U.S. patent application number 13/517773 was filed with the patent office on 2012-11-08 for treating learning deficits with inhibitors of hmg coa reductase.
Invention is credited to Yijun Cui, Steven A. Kushner, Weidong Li, Alcino Silva.
Application Number | 20120283323 13/517773 |
Document ID | / |
Family ID | 35503642 |
Filed Date | 2012-11-08 |
United States Patent
Application |
20120283323 |
Kind Code |
A1 |
Silva; Alcino ; et
al. |
November 8, 2012 |
Treating Learning Deficits with Inhibitors of HMG CoA Reductase
Abstract
The disclosure provides methods of treating cognitive disorders
by administering a HMG CoA reductase inhibitor. Cognitive deficits
treatable with the inhibitor compound include those associated with
Angelman Syndrome, Neurofibromatosis-1, certain forms of X-linked
mental retardation, tuberous sclerosis, Down Syndrome, autism, and
attention deficit/hyperactivity disorder.
Inventors: |
Silva; Alcino; (Sherman
Oaks, CA) ; Cui; Yijun; (Los Angeles, CA) ;
Li; Weidong; (Shanghai, CN) ; Kushner; Steven A.;
(Rotterdam, NL) |
Family ID: |
35503642 |
Appl. No.: |
13/517773 |
Filed: |
June 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11569426 |
Nov 20, 2006 |
8222293 |
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PCT/US05/18129 |
May 23, 2005 |
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13517773 |
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60661764 |
Mar 14, 2005 |
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60574442 |
May 24, 2004 |
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Current U.S.
Class: |
514/460 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 31/366 20130101; A61P 25/28 20180101 |
Class at
Publication: |
514/460 |
International
Class: |
A61K 31/366 20060101
A61K031/366; A61P 25/00 20060101 A61P025/00 |
Goverment Interests
1. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] The United States government may have certain rights in this
invention pursuant to Grant No. AG13622 awarded by the National
Institutes of Health.
Claims
1-56. (canceled)
57. A method of treating a subject with a cognitive deficit,
comprising: administering an effective amount of a
hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitor to a
subject afflicted with a cognitive disorder associated with defects
in the RAS-MAPK pathway.
58. The method of claim 57, wherein the inhibitor comprises a
statin.
59. The method of claim 58, wherein the statin is selected from the
group consisting of atorvastatin, cerivastatin, fluvastatin,
lovostatin, pravastatin, pitavastatin, rovustatin, simvastatin, and
compatible mixtures thereof.
60. The method of claim 57, wherein the inhibitor comprises a
mixture of HMG CoA reductase inhibitors.
61. The method of claim 57, wherein the effective amount of the
inhibitor is dose that does not significantly lower total serum
cholesterol level in the subject.
62. The method of claim 57, wherein the cognitive disorder is
associated with dysregulation of small monomeric GTP binding
protein activity.
63. The method of claim 62, wherein the small monomeric GTP binding
protein activity is that of RAS protein.
64. The method of claim 57, wherein the cognitive disorder is
associated with dysregulation of mitogen activated protein kinase
(MAPK) signaling pathway.
65. The method of claim 57, wherein the cognitive disorder is
associated with increased inhibitory neuronal activity.
66. The method of claim 65, wherein the inhibitory neuronal
activity is associated with increased GABA-mediated inhibition.
67. The method of claim 66, wherein the GABA-mediated inhibition is
associated with activity of GABA.sub.A or GABA.sub.B.
68. The method of claim 57, further comprising administering a
farnesyl transferase inhibitor, a geranylgeranyl transferase
inhibitor, an inhibitor of .gamma.-aminobutyric acid (GABA)
mediated inhibition, or an inhibitor of GABA receptor activity.
69. The method of claim 68, wherein the administering is adjunctive
administration.
70. The method of claim 69, wherein the adjunctive administration
is simultaneous administration or sequential administration.
71. The method of claim 57, wherein the subject has a normal
cholesterol level.
Description
2. TECHNICAL FIELD
[0002] The disclosure provides methods and compounds for treating
learning and memory deficits and other cognitive disorders.
3. INTRODUCTION
[0003] Cognition is a complex neurological process where stimuli
are received and processed by the neuronal circuitry into
perception and memory, and where such processed information becomes
transformed into reasoning, judgment, awareness, and creativity.
Some understanding of the biological basis for the complex cellular
mechanisms that underlie cognition have come from identification of
genes affecting cognitive abilities in various animal models and
the molecular analysis of genetic abnormalities in the human
population that lead to impairment of various cognitive
processes.
[0004] The genetic defects identified as affecting cognitive
function implicate a diverse array of molecular mechanisms. A
number of genes are involved in signaling pathways, including
serine-threonine kinase RSK2 implicated in Coffin-Lowry syndrome,
neurofibromin implicated in Neurofibromatosis-1 (NF-1), and
signaling by small monomeric guanine nucleotide (GTP) binding
proteins implicated in a number of mental retardation (MR)
syndromes. Protein degradation pathways may also be involved, as
indicated by the identification of UBE3A gene affected in Angelman
syndrome. Transcription factors and transcriptional regulation in
cognitive processes are implicated by defects of CREB Binding
Protein (CBP) in Rubinstein-Taybi syndrome, mutations in
transcription repressor methyl-CpG binding protein in Rett
syndrome, and defects in helicase/histone deacetylase XH2 protein
in .alpha.-thalassemia (ATR-X syndrome). Protein synthesis appears
affected by mutations in the FMR1 gene associated with fragile X
mental retardation.
[0005] Although the identified genes affecting cognitive function
have diverse activities, it is suggested that they are related by
their effect on the signaling pathways involved in memory
formation, synaptic development, and synaptic maturation. For
instance, Ras mediated signal transduction may affect the
mitogen-activate protein kinase (MAPK) signaling pathway involving
MEK and ERK, which are part of a pathway involved in regulating the
activity of transcription factor CREB involved in consolidation of
memory and learning. Genes regulated by CREB are believed to affect
long term changes in synaptic properties, such as responsiveness to
neurotransmitters, membrane excitability, and number and size of
synapses. Additional lines of evidence linking such pathways with
cognitive function are provided by the effect of the kinase
activity of RSK2 in Coffin Lowry syndrome and the CBP in
Rubinstein-Taybi syndrome in modulating the activity of
transcription factor CREB.
[0006] Although the underlying cause of other cognitive disorders,
such as autism and attention deficit/hyperactivity disorder (ADHD)
have not been identified, there are indications that the
dysfunction in these conditions may also arise, at least in part,
in the cellular pathways involved in regulating synaptic activity
and functional plasticity. For example, some Rett syndrome patients
display autistic symptoms, while subjects diagnosed with autism
have abnormal expression of the gene associated with Rett syndrome
(Samaco, R. C. et al., Hum Mol. Genet. 13(6):629-39 (2004)).
Furthermore, characteristics of ADHD, which is a heterogeneous set
of dysfunctions characterized by deficits in sustained attention,
behavioral over activity, and impulsivity, are also observed in
some molecularly characterized cognitive disorders such as NF-1
(Barton, B. and North, K., Dev. Med. Child Neurol. 46(8):553-63
(2004)).
[0007] Although an understanding of the molecular basis of
cognitive function has advanced significantly, treatments for the
cognitive deficits associated with disorders of known and unknown
etiology have focused primarily on use of cognitive or physical
therapy to treat the symptoms of the disorder. These include
regimens emphasizing psychomotor development, speech therapy, and
special educational programs. Drug treatments, where available,
typically involve compounds affecting neurotransmitter activity.
For example, one treatment of Rett syndrome patients uses L-Dopa to
improve rigidity. Modulating glutamate receptor activity is the
target of dextromethorphan treatment in Rett syndrome and also the
focus of treatments with benzamide derivatives for fragile X
syndrome (see Danysz, W., Curr. Opin. Investig. Drugs. 3(7):1081-8
(2002)). ADHD has traditionally been treated with phychotropic
drugs, such as methylphenidate and pemoline. Although they may
ameliorate behavioral problems associated with hyperactivity,
improvements in cognitive function may not be significant.
[0008] Although drug therapies targeting neurotransmitters and
their receptors have a place in the treatment of cognitive
disorders, there is a need in the art for therapies targeting the
molecules and cellular pathways involved in cognitive function.
Modulating the underlying molecular basis responsible for a
cognitive deficit may provide longer lasting improvements in
cognitive function in subjects afflicted with these disorders.
4. SUMMARY
[0009] The present disclosure provides methods of treating
cognitive disorders by administering an effective amount of a
hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitor, where the
subject has a level of cholesterol that does not warrant
therapeutic intervention with the inhibitor to lower the
cholesterol levels. Generally, the class of HMG CoA reductase
inhibitor compounds useful in the treatments are statins, which are
normally prescribed to treat hypercholesterolemia. Dosages of the
inhibitor administered may be the dosages generally used to lower
serum cholesterol levels in subjects afflicted with
hypercholesterolemia. In some embodiments, dosages of the
inhibitors may comprise amounts that do not effectively lower
cholesterol levels in hypercholesterolemic patients but which are
effective in treating the cognitive disorder. In some embodiments,
the HMG CoA reductase inhibitors may be used in combination with
other inhibitor compounds, including farnesyl transferase
inhibitors, geranygeranyltransferase inhibitors, and inhibitors of
inhibitory neuronal activity (e.g., antagonists and inverse
agonists of GABA receptors)
[0010] Various disorders that manifest cognitive disorders may be
treated with the HMG CoA reductase inhibitors. These include
cognitive deficits associated with genetic abnormalities such as
Angelman Syndrome, Down Syndrome, neurofibromatosis NF-1, X-linked
mental retardation gene OPHN1, and tuberous sclerosis. In other
embodiments, identifiable cognitive disorders of unknown etiology
but which share disease characteristics with cognitive disorders of
a known genetic basis may be treated. Exemplary disorders of this
type are attention deficit/hyperactivity disorder (ADHD) and
autism.
[0011] In other embodiments, the inhibitor compounds are used to
treat cognitive disorders associated with dysregulation of the
basic cellular processes believed to be responsible for cognitive
function. These include dysregulation of small monomeric GTP
binding proteins implicated in learning and memory, such as Ras,
Rho, Rab, Sarl/Arf and Ran and their associated signaling pathways.
In other embodiments, the cognitive disorders treatable with the
compounds are associated with dysfunction in MAPF pathways and/or
inhibitory neuronal activity.
[0012] In some embodiments, the inhibitor compounds are used to
modulate the cellular correlates of cognitive function, such as
early and late forms of LTP. Because HMG CoA reductase inhibitors
appear to have no measurable effect on subjects with normal
cognitive function, the inhibitors are indicated for systems
displaying an abnormal LTP. Thus, in some embodiments, a neural
system with a depressed LTP response is contacted with an effective
amount of the inhibitor to modulate the LTP response.
[0013] Further provided herein are various compositions of
inhibitor compounds, including combinations of a HMG CoA reductase
inhibitor and a farneysl transferase inhibitor, HMG CoA reductase
inhibitor and a geranylgeranyl transferase inhibitor, and HMG CoA
reductase inhibitor and an inhibitor of GABA receptor activity. In
some embodiments, the compositions comprise a HMG CoA reductase
inhibitor and an excipient, where the HMG CoA reductase inhibitor
is present in an amount that does not significantly lower serum
cholesterol level but which is effective in treating a cognitive
disorder.
5. BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows down-regulation of p21Ras-MAPK activity in
nfl.sup.+/- mice by Lovastatin. a, Lovastatin decreased active MAPK
(phosphor-p44/42) in WT mice in a dose-dependent manner; b,
Lovastatin was effective at decreasing active MAPK in the
nfl.sup.+/- mice; c, Lovastatin also decreased active p21Ras
(21Ras-GTP) in nfl.sup.+/- mice.
[0015] FIG. 2 shows rescue by lovastatin of Nfl.sup.+/- deficits in
long-term potentiation. Percentage of baseline field EPSP (fEPSP)
is plotted over time. A five theta-burst induction protocol was
delivered at time 0 (WT=8, nfl.sup.+/-=7, WT with lovastatin=8,
nfl.sup.+/- with lovastatin=7). For clarity purposes, error bars
(standard error of the mean) are shown in only one direction.
Representative traces are shown from left to right: WT off drug,
nfl.sup.+/- off drug, WT on lovastatin, nfl.sup.+/- on lovastatin.
Horizontal bar represents 2 ms. Vertical bar represents 0.5 mV.
[0016] FIG. 3 shows lovastatin rescue of spatial learning deficits
in nfl.sup.+/- mice. a, Percent time spent in each quadrant during
a water maze probe trial on day 5; b, Percent time spent in each
quadrant during a probe trial on day 7; c, Average proximity to the
exact position where the platform was during training, compared
with proximity to the opposite position in the water maze.
Quadrants are training quadrant (TQ), adjacent left, opposite
quadrant (OP) and adjacent right. (WT=24, nfl.sup.+/-=21, WT with
lovastatin=21, nfl.sup.+/- with lovastatin=20)
[0017] FIG. 4 shows attention deficit in nfl.sup.+/- mice and
reversal of the attention and sensory gating deficit by treatment
with lovastatin. a, Tests in the lateralized reaction task in which
target-stimulus durations are randomly varied within session.
Target durations are 0.5, 1.0, or 2.0 sec. Correct choice rate is
plotted for WT and nfl.sup.+/- mice off lovastatin (WT=10,
nfl.sup.+/-=14); b, Correct choice rate is plotted for WT and
nfl.sup.+/- mice on lovastatin (WT with lovastatin=7, nfl.sup.+/-
with lovastatin=7); c, PPI was examined using prepulses at three
different stimulus intensities (70, 75 and 80 dB) (WT=8,
nfl.sup.+/-=8, WT with lovastatin=9, nfl.sup.+/- with
lovastatin=9).
6. DETAILED DESCRIPTION OF EMBODIMENTS
[0018] The present disclosure provides methods of treating
cognitive deficits by use of inhibitors of hydroxymethylglutaryl
CoA (HMG CoA) reductase. Cognitive deficits that may be treated by
the methods herein include those associated with known genetic
abnormalities and cognitive deficits displaying clinical symptoms
similar to, and in many cases overlapping with the identified
genetic causes of the cognitive dysfunction.
[0019] The compounds and compositions for use in the methods herein
comprise inhibitors of the enzyme HMG-CoA reductase, which
catalyzes the conversion of HMG-CoA to mevalonate, the isoprenoid
intermediate used for cholesterol biosynthesis. An important class
of HMG CoA inhibitor compounds is statins, which are used to treat
subjects with hypercholesterolemia to decrease serum cholesterol
and reduce the risk of associated diseases, such as heart disease
and atherosclerosis. Although the beneficial effects of statins
reside in their ability to lower cholesterol, the effects of the
drug are pleiotropic. Statins appear to affect endothelial cell
function via its effect on NO production and inhibition of reactive
oxygen species, proliferation of smooth muscle cells, inhibition of
platelet function, and suppression of vascular inflammation. In
some instances, statin therapy is linked to peripheral neuropathies
characterized by degeneration of nerves in a progressive and graded
fashion. Sensory nerves, for instance those sensing heat or pain,
appear most sensitive, but motor nerves and nerves involved in
coordination of movement are also involved. Thus, the art suggests
that statins may not be indicated for disorders affecting the
nervous system. The pleiotropic effects of statin are thought to be
associated with its interference with the attachment of lipid
moieties to various regulatory proteins.
[0020] Although statins are generally administered for treating
hypercholesterolemia, it is shown here that subjects suffering from
cognitive deficits associated with specific disorders, but who do
not display abnormal cholesterol levels, may benefit in improved
cognitive function that is adversely affected in the particular
disorder. Dose of statins comparable to the dosage generally
prescribed for hypercholesterolemia is shown to have beneficial
effects, and subjects with normal cognitive function are not
affected upon treatment with statins, suggesting that the statins
are affecting a physiological process that is abnormal or
imbalanced in the afflicted subject. Moreover, the studies herein
show that statins may cross the blood-brain barrier and have
therapeutic effect on neuronal cells to improve cognitive function
in subjects whose blood brain barrier may not be compromised by
traumatic injury, or age related diseases such as Alzheimer's or
other dementias.
