Methods for decreasing beta amyloid protein

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.

BACKGROUND OF THE INVENTION 
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 an 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. 
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. 
It is therefore an object of the present invention to provide methods for 
predicting populations at risk of developing AD. 
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. 
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 
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. 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.. 
Diagnostic kits based on the discovery of these risk factors include 
reagents for measurement of cholesterol, total lipoproteins, and/or Apo 
E4. 
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 
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. 
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: 
(1) individuals with Apo E4 and high cholesterol, defined as a blood 
cholesterol level of greater than 200 mg/dl, 
(2) post menopausal women with high cholesterol, especially those who are 
not taking estrogen, 
(3) young individuals with high blood cholesterol levels who are not obese 
(age 48-65 yrs), 
(4) individuals with high blood cholesterol levels who have a family 
history of AD, 
(5) individuals with high blood cholesterol levels who have a family 
history of AD, and 
(6) all adult individuals with Down's syndrome. 
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). 
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.. 
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. 
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, 34-47). 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. 
Representative CoA reductase inhibitors include the statins, including 
lovastatin, simvastatin, compactin, fluvastatin, atorvastatin, 
cerivastatin, and pravastin. These are typically administered orally. 
Compounds which inhibit cholesterol biosynthetic enzymes, including 
2,3-oxidosqualene cyclase, squalene synthase, and 7-dehydrocholesterol 
reductase, can also be used. 
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. 
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 
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, 
fluvastatin, pravastatin and compactin, significantly inhibit the level of 
A.beta. production in human neuronal cultures. 
Example 1 
Cholesterol Increases the Level of A.beta. in Human Neuronal Cultures. 
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 of very 
low density lipoprotein (VLDL), low density lipoprotein (LDL) or high 
density lipoprotein (HDL) particles isolated from human plasma. These 
lipoprotein particles are 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.125 I-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. 
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. 
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 
Dietary Cholesterol Increases A.beta. Levels in the Brain. 
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. 
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 
HMG CoA Reductase Inhibitors Inhibit the Production of A.beta. by Human 
Neurons. 
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. 
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. 
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. 
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.