Note: Descriptions are shown in the official language in which they were submitted.
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Methods and Systems for Evaluating
Memory Agents
Cross-Reference to Related Applications
This application claims priority under 35 U.S.C. 119 to U.S. Provisional
Patent
Application Serial No. 61/081,845, filed July 18, 2008, the entire content of
which is fully
incorporated by reference herein.
Field of the Invention
The invention relates to methods and systems of evaluating, identifying or
assessing the
effectiveness of memory agent and/or training protocols administered to
subjects.
Background of the Invention
Memory is the recollection of past experiences. One is able to register new
information,
store it and then recall it sometime later. Recollection of facts and events
represents a form of
conscious, or explicit, memory. One also remembers less conscious skills, such
as riding a bike,
playing a musical instrument or associating simple cues with reward or
punishment, which
represent more implicit forms of memory. For both of these cases, the process
of memory
formation appears to proceed through three general stages. Acquisition
(learning) involves the
initial perception of a new experience. Short-term memory of this newly
acquired experience
then appears transient and labile. Under appropriate conditions, often
requiring repetition and
rest, short-term memory of an experience then can be "consolidated" into a
more durable long-
term memory.
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An every-day example of this memory process involves using a phone number.
When
one first looks up a new phone number, they recite it to themselves a few
times to learn it
(learning). One then can dial the number successfully within seconds to
minutes, if not
distracted. Interruptions, on the other hand, almost always cause one to
forget the number.
Minutes to hours after dialing the number, one most likely would not remember
the number
anymore (short-term memory). If the phone number corresponds to that of a
friend's new cell
phone, however, one might find themselves dialing it repeatedly every day or
so. With enough
repetition, one eventually can recall the phone number through long-term
memory.
The human brain is evolutionarily designed to sense its internal and external
environment, to perceive causal relations among stimuli and to change its
response adaptively.
To accomplish this incredible task, a self-regulating network of 40 thousand
genes directs the
development of a cellular network of one hundred billion neurons with 100
trillion connections
among them. Such complexity is staggering. Yet neurobiologists have begun to
understand how
the human brain works in part by studying brain function in other animals.
New experiences obviously can be quite complex, but when reduced in the
laboratory to
simple, controllable studies of "associations" between two identified stimuli
(Pavlovian learning,
for instance), the process of memory formation can be studied in many
different animals ranging
from insects to mammals. These animal studies have contributed to our current
understanding of
how memory formation proceeds, revealing aspects of neuroanatomy and
biochemistry of the
brain. Remarkably, these studies have established that much of the basic
biochemistry and
behavioral phenomenology of memory formation has been evolutionarily conserved
throughout
the animal kingdom, even though neuroanatomy differs appreciably among
species.
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These animal studies have shown that memory formation generally proceeds
through
several functionally distinct temporal phases. Acquisition (LRN) usually
requires repetition and
is thought to recruit maximal participation of the underlying neural circuitry
that normally
registers such an experience. Once learning occurs, early memory tends to
decay rapidly and is
sensitive to disruption by distraction and anesthesia (i.e., anesthesia-
sensitive memory; ASM).
Genetic dissection of ASM in fruit flies has shown that ASM can be further
decomposed into
short-term memory (STM) and intermediate-term memory (ITM).
By contrast, long-lasting memory becomes resistant to disruption (i.e.,
anesthesia-
resistant memory; ARM) and persists for long periods. Importantly, these
animal studies
generally have revealed that protein synthesis is required for the appearance
of long-term
memory (LTM). Once again, fruit fly studies have revealed that (i) LTM, but
not ARM, requires
protein synthesis and (ii) ARM and LTM can be genetically dissected into
functionally
independent phases of memory.
Animal studies also have revealed that the basic wiring diagram of the brain
emerges
during development. Most connections (synapses) among neurons, however, are
crude and non-
functional. Subsequent experience is required to refine these synaptic
connections, thereby fine
tuning the functions of various circuits. A ctivity-dependent synaptic
reorganization continues
within the adult brain, as well. Learning a new experience initiates a neural
activity, which
ultimately produces synaptic reorganization among neurons in the relevant
circuit. These
synaptic changes (synaptic plasticity), distributed throughout the neural
circuit, are considered to
be the physical basis of long-term memory.
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Experience-dependent changes in neural activity initiate within neurons a
complex
biochemical response. The ultimate effects of this biochemistry are to produce
short-term
(transient) and long-term (long-lasting) synaptic plasticity. Basic research
continues to identify
more and more of the signaling pathways and other cell biology that underlie
this synaptic
plasticity. The first of these to be discovered was the cAMP pathway, a
cascade of enzymatic
reactions that transduces extracellular neurotransmitter release into
intracellular changes in the
functional states of many proteins. Neurotransmitters, their corresponding
receptors, and
cytoplasmic PKA appear to regulate short-term plasticity, while activation of
the transcription
factor CREB (cAMP response element binding protein) is required to produce
long-term
plasticity.
In all species studied so far, the CREB gene is expressed in two opposing
forms -- an
activator or a repressor of downstream, CRE-mediated gene transcription. Key
behavioral
experiments in Drosophila originally established a critical role for CREB in
the formation of
long-term memory. Normal fruit flies are capable of learning the association
between a
particular smell and the punishment of foot shock. After one training session,
a majority of flies
will avoid the previously shock-paired odor when given a choice between it and
a neutral odor in
a T-maze. Though such learning initially is quite robust, memory thereafter is
transient,
decaying away within a day. In contrast, a bona fide protein synthesis-
dependent LTM lasting
more than a week can be produced in normal flies when subjected to ten
training sessions with a
15-min rest interval between them. In flies genetically engineered to produce
high levels of
CREB repressor, LTM specifically is blocked with no effects on learning or
early memory. In
flies genetically engineered to produce high levels of CREB activator, LTM
after spaced training
is not affected but, surprisingly, is induced after just one training session.
In short,
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overexpression of CREB activator enhances memory by reducing the number of
training sessions
required to induce the formation of LTM -- the functional equivalent of a
photographic memory.
Molecular studies in several animal models have begun to elucidate further the
biochemistry of
plasticity and to link various aspects of synaptic plasticity with memory
formation, leading to the
following general view of brain plasticity: a new experience is registered in
a specific neural
circuit as a change in neural activity. This neural activity induces a complex
inter- and intra-
cellular biochemical response that still is being discovered. To date,
however, a few key aspects
of the process appear evolutionarily conserved. Stimulation of the NMDA
receptor appears
central to induction of both the local, transient biochemical response at the
synapse and the long-
lasting biochemical response in the cell nucleus (phosphorylation of CREB).
Several
intracellular signaling pathways appear to be induced initially by neural
activity, but CREB
nonetheless appears to be a dominant "downstream" target, regulating the
conversion of early
memory to lasting memory. Down-regulation of CREB suppresses, while up-
regulation of
CREB enhances, a gene transcriptional response underlying a synaptic grow
process that yields
lasting functional and structural plasticity at synapses. In essence, CREB
appears to act as a
master switch that determines when neural activity will give rise to lasting,
structural changes in
a circuit. A key factor influencing this switch is repetition; when the CREB
pathway is up-
regulated, the number of training sessions required for the formation of long-
term memory is
decreased.
Four basic objectives guided the approach to screen for drugs that modulated
the "CREB
pathway." First, for efficiency, the screen had to be high throughput,
amenable to robotic
manipulations in a 96-well plate format. Second, because any identified drug
was ultimately to
be used in humans, the preferable target was an endogenous (neuronal) form of
human CREB.
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Third, when designing the screen, all the relevant upstream signaling pathways
that might
impinge on CREB function during memory formation were not yet known.
Therefore, a broad-
based screen, monitoring CREB-dependent gene expression as a functional
readout of the CREB
pathway seemed more inclusive of any missing, yet potentially important,
biochemical signaling.
Finally, the screen needed to identify drugs that had no effect on CREB
function alone but rather
synergized with co-activation of cAMP signaling. This requirement was designed
to mimic the
original behavioral experiments. In flies, overexpression of CREB alone had no
effect on fly
behavior. Rather, training per se was required to produce enhanced memory,
because
experience-induced increases in neural activity activated the cAMP pathway.
These considerations were incorporated into a high throughput screen (HTS) of
cultured
human neuroblastoma cells, which were stably transfected with a CRE-luciferase
reporter gene.
In this manner, changes in CREB-dependent transcription of luciferase could be
monitored via
fluorescence. To mimic (at the biochemical level) the associative requirement
for memory
formation, drugs were sought that produced no change in luciferase activity on
their own but
synergized (> 2-fold) with a suboptimal dose of forskolin, which activates the
cAMP pathway
(as does neural activity in vivo). Using a robotic system to screen these
cells in 96-well plates,
large libraries of drug compounds have been screened efficiently, yielding
numerous "active
hits." To confirm, these active hits then were assessed at four different
concentrations with or
without forskolin. Several Confirmed Actives emerged from this screening
process.
Interestingly, an efficacious molecular target was discovered to be
phosphodiesterase-4
(PDE4). Generally and specifically, this discovery makes sense. Most
biochemical pathways
function within a well-regulated optimum, with some molecular components
accelerating
(positive regulators), and others retarding (negative regulators), signal
propagation. Given the
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opposing functions of CREB activator and repressor isoforms, this notion
applies to regulation of
the CREB switch during long-term memory formation. Like genetic mutations,
most drug
compounds actually serve as inhibitors of target function ("antagonists" for
drugs and
"hypomorphs" for mutations). Hence, discovering drug compounds that antagonize
negative
regulators of a pathway might be expected often from a screen for enhanced
function. This, of
course, is the case for a PDE inhibitor; decreasing PDE activity leads to an
increase of cAMP
normally synthesized by adenylyl cyclase (AC), and elevated cAMP leads to
activation of CREB
via PKA. Moreover, a PDE inhibitor might be expected to have minimal effect
alone, in the
absence of forskolin-induced activation of AC (or neural activity-induced
activation of AC in
vivo).
An advanced PDE-4 inhibitor, HT-0712, has been shown to be a validated
compound for
clinical development, along with several other CREB pathway enhancers in
addition to HT-0712.
To date, behavioral assessments in rodent models of memory formation and
biochemical
experiments have shown that these compounds effectively enhance memory.
It is important to develop, identify and evaluate such compounds and
therapies. Such
therapies may include particular training protocols and/or administration of
such compound or
compounds, including compounds that serve as memory agents, which may be
memory
enhancers that specifically up-regulate the CREB pathway, as well as memory
impairers.
Summary of the Invention
The present invention includes methods including the steps of assessing the
effectiveness
of a memory agent administered to a subject, wherein said assessing comprises
presenting to the
subject sets of stimuli and evaluating the subject's response to the stimuli.
The assessing may
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include a first period comprising presenting to the subject stimuli associated
into sets and
evaluating the subject's response to the stimuli to establish a criterion; and
a second period
comprising presenting to the subject stimuli associated into sets and
evaluating the subject's
response to the stimuli to establish a long term success measurement. In some
embodiments, the
second period begins at least about one day after the completion of the first
period. The
evaluating of the subject's response may include comparing the criterion to
the long term success
measurement. The sets of stimuli may include a plurality of pairs of stimuli.
In some
embodiments, the each of the plurality of pairs of stimuli can include a
positive element and/or a
negative element. In other embodiments, each of the plurality of pairs of
stimuli may include an
element that has an identifiable association with the other element of the
pair. In some
embodiments, this identifiable association may be a face - name association or
word - word
association. The subject may be an animal. In certain embodiments, the animal
may be a
primate, a mammal, a mouse or a rat. In some embodiments, the primate may be a
human, a
monkey, a lemur, a macaque or an ape. In some embodiments, the first period
may be from
about one to about ten days. In some embodiments, the second period may be
about one day. In
some embodiments, there may be a plurality of subjects, which may include a
control group and
an experimental group. In certain embodiments, the assessing may include
comparing the
control group to the experimental group. In some embodiments, the evaluating
of the subject's
response includes calculating and/or measuring one or more aspects selected
from the group
consisting of. the ratio of the long term success measurement to the
criterion, long term memory
retention, the amount of time required to achieve a particular criterion,
and/or the amount of time
between the first period and the second period while keeping a constant, near-
constant, or
improved ratio of the long term success measurement to the criterion.
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In other embodiments, the present invention may involve a method including
selecting a
memory agent as a drug candidate, wherein said selecting includes the steps
of. administering a
memory agent to a subject; and presenting to the subject sets of stimuli and
evaluating the
subject's response to the stimuli. The assessing may further include: a first
period comprising
presenting to the subject stimuli associated into sets and evaluating the
subject's response to the
stimuli to establish a criterion; and a second period comprising presenting to
the subject stimuli
associated into sets and evaluating the subject's response to the stimuli to
establish a long term
success measurement. In some embodiments, the second period begins at least
about one day
after the completion of the first period. In certain embodiments, the
evaluating of the subject's
response comprises calculating and/or measuring one or more aspects selected
from the group
consisting of. the ratio of the long term success measurement to the
criterion, long term memory
retention, the amount of time required to achieve a particular criterion,
and/or the amount of time
between the first period and the second period while keeping a constant, near-
constant, or
improved ratio of the long term success measurement to the criterion. In some
embodiments, the
sets of stimuli comprise a plurality of pairs of stimuli. In some embodiments,
the subject may be
an animal, including an mammal such as a primate, a mouse and a rat.
In yet other embodiments, the present invention involves a method including:
testing a
memory agent as a long term memory enhancer, wherein said testing includes the
steps of:
administering a memory agent to a subject; and presenting to the subject sets
of stimuli and
evaluating the subject's response to the stimuli. In certain embodiments, the
assessing further
includes: a first period comprising presenting to the subject stimuli
associated into sets and
evaluating the subject's response to the stimuli to establish a criterion; and
a second period
comprising presenting to the subject stimuli associated into sets and
evaluating the subject's
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response to the stimuli to establish a long term success measurement. In some
embodiments, the
second period begins at least about one day after the completion of the first
period. In some
embodiments, the evaluating of the subject's response comprises calculating
and/or measuring
one or more aspects selected from the group consisting of. the ratio of the
long term success
measurement to the criterion, long term memory retention, the amount of time
required to
achieve a particular criterion, and/or the amount of time between the first
period and the second
period while keeping a constant, near-constant, or improved ratio of the long
term success
measurement to the criterion. In certain embodiments, the sets of stimuli
comprise a plurality of
pairs of stimuli. In some embodiments, the subject may be an animal, including
an animal
selected from the group consisting of a primate, a mammal, a mouse and a rat.
In yet further embodiments, the present invention may be a method including:
assessing
the effectiveness of a memory agent, wherein said assessing comprises the
steps of. providing a
first subject and a second subject, wherein the first subject and the second
subject are of the same
species; administering a memory agent to the first subject; presenting the
first subject and the
second subject a plurality of pairs of stimuli during a first period and
evaluating the first subject
and the second subject's responses to the stimuli to establish a criterion for
each of the first
subject and the second subject; presenting the first subject and the second
subject the plurality of
pairs of stimuli during a second period and evaluating the first subject and
the second subject's
responses to the stimuli to establish a long term success measurement for each
of the first subject
and the second subject; comparing the first subject and the second subject's
criterions to their
respective long term success measurements. In some embodiments, the second
period begins at
least about one day after the completion of the first period. In some
embodiments, the second
period begins from about 1 to about 10 days following the completion of the
first period. In
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certain embodiments, the comparing of the criterions to the long term success
measurements
comprises: establishing a first ratio of the first subject's long term success
measurement to the
first subject's criterion; establishing a second ratio of the second subject's
long term success
measurement to the second subject's criterion; and comparing the first ratio
to the second ratio.
In certain embodiments, the sets of stimuli include a plurality of pairs of
stimuli. In some
embodiments, the plurality of pairs of stimuli comprises a positive element
and/or a negative
element. In other embodiments, each of the plurality of pairs of stimuli
comprises an element
that has an identifiable association with the other element. In some
embodiments, the
identifiable association is selected from the group consisting of a face -
name association and a
word - word association. In some embodiments, each of the first subject and
the second subject
is an animal, including an animal is selected from the group consisting of a
primate (including a
human, a monkey, a lemur, a macaque and an ape), a mammal, a mouse and a rat.
In certain
embodiments, the first period comprises from about one to about ten days. In
some
embodiments, the second period comprises about one day. In some embodiments,
each of the
first subject and the second subject is a plurality of subjects.
In some embodiments, the present invention includes methods similar to those
recited
above, but which focus on evaluating an training protocol rather than or in
addition to a memory
agent. Some embodiments involve a method including the steps of. assessing the
effectiveness
of an training protocol administered to a subject, wherein said assessing
comprises presenting to
the subject sets of stimuli and evaluating the subject's response to the
stimuli. Other
embodiments involve a method including: testing a training protocol as a long
term memory
enhancer, wherein said testing includes the steps of: administering an
training protocol to a
subject; and presenting to the subject sets of stimuli and evaluating the
subject's response to the
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stimuli. Further embodiments involve a method including: assessing the
effectiveness of an
training protocol, wherein said assessing includes the steps of. providing a
first subject and a
second subject, wherein the first subject and the second subject are of the
same species;
administering an training protocol to the first subject; presenting the first
subject and the second
subject a plurality of pairs of stimuli during a first period and evaluating
their responses to the
stimuli to establish a criterion for each of the first subject and the second
subject; presenting the
first subject and the second subject the plurality of pairs of stimuli during
a second period and
evaluating their responses to the stimuli to establish a long term success
measurement for each of
the first subject and the second subject; comparing the criterions of the
first subject and the
second subject to their respective long term success measurements.