6.1 Treatment of Cognitive Deficits
[0021] In accordance with the above, the methods disclosed herein
comprise administration of a HMG-CoA reductase inhibitor to
improve, enhance, or restore the cognitive function of subjects
suffering from a cognitive deficit. "Cognitive function" as used
herein refers to the performance of some cognitive activity, such
as memory, perception, learning, and reasoning. "Learning" refers
to acquisition of information and/or knowledge, and is typically
evaluated by exposing a subject to a learning experience and
observing changes in behavior arising from that experience.
Learning may be categorized as non-associative and associative.
Non-associative learning occurs when a subject is exposed to a
single stimulus in the absence of any other connected stimulus.
Habituation and sensitization are two examples of non-associative
learning. Associative learning occurs when a subject is exposed to
a stimulus in association with another stimulus or where a stimulus
is associated with the organism's behavior. Examples of associative
learning are classical conditioning or operant conditioning.
[0022] "Memory" refers to the storage and retrieval of information.
Memory is generally classified into short term memory (also called
working memory) and long term memory, where consolidation into long
term memory is believed occur through a stage involving short term
memory. Short-term memory lasts for period of seconds to minutes,
up to several days but which is subject to disruption and loss.
Long-term memory is durable and can last for years, up to the life
of the subject. As further described below, a correlate of learning
and memory at the cellular level is long term potentiation (LTP),
which is an increase in synaptic strength (i.e., potentiation) that
occurs following a train of stimuli of an afferent neural pathway.
There are different components to LTP that mimic short term and
long term memory. Short-term component of LTP typically follows a
single train of stimuli, is durable for minutes, and is not blocked
by inhibitors of protein synthesis. Long-term component of LTP
(L-LTP) can be induced by multiple trains of stimuli, may last for
hours to weeks, and requires transcription and protein synthesis.
Modulation of LTP is associated with activation of glutamate
receptors as well as activity of inhibitory GABA receptors
(Remondes, M. et al., Learn Mem. 10(4):247-52 (2003)).
[0023] Correspondingly, "cognitive disorder" refers to a disorder
that affects mental processes, including impairments of memory,
learning, awareness, attention, communication, motor coordination,
and/or intellectual capacity. "Impairment of cognition," or
"cognitive deficits" as used herein, are associated with various
disorders, including among others, developmental disabilities, such
as mental retardation, autism, dyslexia, attention
deficit/hyperactivity disorder, ischemic stroke, traumatic brain
injury, Alzheimer's Disease, degenerative dementia, obsessive
compulsive disorder, and schizophrenia. Such disorders are often
accompanied by personality and behavioral differences. However, a
cognitive deficit as used herein specifically excludes impaired
cognitive abilities associated with age related disorders, such as
Alzheimer's and degenerative dementias. An "age related disorder"
refers to a disorder in which the subject exhibits normal cognitive
abilities and function for an extended time period from birth, but
where cognitive function declines with passage of time. For
instance Alzheimer's is considered an age related disease where the
affected subject has normal cognitive abilities for much of the
individual's life until onset of the disease in late stages of
life. Although genetic abnormalities may contribute to a familial
form of Alzheimer's disease characterized by early onset, the time
period for manifestation of cognitive decline still requires about
30-50 years.
[0024] Humans with intellectual disabilities are those who develop
at a below average rate and experience difficulty in learning and
social adjustment. Intellectual disabilities refers to
significantly subaverage general intellectual functioning existing
concurrently with deficits in adaptive behavior and manifested
during the developmental period that adversely affects a subject's
educational performance. General intellectual functioning is
typically measured by an intelligence test that is adjusted for the
developmental level to which the test subject is a member.
[0025] "Subject" as used herein refers to an animal or a patient
for whom is intended the described treatment. Subjects include,
ayes (e.g., chickens, pigeons, owls), and mammals, including by way
of example and not limitation, members of rodentia (e.g., mouse,
rat, guinea pig), lagomorpha (e.g., rabbits, hares), perissodactyla
(e.g., horses, donkeys, etc.), artodactyla (e.g., pigs, cows,
sheep), carnivora (e.g., cats, canines), and primates (e.g., apes,
monkeys, baboons, and humans). Subjects also include animals
modified using recombinant DNA and/or transgenic techniques, such
as animals modified to inactivate, overexpress, or misexpress genes
involved or suspected of involvement in cognitive function.
[0026] In some embodiments, subject as used herein specifically
excludes those within a population for whom HMG CoA reductase
inhibitors are medically prescribed for higher than normal
cholesterol levels, or for elevated cholesterol levels that result
in adverse effects on cognitive function. A normal level of
cholesterol is a level that generally does not warrant therapeutic
use of HMG CoA reductase inhibitors and/or a level that does not
manifest itself in a cognitive deficit in a specified class of
subjects or in the general population. This level will depend on
the subject and variations in cholesterol levels observed with
respect to age, sex, and the population type. Generally,
cholesterol levels are measured when the subject is not suffering
from an acute illness, not under stress, and for a woman, when not
pregnant. The level of cholesterol as used herein refers to the
total serum cholesterol level, which includes the combined
cholesterol found in sera in the form of high density lipoprotein
(HDL), intermediate density lipoprotein (IDL), low density
lipoprotein (LDL) and very low density lipoprotein (VLDL).
[0027] An exemplary normal cholesterol level for a human is that
below about the 95th percentile of the general population pool,
below about the 85th percentile of the general population pool,
below about the 75th percentile of the general population pool,
below about the 50th percentile of the general population pool to
about the 25th percentile of the general population pool. Thus, in
some embodiments, a normal level for a human is below about 240
mg/dL, below about 220 mg/dL, below about 200 mg/dL, below about
190 mg/dL, below about 180 mg/dL, or below about 170 mg/dL, where
the lower limit of cholesterol level is that considered healthy for
the subject, such as about 120 mg/dL, 140 mg/dL, or 150 mg/dL,
depending on various factors, such as the age and sex of the
subject. A level consider healthy for a child or adolescent is
between about 120 mg/dL and about 170 mg/dL. An exemplary normal
level of serum cholesterol for a human adult is a range that is
below about 240 mg/dL or below about 200 mg/dL to about 140 mg/dL.
Thus, in some embodiments, the population of subjects treatable
using the methods herein include children, adolescents, and adults
who do not have abnormally elevated cholesterol levels and who have
not manifested age related cognitive disorders, as described
above.
[0028] In some embodiments, the cholesterol level may be based on
the amount of cholesterol in the LDL fraction. Cholesterol and
triglycerides found in sera fractionate into various components:
HDL, IDL, LDL, and VLDL. The LDL fraction derives from VLDL, and
elevated levels of total serum cholesterol and cholesterol in the
LDL (c-LDL) fraction are correlated with increased risk of
atherosclerosis. In some embodiments, the normal level of c-LDL for
a human is that below about the 95th percentile of the general
population pool, below about the 85th percentile of the general
population pool, below about the 75th percentile of the general
population pool, below about the 50th percentile of the general
population pool, to about the 25th percentile of the general
population pool. Thus, in some embodiments, the c-LDL level is less
than about 160 mg/dL, less than about 130 mg/dL, or less than about
100 mg/dL with the lower limit being a level of c-LDL that is
considered a healthy level.
[0029] In addition to subjects with above-normal levels of serum
cholesterol who are prescribed HMG CoA reductase inhibitors,
another class of subjects for whom the treatment is not intended is
those with certain defects in cholesterol biosynthesis. Defects in
synthesis of intermediates prior to formation of squalene are not
indicated for treatment with statins. For instance, there is a
single human genetic disorder arising from a deficiency of
mevalonate kinase known to affect this portion of the cholesterol
biosynthetic pathway. Subjects with defects in the cholesterol
biosynthetic pathway downstream of the squalene intermediate are
also generally excluded, although it is to be understood that the
cognitive deficits arising from such disorders, such as
Smith-Lemli-Opitz syndrome, might benefit from treatment from
statins.
[0030] A variety of cognitive disorders may be treated using the
inhibitor compounds described herein. In some embodiments, the
cognitive disorder is associated with a known genetic abnormality.
Generally, the types of genetic defects for which the attendant
cognitive disorders are amenable to treatment with the inhibitors
herein are typically those associated with dysregulation of mitogen
activated protein kinase (MAPK) signaling pathway, dysregulation of
signaling pathways involving small monomeric GTP binding proteins,
and/or dysregulation of inhibitory neuronal activity. As used
herein, "dysregulation" or "dysfunction" refers to impaired or
abnormal function of the specified process, including, loss of
normal function, or their overactivation or underactivation. In the
context of genetic abnormalities, dysregulation of a cellular
process may arise from a genetic change that causes a loss of
function, increased dosage, or altered activity of the molecules
involved, directly or indirectly, in the cellular process.
[0031] Generally, genetic defects may be categorized based on the
type of genetic alteration. Segmental aneusomy results from the
deletion or duplication of a specific chromosomal region such that
there is an inappropriate dosage of critical gene(s). The gene
dosage may result from increased or decreased expression at a
single gene (i.e., single locus) or from multiple genes (i.e.,
multi-locus). Examples of segmental aneusomy that display cognitive
disorders treatable with the compounds described herein, include,
among others, Angelman syndrome and Down Syndrome.
[0032] Angelman Syndrome (AS) is associated with the deletion of
chromosomal region 15g11-q13, and although the deletion overlaps
with chromosomal deletions resulting in another form of mental
retardation syndrome termed Prader Willi syndrome (PWS), AS occurs
when the deletion is on a maternally inherited chromosome while PWS
occurs when the deletion is on a paternally inherited chromosome.
Different classes of AS are known based on the location of the
cytogenetic abnormality. Molecular analysis indicates that the
affected gene in one form of AS encodes an ubiquitin ligase, UBE3A,
a protein involved in the ubiquitin mediated protein degradation
pathway (Kishino, T. et al., Nature Genetics 15:74-77 (1997)). In
the normal brain, the copy of UBE3A inherited from the father is
almost completely inactive through genetic imprinting such that the
maternal copy performs most of the UBE3A function in the brain.
Because of this imprinting phenomena, AS phenotype is typically
seen when the maternal copy is affected. Another form of AS is
characterized by biparental inheritance of imprinted gene in the
deleted region with a paternal only methylation pattern. The
deleted region termed IC is hypothesized to act by resetting the
male-female genomic imprint during oogenesis and the female-male
imprint during spermatogenesis. In other words, the IC acts as a
switch that turns on the maternal copy of UBE3A while silencing the
paternal copy of the gene. Mutations and deletions in this critical
region prevent the maternal to paternal imprinting switch in the AS
families. Individuals with mutations in IC inherit the paternal
imprint pattern on the mutant chromosome resulting in the inability
to turn on the maternal UBE3A gene. Another form of AS is paternal
uniparental disomy (UPD), where the child inherits both copies of
chromosome 15 from the father, with no copy inherited from the
mother. In this case, there is no deletion or mutation, but the
child is still missing the active UBE3A gene because the
paternal-derived chromosomes only have brain-inactivated UBE3A
genes. Mouse models of AS have been created by knockout of the
corresponding mouse UBE3A gene. These animals show impairment of
LTP, abnormal levels of p53 activity due to the reduction in its
degradation by the ubiquitin pathway, and a dysregulation of CaMKII
activity (Jiang, Y. H. et al., Neuron 21(4):799-811 (1998); Weeber,
E. J. et al., J. Neurosci. 23(7):2634 (2003)). Studies suggest an
association between CaMKII activity and activation of Ras GTPase
activating protein (Song B. et al., Brain Res. 1005(1-2):44-50
(2004); Oh, J. S. et al., J Biol Chem. 279(17):17980-8 (2004)). In
addition, some of the deletions in Angelman syndrome also removes
the P3 subunit of the GABA receptor, suggestive of dysregulation of
GABA receptor activity for some of the cognitive disorders
associated with AS. Interestingly, autism is also correlated with
polymorphisms of the P3 subunit of the GABA receptor.
[0033] In other embodiments, the compounds and compositions are
used to treat the learning disorders associated with trisomy of
chromosome 21, more commonly known as Down Syndrome (DS), which is
a segmental aneusomy believed to affect expression of multiple
genes. DS is the most common and readily identifiable chromosomal
condition associated with mental retardation and is most often
caused by an abnormality during cell division in gamete formation
called nondysjunction. The extra copy of chromosome 21 appears to
interfere with normal growth and development. The cause of the
mental retardation in DS has not been identified, although the
over-expression of genes located on the trisomic region is assumed
to be responsible for the phenotypic abnormalities of DS. However,
in a mouse model of DS characterized by trisomy for chromosome 16,
there is severe abnormality in the induction of LTP that may result
from over activation of inhibitory pathways that reduce neuronal
activation by metabotropic glutamate receptors (Kleschevnikov, A.
M. et al., J Neurosci. 24(37):8153-8160 (2004)). Importantly,
increased GABA-mediated inhibition is also observed in animal
models of NF-1, and a corresponding inhibition of the Ras activity
in the NF-1 animals attenuates the increased GABA-inhibition and
rescues the decreased LTP (Costa, R. M. et al., Nature
415(6871):526-30 (2002)).
[0034] Another class of identified genetic abnormalities affecting
cognitive processes is single gene mutations that result in mental
retardation. These disorders may be further divided into syndromic
and non-syndromic mental retardation (MR), where in non-syndromic
MR the cognitive impairment is the only identified phenotype
whereas syndromic MR shows other phenotypes, such as unique facial
profiles, underdeveloped limbs, and other physical
characteristics.
[0035] A single gene mutation resulting in a syndromic MR that may
be treated with the inhibitor compounds is Neurofibromatosis-1
(NF-1), a common genetic disorder caused by mutations in the gene
encoding neurofibromin. The protein neurofibromin has several
biochemical functions, including Ras GTPase-activation, adenyl
cyclase modulation, and microtubule binding, and is expressed in a
variety of different cell populations. Activation of Ras in NF-1 is
associated with increased cell proliferation, and mutations in
neurofibromin are shown to predispose the subject to certain types
of cancers. In addition to the increased incidence of cancers, NF-1
affected subjects also show a broad range of both nonverbal and
verbal learning disabilities (Costa R M et al., Trends Mol Med.
9(1):19-23 (2003)). Children with NF-1 display an increased
frequency of mental retardation (Wechsler Full-Scale IQ<70) and
have specific deficits in visual-spatial ability, executive
function, expressive and receptive language, and attentional
skills. The underlying cause of the cognitive deficits in subjects
with NF-1 defects is unclear because of the multiple functions
associated with the protein (see, e.g., U.S. Pat. No. 6,356,126).
Although farnesyl transferase inhibitors have been shown to improve
the learning deficits in animal models of NF-1 (Costa, R. M. et
al., Nature 415(6871):526-30 (2002)), inhibiting the farnesyl lipid
attachment pathway is demonstrated to cause compensating increases
in geranylgeranylation pathway (Du, W. et al., Mol Cell Biol.
19(3):1831-40 (1999)). Thus, results from use of farnesyl
transferase inhibitors are not predictive of the effect HMG CoA
reductase inhibitors, which would affect both farnesylation and
geranylgeranylation of proteins. Evidence presented herein using
HMG CoA reductase inhibitors suggest that the activity of Ras is
responsible for the various cognitive deficits associated with
NF-1.
[0036] In yet other embodiments, the syndromic MR is tuberous
sclerosis complex (TSC), an autosomal dominant disease
characterized by mental retardation, seizures, and tumors of
various organs, including the kidney, brain, heart, and skin. Thus,
TSC appears to act as a tumor suppressor gene. The TSC complex is
composed of TSC1, which encodes hamartin, a protein of unknown
function, and TSC2 gene product termed tuberin, which is a GTPase
activating protein that is known to affect the Ras family GTPases,
Rapl and RabS in vitro. Since deleting TSC may result in over
activation of MAPK (see Karbowniczek et al., J. Biol. Chem.
279(29):29930-7 (2004)), statins, which can decrease MAPK activity,
could be used to treat this disorder.
[0037] Another type of genetic abnormality affecting cognitive
functions is non-syndromic MR, also referred to as non-specific MR.
Affected patients have no distinctive clinical or biochemical
features other than the cognitive deficit. A number of X-linked
chromosomal genes mutated in nonspecific MR have been identified.
