(S)-protopanaxadiol (PPD) on A[i42
generation. 20(S)-
panaxatriol (PT) and 20(S)-panaxadiol (PD) are the artificial derivatives of
PPT and PPPD,
respectively. Treatment with either PPT or PT reduced the production of A042
without
affecting the levels of A042 in Neuro2a cells expressing the human Swedish
mutant form of
APP (Neuro2a-SW, bottom panel), as well as in CHO cells expressing wild-type
human APP
(data not shown). PPD and PD did not confer any inhibitory effects on A040 or
AR42
generation.
[0041] FIG. 17 shows mass spectrometric analysis of A(3 species produced from
CHO-APP cells treated with DMSO (vehicle), Rkl, or (20S)Rg3. Note that
treatment leads
to a decrease in A042 species (1-42), and elevation in both A037 (1-37) and
AJ338 (1-38).
Mass spectrometric analysis of A(3 species were performed as previously
described (Wang R,
Sweeny D, Gandy SE, Sisodia SS. The profile of soluble amyloid [i-protein in
cultured cell
media. J. Bio. Chern. 1996; 271: 31894-31902).
[0042] FIG. 18 depicts analysis of secreted A(3 levels after treatment of CHO-
APP
cells with DMSO (Control 1), naproxen (Control 2), Rlcl, or (20S)Rg3. A[i was
immoprecipitated using 4G8 antibody (Purchased from Senetek), subjected to SDS-
PAGE
using Tricine/Urea gel (the protocol was supplied by Dr. Y. Ihara, University
of Tolcyo), and
analyzed by Western blot analysis using the 6E10 antibody (Senetek). Synthetic
A(340 and
A[342 peptides were used to identify corresponding A(3 species.
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[0043] FIG. 19 shows the effects of the ginsenoside Rkl and (20S)Rg3 on A[i40
and
A042 secretion in primary embryonic cortical neurons derived from Tg2576
transgenic mice.
Treatment of Rkl and Rg3 decreased the level of secreted A040 and A042.
DETAILED DESCRIPTION OF THE INVENTION
5 [0044] As used herein and in the appended claims, the singular forms "a,"
"an," and
"the" include plural references unless the content clearly dictates otherwise.
Thus, for
example, reference to "an agent" includes a plurality of such agents, and
reference to "the
ginsenoside" is a reference to one or more ginsenodies and equivalents thereof
known to
those skilled in the art, and so forth. All publications, patent applications,
patents, and other
10 references mentioned herein are incorporated by reference in their
entirety.
[0045] In accordance with the present invention, compounds and methods for
treating
Alzheimer's disease, neurodegeneration and for modulating the production of
amyloid-beta
protein (A(3) are provided.
[0046] In one aspect, the present invention provides a compound having the
general
15 formula:
R3 R4
R5
RI 4 $-
R2
wherein Rl is selected from the group consisting of a-OH, P-OH, a-O-X, (3-O-X,
a-R6COO-,
[3-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate containing one
or more
sugars or acylated derivatives thereof, and R6 is alkenyl, aryl, or alkyl I;
R2 is selected from
20 the group consisting of H, OH, OAc, and O-X, wherein X is a carbohydrate
containing one or
more sugars or acylated derivatives thereof; R3 is selected from the group
consisting of H,
OH, and OAc; R4 is alkenyl, aryl, or alkyl II; and RS is H or OH. The alkyl I
group may
further contain oxygen, nitrogen, or phosphorus and the alkyl II group may
further contain a
function group, such as hydroxyl, ether, ketone, oxime, hydrazone, imine, and
Schiff base. In
one embodiment, the sugar is selected from a group comprising Glc, Ara(pyr),
Ara(fur), Rha,
and Xyl. In another embodiment, R4 is selected from the group consisting of:
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O
O O O ,,A_~ X
O
N" X' N" X' N" X'
I I _,~I
OR'
OR' OR' OR' ~ I I X
+ O
HO
OR' OR'
O O
\
OH OH
[0047] wherein the configuration of any stereo-center is R or S; X is OR or
NR,
wherein R is alkyl or aryl; X' is alkyl, OR, NR, wherein R is alkyl or aryl;
and R' is H, alkyl,
or acyl. As disclosed herein, the compounds are dammaranes, particularly
ginsenosides and
their analogues. As used herein, the teml "ginsenoside" refers to the class of
triterpene
glycosides which includes, without limitation, the specific compounds Ral,
Ra2, Ra3, Rbl,
Rb2, Rb3, Rc, Rd, Re, Rf, Rgl, (20R)Rg2, (20S)Rg2, (20R)Rg3, (20S)Rg3, Rg5,
Rg6, Rhl,
(20R)Rh2, (20S)Rh2, Rh3, Rh4, (20R)Rg3, (20S)Rg3, Rkl, Rk2, Rk3, Rsl, Rs2,
Rs3, Rs4,
Rs5, Rs6, Rs7, F4, Rgk351, protopanaxadiol (PPD), protopanaxatriol (PPT),
DHPPD-I,
DHPPD-II, DHPPT-I, DHPPT-II, a butanol-soluble fraction of sun ginseng, white
ginseng or
red ginseng or analogues or homologues thereof. The ginsenosides of the
present invention
may be chemically associated with carbohydrates including, but not limited to,
glucopyranosyl, arabinopyranosyl, arabinofuranosyl and rhamnopyranosyl. The
ginsenosides
of the present invention may be isolated ginsenoside compounds or isolated and
further
synthesized ginsenosides. The isolated ginsenosides of the present invention
can be further
synthesized using processes including, but not necessarily limited to, heat,
light, chemical,
enzymatic or other synthesis processes generally known to the skilled artisan.