The present invention also includes systems, including systems for use along
with the
methods disclosed herein. For example, in some embodiments, the system may
include all
elements necessary to perform an embodiment of the methods of the present
invention. Systems
of the present invention may be useful for selecting a memory agent as a drug
candidate,
assessing the effectiveness of a memory agent, assessing the effectiveness of
a training protocol,
and/or testing a memory agent as a long term memory enhancer, and/or testing a
memory agent
along with an training protocol as a long term memory enhancer.
In some embodiments, a system of the present invention includes sets of
stimuli, wherein
said sets of stimuli are configured to permit evaluation of a subject's
response to the sets of
stimuli and thereby assess the effectiveness of an training protocol
administered to the subject.
In some embodiments, the sets of stimuli comprise a plurality of pairs of
stimuli. In certain
embodiments, each of the plurality of pairs of stimuli comprises a positive
element and/or a
negative element. In some embodiments, each of the plurality of pairs of
stimuli comprises an
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element that has an identifiable association with the other element of the
pair. In further
embodiments, the identifiable association is selected from the group
consisting of a face - name
association and a word - word association. In other embodiments, the system
may further
include testing protocols, testing schedules, and/or memory agent dosage and
administration
schedules.
In other embodiments a system of the present invention includes sets of
stimuli, wherein
said sets of stimuli are configured to permit evaluation of a subject's
response to the sets of
stimuli and thereby assess the effectiveness of a memory agent administered to
the subject. In
some embodiments, the sets of stimuli comprise a plurality of pairs of
stimuli. In various
embodiments, each of the plurality of pairs of stimuli comprises a positive
element and/or a
negative element. In other embodiments, each of the plurality of pairs of
stimuli comprises an
element that has an identifiable association with the other element of the
pair. In certain
embodiments, the identifiable association is selected from the group
consisting of a face - name
association and a word - word association. In other embodiments, the system
may further
include testing protocols, testing schedules, and/or memory agent dosage and
administration
schedules.
In some embodiments, the methods and systems of the present invention utilize
a
memory agent that is one or more selected from the group consisting of a
compound having the
structure:
+'>
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wherein each of Y1, Y2, Y3, and Y4 is independently --H; straight chained or
branched Ci-
C7 alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-
C7 alkenyl or
alkynyl; C3-C7 cycloalkyl or C5-C7 cycloalkenyl; --F, --Cl, --Br, or --I; --
NO2; --N3; --CN; --OR4,
--SR4, --OCOR4, --COR4, --NCOR4, --N(R4)2, --CON(R4)2, or --COOR4; aryl or
heteroaryl; or
any two of Y1, Y2, Y3 and Y4 present on adjacent carbon atoms can constitute a
methylenedioxy
group; wherein each R4 is independently --H; straight chained or branched CI-
C7 alkyl,
monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-C7 alkenyl
or alkynyl; C3-
C7 cycloalkyl, C5-C7 cycloalkenyl, aryl or aryl(Ci-C6)alkyl; wherein A is A',
straight chained or
branched CI-C7 alkyl, aryl, heteroaryl, aryl(Ci-C6)alkyl or heteroaryl(Ci-
C6)alkyl; wherein A' is
1
''
wherein Ri and R2 are each independently H, straight chained or branched CI-C7
alkyl, -
F, --Cl, --Br, --I, --NO2, or --CN; wherein R3 is H, straight chained or
branched CI-C7 alkyl, --F,
--Cl, --Br, --I, --NO2, --CN, --OR6, aryl or heteroaryl; wherein R5 is
straight chained or branched
CI-C7 alkyl, --N(R4)2, --OR4 or aryl; wherein R6 is straight chained or
branched CI-C7 alkyl or
aryl; wherein B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,
triazinyl, indolizinyl, indol-
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4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl,
benzo[b]furan-5-yl,
benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl,
benzo[b]thiophen-5-yl,
benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl,
benzo[b]thiazolyl,
purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl,
2,1,3-benzothiazolyl,
furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,
isoxazolyl, isothiazolyl,
oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl,
cinnolinyl, quinoxalinyl, 1,8-
napthyridinyl, pteridinyl, or phthalimidyl; provided however, that the carbon
atom or carbon
atoms ortho to the nitrogen atom of the imine bond may only be substituted
with one or more of
the following --F, -Cl, --Br, --I, --CN, methyl, ethyl or methoxy; wherein n
is an integer from 1
to 4 inclusive; wherein aryl is phenyl or napthyl, including phenyl and
napthyl substituted with
one or more of the following: -F, -Cl, -Br, -I, -NO2, -CN, straight chained or
branched CI-C7
alkyl, straight chained or branched CI-C7 monofluoroalkyl, straight chained or
branched CI-C7
polyfluoroalkyl, straight chained or branched C2-C7 alkenyl, straight chained
or branched C2-C7
alkynyl, C3-C7 cycloalkyl, CI-C7 monofluorocycloalkyl, C3-C7
polyfluorocycloalkyl, C5-C7
cycloalkenyl, -OR4, SR4, -OCOR4, -COR4, -NCOR4, -C02R4, -CON(R4)2 or (CH2)õ-O-
(CH2)mCH3; or a pharmaceutically acceptable salt thereof
In some embodiments, the methods and systems of the present invention utilize
a
memory agent that is one or more selected from the group consisting of a
compound having the
structure:
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CF3
CF3 C
OH
or
O
NH
CH3
OcPent
OMe
wherein Me is methyl, cPent is cyclopentyl and the 3 and 5 carbons are in the
S
configuration.
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BRIEF DESCRIPTION OF THE FIGURES
FIGS. IA - 1B illustrate graphs showing results from an embodiment of the
present
invention described in Example 1. Results are expressed as mean days s.e.m
per treatment,
p < 0.05 versus vehicle.
FIGS. 2A - 2B illustrate graphs showing results from an embodiment of the
present
invention described in Example 1. Results are expressed as mean percent
performance s.e.m.
per treatment.
FIGS. 3A - 3B illustrate graphs showing results from an embodiment of the
present
invention described in Example 1. Results are expressed as mean percent
performance s.e.m.
per treatment.
FIGS. 4A - 4D illustrate exemplary pairs of visual stimuli that may be used in
an
embodiment of the present invention described in Example 1.
FIG. 5 illustrates a graph showing performance of healthy elderly volunteers
in a paired-
associate (face-name) memory assay embodiment described in Example 2. Subjects
were asked
to memorize a set of 40 pairs of faces and names (professions) for 6 days (d2 -
d7). On training
days, subjects went through the list of 40 pairs only once. They were queried
for their recall first
and then given the correct answers to continue training. After training, three-
day memory
retention was quantified using only face-name pairs 1 - 20 (d10). Seven-day
memory retention
was quantified again for these face-name pairs 1 - 20 (d14b). Seven-day
retention also was
quantified for face-name pairs 21 - 40 twice (dl4a and d14a2), with a 1-min
interval between
recall sessions.
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Detailed Description
In the following paragraphs, the present invention will be described by way of
example
with reference to the attached figures. Throughout this description, the
preferred embodiments
and examples shown should be considered as exemplars, rather than as
limitations on the present
invention. As used herein, the "present invention" refers to any one of the
embodiments of the
invention described herein, and any equivalents. Furthermore, reference to
various feature(s) of
the "present invention" throughout this document does not mean that all
claimed embodiments or
methods must include the referenced feature(s).
The terminology used herein is for the purpose of describing particular
embodiments
only, and is not intended to limit the scope of the present invention. As used
herein and in the
appended claims, the singular forms "a," "an," and "the" include the plural
reference unless the
context clearly dictates otherwise. Thus, for example, a reference to "a
compound" is a reference
to one or more compounds and includes equivalents thereof known to those of
ordinary skill in
the art and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Specific methods, devices, and materials are described, although any
methods and
materials similar or equivalent to those described herein can be used in the
practice or testing of
the present invention.
The present invention provides methods and systems for identifying,
evaluating, and
testing various compounds as memory agents and regimens as training protocols.
Such methods
and systems may test any compounds or protocols, including the memory agents
and training
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protocols described herein. The methods can comprise combining training
protocols with the
general administration of memory agents. In some embodiments, the present
invention may
involve identifying, selecting, testing, evaluating or assessing a compound as
a drug candidate
for a memory agent, for example, as one effective for enhancing memory.
A drug candidate may be a compound that has been identified through a drug
discovery
process and has been synthesized, characterized, optimized, and/or screened or
assayed. Once a
compound has been shown to have potential for a given activity or effect, for
example by use of
the methods or systems of the present invention, it may be a drug candidate -
i.e., one that may
be put through clinical trials with the intent that it be ultimately
commercialized.
METHODS OF IDENTIFYING, EVALUATING AND TESTING MEMORY
AGENTS AND TRAINING PROTOCOLS
In some embodiments, the present invention involves the use of certain tasks
to assess
one or more aspects of an animal's memory capabilities. For example, the
animal's learning
capabilities, short term memory, or long term memory retention may be tested
and/or evaluated.
As used herein, the term "memory," without an additional modifier (e.g., short
term memory),
refers to long term memory. As used herein, the term "memory agent" refers to
memory
augmenting agents or memory suppressing agents. Memory augmenting and
suppressing agents
include, but are not limited to, those molecules, compounds, and/or agents
that act on a CREB
pathway, as described, for example, in U.S. Patent Nos. 5,929,223, 6,051,559,
6,689,557, and
6,890,516, and in U.S. Patent Application Serial No. 11/066,125, the
disclosure of each of which
is incorporated by reference in their entirety herein.
As used herein, the term "animal" includes mammals, as well as other animals,
vertebrate, invertebrate (e.g., birds, fish, reptiles, insects (e.g.,
Drosophila species) and mollusks
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(e.g., Aplysia)). The terms "mammal" and "mammalian", as used herein, refer to
any vertebrate
animal, including monotremes, marsupials and placental, that suckle their
young and either give
birth to living young (eutharian or placental mammals) or are egg-laying
(metatharian or
nonplacental mammals). Examples of mammalian species include, without
limitation, humans
and primates (e.g., monkeys, chimpanzees, humans, apes, lemurs, or macaques),
rodents (e.g.,
rats, mice, guinea pigs) and ruminents (e.g., cows, pigs, horses).
The animal can be an animal with some form and degree of memory dysfunction or
an
animal with normal memory performance (i.e., an animal without any form of
memory failure
(dysfunction or loss of any memory)). One or more animals may be used and such
animals may
be placed in one or more groups (e.g., one or more control groups and/or one
or more
experimental groups). The animal may be any suitable age.
The tasks may involve the presentation of certain stimuli to the subjects. The
stimuli may
be any suitable sensory stimulus including, without limitation, auditory,
visual, tactile, olfactory,
gustatory and/or combinations thereof. In some embodiments, the stimuli may be
visual and be
one or more of a symbol, letter, number, line, shape, color, picture or
combinations thereof. The
stimuli may be associated into sets, such that a certain relationship is
established between the
members of the sets. The stimuli may be associated into groups of about 2, 3,
4, 5, 6, 7, 8, 9 or
10. In some such embodiments, the stimuli are associated into pairs. In some
embodiments,
each stimulus in the pair is of the same type (i.e., audio, visual or tactile)
while in other
embodiments the pair may be of different types. In some embodiments, an
element in the pair
will have some identifiable association with the other element of the pair. An
identifiable
association can be a visual, audible, logical or other aspect of the one or
more elements of the
pair that would cause one to associate that element of the pair with its
corresponding element. In
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other embodiments, there may be no identifiable association. For example, the
pair could be a
word-word pair wherein one word does not have an identifiable association with
the other word
(e.g., comb-apple). An example of visual paired stimuli is depicted in FIGs.
4A-4D. Another
example of a stimulus pair with an identifiable association could be a face -
name pairing and/or
an occupation - face/name pairing. In some embodiments, there may also be an
occupation
listed along with the face - name pairing.
In some embodiments, the subjects may be presented one or more pairs of
stimuli. In
some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24,
25, 50, 100, 120, 150, or any other suitable number of pairs of stimuli may be
presented to the
subject. In some embodiments, one element of the pair is designated to be
positive - meaning
that the subject will receive a reward if it selects that element. In other
embodiments, one
element of the pair is designated to be negative - meaning that the subject
will receive negative
feedback if it selects that element. In some embodiments, the subject selects
the element by
touching the element, but in other embodiments any other suitable selection
method may be
used. In some embodiments, the reward may be food, but in other embodiments
the reward may
be a pleasant sound, access to certain toys or items, or anything suitable for
triggering a positive
response from the subject. In some embodiments, the negative feedback is a
shock, a sound, a
punishment or withholding of privileges, or any suitable action or the like
that provides the
subject with negative feedback.
The type of feedback, the selection of the pairs of stimuli, sets of pairs of
stimuli, species
and age of the subjects, and other aspects of the tasks may be modified to
test and/or evaluate
different aspects of a subject's memory. For example, using stimulus pairs
wherein each element
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has an identifiable association to the other element may be used to evaluate
the subject's
relational memory.
In other embodiments, other evaluation methods other than the reward and/or
negative
feedback evaluation methods may be used. For example, in some embodiments,
such as those
where the subject is human, the stimuli may have a relation such that the
subject may determine
if the pairing or other association is correct or incorrect (e.g., are the
stimuli paired such that the
intended identifiable association between one element of a pair and the other
element in the pair
is present). For example, if a face - name pairing has been taught to the
subject, the subject may
be presented pairs of stimuli that have been scrambled such that the correct
face - name pairing
has been disrupted. The subject could then communicate whether the face - name
pairing
presented to it is the correct face - name pairing it has been taught. In such
embodiments, no
discrete reward would be necessary, or the reward could simply be feedback on
the correctness
of the human's responses. The pairs of stimuli may be presented in any
suitable fashion. For
example, the pairs may be presented in random, organized, or a pseudo-random
order during
each testing session.
The subjects may be tested over any suitable period at any suitable frequency.
For
example, the subjects may be tested 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, 365, or any other
suitable number of times
per testing event (e.g., if testing is daily, the subject may be tested once,
twice, three times, etc).
In such testing, subjects may be shown any suitable number of stimuli or pairs
of stimuli.
Subjects may have tests daily, every other day, weekly, bi-weekly, monthly, or
any other suitable
frequency. Moreover any suitable number of subjects may be tested. In some
embodiments,
testing is done in an effort to teach or encode the subject with the
information as to the
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relationship between the paired stimuli. For example, the testing may be done
to teach the
subject which of the elements of the pair is the positive and/or negative
element. In other
embodiments, the testing may be done to teach the subject the correct pairing
of elements within
a set of stimuli (e.g., the correct face - name, occupation-name, or
occupation-face/name
association). In such embodiments, a plurality of phases of testing would
likely be required. A
first teaching phase, followed by a second phase wherein the subject's
knowledge is tested by
asking, through an appropriate method depending on the subject type (e.g.,
human vs. rat), if the
presented pairings are correct.
The subjects may be evaluated in any suitable manner. In some embodiments, the
subjects are presented with one or more pairs of stimuli in which one element
of the pair is
designated positive and/or one element of the pair is designated negative. In
such an
embodiment, the subject, having learned which element of the pair is the
positive and/or negative
element, is evaluated on the number of times it selects the positive and/or
negative element
and/or does not select the positive and/or negative element. In other
embodiments, the pairs of
stimuli may have a relation to one another (e.g., a face - name relation,
occupation-name
relation, occupation-face/name relation, a pair of words, a set of letters or
numbers, parts of a
picture or symbol). In such embodiments, the subject may be evaluated on its
ability, having
learned the correct relation between the elements of the pair, to re-establish
the appropriate pairs
(e.g., placing the name element with the corresponding picture element, or the
like). In some
embodiments, the pairs may be altered in some manner and the subject could be
asked to identify
the manner in which the pair was altered (for example, the response could be
"intact,"
"rearranged," "one new," or "both new").
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In some embodiments, a subject's long-term memory may be tested. In such
embodiments, a criterion result can be established. Such a criterion may be
established in a
variety of manners. For example, in embodiments where a pair of stimuli is
presented to the
subject wherein one element of the pair is positive and/or one element of the
pair is negative, the
subject may be presented with the pair (or sets of pairs) until the subject
identifies the positive
and/or negative element with a certain frequency. That frequency would then
serve as a criterion
against which later results may be evaluated. Similarly, the frequency of the
correct
establishment of the relation between elements of a pair of stimuli may also
be used to establish
a criterion. In some embodiments, a subject is evaluated until it reaches a
certain success rate.