These include, among others, FMR2, GDR, RPS6KA3, IL1RAPL, TM4SF2,
OPHN1 and PAK3. Cognitive deficits associated with mutations in the
gene encoding OPHN1 (Oligopherin) may be treated with the compounds
disclosed herein.
[0038] OPHN1 encodes a protein related to Rho-GTPase-activating
protein (RhoGAP) (van Galen, E. J. et al., Prog Brain Res.
147:295-317 (2005)). By enhancing their GTPase activity, GAP
proteins inactivate Ras and Ras related proteins, such as Rho.
Consequently, inactivation of RhoGAP proteins is believed to cause
constitutive activation of their GTPase targets (Billuart, P. et
al., Nature 392(6679):923-6 (1998)). OPHN1 is expressed in both
glial and neuronal cells and is shown to colocalize with actin at
the tip of growing neurites. In addition to the cognitive deficits,
subjects with OPHN1 mutations display epileptic seizures, ataxia,
and cerebellar hypoplasia.
[0039] Although the various embodiments of cognitive disorders
described above have a known biological foundation, it is to be
understood that the methods disclosed herein may be used for a
recognized and diagnosable cognitive disorders for which there are
no identified biological cause. The cognitive deficits and
associated symptoms seen in the disorders arising from identified
genetic abnormalities appear in some instances to overlap with the
features of cognitive disorders of unknown etiology. For instance,
enhanced sensitivity to startle stimuli is seen in Coffin Lowry
syndrome but also in ADHD. There is also a high incidence of ADHD
in NF-1 patients, suggestive of a correlation of ADHD and the
underlying biological defects in Coffin-Lowry syndrome and/or NF-1
(Schrimsher, G. W, et al., Am. J. Med. Genet. 120(3):326-30
(2003)). Another example of this overlap is seen in Angelman
syndrome, Down syndrome, or TSC patients, who display
characteristic impairments in language ability, adaptive behavior,
and cognition found in autism (Peters, S. U. et al., Clin Genet.
66(6):530-6 (2004); Kent L, et al., Dev. Med. Child Neurol.
41(3):153-8 (1999)).
[0040] Thus, in some embodiments, the cognitive disorder treatable
with the inhibitor compounds may be ADHD. ADHD is a behavioral
condition of childhood, affecting 5-10% of school-age children.
Affected patients exhibit various behavioral problems such as
carelessness, restlessness, disobedience and failure to stay quiet
in class. As noted below, ADHD is diagnosed when the subject
suffers from levels of inattention and/or hyperactivity-impulsivity
that has persisted for more than 6 months and is maladaptive or
inconsistent with the developmental level observed in the general
population. Working memory appears to be impaired in ADHD
(Westerberg, H. et al., Neuropsychol. Dev Cogn. C Child
Neuropsychol. 10(3):155-61 (2004)). The most common pharmacologic
therapy for ADHD is stimulants or stimulant mixtures, such as
Ritaline.RTM. (methyphenidate), Adderall.RTM., pemoline, or
dextroamphetamine. It is believed that stimulants affect
nonepinephrine and dopamine pathways, thereby providing impulse
control and working memory. For those individuals who do not
respond to treatments with stimulants, alternative treatments
include use of antidepressants (e.g., desipramine, imipramine,
nortryptiline, bupropione) and .alpha..sub.2-agonists (e.g.,
clonidine and guanfacine). Use of the inhibitor compounds described
herein may provide an alternative therapy for ADHD.
[0041] In other embodiments, the cognitive disorder that may be
treated is autism. Criteria for the diagnosis of autism is given in
the ICD-10 (International Classification of Diseases, 10th
Revision) and the Diagnostic and Statistical Manual of Mental
Disorders, 4th Ed. (DSM-IV). Though a complex disorder, autism has
identifiable characteristics that include qualitative impairments
in social communication, social interaction, social imagination,
with a restricted range of interests and stereotyped repetitive
behaviors and mannerisms. Affected individuals also show sensory
hyposensitivities or hypersensitivities (Herault J. et al., Am J
Med Genet. 60(4):276-81 (1995)). Epilepsy occurs more commonly than
usual in autism. As discussed above, autism has been associated
with many cytogenetic abnormalities, including Angelman syndrome,
TSC syndrome, and thus provides a basis for extension of the
treatments herein to cognitive deficits associated with autism.
[0042] As is apparent from the foregoing descriptions, dysfunction
of basic cellular processes underlie many known forms of cognitive
disorders. Consequently, any cognitive deficits arising from same
underlying molecular mechanism may also be treated using the
inhibitor compounds described herein. Accordingly, in some
embodiments, the HMG CoA reductase inhibitors and other inhibitor
compounds are used to treat cognitive deficits associated with
dysregulation of small monomeric GTP binding proteins and proteins
that regulate or are the targets of the signal transduction pathway
regulated by these proteins. For instance, the role of Ras mediated
signaling in synaptic plasticity and learning and memory is
underscored by the observed effect of Ras in synaptic plasticity
and a role for Rap, Rab and Rac in LTP (Murray, H. J. et al., Brain
Res. 1000(1-2):85-91 (2004)). Both the overactivation and
underactivation of Ras and related pathways appear to affect
learning and memory.
[0043] "Small monomeric GTP binding protein" as used herein refers
to a protein that binds guanine nucleotides (GTP and GDP),
generally has an associated GTPase activity, and displays homology
to Ras protein sequence. The term "Ras-related protein" will refer
to a small monomeric GTP binding protein with sequence homology to
Ras. The Ras-related proteins typically have a sequence motif
involved in binding to guanine nucleotides, a carboxy terminal
domain for posttranslocation modification with farnesyl,
geranylgeranyl, palmitoyl, or methyl moieties (e.g., a Cys-A-A-X,
where A is aliphatic and X is any amino acid; Cys-X-Cys; and
Cys-Cys), and in some instances, a domain that interacts with
guanine nucleotide exchange factors (GEFs). Small monomeric GTP
binding proteins may be categorized into subfamilies that include,
by way of example and not limitation, the proteins within groups
designated as Ras, Rho, Rab, Sarl/Arf and Ran. The Ras subfamily of
proteins includes c-Harvey (H)-ras, c-Kirsten (K)-ras, and N-ras.
The Rho subfamily includes Rho, Rac, and Cdc42.
[0044] Homology between small monomeric GTP binding proteins is
about 30% or more amino acid identity. For example, Ras proteins
share about 30% amino acid identity with Rab, Rho, Rac, and Cdc42.
Proteins within a particular subgroup may have higher sequence
homology (e.g., more than 50% amino acid identity) than between
subgroups. The terms "percentage of sequence identity" and
"percentage homology" are used interchangeably herein to refer to
comparisons among polynucleotides and polypeptides, and are
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide or
polypeptide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage may be
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity. Alternatively, the percentage may
be calculated by determining the number of positions at which
either the identical nucleic acid base or amino acid residue occurs
in both sequences or a nucleic acid base or amino acid residue is
aligned with a gap to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity. Those of skill in
the art appreciate that there are many established algorithms
available to align two sequences. Optimal alignment of sequences
for comparison can be conducted, e.g., by the local homology
algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by
the homology alignment algorithm of Needleman and Wunsch, J. Mol.
Biol. 48:443 (1970), by the search for similarity method of Pearson
and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the GCG Wisconsin Software Package), or by
visual inspection (see generally, Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (1995 Supplement). Examples
of algorithms that are suitable for determining percent sequence
identity and sequence similarity are the BLAST and BLAST 2.0
algorithms, which are described in Altschul et al. J. Mol. Biol.
215: 403-410 (1990) and Altschul et al. Nucleic Acids Res.
3389-3402 (1977), respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information website. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to
as, the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA
89:10915 (1989)).
[0045] All of the above mentioned algorithms and programs are
suitable for a determination of sequence alignment and % sequence
identity, for determination of % sequence identity in connection
with the small monomer GTP binding proteins. Exemplary programs for
determining the % sequence identity or homology may use the BESTFIT
or GAP programs in the GCG Wisconsin Software package (Accelrys,
Madison Wis.), using default parameters.
[0046] In some embodiments, the present disclosure provides methods
of treating cognitive deficits associated with dysregulation of Ras
protein activity, such as N-ras and K-ras. Ras proteins transmit
extracellular signals that promote the growth, proliferation,
differentiation, and survival of cells. Extracellular signals
generate an intracellular signal that, in some instances, leads to
activation of gunanine nucleotide exchange factors (GEF), and
subsequent activation of Ras. Regulation of MAPK by Ras is believed
to occur through Raf, which is activated on the plasma membrane by
Ras-GTP. Raf phosphorylates mitogen-activated kinase 1/2 (MEK1/2
kinase), which activates the extracellular-regulated kinase 1/2
(ERK1/2 kinase or p44/42 MAPK) by phosphorylation. ERK1/2 kinase
phosphorylates a variety of downstream targets, which results in
changes in gene expression and the activities of other proteins.
Mutations in genes encoding members of the MAPK pathway, such as
MEK, Ras-GRF, and H-Ras, may cause defects in learning and LTP
(Brambilla, R. et al., Nature 390:281-286 (1997); Atkins, C. M. et
al., Nat. Neurosci. 1:602-609 (1998); Manabe et al., J. Neurosci.
20:2504-2511 (2000), and may couple metabotropic glutamate receptor
activity to regulation of CREB transcription factor activity (Tian,
X. et al., EMBO 123(7):1567-1575 (2004)).
[0047] In other embodiments, the cognitive deficit is associated
with dysregulation of Rho protein activity. Rho proteins
participate in various cellular processes such as cytoskeletal
reorganization, membrane trafficking, transcriptional activation,
and cell growth regulation. Mechanistically, Rho protein is thought
to act by binding to target proteins that include Rho-kinase,
myosin light chain, and protein kinases PKN and PRK2. Rho is
believed to be regulated by Rac. Involvement of Rho protein in
actin reorganization may indicate a role in both pre- and
post-synaptic morphological changes. In addition, modulation of Rho
activity appears to reduce or enhance LTP in vitro (O'Kane, E. M.
et al., Neuropharmacology 46(6):879-87 (2004)). Thus, the HMG CoA
inhibitor compounds may be used to treat cognitive deficits that
arise from dysregulation of Rho protein activity.
[0048] In some embodiments, the treatment is directed to cognitive
deficits associated with dysregulation of Rac proteins, which play
a role in stimulating the formation of lamellipodia and membrane
ruffles. Among the effectors of Rac activity are serine/threonine
kinases known as PAKs, one of which (i.e., PAK3) is associated with
X-linked MR. In some instances, targeted inhibition of Rac in vitro
produces enhanced LTP in hippocampal cultures (O'Kane, E. M. et al,
supra). Thus, overactivation or underactivation of Rac may result
in cognitive deficits.
[0049] In other embodiments, the treatment is directed to cognitive
deficits associated with dysregulation of Rab protein, which
regulates vesicle formation, actin- and tubulin-dependent vesicle
movement, and membrane fusion. Rab proteins may be categorized
based on function into two groups: (1) proteins involved in
regulated secretion and (2) proteins involved in vesicle transport.
For instance Rab3A is involved in regulated exocytosis of
neurotransmitters and thus may contribute synaptic plasticity. In
animal models, elimination of Rab3A function affects short and
long-term synaptic plasticity in the mossy fiber pathway and
altered circadian motor activity, but show no effects on spatial
learning. Rab3A deleted animals, however, are moderately impaired
in reference memory, show deficits in spatial working memory, have
increased locomotor activity, and display enhanced exploratory
activity (D'Adamo, P. et al., Eur J Neurosci. 19(7):1895-905
(2004)).
[0050] In further embodiments, the inhibitors compound are used to
treat cognitive deficits associated with dysregulation of Rap
protein, a Ras-like GTPase that is localized in endocytic and
lysosomal vesicle. Rap is a target of protein kinase A and may act
as an antagonist of Ras activity by interacting with and trapping
Raf1, a Ras effector, in an inactive complex. It may also function
independently of Ras to regulate MAPK pathway (Asha, H. et al.,
EMBO J. 18(3):605-15 (1999)). The antagonistic activity of Rapl
suggests that lack of Rapl function may result in enhanced Ras
signaling.
[0051] In additional embodiments, the inhibitor compounds are used
to treat cognitive deficits associated with dysregulation of Ra1
proteins, a downstream effector of Ras. In addition to its role in
Ras pathway, Ra1 may also be activated by Ras independent pathway.
Ra1 GTPases, Ra1A and Ra1B, appear play a role in vesicle
regulation since they are present at high levels in synaptic
vesicles; participate in the regulation of Arf-dependent
phospholipase D (PLD), an enzyme implicated in vesicle function;
and regulate Ra11BP1, which forms a complex with proteins involved
in clathrin-mediated endocytosis. In animals with inactive Ra1
pathway, there is suppression of protein kinase C-mediated
enhancement of glutamate secretion, indicating a role of Ra1 in
modulating synaptic strength, a key component of LTP.
[0052] Activity of small monomeric GTP binding proteins is
regulated by proteins that affect the GTP/GDP bound form.
Accordingly, in some embodiments, the cognitive deficit treatable
by the inhibitor compounds is associated with dysregulation of a
GPTase activating protein (GAP). GAP proteins interact directly
with Ras and Ras-related proteins to enhance the intrinsic rate of
hydrolysis of bound GTP. Loss of function of GAP may result in an
increase in GTP bound forms of guanine nucleotide binding proteins,
thereby increasing the activity of proteins such as Ras, Rac, and
Rho. For example, activity of Rho protein affected by 190Rho-GAP
appears to be involved in memory formation in the amygdala
(Lamprecht, R. et al., Neuron. 36(4):727-38 (2002)). In addition,
the Rho-GTPase activating enzyme MEGAP/srGAP is show to be affected
in X-linked mental retardation (Endris V. et al., Proc Natl Acad
Sci USA 99(18):11754-9 (2002)). In some embodiments, the
dysregulation is in the Ras-GAP protein Neurofibromin-1 (Costa, R.
M. et al., Nature Genetics 27:399-405 (2001)). In other
embodiments, the dysregulation is in Rho-GAP, such as OPHN1 noted
above.
[0053] In other embodiments, the inhibitor compounds are used to
treat cognitive deficits associated with dysregulation of a guanine
nucleotide exchange protein (GEP), also referred to as guanine
nucleotide release factor (GRF) or guanine nucleotide exchange
factor (GEF). GEFs may be specific to certain small monomeric GTP
binding proteins, such as Ras, or have wider specificity, such as
GEFs that active Rho, Rac and Cdc42. These regulators of Ras and
Ras-related proteins enhance the exchange of bound GDP for GTP,
thereby activating the Ras or Ras-related proteins. Thus, loss of
GEF function would result in reduction in Ras or Ras-related
protein activity, which has been correlated with loss of learning
and memory. However, in some instances, Ras can also activate GEFs
that target other Ras-related proteins. For instance, GTP bound
forms of Ras and Rapl interact with Ra1GEF to activate its GEF
activity directed against Ra1, thereby activating Ra1 activity
(Giese, K. et al., Neuropharmacology 41, 791-800 (2001)).
Accordingly, cognitive deficits arising from changes in GEF
activity could be treated with HMG CoA reductase inhibitors and
other inhibitor compounds described herein.
[0054] In other embodiments, the cognitive deficit is associated
with dysregulation of guanine nucleotide dissociation inhibitors
(GDI). This regulator of Ras-like protein inhibits dissociation of
GDP, thereby maintaining a pool of GDP bound small monomeric GTP
binding proteins. GDIs are known to regulate the activities of Ras,
Rab, Ran, and Rho. For instance, GDI affects state of Rab and also
functions in the vesicular transport of Rab GTPases through the
secretory pathway by altering the cytosolic and membrane
localization of Rab. GDIs are know to affect learning and memory.
For example, model animals systems with deletion of GDI1 displays
impairment in tasks requiring formation of short-term temporal
associations, suggesting a defect in short-term memory. The animals
also show lowered aggression and altered social behavior (D'Adamo,
P. et al., Human Molecular Genetics 11(21):2567-2580 (2002)). Thus,
GDI may act to suppress hyperexcitability in neurons since loss of
GDI1 function appears to produce hyperexcitability, a consequence
of which is an increase in epileptic seizures.
