U.S. patent application number 10/198331 was filed with the patent office on 2002-12-05 for kits for determining risk of alzheimer's disease.
This patent application is currently assigned to Children's Medical Center Corporation. Invention is credited to Nadeau, Philip, Yankner, Bruce A..
Application Number | 20020183379 10/198331 |
Document ID | / |
Family ID | 21942345 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020183379 |
Kind Code |
A1 |
Yankner, Bruce A. ; et
al. |
December 5, 2002 |
Kits for determining risk of Alzheimer's disease
Abstract
Blood cholesterol levels are correlated with production of
amyloid .beta. protein (A.beta.), and are predictors of populations
at risk of developing AD. Methods for lowering blood cholesterol
levels can be used to decrease production of A.beta., thereby
decreasing the risk of developing AD. The same methods and
compositions can also be used for treating individuals diagnosed
with AD. Methods include administration of compounds which increase
uptake of cholesterol by the liver, such as the administration of
HMG CoA reductase inhibitors, administration of compounds which
block endogenous cholesterol production, such as administration of
HMG CoA reductase inhibitors, administration of compositions which
prevent uptake of dietary cholesterol, and administration of
combinations of any of these which are effective to lower blood
cholesterol levels, Methods have also been developed to predict
populations at risk, based on the role of cholesterol in production
of A.beta.. For example, individuals with Apo E4 and high
cholesterol, defined as a blood cholesterol level of greater than
200 mg/dl, post menopausal women with high cholesterol
levels--especially those who are not taking estrogen, or
individuals which high blood cholesterol levels who are not obese
are all at risk of developing AD if blood cholesterol levels are
not decreased
Inventors: |
Yankner, Bruce A.; (West
Newton, MA) ; Nadeau, Philip; (Boston, MA) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Assignee: |
Children's Medical Center
Corporation
|
Family ID: |
21942345 |
Appl. No.: |
10/198331 |
Filed: |
July 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10198331 |
Jul 14, 2002 |
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09239387 |
Jan 28, 1999 |
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6440387 |
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09239387 |
Jan 28, 1999 |
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09046235 |
Mar 23, 1998 |
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6080778 |
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Current U.S.
Class: |
514/423 ;
514/460; 514/548 |
Current CPC
Class: |
A61K 31/40 20130101;
A61K 31/4418 20130101; A61K 31/225 20130101; A61K 31/365 20130101;
A61P 43/00 20180101; G01N 2333/4709 20130101; A61P 25/28 20180101;
G01N 33/6896 20130101; A61P 3/06 20180101; A61K 31/22 20130101;
A61K 31/445 20130101; G01N 2800/2821 20130101; A61K 31/00 20130101;
A61K 31/366 20130101; A61K 31/404 20130101; A61K 31/401
20130101 |
Class at
Publication: |
514/423 ;
514/460; 514/548 |
International
Class: |
A61K 031/401; A61K
031/365; A61K 031/225 |
Claims
We claim:
1. A method for decreasing the production of A.beta. comprising
administering a composition which decreases blood cholesterol
levels to a person with elevated cholesterol levels who is at risk
of, or exhibits the symptoms of, Alzheimer's disease.
2. The method of claim 1 wherein the composition is an HMG CoA
reductase inhibitor.
3. The method of claim 2 wherein the composition is selected from
the group consisting of lovastatin, simvastatin, fluvastatin,
pravastatin, atorvastatin, cerivastatin, and compactin.
4. The method of claim 1 wherein the composition inhibits uptake of
dietary cholesterol.
5. The method of claim 1 wherein the composition blocks or
decreases endogenous cholesterol production.
6. The method of claim 1 wherein composition increases cholesterol
metabolism or clearance.
7. The method of claim 1 wherein the person carries the
apolipoprotein E4 gene.
8. The method of claim 1 wherein the person has trisomy 21 (Down's
syndrome).
9. The method of claim 1 wherein the person carries one or more
mutations in the genes that encode amyloid .beta. protein, amyloid
precursor protein, presenilin-1 or presenilin-2.
10. The method of claim 1 wherein the person has a family history
of Alzheimer's disease or dementing illness.
11. The method of claim 1 wherein the person is a post menopausal
woman with high cholesterol.
12. The method of claim 1 wherein the person has high blood
cholesterol levels who is not obese.
13. The method of claim 1 wherein the person is between 48-65 years
of age.
14. A method for predicting if a person is at risk of developing
Alzheimer's Disease comprising determining if they have elevated
blood levels of cholesterol.
15. The method of claim 14 wherein the level is 200 mg/dl or
greater.