For example, a criterion of about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
99%,
100%, or any other suitable percentage or number of correct results could be
used as a target. In
other embodiments, a subject may be tested a certain number of times or over a
certain period of
time, and the last evaluation may be used to establish the criterion. For
example, a period of
about 1-23 hours or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 50, 60, 75, 100, 150, 200, 250, or 365 days, or any other suitable
number of hours or
days may be used to establish the criterion. Also, the subject may be tested
about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50,
60, 75, 100, 150, 200,
250, 365, or any other suitable number of times to establish a criterion. The
criterion may also
be established using a mean, median, or other statistical measure of the
subject's actual results.
In some embodiments, once the criterion is established, the subject is not
evaluated or
shown any of the pairs of stimuli used to establish the criterion for a
certain amount of time.
This amount of time maybe about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 50, 60, 75, 100, 150, 200, 250, 365, or any other
suitable number of days.
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After the delay period, the subject is once again tested using the same pairs
of stimuli used to
establish the criterion. In some embodiments, these tests are repeated under
circumstances
identical, similar, or different to those used in establishing the criterion.
Moreover, the subject's
performance may be evaluated by establishing a long term success measurement
in any suitable
manner. In some embodiments, the manner of establishing the long term success
measurement
may be similar to that described with respect to establishing a criterion,
albeit using a generally
shorter time period such that the long term success measurement is indicative
of the subject's
memory, rather than it having relearned the relevant criteria. The subject
could then be
evaluated by comparing the long term success measurement with the criterion.
Statistical or
similar analysis may be used to evaluate such results.
In some embodiments, the methods of the present invention may be used to
evaluate the
efficacy of a potential memory agent and/or a potential training protocol. For
example, the
methods of the present invention may be used to identify a reduction in the
amount of training
required to reach a chosen performance level. In such embodiments, a subject
may first be
evaluated in the absence of a memory agent and subsequently evaluated having
been
administered a memory agent. Also, a control group of subjects could be tested
using certain
pairs of stimuli and those results compared to a group of subjects having been
administered a
given dose of a memory agent. The evaluation of the memory agent and/or
training protocol
may take a variety of forms. For example, a memory agent may reduce the time
and/or number
of days of training sessions required to establish or meet a certain
criterion, or any other suitable
measure of efficacy.
In some embodiments of the present invention, the memory agent is evaluated to
see if it
alters (e.g., reduces or increases) the number of days or training sessions to
reach a criterion of
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performance. As used herein, the term "criterion" refers to a metric of a
desired result and/or a
desired level of performance such as, for example, a percentage of correct
responses (e.g.,
correctly paired stimuli) from a test subject or group of test subjects. The
criterion may be
changed or chosen as desired by the administrator of the protocol, tests,
tasks, and/or other
embodiments of the present invention. The complexity of the training
administered in these
embodiments may be manipulated by, for example, changing the criterion, the
numbers and/or
types stimuli to be paired, the number of days of training, the quantity of
training, and/or the type
of training. In further embodiments, the training, with or without the memory
agent, is sufficient
to produce a CREB-dependent change in long term memory, for example a CREB-
dependent
enhancement of long term memory.
In some embodiments, the methods of the present invention may be used to
evaluate the
efficacy of a memory impairer. Such embodiments may involve steps similar to
or identical to
the methods for evaluating a memory agent. However, in such an embodiment, the
evaluated
indicia would be different. For example, the memory impairer may increase the
time required to
establish a certain criterion, increase the number of training sessions
required to reach a criterion,
decrease the ratio of the long term success measurement to the criterion, or
any may be evaluated
in any other suitable manner.
Any suitable memory agent or memory impairer may be provided by any suitable
method, via any suitable delivery system and using any suitable delivery
schedule and dose. In
addition, any suitable training protocol may be used. Exemplary training
protocols, memory
agents and memory impairers are specifically discussed in the following
sections.
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The present invention also includes systems, including systems for use along
with the
methods disclosed herein. For example, in some embodiments, the system may
include all
elements necessary to perform an embodiment of the methods of the present
invention. Systems
of the present invention may be useful for selecting a memory agent as a drug
candidate,
assessing the effectiveness of a memory agent, assessing the effectiveness of
a training protocol,
testing a memory agent as a long term memory enhancer, testing a memory agent
as a impairer
of specific or general memories, and/or testing a memory agent along with a
training protocol as
a long term memory enhancer. Such systems may involve use of sets of stimuli
as described
above, including paired stimuli, and testing schedules and protocols as
described above.
TRAINING PROTOCOLS
As described herein, assessment using the methods and systems of the present
invention
can comprise two parts: (1) a specific training protocol and (2)
administration of a memory
agent. In some embodiments, only the training protocol is used. In other
embodiments, both
training and administration of a memory agent occur. This combination can
augment training
protocols by reducing the number of days of training sessions required to
yield a performance
gain relative to that obtained with training alone or by requiring shorter or
no rest intervals
between training sessions to yield a performance gain. This combination can
also augment
training protocols by reducing the duration and/or number of training sessions
required for the
induction in a specific neuronal circuit(s) of a pattern of neuronal activity
or by reducing the
duration and/or number of training sessions or underlying pattern of neuronal
activity required to
induce cyclic AMP response element binding protein (CREB) - dependent long-
term
structural/function (i.e., long-lasting) change among synaptic connections of
the neuronal circuit.
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In this manner, one can improve the efficiency of existing training protocols,
thereby yielding
significant functional and economic benefits.
Training protocols (e.g., massed training, spaced training) are employed in
learning a new
language or in learning to play a new musical instrument. Administration of a
memory agent in
conjunction with training reduces the time and/or number of training sessions
required to yield a
gain in performance. As a result, less practice (training sessions) is
required to learn the new
language or to learn to play the new musical instrument.
Training protocols are employed for repeated stimulation of neuronal activity
or a pattern
of neuronal activity underlying (a) specific neuronal circuit(s) in
individuals. Administration of a
memory agent in conjunction with training reduces the time and/or number of
training sessions
and/or underlying pattern of neuronal activity required to achieve a given
result, such as
induction of CREB-dependent long-term structure/function (i.e., long-lasting)
change among
synaptic connections of the neuronal circuit.
These methods can enhance specific aspects of memory in an animal
(particularly a
human or other mammal or vertebrate) in need thereof by (a) administering to
the animal a
memory agent, for example one that enhances CREB pathway function; and (b)
training the
animal under conditions sufficient to produce an improvement in performance of
a memory task
of interest by the animal. In some embodiments, the memory agent is a CREB
pathway-
enhancing drug.
The described methods can be used to treat a memory deficit associated with a
central
nervous system (CNS) disorder or condition in an animal in need of said
treatment by (a)
administering to the animal a memory agent, for example one that enhances CREB
pathway
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function; and (b) training the animal under conditions sufficient to produce
an improvement in
performance of a particular memory task by the animal. CNS disorders and
conditions include
age-associated memory impairment, neurodegenerative diseases (e.g.,
Alzheimer's disease,
Parkinson's disease, Huntington's disease (chorea), other senile dementia),
psychiatric diseases
(e.g., depression, schizophrenia, autism, attention deficit disorder), trauma
dependent loss of
function (e.g., cerebrovascular diseases (e.g., stroke, ischemia), brain
tumor, head or brain
injury), genetic defects (e.g., Rubinstein-Taybi syndrome, down syndrome),
learning disabilities,
and/or combinations thereof.
The described methods can also be used for repeated stimulation of neuronal
activity or a
pattern of neuronal activity, such as that underlying a specific neuronal
circuit(s), in an animal
comprising (a) administering to the animal a memory agent, for example one
that enhances
CREB pathway function; and (b) training the animal under conditions sufficient
to stimulate or
induce neuronal activity or a pattern of neuronal activity in the animal.
For many tasks in many species, including human, spaced training protocols
(multiple
training sessions with a rest interval between each) produce stronger, longer-
lasting memory than
massed training protocols (multiple training sessions with no rest interval in
between).
Behavior-genetic studies of Pavlovian olfactory learning in Drosophila have
established that
massed training produces a long-lasting memory that nevertheless decays away
in at least four
days, is not protein synthesis-dependent, is not disrupted by over-expression
of a CREB-
repressor transgene, and is disrupted in radish mutants (Tully, T. et al.,
Cell, 79(1):35-47 (1994);
and Yin, J. C. et al., Cell, 79(1):49-58 (1994)). In contrast, spaced training
produces a long-
lasting memory that persists for at least seven days, is protein synthesis-
dependent, is disrupted
by over-expression of a CREB-repressor transgene and is normal in radish
mutants (Tully, T. et
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al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58
(1994)). One day after
spaced training, memory retention is composed of both the protein synthesis-
and CREB-
independent early memory (ARM) and the protein synthesis- and CREB-dependent
long-term
memory (LTM). Additional massed training is insufficient to induce LTM (Tully,
T. et al., Cell,
79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).
A growing body of evidence extends these results from invertebrates to
mammals. For
example, in Aplysia, molecular manipulations of CREB expression, similar to
those in flies,
suppress or enhance (i) LTM of a facilitatory electrophysiological response at
a sensorimotor
monosynapse in cell culture and (ii) the synaptic connections between sensory
and motor
neurons that are normally produced after spaced applications of the
facilitatory stimulus
(Bartsch, D. et al, Cell, 83(6):979-992 (1995)). In rats, injections of
antisense RNA
oligonucleotides into hippocampus or amygdala block LTM formation of two
different tasks that
are dependent on activity in these anatomical regions, respectively (Guzowski,
J. F. et al., Proc.
Natl. Acad. Sci. USA, 94(6):2693-2698 (1997); and Lamprecht, R. et al., J.
Neurosci.,
17(21):8443-8450 (1997)). In mice, LTM formation for both implicit and
explicit tasks is
defective in CREB mutant mice (Bourtchuladze, R. et al., Cell, 79(1):59-68
(1994)).
Training of transgenic mice, carrying a CRE-dependent reporter gene
(betagalactosidase),
in hippocampal-dependent contextual fear conditioning or passive avoidance
tasks induces CRE-
dependent reporter gene expression in areas CAI and CA3 of the hippocampus.
Training of these
mice in an amygdala-dependent fear conditioning task induces CRE-dependent
reporter gene
expression in the amygdala, but not the hippocampus. Thus, training protocols
that induce LTM
formation also induce CRE-dependent gene transcription in specific anatomical
areas of the
mammalian brain (Impey, S. et al., Nat. Neurosci., 1(7):595-601 (1998)).
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With these animal models, three salient cases of LTM enhancement have been
demonstrated. First, over-expression of a CREB-activator transgene abrogates
the requirements
for multiple, spaced training sessions and, instead, induces LTM formation
after only one
training session (which normally produces little or no memory retention 24
hours later (Yin, J. C.
et al, Cell, 81(1):107-115 (1995)). Second, injection of a virally expressed
CREB-activator
transgene into rat amygdala also is sufficient to enhance memory after massed
training for the
fear-potentiated startle response, which abrogates the requirement for a rest
interval in spaced
training (Josselyn, S. A. et al., Society for Neuroscience, Vol. 24, Abstract
365.10 (1998)).
Third, LTM formation in CREB-deficient mice (Bourtchuladze, R. et al., Cell,
79(1):59-68
(1994)) can form normally, if mutant mice are subjected to a different, spaced
training protocol
(Kogan, J. H. et al., Curr. Biol., 7(1):1-l l (1997)).
CREB also appears involved in various forms of developmental and cellular
plasticity in
the vertebrate brain. For example, neuronal activity increases CREB activity
in the cortex
(Moore, A. N. et al., J. Biol. Chem., 271(24):14214-14220 (1996)). CREB also
mediates
developmental plasticity in the hippocampus (Murphy, D. D. et al., Proc. Natl.
Acad. Sci. USA,
94(4):1482-1487 (1997)), in the somatosensory cortex (Glazewski, S. et al.,
Cereb. Cortex,
9(3):249-256 (1999)), in the striatum (Liu, F. C. et al., Neuron, 17(6):1133-
1144 (1996)), and in
the visual cortex (Pham, T. A. et al., Neuron, 22(1):63-72 (1999)).
CREB appears to be affected in human neurodegenerative disease and brain
injury. For
example, CREB activation and/or expression is disrupted in Alzheimer's disease
(Ikezu, T. et al.,
EMBO J., 15(10):2468-2475 (1996); Sato, N. et al., Biochem. Biophys. Res.
Commun.,
232(3):637-642 (1997); and Yamamoto-Sasaki, M. et al., Brain. Res., 824(2):300-
303 (1999).
CREB activation and/or expression is also elevated after seizures or ischemia
(Blendy, J. A. et
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al., Brain Res., 681(1-2):8-14 (1995); and Tanaka, K. et al., Neuroreport,
10(11):2245-2250
(1999)). "Environmental enrichment" is neuroprotective, preventing cell death
by acting through
CREB (Young, D. et al., Nat. Med., 5(4):448-453 (1999)).
CREB functions during drug sensitivity and withdrawal. For example, CREB is
affected
by ethanol (Pandey, S. C. et al., Alcohol Clin. Exp. Res., 23(9):1425-1434
(1999);
Constantinescu, A. et al., J. Biol. Chem., 274(38):26985-26991 (1999); Yang,
X. et al., Alcohol
Clin. Exp. Res., 22(2):382-390 (1998); Yang, X. et al., J. Neurochem.,
70(1):224-232 (1998);
and Moore, M. S. et al., Cell, 93(6):997-1007 (1998)), by cocaine (Carlezon,
W. A., Jr. et al.,
Science, 282(5397):2272-2275 (1998)), by morphine (Widnell, K. L. et al., J.
Pharmacol. Exp.
Ther., 276(1):306-315 (1996)), by methamphetamine (Muratake, T. et al., Ann
N.Y. Acad. Sci.,
844:21-26 (1998)) and by cannabinoid (Calandra, B. et al., Eur. J. Pharmacol.,
374(3):445-455
(1999); and Herring, A. C. et al., Biochem. Pharmacol., 55(7):1013-1023
(1998)).
A signal transduction pathway that can stimulate the CREB/CRE transcriptional
pathway
is the cAMP regulatory system. Consistent with this, mice lacking both
adenylate cyclase 1
(AC1) and AC8 enzymes fail to learn (Wong S. T. et al., Neuron, 23(4):787-798
(1999)). In
these mice, administration of forskolin to area CA1 of the hippocampus
restores learning and
memory of hippocampal-dependent tasks. Furthermore, treatment of aged rats
with drugs that
elevate cAMP levels (such as rolipram and Dl receptor agonists) ameliorates an
age-dependent
loss of hippocampal-dependent memory and cellular long-term potentiation
(Barad, M. et al.,
Proc. Natl. Acad. Sci. USA, 95(25):15020-15025 (1998)). These latter data
suggest that a cAMP
signaling is defective in learning-impaired aged rats (Bach, M. E. et al.,
Proc. Natl. Acad. Sci.
USA, 96(9):5280-5285 (1999)).
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A growing body of evidence suggests that neurons continue to proliferate in
the adult
brain (Arsenijevic, Y. et al., Exp. Neurol., 170: 48-62 (2001); Vescovi, A. L.
et al., Biomed.
Pharmacother., 55:201-205 (2001); Cameron, H. A. and McKay, R. D., J. Comp.
Neurol.,
435:406-417 (2001); and Geuna, S. et al., Anat. Rec., 265:132-141 (2001)) and
that such
proliferation is in response to various experiences (Nilsson, M. et al., J.
Neurobiol., 39:569-578
(1999); Gould, E. et al., Trends Cogn. Sci., 3:186-192 (1999); Fuchs, E. and
Gould, E., Eur. J.
Neurosci., 12: 2211-2214 (2000); Gould, E. et al., Biol. Psychiatry, 48:715-
720 (2000); and
Gould, E. et al., Nat. Neurosci., 2:260-265 (1999)). Experimental strategies
now are underway
to transplant neuronal stem into adult brain for various therapeutic
indications (Kurimoto, Y. et
al., Neurosci. Lett., 306:57-60 (2001); Singh, G., Neuropathology, 21:110-114
(2001); and
Cameron, H. A. and McKay, R. D., Nat. Neurosci., 2:894-897 (1999)). Much
already is known
about neurogenesis in embryonic stages of development (Saitoe, M. and Tully,
T., "Making
connections between synaptic and behavioral plasticity in Drosophila", In
Toward a Theory of
Neuroplasticity, J. McEachern and C. Shaw, Eds. (New York: Psychology Press.),
pp. 193-220
(2000)). Neuronal differentiation, neurite extension and initial synaptic
target recognition all
appear to occur in an activity-independent fashion. Subsequent synaptogenesis
and synaptic
growth, however, then requires ongoing neuronal activity to fine-tune synaptic
connections in a
functionally relevant manner. These findings suggest that functional (final)
integration of
transplanted neural stem cells require neuronal activity. Thus, ACT can be
used to exercise
appropriate neuronal circuits to fine-tune the synaptic connections of newly
acquired,
transplanted stem cells that differentiate into neurons. By "exercise
appropriate neuronal
circuit(s)" is meant the induction in the appropriate neuronal circuit(s) of a
pattern of neuronal
activity, which corresponds to that produced by a particular cognitive
training protocol. The
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cognitive training protocol can be used to induce such neuronal activity.
Alternatively, neuronal
activity can be induced by direct electrical stimulation of the neuronal
circuitry. "Neuronal
activity" and "neural activity" are used interchangeably herein.