[0055] In other embodiments the cognitive deficit is associated
with dysregulation of a target of Ras or Ras-related protein
activity. In some embodiments, the treatments with inhibitor
compounds are directed to cognitive deficits associated with
dysregulation of Raf, a downstream effector of Ras. Raf encodes a
serine threonine kinase and is believed to be activated by direct
interaction with Ras. Activation of Raf1 by Ras leads to activation
of the MAPK pathway, which in hippocampal cultures is thought to be
involved in establishment LTP. Further, as disclosed herein,
hyperactivation of MAPK pathway by Ras signaling is observed in
animal models of NF-1.
[0056] In some embodiments, the present disclosure also provides
use of the inhibitor compounds to treat cognitive deficits arising
from dysregulation of MAPK signaling pathway. Genetic and
biochemical studies implicate components of the MAPK signaling
pathway in cognitive function. For example, Ras in NF-1 appears to
act through modulation of the MAPK pathway. In addition,
extracellular-regulated receptor kinases (ERK) may be involved in
regulating downstream CREB activity and modulating synaptic
structure (Sweatt, J. D. et al., Curr. Opin. Neurobiol. 14(3):311-7
(2004)). As used herein, "MAPK signaling pathway," some of which
have been described in various parts of this disclosure, refers to
a signaling pathway that uses a cascade of three types of kinases,
also referred to as the "MAPK module." These canonical kinases
include a MAP kinase kinase kinase (MAPKKK), which activates a
second kinase, the MAP kinase kinase (MAPKK) by phosphorylation of
serine/threonine residues. MAPKKs are dual specificity kinases
capable of phorphorylating both serine/threonine and tyrosine
residues. Activated MAPKKs modify MAP kinases (MAPK) by
phosphorylation of both threonine and tyrosine residues. In turn,
the MAPKs regulate activity of other protein kinases and numerous
transcription factors to effect the cellular responses triggered by
activation of the signaling cascade. Three distinct pathways form
the superfamily of MAPK pathways, each designated based on the MAPK
involved. The p38/HOG pathway uses p38/HOG MAPKs, which are
activated by dual specificity kinases MEK3/MKK4. The corresponding
MAPKKK for this pathway appears to be TAO-1. The second MAPK
pathway, also known as the stress activated protein kinase pathway,
uses c-jun N-terminal kinase (JNK) MAPKs, which are activated by
dual specificity kinases MEK4/JNK kinase. The corresponding MAPKKK
for the JNK pathway is MEKK. The third pathway and the best
characterized uses a MAPK referred to as extracellular
signal-regulated kinases (ERK), of which ERK1 and ERK2 are members.
Dual specificity kinases of the ERK pathway include MEK1 and MEK2,
which are targets of Raf, a MAPKKK. As explained in the previous
sections, the ERK pathway is believed to be directly involved in
learning and memory via the action of Ras on Raf. Evidence for
involvement of the other MAPK pathways in cognitive function come
from use of selective inhibitors of JNK and p38 pathways. Selective
inhibition of p38 pathway affects associative learning and memory
formation (Zhen, X. et al., J Neurosci. 21(15):5513-9 (2001);
Alonso, M. et al., Neuroreport 14(15):1989-92 (2003)) while
selective inhibition of INK blocks long term memory (Bevilaqua, L.
R. et al., Eur. J. Neurosci. 17(4):897-902 (2003)). Rac and its
downstream effectors, p21 activated kinases (PAK), are known to
regulate the p38 and INK MAPK pathways. Thus, cognitive deficits
arising from dysregulation of p38 and JNK signaling pathways by
altered activity of Rac and other small monomeric GTP binding
proteins may be treated with the inhibitor compounds.
[0057] In other embodiments, the cognitive deficit is associated
with dysregulation of inhibitory neuronal activity. "Inhibitory
neuronal activity" as used herein refers to activity that opposes
or inhibits excitation of a neuron. Generally, inhibitory neuronal
activity may occur presynaptically, such as attenuating or
inhibiting release of excitatory neurotransmitters, or occur
postsynaptically by attenuating or preventing the excitatory
neurotransmitter from activating the postsynaptic neuron. In some
embodiments, the inhibitory neuronal activity is an inhibitory
postsynaptic potential (IPSP), which lowers the membrane potential
of the postsynaptic neuron, thereby reducing the probability of the
postsynaptic neuron from generating an excitatory postsynaptic
potential (ESPS).
[0058] In some embodiments, the dysfunction in the inhibitory
neuronal activity is associated with increased GABA-mediated
inhibition. In the GABA pathway, inhibitory neurons package the
neurotransmitter GABA in synaptic vesicles and release it upon
activation of the inhibitory neuron. GABA discharged into the
synaptic cleft is recognized by GABA receptors, whose activation
inhibits an excitatory signal in the postsynaptic neuron. The
principle GABA receptors are GABA.sub.A and GABA.sub.B, although
other GABA type receptors that are known to act in inhibiting
neuronal activity are to be included within this class. The
GABA.sub.A receptors are members of the Cys-loop superfamily of
ligand gated ion channels that includes the receptors for glycine,
acetylcholine, and 5-HT3. GABA.sub.A receptors are known for their
interaction with benzodiazepine type agonists. Structurally, the
GABA.sub.A receptor is a heteromultimeric protein, generally
composed of five subunits that come from at least four principle
families of subunits .alpha., .beta., .gamma., and .delta., but
which may include other subunits, such as .pi., .theta., and
.epsilon.. Typically, each subunit transverses the postsynaptic
membrane and interacts to form a central pore, which, when opened,
allows for the passage of chloride ions into the neuron. Thus,
GABA.sub.A type receptors are ionotropic receptors. Activation of
the GABA.sub.A receptor by binding of GABA results in increased
inward chloride ion flux and hyperpolarization and subsequent
neuronal inhibition. GABA.sub.B receptors also bind GABA, but are
G-protein coupled receptors (GPCRs) that modulate Ca.sup.+2 or
K.sup.+ ion channel activity and various second messenger pathways.
GABA.sub.B receptors are heteromeric proteins, typically a dimer,
and like other GPCRs, characterized by the presence of seven
transmembrane spanning regions. Of the various families of GPCRs,
GABA.sub.B receptors are categorized within Family 3, the members
of which are defined by the presence of a ligand binding domain in
the large extracellular amino terminal region. In addition to
GABA.sub.B receptors, exemplary members of Family 3 GPCRs include,
by way of example and not limitation, metabotropic glutamate
receptors, Ca.sup.+2 receptors, taste receptors, and odorant
receptors. Without being bound by theory, GABA.sub.B receptors are
believed to mediate neuronal inhibition by activation of inwardly
rectifying potassium channels (GIRKS) resulting in
hyperpolarization in the postsynaptic membrane. In the presynaptic
membrane, GABA.sub.B is thought to inhibit presynapstic Ca.sup.+2
channels, thereby causing inhibition of neurotransmitter release.
Overactivation of GABA-mediated inhibition correlates with impaired
cognitive function in NF-1 and Down Syndrome. For NF-1, the
associated depressed LTP is ameliorated by inhibition of RAS
activity and by attenuation of GABA mediated inhibition by the GABA
antagonist picrotoxin. As shown herein, HMG CoA reductase
inhibitors not only reverse the cognitive deficits in animal models
of NF-1 but also enhances the LTP response, indicative of
attenuation of GABA mediated inhibition.
[0059] In some embodiments, the inhibitor compounds are used to
modulate the underlying cellular processes associated with
cognitive function. As noted above, the phenomena of LTP in neural
cultures, typically a hippocampal system, is widely held as being a
molecular correlate of the processes involved in short term and
long-term memory. LTP occurs at all three major synaptic
connections in the hippocampus, including: the perforant path
synapse to dentate gyrus granule cells, mossy fibers to CA3
pyramidal cells, and the Schaffer collaterals of CA3 cells to CA1
pyramidal cells. There are at least two art-recognized forms of LTP
that are temporally related to each other. An early-phase LTP or
E-LTP has the characteristics of being independent of transcription
and protein synthesis, and decays within 1-3 h of induction. This
short lasting LTP is considered as the molecular correlate to
short-term memory. The second form of LTP, referred to as late
phase LTP or L-LTP, requires transcription and translation and can
persist for hours or days. L-LTP is believed to be cellular
counterpart of long-term memory storage. Both forms of LTP may be
generated in hippocampal cultures by stimulation of a single input
pathway (i.e., homosynaptic) by a train of evoked potentials. E-LTP
is typically induced by a single high-frequency tetanic stimulus
whereas L-LTP is typically induced by multiples (e.g., three to
four) of such tetanic trains (see, e.g., Thomas, M. J. et al., J
Neurosci. 18:7118-7126 (1998)). L-LTP may also be induced by paired
stimulation of multiple input pathways (i.e., heterosynaptic),
where activation of one afferent pathway is paired to a
conditioning stimulus in another afferent pathway in the neural
network (Huang, Y. Y. et al., Proc. Natl. Acad. Sci. USA
101(3):861-864 (2004)). Timing of the paired stimulus appears
critical for generating L-LTP in the heterosynaptic system.
[0060] Since HMG CoA reductase inhibitors appears to show now
measurable effect on subjects with normal cognitive function, the
neural systems on which the inhibitors may be used will typically
have a depressed LTP. As used herein, a "depressed LTP" refers to a
lower LTP response than measured for another subject. An exemplary
depressed LTP is that observed for a subject with a genetic defect
affecting LTP, where the LTP in the affected subject is lower than
what is observed for a subject without the genetic defect.
Similarly, another exemplary depressed LTP is that observed when
the neural system is treated with a pharmacological agent that
reduces the LTP response as compared to a subject that has not been
treated with the pharmacological agent.
[0061] For modulating the LTP of neural networks, the neural system
is contacted with an effective amount of an inhibitor compound.
"Modulate" as used herein refers to inhibition or enhancement of
the LTP in the neural system being examined when compared to the
LTP in the absence of such compounds. In some embodiments, the
neural system may have an underlying deficit in cognitive function,
which may be reflected in the LTP. The LTP affected may be the
early phase E-LTP, but more typically late phase or L-LTP.
Dysregulation of the cellular processes associated with altered LTP
include genetic defects induced in model animal systems or those
found naturally in animals and humans, or various in vitro
manipulations that disrupt a cellular process. Exemplary
manipulations of in vitro systems to alter LTP include
overexpression of a target protein (e.g., small monomeric GTP
binding protein), expression of proteins with dominantly acting
mutations (e.g., dominant negative or dominant active), use of
inhibitors of enzyme activity (e.g., protein kinase inhibitors,
ubiquitin mediate protein degradation inhibitors, toxins to inhibit
small monomeric GTP binding proteins), and silencing of expression
of target genes (e.g., using interfering RNA, anti-sense RNA,
etc.). Other manipulations will be apparent to the skilled artisan.
It is to be understood that the LTP resulting from the dysfunction
or dysregulation of processes described in the preceding sections
may be modulated by using the inhibitor compounds.
[0062] 6.2 HMG CoA Reductase Inhibitors, Compositions, and
Inhibitor Combinations for the Treatment of Cognitive Disorders
[0063] Treating the cognitive deficits associated with the
disorders described above comprises administering a HMG CoA
reductase inhibitor to a subject in an amount effective to improve,
enhance, or restore cognitive function. As used herein, an "HMG CoA
Reductase inhibitor" is any compound or composition, including
prodrugs, salts, solvates and hydrates thereof, that inhibits HMG
CoA reductase activity. An inhibitor includes compound that act via
competitive, non-competitive, or un-competitive mechanisms, as they
are commonly known in the art. One important class of HMG CoA
reductase inhibitors are generally known as statins, which are
prescribed to treat hyperlipidemia characterized by elevated serum
cholesterol levels.
[0064] Various HMG CoA reductase inhibitors, corresponding
prodrugs, salts, solvates and hydrates, are known in the art and
may be used for the methods herein. Atorvastatin and derivatives
thereof are described in U.S. Pat. No. 5,273,995 and EP 409281 and
are available commercially under the tradenames Lipitor.RTM.,
Sortis.RTM., Torvast.RTM., Totalip.RTM., and Xarator.RTM..
Cerivastatin and derivatives thereof are described in U.S. Pat.
Nos. 5,006,530; 5,177,080, and EP 325130 and are available under
the tradenames Rivastatin.RTM., Baycol.RTM., and Lipobay.RTM..
Although the levels of cerivastatin prescribed for hyperlipidemia
has resulted in toxic side effects, lower non-toxic levels may be
appropriate for treatment of cognitive deficits.
[0065] Another of these statin compounds is clofibrate and
derivatives thereof, as described in U.S. Pat. No. 3,262,850 and GB
860303. Clofibrate is available under the tradenames Amotril.RTM.,
Anparton.RTM., Apolan.RTM., Artevil.RTM., Claripex.RTM.,
Liprinal.RTM., Normet.RTM., Regelen.RTM., Serotinex.RTM., and
Xyduril.RTM.. Inhibitor colestipol and derivatives thereof are
described in U.S. Pat. Nos. 3,692,895 and 3,803,237 and published
patents DE 1927336, and DE 2053585. Fluvastatin and derivatives
thereof are described in U.S. Pat. No. 4,739,073 and WO 84/02131
and are available under the tradenames Fluindostatin.RTM., XU
62-320, Lescol.RTM., Lipaxan.RTM. and Primexin.RTM.. Gemfibrozil
and derivatives thereof are described in U.S. Pat. Nos. 3,674,836
and 4,126,637, and published patent DEL 1925423, and are available
under the tradenames Decrelip.RTM., Genlip.RTM., Gevilon.RTM.,
Lipozid.RTM., and Lopid.RTM.. Lovastatin and derivatives thereof
are described in U.S. Pat. No. 4,231,938. and are available under
the tradenames Altocar.RTM., Lovalip.RTM., Mevacor.RTM.,
Mevinacor.RTM., Nevlor.RTM., and Sivlor.RTM.. Pitavastatin and
derivatives thereof are described in EP65835 and U.S. Pat. No.
6,162,798 and are available under the tradenames Itabastatin.RTM.,
Livalo.RTM., Nisvastatin.RTM., Itavastatin.RTM., and Zomaril.RTM..
Pravastatin and derivatives thereof are described in U.S. Pat. No.
4,346,227 and published patent DE 3122499, and are available under
the tradenames Elisor.RTM., Lipostat.RTM., Liprevil.RTM.,
Mevalotin.RTM., Oliprevin.RTM., Pravachol.RTM., Pravasin.RTM.,
Selectin.RTM., and Vasten.RTM.. Rosuvastatin and derivatives
thereof are described in U.S. Pat. Nos. 5,128,366, 6,589,959, and
published application WO 521471, and are available under the
tradename Crestor.RTM.. Simvastatin and derivatives thereof are
described in U.S. Pat. No. 4,444,784 and EP 33538 and are available
under the tradenames Denan.RTM., Liponorm.RTM., Simovil.RTM.,
Sinvacor.RTM., Sivastin.RTM., Zocor.RTM., and Zocord.RTM..
[0066] It is to be understood that while a single inhibitor is
typically prescribed to lower elevated cholesterol levels, mixtures
of HMG CoA reductase inhibitors may be used for the uses described
herein. Compatible mixtures may be made to enhance the efficacy
and/or lower the toxicity of the inhibitors in treating the
cognitive disorders.
[0067] In some embodiments, other compounds targeting the
cholesterol biosynthetic pathway may be used to treat the cognitive
deficit. Thus, in some embodiments, the compound is a modulator of
farnesyl transferase, such as an inhibitor of farensyl transferase
activity. As used herein, a farnesyl transferase inhibitor is an
inhibitor of the enzyme responsible for transfer of farnesyl
pyrophosphate onto protein substrates. Suitable farnesyl
transferase inhibitors include, by way of example and not
limitation, FTI-276 (Calbiochem, San Diego, Calif., USA); SCH66336
(Schering-Plough, (Kenilworth, N.J., USA);
(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlor-
ophenyl)-1-methyl-2(1H)-quinolinone (also known as R115777,
tipifamib, and Zarnestra) (Johnson & Johnson); L-778,123; and
FTI-2148.