[0048] The present invention further provides a method for the synthesis of a
compound having formula:
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R3 R
4
R,
HO
e-
R2
wherein the method comprises the steps of:
(a) treating a compound having formula:
R3 R4
R,
HO
RZ
with an oxidizing agent, to form a compound having formula:
R3 R
4
Rl
R2
(b) treating the compound formed in step (a) with a reducing agent, to form a
compound having formula:
R3 R4
R,
HO
e-
R2
wherein R, is H or OH; R2 is selected from the group consisting of H, OH, OAc,
and O-X,
wherein X is a carbohydrate containing one or more sugars or acylated
derivatives thereof; R3
is selected from the group consisting of H, OH, and OAc; and R4 is alkenyl,
aryl, or alkyl. In
one embodiment, the oxidizing agent is chromic anhydride and the reducing
agent is NaBH4.
[00491 The starting material, i.e. the compound having formula:
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23
R3 R
4
R,
HO
4$-
R2
particularly, betulafolienetriol, may be obtained from plants including,
without limitation,
common birch. The extracts of these plants are rich sources of
betulafolienetriol and are
desired starting materials for making ginsenosides because they cost
significantly less than
ginseng.
[0050] The present invention also provides a method for the synthesis of a
compound
having formula:
R3 R
4
R5
RIe-
R2
wherein the method comprises the steps of:
(a) treating a compound having formula:
R3 R
4
R5
HO
e-
R2
with an oxidizing agent, to form a compound having formula:
R3 R
4
R5
RZ
(b) treating the compound formed in step (a) with a reducing agent, to form a
compound having formula:
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R3 R
4
R5
HO
e-
R2
(c) optionally, treating the compound formed in step (b) with protected Rl
derivative, to form a compound having formula:
R3 R4
R5
R, Protected
e-
R2
(d) treating the compound formed in step (c) with deprotection agent, to form
a
compound having formula:
R3 R
4
R5
Rl
e-
R2
wherein Rl is selected from the group consisting of a-OH, P-OH, a-O-X, (3-O-X,
a-R6COO-
, R-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate containing
one or more
sugars or acylated derivatives thereof, and R6 is alkenyl, aryl, or alkyl I;
R2 is selected from
the group consisting of H, OH, OAc, and O-X, wherein X is a carbohydrate
containing one or
more sugars or acylated derivatives thereof; R3 is selected from the group
consisting of H,
OH, and OAc; R4 is alkenyl, aryl, or alkyl II; and R5 is H or OH. The alkyl I
group may
further contain oxygen, nitrogen, or phosphorus; and the alkyl II group may
further contain a
function group, such as hydroxyl, ether, ketone, oxime, hydrazone, imine, and
Schiff base. In
one embodiment, the oxidizing agent is chromic anhydride and the reducing
agent is NaBH4.
In another embodiment, the protected Rl derivative is a protected Rl halogen
derivative. For
example, the protected RI derivative may be protected by an Ac8- group. The
protected Rj
group may be deprotected using agents such as NaOMe.
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[0051] Additionally, the present invention provides a method for the synthesis
of a
compound having formula:
OH
---
O
GIcGIc H
wherein the method comprises the steps of:
5 (a) treating a compound having formula:
HO
HO OH
H
with an oxidizing agent, to form a compound having formula:
HO
OH
O
H
(b) treating the compound formed in step (a) with a protecting agent, to form
a
10 compound having formula:
HO
OAc
O
H
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26
(c) treating the compound formed in step (b) with a reducing agent, to form a
compound having formula:
HO
OAc
HO 0
H
(d) treating the compound formed in step (c) with Ac8-Glc-Glc-Br, to form a
compound having formula:
HO
OAc
Ac$blcGlcO H
(e) treating the compound formed in step (d) with deprotection agent, to form
a
compound having formula:
HO
OH
O
GIcGIc H
(f) further modifying the compound formed in step (e) to form a compound
having formula:
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27
OH
---
O
GIcGIc H
In one embodiment, the oxidizing agent is chromic anhydride, the reducing
agent is NaBH4,
the compound is deprotected using NaOMe.
[0052] The present invention also provides a method for the synthesis of a
compound
having formula:
HO
HO
H
wherein the method comprises the step of treating a compound having formula:
HO
H
with a reducing agent, such as, NaBH4.
[0053] Also provided is a method for the synthesis of a compound having
formula:
HO
O
GIcGIc H
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28
wherein the method comprises the steps of:
(a) treating a compound having formula:
HO
O
4 H
with a reducing agent, to form a compound having formula:
HO ~
HO
H
(b) treating the compound formed in step (a) with Ac8-Glc-Glc-Br, to form a
compound having formula:
HO ~
Ac$ O
,GlcGc H
(c) treating the compound formed in step (d) with deprotection agent, to forin
a
compound having formula:
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29
HO
O
GIcGIc H
In one embodiment, the reducing agent is NaBH4 and the compound is deprotected
using
NaOMe.
[0054] Additionally, the present invention provides ginsenoside compositions
for use
in modulating amyloid-beta production in a subject, treating or preventing
Alzheimer's
disease and treating or preventing neurodegeneration comprising a mixture of
isolated or
isolated and further synthesized ginsenosides, wherein one or more of the
ginsenosides is
selected from the group consisting of: Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Rc, Rd,
Re, Rf, Rgl,
(20R)Rg2, (20S)Rg2, (20R)Rg3, (20S)Rg3, Rg5, Rg6, Rhl, (20R)Rh2, (20S)Rh2,
Rh3, Rh4,
(20R)Rg3, (20S)Rg3, Rkl, Rk2, Rk3, Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, F4,
protopanaxadiol (PPD), protopanaxatriol (PPT), DHPPD-I, DHPPD-II, DHPPT-I,
DHPPT-II,
a butanol-soluble fraction of sun ginseng, white ginseng or red ginseng or
analogues or
homologues thereof. In an embodiment of the invention, the ginsenoside
composition is
Rgk351.