Memory agents, for example CREB pathway-enhancing drugs, may enhance CREB
pathway function, which is required to consolidate newly acquired information
into LTM. By
"enhance CREB pathway function" is meant the ability to enhance or improve
CREB-dependent
gene expression. CREB-dependent gene expression can be enhanced or improved by
increasing
endogenous CREB production, for example by directly or indirectly stimulating
the endogenous
gene to produce increased amounts of CREB, or by increasing functional
(biologically active)
CREB. See, e.g., U.S. Pat. No. 5,929,223; U.S. Pat. No. 6,051,559; and
International Publication
No. W09611270 (published Apr. 18, 1996), which references are incorporated
herein in their
entirety by reference. Administration of a memory agent may decrease the
training needed to
yield a performance gain relative to that yielded with training alone. By
"performance gain" is
meant an improvement in an aspect of memory performance.
The invention provides methods for evaluating, testing and/or identifying the
enhancement of a specific aspect of memory in an animal (particularly in a
human or other
mammal or vertebrate) (in some embodiments, in need thereof) wherein the
enhancement may be
due to the (a) administering to the animal a memory agent, for example one
that enhances CREB
pathway function; and (b) training the animal under conditions sufficient to
produce an
improvement in performance of a particular memory task by the animal.
Training can comprise one or multiple training sessions and is training
appropriate to
produce an improvement in performance of the memory task of interest. For
example, if an
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improvement in language acquisition is desired, training would focus on
language acquisition. If
an improvement in ability to learn to play a musical instrument is desired,
training would focus
on learning to play the musical instrument. If an improvement in a particular
motor skill is
desired, training would focus on acquisition of the particular motor skill.
The specific memory
task of interest is matched with appropriate training.
The invention also provides methods for evaluating, testing and/or identifying
the
enhancement of memory performance in an animal caused by repeated stimulation
of neuronal
activity or a pattern of neuronal activity, such as that underlying a specific
neuronal circuit(s), in
an animal comprising (a) administering to the animal a memory agent which
enhances CREB
pathway function; and (b) training the animal under conditions sufficient to
stimulate or induce
neuronal activity or a pattern of neuronal activity in the animal. In this
case, training refers to
training appropriate to stimulate or induce neuronal activity or a pattern of
neuronal activity in
the animal.
By "multiple training sessions" is meant two or more training sessions. The
memory
agent can be administered before, during or after one or more of the training
sessions. In a
particular embodiment, the memory agent is administered before and during each
training
session.
Certain training protocols that can be evaluated in accordance with the
present invention
are known and readily available in the art. See for example, Karni, A. and
Sagi, D., "Where
practice makes perfect in text discrimination: evidence for primary visual
cortex plasticity",
Proc. Natl. Acad. Sci. USA, 88:4966-4970 (1991); Karni, A. and Sagi, D., "The
time course of
learning a visual skill," Nature, 365:250-252 (1993); Kramer, A. F. et al.,
"Task coordination and
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aging: explorations of executive control processes in the task switching
paradigm," Acta
Psychol. (Amst.), 101:339-378 (1999); Kramer, A. F. et al., "Training for
executive control:
Task coordination strategies and aging," In Aging and Skilled Performance:
Advances In Theory
and Applications, W. Rogers et al., eds. (Hillsdale, N.J.: Erlbaum) (1999);
Rider, R. A. and
Abdulahad, D. T., "Effects of massed versus distributed practice on gross and
fine motor
proficiency of educable mentally handicapped adolescents," Percept. Mot.
Skills, 73:219-224
(1991); Willis, S. L. and Schaie, K. W., "Training the elderly on the ability
factors of spatial
orientation and inductive reasoning," Psychol. Aging, 1:239-247 (1986);
Willis, S. L. and
Nesselroade, C. S., "Long-term effects of fluid ability training in old-old
age," Develop.
Psychol., 26:905-910 (1990); Wek, S. R. and Husak, W. S., "Distributed and
massed practice
effects on motor performance and learning of autistic children," Percept. Mot.
Skills, 68:107-113
(1989); Verhaehen, P. et al., "Improving memory performance in the aged
through mnemonic
training: a meta-analytic study," Psychol. Aging, 7:242-251 (1992);
Verhaeghen, P. and
Salthouse, T. A., "Meta-analyses of age-cognition relations in adulthood:
estimates of linear and
nonlinear age effects and structural models," Psychol. Bull., 122:231-249
(1997); Dean, C. M. et
al., "Task-related circuit training improves performance of locomotor tasks in
chronic stroke: a
randomized, controlled pilot trial," Arch. Phys. Med. Rehabil., 81:409-417
(2000); Greener, J. et
al., "Speech and language therapy for aphasia following stroke," Cochrane
Database Syst. Rev.,
CD000425 (2000); Hummelsheim, H. and Eickhof, C., "Repetitive sensorimotor
training for arm
and hand in a patient with locked-in syndrome," Scand. J. Rehabil. Med.,
31:250-256 (1999);
Johansson, B. B., "Brain plasticity and stroke rehabilitation. The Willis
lecture," Stroke, 31:223-
230 (2000); Ko Ko, C., "Effectiveness of rehabilitation for multiple
sclerosis," Clin. Rehabil., 13
(Suppl. 1):33-41 (1999); Lange, G. et al., "Organizational strategy influence
on visual memory
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performance after stroke: cortical/subcortical and left/right hemisphere
contrasts," Arch. Phys.
Med. Rehabil., 81:89-94 (2000); Liepert, J. et al., "Treatment-induced
cortical reorganization
after stroke in humans," Stroke, 31:1210-1216 (2000); Lotery, A. J. et al.,
"Correctable visual
impairment in stroke rehabilitation patients," Age Ageing, 29:221-222 (2000);
Majid, M. J. et al.,
"Cognitive rehabilitation for memory deficits following stroke" (Cochrane
review), Cochrane
Database Syst. Rev., CDO02293 (2000); Merzenich, M. et al., "Cortical
plasticity underlying
perceptual, motor, and cognitive skill development: implications for
neurorehabilitation," Cold
Spring Harb. Symp. Quant. Biol., 61:1-8 (1996); Merzenich, M. M. et al.,
"Temporal processing
deficits of language-learning impaired children ameliorated by training,"
Science, 271:77-81
(1996); Murphy, E., "Stroke rehabilitation," J. R. Coll. Physicians Lond.,
33:466-468 (1999);
Nagarajan, S. S. et al., "Speech modifications algorithms used for training
language learning-
impaired children," IEEE Trans. Rehabil. Eng., 6:257-268. (1998); Oddone, E.
et al., "Quality
Enhancement Research Initiative in stroke: prevention, treatment, and
rehabilitation," Med. Care
38:192-1104 (2000); Rice-Oxley, M. and Turner-Stokes, L., "Effectiveness of
brain injury
rehabilitation," Clin. Rehabil., 13(Suppl 1):7-24 (1999); Tallal, P. et al.,
"Language learning
impairments: integrating basic science, technology, and remediation," Exp.
Brain Res., 123:210-
219 (1998); Tallal, P. et al., "Language comprehension in language-learning
impaired children
improved with acoustically modified speech," Science, 271:81-84 (1996), which
references are
incorporated herein in their entirety by reference.
Accordingly, the invention also relates to methods of enhancing long term
memory in an
animal (particularly in a human or other mammal or vertebrate) in need of said
treatment
comprising (a) administering to the animal a memory agent identified, tested
or evaluated by the
methods of assessing, evaluating and/or testing the effectiveness of a memory
agent, selecting a
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memory agent as a drug compound, or testing a memory agent as a long term
memory enhancer,
and (b) training the animal under conditions sufficient to enhance long term
memory in the
animal.
In one embodiment, the invention also relates to a method of treating an age-
associated
memory impairment in an animal in need of said treatment comprising (a)
administering to the
animal a memory agent identified, tested or evaluated by the methods of
assessing, evaluating
and/or testing the effectiveness of a memory agent, selecting a memory agent
as a drug
compound, or testing a memory agent as a long term memory enhancer; and (b)
training the
animal under conditions sufficient to enhance long term memory in the animal.
.
In another embodiment, the invention relates to a method of treating a memory
deficit
associated with a neurodegenerative disease (e.g., Alzheimer's disease,
Parkinson's disease,
Huntington's disease, other senile dementia) in an animal in need of said
treatment comprising
(a) administering to the animal a memory agent identified, tested or evaluated
by the methods of
assessing, evaluating and/or testing the effectiveness of a memory agent,
selecting a memory
agent as a drug compound, or testing a memory agent as a long term memory
enhancer; and (b)
training the animal under conditions sufficient to enhance long term memory in
the animal. .
In another embodiment, the invention relates to a method of treating a memory
deficit
associated with a psychiatric disease (e.g., depression, schizophrenia,
autism, attention deficit
disorder) in an animal in need of said treatment comprising (a) administering
to the animal a
memory agent identified, tested or evaluated by the methods of assessing,
evaluating and/or
testing the effectiveness of a memory agent, selecting a memory agent as a
drug compound, or
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testing a memory agent as a long term memory enhancer; and (b) training the
animal under
conditions sufficient to enhance long term memory in the animal.
In another embodiment, the invention relates to a method of treating a memory
deficit
associated with trauma dependent loss of memory function (e.g.,
cerebrovascular diseases (e.g.,
stroke, ischemia), brain tumor, head or brain injury) in an animal in need of
said treatment
comprising (a) administering to the animal a memory agent identified, tested
or evaluated by the
methods of assessing, evaluating and/or testing the effectiveness of a memory
agent, selecting a
memory agent as a drug compound, or testing a memory agent as a long term
memory enhancer;
and (b) training the animal under conditions sufficient to enhance long term
memory in the
animal.
In another embodiment, the invention relates to a method of treating a memory
deficit
associated with a genetic defect (e.g., Rubinstein-Taybi syndrome, down
syndrome) in an animal
in need of said treatment comprising (a) administering to the animal a memory
agent identified,
tested or evaluated by the methods of assessing, evaluating and/or testing the
effectiveness of a
memory agent, selecting a memory agent as a drug compound, or testing a memory
agent as a
long term memory enhancer; and (b) training the animal under conditions
sufficient to enhance
long term memory in the animal.
The invention also relates to methods of therapy of a memory deficit
associated with a
CNS disorder or condition in an animal having undergone neuronal stem cell
manipulation (a)
administering to the animal a memory agent identified, tested or evaluated by
the methods of
assessing, evaluating and/or testing the effectiveness of a memory agent,
selecting a memory
agent as a drug compound, or testing a memory agent as a long term memory
enhancer; and (b)
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training the animal under conditions sufficient to enhance long term memory in
the animal. By
"neuronal stem cell manipulation" is meant that (1) exogenous neuronal stem
cells are
transplanted into the brain or spinal chord of an animal or (2) endogenous
neuronal stem cells are
stimulated or induced to proliferate in the animal. Methods of transplanting
neuronal stem cells
into the brain or spinal chord of an animal are known and readily available in
the art (see, e.g.,
Cameron, H. A. and McKay, R. D., Nat. Neurosci., 2:894-897 (1999); Kurimoto,
Y. et al.,
Neurosci. Lett., 306:57-60 (2001); and Singh, G., Neuropathology, 21:110-114
(2001)).
Methods of stimulating or inducing proliferation of endogenous neuronal stem
cells in an animal
are known and readily available in the art (see, e.g., Gould, E. et al.,
Trends Cogn. Sci,, 3:186-
192 (1999); Gould, E. et al., Biol. Psychiatry, 48:715-20 (2000); Nilsson, M.
et al, J. Neurobiol.,
39:569-578 (1999); Fuchs, E. and Gould, E., Eur. J. Neurosci., 12:2211-2214
(2000); and Gould,
E. et al., Nat. Neurosci., 2:260-265 (1999)). The particular methods of
transplanting neuronal
stem cells into the brain or spinal chord of an animal and the particular
methods of stimulating or
inducing proliferation of endogenous neuronal stem cells in an animal are not
critical to the
practice of the invention.
The invention further relates to methods of improving or enhancing memory
and/or
performance in an animal with a learning, language or reading disability, or
combinations of any
of the foregoing, comprising (a) administering to the animal a memory agent
identified, tested or
evaluated by the methods of assessing, evaluating and/or testing the
effectiveness of a memory
agent, selecting a memory agent as a drug compound, or testing a memory agent
as a long term
memory enhancer; and (b) training the animal under conditions sufficient to
enhance long term
memory in the animal.
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MEMORY AGENTS
Memory agents, as used herein, are compounds with pharmacological activity
relative to
memory disorders and/or memory dysfunctions, and include drugs, chemical
compounds, ionic
compounds, organic compounds, organic ligands, including cofactors,
saccharides, recombinant
and synthetic peptides, proteins, peptoids, nucleic acid sequences, including
genes, nucleic acid
products, and other molecules and compositions. Memory agents may, for
example, enhance or
impair short or long term memory. For example, memory agents may be any of the
compounds
set forth in United States Patent Application Serial No. 11/679,782 entitled
"Therapeutic
Piperazones" and filed on February 27, 2007; United States Patent Application
Serial No.
11/608,746 entitled "Indolone Compounds Useful To Treat Cognitive Impairment"
and filed on
December 8, 2006; and United States Patent Application Serial No. 11/679,775
entitled
"Therapeutic Compounds" and filed on February 27, 2006, United States
Provisional Patent
Application Serial No. 60/942,992, entitled "Therapeutic Pyrazoloquinoline
Urea Derivatives"
filed on June 8, 2007, each of the above patent applications is hereby
expressly incorporated by
reference herein in its entirety.
A memory impairer, as used herein, are compounds with pharmacological activity
relating to retained memories (for example, post-traumatic stress disorder),
and include drugs,
chemical compounds, ionic compounds, organic compounds, organic ligands,
including
cofactors, saccharides, recombinant and synthetic peptides, proteins,
peptoids, nucleic acid
sequences, including genes, nucleic acid products, and other molecules and
compositions. The
memory impairers and memory enhancers of the present invention can be used
with any of the
present methods and systems.
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For example, memory agents can be cell permeant cAMP analogs (e.g., 8-bromo
cAMP);
activators of adenylate cyclase 1 (AC l) (e.g., forskolin); agents affecting G-
protein linked
receptor, such as, but not limited to adrenergic receptors and opioid
receptors and their ligands
(e.g., phenethylamines); modulators of intracellular calcium concentration
(e.g., thapsigargin, N-
methyl-D-aspartate (NMDA) receptor agonists); inhibitors of the
phosphodiesterases responsible
for cAMP breakdown (e.g., rolipram (which inhibits phosphodiesterase 4), iso-
buto-metho-
xanthine (IBMX) (which inhibits phosphodiesterases 1 and 2)); modulators of
protein kinases
and/or protein phosphatases, which mediate CREB protein activation and CREB-
dependent gene
expression. Memory agents can also be exogenous CREB, CREB analogs, CREB-like
molecules, biologically active CREB fragments, CREB fusion proteins, and/or
nucleic acid
sequences encoding exogenous CREB, CREB analogs, CREB-like molecules,
biologically active
CREB fragments and/or CREB fusion proteins.
Memory agents can also be CREB function modulators, and/or nucleic acid
sequences
encoding CREB function modulators. CREB function modulators, as used herein,
have the
ability to modulate CREB pathway function. By "modulate" is meant the ability
to change
(increase or decrease) or alter CREB pathway function.
Memory agents can be compounds which are capable of enhancing CREB function in
the
CNS. Such compounds include, but are not limited to, compounds which affect
membrane
stability and fluidity and specific immunostimulation. In a particular
embodiment, the memory
agent is capable of transiently enhancing CREB pathway function in the CNS.
CREB analogs, or derivatives, are defined herein as proteins having amino acid
sequences analogous to endogenous CREB. Analogous amino acid sequences are
defined herein
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to mean amino acid sequences with sufficient identity of amino acid sequence
of endogenous
CREB to possess the biological activity of endogenous CREB, but with one or
more "silent"
changes in the amino acid sequence. CREB analogs include mammalian CREM,
mammalian
ATF-1 and other CREB/CREM/ATF-1 subfamily members.
CREB-like molecule, as the term is used herein, refers to a protein which
functionally
resembles (mimics) CREB. CREB-like molecules need not have amino acid
sequences
analogous to endogenous CREB.
Examples of memory agents can include:
CF3
CF3
N --6 ;w~
C C
OH
Examples of memory agents can also include:
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0
QH
CH, /
OePent
OMe
wherein "Me" means methyl and "cPent" means "cyclopentyl." The above formula
embraces both enantiomers and mixtures thereof. In a particular embodiment,
(HT-0712), the 3
and 5 carbons of the above formula are in the S configuration.