16. The method of claim 14 further comprising determining if the
person carries the apolipoprotein E4 gene.
17. The method of claim 14 further comprising determining if the
person has trisomy 21 (Down's syndrome).
18. The method of claim 14 further comprising determining if the
person carries one or more mutations in the genes that encode
amyloid .beta. protein, amyloid precursor protein, presenilin-1 or
presenilin-2.
19. The method of claim 14 further comprising determining if the
person has a family history of Alzheimer's disease or dementing
illness.
20. The method of claim 14 further comprising determining if the
person is a post menopausal woman with high cholesterol.
21. A kit for determining if a person is at risk of developing
Alzheimer's disease comprising reagents for determining if the
blood cholesterol level is in excess of 200 mg/dl.
22. The kit of claim 21 further comprising reagents for determining
at least one of the factors selected from the group consisting of
the apolipoprotein E4 gene or its product, amyloid .beta. protein,
amyloid precursor protein, presenilin-1, and presenilin-2.
23. A composition for decreasing the production of A.beta.
comprising an effective amount of a compound to decrease blood
cholesterol levels.
24. The composition of claim 23 comprising an HMG CoA reductase
inhibitor.
25. The composition of claim 24 wherein the inhibitor is selected
from the group consisting of lovastatin, simvastatin, fluvastatin,
pravastatin, atorvastatin, cerivastatin, and compactin.
26. The composition of claim 23 comprising a compound which
inhibits uptake of dietary cholesterol.
27. The composition of claim 23 wherein the composition blocks or
decreases endogenous cholesterol production.
28. The composition of claim 27 wherein the composition comprises
an inhibitor of the cholesterol biosynthetic enzymes selected from
the group consisting of 2,3-oxidosqualene cyclase, squalene
synthase, and 7-dehydrocholesterol reductase.
29. The composition of claim 23 wherein the composition is selected
from the group consisting of a fibrate, a bile acid binding resin,
probucol, nicotinic acid, garlic or garlic derivative, and
psyllium.
Description
BACKGROUND OF THE INVENTION
[0001] The United States government has certain rights in this
invention by virtue of National Institutes of Health grant number
RO1NS33325 to Bruce A. Yankner.
[0002] Alzheimer's disease (AD) is the most common cause of
dementia in the aged population. The accumulation of large numbers
of senile plaques containing the 40-42 amino acid amyloid .beta.
protein (A.beta.) is a classic pathological feature of AD. Both
genetic and cell biological findings suggest that the accumulation
of A.beta. in the brain is the likely cause of AD (Yankner, B. A.
(1996) Neuron 16, 921-932.; Selkoe, D. J. Science 275, 630-631
(1997)). Strong genetic evidence in support of the pathogenic role
of A.beta. came from the observation that individuals who inherit
mutations in the amyloid precursor protein almost invariably
develop AD at an early age. These mutations increase the production
of a long variant of the A.beta. peptide that forms senile plaques
in the brain (Goate et al., (1991) Nature 349, 704-706). Mutations
and allelic variations in other genes that cause AD, including the
presenilins and apolipoprotein E, also result in increased
production or deposition of the A.beta. peptide. Reiman, et al.
(1996) N.E.J.Med. 334, 752-758, reported that in middle age, early
to mid 50's, individuals who are homozygous for the Apo E4 gene
have reduced glucose metabolism in the same regions of the brain as
in patients with Alzheimer's disease. These findings suggest that
the pathological changes in the brain associated with this gene
start early. Furthermore, individuals with Down's syndrome
overexpress the amyloid precursor protein, develop A.beta. deposits
in the brain at an early age, and develop Alzheimer's disease at
all early age. Finally, the A.beta. protein has been demonstrated
to be highly toxic to nerve cells Thus, it is widely believed that
drugs which decrease the levels of A.beta. in the brain would
prevent Alzheimer's disease.
[0003] The known genetic causes of AD can account for only a small
proportion of the total number of cases of AD. Most cases of AD are
sporadic and occur in the aged population. A major goal of research
is the identification of environmental factors that predispose to
AD that would be amenable to therapeutic measures.
[0004] It is therefore an object of the present invention to
provide methods for predicting populations at risk of developing
AD.
[0005] It is another object of the present invention to provide
diagnostics and pharmaceuticals to decrease the production of
amyloid .beta. protein (A.beta.), and thereby to prevent or reduce
the liklihood of developing AD.
[0006] It is a further object of the present invention to provide
pharmaceutical treatments to treat AD in patients' having the
neuropsychiatric or diagnostic criteria for AD.