[0055] The present invention provides methods and pharmaceutical compositions
for
use in decreasing amyloid-beta production, comprising use of a
pharmaceutically-acceptable
carrier and a ginsenoside compound. Examples of acceptable pharmaceutical
carriers,
formulations of the pharmaceutical compositions, and methods of preparing the
formulations
are described herein. The pharmaceutical coinpositions may be useful for
administering the
dammarane and ginsenoside compounds of the present invention to a subject to
treat a variety
of disorders, including neurodegeneration and/or its associated symptomology,
as disclosed
herein. The ginsenoside compound is provided in an amount that is effective to
treat the
disorder (e.g., neurodegeneration) in a subject to whom the pharmaceutical
composition is
administered. The skilled artisan, as described above, may readily determine
this amount. In
one embodiment, the present invention provides a method for inhibiting 0-
amyloid
production in a subject, comprising administering a compound having the
general formula:
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R3 R
4
R5
R,
4-
R2
to the subject, wherein RI is selected from the group consisting of a-OH, (3-
OH, a-O-X, (3-
O-X, a-R6COO-, (3-R6COO-, a-R6P03-, and (3-R6P03-, wherein X is a carbohydrate
containing one or more sugars or acylated derivatives thereof, and R6 is
alkenyl, aryl, or alkyl
5 I; R2 is selected from the group consisting of H, OH, OAc, and O-X, wherein
X is a
carbohydrate containing one or more sugars or acylated derivatives thereof; R3
is selected
from the group consisting of H, OH, and OAc; R4 is alkenyl, aryl, or alkyl II;
and R5 is H or
OH. As used herein, the term "subject" includes, for example, an animal, e.g.
human, rat,
mouse, rabbit, dog, sheep, and cow, as well as an in vitro system, e.g. a
cultured cell, tissue,
10 and organ.
[0056] The present invention also provides a method for treating
neurodegeneration
in a subject in need of treatment, by contacting cells (preferably, cells of
the CNS) in the
subject with an amount of a ginsenoside compound or composition effective to
decrease
amyloid-beta production in the cells, thereby treating the neurodegeneration.
Examples of
15 neurodegeneration which may be treated by the method of the present
invention include,
without limitation, Alzheimer's disease, amyotrophic lateral sclerosis (Lou
Gehrig's disease),
Binswanger's disease, corticobasal degeneration (CBD), dementia lacking
distinctive
histopathology (DLDH), frontotemporal dementia (FTD), Huntington's chorea,
multiple
sclerosis, myasthenia gravis, Parkinson's disease, Pick's disease, and
progressive supranuclear
20 palsy (PSP). In a preferred embodiment of the present invention, the
neurodegeneration is
Alzheimer's disease (AD) or sporadic Alzheimer's disease (SAD). In a further
embodiment
of the present invention, the Alzheimer's disease is early-onset familial
Alzheimer's disease
(FAD). The skilled artisan can readily determine when clinical symptoms of
neurodegeneration have been ameliorated or minimized.
25 [0057] The present invention also provides a method for treating or
preventing a
pathological condition, such as neurodegeneration and AP42-related disorder,
in a subject in
need of treatment, comprising administering to the subject one or more
ginsenoside
compounds in an amount effective to treat the neurodegeneration. The A(342-
related disorder
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may be any disorder caused by A(342 or has a symptom of aberrant A(342
accumulation. As
used herein, the phrase "effective to treat the neurodegeneration" means
effective to
ameliorate or minimize the clinical impairment or symptoms of the
neurodegeneration. For
example, where the neurodegeneration is Alzheimer's disease, the clinical
impairment or
symptoms of the neurodegeneration may be ameliorated or minimized by reducing
the
production of amyloid-beta and the development of senile plaques and
neurofibrillary tangles,
thereby minimizing or attenuating the progressive loss of cognitive function.
The amount of
inhibitor effective to treat neurodegeneration in a subject in need of
treatment will vary
depending upon the particular factors of each case, including the type of
neurodegeneration,
the stage of the neurodegeneration, the subject's weight, the severity of the
subject's
condition, and the method of administration. This amount can be readily
determined by the
skilled artisan. In one embodiment, the present invention provides a method
for treating or
preventing neurodegeneration in a subject, comprising administering a compound
having the
general formula:
R3 R4
R5
RI
R2
to the subject, wherein Rl is selected from the group consisting of a-OH, (3-
OH, a-O-X, (3-
O-X, a-R6COO-, (3-R6COO-, a-R6P03-, and (i-R6PO3-, wherein X is a carbohydrate
containing one or more sugars or acylated derivatives thereof, and R6 is
alkenyl, aryl, or alkyl
I; R2 is selected from the group consisting of H, OH, OAc, and O-X, wherein X
is a
carbohydrate containing one or more sugars or acylated derivatives thereof; R3
is selected
from the group consisting of H, OH, and OAc; R4 is alkenyl, aryl, or alkyl II;
and R5 is H or
OH.
[0058] In one embodiment of the invention, Alzheimer's disease is treated in a
subject in need of treatment by administering to the subject a therapeutically
effective amount
of a ginsenoside composition, a ginsenoside or analogue or homologue thereof
effective to
treat the Alzheimer's disease. The subject is preferably a mammal (e.g.,
humans, domestic
animals, and commercial animals, including cows, dogs, monkeys, mice, pigs,
and rats), and
is most preferably a human. The term analogue as used in the present invention
refers to a
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chemical compound that is structurally similar to another and may be
theoretically derivable
from it, but differs slightly in composition. For example, an analogue of the
ginsesnoside
(20S)Rg3 is a compound that differs slightly from (20S)Rg3 (e.g., as in the
replacement of
one atom by an atom of a different element or in the presence of a particular
functional
group), and may be derivable fronl (20S)Rg3. The term homologue as used in the
present
invention refers to members of a series of compounds in which each member
differs from the
next member by a constant chemical unit. The term synthesize as used in the
present
invention refers to formation of a particular chemical compound from its
constituent parts
using synthesis processes known in the art. Such synthesis processes include,
for example,
the use of light, heat, chemical, enzymatic or other means to form particular
chemical
composition.