Examples may also include a compound having the structure:
B
Ys
Y2
Yy
A
Y4
wherein each of Yi, Y2, Y3, and Y4 is independently --H; straight chained or
branched Ci-
C7 alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-
C7 alkenyl or
alkynyl; C3-C7 cycloalkyl, or C5-C7 cycloalkenyl; --F, --Cl, --Br, or --I; --
NO2; --N3; --CN; --
OR4, --OCOR4, --COR4, --NCOR4, --N(R4) 2, --CON(R4) 2, or --COOR4; aryl or
heteroaryl; or
any two of Y1, Y2, Y3 and Y4 present on adjacent carbon atoms can constitute a
methylenedioxy
group; wherein each R4 is independently --H; straight chained or branched CI-
C7 alkyl,
monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-C7 alkenyl
or alkynyl; C3-
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C7 cycloalkyl, C5-C7 cycloalkenyl, aryl or aryl(Ci-C6)alkyl; wherein A is A',
straight chained or
branched CI-C7 alkyl, aryl, heteroaryl, aryl(Ci-C6)alkyl or heteroaryl(Ci-
C6)alkyl; wherein A' is
0
o R1
In
n R5; ; n CR2R3; or
(CH2)n R4;
wherein Ri and R2 are each independently H, straight chained or branched CI-C7
alkyl, -
F, --Cl, --Br, --I, --NO2, or --CN; wherein R3 is H, straight chained or
branched CI-C7 alkyl, --F,
--Cl, --Br, --I, --NO2, --CN, --OR6, aryl or heteroaryl; wherein R5 is
straight chained or branched
Ci-C7 alkyl, --N(R4) 2, --OR4 or aryl; wherein R6 is straight chained or
branched Ci-C7 alkyl or
aryl; wherein B is C3-C7 cycloalkyl, C5-C7 cycloalkenyl, adamantyl, aryl,
pyridyl, pyridazinyl,
pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-yl, indo l -5-yl,
indol-6-yl, indol-7-yl,
isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl, benzo[b]furan-6-yl,
benzo[b]furan-7-yl,
benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl, benzo[b]thiophen-6-yl,
benzo[b]thiophen-7-yl,
indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl, imidazo(2,1-
b)thiazolyl, quinolinyl,
isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl, furanyl, thienyl, pyrrolyl,
oxazolyl, thiazolyl,
imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl,
thiadiazolyl, benzoxazolyl,
benzisoxazolyl, cinnolinyl, quinoxalinyl, 1,8-naphthridinyl, pteridinyl, or
phthalimidyl; provided
however, if B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,
triazinyl, indolizinyl, indo 1-4-
yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl,
benzo[b]furan-5-yl, benzo
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[b] furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-
yl,
benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl,
benzo[b]thiazolyl,
purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl,
2,1,3-benzothiazolyl,
furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,
isoxazolyl, isothiazolyl,
oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl,
cinnolinyl, quinoxalinyl, 1,8-
napthyridinyl, pteridinyl, or phthalimidyl the carbon atom or carbon atoms
ortho to the nitrogen
atom of the imine bond may only be substituted with one or more of the
following --F, --Cl, --Br,
--I, --CN, methyl, ethyl or methoxy; wherein n is an integer from 1 to 4
inclusive, and wherein
aryl is phenyl or napthyl, including phenyl and napthyl substituted with one
or more of the
following: -F, -Cl, -Br, -I, -NO2, -CN, straight chained or branched CI-C7
alkyl, straight chained
or branched CI-C7 monofluoroalkyl, straight chained or branched CI-C7
polyfluoroalkyl, straight
chained or branched C2-C7 alkenyl, straight chained or branched C2-C7 alkenyl,
C3C7 cycloalkyl,
C3-C7 monofluorocycloalkyl, C3-C7 polyfluorocycloalkyl, C5-C7 cycloalkenyl, -
OR4, SR4, -
OCOR4, -COR4, -NCOR4, -CO2R4, -CON(R4)2 or (CH2)õ-O-(CH2)m.-CH3.
In some embodiments A is aryl, heteroaryl, or heteroaryl (Ci-C6)alkyl; and
wherein aryl
is substituted with --F, --Cl, --Br, --I, --NO2, --CN, straight chained or
branched CI-C7 alkyl,
straight chained or branched CI-C7 monofluoroalkyl, straight chained or
branched CI-C7
polyfluoroalkyl, straight chained or branched C2-C7 alkenyl, straight chained
or branched C2-C7
alkynyl, C3-C7 cycloalkyl, C3-C7 monofluorocycloalkyl, C3-C7
polyfluorocycloalkyl, C5-C7
cycloalkenyl, --N(R4)2, --OR4, --SR4, --OCOR4, --COR4, --NCOR4, --C02R4, --
CON(R4)2 or --
(CH2)nO(CH2)mCH3. A may also be aryl, heteroaryl, heteroaryl(Ci-C6)alkyl or --
(CH2)ri -CC--
R4; wherein the aryl is substituted with --OH. In other embodiments, wherein A
is A' and A' is
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R~
\V
CRZR3.
In other embodiments each of Y1, Y2, Y3, and Y4 is independently --H; straight
chained
or branched Ci-C7alkyl, --CF3, --F,--Cl, --Br, --I, --OR4, --N(R4)2, or --
CON(R4)2; wherein each
R4 is independently --H; straight chained or branched Ci-C7alkyl, --CF3, or
phenyl; wherein A is
A', straight chained or branched Ci-C7alkyl, aryl, heteroaryl, aryl(Ci-
C6)alkyl or heteroaryl(Ci-
C6)alkyl; and wherein A' is
In certain embodiments, B is C3-C7cycloalkyl or adamantyl. In other
embodiments, B is
pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indol-4-
yl, indol-5-yl, indo l -
6- yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl, benzo[b]furan-5-yl,
benzo[b]furan-6-yl,
benzo[b]furan-7-yl, benzo[b]thiophen-4-yl, benzo[b]thiophen-5-yl,
benzo[b]thiophen-6-yl,
benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl, benzo[b]thiazolyl, purinyl,
imidazo[2,1-
b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 2,1,3-benzothiazolyl,
furanyl, thienyl,
pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,
isothiazolyl, oxadiazolyl,
triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl, cinnolinyl,
quinoxalinyl, 1,8-napthyridinyl,
pteridinyl, or phthalimidyl. In other embodiments B is aryl or is phenyl and
the phenyl is
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optionally substituted with one or more of the following: --F, --Cl, --Br, --
CF3, straight chained
or branched CI-C7 alkyl, --OR4, --COR4, --NCOR4, --C02R4, or --CON(R4)2.
Further examples include compounds have the following structures:
cU
yJ ~
1
-41
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N
CF3
O
CCN'
OH
5"xifl% Y
ti f.l
OIX/
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4 i
Further examples include a compound having the structure:
&~. JI
wherein each of Y1, Y2, Y3, and Y4 is independently --H; straight chained or
branched Ci-
C7 alkyl, monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-
C7 alkenyl or
alkynyl; C3-C7 cycloalkyl or C5-C7 cycloalkenyl; --F, --Cl, --Br, or --I; --
NO2; --N3; --CN; --OR4,
--SR4, --OCOR4, --COR4, --NCOR4, --N(R4)2, --CON(R4)2, or --COOR4; aryl or
heteroaryl; or
any two of Y1, Y2, Y3 and Y4 present on adjacent carbon atoms can constitute a
methylenedioxy
group; wherein each R4 is independently --H; straight chained or branched CI-
C7 alkyl,
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monofluoroalkyl or polyfluoroalkyl; straight chained or branched C2-C7 alkenyl
or alkynyl; C3-
C7 cycloalkyl, C5-C7 cycloalkenyl, aryl or aryl(Ci-C6)alkyl; wherein A is A',
straight chained or branched CI-C7 alkyl, aryl, heteroaryl, aryl(Ci-C6)alkyl
or
heteroaryl(Ci-C6)alkyl; wherein A' is
Fi
wherein Ri and R2 are each independently H, straight chained or branched CI-C7
alkyl, -
F, --Cl, --Br, --I, --NO2, or --CN; wherein R3 is H, straight chained or
branched CI-C7 alkyl, --F,
--Cl, --Br, --I, --NO2, --CN, --OR6, aryl or heteroaryl; wherein R5 is
straight chained or branched
CI-C7 alkyl, --N(R4)2, --OR4 or aryl; wherein R6 is straight chained or
branched CI-C7 alkyl or
aryl; wherein B is aryl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,
triazinyl, indolizinyl, indol-
4-yl, indol-5-yl, indol-6-yl, indol-7-yl, isoindolyl, benzo[b]furan-4-yl,
benzo[b]furan-5-yl,
benzo[b]furan-6-yl, benzo[b]furan-7-yl, benzo[b]thiophen-4-yl,
benzo[b]thiophen-5-yl,
benzo[b]thiophen-6-yl, benzo[b]thiophen-7-yl, indazolyl, benzimidazolyl,
benzo[b]thiazolyl,
purinyl, imidazo[2,1-b]thiazolyl, quinolinyl, isoquinolinyl, quinazolinyl,
2,1,3-benzothiazolyl,
furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,
isoxazolyl, isothiazolyl,
oxadiazolyl, triazolyl, thiadiazolyl, benzoxazolyl, benzisoxazolyl,
cinnolinyl, quinoxalinyl, 1,8-
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napthyridinyl, pteridinyl, or phthalimidyl; provided however, that the carbon
atom or carbon
atoms ortho to the nitrogen atom of the imine bond may only be substituted
with one or more of
the following --F, -Cl, --Br, --I, --CN, methyl, ethyl or methoxy; wherein n
is an integer from 1
to 4 inclusive; wherein aryl is phenyl or napthyl, including phenyl and
napthyl substituted with
one or more of the following: -F, -Cl, -Br, -I, -NO2, -CN, straight chained or
branched CI-C7
alkyl, straight chained or branched CI-C7 monofluoroalkyl, straight chained or
branched CI-C7
polyfluoroalkyl, straight chained or branched C2-C7 alkenyl, straight chained
or branched C2-C7
alkynyl, C3-C7 cycloalkyl, CI-C7 monofluorocycloalkyl, C3-C7
polyfluorocycloalkyl, C5-C7
cycloalkenyl, -OR4, SR4, -OCOR4, -COR4, -NCOR4, -C02R4, -CON(R4)2 or (CH2)õ-O-
(CH2)m
CH3; or a pharmaceutically acceptable salt thereof. In some embodiments, each
of Y1, Y2, Y3,
and Y4 is independently --H; straight chained or branched CI-C7 alkyl, --CF3, -
-F, --Cl, --Br, --I, -
-OR4, --N(R4)2, or --CON(R4)2.
In some embodiments, A is aryl, heteroaryl, heteroaryl(Ci-C6)alkyl or --(CH2)õ-
-CC--R4;
wherein the aryl is substituted with -OH. In other embodiments, A is aryl,
heteroaryl, or
heteroaryl(Ci-C6)alkyl; and wherein aryl is substituted with --F, --Cl, --Br, -
-I, --NO2, --CN,
straight chained or branched CI-C7 alkyl, straight chained or branched CI-C7
monofluoroalkyl,
straight chained or branched CI-C7 polyfluoroalkyl, straight chained or
branched C2-C7 alkenyl,
straight chained or branched C2-C7 alkynyl, C3-C7 cycloalkyl, C3-C7
monofluorocycloalkyl, C3-
C7 polyfluorocycloalkyl, C5-C7 cycloalkenyl, --N(R4)2, --OR4, --SR4, --OCOR4, -
-COR4, --
C02R4, --CON(R4) 2 or --(CH2)nO(CH2)mCH3. A may also be aryl or
aryl(Ci_C6)alkyl. In other
embodiments, A is A', straight chained or branched CI-C7 alkyl, aryl,
heteroaryl, aryl(Ci-
C6)alkyl or heteroaryl(Ci-C6)alkyl; and A' is
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The compounds described herein may be enantiomerically and/or
diastereomerically
pure. The may also be a pure Z imine isomer or a pure Z alkene isome, or a
pure E imine isomer
or a pure E alkene isomer.
Additional examples include:
ti
i Eby
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f
Additional examples may include a compound of the following formula:
Ar
;c 1
fr.~
R.
N R.
R, .7
.:r^-. F
0
or a pharmaceutically acceptable salt thereof,wherein:
R1, R2, Rd, and R4 are each independently selected from the group consisting
of
hydrogen, hydroxy, halo, cyan, -CONRaRb, -NRaRb, hydroxy(Ci-C6)alkyl , aryl,
heteroaryl,
heterocycle, amino(Ci-C6)alkyl, (Ci-C6)alkyl optionally substituted with up to
5 fluoro, and (Ci-
C6)alkoxy optionally substituted with up to 5 fluoro;
each Ra and Rb are independently hydrogen, (Ci-C6)alkyl, aryl, (Ci-
C6)alkylOC(O)-, or
arylOC(O)-, or Ra and Rb are taken together with the nitrogen to which they
are attached to form
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a heterocycle group optionally substituted with one or more Rd; wherein the
heterocycle group
optionally include one or more groups selected from 0 (oxygen), S(O)z7 and
NRC;
each z is an integer selected from 0 , 1, and 2;
each R, is independently hydrogen, (C2-C6)alkenyl, (C2-C6)alkynyl, -C(O)O(Ci-
C6)alkyl,
-C(O)Oaryl, (Ci-C6)alkoxy(Ci-C6)alkyl, (Ci-C6)alkylO(CH2)m, hydroxy(Ci-
C6)alkyl, aryl,
heteroaryl, Heterocycle, arylO(Ci-C6)alkyl, -C(O)NRg(Ci-C6)alkyl, -
C(O)NRgaryl, -S(O),(Ci-
C6)alkyl, -S(O), aryl, -C(O)(Ci-C6)alkyl, aryl C(O)-, (Ci-C6)alkyl optionally
substituted with up
to 5 fluoro, or (Ci-C6)alkoxy optionally substituted with up to 5 fluoro;
each m is an integer selected from 2, 3, 4, 5, and 6;
each Rd is independently selected from the group consisting of hydrogen, halo,
oxo,
hydroxy, -C(O)NRaRb, -NRaRb, hydroxy(Ci-C6)alkyl, aryl, aryl(Ci-C6)alkyl, (Ci-
C6)alkyl
optionally substituted with up to 5 fluoro, and (Ci-C6)alkoxy optionally
substituted with up to5
fluoro;
R, and Rr are each independently selected from the group consisting of
hydrogen, (Ci-
C6)alkyl, aryl, -S(O),(Ci-C6)alkyl, -S(O),aryl, -CONRg(Ci-C6alkyl), (Ci-
C6)alkylC(O)-,
arylC(O)-, (Ci-C6)alkylOC(O)-, and arylOC(O)-;
Rg is hydrogen or (Ci-C6)alkyl;
R5 and R6 are each independently selected from the group consisting of
hydrogen, (Ci-
C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, and aryl, or R5 and R6 are taken
together with the
nitrogen to which they are attached to form a heterocycle group optionally
substituted with one
or more Rd; wherein the heterocycle group optionally include one or more
groups selected from
0 (oxygen), S(O)z, and NRC;
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R7 is selected from the group consisting of hydrogen, hydroxy, halo,
hydroxy(Ci-
C6)alkyl, (Ci-C6)alkyl optionally substituted with up to 5 fluoro, and (Ci-
C6)alkoxy optionally
substituted with up to 5 fluoro;
Ar is aryl, or heteroaryl, each optionally substituted with one or more R8;
and
each Rs is independently hydrogen, halo, CF3, CF2H, hydroxy, cyan, nitro,( Ci-
C6)alkyl,
hydroxy(Ci-C6)alkyl, (Ci-C6)alkoxy, -NRaRb, aryl, heteroaryl or heterocycle.
Other examples include the compound of following formula:
= {ff
N' R7
0 N" R,
and pharmaceutically acceptable salts thereof.
R5 and R6 may, for example, together with the nitrogen to which they are
attached, form a
piperidinyl, pyrrolidinyl, morpholinyl, or thiomorpholinyl ring in the
compound of the above
formula.
Yet further examples include a compound of the following formula:
wref N-- N1
I r/
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and pharmaceutically acceptable salts thereof, wherein: X is N(R,), 0
(oxygen), C(Rd)2,
or S(O),; z is an integer selected from 0, 1, and 2; each Rd is independently
selected from the
group consisting of hydrogen, halo, oxo, hydroxy, -C(O)NRaRb, -NRaRb,
hydroxy(Ci-C6)alkyl,
aryl, aryl(Ci-C6)alkyl, (Ci-C6)alkyl optionally substituted with up to 5
fluoro , and (Ci-C6)alkoxy
optionally substituted with up to 5 fluoro; and n is an integer selected from
0, 1, and 2; with the
proviso that when n = 0 then X is C(Rd)2.
Other examples include a compound of the following formula:
Ar
N---N
k 4 . r..
and pharmaceutically acceptable salts thereof.
Other examples include a compound of the following formula:
Fly N
Z~o
Ne"
r F~
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and pharmaceutically acceptable salts thereof, wherein n is 0, 1, or 2.
In some embodiments, R2 is Methyl, fluro, or OMe. In some embodiments, R3 is
Methyl,
fluro, or OMe. In some embodiments, R2 and R3 are fluoro. In another
embodiment, R2 and R3
are Methyl.
Other examples include a compound of the following formula:
rt ~
~n f ~~" r`' 7lff
'N' 'P'r
P {- j N, R
and pharmaceutically acceptable salts thereof.