[0068] In other embodiments, the other compound may be a modulator
of geranylgeranyl transferase activity, such as an inhibitor of
geranylgeranyl transferase. These compounds may be suitable for
cognitive disorders that are associated with dysregulation of Rac
or Rho activity since these proteins are modified by attachment of
geranylgeranyl groups. Suitable geranylgeranyl transferase
inhibitors include, by way of example and not limitation, GGTI-286
(Calbiochem, San Diego, Calif., USA); GGTI-297; GGTI-2154; and
GGTI-2166. Compounds with inhibitory activities to both farnesyl
transferase and geranylgeranyl transferases are described in Tucker
T. J. et al, Bioorg. Med. Chem. Lett. 12(15):2027-30 (2002)).
[0069] In some embodiments, the compounds that inhibit inhibitory
neuronal activity may be used. A number of different aspects of
inhibitory neuronal activity may be targeted, including, among
others, transport of inhibitory neurotransmitters into synaptic
vesicles, degradation of the inhibitory neurotransmitter, receptors
that are activated by binding to inhibitory neurotransmitters, and
channel proteins that decrease the generation of action
potentials.
[0070] In some embodiments, the inhibitors inhibit GABA mediated
inhibition, and thus are inhibitors of GABA receptor activity. An
"inhibitor of GABA receptor" as used herein refers to a compound
that binds to but does not activate GABA receptors (i.e.,
antagonists), thereby blocking the actions of endogenous GABA and
GABA agonists. Also encompassed within "inhibitor of GABA receptor"
is an inverse agonist, which binds to a region of the GABA receptor
different from the region that interacts with GABA but which
results in inhibition of GABA or GABA agonist binding. Useful
inhibitors may have general activity against various forms of GABA
receptors, or are selective for different GABA receptor types.
Compatible mixtures of selective GABA receptor inhibitors may be
used to generate a general inhibitor of GABA receptor activity.
[0071] Accordingly, in some embodiments, the inhibitor used is
selective for GABA.sub.A. Exemplary embodiments of antagonist
compounds selective for GABA.sub.A receptor include, by way of
example and not limitation, picrotoxin; hydrastine; securinine;
6-(5,6,7,8-tetrahydro-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5-yl)furo[3,-
4-e]-1,3-benzodioxol-8(6H)-one (i.e., bicuculline);
6-Imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid
hydrobromide (i.e., gabazine);
4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol and analogs
thereof (Frolund, B. et al., J. Med. Chem. 48(2):427-39 (2005));
.beta.-carboline-3-carboxylate-t-butyl ester (Rowlett J, et al.,
CNS Spectr. 10(1):40-8 (2005). GABA.sub.A inverse agonists include
the naturally occurring peptide Diazepam Binding Inhibitor (DBI);
methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM);
ethyl-beta-carboline-3-carboxylate (beta-CCE),
N-methyl-beta-carboline-3-carboxamide (FG 7142);
ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5
alpha][1,4]-benzodiazepine-3-carboxylate (Ro 15-4513);
(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-yl-
methoxy)pyrazolo[1,5-d][1,2,4]triazine; and
2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). Other GABA.sub.A
antagonists and inverse agonists applicable to the uses herein will
be apparent to the skilled artisan.
[0072] In other embodiments, the inhibitor used is selective for
the GABA.sub.B receptor. Some exemplary embodiments of antagonist
compounds selective for receptor GABA.sub.B include by way of
example and not limitation,
3-Amino-2-(4-chlorophenyl)propylphosphonic acid (i.e., phaclofen);
3-amino-2-(4-chlorophenyl)propylsulfonic acid (i.e., saclofen);
3-amino-2-(4-chlorophenyl)-2-hydroxypropyl-sulfonic acid (i.e.,
2-hydroxysaclofen); 3-aminopropyl-diethoxymethylphosphinic acid
(CGP 35348);
3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic
acid (CGP 52432);
(2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylme-
thyl)phosphinic acid (CGP 55845);
3-[[1-(S)-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohex-
ylmethyl phosphinic acid (CGP 54626);
(3-aminopropyl)(cyclohexylmethyl)phosphinic acid (CGP 46381); and
(2S)-(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911). Other
GABA.sub.B receptor inhibitors will be apparent to the skilled
artisan.
[0073] In other embodiments, the inhibitors of HMG CoA reductase,
farnesyl transferase inhibitors, and geranylgeranyl transferase,
and inhibitors of inhibitory neuronal activity, (collectively
referred to as "inhibitor compounds") may be used in combination to
treat the cognitive disorder or modulate LTP. Combinations include
a HMG CoA reductase inhibitor and a farnesyl transferase inhibitor,
a HMG CoA reductase inhibitor and a geranylgeranyl transferase
inhibitor, a farnesyl and geranylgeranyl transferase inhibitor, a
HMG CoA reductase inhibitor in combination with farnesyl and
geranylgeranyl tranferase inhibitors, or a HMG CoA reductase
inhibitor in combination with an inhibitor of inhibitory neuronal
activity. Other combinations will be apparent to the skilled
artisan. While the combinations may be used generally for the
cognitive disorders effectively treated by HMG CoA reductase
inhibitors alone, some disorders may be treated with a specific
combination where the molecular basis underlying the disorders is
suggested as a farnesylated protein (e.g., RAS), a
geranylgeranlylated protein (e.g., Rho or Rac), or a GABA receptor
activity. For instance, learning disorders associated with NF-I may
be treated with a combination of HMG CoA reductase inhibitor and a
farnesyl transferase inhibitor or a HMG CoA reductase inhibitor and
a GABA.sub.A receptor inhibitor.
[0074] The inhibitor compounds may be administered in the form of a
composition. In other embodiments the inhibitor combinations are
administered adjunctively, by the same route or by a different
route. Adjunctive administration includes simultaneous or
sequential administration of the inhibitor compounds.
[0075] The amounts of the inhibitor compounds to be administered
will be determined empirically in accordance with conventional
procedures. Generally, for administering the inhibitor compounds,
the subject formulations are given at a pharmacologically effective
dose. A "pharmacologically effective amount" or "pharmacologically
effective dose" is an amount sufficient to produce the desired
physiological effect or an amount capable of achieving the desired
result, particularly for treating the disorder or condition,
including reducing or eliminating one or more symptoms of the
disorder or disease. Thus the compounds and compositions described
herein may be administered therapeutically to achieve a therapeutic
benefit or prophylactically to achieve a prophylactic benefit. By
therapeutic benefit is meant eradication or ameliorating of the
underlying disorder being treated, and/or eradication or
amelioration of one or more of the symptoms associated with the
underlying disorder such that the patient reports an improvement in
cognitive function, notwithstanding that the patient may still be
affected with the underlying disorder.
[0076] In the case of cognitive disorders, administration of the
compounds and compositions to a patient suffering from the
cognitive deficit provides a therapeutic benefit when there is
improvement, enhancement, or restoration in the cognitive function.
The compounds and compositions may also be administered
prophylactically to a patient at risk of being afflicted with the
cognitive disorder. For instance, these include individuals who
have been diagnosed with an inherited disorder that has an
associated disruption of normal cognitive function such that
therapy may be initiated by early diagnosis (e.g., infancy).
[0077] A therapeutically effective dose of the inhibitor compounds
is readily determined by methods well known in the art. Factors to
consider in determining an appropriate dose include, but are not
limited to, size and weight of the subject, the age and sex of the
subject, the type of cognitive disorder, the severity of the
cognitive disorder, method of delivery of the compounds and
compositions, and half-life and efficacy of the inhibitor
compounds.
[0078] An initial effective dose can be estimated initially from
cell culture assays. For example, because the hippocampus is a
model system for learning and memory, in vitro culture systems
using hippocampal slices or cultures may be suitable for initial
determination of an effective dose. The cells may be contacted with
the inhibitor compounds and in the absence of inhibitor to
determine the levels of drug useful for enhancing the cellular
correlates of neural processes associated with cognitive function,
such as LTP.
[0079] Following in vitro studies, a dose can then be formulated in
experimental animal models to generate data on circulating
concentration or tissue concentration, including that of the
IC.sub.50 (i.e., concentration sufficient to affect 50% of the
activity being targeted or measured) as initially determined by the
in vitro culture assays. Suitable experimental animals include, but
are not limited to mouse, rat, guinea pigs, rabbits, pigs, monkeys
and chimpanzees. As with the in vitro studies, initial
determination is made of an effective dose of the inhibitor
compound (e.g., C.sub.max) and the corresponding pharmacokinetic
profile. Useful in this regard are numerous identified animal model
systems (e.g., pure bred animal lines) with associated cognitive
disorder or transgenic (e.g., knockout) animals that mimic or
approximate the genetic disorders that display the cognitive
deficit. Behavioral tests can be conducted on these animal systems
to determine an effective dose.
[0080] In accordance with the above, the dosages of the HMG CoA
reductase inhibitors may be the standard dosages administered to
treat hypercholesterolemia (i.e., an amount sufficient to lower
serum cholesterol levels in a subject with hypercholesterolemia).
Thus, an amount of inhibitor compound is used to lower the
cholesterol level to those observed on or below the 95th
percentile, on or below the 85th percentile, on or below the 75th
percentile, on or below the 50th percentile of the subject
population, to about 25th percentile of the subject population. In
some embodiments, the amount of inhibitor compound is administered
to lower the cholesterol level below about 240 mg/dL, below about
220 mg/dL, below about 200 mg/dL, below about 190 mg/dL, below
about 180 mg/dL, or below about 170 mg/dL.
[0081] In other embodiments, an amount of HMG CoA reductase
inhibitor is administered to lower the c-LDL levels to that below
about the 95th percentile of the general population pool, below
about the 85th percentile of the general population pool, below
about the 75th percentile of the general population, below about
the 50th percentile of the general population, to about the 25th
percentile of the general population pool. Thus, in some
embodiments, an amount of HMG CoA reductase inhibitor is
administered to lower the c-LDL in a human subject to less than
about 160 mg/dL, to less than about 130 mg/dL, to less than about
100 mg/dL, to less than about 70 mg/dL, with the lower limit being
a level of LDL considered healthy, which may range from 40 mg/dL or
50 mg/dL for the human population.
[0082] Exemplary dosages for use of atorvastatin (Lipitor.RTM.) in
the treatment of hypercholesterolemia are from about 10 mg to about
80 mg per day. For subjects of 45 to 100 kg body weight, this
dosage corresponds to about 0.1 mg/kg/day to about 1.8 mg/kg/day.
The recommended dosages of lovastatin (Mevacor.RTM.) is from about
10 mg to about 80 mg/day in one or two dosages, or about 0.1
mg/kg/day to about 1.8 mg/kg/day. The recommended dosage of
rosuvastatin (Crestor.RTM.) is from about 5 mg to about 40 mg/day,
or about 0.05 mg/kg/day to about 0.9/mg/kg/day. The recommended
dosage for pravastatin (Pravachol.RTM.) is from about 10 mg to
about 80 mg/day as a single dose, or about 0.1 mg/kg/day to about
1.0 mg/kg/day. The recommended dosage for simvastatin (Zocor.RTM.)
is from about 5 mg to about 80 mg/day taken once per day, or about
0.05 mg/kg/day to about 1.8 mg/kg/day. Determining corresponding
dosages for other HMG CoA reductase inhibitors are well within the
skill of those in the art.
[0083] In other embodiments, dosages are lower than those
prescribed to treat hypercholesterolemia or are dosages that do not
result in significant lowering of serum cholesterol levels in the
treated subject but which are effective in treatment of the
cognitive deficit. These dosages are referred herein as "low
dosages." In some embodiments, a significant lowering of
cholesterol level is a change of about 5 percentile, 10 percentile,
15 percentile, 20 percentile, 30 percentile, 40 percentile of the
cholesterol level in the general population. In other embodiments,
a significant lowering of cholesterol level is change in serum or
LDL cholesterol level of 20 mg/dL, 30 mg/dL, 50 mg/dL, 75 mg/dL or
more. For atorvastatin or lovastatin, this may correspond to a
dosage of from about 0.1 mg/kg/day to about 0.01 mg/kg/day or
lower. For rosuvastatin and simvastatin, the lower dosage may
correspond to a dosage of from about 0.05 mg/kg/day to about 0.005
mg/kg/day. Determining low dosages of all of the HMG CoA reductase
inhibitors are well within the skill of those in the art.
[0084] The inhibitor compounds may be provided as various
pharmaceutical compositions formulated in pharmaceutical
compositions per se, or in the form of a hydrate, solvate, or
pharmaceutically suitable salts thereof or with a suitable
excipient. Accordingly, in one embodiment, the pharmaceutical
compositions comprise a pharmaceutically acceptable carrier or
vehicle and a pharmacologically effective amount of the inhibitor
compound.
[0085] As described above, pharmaceutically acceptable salts are
intended to include any art recognized pharmaceutically acceptable
salt of the compound or inhibitor which is made with counterions
understood in the art to be generally acceptable for pharmaceutical
uses and which possesses the desired pharmacological activity of
the parent compound. Examples of salts include sodium, potassium,
lithium, ammonium, calcium, as well as primary, secondary, and
tertiary amines, esters of lower hydrocarbons, such as methyl,
ethyl, and propyl. Other salts include organic acids, such as
acetic acid, propionic acid, pyruvic acid, maleic acid, succinic
acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,
salicylic acid, etc.
[0086] As used herein, pharmaceutically acceptable vehicle or
pharmaceutically acceptable carrier comprise any of standard
pharmaceutically accepted carriers used by those skilled in the art
for administering a pharmaceutical composition. Thus, the inhibitor
compounds may be prepared as formulations in pharmaceutically
acceptable excipients suitable for any mode of administration that
include, but are not limited to, oral, topical, transdermal,
cutaneous, subcutaneous, intravenous, intraperitoneal,
intramuscular, nasal, transdermal, vaginal, buccal, and rectal
(e.g., colonic administration) delivery. Choosing the appropriate
route of administration is well within the skill of the art.
[0087] For oral administration, the pharmaceutical compositions may
be prepared with pharmaceutically acceptable excipients such as
binding agents (e.g., starch, carboxymethyl cellulose,
hydroxylpropyl methyl cellulose), fillers (e.g., lactose,
microcrystalline cellulose, calcium phosphate, etc.), lubricants
(e.g., magnesium stearate, talc, silicon dioxide, etc.);
disintegrants (potato starch and sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). Formulations for oral
administrations may take various forms, including, but not limited
to, tablets, capsules, lozenges, powders, etc. Pills, tablets, or
capsules may have an enteric coating that remains intact in the
stomach but dissolves in the intestine. Various enteric coatings
are known in the art, a number of which are commercially available,
including, but not limited to, methacrylic acid-methacrylic acid
ester copolymers, polymer cellulose ether, cellulose acetate
phathalate, polyvinyl acetate phthalate, hydroxypropyl methyl
cellulose phthalate, and the like.
[0088] The inhibitors compounds may be in liquid form prepared in
diluents for administration orally or by injection. These diluents
include, by way of example and not limitation, saline, phosphate
buffer saline (PBS), aqueous ethanol, or solutions of glucose,
mannitol, dextran, propylene glycol, polyethylene glycol (e.g.,
PEG400), and mixtures thereof. Suitable diluents also include
non-aqueous vehicles, including oils and other lipophilic solvents,
such as various vegetable oils, animal oils, and synthetic oils
(e.g., peanut oil, sesame oil, olive oil, corn oil, safflower oil,
soybean oil, etc.); fatty acid esters, including oleates,
triglycerides, etc.; cholesterol derivatives, including cholesterol
oleate, cholesterol linoleate, cholesterol myristilate, etc.;
liposomes; and the like. The compositions for injection may be
prepared directly in a lipophilic solvent or preferably, as
emulsions (see, e.g., Liu, F. et al., Pharm. Res. 12: 1060-1064
(1995); Prankerd, R. J. J., Parent. Sci. Tech. 44: 139-49 (1990);
and U.S. Pat. No. 5,651,991). The formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multidose
containers. The diluents may also contain suspending agents (e.g.,
soribitol solution, cellulose derivatives, or hydrogenated edible
fats) and emulsifying agents (e.g., lecithin or acacia).