[0059] The term "therapeutically effective amount" or "effective amount," as
used
herein, means the quantity of the composition according to the invention which
is necessary
to prevent, cure, ameliorate or at least minimize the clinical impairment,
symptoms or
complications associated with Alzheimer's disease in either a single or
multiple dose. The
amount of ginsenoside effective to treat Alzheimer's disease will vary
depending on the
particular factors of each case, including the stage or severity of
Alzheimer's disease, the
subject's weight, the subject's condition and the method of administration.
The skilled
artisan can readily determine these amounts. For example, the clinical
impairment or
symptoms of Alzheimer's disease may be ameliorated or minimized by diminishing
any
dementia or other discomfort suffered by the subject; by extending the
survival of the subject
beyond that which would otherwise be expected in the absence of such
treatment; or by
inhibiting or preventing the progression of the Alzheimer's disease.
[0060] Treating Alzheimer's disease, as used herein, refers to treating any
one or
more of the conditions underlying Alzheimer's disease including, without
limitation,
neurodegeneration, senile plaques, neurofibrillary tangles, neurotransmitter
deficits,
dementia, and senility. As used herein, preventing Alzheiiner's disease
includes preventing
the initiation of Alzheimer's disease, delaying the initiation of Alzheimer's
disease,
preventing the progression or advancement of Alzheimer's disease, slowing the
progression
or advancement of Alzheimer's disease, and delaying the progression or
advancement of
Alzheimer's disease.
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[0061] Prior to the present invention, the effect of dammaranes and
ginsenosides on
production of beta amyloid protein was unknown. The present invention
establishes that
ginsenosides such as (20S)Rg3, Rkl and Rg5 or their analogues or homologues
can also be
used to prevent and treat Alzheimer's disease patients. This new therapy
provides a unique
strategy to treat and prevent neurodegeneration and dementia associated with
Alzheimer's
disease by modulating the production of A(342. Further, neurodegeneration and
dementias
not associated with Alzheimer's disease can also be treated or prevented using
the
ginsenosides of the present invention to modulate the production of A(342.
[0062] The ginsenosides of the present invention include natural or synthetic
functional variants, which have ginsenoside biological activity, as well as
fragments of
ginsenoside having ginsenoside biological activity. As further used herein,
the term
"ginsenoside biological activity" refers to activity that modulates the
generation of the highly
amyloidogenic A(342, the 42-amino acid isoform of amyloid 0-peptide. In an
embodiment of
the invention, the ginsenoside reduces the generation of A[i42 in the cells of
a subject.
Commonly known ginsenosides and ginsenoside compositions include, but are not
limited to,
Ral, Ra2, Ra3, Rbl, Rb2, Rb3, Rc, Rd, Re, Rf, Rgl, (20R)Rg2, (20S)Rg2,
(20R)Rg3,
(20S)Rg3, Rg5, Rg6, Rhl, (20R)Rh2, (20S)Rh2, Rh3, Rh4, (20R)Rg3, (20S)Rg3,
Rkl, Rk2,
Rk3, Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, F4, Rgk351, protopanaxadiol (PPD),
protopanaxatriol (PPT), DHPPD-I, DHPPD-11, DHPPT-I, DHPPT-11, a butanol-
soluble
fraction of sun ginseng, white ginseng or red ginseng or analogues or
homologues thereof.
In one embodiment of the invention the ginsenoside is Rkl. In another
embodiment of the
invention, the ginsenoside is (20S)Rg3. In a further embodiment, the
ginsenoside is Rg5. In
still another embodiment, the ginsenoside composition is Rgk351, a mixture of
(20S)Rg3,
Rg5 and Rkl.
[0063] Methods of preparing ginsenosides such as Rkl, (20S)Rg3 and Rg5, as
well as
their analogues and homologues, are well known in the art. For example, U.S.
Patent
5,776,460, the disclosure of which is incorporated herein in its entirety,
describes preparing a
processed ginseng product in which a ratio of ginsenoside (Rg3 + Rg5) to (Rc +
Rd + Rbl +
Rb2) is above 1Ø The processed product disclosed in U.S. Patent 5,776,460 is
prepared by
heat-treating ginseng at a high temperature of 120 to 180 C for 0.5 to 20
hours. The
ginsenosides of the present invention may be isolated ginsenoside compounds or
isolated and
further synthesized ginsenoside compounds. The isolated ginsenosides of the
present
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34
invention can be further synthesized using processes including, but not
necessarily limited to,
heat, light, chemical, enzymatic or other synthesis processes generally known
to the skilled
artisan.
[0064] In a method of the present invention, the ginsenoside compound is
administered to a subject in combination with one or more different
ginsenoside compounds.
Administration of a ginsenoside compound "in combination with" one or more
different
ginsenoside compounds refers to co-administration of the therapeutic agents.
Co-
administration may occur concurrently, sequentially, or alternately.
Concurrent co-
administration refers to administration of the different ginsenoside compounds
at essentially
the same time. For concurrent co-administration, the courses of treatment with
the two or
more different ginsenosides may be run simultaneously. For example, a single,
combined
formulation, containing both an amount of a particular ginsenoside compound
and an amount
of a second different ginsenoside compound in physical association with one
another, may be
administered to the subject. The single, combined formulation may consist of
an oral
formulation, containing amounts of both ginsenoside compounds, which may be
orally
administered to the subject, or a liquid mixture, containing amounts of both
the ginsenoside
compounds, which may be injected into the subject.
[0065] It is also within the confines of the present invention that an amount
of one
particular ginsenoside compound and an amount one or more different
ginsenoside
compound may be administered concurrently to a subject, in separate,
individual
formulations. Accordingly, the method of the present invention is not limited
to concurrent
co-administration of the different ginsenoside compounds in physical
association with one
another.
[0066] In the method of the present invention, the ginsenoside compounds also
may
be co-administered to a subject in separate, individual formulations that are
spaced out over a
period of time, so as to obtain the maximum efficacy of the combination.