Other examples include a compound of the following formula:
Y=Y
R, N'-=
R
R~N
and pharmaceutically acceptable salts thereof, wherein each Y is independently
N or
C(R8). In some embodiments, R5 and R6, together with the nitrogen to which
they are attached,
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form a piperidinyl, pyrrolidinyl, morpholinyl, or thiomorpholinyl ring, each
optionally
substituted with one or more Rd.
Further examples include a compound of the following formula:
Y~y
ri ~k
Y Y
k.r 71
and pharmaceutically acceptable salts thereof.
In some embodiments n can be 0, 1 or 2. In some embodiments, R2 may be Methyl,
fluoro or OMe. In some embodiments, R3 can be Methyl, fluoro or OMe. In some
embodiments,
R2 and R3 can be fluoro. In another embodiment, R2 and R3 can be Methyl.
Additional examples include a compounds of the formulas:
Y-Y
3F ~'v
4
y-i7y
.1
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R4 'J
R j......
ff?
q NI
A,j
P : C
and
N Rr
and pharmaceutically acceptable salts thereof.
Examples also include a compound of the following formula:
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RI, "IX W
f't a
z
wherein: Ri is H, (Ci-C6)alkyl, (Ci-C6)alkoxy, (Ci-C6)alkanoyl, (Ci-
C6)alkoxycarbonyl,
(CI-C6)alkanoyloxy, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl((Ci-C6)alkyl, halo(Ci-
C6)alkyl, aryl,
aryl(Ci-C6)alkyl, aryl(Ci-C6)alkoxy, aryl(Ci-C6)alkanoyl, het, het(Ci-
C6)alkyl, het(Ci-C6)alkoxy,
or het(Ci-C6)alkanoyl;
n is 1 or 2;
m is 1 or 2;
W is 0, S, or two hydrogens;
X is 0 or N-Y-R4;
Y is a direct bond, -CH2-, -C(=0)-, -C(=S)-, -0-, -C(=0)O-, -OC(=0)-, -
C(=0)NRa-, -S-, -
S(=0)-, -S(=0)2-, or -S(=0)2NRa-;
R4 is H, (Ci-C6)alkyl, (Ci-C6)alkoxy, (Ci-C6)alkanoyl, hydroxy, (C3-
C8)cycloalkyl, (C3-
Cg)cycloalkyl(Ci-C6)alkyl, halo(Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, (Ci-
C6)alkoxycarbonyl,
carboxy, aryl, aryl(Ci-C6)alkyl, het, NRdRe7 -C(=O)NRdRe7 NRdRe (Ci-C6)alkyl,
or het(Ci-
C6)alkyl;
Ra is H, (Ci-C6)alkyl, (C3-C8)cycloalkyl, (C1-C6)alkoxy(C2-C6)alkyl, or (C3-
Cg)cycloalkyl(Ci-C6)alkyl;
Z is a phenyl ring substituted with one or more substituents independently
selected from
(Ci-C6)alkyl, halo(Ci-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(Ci-
C6)alkyl, (Ci-
C6)alkoxy, halo(Ci-C6)alkoxy, (C3-C8)cycloalkyloxy, and (C3-C8)cycloalkyl(Ci-
C6)alkoxy; or Z
is a phenyl ring that is fused to a saturated, partially unsaturated, or
aromatic, mono- or bicyclic
ring system comprising from about 3 to about 8 atoms selected from carbon,
oxygen, and NRb,
wherein the mono- or bicyclic ring system of Z is optionally substituted with
one or more Rb, and
wherein the phenyl ring that is fused to the mono- or bicyclic ring system is
optionally
substituted with one or more substitaents independently selected from (Ci-
C6)alkyl, halo(Ci-
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C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(Ci-C6)alkyl, (Ci-C6)alkoxy,
halo(C2-C6)alkoxy,
(C3-C8)cycloalkyloxy, and (C3-C8)cycloalkyl(Ci-C6)alkoxy;
Rb is absent, H, (C1-C6)alkyl, (C3-C8)cycloalkyl, (C1-C6)alkoxy(C2-C6)alkyl,
or (C3-
Cg)cycloalkyl(C1-C6)alkyl;
R, is (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkanoyl, (C1-C6)alkoxycarbonyl, (Ci-
C6)alkanoyloxy, (C3-Cg)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, halo(Ci-
C6)alkyl, aryl,
aryl(Ci-C6)alkyl, aryl(C1-C6)alkoxy, aryl(C1-C6)alkanoyl, het, het(Ci-
C6)alkyl, het(Ci-C6)alkoxy,
or het(C1-C6)alkanoyl;
each Rd and Re is independently H, hydroxy, (C1-C6)alkyl, (C1-C6)alkenyl, (C2-
C6)alkynyl, (C1-C6)alkoxy, (C2-C6)alkenyloxy, (C2-C6)alkynyloxy, (C1-
C6)alkanoyl, (Ci-
C6)alkoxycarbonyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, aryl,
aryl(Ci-C6)alkyl,
NRfRg, or aryl(C1-C6)alkoxy; and
each Rf and Rg is independently H, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-
C6)alkanoyl, (Ci-
C6)alkoxycarbonyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, aryl,
aryl(Ci-C6)alkyl, or
aryl(C1-C6)alkoxy; or Rf and Rg together with the nitrogen to which they are
attached form a
pyrrolidino, piperidino , piperazino , morpholino, or thiomorpholino ring;
wherein any aryl or het of Ri and R4 is optionally substituted with one or
more
substitutents independently selected from (C1-C6)alkyl, phenyl, (C1-C6)alkoxy,
(C1-C6)alkanoyl,
(C1-C6)alkoxycarbonyl, (C1-C6)alkanoyloxy, (C3-C8)cycloalkyl, (C3-
C8)cycloalkyl(C1-C6)alkyl,
halo(Ci-C6)alkyl, (C3-C8)cycloalkyloxy, (C3-C8)cycloalkyl(C1-C6)alkoxy,
halo(C2-C6)alkoxy,
cyan, nitro, halo, carboxy or NRdRej
and wherein the ring containing X is optionally substituted on carbon with one
or more
halo, (C1-C6)alkyl, or (C1-C6)alkoxy.
Including any pharmaceutically acceptable salt thereof.
Other examples include a compound of the following formula:
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wherein: R1 is (Ci-C6)alkyl, (C1 -C6)haloalkyl, or aryl , unsubstituted or
substituted with
one or more Re;
one of R2 and R3 is absent and the other is hydrogen, (Ci-C6)alkyl, halo(Ci-
C6)alkyl,
hydroxy(Ci-C6)alkyl, (C3-C8)cycloalkyl, amino(C2-C6)alkyl, or aryl, each
unsubstituted or
substituted with one or more groups selected from alkyl, halo, haloalkyl or
nitro, Het, (C3-
Cg)cycloalkyl(Ci-C6)alkyl, aryl(Ci-C6)alkyl, (C1-C6) or Het(Ci-C6)alkyl;
B is aryl, thiophene, heteroaryl, furan or pyrrole;
X is -C(=0), -C(-S), -C(R4)2, -C(OH)-, or -S(O)zj
each z is independently 0, 1, or 2;
Y is R4, -N(R4)2, -OR4, -SR4, or -C(R4)3;
each R4 is independently selected from the group consisting of hydrogen,
(C1C6) alkyl,
(C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkoxy, (Ci-C6)alkanoyl, (C
1C6)alkoxycarbonyl, (C3-
Cg)cycloalkyl, (C3-C8)cycloalkyl(Ci-C6)alkyl, (Ci-C6)alkoxy(C2-C6)alkyl,
hydroxy(C2-C6)alkyl,
cyano(Ci-C6)alkyl, (Ci-C6)alkthio(Cz-C6)alkyl, aryl, aryl(Ci-C6)alkyl,
aryloxy(C2-C6)alkyl,
halo(C2-C6)alkyl, (Ci-C6)alkoxycarbonyl(CiC6)alkyl, NRaRb, Het, or Het(Ci-
C6)alkyl,
unsubstituted or substituted with one or more or two R4 groups are taken
together with the atom
to which they are attached to form aryl, Het, or a saturated or unsaturated 3-
8 membered
monocyclic or 8-12 membered bicyclic ring system comprising carbon atoms and
optionally
comprising one or more additional heteroatoms selected from 0, S(O), and NRC,
wherein each
ring system is optionally substituted with one or more Rd;
each Ra and Rb is independently hydrogen or (Ci-C6)alkyl;
each Re7 is independently hydrogen, aryl, S(O)2, (Ci-C6)alkanoyl, hydroxy(Ci-
C6)alkyl,
alkoxy(Ci-C6)alkyl, Het, (Ci-C6)alkoxycabonyl or (Ci-C6)alkyl, unsubstituted
or substituted with
one or more subtituents Re;
each Rd is independently halo, hydroxy, cyan, nitro, azido, amino, (Ci-
C6)alkylamino,
amino(Ci-C6)alkyl, amido, (Ci-C6)alkyamido, aryl amido, carboxylic acid, (Ci-
C6)alkyl,
hydroxy(Ci-C6)alkyl, halo(Ci-C6)alkyl, (C1-C6)alkoxy, halo(Ci-C6)alkoxy, (C1-
C6)alkanoyl, (Ci-
C6)alkoxycarbonyl, carboxy, (Ci-C6)alkanoyloxy, Het, aryl, Het(Ci-C6)alkyl, or
aryl(Ci-
C6)alkyl, (Ci-C6)alkylaryl, sulfonyl, sulfonamido, urea, carbamate,
unsubstituted or substituted
with one or more substituents Re, or two Rd come together with the atom to
which they are
attached to form a ketone or spirocyclic carbocyclic or heterocyclic ring, or
two Rd come
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together with the atoms to which they are attached to form a bicyclic
carbocyclic or heterocyclic
ring, wherein each spirocyclic or bicyclic ring isunsubstituted or substituted
with one or more
halo, hydroxy, cyan, nitro, azido, (Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, halo(Ci-
C6)alkyl, (Ci-
C6)alkoxy, halo(Ci-C6)alkoxy, (Ci-C6)alkanoyl, (Ci-C6)alkoxycarbonyl, carboxy,
(Ci-
C6)alkanoyloxy, NRfRg, RfRgNC(=0)-,phenyl, or phenyl(Ci-C6)alkyl, sulfonyl,
sulfonamido,
urea, carbamate, wherein Rf and Rg together with the nitrogen to which they
are attached form a
piperidino, pyrrolidino, morpholino, or thiomorpholino ring, unsubstituted or
substituted with
one or more substituents Re;
each Re, is independently selected from halo, hydroxy, cyan, nitro, azido, (Ci-
C6)alkyl,
Het, aryl, (C1-C6)alkylHet, (C1-C6)alkylaryl, (C1-C6)alkylHet(Ci-C6)alkyl, (C1-
C6)alkylaryl(Ci-
C6)alkyl, (Ci-C6)haloalkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1C6)alkanoyl,
(Ci-
C6)alkoxycarbonyl, carboxy, and (C1-C6)alkanoyloxy;
R5 is H, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, aryl(C1-C6)alkyl; and
each R6 is H, (C1-C6)alkyl, amino, amido, keto, or aryl(C1-C6)alkyl, with the
proviso that
when B is thiophene, R1 is trifluoromethyl, R2 is methyl, R3 and R6 are
absent, R5 is H, X is
C(=0) and Y is N(R4)2, both R4 are not methyl.
Additional examples include a compound of the following formula:
r/ c
Z1 x
Y
wherein R1 is (C1-C6)alkyl, (C1-C6)haloalkyl, or aryl, unsubstituted or
substituted with
one or more Re;
one of R2 and R3 is absent and the other is hydrogen, (Ci-C6)alkyl, halo(Ci-
C6)alkyl,
hydroxy(Ci-C6)alkyl, (C3-C8)cycloalkyl, amino(C2-C6)alkyl, or aryl, each
unsubstituted or
substituted with one or more groups selected from alkyl, halo, haloalkyl or
nitro, Het, (C3-
Cg)cycloalkyl(Ci-C6)alkyl, aryl(Ci-C6)alkyl, (C1-C6) or Het(Ci-C6)alkyl;
each of Z', Z2 , and Z3 is independently C(R6)p, N(R6)q, 0, or S, wherein if
Z' is N(R6)q,
0, or S, at least one of Z' or Z2 must be N(R6)q, 0, or S;
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each p is independently 0, 1, or 2;
each q is independently 0 or 1;
X is -C(=0), -C(=S), -C(R4)2, or -S(O)L;
each z is independently 0, 1, or 2;
Y is R4, -N(R4)z, -OR4, -SR4, or -C(R4)3;
each R4 is independently selected from the group consisting of hydrogen, (Ci-
C6)alkyl,
(C2-C6)alkenyl, (C2-C6)alkynyl, (Ci-C6)alkoxy, (Ci-C6)alkanoyl, (Ci-
C6)alkoxycarbonyl, (C3-
Cg)cycloalkyl, (C3-C8)cycloalkyl(Ci-C6)alkyl, (Ci-C6)alkoxy(C2-C6)alkyl,
hydroxy(C2-C6)alkyl,
cyano(Ci-C6)alkyl, (Ci-C6)alkthio(C2-C6)alkyl, aryl, aryl(Ci-C6)alkyl,
aryloxy(C2-C6)alkyl,
halo(C2-C6)alkyl, (Ci-C6)alkoxycarbonyl(Ci-C6)alkyl, NRaRb, Het, or Het(Ci-
C6)alkyl, wherein
each alkyl, aryl, or Het is unsubstituted or substituted with one or more Rd,
or two R4 groups are
taken together with the atom to which they are attached to form aryl, Het, or
a saturated or
unsaturated 3-8 membered monocyclic or 8-12 membered bicyclic ring system
comprising
carbon atoms and optionally comprising one or more additional heteroatoms
selected from 0,
S(O), and NRC, wherein each ringsystem is optionally substituted with one or
more Rd;
each Ra and Rb is independently hydrogen or (Ci-C6)alkyl;
each Re is independently hydrogen, aryl, S(O)2, (Ci-C6)alkanoyl, hydroxy(Ci-
C6)alkyl,
alkoxy(Ci -C6)alkyl, Het, (Ci-C6)alkoxycabonyl or (Ci-C6)alkyl, unsubstituted
or substituted
with one or more substituents Re;
each Rd is independently halo, hydroxy, cyan, nitro, azido, amino, (Ci-
C6)alkylamino,
amino(Ci-C6)alkyl, amido, (Ci-C6)alkyamido, aryl amido, carboxylic acid, (Ci-
C6)alkyl,
hydroxy(Ci-C6)alkyl, halo(Ci-C6)alkyl, (Ci-C6)alkoxy, halo(Ci-C6)alkoxy, (Ci-
C6)alkanoyl, (Ci-
C6)alkoxycarbonyl, carboxy, (Ci-C6)alkanoyloxy, Het, aryl, Het(Ci-C6)alkyl, or
aryl(Ci-
C6)alkyl, (Ci-C6)alkylaryl, sulfonyl, sulfonamide, urea, carbamate,
unsubstituted or substituted
with one or more substituents Re, or two Rd come together with the atom to
which they are
attached to form a ketone or spirocyclic carbocyclic or heterocyclic ring, or
two Rd come
together with the atoms to which they are attached to form a bicyclic
carbocyclic or heterocyclic
ring, wherein each spirocyclic or bicyclic ring is unsubstituted or
substituted with one or more
halo, hydroxy, cyan, nitro, azido, (Ci-C6)alkyl, hydroxy(Ci-C6)alkyl, halo(Ci-
C6)alkyl, (CI_
C6)alkoxy, halo(Ci-C6)alkoxy, (Ci-C6)alkanoyl, (Ci-C6)alkoxycarbonyl, carboxy,
(Ci-
C6)alkanoyloxy, NRfRg, RfRgNC(=O)-, phenyl, or phenyl(Ci-C6)alkyl, sulfonyl,
sulfonamido,
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urea, carbamate, wherein Rf and Rg together with the nitrogen to which they
are attached form a
piperidino, pyrrolidino, morpholino, or thiomorpholino ring, unsubstituted or
substituted with
one or more substituents Re;
each Re is independently selected from halo, hydroxy, cyan, nitro, azido, (Ci-
C6)alkyl,
Het, aryl, (Ci-C6)alkylHet, (Ci-C6)alkylaryl, (Ci-C6)alkylHet(Ci-C6)alkyl, (C
i-C6)alkylaryl(Cl-
C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (Cl-C6)alkanoyl,
(Cl-
C6)alkoxycarbonyl, carboxy, and (Ci-C6)alkanoyloxy;
R5 is H, (Ci-C6)alkyl, aryl(Ci-C6)alkyl; and
each R6 is H, (Ci-C6)alkyl, amino, amido, keto, or aryl(Ci-C6)alkyl; with the
proviso that
when X is -C(=0), Y is N(R4)z, Zi is 0, Z2 is N, and Z3 is CH, both R4 of Y
are not H.
Biologically active polypeptide fragments of CREB can include only a part of
the full-
length amino acid sequence of CREB, yet possess biological activity. Such
fragments can be
produced by carboxyl or amino terminal deletions, as well as internal
deletions.