SUMMARY OF THE INVENTION
[0007] Blood cholesterol levels are correlated with production of
amyloid .beta. protein (A.beta.), and are predictors of populations
at risk of developing AD. Methods for lowering blood cholesterol
levels can be used to decrease production of A.beta., thereby
decreasing the risk of developing AD. The same methods and
compositions can also be used for treating individuals diagnosed
with AD. Methods include administration of compounds which increase
uptake of cholesterol by the liver, such as the administration of
HMG CoA reductase inhibitors, administration of compounds which
block endogenious cholesterol production, such as administration of
HMG CoA reductase inhibitors, administration of compositions which
prevent uptake of dietary cholesterol, and administration of
combinations of any of these which are effective to lower blood
cholesterol levels. Methods have also been developed to predict
populations at risk, based on the role of cholesterol in production
of A.beta.. For example, individuals with Apo E4 and high
cholesterol, defined as a blood cholesterol level of greater than
200 mg/dl, post menopausal women with high cholesterol
levels--especially those who are not taking estrogen, or
individuals which high blood cholesterol levels who are not obese
are all at risk of developing AD if blood cholesterol levels are
not decreased. In the preferred embodiment, individuals with these
risk factors are treated to lower blood cholesterol levels prior to
developing any mental impairment attributable to AD, based on
accepted neuropsychiatric and diagnostic criteria in clinical
practice. Treatment is based on adminstration of one or more
compositions effective to lower cholesterol blood levels at least
10%, which is believed to be sufficient to decrease production of
A.beta..
[0008] Diagnostic kits based on the discovery of these risk factors
include reagents for measurement of cholesterol, total
lipoproteins, and/or Apo E4.
[0009] The examples demonstrate the use of HMG CoA reductase
inhibitors to treat Alzheimer's disease. Rats fed a high
cholesterol diet show increased levels of the Alzheimer's disease
A.beta. protein in the brain. Cholesterol has been shown to
increase the amount of A.beta. in human neurons in culture. The HMG
CoA reductase inhibitors reduce cholesterol production. Several
different HMG CoA reductase inhibitors, including lovastatin,
simvastatin, fluvastatin, pravastatin and compactin, significantly
inhibit the level of A.beta. production in human neuronal
cultures.
DETAILED DESCRIPTION OF THE INVENTION
I. Methods for Predicting Populations at Risk for AD
[0010] Individuals at increased risk for A.beta. accumulation and
Alzheimer's disease are those who carry a copy of the
apolipoprotein E4 gene (Strittmatter et al., (1993) Proc. Natl.
Acad. Sci. U.S.A. 90, 1977-1981), those with trisomy 21 (Down's
syndrome) (Mann and Esiri, (1989) J. Neurol. Sci 89, 169-179)), and
individuals who carry a mutation in one of the genes that encode
the amyloid precursor protein, presenilin-1 or presenilin-2
(reviewed in Yankner, 1996). In addition, individuals with a family
history of Alzheimer's disease have been documented to be at
increased risk of Alzheimer's disease (Farrer et al., (1989) Ann.
Neurol. 25, 485-492; van Duijn et al., (1991) Int. J. Epidemiol. 20
(suppl 2), S13-S20), and could therefore benefit from prophylactic
treatment with an HMG CoA reductase inhibitor.
[0011] Methods have also been developed to predict populations at
risk, based on the role of cholesterol in production of A.beta..
Several risk factors for developing AD have been identified. These
include:
[0012] (1) individuals with Apo E4 and high cholesterol, defined as
a blood cholesterol level of greater than 200 mg/dl,
[0013] (2) post menopausal women with high cholesterol, especially
those who are not taking estrogen,
[0014] (3) young individuals with high blood cholesterol levels who
are not obese (age 48-65 yrs),
[0015] (4) individuals with high blood cholesterol levels who have
a family history of AD.
[0016] (5) individuals with high blood cholesterol levels who have
a family history of AD, and
[0017] (6) all adult individuals with Down's syndrome.
[0018] These individuals are all at risk of developing AD if blood
cholesterol levels are not decreased. In the preferred embodiment,
individuals with these risk factors are treated to lower blood
cholesterol levels prior to developing any mental impairment
attributable to AD using accepted neuropsychiatric and diagnostic
criteria for probable Alzheimer's disease (McKhahn et al. (1984)
Neurology 34:939-944).
[0019] Individuals can be screened using standard blood tests for
cholesterol, ApoE4, and/or total lipoprotein levels, as well as by
taking a medical and family history. In addition, over the counter
immunoassay tests can be used by individuals who may be at risk, so
that they can seek further medical advise. These immunoassay kits
can be qualitative and/or quantitative for elevated cholesterol,
total lipoproteins, and Apo E4.