Administration of
each therapeutic agent may range in duration from a brief, rapid
administration to a
continuous perfusion. When spaced out over a period of time, co-administration
of
the ginsenoside compounds may be sequential or alternate. For sequential co-
administration,
one of the therapeutic agents is separately administered, followed by the
other. For example,
a full course of treatment with an Rg5 derivative may be completed, and then
may be
followed by a full course of treatment with an Rkl derivative. Alternatively,
for sequential
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co-administration, a full course of treatment with Rkl derivative may be
completed, then
followed by a full course of treatment with an Rg5 derivative. For alternate
co-
administration, partial courses of treatment with the Rkl derivative may be
alternated with
partial courses of treatment with the Rg5 derivative, until a full treatment
of each therapeutic
5 agent has been administered.
[0067] The therapeutic agents of the present invention (i.e., the ginsenoside
and
analogues and analogues thereof) may be administered to a human or animal
subject by
known procedures including, but not limited to, oral administration,
parenteral administration
(e.g., intramuscular, intraperitoneal, intravascular, intravenous, or
subcutaneous
10 administration), and transdermal administration. Preferably, the
therapeutic agents of the
present invention are administered orally or intravenously.
[0068] For oral administration, the formulations of the ginsenoside may be
presented
as capsules, tablets, powders, granules, or as a suspension. The formulations
may have
conventional additives, such as lactose, mannitol, corn starch, or potato
starch. The
15 formulations also may be presented with binders, such as crystalline
cellulose, cellulose
analogues, acacia, cornstarch, or gelatins. Additionally, the formulations may
be presented
with disintegrators, such as cornstarch, potato starch, or sodium
carboxymethyl cellulose.
The formulations also may be presented with dibasic calcium phosphate
anhydrous or sodium
starch glycolate. Finally, the formulations may be presented with lubricants,
such as talc or
20 magnesium stearate.
[0069] For parenteral administration, the formulations of the ginsenoside may
be
combined with a sterile aqueous solution which is preferably isotonic with the
blood of the
subject. Such formulations may be prepared by dissolving a solid active
ingredient in water
containing physiologically-compatible substances, such as sodium chloride,
glycine, and the
25 like, and having a buffered pH compatible with physiological conditions, so
as to produce an
aqueous solution, then rendering said solution sterile. The formulations may
be presented in
unit or multi-dose containers, such as sealed ampules or vials. Moreover, the
formulations
may be delivered by any mode of injection including, without limitation,
epifascial,
intracapsular, intracutaneous, intramuscular, intraorbital, intraperitoneal
(particularly in the
30 case of localized regional therapies), intraspinal, intrasternal,
intravascular, intravenous,
parenchymatous, or subcutaneous.
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[0070] For transdermal administration, the formulations of the ginsenoside may
be
combined with skin penetration enhancers, such as propylene glycol,
polyethylene glycol,
isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like, which
increase the
permeability of the skin to the therapeutic agent, and permit the therapeutic
agent to penetrate
through the skin and into the bloodstream. The therapeutic agent/enhancer
compositions also
may be further combined with a polymeric substance, such as ethylcellulose,
hydroxypropyl
cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to
provide the
composition in gel form, which may be dissolved in a solvent such as methylene
chloride,
evaporated to the desired viscosity, and then applied to backing material to
provide a patch.
[0071] The dose of the ginsenoside of the present invention may also be
released or
delivered from an osmotic mini-pump. The release rate from an elementary
osmotic mini-
pump may be modulated with a microporous, fast-response gel disposed in the
release orifice.
An osmotic mini-pump would be useful for controlling release, or targeting
delivery, of the
therapeutic agents.
[0072] It is within the confines of the present invention that the
formulations of the
ginsenoside may be further associated with a pharmaceutically-acceptable
carrier, thereby
comprising a pharmaceutical composition. The pharmaceutically-acceptable
carrier must be
"acceptable" in the sense of being compatible with the other ingredients of
the composition,
and not deleterious to the recipient thereof. Examples of acceptable
pharmaceutical carriers
include, but are not limited to, carboxymethyl cellulose, crystalline
cellulose, glycerin, gum
arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium
alginate,
sucrose, starch, talc, and water, among others. Formulations of the
pharmaceutical
composition may conveniently be presented in unit dosage.
[0073] The formulations of the present invention may be prepared by methods
well
known in the pharmaceutical art. For example, the active compound may be
brought into
association with a carrier or diluent, as a suspension or solution.
Optionally, one or more
accessory ingredients (e.g., buffers, flavoring agents, surface active agents,
and the like) also
may be added. The choice of carrier will depend upon the route of
administration. The
pharmaceutical composition would be useful for administering the therapeutic
agents of the
present invention (i.e., ginsenosides their analogues and analogues, either in
separate,
individual formulations, or in a single, combined formulation) to a subject to
treat
Alzheimer's disease. The therapeutic agents are provided in amounts that are
effective to
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37
treat or prevent Alzheimer's disease in the subject. These amounts may be
readily
determined by the skilled artisan.
[0074] The effective therapeutic amounts of the ginsenoside will vary
depending on
the particular factors of each case, including the stage of the Alzheimer's
disease, the
subject's weight, the severity of the subject's condition, and the method of
administration.
For example, (20S)Rg3 can be administered in a dosage of about 5 g/day to
1500 mg/day.
Preferably, (20S)Rg3 is administered in a dosage of about 1 mg/day to 1000
mg/day. Rg5
can be administered in a dosage of about 5 g/day to 1500 mg/day, but is
preferably
administered in a dosage of about lmg/day to 1000mg/day. Rkl can be
administered in a
dosage of about 5 g/day to 1500 mg/day, but is preferably administered in a
dosage of about
lmg/day to 1000 mg/day. Further, the ginsenoside composition Rgk351 can be
administered
in a dosage of about 5 g/day to 1500 mg/day, but is preferably administered in
a dosage of
about lmg/day to 1000 mg/day. The appropriate effective therapeutic amounts of
any
particular ginsenoside compound within the listed ranges can be readily
determined by the
skilled artisan depending on the particular factors of each case.