[0089] Formulations for rectal or vaginal administration may be in
the form of salves, tinctures, cremes, suppositories, enemas or
foams. Suppositories for rectal application may contain
conventional suppository bases such as cocoa butter, carbowaxes,
polyethylene glycols, or glycerides, which are solid or semi-solid
at room temperature but liquid at body temperature.
[0090] Additionally, the pharmaceutical compositions may include
bactericidal agents, stabilizers, buffers, emulsifiers,
preservatives, flavoring, sweetening agents, and the like as needed
or desired in the various formulations.
[0091] The pharmaceutical compositions comprising the inhibitor
compounds may be manufactured in a manner well known to the skilled
artisan, such as by conventional means of mixing, dissolving,
granulating, levigating, emulsifying, encapsulating, entrapping or
lyophilization processes. Suitable pharmaceutical formulations and
methods for preparing such compositions may be found in various
standard references, such as Remington's Pharmaceutical Sciences,
17th edition, Mack Publishing Co., Philadelphia, Pa. (1985) and
Handbook of Pharmaceutical Excipients, 3rd Ed, Kibbe, A. H. ed.,
Washington D.C., American Pharmaceutical Association (2000); hereby
incorporated by reference in their entirety.
[0092] Additionally, the inhibitors, either separately or as a
combination, may also be introduced or encapsulated into the lumen
of liposomes for delivery and for extending lifetime of the
compounds. As known in the art, liposomes can be categorized into
various types: multilamellar (MLV), stable plurilamellar (SPLV),
small unilamellar (SUV) or large unilamellar (LUV) vesicles.
Liposomes can be prepared from various lipid compounds, which may
be synthetic or naturally occurring, including phosphatidyl ethers
and esters, such as phosphotidylserine, phosphotidylcholine,
phosphatidyl ethanolamine, phosphatidylinositol,
dimyristoylphosphatidylcholine; steroids such as cholesterol;
cerebrosides; sphingomyelin; glycerolipids; and other lipids (see,
e.g., U.S. Pat. No. 5,833,948).
[0093] Cationic lipids are also suitable for forming liposomes.
Generally, the cationic lipids have a net positive charge and have
a lipophilic portion, such as a sterol or an acyl or diacyl side
chain. Preferably, the head group is positively charged. Typical
cationic lipids include 1,2-dioleyloxy-3-(trimethylamino)propane;
N-[1-(2,3,-ditetradecycloxy)propyl]-N,N-dimethyl-N--N-hydroxyethylammoniu-
m bromide; N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy
ethylammonium bromide;
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride;
3-[N--(N',N'-dimethylaminoethane) carbamoyl] cholesterol; and
dimethyldioctadecylammonium.
[0094] Of particular interest are fusogenic liposomes, which are
characterized by their ability to fuse with a cell membrane upon
appropriate change in physiological condition or by presence of
fusogenic component, particularly a fusogenic peptide or protein.
In one aspect, the fusogenic liposomes are pH and temperature
sensitive in that fusion with a cell membrane is affected by change
in temperature and/or pH (see for example, U.S. Pat. Nos. 4,789,633
and 4,873,089). Generally, pH sensitive liposomes are acid
sensitive. Thus, fusion is enhanced in physiological environments
where the pH is mildly acidic, for example the environment of a
lysosome or endosome. This property allows direct release of the
liposome contents into the intracellular environment following
endocytosis of liposomes (Mizoue, T., Int. J. Pharm. 237: 129-137
(2002)).
[0095] Another form of fusogenic liposomes comprises liposomes that
contain a fusion-enhancing agent. When incorporated into the
liposome or attached to the lipids, the agents enhance fusion of
the liposome with other cellular membranes, thus resulting in
delivery of the liposome contents into the cell. The agents may be
fusion enhancing peptides or proteins, including hemaggulutinin HA2
of influenza virus (Schoen, P., Gene Ther. 6: 823-832 (1999));
Sendai virus envelope glycoproteins (Mizuguchi, H., Biochem.
Biophys. Res. Commun. 218: 402-407 (1996)); vesicular stomatitis
virus envelope glycoproteins (VSV-G) glycoprotein (Abe, A. et al.,
J. Virol. 72: 6159-63 (1998)); peptide segments or mimics of fusion
enhancing proteins; and synthetic fusion enhancing peptides (e.g.,
Kono, K. et al., Biochim. Biophys. Acta. 1164: 81-90 (1993);
Pecheur, E. I., Biochemistry 37: 2361-71 (1998); and U.S. Pat. No.
6,372,720).
[0096] Liposomes also include vesicles derivatized with a
hydrophilic polymer, as provided in U.S. Pat. Nos. 5,013,556 and
5,395,619, hereby incorporated by reference, (see also, Kono, K. et
al., J. Controlled Release 68: 225-35 (2000); Zalipsky, S. et al.,
Bioconjug. Chem. 6: 705-708 (1995)) to extend the circulation
lifetime in vivo. Hydrophilic polymers for coating or derivation of
the liposomes include polyethylene glycol, polyvinylpyrrolidone,
polyvinylmethyl ether, polyaspartamide, hydroxymethyl cellulose,
hydroxyethyl cellulose, and the like. In addition, as described
above, attaching proteins that bind a cell surface protein which is
endocytosed, e.g., capsid proteins or fragments thereof tropic for
a particular cell types and antibodies for cell surface proteins
which undergo internalization (see Wu et al, supra; Wagner et al.,
supra), may be used for targeting and/or facilitating uptake of the
liposomes to specific cells or tissues.
[0097] Liposomes are prepared by ways well known in the art (see,
e.g., Szoka, F. et al., Ann. Rev. Biophys. Bioeng. 9: 467-508
(1980)). One typical method is the lipid film hydration technique
in which lipid components are mixed in an organic solvent followed
by evaporation of the solvent to generate a lipid film. Hydration
of the film in aqueous buffer solution, preferably containing the
subject compounds and compositions, results in an emulsion, which
is sonicated or extruded to reduce the size and polydispersity.
Other methods include reverse-phase evaporation (see, e.g.,
Pidgeon, C. et al., Biochemistry 26: 17-29 (1987); Duzgunes, N. et
al., Biochim. Biophys. Acta. 732: 289-99 (1983)), freezing and
thawing of phospholipid mixtures, and ether infusion.
[0098] In another preferred embodiment, the carriers are in the
form of microparticles, microcapsules, microspheres and
nanoparticles, which may be biodegradable or non-biodegradable
(see, e.g., Microencapsulates: Methods and Industrial Applications,
Drugs and Pharmaceutical Sciences, Vol 73, Benita, S. ed, Marcel
Dekker Inc., New York, (1996); incorporated herein by reference).
As used herein, microparticles, microspheres, microcapsules and
nanoparticles mean a particle, which is typically a solid,
containing the substance to be delivered. The substance is within
the core of the particle or attached to the particle's polymer
network. Generally, the difference between microparticles (or
microcapsules or microspheres) and nanoparticles is one of size.
Typically, microparticles have a particle size range of about 1 to
about >1000 microns. Nanoparticles have a particle size range of
about 10 to about 1000 nm.
[0099] A variety of materials are useful for making microparticles
containing the inhibitor compound. Non-biodegradable microcapsules
and microparticles include, but not limited to, those made of
polysulfones, poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl
acetate, hydroxyethylmethacrylate-methyl-methacrylate copolymers.
These are useful for implantation purposes where the encapsulated
compound diffuses out from the capsules. In another aspect, the
microcapsules and microparticles are based on biodegradable
polymers, preferably those that display low toxicity and are well
tolerated by the immune system. These include protein based
microcapsulates and microparticles made from fibrin, casein, serum
albumin, collagen, gelatin, lecithin, chitosan, alginate or
poly-amino acids such as poly-lysine. Biodegradable synthetic
polymers for encapsulating may comprise polymers such as
polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)
(PLGA), poly(caprolactone), polydioxanone trimethylene carbonate,
polyhybroxyalkonates (e.g., poly(b-hydroxybutyrate)), poly(g-ethyl
glutamate), poly(DTH iminocarbony (bisphenol A iminocarbonate),
poly (ortho ester), and polycyanoacrylate. Various methods for
making microparticles containing the subject compounds are well
known in the art, including solvent removal process (see for
example, U.S. Pat. No. 4,389,330); emulsification and evaporation
(Maysinger, D. et al., Exp. Neuro. 141: 47-56 (1996); Jeffrey, H.
et al., Pharm. Res. 10: 362-68 (1993)), spray drying, and extrusion
methods.
[0100] Another type of carrier is nanoparticles. Submicron and
nanoparticles are generally made from amphiphilic diblock,
triblock, or multiblock copolymers, as is known in the art.
Polymers useful in forming nanoparticles include, but are limited
to, poly(lactic acid) (PLA; Zambaux et al., J. Control Release 60:
179-188 (1999)), poly(lactide-co-glycolide), blends of
poly(lactide-co-glycolide) and polycarprolactone, diblock polymer
poly(l-leucine-block-1-glutamate), diblock and triblock poly(lactic
acid) (PLA) and poly(ethylene oxide) (PEO) (De Jaeghere, F. et al.,
Pharm. Dev. Technol. 5: 473-83 (2000)), acrylates, arylamides,
polystyrene, and the like. As described for microparticles,
nanoparticles may be non-biodegradable or biodegradeable. In
addition, nanoparticles may be made from poly(alkylcyanoacrylate),
for example poly(butylcyanoacrylate), in which the compound to be
delivered is absorbed onto the nanoparticles and coated with
surfactants (e.g., polysorbate 80). Methods for making
nanoparticles are similar to those for making microparticles and
include, by way of example and not limitation, emulsion
polymerization in continuous aqueous phase,
emulsification-evaporation, solvent displacement, and
emulsification-diffusion techniques (see, e.g., Kreuter, J., Nano
particle Preparation and Applications, In Microcapsules and
nanoparticles in medicine and pharmacy, M. Donbrow, ed., pg.
125-148, CRC Press, Boca Rotan, Fla., 1991; incorporated herein by
reference).
[0101] Hydrogels are also useful in delivering the subject agents
into a host. Generally, hydrogels are crosslinked, hydrophilic
polymer networks permeable to a wide variety of drug compounds.
Hydrogels have the advantage of selective trigger of polymer
swelling, which results in controlled release of the entrapped drug
compound. Depending on the composition of the polymer network,
swelling and subsequent release may be triggered by a variety of
stimuli, including pH, ionic strength, thermal, electrical,
ultrasound, and enzyme activities. Non-limiting examples of
polymers useful in hydrogel compositions include, among others,
those formed from polymers of poly(lactide-co-glycolide),
poly(N-isopropylacrylamide); poly(methacrylic acid-g-polyethylene
glycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene)
glycol; and natural compounds such as chrondroitan sulfate,
chitosan, gelatin, or mixtures of synthetic and natural polymers,
for example chitosan-poly(ethylene oxide). The polymers are
crosslinked reversibly or irreversibly to form gels embedded with
the inhibitor compound, or pharmaceutical compositions thereof
(see, e.g., U.S. Pat. Nos. 6,451,346; 6,410,645; 6,432,440;
6,395,299; 6,361,797; 6,333,194; 6,297,337 Johnson, O. et al.,
Nature Med. 2: 795 (1996); incorporated by reference in their
entirety).
[0102] Another pharmaceutical compositions may include those in the
form of transdermal patches for delivery of the compounds through
the skin by diffusion or electrically mediated transport (see,
e.g., Banga, A. K. et al., Int J Pharm. 179(1):1-19 (1999); U.S.
Pat. Nos. 5,460,821, 5,645,854, 5,853,751, 6,635,274, 6,564,093;
all publications incorporated herein by reference).
[0103] In some embodiments, the inhibitors may be provided as a
depot, such as a slow release composition comprising particles, a
polymer matrix (e.g., a collagen matrix, carbomer, etc.) that
maintains release of compounds over an extended period of time, use
of a pump which continuously infuses the inhibitor compounds over
an extended period of time with a substantially continuous rate,
and the like. These and other combinations of administering
effective dosages will be apparent to those skilled in the art.
[0104] The inhibitor compounds may be provided in the form of a kit
or packaged formulation. A kit or packaged formulation as used
herein includes one or more dosages of an HMG CoA reductase
inhibitor, or salts, solvates or hydrates thereof in a container
holding the dosages together with instructions for administration
to a host. For example, the package may contain the HMG CoA
reductase inhibitors along with a pharmaceutical carrier combined
in the form of a powder for mixing in an aqueous solution, which
can be ingested by the afflicted subject. Another example of
packaged drug is a preloaded pressure syringe, so that the
compositions may be delivered intravenously, intramuscularly. The
package or kit includes appropriate instructions, which encompasses
diagrams, recordings (e.g., audio, video, compact disc), and
computer programs providing directions for use of the combination
therapy.
6.3 Methods of Measuring Cognitive Function
[0105] To determine whether a subject is afflicted with a cognitive
deficit and/or to determine improvement or restoration of cognitive
function, a variety of tests may be employed for both animal model
systems and for assessing individual patients. These include tests
ranging from assessments of general cognitive ability to
measurement of specific physiological processes associated with
cognitive function.
[0106] The global examination of cognitive deficits may employ
those commonly used for diagnosing such disorders as described in
various reference works, such as Diagnostic and Statistical Manual
of Mental Disorders, 4.sup.th Ed., American Psychiatric
Association; (2000) (acronym DSM) and the International
Classification of Disease (ICD), 10.sup.th Revision, World Health
Organization (WHO) (2003). The DSM provides a basis for selecting
the disorder from a classification that best reflects the signs and
symptoms displayed by the individual being evaluated (diagnostic
classification); a set of diagnostic criteria that indicates what
symptoms must be present (and for how long) in order to qualify for
a diagnosis (i.e., inclusion criteria) as well as those symptoms
that must not be present (i.e., exclusion criteria) in order for an
individual to qualify for a particular diagnosis (diagnostic
criteria sets); and a description of each disorder that includes
diagnostic features, subtypes of the disorder, culture, age, and
gender features, prevalence, course of the disorder, hereditary
pattern, and differential diagnosis. For instance, in an exemplary
embodiment for diagnosing ADHD, the DSM indicates a diagnosis when
the subject suffers from 6 or more symptoms of inattention that
persists for more than 6 months that is maladaptive and
inconsistent with the developmental level, and/or 6 or more
symptoms of hyperactivity-impulsivity that has persisted for more
than 6 months that is maladaptive or inconsistent with the
developmental level.
[0107] The ICD is a more general reference work for all diseases
and includes classifications diseases and other health problems
recorded on many types of health and vital records, including death
certificates and hospital records. ICD provides descriptions of
mental and behavioral disorders (Chapter V); diseases of the
nervous system (Chapter IV); congenital malformations, and
chromosomal abnormalities (Chapter XVII). The DSM and ICD systems
provide a set of standard criteria for effectively and reliably
diagnosing a broad range of cognitive disorders.
[0108] Exemplary tests for cognitive function may use any number of
procedures used in the art. In some embodiments, the analysis of
cognitive function may use that described in Roid, G.,
Stanford-Binet Intelligence Scale, 5th Ed., Riverside Publishing,
which is a standardized test that assesses intelligence and
cognitive abilities in children and adults, generally of ages of
about 2-85+ years. The test measures four areas that include verbal
reasoning, quantitative reasoning, visual-spatial processing, and
working memory. These areas are covered by subtests for measuring
vocabulary, comprehension, verbal absurdities, pattern analysis,
matrices, paper folding and cutting, copying, quantitative, number
series, equation building, memory for sentences, memory for digits,
memory for objects, and bead memory. The tests identify a distinct
hierarchy of abilities from normal to affected patients.
[0109] In some embodiments, the test for cognitive function may use
the Mini-Mental State Exam (MMSE) and variations thereof (Folstein,
M. F. et al., J. Psych. Res. 12:189-198 (1975)). MMSE is a test of
cognitive status that typically takes between 5 and 10 minutes to
administer. Areas measured on the MMSE include orientation to time
and place, immediate and delayed verbal recall memory, attention,
concentration, naming, repetition, following a 3-step command,
following a written command, sentence writing, and visual-motor
copying. Performance on each of the tasks is numerically graded
with a maximum score of 30, with scores lower than 23 being
considered indicative of cognitive impairment. The MMSE may be used
to identify patients with cognitive disturbance from those without
such disturbance and is also applicable to measuring the changes in
cognitive state upon treatment. This test as well as others
described herein and known in the art may be used in combination
with other tests to substantiate or correlate the results.