Fusion proteins comprise a CREB protein as described herein, referred to as a
first
moiety, linked to a second moiety not occurring in the CREB protein. The
second moiety can be
a single amino acid, peptide or polypeptide or other organic moiety, such as,
without limitation, a
carbohydrate, a lipid or an inorganic molecule.
Nucleic acid sequences are defined herein as heteropolymers of nucleic acid
molecules.
The nucleic acid molecules can be double stranded or single stranded and can
be a
deoxyribonucleotide (DNA) molecule, such as cDNA or genomic DNA, or a
ribonucleotide
(RNA) molecule. As such, the nucleic acid sequence can, for example, include
one or more
exons, with or without, as appropriate, introns, as well as one or more
suitable control sequences.
In one example, the nucleic acid molecule contains a single open reading frame
which encodes a
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desired nucleic acid product. The nucleic acid sequence can be operably linked
to a suitable
promoter.
A nucleic acid sequence encoding a desired CREB protein, CREB analog
(including
CREM, ATF-1), CREB-like molecule, biologically active CREB fragment, CREB
fusion protein
or CREB function modulator can be isolated from nature, modified from native
sequences or
manufactured de novo, as described in, for example, Ausubel et al., Current
Protocols in
Molecular Biology, John Wiley & Sons, New York (1998); and Sambrook et al.,
Molecular
Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University
Press, New York.
(1989). Nucleic acids can be isolated and fused together by methods known in
the art, such as
exploiting and manufacturing compatible cloning or restriction sites.
Typically, the nucleic acid sequence will be a gene which encodes the desired
CREB
protein, CREB analog, CREB-like molecule, CREB fusion protein or CREB function
modulator.
Such a gene is typically operably linked to suitable control sequences capable
of effecting the
expression of the CREB protein or CREB function modulator, preferably in the
CNS. The term
"operably linked", as used herein, is defined to mean that the gene (or the
nucleic acid sequence)
is linked to control sequences in a manner which allows expression of the gene
(or the nucleic
acid sequence). Generally, but not always, operably linked means contiguous.
Control sequences include a transcriptional promoter, an optional operator
sequence to
control transcription, a sequence encoding suitable messenger RNA (mRNA)
ribosomal binding
sites and sequences which control termination of transcription and
translation. In a particular
embodiment, a recombinant gene (or a nucleic acid sequence) encoding a CREB
protein, CREB
analog, CREB-like molecule, biologically active CREB fragment, CREB fusion
protein or
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CREB function modulator can be placed under the regulatory control of a
promoter which can be
induced or repressed, thereby offering a greater degree of control with
respect to the level of the
product.
As used herein, the term "promoter" refers to a sequence of DNA, usually
upstream (5)
of the coding region of a structural gene, which controls the expression of
the coding region by
providing recognition and binding sites for RNA polymerase and other factors
which may be
required for initiation of transcription. Suitable promoters are well known in
the art. Exemplary
promoters include the SV40 and human elongation factor (EFI). Other suitable
promoters are
readily available in the art (see, e.g., Ausubel et al., Current Protocols in
Molecular Biology,
John Wiley & Sons, Inc., New York (1998); Sambrook et al., Molecular Cloning:
A Laboratory
Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); and
U.S. Pat. No.
5,681,735).
Memory agents can enhance CREB pathway function by a variety of mechanisms.
For
example, a memory agent can affect a signal transduction pathway which leads
to induction of
CREB-dependent gene expression. Induction of CREB-dependent gene expression
can be
achieved, for example, via up-regulation of positive effectors of CREB
function and/or down-
regulation of negative effectors of CREB function. Positive effectors of CREB
function include
adenylate cyclases and CREB activators. Negative effectors of CREB function
include cAMP
phosphodiesterase (cAMP PDE) and CREB repressors.
A memory agent can enhance CREB pathway function by acting biochemically
upstream
of or directly acting on an activator or repressor form of a CREB protein
and/or on a CREB
protein containing transcription complex. For example, CREB pathway function
can be affected
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by increasing CREB protein levels transcriptionally, post-transcriptionally,
or both
transcriptionally and post-transcriptionally; by altering the affinity of CREB
protein to other
necessary components of the of the transcription complex, such as, for
example, to CREB-
binding protein (CBP protein); by altering the affinity of a CREB protein
containing
transcription complex for DNA CREB responsive elements in the promoter region;
or by
inducing either passive or active immunity to CREB protein isoforms. The
particular mechanism
by which a memory agent enhances CREB pathway function is not critical to the
practice of the
invention.
Memory agents can be administered directly to an animal in a variety of ways.
In a
specific embodiment, memory agents are administered systemically. Other routes
of
administration are generally known in the art and include intravenous
including infusion and/or
bolus injection, intracerebroventricularly, intrathecal, parenteral, mucosal,
implant,
intraperitoneal, oral, intradermal, transdermal (e.g., in slow release
polymers), intramuscular,
subcutaneous, topical, epidural, etc. routes. Other suitable routes of
administration can also be
used, for example, to achieve absorption through epithelial or mucocutaneous
linings. Particular
memory agents can also be administered by gene therapy, wherein a DNA molecule
encoding a
particular therapeutic protein or peptide is administered to the animal, e.g.,
via a vector, which
causes the particular protein or peptide to be expressed and secreted at
therapeutic levels in vivo.
A vector, as the term is used herein, refers to a nucleic acid vector, e.g., a
DNA plasmid,
virus or other suitable replicon (e.g., viral vector). Viral vectors include
retrovirus, adenovirus,
parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA
viruses such as
orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and
vesicular stomatitis virus),
paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as
picomavirus and
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alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus
(e.g., Herpes
Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and
poxvirus (e.g., vaccinia,
fowlpox and canarypox). Other viruses include Norwalk virus, togavirus,
flavivirus, reoviruses,
papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of
retroviruses include:
avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-
BLV group,
lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In
Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-
Raven Publishers,
Philadelphia, 1996). Other examples include murine leukemia viruses, murine
sarcoma viruses,
mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus,
feline sarcoma virus,
avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus,
Gibbon ape
leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus,
simian sarcoma
virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are
described, for
example, in McVey et al., U.S. Pat. No. 5,801,030, the teachings of which are
incorporated
herein by reference.
A nucleic acid sequence encoding a protein or peptide (e.g., CREB protein,
CREB analog
(including CREM, ATF-1), CREB-like molecule, biologically active CREB
fragment, CREB
fusion protein, CREB function modulator) can be inserted into a nucleic acid
vector according to
methods generally known in the art (see, e.g., Ausubel et al., Eds., Current
Protocols in
Molecular Biology, John Wiley & Sons, Inc., New York (1998); Sambrook et al.,
Eds.,
Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor
University Press,
New York (1989)).
The mode of administration is preferably at the location of the target cells.
In a particular
embodiment, the mode of administration is to neurons.
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Memory agents can be administered together with other components of
biologically
inactive agents, such as pharmaceutically acceptable surfactants or carriers
(e.g., glycerides),
excipients (e.g., lactose), stabilizers, preservatives, humectants,
emollients, antioxidants, carriers,
diluents and vehicles. If desired, certain sweetening, flavoring and/or
coloring agents can also be
added. When combined with various inactive ingredients, a memory agent may
also be referred
to as a pharmaceutical composition.
Memory agents can be formulated as a solution, suspension, emulsion or
lyophilized
powder in association with a pharmaceutically acceptable parenteral vehicle.
Examples of such
vehicles are water, saline, Ringer's solution, isotonic sodium chloride
solution, dextrose solution,
and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed
oils can also
be used. The vehicle or lyophilized powder can contain additives that maintain
isotonicity (e.g.,
sodium chloride, mannitol) and chemical stability (e.g., buffers and
preservatives). The
formulation can be sterilized by commonly used techniques. Suitable
pharmaceutical carriers are
described in Remington's Pharmaceutical Sciences.
The dosage of memory agent administered to an animal is that amount required
to effect
a change in CREB-dependent gene expression, particularly in neurons. The
dosage administered
to an animal, including frequency of administration, will vary depending upon
a variety of
factors, including pharmacodynamic characteristics of the particular memory
agent, mode and
route of administration; size, age, sex, health, body weight and diet of the
recipient; nature and
extent of symptoms being treated or nature and extent of the memory being
enhanced or
modulated, kind of concurrent treatment, frequency of treatment, and the
effect desired.
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Memory agents can be administered in single or divided doses (e.g., a series
of doses
separated by intervals of days, weeks or months), or in a sustained release
form, depending upon
factors such as nature and extent of symptoms, kind of concurrent treatment
and the effect
desired. Other therapeutic protocols or agents can be used in conjunction with
the present
invention.
The teachings of all the articles, patents and patent applications cited
herein are
incorporated by reference in their entirety.
EXAMPLE 1
A study was undertaken to examine the effects of a memory agent (HT-0712).
The study was conducted using 6 aged male macaques (age approximately 22 years
at
onset of the study which is equivalent to approximately 66 human years)
previously trained to
performed certain memory tasks. Assessments of memory performance used the
CANTAB
system (the Cambridge Neuropsychological Test Automated Battery) and a new
task.
Discrimination Learning - General Description of a Long Term Retrieval Task
(DL
LTR)
In this task, animals are presented with 2 pairs of stimuli (e.g. 1 pair of
shapes and 1 pair
of lines) over the course of a daily test session. For each pair of stimuli,
one stimulus is
arbitrarily designated as the positive stimulus (i.e., touch of that stimulus
is rewarded). Each pair
of stimuli is presented 14 times during a daily session that consists of 28
trials. The pairs of
stimuli (shapes or lines) are presented in a pseudo randomized fashion during
each session.
Animals have to touch the "positive" shape or line in order to receive a sugar
pellet reward
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accompanied by a pleasant tone. Touching the alternative shape or line results
in a negative
tone, no sugar pellet and a time out. Animals are tested daily until they
reach a performance
criterion of 85% correct responses during a daily session. When this criterion
is reached,
animals receive no further testing for the next 7 days. On day 8 post-
criterion, animals are tested
again using the same pairs of stimuli that they have seen previously learned
in order to assess
long term retrieval of the learned discriminations.
Drug Administration
All drugs were administered orally in marshmallow while the animal was in a
specially
designed testing cage. Vehicle was marshmallow alone.
The treatment schedule is shown in Table 1. Treatments were administered
sequentially
starting with treatment 1. Each treatment (dose of HT-0712 or vehicle) was
administered once
per day for 4 days before behavioral training began and then continued at 1
treatment (dose of
HT-0712 or vehicle) per day, delivered four hours before testing, for the
duration of the training
period. When each individual animal reached performance criterion of at least
85% correct
responses during a daily session, training and dosing ceased for seven days.
Then, on post-
criterion day 8, animals were tested again on the DL LTR task (retention
testing).
A second vehicle study was conducted at the end of the drug trials using the
first 3
animals that completed the 100mg/kg dosing study.
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Table 1 - Treatment Schedule
1 (N = 6) vehicle 0 P.O.
1 P.O.
2 (N= 6) HT-0712
mg/kg
HT-0712 10 P.O.
3 (N = 6)
mg/kg
100 P.O.
4 (N= 6) HT-0712
mg/kg
(N = 3) vehicle 0 P.O.
Analysis of Behavioral Data
The percent correct responses on the total number of daily trials were
recorded as well as
the number of days needed to reach the criterion (at least 85% correct
responses in a single test
5 session). The percent correct responses on the retention trial was recorded
for the entire
retention trial session and sub-analyzed for performance differences on trials
1-14 versus trials
15-28.
Data from vehicle trials were compared to data from drug trials using a one
way
ANOVA. Post hoc comparisons were made using the Bonferroni t-test.
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Results
There were no noticeable behavioral or physical side effects following
administration of
HT-0712 at any dose given during the study. Five of the six animals used
displayed improved
memory performance as evidenced by a decrease in the number of days needed to
reach
criterion. One animal (old-9) did not show improved task performance at any
dose of HT-0712
administered. Data from this non-responder were removed from the statistical
analysis.
Vehicle Treatment
During the first vehicle study, all animals reached criterion over a period of
26.3 4.6
days. The mean criterion value reached was 89.3 1.8% correct responses. Mean
performance
at the retention trial was of 79.7 5.9% correct responses. When retention
trial performance was
divided between trials 1 to 14 versus trials 15 to 28, animals performed 77.5
5.1 % correct
responses and 82.3 7.5% correct responses, respectively.
The second (post drug study) vehicle study was performed using the first 3
animals that
finished the drug trials. During this second vehicle study, animals reached
criterion over a
period of 20.0 4.5 days. Their mean criterion value was 86.3 1.3% correct
responses.
Performance at the retention trials averaged 64.0 4.0% correct responses.
When retention trial
performance was divided between trials 1 to 14 versus trials 15 to 28, animals
performed 59.3
8.4% correct responses and 69.0 10.4% correct responses, respectively.
No statistically significant differences were found between the results of the
pre-study
and post-study vehicle trials (t = 1.168, (P = 0.363)).
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Memory Agent Treatment
At the lowest dose of HT-0712 used in this study (1 mg/kg), animals reached
criterion
over a period of 12.0 3.6 days, at a performance level of 91.4 1.8%
correct responses). The
mean performance of the animals at the retention trial was of 85.8 4.6%
correct responses.
When retention trial performance was divided between trials 1 to 14 versus
trials 15 to 28,
animals performed at a level of 80.2 6.2% correct responses and 91.6 3.4%
correct
responses, respectively.
At the medium dose of HT-0712 used in the study (1 Omg/kg), animals reached
criterion
(90.0 2.6% correct responses) over a period of 11.8 2.3 days. The mean
performance of
these animals at retention testing was of 79.2 3.5% correct responses. When
retention test
performance was divided between trials 1 to 14 versus trials 15 to 28, animals
performed at a
level of 71.4 3.4% correct and 87.2 4.8% correct, respectively.
At the highest dose of HT-0712 used (100mg/kg), animals reached criterion
(86.4 0.7%
correct responses) over a period of 9.0 2.8 days. The mean performance of
these animals at
retention testing was of 65.0 3.5% correct responses. When retention test
performance was
divided between trials 1 to 14 versus trials 15 to 28, animals performed at a
level of 58.4 4.1%
correct and 71.4 6.1 % correct, respectively.
The effect of treatment on days to criterion was significant (repeated
measures ANOVA,
F(4,3) = 6.327, p = 0.008). Post hoc comparisons using the Bonferroni t-test
showed a
significant difference between vehicle and each dose of HT-0712 (p < 0.050 for
each
comparison).
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There was no significant effect of treatment on the level of performance
attained on the
criterion day (F(4,3) = 1.408, p = 0.289).
There was no significant effect of treatment on the level of performance
attained at the
retention test (F(4,3) = 3.199, p = 0.062). Analysis of treatment effect on
retention trial
performance at trials 1-14 and trials 15-28, also failed to reach statistical
significance (p = 0.058
and p = 0.167, respectively).
This study was conducted using a new task, the Discrimination Learning - Long
Term
Retrieval task (DL LTR), that was designed to assess the effects of an
experimental memory
agent on learning and retention in aged non human primates.
The results of the vehicle control studies indicate that normal aged animals
require a long
time (ex. > 20 days) to reach an 85% correct response criterion on this test.
Additionally, the
post-drug vehicle test results indicate that the increase in performance seen
with HT-0712 was
not due to an increase in the animal's proficiency in the test or a practice
effect as the second
vehicle control test results were not significantly different from the results
of the first vehicle
study.
Five of the six animals tested showed improved performance following
administration of
HT-0712. The improvement was expressed as a significant decrease in the number
of days to
reach criterion, showing improved learning abilities when treated with HT-
0712.
The response of the animals could be separated into 2 general groups. In the
first group
(animals old-5 and old-8), the best dose for improving performance was the
lmg/kg dose of HT-
0712. These animals showed a decrement in performance as the dose administered
increased
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from 1 to 10 to 100mg/kg. That is, with increasing doses of HT-0712, it took
longer for the
animals to reach criterion (i.e., 4, 5, and 10 days to criterion (old-5) and
3, 12, and 18 days to
criterion for (old-8) after administration of 1, 10, or 100 mg/kg,
respectively). The second group
of animals (old-1, old-2 and old-7) showed a more typical dose-response effect
in which
performance improved with increasing doses (21, 15, and 3 days to criterion;
17, 18, and 11 days
to criterion, and 15, 9, and 3 days to criterion for old-1, old2 and old-7
respectively with doses of
1, 10, and 100 mg/kg).
The present animals were previously tested and assessed for cognitive deficits
using both
delayed matching to sample (DMTS) and paired associative learning (PAL) tasks.
These animals
were ranked accordingly to their level of performance on these tasks (i.e.,
animals were ranked
per task from 1 to 6, 1 being the best performer and 6 the worst (see Table
10). This ranking
showed that the animal that did not respond to HT-0712 was ranked 1 on
performance of the
PAL and 2 on performance of the DMTS. This same animal also had the best
performance on
the first vehicle study performed on the current project.
In summary, the results of this study show that HT-0712 significantly improves
the
learning ability and long term memory of normal aged non-human primates using
particular
systems and methods of the present invention.