[0075] The present invention additionally encompasses methods for preventing
Alzheimer's disease in a subject with a pre-Alzheimer's disease condition,
comprising
administering to the subject a therapeutically effective amount of a
ginsenoside compound.
As used herein, "pre-Alzheimer's disease condition" refers to a condition
prior to
Alzheimer's disease. The subject with a pre-Alzheimer's disease condition has
not been
diagnosed as having Alzheimer's disease, but nevertheless may exhibit some of
the typical
symptoms of Alzheimer's disease and/or have a medical history likely to
increase the
subject's risk to developing Alzheimer's disease.
[0076] The invention further provides methods for treating or preventing
Alzheimer's
disease in a subject, comprising administering to the subject a
therapeutically effective
amount of ginsenoside compound.
EXAMPLES
[0077] The following examples illustrate the present invention, which are set
forth to
aid in the understanding of the invention, and should not be construed to
limit in any way the
scope of the invention as defined in the claims which follow thereafter.
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[0078] The inventors have unexpectedly found that at least three Ginsenoside
compounds, Rkl, (20S)Rg3 and Rg5 as well as the mixture Rgk351, lower the
production of
A(342 in cells, thus treating AD and non-AD associated neuropathogenesis
and/or preventing
the progression of AD and non-AD associated neuropathogenesis. Rgk351 and Rkl
were
most effective in reducing AR42 levels. Further, Rkl was shown to inhibit the
A042
production in the cell-free assay using a partially purified y-secretase
complex, suggesting
that Rk1 modulates either specificity and/or activity of the y-secretase
enzyme.
EXAMPLE 1
[0079] The potential effects of ginsenosides and their analogues in treating
AD were
examined. First, a number of ginsenosides were screened based on their effects
on A(3
generation. The effects of various ginsenosides on A(3 (e.g., A[i40 and A(342)
production was
initially accessed by incubating the Chinese hamster ovary (CHO) cells
expressing human
APP (CHO-APP cells) with each ginsenoside purified from unprocessed ginseng
(known as
"white ginseng"). These representative ginsenosides included Rbl, Rb2, Rc, Rd,
Re, Re, Rgl
and Rg2 and differ in their side chains and sugar moieties.
[0080] Tables 1-3 Structure of ginsenosides utilized in the study and their
effects on
A(342 generation. They differ at the two or three side chains attached to the
common
triterpene backbone known as danunarane. The common structure skeleton for
each group of
ginsenosides is shown in the top panel. Ginsenosides that harbor A042-lowering
activity are
indicated in the far right column of the tables: AJ342-lowering activity
("Yes"), no profound
effects ("No"), and non-determined ("ND"). Ginsenosides that affected cell
viability are
indicated as "Cytotoxic." Abbreviation for carbohydrates are as follows: Glc,
D-
glucopyranosyl; Ara (pyr), L-arabinopyranosyl; Ara (fur), L-arabinofuranyosyl;
Rha, L-
rhamnopyranosyl.
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Table 1
R3~
OH
RIO
,,~s~i
R2
A(342-lowering
Ginsenoside Rl R2 R3 activity
PPD (Protopanaxadiol) -H -H -H No
Ral -Glc-Gle -H -Glc-Ara (pyr)-Xyl ND
Ra2 -Glc-Glc -H -Glc-Ara (fur)-Xyl ND
Ra3 -Glc-Glc -H -Glc-Glc-Xyl ND
Rbl -Glc-Glc -H -Glc-Glc No
Rb2 -Glc-Glc -H -Glc-Ara (pyr) No
Rb3 -Glc-Glc -H -Glc-Xyl No
Rc -Glc-GIc-AC -H -Glc-Ara (fur) No
Rd -Glc-GIc-AC -H -Glc No
Rg3 (20R) -Glc-GIc-AC -H -H Yes
Rg3 (20S) -Glc-Gle -H -H Yes
Rh2 (20R,S) -Glc -H -H Yes/Cytotoxic
Rsl -Glc-Glc -H -Glc-Ara (pyr) ND
Rs2 -Glc-Glc -H -Glc-Ara (fur) ND
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Rs3 -Glc-Glc -H -H Yes/Cytotoxic
PPT (Protopanaxatiol) -H -OH -H Yes
Re -H -O-Glc- -Glc No
Rf -H Rha -H ND
Rgl -H -O-Glc- -Glc No
Glc
Rg2 (20R,S) -H -H No
-O-GIc
Rh1 (20R,S) -H -H No
-0-Gle-
Rha
-O-Glc
Table 2
OH
RIO
,,~~
R2
Ginsenoside Ri R2 A(342-lowering activity
DHPPD-I H H ND
(Double-bond PPD)
Rkl -Glc-Glc -H Yes
Rk2 -Glc -H ND
Rs5 -Glc-Glc-Ac -H Yes/Cytotoxic
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DHPPT-I -H -OH ND
(Double-bond PPT)
Rg6 -H -O-GIc-Rha No
Rk3 -H -O-GIc No
Rs7 -H -O-GIc-Ac ND
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Table 3
OH
R1O
R2
Ginsenoside RI R2 A(342-lowering activity
DHPPD-II H -H ND
Rg5 -Gic-Glc -H Yes
Rh3 -Glc -H ND
Rs4 -Glc-Glc-Ac -H ND
DHPPT-II -H -OH ND
F4 -H -O-GIc-Rha ND
Rh4 -H -O-GIc No
Rs6 -H -O-GIc-Ac ND
[0081] After 8 hours of incubation, the media were collected and the levels of
secreted A[i40 and A042 were determined by ELISA. None of the ginsenosides
from the
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43
group Rb 1, Rb2, Rc, Rd, Re, Re, Rg 1 and Rg2 exhibited any inhibitory effects
on A(340 and
A(342 production (Figure 10).