II. Methods of Treatment to Decrease Production of A.beta..
[0020] Methods for lowering blood cholesterol levels can be used to
decrease production of A.beta., thereby decreasing the risk of
developing AD. The same methods can also be used to treat patients
who have already been diagnosed with AD. Methods include
administration of compounds which increase uptake of cholesterol by
the liver, such as the administration of HMG CoA reductase
inhibitors, administration of compounds which block endogenous
cholesterol production, such as administration of HMG CoA reductase
inhibitors, administration of compositions which prevent uptake of
dietary cholesterol, and administration of combinations of any of
these which are effective to lower blood cholesterol levels
[0021] The examples indicate that several different HMG CoA
reductase inhibitors reduce the production of A.beta.. HMG CoA
reductase inhibitors may act to lower cholesterol at several
different levels. For example, HMG CoA reductase inhibitors have
been shown to lower blood cholesterol levels by upregulating
liporprotein clearance receptors in the liver (Brown and Goldstein,
(1986) Science 232, 3447). In addition, HMG CoA reductase
inhibitors will directly inhibit cholesterol synthesis in neurons.
Since every HMG CoA reductase inhibitor tested reduces A.beta.
production, it is anticipated that new members of this class of
drugs will also inhibit A.beta. production. Furthermore, since
increased dietary cholesterol increases A.beta. in the brain, drugs
which act through other mechanisms to reduce cholesterol will also
inhibit A.beta. production.
[0022] Representative CoA reductase inhibitors include the statins,
including lovastatin, simvastatin, compactin, fluvastatin,
atorvastatin, cerivastatin, and pravastin. These are typically
administered orally.
[0023] Compounds which inhibit cholesterol biosynthetic enzymes,
including 2,3-oxidosqualene cyclase squalene synthase, and
7-dehydrocholesterol reductase, can also be used.
[0024] Representative compositions which decrease uptake of dietary
cholesterol include the bile acid binding resins (cholestryramine
and colestipol) and the fibrates (clofibrate). Probucol, nicotinic
acid, garlic and garlic derivatives, and psyllium are also used to
lower blood cholesterol levels. Probucol and the fibrates increase
the metabolism of cholesterol-containing lipoproteins. The
cholesterol-lowering mechanism of nicotinic acid is not
understood.
[0025] Although the preferential route of administration of HMG CoA
reductase inhibitors would be oral, the drugs could also by
administered by intravenous, subcutaneous or intramuscular routes.
In some cases, direct administration into the cerebrospinal fluid
may be efficacious.
III. EXAMPLES
[0026] Prior to the studies described in the following examples,
the relationship between cholesterol and A.beta. levels in the
brain was unknown. In one study, rabbits which were fed a high
cholesterol diet showed increased immunocytochemical staining of
brain neurons with an antibody to A.beta.. However, this antibody
was not specific for A.beta., and could cross-react with other
metabolites of the amyloid precursor protein (Sparks, D. L. (1996)
Neurobiology of Aging. 17, 291-299). The studies in the following
examples demonstrate that: rats fed a high cholesterol diet show
increased levels of the Alzheimer's disease A.beta. protein in the
brain; cholesterol increases the amount of A.beta. in human neurons
in culture; HMG CoA reductase inhibitors reduce cholesterol
production; and several different HMG CoA reductase inhibitors,
including lovastatin, simvastatin. Iluvastatin, pravastatin and
compactin, significantly inhibit the level of A.beta. production in
human neuronal cultures.
Example 1
[0027] Cholesterol Increases the Level of A.beta. in Human
Neuronial Cultures.
[0028] Busciglio et al., (1993) Proc. Nat. Acad. Sci. 90,
2092-2096, described the production of A.beta. by human cortical
neurons in culture. To determine whether cholesterol can affect the
production of A.beta., primary human brain cultures were
established from the cortex of 16-20 week fetal abortuses, and the
neurons incubated in the absence or presence or very low density
lipoprotein (VLDL), low density lipoprotein (LDL) or high density
lipoprotein (HDL) particles isolated from human plasmas These
lipoprotein particles arc the physiological vehicles for the
transport of cholesterol to cells. The effects of the different
lipoprotein particles on the levels of A.beta. in the human
cortical cultures was determined. The human cortical cultures were
maintained in serum-free Dulbecco's Modified Eagle's Medium (DMEM)
with N2 supplements (a serum-free supplement that supports neuronal
viability). The medium was then changed to the same medium
(controls) or medium supplemented with VLDL, LDL, or HDL particles.