Tables
Table 2: Descriptive statistics for the entire study group, including NHP old-
9
Mean Criterion Criterion
Deviation Error
vehicle day 26.333 11.237 4.587
toC
vehicle C 89.333 4.274 1.745
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value
vehicle 79.667 14.459 5.903
retention test
vehicle RT 77.500 12.486 5.097
1-14
vehicle RT 82.333 18.468 7.540
15-28
vehicle2 20.000 7.810 4.509
day to C
vehicle2 C 86.333 2.309 1.333
value
vehicle 64.000 7.000 4.041
retention test
vehicle2 59.333 14.572 8.413
RT 1-14
vehicle2 69.000 18.083 10.440
RT 15-28
l mg day to 12.667 7.394 3.018
C
I mg C 91.000 3.633 1.483
value
1 mg 82.833 11.788 4.813
retention test
1 mg RT 1- 77.500 14.068 5.743
14
l mg RT 88.167 10.852 4.430
15-28
mg day 14.000 7.043 2.875
toC
10mg C 89.333 5.428 2.216
value
10 mg 80.333 7.528 3.073
retention test
10 mg RT 73.833 8.976 3.664
1-14
10 mg RT 87.000 9.633 3.933
15-28
100 mg day 10.333 6.501 2.654
toC
100 mg C 86.333 1.366 0.558
value
100 mg 66.000 7.430 3.033
retention test
100 mg RT 59.333 8.477 3.461
1-14
100 m RT 72.667 12.580 5.136
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15-28
Legends: C: criterion; RT: retention test, RT 1-14 Retention test trials 1 to
14; RT 15-28:
retention test trials 15 to 28
Table 3: Descriptive statistics for the entire study group, excluding NHP old-
9
Mean Criterion Criterion
Deviation Error
Vehiclel 29.000 10.223 4.572
day to C
Vehiclel C 88.600 4.336 1.939
value
Vehiclel 79.200 16.115 7.207
retention test
Vehiclel 77.200 13.936 6.232
RT 1-14
Vehiclel 81.600 20.550 9.190
RT 15-28
vehicle2 22.000 9.899 7.000
day to C
vehicle2 C 85.000 0.000 0.000
value
Vehicle2 64.000 9.899 7.000
retention test
vehicle2 67.500 4.950 3.500
RT 1-14
vehicle2 60.500 14.849 10.500
RT 15-28
1 mg day to 12.000 8.062 3.606
C
1 mg C 91.400 3.912 1.749
value
1 mg 85.800 10.378 4.641
retention test
1 mg RT 1- 80.200 13.882 6.208
14
1 mg RT 91.600 7.668 3.429
15-28
mg day 11.800 5.070 2.267
toC
10mg C 90.000 5.788 2.588
value
10 mg 79.200 7.823 3.499
retention test
10 m RT 71.400 7.503 3.356
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1-14
mg RT 87.200 10.756 4.810
15-28
100 mg day 9.000 6.285 2.811
toC
100 mg C 86.400 1.517 0.678
value
100 mg 65.000 7.842 3.507
retention test
100 mg RT 58.400 9.127 4.082
1-14
100 mg RT 71.400 13.631 6.096
15-28
Legends: C: criterion; RT: retention test, RT 1-14 Retention test trials 1 to
14; RT 15-28:
retention test trials 15 to 28
5 Raw data per treatment
Table 4: Vehicle 1
Tr D C RT RT RT trials
eatment ays to C value overall trials 1-14 15-28
01 V 43 86 54.0 57.00 50.00
d-1 ehicle 1 .00 0
01 V 18 86 89.0 79.00 100.00
d-2 ehicle 1 .00 0
01 V 36 96 96.0 93.00 100.00
d-5 ehicle 1 .00 0
01 V 25 86 82.0 86.00 79.00
d-7 ehicle 1 .00 0
01 V 23 89 75.0 71.00 79.00
d-8 ehicle 1 .00 0
01 V 13 93 82.0 79.00 86.00
d-9 ehicle 1 .00 0
Table 5: 1mg/kg HT-0712
Tr D C RT RT RT trials
eatment ays to C value overall trials 1-14 15-28
01 1 21 86 93.0 93.00 93.00
d-l mg/kg .00 0
01 1 17 93 86.0 79.00 93.00
d-2 mg/kg .00 0
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O1 1 4 89 93.0 86.00 100.00
d-5 mg/kg .00 0
O1 1 15 93 68.0 57.00 79.00
d-7 mg/kg .00 0
01 1 3 96 89.0 86.00 93.00
d-8 mg/kg .00 0
01 1 16 89 68.0 64.00 71.00
d-9 mg/kg .00 0
Table 6: 10mg/kg HT-0712
Tr D C RT RT RT trials
eatment ays to C value overall trials 1-14 15-28
O1 10 15 86 75.0 64.00 86.00
d-l mg/kg .00 0
O1 10 18 86 71.0 71.00 71.00
d-2 mg/kg .00 0
O1 10 5 10 86.0 79.00 93.00
d-5 mg/kg 0.00 0
01 10 9 89 75.0 64.00 86.00
d-7 mg/kg .00 0
O1 10 12 89 89.0 79.00 100.00
d-8 mg/kg .00 0
01 10 25 86 86.0 86.00 86.00
d-9 mg/kg .00 0
Table 7: 100mg/kg HT-0712
Tr Da C RT RT RT trials
eatment s to C value overall trials 1-14 15-28
01 10 3 8 57.0 50.00 64.00
d-1 0 mg/kg 9.00 0
O1 10 11 8 68.0 50.00 86.00
d-2 0 mg/kg 6.00 0
O1 10 10 8 57.0 57.00 57.00
d-5 0 mg/kg 6.00 0
O1 10 3 8 68.0 71.00 64.00
d-7 0 mg/kg 6.00 0
O1 10 18 8 75.0 64.00 86.00
d-8 0 mg/kg 5.00 0
O1 10 17 8 71.0 64.00 79.00
d-9 0 mg/kg 6.00 0
Table 8: Vehicle 2
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Tr Da C RT RT RT trials
eatment s to C value overall trials 1-14 15-28
01 Ve 15 8 71.0 71.00 71.00
d-l hicle 2 5.00 0
01 Ve 29 8 57.0 64.00 50.00
d-5 hicle 2 5.00 0
O1 Ve 16 8 64.0 43.00 86.00
d-9 hide 2 9.00 0
Legends: C: criterion; RT: retention test, RT 1-14 Retention test trials 1 to
14; RT 15-28:
retention test trials 15 to 28
Table 9: Description of animals used in the study
ID Weight at start of Estimated age Sex
study
Old-1 9.9 kg 22 years old Male
Old-2 9.65 kg 22 years old Male
Old-5 8.4 kg 22 years old Male
Old-7 10.6 kg 22 years old Male
Old-8 9.8 kg 22 years old Male
Old-9 11.15 kg 22 years old Male
Table 10: Ranking of animals used in the study on PAL and DMTS
of of of of of of
d-1 d-2 d-5 d-7 d-8 d-9
PAL 4 5 6 2 3 1
DM
TS 6 1 5 4 3 2
mea 5. 1.
n rank 5 3 5 3 3 5
Rankings were calculated for PAL and DMTS task as follows:
1. PAL: Mean number of trials needed to complete the most difficult level of
the task (3
stimuli / 3 locations).
2. DMTS: Mean number of correct response at the longer delay.
Animals were ranked per task from 1 to 6, 1 being the best performer and 6 the
worst.
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EXAMPLE 2: SINGLE-BLIND STUDY IN HEALTHY, ELDERLY, HUMAN
SUBJECTS
Study Design
The study using the general protocol and drug administration as set forth in
Example 1
can be performed using humans. As described below, a long term retrieval task
can utilize
stimuli in the form of face - name and/or occupation pairings. The human
subjects can be shown
a picture of a face along with the name and/or occupation of a person. An
occupation may be
listed for each name-face pair. During encoding, the human subject is asked to
decide if the
name fits with the occupation. During the test, the human subject is shown
intact, rearranged,
and novel face-name pairs (presented without the occupation) and asked to
decide if the face and
name pairings are intact, rearranged, or novel. Using this testing, human
subjects can then be
evaluated in a manner as set forth in Example 1.
In one embodiment, a single site, double-blind, three-way cross-over study was
performed that tested the following three dosing regimens: (1) HT-0712 45 mg
po for 7 days;
(2) HT-0712 45 mg po plus warfarin 2.5 mg po for 7 days; and (3) Warfarin 2.5
mg po for 7
days. There was a seven-day washout periods between dosing periods. Subjects
remained in-
house for three (72 hours) of the seven days washout days, for study related
procedures.
Subjects were furloughed from the clinic for four of the seven washout days.
With regard to
subject selection criteria, there were no inclusion and/or exclusion criteria
for the paired
associate test.
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Study Schedule
Group I subjects received 45 mg doses of HT-0712 on Days 1,2,3,4,5,6, and 7;
45 mg
doses of HT-0712 plus 2.5 mg doses of warfarin on Days, 15, 16, 17, 18, 19,
20, and 21; and 2.5
mg doses of warfarin on Days 29, 30, 31, 32,33, 34, and 35. Group II subjects
received 45 mg
doses of HT-0712 plus 2.5 mg doses of warfarin on Days 1,2,3,4,5,6, and 7; 2.5
mg doses of
warfarin on Days 15, 16, 17, 18, 19, 20, and 21; and 45 mg doses of HT-0712 on
Days 29, 30,
31, 32,33, 34, and 35. Group III subjects received 2.5 mg doses of warfarin on
Days 1, 2,3,4,5,6,
and 7; 45 mg doses of HT-0712 on Days 15, 16, 17, 18, 19, 20, and 21; and 45
mg doses of
HT-0712 plus 2.5 mg doses of warfarin on Days 29, 30, 31, 32, 33, 34, and 35.
Table 11: Unblinded Subject Codes
G G G
roup 1 rou 2 roup 3
0 0 0
02 01 03
0 0 0
05 04 06
0 0 0
08 07 09
0 0 0
12 11 10
0 0 0
13 14
0 0 0
16 18 17
0 0 0
19 20 21
Paired Associates Testing
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Materials: Materials consisted of 21 Lenovo notebook computers. Each notebook
was
configured to eliminate all auto-configuration software and reduce settings
which might interfere
with the internal clock settings and E-Run software. E-Run (vl.2.1.68) was
installed on all the
notebook computers to administer the paired associate test. 120 grayscale
faces (male and
female) were paired with a two-word professions, such as "fire fighter."
Grayscale face images
were drawn from a face database from Lund University Cognitive Science Lab.
Only two-word
or compound-word professions were used. The 120 face-name pairs were divided
into three
groups of 40. During each round the same 40 face-name pairs were used
throughout the round.
Procedure - Timing of Administration: Memory testing commenced approximately 6
hours post-drug administration. All memory testing was completed prior to
daily blood samples
being taken. The training was performed in three Rounds (or Segments)
consisting of 14 days
as shown in Table 12. The testing was performed over 42 days. In a given round
on day 1,
Subjects were familiarized with the computers and trained how to use the test
software. On day
2 subjects were given an encoding and retrieval session. On days 3-7, subjects
were tested daily
for recall. On day 10 subjects were tested on half the face-name pairs, and on
day 14, subjects
were tested on the remaining half of the face-name pairs followed by a
complete test of all face-
name pairs used in that round.
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Table 12: Training and Testing
Round 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Day
Procedure Training Encoding Testing Testing Testing Testing Testing _Testing
Testin
40 40 40 40 40 Pairs Part I:
# of Pairs 40 Pairs Pairs Pairs Pairs Pairs Pairs 1-20 Pairs
21-40;
Part II:
Repeat
all 40
Pairs
Testing Procedure Familiarization: On day 1, subjects were familiarized with
the
computers and trained how to use the test software by receiving a mock
encoding and retrieval
session consisting of nine face-name pairs. These pairs were never observed
again.
Encoding and Retrieval: On Day 2, each participant received a single encoding
and
retrieval session:
Encoding: During the encoding session, participants see 40 faces paired with
professions
one at a time on a computer screen. For instance they see a face, and to the
right, the profession
"ice skater." Their task for each face-profession pair is to form a vivid
mental image in which
the person pictured is carrying out the profession appearing to the right.
Thus, for a face paired
with ice skater, they might imagine that person doing a difficult jump and
falling, or imagine
them very happy because they've won the gold medal. After 5 seconds of viewing
the face
profession pair, they were prompted to rate how easy it was to form the mental
image on a scale
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of 1-3: 1 if it was easy, 2 if it was difficult, 3 if they were unsuccessful
at creating a mental
image. After 2 seconds, the next face profession pair was displayed.
Retrieval: In this part of the experiment, participants were administered a
cued-recall test
for the 40 face-profession pairs they had seen in during Encoding. During the
retrieval test
subjects were presented face without the associated profession. Next to the
face there was a box
for them to type in the profession that was paired with that face during
encoding. Once the
participant types a response, they received feedback as to whether the
profession they typed was
correct or incorrect. If the response was correct, "Correct!" appeared on the
screen with the
appropriate face and profession pairing, followed by the next stimulus pair.
If it was incorrect,
the correct face and profession pairing was presented as feedback on the
screen for three
seconds, prior to the presentation of the next stimulus pair. The session
ended once all forty
face-profession pairs were presented. This retrieval session was presented
once, each day 3-7, of
the study.
Long-term memory assessment: Three days after each final drug dose and prior
to being
discharged (day 10, 24, and 38) participants were administered a cued-recall
test consisting of 20
face-profession pairs to measure long term stability of the face-profession
memories. Seven days
after each drug treatment (dayl4 and 28, and 42), participants were
administered the remaining
face-profession pairs which were not previously tested. On days 14 and 28, and
42, a minute
rest period followed the presentation of the 20 face-profession pairs.
Following this one minute
20 of rest, participants were administered the complete set of 40 face-
profession pairs.
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Results: Data reflecting participant recall was collected and graphed as shown
in Figure
5. These data show that long-lasting memory of a paired-associate task can be
established with sufficient
daily repetitions of training.
Blinding Procedure: The study was a double-blind placebo controlled trial. The
pharmacist had responsibility for maintaining subject treatment codes.
Thus, it is seen that various methods are provided. One skilled in the art
will appreciate
that the present invention can be practiced by other than the various
embodiments and preferred
embodiments, which are presented in this description for purposes of
illustration and not of
limitation, and the present invention is limited only by the claims that
follow. It is noted that
equivalents for the particular embodiments discussed in this description may
practice the
invention as well.
While various embodiments of the present invention have been described above,
it should
be understood that they have been presented by way of example only, and not of
limitation.
Likewise, the various figures may depict an example configuration for the
invention, which is
done to aid in understanding the features and functionality that may be
included in the invention.
The invention is not restricted to the illustrated example configurations, but
the desired features
may be implemented using a variety of alternative configurations. Indeed, it
will be apparent to
one of skill in the art how alternative functional, logical or physical
configurations may be
implemented to implement the desired features of the present invention.
Additionally, with
regard to method claims, the order in which the steps are presented herein
shall not mandate that
various embodiments be implemented to perform the recited functionality in the
same order
unless the context dictates otherwise.
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Although the invention is described above in terms of various exemplary
embodiments
and implementations, it should be understood that the various features,
aspects and functionality
described in one or more of the individual embodiments are not limited in
their applicability to
the particular embodiment with which they are described, but instead may be
applied, alone or in
various combinations, to one or more of the other embodiments of the
invention, whether or not
such embodiments are described and whether or not such features are presented
as being a part of
a described embodiment. Thus the breadth and scope of the present invention
should not be
limited by any of the above-described exemplary embodiments.
Terms and phrases used in this document, and variations thereof, unless
otherwise
expressly stated, should be construed as open ended as opposed to limiting. As
examples of the
foregoing: the term "including" should be read as meaning "including, without
limitation" or the
like; the term "example" is used to provide exemplary instances of the item in
discussion, not an
exhaustive or limiting list thereof; the terms "a" or "an" should be read as
meaning "at least
one," "one or more" or the like; and adjectives such as "conventional,"
"traditional," "normal,"
"criterion," "known" and terms of similar meaning should not be construed as
limiting the item
described to a given time period or to an item available as of a given time,
but instead should be
read to encompass conventional, traditional, normal, or criterion technologies
that may be
available or known now or at any time in the future. Likewise, where this
document refers to
technologies that would be apparent or known to one of ordinary skill in the
art, such
technologies encompass those apparent or known to the skilled artisan now or
at any time in the
future.
A group of items linked with the conjunction "and" should not be read as
requiring that
each and every one of those items be present in the grouping, but rather
should be read as
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"and/or" unless expressly stated otherwise. Similarly, a group of items linked
with the
conjunction "or" should not be read as requiring mutual exclusivity among that
group, but rather
should also be read as "and/or" unless expressly stated otherwise.
Furthermore, although items,
elements or components of the invention may be described or claimed in the
singular, the plural
is contemplated to be within the scope thereof unless limitation to the
singular is explicitly
stated.
The presence of broadening words and phrases such as "one or more," "at
least," "but not
limited to" or other like phrases in some instances shall not be read to mean
that the narrower
case is intended or required in instances where such broadening phrases may be
absent. As will
become apparent to one of ordinary skill in the art after reading this
document, the illustrated
embodiments and their various alternatives may be implemented without
confinement to the
illustrated examples.
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