[0110] In other embodiments, the test for cognitive function is the
Wechsler Intelligence Scale for Children or Adults. The test for
adults has two sections, a verbal and a performance measurement.
The verbal section has a general knowledge test, a digit span test
in which subjects are given sets of digits to repeat initially
forwards then backwards (auditory recall and short term memory), a
vocabulary test to measure expressive word knowledge, an arithmetic
tests that measures distractibility as well as numerical reasoning,
a comprehension test that focuses on issues of social awareness,
and a similarities test for measuring concept formation that asks
subjects to specify how two seemingly dissimilar items might in
fact be similar. The performance section involves picture
completion test (small pictures that all have one vital detail
missing) that measures attention to detail, picture arrangement
test where the subject is required to arrange them into a logical
sequence, a block design test that involves putting sets of blocks
together to match pattern on cards, digit symbol test that involves
copying a coding pattern, and object assembly test that involves
solving jig-saw type puzzles. The scores on both sections are
processed to arrive at a numerical intelligence quotient (IQ).
[0111] The Wechsler Intelligence Scale for Children is similar to
the adult test, having a verbal section and a performance section.
The verbal sections involve general knowledge test (oral, general
information questions), a similarities test that requires
explaining how two different things or concepts are similar, an
arithmetic test that uses verbally framed math applications
problems without paper, a vocabulary test that requires giving oral
definitions of words, a comprehension test that measures social and
practical understanding, and a digit span test that requires
repeating dictated series of digits forwards and backwards. The
performance section involves a picture completion test (identifying
missing parts of pictures, coding A test (marking rows of shapes
with different lines according to a code as quickly as possible),
coding B test (transcribing a digit-symbol code as quickly as
possible), a picture arrangement test (sequencing cartoon pictures
to make sensible stories), a block design test (copying small
geometric designs with four or nine larger plastic cubes), an
object assembly test (puzzles of cut-apart silhouette objects with
no outline pieces), symbol search test (deciding if target symbols
appear in a row of symbols), and maze tests (no pencil lifting,
points off for entering blind alleys). As with the adult version,
full scale IQ is based on the tests in the verbal and performance
scales.
[0112] Other embodiments for measuring cognitive function include,
among others, Test of Nonverbal Intelligence and Comprehensive Test
of Nonverbal Intelligence. Related tests may be used to assess
specific brain areas as they relate to attention, executive
function, language, memory and visual-spatial and visual-motor
skills. Non-limiting examples of these types of tests include
NEPSY: A Development Neuropsychological Assessment; Delis-Kaplan
Executive Function System (D-KEFS); Comprehensive Test of
Phonological Processing (CTOPP); Rey-Osterrieth Complex Figure
Test; Children's Memory Scale, Wechsler Memory Scale--Third Edition
(WMS-III); Woodcock-Johnson (WJIII) Tests of Cognitive Abilities;
Beery-Buktenica Developmental Test of Visual Motor Integration;
Wisconsin Card Sorting Test (WCST); Children's Category Test,
Judgment of Line Orientation; Behavior Rating Inventory of
Executive Function; and Wide Range Assessment of Memory and
Learning (WRAML).
[0113] Some tests of cognitive function have been developed that
are useful extrapolations to animal model systems. Many of these
tests are based on operant and non-operant problem solving tasks.
General tests include delayed matching sample to sample (short term
memory), repeated acquisition (learning), temporal discrimination
(timing ability), condition and position response, and progressive
ratio (see Slikker et al., Toxicological Sciences 58:222-234
(2000)).
[0114] In some embodiments, the test for cognitive function in some
animal model systems is a water maze test, generally known as the
Morris water maze test, typically used to test learning and memory
in small animals such as rats and mice. The Morris water maze
consists of a round tank (pool) of water with a submerged hidden
escape platform from the water. Extra-maze cues, to test spatial
learning, may be placed around the tank at positions visible to the
test animal. The ability of the test animal to find the submerged
platform provides a measure of the learning and memory function.
Malperformance in the Morris water maze test has been associated
with impaired LTP.
[0115] In other embodiments, the cognitive test is a fear
conditioning test, which allows for the assessment of learning and
memory of aversive events. Fear conditioning typically relies on
the ability of normal animals to learn to fear a previously neutral
stimulus because of its temporal association with an aversive
stimulus, such as an electric shock, noxious odor, or a startling
noise. Typically, the test animal is placed in a conditioning
chamber (context) before the onset of a discrete stimulus (the
conditioned stimulus or CS), such as a discrete tone. The tone is
followed by the aversive stimulus, such as an electrical shock to
the foot. The task allows for the simultaneous assessment of
learning about simple, unimodal cues and learning about complex,
multimodal stimuli such as context. A related test is the startle
test, which is used to measure a number of behaviors, including
basic startle, pre-pulse inhibition, and fear potentiation of the
startle response.
[0116] Another type of cognitive test for experimental systems is
the Radial Arm Maze. An exemplary maze of this type has a number of
arms (e.g., 8) that extend outward from a circular central arena.
One or more of the arms is baited to contain a reward and the
animal tested for their ability to consume the bait as a function
of time. This cognitive test is used to measure spatial learning
and memory. Some versions of the task can be used to examine both
working and reference memory, such as by measuring the number of
reference memory errors (entering an arm that does not contain the
reward) and working memory errors (entering an arm containing the
reward but previously entered). Like the water maze, this task is
sensitive to hippocampal function.
[0117] In other embodiments, the cognitive test is a social
recognition test that is used to measure social learning and
memory. Animals are tested for their ability to remember
conspecifics over various time intervals. This may test a variety
of cognitive tasks, such as the ability to learn about the safety
of food from its conspecifics by sampling those food odors on the
breath of littermates. This test may also provide information on
aggression and social interaction with non-littermate conspecifics.
Memory components can be assessed by repeated exposures to the
different stimulus at various frequencies.
[0118] In further embodiments, the cognitive test may be an open
field test, which evaluates the subject for hyperactivity,
exploratory activity, and stereotyped rotation in a test chamber.
Additional behavior in this type of test includes, among others,
time taken to move to the edges of the open field apparatus, total
activity in the open field, and percentage of time spent in the
periphery. Versions of the task are used to assess anxiety and
memory for context.
[0119] In yet other embodiments, the cognitive test is the SHIRPA
Primary Screen, as described in Rogers, D. C. et al., Mamm. Genome
8:711-713 (1997)). This test examines the behavioral and functional
profile of the animal by an initial evaluation of the undisturbed
behavior in a testing chamber and then under a series of
manipulations to elicit a behavioral response from the animal. In
the test, observations are made of gait or posture, motor control
and co-ordination, changes in excitability and aggression,
salivation, lacrimation, piloerection, defecation and muscle tone.
In addition to these scored behaviors, the animal is evaluated for
other types of stereotyped behavior including, convulsions,
compulsive licking, self-destructive biting, retropulsion and
indications of spatial disorientation. Initial observations are
followed by a sequence of manipulations using tail suspension and
the grid across the width of the arena. To complete the assessment,
the animal is restrained in a supine position to record autonomic
behaviors prior to measurement of the righting reflex. Throughout
this procedure vocalization, urination and general fear,
irritability or aggression are recorded.
[0120] Where a biochemical or molecular defect, such as a genetic
abnormality is suspected, the cognitive tests may be used in
conjunction with tests used to determined existence of the
biochemical or genetic abnormality. Tests include analysis for
gross chromosomal abnormalities (e.g., metaphase chromosome), and
techniques for determining specific genetic defects, which include
as non-limiting examples, polymerase chain reaction, nucleic acid
sequencing, nucleic acid hybridization, restriction fragment length
analysis (for RFLP), single stranded conformational polymorphism,
and fluorescence in situ hybridization (FISH). For example, defects
in NF-1 gene may be based on RFLP (Jorde, L. B. et al., Am J Hum
Genet. 53(5):1038-50 (1993)); polymerase chain reaction (Abernathy,
C. et al., Clin Genet. 45(6):313 (1994)); and single stranded
conformational polymorphism (Gomez, L., Cancer Genet Cytogenet.
81(2):173-4 (1995)). Corresponding physiological (facial and limb
features) and developmental characteristics may also be assessed to
supplement the diagnosis.
[0121] In some embodiments, the test for the cognitive defect is an
in vitro test that measures molecular correlates of the processes
thought to be involved in cognitive function. In some embodiments,
the test is an electrophysiology test for LTP (see, e.g., Bliss and
Collingridge, Nature 361: 31-39 (1993)). In its basic format,
slices of the hippocampus containing the CA1 region, or other
suitable neural systems, are removed and a train of stimuli used to
evoke action potentials in presynaptic neurons. With certain types
of presynaptic stimulation, enhancement of the excitatory
postsynaptic potentials (EPSPs) is observed that can last for day
or weeks. Induction of LTP is dependent on Ca.sup.2+ entry into the
postsynaptic neuron triggered by N-methyl-D-aspartate receptor
activation (see, e.g., Tsien, R. et al. Cell 87:1327-1338 (1996)).
As discussed above, LTP may be generated in hippocampal cultures by
stimulation of a single input pathway (i.e., homosynaptic) by a
train of evoked potentials. Early phase or E-LTP may be induced by
a single high-frequency tetanic stimuli while late phase or L-LTP
is typically induced by multiples of such tetanic trains (see,
e.g., Thomas, M. J. et al., J Neurosci. 18:7118-7126 (1998)). L-LTP
may also be induced by paired stimulation of multiple input
pathways (i.e., heterosynaptic), where activation of one afferent
pathway is paired to a conditioning stimulus in another afferent
pathway in the neural network (Huang, Y. Y. et al., Proc. Natl.
Acad. Sci. USA 101(3):861-864 (2004)).
[0122] To determine whether the LTP is the early phase or the
longer lasting phase, various pharmacological agents may be added
to the cultures. These include as non-limiting examples,
transcription inhibitors, protein synthesis inhibitors, and
inhibitors of enzymes thought to be critical for establishment of
LTP. Transcription inhibitors include, among others, alpha
amanitin, actinomycin D, cordycepin, and
5,6-dichloro-1-D-ribofuranosylbenzimidazole. Protein synthesis
inhibitors useful in these in vitro tests include anisomycin,
cycloheximide, emetine, rapamycin (Cammalleri, M. et al., Proc Natl
Acad Sci USA 100(24):14368-73 (2003)), and puromycin, Enzyme
inhibitors may include enzymes involved in formation of LTP,
including protein kinase A inhibitors (e.g., KT5720), protein
kinase C inhibitors (e.g., chelerythrine); tyrosine kinase
inhibitors (e.g., genistein); calmodulin kinase (CaMK) inhibitors
(e.g., autocamtide-2-related inhibitory peptide (AIP)
(KKALRRQEAVDAL). These compounds may be used in combination with
the HMG CoA reductase inhibitors (or other modulators of the
isoprenoid pathway) to determine the effect of inhibitors on
LTP.
[0123] It is to be understood that other types of tests known in
the art may be used for the purposes described above, and are to be
included within the scope of the methods described herein.
7. EXAMPLES
7.1 Example 1
Treatment of Learning Deficits in an Animal Model of
Neurofibromatosis-1
[0124] 7.1.1 Animal Experiments
[0125] All animal protocols were approved by the Chancellor's
Animal Research Committee at the University of California at Los
Angeles, in accordance with the National Institutes of Health
guidelines. All the animals were 129T2/SvEmsJ-C57BL/6N F1 hybrids
generated by an F1 cross between nfl+/- mice (maintained in the
C57BL/6N background for more than 11 generations) and wild-type
mice on the 129T2/SvEmsJ background. In every experiment, the
controls were the littermates of the mutants. All experiments were
carried out blind with respect to genotype and treatment.
[0126] 7.1.2 Western Blot Analysis for p44/42 MAP Kinase
Phosphorylation and p21Ras Activity
[0127] Hippocampus from control and lovastatin-treated mice were
isolated and homogenized in protein extraction buffer, with 1%
Triton X-100, 25 mM HEPES pH 7.5, 150 mM NaCl, 10% Glycerol, 2 mM
EDTA, 1 ug ml-1 leupeptin (Sigma), 100 ug ml-1 PMSF (Sigma), 10 mM
NaF (Sigma), 25 mM Na glycerophosphate (Sigma) and 1 mM
Na.sub.3VO.sub.4 (Sigma). Supernatant was collected after 10 min of
13,000 rpm centrifugation. Protein concentrations were determined
by bicirchoninic acid protein assay (Pierce). Lysates were added to
SDS loading buffer and boiled 2 min. Products were separated by
electrophoresis on a 4-15% SDS--PAGE gradient gel (Bio-Rad
Laboratories Inc.). Gels were blotted to Nitrocellulose membranes
(Bio-Rad) at 15 V in 25 mM Tris, 192 mM glycine and 20% (v/v)
methanol, then blocked for 1 h at room temperature with
Tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 and 5%
(w/v) non-fat dry milk. After washing in TBST, membranes were
hybridized 1 h at room temperature with anti-phospho-p44/42(Cell
Signaling) antibody diluted 1:1,000 in TBS, 0.1% (v/v) Tween 20 and
5% (w/v) non-fat dry milk. The membranes were then processed by ECL
Plus protocol (Amersham BioSciences, Inc.) for visualization of the
bands. Membranes were stripped in Strip buffer (Pierce) for 15 min
at room temperature, then probed with anti-p44/42 (cell Signaling)
as a control to normalize equal protein loading. Phosphorylated
p44/42 and p44/42 migrated at a relative molecular mass of 42,000
44,000.
[0128] 7.1.3 p21Ras Activity Assay
[0129] For p21Ras activity assay, p21Ras pull-down experiments were
performed with the EZ-detect p21Ras activation kit (Pierce
Biotechnology) according to the manufacturer's protocol.
Hippocampal lysates (200 mg) from different groups were incubated
with 40 mg GST-Raf1-RBD and one Swell Gel Immobilized Glutathione
Discs at 4.degree. C. for 2 h. The resin was washed 4 times with
Lysis/Binding/Wash buffer and 50 ul of 2.times.SDS Sample buffer
(125 mM Tris-HCl, pH 6.8, 2% glycerol, 4% SDS, 0.05% bromophenol
blue, and 5% 2-mercaptoethanol) was added. The spin columns were
centrifuged at 7,200.times.g for 2 min and the collected solution
was boiled for 5 min. Samples were applied 25 ml per lane on
SDS-PAGE. p21Ras was detected by Western blotting as described
using an anti-pan-p21Ras antibody (Sigma).
[0130] 7.1.4 Hippocampal LTP.
[0131] Transverse hippocampal slices (400 mm thick) were place in a
submerged recording chamber perfused (2 ml/min) with artificial
cerebrospinal fluid containing 120 mM NaCl, 3.5 mM KCL, 2.5 mM
CaCl.sub.2, 1.3 mM MgSO.sub.4, 1.25 mM NaH.sub.2PO.sub.4, 26 mM
NaHCO.sub.3 and 10 mM D-glucose at 34.degree. C. Extracellular
excitatory postsynaptic field potentials (EPSPs) were recorded with
a Pt/Ir electrode (FHC, Bowdoinham, Me.) from the stratum radiatum
layer of the area CA1, and the Schaffer collateral/commissural
afferents were stimulated with two bipolar electrodes placed one on
either side of the recording electrode (300 microns from the
recording electrode). Test pulses were alternated each minute
between the two electrodes throughout the duration of the
experiment. The stimulation intensity used during the experiment
was 60 mA. After the responses were monitored at least for 20 min
to ensure a stable baseline, LTP was induced with a single tetanus
delivered to one pathway (the test pathway) using a five-theta
burst stimulation (TBS) protocol (five bursts, each burst 4 pulses
at 100 Hz, 200 ms inter-burst interval). The untetanized pathway
served as a control pathway. Slices in which there was significant
drift in the control pathway were excluded from further analysis.