After incubation for 48-72 hours, A.beta. was measured by
immunoprecipitation of the culture medium with a polyclonal
antibody to A.beta. (B12), followed by Western blotting with a
monoclonal antibody to A.beta. (6E10). The Western blots were
developed either by the enhanced chemiluminescence method or by
addition of an .sup.125I-labeled secondary antibody and
phosphorimager scanning. The bands corresponding to the 40 and 42
amino acid form of A.beta. were analyzed quantitatively using a
computer software program Control human cortical cultures produced
basal levels of A.beta.. Exposure of the human cortical cultures to
VLDL, LDL or HDL particles increased the levels of both the 40 and
42 amino forms of A.beta.. These results suggest that the major
classes of cholesterol-containing lipoproteins all act to increase
production of A.beta. in human neurons.
[0029] It was then determined whether lipoprotein particles
containing apolipoproteins E or A1 were able to increase A.beta.
production. To address this question, synthetic lipoprotein
particles containing these proteins were created. Particles
containing either apolipoprotein E or A1 increased the level of
A.beta. in the human cortical cultures.
[0030] These results indicate that a variety of different
cholesterol carrying lipoprotein particles can increase the
production of A.beta. in primary human neuronal cultures.
Example 2
[0031] Dietary Cholesterol Increases A.beta. Levels in the
Brain.
[0032] After establishing that cholesterol-carrying lipoprotein
particles increase A.beta. in cultures of human neurons, it was
determined whether dietary cholesterol increases the level of
A.beta. in the brain in vivo. Increased dietary intake of
cholesterol is known to increase circulating levels of lipoprotein
particles, which in turn increases the delivery of cholesterol to
cells. These experiments were performed on 20 month old rats The
rats were fed a low cholesterol diet (0.1% cholesterol) or a high
cholesterol diet (5% cholesterol). After 10 weeks, the animals were
sacrificed and the cortex was removed for measurement of A.beta.
levels. A.beta. was assayed by immunoprecipitating cortical
homogenates with the A.beta. antibody B12, followed by Western
blotting with the commercially available A.beta. monoclonal
antibody 4G8.
[0033] Resolution of the A.beta. isolated from rat cerebral cortex
by electrophoretic separation on gels showed that A.beta. levels
were significantly increased by about 50% in the group of rats fed
the high cholesterol diet relative to the group of rats fed the low
cholesterol diet. These findings indicate that dietary cholesterol
increases the amount of A.beta. in the brain. It is noteworthy that
the approximately 50% increase in A.beta. in the brain induced by a
high cholesterol diet is similar to the increase in A.beta. which
occurs in Down's syndrome, which is known to predispose to the
development of Alzheimer's disease.
Example 3
[0034] HMG CoA Reductase Inhibitors Inhibit the Production of
A.beta. by Human Neurons.
[0035] The HMG CoA reductase inhibitors have been used in humans to
decrease plasma levels of cholesterol in patients at risk for heart
disease The discovery that cholesterol increases the amount of
A.beta. in the brain led to this investigation to determine whether
the HMG CoA reductase inhibitors may be therapeutically efficacious
for Alzheimer's disease by inhibiting the production of A.beta..
Human cortical neuronal cultures were established from 18 weeks
gestation normal fetal cortical tissue as described above and
maintained in a culture medium comprised of DMEM containing N2
supplements. After one week, the culture medium was changed to
DMEM+N2 supplements (control), or DMEM+N2 supplements+either 100
.mu.M lovastatin, 100 .mu.M simvastatin, 100 .mu.M compactin, 100
.mu.M fluvastatin, or 1 mM pravastatin. after incubation for 48
hours, the cultured cells were harvested and the levels of A.beta.
were assayed, as described above.
[0036] A.beta. was isolated from the culture medium from human
cortical neuronal cultures and resolved by electrophoresis in gels.
These results demonstrate that human neurons treated with either
lovastatin, simvastatin compactin, fluvastatin or pravastatin have
significantly decreased levels of A.beta. relative to controls.
These results indicate that HMG CoA reductase inhibitors decrease
the production of A.beta. by human neurons.
[0037] The finding that HMG CoA reductase inhibitors inhibit
A.beta. production by human cortical cells supports the use of this
class of drugs for reducing the levels of A.beta. in individuals
with Alzheimer's disease or at risk of developing Alzheimer's
disease.
[0038] Modifications and variations of the methods and compositions
for prediction of the liklihood of developing AD, and for
preventing and/or treating AD, will be obvious to those skilled in
the art. These modifications and variations are intended to come
within the scope of the appended claims.
* * * * *