[0082] Steaming ginseng at high temperature gave rise to additional
ginsenosides
with enhanced pharmacological activity, including (20S)Rg3, Rkl and Rg5 (22-
25). Next,
the effects of these heat-processing derived ginsenosides (e.g., (20S)Rg3,
Rh1, Rh2, Rkl,
Rg6, Rg5) on AP40 and AP42 generation were tested. Initial screening
identified three
structurally related ginsenosides, Rkl, (20S)Rg3, and Rg5, which selectively
lowered the
secretion of Aj342 (Figure 11). In contrast, AJ342 levels were not affected by
(20R)Rg3, Rhl,
and Rg6. A(340 levels were not changed by treatment with any of the
ginsenosides tested.
The potency of A(342-lowering activity was highest with Rkl and (20S)Rg3. Rg5
was a less
effective A(342-lowering reagent as compared to Rkl or (20S)Rg3 (Figure 2).
The secretion
of AP40 was affected by treatment with Rkl only at very high concentration (-
100 M) and
cell viability was not affected by treatment of Rk1 under these conditions (up
to 100 M, 8
hour treatment; data not shown). Interestingly, the P S I AE9 FAD mutation
diminished A[i42-
lowering response to (20S)Rg3, Rkl and Rg5 treatment (Figure 11B) as compared
to PS1
wild-type expressing cells (Figure 11A). Further analyses revealed that Rkl
and Rg5 lower
A042 in a dose-dependent manner (Figure 12A). Overnight treatment with Rgk351,
Rkl, and
Rg5 also reduce A042 production in CHO-APP cells (Figure 12B). A(342-lowering
activity
of Rkl was similar to that of sulindac sulfide, one of the known A(342-
lowering NSAIDs.
During overnight treatment, A040 production was also slightly affected by
treatment with
Rkl or sulindac sulfide (Figure 12B). These studies provide a structure-
activity relationship
between the chemical structures of ginsenosides and A042-lowering activity,
further
providing the basis for designing additional A[i42-lowering analogues as well
as for defining
a class of compounds that harbor A(342-lowering activity.
[0083] Rkl did not affect steady-state levels of full-length APP in both CHO-
APP
and Neuro2a-APPsw cells (Figure 13), suggesting that the reduction of A042 is
likely due to
altered post-translation processing of APP. In contrast to the full-length
form, the steady-
state levels of C-terminal APP fragments were up-regulated by treatment with
Rkl (Figure
13). These data suggest that Rkl may affect the g-secretase cleavage step
(e.g., A(342
cleavage), therefore causing the accumulation of APP C-terminal fragments, as
has been
shown for a general y-secretase inhibitor Compound E. A(342 levels in the
medium of each
corresponding samples are shown in the bottom panel.
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[0084] Since the effect of Rkl was rather selective to A[342 (but not A[i40)
in a cell-
based assay, the question of whether Rkl affects other y-secretase-mediated
cleavage events,
including the generation of AICD resulted from a transmembrane cleavage of APP
distal
from either A(340 or A042 site, and y-secretase-mediated intramembrane
cleavage of Notchl
or p75 neurotrophin receptor (p75NTR) to yield Notchl or p75NTR intracellular
domains
(NICD or p75-ICD, respectively) was tested. The cell-free generation of AICD,
NICD and
p75-ICD was not affected by incubation with Rgk351 or Rkl (Figure 5). Under
these
conditions, Compound E efficiently inhibited the cell-free generation of ICDs
and sulinac
sulfide did not affect ICD generation from APP, Notchl or p75NTR. These data
indicate that
Rkl is not a general inhibitor of y-secretase cleavage and does not affect the
intramembrane
cleavage of other y-secretase substrate, such as Notchl or p75NTR.
[0085] Next, the inhibitory effects of Rkl and (20S)Rg3 on A(3 generation in
an in
vitro y-secretase assay was studied. Both Rkl and sulindac sulfide potently
inhibited A[342
generation in vitro (Figure 15). In contrast, naproxen, an NSAID without A(342-
lowering
activity, had no effects on A042 production (Figure 15A). Similar to what has
been reported
for A042-lowering NSAIDs (Weggen, et al., Evidence that nonsteroidal anti-
inflammatory
drugs decrease amyloid beta 42 production by direct modulation of gamma-
secretase activity,
J. Biol. Chem. 278:3183-3187 (2003)), A(342-lowering ginsenosides (e.g., Rkl
and
(20S)Rg3) inhibited both A[i40 and AP42 with a similar potency in a cell-free
y-secretase
assay (Figure 15B), although both compounds primarily affect A(342 production
in cell-based
assay.
[0086] Ginsenosides are metabolized by human intestinal bacteria after oral
administration of ginseng extract (Kobayashi K., et al., Metabolism of
ginsenoside by human
intestinal bacteria [II] Ginseng Review 1994; 18: 10-14; Hasegawa H., et al.,
Main ginseng
saponin metabolites formed by intestinal bacteria. Planta Med. 1996; 62: 453-
457.).
Therefore, the effects of two major metabolites of ginsenosides, including
20(S)-
protopanaxatriol (PPT) and 20(S)-protopanaxadiol (PPD) on A(342 generation
were tested.
20(S)-panaxatriol (PT) and 20(S)-panaxadiol (PD) are the artificial
derivatives of PPT and
PPPD, respectively. Treatment witli either PPT or PT reduced the production of
A(342
without affecting the levels of AJ342 in Neuro2a cells expressing the human
Swedish mutant
form of APP (Neuro2a-SW) as well as in CHO cells expressing wild-type human
APP
(Figure 16). PPD and PD did not confer any inhibitory effects on A040 or A042
generation.
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[0087] In summary, A042-lowering natural compounds that originate from heat-
processed ginseng have been identified. A042-lowering ginsenosides, including
Rkl and
(20S)Rg3, appear to specifically modulate y-secretase activity that is
involved in A[i42
production. Structure-activity defines a class of compounds that could serve
as a foundation
5 for development of effective therapeutic agents for treatment of AD.
EXAMPLE 2
[0088] The benefits of ginsenoside therapy for treating AD associated
neurodegeneration can be demonstrated in a murine model of AD. Specifically,
the
ginsenoside compounds (20S) Rg3, Rkl, Rg5 and Rgk351 can be used to treat mice
suffering
10 from AD associated neurodegeneration.
[0089] Mice expressing human APP as well as mice expressing the Swedish
familial
Alzheimer's disease mutant form of APP can be obtained from the Jackson
Laboratory, 600
Main Street, Bar Harbor, Maine 04609. Four groups of mice can then be studied:
(1) APP
mice without ginsenoside treatment (placebo); (2) Swedish mice without
ginsenoside
15 treatment (placebo); (3) APP mice + RgS (100 g/ l/day); and (4) Swedish
mice + Rg5 (100
g/ l/day). After approximately 16 weeks of injection therapy, amounts of A(342
in the
serum of the mice can be measured. It is expected that the results of this
study will
demonstrate the general benefits of ginsenoside therapy for treating AD
associated
neuordegeneration. APP and Swedish mice without ginsenoside treatment should
have
20 significantly higher levels of serum A042 and demonstrate behavior
characterisitic of
neurodegeneration, as compared with APP and Swedish mice receiving ginsenoside
treatment.
EXAMPLE 3
[0090] The genuine sapogenines of the ginseng glycosides are structurally
similar to
25 some chemical constituents of other plants. Betulafolienetriol [dammar-24-
ene-
3a,12(3,20(S)-triol}] isolated from birch leaves differ from the genuine
sapogenin of ginseng
glycosides, 20(S)-protopanaxadiol, in the configuration at C-3 only.
Therefore,
betulafolienetriol, cheap and relatively accesable, makes a desirable sustrate
to prepare
20(S)-protopanaxadiol and its glycoside Rg3, Rg5, and Rkl.
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46
Scheme 1
OH HO / HO
[01 AczO
-~ --
HO O OH
H H l
OAc 0 OAc 0
NaBH4 0 3~Acs-Glc-Gllc-Br
--
O HO
H 2 H 3
OAc O OH HO
NaOMe
-- --
AcB O
H Rg3
,GicGlc H 4 GIcGIcO
e"-
-
0
GIcGIc H Rkl or Rg5
[0091] Betulafolienetriol was isolated from an ethereal extract of the leaves
Btula
pendula, followed by chromatography on silica gel and crystallization from
acetone: mp 195-
1950, lit. 197-198 (Fischer et al. (1959) Justus Liebigs Ann. Chem. 626:185).
[0092] The 12-O-acetyl derivative of 20(S)-protopanaxadiol (3) is prepared
from
betulafolienetriol by the sequence of reactions showen in Scheme 1.
Betulafolienetriol is
oxidized to ketone 1, dammar-24-ene-12[i, 20(S)-diol-3-one, mp 197-199 , lit
196-199 ,
CA 02573699 2007-01-11
WO 2006/019685 PCT/US2005/024533
47
(yield: 60%), which is acetylated with acetic anhydride in pyridine to give
compound 2, 12-
O-Acetyl-dammar-24-ene-12(3, 20(S)-diol-3-one (yield: 100%?) (Nagal et al.,
(1973) Chem.
Pharm. Bull. 9:2061). 1H NMR (CDC13) of the compound 2: 0.90 (s, 3 H), 0.95
(s, 3 H), 1.0
(s, 6 H), 1.1 (s, 3 H), 1.1 (s, 3 H), 1.65 (s, 3 H), 1.72 (s, 3 H), 2.1 (s, 3
H), 3.04 (s, 1 H), 4.73
(td, 1 H), 5.17 (t, 1 H). Sodium borohydride reduction of the compound 2 in 2-
propanol
affords compound 3, 12-O-Acetyl-dammar-24-ene-3 (3, 12(3, 20(S)-triol (yield:
90%). 1 H
NMR (CDC13) of the compound 3: 0.78 (s, 3 H), 0.86 (8, 3 H), 0.95 (s, 3 H),
1.0 (s, 3 H),
1.02 (s, 3 H), 1.13 (s, 3 H), 1.64 (s, 3 H), 1.71 (s, 3 H), 2.05 (s, 3 H,
OAc), 3.20 (dd, 1 H, H-
3a), 4.73 (td, 1 H, H-12(x), 5.16 (t, 1 H, H-24).
[0093] Condensation of compound 3 with 0-acetylate-sugar bromide in the
presence
of silver oxide and molecular sieves 4A in dichloroethane results in formation
of compound 4
(yield: 50%). Specifically, a mixture of compound 3 (1.08 g, 2 mmol), silver
oxide (1.4 g, 6
mmol), a-acetobromoglucose (2.47 g, 6 mmol), molecular sieves 4A (1.0 g) and
dichloroethane (20m1) was agitated at ambient temperature until the
acetobromoglucose had
reacted (TLC). The reaction mixture was then diluted with CHC13 and filtered.
The solvent
was evaporated and the residue was washed with hot water to remove the excess
of glucose
derivatives. Silica gel column chromatography (8:1 n-hexane-acetone) gave
compound 4
(853 mg). Deprotection of the glucoside 4 gives ginsenoside Rg3 which is
concerted to Rkl
or Rg5 in 2 steps.
Scheme 2
HO HO ~
0 NaBH4
-
O HO
H 5 H 6
HO
0 7
Ac8-Glc-Glc-Br NaOMe
O
GIcGIc H 7
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48
[0094] While the foregoing invention has been described in some detail for
purposes
of clarity and understanding, it will be appreciated by one skilled in the
art, from a reading of
the disclosure, that various changes in form and detail can be made without
departing from
the true scope of the invention in the appended claims.