When multiple slices were used from a single animal, data were
averaged and then entered into analysis as a single subject. Thus,
all data reported reflect individual mice rather than individual
slices. To determine whether the magnitude of LTP differed
significantly between the groups, responses from the last 10 min
block of recordings (40-50 min) were compared. Mice were injected
with 10 mg/kg of lovastatin subcutaneously once per day for 4 days
and sacrificed on the 4th day, 6 hours following the final
injection. Slices were then prepared as described above.
[0132] 7.1.5 Water Maze Test.
[0133] The basic protocol for the water maze experiments has been
previously described. Mice from the 129T2/SvEmsJ-C57B/6N F1 genetic
background were given two trials per day (30-s inter-trial
intervals) with a probe trial (60 s) at the end of training day 5
and 7. Mice were given subcutaneous injections of 10 mg/kg
lovastatin or vehicle for 3 days before the 1st training day and
then 6 hours before training every day.
[0134] 7.1.6 Lovastatin Solution and Pellet.
[0135] Because of the extended nature of the lateralized reaction
time task (see below), lovastatin was administered orally as
pellets. 100 mg mevinolin (lovastatin, Sigma Inc.) in the lactone
form was dissolved in 2 ml of warm (55.degree. C.) ethanol, then
0.6 ml 1N NaOH and 20 ml water were added. The solution was
incubated at room temperature for about 30 min to complete the
conversion of mevinolin to the sodium salt. The final mevinolin
solution (4 mg/ml) was adjusted to pH7.5 with HCl and the volume
was brought to 25 ml 10. Vehicle was prepared in the same way
except that mevinolin was omitted. Lovastatin tablets (Eon Labs)
(prescription formulation) were crushed into powder and mixed with
melted peanut butter chips (H.B. Reese Candy Co.) and molded to 200
mg pellet. Each pellet contained 0.15 mg Lovastatin. The pellet was
administered orally (10 mg kg-1 dose) to mice once daily.
[0136] 7.1.7 Lateralized Reaction Time Task
[0137] Mice (Placebo-treated: nfl+/-=14, WT=10; lovastatin treated:
nfl+/-=7, WT=7) subjects were initially deprived of food to 90% of
their free-feeding weights. Mice were fed 1.5 gm of chow every day
in their home cages (1 hr after experiment). Lovastatin animals
received 1.1 gm of chow plus 400 mg pellets contain 0.3 mg
lovastatin (10 mg/kg) every day. Mice were trained in miniaturized
versions of a "5-choice" box (Med Associates Inc., St Albans Vt.)
that was equipped with a curved wall with horizontal array five
apertures that could be internally illuminated. The opposite wall
was fitted with a food receptacle where pellets were delivered as
reinforcers. The animals were shaped to produce a "poke and hold"
response in the central aperture. A correct response was scored
when the animals correctly poked a side aperture that had been
indicated during the poke and hold response. The side apertures
were initially illuminated for 30 seconds, which was gradually
decreased to 1 second over a period of weeks. When animals
performed at 75% accuracy at 1 second target stimulus duration they
entered the test phase. Mice were tested on a variable duration
condition in which the target aperture was illuminated for 0.5, 1.0
or 2.0 sec (varied from trial to trial within the session). Correct
responses/total trials were measured, which vary as a function of
the target stimulus duration and was therefore analyzed with
repeated measures analysis of variance (the repeated measure being
stimulus duration).
[0138] 7.1.8 Prepulse Inhibition
[0139] Mice subjects were initially deprived of food to 90% of
their free-feeding weights and subsequently fed 1.5 gm of chow or
1.1 gm of chow plus 400 mg of pellets containing 0.3 mg lovastatin
(10 mg/kg) every day in their home cages for 3 months. Following an
acclimation period of 5 min, mice were presented with a total of 20
noise bursts (40 ms duration, 120 dB, <1 ms rise/fall time). In
the prepulse inhibition phase, mice were presented with a total of
90 trials. Three prepulse intensities were tested: 70, 75 and 80
dB. Prepulses were 20 ms in duration with a rise/fall time of less
than 1 ms. For each prepulse intensity, there were three types of
trial: prepulse alone, prepulse/startle stimulus and startle
stimulus alone. In the prepulse/startle stimulus trial, the onset
of the prepulse preceded the onset of the startle stimulus by 100
ms. Background noise levels were maintained at 68 dB throughout
testing, and the trials were spaced 15 s34.
[0140] 7.1.9 Statistical Analysis
[0141] Data acquired from the water maze were analyzed by
repeated-measures ANOVA. Percent time in training quadrant for the
different genotypes was analyzed using 2-way ANOVA. Planned
comparisons using a paired t-test were used to analyze the
proximity data. Attention data was analyzed using three-way
repeated-measures ANOVA on the average of correct response rate.
PPI data was analyzed using two-way repeated-measures ANOVA. For
the electrophysiological experiments, the significance of
differences between the groups was determined by two-way ANOVA.
Post-hoc comparisons (Fisher's PLSD) between groups were carried
out where appropriate.
[0142] 7.1.10 Results of Lovastatin Treatment
[0143] Lovastatin, a specific inhibitor of the rate-limiting enzyme
in cholesterol biosynthesis (HMG-CoA reductase), is widely used to
treat hyperlipidemia in humans. Interestingly, previous studies
have shown that lovastatin can inhibit p21Ras isoprenylation and
activity. Since the cognitive deficits caused by mutations in the
NF1 gene may result from increased p21Ras activity, studies were
conducted to determine whether lovastatin could rescue these
deficits. Pharmacokinetic data in mice indicate that the dose used
for most of the mouse experiments described here (10 mg kg-1)
results in total plasma drug levels similar to those normally
present in patients taking lovastatin (data distributed by Merck
& CO., Inc; available at world wide web (www)
druginfonet.com/mevacor.htm). The biochemical studies show that
this dose was effective at ameliorating the abnormally high
p21Ras/MAPK activity in nfl+/- mice (FIG. 1).
[0144] The effect of lovastatin treatment on p21Ras/MAPK was
determined using western blotting. Mice were injected with 0-50
mg/kg lovastatin subcutaneously once per day for 4 days, and
sacrificed on the 4th day, 6 hours after the final injection.
Hippocampal extracts were prepared; proteins were resolved by
SDS-PAGE and transferred to membranes, hybridized with anti-phospho
p44/42 MAPK (Cell Signaling) antibody and visualized with ECL-Plus
(Amersham Biosciences). The results showed that lovastatin
decreased the amount of phosphorylated p44/42 MAPK (the active
form) in a dose-dependent fashion (FIG. 1a). The results in FIG. 1b
demonstrate that 10 mg/kg of lovastatin, the dose used in the
electrophysiological and behavioral experiments described below,
decreased the levels of phosphorylated p44/42 MAPK in nfl+/- mice.
Additionally, the results also showed that the levels of
phosphorylated p44/42 MAPK in nfl+/- mice are higher than in
wild-type littermates (WT). The nitrocellulose membranes used for
the analysis just described were also re-probed with an anti-p44/42
MAPK antibody to control for sample loading.
[0145] Neurofibromin functions as a p21Ras GTPase activating
protein which catalyzes the conversion of active GTP-bound p21Ras
to the inactive GDP-bound form. The impact of lovastatin treatment
on p21Ras activity was assessed directly. Hippocampal extracts were
reacted with GST-Raf1-RBD beads (Pierce Bio), which specifically
bind p21Ras-GTP, the active form of p21Ras. p21Ras-GTP was resolved
by SDS-PAGE and visualized with an anti-pan p21Ras antibody
(Sigma). The results showed that lovastatin decreased the
hippocampal levels of p21Ras-GTP in WT (FIG. 1c), just as it
decreased the levels of MAPK activity, and that these levels were
higher in nfl+/- mice. Altogether these data demonstrate that
lovastatin can decrease p21Ras/MAPK activity in the hippocampus and
may therefore be useful to treat the hippocampal LTP and cognitive
deficits of the nfl+/- mice.
[0146] A previous study had shown that the learning deficits of the
nfl+/- mice are likely caused by impairments in LTP, a stable
long-lasting change in synaptic strength widely believed to be a
key cellular mechanism for learning and memory. Therefore,
experiments were conducted to determine whether the LTP deficits in
nfl+/- mice could be reversed by lovastatin. Mice were injected
with 10 mg/kg of lovastatin subcutaneously once per day for 4 days
and sacrificed on the 4th day, 6 hours following the final
injection. LTP in hippocampal slices at the Schaffer collateral/CA1
synapse were examined since LTP at this synapse has been implicated
in hippocampal learning and memory. LTP was measured after a five
theta-burst stimulation protocol (TBS, five bursts 200 ms apart,
each burst of 4 pulses at 100 Hz), which mimics in vivo activity of
hippocampal neurons during exploratory behavior. FIG. 2 shows that
there was a difference among the genotypes and treatments (ANOVA,
F1, 26=8.55, P<0.05). The LTP measured in nfl+/- mutants was
significantly lower than in WT mice (PLSD, P<0.05; FIG. 2), a
result consistent with previously published findings. The amount of
LTP induced in nfl+/- mutants treated with lovastatin was
significantly higher than that induced in mutants (PLSD, P<0.05;
FIG. 2), and equivalent to that of WTs (PLSD, P=0.602; FIG. 2).
These data demonstrate that the lovastatin treatment completely
reversed the LTP deficits of the nfl+/- mice. Thus, treated animals
were further examined to determine whether statins could reverse
the cognitive deficits associated with NF- in mice.
[0147] Spatial problems are among the most common cognitive
deficits in individuals affected with NF1. It was previously shown
that nfl+/- mice have abnormal spatial learning tested in the
hidden version of the water maze, a task that is sensitive to
hippocampal lesions. To test the hypothesis that lovastatin can
rescue the deficits of nfl+/- mice in this hippocampal-dependent
task, just as it rescued their hippocampal p21Ras/MAPK and LTP
abnormalities, animals were injected with 10 mg/kg lovastatin
subcutaneously for 3 days before the 1st training day, and then 6
hours before behavioral training daily. Mice were trained with two
trials per day. No differences were observed between genotypes
and/or treatment groups in measures of acquisition, floating,
thigmotaxic behaviour or swimming speed (data not shown),
confirming that just as in humans nfl mutations in mice cause
selective deficits in cognitive function.
[0148] Spatial learning was assessed in probe trials given at the
end of water maze training on days 5 and 7 since previous studies
showed that probe trial performance is the most faithful measure of
spatial learning in the Morris maze. In the probe trials the
platform was removed from the pool and the mice were allowed to
search for it for 60 seconds. There was no significant difference
between WT and nfl+/- mice in the day 5 probe trial, because at
this time neither group showed clear evidence of having learned the
task (FIG. 3a). After two more days of training, it was observed
that the time spent searching in the training quadrant during the
day 7 probe trial was different among the different genotypes and
treatments (ANOVA, F1,82=4.415, P<0.05). WT mice spent
significantly more time searching in the training quadrant than
nfl+/- mice (PLSD, P<0.05; FIG. 3b), confirming that the nf1+/-
mutants have impaired spatial learning. In contrast, the mutants
treated with lovastatin spent as much time as WTs in the training
quadrant (P=0.862; FIG. 3b), and significantly more time than
mutants given placebo (P<0.05).
[0149] The lovastatin-mediated rescue of the spatial learning
deficits in nfl+/- mice was confirmed using another measure of
learning during the probe trial (proximity). All groups, except
nfl+/- mice on placebo (t21=0.313; P=0.757), searched closer to the
exact platform position than to the opposite position in the pool
(WT paired t-test, t22=6.274, P<0.0001; WT on lovastatin
t19=2.159, P<0.05; nfl+/- mice on lovastatin t20=2.170,
P<0.05; FIG. 3c). These results demonstrate that the spatial
learning deficits of the nfl+/- mice are not caused by irreversible
developmental abnormalities since they are reversed with acute
lovastatin treatment in adult mutant mice.
[0150] Besides spatial impairments, NF-1 patients also show
attention deficits. Thus, it was investigated whether nfl+/- mice
also exhibit impairments in attention, and whether lovastatin could
rescue those deficits. For this purpose, a lateralized
reaction-time task, a test that measures divided visuo-spatial
attention, was used. In this task, animals produce a fixation
response that triggers the delivery of a variable duration visual
target stimulus in one of their visual fields; the spatial location
and time of onset of the target is unpredictable. This task
therefore requires sustained (over time) and divided (across space)
attention. WT and nfl+/- mice were tested with lovastatin
(nfl.sup.+/-=7, WT=7) or placebo (nfl+/-=14, WT=10). The rate of
correct responses (an index of attention accuracy) revealed a
Genotype X Treatment X Target Stimulus Duration interaction (ANOVA,
F2, 70=3.200, P<0.05). At the most difficult stimulus duration
(0.5 sec), the correct response rate of WT mice is significantly
higher than that of nfl.sup.+/- mice (PLSD, P<0.05; FIG. 4a),
indicating that the nfl+/- mice have impaired attention. In
contrast, the correct response rate of nfl+/- mice treated with
lovastatin is indistinguishable from that of WT mice at target
stimulus duration of 0.5 sec (PLSD, P=0.148; FIG. 4a), and
significantly higher than nfl+/- given placebo (PLSD, P<0.05).
These data demonstrate that nfl.sup.+/- mice exhibit substantial
attention deficits and that lovastatin treatment can reverse these
deficits.
[0151] Children with attention-deficit hyperactivity disorder
(ADHD) are reported to have significantly reduced pre-pulse
inhibition (PPI). This task assays sensory "gating" of
environmental stimuli. A powerful and sudden acoustic stimulus will
elicit a whole body startle response. When the startle producing
stimulus is preceded by a weak pre-stimulus (by approximately 100
milliseconds) the startle response is inhibited in normal persons
and animals. Previous studies show a high incidence of ADHD in NF1
and support an association between ADHD and learning problems in
these children. Thus, nfl.sup.+/- mice were tested for deficits in
this task and whether these deficits could also be reversed by
lovastatin using the same treatment regimen described for the other
two behavioral experiments described above. A two-way repeated
measures ANOVA revealed significant main effects of Genotype and
Treatment (FIG. 4b). The nfl+/- animals have deficient PPI (F1,
30=7.42, P<0.05) and lovastatin treatment resulted in an
increase in performance (F1, 30=6.61, P<0.05). Importantly, the
performance of nfl+/- animals on lovastatin is indistinguishable
from that of WT animals on placebo (PLSD, P=0.877), demonstrating
that lovastatin can reverse the PPI deficits of these mutants.
[0152] The present results demonstrate that lovastatin treatment
can reverse the biochemical, electrophysiological, and cognitive
deficits observed in a mouse model of NF1, and that these deficits
are not due to irreversible developmental changes. Previous studies
have shown that an increase in p21Ras/MAPK activity is central to
the pathophysiology associated with NF1, and our biochemical data
demonstrate that lovastatin reverses the abnormally elevated
p21Ras/MAPK activity in an animal model of NF1. Together with
previous findings, these results indicate that the ability of
lovastatin to dampen the elevated p21Ras/MAPK signaling of the
nfl+/- mice rescues their deficits in a cellular mechanism of
learning and memory (LTP), and that this reverses the cognitive
impairments of these mutants. Importantly, the studies herein
demonstrate that the dose of lovastatin that is effective in nfl+/-
mice did not affect cognitive function in control mice, a result
consistent with randomized studies performed with human subjects
that did not identify a reliable effect of lovastatin on cognitive
function. Although it is worth noting that there are sporadic
reports that statins can be associated with mild cognitive
impairment, this study used doses of lovastatin which, based on
pharmacokinetic data, should produce a total plasma drug exposure
of less than 0.3 times that of typical human doses (i.e., an 80
mg/day dose). Altogether, the studies reported here demonstrate
that the cognitive deficits associated with NF-1 can be reversed by
treatments with lovastatin, a widely prescribed drug that is known
to be well-tolerated even in long-term treatments. Thus, these data
suggest that lovastatin could be used to treat the cognitive
impairments associated with NF-I in humans.
[0153] The foregoing descriptions of specific embodiments of the
present invention have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
Claims appended hereto and their equivalents.
[0154] All patents, patent applications, publications, and
references cited herein are expressly incorporated by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *